HK1186117A - Compositions and methods to inhibit stem cell and progenitor cell binding to lymphoid tissue and for regenerating germinal centers in lymphatic tissues - Google Patents

Description

Compositions and methods for inhibiting stem and progenitor cell binding to lymphoid tissue and for regenerating germinal centers in lymphoid tissue

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 61/374,943 filed on 18/2010, 61/441,485 filed on 10/2/2011, and 61,449,372 filed on 4/3/2011. All of the above applications are hereby incorporated by reference in their entirety.

Technical Field

The presently disclosed subject matter relates to methods and compositions for modulating stem cell binding to organs and tissues and for regenerating germinal centers in lymphoid tissues.

Background

Regenerative medicine is a method of creating living functional tissue to repair or replace tissue or organ function lost due to injury or congenital defects. By stimulating the self-healing of previously irreparable organs, the art has hopefully developed for the regeneration of damaged tissues and organs within the body.

One method for regenerating tissue or organ function is to deliver stem cells to the affected organ or tissue. However, even when stem cells are directly injected into the tissue of a damaged organ, the stem cells are not well retained in the target organ for tissue regeneration. Imaging studies in humans and animals demonstrated that within one hour after injection of stem cells, the majority of the stem cells delivered can be found in the spleen. Animal studies have also demonstrated that: surgical removal of the spleen after induction of myocardial infarction and prior to stem cell therapy improves functional recovery of the injured heart (Blood, Vol.84, No. 5: 1482-. Splenectomies have also been shown to improve engraftment in human patients following bone marrow transplantation (Stem Cells Dev13(1):51-62,2004; Transplant Proc.28(2): 736-83, 1996; Am J Hematol.22(3):275-83, 1986). However, splenectomy has also been associated with surgical mortality, sepsis and life-long thrombotic complications (Blood Rev.14(3): 121-.

Therefore, there is a need to develop methods and compositions for preventing stem cells from being localized in the spleen and other lymphoid tissues without removing the spleen.

Disclosure of Invention

SUMMARY

The present invention meets this need by providing methods and compositions for inhibiting the binding of stem cells to lymphoid tissue, comprising administering stem cells to an individual along with one or more therapeutic agents that inhibit the binding of stem cells to germinal centers in lymphoid tissue, particularly lymph nodes and germinal centers in the spleen. The term 'in conjunction with' means together with, before or after stem cell processing. By 'stem cell processing' is meant the following actions: administering stem cells to an individual, mobilizing stem cells from an individual's endogenous stem cell reserve, or relying on the spontaneous release of stem cells from an individual's endogenous stem cell reserve.

For example, patients treated with stem cells to cause organ regeneration have demonstrated decreased mortality and improved function following stem cell therapy, although stem cell treatment often fails to restore patients to their functional state prior to organ damage. Reducing stem cell binding to the spleen and other lymphatic vessels increases the number of circulating stem cells that can be attracted to the damaged organ and thereby increases the degree of functional recovery induced by stem cell treatment of the patient.

In administering a therapeutic agent that inhibits binding of stem cells to lymphoid tissue, the therapeutic agent is preferably administered 1-14 days prior to treatment, more preferably 3-7 days and most preferably 3-4 days prior to treatment or mobilization of stem cells with stem cells. In administering a therapeutic agent that inhibits binding of stem cells to lymphoid tissue in conjunction with stem cells that are spontaneously released from an individual's endogenous stem cell reserve, the therapeutic agent is preferably administered over a period of 1-60 days, more preferably 1-30 days, and most preferably 1-14 days.

Agents that inhibit the binding of stem cells to lymphoid tissues, particularly the germinal centers of lymphoid tissues, include radiation, chemotherapeutic agents, immunosuppressive agents, and CD45 antagonists and CD26 antagonists. Stem cells found within mononuclear fractions from whole blood or bone marrow or purified stem cells from whole blood or bone marrow bind to germinal centers in the white marrow region in the spleen, more particularly in the white marrow of lymphoid tissues including the spleen, and even more particularly active germinal centers in the white marrow of the spleen. Antibodies to the epitope recognized by CD45, particularly 30-F11 rat IgG2b anti-mouse anti-CD 45 monoclonal antibody, reduce binding of stem cells to the identified site in the spleen, thereby making more stem cells available for biodistribution to the targeted damaged organ and enhancing tissue regeneration and functional recovery.

In another embodiment of the invention, a therapeutic agent that reduces, destroys, or ablates active germinal centers in lymphoid tissue is administered, thereby reducing binding of stem cells to lymphoid tissue. Another embodiment of the invention describes a method of reducing the number of active germinal centers in the spleen to reduce binding of stem cells to the spleen, thereby increasing the number of circulating stem cells available for delivery or homing to damaged organs in need of repair. Agents that inhibit the immune response may reduce the number of active germinal centers in the spleen and other lymphoid tissues. General classes of immunomodulators include agents that interfere with purine synthesis, antimetabolites, radiation to the spleen, immunosuppressive agents, glucocorticoids, anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents, anti-eotaxin, anti-T Cell Receptor (TCR) agents, anti-interferon alpha agents, anti-interferon beta agents, anti-interferon gamma agents, anti-TGF alpha agents, anti-TGF beta agents, anti-integrin agents, anti-alpha 4 agents, anti-interleukin 1 agents, anti-interleukin 2 agents, anti-interleukin 4 agents, anti-interleukin 5 agents, anti-interleukin 6 agents, anti-interleukin 12 agents, anti-interleukin 13 agents, anti-interleukin 23 agents, anti-IgE agents, anti-Vascular Adhesion Protein (VAP) agents, anti-B7 agents, anti-Vascular Endothelial Growth Factor (VEGF) agents, anti-baff (blys) agents, anti-CTLA 4 agents, anti-complement agents, anti-CD 2 agents, anti-CD 3 agents, anti-CD 4 agents, anti-CD 5 agents, anti-CD 20 agents, anti-CD 23 agents, anti-CD 25a agents, anti-CD 40 agents, anti-CD 154(CD40L) agents, anti-CD 62L agents, anti-CD 80 agents, anti-CD 147 agents, anti-LFA 1 agents, anti-CD 11a agents, anti-CD 18 agents, purine synthesis inhibitors, pyrimidine synthesis inhibitors, anti-proliferative agents, anti-metabolic agents, anti-folate agents, and anti-mTOR agents.

As with agents that trigger the accumulation of deoxyATP, adenosine deaminase deficiency also results in a reduction in the formation of active germinal centers (J Immunol171:5562-5570, 2003). Similarly, agents that enhance CCR7 expression or activate CCR7 result in a decrease in the formation of active germinal centers.

Chemotherapeutic agents may also be used to inhibit the formation of germinal centers in lymphoid tissues or to disrupt or ablate germinal centers. Representative examples include alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antineoplastic agents, and arsenic trioxide.

Examples of alkylating agents include cisplatin (cissplatin) and carboplatin (carboplatin), as well as oxaliplatin (oxaliplatin), which are alkylating agents. They impair cellular function by forming covalent bonds with amino, carboxyl, sulfhydryl and phosphate groups in biologically important molecules.

Examples of antimetabolites are azathioprine, mercaptopurine, capecitabine (capecitabine), fluorouracil, which are building blocks of DNA. They prevent these substances from being incorporated into DNA during the "S" phase (of the cell cycle), thereby terminating normal development and division. They also affect RNA synthesis. Because of their effectiveness, these drugs are the most widely used cytostatics.

The alkaloids include vinca alkaloids and taxanes. Vinca alkaloids include vincristine (vinchristin), vinblastine (vinblastin), vinorelbine (vinorelbine) and vindesine (vindesine). Taxanes include paclitaxel (taxol), taxol (paclitaxel), and docetaxel (docetaxel).

Topoisomerase is an enzyme essential to maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with DNA transcription and replication by disturbing the correct DNA supercoils. Some type I topoisomerase inhibitors include camptothecin (camptothecin): irinotecan (irinotecan) and topotecan (topotecan). Examples of type II inhibitors include amsacrine (amsacrine), etoposide (etoposide), etoposide phosphate and teniposide (teniposide).

Antineoplastic agents include dactinomycin (dactinomycin), doxorubicin (doxorubicin), epirubicin (epirubicin), and bleomycin (bleomycin).

Definition of

Definitions used to describe embodiments of the invention:

the term agonist as used herein means any entity that activates a specific receptor or downstream signaling pathway necessary to mediate the action of the receptor. Agonists may include, but are not limited to, antibodies, antibody fragments, soluble ligands, small molecules, cyclic peptides, cross-linking agents.

The term antagonist as used herein means any entity that interferes with the binding of the inverse structure (counter structure) of the receptor or with the activation of a specific receptor or downstream signaling pathway necessary to mediate the action of the receptor. Antagonists may include, but are not limited to, antibodies, antibody fragments, soluble ligands, Fc fusion receptors, chimeric receptors, small molecules, cyclic peptides, peptides.

The term inhibitor as used herein means any entity that reduces the targeted effect of a specific ligand or its receptor. The inhibitor may be a small molecule, an antisense agent, a nucleic acid including siRNA and micro rna (microrna).

Lymphoid tissue, lymph nodes, spleen and germinal center

Lymphoid tissue is a specialized form of reticular connective tissue in the lymphatic system that contains a large number of lymphocytes. This tissue type constitutes the spleen, thymus and tonsil, together with the visceral node, peyer's patch and all chylomicrons associated with the gastrointestinal mucosa.

Lymph nodes are small spherical organs of the immune system, widely distributed throughout the body including the axilla and stomach/internal organs and connected by lymphatic vessels. Lymph nodes are the premises for B cells, T cells and other immune cells (garrison). Lymph nodes are distributed throughout the body and act as filters or traps for foreign particles. The lymph nodes are surrounded by fibrous sacs that extend to form trabeculae within the lymph nodes. The lymph node body (substancece) is divided into the outer cortex and the inner medulla, which is entirely surrounded by the former except at the hilum where it is in direct contact with the surface. The outer cortex consists mainly of B cells arranged as follicles (folliculules), which can develop into germinal centers when challenged with antigen, and the deeper cortex consists mainly of T cells. There is a region, called the subcortical region, in which T cells (or primarily erythrocytes) interact primarily with dendritic cells, and in which the reticular network is dense.

The spleen is the intraperitoneal organ located on the left side of the abdomen, between the stomach and the diaphragm. This organ is the main site of the immune system for emptiness. It is a vascular organ (vascular organ) with a large arterial blood supply. Upon entering the spleen, blood flows into the reticulum or "sinus" of dilated blood vessels (located between a large number of lymphocytes). The sinus wall contains phagocytic cells that are capable of phagocytosing dead cells and foreign particles in the blood and removing them from the systemic circulation. Like lymph nodes, spleen is an important source of antibodies, however, it is to a greater extent than lymph nodes, which are involved in their removal from circulation by destroying abnormally or normally depleted ("dead") red blood cells.

The spleen contains white and red pith. The red marrow of the spleen holds macrophages that normally filter from circulation and remove senescent or defective Red Blood Cells (RBCs) and antibody-coated bacteria or red blood cells. The white marrow of the spleen contains lymphoid compartments (lymphoid complexes) and is critical for immune surveillance and response: it synthesizes antibodies against invading pathogens and releases platelets and neutrophils in response to bleeding or infection. The spleen is thought to have multiple roles during development, including as a hematopoiesisThe first part of (at the sixth week of gestation). Although it has long been thought that spleen loses its hematopoietic function during early developmental myeloid hematopoietic replacement, recent studies have identified adult spleen as the site of stem cell production, stem cell differentiation into different lineages, and stem cell storage (Trends Mol Med11(6):271-276, 2005). However, the site of accumulation of exogenous stem cells within the spleen and the molecular mechanism by which exogenous stem cells bind to the spleen are not known.

Periarterial lymphoid sheaths (PALS) of the splenic white marrow are predominantly populated by T cells, while the lymphoid fraction is predominantly populated by B cells. Germinal Centers (GCs) are sites within lymph nodes or lymph nodules in peripheral lymphoid tissues and in the white marrow of the spleen where dense mature B lymphocytes (otherwise known as central cells) rapidly proliferate, differentiate, mutate by somatic hypermutation and undergo class switching (class switch) during antibody responses. Germinal centers are an important part of the B cell humoral immune response. They develop dynamically after B cells are activated by T-dependent antigens. Histologically, GC describes microscopically resolvable fractions in lymphoid tissue. Activated B cells migrate from the primary aggregation center into the primary follicular system (primary folliculular system) and begin monoclonal expansion in the context of Follicular Dendritic Cells (FDCs).

After several days of expansion, B cells mutate their antibody-encoding DNA and thus produce multiple clones in germinal centers. This involves random substitutions, deletions and insertions resulting from somatic hypermutations. Under some unidentified stimuli from FDC, mature B cells (central blasts) migrate from dark to light regions and begin to expose their antibodies to their surface, and are called central cells at this stage. The central cell is in an activated apoptotic state and competes for survival signals from the antigen-presenting FDCs. This rescue process is believed to be dependent on the affinity of the antibody for the antigen. Functional B cells then interact with helper T cells to derive the final differentiation signal. This also involves isotype switching, for example from IgM to IgG. Interaction with T cells is thought to prevent the production of self-reactive antibodies. B cells become antibody-transmitting plasma cells or memory B cells that are activated in subsequent contact with the same antigen. They can also restart the entire process of proliferation, mutation and selection according to the cycle hypothesis.

The B cells contained within the splenic white marrow region can be further divided into specific regions, which are identified by staining with specific molecular markers. The marginal zone of the spleen contained non-circulating mature B cells that were bounded by white and red marrow, forming a space between the white and red marrow and expressing high levels of CD21 and IgM and CD24 and CD79a, and measurable levels of CD9 and CD 22. The coat region surrounds the normal germinal central follicle and expresses CD21, CD23, and CD 38. The follicular region is contained within the germinal center and expresses high levels of IgD and CD23, moderate levels of CD21 and CD24, and can also be identified by PNA staining. Germinal centers are best distinguished by PNA binding and express higher levels of CD54 than the follicular region. Germinal centers have a particular population of helper T cells that appear to be evenly distributed among all germinal centers. Germinal centers have traditionally been associated with immune responses requiring T helper cells, although this is not absolute. The germinal center is where hypermutable gene mutations occur and B cells produce high affinity IgG. Active germinal centers have tangible (tandiable) macrophages and dendritic cells expressing CD 21. Follicular centers can also be identified by expression of CD45R (B220) [ clinical Pathology,35:366-375,2007 ]. The CD45R follicular center was found to surround the germinal center expressing Bcl6 and Bcl 2. BioEssays29:166-177, 2007; toxicol Pathol34(5):648-

CD45 is a common leukocyte antigen, also known as PTPRC (protein tyrosine phosphatase, receptor type C), found on all differentiated hematopoietic cells except erythrocytes and plasma cells. It is also expressed in lymphomas, B-cell chronic lymphocytic leukemia, hairy cell leukemia, and acute non-lymphocytic leukemia. It has been shown to be essential in B cell and T cell antigen receptor signaling. The CD45 family consists of multiple members that are all the products of a single complex gene. This gene contains 34 exons, and the three exons of the primary transcript can be selectively spliced to produce up to eight different mature mrnas, and eight different protein products after translation. These three exons produce the RA, RB and RC isoforms.

There are different isoforms of the CD45 antigen. CD45 antigen isoforms include CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45RA is located on native T cells and CD45RO is located on memory T cells. CD45 is also highly glycosylated. CD45R is the longest protein and migrates at 200kDa when isolated from T cells. B cells also express CD45R with a greater degree of glycosylation, bringing the molecular weight to 220kDa, hence the name B220, a 220kDa B cell isoform. B220 expression is not limited to B cells and may also be expressed on activated T cells, subsets of dendritic cells, and other antigen presenting cells. Native T lymphocytes express a large number of the CD45 isoform and are generally positive for CD45 RA. Activated and memory T lymphocytes express the shortest CD45 isoform, CD45RO, which lacks RA, RB, and RC exons. This shortest isoform contributes to T cell activation. The cytoplasmic domain of CD45 is one of the largest known and it has intrinsic phosphatase activity that removes the inhibitory phosphate group on tyrosine kinases called Lck (in T cells) or Lyn/Fyn/Lck (in B cells) and activates the tyrosine kinase. CD45 may exist as monomers and dimers, and dimerization may down-regulate CD45 phosphatase activity (Blood, Vol. 103 (9):3440-3447, 2004).

Since CD45 is expressed on and is most widely expressed among all hematopoietic Cells, it has been used in other models of transplantation and Stem cell reconstitution to isolate cell populations also containing hematopoietic Stem Cells, however, mesenchymal Stem Cells, although derived from a population of CD45+ early precursors, are generally found to be CD45 negative (Stem Cells28: 140-. The complete absence of all CD45 isoforms has been demonstrated in mice to affect stem cell retention, motility and homing into the bone marrow and to play a role in the generation of functional B cells in the spleen from early stem cells (J.exp. Med.205: 2381-. Interestingly, the number of cKit + Lin hematopoietic progenitors decreased in bone marrow but increased in spleen in CD45 knockout mice that lack all CD45 isoforms.

30-F11 was a rat monoclonal IgG2b cultured against thymus and spleen of mouse origin. Clone 30-F11 reacted to both the CD45 alloantigen (CD45.1 and CD45.2) and all isoforms. 30-F11 and clone 30-F4 each blocked binding of the other to CD45, indicating that they bound the same or overlapping epitope on CD45 (J Immunol127(3):982-986, 1981). Again, both clones cross-blocked the antibody described by Dennerrt et al, however, anti-CD 45 antibody 55-6.1 did not cross-block 30-F11 or 30-F4 (Cell Immunol53:350-364, 1980).

The radiolabeled 30-F11 antibody showed the highest accumulation in mouse spleen (60%) but only 20% accumulation in bone marrow (Blood93(2):737-745,1999) and has been used in mice for delivery of targeted hematopoietic irradiation. The 30-F11 antibody has been used to identify stem cell fractions from mouse muscle along with the Sca1 antigen because CD45 is expressed on all hematopoietic cells (PNAS991341-1346,2002). Radiolabeled F (ab)' 2 fragments of 30-F11 and 30-F11 have been evaluated as a method of delivering radiation therapy. 30-F11 uptake was most pronounced in the mouse spleen, which was the axillary lymph node (Cancer Res52(5):1228-34, 1992).

CD45 polypeptides can be produced by published procedures. Methods for preparing polyclonal and monoclonal Antibodies against CD45 are well known in the art (see, e.g., Sambrook et al, Molecular Cloning: A laboratory Manual, second edition (Cold Spring Harbor, N.Y.,1989) and Hurrell, J.G.R., Ed., Monoclonal hybrids Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, Fla.,1982), which are incorporated herein by reference). It will be apparent to one of ordinary skill in the art that polyclonal antibodies can be produced from a variety of warm-blooded animals, such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice, and rats. The production of humanized antibodies is well known. See, Riechmann L, Clark M, Waldmann H, Winter G (1988), "rehaping human antibodies for therapy". Nature332(6162):332: 323; queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA. (12.1989.) "A humanized antibody bits to the interleukin2 receiver.". Proc Natl Acad Sci U S.A. 86(24): 10029-33; norderhaug L, OlafsenT, Michaelsen TE, Sandlie I. (5 months 1997), "Versatile vectors for transfer and stablexpression of recombinant antibody molecules in mammalian cells." J Immunol Methods204(1): 77-87.

Methods and reagents for preventing stem cells from binding to lymphoid tissue

Other particular embodiments provide methods for moderating binding of stem cells to the spleen by exposing the spleen to a solution of a cluster of antagonists of the differentiation 45(CD45) antigen. Solutions of antagonists of CD45 antigen can be formulated or formulated to bind the 30-F11 epitope. Solutions of antagonists to CD45 antigen can be formulated to promote therapeutic regeneration by increasing the delivery of stem cells to damaged tissues or organs. Solutions of antibodies containing the CD45 antigen can be formulated to bind the 30-F11 epitope and the human equivalent of the 30F-11 epitope.

The present invention describes methods of reducing stem cell binding to lymphoid tissues (e.g., lymph nodes and spleen) and the use of these methods to treat human patients. The present invention describes specific sites within the spleen where circulating stem cells bind in the spleen and methods of increasing or decreasing stem cell binding to such sites. Immunofluorescence and histological analysis of fresh thick spleen sections demonstrated that stem cells circulating in the vascular system bind to active germinal centers in the spleen when administered in vivo or ex vivo as shown in examples 1,2, 3 and 4. One method of reducing the amount of stem cells that bind to the spleen is to deliver an agent that blocks the administered stem cells from binding to molecular targets on the active germinal centers of the spleen. According to the methods of the invention, antibody 30-F11 binds to mouse CD45 antigen and blocks stem cell binding to the germinal center of mouse spleen. Anti-human anti-CD 45 antibodies that may be equivalent to 30-F11 rat IgG2b anti-mouse CD45 include: YAML568 recognizing epitope P of human CD45 (J Nucl Med47:1335-1341, 2006; in Leucocyte Typing III: White cell differentiation antibodies 811-814, 1987; Transplantation40:538-544,1985), anti-CD 45 clones HI30 or YTH-24 and YTH-54 anti-human anti-CD 45 antibodies.

Another embodiment of the invention is the use of an antagonist of CD26, such as an antibody to CD26, to inhibit stem cell binding to germinal centers. Since CD26 is expressed by stem cells and is an antigen present on stem cells that attach to germinal centers in lymphoid tissues, particularly lymphoid tissues. By blocking CD26 on stem cells, stem cells are unable to bind to lymphoid tissues.

Agents for inhibiting, down-regulating the formation of, destroying or ablating germinal centers

Another embodiment of the invention is to inhibit stem cell binding to lymphoid tissue or to destroy or ablate germinal centers by inhibiting or down-regulating germinal center proliferation. Germinal Centers (GCs) develop dynamically after B cells are activated by T-dependent antigens. The T cell antigen that activates B cells and thus induces germinal center proliferation is CD40L (also known as CD154) that binds to the CD40 receptor present on B cells. This binding of CD40L to the CD40 receptor not only activates B cells but also induces proliferation of hair centers. Accordingly, another embodiment of the invention comprises administering to an individual an agent that inhibits the binding of CD40L to CD 40. Examples of such agents are antagonist antibodies to CD40 or CD 40L.

Another protein important for the development of germinal centers is the ` Signaling lymphocyte activating molecule-associated protein ` (SAP) (Hai Qui et al, Nature,455: 764-.

IL-21 is another polypeptide important for the differentiation and proliferation of B cells in the center of development through B cell-intrinsic mechanisms. The absence of IL-21 signaling significantly affects B cell responses to protein antigens, thereby reducing spleen and bone marrow plasma cell formation and GC persistence and function, affecting their proliferation, transitioning to memory B cells and affinity maturation [ Zotos, D. et al, JEM207: 365-. Thus, by administering an antibody to an antagonist such as IL-21 to an individual, germinal centers can be down-regulated and their formation inhibited. This would inhibit stem cell binding to lymphoid tissue and spleen due to the lack of germinal centers in lymphoid tissue.

Chemotherapeutic agents can inhibit the binding of stem cells to the germinal center of lymphoid tissues including lymph nodes, Peyer's patches and spleen white marrow. Likewise, agents that inhibit the immune response may reduce the number of active germinal centers in the spleen. These agents include:

azathioprine (Azathioprine)Prometheus Laboratories, San Diego, Calif.), preferably 3-5mg/kg per day 3-4 days before administration of the stem cells. Azathioprine interferes with the synthesis of purines (adenine and guanine) required for DNA synthesis. Rapidly growing cells (including T cells and B cells) are particularly affected by inhibition of purine synthesis.

Corticosteroids such as dexamethasone (dexamethasone), prednisolone (prednisolone), methylprednisolone (methylprednisolone), dexamethasone sodium phosphate, and betamethasone (betamethathasone). Dexamethasone tablets (Merck) and dexamethasone sodium phosphate injection may be administered 1-14 days before treatment with stem cells, more preferably 3-7 days before treatment with stem cells and most preferably 3-4 days. The total amount of dexamethasone administered is an amount sufficient to down-regulate germinal centers in the lymphoid tissue such that stem cells cannot bind to the lymphoid tissue. The total amount of dexamethasone during this period may range from 2mg to 3g, preferably a total of 27 mg. The daily dose of dexamethasone can range from 0.75mg to 700mg per day, preferably 7mg per day. Like other glucocorticoid steroids, dexamethasone inhibits the formation and proliferation of hair-growth centers in lymphoid tissues.

Mycophenolic acid (Mycophenolic acid) (II)Delayed release capsules, Novartis pharmaceuticals corporation East Hanover, New Jersey, 720mg, administered twice daily on an empty stomach (total daily dose 1440mg), either before or after one hour of ingestion of the dry cells), preferably 3-4 days before administration of the dry cells. (Roche Labs, Nutley, NJ, mycophenolate mofetil) tablets and capsules, oral suspension, mycophenolate mofetil hydrochloride) for intravenous injection, 2-morpholinoethyl ester of mycophenolic acid (MPA), IV administered twice daily at 1g, orally administered twice daily at 1.5g, preferably 3-4 days prior to administration of the stem cells. It inhibits inosine monophosphate dehydrogenase, an enzyme that controls the rate of synthesis of guanine monophosphate in the purine de novo synthesis pathway used in the proliferation of B and T lymphocytes. Mycophenolate mofetil is potent and can be used in place of earlier antiproliferative azathioprine. It is commonly used as part of a three-compound regimen of immunosuppressants, also including calcineurin inhibitors (cyclosporine or tacrolimus) and prednisolone.

Leflunomide (Leflunomide) (Sanofi-Aventis u.s.llc, Bridgewater, NJ), 100mg daily for three days, 3-4 days before stem cell administration. Leflunomide is a pyrimidine synthesis inhibitor belonging to the DMARD (disease modifying antirheumatic drug) class of drugs, which are very heterogeneous chemically and pharmacologically. Leflunomide is an immunomodulatory drug that inhibits dihydroorotate dehydrogenase (an enzyme involved in the de novo synthesis of pyrimidines), abbreviated DHODH.

Teriflunomide (Teriflunomide) is an active metabolite of leflunomide (Sanofi-Aventis u.s.llc, Bridgewater, NJ) 100mg per day for three days, preferably 3-4 days before administration of stem cells.

Methotrexate (Methotrexate) is an antimetabolite and antifolate. It acts by inhibiting folate metabolism. Preferably, it is administered orally or intramuscularly at a dose of 15 to 30mg per day for up to five days 3-4 days before the administration of the stem cells. Mylan Pharmaceuticals, Morganown, West Virginia. Immunosuppressive macrolides:

tacrolimus (Tacrolimus) reduces the production of interleukin-2 (IL-2) by T cells in the form of capsules or injection, 0.10-0.15 mg/kg/day, preferably 3-4 days before the administration of stem cells. (Astellas Pharma US, Inc. Deerfield, IL).

Cyclosporin (Ciclosporin) -a cytoplasmic protein cyclophilin (immunophilin) which is believed to bind to immunocompetent lymphocytes, particularly T lymphocytes. AsSold in the form of capsules, oral solutions or injections, preferably administered at 14-18 mg/kg/day 3-4 days prior to administration of the stem cells. (Novartis pharmaceuticals corporation, East Hanover, New Jersey).

Pimecrolimus (Pimecrolimus) (Elidel) is an ascomycin macrolactam derivative. Pimecrolimus has been shown to bind to penphilus protein-12 and inhibit calcineurin in vitro. Pimecrolimus therefore inhibits T cell activation by inhibiting cytokine synthesis and release from T cells. Pimecrolimus also prevents the release of inflammatory cytokines and mediators from mast cells. Preferably pimecrolimus is used as a topical 1% cream for up to 6 weeks prior to stem cell therapy.

Guanolimus (Guspermimus) is a derivative of the antitumor antibiotic Segal (spergualin) and inhibits the maturation of interleukin-2 stimulated T cells to the S and G2/M phase and the polarization of T cells into IFN- γ -secreting Th1 effector T cells, resulting in the inhibition of growth of activated native CD4T cells. It is SC administered, 0.5 mg/kg/day, for 21 days, preferably 3-4 days prior to administration of the stem cells. Nippon Kayaku co., Ltd.

Everolimus (Everolimus) (RAD-001), preferably administered orally at a dose of 10 mg/day 3-4 days prior to stem cell administration. Novartis, East Hanover, NJ under the trade name Zortress (USA).

Thalidomide (Thalidomide) -Thalidomide can reduce levels of TNF alpha, ((S) (), (Celgencorporation, Summit, NJ). An acceptable dose is 100-300 mg/day, preferably at bedtime 1 hour after dinner, preferably 3-4 days before stem cell administration.

Lenalidomide (Lenalidomide) is a derivative of thalidomide, is 50,000 times more potent in inhibiting tumor necrosis factor-alpha than thalidomide, and has fewer severe adverse drug reactions. Celgene Corporation, Summit, NJ), 25mg is preferably administered orally once daily on days 1-21, 3-4 days prior to administration of the stem cells.

Anakinra (Anakinra) is a recombinant, non-glycosylated form of human IL-1RA (RA refers to receptor antagonist) ((RA))Biovitrum, Stockholm, Sweden), is delivered as an injection concentrate containing 100mg per single dose within 7-14 days, and preferably within 3-4 days, prior to administration of the stem cells.

Infliximab (Infliximab) (trade name)) Is a monoclonal antibody directed against tumor necrosis factor alpha (TNF α). Centocor Ortho Biotech, Horsham, PA, is administered by intravenous infusion at a dose of 3mg/kg up to 10mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Golimumab (Golimumab) (CNTO148) is a human monoclonal antibody and is sold under the trade name Simponi. The golimumab targets TNF-alpha. Centocor Ortho Biotech, Horsham, PA, is administered as a subcutaneous injection of 50mg in 0.5ml within 7-14 days and preferably within 3-4 days before the stem cells are administered.

Adalimumab (Adalimumab) (HUMIRA, Abbott Laboratories, North Chicago, IL) is a TNF inhibitor, Adalimumab binds TNF α, preventing it from activating TNF receptors; adalimumab was constructed from a fully human monoclonal antibody and sold pre-filled with 0.8mL syringes and pre-filled pen devices each containing 40mg adalimumab. To down-regulate germinal centers prior to administration of stem cells, at least 40mg of adalimumab should be administered within 7-14 days, and preferably within 3-7 days, prior to administration of stem cells. Preferably, two 40mg doses of adalimumab should be administered within 7-14 days and preferably within 3-7 days prior to administration of stem cells.

Certolizumab pegol (Certolizumab pegol) is a monoclonal antibody directed against tumor necrosis factor alpha. More precisely, it is a pegylated Fab' fragment of a humanized TNF inhibitor monoclonal antibody. It is administered as two subcutaneous injections of 200mg injection within 7-14 days and preferably within 3-4 days prior to the administration of the stem cells. (UCB Inc., Atlanta, Georgia).

Temsirolimus (Temsirolimus) (Pfizer Corp.) is a specific inhibitor of mTOR (a mammalian target of rapamycin) and interferes with the synthesis of proteins that regulate the proliferation, growth and survival of tumor cells. The recommended dose of temsirolimus is an IV infusion of 25mg for 30-60 minutes within 7-14 days and preferably within 3-4 days prior to stem cell administration.

Azololimus (Zotarolimus) is a semisynthetic derivative of rapamycin, Abbot Laboratories, North Chicago, IL).

Biolimus (Biolimus) a 9: biosensors International, Singapore

Eculizumab (ecilizumab) (trade name Soliris) is a monoclonal antibody directed against complement protein C5. This antibody blocks C5 cleavage and interrupts the complement-mediated cell destruction process. Within 7-14 days and preferably within 3-4 days prior to administration of the stem cells, Soliris is administered as an IV infusion administered at a dose of 600mg or 900 mg. (Alexion pharmaceuticals Cheshire, CT)

Mepolizumab (Mepolizumab) (proposed trade name Bosatria) is a humanized monoclonal antibody that recognizes interleukin-5 (IL-5) and is administered as an infusion of 750mg within 7-14 days and preferably within 3-4 days prior to the administration of stem cells. GlaxoSmithKline, King of Prussia, Pa.

Omalizumab (Omalizumab) (trade name Xolair, Genentech/Novartis) is a humanized antibody. Omalizumab is a humanized IgG1k monoclonal antibody of recombinant DNA origin that selectively binds to human immunoglobulin e (ige). Subcutaneous injections are administered at doses of 150 to 375mg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Nerrimumab (BAYX) is a mouse anti-TNF antibody and can be administered at 10mg/kg within 7-14 days and preferably within 3-4 days prior to administration of stem cells.

Faradalimumab (Faralimomab) is a mouse anti-TNF antibody and can be administered at 10mg/kg within 7-14 days and preferably within 3-4 days prior to administration of stem cells.

Exemplar (Elsilimomab) (also known as B-E8) is a mouse monoclonal antibody and immunosuppressive drug. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Lyratizumab (Lebrikizumab) is a humanized monoclonal antibody designed to specifically bind IL-13 and may be administered at 10mg/kg within 7-14 days and preferably within 3-4 days prior to administration of stem cells. Genentech, South san francisco, California.

Ultekinumab (Experimental name CNTO1275, patent name Stelara, Centocor) is a human monoclonal antibody. It is directed against interleukin 12 and interleukin 23, naturally occurring proteins that regulate the immune system and immune-mediated inflammatory disorders. 90 or 45mg injections or a single 45mg injection are made within 7-14 days and preferably within 3-4 days of the administration of the stem cells, 2 at one month intervals.

Moluomab (Muromonab) -CD3 (trade name Orthoclone OKT3, sold by Janssen-Cilag) is a monoclonal antibody that targets the CD3 receptor (membrane protein on the surface of T cells). It is administered at 5 mg/day by a single intravenous bolus for 10 to14 days. Administration should be completed within 7-14 days and preferably within 3-4 days prior to administration of the stem cells. Children weighing less than 30lb should receive 2.5 mg/day. (Ortho Biotech, Raritan, NJ).

Oxzezumab (Otelixizumab) is a monoclonal antibody that targets the epsilon chain of CD 3. It is administered at 5 mg/day by a single bolus intravenous injection for 10 to14 days. Administration should be completed within 7-14 days and preferably within 3-4 days prior to administration of the stem cells. Children weighing less than 30lb should receive 2.5 mg/day. The antibodies were developed by the cooperation of Tolerx, inc. and GlaxoSmithKline and are being manufactured by Abbott Laboratories.

Tilizumab (Teplizumab) is a humanized Fc engineered monoclonal antibody, also known as MGA031 and hcokt 3 γ 1 (Ala-Ala). It is an anti-CD 3 antibody. It may be administered according to the invention by a single intravenous bolus at a dose of 5 mg/day for 10 to14 days. Administration should be completed within 7-14 days and preferably within 3-4 days prior to administration of the stem cells. Children weighing less than 30lb should receive 2.5 mg/day (Eli Lilly, Indianapolis, IN).

Vislizumab (Visilizumab) (provisionally under the tradename Nuvion, PDL BioPharma Inc.) is a humanized monoclonal antibody that targets CD3 on activated T cells. It may be administered according to the invention by a single intravenous bolus at a dose of 5 mg/day for 10 to14 days. Administration should be completed within 7-14 days and preferably within 3-4 days before administration of the stem cells. Children weighing less than 30lb should receive 2.5 mg/day.

Clinicimab (Clenoliximab) is a monoclonal antibody directed against CD 4. It may be administered according to the invention by a single intravenous bolus at a dose of 5 mg/day for 10 to14 days. Administration should be completed within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Keliximab (Keliximab) is a monoclonal antibody that binds to leukocytes via the protein CD 4. It is administered at an infusion dose of 3mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Zamumab (Zanolimumab) (expected trade name HuMax-CD4) is a human monoclonal antibody targeting CD4 and is administered at a dose of 20 mg/kg/day within 7-14 days and preferably within 3-4 days prior to stem cell administration. (Genmab, A/S COPENHAGEN/TenX Biopharma, Inc., Philadelphia, Pa.).

Efavirenzumab (Efalizumab) (trade name Raptiva, Genentech, Merck Serono) is a recombinant humanized monoclonal antibody. Efuzumab binds to the CD11a subunit of antigen 1, which is associated with lymphocyte function. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells.

Erlizumab (Erlizumab) (also known as rhuMAb) is a recombinant humanized monoclonal antibody developed by Genentech in partnership with Roche. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells. The drug acts by blocking growth factors in blood vessels. Specifically, erlizumab targets CD18 and LFA-1 integrin.

Atorvastatin (Affutuzumab) is an anti-CD 20 monoclonal antibody. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells. (Hoffmann-La Roche Inc.)

Ocrelizumab (Ocrelizumab) is a humanized anti-CD 20 monoclonal antibody. It targets mature B lymphocytes. It is being developed by the subsidiary companies Genentech and Biogen Idec of Hoffmann-La Roche. According to the invention, it is administered at doses of 200mg and 600mg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Palivizumab (Pascolizumab) is an anti-IL-4 humanized monoclonal antibody. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells.

Luximab (Lumiliximab) is a monoclonal antibody targeting CD 23. According to the invention, it can be administered within 7-14 days and preferably within 3-4 days before the administration of the stem cells at 50mg/m2 to 450mg/m2 to 500mg/m 2. The drug is a chimeric antibody from macaque (Macaca irus) and Homo sapiens (Homo sapiens). (Biogen IDEC)

Tenecteximab (Teneliximab) is a chimeric monoclonal antibody that binds to the immunostimulatory protein CD 40. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells.

Tollizumab (Toralizumab) (IDEC131) is a humanized monoclonal antibody. According to the invention, it can be administered by subcutaneous injection at a dose of 20mg/kg, once a week, within 7-14 days and preferably within 3-4 days before the administration of the stem cells. (IDEC Pharmaceuticals Corporation)

Aselizumab (Aselizumab) is anti-CD 62L administered by I.V. infusion at doses in the range of 0.5mg/kg, 1.0mg/kg, and 2.0mg/kg within 7-14 days and preferably within 3-4 days prior to administration of stem cells.

Galiximab is an anti-CD 80(Biogen Idec) monoclonal antibody administered at a dose IV of 500mg/m2 within 7-14 days and preferably 3-4 days prior to administration of stem cells. It is a chimeric antibody from cynomolgus monkey and homo sapiens.

Gavimomab (Gavilimomab) is a mouse monoclonal antibody developed by Abgenix (also known as ABX-cbl. it binds antigen cd147. according to the present invention, it can be administered by i.v. infusion at a dose of 20mg/kg within 7-14 days and preferably within 3-4 days prior to administration of stem cells.

BG9588 (humanized anti-CD 40L) was administered at 20mg/kg 7-14 days and 3-4 days prior to stem cell administration. Antibodies to CD154 (also known as CD40 ligand or CD40L) are administered, CD154 being a protein expressed predominantly on activated T cells and being a member of the TNF superfamily of molecules. It binds to CD40 on Antigen Presenting Cells (APCs), which causes a number of effects depending on the type of target cell. Generally, CD40L functions as a costimulatory molecule and stimulation of the T cell receptor by MHC molecules bound to the APC induces activation of the APC. CD40L has a total of three binding partners: CD40, α 5 β 1 integrin, and α IIb β 3.

(Hu5c8)5c8 is a monoclonal antibody that binds CD154(CD40 ligand), thus blocking the interaction between CD40 and CD154, administered at 20mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Belimumab (Belimumab) (registered name Benlysta, previously known as LymphoStat-B) is a fully Human monoclonal antibody (Human Genome Sciences) that specifically recognizes and inhibits the biological activity of B lymphocyte stimulating factor (BLyS), also known as the B cell activating factor of the TNF family (BAFF). According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Yipriumamab (Iplilimumab), also known as MDX-010 or MDX-101, is an anti-CTLA-4 (cytotoxic T cell lymphocyte-associated) human monoclonal antibody developed by Bristol-Myers Squibb. According to the invention, it is administered at 10mg/kg of active drug within 7-14 days and preferably within 3-4 days before the administration of the stem cells.

Trimetumab (Tremelimumab) (formerly Temmimumab (ticilimumab), CP-675,206) is a fully human IgG2 monoclonal antibody produced by Pfizer. It binds to the protein CTLA-4, CTLA-4 expressed on the surface of activated T lymphocytes. Tramadol blocks binding of antigen presenting cell ligands B7.1 and B7.2 to CTLA-4, thereby causing inhibition of T cell activated B7-CTLA-4 mediated downregulation; subsequently, either B7.1 or B7.2 can interact with another T cell surface receptor protein (CD28), resulting in B7-CD28 mediated T cell activation against B7-CTLA-4 mediated inhibition. Tramelimumab is administered by IV infusion at 3mg/kg, 6mg/kg, 10mg/kg, 15mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Betilimumab (Bertillimumab) is a human monoclonal antibody that binds eotaxin-1. (iCotheriapeutics Inc. Vancouver, B.C.). According to the invention, it is administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Ledellimumab (Lerdelimumab) (CAT-152) is an anti-TGF β -2 developed by Cambridge Antibody Technology. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Metelimumab (CAT-192) is a human IgG4 monoclonal Antibody developed by Cambridge Antibody Technology that neutralizes TGF β 1. According to the present invention, it is preferable that it is administered at a dose of 10mg/kg 3 to 4 days before the administration of the stem cells. Natalizumab (natalizumab) is a humanized monoclonal antibody directed against the cell adhesion molecule α 4-integrin. It is commonly sold by Biogen Idec and Elan as Tysabri, and was named altegren (Antegren) earlier. Natalizumab is administered within 7-14 days and preferably within 3-4 days prior to administration of the stem cells at a dose of 300mg infused intravenously over approximately one hour.

Tolizumab (Tocilizumab) or Attitumumab (atlizumab) (developed by Hoffmann-La Roche and Chugai under the trade names Actemra and RoActemra) is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R). According to the invention, it can be administered by intravenous infusion at 8mg/kg within 7-14 days and preferably within 3-4 days before the administration of the stem cells.

Edolimumab (Odulimomab) is a mouse monoclonal antibody directed against the alpha chain of antigen 1 associated with the lymphocyte function of the protein involved in the immune response. It is administered at 10mg/kg of active drug 7-14 days and 3-4 days prior to administration of the stem cells.

Basiliximab (Basiliximab) (trade name Simulect) is a chimeric mouse-human monoclonal antibody directed against the alpha chain of the T-cell IL-2 receptor (CD 25). The dose is 20mg, twice within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Daclizumab (Daclizumab) (trade name Zenapax) is a therapeutic humanized monoclonal antibody directed against the alpha subunit of the T-cell IL-2 receptor. Roche Pharmaceuticals, Hoffmann-La Roche Inc,340KingslandStreet, Nutley, New Jersey. It is administered at 10mg/kg of active drug within 7-14 days and preferably 3-4 days prior to administration of the stem cells.

Itumomab (inolomab) is a mouse monoclonal antibody that targets the alpha chain of the interleukin-2 receptor. OPi (formerly Orphan Pharma International). It is administered at 10mg/kg of active drug within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Adozomumab (zolomab aritox) is a mouse monoclonal antibody and is an anti-CD 5 antibody linked to the a chain of ricin protein (reflected by atropis (aritox) in the drug name). According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Atosamumab (Atorolimumab) is a mouse monoclonal antibody directed against rhesus factor. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Celizumab (Cedelizumab) is an anti-CD 4 monoclonal antibody. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Dalizumab (Dorlixizumab) is a chimeric/humanized monoclonal antibody and immunosuppressive drug. It is administered at a dose of 10mg/kg of active drug within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Artuzumab ozolomab (Fontolizumab) (trade name HuZAF) is an anti-interferon gamma humanized monoclonal antibody. According to the invention, it may be administered within 7-14 days and preferably within 3-4 days before the administration of the stem cells, at an i.v. dose of ar-tuzumab administered at 4.0mg/kg or 10.0 mg/kg. (PDL Biopharma)

Rochlomib (Gantenerumab) is an anti-beta amyloid monoclonal antibody (Roche). It is administered at a dose of 10mg/kg of active drug within 7-14 days and preferably 3-4 days prior to administration of the stem cells.

High mifiximab (Gomiliximab) is a monoclonal antibody that targets the low affinity IgE receptor (Fc epsilon RII or CD 23). According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Masseculizumab (massimomab) is a mouse monoclonal antibody that targets the T cell receptor. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Moluumab (Morolimumab) is a human monoclonal antibody directed against human rhesus factor. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Pexelizumab (Pexelizumab) is a single-chain variable fragment of a monoclonal antibody that targets component 5 of the complement system. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Rayleigh mab (Reslizumab) is an anti-IL-5 humanized monoclonal antibody. According to the invention, it can be administered by infusion within 7-14 days and preferably within 3-4 days before the administration of the stem cells at a preferred dose of 3.0mg/kg Rayleigh mab. (fractionation Therapeutics Inc).

Rovelizumab (also known as LeukArrest and Hu23F2G) is an anti-CD 11/CD18 humanized monoclonal antibody that inhibits leukocytes. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Celizumab (Silizumab) (MEDI-507) is an anti-CD 2 monoclonal antibody with human IgG1 κ directed to CD 2. The agents exhibit potent immunomodulating effects, selectively inhibiting the function of T and NK cells. The cetilizumab binds to CD2, a specific receptor found in T cells and NK cells, triggering a host immune response that causes CD2+ cytolysis, selective suppression of the immune system, and control of the growth of activated T cells. According to the invention, the Celizumab can be administered by subcutaneous injection at a preferred dose of 0.04mg/kg i.v. and 5 or 7mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells. (Medmimmune)

Tallizumab (Talizumab) (TNX-901) is a humanized monoclonal antibody developed by Tanox of Houston, Texas. It is designed to specifically target immunoglobulin E (or IgE) and IgE-expressing B lymphocytes, without binding IgE that has been bound by IgE receptors on mast cells and basophils. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Omalizumab (Omalizumab) is an anti-IgE monoclonal antibody developed by Tanox, Novartis, and Genentech. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Attentiomab aritox (Telimomab aritox) is a mouse monoclonal antibody. The antibody was linked to the a chain of ricin protein (this is reflected by the attorney in the drug name). According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Valliximab (Vapaliximab) is an anti-VAP-1 chimeric monoclonal antibody. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Vepamomab (Vepalimomab) is an anti-VAPI mouse monoclonal antibody. According to the invention, it can be administered at a dose of 10mg/kg within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells.

Abatacept (marketed as Orencia) is a fusion protein consisting mainly of an immunoglobulin fused to the extracellular domain of CTLA-4, a molecule capable of binding B7 and thus preventing complete activation of T cells. The albuterol should be administered according to a prescribed dosing regimen based on body weight, following 30 minutes of intravenous infusion. Within 7-14 days and preferably within 3-4 days before administration of the stem cells, the dose should preferably be: 500mg for <60 kg; 750mg for 60kg-100kg and 1 gram for >100 kg.

Belatacept (Belatacept) (Bristol-Myers-Squibb) is a fusion protein consisting essentially of an Fc fragment of human IgG1 immunoglobulin linked to the extracellular domain of CTLA-4, a molecule critical to T cell co-stimulation, selectively blocking the T cell activation process. It was developed. It differs from the aflibercept (Orencia) only by 2 amino acids. According to the invention, it can be administered according to a prescribed dosing regimen based on body weight, according to an intravenous infusion of 30 minutes, within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells, at the following preferred doses: 500mg for <60 kg; 750mg for 60kg-100kg and 1 gram for >100 kg.

Etanercept (trade name Enbrel, Amgen, Thousand Oaks, CA) is a drug that treats autoimmune diseases by interfering with tumor necrosis factor (TNF, a part of the immune system) acting as a TNF inhibitor. Etanercept may be administered subcutaneously (s.c.) at a dose of 25mg or 50mg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Pinacept (pegsunecrcept) is a pegylated soluble tumor necrosis factor receptor. According to the invention, it can be administered subcutaneously in the preferred dose of 9mg/kg s.c. within 7-14 days and preferably within 3-4 days before the administration of stem cells.

Aflibercept (Aflibercept) is a protein comprising extracellular domain segments of human vascular endothelial growth factor receptors 1(VEGFR1) and 2(VEGFR2) fused to the constant region (Fc) of human IgG1, which has potential anti-angiogenic activity, and is being co-developed by Sanofi-Aventis and Regeneron Pharmaceuticals. Aflibercept (VEGFTrap), an anti-angiogenic agent, is a fusion protein designed specifically to bind all forms of vascular endothelial growth factor-a (known as VEGF-a). In addition, aflibercept binds placental growth factor (PLGF), which is also implicated in tumor angiogenesis. Aflibercept may be administered by injection or IV infusion at a preferred dose of 2 milligrams per kilogram (mg/kg) or 4mg/kg within 7-14 days and preferably within 3-4 days prior to administration of the stem cells.

Alexidipt (Alefacept) is a fusion protein: it combines a portion of the antibody with a protein that blocks the growth of some types of T cells.(Alicept) is an immunosuppressive dimeric fusion protein consisting of an extracellular CD2 binding portion of human leukocyte functional antigen-3 (LFA-3) linked to the Fc (hinge, CH2, and CH3 domains) portion of human IgG 1. A preferred dose is 7.5mg IV or 15mg IM, preferably within 7-14 days and preferably 3-4 days prior to administration of the stem cells. Astellas Pharma US, inc. deerfield, IL 60015.

Riloncept (Rilonacept), also known as IL-1Trap (sold under the trade name Arcalyst), is a dimeric fusion protein consisting of the extracellular domain of the human interleukin-1 receptor and the Fc domain of human IgG1 that binds to and neutralizes IL-1 h. Treatment should be initiated with a 320mg loading dose delivered in two (2mL) subcutaneous injections of 160mg, each given at two different sites on the same day, within 7-14 days and preferably within 3-4 days prior to administration of the stem cells. Pediatric patients aged 12 to 17 years: treatment should be started with a loading dose of 4.4mg/kg, up to a maximum of 320mg, delivered as one or two subcutaneous injections, with a maximum single injection volume of 2mL, within 7-14 days and preferably within 3-4 days before administration of the stem cells. Produced by Regeneron.

Daclizumab (Dacetuzumab), also known as SGN-40 or huS2C6, is an anti-CD 40 humanized monoclonal antibody. The CD40 antigen is highly expressed on most B lineage hematological malignancies, including multiple myeloma, non-Hodgkin lymphoma, and chronic lymphocytic leukemia. CD40 is also found on many types of solid tumors (including bladder, kidney, and ovarian cancers) and on cells that play a role in immune disorders. It is administered within 7-14 days and preferably within 3-4 days prior to administration of the stem cells at a preferred dose of 10mg/kg of active drug. Seattle Genetics, Inc.

HCD122 is the fully human antagonist anti-CD 40 monoclonal antibody. CD40 is a cell surface receptor that plays a key role in immune response as well as cell growth and survival signaling by being activated by CD40 ligand (CD 40L). It is usually overexpressed and activated in B cell malignancies. According to the invention, it can be administered within 7 to14 days and preferably within 3 to 4 days before the administration of the stem cells at a dose of 10mg/kg of active drug. It is being developed by XOMA/NOVARTISONCOLOGY.

Rituximab (Rituximab) (sold under the tradenames Rituxan and MabThera, Genentech, inc., san francisco, CA) is a chimeric monoclonal antibody against the protein CD20, found primarily on the surface of B cells. It can therefore destroy B cells. CD20 is widely expressed on B cells (from early pre-B cells to late differentiated cells), but is not present on late differentiated plasma cells. Rituxan is provided in a single use vial of 100mg (10mL) or 500mg (50mL) at a concentration of 10 mg/mL. It may be administered as an infusion at a rate of 50 mg/hr. In the absence of infusion toxicity, the infusion rate is increased in increments of 50mg/hr every 30 minutes up to a maximum of 400mg/hr, preferably within 7-14 days and preferably 3-4 days prior to administration of the stem cells. The preferred recommended dosage is 375mg/m as an IV infusion²Preferably 3-4 days before the stem cells.

Rituximab also acts as a drugThe administration of the components of (a), at a preferred dose of 250mg/m2, by infusion of rituximab within 4 hours before the administration of indium-111- (In-111-) Zevalin and within 4 hours before the administration of yttrium-90- (Y-90-) Zevalin, should be carried out within 7-14 days and preferably within 3-4 days before the administration of stem cells. Rituxan can also be administered in combination with methotrexate, preferably 3-4 days prior to administration of the stem cells. Biogen Idec Inc and GenentechUSA, Inc.

Ibritumomab tiuxetan (sold under the trade name Zevalin) is a B-cell targeted monoclonal antibody radioimmunotherapy. The drug uses the monoclonal mouse IgG1 antibody, ibbemomab (ibritumomab), in combination with the chelator titatan (tiuxetan), to which a radioisotope (yttrium-90 or indium-111) was added. Titan is a modified form of DTPA, the carbon backbone of which contains isothiocyanatobenzyl and methyl groups.

As with agents that trigger the accumulation of deoxyATP, adenosine deaminase deficiency also leads to a reduction in active germinal center formation (JImmunol171:5562-5570, 2003). Similarly, agents that enhance CCR7 expression or activate CCR7 will result in a decrease in active germinal center formation.

Stem cells: defining, isolating, delivering and therapeutic uses

The term stem cell within the scope of the present invention includes any cell capable of differentiating into the desired tissue. The cells include pluripotent stem cells, embryonic stem cells, multipotent adult stem and progenitor cells, and precursor cells. "Stem cells" are cells from embryos, fetuses or adults that are capable of self-replication throughout the life of an organism under certain conditions, either long-term or in the case of adult stem cells. It can also produce specialized cells that make up body tissues and organs.

"pluripotent stem cells" have the ability to produce cell types that develop from three germ layers (mesoderm, endoderm and ectoderm), from which all cells of the body originate. The natural source of known human pluripotent stem cells are those cells isolated and cultured from early human embryos from fetal tissue destined to be part of the gonads.

"embryonic stem cells" are derived from a group of cells called the inner cell mass, which is part of an early (4 to 5 day) embryo called the embryo sac. Once removed from the embryo sac, the cells of the inner cell mass may be cultured into embryonic stem cells.

An "adult stem cell" is an undifferentiated (non-specialized) cell that appears in a differentiated (specialized) tissue, which self-renews and when transferred to the appropriate tissue specializes to produce all the specialized cell types in the tissue in which it resides. Adult stem cells are able to produce identical copies of themselves throughout the life of the organism. This property is called "self-renewal". Adult stem cells typically divide to produce progenitor or precursor cells, which then differentiate or develop into "mature" cell types with characteristic shapes and specialized functions (e.g., muscle cell contraction or nerve cell signaling). Sources of adult stem cells include, but are not limited to, bone marrow, blood, cornea and retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, gastrointestinal tract, and the lining of the pancreas.

Delivering or administering stem cells to an individual includes delivering or administering exogenous stem cells as well as mobilizing endogenous stem cells, as well as increasing the bioavailability of spontaneously released endogenous stem cells.

Stem cells from bone marrow are the most studied adult stem cell type. Currently, they are used clinically as various blood and immune components for bone marrow repair via transplantation. Two major types of stem cells found in bone marrow are currently identified: hematopoietic stem cells (HSC or CD34+ cells), which are typically considered to form blood and immune cells, and stromal (mesenchymal) stem cells (MSC), which are typically considered to form bone, cartilage, muscle and fat. However, two types of bone marrow-derived stem cells have recently been demonstrated to have broad plasticity and pluripotency in terms of their ability to form the same tissue. Bone marrow (located in the medullary cavity of the bone) is the major site of hematopoiesis in adults. It produces about sixty billion cells per kilogram of body weight per day. The hematopoetic (red) bone marrow regresses after birth until late adolescence, after which it concentrates in the lower cranial vertebrae (lower skull vertebrae), shoulder and pelvic girdle, ribs and sternum. Adipocytes replace hematopoietic cells (yellow bone marrow) in the bones of the hands, feet, legs, and arms. Fat occupies about 50% of the red bone marrow space in adults, and further fat metamorphosis slowly continues with aging. In very elderly individuals, a gelatinous conversion of fat to a mucoid material (white bone marrow) may occur. If there is a long-term need, such as in the case of hemolytic anemia, yellow bone marrow can be restored to hematopoietically active bone marrow. Hematopoiesis can therefore be expanded by increasing the volume of the red bone marrow and decreasing the time for progenitor cells to develop (transform) into mature cells.

The bone marrow stroma is composed primarily of a network of sinuses that originate in the endosteal lining of cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation. The three layers of the sinus wall are composed primarily of endothelial cells, underdeveloped basement membranes, and adventitial reticulocytes, which are fibroblasts that are capable of transforming into adipocytes. Endothelial and reticulocytes are a source of hematopoietic cytokines. Hematopoiesis occurs in the intersinus space and is controlled by a complex series of actions of stimulating and inhibiting cytokines, cell-to-cell contact, and extracellular matrix components on neighboring cells. In this unique environment, lymphoid hematopoietic stem cells differentiate into all blood cell types. Mature cells are produced and released, thereby maintaining a steady blood cell level. The system can meet the increased demand for additional cells due to blood loss, hemolysis, inflammation, immune cytopenia, and other conditions.

"progenitor or precursor cells" are partially specialized; it self-renews and also produces differentiated cells. When a stem cell divides, one of the two new cells is often a stem cell that is able to self-replicate again, so researchers often distinguish between precursor/progenitor cells and adult stem cells. In contrast, when a progenitor/precursor cell divides, it may form more progenitor/precursor cells or it may form two specialized cells. Progenitor/precursor cells can replace damaged or dead cells, thus maintaining the integrity and function of tissues such as the liver or brain.

Methods of isolating and culturing stem cells useful in the present invention are well known. Cord blood is a rich source of hematopoietic stem cells. For transplantation use, stem cells obtained from umbilical cord blood and stem cells obtained from bone marrow or peripheral blood appear to be very similar. Placenta is an excellent, readily available source of mesenchymal stem cells. In addition, it has been shown that mesenchymal stem cells can be derived from adipose tissue and bone marrow stromal cells, and they are presumed to be present in other tissues. Amniotic fluid and tissue are another excellent source of stem cells. Although organs from which adult stem cells are available vary significantly in quality and quantity, the original differences between cells may be relatively superficial and may be balanced by the range of similar plasticity they exhibit. For example, adult hematopoietic stem cells and adult mesenchymal stem cells can become cardiomyocytes under appropriate conditions. The description of the whole potential range of adult stem cells has just begun. Known methods can be used to isolate and differentiate stem cells. For example, in mice, bone marrow cells are isolated by killing the mice, cutting the leg bone with a pair of scissors and washing out the stem cells. Stem cells can also be isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind to unwanted cells (e.g., CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes)). An example of such a scheme is found in Inaba et al, J.Exp.Med.176:1693-1702 (1992).

FIG. 1 shows a typical view of a layer resulting from gradient centrifugation of whole blood.1 shows platelets; 2 buffy coat with MNC and stem cells; 3, sucrose polysaccharide; and 4RBC pellet and stem cells.

In humans, CD34+ hematopoietic stem cells can be obtained from a variety of sources including cord blood, bone marrow, and mobilized peripheral blood. Purification of CD34+ cells can be achieved by antibody affinity procedures. Ho et al, Stem Cells13 (suppl 3):100-105(1995) describe affinity column separation procedures for the separation of CD34+ Cells. See also, Brenner, Journal of Hematotherapy2:7-17 (1993). Methods for isolating, purifying, and culturing expanded mesenchymal stem cells are known. Specific antigens of MSCs are also known (see, U.S. Pat. nos. 5,486,359 and 5,837,539).

Stem cells are characterized by the ability to self-renew and differentiate into a diverse range of specialized cell types through mitotic cell division. There is a range of potential stem cells. Totipotent stem cells are cells that can give rise to all the tissues required for the development of the whole organism, such as fertilized eggs. Pluripotent Stem cells are cells that can produce Stem cells of all 3 germ layers and include cells such as embryonic Stem cells, spermatogonial Stem cells (cell.119(7): 1001) -. Very small embryonic-like stem cells are found in adult bone marrow, blood, heart and other tissues, which can give rise to cells from cells of all 3 embryonic lineages, however, they have not been shown to give rise to a complete organism in tetraploid complementation assays, and thus, it is unclear whether they are true pluripotent stem cells, whether the somatic blotting prevents their activation in tetraploid complementation, or whether they are more restricted but extremely plastic multipotent stem cells (DEVELOPMENT DYNAMICS236: 3309) -3320, 2007). Pleiotropic Stem cells are cells that can produce a variety of functional cells within a defined lineage, such as Hematopoietic Stem Cells (HSCs) (J Exp Med.207(6): 1127. sup. 1130,2010), Adipose Stem Cells (ASCs) (Stem Cell sDev.2010[ electronic edition before printing plate ]) (6) or Mesenchymal Stem Cells (MSC) (StemBook, Cambridge (MA): Harvard Stem Cell Institute; 2008. sup. 2009).

Stem cells may be further characterized by the extent to which they can differentiate, and by their potency. Potency explains the differentiation potential (potential to differentiate into different cell types) of stem cells.

Totipotent (a.k.a. universal) stem cells can differentiate into embryonic and extra-embryonic cell types. The cells can construct a whole living organism. These cells result from the fusion of egg cells and sperm cells. The cells resulting from the first few divisions of the zygote are also totipotent.

Pluripotent stem cells are progeny of totipotent cells and can differentiate into almost all cells, i.e., cells derived from any of the three germ layers.

Pleiotropic stem cells can differentiate into many cells, but only those of a closely related cell family.

The oligopotent stem cells can differentiate into a few cells, such as lymphoid or myeloid stem cells.

Unipotent cells can only produce one cell type, themselves, but have the property of self-renewal, which distinguishes them from non-stem cells (e.g., muscle stem cells).

Two broad types of mammalian stem cells are: embryonic stem cells isolated from the inner cell mass of the embryo sac and adult stem cells found in adult tissues.

Adult stem cells are undifferentiated cells that proliferate through cell division to replenish dead cells and regenerate damaged tissues, which can be found throughout the body after embryonic development. They are also known as somatic stem cells and can be found in young as well as in adult animals and humans. Types of adult stem cells include hematopoietic stem cells, mammary stem cells, mesenchymal cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, adipose-derived stem cells, neural crest stem cells, and testicular stem cells.

Progenitor cells have a tendency to differentiate into a specific cell type. However, they have been more specific than stem cells: they are forced to differentiate into their "target" cells. The most important difference between stem and progenitor cells is that stem cells can replicate indefinitely, while progenitor cells can divide only a limited number of times. There is still a contention for accurate definition and this concept is still under development.

The terms "progenitor cell" and "stem cell" are sometimes equivalent.

Stem cells found in mononuclear fractions of whole blood, bone marrow, adipose tissue, cord blood, and other tissues, and stem cells isolated from these mononuclear fractions, have been shown to provide benefits to human patients. Peripheral Blood Mononuclear Cells (PBMC) are any blood cells with a circular nucleus. For example: lymphocytes, monocytes or macrophages. These blood cells are key components in the immune system to fight infections and adapt to invaders. The lymphocyte population consists of T cells (positive for CD4 and CD8, about 75%), B cells, and NK cells (about 25% in combination). PBMCs are often extracted from whole blood using saccharlycan, a hydrophilic polysaccharide that stratifies the blood, while monocytes and lymphocytes form a buffy coat beneath the plasma layer. This buffy coat contains PBMCs. In addition, PBMCs can be extracted from whole blood using a hypotonic lysis solution that preferentially lyses red blood cells. This approach produces neutrophils and other Polymorphonuclear (PMN) cells that are important in the acquired innate immune defense. The PBMC fraction of bone marrow aspirates has been used to treat patients following myocardial infarction and has been shown to reduce subsequent mortality and slightly improve cardiac function in these patients (Eur Heart J27:2775-2783, 2006). Although these types of treatment significantly reduced mortality, there was only a slight improvement in cardiac function. Nuclear imaging studies in these patients have shown that within 60 to 90 minutes after injection, the majority (up to 97%) of the injected mononuclear fraction of stem cells in the coronary arteries do not remain in the heart, but can be found primarily in the spleen and liver (Circulation111:2198-2202, 2005). Other imaging studies have also demonstrated that stem cells found in whole blood, bone marrow, adipose tissue, umbilical cord blood, placenta, amniotic fluid and other tissues, as well as stem cells isolated from these mononuclear fractions, accumulate in the spleen in many different species (STEM CELLS24: 2279-.

Animal studies have demonstrated that administration of higher numbers of bone marrow mononuclear cells results in improved cardiac repair and functional recovery (Circulation114:2163-2169, 2006). However, for clinical patients this requires aspiration of large amounts of bone marrow (up to 200ml) under general anesthesia and is considered highly undesirable for post-infarction patients whose cardiac function is still under stress. In order to obtain better organ targeting and retention, researchers have attempted to concentrate injected stem cells. Although enriching the number of injected CD34+ cells leads to a higher accumulation of CD34+ cells in the heart after intracoronary injection, this method of increasing stem cell delivery is not considered optimal because it is not known which specific stem cells contained in the monocyte fraction are necessary for tissue regeneration at this time (Circulation111:2198-2202, 2005). In addition, purified human Mesenchymal Stem Cells (MSCs) have been shown to increase engraftment of human cord blood CD34+ stem cells (Hematology VOL14NO3: 125-.

The stem cells may be autologous or derived from an unrelated donor. The stem cells may be contained within a mononuclear cell fraction from bone marrow, whole blood, cord blood, adipose tissue, or other sources, or they may be purified by selection for CD34, CD133, CD105, CD117, SSEA1-4, dye exclusion (dye exclusion), or other specific stem cell antigens. Stem cells can be isolated from whole blood, bone marrow, cord blood, adipose tissue, tissue scrapings from olfactory mucosa, and other sources of stem cells (e.g., umbilical cord tissue) that can be dissociated into single cell suspensions by density gradient centrifugation using Ficoll-Hypaque or other commercially available gradients. Stem cells can be recovered from the mononuclear cell fraction produced by the procedure. Alternatively, Stem Cells can be found in other parts after density gradient centrifugation (Stem Cells and Development2011 Bhatiya et al). For example, cord blood may be diluted 1:1 in PBS, carefully overlaid on Histopaque1077(Sigma) and centrifuged at 1500rpm for 30 minutes at room temperature. The resulting layer as depicted in fig. 1 can be further processed for stem cell isolation. Layer 1 is a platelet layer, layer 2 is a buffy coat layer containing mononuclear cells, layer 3 is a sucrose layer, and layer 4 is a red blood cell pellet layer. Layers 1,2 and 3 can be collected, diluted with a suitable medium such as dmef 12 with or without FBS and centrifuged again to obtain cell pellets. Layer 4 may be diluted with an appropriate medium such as DMEM F12 and centrifuged at 800rpm for 15 minutes at room temperature in a standard tabletop centrifuge. Stem cells can be recovered mainly from layer 2 (buffy coat) and layer 4(RBC pellet).

Stem cells can be further characterized and isolated by specific antigens expressed on the surface using cell classifiers such as ARIA from BD, using magnetic columns such as those obtained from Miltenyi, using magnetic beads and DYNAL magnets, and other antibody/antigen-based isolation methods known to those skilled in the art. Stem cells can also be identified and isolated by their ability to bind to other cells as described in the present disclosure.

Pluripotent stem cells may be characterized by expressing stage-specific embryonic antigen (SSEA), the transcription factors Oct4 and Nanog, as well as other markers. Hematopoietic stem cells are characterized by expression of markers such as CD34, CD133, ckit, Sca1 and are also CD45 positive. The abbreviation CD refers to the antigen family and means "cluster of differentiation".

Hematopoietic Stem Cells (HSCs) are multipotent stem cells that produce all blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). Hematopoietic tissues contain cells with long-term and short-term regenerative capacity and specialized multipotent, oligopotent, and unipotent progenitor cells. HSCs constitute 1:10.000 of the cells in myeloid tissues. HSC express the following antigens: CD34, CD90(Thy1), CD45, CD41, CD105, CD117(c-kit), SCF (kit ligand), Ly6A/E (sca-1), CD127, CD44, CD33, CD38, CD14, CD106, CD84, CD90, Flk-1, CD164, Notch1, CD338(ABCG2), CD202b, CD184, AC133(= CD133) and CXCR 4.

Mesenchymal stem cells or MSCs are multipotent stem cells that can differentiate into a variety of cell types, including: osteoblasts (osteocytes), chondrocytes (chondrocytes, cartilage cells), and adipocytes (adipocytes, fatcell). Mesenchymal stem cells are characterized by expressing: CD45, CD90, CD105, CD34, CD31, CD29, CD106, CD44, CD51, CD166, Ly6A/E (sca-1), CD117, CD71, CD10, CD49D, CD49E, TNAP, PTP LAR, W5C5 antigen, W3D5 antigen, W4a5 antigen, and CXCR 4.

Endothelial stem cells (or endothelial progenitor cells or precursor cells) are multipotent stem cells. They are one of three stem cell types found in bone marrow and express the following antigens: CD45, CD31, CD34, CD105, CD146, CD106, CD54, CD117, CD102, CD120a, CD120b, CD14, CD29, CD49d, CD49e, CD49f, CD62P, CD62L, and CXCR 4.

Neural Stem Cells (NSCs) are self-renewing, pleiotropic cells that produce the major phenotype of the nervous system. Neural progenitor and stem cells have been isolated from striatal tissue (including the subventricular region of adult mouse brain tissue, one of the neurogenic regions) and various regions of the adult brain (including non-neurogenic regions, such as the spinal cord), as well as from a variety of species including humans. NSCs express the following antigens: CD29, CD146, Notch1, Ki67, CD24, CD49f, vimentin, CD81, and CXCR 4. Neural progenitor cells express the following antigens: 57D2 antigen, W4a5 antigen, and CXCR 4.

Embryonic stem cells, spermatogonial stem cells, testicular stem cells, and pluripotent stem cells, such as those from iPS, SCNT, ANT-OAR, express the following antigens: CD24, CD9, Nanog, Smad, Runx2, c myc, CD30, GSC, Oct3/4, Sox2, SSEA1(CD15), SSEA4, CD324, CD29, Tra-1-60, Tra-1-81, CD338(ABCG2), CD49f, FoxD3, Stat3, Hox11 and CXCR 4.

VSELs were positive for SSEA1, Oct4, Nanog, Rex1 and other pluripotent stem cell markers and for CD133, CD34, AP, cMet, LIF-R and CXCR 4. (J Am Coll Cardiol53(1):10-20,2009; Stem Cell Rev4:89-99,2008). In addition, novel stem cells characterized by distinct markers, such as Hox11+ stem cells found in adult spleens, are often identified. (Horm Metab Res40:137-146, 2008). Fetal stem cells still present in the adult spleen have been identified which are capable of regenerating islet cells, however, such cells are present in the CD 45-negative portion of the spleen (Mol Cell Proteomics4(10): 1459-. Hox11+ splenocytes, although negative for CD45, did express OCT3/4, SOX2, KLF4, c-MYC, and NANOG, making them potentially equivalent to embryonic stem cells and induced pluripotent stem cells (iPS) (Int J Biochem Cell biol.2009Dec18).

Therapeutic uses of stem cells

Stem cell therapy is under investigation and is well established for the treatment of many diseases. Conditions that may benefit from stem cell therapy include: eye diseases, neurological diseases, GI diseases, musculoskeletal diseases, metabolic diseases, endocrine diseases, vascular diseases, pulmonary diseases, heart diseases, cardiovascular diseases, immune-mediated diseases, autoimmune-mediated diseases, cardiovascular diseases, and all diseases for which regenerative therapy is beneficial. The clinical trial information contained on NIH website www.clinicaltrials.gov lists over 3000 stem cell studies. Diseases in the evaluation included: vasculitis, rheumatism using endothelial progenitor cells, therapeutic neovascularization by implantation of autologous monocytes in patients with connective tissue disease, repeated administration of granulocyte colony stimulating factor for blood stem cell mobilization, corticobasal degeneration, and multiple system atrophy in patients with progressive supranuclear palsy; hematological malignancies, leukemias, lymphomas, cancers, osteopetrosis, aplastic anemia and cytopenia, sickle cell disease and thalassemia, limbal Stem cell deficiency, breast cancer, acute myocardial infarction (see U.S. Pat. No. 7,862,810, isolating and trimming cardiac cell present c-kit positive), coronary artery disease (see U.S. Pat. No. 7,470,538, isolating and characterizing by infusion into the coronary artery expressed CD133+/CD34+/CXCR 4-cellular induced cardiac tissue present), peripheral vascular disease, heart failure, type I Diabetes (see U.S. Pat. Pub. No. 2011000830, Human induced tissue culture Derived from Diabetes Mellitus), type 2 Diabetes, spinal cord injury, multiple sclerosis Derived neural stroke (see U.S. Pat. Pub. No. 20100166712, multiple sclerosis Derived from Diabetes Mellitus), spinal cord injury, multiple sclerosis Derived from Diabetes, and multiple sclerosis Derived from Diabetes, Systemic sclerosis, lupus erythematosus, chronic wound healing, burns, fracture healing, cartilage repair, central nervous system tumors, osteoarthritis, renal failure, Parkinson's Disease (see U.S. patent application publication No. 20100010087, Methods for Inducing Stem cell differentiation and specificity with EC-18), myeloma, diabetic foot, liver and biliary cirrhosis, dilated cardiomyopathy, anemia, retinitis pigmentosa, Crohn's Disease, diabetic neuropathy, mast cell proliferative Disease, ovarian cancer, epilepsy, myasthenia gravis, autoimmune Disease, granulomatous Disease, osteonecrosis, liver failure, PMD Disease, lipodystrophy (lybistrophy), demyelinating Disease, cartilage defects, retinal Disease, lupus nephritis, Alzheimer's Disease, traumatic brain injury, sarcoma, myositis, hyperglycemia, and Parkinson's Disease, Macular degeneration, ulcerative colitis, muscle degeneration, and others. Limitations of these stem cell therapies include: stem cells are not optimally delivered and engrafted, either to specific damaged organs or to hematopoietic centers in the bone marrow and spleen.

Delivery of exogenous stem cells

Stem cells can be delivered to a patient by a number of routes. For example, stem cells (in a suitable vehicle to optimize stem cell viability and eliminate cell clumping) can be administered by needle, catheter or other minimally invasive methods in the following manner: intravenous, intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intrapericardial, intraocular, transvascular, endocardial, epicardial, transseptal, epicardial, by coronary vein, by percutaneous myocardial revascularization, intrathecal, intraorgan, intranasal, intraventricular, or epidural. Stem cells can also be administered by these routes in 'matrix' mixtures or suspension mixtures designed to help retain stem cells at the injection site, including, for example, collagen, fibrinogen, fibronectin, laminin, alginate, agarose, methylcellulose, liposomes, nanoparticles, micelles, albumin vesicles, fatty acids, or other semi-solid suspension formulations.

Catheter-based delivery systems that may be used to deliver stem cells include standard balloon angioplasty infusion catheters (standard balloon angioplasty infusion catheters), percutaneous coronary delivery catheters, stop flow inflections of over-the-wire balloon catheters, stent-Ganz type catheters, Hickman type catheters, Foley type catheters, central venous catheters, pig-tail type catheters, SmartPort type catheters, and the like^TMSystems, metal-tipped magnetically permeable catheters (metal-tipped magnetically guided catheters), such as the Gentle Touch Magnetic Navigation System, Accucich System developed by Stereotaxis Inc or Mitralign, and catheters for direct injection into organs, such as HELIX^TM、MyoCath^TMNOGA R-guided Myostat^TM、Stiletto^TMOr intravascular ultrasound (IVUS) guided TransAccess Delivery System^TMOr a catheter delivered via arterial route such as OpenSail^TM、Concerto^TMMicro-syringe infusion catheter from Mercator and Maverick^TMOr via implantable devices, such as Left Ventricular Assist Devices (LVAD), biventricular assist devices (BiVAD), optimizers^TMA cell delivery catheter as described in US 2009/0299269.

Stem cells can also be administered to a patient using invasive surgical means and then injected directly into or applied to an organ. Applications of stem cell compositions to organs include, inter alia, collagen matrices, extracellular matrix compositions, biopolymer microwires made of fibrin or other extracellular matrix materials, patches containing extracellular matrix and biodegradable materials, fibrin patches, alginate (alginator) agarose based patches, scaffolds consisting essentially of extracellular matrix materials and a bio-physiologically degradable inert material that may contain components such as dextran, coated stem cells with organ-specific antigens or binding molecules, residual extracellular matrix (also known as scaffolds or decellularized organs, from ex vivo digested organ donors or remains), contact lenses, and the like.

Mobilization of endogenous stem cells

Another method of treating patients with stem cells involves mobilizing their own stem cells away from organs (e.g., bone marrow) and into the circulation. For example, therapeutic agents such as granulocyte colony stimulating factor (G-CSF; Filgrastim) sold as Neupogen or longer acting forms such as Neulasta, granulocyte-macrophage colony stimulating factor (GM-CFS; Sargrastim) sold as Leukine, AMD3100 sold as Mozobil/Plerixafor by Genzyme Corporation, cause an increase in the number of stem cells in the circulation. Neupogen is provided as a single use vial or single use syringe containing 300 or 480 micrograms of filgrastim. The excipient mainly comprises acetate, sorbitol, polysorbate 80, sodium and water for injection. Neupogen is used clinically as an intravenous twice daily dose, a subcutaneous once daily dose, or as a chronic subcutaneous treatment. Neupogen has been approved for accelerating recovery of neutrophil counts in cancer patients receiving myelosuppressive chemotherapy, acute myelogenous leukemia patients receiving induced or consolidated chemotherapy, cancer patients receiving bone marrow transplantation, severe chronic neutropenic patients, and patients undergoing peripheral blood progenitor collection and treatment. Neupogen is typically administered between 3 and 69 micrograms per kilogram of body weight per day on a daily basis beginning 4 days after chemotherapy for 2 to 20 days. According to Neupogen's package insert, G-CSF regulates neutrophil production within the bone marrow and affects neutrophil progenitor cell proliferation, differentiation, and selective end-cell functional activation (including increased phagocytic capacity, cellular metabolic challenges associated with respiratory bursts, antibody-dependent killing, and increased expression of some functions associated with cell surface antigens). G-CSF has also been shown to mobilize stem cells into the circulation by: it reduces the expression of CXCL12 on bone marrow stroma and reduces the effect of CXCR4 expression, by causing truncation of the N-terminus of CXCR 4(1), by reducing the expression of VCAM in bone marrow (2). G-CSF has been shown to increase CXCL2 (a cognate ligand for CXCR 2). G-CSF has also been shown to increase the number of very small embryonic-like stem cells in circulation, as approved for clinical treatment.

According to the packaging instructions, LEUKINE is indicated for mobilizing hematopoietic progenitor cells into peripheral blood for collection by leukapheresis. Mobilization allows for the collection of an increased number of progenitor cells that can be used for engraftment as compared to collection without mobilization. Transplanting an increased number of progenitor cells after myeloablative chemotherapy can result in more rapid engraftment, which can lead to a reduced need for supportive care. Administration of leukin after peripheral blood progenitor cell transplantation further accelerated the reconstruction of myeloid cells. The recommended dose of leukin is 250 micrograms per square meter of body surface area per day, administered as a 24 hour intravenous infusion or subcutaneously once per day. For mobilizing stem cells into circulation, the optimal treatment time with LEUKINE is obviously 5 days. Leukine in combination with G-CSF has also been shown to be effective in patients who respond to G-CSF alone, as a poor mobilizer.

The Mobozil chemical name is 1,1' - [1, 4-phenylenebis (methylene) ] -di-1, 4,8, 11-tetraazacyclotetradecane. Its molecular formula is C28H54N 8. Mobozil is an inhibitor of chemokine receptor CXCR4 and blocks the binding of its cognate ligand, SDF-1(CXCL 12). Mobozil results in an increase in the number of circulating stem cells by disrupting the binding of CXCR4 expressed by stem cells to SDF-1(CXCL12) expressed by the stroma of the bone marrow and other cells. Optimal mobilization following Mobozil treatment is dependent on complement activation. The subcutaneous injection of Mobozil has been approved for use in combination with Neupogen in order to mobilize hematopoietic stem cells into peripheral blood for collection and subsequent autologous transplantation in patients with non-hodgkin's lymphoma (NHL) and Multiple Myeloma (MM). Mozobil is sold as a single use vial containing 1.2mL of a 20mg/mL solution. Patients were treated with Mozobil according to the following recommended schedule as detailed in the package insert: initiating Mozobil treatment after the patient receives G-CSF once a day for 4 days; repeating the Mozobil administration for up to 4 consecutive days; selecting a dose based on 0.24mg/kg actual body weight; administration by subcutaneous injection was approximately 11 hours before starting plasma separation (apheresis). It has been shown that the combination of G-CSF with Mobozil mobilizes more primitive stem cells into the circulation than G-CSF alone.

Other agents known to mobilize stem cells, including hematopoietic stem cells, into the circulation include Hepatocyte Growth Factor (HGF), erythropoietin, parathyroid hormone, Flt 3-ligand, Stem Cell Factor (SCF). Other agents known or expected to result in increased mobilization of stem and progenitor cells into the circulation include, inter alia: agents that increase stem cell proliferation, such as colony stimulating factor; agents that increase endogenous G-CSF production, such as grifola frondosa (Maitake) β -glucan; agents that reduce expression of SDF-1 or CXCR4, including CXCR4 down-regulated agonists; an agent that reduces the binding affinity of SDF-1 or CXCR 4; an agent that reduces signaling of CXCR 4; agents that block bioaccumulation of stem cells out of circulation; agents that increase stem cell entry into the circulation, such as complement activation or increase plasma sphingosine-1-phosphate; agents that upregulate the expression of CXCR2 in bone marrow or its cognate ligand CXCL 2; agents that reduce VCAM expression in bone marrow, such as the chemotherapeutic agents cyclophosphamide, retinoic acid receptor agonists, small molecule inhibitors of VLA-4 (such as BIO5192) or other blockers of VLA4, metalloproteases or carboxypeptidase activators that degrade CXCL12 expressed in bone marrow, or selected chemotherapeutic regimens, or regimens that add cyclophosphamide or the topoisomerase inhibitor etoposide to G-CSF treatment, ingest fucoidan, via the chemokines CXCL2 and colominic.

Spontaneously released endogenous stem cells

Another method of treating patients with stem cells involves preventing the sequestration of spontaneously released endogenous stem cells in lymphoid tissues. Stem and progenitor cells are released into the blood stream spontaneously on a daily basis. Experiments with conjoined mice (paragenic mice) demonstrated that spleen readily swaps stem and progenitor cells in the circulation. Furthermore, it has been demonstrated that disease conditions, such as, inter alia, hypercholesterolemia, heart attack, STEMI or CAD, arterial ligation or transient cerebral ischemia, sojourn at moderate altitude, primary hyperparathyroidism, and retinal pigment epithelium damage, result in increased levels of circulating stem and progenitor cells. Preventing the encapsulation of spontaneously released or disease-induced stem cells in lymphoid tissues would make more stem cells available for regeneration of damaged tissues or organs.

Compositions and methods for regenerating germinal centers in lymphoid tissues

The present invention fills this need by providing methods and compositions for regenerating, restoring, and increasing the number of germinal centers in lymphoid tissue following radiation or chemotherapy. Therapeutic agents that restore or regenerate germinal centers in lymphoid tissues according to the present invention include immune activators, co-stimulatory molecules, immune adjuvants, and combinations thereof.

In one embodiment of the invention, germinal centers in lymphoid tissue are regenerated by administering an adjuvant. Examples of such adjuvants include pathogen-associated molecular patterns (molecular patterns), liposomes, lipopolysaccharides, molecular cages for antigens, bacterial cell wall components, endocytosed nucleic acids such as double-stranded rna (dsrna), single-stranded DNA (ssdna), and DNA containing unmethylated CpG dinucleotides, mineral salts such as aluminium hydroxide (alum), aluminium phosphate, calcium phosphate, aluminium hydroxide, aluminium potassium phosphate, aluminium sodium hydrogen phosphate, and aluminium hydroxyphosphatesulfate. Other adjuvants include oil-in-water emulsions such as squalene, Montreade ISA720 (squalene) or ISA51(Drakeol), MF59(Novartis) and SBAS 2. Another class of adjuvants that can be used according to the invention are particulate adjuvants such as virosomes, saponins and lipids, including microbial derivatives such as monophosphoryl lipid a CpG motifs, known as BCG-CWS (Mycobacterium bovis) immunization.

Lipopolysaccharides and mitogens such as canavalin a, components of bacterial cell walls, archaeological liposomes (archaeosomes) (etherglycerides of archaebacteria methanobacterium smithii), the TLR4 agonist GLA glucopyranosyl lipid adjuvant, LPS and BCG (immune design), the TLR2 agonist BCG, peptidoglycan and gram positive bacteria, the TLR5 agonist flagellin, the blood Sucker Egg Antigen (SEA), Listeria Monocytogenes (LM). Other adjuvants include bell-like receptor agonists and activators including CpG oligonucleotides up to 100 bases in length (most preferably 20 bases in length), TLR1 agonists such as Pam3Cys, TLR2 agonists such as Pam3 Cys; TLR3 agonists such as dsRNA and poly I: C; TLR7 agonists such as imidazoquinolines, for example Imiquimod (Imiquimod) (R-839) and Rasimmod (Resiquimod) (R-848); a TLR8 agonist such as Rasimotene (R-848); and TLR9 agonists such as poly I: C and CpG. The invention also includes the use of plant derivative adjuvants beta-glucan, saponin-based QS21, and concanavalin a.

Another embodiment of the invention includes increasing the number of active germinal centers in the spleen to enhance stem cell transplant engraftment and blood recovery in patients undergoing cancer therapy, non-myeloablative therapy or myeloablative therapy, including chemotherapy, radiation and combination therapy. An increase in the number of active germinal centers leads to a regimen of chemotherapy modulation and an enhanced rate of stem cell binding and engraftment in the spleen and subsequent hematopoietic recovery following cancer treatment. In addition to the treatment of cancer and leukemia, non-myeloablative therapies are used to treat autoimmune diseases (Pediatr Clin North am.57(1): 239-. The present invention provides methods for enhancing hematopoietic recovery in cancer or autoimmune patients undergoing myeloablative or non-myeloablative modulation by increasing the number of spleen germinal centers and thereby increasing transplanted stem cell binding, engraftment, and proliferation. For example, patients undergoing stem cell therapy following myeloablative or non-myeloablative conditioning regimens are at risk of infection and death during the time required for engraftment and hematopoietic regeneration of the administered stem cells. Accelerating and increasing stem cell engraftment by increasing the number of germinal centers available for stem cell binding in the spleen can reduce the time required for hematopoietic recovery and thereby reduce the risk of infection and death in these patients.

The stem cells may be autologous or derived from an unrelated donor. The stem cells may be contained within a mononuclear cell fraction from bone marrow, whole blood, cord blood, adipose tissue, or other sources, or they may be purified by selection for CD34, CD133, CD105, CD117, SSEA1-4, dye exclusion, or other specific stem cell antigens.

Spleen hair growth centers can be specifically increased by treatment that activates the CD40 receptor (blood.104: 4088-. The functional receptor is a CD40 trimer or multimer having a TNFR1 or TNFR2 component. Examples of treatments that activate the CD40 receptor include: agonistic antibodies to CD40 activate the CD40 receptor, appropriate conformation of solCD40L may activate the CD40 receptor, and agents that increase expression of the CD40 receptor by altering transcription rate, such as via the AT-hook transcription factor AKNA, mRNA stability, or protein stability, may also cause increased activity and signaling. Alternatively, members of the TRAF and TTRAP families interact with the CD40 receptor and mediate its signaling, thereby causing an increase in the number of active germinal centers. Agents that activate germinal center B cell cyclooxygenase 2 or EP2 receptors replicate CD40 receptor engagement and may cause increased formation of active germinal centers. Other means of activating the formation and persistence of germinal centers include inhibition or loss of CCR7(J.Leukoc.biol.85: 409-. Other means of activating the formation and persistence of germinal centers include inhibition or loss of CCR7(J.Leukoc.biol.85: 409-.

In another embodiment of the invention comprises administering an immunostimulatory molecule to promote regeneration of germinal centers in lymphoid tissue. The immunostimulatory molecule can be an antibody, a fusion protein, a soluble ligand, a small molecule, a transcriptional modulator, an mRNA or protein stabilizer, and other immunostimulatory moieties. For example, the co-stimulatory CD28 pathway can be activated with soluble B7 protein and an antibody such as a compound with TeGenero 1412.

TGN1412 is a humanized monoclonal antibody designed as an agonist of the CD28 receptor on T lymphocytes that stimulates the production and activation of T lymphocytes. TGN1412 is manufactured by Boerhinger Ingelheim.

Other costimulatory molecules and pathways include OX40/OX40 ligand, 4-1BB/4-1BB ligand, the B7/CD28 family; B7-1/CD80, CD28, B7-2/CD86, CTLA-4, B7-H1/PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2/B7-DC, B7-H4, PDCD6, BTLA, co-stimulatory TNF superfamily molecules; 4-1BB/TNFRSF9/CD137, 4-1BB ligand/TNFRSF 9, BAFF/BLyS/TNFRSF 13B, BAFFR/TNFRSF13C, CD27/TNFRSF7, CD27 ligand/TNFRSF 7, CD30/TNFRSF8, CD30 ligand/TNFRSF 8, CD40/TNFRSF5, CD40 ligand/TNFRSF 5, GITR/TNFRSF18, GITR ligand/TNFRSF 18, HVEM/TNFRSF14, LIGHT/TNFRSF 14, OX40/TNFRSF4, OX40 ligand/TNFRSF 4, and TACI/TNFRSF13B, SLAM family; 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and SLAM/CD150, as well as other costimulatory molecules; CD2, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, Ikaros, integrin alpha 4/CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7/LPAM-1, LAG-3, LMIR1/CD300A, CRTAM, DAP12, dendritic cell-associated lectin-1/CLEC 7A, DPPIV/CD26, EphB6, TCL1A, TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, HLA-DR, and erythropoietin hepatocyte receptor ligands.

Other agents that can enhance germinal center regeneration are agents known to cause cytokine outbreaks, such as anti-CD 20 (rituximab).

Other immune activators may include IL21 receptor agonists, i.e., agonistic antibodies.

Other adjuvants used preclinically or clinically include:

endocytic nucleic acids such as double-stranded rna (dsrna), single-stranded DNA (ssdna), and DNA containing unmethylated CpG dinucleotides.

Adenoviruses and adenoviral components such as adenovirus type 5 (Ad 5).

Agonists and activators of retinoic acid inducible gene (RIG) -1-like receptor (RLR).

A P2X1, P2X4, or P2X7 activator or agonist.

Agonists and activators of nucleotide-binding oligomerization domain-like receptors (NLRs).

Advax, liposomes, chitosan microspheres, and dimethyl-dioctadecyl ammonium bromide (DDA).

The most recent human adjuvants in development are ISCOMS, QS21, AS02 and AS 04.

ASO4 is deacetylated monophosphoryl lipid A MPL plus aluminum.

The CpG oligonucleotide may be an oligonucleotide up to 100 bases in length, most preferably 20 bases in length.

anti-CD 3OKT3 may also be administered intravenously (i.v.) to induce regeneration of germinal cells within lymphoid tissues.

The invention also includes administering two or more therapeutic agents to create germinal centers within lymphatic tissue. In another embodiment of the invention, the stem cells are administered in conjunction with a therapeutic agent that regenerates germinal centers.

Administration of adjuvants

The potassium aluminum phosphate may be administered at a dose of about 0.17 mg.

The aluminum phosphate may be administered at a dose of about 1.5 mg.

The aluminum hydroxide may be administered in a dose of about 0.15mg to about 0.3 mg. Aluminum salts may generally be administered at doses up to 0.85 mg.

Aluminum hydroxyphosphate can be administered at a dose of about 0.225 mg.

The aluminum hydroxide and aluminum phosphate as a combination may be administered in a combined dose of about 0.45 mg.

Administration of oil emulsions

SBAS-2 is an oil-in-water emulsion of MPL and QS 21.

Oil-in-water emulsions such as Monteda ISA720 (squalene) or ISA51 (Drakeol).

Squalene is in MF59 adjuvant used by Novartis and is administered at 4 grams per hundred 1.95% or 2 parts or per 100ml for 0.5 to 1ml injection for adjuvant activity.

MF59 adjuvant contains (Lipotant; 4-5% w/v squalene, 0.5% w/v Tween 80, 0.5% span 85 and optionally various amounts of the muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE), which activates the non-TLR sensory receptor known as NOD-LRR).

Administration of microbial derivatives

BCG-stimulated immunity called BCG-CWS (M. bovis): adult human being 1 to 8X 10 per vial⁸Individual Colony Forming Units (CFU), the child dose was halved.

The archaeal liposomes (etherglycerides of the archaeal methanobacterium brevibacterium smithii) are administered at 0.1 to 500 micrograms per gram of body weight and most preferably at 38 micrograms per mg of body weight.

Administration of toll-like receptor agonists and activators

CpG oligonucleotides up to 100 bases in length, most preferably 20 bases in length, are administered at 1, 10, 100, 500 micrograms per 20-25 grams body weight. The preferred dose is 10. mu.g per 20-25mg body weight.

The co-stimulatory CD28 pathway can be activated with soluble B7 protein and an antibody such as a compound with TeGenero 1412. TGN1412 is a humanized monoclonal antibody designed as an agonist of the CD28 receptor on T lymphocytes that stimulates the production and activation of T lymphocytes. TGN1412 is manufactured by Boerhinger Ingelheim. For the purpose of immune activation to regenerate lymphatic germinal centers, a preferred dose of TGN1412 is given as less than 0.1mg/kg body weight administered by infusion over 3-6 minutes. An effective dose of TGN1412 for germinal center regeneration is between 0.001mg/kg and 0.1mg/kg, preferably 0.01 mg/kg.

Other agents that can enhance germinal center regeneration are agents known to cause cytokine outbreaks such as anti-CD 20 (rituximab), which is administered at 50mg/mm2 or 150mg/mm2 but less than 375mg/mm 2; anti-CD 3OKT3 administered intravenously (i.v.) at a preferred dose of less than 5 mg/day for 10 to14 days; an anti-CD 52(CAMPATH) antibody administered as a 30mg infusion over 2 hours, given three times per week for up to12 weeks.

Other immune activators may include agonists of the IL21 receptor (agonistic antibodies), administered at between 0.001mg/kg and 50mg/kg, preferably between 0.01 and 0.1 mg/kg. The protein ligand agonist may be administered between 0.0001mg/kg and up to 50mg/kg per day for up to 28 days following myeloablative or non-myeloablative treatment. Generally, depending on the molecular weight and biodistribution of the agonist, the protein ligand agonist is effective at 1/10 doses of the antibody therapeutic.

The small molecule immune activator can be delivered orally in a single oral dose between 1mg and 1000mg per day, or at specific intervals, which can include an interval period of every 2 hours, every 4-6 hours, or longer.

ASO₄Is deacetylated monophosphoryl lipid A MPL plus aluminium administered as 50 microgram in combination with 0.5mg aluminium phosphate in Fendrix (GSK) at a dose of 0.5 ml.

CpG oligonucleotides up to 100 bases in length, most preferably 20 bases in length, are administered at 1, 10, 100, 500 micrograms plus alum per 20-25 grams body weight. The preferred dosage is 10. mu.g per 20-25mg body weight.

Administration of

Administration of a therapeutic agent that induces regeneration of germinal centers within lymphatic tissue may be performed by any method including: intravenous, intraarterial, oral, intramuscular, nebulization, inhalation, intradermal, subcutaneous, intraperitoneal, intrapericardial, intraocular, transvascular, endocardial, epicardial, transseptal, epicardial, by coronary vein, percutaneous transmyocardial revascularization, intrathecal, intraorgan, intranasal, intraventricular, or epidural via needle, catheter, or other minimally invasive means.

Detailed Description

The following examples illustrate the experimental results of methods and compositions for modulating or moderating the number of binding sites available for participation in stem cell binding.

Example 1

Bone marrow and whole blood mononuclear fraction enriched in stem cells and B of the margin of white marrow of spleen when administered to allogeneic mice Cell zone binding

Bone marrow and whole blood mononuclear cell fractions were isolated from male 129S1/SvlmJ mice using Histopaque and pooled. The stem cell fraction in monocytes was concentrated by incubating the cells at 37 ℃ for 4 days with 5% CO2 to deplete differentiated somatic cells. The resulting cells were then labeled with cell tracer orange (CTO Invitrogen) according to the manufacturer's instructions. Approximately 1 million labeled cells were administered to recipient pups by retrobulbar injection, and after 90 minutes the mice were bled and blood collected, residual red blood cells of the vascular system were flushed and the spleen was harvested. Spleens were fixed in 1% PFA overnight and then embedded in low melting, low gelation temperature agarose and sectioned at a thickness of 200 microns per slice. Binding of MNC stem cells to spleen was visualized using immunofluorescence. Labeled MNCs containing a stem cell fraction bind to the B cell region of the margin of the white marrow of the spleen. Fluorescence immunoassay of MNC fraction from whole blood collected during exsanguination demonstrated that approximately 40,000 of 1 million injected labeled cells were continuously found in the circulation 90 minutes after injection.

Results and conclusions: MNC cells enriched for CTO-labeled stem cells bind peripherally to the white marrow region. The binding of the cells is histologically evident on the B cell region. This shows that when administered to allogeneic mice, the bone marrow and whole blood mononuclear fraction, which is rich in stem cells, binds to the B cell region of the margin of the white marrow of the spleen

Example 2

CD34+, CD105+, and CD117+ purified stem cells bind to the fraction of MNC stem cells containing the label Spleen regions of the same size

Bone marrow and whole blood mononuclear cell fractions were isolated from male mice using Histopaque and pooled. MNC cells labeled with cell tracer green (CTG, Invitrogen) were then incubated with biotin-labeled antibodies to CD34, ckit, and CD105 and purified using a Miltenyi magnetic separation column according to the manufacturer's recommendations. A portion of the isolated MNCs were individually labeled with CTO. One million CTO-labeled MNCs were incubated with 205,000 CTG-labeled purified stem cells at 4 ℃ for 12-18 hours on fresh spleen sections 100-200 microns thick. Spleen sections were washed thoroughly to remove unbound cells, fixed in 1% PFA for one hour and then wet-mounted (wet mounted) for fluorescence imaging. The resulting red (MNC) and green (stem cell) binding was captured and superimposed using MetaMorph software.

Results and conclusions: the purified stem cell population combined with the same spleen regions into MNC fractions. This indicates that CD34+ CD105+ and CD117+ purified stem cells bind to the same spleen region as the fraction of labeled MNC stem cells.

Example 3

Bone marrow and whole blood mononuclear fraction stem cells bound to PNA positive regions in spleen germinal center

Bone marrow and whole blood mononuclear cells (MNC) were isolated from adult mice using Histopaque. The resulting monocytes are stained with cell-tracing dyes such as cell-tracing orange, green or blue, DiI or calcein orange or blue according to the manufacturer's recommendations. Specific B cell regions in spleen white marrow were identified using FITC labeled PNA (10ug), IgD (10 μ g) or anti-CD 21(10 μ g). PNA marks the germinal center, IgD marks the follicular region, and anti-CD 21 marks the limbic and mantle regions (mantle zones). FITC labeled anti-CD 3(200ng to1 μ g) was used to identify T cell regions in spleen white marrow. After thorough washing, the bound cells and antibody were fixed on spleen sections using 1% PFA at 4 ℃ for 1 hour. The wet sections were observed for fluorescence and photographs were taken using MetaMorph software.

[0222] Results and conclusions: after incubation for 15 hours at 4 ℃, CTO-labeled MNCs bound to active PNA + development centers. No co-localization of CTO-labeled MNC binding with IgD or anti-CD 21 was observed (colocalized). This indicates that bone marrow and whole blood mononuclear fraction stem cells bind to the PNA-positive IgD-negative CD 21-negative region in the spleen germinal center.

Example 4

Separation of CD34+, CD105+, CD117+ stem cells from whole blood and bone marrow mononuclear cell fractions from spleen white marrow PNA + hair growth center binding

The mononuclear cell fractions from mouse whole blood and bone marrow were combined, labeled with CTO and then incubated with biotinylated anti-CD 34, anti-CD 117, and anti-CD 105, and then the antibody-bound cells were isolated using a Miltenyi magnetic cell separation column according to the manufacturer's instructions. The number of recovered CD34+ CD105+ CD117+ stem cells ranged from 0.3% to 3% of the starting MNC fraction.

The resulting CD34+ CD105+ CD117+ cells were co-incubated with 10. mu.g PNA for 15 hours on fresh mouse spleen sections. Positively selected cells were added at 100,000(a), 50,000(B), 25,000(C) or 10,000(D) cells per spleen section. Similar to MNC incubation, purified stem cells bound to PNA-positive germinal centers of splenic white marrow. Stem cells bind in a concentration-dependent manner. Cells bind to discrete niches in the germinal center, and a large number of added cells greater than about 100,000 produce stronger binding signals, rather than extending to additional niches (see example 2).

Results and conclusions-these results indicate that CD34+, CD105+, CD117+ stem cells isolated from whole blood and bone marrow mononuclear cell fractions bind to the PNA + hair center of the splenic white marrow.

Example 5

Binding of stem cells in MNC moieties is blocked by anti-CD 45 antibodies

Binding of MNCs and stem cells to spleen sections was blocked by rat monoclonal IgG2b anti-mouse anti-CD 45(800 nanograms to 4 micrograms), but not by anti-CD 45R (10 micrograms) or anti-CD 3 antibody (1 microgram). 30-F11 rat anti-mouse anti-CD 45 antibody (Santa Cruz Biotechnology) or 17A2 anti-CD 3 antibody (Santa Cruz Biotechnology) was diluted 1:50 or 1:10 and incubated with 250,000 CTO-labeled MNC for one hour. MNC binding was counted visually by the number of bound niches and the size of the niches. CTO-MNC control fresh spleen sections each had 3-6 MNC binding niches ranging from medium to large size. The anti-CD 3 antibody did not affect the number or size of MNC binding niches. The anti-CD 4530-F11 antibody diluted 1:50 reduced MNC binding to niches by half in both number and size. anti-CD 4530-F11 antibody diluted 1:10 completely abolished binding of CTO-MNC to fresh spleen sections.

In another experiment, labeled MNC were incubated for 1 hour on spleen sections in the presence of 30-F11 anti-CD 45(4 micrograms) or PC3/188A anti-CD 3(1 microgram) (Santa Cruz Biotechnology).

In another experiment, antibodies to cell-labelled orange (CTO) labelled MNCs were incubated with CD45R or 30-F11 anti-CD 45 for 15 hours at 4 ℃. 1:10 dilution (5. mu.g) of CD45R antibody (Miltenyi Biotec) did not block CTO-MNC binding to fresh mouse spleen sections. In contrast, a 30-F11 anti-CD 45 antibody (Santa Cruz) at 1:10 (4. mu.g) reduced binding of CTO-labeled MNCs to fresh spleen sections. CD45R bound B cells in the follicular region but not the active germinal centers.

Incubation with 1:10 of 30F-11 anti-CD 45 reduced binding of CTO-labeled MNCs to fresh spleen sections by approximately 75%.

Results and conclusions: these data indicate that binding of stem cells in MNC fraction to spleen was blocked by anti-CD 45 antibody.

Example 6

The CD45 epitope was bound by the 30-F11 rat IgG2b anti-mouse anti-CD 45 antibody. 30-F11 binding to mouse CD45 All isoforms of

The precise binding of the 30-F11 epitope has never been mapped (mapped). 30-F11 was generated by immunization with mouse spleen and thymocytes. The extracellular domain of mouse CD45 isoform 1 comprises amino acids 24 through 564. Isoform 2 loses amino acids 31 to 73, while isoform 3 loses amino acids 31 to 169. Since 30-F11 is reported to bind to all isoforms of mouse CD45, the binding epitope should therefore be between amino acids 170 to 564 in isoform 1. Antigenic regions of proteins can be predicted using the hydrophobic (Kyte Doolittle) and accessibility (accessibility) algorithms found on the Swisport website. Antigenic regions are most likely to be found in regions of low hydrophobicity and high accessibility. Amino acid residues in the human sequence close to 501 to 521 have low hydrophobicity prediction values, indicating that this region of the protein is a potential antigenic site. This region is also quite conserved from mouse to human. (see Okumura M. et al, 1996Aug15;157(4): 1569-75).

Example 7

Immune adjuvants increase germinal center formation and increase binding of mononuclear stem cell fractions to the spleen

Active germinal centers were elicited in normal mice by purposeful immunization with Incomplete Freund's Adjuvant (Incomplate Freund's Adjuvant) or Ribi. Mice were injected Intraperitoneally (IP) with 0.5ml incomplete freund's adjuvant (FIA) mixed with PBS or 0.5ml RIBI adjuvant at 1:1 on day 0. On day 7 or 14 after immunization, mice were heparinized with 100U intraperitoneal heparin for 30 minutes before avertin (avertin) anesthesia. Mice were bled by retrobulbar ocular bleeding, yielding a total of 1.5 to 1.8ml of whole blood, which was added to 200 μ l of 5U/ml heparin in a 15ml conical tube. The abdominal aorta and vena cava were then severed and residual blood was completely flushed from the vascular system by slow bolus injection of 10mL of 5U/mL heparin through the ascending thoracic vena cava (asciding thoracicvena cava). Spleens were removed and placed in growth medium and then embedded in soft agarose and sliced to obtain uniform slices 200 microns thick. Sternal and femoral bone marrow were washed out of bone with Hanks buffered saline solution and whole blood and mononuclear stem cell fractions from bone marrow were separated and pooled using Histopaque. Germinal centers on spleen sections were identified by incubation overnight at 4 ℃ using FITC-labeled PNA (10 μ g). After overnight incubation with PNA, without washing, CTO-labeled mononuclear stem cell fractions were added to the sections at 4 ℃ for 1 hour. After thorough washing, bound cells and labeled PNA were fixed on spleen sections using 1% PFA for 1 hour at 4 ℃. The wet sections were observed for fluorescence and photographs were taken using MetaMorph software.

PNA binding 7 days after immunization was similar in FIA and RIBI treated mice, both almost double the binding of controls. However, FIA treated mice were healthier than RIBI treated mice, so all subsequent experiments were performed with FIA. Compared to days 7 and 14 after FIA treatment, PNA brightness was higher at day 7, however, the active germinal centers at day 14 appeared to be more tightly organized and compact. FIA immunization doubled the number of active germinal centers in the mouse spleen compared to controls. The addition of a CTO-labeled monocyte fraction to the spleen (isolated from control mice) demonstrated that the increased formation of active germinal centers observed in FIA-treated mice also caused significantly greater binding of the monocyte to the spleen. Monocyte binding at day 7 was increased approximately 3-5 fold compared to binding to the control spleen, while monocyte fraction binding was increased as much as, and in some cases, more than 10 fold, compared to binding to the control spleen.

Results and conclusions: these data indicate that the immunoadjuvant enhances germinal center formation and increases the binding of the mononuclear stem cell fraction to the spleen.

Example 8

Inhibition of active germinal center formation reduces ex vivo stem cell binding to spleen

Mice were treated with 1mg dexamethasone dissolved in ethanol (1 part) and PBS (9 parts) by intraperitoneal injection 7 days before collection of mouse spleen and stem cells for ex vivo analysis of stem cell binding to spleen sections. Control mice were treated with ethanol (1 part) and PBS (9 parts) only in a total volume of 1 ml. On day 7, mice were harvested and treated as detailed in example 7. The mononuclear stem cell fraction from control mice was used for binding studies on control and dexamethasone-treated spleen sections. Monocyte binding decreased by 30-40% on day 7, 7 days prior to the experiment, after a single treatment with 1mg dexamethasone.

A single 1mg dexamethasone treatment 7 days prior to collection reduced spleen weight by an average of 22%, reduced mean circulating MNC number by 34% and reduced PNA-labeled germinal centers by up to 24%, however, the percentage of stem cells within MNC fraction from whole blood or bone marrow did not decrease and actually increased by an average of 32% over the control.

Results and conclusions: these data indicate that the immunosuppressive agent reduces the number of monocyte fraction-binding cells in the spleen of treated mice.

Example 9

Inhibition of active germinal center formation reduces in vivo stem cell binding to spleen

The primary control mice were heparinized with 100U intraperitoneal heparin for 30 minutes prior to avertin anesthesia. Mice were bled by retrobulbar ocular bleeding, yielding a total of 1.5 to 1.8ml of whole blood, which was added to 200 μ l of 5U/ml heparin in a 15ml conical tube. The abdominal aorta and vena cava were then severed and residual blood was completely flushed from the vascular system by slow infusion of 10mL5U/mL heparin through the ascending thoracic vena cava. Sternal and femoral bone marrow were washed out of bone with hanks buffered saline solution and whole blood and mononuclear stem cell fractions from bone marrow were separated and pooled using Histopaque. The mononuclear stem cell fraction was resuspended in growth medium (DMEM plus 10% FBS) and incubated overnight at 37 ℃ under 5% CO 2. The next morning mononuclear stem cells were labeled with Cell Tracer Green (CTG) according to the manufacturer's instructions.

On day 7, the recipient mice were injected through the retrobulbar sinus with approximately 6M CTG MNC in 100ul immediately after staining of the native control mononuclear stem cells (MNC) with CTG. Five control mice received intraperitoneal injections of 100ul ethanol and 900ul PBS on days 0 and 4; 3 mice were treated with peritoneal injections of 1mg dexamethasone on days 0 and 4; and two mice were treated with peritoneal injections of 1mg dexamethasone on days 0,2 and 5. One hour later, mice were bled by retrobulbar ocular hemorrhage. The abdominal aorta and vena cava were then severed and residual blood was flushed from the intravascular system completely by slow bolus injection of 10mL5U/mL heparin through the ascending thoracic vena cava. Spleens were removed and kept on ice in RPMI without phenol red plus 1% BSA. Sternal and femoral bone marrow were washed out of bone with hanks buffered saline solution and whole blood and mononuclear stem cell fractions from bone marrow were separated using Histopaque.

Spleens were dissociated into single cell suspensions and the extent of injected MNC CTG sequestration in spleens was determined using flow cytometry. Dexamethasone (2 mg total) reduced the accumulation of total MNC CTG cells in the spleen by 33-43%, while a dose of 3mg total dexamethasone reduced the accumulation of total MNC CTG cells in the spleen by approximately 70%.

Results and conclusions: these data indicate that 7-day immunosuppressant treatment reduced the number of exogenously injected MNC stem cells sequestered in the spleen.

Example 10

Inhibition of active germinal center formation reduces stem cell binding to spleen

(prophetic)

Human volunteers are treated prophylactically with commercially available common immunosuppressive agents such as prednisone (prednisone) according to established clinical protocols, using dosages selected to limit or avoid all side effects of the agents altogether. Another group of human volunteers was treated with antagonistic antibodies to CD40 (e.g., HCD-122 anti-CD 40mAb) at a dose of between 5 and 100 mg/kg.

Autologous stem cells from 50ml of iliac crest (iliac crest) bone marrow or from the mononuclear cell fraction of whole blood isolates were isolated before or on the day of the last prophylactic immunosuppression. The resulting stem cell-containing MNC moiety is labeled with 2- [18F ] -fluoro 2-deoxy-D-glucose (18F-FDG) for subsequent 3D-PET imaging or with an appropriate nuclear imaging tag for subsequent SPECT imaging. Fractions containing labeled MNC stem cells (with between 2 million and 1 million stem cells) were injected intravenously and their biodistribution was determined by PET or SPECT imaging 60-90 minutes and up to 48 hours after administration. Within 90 minutes after injection, stem cells in MNCs accumulate predominantly in the spleen of normal volunteers. In contrast, spleen accumulation was reduced in MNC stem cell fraction of immunosuppressed and HCD-122 treated volunteers.

Example 11

Inhibiting or reducing active germinal center formation increases stem cell delivery to the heart and promotes functional recovery in mice Compound medicine

(prophetic)

3-and 12-month-old PN and 129S1/SvlmJ mice were injected intravenously with anti-CD 40L mAb (PharMingen) or control hamster Ig (Pierce, Rockford, Ill.) (i.v.; on days 0,2, and 4, 250 mg/injection). Whole blood and bone marrow mononuclear fraction stem cells are collected from primary pup mice, labeled with a cell tracking dye such as CTO, and then purified with a Miltenyi magnetic separation column using biotinylated anti-CD 34, anti-CD 105, anti-SSEA 1, and anti-CD 117 antibodies. Experimental mice were injected retrobulbar or intravenously on day 5 with between 100,000 and 1M purified stem cells. After 15 to 24 hours, mice were bled and blood collected, residual red blood cells in the vascular system were washed and spleens were collected for fluorescence imaging of stem cell accumulation. Stem cell accumulation in active PNA + germinal centers was evident, and control IG-treated PN mice demonstrated significantly higher active germinal center numbers and consequent elevated stem cell binding than 129S1 mice. anti-CD 40L mAb treated 129S1 mice had little, if any, apparent active germinal centers and negligible to no stem cell binding. anti-CD 40L mAb treated PN mice showed a reduction in the number of active germinal centers, and thus, a reduction in stem cell binding associations, compared to control Ig treated PN mice.

To study cardiac regeneration in these mice, 3-and 12-month-old PN and 129S1/SvlmJ mice were injected intravenously with anti-CD 40L mAb (PharMingen) or control hamster Ig (Pierce, Rockford, IL) (i.v.; 250 mg/injection on days 0,2, and 4). On day 4, mice were anesthetized, echocardiography was performed for baseline cardiac function and volume, and then the LAD coronary artery was permanently ligated via thoracotomy to infarct approximately 70% of the left ventricular free wall.

Whole blood and bone marrow mononuclear fraction stem cells were collected from naive pup mice and purified using a Miltenyi magnetic separation column using biotinylated anti-CD 34, anti-CD 105, anti-SSEA 1, and anti-CD 117 antibodies. Three days after permanent ligation of LAD, experimental mice were injected retrobulbar or intravenously with between 100,000 and 1M purified stem cells on day 7. Serial echocardiography was performed on mice on day 14, day 21, and day 28.

Mice treated with no Ig control receiving stem cell injection showed significant cardiac function decline and increased end diastolic volume, end systolic volume, and increased non-infarct wall thickness, with 50-100% of the mice dying from heart failure before day 28. The 129S1 mice treated with Ig controls receiving stem cell injections showed reduced heart failure death and slightly improved cardiac function compared to those not receiving stem cell injections. The Ig control treated PN mice receiving stem cell injection showed no improvement in survival or function compared to 129S1 mice. In contrast, 129S1 mice injected with anti-CD 40L mAb-treated stem cells showed significant improvement in survival and cardiac function compared to all other groups of mice, while PN mice injected with anti-CD 40L mAb-treated stem cells had improved survival and cardiac function compared to control Ig-treated stem cell-injected PN mice.

Example 12

Inhibiting or reducing active germinal center formation increases stem cell delivery to the heart and promotes functional recovery in humans Compound medicine

(prophetic)

Patients with acute ST elevation MI successfully treated with stenting in the acute phase of infarction by percutaneous coronary intervention were eligible for the study. The patients were treated with 5, 10, 20 or 100mg/kg of anti-CD 40L mAb rayleigh monoclonal antibody (Replizumab) by IV infusion for 30 minutes on day 1.

Monocytes were recovered from 50mL of bone marrow aspirated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) 3 to 15 days after MI. The entire process from bone marrow aspiration to final product is performed according to Good Manufacturing Practice guidelines.

Cardiac function is followed by MRI and other outcomes including death, recurrent MI, and subsequent revascularization or hospitalization (event-free survival). Stem cell treated patients historically have a 80% versus 55-60% event-free survival reduction of one year compared to placebo patients. anti-CD 40L and stem cell treated patients had improved event-free survival compared to patients treated with stem cells alone. In addition, patients treated with anti-CD 40L stem cells showed a further reduction in ventricular volume and improved cardiac ejection parameters compared to patients treated with stem cells alone.

Example 13

Identification and isolation of monocyte fraction stem cell binding partners in mouse spleen

Bone marrow and whole blood mononuclear cell fractions from male 129S1/SvlmJ mice were isolated using Histopaque, pooled and labeled with CTO according to the manufacturer' S instructions. Spleens were dissociated into single cell suspensions and labeled with CTB. Obtaining stem cells by: MNCs were cultured in growth medium for 7 days, followed by 7 days in FBS-free growth medium supplemented with 120ng/ml stem cell factor and 25% horse serum, and non-adherent cells were harvested. Typically, up to 40% of non-adherent cells express CD34, CD105, SSEA1, and/or CD 117. Stem cells were labeled with CTO.

CTB-labeled splenocytes were incubated with CTO-labeled MNCs at a ratio of 1: 1(1 million CTB-labeled splenocytes and 10M MNCs in 500ul volume of PBS) or at a ratio of 100: 1(1 million CTB-labeled splenocytes and 100,000 CTO-labeled stem cells in 500ul volume of PBS) at 37 ℃ with 5% CO2 on a shaker. Aliquots were run on a Beckman Coulter Gallios flow cytometer to capture FL2 and FL9 fluorescence. In the Upper Right (UR) quadrant of the scatter plot, cell-cell binding was evident with a time-dependent increase in CTB + CTO + common positive signals. The background UR is 0.15%. The maximum binding of splenocytes and stem cells was 2.6% UR at 50 min, and the maximum binding of splenocytes and MNCs was 5% UR at 30 min.

Results and conclusions: these data indicate that splenocytes involved in the binding of stem cells to the spleen can be identified and isolated using flow cytometry.

While the preferred embodiments of the invention have been illustrated and described, as noted above, many changes can be made without departing from the spirit and scope of the invention. Accordingly, the scope of the invention is not limited by the disclosure of the preferred embodiments. Instead, the invention should be determined entirely by reference to the claims that follow.

The claims (modification according to treaty clause 19)

1. A method of delivering stem cells to a target tissue or organ and inhibiting binding of the stem cells to the spleen, peyer's patches, lymph nodes or other secondary lymphoid tissue comprising:

administering the stem cells in conjunction with a therapeutic agent that inhibits binding of the stem cells to the lymphoid tissue, but does not inhibit binding of the stem cells to the target tissue or organ.

2. And (5) deleting.

3. The method of claim 1, wherein the therapeutic agent inhibits binding of the stem cells to germinal centers within the lymphoid tissue.

4. The method of claim 3, wherein said germinal center is present in a lymphoid tissue selected from the group consisting of spleen, Peyer's patches, and lymph node tissue.

5. The method of claim 3, wherein the germinal center is active.

6. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of:

agents that interfere with purine synthesis, antimetabolites, radiation to the spleen, chemotherapeutic agents, immunosuppressive agents, glucocorticoids, anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents, anti-eotaxin, anti-T Cell Receptor (TCR) agents, anti-interferon alpha agents, anti-interferon beta agents, anti-interferon gamma agents, anti-TGF alpha agents, anti-TGF beta agents, anti-integrin agents, anti-alpha 4 agents, anti-interleukin 1 agents, anti-interleukin 2 agents, anti-interleukin 4 agents, anti-interleukin 5 agents, anti-interleukin 6 agents, anti-interleukin 12 agents, anti-interleukin 13 agents, anti-interleukin 23 agents, anti-interleukin 2 agents, anti-Vascular Adhesion Protein (VAP) agents, anti-B7 agents, IgE agents, anti-TNF agents, anti, anti-Vascular Endothelial Growth Factor (VEGF) agents, anti-baff (blys) agents, anti-CTLA 4 agents, anti-complement agents, anti-CD 2 agents, anti-CD 3 agents, anti-CD 4 agents, anti-CD 5 agents, anti-CD 20 agents, anti-CD 23 agents, anti-CD 25a agents, anti-CD 40 agents, anti-CD 154(CD40L) agents, anti-CD 62L agents, anti-CD 80 agents, anti-CD 147 agents, anti-LFA 1 agents, anti-CD 11a agents, anti-CD 18 agents, purine synthesis inhibitors, pyrimidine synthesis inhibitors, anti-proliferative agents, anti-metabolic agents, anti-folate agents, and anti-mTOR agents.

7. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of: azathioprine, mycophenolic acid, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, acipimox, guanolimus, thalidomide, lenalidomide, anakinra, sirolimus, rephosmus, everolimus, temsirolimus, zololimus, biolimus A9, eculizumab, infliximab, adalimumab, certolizumab, alftemab, golimumab, meprolimus, omalizumab, nemoruimumab, faromamab, eimimazezumab, lexezumab, securitzumab, eculizumab, rituximab-CD 3, oxpoclizumab, temlizumab, virlizumab, crilizumab, keliximab, zalimumab, efuzumab, eculizumab, atrozumab, omab, rituximab, and rituximab, Tollizumab, aselizumab, galiximab, gavimumab, lulizumab, belimumab, yiprizumab, tremelimumab, batimumab, securitmumab, metumumab, natalizumab, tositumomab, obiumumab, basiliximab, dallizumab, itumomab, adozelimumab, atostemab, cetimumab, rillizumab, dallizumab, trastuzumab, rituximab, rocumab, rocatuzumab, basiliximab, momab, pezelizumab, rayleigh-lizumab, rolizumab, cetizumab, cetilizumab, talizezumab, atimumab, vaclizumab, vepamitumumab, abacept, belicept, etanercept, pezecept, abixib, alexib, rituximab, lymphotoxin alpha and beta inhibitors, darunavizumab 40 sgs, HCD-12.

8. The method of claim 1, wherein binding of stem cells to the lymphoid tissue is inhibited by administering a therapeutic agent that down-regulates or blocks the CD45 antigen.

9. The method of claim 1, wherein the therapeutic agent is radiation.

10. The method of claim 1, wherein the stem cell is an exogenous or endogenous stem cell.

11. The method of claim 1, wherein the therapeutic agent is a chemotherapeutic agent.

12. The method of claim 1, wherein the stem cells are administered to treat a disease selected from the group consisting of: hematological malignancies, leukemias, lymphomas, cancer, osteopetrosis, aplastic anemia and cytopenia, sickle cell disease and thalassemia, limbal stem cell deficiency, breast cancer, acute myocardial infarction, coronary artery disease, peripheral vascular disease, heart failure, type I diabetes, type 2 diabetes, stroke, spinal cord injury, neuroblastoma, multiple sclerosis, systemic sclerosis, lupus erythematosus, chronic wound healing, burn injury, fracture healing, cartilage repair, CNS tumors, osteoarthritis, renal failure, Parkinson's disease, myeloma, diabetic foot, liver and biliary cirrhosis, dilated cardiomyopathy, anemia, retinitis pigmentosa, Crohn's disease, diabetic neuropathy, mastocytosis, ovarian cancer, epilepsy, myasthenia gravis, autoimmune diseases, granulomatosis, osteonecrosis, and sarcopenia, Liver failure, PMD disease, lipodystrophy, demyelinating disease, cartilage defects, retinal disease, lupus nephritis, alzheimer's disease, traumatic brain injury, sarcoma, myositis, hyperglycemia, macular degeneration, ulcerative colitis, and muscle degeneration.

13. A method for inhibiting binding of stem cells to lymphoid tissue in an individual comprising inhibiting formation of germinal centers present in the lymphoid tissue or disrupting or ablating germinal centers in the lymphoid tissue.

14. The method of claim 13, wherein the individual is administered a therapeutic agent that inhibits formation of the germinal cells or promotes destruction or ablation of the germinal cells, wherein the therapeutic agent is selected from the group consisting of: agents that interfere with purine synthesis, antimetabolites, radiation, immunosuppressive agents, glucocorticoids, anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents, anti-eotaxin, anti-T Cell Receptor (TCR) agents, anti-interferon alpha agents, anti-interferon beta agents, anti-interferon gamma agents, anti-TGF alpha agents, anti-TGF beta agents, anti-integrin agents, anti-alpha 4 agents, anti-interleukin 1 agents, anti-interleukin 2 agents, anti-interleukin 4 agents, anti-interleukin 5 agents, anti-interleukin 6 agents, anti-interleukin 12 agents, anti-interleukin 13 agents, anti-interleukin 23 agents, anti-IgE agents, anti-Vascular Adhesion Protein (VAP) agents, anti-B7 agents, anti-Vascular Endothelial Growth Factor (VEGF) agents, anti-tumor-cancer agents, anti-tumor, Anti-baff (blys) agents, anti-CTLA 4 agents, anti-complement agents, anti-CD 2 agents, anti-CD 3 agents, anti-CD 4 agents, anti-CD 5 agents, anti-CD 20 agents, anti-CD 23 agents, anti-CD 25a agents, anti-CD 40 agents, anti-CD 154(CD40L) agents, anti-CD 62L agents, anti-CD 80 agents, anti-CD 147 agents, anti-LFA 1 agents, anti- (CD11a) agents, anti-CD 18 agents, purine synthesis inhibitors, pyrimidine synthesis inhibitors, antiproliferative agents, antimetabolite agents, anti-folate agents, and anti-mTOR agents.

15. The method of claim 13, wherein the therapeutic agent is a chemotherapeutic agent.

16. A method for regenerating germinal centers in lymphatic tissue, wherein said germinal centers have been damaged by a chemical agent, a biological agent, or radiation, comprising administering one or more agents that stimulate regeneration of said germinal centers.

Claims (16)

1. A method of inhibiting binding of stem cells to lymphoid tissue comprising:

administering the stem cells in conjunction with a therapeutic agent that inhibits binding of the stem cells to the lymphoid tissue.

2. The method of claim 1, wherein the lymphoid tissue consists of spleen, Peyer's patches and lymph nodes.

3. The method of claim 1, wherein the therapeutic agent inhibits binding of the stem cells to germinal centers within the lymphoid tissue.

4. The method of claim 3, wherein said germinal center is present in a lymphoid tissue selected from the group consisting of spleen, Peyer's patches, and lymph node tissue.

5. The method of claim 3, wherein the germinal center is active.

6. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of:

agents that interfere with purine synthesis, antimetabolites, radiation to the spleen, chemotherapeutic agents, immunosuppressive agents, glucocorticoids, anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents, anti-eotaxin, anti-T Cell Receptor (TCR) agents, anti-interferon alpha agents, anti-interferon beta agents, anti-interferon gamma agents, anti-TGF alpha agents, anti-TGF beta agents, anti-integrin agents, anti-alpha 4 agents, anti-interleukin 1 agents, anti-interleukin 2 agents, anti-interleukin 4 agents, anti-interleukin 5 agents, anti-interleukin 6 agents, anti-interleukin 12 agents, anti-interleukin 13 agents, anti-interleukin 23 agents, anti-interleukin 2 agents, anti-Vascular Adhesion Protein (VAP) agents, anti-B7 agents, IgE agents, anti-TNF agents, anti, anti-Vascular Endothelial Growth Factor (VEGF) agents, anti-baff (blys) agents, anti-CTLA 4 agents, anti-complement agents, anti-CD 2 agents, anti-CD 3 agents, anti-CD 4 agents, anti-CD 5 agents, anti-CD 20 agents, anti-CD 23 agents, anti-CD 25a agents, anti-CD 40 agents, anti-CD 154(CD40L) agents, anti-CD 62L agents, anti-CD 80 agents, anti-CD 147 agents, anti-LFA 1 agents, anti-CD 11a agents, anti-CD 18 agents, purine synthesis inhibitors, pyrimidine synthesis inhibitors, anti-proliferative agents, anti-metabolic agents, anti-folate agents, and anti-mTOR agents.

7. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of: azathioprine, mycophenolic acid, leflunomide, teriflunomide, methotrexate, tacrolimus, cyclosporine, pimecrolimus, acipimox, guanolimus, thalidomide, lenalidomide, anakinra, sirolimus, rephosmus, everolimus, temsirolimus, zololimus, biolimus A9, eculizumab, infliximab, adalimumab, certolizumab, alftemab, golimumab, meprolimus, omalizumab, nemoruimumab, faromamab, eimimazezumab, lexezumab, securitzumab, eculizumab, rituximab-CD 3, oxpoclizumab, temlizumab, virlizumab, crilizumab, keliximab, zalimumab, efuzumab, eculizumab, atrozumab, omab, rituximab, and rituximab, Tollizumab, aselizumab, galiximab, gavimumab, lulizumab, belimumab, yiprizumab, tremelimumab, batimumab, securitmumab, metumumab, natalizumab, tositumomab, obiumumab, basiliximab, dallizumab, itumomab, adozelimumab, atostemab, cetimumab, rillizumab, dallizumab, trastuzumab, rituximab, rocumab, rocatuzumab, basiliximab, momab, pezelizumab, rayleigh-lizumab, rolizumab, cetizumab, cetilizumab, talizezumab, atimumab, vaclizumab, vepamitumumab, abacept, belicept, etanercept, pezecept, abixib, alexib, rituximab, lymphotoxin alpha and beta inhibitors, darunavizumab 40 sgs, HCD-12.

8. The method of claim 1, wherein binding of stem cells to the lymphoid tissue is inhibited by administering a therapeutic agent that down-regulates or blocks the CD45 antigen.

9. The method of claim 1, wherein the therapeutic agent is radiation.

10. The method of claim 1, wherein the stem cell is an exogenous or endogenous stem cell.

11. The method of claim 1, wherein the therapeutic agent is a chemotherapeutic agent.

12. The method of claim 1, wherein the stem cells are administered to treat a disease selected from the group consisting of: hematological malignancies, leukemias, lymphomas, cancer, osteopetrosis, aplastic anemia and cytopenia, sickle cell disease and thalassemia, limbal stem cell deficiency, breast cancer, acute myocardial infarction, coronary artery disease, peripheral vascular disease, heart failure, type I diabetes, type 2 diabetes, stroke, spinal cord injury, neuroblastoma, multiple sclerosis, systemic sclerosis, lupus erythematosus, chronic wound healing, burn injury, fracture healing, cartilage repair, CNS tumors, osteoarthritis, renal failure, Parkinson's disease, myeloma, diabetic foot, liver and biliary cirrhosis, dilated cardiomyopathy, anemia, retinitis pigmentosa, Crohn's disease, diabetic neuropathy, mastocytosis, ovarian cancer, epilepsy, myasthenia gravis, autoimmune diseases, granulomatosis, osteonecrosis, and sarcopenia, Liver failure, PMD disease, lipodystrophy, demyelinating disease, cartilage defects, retinal disease, lupus nephritis, alzheimer's disease, traumatic brain injury, sarcoma, myositis, hyperglycemia, macular degeneration, ulcerative colitis, and muscle degeneration.

13. A method for inhibiting binding of stem cells to lymphoid tissue in an individual comprising inhibiting formation of germinal centers present in the lymphoid tissue or disrupting or ablating germinal centers in the lymphoid tissue.

14. The method of claim 13, wherein the individual is administered a therapeutic agent that inhibits formation of the germinal cells or promotes destruction or ablation of the germinal cells, wherein the therapeutic agent is selected from the group consisting of: agents that interfere with purine synthesis, antimetabolites, radiation, immunosuppressive agents, glucocorticoids, anti-beta amyloid agents, anti-rhesus factor, anti-TNF agents, anti-eotaxin, anti-T Cell Receptor (TCR) agents, anti-interferon alpha agents, anti-interferon beta agents, anti-interferon gamma agents, anti-TGF alpha agents, anti-TGF beta agents, anti-integrin agents, anti-alpha 4 agents, anti-interleukin 1 agents, anti-interleukin 2 agents, anti-interleukin 4 agents, anti-interleukin 5 agents, anti-interleukin 6 agents, anti-interleukin 12 agents, anti-interleukin 13 agents, anti-interleukin 23 agents, anti-IgE agents, anti-Vascular Adhesion Protein (VAP) agents, anti-B7 agents, anti-Vascular Endothelial Growth Factor (VEGF) agents, anti-tumor-cancer agents, anti-tumor, Anti-baff (blys) agents, anti-CTLA 4 agents, anti-complement agents, anti-CD 2 agents, anti-CD 3 agents, anti-CD 4 agents, anti-CD 5 agents, anti-CD 20 agents, anti-CD 23 agents, anti-CD 25a agents, anti-CD 40 agents, anti-CD 154(CD40L) agents, anti-CD 62L agents, anti-CD 80 agents, anti-CD 147 agents, anti-LFA 1 agents, anti- (CD11a) agents, anti-CD 18 agents, purine synthesis inhibitors, pyrimidine synthesis inhibitors, antiproliferative agents, antimetabolite agents, anti-folate agents, and anti-mTOR agents.

15. The method of claim 13, wherein the therapeutic agent is a chemotherapeutic agent.

16. A method for regenerating germinal centers in lymphatic tissue, wherein said germinal centers have been damaged by a chemical agent, a biological agent, or radiation, comprising administering one or more agents that stimulate regeneration of said germinal centers.