WO2002078743A1 - Compositions and methods for reducing tranfusion reactions - Google Patents

Abstract

The present invention is based, in part, on the discovery that febrile responses and transfusion immunomodulaton can occur in patients who have received platelets and, further, that these adverse effects are due to CD40 ligand (CD40L; also known as CD154) presentation and release (e.g. relaease during the collection, centrigugation, and storage of platelets or other CD40L-bearing cells or substances). Accordingly, the invention features compositions and methods for reducing the risk that patients who receive platelets (or other DC40L-bearing cells or substances) will experience an adverse reaction. The invention also features methods for diagnosing an adverse response in a patient receiving a transfusion. In addition, the invention features compositions and methods of immunotherapy through modulation of a patient's immune response.

Description

COMPOSITIONS AND METHODS FOR REDUCING TRANSFUSION REACTIONS

TECHNICAL FIELD

The field of this invention is reducing adverse responses to transfusions.

BACKGROUND

The CD40-CD40L system is a diverse communication pathway for interaction among bone marrow-derived cells of the immune system. This pathway is necessary for B lymphocyte imrnunoglobulin class switching and for activation of antigen presenting cells that, in turn, incite T cell activation. More recently, the CD40L (i.e., CD40 ligand) system was found to be critical for activating tissue structural cells, such as fibroblasts, epithelial cells, and endothelial cells (Phipps, Proc. Natl Acad. Sci. USA 97:6930-3932, 2000). Engagement of CD40 on the surface of these cells induces production of several proinflamrnatory cytokines, including LL-1, IL-6, and IL-8. Platelets, including human platelets, contain preformed CD40L (also referred to in the art as CD 154), which can be expressed on the surface of the cell and expelled after activation (Henn et al, Nature 391:591-594, 1998). This activity may be necessary to initiate the wound healing process.

In the United States and the United Kingdom alone, millions of platelet transfusions are performed each year, and febrile non-hemolytic reactions occur in about 30% of the patients who receive them (Heddle et al, N. Engl J. Med. 331:625-628, 1994). The actual incidence is likely to be higher than 30%, however, because mild adverse reactions are often not reported. In addition, life-threatening pulmonary failure occasionally occurs as part of the transfusion-related acute lung injury syndrome. There is a statistical association between the concentrations of cytokines (e.g. interleukins such as IL-1 and IL-6) in stored platelet supernatants and the occurrence of febrile non-hemolytic transfusion reactions (Muylle et al, Transfusion 33:195-199, 1993). While it is known that removal of the supernatant plasma partially ameliorates these reactions (Heddle et al., N. Engl J. Med. 331:625-628, 1994), it was not known which, or which combination of, substances within the supernatant caused the adverse reactions. SUMMARY

The present invention is based, in part, on the discovery that the febrile responses and transfusion immunomodulation that can occur in patients who have received platelets are due, at least in part, to the presentation and/or release of CD40 ligand (CD40L; also known as CD154; see, e.g., Armitage et al, Nature 357:80-82, 1992). Release can occur, for example, during the collection, centrifugation, and storage of platelets or other CD40L-bearing cells or substances. Accordingly, the invention features compositions and methods for reducing the risk that patients who receive platelets (or other CD40L-bearing cells or substances) will experience an adverse reaction.

The methods of the invention can be carried out by reducing the amount of, or the activity of, CD40L that is administered to a patient (the term "reducing the expression of CD40L," and similar phrases, are meant to encompass a reduction in the amount of CD40L that is present (i.e., expressed) or the level of CD40L activity). The method can be carried out so the reduction occurs either before, during, or after CD40L (e.g., a

CD40L-bearing cell, cell fragment, supernatant or other substance) is administered to the patient. Moreover, the method can be carried out so that one reduces either (or both) the expression of CD40L in the substance being administered or the expression of CD40L in the patient (i.e., the CD40L that may naturally occur in a patient). For example, the amount of CD40L in the patient can be reduced before additional CD40L is administered (by way of, for example, a blood transfusion). Alternatively, or in addition, samples of cells can be screened to identify those that contain CD40L. CD40L is undetectable in samples of platelets freshly obtained from healthy individuals, and any elevation in its expression or activity can be detrimental. The more CD40L present in a sample (or the more active that CD40L is), the more likely it is that that sample will cause an adverse response when administered to a patient. Thus, whenever there is a choice among samples to administer, the clinician can reduce the risk of an adverse reaction by screening for CD40L expression or activity and administering the sample with the lowest CD40L expression (or content) or activity. Such screening can be done prior to actively reducing the amount of CD40L.

Regardless of the manner in which CD40L is reduced, one may elect to begin with a sample that contains less, rather than more, CD40L. To reduce the amount of CD40L, samples of CD40L-bearing cells (or cell fragments, lysates, or supernatants), or samples of other CD40L-bearing substances, are treated so that the expression, amount, or activity of CD40L is reduced. For example, CD40L-bearing cells can be washed according to methods presently available to those of ordinary skill in the art and then administered to a patient. In more complex embodiments, the expression, amount, or activity of CD40L can be reduced by exposing the CD40L-bearing cell or substance to an agent that inhibits (directly or indirectly) the expression, amount, or activity of CD40L. Preferably, the agent is used to inhibit CD40L before a given procedure (i.e., before administration of CD40L-bearing cells or substances), but the agents can also be administered to the patient during or after the procedure (e.g. during or after a transfusion that includes platelets). For example, CD40L-bearing cells or substances can be exposed to an anti-CD40L antibody or other CD40L antagonist (e.g. a small molecule, a CD40 molecule, or a fragment or mutant thereof). Those of ordinary skill in the art are well able to determine whether a molecule functions to antagonize CD40L.

The invention encompasses the use of CD40L antagonists regardless of their mechanism of action. For example, antagonists that block: transcription of the gene encoding CD40L; translation of the mRNA encoding CD40L; transport of CD40L to the cell surface; expulsion of CD40L from the cell; or transduction of a signal (generated by CD40L's binding to CD40) across the plasma membrane are all useful in the methods of the present invention. Of course, more than one type of antagonist can be used, and the methods of the invention can be combined. For example, CD40L can be reduced by washing the cells and, in addition, exposing the cells to one or more CD40L antagonists. Moreover, the methods can be carried out in conjunction with other methods whose aims are to reduce the risk, severity, or duration of an adverse reaction triggered by CD40L. For example, a patient can be treated with a CD40L antagonist and an agent that inhibits the activity of the cyclooxygenase enzyme or the activity of prostaglandins.

The invention also features an assembly of compositions (i.e., a kit) that can contain one or more reagents that can be used to detect the presence of, or determining the expression or activity of, CD40L. The kit optionally contains instructions (on any conventional medium) for using the reagent(s) to detect or examine the expression of CD40L (thereby assessing or reducing the risk of an adverse reaction to CD40L. For example, the invention features a kit that contains a detectable agent (e.g. a detectable antibody or ligand) that specifically binds CD40L and instructions for using that agent to screen blood or platelet samples for the presence of CD40L. The agent can be provided alone or conjugated to a substrate, such as a bead (e.g. a magnetic bead), plastic matrix, or solid support. Alternatively, or in addition, the kit can contain a CD40L antagonist and instructions for using that antagonist to reduce the expression of, or the activity of, CD40L in a sample of, for example, CD40L-bearing cells or substances. The invention also features a method of reducing an adverse response to a transfusion, the method including providing a transfusion composition comprising CD40L (e.g., as soluble CD40L, or where at least part of the CD40L is associated with cells (e.g., platelets) in the transfusion composition), transfusing the composition into a patient and, prior to or after the composition is transfused into the patient, treating the composition with an amount of a CD40L antagonist (e.g., antibody; monoclonal antibody; anti-CD40L antibody; antibody produced by a hybridoma selected from the group consisting of clone # MK13A4, clone # TRAP-1, clone # B-B29, clone # 24-31, and clone # 5c8; small molecule; polypeptide; oligonucleotide; antisense oligonucleotide that inhibits the expression of CD40L; ribozyme; RNAi) effective to reduce the amount or activity of CD40L in the transfused patient.

The invention also features a method of identifying a compound useful for reducing an adverse response to a transfusion, the method including providing a composition comprising CD40L (e.g., as soluble CD40L, or where at least part of the CD40L is associated with cells (e.g., platelets) in the composition), contacting the composition with a test compound, identifying a test compound that reduces the ability of CD40L to bind to CD40, and determining whether the test compound so identified reduces an adverse (e.g., febrile) response to a transfusion in an animal. .

The invention also features a method of diagnosing an adverse response to a transfusion in a patient who has undergone a transfusion, the method including obtaining a sample of blood from the patient, measuring the level of CD40L in the patient's blood, comparing the post-transfusion level of CD40L so measured to a normal pre-transfusion level of CD40L, in which a post-transfusion level of CD40L above the normal pre- transfusion level of CD40L indicates an increased risk of an adverse reaction to the transfusion. The invention also features a method of treating a patient suffering from acute myeloid leukemia, the method including providing a transfusion composition including platelets, treating the composition with a CD40L antagonist (e.g., an antibody), thereby producing a treated composition comprising platelets, and introducing the treated composition into the patient.

The invention also features the use of a CD40L antagonist in the preparation of a transfusion composition by a method including providing a transfusion composition including platelets and CD40L, treating the composition with an amount of the CD40L antagonist effective to reduce the amount or activity of CD40L in the transfusion composition, thereby producing a treated composition, and transfusing the treated composition into a patient.

The invention also features a transfusion composition that includes platelets and an exogenous CD40L antagonist for use in treatment. "Exogenous" refers to a substance that does not occur naturally in the transfusion composition.

The invention also features the use of a CD40L-depleted composition in the preparation of a transfusion composition by a method that includes providing a transfusion composition that includes platelets, treating the transfusion composition with a CD40L antagonist, thereby producing a treated composition, and introducing the treated composition into the patient.

The invention also features a method of immunotherapy, the method including identifying a patient (e.g. , neonate; elderly; immunocompromised; organ transplant recipient; suffers from an autoimmune disease; suffers from an inflammatory disease; suffers from Crohn's Disease or rheumatoid arthritis; woman at risk for spontaneous abortion) in need of immunotherapy, providing a composition (e.g., including platelets and/or supernatant from spun-down platelets) that includes CD40L derived from blood of the patient, and introducing into the patient an amount of the composition effective to modulate the patient's immune system (e.g., CD40L stimulates anti-tumor immunity; CD40L fosters humoral (Th2) immunity and down-regulates cellular (Thl) immune responses). In addition, this method can include the further step of, before, after, or during the introducing step, introducing a vaccine (e.g., antiviral vaccine, anti-hepatitis B virus vaccine, anti-influenza virus vaccine, anti-HIV vaccine) into the patient. Enzyme-linked immunosorbent assay ("ELISA") refers to a variety of immunological methods that employ an enzyme-labeled immunoreactant (an antigen or antibody) and an immunosorbent (an antigen or antibody bound to a solid) to identify specific serum or tissue antibodies or antigens. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS Fig. 1 is a bar graph showing the amount of sCD40L (soluble CD40L) (pg/ml) in five samples of platelet concentrates prepared for transfusion.

Fig. 2 is a pair of bar graphs illustrating the effect of platelet supernatants on IL-6 expression (left-hand graph) and PGE expression (right-hand graph) in fibroblasts.

Fig. 3 is a bar graph illustrating the expression of PGE by fibroblasts exposed to intact platelets, either unstimulated, stimulated, or stimulated plus anti-CD40L antibody.

Fig. 4 is a bar graph illustrating the depletion of CD40L from platelet rich plasma using the "depletion protocol" described herein.

Fig. 5 is a graph illustrating survival in patients with AML, age <50, in a randomized trial of washed-filtered versus filtered only (unwashed) blood transfusions.

DETAILED DESCRIPTION

As described further below, supernatant obtained from stored platelets contains substantial amounts of soluble CD40L (sCD40L), and stored platelets typically express CD40L on their surface (CD40L increases, peaking and remaining high, after 2-3 days under typical blood or platelet storage conditions). These findings indicate that platelets routinely prepared from donated whole blood have been at least partially activated. Moreover, platelet supernatant activates human fibroblasts, causing them to produce prostaglandin E₂ (PGE₂), a primary inducer of fever.

Our discovery of high concentrations of bioactive CD40L in platelets routinely prepared for transfusion led us to study its role in febrile and other undesirable responses and to conclude that CD40L can generate adverse transfusion reactions (which may persist even when white blood cells have been removed prior to platelet storage and administration). While one normal function of platelets is to ensure clotting, they also play a major role in inciting wound healing and inflammation. It has now been shown that the release and/or surface expression of CD40L is responsible for transfusion immunomodulation. This reaction is known in the art as a serious condition that can culminate in multi-organ failure and death. The intensity of the adverse response can depend not only upon the level of CD40L in the preparation administered to the patient, but also on the sensitivity of a patient to CD40L and concomitant pharmaceutical regimes (e.g. whether or not a patient is talcing steroids or nonsteroidal anti-inflammatory drugs that would inhibit the activity of the cyclooxygenase enzyme or prostaglandin synthesis).

Patients amenable to treatment

Any mammal (including domestic animals such as dogs and cats, farm animals such as horses, cows, pigs, goats, and sheep, and humans) that receives, by injection, infusion, or otherwise, a sample that includes CD40L can benefit from the methods of the present invention. Those patients are susceptible to adverse reactions that include, but are not limited to, febrile responses (a rise in body temperature of 1 degree Celsius or more), inflammatory responses, autoimmune responses, adverse vascular effects (e.g., hemorrhage, thrombosis, and platelet aggregation), and the syndrome known as transfusion immunomodulation. Specific diseases or medical conditions are associated with additional adverse reactions. For example, in a patient with leukemia (e.g., acute myeloid leukemia (AML)), these reactions can include pulmonary dysfunction, infections (including bacterial, viral, and fungal), failure promptly to enter and remain in complete remission, respiratory problems (including transfusion related acute lung injury (TRALI)), and changes in anti-tumor immunity during recovery from aplasia. The methods of the invention can be used to treat mammals (e.g. human patients) receiving a transfusion that includes platelets. "Treatment" encompasses reducing the risk, severity, or duration of an adverse reaction. Thus, the methods of the invention can be carried out prophylactically or, if need be, in the context of a treatment if an adverse reaction has begun. CD40L Antagonists

Any substance that reduces the expression or amount or activity of CD40L can be used to carry out the methods of the invention. As shown in the Examples below, CD40L can be substantially removed (e.g. , reduced by 50%, 60%, 70%, 80%, 90% or more (e.g., below a detectable level)) from a sample, such as human plasma, in which platelets are normally suspended. The method can involve using a solid substrate, such as a bead coated with protein (e.g., protein G), which binds an anti-CD40L antibody (e.g., a monoclonal anti-CD40L antibody, many of which are commercially available). The beads are then suspended in the sample where any free, soluble CD40L binds to the antibody-coated beads. Thus, antibodies that specifically bind CD40L are antagonists of CD40L (whether used in free form or associated with a solid support).

In addition to the antibody antagonists used and described herein (see, e.g., Lederman et /., J Exp. Med. 175:1091-1101, 1992; see also Kirk et al, Nature Med. 5:686-693, 1999), those of ordinary skill in the art are aware of and can employ other CD40L-specifϊc antibodies. For example, one can use the commercially available hybridoma clone # MK13A4 (available from Alexis Biochemicals and Serotec), clone # TRAP-1 (available from Serotec), clone # B-B29 (available from Biosource), clone # 24-31 (available from Calbiochem), clone # 5c8 (a hybridoma available from the American Type Culture Collection, Manassas VA), and anti-CD40L (available from Santa Cruz Biotechnology), or those described in U.S. Patent Nos. 5,961,974 and 5,683,693. When necessary or desired, antibody antagonists can be humanized antibodies (i.e., molecules having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin; the antigen binding site may comprise either complete variable regions fused onto human constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate human framework regions in the variable domains (see, e.g., U.S. Patent No. 6,180,377). Methods of humanizing antibodies are well known in the art. Small molecules (e.g. , compounds having a molecular weight at or below

1000 daltons) can also be used as CD40L antagonists. Peptides that consist of all or part of, or that mimic, the normal binding partner of CD40L (i.e., CD40) can also be used in the methods of the invention. Substantial information is available regarding the normal binding partner of CD40L, and this information can be used to make CD40L antagonists. Human CD40 protein (CD40) is a peptide of 277 amino acids having a molecular weight of 30,600, and a 19 amino acid secretory signal peptide comprising predominantly hydrophobic amino acids (Stamenkovic et al, EMBOJ. 8:1403-1410, 1989). A cDNA encoding human CD40 was isolated from a cDNA library prepared from Burkitt lymphoma cell line Raji and thus is available to those of ordinary skill in the art.

The invention also includes treatment regimes based on an "antisense" approach that involves the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA (e.g., CD40L mRNA). These oligonucleotides are believed to bind to the complementary mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarily to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarily and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Oligonucleotides that are complementary to the 5' end of the message, e.g., the

5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs recently have been shown to be effective at inhibiting translation of mRNAs as well (Wagner, Nature 372:333, 1984). Thus, oligonucleotides complementary to either the 5 ' or 3' non-translated, non-coding regions of the gene or mRNA could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.

Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.

Whether designed to hybridize to the 5', 3', or coding region of an mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides. Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence. "Gene walk" experiments to identify useful antisense oligonucleotides are routine in the art. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (as described, e.g., in Letsinger et al, Proc. Natl Acad. Set USA 86:6553, 1989; Lemairre et al, Proc. Natl Acad. Sci. USA 84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, for example, PCT Publication No. WO 89/10134), or hybridization-triggered cleavage agents (see, for example, Krol et al., BioTechniques 6:958, 1988), or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539, 1988). To tins end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 2-(3-amino-3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal, or an analog of any of these backbones.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β -units, the strands run parallel to each other (Gautier et al, Nucl. Acids. Res. 15:6625, 1987). The oligonucleotide is a 2'-0- methylribonucleotide (Inoue et al. , Nucl. Acids Res. 15:6131, 1987), or a chimeric RNA- DNA analog (Inoue et al, FEBSLett. 215:327, 1987).

Antisense oligonucleotides for use in the invention can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Nucl. Acids Res. 1^:3209, 1988), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Natl. Acad. Set USA 85:7448, 1988).

The antisense molecules should be delivered to cells that express nucleic acids or polypeptides of interest in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrations of the antisense molecule sufficient to suppress translation of endogenous mRNAs. Therefore, a preferred approach uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous transcripts of CD40L nucleic acids and thereby prevent translation of the endogenous mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.

Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist et al, Nature 290:304, 1981); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, Cell 22:187-191, 1988); the herpes thymidine kinase promoter

(Wagner et al, Proc. Natl Acad. Sci. USA 78:1441, 1981); the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296:39, 1988), or a.poRU or poHI promoter.

Similarly, ribozymes and RNAi triplexes can be used. Their use is well known in the art. Ribozymes can include a catalytic sequence that cleaves mRNA (see

U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334:585-591, 1988). For example, a derivative of a tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a desired mRNA (see, e.g., Cech et al. U.S. Patent Nos. 4,987,071 and 5,116,742). Alternatively, mRNA can be used to select from a pool of RNA molecules a catalytic RNA having a specific ribonuclease activity (see, e.g., Barrel and Szostak, Science 261:1411-1418, 1993; see also Krol et al, Bio-Techniques 6:958-976, 1988). Other CD40L antagonists can also be used: e.g., transcription factors or other compounds that decrease CD40L expression.

The ability of a potential CD40L antagonist to inhibit the biological activity of CD40L can be assessed in any number of standard assays. For example, potential CD40L antagonists can be assessed in competitive binding assays (measuring binding to CD40L or alternatively, measuring inhibition of CD40L's binding to CD40) or those that reflect the biological activity of CD40L (e.g. a B cell proliferation assay) or an in vivo assay detecting adverse reactions to transfusion, such as febrile non-hemolytic reactions.

EXAMPLES

Example 1. Expression of CD40L in platelet concentrates Nine samples of human platelets, routinely prepared for transfusion, were analyzed over a five-month period. Platelets intended for transfusion were obtained from the regional American Red Cross Blood Services. The samples were randomly selected, routinely prepared concentrates derived from donated whole blood by the platelet rich plasma method widely used in North America. The experiments were conducted within the five-day clinical storage period for platelet concentrate. The supernatant of every sample of stored platelet concentrate examined contained substantial amounts of sCD40L, which was detected by a sensitive and specific ELIS A. The amounts varied from 3000-7000 pg/ml (Fig. 1). This level of CD40L is found in human serum during chronic inflammation. The stored platelets also typically expressed CD40L on their surface. These results indicate that the stored platelets have been at least partially activated. Even platelet preparations that were carefully washed continued to release ng/ml quantities of CD40L into the supernatant. In another study, employing supernatants from eight whole blood derived platelet concentrates, it was demonstrated that levels of CD40L after five days of storage were in the range of thousands of ng per liter and vary three to four fold. Platelet surface CD40L was also increased during storage with the per cent platelets expressing elevated levels of CD40L varying two to three fold. 27% to 60% of stored platelets express surface CD40L. Soluble CD40L from platelet concentrate supernatants induced PGE₂ production even at dilutions of 1/200 fold. PGE₂ production was completely abrogated by pretreatment with an excess of anti-CD40L monoclonal antibody, but not with a control antibody. These results show that platelet surface and supernatant soluble CD40L are mediators of febrile, non-hemolytic transfusion reactions in patients. The findings indicate that the processing of human platelets from whole blood using either apheresis (Vanderlinde et al, Blood 9S:ll2b (Abstract), 2001) or standard centrifugation processes partially "activates" the platelets, resulting in release of their internal stores of CD40L. The transfused platelets and the plasma supernatant contain very large amounts of bioactive CD40L that can induce fever and can be immunomodulatory as well.

Example 2. Human platelet-derived CD40L is a potent activator of lung fibroblast synthesis of PGE and IL-6 The sCD40L in the platelet supernatant activates human fibroblasts to produce

PGE₂, a primary inducer of fever. Fresh human lung fibroblasts were unstimulated or were exposed up to 72 hours with stored platelet supernatants in the presence of an isotype control antibody or an antibody that neutralizes CD40L (5C8). Massive amounts of PGE₂ were produced following exposure to CD40L-containing supernatants. The anti-CD40L antibody completely abrogated the induction of PGE₂.

Example 3. Surface marker analysis shows that platelets prepared for transfusion are at least partially activated Platelets prepared by standard methods for transfusion were stained with anti-

CD42a (a platelet marker), with PAC1 (which detects activated platelets) or with the humanized antibody 5C8 (which binds CD40L). This experiment showed that these platelets were at least partially activated.

In another series of experiments, platelet supernatants were incubated for 72 hours with human fibroblasts, with and without 5C8. PGE₂ and IL-6 were assayed using specific ELIS A. IL-6 is a key mediator of inflammation. As shown in Fig. 2, both IL-6 and PGE₂ are induced by CD40L and blocked by 5C8. Induction of PGE₂ and IL-6 occurred with other fibroblast strains as well.

Example 4. Intact platelets are also potent inducers of PGE in fibroblasts

Intact platelets (as opposed to supernatants) were also tested for their ability to induce PGE expression in fibroblasts. These intact cells were found to be potent inducers. Moreover, PGE₂ production by platelet-stimulated fibroblasts could be inhibited by approximately 50% with the monoclonal antibody 5C8 (see Fig. 3). That there was not a greater degree of inhibition may be due to the conformation of CD40L on the surface of the platelet (which may vary from the conformation when secreted). Alternatively, it may have been the result of further platelet activation and the expulsion of other mediators of PGE₂ expression, such as PDGF or TGF-β.

Example 5. A protocol for depleting platelet-rich plasma of CD40L ("the depletion protocol")

To preclear plasma: (a) combine 500 μl of platelet-rich plasma with 200 μl of Protein-G™ sepharose (50% slurry; Pharmacia Biotech #17-0618-01), which binds immunoglobulins and is used in this step to remove all of the immunoglobulins in the plasma sample so as not to interfere with the rest of the protocol; (b) incubate the plasma/sepharose slurry at 4°C for 2 hours on a rotator set to 40 rotations per minute; (c) centrifuge at 14,000 x g at 4°C for 5 minutes to pellet the Protein-G™ sepharose; and (d) transfer the supernatant (pre-cleared plasma) to a clean tube.

To form and capture antibody-antigen complexes: (a) to the precleared plasma, add 5 μg of mouse anti-human CD 154 antibody (Calbiochem #217595); (b) incubate at 4°C for 2 hours on a rotator set to 40 rotations per minute; (c) add 30 μl of Protein-G™ sepharose (50% slurry; in this step, protein-G™ is used to bind up the specific antigen- antibody complexes); (d) incubate at 4°C for 1 hour on a rotator set to 40 rotations per minute; (e) transfer the suspension to a spin filter (Cytosignal #C00-100) and centrifuge at 14,000 x g for 2 minutes at room temperature (the filter is made up of low-protein binding, porous material that retains Protein-G™ sepharose and the antigen-antibody complex to which the Protein-G™ sepharose is bound, but allows non-bound material to pass through easily; and (f) collect the CD40L-depleted plasma that flows through.

To test the various plasma preparations for the presence of soluble CD40L: (a) test samples of CD40L-depleted plasma and non-depleted plasma using the CD40L (sCD154) ELISA module set (Bender MedSystems #BMS239MST); and (b) confirm that CD40L is present in the original platelet-rich plasma and pre-cleaned plasma, but undetectable in the CD 154-depleted plasma.

The results of an experiment carried out according to this protocol are shown in Fig. 4. The original plasma was found to contain 14,444 pg/ml CD40L. Following the depletion protocol, CD40L was no longer detectable in the plasma. To prove that CD40L from plasma is bound to the antigen-antibody-Protein-G™ sepharose complex: (a) wash the antigen-antibody-Protein-G™ complex that remains on the filter with 500 μl RIPA buffer (50 M Tris-HCl (pH 7.4), 150 mM NaCl, 1% Igepal™, 0.25% Na-deoxycholate, 1 mM EDTA) and centrifuge at 14,000 x g for 2 minutes at room temperature; (b) repeat the washing step three times; (c) move the spin filter to a clean collection tube; (d) add 40 μl SDS-PAGE reducing sample buffer to cover the antigen-antibody-Protein-G™ complex on the filter; (e) incubate for 5 minutes at 70°C followed by 10 minutes at room temperature (this will release the antigen- antibody- Protein-G™ complex from the filter; (f) centrifuge at 14,000 x g for 2 minutes at room temperature; (g) collect the flow-through and boil the sample for 5 minutes at 100°C; and (h) electrophorese the sample on an SDS-polyacrylamide gel, transfer to nitrocellulose, and probe the blot for CD40L by Western blotting.

Example 6. Use of autologous platelets and platelet supernatant in immunotherapy

The extraordinarily high levels of platelet membrane CD40L and supernatant soluble CD40L (sCD40L) that accumulate in platelets collected for transfusion represent an opportunity to use the patient's own stored autologous platelets and platelet supernatant for immunotherapy. Infusions of the patient's own stored platelets and platelet supernatant can be used therapeutically to modulate immune responses, with minimal issues of safety and no risks of disease transmission. Autologous platelet transfusion is well established as a safe and efficacious way of evaluating the hemostatic properties of new devices and disposables for collecting and storing platelets for transfusion. The infusion of CD40L from autologous stored blood acts as an adjuvant for stimulating the production of immunoglobulin (i.e., humoral immunity, including class switching from IgM to IgG production). It is well known that neonates, the elderly, and patients who are immunocompromised often have poor responses to conventional vaccines, such as those for hepatitis B, influenza A, etc.. Stored autologous platelet transfusions can be used as an adjuvant to stimulate patients with poor responses to conventional vaccines and adjuvants. Similarly, stored platelet CD40L and sCD40L can be used stimulate anti-tumor immunity, with or without concomitant use of an anti-tumor vaccine. The platelet material or purified sCD40L can be administered to the patient before, after, or at the same time as the vaccine, provided that it is sufficiently near enough in time for the adjuvant effect to be seen.

In some situations, infusion of the patient's stored autologous platelet products rich in CD40L and sCL OL not only fosters humoral (Th2) immunity, as in the vaccine adjuvant example cited above, but also down-regulates inappropriate cellular (Thl) immune responses. Pathologic or undesirable Thl cellular immune responses are seen, for example, in solid organ transplant rejection, Crohn's disease, rheumatoid arthritis, and women with repetitive spontaneous abortions. Infusion of a patient's stored autologous platelets and/or supernatant can mitigate disease activity and act as a non- toxic replacement for toxic pharmacologic agents currently used to down-regulate cellular immunity in these, and other, settings.

Example 7. Role of transfusing platelet CD40L in adult acute myeloid leukemia The dose of platelet derived CD40L transfused influences the incidence of non- hemolytic febrile transfusion reactions, pulmonary dysfunction, infection and poor post- transfusion platelet count increments in adults with acute myeloid leukemia (AML) undergoing induction chemotherapy.

Patients undergoing induction chemotherapy for AML experience numerous complications. Some of these are known to be transfusion related, most commonly including febrile, non-hemolytic transfusion reactions. Other complications such as pulmonary dysfunction, infections and failure to promptly enter and remain in complete remission are potentially related to transfusion therapy.

Non-hemolytic febrile transfusion reactions Platelet transfusion therapy is almost universally employed in the care of patients with AML to prevent thrombocytopenic hemorrhage. Approximately 10,000 patients nationwide and 30 patients each year in the University of Rochester Medical Center system are treated with curative intent for AML. There, each patient receives a mean of 15 platelet transfusions during induction therapy. Acute febrile reactions, which occur in up to 30% of AML patients following platelet transfusions, can be reduced somewhat by leukoreduction. Interestingly, removal of supernatant plasma from platelet concentrates appears to be important in abrogating reactions in some patients. The invention is based, in part, on the discovery that CD40L that is found in very high concentrations both on the platelet surface and in soluble secreted form in stored platelet concentrates. It was demonstrated that platelet- derived CD40L induces cyclo-oxygenase-2 (COX-2) activity and PGE₂ production by human lung fibroblasts in vitro. It was also shown that this activity was wholly or largely abrogated by use of a neutralizing anti-CD40L monoclonal antibody. PGE₂ is the major mediator of fever in humans. CD40L is a good candidate for being a platelet plasma supernatant mediator of febrile reactions after platelet transfusion.

Pulmonary dysfunction and infection There are other important complications that are mediated in part by platelet transfusion therapy. Amongst these are respiratory problems (Clarke et al, Blood 84 (Supplement) :465a, 1994; Gordon et al, Bone Marrow Transplantation 22:999-1003, 1998), including TRALI (transfusion related acute lung injury), bacterial, viral and fungal infections (Rios et al, Transfusion 41:873-7, 2001), and changes in anti-tumor immunity (Heal et al, Am.J.Hematol 45:189-190, 1994) during recovery from aplasia. CD40L is a mediator of pulmonary dysfunction after platelet transfusion. Conversely, CD40L has pro-inflammatory activity that can stimulate host immunity against infection, and thus reduce the likelihood of this complication.

Generally, adult patients with AML will benefit from having their platelet transfusions depleted for active CD40L, including via the use of CD40L antagonists. However, patients at unusual risk of infection (including, in particular, children, the elderly, and immunocompromised patients) may benefit from the pro-inflammatory effects of CD40L.

Clinical significance The treatment of AML has changed dramatically over the last few decades (Greer et al, In: Lee et al, ed. Wintrobe's Clinical Hematology. Baltimore, MD: Williams & Wilkins, pp. 2272-2319, 1999). Until fairly recently, long term survival in adults with AML was close to zero. Over the last twenty years, improvements in (a) remission induction and consolidation chemotherapy, (b) transplantation, and (c) supportive care have yielded improved remission rates. Unfortunately, the majority of adult patients still die of their leukemia. In addition to these dismal long term results, patients undergoing induction, consolidation and transplant therapy for AML experience a very high rate of complications due to the side effects of myeloablative chemotherapy. Almost all patients experience one or more of the following events during induction therapy: neutropenic fever, mucositis, infection, or transient pulmonary, renal or hepatic dysfunction.

Platelet transfusion continues to play a key role in causing febrile, non-hemolytic transfusion reactions even after the introduction of leukoreduced transfusions (Mangano et al, Am.J.Clin.Pathol 95:733-738, 1991; Goodnough et al, Vox Sang. 65:103-107, 1993). While such reactions are usually not life threatening, they are often a source of significant distress to patients and their families, and on occasion are accompanied by more severe systemic symptoms including pulmonary dysfunction or hypotension (Gordon et al, Bone Marrow Transplantation 22:999-1003, 1998; Hume et al, Transfusion 36:904-909, 1996). A major problem is that even leukoreduced platelet concentrates induce febrile responses. A number of reports suggest that removal of supernatant plasma from platelet concentrates can reduce fevers and rigors (Heddle et al, Transfusion 33:794-797, 1993; Heddle et al. N.Engl.J.Med. 331:625-628, 1994; Heddle et al, Transfusion 39:231-238, 1999). One hypothesis to account for these observations is that cytokines present in the supernatant of stored platelet concentrates mediate these reactions (Heddle et al, Transfusion 33:794-797, 1993; Heddle et al, N.Engl.J.Med. 331:625-628, 1994; Heddle et al, Transfusion 39:231-238, 1999; Muylle et al,

Transfusion 33:195-199, 1993; Muylle et al, Transfusion 33:962-963, 1993; Muylle and Peeiermans, Vox Sang. 66:14-17 , 1994; Muylle, Blood Rev. 9:77-83, 1995; Muylle et al, Transfusion 36:886-890, 1996; Snyder, Immunol. Invest. 24:333-339, 1995; Stack and Snyder, Transfusion 34:20-25, 1994). In accordance with the present invention, it is now known that the adverse transfusion reaction is attributable to CD40L on the surface of, or released by, platelets, and that removing or neutralizing the CD40L can prevent or reduce the adverse reaction.

CD40L infusion Extensive transfusion is considered essential to the delivery of curative doses of chemotherapy in AML in both children and adults. There is a growing interest in cellular immunity in the control of leukemia, largely due to the observed graft versus leukemia effect in allogeneic stem cell transplantation, and the anti-leukemic efficacy of donor leukocyte infusion (DLI). However, in adult patients with acute leukemia, larger numbers of platelet transfusions during successful remission induction has been associated with earlier recurrence and death due to leukemia. This relationship was not true for increasing doses of red cell transfusion. The same effect was seen if the analysis

5 was limited to patients who required only one course of induction therapy to achieve a complete remission. This shows that platelet transfusion dose was not merely acting as a surrogate measure of responsiveness of disease, or rate of marrow recovery after aplasia. To further investigate this issue, a randomized pilot study of plasma (washed) and leukocyte reduced (filtered) transfusions versus leukocyte-reduced only was completed, o to assess whether removal of stored plasma would alter clinical outcomes in the treatment of adult ALL and AML. In subset analysis of these data, patient age was examined because younger patients might be expected to mount better anti-leukemic immunity. When patients 18 to 50 years of age were examined, a statistically significant advantage (p=0.04) to receipt of washed-leukoreduced transfusions was found, (see 5 Fig. 5). Five of 10 patients in the washed group had unfavorable cytogenetics versus only 2 of 9 in the unwashed group. The survival in complete remission beyond four years of 90% of a group of patients with AML shows that it is beneficial to remove plasma from stored red cells and platelets prior to transfusion.

0 Washing of platelets as a means of reducing febrile, nonhemolytic transfusion reactions Washing of platelets is an effective means of reducing febrile, nonhemolytic transfusion reactions (Vo et al, Transfusion Medicine 1 L45-47, 2001). In the University of Rochester Medical Center system, patients who experience severe or 5 repeated reactions to post-storage leukocyte-depleted platelets are given saline washed platelets to attempt to minimize such reactions. A retrospective medical record review was performed of hematology/oncology patients during 1997 who received leukocyte- depleted platelet transfusions yet experienced at least one significant "fever/chill" type reaction and who had been placed on a washed transfusion protocol. Using the last 0 transfusion before initiating the washed protocol as the index, reactions were examined in up to 10 transfusions before and 20 transfusions after the index for each patient. A reaction was defined as rigors, chills, fever, pruritus, headache, and/or nausea/vomiting not present prior to the transfusion. Pre- and post-transfusion platelet counts and the time elapsed between transfusion and post-transfusion count were also recorded. Twenty-four patients, 12 males and 12 females, with a mean age of 32.3 years [range 9 to 63 years] received a total of 522 transfusions [331 washed and 191 unwashed]. Reactions occurred in 20% of the unwashed but in only 0.6% of the washed transfusions.

5 (pθ.0001). The mean platelet count increment was significantly lower for the washed group than the unwashed group, 10.3 (x 1000/ml) versus 14.2, respectively. Mean platelet recovery after washing in our current quality control data is 82% (range 68 to 92%) (n =32). No clinically evident bleeding occurred in any patient. Virtually all transfusions were preceded by pre-medication with an antipyretic. The study concluded o that patients with a history of transfusion reaction to leukocyte-depleted platelets experience a 30 fold reduction in the incidence of complications when the platelets are washed.

Other Embodiments 5 It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 0

Claims

WHAT IS CLAIMED IS:

I . A method of reducing an adverse response to a transfusion, the method comprising a. providing a transfusion composition comprising CD40L; b. transfusing the composition into a patient; and c. prior to or after the composition is transfused into the patient, treating the composition with an amount of a CD40L antagonist effective to reduce the amount or activity of CD40L in the transfused patient.

2. The method of claim 1, wherein the CD40L comprises soluble CD40L.

3. The method of claim 1, wherein at least part of the CD40L is associated with cells in the transfusion composition.

4. The method of claim 3, wherein the cells are platelets.

5. The method of claim 1, wherein the antagonist is an antibody.

6. The method of claim 5, wherein the antibody is a monoclonal antibody.

7. The method of claim 6, wherein the monoclonal antibody is an anti-CD40L antibody.

8. The method of claim 7, wherein the monoclonal antibody is produced by a hybridoma selected from the group consisting of clone # MK13 A4, clone # TRAP-1, clone # B-B29, clone # 24-31, and clone # 5c8.

9. The method of claim 1, wherein the antagonist is a small molecule.

10. The method of claim 1, wherein the antagonist is a polypeptide.

II. The method of claim 1, wherein the antagonist is an oligonucleotide.

12. The method of claim 1, wherein the antagonist is an antisense oligonucleotide that inhibits the expression of CD40L.

13. The method of claim 1, wherein the antagonist is a ribozyme.

14. The method of claim 1, wherein the antagonist is RNAi.

15. A method of identifying a compound useful for reducing an adverse response to a transfusion, the method comprising a. providing a composition comprising CD40L; b. contacting the composition with a test compound; c. identifying a test compound that reduces the ability of CD40L to bind to CD40; and d. determining whether the test compound identified in (c) reduces an adverse response to a transfusion in an animal.

16. The method of claim 15, wherein the CD40L comprises soluble CD40L.

17. The method of claim 15, wherein at least part of the CD40L is associated with cells in the composition.

18. The method of claim 17, wherein the cells are platelets.

19. A method of diagnosing an adverse response to a transfusion in a patient who has undergone a transfusion, the method comprising a. obtaining a sample of blood from the patient; b. measuring the level of CD40L in the patient's blood; and c. comparing the post-transfusion level of CD40L measured in (b) to a normal pre-transfusion level of CD40L, wherein a post-transfusion level of CD40L above the normal pre-transfusion level of CD40L indicates an increased risk of an adverse reaction to the transfusion.

20. A method of treating a patient suffering from acute myeloid leukemia, the method comprising a. providing a transfusion composition comprising platelets; b. treating the composition with a CD40L antagonist, thereby producing a treated composition comprising platelets; and c. introducing the treated composition of (b) into the patient.

21. The method of claim 20, wherein the CD40L antagonist is an antibody.

22. Use of a CD40L antagonist in the preparation of a transfusion composition by a method comprising a. providing a transfusion composition comprising platelets and CD40L; b. treating the composition with an amount of the CD40L antagonist effective to reduce the amount or activity of CD40L in the transfusion composition, thereby producing a treated composition; and c. transfusing the treated composition into a patient.

23. A transfusion composition comprising platelets and an exogenous CD40L antagonist for use in treatment.

24. Use of a CD40L-depleted composition in the preparation of a transfusion composition by a method comprising a. providing a transfusion composition comprising platelets; b. treating the transfusion composition with a CD40L antagonist, thereby producing a treated composition; and c. introducing the treated composition of (b) into the patient.

25. A transfusion composition that comprises a CD40L antagonist and platelets for use in treatment.

26. A method of immunotherapy, the method comprising a. identifying a patient in need of immunotherapy; b. providing a composition comprising CD40L derived from blood of the patient; and c. introducing into the patient an amount of the composition effective to modulate the patient's immune system.

27. The method of claim 26, wherein the patient is neonate, elderly, or immunocompromised.

28. The method of claim 26, wherein the CD40L stimulates anti-tumor immunity.

29. The method of claim 26, wherein the CD40L fosters humoral (Th2) immunity and down-regulates cellular (Thl) immune responses.

30. The method of claim 26, wherein the patient is an organ transplant recipient.

31. The method of claim 26, wherein the patient suffers from an autoimmune disease.

32. The method of claim 26, wherein the patient suffers from an inflammatory disease.

33. The method of claim 26, wherein the patient suffers from Crohn's Disease or rheumatoid arthritis.

34. The method of claim 26, wherein the patient is a woman at risk for spontaneous abortion.

35. The method of claim 26, wherein the composition comprises platelets.

36. The method of claim 26, comprising the further step of, before, after, or during step (c), introducing a vaccine into the patient.

37. The method of claim 36, wherein the vaccine is an antiviral vaccine.

38. The method of claim 36, wherein the vaccine is an anti-hepatitis B virus vaccine.

39. The method of claim 36, wherein the vaccine is an anti-influenza virus vaccine.

40. The method of claim 36, wherein the vaccine is an anti-HIV vaccine.

41. The method of claim 36, wherein the composition comprises platelets.