WO2005058359A2

WO2005058359A2 - Method for neutralizing effects of crp for increasing immune reactions to hiv

Info

Publication number: WO2005058359A2
Application number: PCT/EP2004/053496
Authority: WO
Inventors: Ahmed Sheriff; Birgit Vogt
Original assignee: Theralogic Gmbh & Co. Kg
Priority date: 2003-12-15
Filing date: 2004-12-15
Publication date: 2005-06-30
Also published as: WO2005058359A3

Abstract

A pharmaceutical composition for reducing the concentration of pentameric C-reactive protein (CRP) blocking or neutralizing pentameric CRP, the pharmaceutical composition comprising a therapeutically effective amount of an antibody having an antigen binding site to pentameric CRP or an antibody fragment or derivative having an antigen binding site to pentameric CRP and a pharmaceutically acceptable carrier.

Description

Method for neutralizing effects of CRP for increasing im m une reactions to H I V

The invention pertains with a compound neutralizing effects of CRP, a host cell producing the compound, a host cell producing the compound, a pharmaceutical composition for reducing the CRP concentration and uses of the compound.

The present invention deals with the disciplines of therapeutic proteins, HIV infection, and pharmacology. Specifically, the present invention is related to decreasing anti- inflammatory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients associated with increased levels of C- reactive protein (CRP) by administering molecules that bind CRP.

AIDS is a major cause of death in the United States and a major source of morbidity, medical cost, and economic loss to m illions of people.

Lym ph Node Fibrosis I m pedes Peripheral CD4+ T-cell Count Fibrosis could be better predictor of ability to recover after HAART Because HIV preferentially targets CD4+ T cells, their numbers, along with other metrics like HIV RNA levels, traditionally are used to indicate the infection's severity. Moreover, clinicians use these numbers to predict the efficacy of future immunological reconstitution treatment in first-time patients undergoing antiretroviral therapy.

But a recent finding shows that highly active antiretroviral therapy (HAART) failed to markedly increase the peripheral CD4+ count in 25% of patients, despite sometimes being able to reduce HIV RNA in the blood to undetectable levels. This finding has brought into question the utility of these factors as recovery predictors. There are factors beyond suppression of viral replication.

The amount of lymph node fibrosis prior to HAART initiation is perhaps a better indicator of a patient's ability to recover peripheral CD4+ T cells following HAART. The damage to the lymph nodes, where nearly all HIV replication takes place in the activated CD4+ cells that reside there, has already occurred before therapy has even started.

An ever-expanding body of work is showing the importance of lym ph node architecture in providing a suitable microenvironment for the immune processes. Here, T cells interact with B cells, antigen-presenting cells, stroma, and each other, as well as receive soluble messages through cytokines and other growth factors. So, if the structure is com promised, the lymph nodes' ability to support a viable immune system may be severely compromised as well.

PERPETUA L I NFL A MM A TI ON

.Examination of the T-cell zones of lymph nodes from treatment -naive patients at various stages of HIV infection, from presym ptomatic to full-blown AI DS was done. The number of CD4+ T cells found there did not correlate with either peripheral CD4+ cells or with detectable amounts of viral RNA in the plasma. But the nodes had considerably more collagen deposition than HIV- negative controls. The collagen showed an inverse relationship to the nodal CD4+ T-cell population; the number of CD4+ cells decreased as the amount of fibrosis increased. Similarly, the potential for imm unological reconstitution as measured by the peripheral CD4+ T-cell count after therapy showed an inverse relationship with the amount of nodal collagen deposition.

It is speculated that lymph nodes are likely damaged because of perpetual inflammation. I n the long struggle between immune defenses and HIV- 1 that partially controls replication, the immune system is maintained in a state of chronic activation.

The model is not unprecedented. The situation is analogous to what happens to the liver in a chronic hepatitis infection. In such cases, ongoing viral replication leads to chronic inflammation and fibrosis, eventually replacing functional hepatic tissue with collagen; the end result is cirrhosis.

While it is unclear exactly what mechanism is operating here damage to lymph node structure could have several consequences for the immune response, including the inability of the lym ph nodes with large amounts of collagen to physically house T cells, or to alter positioning of T cells such that proper activation, growth or chemotactic signals are not received. Excessive deposition of collagen and other extracellular m atrix components within the T zone might be expected to disrupt T-cell interactions with dendritic cells or local production of I L-7 [the T-cell survival factor interleukin 7] .

Damage and disruption to the lymphatic tissue microenvironment results in the impaired recruitment, retention, and proliferation of CD4+ cells. The most significant impact would be on naive CD4+ T cells, which are known to require greater external signaling to proliferate and remain viable than do activated or memory cells.

Presuming that chronic inflammation is responsible for damaging the lymph node architecture, anti- inflammatory therapy would essen, prevent, or even reverse some of the fibrosis, perhaps leading to an improved im munological recovery. This can easily be achieved by reducing CRP levels or neutralizing CRP.

Apoptotic signals from HI V vesicles transform APC into an anti- inflam m atory m ood

It has been shown that HIV infects antigen- presenting cells (APC: dendritic cells [DC] , macrophages, monocytes, and B cells) . It has also been demonstrated that HIV patients do have a compromised cellular immunity. It has been claimed that the DC from asymptomatic HIV infected individuals (lacking lymphadenopathy and without treatment) cause low levels of stimulation of allogeneic lymphocytes in the MLR (mixed leucocyte reaction). By contrast, lymphocytes from these patients respond to norm al allogeneic DC. The failure of T cell stimulation by DC in HIV infection therefore shows an incapacity of these DC to transfer stim ulatory signals to activate T cells. Also, phosphatidylserine on the HIV envelope is a cofactor for infection of APC.

It is suggested that both observations may be related and that the anti- inflammatory signal exerted by phosphatidylserine (PS) may be the reason for the observations. PS is also a target for secretory phospholipase A2 (sPLA2) which binds PS on the outer membrane leaflet of cells. After interaction with sPLA2, cells are left with an increased proportion of lysophospholipids (phospholipids that have lost free fatty acids at the sn-2-ester bond) like lysophosphatidylcholine (lyso-PC) in the outer membrane leaflet. This modification disturbs the packing of the phospholipids and generates binding sites for the pentraxin C-reactive protein (CRP) in the outer leaflet.

CRP is capable of binding to cells provided that they contain a substantial amount of lyso-PC in the outer leaflet of their membranes. Thus, in inflamed tissues, it can be proposed that binding of CRP initially occurs to cells containing a significant proportion of lysophospholipids in the outer leaflet of their membranes due to the activity of sPLA2. Once bound, CRP induces complement activation via the classical pathway, which in turn triggers the influx of neutrophils, decorates the surface of the ligand with opsonising complement fragments and enhances phagocytosis of the cells that have bound CRP and complement. CRP also interacts with Fc receptors on phagocytic cells and acts as an opsonin. Because the occurence of lyso-PC is dependent on the exposure of PS, CRP, in this chronological view, efficiently decorates late apoptotic cells. This way, decoration with CRP can be an apoptotic signal.

The macropinocytosis of apoptotic cells by macrophages triggers the production of transforming growth factor β (TGFβ), a cytokine that suppresses inflammatory processes. When monocytes or immature DC encounter apoptotic cells they are triggered by CD36 to produce I L-10 which is also an anti- inflammatory cytokine. In addition to the enhanced secretion of anti- inflammatory cytokines, the production of proinflammatory cytokines such as TNF alpha, IL-1 and IL- 12 is markedly inhibited in the presence of apoptotic cells. APC which produce anti- inflammatory cytokines do suppress T cells.

I n this case, the treatment with CRP-binding molecules prevents the decoration of HIV particles with CRP. This way, the anti-inflammatory signal can be abrogated. Of particular interest to the methods of the present invention is the reduction of anti- inflammatory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients associated with CRP.

CRP is produced by the liver in response to cytokine production. Cytokines are produced as part of an inflammatory response in the body. Thus, CRP levels are a marker of systemic inflam matory activity. Chronic inflammation is thought to be one of the underlying and sustaining pathologies in lymph node fibrosis.

One object of the invention is to provide a new therapeutic agent which lowers the risk factors mentioned above by neutralization of CRP.

Another object is to provide tools for decreasing levels of CRP in humans comprising administering to a human in need thereof an effective amount of a compound containing at least a molecule which binds CRP or a pharmaceutical salt or solvate thereof.

Still another object is to provide molecules and methods for decreasing levels of CRP in humans com prising administering to a human in need thereof an effective amount of a compound containing at least a molecule which binds CRP or a pharmaceutical salt or solvate thereof.

Further, the present invention relates to a method for inhibiting conditions or detrimental effects caused accidentally by CRP co prising administering to a human in need thereof, an effective amount of a com pound containing at least a molecule which binds CRP, or parts of it and more preferably human CRP or a pharmaceutical salt or solvate thereof.

The present invention is based on the finding that molecules that bind CRP, i.e., antibodies, a recombinant antibody (as e.g. single chain antibody - scAb or scFv; bispecific antibody, diabody), monoclonal antibodies, are useful for lowering the levels of CRP and/or blocking and/or neutralizing CRP.

As used herein, the term "effective amount" means an amount of a compound of molecules which bind CRP which is capable of decreasing levels or blocking CRP and/or hhibiting conditions or detrimental effects accidentally caused by CRP.

The term "inhibiting" in the context of inhibiting conditions or detrimental effects accidentally caused by CRP includes its generally accepted meaning, i.e., blocking, prohibiting, restraining, alleviating, ameliorating, slowing, stopping, or reversing the progression or severity of an increase of CRP and the pathological sequelae, i.e., symptoms, resulting from that event.

The term "pharmaceutical" when used herein as an adjective, means substantially non-toxic and substantially non-deleterious to the recipient.

By "pharmaceutical formulation" or "medicament" or "pharmaceutical composition" it is further meant that the carrier, solvent, excipients and salt must be compatible with the active ingredient of the formulation (a compound of at least a molecule, which binds CRP) .

The term "solvate" represents an aggregate that comprises one or more molecules of the solute, with one or more molecules of a pharmaceutical solvent, such as water, buffer, physiological salt solution, and the like.

The present invention claims a pharmaceutical composition for reducing the concentration of pentameric Oreactive protein (CRP) blocking or neutralizing pentameric CRP, the pharmaceutical composition comprising a therapeutically effective amount of an antibody having an antigen binding site to pentameric CRP or an antibody fragment or derivative having an antigen binding site to pentameric CRP and a pharmaceutically acceptable carrier.

In the following, the term CRP also encom passes pentameric CRP in particular human pentameric CRP.

Native antibodies are multi-subunit animal protein molecules with highly specific antigen-binding properties. Animals make multiple classes of antibodies. There are five major dasses ( IgA, IgD, IgE, IgG and IgM) and a variety of subclasses. Native antibodies are made up of two or more heterodimeric subunits each containing one heavy (H) and one light (L) chain. The differences between antibody classes derive from their different H chains. H chains have a molecular weight of about 53 kDa, while L chains are about 23 kDa in mass.

Every individual native antibody has one type of L chain and one type of H chain, which are held together by disuifide bonds to form a heterodimeric subunit. Typically a native antibody (e.g., an IgG) has two such subunits, which are also held together by disuifide bonds. Within each chain, units of about 110 amino acid residues fold so as to form compact domains. Each domain is held together by a single intrachain disuifide bond. L chains have two domains, while H chains have four or five. Most H chains have a hinge region after the first (i.e., most amino-terminally located) two domains. The disuifide bonds linking together the heterodimeric subunits are bcated at the hinge regions. The hinge region is particularly sensitive to proteolytic cleavage, such proteolysis yielding two or three fragments (depending on the precise site of cleavage), a non-antigen binding fragment containing only H chain C regions (Fc) and one bivalent (Fab'2) or two monovalent (Fab) antigen binding fragments. The hinge region allows the antigen binding regions (each made up of a light chain and the first two domains of a heavy chain) to move freely relative to the rest of the native antibody, which includes the remaining heavy chain domains.

The first domain of each chain is highly variable in amino acid sequence, providing the vast spectrum of antibody binding specificities found in each individual. These are known as variable heavy (VH) and variable light (VL) domains. The second and subsequent (if any) domains of each chain are relatively invariant in amino acid sequence. These are known as constant heavy (CH) and constant light (CL) domains.

Each variable region contains three loops of hypervariable sequence that provide a complementary structure to that of the antigen and are critical in determining the antigen binding specificity of the antibody, as they are the contact sites for binding to the antigen. These loops are known as complementarity determining regions, or CDRs. Each variable domain is made up of three CDRs embedded in four much less variable framework segments (FRs). Together, the sets of collinear CDRs and FRs are in large part responsible for determining the three dimensional conformation of the variable regions of antibody molecules.

CDRs and FRs are features that have been deduced from structural properties of antibody variable regions. Both amino acid sequence (primary structure) and three dimensional modeling (deduced secondary and tertiary structure) of antibody variable regions have been used by various researchers to define CDRs and, by default, FRs. While the positions of the CDRs are beyond question, not all workers in the art agree upon the precise locations of the boundaries of each CDR in VH or VL regions; there is no clear cut structural marker delineating CDR/ FR boundaries.

Two definitions of CDR location are currently in general use in the art. These are the "sequence variability" definition of Kabat et al. ("Sequences of Proteins of I mmunological I nterest," 4th ed. Washington, D.C. : Public Health Service, N. I .H.) and the "structural variability" definition of Chothia and Lesk (J. Mol. Biol. 1987, 196: 901 ) . As used herein, the terms VL CDR1 , VL CDR2, VL CDR3, VH CDR1 , VH CDR2, and VH CDR3 refer m inimally to the region of overlap between the regions designated for each CDR by each of these two definitions, and maximally to the total region spanned by the combination of the regions designated for each CDR by each of these two definitions.

One problem that antibody engineering attempts to address is the imm une activity of a human patient that occurs in response to a native murine (or other non-human anim al) antibody, typically a mAb, that is being administered to the patient for therapeutic purposes. This activity against murine antibodies is characterized by a human anti-mouse antibody (HAMA) response that can have deleterious effects on treatment efficacy and patient health. It has been found that almost all such human anti-non-human antibody ("HAMA type") activity is directed at the constant domains and at the FR regions of the variable domains of native non-human antibodies.

By manipulating the nucleic acid molecules encoding antibody H and L chains it is possible to incorporate non-human variable regions into antibodies otherwise made up of human constant regions. The resulting antibodies are referred to as "chimeric antibodies," and are typically less prone to eliciting HAMA type responses than are the non-human antibodies from which the variable regions are derived.

An even more effective approach to eliminating the potential of a non-human antibody to elicit a HAMA type response is to "humanize" it, i.e., to replace its non-human framework regions with hum an ones. One way of achieving such humanization involves the insertion of polynucleotide fragments encoding the non-human CDRs of the antibody to be humanized into a nucleic acid molecule encoding an otherwise human antibody (with human constant regions if desired) so as to replace the human CDRs and to use the resulting nucleic acid molecule to express the encoded "humanized" antibody.

Unfortunately, however, humanization of non-human antibodies has unpredictable effects on antibody antigen interactions, eg., antigen binding properties. Some of this unpredictability stems from the properties of the CDRs. Certain CDRs may be more amenable to the construction of humanized antibodies that retain the properties of the non-human CDR donor antibody than others. While the CDRs are key to tfcte antigen binding properties of an antibody, CDRs and FRs must interact appropriately if the antigen specificity of an antibody is to be retained following humanization. The effects of combination with particular human FRs on uncharacterized non-human CDRs cannot be reliably predicted by any known method. However, the successful humanization of an antibody provides information that, in general, facilitates the successful humanization of the CDRs of that antibody using other human or altered human FRs. In addition, approaches are available that facilitate tailoring human FRs to enhance the likelihood of successful humanization.

Other problems addressed by antibody engineering include efficient antibody production and alteration of antibody pharmacokinetics. Recombinant protein production is generally most efficiently carried out in bacterial hosts. The large size and multimeric nature of native antibodies makes their production in bacteria difficult. One approach to dealing with production problems is to use recombinant DNA methods to construct antibodies that have their H and L chains joined by a linker peptide to form a single chain (sc) antibody. As described below, there are several types of sc antibodies that can be constructed.

As is the case for humanization, the effects on antigen binding properties of constructing a particular type of sc antibody using H and L chains that have not been characterized with regard to their ability to function as part of an sc antibody cannot be reliably predicted by any known method. However, the successful construction of any one type of sc antibody from a particular native antibody provides information that, in general, facilitates the successful construction of other types of sc antibodies from that native antibody.

Single chain antibodies may include one each of only VH and VL domains, in which case they are referred to as scFv antibodies; they may include only one each of VH, VL, CH, and CL domains, in which case they are referred to as scFab antibodies; or they may contain all of the variable and constant regions of a native antibody, in which case they are referred to as full length sc antibodies.

The differing sizes of these antibodies im parts each with differing pharmacokinetic properties. I n general, smaller proteins are cleared from the circulation more rapidly than larger proteins of the same general composition. Thus, full length sc antibodies and native antibodies generally have the longest duration of action, scFab antibodies have sho rter durations of action, and scFv antibodies have even shorter durations of action. Of course, depending upon the illness being treated, longer or shorter acting therapeutic agents may be desired. For example, therapeutic agents for use in the prevention of immune and hemostatic disorders associated with extracorporeal circulation procedures (which are typically of brief duration) are preferably relatively short acting, while antibodies for the treatment of long term chronic conditions (such as inflam matory joint disease or GN) are preferably relatively long acting.

Detailed discussions of antibody engineering may be found in numerous recent publications including: Reichmann, et al., Nature, 332:323-327, 1988; Winter and Milstein, Nature, 349:293-299, 1991 ; Clackson, et al., Nature, 352: 624- 628, 1991 ; Morrison, Annu Rev I mmunol, 10:239-265, 1992; Haber, I mm unol Rev, 130: 189-212, 1 992; Borrebaek, "Antibody Engineering, A Practical Guide," 1992, W.H. Freeman and Co. NY; Rodrigues, et al., J Immunol, 151 :6954-6961 , 1 993; and Borrebaek, "Antibody Engineering," 2nd ed. 1 995, Oxford University Press, NY, Oxford

The pharmaceutical composition of the invention containing an the antibody having an antigen binding site to pentameric CRP or said antibody fragment is in particular combined with therapeutics for HIV or anti- inflammatory substances such as anti- I L-6-molecules, anti- 1 L- 1 β- molecules, anti-sPLA2 molecules especially sPLA2 HA, anti-complement molecules, blocking agents or inhibitors for I L-6, CRP, I L- 1 β, sPLA2 especially sPLA2 MA, complement blockers or combinations thereof.

According to the invention the antibody fragment or derivative is obtainable by a process including immunizing vertebrates or transgenic vertebrates with CRP, mice, rats, guinea pigs, hamsters, monkeys, pigs, goats, chicken, cows, horses and rabbits.

According to the invention the pharmaceutical composition com prises a monoclonal antibody which has been produced after immunizing humanized vertebrates having a humanized immune system , most preferably mice, rats, guinea pigs, hamsters, monkeys, pigs, goats, chicken, cows, horses and rabbits or by immunizing imm une defective mice such as SCI D or nude mice repopulated with vital im mune cells e.g. of human origin such as SCI D-hu mice.

The antibody having an antigen binding site to pentameric CRP said antibody fragment or derivative is in particular a recombinant antibody selected from the group consisting of single chain antibodies - scAb or scFv, bispecific antibody, diabody and combination thereof, capable of binding to pentameric CRP, in particular by containing the antigen-binding site of an antibody which is cross- reactive with pentameric CRP. It is advantageous to employ a humanized or human antibody. Subject matter of the invention is also the use of an antibody having an antigen binding site to pentameric CRP said antibody fragment or derivative for the manufacturing of a medicament for inhibiting anti- inflammatory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients as well as the use of an antibody having an antigen binding site for pentameric CRP, said antibody fragment or derivative for the manufacturing of a medicament for reducing anti- inflammatory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients by reducing the CRP concentration and/or neutralizing CRP.

The antibody having an antigen binding site to pentameric CRP, said antibody fragment or derivative can be used for the manufacturing of a medicament for treatment of HIV- infected patients.

Furthermore, subject matter of the invention is at least one recombinant vector comprising the nucleotide sequences encoding the binding molecule fragments according to the invention, operably linked to regulating sequences capable of expressing the antibody molecule in a host cell, preferably as a secretory protein.

A host cell producing an antibody having an antigen binding site to pentameric CRP, said antibody fragment or derivative is also subject matter of the invention. The skilled person knows how to generate the respective host cell

The present invention provides recombinant expression vectors which include the synthetic, genomic, or cDNA-derived nucleic acid fragments of the invention, i.e. polynucleotides encoding the mAbs of the invention. The nucleotide sequence coding for any of the mAbs of the invention can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native or source gene and/or its flanking regions. A variety of host vector systems may be utilized to express the recombinant expression vectors of the invention. These include, but are not limited to, mammalian cell systems infected with recombinant virus (e.g., vaccinia virus, adenovirus, retroviruses, etc.) ; mammalian cell systems transfected with recombinant plasmids; insect cell systems infected with recombinant virus (e.g., baculovirus) ; microorganisms such as yeast containing yeast expression vectors, or bacteria transformed with recombinant bacteriophage DNA, recombinant plasmid DNA, or cosmid DNA (see, for example, Goeddel, 1990) .

Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (American Type Culture Collection-'ΑTCC"-, 10801 University Boulevard, Manassas, Va. 201 10-2209, United States of America; ATCC Accession No. 37017) . These pBR322 "backbone sections," or functionally equivalent sequences, are combined with an appropriate promoter and the structural gene to be expressed.

Preferred bacteria for expression of recombinant mAbs include Bacillus subtilis and, most preferably, Escherichia coli.

The recombinant mAbs of the invention may also be expressed in fungal hosts, preferably yeast of the Saccharomyces genus such as S. cerevisiae. Fungi of other genera such as Aspergillus, Pichia or Kluyveromyces may also be employed. Fungal vectors will generally contain an origin of replication [autonomously replicating sequence (ARS)] , a promoter, DNA encoding a mAb of the invention, sequences directing polyadenylation and transcription termination, and a selectable marker gene, and optionally a secretion signal. Preferably, fungal vectors will include origins of replication and selectable markers permitting transformation of both E. coli and fungi.

Various mammalian or insect cell culture systems can be employed to express recombinant mAbs. Examples of suitable mammalian host cell lines include the COS cell of monkey kidney origin, mouse L cells, murine C127 mam mary epithelial cells, mouse Balb/3T3 cells, Chinese hamster ovary cells (CHO), human 293 EBNA and HeLa cells, myeloma, and baby hamster kidney (BHK) cells, with myeloma cells, CHO cells, and human 293 EBNA cells being particularly preferred.

Mammalian expression vectors may comprise non-transcribed elements such as origin of replication, a suitable promoter and enhancer linked to the recombinant mAb gene to be expressed, and other 5' or 3' flanking sequences such as ribosome binding sites, a polyadenylation sequence, splice donor and acceptor sites, and transcriptional termination sequences, and optionally a secretion signal.

Purified recombinant mAbs are prepared by culturing suitable host/vector systems to express the recombinant mAb translation products of the nucleic acid molecules of the present invention, which are then purified from the culture media or cell extracts of the host system, e.g., the bacteria, insect cells, fungal, or mammalian cells. Fermentation of fungi or mammalian cells that express recombinant mAb proteins containing a histidine tag sequence (a sequence comprising a stretch of at least 5 histidine residues) as a secreted product greatly simplifies purification. Such a histidine tag sequence enables binding under specific conditions to metals such as nickel, and thereby to nickel (or other metal) columns for purification. Recombinant mAbs may also be purified by protein G affinity chromatography (Proudfoot et al., 1992, Protein Express. Purif. 3:368) .

Also a host cell is subject matter of the invention which host cell is producing antibody fragments having an antigen binding site to pentameric CRP, said antibody fragment or derivative operably linked to regulating sequences capable of expressing the antibody in a host cell, preferably as a secretory protein. Preferably the host is comprising, in particular stably transgenic, a vector, a prokaryotic or eukaryotic cell line producing a recombinant antibody of the invention as well as a eukaryotic organism, most preferably an animal, a plant or a fungus, producing a recombinant antibody according to the invention. The invention is suitable for a method of treating HIV infected patients by administering an antibody having an antigen binding site to pentameric CRP, an antibody fragment having an antigen binding site to pentameric CRP, a derivative of an antibody having an antigen binding site to pentameric CRP or a combination thereof (to a HIV infected patient) .

I n one embodiment the compound of the invention is a polypeptide com prising a binding site to CRP, preferably an antibody containing an antigen- binding site to CRP. The compound of the invention is in particular a poly- or monoclonal antibody comprising an antigen-binding site to CRP.

I n a further embodiment the antibody of the invention is a recom binant antibody (as e.g. single chain antibody - scAb or scFv; bispecific antibody, diabody etc.) capable of binding to CRP, in particular by containing the antigen-binding site of an antibody which is cross- reactive with CRP. The antibody molecule of the invention is a humanized or human antibody. Subject matter of the invention is also a host cell, preferably a stable host cell, producing the compound of the invention.

The pharmaceutical composition of the invention may contain in addition the use of other clinically relevant agents administered to treat the pathological conditions embodied in the present invention in combination with a compound of at least the antbodies, fragments or derivatives binding pentameric CRP.

A preferred pharmaceutical formulation is a fluid as e.g. a physiological salt solution. This will be used for injection. Pharmaceutical form ulations can be prepared by procedures known in the art, such as, for exam ple, a compound of at least a molecule which binds CRP can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, and the like.

Exam ples of excipients, diluents, and carriers that are suitable for formulation include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrroiidone; moisturizing agents such as glycerol; disintegrating agents such as agar, calcium carbonate, and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonire; and lubricants such as talc, calcium and magnesium stearate and solid polyethyl glycols. Final pharmaceutical forms may be: pills, tablets, powders, lozenges, syrups, aerosols, saches, cachets, elixirs, suspensions, emulsions, ointments, suppositories, sterile injectable solutions, or sterile packaged powders, depending on the type of excipient used.

Additionally, the antibodies, fragments or derivative binding pentameric CRP are well suited to formulation as sustained release dosage forms. The formulations can also be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. Such formulations would involve coatings, envelopes, or protective matrices, which may be made from polymeric substances or waxes.

The particular dosage of a compound containing molecules which bind CRP required to decrease levels of homocysteine and/or CRP according to this invention will depend upon the particular circumstances of the conditions to be treated. Considerations such as dosage, route of administration, and frequency of dosing are best decided by the attending physician. Generally, an effective minimum dose for oral or parenteral administration of the antibodies, fragments or derivatives which bind CRP is about 0,01 to 20,000 mg/l. Typically, an effective maximum dose is about 20,000, 6,000, or 3,000 mg. Such dosages will be administered to a patient in need of treatment as often as needed to effectively decrease levels of CRP and/or inhibit conditions or detrimental effects caused by an excess of CRP.

The dose range and frequency of administration of the individuals depends on the disease to be treated. It is recommended not to neutralize the CRP level completely, but to lower it to the normal human CRP-level. It is desirable to measure the actual CRP-value of the respective patient and to calculate the amount of anti-CRP antibody needed. In the following some typical dose ranges are illustrated by way of examples.

Regarding cardiovascular diseases high amounts of antibodies are required, e.g. in the range of 10 - 20,000 mg/L For treating bacterial infections the dosage is lower typically, e.g. in the range of about 1 ,000 mg/L. For treatment of viral infections the range can be still lower, e.g. 3 - 100 mg/L. The absolute amount of anti-CRP antibodies administered depends on the total blood volume in the individual patient. The duration of anti-CRP antibodies administration depends also on the kind of disease. In case of cardiovascular diseases a single administration of relative high amount can be sufficient, whereas in case of infections the administration can be repeated preferably under consideration of the actual CRP-level of the treated patient, typically until the CRP-level is about the normal CRP-value.

CRP experim ents

Type I I secretory phospholipase A2 I IA (sPLA2 I IA) hydrolyses the sn-2-ester bond of phospholipids to produce free fatty acids and lysophospholipids (e.g. lysoPC) . C- reactive protein (CRP) binds lysoPC and subsequently complement (for example as the first complement protein C1 q) binds to CRP. ^s

CRP and increased cell death

I n Jurkat cells apoptosis can be induced chemically. Addition of sPLA2 I IA, CRP and C1 q to the medium will lead to binding of CRP and C1q to these cells. Further addition of antibodies specific for CRP will inhibit this binding. Figure 1 shows typical results.

Fig. 1 : Binding of CRP and C1 q to apoptotic Jurkat cells. Panel A: Binding of CRP. Panel B: Binding of C1 q. Control: no sPLA2 added (upper row). Addition of sPLA2 I IA (middle row) leads to binding of CRP and C1 q. Addition of antibodies (AB, lower row) specific for CRP inhibits binding of CRP and C1 q.

Peripheral blood mononuclear cells (PBMC) were separated from buffy coats of healthy HIV-1 seronegative male donors by Ficoll density gradient centrifugation and cultured on hydrophobic Teflon foils (Heraeus, Hanau, Germany) for 7 days in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco, Berlin, Germany) , L-glutamine (2 mM; Gibco) , and 4% pooled human AB-group serum . Monocyte-derived macrophages (MO-MAC) were separated by adhesion. Briefly, PBMC were plated on chamber slides (NUNC, Wiesbaden, Germany), after 1 hour the nonadherent peripheral blood lymphocyte (PBL) containing cell fraction was removed by repeated washing and MO-MAC were further cultivated for 24 hours in fresh m edium (V. Eisert, M. Kreutz, K. Becker, C. Konigs, U. Alex, H. Rubsam en-Waigmann, R. Andreesen, H. von Briesen, Analysis of cellular factors influencing the replication of human imm unodeficiency virus type I in human macrophages derived from blood of different healthy donors, Virology. 286 (2001 ) 31 -44) . Human monocyte-derived macrophages from peripheral blood of healthy donors were CD14 and MAX.1 positive. The purity of the cultures is between 95-98% . A monocytotrope ( R5) HIV stem from a patient (HIV- 1 D1 1 7I I I) [ Rubsamen-Waigmann, H., Willems, W. R., Bertram , U., von Briesen, H. (1989) : Reversal of HIV-phenotype to fulm inant replication on macrophages in perinatal transmission. Lancet ii, 1 155-1 156.] was used for the infection of the macrophages. The infection took place on day 7. Anti-CRP antibodies (aCRP; 1 0 or 50 μg/m l) were either added on day 7 or day 13. HIV production was measured with the p24 antigen assay. A positive (pos.) control was cultured without addition of aCRP and a negative (neg.) control was cultured without the addition of HIV or aCRP.

CRP also binds to early apoptotic human T lym phocytes. This binding can be prevented by antibodies (AB) specific for CRP. C1 q binding will also be reduced. A typical experiment will give the following results.

Table 1 : Binding of sPLA2 I IA, CRP and C1 q to apoptotic human T-cells. Addition of antibodies specific for CRP will also inhibit binding of C1 q. Conditions Binding of sPLa2 Binding of CRP Binding of C1 q

CRP and H I V

APC are typical HlV-reservoirs and therefore, one of the most important targets of HIV. In another setting, human macrophages and dendritic cells are infected with HIV. Two weeks later the amount of virus is quantified using an ELISA for p24. Addition of sPLA2 and CRP will enhance the infection process and therefore, the amount of p24. Treatment with antibodies specific for CRP will hamper the infection process and reduce the amount of p24. A typical experiment will give the following results as exemplified in table (Tab. 2) and figure (fig. 2) .

Table 2 : Amounts of p24 after two weeks of infection in macrophages/dendritic cells in the presence of sPLA2 and CRP and antibodies. The amount of p24 in infected, normally cultured cells is set as 1 .

HIV infection with sPLA2 and CRP with AB < 1 against CRP

Fig . 2 : Effect of CRP and specific antibodies on the growth of HIV in infected human monocytes and macrophages.

I n hibition of CRP and C1 q binding to apoptot ic cells Jurkat cells (ACC 282) were cultured in RPMI with 10% FCS. Apoptosis was induced by culturing 10⁶ cells in 1 ml medium with 2 μM Staurosporin for 4 hours. Cells were washed and incubated in 1 ml medium with 10 μg/ml CRP for 30 min. To some samples 50 μg of murine anti-CRP- IgG antibody (mixture of 5 different antibodies) was added. After washing the samples were divided into two portions. Half were stained with goat-anti-m ouse- lgG-FITC. The other half was incubated in 1 ml medium with 10 μg/ml C1 q for 30 min. Cells were washed afterwards and stained with anti-C1 q FITC antibody. All samples were analysed for CRP or C1 q using a FACS Caiibur (Becton- Dickinson). Dead cells were excluded using PI staining. Apoptosis was confirmed by Annexin-V-FITC staining. I ncubation with anti-CRP antibody blocked CRP and subsequently C1 q binding.

Claims

1 . A pharmaceutical composition for reducing the concentration of pentameric C-reactive protein (CRP) blocking or neutralizing pentameric CRP, the pharmaceutical composition comprising a therapeutically effective amount of an antibody having at least one antigen binding site to pentameric CRP or an antibody fragment or derivative having at least one antigen binding site to pentameric CRP and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1 wherein the antibody having an antigen binding site to pentameric CRP or said antibody fragment is combined with therapeutics for HIV or anti-inflammatory substances such as anti- IL-6-molecules, anti- IL- 1 β-molecules, anti-sPLA2 molecules especially sPLA2 I IA, anti-complement molecules, blocking agents or inhibitors for I L-6, CRP, I L- 1 B, sPLA2 especially sPLA2 I IA, complement blockers or combinations thereof.

3. The pharmaceutical composition according to claim 1 and/or 2 wherein the antibody having an antigen binding site to pentameric CRP said antibody fragment or derivative is obtainable by a process including immunizing vertebrates or transgenic vertebrates with CRP, mice, rats, guinea pigs, hamsters, monkeys, pigs, goats, chicken, cows, horses and rabbits.

4. The pharmaceutical composition according to claim 1 -3 wherein the antibody having an antigen binding site to pentameric CRP, said antibody fragment or derivative is a monoclonal antibody which has been produced after immunizing humanized vertebrates having a humanized immune system, most preferably mice, rats, guinea pigs, hamsters, monkeys, pigs, goats, chicken, cows, horses and rabbits.

5. The pharmaceutical composition according to claim 1-4 wherein the monoclonal antibody containing an antigen-binding site to pentameric CRP said antibody fragment or derivative has been produced by im munizing immune defective mice such as SCI D or nude mice repopulated with vital immune cells e.g. of human origin such as SCI D- hu mice.

6. The pharmaceutical composition according to claim 1-5 wherein the antibody having an antigen binding site to pentameric CRP said antibody fragment or derivative is a recombinant antibody selected from the group consisting of single chain antibodies - scAb or scFv, bispecific antibody, diabody and combination thereof, capable of binding to pentameric CRP, in particular by containing the antigen-binding site of an antibody which is cross- reactive with pentameric CRP.

7. The pharmaceutical composition according to claim 1-6 wherein the antibody having an antigen binding site to pentameric CRP, said antibody fragment or derivative, is a humanized or human antibody.

8. Use of an antibody having an antigen binding site to pentameric CRP said antibody fragment or derivative for the manufacturing of a m edicament for inhibiting anti- inflamm atory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients.

9. Use of an antibody having an antigen binding site for pentameric CRP, said antibody fragment or derivative for the manufacturing of a medicament for reducing anti- inflammatory effects of HIV and/or pathophysiological responses to lymph nodes in response to continuos HIV infection in HIV patients by reducing the CRP concentration and/or neutralizing CRP.

1 0. Use of an antibody having an ant igen binding site to pentameric CRP, said antibody fragment or derivative for the manufacturing of a medicament for treatment of HIV-infected patients.

1 1 .A host cell producing an antibody having an antigen binding site to pentameric CRP, said antibody fragment or derivative.

2.A host cell producing antibody fragments having an antigen binding site to pentameric CRP, said antibody fragment or derivative operably linked to regulating sequences capable of expressing the antibody in a host cell, preferably as a secretory protein.3.Method of treating HIV infected patients by administering an antibody having an antigen binding site to pentameric CRP, an antibody fragment having an antigen binding site to pentameric CRP, a derivative of an antibody having an antigen binding site to pentameric CRP or a combination thereof (to a HIV infected patient) .