key: cord-0007936-8x4rnkpm
authors: Giefing, Carmen; Nagy, Eszter; von Gabain, Alexander
title: The Antigenome: From Protein Subunit Vaccines to Antibody Treatments of Bacterial Infections?
date: 2009-12-30
journal: Pharmaceutical Biotechnology
DOI: 10.1007/978-1-4419-1132-2_9
sha: ee8bdbed9a9bf20bb1e4922c70f85680d6f00f71
doc_id: 7936
cord_uid: 8x4rnkpm

New strategies are needed to master infectious diseases. The so-called “passive vaccination”, i.e., prevention and treatment with specific antibodies, has a proven record and potential in the management of infections and entered the medical arena more than 100 years ago. Progress in the identification of specific antigens has become the hallmark in the development of novel subunit vaccines that often contain only a single immunogen, frequently proteins, derived from the microbe in order to induce protective immunity. On the other hand, the monoclonal antibody technology has enabled biotechnology to produce antibody species in unlimited quantities and at reasonable costs that are more or less identical to their human counterparts and bind with high affinity to only one specific site of a given antigen. Although, this technology has provided a robust platform for launching novel and successful treatments against a variety of devastating diseases, it is up till now only exceptionally employed in therapy of infectious diseases. Monoclonal antibodies engaged in the treatment of specific cancers seem to work by a dual mode; they mark the cancerous cells for decontamination by the immune system, but also block a function that intervenes with cell growth. The availability of the entire genome sequence of pathogens has strongly facilitated the identification of highly specific protein antigens that are suitable targets for neutralizing antibodies, but also often seem to play an important role in the microbe’s life cycle. Thus, the growing repertoire of well-characterized protein antigens will open the perspective to develop monoclonal antibodies against bacterial infections, at least as last resort treatment, when vaccination and antibiotics are no options for prevention or therapy. In the following chapter we describe and compare various technologies regarding the identification of suitable target antigens and the foundation of cognate monoclonal antibodies and discuss their possible applications in the treatment of bacterial infections together with an overview of current efforts.

Infectiousdiseases remain a major threat againsthuman life.Microbialinfectionsare stillout ofcontrol in manyparts of the less developedworldwhere they count for most of the deaths,but alsocausean often underestimatedtoll ofdeath [e.g.,communityacquiredPneumococcal diseases and Pseudomonas infectionsin patients in intensivecare), life-longmutilation (infertilitydue to Chlamydia trachomatis), medicalcomplicationdue to nosocomialinfectionscausedmostoftenby Staphylococcus aureus, Enterococcusfaecalis,Klebsiella sspand fungi.It isestimatedthat nosocomial infectionsannuallyadd US$5-10 billionto the costof the nationalhealthcaresystem in the United States,' Apart from infections causedby viruses and protozoa that only in specific instancescan be treated with suitable pharmaceuticals, the emergence of antibiotic-resistant strains of nearly all kinds ofbacterial pathogens in the community and in hospitals is occurring at an increasingly alarming rate. 2,3 The increase of nosocomial infections, the comeback of bacterial infections in immune suppressed individuals, e.g., TB in AIDS patients," and the lately appeared scenario of bio-terrorism, e.g., in the context of anthrax,S,6 are reminders that new strategies are needed to master infectious diseases in prophylactic and therapeutic settings.

Vaccination is undeniable the most successful medical intervention in the control ofinfectious diseases. However, since vaccine-induced immune protection against specific microbes takes more than a couple of weeks to develop and postexposure vaccination is only exceptionally a useful tool, combination with passive immunization is indispensable (e.g., treatment against the rabies virus. reviewed in ref 7) . when instant protection or treatment is required. Therapeutic vaccines are still in the exploratory stage of development and more prone to find their application in the treatment ofchronic infectious diseases,8.9rather than to become an immediate measure against a sudden infectious threat. On the other hand. most vaccines seem to confer protective immunity to the vaccinated individuals by the means to induce specific antibodies that capture the invading microbe. prior it had an opportunity to colonize in the exposed host. The so-called "passive vaccination" i.e., prevention and treatment with specific antibodies, has a proven record and potential in the management of infections. Already the pioneers of early microbiology and immunology in the late 19th century, led by their prominent proponents. Emile Roux and Emil von Behring, have realized the concept of "passive vaccination", namely that sheep and horses inoculated with filterable toxin extracts derived from Corynebacterium diphteriae cultures were able to mount an "anti -toxin" in their blood. Serum derived from the animals' blood was able to rescue children in the lethal stage of the infection caused by the same pathogen. Revisiting this historical landmark therapy of diphtheria, it was realized that the "anti-toxin" in the serum ofinoculated animals is synonymous with a protein species coined today antibodies and the "toxin" with a virulence factor secreted by the pathogen during infection. Thus , the remarkable and groundbreaking therapy concept explored more than 100 years ago, has paved the way to "passive immunization". i.e., all kind of serum-treatments that have found their broad medical applications in prevention of e.g.•viral infections or in emergency treatments against e.g., snake venoms." :" Serum antibodies against microbes and even isolated antigens, like the diphtheria toxin. are polyclonal, meaning that they bind-in case of a specific antigen molecule-to a variety of sites or-in case of a microbe-to multiple surface structures.

The advent of the monoclonal antibody technology launched by Georg Kohler and Cesar

Milstein nearly 30 years ago, has enabled biotechnology to engineer specific antibody species that bind with high affinity to only one specific site of a given antigen and can be produced in unlimited quantities. Follow-up technologies made it possible to produce monoclonal antibodies that are more or less identical to their human counterparts, employing microbial and tissue culture resources for manufacruring." During the last decade, monoclonal antibodies have infiltrated the therapeutic arena with great success and thereby provided a plethora of novel treatments against a variety of typically devastating diseases including specific cancers, autoimmune diseases and other pathological conditions." The common denominator of all monoclonal antibodies used in therapy is to bind to highly specific sites of typically well characterized protein targets and thereby intervene with biological functions involved in the pathogenic condition; e.g., to growth hormone receptors expressed at the surface of malignant cells. 14 • l s Interestingly, monoclonal antibodies engaged in the treatment of specific cancers seem to work by a dual mode; they mark the cancerous cells for decontamination by the immune system , but also block a function that intervenes with cell growth. 16 Progress in the identification ofspecific antigens has become the hallmark in the development of novel subunit vaccines that only contain single specific structures derived from the microbe in order to induce protective immunity. The first viral subunit vaccine on the market that has become a great success is directed against Hepatitis B virus and based on recombinant protein technology. Alsopathogen-specific glycosides coupledto carrierproteins aresuccessfully usedin so-calledconjugatedvaccines directedagainstbacterialinfections; an example is"Prevnar" a registered vaccine againstPntumococcus. 17The successful development ofsubunitvaccines comprising isolatedmicrobialcomponentsas antigens has supported the notion that antibodiesper se, may suffice to neutralizepathogensin the body evenin a setting of"passive vaccination". So far only one anti-infective monoelonalantibody,whichisdirectedagainstthe Respiratory Syncytial Virus (RSV) (Palivizurnab), has entered the therapeuticarena." A number of anti-infective antibodies based on specific antigensagainstbacterialinfectionsare in the stageof clinicaland preclinical development(Table1).

Theavailability of the entiregenomesequence ofpathogensand subsequently the application ofproteome and genomebasedtechnologies havefacilitatedthe identificationofhighlyspecific protein antigens suited for the development of novel bacterial subunit vaccines," One of the recentlydescribedmethods designedto comprehensively mine bacterialgenomesfur protective antigens, has taken advantageof antibodies derived from humans who have encountered the target pathogen with positiveoutcome. The sum of all protein antigens that are recognizedby cognateantibodiesfrom individuals exposed to the pathogenhasbeendefinedasantigenome. 2 ll-22 Typicallythe antigenome comprises100 to 200 antigens. Applyinga number ofselective filters and criteria to the antigenome, in vitro validation makes it possibleto reduce the number of best-suitablecandidateantigensfor vaccinedevelopmentto about IS to 30 (unpublished data). Such antigensare presentlytested in advancedpreclinicaland earlyclinicaltrials (Kuklin et al 23 and unpublished data).The availability of bacterialprotein antigenswith promisingprofilesfor vaccinedesign,but also the identification of specific host targets,haveprovided novelgates to developmonoelonalantibodiesfor protection and treatment againstspecific infectiousdiseases.

In the followingchapter we willdiscuss the impact of discoveryand characterizationofspecific antigens on the development of novelvaccines and antibody treatments.

The capabilityof the human immune system to identify and eliminatepathogensand pathogen-infected cells is the cornerstone of immunization, the most effective strategy to prevent infectious disease. However, vaccines are still not available against major pathogens including Meningococcus serogroup B, Gonococcus, Helicobacter pylori and Shigtlla.Traditional vaccines are mainly based on inactivated or attenuated microbesor more recentlyon polysaccharides of a particular pathogen. Due to the fact that such vaccines cannot prevent numerousdiseases, or evenworse,inducesevere sideeffects,noveland definedvaccines arebeingdeveloped to overcome these limitations. Improvedvaccines are needed to combat diseases for which current vaccines are inadequate (e.g., tuberculosis) or against pathogens that had not been on the rarget list fur immunization,such asStaphylococci and Enterococci both with an enormouspotential to develop drug resistance. 24 ,2SThe recentlyemergingthreat ofbioterrorism booststhe need for newvaccines further.

Most of the newgenerationvaccines comprise subunitsofpathogens(purifiedprotein, toxoid, polysaccharide with or without conjugation) and havemademajorheadways in controllingserious diseases. At present, thereareonlytwovaccines basedon recombinantproteins(againstHepatitis B and Lyme disease) that areshownto be effective in preventinghuman infections. Nevertheless, protein based recombinantvaccines are considered to be the most promisingapproach to meet the demandsoffuture vaccinology.

In order to designnovelsubunit vaccines, the proper antigenshaveto be identifiedand subsequentlyevaluatedin experimental animal models mimickinghuman diseases. While vaccine developmentfor obligatepathogenswith well-defined virulence mechanisms has progressed well, those bacteriathat are in the focus of current vaccine efforts (e.g., opportunistic pathogensand those with multipleserorypes) havemore complexpathogenesis.

Vaccinologists are witnessinga remarkable revolution in technologies that now contribute to rapid identificationof novelvaccinecomponentsagainstmanyImportant human pathogens. --Data w ere co llec ted fro m the Ad is R&D Insight database and homepages of listed companies in July 2006 using the wo rld w ide web. Antibodies were sorted according to antigen target: first, antibo dies targeting pathogen surface structures are listed, fo llowe d by anti -tox in anti bodies and last antibodies against TNFo..

The availability of complete genome sequences of pathogens has dramatically changed the perspectives for developing improved and novel vaccines by increasing the speed of target identification. Genomics-based technologieshave many advantages compared to conventional approaches.which are time-consumingand usuallyidentify only abundant antigensexpressible under in vitro culture conditions. Strategiesbasedon genomicshavemade major contributions to the identification and selection of novel vaccine candidates to combat bacterial infections by exploiting genome sequence information in alliance with adjunct technologies. including in silico prediction (bioinformatics). expression analyses (random mutagenesis. microarrays, in vivoexpressiontechnologies). or protein/peptide based selection methods (proteomics and immune-selection usingpeptide expression libraries). Although. most technologiescan be readily applied to most pathogens. certain strategiesare more suitable than others due to distinct advantages and limitations. The most promising candidate antigens haveto be (1) expressed during human disease; (2) accessible (surfacebound or secreted) for functional antibodies or effector immune cells; (3) conservedamongstrains; (4) essential for in vivosurvival in order to avoidcounter selection;and (5) protective in animal models mimickingthe relevant human disease. There is no technology available today that can selectantigen candidatesfulfilling all five attributes.However. a comprehensiveselectionprocedure meetingthe keycriteriacan be combinedwith a validationscreening that addresses the remainingrequirements.

To date. approximately 300 pathogen genomesequences havebeen determined (http://www. tigr.orglcmr). Genome sequences of bacterialpathogens contain an average of 2700 genes. thus appropriate selectioncriteriahaveto be applied to reduce the number of antigen candidatesfor empiricaltesting.Bioinformatics has been successfully employedfor the prediction ofcandidate antigensofextracellular pathogens.due to the specific features easingthe predictionof cellsurface and secreted proteins and/or the identificat ion of genes that show sequenceand/or structural homology to known virulencefactors.26This type of genome-based systematic searchfor vaccine candidateswastermed"reverse vaccinology".The validityof thisapproachwasfirstconfirmedbythe identificationofprotectiveantigensfromMeningococcus serogroupBand laterfromPneumococcus (reviewedin ref 27) . "Reverse vaccinology in silicoprediction" typicallytargetsup to 25%ofall genome-encoded proteinsand. thus,necessitates subsequenthighthrough-putcloningand recombinant protein expression.Inclusionof more restrictive selectioncriteriabecamepossiblethrough the availability of several genomesfor individual pathogenic species. Comparative genomicsis another suitabletool to identifygenes sharedamongspecies of relatedpathogensor. alternatively. to identify genespresent only in pathogenic, but not in attenuated or narurallynonpathogenic strains or species. Such approaches havebeen successfully applied to Group A Streptococcus and Mycobacterium, [28] [29] [30] Thehallmarkofeffective vaccine antigensistheir abilityto induceantibodiesand/or to activate immune cells. Regardingthis feature, in silicoprediction of antigenicityis stillin infancy. It is anticipatedthat with the wealthof knowledge currentlybeinggenerated. it willbepossible to develop prediction algorithmsto pinpoint proteins likely to be immunogenicand/or protective." More advancedis the strategyto mine genomicsequencedatabases of intracellularpathogensfor predicted T-cellepitopesand validatethem experimentally basedon immunerecognition. 32J 3 Despite all successes. the bioinformaticgenomemining approachhas limitationsdue to the inaccuracy of available algorithms. regardingthe prediction of (1) open-readingframes that encode proteins;

(2) surfaceand secretedproteins; (3) genefunction basedon homologysearches. Moreover. it is almost impossible to predict the conditions under which candidateantigensareexpressed. unless the genesare equipped with well-definedregulatorysequences and promoters.

The availability of complete pathogen genome sequences stimulated the development and wide-spread application of high density DNA-arrays. Comparative microarray analysis identifies genomic diversityand conservationpatterns among bacteria.The developmentof vaccines cross-protective among serotypes and variantsof pathogenicspecies specifically profitsfrom this analysis, as it was demonstrated by the identification of common genes and protective antigens from major serotypes ofStreptococcus agalactiae. 34 ,35 Profiling of genomic expression with microarrays has revolutionalized the analysis of genes involved in microbial pathogenesis (reviewed in ref. 36) . Considering its value in vaccine development, the emphasis is focused on pathogen-host interactions. In several studies novel vaccine candidates were identified, based on requirement for infectious state and dissemination, adhesion or evasion of innate defense mechanisms.Fr'?This approach-that heavily relies on genome annotation and bioinformatics-is most powerful in providing a global view on integrated cellular processes active during infection. Again, it has to be followed by combined application of gene cloning, recombinant protein technology and in vitro functional assaysto validate target selection for vaccine development.

Proteome analysis has rapidly developed in the postgenome era and is now widely accepted as a complementary technology to genetic profiling (reviewed in refAI). The most direct way of using proteomics technologies for antigen identification is the combination of conventional proteome analysis with serology. There have been a number of recent studies investigating the "immunoproteome" ofirnportant human pathogens (for an example see Haas et al 42 ) . Combining "reverse genomics" and proteomics isespeciallyuseful for confirmation ofbioinformatic prediction of0 RFs and surface location. Moreover, a strong asset ofproteomic studies is the identification of surface located proteins that cannot bepredicted by bioinformatic means. 43 ,44 Serologicalproreome analysis of enriched membrane and cell wall fractions from several pathogens, such as S. aureus, Bacillus antbracis and S. agalactiae has indeed demonstrated to identify novel surface antigens and protective vaccine candidates without sequence features that could have been recognized by in silico prediction algorirhms.v"

The design ofproteome-based studies has to be carefully performed, since there is an inherent risk to preferentially detect abundant proteins and to miss those that are expressed only under in vivo conditions and have lower solubility (e.g., membrane and surface proteins). Another need, not necessarilymet by proteome analysis,is that protective vaccine components have to be derived from proteins expressed under disease conditions against which prevention is directed. As many virulence factors and antigens are only expressed in vivo, approaches that solely rely on in vitro grown bacteria are likely to miss important protective antigen s.

Evaluation of immune responses against any candidate antigen is a crucial validation task and cannot be circumvented. Therefore, techniques using human immunogenicity as their primary screening and selecting parameter on a genome-wide basis seem to be especially valuable for vaccine development. Recently a novel approach combining the advantages of full genome coverage and serological antigen identification was published. The method was first applied to the genome-wide identification of in vivo expressed antigens from S. aureusby using antibodies from human serum and comprehensive small-fragment genomic surface display libraries .P Subsequently, the technology was extended with an integrated approach for antigen validation as selected clones are directly subjected to generation ofepirope-specific immune sera for surface localization and in vitro functional assays. This feature allows the analysis of antigens without the demanding task ofhigh through-put recombinant protein production. This method, named antigenome technology, has been extended to many important human pathogens and validated by the discovery ofnovel and highly protective antigens, in addition to the identification ofthe majority of the one s that have been previously described.P Since the antigenome technology provides a subset of all genome-encoded proteins, which are expressed by the pathogen in vivo and induce antibodies in humans, the identified antigens fulfill major requirements ofvaccine candidate antigens. It is interesting to note that the antigens confined by the antigenome seem ofien to be involved , as secreted and surface bound proteins, in virulence functions and , thus , being attributed to the "parhosphere" that has been defined as the growing gene pool in which pathogens meet and mingle to cause diseases.48lt is observed that many ofthe identified antigens from various pathogens were not or only very weakly expressed under in vitro growth conditions, indicating that a proteomic approach that preferentially selects abundant proteins would likely fail to identify them. As the bioinformatic genome mining approach depends on the accuracy of available algorithms, potential vaccine candidates can be missed due to a misleading or not existing annotation. Based on the analysis of the antigenomes of fifteen pathogens. approximately 25% ofall identified antigenic proteins can only be assigned to hypothetical proteins or proteins with unknown function. Many ofthe identified antigens would, thus. be not be found by a bioinformatic approach. The cumulative data obtained fur the fifteen antigenomes showed that a large fraction ofthe antigens identified by this method represents cell surface or secreted proteins. Nearly fifty percent of all antigens fell into four cellular role categories: cell wall, cellular processes, transport and binding proteins and determinants of protein fate. In order to pinpoint candidates for vaccine development, a comprehensive and rapid validation strategy to retrieve the most promising antigens from the 100-200 antigens was implemented. Clones selected from peptide display libraries are directly subjected to generation of epitope-speci6c immune sera used fur testing of surface localization and in in vitro bactericidal assays.The human immunogenicity of identified antigens is evaluated with synthetic peptide epitopes. The application ofthese major selection criteria combined with traditional gene conservation studies reduces the antigenome to a small number ofcandidate proteins that can be rapidly expressed in recombinant form fur subsequent in vivo studies. The re-idenrification ofmost ofthe previously identified protective antigens of Staphylococci and Streptococci. such as PspA. M1 protein. Sip and ClfA gives further supports the power of the antigenome technology. Most importantly. novel protective proteins yielding animal protection in animal vaccine models. were found in the prioritized groups of antigens derived e.g., from S. aureus 23 and Streptococcus pneumoniae (unpublished data) , respectively. Thus, the utilization ofprotective antigens-included in subunit vaccines-as targets for monoclonal antibodies, provides an attractive strategy to develop novel treatments against life threatening infections. Such a notion is supported by recent data showing that protection can be conferred to naive animals, using serum directed against target antigens that have been validated in vaccine models (Nagy et al, personal communication).

The renaissance of antibody therapy since the mid-1990s was mainly possible through significant improvements in antibody generation and purification (Fig. 1) . The firststep towards nowadays production technologies was the description ofthe unlimited generation ofmonoclonal antibodies by Georges Koehler and Cesar Milstein in 1975,49for which they were awarded the Nobel Prize in 1984. They fused mou se myeloma cells with normal antibody-producing splenic B-cells isolated from mice that were immunized with sheep red blood cells as antigen. The resulting hybridoma cells possessed the immortal propagation potential of the myeloma cells and secreted anti-sheep red blood cell antibodies. Selected clones could then be cultured indefinitely and secreted large quantities ofmonoclonal antibodies.

Despite their success as research tools, mouse monoclonal antibodies as human therapeutics are limited fur various reasons. The main problem is the high immunogenicity of these foreign proteins in humans resulting in fast clearance (short halflife) and toxicity by human anti-mouse antibodies (HAMAs).50 Moreover, mouse antibodies have a reduced effect in human recipients due to their nonoptimal interactions with human complement and F, receptors,"

In the early 1980s strategies for chimerization and humanization were ensued to overcome the limitations ofmouse monoclonal antibodies. Chimerization demands the joining ofthe variable regions of mouse antibodies with the constant domains of human immunoglobulins that takes advantage ofrecombinant DNA techniques resulting in chimeric antibody derived from mouse and human antibody genes." Although being less immunogenic than murine monoclonal antibodies, human antichirneric antibody responses have even been reported for chimeric antibodies. 53 To further reduce the undesirable immune response and confined inactivation, the mouse segment within the humanized monoclonal antibodies has been restricted to the complementarity determining regions (CDR) in CDR-grafted "humanized" anribodies.Yln order to humanize a mouse monoclonal antibody, the closest matching human immunoglobulin allotype is first identified by structural comparison. 55.56 Then recombinant approaches are used to graft the CDRs from mouse hybridomas to the corresponding selected human immunoglobulin framework. As a result, the '" While routine mousemonoclonal antibody production has been established. human monoclonal antibodies cannot be generated by conventionalhybridoma technology, sinceit was not possibleto found human celllines that secreteconstantlyhigh levels of antibodiesand, furthermore. humans cannot bechallengedwith all kind ofantigens,due to ethical and safetyreasons.

Nowadays. phagedisplaytechnology(reviewed in refs. [57] [58] [59] and transgenicmicewith a human antibody locus (reviewed in ref 60) represent established, widespread and robust technologies that allowthe generationof potent human antibodies.

Phage displaytechnologies enable in a simple to use and highly versatile procedure for the selectionof antibodiesagainstknown or novelantigens.The phage displaylibrary (firstdescription by McCaffertyet al 61) represents a collectionof independent clonescarryinga foreignDNA sequence encoding an antibody domain expressed as a fusion with the coat protein of mainly filamentous bacteriophages. as M13 or Fd (reviewed in ref 62) . Monoclonal antibody libraries can be recruited from immune fragments that are already biased towards certain specificities (encoded in the genome of immunized or infected animalsor humans),or naiveunbiasedfragments that can be derivedfrom nonimmune natural or semi-synthetic sources, bypassing the need for previousimmunization. Byapplyingthe best suitableselectionprocedures, those phagesthat bind to the target antigen with highest affinityare retained.The phagesareenriched by selective adsorption to an immobilizedantigen ("panning") (reviewed in ref. 12); howevervariousspecialized screening techniques exist.57.6~5 Phage display provides the opponunity to mimic human immune response, alsobecauseof the high degreeof natural variationsfound in the replicationof the phagegenomes. 66 B-cellmaturation in vivorequiresrecombinationofgermlinegenesegments accompaniedwith changes and mutations that can be imitated in vitro by DNA random cloning ofVH and VL chain genes. 67The somatichypermutation processthat naturally contributes to the affinitymaturation of antibodiescanbe achieved artificially byinsertingpoint mutations into genesegmentsofcomplementaritydetermining regions. 68 .69 A method to circumvent the laborious steps of founding humanized and to obtain directly human monoclonal antibodies wasdevelopedbyengineeringtransgenicmicewith a human immunoglobulin locusassourcefor antibodyproducing hybridomacelllines.(reviewedin ref.60). Already in 1985Alt et al proposed to exploit transgenicmice for the generation oftherapeutic antibodies." and as soon as 1994 the Xenolvlouse" (Abgenix, Inc.FI and the HuMAb Mouse" (Gen-Pharm-Medarex}" were reported to be the first mice carryingboth the human VH and VL repertoire createdvia pronuclear microinjectionor yeastprotoplast fusion with embryonic stem cells, respectively. For monoclonal antibody generation B-cells are isolated from immunized mice and fused to hybridomas,in a similarmanner to the traditional mouse monoclonal antibody production. By employing rnicrocell-mediared chromosome transfer-a technique capable to transfer very large fractions of the human germline-Tomizuka et al generated a chimeric mouse-TransChromo Mouse" (TC Mouse™) carryinghuman chromosomes2 and 14regionscontaining the human K-light-chainand heavy-chain loci?3,74 In order to increasethe lowefficiency of hybridoma production due to instabilityof the IgKlocus,the KM MouscTM was created by cross-breeding the Kirin TC Mouse™ with the MedarexYAC-transgenic mouse." These mice possess the capability to carry out VDJ recombination. heavy-chain class switching and even somatic hypermutation of human antibody genesin a normal mode to generate high-affinityantibodies with completelyhuman sequences.f The resultingantibodies exhibit a half-lifesimilar to natural human antibodies" and show only differences in glycosylation patterns, thereby representinga major improvement in hybridoma technology." Although human monoclonal antibodies derived from transgenic mice havenot yet paved their wayup to FDA approvaland registration.so far clinicaltrialswith them havenot revealed adverse immunogenic sideevents in patients. 71l • w in contrast to chimeric,CDR graftedor phagedisplayderivedmonoclonal antibodies." However there is still a need for confirmingthese promisingdata by testing transgenic mousederivedantibodies in largersubject cohorts.

On the other side,the success of phagedisplaytechnologies in mimickingthe in vivoantibody selectionprocessin essence has led to intensive explorationof possible improvements, mainlyin the fieldof new display techniques. All these new selectionplatformssharefour major steps: (1) the creation of genotypicdiversity; (2)the linkagebetweengenotypeand phenotype; (3) the application of a screeningprocedure; and (4) the amplification of the selectedbinding sites.

In the Ribosome and mRNA display method,82 the antibody and its encoding mRNA are linkedbythe ribosomewhichismadeto stop without releasing the polypeptide. 8 lo85 Theuseofe.g., nonproof-readingpolymerases providesadditional diversitybetween generations and therefore represents a verysuccessful technique in the fieldof antibody affinitymaturation.f Theattempt in displaying antibodieson the surface ofdifferentmicrobes hasonlybeensuccessful so far, when employing the yeastSaccharomyces cerevisiae. 87 Antibodiesaredisplayed viafusion to the a-agglutinin yeastadhesionreceptoron the cellwalland selectioncan be accomplished via flowcytometriccellsorting.Besides yeast display, alatelydescribed.&cherichia coli basedapproach is currentlyunder development/"

Recently developedantibodyplatformtechnologies includeretroviraldisplay,"protein-DNA display,90 microbead displayby in vitro cornpartmenralizarion," in vivo growth selectionbased on protein fragment complementation'?and other techniques." However, their advantages over more establishedsystems remainto be demonstrated.

One problem in the applicationof monoclonal antibodies lies in their restriction to a single specific epirope,limiting their ability in eliminatingdynamicand evolving targets and retaining activityin the event of antigen mutation. A new generation of therapeutic antibodies that may overcome the restriction of monoclonal antibodies is the development of a recombinant polyor oligoclonal antibody technology," For the generation of "Syrnphobodies"-fully human, antigen-specific recombinant polyclonal antibodies-antibody producing cells are isolatedfrom naturally immune donor blood. cDNA encoding human heavyand light chains are amplified and linked together by Symplex PCRTM; pooled PCR products are then inserted into an expression vector and screenedfor antigen binding. Constructs expressing the selectedantibodies are cloned into Chinese hamsterovarycells wherethey are site-specific integratedinto the genome. " Thus, such adevelopmentof a human antibody repertoiremirrorsthe human polyclonalimmune response againstspecific antigens.

Besides the fact that the recombinant expression of antibody genesis often difficultbecause of their large size, the usage of whole immunoglobulins sometimes causes undesiredside effects that are mediated by the Fcpart of the antibody.To overcome such problemsantibodyfragments such as Fab, scFv, diabodiesand minibodieshavebeen engineeredby removing either the entire constant regionor the Fcportion.'! Advantages sharedby theseantibody fragments includetheir better clearance fromwholebody, better tissue/tumor penetration characteristics and their simple and straightforward production in bacteria bypassing mammalian cell based production. The smallestfragments are singlechain fragment variables (scFv) formed by tandem arrangementof the VH and VL domains joined by a flexible linker peptide exhibitinga comparable affinityof a Fab. 95 . 96 Their biological effects can be enhanced through linker length reduction that generates noncovalent scFv dimers "diabodies'j'" by further shortening trirners" or even tetramers can be formed. " ScFvs havealso been modified to delivertoxins and chemotherapeutics to various tumors by binding to cancer-associated antigens, e.g., by coupling the Pseudomonas aeruginosa exotoxin A to scFv. 1OO Linking of scFvs of different specificity creates bispeclfic antibodies that bind two different structures on single or different cells.'?' Other truncated antibody variants are Minibodies-homodimers of scFv-CH3 fusion proteins-and Flexminibodies-scFv-IgG 1 hinge region fusion proteins. 102 Whole antibody molecules can be modified as well by coupling with anti-microbial drugs. Antibodies possessing specificity to microbial antigens can be simultaneously linked to toxins, acting as immunotoxins that way. For example, Human Immunodeficiency Virus (HIV) and Cytomegalovirus specific antibodies have been linked to the ricin A chain or the Pseudomonas Exotoxin A.103.105 Unfortunately toxins can elicit immune responses limiting their repeated therapeutic use. An alternative represents the linking of radionuclides to specific antibodies that do not need to be internalized. liketoxinsand are unlikely to producesignificant immune responses. Radionuclide-labeled antibodieshave been testedagainst Cryptococcus neoformans and pneumococcal infections in mice. I 06.107 Anotherdevelopment in modifyingthe antibodymolecule wasthe creationofbispecific antibodiescarrying two different Fabfragments and recognizing a microbialepitope for pathogen binding and at the same time a host immune component.This strategywasshown to be successful in animalmodels for the clearance ofbacteriophages'P and p.aeruguinosa. I 09

The applicationof humanizedand evenfullyhuman antibodies-is associated with low toxicity and high specificity. The benefit of high specificity is that only disease-causing pathogens are targeted and therefore the host flora should not be altered or resistant microorganisms be selected. A caveat is that pathogens with high antigenic variation may require more than one monoclonalantibody for therapyand mutants lacking the antibodydeterminant could emerge during treatment. Antibody molecules are highlyversatile; by binding to a single determinant theycanmediatevarious biological effects includingtoxinneutralization. microbial opsonization, complementactivation and antibody-directed cellular cytotoxicity (Fig. 2) . Antibodiescan also be usedto targethost cells and enhanceimmunefunctionsespecially desirable againstinfectious diseases and tumors or to suppress immuneresponses by reducing the number of immunecells, neutralizingcytokines or blockingreceptors. The major disadvantages of antibody based therapies are high costs associated with production, storage and administration. Since antibodies have to be produced in live expression systems, the risk of contamination with prions or viruses requires continuous monitoring and testing. Additionally, antibodies have to be administered shortly after infection to be efficient, requiring rapid microbiological diagnosis . Additionally manufacturing ofSymphobodies, mimicking polyclonal antibodies in human immune response , may still have to prove that they can be obtained without chance in their composition under stable GMP conditions.

In the late 19th century Behring and Kisato discovered the efficacyofimmune sera in treating infectious diseases, such as diphtheria and tetanus yo In 1891 the Klemperers already protected rabbits against S.pneumoniae with immune sera showing the potential usefulness of passively administered antibodies for the treatment of pneumococcal infections.'!' However reliable anti-pneumococcal therapy was not availableuntil the mid 1920s, since the development ofsuccessful serum therapy required the discovery that pneumococci are genetically diverse and only typespecific sera provide protection. Improved vaccination schedules for serum donors to generate good immune responses and advanced antibody purification techniques, as well as the standardization ofserum potency were necessary steps in the introduction ofserum therapy (reviewed in ref. 112 ) . The high death rate associated with meningococcal meningitis lead to fast developments also in this sector; a significant reduction of the case fatality rates was already achieved with horse sera in the early 20th century. ll2·m

Serum therapy reached its heyday in the 1920s to the mid 1930swhen it was standard clinical practice in the treatment ofa variety ofinfectious diseases caused by S. pneumoniae, C.dipbteriae, Neisseria meningitidis, Haemopbilus influenzae, Streptococcuspyogenesand Clostridium tetani.The broad application ofserum as treatment for pneumococcal disease can be estimated regarding advertisements ofthat time in medical journals (Fig. 3) . However, with the advent ofanti-microbial chemotherapy passive immunization with serum was largely abandoned for the treatment of bacterial infections due to major advantages in being less toxic, more effective and cheaper. Serum th erapy was often associated with severe side effects including fever, chills and allergic reaction s and delayed toxicity called "serum sickness" a syndrome associated with rash, proteinuria and arthralgia. Moreover, for satisfying efficacyprecise diagnosis , appropriate and nondelayed dosage was necessary asking for physicians with considerable experience . The production of horse or rabbit therapeutic sera was very expensive because of the need for animal facilities, purification techniques, adequate storage and standardization. Nevertheless lot-to-lor variation could not be fully eliminated (reviewed in ref. 114) .

Upon the arrival ofthe antibiotic era, anti-sera were still used for toxin -mediated disease such as botulism, tetanus and diphtheria in addition to anti -toxin therapy in the treatment ofvenomous snake bites.I I The lack ofefficient anti -viral treatments also stimulated the use ofantibody preparations as postexposure prophylaxis in e.g., rabies or hepatitis B (reviewed in ref. 9 ).

In spite ofthe previously experienced shortcomings, long-time neglected antibody based therapies face a renaissance today. The description ofhybridoma technology in 1975 49 fired researcher's imagination in developing new therapies against cancer, autoimmune or infectious diseases. As early as at the dawn ofthe 20th century Paul Ehrlich already dreamed about the use ofantibodies as "magic bullet" for the treatment of cancer. Indeed, in the mid' 1980sth e first efficient use of a monoclonal antibody for the treatment of refractory lymphoma was reported. II S The anti-tumor effect was only temporary, since murine monoclonal antibodies have only short in vivo halflife and are immunogenic in humans; moreover the y don 't kill target cellsforcefully due to low efficiency in complement activation and antibody dependent cell cytotoxicity. The first FDA approved murine monoclonal antibody for clinical use was OKT3 targeting CD3 in 1986 and was designed for prevention and treatment oforgan rejection .' !"

Fortunately, monoclonal antibody techniques underwent continuous and tremendous improv ements in reducing the mouse derived portion ofthe protein and enabling the production of . . In spite of the incredibleeffortsundertaken to developnovelantibody basedtreatmentswith hundredsof monoclonalantibodiesbeingcurrentlyunder preclinical developmentor clinicaltesting,onlythe minorityof theseeffortsaredirectedagainstinfectious targets.Amongviralinfections, AIDS is far the most exploredarea(Table2). Due to the extremevariabilityof neutralizingHIV epitopes,in addition to those combatingthe virusparticleitself,117.118 manymonoclonalantibody approaches target host molecules (such as CTLA-4, CD4, LFA-1 , CCRS) to hinder viralentry (reviewed ref 119) . Emergingviral infections causedby the SARS corona virus and West Nile Virusalsoattracted the attention of monoclonalantibodydevelopers and several preclinical efforts are expectedto enter clinicaldevelopment (Table2).

Due to the widespread appearance of multi-drug resistant bacterial pathogens and the increasing population of immuno-compromisedpatients, more and more efforts are focusedon antibody-basedstrategies againstpathogenic bacteriaand fungi. Especially considerable effortswere and arestillundertakento treat septicshockcausedbygram-negative bacteriavianeutralizationof endotoxin and of TNF-a induced earlyin the disease, unfortunatelywith no successful outcome sofary o.123The mostfrequentmicrobial targetsof newdevelopments areopportunisticnosocomial pathogens, suchasS.aureus and epidermidis, P. aeruginos« and Candidaspecies (Table 1and Table   3 ).The moleculartargetsfor thesemonoclonalantibodiesaresurface structuresof thesepathogens, includingcapsular polysaccharides, cellwallglycolipids and surface proteins.Theprimaryaimisto increase opsonophagocytic eliminationof the respective organisms with the help of the host'simmunecells. Unfortunately, in immune-compromisedpatients (under anti-tumor treatment,organ transplantation, old age), the number of effective phagocytic cells is significantly lower than in healthypeopleand relying onlyon opsonophagpcytosis maynot besufficient for cure.Monoclonal antibodies that target surface proteins and that also have essential functions in in vivo survival, multiplication(celldivision, nutrient acquisition) and pathomechanisms (adhesion, cytoroxiciciry, immuneevasion), offeranother opportunity to reducebacterialgrowth and ameliorateinfectious damageto the host. 124 . 127 A single chain anti-fungalantibody that wasselectedby the hsp90protein of Candida albicans from antibody cDNA librariesof patients who recovered from invasive candidiasis isbeingdeveloped(Mycograb"). It consists of the antigen-bindingvariable domainsof antibody heavyand light chainslinked together to a recombinant protein that is expressed in E. coli. Mycograb" is not dependent on recruitment of white blood cells or complement,but simply acts by binding and inhibiting hsp90 of Candida.!" Current fearof bioterrorismusingbiological weapons encourages the development of antibody therapiesagainst anthrax, botulism, ebola or smallpox virus infections and aims to provide immediate immune protection through antibodies that either neutralize the pathogensand toxins themselves,or targetthe host byblockingcorrespondingreceptorsto preventinfectionor toxicity. Recently Cohen and coworkers demonstrated the inhibition of the lethal effectof anthrax toxin viablockingof its human coreceptor, LRP6 with LRP6 specific antibodies.F'

In spite of the historicallandmark therapyagainstdiphtheria, antibody therapyagainstbacterial infections,only exceptionally, has entered the medical arena in the last 70 years. The advent of antibioticsduring the fortieshascertainlydiscouraged the developmentof further serumtreatments againstbacterialpathogens.

Antibiotics haveseemingly become a relatively cheep and mostlyreliable weapon to control most bacterial infections and epidemics. Alongside with the increase of hygienic standards, the penetrations of mandatory childhood vaccinations and antibiotic treatment, bacterialinfections seemed to be a medical problem confined only to less developed parts of the world. The costefficient availability, the seemingly evergrowing pipelineof novel antibiotics with increasingefficacy Table 2 . continued on next page ..

Data were collected from the Adis R&D Insight database and homepages of listed companies in July 2006 using the world wide web. Data were grouped into:

first, antibodies targeting the virus particle itself and then second, those targeting host structures. Monoclonal Antibodies are abbreviated with mAbs. This is not a complete list. invadingthe market has establishedthe attitude in the medicalcommunityup into the 1970sof the last century that bacterialdiseases maybelong to the past. However. the emergingpattern of rnuleidrug-resisrant strains of an increasing number of pathogens in hospitals and communities has quicklyended the optimisticbeliefthat the repertoireof anti-bacterial treatmentswill suffice the challenges in the infectiousdisease arena (for reviewseeref 130) . Also the discovery, development and registrationof novelantibioticshavenot fulfilled the too optimistic expectationsthat new registrations of treatments maybounceoff the threat ofuntreatablebacterialinfections (see commentariesby Clarke!" and in Blocenrury'Pj.The infiltrarionofgenomics.P' intelligentdrug designand molecularstudiesofbacterialhost interactionsin antibioticdevclopmenr' " has rather ledto the soberingrecognitionthat the numberof suitabletargetsfor newanti-bacterial drugsmay be ratherlimited. 135 • 137 Furthermore. the oftensevere sideaffects, includingallergic reactions against specific antibiotics.isrestrictingtheir applications, sometimes in criticalmedicalconditionswhen they are most needed. Last, not least antibioticsoften lead to lysis of bacterialcellsand thereby freeingendotoxins at high levels, therebycausingovershootingimmunity includingsepsis. 138 On the other hand before the advent of the monoclonal antibody technology. treatments of bacterialdiseases with antibodieshavebeenout of the reachof economicalfeasibility. Production of antibodies by immunizinganimalsas resorts to obtain serum is not a trivialprocessregarding quality, reproducibilityand unwanted contaminations. Also as one has experienced with whole cellbacterialvaccines. immunization with in vitro grown pathogens maynot lead to the type of specific antibodies that neutralize them, since they may not display the proper antigens at the surface. Thus,the progress madein definingdisease specific antigensfor vaccinedevelopmenthas provided noveltools to raisehighlyspecific antibodiesthat mayprevent or block bacterialinfections or at leastsupporting the recovery process.

Theskepticism in the medicaland scientificcommunitytowardsthe paradigmof anti-infective anti-bacterialmonoclonalantibodiesis nurtured by multiple linesof thoughts:

1. Existingtreatments are sufficient to control bacterialdiseases. 2. Monoclonal antibody therapymaynot find its wayinto treatment schedules that would justify the costs.

A singlemonoclonalantibody directed against a specific antigen per se maynot be able to counteract the pathogeniccourseof a bacterialinfection. While all three argumentsare widdy accepted, a closerlook into the paradigmdiscloses that they are not necessarily substantiated. if one considers the medical need, the progressmade in identificationofsuitableantigenictargets and the positiveexperience of usingmonoclonal antibodies againste.g., malignantdiseases (reviewed in ref 139) .

The medical need is given, wheneverconventionaltreatment and prophylaxis are not available. S. aureusin context with nosocomialinfections is equallya target for antibody treatments asPneumococcus, both representing problem germsin intensivecare (Table4).

Moreover, costsfor antibodytreatmentsin connectionwith abovedescribedinfectiousdisease outbreaksoften missingadequatemedicaltreatmentsappearto be not too dramatic,if one relates them to the hospital conditions and the underpinning economical efforts spent. Last but not least, the increasingly wide usageof monoclonalantibodiesoutside of the infectionsdisease area has certainlyaided in loweringthe costsof developmentand manufacturing,therebypavingthe wayto noveltreatments.! Thequestion remainswhat kind of features form the prerequisites for a monoclonalantibody in order to be able to counteract a bacterialinfection?The answerto this problem lies-to our opinion-in the sdection of the bestsuitableantigenictargetsfor the developmentofmonoclonal antibodies. Theantigensshouldbeexpressed on the pathogensurface duringthe infectiousprocess; preferredthroughout the mostimportant stages ofdisease manifestation: i.e., duringcolonization, spreading and invasion. Also the antigens of choice should have a proven record to be a target of antibodies from individuals who haveencountered the pathogen with positiveor protective outcome. In addition, the selectedantigensshould be conservedamong all clinicalstrains of the germ causingthe underpinning infections. All up to here listed features of target antigens may suffice the need to detect the intruder with a monoclonal antibody. particularly ifthe bound antibody funnels the bacterium into the immunological decontamination program, e.g.• opsonization. On the other hand, the lesson learnt from antibiotics is that they have to kill the pathogen or at least disable bacterial growth in the host. In the light of the notion that prevention or treatment ofa bacterial infection with monoclonal antibodies may be restricted to a single antibody. one would aim the target antigen also to exert a function needed for bacterial survival in the host . Thus, the antibody will neutralize a virulence factor or an enzyme needed in the infections life cycle of the pathogen. Such a dual mode of action resembles the features of monoclonal antibodies employed in cancers therapy: these antibodies seem to block cancerous cells by marking them for the immunological destruction, but also by blocking their growth. Thus. monoclonal antibodies need to bedirected against carefully selected antigenic targets in order to achieve an optimum ofinterference with bacterial survival in the host.

The recently invented antigen identification procedure that is designed to establish the "antigenorne" of pathogens has been instrumental in the development of novel bacterial subunit vaccines. 2()' 22 Characterization ofas. aureus antigen derived from the antigenome-that is presently used in preclinical and clinical programs-has indeed revealed its involvement in virulence and survival function. 23,128.14O The feasibility of antigens to serve as targets for monoclonal antibody treatments can be pretested in vaccine models where protection of pathogen-challenged animals is accessed,t06, 107.109.124.129.141.152 There is no doubt in mind that antigens giving the wanted protection in a vaccine model may not be sufficient when employed for the development ofanti-infective antibodies. However. the potency of an antigen in providing protective immunity as vaccine may be a positive and sufficient selective criterion, alongside with all the other features that have been described for antigens selected for subunit vaccine development.

Immunotherapeutics for nosocomial infections

Mechanisms of anti-microbial resistance in bacteria

Antimicrobial use and anti-microbial resistance: a population perspective

The growing burden of tuberculosis: global trends and interactions with the HIV epidemic

Passive antibody administration (immediat e immunity) as a specific defense against biological weapons

Anthrax: a continuing concern in the era of bioterrorism

Rabies reexamined

Passive antibody therapy for infectious diseases

Passive immunity in prevention and treatment of infectious diseases

Effectiveness of Snake Antivenom : Species and Regional Venom Variation and Its Clinical Impact

Poisonous snakebite treatment in the United States

Antibody engineering for the development of therapeutic antibodies

Potent antibody therapeutics by design

Epidermal growth factor receptor inhibitors in cancer treatment

The EGF receptor system as a target for anti-rumor therapy

Monoclonal antibody therapy of cancer

Immunogen icity of heptavalent pneumococcal conjugate vaccine in infants

Prevention of respiratory syncytial virus infections : indications for the use of palivizumab and update on the use of RSV-IGIV

Bacterial genomes pave the way to novel vaccines

Antigenome technology : a novel approach for the selection of bacterial vaccine candidate antigens

Pfeifer U et at Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens

Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model

The progressive intercontinental spread of methicill in-resistant Staphylococcus aureus

Resistant enterococci: a prospective study of prevalence, incidence and factors associated with colonization in a university hospital

Bioinformatics: how it is being used to identify bacterial vaccine candidates

Two years into reverse vaccinology

Identification of new candidate vaccine antigens made by Streptococcus pyogenes: purification and characterization of 16 putative extracellular lipoproteins

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains

Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics

Analysis of known bacterial protein vaccine antigens reveals biased physical properties and amino acid composition

Imrnuno-informatics: Mining genomes for vaccine components

Towards in silic o prediction of immunogenic epiropes

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococ cus agalactiae

Identification of a universal Group B streptococ cus vaccine by multiple genome screen

Microarray expression profiling : capturing a genome-wide portrait of the rranscriprome

Host-induced epidemic spread of the cholera bacterium

Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes

Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic cond itions found in phagosomes

Gene expression profile in Neisseria meningitidis and Neisseria lactamica upon host-cell contact : from basic research to vaccine development

Proreomics and regulomics: the yin and yang of functional genomics

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

Surface analyses and immune reactivities of major cell wallassociated proteins of group a strepto coccus

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

Vytvytska 0, NagyE, Bluggel Metal. Identification ofvaccine candidate antigens of Staphylococcus aureus by serological proteome analysis

Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupledwith proteomic identification of surface-associated antigens

Identification of major outer surface proteins of Streptococcus agalactiae

Pathosphere, New Scientist2005

Continuous cultures of fused cellssecretingantibody of predefinedspecificity

Human anti-mouseantibodies

Differences in promiscuity for antibody-FeRn interactions across species: implications for therapeutic antibodies

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains

Review article: the clinical role of anti-TNFalpha antibody treatment in Crohns disease

Replacing the complementarity-determining regions in a human antibody with those from a mouse

Designofhumanizcdantibodies: from anti-Tac to Zenapar

Use of human germline genes in a CDR homology-based approach to antibody humanization

Antibody phage display technologyand its applications

Overview of antibody phage-display technologyand its applications

Selecting and screening recombinant antibody libraries

Human antibodies from transgenic animals

Phage antibodies: filamentous phage displaying antibody variable domains

Antibody phage display technologies with special reference to angiogenesis

Clonal selection and amplification of phagedisplayed antibodiesby linking antigen recognition and phage replication

Strategies for selection of antibodies by phagedisplay

Selectively infective phages (SIP)

Makingantibodiesby phage display technology

Molecular evolutionof proteinson filamentous phage. Mimickingthe strategy of the immune syst em

Human antibodies with subnanomolaraffinity against a markerof angiogenesis eluted from a two-dimensional gel

Improving antibody affinity by mimicking somatic hypermutation in vitro

Immunoglobulin genesin transgenic mice

Antigen-specific human monoclonal antibodies from mice engineeredwith human Ig heavy and light chain YACs

Antigen-specific human antibodies from mice comprising four distinct genetic modifications

Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies

Production of human monoclonal and polyclonal antibodies in TransChromo animals

Class switching in human immunoglobulin transgenic mice

Fully human anri-interleukin-B monoclonal antibodies : potential therapeutic s for the treatment of inflammatory disease states

Preclinical and clinical evaluations of ABX-EGF. a fully human anti-epidermal growth factor receptor antibody

A single-dose placebo-controlled study of AMG 162. a fully human monoclonal antibod y to RANKL. in postmenopausal women

Cancer regression and autoimmun ity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma

Immunogenicity of therapeutic monoclonal antibodies

In vitro protein evolution by ribosome display and mRNA display

An in vitro polysome display system for identifying ligands from very large peptide libraries

ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites

In vitro selection and evolution of functional proteins by using ribosome display

Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display

Yeast surface display for screening combinatorial polypeptide libraries

Anchored periplasmic expression. a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries

Selection of functional human ant ibodies from retroviral display libraries

Covalent antibody display-an in vitro antibody-DNA librar y selection system

Microbead display by in vitro compartmentalisation : selection for binding using flow cyro metry

Fast selection of antibodie s without antigen purification : adaptation of the protein fragment complementation assay to select antigen-antibody pairs

Cell growth selection system to detect extracellular and transmembrane protein interactions

Recombinant polyclonal antibodies : therapeutic antibody technologies come full circle

Single chain antibody variable regions

Recombinant anti-sialidase single-chainvariable fragment antibody. Char acterization. formation of dimer and higher-molecular-massmultimers and the solution of the crystal structure of the single-chain variable fragmenr/sialidase complex

Diabodies": small bivalent and bispecific antibody fragments

Single-chain Fv fragments of ant i-neuraminidase antibody NCI0 containing five and ten-residue linkers form dimers and with zero-residue linker a trimer

Dt-, tri-and tetrarneric single chain Fv antibody fragments against human CDI9: effect of valency on cell binding

A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective anti-tumor activity

Kontermann RE.Bispecific single-chain diabody-mediared killing of endoglin-posirive endothelialcdls by cytotoxic T lymphocytes

Minibody: A novel engineered anti-carcinoembryonic antigen antibodyfragment (single-chainFv-CH3)whichexhibits rapid. high-level targeting of xenogrsfis

Selective cytotoxicity of ricin A chain irnrnunotoxins towards murine cytomegalovirus-infected cells

Human immunodeficiency virus-infected T cells and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain

Increased affinity and stability of an anti-HIV-l envelope irnmunotoxin by structure-based mutagenesis

Evaluation of acute hematologic and long-term pulmonary toxicities of radioimmunotherapy of Cryptococcus neofonnansinfectionin murinemodels

Feasibility of radioimmunotherapy of experimental pneumococcal infection

Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulatepathogen in a monkey model

Targetingof Pseudomonas aeruginosa in the bloodstream with bispecific monoclonal antibodies

Ueberdas zustandekommen der diphterie-immunirar und der tetanus-immunitat bei thieren

Versuche ueberimmunisirung und heilungbei der pneumokokkeninfection

Serum therapy revisited: animal models of infection and development of passive antibody therapy

Serumtreatmentof epidemic cerebro-spinal meningitis

Antibody-based therapies for emerging infectious diseases

Serotherapy of a patient with a monoclonal antibodydirected against a human lymphoma-associated antigen

Muromonab CD3: a reappraisal of its pharmacology and use as prophylaxis of solid organ transplant rejection

Therapeutic potential of neutralizing antibodies in the treatment of HIV-l infection

Neutralizing antibodies take a swipe at HIV in vivo

Recent advances in understanding the molecular mechanisms of HIV-I entry and fusion: revisiting current targets and considering new options for therapeutic intervention

Anti-endotoxin monoclonal antibodies

Why irnmunomodulatory therapies have not worked in sepsis

Anti-rumornecrosis factor therapy in sepsis: updateon clinical trialsand lessons learned

Novel therapies for sepsis: antiendotoxin therapies

Defining antibody targets in Streptococcus oralis infection

Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections

Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens

Fungal heat-shock proteins in humandisease

Recombinant antibodies: a naturalpartner in combinatorial anti-fungal therapy

The LDL receptor-related protein LRP6 mediate s internalization and lethality of anthrax toxin

Drug companies snub antibiotics as pipeline threatens to run dr

Use of genomics to select anti-bacterial targets

Can virulence factors be viable anti-bacterial targets ?

Robust Salmonella metabolism limits possibilities for new anti-microbials

The challenge of multidrug resistance: actual strategies in the development of novel antibacterials

Exploiting genomics, genetics and chemistry to combat antibiotic resistance

Antibiotic induced endotoxin release and clinical sepsis: a review

Overview of monoclonal antibodies in cancer therapy : present and promise

Passageof heme-iron across the envelope of Staphylococcus aureus

A human IgM monoclonal antibody prolongs survival of mice with lethal cryptococcosi s

Human monoclonal antibodies to Pseudomonas aeruginosa alginate that protect against infection by both mucoid and nonmucoid strains

Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa Ilagellin for treating infections in a murine burn wound model

Harnmer-Munz 0 er al. Antibodies raised against N '-terminal Pseudomonas aeruginosa Ilagellin prevent mortality in letha l murine models of infection

Characterization of two distinct opson ic and protective epiropes within the alpha C protein of the group B Streptococcus

Mouse-protective antibodies against the Ibc proteins of group B streptococci

Cloned alpha and beta C vprotein antigens of group B streptococci elicit protective immunity

Protection from group B streptococcal infection in neonatal mice by maternal immunization with recombinant Sip protein

Active and passive intranasal immunizations with streptococcal surface protein C5a peptidase prevent infection of murine nasal mucosa-associated lymphoid tissue, a functional homologue of human tonsils

Cutting edge: th e spirochetemia of murine relapsing fever is cleared by complement-independent bacter icidal antibodies

Passive tran sfer of protection against Bacillus anthraci s infection in a murine model

Passiveprotection by polyclonal ant ibodies against Bacillus anthracis infection in guinea pigs

CCR5 and HIV infection

Human immunoglobulin G recognizing fibrinogen-binding surface proteins is protective against both Staphylococcus aureus and Staphylococcus epidermidis infections in vivo