Patent Application: US-93643304-A

Abstract:
immunoglobulin light chains are produced in excess in animals compared to heavy chains . the present invention implicates ig - lc in hypersensitivity responses and provides ways for manipulating the responses . the invention further provides a common gamma chain - independent receptor on mast cells capable of mediating the mentioned effects of ig - lc . in response to activation of the pathway of which the found receptor is a part , a mast cell is activated and stimulated to degranulate .

Description:
in one aspect , the invention provides evidence for a novel role for free ig - lc . we show that ig - lc transfer hapten sensitivity into naive animals and subsequent antigen challenge elicits an immediate hypersensitivity - like response , which appears to be mast cell dependent . mast cells have been implicated in a wide variety of biological responses including immediate hypersensitivity reactions , bacterial sepsis and also in t - cell - dependent reactions such as contact hypersensitivity reactions , experimental autoimmune encephalomyelitis ( eae ), or non - allergic asthma . the diseases like contact sensitivity , eae and non - allergic asthma can be induced by local delayed - type hypersensitivity ( dth ) reactions and are independent of ige or igg1 . studies in mast cell - deficient animals show that mast cells are crucial in orchestrating a full dth response . humoral factors released by b - cells seem important in contact hypersensitivity reactions since hypersensitivity is impaired in b - cell - deficient animals . in ongoing studies , we have found that tnp - specific ig - lc are capable of rescuing impaired contact hypersensitivity reactions as a result of hapten application in b - cell - deficient mice . in addition , passive sensitization with ig - lc can give rise to a rapid and profound airway bronchoconstriction in mice after intra - airway antigen challenge , a reaction dependent on mast cell activation . importantly , ig - lc are involved in delayed - type hypersensitivity reactions leading to bronchoconstriction , cellular influx in bronchoalveolar lavage , and airway hyper reactivity after active sensitization with low - molecular weight compounds followed by intranasal challenge ( manuscript in preparation ). it is of interest that the secretion of ig - lc is augmented under pathological conditions such as multiple sclerosis , sjögren &# 39 ; s disease , systemic lupus erythematosus , and other neurological disorders ( 18 - 21 ). for example , production of ig - lc in patients with multiple sclerosis is greatly enhanced ( 20 , 22 ). this production of ig - lc is associated with recent antigenic stimulation and correlates with severity of the disease . in concord with our hypothesis , it has been demonstrated that mast cells play an important role in the pathogenesis of ms or eae ; substantially reduced disease symptoms are found in mast cell - deficient animals . from our studies , it is clear that mast cells are crucial for the development of acute responses in skin and airways leading to ear swelling and acute bronchoconstriction , respectively . thus far , triggering the high - affinity ige receptor ( fcεri ) and the low - affinity igg - receptor ( fcγriii ) are the only routes known to activate mast cells in an antigen - specific manner . we investigated whether ig - lc exerted their action via activation of the fcγriii or fcεri receptors . both receptors signal via the common gamma chain and can trigger hypersensitivity reactions via activation of mast cells . passive sensitization of animals deficient in the common gamma chain ( fcrγ −/−) resulted in similar ear swelling responses after hapten challenge as compared to wild - type ( c57bl / 6 ) animals ( fig1 ). this indicates that ig - lc interacts with a receptor that does not need the common gamma chain for signaling and thereby excludes fcεri and fcγriii but also other gamma chain - associated receptors such as pir - a and - b as effector molecules in ig - lc - induced hypersensitivity reactions . preliminary experiments showed that ig - lc bind to mast cells and recognize antigen in rosetting assays with hapten - conjugated red blood cells . facs analysis indicated that both ige and igg1 do not compete with the binding of ig - lc to murine bone marrow - derived mast cells , confirming our results in the fcrγ −/− mice . various attempts to directly activate murine bone marrow - derived mast cells in vitro to release prestored mediators after cross - linking of surface - bound ig - lc were not successful . although in some experiments a significant degree of degranulation was found , results were highly variable . the latter may be explained by a possible lack of co - stimulatory factor in vitro , a maturation - dependent expression of cell surface proteins involved in ig - lc - mediated activation , or the simultaneous presence of inhibitory and activator receptors . chemical cross - linking of ligand to cellular membrane was the method employed to isolate and identify cell surface proteins as putative receptors for various ligands . magnetic beads were coupled to ig - lc . these beads were then incubated with murine bone marrow - derived mast cells and after washing the cells , ig - lc ( bait ) were chemically cross - linked to cell surface proteins in immediate proximity to the binding site . after lysing the cells , ig - lc cross - linked cell surface proteins were separated from non - bound proteins using a magnetic device . proteins were washed and separated using sds - page ( 1 - d or 2 - d ), followed by silver staining . proteins were further characterized and identified with maldi - tof mass spectrometry and / or edman degradation . binding of ig - lc - conjugated magnetic beads coupled to mast cells were visualized by light microscopy . binding was specific for light chain , since no binding was detected when beads were conjugated to bovine serum albumin ( bsa ) ( fig2 a - 2d and 19 ). expression cloning was used to clone an ig - lc receptor . a cdna library from primary cultured murine mast cells ( bmmc ) was constructed . this cdna was transfected into mammalian cells . transfected cells were compared in their binding capacity for ig - lc using a flow cytometer . cells binding ig - lc above background were collected by the facs sorter . transfected dna from these cells were isolated and used for succeeding transfection rounds . after several transfection / sorting rounds , a single ig - lc receptor - expressing cell population was isolated . the transfected dna from this population encodes for the putative ig - lc receptor . a receptor present on mast cells of specific interest for binding ig - lc is cd 63 , a transmembrane - 5 ( tm5 ) membrane protein . this receptor is expressed by , for example , mast cells , granulocytes and leucocytes and cross - linking of this receptor results in mast cell activation and mediator release . facs experiments showed the presence of cd63 on mast cell line rbl - 2h3 , but not on cos cells ( fig3 ). cd63 seems widely expressed on different mammalian cells . in mast cell studies , cd63 has been used as an activation marker , since expression is increased after degranulation . on the other hand , a study in human cd63 - transfected rat basophilic cells showed cross - linking of cd63 results in mast cell activation and degranulation . thus far , no ligand for cd63 has been described yet ( i . e ., orphan receptor ). in our current studies , we have shown that ig light chain - mediated hypersensitivity responses can be effectively blocked by a 9 - mer peptide ( ahwsghccl ). this peptide binds free light chains and thus prevents binding of the light chain to its receptor . it is , therefore , likely that the prominent binding sequence of this peptide , which is determined by the ccl part of the peptide , is present in the receptor for this ligand . sequence analysis revealed that a sequence ccl or cci is conserved in tm4 proteins such as cd63 . importantly , this sequence is present in an extracellular loop of the protein and , therefore , a putative docking site for receptor ligands . cd63 is the most likely candidate of this tm4 family because of its known expression in mast cells and its demonstrated capability to activate mast cells . the involvement of mast cells in eliciting ear swelling responses after topical challenge of ig - lc - sensitized mice was investigated using mast cell - deficient mice ( w / wv ). as shown in fig4 , passive sensitization of mast cell - deficient mice with tnp - specific ig - lc followed by topical application of tnp did not result in a significant ear swelling at two hours after challenge . as expected , similarly treated control mast cell - sufficient littermates (+/+) showed normal ear swelling responses . to further prove that the absence of mast cells alone was responsible for the completely reduced ear swelling response in the mast cell - deficient mice , we reconstituted these animals with bone marrow - derived mast cells from wild - type animals . reestablishment of the mast cell population by local injection of bone marrow - derived mast cells restored the ear swelling responses in ig - lc - sensitized and hapten - challenged animals ( fig4 ). indeed , hapten challenge of ig - lc - sensitized animals was accompanied with a rapid increase in plasma histamine levels in vivo and direct proof for mast cell activation was obtained after histological and ultrastructural analysis of biopsies of the ears at one hour after topical hapten challenge . mast cells in tissue sections of mice intravenously sensitized with tnp - specific ig - lc showed marked signs of degranulation after re - exposure to hapten . electron microscopy revealed that degranulation was characterized by swelling of intracytoplasmic granules , decrease of electron - density of the granules and extrusion of membrane - free granules from the mast cells ( fig5 ). in general , it is acknowledged that both heavy chains and light chains contribute to binding of antigens by immunoglobulins , which are monomers or multimers of a tetrameric structure consisting of two light chains and two heavy chains . the structure of an ig - lc can be separated in a constant and variable region . the latter region contains hypervariable domains ( cdrs ) that are responsible for antigen recognition . the genes encoding these regions undergo rearrangement / mutation to effect affinity maturation and gain specificity for a certain antigen . similar mechanisms play a role in shaping the antigen recognition by ig heavy chains . in order to prove that free ig - lc have the capability to recognize and bind to antigen , we have performed the following experiments . first , two ig - lcs with different antigen specificity were generated by separation of immunoglobulin heavy and light chains from oxazolone - and tnp - specific iggs . naive mice were sensitized with the isolated ig - lc and subsequently topically challenged with tnp or oxazolone . two hours after challenge , ear thickness was measured . as shown in fig6 , when mice were sensitized with oxazolone - specific ig - lc , only an increase in ear thickness was measured when the ears were challenged with oxazolone and not after application of picryl chloride , i . e ., tnp . vice versa , when animals were sensitized with tnp - specific ig - lc , ear swelling was only induced by tnp and not by oxazolone . in a second experiment , we investigated if ig - lc , when bound to mast cells , is able to recognize antigen in a proper way . in in vitro experiments , bone marrow - derived mast cells were sensitized with tnp - specific ig - lc . next , sensitized cells were co - incubated with either unlabeled , tnp - labeled or oxazolone - labeled sheep red blood cells ( srbc ). binding ( resetting ) of the srbc was scored under a light microscope . as evidenced in fig7 , tnp - specific ig - lc - sensitized mast cells only show significant binding to tnp - labeled srbc , but not to unlabeled or with an unrelated hapten ( ox )- coupled srbc . this experiment confirms that ig - lc are indeed able to specifically recognize antigen . induction of immediate hypersensitivity by ig - lc may be physiologically relevant . ig - lc are produced and secreted upon antigen exposure . to test this hypothesis , spleen and lymph node cells from mice that had been sensitized epicutaneously to pcl , dnfb or oxazolone four days before , were cultured in vitro for one day in the absence of hapten . hapten - binding proteins from culture supernatant were isolated by hapten - affinity chromatography . western analysis showed that the hapten - binding factors obtained this way contained ig kappa light chain ( fig8 ), but no ig heavy chains were detected ( data not shown ). importantly , tnp - binding factor could be isolated from culture supernatant of spleen and lymph node cells isolated from mice as early as one day after topical sensitization with pcl . in this preparation , the presence of ig - lc was confirmed by western blot analysis ( data not shown ). the n - terminal amino acid sequence of this protein showed almost complete homology with mouse ig kappa light chain . free ig - lc can be detected in various human body fluids , e . g ., liquor , urine and serum . using an ig - lc - specific elisa , we were able to detect significant levels of ig - lc in serum ( fig9 ). the detected levels are comparable to those reported earlier by other groups . f991 is developed as an antagonist of ig light chain . the working hypothesis for this compound is that it binds to ig light chains and thereby prevents binding of light chains to their putative receptors . f991 is a 9 - mer peptide derived from the endogenous protein uromodulin . the potency of f991 to inhibit ig - lc - induced cutaneous reactions ( ear swelling responses ) was studied . our working hypothesis for this compound is that it is an antagonist of ig - lc and binds to ig - lc and thereby prevents binding of ig - lc to their putative receptors . f991 was administered in different dosages , via various administration routes . indicated amounts of f991 per mouse were intravenously injected at 30 minutes before hapten challenge . at that same time point , mice were injected ( passively sensitized ) with tnp - specific ig - lc or vehicle ( pbs ). two hours after hapten challenge , increase in ear thickness was monitored . fig1 shows a clear dose - dependent inhibition of the ear swelling response by f991 . amounts of 2 μg per mouse ( 1 . 9 nmole !!) were sufficient to completely block the ig - lc - induced ear swelling ( fig1 ). mice were injected with 50 μg of f991 intraperitoneally at four or 24 hours before challenge with hapten . thirty minutes before challenge , the animals were passively ( i . v .) sensitized with tnp - specific ig - lc and two hours after hapten application onto the ears , ear thickness was measured . as demonstrated in fig1 , i . p . administration of f991 , even at 24 hours before hapten challenge , completely prevented the induction of an ear swelling response ( fig1 ). protocol ( see above ) for i . p . administration , except f991 , was injected subcutaneously . similar to the i . p . administration of f991 when injected subcutaneously , f991 again completely inhibited ig - lc - induced ear swelling after hapten challenge ( fig1 ). a . f991 was prepared as an ointment in cremor cetomacrogolis fna and topically applied on the ear at four hours before hapten challenge . subsequently , mice were passively sensitized with 2 μg tnp - specific ig - lc at 30 minutes before and ear thickness was measured two hours after hapten challenge of the ears . as shown in fig1 , topical application of f991 in an ointment resulted in a dose - dependent inhibition of the ear swelling induced after hapten challenge of ig - lc - sensitized animals ( fig1 ). b . we further investigated if local application of f991 as an ointment resulted in systemic inhibition of the ig - lc - induced effects . therefore , f991 was applied on the back of the mice instead of at the site of hapten challenge ( ear ). mice were again sensitized and challenged as described above . fig1 shows that topical application of f991 does not result in systemic inhibition of the ig - lc effects . this means that epicutaneous treatment should be done at the site of challenge ( fig1 ). to determine the stability of f991 in ointment , different preparations of f991 in cremor cetamacrogolis fna were tested for their activity after storage for two to three months at room temperature and at 4 ° c . the different preparations were applied at the ear as described in a ( above ) and subsequently , ear swelling was monitored after hapten challenge of ig - lc - sensitized animals . as shown in fig1 , f991 stored under all different conditions retained its activity ( fig1 ). d . next , we investigated if topical treatment with f991 also inhibited the development of contact sensitivity reactions induced after active sensitization . mice were sensitized with a low - molecular weight compound dnfb on the skin and footpads ( on day 0 and 1 ). five days after the start of sensitization and four hours before local challenge with hapten , mice were treated with f991 on the ears . two and 24 hours after hapten challenge , the increase in ear thickness was determined . as shown in fig1 , topical treatment with f991 completely inhibited the ear swelling at both two and 24 hours after challenge . this experiment indicates that topical treatment with f991 may be of therapeutic use in the treatment of contact dermatitis , a disease with similar characteristics ( fig1 ). e . next , we investigated if topical treatment with f991 also inhibited or decreased the development of contact sensitivity reactions induced after active sensitization . mice were sensitized with a low - molecular weight compound dnfb on the skin and footpads ( on day 0 and 1 ). five days after the start of sensitization and two hours after local challenge with hapten , mice were treated with f991 on the ears . ear swelling was measured 22 hours after application of the cream . subsequently , mice were treated daily with the f991 cream , two hours after measuring of the ear swelling . control mice were treated with the vehicle cream without f991 . two , 24 , 48 , 72 and 96 hours after hapten challenge , the increase in ear thickness was determined . as shown in fig1 , topical treatment with f991 diminished the ear swelling at all time - points after two hours after challenge . this experiment indicates that topical treatment with f991 may be of therapeutic use in the treatment of contact dermatitis , a disease with similar characteristics . it is well documented that production of ig - lc in patients with multiple sclerosis is greatly enhanced . elevated levels of free ig - lc can be detected in cerebrospinal fluid and urine of ms patients . this production of free ig - lc is associated with recent antigenic stimulation and correlates with the severity of the disease . in concord with our hypothesis , it has been demonstrated that mast cells play an important role in the pathogenesis of ms ; substantially reduced disease symptoms are found in mast cell - deficient animals ( 23 ). further , mast cells are observed in cns plaques , and histamine and tryptase levels are elevated in the liquor of ms patients , whereas treatment with mast cell stabilizers or antagonists of histamine and serotonin seem to ameliorate ms . if light chains were involved in the activation of mast cells in patients with ms , the prediction is that f991 may be of therapeutic interest in the treatment of ms . we tested to see if f991 was able to prevent or reduce development of clinical signs in an established mouse model for ms , i . e ., myelin oligodendrocyte glycoprotein ( mog )- induced experimental allergic encephalomyelitis ( eae ). mice were sensitized with antigenic peptide 35 - 55 from mog in complete freund adjuvant . at the day of sensitization and three days later , mice also received an injection with pertussis toxin . in general , ten to twelve days after the start of sensitization , mice developed clinical signs of ms , which can be scored in degree of paralysis ( 0 = no paralysis , 1 = tail flaccidity , 2 = hind limb weakness , 3 = hind limb paralysis , 4 = forelimb paralysis or loss of ability to right supine , 5 = death ). to investigate if f991 is able to prevent development of clinical signs of ms , mice were daily injected with 50 μg f991 / animal i . p . starting at day − 1 until day 21 . as shown in fig1 , treatment with f991 completely prevented the development of clinical signs of paralysis . control mice developed disease with a mean day of onset of 11 . 8 and a mean clinical score of 2 . 3 . there was a significant difference in disease burden ( mean cumulative eae score ) between the f991 - treated group ( mean 4 . 2 ) and the control group ( mean 18 . 3 ). termination of the treatment with f991 at 21 days after sensitization , resulted in the development of some minor clinical symptoms (“ silly walk ”), but not in a clear manifestation of eae / ms ( data not shown ) ( fig1 ). a rt - pcr reaction was performed to detect fcrn mrna in mouse bone marrow - derived mast cells ( bmmc ) and pulmonary mast cells ( pmc ). trizol reagent ( gibco ) was used to isolate total rna from four - week - old bmmcs and pmcs and cultured k562 erythroleukemia cells . the rna isolations and subsequent amplification steps from bmmcs and pmcs were performed as duplicates . first strand cdna was synthesized from 1 . 6 μg total rna by using superscript reverse transcriptase ( invitrogen ) and an oligo dt primer . pcr reactions were done with 1 μl and 3 μl of first strand cdna per 50 μl reaction volume . the forward and reverse primers for the pcr reactions had the following dna sequences , respectively : cctgctgggctgtgaactgg ( seq id no : 2 ) and gctccggdgggtagaaggag ( seq id no : 3 ). using these primers , a dna fragment of 347 basepairs ( bp ) was expected to be amplified from both mouse and human sequences . dna sequences were run on 1 . 5 % agarose gels and visualized by ethidium bromide staining . the main amplified dna fragment that was obtained after doing the rt - pcr reaction on the rna isolated from bmmcs , pmcs , k562 cells , and a control plasmid that contained cloned human fcrn cdna as an insert , had the expected size of approximately 347 bp ( fig2 ). the results indicate that mrna encoding fcrn is present in pmcs as well as in the bmmcs , although the concentration in bmmcs is a little higher . in k562 cells , even more amplification product was obtained , suggesting that these cells express relatively high levels of fcrn . it should be noted that these experiments are not conclusive about the mrna expression levels , since no household mrna level has been determined to ensure that equal amounts of cdna have been added to each pcr reaction . however , the experiments confirm the presence of fcrn mrna in mouse bmmcs and pmcs . from literature , it is known that expression of fcrn in cells does not guarantee the presence of the protein on the outside of the cell . the co - expression of β - 2 - microglobulin is required to transport fcrn to the plasma membrane . the lack of an antibody against mouse fcrn kept us from confirming the presence of fcrn on the outside of the bmmcs . immunofluorescence microscopy has been used to show that fcrn is present intracellularly , as well as on the outside of the plasma membrane of k562 cells . flow - cytometric analysis shows that k562 cells are able to bind human kappa ig - lc ( fig2 ). the presence of fcrn on the outside of the cell in combination with the ability to bind ig - lc is still in agreement with the proposed role of fcrn as the lc - r , although it does not prove this . flow cytometry was also used to show binding of ig - lc to freshly isolated human neutrophils ( pmns ) ( fig2 ). this binding was enhanced after stimulation of pmns by pma or fmlp . it is known from literature that most fcrn is present in intracellular vesicles , which fuse with the plasma membrane after stimulation . this finding again matches with the proposed role of fcrn as the lc - r , but does not prove it . an elisa was used to study a possible interaction between human fcrn ( hfcrn ) and human κ - ig - lc . two different types of human κ - ig - lcs ( κ1 and κ3 ), a λ - ig - lc , and a mixture of iggs were chemically linked to biotin . the igs were dissolved in pbs at a concentration of 1 mg / ml . the ig - lcs were incubated in the presence of 426 μg / ml ez - link sulfo - nhs - lc - biotin ( pierce ) at room temperature in the dark for 90 minutes . the reaction of the iggs was performed with 220 μg / ml biotinylation agent for 30 minutes . the labeled igs were dialyzed against pbs for 12 hours . an elisa plate was coated with 100 ng / ml recombinant soluble hfcrn ( a kind gift of p . bjorkman ) in pbs for 15 hours at 4 ° c . blocking of the plate was done with 1 % bsa in pbs / 0 . 5 % tween - 20 for 60 minutes at room temperature , followed by three washes with washing buffer ( pbs containing 0 . 05 % tween - 20 ). the plate was incubated with various amounts of biotinylated igs in assay buffer ( washing buffer with 0 . 1 % bsa ) for 60 minutes at room temperature . after three washes , the plate was incubated with streptavidin poly - hrp ( 100 ng / ml in assay buffer ) for 30 minutes , followed by six washes . the plate was assayed with o - phenylenediamine according to the protocol of the manufacturer . several elisa experiments showed a significant binding of ig - lcs and igg to the hfcrn . an example is shown in fig2 . no binding was seen when the hfcrn was omitted from the coating reaction , suggesting that the interaction is specific ( not shown ). the amount of signal differs between the igs , but this is not conclusive since the biotinylation efficiency of each sample has not been determined . however , there are some differences in the shape of the binding curves , with igg and κ3 reaching a plateau at lower protein concentrations than κ1 and λ . it should be noted that , despite the observed specificity , there were no differences in binding between incubations at ph 7 . 4 or 6 . 5 ( not shown ). this contradicts findings in literature , where several groups independently showed binding of igg at ph 6 . 5 , but not at ph 7 . 4 . in the described elisa setup , a significant binding of ig - lcs to hfcrn was shown . however , further research using another experimental method will be required to confirm and validate this observation . clinical safety study of a phase 1 single , rising dose , double - blind , placebo - controlled study with f991 in healthy male volunteers primary objective : to study the safety and tolerability of f991 at increasing single dose levels in healthy male volunteers secondary objective : to study the pharmacokinetics of f991 in healthy male volunteers tertiary objective : to study the pharmacodynamics of f991 in healthy male volunteers ( exploratory ) design : this study was a double - blind , placebo - controlled , single rising dose study in two alternating panels of eight healthy male volunteers . in each panel , two volunteers received placebo throughout the study and the other six volunteers received three increasing single doses of f991 . screening and follow - up : clinical laboratory , full physical examination , ecg ; at eligibility screening : medical history , drug screen including alcohol , hbsag , anti - hcv and anti - hiv 1 / 2 observation period : each period in clinic from − 17 hours prior to drug administration up to 24 hours after drug administration panel i period 1 : pre - dose and at 5 , 10 , 15 , 20 , 30 and 45 minutes and 1 , 1 . 5 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 16 and 24 hours post - dose panel i period 2 and panel ii periods 1 and 2 : pre - dose and at 15 , 30 , 35 , 40 and 50 minutes and 1 , 1 . 5 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 16 and 24 hours post - dose panel i period 3 : pre - dose and at 15 , 30 , 45 , 50 , 55 and 65 minutes and 1 . 5 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 16 and 24 hours post - dose panel ii period 3 : pre - dose and at 15 , 30 , 55 , 60 and 65 minutes and 1 . 5 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 16 and 24 hours post - dose for pharmacodynamics : tryptase and ig - lc ( immunoglobulin light chains ) pre - dose and one hour and eight hours post - dose safety assessments : vital signs ; adverse events ( including eyesight ); ecg four times each period ; clinical laboratory : on admission for each period weight : within ± 15 % deviation from normal range , from 60 to 100 kg , inclusive strength : freeze - dried material ( 8 . 4 mg ) to be reconstituted with 10 ml water / 0 . 9 % nacl : 0 . 84 mg f991 - peptide / ml , 10 mm citrate ph 5 , 2 . 25 % mannitol , 0 . 45 % nacl the following treatments were administered , as a single dose in alternating panels , according to the randomization schedule . panel i : period 1 : 0 . 08 mg / kg f991 or placebo iv infusion period 2 : 0 . 8 mg / kg f991 or placebo iv infusion period 3 : 3 . 2 mg / kg f991 or placebo iv infusion panel ii : period 1 : 0 . 32 mg / kg f991 or placebo iv infusion period 2 : 1 . 6 mg / kg f991 or placebo iv infusion period 3 : 6 . 4 mg / kg f991 or placebo iv infusion safety : adverse events , clinical laboratory test results , ecg recordings , vital signs and physical examination pharmacokinetics : pharmacokinetic parameters derived from f991 plasma concentration - time data are : c max , t max , k el , t 1 / 2 , auc last , auc 0 - inf , cl and v d no correlation was observed between the dose of f991 and the number , frequency and intensity of aes . there were no saes during the study . all possibly related aes were of mild intensity , except one , which was a headache of moderate intensity that lasted about 14 . 5 hours and required treatment with a single dose of 500 mg paracetamol . the most frequently reported ae with a possible relationship to the study drug was headache , however , this occurred mainly in the placebo group . for volunteers on active drug , the most frequently reported aes were eye disorders . with regard to clinical laboratory data , vital signs , ecg and physical examination , no clinically significant abnormalities were observed . i . v . administration of single rising doses up to 6 . 4 mg / kg of f991 was safe and well tolerated in healthy male volunteers . the effect of f91 1 on allergen reaction to house dust mite in mice next , we investigated if topical treatment with f991 also inhibited the development of contact sensitivity reactions induced after passive sensitization with house dust mite allergen der p 2 - specific ig - lc . mice were sensitized with a der p 2 - specific ig - lc in the ear skin . sixteen hours after sensitization and four hours before systemic challenge with house dust mite extract or recombinant der p 2 , mice were treated with f991 on the ears . sixty minutes after allergen challenge , the thickness of the ear was determined . as shown in fig2 and 25 , topical treatment with f991 completely inhibited the ear swelling 60 minutes after challenge . this experiment shows that topical treatment with f991 may be of therapeutic use in the treatment of , for example , cutaneous anaphylaxia and contact dermatitis . 1 . van loveren , h ., r . e . ratzlaff , k . kato , r . meade , t . a . ferguson , g . m . iverson , c . a . janeway , and p . w . askenase . immune serum from mice contact - sensitized with picryl chloride contains an antigen - specific t - cell factor that transfers immediate cutaneous reactivity . eur . j . immunol . 16 ( 10 ): 1203 - 8 ( 1986 ). 2 . hopper , j . e ., and e . papagiannes . evidence by radioimmunoassay that mitogen - activated human blood mononuclear cells secrete significant amounts of light chain ig unassociated with heavy chain . cell immunol 101 ( 1 ): 122 - 31 ( 1986 ). 3 . shapiro , a . l ., m . d . scharff , j . v . maizel , and j . w . uhr . synthesis of excess light chains of gamma globulin by rabbit lymph node cells . nature 211 ( 46 ): 243 - 5 . 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