Vol. 55, No. 8INFECTION AND IMMUNITY, Aug. 1987, p. 1867-18720019-9567/87/081867-06$02.00/0Copyright © 1987, American Society for Microbiology

Characterization of Murine Monoclonal Antibodies That RecognizeNeutralizing Epitopes on Human C5a

JAMES W. LARRICK,l* JEFF WANG,' BRIAN M. FENDLY,' DENNIS E. CHENOWETH,2STEVEN L. KUNKEL,3 AND TRACY DEINHART'

Cetus Immune Research Labs, Palo Alto, California 943031; RLT-02, Round Lake, Illinois 600732; and Department ofPathology, University of Michigan School of Medicine, Ann Arbor, Michigan 481093

Received 9 December 1986/Accepted 24 April 1987

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complementfragment C5a. These MAbs were characterized for their ability to immunoprecipitate '251-labeled C5a, bindC5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes.Four of these MAbs had affinity constants for C5a in the 1 x 109 to 3 x 109 M-' range. These MAbs blockedC5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively adminis-tered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeuticuse in states of complement activation.

C5a is a 74-amino-acid glycoprotein with a molecularweight of 11,300, which is generated as a cleavage product ofcomplement protein C5 in both the classical and alternativepathways of complement activation (10, 17). C5a possessesanaphylatoxic, chemotactic, contractile, and permeability-enhancing activities. A specific C5a receptor has been iden-tified on human polymorphonuclear leukocytes (PMNs) anda chemotactic response of these granulocytes to purifiedhuman C5a has been observed at concentrations of 0.4 to 17nm (3-5, 32). Binding of C5a to this receptor is thought to bethe causal event mediating complement-induced granulocyteaggregation (14) and superoxide production (7, 8). Suchaggregation in vivo has been postulated as a mechanism oftissue damage in such clinical conditions as pulmonarydysfunction and leukostasis in hemodialysis patients, suddenblindness with retinal infarction after trauma or pancreatitis,myocardial infarction, postpump syndrome after cardiopul-monary bypass surgery, systemic lupus erythematosis, burninjury, lung injury, and the adult respiratory distress syn-drome (ARDS) (2, 26, 29). In vivo studies of mice geneticallydeficient in C5 have established that C5a is required forpulmonary edema formation in response to pneumococcalsepsis, scald, wounds, and hypoxia (12, 16, 25). Detection ofelevated levels of C5a has been used to predict the onset ofARDS in patients at high risk who have been followedprospectively (15). Recent work from Stevens et al. (28)suggests that rabbit anti-C5a antibodies can attenuate ARDSand sepsis in nonhuman primates.We generated a panel of murine anti-C5a antibodies to

investigate the potential role of CSa in clinical syndromes ofcomplement activation. These antibodies bind to des-ArgC5a with high affinity, immunoprecipitate 125I-labeled C5a,block binding of CSa to granulocytes, block C5a-inducedPMN polarization, and protect rabbits from neutropeniainduced by systemic infusion of human C5a. The potentialtherapeutic use of these reagents is discussed below.

* Corresponding author.

MATERIALS AND METHODS

MAb production. BALB/c mice were immunized intraperi-toneally with 3 to 10 ,ug of purified C5a in complete Freundadjuvant. The human C5a antigen was affinity purified aspreviously described (19). The mice were boosted withimmunogen in incomplete Freund adjuvant and then inphosphate-buffered saline (PBS) at 2-week intervals. Micewere boosted intravenously (i.v.) with 5 Rg of CSa 3 daysprior to fusion. Monoclonal antibodies (MAbs) were gener-ated by standard methods (24). Spleen cells were fused to themouse myeloma parent SP2/OAgl4 (27) at a ratio of 5:1, andhybridomas were selected in hypoxanthine-azaserine selec-tion media (11). Positive wells were cloned by limitingdilution.C5a enzyme-linked immunosorbent assay. Immulon 2 96-

well flat-bottom plates were coated with 100 ,ul of 200 ng ofhuman C5a per ml in 0.5 M bicarbonate buffer for 1 h at 37°C.Antigen-coated plates were washed three times with washbuffer (PBS plus 0.1 g of MgCl2 per liter, 0.2 g of CaCI2 perliter, 0.05% Tween 20, 0.01% thimerosal) with an automatedplate washer. Wells were blocked with 100 ,ul of dilutionbuffer (wash buffer plus 1% bovine serum albumin). Testsupernatant (100 ,ul) or purified antibody was added to eachwell and incubated for 30 min at room temperature. Wellswere washed three times as described above, and then 100 ,ulof peroxidase-conjugated rabbit anti-mouse immunoglobulinG (IgG) or IgM (Zymed, San Francisco, Calif.) diluted1:4,000 or 1:1,000 in dilution buffer was added to each well.Wells were incubated for 30 min at room temperature andwashed five times. The peroxidase substrate (200 pI), 2-azino-di(ethylbenzthiazoline sulfonic acid) in citrate bufferwith 1:103 30% H202, was added for 30 min at 37°C in thedark. Plates were read on a Multiscan plate reader at 405 nm(Flow Laboratories, Inc., McLean, Va.).

Multiwell immunoprecipitation. Wells of a 96-well polyvi-nyl chloride microtiter plate (Dynatech Laboratories, Inc.,Alexandria, Va.) were blocked with 1% bovine serum albu-min in PBS (BPBS) (pH 7.2) for 60 min at 37°C. The plateswere then flicked, and 50 pu1 of MAb supernatant or purifiedMAb was added to the wells. A 100,000-cpm sample of

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251I-labeled C5a (50 pIl) was then added to each well, and themixture was incubated for 60 min at room temperature. Afterthis incubation, 50 p.1 of a 1:5 dilution of rabbit anti-mouseimmunobeads (Bio-Rad Laboratories, Richmond, Calif.)was added to each well and incubated for 60 min at roomtemperature with agitation. The immunobeads were thenwashed three times with PBS plus 0.1% Tween 20, and theplates were cut. The individual wells were counted on agamma counter (LKB Instruments, Inc., Wallac, Finland).

After being counted, the immunobeads were suspended in30 p.1 of sample buffer (0.06 M Tris [pH 7.0], 5% 2-mer-captoethanol, 2% sodium dodecyl sulfate, 1.5% glycerol).This suspension was boiled for 3 min, and the supernatantwas then analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis according to the stacking-gel pro-cedure of Laemmli (18). The gels were dried and exposed toCronex X-ray film (Du Pont Co., Wilmington, Del.) at -70°Cwith intensifying screens (1).MAb purification. Pristane (tetramethylpentadecane; Al-

drich Chemical Co., Inc., Milwaukee, Wis.)-primed BALB/cmice were injected intraperitoneally with 5 x 106 clonedhybridoma cells. Ascites fluid was collected 14 to 28 dayslater, centrifuged, and stored at -76°C until needed. Ascitesfluid was filtered and applied to an AffiGel Protein A Mono-clonal Antibody Purification System (Bio-Rad) according tothe instructions of the manufacturer. Active fractions werecollected and dialyzed against PBS. Fractions were thenfiltered through an Acrodisc filter (0.2-pm pore size; GelmanSciences, Inc., Ann Arbor, Mich.) and stored in the samebuffer at 4°C.

Determination of MAb binding affinities for C5a. Dilutionsof the MAbs giving 75% of maximum C5a immunoprecipita-tion (see Table 2) were determined. Various concentrationsof cold CSa (31 ng/ml to 1 p.g/ml) diluted in 200 p.1 of PBS and5% bovine serum albumin were incubated with the 200 p.l ofMAbs for 2 h. All procedures were carried out at roomtemperature. A sample of 105 cpm of 125I-labeled CSa (100 p.l)was added for 1 h, followed by 100 p.1 of rabbit anti-mouseIgG immunobeads for an additional hour. The beads werewashed with PBS and transferred to an LKB gammacounter. A 50% inhibition of binding was determined, andthe affinity constant was calculated by the method of Muller(20).

Isotype determination. A MonoAb-ID enzyme immunoas-say kit (Zymed) was used according to the instructions of themanufacturer for isotype determination. Briefly, a 96-wellImmulon 1 plate was coated with 100 pL. (200 ng/ml) of C5aand then blocked with 1% BPBS (pH 7.2). Then 50 p.1 ofculture supernatant was added to the wells and incubated for60 min at room temperature. Plates were washed, and 50 p.1of each subclass-specific rabbit anti-mouse IgG antibody wasadded to separate wells. After the plates were incubated for60 min at room temperature and washed four times, 50 p.l ofperoxidase-conjugated goat anti-rabbit IgG was added toeach well and incubated for 60 min at room temperature.Plates were washed four times, and 0.1 ml of substratesolution was added to each well and incubated for 30 min at37°C. Results were read with a Multiscan spectrophotome-ter.PMN polarization. Polarization of human PMNs was per-

formed essentially as described previously (6), with minormodifications. PMNs were isolated from fresh heparinizedblood by dextran sedimentation followed by Ficoll-Hypaquegradient centrifugation. The PMNs were adjusted to 1.25 x106 cells per ml of Hanks balanced salt solution (HBSS), and0.9 ml was added to each duplicate polypropylene tube (12

by 75 mm) containing 0.1 ml of stimulant or HBSS. Thesamples were incubated for 10 min in a 37°C shaking waterbath. To end the reaction, 1 ml of ice-cold 0.1 M phosphate-buffered formaldehyde (10%, vol/vol [pH 7.2]) was added toeach tube. Cells were kept cold (4°C) until they wereexamined by phase-contrast microscopy. Tubes were codedand read blindly. The percentage of cells polarized (i.e.,elongated morphology) was determined by counting 200 cellsper tube.Measurement of PMN chemiluminescence. PMNs were

isolated from heparinized blood on Mono-Poly resolvingmedium (Flow). Cells were washed twice in HBSS afterhypotonic lysis of erythrocytes. Cells were incubated in 5-mlscintillation vials at 106/ml in BPBS and 25 p.M lucigenin(Sigma Chemical Co., St. Louis, Mo.). Various stimuli orinhibitors were added, and counts per minute were recordedin an LKB scintillation counter equipped for chemilumi-nescence.Measurement of binding in molar excess of C5. Polyvinyl

chloride plates (96 well; v-bottom) were blocked for 1 h withBPBS at 37°C. To obtain CSa-containing complement-activated sera, various serum samples were incubated with 5mg of zymosan per ml. The mixture was stirred and incu-bated for 30 min at 37°C and then spun in an Eppendorfcentrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.)for 3 min. The pellet was discarded, and the activated serawere removed to a new tube. A total of 50 p.l of the sera wasused neat for blocking each test sample. For tests usingnormal nonactivated sera, 50 p.1 of neat sera (from the samesource as that used for activation) was used to block.For immunoprecipitation, 50 p.1 of the MAb supernatant or

purified MAb was diluted in 1% BPBS to give about 75% ofmaximum binding. The 50 p. of the neat sera (one each of theactivated or nonactivated sera) was added, and incubationwas carried out at room temperature for 1 h. lodinated CSa(50 p.; 105 cpm) in 1% BPBS was added. This solution wasincubated for 1 h at room temperature.

Rabbit anti-mouse IgG immunobeads (1:5 stock beads inPBS) was added, and the mixture was agitated for 1 h atroom temperature. The plates were spun for 2 min at 2,000rpm to bring down the complex. The supernatant wasaspirated with an eight-pronged manifold and washed threetimes with a solution of PBS plus 0.1% Tween 20. The wellswere cut into tubes, and the pellets were read on an LKBgamma counter. The percentage of immunoprecipitation wasdetermined by dividing counts bound by input counts times100%.MAb 260-114G, with no serum, was used as a positive

control. The negative control was PBS or an isotype controlMAb [IgGl(K)]. An anti-tumor necrosis factor MAb, ananti-Pseudomonas lipopolysaccharide MAb, or an anti-ricintoxin A MAb was used for this purpose.For the MAb designated 260-114G, a graph of percent

input counts bound as a function of serum dilution (1 perdilution) indicated that there was substantial binding of theMAb to CSa, which decreased as the MAb supernatant wasdiluted from 1:2 to 1:128. The binding of the anti-tumornecrosis factor control MAb was negligible.

In vivo studies. Purified anti-CSa MAbs were injected i.v.into an ear vein of 1-kg rabbits. A blood sample was takenfrom each rabbit at time zero (before CSa or the MAb wasinjected into each rabbit). The MAb (4 mg) was then injectedi.v. A second blood sample was withdrawn after 1 h, and 2.5p.g of CSa was injected i.v. in the contralateral ear. Bloodsamples were taken at 1 and 5 min post-CSa injection. Eachblood sample was analyzed for PMNs and total leukocytes.

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TABLE 1. Anti-C5a MAbs and CSa ELISA titers"

MAb Ascites C5aELISA titer"

269-10F7.... 1:100,000260-114G.... 1:65,000269-3E5.... 1:500,000269-9H3.... 1:2,000,000260-91H.... 1:328,000260-11G5.... 1:100,000269-12F8.... 1:500,000261-9F11..... 1:200,000261-174D.... 1:40,000260-7C9.... 1:100,000Control'.... 1:10

" All MAbs were isotype IgGl(K). ELISA. Enzyme-linked immunosorbentassay.

Scale 1.0; 50 ng per well.Anti-tumor necrosis factor MAb.

The differential between total leukocytes and the PMNs (%PMN) was calculated.

RESULTS

Characterization of anti-C5a MAbs. We generated a panelof 10 murine anti-human C5a MAbs (Table 1). The C5a-specific enzyme-linked immunosorbent assay binding titersof ascites ranged from 1/40,000 to 2 x 106. All of these MAbsare of the IgGl(K) isotype. The panel of MAbs was alsotested for the ability to immunoprecipitate soluble radioiodi-nated des-Arg C5a. The percent of input counts immunopre-cipitated by 10 ,ug of purified MAbs per ml and the concen-tration of MAbs giving 75% of maximal immunoprecipitationare shown in Table 2. The affinity constants of the panel ofMAbs for soluble C5a ranged from 2.0 x 108 to 3.8 x 109M-1 (Table 3).

Specificity of MAbs for human C5a. The specificity ofbinding of the MAbs for C5a versus C5 was determined inthree ways. The panel of MAbs did not immunoprecipitateiodinated human C5 (Table 4). In addition, Ouchterlonyimmunodiffusion analysis demonstrated a precipitation linebetween the MAb and human C5a, while human C5 was notimmunoprecipitated (Fig. 1). Finally, the relative inhibitionof C5a-specific immunoprecipitation by normal human se-rum and zymosan-activated human sera containing C5a wasmeasured. The zymosan-activated sera provided much more

TABLE 2. Plate immunoprecipitations of 251I-labeled CSa byanti-CSa MAbs"

Concn (,ug/mi )Maximum cpmMAb bound giving 75%

(% of input) maximumimmunoprecipitation

269-10F7 40 0.55260-114G 14 0.94269-3E5 26 0.5269-9H3 38 0.5260-91H 20 0.5260-11G5 14.5 0.94269-12F8 26 0.5261-9F11 20 0.94260-174D 16 1.9260-7C9 16 0.94

a These studies were performed as described in Materials and Methods byusing affinity-purified MAbs. An input of 100,000 cpm of 125I-labeled C5a wasused for each immunoprecipitation.

TABLE 3. Affinity constants of anti-CSa MAbs

AffinityMAb constant

(M ')

269-10F7..... 3.8 x 109260-114G..... 2.5 x 109269-3E5....... 1.1 x 109260-llG5....... 1.1 x 109260-91H..... 6.3 x 108260-7C9..... 5.8 x 108269-9H3..... 2.9 x 108269-12F8..... 2.6 x 108261-9F11..... 2.5 x 108261-174D..... 2.0 x 108

effective inhibition (Table 5). (Zymosan-activated serum forother species [dog, rabbit, rhesus monkey, pig, and guineapig] did not inhibit specific human CSa immunoprecipitation[data not shown].)

Neutralization of C5a binding in vitro. A subset of theoriginal 10 MAbs was selected for functional characteriza-tion. The quantity of antibody giving a 50% inhibition ofI251-labeled C5a binding to purified granulocytes is shown inTable 6. The highest-affinity MAbs, 269-10F7, inhibited at 25ng/ml.

Neutralization of CSa-induced chemiluminescence. HumanC5a produced a rapid transient increase in PMN chemilu-minescence. Chemiluminescence of PMNs stimulated byC5a incubated with various MAbs is shown in Fig. 2. Anonbinding isotype control MAb did not diminish the C5a-induced signal. Each of the five MAbs tested blocked theability of C5a to stimulate PMN chemiluminescence. TheMAb with the highest affinity for C5a, 269-10F7, demon-strated the best inhibition of C5a-induced chemiluminesc-ence.

Neutralization of C5a-induced PMN polarization. Mem-brane binding of C5a causes PMNs to undergo a dramaticchange in shape called polarization. This phenomenon isdose dependent. At a concentration of C5a of 5 ng/ml, 50%of the cells became polarized. MAb 269-10F7 demonstrateda near 100% inhibition of C5a-induced PMN polarization(Table 7). Four other antibodies with lower affinities showedless inhibition (10 to 50%).

Neutralization of CSa activity in vivo. C5a administered torabbits produces a rapid, although transient, fall in circulat-ing PMNs (21, 33). We used this method to demonstrate in

TABLE 4. Anti-C5a MAb cross-reactivity: immunoprecipitationwith t25"'labeled C5 and I-C5a

cpm bound (% of input)MAb" '5-I-C5 'I51-C5a

260-114G 1.6 25.0260-91H 1.5 25.8260-7C9 1.0 26.3260-11G5 1.7 23.9261-9F11 1.0 20.1261-174D 1.1 18.1269-12F8 0.8 26.2269-10F7 1.3 36.3269-9H3 1.0 22.5269-3E5 1.3 26.8L113b 1.6 1.0

a MAb concentration was 1.25 ,ug/ml. An input of 100,000 cpm of 1251_labeled C5a was used for each immunoprecipitation.bL113 is a control antipseudomonad MA6 [IgGl(K)].

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TABLE 6. Inhibition of '21I-labeled C5a binding to PMN byanti-C5a MAbs

Concn (ng/ml) ofMAb giving 50%MAb inhibition of 1251_

labeled C5abinding to PMN

269-10F7 ...................................... 25260-llG5...................................... 75269-3E5 ....................................... 63261-9F11....................................... 250269-12F8....... . 2 ,ug/ml

FIG. 1. Ouchterlony immunodiffusion using anti-C5a MAb269-10F7. Wells: A, human C5a; B, human C5a des-Arg; C, MAb;D, human CS. Note the lines of identity with human C5a and humanC5a des-Arg. No reaction with human CS is seen.

vivo neutralization of C5a with the panel of C5a MAbs(Table 8). Nine of the MAbs administered prior to C5ablocked the C5a-induced neutropenia as compared withnonbinding control MAb.

DISCUSSIONThis report describes the generation of a panel of murine

MAbs that bind to and neutralize the in vitro and in vivoeffects of complement fragment C5a. They were generatedby a screening procedure we call plate immunoprecipitation.This technique, which uses 1251-labeled antigen, selectsMAbs that recognize the C5a in a native, soluble form. Inprevious studies, we have found that antigen binding toplastic could alter its antigenicity; i.e., some MAbs that bindsolid-phase antigen do not bind soluble antigen (J. W. Lar-rick, unpublished data). Among the panel of MAbs we

generated are several with a high affinity for C5a. Four ofthese (269-10F7, 260-114G, 269-3E5, and 260-11G5) haveaffinities of 109 M-1 or better.

Several pieces of information suggest that the highestaffinity MAb, 269-10F7, blocks the receptor binding of C5a.It blocked 1251I-labeled C5a binding to granulocytes, blockedC5a-induced PMN polarization, and blocked C5a-inducedPMN chemiluminescence. The lower-affinity MAbs had lessability to neutralize these in vitro effects of C5a.In vivo passively administered C5a is known to provoke arapid, although transient, neutropenia (13, 22). It has beenpostulated that the continued production of this molecule

610

(PM

TABLE 5. Inhibition of binding of 125I-labeled human C5a to anti-human C5a MAb by zymosan-activated human sera'

MAb % Increasein inhibitionh269-10F7 ........................................... 44260-114G........................................... 44269-3E5 ............................................ 39260-1llGS ........................................... . 5 1260-91H............................................ 63260-7C9 ............................................ 65269-9H3 ............................................ 41269-12F8 ........................................... 47261-9Fll ........................................... . 27261-174D ........................................... . 40

a The antibody concentration used was the amount that precipitated 75% ofthe maximum possible precipitable counts (Table 2).b The percent increase in inhibition (I) of binding of 125I-labeled human C5awith the anti-human C5a MAbs when using zymosan-activated sera (ZS)compared with normal sera (NS): % increase in I = [(% I with ZS) - (% I withNS)]/(% I with NS). A nonbinding MAb of the IgGl(K) isotype did not bindC5a in the presence of zymosan-activated sera or normal sera.

105

non specific MABCSa alone3E59F1111G5

,- 91H- 10F7

- cells alone

l0

1 2 3

TIME

14min

FIG. 2. MAbs blocked C5a-induced granulocyte chemilumines-cence. C5a was used at a final concentration of 10 ng/ml. MAbs weremixed with C5a 45 min prior to assay at a final concentration of 15,ug/ml. The mixture was added to cells at time zero (arrow). Theexperiment was performed four times with essentially the sameresults as shown. Anti-CSa MAbs blocked the C5a-inducedchemiluminescence signal, whereas a nonbinding monoclonal hadno effect on the assay.

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TABLE 7. Inhibition of C5a-generated polarization ofgranulocytes by anti-CSa MAbs'

% Polarized cellsSample Test 1 Test 2 Test 3

Media only (HBSS) 2 2 2C5a only' 98 97 98269-10F7 only 2 2 2

15 pLg + C5a 4 3 230,ug + t5a 4 NT NT

260-91H only 2 2 215lg + C5a 86 58 6230 ,ug + C5a 86 NT NT

269-3E5 only 2 2 215 ,ug + C5a 90 54 5130 ,ug + C5a 88 NT NT

269-7C9 only NT 2 2269-7C9, 15 ,ug + C5a NT 68 65261-9F11 only 2 NT NT

15 ,ug + C5a 91 NT NT30 p.g + C5a 89 NT NT

260-llG5 only 2 NT NT15 l.Lg + C5a 89 NT NT30 jig + C5a 83 NT NT

L113 only' 2 NT NT15l.g + C5a 98 NT NT30 ,ug + C5a 97 NT NT

a All MAb dilutions were in HBSS. Blocking of MAb and C5a as well ascontrols was performed for 1 h at room temperature. NT, Not tested.

10 ng of human C5a in HBSS.Non-C5a-binding MAb.

during sepsis and other states of complemnent activationleads to PMN aggregation and activation (7, 8). Rabbitspassively preimmunized with several of the MAbs wereprotected from subsequent C5a-induced neutropenia.A number of workers have generated data supporting the

role of activated complement in the pathogenesis of ARDS(7, 9, 16, 30, 31). The major mediator of damage is the PMNactivated by C5a, although studies of ARDS developing inneutropenic patients suggest that other factors may alsocontribute to the syndrome (23). Although we have notdirectly tested the MAbs in a model of sepsis, it is reasonableto expect that they would give protection in human patientsbecause they neutralize passively administered human C5ain rabbits. A rabbit anti-human C5a serum used by Stevensand co-workers (28) shared enough cross-reactivity with C5afrom Cygnomolus spp. monkeys to show protection in thisprimate Escherichia coli sepsis-ARDS model. This antise-

TABLE 8. Anti-CSa MAbs provide protection against C5a-induced neutropenia in rabbits

Total PMNs/mm3 of blood at time (min):MAb

oa 60 71 75

269-10F7 1,250 2,280 1,764 1,938261-9F11 3,456 2,392 1,496 1,440260-llG5 3,536 2,300 1,976 2,560260-91H 1,872 1,632 1,024 1,980269-3E5 1,480 1,406 638 1,470260-7C9 2,160 1,840 864 1,900260-114G 4,180 3,800 1,920 4,100261-174D 1,260 1,120 720 1,008269-9H3 1,410 1,692 984 2,860Negative control' 770 1,204 26 450

"The MAb was injected at 10 min after time zero.Human C5a was injected at 70 min after time zero.The control was an anti-ricin toxin A MAb (IgGl).

rum also neutralized C5a-induced PMN aggregation in vitro.Our panel of MAbs showed only weak cross-reactivity withC5a generated by zymosan activation of serum from severalspecies, including rabbit, guinea pig, dog, pig, and rhesusmnonkey (data not shown). Generation of species-specificanti-C5a MAbs should permit us to dissect the role ofcomplement activation in animal models of sepsis andARDS.At the present time we are generating high-affinity MAbs

to C5a derived from various species used for sepsis andARDS models. It is anticipated that the anti-human C5aMAbs described in this report will have therapeutic usebased on their capacity to neutralize the biological effects ofhuman C5a both in vitro and in vivo.

ACKNOWLEDGMENTSWe wish to acknowledge the expert word processor help provided

by Joan Murphy. Helpful discussions were provided by Tom Raffin,Tom White, and Judy Blakemore. The technical assistance ofGeorge Senyk, Mark Jahnsen, Jon Silver, and Pam Cato is gratefullyacknowledged.

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