CA2094242A1 - Antibodies to streptolysin o derivatives and variants - Google Patents

Abstract

Disclosed are antibodies to derivatives and variants of Streptolysin O ("SLO"). Methodologies for generating such antibodies are further disclosed, as are purification protocols for wild-type SLO
and assaying techniques for wild-type SLO.

Description

WO !~3~05152PCI-/US92/05122 2~24~

ANTIBODIES T~) STREPTOLYSIN O
DERIVATIVES AND VARIANTS

~l2~ATED APPLI CATI ONS
The application ls related to United States Serial No.(Beckman Docket No. 12aD-104) en~i~led ~Streptolysin O Derivatives" by Craig ~'. Adams and Eva ~. Wang and Unlted States Serial No.
(~eckman Docket No. 128D-122) entitled "Streptolysin O
Variants~, by Craig W. Adams. ~oth applications are filed simultaneously herewith, and both are incorporated herein by reference.

FIELD OF TRE INVENTION

The present invention i9 generally related to St-eptolysin O and particularly to Streptolysin O
ant bodies Or _he monoclonal, polyclonal and recomDinant-DNA derived types. In its most particular a3pect, thepresent invention relates to antibodies to Streptolysin O
derivatives and variants produced by recombinant DNA
technology.

~AC~G~O~ND OP T~E INVENTION

I. Stse~tolvsin o Strep~oiysin O ("SLO") has an approximate moiecular weight of between about 65,000 and about 70,000 daltons. SLO belongs to a class o~ cxygen sensiti~e W093tO5152 PCT/US92/05122 ~ 2-("thiol-activated"), cell destroying ~"cytolytic") toxin (~cyto~oxin~) whic~ are produced by gram-positive bacterial species belonging to four dif-erent genera (streptococcus, bacfllus, clostridium and listeria).
, SLO ir.teracts wlth mem~rane cholesterol and exerts cytolytic-cytotoxic effects on a broad range o~
mammalian cells. Additionally, SLO ha3 very potent cardiotoxic properties. One of the toxic and patnogenic properties associated with SLO is its hemolytic activity, i.e. SLO will lyse red blood cells, resulting in the release of hemoglobin. S~O can be lethal to laboratory animals in relatively small doses. Injection of SLO
into an animal typically results in its immediate death.
Most typically, SLO is associated with rheuma~ic fever in humans, an acute infectious disease characterized by fever, profuse perspiration, painful inflammation and swelling of the joints, and often inflammation of the lining membrane of the heart ("endocarditis").
~ ecause SLO i9 produced by specified bacterial species, when these species ~invade~ a mammalian host, the SLO released by the bacteria is treated by the hoYt as a foreign protein. SLO, then, is an antigen.
"Antigens" are high molecular weight compounds which upon entry into the blood stream of a vertebrate stimulate the transformation of the small lymphocytes of the B-type into lymphoblasts. The lymphoblasts secrete antibodies specific to the antigen stimulator. The antibodies are proteins possessing reactive sites specifically complementary to a reactive feature or site on the stimulating antigen. Antibodies generally have~the property of rendering the antigen harmless to the host organism by occupying the immunologically active sites, or "epitopes", on the antigen particles or molecules.
Anti-SLO antibodies ("ASO") are therefore produced by the Pateni 209~24~ 128Dl23 host in response to the secretion of SLO into the hos~.
Approximately 80-85~ of individuals with current streptococcal infection or their sequelae (an after effect of a disease or injury) will demonstrate elevated levels of ASO.

Determination of previous and/or current infection by the specified bacterial species which secretes SLO is possible using immunodiagnostic assaying techniques which, e.g., rely upon the hemolytic proper-ties of SLO and the binding of ASO to SLO. Focusing on hemolytic immunodiagnostic assays for SLO, a patient sample is added to a known amount of SLO derived from a source other than the patient and this mixture is added to a known amount of red blood cells such as, for example, rabbit red blood cells. Because SLO has hemolytic properties, it will lyse these red blood cells.
However, when ASO binds to SLO, the hemolytic properties of SLO are neutralized. Thus, if the sample is obtained from a patient having current streptococcal infection or their sequelae, there will be elevated levels of ASO in the sample. Accordingly, if the mixture results in high levels of hemolytic activity, this indicates that there is little, if any, ASO in the serum sample (and hence little, if any, infection from the SLO secreting bacteria) because the known quantity of SLO in the mixture is capable of lysing the known quantity of red blood cells in the mixture. If the mixture does not lead to hemolytic activity, this is indicative of an amount of ASO in the sample sufficient to inactivate the known quantity of SLO in the mixture. Investigators refer to such an amount of ASO as a "titer". Typically, an ASO
titer of greater than about 300 International Units/ml is indicative of infection by a bacterial source capable of secreting SLO. Other immunodiagniostic assays for J: \039~11990 . 1 W O 93/05152 9~ P ~ /US~2/05122 9,~

determination of in~ection by SL~ seoreting bacteria involve nephelometric and turbidime~ric protocols.

In order to ut- lize these i~;ununodiagnos~ic S assaying techniques, it is necessary to have access to suf icient S~O to be added to the mixture. One source of SLO is the bac~eria Streptococcus pyogenes ("S.
pyogenes"), whereby the SLO i9 secreted from S. pyogenes into the culture broths. Experience has demonstrated that the purification of SLO from its natural host i9 difficult and expensive.

II. Antlbodie3: Monoclonal. Polyclonal and Recomblnant DNA Technology_Ba~ed Monoclonal antibodies have a single antibody specificity and affinity, as well as a single immunoglobulin isotype (a protein fraction containing antibody activity is referred to as an "immunoglobulin").
Polyclonal antibodies, on the other hand, contain a ~ariety of antibody molecules directed against an antigen of interest, as well as antibodies which do not react with the antigen of intere~t. ~ecause of the specificity and affinity associated with monoclonal antibodies, these are generally preferred over polyclonal antibodies when such specificity i9 needed, e.g., in purification protocols. The specificity of monoclonal antibodies allows one to identify and screen for monoclonal antibodies which comprise the specific binding characteristics of interest. For example, two monoclonal antibodies can be selected to bind to the same antigen, but at different locations. Such antibodies. can be of significant value in, e.g., sandwich-based immunoas~ays.

W O 93/OSt52 2 ~ 9 ~ 2 ~ 2 P ~ /US92/05122 3riefly, the production of monoclonal ~ntibodies comprises the following steps: Immunization;
Hyb-ldization; ProDagatior.; Screening; and Cloning.

The immune ~esponse cf an animal, e.g. a mammal such as a mouse, injected with an antigen which it has never encountered beforè will elicit a "Primary Response", i.e., the animal's immune system will generate a small amount of antibody against the antigen. If after a time period the animal i9 reinjected with the antigen, the immune system will elicit a "Secondary Response", i.e., the response is faster, stronger (more antikody is made), and qualitatively different from the Primary Response (antibody which binds with a higher affinity to the antigen will be generated). Generally, it i9 preferred that a pure version of the anti~en be used in order to reduce the number of irrelevant antibodies produced. For soluble antigens, it i9 preferred to immunize the animal with a mixture of the antigen and an adjuvant. Adjuvants provide a pool of the emulsified antigen at the injection site, and this allows the antigen to be released slowly over an extended period of time. Immunization may be by the intraperitoneal, intravenous, subcutaneous or intramuscular routes.
Hybridization consists of the preparation of a myeloma cell line, spleen cells and the process of fusing these cell lines. The myeloma cell line confers "immortality" to the antibody; this cell line has the ability to multiply indefinitely and secrete immunoglobulin. The spleen cells are derived from the spleen of the immunized animal. The fusi~on_~rocess, in essence, merges the immortal myeloma cells with the spleen cells containing antibody to the antigen. The result of the fusion process i9 typically referred to as a "hybrid" or, "hybridoma".

W093/OSlS2 ~ ~ PCT/US92/05122 The propagation phase, n essence, encourages the growth of hybridoma colonies. Typically, only hybridomas will grow- unfused myeloma cells and unfused ~pleen cells cannot propagate. Hybridoma colonies are placed in.o growth-st~mulatlng medlum and incubated unti' suf~icien~ colonies have propagated.

In the screening phase, hybridoma colonies producing antibody against the~antigen are identified.
Generally, any assay for antibody against the antigen can be used for screening. For example, a labelled anti-immunoglobulin can be used to detect the presence of immunoglobulin.

Cloning ensures that the cells producing antibody comprise a monoclonal population. I.e., the hybridomas selected in the screening phase may comprise antibodies which lack the requisite monoclonality. In essence, cloning involves the setting up of sample-cell cultures, each cell intended to grow into a colony of identical cells. Three different approaches may be used:
limiting dilution; semisolid agar culturing; and selective isolation by flow sorting. Limiting dilution i9 typically preferred. This process comprises plating out in individual culture wells a suspension of hybridoma cells at a dilution such that, statistically, the number of cells in any particular well is from about O to about 10 cells. ~e-cloning (i.e. repeating the foregoing) i9 typically recommended. In order to ensure that the clones all produce antibody of the same specificity, the antibody is analyzed by, e.g., electrophoresis or rate nephelometry. r. _--The production of polyclonal antibodies comprises the following steps: Immunization andPurification. Immunization is substantially the same as W093/05152 ~ V Y '12 4 2 PCT/US92/05122 with monoclonal antibody immunization. Pu_ification involves the removal of a sample of the immunized animal~s bodv fluid and purifying lt by nsolubilizing ~the antigen on an approprlate af.inity gel and pouring the body fluid over such gel; bound antibody can then be removed by, e.g., elution or displacement chromatography.
Goats, rabbits o- mice can ~e used as the animal, with rabbits being preferred.

Antibodies can also be derived by recombinant DNA technology as outlined above. I.e., the gene or relevant portion of the yene of a cell immunized against an antigen can be removed and inserted into a suitable cloning vector. The recombinant vector can then be transfected into an appropriate host under conditions whereby recombinant antibodies are secreted.

Heretofore, the intentional production of antibodies to SLO (i.e., sther than those that are produced by a host in response to infection by SLO-secreting bacteria), has been hampered because of the lethal affects of wild-type SLO on laboratory animals. as used herein, the term "wild-type SLO" is meant to indicate SLO that is naturally secreted by bacterial sources such as, for example, S. pyogenes. A need therefore exists for such antibodies, which could be used, for example, for the purification of wild-type, derivative or variant SLO, for immunodiagnostic assayq, for therapeutic purposes and for identification purposes.
SUMMA~Y OF TRE INVENTION
, .
The present invention satisfies the above needs. Monoclonal, polyclonal and recombinant-derived antibodies can be generated using the SLO deri~atives and variants disclosed in the above-referenced and co-pending P~r/US92/05122 applications. Such antibodies are particularly applicable in at least the following areas: the purification o_ ~oth wild-type SL0 derived from, e.g., pacterial sources, as well as SL0 derived by recombinant DNA technology whereby,the antibodies are used to bind SL0 from the solution~ from which the antigenic material was generated; immunodiagnostic assays, including, for example, competiti~e assays, two-site or "sandwich"
assays, and non-competitive assays, whereby the presence of SL0 is examined; identification and localization of points of SL0 infection whereby labelled antibodies are introduced to a host infected by SL0 to determine the point~s) of SL0 interaction; and therapeutic areas whereby the antibodies are introduced ~ia a suitable pharmaceutical carrier to a host infected by S. pyogenes in an effort to neutralize the resulting secretion of SL0. As further disclosed, an improvement in generating such monoclonal antibodies i9 provided whereby prior to the immunization step, the rSL0 or mSL0 is complexed with high density lipoprotein, or introduced to an agent capable or reducing a cysteine amino acid residue.

Ideally, the polyclonal antibodies should ha~e substantially no cross-reactivity to antigenic substances secreted by S. pyogenes other than SL0, and most preferably, substantially no cross reactivity to antigenic substances other than rSL0 and mSL0. Ideally, the monoclonal antibodies have an affinity for wild-type SL0, mSL0, and rSL0 of greater than about 10~, preferably greater than about 107, and more preferably greater than about 10~.
,, . _ ~
These and other uses and advantages will become apparent as the disclosure proceeds.

W O 93/05152P ~ /US92/05122 9 2~9~12~2 DETAILED D~SCRIPTION OF
PRE~ERRED EM30DIMENTS

~Her~tofore, the produc~ion of antibodies to Streptolysin 0 for use,in pur1_ication, diagnostic or therapeutic methodologie3 ha~ been restricted because SL0 is lethal when injected in small àoses in the animals from which such antibodies are ordinarily derived ~i.e., mice, rabbits, etc.). Thus, attempting to obtain such antibodies using wild-type SL0 is not a realistic option.

In the co-pending applications referenced above, soluble SL0 derivatives which are hemolytically active, recosnized by AS0 antibodies against wild-type SL0 and which are derived via recombinant DNA techniques (hereinafter referred to as "rSL0"), and soluble SL0 variants which are non-hemolytically active, recognized by AS0 antibodies against wild-type SL0 and which are derived via recombinant DNA techniques (hereinafter "mSL0"), are di~closed. The antibodies disclosed herein are based upon rSL0 and mSL0.

As used in this disclosure, rSL0 indicates an SL0 derivative as defined above and having a percent wild-type SL0 specific activity of less than about 75~, more preferably between about 5~ and 50~, and most .
preferably about 9~, based upon a wild-type SL0 specific activity of 4x105 hemolytic unit9/mg wild-type SL0. As used in this di~closure, mSL0 indicates an SL0 variant as defined above and having a percent wild-type SL0 9pecific activity of less than about 1.5~, more preferably less than about 0.5~, and most.preferably less than about 0.11~, based upon a wild-type SL0 specific activity of 4x105 hemolytic units/mg wild-type SL0. These values are relative; thus, if percent wild-type SL0 specific activity is based upon a wild-type SL0 specific W093/05152 ~ PCT/US92/05122 ~9~ o-activity of lx106 hemolytic units/mg, the above values are decreased by a factor or 2.s (i.e., 75~ becomes 30~; 9 becomes 3.6~; etc.).

The precedi~g i9 detailed because commercially available wild-typ~ SL0 is inherently impure such that an actual "specific activity" of wild-type SL0 is elusive.
Stated again, the specific activity of wild-type SL0 presupposes that the material being analyzed is of su~ficient purity to establish àn accurate specific activity; however, because commercially available wild-type SL0 is inherently imDure, a single "correct"
specific activity cannot be established.

The specific activity of wild-type SL0 has been described as being as high as 1x106 hemolytic units/mg, although specific activity of 4x105 hemolytic units/mg have also been described. Alouf, J. E. "Streptococcal Toxins (Streptolysin 0, Streptolysin S, Erythrogenic Toxin)." Pharmac. Ther. II: 661-717 (1980), which i9 incorporated herein by reference. As noted in our co-pending applications, the specific activity and percent hemolytic activity of specific ver~ions of rSL0 and mSL0 (rSL0.3 and mSL0.3/6 respectively), based upon the "specific activity" of wild-type SL0, are as followR:

TABLE I
Wild-Type rSL0.3 mSL0.3/6 S~0 Specific Activity (Hemolyt~c Activity a) 1x106 in Hemolytic Units/mg) b)-4x105 3.6x104 14 , _ Percent Hemolytic a) 100 3.6 1.4x103 Activity of Wild-Type SL0 b) 100 9 3.5x10-3 -11- 2~9~2~

The variant, mSLO.3/6, differs from the derivative, rSLO.3, by a single amino acid. The foregoing indicates that purified rSLO.3 is about 3.6~ less hemolytically ~active than wild-type SLO and that pur~fied mSLO.3/6 is about '.~x10 3~ iess h~molytically active ~han wild-type SLO.

EXAMP~E 1 In Vivo Toxlcity E~fects of rSLO and mSLO

In order to evaluate n vlvo toxici~y effects of rSLO.3 and mSLO.3/6, Balb/c mice were administered undiluted and diluted intravenous injections of rSLO.3 and mSLO.3/6. Undiluted and diluted control suspension buffer was administered to an equivalent number of mice.
To improve the intravenous injections, the mice were warmed under a heat lamp for 20-30 minutes of pre-injection. Approximately 20 mice were used for each condition.

For the undiluted rShO.3 and mSLO.3/6, each mouse received an approximate dosage of 17 mg/kg, while for the diluted rSLO.3 and mSLO.3/6, each mouse received an approximate do~age of 1000 ~g/kg. Control solution buffer did not affect the control mice.

Aside from minor ruffling for several minutes after injection, none of the mice receiving either diluted or undiluted rSLO.3 or ~SLO.3/6 showed any ill effects from the intravenous administrations.
, . . _ ., , The foregoing toxicity results indicate that rSLO and mSLO can be utilized in the production of W093/05152 PCT/US92/0~122 ~ ?~ 12-monoclonal, poiyclonal and recomblnant DNA derived antibodies to SL0.

~ In the course of generating monoclonal ant bodies to rSL0 and,mSL0, Applicants ha~e also discovered an improvement in the process used for the production of such antibodiès. That improvement relies upon interaction of the rSL0 or mSL0 to high density lipoproteins or in introducing-~the rSL0 or mS~0 to an agent capable of reducing a cyqteine amino acid residue.
These improvements are set forth in detail in Example 2, - Preparation of Hybridomas.

Preparatlo~ of ~ybridomas Hybridomas capable of making monoclonal antibody with a specific affinity for rSL0 and mSLO were prepared. The materials utilized were as follows. The myeloma cells used were derived from P3X63-AG8.653 myeloma cell line, a non-secreting mouse myeloma line described by Kearny et al. J. Immunol, 123:1548 (1970).
Myeloma cells can be derived from interspecie~ hybrids, such as, for example SMH-D33, a mouse-human "heteromyeloma~' fusion partner. Teng et al. Proc. Natl.
Acad. Sci. USA 80:7308 (19~3). Alternati~ely, myeloma cell lines deri~ed from humans offer the opportunity to de~elop human monoclonal antibodies; such would be preferred for use in, for example, therapeutic situations. Human myeloma cell lines are described in Nilsson et al J. Clin. Ex~! Immunol. 7:477 (1970) ("U-266") and Matsuoka et al Proc, Soc. Exp.,Biol Med.
125:1246 (1969) ("RPMI-8226"). The foregoing references are incorporated herein by reference.

-13- 2~9~24~

Splee~ cells used were obtained from Balb/c mice immunized according to the procedure disclosed below. The growth media was DME low glucose (I~vine ~Scientific, Santa Ana, CA.), sup?lemented with 10~ fetal calf serum (Gemlni ~io,Products, Calabasas, CA) and 2~M
~-glutamine (Irvine Scient ic). The used media was growth media from three-day culture of 6;3.1 cells, centrifuged and filtered through a .22~ filter to remove cells. Conditioned Hypoxanthine Aminopterin Thymidine ("CXAT") media was 50~ growth media and 50~ used media with 100 units/ml of penicillin-streptomycin solution (Irvine Scientific), 4x107M aminop~erin (Sigma, St.
Louis, M.O.), lOx104M hypoxanthine and 1.6xlOsM thymidine (both from GIBCO, Grand Island, ~.Y.), and 10 units/ml insulin (Eli Lilly, Indianapolis, Ind.). The conditioned media was 50~ growth media - 50~ used media and 2.5x105M
b-mercaptoethanol (Sigma). Polyethylene glycol ("PEG") with a molecular weight between about 1300 and 1600 (Sigma) was used. Injection media was DME low glucose with 100 units/ml penicillin-streptomycin solution (both from Irvine Scientific). One-half milliliter of Pristane~ solution (2,6,10,14-tetramethylpentadecane;
Aldrich) was injected intraperitoneally into each Balb/c mouse two weeks prior to hybridoma i~jection.
Hybridomas were constructed in accordance with the method described by Kohler and Milstein, Nature 256:495 (1975). The spleen from the immunized mouse was aseptically removed after cervical dislocation and was ground in a tissue sieve until a single-cell suspension was obtained. After washing, the cells were mixed with the washed 653.1 myeloma cells in a 2:1 rat~o.of spleen to myeloma cells and then pelleted. The supernatant was removed and the PEG added slowly over one minute. P~S
was added to brins the total volume to 22ml and the cells were then pelleted after 8 minute~ from the initiation of ~ ~L r~ ~ 14 ~
C~
PEG additlon. The pellet was resuspended in 200 ml of C~AT media and 0.2ml of the suspen3ion was added to each well of ten 96-well (microtiter plates (960 wells total).
The wells were supplied with fresn CHAT on day 6 or 7 post-fusion.

Testl~.g the wells for srowth began on day 10 and continued over the next 3-4 days. One-fifth milliliter of rSLO.3 (final c~ncentration: 1.5~g/ml coating buffer, 1.59g N~CO3, 2.93g NaHCO3, 1~ triple distilled H2O, pH 9.6) was dispensed into an appropriate number of microtiter plate wells; these plates, covered and sealed with parafilm, were stored at 5-10C for at least 3 days prior to use. Hybridoma candidate supernatants were standardized in PBS-0.05~ TWEEN 20 (polyoxyethylene sorbitan monolaurate; Sigma), pH 7.2.
Antigen-coated microtiter well plates were washed with P~3S 0.05~ TWEEN 20, followed by aspiration of all liquid from the individual wells. Each well was then filled with 200~1 of the supernatant from hybridoma candidates, and covered. Plates were then covered and incubated for 2 hrs. in a 37~C incubator, followed by washing (three times) with P~S-0.05~ TWEEN 20, and followed by addition of a working dilution of rabbit an~i-mouse conjugatè
(Sigma). Each well was then aspirated to dryness, followed by addition of 200~1 of the alkaline phosphatase substrate. Alkaline phosphate substrate was prepared as follows. 10mg p-nitrophenyl phosphate (Sigma~ was di~solved for each 1 ml of diethanolamine buffer.
Diethanolamine buffer was prepared by dissolving .203 g MgCl2 (Mallinckrodt) in 100 ml double di~tilled water, followed by the addition of 95.87 ml of diethanolamine ~Mallinckrodt) in 750 ml double distilled water; Ph was adjusted to 9.8 with HCl and NaOH. Final volume of one liter wa~ achieved by the addition of double distilled water. Working dilution i5 defined as lmg p-nitrophenyl W O 93/05152 P ~ /US92/05122 2as-~2~2 pAosphate per lml of diethanolamine bu~fer and was achieved by diluting the p-nitrophenyl phosphate-diethanoia~ine buffer in diethanolamine buffer.

Plate~ wer~ then covered and incubated for 30 minutes in a 37OC incubato- Thereafter, 50~1 of 2N NaOH
was added to each well, followed by gentle shakins, to stop the reaction. Thereafter, optical density readings from microtiter wells were obtained with a Molecular Devices reader set at 405 nM.

Wells with an optical density reading greàter than negative con~rols were retested the following day.
If the reading remained greater than the negative control on the second day of testing, the colony was considered positive and cloned.

Cloning was carried out by the limiting dilution method, whereby two 96^well plates were utilized, one with 5 cells per well in conditioned media and one with 1 cell per well in conditioned media. One week a~ter cloning, single-colony wells were tested by enzyme immunoassay ("EIA"), as described above. If all wells were positive, the line was considered pure and was recloned a second time for stability. If all the wells did not test 100~ positive, a positive well was used for the second cloning. The plates were again tested 7 days after the cloning. This procedure was repeated until all the clones tested 100~ positive. The cells were then expanded in growth media and injected in injection media into the peritoneal cavity of Pristane-primed ~alb/c mice at a concentration of 3x106 hybridoma cells_per mouse.

Prior to injection, supernatant from the cultured cells was used for isotyping by the Ouchterlony gel diffusion method, Ac~a. Path. Microbiol. Sçand.

W O 93/05152 ~9~ PC~r/US92/05122 26:s07 (1949). Ascites fluid was harvested from the mlce about 10 day~ after the mice had been injected with the hybridoma c5119 . The ascites ~'uid was then titered by EIA, and the IgG content was measured using an ICS~ rate nephelometer (~ec~man Instruments, Inc., Fullerton, CA.).

Immuniza~ion protbcols were as follows. For the mice designated SL0 AS11 and SL0 AS12, 20~g of rSL0.3-RA (to be described inf~;_) in Freund' 9 Complece Adjuvant ("FCA") (1.5 ml Aracel A (Mannide Monooleate);
8.5 ml 3ayol F (paraffin oil); 5 mg mycobacterium butyrieum, killed and dried) was injected intraperitoneally followed at slx weeks by intraperitoneal injection of 20~g or rSL0.3-RA in Freund's Incomplete Adjuvant ("FICA"); FICA differs from FCA in that it does not include therein mycobacterium butyricum.. Approximately one month later, lO~g of rSL0.3-RA in FICA was injected intraperitoneally. Three weeks later (three days prior to fusion~, 5~g of rSL0.3-RA in PBS was injected intraperitoneally. For mice designated S~0 AS13-AS21 (inclusive, without an AS18), lO~g of rSL0.3-HDL (to be described in~ra) in FCA
was injected intraperitoneally, followed at six weeks by intraperitoneal injection of 20~g of rSL0.3-HDL in FICA.
Approximately nine weeks later (three days prior to fusion), 5~g of rSL0.3-HDL in PBS was injected intraperitoneally.

Two of the monoclonal antibody candidates were ~0 identified as capable of blocking the hemolytic activity of rSL0.3. These two antibodies were derived from SL0 AS16 and S~0 AS21, respectively. Identi$ication protocol was as follows. To one-half milliliter of PBS (pH 7.0) was added .O~g of rSL0.3 and .2~1 of delipidated ascites fluid. Delipidated ascites fluid was prepared by mixing in a 1:1 concentration ratio the ascites fluid from the W093/OSlS2 -i7- 2 PCT/US92/0~122 above lndicated hybridomas and ~3eckman Lipid Clearing Solution (3eckman Instruments, Inc.); this mixture was vortexed for at least l min. After 5 min. at room temperature, one-half millili~er of rabbit red blood cells (pre-washed once with PBS) was added to tne foregoing. Af~er lO min. at room temperature, the mixture was centrlfuged in a ~eckman micro-cent-l~uge at 12,000 rpm for 5 minutes.

A positive result, a result indicating blockage of hemolytic activity by binding of the monoclonal antibody to rSLO.3, was indicated by a clear supernatant and a pellet having a red color (i.e. the pellet comprised red blood cells); a negative result, a result not indicating blockage of hemolytic activity, was indicated by a reddish colored supernatant (i.e. lyses had occurred). The microcapsules containing antibody secreted from the SLO AS16 and SLO AS21 evidenced a clear supernatant, i.e. a positive result.
As noted above, the mice were immunized with either rSLO.3-RA or rSLO.3-HD~. Manipulation of rSLO.3 prior to immunization was conducted based on two properties of wild-type SLO. First, secreted wild-type SLO tends to initially bind to cholesterol within the host. Second, SLO has a single Cys amino acid. These manipulations involved the formation of rSLO-High Density Lipoprotein fractions ("rSLO-HDL") mixtures, and introduction of rSLO to a reducing agent ("rSLO-RA").
These manipulations are also applicable to rSLO, mSLO, and mSLO.3/6.

While not wishing to be bound to any particular theory, Applicants postulated that immunization with rSLO.3-HDL would lead the immune system of the host to recognize the HDL portion of the complex as a foreign W O 93/05152 ~ PC~r/US92/05122 ~ 18-substance, thus provoking an immune response. Along with the response to the HDL, the immune system of the host would be ~sensitized~ to the presence of rSL0.3 in the complex and thus antibodies would also be generated by the host relative to the,epitop1c sites on the rSL0.3.
Applicants similarly postulated that the binding of rSL0.3 to HD~ leads to a different conformational structure which leads to an immunogenic response to the rs~o. 3 epitopic sites. With respect to rSL0-RA, Applicants theorized that the reducing agent interacts with the Cys amino acid of rSL0.3, thus leading to the separation of two rSL0.3 protein structures joined via a single disulfide bond and the formation o~ -SH groups on each of the two rSL0.3 structures. These rSL0.3 structures would then be capable of forming mixed di-sulfide bond~ between a single rSL0.3 protein structure and material in the adjuvant comprising an -SH group.
Such an rSL0.3-adjuvant complex would be similar to the rSL0.3-HDL complex described. I.e., "similar" in the sense that the adjuvant portion of such a complex would be recognized by the host's immune system as a foreign Yubstance, thu~ leading to sensitization of that system to the rSL0.3 portion of the complex.

High density lipoprotein fractions can be from any mam alian source, and most preferably, the mammalian source i9 not the same as the mammalian host to be immunized. Accordingly, any HDh is app].icable to the present invention, with Bovine HDL Cholesterol and Human HDL Cholesterol being particularly preferred. A most preferred HDL is Pentex~ Human Chole3terol Concentrate II
~Miles Laboratories, Xankakee, Ill. Source: human plasma, about 24~ HD~/cholesterol). The weight ratio of rSL0 to HDL (rSLO:HDL) is between about 10:1 to about 1:1, preferably between about 6:1 and about 1:1, and most preferably about 2:1.

W093/05152 PCT/~S92/05122 -19- 2~9~2~2 Reduclng agenes wnich can be utllized l~clude mercaptoethanol, sodium borohydride, sodium cyanoborohydride, sodium bisulfite, sodium thiosul~ate, ascorbic acid (~itamin C), uric acid, dithioerythreitol, and dithiothretiol. Of these, dithio~hretiol i9 a particularly prererred red1~cing agent. The concentration of the reducing agent is preferably between about 2.0mM
about 8.0mM, more preferably between about 3.OmM and about 7.0mM, and most preferably about 5.OmM.
For rSLO.3-RA, 20~g of rSLO.3 was dissolved in 5.0 mM DTT in PBS. This mixture was heated at 37~C for 30 minutes prior to immunizaticn. For rSLO.3-XDL, rSLO.3 (1 mg/ml) was mixed with of Pentex~ Human Cholesterol Concentrate II in a 6:1 volume to volume ratio (rSLO.3:Pentex~) prior to immunization.

The hybridomas prepared by this method were capable of producing monoclonal antibody with a specific affinity for rSLO.

~ind~ny of SLO Monoclonal Antlbodles to Wlld-Type SLO
While the monoclonal antibodies derived from the SLO AS16 and SLO AS21 hybridomas were able to bind to rSLO.3, it was not an absolute that these would also be capable of binding to wild-type SLO. Stated again, while it is expected that a monoclonal antibody having the requisite monoclonality will have a specific affinity for the antigenic material to which it i9 specific,;there i5 - no guarantee that such a monoclonal antibody will have a specific affinity for any other substance. Accordingly, investigations were conducted to determine if these W093/051~2 ~J PCTtUS92/05122 ?,Q9 - 20-monoclonal antlbodies also had a specifi~ affinlty for wild-type SL0.

~ Wild type SL0 was obtained f.cm DIFC0 Laboratories (Detroit,~Michigan, Code 0482). The DIFC0 SL0 is a desiccated, standardized f~ltrate of Streptolysin 0, in reduced form, prepared from group A
streptococcus. The DIFC0 S~0 was rehydrated in water according to supplier directions just prior to use.

To 0.5 milliliter of rehydrated DIFC0 SL0 was added 20 ~l of a 1/100 dilution of delipidated ascites fluid that comprised the designated monoclonal antibodies in P~S. The mixture was vortexed for at least about 1 sec. After 5 min. at room temperature, one-half milliliter of rabbit red blood cells (pre-washed once with PBS) was added to the foregoing. After 10 min. at room temperature to mixture was centrifuged in a ~echman micro-centrifuge at 12 rpm for 5 minutes.
A positive result, that is, a result indicating blockage of hemolytic activity by binding of the designated monoclonal antibodies to the wild-type SL0, was indicated by a clear supernatant and a red-colored pellet. A negative result, one not evidencing blockage of hemolytic activity, was indicated by a reddish-colored supernatant. Microfuge tubes containing the designated monoclonal antibodies evidenced a clear supernatant, i.e.
a positive result.
The results indicate that the designated monoclonal antibodies derived from the S~O ~iS16-and S~0 AS21 hybridomas and which are specific as to rS~0.3, were capable of sufficiently binding to wild-type SL0 to prevent lysis of the red blood cells. Thus, not only do these monoclonal antibodies have a specific affinity for W093/05152 2 ~ 2 ~ .2 PCTIUS92/05122 rSLO.3, they additionally have a specific affinity for wild-type SLO. Upon binding thereto, the hemolytic act'vity of wild-type SLO is substantially prevented by ~the monoclonal antibodies. Thus, the hybridomas prepared in Example 2 were capable of producing monoclonal antibody with a specific affinity for wild-type SLO.

These results indicate that there are a variety of uses for such SLO monoclonal antibodies. For example, such SLO monoclonal antibodies can be used in the purification of wild-type SLO and in assays to determine SLO. It is to be understood that the following are not limited solely to SLO monoclonal antibodies; SLO
polyclonal antibodies and recombinant DNA derived SLO
antibodies are equally applicable. Most preferably, monoclonal antibodies specific to SLO derivatives or SLO
variants are utilized, more preferably monoclonal antibodies specific to rSLO.3, and most preferably, the monoclonal antibodies derived from the SLO AS16 and ShO
AS21 hybridomas.

A) Purification of Wild-Type SLO

~ Purification of wild-type SLO can be accomplished using monoclonal antibodies specific to, e.g. SLO derivatives, by coupling such monoclonal antibodies to a suitable solid support.

SLO monoclonal IgG, either freshly prepared or previously frozen and thawed, may be utilized. Althouyh the monoclonal antibody may be bound to any material which itself does not have a high affinity ~or protein, materials such as glass beads, agarose and derivati~es thereof are preferred. Most preferred is ~io-Rad Affi-Gel 10.~ Methods for coupling monoclonal antibodies tosuch materials are known. For example, lOOmg of such W093/051~2 ~...?~ PCT/US92/05122 ~ -22-monoclonal antibcdies in 3Oml of O.~M MOPS buffe-, p~.
7 . 5 i9 prepared and ten mllliliters of unwashed Bio-Rad Af_i-Gel 10 is t:~er. added t;~ereto. The resul~ ng slur-y ~i9 allowed to react a~ roo~ temperature ~or 2 hours, followed by cens~ gation at 500 rpm lr, for example, a 3eckman TJ-5 cent.lfuge. The supernatan~ is then removed and the remaining gel-pelle~ q washed e~haustively with L, e.g., distillea water, and then with PBS.

Five milliliters of the monoclonal antibody bound to affinity gel is then incubated at 4C for 4 hours with 200ml o' fresh or rehydrated wild-type SLO
derived from, e.g., S. pyogenes culture broths. The resulting slurry consisting ol the wild-type SLO bound to monoclonal antibody bound to the affinity gel is loaded onto a small Bio-Rad Econo-Column and washed with lOOml of PBS at 4C.

Removal of the bound wild-type SLO can be accomplished by methods well known to those in the art, although elution chromatography is preferred. For example, by flushing the column with deionized water, the ionic strength of the column is lowered such that the wild-type SLO is eluted. Similarly, use of buffer having a pH of about 6.0 or less can be used for the elution of the wild-type SLO although a buffer having a pH of greater than about 6.0 may achieve similar results.
Elution reagents are well known and can include, for example, high pH reagents (lOOmm triethylamine, pH 11.5;
lOOmm phosphate acid, pH 12.5); low pH buffers ~lOOmm glycine, pH 2.5; lOOmm glycine, pH 1.~); high salt buffers (5m LiC1, lOmm phosphate, pH 7.2;.3 5_MgCl2, lOmm phosphate, pH 7.2); ionic detergents (1~ SDS; 1~ DOC);
disassociating agents (2m vrea; ~m vrea; 2m guanidine HCl); chaotropic agents (3m thiocyanate); and organic W O 93/05152 PC~r/US92/05t22 solvants (10~ dioxane; 50~ ethyiene glycol, pH 11.,; ,0 ethylene glycol, pu 8.0).

For ul~raourifi-arion, .h~ result~ng purified wild-type S~0 can then~be re-3ubjected ~o the same purification protocol.

~) I~munodiagnostic Assays to Determine Levels of Streptoly~in 0.
Examples of immunodiagnostic assays includes nephelometric or turbidimetric assays wherein at least ~wo SL0 monoclonal antibody specific rOr different epitopic regions o, SL0 are biotir.ylated. These biotinylated monoclonal antibodies (or frayments thereof) are admixed with a sample suspected of containing SL0, and avidin.

Avidin i9 a relatively large macromolecular protein found in egg whites, and contains four subunits.
Biotin is a relatively small, stable, water soluble vitamin. Each of the four avidin subunits of an avidin molecule is capable of specifically binding to a molecule of biotin. Thus, the biotinylated SL0 monoclonal antibody conjugates can become bound to a molecule of avidin.

In turbidimetry, the reduction of light transmitted through the suspension of particles, or aggregates, is measured. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In nephelometry, it i9 light.scattered or reflected toward a detector set not in the direct path of light which is measured. Therefore, if biotinylated SL0 35j monoclonal antibodies, avidin and a sample suspected of containiny SL0 are admixed, and the mixture i9 subjected WO93/05152 ~ PCT/US92/0512 ~ 24-to turbiàimetr-- c~ nephelometric -rCLOCO1S~ ~he amour.
of SLO presen~ n such sampie can be àetermined.
Turbidimetr_c zr.a nephelometr__ ~ro~ocols are we~ .~now-.
anà wiil not ~e se~ o-th :l àeta ' here. See, Sternberg, J. ~ atQ Nevhelome-e- for Measur-nc Specific Proteins bv lmmunoprecip.taLion for Measurlns Specific Proteins by Immunoprecipi.ation Reactlons,"
Clin. Chem., 23:8, 1456-1464 (1977). A particularly useful nephelometer is the Beckman ICS~ nephelometer (Leckman Instruments, Inc.).

I-respective of the presence o~ SLO in the sample, the biotinylated SL~ monoclonal antibodies wil' bind to avidin. lf SLO i9 not present in the samDle or 15 i9 present in a limited titer, the formation of aggregates will be limited. However, if ShO i9 present in the sample, then the SLO monoclonal antibodies can bind thereto, thus increasing the formation of aggregates as îollows:
{Avidin:3iotin - MAb~:SLO:MAb2 - Biotin}~

wherein "MAb~" and "MAb2" are SLO monoclonal antibodies specific for different SLO epitopic sites, and "n"
indicates that the parenthetical i9 repetitive. It should be noted that the aforementioned technique may also be performed without the necessity of the use of avidin and biotin.

ShO monoclonal antibodies can be coupled to, for example, latex particles for use in latex enhanced nephelometry. Such a protocol is similar..t3-thë above described nephelometric assay whereby the SLO monoclonal antibody i9 coupled to latex partic~es (instead of biotin). These can then bind to SLO in a sample, and thus increase the number of scatter centers. For -2,- 2 Q9~ 4 2 example, la~e~ beads havinc carboxy~ g~oups on the surface thereof (17-29n~" availabie from I~C` can be coupled to, e.c., SL0 monoclonal ant_boay. mhe carboxyl groups can be activa~e~ w r- l e.C. ~ a solubie -arbodiim-de, :-ethyl-3-~,-dimeth~ylaminopro?yl~
carbodiimide (I~E3Cl') and stabilized with, e.s., n-hydroxysucAinami~e ("NHS"); the concentrations thereof are lOmM and 70mM respectively. Excess untreated EDC and NHS are removed by centrifugation and the "activated"
beads are resuspended in an appropriate "attachment"
mixture. An appropriate attachment mixture can comprise 5mg/ml of SLO monoclonal antibody in PBS and l~ (v/v) TWEEN-20. After an approprlate attachment period, the SLO monoclonal antiboay:latex beads are washed twice with PBS by centrifugation, followed by resuspension in PBS.

In a nephelometric procedure similar to that descxibed above, an inhibition assay to determine levels of anti-Streptolysin 0 can also be performed. In such a protocol, a fixed nephelometric signal is determined by combining a fixed amount of SL0 (preferably rSLO or mSL0) and a fixed amount of an SL0 monoclonal antibody.
Thereafter, a serum sample su~pected of containing ASO
would be combined with the ~ixed amoun~ of SLO such that 2~ an immuno-reaction takes piace. This i9 followed by the addition of a fixed amount of SLO monoclonal antibody would be added to the immuno-reactive mixture. An inhibition signal i9 obtained different from the fixed signal to the degree that AS0 is in the sample; i.e., as the amount of AS0 in the sample increases, the signal for the immuno-reaction will be different than the fixed signal obtained from the SLO-SL0 monoclonal_antibody signal.

Alternatively, the Fab~ or Fab portions of the SLO monoclonal antibodies can be incorporated, or coated W093~05152 ~ PCT/US92/05122 ~ 99 ~ - 26-onto a firs~ region of a so-caiied "dip-s~ick"; these are capable of travelllr.g along t:~e regions cf the aips~ck.
SLO der-vatives, sucn as, _o- example, rSLG.3 can be ?ermanent y aC~lxed onto a second region above that o;~
_ the Clrs~ regicn, and labelled antibodies tc the SLO
monoclonal an.ibodies can be incorporated, coated, or permanently a_'ixe~, onto a thlrd region above the second region. The dipstick, which operate by capillary action, is inserted into a sample suspected of containing SLO
such that the sample is able to travel from the first region, throuyh the second region, and then the third region. If SLO i9 in the sample, it will become bound to the SLO monocional antioodies; capillary ac~ion will carr~ both bound and unbound SLO monocional antlbodies into the second region. Tho3e that are unbound, however, will become bound to the immobilized SLO in the second region; thus, only SLO-SLO monoclonal antibody complexes will be capable of travelling to the third region. In this region, the labelled anti-ShO monoclonal antibodies will bind to those complexes such that, depending upon the label (e.g., radioactive, chemiluminescent, bioluminescent, enzymatic,etc.), the labelled complexes can be either read directly or subjected to further chemical analysis prior to reading the label. Suitable dipsticks which can be utilized in conjunction with such monoclonal antibodies are well-known and will not be discussed herein in detail. See, for example, U.S.
Patent No. 5,013,669.

Competitive binding assays are also practical whereby SLO (wild-type, ShO derivatives such as , for example, rShO.3, or SLO variants, such as, ~or ëxample, mSLO.3/6) is labelled with, for example, radioactive label, chemiluminescent label, bioluminescent label, fluorophore, enzyme, biotin, etc. using known labelling techniques. Preferably, the SLO derivative i9 rSLO.3.

W O 93/051S2 P ~ /US92/05122 -27- 2 ~ 9 f~ 2 ~ ~

In such an assay, a k~own amount of the labelled SL0 derivative is admixed with insolubili7e~ SL0 monoclonai antlbody and a sample suspected o~ containir.g SL0 Under these parameters, the iabelled SL0 and sample SL0 wil' compete wit~. bi.. ding tr3 the insolubilized SL0 monoclonal antibody such that the amour.t of insolubilized label i9 in~ersely propor~ional to the amoun~ of SL0 in the sample.

Two-site called sandwich assays can also be utilized whereby at least one from each of the following reagent Groups A, B and C are admixed: Group A comprises insolubilized SL0 polyclonal antibody, SL0 antibody produced by recombinant DNA techniques, or SL0 monoclonal antibody specific for a first SL0 epitopic site; Group comprising a known quantity of labelled SL0 polyclonal antibody, SL0 monoclonal antibody specific for a different SL0 epitopic site other than the first site, or SL0 antibody produced by recombinant DNA techniques; and Group C comprises sample suspected of containing SL0.
Insolubilized ternary complexes comprising insolubilized SL0 antibody, SL0 from the sample, and labelled SL0 antibody are then formed such that the amount of insolubilized label is directly proportional to the amount of SL0 in the sample.

The foregoing is by no means exhausti~e.
Rather the described assays for SL0 are exemplars of SL0 assays utilizing SL0 antibodies. Similar assays will be apparent to those skilled in the art.

,, . _ 9~ 2~-Preparatlon of Polyclonal Antlbod~es To rSLO

-a or~er to prepa~e ar.sibodies to rSLO, 25ug o~
rSLO.3 was aamir.istered intramuscularly to each of two legs of a rabbit. This was accomplished by mixing equal ~olumes of -SLO.3, at an initial concentration of 0.2 mg/dl in phosphate-buffered saline ("P~3S"), with FCA.
This inoculum was then emulsified until a frothy-texture was achievec.

One-hai, mllliliter cr the inoculum was administered as described. One-f_fth milliliter of the inoculum was also administered intradermally to each of ten dorsal sites of the rabbit.

Approximately one month later, the identical protocol was followed with one exception. For the secondary immunization, the rSLO.3 was mixed with an equal volume of 50~ FCA and 50~ FICA.

300ster injections of 25~g of rSLO.3 were given monthly for se~eral months. The booster injection 2S protocol i9 identical to that described above, with two exceptions. For booster injections, rSLO.3 i9 combined in equal volumes of FICA, and one-fifth milliliter of the inoculum was administered intradermally to fifteen dor~al sites of the rabbit.

Preparatlon of Polyclonal. _ -Antibodles to mSLO

Polyclonal antibodies to mSLO were also prepared, .ollowing the protocol described in Example 4 W O 93/05t52 PCT/US92/05122 -29- 2 d 9 l' 2 4 2 with the following exceptions: a) 100yg c- mSLC.3/6 was administered intramuscularly to each of two iegs o two rabbits; and b) the fnitial concentration c_ mSLO.3/5 was 7.0 mg/dl.
~ EXAMPLE 6 Furification of rSLO
Polyclonal Antibody The rabbit anti-rSL~.3 polyclonal antibody prepared by the method of Example 4 i9 purified according to procedures well know in the art. A part cularly preferred purification protocol is affinity purification.

SLO ~wild-type, an SLO der~ative such as, for example, rSLO or an SLO variant, such a3, for example, mSLO) bound to affinity gel, is prepared by first solubilizing 100 mg of SLO in 30 ml of 0.1 M MOPS buffer, pH 7.5. Ten milliliters of unwashed ~3io-Rad Affi-Gel 10 affinity gel i9 then added to the SI.O solution. The resulting slurry is allowed to react at room temperature for 2 hours. The reacted slurry i9 centrifuged at 1500 rpm; a preferred centrifuge i9 a Beckman TJ-6~
centrifuge. The superna~ant is removed and the remaining gel-pellet is washed exhaustively with dist 11ed water and then with PBS.

Five milliliters of the S~O bound to the affinity gel i9 then incubated at 4C for 4 hours with 200 ml of pooled rabbit anti-rSLO.3 antisera obtained from the method of Example 2. The resulting slurry consisting of the rabbit anti-rSLO.3 antibodies bound to SLO bound to the affinity gel i9 loaded ontD.-a small Bio-Rad Econo-Column and washed with 100 ml of PBS at 4C.

Methods for releasing bound polyclonal antibody are well known in the art. Particularly preferred i9 ~ 30-elution chromatograpny, as previously described in Example 3,A.

~ The foregoing method ~ovides purl,ied polyclonal ant-bodies to rSLO. Preîerably, the polyclonal antibodies to rSLO should have substantially no cross reac_lvlty with S. pyogenes antigens other than SLO, and most preferably, substantially no cross reactivity with antigens other~than rSLO.

Purlflcatlon of mSLO
Polyclonal A~tibody The purification protocol of Example 6 is also applicable to pooled rabbit anti-mSLO.3/6 antisera obtained from the method of Example 5, such that purified polyclonal antibodies to mShO are provided. Preferably, the polyclonal antibodies to mShO should have substantially no cross reactivity with S. pyogenes antigens other than ShO, and most preferably, substantially no cross reactivity with antigens other than mShO.

SLO Antibodles by Recomblnant DNA Technique~

ShO antibodies can be generated using recombinant DNA techniques. Under this approach, wild-type SLO, SLO derivatives or SLO variants (as defined) can be utilized to obtain ShO antibodies,usLng standard immunization protocols such as those described above.
Thereafter, the B-cell proclucing organ (e.g., spleen, PBh 3; or lymph modeq) is removed from the source of immunization. The lymphocytes are then isolated, W093/05~52 2 0 ~ ~ 2 4 ~ PCT/US92/0sl22 followed by isolation of mRNA. cD~A is then synthesized .-om the isolated mRNA such that cDNA iibraries are obtalned. Methodologies fo- c~ cioning, suc~ as t;~ose set ~or~hl ir. Sambrook, J. et al Molecular Clor.inc: A
Labcra.ory Manua' 2d ~dition, Cold Spring Harbor Laboratory Press (1589) Vol. 1-3, and Perbal, Bernard, Pract_~al Guide to Molecular Cloninc, 2d Edition, John Wiley & Sons, New York (1988), which are incorporated herein by reference, can be utilized. Once the cDNA is obtained, an antibody expression library can be generated, using, for exampie, ImmunoZap~ cloning and expression systems (available from Stratacyte, La Jolla, CA., USA). See also, Sastry, L. "Cloning of the immunological ~epertoire in Escherichia coli for generation of monoclonal catalytic antibodies:
construction of a heavy chain variable region-specific cRNA library," PNAS, USA; 86:5728-S732 (1989), which i9 incorporated herein by reference.

An alternative procedure can similarly be utilized. Antibodies, including SLO antibody, have an approximate molecular weight of between about 100,000 to 130,000 kD. With an average molecular weight for each amino acid of about 110 kD, a 100,000 ~D protein would be encoded by approximately 909 codons, which would be encoded by messages about 2727 bases long; a 130,000 kD
protein would be encoded by approximately 1182 codons, which would be encoded by messages about 3545 bases long.
Thus, from a conservative perspective, purification of cDNA made from the B-cell producing organ, expressing SLO
antibody and which are larger than about 3545 base pairs is preferred; purified fragments greater,than.about 2727 base pairs can be also be utilized. Thereafter, the isolated cDNA fragments are purified using standard techniques and ligated into an appropriate expression vector (such as, 'or example, ~gtll) which has W O 93/05152 ~ 32- PCT/US92/05122 corresponding ends capable of annealing ~o sucn CragmeIltS, m~ he vector is ~hen trans ec~ed into an appro?,iate cell, such as fo- examDls, _. coli, under cond t-ons su-~able for srowth of a~ SLO antibody ~enomic library.

Screen ng can be accompiished by any me~hod known to those in the art. For example, the antibody proteins can be "lifted" from ~laques using protein binding filters (e.g. PVDF membranes, Millipore). The filter is then incubated with labelled SLO, or with SLO
followed by washing and incubation with labelled anti-SLO
antibody. Identificatlon of the plaques expressir,g the SLO antibodies having affinity for SLO i9 possible. mhis method of screening i9 preferred in that thousands of plaques can be screened per membrane, such that millions of clones can be screened in a relatively short time period.

~odlflcatlon of SLO
Antibodles with Enzymes, Biotln Pluorochromes, and Drug Dell~ery Partlcles SLO antibodies as described can be labelled with enzymes, biotin and fluorochromes. Such labelled SLO monoclonal antibody can be used for, e.g., diagnostic purposes (such as those disclosed above), screening purposes, or therapeutic purposes.
SLO monoclonal antibodies are preferably used, and most preferably, the IgG isotopes thereof. ;Prior to such labelling, the SLO antibodies are preferably purified. Purification can be by affinity chromatography ~5 with either protein A or SLO as the bonded ligand.
Purification by high-performance ion-exchange W O 93/05152 PC~r/US92/05122 33 ~9'~2~

chromatograpny can aiso be ut l zed. rrane, ~.E.
"~urification o_ ~cr.oclonal Antibodies by Hign-Performance Ion-~~cr.ange Chromatog.apny." Chp_. 9, onoclonal ~-ti~cd~ ?roduction ~echni~ues anc A~plica~_ion3, ~. E. ~chook, Ed. ~-la-cel Dekker, Inc. N.Y., .Y. (1987) (herelnafte- "Monoclona' ~ntibody Produc~ion Technique5."` The -~egoing chap~e- is incor?orated herein by reference. Purified IgG isotopes are digested with pepsin to yield F(ab' )2~ followed by reduction to yield Fab'.

The Fab' 'ragments may then be labeiled in accordance with tne procedures outl ned n e.g., Ishikawa, ~. et a ~'~odification of Monoclonal Ant-bodies with Enzymes, Biotir., and fluorochromes and their Applications." Chpt. 8, Monoclonal Antibody Production Techniques. The foregoing chapter is incorporated herein by reference.

Targeted delivery of particles functioning as reservoir or monoli~hic devices for important pharmacologic agents i9 a primary goal for many therapeutic treatments. These particles, typically nanometer ~o micrometer sized colloidal par~icles, are typically biodegradable and nontoxic, bioadsorptive, retained by tissues, and exhibit sustained or controlled-release of pharmacologic agent(s). A monolithic device i9 one in which the agent (9) i9 dispersed in the particle matrix, while a reservoir device i9 one in which the agent (9) i9 encapsulated by the particle. Natural polymers used in the preparation of particles include polysaccharides, e.g., starch and cellulose_derlvatives, and proteins, e.g., albumin, collagen and gelatin.
Synthetic biodegradable polymers include polylactide, polyamino acids and copolymers of lactide-co-glycolide, lactide-co-E-caprolactore, N-(2-hydroxy-propyl)-~9 ~ - 34-methacrylamide, polyortho esters and polyanhydrides.
Liposomes and lipoproteins are also available ~or delivery vehicles. Shaw, J. M. et al. ~Drug Delivery ?articles and Monoclonal Antibodies," Chpt. 15, ~.~noclonal Antibody Prqdu~~ on Techniques. The foregoing chapter is incorporated herein by reference.

The particles can be coupled directly to the SLO antibody or to F(ab' )2 and-Fab' fragments thereof.
Such coupling methodologies are deqcribed in Shaw, J. M., supra although after such coupling procedures will be readily apparent to the skilled artisan. Purification f~llcwing such coupling is recommended in order to remove uncoupled SLO antibodies or F(ab' )2 and Fab' fragments.
Following purification, pharmaceutical agents can be entrapped in the particles. Additionally radioactive isotQpes which are typically used for imaging processe~
in patients can be entrapped in the particles.

The specific pharmaceutical agent(s) utilized depend principally on the target of the monoclonal antibody. With respect to SLO, cardiotoxic effects thereof are well known, as well as the a~sociation with, e.g. rheumatic fever. Accordingly, one application of SLO monoclonal antibody coupled to particles comprising radioactive isotopes i9 the identification of specific compartmental and regional areas where SLO specifically interacts. This would then allow for the delivery of specific agents to such areas. For example, if the cardiotoxic effects of ShO are associated with the direct binding of SLO to myocardial ~issue, pharmaceutical agents could be appropriately and readily selected to be delivered to such tissue. Fur~hermore, while the relatianship between SLO and rheumatic fever is well documented, the specific interaction is not well understood. If, however, the inflammation of joints WO93/05152 2 ~ ~ 4 ~ ~ 2 PCT/US92/05122 assoclated with rheumatlc fever i3 caused by SLO binding to the tissues and muscles of the joints, pharmaceutical ag2n.s couid be appropriatei~ and -eadily selec~ed to be aelivered .o such an area. .he selec~on of such S isotopes .o. the ldentl'icaslon o~- the binding areas o_ S~O are considered to be within the purview of those in the art. Additionally, once such a localized region is identified, the choice of specified pharmaceutical agents to be delivered to such areas for a specified purpose i9 also considered to be within the purview of the art.

The Examples herein are not to be construed as limited to speci.ic hybridoma cel1 lines which are preferred. The methodologies desc-ibed for generatins hybridoma cell lines capable of producing monoclonal antibody with an affinity to rSL0, mSL0 and/or wild-type SL0 are not to be construed as limited solely to preferred hybridoma cell lines. Similarly, the preferred hybridoma cell line~ disclosed above in no way constitute an admission, either actual or implied, that these are the only hybridoma cell lines to which Applicants are entitled. They are entitled to the full breadth of protection under applicable patent laws. Additionally, the monoclonal antibodies generated from such cell lines are similarly not limited. Preferred hybridoma cell lines have been identified by Applicants as SLO AS11, SL0 AS12, SL0 AS16 and SLO AS21. For purposes of claiming the particularly cell lines by designation, the hybridoma cell lines were deposited on July 16, 1991 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The hybridoma cultures were tested by the ATCC on July 19, 1991, and determined to all be viable cultures. The ATCC has assigned the four WO93/05152 ~ PCr/US92/05l22 ?,~
;nyD-~doma cel' ~ nes ATC~ aepos~. numbers HB 10825, H~
10~27, r:3 ;0828 anc ~B 1~82, -espect_vely.

~ h~ugs ~he ~reser.~ nvention has been desc~ibeà _n co~slderable deta-i with regard to ce-tain prererred embodlments thereof, o~her embodiment~ within the scope c the ~eachings of the present invention are possible. For example, although certain of the disclosed antibodies are based upon the SLO derivative rSLO.3 and the SLO variant mSLO.3/6, the invention i9 not limited thereby. As such, while the production of SLO antibodies to these specific SLO derivative and variant have been desc-ibed r. de~ail, these are ~o be construed as exemplars such that othe_ -SLO cr mSLO antlgenic candidates can be used to generate monoclonal, polyclonal and recombinant DNA derived antibodies within the spirit and scope of the disclosure. Accordingly, neither the disclosure, nor the claims to follow, are intended, nor should be construed to be, limited by the descriptions of the preferred embodiments contained herein.

Claims (67)

What is claimed is:

1. A monoclonal antibody with an affinity for rSLO of at least 10-6.

2. A monoclonal antibody with an affinity for mSLO of at least 10-6.

3. A monoclonal antibody with an affinity for rSLO and wild-type SLO of at least 10-6.

4. A polyclonal antibody with an affinity for rSLO.

5. A polyclonal antibody with an affinity for mSLO.

6. A polynucleotide sequence encoding an antibody to rSLO.

7. A polynucleotide sequence encoding an antibody to mSLO.

8. A hybridoma cell line capable of secreting antibody for rSLO.

9. The hybridoma cell line of claim 8 wherein the hybridoma is a hybrid of a B-cell source from an animal immunized with an adjuvant comprising rSLO and an animal myeloma cell.

10. The hybridoma cell line of claim 9 wherein the B-cell source is an animal spleen cell.

11. The hybridoma cell line of claim 10 wherein the animal is a mouse.

12. The hybridoma cell line of claim 9 wherein the myeloma cell is derived from an animal selected from the group consisting of mouse and human.

13. The hybridoma cell line of claim 9 wherein the B-cell source animal is a mouse and the myeloma cell source animal is a mouse.

14. A hybridoma cell line designated by the ATCC identifier HB 10926.

15. A hybridoma cell line designated by the ATCC identifier HB 10827.

16. A hybridoma cell line designated by the ATCC identifier HB 10828.

17. A hybridoma cell line designated-by the ATCC identifier HB 10829.

18. An antibody secreted by a hybridoma cell lines selected from the group consisting of hybridoma cell line designated by the ATCC identifiers HB 10826, HB
10827, HB 10828 and HB 10829.

19. A hybridoma cell line capable of secreting antibody to wild type SLO.

20. The hybridoma cell line of claim 19 wherein the hybridoma is a hybrid of a B-cell source from an animal immunized with an adjuvant comprising rSLO and an animal myeloma cell.

21. The hybridoma cell line of claim 20 wherein the B-cell source is an animal spleen cell.

22. The hybridoma cell line of claim 21 wherein the animal is a mouse.

23. The hybridoma cell line of claim 19 wherein the myeloma cell is derived from an animal selected from the group consisting of mouse and human.

24. The hybridoma cell line of claim 19 wherein the B-cell source animal is a mouse and the myeloma cell source animal is a mouse.

25. A hybridoma cell line capable of secreting antibody for mSLO.

26. The hybridoma of claim 25 wherein the hybridoma is a hybrid of a B-cell source from an animal immunized with an adjuvant comprising rSLO and an animal myeloma cell.

27. The hybridoma of claim 26 wherein the B-cell source is an animal spleen cell.

28. The hybridoma of claim 27 wherein the animal is a mouse.

29. The hybridoma of claim 26 wherein the myeloma cell is derived from an animal selected from the group consisting of mouse and human.

30. The hybridoma of claim 25 wherein the B-cell source animal is a mouse and the myeloma cell source animal is a mouse.

31. An antibody to rSLO.

32. The antibody of claim 31 wherein the antibody has an IgG isotype.

33. An antibody to mSLO.

34. The antibody of claim 33 wherein the antibody has an IgG isotype.

35. A process for purifying wild-type SLO
comprising the following steps:

a) obtaining a source comprising wild-type SLO;

b) introducing said source to an insolubilized antibody to rSLO having a specific affinity for wild-type SLO such that said wild-type SLO becomes bound to said insolubilized antibody;

c) removing undesired materials from said insolubilized antibody to rSLO; and d) displacing said wild-type SLO by elution thereof wherein the eluate contains purified wild-type SLO.

36. The process of claim 35 wherein the process is repeated at least once.

37. The process of claim 35 wherein an elutinator capable of displacing said wild type SLO is selected from the group consisting of deionized aqueous solutions, solutions having a pH less than the pH of said source comprising wild-type SLO, organic solvants, chaotropic agents, dissociating agents, ionic detergents and buffers comprising high salt concentration.

38. A process for purifying wild-type SLO
comprising the following steps:

a) obtaining a source comprising wild-type SLO;

b) introducing said source to an insolubilized antibody to mSLO having a specific affinity for wild-type SLO such that said wild-type SLO becomes bound to said insolubilized antibody;

c) removing undesired materials from said insolubilized antibody to mSLO; and d) displacing said wild-type SLO by elution thereof wherein the eluate contains purified wild-type SLO.

39. The process of claim 38 wherein the process is repeated at least once.

40. The process of claim 39 wherein an elutinator capable of displacing said wild type SLO is selected from the group consisting of a deionized aqueous solution solutions having a pH less than the pH of said source comprising wild-type SLO, organic solvants, chaotropic agents, dissociating agents, ionic detergents and buffers comprising a high salt concentration.

41. The IgG isotype of claim 27 wherein said isotype is manipulated to yield Fab' fragment portions thereof.

42. The Fab' fragment of claim 41 comprising an agent conjugated thereto.

43. The Fab' fragments of claim 34 wherein the agent is selected from the group consisting of enzymes, biotin, fluorochromes, latex, particles, radioactive isotopes, chemiluminescent labels, bioluminescent labels and particles capable of incorporating pharmaceutical agents therein.

44. The monoclonal antibody of claims 1, 2, and 3 comprising an agent conjugated thereto.

45. The monoclonal antibody of claim 44 wherein said agent is selected from the group consisting of enzymes, biotin, fluorochromes, latex particles, radioactive isotopes, chemiluminescent labels, bioluminescent labels and particles capable of incorporating pharmaceutical agents therein.

46. In a process for producing monoclonal antibody having a specific affinity for rSLO, the improvement comprising complexing rSLO to high density lipoprotein and immunizing an animal with the rSLO-high density lipoprotein complex.

47. In a process for producing monoclonal antibody having a specific affinity for mSLO, the improvement comprising complexing mSLO to high density lipoprotein and immunizing an animal with the mSLO-high density lipoprotein complex.

48. In a process for producing monoclonal antibody having a specific binding affinity for rSLO, the improvement comprising immunizing an animal with an adjuvant comprising rSLO and an agent capable of reducing a cysteine amino acid residue.

49. In a process for producing monoclonal antibody having a specific binding affinity for mSLO, the improvement comprising immunizing an animal with an adjuvant comprising mSLO and an agent capable of reducing a cysteine amino acid residue.

50. The process of claim 48 wherein said reducing agent is selected from the group consisting of mercaptoethanol, sodium borohydride, sodium cyanoborohydride, sodium bisulfite, sodium thiosulfate, ascorbic acid, uric acid, dithioerythreitol and dithiothretiol.

51. The process of claim 48 wherein said reducing agent is dithiothretiol.

52. The process of claim 48 wherein the concentration of said reducing agent in said adjuvant is between about 2.0mM and about 7.0mM.

53. The process of claim 48 wherein the concentration of said reducing agent in said adjuvant is between about 3.0mM and about 7.0mM.

54. The process of claim 48 wherein concentration of said reducing agent in said adjuvant is about 5.0mM.

55. The process of claim 49 wherein said reducing agent is selected from the group consisting of mercaptoethanol, sodium borohydride, sodium cyanoborohydride, sodium bisulfite, sodium thiosulfate, ascorbic acid, uric acid, dithioerythreitol and dithiothretiol.

56. The process of claim 49 wherein said reducing agent is dithiothretiol.

57. The process of claim 49 wherein the concentration of said reducing agent in said adjuvant is between about 2.0mM and about 8.0mM.

58. The process of claim 49 wherein the concentration of said reducing agent in said adjuvant is between about 3.0mM and about 7.0mM.

59. The process of claim 49 wherein concentration of said reducing agent in said adjuvant is about 5.0mM.

60. The process of claim 46 wherein the high density lipoprotein fractions is from a mammalian source selected from the group consisting of bovine and human.

61. The process of claim 46 wherein the weight ratio of rSLO to high density lipoprotein fraction is between about 10:1 and about 1:1.

62. The process of claim 46 wherein the weight ratio of rSLO to high density lipoprotein fraction is between about 6:1 and about 1:1.

63. The process of claim 46 wherein the weight ratio of rSLO to high density lipoprotein fraction is about 2:1.

64. The process of claim 47 wherein the high density lipoprotein fraction is from a mammalian source selected from the group consisting of bovine and human.

65. The process of claim 47 wherein the weight ratio of rSLO to high density lipoprotein is between about 10:1 and about 1:1.

66. The process of claim 47 wherein the weight ratio of rSLO to high density lipoprotein is between about 6:1 and about 1:1.

67. The process of claim 47 wherein the weight ratio of rSLO to high density lipoprotein is about 2:1.