CA2347734A1 - Methods and products for regulating lectin complement pathway associated complement activation - Google Patents

Abstract

The invention relates to methods and products for regulating lectin compleme nt pathway associated complement activation. The methods include both in vitro and in vivo methods for inhibiting lectin complement pathway associated complement activation. The methods are accomplished by contacting a mammalia n cell having surface exposed MBL ligand with an effective amount of a mannan binding lectin inhibitor to inhibit lectin complement pathway associated complement activation. The mannan binding lectin inhibitor may be administer ed to a subject to prevent cellular injury mediated by lectin complement pathwa y associated complement activation. The products of the invention include compositions of a mannan binding lectin inhibitor. The mannan binding lectin inhibitor is an isolated mannan binding lectin binding peptide that selectively binds to a human mannan binding lectin epitope and that inhibits lectin complement pathway associated complement activation. The products als o include hybridoma cell lines and pharmaceutical compositions.

Description

VI~'O 00/35483 PCT/US99/29919 METHODS AND PRODUCTS FOR REGULATING LECTIN COMPLEMENT
PATHWAY ASSOCIATED COMPLEMENT ACTIVATION
s Related Applications This application claims priority under 35 U.S.C. ~119 to US Provisional Patent Application No. 60/112,390, filed December 15, 1998, the entire contents of which is hereby incorporated by reference.
Government Support ~ o The present invention was supported in part by grants from the National Institutes of Health HI,56086, HL52886., and Ci1VI07592. The U.S. Government may retain certain rights in the invention.
Field of the Invention The present invention relates to methods and products for regulating lectin t s complement pathway (LCP,I associated complement <~ctivation. In particular, the invention relates to methods for inhibiting LCP associated complement activation by contacting a m~unmalian cell having a mannose binding lectin. (MBL) ligand with an MBL
inhibitor.
The invention also relates t~~ products which are MBL inhibitors, such as an MBL binding pe ptide.
Background Of The Invention The immune system functions to defend the body against pathogenic bacteria, viruses and parasites. Immunity against foreign pathogens usually involves the complement sy item. The complement ~;ystem is a cascade of 18 sequentially activated serum proteins 2s which functions to recruit and activate other cells of the immune system, effect cytolysis of target cells and induce opsonization of foreign pathogens. Complement can be activated by the: presence of either antibody/antigen complexes, as in the classical complement pathway, or microbial surfaces, as iri the alternative complement pathway. Complement activation can also occur via the lectir, complement pathway (LCP). Lectins are carbohydrate-binding 3o proteins that recognize oligosaccharide structures :present on cell surfaces, the extracellular m~~trix, and secreted glycoproteins. As shown i:n Figure 1, these distinct activation pathways ultimatel~~ converge at the common enzymatic step of serum protein C3 cleavage to C3b and C3a. This in turn initiates the terminal steps of complement function including V1'O 00/35483 PCT/US99/29919

-2-the cleavage of CS to CSb and CSa and subsequent deposition of CSb-C9 onto the target cell membrane.
The LCP is an antil~~dy-independent cascade that is initiated by binding of mannan (or mannose) binding lectin (MBL) to cell surface carbohydrates on bacteria, yeasts, s parasitic protozoa, and viruses (Turner MW, "Marmose-binding lectin: The pluripotent molecule of the innate immune system", Immunol. Today, 1996;17:532-540). MBL

kl;~a) is a member of the ce~llectin protein family and is structurally related to the classical complement C 1 subcomponent, C I q. Associated with MBL are two serine proteases, Mannose binding lectin associated serine protease, MASP-1 and MASP-2, which show ~ o striking homology to the two C 1 q-associated serine proteases of the classical complement pa~:hway, C 1 r and C 1 s (Thief S, et aL, "A second serine protease associated with mannan-binding lectin that activate: complement", NaturES 1997;386:506-510 ). The selectivity of M~3L sugar binding is: N-acetyl-D-glucosannine (GIuNAc) > mannose > N-acsaylmannosamine and fucose > maltose > glucose » galactose and N-~s ac~;tylgalactosamine (Thief S, et al., "A second serine protease associated with mannan-binding lectin that activates complement", Nature 1997;386:506-510; Turner MW, "Mannose-binding lectin: The pluripotent molecule of the innate immune system", Immunol. Today, 1996;17:532-540). Binding of the MBL/MASP complex to cell surface carbohydrates activates the LCP, which in turn activates the classical complement pathway 2o independently of C 1 q, (: I r, C 1 s or antibodies (Fig. 1 ). Most if not all the carbohydrate moieties to which MBL birn~s are not normally expressed by unperturbed human tissue.
Summary Of The Invention The present invention relates to methods and products for regulating lectin 2s complement pathway (LCP) associated complement activation. Prior to the instant invention, it was known that LCP associated complement activation was a mechanism used by the body to recognize and destroy an invading microorganism. LCP activation normally occurs through the binding ~~f mannan-binding lecti.n (MBL) and its two associated serine pr~~teases, MASP-1 and M~,SP-2, to carbohydrates on the surface of microorganisms. Once 3o MSL and MASP-l and MA.SP-2 are localized to th.e surface of the microorganism, complement begins to assemble, ultimately killing the microorganism. These prior art

3 PCT/US99/29919 teachings demonstrate that I~~IBI. is an important cellular component in the process of the eradication of infectious microorganisms. In fact, MBL deficiencies can result in medical disorders. A disease known as MBL deficiency, in which children are deficient in MBL, renders the children prone to the development of ini:ectious diseases.
s The present invention is based upon the surprising discovery that MBL
recognizes specific carbohydrates or pc;ptides on the surface of mammalian endothelial cells, causing complement deposition through activation of the L,CP. According to U.S. Patent No.
5,270,199 issued to Ecekowitz, MBL does not recognize the cell wall of human and animal cells. In contrast to these prior art teachings, it has been discovered, according to the ~o invention, that MBL does rc;cognize specific sequences on the surface of mammalian cells.
It has also been discovered that MBL deposition on the surface of mammalian cells results in ;activation of LCP., contributing to the development of diseased or damaged tissue.
In one aspect, the invention is a method for inhibiting LCP-associated complement activation. The method includes the step of contacaing a mammalian cell having a surface ~ s exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit cellular MBL
deposition and LCP-associated complement activation. In one illustrative embodiment, the method is an in vitro screening assay.
In another aspect, the invention is a method for inhibiting a cellular injury mediated by LCP-associated complerzent activation. The method includes the step of administering zo to a subject in need thereof an effective amount of an MBL inhibitor to inhibit LCP
as<.~ociated complement activation.
In one embodiment of the methods of the invention, the MBL inhibitor is an isolated MJ3L binding peptide. In an illustrative embodiment, the isolated MBL binding peptide has an MBL binding CDR3 region or functional variant thereof. In some embodiments, the zs isolated MBL binding peptide is an antibody fragment. In other embodiments, the isolated M BL binding peptide is an ;antibody.
According to anotr~er embodiment of the methods of the invention, the MBL
inhibitor is an isolated MASP binding peptide. The: isolated MASP binding peptide may bind to either MASP-1 or MASP-2 or both, preventing MASP from participating in the 3o L(:P.

W~~ 00/35483 PCT/US99/29919

-4-The cellular injury mediated by LCP-associated complement activation may contribute to the development of injured tissue associated with a variety of disorders. In one embodiment, the cellular injury is associated with atherosclerosis. In another em'~odiment, the cellular injury is associated with arthritis, myocardial infarction, ischemia s and reperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, or dialysis.
The MBL inhibitor rnay be administered to the subject by any route known in the art. When the cellular injury is associated with the pulmonary system, the MBL
inhibitor may be administered to the subject by an aerosol route of delivery.
According to another aspect of the invention, an MBL inhibitor is provided.
The io MBL inhibitor is an isolated peptide that selectively binds to a human MBL
epitope and inhibits LCP-associated complement activation.
In another aspect, l:he invention is a hybridoma cell line. In one illustrative embodiment, the hvbridoma cell line is the cell line deposited under ATCC
accession nwnber HB-12621. In another embodiment, the hybridoma cell line is the cell line ~s deposited under ATCC a~~cession number HB-12620. In another embodiment, the hybridoma cell line is the cell line deposited under ,ATCC accession number HB-12619.
According to vet another aspect, the invention is a composition of an MBL
inhibitor, wherein the MBL inhibitor is an isolated binding peptide that selectively binds to a human M'.BL epitope and that inhibits LCP-associated complement activation. In an illustrative 2o err~bodiment the composition is a pharmaceutical composition including an effective amount for treating an MBI~ mediated disorder of the isolated MBL binding peptide and a pharmaceutically acceptable: carrier. In one embodiment, the composition also includes a drag for the treatment of an MBL mediated disorder.
In one embodiment the isolated MBL binding peptide has an MBL binding CDR3 ~
2s re;~ion or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line 3Fg deposited under A~CG(: accession number HB-12621. In another embodiment the isolated MBL binding peptide has an MBL binding CDR32 region or a functional variant thereof of a monoclonal antibody produced by lzybridoma cell line an9 deposited under ATCC accession number 1-IB-12620. In another embodiment the isolated MBL
binding 3o pf;ptide has an MBI_ binding C:DR32 region or a functional variant thereof of a monoclonal Wf) 00/35483 PCT/US99/299i9

-5-antibody produced by hybridoma cell line nMBm.2 deposited under ATCC accession number HB-12619.
The isolated peptide; may be an intact soluble monoclonal antibody. In one embodiment the isolated peptide is monoclonal antibody ~3Fg~ produced by the hybridoma s cell line deposited under A'TCC Accession No. H:B-12621. In another embodiment the isolated peptide is monoclonal antibody~2Ay~ produced by the hybridoma cell line deposited under ATCC Accession No. HB-12620. In another embodiment the isolated peptide is monoclonal antibody hMBLl.2 produced by the hybridoma cell line deposited under ATCC
Ac~~ession No. HB-12619. In an illustrative embodiment the isolated peptide is a ~o humanized monoclonal anti~~ody.
According to some embodiments the isolated peptide is an antibody fragment.
The isolated peptide, for instance, may be a monoclonal antibody fragment selected from the grcup consisting of an F(ab')z fragment, Fd fragment, and an Fab fragment. The isolated peptide may also be a peptide having a light chain C'DR2 region selected from the group ~s consisting of a CDR2~3F8t of a monoclonal antibody produced by hybridomat3F8~ deposited under ATCC Accession No. HB-12621, a CDR2t2A~~t of a monoclonal antibody produced by hyhridoma Za,9 deposited under ATCC Accession No,. HB-12620, and a CDR2~nMBLl.2) of a monoclonal antibody produced by hybridoma~nMBLi.2~ deposited under ATCC
Accession No.
Hl=4-12619. In another em'oodiment the isolated peptide has a light chain CDR1 region 2o selected from the group consisting of a CDRI~3Fg~ of a monoclonal antibody produced by hybridomat3f~g? deposited under ATCC Accession No. HB-12621, a CDR1~2A9~ of a monoclonal antibody produced by hybridomatZA9~ deposited under A'TCC Accession No.
HB-12620, and a CDRI~nMBL~.z> of a monoclonal antibody produced by hybridomatnMSr.A.2~
deposited under ATCC Accession No. HB-12619.
2s In another aspect, the invention is a composition, wherein the MBL
inhibitor is an anti-MBL antibody that: (i;1 selectively binds to a human MBL epitope, and (ii) prevents L(:P activation.
In yet another aspect, the invention is a method for screening a subject for susceptibiliy to treatment with an MBL inhibitor. The method includes the steps of so isolating a mammalian cell from a subject, and .detecting the presence of an MBL on a surface of the mammalian cell, wherein the presence of the MBL indicates that the cell is V1'O 00/35483 PCT/US99/29919

-6-susceptible to LCP-associated complement activation and that the subject is susceptible to treatment with an MBL inhibitor. In one embodiment, the method includes the step of co:ltacting the MBL with a detection reagent that selectively binds to the MBL
to detect the prcaence of the MBL. The detection reagent in one embodiment is an isolated MBL
s binding peptide.
A method for screening a subject for susceptibility to treatment with MBL
inhibitor is provided in another aspect of the invention. The; method includes the steps of contacting a mammalian cell from a subject with a labeled isolated MBL binding peptide, and detecting the presence of .an MBL on the surface of the mammalian cell, wherein the Io pr~aence of the MBL indicates that the cell is susceptible to LCP-associated complement ac:ivation and that t:he subject is susceptible to treatment with an MBL
inhibitor. In one embodiment, the mammalian cell is an endothelial hell.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving ~ s any one element or combinations of elements can be included in each aspect of the invention.
Brief Description Of 'The Drawings Figure 1 is a schematic depicting the antigen/antibody-dependent classical complement pathway and the antibody-independent alternative and lectin complement 2o pathways. All three path~Nays merge at C3 and lead to the formation of the terminal complement complex (CSb-9).
Figure 2 depicts a flow cytometry printout to demonstxate MBL deposition on H1JVECs. MBL deposition on HUVECs subjecaed to zero (normoxia) or 24 hours of hypoxia was studied by flow cytometry using a monoclonal anti-human MBL
antibody.
is MBL deposition (MFI =- 40 ~ 3) was significantly increased on hypoxic HUVECs re~~xygenated for 3 hours in 30% human serum compared to normoxic HUVECs (MFI
= 8 t 2). where MFI = mean fluorescent intensity.
Figure 3 is a graph depicting MBL deposition on HUVECs (ELISA). MBL
deposition on HUVECs subjected to zero (normoxia) or 24 hours of hypoxia followed by 3 3o hours of reoxygenation was examined by ELISA using a monoclonal anti-human MBL
artibody. MBL deposition on hypoxic I-iUVECs reoxygenated in the presence of 30%

WcJ 00/35483 PCTNS99/29919 human serum (vehicle) was significantly greater than normoxic HUVECs or hypoxic HUVECs reoxygenated in 30% human serum treated with 30 mmol/L GIuNac.
Figure 4a is a graph depicting iC3b deposition following competitive inhibition of MEtL. iC3b deposition was studied by ELISA on HUVECs reoxygenated in the presence of s 30°,io human serum treate~~ with 30 mmol/L GIuNAc, D-mannose, or L-mannose.
Deposition of iC3b on hypoxic HUVECs reoxygenated in 30% human serum (vehicle) or 30~~o human serum treated. with L-mannose was significantly greater than normoxic HL~VECs. iC3b deposition, however, on HUVECs reoxygenated in 30% human serum treated with GIuNAc or D-rr~annose did not significantly differ from normoxic controls.
~o Figure 4b is a grapl-., depicting iC3b deposition following depletion of MBL from human serum. HUVECs were reoxygenated in the presence of MBL-depleted human serum to inhibit the lectin complement pathway. Deposition of iC3b (ELISA) on hypoxic HLJVECs reoxygenated in 30% human serum was significantly greater (p < 0.05) than noomoxic HUVECs. iC3b deposition, however, on hypoxic HUVECs reoxygenated in 30%
is Ml3L-depleted human cell was significantly less (p < 0.05) than hypoxic HUVECs rec~xygenated in 30% humem serum. When MBL, was added back to the MBL-depleted human serum, iC3b deposition on the hypoxic/reoxygenated HUVECs was significantly greater than normoxic HUV ECs.
Figure S is a graf~h depicting percent hemolysis as an indicator of classical 2o co::nplement pathway actimity. No significant differences in the serum complement hemolytic assay (CH;o) were observed between human serum or MBL-depleted human semm, indicating that depletion of MBL did not inhibit or deplete classical complement pathway activity;
Figure 6 depicts a Western blot analysis of C3 activation following 2s hypoxia/reoxygenation using purified C2, C3, C4, and MBL. Western blot analysis of the C_~ and C3b a'-chain was performed under reduced conditions with a polyclonal anti human C3 antibody on thc: supernatants of norrr~oxic and hypoxic (12 hours) HUVECs reoxygenated for 3 hours ir, the presence of purified C2, C3, C4, and MBL.
Lanes 1 and 2 revresent normoxic HUVECs supernatant, lanes :3 and 4, hypoxic HUVECs supernatant, 30 lane 5, purified C3 standard. and lane 6, purified C:3b standard. The results demonstrate an _8_ increased band density of C3b a'-chain in the hypoxic/reoxygenated supernatants compared to the normoxic supernatants. The Figure is representative of five experiments.
Figure 7 is a scan of a Western blot analysis of human MBL. Monoclonal antibodies 3F8, hMBL 1.2, A9 or 1 C 10 were used for western blot analysis of reduced s M)=.L. Lanes l, 2, 3 and 4 represent staining of reduced human MBL with 10 p.g/ml of mAb 2A'~, hMBL 1.2, 1 C 10 or 3F8, respectively. A single band with an approximate molecular weight (MW) of 32 kDa (i.~~., consistent with MB:L) was observed with each mAb. This figure is representative of three separate experiment's.
Figure 8 is a graph depicting C3 deposition with inhibitors.
~o Figure 9 is a graph df;picting inhibition of VCAM-1 expression.
Reoxygenation of hyhoxic HUVECs in 30% HS treated with PBS (Vehicle) induced a significant increase in VCAM-1 expression compared to normoxic cells incubated with 30% HS. Treatment of the 30°,io HS with 3F8 (~ pg/ml) significantly inhibited'VC',AM-1 expression. The bars represent the mean of 3 individual experiments. Brackets represent SEM. * represent p<0.05 t s compared to the respective normoxia control. * * represent p<0.05 compared to vehicle tre~~ted hypoxia group.
Dfaailed Description Of The Invention The invention relate, to methods and products for regulating and manipulating lectin 2o complement pathv-av (LCP)-associated complement activation. As discussed above, the invention is based on the finding that LCP-associated complement activation plays a role in complement induced cellular injury of mammalian cells. It was discovered according to the invention that MBL interac~a with carbohydrates o~r peptides on the surface of mammalian cells in vitro and in vivo. The surface associated MBL leads to the accumulation of 2s complement on the surface ~af the cell, ultimately leading to cell injury or death. According to the prior art, LCf-associated complement activation was predominantly associated with infectious microorganisms, suggesting that MBL deposition should be promoted in order to enhance the killing of infectious microorganism:>. It was discovered, according to the imrention, that in mammal~~ it is preferable to block MBL cellular association, preventing LC'.P-associated complement activation rather than to promote it. The LCP is not necessary for eradication of ini:ectious microorganisms i;n adu.lt mammals, and in fact, it contributes to cellular injury associated with several types of disorders, such as atherosclerosis, arthritis, myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke, ARDS, SLE, s Lupus, or dialysis.
In one aspect, the invention is a method for inhibiting LCP-associated complement activation. The method includes the steps of contacting a mammalian cell having surface exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit LCP-associated complement activation.
~o The methods of the invention are useful for inhibiting LCP-associated complement ac:ivation on the surface of a mammalian cell having surface exposed MBL
ligand (c~~rbohydrate or peptide groups) recognized by ME3L.. The mammalian cell may be any cell in which the cell surface c~;rbohydrates or peptides interact with MBI,. In one illustrative embodiment, the mammalian cell is an endothelial cell having a surface exposed MBL
~s ligand. For instance, vascular endothelial cells have been shown in subjects that have sustained ischemic/reperfusion injury to express an MBL ligand. Mammalian cells having MBL ligands can easily be identified. For instance, an MBL binding assay (e.g., such as those described below) can be used to identify MBI:. ligands.
The method for inhibiting LCP-associated complement activatian may be used for a 2o variety of in vitro and in vivo purposes. The method may be used, for instance, as an in vi~ro screening assay. The o vitro screening assay may be used to identify compounds which function as an MBL inhibitor, such as the assay described above, to identify m;~mmalian cells having surface exposed MBL ligands, or to detect susceptibility of a subject to treatment with MBL inhibitor. In order to screen a subject for susceptibility to 2s treatment with an MBL inhibitor, a cell is isolated from the subject and the presence of MBL or the ability of MBL to bind to the surface is detected. If MBL is present on the surface of a cell or is able to bind to the surface of a cell, then the cell is susceptible to LCP-associated complement activation. If this is the case, then the subject is susceptible to trf~atment with an MBL inhibitor.
3o The methods of the invention are also useful in vivo when it is desirable to inhibit MBL deposition on a mammalian cell surface. For instance, the methods of the invention are useful for treating an MBL mediated disorder. 'the MBL inhibitors can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.
The mammalian cell is contacted with an MBL inhibitor. The step of "contacting"
s as used herein refers to tle addition of the MBL inhibitor to a medium containing a mammalian cell. The medium may be an in vitro tissue culture or a biological specimen, an ex vivo sample, or in vivo. The step of contacting refers to the addition of the MBL
inriibitor in such a manner that it will prevent L(JP-associated complement activation as:.ociated with the mammal ion cell.
~o An "MBL mediated disorder" as used herein is a disorder which involves cellular inj ury caused by LCP-associated complement activation. MBL disorders include, for instance, atherosclerosis, arthritis, myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke:, ARDS, SLE, Lupus, or dialysis. Each of these disorders is well-known in the art and is described, for instance., in Harrison's principles of Internal is ME~dicine (McGraw I-Iill, Inc;., New York).
Atherosclerosis and myocardial infarction can lead to ischemia-reperfusion (I/R) injury. One of the underlying mechanisms for I/R-induced injury is the hypoxic and reoxygenated environments created in affected tissues. Fluctuations in oxygen content as observed in these instances can create oxygen free radicals which have been reported to, 2o among other things, modulate endothelial cell surface profile.
The invention also i;; useful for treating cellular injury arising from ischemia/reperfusion associ;~ted with atherosclerosis and/or cardio-vascular remodeling.
Injury to the vascular system can lead to a number of undesirable health conditions, including, for example, forms of atherosclerosis an<I arteriosclerosis that are associated with 2s unwanted vascular smooth muscle cell proliferation. ,A common injury to the vascular sy,~tem occurs as a side effe~~t of a medical procedure for treating ischemic heart disease.
Isc:hemia refers to a lack of oxygen due to inadequate perfusion of blood.
Ischemic heart di:;ease is characterized by a disturbance in cardiac function due to an inadequate supply of oxygen to the heart. The m~~st common form of this disease involves a reduction in the 30 lumen of coronary arteries, which limits coronary blood-flow. Under these conditions the carbohydrate or peptide residues of the cell surface become exposed or an MBL
ligand is synthesized, allowing MBL to associate with the cell surface and initiate the LCP associated complement activation.
When ischernic heart disease becomes very serious, then management must be invasive. Until recently, ischemic heart disease was treated by coronary-artery, bypass s surgery. Less invasive procedures, however, now ihave been developed. These procedures involve the use of catheters introduced into the narrowed region of the blood vessel ("the stenosis") for mechanically disrupting, laser ablating or dilating the stenosis.
The compositions rnay be administered in combination with other therapeutic tre;~tments. The most widely used method to achieve revascularization of a coronary artery ~ o is percutaneous transluminal coronary angioplasty. A flexible guide wire is advanced into a coronary artery and positioned across the stenosis. A balloon catheter then is advanced over the guide wire until the balloon is positioned across the stenosis. The balloon then is reF~eatedly inflated until the stenosis is substantially eliminated. This procedure, as compared to heart surgery, is relatively noninvasive and can result in hospital stays of only is three days. The procedure is an important tool :in the management of serious heart conditions.
An "MBL inhibitor" as used herein is a compound that prevents LCP-associated complement activation. The MBL inhibitor may function by blocking MBL
deposition on the surface of a mammalian cell or by blocking the association of MASP-1 or MASP-2 or 2o C3b associated with MBL, deposition. The ability of an MBL inhibitor to block MBL
deposition or prevent association of MASP-1, MA~SP-2, or C3b with MBL can be detected using routine in vitro binding assays, such as the following assay (also described in the Examples).
MBL deposition (or association with MASP-1, MASP-2, or C3b) can be measured is by ELISA on normoxic H1JVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 r~r of reoxygenation in the presence of 30% human serum {HS) or 30% HS
treated with 3, 30, or 300 mmol/L of N-acetyl-D-glucosamine (GIuNAc) or with the putative binding peptide to inhibit competitively MBL deposition.
C3 and MBL specific cell surface ELISAs can be performed using peroxidase 3o conjugated polyclonal goat anti-human C3 antibody (Cappel, West Chester, PA) and monoclonal anti-human 1V1BL antibody (Biodesign, Kennebunk, ME, clone #131-1), respectively. HUVE;Cs are grown to confluence on 0.1% gelatinized 96-well plastic plates (C~~rning Costar, Cambridge, MA). The plates are then subjected to 0 (normoxia) or 24 hr of hypoxia. Hypoxic strc;ss is maintained using a humidified sealed chamber (Coy Laboratory Products. Inc., Grass Lake, MI) at 37 "C gassed with 1% OZ, 5% C02, balance s NZ (Collard CD, et al., "Re~~xygenation of hypoxic; human umbilical vein endothelial cells ac~.ivates the classical cornp:lement pathway", Circadation, 1997;96:326-333).
Following the specified period of normoxia or hypoxia, the cell media are aspirated and 100 ~.1 of one the following is added to each well: 1) 30% HS, 2) Hank's balanced salt solution, 3) 30% HS +
3, 30, or 300 mmol/L GIuNAc, 4) 30% HS + 3, 30., or 300 mmol/L D-mannose, 5) 30% HS
~o + :3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depleted HS 7) 30% MBL-depleted HS +
O.fi ~.g/ml MBL or 8) 30°/<. HS + 3, 30, or 300 :mmol/L putative MBL
binding peptide.
Additionally, 100 ~1 of purified MBL (3-300 ng,/ml) is added to select wells to form a standard curve for quantitative analysis of MBL deposition. 'the cells are then reoxygenated for 3 hr at 37°C in 95% air and 5% C'.O_Z. The cells are washed and then fixed ~s with 1% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) for 30 min.
The cells axe thc;n washed and izicubated at 4 °C for 1.5 hr with 50 pl of peroxidase-conjugated polyclonal goat anti-human C3 antibody (1:1000 dilution) or monoclonal anti-human MBL
antibody (1:1000 dilution). The MBL ELISA plates are then washed and incubated for 1 hr at 4 °C with 50 ~1 of pernxidase-conjugated polyclonal goat anti-mouse IgG secondary 2o antibody (1:1000 dilution). After washing the cells, the plates are developed with SO ~l of ARTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfi~n:ic acid)), and read (Molecular Devices, SLnnyvale, CA) at 405 nrn. Background control~.s for the C3 ELISA consist of cells to which only the anti-human C3 antibody is added (i.e., no HS) or cells incubated with 30%
heat-inactivated HS. Background controls for thc; MBL ELISA consist of cells to which 2s only secondary antibody and an isotype control monoclonal antibody to porcine CSa are added. Background optical density is subtracted from all groups. ELISA
experiments are generally performed 3 times using 6 wells per experimental group. Endothelial C3 and MBL deposition on normoxic vs. hypoxic HUVECs is analyzed by two-way analysis of variance (ANOVA).
3o The MBL inhibitor prevents LCP-associated complement activation. Whether a particular compound can inhibit LCP-associated complement activation can also be W~~ 00/35483 PCTNS99/29919 ass~~ssed using routine in vitro screening assays. For instance, the Complement hemolytic ass;~y (CHSO) described in t:he Examples below can be performed on MBL-depleted HS in order to demonstrate that MBL depletion inhibit :LC'.P-associated complement activation.
Thf; assay may be performed, however, using MBL containing HS and adding an MBL
s binding peptide and/or a control peptide.
In one illustrative embodiment, the MBL inhibitor is an isolated MBL binding peytide. An "isolated MBL binding peptide " as used herein is a peptide which binds to MI=4L and inhibits LCP ass~~ciated complement activation. One method by which MBL
binding peptides inhibit LC:I? associated complement activation is by binding to MBL and ~o inhibiting MBL association with surface exposed MBL ligands. Additionally, the MBL
binding peptide may bind to MBL and inhibit the association between MBL and MASP-1 or -2 ;and/or C3b. Several pepi;ides which bind to MBL or MASP have been described in the art. including Lanzrein, A.S. et al., "Mannan-binding lectin in human serum, cerebrospinal flu:.d and brain tissue and i~a role in Alzheimer's .disease", Department of Pharmacology, is University of Oxford, UK, May I1, 1998, Neuroreport, 9(7):1491-5; Jack, D.L. et al., "A~tivation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B", Immunobiology Unit, Institute of Child Health, London, UK, J
Immunol, February 1, 1998,160(3):1346-53, Terai, I. et al., "Human serum mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of levels in 2o body fluids and identification of two forms in serum", Division of Clinical Pathology, Hokkaido Institute of Public Health, Sapporo, Japan, Clin. Exp. Immunol., Nov., 1997, 110(2):317-23; Endo, M. et al., "Glomerular depo;>ition of mannose-binding lectin (MBL) inc.icates a novel mechanism of complement activation in IgA nephropathy [In Process Citation]", Second Department of Internal Medicine, hlihon University School of Medicine, 2s Tokyo, 3apan, Nephrol Dial Transplant, August 13, 1998, (8):1984-90;
Valdimarsson, H. et al.. "Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to Ml3L-deficient humans", Department of Immunology, University of Reykjavik, Iceland, Sc~nd. J. Immunol., August 1998, 48(2):116-23; T'hiel, S. et al., "The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase 3o re~;ponse", Clin Exp. Immunol., Oct. 1992, 90(1):31-5. These peptides can be tested for their ability to inhibit the association between MBL, and MASP-1 or -2 and/or C3b.

Wc~ 00/35483 PCT/US99/29919 The preferred compositions of the invention include an MBL inhibitor which is an isolated binding peptide that selectively binds to a human MBL epitope and that inhibits LC:P-associated complemem: activation. A ''human MBL epitope" as used herein is a portion of MBL which when contacted with an MBL-binding peptide inhibits LCP-s associated complement activation by preventing the association between MBL
and the MBL
lig~.nd or MASP-1 or - 2 and/or C3b. Preferably the MBL epitope is a region of the MBL
which interacts with any of t:ze three deposited monoclonal antibodies.
In another embodiment, the MBL inhibitor is an isolated MASP binding peptide.
An "isolated MASP binding peptide" as used herein refers to a peptide which binds to co M~.SP-I or MASP-2 and prevents LCP-associated complement activation by preventing M~~SP-1 or MASP-2 from forming a complex with MBL on the surface of a cell thereby preventing the resulting C3b deposition associated with the MBL-MASP complex.
In another embodimewt the MBL inhibitor is, a mannan binding peptide. A
"mannan binding peptide" as used herein is a peptide which binds to the MBL ligand on the surface i s of a mammalian cell, preventing its interaction with the MBL-MASP complex.
The MBL
inhibitors may easily be prepared or identified by those of ordinary skill in the art using routine experiments since 1V1BL,, MASP, mannan and. C3b are all well known compounds which have been characterized and described extensively in the prior art.
The MBL, MASP, and mannan binding peptides of the invention can be identified zo using routine assays, such a;~ the binding and LCP complement activation assays described above and elsewhere throughout this patent application.
The peptides of the invention are isolated peptides. As used herein, with respect to peytides, the term "isolated ;peptides" means that the peptides are substantially pure and are essentially free of other suostances with which they may be found in nature or in vivo 2s systems to an extent practical and appropriate for their intended use. In particular, the peptides are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be Laeful in, for example, producing pharmaceutical preparations or secuencing. Because an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may 3o comprise only a small percentage by weight of the preparation. The peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it rnay be associated in living systems.
MBL binding peptides also may easily be :>ynthesized or produced by recombinant means by those of skill in the art. Methods for preparing or identifying peptides which bind s to a particular target are well known in the art. Molecular imprinting, for instance, may be used for the de novo constn::ction of maeromoleeular structures such as peptides which bind to a particular molecule. See for example Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, ~o Trends in Biochenz Sci., 1~~(9) January 1994; andl Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., E~i.) AC.S Symposium Series No. 308, pp 186-230, American Chemical Society (1986). One method for prep~~ring mimics of MBL binding peptides involves the steps of: (i) polymerization of functional monomers araund a known MBL
binding peptide or the bin~~ing region of an anti-MBL antibody (such as the deposited t s am:ibodies) (the template) that exhibits a desiredl activity; (ii) removal of the template molecule; and then I,iii) polymerization of a second class of monomers in the void left by the. template, to provide a new molecule which exhibits one or more desired properties wl-~ich are similar to that of the template. In addition to preparing peptides in this manner other MBL binding molecules which are MBL inhibitors such as polysaccharides, 2o nucleosides, drugs. nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can ;also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural co anterparts, because they are typically prepared by the free radical polymerization of functional monomers. resul:ing in a compound with a nonbiodegradable backbone.
Other zs mf;thods for designing such molecules include for example drug design based on structure acrivity relationships which require the synthesis aaad evaluation of a number of compounds and molecular modeling.
Peptides which bind to the MBL may also be identified by conventional screening mcahods such as phage display procedures (e.g., met.hods described in Hart, et al., J. Biol.
3o Chem. 269:12468 ( 1994);1. Hart et al. report a. filamentous phage display library for idE:ntifying novel peptide ligands for mammalian cell receptors. In general, phage display libraries using, e.g., M13 or fd phage, are prepared using conventional procedures such as those described in the foregoing reference. The libracries display inserts containing from 4 to 80 amino acid residues. The inserts optionally rc;present a completely degenerate or a biased array of peptides. I,igands that bind selectively to MBL are obtained by selecting s those phages which express on their surface a liga~ld that binds to the MBL.
These phages thc;n are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (e.g., highest affinity) arc: further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the ~ o phage surface and the optirrmm length of the expressed peptide to achieve optimum binding to the MBL. Alternatively, such peptide ligands can be selected from combinatorial li>"~raries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation com,aared to their naturally-occurring counterparts.
i s To determine whether a peptide binds to MBL any known binding assay may be employed. For example, the peptide may be immobilized on a surface and then contacted with a labeled MBL. The ;mount of MBL which interacts with the peptide or the amount which does not bind to the peptide may then be quantitated to determine whether the peptide bi nds to MBL. A surface having the deposited monoclonal antibody immobilized thereto 2o m ay serve as a positive com.rol.
Screening of peptides of the invention., also can be carried out utilizing a ccmpetition assay. If the peptide being tested competes with the deposited monoclonal antibody, as shown by a decrease in binding of the deposited monoclonal antibody, then it is lil:ely that the peptide and the deposited monoclonal antibody bind to the same, or a closely 2s related, epitope. Still another way to determine whether a peptide has the specificity of the deposited monoclonal antibody of the invention is 'to pre-incubate the deposited monoclonal antibody with MBL with which it is normally reactive, and then add the peptide being tested to determine if the peptide being tested is inhibited in its ability to bind MBL. If the peptide bc;ing tested is inhibited thc;n, in all likelihood, it has the same, or a functionally equivalent, 3o epitope and specificity as the deposited monoclonail antibody.

Using routine procedures known to those of ordinary skill in the art, one can determine whether a peptide which binds to MBI, is useful according to the invention by determining whether the peptide is one which bIoc:ks MBL from binding to an MBL ligand.
Such assays are described above and in the Examples section. Other assays will be apparent s to those of skill in the art, having read the present specification, which are useful for determining whether a peptide which binds to MBL also inhibitors LCP
associated complement activation.
By using the deposi~.ed monoclonal antibodies of the invention, it is now possible to produce anti-idiotypic antibodies which can be used to screen other antibodies to identify ~ o whether the antibody has the same binding specificity as the deposited monoclonal antibodies of the invention.. In addition, such a~zti~-idiotypic antibodies can be used for active immunization (Herlyn, et al., Science, 232:100., 1986). Such anti-idiotypic antibodies ca:n be produced using well-known hybridoma techniques (Kohler and Milstein, Nature, 256:495, 1975). An anti-idiotypic antibody is an antibody which recognizes unique is determinants present on the deposited monoclonal antibodies. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, is respc.nsible for the specificity of the antibody. An anti-idiotypic antibody can be prepared by immunizing an animal with the deposited monoclonal antibodies. The immunized animal will recognize and respond to the idiotypic determinants 20 of the immunizing depos,.ted monoclonal antibodies and produce an antibody to these idiotypic determinants. By using the anti-idiotypic antibodies of the immunized animal, which are specific for the deposited monoclonal antibodies of the invention, it is possible to idc;ntify other clones with the same idiotype as the deposited monoclonal antibody used for immunization. Idiotypic identity between monoclonal antibodies of two cell lines 2s de monstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using anti-idiotypic antibodies, it is possible to identify other h.ybridomas expressing monoclonal antibodies having the same ep itopic specificity.
It is also possible: to use the anti-idiotype technology to produce monoclonal 3o antibodies which mimic an epitope. For example:, an anti-idiotypic monoclonal antibody made to a first monoclonal ;antibody will have a binding domain in the hypervariable region which is the image of the e~~itope bound by the first monoclonal antibody.
Thus, the anti-idiotypic monoclonal antih~ody can be used for immunization, since the anti-idiotype monoclonal antibody binding domain effectively acts as an antigen.
Activation assays al~.o can be used to assess the relative inhibitory concentrations of s a peptide in an activation assay and to identify those peptides which inhibit activation by at least, e.g., 75%.
Other assays will be: apparent to those of skill in the art, having read the present specification, which are useful for determining whether a peptide which binds to MBL also inl:.ibits MBL activation.
t o In one embodiment the peptide that inhibits the activation of MBL is an antibody or a functionally active antibody fragment. Antibodies are well known to those of ordinary skill in the science of immunology. As used herein., the term "antibody" means not only int:~ct antibody molecules but also fragments of antibody molecules retaining MBL binding ability. Such fragments are also well known in the art and are regularly employed both in i s vit~~o and in vivo. In particular, as used herein, the term "antibody"
means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab.
F(ab')2, and Fab fragments vrhich lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and rr,ay have less non-specific; tissue binding of an intact antibody (V4'ahl et al., J. Nucl. Mecl. 24:316-325 (1983)). As is well-known in the art, the zo complementarity determini:zg regions (CDRs) of an antibody are the portions of the anl:ibody which are largely responsible for antibody specificity. The CDR's directly interact with the epitope of the antigen (see, in general, Clark, 1986; Roitt, 1991 ).
In both the heavy ch~~in and the light chain variable regions of IgG imrnunoglobulins, there are four framework regions (FRI through FR4) separated respectively by three complementarity 2s derermining regions (CDRI through CDR3). The: framework regions (FRs) maintain the tertiary structure of the paratope, which is the portion of the antibody which is involved in thc; interaction with the antigen. The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3 contribute to antibody specificity. Because these CDR
regions and in particular th~~ CDR3 region confer .antigen specificity on the antibody these 3o rel;ions may be incorporated into other antibodies or peptides to confer the identical antigen specificity onto that antibody or peptide.

As discussed above ~:he MBL inhibitors of the present invention encompass in some embodiments of the invention MBL binding peptides which include a MBL binding region which specifically binds to human MBL and inhibits LCP assaciated complement activation, e.g., by preventing MBL from interacting with MBL ligands. "MBL
ligands" as s used herein are carbohydra~es or peptides with which MBL can interact.
Optionally the MBL binding region is a MBL binding CDR3 region. A "MBL binding CDR3 region"
as used herein is a CDR3 peptide sequence derived from the monoclonal antibodies produced by the hybridomas deposited with the ATCC under ATCC Accession No. (HB-12621 ), ATCC Accession No. (HB-12620), and ATCC Accession No. (HB-12619).
~o Three antibody producing hybridoma cell lines (3F8, 2A9, hMBLl.2) were deposited by Applicants with the ATCC on December 15, 1998. Hybridoma 3F8 produces monoclonal antibodoy ;FBA having binding specificity for MBL. Monoclonal antibody 3Fa includes the CDR3;FS regicm within its sequence. .As used herein "CDR3~3Fg~"
includes the CDR3 region of monoclonal antibody~3Fx~. H;ybridomat2,a9> produces monoclonal ~s antibody~2A9> having binding specificity for MBL. Monoclonal antibody~2A9~
includes the CLtR3~2A9~ region within its sequence. As used ',herein "CDR3~2A9~" includes the CDR3 region of monoclonal <~ntibody 2p9. HybTl(IOma~hMBLl.2) produces monoclonal antibody~nMem.z~ having binding specificity for MBL. Monoclonal antibody~hMaL~.z>
includes the CDRJ~h~~BLl.2) region within its sequence. As used herein "CDR3~,,MBL1.2~"
2o includes the CDR3 region of monoclonal antibody~hMSL~.2~. Each of monoclonal antibody 3F8~ monoclonal antibody f'9, and monoclonal antlbady~hMBLl.2) specifically bind to MBL
an~i prevent MBL from interacting with an MBL ligand.
The "MBL binding; C:DR3 region" refers to the CDR3(3Fg~, CDR3(zA9~ and CI)R3~hMBt.~.2~ peptide sequesnces. In one embodiment the peptides of the invention include 2s functional variants of the M:BL binding CDR3 region. A "functional variant"
as used herein is ~t peptide having the sequence of the CDR3~3Fg~, CDR3(2A9), or CDR3~hMaLl.z> regions with conservative substitutions therein. As used herein., ".conservative substitution" refers to an arr;ino acid substitution whi~~h does not alter the relative charge or size characteristics of the pe;~tide in which the amino acid substitution is made. Conservative substitutions of amino 3o acids include substitutians made amongst amino acids with the following groups: (1) M.I,L,V; (2) F,Y,Vv': (3) K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art.
For example, amino-acid substitutions may be made by PCR-directed mutation, site-directed rnutagenesis according to the method oKunkel (Kunkel, Prc~c. Nat. Acad. Sci. U.S.A. 82: 488-492, 19$5), or by chemical synthesis of a gene encoding; the CDR3 region. These and other s methods for altering a CDR3 region peptide will b~e known to those of ordinary skill in the art and may be found in rc;ferences which compile such methods, e.g. Sambrook.
et al., Molecular Cloning.' A Lab~~ratory Manual, 2nd e;di ion, Cold Spring Harbor Laboratory Prcas, 1989. The activity of functionally equivalent variants of the MBL
binding CDR3 region can be tested by the Minding and activity assays discussed above.
~o For purposes of bre~rity the term "ATCC deposited hybridoma" is used throughout the' specification to refer to the three hybridomas deposited with the ATCC on December 15, 1998. The term "deposited monoclonal antibody" is used to refer to each of the monoclonal antibodies (monoclonal antibodyt3Fgt, :monoclonal antibody~2A9~, or monoclonal an°.ibody~hMSL~.2> produced by the ATCC deposited hybridomas. For purposes of i s de Eniteness in the attached claims each of the hybridomas and monoclonal antibodies is specifically recited.
According to one embodiment, the peptide of the invention is an intact soluble anti-M:BL monoclonal antibody in an isolated form or in a pharmaceutical preparation. An intact soluble monoclonal antibody, as is well known in the art, is an assembly of 2o polypeptide chains linked by disulfide bridges. Two principle polypeptide chains, referred to as the light chain and heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chair..s and light chains are further divided into subregions referred to as variable regions and constant regions. As used herein the term "monoclonal antibody"
re~~ers to a homogenous population of immunog;lobulins which specifically bind to an 2s epitope (i.e. antigenic determinant) of human MBL.
The peptide of the invention in one embodiment is, for example, the deposited monoclonal antibody. The preparation and use of the deposited monoclonal antibody is described more fully in the attached Examples. In another embodiment the peptide of the invention is an intact antibody having the binding characteristics of the deposited 3o m~~noclonal antibody. An antibody having the binding characteristics of the deposited monoclonal antibody is one: which binds to MBL and inhibits MBL from interacting with V1'O 00/35483 PC'T/IlS99/29919 M13L ligands. One of ordinary skill in the art can easily identify antibodies having the binding characteristics of the deposited monoclonal antibody using the screening and binding assays set forth in detail below.
In one set of embodiments, the peptide useful according to the methods of the s present invention is an intact humanized anti-MBL monoclonal antibody in an isolated form or in a pharmaceutical preparation. The following examples of methods for preparing humanized monoclonal antibodies that interact~with MBL and inhibit LCP
associated complement activation are exemplary and are provided for illustrative purposes only.
A "humanized monoclonal antibody" as used herein is a human monoclonal ~ o antibody or functionally act:,ve fragment thereof having human constant regions and a MBL
binding CDR3 region from a mammal of a species other than a human. Humanized monoclonal antibodies rna;y be made by any method known in the art. Humanized monoclonal antibodies, for c:xarnple, may be constructed by replacing the non-CDR regions of a non-human mammali~~n antibody with similar regions of human antibodies while ~s retaining the epitopic specificity of the original antibody. For example, non-human CDRs and optionally some of thc; framework regions may be covalently joined to human FR
and/or Fc/pFc' regions to produce a functional antibody. There are entities in the United States which will synthesize humanized antibodies; from specific murine antibody regions commercially, such as Protein Design Labs (Mountain View California).
2o European Patent Application 0239400, the entire contents of which is hereby incorporated by reference, provides an exemplary teaching of the production and use of humanized monoclonal antibodies in which at least the CDR portion of a murine (or other non-human mammal) antibody is included in the humanized antibody. Briefly, the following methods are useful for constructing a humanized CDR monoclonal antibody 2s including at least a portion ~~f a mouse CDR. A first replicable expression vector including a suitable promoter operabhr linked to a DNA sequence encoding at least a variable domain of an Ig heavy or light chain and the variable domain comprising framework regions from a human antibody and a CDF: region of a murine antibody is prepared. Optionally a second replicable expression vector is prepared which includes a suitable promoter operabiy linked 3o to a DNA sequence encoding at least the variable: domain of a complementary human Ig light or heavy chain respectively. A cell line is then transformed with the vectors.

W O 00/35483 PCT/IlS99/29919 Preferably the cell line is an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma, trio ma, or quadroma cell line, or is a normal lymphoid cell which has been immortalized by transformation with a virus. The transformed cell line is then cultured under conditions I<:nown to those of skill in the art to produce the humanized s antibody.
As set forth in European Patent Application 0239400 several techniques are well known in the art for creating the particular antibody domains to be inserted into the replicable vector. (Preferre~~ vectors and recombinant techniques are discussed in greater detail below.) For example, the DNA sequence encoding the domain may be prepared by ~o olit;onucleotide synthesis. ~.lternatively a synthetic gene lacking the CDR
regions in which four framework regions are fused together with suitable restriction sites at the junctions, such that double stranded synthetic or restricted sulbcloned CDR cassettes with sticky ends could be ligated at the juncv:ions of the framework regions. Another method involves the preparation of the DNA sequence encoding the variable CDR containing domain by ~ s olil;onucleotide site-directed mutagenesis. Each of these methods is well known in the art.
Th~:refore, those skilled in the art may construct humanized antibodies containing a marine CIJ~R region without destroying the specificity of they antibody for its epitope.
In preferred embodiments, the humanized antibodies of the invention are human monoclonal antibodies including at least the MBL binding CDR3 region of the deposited zo monoclonal antibody. As noted above, such humanized antibodies may be produced in which some or all of the FR regions of deposited monoclonal antibodies have been replaced by homologous human FR :°egions. In addition, the Fc portions may be replaced so as to produce IgA or IgM as wel:: as human IgG antibodies bearing some or all of the CDRs of the deposited monoclonal antibody. Of particular importance is the inclusion of the is deposited monoclonal antibody MBL binding CDR3 region and, to a lesser extent, the other CIr~Rs and portions of the framework regions of the deposited monoclonal antibody. Such humanized antibodies will have particular clinical utility in that they will specifically recognize human MBL buy: will not evoke an immune response in humans against the antibody itself. In a most preferred embodiment, a marine CDR is grafted into the 3o framework region of a humor antibody to prepare thf: " humanized antibody."
See, e.g., L.

V~~O 00/35483 PCT/US99/29919 Riechmann et al., Nature :3:32, 323 (1988); M. S. l~leuberger et al., Nature 314, 268 (1985) and EPA 0 239 400 (published Sep. 30, 1987).
In addition to the deposited monoclonal antibodies, other antibodies (e.g., anti-MBL, anti-MASP, anti-mannan-like antibodies) can be generated. The following is a description s of a method for de~-elopinl; a monoclonal antibody specific for MBL (MASP-1 or -2, or m;mnan). The description i;~ exemplary and is provided for illustrative purposes only.
Murine monoclonal antibodies may be made by any of the methods known in the art utilizing MBL as an immunogen. An example of a method for producing murine monoclonals useful according to the invention is the following: Female Balb/C
mice were to initially inoculated (i.p.) wish 250 ul of the following mixture: 250 pl Titermax mixed with 100 p,g human MBL in 25G pl PBS. The following week and for three consecutive weeks thc; mice were injected with 5() p.g hMBL in 250 ~IPBS. On the 4th week the mice were injected with 25 ~g h-IBL in 250 p.l PBS and the mice were fused 4 days later.
The fusion protocol is adapted from Current Protocols in Immunology. The is splenocytes were fused l:l with myelinoma fusion partner P301 from ATCC
using PEG
150 at 50% w/v. The fusec: cells were plated at a density of 1.25x 106/ m.
with 100 ~1/well of a 96 well microtiter plate. 'The fusion media consisted of Deficient DME
high glucose, Pen/Strep (50,000 LJ pen, 50,000 ~g strep per lite:r), 4 mM L-glutamine, 20%
fetal bovine seoum, 10% thyroid enriched media, 1 % OPI, 1 °ro NEAA, 1 % HAT, and 50 p.M 2 2o me:rcaptoethanol. The cells were fed 100 pl/well on day one and 100/well media were exchanged on days '_'. 3, ~I, 7, 9, 11, and 13. The last media change before primary sc;.-eening consisted of HAT' substituted for the 1 % HT. All subsequent feedings were done with fusion media minus the minus HT or HAT. Screening was done with human MBL
pl;rted to plastic EL1S A plates (96 well plates). Purified hMBL was plated in each well at 2s 50 p,l volume containing 'Z ~g/ml MBL in 2% sodium carbonate buffer. The plates were th~.n blocked with 3°,~o BSA. in PBS. Tissue culture media (50 pl) was placed in the wells and incubated for 1 hour at room temperature. The plates were washed and a secondary H1RP labeled goat anti-mouse IgG antibody was used for detection. Colorimetric analysis was done with ABTS and read at a405 nm. Positive controls consisted of a polyclonal 3o antibody to human ~tBL. Cells are then grown in media consisting of the following:
DiVIEM high glucose no-I-glut, sod, pyruvate 500m1 (Irvine Scientific #9024), heat VliO 00/35483 PCT/US99/29919 inactivated Hyclone 10%, 1 % Non-essential amino acids, 4mM L-gluamine, 100 U/ml penicillin and 100 pg/ml streptomycin. All positive wells were then screened for function in a secondary screen.
Human monoclonal antibodies may be made: by any of the methods known in the art, s su~~h as those disclosed in L1S Patent No. 5,567,610, issued to Borrebaeck et al.. US Patent No. 565,354, issued to Ostt~erg, US Patent No. 5,571,893, issued to Baker et al, Kozber, J.
Immunol. 133: 3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, p. 51-63 (Marcel Dekker, Inc, new York, 19$7), and Boerner el al., J.
Immunol., 147: 86-95 (1991 ). In addition to the conventional methods for preparing human ~ o monoclonal antibodies, such antibodies may also bc: prepared by immunizing transgenic animals that are capable of o~roducing human antibodies (e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993), Jakobovits et ai., Nature, 362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 (1993) anti US Patent No. 5,569,825 issued to Lonberg).
An example of one method for producing :human monoclonals useful according to ~s thc; invention is the following: Peripheral Bloocl Lymphocytes (PBL) are isolated from he;~lthy human donors using density centrifugation, and further separated into B, T and accessory (A) cells, described methods such as (Danie:lsson, L., Moller, S. A.
& Borrebaeck, C..A.K. Immunology 61, 51-55 (1987)). PBL are fractionated into T and non-T
cells by ro~~etting with 2-amino ethyl {isothiouronium brorrride) - treated sheep red corpuscles, and 2o thc; latter cell population is incubated on Petri dishes coated with fibronectin or autologous plasma. Non-adherent cells (B-cells) are decanted" and adherent cells (accessory cells) are removed by IOmM EDTA. 'the B cells are stimulated with 50 p g Staphylococcus aureus Cowan I/ml and irradiated (2000R) T cells with 10 p g PWM/ml overnight. The accessory cells are stimulated with 5 1U gamma interferon/ml and 10 p m indomethacin.
The cell 2s populations are cultured in supplemented RPMI 1 fi40 which contains 10%
human AB
semm at a cell ratio of 2:1:0.4 (Ti:B:A) for a total of 6 days. The antigenic dose of MBL is 1 la g/ml. The culture is supplemented with recombinant IL-2 (5 U/ml) and sPWM-T (25%
by vol.), produced by described methods such. as (Danielsson, L., Moller, S.A.
&
Borrebaeck C.A.K. Immamology 61, 51-55 (198'1)). T cells (10 cells/ml) suspended in 3o serum-free RPMI 1640 are incubated with 2.5 mM freshly prepared Leu-OMe for 40 min at room temperature. The cells are then washed 3 times in RPMI 1640 containing 2%
human antibody serum.
In one embodiment of the invention the peptide containing a MBL binding region is a functionally active antibody fragment. Significantly, as is well-known in the art, only a s small portion of an antibody molecule, the parai:ope, is involved in the binding of the antibody to its epitope {see, in general, Clark, W.P;. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Black:well Scientific Publications, Oxford). The pFc' and Fc regions of the antibody, for examphs, are effectors of the complement cascade but are not involved ~o in antigen binding. An antibody from which the pFe' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the: antigen binding sites of an intact antibody. An isolated F(ab')Z
fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sitc;s. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or ~ s which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd (heavy chain variable region). The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light 2o chs~ins without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. The terms Fab, Fc, pFc', F(ab')2 and Fv are used consistently with their standard immunological me,~nings [Klein, Immunology (John Wiley, New York, NY, 1982);
Cl;~rk, W.R. (1986) The Experimental Foundations of Modern Immunology (Wiley &
Sons, Inc., New York); Roitt, 1. ( 1991 ) Essential Immunology, 7th Ed., (Blackwell Scientific 2s Publications, Oxford)].
As used herein the term "functionally active antibody fragment" means a fragment of an antibody molecule including a MBL binding peptide of the invention which retains the LC'.P associated complement inhibitory activity of an intact antibody having the same sp~.cificity such as the deposited monoclonal antibodies. Such fragments are also well 3o known in the art and are regularly employed both ira vitro and in vivo. In particular, well-known functionally active antibody fragments include but are not limited to F(ab')z, Fab, Fv WO 00/35483 PCTNS99/299i9 ancl Fd fragments of antibodies. These fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucf. Med. 24:316-325 (1983)). For ex~unple, single-chain antibodies can be constructed in accordance with the methods s described in U.S. Patent No. 4,946,778 to Ladner et al. Such single-chain antibodies include the variable regions of the light and heavy chains joined by a flexible linker moiety.
Mc;thods for obtaining a sinl;le domain antibody ("Fd") which comprises an isolated variable heavy chain single domain, also have been reported (see, for example, Ward et al., Nature 341:644-646 (1989), disclosing a method of'sc:reening to identify an antibody heavy ~o chstin variable region (VH single domain antibody) with sufficient affinity for its target epitope to bind thereto in isolated form). Methods for making recombinant Fv fragments ba >ed on known antibody heavy chain and light chain variable region sequences are known in the art and have been described, e.g., Moore et al., I;JS Patent No.
4,462,334. Other references describing the use and generation of antibody fragments include e.g., Fab ~ s fragments (Tijssen. Practice and Theory of Enzyme Immunoassays (Elsevieer, Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistrw 12: 1130 (1973); Sharon et al., Bi~xhemistry I5: 1591 (1976); Ehrilch et al., U.S. Patent No. 4,355,023) and portions of am.ibody molecules ('Audilore-Hargreaves, U.S. patent No. 4,470,925). Those skilled in the art rnay construct amibody fragments from various portions of intact antibodies without Zo de;~troying the specificity of the antibodies for the MBL epitope.
Functionally active antibody fragments also encompass "humanized antibody fra.gments." As one skilled in the art will recognize, such fragments can be prepared by traditional enzymatic cleavage of intact humanized antibodies. If, however, intact antibodies are not susceptible to such cleavage, because of the nature of the construction 2s involved, the noted constru~~tions can be prepared with immunoglobulin fragments used as thc: starting materials: or, if recombinant techniques are used, the DNA
sequences, th~;mselves, can be tailored to encode the desired "i:ragment" which, when expressed, can be combined in vivo or in vitr~~. by chemical or biological means, to prepare the final desired intact immunoglobulin frag:_nent.
3o In addition to the identification of peptides from libraries etc. the peptides of the invention including those containing the MBL binding CDR3 region may easily be synthesized or produced by recombinant means. Such methods are well known to those of orJinary skill in the art. Peptides can be synthesi2;ed for example, using automated peptide synthesizers which are commercially available. The peptides can be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression s vector and transforming cells with the expression vector to produce the peptide.
The sequence of the CDR regions, for instance, for use in synthesizing peptides of thc.~ invention, may be determined by methods known in the art. The heavy chain variable region is a peptide which generally ranges from lU0 to 150 amino acids in length. The light chain variable region is a peptide which generally ranges from 80 to 130 amino acids in ~o length. The CDR sequences within the heavy .and light chain variable regions which include only approximately 3-25 amino acid sequences may easily be sequenced by one of ordinary skill in the art. The: peptides may even be synthesized by commercial sources such as by the Scripps Protein ann Nucleic Acids Core Sequencing Facility (La Jolla California).
The sequences responsible for the specificity of the deposited monoclonal antibody is can easily be determined by one of ordinary skill in the art so that peptides according to the invention can be prepared using recombinant DN,~1 'technology. There are entities in the United States which will perform this function commercially, such as Thomas Jefferson University and the Scripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla California). For example, the variable region cDNA can be prepared by polymerase chain 2o reaction from the deposited hybridoma RNA using degenerate or non-degenerate primers (derived from the amino acid sequence). The cDNA can be subcloned to produce sufficient quantities of double stranded DNA for sequencing by conventional sequencing reactions or equipment.
Once the nucleic acid sequences of the heavy chain Fd and light chain variable 2s domains of the deposited MBL monoclonal antibody are determined, one of ordinary skill in the art is now enabled to produce nucleic acids which encode this antibody or which encode the various antibody fragments, humanized antibodies, or peptides described above.
It is contemplated that such nucleic acids will be operably joined to other nucleic acids forming a recombinant vector for cloning or for expression of the peptides of the invention.
3o The. present invention includes any recombinant vector containing the coding sequences, or part thereof, whether for prokaryotic or eukaryotic transformation, transfection er gene therapy. Such vectors may be prepared using conventional molecular biology techniques, known to those with skill in the ari, and would comprise DNA coding sequences for the CC~R3 region and additional variable sequences contributing to the specificity of the antibodies or parts thereof, as well as other non-specific peptide sequences and a suitable s promoter either with (Whittle et al., Protein Eng. 1:499, 1987 and Burton et al., Science 26fi:1024-1027, 1994) or w~.thout (Marasco et al., Proc. Natl. Acad. Sci.
(USA) 90:7889, 1993 and Duan et al., Proc. Natl. Acad. Sci. (USA) 91:5075-5079,1994) a signal sequence for export or secretion. Such vectors may be transformed or transfected into prokaryotic (Hose et al., .Science 246:12'75, 1989, Ward et al., Nature 341: 644-646, 1989; Marks et al., ~o J. l~ol. Biol_ 222:581, 1991 and Barbas et al., Proc, Ncxtl. Acad. Sci.
(U~fA) 88:7978, 991) or eul<:aryotic (Whittle et al., 1987 and Burton et al., 1994) cells or used for gene therapy (Marasco et al., 1993 and Cnan et al., 1994) by conventional techniques, known to those with skill in the art.
As used herein, a "vector" may be any of a number of nucleic acids into which a is desired sequence may be inserted by restriction and Iigation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DT A although RNA vectors are also available. Vectors include, but are not limited to, pla~mids and phagemids. A cloning vector is one which is able to replicate in a host cell, anci which is further characterized by one or more endonuclease restriction sites at which zo the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the: new recombinant vector retains its ability to replicate in the ho:.t cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per ho~,t before the host reproduces by mitosis. In the ease of phage, replication may occur 2s act:.vely during a lytic phase or passively during a lysogenic phase. An expression vector is one: into which a desired I):VA sequence may be inserted by restriction and ligation such that it is opexably joined v.o regulatory sequence, and may be expressed as an RNA
transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the 3o vector. Markers include, fo;- example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode en:ymes whose activities are detectable by standard assays known in the art (e.g., 13-~;alactosiidase or alkaline phosphatase), and genes which visibly affect the phenotype of tra:lsformed or transfected cells, hosts, colonies or :plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in s the DNA segments to which they are operably joined.
The expression vectors of the present invention include regulatory sequences operably joined to a nucleotide sequence encoding one of the; peptides of the invention. As used herein, the term "regulatory sequences" means nucleotide sequences which are necessary for or conducive to the transcription of a nucleotide sequence which encodes a desired ~ o peptide and/or which are necessary for or conducive to the translation of the resulting transcript into the desired peptide. Regulatory sequences include, but are not limited to, S' sequences such as operator, promoters and riboso~mc: binding sequences, and 3' sequences such as polyadenylation signals. The vectors of the invention may optionally include 5' leader or signal sequences, S' or 3' sequences encoding fusion products to aid in protein is purification, and various markers which aid in the identification or selection of transformants. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. The subsequent purification of the peptides ma.y be accomplished by am/ of a variety of standard means known in the art.
A preferred vector for screening peptides, but not necessarily preferred for the mass 2o production of the peptides of the invention, is a recombinant DNA molecule containing a nu~~leotide sequence that codes for and is capable of expressing a fusion peptide containing, in the direction of amino- to carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) a peptide of the invention, and, optionally, (3) a fusion protein domain.
The vector includes DNA regulatory sequences for expressing the fusion peptide, preferably 2s prokaryotic regulatory sequences. Such vectors can be constructed by those with skill in the art and have been described by Smith et al. (Science 228:1315-1317, 1985), Clackson et al.
(:~'~ture 352:624-628, 1991 ); Kang et al. (in "Methods: A Companion to Methods in Enzymology: Vol. 2", R.A. Lerner and D.R. Burton, ed. Academic Press, NY, pp 118,1991 ); Barbas et al. (Pnoc. Natl. Acad. S'ci. (L'S~l) 88:7978-7982, 1991 ), Roberts et al.
30 (P.roc. Natl. Acad. Sci. (USfI) 89:2429-2433, 1992) V4'O 00/35483 PCT/US99/29919 A fusion peptide ma:r be useful for purification of the peptides of the invention. The fusion domain may, for example, include a poly-His tail which allows for purification on Ni-~- columns or the maltose: binding protein of the commercially available vector pMAL
(New England BioLabs, Be~rerly, MA). A currently preferred, but by no means necessary, s fusion domain is a filamentous phage membrane anchor. This domain is particularly useful for screening phage display libraries of monoclonal antibodies but may be of less utility for the mass production of antibodies. The filamentouso phage membrane anchor is preferably a domain of the cpIII or cp'1III coat protein capable of associating with the matrix of a filamentous phage particle, i:hereby incorporating the fusion peptide onto the phage surface, io to enable solid phase binding to specific antigens or epitopes and thereby allow enrichment and selection of the specific antibodies or fragments. encoded by the phagemid vector.
The secretion signal is a leader peptide domain of a protein that targets the protein membrane of the host cell, such as the periplasmic membrane of gram negative bacteria. A
preferred secretion signal fc~r E. coli is a pelB secretion signal. The predicted amino acid is residue sequences of the secretion signal domain :from two pelB gene producing variants from Erwirria carotova are described in Lei, et al. (Nature 381:543-546, 1988). The leader secuence of the pelB protein has previously been used as a secretion signal for fusion proteins (Better, et al., Science 240:1041-1043, 1988; Sastry, et al., Proc.
Natl. Acad. Sci (USA) 86:5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad S'ci. (USA) 87:8095-20 80!9, 1990). Amino acid re;~idue sequences for other secretion signal peptide domains from E. coli useful in this invention can be found in Olives, In Neidhard, F.C.
(ed.), Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington, D.C., 1:56-69 (1987).
To achieve high levels of gene expression in E. coli, it is necessary to use not only 2s strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is effciently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located nucleotides upstream from the initiation codon (Shine, et al., Nature 254:34, 1975). The sequence, AGGAGGU. which is called the Shine-Dalgarno (SD) sequence, is 3o complementary to the 3' end of E. coli 16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors:

(I) The degree oi' complementarity between the SD sequence and 3' end of the 16S rRNA.
(ii) The spacing and possibly the DNA sequence lying between the SD sequence and the AUG (Roberts, et al., Proc. l1~'atl. Acad. Sci. (LISA) 76:760.,1979a:
s Roberts, et al., Proc. Natl. Acad. Sci. (USA) 76:5596, 1979b; Guarente, et al., Science :09:1428, 1980; and Guarente, et al., C.'ell 20:543, 1980).
Optimization is achieved by measuring the level of expression of genes in plasmids in which this spacing is systematically altered. Comparison of different mRrJAs shows that there a:re statistically preferred sequences from positions -20 to +13 (where the A of the AUG is position 0) (Gold, et al., Arrnu. Rev. Microbiol. 35:365, 1981). L eader sequences have been shown to influence translation dramatically (Roberts, et al., 1979a, b supra).
(iii) The nucleotide sequence following the AUG, which affects ribosome binding (Taniguchi, et al., J. Mol. Binl_, 118:533, 1978).
i s The. 3' regulatory sequences define at least one termination (stop) codon in frame with and ope;rably joined to the heterologous fusion peptide.
In preferred embodiments with a prokaryotic: expression host, the vector utilized includes a prokayotic origin of replication or replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule 2o extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such origins of replication are well I<;nown in the art. Preferred origins of replication are those that are efficient in the host organism. A preferred host cell is E. toll.
Fo:- use of a vector in E. toll, a preferred origin of replication is ColE1 found in pBR322 and a variety of other common plasmids. Also preferred is the plSA origin of replication 2s found on pACYC and its derivatives. The ColEl and plSA replicons have been extensively utilized in molecular biology, are available on a variety of plasmids and are described by Sa~nbrook. et al.. :Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).
In addition. those embodiments that include a prokaryotic replicon preferably also 3o include a gene whose expret~sion confers a selective advantage, such as drug resistance, to a banterial host transformed therewith. Typical bacterial drug resistance genes are those that W~~ 00/35483 PCT/US99/29919 confer resistance to ampicillin, tetracycline, neo:mycin/kanamycin ar chloramphenicol.
Vectors typically also contain convenient restriction sites for insertion of translatable DNA
seq uences. Exemplary vectors are the plasmids pUC 18 and pUC 19 and derived vectors such as pcDNAII available fiom Invitrogen, (San Diego, CA).
s When the peptide of the invention is an antibody including both heavy chain and light chain sequences, these sequences may be encoded on separate vectors or, more conveniently, may be expressed by a single vector.. 'the heavy and light chain may, after translation or after secretion, form the heterodirneric structure of natural antibody molecules. Such a heterodirneric antibody may or rnay not be stabilized by disulfide bonds ~o between the heavy and light chains.
A vector for expression of heterodimeric antibodies, such as the intact antibodies of the invention or the F(ab')2, Fab or Fv fragment antibodies of the invention, is a recombinant DNA molecule adapted for receiving and expressing translatable first and second DNA sequences. That is, a DNA expression vector for expressing a heterodimeric is antibody provides a system :for independently cloning (inserting) the two translatable DNA
sequences into two separate cassettes present in the vector, to form two separate cistrons for exyressing the first and second peptides of a heterodirneric antibody. T'he DNA expression vector for expressing two cis,trons is referred to as a dicistronic expression vector.
Preferably, the vecv:or comprises a first cassette that includes upstream and zo downstream DNA regulatory sequences operably joined via a sequence of nucleotides adapted for directional ligation to an insert DNA.. The upstream translatable sequence preferably encodes the secretion signal as described above. The cassette includes DNA
regulatory sequences for expressing the first antic>ody peptide that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the cassette via zs the sequence of nucleotides ;adapted for directional ligation.
The dicistronic exprcasion vector also contains a second cassette for expressing the second antibody peptide. The second cassette includes a second translatable DNA sequence that preferably encodes a secretion signal, as described above, operably joined at its 3' terminus via a sequence of nucleotides adapted far directional ligation to a downstream 3o DrdA sequence of the vector that typically defines at least one stop codon in the reading frame of the cassette. The second translatable DNA sequence is operably joined at its 5' WI) 00/35483 PCTNS99/29919 tern..inus to DNA regulatory sequences forming the 5' elements. The second cassette is cap2~ble, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second fusion peptide comprising a secretion signal with a peptide coded by the insert DN~~.
s The peptides of the present invention rnay also, of course, be produced by eukaryotic cell; such as CHO cells, human hybridomas, immortalized B-lymphoblastoid cells, and the like. In this case, a vector is constructed in which eukaryotic regulatory sequences are operably joined to the nucleotide sequences encoding the peptide. The design and selection of an appropriate eukaryotic vector is within the ability and discretion of one of ordinary o skill in the art. The subsequent purification of the peptides may be accomplished by any of a variety of standard means known in the art.
In another embodiment, the present invention provides host cells, both prokaryotic and eukaryotic, transformed ~~r transfected with, and therefore including, the vectors of the present invention.
t s As used herein, a coding sequence and regulatory sequences are said to be "operably joined" when they are covulently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional peptide, two DNA sequences are said to be operably joined if induction of a promoter in the 5' 2o regulatory sequences results in the transcription of tlhe coding sequence and if the nature of the linkage between the two DNA sequences does not ( 1 ) result in the introduction of a frame-shift mutation, (2) i:m:erfere with the ability of the promoter region to direct the transcription of the coding sc;quences, or (3) interfere with the ability of the corresponding RN.~ transcript to be translated into a protein. Thus, a promoter region would be operably zs joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such tlmt the resulting transcript might be translated into the desired peptide.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell type., but shall in general include, as necessary, 5' non-transcribing 3o and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.

W~0 00/35483 PCT/US99/29919 Especially, such 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
Regulatory sequences may also include enhanc;er sequences or upstream activator sequences, as desired.
s According to the methods of the invention, the compositions may be administered in a ~~harmaceutically acceptable composition. In general, pharmaceutically-acceptable carriers for monoclonal antibodies, antibody fragments, and peptides are well-known to tho;~e of ordinary skill in the art. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere 'with the effectiveness of the biological ~o activity of the active ingredients, i.e., the ability of the MBL inhibitor to inhibit LCP
associated complement activation. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers. stabilizers, solubilizers and other materials which are well-known in the art. Exemplary pharmaceutically acceptable carriers for peptides in particular are des~,ribed in U.S. Patent No. 5,211,657. The peptides of the invention may be formulated ~s rote preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants (e.g., aerosols) and injections, and usual ways for- oral, parenteral or surgical administration.
The invention also embraces locally administering the compositions of the invention, including as implants.
According to the methods of the invention the compositions can be administered by 2o injection by gradual infusion over time or by any other medically acceptable mode. The administration may, for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. Preparations for pa.renteral administration includes sterile aqueous or nonaqueous solutions, suspensions andl emulsions. Examples of nonaqueous sohrents are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an 2s injeetable organic esters such as ethyloliate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions., including saline and buffered media.
Par~nterai vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Kinger's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, (such as those based on Ringer's dextrose), 3o and the like. Presen~atives ;end other additives may also be present such as, for example, ant:.microbials, antioxidants, chelating agents, and inert gases and the like.
Those of skill in w0 OOI35483 PCTNS99/29919 the art can readily determine the various pararneters for preparing these alternative pharmaceutical compositions without resort to undue experimentation. When the compositions of the inventio n are administered for the treatment of pulmonary disorders the compositions may be delivered for example by aerosol.
s The compositions o:f the invention are administered in therapeutically effective am~~unts. As used herein, an "effective amount" of the inhibitor of the invention is a dosage which is sufficient to inhibit the increase in, m<~intain or even reduce the amount of undesireable LCP associated complement activation. The effective amount is sufficient to produce the desired effect of inhibiting associated cellular injury until the symptoms ~o ass~~ciated with the MBL mediated disorder are ameliorated or decreased.
Preferably an effc;ctive amount of the peptide is an effective .amount for preventing cellular injury.
Generally, a therapeutically effective amount may vary with the subject's age, condition, and. sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. The dosage may be adjusted by the: individual physician or veterinarian in i s the event of any complication. A therapeutically effective amount typically will vary from about 0.01 mg/kg to about _'~00 mg/kg, were typically from about 0.1 mg/kg to about 200 mg.~kg, and often from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days (.depending of course of the mode of administration and the factors discussed above). A preferred concentration of the inhibitor 2o is a concentration which is equimolar to the concentration of MBL in the plasma of a subject. The normal plasma concentration of MBI_, c;an be assessed clinically.
A normal range of MBL is 1-2yg/ml MBL,/plasma.
One of skill in the art can determine what an effective amount of an inhibitor is by scr~:ening the ability of the inhibitor to inhibit the LCP associated complement activation in 2s an in vitro assay. The activity of the inhibitor canr be defined in terms of the ability of the inhibitor to inhibit LCP associated complement activation. An exemplary assay for measuring the ability of a putative inhibitor of the invention to inhibit LCP
associated complement activation is provided in the Examplc;s and has been discussed above. The exemplary assay is predictv.ve of the ability of an inhibitor to inhibit LCP
associated 3o complement activation in vivo and, hence, can be used to select inhibitors for therapeutic applications.

The MBL inhibitors may be administered in a physiologically acceptable carrier.
The: term "physiologically-a~~ceptable" refers to a non-toxic material that is compatible with the biological systems such of a tissue or organism, The physiologically acceptable carrier must be sterile for in viva administration. The characteristics of the carrier will depend on s the route of administration. The characteristics of the carrier will depend on the route of adrninistration.
The invention further provides detestably labeled, immobilized and toxin conjugated forms of the peptides, antibodies and fragments thereof. The antibodies may be labeled using radiolabels, fluorescent labels, enzyme labels, fivee radical labels, avidin-biotin labels, ~o or bacteriophage labels, using techniques known to the art (Chard, Laboratory Technigues in Biology, "An lntroduct.on to Radioimmunoassay and Related Techniques,"
North Holland Publishing Company (1978).
Typical fluorescern: labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin. allophycocyanin, and fluorescamine.
~ s Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazole~, and the oxalate esters.
Typical biolumines<;ent compounds include luciferin, and luciferase. Typical en.,ymes include alkaline phosphatase, 13-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
2o The invention also includes methods for screening a subject for susceptibility to tre;~tment with an MBL inhibitor. In one aspect, the method is accomplished by isolating a mammalian cell from a subject and detecting the presence of an MBL or an MBL
ligand on a ~~urface of the mammalian cell. The presence o:f the MBL indicates that the cell is su~;ceptible to LCP-associated complement activiation, and that the subject is susceptible to 2s treatment with an MBL inhibitor. The mammalian cell may be isolated by any method known in the art, for instance by a biopsy. Another method for accomplishing the screening as~,ay involves the steps of contacting a mammalian cell from the subject with a labeled isolated MBL binding peptide and detecting the presence of an MBL on the surface of the mammalian cell. This assay may be performed in vitro, ex vivo, or in vivo.
Many labels 3o v~~l~iich can be used to observe the MBL binding peptide interacting with the mammalian cell area known in the art under each of these conditions. For instance, radioactive compounds V1'O 00/35483 PCT/US99/299I9 can be used in vitro, and other biocompatible labels can be used ex vivo or in vivo. Once the subjects are identified which are susceptible to treatment with an MBL
inhibitor, the subjects can then be treated ;according to the methods of the invention.
The following examples are provided to ilhustrate specific instances of the practice s of the present invention and are not to be construed as limiting the present invention to these ex~unples. As will be apparent to one of ordinary skill in the art, the present invention will fine application in a variety ~f compositions and methods.
Examples ~o Ex,~mple 1:MBL and Complement Deposition on Human Coronary Arteries.
Isolation and Purification ofMBL. MBL and associated MASPs were purified from human plasma. MBL was isolated from human plasma as previously described{Tan, Chung, et al. 1996 Biochem. J. 319, 329-332}. Briefly, human plasma was mixed with 7%
PEG3500 (w:v). The pellet was collected by centrifugation and resuspended in TBS-Ca2+
is [50 mM Tris, 150 mM N;~CI, 0.05% Tween 20 and 20 mM CaCl2 at pH 7.8]. The supernatant was applied to a mannan-Sepharose column (25 ml, Sigma). The column was washed with TBS-C'.a2+ with 109mM EDTA]. "I'he protein containing supernatant was calcified to 40 mM calcium and then applied to a maltose-Sepharose column (5 ml). The column was washed with T'I3S-Ca2+ and then eluted. with TBS-Ca2+ containing 100 mM N-2o ace~tylglucosamine. Western analysis and SDS-PAGE established purity for MBL, and the ab:;ence of IgG and IgM. Purified MBL and associated MASPs were analyzed by SD S/PAGE. Western blotting was performed to rule out IgG andlor IgM
contamination.
Production of Anti-.~luman MBL Antibodies. Purified human MBL was used to immunize rabbits to produ~~e polyclonal anti-human. MBI. antibodies (Harlow E, et al., 2s An!ibodies: A laboratoy manual. Cold Spring Harbor, NY, Cold Spring Harbor La~oratory, 1988). Adult rabbits were injected with 100 p,g of MBL emulsified in complete Fre;und's adjuvant. Booster immunizations ( 100 ~g of MBL in incomplete Freund's adjuvant) were started 4 wk after the priming immunir..ation and continued at 4 wk intervals.
Polyclonal IgG anti-human MBL antibody (R2.2) was purified from sera by protein G
3o affinity chromatography.

Human Coronary Ar,!ery Immunohistochemistry. Immunohistochemical analysis of MH~L, C3d, IgG, IgW, transferrin, and haptoglobin deposition was performed on tissue spe~~imens from normal (rn-=14) and atherosclerotic human epicardial coronary arteries (n=18) obtained at autopsy (Department of Pathology, University of Helsinki, Finland) from s patients who expired secor,,dary to an acute myocardial infarction (MI). The control spe~eimens were histologically normal coronary arteries obtained from patients who died from non-cardiovascular causes. The mean (~ SD) MI age (time difference between the beginning of the clinical episode and death) was :5 =~ 5 days. The mean (~ SD) age of patients suffering from acute MI was 65 t 15 years compared to 66 ~ 24 years for the to control patients. Infarcted myocardium was identified macroscopically at autopsy by dis<;olor, pallor, and hyperemia. To improve macroscopic diagnosis, a slice of non-fixed myocardium was incubated in nitroblue tetrazolium solution that leaves the damaged my~~cardium unstained. Histopathological first signs of infarction were wavy myocardial fibers and myocytolysis followed by signs of coagulation necrosis (i.e., edema, hemorrhage, is neutrophil infiltration and pyknosis of nuclei). Infarcts older than 24 hr showed signs of total coagulative necrosis with loss of nuclei and striations together with heavy interstitial neutrophil infiltration. Coronary blood vessel samples for indirect imrnunofluorescence (IFI~) microscopy were snap frozen in liquid nitrogen and stored at -80 "C until analyzed. Frozen sections (4 pm) were air dried and fixed in -20 °C acetone for 20 10 ~nin. The tissue samples were then incubated for 30 min at 22 °C
with either polyclonal rab'oit anti-human C3d (Dakopatts, Glostrup, Denmt~rk), MBL (polyclonal R2.2), IgG, IgM, trar~sferrin, or haptoglobin antibody (all from Behringwerke AG, Germany).
After washing wit:1 PBS, the specimens were then stained with an appropriate fluorescein isothiocyanate (FI'rC)-conjugated secondary antibody. Controls consisted of specimens incubated with 2s nor.immune sera or the secondary antibody alone. The slides were then mounted with Mowiol and examined with au~ Olympus Standard microscope equipped with a filter specific for FITC-fluorescence.
Results. Atherosclerotic coronary arteries obtained from patients suffering from acute MI demonstrated specific MBL and C3d deposits on the endothelium, intima, and 3o media Immunohistochemical. analysis of human coronary arteries, and in particular, the IFL
mic;roscopical demonstration of MBL and C3d deposition in an atherosclerotic human V1~0 00/35483 PCT/US99/Z9919 coronary artery was performed. MBL and C3d were observed co-localized within the atherosclerotic lesion. M>3L staining of a normal coronary artery was also performed.
An.tisera against human transferrin, haptoglobin, IgG, and IgM did not stain normal or atherosclerotic human coronary arteries.
s Additionally, MBL was observed to co-localize with C3d, with staining intensity being greatest in ruptured atherosclerotic plaques. Specifically, MBL and C3d deposition appeared to be greatest in the lipid core and surrounding areas of this core in atherosclerotic lesions. No MBL deposits were seen on normal coronary arteries, although the basement membrane sometimes appeared to stain lightly for MBL. Further, antisera against human ~o transferrin, haptoglobin, Ig~3, and IgM did not stain normal human coronary arteries or ath.erosclerotic lesions in weasels obtained from acute MI patients.
Similarly, no staining was observed in control experiments in which human coronaries were stained with non immune rabbit serum or with the secondary antibody only. These data demonstrated that M13L co-localized with complement in human coronary atherosclerotic lesions in patients t s wl-.o have died of acute MI.
Example 2: Endothelial Hy~~oxia/Reoxygenation Effects MBL Deposition.
Cell Culture. Hum;~n umbilical vein endothelial cells (HUVECs) were harvested with 0.1% collagenase (Worthington Biochemical Corp., Freehold, NJ) and suspended in 2o Mc;dia 199 containing 20% heat-inactivated bovine calf serum (Gibco Life Technologies Inc., Grand Island, NY). The cells were initially seeded in either 75 cmz flasks or 100 mm Petri dishes (Corning Costar, Cambridge, MA), and incubated at 37°C in 95% air and 5%
CO2. When confluent, the endothelial cells were passaged with 0.5% trypsin-EDTA.
Endothelial cell purity was assessed by phase microscopic "cobblestone appearance", 2s uptake of fluorescent acetylated low-density lipoprotein and the presence of won Willebrand facaor. All experiments were conducted on HUVE(~s during passages 1-3.
MBL-depleted Human Serum (HS). HS was depleted of MBL by affinity chromatography using manr~an cross-linked to 4% beaded agarose (Sigma Chemical Co., St.
Lcuis, MO). All operations were performed at 4 '°C. HS was treated with 2 mmol/L
3o ethylenediamine tetraacetai:e (EDTA) and phen,ylrnethanesulfonyl fluoride (PMSF) to inhibit complement activation and was applied to a mannan column equilibrated with V1'O 00/35483 PCT/US99/299i9 loading buffer (1.25 mmol/I, NaCI, 10 mmol/L imidazole, 20 mmol/L CaCl2, pH

7.8). The resultant eluent was dialyzed overnight in Hank's buffered salt solution containing Mg2+
anti Ca2+.
Flow Cytometry. HUVECs were grown to confluence in 100-mm Petri dishes s coated with gelatin. MBI, deposition was measured by flow cytometry in normoxic HL~VECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30% HS. After washing the cells in Ca2+ free or sufficient buffer, the cells we re fixed, scraped, and then incubated with 20 ~,g,~ml of monoclonal anti-human MBL
antibody (Biodesign, Kenr~ebunk, ME, clone # l!. 31-1 ) or isotype control monoclonal ~o antibody to porcine CSa for 1.5 hr at 4 °C. The cells were then washed and incubated with a FITC-conjugated goat anti-mouse IgG secondary antibody for 1 hr at 4 °C. MBL
deposition on HUVECs was measured by florescence activated cell sarting (FACS) using the FACSort flow cytometer (Becton Dickinson, San Jose, CA). All flow cytometry experiments were performed in triplicate.
~s Enryme-LinlcEd Imrr~unoabsorbent Assay (ELISA) Experiments. C3 and MBL
specific cell surface ELISA;~ were developed using; peroxidase-conjugated polyclonal goat anti-human C3 antibody (C'.appel, West Chester, lPA) and monoclonal anti-human MBL
antibody (Biodesign, Kennebunk, ME, clone #131-l ), respectively. HUVECs were grown to confluence on 0.1 % gelatinized 96-well plastic plates (Corning Costar, Cambridge, MA).
2o Th~~ plates were then subjecaed to 0 (normoxia) or 24 hr of hypoxia.
Hypoxic stress was maintained using a humidified sealed chamber (Cov Laboratory Products, Inc., Grass Lake, MI) at 37 °C gassed with 1°,'° 02, 5% C02, balance N(Collard CD, et al., "Reoxygenation of hypoxic human umbilical vein endothelial cells activates the classical complement pathway", Circulation 199'x;96:326-333). Following the specified period of normoxia or Zs hypoxia, the cell media were aspirated and 100 ~.1 o:f one of the following was added to each well: 1) 30% HS, 2,1 Hank's balanced salt solution, 3) 30% HS + 3, 30, or 300 mmol/L
GIoNAc, 4) 30% HS + 3, 30, or 300 mrnol/L D-mannose, 5) 30% HS + 3, 30, 300 mmol/L
L-mannose, 6) 30% MBL-depleted HS + 3F8 (0, 20, 50 ~g/ml)or 7) 30% MBL-depleted HS
+ (f.6 ~g/ml MBL. Additionally, 100 pl of purified MBL (3-300 ng/ml) was added to select 3o wells to form a standard curve for quantitative analysis of MBL deposition.
The cells were then reoxygenated for 3 hr at 37°C in 95% air and _'>% COZ. The cells were washed and then fixed with 1% paraforrnaldehyde (Sigma Chemical Co., St. Louis, MO) for 30 min.
The cells were then washed and incubated at 4 °C l:or 1.5 hr with 50 p.l of peroxidase-conjugated polyclonal goat anti-human C3 antibody (1:1000 dilution) or monoclonal anti-human MBL antibody ( 1:1000 dilution). The MB:L ELISA plates were then washed and s incubated for 1 hr at 4 °C', with 50 pl of peroxidase-conjugated polyclonal goat anti-mouse IgG secondary antibody (1:1000 dilution). After washing the cells, the plates were developed with 50 p.1 of ABTS (2,2'-azino-bis(3-ethy:lbenzthiazoline-6-sulfonic acid)), and react (Molecular Devices, S unnyvale, CA) at 405 nm. Background controls for the C3 EL1SA consisted of cells to which only the anti-hutrian C3 antibody was added (i.e., no HS) ~o or cells incubated with 30% .'teat-inactivated HS. Background controls for the MBL ELISA
consisted of cells to which only secondary antibody and an isotype control monoclonal antibody to porcine C~a were added. Background optical density was subtracted from all groups. All ELISA experiments were performed _'3 times using 6 wells per experimental group. Endothelial C3 and MBL deposition on n~ormoxic vs. hypoxic HUVECs was ~s analyzed by two-way analysis of variance (ANOVA).
Results. Flow cytc~metric analysis (Figure 2) of endothelial MBL deposition rev~:aled that the mean fluorescent intensity (MFI) of hypoxic HUVECs (24 hr) reo:~cygenated (3 hr) in 30°,~o HS was significantly greater than normoxic HUVECs or hyf~oxic HUVECs reoxygenated in buffer alone.. Further, MBL deposition was not 20 observed following hypoxia/reoxygenation if the cells were washed in Caz+-free buffer.
Thus, MBL deposition on hypoxic/reoxygenated HLJVECs was Ca2+-dependent.
In order to further confirm these findings, MBI~ deposition was measured by ELISA
on normoxic HUVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30% HS or 30% I-1S treated with 3, 30, or 300 mmol/L of 2s N-acetyl-D-glucosarnine (GIuNAc) to competitively inhibit MBL deposition.
MBL
def~osition on hypoxic HL~VECs reoxygenated i.n 30% HS was significantly greater (approximately 3-fold increase; p<0.05) than on normoxic HUVECs or HUVECs reoxygenated in HS treated with GIuNAc (Figure 3). Addition of GIuNAc to the HS
significantly inhibited MBI. deposition on hypoxic/reoxygenated HUVECs in a dose-3o deyendent manner with 3, 30 and 300 mmol/L of GIuNAc attenuating MBL
deposition 40==4%, 715% and 9613°/~, respectively. Finally, quantitative analysis of the standard V1'O 00/35483 PCT/US99/29919 curve formed by the addi~.ion of purified human MBL (3-300 ng/ml) revealed that approximately 3 ng or 8.3x10~s fmol of MBL. maximally deposits per well (e.g., 48"?001000 moleculeslcell) of hypoxic/reoxygenated HUVECs assuming 2x10s HUVECs/well and a MBL MW of 600 kDa. 'Chas, hypoxia/reoxygenation increased s endothelial MBL deposition.
Exaunple 3: Deposition of iC'3b Following Competitive Inhibition of MBL.
HUVEC cell culture ;end quantitation of iC3b deposition by ELISA were performed as outlined in Example 2.
~o Results. HUVECs vrere subjected to 0 or 24 hr of hypoxia followed by 3 hr of reo:~cygenation in the presen<;e of 30% HS or 30% HS treated with 3, 30, or 300 mmol/L
GIuNAc, D-mannose or L-mannose in order to inhibit MBL deposition, LCP
activation and iC3b deposition. Deposition of iC3b on hypoxic H1:IVECs reoxygenated in 30% HS
or 30°~o HS treated with L-:nannose was signific;amtly greater (approximately 3-fold;
~ s OD4os=0.140.01; p<0.05) than normoxic HUVECs (OD4os=O.OSt0.01 ) or hypoxic HUVECs reoxygenated in H~~ treated with GIuNAc or D-mannose (Figure 4a).
Further, D-matrrrose, but not I,-mannose, inhibited iC3b deposition on hypoxic/reoxygenated HUVECs in dose-dependent manner with 3, 30 and 300 m~mol/L of D-maumose attenuating iC3b deposition 192%, ~:?t3% a,nd 962%, respectively. Thus, these data demonstrated that 2o inhibition of MBL deposition using GIuNAc or D-mannose during reoxygenation significantly attenuated complement activation and iC3b deposition following reo:cygenation of hypoxic endothelial cells. Further., inhibition of iC3b deposition with maumose was stereospecifrc as L-mannose in concentrations up to 300 mmol/L did not inhibit iC3b deposition (Figure 4a).
Example 4: Deposition of iC3b Following MBL Depletion and Reconstitution.
HUVEC cell culture and quantitation of iC3b deposition by ELISA were carried out as in Example 2.
Results. HU~'ECs vrere subjected to 0 or 24 hr of hypoxia followed by 3 hr of 3o reo:~ygenation in the presenr.e of 30% HS, 30% MBL,-depleted HS or 30% MBL-depleted HS to which MBL was added back (Figure 4b). Deposition of iC3b on hypoxic HUVECs reoxygenated in HS was significantly greater (p<0.05) than on normoxic HUVECs.
However, iC3b deposition cm hypoxic HUVECs reoxygenated in MBL-depleted HS was significantly less (p<0.05) than on hypoxic HUVECs reoxygenated in HS. When MBL was adc.ed back to the MBL-depleted HS, iC3b deposition on HUVECs following 24 hr of s hypoxia and 3 hr of reoxygenation was restored. These data demonstrated that reoxygenation of hypoxic human endothelial cells activated the LCP leading to increased deposition of iC3b.
Ex~unple 5: Complement hemolytic assay (CHSOLof"MBL-depleted HS.
~o Methods. Hemolytic assays were completed as previously described by us {A::nsterdam, Stahl, et al., Limitation of reperfusion injury by a monoclonal antibody to CSa during myocardial infarction. in pigs, Am. J. Physiol. Heart Circ. Physiol.
1995; 268:H448-H457} {Lennon, Collard, et al., Complement-induced endothelial dysfunction in rabbits:
me~~hanisms, recovery, and ;sender differences, Am. J. Physiol. Heart Circ.
Physiol., 1996;
is 27(I:H1924-H1932}{Vakeva, Agah, et al. Myocardial infarction and apoptosis after myocardial ischemia and re perfusion. Role of the terminal complement components and inhibition by anti-CS therapy., Circulation 1998; 97:2259-2267}. Briefly, chicken red blood cel;,s were sensitized with sl:.eep anti-chicken antibodies. Serial dilutions of sera were then used to lyse the cells. Hemolytic activity was calculated by using 0.1% Triton X100 and Zo PB S as positive and negative controls, respectively. C>ptical density was read at 550 nm on a plate reader. Percent hemolytic activity was calculated as follows:
(Sample OD - PBS) / (Triton. OD - PBS) X 100 = % hemolytic OD
Samples were run in triplicate and at three determinations per group were performed.
Results. Complement hemolytic assay (CH<;o) was performed on the MBL-depleted 2s HS in order to demonstrate; that MBL depletion did not alter the classical complement pathway. CHso of the MBI,-depleted HS revealed classical complement pathway activity similar to that of complete 1-1S (Figure 5). Similar :findings were observed when antibodies 3F3, and 2A9 were used. 'thus, the decrease in iC',3b deposition on hypoxic HUVECs reoxygenated in MBL-depleted serum was not a result of altered classical pathway 3o complement components (i.e., C: l q, C 1 r, and C 1 s).

W~D 00/35483 PCT/US99/29919 Ex~unple 6: Western blot analysis of C3 activation following hypoxia/reoxygenation using purified C2, C3, C4, and ME~L.
Western Blot. HU~~ECs were grown to confluence in 96 well plates and then subjected to normoxia or h~rpoxia (24 hr). The cells were then washed with GVB+ and s reoxygenated for 3 hr in the presence of 50 Pl of the following complement cocktail: MBL
(1.~! p,g/ml), C2 (8 p.g/ml), C3 (400 ~g/ml), and C~4 (200 p,g/ml) (C2, C3, and C4 were purchased from Advanced Research Technologies:; San Diego, CA). These complement concentrations were represc;ntative of the concentrations normally present in 30% HS.
Following reoxygenation, the supernatants were collected and the protein concentration io determined (BioRad, Hercules, CA). Five p.g of total protein was then resolved by 9%
SDS-PAGE under reduced conditions. The gel was then transferred to nitrocellulose, blocked, and probed for the C3 and C3b a'-chain by western blot (Collard CD, "Rc:oxygenation of hypoxic human umbilical vein endothelial cells activates the classical complement pathway", Circ:ulation 1997;96:326-333). Purified C3 and C3b {Advanced ~s Re:;earch Technologies; San Diego, CA) served as internal standards for MW
comparisons of the cleaved C3 a'-chain. This experiment was performed 5 times (n=5).
Results. Western blot analysis of the C3 and C3b a'-chain was performed under reduced conditions on the supernatants of normo:Kic and hypoxic (12 hr) HUVECs reoxygenated (3 hr) in the presence of purified C2, C3, C4, and MBL (Figure 6). A
2o sig:zificant increase in the C3b a'-chain band density was observed in the hypoxic/reoxygenated supernatants (Lanes 2 and 4) compared to the normoxic supernatants (Lz.nes 1 and 3). These results demonstrated LC'.P-mediated activation of C3 following endothelial hypoxic/reoxygenation independent of natural antibody or Cl. Thus, complement activation following endothelial h;ypoxia/reoxygenation appeared to be 2s mediated by the LCP and not the classical complement pathway.
Ex;~mple 7: Microphysiometer evaluation of HUVF;C receptor-ligand activation.
Microphysiometry. C'.hanges in 1-IUVEC ext:racellular acidification rate (EAR) were evaluated by use of a Cytosensor micraphysiometer (Molecular Devices, Sunnyvale, CA).
3o HtIVECs were grown to 75% confluence on gelatin-coated (I%) transwell capsules and subjected to 24 hr of hypo:Kia followed by 3 hr o~f rreoxygenation. Following 30 min of W'O 00/35483 PCTNS99/29919 equilibration in modified RF'MI containing 1 mmol/L phosphate buffer (Molecular Devices, Sunnyvale, CA), the EARS were determined (Gronert K, et al., "Characterization of human neutrophil and endothelial cell ligand-operated extacellular acidification rate by mi~.rophysiometry: Impact of reoxygenation", J.Pharmacol.Exp.Ther.
1998;285:252-261).
s HLfVECs were perfizsed with 300-1500 ng/ml of purified MBL (dialyzed in the modified RPMI) for 30 sec before the first rate measurement and perfusion was maintained for 40 min. As a positive control, the HUVECs were perfused with media alone for 15 min following MBL exposure ar,.d then stimulated with histamine (1 pmol/L, 15 min perfusion) to evoke extracellular acidvfication. Each concentration of MBL was analyzed in two ~o independent chambers containing normoxic or hypoxic/reoxygenated HUVECs.
The HLfVEC response to each MBL concentration was evaluated in 3 separate experiments (n==3).
Results. Microphysiometry was performed on normoxic and hypoxic/reoxygenated HLfVECs in order to determine if MBL evoked receptor-mediated changes in the ~s endothelial EAR. Neither perfusion (40 min) of normoxic or hypoxic {12 hr) /
reoxygenated (3 hr) I~IJVEC'.s with purified MBL (30(I -1500 ng/ml) evoked a change in the EAR, whereas all cells rem~~ined responsive to the agonist histamine. Thus, MBL did not evoke receptor-mediated changes in the EAR in normoxic or hypoxic/reoxygenated HL~VECs. These data indicated that MBL binding t:o reoxygenated HUVECs occurred via a 2o MI3L ligand and not a classi~~al receptor.
Ex;~mple 8: Preparation and Characterization of Monoclonal Antibodies to Human MBL.
Female Balb/C mice were initially inoculated (i.p.) with 250 ul of the following mi;tture: 250 pl Titermax mixed with 100 pg human MBL in 250 pl PBS. The following 2s week and for three consecutive weeks the mice were injected with 50 pg hMBL
in 250 pIPBS. On the 4th r~-eek the mice were injected with 25 pg MBL in 250 pl PBS
and the mi~~e were fused 4 days later. 'The fusion protocol was adapted from Current Protocols in Immunology. The splenocytes were fused 1:1 with myelinoma fusion partner P301 from ATCC using PEG 150 at 50'% w/v. The fused cells were plated at a density of 1.25x106/ m.
3o with 100 p,l/well of a 96 well microtiter plate. The: fusion media consisted of Deficient DME high glucose, Pen/Strep (50,000 U pen, 50,000 ~g strep per liter), 4 mM L-glutamine, 20~'i° fetal bovine serum, 10'% thyroid enriched media, 1 % OPI, 1 %
NEAA, 1 % HAT, and 50 p.M mercaptoethanol. 7'he cells were fed 100 ul/ well on day one and 100/well media were exchanged on days 2, 3, 4, 7, 9, 11, and 13. T'he last media change before primary screening consisted of HAT substituted for the 1 % HT . All subsequent feedings were done s with fusion media minus the minus HT or HAT. S<;reening was done with human MBL
plated to plastic ELISA plates (96 well plates). Purified hMBL was plated in each well at 50 pl volume containing 2 ug/ml MBL in 2% sodium carbonate buffer. The plates were then blocked with 3% BSA in PBS. Tissue culture media (50 pl) was placed in the wells and incubated for 1 hour at room temperature. Tlle plates were washed and a secondary to HR.P labeled goat anti-mouse IgG antibody was used for detection.
Colorimetric analysis was done with ABTS and read at a405 nm. Positive controls consisted of a polyclonal antibody to human MBL. Cells are then grown in media consisting of the following:
DWEM high glucose no-I-glut, sod, pyruvate :500m1 (Irvine Scientific #9024), heat inactivated Hyclone 10%, 1 % Non-essential amino acids, 4mM L-gluamine, 100 U/ml ~ s penicillin and 100 ~tg/ml streptomycin. All positive wells were then screened for function in ~i secondary screen.
Fu.netional Screen fon Anti-II~IBL antibodies.
Methods. The functional screen for inhibition ~of MBL function by anti-human MBL
2o antibodies was adapted from the literature{Super, Lc;vinsky, et al., The level of mannan-binding protein regulates the binding of complc;ment-derived opsonins to mannanand zymosan at low serum concentrations, Clin. Exp. Immunol. 1990; 79:144-150}.
Briefly, 100 pl of meannan (0.5 mg/ml in sodium carbonate/bicarbonate buffer, pH 9.6) was added to R:LA/EIA plates at 4(: overnight. The plates were then washed 3 times in PBS/0.5% Tween 2s pH 7.3, once in PBS and finally in veronal-buffered saline. Human serum is diluted to 4%
in VBS containing 5 mM (".a2~ and Mgz+. Diluted sera and tissue culture supernatant or purified antibody (~~arious concentrations) are then 1:1 to a mannan-coated well to yield a final volume or 100 ~l at a concentration of 2% human sera. The plate is then incubated at 37~~ for 30 min. Positive ar,~d negative controls consist of human sera without and with 100 3o mr~I N-acetlglucosamine (GluNac). The plates are then washed four times in PBS/Tween.

V4'O 00/35483 PCTNS99/29919 The plates are then incubated with an anti-human C3 polyclonal antibody coupled with HRP
( 1 hours at RT), washed and developed with ABTS and read at 405 nm.
Results. Antibody production and characl:erization. Following a primary screen using a solid phase antibody-capture ELISA, we identified 1 I clones that recognized human s M13L. After limiting dilution and isotyping, we identified eight mAbs that recognized human MBL in an antibody~~capture ELISA. Clones 3F8, 2A9, and hMBLl.2 were isotyped as mouse IgG~k, where as clone 1C10 was a mouse IgG2b. The other hybridomas produced Iglvl antibodies and were not included in this study.
Western blot analysis was used to determine that the mAbs recognized MBL. As ~o shown in Figure 7, antibodies 2A9 (Lane 1), hM:BI,1.2 (Lane 2), 1C10 (Lane 3) or 3F8 (L;~ne 4) recognized purified and reduced human )VIBL [i.e., molecular weight (MW) ~32 kl;~]. Thus, these antibodie~~ are specific for human MBL. Clones hMBLl.2, 2A9 and 3F8 have been deposited at the International Desposito~y Authority with A'TCC
designations of HI3-12619, HB-12620 and I-IB-12621, respectively.
~ s The most potent inhibitor of MBL induced complement activiation, N-acc;tylglucosamine (GIuNAc) inhibited C3 deposition to plastic in mannan coated plates in a dope-dependent manner with an EC50 of approxirr~ately 1 nM. Similarly, 2A9 and hMBL
l.a; inhibited C3 deposition with and EC50 of approximately 30 and 50 nM, respectively.
Are isotype control antibody that recognizes MBh by solid phase ELISA did not inhibit 2o M13L dependent C3 deposition. Thus, these antibodies are approximately 105 -106 times more potent than GIuNAc. The data represent 3 separate experiments with at least 4 observations per experiment. HUBECs were hypoxic for 24 hours and then reoxygenated in 30% human sera. iC3b deposition was then normalized to normoxic cells. An approximate 190% increase in iC3b deposition on hypoxic cells was observed following reoxygenation zs (Fi gore 8). 3F8 attenuated iC3b deposition on hypoxic/reoxygenated HUVECs in a dose-de pendent manner. These: data demonstrate thz~t specific inhibition of MBL
with an antibody attenuates complement activation and iC3b deposition following hypoxia/reoxygenation of human endothelial calls. *p<0.05 compared to all groups; n+2.
3o En.ample 9:

Complement activation and deposition following HUVEC oxidative stress. To chz~racterize further the functional properties of these novel mAbs and to demonstrate specifically the role of MBI, in complement activation following oxidative stress of human endothelial cells, we assessed MBL and C3 deposition on hypoxic human endothelial cells s fol owing reoxygenation in human sera.
Western blot analysis. To demonstrate the complement inhibitory action of these anti-human MBL mAbs, hypoxic HUVECs were reoxygenated in human sera treated with PBS (vehicle), 3F8, hMB:Ll.2, 2A9, or 1C10 (50 pg/ml final concentration).
Cell membrane bound proteins were resolved by SDS-PAGE under reduced conditions, ~o transferred to membranes, and analyzed for human C3dg (i.e., part of the a-chain of iC3b).
Th~~ a- and [3-chain of iC;sb were the only C3 stainable bands present on the cellular membranes. A representative C3dg band for vehicle, 3F8-, hMBL 1.2-, 2A9- and 1 tre~~ted cells was observed. We observed a significant decrease in C3dg band intensity on cells reoxygenated in human sera treated with either 3F8, 2A9 or hMBLl.2.
However, the is non-functional clone, 1C10, did not decrease iC3b deposition (i.e., C3dg band intensity) on the endothelial membranes. These data further support the role of MBL-dependent complement activation following reoxygenation of hypoxic HUVECs. Further, these data confirm that clone 1 C10 is an isotype control mAb that does not functionally inhibit MBL.
Confocal microscopy studies. Dual labeling for MBL and C3 deposition on 2o normoxic and hypoxic HUVECs was performed to demonstrate co-Localization of these complement components and MBL-dependent complement pathway activation.
Normoxic and hypoxic HUVE(a were reoxygenated in 30% HS treated with and without mAb 3F8 (5 pg.~ml) or 1 C 10 (50 p,g/ml). MBL (blue), C3 (green) and nuclei (red) were then stained on the same slide and anzlyzed by immunofluorescent confocal microscopy. Small amounts of 2s C3 and MBL staining were observed under normoxic conditions, confirming our finding of low level C3 deposition under normoxic conditions, confirming our fording of low level C3 deposition under normoxic conditions. C3 and IvIBL staining on hypoxic/reoxygenated HtJVECs was significantly greater than normoxic H1:IVECs. Clone 1C10 failed to inhibit C3 or MBL deposition following oxidative stress. C3 and MBL staining was significantly 3o de~~reased on hypoxic/reox:/genated HUVECs treated with mAb 3F8 (5 p.g/ml) to levels below those observed under normoxic conditions (similar results were observed with mAbs V1~'O 00/35483 PCT/US99t29919 hMBL 1.2 or 2A9). It was observed that MBL and C3 co-localize on human endothelial ce:.ls under the conditions outlined above. These data demonstrate that functional inhibition of MBL with a mAb attenuates C3 deposition following oxidative stress of human endothelial cells.
s Example 10:
Methods: VCAM-1 ELISA. Briefly, HUVECs were grown to confluence on 0.1%
gelatinized 96-well plastic plates and then subjected to 0 or 12 hr of hypoxia. The cell media was them aspirated and HBSS, 30°/~ HS or 30% HS treated with 3F8 (5 p.g/ml) was added to each well.
t 0 The cells were then reoxygenaied for 3 hr at 37°C in 95'% air and 5% C02. The cells were washed, fixed, washed again, and incubated at 4°C for 1.5 hr with the anti-human VCAM-1 mAb (clone 6G 10 obtained from the Developmental Studies Hybridorria Bank, University of Iowa, Iowa City, IA). A peroxidase-conjugated goat anti-mouse secondary antibody (Cappel, West Chester, PA) was then used. An inappropriate isotype control antibody (nnA.b GSI to porcine CSa) was used to assess t 5 bankground optical density an~~ fluorescence was subtracted from the data.
These experiments (6 wells per experimental group) were performed 3 times (n=~3).
Results: Inhibition of VCAM-I expression following oxidative stress. We have demonstrated that oxidative stress of HUVECs activate, complement and results in CSb-9 dependent VCAM-1 induction. Thus, we: examined VCAM-1 expression by ELISA to demonstrate further the 20 functional significance of MBL inhibition. As shown in Figure 9 and confirming our own findings, reoxygenation of hypoxic 1-II1'VECs in 30% HS treated 'with PBS (vehicle) resulted in a significant increase in VCAM-I protein expression. Treatment of 30'% HS with 3F8 (S pg/ml) significantly attenuated VCAM-1 expression. Since VCAM-1 expression in this model is mediated by CSb-9, these data demonstrated that C:Sb-9 formation is dependent on MBL depositian and lectin pathway 25 activation.
The foregoing writven specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in sc~~pe by examples provided. since the examples are intended as a single illustration of one asy~ect of the invention and other functionally equivalent embodiments are within the scope 30 of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of th~~ invention are not necessarily encompassed by each embodiment of the invention.

V1'O 00/35483 PCT/US99/29919 All references, patents and patent publications that are recited in this application are incorporated in their entiret~~ herein by reference.
W~~ claim:

Claims (41)

1. A method for inhibiting LCP associated complement activation, comprising contacting a mammalian cell having surface exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit LCP-associated complement activation.

2. The method of claim 1, wherein the MBL inhibitor is an isolated MBL binding peptide.

3. The method of claim 2, wherein the isolated MBL binding peptide has an MBL
binding CDR3 region or functional variant thereof.

4. The method of claim 2, wherein the isolated MBL binding peptide is an antibody fragment.

5. The method of claim 2, wherein the isolated MBL binding peptide is an antibody.

6. The method of claim 1, wherein the MBL inhibitor is an isolated MASP
binding peptide.

7. The method of claim 2, wherein the method is a screening assay.

8. The method of claim 1, wherein the MBL inhibitor is administered to a subject in an amount effective to inhibit LCP-associated complement activation.

9. The method of claim 8, wherein the MBL inhibitor is an isolated MBL binding peptide.

10. The method of claim 9, wherein the isolated MBL binding peptide has an MBL
binding CDR3 region or functional variant thereof.

11. The method of claim 9, wherein the isolated MBL binding peptide is an antibody fragment.

12. The method of claim 9, wherein the isolated MBL binding peptide is an antibody.

13. The method of claim 8, wherein the MBL inhibitor is an isolated MASP
binding peptide.

14. The method of claim 8, wherein the cellular injury mediated by LCP
associated complement activation contributes to tissue injury associated with atherosclerosis.

15. The method of claim 8, wherein the cellular injury mediated by LCP
associated complement activation contributes to tissue injury associated with the pulmonary system.

16. The method of claim 15, wherein the MBL inhibitor is administered to the subject by an aerosol route of delivery.

17. The method of claim 8, wherein the cellular injury mediated by LCP
associated complement activation contributes to tissue injury associated with a disorder selected from the group consisting of arthritis, myocardial infarction, ischemia, repertusion, transplantation, CPB, stroke, ARDs, SLE, lupus, and dialysis.

18. A composition, comprising an MBL inhibitor, wherein the MBL inhibitor is an isolated binding peptide that selectively binds to a human MBL epitope and that inhibits LCP associated complement activation.

19. The composition of claim 18, wherein the isolated MBL binding peptide has an MBL binding CDR3, region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line (3F8) deposited under ATCC accession number HB-12621.

20. The composition of claim 18, wherein the isolated MBL binding peptide has an MBL binding CDR3 2 region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line (2A9) deposited under ATCC accession number HB-12620.

21. The composition of claim 18, wherein the isolated MBL binding peptide has an MBL binding CDR3 1 region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line (hMBL 1.2) deposited under ATCC accession number HB-12619.

22. The composition of claim 18 wherein the isolated peptide is an intact soluble monoclonal antibody.

23. The composition of claim 18 wherein the isolated peptide is monoclonal antibody (3F8) produced by the hybridoma cell line deposited under ATCC
Accession No.
HB-12621.

24. The composition of claim 18 wherein the isolated peptide is monoclonal antibody(2A9) produced by the hybridoma cell line deposited under ATCC
Accession No.
HB-12620.

25. The composition of claim 18 wherein the isolated peptide is monoclonal antibody(hMBL1.2) produced by the hybridoma cell line deposited under ATCC
Accession No. HB-12619.

26. The composition of claim 18 wherein the isolated peptide is a humanized monoclonal antibody.

27. The composition of claim 18 wherein the isolated peptide is a monoclonal antibody fragment selected from the group consisting of an F(ab')2 fragment, an Fd fragment, and an Fab fragment.

28. The composition of claim 18 wherein the isolated peptide has a light chain CDR2 region selected from the group consisting of a CDR2(3F8) of a monoclonal antibody produced by hybridoma(3F8) deposited under ATCC Accession No. HB-12621, a CDR2(2A9) of a monoclonal antibody produced by hybridoma(2A9) deposited under ATCC
Accession No. HB-12620, and a CDR2(hMBL1.2) of a monoclonal antibody produced by hybridoma(hMBL1.2) deposited under ATCC Accession No. HB-12619.

29. The composition of claim 18 wherein the isolated peptide has a light chain CDR1 region selected from the group consisting of a CDR1(3r8) of a monoclonal antibody produced by hybridoma(3F8) deposited under ATCC Accession No. HB-12621, a CDR1(2A9) of a monoclonal antibody produced by hybridoma(2A9) deposited under ATCC
Accession No. HB-12620, and a CDR1(hMBL1.2) of a monoclonal antibody(hMBL1.2) produced by hybridoma hMBL1.2 deposited under ATCC Accession No.HB-12619.

30. A hybridoma cell line deposited under ATCC Accession No. HB-12621.

31. A hybridoma cell line deposited under ATCC Accession No. HB-12620.

32. A hybridoma cell line deposited under ATCC Accession No. HB-12619.

33. The composition of claim 18, wherein the composition is a pharmaceutical composition including an effective amount for treating an MBL mediated disorder of the isolated MBL binding peptide; and, a pharmaceutically acceptable carrier.

34. The composition of claim 33, further comprising a drug for the treatment of an MBL mediated disorder.

35. A. composition, comprising an MBL inhibitor, wherein the MBL inhibitor is an anti-MBL antibody that: (i) selectively binds to a human MBL epitope and (ii) prevents LCP activation.

36. A method for screening of a cell for susceptibility to treatment with a MBL
inhibitor comprising:
detecting the presence. of a MBL on a surface of a mammalian cell, wherein the presence of the MBL indicates that the cell is susceptible to LCP associated complement activation and that the subject is susceptible to treatment with an MBL
inhibitor.

37. The method of claim 36, wherein the mammalian cell is isolated from the subject.

38. The method of claim 36, wherein the mammalian cell is an endothelial cell.

39. The method of claim 36, wherein the method comprises the step of contacting the MBL with a detection reagent that selectively binds to the MBL to detect the presence of the MBL.

40. The method of claim 39, wherein the detection reagent is an isolated MBL
binding protein.

41. The method of claim 39, wherein the detection reagent is a labeled isolated MBL binding peptide.