CA2088496A1 - Recombinant bpi proteins, uses of bpi proteins, and methods of preparing same - Google Patents

Abstract

The present invention provides a composition comprising a BPI
Protein and an anionic compound which composition exhibits (1) no bactericidal activity and (2) endotoxin neutralizing activity.
Also, this invention provides methods for using BPI Proteins.

Description

W~ !~2/03:~35 ~ 9 ~ P~/US91/05758 RECO~IBINAN5 BPI PROT13IN8, ~J8EQ OF BPI PROTE~IN~3, AND
N~STIIOD8 OF PR3PARIl!J~3 8~

~his applica~ion is a continuation-in-part of U.S. Serial No. 567,016, filed August 13, 1990 and U.S. Serial No.
681,551, filed April 5, 1g91. U.S. Serial No. 681,551, filed April 5, 1991 is a continuation-in-part of U.S.
Serial No. 567,016, filed August 13, 1990, which is a continuation-in-part of U.S. Serial No. 468,696, filed January 22, 1990, which is a continuation~in-part of U.s.
Application Serial No. 310,842 filed February 14, 1989, the contents of all of which ar~ hereby incorporated by "~
reference.
:
Bac~qroun~ o~ the Inventio~ ~-., .
Gram negative infections are a major cause of morbidity and mortality especiall~ in hospitalized and immunocompromised patients. [Duma, R.J., Am. J. of Med., 78 (Suppl. 6A): 154-164 (1985); and Kreger B.E., D.E.
Craven and W.R. McCabe, Am. J. Med., 68: 344-355 (1980)].
Although available an ibiotics are generally effective in containing the ~infection, they do nothing to neutralize the pathophysiological effects associated with lipopolysaccharide (LPS).

LPS is a major component of the outer membrane of gram negative bacteria and is released when the organisms are ly~ed. ~Shenep, ~.L. and K.A. MQrgan, ~. Infect. Dis., 150 (3): 380-3~8 (1984)~ :

LPS released during antibiGtic therapy is a potent stimulator o~ the inflammatory response. Many detrimental effects of LPS in vivo result from soluble , ,~. ,,, ","~, ""~,~, ~,, .,.,., ~,~!n~,~,~-'~ ''~

W0~2/03~35 2 ~ PCT/US91/05758 ~ediators released by in~lammatory cells. ~Morrison D.C.
and R.J~ Ulevich, Am. ~. Pathol., 93 (2): 527-617 (1978)]
LPS induces the release of mediators by host inflammatory cells which may ultimately result in disseminated intraYascular coagulation (DICl, adult respiratory distress syndrome tARDS), cardiac dys~unction, organ failure, liver failure (hepatobiliary dysfunction), brain failure (CNS dysfunction), renal failure, multi-organ ' failure and shock.
`' 10 Soluble LPS causes decreased neutrophil chemotaxis, increased adhesiveness, elevated hexose monophosphate shunt activity and 2 radical production, upregulation of surface receptors for complement, and reIease of granule proteins into the surrounding medium. [Morrison and Ulevich (1978)]
~.

Endotoxemia is a condition associated with the presence o~ endotoxins, i.e. heat stable bacterial toxins, in the blood. Endotoxins elicit an inflammatory response that is beneficial iN fighting the infection but can be damaging : `
to the host if uncontrolled. Endotoxemia induces productio~ of endotoxin binding proteins from the liver and causes release of microbicidal proteins from leukocytes. our studies show that one of these leukocytes proteins, i.e. BPI, previously known only for its bactericidal activity in vitro, inhibits the ability of endotoxin to stimulate neutrophils and monocytes 'n vitro and reduces death due to endotoxin or bacterial challenge when given i~ yo. Further, BPI has been shown to possees antibiotic ~unctions but not cy~otoxin functions against the host cell. ~-Monocytes and neutrophilic granulocytes play a key role ''' '''' '~, , ' '~' ' ' , . . .
. "

W092/n3a3a 2 ~ 8 ~ PCT/US9l/057~8 ~` 3 - in host defense against bacterial in~ections and also participate in the pathology of endotoxemia. These cells ingest and kill microorganis~s intracellularly and also respond to endotaxin in vivo and in vitro by releasing soluble proteins with microbicidal, proteolytic, opsonic, pyrogenic, complement activatiny and tissue damaging effects.
. .
Tumor necrosis factor (TNF), a cytokine released by ,` 10endotoxin stimulated monocytes mimics some of the toxic effects of endotoxin in vivo. Injecting animals with TNF
causes fever, shock and alterations in glucose metabolism. TNF is also a potent stimulator of neutrophils. Other cytokines such as IL-l, IL-6, and IL-8 also mediate some of the pathophysiologic effects of LPS.
':
Despite improvements in antibiotic therapy, morbidity and mortality associated with endotoxemia remains high.
Antibiotics alone are not effective in neutralizing the toxic effects of LPS. Therefore, the need arises ~or a therapy with direct endotoxin neutralizing activity.
Current methods for treatment of endotoxemia use antibiotics and supportive care. Most available adjunct therapies treat symptoms of endotoxic shock such as low blood pressure and fever but do not inactivate endotoxin.
Other therapies inhibit inflammatory host responses to LPS. As indicated helow, present therapies have major limitations due to toxicity, immunogenicity, or irreproducible e~ficaay betwe~n animal models and human trials.

Polymyxin B (PMB) is a basic polypeptide antibiotic which has been shown to bind to, and structurally disrupt, the most toxic and biologically active component of ~ WO9~/03~35 PCT/US9l/~5758 (J ~ . :

endotoxin, Lipid A. PMB has been shown to inhibit endotoxin activation of nautrophil granule release in vitro and is a potential treatment for gram negative in~ections in humans. However, because of its systemic toxicity, this drug has limited use except as a topical agent.

Combination therapy using antibiotics and high doses of methylprednisolone sodium succinate (MPSS) has been shown to prevent death ln an experimental model of gram negative sepsis using dogs. Another study using MPSS
with antibiotics in a multicenter, double blind, placebo-controlled, clinical study in 223 patients with clinical signs of systemic sepsis concluded that mortality was not significantly different between the treatment and placebo groups. Further, the investigators found that resolution of secondary infection within 14 days was significantly higher in the placebo group.

A relatively new approach to treatment of endotoxemia is passive immunization with endotoxin neutralizing antibodies. Hyperimmune human immunoglobulin against E.
Coli J5 has been shown to reduce mortality in patients with gram negative bacteremia and shock by 50~. Other groups have shown promising results in animal models using mousel chimeric, and human monoclonal antibodies.
Although monoclonal antibodies have advantages over hyperimmune sera, e.g. more consistent drug potency and decreased transmission of human pathogens, there are still many pxoblems associated with administering immunoglobulin to neutralize LPS. Host responses to the immunoglobulins themselves can result in hypersensitivity. Tissue damage following complement activation and deposition of immune complexes-is another concern in the u,e of tberapies involving anti-endotoxin '''.:
~ ' W092/03S3~ 2 ~ ~ S ;~ ~ ~ PCT/~S91/0575~

antibodies in septic patier,ts.
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~^ BPI is a neutrophil granule protein first discovered in 1975 CWeiss, J., R.C. Franson, S. Becherdite, K.
Schmeidler, and P. Elsbach J. Clin. Invest., 55:33 ; (1975)]. BPI was obtained in highly purified form from human neutrophils in 1978 and was shown to increase membrane permeability and have bactericidal activity against Gram negative bacteria when assayed in phosphate buffered saline in vitro [Weiss, J., et al. J. Biol.
Chem, 253(8): 2664-2672 (1978)]. Weiss et al. ~J. Biol.
Chem. 254(21): 110010-1101~ (1379)], furth~r showed that BPI increased phospholipase A2 activity suggesting a , proinflammatory activity for BPI in addition to its ln vitro bactericidal activity.
....
Rabbit BPI was purified in 1979 [Elsbach et al. J. Biol.
Chem 254(21): 11000-11009] and shown to have identical bactericidal and permeability increasing properties as BPI from humans providing a further source of material for study. Both BPI from rabbit and human were shown to be effective against a variety of Gram negative bacteria j in vitro, including X}-encapsulated E. coli [Weiss et al.
Infection and Immunity 38(3): 1149-1153, (1982)].
A role for lipopolysaccharide in the m _ vitro bactericidal action of BPI was proposed in 1984 by Weiss et al. [J. Immunol. 1~2(6): 3109-3115, (1984)J. These investigators demonstrated that BPI bound to the outer membrane o~ gram-negative bacteria, caused extracellular release o~ LPS, and selectively stimulated LPS
biosynthesis. In 1984 a proteir. ~ith similar properties was isolat~i from human neu~-ophils and designated cationic an.lmicrobial protein 57 (CAP 57) [Shafer, W.M., C.E. Martin and J.K. Spitznagel, Infect. Immun., 45:29 : - - . .
. - , - , .

1 W092/~3~35 PCT/US91/057~8 ~
~g~9~ 6 ``(i984)] This protein is identical to BPI as determined by the N-terminal amino acid saquence, amino acid composition, molecular weight and source [spitznagel et al., Blood 76:825-834, 1990]. Another group, Hovde and Gray, reported a bactericidal glycoprotein with ~irtually identical properties to BPI in 1986 rHovde and Gray, In~ection and Immunity 54(1): 142-148 (1g~6)].

In 1985 Ooi et al. reported that~BPI retains its in vitro bactericidal activity after cleavage with neutrophil proteases su~gesting that fragments of the molecule retain activity (ooi and ~lsbach, Clinical Research 33(2):567A (1985)]. All of the in vitro bactericidal and permeability increasing activities of BPI were present in the N-terminal 25kD fragment of the protein (ooi~ C.E., et al. J. Biol. Chem. 262: 14891 (1987)]

Evidence that BPI binds to a structure associated with endotoxin on the outer membrane of bacteria is as follows: (1) increased sensitivity of rough strains of E.
coli relative to smooth strains to the permeability increasing activitie~ of BPI [Weiss, J. et al. Infect.
Immun. 51:594 tl986)]; (2) the Prm A mutation which results in altered endotoxin structure caused decreased binding of both polymyzin b. and BPI ~Farley, M. M. et al. Infect. Immun. 56:1536-1539 (1987) and Farley et al.
Infect. Immun. 58:1589-1592 (1988~]; (3) polymyxin B
tP~B) completed with BPI ~or binding to S. typhimurium [Faxley 198~]; and (4) BPI shared amino acid sequence homology and immunocrossreactivity to another endotoxin binding protein termed Lipopolysaccharide Binding Protein (LBP) rTobias et al., J. Biol. Chem. 263(27): 13479-13481 (1988)).

LBP-LPS complexes bind to a cell surface receptor on '',' -''''"','''""';'''.'''''~'"''-~''' '."'',"'' ' ';

W092t03~3S ~ PCT/US91/05758 .
monocytes (CD 14) which results in increased synthesis and release of the inflammatory cytokine tumor necrosis fac~or (TNF) ~Schumann et al. Science ~2:1429~1431].
Thus, LBP promotes the immunost.imulatory activities of LPS. BPI has exactly the opposite effect of LBP. BPI
binds LPS and inhibits neutrophil and monocyte activation [Marra et al., J. Immunol. 144:662_666 (l990); Marra and Scott, WosO/09183, published 23 August l990; C.J. Fisher et al. Circulatory Shock 34: 120 (1991). ~
, A cDNA encoding BPI was obtained and sequenced by Gray et al. [Gray et al. Clin. Res. 36.620A (1988) and Gray et al. J. Biol. Chem. 264(16): 9505-9506 (I989)]. They reported that BPI is a membrane protein which can be cleaved and released in soluble form as a 25 kDa fragment.

BPI binding to gram negative bacteria was reported originally to disrupt LPS structure, alter microbial permeability to small hydrophobic molecules and cause cell death (Weiss, et al., 1978). More recently these same authors have demonstrated that such effects occur only in the absence of serum albumin. BPI has no bactericidal activity when added to bacteria cultured in the presence o~ serum albumin, thus suggesting that BPI
does not kill bacteria in _vivo where albumin is ; ,~
ubiguitous [Mannion et al. J. Clin. Invest. 85: 853-860 (1990) and Mannion et al J. clin. Invest. 86: 631-641)].
Thus it has been previously understood in the art that the beneficial e~fects of BPI are limited to in vitro bactericidal e~fects.

Here we show that BPI binds endotoxin in the presence of serum and plasma and, unlike other known endotoxin binding proteins such as LBP, BPI inhibits the . - .., : ~ .
- . . . .. .

2~`D 3 i~ 9 ~ PCT/US91/05758 immunostimulatory and toxic activities of endotoxin both .~. in vi.~ro and in vivo respectively. Thus BPI has a noval and distinct use i~ the therapeutic and prophylactic treatmen~ of endotoxin-related disorders includiny endotoxemia and endotoxic shock. . ~.

Further, BPI is described by Gray et al. [J. Biol. Chem~
264 (16): 9505-9509 (1989)] as a membrane protein which must be cleaved to the 25kDa fragment to be released from the neutrophil granule membrane in soluble form. The present invention provides for a method of producing full length soluble BPI in active form. Further the present .:
invention separates for the first time two molecular . forms of the molecule apparently unresolved by Gray et ; 15 al. representing glycosylated and nonglycosylated forms : . of the molecule which appear to have different serum . half-life profiles in vivo and thus different therapeutic ;~ : potential. BPI from neutrophils is a mixture of the glycoslyated end nonglycosylated ~orms.

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` W092/n3535 ~ ~ g ~ PCT/US91/05758 g 8ummary o~ the Invant~o~

The present invention provides a composition comprising a BPI Protein and an anionic compound which composition exhibits (l) no bactericidal activity and (2) endotoxin - neutralizing activity.

This invention also provides a biologically active variant of BPI which (l) specifically'inds to endotoxin, (2) competes with BPI Protein for bi. ing to endotoxin, and (3) inhibits endotoxin-induced lethality.

The present invention further provides a method for producing and secreting a recombinant BPI Protein from a cell. This method comprises (a) constructing a vector comprising DNA encoding BPI Protein; (b) transfecting the cell with the vector, and (c) culturing the cell ~o transfected in culture medium under conditions such thc recombinant BPI Protein is secreted Also, the present invention provides a method for producing a recombinant BPI Protein from a bacterial cell. This method comprises ~a) constructing a vector without a signal sequence and comprising DNA encoding BPI
Protein; (b) transfecting the bacterial cell with the vector: and (c) culturing the bacterial cell so transfected in culture medium under conditions such that recombinant BPI Protein is produced.
The subject invention ~urther provides a method for producing a recombinant BPI Protein from an insect cel~.
This method comprises (a) constructing a vector comprising DNA encoding BPI Protein; (b) transfecting the insect cell with the vector; and (c) culturing the insect ~, . . , . ~ . . .

W09~03~3~ 9 ~ PCT/US91/05758 cell so transfec~ed in culture medium under conditions such that BPI Protein is produced.

Also, this invention provides a method for determining the amo~nt o~ endotoxin in a sample from a sub~ect which comprises contacting the sample with a BPI Protein under conditions such that an andotoxin-BPI Protein complex is formed, detecting the amount of the complex so formed thereby determining the amount of endotoxin in the lo sample. ~-.
Additionally, the present invention provides a method for determining the amount of endotoxin in a sample containing bound and unbound endotoxin from a subject.
This method comprises (a) treating the sample so as to denature any endotoxin binding protein to which the endotoxin may be bound thereby obtaining unbound ~-endotoxin; (b) contacting the treated sample with a BPI
Protein under conditions such that the BPI Protein binds to unbound endotoxin of step (a) so that a endotoxin-BPI
Protein complex is formed; (c) detecting the amount of the complex so formed thereby determining the amount of endotoxin in the sample.
~-..~.
This present invention also provides a method of detecting endotoxin in a sample which comprises contacting the sample with a BPI Protein such that the endotoxin binds to the BPI Protein and forms a complex therewith; and detecting such complex.
,~
The present invention further provides a metho~ for coating a surgical tool with a BPI Protein so that the BPI Protein will complex with endotoxin which method comprises attaching BPI Protein onto a surface of the tool which surface is designed for contact with a , .. .
. , .
:.
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~YO92/03~3~ 2~ ~ ~ PCT/U~9~/05758 biolo~ical sample. ;

Also, this invention provides a method for coating an implantable, invasive device with a BPI Protein so that ` 5 it will form a complex with endotoxin which method comprises attaching BPI Protain onto a surface of the device which surface is designed for contact with a biological sample.
::
The present invention further provides a method for decontaminating a fluid containing endotoxin prior to - administration of the fluid into a subject which comprises contacting the fluid with BPI Protein prior to administration, under conditions such that endotoxin forms a complex with BPI Protein, thereby decontaminating the fluid. The fluid may be blood, plasma, blood serum, an isotonic solution, a pharmaceutical agent, a cell culture reagent, or bone marrow.

This invention also provides a kit for detecting the presence of BPI Protein in a biological fluid sample which comprises ~a) an assay buffer containing polymyxin B which binds unbound endotoxin molecules; ~) a first antibody attached to a surface, which antibody (1) binds to a portion of active BPI Protein and (2) does not compete with BPI Protein for an endotoxin binding domain;
and (c~ a second antibody labeled with a detectable moiety which antibody ~1) does not compete with the first antibody for ~PI Pro~ein binding and (2) speci~ically binds to BPI Protein at or near the endotoxin binding site, so that when the biological ~luid sample is contacted with the ~irst and second antibody in assay buffer, an active BPI Protein contained in the biological fluid sample is bound by the first and second antibody thus forming a first antibody-BPI Protein second antibody W092/03~35 ~ 12 PCT/US91/057~8 complex, detecting such complex, and thereby detecting BPI Protein in the biological fluid sample.

Also, this invention provides a kit for determining the amount of BPI Protein in a biological fluid sample which comprises ~a) an assay buffer containing polymyxln B
which binds unbound endotoxin molecules; ~b) a first antibody attached to a surface, which antibody (l) binds to a portion of active BPI Protein and (2) does not compete with BPI Protein for an endotoxin binding domain;
and (c) a second antibody labeled with a detectable moiety which antibody (l) does not compete with the first antibody for BPI Protein binding and (2) specifically binds to BPI Protein at or near the endoto~in binding site, so that when the biological fluid sample is contacted with the first and second antibody in assay buffer, an active BPI Protein contained in the biological fluid sample is bound by the first and second antibody thus forming a first antibody-active BPI Protein-second antibody complex, detecting such complex, and determining the amount of active BPI Protein in the biological fluid sample.
. .
Additionally, this invention provides a method for preventing endotoxemia in a subject which comprises administering to the subject an amount of a BPI Protein effective to bind to endotoxin so as to prevent : .
endotoxe~ia in the subject. ~:

The present invention provides a method for traating a ::-subject suffering from endotoxemia which comprises administering to the subject an amount of a BPI Protein effective to bind endotoxin so as to treat the subject ::
suffering from endotoxemia.
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wo 92/n3s3; ~ ~ 8 ~?,~ PCT/US91/05758 . ~ .

; Br~e~ Des~r~ptio~ of the Fiqures . I , Figur~ 1: Photographs of transformed plates of JMlO9(DE3~ with the T7 promoter/BPI Protein plasmid constructs. Photographs were taken with f8 at 1/125 second exposure. ;~
A. pT7BPI-F ~+~ contains the full-length BPI Protein sequence (including the signal sequence) placed in the correct orientation behind the T7 ` promoter for expression.
B. pT7BPI-F (-) contains the full-length BPI Protein sequence (including the signal sequence) placed in the incorrect orientation behind the T7 promoter (resulting protein is a fusion protein with the 260 amino acid leader peptide of T7 gene 10).
C. pT7BPI-S contains the full-length BPI Protein -~
seouence (without the signal sequence) placed in the correct orientation behind the T7 promoter for expression.
D. pT7212-F contains the proline-212 truncated BPI
Protein se~uence (including the signal sequence) placed in the correct orientation behind the T? promoter for expression.
E. pT7212-S contains the proline-212 truncated BPI
Protein sequence (without the signal sequence) placed in the correc~ orien~ation behind the T7 promoter for expression.
Figure 2: Schematic of the pT7BPI Protein plasmid construct.

Figure 3: Standard curve showing BPI Protein activity in ELISA Assay.

' . ' ': ,' ~ . : ' WO9~/O~S3~ P~,~/VS91/05758 Figure 4: BPI Protein Sandwich EI.ISA + endotoxin +
Polymyxin B. The pro~ocol is as follows: BPI Protein was performed în the presence and absence of l~g/ml of polymyxin B sulfate and the presence or absence of l~g/ml "
E. coli Olli B4 endotoxin using PBS ~ 1% BSA as diluent. ;~-Figure 5: Schematic drawing of cDNA encoding BPI.

Figure 6: A nucleotide and amino acid sequence of BPI
Protein mutagenic primer 25 kDa Pro 212 TGA which is a C-terminal truncation of BPI Protein. ~--Figurs 7: A nucleotide and amino acid sequence of BPI
Protein mutagenic primer 38 kDa Pro 337 TGA which is a C-terminal truncation of BPI Protein.

Figure 8: A nucleotide and amino acid sequence of BPI
Protein mutagenic primer: Preferrad ATG 5' ~indIII which is a C-terminal truncation of BPI Protein. -Figuro 9: A schematic drawing of pSVBPIMDH.

Figure 10: A schematic drawing of pAc373.
'', . .~,:
Yigure 11: SDS-PAGE analysis of (1) nBPI Protein (Lot No. ~148104), (2) rBPI Protein (Lot No.#148159), and (3) rBPI Protein (Lot No. #148179).

Figur0 12: cDNA sequence of BPI.
Figure 13: Protein sequence for p337.

~lguro 14: Protein sequence for p212.

W092/03535 ~ PCT/US91/05758 ` ~igus~ 15: Line graph showing BPI efficacy using `;; neutropenic rat models.
~ .
F~guro 16: Bar graph showing BPI efficacy in vivo.

~iguru 17: Bar graph showing BPI efficacy.

F~gur0 lB. Line graph showing BPI serum half life.

Figure 19: Line graph showing BPI binding to endotoxin.
BPI binding was assayed on endotoxin coated wells which were treated with varying concentrations of polymyxin B
sulfate. Results show absorbance (O.D. 405) for buffer control (closed circles). lO~g/ml polymyxin B (open circles). lOO~g/ml polymyxin B (closed triangles).
lmg/ml polymyxin B (open triangles). Data is represented - as the mean + SX of qua~ruplicate values.

Figur~ 20: Line graph showing BPI endotoxin binding.
BPI was diluted in buffer (closed circles) or neat plasma (open circles) and assayed for endotoxin binding.

Figure 2': Line graph showing BPI endotoxin binding.
BPI was diluted in increasing concentrations (expressed - 25 as ionic strength, m~) of ~aC1 (closed circles), MgCl2 (open circles), or CACl2 (closad triangles), and assayed for endotoxin binding as described.

~iguro 22: A schematic diagram showing the role of BPI
and LBP in regulating endotoxin activity.

Figura 23: A biologically active variant designated LBP/BPI Chimera.

W(~ 92/~)3~3~ PCT/US91/05758

3`3;~l3 16 ~lgur~ 2~: A biologically active variant designated C}~O~
BPI.

~ igura 25: A biologically active variant designated BPI
5 ( DP l inkage ) .
; ' ", Figuro 26: A construct for making biologically active : variants of BPI.
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'"'```'' ' ,''',,,', ,',;-,, , ,..', .'.,''',:. '' ~- ''' '"'..:.' W0~03535 ~ ~ 8 3 ~ PCT/US9l/05758 Deta~lafl Da3criptio~ o~ the Invention ~ , As used in this application, the following words or phrases have the meanings specified.

As used herein, 'IBPI" means a native or naturally occurring biologically active human 57kd protein which binds to the outer membrane of susceptible gram negative bacteria. -As used herein, "biologically active polypeptide fragment of BPI" means a polypeptide of molecular weight less than 57~d, having the biological activity of, and an amino acid sequence present within, BPI.

As used herein, "biologically active polypeptide analogs of BPI" means a polypeptide which has substantially the same amino acid sequence as, and the biological activity of, BPI. Biologically active polypeptide analogs of BPI
include polypeptide, th~ sequence of which varies from the sequence of BPI by a changed amino~ acid within the BPI sequence, e.g. a mutation, or by the addition of one or more amino acids at the amino- or carboxy- terminus, or both, of the BPI sequence.

As used herein, "biologically active variant of BPI"
means a polypeptide that (1~ includes a portion of the ami~o acid sequence which is present within BPI and an amino acid sequence which is not present within BPI, and (~) has substantially the same biological activity, i.e.
endotoxin-neutralizing activity, as BPI.

As used herein, "recombinant" means a polypeptide produced by genetic engineèring methods. Thus, each of ~"
. ~

W092J03~35 2 ~ g ~ ~ 9 ~ 18 PCT/US9l/0~7~8 BPI, biologically active polypeptide fragments o~ BPI, - biologically active polypeptide analogs of BPI, and biolog.ically active variants of BPI may be recomhinant.
However, in the context o~ this application, BPI is not the same as recombinant BPI, the latter dif~ering in some molecular characteristic ~rom tbe native or naturally ~- occurring polypeptide, P.g. in glycosylation pattern.
.
As used herein, BPI Protein means (1) BPI, (2) a biologically active fragment of BPI, (3) a biologically active polypeptide analog of BPI, or (4) a biologically active variant of BPI, each of which may be either ;
recombinant or nonrecombinant. ~ -' :. .
The present invention provides a composition comprising a BPI Protein and an anionic compound which composition exhibits (l) no bactericidal activity and (2) endotoxin neutralizing activity.

In accordance with the practice of this invention, the anionic compound could be a protein, a proteoglycan (for example heparinJ or a synthetic polymer (for example dextran sulfate or polyglutamic acid). Preferably, the anionic compound is a protein such as serum albumin.
This invention also provides a biologically active variant of BPI which (l) specifically binds to endotoxin, (2) competes with BPI Protein ~or binding to endotoxin, and (3) inhibits endotoxin-induced lethality.
As used in this application the term "endotoxinl' means a bacterial toxin which is pyrogenic.

One example of a biologically active ~ragment of BPI is shown in Figure 13. Another example of a biologically , , :, , - . - . , ~ :, - . . . ., . -, ... ~ .. : .. . . .. . .

~ WOg2/0353~, PCT/US91/~5758 active fragm,ent of BPI is shown in Figure 14.

Additionally, examples of a biologically active Yariant of ~PI is shown in Figure 23. Another example o~ a `5 biologically active variant of BPI is shown in Figure 24.
Further, yet another example of a biologically active variant of BPI is shown in Figure 25.

The present invention further provides a method for producing and secreting a recombinant BPI Protein from a cell. This method comprises (a) constructing a vector comprising DNA encoding BPI Protein; (b) transfect~ng the cell with the vector; and (c) culturing the cell so transfected in culture medium under conditions such that BPI Protein is secret-ed. In accordance with this invention, the vector further comprises a signal sequence.
.
In accordance with this method, mammalian cells are preferred. Examples of a mammalian cell includes, but is not limited to, HeLa, CH0, DUX Bll, Sp2/0, W138, DHK, HEPG2, and COS-1 cells.

This invention also provides a BPI Protein produced by the above-described method. In one embodiment the BPI
Protein is a recom~ ant BPI Protein designated 148159 rBPI protein shown in Figure 11. Additionally, the invention provides a recombinant BPI protein designated as 148179 rBPI protein shown in Figure 11.
~ -~
Interestingly, recombinant BPI Protein produced in mammalian cells such as Chinese hamster ovary (CH0) cells exhibit a slightly altered migration pattern on sodiu.m dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicating that the molecule may also be proc~ssed ,.'''' ~

',: .

W092/03535 ~ 3 ~ ~ ~ PCT/US91/05758 differently in mammalian cells than in neutrophils or HL60 cells. Such processing may be either responsible ~or, or a result of, the molecule being secreted rather than packaged into granule membranes.
This invention also provides a glycosylated BPI Protein.
. .:
In accordance with the above described method, the BPI
Protein so secreted may be a full length soluble BPI
Protein. -Also, the present invention provides a method for producing a recombinant BPI Protein from a bacterial cell. This method comprises (a) constructing a vector without a signal sequence and comprising DNA encoding BRI
Protein; (b) transfecting the bacterial cell with the vector; and (c) culturing the bacterial cell so transfected in culture medium under conditions such that recombinant BPI Protein is produced. An example of a bacterial cell includes but is not limited to E. coli.

BPI Protein has been shown to be toxic to bacteria, however, the toxic effects of the BPI Protein so produced against bacteria may be overcome by deleting the normal leader sequence in the vector comprising the BPI protein cDNA.

Apparently, when the signal sequence is included in the expression plasmid as provided in a full length clone and reported by Gray et al. ((1989) ~ourn. of Biol. Chem., 264:9505) no bacterial colonies are obtained, whereas, numerous colonies can be obtained if the signal sequence is deleted. Further, the method described hereinabove provides for expression of full length BPI Protein in a nongl~cosylated form. The invention further provides for :' '~

W092/03~3~ ~3~ PCT/US91/0~758 a nonglycosylated form of BPI Protein which is free from glycosylated BPI Protein.

The subjact invention further provides a method for producing a reaom~inant BPI Protein from an insect cell.
This ~ethod comprises ~a) constructing a vector without a signal sequence and comprising DNA encoding BPI
Protein; (b) transfecting the insect cell with the vector; and (c) culturing the insect cell so transfected in culture medium under conditions such that BPI Protein ` is secreted.
.
In one example of the above-described method, insect cells function as hosts for a baculovirus vector r 15 containin~ a sequence encoding the BPI Protein. Also, BPI protein derived from insect cells exhibit a different migration pattern on SDS-PAGE than that derived from ' either mammalian cells or the BPI protein found naturally-occurring in neutrophils. Thus, the invention provides for a new mole~ular species of BPI Protein as ~-processed by baculovirus infected insect cells.
' Further, this invention provides a biologically active variant of BPI produced by the above-described method.
Also, this invention provides a method for determining the amount of endotoxin in a sample from a subject which comprises contacting the sample wit a BPI Protein under conditions such that an endotoxin-BPI Protein complex is ;
for~ed, detecting the amount of the complex so formed thereby determining the amount of endoto~in in the sample. `

Additionally, thé present invention provides a method for determining tha amount of endotoxin in a sample ~:,.
,~
" ' ' ;W092~0353~ PCT/US9l/05758 ?~ 22 containing bound and unbound endotoxin from a subject.
This me~hod comprises (a) treating ~he sample so a~ to denature any endotoxin binding protein to which the endotoxin may be bound théreby obtaining unbound endotoxin; tb) contacting the treated sample with a BPI
Protein under conditions such that the BPI Protein binds to unbound endotoxin of step (a) so that a endotoxin-~PI
Protein complex is formed; (c) detecting the amount of ~ ;
th~ complex so formed thereby determining the amount of endotoxin in the sample.

In accordance with the invention, denaturation in step (a) may be effected using an elevated temperature. For example, the elevated temperature may be ~5 degrees centigrade. Alternatively, denaturation may be effected with an acid.

This present invention also provides a method of detecting endotoxin in a sample which comprises contacting the sample with a BPI Protein such that the endotoxin binds to the BPI Protein and forms a complex therewlth;~and detecting such complex. ~-In one example of the invention, the sample containing endotoxin is transferred onto a suitable support under conditions permitting endotoxin in the sample to attach to the support prior to contacting the sample with BPI
Protein labeled with a detectable moiety.
:, ", This invention ~urther provides a method for diagnosing endotoxemia in a subject which comprises obtaining from the subject a biological fIuid sample, detecting endotoxin in such sample using the above-described method and thereby diagnosing such disorder. The sample may be a cellular sample. Alternatively, the sample may be a : .

W092/0353~ f~ ~ ~4 ~ f ~ ~ ~r ~ ~ PCI/US91/05758 biological fluid sample such as serum, urine, blood, a tissue extract, or sputum.

In accordance with the practice o~ the invention, the BPI
Protein may be labeled with a fluorescent label and detection may be effected by a fluorometer.
Alternatively, the BPI Protein mav be labeled with a radioactive label and detection may be effected by a radiograph. Further, the BPI Protein may be labeled with an enzyme and detection may be effected by a spectrophotometer.

The present invantion further provides a method for coating a surgical tool with a BPI Protein so that the BPI Protein will complex with endotoxin which method comprises attaching BPI Protein onto a surface o~ the tool which surface is designed for contact with a biological sample.

Also, this invention provides a method for coating an implantable, i~vasive device with a BPI Protein so that it will form a complex with endotoxin which method - comprises attaching BPI Protein onto a surface of the device which surface is designed for contact with a biological sample.

In accordance with the practice of the present invention, the biological sa~ple may be blood. Alternatively, the biological sample may be a tissue sample. Further, the biological sample may be a muscle sample. Also, the biological sample may be cartilage. Additionally, the biological sample may be bone.

Also, in accordance with the practice of this invention, the surgical tool may be a catheter tubing.

' .- : . - .. . : . .- ,: :, : ... .,... .. , , .. -. : : . , W09~/03535 PCT/US91/05758 8i~ ~ 24 Alternatively, the surgical tool may be a surgical staple.
.
Further, in accordance with the practice o~ this invention, the device may be a surgical implant.
.
The present invention further provides a method for decontaminating a fluid containing endotoxin prior to administration of the fluid into a subject which comprises contacting the fluid with BPI Protein prior to administration, under conditions such that endotoxin . forms a complex with BPI Protein, thereby decontaminating the fluid. The fluid may be blood, plasma, blood serum, an isotonic solution, a pharmaceutical agent, a cell culture reagent, or bone marrow.

This invention also provides a kit for detecting the presence of BPI Protein in a biological fluid sample which comprises (a) polymyxin B in an assay buffer which binds unbound endotoxin molecules; tb) a first antibody attached to a surface area containing the assay buffer, which antibody (1) binds to a portion of active BPI
Protein and (2) does not compete with BPI Protein for an endotoxin binding domain; and (c) a second antibody labeled with a detectable moiety which antibody (1) does not compete with the first antibody for BPI Protein binding and (2) specifically binds to BPI Protein at or near the endotoxin binding site, so that when the biological ~luid sample is contacted with the first and second antlbody an aative BPI Protein contained in the biological ~luid sample is bound by the first and second antibody thus forming a first antibody-BPI Protein-second antibody complex, detecting such complex, and thereby detecting BPI Protein in the biological fluid sample.
.~.:' ','.

- : :. , - - - .. . . - . . . ..

W092~03~35 ~ PCT/US91/05758 Also, this invention provides a kit for determining the amount of BPI Protein in a biological fluid sample which comprises ~a) polymyxin B in an assay buffer which binds unbound endotoxin molaculas; ~b) a first antibody attached to a surface area containing the assay buffer, which anti~ody (1) bi ~s to a portion of active BPI
Protein and (2) does nol compete with B?I Protein for an endotoxin binding domain; and (c~ a second antibody label~d with a detectable moiety which antibody (1) does not compete with the first antibody for BPI Protein binding and (2) specifically binds to BPI Protein at or - near the endotoxin binding site, so that when the biological fluid sample is contacted with the first and second antibody an active BPI Protein contained in the biological fluid sample is bound by the first and second antibody thus forming a first antibody-active BPI
Protein-second antibody complex, detecting such complex, and determining the amount of active BPI Protein in the biological fluid sample.
Additionally, this invention provides a method for preventing endotoxemia in a subject which comprises administering to the subject an amount of a BPI Protein effective to bind to endotoxin so as to prevent endotoxemia in the subject.

The present invention provides a method for treating a subject suffering from endotoxemia which comprises adminlstering to the subject an amount of a BPI Protein ef~ective to bind endotoxin so as to treat the subject suf~ering from endotoxemia.

In accordance with the practice of the invention, the effective amount of the BPI Protein for preventing endotoxemia or treating a subject suffering from ~092/Q3~3~ ~ PCT/US91/05758 2~ ~ 26 endotoxemia may be between about 0.1 and about lOmg/kg body weight of subject. Also, the e~fective amount may ; be an amount batween about 1 and a~out lO mg~kg body weight of subject.
S
This invention is illustrated in the Experimental Details section which follows. This section is set forth to aid an understand of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.

.... ... , . , ... , ..... . .~ ........ .. .. . .

- , - . , -~V0~2/~3~35 ~ 5 PCT/US91/05758 Ex~ril,~nental ~etails :
~nmpla_1~

Materials and Methods Reaaents and $olutions: endotoxin from E. coli OL11:~4 and from S. typhimurium RE mutant were purchased from RIBI Immunochem Research, Inc., Hamiltsn, MT. F~LP, cytochalasin B and polymyxin B sulfate (7900 U/mg) was --purchased from Sigma Chemical Co., St. Louis, MO. `
Natural human tumor necrosis factor was purchased from Endogen Inc., Boston MA. HBSS without calcium and magnesium and RPMI 1640 were purchased from Gibco BRL, Grand Island, N.Y.
.
BPI Purification: BPI was purified from neutrophil granule preparations as previously described (Marra, M.N.
et al. J. Immunol. 144: 662, 1990) with the exception that the purification was performed under rigorously pyrogen-free conditions using new, pyrogen-free columns `~
and de-pyrogenated buffers. Buffers wer deyprogenated -`
using a Pyrosart filter (Sartorius Filters, Hayward, CA). -`
Purification of BPI under these conditions resulted in material with approximately four-fold greater activit~
for neutralizing endotoxin-mediated neutrophil stimulation than previously reported ~Marra, ~.N. et al.
J. Immunol. 144: 662, l990).
:
Imm4~oaff~r~itY purification of anti-BPI antibodies:
Sera was collected from rabbits immunized with a 20 amino acid peptide corresponding to the N-Terminal 20 amino acids of the BPI molecule (BPI peptide 1-20). The IgG
fraction of pooled sera was purified using Protein A
Sepharose (Pharmacia, Piscataway, N.J.). Specific anti-'' .,'` ' W092/03535 PCT/US91/0~758 2 ~ & ~3 ~ 28 peptide IgG was purified from this ~raction using BPI
peptide 1-20 coupled to activated CNBr Sepharose ~Pharmacia). Bound IgG was collected and pooled, and the adsorbed IgG was further depleted of residual specific anti~ody by passing over the peptide column three additional times to generate immunoadsorbed negative control. Antibody concentration was determined~ by optical density at 280 nm. Immunoaffinity puri~ied and adsorbed IgG were tested for specificity by Western blotting. No activity was observed in the immunoadsorbed control IgG, even at concentrations 103-fold greater than that utilized for the immunoaffinity purified antibody.

Endotoxin Bindinq Assay: BPI binding to endotoxin immobilized on microtiter plates was performed using a modified procedure described by Tobias, P.S. et al. J.
Biol. Chem. 264:10867, 1989. Briefly, Immulon 2 96 well microtiter plates ~Dynatech Biotechnology Products, Chantilly, VA) were coated with 4~gJwell glycolipid from Salmonella typhimurium RE mutant in 50mM borate pH 9 + 20 mM EDTA overnight at 37 C. Plates were then washed extensively under running distillëd deionized water, then dried at 37~C. Assay plates were blocked for 30 minutes at 37-C with 5mg/ml very low endotoxin BSA (Sigma, St.
Louis, M0) prepared in pyrogen-free PBS. Plates were flicked, and in some experiments polymyxin B was added to the wells and incubated for an additional 30 minutes at 37-C. Plates were flicked again, and BPI samples were added. All buf~ers containing BPI or polymyxin B were prepared in pyrogen-~ree PBS. BPI samples diluted in pyrogen free buffer, or in some experiments, serum or plasma ~rom normal human volunteers, were incubated for 3 hours at 37C with shaking. The plates were washed with PBS containing lmg/ml pyrogen free BSA, then O9~/03~35 ~ L~ a ~ J PCT/US~1/057~8 developed, using rabbit polyclonal anti-BPI peptide lgG
antibody as described followed by goat-anti-rabbit IgG-alkaline phosphatase conjugate (Gibco BRL Life Technologias, Inc., Grand Island, N.Y.). AbsorbancPs were read at 40S nm on a Vmax kinetic microplate reader (Molacular Devices Inc , Menlo Park, CA).

BPI inhibition of endotoxin mediated TNF_induction by human adherent mononuclear cells: Blood collected in acid citrate dextrose containing vacutainer tubes (Becton Dickinson, Rutherford, N.J.) was diluted in Hank's balanced salt solution (HBSS) minus Ca2+ and Mg2~
Mononuclear cells were separa~ed using Ficol-Paque (Pharmacia Inc., Piscataway, N~Jo ), cGllected and washed three time in HBSS, and the proportion of monocytes was estimated by microscopic examination. Cells were brought up to an appropriate volume of RPMI 1640 with glutamine and antibiotics and without serum to give approximately l X lO6 monocytes/ml. Cells were plated into 96 well flat bottom tissue culture plates (Costar, Cambridge, MA), 200 ~l/well, and incubated for 2 hours at 37-C in a humidified incubator with 7~ 2- Cells were then washed three times in warm RPMI 1640 without serum. After the last wash was aspirated, 200 ~l/well RPMI 1640 with 10%
autologous heat inactivated serum was added. To each well was then added the 22~1 of lOX solution of E. coli Endotoxin preinc~oated in bu~fer, polymyxin B, or BPI.
Cells were incubated with the endotoxin mixture for 4 hours at 37'C, then the supernatants were collecte~ and assayed ~or TNF~ antigen by ELISA (Endogen Inc., Boston, MA) ' :
Inhibition of endotoxin-induced TNF~ secretion by murine broncheoalveolar macrophaqes: Normal anesthetized Swiss-- . ' :
'~ .

' '-'- ' ~V092/03-3- PC~/US91/0575~

Webester mice were challanged by the intranasal route with lOng ~.~coZi Olll:B4 endotoxin (List, ~ampbell, CA).
Twenty minutes before challenge, anasthetized mice were treatRd by the intranasal ~oute with 50 ~1 saline, BPI or polymyxin B solution. At one hour after endotoxin challen~e, mice were re-anesthetized, and 0.7 ml o~
; saline containing 1% human serum albumin was added to the lungs via the trachea. The lungs were gently kneaded.
A 0.5 ml volume bronchoalveolar lavage (BAL) fluid was - 10 aspirated, cells were pPlleted by centrifugation, and the BAL sample was stored at -70C. The TNF~ level in the BAL fluid was determined by measuring cytotoxicity towards WEHI clone 13 mouse fibrosarcoma cells. Human rTNF~ (Chiron, Emeryville, CA) was used as the standard.
RESULTS

BPI binds to bacterial lipopolysaccharide: Binding of BPI to endotoxin was demonstrated using a modified ELISA
protocol to detect BPI bound to immobilized S.
typhlmurium Re endotoxin as described in Methods above.
BPI had bound endotoxin in a concentration dependent manner and binding was inhibited by polymyxin B/
suggesting that BPI binds at or near lipid A (Figure 19).
Significant binding of BPI to endotoxin was retained in the presence of plasma (Figure 20) or serum, thus indicating that BPI binds to endotoxin in ths presence of blood proteins as well as physiologic salts. This date is consistent with the observation by Mannion, B.A. et al. ~ lin, Invest. 86:631 l99O) that BPI binds to bacteria in the presence of serum albumin, although under th~se conditions BPI is not bactericidal. Also, concentrations of Ca2+ and Mg2+ which can rescue bacteria from the lethal actions of BPI (20-80mM) do not : , , ~ ' . ,: . ' ': .. . ~: . ' . , ...... ' ', ' '' : . , -' :
. ' .. : . . ' , :, , ' . ' W092/03535 2 ~ 3 ~ PCTtUS~ 5758 significantly reduce binding of BPI to endotoxin (Figure :

: . ~
BPI bl ocks,, ,endo~ox i n -medi a ted~_ TNF ",~cret~ on ~ vi tro: ; , ~elease in TNF in response to endotoxin in_Yiv~ may play : .
an important role in pathogenesis of endotoxic shock. To investigate the role of BPI in regulating endotoxin- :
mediated TNF secretion, we measured TNF secretion by ~
human adherent peripheral blood mononuclear cells in :.: :
response to endotoxin and to endotoxin preincubated with ::
BPI (Table 1). BPI specifically prevented endotoxin- ~ .
stimulated TNF secretion by these cells in a ~ ::
concentration dependent manner. In addition, inhibition ::
by BPI could be overcome by a Iarge excess of endotoxin (100-lOOo ng/ml) or 0.1% killed S. aureus indicating ~-that BPI did not interfere with monocyte function but :~
rather blocked specific activation of monocytes by ::
endotoxin.

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W~92/03~35 PCT/US91/0~758 3~
Table 1 . . ' .

Inhibition of endotoxin-Induced TNF Production by BPI

TNF(pg/~l) Endotoxin Buffer Polymyxin BPI BPI
ng/ml Control l.0 ~Og/ml O . 4 ~g/ml 0.1 ~g/ml - - - -- - - -- - - - -100 823 + 67 400 +148 530 +16 746 + 48 756 + 116 76 ~ 25 60 + 9 182 + 42 598 + 89 0 0 o -- - -- - ---- - -- -- --- - '. ' Human peripheral blood mononuclear cells were stimulated ;~
with E. _ coli Olll:B4 endotoxin which had been preincubated for 30 minutes at 37C with buffer, BPI or polymyxin B. Supernatants were harvested ~our hours after endotoxin mixtures were added. Secretion of TNF~
was quantitated by ELISA.
BPI bloc~s in vivo pyroqenicity of endotoxin: Cytokines released in response to experimental endotoxin in~usion cause physiologic changes including fever induction. We studied the ef~ects of BPI on endotoxin pyroyenicity by injecting rabbits with endotoxin or endotoxin preincubated with BPI. Resulting changes in temperature were monitored at three one-hour intervals post injection. The greatest temperature increase was used to calculate ~(~T) for the three animals test in each group.
A value of > 1.4C is considered pyrogenic (U.S.

WO9~/~3~35 2 i~ ~ g `~ 3 ~ PCT/~S9l/05758 Pharmacopeal Convention, Inc., 1990 Rockville, MD, Test 151, p. 1515). While a total temperature rise o~ 3.9C
was observed in the gro~p in~ected with 400EU of FDA
reference standard endotoxin alone, endotoxin pre-treated with 2~g BPI was not pyrogenic, showing a total ~ ;
te~perature rise of only l.l-C. ~o res~nse was observed in buffer treated control animals or BPI :reated animals.
~ ::'.' BPI blocks endotoxin-mediated TNF secretion ln vivo: To determine whether BPI could inhibit endotoxin-mediated TNF~ secretion n vlvo, we tested BPI neutralization of -endotoxin in the murine lung. Administration af BPI into the lung twenty minutes prior to endotoxin challenge significant~y reduced the amount~ of TNF secreted into bronchoalveolar lavage fluid by alveolar macrophages (Table 2). Four out of five saline treated mice had TNFQ
levels greater than 1,000 pg/ml, versus one of five for BPI. Overall, BPI reduces endotoxin-mediated TNF~
secretion by murine lung alveolar macrophages by 8.2-fold. Relative to the saline control, reduction of TNF
secretion by BPI was significant (using the Student's t-test) at the p<0.05 level. (Geometric mean + SD of saline control: 3.364+0.402, BPI treated group 2.1Q9+0.764). Polymyxin B was slightly more effective in reducing TNF~ secretlon relative to the saline control (p<0.02) although the dose of PMB was S0-fold greater on a molar basis than that used for BPI. These data indicate the soluble BPI neutralizes endotoxi~ in_Yi~Q-,, , : .

~, .
'.: ' ' '' , ~092/~3~3j PCT/US91/0575 Table 2 E~fect o~ BPI on endotoxin-Mediated TNF secretion by ~urine BAL
TNF (pg/ml) . _ . . _ Mouse Saline BPI Polymyxin B -Control 0.86~g l.O~g -(15 pmol) (782 pmvl) 1 1200 15 74 - ;

4 2800 67 370

5 5250 1310 640 Mean ' SD 3097 + 2250 376 + 547 253 + 251 Normal anesthetized mice were challenged by the intranasal route with 10 ng E._coli Olll:B4 endotoxin.
Twenty minutes before challenge, anesthetized mice were treated by the intranasal route with 50~1 saline, BPI or i~ -polymyxin B solution. Bronchoalveolar lavage (BAL) fluid was assayed for TNF~ by measuring cytotoxicity towards WEHI as clona 13 mouse ~ibrosarcoma cells. Human rTNF~ ;
was used at the standard.
. _ .. . . . ._ . :

our data show that BPI specifically prevented endotoxin-stimulated TNF secretion in vi tro by human adherent mononuclear cells in a concentration dependent manner.
Inhibltion of endotoxin-induced TNF secretion distinguishes BPI from LBP. LBP, a 60 kDa acute phase protein synthesized by hepatocytes, has 44~ amino acid sequence homology to BPI and binds to endotoxin in vivo ; W092/03535 ~ V PCT/VS91/057~8 and in vi~o (Tobias, P.S., X. Soldau, and R. J.
Ulevitch. 1986. Isolation of ~ lipopolysaccharide-binding acute phase reactant from rabbit serum. J. Exp.
~ed. 164:777) (Schuman, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, 5. D. Wright, J. C. Mathison, P. S. Tobias, and R. J. Ulevitch. 1990. Structure and function of lipopolysacchride binding protein. Science. 249:1429).
Despite their structural similarities, BPI and 1BP are functionally antagonistic. LBP-endotoxin complexes prime - lo neutrophils for the oxidative burst response to FMLP and cause accelerated and increased TNF production by monocytes in vitro (Vosbeeck, K., L. Sklar, H. Muller, C.
Lundberg, c. Hanson, K. Arfors, R. Ulevitch, and P.
Tobias. 1988. Nodulation of lipopolysaccharide (LPS) induced neutrophil priming by an acute phase reactant, lipopolysaccharide binding protein, LBP. Eur. J. Clin Invest. 18A50) (Wright. S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 249:1931) (Tobias, P. S., J. C., Mathison, and R. J. Ul~vitch. 1988. A family of , -lipopolysaccharide binding proteins involved in responses to Gram-negative sepsis. J. Biol. Chem. 263:13479). In contrast, BPI blocks LPS-mediated stimulation of both neutr~phils (Marra, M. N., C. G. Wilde, J. E. Griffith, ~i J. L. Snable and R. W. Scott. 1990. ~-Bactericidal/permeability increasing protein has endotoxin-neutralizing activity. J. Immunol. 14~:662) !~
and macrophages in vi~ro. Since BPI-endotoxin complexes fail ko stimulate inflammatory cells in vitro one would not expect such complexes to elicit a pyrogenic response when administered ~n vivo. Small quantities of endotoxin alone induce a strong pyrogenic response resulting from the release of endogenous pyrogens such as TNF, IL-l, and -gamma IFN (Farley, M. M., W. M. Shafer, ~nd J. K.
.

.
:. :
: .

W092/0353~ PCT/US91/057~8 `3'~ 36 Spitznagel. 1988. Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein. InPect.
I~mun. 56:1589)~ Rabbits are exquisitely sensitive to trace quantities of endotoxin, and respond with a dose dependent and reproducible elevation o~ core temperature.
In complex with BPI, endotoxin was unable to stimulate a pyrogenic response in rabbits. Thus, BPI is an effective inhibitor of endotoxin in _iyQ presumably a result of BPI
blocking endotoxin-mediated cytokine secretion.

The bactericidal and permeability increasing activities of BPI in v ro are associated with the N-terminal half of the molecule which shares extensive homology with LBP
(Schuman, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison P. S. Tobias, and R. J.
Ulevitch. 1990. Structure and function of lipopolysaccharide binding protein. Science. 249:1429).
No function has been ascribed to the carboxy-terminal region, other than a membrane spanning domain. Gray and colleagues (Gray, P. W., G. Flaggs, S. R. Leong, R. J.
Gumina, J. Weiss, C. E. Ooi, and P. Elsbach. 1989.
Cloning of the cDNA of a human neutrophil bactericidal , protein. Structural and functional correlations. J.
Biol. Chem. ~64:9505) suggest that the carboxy-terminal half of BPI is associated with the azurophil granule membrane. In their model, when neutrophils are stimulated, proteolytic enzymes such as elastase cleave the molecule releasing the active, bactericidal N-terminal half into the phagolysosome. Several lines of evide~ce, however, argue against BPI as an integral membrane protein. BPI can be extracted from isolated azurophil granules in the absence of detergents. BPI is soluble in aqueous solutions and soluble BPI is active in tests for both endotoxin binding and inhibition. Also, -, .

W092/03~3~ 8 ~ ~ `S PCT/US91/05758 BPI is released ~y FMLP~cytochalasin B stimulated neu~rophils (71~ of total cellular BPI) as a full-len~th, protein, arguing against release o~ the N-terminus by neutrophil proteases upon deyranulation.

In vivo. BPI likely functions to suppress endotoxin toxicity and not as a bactericidal protein. Endotoxin binding proteins such as LBP and BPI may function respectively as a receptor/receptor-antagonist system to regulate the host response to endotoxin (Figure 22). LBP
acts as a soluble receptor for endotoxin and amplifies the effects of endotoxin on both neutrophils and macrophages. The ability of BPI to limit the host response to endotoxin indicates that BPI may have an important role in blocking lethal effects of endotoxin L
vivo. Preliminary results in animals (see Example 4) show that treatment with recombinant BPI markedly reduces endotoxin-induced lethality. Thus, use of E~l to neutralize endotoxin, in conjunction with conventional antibiotics to limit bacterial growth, may be a useful therapy against endotoxic shock.

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. ':, W092/03535 PCT/US9l/05758 M~ PRE88~0N OF ~PI RRo~EIN8i~ND BPI~5~5~En ~0R~8 A. G~E~IC~Y ~NGIN~ERED ~MNALI~N CEL~ ~XPR~8 BP~
In order to produce BPI protein and/or BPI protein variants in mammalian cells, the cDNA sequences must be inserted into a suitable plasmid vector. A suitable vector for such an application is pSV 1, which contains the origin of replication and early and late promoters of SV40, followed by multiple insert cloning sites, followed ; by the termination sequences from the hepatitis B surface antigen gene. Also cont~ined within the plasmid are an origin of bacterial DNA replication, and the genes encoding ampicillin resistance and dihydrofolate reductase. Similar vectors have been used to express other foreign genes (McGrogan, et.al. Biotechnology 6, 172-177). Vector DNA was prepared for acceptance of BPI
protein cDNA sequences by digestion with HindIII and Bam HI, and dephosphorylation with alkaline phosphatase.

` Several BPI protein cDNA-containing inserts were prepared for insertion into pSV-l. First, an insert encoding full-length BPI protein was prepared by digestion of the parent plasmid with appropriate restriction enzymes for ex. EcoRI and Bgl II, yielding two DNA fragments containing portions o~ the BPI protein coding sequence.
These two fragments were ligated together into prepared SV-l, and the recombinant clones obtained were screened by restriction enzyme digestion ~or the presence of the two inserts in the proper orientation. Two cDNAs encoding truncated forms of BPI protein were generated using oligonucleotide-directed DNA amplification of the parent BPI protein insert DNA. The amplifying oligos were designed to replace codons 212 (oligo 459) (Figure w~ g2~3~3~ h ~ ~ 3 ;~ PCT/US91~OS758 7) and 337 (oligo 50) (Figure 8) with stop codons, in addition to a BamK loning site (Figure 5). ~t the 5'-end o~ both constructs, oligo 458 was used in the ampli~ications to create a HindIII site immediately upstream of the translational start codon ATG (Figure 8).
Thus, three BPI-encoding inserts were created, each encoding 55 kDa, 38 kDa, and 25 kDa forms of BPI, and each was ligated separately into prepared vector DNA.

Each of the three constructs was verified by restriction digest analysis, and then prepared in amounts sufficient for transfection into CHO cell line DUXBll cells.
Transfection was performed using lipofectin, and the resulting transformed cells were selected in the presence of increasing amounts of methotrexate using standard protocols (Figure 3).

Supernatants from either transfected pools or clones derived from the pools were assayed for the presence of endotoxin binding activity by inhibition of TNr release.
BPI was negligible in the vast majority of the selected ~
cell lines. We found that only cell lines established .!,'1 ~ .`' from a 500 nM methotrexate bulk amplification produced commercially reasonable quantities of BPI. Two such cell lines are designated 3Al and 4D6. It was unexpected that only the bulk amplification resulted in such cell lines.

B. BAC~LOVIR~8 EXPRE8~ION o~ rBPI IN I~BEC~ CEL~8 ~0 ' -Construction o~ pl~smi~ expressio~ veotor: In order to ~-produce BPI protein and/or BPI protein variants in insect cells, the cDNA sequence must first be inserted into a suitable plasmid expression vector, such as pAC373 (Figure 10). Appropriate restriction sites for this `~
`"' : ' ' . , , . , ~ . , ~ , , . . . . - . . . .

~V092/03S3~ PCT/US91/05758 insertion were created by standard site-directed mutagenesis procedures. The essential properties of a suitable expression vector include a transcriptional promoter such as the polyhedron gene promoter o~ pAC373, and flanking homologous sequences to direct recombination ~`i into the baculovirus genome. A polyadenylation signal, such as the one from the polyhedron gene present in this plasmid vector, may or may not be necessary for expression of the recombinant gene. A marker gene such as the beta-galactosidase gene of E. coli, juxtaposed to regulatory sequences including a transcriptional promoter and possibly a polyadenylation signal, may be included in the vector but is not essential for BPI protein expression. A typical vector for such purposes pAC373, is shown in Figure 10.

Creation of r~combinant baculavirus: A chimeric baculovirus was created by homologous recombination between the expression plasmid containing the ~PI protein target gene (or truncations thereof derived as described in Section A) and wild type baculovirus DNA. Plasmid and wild type baculovirus DNA were co-precipitated by the calcium phosphate technique and added to uninfected Spodoptera frugiperda (Sf9) insect cells. Four to seven days following transfection, cells exhibited a cytopathic morphology and contained the nuclear occlusion bodies typically produced by viral infection. The cell-free culture media containing both wild type and recombinant virus was harvested and assayed for BPI activity.
Ide~tif$cation and i~olatio~ o~ ohi~eri~ baculavirus:
Clonal isolates o~ virus was obtained from this co-transfection stock by plaque purification on Sf9 cell monolayers overlaid with agarose. Candidate plaques for analysis will be identified by a plaque morphology W09~/03~3~ $ ~ ~ ~ PCT/US91/~575 ,~ 1 negative for occlusion bodies. If the expression plasmid contains a marker gene such as beta galactosidase, recombinant plaques will be indicated by the blue color produced fro~ a chromogenic substrate such as 5-bromo-4~
chloryl-3-indolyl-B~D-galactopyranoside (X-gal) in the agarose plating medium. Picked plaques will be used for inoculation of cells in multiwell dishes. The resulting cell lysates and infected cell supernatants can he evaluated for expression of recombinant BPI, using standard activity or immunological assays. Positive wells may require additional rounds of plaque purification to obtain pure recombinant virus stocks free from wild type contamination.
, .
B~tch production of BPI: Sfg cells are adapted to growth in serum-free, low protein medium such as Ex~ell (J.R.
Scientiric). Cells are collected from suspension culture by gentle centrifugation and resuspended in fresh medium containing the viral inoculum at a concentration of ten million cells per ml, using a multiplicity of infection of one virus plaque forming unit per cell. After a period of two hours, the culture is diluted five fold with fresh medium and incubated two to three days. At the end of that time, the cells were pelleted by centrifugation and the conditioned medium was harvested.
BPI protein was purified from the cell-free supernatant by standard means.
.. ..

Char~cterisation o~ l~s~¢t cell ~erive~ BPI: BPI pro~ein produced in insect cells using a baculovirus expression system is a glycosylated protein o~ a~;roximate molecular weight o~ 55,000 kd. The N-ter~inal amino acid sequence is identical to that of mature mammalian cell BPI, indicating correct processing of the signal sequence.
The specific activity of endotoxin binding of recombinant WOg2/03~35 PCT/US9l/057~8 protein was indistinguishable from BPI.

Co~struction o~ p~7BPI protein Pl~mi~:
Oligonucleotides were prepared on an Applied Biosystems 380B DNA Synthesizer ~or use in oligonucleotide directed DNA ampli~ication. The oligonucleotides created Nde I
and BamHI restriction sites at the 5' and 3' ends, respectively, of the BPI protein DNA. In addition, another oligonucleotide containing a BamHI restriction site was used to create the truncated proline-212 version of the BPI protein DNA.

Following the amplification reactions, fragments were purified and digested with Nde I and BamHI. The plasmid, pGEMEX-l, (available from Promega) was selected as the vector for the constructions. pGEMEX-l contains a T7 promoter which can be used for the expression of downstream sequences when placed into the proper host.
The vector was cleaved with BamHI and, following purification, partially digested with Nde I to generate a vector with a single Nde I site and a single BamHI
site. The fragments were ligated and transformed into the E. coli strain JM101 using the Hanahan transformation protocol (DNA Cloning Volume I, A Practical Approach, Edited by D. M. Glover, IRL Pre s). The transformed bacteria were plated on LB plates containing carbamicillin and incubated overnight at 37c. Resistant colonies were selected and analyzed by preparing mini-plasmid preparations and digesting with the appropriate restriction enzymes. Digests were analyzed on both 1 agarose gels and 5% polyacrylamide gels.

The expression host, E. coll strain JMlO9(DE3), was transformed using 1 ~l of the mini-plasmid preparation and the ~anahan transformation protocol. JMlO9(DE3) _ ' , ' ' . ' . ' ' , , ' ' : ' ,.', , , . '.. 1 ' . . ' , . . . . " , . . : ,' . ' ' .

. . , ' . ' . ' . ' . . . ' . .

W092~0353~ 53~k)~ PCT~US91/0~758 contains a chromosomal copy of the gene for T7 RNA
polymerase which can be induced with IPTG. The transformed bacteria were plated on LB plates containing carbamicillin and incubated overnight at 37C. Results S are shown in Figure lA~lE.
:~ .
Since the full-length and proline-212 truncated forms of BPI protein containing the signal peptide do not give colonies while those forms that do not contain the signal peptide do give colonies, the BPI protein was expressed in an active form and is processed correctly, sending the protein to the periplasmic space of the bacteria (the location in bacteria that proteins possessing a signal peptide are sent to) where the bactericidal activity kills the cell. This also implies that both the full-length form and the proline-212 truncated form are active and capable of bactericidal activity.
:, Whether the forms of BPI protein which do not contain the signal peptide are active or are prevented from exhibiting their bactericidal activity by being sequestered in the cell (either by the formation of inclusion bodies or by the inability to gain access to the plasma membrane due to the absence of the signal peptide or both) is not known.
:
~BPI was purified as ~ollQws: Conditioned media containing recombinant BPI ~rBPI) was puri~ied to 95%
homogeneity in a single step over CM-Sepharose. I'he CM-Sepharose column ~Pharmacia, Piscataway, N.J.) was firstwashed in ~ive column volumes of 0.5 M NaOH followed by rinsing with pyrogen free bu~fers or water until no pyrogen could be detected by the Limulus Amebocyte Lysate Assay (Whittaker, Walkersville, MD). The column was then equilibrated in 50mM Tris pH 7.4. The conditioned media WO9~/03535 . PCT/US9~/0S7S8 was then loaded and the bound protein was eluted in 50mM
Tris lM NaCl pH 7.4. rBPI was concentrated and further purified by loading onto a second CN-Sepharose column equillbrated in 50mM Tris pH 7.4 and eluted using a gradiant ~f O~O-l.OM NaCl. BPI elutes at approximately 0.75M NaCl. rBPI thus puri~ied appeared as a single band on SDS-polyacrylamide gel electrophoresis and as a single peak on reverse phase HPLC.

WO 92~03~3~ ) r~ ~ $ ~ PCI'/US91/057S8 $~ 3 In~ibi~lo~ o~_ en~oto~in-in~uce~ ~NF
p~oductlo~Lby BPI
:~ .
Human peripheral blood mononuclear cell~ were isolated on Ficoll-Paque (Pharmacia) gradients, washed 2X in pyrogen free HBSS (Hazelton), and resuspended at 5 X 106/ml in RPMI (Gibco) media without serum. Two hundred ~l of this cell suspension was incubated in each well of flat-bottom 96 well tissue culture dishes (Costar) for 2 hours at lo 370C. Nonadherent cells were removed by washing 2X with RPMI + 10% autologous heat inactivated serum. Adherent mononuclear cells were stimulated with E. coli Olll:B4 endotoxin which had been preincubated for 30 minutes at 37C with buffer, BPI protein or polymyxin B (Gibco; 7900 - U/ml). Supernatants were harvested four hours after endotoxin mixtures were added. Secretion of TNF~ was quantitated by ELISA (Endog~n) (results at Table 3).
Several lots of natural and recombinant BPI from CHO
cells were tested.
-~
. ~

ENDOTOXIN DOSE
10 ng/ml 1 ng/ml O ng/ml endotoxin endotoxin endotoxin BPI
Protein 0 7.3 1.4 0.3 0 7.3 1.4 0.3 0 (n~):
Control 626 ~ - - 334 - - - 113 78038n - 129 159 203 - 153 187 165 148104n - 98 104 162 - 98 119 162 148113n - 92 114 151 ~ 71 129 155 14~159r - 82 158 155 - 87 136 147 r 148165r - 124 128 138 - 116 129 146 148179r - 85 139 134 - 93 131 166 n=natural r=recombinant '~
,:
~.

W092/03~35 PCT/US91/0;758 ~ 46 MP~

The pakhophysiologic consequences of gram negative sepsis are primarily mediated by the release of bacterial endotoxin ~LPS). Since BPI Protein has endotoxin neutralizing activity in v~tro, the effects of BPI
Protein in vivo were studied in experimental models of endotoxic shock.
Specifically, in one experiment one group of 8 rats : (Sprague Dawley rats) was given a single, bolus injection of 1 mg BPI Protein per kg body weight four hours before a single intravenous bolus of 0.5 mg/kg ~ody weight Olll:B4 endotoxin obtained from Sigma. In the same experiment, a second group of 8 rats was given a single bolus injection of 1 mg BPI Protein per kg body weight simultaneously with a single intravenous bolus of 0.5 mg/kg body weight Olll:B4 LPS. Further, a third group of 5 rats was given a single bolus injection of 1 mg BPI
Protein per kg body weight four hours after a single intravenous bolus of 0.5 mg/~g body weight Oll:B4 LPS.
Finally, a fourth group of 10 rats was treated with endotoxin alone. The rats were observed for 48 hours and the survival recorded for each group. The results of this experiment are shown in Table 4. Rats to which ehdotoxin alone was administered exhibited a mortality rate of 80%. Rats which received both BPI Protein and endotoxin showed a significantly reduced mortality rate.
The results set forth in Table 4 establish BPI Protein is useful in vivo both to prevent and to treat disorders associated with the presence of endotoxin. High dose BPI
Protein toxicity studies revealed no evidence of toxicity when the animals were sacrificed at 7 days. We conclude BPI is a non-toxic naturally occurring protein which W092/03535 ~ 6 PCT/V~91/057~8 bindq LPS, inhibits release o~ TNF a~d reduces mortality in both endotoxin and GNB experimental ~epsis models tFigure 17). We believe BPI Protein offers a novel immunotherapeutic approacA to the management of septic 5 shock.

10 INVE~IGATION OF ~ PO~EN~IAI PROTECTI~E EF:E ICACY OP' BPI
IN THE: R~T ENDOTOXIN CI~I L~NG13 NODEL : ` -.:
Endotoxin BPI Protein BPI Protein Dose DoseAdministration Reqimen %
Survival ~-0.5 mg/Xg (2/10) 0.5 mg/kg lmg/kg4 hr pre-injection (6/8) 0.5 mg/Xg lmg/kgsimultaneous ~4/8) 0.5 mg/kg lmg/kg4 hr post-i~jection (4/5) ;
Additionally, in asecond experiment with Bactericidal/Permeability Increasing Protein (BPI) neutropenic rats were challenged with Pseudomonas (PA1244) during a period of neutropenia. One group of rats was treated with lOmg BPI/kg of body weight ~y intravenous administration at the onset of fever at day 5 and observed through day 11. A second group of rats was treated at tha onset of fever with buffer containing saline at day 5 and observed until day 11. After day 8, the rat group treated with buffer was found dead;
however, the rat group treated with BPI Protein exhibited 60% survival. The rats were observed for 11 days and the ,: .

W~92/03i3~ PCT/US91/05758 ~8 survival recorded for each group. At day ll, no additional deaths occurred for the rat group treated with BPI. The rasults of this experiment are ~hown in F'igure lS. Figure 15 is a line graph showing khat ~l) during and after day 8 the rat group treated with buffer experienced a 100% mortality rate and (2) during and a~ter day 7 the rat group treated with BPI Protein ` exhibited about a 40% mortality rate. The rats which received BPI Protein showed a significantly reduced mortality rate.
.
Human BPI Protein at does up to lo mg/kg intravenously (IV) produced no acute hexatologic, biochemical, or pathologic abnormalities in outbred CD-l mice or Sprague-Dawley rats (Table 5). Infusion of 1 mg/kg of E. coliolll:B4 endotoxin IV in 6 CD-} mice resulted in a 100%
(6/6) survival rate in control CD~1 mice. The survival rate in BPI Protein treated mice in~used with 1 mg/kg of E. coli Olll:B4 endotoxin IV at l mg/kg BPI Protein IV, 2 mg/kg BPI Protein IV, and 10 mg/kg BPI Protein IV was 100% (4/4), 100% ~4/4) and lO0 (5/5), respectively.
.
Infusion of 10 mg/kg of E. coli Olll:B4 endotoxin IV in

6 CD-1 mice resulted in a 17~ (1/6) survival rate in -~
control CD 1 mice. The survival rate in BPI Protein treated mice infused with 1 mg~kg of '~ .

, .:

t ; ~ ~;

~O 92/03a3a ~ 3 ~ PC~/US91/057S8 ~3PI PROTEIN P~OTECq'l3 AGAIN8T LE~ Y FROM
~ND,O?OXI~ ~.-~HO~

% SURVIVAL ¢NO. SURVIVORS~TOTAL NO. ~NIMAI.S

Control BPI BPI BPI
(Saline) lmg/kgIV 2 mg/kgIV 1 0 mg/kg IV

Endotoxin ChallPnge (E. Coli Olll: B4 ) * 1 mg/kg IV 100 (6/6) 100 (4/4) 100 ~4/4) 100 (5/5) * 10 mg/kg IV 17 (1/6) 50 (2/4) 100 (4/4) 100 (5/5) * 50 mg/kg IV - 0 (0/6) 25 (1/4) 25 (1/4) 100 (5/5) * 100 mg/kg IV 0 (0/6) 0 (0/4) o (0/4) 80 (4/5) 200 mg/kg IV o (o/6j o (0/4) 0 (0/4) 20 (1~5) : , - ''~'--' , , ~
,:
-.

WO9~/0353~ PCT/US91/0575~

$ E ~ ÇDli. 0111: B4 endotoxin IV at l mg/kg BPI Pxotein IV, 2 mg/kg BPI Protein IV, and lO mg/~g BPI Protein IV was 50% (2/4), 100% (4/4) and lO0 (5/5), respectively.

Infusion of 50 ~g/kg of ~. co~i Olll:B4 endotoxin IV in 6 CD-l mice resulted in a 0~ (0/6) survival rate in control CD-l ~ice. The survival rate in BPI Protein treated mice infused with l mg/kg of ~. coli Olll:B4 endotoxin IV at l mg/kg BPI Protein IV, 2 mg/kg BPI
Protein IV, and lO mg/kg BPI Protein IY was 25~ (l/4), 25~ (l/4) and 25 (5/5), respectively.

Infusion of lOO mg/kg of E. coli Olll:B4 endotoxin IV in 6 CD-l mice resulted in a 0% (0/6) survival rate in control CD-l mice. The survival rate in ~PI Protein treated mice infused with l mg/kg of E. coli Olll:B4 endotoxin IV at l mg/kg BPI Protein IV, 2 mg/kg BPI
Protein IV, and lO mg/kg BPI Protein IV was 0% (0/4), 0~ (~
(o/4) and 80% (4/5), respectively.
Infusion of 200 mg/kg of E. coli Olll:B4 endotoxin IV in ~-6 CD-l ~ice resulted in a 0~ (0/6) survival rate in control CD-l mice. The survival rate in BPI Protein treated mice infused with l mg/kg of E. _coli Olll:B4 endotoxin IV at l mg/kg BPI Prot~in IV, 2 mg/kg BPI
Protein IV, and lO mg/kg BPI Protein IV was 0% (0/4), o%
~0/4) and 20% (l/5), respectively.

In conclusion, Table 5 demonstrates that ~PI Protein is :
non-toxic in experimental animals and provides ~; -significant protection from lethality following endotoxin challenge ~Figure l6). This naturally occurring, :-neutrophil derived, antimicrobial protein provides a new ~ -~
therapeutic strategy in the treatment of septic shock. -`

- ' ;' , :
-; -W092/03~3~ PCT/US91/05758 Human BPI Protain at doe~ up to lo mg/kg intravenously(IV) produced no acute hexatologic, biochemical, or pathologic abnormalities in outbred CD-1 mice (Table 6).
Using CD-l mice, the ~IL~YiYn e~`ficacy of BPI Protein against endotoxin was tested by infusing 50 mg/kg of coli Olll:B4 endo~oxin IV in lO mice resulted in a 100%
tO/10) survival rate in control CD-l mice. The survival rate for BPI Protein treated mice infused with 50 mg/kg of E. coli Olll:B4 endotoxin IV at lO mg/kg BPI Protein IV was 0% (0/10). The p value is p<0.001. Further, 5 mice were infused with 50 mg/kg of 055 IV (as control~
which resulted in a 0% (0/5) survival rate. The survi~l rate for BPI Protein treated mice infused with of ~0 mg/kg 055 IV at 10 mg/kg BPI Protein IV was 100% (5/5).
The p value is p<0.01. Additionally, 5 mice wexe infused with 25 mg/kg of Rc rough mutant ~core glycolipid) IV (as control) which resulted in a 0~ ~0/5) survival rate. The survival rate for BPI Protein treated mice infused with 25 mg/kg of Rc rough mutant (core glycolipid)IV at 10 mg/kg BPI Protein IV was 100% (5j5). The p value is p<0.01. Also, 4 mice were infused with 25 mg/kg of Lipid A IV (as control) which resulted in a 0% (o/4) survival rate. The survival rate for BPI Protein treated mice infused with 25 mg/kg of Lipid A IV at l0 my/kg BPI
Protein IV was 100% ~5/5). The p value is p<0.05.

BPI is a non-toxic naturally occurring protein with endotoxin neutralizing activity which reduces mortality in both endotoxic and bacteremic models of sepsis syndrome and may be a use~ul immunotherapeutic approach to the management of the septic shock.

. . :
. ;

. . . ~ ... . . . . : . . . . - - .~

W092/n35~ PCT/US91/057~8 2~ 52 TABT.~_6 ~FF~C~_OF BPI PRO~BIN ON LE~ OF VARIO~S ~NDOTO~IN P~NOTYPE8 ~ Survival 10 (No. Surviving/No, Animals ~ested) l0mg/kg ontrol ~ Value endotoxin Phenotype -* 0lll:B4 50mg/kg l00 (l0/l0) 0 (l/l0) p < 0.00l -* 05S
50mg/kg l00 (5/5) 0 (0/5) P = 0.0l -* Rc Rough mutant ~ - .-(core glycolipid) -.:-25 mg/kg l00 (5/5) 0 (0/5~ p = 0.0l :.
* Lipid A
25 mg/kg l00 (5/5) 0 (0/4) p < 0.05 :
''':.
' ': '' . ' ;, ~ .

..

~"''' ~

W092/0353~ PCT/US91/05758 _ In order to generate a non-glycosylated form of the BPI
molecule, the (CH0) cell line which normally expresses glycosylated racombinant BPI Protein (clone 3Al), was grown to con~luenca in roller bottles (Costar, Cam~,ridge, ; 5 MA) in REM 020 (Haæelton, Inc. Denver, PA) containing

7.5% dialyzed boYine serum (Gibco) + 2~g/ml tunicamycin (Boehringer Mannheim, Indianapolis, IN). After 24 hours, the medium was discarded, and replaced with fresh complete medium containing 2~/ml tunicamycin.
Conditioned medium was collected and replaced every 24 hours for three daysO Non-glycosyl.ated recombinant BPI
Protein was purified as described in Example 3 above for recombinant BPI Protein and further s~parated from residual glycosylated reco~binant BPI Protein by Superose 12 (Phar~acia) size e~lusion chromatography in 20mM
glycine + 100 mM NaCl at PH 2. Fractions containing nonglycosylated BPI Protein (identified by polyacrylamide gel electrophoresis) were pooled.

Glycosylated or nonglycosylated recombinant BPI Protein was injected into mice at lOmg/kg. Blood was collected at the indicated times through the retroor~ital plexus.
Blood samples ~ere then allowed to clot, the fi.brin clot was removed by centrifugation, and the recombinant BPI
Protein levels were determined by ELISA assay (results are shown in Figure 18).

- . . .. .- , - . . . .. . .

W ~ 92/03535 PC~r/US91/05758 LI8A ~UB~AY

~Q~IPNE~rr . 5 Immulon-2 96 well plates (Dynatech) - 12-channel 50-200~1 pipettor P20, P200, P1000 pipettors Reagent reservoirs (Costar) Racked 1 ml tubes (BioRad) polypropylene 15 ml conical tubes R~G~I~T8 : . ' SOLUTIONS . : .
-::
25Mm Borate pH 9.S : :
Blocking solution = 5~ BSA (Sigma Fraction V, Low ::.
Endotoxin) in PBS ~ :
Wash/Sample Buffer: 50 mM Tris pH 7.4 500mM NaCl ...
1 mg/ml BSA : ::
0.05% Tween 20 1 ~/ml Polymyxin B Sulfate (Gibco/BRL, 7900 U/mg) . ~:
BPI standard (aliquots stored ~ - 70C) .
NOTE: BPI standards and samples should be diluted in polypropylene ' ' Standard and sample diluent = appropriate solution for unknowns (e.g. if testing tissue culture supernatants, -use REM + 7.5~ dFBS) ''.

W092/03~3S ~ ~ g~;Q~ V PCT/US9l/~57~8 Substrate Bu~er: (makes 500 ml) 24.5 mg MgC1 48 ml ethanolamine bring up to - 400ml with Lab V H2O ;~
Adjust to pH 9.8 Bring up to 500 ml with Lab V H2O ;, PNPP substrate tablets (5mg/tablet: Sigma) ANTIBODIES ~-~
.: . .
Capture (lst) Antibody (100~1/well) A. INVN 1-2 (rabbit polyclonal anti-human BPI
Protein) IgG l~g/ml, or, B. MM-1 (rabbit anti N-terminal 20 amino acid BPI -peptide) 3~g/ml.

Reporter (2nd) Antibody A. INVN 1-2-Biotin (Use Q 1:~1000) B. PIG8 (murine monoclonal anti-BPI which blocks BPI binding to bacteria Third;(developing) reagent A. Streptavidin/Alkaline Phosphatase (BioRad) (use Q 1:2000) B. Goat anti-mouse Ig/Alkaline Phosphatase con~ugate (BioRad) (use ~ 1:2000) ~ :

.:

~`.

,. ..

` W092/0353~ PCT/US91/05758 X~ ~ 56 PROCiEDURB
,' '', 1. COATING PL~TES

'' 5 Note: Coat plates up to 1 month in advance. ;
; Store plates at 4C until needed.

Dilute capture antibody as directed in 25mM Na Borate p~ 9.5 (lOml/plate). ::~
, lo Add 100~1 ~o cach well of 96 well plate :~
, (Immulon-2). ~ -~
: Incubate overnight at 37C.
`~ . Refrigerate until used. ~ ~ .
~. .
~: 15 2. BLOCKING

,: Flick coating antibody out of plates. ~ :
Add 200~1 5~ BSA in PBS to each well. . : .
Incubate 2-4 hours 37C or overnight at 4C.
Wash 4X with wash solution and blot on paper towels.

~ . 3. BPI STANDARDS AND UNKNOWNS
; : STANDARDS ~ ~;
Thaw new standard aliquot (O.S;ml ~ 1 mg/ml) ~ ~
every 2 months. ~ ~ .
1. Make lml stock solution of purified or BPI at lOOng/ml ', -, ......

~ .
. . .:
~ j . '", ~

.. . .

WO~/03535 ~ PCT/~S91/05758 57 .
2. Make 500 ~l of each o~ the following standard concentrations as follows:
, , ~l lOO ng/~l BPI ~ diluent final [BPI] ng/ml lO~ 400 ~0 .
425 15 ~ ~
450 lO ~ .

o 50~ 0 Add lOO ~l standard (unknown) / well and incubate at RT ` .
for 2-4 hours, or overnight at 4C.

wash 4X

: 2nd ANTIBODY
After final wash, blot plate vigorously, and add 100~1 of INVN1-2-Biotinylated @ l:lOOO (=lO~l in lOml of wash/sample bu~fer~ to each well.
;
- Incubate 37C l hour :~
Wash 4X

3rd ~NTIBODY
After final wash, blot plate vigoxously, and add lOO~l developing reagent to each well.

Incubate 37C 30 minutes Wash 4X
~t~, ~'',";
.

W092/03~35 PCT/US9l/05758 ; s~ . 58 SUBSTRATE
NOTE: Add substrate tablets to substrate buf~er just :: ~
before adding to plate. ~ :

After final wash, bl~t plate vigorously, and add 100~1 substrate solution (2X 5~g PNPP substrate tablet~lOml substrate bu~fer) .~

Read plate at 405 nm. Xeep plate in the dark :.
between readings.

'' ",.

,' . .

.

', ;, -, ~ . .
~ ~ . ...

, ,: :, ~, .
''``"' `"

-,:
.': ': .

. ::

- - " ~

W092~03S3S ~ ~5~ PCT/US91/0~758 .
; 59 _E~ampl~ 5 ,: :
~iQl~qi~ y Ac~ve Vax~iants of BpI: Several clas~es of variants of BPI were constructed to alter some of the di~ferent properties of the native molecule. In the first type of construct, variants were designed to extend the molecular half-life in serum. In one of such constructs, the single glycosylation site at Ser351 was altered by making a single base pair change at position -`
1175 (Figure 12) so that it encodes Ala and would not support N-glycosylation (i.e. Ser351->AlaBPI(nonglycosylated) at Table 7). This change was made by amplifying this particular segment of the molecule by PCR using amplimers containing the desired sequence, and then replacing the native segment with the corrected segment by virtue of convenient restriction sites (the SphI site at base 1202 in this case). such molecule was expect~d to possess similar properties as BPI but may be cleared less rapidly by the liver since it would lack the mannose residues recognized by hepatic clearance receptors. Other constructs were designed to take ad~antage of the apparent high stability of LBP, a homolog of BPI. For instance, the amino-terminal 25 kDa segment (presumably the endotoxin-binding domain) of 1BP
; 25 was com~ined with the carboxy-terminal 30 kDa portion of BPI to create a chimeric molecule with the greater serum half-life of LBP but the functionality of BPI (i.e.
LBP25K/BPI30K chimeric at Table 7).

A third type o~ construct utilized the extraordinary seru~ stability of immunoglobin to extend the stability of BPI. The amino-terminal 25 kDa tLPS-binding) portion of BPI was linked to cDNA encoding the constant domain of IgGl. The resultant chimeric molecule could be expected to bind endotoxin and inactivate it like the anti-. . .

W0~2~03~3s PCT/US91/0$758 ~
~ 3''~ 60 endotoxin antibodies currently under development.

A second class o~ molecules were d~signed to enhance the therapeutic index of BPI Protein. For example, the amino terminal domain of BPI Protein contains a very high proportion of positively-charged residues (approximately 14%). In several of the variants, one or more of the amino tsrminal cationic residues were changed to neutral or negatively charged residues by the methods described below. Such redesigned molecules may prove less disruptive to biological membranes and therefore be less cationic. Also, the LBP/BPI Protein chimeric molecule described below (i.e. LBP25K/BP130X chimeric at Table 7) may also be less toxic due to the xeduced cationicity of the LBP amino terminal domain relative to BPI.
.
A third class of variants were intended to increase the affinity, specificity, and/or valency of endotoxin binding to BPI. For example, recombinant BYI Protein containing single base pair changes within the 25 kDA
portion were produced and tested for their ability to .~
bind endotoxin in v tro. Changing certain key amino acids, particularly cationio residues, may enhance the a~finity of BPI Protein for its ligand, i.~e. endotoxin.
Also, the LBP/BPI Protein chimeric molecule designated LBP25X/BPI30K himeric may have the affinity of LPB for endotoxin, while possessing the functionality of BPI.
Other constructs added a second endotoxin-binding domain to BPI, under the expectation that it may bind twice the amount o~ ondotoxin per BPI Protein. ~ ~

A fourth class of mutants were designed to modify the ~ -binding a~finity of BPI and/or BPIfendotoxin complexes for the receptors with which it normally interacts to downregulate maorophage activation. Examples of this ..~,. .
- .
: ~ .

W092/03535 ~ 6 PCT/US91/05758 include BPI Protein with single amino acid change~ within the 30 kDa portion of BPI, created by in vi~rQ
mutagenesis as described below. one such mutant designated BPI25K/DP/BPI30K (Table 7) created a variant from which the intact 25kDA domain could be liberated by treatment with formic acid.

Methods used to create the biologically active variants of BPI were standard as practiced in the art. Relevant portions of key molecules were re_ombined to form chimeric molecules through commonly used methods. For example, the amino-terminal 25'~a portion of LBP was linked to the carboxy-terminal p~;tion of BPI Protein by virtue of an engineered ClaI site within the coding sequence~ as shown in Figure 26. Oligonucleotide amplimers containing cloning sites as well as a segment of DNA sufficient to hybridize to stretches at both ends of the target cDNA (25 bases) were synthesized chemically by standard methods. These primers were then use~ to amplify the desired gene segments by Polymerase Cn~in ~-;
Reaction. The resulting new gene segments were digested with the corresponding restriction enzyme under standard conditions, and were isolated by gel electrophoresis. ~`i Alternately, similar gene segments were produced by digestion of the cDNA itself with appropriate restriction enzymes, and filling in missing gene seg~ents with chemically synthesized oligonucleotides. These seyments of coding sequence were ligated together and cloned into appropriate expression vectors which would allow recombinant production of the encoded sequence. ~elevant expresslon systems for such chimeric molecules include but are not limited to mammalian cells such as CH0, fungi such as yeast, insect viruses such as baculavirus, and - bacteria such as E. coli. For each of these systems, a useful expression vector would include antibiotic ', . . . - ',-: : .-- ' ' ' "': .. ' . : ' :

W092/03~3~ PCT/~S91/05758 2 ~ G 62 resistance gene such as ampicillin which would allow selection of recombinant clones; a second antibiotic resistance gene to allow selection on the host cells, such as neomycin; a bacterial replication origin to allow propagation in bacteria; a hiqh level promoter upstream of the qene insertion site, such as the MMTV, SV40, or metallothionine promoter for CHo cells, the trp, lac, tac or T7 promoter for bacterial hosts, or the alpha factor, gal, or PGDH promoters in yeast; transcription enhancers for the mammalian hosts, such as the RSV enhancer; and a polyadenylation site, AATAAA, if one does not exist within the cDNA sequence. Once homogeneous cultures of recombinant cells were obtained through standard culture methods, large quantities of recombinant chimeric molecules were recovered and analyzed from the conditioned medium through standard chromatographic methods.

As examples, three of the constructs described above were constructed in vector pMamNeo, a commercially available expression vector (Clontech, Mountain View, CA.), and used to transform mammalian cell host DUXBll. After transformation using lipofectin, a commercially available reagent (BRL/Gibco Gaithersberg, MD~), the c~lIs were cultured in standard tissue culture medium to allow recovery and phenotypic expression of neomycin resistance. After 24 hours of recovery, the salective agent G418 was added to the medium to select for cells expressing the introduced genes. After three weeks of culture in selective media, drug resistant cell pools were obtained and grown to confluent densities. At this time, media was removed and assayed for the presence of immunoreactivity to anti-BPI Protein by ELISA, and for binding to endotoxin prebound to multiwell plates. In some cases, 160 nM dexamethasone was added to the medium '''~:' ' W O 92/03535 2 ~ a~ PC~r/US9~/05758 to enhance expression because the vector also contained a glucocorticoid binding site in the promoter region.
Tha level3 of BPI Protein produced were monitored in each supernatant sample taken, and representative date is shown below:
Culture Description ELISAEndotoxin Binding ng/mlng/ml A LBP25K/BP130K chimeric 4.3 7 8 B Serl82->AlaBPI(nonglycosylated) 8.3 10 8 C BP125K/DP/BPI3QX 5.8 5.2 D BPI 3.9 3.7 Therefore, these three biologically active variants of BPI were shown to be produced in CH0 hosts, were immunologically crossreactive with anti-BPI Antibody, and able to bind endotoxin at levels similar to BPI. The -;
same vector was transfected into alternate cell host lines to see of improved levels could be achieved.
Constructs from which large quantities of recombinant protein was desired were also recloned into an ampliryable vector such as pSE, containing the gene encoding dihydrofolate reductase. ~,~

~;~
.

.

:- . . :: ~ ... .. ..

Claims (52)

What is claimed is:

1. A composition comprising a BPI Protein and an anionic compound which composition exhibits (1) no bactericidal activity and (2) endotoxin neutralizing activity.

2. A composition of claim 1, wherein the anionic compound is a serum album.

3. A biologically active variant of BPI which (1) specifically binds to endotoxin, (2) competes with BPI Protein for binding to endotoxin, and (3) inhibits endotoxin-induced lethality.

4. A biologically active variant of claim 5 as shown in Figure 23.

5. A biologically active variant of claim 5 as shown in Figure 24.

6. A method for producing and secreting a recombinant BPI Protein from a cell which comprises (a) constructing a vector comprising DNA encoding BPI
Protein; (b) transfecting the cell with the vector;
and (c) culturing the cell so transfected in culture medium under conditions such that recombinant BPI Protein is secreted.

7. A method for producing a recombinant BPI Protein from a bacterial cell which comprises (a) constructing a vector without a signal sequence and comprising DNA encoding BPI Protein; (b) transfecting the bacterial cell with the vector;

and (c) culturing the bacterial cell so transfected in culture medium under conditions such that recombinant BPI Protein is produced.

8. A method for producing a recombinant BPI Protein from an insect cell which comprises (a) constructing a vector comprising DNA encoding BPI
Protein; (b) transfecting the insect cell with the vector; and (c) culturing the insect cell so transfected in culture medium under conditions such that recombinant BPI Protein is produced.

9. A recombinant BPI Protein produced by the method of claim 6, 7, or 8.

10. A glycosylated, recombinant BPI Protein of claim 6.

11. A nonglycosylated, recombinant BPI Protein of claim 7.

12. A method for determining the amount of endotoxin in a sample from a subject which comprises cont??ting the sample with a BPI Protein under condition such that an endotoxin-BPI Protein complex is formed, detecting the amount of the complex so formed thereby determining the amount of endotoxin in the sample.

13. A method for determining the amount of endotoxin in a sample containing bound and unbound endotoxin from a subject which comprises (a) treating the sample so as to denature any endotoxin binding protein to which the endotoxin may be bound and thereby obtaining unbound endotoxin; (b) contacting the treated sample with a BPI Protein under conditions such that the BPI Protein binds to unbound endotoxin of step (a) so that a endotoxin-BPI Protein complex is formed; (c) detecting the amount of the complex so formed thereby determining the amount of endotoxin in the sample.

14. A method of claim 13, wherein in step (a) denaturation is effected using an elevated temperature.

15. A method of claim 13, wherein the elevated temperature is 95 degrees centigrade.

16. A method of claim 13, wherein in step (a) denaturation is effected with an acid.

17. A method of detecting endotoxin in a sample which comprises contacting the sample with a BPI Protein such that the endotoxin binds to the BPI Protein and forms a complex therewith; and detecting such complex.

18. A method of claim 17, wherein the sample containing endotoxin is transferred onto a suitable support under conditions permitting endotoxin in the sample to attach to the support prior to contacting the sample with BPI Protein labeled with a detectable moiety.

19. A method of claim 17, wherein the sample comprises serum, urine, blood, a tissue extract, or sputum.

20. A method for diagnosing endotoxemia in a subject which comprises obtaining from the subject a biological fluid sample, detecting endotoxin in such sample using the method of claim 17 and thereby diagnosing such disorder.

21. A method of claim 17, wherein the sample is a cellular sample.

22. A method of claim 17, wherein the sample is a biological fluid sample.

23. A method of claim 17, wherein the label is a fluorescent label and detection is effected by a fluorometer.

24. A method of claim 17, wherein the label is a radioactive label and detection is effected by a radiograph.

25. A method of claim 17, wherein the label is an enzyme and detection is effected by a spectrophotometer.

26. A method for coating a surgical tool with a BPI
Protein so that the BPI Protein will complex with endotoxin which method comprises attaching BPI
Protein onto a surface of the tool which surface is designed for contact with a biological sample.

27. A method for coating an implantable, invasive device with a BPI Protein so that it will form a complex with endotoxin which method comprises attaching BPI Protein onto a surface of the device which surface is designed for contact with a biological sample.

28. A method of claim 27, wherein the biological sample is blood.

29. A method of claim 27, wherein the biological sample is a tissue sample.

30. A method of claim 27, wherein the biological sample is a muscle sample.

31. A method of claim 27, wherein the biological sample is a cartilage.

32. A method of claim 27, wherein the biological sample is bone.

33. The method of claim 26, wherein the surgical tool is a catheter tubing.

34. The method of claim 26, wherein the surgical tool is a surgical staple.

35. The method of claim 26, wherein the device is a surgical implant.

36. A method for decontaminating a fluid containing endotoxin prior to administration of the fluid into a subject which comprises contacting the fluid with BPI Protein prior to administration,, under conditions such that endotoxin forms a complex with BPI Protein, thereby decontaminating the fluid.

37. The method of claim 36, wherein the fluid is blood.

38. The method of claim 36, wherein the fluid is plasma.

39. The method of claim 36, wherein the fluid is blood serum.

40. The method of claim 36, wherein the fluid is an isotonic solution.

41. The method of claim 36, wherein the fluid is a pharmaceutical agent.

42. The method of claim 36, wherein the fluid is a cell culture reagent.

43. The method of claim 36, wherein the fluid is bone marrow.

44. A kit for detecting the presence of BPI Protein in a biological fluid sample which comprises (a) an assay buffer containing polymyxin B which binds unbound endotoxin molecules; (b) a first antibody attached to a surface area containing the assay buffer, which antibody (1) binds to a portion of active BPI Protein and (2) does not compete with BPI Protein for an endotoxin binding domain; and (c) a second antibody labeled with a detectable moiety which antibody (1) does not compete with the first antibody for BPI Protein binding and (2) specifically binds to BPI Protein at or near the endotoxin binding site, so that when the biological fluid sample is contacted with the first and second antibody an active BPI Protein contained in the biological fluid sample is bound by the first and second antibody thus forming a first antibody-BPI
Protein-second antibody complex, detecting such complex, and thereby detecting BPI Protein in the biological fluid sample.

45. A kit for determining the amount of BPI Protein in a biological fluid sample which comprises (a) an assay buffer containing polymyxin B which binds unbound endotoxin molecules; (b) a first antibody attached to a surface area containing the assay buffer, which antibody (1) binds to a portion of active BPI Protein and (2) does not compete with BPI Protein for an endotoxin binding domain; and (c) a second antibody labeled with a detectable moiety which antibody (1) does not compete with the first antibody for BPI Protein binding and (2) specifically binds to BPI Protein at or near the endotoxin binding site, so that when the biological fluid sample is contacted with the first and second antibody an active BPI Protein contained in the biological fluid sample is bound by the first and second antibody thus forming a first antibody-active BPI Protein-second antibody complex, detecting such complex, and determining the amount of active BPI Protein in the biological fluid sample.

46. A method for preventing endotoxemia in a subject which comprises administering to the subject an amount of a BPI Protein effective to bind to endotoxin so as to prevent endotoxemia in the subject.

47. The method of claim 46, wherein the effective amount of the BPI Protein is between about 0.1 and about 10mg/kg body weight of subject.

48. The method of claim 47, wherein the effective amount is an amount between about 1 and about 10 mg/kg body weight of subject.

49. A method for treating a subject suffering from endotoxemia which comprises administering to the subject an amount of a BPI Protein effective to bind endotoxin so as to treat the subject suffering from endotoxemia.

50. The method of claim 45, wherein the effective amount of the BPI Protein is between about 0.1 and about 10 mg/kg body weight of subject.

51. The method of claim 50, wherein the effective amount is an amount between about 1 and about 10 mg/kg body weight of subject.

52. A biologically active variant of claim 5 as shown in Figure 25.