WO 94/14~37 21513 8 6 PCT/US93112381
PREVENTION AND TREATMENT OF SEPSIS
RELATED APPLICATION DATA
~,, This Application is a Continuation-in-Part Application of Co-Pending Application
Serial No. 07/995,388, filed on December 21, 1992.
5 FIELD OF THE INVENTION
The present invention relates to therapeutics for the prevention and treatment of blood-
borne and toxin mediated ~ e~ec~ and in particular the prevention and treatment of sepsis in
humans as well as other ~nim~ls
BACKGROUND OF THE INVENTION
10 I. Sepsis
Sepsis is a major cause of morbidity and mortality in hllm~n~ and other ~nim~lc It is
çstim~ted that 400,000-500,000 episodes of sepsis resulted in 100,000-175,000 human deaths
in the U.S. alone in 1991. Sepsis has become the leading cause of death in intensive care
units among patients with non-traumatic illnPs~es [G.W. Machiedo et al., Surg. Gyn. &
15 Obstet. 152:757-759 (1981).] It is also the leading cause of death in young livestock,
affecting 7.5-29% of neonatal calves [D.D. Morris e~ ., Am. J. Vet. Res. 47:2554-2565
(1986)], and is a common medical problem in neonatal foals. [A.M. Hoffman et al., J. Vet.
Int. Med. 6:89-95 (1992).] Despite the major advances of the past several dec~des in the
treatment of serious infections, the incidence and mortality due to sepsis continues to rise.
20 [S.M. Wolff, New Eng. J. Med. 324:486-488 (1991).]
Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis,
decreased systemic vascular resi~t~nce, tachypnea and organ dysfunction. Sepsis can result
from septicemia (i.e., org~ni~mc, their metabolic end-products or toxins in the blood stream),
,. including bacteremia (i.e.. bacteria in the blood). as well as toxemia (i.e., toxins in the blood),
25 including endotoxemia (i.e., endotoxin in the blood). The term "bacteremia" includes occult
~^ bacteremia observed in young febrile children with no ap~ L foci of infection. The term
"sepsis" also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus
particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood).
WO 94/14437 PCT/US93/12381
2 `~ 3 8 ~ --
Thus, septicemia and septic shock (acute circulatory failure resulting from septicemia often
associated with multiple organ failure and a high mortality rate) may be caused by a number
of org~ni~m~
The systemic invasion of microorg~ni~m~ presents two distinct problems. First, the ,"
5 growth of the microorg~ni~mc can directly damage tissues, organs, and vascular function.
Second, toxic components of the microorg~ni~m~ can lead to rapid systemic infl~mm~t~ry
responses that can quickly damage vital organs and lead to circulatory collapse (i.e., septic
shock) and o*entimec, death.
There are three major types of sepsis characterized by the type of infecting organism.
10 Gram-negative sepsis is the most common and has a case fatality rate of about 35%. The
majority of these infections are caused by Escherichia coli, Klebsiella pneumoniae and
Pseudomonas aeruginosa. Gram-positive pathogens such as the staphylococci and
streptococci are the second major cause of sepsis. The third major group includes the fungi,
with fungal infections causing a relatively small pt;.c~l,~ge of sepsis cases, but with a high
15 mortality rate.
Many of these infections are acquired in a hospital setting and can result from certain
types of surgery (e.g, abdomin~l procedures), immlme ~u~ple~ion due to cancer ortransplantation therapy, immunr deficiency ~ e~ec, and exposure through intravenous
catheters. Sepsis is also commonly caused by trauma, difficult newborn deliveries, and
20 intestin~l torsion (especially in dogs and horses).
A well established merh~ni~m in sepsis is related to the toxic components of gram-
negative bacteria. There is a common cell-wall structure known as lipopolysaccharide (LPS)
that is widely shared among gram-negative bacteria. The "endotoxin" produced by gram-
negative org~ni~m~ is compri~e~l of three major structures: a lipoy~teill; a lipid (lipid A),
25 thought to be responsible for most of the biological ~,opc;lLies of endotoxin; and
polysaccharide structures unique to each species and distinct strains of bacteria. [D.C.
Morrison, Rev. Infect. Dis. 5(Supp 4):S733-S747 (1983).] Research over the past decade or .
so has demonstrated that purified endotoxin can elicit all of the features of full-blown gram-
negative bacteremia. Furthermore, several of the host responses to endotoxin have been -~
30 identified. Two key mediators of septic shock are tumor necrosis factor (TNF) and
interleukin-1 (IL-1) which are released by macrophages and appear to act synergistically in
wo 94/14~37 2 1 5 13 8 6 PCT/US93/12381
causing a cascade of physiological changes leading to circulation collapse and organ failure.
[R.C. Bone, Ann. Intern. Med. 115:457-469 (1991).] Indeed, large doses of TNF ~K.J.
Tracey et al., Science 234:470-474 (1986)] and/or IL-I [A. Tewari e~ al., Lancet 336:712-714
, (1990)] can mimic the symptoms and outcome of sepsis.
It is generally thought that the distinct cell wall substances of gram-positive bacteria
and fungi trigger a similar cascade of events, although the structures involved are not as well
studied as grarn-negative endotoxin.
Regardless of the etiologic agent, many patients with septicemia or suspected
septicemia exhibit a rapid decline over a 24-48 hour period. Thus, rapid methods of diagnosis
and treatment delivery are es.cen~i~l for effective patient care. Unfortunately, a confirmed
diagnosis as to the type of infection kaditionally requires microbiological analysis involving
inoculation of blood cultures, incubation for 18-24 hours, plating the causative organism on
solid media, another incubation period, and final identification 1-2 days later. Therefore,
therapy must be initiated without any knowledge of the type and species of the pathogen, and
with no means of knowing the extent of the infection.
II. Prevention And Treatment
A. Antibiotics
Antibiotics of enormously varying skucture rBérdy in Advances in Applied
Microbiolog~, (D. Perlman, ed.), Academic Press, New York, 18:309-406 (1974)] are widely
used to prevent and conkol infections. Nonetheless, up to one half of the patients in whom
bacteremia develops in the hospital die (i.e., nosocomial or iakogenic bacteremia). [D.G.
Maki, Am. J. Med. 70:719-732 (1981).] The causes for this are many-fold. First, for many
commonly used antibiotics, antibiotic resist~n~e is common among various species of bacteria.
This is particularly true of the microbial flora resident in hospitals, where the org;mism.s are
under constant selective pressure to develop rcsist~nce. Furthermore, in the hospital setting~
spread of antibiotic-resistant org~nisms is facilitated by the high density of potentially infected
patients and the extent of staff-to-staff and staff-to-patient contact. Second, those antibiotics
that are the most economical, safest, and easiest to ~mini.st~r may not have a broad enough
speckum to suppress certain infections. For example, many antibiotics with broad spectra are
not deliverable orally and physicians are reluctant to place patients on intravenous lines due to
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the enhanced risk of infection. Third~ antibiotics can be toxic to varying degrees including
causing allergy, untoward interactions with other drugs, and direct damage to major organs
(e.g, kidneys, liver). Many potent antibiotics are elimin~tPd from routine use due to the
probability of adverse reactions at therapeutic doses. Fourth, many antibiotics alter the ,~
normal intestin~l flora and frequently cause diarrhea and nutritional malabsorption; some may
even unleash opportunistic org~ni.cmc which can cause life-thre~tening infections of the
gastrointestinal (GI) tract such as Clostridium difficile. For example, antimicrobial-associated
pseudomembranous colitis caused by C. diJ~icile is a potentially serious complication
associated with ~lminictration of certain antimicrobials. Physicians must therefore consider
the impact of prophylactic antibiotic use on the development of resistant org~nicmc~ on overall
patient health, and on the economics of health care.
While many infections are controlled by antibiotics, gram-negative bacteremia presents
some special challenges. It has been shown that tre~tment of bacteria with antibiotics may
catalyze the release of endotoxin from dying cells as their cell walls rlicintegrate. In
experimental E. coli sepsis in rabbits, antibiotics cause a 10 to 2,000 fold increase in
endotoxin levels despite decreasing levels of bacteremia. [J.L. Shenep and K.A. Morgan, J.
Infect. Dis. 150:380-388 (1984).] Thus, once gram-negative bacteremia is established, there is
justifiable concern that antibiotic therapy may allgment symptoms while mitig~ting the
infection.
Fortunately, certain antibiotics are known to neutralize the action of endotoxin. The
polymyxin antibiotics, most notably polymyxin B and polymyxin E (also known as colistin)
are cyclic polypeptide compounds produced by certain strains of Bacillus polymyxa. These
antibiotics bind to the lipid A portion of endotoxin [D.C. Morrison and D.M. Jacobs,
Immunochem. 13 :813-818 (1976)] and neutralize its activity as measured by lethality tests in
~nim,.lc [D. Rifkind and J.D. Palmer, J. Bacteriol. 92:815-819 (1966)], activation of serum
complement [D.C. Morrison and D.M. Jacobs, Infect. Immun 13:298-301 (1976)], and the
Limulus amebocyte lysate (LAL) assay. [M.S. Cooperstock, Antimicrob. Agents Chemother. ~,
6:422-425 (1974).] Unfortunately, the polymyxins are not absorbed from the GI tract and
must be ~flminictered parenterally. At the recommended therapeutic dose for systemic t~
infection by P. aeruginosa (1-2.5mg/kg body weight/day), there is a significant risk of renal
illlpaillllent. [Physicians' Desk Reference, 47th Ed., pp. 818-819 (1993).] This is a major
~ WO 94/14437 2 1 5 1 3 8 6 PCT/US93/12381
concern in patients already suffering from kidney disease. In addition to nephrotoxicity,
neurotoxic reactions have been observed, the most severe being lejl,i,atory paralysis when
given soon after anesthesia and/or muscle relaxants. Polymyxin B, in its intravenous form, is
,. only given to hospitalized patients under constant supervision and monitoring of renal
function. As such, polymyxins are not used routinely for systemic infections (but they are
f quite common as components of topical ointments).
Several approaches have been taken to reduce the toxicity of polymyxins. Colistin
exhibits a lower systemic toxicity, and when complexed as meth~nesulfonate salt, the locally
severe pain experienced at intramuscular injection sites is tlimini~hed. The toxicity of
10 polymyxin B is also reduced by ~tt~chment to dextran, a high molecular weight carrier.
[D.A. Handley, Eur. Patent Appl. Pub. No. 428486.] Conjugation to dextran is often used in
an attempt to decrease the toxicity and/or increase the circulating half-lives of drugs. [P.E.
Hallaway et al., Proc. Natl. Acad. Sci. USA 86:10108-10112 (1989); M.J. Poznansky and
L.G. Cleland in Drug Delivery Systems: Characteristics and Biomedical Applications, (R.L.
15 Juliano, ed.), Oxford University Press, New York, pp. 253-315 (1980); L. Molteni in Drug
Carriers in Biology and Medicine, (G. Gregoriadis, ed.), Ac~-lemic Press, New York, pp. 107-
125 (1979); C. Larsen, Adv. Drug Delivery Rev. 3:103-154 (1989); A.D. Virnik et al.,
Russian Chem. Rev. 44:588-602 (1975); and Hager et al., French Patent No. 2,342,740
(1977).] Alone, polymyxin B has a half-life of only a few hours [G. Brownlee et al., Brit. J.
20 Pharmacol. 7:170-188 (1952)], while dextran (M.W. 70,000) has a half-life in hllm~n~ of
about a day, depending upon the dose infused. [Reynolds et al., in Martindale - The Extra
Pharmacopoeia, 28th Ed., The Ph~ reutical Press, London, pp. 512-513 (1982); and W.~.
Gibby et al., Invest. Radiol. 25:164-172 (1990).]
Polymyxin B has been inve~tig~tç~l as a specific therapy for grarn-negative sepsis or
2~ endotoxemia over the past 20 years in both animal models and human trials but with mixed
results. For example, endotoxin-induced ~ çmin~tecl intravascular coagulation (DIC) was
not prevented in rabbits ~lmini~tered polymyxin B fifteen (15) min~ltes after endotoxin
challenge. [J.J. Corrigan, Jr. and B.M. Bell, J. Lab. Clin. Med. 77:802-810 (1971).] In fact,
most experiment~l studies have shown a requirement for ple,~ L~Ire of endotoxin and
30 polymyxin B, or ~1mini~tration of polymyxin B prior to endotoxin challenge to reduce or
abolish the effects of endotoxin. [D. Rifkind and J.D. Palmer, J. Bact. 92:815-819 (1966);J.J.
WO 94/14437 , PCT/US93/12381
2~5~ 3~6
Corrigan. Jr. and B.M. Bell, J. Lab. Clin. Med. 77:802-810 (1971); B. Hughes et al.,
Br. J. Pharmac. 74:701-707 (1981); J.J. Corrigan, Jr. and J.F. Kiernat, Pediat. Res. 13:48-51
(1979); G. Ziv and W.D. Schult_e, Am. J. Vet. Res. 44:1446-1450 (1982); and G. Baldwin et
al. J. Infect. Dis. 164:542-549 (1991).] Some studies have found little benefit in polymyxin ..
5 B, even as a ~ e~ lent. [A.H.L. From et al., Infect. Immun. 23:660-664 (1979).]
Importantly, clinical studies on endotoxemia in human obstructive jaundice found no benefit
in polymyxin B therapy [C.J. Ingoldby et al., Am. J. Surgery 147:766-771 (1984)], consistent
with results in animal models. [C.J.H. Ingoldby, Br. J. Surg. 67:565-567 (1980).]
Low dose polymyxin B therapy has also been investig~tçcl in ~nim~l~ and hum~n~. In
10 the infant rat, subinhibitory doses of polymyxin B, ~rlmini~tered 12 hours after infection with
live Haemophilus influenzae Type B org~ni~mc alone or in combination with a large dose of
ampicillin, significantly reduced mortality due to the infection. The theory here is that the
polymyxin B neutralizes endotoxin released by org;~ni~m~ killed by other antibiotics. [J.W.
Walterspiel et al., Pediat. Res. 20:237-241 (1986).] It should be noted that the design of this
15 experiment differed from the endotoxin challenge experiments, in that live org~ni~mc, not free
endotoxin were the starting materials for the challenge. In hum~n~, continuous infusion of
subtherapeutic doses of polymyxin B (10-50% of normal dosage) was found to reduce
endotoxin levels, restore some imml-ne functions, and appalelllly (i.e., results were not
statistically significant) reduce wound infection in burn patients. [A.M. Munster et al., J.
20 Burn Care Rehab. 10:327-330 (1989).]
B. lmm~ tion
In addition to antibiotic research and development, the effort to control bacterial
infections has focused on the role of host defenses, and in particular, the humoral immlme
system. The role of active immlmi7~tion against bacterial components and the utility of
25 passive immuni_ation with antibodies or plasma derived from immllni7ç~1 donors is a highly
controversial area. While there is abundant experimental evidence that specific antibodies can
protect experimental ~nim~l~ from infections and toxin challenge, the nature and degree of
this protection and its relevance to in vivo infection is not clear despite the large volume of
literature on the subject. [J.D. B~llmg~rtner and M.P. Glauser, Rev. Infect. Dis. 9:194-205
30 (1987); and E.J. Ziegler, J. Infect. Dis. 158:286-290 (1988).] Disease progression in the
~ WO 94/14437 21~ 13 8 6 PCT/US93/12381
critically ill patient, and its prevention, involves a myriad of factors which complicate the
design and interpretation of human clinical trials.
In gram-negative bacteremia and endotoxemia. it was found that the frequency of
septic shock was inversely related to the titer of antibodies cross-reactive with shared antigens
5 of bacterial LPS. [W.R. McCabe et al., New Eng. J. Med. 287:261-267 (1972).] Given this
correlation, an enormous effort has been expended to develop a means of raising endotoxin
antibody titers and/or passively transferring endotoxin antibody from donors to experimental
subjects and patients.
Antibodies to endotoxin have two important functions. First, by binding free
10 endotoxin, antibodies may block endotoxin activity or remove it from the circulation. Second,
immunoglobulin effector functions such as complement fixation and binding to Fc receptors
on phagocytes can mediate killing and opsonophagocytosis of bacteria. Thus, endotoxemia,
bacteremia, and the onset of sepsis, may be thwarted by such antibodies.
i) Active Imm~ on
One approach to protecting z~nim~l~ and hllm~n~ from endotoxin-mediated effects is by
immunization with bacteria or LPS. For example, it has been shown that immunization of
rabbits with a mutant E. coli strain (J5) which lacks certain polysaccharide side chains but
possesses a widely shared core lipid A structure can protect the 71nim~1~ from challenge with
live Pseudomonas. [A.I. Braude et al., J. Infect. Dis. 136(Supp):S167-S173 (1977).] The J5
20 vaccine was found to be only weakly protective in a guinea pig model of Pseudomonas
pneumonia, whereas a species-specific Pseudomonas LPS was greatly protective. [J.E.
Pennington and E. Menkes, J. Infect. Dis. 144:599-603 (1981).] These results suggest that
species-specific vaccines may be superior to cross-protective antigens for immnni7~tion of
hllm~n.~ and other ~nim~l~ against endotoxin. Unfortunately, the vast diversity of LPS
25 antigens makes the forrner an unlikely prospect.
While active immunization against endotoxin continues to be investig~te-l there are
some important limitations to this approach. First, endotoxin is weakly immunogenic,
eliciting only a three- to five-fold increase in antibody titers to LPS with virtually no booster
response. [E.J. Ziegler et al., New. Eng. J. Med. 307:1225-1230 (1982).] Second, many
30 patients at risk for sepsis are immunoconll,lolllised and may not be capable of mounting
WO 94/14437 PCT/US93112381
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2 ~ 8 ~
and/or su~t~ining a sufficient response to be protective upon ~lmini.~tration of vaccine. And
third, the degree of cross-protection afforded by immnni7~tion with one or more core
glycolipid antigens is not well understood clinically.
ii) Passive Immunization
In order to overcome some of the limitations inherent to active immuni_ation, various
techniques have been used to produce endotoxin-binding antibodies that could be passively
transferred to experimental ~nim~lc or human subjects. A large number of endotoxin
antibodies have been prepared by: (i) immuni_ation of ~nim~l~ or humans with bacteria~ LPS,
or derivatives thereof and collection of immune serum or plasma; or (ii) production of
monoclonal murine or human antibodies and collection and purification of these antibodies by
established methods.
The two major antibody types elicited by either procedure are lgM and IgG antibodies.
These antibodies differ in important aspects of their structure and effector functions as well as
their titer in normal and hy,ut;~ lne plasma. Most studies suggest that IgM antibodies, by
virtue of their greater avidity are more effective than IgG antibodies at protecting ~nim~
[W.R. McCabe e~ aZ., J. Infect. Dis. 158:291-300 (1988)] and humans [Id.; E.J. Ziegler et al.,
New. Eng. J. Med. 307:1225-1230 (1982)] from gram-negative bacteremia or endotoxin
challenge. However, it should be noted that numerous IgG ~l~dlions from immnni7~cl
animal donors have been developed and demonstrated to have some protective effect in
experimental studies. [D.L. Dunn et al., Surgery 96:440-446 (1984); and S.J. Spier et al.,
Circulatory Shock 28:235-248 (1989).] The advantage to IgG ~ ~dlions is that IgG titers
may increase in response to repeated immuni7~tion whereas IgM titers are relatively constant.
No matter what the hlllllLLIli~dlion course, however, the total amount of bacterially-reactive or
endotoxin-reactive antibodies in hyperimmnne plasma or serum is only a small fraction of
total antibody and is highly variable from donor to donor.
In order to develop more con~i~ent plel)~dLions of therapeutic antibodies. numerous
LPS-reactive monoclonal antibodies have been developed to both shared and unique epitopes.
Since gram-negative sepsis can be caused by a number of species, emphasis has been placed ~
on widely cross-reactive antibodies as potential therapeutics. Two IgM monoclonal antibodies
have received the most study. A human-derived antibody now known as Centoxin-HA-lA
- 8 -
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2~ 51~86
[N.N.H. Teng et al., Proc. Natl. Acad. Sci. USA 82:1790-1794 (1985)] and a mouse-derived
antibody now known as XOMEN-E5 [Young and Alam~ U.S. Patent No. 4,918.163] have
been tested in both ~nim~l~ and humans. The animal data suggest that both antibodies are
capable of binding endotoxin, neutralizing its biological activity, and suppressing gram-
S negative bacteremia. Unfortunately, the human clinical studies have not yielded clear benefits[E.J. Ziegler et al., New. Eng. J. Med. 324:429-436 (1991); R.L. Greenman et al.~ JAMA
266:1097-1102 (1991)] despite the optimism of the authors and sponsors of these trials. The
U.S. Food and Drug A-lmini~tration has refused to approve either antibody for the treatment
of sepsis based upon the extensive clinical trials performed to date.
It should be noted that each antibody was tested in humans after the onset of
symptoms of sepsis and when the type of organism was uncertain. It is widely believed that
anti-endotoxin antibody treatment ~(lmini~tered after sepsis is established may yield linle
benefit because these antibodies cannot reverse the infl~mm~tory c~cc~e initiated by
endotoxin and the ~tten~l~nt triggering of meAi~tQrs such as TNF and IL 1. In addition, the
15 high cost of each antibody (Centoxin HA-lA was expected to cost $3700 per 100 mg dose)
would limt physicians' use of aproduct where no clear benefit has been demonstrated. [K.A.
Schlllm~n et al., JAMA 266:3466-3471 (1991).] Of course, these endotoxin antibodies only
target gram-negative sepsis; no equivalent antibodies exist for the array of gram-positive
org~ni.cm~ and fungi.
20 III. Inhibiting Cytokines Rele~eA During Sepsis
With new knowledge regarding the effects of endotoxin on host infl~mm~tory
responses, other therapies are being targeted towards blockage of IL-1 and TNF functions.
For example, an IL-l receptor antagonist has been identified that occupies the same receptor
site as IL-1, but mediates no biological effect. Blockage of the IL-1 receptor with this
25 molecule can reduce mortality from endotoxin shock. [K. Ohlsson et al., Nature 348:550-552
(1990).] While the IL-1 receptor antagonist appears to be well-tolerated, the required dosage
is extremely large (over 100 mg of recombinant protein per kg of body weight is infused over
a period of hours to days). For human therapy, the 8-10 grams of recombinant protein
anticipated to be required is likely to be extremely costly (several thousand dollars).
WO 94/14437 PCT/US93/12381
2 TNF therapies target removal of this mediator from the circulation. Monoclonal
antibodies have been found to offer some protection in experimental ~nim~l~ [S.M. Opal et
al., J. Infect. Dis. 161:1148-1152 (1990)] but studies in human patients with sepsis have not
been conclusive. Once again, these antibodies are likely to be expensive therapeutic agents
~lmini~tçred only when signs of sepsis are present.
IV. Prophylaxis
Since the treatrnent of ongoing septicemia presents so many challenges. there have
been several aLL~ at prevention. These ~u~ have provided mixed results. One
promising study utilized hy~lhlllllune plasma against core glycolipid in surgical patients at
high risk of infection. While antibody prophylaxis did not lower the infection rate, it did
reduce the severity of grarn-negative infections and improved the survival of such patients.
[J.-D. Ranmg~rtner et al.~ Lancet 2:59-63 (1985).] Nurnerous studies using intravenous
immunoglobulin, collected from large numbers of normal donors and cont~inin~ a wide range
of antibodies, have given mixed results. [J.D. ~nnn~rtner and M.P. Glauser, Rev. Infect.
Dis. 9:194-205 (1987).] The primary limit~titlns to these studies would appear to be the
variable and relatively low potency of pooled immunoglobulin ~repaldlions that were used.
[T. Calandra et al., J. Infect. Dis. 158:312-319 (1988).]
Monoclonal antibodies have also been made. While these prepa.~ions should possess
greater potency, their high cost, immunogenicity [S. Harkonen et al., Antimicrob. Agents
Chemother. 32:710-716 (1988)] and nnllcll~lly short circulating half-lives (less than 24 hr) [S.
H~rkonen et al., Antimicrob. Agents Chemother. 32:710-716 (1988); and C.J. Fisher et al.,
Clin. Care Med. 18:1311-1315 (1990)] make them unattractive c~n~ t~s for prophylaxis.
Clearly, there is a great need for agents capable of preventing and treating sepsis.
These agents must be capable of neutralizing the effects of endotoxin in gram-negative sepsis
as well as controlling and reducing bacteremia. It would be desirable if such agents could be
a-lmini~tered prophylactically in a cost-effective fashion. Furtherrnore, approaches are needed
to combat all forms of sepsis, not just gram-negative cases.
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SUMMARY OF THE INVENTION
The present invention relates to therapeutics for the prevention and treatment of blood-
borne and toxin-mediated ~ e~e~, and in particular the prevention and treatment of sepsis in
humans as well as other ~nim~l~. In one embodiment, the present invention relates to
compositions and methods for preventing sepsis in high-risk patients (e.g.,
immunocompromised patients such as surgical and other hospitalized patients, low birth
weight infants, and burn and traurna victims). In another embodiment, the present invention
contemplates treatment of hllm~n.~ and ~nim~l~ having symptoms of a systemic septic reaction.
In accordance with the present invention, a member from the class of compounds
broadly described as antibody-antibiotic conjugates or "antibodiotics" is employed for
intravenous, intramuscular, intrathecal or topical ~flmini.~tration. Antibodiotics are comprised
of antibody (e.g, IgG, IgM, IgA) to which an antibiotic is covalently ~tt~rhed to make an
antibody-antibiotic conjugate. Preferably, the antibody is non-specific IgG. By non-specific,
it is meant that no single specificity within the antibody population or pool is dominant.
Thus, it is to be contrasted with the use of antigen-specific antibodies.
In one embodiment, the present invention contemplates an antibiotic-antibody
conjugate, comprising antibiotic covalently bound to non-specific immnnoglobulin. It is
plcfcllcd that the immunoglobulin is IgG having an Fc region and is capable of binding to
phagocytic cells via the Fc region.
In one embodiment, the conjugate is capable of binding to bacteria via the antibiotic.
The conjugate may be bacteriostatic, bactericidal, neither, or both.
However, the antibiotics contemplated are not limited to ~ntib~ct~rial agents; antifungal
agents and antiviral agents are also contemplated. Where antibacterial antibiotics are used,
agents effective against both gram-positive and gram-negative org~ni~m~ are contemplated.
The present invention contemplates conjugates capable of binding lipopolysaccharide
on gram negative bacteria as well as conjugates capable of binding free endotoxin and
neutralizing free endotoxin.
Preferred antibiotics include polymyxins, specifically polymyxin B. Polymyxin is a
known endotoxin-binding compound capable of binding free endotoxin.
The present invention also cont~mpl~t~s a therapeutic ulcp~dlion~ comprising
antibiotic covalently bound to non-specific immunoglobulin, wherein the preparation is
WO 94/14437 PCT/US93/12381
3 8 ~ ~
bactericidal for both gram-positive and gram-negative org~ni~m.s. In one embodiment of the
therapeutic preparation, the antibiotic is selected from the group comprising cephalosporins
and penicillins. In another embodiment~ the therapeutic preparation further comprises: (i) a
first conjugate con~i.cting of a first antibiotic covalently bound to non-specific ~,
S immunoglobulin; and (ii) a second conjugate consisting of a second antibiotic covalently
bound to non-specific immunoglobulin (e.g, where the first antibiotic is polymyxin and the
second antibiotic is vancomycin or bacitracin). In still another embodiment of the therapeutic
~,epa~d~ion, two different antibiotics are covalently bound to the same immunoglobulin
molecule, one capable of binding to grarn-positive organisms and the other capable of binding
to gram-negative org~ni~m~
The present invention contemplates a method of treatment~ comprising: (a) providin~
a m~mm~l for treatment; (b) providing a therapeutic p~e~aldlion, comprising an endotoxin-
binding compound covalently bound to protein; and (c) ~lmini~tering the l~le~ Lion to the
m~mm~l (e.g, intravenous). The endotoxin-binding compound may be polymyxin and the
protein is preferably non-specific immunoglobulin such as IgG.
The tre~tment with the antibodiotic is expected to have many of the effects of the
antibiotic alone -- however, without the toxicity and short half-life typically associated with
these agents. Furthermore, these conjugates are expected to possess the opsonizing function
of immunoglobulin which may f~.ilit~te clearance of both the toxin and org~ni~m.The plesent invention conl~l"l)lates a method of tre~tment of m~mm~l~ at risk for
developing sepsis, in which a therapeutic ~lel)~dlion comprised of an antibiotic capable of
binding to a microorganism covalently bound to a non-specific immunoglobulin is
~tlmini.~tered to the at-risk animal prior to the onset of any septic symptoms. In a preferred
embodiment, it is contemplated that the method of the present invention will be ~tlmini.~tered
intravenously.
The present invention collLelllplates that the method will be used for such ~nimzll~ as
neonatal calves and foals, as well as human and veterinary surgical patients, trauma, and burn
victims. It is contemplated that the method will be used to treat immllnocompromised
patients.
It is contemplated that the present invention will be useful for the treatment of
m~mm~l~ potentially exposed to gram-negative and/or grarn-positive bacteria. It is
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wO 94114437 2 1~ 13 ~ ~ PCT/US93/12381
contemplated that the therapeutic preparation used in the method of the present invention is
capable of binding endotoxin.
The present invention further contemplates a method of treatment of m~mm~l~ infected
with a pathogenic org~ni~m~ wherein a therapeutic preparation, comprising a surface-active
S antibiotic covalently bound to a non-specific immunoglobulin G having an Fc region capable
of me~ ting opsonization of the pathogenic organism is ~-lmini~tered It is contemplated that
the infecting pathogen is a gram-negative or grarn-positive bacterial org~ni~m It is
contemplated that the surface-active antibiotic used in the therapeutic p~ a~alion is a
polymyxin (e.g, polymyxin B).
One embodiment of the present invention contemplates a method of diagnosis,
comprising: (a) an antigen associated with the surface of a pathogenic organism immobilized
to a solid support; (b) a conjugate comprising a surface-active antibiotic covalently bound to a
non-specific immunoglobulin; and (c) a competitor comprising the surface antigen present in
solution. The immobilized antigen is incubated with the conjugate in the presence of the
15 competitor, washed to remove unbound conjugate and competitor, followed by detection of
the conjugate bound to the immobilized surface antigen.
In a ~le~ d embodiment~ the present method of diagnosis comprises immobilizationof surface antigen in the well(s) of a microtiter plate. It is also contemplated that the surface
antigen of the method is isolated from bacterial org~nicm.~ It is col~Lelll~lated that the surface
20 antigen be isolated from such gram-negative bacteria as Escherichia coli, (e.g,
lipopolysaccharide). It is also contemplated that the competitor in the present method of
diagnosis is comprised of lipopolysaccharide from gram-negative bacteria. It is further
contemplated that the competitor will be comprised of lipopolysaccharide from such gram-
negative bacteria as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
25 Vibrio cholerae, Shigellaflexneri, Klebsiella pneumoniae, Salmonella enteritiditis, Serratia
marcescens and Rhodobacter sphaeroides.
The present invention also contemplates a method of synth~si7ing a conjugate
comprising the steps of: a) reacting an antibiotic with a cro.~linking agent to form a
- derivatized antibiotic; and b) reacting the derivatized antibiotic with non-specific
30 immunoglobulin, to form a conjugate. It is further contemplated that the antibiotic will bind
to the surface of microorg~ni.~m~. In a preferred embodiment, the antibiotic is a peptide. In
WO 94/14437 PCT/US93/12381
2~ 51~8~ ~
another preferred embodiment, the peptid-e is. Limulus antilipopolysaccharide factor, in
another, the peptide is a D-amino acid-cont~ining peptide. It is also contemplated that the
peptide binds endotoxin. In a preferred embodiment, the antibiotic is a polymyxin such as
polymyxin B.
In an alternative ~l~r~ d embodiment, the antibiotic used is bactericidal to gram-
negative bacteria. In another, the antibiotic is bactericidal to gram-positive bacteria. In one
embodiment. the antibiotic is vancomycin. In one embodiment, the non-specific
immunoglobulin consists of an Fc region.
The present invention also contemplates a method of synthesizing a conjugate
comprising the steps of: a) reacting an antibiotic with a first bifunctional croc~linking agent to
form a derivatized antibiotic; b) reacting non-specific imml-noglobulin with a second
bifunctional cro~linking agent to form a derivatized immllnoglobulin; and c) reacting the
derivatized antibiotic with the derivatized immunoglobulin to form a covalent bond between
the derivatized antibiotic with the derivatized immlmoglobulin to form a conjugate.
In one embodiment of this method, the antibiotic binds to the surface of
microorg;~ni~m.c. In pler~ d embo-liment~, the antibiotic is a peptide, such as (but not
limited to) Limulus antilipopolysaccharide factor In another preferred embodiment, the
peptide is a D-amino acid-co.,t;~ g peptide. In an ~lt~rn~tive embodiment, the peptide binds
endotoxin. In a pler~..ed embodiment, the antibiotic is a polymyxin, such as (but not limited
20 to) polymyxin B.
In one embodiment of the method, the antibiotic is bactericidal to gram-negativebacteria. In another embodiment, the antibiotic is bactericidal to gram-positive bacteria. In a
p~c;r~;lled embodiment, the antibiotic is vancomycin. In an alternative ~refcl.ed embodiment,
the first and second bifunctional cro~linking agents are N-succinimidyl 3-(2-pyridyldithio)
25 propionate. In another embodiment of the method, the first bifunctional cro~linkin~ agent is
S-acetylmercaptosuccinic anhydride. In an additional embodiment, the second bifunctional
cro~linkin~ agent is sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
The present invention also contemplates a method of synth~i7ing a conjugate
comprising the steps of: a) providing in any order: i) a cro~linking agent having first and
30 second reactive sites, with the first site being exposed, active, and reactive with primary
amino groups, and the second site being blocked by a cleavable group; ii) an antibiotic having
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one or more primary amino groups; and iii) non-specific immunoglobulin having one or
more primary amino groups; b) reacting in any order: i) the cros~linking agent with the
antibiotic, forming a blocked derivatized antibiotic; and ii) the cro~linking agent with
immunoglobulin, forming a blocked derivatized immunoglobulin; c) reacting in any order:
5 i) the blocked derivatized antibiotic with a modifying reagent, forming a free derivatized
antibiotic; ii) the blocked derivatized immunoglobulin with a modifying reagent, forming a
free derivatized immunoglobulin; and d) reacting the free derivatized antibiotic with the free
derivatized immunoglobulin to form a conjugate.
In a preferred embodiment of this method, the antibiotic binds to the surface of10 microorg~ni.~m.~ In another preferred embodiment, the antibiotic is a peptide~ such as (but
not limited to) Limulus antilipopolysaccharide factor. In one embodiment, the peptide is a D-
amino acid-cont~inin~ peptide. In one embodiment~ the peptide binds endotoxin. In a
preferred embodiment, the antibiotic is a polymyxin, including (but not limited to) polymyxin
B.
l S In a preferred embodiment, an antibiotic bactericidal to gram-negative bacteria is used
in the method of the present invention. In another embodiment, the antibiotic is bactericidal
to gram-positive bacteria. In a ~l~r~l,ed embo-liment . the antibiotic is vancomycin. In an
alternative embodiment, the non-specific immunoglobulin consists of an Fc region. In one
embodiment of the method, the modifying reagent is a re~lcing agent. In a p,efe"~d
20 embodiment, the re~ cing agent is dithiothreitol.
The present invention further contemplates a method of synthesi7ing a conjugate
comprising the steps of: a) providing in any order: i) a first cros.~linking agent having first
and second reactive sites, with the first site being reactive with primary arnino groups, and the
second site being reactive with maleimide groups; ii) an antibiotic having one or more
25 primary amino groups; iii) a second cro~.clinking agent having first and second reactive sites,
with the first site being reactive with primary amino groups, and the second site being
reactive with sulfhydryl groups; and iv) non-specific immunoglobulin having one or more
primary amino groups; b) reacting in any order: i) the first cro~slinking agent with the
antibiotic, forming a derivatized antibiotic; and ii) the second cro~linking agent with the
30 immllnt)globulin, forming a derivatized immunoglobulin; and c) reacting the derivatized
antibiotic with the derivatized immunoglobulin to forrn a conjugate.
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WO 94/14437 PCT/US93/12381
2~13~ ~
In one embodiment of this method, the first cro.~1inkinE agent is bifunctional. In a
plcf~-lcd embodiment, the first bifunctional cros~linkinE agent is S-acetylmercaptosuccinic
anhydride. In an alternative plcrellcd embodiment, the second cro~linkinE agent is
bifunctional. In one embodiment, the second bifunctional cro.~1inkinE agent is
S sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate.
The present invention also contemplates a method of synthe~i7.inE a conjugate
comprising the steps of: a) reacting a non-specific immunoglobulin with a first modifying
reagent to form an oxidized immunoglobulin preparation;and b) reacting the oxidized
immunoglobulin preparation with an antibiotic and a second modifying reagent to forrn an
antibiotic-imm11noglobulin conjugate. In a preferred embodiment, the immunoglobulin
consists of an Fc region.
ln one embodiment of this method, the first modifying reagent is an oxidizing agent.
In a preferred embodiment, the oxidizing agent is periodate. In an ~1tern~tive embodiment,
the method includes a second modifying reagent which is a reducing agent. In a preferred
embodiment, the reducing agent is sodium borohydride.
The present invention also contemplates a method of synthe~i7inE a conjugate
comprising the steps of: a) reacting an antibiotic precursor with a first cro.c.~1inkinE agent, the
antibiotic precursor posses~inE limited bactericidal activity to form a derivatized antibiotic
precursor, and a derivatized antibiotic plc~ ol pos.ce~.~inE increased bactericidal activity; b)
reacting non-specific immunoglobulin with a second cro~1inkinE agent, to form a derivatized
imm~lnoglobulin; and c) reacting the derivatized antibiotic precursor with derivatized
imm1moglobulin to form a covalent bond between the derivatized antibiotic precursor and
derivatized immunoglobulin to form a conjugate.
In one preferred embodiment, the antibiotic precursor is selected from the groupcon~i~tin~ of 7-aminocephalosporanic acid and 6-aminopenicillanic acid. In another
embodiment. the first cro~1inkinE agent is bifunctional. In one plcfellcd embodiment, the
first bifunctional cro.s.~linking agent is m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester.
In another embodiment the second cro~ss1inkinE agent is bifunctional. In one preferred
embodiment, the second bifunctional cro~1inkinE agent is iminothiolane.
The present invention further contemplates a method of synthe~i7.inE a conjugatecomprising the steps of: a) providing in any order: i) a first cro~1inkinE agent having first and
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WO 94/14~37 21~ 13 8 6 PCT/US93/12381
second reactive sites, the first site being reactive with primary amino groups, and the second
site being reactive with sulfhydryl groups; ii) an antibiotic precursor having one or more
primary amino groups, with the antibiotic precursor po~es~ing limited bactericidal activity;
iii) a second cro.c~linkin~ agent having first and second reactive sites, the first site being
reactive with primary amino groups, and the second site being reactive with maleimide
groups; and iv) non-specific immunoglobulin having one or more primary amino
groups; b) reacting in any order: i) the first cro.s~linking agent with the antibiotic precursor?
forming a derivatized antibiotic precursor; ii) the second cro~linking agent with the
immunoglobulin, forming a derivatized immunoglobulin; c) reacting the derivatized antibiotic
precursor with the derivatized immunoglobulin to form a conjugate.
ln a preferred embodiment, the antibiotic precursor is selected from the group
consisting of 7-aminocephalosporanic acid and 6-aminopenicillanic acid. In another ~ f~,led
embodiment, the first cro~clinking agent is bifunctional. In an additional embodiment, the
first bifunctional cros~linkin~ agent is m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester.
In an alternative preferred embodiment, the second cro~linking agent is bifunctional. In
another preferred embodiment, the second bifunctional cros~linkin~ agent is iminothiolane.
DESCRIPTION OF THE DR~WINGS
Figure lA schem~tically shows the design of an antibodiotic of the present invention.
Figure lB scht-m~tically shows the design of another antibodiotic of the present invention.
Figure 2 sçhem~lically shows a means of s~;le~ g modified antibiotics for anti-
bacterial activity.
Figure 3 outlines an alternative method by which new antibiotics can be screened for
use as compounds for conjugation with immunoglobulins. Figure 3A shows a means by
which the minimum concentration for bacterial growth inhibition is established. Figure 3B
shows a means by which a new antibiotic can be assessed for bactericidal activity.
Figure 4 describes solid phase assays for determining the level of binding of
antibodiotics of the present invention. Step 1 shows toxin or org~ni~mc in a testing
~- microwell. Step 2 schematically represents the binding of antibodiotic. Step 3 schematically
shows the binding of secondary reagents.
WO 94/14437 PCT/US93/12381
2 ~ 8 ~
Figure S shows conjugates of the present invention binding to LPS. as measured by
ELISA.
Figure 6 shows additional conjugates of the present invention binding to LPS, asmeasured by ELISA.
Figure 7 shows inhibition of LPS binding of conjugates of the present invention using
free polymyxin (PMB). as measured by ELISA.
Figure 8 shows periodate conjugates of the present invention binding to LPS, as
measured by ELISA.
Figure 9 shows inhibition of LPS binding of conjugates of the present invention using
LPS of various bacterial species, as measured by ELISA.
Figure 10 shows the binding of conjugates of the present invention to phagocytic cells
in a radioactive competition assay.
Figure 11 shows the pharmacokinetic profile of intravenously ~mini~t~red PMB-HlgG
and HIgG in rabbits expressed in absorbance at 410 nm.
Figure 12 shows the ph~rm~okinetic profile of intravenously ~-lmini.~tered PMB-HIgG
and HIgG in rabbits ~ es~ed in IgG concenlldlion.
Figure 13 shows the sequence of Limulus antilipopolysaccharide factor (LALF), a
single chain peptide known to bind and neutralize endotoxin.
DESCRIPTION OF THE INVENTION
The present invention relates to therapeutics for the prevention and trç~tment of blood-
borne and toxin mediated rli.~ç~es, and in particular the prevention and tre~tment of sepsis
caused by various types of org~ni.~m.c in hllm~n~ as well as other ~nim~l~ The present
invention is particularly suited for the in vivo neutralization of the effects of endotoxin.
However, it is contemrl~te~l that the present invention will be used in the treatment of gram-
negative and gram-positive sepsis. Although the invention may be used for tre~tment of
sepsis due to one org~ni.cm, it may also be used to treat sepsis caused by multiple org~ni.~m~ _
(e.g, sepsis and/or bacteremia due to gram-negative and gram-positive org~ni~m~). The
present invention also contemrl~t~?s tre~tment comprising multiple antibody-antibiotic
conjugates used in combination. It is also contemplated that the present invention will be
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WO 94/14437 PCT/US93/12381
215138~
used to treat bacteremia, viremia or fungemia, by enhancing the removal of org~ni~m~ by
opsomzatlon.
In accordance with the present invention, soluble antibody-antibiotic conjugates or
"antibodiotics" are ~lmini.~tered intravenously, intra-muscularly, subcutaneously,
S intradermally, intraperitoneally, inkapleurally, intrathecally or topically. The conjugate is
water-soluble if it has a solubility in physiologic saline of at least 0.1 mg/ml, and preferably
of at least 1.0 mg/ml, when measured at room temperature. The present invention
contemplates the use of antibodiotics in a therapeutic pr~aldlion for both prophylactic and
acute treatment.
While the benefit conveyed by treatment according to the present invention is not
dependent on the underst~ntling of the merh~ni~m(s) by which soluble antibody-antibiotic
conjugates achieve a therapeutic result, it is believed that, in the case of bacteria, success is
accomplished by: (i) binding and opsonization of bacteria; (ii) bacterial killing (direct killing
by the conjugate and/or complement-me~ t~cl); and (iii) neutralization and removal of free
15 bacterial toxins (e.g., gram-negative endotoxin, thereby preventing initiation and/or escalation
of the septic reaction).
It is believed that antibodiotics provide a low cost, reasonably effective and needed
preventive as well as tre~tment Antibodiotics can ~u~lJleS~ fungal and viral infection.
Furthermore, antibodiotics suppress bacteremia as well as endotoxin-mediated effects.
20 Antibodiotic$ with long (e.g, days to weeks) duration of action are easily ~-lmini~tered.
Furtherrnore? since the invention encomp~.ces antibodiotics with reactivity against gram-
negative org~ni~m.~ as well as antibodiotics with reactivity to gram-positive org~ni~m.~, a
wider spectrum of protection is expected than any other known approach.
It is also contemplated that this invention will be used in diagnostic applications.
25 These diagnostic applications include methods to detect LPS from particular org~ni~m~ or the
surface structures present on org~ni.~m~ which are recognized by antibiotics (i,e., the receptors
expressed on cell surfaces which bind antibiotic).
The description of the invention involves: (I) Antibodiotic Design and
Characterization; (II) Antibodiotic in vitro and in vivo Efficacy; (III) Antibodiotic
30 Applications; and (IV) Therapeutic Pl~l.~dLions and Combinations. Section III describes the
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WO 94/14437 PCT/US93/12381
2i~l38~ --
use of antibodiotics for: (A) Prophylactic Use in Humans; (B) Acute Therapy in Humans;
and (C) Veterinary Care.
I. Antibodiotic Design And Chara~ ~,.tion
A. Antibodies
S In clesi~ning antibodiotics, all types of antibody (e.g, IgG, pentameric and monomeric
IgM, secretory and monomeric IgA, IgE and IgD) are contempl~tec1 Nonetheless, there are
advantages to using a particular class of antibody. Table 1, for example, co~llpales the
characteristics of IgG and IgM. While IgM has the advantage of better opsonization and
complement activation, IgG has a longer half-life in vivo and can be raised to higher titers
because of the fact that it is the primary antibody raised during secondary responses to
antigen. Consequently, the preferred antibody for conjugation according to the present
invention is IgG.
While antigen-specific IgG can be employed (e.~., bacteria^seeking antibodies),
antigen-specificity may result in a shorter half-life of the compound (and/or greater cost).
Consequently, the pl~rt;l~ed antibody is non-specific. [Contrast C.H.J. Ford et al., Indian J.
Pediatr. 57:29-46 (1990).]
Goers et al. (U.s. Patent No. 4,867,973) describe the use of antibody conjugated to
antimicrobials, but with antigen-specific antibody. In contrast, the conjugates of the present
invention utilize non-specific antibody. Goers et a/. describe in particular the conjugation to
antigen-specific monoclonal antibodies. Monoclonal antibodies have not been a step forward
in the prevention and/or treatment of bacteremia and sepsis. While these ~ dlions should
possess greater potency and specificity than polyclonal sera, they are: a) prohibitively
expensive; b) frequently immllnogenic; and c) exhibit llnn~u~lly short circ~ tin~ half-lives
(typically less than 24 hours).
With respect to cost, Centoxin (a commercially produced antigen-specific monoclonal
antibody) serves as a real life example; the price was a~ xhllately $3,700.00 per 100 mg
dose. Ph~rm~roeconomic analysis indicated that - even if the product was used under strict
guidelines for acute cases - "its use could add $2.3 billion to the nation's health care budget."
[K.A. Schlllm~n et al., JAMA 266:3466-3471 (1991).] The expense of Centoxin is such that
it simply could not be used prophylactically. The conjugates of the present invention, on the
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~ 2 ~ 8 ~
other hand, are produced from materials costing a fraction of this figure (e.g, $2.00 per 100
mg dose) because of the readily available inexpensive source of pooled donor IgG.
Also, human monoclonals while perhaps lessening the chance of immunogenicity~ donot overcome the problem of short circulating half-lives. In a study using human monoclonal
anti-lipid A antibody in patients with sepsis syndrome, the mean serurn half-life was
approximately sixteen (16) hours. ~See C.J. Fisher e~ al., Clin. Care Med. 18: 1311 - 1315
(1990).] To m~int~in a protective level of antibody, this reagent would need to be given
repeatedly. Again, the cost of such an approach would be staggering.
From the above, it should now be clear why the limitation to "non-specific
immunoglobulin" is a critical limitation that is unique to the present invention. Non-specific
IgG is easily and cheaply obtained, requiring no im~nunization and eliciting no immune
response in a syngeneic setting. Non-specific IgG does not have the standardization problems
of antigen-specific antibody. Simply put, there is no antigen-specific titer to be concerned
about (let alone variability in the titer from unit to unit~. Rather, standardization comes from
the conjugated ligand; conjugation of non-specific IgG results in >1000-fold increase in LPS-
binding titer and by standardization of the ligand that is ~tt~c.h~-l, one standardizes the activity
of the therapeutic. Finally, non-specific IgG, unlike monoclonals, has a long half-life needed
for a prophylactic (compare the >21 day half-life of pooled polyclonal human IgG with the
mean serum half-life of 16 hours for the human monoclonal antibodies discussed above).
For purposes of expense, IgG from donors (i.e., human and animal) rather than cell
lines is desirable. In this regard, typically large pools of plasma are used as starting material.
Large scale fractionation techniques known in the art include ethanol l,lcci~,i~lion and
precipitation with high concentrations of salt. [See H.F. Deutsch in Methods in Immunology
and Immunochemist7y, (C.A. Williams and M.W. Chase, eds.), ~c~(lemic Press, New York,
pp. 315-321 (1967).] There is also the somewhat complicated procedure where the
immunoglobulin is isolated from Cohn Effluent III by diafiltration and ultrafiltration. [See
- E.J. Cohn et al., J. Am. Chem Soc. 68:459-475 (1946).]
This latter procedure is used to make a commercially available human IgG ~ lion
called G~mmimlme~) N (Miles, lnc., West Haven, CT). Of course, each individual donor
used to make the product must be tested and found nonreactive in tests to determine exposure
to or the presence of pathogens. In this product~ which is intt ntle~l for intravenous
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WO 94/14437 PCT/US93/12381
215~386
~lmini~tration, the protein (as a 4.5-5.5~O solution) has not been chemically modified other
than in the adjustment of the pH of the solution to 4.0-4.5. Isotonicity is achieved by the
addition of (9-11%) maltose.
Each milliliter (ml) contains approximately 50 mg of protein, of which not less than
5 98% has the electrophoretic mobility of gamma globulin. Not less than 90% of the gamma
globulin is monomeric. There are traces of IgA and IgM. The distribution of IgG subclasses
is similar to that found in normal serum.
The commercial product displays a broad spectrum of opsonic and neutralizing
antibody activities. When ~lmini.~tered intravenously, e~senti~lly 100% of the infused
IgG antibodies are immediately available in the recipient's circulation. The in vivo
half-life equals or exceeds the three week half-life reported for IgG in the literature. It is
therefore quite acceptable for use in the ~Lc~Lion of antibody-antibiotic conjugates of the
present invention.
Of course, the infusion of large arnounts of antibody in hl~m~n~ is contraindicated in
15 individuals who are known to have had previous anaphylactic or severe systemic responses to
IgG. Care must also be taken to confirm that there is no sensitivity to the trace amounts of
other antibody (e.g, IgA).
Before ~q-lmini~tration of the antibody-antibiotic conjugates of the present invention to
hllm~n.c, it may be good medical practice to have an antibodiotic sensitivity test performed.
20 This can be done by subcutaneously injecting a small arnount of the conjugate in the arm of
the patient. A salt solution is injected in the other arm as a control. Normally, a positive
TABLE 1
IgM IgG
Structure Pent~meric Monomeric
C' Fixation l l I +
Opsonophagocytosis I I I +
Half-life 5 days 25 days
Biodistribution Slow Fast
Secondary Response Minimal Large
WO 94/14437 215 13 8 ~ PCT/US93/12381
hypersensitivity test is indicated by no more than formation of a welt on the skin surface with
surrounding swelling. Some patients, however, develop anaphylactic shock (i.e., a full-blown
immediate hypersensitivity reaction). It is recommended that adrenalin be available for these
cases.
The usual dosage of the commercial intravenous immunoglobulin product is 100-200mg/kg (2-4 ml/kg) of body weight ~rlmini~tered approximately once a month by intravenous
infusion. The dosage may be given more frequently or increased as high as 400 mg/kg (8
ml/kg) body weight, if the clinical response is inadequate, or the level of IgG achieved in the
circulation is felt to be insufficient.
The present invention contemplates a typical dosage for antibodiotics that is much less
than that given for the commercial immunoglobulin ple~ dLions. This is particularly true
where the number of conjugated antibiotic molecules exceeds one (1) per immllnoglobulin
molecule. The present invention contemplates a conjugate dosage range of 0.1-100 mg/kg,
and a preferred range of 1-20 mg/kg. The amount of PMB (~csllming 3 molecules per IgG
molecule) contained in a dose for this preferred range will be 0.025 - 0.5 mg/kg.
B. Antibiotics
Thousands of natural, synthetic, and semi-synthetic compounds have been identified
that possess antib~t~ri~ antifungal, antiviral, or anLip~dsitic activity.
In the design of antibody-antibiotic conjugate, a primary consideration is the mode of
action of the antibiotic. Since the conjugates will be much larger molecules than the parent
antibiotics, only antibiotics that bind to exposed or secreted components (e.g, toxins) of the
bacteria, fungus, virus, or parasite are likely to target the antibody carrier to the pathogen or
its products. For example, penicillin antibiotics disrupt bacterial cell wall synthesis and bind
to surface-exposed components of certain bacteria whereas aminoglycoside antibiotics
comrnonly bind to ribosome subunits in the cell cytoplasm. The former is a much better
candidate for effective antibody-antibiotic conjugates than the latter.
Antibiotics vary greatly in the type and species of org~ni~m.~ upon which they are
active. For example~ certain antibiotics such as the polymyxins are far more effective against
gram-negative bacteria, whereas other antibiotics such as vancomycin tend to be more
effective against gram-positives. Some, like the cephalosporins, and broad-spectrum
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WO 94tl4437 PCTIUS93/12381
2151~8~ ~
penicillins are comparably effective against both types. Other antibiotics, such as
arnphotericin are primarily antifungal agents whereas amantadine exhibits activity against
certain influenza viruses. In cle~igning antibody-antibiotic conjugates for the prevention or
treatment of disease one must consider the spectrurh of antibiotic activity desired and select
S those antibiotic(s) that are active against the target pathogen(s) and, as described above, act
primarily on exposed components of the pathogen(s).
As used herein, the term "pathogen" refers to any organism which is associated with
infection or tli~ç~e, whether that organism is among those traditionally considered pathogens
(e.g, S. aureus, S. pyogenes, S. dysenteriae, S. flexneri, etc.) or is an opportunistic pathogen
(e.g., P. aeruginosa, S. marcesens, 5. mitis, etc.).
Within a family of antibiotics (e.g, penicillins, cephalosporins, polymyxins) there are
structural features common to all members. However, there often exists a wide variety of
natural and synthetic variations on this common structure that may influence the activity
spectrum, ph~rm~cokinetics, or other properties of the antibiotic. In the design of antibody-
antibiotic conjugates, these structural differences within an antibiotic family are important
from two perspectives. First, the activity spectrum may influence the choice of antibiotic;
and, second, the chemical dirr~,le.~ces between antibiotics will influence the range of cross-
linking ~hemi~tries available to conjugate the antibiotic. For example, the variable side chain
component of penicillin antibiotics is a methyl benzyl group in penicillin G but the variable
side chain group is a phenolic group with a primary amine side chain in amoxicillin. The
latter antibiotic ~-es~ a wider array of potential modes for cross-linking than does penicillin
G.
In Table 2, several families of antibiotics are disclosed that possess surface- and/or
product-reactive activities against various pathogens. This is just for illustration and by no
means is int~n~l~cl to limit the invention to these compounds alone.
A ~ r~llc;d antibiotic of the present invention is polymyxin B (PMB). As noted
above, this antibiotic binds to and neutralizes endotoxin. However, when used in vivo, PMB
is short-lived, and furthermore, at the recommen-lecl therapeutic dose for systemic infections,
there is a significant risk of nephrotoxicity.
The level of protection achieved by the present invention is best understood when
compared with other known approaches (see Table 3). For exarnple, the widely-tested and
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Wo 94/14437 21513 8 6 PCT/US93112381
TABLE 2
Antibiotics That May Be Conjugated To Antibodies
TYPE EXAMPLES ACllVITY SPECTRUM
penicillin G,: 1~ inhibition of cell h.l, grarn-posilivc
Penicillins' nafcillin, arnpicillin ticarcillin, wall synthesis and ,. , ~,
penicillin V
S Cl ~ ~ ; 2 cefoxitin, ceforanide t;l~ l, inhibition of cell ~;n ~.i l, gram-positive
lla wall synthesis and t~ull , ' v~.
Polymyxin polymyxin B, colistin ' idl binds and inhibits al~tib~l.,t~"h.l, primarily gram-
cell wall synthesis negative
Pirop~ n, ;al, binds lo cell wall ~ l, primarily gram-
V ~ ristocetin precursor, inhibits synthesis positive
circulin, EM49, polypeptin, surface-activc <ultil,act.lidl
brecistin, cerexin, l.id~ ,'
L` Il.. ,l~lb~ suriàctin
surt`actin, subsporin. surface-activc iungicidal
viomycin, , . .~chl not known ycul,.. ,l~,li.,
(t~
Other Peptide Antibioticsa
bacitracin, G ~ ti~ a,lltibCl~tlial
gramicidin S, tyrocidine
Amantadine6 ' blocks ion channel antiviral (Influenza A)
Polyene macrolide7 surface activity on membrane antifungal
sterols
Ll~ aillK surface active ' i"l
Limulus anti-LPS factor5 LPS-binding
Endotoxin binding proteins LPS binding protein (human)~' LPS-binding : '
b~,t~,lil,id~d I ' ~ LPS-binding _ '
increasing protein"
G.L. Mandell and M.A. Sande in Goodman and Gilman 's: The P ' ~,;.,ul Basis of T) , 8th Ed.,
(Gilman, Rall, Nies. and Taylor, eds.), Pergamon Press, New York, pp. 1065-1097 (1990).
M.A. Sande and C.L. Mandell in Goodman and (iilman's: The P' ~ ul Basis of Th_, r ' , 8th Ed.,
(Gilman, Rall, Nies. and Taylor. eds.), Pergamon Press, New York, pp. 1117-1145 (1990).
A. Fiechter. Trends in Biotech. 10:208-217 (1992).
G.L. Mandell and M.A. Sande in Coodman and Gilman's: The F' ~,;~al Basis of Th_,, . 8th Ed.,
(Gilman. Rall, Nies. and Taylor. eds.), Pergamon Press, New York, pp. 1146-1164 (1990).
R.G. Douglas in Coodman and Gilman 's: The Ph.., ' ~;~1 Basis of T~,, u~c r;~O, 8th Ed., (Gilman, Rall, Nies
and Taylor, eds.), Pergamon Press, New York, pp. 1182-1201 (1990).
7 J.E. Bennett in Goodman and Gilman's: The Ph.. , ' .g;.ul Basis of T~ .r . 8th Ed., (Gilman. Rall, Nies, and
Taylor, eds.), Pergamon Press, New York pp. 1165- 1181 (1990)
25 K T. Nakamura et al., J. Biol. Chem. 263:16709-16713 (1988).
G. Alpert et al.. J. Infect. Dis. 165:494-500 (1992).
' ' R.R. Schumann et al., Science 249:1429-1431 (1990).
" M.N. Marra et al., J. Immunol. 148:532-537 (1992).
- 25 -
WO 94/14437 PCT/US93/12381
~, 15 1 3 ~ ~ TABLE 3
CENTOXIN IgG-PMB
Dosage 100 mg 100-500 mg
Raw Material Cost $300 $2-10
Endotoxin Affinity low high
Half-life short (<24 hr) long (>20 days)
Safety good good
publicized monoclonal antibody Centoxin-HA-lA is capable of binding endotoxin and
neutralizing its biological activity. However~ when co~ ,aled to an IgG-PMB conjugate of
the present invention, the monoclonal antibody is costly and suffers from low affinity and
short half-life. The latter characteristics may explain why the human clinical studies have yet
to yield clear benefits.
Others have attempted to reduce the toxicity of polymyxin B by ~ .hment to dextran.
[D.A. Handley, Eur. Patent Appl. Pub. No. 428486.] However, dextran has a half-life in
hl]m~n~ of only about a day. By use of immunoglobulin according to the present invention, a
much longer half-life is achieved (see Table 4 and Examples 24 and 25). Dextran, having no
Fc receptor ~FcR), also has no known capacity to promote opsonization or activate
complement (C').
As it important that the antibodiotics be non-toxic to the host animal, the present
invention contemplates the use of conjugates which are effective against the org~qnism~ of
interest, yet are non-toxic to the host. The non-toxic character of IgG-PMB is demonstrated
in Example 27.
As noted previously, the present invention also contemplates antibodiotics having
reactivity with gram-positive org~nism~ and their toxins. In one embodiment, the present
invention contemplates the use of bacitracin conjugated to immunoglobulin.
In another embodiment, the present invention contemplates the use of vancomycin
conjugated to immunoglobulin.
- 26 -
~WO 94/14437 ~ I S 13 8 6 PCT/US93/12381
Bacitracin i~ a polypeptide produced by a strain of Bacillus subtilis (Tracy strain),
which is primarily bactericidal for gram-positive org~ni~m~ including Streptococcus pyogenes~ -
other ~-haemolytic streptococci, Pneumococcus pneumoniae, and certain strains of Clostridiu~
species. Bacitracin exerts its effect by inhibiting early steps in the biosynthesis of
5 peptidoglycan interfering with cell wall synthesis. Commercially available bacitracin is stable
and poorly absorbed from the intestinal tract or wounds. Because of the proteinuria,
hematuria and nitrogen retention observed upon systemic ~tlmini~tration, its use is usually
restricted to topical application. [See e.g., R. Berkow and A.J. Fletcher (eds.). The Merck
Manual, 16th ed., 1992, p. 46; and G.F. Brooks et al., Jawet_, Melnick & Adelberg'.s Medical
10 Microbiology, l9th ed., 1991, pp. 172-173).]
Despite the unacceptable occurrence of nephrotoxicity associated with systemic
~rlmini~tration of free bacitracin, when it is conjugated to immunoglobulin according to the
present invention, the advantages of bacitracin can be achieved without this side-effect. lt is
not intended that the present invention be limited by the mech~ni~m of action of any
15 particular antimicrobial.
Vancomycin is active, principally, against grarn-positive org~ni~m.c including
Staphylococcus aureus and Clostridium difficile . While it is not intentle-l that the present
invention be limited by the mech~ni.cm~ of action, it is believed that vancomycin exerts its
bactericidal action by hllelr~;lillg with cell-wall synthesis. This invention contemplates
20 conjugates synthe~i~e~l from vancomycin and non-specific human immunoglobulin using a
variety of cro.~.clinking agents and scheme~ These conjugates, like the ones previously listed,
SUpl)leS:j bacteremia as well as toxin-mediated effects for gram-positive org~ni~m~.
In one embodiment, the method involves conjugating the vancomycin to non-specific
immunoglobulin by first treating the vancomycin and the immunoglobulin with different
25 heterobifunctional cro.~linkin~ agents, and second reacting the derivatized species with each
other to form a conjugate. In a second embodiment, the method involves conjugating the
- vancomycin to non-specific immunoglobulin by first reacting the same heterobifunctional
cro~linking agent with both the vancomycin and the non-specific immunoglobulin, then
second reacting both derivatized species with each other forming a conjugate. For both
30 synthetic schemes a variety of crosslinker combinations have been contemplated and tested.
Below is a table which lists the cro~linking compounds which have been tested to date for
- 27 -
WO 94/14437 PCT/US93/12381
2~ g~
TABLE 4
DEXTRAN-PMB Ig-PMB
Carrier Polysaccharide Protein
Conjugation ChemistryCarbonyl, amide-SH, CHO, NH~
Cross-linkers? No Yes
Bactericidal ? Yes
Expected Half-life ~ 24 hr > 20 days
Effector for C' No Yes
FcR No Yes
Additional Reactivities No Yes (IV Ig has additional
reactivities)
10 reaction with vancomycin. Some of the cro~linking agents, upon reaction with vancomycin,
were insoluble in aqueous solution and were not further pursued. It is recognized, however,
that should steps be taken to render them soluble (e.g. addition of solvents, further side group
modification of the base ~ conlycin structure, etc.) that such cro~linking agents could prove
useful.
The table describes the cro~linking approach, the group on the modified vancomycin
that is reactive ("reactive group") with either immunoglobulin or the corresponding linker on
immunoglobulin (if any), the solubility, and the biological activity of the conjugate. The
following examples describe representative reactions set forth in the table.
C. Conjugates And Cross-linking
Numerous agents have been developed for the cross-linking of biological molecules.
[Pierce Chemical Co., (Rockford, IL), General Catalog, pp. E-10 - E-39 (1992).] In general,
these agents possess functional groups that are reactive with the side chains of different amino
acids found in proteins or peptides. As snmm~rized in Table 5, various functional groups will
react with primary amino groups, carboxyl groups, hydroxyl groups, or thiol groups of
28
~wo 94/14437 2 i ~i l 3 ~ 6 PCT/US93/12381
proteins or other compounds. In the design of antibody-antibiotic conjugates, the reactive
groups of both the antibody and antibiotic must be considered. In general, antibodies have
many reactive groups that can be used in direct conjugation schemes (amino acids cont~ining
primary amine, carboxyl, hydroxyl, thiol [after reduction]) or modified groups (glycosylated
5 amino acids that can be oxidized to aldehyde; or primary amines that can be made thiol-
reactive) for conjugation schemes. Individual antibiotics will not, in general, possess very
many different reactive groups and offer fewer choices for conjugation to antibodies. The
selection of an antibiotic from a family of related compounds and the selection of a cross-
linking scheme must take into consideration the reactive groups on an antibiotic.
A key concern in modifying an antibiotic is the preservation of its ability to bind to
the surface or secreted products of a pathogen. The modification of individual reactive
groups or excessive modification of more than one reactive group with cross-linking agents,
or the steric hindrance created by ~tt~chment to a large protein such as immnnnglobulin may
abolish antibiotic activity. Therefore, before conjugate activity is considered, conditions for
15 preservation of antibiotic activity must be detPrminPd by e~,.,;.,;-,g the biological activity of
the modified or cross-linked antibiotic in simple antimicrobial assays. Preferably, one chooses
a cross-linker type and concentration that preserves antibiotic activity.
29
WO 94/14437 PCT/US93112381 ~
3~;
cn
O G O ~_)C m O o o o o o
y C~ Z Z Z Z Z
~ C
cn cn
E
~ o o _ o o o o o
CD z zr~ er z
~ .
g o o~t ~ o o o o o
z ~ ~iz z z z z
cL
u~
~ D V~ Zvl V~ zo o O ZO O
8 CQ
U
-
O r
r ~ c cn cn cn - -
. ~ o
~ c~
c
cn ~ ~ ~ I
c cn c
cO, c c
cn
r
.
: ~ ' a -
r z ` ~ ~ ; IA _~
o , ; ~ -
r~ . .
cn c
c c
WO 94/14~37 PCT/US93/1238i
~ 2~S~13~6
Different cross-linkers may influence the activity of individual antibiotics and the
efficiency with which they are conjugated to antibodies. In the design of antibody-antibiotic
conjugates, the discovery of more optimal cross-linkers relies on the empirical analysis of
conjugates prepared using varying concentrations of different cross-linkers.
S The in vivo safety and efficacy of antibody-antibiotic conjugates will depend upon
their activity, toxicity and stability. The selection of the cross-linking agent may also affect
these aspects of conjugate performance. For example, in addition to inflllencing the activity
of the conjugate imparted by the antibiotic, the cross-linker employed may affect the
properties of the antibody. Effector functions dependent upon the Fc region of the antibody
such as opsonization or complement fixation may be influenced by which reactive groups are
utilized and their location on the antibody molecule. Furthermore, some cross-linkers may
cause adverse reactions by eliciting an immune response to the haptenic groups on the cross-
linker. Finally, the in vivo stability of the bonds created by the cross-linking scheme may
vary in important ways. Disulfide bonds linking the antibiotic and antibody may not be as
stable, for example, as amide bonds created by other cross-linkers. Dissociation between
antibody and antibiotic may not be tolerable in cases where long-term prophylaxis is desired.
D. Analogues
The present invention contemplates the use of antibody analogues. Antibody
analogues are those compounds which act in an analogous manner to antibodies. In one
embodiment, the present invention conte,l,~lates fr~gment~ of antibodies (e.g, Fc fractions) to
make antibody-antibiotic conjugates. As herein used, the terms "antibody" and
"immunoglobulin" are meant to include antibody analogues.
E. New Antibiotics And Conjugates
Antibiotic compounds have been isolated from many different microbial, plant, and
animal sources and new promising compounds continue to be discovered. In addition,
synthetic derivatives of natural compounds as well as wholly synthetic compounds such as
small peptides are also being screened for antibiotic activities in many laboratories. As used
herein, the term "antibiotic" refers to any chemical compound which destroys, inhibits the
growth of, or binds to microorg~ni~m.c (i.e., "antimicrobials"). It is not intended that the term
31
Wo 94/14437 PCT/US93/12381 ~
2~ 3~
TABLE ~
Conjugates
Functional Groups Reacts With:
Aldehyde Primary amines
S Imide Primary amines
Amino Aldehyde
Cyano Hydroxyl groups
Halogen (e.g., Bromine) Thiol groups
Carboxyl groups Primary amines
10Activated carboxyl groups (e.g, N-
Primary amines or hydroxyl groups
succinimidyl esters of carboxylic acids)*
Anhydrides (e.g, succinic anhydride
Primary amines
and maleic anhydride)
Maleimide derivatives Thiol groups
15 * e.g~ N-hydroxyl succinimide ester of N-(-4-carboxycyclo-hexyl methyl)
maleimide.
be limited only to those compounds which are produced by microorg~ni~m.c "Antibiotic"
therefore includes compounds which are produced synthetically, as indeed many of the
antibiotics are now produced in the chemi~try lab rather than by microorg~ni~m~ Polymyxin
20 and other compounds discussed herein may be produced synthetically or obtained from
"natural" sources (e.g B. polymyxa). Therefore, the invention co~ plates the design and
synthesis of a variety of antibody-antibiotic conjugates utili7inp antibiotics from all sources.
Figures 2-4 outline the methods by which new antibiotics can be screened for use as
compounds for conjugation with immunoglobulins. The "Screening Modes" consist of the
25 following temporal steps:
32
_WO 94/14437 PCT/US93/12381
~ 2~3~6
Mode I: Conjugate the antibiotic to a cross-linker only and then assess for inhibition
of organism growth in liquid culture and on a disc inhibition lawn assay (e.g, Kirby-Bauer).
Mode IIA: Conjugate the antibiotic via the cross-linker to immunoglobulin and then
assess for binding to bacteria and bacterial toxin by a solid phase assay.
S Mode IIB: Conjugate the antibiotic to immunoglobulin without the use of a cross-
linker (e.g., periodate oxidation of the carbohydrate groups ["CHO"] of IgG) and then assess
for binding to bacteria and bacterial toxin by a solid phase assay.
Mode III: Check specificity of the antibodiotic by inhibition of bacterial toxin binding
with the antibiotic.
Mode IV: Assess the antibodiotic for inhibition of org~ni~m~ growth in liquid culture.
By using this approach, a new antibiotic ("X") can be evaluated for use in the present
invention. Although it is not required for research use, it is contemplated that in the clinical
setting, the current protocols for broth dilution, disk diffusion, and other methods developed
by the National Committee for Clinical Laboratory Standards (NCCLS) will be followed.
For example, antibiotic X may initially be evaluated by Mode I. In this Mode, X is
only conjugated to a cross-linker "c" to create "X-c"; this compound is then added to a liquid
or solid phase culture. By creating only part of antibodiotic, the question of compatibility
with immllnoglobulin is avoided; Mode I only addresses compatibility of "X" with the
conjugation ch~?rni~ry. The assay is performed and the results are co~ ,ared to an identical
assay of unconjugated antibiotic X.
For the lawn assay colllp~;son in Mode I, an agar-filled petri dish is inoculated with
the organism (Step 1, Figure 2). A small filter-paper disc cont~ining a known amount of
antibiotic X or X-c is placed on the agar surface and allowed to diffuse into the medium over
an 18- to 24-hr period (Step 2, Figure 2). After this incubation, a zone of growth inhibition
is apparent with X and this is colllpal~d to the zone (if any) achieved with X-c (Step 3,
Figure 2).
Alternatively for Mode I, known concentrations of X or X-c are diluted in broth in a
test tube, which is then inoculated with an organism susceptible to X (Figure 3). After
incubation, the concentration that inhibits growth (i.e., no visible growth, as indicated by lack
of turbidity) is determined. This value corresponds to the minimum inhibitory concentration
("MIC") (Figure 3A). To assess bactericidal activity, an aliquot is taken from a tube showing
bacteriostatic activity, and this aliquot is added to agar plates (Figure 3B). If growth occurs,
33
WO 94/14437 PCT/US93/12381 ~
215~L3~
then the agent is bacteriostatic; if no growth occurs, the agent is bactericidal. The minim~
bactericidal (lethal) concentration is the lowest concentration of X-c or X that produces a
99.9% reduction in org~ni~m~ from the original inoculum of approximately 100,000org~ni~mc In this manner the minimum bactericidal concentration ("MBC") is established.
[I.S. Snyder and R.G. Finch in Modern Pharmacology, 2d Ed. (C.R. Craig and R.E. Stitzel,
eds.), Little, Brown and Company, Boston, pp. 631-640 (1986); J.E. Conte, Jr. and S.L.
Barriere, Manual of Antibiotics and Infectious Diseases, 6th Ed., Lea and Febiger,
Philadelphia, pp. 135-152 (1988).~
When comparing X-c with X, some reduction in activity is expected. However, the
more potent X is, the greater the reduction in X-c activity permissible. Overall, a range of
0.01 to 50 llg/ml for both the MIC and the MBC is practical.
If the activity of X-c is good, it is further evaluated in Mode IIA. If the activity of X-
c is poor, X is evaluated in Mode IIB. Both Modes IIA and IIB contemplate covalent
rllment Mode IIA uses a cross-linker to create "X-c-Ig", while Mode IIB does not use a
cross-linker and gencl~es "X-CHO-Ig." In both cases, the antibody-antibiotic conjugate, or
simply the "antibodiotic", is assayed on a solid phase assay such as shown sch~m~tically in
Figure 4.
Toxin or or~ni~m~ may be used in the solid phase assay to coat a microwell or other
a,ulJlol~liate surface (Step 1, Figure 4A). The antibodiotic is then added to test for binding
(Step 2, Figure 4A). Standard washing procedures are used to avoid non-specific binding.
The antibody portion of the conjugate may thereafter serve as a target for secondary reagents
(e.g., goat anti-human IgG antibody having an enzyme reporter group such as horseradish
peroxidase) (see Step 3, Figure 4A). An ~ ;ate substrate for the enzyme may then be
added (not shown) to generate a colorimetric signal.
Where toxin is used in the solid phase assay, X-c-Ig binding may be compared with
that of X-CHO-Ig. Where the organism is used, care must be taken that binding is not via the
Fc l`eCep10l of Ig. Unconjugated Ig can be used as a control for this purpose.
To avoid any binding due to d~:n~ ion or other artifact. conjugates showing
reactivity in Mode IIA or Mode IIB should be evaluated in Mode III. As shown in Figure
4B, this simply involves adding free antibiotic to show that it will compete specifically for
binding.
34
WO 94/14437 PCT/US93/12381
~ 21~138~
The next portion of the evaluation involves testing the antibody-antibiotic conjugate
for growth inhibition and/or bactericidal activity (Mode IV). This is the same assay as shown
in Figure 2~ the difference being that now the complete conjugate X-c-lg (or X-CHO-Ig) is
evaluated rather than just the antibiotic (X-c).
S Both X-c-Ig and X-CHO-Ig may show good toxin binding in Mode II but poor anti-
bacterial activity in Mode IV. If the specificity of the binding is nonetheless confirmed in
Mode III, these compounds are candidates for diagnostic reagents. Alternatively, they may be
used in vivo simply to bind free toxin and thereby reduce toxin load.
Thoughtful consideration of the results of each of these steps allows any antibiotic to
be analyzed for potential use in the form of an antibodiotic. Following these in vitro tests.
the antibiotic can then be evaluated in vivo for reduced toxicity and
ph~rm~r.okinetics."antibody" and "immlmoglobulin" are meant to include antibody analogues.
F. Antibiotic Precursors And Conjugates
"Antibiotic precursors" as herein defined are rc~ct~nt~ used in the synthesis of semi-
lS synthetic antibiotics that possess limited in vitro and in vivo anti-microbial activity.
Therefore one would not typically use such compounds as anti-microbial agents. The present
invention contemplates, however, that the "latent" anti-microbial potential of such compounds
can be activated upon conjugation, according to the methods herein described, with
immunoglobulin .
In one embodiment, the method involves, as a first step, the unlocking of the anti-
microbial potential of antibiotic precursors by derivatizing them with a cro~linkin~ agent.
These derivatized antibiotic precursors by themselves possess increased anti-microbial effects
co~ .~ed to the free antibiotic precursors. As a second step, conjugates are made between
the derivatized antibiotic precursors and human immllnoglobulin. Through this latter step, a
conjugate is created that possesses the benefits exhibited by the earlier described conjugates
(increased half life, reduced nephrotoxicity, etc.).
-
WO 94/14437 PCT/US93112381 ~
2~ ~ ~ 3g~ .
II. Antibodiotic In Vitro And In Vivo Efficacy
A. In Vitro Reactivity Of The Fc Region
In the previous section~ the key question was whether the antibiotic portion of theconjugate shows the same or similar reactivity as the native antibiotic. However, it must be
5 emph~si7to~ that immunoglobulin is not simply an inert carrier. The Fc portion of the
antibody can mediate pathogen eliminzltion by two merh~nicm~ that are distinct from the
effects of the antibiotic. First, it is known that following binding of antibody to antigen, the
Fc region can activate the classical pathway of complement, ultimately resulting in the Iysis of
org~ni~mc Second, binding of the conjugate to bacteria can lead to the ingestion or
10 opsonization of the organism by recognition of the Fc region by phagocytes (e.~
macrophages) and/or Iysis by killer cells. [See L.E. Hood et al., Immunology, 2d Ed., The
Benjamin/CI-mming~ Publishing Company, Inc., Menlo Park, pp. 339-340 (1984).]
The present invention contemplates antibody-antibiotic conjugates with the capability
of binding Fc receptors on phagocytes. It is preferred that in competition binding, the binding
15 of the antibody-antibiotic conjugates of the present invention to such cells is substantially
similar to that of normal IgG.
The present invention contemplates antibody-antibiotic conjugates which, while not
activating complement systemically, are capable of binding complement to facilitate pathogen
killing. Furthermore, conjugates are contemplated which bind phagocytes via the Fc region to
20 facilitate pathogen elimin~tion. Thus, it is contemplated that the antibody-antibiotic
conjugates will mediate or enhance opsonization and removal (opsonophagocytosis) of the
etiologic agent(s) of sepsis in the treated patient.
B. Efficacy Of The Conjugate In Vivo
Regardless of the manner in which the conjugate is used in vzvo (acute, prophylactic.
25 etc.), the conjugate will be present in a background of the entire repertoire of host immune
mediators. These immune mediators include, of course, humoral immune mediators such as
endogenous antibodies directed against bacteria and their toxins.
In this regard, several studies have suggested a causal relationship between a person s
humoral immune status and the susceptibility to gram-negative infections. In patients who
30 survived Pseudomonas aeruginosa septicemia~ both total IgG levels and the circulating titer of
36
WO 94/14437 PCT/US93112381
21 513~B
core antigen-specific anti-LPS levels were significantly higher than in those patients who
succumbed. [M. Pollack et al., J. Clin. lnvest. 72:1874-1881 (1983).] Similarly, a correlation
has been found between the titer of IgG against the patient's infecting organism and the
frequency of shock and death. [S.H. Zinner and W.R. McCabe, J. Infect. Dis. 133:37-45
(1976)-]
These studies suggest that patients at risk of gram-negative sepsis and endotoxemia
may be so because of we~ken~d humoral immune defenses. For this reason, the present
invention contemplates, in one embodiment, determining the immune status of the host prior
to ~lminictration of the antibodiotic. This determination can be made by screening potential
risk groups for total and endotoxin core antigen-specific IgG and IgM levels. [B.J. Stoll et
al., Serodiagnosis and lmmunotherapy 1:21-31 (1987).] Screening is believed to be
particularly important with the elderly. full-terrn and pre-term neonates [W. Marget et al.,
Infection 11:84-86 (1983)], patients with malignancies [C. Stoll et al., Infection 13:115-119
(1985)]. abdominal surgery candidates, individuals under long-term catheterization or artificial
ventilation, and burn and other trauma victims.
Where the immune status is poor (e.g., low total IgG levels and low levels of anti-
bacterial antibodies), the efficacy of the antibody-antibiotic conjugate is expected to be most
dramatic. Where the host's immune status is good, use of the conjugate will support the
endogenous anti-bacterial defenses.
For optimal in vivo treatment, the conjugate itself must be effective against clinically
relevant org~ni~m~, non-toxic and non-immunogenic. Thus, it is contemplated that the
conjugates of the present invention will be effective against gram-positive and gram-negative
or3~ ni~m.~ which are commonly associated with sepsis (e.g, E. coli, K pneumoniae, P.
aeruginosa, S. pyogenes, S. aureus, 5. epidermidis, etc.). It is also contemplated these
conjugates will be non-toxic to the host animal. As with any chemotherapeutic~ the conjugate
must be effective against the infecting org~ni~m.c but not harm the host. In addition, in order
to enhance the host's response to the infecting organism and to prevent such complications as
serum sickness upon subsequent ~lmini~tration of conjugate, the conjugates themselves must
be non-immllnogenic. This characteristic permits the immune system of the host to focus on
battling the infecting org~ni~m~, rather than attack the conjugates int~n~le~l as treatment. As it
is contemplated that these conjugates may be ~iminict~red to the same animal multiple times
(i.e., upon subsequent exposures to potentially pathogenic org~ni~m~) it is important that the
37
Wo 94/14437 PCT/US93/12381 ~
2~ 3~
host not produce antibodies against the conjugates themselves. Such antibody production
would be likely to lead to rapid clearance of the conjugate upon subse~luent a~1mini~tration or
result in a serious, potentially life-thre~tening hypersensitivity response.
Conjugates which are non-immunogenic or p~orly immunogenic due to high
5 concentrations of D-configuration amino acids are also contemplated. Synthetic polypeptides
entirely comprised of D-amino acids are generally unable to elicit an immune response. [M.
Sela, in Advances in Immunology, Vol. 5, (F.Dixon and J. Humphrey. eds,), pp. 29-129
(1966).] Thus, conjugation of a synthetic antimicrobial comprised entirely of D-amino acids
to the antibody would be beneficial in the present invention.
10 III. Antibodiotic Applications
A. Prophylactic Use In Humans
The diagnosis of sepsis is problematic. First, the development of sepsis does not
require the persistent release of toxin(s) into, nor the presence of org~ni~m.c the circulation.
Thus, many patients who die of sepsis are never shown to be bacteremic. rR.C. Bone, Ann.
15 Intern. Med. 115:457-469 (1991).] Second, even if bacteria are ~letecte~l, the amount of time
needed for this detection is often too great to be practical.
For these reasons and others, the present invention co~ "pl~tes the use of
antibodiotics in hllm~n.~ prior to the onset of ~y~ tonls (e.g, prophylactically). In particular.
the present invention contemplates the use of antibodiotics as a prophylactic in patients at
20 high risk for infection, as well as sepsis.
High risk patients include surgical patients (particularly the elderly), low birth weight
infants, burn and trauma. Trauma patients are particularly difficult to examine because of the
multitude of invasive procedures that they have undergone. Trauma patients are also typically
hooked up to a number of devices, including intravascular lines, mechanical ventilators and
25 Foley catheters. While every attempt is made to change intravascular lines, this is frequently
impossible because of the extent of trauma and the lack of venous accessibility. [E.S. Caplan
and N. Hoyt, Am. J. Med. 70:638-640 (1981).]
Most patients with multiple trauma have fever, as well as increased white cell counts
due to the stress of the trauma itself. The classic indicators of infection, therefore, may or
may not reflect an ongoing infection. 38
wo 94/14437 PCT/US93tl2381
3 8 6
Because of this~ current clinical practice involves treating patients with antibiotics only
for specific indications, and for as short a period of time as possible. Generally, the average
course for any documented infection is seven to ten days. Prophylactic antibiotics are used in
only three instances: open fractures, penetrating abdominal injuries and penetrating facial
injuries in which there is injury to the respiratory mucosa. Even in these situations,
antibiotics are used for only three to five days, depending on the injury.
In contrast, the present invention contemplates treating all trauma patients
prophylactically with antibodiotics. Because of the reduced toxicity of the conjugates and
their longer residence time in the circulation, the present invention contemplates ~-lmini~tering
antibodiotics immediately to the trauma patient upon admission. Indeed, the antibodiotics
may successfully be used at the first moment that clinical care is available (e.g., emergency
mobile care).
Rather than the short (i.e., three to seven day) period of protection provided by native
antibiotics, the use of the antibiotic-antibody conjugates of the present invention should
protect the trauma patient during the entire period of risk.
Burn patients have many of the sarne problems with respect to the diagnosis and
therapy for infection. Since the magnitude of thermal injury is related to the level of trauma
in a burn victim, this even becomes more of a problem with acute cases.
It is reported that septicemia appears in the blood cultures of burn patients almost four
days after a septic state. [M. Meek et al., J. Burn Care Rehab. 12:564-568 (1991).]
Consequently, therapy with the conjugates of the present invention is particularly applo~liate
immediately after the burn injury as a means of preventing a septic reaction. Furthermore, in
severe cases, consideration should be given to the topical ~tlmini.~tration of antibodiotics to
prevent wound sepsis.
2S Importantly, burn victims are exposed equally to both gram negative and gram positive
org~ni~m.s Burn victims are particularly good candidates for therapeutic plepald~ions having
bactericidal activity for both gram-positive and gram-negative org~ni~m~ This includes
conjugates using a single antibiotic with reactivity for both groups of org~ni~m.~ (e.g,
antibiotics such as a cephalosporin or broad-spectrum penicillin) and well as therapeutic
"cocktail" ~ ~aLions comprising: (i) a first conjugate consi~ting of a first antibiotic
covalently bound to non-specific immunoglobulin; and (ii) a second conjugate con~i~fin~; of a
second antibiotic covalently bound to non-specific immllnoglobulin (e.g, where the first
39
WO 94/14437 PCT/US93/12381
2~ 5~3~
antibiotic is polymyxin and the second antibiotic is bacitracin). Alternatively, two different
antibiotics can be covalently bound to the same immunoglobulin molecule.
The use of blood cultures and the like has also been shown to be unreliable in the
diagnosis of neonatal sepsis. Indeed, in practice cultures appear to have little or no influence
5 on antibiotic therapy decision-making for at-risk infants. [T.J. Zuerlein et al., Clin. Ped.
29:445-447 (1990).] For this reason, the conjugates of the present invention can be applied
with great advantage (i.e., antibiotics can be used without the concern of toxicity, and the
longer circulating half-life allows for antibiotic therapy without n~ces~rily prolonging
hospitali~ation) .
Finally, surgical patients also r~ csent a risk group where the conjugates of the
present invention can be used successfully. Current practice involves the prophylactic use of
antibiotics in a very narrow category of cases (e.g., elective colorectal procedures,
cholecystectomy, hysterectomy and Caesarean sections). [R.L. Nichols in Decision Making in
Surgical Sepsis, B.C. Decker, Inc., Philadelphia, pp. 20-21 (1991).] One to two grams of a
15 broad-spectrum antibiotic are ~rlmini~t~red intravenously at the induction of anesthesia. An
additional dose may be given during an extensive procedure or post-operatively but
prophylaxis beyond 24 hours is not indicated. Twenty-four hours of antibiotic prophylaxis is
considered to be sufficient to control co~ ".il-~tion. Continll~n~e of antibiotic prophylaxis
beyond 24 hours is an added expense, particularly when using an antibiotic with short serum
20 and tissue half-lives. Most i~ ol ~ ly, co,~ ion of antibiotic prophylaxis also runs an
excessive risk of drug toxicity and emergence of resistant strains.
By contrast as shown in rabbits in the accompanying Examples, the longer serum half-
life of the conjugates of the present invention provide ~xten~led protection against sepsis
without the expense of multiple dosing. Furthermore, since the distribution of
25 immunoglobulin is predomin~ntly to vascular conl~ ents, the use of the conjugates of the
present invention may reduce the risk of disruption of endogenous flora. Consequently, the
conjugates of the present invention may be used liberally (e.g, in more categories of surgical
procedures).
WO 94/14~37 PCT/US93/12381
2 i~386
B. Acute Therapy In Humans
As noted previously~ the present invention also contemplates the use of antibodiotics in
a therapeutic preparation for acute treatment. In this case, treatment involves a-lrnini~tration
of the antibody-antibiotic conjugates after infection is detected and/or sepsis is suspected.
Evidence suggestive of gram-negative infection includes the following: (1) core
temperature higher than 38C or lower than 35C; (2) peripheral blood leukocyte count
greater than 12 x 109/L or less than 3 x 109/L (not due to chemotherapy), or at least 20%
imm~tllre forms; (3) growth of grarn-negative org~ni~m.~ from a blood culture drawn within
the preceding 48 hours; or (4) documented or suspected site of gram-negative infection.
Current medical practice accepts sepsis as having no specific pharmacotherapy
available. ~R.L. Greenman et a/., JAMA 266:1097-1102 (1991).] A systemic septic reaction
is characterized by at least one of the following: arterial hypotension (systolic blood pressure
<90 mm Hg or an acute drop of 30 mm Hg); metabolic acidosis (base deficit >5 mEq/L);
decreased systemic vascular resi.~t~nce (systemic vascular resistance <800 dynes/s cm5);
tachypnea (respiratory rate >20/min or ventilation >10 L/min if mechanically ventil~te~l); or
otherwise unexplained dysfunction of the kidney (urine output <30 ml/h), or lungs.
It must be stressed that the antibodiotics of the present invention should ideally be
used prior to a systemic infection, if possible. For example, the conjugates can be
~Amini~tered immediately after bacteremia or fungemia is AetecteA Similarly, conjugate(s)
can be ~Amini~tered where there is an obvious sign of infection at a particular site (e.g.,
wounds, sinusitis, meningiti~, respiratory, ga~Lloi,.le~ l, or urinary tract infections, etc.).
Primary bacteremia is typically defined as two or more blood cultures with the same
bacterial organism occurring in a patient with no other obvious site of infection. Sinusitis is
diagnosed in a patient`who has at least two of the following: purulent nasal discharge,
roentgenographic evidence of sinusitis or purulent material aspirated from the sinuses.
The lower ~ tory tract is a common site of infection. Pneumonia in the int~lb~ted
patient is diagnosed in a patient when there is fever, leukocytosis and a Gram stain with many
polymorphonuclear leukocytes. Pneumonia may also be diagnosed in the patient with a new
infiltrate that has not cleared with intensive physical therapy (this last criterion helps rule out
atelectasis).
41
WO 94/14437 PCT/US93/12;~81
8 ~
C. Veterinary Care
Septicemia and sepsis are by no means limited to human beings. Infection by gram-
negative bacteria accounts for significant morbidity and mortality in neonatal livestock~ such
as calves. [D.D. Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986).] Interestingly,
5 humoral immune status is again related to susceptibility to sepsis and this is largely dependent
on passive transfer from colostrum. For this reason, the present invention contemplates, in
one embodiment, determining the immllne status of the animal prior to a-lmini~tration of the
antibodiotic. This determin~tion can be made by screening neonatal calves for total
circulating serum immunoglobulin ~e.g, by ELISA).
Where the immlme status is poor (e.g., low total IgG levels), the conjugate should be
used prophylactically. Where the animal's immune status is good, use of the conjugate may be
needed for acute therapy of gram-negative bacterial sepsis, which remains prevalent in
neonatal calves even with high antibody levels.
The present invention conten rl~tes the tre~tment of other ~nim~l.c as well. Among
foals less than 10 days of age in critical distress, sepsis is the most serious problem. [A.M.
Hoffman et al., J. Vet. Int. Med. 6:89-95 (1992).] Symptoms highly indicative of sepsis risk
include we~kn( s~, metabolic disturbance and dehydration. In one embodiment, the invention
contemplates using antibodiotics for prophylactic treatment of foals less than 10 days of age
having these indicators, or those at risk of infection.
While positive blood cultures are found in less than half of the cases, those ~nim~
found positive have a very poor chance of survival. The present invention therefore
contemplates using antibodiotics for acute tre~tmp-nt of any animal with evidence of
septicemia, with or without culture-proven cases.
IV. Therapeutic Preparations And Combinations
~ The present invention contemplz~te~ using therapeutic compositions of soluble
antibodiotics. It is not intçn-lçd that the present invention be limited by the particular nature
of the therapeutic preparation. For example, such compositions can be provided together with
physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients. In
addition, antibodiotics may be used together with other therapeutic agents, including
unconjugated immunoglobulin.
42
WO 94/14437 PCT/US93/12381
21S1386
As noted above, these therapeutic preparations can be ~tlmini~tered to m~mm~ forveterinary use, such as with domestic ~nim~ , and clinical use in humans in a manner similar
to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary
according to the type of use and mode of ~(lmini~tration, as well as the particularized
5 requirements of individual hosts.
With respect to the mode of Q~lmini~tration, the antibodiotics may be employed for
intravenous, intramuscular, intrathecal or topical (including topical ophth~lmic) ~mini~tration.
Formulations for such ~-lmini.~trations may comprise an effective amount of antibodiotic in
sterile water or physiological saline.
On the other hand, formulations may contain such normally employed additives as
binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients
as, for example, pharmaceutical grades of mannitol, lactose, starch, m~gn~?~ium stearate,
sodium saccharin, cellulose, m~gnecium carbonate, and the like. These compositions typically
contain 1%-95% of active ingredient, preferably 2%-70%.
The compositions are preferably prepared as injectables, either as liquid solutions or
suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection
may also be prepared.
The antibodiotics of the present invention are often mixed with diluents or excipients
which are compatible and physiologically tolerable. Suitable diluents and excipients are, for
20 example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition,
if desired the compositions may contain minor amounts of auxiliary substances such as
wetting or emulsifying agents, stabilizing or pH buffering agents.
Where repeated ~rlmini.~trations are required, it may be beneficial to first clear any
anti-hapten antibodies by ~lmini~t~ring free antibiotic. This can then be followed by
25 ~lmini~tration of the antibodiotic.
EXPERIMENTAL
The following examples serve to illustrate certain preferred embodiments and aspects
of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: eq
30 (equivalents); M (Molar); ,uM (micromolar); N (Normal); mol (moles); mmol (millimoles);
,umol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); ~lg (micrograrns); ng
43
WO 94/l4437 PCT/US93/12381
2~5~ ~8~
(nanograms); L (liters); ml (milliliters); ,ul (microliters); cm (centimeters); mm (millimeters);
,um (micrometers); nm (nanometers); C (degrees Centigrade); AUFS (absorption units full
scale); hr (hour); sec (second); min (minutes); IV (intravenous); lg (immnnQglobulin); FCS
(fetal calf serum); CFU (colony forming units); ELISA (Enzyme-Linked Immunosorbent
5 Assay); Aldrich (Aldrich Chemical Co., Milwaukee, WI); Amicon (Beverly, MA); Baxter
(Deerfield, IL); BBL (Becton Dickinson Microbiology Systems, Cockeysville, MD); Bio-Rad
(Richmond, CA); Corning (Corning, Inc., Corning, NY); Falcon (Lincoln Park, NJ); Lee (Lee
Laboratories, Grayson, Georgia); Harlan Sprague-Dawley (Harlan Sprague-Dawley,
Intli~n~polis, IN); ICN (ICN Biomedicals, Costa Mesa~ CA); Mallinckrodt (Mallinckrodt, St.
10 Louis, MO); Pharmacia (Pharmacia, Inc., Piscataway, NJ); Pierce (Pierce Chemical Co..
Rockford, IL.); Prochem (Prochem, Inc., Rockford, IL); Scientific Products or S/P (Scientific
Products, McGraw Park, IL); S & S (Schleicher & Schuell, Inc., Keene, NH); Sigma (Sigma
Chemical Co., St. Louis, MO.);Spectrum (Spectrum, Houston, TX); Whatman (Wh~tm~n,
Inc., Clifton, NJ).
In some of the examples below, purification of products from re~ct~nt~ is performed
using various types of chromatography. Standard terms understandable to those skilled in the
art are employed to describe this purification. For example. "eluent" is a chemical solution
capable of dissociating desired products bound to the column matrix (if any) that passes
through the column matrix and comprises an "eluate". Products that are dissociated (if any)
20 are freed from the column matrix and pass by elution with the "eluent" into the "eluate".
EXAMPLE 1
~tt~rhmtont Of An Antibiotic To
Human IgG Using A Carbodiimide Cross-Linker
This example describes attempts to attach antibiotics to a carrier (i.e., in this case
25 antibodies). In this regard, K. Hanasawa et al. describe the ~tt~chment of PMB to an
immobilized fiber via carbodiimide chemi~try. [Surg. Gyn. & Ob. 168:323-331 (1989).] In
this example, the ability of a carbodiimide cross-linker to conjugate polymyxin B (PMB) to
human IgG was analyzed.
It is known that l-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
30 cross-links proteins and peptides between amine and carboxylic acids. The example involved:
44
~WO 94/14437 2 ~ ~ ~L 3 8 6 PCT/USg3/12381
(a) EDC-mediated cross-linking of PMB and IgG; and (b) enzyme-linked immunoassay(ELISA) of conjugate binding to LPS.
a) EDC-Me~ te-l Cross-l,inkin~ Of PMB To IgG
In this and in all examples, measures were taken to make glassware, solutions, and
5 other materials and reagents pyrogen-free because adventitious pyrogen (e.g~, LPS) could
inhibit conjugation reactions, absorb PMB or conjugates~ or block the activity of PMB
conjugates.
Human IgG (Sigma) and PMB (Sigma) were each dissolved at a concentration of 8
mg/ml in pyrogen-free MES (2-[N-Morpholino]eth~nf sl~lfonic acid) buffer (0.lM MES, 0.09
10 M NaCl pH 4.7). 0.5 ml of each solution were mixed together and 0.15 ml of the mixture
was incubated with 0.15 ml of either 0.4 M EDC, 0.2 M EDC, 0.1 M EDC (Pierce), or
control solution in MES buffer, for 2 hrs at room temperature. The reactions were stopped by
the addition of 2.7 ml of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.2). The five mixtures
were dialyzed separately (molecular weight cut-off of dialysis tubing 12-14,000, Scientific
15 Products) at 4C against four changes of 1500 ml of PBS over a 36 hr period. The samples
cont~ining human IgG at 0.2 mg/ml were stored at 4C.
b) Enzyme-~.irk~l Immunoassay Of EDC-PMB Conjugate
Binding To LPS
In order to determine whether the ~tt~r.hment of PMB facilitated the binding of IgG to
20 LPS, a simple indirect binding assay was performed. To each well of a 96-well microtiter
plate (~alcon), 100 111 of a 2.0 ,ug/ml solution of E. coli 011 l:B4 LPS (Sigma) in PBS was
coated, except for those control wells in which PBS but no LPS was added. After an
overnight incubation at 4C, the coating solutions were clec~ntç~l and all wells were washed
three times with PBS. Non-specific binding sites were blocked by the addition of 100 1l1 of
25 PBS cont~ining 5 mg/ml bovine serum albumin (BSA, Sigma) for 2 hrs at room temperature.
After ~lçç~nting the blocking solution, samples of the conjugates prepared in (a) above were
diluted in PBS cont~ining 1 mg/ml BSA to an initial concentration of 10 ~lg/ml IgG followed
by five-fold dilutions. A positive control antiserum of commercially prepared rabbit anti-E.
coli 0111:B4 antiserum (Lee Laboratories, Lot M25082) was initially diluted 1:100. One0 hundred (100) ~11 of each sample was incubated in duplicate for two hours at room
WO 94/14437 PCT/US93/12381
~138~
temperature and the plates were washed three times with BBS-Tween 20 (0.1 M boric acid,
0.025 M Na borate, 1.0 M NaCI, 0.1% Tween 20. p~ 8.3), followed by two washes with
PBS-Tween 20 (0.1% Tween 20 (v/v)), an~ finally, two washes with PBS.
In order to detect bound antibodies, the wells incubated with the hurnan antibody
5 conjugates were incubated with 100 ,ul of a 1:500 dilution of goat anti-human IgG (whole
molecule)-~lk~line phosphatase conjugate (Sigma) and the wells incubated with the rabbit
serum were incubated with 100 ~l of a 1 :500 dilution of goat anti-rabbit IgG (whole
molecule)-alkaline phosphatase conjugate (Sigma) for 2 hours at room temperature. The
secondary antibody solutions were discarded, the plates were washed with BBS-Tween 20,
10 and PBS-Tween 20 as above and then twice with 50 mM Na7Co3, 10 mM MgCI7, pH 9.5.
~fter 45 minl~te~ at room temperature, the absorbance of each well was measured at 410 nrn
on a Dynatech MR700 plate reader using diluent control wells as blanks. Tables 6 and 7
show the results for the rabbit control serurn and EDC-conjugates.
The results in Table 6 show that the positive control serum, as expected, bound to
15 LPS-coated wells in a specific manner. These data validate the ELISA design as being
capable of detecting LPS binding antibodies.
The results in Table 7 appear to indicate that EDC cross-linking caused the IgG-PMB
to bind to the LPS. However, the titration of the conjugates drops off rather abruptly between
S and 1 ~Lg/ml. To verify that the observed binding is specific, it must be determin~d that the
20 binding is inhibitable by PMB and antigen-dependent.
TABLE 6
Binding Of Rabbit Anti-E. coli 0111 :B4 Antiserum
To E. coli 0111:B4 LPS (OD4l0 Values)
Serum Dilution LPS Coated No Antigen
1:1 1.801 0.032
1 :5 1.817 0.028
1 :25 1.648 0.024
1:175 0.308 0.026
1 :625 0.070 0.027
1:3125 0.021 0.028
1:15,625 0.014 0.018
46
WO 94/14437 PCT/US93/12381
2 ~ 8 ~
The ELISA described above was repeated; however, in this instance, a fixed
concentration of the EDC-conjugate (10 ~lg/ml) that yielded the highest binding by ELISA
was incubated with five-fold dilutions of polymyxin B (beginning with 10 mg/ml) and the
LPS-binding activity was determined. In addition, the binding was
-
5 TABLE 7
Binding Of EDC-Mediated Human IgG-PMB Conjugates
To E. coli 0111 :B4 LPS (OD4,0 Values)
EDC Conjugation Conc. (M)
Conjugate IgG Conc. (llg/ml) 0.2 0.1 0.05 0.025 0
0.638 0.369 0.306 0.464 0.015
/2 0.010 0.012 0.026 0.054 0.008
0.4 0.000 0.000 0.002 0.009 0.007
0.008 0.000 0.000 0.000 0.006 0.005
tested in control wells co~ ;"i"g no antigen. The results are shown in Tables 8 and 9. Since
the binding of the conjugate is only inhibited at the very highest concentration of PMB tested
15 and because the conjugate exhibited significant binding to wells that contained no antigen, it
is clear that most of the binding observed is was not specific. This may reflect an
il1al~pl~ iate type or number of bonds between PMB and IgG and, since IgG that was not
treated with a cross-linker shows very little binding to LPS, it suggests that the cross-linking
of the IgG molecule is causing nonspecific binding.
47
Wo 94/14437 PCT/US93/12381 ~
2~5~ 38g
TABLE 8
Specificity Of EDC-Conjugates Of IgG-PMB Binding
To LPS: PMB Inhibition Test (OD4,0 Values)
PMB Concentration (mg/ml) 0.2 M EDC-Conjugate Binding
0 1.757
0.755
2 1.775
0.4 1.785
0.08 1.770
100.016 1.766
0.0033 1.775
Clearly, carbodiimide chemi~try does not work at a level which is practical. Indeed. it
is evident that the interactions of the cross-linking reagent with the antibiotic are somewhat
complex. It is to be remembered that three reactions arepossible: PMB to PMB; IgG to IgG;
15 and PMB to IgG. Only the latter reaction is productive.
TABLE 9
Antigen-Dependent Binding Of EDC-Conjugate
Of IgG-PMB To LPS (OD4,0 Values)
Conjugate Conc. (~g/ml)LPS Coated Wells No Antigen Wells
20 10 1.770 1.766
2 0.976 0.552
0.4 0.347 0.045
0.08 0.034 o.oo
0.016 0.062 0.00
48
WO 94/14437 PCT/US93/12381
21~13~6
EXAMPLE 2
Attachment Of An Antibiotic To Human IgG Using A Disuccinimide Ester
ln an attempt to remedy the difficulties observed with EDC conjugates. different cross-
linkers and chemistries were invcstig~tecl Talmadge and Siebert describe the ~ chment of
5 PMB via a hydroxysuccinimide ester reagent. [J. Chrom. 476:175-185 (1989).] Along the
lines of this approach, this example ex~mines the ability of a homobifunctional cross-linking
agent suberic acid bis-(N-hydroxysuccinimide ester) (DSS), which cross-links peptides and
proteins via their amine groups~ to conjugate PMB to IgG. The example involved: (a) DSS-
mediated cross-linking of PMB and IgG, and (b) ELISA of conjugate binding to LPS.
~o a) DSS-Mediated Cross-~ kir~ Of PMB To IgG
Pyrogen-free PBS was prepared in pyrogen-free water (Baxter), and stock solutions of
human IgG (40 mg/ml) and PMB (40 mg/ml) were dissolved in pyrogen-free PBS. A 60 mM
stock solution of DSS was prepared in 100% dimethylsulfoxide (DMSO). This solution was
diluted to 6.0 mM DSS in PBS where some precipitation was noted. A stock solution of
15 human IgG and PMB was prepared cont~ining 20 mg/ml IgG and 20 mg/ml PMB in PBS.
Five dirr~,lc"t conjugates were prepared by mixing two-fold dilutions of the stock DSS
solution (0.15 ml) with a constant (0.15 ml) volume of the IgG/PMB stock solution. The five
resulting DSS concentrations were 3.0 mM, 1.5 mM, 0.75 mM, 0.375 mM, and 0.0 mM
DSS. After incubation for 1 hour at room temperature, the reactions were stopped by the
20 addition of 2.7 ml of TBS. The five ~ Lul~:s were dialyzed against PBS as described in
Example I for the EDC conjugates. The resulting dialyzed conjugates contained a final
concentration of I mg/ml IgG and were stored at 4C.
49
2 ~W~ 7 PCT/US93/1238
b) ELISA Of DSS Conjugated Binding to LPS
The ELISA was performed es~enti~lly as in Example l(b) using the DSS conjugates at
starting concentrations of 10 ~g/ml and the same control rabbit anti-E. coli Ol l l :B4
antiserum. The results of the initial binding assay are shown in Table 10.
TABLE 10
Binding Of DSS Conjugates Of IgG-PMB To LPS (OD4,0 Values)
DSS Concentration (mM)
Conjugate IgG Conc.
3.0 1.5 0.75 0.375 0.00
(!lg/ml)
0.098 0.032 0.014 0.01 1 0.015
2 0.026 0.003 0.007 0.005 0.007
0.4 0.01 l 0.001 0.00 0.002 0.002
0.08 0.010 0.00 0.002 0.004 0.004
The results indicate a low level of binding that is correlated with the concentration of
DSS utili7~tl The specificity of this binding was then tested by eX~mining the ability of
15 PMB to inhlbit binding and its dependence on antigen. The assays were performed exactly as
described for the EDC conjugates in Example 1(b). The results are shown in Tables 11 and
12.
These results indicate that the DSS conjugate binds somewhat non-specifically. The
pattern of PMB inhibition is erratic in that the highest concentration shows no inhibition of
20 binding but intermediate PMB concentrations do ~ar~ ly inhibit.
These results indicate some level of specific binding above a significant amount of
non-specific binding. The binding of the control rabbit antiserurn at 1:500 and 1:12,500
dilution was 1.766 and 0.380, respectively and was virtually all antigen-dependent. The
relatively low level of binding here suggests that hydroxysuccinimide ester reagents such as
25 DSS are not very effective cross-linkers for PMB and IgG. This could be due to the amine-
amine chemistry employed, or the properties of the DSS agent. We did note some
insolubility of DSS in PBS, perhaps a more water soluble form of DSS would perform better.
~ wO 94/14~37 2 L 5 ~ 3 8 6 PCT/US93/12381
TABLE 11
Inhibition Of IgG-PMB Binding To LPS By Free PMB (OD4,0 Values)
PMB Conc. (mg/ml)3.0 mM DSS Conj. of IgG-PMB (50 mg/ml)
0 0.144
0.182
2 0.054
0.4 0.059
0.08 0.097
0.016 0.128
0.0033 0.213
TABLE 12
Antigen-Dependent Binding Of DSS Conjugate
Of IgG-PMB To LPS (OD4,0 Values)
Conjugate Conc. (,ug/ml) LPS Coated Wells No Antigen Wells
0.268 0.096
0.168 0.043
2 0.094 0.007
0.4 0.016 0.010
0.08 0.009 0.00
20 In addition, in both cases of the EDC and DSS conjugates, the PMB was exposed to a vast
excess of cross-linker which could inhibit the ability of PMB to bind to LPS.
WO 94/14437 PCT/US93/12381 ~
215l~
EXAMPLE 3
Two-Step Conjugation Of PMB To IgG
Using EDC And A Water Soluble Analogue Of DSS
In the previous two examples, cross-linkers were present in molar excess over IgG and
5 were mixed simultaneously with both antibody and antibiotic. In this example, IgG was first
modified with the cross-linker, the cross-linker removed, and then PMB added to the coupling
reaction. In this way, the binding activity of PMB might be improved and the non-specific
binding of the IgG reduced. In order to have an amine to arnine coupling reagent that was
more water soluble, BS3 (Pierce). a water soluble analogue of DSS was employed. The
10 example involved: (a) two-step conjugation of IgG-PMB with EDC; (b) two step conjugation
of IgG-PMB with BS3: and (c) ELISA of conjugate binding to LPS.
a) Two-Step Conjugation Of IgG-PMB With EDC
A 0.75 ml of a 4 mg/ml IgG solution in MES buffer was prepared as described in
Example 1, and mixed with 0.75 ml of a 0.4 M EDC solution in MES buffer at room
15 tem~dLul~ for 2 hours. The unreacted cross-linker was removed by passing the 1.5 ml
reaction lllixLu~e over a Sephadex G-10 (Pharmacia) column that was poured into a sterile l0
ml pipette and equilibrated with pyrogen-free MES buffer. The void volume was collected
and the IgG content was determined by measuring the OD280 of a 1:40 dilution of each
fraction. The peak fraction cont~ining 2.37 mg IgG/ml was divided into two fractions: 1.5
20 mg of PMB was added and dissolved in one volume; nothing was added to the other
(control). After incubation at room t~ el~Lule overnight, the reaction was stopped with TBS
and the final Ig(~ concentration was adjusted to 0.2 mg/ml. Both samples were dialyzed as in
Example l(a) and stored at 4C.
wo 94/14437 2 i 5 13 8 6 PCT/US93/12381
b) Two-Step Conjugation Of IgG To PMB With BS3
A 0.75 ml of a 20 mg/ml IgG solution was mixed with 0.75 ml of a 6.0 mM BS3
solution, each prepared in PBS and incubated at room temperature for l hr. Unreacted cross-
linker was removed as in Example 3(a) above and the peak IgG fractions identified and
5 pooled. Two equal fractions of IgG at 8.35 mg/ml were made and 7.5 mg of PMB was added
and dissolved in one while nothing was added to the other. After overnight incubation at
room temperature, the reactions were stopped with TBS, the conjugates dialyzed and the final
IgG concentration adjusted to 1.0 mg/ml.
c) ELISA Of Conjugate Binding To LPS
This LPS-binding assay was performed as described in Example (I) except that theBBS-Tween 20 washes were elimin~te~ and the Tween 20 concentration in the PBS-Tween 20
wash was lowered to 0.05% (v/v). The results are shown in Tables 13 and 14.
The 0.2 M EDC IgG-PMB conjugate exhibited a high level of binding but this was
partly due to non-specific binding as evidenced by the binding to control wells COt~t~ g no
15 LPS. Further evidence of non-specific binding created by EDC cross-linlcing is shown by the
results for the conjugate collt~ g no PMB (which exhibited somewhat comparable levels of
binding to the wells regardless of whether antigen was present or not).
TABLE 13
Binding Of Two-Step EDC Conjugates To LPS (OD4,0 Values)
0-2 M EDC (No PMB)
0-2 M EDC IgG-PMB Conj.
IgG Control
20Conjugate IgG Conc.
LPS
LPS Coated No Antigen No Antigen
Coated
1 0 1 .790 1 .790 1 .7841 .790
2 1.520 0.886 0.6760.522
0.4 0.092 0.146 0.0880.079
0.08 0.024 ND 0.028 ND
0.016 0.046 ND 0.030 ND
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TABLE 14
Binding Of Two-Step BS3 Conjugate To LPS (OD4,0 Values)
6.0 mM BS3 IgG-PMB 6.0 M BS3 IgG
Conjugate IgG Conc. (~lg/ml) No
LPS LPSNo Antigen
Antigen
1 0 0.037 0.040 0.0280.00
2 0.01 60.00 0.02~0.00
0.4 0.044 0.00 0.0440.00
0.08 0.040 ND 0.076 ND
0.016 0.038 ND 0.024 NI~
The BS3 conjugates exhibited no specific binding to LPS whatsoever at the
10 concentrations tested. However, they did not exhibit much non-specific binding either,
indicating that this cross-linker may not be as problematic as EDC in causing non-specific
binding of IgG.
Given the low level of BS3 conjugate background binding, the ELISA was performedagain using higher concentrations of the conjugates and a tenfold higher concentration of LPS
15 coated onto the wells (2 ,ug LPS/well). This increased the assay sensitivity. The results
shown in Table 15 indicate that the BS3 conjugates do possess LPS-binding activity above
background.
Together, the results of the two-step conjugations described in this example indicate
that EDC creates an unacceptable level of non-specific binding, while BS3, the water soluble
20 analogue of DSS, effects a modest level of specific binding and causes very little non-specific
binding in this two-step conjugation format. Additional two-step conjugation procedures
using other cross-linkers were investip.~te~l to cletermine whether higher levels of LPS-binding
activity could be achieved than those obtained with BS3.
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TABLE 15
More Sensitive Detection Of BS3 Conjugates
Of IgG-PMB Binding To LPS (OD4,0 Values)
6.0 mM BS3 IgG-PMB 6.0 M BS3 IgG (Control)
Conjugate IgG Conc. (~lg/ml)
LPSNo antigen LPSNo antigen-
5 50 0.0980.01 0 0.0060.006
I 0 0.0580.006 0.0060.008
2 0.0200.005 0.0040.004
0.4 0.0090.005 0.0040.004
0.08 0.005 ND 0.004 ND
EXAMPLE 4
Three Step Conjugation Of PMB To IgG Using An
Amine To Sulfhydryl Coupling Chemistry With SMCC
Because of the unsatisfactory results of previous examples in obtaining high specific-
binding of IgG-PMB conjugates to PMB, an alternative cross-linking method was investigated
using sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate [sulfo-SMCC] in a
three-step procedure similar to that of T. Kitagawa et al. [J. Assoc. Anal. Chem. (1985).]
The example involved: (a) three-step conjugation of PMB to reduced IgG with sulfo-SMCC;
and (b) ELISA of conjugate binding to LPS.
a) Three-Step Conjugation Of PMB To IgG
ln the first step of this procedure, reactive thiol groups were created in the IgG by
treatment with 2-mercaptoethanol. In this procedure, 4.0 mg of IgG was first dissolved in
0.45 ml of pyrogen-free O.lM NaPO4 pH 6Ø Fifty ,ul of 0.1 M 2-mercaptoethanol in 5 mM
EDTA, 0.1 NaPO4, pH 6.0 was then added and incubated at 37C for 1.5 hours. The free 2-
mercaptoethanol was separated by applying the 0.5 ml sample to a 5 ml Sephadex G-10
WO 94/14437 PCT/US93/12381
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colurnn equilibrated in 0.1 M NaPO4, 5 mM EDTA, pH 6.0 and the IgG cont~ining fractions
identified and pooled.
The second step of this procedure involved preparation of malemide-activated PMB.
This involved mixing 1.5 ml of a 0.16 mg/ml PMB solution in 50 mM sodium borate buffer,
5 pH 7.6 (pyrogen-free) and 1.5 ml of a 0.46 mg/ml sulfo-SMCC (Pierce) solution in the same
borate buffer (creating a final concentration of 0.053 mM of each reactant). This "SMCC-
activated" PMB was incubated at 30C for 60 minutes.
The third step of the procedure involved incubation of 0.65 ml of the reduced IgG
with 0.65 ml of the SMCC-activated PMB.
The concentrations of the two re~ct~nt.~ were 0.0265 mM PMB and 0.013 mM IgG (a
2:1 molar ratio). After incubation at 4C for 20 hrs~ 8.7 1ll of a fresh solution of 0.1 M ~-
mercaptoethanol was added and incubated at room temperature for 20 minut~s The IgG
concentration was adjusted to 1.0 mg/ml with an equal volume of PBS. Sarnples of the
conjugates were purified by dialysis against two 800 ml volumes of PBS over a 20 hour
15 period or by gel filtration on a Sephadex G-10 column equilibrated in PBS. A control
reduced human IgG fraction was prepared from the reduced IgG pool and the three
al~lions stored at 4C.
b) ELISA Of Conjugate-Binding To LPS
The LPS binding assay procedure was the same as that described in Example l(b)
20 except that the LPS was coated at 2 ~g/well, the BBS-Tween 20 washes were elimin~t~-l and
the Tween 20 concentration in the PBS-Tween 20 wash was lowered to 0.05%. The blocking
solution and sample diluent were prepared using pyrogen-free PBS and low-endotoxin BSA
(Sigma). The results are shown in Table 16.
56
wo 94114437 PCT/US93/12381
2~51386
TABLE 16
Binding Of An SMCC Conjugate Of IgG-PMB To LPS (OD4,0 Values)
SMCC l ,G-PMB SMCC IgG Control
Conjugate IgG
Concentration (,ug/ml) LPS No LPS No Antigen
4 AIltigell
5100 0.084 O.Oll 0.038 0.00
0.013 0.00 0.012 0.00
4 0.00 0.005 0.00 0.00
0.8 0.00 0.028 0.00 0.00
0.016 0 00 0.00 o oo o oo
The SMCC IgG-PMB exhibited slightly higher binding to LPS than the control but the
overall level of binding was far below that of the positive control rabbit anti-~. coli Olll:B4
antiserum (1.097 at a 1:25,000 dilution). It is possible that reduced IgG possesses only a few
thiol groups available for cross-linking and that higher concentrations of activated PMB might
drive the reaction more effectively.
E~M PLE 5
-. Conjugation Of An Antibiotic To IgG
Without Using A Bifunctional Cross-linker
In all of the previous examples, free bifunctional cross-linkers were employed in
attempts to covalently attach the antibiotic polymyxin to IgG. The configurations failed to
yield a conjugate with LPS-binding activity comparable to that of an immune serum. Because
of the binding observed in the absence of antigen, there were probably conjugates having less
- than one active PMB molecule to each molecule of IgG. To investigate means of ~tt~t~hing
antibiotics to IgG without the involvement of a bifunctional cross-linker~ periodate oxidation
of the carbohydrate groups of IgG [D.A. Handley, Eur. Patent Appl. Pub. No.428486] was
used to create amine-reactive aldehyde groups that could potentially react with PMB and be
reduced to establish a stable covalent linkage.
WO 94/14437 PCT/US93/12381
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2~13
The example involved: (a) periodate oxidation of IgG in pH 4.0 sodium acetate
buffer; and (b) conjugation of polymyxin B to periodate oxidized IgG.
a) Periodate oxidation of IgG in pH 4.0 sodiurn acetate buffer was achieved by
dissolving 5 mg human IgG in 1 ml of water and mixing this solution with 200 ,ul of sodium
5 acetate pH 4.0 (0.3 g sodium acetate and 960 ,ul glacial acetic acid in 100 ml H~O) and 200
,ul of 0.2 M NaIO4. [Modification of J.W. Goding, Monoclonal Antibodies: Principles and
Practice, Ac~ c Press, New York, p. 84 (1986).] After 15 minlltec at room telllp~ldl~lre
in the dark, the periodate solution was removed by gel filtration on a P-10 column in 50 mM
Na,CO3, pH 9.5.
b) Conjugation of periodate-oxidized IgG with PMB was carried out by adding 10
mg of PMB to the IgG prepared in (a) and incubating for 1 hour at room tem~eldLu.e,
followed by the addition of 100 ~11 of NaBH3CN (4 mg/ml) and room tel~ ule incubation
for another hour and dialysis against PBS overnight at 4C.
Inspection of the LPS binding activity (not shown) revealed that the prepared
conjugate was inactive. These results suggest that the periodate-oxidation of IgG, without the
use of a cross-linker, is an ineffective means of covalent conjugation of antibiotics to
antibody.
EXAMPLE 6
The Derivatization Of Antibiotics With
Cross-Linkers: Preservation Of Antibiotic Activity
A significant concern with either one-step or multi-step schemes for conjugatingantibiotics to antibodies is whether the conjugation scheme reduces or inactivates antibiotic
function. In order to determine the best cross-linker concentration for derivatization of PMB
in a multi-step conjugation sch~me, the effect of the concentration of cross-linker on antibiotic
25 activity was clet~rrnined (see discussion of Mode IA above). The exarnple involved: (a)
modification of PMB with SPDP and the separation of free cross-linker; and (b) assay of
derivatized PMB antibacterial activity.
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WO 94/14437 PCT/US93/12381
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a) Modification of PMB with SPDP and the separation of free cross-linker was
carried out by mixing three different molar ratios of SPDP (2:1, 3:1 and 4:1) to PMB. First,
2.1 mg, 3.15 mg or 4.2 mg of SPDP (Pierce) dissolved in dimethyl sulfoxide was added to 5
mg of PMB in 0.5 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0 and incubated for 30
5 minutes at room telll~Ue~ UlC~ with occasional .ch~king. Free cross-linker was then removed
from each sample by chromatography on a 15 ml Swift desalting column equilibrated with
PBS-EDTA. The peak fractions cont~ining the derivatized PMB were collected and pooled.
b) Assay of derivatized PMB antibacterial activity was carried out in a disc
inhibition assay (see Figure 2). E. coli HB101 was plated on Trypticase Soy Agar (TSA;
10 BBL) to create a confluent lawn of bacteria. One-quarter inch blank paper discs (BBL) were
then applied to the surface of the lawn and 20 ~1 of each test solution applied. After
incubation at 37C overni~ht zones of inhibition surrounding the disc were observed. The
results (not shown) indicate that PMB derivatized at 2:1 or 3:1 molar ratios of SPDP-PMB
were still active whereas antibiotic derivatized at a 4:1 molar ratio was inactive. Therefore,
15 derivatization of PMB with SPDP should be carried out at ratios of SPDP to PMB of less
than or equal to 3:1.
EXAMPLE 7
Conjugation Of SPDP-PMB To IgG
Having determined an SPDP cross-linker concentration that preserved the antibiotic
20 activity of polymyxin B in Example 6, conjugates were prepared between SPDP-PMB and
IgG by reacting the derivatized antibiotic with IgG to which sulfhydryl (-SH) groups were
introduced with Traut's reagent.
The example involved: (a) derivatization of PMB with SPDP; (b) derivatization ofIgG with Traut's reagent; (c) conjugation of Traut-IgG with SPDP-PMB; and (d) conjugate
25 LPS-binding activity ~Sec.~ment.
a) Derivatization of PMB with SPDP was carried out by adding 7 ,umoles of
SPDP (2.1 mg) in 50 ,ul of dimethyl-sulfoxide to 10 mg of PMB in 1 ml of 50 mM sodium
borate~ 300 mM NaCl, pH 9.0 and incubating at room temperature for 30 minutes on a
59
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2~138~ -
rotating shaker. The unconjugated cross-linker was removed by applying the sample to 15 ml
Swift desalting column (Pierce) equilibrated with 20 mM NaPO4, 150 mM NaCl, I mMEDTA, pH 7.2 (PBS-EDTA). Peak fractions were pooled and stored at 4C.
b) Derivatization of IgG with Traut's reagent was carried out by adding a five-
5 fold molar excess (100 ~Ll of a 0.2 mg/ml stock) of Traut's reagent (Pierce) to S mg of IgG
dissolved in 1 ml of 50 mM triethanolamine, 0.15 M NaCl, 1 mM EDTA, pH 8.0 and
incubating under nitrogen for 45 minlltes at room temperature. The excess Traut's reagent
was removed by gel filtration on a P-10 column equilibrated with PBS-EDTA. The peak
fractions were combined.
c) Conjugation of Traut-IgG with SPDP-PMB was carried out by adding 3.5 mg
Traut-IgG and 2 mg SPDP-PMB (77 fold molar excess of PMB) and incubating for 18 hours
at room t~ ldLul~. The conjugates were sep~d~d from free SPDP-PMB by gel filtration
on a P-10 column (50 ml) equilibrated with PBS-EDTA and the peak fractions co,.l~ i,.g the
IgG were collected, pooled, and stored at 4C.
d) Conjugate LPS-binding activity ~es~",ent was carried out by evaluating the
ability of each conjugate in (c) to bind LPS in an ELISA assay (see Figure 4). The results
indicated that the Traut IgG-PMB conjugate possessed limited binding activity (not shown).
EXAMPLE 8
Conjugation Of SPDP-PMB To SPDP-IgG
Having rletermined that Traut's reagent does not generate a conjugate with preserved
antibiotic activity in Example 7, conjugates were prepared between SPDP-PMB and IgG by
reacting the derivatized antibiotic with IgG in which amino (NH2) groups were convered to
sulfhydryl (-SH) groups by activation with SPDP.
The example involved: (a) derivatization of PMB with SPDP; (b) derivatization ofIgG with SPDP; (c) conjugation of SPDP-IgG with SPDP-PMB; and (d) conjugate LPS-binding activity ~e~ment
WO 94/14437 PCT/US93/12381
21~1~86
a) Derivatization of PMB with SPDP was carried out as in Example 7.
b) Derivatization of IgG with SPDP was carried out by adding 20 ~l of 20 mM
SPDP to lO mg of IgG in 1 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0 and
incubating 30 minutes at room t~ pelaLIlre with ~h~kin~. The free cross-linker was removed
5 by chromatography on a 15 ml Swift desalting column equilibrated in lO0 mM sodium
acetate, 100 mM sodium chloride pH 4.5. The peak fractions were collected and concentrated
on C~llllip-el)-30 concentrator (Amicon). To this sample, 7.7 mg of dithiothreitol in 250 ~11
of 100 mM sodium acetate, 100 mM sodium chloride, pH 4.5 was added and incubated at
room temperature for 30 mimltes The sample was again applied to a 15 ml Swift desalting
10 column equilibrated with PBS-EDTA and peak fractions with the highest OD280 were
collected, pooled, and concentrated on a C~IlL~ cp-30 concentrator (Amicon).
c) Conjugation of SPDP-IgG with SPDP-PMB was carried out by adding the
following combinations of re~ct~nt~
- 5 mg SPDP-IgG and 2 mg SPDP-PMB (43 fold molar excess of PMB)
- 2 mg SPDP-IgG and 2 mg SPDP-PMB (107 fold molar excess of PMB)
and incubating for 18 hours at room t~ dlule. The conJugates were each separated from
free SPDP-PMB by gel filtration on a P-10 column (50 ml) equilibrated with PBS-EDTA.
Fractions cont~inin~ PMB-IgG conjugate were collected, pooled, and stored at 4C.
d) Conjugate LPS-binding activity ~ c~ment was carried out by evaluating the
ability of each conjugate in (c) to bind LPS in an ELISA assay and co~"p~ E them with the
Traut conjugate produced in Example 7. The results (Figure 5) in~ te~l that both SPDP-
IgG-PMB conjugates possessed considerable activity -- a much higher activity than that of the
Traut IgG-PMB conjugate.
EXAMPLE 9
Conjugation Of PMB To IgG Using A Long Chain SPDP Cross-Linker
Since SPDP proved to be an effective agent for the derivatization and cross-linking of
IgG and PMB. a long chain form of SPDP (sulfo-LC-SPDP) was then examined to see if the
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addition of a larger spacer arm between the IgG and PMB enhanced the activity of the
conjugate. This example involved: (a) derivatization of PMB with sulfo-LC-SPDP;
(b) derivatization of IgG with sulfo-LC-SPDP; (c) conjugation of derivatized IgG with
derivatized PMB; and (d) conjugate activity ~es~ment by ELISA.
S a) Derivatization of PMB with sulfo-LC-SPDP was carried out by adding 35 ~11 of
a 9 mg/ml solution of sulfo-LC-SPDP to 10 mg of PMB in 1 ml of 50 mM sodium borate,
300 mM NaCl, pH 9.0 and inc~lh~ting for 30 minlltes at room temperature. Free cross-linker
was removed by gel filtration on a 1.5 x 35 cm P-2 column equilibrated in PBS-EDTA. Peak
fractions containing derivatized PMB were combined and stored at 4C.
b) Derivatization of IgG with sulfo-LC-SPDP was carried out by adding 0.3 mg of
sulfo-LC-SPDP to 10 mg of IgG in 50 mM sodiurn borate, 300 mM NaCl pH 9.0 and
incubating for 30 minlltes on a rotating shaker. The derivatized IgG was ~,epd,dL~d from free
cross-linker on a S ml Swift de~lting column (Pierce) equilibrated with 100 mM sodium
acetate, 100 mM sodium chloride, pH 4.5 and the peak fractions collected and pooled. This
sample was then reduced by adding 7.7 mg of dithiothreitol in 250 ~1 of the same sodium
acetate buffer and incubated for 30 ~ PS at room temperature. Excess reducing agent was
removed by gel filtration on a 10 ml P-10 column equilibrated in PBS-EDTA. The peak
fractions were collected and pooled.
c) Conjugation of derivatized IgG with derivatized PMB was carried out by
adding 2.5 mg of IgG to 2.5 mg of PMB (107-fold molar excess of PMB) and 3.5 mg of IgG
to 1.4 mg of PMB (43-fold molar excess of PMB), and incubating for 18 hours at room
t~ ld~Ult;. The IgG-PMB conjugate was sep~a~ed from the rest of the reaction mixture on
a 50 ml P-10 gel filtration column equilibrated with PBS-EDTA.
d) Conjugate activity ~se~ment by ELISA indicated that the sulfo-LC-SPDP
conjugates did not possess greater activity than the shorter SPDP molecule (Figure 6).
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EXAMPLE 10
Inhibition Of Specific Binding Of Antibodiotic To LPS By ~ree Antibiotic
In order to determine that the antibody-antibiotic conjugate binding observed in Figure
5 is specific, free antibiotic was used to block conjugate binding (see Mode III discussion,
S above). This example involved: (a) mixing of the antibodiotic with free antibiotic; and (b)
assaying the degree of conjugate binding to LPS in the presence of different concentrations of
free antibiotic.
a) Mixing of antibodiotic with free antibiotic was performed by adding an equal
volume of a 1:125 dilution (32 !lg/ml) of the SPDP IgG-PMB conjugate in PBS-Tween 20
10 (0.05%) cont~ining 1 mg/ml BS~ with polymyxin at 0-20 ,ug/ml in the same buffer. Two
hundred (200) ~11 of this mixture co~ g 0-2 llg of PMB and 3.2 ,ug of conjugate was then
assayed for binding activity.
b) Assaying the degree of conjugate binding to LPS in the presence of different
concentrations of free antibiotic was performed by adding 200 ~1 of the antibodiotic/free
15 antibiotic IlliX~ to wells of a 96-well microtiter plate that was coated with 2 ~g of E. coli
Ol l l :B4 LPS and blocked as described in Example 1. The wells were washed, goat anti-
human Ig-~lk~line pho~ph~t~ce was added, and the binding assayed ~u~lliL~ ely on a
MicroELISA reader exactly as described in Example 1.
The results are shown in Figure 7 and demonstrate that free polymyxin competitively
20 inhibits IgG-PMB binding to LPS. Clearly, the antibodiotic is binding specifically to LPS
(i.e., via the conjugated PMB moieties).
Inspection of the inhibition curve gives some indication of the extent of active PMB
conjugation, in that a 16 ~lg/ml solution of antibody (1.1 x 10-7M) is 50% inhibited in its
binding to LPS by a concentration of 40 ng/ml PMB (2.6 x 10-8M). If one molecule of PMB
25 was present on each PMB (making the PMB concentration on IgG equal to 1.1 x 10-7) one
would expect that an equimolar concentration of free PMB would inhibit binding by 50%.
Since it requires one fourth the concentration of free PMB to inhibit this antibodiotic, one
may conclude that there is at least one PMB molecule per four IgG molecules. In fact, since
SPDP-modified PMB has a four-fold lower antibiotic activity than free PMB, the actual
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WO 94/14~37 PCTIUS93/12381
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degree of IgG conjugation with PMB is probably at least four-fold higher than that calculated
above (i.e., there is probably at least one PMB conjugated to each IgG molecule).
EXAMPLE 11
Conjugation Using Periodate Oxidation Of IgG In NaPO4
In Example 5, a means of ~tt~rhing antibiotics to IgG without the involvement of a
bifunctional cross-linker (i.e., periodate oxidation of the carbohydrate groups of IgG) was
attempted. This involved, in part, periodate oxidation of IgG in pH 4.0 sodium acetate buffer
and failed to yield a conjugate with significant activity. Because this failure may have been
due to the reaction conditions, different reaction conditions were explored. This example
involves. (a) periodate oxidation of IgG in phosphate buffer; and (b) conjugation of
polymyxin B to periodate oxidized IgG.
a) Periodate oxidation of IgG in phosph~te buffer was achieved by dissolving 10
mg of human IgG in I ml of 50 mM NaPO4, pH 7.2 and adding 0.011 g sodium
metaperiodate (final concentration 50 mM). After 30 minllt~s at room temperature, the
periodate was removed by gel filtration on a 10 ml P-10 gel filtration column equilibrated in
50 mM NaPO4, pH 7.2. The peak fractions co,.~ -g antibody were pooled and concentrated
to 1.5 ml.
b) Conjugation of periodate-oxidized IgG with PMB was carried out by adding 10
mg of PMB to either 5 mg or 3 mg of IgG prepared in (a) at 4C overnight with gentle
~h~king, followed by reduction with 0.1 mg/ml NaBH3CN in 20 mM NaPO4, pH 6.5 for 3
hours at room ~ claL~ . The IgG-PMB was separated from the rest of the reaction
products by gel filtration on a 10 ml P-10 column.
Inspection of the LPS binding activity (Figure 8) revealed that the conjugates prepared
were active. This is in contrast to the conjugate prepared in Example 5.
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EXAMPLE 12
Antibacterial Activity Of IgG-PMB Conjugates
Having determined which conjugates of IgG-PMB possessed LPS binding activity, the
biological activity of the conjugates were ç~mined (see discussion of Mode IV~ above).
5 Since polymyxin possesses direct antibiotic activity, it was possible that the conjugated
polymyxin was also active. To determine whether the conjugates had any antibacterial
activity, the minimum inhibitory concentration (MIC) and minimllm bactericidal concentration
(MBC) for the SPDP-conjugated IgG-PMB (107-fold molar excess of PMB, Exarnple 8) and
the periodate mediated IgG-PMB conjugate (3:1 ratio of PMB, Example 11) were determined.
10 The example involved: (a) ~ule~ ion of an E. coli bacterial inoculum; (b) determin~tion of
the MIC; and (c) determination of the MBC.
a) P.~ ion of an ~. coli bacterial inoculum was initiated by first culturing E.
coli HB101 overnight on TSA agar at 37C. Colonies were suspended in sterile saline at 1.2
x lo8 org~ni~m~/ml then diluted to 5 x 105 org~ni~m~/ml in Trypticase-Soy Broth (TSB;
15 BBL). This concentration was co~ ..l.ed by dilution plating.
b) D~L~ ion of MIC for each conjugate and a native polymyxin B control
was made by mixing 0.5 ml of the 5 x 105 org~ni~m.s/ml inoculum with 0.5 ml of a two-fold
dilution series of each conjugate and incub~ting overnight in sterile 12 x 75 mm culture tubes
at 37C. The MIC was defined as the lowest concentration of the conjugate or PMB which
20 resulted in complete inhibition of visible growth.
For the PMB control, the MIC was found to be 0.031 ~lg/ml while for the SPDP
conjugate, the MIC was found to be 0.25 mg/ml. For the 3:1 (PMB:IgG) periodate conjugate,
the MIC was found to be 0.031 mg/ml, which is approximately 1000-fold higher than for
native PMB and eight-fold lower than for the SPDP IgG-PMB conjugate. Thus, both IgG-
25 PMB conjugates do indeed retain antibacterial activity with the periodate conjugate exhibitingthe highest degree of activity. The difference between the PMB and IgG-PMB values reflect
in part, the greater size of IgG (about 100 times that of PMB) in that if PMB activity was
perfectly preserved during conjugation and one PMB molecule was conjugated to each IgG
molecule, the MIC would increase 100-fold due to the size of the IgG. The 1000-fold shift
WO 94/14437 PCTJUS93/12381
215138~ :
observed suggests that the activity of PMB is reduced by conjugation, and/or not all IgG
molecules are conjugated. Nonetheless, it is surprising that a small surface-active antibiotic
can still inhibit bacterial growth when conjugated to a much larger protein.
c) Determination of the MBC for each conjugate was made by plating serial
5 dilutions of the nli~Lu,es in (b) above that exhibited no growth on TSA agar ovemight at
37C. The MBC was defined as the lowest concentration of conjugate of PMB which
resulted in 99.9% or more of the viable org~ni~m.c in the primary inoculum being killed. The
MBC for the PMB was found to be 0.031 ~g/ml, for the SPDP IgG-PMB it was 0.5 mg/ml,
and for the periodate it was 0.031 mg/ml. The ability of the IgG-PMB conjugates to suppress
10 bacterial growth and to kill bacteria on contact suggests that these compounds may be
effective in preventing or treating bacteremia.
EXAMPL~ 13
The Effect Of IgG-PMB Conjugate On
Complement And Its Activation By LPS
Both immlmnglobulin and LPS have the potential to interact with complement. The
interaction of LPS with complement can exacerbate the infl~mm~tory response to
endotoxemia or bacteremia. In this example, the ability of IgG-PMB conjugate to block
complement activation by LPS was investig~te~l In addition, since immllnoglobulin can also
trigger adverse complement reactions [S. Barandun et al., Vox Sang. 7:157-174 (1962)], the
20 ability of conjugate alone to activate complement was also ~i~termin~ The example
involved: (a) ~ tion of the LPS concentrations sufficient to activate complement; and
(b) blocking Lps-inrlllce~l complement activation with IgG-PMB.
a) Det~rmin~tion of the LPS concentrations sufficient to activate complement wascarried out by adding varying concentrations of LPS to a standard quantity of a complement
25 source (CH50 Reference Standard; Sigma) and m~llring the amount of complementconsumed by titration on sen~iti7.p~l sheep red blood cells (SRBCs). [Modification of A.
Chonn et al., J. Immunol. 146:4234-4241 (1991).]
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WO 94/14437 PCTIUS93/12381
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To 40 ~11 of the Reference Standard, 40 ,ul of solution contslining 80 ~g, 8,ug, 0.8 ~lg,
0.0 ~g of E. coli LPS or GVB+2 buffer (Sigma) were added, mixed, and incubated for 30
minlltes at 37C. Five or 10 ,ul aliquots of each mixture or a blank control were then added
to CompQuick CH50 tubes (Sigma), mixed by repeated inversion and incubated for 60
5 minutes at room temperature with occasional mixing. The tubes were then centrifuged at 600
x g for 10 minutes at 4C and the hemolysis present in the s~ ldl~t measured at 415 nrn
versus the lysis control blank solution. The CH50 value of each mixture was calculated as
follows:
Absorbance of Sample
CH50 of Sample = x CH50 of Standard
Absorbance of Standard
The results are shown in Table 17.
TABLE 17
Activation Of Complement By LPS
Sample Tested Abs. ~ 415 nm CH50 Value % Decrease
1.0 mg/ml LPS + Ref. StdØ124 114.7 51.2
0.1 mg/ml LPS + Ref. StdØ170 157.8 33.1
0.01 mg/ml LPS + Ref. Std. 0.215 198.9 15.4
Reference Standard 0.254 235.0 0.00
These results show that preincubation of a complement source with LPS consumes
complement which is then unavailable for action on SRBCs in the second phase of the assay.
The LPS effect was concentration-dependent.
b) Blocking of LPS-incl~lcecl complement activation with IgG-PMB was carried
out by mixing SPDP-conjugated IgG-PMB prepared as described in Example 7 with LPS and
then ~x;..,.i~ g the effect of ~ Ll~dled LPS on complement activation. To 1.5 ~g of E. coli
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WO 94/14437 PCT/US93/12381
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026:B6 LPS, 7.5 llg of IgG PMB or 15 ,ul of a buffer control was added and incubated at
37C for 60 minl1tes Thirty (30) ~11 of complement (Ref-Std as in (a) above) or GVB+2
buffer was added to each sample and incubated for 60 minlltes at 37C. Twenty (20) ~1 of
each mixture was added to CompQuick CH50 tubes (Sigma), mixed and incubated for 60
5 minutes at room temperature. The tubes were centrifuged as in (a) above and hemolysis
4ua~ ed at 415 nm. The results are shown in Table 18.
The results show that preincubation of IgG-PMB with LPS blocks the effect of LPSon complement activation. Furthermore, the IgG-PMB conjugate has no effect on
complement activation on its own, suggesting that cross-linking with PMB has not perturbed
10 IgG structure such that it would have a deleterious effect through
TABL~: 18
Inhibition Of LPS-Mediated Complement Activation By IgG-PMB
Sample Tested Abs. (~ 415 nm CH50 Value %
IgG-PMB only 0.002 0.40
IgG-PMB + Complement 1.273 253.1 +7.7
LPS Only 0.008 1.59
Complement Reference Standard 1.182 235 0.00
LPS + Complement 0.806 160.2 -31.8
IgG-PMB + LPS + Complement 1.237 245.9 +4.6
20 spontaneous complemçnt reactions. The ability to block LPS effects and the ~~ safety
of the IgG-PMB conjugate suggests that it could possess both prophylactic and therapeutic
value against bacteremia and endotoxemia.
WO 94/14437 PCT/US93/12381
2~13~ `
EXAMPLE 14
Improved IgG-PMB Conjugates
Methods were invçstig~te~l for improving the activity of the IgG-PMB conjugates
prepared by the methods of Example 7 (SPDP) and Example 11 (periodate oxidation/Schiff
5 base reduction). Since both families of conjugates exhibited much higher levels of LPS-
binding than conjugates prepared with other ch~mi~tries, it was possible that even higher
levels of binding could be achieved by increasing the degree of PMB-substitution on the lgG.
The two mech~ni~m.~ employed for achieving greater substitution were to increase the reactant
(IgG and PMB) concentration at the conjugation step and to use more highly derivatized
10 SPDP-PMB. The example involved: (a) ~lel)~dlion of a new periodate IgG-PMB conjugate;
(b) prepalalion of new SPDP IgG-PMB conjugates; (c) ELISA of conjugate binding to LPS;
(d) ~lele~ tion of conjugate MICs and MBCs; and (e) determination of the degree of
conjugation by amino acid analysis.
a) Pl~paldlion of a new periodate IgG-PMB conjugate was carried out by
15 oxidizing 30 mg of IgG dissolved in 1 ml of 50 mM NaPO4, pH 7.2 with 10.7 mg of sodium
periodate ~Sigma) for 30 ~ es at room Lt;lll,u~ldlult;. The 1 ml reaction l~ Lu~e was
applied to a 15 ml Swift desalting colD equilibrated in 50 mM NaPO4, pH 7.2 and the peak
IgG fractions were pooled to an IgG concentration of 7.1 mg/ml. To 1 ml of this Ig mixture
co"~ g 0.0476 ~Lmoles of IgG, 20 mg of PMB (14.44 ~lmoles) was added and incub~te(l
20 overnight at 4C. The reaction ~ Lule was adjusted to pH 6.5 with 1.0 N HCI, and 10 ~LI of
a 10 mg/ml NaBH3CN solution was added and incubated at room temperature for 4 hours.
The conjugate was then chromatographed on a 10 ml P-10 column and stored at 4C.
b) Plel)~dlion of new SPDP IgG-PMB conjugates was carried out by first
derivatizing PMB at 2:1 and 3:1 molar ratios of SPDP:PMB as described in Example 6. For
25 each reaction, 5 mg of IgG in 0.5 ml was derivatized with 15 ~1 of 20 mM SPDP solution in
DMSO by inc~lb~ting for 30 minutes at room telllp~ldlule with intermittent .~h~king The
derivatized IgG was purified on a 15 ml Swift desalting column equilibrated with acetate
buffer and the peak fractions were pooled and concentrated on a Cenlliplep-30 concentrator
(Amicon). To the 5 mg of IgG in 1.8 ml of acetate buffer, 7.7 mg of dithiothreitol in 250 ~11
69
WO 94/14437 PCT/US93/12381
2~5~ 3~
of acetate buffer was added and incubated at room te~ ld~ule for 30 minlltt~s Each sample
was then purified on a 15 ml Swift desalting column equilibrated in PBS-EDTA. To each
sample cont~ining approximately 5 mg of SPDP derivatized IgG, 5 mg of PMB derivatized at
either a 2:1 or 3:1 molar ratio of SPDP was added and inc~lb~tecl for 18 hours at room
S te-~p~ re. Each conjugate was then separated from free SPDP-PMB by gel filtration on a
P-10 column (50 ml) equilibrated with PBS-EDTA, and the peak fractions were collected,
pooled, and stored at 4C.
c) ELISA analysis of conjugate binding to LPS was performed as described in
Example 1 using E. coli Ol l l :B4 LPS (Sigma). The binding of different dilutions of the
10 periodate conjugate made in (a) above, and the two SPDP conjugates made in (b) above to
LPS coated and uncoated wells of a 96 well microtiter plate are shown as averages of
duplicate samples in Table 19.
The results show that the 3:1 SPDP:PMB conjugate had the highest specific LPS
binding activity, applo~i",ately 2-4 times the binding exhibited by the 2:1 SPDP:PMB
15 conjugate and the periodate COlljU~ at concentrations of 0.8-4.0 ~lg/ml.
d) D~L~l.llin~lion of conjugate MICs and MBCs was carried out exactly as
described in Example 12 using E. coli HB101 as the ~usct;~Lible test strain. The results are
shown in Table 20.
When colllpa.ed with these ~ ..,.i"~lions for the conjugates ex~min~l in Example 12,
20 the new periodate conjugate is four times as potent, and the 3:1 SPDP-PMB conjugate is
twice as potent. Surprisingly, the periodate conjugate exhibits lower LPS-binding activity by
ELISA but stronger antibacterial activity than the 3:1 SPDP-PMB conjugate. Perhaps the
modification of PMB and IgG with SPDP improves the conjugation efficacy but decreases the
antibiotic activity compared to the conjugation of native PMB to periodate-treated IgG.
e) D;;~ ion of the degree of conjugation by amino acid analysis was carried
out by e~mining the a~nino acid composition of 2:1 SPDP Ig-PMB, 3:1 SPDP Ig-PMB, and
the periodate Ig-PMG conjugates above, compared with control samples of native human IgG
and free polymyxin B. The novel amino acid ~ minobutyric acid (DAB) which constitutes 6
of the 10 residues of PMB was the key component that was detected and ~lu~~ d.
Wo 94/14437 PCT/US93/12381
~ 2151~6
Five samples in all were analyzed, including:
1. Free PMB (25 nmoles in 50 ~11 H~0)
2. Periodate Ig-PMB (600 pmoles in 100 ~I PBS)
3. SPDP 3:1 lg-PMB (600 pmoles in 100 ~I PBS)
4. SPDP 2:1 Ig-PMB (600 pmoles in lO0 ,ul PBS)
5. Human IgG (600 pmoles in 100 ,ul PBS)
TABLE 19
LPS-Binding Activity Of New IgG-PMB Conjugates
Abst"~ ce at 410 nm
Conjugate TestedConjugate Dilution
wlAgw/o Ag
1: 10 (=0.1 mg/ml) I .7881.694
I :50 1.3920.632
10IgG-PMB (104) 1:250 0.4400.096
1:1250 0.1210.039
I :6250 0.0350.009
1: 10 (=0.09 mg/ml) 1.7260.718
I :50 1.6500.156
IgG-PMB (SPDP) 3:1 1:250 0.9790.167
1:1250 0.5200.013
1:6250 0.1200.007
1:10 (=0.1 mg/ml) 1.5920.375
I :50 1.2560-057
IgG-PMB (SPDP) 2:1 1:25Q 0.5780.015
1: 1250 0.1510.008
1:6250 0.0280.010
WO 94/14437 PCT/US93/12381
2~ ~ 13~
TABLE 20
MIC And MBC For The New IgG-PMB Conjugates
ConjugateMIC :` MBC
Periodate IgG-PMB 7.8 ~lg/ml 7.8 ~g/ml
2:1 SPDP IgG-PMB 250 ~g/ml 250 !lg/ml
3:1 SPDP IgG-PMB 125 ~Lg/ml > 125 llg/ml
PMB Control 0.039 ~lg/ml 0.039 ~lg/ml
The samples were prepared by transferring each to a glass hydrolysis tube using three
rinses of 100 ,ul of pure water and then concentrated to dryness in a vacuum centrifuge. To
10 each of the sample tubes, 500 ~11 of distilled 6N HCl, 10 ,ul of 2-mercaptoethanol, and ] O Ill
of a 50% aqueous phenol solution were added. The tubes were then purged with nitrogen gas
and capped. The sarnples were hydrolyzed by heating at 110C for 22 hours and then
concentrated again to dryness. The PMB sample was suspended in 500 ~l of 0.2 N sodium
citrate buffer, pH 2.2 while the other four sarnples were suspended in 250 ~ul of this buf~er.
15 After thorough mi~ing, the sample solutions were passed through a 0.2 ~lm pore nylon
membrane syringe filter.
A Beckman Instruments 6300 Amino Acid Analyzer was used to analyze 20 ,ul of each
filtered hydrolysate solution. The m~rhine was equipped with a Beckman 10 cm cationic
exchange HPLC column, a Beçkm~n sodium buffer system, a 60 minute analysis
20 methodology? and a Be~m~n ninhydrin reagent detection system with absorbance measured at
the 570 nm and 440 nm wavelengths. The detector sensitivity was set at 1.0 AUFS for the
PMB sample and 0.5 AUFS for the other four samples.
All data collection and peak integration calculations were performed with a Gilson
HPLC System Controller 712 v. 1.1 software package (Middleton, WI). Sample peak
25 identification and amino acid concentrations were determined by comparison to analyses made
at known concentrations of a l 7 amino acid standard mixture (Beckman Standard, Lot
#A108039) and (S) - (+) 2,4 - Diaminobutyric acid dihydrochloride (Aldrich Chemical, Lot
#07301CY). The results of the amino acid analyses are shown in Table 21.
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wo 94/14437 21~1~ 8 6 PCT/US93/12381
The values represent the estim~tP~ amino acid composition of each sample, determined
by multiplying the percentage of each amino acid measured by the expected total number of
amino acids (1320 for human IgG~ for example). The moles of PMB/mole IgG were
calculated by dividing the number of unique DAB residues detected by 6 (the number of
S DAB residues/PMB).
The results show that the 3:1 SPDP conjugate possessed the highest degree of
conjugation (avg. 3.7 PMB molecules per IgG molecule). This is consistent with this
conjugate po.~es~ing the highest LPS-binding activity as measured by ELISA (see (b) above).
The 3:1 SPDP conjugate contained, on average,.twice the number of PMB molecules than the
10 2:1 SPDP conjugate, which would explain the two-fold greater activity of the 3:1 SPDP
conjugate in the LPS-binding ELISA. The periodate Ig-PMB is also well conjugated and it
exhibited the highest degree of antibacterial activity. It appears that the SPDP linkage affords
the highest degree of LPS-binding activity while the periodate linkage provides greater
antibacterial activity. This may reflect steric differences in the way PMB is ~tt~hed to the
15 IgG and/or the different effects of the two conjugation ch~mi~tries on PMB activity.
EXAMPLE 1~
The Use Of IgG-PMB Conjugates As A Diagnostic: Cross-
Reactivity Of Dirr~rellt Gram-Negative LPS Antigens with IgG-PMB
Since the IgG-PMB conjugates exhibited binding to E. coli ()lll:B4 LPS, and this20 species is only one of many potential gram-negative agents of endotoxemia and bacteremia, it
was of interest to determine whether the IgG-PMB conjugate was capable of detecting other
species of LPS in a diagnostic format using a competitive ELISA. The example involved:
(a) coating of E. coli 011 l:B4 LPS to microtiter wells; (b) incubation of IgG-PMB conjugates
with different concentrations of several species of LPS; and (c) assay of conjugate binding to
25 E. coli Ol l l :B4 LPS in the presence of competitor.
a) Coating of E. coli 0111 :B4 LPS to the wells of 96-well microtiter ELISA
plates was performed as described in Example 1, (100 ~Ll/well of pyrogen-free PBS cont~ining
1 mM EDTA and 2 ~Lg of LPS was coated onto the wells and allowed to incubate overnight
73
WO 94/14437 PCT/US93/12381
21~ ~ 3~ --
at 4C). The wells were washed with PBS-0.05% Tween 20 and blocked with PBS
cont~ining 10 mg/ml endotoxin-free BSA at 37C for 90 minlltç~
TABLE 21
Amino Acid Composition Of Ig-PMB Conjugates
Amino Letter Human IgG- IgG- IgG-
Acid Code IgG PMB PMB 104. PMB 1:2 PMB 1:3
Asp D 111 107 105 111
Thr S 117 2 120 115 122
Ser S 177 188 208 176
Glu E 133 129 129 136
Pro P 119 105 120 123
Gly G 95 101 102 101
Ala A 75 75 78 80
Val V 116 112 107 112
Met M 9 8 9 9
Ile 1 26 28 25 26
Leu L 99 1 105 96 100
Tyr ~ Y 51 53 51 51
Phe F 42 1 47 44 45
His H 24 24 23 23
Lys K 83 87 79 82
Arg R 41 44 38 39
DAB 6 20 11 22
TOTAL 1318 10 1353 1340 1358
Moles PMB/Mole IgG 3.3 1.8 3.7
b) Incubation of IgG-PMB conjugates with different
concentrations of LPS purified from E. coli 0111 :B4 (control standard) Salmonella
typhimurium, Pseudomonas aeruginosa, Vibrio cholerae, Shigella flexneri, Klebsiella
pneumoniae, Salmonella enteritidis, Serratia marcescens, and ~hodobacter sphaeroides (all
74
WO 94114437 PCT/US93/12381
21S~;38~
from Sigma? except the Rhodobacter, which was obtained from List Biologicals Laboratory,
Campbell. CA) was carried out by adding 250 !11 of a 7.2 ,~Lg/ml solution of IgG-PMB
conjugate prepared with a 3:1 molar ratio of SPDP:PMB (Example 13) to 250 ~Ll of PBS-
0.05% Tween 20 cont~ining 1 mg/ml BSA followed by incubation at 37C; 100 ~ll of these
5 mixtures was added per well.
c) Assay of conjugate binding to E. coli 0111 :B4 LPS in the presence of
competitor was measured by incubating 100 ~1 of the mixtures from (b) above at 37C for 1
hour. The plates were then washed and the wells incubated with alkaline phosphate-
conjugated goat anti-human IgG (Sigma) diluted 1 :500 in PBS with 0.05% Tween 2010 cont~ining l mg/ml BSA, incubated 37C for 1 hour, washed again and incubated in p-
nitrophenyl phosphate for 30 minlltes and read at 410 nm, as described in Example 1. The
results are shown in Figure 9 and demoll~L~ate that LPS antigens from all nine species
representing four different orders of gram-negative bacteria compete effectively for IgG-PMB
binding to E. coli Olll:B4 LPS. These results show that IgG-PMB conjugates can be used to
15 detect and quantitate a LPS from a variety of bacterial species, and suggest that the IgG-PMB
conjugate will be therapeutically effective against a broad spectrum of gram-negative
or~ni~m~ and endotoxins.
EXAMPLE 16
Neutralization Of The In Vivo Effects Of Endotoxin By IgG-PMB
Endotoxin (LPS) can trigger a lethal reaction in vivo. In order to ~lettormine whether
IgG-PMB conjugate is capable of neutralizing the lethal effects of endotoxin, a well-
characterized and accepted murine model of endotoxic shock was lltili7~.1 [C. Galanos et al.,
Proc. Natl. Acad. Sci. USA 76:5939-5943 (1979).] The example involved: (a) determination
of a minimum lethal dose of endotoxin in galactosamine-sensitized mice; and (b)
neutralization of endotoxin lethality by premixture with Ig-PMB.
a) Dete, ."i"~tion of a ",illi",ll", lethal dose of endotoxin in galactosamine-
sensitized mice was performed by ~(lmini~tPring dirrerell~ doses of E. coli 0111 :B4 LPS to
C57Bl/6 mice that were co-~lmini~tered 20 mg of D-galactosamine-HCl in 400 ,ul of PBS.
WO 94/14437 PCT/US93l12381
2 ~ 8 ~
The latter compound is a specific hepatotoxic agent that increases the sensitivity of
experimental ~nimz~l~ to endotoxin several thousand-fold. [C. Galanos et al.~ Proc. Natl.
Acad. Sci. USA 76:5939-5943 (1979).] To accomplish this determination, 1-500 ng of E. coli
Olll:B4 LPS (List Biological Laboratories, Campbell, CA) in PBS was injected
5 intraperitoneally, along with 20 mg of D-galactosamine (Sigma). It was found that 10-25 ng
of endotoxin was usually sufficient to kill most or all mice within 24 hr. The variability in
endotoxin lethality may be related to the dirr~lellt ages of the mice used. Since 10 ng was the
minim~l effective lethal dose, this amount of LPS was utilized in neutralization experiments.
b) Neutralization of endotoxin lethality by plelllixlul~ with Ig-PMB was
10 performed by incubating 50 ng of E. coli Olll:B4 LPS with 5 mg of periodate conjugated
IgG-PMB (prepared as described in Example 14), or 5 mg of unconjugated control human
IgG (Sigma) and 100 mg D-galactosamine in PBS and injecting a portion of each mixture
intraperitoneally into C57BL/6 mice. The results are shown in Table 22. Survival was
~ ec~e-l 24 hours later.
TABLE 22
Neutralization Of Endotoxin Lethality By IgG-PMB (Therapeutic Prophylactic)
Tre~tmçnt Survivors/Total
1 mg Human IgG and 20 mg D-galactosamine 5/5
1 mg Human IgG, 10 ng LPS and 20 mg D-galactosamine 1/4
1 mg Periodate IgG-PMB, 10 ng LPS and 20 mg D-galactosamine 5/5
Since the number of ~nim~l~ used in this experiment was small, the trial was repeated
using: a) 12 mice in the control group treated with endotoxin, D-galactosamine, and normal
human IgG and b) 12 mice in the experimental group that received endotoxin~ D-
galactos~mine, and the periodate IgG-PMB. The per mouse dosage of each component was
25 the same as above and the experiment was repeated exactly as above. The results are shown
in Table 23.
76
WO 94tl4437 PCT/US93/12381
21~3~
TABLE 23
Neutralization Of Endotoxin Lethality By IgG-PMB
Tre~tm~nt Survivors/Total
1 mg Human IgG, 10 ng LPS and 20 mg D-galactosamine 0/12
1 mg Periodate IgG-PMB~ 10 ng LPS and 20 mg D-galactosamine 11/12
The results of these two trials prove that IgG-PMB neutralizes the lethal effect of
endotoxin in vivo and suggest that Ig-PMB conjugates will be useful in preventing or treating
sepsis due to gram-negative bacteria.
EXAMPLE 17
Prevention Of Endotoxin Lethality By
Prophylactic ~rlmini.ctration Of IgG-PMB Conjugate
In the previous example, the ability of IgG-PMB conjugate to neukalize endotoxinlethality in vivo was investig~te~l by mixing conjugate or control IgG with endotoxin and
~ mini~tering the mixture with D-galactosamine into mice. The results showed that the
15 conjugate neutralized the endotoxin. A more strenuous test of the ability of the conjugate to
neukalize endotoxin lethality is to ~flminicter the conjugate at a separate time and via a
s~ L~ route than that used to ~lminicter endotoxin. In addition, to demonstrate its
prophylactic value~ lower doses of conjugate were utili7~1 The Example involved the
inkavenous ~lminictration of IgG-PMB or control IgG followed 1 hr later by the
20 inkaperitoneal ~-lminictration of a lethal dose of endotoxin and D-galactosamine.
Twenty (20) CS7BL/6 mice weighing twenty (20) grams each were a-lmini~tto.red 200
llg (5 mice) or 400 ~lg (8 mice) of IgG-PMB conjugate (periodate conjugate prepared as in
Example 14) or 400 ~lg conkol human IgG (7 mice) in 100 ~LI of PBS through their tail vein.
Ninety (90) mimlte,c later, each mouse received 10 ng E. coli 0111 :B4 endotoxin and 20 mg
25 D-galactosamine in 200 111 of PBS ~lminictered hlLIdp~liLoneally. After 24 hrs, the number of
mice surviving in each group was recorded. The results are shown in Table 24.
WO 94/14437 PCT/US93/12381
.
8 6
TABLE 24
Prophylaxis Against Endotoxin In Challenge With IgG-PMB Conjugate
Tre~tment Survivors/Total
400 ,ug Human IgG, 10 ng Endotoxin and 20 mg D-galactosamine 0/7
200 ~lg IgG-PMB, 10 ng Endotoxin and 20 mg D-galactosamine 5/5
400 ~lg IgG-PMB, 10 ng Endotoxin and 20 mg D-galactosamine 8/8
The results show that a 10-20 mg/kg dose of IgG-PMB ~lmini.ctered intravenously is
sufficient to protect against a subsequent lethal challenge of endotoxin ~mini.~tered
intraperitoneally. These fintling.~ suggest that the IgG-PMB conjugate given prophylactically
10 will prevent endotoxin-mediated effects and that the conjugate is capable of neutralizing
endotoxin outside of the vascular conlp~Ll.,ent.
EXAMPLE 18
Preservation Of IgG Effector Functions In
IgG-PMB Conjugates: Fc Receptor Binding
One of the functions of IgG is to opsonize and facilitate clearance of org~ni~m.c,
toxins, antigens, etc. by phagocytic cells. In order to determine whether this ~lo~oelLy of IgG,
which is facilitated by the Fc region of the native molecule, remains intact in IgG conjugates
that have been prepared with SPDP or periodate, the binding of IgG-PMB to human
monocyte/macrophage cells was e~mined in a competition assay. This assay is similar to
20 that employed to e~mine the Fc receptor binding activity of hybrid recombinant antibody
fragments fused to cell surface viral receptors. [D.J. Capon el al., Nature 337:525-531
(1989); A. Traunecker e~ al., Nature, 339:68-70 (1989).] The example involved: (a)
ala~ion of a control conjugate of PMB to human albumin (a non-Fc receptor binding
human protein-PMB conjugate); and (b) assay of IgG-PMB conjugate binding to Fc receptors
25 of the human U937 monocyte/macrophage cell line.
WO 94/14437 PCT/USg3/12381
2151~86
a) In order to compare the specific properties of IgG-PMB conjugates with other
protein-PMB conjugates, human albumin was conjugated with PMB using the SPDP
chemistry of Example 7 (because albumin is not glycosylated, the periodate chemistry of
Example S was not applicable to albumin). Conjugation of albumin with PMB was carried
5 out in three steps similar to the scheme described in Example 7. The first step involved
derivatization of 10 mg of PMB in 50 mM sodium borate, 300 mM NaCl, pH 9.0 with 2:1
mg of SPDP dissolved in 50 ~Ll of dimethylsulfoxide for 30 minutes at room te~ eldlule.
The free cross-linker was removed on a 15 ml Swift desalting column as described in
Example 7.
Ten (10) mg of human serum albumin was derivatized with 1.2 mg of SPDP (in 2S ~11
DMSO), dissolved in 1 ml of 50 mM sodium borate, 300 mM NaCI, pH 9.0, and mixed for
30 minutes at room temperature. The free cross-linker was removed by gel filtration on a 15
ml Swift desalting column equilibrated with PBS-EDTA, pH 7.5 and the peak fractions
cont~ininp SPDP-albumin were collected, pooled and concentrated on a C~ lcp-30
15 concentrator. The pH of the sample was raised to 8.0 with 10 ~11 of 10N NaOH, and reduced
with 15.4 mg dithiothreitol dissolved in 200 ~11 of pyrogen-free water for 30 mimltes at room
temperature. The reclllce-l, derivatized albumin was purified by gel filtration on a 15 ml
rle~lting colurnn and concentrated on a Ct;llLIi~le~-30 concentrator.
The re(l~lce~l. derivatized albumin was conjugated with SPDP-PMB by mixing the two
20 solutions prepared above and incubating overnight at room t~;lllpt;lalule. The conjugate was
separated from SPDP-PMB by gel filtration on a 50 ml P-10 column.
b) IgG-PMB conjugate binding to Fc lec~Lol, of the human U937
monocyte/macrophage cell line was assayed in a manner similar to that described by Capon e~
al. [Nature 337:525-531 (1989).] First, a saturation curve of the binding of l25I-labelled
25 human IgG [the l25I-IgG stock concentration was 16 ~lg/ml = 1.07 x 10-7 M] (New England
Nuclear, Boston, MA) was performed by inc~lb~ting 1 x 10-8 M to 1 x 10-12 M l25I-IgG with 2
x 105 U937 cells in 0.5 ml of PBS cont~ining 2 mg/ml BSA and 0.1% sodium azide. The
cell suspensions were incubated for 60 minnte~ at 37C, centrifuged for 3 minutes at 1500 x g
and washed three times with incubation buffer. The cell pellets were then counted for
30 radioactivity with a Bioscan "Quick Count" benchtop radioisotope counter (Bioscan, Inc.,
79
Wo 94/14437 PCT/US93/12381
2~513~ `
Washington D.C.). The binding was found to saturate at I x 10-8 M ''sI-Ig so this
concentration was used for the competition assay described below.
For the competition experiment, a constant quantity of '75I-IgG (1 x 10-8 M) wasincubated with 2 x 105 U937 cells in 0.5 ml of PBS cont~ining 2 mg/ml BSA, 0.1% sodium
azide and varying concentrations of the unlabelled competitor proteins: human IgG, IgG-PMB
(SPDP), IgG-PMB (periodate), and human albumin-PMB from (a) above. The cells were
incubated, washed, and the amount of bound radioactive l75I-IgG was quantitated as described
above. In the absence of any of the human competitor proteins, 12?029 cpm of labelled IgG
was bound to the cells. The results of the competitor assay are plotted in Figure 10. It is
clear that human IgG and both IgG-PMB conjugates have similar binding ~.iop~llies to the
U937 cells in that all three compete comparably well at 10-8 M and 10-6 M. This result shows
that the modification of the IgG with SPDP and PMB or by periodate oxidation of the
carbohydrate side chains does not impair the ability of IgG to bind to Fc receptors. This
suggests that the conjugates can facilitate Fc receptor-mediated opsonization ofantigen/org~ni.sm.s by phagocytic cells. As expected, the human albumin-PMB exhibited no
con~ ilive binding activity at concentrations up to 10-6 (data not shown) and is therefore
unable to facilitate opsonization.
EXAMPLE 19
P,~p~dLion Of An Antibody-Antibiotic Conjugate With
Activity Against Gram-Positive Bacteria: IgG-Bacitracin
Gram-positive org~ni.sm.s are responsible for approximately one-third of sepsis cases.
It would be desirable to have IgG-antibiotic conjugates with activity against these org~ni~m.s.
To this end, conjugates were made between IgG and bacitracin and vancomycin, two surface-
active gram-positive antibiotics. The example involved: (a) periodate activation of IgG, and
25 (b) conjugation to bacitracin and vancomycin.
a) Periodate activation of IgG was carried out as described in Exarnple S(b), using
30 mg of human IgG and 50 mM sodium periodate in 1 ml of 50 mM NaPO4, pH 7.2 for 30
minutes at room temperature. The activated IgG was purified on a 15 ml Swift ~les~lting
column (Pierce) and the peak fractions pooled.
~ WO 94/14437 2 ~ 5 13 8 6 PCT/US93/12381
b) Conjugation to bacitracin and vancomycin was carried out by adding 18.6 mg
of bacitracin to 7.1 mg of activated IgG and 19.7 mg of vancomycin to 7.1 mg of activated
IgG and each solution was incubated overnight at 4C. The mixtures were then clarified by
centrifugation to remove any precipitates formed during incubation. The reaction mixtures
5 were adjusted to pH 6.5 with 1.0 N HCl, and 10 ~l of a NaCNBH3 solution (10 mg/ml) was
added and incubated for 4 hours at room temperature. The conjugate was then purified on a
15 ml Swift desalting column equilibrated in PBS-EDTA, pH 7.2.
EXAMPLE 20
Antibacterial Activity Of IgG-
Antibiotic Conjugate On Gram-Positive Bacteria
To deterrnine if the conjugates prepared in Example 19 possessed anti-bacterial
activity, the MIC and MBC of these conjugates was assayed against Staphylococcusepidermidis obtained from Dr. Edward Balish, Department of Medical Microbiology,University of Wisconsin. The strain is gram- positive, DNase negative, mannitol salt
15 negative, coagulase negative and novobiocin sensitive. The example involved:
(a) ~ Lion of an S. epidermidis inoculum; and (b) determination of the MIC and MBC of
free and conjugated antibiotics.
a) Pl~aLion of an S. epidermidis inoculum was carried out by plating org~ni~m~
on TSA overnight at 37C, and suspending bacteria at 5 x 105 org;~ni~m~/ml in TSB.
b) Det~rmin~ion of the MIC and MBC of the free and conjugated antibiotics was
carried out by mixing 0.5 ml of the S. epidermidis inoculum with 0.5 ml of solutions
co~ i"~; 0.3125 to 10 ~Lg/ml of free antibiotic or 12.5 to 250 ~lg/ml of each conjugate. The
MIC was defined as the minimllm concentration of the compounds that inhibited visible
growth and the MBC defined as the concentration that killed 99.9% or more of the initial
org~ni.cm~ present in the inoculum (measured by plating those solutions that do not exhibit
visible growth; see Example 10). The results are shown in Table 25.
81
WO 94/14437 PCT/US93/123~1
2~ ~1386
TABLE 25
MIC And MBC Of Free And IgG-Conjugated Antibiotics On S. epidermidis
Compound MIC(~lg/ml) MBC (~Lg/ml)
Bacitracin 25 50
S IgG-Bacitracin 125 250
Vancomycin 1.25 2.5
IgG-Vancomycin >50 N.D.
The results show that the IgG-bacitracin conjugate was indeed active against S.
epidermidis and suggest that this compound could be useful in the prevention and treatment of
10 gram-positive sepsis.
EXAMPLE 21
Treatment Of Persons Susceptible To Gram-Negative
Sepsis And Endotoxemia With An Antibody-Antibiotic Conjugate
As noted earlier, studies have suggested a causal relationship between a person's
15 humoral immllne status and the susceptibility to gram-negative infections. The present
invention contemplates screening for patients having a poor immllne status for determining a
subpopulation having the greatest need for antibodiotics. The example involves: (a) assay of
patient total IgG and IgM levels, (b) assay of patient endotoxin core antigen-specific IgG and
IgM levels; (c) comparison of patient immunoglobulin levels to healthy normal controls; (d)
20 a-lminictration of immllnoglobulin and/or immunoglobulin-antibiotic conjugate to patients with
significant deficiencies in total or core antigen-specific immunoglobulin levels.
(a) Assay of patient total IgG and IgM levels is perforrned by nephelometry using
the Beckman Automated immunochemistry system (Beckman Instruments, Inc., Brea, CA) as
described by Stoll et al., Serodiagnosis and Immunotherapy 1:21-31 (1987).
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(b) Assay of endotoxin in core-antigen specific IgG and IgM levels is performed
by ELISA. Plasma or sera are diluted and the level of binding of different sample dilutions
to purified E. coli J5 endotoxin and Salmonella minnesota R595 endotoxin are quantitated and
compared with known standards of purified anti-endotoxin antibodies. ~B.J. Stoll et al.,
Serodiagnosis and Immunotherapy 1:21-31 (1987); and M. Pollack et al., J. Clin. Invest.
72:1874-1881 (1983).]
c) Comparison of patient irnmunoglobulin levels to healthy controls is performedby analyzing the total IgG and IgM levels (as mg/ml of sample) in the patient vs. the control
group and the endotoxin core antigen-specific IgG and IgM levels (as ~g/ml of sample)
between these same two groups. Patients with ~ 80% of the normal control level of total IgG
and/or S 60% of the normal control level of endotoxin core antigen-specific IgG and IgM are
defined as at risk for gram-negative infection and endotoxemia.
d) Atlmini~tration of immunoglobulin and/or immunoglobulin antibiotic conjugate
to patients with significant deficiencies in total or core antigen-specific immunoglobulin levels
is carried out to restore normal or near normal total and antigen-specific hDoral defenses.
To restore normal IgG levels, a 3% solution of intravenously injectable immunoglobulin
(available from Sandoz Fors~ .-gsin.~tit~t, Vienna, Auskia; Hyland Therapeutics, Duarte, CA;
or Cutter Laboratories, Berkeley, CA) is ~-lmini.ctPred twice daily until immunoglobulin levels
rise to within 10% of normal levels.
Because the IgG-PMB conjugates of the present invention comprise a population ofantibody molecules all of which are capable of binding to endotoxin, much less IgG-PMB
conjugate is required than total IgG to restore or increase levels antigen-specific antibody. A
single intravenous dose con~i~ting of 1-20 mg of IgG-PMB conjugate per kg of body weight
is ~lmini~tered to restore endotoxin-specific antibody levels to 2 100% of normal levels.
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EXAMPLE 22
Treatment Of Persons Susceptible To Gram-Negative Sepsis~ Endotoxemia,
And Gram-Positive Sepsis With A Cocktail Of Antibody-Antibiotic Conjugates
Since there is a causal relationship between a person's humoral status and their5 susceptibility to infection, there is also a need to restore antibody levels against gram-positive
org~ni.cm~ as well as the levels against gram-negative org~ni~m~ and endotoxin. This is
achieved by ~(lmini.~tration of a cocktail of antibody-antibiotic conjugates with activity against
both classes of bacteria as well as endotoxin. The example involves: (a) identification of
persons at risk of infection; and (b) ~lmini~tration of a cocktail of antibody-antibiotic
l O conjugates and, if necessary, total pooled human immunoglobulin to restore antigen-specific
and total immunoglobulin levels.
a) Identification of persons at risk of infection is carried out by the means defined
in Example 21.
b) A~lmini~tration of a cocktail of antibody-antibiotic conjugates and, if necessary,
15 total pooled hDan immnnnglobulin to restore antigen-specific and total immunoglobulin
levels is carried out by injecting a single intravenous dose of IgG-PMB (1-20 mg/kg) and a
single intravenous dose of IgG-bacitracin conjugate (1-20 mg/kg) to increase the levels of
grarn-negative and gram-positive-reactive antibodies, respectively. If total immlmoglobulin
levels are also < 80% of normal, a 3% solution of intravenously injectable immunoglobulin
20 (available from Sandoz Forsçllllngin~titut~ Vienna, Austria; Hyland Therapeutics, Duarte, CA;
or Cuter Laboratories, Berkeley, CA) is ~rlmini~tered twice daily until immllnnglobulin levels
rise to within 10% of normal levels.
EXAMPLE 23
Long-Term Prophylaxis Against Endotoxin Lethality By IgG-PMB Conjugates
The long-term prophylactic effect of the IgG-PMB conjugate was exarnined in the
D-galactosamine-sensitized mouse model. [C. Galanos et al., Proc. Natl. Acad. Sci.
76:5939-5943 (1979); V. Lehmann et al.? J. Exp. Med. 165:657-663 (1987); and
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M.A. Freudenberg and C. Galanos, Infect. Imrnun. 56:1352-1357 (1988).] One mg of human
IgG-PMB conjugate in PBS (prepared as described in Example 14) was given intravenously to
each of 10 male C57Bl/6 mice. Ten control mice received I mg of unconjugated human IgG
in PBS, again intravenously. Both the conjugate and control IgG solutions were at 8 mg/ml.
S the injection volumes were therefore 125 ,ul/mouse.
The in vivo experiments described in Examples 16 and 17 showed that as little as 25
,ug of the IgG-PMB conjugate could provide complete protection when ~imini~teredintravenously I hour prior to endotoxin challenge. Here, we investig~tecl the protective
window at 24 hours.
Twenty-four hours after the ~(lmini~tration of PMB-conjugated or control IgG, both
groups of mice were challenged intraperitoneally with a lethal dose of E. coli 0111:~4
endotoxin (# 201; List Biological Laboratories, Campbell~ CA), prepared as described below.
A 1 mg/ml stock solution of endotoxin was sonicated for 2 minutes in a Branson 2000 water
bath sonicator and diluted 100-fold in PBS to make a 10 ng/~l working solution. Two
15 hundred and forty mg of D-galactosamine hydrochloride (# G-1639; Sigma Chemical Co., St.
Louis, MO) was weighed into 2 siliconized Reacti-vials (Pierce) and dissolved in 2.4 ml of
PBS cont~ininp 0.1 mg/ml bovine serum albumin (BSA) as a carrier protein. Twelve ~ll of
the 10 ng/,ul endotoxin solution (120 ng) was added to each vial, and the solutions were
mixed for 15 minllf.~s at room tt;l~ Lule. Each vial contained enough solution for twelve
20 200 ,ul injectlons, con~i~ting of 10 ng endotoxin and 20 mg galactosamine/injection. Each
mouse in both groups was injected intraperitoneally with 200 ,ul of the mixture. The mice
were given food and water ad libitum, and observed for 24 hours, using mortality as the
endpoint. The results were analyzed by Fisher's exact method for estim~ting probabilities [F.
Mosteller, et al., in Probabilty With Statistical Applications, Addison Wesley, Re~tlin~, MA
25 (1970)]; significant protection was defined as a p value <0.05 when the experimental and
control groups were compared.
The results of the prophylactic study are summarized in Table 26.
wo 94/14437 PCT/US93112381
.
2~13~6
TABLE 26
Group # Survivors/Total % Survival p Value
Control 2/10 20 --
Experimental 7/10 70 0.03215
SThese results show that the IgG-PMB conjugate can be given intravenously as a
prophylactic, and significant protection from endotoxin lethality can be obtained for at least
24 hours after ~mini~tration of the conjugate.
EXAMPLE 24
Det~rmin~tion Of The Relative Half-Life
10Of PMB-HIgG Conjugate And HIgG In Rabbits
This example describes experiments to determine if there was any effect on the
half-life of HIgG in rabbits when conjugated to PMB. The pharmacokinetic study was
conducted using male New Zealand White rabbits (10 Ib, 12 months old). Two rabbits each
received a single dose of 3 mg of PMB-HIgG conjugate in 10 mM phosphate buffer (pH 7.1 )
5 CO~ ill;llg 150 mM sodium chloride intravenously on day 0. At the same time, two control
rabbits received intravenous injections of 3 mg of HIgG in the same buffer. Both the test
samples and control samples were tested and found to be pyrogen-free. Rabbits were bled at
one hour and 5 hours after the initial injection and then at days 1, 2, 3, 4, 7, 10, and 14 after
the initial injection. Serum samples were collected and stored at -70C until tested for the
20 presence of HIgG.
A sandwich ELISA was developed in order to detect the presence of HIgG in rabbitserum samples. Each well of a microtiter plate (Corning) was coated with 100 ~1 of a
solution cont~ining 25 ~lg/ml of goat-anti human IgG (Sigma) in 50 mM carbonate buffer, pH
9.5. Af~er an overnight incubation at 4C, the coating solutions were removed and all wells ~.
25 were washed three times with PBS-Tween-20 (.05% Tween-20 in PBS). The rem~ining
antigen binding sites were blocked by the addition of PBS cont~ining IO mg/ml BSA (Sigma)
for one hour at 37C.
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The test serum samples, which were stored at -70C, were thawed just prior to assay
and diluted 1:10 in PBS-Tween-20 Cont~ining 0.1% BSA. All samples were added (200
,ul/well) as duplicate aliquots to wells of the microtiter plate. Negative control wells were
prepared by adding 200 ~l/well of 1:10 diluted normal rabbit serum in the same diluent as
used with the test serum samples. As a positive control, normal HIgG was diluted in the
same diluent at 20 ~g/ml and subsequently underwent eight serial 1:4 dilutions up to 0.00031
~lg/ml. The corresponding O.D. values were used to generate a standard curve from which
corresponding HIgG levels from test serum samples were determined. Plates were incubated
at 37C for I hour and washed three times in PBS-Tween-20. Alkaline phosphatase
conjugated goat anti-human IgG (Sigma), diluted 1:500 in 0.1% BSA in PBS-Tween-20 was
added to the wells and incubated at 37C for one hour. After washing the wells four times
with PBS-T, 100 ,ul of 1 mg/ml p-nitrophenylphosphate (Sigma) in 50 mM Na.CO3, pH 9.5,
and 1 mM MgCI2 was added to all wells. Plates were shielded from light and allowed to
develop at room temperature for 20-30 minut~c Absoll,ance at 410 nm was determined using
a Dynatech MR 700 microplate reader.
The absorbances of duplicate wells were averaged and corrected for background bysubtracting the absorbance of the blank wells, which contained only conjugate and substrate.
A standard curve of absorbance versus log concentration of HIgG was plotted. Absorbances
from test samples were qll~ntified from the linear portion of the standard curve.
The serum clearance curve for PMB-HIgG (R3 and R4)and HIgG (Rl and R2) are
shown in Figures 11 and 12. The absorbance at 410 nm which directly corresponds to the
co,lcen~ ion of HIgG is plotted against days in Figure 11. Figure 12 shows a graph of HIgG
in ,~Lg/ml serum over time. From both Figures 11 and 12, it is clear that the serum half life of
PMB-HIgG is similar to that of unconjugated human IgG. Since the half-life of human IgG
in humans is on the order of 21 days, these experiments suggest that the conjugate half-life
will be long. Therefore, in this Example, we have demonstrated that active conjugate is still
detectable in rabbit sera two weeks after conjugate ~(lmini~tration.
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2~5~ ~g~ --
EXAMPLE 25
Detection Of LPS-Binding Of The PMB-HlgG
Conjugate After Two Weeks Of Circulation In Rabbits
This example describes an experiment to determine if.PMB-HIgG LPS-binding activity
is still present after two weeks of circulation in rabbits. The study was conducted using male
New Zealand White rabbits (10 Ib, 12 months old). Two rabbits each received a single dose
of 3 mg of PMB-HI~G conjugate in 50 mM phosphate buffer cont~ining 150 mM sodiumchloride intravenously on day 0. At the sarne time, two control rabbits received intravenous
injections of 3 mg of HIgG in the same buffer. Both the test samples and control samples
were tested and found to be pyrogen-free. Rabbits were bled at one hour and 5 hours after
the initial injection and then at days 1, 2, 3, 4, 7, 10, and 14 after the initial injection. Serum
samples were collected and stored at -70C until tested for the activity of PMB-HIgG
conjugate.
In order to detect the activity of PMB-HIgG conjugate from rabbit serum (i.e., ability
to bind to LPS), a simple indirect binding assay was lltili7P~ Each well of a 96-well
microtiter plate (Corning) was coated with 100 ~11 of a 20 ~lg/ml solution of LPS from E.coli
O11 :B4 (Sigma) in PBS. Control wells were coated with PBS only (no LPS). After an
overnight inrllb~tion at 4C, the coating solutions were removed and all wells were washed 3
times with PBS-Tween-20. The rem~ining antigen binding sites were blocked by the addition
of PBS co,.~ g 10 mg/ml BSA (Sigma, tissue culture grade) for 1 hour at 37C. The
blocking solution was removed and test rabbit serum samples diluted 1:10 in PBS-Tween-20
were added. As a positive control PMB-HIgG conjugate was also diluted in 10% normal
rabbit serum and added to the wells. Samples wcre incubated in duplicate at 37C for 1 hour
and the plates were washed three times with PBS-Tween-20.
In order to detect bound antibodies, the wells were inc~lb~te~l with 100 ~l of a 1:500
dilution of goat anti-human IgG-~lk~line phosph~t~e labeled antibody (Sigma) and incubated
for 1 hour at 37C. After removing the secondary antibody solutions, the wells were washed
4 times with PBS-Tween-20. Substrate [p-nitrophenylphophate (Sigma)] at 1 mg/ml in 50
mM Na7CO3, and 10 mM MgCI7 was added to each well. The color developed after 15-20
minlltes of incubation at room L~;lllpelaLuL~ was measured at 410 nm using a Dynatech MR700
microplate reader
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WO 94114437 PCT/US93/12381
2:1~1386
The results of the LPS binding assay are as shown in Table 27. The conjugate from
rabbit sera collected on day 14 bound to the LPS coated wells indicating that the conjugate
was still active after circulating for two weeks in rabbits.
TABLE 27
S Binding Of PMB-HIgG Conjugate From Rabbit Serum To LPS
Absorbance At 410 nrn
Dilution Of Sera From Sera From Experimental
Bleeding Date
Anti-Serum Control Rabbits Group
1:10 0.016 0.166 Day 14
EXAMPLE 26
PMB-IgG Conjugates Do Not Elicit An lmmllne
Response When A~lmini~tered Intravenously Into Rabbits
This example describes an experiment to determine if anti-PMB antibodies are elicited
in rabbits by conjugate ~mini~tration. Two rabbits were each given 3 mg of PMB-HIgG
conjugate intravenously on day 0. These rabbits received additional injections (boosts) at 2
weeks? 4 weeks and 7 weeks. As a control, 2 rabbits each received 3 mg of HIgG alone at
the same scheduled day and time as with the experimental group. All rabbits were bled every
two weeks after receiving either conjugate or IgG alone. Sera were collected and stored at
-70C until tested for anti-PMB antibodies.
In order to detect anti-PMB antibodies in rabbit serum, a simple indirect binding assay
was developed. Each well of a 96-well microtiter plate (Corning) was coated with 100 ~Ll of
a 200 ,ug/ml solution of PMB (Sigma) in endotoxin-free PBS. Control wells were coated
with PBS only (no PMB). After an overnight incubation at 4C, the coating solutions were
r removed and all wells were washed 3 times with endotoxin-free PBS-Tween-20. The
rem~ininp~ antigen binding sites were blocked by the addition of PBS col~t~;"il-~ 10 mg/ml
BSA (Sigma, tissue culture grade) for 1 hour at 37C. The blocking solution was removed
and test rabbit serum samples diluted in 2% normal rabbit serum at dilutions of 1:10, 1:100,
1:1000 and 1:10,000 were added. A positive control antiserum (chicken anti-PMB
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2~51386
immunoglobulin, Ophidian Pharmaceuticals Inc., Madison, WI) was also diluted as for the test
rabbit serum samples. Samples were incubated in duplicate at 37C for 1 hour. Following
this incubation, the plates were washed three times with PBS-Tween-20.
In order to detect bound antibodies, the wells incubated with rabbit serum were
5 incubated with 100 ,ul of a 1:500 dilution of goat anti-rabbit IgG-alkaline phosph~t~se labeled
antibody (Sigma) and the wells incubated with chicken antibody were incubated with 100 ~L]
of 1 :500 dilution of goat anti-chicken IgG (whole molecule)-~lk~line phosphatase conjugate
(Sigma) for 1 hour at 37C. After removing the secondary antibody solutions, the wells were
washed 4 times with PBS-Tween-20 and p-nitrophenylphosphate (Sigma) at 1 mg/ml in 50
10 mM Na~CO3, 10 mM MgCl~ was added to each well. The color developed after 15-20
minntes of incubation at room temperature was measured at 410 nm using a Dynatech MR700
microplate reader.
The results in Table 28 show that the positive control antibody, as expected, bound to
PMB. This validates that the design of ELISA is capable of detecting PMB-binding15 antibodies. The results in Table 29 (shown as A4l0 readings of duplicate samples) indicate
that none of the rabbit serum samples bound to PMB, indicating the absence of anti-PMB
antibodies. These results demonstrate that PMB is not immnnQgenic, even on an heterologous
protein carrier with repeated injections when given intravenously.
The lack of immllnc)genicity of this peptide may be related to its D-amino acid
20 content, as these residues may not be recognized by the immune system.
TABLE 2X
Binding Of Chicken Antibodies To PMB
Abso~ ce At 410 nm
Dilution Of Antibody Preimmllne Egg Anti-PMB Egg
Yolk Antibodies Yolk Antibodies ,~
1 :10 0.149 1.741
1: 100 0.083 1.732
1: 1,000 0.026 1.700
1 :10,000 0.015 0.686
1:100,000 0.006 0.100
WO 94/14437 PCT/US93112381
~151386
EXAMPLE 27
IgG PMB Is Not Toxic
In order to investigate the safety with which IgG-PMB conjugate can be lltili7~.1 a
toxicity study was performed. Female Sprague-Dawley rats (Harlan Sprague- Dawley)
5 weighing 250-300 g were given 2 relatively high doses of conjugate (approximately 16
mg/kg) intravenously. Serum samples, taken at various times during the study. and major
organs were then analyzed for any indication of pathology.
TABLE 29
Binding Of Rabbit Antiserum To PMB Coated Wells As Determined By ELISA
Ab~nballcc At 410 nm
10Rabbit Dilution of BleedingDates
IdentificationAntiserum
.çWeek 2Week 4 Week 7 Week 9
Rabbit #1 1:10 0.000 0.005 0.004 0.015 0.018
Control 1: 100 0.004 0.008 0.000 0.001 0.005
Group 1: 1,000 0.008 0.009 0.000 0.000 0.004
153 mg HlgG 1:10,000 0.012 0.012 0.004 0.004 0.002
Rabbit #2 1:10 -- 0.000 0.003 0.013 0.005
Control 1: 100 -- 0.009 0.004 0.009 0.000
Group 1: 1,000 -- 0.014 0.002 0.005 0.000
3 mg HlgG 1:10,000 -- 0.012 0.000 0.009 0.001
20Rabbit #3 1:10 0.013 0.004 0.105 0.111 0.026
E~.c.i~c~ l 1:100 0.010 0.006 0.013 0.012 0.000
Group 3 mg 1:1,000 0.008 0.006 0.002 0.007 0.000
PMB-HlgG 1:10,000 0.007 0.007 0.000 0.004 0.002
Rabbit #4 1:10 -- 0.000 0.061 0.039 0.001
25Experimental1: 100 -- 0.009 0.006 0.007 0.003
Group 3 mg 1:1,000 -- 0.007 0.007 0.007 0.001
PMB-HlgG 1:10,000 -- 0.000 0.001 0.007 0.005
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Ig4437 PCT/US93/12381
The study consisted of 5 groups, 3 rats per group. After a 7 day acclimation period,
rats in groups 1, 2, and 3 received intravenous injections of 4 mg of rat IgG conjugated to
PMB (see (E) below for conjugation of PMB to rat IgG) on day 0 and again on day 2; group
4 rats received 4 mg of unconjugated rat IgG intravenously on day 0 (IgG control); and group
5 5 rats served as a normal control (no injection of either conjugate nor normal rat IgG). The
rats were bled by cardiac puncture and sacrificed for organ pathology as indicated in Table
30.
TABLE 30
Study Design
Group Day O Day 2 Day 5 Day 7 Day 14
Bleed; Remove
4 mg IgG-PMB,4 mg IgG-PMB
Kidney, Liver,
I.V. I.V.
Spleen
Bleed; Remove
4 mg IgG-PMB,4 mg IgG-PMB, Kidney, Live~,
I.V. I.V.
Spleen
4 mg IgG-PMB,4 mg IgG-PMB,
3 BleedBleed
l.V. I.V.
4 mg Rat IgG, Bleed; Remove
4 .I.V. (IgG Kidney, Liver,
Control) Spleen
Bleed; Remove
Normal Control Kidney, Liver,
Spleen
Tmme~ tely following cardiac puncture, blood smears (2 slides/rat) were prepared and
stained with Diff-Quik (Baxter Healthcare, McGaw Park, IL) for white blood cell differential
counts. The rem~inin~ blood (2-7 mls) was allowed to clot at 2-8C. The clot was pelleted
by centrifugation at 2000 rpm for 10 minl-tes, and the serurn was removed and frozen at
20 -70C in 2 aliquots for blood chemi~try analysis (SMAC 12) and measurement of complement
activation by the CH50 EZ Complement assay (Diamedix Corporation, Miami, FL). The organs
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of interest (kidney, liver, and spleen) were removed from each rat as indicated in Table 30,
and fixed imrnediately in phosphate buffered formalin (50 mM sodium phosphate, 10%
formaldehyde) until sections were made for histopathology slides.
A. Biochemical Serum Analysis For Liver And Kidney Function
All rat serum samples (18 total: 3 from groups 1, 2, 4~ and 5; and 6 from group 3)
were analyzed on the DuPont Dimension AR (DuPont Co., Wilmington, DE) for the
following 12 tests (SMAC 12): glucose, blood urea nitrogen (BUN), cle~ P, uric acid,
calcium, alburnin, total protein, cholesterol, total bilirubin, ~Ik~line phosphatase, aspartate
transferase (SGOT/AST), and lactate dehydrogenase (LDH). The values for each group were
averaged (Table 31) and the ~xl~c;l;lllental groups (1, 2, and 3) were compared with the
control groups (4 and 5) to detect any significant differences. The laboratory results were
also compared to the normal ranges for each assay, deterrnined by analyzing laboratory data
for 20 female Sprague-Dawley rats (data provided by Harlan Sprague-Dawley).
The standard laboratory tests for liver disease include measurement of serum levels of
bilirubin, AST, alkaline phosph~t~e, LDH, albumin, and, to a lesser extent, glucose. Kidney
function can be ~e~ed by measuring plasma levels of urea, ~ P, and calcium. [J. F.
Zilva, P.R. Pannall, Clinical Chemistry in Diagnosis and Treatment, Yearbook Medical
Publishers, Chicago, IL (1984).] With the exception of the lactate dehydrogenase value
(LDH), which will be tli~c~ e~l below, Table 31 shows no significant differences when the
values for the ~ ,.hllental groups 1, 2, and 3 are co~ )~ed with the control groups 4 and 5.
Furthermore, all the values are within or close to the normal ranges for each assay for this
strain of rat.
The values for LDH vary considerably from group to group, and most of the valuesalso exceed the norrnal range for rat serum LDH (data from Harlan Sprague-Dawley).
Lactate dehydrogenase is found in high concentrations in the liver, heart, skeletal muscle,
brain, kidney and in erythrocytes. Elevated values of particular isozymes can indicate liver or
" cardiac muscle damage. however in this study the control rats also show elevated LDH values,
suggesting the elevated LDH values are not associated with the IgG-PMB conjugate.
Hemolysis, which may have occurred in vitro (as the blood samples were being drawn, or if
the serum was not separated from the blood cells soon enough), also increases serum LDH
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2 ~ 8 ~
values (J. F. Zilva and P. R. Pannall, Clinical Chemistry in Diagnosis and Treatment, supra) .
and may explain the elevated values in this study.
It is also worth noting that the measurements for serum samples drawn on day 5 (3
days after the second injection), day 7 (5 days after the second injection) and day 14 (12 days
5 after ~he second injection) show no significant differences that can be attributed to the
~lmini.~tration of the IgG-PMB conjugate. In other words~ the day S, 7 and 14 values for all
of the serum components measured are ~vithin or close to their respective norrnal ranges
(except LDH) and show little or no significant change over time~ as would be expected if the
conjugate brought about any acute changes in the condition of the test rats.
TABLE 31
Blood Chemistry Analysis*
Group 1Group 2Group 3Group 3Group 4Group 5
Test
Day 5 Day 14 Day 7 Day 14 Day 14 Day 14
Glucose (mg/dl) 121 111 135 92 100 85
BUN (mg/dl) 16 19 15 16 19 21
15Creatinine (mg/dl) 0.2 0.1 0.2 0.1 0.1 0.1
Uric Acid (mg/dl) 2.4 3.0 2.2 3.4 2.8 2.8
Calcium (mg/dl) 10.2 9.9 9.9 9.7 9.6 9.8
Albumin (g/dl) 1.3 1.4 1.5 1.4 1.4 1.4
Total Protein (g/dl) 5.6 5.8 5.9 6.0 5.8 6.0
20Cholesterol (mg/dl) 79 81 81 82 83 80
Total Bilirubin (mg/dl) 0.1 0.2 0.1 0.1 0.1 0.1
Alkaline Pho~ e (U/L) 112 100 99 112 101 111
SGOT/AST (U/L) 194 203 180 215 362 207
LDH (U/L) 880 3083 939 3333 2830 2660
25 * The Value For Each Measurement Rel),ese~ The Average For All 3 Rats In
Each Group.
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B. White Blood Cell Differential
Blood smears from each rat were examined under oil immersion (lOOOX) to determine
the white blood cell differential. At least one hundred white blood cells were counted on
each slide~ and the percentages of each cell type (Iymphocyte, monocyte, neutrophil,
5 eosinophil, and basophil) were calculated to determine the white blood cell differential for
each rat. The differentials for rats in each group were averaged and dirr~lclllials from groups
1, 2, and 3 were compared with the dirrer~ ials from groups 4 and 5, to detect any
significant differences in white blood cell populations. In addition, the red blood cells on
each slide were examined for morphology, and a crude estim~te of the nurnber of platelets
10 present was made. The results of the differential cell counts are summarized in Table 32.
TABLE 32
White Blood Cell Differentials*
Group 1 Group 2 Group 3 Group 3 Group 4 Group 5
Cell Type
Day 5 Day 14 Day 7 Day 14 Day 14 Day 14
Lymphocytes 80 84 81 86 89 84
15Monocytes 4 7 1 1 8 5 7
Neutrophils 16 8 6 4 3 6
Eosinophils -- 1 2 2 3 2
Basophils -~
* Numbers Given R~plesellt The Percentage For Each Cell Type (Mean Values
for Each Group).
Table 32 shows no significant differences in the percentages of each cell type from
group to group. The dirrerc;-llials do show some variation from the normal reference values
for rats obtained from Harlan Sprague-Dawley (about 5-10% more Iymphocytes and 5-10 %
fewer neutrophils than expected), however this is found in both the normal control and
25 ~t;l;.l.ental groups, suggesting this finding is not related to the ~tlrnini~tration of the
WO 94/14437 PCT/US93/12381
IgG-PMB conjugate. The red blood cell morphology appeared normal, and platelets were
abundant on all slides examined.
C. Organ Histopathology
The organs of interest were removed and fixed in phosphate buffered formaldehyde.
5 Sections were made as described below and stained with hematoxylin and eosin.
Kidney: Full length mid-longitudinal section through center
Liver: Transverse section through hepatic lobule
Spleen: Transverse section
The slides were examined for organ pathology and no abnormalities were found.
D. Analysis Of Serum Complement Activity
Immunoglobulin and immlln~globulin complexes have the potential to activate the
complement system. Complement activation of this type, mediated by IgG-PMB conjugates.
would exacerbate the infl~mm~tory response to endotoxemia or bacteremia. In addition,
inhibition of normal complement function would impair complement-m~ te-l host defense
15 mech~ni~m~. In this example, the in vivo effect of IgG-PMB conjugate on serum complement
activity was investig~teA
Rat serum samples were analyzed for total hemolytic complement activity (CH50) using
the EZ Complement CHso Assay (Diamedix Corp., Miami, FL). In order to determine the
effect of the conjugate on complement activity, the resulting CH50 values obtained from
20 u~ e~d control rats were compared to the CHso values obtained from the IgG and IgG-PMB
conjugate-treated rats.
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TABLE 33
Analysis Of Serum Complement Activity*
GroupTreatment (I.V.) Day 5 Day 7 Day 14
4 mg Conj. on Days 0,2 303.8
2 4 mg Conj. on Days 0,2 -- -- 312.2
3 4 mg Conj. on Days 0,2 -- 290.4 299.8
4 4 mg Rat IgG on Day 0 -- -- 338.6
Untreated Control -- -- 298.2
* Each Measurement Represents The Mean CH50 Value Determined For The 3
Rats In Each Group.
Referring to Table 33 above, there were no significant differences in the CH50 values
between any of the groups tested. If IgG-PMB conjugate-merli~t~cl complement activation
was occurring in vivo, this effect would have been reflected as a decrease in the CH50 values
of the conjugate-treated rats (groups 1, 2, and 3), as compared against the untreated control
15 rats (group 5), due to depletion of complement components in the treated ~nim~l~. Similarly,
inh~bition of normal complement function would have been indicated by a decrease in CH50
values in the conjugate-treated groups as compared with the untreated control group.
Unconjugated IgG was also found to have no effect on serum complement activity (group 4).
Therefore, these results show that intravenous ~lmini~tration of IgG-PMB conjugate has no
20 adverse effect on serum complement activity.
E. Conjugation Of PMB Rat IgG Using Periodate Oxidation Of
IgG In NaPO4
PMB was conjugated to rat IgG using periodate oxidation of IgG. This involved a)periodate oxidation of IgG in phosphate buffer followed by conjugation of PMB to the
25 periodate-oxidized IgG.
a) Periodate oxidation of IgG in phosphate buffer was achieved by dissolving 25
mg rat IgG (Sigma) in 1 ml of 50 mM NaPO4, pH 7.2 buffer and adding 10.7 mg of sodium
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metaperiodate (final concentration 50 mM ). After 30 minutes of incubation at room
temperature with gentle vortexing every S minutes, the periodate was removed by gel
filtration on a 15 ml Swift desalting column (Pierce) equilibrated with 50 mM NaPO4, pH 7.2
buffer. The peak fractions cont~ining highest amount of antibody as monitored by A,80
5 absorbance were pooled.
b) Conjugation of periodate-oxidized IgG with PMB was carried out by adding 75
mg PMB to oxidized IgG at 4C overnight with gentle ~h~king followed by reduction with
0.1 mg/ml of NaBH3CN in 20 mM NaPO4, pH 6.5 for 2-3 hours at room temperature. The
PMB-IgG was separated from the rest of the reaction products by gel filtration on a 15 ml
10 Swift desalting column equilibrated with 50 mM phosphate cont~ining 150 mM NaCL pH 7.5
(PBS).
The activity of PMB-rat IgG conjugate was detçnninç~ by LPS binding assay as
described previously. Results in Table 34 indicate that PMB-rat IgG conjugate had excellent
LPS binding activity.
TABLE 34
LPS Binding Activity Of PMB-Rat IgG Conjugate As Determin~-l By ELISA (A28o)
Conjugate IgG ConcentrationLPS-Coated Wells No Antigen Wells
100 ~g/ml 1.756 0.110
20,ug/ml 1.756 0.079
20 4 llg/ml 1.737 0.017
0.8,ug/ml 1.521 0.036
0.16 ,ug/ml 0.998 0.021
0.032 ~lg/ml 0.506 0.016
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EXAMPLE 28
Demonstration Of Opsonophagocytic Activity Of IgG-PMB Conjugates
Opsonic IgG class antibodies mediate an important immune effector function by
enhancing the phagocytic clearance of extracellular bacteria [Raff, e~ al., J. Infect. Dis.
5 163:346-354 (1991).] In this way, opsonic IgG plays a critical role in host defense
meçh~ni~m.~ against bacterial pathogens. [Rozenberg-Arska, et al. J. Med. Microbiol.
22:143-149 (1991).] The purpose of this example was to investigate whether the IgG
component of IgG-PMB conjugates retains this important effector function. This was done by
~c~es.cing whether the pre-treatment of E. coli org~ni.~m~ with IgG-PMB conjugate potentiates
10 phagocytic uptake (opsonophagocytosis) by the human monocytic cell line U937.Opsonophagocytosis assays provide a useful means by which the potential therapeutic efficacy
of immunoglobulin ple~,aldlions, used for the tre~tment of bacterial infection, can be assessed.
[Hill, et al. Am. J. Med. 61-66 (1984).] This example involved (a) Assay for
opsonophagocytic activity of IgG-PMB conjugate, and (b) Determin~tion of the minimllm
15 effective concentration of IgG-PMB conjugate.
A. Assay For Opsonophagocytic Activity Of IgG-PMB
Conjugate
Opsonophagocytic activity of IgG-PMB conjugates was measured using an assay
procedure which was modified from published methods. [Gemmell, et al., J. Clin. Invest.
20 67:1249-1256 (1981) and Bohnsack, et al., J. Tmmllnol. 143(10):3338-3342 (1989).] E. coli
strain HB101 was grown for ~ oxhnately 20 hours at 37C on TSA ~BBL). The or~ni~m~
were then suspended in PBS, pH 7.2, at a concentration of 1 x 108 CFU./ml. Aliquots of 1.0
ml volumes of this suspension were placed into separate microfuge tubes and the tubes
centrifuged at approximately 14,000 x g for 5 min. at 4C. Each of the resulting pellets was
25 then resuspended in a 1.0 ml volume of one of the following opsonin or control solutions:
1. IgG-PMB Conjugate (prepared by periodate oxidation, as
described in Example 14(a)) at the MIC for E. coli HB101 (0.062mg/ml) (The
MIC was deterrninecl as described in Example 12).
2. IgG-PMB Conjugate (same as above) at 2x the MBC for E. coli
HB101 (0.25 mg/ml) (The MBC was ~letermined as described in Example 12).
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3. IgG Control (unconjugated) at 0.062mg/ml (control for #1 above; s
this was the same IgG as that used for production of the conjugate).
4. IgG Control (unconjugated) at 0.25mg/ml (control for #2 above).
5. PBS Control (no IgG or conjugate). PBS, pH 7.2 only.
The five suspensions were opsonized by incubation at 37C for 60 min. with periodic
mixing. Following opsonization, the suspensions were centrifuged as above~ and the resulting
pellets were each resuspended in 0.5ml of RPMI 1640 medium which was supplemented with
10% FCS (this will be referred to as "medium" for the r~m~in(ler of this example). Into each
of 5 separate polypropylene culture tubes (S/P) was placed 1.0 ml of a U937 cell suspension,
which was prepared in medium, and contained 1 x 10~ U937 cells/ml. To each tube, O.lml of
one of the opsonized E. coli suspensions prepared above was also added. A sixth control
group was also prepared which contained 1.0 ml of the U937 cell suspension and 0.1 ml of
PBS(PBS control). At this point, each tube contained 1 x 106 U937 cells, and 2 x 107 E. coli
org~ni~m.~, thus providing an E. coli to U937 cell ratio of 20:1. The 6 tubes were then
incubated at 37C for 60 min. with constant ~h~king, in order to allow phagocytosis to occur.
Following incubation, the tubes were placed on ice for several minl1tes to prevent further
phagocytosis. The 6 tubes were then centrifuged for 10 min. at 500 x g at 4C. The
resulting pellets were washed three times (centrifuging as in the previous step) with chilled
PBS, to remove extracellular E. coli org~ni~m.c. The final pellets were each resuspended in
0.2 ml of chllledPBS, and smears were ~ parcd by applying 40 ~11 volumes of the
suspensions to glass microscope slides. The smears were allowed to air-dry, and were then
fixed by immersion in 100% methanol for 5 sec. and again allowed to air-dry.
The smears were stained using a modified version of the Sowter-McGee staining
procedure [Sowter and McGee. J. Clin. Pathol. 29:433-437 (1976)], which chromatically
differentiates between intracellular bacteria and the surrounding cytoplasm of the host cells.
The slides were hydrated by immersion in water for approximately 60 sec., and were then
placed in a methyl green-pyronin (MGP) solution (Sigma) for 5 min. The slides were washed
in water for 15-20 sec. and then immersed in light green counterstain (0.25% Sigma Light
Green SF Yellowish in distilled H~O) for 3-5 sec. Following a 15-20 sec. rinse in water, the
slides were dipped in 100% ethanol for 5 sec., and then in xylene for 5 sec. The slides were
allowed to air-dry, and were then mounted with glass coverslips.
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Seoring of opsonophagocytosis was performed in a blind manner, by light mieroseopy..
For eaeh experimental group. a total of 100 U937 eells were randomly counted to determine
the pereentage of those eells whieh contained one or more intraeellular E. coli org~ni.~m.~.
The results of this study are presented in Table 35.
,.
TABLE 35
U937 Cells Whieh Contained One Or More
Opsonization Tre~tme~t
E. coli
IgG-PMB ~ MIC (0.062 mg/ml) 65%
IgG-PMB @ 2 x MIC (0.25 mg/ml) 56%
IgG (~ 0.062 mg/ml 0%
10IgG ~ 0.25 mg/ml 3%
PBS Control 0%
U937 Cells Only (No E. coli) 0%
Trç~tment of E coli org~ni.em.c with IgG-PMB conjugates at eoneentrations that were
equivalent to the MIC and 2x the MBC for that organism resulted in the phagocytic uptake of
15 the org~ni.~m.~ by greater than 50% of the U937 cells. Tre~tment of the org~ni~m~ with
comparable coneent~ations of the uneonjugated form of the same IgG resulted in minim~l to
no uptake. In the absenee of IgG-PMB eonjugate or IgG, no phagoeytic uptake of the E. coli
org~ni~m~ occurred (PBS eontrol group). These results demonstrate that the IgG portion of
the IgG-PMB conjugate retains opsonic effeetor function, and that IgG-PMB conjugates
20 potentiate the phagocytic clearanee of baeterial org~ni~m~
B. Determination Of The Minimum Effective Concentration Of
IgG-PMB ConJugate
The minimum concentration of IgG-PMB required to mediate opsonophagocytosis was
let(~.rmined by testing the eonjugate at the MIC and at fraetional eoncenkations of the MIC
25 (sub-MIC). As an additional control, a parallel series of albumin-PMB (Alb-PMB) conjugate
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solutions were also tested at concentrations comparable to the IgG-PMB conjugate. The
following conjugate and control solutions were assayed for opsonophagocytic activity by the
procedure described in part (a) of this example: ~
1. IgG-PMB Conjugate (same as that used in part (a) of this
Example) at the MIC for E. coli HB101 (0.062 mg/ml).
2. IgG-PMB Conjugate (same as above) at 1/2 the MIC for E. coli
HB101 (0.031 mg/ml).
3. IgG-PMB Conjugate (same as above) at 1/4 the MIC for E. coli
HB101 (0.0155 mg/ml).
4. IgG-PMB Conjugate (same as above) at 1/8 the MIC for E. coli
HB101 (7.75 ~Lg/ml).
5. Alb-PMB Conjugate (prepared as described in Example 18(a)) at
0.062 mg/ml (this group served as a control for #1 above).
6. Alb-PMB Conjugate (same as above) at 0.031 mg/ml (this group
served as a control for #2 above).
7. Alb-PMB Conjugate (same as above) at 0.0155 mg/ml (this
group served as a control for #3 above).
8. Alb-PMB Conjugate (same as above) at 7.75 ~Lg/ml (this group
served as a control for #4 above).
9 IgG Control (unconjugated) at 0.062 mg/ml.
10. PBS Control (no IgG or conjugate). PBS, pH 7.2 only.
The results of this study are presented in Table 36.
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TABLE 36
U937 Cells Which
Opsoni~ation Tre~tment
Contained One Or More E. coZi
IgG-PMB (~ MIC (0.062 mg/ml) 38%
IgG-PMB (~1/2 MIC (0.031 mg/ml) 41%
5lgG-PMB ~1/4 MIC (0.0155 mg/ml) 14%
IgG-PMB ~1/8 MIC (7.75 ~lg/ml) 10%
Alb-PMB (~ 0.062 mg/ml 2%
Alb-PMB (~ 0.031 mg/ml 0%
Alb-PMB (~10.0155 mg/ml 2%
10Alb-PMB (~ 7.75 llg/ml 5%
IgG (~ 0.062 mg/ml 2%
PBS Control 0%
Tre~tment of E. coli org~ni~m~ with IgG-PMB conjugates, using collcellll~lions at or
below the MIC for the conjugate, resulted in the increased phagocytic uptake of the org~ni~m~
15 by the U937 cells at all IgG-PMB concentrations tested. The parallel series of Alb-PMB
conjugate concentrations tested did not r~ nol,~l,dl~ significant levels of opsonophagocytic
activity, nor did the unconjugated IgG control. These results demon~ te that IgG-PMB
conjugates possess significant levels of opsonophagocytic activity at clinically-relevant,
sub-MIC concentrations, and that both the functional PMB and IgG portions of the conjugate
20 are required ~imlllt~neously in order for the conjugate to be capable of me~ ting
opsonophagocytosis .
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EXAMPLE 29
Antimicrobial Activity Of IgG-PMB
Conjugates Against Clinically-Relevant Bacterial Strains
MIC and MBC values were determined for IgG-PMB conjugate and native PMB
5 control against bacterial strains which are known to be human pathogens (see Exarnple 29
below). This exarnple involved (a) Plcpald~ion of the Conjugate, (b) Preparation of the
Bacterial Inocula, and (c) Deterrnination of the MIC and MBC.
A. Preparation Of The Conjugate
The IgG-PMB conjugate was prepared by periodate oxidation as described in Exarnple
10 14(a) with the following modification, which was pclro~ ed in order to more effectively
remove free (unconjugated) PMB from the final conjugate pl~dldLion. The final conjugate
solution was adjusted to contain 1.0% Tween-20, and then was chromatographed on a P-10
column using PBS co~ i"g 0.1% Tween-20 as the eluent. The material in the void volume
was concentrated and then further purified by column chromatography as described in the
15 previous sentence.
B. Preparation Of The Bacterial Inocula
Org~ni~m.c were grown, and ~ep~d~e inocula were prepared for the following test
org~ni~m~, as described in Exarnple 12 (a): E. coli strain EC 5; Pseudomonas aeruginosa
strain ATCC 27312; Pseudomonas aeruginosa strain Strong; and Pseudomonas aeruginosa
20 strain 3.
C. Deter~ir~tion Of The MIC And MBC
The MIC and MBC of IgG-PMB conjugate and native PMB control were determined
for each of the test org~ni~m~, as described in Examples 12(b) and 12(c).
The results of the MIC ~ ""i"~tion are shown in Table 37.
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TABLE 37
Test OrganismConjugate MIC PMB Control MIC
E. coli, EC 50.03125 mg/ml 0.156 ~lg/ml
P~ aeruginosa, ATCC 27312 0.25 mg/ml not done
S P. aeruginosa, Strong 0.25 mg/ml not done
P. aeruginosa, Strain 3 0.25 mg/ml 1.0 ~lg/ml
The results of the MBC determinations are shown Table 38.
TABLE 38
Test Organism Conjugate MBC PMB Control MBC
10E. coli, EC 5 0.0625 mg/ml 0.156 ~Lg/ml
P. aeruginosa, ATCC 27312 >0.5 mg/ml not done
P. aeruginosa, Strong 0.25 mg/ml not done
P. aer~ginosa, strain 3 0.25 mg/ml 1.0 ~g/ml
The bacteriostatic and bactericidal activity of IgG-PMB conjugates against pathogenic
15 bacterial strains demonstrate that these compounds may be effective for the prophylaxis and/or
tre~tment of bacteremia.
EXAMPLE 30
Prophylactic A-lminictration Of IgG-PMB Conjugate
Protects Rats Against An Escherichia coii Bacteremia
Gram negative bacteremia and endotoxic shock can trigger a lethal reaction in vivo.
Indeed, overwhelming gram negative bacteremia has become a leading cause of death from
infection in the hospital. [S.M. Wolf, N. Eng. J. Med. 307;1267-1268 (1982).] In particular,
E. coli sepsis cl ntinll~s to be associated with an unacceptably high mortality rate, despite the
availability of potent antibiotics. E. coli strains with the Kl c~rsul~r type have been
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identified as the etiologic agent in up to 24% of blood culture isolates [G. W. Count and M. ~ e
Turck, J. Clin. Microbiol. 5:490 (1977)], 80% of the cases of neonatal meningitis. [L.D.
Sarff et al, Lancet 1:1099 (1975).] It is the most frequent cause of nosocomial gram negative
bacteremia in adults [M.P. Weinstein et al., Rev. Infect. Dis. 5:35-53 (1983)] and
5 pyelonephritis in children. [G.H. McCracken et al., Hosp. Pract. 9:57 (1974).] In addition to
the Kl type, human blood E. coli isolates also possess an O antigen serotype, with 018
lipopolysaccharide being most frequently associated with bacteremia. [A.S. Cross et al., J.
Infect. Dis. 149:184-193 (1984).] E. coli serotype 018:Kl is a very virulent human pathogen,
as defined by either D. Rowley, Br. J. Exp. Pathol. 35:528-538 (1954) or H. Smith, J. Gen.
10 Microbiol. 136:377-383 (1990), they can grow in vivo from a small inoculum, evade host
defenses and cause ~xL~ stin~l infections.
To test the IgG-PMB conjugate for in vivo efficacy against bacteremia caused by a
virulent bacterium such as E. coli 018:Kl, the established animal infection model described
by D.E. Schiff et al., Infect. Tmmlln 61:975-980 (1993) was l-tili7~-1 This model fulfills
- 15 many criteria important in evaluating the toxicity, efficacy and safety of immunoth-,.dpculics,
some of which have been outlined by A.S. Cross et al., Infect. Immun. 61:2741-2747 (1993).
Specifically, the model is an infection rather than an intoxication model in which rats are
challenged with low doses of a virulent bacteria instead of using large doses of an avirulent
strain. Experimental evidence indicates that models of infection, in contrast to intoxication
20 models, more accurately reflect the course of human sepsis. Infection models: a) use bacteria
that cause human sepsis pos~es~ing an invasive phenotype with virulent factors (i.e., a
particular K antigen or smooth LPS antigen) [I. Orskov and F. Orskov, J. Hyg. Camp.
95:551-575 (1985)]; b) mimic the normal progression of sepsis from a focal site to
colonization; c) generate levels of circulating bacteria c-~n~i~t~nt with clinical bacteremia [D.E.
25 Dietzman et al., J. Pediatr. 85:128-131 (1974)]; and d) produce endotoxin levels and induce
physiological cytokine responses such as triggering TNF kinetics similar to the clinical
experience. [Reviewed by A.S. Cross et al., Infect. Tmm~n 61:2741-2747 (1993).]
In this Example, we tested whether the prophylactic tre~tment of neonatal rats with
IgG-PMB could protect against bacteremia and death caused by E. coli 018:Kl. This
30 example involved: (a) determin~tion of the lethal dosage of E. coli 018:Kl in rat pups; and
(b) in vivo protection against E. coli 018:Kl using IgG-PMB.
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A. Determination Of Lethal Dosage Of E. coli 018:K1 In
Newborn Rats
Five day old pathogen-free Sprague-Dawley rats (Charles River Laboratories,
Wilmington, MA) were inoculated subcutaneously with different doses of E. coli 01 8:K1.
The E. coli (strain de~ign~tion C5) obtained from K.S. Kim, Children's Hospital (Los
Angeles, CA), was isolated from the cerebrospinal fluid of a child. An overnight culture of
E. coli C5 in brain heart infusion (BHI) medium was diluted 1 :40 in fresh medium and grown
to early log phase to an OD620 of 0.25 which replese~ approximately 1 x 108 bacteria/ml.
The cells were washed twice by centrifugation with sterile saline (0.9% NaCl) and diluted to
different cell densities in saline. Each dilution was streaked onto a BHI agar plate and
incubated at 37C to l~t~rmine actual cell number.
A~ v~hllately 370 to 2620 bacteria in 100 ,ul of sterile saline were inoculated
subcutaneously into the rats. A typical lethal infection with E. coli 018:KI produced a
bacteremia within 18 hours, and death within 24-72 hours post-infection. We found that
about 300 bacteria lc~cst;ll~:d an LD50 and killed about 50% of the pups, whereas, 1500
bacteria was usually sufficient to kill most or all rats. Since between 1500-2500 bacteria
represent the minim~l effective lethal dose, this concentration of E. coli was utilized in the in
vivo bacteremia protection studies using IgG-PMB.
B. In vivo P~ lion Against E. coli 018:Kl (C5) Using
IgG-PMB Conjugate
To ~iet~rmine if the ~lmini.~tration of IgG-PMB conjugate can protect in vivo, rat pups
were ~lcLlc~Led with IgG-PMB or control IgG followed by an ~-lmini~tration of a lethal dose
of E. coli C5. The IgG used as the carrier in the conjugate and as the control was a human
myeloma protein which was shown to be unreactive to the E. coli CS by ELISA. Themyeloma IgG was used as the carrier to produce the PMB-conjugate, in order to ensure the
reactivity between the E. coli and IgG-PMB conjugate was exclusively due to binding
between PMB and lipopolysaccharide.
Eleven to twelve 4-5 day-old Sprague-Dawley rats (weighing 10 gm) in each group
were given 30 ,ug, 100 ~Lg or 300 ~Lg of an IgG-PMB conjugate or control IgG in 100 ~Ll of
endotoxin-free PBS intraperitoneally. An untreated group was given only PBS (conjugate
diluent). Two hours later, each rat received approximately 2560 E. coli C5 bacteria in 100 ~1
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of PBS subcutaneously behind the head. After 24 hours post-infection, the nurnber of
survivors, survivors with focal lesions and dead were recorded. The results are shown in
Table 39. The focal lesions appeared very hemorrhagic and were located at or near the site of
inoculation. All pups displaying focal lesions were found to be bacteremic and subsequently
5 died within 24-48 hours of apl)e~ce of the lesion. Bacteremia was detected by collecting
blood from the tail vein, diluted 20-fold in BHI broth and 20 ~11 aliquots were plated onto
(5% sheep red cells) blood agar plates.
The results show that 30 ~g of IgG-PMB conjugate/pup or a 300~1g/Kg dose was
sufficient to completely protect all the ~nim~ against the generation of a focal lesion or
10 death by E. coli for 24 hours post-infection. In contrast, no protection was afforded by doses
of 30 or 100 ,ug of IgG/pup co~ ared to the untreated group. Pups treated with IgG at the
highest dose (300 ~lg) were protected (probably leplesP~ g a nonspecific phenomenon).
These fin~ling~ demonstrate that IgG-PMB conjugate given prophylactically can
prevent the progression of sepsis due to E. coli. This was shown by the prevention of death
15 in the pups and also the protection against focal lesions. These results imply that the
conjugate is capable of systemic distribution throughout the body to regions outside the
vascular cc",-p~l",ent and inhibiting infection.
EXAMPLE 31
D~.;vali~lion Of 7-Aminocephalosporanic Acid With Sulfo-MBS
This example describes the ~tt~l~hmPnt of a heterobifunctional cros~linkin~ agent to an
antibiotic precursor. This example outlines the derivatization of 7-aminocephalosporanic acid,
an antibiotic ~,e~ or exhibiting no significant anti-microbial plol)~,lies, with m-
Maleimidobenzoyl-N-hydroxysulfosuccinimide ester ("sulfo-MBS").
For the derivatization of 7-aminocephalosporanic acid, 2.9 mg of 7-
aminocephalosporanic acid (Sigma) were dissolved in 1.0 ml of 50 mM phosphate buffer (pH
6.65) and 10.7 ~LI of 1.0 N NaOH were added during mixing to return the pH to 6.65. Then
4.6 mg of sulfo-MBS (Pierce) were added and dissolved with mixing. The mixture was
incubated at ambient temperature with agitation for 4.5 hours. 10 mg of ethanolamine were
added and the incubation was continued for 17 additional minntec
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The reaction mixture (0.5 ml) was applied to a 1.5 x 20 cm column of Bio-Rad P2
resin. The eluent was 50 mM phosphate buffer (pH 6.65) flowing at 0.5 ml/minute. The
eluate (i.e. the liquid collected at the bottom of the column) was monitored for absorbance at
280 nm. 0.5 ml fractions were collected. Two major absorbance peaks were evident - one
S centered at 42 minutes and the other at 47.5 minlltes (aminocephalosporanic acid and reaction
products, respectively). Fractions corresponding to the leading edge of the first peak were
pooled (2.0 ml, 3.42 A 280). 1.4 ~Ll of 100% beta-mercaptoethanol was added to the mixture
which was then filtered using a sterile Whatman 0.45 micron Puradisc. The MIC of the
putative S-
TABLE 39
Protection Against A Lethal E. coli 018:Kl
Bacteremia Using IgG-PMB Conjugate Treatment
Number o~ Focal
Tre~tm~:nt Number Dead Number Alive
Lesions
Untreated (PBS) 4 3 4
15IgG (30 ,ug/lOO~l) 4 4 4
IgG (lOO~lg/lOO,ul) 1 9 2
IgG (300~1g/100~l1) 1 2 9
IgG-PMB (30~1g/100~1) 0 0 12
IgG-PMB (lOOIlg/lOO,ul) 1 0 11
20IgG-PMB (300~1g/100~1) 0 0 12
hydroxyethylthio-maleimidoben_oyl-N-aminocephalosporanic acid ester was determined to be
0.86 A280 with Staph. aureus compared to 3.7 A280 for 7-aminocephalosporanic acid.
Controls were also tested for the above deriv~i7~ted precursor. Sulfo-MBS, beta-mercaptoethanol and ethanolamine were plcp~-,d at concentrations used above and assessed
25 for activity against S. aureus. The compounds were inactive.
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EXAMPLE 32
Derivatization Of 6-Aminopenicillanic Acid With Sulfo-MBS
This example describes the ~tt~rllment of the heterobifunctional crosslinkinp agent of
Example 31 to a different antibiotic precursor. This example outlines the derivatization of 6-
aminopenicillanic acid, an antibiotic precursor exhibiting no significant anti-microbial
~,.ol,c"ies, with sulfo-MBS.
For the derivatization of 6-aminopenicillanic acid, 12.1 mg of 6-aminopenicillanic acid
(ICN) was dissolved in 2.5 ml of 50 mM phosphate buffer (pH 6.65). The solution was
continuously mixed with a stir bar and m~gnP~tic stirrer and the pH was monitored. Sulfo-
MBS (24.1 mg, Prochem, Inc.) was added and the pH was adjusted to 6.85 with a 1.0 N
sodium hydroxide solution. The lnixLulc was incl-b~ted at ambient temperature for 2.5 hours.
The reaction mixture was applied to a 1.5 x 20 cm column of Whatman LRP-2 resin
(C18 reverse phase), equilibrated with 10% methanol in water. The column was developed at
1.0 ml/min. with 10% methanol for 5 min., followed by a linear, 30 min. gradient of 10 to
90% methanol in water. The eluate co.~L~;,.i-.g the last peak of material absorbing at 280 nm
(eluted at 26 min.) was collected and conccllL.dl~d to dryness under reduced ~les~ulc using a
Labconco Cc~lLldVd~l conccl.~,dlor. The derivatized aminopenicillanic acid was dissolved in
1.0 ml of 50 mM phosphate buffer plus 1.0 mM EDTA, pH 6.65. The MIC of the
derivatized aminopenicillanic acid was let~rmined to be 8~lg/ml against S. aureus, compared
to 250 ~Lg/ml for the native aminopenicillanic acid.
Purified human IgG (40 mg, Sigma) was dissolved in 2.5 ml of 50 mM
triethanolamine, 1.0 mM EDTA, pH 8.0 and continuously stirred with a m~gnPtic stir bar and
stirrer. 100 ,ul of 13 mg/ml iminothiolane (Traut's Reagent, Prochem) in water was added.
The pH was monitored and adjusted to 8.0 with 1.0 N. sodium hydroxide. The llli~Lule was
incubated at ambient lclllpcl~c for 2 hours. The mixture was then applied to a 2.5 X 20
cm column of Spectra/Gel ACA 202 (Spectrum). The column was eluted at 2.0 ml/min with
50 mM sodium phosphate buffer, 1.0 mM EDTA, pH 6.5. The absoll,~lce at 280 nm was "
monitored. The material in the void volume, co,~ g iminothiolated IgG, was collected
and pooled. The concentration of the iminothiolated IgG was 5.0 mg/ml.
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The derivatized aminopenicillanic acid (0.5 ml, 9.0 mg/ml) was mixed with 1.75 ml of
iminothiolated IgG and incubated at ambient temperature for 10 min with mixing and then at
2^8C overnight. The mixture was transferred to ambient temperature and incubated with
agitation. After 20 minutes, 146 ~1 of 10 mM N-ethylmaleimide (Pierce) was added and the
5 incubation was continued for 4 hours. The reaction mixture was passed through a Uniflo-Plus
filter (S&S) and applied to a 2.5 X 20 cm column of Spectra/Gel ACA 202 (Spectrum). The
column was eluted at 2.0 ml/min with 50 mM sodium phosphate buffer, pH 6.5. The
absorbance at 280 nm was monitored. The material in the void volume, cont~ining MBS
aminopenicillanic acid:IgG was collected, pooled, concentrated using a Centricon concentrator
10 (Amicon) and passed through a Uniflo-Plus filter (S&S). The MBS aminopenicillanic
acid:IgG was found to be inactive at 0.65 mg/ml against S. aureu,s.
Controls were also tested for the above deriv~ti7~tPcl precursor. Sulfo-MBS~ beta-
mercaptoethanol and ethanolamine were prepared at concentrations used above and ~essed
for activity against S. aureus. The compounds were inactive.
EXAMPLE 33
Derivatization Of Amoxicillin with Sulfo SMCC
This example outlines the derivatization of amoxicillin, an antibiotic exhibiting
significant anti-microbial properties, with sulfo-SMCC. For the derivatization of amoxicillin,
23 mg of amoxicillin trihydrate (ICN) was added to 2.5 ml of 50 mM phosphate buffer (pH
6.65). The suspension was continuously mixed with a stir bar and magnetic stirrer and the
pH was monitored. Sulfo SMCC (23 mg, Prochem, Inc.) was added and the pH was adjusted
to 7.0 (and periodically readjusted to 7.0) with 1.0 N sodium hydroxide. The mixture was
incubated at ambient temperature for 4 hours. (The reaction mixture was initially turbid due
to suspended amoxicillin, but the mixture became clear with time.)
The reaction mixture was applied to a 1.5 X 20 cm column of Whatman LRP-2 resin
(C18 reverse phase), equilibrated with 10% methanol in water. The column was developed at
1.0 ml/min. with 10% methanol for 5 min., followed by a linear, 30 min. gradient of 10 to
90% methanol in water. The eluate cont:~ining the last peak of material absorbing at 280 nm
(eluted at 27.5 min.) was collected and concentrated to dryness under reduced pressure in a
Labconco Centravap concentrator. This derivatized amoxicillin was dissolved in 1.0 ml of 50
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mM phosphate buffer plus 1.0 mM EDTA, pH 6.65. The MIC of the derivitized amoxicillin
was determined to be 8 ug/ml against S. aureus, compared to 250 ug/ml for native amoxicillin
EXAMPLE 34
Derivatization Of Cefadroxil With Sulfo-SMCC
In the following example, cefadroxil, an antibiotic active against gram-positivebacteria, was reacted with the cros~linkin~ agent sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate ("sulfo-SMCC").
Cefadroxil (Sigma) was dissolved at 3.0 mg/ml in 50 mM phosphate (pH 6.65). sulfo-
SMCC was added and dissolved at 2.6 mg/ml. After a 1 hour and 55 minute incubation at
ambient temperature with agitation, ethanolamine was added at 3.4 mg/ml and the incubation
was contim~ for an additional 42 mimltes
The reaction mixture (0.5 ml) was applied to a 1.5 X 13 cm column of Sephadex G10
resin (Pharmacia). The eluent was 50 mM phosphate buffer, pH 6.65, flowing at 0.5
ml/minute. The eluate was monitored for absorbance at 280 nm. 1.0 ml fractions were
collected. Two major absorbance peaks were evident - one centered at 22 minlltes and the
other at 36 minl-tes (reaction products and cefadroxil, respectively). Fractions corresponding
to the leading edge of the first peak were pooled (3.0 ml, 3.5 A 280). 300 ~11 of 100 mM
beta-mercaptoethanol was added to the mixture which was then filtered using a sterile
Whatman 0.45 micron Puradisc. The MIC of the putative S-hydroxyethylthio
maleimidomethyl cyclohexane carboxyl-N-cefadroxil ester was determin~d to be 1.6 A280
against Staph. aureus co",~ed to a MIC of 0.028 A280 for native cefadroxil. Thus, the
derivatized cefadroxil was relatively inactive.
EXAMPLE 35
Derivatization Of Vancomycin With Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB, And Sulfo-
SMPB.
In the following example vancomycin, an antibiotic active against gram-positive
bacteria, was reacted with four different heterobifunctional cro~slinking agents.
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Vancomycin in phosphate buffer was reacted with each of the following compounds:sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamide] hexanoate ("sulfo- LC- SPDP"), m-
maleimidobenzoyl-N-hydroxysulfosuccinimide ester ("sulfo-MBS"), sulfosuccinimidyl (4-
iodoacetyl) aminobenzoate ("sulfo-SIAB"). and sulfosuccinimidyl 4-(p-maleimidophenyl)
butyrate ("sulfo-SMPB"). All four cro~linkin~ agents react with a primary amino group on
the vancomycin molecule, resulting in the formation of an amide bond. Vancomycinderivatized by sulfo-LC-SPDP possesses a sulfhydryl group which can be exposed under the
proper conditions and can be reacted with a maleimide on derivatized IgG. Vancomycin
derivatized by sulfo-MBS possesses a maleimide group which can react with a sulfhydryl
group on either reduced IgG or derivatized IgG, by addition to the maleimide's carbon-carbon
double bond. Finally, vancomycin derivatized by sulfo-SIAB posesses an iodo group, which
can also react with a sulfhydryl group on either reduced IgG or derivatized IgG, by
nucleophilic substitution for the iodo group. Sulfo MBS, sulfo SIAB, sulfo SMPB or sulfo
SMCC (Pierce) were dissolved with mixing at a concentration of 20 mM in a solution of lO
mM vancomycin (ICN) in 50 mM sodium phosphate buffer, pH 7.l5. The ~ Lules were
incubated with agitation at ambient te--.pe.~LIlre. P.~ci~ Les formed in all four mixtures and
the products of the reaction were not further pursued.
EXAMPLE 36
Conjugation Of Vancomycin To IgG With Iminothiolane And Sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane- l -carboxylate
Because of the lln~ti~f~tory results of the previous example in obtaining a soluble
derivatized vancomycin suitable for further conjugation to immunoglobulin, an alternative
cro~linking method was investigated using iminothiolane ("Traut's Reagent") to derivatize the
vancomycin and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate ("sulfo-
25 SMCC") to derivatize the IgG. The reaction proceeds in three steps which are outlinedbelow. First, vancomycin is derivatized with Traut's Reagent. Second, non-specific
immunoglobulin is derivatized with sulfo-SMCC. Third, the derivatized vancomycin and the
derivatized IgG are reacted with each other forming a conjugate.
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a) Reaction Of VancomYcin With Iminothiolane
33.4 mg of vancomycin (ICN) was dissolved in 2.0 ml of freshly (leg~c~ed 50 mM
triethanolamine, 1.0 mM EDTA, pH 8.0 buffer. 34.5 mg of Traut's Reagent (Pierce) was
dissolved~ and N2 gas was blown into the vial which was then tightly capped. The vial was
5 incubated with agitation for 1 hour and 15 minl-te~
The mixture (0.5 ml) was applied to a 1.5 x 13 cm column of Sephadex G10 (Pharmacia).
The eluent was freshly ~leg~se~l 50 mM phosphate, 1.0 mM EDTA, pH 6.65 buffer flowing
at 0.5 ml/min. 1.0 ml fractions were collected and the fractions in the first peak (void volume
of column) were pooled (3.0 ml).
The pool from the G10 column was applied to a 4 ml, 1.0 cm diameter column of
Bio-Rad Affi-Gel 501, an organomercury resin. The 501 resin had been previously washed
with 25 ml 50 mM sodium acetate, pH 5.0 (acetate buffer), 25 ml of 4.0 mM mercuric acetate
in acetate buffer, 25 ml of acetate buffer and the equilibrated to 50 mM phospha te, 1.0 mM
EDTA, 0.5% Tween 20, pH 6.65. The pool was applied at 0.2 ml/min using 50 mM
15 phosphate, 1.0 mM EDTA, 0.5% Tween 20, pH 6.65 to wash the column. The flow was
increased to 1.0 ml/min after 13 min~ltes Washing continued for a total time of 85 minlltPc
to remove native, nonthiolated vancomycin. Thiolated vancomycin was eluted from the Affi-
Gel 501 resin at 1.0 ml/min using freshly prepared 10 mM beta-mercaptoethanol in 50 mM
phosphate, 1.0 mM EDTA, 0.5% Tween 20, pH 6.65. The first 5 fractions, 1.0 ml each, had
20 A280 significantly greater than baseline and were pooled (0.116 A280 or 29 ug/ml, 5.0 ml). The
MIC for the putative iminothiolated vancomycin was clet~?rmined to be 2.4 ug/ml with
S. aureus. The MIC of vancomycin is 1-2 ~Lg/ml.
The derivatized vancomycin was concentrated on a Labconco Centravap, 1.0 ml of
water was added, and the material was applied to a 1.5 X 13 cm column of Sephadex G10
25 (Pharmacia). The column was eluted with 50 mM sodium phosphate buffer, 1.0 mM EDTA,
0.5% TWEEN 20, pH 6.65. The material in the void volume was collected and pooled.
b) Reaction Of I~G With Sulfo-SMCC
In general, 15 moles of sulfo-SMCC were used per mole of IgG. Higher molar ratios
of crosslinker than this resulted in precipitation of IgG. 40 mg human IgG (Sigma? Cat #
30 I4506, Lot # 063H-8875I) was dissolved in 2 ml of 50 mM phosphate pH 7.1 buffer and 1.6
mg sulfo-SMCC (Prochem, Cat # CL207, Lot # 03092) was added with mixing. The llli?~iUl~
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was incubated at room temperature for 45 min and excess crosslinker was removed by
chromatography on an AcA 202 gel filtration column. A 2 ml sample was applied to a 2.5 x
20 cm ACA 202 gel filtration column (Spectrum) equilibrated and eluted with 50 mM MES,
0.5% Tween-20, pH 6.5 buffer. The first peak corresponding to the activated IgG was
collected and absorbance at 280 nm was monitored.
c) Coniu~ation Of Derivatized Vancomycin And Derivatized I~G
For the final conjugation of the derivatized vancomycin and the derivatized IgG? 2.95
ml of 4.4 mg/ml SMCC:IgG (from step b above) was added to 14 ml of 0.74 mg/ml
iminothiolated vancomycin (from step a above). The solution was incubated at ambient
temperature on a rotary shaker at 200 rpm. After one hour and five minutes~ lO0 ~ll of 3.5
mg/ml 2-mercaptoethanol was added and the incubation was continued for an additional 25
min~-tes The solution was concentrated to approximately 2 ml using an Amicon Centricon
30.
The sample was loaded onto a 2.5 x 20 cm column of Spectra Gel ACA 202 resin
lS (Spectrum) and eluted, at l.0 ml/min, with PBS plus 0.1% Tween 20. 1.5 ml fractions were
collected. The fractions in the void volume were pooled and sterile filtered. The activity of
the conjugate was (l~t~rrninçd by standard MIC testing against S. aureus. The conjugate
against S. aureus was found to be inactive at l.l mg/ml.
EXAMPLE 37
Conjugation Of Vancomycin To IgG With S-Acetyl Mercapto Succinic Anhydride
("SAMSA") And Sulfosuccinimidyl 4-(N-Maleimidomethyl)cyclohexane-l-Carboxylate
("Sulfo-SMCC")
Because of the l1n~ti~f~çtory results of the previous example in obtaining a
vancomycin-IgG conjugate with antibacterial activity, an alternative cro~linkin~ method was
investig~ted using S-acetyl mercapto succinic anhydride ("SAMSA") to derivatize the
vancomycin and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate ("sulfo-
SMCC") to derivatize the IgG. The reaction proceeds in five steps which are outlined below.
First, vancomycin is derivatized with SAMSA. Second, both derivatized vancomycin and free
unreacted vancomycin are separated from any unreacted cro~linking agent. Third~ the
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derivatized vancomycin is separated from the free unreacted vancomycin. Fourth~ non-
specific immunoglobulin is derivatized with sulfo-SMCC. Fifth, the purified derivatized
vancomycin and the derivatized IgG are reacted with each other forming a conjugate.
a) Reaction Of Vancomycin With SAMSA
20.4 mg vancomycin (Sigma, Cat # V2002, Lot # 43H1090) ~14 ~lmoles] was
dissolved in 200 ~11 of water. 1.2 ml of saturated sodium succinate was added slowly with
stirring and this mixture was cooled to 4C by placing the reaction mixture on ice. The
mixture appeared slightly cloudy. To this mixture was added 121.8 mg of SAMSA (Sigma,
Cat # A1251, Lot 3 # 120H5017) dissolved in 200 ,LI of dimethyl sulfoxide (DMSO,Mallinckrodt, Cat # 5507, Lot # 5507 KLDL). The pH was monitored. The beginning pH
was 8.1, and after addition of the SAMSA, it was 6.8. The reaction mixture was incubated at
4C for one hour followed by another one hour incubation at room temperature, while stirring
constantly.
b) Separation Of Excess Crosslinker
To remove excess crosslinker from modified and unmodified vancomycin, the lni~Lule
was applied to a G-10 colurnn (2.5 x 20 cm, Pharmacia) equilibrated with 50 mM sodium
phosphate, pH 7.1. The first peak which contained both modified and unmodified
vancomycin was collected and stored at 4C.
c) Purification Of Modified Vancomycin From Unmodified Vancomycin BY AffinitY
Chromato~raphY On Or~anomercurical Column
A 5ml Affi Gel 501 Organomercurial agarose column (Bio-Rad) was prepared
according to the m~nnf~cturer's instructions. The column was equilibrated with 50 mM
phosphate, pH 7.1. SAMSA modified vancomycin contains protected sulfhydryl groups
which were deprotected with hydroxylamine hydrochloride before applying to the column.
Hydroxylamine hydrochloride was added to the modified vancomycin solution to a final
concentration of 0.2 M and the mixture was incubated at room temperature for five minlltes
The sample was applied to the Affi Gel 501 column at a flow rate of 0.5 ml/min and the
column was then washed with 10 mM 2-(N-Morpholino) ethane sulfonic acid, 1 mM EDTA,
0.5% Tween-20, pH 6.5 buffer until the baseline A280 was obtained. The bound modified
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vancomycin was eluted with the same wash buffer cont~inin~ 20 mM 2-mercaptoethanol. The
activity of the modified vancomycin was determined to be 2.6 ~g/ml by MIC testing against
S. aureus.
d) Reaction Of I~G With Sulfo-SMCC
This reaction was carried out as described in example 35, step b (above).
e) Conju~ation Of Derivatized Vancomycin And Derivatized I~G
Modified vancomycin was in a buffer cont~ining 20 mM 2-mercaptoethanol which wasremoved by dialysis, using benzoylated dialysis tubing (Sigma Cat # D7884, Lot # 43H7085).
750 ~g vancomycin (~0.5 ,umole) and 7.5 mg (0.05 ,umole) maleimide activated IgG was used.
10 The mixture was incl-hate-l at room temperature for one hour. Unreacted maleimide sites
were blocked by adding 30 moles of 2-mercapto ethyl amine per mole of IgG and incubating
the mixture at room temperature for 20 min. The conjugate was purified ~rom excess 2-
mercapto ethyl arnine and unreacted vancomycin by gel filtration chromatography. The
sample was applied to a AcA 202 gel filtration colurnn (2.5 x 20 cm, Spectrurn) equilibrated
15 with 0.01 M phosphate buffered saline, pH 7.2 with 0.1% Tween-20. Absorbance at 280 nm
was monitored. The first peak co.,~ g vancomycin-IgG conjugate was collected. The
activity of the conjugate was (letennined by MIC testing against S. aureus. MIC of this
conjugate against S. aureus was found to be 0.438 mg/ml. This example demollsllales that
conjugation of vancomycin derivatized by SAMSA with IgG derivatized by sulfo-SMCC
20 results in an active conjugate. This conjugate was found to be an effective anti-microbial
agent when tested against S. aureus.
EXAMPLE 38
Conjugation Of Limulus Antilipopolysaccharide Factor to IgG By Periodate Oxidation of IgG
This exarnple describes the conjugation of Limulus antilipopolysaccharide factor25 (LALF) to hurnan immunoglobulin by periodate oxidation of the IgG. LALF is a single chain
peptide known to bind and neutralize endotoxin. See H.S. Waver et al. Infection and
T--.---l-..ily 60:2506 (1992). The sequence of the peptide is shown in Figure 13. After
conjugation of the LALF to the IgG was accomplished, the conjugate was tested for binding
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to both E.coli Olll:B4 lipopolysaccharide (LPS) and E.coli HB101. The binding of LALF ~-
and PMB conjugates to LPS was also compared.
a) P~ Lion of LALF-I~G Conju~ate
Purification is achieved by using a speckophotometric LAL assay to monitor
S inhibition of LPS-in-luced Iysate activation. Briefly, amoebocytes from L. polyphemus are
collected under endotoxin-free conditions, Iysed by the addition of ~ tillecl water, and
ce,~lliruged at 5,000 x g for 30 min. The pellet is extracted with 3 M urea. The extract is
filtered through a membrane with a 30,000-Da cutoff and conræ..~ ed by a membrane with a
8,000-Da cutoff. The retentate is applied to a cation exchange column (CM Sepharose)
equilibrated with 3 M urea-10 mM ammonium acetate (pH 5.5) and step eluted with NaCI at
0.15, 0.25, 0.5M. The 0.5 M NaCl peak is directly applied to a C-4 reversed-phase column
(Vydac, Hesperia, Calif.) equilibrated with water-0.2% trifluoroacetic acid. The column is
step eluted with 25, 35, and 50% iSc~lU~ lOl CO1ll;~ ;11g 0.2% trinuoroact;lic acid. The 50%
iSOpl`OIJallOl peak is lyophili7tod and lcco~ iLI.~ed immecli~tely before use. The final product is
typically found to be >95% pure by reversed-phase high-p~.ro,ll,ance liquid chromatography
and SDS-PAGE.
In this case, purified LALF was obtained from Associates of Cape Cod. Lyophili7~LALF was dlssolved at 15 mg/ml in 50 mM sodium acetate, pH 5Ø The MIC of this native
LALF against E; coli HB101 was found to be 16 ug/ml.
10 mg of purified, human IgG (Sigma) was dissolved in 1.0 ml of 50 mM sodium
phosph~te7 pH 7.1. 10.7 mg of sodium meta-periodate was added to the IgG solution and
dissolved with mixin~. The llli~lur~ was in~llb~teA at ambient tt;lll~ Lure on a rotary shaker
at 180 rpm for 30 minlltes
The mixture was applied to a 2.5 x 20 cm column of Spectra-Gel ACA 202
(Spectrum) resin and eluted at 2.0 ml/min with 50 mM sodium phosphate, pH 6.7. The
abso,l~1ce at 280 nm was monitored and 2.0 ml fractions were collected. The fractions
corresponding to the void volume of the column (oxidized IgG) were collected.
0.66 ml of the above 15 mg/ml LALF solution was added dropwise with mixing to 1.3
ml of the oxidized IgG (2.1 mg/ml). The n,ix~ul~ was incubated on a rotary shaker at 180
rpm for ~plu~ ately 18 hours. 39 ,ul of 1.0 N HCl was added with mixing The mixture
was incubated at 180 rpm for 3.0 hours at ambient t~l"peld~LIre.
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~ 21~138~
The mixture was applied to a 1.5 x 20 cm column of Spectra-Gel ACA 202 resin andwas eluted at 1.0 ml/min. with PBS plus 0.1% Tween 20. The eluent corresponding to the
void volume, cont~ining the LALF:IgG conjugate, was collected. The MIC of the LALF:lgG
conjugate against E. coli HBIOI was determined to be 300 mg/ml.
b) Assav of LPS and E. coli HBIOI Bindin~ of LALF-I~G Conju~ate
E. coli 0111 :B4 lipopolysaccaride (LPS) was obtained from Sigma and was dissolved
at 0.02 mg/ml in PBS plus 0.005% thimerosol. E. coli HBIOI was diluted to 10,000,000
CFU/ml in PBS. 100 ~1 aliquots of LPS solution, E. coli HBIOI suspension or PBS were
added to wells of Falcon Pro-Bind 96 well microtiter plates. The plates were incubated for
18 hours at 2-8C . The wells were washed 3 times with PBS. 100 ,ul of PBS plus S mg/ml
BSA (Sigma Chemical Co.) was added to each well of the plates and the plates were
incubated for 2.0 hours at room temperature. The plates were dec~nted and 100 ,ul of sample
(e.g. conjugate, antibody, etc.) was added per well and the plates were incubated at arnbient
temperature for 2.0 hours. The wells were washed 6 times with BBS (0.125 sodium borate,
1.0 M NaCl, pH 8.3) plus 0.5% Tween 20, 3 times with 50 mM sodium carbonate, pH 9.5.
Three Sigma 104 phosphatase substrate tablets were dissolved in 15 ml of 50 mM sodium
carbonate buffer plus 10 mM MgCl~ and added at 100 ml per well. After approximately 20
min~l~ec at ambient temperature, the abso~ ce at 410 nm of each well was determined. The
results are shown in Table 40.
Table 40
Absorbance at 410 nm.
Concentration of A4,0
LALF:IgG, ~g/ml LPS PLATES E. coli HB101 Plates
1.52 057
4.0 1.40 0.32
0.8 0.08 0.06
$Net A4l0 = A4l0 Sample - A4l0 of PBS Coated Plate
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Another LALF:IgG and a PMB:IgG conjugate were prepared as described above.
Binding data for PMB:IgG, LALF:IgG and control IgG to LPS-coated and uncoated (PBS)
plates is given in the table below. The binding of both conjugates to the LPS-coated plates is
significant~ with greater binding of the LALF:IgG to LPS at 4 and 0.8 ~lg/ml. The LALF:IgG
5 and PMB:IgG conjugates bind LPS in a specific manner, since binding of controlunconjugated IgG to LPS-coated plates and binding of both conjugates to PBS-coated plates
are low. Thus, binding is me~ ted by the LALF and PMB portions of the conjugates.
Table 41
Binding of Conjugates to LPS
1 0 Conjugate
Conjugate A4,0
C~ c~ aLion
PMB: IgG LALF: IgG Control IgG
,uglml
LPS Plates PBS Plates LPS Plates PBS Plates LPS Plates PBS Plates
1.56 0.13 1.72 0.25 0.03 0.02
1S 4 0.18 0.23 0.95 0.04 O.Ol 0.02
0.8 0.02 0.04 0.12 0.02 0.01 0.02
0. 1 6 0.0 1 0.02 0.02 0.0 1
EXAMPLE 39
Neutralization of the in vivo Effects of Endotoxin
by LALF-IgG
In order to ~letermine whether LALF-IgG conjugate is capable of neutralizing thelethal effects of endotoxin, the murine model of endotoxic shock discussed in Example 16
(see above) was utili7e~1. Neutralization of endotoxin lethality was assessed by LALF-IgG.
The minim~l effective lethal dose of endotoxin was 15 ng, and the injection volume was 200
25 ~11 per mouse.
E. coli Ol l l :B4 endotoxin (Sigma) was prepared as described in Example 23 (see
above)~ except that the diluent used was PBS and Ø1% Tween-20 (without BSA). The
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endotoxin and variable amounts of LALF-IgG were incubated in varying amounts as in
Example 16(b) (see above). The results are shown in Table 42. As shown in Table 42, the
LALF-IgG conjugate was 100% effective at 5 ~g per mouse in neutralizing the lethal dose of
15 ng endotoxin per mouse.
Table 42
Results of In Vivo Neutralization of Endotoxin
#Survivors/Total p Value
125 ,ug Control IgG 0/5 <0.004
125 ,ug LALF: IgG 5/5 <0.003
25 ~lg LALF: IgG 6/6 <0.004
5 ~lg LALF: IgG 5/5 <0.004
121
