U.S. patent application number 12/555701 was filed with the patent office on 2011-03-10 for human anthrax toxin neutralizing monoclonal antibodies and methods of use thereof.
This patent application is currently assigned to IQ Therapeutics BV. Invention is credited to Christine Cool-Kebbedies, Herman Groen, Kunja S. Slopsema, Hans Westra.
Application Number | 20110059098 12/555701 |
Document ID | / |
Family ID | 35276110 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059098 |
Kind Code |
A1 |
Groen; Herman ; et
al. |
March 10, 2011 |
Human Anthrax Toxin Neutralizing Monoclonal Antibodies and Methods
of Use Thereof
Abstract
The invention fully humanized monoclonal antibodies that
neutralize Bacillus anthracis. Also provided are methods of
treating or preventing a Bacillus anthracis infection. The
invention also provides methods of passive vaccination of a subject
against Bacillus anthracis.
Inventors: |
Groen; Herman; (Groningen,
NL) ; Cool-Kebbedies; Christine; (Groningen, NL)
; Slopsema; Kunja S.; (Drachten, NL) ; Westra;
Hans; (Zwaagwesteinde, NL) |
Assignee: |
IQ Therapeutics BV
Groningen
NL
|
Family ID: |
35276110 |
Appl. No.: |
12/555701 |
Filed: |
September 8, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11072102 |
Mar 3, 2005 |
7658925 |
|
|
12555701 |
|
|
|
|
60549641 |
Mar 3, 2004 |
|
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Current U.S.
Class: |
424/142.1 ;
530/388.15; 530/389.5 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/56 20130101; C07K 2317/76 20130101; C07K 16/1278
20130101; C07K 2317/21 20130101; C07K 2317/565 20130101; A61P 31/04
20180101; A61K 2039/505 20130101 |
Class at
Publication: |
424/142.1 ;
530/388.15; 530/389.5 |
International
Class: |
A61K 39/40 20060101
A61K039/40; C07K 16/12 20060101 C07K016/12; A61P 31/04 20060101
A61P031/04 |
Claims
1. An isolated fully human monoclonal antibody or an
antigen-binding fragment thereof, comprising a heavy chain variable
(VH) domain and a light chain variable (VL) domain, wherein the VH
domain and the VL domain each comprises three complementarity
determining regions 1 to 3 (CDR1-3), and wherein each CDR comprises
the following amino acid sequences: TABLE-US-00018 VH CDR1:
KKPGA[[;]], (SEQ ID NO: 11) VH CDR2: SNAIQWVRQAPGQRLEW[[;]], (SEQ
ID NO: 12) VH CDR3: YMELSSLR[[;]], (SEQ ID NO: 13) VL CDR1:
LTQSPGTLSLS[[;]], (SEQ ID NO: 14) VL CDR2: SYSSLAW[[;]], (SEQ ID
NO: 15) and VL CDR3: GPDFTLTIS[[;]], (SEQ ID NO: 16)
and wherein the antibody binds to an epitope on a region of the
protective antigen polypeptide of Bacillus anthracis.
2. An isolated antibody comprising: a heavy chain comprising a
polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1, and
a light chain comprising a polypeptide encoded by the nucleotide
sequence of SEQ ID NO: 3, wherein the antibody binds protective
antigen polypeptide and neutralizes Bacillus anthracis lethal
toxin.
3. An isolated antibody comprising: a heavy chain comprising the
amino acid sequence of SEQ ID NO: 2, and a light chain comprising
the amino acid sequence SEQ ID NO: 4, wherein the antibody binds
protective antigen polypeptide and neutralizes Bacillus anthracis
lethal toxin.
4. The monoclonal antibody of claim 1, wherein said monoclonal
antibody comprises the amino acid sequences encoded by the
nucleotide sequences selected from SEQ ID NO: 1 and SEQ ID NO:
3.
5. The monoclonal antibody of claim 1, wherein said monoclonal
antibody comprises the amino acid sequences selected from SEQ ID
NO: 2 and SEQ ID NO: 4.
6. A pharmaceutical composition comprising the monoclonal antibody
of any one of claims 1-5, and a pharmaceutically acceptable
carrier.
7. A passive vaccine against Bacillus anthracis, comprising the
pharmaceutical composition of claim 6.
8. A kit comprising, in one or more containers, the monoclonal
antibody of any one of claims 1-5.
9-10. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
11/072,102, filed on Mar. 3, 2005, allowed, which claims priority
to U.S. Provisional Application No. 60/549,641, filed on Mar. 3,
2004. The contents of each of these applications are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to anti-anthrax antibodies
as well as to methods for use thereof.
BACKGROUND
[0003] Bacillus anthracis, the virulent, endospore-forming
bacterium notorious for its recent use as a bioterror weapon, has
plagued humans and livestock from antiquity (Friedlander 2000). The
bacterium was associated with the founding of the sciences of
bacteriology and immunology, highlighted by Pasteur's famous
demonstration of vaccine protection of sheep at Pouilly-le-Fort,
France. Since then, the attention Bacillus anthracis has received
has largely revolved around its properties that make it ideally
suited as a biological weapon as it forms heat resistant spores
that are easy to produce and to transport, and can infect via the
aerosol route.
SUMMARY OF THE INVENTION
[0004] The invention is based in part upon the discovery of fully
human anthrax toxin neutralizing monoclonal antibodies. The
monoclonal antibody (mAb) binds to Bacillus anthracis protective
antigen (PA) polypeptide or the lethal factor (LF) polypeptide and
neutralizes lethal toxin (LeTx). Exemplary monoclonal antibodies
include IQNPA and IQNLF described herein.
[0005] An IQNPA antibody contains a heavy chain polypeptide having
the amino acid sequence of SEQ ID NO:2 or fragment thereof and
nucleic acid sequence of SEQ ID NO:1 or fragment thereof.
Preferably, the IQNPA antibody heavy chain polypeptide has the
amino acid sequence of amino acid residues 1-106 of SEQ ID NO:2 and
more preferably amino acid residues 31-106 of SEQ ID NO:2. An IQNPA
antibody contains a light chain polypeptide having the amino acid
sequence of SEQ ID NO:4 or fragment thereof and nucleic acid
sequence of SEQ ID NO:3 or fragment thereof. Preferably, the IQNPA
antibody light chain polypeptide has the amino acid sequence of
amino acid residues 1-97 of SEQ ID NO:2 and more preferably amino
acid residues 24-97 of SEQ ID NO:2.
[0006] An IQNLF antibody contains a heavy chain polypeptide having
the amino acid sequence of SEQ ID NO:6 or fragment thereof and
nucleic acid sequence of SEQ ID NO:5 or fragment thereof.
Preferably, the IQNLF antibody heavy chain polypeptide has the
amino acid sequence of amino acid residues 1-106 of SEQ ID NO:6 and
more preferably amino acid residues 31-106 of SEQ ID NO:6. An IQNLF
antibody contains a light chain polypeptide having the amino acid
sequence of SEQ ID NO:8 or fragment thereof and nucleic acid
sequence of SEQ ID NO:7 or fragment thereof. Preferably, the IQNLF
antibody light chain polypeptide has the amino acid sequence of
amino acid residues 1-97 of SEQ ID NO:2 and more preferably amino
acid residues 24-97 of SEQ ID NO:2.
[0007] Also included in the invention is an isolated fully human
monoclonal antibody or fragment thereof having a heavy chain with
three CDRs containing the amino acid sequence of KKPGA (SEQ ID
NO:11); SNAIQWVRQAPGQRLEW (SEQ ID NO:12); YMELSSLR (SEQ ID NO:13)
or a light chain with three CDRs containing the amino acid of
LTQSPGTLSLS (SEQ ID NO:14); SYSSLAW (SEQ ID NO:15); GPDFTLTIS (SEQ
ID NO:16). The antibody binds to an epitope on a region of the
protective antigen polypeptide and neutralizes Bacillus anthracis
lethal toxin polypeptide.
[0008] Additionally, the invention provides an isolated fully human
monoclonal antibody or fragment thereof, having a heavy chain with
three CDRs containing the amino acid sequence of VQPGG (SEQ ID
NO:17); SYAMSWVRQAPGKGLEW (SEQ ID NO:18); YMQMNSL (SEQ ID NO:19),
or a light chain with three CDRs containing the amino acid sequence
of TQSPDFQSVSP (SEQ ID NO:20); SSLHWYQ (SEQ ID NO:21); DFTLTINSL
(SEQ ID NO:22). The antibody binds to an epitope on a region of the
lethal factor polypeptide and neutralizes the Bacillus anthracis
lethal toxin.
[0009] Alternatively, the monoclonal antibody is an antibody that
binds to the same epitope as IQNPA, or IQNLF. For example, the
antibody competes with the binding of monoclonal antibody IQNPA to
domain 4 of a protective antigen polypeptide or with IQNLF to a LF
polypeptide.
[0010] By binding to protective antigen is meant that the
monoclonal antibody specifically interacts with a portion of the
protective antigen polypeptide. For example, the monoclonal
antibody binds to domain 4 of a protective antigen polypeptide. By
binding to LF is meant that the mAb specifically interacts with a
portion of the LF polypeptide. By specific interaction it is meant
that the monoclonal antibody has a binding affinity said binding
affinity from about 10.sup.-6 M to about 10.sup.-14 M. Preferably,
the binding affinity is from about 10.sup.-8M to about 10.sup.-12
M. For example, the binding affinity is about 10.sup.-1.degree. M.
Binding affinity is measured by methods known in the art.
[0011] Neutralizing lethal toxin is defined by an increase in cell
survival after exposure to Bacillus anthracis. For example a the
monoclonal antibody decreases complex formation between protective
antigen and lethal factor (LF) or edema factor (EF), thereby
decreasing the translocation of LF and EF into the cellular
cytosol.
[0012] Optionally, the monoclonal antibody inhibits binding of (i)
PA to target cells, (ii) PA to the anthrax toxin receptor (ATR) or
(iii) lethal factor to protective antigen. Alternatively, the mAb
inhibits the binding of PA to PA thus preventing
heptamerization.
[0013] The monoclonal antibody is 2, 4, 8, 10, 15, 20, 25 or more
times effective at neutralizing Bacillus anthracis lethal toxin
polypeptide compared to a naturally occurring Bacillus anthracis
antisera. A naturally occurring Bacillus anthracis antisera is for
example derived from subjects immunized with an anthrax vaccine
such as AVA or AVP. Exemplary Bacillus anthracis antisera includes
AVR414.
[0014] The invention also features methods of preventing or
reducing the risk of developing a Bacillus anthracis infection in a
subject by identifying a subject at risk of developing Bacillus
anthracis infection and administering to the subject a composition
containing a human monoclonal antibody of the invention such as
IQNPA, IQNLF or a combination thereof.
[0015] The invention further features methods of alleviating a
symptom of a Bacillus anthracis infection in a subject by
identifying a subject suffering from a Bacillus anthracis infection
and administering to the subject a composition containing a human
monoclonal antibody of the invention such as an IQNPA or IQNLF
antibody. Optionally, the subject is further administered an
antibiotic such as ciprofloxacin, doxycycline, amoxicillin, or
penicillin G procaine.
[0016] Also included in the invention is a method for passive
immunization a subject against Bacillus anthracis, by administering
to a subject a composition containing the monoclonal antibody of
the invention such as IQNPA or IQNLF mAbs.
[0017] The monoclonal antibody is administered before exposure to
Bacillus anthracis. For example, the monoclonal antibody is
administered at least 1 year before Bacillus anthracis exposure.
The monoclonal antibody is administered 1 day, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months,
3 months or more before exposure to Bacillus anthracis.
Alternatively, the monoclonal antibody is administered after
exposure to Bacillus anthracis. For example, the monoclonal
antibody is administered at least 1 hour, 2 hours, 3 hours, 4
hours, 8 hours or more after Bacillus anthracis exposure. The
monoclonal antibody is administered 1 day, 2 days, 3 days, 4 days
or more after exposure to Bacillus anthracis.
[0018] The subject is suffering from or at risk of developing to
Bacillus anthracis infection. The subject is a mammal such as a
human, a primate, mouse, rat, dog, cat, cow, horse, pig.
[0019] A subject suffering from or at risk of developing Bacillus
anthracis is identified by methods known in the art, e.g., by
isolating B. anthracis from the blood, skin lesions, or respiratory
secretions or by measuring specific antibodies in the blood.
Symptoms of B. anthracis infection include fever (temperature
greater than 100 degrees F.), chills or night sweats, flu-like
symptoms, cough, usually a non-productive cough, chest discomfort,
shortness of breath, fatigue, muscle aches, sore throat, followed
by difficulty swallowing, enlarged lymph nodes, headache, nausea,
loss of appetite, abdominal distress, vomiting, or diarrhea or in
the case of cutaneous contraction, a sore, especially on the face,
arms or hands, that starts as a raised bump and develops into a
painless ulcer with a black area in the center.
[0020] The invention further provides a method of detecting the
presence of a Bacillus anthracis bacterium in a sample, by
contacting a sample known to or suspected of containing a Bacillus
anthracis bacterium with the monoclonal antibody according to the
invention and detecting the presence or absence of an
antibody-bacterium complex. The presence of an antibody-bacterium
complex indicates the sample contains a Bacillus anthracis
bacterium. In contrast, the absence of an antibody-bacterium
complex indicates the sample does not contains a Bacillus anthracis
bacterium. The sample is for example blood, a skin lesion, a
respiratory secretions, vesicular fluid or cerebrospinal fluid. The
sample is contacted with the monoclonal antibody in vitro or in
vivo. The monoclonal antibody is for example IQNPA or IQNLF.
Optionally, the monoclonal antibody is labeled.
[0021] Also provided is a composition, and a passive vaccine
composition containing the monoclonal antibody according to the
invention and a carrier. The invention also includes a kit
containing in one or more containers, the monoclonal antibody
according to the invention.
[0022] In another aspect, the invention provides an isolated
nucleic acid molecule that includes the sequence of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or fragment, homolog,
analog or derivative thereof. The nucleic acid can include, e.g., a
nucleic acid sequence encoding a polypeptide at least 99% identical
to a polypeptide that includes the amino acid sequences of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 or a nucleic acid
sequence encoding a polypeptide at least 95% identical to a
polypeptide that includes the amino acid sequences of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. The nucleic acid can be,
e.g., a genomic DNA fragment, or a cDNA molecule. Preferably, the
nucleic acid is naturally occurring. The invention also provides a
nucleic acid sequence that is complementary to the nucleic acid
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:7.
[0023] Also included in the invention is a vector containing one or
more of the nucleic acids described herein, and a cell containing
the vectors or nucleic acids described herein.
[0024] In another aspect, the invention provides an isolated
polypeptides that includes the sequence of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6 or SEQ ID NO:8 or fragment, homolog, analog or
derivative thereof. The polypeptide can include, e.g., an amino
acid sequence at least 99% identical to a polypeptide of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 or a a polypeptide at
least 95% identical to a polypeptide that includes the amino acid
sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID
NO:8.
[0025] The invention is also directed to host cells transformed
with a vector comprising any of the nucleic acid molecules
described above.
[0026] Also included in the invention is the hybridoma cell line
which was deposited at the American Type Tissue Collection and
assigned ATCC designation ______, ______, ______, or ______ and the
monoclonal antibodies produced the hybridoma cell lines.
[0027] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and are not intended to be
limiting.
[0028] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a bar chart depicting the results of an anthrax
toxin neutralization assay (first experiment) showing that IQNPA-1
and IQNPA-2 are effective at neutralizing toxin in vitro.
[0030] FIG. 2 is a bar chart depicting the results of an anthrax
toxin neutralization assay (second experiment) showing that IQNPA-1
and IQNPA-2 are effective at neutralizing toxin in vitro.
[0031] FIG. 3 is a bar chart depicting the results of an anthrax
toxin neutralization assay showing that IQNPA-1 and IQNPA-2 are
effective at neutralizing toxin in vitro after PA had been allowed
to bind target cells for 2 hours.
[0032] FIG. 4 is a bar chart depicting the results of an anthrax
toxin neutralization assay showing that IQNPA-1 and IQNPA-2 are
effective at neutralizing toxin in vitro after PA had been allowed
to bind target cells for 3 hours.
[0033] FIG. 5 is a bar chart depicting the results of an in vitro
anthrax toxin neutralization assay showing that IQNLF-1 and IQNLF-2
are effective at neutralizing toxin in vitro.
[0034] FIG. 6 is a bar chart depicting the results of an in vivo
anthrax toxin neutralization assay measured after pre-incubation of
target cells and protective antigen showing that IQNPA-1 and
IQNPA-2 are effective at neutralizing toxin in vivo.
[0035] FIG. 7 is a line graph depicting the effect of dilution of
IQNPA-1 on survival of mice passively immunized 2.5 hours before
30.times.MLD anthrax spore challenge.
[0036] FIG. 8 is a line graph depicting the effect of dilution of
IQNPA-2 on survival of mice passively immunized 2.5 hours before
30.times.MLD anthrax spore challenge.
[0037] FIG. 9 is a line graph depicting the effect if dilution of
Control Anthrax sera on survival of mice passively immunized 2.5
hours before 30.times.MLD anthrax spore challenge.
[0038] FIG. 10 is a is a line graph showing the effect of dose on
survival of passively immunized mice measured until 10 days post
challenge.
[0039] FIG. 11 is a probit graph for the HuMabs 10 days post
challenge.
[0040] FIG. 12 is a graph showing the effect of survival of mice
treated once with IQNPA-1, IQNPA-2, or control anthrax serum at
different time points post challenge.
[0041] FIG. 13 is a graph showing the effect of survival of mice
treated once with IQNPA-2, or control anthrax serum at different
time points post challenge.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention is based in part upon the discovery of
fully human monoclonal antibodies, IQNPA-1 and IQNPA-2, which are
specific for B. anthracis protective antigen (PA) and IQNLF-1 and
IQNLF-2 which are specific for B. anthracis lethal factor (LF).
Sequence analysis of IQNPA-1 and IQNPA-2 revealed that the two
antibodies have identical nucleic acid sequences, thus are derived
from the same primary clone. Similar results were found for IQNLF-1
and IQNLF-2. Accordingly, the terms IQNPA-1 and IQNPA-2 and IQNLF-1
and IQNLF-2 are used interchangeably and the antibodies are
respectively referred to herein is IQNPA and IQNLF antibodies.
Hybridoma cells lines producing IQNPA-1 and IQNPA-2 and IQNLF-1 and
IQNLF-2 human monoclonal antibodies are designated IQNPA-1 and
IQNPA-2 and IQNLF-1 and IQNLF-2 hybridomas respectively. The IQNPA
and IQNLF antibodies are collectively referred to herein as IQN
antibodies or huMabs. A deposit of the hybridoma cell lines IQNPA-1
and IQNPA-2 and IQNLF-1 and IQNLF-2 was made at the American Type
Tissue Collection, 12301 Parklawn Drive, Rockville Md. 20852, on
______, and given the ATCC designations ______, ______, ______, and
______ respectively.
[0043] IQNPA antibodies neutralize Bacillus anthracis' Lethal Toxin
in vivo and in vitro. Similarly, IQNLF antibodies neutralize
Bacillus anthracis' Lethal Toxin in vitro. In addition, the IQNPA
antibodies bind to the anthrax toxin receptor (ATR) binding site of
the PA polypeptide. In mice exposed to the IQNPA antibodies prior
to B. anthracis challenge it was found that the antibody treatment
protected 50% of the animals exposed at dosages as low as 2.7 and
4.8 .mu.g/mL, which approximately compares to 0.125 and 0.25 mg/kg
respectively. Moreover, IQNPA antibodies provided 100% protection
when administered up to 36 hrs after exposure to 25-40.times.MLD
Bacillus anthracis spores. In contrast, in untreated mice the
average time-to-death after exposure to these spores is about 55
hrs (2.3 days). These results demonstrate that IQNPA antibodies are
useful for both post-exposure and prophylactic treatment of anthrax
infection.
[0044] B. anthracis is a Gram-positive aerobic, spore-forming,
rod-shaped bacterium. B. anthracis has two major virulence factors,
a tripartite toxin and an anti-phagocytic capsule. The three
proteins of the exotoxin are oedema factor (EF), lethal factor (LF)
and protective antigen (PA). EF and LF enzymatically modify
substrates within the cytosol of mammalian cells; EF is an
adenylate cyclase that causes fluid loss from affected tissues and
inhibition of phagocytosis and LF is a zinc-dependent protease that
cleaves mitogen-activated protein kinase and causes lysis of
macrophages PA derives its name from the fact that it is the key
protective immunogen in the current human vaccines. PA binds to the
anthrax toxin receptor (ATR), whereupon a 20 kDa fragment is
cleaved off, allowing the remaining 63 kDa carboxy-terminal part to
form a membrane-inserting heptamer that binds one to three of the
toxic enzymes, LF, to form lethal toxin (LeTx) or EF, to form edema
toxin (ET), and translocate the toxic enzymes into the cytosol.
LeTx is the major contributor to virulence in infected animals,
which appears to be the central effector of shock and death from
systemic anthrax
[0045] Man generally acquires the disease directly, from contact
with infected livestock or indirectly in industrial occupations
concerned with processing animal products. There are three forms of
the disease that are recognised in humans: cutaneous, inhalational
and gastrointestinal infection. The inhalational form is of most
concern in the context of biological attack. Following inhalation,
spores are phagocytosed by alveolar macrophages and transported to
draining lymph nodes, where the spores germinate and multiplication
of vegetative bacilli occurs. Fatal bacteraemia and toxaemia then
ensue, with a mortality rate in untreated individuals of >80%.
Early treatment is essential as animal studies suggests that the
disease reaches a point at which antibiotics are no longer
effective due to the accumulation of a lethal level of toxin even
though the organism is sensitive to the agent.
[0046] Currently, the US licensed human vaccine (AVA) stimulates
antibodies which neutralize the activity of anthrax toxin (Ivins et
al. 1998). However, it has been shown that it can take several
weeks to mount a significant toxin neutralizing antibody response.
Thus, active immunization is unlikely to be affective within the
time frame on an infection. An alternative approach would be to
administer preformed lethal toxin neutralizing antibodies. It has
been demonstrated across a number of animal studies that preformed
antibodies from animals (e.g., horse) immunized with anthrax
vaccine or PA can passively protect recipients including humans.
However, there are disadvantages to using animal derived sera.
Access is dependant on the continuous availability of
well-immunized, well-maintained and well-controlled animals. The
concentration, efficacy and safety of the material is variable and
uncontrollable. Furthermore, animal derived sera can only be used
effectively for a limited period of time as a neutralizing antibody
response will be mounted against the animal antibodies after
prolonged or repeated usage. More seriously, is the fact that human
recipients may react adversely to the serum, ranging from serum
sickness to anaphylactic shock, or may contract one of a number of
animal pathogens lethal to humans.
[0047] An improvement would be to use polyclonal antibodies
collected from humans who have been immunized with the licensed
human anthrax vaccine (AVA). The advantage of using human-derived
sera is that human sera are less immunogenic than animal sera, thus
there will be a reduced neutralizing antibody response enabling
prolonged and repeated usage and a probable reduction in adverse
responses. In addition, human IgG has a serum half life of 20 days,
one infusion of human antibody could theoretically protect an
exposed individual for several weeks However, the major
disadvantages of this approach, are the risk of disease
transmission, the batch to batch variations in concentrations of
the active ingredients and therefore efficacy of the material and
the inability to raise sufficient amounts of protective sera (with
sufficiently high titers) to protect all people exposed in the
event of a biological warfare or bioterrorist attack.
[0048] Alternatively, the best approach would be to develop human
anthrax toxin neutralizing monoclonal antibodies which could be
used to treat infected individuals and/or to provide "short term
cover" to unprotected individuals who are in or will be deployed
into high risk environments. Further advantages would be the
reduced need to take prophylactic antibiotics, the long term use of
which can cause considerable gastrointestinal dysfunctions well as
the antibodies being effective against antibiotic resistant anthrax
strains. Accordingly, human monoclonal antibodies have all the
advantages of human polyclonal antibodies, but none of the
disadvantages mentioned above and as such would constitute optimal
anti-toxins.
[0049] Accordingly, the invention provides human monoclonal
antibodies that neutralize Bacillus anthracis' Lethal Toxin which
are useful in both reducing the risk of a B. anthracis infection
and for treating an B anthracis infected subject. For example,
monoclonal antibody IQNPA-1 and IQNPA-2 were identified as
antibodies capable of neutralizing Bacillus anthracis' Lethal Toxin
both in vivo and in vitro.
[0050] The IQNPA antibody includes a heavy chain region (SEQ ID
NO:2) encoded by the nucleic acid sequence shown below in SEQ ID
NO:1, and a light chain (SEQ ID NO:4) encoded by the nucleic acid
sequence shown in SEQ ID NO:3 The stop and start codons, defining
the coding region are underlined in SEQ ID NO:1 and SEQ ID
NO:3.
TABLE-US-00001 >IQNPA H.gamma. nucleotide sequence: (SEQ ID NO:
1) GGCCCAGCCGGCCATGGACTGGATCTGGAGGATCCTCTTTTTGGTGGC
AGCAGCCACAGGTGCCCACTCCCAGGTCCAGCTTGTGCAGTCTGGGGC
TGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCCTC
TGGATACACCTTCACTAGCAATGCTATACAATGGGTGCGCCAGGCCCC
CGGACAAAGGCTTGAGTGGGTGGGATGGATCAACGGTGGCGATGGTAA
CACAAAATATTCACAGAAGTTCCAGGGCAGAGTCACCATTAGTAGGGA
CATATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGA
AGACACGGCTGTGTATTACTGTGCGAGACATCGTTTGCAAAGAGGGGG
GTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTC
CACCAAGGGCCCATCGGTCTTCCCCCTGGCACCTTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGAC
CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA
TAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT
CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
GAGCCTCTCCCTGTCTCCGGGTAAATGAGGCCTCCGAGGC >IQNPA H.gamma. amino
acid sequence: (SEQ ID NO: 2)
MDWIWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTF
TSNAIQWVRQAPGQRLEWVGWINGGDGNTKYSQKFQGRVTISRDISAS
TAYMELSSLRSEDTAVYYCARHRLQRGGFDPWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >IQNPA L.kappa. nucleotide
sequence: (SEQ ID NO: 3)
GGCCCAGCCGGCCATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCT
ACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCC
AGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAG
GGCCAGTCAGAGTGTTAGCTACAGCTCCTTAGCCTGGTACCAGCAGAA
ACCTGGCCAGGCTCCCAGCCTCCTCATCTATGGTGCATCCAGCAGGGC
CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGCCAGACTT
CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTA
CTGTCAGCACTATGGTAACTCACCGTACACTTTTGGCCAGGGGACCAA
GCTGGAGATCACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC
CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA
ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA
GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGG GCCTCCGAGGC
>IQNPA L.kappa. amino acid sequence: (SEQ ID NO: 4)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATISCRASQS
VSYSSLAWYQQKPGQAPSLLIYGASSPATGIPDRFSGSGSGPDFTLTI
SRLEPEDFAVYYCQHYGNSPYTFGQGTKLEIKRTVAAPSVFIFPPSDE
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0051] The IQNLF antibody includes a heavy chain region (SEQ ID
NO:6) encoded by the nucleic acid sequence shown below in SEQ ID
NO:5, and a light chain (SEQ ID NO:8) encoded by the nucleic acid
sequence shown in SEQ ID NO:7 The stop and start codons, defining
the coding region are underlined in SEQ ID NO:5 and SEQ ID
NO:7.
TABLE-US-00002 >IQNLF H.gamma. nucleotide sequence: (SEQ ID NO:
5) GGCCCAGCCGGCCATGGAGTTGGGGCTGTGCTGGCTTTTTCTTGTGGC
TATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGTTGGAGTCTGGGGG
AGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTTCTGGCTC
TGGATTCATGTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCC
AGGGAAGGGGCTGGAGTGGGTCTCAGGAATTAGTGGTAGCGGTGGTAC
TACAAACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATATGCAAATGAACAGCCTGAGAGCCGA
GGACACGGCCGTATATTACTGTGCGAAAGATGGGGTATATGGCCGACT
GGGGGGTTCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC
AGCCTCCACCAAGGGCCCATCAGTCTTCCCCCTGGCACCCTCCTCCAA
GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC
CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC
CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA
GAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA
GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA
CAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA
GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA
CGTCTTCTCATGCTCCGTGATGCATGAGGGTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGCCTCCGAGGC >IQNLF H.gamma.
amino acid sequence: (SEQ ID NO: 6)
MELGLCWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCSGSGFMF
SSYAMSWVRQAPGKGLEWVSGISGSGGTTNYADSVKGRFTISRDNSKN
TLYMQMNSLRAEDTAVYYCAKDGVYGRLGGSDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTITSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCDKTIITCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNITFSCSVMHEGLHNHYTQKSLSLSPGK >IQNLF L.kappa.
nucleotide sequence: (SEQ ID NO: 7)
GGCCCAGCCGGCCATGTTGCCATCACAACTCATTGGGTTTCTGCTGCT
CTGGGTTCCAGCCTCCAGGGGTGAAATTGTGCTGACTCAGTCTCCAGA
CTTTCAGTCTGTGAGTCCAAAGGAGAAAGTCACCATCACCTGCCGGGC
CAGCCAGAGCGTTGGTAGTAGCTTACACTGGTACCAGCAGAAACCAGA
TCAGTCTCCAAAGCTCCTCATCAAGTATGCTTCCCAGTCCTTCTCAGG
GGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCCT
CACCATCAATAGCCTGGAAACTGAAGATGCTGCAACGTATTACTGTCA
TCAGAGTAGTAGTTTACCTCTCACTTTCGGCGGAGGGACCAAGGTGGA
GATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATC
TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGCCT CCGAGGC >IQNLF
L.kappa. amino acid sequence: (SEQ ID NO: 8)
MLPSQLIGFLLLWVPASRGEIVLTQSPDFQSVSPKEKVTITCRASQSV
GSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINS
LETEDAATYYCHQSSSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0052] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically bind" or "immunoreacts with" is meant that the
antibody reacts with one or more antigenic determinants of the
desired antigen and does not react (i.e., bind) with other
polypeptides or binds at much lower affinity (K.sub.d>10.sup.-6)
with other polypeptides.
[0053] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs contain an antigen
binding site capable of immunoreacting with a particular epitope of
the antigen characterized by a unique binding affinity for it.
[0054] In general, antibody molecules obtained from humans relate
to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from
one another by the nature of the heavy chain present in the
molecule. Certain classes have subclasses as well, such as
IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans, the light
chain may be a kappa chain or a lambda chain.
[0055] The term "antigen-binding site," or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0056] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin, an
scFv, or a T-cell receptor. Epitopic determinants usually consist
of chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Antibodies may be raised against N-terminal or
C-terminal peptides of a polypeptide. For example, the antibody is
raised against lethal factor, edema factor or protective antigen.
Optimally, the antibody is raised against domain 4 of a protective
antigen peptide. For example, the antibody binds to amino acid
residues 608-735 of a protective antigen peptide. The antibody
binds to an amino acid sequence of: NNIAVGADES VVKEAHREVI
NSSTEGLLLN IDKDIRKILS GYIVEIEDTE GLKEVINDRYDMLNISSLRQ DGKTFIDFKK
YNDKLPLYIS NPNYKVNVYA VTKENTIINP SENGDTSTNG IKKILIFSKK GYEIG (SEQ
ID NO:9) or TNIYTVLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN
SSTEGLLLNI DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY
NDKLPLYISNPNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG (SEQ ID
NO:10).
[0057] As used herein, the terms "immunological binding," and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
The strength, or affinity of immunological binding interactions can
be expressed in terms of the dissociation constant (K.sub.d) of the
interaction, wherein a smaller K.sub.d represents a greater
affinity. Immunological binding properties of selected polypeptides
are quantified using methods well known in the art. One such method
entails measuring the rates of antigen-binding site/antigen complex
formation and dissociation, wherein those rates depend on the
concentrations of the complex partners, the affinity of the
interaction, and geometric parameters that equally influence the
rate in both directions. Thus, both the "on rate constant"
(K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to an anthrax epitope when
the equilibrium binding constant (K.sub.d) is .ltoreq.1 .mu.M,
preferably .ltoreq.100 M, more preferably .ltoreq.10 nM, and most
preferably .ltoreq.100 pM to about 1 pM, as measured by assays such
as radioligand binding assays or similar assays known to those
skilled in the art.
[0058] As used herein, the term "fragment" when used in reference
to a nucleic acid encoding IQNLF or IQNPA is intended to mean a
nucleic acid having substantially the same sequence as a portion of
a nucleic acid encoding IQNLF or IQNPA The nucleic acid fragment is
sufficient in length and sequence to selectively hybridize to an
IQNLF or IQNPA antibody encoding nucleic acid or a nucleotide
sequence that is complementary to an IQNLF or IQNPA antibody
encoding nucleic acid. Therefore, fragment is intended to include
primers for sequencing and polymerase chain reaction (PCR) as well
as probes for nucleic acid blot or solution hybridization. The
meaning of the term is also intended to include regions of
nucleotide sequences that do not directly encode IQNLF or IQNPA
polypeptides such as the introns, and the untranslated region
sequences of the IQNLF or IQNPA encoding gene.
[0059] Those skilled in the art will recognize that it is possible
to determine, without undue experimentation, if a human monoclonal
antibody has the same specificity as a human monoclonal antibody of
the invention (e.g., monoclonal antibody IQNPA and IQNLF) by
ascertaining whether the former prevents the latter from binding to
an anthrax protective antigen polypeptide, lethal factor
polypeptide or a anthrax toxin receptor. If the human monoclonal
antibody being tested competes with the human monoclonal antibody
IQNPA or IQNLF, as shown by a decrease in binding by the human
monoclonal antibody IQNPA or IQNLF, then the two monoclonal
antibodies bind to the same, or a closely related, epitope. Another
way to determine whether a human monoclonal antibody has the
specificity of a human monoclonal antibody of the IQNPA or IQNLF is
to pre-incubate the human monoclonal antibody of the invention with
the protective antigen polypeptide, with which it is normally
reactive, and then add the human monoclonal antibody being tested
to determine if the human monoclonal antibody being tested is
inhibited in its ability to bind the protective antigen
polypeptide. If the human monoclonal antibody being tested is
inhibited then, in all likelihood, it has the same, or functionally
equivalent, epitopic specificity as the monoclonal antibody of the
invention. Screening of human monoclonal antibodies of the
invention, is also carried out by utilizing B. anthracis and
determining whether the test monoclonal antibody is able to
neutralize B. anthracis.
[0060] Various procedures known within the art are used for the
production of the monoclonal antibodies directed against a protein
of anthrax, or against derivatives, fragments, analogs homologs or
orthologs thereof. (See, for example, Antibodies: A Laboratory
Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., incorporated herein by reference).
Fully human antibodies are antibody molecules in which the entire
sequence of both the light chain and the heavy chain, including the
CDRs, arise from human genes. Such antibodies are termed "human
antibodies", or "fully human antibodies" herein. Human monoclonal
antibodies can be prepared by using trioma technique; the human
B-cell hybridoma technique (see Kozbor, et al., 1983 Immunel Today
4: 72); and the EBV hybridoma technique to produce human monoclonal
antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND
CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal
antibodies may be utilized and may be produced by using human
hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus
in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND
CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0061] Also included in the invention are functional fragments of
IQNLF and IQNPA antibodies. By functional fragment is meant an
antibody molecule or fragment thereof having substantially the same
heavy and light chain CDR amino acid sequences as found in IQNLF or
IQNPA. The functional fragment which still retains some of all or
LF or PA binding activity. Such functional fragments can include,
for example, antibody functional fragments such as Fab,
F(ab).sub.2, Fv, single chain Fv (scFv). Other functional fragments
can include, for example, heavy or light chain polypeptides,
variable region polypeptides or CDR polypeptides or portions
thereof so long as such functional fragments retain binding
activity, specificity, LF or PA binding activity or neutralizing
activity. The term is also intended to include polypeptides
encompassing, for example, modified forms of naturally occurring
amino acids such as D-stereoisomers, non-naturally occurring amino
acids, amino acid analogues and mimetics so long as such
polypeptides retain functional activity as defined above. When used
in reference to a functional fragment, not all IQNLF or IQNPA CDRs
need to be represented. Rather, only those CDRs that would normally
be present in the antibody portion that corresponds to the
functional fragment. Similarly, the term "functional fragment when
used in reference to an encoding nucleic acid is intended to refer
to a nucleic acid encoding a antibody or functional fragment being
absent of the substitution of IQNLF or IQNPA amino acids outside of
the CDRs and having substantially the same nucleotide sequence as
the heavy and light chain CDR nucleotide sequences and encoding
substantially the same CDR amino acid sequences as found in IQNLF
or IQNPA The meaning functional fragment is intended to include
minor variations and modifications of the antibody so long as its
function remains uncompromised. Such functional fragments are well
known to those skilled in the art. Such terms are described in, for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York (1989); Molec. Biology and
Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.),
New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics,
22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol.,
178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry,
Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990).
[0062] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "CDR" to describe the non-contiguous antigen
combining sites found within the variable region of both heavy and
light chain polypeptides. This particular region has been described
by Kabat et al., supra, and by Chothia et al., J. Mol. Biol.
196:901-917 (1987) and by MacCallum et al., J. Mol. Biol.
262:732-745 (1996) where the definitions include overlapping or
subsets of amino acid residues when compared against each other.
Nevertheless, application of either definition to refer to a CDR of
IQNLF or IQNPA or variants thereof is intended to be within the
scope of the term as defined and used herein. The amino acid
residues which encompass the CDRs as defined by each of the above
cited references are set forth below in Table A as a
comparison.
TABLE-US-00003 TABLE A CDR Definitions Kabat.sup.1 Chothia.sup.2
MacCallum.sup.3 V.sub.H CDR1 31-35 26-32 30-35 V.sub.H CDR2 50-65
53-55 47-58 V.sub.H CDR3 95-102 96-101 93-101 V.sub.L CDR1 24-34
26-32 30-36 V.sub.L CDR2 50-56 50-52 46-55 V.sub.L CDR3 89-97 91-96
89-96 .sup.1Residue numbering follows the nomenclature of Kabat et
al., supra .sup.2Residue numbering follows the nomenclature of
Clothia et al., supra .sup.3Residue numbering follows the
nomenclature of MacCallum et al., supra
[0063] Sequences corresponding to the IQNPA CDRs include, for
example, those regions defined by Kabat et al., supra, and/or those
regions defined by Chothia et al., supra, as well as those defined
by MacCallum et al., supra. The IQNPA CDR fragments for each of the
above definitions correspond to the nucleotides set forth below in
Table B when numbered in accordance to SEQ ID NO: 1 and 3. The
nucleotide sequence numbering is taken from the primary sequence
shown in SEQ ID NOS:1 and 3 and conforms to the definitions
previously set forth in Table A.
TABLE-US-00004 TABLE B IQNPA CDR Nucleotide Residues Kabat Chothia
MacCallum V.sub.H CDR1 104-118 89-109 101-118 V.sub.H CDR2 161-211
170-181 152-190 V.sub.H CDR3 308-331 311-328 302-328 V.sub.L CDR1
83-115 89-109 101-121 V.sub.L CDR2 161-181 161-169 149-178 V.sub.L
CDR3 278-304 284-301 278-301
[0064] Similarly, the IQNPA CDR fragments for each of the above
definitions correspond to the amino acid residues set forth below
in Table C, when numbered in accordance to SEQ ID NO: 2 and 4. The
amino acid residue number is taken from the primary sequence shown
in SEQ ID NOS:2 and 4 and conforms to the definitions previously
set forth in Table A.
TABLE-US-00005 TABLE C IQNPA CDR Amino Acid Residues Kabat Chothia
MacCallum V.sub.H CDR1 Lys31-Ala35 Ser26-Lys32 Val30-Ala35 V.sub.H
CDR2 Ser50-Trp66 Ile53-Val56 Thr47-Ala59 V.sub.H CDR3 Tyr99-Arg106
Met100-Leu105 Thr97-Leu105 V.sub.L CDR1 Leu24-Ser34 Gln26-Ser32
Thr30-Gly36 V.sub.L CDR2 Ser50-Trp56 Ser50-Ser52 Ser46-Ala55
V.sub.L CDR3 Gly89-Ser97 Asp91-Ile96 Gly89-Ile96
[0065] Thus, the invention also provides nucleic acid fragments
encoding substantially the same amino acid sequence as a CDR of a
IQNLF heavy or light chain polypeptide.
[0066] Sequences corresponding to the IQNLF CDRs include, for
example, those regions defined by Kabat et al., supra, and/or those
regions defined by Chothia et al., supra, as well as those defined
by MacCallum et al., supra. The IQNLF CDR fragments for each of the
above definitions correspond to the nucleotides set forth below in
Table D when numbered in accordance to SEQ ID NO: 5 and 7. The
nucleotide sequence numbering is taken from the primary sequence
shown in SEQ ID NOS:5 and 7 and conforms to the definitions
previously set forth in Table A.
TABLE-US-00006 TABLE D IQNLF CDR Nucleotide Residues Kabat Chothia
MacCallum V.sub.H CDR1 Lys31-Ala35 Ser26-Lys32 Val30-Ala35 V.sub.H
CDR2 Ser50-Trp66 Ile53-Val56 Thr47-Ala59 V.sub.H CDR3 Tyr99-Arg106
Met100-Leu105 Thr97-Leu105 V.sub.L CDR1 Leu24-Ser34 Gln26-Ser32
Thr30-Gly36 V.sub.L CDR2 Ser50-Trp56 Ser50-Ser52 Ser46-Ala55
V.sub.L CDR3 Gly89-Ser97 Asp91-Ile96 G1y89-Ile96
[0067] Similarly, the IQNLF CDR fragments for each of the above
definitions correspond to the amino acid residues set forth below
in Table D, when numbered in accordance to SEQ ID NO: 6 and 8. The
amino acid residue number is taken from the primary sequence shown
in SEQ ID NOS:6 and 8 and conforms to the definitions previously
set forth in Table A.
TABLE-US-00007 TABLE E IQNPA CDR Amino Acid Residues Kabat Chothia
MacCallum V.sub.H CDR1 Val31-Gly35 Ser26-Gln32 Leu30-Gly35 V.sub.H
CDR2 Ser50-Trp66 Met53-Val56 Met47-A1a59 V.sub.H CDR3 Tyr99-Arg106
Met100-Leu105 Thr97-Leu105 V.sub.L CDR1 Thr24-Pro34 Ser26-Val32
Gln30-Glu36 V.sub.L CDR2 Ser50-Gln56 Ser50-Leu52 Gln46-Tyr55
V.sub.L CDR3 Asp89-Thr97 Thr91-Ser96 Asp89-Ser96
[0068] Thus, the invention also provides nucleic acid fragments
encoding substantially the same amino acid sequence as a CDR of a
IQNPA heavy or light chain polypeptide.
[0069] As used herein, the term "substantially" or "substantially
the same" when used in reference to a nucleotide or amino acid
sequence is intended to mean that the nucleotide or amino acid
sequence shows a considerable degree, amount or extent of sequence
identity when compared to a reference sequence. Such considerable
degree, amount or extent of sequence identity is further considered
to be significant and meaningful and therefore exhibit
characteristics which are definitively recognizable or known. Thus,
a nucleotide sequence which is substantially the same nucleotide
sequence as a heavy or light chain of IQNLF or IQNPA and fragments
thereof, refers to a sequence which exhibits characteristics that
are definitively known or recognizable as encoding or as being the
amino acid sequence of IQNLF or IQNPA. Minor modifications thereof
are included so long as they are recognizable as a IQNLF or IQNPA
antibody sequence. Similarly, an amino acid sequence which is
substantially the same amino acid sequence as a heavy or light
chain of IQNLF or IQNPA or functional fragment thereof, refers to a
sequence which exhibits characteristics that are definitively known
or recognizable as representing the amino acid sequence of IQNLF or
IQNPA and minor modifications thereof.
[0070] In addition to conservative substitutions of amino acids,
minor modifications of the IQNLF or IQNPA encoding nucleotide
sequences which allow for the functional replacement of amino acids
are also intended to be included within the definition of the term.
The substitution of functionally equivalent amino acids encoded by
the IQNLF or IQNPA nucleotide sequences is routine and can be
accomplished by methods known to those skilled in the art. Briefly,
the substitution of functionally equivalent amino acids can be made
by identifying the amino acids which are desired to be changed,
incorporating the changes into the encoding nucleic acid and then
determining the function of the recombinantly expressed and
modified IQNLF or IQNPA polypeptide or polypeptides. Rapid methods
for making and screening multiple simultaneous changes are well
known within the art and can be used to produce a library of
encoding nucleic acids which contain all possible or all desired
changes and then expressing and screening the library for IQNLF or
IQNPA polypeptides which retain function. Such methods include, for
example, codon based mutagenesis, random oligonucleotide synthesis
and partially degenerate oligonucleotide synthesis.
[0071] Identification of amino acids to be changed can be
accomplished by those skilled in the art using current information
available regarding the structure and function of antibodies as
well as available and current information encompassing methods for
CDR grafting procedures. For example, CDRs can be identified within
the donor antibody by any or all of the criteria specified in Kabat
et al., supra, Chothia et al., supra, and/or MacCallum et al.,
supra, and any or all non-identical amino acid residues falling
outside of these CDR sequences can be changed to functionally
equivalent amino acids. Using the above described methods known
within the art, any or all of the non-identical amino acids can be
changed either alone or in combination with amino acids at
different positions to incorporate the desired number of amino acid
substitutions at each of the desired positions. The IQNLF or IQNPA
polypeptides containing the desired substituted amino acids are
then produced and screened for retention or augmentation of
function compared to the unsubstituted IQNLF or IQNPA polypeptides.
Production of the substituted IQNLF or IQNPA polypeptides can be
accomplished by, for example, recombinant expression using methods
known to those skilled in the art. Those IQNLF or IQNPA
polypeptides which exhibit retention or augmentation of function
compared to unsubstituted IQNLF or IQNPA are considered to contain
minor modifications of the encoding nucleotide sequence which
result in the functional replacement of one or more amino
acids.
[0072] The functional replacement of amino acids is beneficial
since it allows for the rapid identification of equivalent amino
acid residues without the need for structural information or the
laborious procedures necessary to assess and identify which amino
acid residues should be considered for substitution in order to
successfully transfer binding function from the donor. Moreover, it
eliminates the actual step-wise procedures to change and test the
amino acids identified for substitution. Essentially, using the
functional replacement approach described above, all non-identical
amino acid residues between the donor and the human framework can
be identified and substituted with any or all other possible amino
acid residues at each non-identical position to produce a
population of substituted polypeptides containing all possible or
all desired permutations and combinations. The population of
substituted polypeptides can then be screened for those substituted
polypeptides which retain function. Using the codon based
mutagenesis procedures described above, the generation of a library
of substituted amino acid residues and the screening of
functionally replaced residues has been used for the rapid
production of grafted therapeutic antibodies as well as for the
rapid alteration of antibody affinity. Such procedures are
exemplified in, for example, Rosok et al., J. Biol. Chem.
271:22611-22618 (1996) and in Glaser et al., J. Immunol.
149:3903-3913 (1992), respectively
[0073] Antibodies are purified by well-known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0074] It is desirable to modify the antibody of the invention with
respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating B. anthracis. For
example, cysteine residue(s) can be introduced into the Fc region,
thereby allowing interchain disulfide bond formation in this
region. The homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
(See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes,
J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can
be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. (See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989)).
[0075] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate).
[0076] Enzymatically active toxins and fragments thereof that can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are available for the
production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
[0077] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene tri aminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. (See WO94/11026).
[0078] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the resultant
antibodies or to other molecules of the invention. (See, for
example, "Conjugate Vaccines", Contributions to Microbiology and
Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press,
New York, (1989), the entire contents of which are incorporated
herein by reference).
[0079] Coupling is accomplished by any chemical reaction that will
bind the two molecules so long as the antibody and the other moiety
retain their respective activities. This linkage can include many
chemical mechanisms, for instance covalent binding, affinity
binding, intercalation, coordinate binding and complexation. The
preferred binding is, however, covalent binding. Covalent binding
is achieved either by direct condensation of existing side chains
or by the incorporation of external bridging molecules. Many
bivalent or polyvalent linking agents are useful in coupling
protein molecules, such as the antibodies of the present invention,
to other molecules. For example, representative coupling agents can
include organic compounds such as thioesters, carbodiimides,
succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes
and hexamethylene diamines. This listing is not intended to be
exhaustive of the various classes of coupling agents known in the
art but, rather, is exemplary of the more common coupling agents.
(See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984);
Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta
et al., Science 238:1098 (1987). Preferred linkers are described in
the literature. (See, for example, Ramakrishnan, S. et al., Cancer
Res. 44:201-208 (1984) describing use of MBS
(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S.
Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide
derivative coupled to an antibody by way of an oligopeptide linker.
Particularly preferred linkers include: (i) EDC
(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii)
SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. 21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.
#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce
Chem. Co., Cat. #24510) conjugated to EDC.
[0080] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NHS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, the linker SMPT contains a sterically
hindered disulfide bond, and can form conjugates with increased
stability. Disulfide linkages, are in general, less stable than
other linkages because the disulfide linkage is cleaved in vitro,
resulting in less conjugate available. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
Methods of Treatment
[0081] The invention provides for both prophylactic, post-exposure
prophylactic and therapeutic methods of treating a subject at risk
of (or susceptible to) a Bacillus anthracis infection. Passive
immunization has proven to be an effective and safe strategy for
the prevention and treatment of infectious diseases. Passive
immunization using a neutralizing human monoclonal antibody
provides an immediate treatment strategy for emergency prophylaxis
and treatment of Bacillus anthracis infection. In addition, passive
immunization in a subject exposed to anthrax using a neutralizing
human monoclonal antibody enables the subject to mount an
endogenous protective response against the infecting agent, thereby
rendering active immunization with an anthrax vaccine for future
protection unnecessary. The infecting agent, in all its immunogenic
and non-immunogenic components, will be processed and presented to
the specific immune system, giving rise to long-term protective
immunity.
[0082] By binding to the Bacillus anthracis the IQNPA and IQNLF
antibodies modulate the recognition of the antigen by a subject
immune system, thus inducing the subjects natural immunity.
[0083] A Bacillus anthracis infection is prevented or the risk of
developing a Bacillus anthracis infection is reduced in a subject
by administering to the subject an IQNPA or IQNLF antibody. A
subject at risk for Bacillus anthracis infection includes
individuals who have been, or suspected of having been, or may come
into contact (i.e., exposed) with spores or vegetative of a cells
Bacillus anthracis strain in any way. For example, this may be by
becoming exposed to a deliberately or undeliberately disseminated
cloud of anthrax spores, by touching infected soil, animals, animal
products or by working with spores or vegetative cells in a
laboratory. Administration of a prophylactic agent occurs prior to
the manifestation of symptoms characteristic of the Bacillus
anthracis such that a disease or disorder is prevented or,
alternatively, delayed in its progression or severity.
[0084] Alternatively, the IQNPA or IQNLF antibodies are
administered therapeutically. For example, the IQN antibodies are
administered to a subject after the manifestation of a symptom of a
Bacillus anthracis infection. Optionally, the IQN antibodies are
administered with an antibiotic treatment regime. Treatment reduces
the severity or alleviates a symptom of a Bacillus anthracis
infection Efficaciousness of treatment is determined in association
with any known method for diagnosing or treating a Bacillus
anthracis infection. Alleviation of one or more symptoms of the
Bacillus anthracis infection indicates that the compound confers a
clinical benefit.
[0085] Symptoms of Bacillus anthracis vary depending on how the
disease was contracted (e.g., cutaneous, inhalation or intestinal),
but symptoms usually occur within 7 days. Most (about 95%) anthrax
infections occur when the bacterium enters a cut or abrasion on the
skin, such as when handling contaminated wool, hides, leather or
hair products (especially goat hair) of infected animals. Skin
infection begins as a raised itchy bump that resembles an insect
bite but within 1-2 days develops into a vesicle and then a
painless ulcer, usually 1-3 cm in diameter, with a characteristic
black necrotic (dying) area in the center. Lymph glands in the
adjacent area may swell. About 20% of untreated cases of cutaneous
anthrax will result in death. Initial symptom of inhalation anthrax
may resemble a common cold. After several days, the symptoms may
progress to severe breathing problems and shock. Inhalation anthrax
is usually fatal. The intestinal disease form of anthrax may follow
the consumption of contaminated meat and is characterized by an
acute inflammation of the intestinal tract. Initial signs of
nausea, loss of appetite, vomiting, fever are followed by abdominal
pain, vomiting of blood, and severe diarrhea. Intestinal anthrax
results in death in 25% to 60% of cases.
[0086] Bacillus anthracis infection is diagnosed by isolating B.
anthracis from the blood, skin lesions, or respiratory secretions
or by measuring specific antibodies in the blood of persons with
suspected cases.
[0087] The IQNPA or IPNLF antibodies are administered before
exposure to a Bacillus anthracis. For example, the monoclonal
antibody is administered at least 1 year before Bacillus anthracis
exposure. For example, the monoclonal antibody is administered 1-7
days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or more
before exposure to Bacillus anthracis.
[0088] Alternatively, the monoclonal antibody is administered after
exposure to Bacillus anthracis. For example, the monoclonal
antibody is administered at least 1 hour, 2 hours, 3 hours, 4
hours, 8 hours or more after Bacillus anthracis exposure. The
monoclonal antibody is administered 1 day, 2 days, 3 days, 4 days
or more after exposure to Bacillus anthracis. The IPNPA or IPNLF
antibodies are administered as a single dose. Alternatively, the
IQNPA or IPNLF antibodies are administered in multiple doses. For
example, the IQNPA or IPNLF antibodies are administered in two,
three, four or five doses. The doses are for example, 1, 2, 3, 4 or
more days apart.
[0089] Optionally, the IQNPA or IPNLF antibodies are
co-administered with an antibiotic. Alternatively the IQNPA or
IPNLF antibodies are administered prior to or after administration
of an antibiotic. Antibiotics include for example ciprofloxacin,
doxycycline, amoxicillin, and penicillin G procaine.
[0090] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target. The
amount required to be administered will furthermore depend on the
binding affinity of the antibody for its specific antigen, and will
also depend on the rate at which an administered antibody is
depleted from the free volume other subject to which it is
administered. Common ranges for therapeutically effective dosing of
an antibody or antibody fragment of the invention may be, by way of
nonlimiting example, from about 0.1 mg/kg body weight to about 50
mg/kg body weight. Common dosing frequencies may range, for
example, from twice daily to once a week.
Drug Compositions
[0091] Therapeutic or prophylactic compositions are provided
herein, which generally comprise mixtures of one or more IQNPA or
IPNLF monoclonal antibodies and combinations thereof. The
prophylactic compositions are used to prevent Bacillus anthracis
infection and the therapeutic compositions are used to treat
individuals following Bacillus anthracis infection. Prophylactic
uses include the provision of increased antibody titer to Bacillus
anthracis in a treated subject. In this manner, subjects at high
risk of contracting Bacillus anthracis are provided with passive
immunity to Bacillus anthracis.
[0092] Optionally, the composition is administered in conjunction
with ancillary immunoregulatory agents. For example, cytokines,
lymphokines, and chemokines, including, but not limited to, IL-2,
modified IL-2 (Cys125.fwdarw.Ser125), GM-CSF, IL-12,
.gamma.-interferon, IP-10, MIP1.beta., and RANTES.
[0093] The antibodies or agents of the invention (also referred to
herein as "active compounds"), and derivatives, fragments, analogs
and homologs thereof, are incorporated into pharmaceutical
compositions suitable for administration. Such compositions
typically comprise the antibody or agent and a pharmaceutically
acceptable carrier. As used herein, the term "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Suitable carriers are described
in the most recent edition of Remington's Pharmaceutical Sciences,
a standard reference text in the field, which is incorporated
herein by reference. Preferred examples of such carriers or
diluents include, but are not limited to, water, saline, ringer's
solutions, dextrose solution, and 5% human serum albumin.
[0094] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0095] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0096] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0097] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0098] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives.
[0099] Transmucosal administration can be accomplished through the
use of nasal sprays or suppositories. For transdermal
administration, the active compounds are formulated into ointments,
salves, gels, or creams as generally known in the art.
[0100] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0101] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0102] The pharmaceutical compositions can be included in a
container, pack, kit, or dispenser together with instructions for
administration.
Screening for Bacillus anthracis Bacterium
[0103] Antibodies directed against a Bacillus anthracis are useful
in methods known within the art relating to the localization and/or
quantitation of a Bacillus anthracis bacterium in a sample. A
Bacillus anthracis bacterium is detected in a sample by contacting
a sample known to or suspected of containing the bacterium with an
IQPN antibody and detecting the presence or absence of a
antibody-bacterium complex. The presence of a complex indicates
that the sample contains Bacillus anthracis. Conversely the absence
of a complex indicates that the sample does not contain Bacillus
anthracis. The sample is contacted with the IQN antibody in vitro.
Alternatively, the sample is contacted with the IQN antibody in
vivo.
[0104] An IQN antibody is used to isolate, i.e., detect, a Bacillus
anthracis bacterium in a sample by standard techniques, such as
immunoaffinity, chromatography or immunoprecipitation. An IQN
antibody is also used diagnostically to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to, for
example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a detectable label. The term "labeled", with regard
to the probe or antibody, is intended to encompass direct labeling
of the probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
Indirect labeling includes detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin.
[0105] The term "biological sample" is intended to include tissues,
cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids present within a subject. Included within
the usage of the term "biological sample", therefore, is blood and
a fraction or component of blood including blood serum, blood
plasma, or lymph, skin lesion, respiratory secretions, vesicular
fluid or cerebrospinal fluid.
[0106] Also included in the invention is a kit for detecting the
presence of Bacillus anthracis bacterium in a sample. For example,
the kit can comprise: a labeled compound or agent capable of
detecting Bacillus anthracis (e.g., IQNPA-1, IQNPA-2, IQNLF-1 or
IQNLF-2) in a sample; means for determining the amount of Bacillus
anthracis in the sample; and means for comparing the amount of
Bacillus anthracis in the sample with a standard. The compound or
agent is packaged in a suitable container. The kit further comprise
instructions for using the kit to detect Bacillus anthracis
bacterium in a sample.
[0107] The invention will be further illustrated in the following
non-limiting examples.
Example 1
Preparation of Human Monoclonal Antibodies Against Anthrax
[0108] The following general methods were used to prepare the human
monoclonal antibodies of the invention.
Reagents
[0109] Culture medium DMEM/HAM's F12 (Cambrex Biosciences 12-719F)
is prepared with 1300 mg/l sodium bicarbonate (Merck), 55 mg/l
sodium pyruvate (Fluka), 2.3 mg/12-mercaptoethanol (Merck). 60 mg/L
Gentamycin (Sigma), and 8% Fetal Bovine Serum (Wisent). In fusion
experiments, the medium is further supplemented with 13.61 mg/l
hypoxanthine (Fluka) and 3.83 mg/l thymidine (Fluka). This medium
is referred to as DMEM/HAM's F12/HT.
[0110] Selection of hybridomas is performed in DMEM/HAM's F12/HT
supplemented with 1% of IL-6 containing supernatant of a human
bladder carcinoma cell line T24 (T24CM) and 0.4 M aminopterin
(Sigma). Fusion medium: Ready to use hypo-osmotic buffer
(eppendorf)
Cell Cultures
[0111] Mutant EL-4 thymoma cells, EL-4/B5 are routinely cultured in
DMEM/HAM's F12 supplemented with 8% FCS) between cell
concentrations of 1.times.10.sup.4 to 1.times.10.sup.6 c/ml. If
cells overgrow 1.times.10.sup.6 c/ml, they may lose their B-cell
stimulating activity. Murine myeloma cells NS-1, or xenohybrids
K6H6B5 and PAI-1 were used as fusion partners for murine and human
B-cells respectively. Cells are routinely cultured in DMEM/HAM's
F12/HT supplemented with 10% FCS in concentrations between
5.times.10.sup.4 and 15.times.10.sup.5 cells per ml. One day before
fusion, cultures were split 1:3 to create a log-phase culture at
the day of fusion.
[0112] Preparation of Human T-Cell/Macrophage Supernatant (TSN)
[0113] Freshly isolated mononuclear cells were centrifuged for 10
minutes at 2000 N/kg. Subsequently, B- and T-cells were separated
according to a modification of the method described by Gutierrez et
al. (1979). The pellet was resuspended in 5 ml 100% SIP. Then, a 10
ml layer of 70% SIP followed by a 25 ml layer of 50% SIP were
layered onto the 100% SIP. The gradient was centrifuged for 10 min.
at 25,000 N/kg. The enriched T-cell fraction remaining at the
interface between 70% and 50% SIP is collected and washed twice
with DMEM/HAM's F12 supplemented with 10% FCS. Washed cells are
stimulated for 40-45 h in DMEM/HAM's F12 supplemented with 10% FCS,
5 g/ml PHA (Wellcome) and 10 ng/ml PMA (Sigma). Finally,
supernatant is harvested, filtered through a 0.2 m membrane filter
and stored in aliquots at -70.degree. C.
[0114] EL-4/B-Cell Cultures
[0115] EL-4/B-cell cultures are prepared as described by Zubler et
al. Briefly, crude or purified B-cells are mixed with TSN and
50,000 irradiated (2500 RAD) EL-4/B5-cells in a final volume of 200
l DMEM/HAM's F12 supplemented with 10% FCS in 96-well flat bottomed
tissue culture plates. The optimal amount of TSN is established for
each batch by titration. Usually 10% TSN was sufficient for optimal
stimulation of human B-cells whereas 20% TSN is usually required
for murine B-cells. The cultures are incubated at 37.degree. C.,
with 5% CO2 and 100% humidity. Between Day 8 and Day 12,
supernatants are tested for immunoglobulin production.
[0116] Isolation of Mononuclear Cells
[0117] Blood was drawn from an Anthrax vaccine, 5-10 days after the
latest booster injection. The blood was diluted 50/50 v/v with
sterile PBS and spun down on Isopaque Ficoll (45 min. 400 g). The
mononuclear cells resulting from this procedure were either used
fresh, or frozen into liquid N.sub.2.
[0118] Enrichment of Human B-Cells
[0119] The isolated mononuclear cells (fresh or thawed) were
enriched for B lymphocytes with `untouched B cell` protocol of an
AutoMACS apparatus (Miltenyi Biotec Inc. Auburn, Calif.). These
enriched B cell suspension were used either fresh or thawed from
liquid N.sub.2.
[0120] CD40 Expansion of Lymphocytes
[0121] Enriched B-lymphocytes are expanded using 3T6CD40L expansion
system. Briefly, 3TCD40L cells were harvested at .about.80%
confluence. The culture medium was discarded and EDTA buffer was
added (6 ml in T75 or 3 ml in T25). The cells were resuspended and
irradiated with 100 Gy with a Cs137 source. The cell are washed in
linolea medium and counted. The required concentration in 24 wells:
8.times.10e4 ml; in 96 wells: 2.times.10e5/ml. A similar amount and
volume B cells are added to the to radiated 3T6CD40L cells (i.e.,
1:1). 10 ng/ml rhIL-4 was added to the culture.
[0122] The culture medium was refreshed, -half of the medium+IL-4
every 3 days. Every 7 days freshly radiated 3T6CD40L cells
(2.times.10.sup.5 in 24 wells; 5.times.10e3 in 96 wells) were added
or B cells were harvested and transferred to new plate with freshly
radiated 3T6CD40L (same concentration as start culture). After
.about.5 to 7 days characteristic B cell clumps were visible in
culture. Cultured B cells were harvested between days 5 and 11 by
carefully resuspending cells with a Pasteur pipette.
Panning Procedure
[0123] Six-well culture plates were incubated overnight with 4 ml
per well of a solution containing 1 to 10 ug antigen in 0.05 M
sodium carbonate buffer pH=9.6. Subsequently, the wells were washed
with PBS and directly used for panning experiments or stored at
-20.degree. C. Panning was performed by incubating enriched B-cells
on antigen coated wells for 1 to 2 hour at 37.degree. C., 5%
CO.sub.2 and 100% humidity. After this incubation, the unattached
cells were removed gently by three subsequent washes with PBS.
Then, the antigen-bound, specific B-cells were recovered by
incubating each well with 250 ul PBS containing 1.1 mM Na2EDTA and
0.05% trypsin (Flow, cat no. 16-893-49) pH=7.5 for 2 minutes.
Trypsin treatment was stopped by addition of 5 ml DMEM/HAM's F12
supplemented with 10% FCS. Finally, the entire surface of the wells
was flushed with the medium using a pasteur pipette in order to
remove residual attached B-cells mechanically.
[0124] Electrofusion
[0125] Electrofusion of lymphocyts to K6H6/B5 myeloma cells occurs
in a ratio's ranging from 1:0.5 to 1:10 in 60 .mu.L of fusion
medium in a micro chamber. B-cell cultures were mixed myeloma cells
in 2-ml centrifuge tubes. The cells were rendered serum-free by
washing once with fusion medium. Then, the cell suspension was
centrifuged and the pellet was resuspended in 60 .mu.L fusion
medium at room temperature. The complete cell suspension was
pipetted into the internal space of a fusion chamber. This chamber
consists of two stainless steel, disc-shaped electrodes embedded in
a perspex box. The electrodes are separated by a teflon spacer of
varying diameter and 0.50 mm thickness. Alignment occurs by an
alternating electric field of 1 MHz and 150 V/cm for 30 s,
immediately followed by a peek pulse of 1500 V/cm for 15 .mu.s
Then, immediately a square, high field pulse of 3 kV/cm and 10 s
duration was applied causing cell membrane breakdown. The
alternating field was applied again for 30 s in order to allow
intermingling of cells and resealing of membranes. The contents of
the fusion chamber were transferred to 20 ml selection medium (HAT)
and plated into a 96-well microculture plate. At Day 9, the
cultures were examined for hybridoma growth and the supernatants
were tested for immunoglobulin production.
[0126] PEG Fusion:
[0127] PEG fusion to K6H6/B5 myeloma cells occurs in a 1:1 ratio in
1-1.5 ml PEG 4000 (50%) solution for 3 minutes. After two washing
steps (step one with DMEM/F12 and step two with HT-medium), these
fusion products were seeded into microtiter plates and cultured in
selection medium (HAT) for 9 days.
[0128] Monoclonal antibodies specific for B. anthracis were
screened by methods known in the art.
Example 2
In Vitro Evaluation of the Neutralization Activity of Human
Monoclonal Antibodies Against Anthrax
[0129] The ability hMabs IQNPA-1 and IQNPA-2 to neutralize anthrax
toxin in vitro was determined. Target cells were exposed to B.
Anthracis protective antigen (PA) that had been pre-incubated with
hMabs IQNPA-1 and IQNPA-2 (pre-exposure). Alternatively, target
cells were incubated with PA prior to in exposure to hMabs IQNPA-1
and IQNPA-2 (post-exposure)
[0130] Pre-Exposure
[0131] Briefly, 50,000 RAW cells/well were plated (target cells).
PA and hMab were pre-incubated for 1 hr and then added to RAW cell
culture. Lethal factor (LF) was added and the culture was incubated
for 12 to 15 hrs at 37.degree. C. WST-1 was added and OD450 was
measured at 1 and 2 hrs. This experiment was repeated twice.
Results are shown in FIGS. 1 and 2.
[0132] Post-Exposure
[0133] RAW264.7 cells (target cells) were incubated with PA for
either 2 hours or three hours prior to addition of hMabs IQNPA-1 or
IQNPA-2. LF and IQNPA-1 or IQNPA-2 was added and the culture was
incubated for 12 to 15 hrs at 37.degree. C. WST-1 was added and
OD450 was measured at 1 and 2 hrs. This experiment was repeated
twice. As shown in FIGS. 3-4, hMabs IQNPA-1 and IQNPA-2 are able to
fully neutralize lethal toxin after protective antigen had bound to
the target cells.
[0134] In another experiments, target cells were exposed to PA.
After 1 hour LF-recognizing hMabs IQNLF-1 or IQNLF-2 and LF were
added to the culture and incubated for 15 hours at 37.degree. C.
WST-1 was added and OD450 was measured at 1 hr. The results are
shown in FIG. 5.
Example 3
In Vitro Comparison of Neutralization Activity of Human Monoclonal
Antibodies Against Anthrax to AVR414 Sera
[0135] The toxin neutralization activity of hMabs IQNPA-1 and
IQNPA-2 was compared to the activity of sera from individuals
immunized with the human anthrax vaccine (AVA). This sera is
designated AVA414. The assay was performed at the Center for
Disease Control (Atlanta, Ga.). As shown in FIG. 7, both of hMabs
IQNPA-1 and IQNPA-2 were 25.times. more effective in neutralizing
anthrax toxin in the 99% protection assay.
Example 4
Determination of Affinity of Human Monoclonal Antibodies Against
Anthrax
[0136] The binding kinetics and affinity of neutralizing hMabs
IQNPA-1 and IQNPA-2 to the purified B. anthracis Protective Antigen
were analyzed by surface plasmon resonance (BIAcore 3000, Sweden).
The B. anthracis Protective Antigen was covalently immobilized to a
CM5 sensor chip via amine group using the amine coupling kit
(BIAcore) Binding kinetic parameters were measured with antibodies
at different molar concentrations, and evaluated with the
BIA-evaluation software. The results are shown in Table 1.
TABLE-US-00008 TABLE 1 Kinetic rates and binding affinity of HuMabs
IQNPA-1 and IQNPA-2 ka kd Rmax KA KD (1/Ms) (1/s) (RU) (1/M) (M)
Chi2 IQNPA-1 1.6e5 1.93e-5 939 8.31e9 1.2e-10 89.5 IQNPA-2 1.78e5
1.81e-5 1090 9.86e9 1.01e-10 211
Example 5
Identification of the Epitope Recognized by the Monoclonal
Antibodies Against Anthrax
[0137] To determine the epitopes recognized by HuMabs IQNPA-1 and
IQNPA-2 were screened against domains of recombinant protective
antigen. The following PA domains were screened GST-1; GST1-2;
GST1-2-3; GST1-2-3-4; GST3-4; and GST-4. The starting concentration
of HuMabs were: IQNPA-1, 0.77 mg/ml and IQNPA-2, 0.65 mg/ml.
[0138] Briefly, one microtitre plate was coated for each domain or
combination of domains. Coating concentration were determined for
the molar concentration of each domain. Each Mab was assayed in
duplicate at two starting dilutions of 1:1000 and 1:10 000. The
assay was performed at follows:
1. Plates coated with domains (50 .mu.l per well) and inc. 0/N at
4.degree. C. 2. Wash and block with 5% Blotto for 2 hrs at
37.degree. C. 3. Wash and add Mabs diluted 1:1000 (2 .mu.l into
1.998 ml) and 1: 10 000 (200 .mu.l of 1:1000 into 1.8 ml) in 1%
Blotto. 100 .mu.l into first two wells and serially diluted down
the plate in 50 .mu.l. Incubate overnight at 4.degree. C. 4. Wash.
Add goat anti-human IgG HRP diluted 1:6000. Incubate 1 hr at
37.degree. C. 5. Wash. Add ABTS and read at 10, 20 and 30 minute
intervals.
Plate Coating Concentrations
[0139] Full length rPA plate coating was 5 .mu.g/ml. As it is an 83
kDa protein this equates to 6.02.times.10.sup.-11 moles per ml.
Therefore equimolar plate coatings for each domain have been
calculated from their molecular weight (assuming insignificant
binding of GST tag).
TABLE-US-00009 TABLE 2 Coating Domain conc.(.mu.g/ml) protein vol.
of stock Mwt for 6.02 .times. stock conc. required to Domain
Daltons 10.sup.-11 moles/ml (mg/ml) coat one plate GST 1 57400 3.45
4.4 3.9 .mu.l in 4.9961 ml GST 1 82600 4.97 1.6 15.5 .mu.l in
4.9845 ml to 2 GST 1 94500 5.69 0.9 31.6 .mu.l in 4.9684 ml to 3
GST 1 109000 6.56 1.3 25.2 .mu.l in 4.9748 ml to 4 GST 3 53300 3.21
4.4 3.6 .mu.l in 4.9964 ml to 4 GST 4 41400 2.49 4.6 2.7 .mu.l in
4.9973 ml
[0140] As shown in Table 3, both HuMabs only recognised the PA
domains proteins containing domain 4. The assays against GST-1, 1
to 2 and 1 to 3, were repeated with previously unused plate coating
proteins, at a sample starting dilution of 1:100 and with a
positive control sample to determine that there was not a problem
with the assay. As the end point titres for the three domain
proteins containing domain 4 are approximately the same, this
suggests that the HuMabs specifically bind only to the domain 4
portion of the protective antigen proteins.
TABLE-US-00010 TABLE 3 End point titre Domain IQNPA1 IQNPA2 GST 1
<1:100* <1:100* GST 1 to 2 <1:100* <1:100* GST 1 to 3
<1:100* <1:100* GST 1 to 4 1:256000 1:128000 GST 3 to 4
1:256000 1:256000 GST 4 1:256000 1:128000 *these assays repeated
with Mabs at starting dilution of 1:100
Example 6
In Vivo Evaluation of the Pre-Exposure Protective Efficacy of Human
Monoclonal Antibodies Against Anthrax
[0141] The HuMabs, IQNPA-1 and IQNPA-2 recognized Bacillus
anthracis protective antigen. The HuMabs have been demonstrated to
neutralise the cytotoxicity induced by lethal toxin on a eukaryotic
cell line in vitro. The objective of this study was to determine
whether the inhibition of cytotoxicity seen in vitro correlates
with protective efficacy in viva.
[0142] The HuMabs were each administered to groups of 5 A/J mice
(Harlan UK) intra-peritoneally at a standard dose of 200 .mu.g in
0.1 ml PBS (equates to 10 mg/kg body weight, assuming 20 g mouse).
A reference serum comprising pooled aliquots of rhesus macaque
antiserum (animal references 221 and 224) to rPA was administered
to groups of 5 mice at dose-levels of 200 .mu.g and 500 .mu.g, each
in 0.1 ml PBS. The challenge was administered 2.5 hours after
passive immunisation by the intra-peritoneal route of injection.
The challenge consisted of STI strain of B. anthracis, given at a
dose of 4.18.times.10.sup.4 spores/0.1 ml.
[0143] Initial observation after immunisation showed that the mice
did not react adversely to the foreign IgG administered. Each of
the HuMabs administered at the 200 .mu.g dose-level fully protected
the mice against injected anthrax challenge (Table 4). The macaque
reference serum conferred 40% protection at either the 200 .mu.g or
500 .mu.g dose-levels.
TABLE-US-00011 TABLE 4 Numbers of A/J mice surviving 10 days post
challenge. Treatment Groups Survivors/Number Recipient IgG
Concentration Challenged (%) Mice Item (.mu.g/mouse) Day 10 1
IQNPA-1 200 5/5 (100) 2 IQNPA-2 200 5/5 (100) 3 Reference Item 200
2/5 (40) 4 Reference Item 500 2/5 (40) 5 Naive -- 0/5 (0)
Example 7
In Vivo ED.sub.50 Determination of the Human Monoclonal Antibodies
Against Anthrax
[0144] The objective of this study was to determine the
relationship between dose of HuMab administered and the protection
conferred by passive transfer, in the mouse model and to identify a
50% effective dose (ED50).
[0145] The study was conducted in age-matched female A/J mice
(Harlan UK). Each HuMab was titrated in a dose-response curve to
determine the ED50. Each hMab was diluted as described in Table 5,
to achieve the dose-levels required. Dose-levels of each hMab in
the range 100 .mu.g to 2.5 .mu.g were administered
intra-peritoneally (i.p.) to groups of 5 mice, at 2.5 hours prior
to challenge with 4.64.times.10.sup.4 spores/0.1 ml (approximately
30MLD) of B. anthracis STI strain. A reference serum comprising
pooled aliquots of rhesus macaque antiserum to rPA (animal
references 221 and 224) was administered to groups of 5 A/J mice in
the same dose range. The survival of mice at 10 days post-challenge
was determined.
TABLE-US-00012 TABLE 5 Preparation of dilution series from the Test
and Reference Items to achieve the working dilutions. Item PBS Item
Concentrations (ml) (ml) Dose IQNPA-1 0.77 mg/ml 1.00 0.54 100
.mu.g/0.2 ml 100 .mu.g/0.2 ml 0.50 0.50 25 .mu.g/0.1 ml 100
.mu.g/0.2 ml 0.20 0.80 10 .mu.g/0.1 ml 25 .mu.g/0.1 ml 0.10 0.90
2.5 .mu.g/0.1 ml IQNPA-2 0.65 mg/ml 1.00 0.30 100 .mu.g/0.2 ml 100
.mu.g/0.2 ml 0.50 0.50 25 .mu.g/0.1 ml 100 .mu.g/0.2 ml 0.20 0.80
10 .mu.g/0.1 ml 25 .mu.g/0.1 ml 0.10 0.90 2.5 .mu.g/0.1 ml
Reference - 5.92 mg/ml 0.10 0.49 100 .mu.g/0.1 ml 221 Reference -
6.09 mg/ml 0.10 0.51 100 .mu.g/0.1 ml 224 Pooled 100 .mu.g/0.1 ml
0.20 0.60 25 .mu.g/0.1 ml Reference 100 .mu.g/0.1 ml 0.10 0.90 10
.mu.g/0.1 ml Item (221 + 224) 25 .mu.g/0.1 ml 0.10 0.90 2.5
.mu.g/0.1 ml
[0146] Initial observation after immunisation showed that the mice
did not react adversely to the HuMabs, although there was a
transient adverse reaction to the macaque reference serum
(attributed to the urea content). All the mice had recovered prior
to challenge. Protection was afforded by both HuMabs in the 10 day
assay, in a dose-related manner. The protection afforded was
superior to that of the reference serum which protected 60% of
animals at the top dose-level (Table 6). The effect of dilution of
the HuMabs and the reference serum on survival over ten days is
shown in FIGS. 7-9. The HuMabs performed very similarly, with the
100 .mu.g and 25 .mu.g dose-levels providing 80% protection, and
the 2.5 .mu.g dose-level providing 60% protection. Suprisingly, the
10 .mu.g dose-level of each hMAb provided the minimum protection,
although the group sizes are too small to identify this as a
significant different result from the 2.5 .mu.g dose-level. A
dose-response curve for survival rate for the HuMabs and the
reference antiserum is shown (FIG. 10).
TABLE-US-00013 TABLE 6 Numbers of A/J mice surviving 10 days post
challenge. Treatment Groups Survivors/Number Recipient IgG
Concentration Challenged (%) Mice Item (.mu.g/mouse) Day 10 1
IQNPA-1 100 4/5 (80) 2 25 4/5 (80) 3 10 1/5 (20) 4 2.5 3/5 (60) 5
IQNPA-2 100 4/5 (80) 6 25 4/5 (80) 7 10 2/5 (40) 8 2.5 3/5 (60) 9
Reference 100 3/5 (60) 10 Item 25 0/5 (0) 11 10 0/5 (0) 12 2.5 0/5
(0)
[0147] There was little difference in times to death for each of
the HuMabs. At the 25 .mu.g dose-level, the IQNPA-1 HuMab had a
delayed time to death compared with the IQNPA-2 HuMab, but the
converse held at the 100 .mu.g dose-level.
[0148] The assay design used in this study is a parallel line assay
where the efficacy of the HuMab has been compared with the efficacy
of the reference antiserum. Since the reference antiserum only
protected mice at the highest dose level, the data could not be
included in the statistical analysis. When the data from the HuMabs
was compared for linearity, the slopes generated by titration of
each HuMab did not differ significantly (p>0.05) (Table 7).
Probit analysis has been carried out on the slopes for each HuMAb
(FIG. 11). The ED50 values have been derived for each hMab and
their relative potency has been calculated (Table 8). From this
calculation, it can been seen that the IQNPA-1 HuMab is half as
potent as the IQNPA-2 hMAb.
TABLE-US-00014 TABLE 7 Summary of statistical analysis of the
vaccine dilution slopes with P values. Statistical Test P value
.dagger. Constant 0.476 Dilution 0.144 1 compared with 2 0.741
Equal Slopes 0.932 .dagger. P value < 0.05 significantly
different
TABLE-US-00015 TABLE 8 Calculation of ED50 values and relative
potency of the HuMabs Relative potency Item ED.sub.50 (.mu.g/ml)
IQNPA-1 vs IQNPA 2 IQNPA-1 4.8511 0.5503 IQNPA-2 2.6696
[0149] In summary, the result of this study demonstrate that both
hMab's provide protection against challenge with anthrax in the
mouse model. Probit analysis indicates that IQNPA-2 is twice as
potent as IQNPA-1. (FIG. 11)
Example 8
In Vivo Evaluation of the Post-Exposure Protective Efficacy of
Human Monoclonal Antibodies Against Anthrax
[0150] The objective of this study was to determine whether the
HuMabs could be efficacious when administered by a post-exposure
therapy and if so, to determine the therapeutic window of
post-exposure.
[0151] A/J mice were administered 200 .mu.g of HuMab
intra-peritoneally at 4 hours, 8 hours, 12 hours and 24 hours post
exposure to the STI strain of B. anthracis, given at a dose of
5.6.times.10.sup.4 spores/0.1 ml, equivalent to approximately 40
median lethal doses (MLD). As shown in Table 9 and FIG. 12,
protection against infection was afforded by both HuMabs. The
slight breakthrough that was observed at days 10 and 20 in the
groups dosed with IQNPA2 and IQNPA1 respectively at 8 h
post-challenge is not significant (1 animal out of 5).
[0152] The HuMab IQNPA antibodies tested did not differ
significantly from one another in the protection conferred on
recipient mice by passive transfer. Both IQNPA1 and IQNPA2 are
protective in the mouse model when given at up to 24 h post
exposure to B. anthracis STI strain and protection was maintained
over 20 days post-exposure, when titres of the passively
transferred antibodies would be expected to be in decline. The
Reference Item offered less protection to the mice over time.
[0153] In summary, these studies demonstrated that the fully human
monoclonal antibodies IQNPA-1 and 2 are useful drugs for the
post-exposure and prophylactic treatment of anthrax.
TABLE-US-00016 TABLE 9 Numbers of A/J mice surviving 20 days post
challenge. Recipient Survivors/ Survivors/ Mice Treatment Number
Number Cage No Treatment time post Challenged Challenged (n = 5)
Groups exposure (%) Day 10 (%) Day 20 1 IQNPA-1 +4 h 5/5 5/5 2 200
.mu.g/mouse +8 h 5/5 4/5 3 +12 h 5/5 5/5 4 +24 h 5/5 5/5 5 IQNPA-2
+4 h 5/5 5/5 6 200 .mu.g/mouse +8 h 4/5 4/5 7 +12 h 5/5 5/5 8 +24 h
5/5 5/5 9 Reference Item +4 h 4/5 4/5 10 (Pooled samples +8 h 4/5
3/5 11 221 + 224) +12 h 3/5 2/5 12 +24 h 1/5 0/5
[0154] In a second study A/J mice were administered 180 .mu.g of
HuMab intra-peritoneally at 24 hours, 36 hours, and 48 hours or
1000 .mu.g of HuMab intra-peritoneally at 4 hours, 8 hours, 12
hours, and 24 hours post exposure to the STI strain of B.
anthracis, given at a dose of 3.4.times.10.sup.4 spores/0.1 ml,
equivalent to approximately 25 median lethal doses (MLD). As shown
in Table 10 and FIG. 13, protection against infection was afforded
by IQNPA-2 over 10 days post-challenge. Total protection was
observed with the mice dosed with 100 .mu.g of the Test Item at 24
hours post challenge. Total protection was extended to 36 hours
post challenge when the mice were dosed with 180 .mu.g of the Test
Item (Table 10). FIG. 13 shows the effect on mouse survival when
vaccinated at different timepoints post challenge. The Reference
Item offered less protection to the mice over time.
TABLE-US-00017 TABLE 10 Numbers of A/J mice surviving 10 days post
challenge. Recipient Mice Survivors/Number Cage No Treatment
Treatment time Challenged (%) (n = 5) Groups post exposure Day 10 1
IQNPA-2 +24 h 5/5 2 180 .mu.g/mouse +36 h 5/5 3 +48 h 3/5 4 IQNPA-2
+4 h 4/5 5 100 .mu.g/mouse +8 h 5/5 6 +12 h 5/5 7 +24 h 5/5 8 +36 h
2/5 9 +48 h 3/5 10 Reference Item +8 h 3/5 11 (Pooled samples +12 h
2/5 12 221 + 224) +24 h 1/5
Other Embodiments
[0155] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. Other aspects, advantages, and modifications considered
to be within the scope of the following claims. The claims
presented are representative of the inventions disclosed
herein.
* * * * *