U.S. patent application number 10/334152 was filed with the patent office on 2003-09-11 for detection and treatment of infections with immunoconjugates.
Invention is credited to Goldenberg, David M..
Application Number | 20030170697 10/334152 |
Document ID | / |
Family ID | 27016671 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170697 |
Kind Code |
A1 |
Goldenberg, David M. |
September 11, 2003 |
Detection and treatment of infections with immunoconjugates
Abstract
A method of targeting a diagnostic or therapeutic agent to a
focus of infection comprises injecting a patient infected with a
pathogen parenterally with an antibody conjugate which specifically
binds to an accessible epitope of the pathogen or of a
pathogen-associated antigen accreted at the focus of infection, the
antibody conjugate further comprising a bound diagnostic or
therapeutic agent for detecting, imaging or treating the infection.
Polyspecific composite conjugates enhance the efficacy of the
method, which is especially useful for treating infections that are
refractory towards systemic chemotherapy.
Inventors: |
Goldenberg, David M.;
(Medham, NJ) |
Correspondence
Address: |
Stephen B. Maebius
Foley & Lardner
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5143
US
|
Family ID: |
27016671 |
Appl. No.: |
10/334152 |
Filed: |
December 31, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10334152 |
Dec 31, 2002 |
|
|
|
10125342 |
Apr 19, 2002 |
|
|
|
6548275 |
|
|
|
|
10125342 |
Apr 19, 2002 |
|
|
|
09935567 |
Aug 24, 2001 |
|
|
|
09935567 |
Aug 24, 2001 |
|
|
|
08037659 |
Mar 22, 1993 |
|
|
|
5332567 |
|
|
|
|
08037659 |
Mar 22, 1993 |
|
|
|
07840591 |
Feb 18, 1992 |
|
|
|
07840591 |
Feb 18, 1992 |
|
|
|
07399566 |
Aug 24, 1989 |
|
|
|
10334152 |
Dec 31, 2002 |
|
|
|
08158782 |
Dec 1, 1993 |
|
|
|
6319500 |
|
|
|
|
Current U.S.
Class: |
435/6.16 ;
424/1.49; 424/159.1; 424/164.1; 424/9.34 |
Current CPC
Class: |
A61K 51/10 20130101;
A61K 47/68 20170801; A61K 2123/00 20130101; A61K 51/1018
20130101 |
Class at
Publication: |
435/6 ; 424/1.49;
424/9.34; 424/159.1; 424/164.1 |
International
Class: |
A61K 051/00; A61K
039/42; A61K 039/40; A61K 049/00; A61M 036/14; C12Q 001/68; A61B
005/055 |
Claims
What is claimed is:
1. A method of targeting a diagnostic or therapeutic agent to a
focus of infection, which comprises injecting a patient infected
with a pathogen parenterally with an antibody conjugate which
specifically binds to an accessible epitope of said pathogen or of
a pathogen-associated antigen accreted at said focus of infection,
said antibody conjugate further comprising a bound diagnostic or
therapeutic agent for detecting, imaging or treating said
infection.
2. The method of claim 1, wherein said agent is a diagnostic agent
selected from the group consisting of a radioisotope and a magnetic
resonance image enhancing agent.
3. The method of claim 1, wherein said agent is a therapeutic
radioisotope or boron addend.
4. The method of claim 1, wherein said agent is an anti-pathogenic
drug or cytotoxic agent.
5. The method of claim 1, wherein said antibody conjugate
specifically binds to an accessible epitope of said pathogen or
pathogen-associated antigen which is not saturated or blocked by
the patient's native antibodies.
6. The method of claim 5, wherein said antibody conjugate comprises
a monoclonal antibody.
7. The method of claim, 1, wherein said antibody conjugate is a
polyspecific conjugate which specifically binds to a plurality of
accessible epitopes of said pathogen or antigen.
8. The method of claim 7, wherein said polyspecific antibody
conjugate is an antiserum.
9. The method of claim 8, wherein said antiserum is affinity
purified by removal of antibodies which bind to antigens associated
with said pathogen circulating at a significant level in the
patient's bloodstream.
10. The method of claim 8, wherein said antiserum is affinity
purified by contact with bound pathogen or pathogen-associated
antigens, and subsequent recovery of antiserum enriched in
antibodies that bind to said pathogen or pathogen-associated
antigens.
11. The method of claim 7, wherein said polyspecific conjugate is a
mixture of monoclonal antibodies.
12. The method of claim 7, wherein said polyspecific conjugate is a
chemically linked molecule having a plurality of antigen binding
sites for said plurality of epitopes.
13. The method of claim 1, wherein said pathogen is a virus.
14. The method of claim 13, wherein said virus is an RNA virus.
15. The method of claim 13, wherein said virus is a DNA virus.
16. The method of claim 13, wherein said virus is selected from the
group consisting of human immunodifficency virus (HIV), herpes
virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B
virus, Sendai virus, feline leukemia virus, Reo virus, polio virus,
human serum parvo-like virus, simian virus 40, respiratory
syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,
Dengue virus, rubella virus, measles virus, adenovirus, human
T-cell leukemia viruses, Epstein-Barr virus, murine leukiemia
virus, mumps virus, vesicular stomatitis virus, Sindbis virus,
lymphocytic choriomeningitis virus, wart virus and blue tongue
virus.
17. The method of claim 1, wherein said pathogen is a
bacterium.
18. The method of claim 17, wherein said bacterium is selected from
the group consisting of Streptococcus agalactiae, Legionella
pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria
gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis
influenzae-B, Treponema pallidum, Lyme disease spirochetes,
Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus,
Mycobacterium tuberculosis and Tetanus toxin.
19. The method of claim 1, wherein said pathogen is a
protozoan.
20. The method of claim 19, wherein said protozoan is selected from
the group consisting of Plasmodium falciparum, Plasmodium vivax,
Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,
Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,
Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca
volvulus, Leishmania tropica, Trichinella spiralis, Onchocerca
volvulus, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia
saginata, Echinococcus granulosus and Mesocestoides corti.
21. The method of claim 1, wherein pathogen is a helminth.
22. The method of claim 1, wherein said pathogen is mycoplasma.
23. The method of claim 22, wherein said mycoplasma is selected
from the group consisting of Mycoplasma arthritidis, M. hyorhinis,
M. orale,. M. arginini, Acholeplasma laidlawii, M. salivarium and
M. pneumoniae.
24. The method of claim 1, which further comprises administering to
said patient, at a time after administration of said conjugate
sufficient to optimize uptake of said conjugate at the site of said
infection, an amount of a second antibody that specifically binds
to said conjugate sufficient to reduce the amount of said conjugate
in circulation by 10-85% within 2-72 hours.
25. The method of claim 1, wherein said agent is a therapeutic
antibiotic or cytotoxic agent that causes hematopoietic toxicity as
a side effect of its administration, and wherein said method
further comprises administering to said patient, at a time prior
to, concomitant with or subsequent to administration of said
therapeutic conjugate, an amount of a cytokine sufficient to
mitigate or prevent the hemtopoietic toxicity of said agent.
26. An antibody conjugate for targeting a focus of infection,
comprising an antibody or antibody fragment which binds to an
epitope on a single species of pathogen or an antigen derived
therefrom, said antibody or antibody fragment being conjugated to
at least one diagnostic or therapeutic agent.
27. An antibody conjugate for targeting a focus of infection,
comprising an immunoreactive composite of a plurality of chemically
linked antibodies or antibody fragments which bind to a plurality
of epitopes on a single species of pathogen or an antigen derived
therefrom, said composite being conjugated to at least one
diagnostic or therapeutic agent.
28. A sterile injectable preparation for targeting a focus of
infection in a human patient, comprising the antibody conjugate of
claim 26, in combination with a pharmacologically acceptable
sterile injection vehicle.
29. A kit for use in preparing a sterile injectable preparation for
targeting a focus of infection in a human patient, comprising in
suitable containers, the antibody conjugate of claim 26 and a
pharmacologically acceptable sterile injection vehicle.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to reagents and methods for targeting
a diagnostic and/or therapeutic agent to a focus of pathogenic
infection by using as the targeting vehicle an antibody conjugate
that specifically binds to one or more accessible epitopes of the
pathogen or of a pathogen-associated antigen.
[0002] Drug therapy against pathogens is conventionally effected by
means of systemic administration of the drug in order to achieve a
blood level which is toxic to the pathogen wherever it is harbored
in the body. Thus, a certain blood level is necessary in order to
provide the proper concentration of the drug at the site of
infection. This requires high doses and often does not achieve the
desired toxicity without resulting in unacceptably adverse
side-effects to the patient, since many of these drugs have general
cytotoxic properties.
[0003] The development and description of murine monoclonal
antibodies (MAbs) against infectious organisms has been the subject
of a number of reviews (e.g., M. C. Harris et al., Indian J.
Pediatr., 54:481-488, 1987; S. Cohen, Brit. Med. Bull., 40:291-296,
1984; R. A. Polin, Eur J. Clin. Microbiol., 3:387-398, 1984; R. C.
Nowinski et al., Science, 219:637-644, 1983; Part V, Monoclonal
Antibodies to Microorganisms, Chapters 17-20, inclusive, In: R. H.
Kennett et al., (eds.), Monoclonal Antibodies. Hybridomas: A New
Dimension in Biological Analyses, New York and London, Plenum
Press, 1980, pp. 295362). These papers, and others in this area,
have been concerned with the use of such monoclonal antibody
reagents for improved diagnostic tests for the infectious
microorganisms, including bacteria, viruses, protozoa and
helminths.
[0004] It has been proposed that these MAbs can be used as such for
both the diagnosis and therapy of certain bacterial diseases, such
as group B streptococcal infections (Harris, cited above), but
exclusively as diagnostic agents in viral diseases (Harris, cited
above). In the case of group B streptococcal infections, MAbs were
used in rodents to treat the infection, and it was found in these
limited trials that only when the MAbs were infused early after
infection was an effect achieved; at 6 hours or later, no survival
of the animal occurred (Christensen et al., Pediatric Res.,
18:1093-1096, 1984). In the case of malarial parasites, it has been
shown that the Fab fragments of a monoclonal antibody directed
against the surface coat of malaria sporozoites is active in
protecting mice against malarial infection, indicating that it
blocks attachment of sporozoites to host receptor cells (P.
Potocnjak et al., J. Exp. Med., 151:1504-1513, 1980). This further
indicates, since it is achieved by the immunoglobulin molecule
lacking the Fc portion, that the protective antibody action is
independent of complement or cells.
[0005] These animal experiments indicate that early infections can
be affected by the use of organism-specific MAbs in well-controlled
laboratory experiments involving certain bacteria and parasites.
Despite these reports a number of years ago, MAbs have not been
shown to have a therapeutic role in infectious diseases in humans.
One major reason has been that such MAbs exert a protective action
only in specific, usually early stages of infection, being less
able to interact with the infectious organisms when they have
disseminated into tissue reservoirs that are less accessible to
interaction with the injected MAbs. Use of such MAbs to form
therapeutic conjugates is not suggested by the references.
[0006] A need therefore exists for a method of targeting a
diagnostic agent, e.g., an imaging agent, or a therapy agent, e.g.,
a drug or radioisotope, to a focus of infection with higher
efficiency and an enhanced therapeutic index to permit more
effective diagnosis and/or treatment of the infection.
OBJECTS OF THE INVENTION
[0007] One object of the present invention is to provide an
effective and selective method of targeting a focus of
infection.
[0008] Another object of the invention is to provide diagnostic and
therapeutic agents with high specificity for foci of infection.
[0009] Another object of the invention is to provide an alternative
or adjunct to chemotherapy for treatment of certain microbial and
parasitic infections that are not amenable or relatively
unresponsive to chemotherapy and which cause debilitating or
life-threatening illness.
[0010] Another object of the invention is to improve the
therapeutic index of a chemotherapeutic agent and/or
radiopharmaceutical.
[0011] Other objects of the present invention will become more
apparent to those of ordinary skill in the art in light of the
following discussion.
SUMMARY OF THE INVENTION
[0012] These and other objects of the present invention are
achieved by providing a method of targeting a diagnostic or
therapeutic agent to a focus of infection, which comprises
injecting a patient infected with a pathogen parenterally with an
antibody conjugate which specifically binds to an accessible
epitope of said pathogen or of a pathogen-associated antigen
accreted at said focus of infection, said antibody conjugate
further comprising a bound diagnostic or therapeutic agent for
detecting, imaging or treating said infection.
[0013] The invention further provides polyspecific or monospecific
antibody conjugates for targeting foci of infection, comprising an
immunoreactive component including at least one substantially
monospecific antibody or antibody fragment, conjugated to at least
one diagnostic or therapeutic agent, wherein the antibody or
antibody fragment specifically binds to an accessible epitope of
the pathogen or of a pathogen-associated antigen. These can be
provided in the form of sterile injectable preparations and kits
for use in practicing the foregoing method.
DETAILED DESCRIPTION
[0014] Antimicrobial agents are conventionally classified into four
main groups, based upon their affecting (1) bacterial cell-wall
synthesis, (2) the cytoplasmic membrane, (3) protein synthesis, and
(4) nucleic acid synthesis, and often each of these groups can be
subdivided into several classes. Reviews of antimicrobial
chemotherapy can be found in the chapter by M. P. E. Slack (In:
Oxford Textbook of Medicine, Second Ed., Vol. I, edited by D. J.
Weatherall, J. G. G. Lidingham, and, D. A. Warrell, pp. 5.35-5.53;
Oxford University Press, Oxford/Melbourne/New York, 1987) and in
Section XII, Chemotherapy of Microbial Diseases (In: Goodman and
Gilman's The Pharmacological Basis of Therapeutics, 6th Ed.,
Goodman et al., Eds., pp. 1080-1248; Macmillan Publishing Co., New
York, 1980).
[0015] As indicated in these texts, some antimicrobial agents are
selective in their toxicity, since they kill or inhibit the
microorganism at concentrations that are tolerated by the host
(i.e., the drug acts on microbial structures or biosynthetic
pathways that differ from those of the host's cells). Other agents
are only capable of temporarily inhibiting the growth of the
microbe, which may resume growth when the inhibitor is removed.
Often, the ability to kill or inhibit a microbe or parasite is a
function of the agent's concentration in the body and its
fluids.
[0016] Whereas these principles and the available antimicrobial
drugs have been successful for the treatment of many infections,
particularly bacterial infections, other infections have been
resistant or relatively unresponsive to systemic chemotherapy,
e.g., viral infections and certain fungal, protozoan and parasitic
infections.
[0017] As used herein, "microbe" denotes virus, bacteria,
rickettsia, mycoplasma, protozoa and fungi, while "pathogen"
denotes both microbes and infectious multicellular invertebrates,
e.g., helminths, spirochetes and the like.
[0018] Virus can infect host cells and "hide" from circulating
systemic drugs. Even when viral proliferation is active and the
virus is released from host cells, systemic agents can be
insufficiently potent at levels which are tolerated by the
patient.
[0019] Similarly, a number of fungal, protozoan and parasitic
infections have been resistant to systemic drug therapy, at least
in part because an effective antipathogenic dose of a drug has been
above the level which is tolerated by the patient or because the
infection was difficulty accessible to conventional systemic routes
of drug administration.
[0020] The present invention resolves many of the problems involved
in the treatment of infections that are refractive to conventional
drug therapy by using very specific antibodies made against
microbial or parasitic antigens in order to target an effective
radionuclide and/or chemical agent to foci of infection, thereby
selectively killing the pathogen. A targeted drug can have enhanced
effectiveness due to significantly increased concentration at the
target site relative to the rest of the body. The targeting
antibody is able to bind to an accessible epitope of the pathogen
or to antigens shed by the pathogen or resulting from its
fragmentation and/or destruction, and which accrete at a focus of
infection. The epitope can be on the surface of the pathogen or
antigen or at an accessible locus in the pathogen. The therapeutic
component of the conjugate is thereby localized at the target site
with higher efficiency and an enhanced target to non-target
ratio.
[0021] Targeting is also effective for diagnostic agents,
especially agents for scintigraphic imaging or magnetic resonance
imaging (MRI) of sites of infection. This is helpful to the
treating physician for evaluation of the patient's level and stage
of infection and for designing and monitoring treatment
protocols.
[0022] The antibody component of the conjugate can be a single
monospecific antibody reacting with one epitope of the pathogen or
its antigen. In such a case, it is preferable for the antibody to
bind to an epitope that is different and separate from epitopes to
which the patient's own antibodies bind. This will avoid the
problem-of blocking due to saturation of the pathogen or its
antigen with native antibodies, and consequent inhibition of
targeting.
[0023] Alternatively, the antibody component can be polyspecific,
i.e., it can include a plurality of antibodies that bind to a
plurality of epitopes on the pathogen or its antigen. The
polyspecific antibody component can be a polyclonal antiserum,
preferably affinity purified, from an animal which has been
challenged with an immunogenic form of the pathogen or its antigen
and stimulated to produce a plurality of specific antibodies
against the pathogen or its antigen. Another alternative is to use
an "engineered polyclonal" mixture, which is a mixture of
monoclonal antibodies with a defined range of epitopic
specificities.
[0024] In both types of polyclonal mixtures, it can be advantageous
to chemically link polyspecific antibodies together to form a
single polyspecific molecule capable of binding to any of several
epitopes. Conjugation of such a polyspecific targeting molecule
with a diagnostic or therapeutic agent increases the likelihood
that the agent will reach the site of infection, thereby increasing
the target to non-target ratio and the efficacity of the vehicle.
One way of effecting such a linkage is to make bivalent
F(ab').sub.2 hybrid fragments by mixing two different F(ab').sub.2
fragments produced, e.g., by pepsin digestion of two different
antibodies, reductive cleavage to form a mixture of Fab' fragments,
followed by oxidative reformation of the disulfide linkages to
produce a mixture of F(ab').sub.2 fragments including hybrid
fragments containing a Fab' portion specific to each of the
original antigens. Methods of preparing such hybrid antibody
fragments are disclosed in Feteanu, "Labeled Antibodies in Biology
and Medicine" pages 321-323 (McGraw-Hill Int. Bk. Co., New York et
al, 1978); Nisonoff et al, Arch Biochem. Biophys., 93, 470 (1961);
and Hammerling et al, J. Exp. Med., 128, 1461 (1968); and in U.S.
Pat. No. 4,331,647.
[0025] Other methods are known in the art to make bivalent
fragments that are entirely heterospecific, e.g., use of
bifunctional linkers to join cleaved fragments. Recombinant
molecules are known that incorporate the light and heavy chains of
an antibody, e.g., according to the method of Boss et al., U.S.
Pat. No. 4,816,397. Analogous methods of producing recombinant or
synthetic binding molecules having the characteristics of
antibodies are included in the invention. More than two different
monospecific antibodies or antibody fragments can be linked using
various linkers known in the art.
[0026] The immunological profile of the substantially monospecific,
preferably monoclonal, antibodies used to make the polyspecific
conjugates of the present invention can be adjusted to ensure
optimal binding to the pathogen or its antigens by mixing the
antibody specificities for different antigens and their epitopes in
particular cases of infections, as well as of binding constants for
the target epitopes, so as to fine tune the selectivity and
targeting efficiency of the reagent according to the invention.
[0027] An imaging reagent according to the invention can comprise
bispecific, trispecific or, more generally, polyspecific
antibody/fragment conjugates, further comprising an imaging
radioisotope or paramagnetic species.
[0028] The antibody component of the conjugate can include whole
antibodies, antibody fragments, or subfragments. Use of the term
"antibody" herein will be understood to embrace whole antibodies,
antibody fragments and subfragments and thus to be equivalent to
the term "antibody/fragment" which is used interchangeably therefor
in this discussion, unless otherwise noted. Antibodies can be whole
immunoglobulin (IgG) of any class, e.g., IgG, IgM, IgA, IgD, IgE,
chimeric antibodies or hybrid antibodies with dual or multiple
antigen or epitope specifities, or fragments, e.g., F(ab').sub.2,
Fab', Fab and the like, including hybrid fragments, and
additionally includes any immunoglobulin or any natural, synthetic
or genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex.
[0029] Antibodies can include antiserum preparations from a variety
of commonly used animals, e.g., goats, primates, donkeys, swine,
rabbits, horses, hens, guinea pigs, rats or mice, and even human
antisera after appropriate selection and purification. The animal
antisera are raised by inoculating the animals with an immunogenic
form of the pathogen or its antigen, by conventional methods,
bleeding the animals and recovering serum or an
immunoglobulin-containing serum fraction.
[0030] The antiserum is preferably affinity-purified by
conventional procedures, e.g., by binding antigen to a
chromatographic column packing, e.g., Sephadex, passing the
antiserum through the column, thereby retaining specific antibodies
and separating out other immunoglobulins and contaminants, and then
recovering purified antibodies by elution with a chaotropic agent,
optionally followed by further purification, e.g., by passage
through a column of bound blood group antigens or other
non-pathogen species. This procedure may be preferred when
isolating the desired antibodies from the serum of patients having
developed an antibody titer against the pathogen in question, thus
assuring the retention of antibodies that are capable of binding to
exposed epitopes.
[0031] Hybridoma-derived monoclonal antibodies (human, monkey, rat,
mouse, or the like) are also suitable for use in the present
invention and have the advantage of high specificity. They are
readily prepared by what are now generally considered conventional
procedures for immunization of mammals with an immunogenic antigen
preparation, fusion of immune lymph or spleen cells, with an
immortal myeloma cell line, and isolation of specific hybridoma
clones. More unconventional methods of preparing monoclonal
antibodies are not excluded, such as interspecies fusions and
genetic engineering manipulations of hypervariable regions, since
it is primarily the antigen specificity of the antibodies that
affects their utility in the present invention. Human lymphocytes
can be fused with a human myeloma cell line to produce antibodies
with particular specificities, preferably to epitopes which are not
masked by circulating antibodies to the major antigenic sites on
the pathogen.
[0032] The present invention also envisions the use of
antigen-specific fragments to create the polyspecific antibody
conjugate. Antibody fragments can be made by pepsin or papain
digestion of whole immunoglobulins by conventional methods. It is
known that antibody fragments may be produced by enzymatic cleavage
of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepain produces two monovalent Fab fragments and an
Fc fragment directly. These methods are described, inter alia, by
Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647 and
references contained therein, which patents are incorporated herein
in their entireties by reference, and in Nisonoff et al, Arch.
Biochem. Biophys., 89, 230 (1960); Porter, Biochem. J., 73, 119
(1959); and Edelman et al, in "Methods in Immunology and
Immunochemistry", Vol. 1, 422 (Acad. Press, 1967), and are
conventional in the art.
[0033] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments
retain specificity to the pathogen or antigen against which their
parent antibodies are raised.
[0034] Antibodies to virus or viral antigens may be made by
inoculating a host with crude or purified, live, attenuated or
killed virus or with antigens shed by virus, e.g., coat protein,
portions thereof, or fragments resulting from destruction of virus.
Monoclonal antibodies may be made by immunizing mice or other
mammalian species with the virus or viral antigens, isolating
splenocytes from the immunized host and fusing them with a suitable
myeloma cell line using somatic cell hybridization techniques to
produce hybridomas that produce antiviral antibodies. These
hybridomas may be isolated, subcloned and cultivated to produce
monoclonal antibodies. The hybridoma derived monoclonal antibodies
to viral antigens are typically of murine or rat origin and
typically are IgGs or IgMs, although suitable antibodies for use in
preparing conjugates according to the invention are not intended to
be limited as regards species or Ig class.
[0035] In general, antibodies can usually be raised to most
antigens, using the many conventional techniques now well known in
the art. Thus, antibodies that specifically bind to other microbial
and parasitic antigens, either on the organism itself or on
fragments or excreted or accreted antigens can be raised by
adapting the foregoing methodology in ways that are now
conventional in the art. Any antibody that binds to a pathogen or
its antigen which is found in sufficient concentration at a focus
of infection in the body of a mammal can be used to make the
targeting conjugate for use in the present invention.
[0036] A wide variety of monoclonal antibodies against infectious
disease agents, have been developed, and are summarized in a review
by Polin, in Eur. J. Clin. Microbiol., 3(5):387-398, 1984, showing
ready availability. However, the principal interest in such
antibodies in the past has been their incorporation into in vitro
diagnostic assays. A few unconjugated antibodies have been tried as
therapeutic agents in animal models, but with only limited
success.
[0037] The value of conjugating such antibodies with radioisotopes
and/or drugs or toxins to achieve targeted detection, imaging and
therapy of infection has not been appreciated and the efficacy of
such agents could not be predicted from such prior disclosures.
[0038] Among the monoclonal antibodies (MAbs) against pathogens and
their antigens cited by Polin, supra, are:
[0039] Anti-Bacterial Mabs
[0040] Streptococcus agalactiae
[0041] Legionella pneumophilia
[0042] Streptococcus pyogenes
[0043] Escherichia coli
[0044] Neisseria gonorrhosae
[0045] Neisseria meningitidis
[0046] Pneumococcus
[0047] Hemophilis influenzae B
[0048] Treponema pallidum
[0049] Lyme disease spirochetes
[0050] Pseudomonas aeruginosa
[0051] Mycobacterium leprae
[0052] Brucella abortus
[0053] Mycobacterium tuberculosis
[0054] Tetanus toxin
[0055] Anti-Viral MAbs
[0056] Rabies virus
[0057] Influenza virus
[0058] cytomegalovirus
[0059] Herpes simplex I and II
[0060] Human serum parvo-like virus
[0061] Respiratory syncytial virus
[0062] Varicella-Zoster virus
[0063] Hepatitis B virus
[0064] Measles virus
[0065] Adenovirus
[0066] Human T-cell leukemia viruses
[0067] Epstein-Barr virus
[0068] Murine leukiemia virus *
[0069] Mumps virus
[0070] Vesicular stomatitis virus
[0071] Sindbis virus
[0072] Lymphocytic choriomeningitis virus
[0073] Wart virus
[0074] Blue tongue virus
[0075] Sendai virus
[0076] Feline leukemia virus *
[0077] Reo virus
[0078] Polio virus
[0079] Simian virus 40 *
[0080] Mouse mammary tumor virus *
[0081] Dengue virus
[0082] Rubella virus
[0083] * Animal virus
[0084] Anti-Protozoan MAbs
[0085] Plasmodium falciparum
[0086] Plasmodium vivax
[0087] Toxoplasma gondii
[0088] Trypanosoma rangeli
[0089] Trypanosoma cruzi
[0090] Trypanosoma rhodesiensei
[0091] Trypanosoma brucei
[0092] Schistosoma mansoni
[0093] Schistosoma japanicum
[0094] Babesia bovis
[0095] Elmeria tenella
[0096] Onchocerca volvulus
[0097] Leishmania tropica
[0098] Trichinella spiralis
[0099] Theileria parva
[0100] Taenia hydatigena
[0101] Taenia ovis
[0102] Taenia saginata
[0103] Echinococcus granulosus
[0104] Mesocestoides corti
[0105] AntimYcoPlasmal MAbs
[0106] Mycoplasma arthritidis
[0107] M. hyorhinis
[0108] M. orale
[0109] M. arginini
[0110] Acholeplasma laidlawii
[0111] M. salivarium
[0112] M. pneumoniae
[0113] Additional examples of MAbs generated against infectious
microorganisms that have been described in the literature are noted
below.
[0114] MAbs against malaria parasites can be directed against the
sporozoite, merozoite, schizont and gametocyte stages. Monoclonal
antibodies have been generated against sporozoites
(circumsporozoite antigen), and have been shown to neutralize
sporozoites in vitro and in rodents (N. Yoshida et al., Science
207:71-73, 1980).
[0115] Several groups have developed MAbs to T. gondii, the
protozoan parasite involved in toxoplasmosis (Kasper et al., J.
Immunol. 129:1694-1699, 1982; Id., 130:2407-2412, 1983).
[0116] MAbs have been developed against schistosomular surface
antigens and have been found to act against schistosomulae in vivo
or in vitro (Simpson et al. Parasitology, 83:163-177, 1981; Smith
et al., Parasitology, 84:83-91, 1982; Gryzch et al., J. Immunol.,
129:2739-2743, 1982; Zodda et al., J. Immunol. 129:2326-2328, 1982;
Dissous et al., J. Immunol., 129:2232-2234, 1982).
[0117] Trypanosoma cruzi is the causative agent of Chagas' disease,
and is transmitted by blood-sucking reduviid insects. A MAb has
been generated that specifically inhibits the differentiation of
one form of the parasite to another (epimastigote to trypomastigote
stage) in vitro, and which reacts with a cell-surface glycoprotein;
however, this antigen is absent from the mammalian (bloodstream)
forms of the parasite (Sher et al., Nature, 300:639-640, 1982).
[0118] Suitable MAbs have been developed against most of the
microorganisms (bacteria, viruses, protozoa, helminths) responsible
for the majority of infections in humans, and many have been used
previously for in vitro diagnostic purposes. These antibodies, and
newer MAbs that can be generated by conventional methods for
further improvement of targeting by use of MAb combinations, are
appropriate for in vivo use as imaging and therapy reagents when
they are conjugated with suitable radionuclides and drugs.
[0119] It is generally desirable to use antibodies having a
relatively high immunoreactivity, i.e., a binding constant of at
least about 10.sup.5/mole, preferably at least about 10.sup.7/mole,
and high immunospecificity, i.e., at least about 40%, preferably at
least about 60%, more preferably at least about 70-95% for pathogen
antigens.
[0120] However, it may be preferable for certain applications,
e.g., for imaging, to use antibodies having a somewhat lower
binding constant in the present invention. Antibodies with high
binding constants are likely to bind tightly not only to pathogens
and their antigens at the site of infection, but also to such
pathogens and/or antigens present in the circulatory system. On the
other hand, antibodies with a lower binding constant will tend to
accrete mainly at concentrated pathogen/antigen foci by virtue of a
type of mass action effect. This will reduce premature clearance
and nontarget accretion of the imaging label and thus increase the
effective amount for targeting the focus of infection.
[0121] Antibody conjugates for imaging can be prepared by a variety
of conventional procedures, ranging from simple glutaraldehyde
linkage to more elegant and specific linkages between functional
groups. The antibodies and/or antibody fragments are preferably
covalently bound to one another, directly or through a short or
long linker moiety, through one or more functional groups on the
antibody/fragment, e.g., amine, carboxyl, phenyl, thiol or hydroxyl
groups. Various conventional linkers in addition to glutaraldehyde
can be used, e.g., diisiocyanates, diisothiocyanates,
bis(hydroxysuccinimide) esters, carbodiimides,
maleimide-hydroxysuccinimide esters and the like.
[0122] A simple method is to mix the antibodies/fragments in the
presence of glutaraldehyde to form an antibody composite. The
initial Schiff base linkages can be stabilized, e.g., by
borohydride reduction to secondary amines. This method is
conventionally used to prepare other conjugates of proteins, e.g.,
peroxidase-antibody conjugates for immunohistochemical uses or for
immunoassays. A diisothiocyanate or a carbodiimide can be used in
place of glutaraldehyde as a non-site-specific linker.
[0123] Bispecific antibodies can be made by a variety
of-conventional methods, e.g., disulfide cleavage and reformation
of mixtures of whole LgG or, preferably F(ab').sub.2 fragments,
fusions of more than one clone to form polyomas that produce
immunoglobulins having more than one specificity, and by genetic
engineering. The bispecific antibodies can bind to one or more
viral epitopes. Bispecific ("hybrid") antibody fragments have been
prepared by oxidative linkage of Fab' fragments resulting from
reductive cleavage of different antibodies. A portion of these will
contain fragments specific to both of the antigens to which the
original antibodies were raised.
[0124] More selective linkage can be achieved by using a
heterobifunctional linker such as a maleimide-hydroxysuccinimide
ester. Reaction of the latter with an antibody/fragment will
derivatize amine groups on the antibody/fragment, and the
derivative can then be reacted with, e.g., an antibody Fab fragment
with free sulfhydryl groups (or a larger fragment or intact
immunoglobulin with sulfhydryl groups appended thereto by, e.g.,
Traut's Reagent). Such a linker is less likely to crosslink groups
in the same antibody and improves the selectivity of the
linkage.
[0125] It is advantageous to link the antibodies/fragments at sites
remote from the antigen binding sites. This can be accomplished by,
e.g., linkage to cleaved interchain sulfhydryl groups, as noted
above. Another method involves reacting an antibody whose
carbohydrate portion has been oxidized with another antibody which
has at least one free amine function. This results in an initial
Schiff base (imine) linkage, which is preferably stabilized by
reduction to a secondary amine, e.g., by borohydride reduction, to
form the final composite. Such site-specific linkages are
disclosed, for small molecules or polypeptides or for solid phase
polymer supports, in U.S. Pat. No. 4,671,958 and for larger addends
in U.S. Pat. No. 4,699,784.
[0126] Similar reactions can be used to bind a plurality of
antibodies and/or antibody fragments, e.g., Fab or F(ab').sub.2
fragments, to one another to form polyspecific conjugates or
conjugates with more than one epitopic specificity for a pathogen
or its antigen to increase its binding affinity or efficiency to
the target site. Bispecific conjugates can be linked to an
antibody/fragment specific to a third, fourth or further epitope
using, e.g., a heterobifunctional maleimide-hydroxysuccin- imide
ester linker to derivatize an amine group, followed by reaction of
the derivative with a fragment having a free sulfhydryl group,
optionally introduced with a reagent such as 2-iminothiolane.
Alternative linkage modes will be readily apparent to the ordinary
skilled artisan based on the disclosures for bispecific composite
formation, and will require only minor variation and adaptation of
such methods.
[0127] The antibody component of the conjugate can be labeled with
or conjugated or adapted for conjugation to, a radioisotope for
scintigraphic imaging or a magnetic resonance image enhancing
agent, for use as a diagnostic imaging agent. Any conventional
method of radiolabeling which is suitable for labeling proteins for
in vivo use will be generally suitable for labeling the composite.
This can be achieved by direct labeling with, e.g., a radioisotope
of a halogen or a metal ion, using conventional techniques or more
sophisticated methodologies, or by attaching a chelator for a
radiometal or paramagnetic ion. Such chelators and their modes of
attachment to antibodies are well known to the ordinary skilled
artisan and are disclosed inter alia in, e.g., Childs et al, J.
Nuc. Med., 26:293 (1985); and in Goldenberg U.S. Pat. Nos.
4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, and
4,624,846. Typical are derivatives of ethylenediaminetetraacetic
acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These
typically have groups on the side chain by which the chelator can
be attached to an antibody. Alternatively, carboxyl or amine groups
on a chelator can be activated and then coupled to an antibody by
well known methods. For example, deferoxamine, which is a chelator
for Ga-67, has a free amine group that can be activated with a
suitable linker to contain an activated carboxyl, isothiocyanate or
like group, and then coupled to amines on an antibody.
[0128] The chelator may be bound to the antibody, directly or
through a short or long chain linker moiety, through one or more
functional groups on the antibody, e.g., amine, carboxyl, phenyl,
thiol or hydroxyl groups. Various conventional linkers can be used,
e.g., diisocyanates, diisothiocyanates, carbodiimides,
bis-hydroxysuccinimide esters, maleimide-hydroxysuccinimide esters,
glutaraldehyde and the like, preferably a selective sequential
linker such as the anhydride-isothiocyante linker disclosed in U.S.
Pat. No. 4,680,338.
[0129] Labeling with either Iodine-131 (I-131) or Iodine-123
(I-123), is readily effected using an oxidative procedure wherein a
mixture of radioactive potassium or sodium iodide and the antibody
is treated with chloramine-T, e.g., as reported by Greenwood et al,
Biochem. J., 89, 114 (1963) and modified by McConahey et al, Int.
Arch. Allergy Appl. Immunol., 29, 185 (1969). This results in
direct substitution of iodine atoms for hydrogen atoms on the
antibody molecule, presumable on tyrosine residues, possibly also
on tryptophan and even on phenylalanine residures, depending on the
proportions of reagents and the reaction conditions. Alternatively,
lactoperoxidase iodination may be used, as described by Feteanu,
supra, page 303, and references cited therein.
[0130] Some more advanced methods of labeling are disclosed in
pending applications U.S. Serial Nos. 742,436 (6-7-85), 084,544
(8-12-87), and 176,421 (41-88). The disclosures of all of the
foregoing patents and applications are incorporated herein in their
entireties by reference. A wide range of labeling techniques are
disclosed in Feteanu, "Labeled Antibodies in Biology and Medicine",
pages 214-309 (McGraw-Hill Int. Book Co., New York et al, 1978).
The introduction of various metal radioisotopes may be accomplished
according to the procedures of Wagner, et al, J. Nucl. Med., 20,428
(1979); Sundberg et al, J. Med. Chem., 17, 1304 (1974); and Saha et
al. J. Nucl. Med., 6, 542 (1976). The foregoing are merely
illustrative of the many methods of radiolabeling proteins known to
the art.
[0131] Examples of compounds useful for MRI image enhancement
include paramagnetic ions, e.g., Gd(III), Eu(III), Dy(III),
Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II),
Ti(III), and V(IV) ions, or radicals, e.g., nitroxides, and these
would be conjugated to a substrate bearing paramagnetic ion
chelators or exposed chelating functional groups, e.g., SH,
NH.sub.2, COOH, for the ions, or linkers for the radical addends.
The MRI enhancing agent must be present in sufficient amounts to
enable detection by an external camera, using magnetic field
strengths which are reasonably attainable and compatible with
patient safety and instrumental design. The requirements for such
agents are well known in the art for those agents which have their
effect upon water molecules in the medium, and are disclosed, inter
alia, in, e.g., Pykett, Scientific American, 246:78 (1982); and
Runge et al., Am. J. Radiol, 141:1209 (1987).
[0132] It is well understood that many of the same methods for
introducing metals, directly or in the form of chelates, into
antibodies will be suitable for introduction of MRI agents into the
antibody conjugates of the invention to form imaging agents for
infections. MRI agents advantageously have a large number of
paramagnetic ions or radicals for enhanced imaging. One method for
introducing a plurality of such ions is to load a carrier polymer
with chelates and link the carrier to the antibody composite,
preferably site-specifically at a site remote from the antigen
binding sites of the conjugate. This has the advantage that larger
numbers of chelators can be attached to the antibody at fewer sites
on the antibody itself, so that immunoreactivity is not as
seriously compromised. Examples of polymers that are useful for
loading the antibody with chelator include, e.g., polyols,
polysaccharides, polypeptides and the like, such as those disclosed
in, e.g., U.S. Pat. Nos. 4,699,784 (Shih et al) and 4,046,722
(Rowland).
[0133] One type of polysaccharide is dextran. The chelator can be
functionalized to contain reactive groups towards the dextran
hydroxyls, e.g., anhydrides, isocyanates or isothiocyanates and the
like. Alternatively, dextran can be derivatized in a number of
ways, e.g., by conversion to an aminodextran. It will be
appreciated that similar methods will be useful for loading a
plurality of drug molecules on an antibody or antibody conjugate,
as will be discussed more fully hereinafter.
[0134] The process for preparing an antibody conjugate with an
aminodextran (AD) carrier normally starts with a dextran polymer,
advantageously a dextran of average molecular weight (MW) of about
10,000-100,000, preferably about 10,000-40,000, and more preferably
about 15,000, The dextran is then reacted with an oxidizing agent
to effect a controlled oxidation of a portion of its carbohydrate
rings to generate aldehyde groups. The oxidation is conveniently
effected with glycolytic chemical reagents, e.g., NaIO.sub.4,
according to conventional procedures.
[0135] It is convenient to adjust the amount of oxidizing agent so
that about 50-150, preferably 100 aldehyde groups are generated,
for a dextran of MW of about 40,000, with about the same proportion
of aldehyde groups for other MW dextrans. A larger number of
aldehyde groups, and subsequent amine groups, is less advantageous
because the polymer then behaves more like polylysine. A lower
number results in less desirable loading of the chelator or boron
addend, which may be disadvantageous.
[0136] The oxidized dextran is then reacted with a polyamine,
preferably a diamine, and more preferably a mono- or poly-hydroxy
diamine. Suitable amines include, e.g., ethylenediamine,
propylenediamine or similar polymethylendadiamines,
diethylenetriamine or like polyamines, 1,3-diamino-2-hydroxypropane
or otherwise like hydroxylated diamines or polyamines, and the
like. An excess of the amine relative to the aldehyde groups can be
used, to insure substantially complete conversion of the aldehyde
functions to Schiff base (imine) groups.
[0137] Reductive stabilization of the resultant intermediate is
effected by reacting the Schiff base intermediate with a reducing
agent, e.g., NaBH.sub.4, NaBH.sub.3CN, or the like. An excess of
the reducing agent is used to assure substantially complete
reduction of the imine groups to secondary amine groups, and
reduction of any unreacted aldehyde groups to hydroxyl groups. The
resultant adduct can be further purified by passage through a
conventional sizing column to remove cross-linked dextrans. An
estimate of the number of available primary amino groups on the AD
can be effected by reaction of a weighed sample with
trinitrobenzenesulfonic acid and correlation of the optical density
at 420 nm with a standard. This method normally results in
essentially complete conversion of the calculated number of
aldehyde groups to primary amine groups on the AD.
[0138] Alternatively, the dextran can be derivatized by
conventional methods for introducing amine functions, e.g., by
reaction with cyanogen bromide, followed by reaction with a
diamine. The AD should be reacted with a derivative of the
particular drug or chelator, in an activated form, preferably a
carboxyl-activated derivative, prepared by conventional means,
e.g., using dicyclohexylcarbodiimide (DCC) or a water soluble
variant thereof.
[0139] It will be appreciated that the foregoing is merely
illustrative of art-recognized methods for appending radioactive
labels and/or drugs or toxins to antibodies/fragments and that
other methods can be used to prepare conjugates according to the
invention.
[0140] The scintigraphic imaging method of the invention is
practiced by injecting a human patient parenterally with an
effective amount for scintigraphic imaging of the radiolabeled
antibody conjugate. By parenterally is meant, e.g., intravenously,
intraarterially, intrathecally, interstitially or intracavitarily.
It is contemplated that a subject will receive a dosage of from
about 1 mCi to 50 mCi of radiolabeled conjugate, the amount being a
function of the particular radioisotope and mode of administration.
For intravenous injection, the amounts are normally: about 2-10
mCi, preferably about 2-5 mCi, of I-131; about 5-10 mCi, preferably
about 8 mCi, of I-123; about 10-40 mCi, preferably about 20 mCi of
Tc-99m; about 2-5 mCi, preferably about 4 mCi of In-111 or Ga-67.
Amounts of other imaging radionuclides will be readily determined
by the ordinary skilled artisan, by reference to the above isotopes
and in view of the half-life of the nuclide and the size of the
antibody/fragment/composite to which it is to be conjugated. The
radiolabeled antibody composite is conveniently provided as an
injectable preparation for mammalian use, preferably a sterile
injectable preparation for human use, for targeting a scintigraphic
imaging agent to a focus of infection, preferably comprising: a
sterile injectable solution containing an effective amount of the
radiolabeled conjugate in a pharmaceutically acceptable sterile
injection vehicle, preferably phosphate-buffered saline (PBS) at
physiological pH and concentration other conventional
pharmaceutically acceptable vehicles may be utilized as required
for the site of parenteral administration.
[0141] A representative preparation to be parenterally administered
in accordance with this, invention will normally contain about 0.1
to 20 mg, preferably about 2 mg, of radiolabeled antibody
conjugate, in a sterile solution which advantageously also
contains, e.g., about 10 mg of human serum albumin (1% USP:
Parke-Davis) per milliliter of 0.04M phosphate buffer (pH 7.4
Bioware) containing 0.9% sodium chloride.
[0142] Once enough isotope has deposited at the target site,
scanning is effected with either a conventional planar and/or SPECT
gamma camera, or by use of a hand held gamma probe used externally
or internally to localize the infection. The scintigram is normally
taken by a gamma imaging camera having one or more windows for
detection of energies in the 50-500 KeV range. The target site can
be any site having the pathogen or its antigens present in a
relatively concentrated focus.
[0143] Magnetic resonance imaging (MRI) is effected in an analogous
method to scintigraphic imaging except that the imaging agents will
contain MRI enhancing species rather than radioisotopes. It will be
appreciated that the magnetic resonance phenomenon operates on a
different principle from scintigraphy. Normally, the signal
generated is correlated with the relaxation times of the magnetic
moments of protons in the nuclei of the hydrogen atoms of water
molecules in the region to be imaged. The magnetic resonance image
enhancing agent acts by increasing the rate of relaxation, thereby
increasing the contrast between water molecules in the region where
the imaging agent accretes and water molecules elsewhere in the
body. However, the effect of the agent is to increase both T.sub.1,
and T.sub.2, the former resulting in greater contrast, while the
latter results in lesser contrast. Accordingly the phenomenon is
concentration-dependent, and there is normally an optimum
concentration of a paramagnetic species for maximum efficacy. The
optimum concentration will vary with the particular agent used, the
locus of imaging, the mode of imaging, i.e., spinecho,
saturation-recovery and for various other strongly T.sub.1
dependent or T.sub.2 dependent imaging techniques, and the
composition of the medium in which the agent is dissolved or
suspended. These factors, and their relative importance are known
in the art. See, e.g., Pykett, op.cit., and Runge et al.,
op.cit.
[0144] The MRI method of the invention is practiced by injecting a
mammal, preferably a human, parenterally with an effective amount
for magnetic resonance imaging of a conjugate according to the
present invention of an antibody conjugate including an MRI
enhancing agent. It is contemplated that a subject will receive a
dosage of labeled conjugate sufficient to enhance the MRI signal at
the site of infection by at least about 20%, preferably 50-500%,
the amount being a function of the particular paramagnetic species
and the mode of administration.
[0145] Again, the labeled antibody conjugate is conveniently
provided as an injectable preparation for mammalian use, preferably
a sterile injectable preparation for human use. A typical
preparation for targeting a MRI agent to a focus of infection
preferably comprises: a sterile injectable solution containing an
effective amount of the labeled conjugate in a pharmaceutically
acceptable sterile injection vehicle, preferably phosphate-buffered
saline (PBS) at physiological pH and concentration.
[0146] Other conventional pharmaceutically acceptable vehicles for
parenteral administration may be utilized as required for the site
of parenteral administration.
[0147] A representative preparation to be parenterally administered
in accordance with this invention will normally contain about 0.1
to 50 mg, preferably about 5 mg, of labeled polyspecific antibody
composite, in a sterile solution which advantageously also
contains, e.g., about 10 mg of human serum albumin (1% USP:
Parke-Davis) per milliliter of 0.04M phosphate buffer (pH 7.4
Bioware) containing 0.9% sodium chloride. Once enough of the MRI
agent has deposited at the target site, scanning is effected with a
conventional MRI camera to image the infection.
[0148] In a preferred embodiment of this invention, the
localization ratio of the primary labeled antibody conjugate is
enhanced through the use of a nonlabeled second antibody to
scavenge non-targeted circulating conjugate and promote its
clearance, as disclosed for related imaging agents in Goldenberg,
U.S. Pat. No. 4,624,846, the disclosure of which is incorporated
herein in its entirety by reference.
[0149] This technique is likewise applicable to an antibody or
antibody composite conjugated to a therapeutic drug, as will be
discussed hereinafter. The term "localization ratio" is utilized in
its conventional sense, i.e. the ratio of target to nontarget
antibody conjugate. In general, the second antibody is used in an
amount that will enhance the localization ratio of the primary
antibody conjugate by at least about 20 percent and typically by 50
percent or more.
[0150] The second antibody may be whole IgG or IgM, or a fragment
of IgG or IgM, so long as it is capable of binding the primary
antibody conjugate to form a complex which is cleared from the
circulation and the non-target spaces more rapidly than the primary
antibody conjugate by itself. Preferably, the second antibody will
be whole IgG or IgM. If the primary antibody is a fragment of IgG
or IgM, it is preferable that the second antibody be whole IgG or
IgM so that the primary/secondary complex retains the capability of
activating the complement cascade conversely, where the primary
antibody is whole IgG, the second antibody may be a fragment if the
complex still retains complement-fixing capability.
[0151] It is preferred that at least one of the primary/secondary
pair be whole IgG or IgM. One advantage of using IgM is that it
forms a higher molecular weight complex with primary antibody or
with detached conjugates, ie., diagnostic and/or therapeutic
principles such as drugs, chelating agents, radionuclides, and the
like. This will increase the rate and effectiveness of clearance of
non-target primary antibody and/or principle, especially from
blood.
[0152] The second antibody can be prepared by methods disclosed in
the aforementioned Goldenberg '846 patent. Monoclonal anti-species
IgG is also available and is advantageously used as second antibody
in the present process. Non-metallic conjugates, e.g.,
radioiodinated linking groups or organic paramagnetic species such
as nitroxides, can also be haptens to which the second antibody is
specific.
[0153] The second antibody is injected into the subject after a
sufficient time has elapsed following parenteral administration of
the primary polyspecific antibody conjugate to permit maximum
uptake thereof by foci of infection, typically about 2-72 hours
following the initial administration, preferably at about 4-48
hours post-administration. If the primary antibody is not
administered intravenously, it may be advantageous to administer at
least a portion of the second antibody by the same parenteral
route. It is advantageous however, to inject at least a portion of
the second antibody intravenously to accelerate clearance of
primary antibody which has diffused into the circulatory
system.
[0154] An alternative or adjunct to the use of second antibody to
clear circulating labeled primary antibody and enhance the
localization ratio of the primary antibody is utilization of
image-enhancing subtraction techniques as disclosed in the
foregoing Goldenberg patents as well as the references cited
therein. This is an art-recognized technique wherein an indifferent
antibody or fragment is labeled with a radionuclide capable of
independent detection, and the labeled indifferent antibody is
injected into the subject This antibody has substantially the same
kinetics of distribution and metabolism as the primary antibody
during the period required for imaging. The injection of such
antibodies is preferred over conventional subtraction agents, such
as Tc-99m-labeled serum albumin, which are nevertheless suitable
for use to enhance image processing by compensating for background.
The use of the radiolabeled indifferent antibody as a subtraction
agent permits computerized correction for nontarget background
radiation from organs which effect clearance of antibodies from the
circulatory system.
[0155] It will be appreciated by those of ordinary skill in the art
that the primary monoclonal antibody and the indifferent antibody
utilized as a subtraction agent are preferably from the same
species or myeloma/hybridoma so that the second antibody will clear
the primary monoclonal antibody and the indifferent antibody
immunoglobulin from untargeted areas at substantially the same
rate. It is further preferred that the second antibody be specific
to a constant region of the primary and indifferent immunoglobulin
species.
[0156] The amount of second antibody introduced will generally be
that amount which can decrease the circulating primary antibody by
10-85% within 2-72 hours. The ratio of second antibody to primary
antibody which will affect the clearance will depend upon the
binding properties of the primary and secondary antibody pair.
Preliminary screening of patient blood in vitro can be used to
provide an initial estimate of the appropriate ratio. The screen
will be used to determine the ratio of second antibody to primary
antibody required to obtain a precipitin band in, e.g., a gel
diffusion test. This indicates the general range of the molar ratio
of second antibody to primary antibody, which serves as a measure
of the lower limit for the ratio, since in vivo application may
require a higher ratio of second antibody to primary antibody than
is indicated by such in vitro tests.
[0157] In practice, the molar ratio of second antibody to primary
antibody will generally be in the range of about 5-50, although the
range should not be considered limitative. Molar ratios of second
antibody to primary antibody of 15-25, and preferably 20-25, have
been found to be advantageous where both the primary and the second
antibody are whole IgG.
[0158] Imaging preparations and kits can include second antibody,
in a separate container, for injection at an appropriate time after
administration of the antibody conjugate.
[0159] Many drugs and toxins are known which have a cytotoxic
effect on pathogens microbes that may infect a human. They can be
found in any of the readily available art-recognized compendia of
drugs and toxins, such as the Merck Index and the like. Any such
antibiotic or cytotoxic drug can be conjugated to an anti-pathogen
antibody or antibody composite to form a therapy agent according to
the present invention, and the use of such a conjugate to improve
the targeting of an antibiotic or cytotoxic drug to a focus of
infection so as to increase its effective concentration at the site
is a part of the present invention.
[0160] One or more antibiotic or cytotoxic drugs can be conjugated
to a polymeric carrier which is then conjugated to the antibody or
antibody composite, for therapeutic use. In certain cases, it is
possible to partially or completely detoxify a drug as part of the
antibody conjugate, while it is in circulation, which can reduce
systemic side effects of the drug or toxin and permit its use when
systemic administration of the drug would be unacceptable.
Administration of more molecules of the drug conjugated to a
polymer which is further conjugated to the antibody, permits
therapy while mitigating systemic toxicity. The methodology of this
invention is applicable to the therapeutic treatment of infections
by conjugating the primary antibody or antibody composite to an
antibiotic or cytotoxic drug or toxin. Art-recognized methods of
conjugating drugs or toxins to immunoglogulins are described, e.g.,
in: the chapter by O'Neill, entitled "The Use of Antibodies as Drug
Carriers," in Drug Carriers in Biology and Medicine, G.
Gregoriadis, ed., Academic Press London, 1979; Arnon et al., Recent
Results in Cancer Res. 75: 236, 1980; and Moelton et al.,
Immunolog. Res. 62:47, 1982, showing art awareness. These methods
are quite similar to the methods employed for coupling drugs
effective against various disease-causing microorganisms, such as
against bacteria, viruses, fungi and diverse parasites to
antibodies developed against these microorganisms, their products
or antigens associated with their lesions.
[0161] Such antibiotic or cytotoxic drugs, including, e.g.,
tetracyclines, chloramphenicol, piperazine, chloroquine,
diaminopyridines, metroniazide, isoniazide, rifampins,
streptomycins, sulfones, erythromycin, polymixins, nystatin,
amphotericins, 5-fluorocytosine, 5-iodo-2'deoxyuridine,
1-adamantanamine, adenine arabinoside, amanitins and azidothymidine
(AZT), are preferred for coupling to appropriate specific
antibodies/fragments and antibody/fragment composites. Various
other potential antibiotic/cytotoxic agents for use in this
invention are listed in Goodman et al., "The Pharmacological Basis
of Therapeutics," Sixth Edition, A. G. Gilman et al, eds.,
Macmillan Publishing Co., New York, 1980, showing general art
awareness. Various conditions appropriate and desirable for
targeting drugs to specific target sites have been reviewed e.g. by
Trouet et al., in Targeting of Drugs, G. Gregoriadis et al., eds.,
Plenum Press, New York and London, 1982, pp. 19-30, showing
clinical knowledge of how such targeting would benefit patients
suffering from infectious lesions.
[0162] The use of a second antibody, as described above in an
imaging context, will increase the effectiveness of the therapeutic
agent according to the invention in the same manner as for the
diagnostic imaging conjugate. The effectiveness of the therapeutic
agent is expressed in terms of its therapeutic index which,
utilized in the conventional sense, is defined as the ratio of
therapeutic effects to undesirable side effects. It is often
defined in terms of a quantitative measure of efficacy vs. toxicity
in a standard model system, e.g., the ratio of the median lethal
dose (LD.sub.50) to the median effective dose (ED.sub.50). The use
of second antibody as described herein produces an increase in the
therapeutic index of antiviral antibody and antibody composite
conjugates by clearing nontarget primary antibody and/or detached
therapeutic principle. In addition to being specific to the primary
monoclonal antibody as discussed above, in the instance of the
therapeutic preparation, the second antibody can be specific to the
therapeutic agent. It can also be specific to a carrier for the
therapeutic agent.
[0163] Therapeutic preparations contemplated herein comprise
monospecific anti-pathogen antibodies/fragments as defined above,
conjugated to a therapeutically effective radioisotope and/or
antibiotic/cytotoxic drug, in a suitable vehicle for parenteral
administration. A therapeutic preparation may likewise comprise a
polyspecific anti-pathogen antibody/fragment composite conjugated
to a radioisotope and/or antibiotic/cytotoxic drug.
[0164] It is advantageous in certain cases to combine a drug with a
radionuclide, especially where the pathogen. "hides", or is
somewhat inaccessible. The longer range action of radionuclides can
reach hidden pathogen so long as some antigen is accessible to the
conjugate. Also, radiation can cause lysis of an infected cell and
expose intracellular pathogen to the antimicrobial drug component
of the conjugate.
[0165] Therapeutic preparations may also include a separately
packaged second antibody as described above. Suitable vehicles are
well known in the art and can include, e.g., analogous sterile PBS
solutions to those used for administration of diagnostic imaging
agents, as discussed hereinabove.
[0166] The anti-microbial polyspecific imaging conjugates and
monospecific or polyspecific therapeutic conjugates according to
the invention also can be conveniently provided in a therapeutic or
diagnostic kit for antibody targeting to a focus of infection.
Typically, such a kit will comprise a vial containing the antibody
conjugate of the present invention, either as a lyophilized
preparation or in an injection vehicle. If the conjugate is to be
used for scintigraphic imaging or for radioisotope therapy, it will
generally be provided as a cold conjugate together with reagents
and accessories for radiolabeling, in separate containers, while
MRI agents and therapeutic drug/toxin conjugates will generally be
supplied with a paramagnetic species or an antibiotic/cytotoxic
agent already conjugated to the antibody/fragment composite or
monospecific antibody/fragment. The kit may further contain a
second, separately packaged, unlabeled antibody or antibody
fragment specific against the antibody or fragment or the
therapeutic agent, a carrier therefor, or a chelating agent for the
radionuclide or paramagnetic ion.
[0167] The imaging preparations and methods of this invention are
able to detect and image relatively small foci of infection and are
easy and safe to use. The therapeutic reagents and methods of the
invention provide a means to target sites of infection with
radioisotopes and drugs to improve the therapeutic index thereof,
reduce their systemic side effects and enhance their efficacy.
[0168] Radionuclide immunoconjugates are particularly effective for
microbial therapy. After it has been determined that labeled
antibodies are localized at infectious sites in a subject, higher
doses of the labeled antibody, generally from 20 mCi to 150 mCi per
dose for I-131, 5 mCi to 30 mCi per dose for Y-90 or 5 mCi to 20
mci Re-186, each based on a 70 kg patient weight, are injected.
Injection may be intravenous, intraarterial, intralymphatic,
intrathecal, or intracavitary (i.e., parenterally), and may be
repeated. It may be advantageous for some therapies to administer
multiple, divided doses of antibody or antibody composite, thus
providing higher microbial toxic doses without usually effecting a
proportional increase in radiation of normal tissues.
[0169] A variety of radionuclides are useful for therapy, and they
may be incorporated into the specific antibody by the labeling
techniques discussed above, as well as other conventional
techniques well known to the art. Preferred therapeutically
effective radionuclides are astatine-211, bismuth-212, yttrium-90,
rhenium-186, rhenium188, copper-67, iodine-131, and iodine-125,
although other radionuclides as well as photosensitizing agents are
also suitable.
[0170] A further aspect of the present invention relates to the use
of antibodies containing a significant number of boron atoms,
having at least the 20% natural abundance of boron-10 isotope. The
boron-containing addend may be introduced by a variety of methods,
such as described in U.S. Pat. No. 4,824,659 (Hawthorne),
incorporated herein in its entirety by reference. The
boron-10-containing antibody can be radiolabeled according to one
or more of the above procedures to produce an antibody containing
both one or more radiolabels for infection detection and/or therapy
and a high content of boron-10 atoms for the absorption of thermal
neutrons. Alternatively, the boron-labeled antibody can be used
without the attachment of a gamma-emitting isotope to the antibody.
The infectious lesions are then irradiated with a well collimated
beam of thermal neutrons, which are preferentially absorbed by
boron-10 nuclei on the boron-containing addends, and the activated
nucleus decays rapidly to lithium-7 and an alpha-particle. These
resultant alpha-particles are toxic, and their production kills
adjacent microorganisms and cells.
[0171] A particularly effective application of the methods and
compositions of the present invention is the treatment of acquired
immune deficiency syndrome (AIDS) and the prodromal
immunodeficiency known as AIDS-related complex (ARC), due to HIV
infection. While there is no cure for AIDS or ARC, drugs that block
reverse transcriptase activity, which is a unique feature of the
HIV retrovirus, are being investigated in AIDS patients. However,
patients eventually become resistant and relapse, thus requiring
other therapeutic modalities.
[0172] AIDS patients develop circulating antibodies to different
HIV components, such as viral core antigens, envelope glycoprotein
antigen complex, and transmembrane protein. The major reactivity in
AIDS patients is directed against a possible viral envelope
glycoprotein of molecular weight 41,000 (gp41). It would not have
been predictable that antibodies could be useful for targeting HIV
in humans, since it might have been expected that the patient's own
antibodies would saturate the target sites needed for targeting of
the exogenous HIV antibodies.
[0173] It is now found that exogenous monoclonal antibodies to HIV,
particularly antibodies specific to certain envelope glycoprotein
epitopes, have high selectivity and affinity for the virus, and in
fact can be distinguished in terms of epitope specificity from the
naturally occurring human HIV antibodies. Immunization of mice with
HIV envelope glycoprotein antigen extracts have generated
monoclonal antibodies that react with different envelope antigen
epitopes. Single such monoclonals, but preferably a combination of
such antibodies, are preferred antibody components of the antibody
conjugate according to the invention, for use in HIV infection
detection, imaging and therapy. Alternatively, human or simian
antibodies can be isolated from their hosts by conventional
immunoglobulin isolation and purification methods, and selected as
targeting agents by their ability to target (e.g., by
immunofluorescent staining methods) HIV-infected cells in vitro.
Human antibodies are preferably those which target epitopes on the
virus that are different and separate from major antigenic sites.
Simian antibodies are preferably produced in animals in which
successful models of HIV infection have been achieved. Monoclonal
human or simian antibodies can be produced by known techniques,
involving, e.g., transfection of mouse myeloma cells with human or
simian DNA for antibody production, as disclosed, e.g., in Gillies
et al., Biotechnology, 7(8):799-804, 1989; Nakatani et al.,
Biotechnology, 7(8):805-810.
[0174] Thus, a composite HIV antibody preparation, conjugated to a
radionuclide, e.g., bismuth-212 or another short-range
alpha-emitter, or to an inhibitor of reverse transcriptase, can be
used at therapeutic doses of the radionuclide or drug to
effectively treat patients with AIDS.
[0175] Other diseases also have proven to be resistant or
refractory towards systemic chemotherapy. These include various
viral, fungal, bacterial and protozoan infections, as well as
particular parasitic infections. Other viral infections include
those caused by influenza virus, herpes virus, e.g., Epstein-Barr
virus and cytomegalovirus, rabies virus (Rhabdoviridae) and
papovavirus, all of which are difficult to treat with systemic
antibiotic/cytotoxic agents. Use of antibody conjugates, especially
conjugates of antibody/fragment composites, to target such virus
provides a significantly higher therapeutic index for antiviral
drugs and toxins, thus enhancing their efficacy and reducing
systemic side effects. Targeted radioimmunotherapy with conjugates
of antibodies/fragments and/or composites thereof with therapeutic
radioisotopes (including boron addends activatable with thermal
neutrons) offers a new approach to antiviral therapy
[0176] Protozoans that are relatively resistant to systemic
chemotherapy include, e.g., Plasmodia (especially P. falciparum,
the malaria parasite), Toxoplasma gondii (the toxoplasmosis
infectious agent), Leishmaniae. (infectious agent in
leishmaniasis), and Escherichia histolytica. Detection and
treatment of malaria in its various stages is significantly
enhanced using the antibody/fragment conjugates of the invention.
As noted above, MAbs that bind to sporozoite antigens are known.
However, since sporozoite antigens are not shared by blood stage
parasites, the use of MAbs against sporozoite antigens for
targeting is limited to a relatively short period of time in which
the sporozoites are free in the circulation, prior to and just
after injection of and development in the host's hepatocytes. Thus,
it is preferable to use a mixture of MAbs or a MAb composite
against, e.g., more than one parasite stage of P. falciparum, as a
targeting agent for the more effective treatment of malaria in
humans that are not responding to conventional chemotherapy. The
MAbs are conjugated to a suitable radionuclide for imaging (e.g.,
Tc-99m) or for therapy (e.g., astatine-211; rhenium-186), or with
an anti-malarial drug (e.g., pyrimethamine) for more selective
therapy.
[0177] Toxoplasmosis is also resistant to systemic chemotherapy. It
is not clear whether MAbs that bind specifically to T. gondii, or
natural, host antibodies, can play a role in the immune response to
toxoplasmosis but, as in the case of malarial parasites,
appropriately targeting MAbs are effective vehicles for the
delivery of therapeutic agents.
[0178] Schistosomiasis, a widely prevalent helminth infection, is
initiated by free-swimming cercariae that are carried by some
freshwater snails. As in the case of malaria, there are different
stages of cercariae involved in the infectious process. MAbs that
bind to a plurality of stages of cercariae, optionally to a
plurality of epitopes on one or more thereof, and preferably in the
form of a polyspecific composite, can be conjugated to an imaging
or therapy agent for effective targeting and enhanced therapeutic
efficacy.
[0179] MAbs that bind to one or more forms of Trypanosoma cruzi,
the causative agent of Chagas' disease, can be made and used for
detection and treatment of this microbial infection. The MAb noted
above which reacts with a cell-surface glycoprotein, as well as
MAbs reactive with other surface antigens on differentiation stages
of the trypanosome, are suitable for directing imaging and
therapeutic agents to sites of parasitic infiltration in the
body.
[0180] Another very difficult infectious organism to treat by
available drugs is the leprosy bacillus (Mycobacterium leprae).
Antibodies that specifically bind to a plurality of epitopes on the
surface of M. leprae can be made, e.g., by challenging a mouse with
attenuated or fragmented M. leprae or its surface antigens, and
these can be used, alone or in combination, to target imaging
agents and/or antibiotic/cytotoxic agents to the bacillus.
[0181] Helminthic parasitic infections, e.g., Strongyloidosis and
Trichinosis, themselves relatively refractory towards
chemotherapeutic agents, are suitable candidates for
antibody-targeted diagnosis and therapy according to the invention,
using antibodies that bind specifically to one or, preferably, to a
plurality of epitopes on the parasites.
[0182] Antibodies are available or can easily be raised that
specifically bind to most of the microbes and parasites responsible
for the majority of infections in humans, as illustrated by the
foregoing disclosure and citation of references. Many of these have
been used previously for in vitro diagnostic purposes and the
present invention shows their utility as components of antibody
conjugates to target diagnostic and therapeutic agents to sites of
infection. Microbial pathogens and invertebrate parasites of humans
and mammals are organisms with complex life cycles having a
diversity of antigens expressed at various stages thereof.
Therefore, targeted treatment can-best be effected when antibody
conjugates which recognize antigen determinants on the different
forms are used in combination, either as mixtures or as
polyspecific conjugates, linked to the appropriate therapeutic
modality. The same principle applies to using the MAb reagents for
detecting sites of infection by attachment of imaging agents, e.g.,
radionuclides and/or MRI enhancing agents.
[0183] To the extent that the therapeutic radioisotopes, drugs,
toxins and other cytotoxic agents produce hematopoietic toxicity as
a side effect of their administration, administration of an
effective amount of a cytokine, especially a lymphokine or other
growth factor, to mitigate or prevent such toxicity and to
stimulate marrow production, is advantageous and is a part of the
invention. Such administration will be effected analogously to that
disclosed in commonly assigned and copending U.S. patent
application Ser. No. 174,490, the entire disclosure of which is
incorporated herein by reference.
[0184] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are therefore to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever.
EXAMPLES
[0185] In the following examples, all temperatures are set forth
uncorrect in degrees Celsius. Unless otherwise indicated, all parts
and percentages are by weight.
Example 1
[0186] Specific Murine Monoclonal Antibody to HIV-qp160
[0187] Mice are hyperimmunized with the gp160 envelope
precursor-protein of HIV-1. Spleen lymphocytes from the
hyperimmunized animals are fused with SP2/0 myeloma cells and the
fused cells diluted in HAT in microtiter plate wells. After 10 days
the supernatant from wells containing growing hybrids are tested by
ELISA for reactivity with gp160. Wells containing monoclonal
antibodies reactive with gp160 are subsequently screened for
reactivity with HIV-1 envelope proteins gp120 and gp41 (gp160
products). Clones specific for gp160 which are not blocked by
antibodies in human serum from seropositive AIDS patients, are
subcloned. Four clones with high specificity and affinity, at least
two of which can bind to HIV in the presence of one another
(hereinafter, collectively MAb-160s, and individually MAb160s1,
MAb-160s2, MAb-160s3, MAb-160s4), are each expanded in culture, and
used to produce ascites fluid in Balb-c mice. Each MAb-160s is
purified from the ascites fluid by affinity chromatography on
protein A. The clones are determined to be of IgG.sub.1 subclass
and are used to prepare conjugates.
[0188] Monoclonal antibodies to other HIV-antigens can be used in
patients that do not have significant blood levels of the antigen
or significant blood levels of antibody that block binding of the
anti-HIV monoclonal antibody.
Example 2
[0189] Preparation of 99m-Tc-MAb-160s1-FAb' imaging agent
[0190] Purified MAb-160s1, prepared according to Example 1, is
converted to the F(ab').sub.2 fragment with pepsin and the divalent
fragment converted to Fab' by reduction with cystine. After removal
of cystine by gel filtration, the Fab' is compounded with a
buffered reducing agent, preferably SnCl.sub.2, and lyophilized.
99m-Tc-MAb-160s1-Fab' is prepared just prior to patient injection
by adding 20 mCi of 99 mTc pertechnetate in sterile saline to the
vial containing the lyophilized MAb-160s1-Fab'.
Example 3
[0191] Preparation of 131-I-MAb-160s1+2-F(ab').sub.2
[0192] Purified MAb-160s1 and MAb-160s2, prepared according to
Example 1, are each converted to Fab' fragments as described in
Example 2. The thiol groups of the MAb-160s2 fragments are capped
with iodoacetamide and the capped fragment is derivatized with
maleimide-hydroxysuccinimide p-nitrobenzoate ester, and the
fragments are separately purified by gel filtration. The purified
fragments are reacted with one another to form a chemically linked
bivalent composite with dual specificity for the gp-160 antigen.
The composite is radioiodinated with I-131 by the chloramine-T
method, to achieve an activity of 180 mCi per dose.
Example 4
[0193] Diagnostic Imaging
[0194] A 24 year old male patient is being treated with AZT and
becomes resistant to the drug, expressing HIV-p24 antigen in his
blood. He exhibits malaise and has daily episodes of chills and
fever. An immunoscintigraphy study is performed using an imaging
agent prepared according to Example 2. To the vial containing 1 mg
of lyophilized Fab' is added 20 mCi of generator-produced sodium
pertechnetate in PBS. After 5 minutes, the imaging agent is
injected and the patient is scanned 3 hours later with a gamma
camera in SPECT/mode. Intense foci of bound Tc-99m are observed in
numerous lymph nodes and in the spleen.
[0195] Analogous detection and imaging of other viral infections
can be effected using single antibody or multiple antibody
radiolabeled or MRI enhancer-labeled conjugates, according to the
general methods illustrated in the foregoing examples.
Example 5
[0196] AIDS Therapy
[0197] The patient of Example 4 is given a 5 mCi dosimetry
injection of 131-I-MAb-160s1+2-F(ab').sub.2, prepared from an
aliquot of a standard dose according to Example 3. Planar imaging
using a gamma camera shows intense accumulation of I-131 in the
same sites imaged with 99m-Tc-MAb-160s-Fab'. Blood pharmacokinetics
indicate that the patient can be safely treated with 180 mCi of the
radioiodinated MAb. He is injected with 180 mCi of
131-1-MAb-160s-1+2F(ab').sub.2. Over the course of the next two
weeks, blood levels of p24-HIV antigen drop rapidly and antigen is
undetectable after 3 weeks. A second imaging study after four weeks
with 99m-Tc-MAb-160sFab' is negative, failing to show localization
of bound Tc-99m in lymph nodes or spleen. At this time, the patient
shows other signs of improvement, with abatement of fever.
[0198] Analogous anti-viral therapeutic conjugates can be made for
treatment of other viral infections, using the general methodology
illustrated in the foregoing examples.
Example 6
[0199] Anti-Malarial Antibodies
[0200] Mice are hyperimmunized with merozoites from Plasmodium
falciparum. Spleenocytes from the hyperimmunized animals are fused
with. SP2/0 myeloma cells and the fused cells diluted in HAT in
microtiter plate wells. After 10 days, the supernatant from wells
containing growing hybrids are tested for specific binding to
merozoites bound to polyacrylamide beads with glutaraldehyde, using
an I-125-labeled rabbit anti-mouse IgG. Hybridoma clones from wells
containing merozoite-binding monoclonal antibodies are subcloned.
Three of the 20 positive clones, each of which can bind to
merozoites in the presence of one another (hereinafter,
collectively .alpha.-mer-MAb, and individually .alpha.-mer-MAb1,
.alpha.-mer-MAb2 and .alpha.-mer-MAb3), are each expanded in
culture, and used to produce ascites fluid in Balb-c mice. Each
.alpha.-mer-MAb is purified from the ascites fluid by affinity
chromatography on protein A. The clones are determined to be of
IgG.sub.1 subclass and are used to prepare conjugates.
[0201] By a completely analogous route, three clones that bind
specifically to P. falciparum sporozoites (hereinafter,
collectively .alpha.-spo-MAb, and individually .alpha.-spo-MAb1,
.alpha.-spo-MAb2 and .alpha.-spo-MAb3) are made, expanded and
ascites-produced monoclonals are purified. They are also determined
to be of the IgG.sub.1. subclass.
Example 7
[0202] Preparation of Anti-Malarial Conjugate
[0203] Each of the purified a-mer-MAbs and a-spo-MAbs prepared
according to Example 6 is converted to a Fab' fragment with pepsin,
followed by cystine reduction, analogously to the procedure of
Example 2, and the fragments are capped with excess iodoacetamide.
To an equimolar mixture of the six different capped Fab' fragments,
in aqueous solution, at pH 4.5, is added a 50-fold molar excess of
glutaraldehyde, followed about. 5-15 minutes later by a 30-fold
molar excess of pyrimethamine (each relative to the total number of
moles of antibody fragments), and the mixture is incubated for 6 hr
at 37.degree. C. The resultant conjugate has an average of 2-3 Fab'
fragments and 510 pyrimethamines per conjugate molecule. The
conjugate is freed of low molecular weight reagents on a short
polyacrylamide gel column and sterile filtered.
Example 8
[0204] Malaria Therapy
[0205] A patient suffering from a late stage attack of P.
falciparum malaria and experiencing chills and fever is infused
with a solution of the anti-malarial conjugate according to Example
7, in physiological saline. A rapid drop in blood levels of
merozoites is observed and the chills and fever subside within a
few hours. The patient's liver receives both merozoite-conjugate
complexes and uncomplexed conjugate, both of which release
pyrimethamine to sporozoites, thereby inhibiting recurrence of the
attack. In addition, slow hydrolytic cleavage of the Schiff base
linkages to pyrimethamine produces a prolonged plasma level of the
drug which also effects a suppressive cure of the infection.
Frequent monitoring of the patient's blood permits the infusion to
be adjusted to achieve optimal drug levels and therapeutic
effect.
[0206] Analogous conjugates using single or multiple
antibody/fragment conjugates of drugs or radionuclides that bind to
toxoplasmosis protozoan antigens, schistosomal antigens,
trypanosomal antigens, bacterial, fungal and other microbial or
parasitic antigens can be produced by variation of the foregoing
illustrative methods in ways that the skilled artisan will
appreciate, and the infections caused by such pathogens can be
treated using these conjugates.
Example 9
[0207] Specific Monoclonals to Mycobacterium leprae
[0208] A series of monoclonal antibodies that specifically bind to
leprosy bacilli are produced by hyperimmunization of mice with a
sonicate of Mycobacterium leprae, fusion of resultant splenocytes
and screening of clones for specific binding to the bacilli by
conjugating supernatant from wells containing growing hybrids with
fluorescein, incubating the conjugates with fixed M. leprae,
washing, and detecting bound antibodies under u.v. light. Four
positive clones are subcloned, expanded and ascites-produced
antibodies are purified according to procedures analogous to those
of Example 1.
Example 10
[0209] Preparation of Leprosy Therapeutic Conjugate
[0210] A mixture of the four monoclonal antibodies produced
according to Example 9 is gently oxidized with periodate to cleave
an average of one sugar residue in the carbohydrate region. An
aminodextran to which an average of twenty carboranes are attached
is reacted with the oxidized antibody, and the Schiff base
conjugate is stabilized with borohydride. The resultant conjugate
is radioiodinated with I-131, analogously to the procdure of
Example 3, to achieve an activity of 70 mCi per dose.
Example 11
[0211] Leprosy Therapy
[0212] A patient suffering from acute, disseminated leprosy, with
high fever and numerous skin lesions, that has been refractory to
conventional chemotherapy, is infused intravenously with a 70 mCi
dose in saline of the conjugate produced according to Example 10.
Gradual reduction in fever occurs, with localization of the
conjugate at the sites of subcutaneous lesions and in other foci,
which are detectable by gamma scintigraphy. After five days,
non-localized conjugate is substantially cleared and excreted, but
lesions and foci of infection still contain bound conjugate. The
patient is then exposed to a collimated thermal neutron beam,
focussed on the scintigraphically detected lesions and foci of
infection. Within the following week, significant necrosis at the
site of the lesions is observed, and regeneration of tissue
commences at the borders of the lesions. Conventional chemotherapy
is then resumed, with further improvement shown, permitting
eventual successful management of the patient.
[0213] The preceding examples can be repeated with similar success
by substituting other described reactants and/or operating
conditions of this invention for those used in the preceding
examples. Thus, antibodies to other human disease-producing
pathogens and/or their antigens, e.g., any of the other
illustrative pathogens enumerated herein, can be produced and
incorporated into imaging and therapy agents according to the
invention, and can achieve successful diagnostic and therapeutic
results in patients.
[0214] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention
and, without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
* * * * *