U.S. patent application number 09/899384 was filed with the patent office on 2001-12-06 for targeted immunostimulation with bispecific reagents.
This patent application is currently assigned to Medarex, Inc.. Invention is credited to Fanger, Michael W., Gosselin, Edmund J., Guyre, Paul M., Romet-Lemonne, Jean Loup.
Application Number | 20010048922 09/899384 |
Document ID | / |
Family ID | 27081614 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010048922 |
Kind Code |
A1 |
Romet-Lemonne, Jean Loup ;
et al. |
December 6, 2001 |
Targeted immunostimulation with bispecific reagents
Abstract
Disclosed are methods of stimulating in a subject an immune
response to an antigen to which the immune response is targeted.
This method includes the step of administering to the subject a
binding agent which binds a surface receptor of an
antigen-presenting cell, in some instances without being blocked
substantially by the natural ligand for the surface receptor, and
an antigen to which the immune response is targeted, in a
physiologically acceptable medium to the subject. Also disclosed
are molecular complexes including the binding agent coupled to an
antigen.
Inventors: |
Romet-Lemonne, Jean Loup;
(Gif-Sur-Yvette, FR) ; Fanger, Michael W.;
(Lebanon, NH) ; Guyre, Paul M.; (Hanover, NH)
; Gosselin, Edmund J.; (North Haverhill, NH) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
28 State Street
Boston
MA
02109
US
|
Assignee: |
Medarex, Inc.
|
Family ID: |
27081614 |
Appl. No.: |
09/899384 |
Filed: |
July 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09899384 |
Jul 3, 2001 |
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08453500 |
May 30, 1995 |
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6258358 |
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08453500 |
May 30, 1995 |
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08249669 |
May 26, 1994 |
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6248332 |
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08249669 |
May 26, 1994 |
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07874622 |
Apr 27, 1992 |
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07874622 |
Apr 27, 1992 |
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07593083 |
Oct 5, 1990 |
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Current U.S.
Class: |
424/136.1 ;
530/388.2 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 16/468 20130101; C07K 2317/77 20130101; C07K 16/283 20130101;
A61K 47/68 20170801 |
Class at
Publication: |
424/136.1 ;
530/388.2 |
International
Class: |
A61K 039/395; A61K
039/40; A61K 039/42; C07K 016/08; C07K 016/12 |
Claims
1. A method of stimulating an immune response in a subject to an
antigen, comprising the step of administering a binding agent,
which binds a surface receptor of an antigen-presenting cell, and
an antigen, to which the immune response is targeted, in a
pharmacologically acceptable medium to the subject, whereby the
antigen is targeted to the receptor of the antigen-presenting
cell.
2. The method of claim 1 wherein the binding agent binds the
surface receptor of the antigen-presenting cell without being
blocked substantially by the natural ligand for the surface
receptor.
3. The method of claim 1, wherein the antigen is coupled to the
binding agent.
4. The method of claim 1, wherein the binding agent is an antibody,
or fragment thereof comprising at least one complementarity
determining region.
5. The method of claim 1, wherein the binding agent is bispecific,
having a binding affinity for the receptor and for the antigen.
6. The method of claim 4, wherein the bispecific binding agent is
selected from the group consisting of heteroantibodies, bispecific
antibodies, and bispecific molecules.
7. The method of claim 1, wherein the antigen is selected from the
group consisting of viral, bacterial, parasite, allergen, venom,
and tumor-associated antigens.
8. The method of claim 7, wherein the antigen is derived from
hepatitis virus.
9. The method of claim 7, wherein the antigen is an HIV
antigen.
10. The method of claim 1, wherein the surface receptor is selected
from the group consisting of Fc.gamma.RI, Fc.gamma.RII, and
Fc.gamma.RIII.
11. The method of claim 1, wherein the antigen-presenting cell is a
macrophage.
12. The method of claim 1 wherein the administering step comprises
administering the binding agent and the antigen as a molecular
complex, the binding agent being a bispecific antibody,
heteroantibody, or bispecific molecule including: (i) a first
antibody, or fragment thereof, which specifically binds the Fc
receptor for immunoglobulin G (IgG) on the macrophage surface
without being blocked substantially by IgG; and (ii) a second
antibody, or fragment thereof, which specifically binds the
antigen.
13. The method of claim 12, wherein the heteroantibody comprises an
Fab-Fab conjugate.
14. A method of treating hepatitis B infection comprising
administering to an individual infected with the virus a molecular
complex comprising: (a) a hepatitis B surface antigen, or
surface-exposed portion thereof; and (b) an Fab-Fab heteroantibody,
wherein the first Fab binds the high affinity Fc receptor for
immunoglobulin G without being blocked substantially by IgG, and
the second Fab binds the antigen.
15. A molecular complex comprising: (a) an antigen; complexed to
(b) a binding agent which binds a surface receptor of an
antigen-presenting cell.
16. The molecular complex of claim 15 wherein the binding agent
binds a surface receptor of an antigen-presenting cell without
being blocked substantially by the natural ligand for the
receptor.
17. The molecular complex of claim 15, wherein the binding agent is
selected from the group consisting of a monoclonal antibody, a
heteroantibody, a bispecific antibody, and a bispecific
molecule.
18. The molecular complex of claim 15 wherein the binding agent is
chemically coupled to the antigen.
19. The molecular complex of claim 15 wherein the binding agent is
peptide-linked to the antigen, the molecular complex produced by
recombinant DNA techniques.
20. The molecular complex of claim 15, wherein the antigen is
selected from the group consisting of viral, bacterial, parasite,
allergen, venom, and tumor-associated antigens.
21. The molecular complex of claim 20, wherein the antigen is a
hepatitis antigen.
22. The molecular complex of claim 20, wherein the antigen is an
HIV antigen.
23. The molecular complex of claim 20, wherein the antigen is
selected from the group consisting of bee venom, pollen, and dust
mite antigen.
24. The molecular complex of claim 15, wherein the
antigen-presenting cell is a macrophage.
25. The molecular complex of claim 15, wherein the surface receptor
is a receptor on a macrophage selected from the group consisting of
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII.
26. The molecular complex of claim 25, wherein the surface receptor
is the high affinity Fc.gamma.RI for immunoglobulin G on a
macrophage.
27. The molecular complex of claim 20 wherein the antigen comprises
an allergen which binds to IgE on mast cells and basophils, thereby
causing a type I hypersensitivity reaction, and the bispecific
binding agent is a heteroantibody that binds the high affinity Fc
receptor without being blocked by IgG binding to the receptor.
28. The molecular complex of claim 15 wherein the binding agent
comprises an antibody fragment including at least one
complementarity determining region
29. The method of claim 28 wherein the antibody fragment is
selected from the group consisting of Fab, Fab', and Fv
fragments.
30. The molecular complex of claim 28 wherein the binding agent
comprises an Fab-Fab heteroantibody, the first Fab binding the
Fc.gamma. receptor for immunoglobulin G (IgG) without being blocked
by IgG, and the second Fab binding the antigen.
31. The molecular complex of claim 30 wherein the first Fab binds
the Fc.gamma.RI.
32. A vaccine composition comprising the molecular complex of claim
15 in a pharmaceutically acceptable medium.
33. A binding agent specific for an Fc.gamma. receptor,
incorporated into a carrier for targeting antigen to an
Fc.gamma.R-expressing cell, the carrier including the antigen.
34. The binding agent of claim 33, wherein the carrier is a
liposome having an inner layer and an outer layer and containing an
antigen within the liposome, the binding agent being incorporated
into the outer layer of the liposome.
35. The binding agent of claim 33 which is an anti-Fc receptor
antibody selected from the group consisting of an anti-Fc.gamma.RI
antibody, an Fc.gamma.RII antibody, and an Fc.gamma.RIII
antibody.
36. The binding agent of claim 35 comprising an anti-Fc.gamma.RI
antibody.
37. The binding agent of claim 33 wherein the carrier contains an
antigen selected from the group consisting of viral, bacterial,
parasite, allergen, venom, and tumor-associated antigens.
38. A bispecific antibody that is reactive with a surface receptor
selected from the group consisting of Fc.gamma.RI, Fc.gamma.RII,
and Fc.gamma.RIII.
39. A method of depleting antigen in the circulation of a subject
comprising the step of administering to the subject the bispecific
antibody of claim 38 in a pharmacologically acceptable medium.
40. A method of decreasing hypersensitivity in a subject comprising
the step of administering a molecular complex in a
pharmacologically acceptable medium to the circulation of a patient
in an amount sufficient to induce an immune response in the
subject, the complex comprising: (i) an allergen which binds to IgE
on mast cells and basophils, thereby causing a type I
hypersensitivity reactions; complexed to (ii) a binding agent
selected from the group consisting of a heteroantibody, bispecific
antibody, and monoclonal antibody, the binding agent binding the
high affinity Fc receptor without being blocked by IgG binding to
the receptor.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of pending patent
application Ser. No. 593,083, filed Oct. 5, 1990.
BACKGROUND OF THE INVENTION
[0002] Antigen molecules are recognized by the immune system after
internal processing by antigen-presenting cells, generally
mononuclear phagocytic cells such as macrophages and B lymphocytes.
In order to present a proteinaceous antigen, the antigen-presenting
cell first internalizes the antigen which is then broken down into
small peptidic fragments by enzymes contained in vesicles in the
cytoplasm of the antigen-presenting cells. After fragmentation, the
peptides are linked to cellular major histocompatibility complex
(MHC) molecules and presented on the presenting cell's surface to
the immune system. Peptides presented in this way are recognized by
the T-cell receptor which engages T-lymphocytes into the immune
response against this antigen. This antigen presentation also
stimulates the B lymphocytes for specific antibody production.
[0003] Complexes of antibody and antigen have been used to
stimulate an immune response against the antigen. Antigen uptake
through antigen-antibody complexes bound to Fc receptors for IgG
(Fc.gamma.R) increases the efficiency of antigen presentation and
thereby antigen-specific T-cell activation by human and mouse
macrophages, (Celis et al (1984) Science 224:297-299; Chang (1985)
Immunol. Today 6:245-259; Manca et al. (1988) Immunol.
140:2893-2898; Schalke et al. (1985) J. Immunol. 134:3643-3648; and
Snider et al (1987) J. Immunol. 139:1609-1616). The binding of
these complexes to Fc.gamma.R is mediated by the Fc region of the
antibody. This binding is susceptible to inhibition by
physiological concentrations of IgG.
SUMMARY OF THE INVENTION
[0004] This invention pertains to a binding agent which binds a
surface receptor of an antigen-presenting cell in some instances
without being blocked substantially by the natural ligand for the
receptor and which binds the antigen.
[0005] In one aspect of the invention, the binding agent employed
is bispecific agent such as a heteroantibody, bispecific antibody,
or other bispecific molecule having a binding specificity for the
antigen and a binding specificity for a surface receptor of an
antigen-presenting cell, such as a mononuclear phagocyte (e.g., a
macrophage).
[0006] As used herein, the term "heteroantibody" refers to a
conjugate of at least the antibody binding sites of two or more
antibody molecules of different specificities.
[0007] An "antibody binding site" is that portion of the antibody
molecule which binds a particular antigenic site. This antibody
binding site includes an immunoglobulin variable domain that
comprises three hypervariable regions flanked by four relatively
conserved framework regions. The hypervariable regions are believed
to be responsible for the binding specificity of individual
antibodies.
[0008] The term "bispecific antibody" refers to a single, divalent
antibody which has two different antigen binding sites (variable
regions).
[0009] A "bispecific molecule" is one which has two different
binding specificities and which can be bound to two different
molecules or two different sites on a molecule concurrently.
[0010] The bispecific binding agent binds the cellular receptor,
such as an Fc receptor, and targets the antigen to the cell. In
some embodiments, this bispecific binding agent binds the cellular
receptor without substantially being blocked by the natural ligand
for the receptor. In a preferred embodiment, the bispecific binding
agent specifically binds an Fc receptor of an antigen-presenting
cell for immunoglobulin G (IgG) without being blocked by IgG.
Preferred binding agents are specific for Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII. In a particularly preferred
embodiment, the agent specifically binds the high affinity Fc
receptor for immunoglobulin G (Fc.gamma.RI) on macrophages.
[0011] In another aspect of the invention, a preferred binding
agent is an antibody or binding fragment thereof which includes one
or more complementarity determining regions.
[0012] As used herein, "complementarity determining region"
includes one hypervariable region of an immunoglobulin molecule and
selected amino acids disposed in the framework regions which flank
that particular hypervariable region in an immunoglobulin
molecule.
[0013] In some aspects of the invention, the binding agent includes
at least two antibody binding fragments linked together by chemical
methods or genetically linked via recombinant DNA techniques. One
preferable binding agent is a Fab-Fab conjugate, wherein the first
Fab binds the high affinity Fc receptor as described above, and the
second Fab binds the antigen.
[0014] The binding agent of the invention is used to stimulate in a
subject an immune response to an antigen. In this method a binding
agent and an antigen are provided and administered in a
pharmacologically acceptable medium to the subject. The binding
agent targets the antigen to the antigen-presenting cell in the
subject.
[0015] The antigen to be targeted can be derived from a foreign
pathogen such as a viral, bacterial, or parasite antigen, or it can
be derived from endogenous diseased host cells such as tumor
associated antigens on tumor cells. Preferred binding agents
include antibodies specific for antigens derived from hepatitis
virus such as the hepatitis surface antigen, or an HIV antigen.
Other binding agents bind an epitope on bee venom, pollen, or dust
mite antigen.
[0016] Generally, the antigen is administered as a preformed,
chemically coupled complex with the binding agent. Alternatively
the antigen is incorporated into the binding agent through
recombinant DNA techniques to create a genetic hybrid that codes
for a fusion product including the binding agent and antigen. In
some cases, however, the antigen and the bispecific binding agent
are administered separately or the binding agent may be
administered alone.
[0017] In another embodiment of the invention, the antigen is
directly bound to a receptor-binding agent to create bispecific
molecules (e.g., receptor-specific antigens). For example, the
antigen can be covalently coupled to an antibody which binds the Fc
receptor without being blocked by IgG.
[0018] The binding agent which binds an Fc receptor may also be
incorporated into a lipid emulsion or the outer layer of a liposome
which contains the antigen. Preferably, the binding agent is an
antibody which recognizes the Fc.gamma.RI receptor. An additional
aspect of the invention is a vaccine including the molecular
complex of the invention in a pharmacologically acceptable
medium.
[0019] The compositions of this invention can be used to treat or
prevent infectious diseases such as hepatitis B, to neutralize the
acute phase of an infection by a pathogenic organism, to stimulate
the immune system in instances of chronic infection of such an
organism, to deplete antigen in the circulation of a subject, and
to treat tumors.
[0020] This invention also relates to methods and compositions used
to induce IgG responses against allergens to effect tolerance in
the case of IgE-mediated type I hypersensitivity, and to induce a
state of T cell tolerance to allergens which would interfere with
the development of IgE mediated responses. In these methods a
molecular complex is administered which consists of an allergen
which binds to IgE on mast cells and basophils, complexed to a
heteroantibody that binds the high affinity Fc receptor without
being blocked by IgG binding to the receptor. Enough of the complex
is administered to the circulation of a subject such that an immune
response is induced, which may include the production of
allergen-specific IgG, thereby inhibiting the binding of the
allergen to IgE on mast cells and basophils. Alternatively, the
administration of the complex induces a state of T cell tolerance
to the allergen by binding to naive B cells, thereby interfering
with an IgE-mediated type I reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The invention itself can be more fully understood from the
following description when read together with the accompanying
drawings in which:
[0022] FIGS. 1A, 1B, and 1C are graphs demonstrating the
enhancement of antigen presentation by monocytes to T cells using
anti-Fc.gamma.R-Ag conjugates or human IgG.sub.1 (HIgG.sub.1)
anti-Ag mAb. FIG. 1A shows T cell proliferation in response to
monoclonal antibody-tetanus toxoid (mAb-TT) conjugates; FIG. 1B
shows T cell proliferation in response to HIgG anti-TT; and FIG. 1C
shows T cell proliferation in response to mAb-TT conjugate as
compared to that of IgG.sub.1 anti-TT mAb+TT and TT alone;
[0023] FIGS. 2A and 2B are bar graphs demonstrating the ability of
the dominant HIgG isotype, HIgG.sub.1, and anti-Fc.gamma.RI
(22.2)-TT to target enhanced antigen presentation to human
Fc.gamma.RI. FIG. 2A shows that an anti-Fc.gamma.RI mAb (197)
blocks HIgG.sub.1 and anti-Fc.gamma.RI (22.2)-TT-enhanced antigen
presentation; while anti-Fc.gamma.RII (IV.3) and anti-Fc.gamma.RIII
(368) mAbs do not. FIG. 2B shows that mAb 197 does not block
enhanced T cell proliferation by anti-Fc.gamma.RIII (368)-TT
conjugates; and
[0024] FIGS. 3A and 3B show the ability of
anti-Fc.gamma.RI-(22.2)-TT to overcome blocking of
Fc.gamma.RI-enhanced antigen presentation by HIgG. FIG. 3A is a
graph showing the amount of HIgG.sub.1 required to saturate
Fc.gamma.RI at 4.degree. C. and at 37.degree. C.; and FIG, 3B is a
bar graph showing the effect of varying concentrations of
HIgG.sub.1 on anti-Fc.gamma.RI (22.2)-TT-enhanced antigen
presentation, as measured by T cell proliferation.
DETAILED DESCRIPTION OF THE INVENTION
[0025] An optimal antibody response to a thymus-dependent antigen
requires that the B cell obtain help from a CD4+helper T cell. The
B cell is uniquely designed to accomplish this in that it contains
antigen-specific immunoglobulin on its surface which allows it to
bind, internalize and process-antigen for presentation very
efficiently. Other antigen presenting cells, such as the macrophage
and dendritic cell, lack antigen-specific receptors, and therefore
also lack this highly efficient mechanism for processing and
presenting antigen. However, the apparent requirement for adjuvants
when administering vaccines suggests a need for an antigen
presenting cell in addition to the B cell. Also, it appears that
antigen presentation by resting B cells to resting T cells does not
lead to a T cell activation, but rather to T cell tolerance (Eynon
et al. (1992) J. ExP. Med. 175:131). This is due to the failure of
the resting B cell to deliver all the signals required for
activation of the resting T cell. On the other hand, it appears
that induction of T cell tolerance by the resting B cell could be
averted if the resting T cell first responds to antigen on the
antigen presenting cell such as the macrophage or dendritic cell
(Parker et al. (1991) FASEB J. 5:2777). This implies that in the
naive individual, the resting T cell must first interact with a
macrophage or dendritic cell before interacting with the resting B
cell.
[0026] These considerations have lead to the conclusion that the
optimal immunogen requires two major components: antigen which can
be recognized by the antigen-specific B cell; and a component which
directs antigen for efficient processing and presentation to an
antigen presenting cell other than the resting B cell (Parker et
al., ibid.; Germain (1991) Nature 353:605). Attaching antigens to
anti-Fc receptor antibodies satisfies these criteria since antigen
directed to Fc receptors on the macrophage enhances antigen
presentation at least 100 fold (Immunol. Today (1985) 6:245).
Studies in vivo support the efficacy of such a vaccine. For
example, a substantial increase in antibody production has been
observed following immunization of mice with bispecific antibody
which directed antigen to MHC class II or Fc.gamma.RII (Snider et
al. (1990) (J. Exp. Med. 171:1957-1963). In addition, the
requirement for adjuvant was eliminated. The ability to use
substantially lower doses of immunogens is especially valuable when
administering immunogens such as allergens that are potentially
toxic at higher doses. Tolerance against some allergens can be
obtained by repeated low dose administration of the allergen.
Tolerance may result from the production of IgG against the
allergen, which competes with allergen-specific IgE, removing the
allergen so that it will not interact with IgE-coated mast cells.
Allergen-anti-Fc receptor conjugates have the potential to both
reduce the amount of allergen administered, thereby further
reducing toxicity, and, at the same time, increase the production
of allergen-specific IgG.
[0027] To construct an immunogen for human use which would satisfy
the above criteria, the observation that antigen-antibody complexes
can significantly enhance antigen presentation was expanded. When
antigen-antibody complexes bind to Fc.gamma.R on the monocyte or
macrophage, the antigen is internalized and its subsequent
presentation and thus T cell activation, is dramatically enhanced
in vitro (Chang (1985) Immunol, Today 6:245), decreasing the
antigen concentration required for T cell activation by 10 to
100-fold. The data presented here demonstrate the potential for
using Fc.gamma.R-targeted immunogens as vaccines and show that all
three Fc.gamma. receptors function to enhance antigen
presentation.
[0028] In the method of this invention, an antigen is targeted to
an antigen-presenting cell to enhance the processes of
internalization and presentation by these cells, and ultimately, to
stimulate an immune response therein.
[0029] In one embodiment of the invention, a bispecific binding
reagent is employed to target the antigen to the cell. The
bispecific binding agent specifically binds the antigen (either
directly, to an epitope of the antigen, or indirectly, to an
epitope attached to the antigen) and, at the same time, binds a
surface receptor of an antigen-presenting cell which can
internalize antigen for processing and presentation. The
receptor-binding component of the bispecific binding agent (and
thus the bispecific binding agent, itself) binds the receptor of
the antigen-presenting cell. In some instances, binding of the
agent occurs without the agent substantially being blocked by the
natural ligand for the receptor. As a result, targeting of the
antigen to the receptor will not be prevented by physiological
levels of the ligand and the targeted receptor will remain capable
of binding the ligand and functioning.
[0030] The preferred surface receptors of antigen-presenting cells
for targeting are the receptors for the Fc region of IgG
(Fc.gamma.R). These receptors mediate internalization of
antibody-complexed antigens. The Fc receptors include Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII. The most preferred target is the
high affinity Fc receptor (Fc.gamma.RI).
[0031] As described in more detail below, the bispecific binding
agents are generally made of antibodies, antibody fragments, or
analogs of antibodies containing at least one complementarity
determining region derived from an antibody variable region.
[0032] Antibodies that bind to Fc receptors on antigen-presenting
cells can be produced by conventional monoclonal antibody
methodology e.g., the standard somatic cell hybridization technique
of Kohler and Milstein (Nature (1975) 256:495). Although somatic
cell hybridization procedures are preferred, in principle, other
techniques for producing monoclonal antibodies can be employed
e.g., viral or oncogenic transformation of B lymphocytes.
[0033] In general, an animal is immunized with an
Fc.gamma.R-bearing cell, a receptor-bearing portion thereof, or the
Fc receptor molecule in purified or partially purified form.
Antibodies are selected which bind an epitope of the Fc.gamma.R
which is located outside of the ligand (i.e., Fc) binding domain of
the receptor. This binding is not inhibited by IgG and, in turn,
does not inhibit the binding of IgG and the function of the Fc
receptor.
[0034] The production and characterization of monoclonal antibodies
which bind Fc.gamma.RI without being blocked by human IgG are
described by Fanger et al. in PCT application WO 88/00052 and in
U.S. Pat. No. 4,954,617, the teachings of which are incorporated by
reference herein. These antibodies bind to an epitope of
Fc.gamma.RI which is distinct from the Fc binding site of the
receptor and, thus, their binding is not blocked substantially by
physiological levels of IgG. Specific anti-Fc.gamma.RI antibodies
useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb
197. The hybridoma producing mAb 32 is available from the American
Type Culture Collection, Rockville, MD, ATCC No. HB9469.
[0035] The bispecific binding agent for targeting the antigen can
be a heteroantibody, a bispecific antibody, a bispecific molecule,
or an analog of any of these agents. Bispecific antibodies are
single, divalent antibodies which have two different antigen
binding sites (variable regions). In the bispecific antibodies of
this invention, one of the antigen binding sites is specific for
the receptor of the antigen-presenting cell and has the
characteristics set forth above, and the other binding site is
specific for the antigen to be targeted to the antigen-presenting
cell. These antibodies can be produced by chemical techniques (see
e.g., Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), by
"polydoma" techniques (See U.S. Pat. No 4,474,893), or by
recombinant DNA techniques.
[0036] The heteroantibodies of the invention are two or more
antibodies or antibody-binding fragments (Fv, Fab, Fab' or
F(ab').sub.2) of different binding specificity linked together.
Heteroantibodies comprise a first antibody (or antigen-binding
fragment thereof) specific for the receptor of the
antigen-presenting cell, coupled to a second antibody (or
antigen-binding fragment thereof) specific for the antigen to be
targeted.
[0037] Heteroantibodies can be prepared by conjugating together two
or more antibodies or antibody fragments. Preferred
heteroantibodies are comprised of crosslinked Fab fragments
(Fab-Fab). A variety of coupling or crosslinking agents can be used
to conjugate the antibodies. Examples are protein A, carboimide,
N-succinimidyl-S-acetyl-thioacetate (SATA) and
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). See e.g.,
Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M.A. et al.
(1985) Proc. Natl. Acad. Sci. USA 82:8648. Other methods include
those described by Paulus (Behring Inst. Mitt. (1985) No. 78,
118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et
al. (J Immunol. (1987) 139:2367-2375).
[0038] Bispecific binding agents can also be prepared via
recombinant DNA techniques from single chain antibodies. See e.g.,
Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879; Skerra et
al. (1988) Science 240:1038. These are analogs of antibody variable
regions produced as a single polypeptide chain. To form the
bispecific binding agent, the single chain antibodies may be
coupled together chemically or by genetic engineering methods.
[0039] As used herein, the term "antigen" means any natural or
synthetic immunogenic substance, a fragment or portion of an
immunogenic substance, a peptidic epitope, or a hapten. Suitable
antibodies against wide variety of antigens for construction of the
bispecific binding agents are available or can be readily made by
standard techniques.
[0040] One type of antigen to which a bispecific binding agent
(such as an antibody) can be produced is an allergen. Many
allergens are found in airborne pollens of ragweed, grasses, or
trees, or in fungi, animals, house dust, or foods. As a class, they
are relatively resistant to proteolytic digestion. Preferable
allergens are those which bind to IgE on mast cells and basophils,
thereby causing a type I anaphylaxis hypersensitivity reaction.
When the second specificity of the bispecific binding agent is for
an epitope of the high affinity Fc receptor that is outside the
ligand binding domain for IgG, this bispecific binding agent can
decrease hypersensitivity in a subject. This is accomplished when
the bispecific binding agent competes for an IgE-binding allergen
before the allergen binds to IgE on a mast cell or basophil,
thereby reducing the possibility of a type I hypersensitivity
reaction. In addition, as a result of directing allergen to
Fc.gamma.R, a state of T cell tolerance to the allergen may be
induced which interferes with IgE-mediated type I reactions.
Tolerance can be accomplished by inducing IgG which competes with
IgE for binding to allergen using doses of allergen substantially
lower than those currently used.
[0041] In some cases, it may be desirable to couple a substance
which is weakly antigenic or nonantigenic in its own right (such as
a hapten) to a carrier molecule, such as a large immunogenic
protein (e.g., a bacterial toxin) for administration. In these
instances, the bispecific binding reagent can be made to bind an
epitope of the carrier to which the substance is coupled, rather
than an epitope of the substance itself.
[0042] In another embodiment of the invention, the antigen can be
coupled directly to the binding agent for the receptor. In these
instances, the antibody itself can serve as the carrier protein.
For example, an antigen can be coupled to an antibody, or fragment
thereof, specific for an Fc receptor of an antigen-presenting cell.
Proteinaceous antigens can be biochemically coupled by the methods
described above or by other methods known by those with skill in
the art. Alternatively, a fusion protein may be produced by the
expression of an immunoglobulin gene genetically engineered to
include a gene encoding the antigen (Zanetti (1992) Nature
355:476-477). Such methods are described in detail in Sambrook et
al. (Molecular Cloning, A Laboratory Manual (Second Edition), Cold
Spring Harbor Press, 1989), herein incorporated by reference.
[0043] In another aspect of the invention, the antigen is targeted
to a cell via a carrier which contains antigen. Useful carriers
include lipid emulsions or synthetic lipid vesicles, i.e.,
liposomes, having incorporated into the outer layer of the liposome
the binding agent of the invention (Nair et al. (1992) J. Exp. Med.
175:609-612; and Reddy et al. (1992) J Immunol. 148:1585-1589). The
allergen may be encapsulated within the internal aqueous space, or
entrapped within the lipid bilayer(s), of the liposome.
Antigen-carrying liposomes can be fabricated according to
procedures known in the art, such as those described by Bangham et
al. (J. Mol. Biol. (1965) 12:238-252), and by Papahadjopoulos et
al. (U.S. Pat. No. 4,241,046), herein incorporated by reference. In
other embodiments of the invention, the binding agent is
incorporated into a lipid emulsion or the outer layer of a liposome
containing antigen
[0044] Alternatively, the binding agent may be incorporated into a
biodegradable hydrogel containing an allergen.
[0045] The antigen targeted by the methods of this invention can be
soluble or particulate; it may carry B cell epitopes, T cell
epitopes or both. The antigen can be bacterial, viral or parasitic
in origin. Often, the antigen will comprise a component of the
surface structure of a pathogenic organism. For example, the
antigen can comprise a viral surface structure such as an envelope
glycoprotein of human immunodeficiency virus (HIV) or the surface
antigen of hepatitis virus. In addition, the antigen can be
associated with a diseased cell, such as a tumor cell, against
which an immune response may be raised for treatment of the
disease. The antigen can comprise a tumor-specific or
tumor-associated antigen, such as the Her-2/neu proto-oncogene
product which is expressed on human breast and ovarian cancer cells
(Slamon et al. (1989) Science 244:707).
[0046] Targeted immunostimulation can be performed in vitro or in
vivo. The bispecific binding agent can be used to target antigen to
antigen-presenting cells in culture. Immunocompetent cells are
separated and purified from patient blood. The cells are exposed to
the antigen and the binding agent. Targeted antigen-presenting
cells will process the antigen and present fragments on their
surface. After stimulation, the cells can be returned to the
patient.
[0047] To elicit an immune response in vivo, the antigen can be
administered to a subject in conjunction with the binding agent.
Although in some circumstances the two may be administered
separately, typically, they are administered as a preformed
immunochemical complex. The complex is formed by incubating the
antigen and the bispecific binding agent at a desired molar ratio
under conditions which permit binding of the two species. For
example, the antigen and the bispecific binding reagent can be
incubated in saline solution at 37.degree. C. In some embodiments,
for therapy of a pre-existing condition, the bispecific binding
agent may be given without accompanying antigen.
[0048] The complex is administered in a pharmacologically
acceptable solution at a dosage which will evoke an immune response
against the antigen. The optimum dose of antigen, as well as the
molar ratio of antigen and binding agent, may vary dependent upon
factors such as the type of antigen, the immune status of the host,
the type of infection or other disease being treated, etc. In most
cases, the dose of antigen required to elicit an immune response
(as determined by any standard method for assessment of immune
response) should be lower than that which would be required if the
antigen were given alone or as a complex with a monospecific
antibody for the antigen (Snider et al., ibid.). Of course, the
dose should also be lower than that which elicits an allergic
response.
[0049] The method of this invention can be used to enhance or
reinforce the immune response to an antigen. For example, the
method is valuable for the treatment of chronic infections, such as
hepatitis and AIDS, where the unaided immune system is unable to
overcome the infection. It can also be used in the treatment of the
acute stages of infection when reinforcement of immune response
against the invading organism may be necessary.
[0050] The method can be used to reduce the dose of antigen
required to obtain a protective or therapeutic immune response or
in instances when the host does not respond or responds minimally
to the antigen. Although generally desirable, the lowering of
effective dose can be especially desirable when the antigen is
toxic to the host such as is the case for allergies.
[0051] The method of targeted immunostimulation can also be used in
disease therapy. For example, the bispecific binding agent can be
used to target a tumor-associated (or tumor-specific) antigen to an
antigen-presenting cell in order to cause or to enhance an immune
response against the tumor.
[0052] The invention is illustrated further by the following
nonlimiting exemplification.
EXAMPLE 1
Anti-Human Erythrocyte, Anti-Fc.gamma.RI Heteroantibody
[0053] A. Procedure
[0054] A bispecific heteroantibody was prepared from a monoclonal
antibody against human erythrocytes (mono-D, a human anti-RhD
antibody) and anti-Fc.gamma.RI antibody 32, by a protocol
previously described. Shen et al. (J. Immunol. (1986) 137:3378).
Briefly, human erythrocytes were washed three times in buffer
solution and then incubated for 60 minutes at 37.degree. C. in
solution of the heteroantibody. After the incubation and three
washings, erythrocytes coated with heteroantibody were diluted at
5.times.10.sup.7 cells per millimeter in Hank's buffer and then
incubated with adherent monocytes (macrophages) at the ratio of
100:1 for one hour at 37.degree. C. Cells were then washed in
phosphate buffered saline (PBS), fixed for one minute in ethanol
and stained with Giemsa for observation through a light
microscope.
[0055] B. Results
[0056] Internalization of erythrocytes was easily observed as
unstained spheres in the macrophage cytoplasm. The number of
macrophages that internalized at least one erythrocyte were
counted. This experiment was repeated numerous times with and
without the heteroantibody present. In each experiment, no
erythrocyte internalization was observed in macrophages which were
incubated with erythrocytes in the absence of the
heteroantibody.
[0057] In addition, experiments were performed after treatment of
adherent monocytes (macrophages) with various concentrations of
interferon-gamma which is known to increase the number of
Fc.gamma.RI receptors on the macrophage surface (Guyre et al.
(1988) J. Steroid Biochem. 30:1-6). As shown in TABLE 1 below, the
number of macrophages that internalized erythrocytes increased in a
direct relation to the concentration of interferon-gamma.
1 TABLE 1 % macrophages gamma interferon having internalized at
least concentration (.mu.g/ml) one erythrocyte 1000 40 100 25 10
6
[0058] These data show that the heteroantibody can trigger
internalization of antigen by macrophages.
EXAMPLE 2
Enhancement of Antigen Presentation by Anti-Fc.gamma.R-Ag and
HIgG.sub.1
[0059] A. Procedure
[0060] 1. Cell Preparation
[0061] Monocytes used in the assay were purified from peripheral
blood using techniques which minimize contamination with endotoxins
(Menzer et al. (1986) Cell. Immunol. 101:312-319). Monocyte purity
was approximately 85-95% as judged by morphology and expression of
the CD14 surface antigen.
[0062] CD4+ T cells used in the assay were isolated following a
primary stimulation of donor mononuclear cells with tetanus toxin.
Briefly, mononuclear cells were isolated from peripheral blood
using Ficoll-Hypaque (Winthrop Pharmaceuticals, New York, NY).
30.times.10.sup.6 mononuclear cells were stimulated in 50 ml of AIM
V medium (Gibco, Grand Island, NY) with 5 .mu.g/ml tetanus toxin
(Accurate Chemical Co., Westbury, N.Y.).
[0063] AIM V is a defined (serum free) medium for the growth of
human cells. The use of AIM V reduces non-specific T cell responses
while maintaining Ag-specific responses equal to those observed
with other media tested. This medium allows more definitive studies
of Fc receptor-enhanced antigen presentation in vitro. If antigen
is directed to Fc receptors using monoclonal antibodies that bind
to Fc receptors regardless of the presence of human IgG, this
medium is not a requirement to see enhanced antigen
presentation.
[0064] After three days at 37.degree. C., unbound cells were
removed by washing flasks 3.times. with Hepes-buffered RPMI-1640
(HRPMI). 40 ml of AIM V were added back to each flask along with 10
units/ml recombinant human interleukin IL-2 (Immunex, Seattle,
Wash.) and 2.5% autologous serum. After 10 to 14 days total
incubation time, T cells were harvested and dead cells were
pelleted through Ficoll Hypaque, yielding a highly enriched
population (90-95%) of CD4+, antigen-specific T cells. Use of this
primed polyclonal population of T cells minimizes non-specific
responses and xenogenic responses to mouse immunoglobulin, and
reduces the potential (which would exist using T cell clones) that
T cell responses will be sensitive to tetanus toxin modification as
a result of monoclonal antibody-tetanus toxin (mAb-TT) conjugation
or antibody binding to tetanus toxin.
[0065] 2. Antibody Preparation
[0066] The mAb-TT conjugates used in the assay were made by
inducing sulfhydryl groups on TT using
N-succinimidyl-S-acetyl-thioacetate, and linking TT to
sulfosuccinimidyl 4-(N maleimidomethyl) cyclohexane-I-carboxylate
treated (Fab').sub.2 mAb at a 1:1 molar ratio of TT:mAb (Partis et
al. (1983) J. Protein Chem. 2:263. HIgG anti-TT was produced by a
hybridoma (SA13) which was obtained from ATCC. The IgG anti-TT mAb
was purified with DEAE HPLC. This isotype of the IgG anti-TT mAb
was determined by ELISA to be IgG.sub.1.
[0067] 3. Antigen Presentation Assays
[0068] Antigen presentation assays were done as follows:
5.times.10.sup.4 T cells and 5.times.10.sup.4 monocytes, each in 50
.mu.l of AIM V medium, were added to wells of a 96 well microtiter
plate. Monocytes were treated with mitomycin C before addition to
wells to prevent proliferation of the antigen presenting cells and
the few contaminating lymphocytes. The volume of AIM V/well was
then increased such that once mAb and/or TT or mAb-TT conjugates
were added, the final volume was 200 .mu.l. Monoclonal antibody
[(Fab').sub.2 anti-Fc.gamma.RI (22.2), Fab anti-Fc.gamma.RII
(IV.3), (Fab').sub.2 anti-Fc.gamma.RIII (3G8)]-TT conjugates, or TT
with or without whole HIgG.sub.1, anti-TT, was added. Monoclonal
antibody 22 (mAb 22) is specific for the high affinity Fc.gamma.
receptor, and its binding to the receptor is not blocked by IgG Fc
(see U.S. Pat. No. 4,954,617). mAb IV.3 and 3G8 are specific for
the ligand binding domains of Fc.gamma.RII and Fc.gamma.RIII (Van
de Winkel et al. (1991) J. Leukocyte Biol. 49:511). Following
addition of cells and antigen to wells, plates were incubated for
72 hours (h) at 37.degree. C. in a CO.sub.2 incubator. After 72 h,
[.sup.3H]-thymidine was added in order to detect T cell
proliferation, according to the method of Lanzavecchia (Nature
(1985) 314:537).
[0069] B. Results
[0070] To determine which Fc.gamma.R types best participate in
enhancing antigen presentation, two approaches were used. In the
first, tetanus toxin was attached to (Fab').sub.2 anti-Fc.gamma.RI,
Fab anti-Fc.gamma.RII, or (Fab').sub.2 anti-Fc.gamma.RIII
monoclonal antibodies (mAb). In the second approach, TT plus whole
monomeric human IgG.sub.1 (HIgG.sub.1) anti-TT was added to
cultures. It was expected that a HIgG.sub.1-dependent response
would involve Fc.gamma.RI, since human Fc.gamma.RI binds monomeric
HIgG (Unkeless (1989) J. Clin. Invest. 83:355). In both systems,
responses were compared to those of tetanus toxin alone.
[0071] Both methods produced enhanced presentation of tetanus toxin
(FIG. 1A, 1B), and all three human Fc.gamma.R types participate
(FIG. 1A). Anti-Fc.gamma.RI-TT and anti-Fc.gamma.RII-TT conjugates
enhanced antigen presentation the greatest (100-fold) as compared
to anti-Fc.gamma.RIII-TT conjugates which enhanced antigen
presentation the least (10-fold). This difference is likely due to
the smaller percentage (10 to 15%) of monocytes which express
Fc.gamma.RIII on their surface, as opposed to those expressing
Fc.gamma.RI and Fc.gamma.RII (100%) (Van de Winkel et al. (1991) J.
Leukocyte Biol. 49:511). In addition, the anti-Fc.gamma.RI-TT
conjugate was consistently more effective than the HIgG.sub.1
anti-TT+TT complex (FIG. 1C).
[0072] In three separate, but similar experiments, including the
experiment depicted in FIG. 1C, enhancement of the response with
the anti-Fc.gamma.RI-TT conjugate was consistently 100 to 150-fold,
but only 30 to 50-fold for the HIgG.sub.1-anti-TT +TT complex. A
possible explanation for this observation is the following.
Analysis by high pressure liquid chromatography (HPLC) indicates
that aggregates of tetanus toxin were present in all tetanus toxin
preparations. Therefore, the presence of large IgG.sub.1-anti-TT+TT
complexes might result in some tetanus toxin enhancement through
the less effective Fc.gamma.RIII.
EXAMPLE 3
HIgG.sub.1 Targeting of Enhanced Antigen Presentation to Human
Fc.gamma.RI
[0073] A. Procedure
[0074] Experiments were done as in EXAMPLE 1, except that the
blocking anti-Fc.gamma.R mAb listed in EXAMPLE 1 or whole
IgG.sub.2a anti-Fc.gamma.RI (197) were added to wells at 37.degree.
C. for 2 h prior to addition of IgG.sub.1anti-TT+TT,
anti-Fc.gamma.RI-TT conjugate, or TT, alone. Monoclonal Ab 197 is a
mouse IgG.sub.2a which binds to human Fc.gamma.RI by both its Fc
and Fab binding domains (Guyre et al. (1989) J. Immunol.
143:1650).
[0075] B. Results
[0076] To confirm the specificity of the IgG.sub.1 anti-TT
enhancement for Fc.gamma.RI, enhancement was eliminated with
blocking concentrations of anti-Fc.gamma.R mAb against each
Fc.gamma.R type. The results are shown in FIGS. 2A and 2B, where T
cell proliferation is expressed as the mean counts/min (CPM) of
three replicates .+-. standard deviation (SD). (Fab').sub.2 mAb
alone or added in combination with tetanus toxin had no effect on T
cell proliferation (FIG. 2A). Only the anti-Fc.gamma.RI mAb 197
blocked IgG.sub.1 anti-TT-enhanced antigen presentation (FIG. 2A).
Monoclonal Ab 197 binds to Fc.gamma.RI by both its Fc and Fab
domains (Guyre et al. (1989) J. Immunol. 143:1650). At 37.degree.
C., this results in crosslinking and capping of Fc.gamma.RI such
that it is no longer available for ligand binding (Partis et al.
(1983) J. Protein Chem. 2:263). A role for the Fc domain of mAb 197
in blocking Fc.gamma.RII or Fc.gamma.RIII enhanced Ag presentation
was excluded by showing that mAb 197 blocks enhanced presentation
with anti-Fc.gamma.RI-TT conjugates, but does not block enhanced
presentation using anti-Fc.gamma.RII-TT and anti-Fc.gamma.RIII-TT
conjugates (FIG. 2B).
EXAMPLE 4
Blockage Inhibition of Fc.gamma.RI-Enhanced Antigen Presentation by
HIgG
[0077] A. Procedure
[0078] Flow-cytometric analysis was performed essentially as
described by Gosselin et al. (J. Immunol. (1990) 144:1817) to
determine the amount of HIgG.sub.1 required to saturate
Fc.gamma.RI. Briefly, cells were diluted in AIM V. To individual
wells of a 96 well pate, 30 .mu.l of 5.times.10.sup.4 monocytes
from the same donor as in EXAMPLE 2 were added. This was followed
by 30 .mu.l of AIM V containing 2.times.(twice concentrated)
HIgG.sub.1. HIgG.sub.1 was purified from the serum of a myeloma
patient using a sizing column and HPLC. The plates were then
incubated for 2 h at 4.degree. C. Plates were centrifuged, the
supernatants discarded, and the cells washed 3.times. with PBS/BSA
at 4.degree. C. Cells were then incubated for 1.5 h with 40
.mu.l/well of FITC-labeled (Fab').sub.2 goat anti-HIgG (Caltas, San
Francisco, Calif.), followed by 3 washes with PBS/BSA and
resuspension in PBS/BSA containing 1% formaldehyde (Kodak,
Rochester, N.Y.). Similar studies were done on monocytes incubated
for 1 h at 37.degree. C. in the presence of 0.1% sodium azide
(Sigma, St. Louis, Mo.).
[0079] Another assay was done as described in EXAMPLE 1 except that
HIgG.sub.1 was added first on ice followed by 22.2-TT conjugates or
HIgG1 anti-TT +TT.
[0080] B. Results
[0081] The results are shown in FIGS. 3A and 3B. In FIG. 3A,
negative controls are cells and FITC goat anti-HIgG preincubated
with 6 mg/ml HIgG at 4.degree. C. (open circles), and 37.degree. C.
(open square). All samples were read on a FACSan. Data are
expressed as mean fluorescence of three replicates .+-.S.D.
[0082] Monocytes used in these antigen presentation assays had HIgG
present on their surface (see FIG. 3A). These results are
consistent with those of Van de Winkel et al. which show that Human
Fc.gamma.RI binds HIgG.sub.1 with high affinity, while Fc.gamma.RII
and Fc.gamma.RIII do not (Van de Winkel et al. (1991) J. Leukocyte
Biol. 49:511). In addition, concentrations of IgG1 anti-TT+TT which
induce a 50% maximal response (shown in FIG. 1) were completely
blocked by monomeric HIgG.sub.1 added at concentrations which
saturate Fc.gamma.RI (FIG. 3A). Under similar conditions HIgG.sub.1
had no significant effect on anti-Fc.gamma.RI(22.2)-TT-enhanced
antigen presentation at concentrations of HIgG five-fold higher
than those that block IgG.sub.1 anti-TT-enhanced antigen
presentation (FIG. 3B). In fact, mAb 22.2 is known to bind outside
the ligand binding domain of Fc.gamma.RI (Guyre et al. (1989) J.
Immunol. 143:1650).
[0083] These results suggest an alternative explanation for reduced
enhancement of antigen presentation through Fc.gamma.RI when using
HIgG.sub.1 anti-TT+TT, which is that IgG.sub.1-enhanced
presentation is inhibited by serum IgG still bound to
Fc.gamma.RI.
[0084] Equivalents
[0085] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *