U.S. patent application number 09/853300 was filed with the patent office on 2002-04-25 for therapeutic method and composition utilizing antigen-antibody complexation and presentation by dendritic cells.
Invention is credited to Noujaim, Antoine, Schultes, Birgit.
Application Number | 20020048583 09/853300 |
Document ID | / |
Family ID | 27394574 |
Filed Date | 2002-04-25 |
United States Patent
Application |
20020048583 |
Kind Code |
A1 |
Schultes, Birgit ; et
al. |
April 25, 2002 |
Therapeutic method and composition utilizing antigen-antibody
complexation and presentation by dendritic cells
Abstract
Disclosed are methods and compositions for use in immunotherapy.
These methods and compositions are particularly useful for
exploiting dendritic cells to present an antigen to a patient,
particularly where the patient has a disease associated with the
antigen. The invention provides methods for treating a patient
having a disease associated with an antigen. The methods according
to the invention comprise combining ex vivo an antigen and an
antigen-presenting cell binding agent specific for the antigen, and
administering the composition to a patient suffering from a disease
associated with the antigen, wherein the patient receives a
therapeutic benefit.
Inventors: |
Schultes, Birgit;
(Arlington, MA) ; Noujaim, Antoine; (Edmonton,
CA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
27394574 |
Appl. No.: |
09/853300 |
Filed: |
May 11, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60203635 |
May 11, 2000 |
|
|
|
60253956 |
Nov 28, 2000 |
|
|
|
60253671 |
Nov 28, 2000 |
|
|
|
Current U.S.
Class: |
424/153.1 ;
424/154.1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61P 11/00 20180101; A61P 25/28 20180101; A61P 31/20 20180101; A61P
31/18 20180101; A61P 31/10 20180101; C07K 16/18 20130101; A61K
39/39558 20130101; A61P 37/00 20180101; A61P 37/04 20180101; A61P
31/00 20180101; A61K 2039/505 20130101; A61P 15/00 20180101; A61P
13/08 20180101; A61P 43/00 20180101; A61K 39/001194 20180801; A61K
39/39533 20130101; A61P 31/04 20180101; A61P 1/18 20180101; A61P
33/00 20180101; A61P 1/00 20180101; A61P 31/12 20180101; A61P 35/00
20180101; A61K 2039/5154 20130101; A61P 31/22 20180101; A61K
39/39533 20130101; A61K 2300/00 20130101; A61K 39/39558 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/153.1 ;
424/154.1 |
International
Class: |
A61K 039/395 |
Claims
We claim:
1. A method for treating a patient suffering from a disease
associated with an antigen, comprising administering to the patient
suffering from the disease a composition comprising an antigen
associated with the disease and a dendritic cell binding agent
specific for the antigen, wherein the antigen is complexed to the
binding agent and wherein the patient administered the composition
receives a therapeutic benefit.
2. The method of claim 1, wherein the patient is human.
3. The method of claim 1, wherein a CD8+ IFN.gamma. producing T
cell is activated to induce a CTL immune response in the patient
administered the composition.
4. The method of claim 1, wherein a CD4+ IFN.gamma. producing T
cell is activated to induce a helper T cell immune response in the
patient administered with the composition.
5. The method of claim 1, wherein a humoral immune response is
activated in the patient administered the composition.
6. The method of claim 1, wherein the dendritic cell binding agent
specifically binds to the antigen and binds to an Fc.gamma. Type I
(CD64) receptor on a dendritic cell in the patient administered the
composition.
7. The method of claim 1, wherein the dendritic cell binding agent
specifically binds to the antigen and binds to an Fc.gamma. Type II
(CD32) receptor on a dendritic cell in the patient administered the
composition.
8. The method of claim 1, wherein the dendritic cell binding agent
specifically binds to the antigen and binds to an Fc.gamma. Type II
CD16 (Fc.gamma.RIII) receptor on a dendritic cell in the patient
administered with the composition.
9. The method of claim 1, wherein the dendritic cell binding agent
is an antibody.
10. The method of claim 3, wherein the dendritic cell binding agent
is a xenotypic antibody to the patient.
11. The method of claim 10, wherein the xenotypic antibody elicits
a human anti-xenotypic antibody response in the patient.
12. The method of claim 11, wherein host anti-xenotypic antibodies
(HAXA) are present in the patient's blood prior to administering
the composition.
13. The method of claim 11, wherein the xenotypic antibody is a
monoclonal antibody.
14. The method of claim 14, wherein the monoclonal antibody is a
murine monoclonal antibody.
15. The method of claim 15, wherein the murine monoclonal antibody
is selected from the group consisting of Alt-1, Alt-2, Alt3, Alt-4,
Alt-5; and Alt-6.
16. The method of claim 3, wherein the composition further
comprises human anti-xenotypic antibodies (HAXA).
17. The method of claim 1, wherein the antigen is a
tumor-associated antigen.
18. The method of claim 1, wherein the antigen is from a
pathogen.
19. A method for treating a patient suffering from a disease
associated with an antigen, comprising administering to the patient
suffering from the disease a composition comprising a host
anti-xenotypic antibody and a xenotypic antibody specific for the
antigen associated with the disease, wherein the patient
administered the composition receives a therapeutic benefit.
20. The method of claim 19, wherein the antigen is present in the
patient.
21. The method of claim 19, wherein the patient is human.
22. The method of claim 19, wherein a CD8+ IFN-.gamma. producing T
cell is activated to induce a CTL immune response in the patient
administered the composition.
23. The method of claim 19, wherein a CD4+ IFN-.gamma. producing T
cell is activated to induce a helper T cell immune response in the
patient administered the composition.
24. The method of claim 19, wherein a humoral immune response is
activated in the patient administered the composition.
25. The method of claim 19, wherein the xenotypic antibody
specifically binds to the antigen and binds to an Fc.gamma. Type I
(CD64) receptor on a dendritic cell in the patient administered the
composition.
26. The method of claim 19, wherein the xenotypic antibody
specifically binds to the antigen and binds to an Fc.gamma. Type II
(CD32) receptor on a dendritic cell in the patient administered the
composition.
27. The method of claim 19, wherein the xenotypic antibody
specifically binds to the antigen and binds to an Fc.gamma. Type II
CD 16 (Fc.gamma.RIlI) receptor on a dendritic cell in the patient
administered with the composition.
29. The method of claim 19, wherein the xenotypic antibody is a
monoclonal antibody.
30. The method of claim 29, wherein the monoclonal antibody is a
murine monoclonal antibody.
31. The method of claim 30, wherein the murine monoclonal antibody
is selected from the group consisting of Alt-1, Alt-2, Alt3, Alt-4,
Alt-5; and Alt-6.
33. The method of claim 1, wherein the antigen is a
tumor-associated antigen.
34. The method of claim 1, wherein the antigen is from a
pathogen.
35. A therapeutic composition comprising a purified dendritic cell
binding agent that is specific for an antigen associated with a
disease and the antigen associated with the disease.
36. The composition of claim 35, wherein binding of the dendritic
cell binding agent to a receptor on a dendritic cell blocks binding
of a natural ligand to the receptor.
37. The composition of claim 33, wherein administration of the
composition to a patient suffering from the disease provides the
patient a therapeutic benefit.
38. The composition of claim 33, wherein the patient is human.
39. The composition of claim 33, wherein the dendritic cell binding
agent is an antibody.
40. A therapeutic composition comprising a host anti-xenotypic
antibody and a xenotypic antibody specific for the antigen
associated with the disease.
41. The composition of claim 40, wherein administration of the
composition to a patient suffering from the disease provides the
patient a therapeutic benefit.
42. The composition of claim 40, wherein the patient is human.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Serial No. 60/203,635 filed on May 11, 2000; U.S.
Provisional Application Serial No. 60/253,956 filed Nov. 28, 2000;
and U.S. Provisional Application Serial No. 60/253,671 filed Nov.
28, 2000 the entire contents of each of which are fully
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to immunotherapy. More particularly,
the invention relates to the use of antigen presenting cells, in
particular dendritic cells, in immunotherapy.
[0004] 2. Summary of the Related Art
[0005] T lymphocytes (i.e., T cells), unlike B lymphocytes (i.e., B
cells), typically recognize their target antigen only when the
antigen is presented in the context of the major histocompatibility
complex (MHC). Thus, to present antigen to T lymphocytes, which
include T helper cells and cytotoxic T cells, the antigen must be
presented in context of an MHC molecule on the surface of an
antigen presenting cell.
[0006] In particular, one type of antigen presenting cell,
dendritic cells, has recently become of interest in the area of
cancer immunotherapy. Steinman, Annu. Rev. Immunol. 9: 271-296
(1991) teaches that dendritic cells are rare leukocytes that
originate in the bone marrow and can be found distributed
throughout the body. Bjork, Clinical Immunology 92: 119-127 (1999)
teaches that dendritic cells receive increasing attention due to
their potential inclusion as biological adjuvants in tumor
vaccines. Dendritic cells express several receptors for the Fc
portion of immunoglobulin IgG, which mediate the internalization of
antigen-IgG complexes (ICs). In this capacity, dendritic cells are
used to present tumor antigens to T cells. Avigan, Blood Reviews
13: 51-64 (1999) teaches that several approaches have been adopted
to directly load tumor antigens onto dendritic cells, including the
pulsing of tumor peptides onto mature dendritic cells. Timmerman et
al., Annu. Rev. Med. 50: 507-529 (1999) teaches that isolated
dendritic cells loaded with tumor antigen ex vivo and administered
as a cellular vaccine have been found to induce protective and
therapeutic anti-tumor immunity in experimental animals. European
Patent No. EP0553244 describes an antigen/dual-specific binding
agent complex for stimulating a response to the antigen, where the
binding agent specifically binds both the antigen and a cell
surface receptor on an antigen-presenting cell, but where binding
of the binding agent to the cell surface receptor does not block
the natural ligand for the receptor.
[0007] The mechanism of action for dendritic cell antigen
presentation has also been explored. Coughlan et al., Veterinary
Immunology and Immunopathology 49: 321-330 (1996) discloses that
antigen uptake by dendritic cells via Fcy receptors results in
functional augmentation of antigen presentation and T cell
proliferation in an in vitro sheep system. Regnault et al., J. Exp.
Med. 189: 371-380 (1999) teaches that Fc.gamma. receptors induce
dendritic cell maturation and promote efficient MHC class
I-restricted presentation of peptides from exogenous,
immunoglobulin (Ig) complexed antigens in the mouse system.
[0008] Thus, there remains a need to discover methods for utilizing
dendritic cells to treat human diseases. The promise of dendritic
cell-based approaches to treat diseases, such as cancer,
underscores the need to actually develop such approaches as
effective therapeutic treatments.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides a therapeutically effective dendritic
cell-based approach to the treatment of diseases associated with an
antigen. The methods according to the invention comprise combining
ex vivo an antigen associated with a disease and a dendritic cell
binding agent specific for the antigen, with or without a dendritic
cell, to provide a composition, and administering the composition
to a patient having a disease associated with the antigen, wherein
the composition-administered patient receives a therapeutic
benefit.
[0010] Accordingly, in a first aspect, the invention provides a
method for treating a patient suffering from a disease associated
with an antigen, comprising administering to the patient suffering
from the disease a composition comprising an antigen associated
with the disease and a dendritic cell binding agent specific for
the antigen, wherein the antigen is complexed to the dendritic cell
binding agent and wherein the patient administered the composition
receives a therapeutic benefit. Preferably, the patient is
human.
[0011] In a second aspect, the invention provides a method for
treating a patient suffering from a disease associated with an
antigen, comprising administering to the patient a composition
comprising an antigen associated with the disease, a dendritic cell
binding agent specific for the antigen, and a dendritic cell
autologous to the patient, wherein the patient administered the
composition receives a therapeutic benefit. Preferably, the patient
is a human.
[0012] In a third aspect, the invention provides a method for
treating a patient suffering from a disease associated with an
antigen, comprising administering to the patient suffering from the
disease a composition comprising a host anti-xenotypic antibody and
a xenotypic antibody specific for the antigen associated with the
disease, wherein the patient administered the composition receives
a therapeutic benefit.
[0013] In a fourth aspect, the invention provides a therapeutic
composition comprising a purified dendritic cell binding agent that
is specific for an antigen associated with a disease and the
antigen associated with the disease. In preferred embodiments,
binding of the dendritic cell binding agent to a receptor on a
dendritic cell blocks binding of a natural ligand to the receptor.
In certain embodiments of the fourth aspect of the invention,
administration of the composition to a patient suffering from the
disease provides the patient a therapeutic benefit. Preferably, the
patient is a human. Preferably, the dendritic cell binding agent is
an antibody.
[0014] In a fifth aspect, the invention provides a therapeutic
composition comprising a purified dendritic cell binding agent that
is specific for an antigen associated with a disease, a dendritic
cell, and the antigen associated with the disease. In preferred
embodiments, binding of the dendritic cell binding agent to a
receptor on the dendritic cell blocks binding of a natural ligand
to the receptor. In certain embodiments, administration of the
composition to a patient suffering from the disease provides the
patient a therapeutic benefit, wherein the dendritic cell is
autologous to the patient. Preferably, the patient is human
[0015] In a sixth aspect, the invention provides a therapeutic
composition comprising a purified xenotypic antibody that is
specific for an antigen associated with a disease and a host
anti-xenotypic antibody. In certain embodiments, administration of
the composition to a patient suffering from the disease provides
the patient a therapeutic benefit, wherein the dendritic cell is
autologous to the patient. Preferably, the patient is human
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A and 1B show the results of antigen binding studies
with monocytes (blue bars) and immature dendritic cells (red bars).
FIG. 1A is a bar graph showing the percentage of positive events of
monocytes or immature dendritic cells following incubation with
1000 U/ml FlTC-labeled CA125 antigen and the indicated amount of
Alt-2 antibody. FIG. 1B is a bar graph showing the mean channel
intensity of monocytes or immature dendritic cells following
incubation with 1000 U/ml FITC-labeled CA125 antigen and the
indicated amount of Alt-2 antibody.
[0017] FIG. 2 is a bar graph showing the uptake of 1000 U/ml
FITC-labeled CA125 antigen in the presence of 0, 0.2, or 0.4
.mu.g/ml Alt-2 antibody (red bars) or control MOPC-21 antibody
(yellow bars).
[0018] FIGS. 3A and 3B are bar graphs showing the percentage of
positive events (FIG. 3A) and mean channel intensity (FIG. 3B) of
monocytes (striped bars) or immature dendritic cells (solid bars)
incubated with 1000 U/ml FITC-labeled CA125 antigen and the
indicated amounts of murine Alt-2 (blue solid and striped bars) or
chimeric Alt-2 (red solid and striped bars).
[0019] FIGS. 4A, 4B, and 4C are bar graphs showing the effect of
complexation with HAMA (FIG. 4A), specific antigen (FIGS. 4A and
4B), or both (FIG. 4C) on antibody binding to dendritic cells. FIG.
4A shows the binding of FITC-labeled anti-CA125 antibody, Alt-2, or
FITC-labeled anti-PSA antibody, Al-6, to dendritic cells in the
presence or absence antigen or in the presence or absence of HAMA.
FIG. 4B shows the binding of the FITC labeled Alt-2 in the presence
of 8000 U/ml CA125 when the concentration of Alt-2 is 0, 0.313,
0.625, 1.25, and 2.5 .mu.g/ml. FIG. 4C show the binding of 1
.mu.g/ml FITC labeled Alt-2 in the presence of 8000 U/ml CA125 with
or without human anti-mouse antibody (HAMA) at 0, 0.33, 1, and 2
.mu.g/ml.
[0020] FIGS. 5A and 5B are bar graphs showing the uptake of
CA125-Alt-2 immune complexes in monocytes and immature dendritic
cells in the presence (red solid and striped bars) and absence
(blue solid and striped bars) of HAMA as measured by the percentage
of positive events (FIG. 5A) and mean channel intensity (FIG. 5B)
for monocytes (striped bars) or immature dendritic cells (solid
bars).
[0021] FIG. 6 is a bar graph showing the in vitro T cell activation
(both CD4+ (white bars) and CD8+ T cells (black bars)) by dendritic
cells "armed" by the indicated primary stimulation and
restimulation as determined by the number of IFN.gamma. producing
cells per 10.sup.6 cells.
[0022] FIG. 7 is a bar graph showing IFN-.gamma. release from T
cells stimulated with DC loaded with CA125, Alt-2, or CA125/Alt-2
complex (i.e., CA125/.alpha.CA125 complex) at the indicated
concentrations for a seven day incubation period (round 1; blue
bars) or for an initial seven days and an additional seven days
with freshly loaded DCs (round 2; red bars).
[0023] FIGS. 8A and 8B are bar graphs showing intracellular
IFN-.gamma. production from CD4+ T cells (red bars) or CD8+ T cells
(blue bars) stimulated with DC loaded with CA125, Alt-2, or
CA125-Alt-2 complex (i.e., CA125/.alpha.CA125 complex) at the
indicated concentrations for a seven day incubation period (FIG.
8A) or for an initial seven days and an additional seven days with
freshly loaded DCs (FIG. 8B).
[0024] FIG. 9 is a bar graph showing the in vitro T cell activation
(both CD4+ and CD8+ T cells) to peptides generated by dendritic
cells "armed" by the indicated primary stimulation and
restimulation. T cell activation was determined by the number of
IFN.gamma. producing cells per 10.sup.6 cells.
[0025] FIGS. 10A and 10B show the rate of complexation of
anti-CA125 antibody, Alt-2, to circulating CA125 following
injection of Alt-2. Specifically, FIG. 10A is a bar graph comparing
the levels of CA125/aCA125 complex (black bars) compared to the
level of free circulating CA125 (dotted bars). FIG. 10B is a line
graph showing the rate of complexation of anti-CA125 antibody,
Alt-2, to circulating CA125 following injection of Alt-2 (left side
of the graph), and the amount of free Alt-2 antibody (green
circles) cleared following injection time, as measured in
percentage of the injected dose (% ID). With time, the percentage
of free circulating CA125 decreases as the amount of complexed
CA125 increases.
[0026] FIGS. 11A, 11B, and C are graphs showing the relationship
between CA125 antigen specific B cell (FIG. 11A) and T cell (FIGS.
11B and 11C) responses, CA125 level, and patient survival.
Specifically, FIG. 11A shows the induction of humoral anti-CA125
response (y-axis) as compared to the level of circulating CA125
antigen present at the time of injection of the anti-CA125
antibody, Alt-2 (x-axis). FIG. 11B shows the induction of a
cellular anti-CA125 response (y-axis) as compared to the level of
circulating CA125 antigen (x-axis), comparing pre-injection of
Alt-2 (open triangles) to post-injection of Alt-2 (closed
triangles). FIG. 11C is a scatter graph showing the stimulation
index of T cells (CA125-specific T cell proliferation) in patient
before (pre) or after (post) Alt-2 injection, where patients having
greater than 105 U/ml CA125 had the greatest T cell response.
[0027] FIG. 12 is a bar graph showing the multi-epitopic nature of
the response from anti-CA125 antibodies produced by thirteen
patients.
[0028] FIGS. 13A and 13B are survival curves of patients receiving
injection of Alt-2. Specifically, FIG. 13A is a survival curve
showing an increased survival in patients who developed a 3x
increase in anti-CA125 antibody response following injection of
Alt-2 (dotted line) as compared to those with developed a less than
3x increase in anti-CA125 antibody response following injection of
Alt-2 (solid line). FIG. 13B is a survival curve showing an
increased survival in patients who developed a CA125-specific T
cell response following injection of Alt-2 (dotted line) as
compared to those who did not develop a CA125-specific T cell
response following injection (solid line).
[0029] FIGS. 14A-14B show the preferential presentation of antigen
complexed with antibody by antigen presenting cells such as
macrophages and B cells. Specifically, FIG. 14A is a bar graph
showing the level of T cell proliferation as measured by the
Stimulation Index (SI) using macrophages as antigen-presenting
cells following stimulation by the CA125, Alt-2, B27.1, mIgG1,
CA125 plus Alt-2, CA125 plus B27.1, and CA125 plus mIgG1 at 0.1
.mu.g/ml (black dots on white bars), 1 .mu.g/ml ((white dots on
black bars), and 10 .mu.g/ml (solid black bars). FIG. 14B is a bar
graph showing the level of T cell stimulation as measured by the
Stimulation Index (SI) using B cells as antigen-presenting cells
following stimulation by the CA125, Alt-2, B27.1, mIgG1 , CA125
plus Alt-2, CA125 plus B27.1, and CA125 plus mIgG1 at 0.1 .mu.g/ml
(black dots on white bars), 1 .mu.g/ml ((white dots on black bars),
and 10 .mu.g/ml (solid black bars).
[0030] FIG. 15 is a bar graph showing the different levels of human
IgG in reconstituted SCID/bg mice.
[0031] FIG. 16 is a bar graph showing the different tumor volumes
in mice treated with control IgG1 (yellow bars), Alt-6 only (green
bars), PSA only (blue bars), and the PSA/.alpha.-PSA immune complex
(red bars).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] The invention relates to immunotherapy. More particularly,
the invention relates to the use of antigen-presenting cells, in
particular dendritic cells, in immunotherapy. The invention
provides a therapeutically effective dendritic-based approach to
the treatment of diseases associated with an antigen. The patents
and publications cited herein reflect the level of skill in this
field and are hereby incorporated by reference in their entirety to
the same extent as if each was specifically and individually
indicated to be incorporated by reference. In the case of any
conflict between a cited reference and this specification, this
specification shall prevail.
[0033] The invention provides methods for treating a patient having
a disease associated with an antigen. The methods according to the
invention comprise combining ex vivo the antigen associated with
the disease with a dendritic cell binding agent specific for the
antigen to provide a composition, and administering the composition
to a patient suffering from the disease associated with the
antigen, wherein the patient receives a therapeutic benefit.
[0034] Accordingly, in a first aspect, the invention provides a
method for treating a patient suffering from a disease associated
with an antigen, comprising administering to the patient suffering
from the disease a composition of an antigen associated with the
disease and a dendritic cell binding agent specific for the
antigen, wherein the antigen is complexed to (i.e., specifically
bound by) the dendritic cell binding agent, and wherein the patient
administered the composition receives a therapeutic benefit.
[0035] In certain embodiments, an antigen/dendritic cell binding
agent complex may be formed, for example, by combining ex vivo the
antigen and the dendritic cell binding agent. By "combining ex
vivo" means bringing into physical proximity outside of the
body.
[0036] In certain preferred embodiments, the patient is a human. In
other embodiments, the patient is preferably a non-human mammal,
particularly a laboratory animal. Preferred non-human patients of
the invention include, without limitation, mice, rats, rabbits,
non-human primates (e.g., chimpanzees, baboons, rhesus monkeys),
dogs, cats, pigs, and armadillos.
[0037] The methods according to the invention are useful for
therapeutically treating patients having a disease associated with
an antigen. As used herein, the term "disease associated with an
antigen" means a condition in which signs or symptoms of illness in
a majority of patients are present when the antigen is present in
the patient's body at a certain concentration, but in which signs
or symptoms of illness are absent or reduced when the antigen is
absent from the patient's body or present in the patient's body at
a lower concentration. "Signs or symptoms of illness" are
clinically recognized manifestations or indications of disease.
[0038] It will be appreciated that a "patient suffering from a
disease associated with an antigen" of the invention may not yet be
symptomatic for the disease. Accordingly, a patient with
circulating BRCA-1 is a patient according to the invention even
though that patient may not yet be symptomatic for breast cancer or
other adenocarcinoma.
[0039] Some non-limiting examples of such antigens associated with
a disease include the prostate specific antigen (associated with
prostate cancer), BRCA-1 and BRCA-2 antigens (associated with many
adenocarinomas, including breast cancer, lung cancer, and
pancreatic cancer), CA125 (associated with ovarian cancer),
aberrant myelin basic protein (associated with Alzheimer's
disease), gpl20 (associated with HIV infection and AIDS), MUC-1
(associated with breast cancer), EBNA-1 (associated with Epstein
Barr Virus infection), CA19.9 (associated with colorectal, stomach,
and pancreatic cancers), and TAG-72 (associated with ovarian,
stromal, and pancreatic cancers), p53 (associated with various
cancers).
[0040] Thus, in certain preferred embodiments, the antigen is a
tumor-associated antigen. A "tumor associated antigen" is an
antigen in the patient's body that is made by tumor cells, and
which may be presented on the tumor surface, or circulating, or
both. Preferred tumor-associated antigens include, without
limitation, CA125, PSA, MUC-1, CA19.9, and TAG-72. Generally from
about 0.1 to about 50 .mu.g antigen are used.
[0041] In certain preferred embodiments, the antigen is from a
pathogen. A "pathogen" is an etiolytic agent capable of causing
disease. Preferred pathogens include, without limitation, viruses
(e.g. hepatitis B, hepatitis C, herpes, and HIV-1), viroids,
bacteria, fungi, prions, and parasites.
[0042] "Specifically bound to the antigen" or "specific for the
antigen" means that the dendritic cell binding agent binds to the
antigen with greater affinity than it binds unrelated proteins.
Preferably such affinity is at least 10-fold greater, more
preferably at least 100-fold greater, and most preferably at least
1000-fold greater than the affinity of the binding agent for
unrelated proteins. Preferably, an antigen presenting cell binding
agent that is specific for an antigen forms an association with
that antigen with an affinity of at least 10.sup.6 M.sup.-1, more
preferably, at least 10.sup.7 M.sup.-1, even more preferably, at
least 10.sup.8 M.sup.-1, even more preferably, at least 10.sup.9
M.sup.-1, and most preferably, at least 10.sup.10 M.sup.-1 either
in water, under physiological conditions, or under conditions which
approximate physiological conditions with respect to ionic
strength, e.g., 140 mM NaCl, 5 mM MgCl.sub.2.
[0043] The injected dendritic cell binding agent complexed to a
circulating antigen is targeted in vivo to dendritic cells (which
are preferably immature dendritic cell) through Fc receptors
present on the surface of these dendritic cells. By targeting
antigen to preferably immature dendritic cells and presentation of
these antigens on both MHC class I and class II molecules, the
immune complex of the binding agent/antigen efficiently sensitize
dendritic cells to induce activation of both CD4(+) helper and
CD8(+) cytotoxic T cells in vivo.
[0044] A "dendritic cell binding agent" of the invention binds to
the ligand-binding site of a receptor on the surface of a dendritic
cell, at any stage of development of the dendritic cell.
Preferably, once the dendritic cell binding agent is bound to the
ligand-binding site of the dendritic cell receptor, the natural
ligand cannot bind to the receptor at the same time that the
dendritic cell binding agent binds to the receptor. Preferably, the
dendritic cell binding agent binds to the receptor on the surface
of a dendritic cell when the binding agent is specifically bound to
an antigen. Preferably, such binding causes internalization of the
binding agent/antigen complex. Even more preferably, binding and/or
internalization of the binding agent/antigen complex by an immature
or precursor dendritic cell causes maturation and/or activation of
the dendritic cell.
[0045] Preferably, the dendritic cell binding agent of the
invention binds to an activating Fcy receptor, such as CD64
(Fc.gamma.RI) or CD32 (Fc.gamma.RIIA) that is not abundant on
neutrophils.
[0046] As used herein, by "ligand-binding site of a receptor" is
meant the site on the receptor to which the natural ligand of the
receptor binds. For example, if the receptor is a Fc.gamma. type II
receptor, the natural ligand for the receptor is an IgG antibody. A
dendritic cell binding agent of the invention, when bound to a
receptor, blocks the ligand-binding site of the receptor such that
the natural ligand for that receptor cannot bind the receptor. In
one non-limiting example, if the receptor is a Fc.gamma. type II
receptor and the dendritic cell binding agent of the invention is
an IgG antibody, then binding of the dendritic cell binding agent
of the invention to the receptor prevents other IgG antibodies from
binding to the receptor.
[0047] Accordingly, dendritic cell binding agents of the invention
are readily identified by art-known methods. In one non-limiting
exarnple, where the dendritic cell binding agent is an IgG
antibody, a precursor, immature, or mature dendritic cell is
purified as described below. Next, the cell is incubated with the
FITC-labeled IgG antibody (with or without the antigen to which the
antibody specifically binds). Next, the phycoerythrin (PE)-labeled
natural ligand (i.e., another IgG antibody) is added to the cell.
The cell can then be subjected to analysis by flow cytometry to
determine if the FITC-labeled IgG antibody of the invention is able
to block binding of the PE-labeled antibody to the receptor on the
dendritic cell.
[0048] In certain preferred embodiments, the dendritic cell binding
agent is not a bispecific antibody which has two antigen binding
sites, one that is specific for the antigen of the invention and
the other that is specific for the receptor, e.g., at its
ligand-binding site, on the surface of a dendritic cell.
[0049] "Administering the composition to a patient" means providing
the composition to the patient in a manner that results in the
composition being inside the patient's body. Such an administration
can be by any route including, without limitation, sub-cutaneoous,
intradermal, intravenous, intra-arterial, intraperitoneal, and
intramuscular. "Receives a therapeutic benefit" means that the
patient experiences alleviation or reduction of signs or symptoms
of illness, and specifically includes, without limitation,
prolongation of survival. In certain preferred embodiments of the
methods according to the invention, a CD8+ IFN-.gamma. producing T
cell is activated to induce a cytotoxic T lymphocyte (CTL) immune
response in the patient administered the composition. In certain
embodiments of the methods according to the invention, a CD4+
IFN-.gamma. producing T cell is activated to induce a helper T cell
immune response in the patient administered with the composition.
These activated CD4+ IFN-.gamma. producing T cells (i.e., helper T
cells) provide necessary immunological help (e.g., by release of
cytokines) to induce and maintain not only CTL, but also a humoral
immune response mediated by B cells. Thus, in certain embodiments
of the methods according to the invention, a humoral response to
the antigen is activated in the patient administered with the
composition.
[0050] Activation of a CD8+ and/or CD4+ IFN-.gamma. producing T
cells means causing T cells that have the ability to produce
IFN-.gamma. to actually produce IFN-.gamma., or to increase their
production of IFN-.gamma.. "Induction of CTL" means causing
potentially cytotoxic T lymphocytes to exhibit antigen-specific
cytotoxicity, or to increase such antigen-specific cytotoxocity.
"Antigen-specific cytotoxicity" means cytotoxicity against a cell
that is presenting the antigen that is greater than cytotoxicity
against a cell that is not presenting the antigen. "Cytotoxicity"
refers to the ability of the cytotoxic T lymphocyte to kill the
target cell. Preferably, such antigen-specific cytotoxicity is at
least 3-fold, more preferably 10-fold greater, more preferably more
than 100-fold greater than cytotoxicity against a cell that is not
presenting the antigen.
[0051] In certain preferred embodiments, the dendritic cell binding
agent of the invention binds to the antigen and an Fc.gamma. type
II or type I receptor on the dendritic cells. Preferably, binding
of the binding agent to the Fc.gamma. type II or type I receptor
blocks the binding of the natural ligand to, respectively, the
Fc.gamma. type II or type I receptor. Accordingly, in certain
embodiments, the dendritic cell binding agent binds to the antigen
and to an Fc.gamma. Type I (CD64) receptor on a dendritic cell in
the patient administered with the composition. In certain
embodiments, the dendritic cell binding agent binds to the antigen
and to an Fc.gamma. Type II (CD32) receptor, such as a Fc.gamma.
Type IIA (CD32A) receptor or a Fc.gamma. Type IIB (CD32B) receptor
on a dendritic cell in the patient administered with the
composition. In certain embodiments, the dendritic cell binding
agent binds to the antigen and to an Fc.gamma. Type II CD 16
(Fc.gamma.RIII) receptor on a dendritic cell in the patient
administered with the composition.
[0052] In certain preferred embodiments, the dendritic cell binding
agent of the invention is an antibody. Preferably the antibody is
provided at a concentration of from about 1-10 .mu.g/ml. An
"antibody" includes a molecule comprising an active portion of an
antibody. "An active portion of an antibody" is a molecule that
includes an antigen binding site that is specific for an antigen
and a receptor binding site that binds an Fc receptor on its
ligand-binding site (e.g., the Fc portion of the antibody included
the heavy chain constant region). Accordingly, an antibody of the
invention may be, e.g., chimeric, single chain, mutant, or antibody
fragment so long as the antibody is able to specifically bind an
antigen and so long as the antibody includes a portion that binds
an Fc receptor on its ligand-binding site.
[0053] Accordingly, an antibody of the may be encoded by an
immunoglobulin gene having specific point mutation in the part of
the gene encoding the receptor binding site. In one non-limiting
example, the gene encoding the anti-PSA antibody, Alt-6, can be
subjected to point mutation in the portion of the gene encoding the
receptor binding site. The resulting antibody mutants can be
screened on cells (e.g., COS or HeLa cells) transfected with a gene
encoding the Fc.gamma. Type I (CD64) receptor, the Fc.gamma. Type
II (CD32) receptor, or the Fc.gamma. Type II CD16 (Fc.gamma.RIII)
receptor. A preferred mutant Alt-6 antibody of the invention is one
which binds better (i.e., by greater numbers or with higher
affinity) to a cell expressing the Fc.gamma. Type I (CD64)
receptor, Fc.gamma. Type II (CD32) receptor and/or the Fc.gamma.
Type II CD16 (Fc.gamma.RIII) receptor as compared to a cell not
expressing one of these receptors.
[0054] In certain embodiments, the dendritic cell binding agent of
the composition includes a portion that elicits a human
anti-xenotypic antibody (HAXA) response.
[0055] In certain embodiments, the dendritic cell binding agent is
a xenotypic antibody. A "xenotypic antibody" is an antibody from a
species other than the patient's species. For example, if the
patient is a human, a dendritic binding agent of the invention that
is a murine antibody is a xenotypic antibody. Similarly, if the
patient is a mouse, a dendritic binding agent of the invention that
is a rat antibody is a xenotypic antibody. Preferred xenotypic
antibodies include monoclonal antibodies, including without
limitation murine monoclonal antibodies. Particularly preferred
murine monoclonal antibodies include Alt-1 (murine IgG1,
specifically binds to MUC-1; ATCC No. PTA-975; American Type
Culture Collection, Manassas, Va.), Alt-2 (murine IgG1,
specifically binds to CA125; ATCC No. PTA-1883), Alt3 (murine IgG3,
specifically binds to CA9.9; ATCC No. PTA-2691), Alt-4 (murine IgM,
specifically binds to CA19.9; ATCC No. PTA-2692), Alt-5 (murine
IgG1, specifically binds to CA19.9; ATCC No. PTA-2690); and Alt-6
(murine IgG1 , specifically binds to prostate specific antigen
(PSA); ATCC No. HB-12526).
[0056] In certain embodiments, the xenotypic antibody elicits a
host anti-xenotypic antibody (HAXA) response in the patient.
[0057] In certain preferred embodiments, the composition
administered to the patient further includes host anti-xenotypic
antibodies (HAXA). "Host anti-xenotypic antibodies (HAXA)" are
antibodies of the host animal species that bind to the xenotypic
antibody contained in the composition of the invention. For
example, if the patient is a human and the xenotypic antibodies are
rabbit antibodies, the HAXA is human anti-rabbit antibodies.
Preferably the HAXA is provided in the composition at a
concentration of from about 1-10 .mu.g/ml. Preferred HAXA include,
without limitation, human anti-mouse antibody (HAMA).
[0058] In certain embodiments, if the composition administered to
the patient does not include HAXA, HAXA is preferably already
present in the patient's blood. Such a patient may already have
HAXA if, for example, the patient has been previously treated with
a non-specific antibody of the same species as the dendritic cell
binding agent of the invention. For example, prior to the
administration of the composition of the invention comprising the
dendritic cell binding agent and the antigen, a human patient may
be administered with a polyclonal murine antibody with no defined
specificity (or with a murine monoclonal antibody that specifically
binds to, for example, an egg shell protein not expressed in
humans). Once HAMA is detectable in the patient's blood, the
composition is administered to the patient.
[0059] In further embodiments, the composition of the first aspect
further comprises a dendritic cell. Preferably, the dendritic cell
is autologous to the patient.
[0060] As used herein, by "dendritic cell" is meant a bone
marrow-derived cell that can internalize antigen and process the
antigen such that it (or a peptide derived from the antigen) is
presented in the context of both the MHC class I complex and the
MHC class II complex. Accordingly, a dendritic cell of the
invention is able to activate both CD8+ T cells (which are
primarily cytotoxic T lymphocytes) and CD4+ T cells (which are
primarily helper T cells). It should be understood that any cell
capable of presenting a peptide derived from an internalized
antigen on both class I and class II MHC is a dendritic cell of the
invention. Preferably, a dendritic cell of the invention has the
phenotype and characteristics of the dendritic cells described in
Steinman, Annu. Rev. Immunol. 9: 271-296 (1991).
[0061] In certain preferred embodiments, the dendritic cell, when
added to the composition, is an immature dendritic cell. As used
herein, by "immature dendritic cells" means a population of
dendritic cells preferably having one or more of the following cell
surface antigens at the indicated level of expression: CD11c
present on greater than about 90% of the dendritic cells in the
population, HLA-DR present on fewer than about 90% of the dendritic
cells in the population but on greater than about 70% of the
dendritic cells in the population, HLA-ABC present on from about
80% to about 90% of the dendritic cells in the population, CD 14
present on fewer than about 20% of the dendritic cells in the
population, CD16 present on greater than about 10% to about 40% of
the dendritic cells in the population, CD80 present on about 50% to
about 70% of the dendritic cells in the population, CD86 present on
greater than about 40% to about 70% of the dendritic cells in the
population, CD83 present on greater than about 10% to about 20% of
the dendritic cells in the population, CD64 present on greater than
about 40% to about 60% of the dendritic cells in the population,
and CD32 antigen present on from about 70% to about 95% of the
dendritic cells in the population, and about less than 10% of the
dendritic cells in the population are CD3/CD19 positive (i.e.,
about less than 10% have CD3/CD19 expression).
[0062] In certain preferred embodiment, the dendritic cell, when
added to the composition, is a precursor dendritic cell. As used
herein, by "precursor dendritic cells" means a population of cells,
each of which is capable of becoming a dendritic cell, where
greater than 80% of the population have CD64 and CD32 antigen
present and about 70% of the population is positive for CD 14.
[0063] In the embodiments of the invention where the dendritic
cell, when added to the composition, is either an immature
dendritic cell or is a precursor dendritic cell, the composition is
preferably incubated ex vivo under conditions (e.g., in cell
culture) that allow for maturation of the immature dendritic cell
or precursor dendritic cell prior to administering the composition
to the patient. Such conditions that allow the formation of mature
dendritic cells from immature or precursor dendritic cells are
described below in the examples.
[0064] In the embodiments of the invention where the dendritic cell
is included in the composition, and where the patient is human, the
dendritic cell preferably expresses the cell surface molecules
described below in Table I at its different maturation stages. Note
that expression of the Fc receptors, particularly the CD64
(Fc.gamma.RI) typically decreases as the DC mature.
1TABLE I Human Dendritic Cell Surface Markers Day 0 Day 4 Day 7
Monocytes immature DC mature DC Marker (all cells) HLA-DR 70-85%
80-85% 95-99% HLA-ABC 70-85% 85-90% 95-99% CD3 1-5% ND ND CD4 2-3%
ND ND CD8 2-3% ND ND CD16 3-15% 15-40% 0.5-5% CD19 5-10% ND ND CD14
75-80% 0.4-0.5% 0.1-0.2% CD11c 75-80% 95-99% 99-100% Marker (gated
on DC) CD86 85-90% 40-70% 95-99% CD80 30-50% 55-80% 85-90% CD40
40-50% 55-60% 55-60% CD83 10-15% 10-15% 55-60% CD32 89-98% 70-95%
40-45% CD64 92-99% 28-60% 4-10%
[0065] One non-limiting method to obtain dendritic cells according
to the invention is described below in Example I.
[0066] Accordingly, in one non-limiting method, 10 .mu.g of CA125
and 5 .mu.g of the murine monoclonal antibody that specifically
binds to CA125, Alt-2, are combined together ex vivo. In a
variation of the method, human anti-murine antibodies are added to
the mixture. Then, the mixture is added to immature dendritic cells
prepared as described below from the patient suffering from the
disease. The addition of the antigen (in this case CA125 plus
.alpha.CA125 antibody, Alt-2) promotes maturation of the immature
dendritic cells. (Note that as used throughout, the symbol
".alpha." means "anti-". Thus, ".alpha.CA125" means "anti-CA125").
Next, the matured dendritic cells "loaded" or "armed" with CA125
and Alt-2 (and, in some cases, HAMA) are removed from culture and
administered to the patient.
[0067] Note that the dendritic cell used in the invention is
preferably autologous to the patient to whom the composition of the
invention is administered. By "autologous" is meant having
identically matched MHC loci (both class I and class II). Thus, an
identical sibling can provide autologous dendritic cells for a
patient. Similarly, a close relative can provide autologous
dendritic cells for a patient, so long as the patient and the close
relative have identically matched MHC loci. Of course, two
individuals of an inbred strain of laboratory animal (e.g., inbred
Balb/c mice) are autologous to one another.
[0068] In certain preferred embodiments, if the patient to whom the
composition of the invention is administered already had an immune
response to the antigen, following administration, the immune
response is shifted predominantly from a helper to a cytotoxic T
cell response, thus providing the patient, following
administration, a therapeutic benefit
[0069] Thus, in one non-limiting example, a patient of the
invention with prostate cancer may already have either antibodies
that are specific for prostate specific antigen (PSA) and/or helper
T cells that are specific for PSA. However, following
administration of the composition of the invention, the PSA of the
composition is internalized and presented on antigen presenting
cells in such a way (e.g., in context of MHC class I) that
cytotoxic T cells that are specific for PSA are stimulated, thereby
providing the patient a therapeutic benefit as compared to the
patient's condition prior to administration of the composition.
[0070] In a second aspect, the invention provides a method for
treating a patient suffering from a disease associated with an
antigen, comprising administering to the patient a composition
comprising an antigen associated with the disease, a dendritic cell
binding agent specific for the antigen, and a dendritic cell
autologous to the patient, wherein the patient administered the
composition receives a therapeutic benefit. Preferably, the patient
is a human
[0071] In some embodiments, the dendritic cell, when combined with
the antigen and the dendritic cell binding agent, is either a
precursor dendritic cell or an immature dendritic cell. In these
embodiments, the composition is incubated ex vivo under conditions
that allow for maturation of the immature dendritic cell prior to
administering the composition to the patient.
[0072] In some embodiments, the antigen and dendritic cell binding
agent may be combined simultaneously with the dendritic cell to
make the composition. In some embodiments, the dendritic cell
binding agent and the antigen are combined with one another before
they are combined with the dendritic cell to make the final
composition. In these embodiments, the composition including the
dendritic cell is then administered to the patient suffering from
the disease.
[0073] In certain embodiments, where the dendritic cell binding
agent is a xenotypic antibody and the composition further comprises
human anti-xenotypic antibodies.
[0074] In a third aspect, the invention provides a method for
treating a patient suffering from a disease associated with an
antigen, comprising administering to the patient suffering from the
disease a composition comprising a host anti-xenotypic antibody and
a xenotypic antibody specific for the antigen associated with the
disease, wherein the patient administered the composition receives
a therapeutic benefit.
[0075] In a fourth aspect, the invention provides a therapeutic
composition comprising a purified dendritic cell binding agent that
is specific for an antigen associated with a disease and the
antigen associated with the disease. Preferably, binding of the
dendritic cell binding agent to a receptor on a dendritic cell
blocks binding of a natural ligand to the receptor.
[0076] In a fifth aspect, the invention provides a therapeutic
composition comprising a purified dendritic cell binding agent that
is specific for an antigen associated with a disease, a dendritic
cell, and the antigen associated with the disease. Preferably,
binding of the dendritic cell binding agent to a receptor on the
dendritic cell blocks binding of a natural ligand to the receptor.
Preferably, the dendritic cell of the composition is autologous to
a patient to whom the composition is administered.
[0077] In a sixth aspect, the invention provides a therapeutic
composition comprising a purified xenotypic antibody that is
specific for an antigen associated with a disease and a host
anti-xenotypic antibody.
[0078] As used herein, by "purified" is meant that the indicated
agent (e.g., a purified dendritic cell binding agent or xenotypic
antibody) has been separated from components which naturally
accompany it. For example, in the case of a protein (e.g., a
dendritic cell binding agent), the purified protein is separated
from components, such as other proteins or fragments of cell
membrane, that accompany it in the cell. Of course, those of
ordinary skill in molecular biology will understand that water,
buffers, and other small molecules may additionally be present in a
purified protein preparation. A purified protein (e.g., a purified
dendritic cell binding agent) of the invention is at least 95% by
weight, more preferably at least 98% by weight, even more
preferably at least 99% by weight, and most preferably 100% by
weight free of components which naturally accompany the nucleic
acid molecule or polypeptide.
[0079] According to the invention, a purified dendritic cell
binding agent of the invention may be generated, for example, by
recombinant expression of a nucleic acid molecule encoding the
dendritic cell binding agent in a cell in which the dendritic cell
binding agent does not naturally occur. Of course, other methods
for obtaining a purified dendritic cell binding agent of the
invention include, without limitation, artificial synthesis of the
dendritic cell binding agent on a peptide synthesizer and isolation
of the dendritic cell binding agent from a cell in which it
naturally occurs using, e.g., an antibody that specifically binds
to the dendritic cell binding agent. In the case where the
dendritic cell binding agent is a monoclonal antibody, a purified
dendritic cell binding agent can be obtained from the culture
supernatant of the hybridoma which secretes the dendritic cell
binding agent, or from ascites fluid from an animal injected with
the hybridoma.
[0080] Preferably, the therapeutic compositions of the fourth,
fifth, and sixth aspects of the invention further comprise a
pharmaceutically acceptable carrier. By "pharmaceutically
acceptable carrier" is meant a carrier that is physiologically
acceptable to the administered patient and that retains the
therapeutic properties of the dendritic cell binding agent and
antigen (and/or dendritic cell) with which is it administered. One
exemplary pharmaceutically acceptable carrier is physiological
saline. Other pharmaceutically-acceptable carriers and their
formulations are well-known and generally described in, for
example, Remington's Pharmaceutical Sciences (18th Edition, ed. A.
Gennaro, Mack Publishing Co., Easton, Pa., 1990).
[0081] Preferably, administration of the therapeutic compositions
of the fourth, fifth, and sixth aspects of the invention to a
patient suffering from the disease provides the patient a
therapeutic benefit. Preferably, the patient is a human.
[0082] The following examples are intended to further illustrate
certain particularly preferred embodiments of the invention and are
not intended to limit the scope of the invention.
EXAMPLE I
Isolation of Dendritic Cells
[0083] To isolate dendritic cells, peripheral blood mononuclear
cells (PBMC) were isolated from the apheresis products from normal
volunteers by ficoll-hypaque (Histopaque 1.077, commercially
available from Sigma, St. Louis, Mo.) gradient centrifugation, and
viably frozen using an automated cell freezer (commercially
available from Gordinier Electronics, Roseville, Mich.) in RPMI
(commercially available from Life Technologies, Frederick, Md.)
containing 40% human antibody serum (commercially available from
Gemini Bio-Products, Woodland, Calif.) and 10% DMSO (Sigma). The
cells were stored in the vapor phase of liquid nitrogen until used.
DNA was prepared from a portion of the cells and used for molecular
HLA typing.
[0084] Next, dendritic cells (DC) were isolated by negative
selection. To do this, DC precursors were prepared from
freshly-thawed PBMC by negative selection using immunomagnetic bead
depletion. Specifically, PBMC were placed into a tube and incubated
on ice for 30 min. with mouse anti-human CD3, CD16, and CD19
antigens (commercially available from Caltag, Burlingame, Calif.).
Excess antibody was removed by washing the cells with phosphate
buffered saline containing 1% of bovine serum albumin (PBS/0.1%
BSA), and the washed cells were next incubated with Pan Mouse IgG
immunomagnetic beads (commercially available from Dynal, Lake
Success, N.Y.) for 30 min. on ice. The tube containing the cells
plus specific mouse anti-human antigens and the Pan Mouse IgG
immunomagnetic beads was placed against a magnet to remove the
cell:bead complexes. The cells that bound to the magnet were either
T cells, B cells, or Natural Killer (NK) cells. Accordingly, the
supernatant contained the lineage-depleted DC precursors (i.e., the
monocytes remaining in the fluid in the tube not expressing CD3, CD
16, or CD19 antigens and so not bound by the magnet). These
negatively selected cells were approximately 70% pure monocytes as
characterized by Flow cytometry using a broad CD marker panel (see
Table I above) were collected.
[0085] Next, the negatively selected cells were washed, resuspended
in cRPMI (RPMI supplemented with 1% glutamine and 10%
heat-inactivated human serum (from a person with blood type AB))
containing GM-CSF (1000 U/mil) and IL-4 (1000 U/mil) (both
commercially available from R & D Systems, Minneapolis, Minn.)
and cultured at 37.degree. C. in 5% CO.sub.2 at 0.5.times.10.sup.6
cells/well in 24 well plates for four days. These cells were
immature dendritic cells by day four, and analyzed for surface
expression of numerous cell surface antigens by flow cytometry see
Table I above).
[0086] On the fourth day of culture, the cells were pulsed with
antigen (e.g., Prostate Specific Antigen (PSA)) and incubated for
an additional three days. (Note that the antigen which the DC cells
were pulsed with was dependent upon what the DC cells would be
eventually used for. For example, if the DC cells were to be used
to generate a T cell response to PSA, the DC cells would be pulsed
with PSA antigen.) Several agents which are known to cause DC
precursors to mature into mature DC cells were added to the
cultures eight hours after addition of the antigen. These agents
included TNF.alpha. (10 .mu.g/ml) and/or IFN.alpha. (50 .mu.g/ml).
The matured DC were harvested on the seventh day of culture,
analyzed for phenotypic markers by flow cytometry and used in
functional studies.
[0087] To analyze DC for cell surface marker expression by flow
cytometry, standard methods were employed. Briefly, the cells were
aliquotted into polystyrene tubes and stained for surface markers
with fluorochrome-labeled murine antibodies. The complete DC cell
surface marker panel included: HLA-A, HLA-A B, HLA-A C, HLA-DR,
CD14, CD11c, CD123, CD4, CD40, CD83, CD86, CD80, CD16, CD32, CD64
(specific detectably labeled antibodies to which are commercially
available from Becton Dickinson, San Jose, Calif.). Following a 30
min. incubation on ice, the cells were washed with PBS and pelleted
by centrifugation. The cell pellets were resuspended in 250 .mu.l
of fixative (2% parafornaldehyde). The data was acquired using a
FACSCan flow cytometer (Becton Dickinson, San Jose, Calif.) and
analyzed with Cellquest software (Becton-Dickinson, San Jose,
Calif.).
EXAMPLE II
Phenotypic Markers on Dendritic Cells
[0088] Initial studies were focused on the uptake of CA125 in
monocytes and dendritic cells in the presence or absence of Alt-2.
For this purpose, CA125 was purified from tissue culture
supernatant of NIH:OVCAR-3 cells (AltaRex Corp.). Highly purified
CA125 was labeled with Fluorescein (Flourescein-EX, Molecular
Probes), and incubated with monocytes, immature dendritic cells,
and mature dendritic cells under various conditions. In some
experiments, the uptake of MAb-Alt-2 was also followed, where the
MAb-Alt-2 was labeled with Fluorescein-EX or Cy-3.
[0089] As shown above in Table I, monocytes, immature DC, and
mature DC could be easily distinguished based on their expression
of various cell surface antigens.
EXAMPLE III
Comparison of CA125 Antigen Uptake in Monocytes to Immature
Dendritic Cells
[0090] In these studies, highly purified CA125 was labeled with
Fluorescein and incubated with monocytes or immature dendritic
cells at 1000 U/mL for 1 hour at 37.degree. C. In some cases,
unlabeled antibody was added simultaneously with the labeled
antigen to study the effect of complex formation on the uptake by
the two antigen-presenting cells. The binding to cells was
quantified by flow cytometry and the results are shown in FIGS. 1A
(percent positive events) and 1B (mean channel intensity). As shown
in FIGS. 1A and 1B, immature dendritic cells showed much higher
uptake of the antigen, with MAb Alt-2 as compared to without MAb
Alt-2. Both monocytes and immature dendritic cells showed an
increase of CA125 uptake in the presence of MAb Alt-2 (see FIGS. 1A
and 1B, respectively). For the immature dendritic cells, the CA125
concentration needed to be lowered to 1000 U/mL to allow the
detection of an antibody effect (concentration below the saturation
point for CA125 uptake).
[0091] It is interesting to note that immature DCs showed an
increase in the number of cells capable of taking up the CA125
antigen (FIG. 1A, % positive cells) as well as the concentration of
antigen within each cell (FIG. 1B, mean fluorescence intensity)
with increasing concentrations of MAb Alt-2. In monocytes,
increasing antibody concentrations could only enhance the
percentage of cells capable of taking up the CA125 antigen. These
results may indicate that the immune complexes are either taken up
by different receptors in monocytes and immature DCs or that the
immune complexes are taken up by the same receptors in monocytes
and immature DCs, but the receptor is recycled at a higher
frequency in immature DCs. The receptor is certainly more abundant
and/or is internalized more rapidly in immature DCs than monocytes,
demonstrated by the higher percentage of targeted cells in DCs (see
FIG. 1A).
[0092] Next, the requirement for antibody specificity was tested.
To do this, highly purified CA125 was labeled with Fluorescein and
incubated with immature dendritic cells at 1000 U/mL for 1 hour at
37.degree. C. in the absence and presence of unlabeled MAb Alt-2 or
MOPC-21 (a control murine IgG1 that does not bind to the CA125
antigen). The binding to cells was assessed by flow cytometry, and
the results are shown in FIG. 2. As FIG. 2 shows, antibody-enhanced
uptake of CA125 was specific for MAb Alt-2 and could not be
achieved with a control antibody that does not bind to CA125.
Consequently, the antibody-enhanced uptake is due to a more
efficient uptake route in DCs and not due to stimulation of the
endocytotic activity of dendritic cells upon receptor engagement by
the binding MOPC-21 antibody
EXAMPLE IV
Effect of Murine Alt-2 and Chimeric Alt-2 on CA125 Antigen
Uptake
[0093] Other binding studies have compared the uptake of CA125 in
the presence of a murine and humanized form of MAb Alt-2. In this
study, fluorescein-labeled CA125 was incubated with monocytes and
immature DCs at 1000 U/mL for 1 h at 37.degree. C. in the presence
of murine (mAlt-2) and chimeric Alt-2 (cAlt-2 (chimeric with a
human IgG3 constant region)). The binding to cells was assessed by
flow cytometry.
[0094] As shown in FIGS. 3A and 3B, the chimeric antibody showed
slightly better enhancement of CA125 binding to monocytes than the
murine Alt-2; on immature dendritic cells, the murine and chimeric
antibody were equally effective. As for the murine antibody, the
chimeric Alt-2 increased the percentage of CA125-targeted cells
within the monocytes (FIG. 3A) and dendritic cell populations, but
increased the amount of CA125 per cell only in dendritic cells
(FIG. 3B).
EXAMPLE V
Effect of HAMA on CA125 Antigen Uptake
[0095] Complexation of antibody with specific antigen or with HAMA
and binding to dendritic cells was measured. To do this, human
anti-mouse antibody (HAMA) was purified from patient serum samples
with high HAMA concentrations after MAb Alt-2 injection via
affinity chromatography on Protein G, and a MAb-AR20.5 column,
followed by negative selection on a MAb Alt-2 column to eliminate
Ab2 (i.e., human antibody that binds to the idiotype of the MAb
Alt-2 antibody).
[0096] Five micrograms (5 .mu.g) FITC-labeled Alt2 (anti-CA125) or
Alt-6 (anti-PSA) murine monoclonal antibody was incubated together
with the corresponding antigen (1 .mu.g) and/or HAMA (2.5 .mu.g)
and 5.times.10.sup.5 dendritic cells at 37.degree. C. for 60
minutes. The mixture was then washed once and resuspended in 0.5%
formalin+PBS and subjected to FACScan on a FACSCalibur machine
(Beckton-Dickinson). The results for Alt-2 and CA125 are shown in
FIGS. 4A, 4B, and 4C. These results demonstrate that binding of
antibody to dendritic cells is enhanced by complexation of antibody
to its antigen, complexation of antibody with HAMA, or complexation
of antibody to its antigen in the presence of HAMA.
[0097] Further binding studies were conducted to compare the uptake
of the CA125-MAb-Alt-2 complex by monocytes or immature dendritic
cells in the presence and absence of HAMA. In this study,
fluorescein-labeled CA125+murine MAb-Alt-2 was incubated at 1000
U/ml of CA125 and a range of MAb-Alt-2 concentrations with
monocytes or immature dendritic cells for 1 hour at 37.degree. C.
The binding studies were conducted in the absence and presence of
human anti-mouse antibodies (HAMA). The HAMA concentrations were
equivalent to the MAb-Alt-2 concentrations to form equimolar
complexes of HAMA and MAb-Alt-2. The binding to cells was assessed
by flow cytometry.
[0098] As shown in FIGS. 5A (percent positive cells) and 5B (mean
fluorescence intensity), HAMA further enhanced the binding of CA125
to human monocytes and dendritic cells. Immune complexes with HAMA
increased the percentage of CA125-targeted cells within the
monocytes and dendritic cell populations (FIG. 5A), but increased
the CA125 concentration per cell mainly in dendritic cells (FIG.
5B).
EXAMPLE VI
Alt-6 Antibody and Alt-6-PSA Immune Complex Binding to DC at
Different Stages of Development and Maturation
[0099] DC of different stages of development and maturation were
first analyzed by flow cytometry to determine their level of
expression of Fc.gamma. receptor. To do this, DC were isolated from
PBMC as described above and tested for Fey receptor expression with
fluorescence labeled antibodies at day 0, after 4 days in culture
with GM-CSF/IL-4 and after maturation with TNF.alpha. and
IFN.alpha. (7 days). Binding was determined on CD11c+, HLA-DR+
cells and is expressed in Table II (below) as percentage of
positive cells within the gate.
[0100] As shown below on Table II, DC express Fc.gamma. type I, and
type II receptors (CD64 and CD32), and low levels of Fc.gamma.R III
(CD16).
2TABLE II Distribution of Fc.gamma. Receptors on DC at Different
Stages of Maturation Days in culture 0 4 7 Fc.gamma. Receptor
Positive Cells [%] (gated on DC) CD16 (Fc.gamma.RIII) 3-15 15-40
0.5-5 CD32 (Fc.gamma.RII) 89-98 70-95 40-45 CD64 (Fc.gamma.RI)
92-99 28-60 4-10
[0101] The percent of DC expressing these Fc.gamma. receptors
differed depending on their stage of maturation/differentiation. As
shown on Table II, the number of DC expressing CD16 was highest
after culture for 4 days with GM-CSF/IL-4 and was nearly absent on
DC after maturation (i.e., after 7 days in culture with
GM-CSF/IL-4). CD32 and CD64 were found on the majority of DC
precursors. CD32 was expressed on 70-95% of immature DC and
declined to about 40% on mature DC. In addition, the number of CD64
expressing cells declined as the DC differentiated and matured.
[0102] Next, to determine if the anti-PSA monoclonal antibody Alt-6
and Alt-6-PSA immune complexes would bind to DC and if the stage of
DC differentiation and maturation affected binding, a range of
concentrations of the fluorescein-labeled Alt-6 as well as
Alt-6-PSA-FITC-complexes were added to three different populations
of DC: I) freshly isolated myloid DC precursors (surface
phenotype-CD11+, HLA-DR+, CD14+), II) DC after culture for 4 days
in GM-CSF/IL-4 (surface phenotype-CD11c+, HLA-DR+, CD 14-, CD86+)
and III) DC that had been further matured by culturing for an
additional 3 days with TNF.alpha. and IFN.alpha. (surface
phenotype-CD11c+, HLA-DR+, CD86+, CD40+, CD80+, CD83+) (see Table I
above).
[0103] To do this, PSA was purchased from Scripps Laboratories (San
Diego, Calif.) and the murine monoclonal anti-PSA antibody Alt-6
(ProstaRex.TM.) was kindly provided by AltaRex Corporation,
Edmonton, Alberta, Canada (ATCC No. HB-12526; American Type Culture
Collection, Manassas, Va.). Alt-6 is an IgG1 antibody that reacts
specifically with an epitope that maps to the region of amino acid
residues 137-146 (sequence EPEEFLTPKK) of PSA. PSA and Alt-6 were
diluted in cRPMI to concentrations of 5 and 25 .mu.g/ml,
respectively. When mixed at these concentrations, approximate
equimolar amounts of PSA and anti-PSA were achieved.
[0104] Alt-6 was labeled with Fluorescein-EX (commercially
available from Molecular Probes, Eugene, OR) following
manufacturers instructions. Equimolar concentrations of PSA were
mixed with 1.25 .mu.g/ml, 2.5 .mu.g/ml, 5.0 .mu.g/ml, and 25
.mu.g/ml of Alt-6-FITC and added to freshly isolated DC precursors,
to DC that had been cultured for 4 days in the presence of
GM-CSF/IL-4 and to DC that had been matured.
[0105] The antibody-PSA composition was incubated for 1 hour at
37.degree. C., washed, pelleted, fixed in 2% paraformaldehyde, and
analyzed by flow cytometry. DC were gated in the sideward/forward
scatter based on DC preparations stained in parallel with the
complete DC marker panel (see Example I). The percentage of cells
that reacted with Alt-6 and Alt-6-PSA complexes were calculated
based on binding to CD11c+, HLA-DR+ cells. Table III (below) shows
percentage of positive cells within the gate.
3TABLE III PSA-Specific Monoclonal Antibody Alt-6 and Alt-6-PSA
Immune Complex Binding to DC at Different Stages of Maturation Days
in culture Antibody 0 4 7 Concentration Positive Cells (gated on
DC) [%] Alt-6-FITC 1.25 .mu.g/ml 0.13 0.68 2.5 .mu.g/ml 0.16 1.56 5
.mu.g/ml 1.84 9.95 1.19 25 .mu.g/ml 7.33 28.1 Alt-6-FITC + 1.25
.mu.g/ml 1.82 2.41 PSA 2.5 .mu.g/ml 3.91 8.70 5 .mu.g/ml 5.38 23.40
1.56 25 .mu.g/ml 18.00 72.11
[0106] As shown in Table m, at the concentrations tested, Alt-6
bound to a low percentage of freshly isolated DC precursors. The
number of cells binding the antibody increased after culture for 4
days in GM-CSF and IL-4. Interestingly, the number of cells binding
Alt-6-FITC was significantly higher in the presence of PSA,
indicating that the Fc receptors have enhanced binding to immune
complexed antibody over the antibody alone, or that immune
complexes bind to different and/or more abundant receptors on DC
precursors and immature DC. No binding of the Alt-6 or the immune
complexes occurred to mature DC.
EXAMPLE VII
T Lymphocyte Responses to DC armed with PSA or PSA/anti-PSA
[0107] T lymphocytes were stimulated with DC armed with PSA or a
combination of PSA/anti-PSA antibody.
[0108] To do this, monocytes were generated from leukaphoresis
samples from healthy donors (Biological Specialty Corp., Colmar,
Pa.) and depleted from lineage cells by incubation with anti-CD3,
CD19 and CD 16 antibodies, followed by incubation with magnetic
bead conjugated anti-mouse Ig and separation on a magnet (Dynal).
Negatively selected cells were approximately 70% pure monocytes as
characterized by flow cytometry using a broad CD marker panel (see
Example I). Monocytes were incubated with IL-4 and GM-CSF (R&D
Systems) for 4 days in RPM11640+10% matched human serum to generate
immature DC. Again, an aliquot of the cells was stained with a
broad CD marker panel to ensure purity and identity of the cells.
These cells were then harvested and loaded with antigen
combinations (e.g., PSA, the anti-PSA monoclonal antibody and the
antibody-PSA combination) for 2-8 hours at 37.degree. C., and then
matured with IFN.alpha. and TNF.alpha. for three days. Dendritic
cell were checked again via flow cytometry for an array of CD
markers to ensure proper maturation of the cells.
[0109] Specifically, the immature DCs were loaded with PSA (25
.mu.g/mL), Alt-6 (5 .mu.g/IL) and PSA+Alt-6 (25 .mu.g/mL of PSA; 5
.mu.g/mL of Alt-6). These immature DCs were added to T cells that
were generated from the same monocytes as the DCs via negative
selection (i.e., autologous T cells), using a magnetic T cell
isolation kit (commercially available from Dynal). Briefly, to
isolate T cells, CD3+ T lymphocytes were isolated from thawed PBMC
(see Example I) by negative selection (using magnetic beads
commercially available from Dynal, Lake Success, N.Y.). Briefly,
the cells were incubated on ice for 30 min with a mixture of
antibodies to CD14, CD16, CD56 and HLA Class II DR/DP. Excess
antibodies were removed by washing with PBS/0.1% BSA. The cells
were incubated for 30 min. at room temperature with immunomagnetic
beads coated with an anti-mouse IgG antibody (Dynal). The cells
were placed against a magnet and the T lymphocytes, which did not
bind the magnet (i.e, did not express CD14, CD16, CD56, and HLA
class II DR/DP) were isolated from the supernatant.
[0110] T cells and DCs were incubated for 7 days (primary
stimulation), re-stimulated with loaded and matured DCs and
incubated for another 7 days. For stimulation, T lymphocytes were
plated in twenty-four well plates at a concentration of
1.times.10.sup.6 cells/well and to which were added
5.times.10.sup.4 DC that were antigen naive or that had been
exposed to PSA, the anti-PSA monoclonal antibody or the combination
of PSA and the monoclonal antibody. An aliquot of the cells was
taken 24 hour later and prepared for intracellular cytokine
staining (Day 15; primary stimulation), whereas the remaining cells
were incubated for another 7 days (secondary stimulation) and
prepared for intracellular cytokine staining on Day 22. Prior to
adding the second or third DC preparation, an aliquot of the cell
supernatant was taken for testing in an IFN-.gamma. ELISA
(Pharmingen DouSet).
[0111] For the intracellular cytokine staining, cells were
incubated with Golgi Plug (R&D Systems) 2 hours after DC
addition and incubated for another 16-18 h. Cells were stained with
anti-CD3-FITC and anti-CD8-Cy-Chrome for 30 min. on ice, washed,
permeabilized, and stained with anti-IFN-.gamma.-PE for 30 min. on
ice. Cells were washed, fixed and analyzed by flow cytometry (FACS
Calibur, Becton Dickinson).
[0112] T cell responses were measured as numbers of CD4+ and CD8+ T
cells producing intracellular IFN.gamma.. To do this, flow
cytometry was used. Briefly, Brefeldin A (10 .mu.g/ml)
(commercially available from Pharmingen, San Diego, Calif.) or
Golgi Plug (commercially available from R&D Systems) was added
to the T cell cultures 2 hours after restimulation with antigen
armed DC. After an additional 16-18 hours of culture, cells were
stained with anti-CD3-FITC and anti-CD8-Cytochrome for 30 min. on
ice, washed, permeabilized (e.g., by incubating in a perm/fix
solution (Pharmingen) for 20 min. on ice), and stained with
anti-IFN-.gamma.-PE for 30 min. on ice in staining buffer (PBS with
1% human AB serum (i.e., human sera from a person with type AB
blood) (antibodies commercially available from Becton Dickinson,
San Jose, Calif.) added. The cells were washed, resuspended in
staining buffer containing 2% paraformaldehyde and analyzed by flow
cytometry (FACS Calibur, Becton Dickinson).
[0113] In this experiment, mature "armed" or "un-armed" DC (i.e.,
matured with cytokines alone) were co-cultured with autologous T
cells, restimulated with armed or un-armed DC, and the number of
CD4+ and CD8+ cells producing IFN.gamma. was determined. The
results of a representative experiment are shown in FIG. 6.
[0114] As shown in FIG. 6, little to no difference in number of
IFN.gamma. producing CD4+ or CD8+ T cells were found in co-cultures
with DC that were not exposed to antigen (negative control) or T
cells cultured with DC that had been exposed to the monoclonal
antibody (i.e., .alpha.-PSA as primary stimulated) and were then
restimulated with armed or un-armed DC. In all combinations of
primary stimulation and restimulation, PSA armed DC stimulated CD4+
T cell responses were consistently greater than CD8+ T cell
responses. Notably, strong CD8+ T cell responses were observed on
restimulation with immune complex armed DC (i.e., where
restimuation was with the complex and primary stimulation was with
the complex of PSA/.alpha.-PSA). When T cells were cultured with
antigen-antibody armed DC (i.e., primary stimulation is
PSA/.alpha.PSA) and restimulated with PSA armed DC, CD4+ T cell
responses were substantially greater than CD8+ responses.
[0115] On the other hand, consistent CD4+ and CD8+ IFN.gamma.
responses were only generated in T cells exposed to and
restimulated by antigen-antibody armed DC (i.e., where both primary
stimulation and restimulation was PSA/.alpha.-PSA). Since IFNY T
cell responses were not generated to the Alt-6 antibody (i.e.,
.alpha.-PSA alone as primary stimulation or restimulation), it is
expected that the response to the complex is directed at the PSA.
The increase in CD8+ T cell responses to PSA presented in
combination with the antibody compared to the responses with free
PSA alone indicate that the immune complex enhances antigen
processing, in particular, through the HLA Class I pathways.
EXAMPLE VIII
T Lymphocyte Responses to DC Armed With CA125 or CA125/.alpha.CA125
Complex
[0116] After identifying broad ranges of functional CA125 and
MAb-Alt-2 concentrations for T cell stimulation in small
checkerboard assay for IFN-.gamma. release into the supernatant,
intracellular cytokine staining assays were performed using 50, 500
and 5000 U/mL of CA125 in the absence and presence of 2.5 .mu.g of
MAb-Alt-2. Matured DC cells and T cells were incubated for 7 days
and then re-stimulated with loaded and matured DCs. Seven days
after stimulation with loaded DCs (FIG. 7, round 1), and after the
restimulation (FIG. 7, round 2), an aliquot of the cell supernatant
was taken and tested in an IFN-.gamma. ELISA (Pharmingen
DouSet).
[0117] As a positive control, PMA +lonomycin was used for
stimulation of the T cells in culture. DCs loaded in medium only
were used to determine the background of the assay.
[0118] As shown on FIG. 7, stimulation with CA125 alone did not
result in substantial IFN-.gamma. release, detected by IFN-y ELISA
of the cell supernatants (FIG. 7).
[0119] For intracellular staining studies, immature DCs were loaded
with CA125 (50, 500, 5000 U/mL), Alt-2 (2.5 .mu.g/mL) and
CA125+Alt-2 (50, 500, 5000 U/mL of CA125; 2.5 .mu.g/ml of Alt-2)
and matured. T cells and DCs were incubated for 7 days, and
restimulated with loaded and matured DCs. An aliquot of the cells
was taken 24 hours later and prepared for intracellular cytokine
staining (Day 15; FIG. 8A), whereas the remaining cells were
incubated for another 7 days. Those cells were stimulated with
another batch of loaded and matured DCs and prepared for
intracellular cytokine staining on Day 22 (FIG. 8B). For the
intracellular cytokine staining, cells were incubated with Golgi
Plug (R&D Systems) 2 hours after DC addition and incubated for
another 16-18 hours. Cells were stained with anti-CD3-FITC and
anti-CD8-Cy-Chrome for 30 min. on ice, washed, permeabilized, and
stained with anti-IFN-.gamma.-PE for 30 min. on ice. Cells were
washed, fixed and analyzed by flow cytometry (FACS Calibur, Becton
Dickinson).
[0120] As shown on FIGS. 7, 8A, and 8B, responses to CA125 could be
detected only after two stimulation rounds and at the highest
concentration tested. As shown in FIGS. 8A and 8B, antigen alone
mainly stimulated CD4 responses as shown by the intracellular
staining results. As shown on FIG. 7, substantially increased
IFN-.gamma. release was observed in responses to CA125 presented in
a complex with MAb-Alt-2 by human DC. IFN-.gamma. release into the
cell supernatant, detected by IFN-.gamma. ELISA, as well as
intracellular cytokine production (ICC) could be detected already
after one stimulation round and was further increased after the
second round of stimulation (FIGS. 7 and 8B, respectively). CA125
concentrations as low as 50 U/mL were able to stimulate T cells,
with an optimum of 500 U/mL at two stimulation rounds (FIGS. 7, 8A,
and 8B).
[0121] Of particular note is the finding that the CA125-MAb Alt-2
immune complexes induced initially strong CD4 responses (FIG. 8A)
and with additional stimulation also strong CD8 responses (FIG.
8B). These observations indicate that CA125 can be presented on MHC
class I and II if presented in the form of an immune complex, but
only on MHC class II molecules, if taken up alone, as expected for
extracellular antigens. The receptor responsible for the uptake of
the antigen or antibody-antigen complex may influence the route for
processing of the internalized protein. Antigen-antibody complexes
may have increased leakage into the cytosol and therefore better
chance of being processed by the proteasomes, the processing
machinery that transports peptides onto MHC class I molecules via
the invariant chain.
EXAMPLE IX
PSA/anti-PSA Complex-Armed DC Generate HLA-A*0201 Peptide-Specific
T Lymphocyte Responses
[0122] The ability of PSA/anti-PSA immune complexes and PSA armed
DC to generate T lymphocyte responses to two PSA peptides that are
known to be restricted by the HLA-A*0201 allele was next compared.
To do this, two HLA-A2 specific PSA peptides (pep1, FLTPKKLQCV;
pep2, KLQCVDLHV) and an HIV-1 peptide (SYNTVAVL) were synthesized
by the Biopolymer Laboratory, University of Maryland, Baltimore,
Md., diluted in cRPMI, added to the DC preparations at
concentrations of 5 .mu.g/ml, and incubated for approximately 1
hour.
[0123] DC were prepared as described in Example I and exposed to
PSA or PSA/anti-PSA complex after 4 days of culture in GM-CSF/IL-4.
Following maturation with CD40L or TNF.alpha. and IFN.alpha., the
armed DC were cultured with autologous CD3+ selected T lymphocytes
for 7 days. The T cells were next harvested, newly armed DC added
to the cell culture, and the cells cultured for an additional 7
days in media supplemented with IL-2 (10 U/ml) and IL-7 (5 ng/ml)
(see Example III).
[0124] At day 14, the T cells were harvested and restimulated with
matured DC that were pulsed with the two PSA-derived peptides and
analyzed for IFN.gamma. release.
[0125] Results of a representative experiment (1 of 3) are shown in
FIG. 9. In T cells cultured with PSA-armed DC for 2 weeks, low
levels of CD4+ responses were detected upon restimulation with the
PSA-derived peptides. Peptide 2 (KLQCVDLHV) also induced small CD8+
responses when the primary stimulation was PSA alone. Consistent
with the previous results described in Example III, restimulation
of T cells with PSA-armed DC (where the primary stimulation was
PSA) resulted in CD4+ T cell responses, and low levels of
responding CD8+ T cells. Restimulation with the PSA/anti-PSA
complex (where the primary stimulation was PSA) showed similar
activation of CD4+ as well as CD8+ INF.gamma. responses.
[0126] As the lower half of FIG. 9 shows, T cells cultured with
PSA/anti-PSA armed DC for two weeks were able to respond to both
PSA-derived peptides. Peptide-restricted responses were
approximately twice as strong in T cells cultured with PSA/anti-PSA
armed DC before restimulation as compared to the responses to
peptide by PSA-armed DC (i.e., T cells whose primary stimulation
was PSA alone).
[0127] Note that T cells propagated in the presence of PSA-armed DC
were able to respond weakly to peptide 2 (KLQCVDLHV), but the CD8+
T cells did not release IFN.gamma. upon restimulation with
PSA-armed DC. This results suggests that this peptide may be
presented by HLA Class II molecules as well as by Class I, but that
the generation of T cells specific for this peptide are not
effectively generated by DC loaded with PSA. No responses were
found when the T cells that had been cultured with immune complex
were stimulated with mature DC that had been pulsed with an
HLA-A*0201 restricted HIV-1 gag peptide (data not shown).
EXAMPLE X
T cell responses to DC armed with PSA or PSA-m
[0128] DC express receptors that bind mannose and related
carbohydrates. Accordingly, experiments were conducted to compare
the T cell responses to DC that had been armed with PSA or PSA that
had been conjugated to mannose. To do this, PSA was purchased from
Scripps Laboratories (San Diego, Calif.). PSA was mannosylated as
follows: 100 .mu.g of PSA was combined with 100 .mu.g
.alpha.-D-mannopyranosylphenylisothiocyanate (a-D-M) and 2 .mu.l
N-methylmorpholine in 460 .mu.l of PBS and stirred overnight at
room temperature. Excess .alpha.-D-M was hydrolyzed by the addition
of 100 .mu.l of 1 M Trizma-base (pH 9.5). Unconjugated mannose
residues were removed by dialysis against PBS and stored at
4.degree. C.
[0129] The T cells were exposed through two weekly rounds of
stimulation to DC armed with PSA or PSA-m and CD4+ and CD8+
IFN.gamma. responses measured after re-exposure to armed DC. To do
this, as described in Example III, T cells were exposed to DC armed
with PSA or mannosylated-PSA (PSA-M) and cultured. At day 14, T
cells were restimulated with unarmed or antigen armed DC and
numbers of CD4+ and CD8+ T cells producing IFN.gamma. determined by
flow cytometry. The results are shown in Table IV.
4TABLE IV Comparison of T Cell Responses to Dendritic Cells Armed
With PSA or Mannosylated PSA IFN.gamma.+ cells/10.sup.6 Primary
stimulation Restimulation CD4+ CD8+ O PSA 33 42 PSA O 109 41 PSA
PSA 438 88 PSA PSA-M 566 92 O PSA-M 80 61 PSA-M O 111 82 PSA-M
PSA-M 894 122 PSA-M PSA 504 113
[0130] As show in Table IV, in three separate experiments, CD4+ T
cell responses predominated within the activated cell population
and were consistently higher when DC were loaded with PSA-m
compared to responses to PSA armed DC. Small to modest increases
(over controls) in number of activated CD8+ T cells were observed.
However, the CD8+ T cell response to PSA-m was far below responses
seen with Alt-6-PSA immune complexes (see FIG. 6).
EXAMPLE XI
Clinical Studies
[0131] Patients suffering from recurring ovarian cancer were
injected with 2 mg monoclonal antibody Alt2 per day, with a total
of 1 to 10 such treatments and followed for disease progression and
survival. Alt2 is a murine monoclonal IgG1 antibody to CA125. The
antibody has a high affinity (1.times.10.sup.10 M.sup.-1) and was
modified, such as by photoactivation (partial reduction of
disulfide bonds, see U.S. Pat. No. 6,086,873). All patients were
tested for HAMA and CA125 before and after each injection by ELISA
(HAMA, commercially available from Medac, Germany; CA125,
commercially available from Centocor, USA). Seventy-five patients
were tested for anti-idiotype antibodies (Ab.sub.2) to Alt2 by
ELISA (commercially available from AltaRex Corp., USA) and
anti-CA125 antibodies by ELISA (AltaRex; see also Schultes et al.,
Cancer Immunol. Inmunother. 46: 201 (1998)). For 17 patients,
peripheral blood mononuclear cells (PBMC) were available before and
after injection.
[0132] To determine how quickly the injected Alt2 antibody could
form immune complexes with free CA125, CA125 was captured from
serum samples (that were obtained at various time points after MAb
Alt-2 injection) with the anti-CA125 antibody MAb-B27.1, which
recognizes an epitope distinct from MAb Alt-2. After washing, tubes
were incubated with .sup.125I-MAb-Alt-2 or .sup.125I-MAb OC125.
This procedure allows for detection of CA125 bound MAb Alt-2 in the
serum, indicated by decreased tracer binding of .sup.125I-MAb Alt-2
in comparison to preinjection samples as well as decreased binding
in comparison to .sup.125I-MAb OC125. As shown in FIGS. 10A and
10B, MAb-Alt-2 was found to form complexes with CA125 within 30
minutes of injection of the antibody. These complexes cleared very
slowly from circulation at the first MAb Alt-2 injection (see right
half of FIG. 10B). The results show that MAb Alt-2 can bind to
CA125 in circulation and form long circulating immune complexes.
Consequently there is an opportunity of such complexes to be taken
up by the immune system and undergo processing and presentation to
T cells.
[0133] In addition, PBMC of the antibody-injected patients were
analyzed for T cell proliferation to CA125 in a standard
.sup.3H-thymidine uptake assay without in vitro sensitization (for
assay methods see, e.g., Current Protocols in Immunology, ed. John
E. Coligan, John Wiley & Sons, Inc. 1993; Current Protocols in
Molecular Biology, eds. Ausubel et al., John Wiley & Sons, Inc.
2000). The results are shown in FIGS. 11A-13B.
[0134] Induction of humoral (Pearson r=0.8335, p<0.001) and
cellular (p=0.037) anti-CA125 responses showed a correlation with
the amount of circulating CA125 antigen present at the time of Alt2
injection (FIGS. 11A-11C). Neither B nor T cell responses specific
for CA125 were detected in the pre-injection samples. Analysis of
the patients' anti-CA125 antibodies revealed that they were
directed against multiple epitopes of CA125 (FIG. 12). This
analysis was done by inhibiting human .alpha.-CA125 antibody by
various .alpha.CA125 monoclonal antibodies that specifically bound
to distinct epitopes on CA125.
[0135] In addition, survival from the time of first antibody
injection correlated with the generation of anti-CA125 antibodies
(median survival 22.9 v. 13.5 months, p=0.0089; FIG. 13A) and
generation of CA125-specific T cells (84 v. 13.2 months, p=0.0202;
FIG. 13B) after Alt2 injection for anti-CA125 responders v.
non-responders. These results demonstrate the following. The levels
of circulating CA125 at time of Alt2 injection showed influence on
the frequency and amount of immune responses induced to CA125 after
injection of the antibody. Patients' anti-CA125 antibodies were
found to be multi-epitopic (See FIG. 12). As FIGS. 13A and 13B
show, survival after the first injection of Alt2 (plotted in
Kaplan-Meier curves) was significantly longer in patients with
CA125-specific antibodies (increase in titer>3 times
pre-injection value), and in patients with CA125-specific T cells
(SI>1.5) (log-rank test, p<0.0l and p<0.05
respectively).
EXAMPLE XII
Effect of complex formation on antigen presentation
[0136] CA125 was purified from tissue culture supernatants of
NIH:OVCAR-3 cells (commercially available from AltaRex Corp.) and
PSA was purified from human seminal plasma (commercially available
from Scripps, La Jolla, Calif.) using standard procedures.
Photoactivated Alt2 antibody was as described previously (see
Example VII). The anti-PSA antibody, Alt6 (commercially available
from AltaRex Corp.), is a mouse IgG1 that binds to the region of
amino acids 135 to 150 of PSA. HAMA was as described previously
(see Example VI). Human dendritic cells were prepared from buffy
coats by Ficoll-Hypaque and negative selection with anti-CD3, CD16
and CD 19, followed by anti-mouse-IgG magnetics beads (Dynal).
Cells were cultured in 1000 U/ml GM-CSF and 1000 U/ml IL-4 for 4
days.
[0137] Murine macrophages were isolated from the peritoneal cavity
of Balb/c mice. Specific B cells were isolated from immunized mice
by panning on antibody-coated petri dishes. Dendritic cells were
loaded with antigen, antibody, or antigen-antibody complex at day 4
and matured with 10 ng/ml TNF-.alpha. and 50 U/ml IFN-.alpha. 4
hours later. Two stimulation rounds were performed before analyzing
the cells for intracellular IFN-.gamma. staining for either CD4 and
CD8 T cells or for the release of IFN-.gamma. into the culture
supernatant.
[0138] The results are shown in FIGS. 14A-14B. These results show
that antigen complexed with specific antibody can be preferentially
presented by professional APC such as macrophages (FIG. 14A) and B
cells (FIG. 14B). Results from the antigen-antibody system with PSA
and CA125 in dendritic cells support an enhanced presentation of
the extracellular antigen on MHC class I when offered as an immune
complex (see FIGS. 6, 8A, and 8B). Also, these results further
confirmed the results shown in Table IV (above); namely that while
mouse IgG1 alone binds weakly to dendritic cells (FIG. 2), the
binding is enhanced by binding of specific antigen, and
substantially enhanced in the presence of HAMA (FIGS. 3A-5B).
Finally, these results demonstrate that dendritic cells present
immune complexes better than non-complexed antigen (FIGS. 6, 8A,
and 8B) and, upon repeated stimulation, the immune complexes
shifted the immune response from a helper (CD4+) to a cytolytic
(CD8+) T cell response (FIGS. 6 and 8B), indicating presentation of
antigen-derived peptides on both MHC class I and class II.
EXAMPLE XIII
Ex vivo therapeutic treatment
[0139] A human-PBL-SCID/BG mouse model is used generally as
described in Schultes et al., Hybridoma 18: 47-55 (1999). Human
ovarian cancer cells NIH:OVCAR-NU-3 are passaged through nude mice
and maintained at 37.degree. C. and 5% CO.sub.2 using RPMI 1640
medium supplemented with 2 mM L-glutamine and 10% fetal bovine
serum Life-Technologies, Gaithersburg, Md.) SCID/BG mice are
obtained from Taconic (Germantown, N.Y.). Tumors are developed in
the SCID/BG mice by subcutaneous injection of 4.times.10.sup.6
NIH:OVCAR-NU-3 tumor cells and three weeks incubation. Dendritic
cells are loaded with CA125 antigen or with CA125 antigen and Alt2
antibody as described in Example III, with or without HAMA. The
combination is then administered to the mouse either
intraperitoneally or intravenously. This treatment is repeated
every 2 to 3 weeks. Tumor burden and days of survival are
monitored. It is expected that the dendritic cells loaded with the
antigen-antibody complex have a greater anti-tumor effect than the
dendritic cells loaded with antigen alone, and that the greatest
effect is observed when the dendritic cells loaded with the
antigen-antibody-HAMA complex are administered.
EXAMPLE XIV
In vivo Efficacy of an Antigen-Specific
Monoclonal Antibody in Murine Animal Model
[0140] The efficacy of the Altl, Alt2 and Alt2 monoclonal
antibodies (MAb) in vivo and the role of antibody-antigen complexes
in the induction of an immune response and anti-tumor responses
were studied in murine animal models. The immunization of mice with
Alt1, Alt2 and Alt6 MAb was studied to determine whether Altl, Alt2
and Alt6 can (a) induce a specific immunity against the specific
antigen, MUC1, CA125 or PSA, respectively, (b) protect the mice
against subsequent tumor challenge; and (c) eradicate established
tumors and/or increase survival.
[0141] These experiments were performed using DBA and Balb/c and
human PBL-SCID/bg mice which allowed one to choose the best animal
model for future experiments. Five groups of mice were immunized
respectively with MAb, control MAb, and MAb/antigen complexes.
Antigen and PBS controls were studied. The immunization and tumor
induction procedures were identical to the ones used for previous
studies. Survival curves were plotted using the Meir Kaplan
Algorithm. Comparison of responses between various groups were
analyzed using standard statistical procedures.
[0142] Specifically, to do these studies, the treatment groups
described in Table V were set up using SCIDI/bg mice:
5 TABLE V Group Mice Number Treatment 1 9 phosphate buffered saline
(PBS) i.v. 2 9 MOPC-21 i.v. 3 9 Alt-6 i.v. 4 9 Alt-6 plus PSA i.v.
5 8 PSA i.v. 6 8 Alt-6 s.c 7 8 MOPC-21 s.c. i.v. (intravenous)
injection: 50 .mu.g of antibody and/or 10 .mu.g of PSA i.p.
(intraperitoneal) injection: 100 .mu.g of antibody and/or 20 .mu.g
PSA s.c. (subcutaneous) injection: 50 .mu.g of antibody
[0143] Groups 1-5 received the first two injections
intraperitoneally., and the next four intravenously Groups 6-7
received the first two injection intraperitoneally, and the next
four subcutaneously with the adjuvant Quil A.
[0144] The treatment schedule was as follows in Table VI:
6TABLE VI Day procedure comments 1 PBL (1 .times. 10.sup.7 human
PBL/mouse) i.v. PBL (peripheral blood plus immunization, i.p.
leukocytes) from one HLA-A2+ donor 8 immunization, i.p. Dose 2 12
Bleeding for hIgG testing 3 .times. 10.sup.6/mouse with 75%
Matrigel 18 LnCap (tumor) inoculation s.c. 21 immunization
i.v./s.c. Dose 3 29 immunization i.v./s.c. Dose 4 35 immunization
i.v./s.c. Dose 5 42 immunization i.v./s.c. Dose 6 48 Terminate
mice/weigh tumor When biggest tumor reached 10 .times. 19 mm
[0145] On Day 22, human IgG levels were measured (as mg/ml). As can
be seen on FIG. 15, those mice injected with PBS only (i.e., Group
1) had the highest level of an IgG antibodies.
[0146] To measure successful reconstitution of human PBLs in the
mice, serum human was measured. These results are shown on Table
VII.
7TABLE VII Number of mice Percentage of mice Number of with greater
than with greater than Group mice 0.1 mg/ml hIgG 0.1 mg/ml hIgG PBS
i.v. 9 5 56 MOPC-21 i.v. 9 7 78 Alt-6 i.v. 9 0 0 Alt-6 + PSA i.v. 9
2 22 PSA i.v. 8 6 75 Alt-6 s.c 8 4 50 MOPC-21 s.c. 8 3 38
[0147] All off the mice in all groups grew tumor (i.e., from the
LnCap inoculation). se tumors were palpable 17 days after
inoculation (i.e., Day 35). The tumors were sured twice a week.
[0148] As shown in FIG. 16, the mouse group injected with Alt-6
plus PSA i.v. onstrated the best tumor suppression (Alt-6 plus PSA
i.v. versus PSA iv; P=0.0477). Alt-6 i.v. group also showed tumor
suppression (as measured by tumor volume).
[0149] These results demonstrated that best anti-tumor effects were
achieved in mice ted with the antibody-antigen complexes.
[0150] The humoral immune response was monitored by measuring the
serum levels of Ab2 and Ab3 in ELISAs.
[0151] The cellular immune response was monitored according to
routine laboratory procedures by measuring lymphocyte
proliferation.
EXAMPLE XV
Histopathological and Toxicological Studies
[0152] The tissue antigen specificity of ALt1 and Alt2 was examined
by histopathological reactivity with both normal and tumor human
tissues. The degree of heterogeneity in reactivity was recorded.
This study was conducted by a commercial organization (Impath Inc.)
under GLP conditions.
[0153] Acute and subacute toxicity of naked Alt1and Alt2 was
conducted in two species (rat and rabbits) by the division of the
animal services at the University of Alberta. There was toxicity
observed in acute or subacute studies.
EXAMPLE XVI
Phase I Clinical Trial
[0154] Because there was data available on the "safety" of murine
antibodies administered to humans, it was not expected that side
effects were a dose limiting problem. However, standard criteria
such as major organ toxicity and patient symptoms were followed
utilizing GCP. As antibody doses did not have to be pushed to
toxicity (the maximally tolerated dose) the major outcome became
defining the most effective dose of antibody that elicited the
desired defined immune response.
[0155] The patient population to be studied needed to be immune
competent and have the target disease. A group of patients with
MUC1 expressing tumor were enrolled in a three-dose Phase I trial
with Alt-1
[0156] Based on studies with other immune therapies it was expected
that the effective dose was in the 1-4 mg dose. Doses of 1, 2, and
4 mg were studied in the stated patient population. Toxicity
criteria were monitored along with the immune response (primary
endpoint). Evidence of therapeutic activity was detected by
monitoring patient MUC1 levels (secondary endpoint). Six patients
per dose level were treated. The 2 mg dose was most effective in
inducing HAMA, Ab2, anti-MUC1 antibodies and MUC1-specific T cells,
and showed the highest incidence of MUC1 serum level stabilization
or decrease.
Equivalents
[0157] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *