U.S. patent application number 13/940558 was filed with the patent office on 2014-01-09 for vaccine immunotherapy for immune suppressed patients.
This patent application is currently assigned to IRX Therapeutics, Inc.. The applicant listed for this patent is IRX Therapeutics, Inc.. Invention is credited to John W. Hadden.
Application Number | 20140010779 13/940558 |
Document ID | / |
Family ID | 22920630 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140010779 |
Kind Code |
A1 |
Hadden; John W. |
January 9, 2014 |
VACCINE IMMUNOTHERAPY FOR IMMUNE SUPPRESSED PATIENTS
Abstract
A method for overcoming mild to moderate immune suppression
includes the steps of inducing production of naive T-cells and
restoring T-cell immunity. A method of vaccine immunotherapy
includes the steps of inducing production of naive T-cells and
exposing the naive T-cells to endogenous or exogenous antigens at
an appropriate site. Additionally, a method for unblocking
immunization at a regional lymph node includes the steps of
promoting differentiation and maturation of immature dendritic
cells at a regional lymph node and allowing presentation of
processed peptides by resulting mature dendritic cells, thus, for
example, exposing tumor peptides to T-cells to gain immunization of
the T-cells. Further, a method of treating cancer and other
persistent lesions includes the steps of administering an effective
amount of a natural cytokine mixture as an adjuvant to endogenous
or exogenous administered antigen to the cancer or other persistent
lesions.
Inventors: |
Hadden; John W.; (Cold
Spring Harbor, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IRX Therapeutics, Inc. |
New York |
NY |
US |
|
|
Assignee: |
IRX Therapeutics, Inc.
New York
NY
|
Family ID: |
22920630 |
Appl. No.: |
13/940558 |
Filed: |
July 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11582063 |
Oct 16, 2006 |
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13940558 |
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10430506 |
May 5, 2003 |
7153499 |
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11582063 |
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10015123 |
Oct 26, 2001 |
6977072 |
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10430506 |
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60243912 |
Oct 27, 2000 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 31/664 20130101;
A61P 35/00 20180101; A61K 38/2053 20130101; A61K 31/496 20130101;
A61P 37/08 20180101; A61K 38/2013 20130101; A61K 38/20 20130101;
A61P 37/02 20180101; A61P 37/06 20180101; A61K 38/21 20130101; A61K
38/217 20130101; A61K 38/193 20130101; A61K 2039/5154 20130101;
A61P 31/04 20180101; A61P 43/00 20180101; A61K 31/5383 20130101;
A61K 38/204 20130101; A61K 31/675 20130101; A61K 38/2006 20130101;
A61K 39/0011 20130101; A61K 31/405 20130101; A61K 45/06 20130101;
A61K 2039/55522 20130101; A61K 38/2086 20130101; A61K 39/39
20130101; A61P 37/04 20180101; A61K 2039/5158 20130101; A61P 31/00
20180101; A61K 38/191 20130101; A61K 38/20 20130101; A61K 2300/00
20130101; A61K 38/191 20130101; A61K 2300/00 20130101; A61K 38/21
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 38/19 20060101 A61K038/19; A61K 31/664 20060101
A61K031/664; A61K 38/20 20060101 A61K038/20 |
Claims
1.-29. (canceled)
30. A method for inducing an immune response to at least one
exogenous antigen in a patient, the method comprising:
administering to the patient an effective amount of a natural
cytokine mixture comprising the cytokines IL-1beta, IL-2, IL-6,
IL-8, IFN-gamma, and TNF-alpha; and administering to the patient an
effective amount of at least one exogenous antigen, wherein the
natural cytokine mixture acts as an adjuvant with the at least one
exogenous antigen and stimulates an immune response to the at least
one exogenous antigen in the patient.
31. The method of claim 30, wherein the at least one exogenous
antigen is at least one exogenous tumor antigen.
32. The method of claim 30, wherein the natural cytokine mixture is
administered to the patient perilymphatically.
33. The method of claim 30, wherein the method further comprises:
administering to the patient an effective amount of
cyclophosphamide.
34. The method of claim 30, wherein the method further comprises:
administering to the patient an effective amount of
cyclophosphamide; and administering to the patient an effective
amount of a nonsteroidal anti-inflammatory drug (NSAID).
35. The method of claim 30, wherein the natural cytokine mixture
further comprises GM-CSF and G-CSF.
36. The method of claim 31, wherein the at least one exogenous
tumor antigen is at least one tumor peptide.
37. The method of claim 36, wherein the at least one tumor peptide
is at least one prostate specific membrane antigen (PSMA)
peptide.
38. A composition comprising: a natural cytokine mixture comprising
the cytokines IL-1beta, IL-2, IL-6, IL-8, IFN-gamma, and TNF-alpha;
and at least one exogenous antigen.
39. The composition of claim 38, wherein the at least one exogenous
antigen is at least one exogenous tumor antigen.
40. The composition of claim 39, wherein the at least one exogenous
tumor antigen is at least one tumor peptide.
41. The composition of claim 40, wherein the at least one tumor
peptide is at least one prostate specific membrane antigen (PSMA)
peptide.
Description
CROSS-RELATED REFERENCE SECTION
[0001] This application is a continuation which claims the benefit
under 35 U.S.C. .sctn.120 of U.S. application Ser. No. 11/582,063,
entitled "VACCINE IMMUNOTHERAPY FOR SUPPRESSED PATIENTS" filed on
Oct. 16, 2006, which is herein incorporated by reference in its
entirety. application Ser. No. 11/582,063 is a divisional which
claims the benefit under 35 U.S.C. .sctn.120 of U.S. application
Ser. No. 10/430,506, now U.S. Pat. No. 7,153,499, granted Dec. 26,
2006, entitled "VACCINE IMMUNOTHERAPY FOR IMMUNE SUPPRESSED
PATIENTS" filed on May 5, 2003, which is herein incorporated by
reference in its entirety. application Ser. No. 10/430,506 is a
continuation which claims the benefit under 35 U.S.C. .sctn.120 of
U.S. application Ser. No. 10/015,123, now U.S. Pat. No. 6,977,072,
granted Dec. 20, 2005, entitled "VACCINE IMMUNOTHERAPY FOR IMMUNE
SUPPRESSED PATIENTS" filed on Oct. 26, 2001, which is herein
incorporated by reference in its entirety. application Ser. No.
10/015,123 claims priority under 35 U.S.C. .sctn.119(e) to U.S.
Provisional Application Ser. No. 60/243,912, entitled "VACCINE
IMMUNOTHERAPY FOR IMMUNIZING CANCER PATIENTS TO CANCER ANTIGENS"
filed on Oct. 27, 2000, which is herein incorporated by reference
in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates to vaccine therapy for cancer
patients. More specifically, the present invention relates to a
vaccine immunotherapy which immunizes cancer patients, having
immune suppression, to both endogenous and exogenous tumor peptides
or proteins.
[0004] 2. Background Art
[0005] It has become increasingly apparent that human cancers have
antigens which, if reacted upon by the host's immune systems, lead
to tumor regression. These antigens have been defined by both
serological and cellular immune approaches. This has led to the
definition of both B and T cell epitopes (Sahin U, et al, Curr Opin
Immunol 9:709-715, 1997; Van der Eynde, B, et al. Curr Opin Immunol
9:684-693, 1997; Wang R F, et al., Immunologic Reviews 170:85-100,
1999). Based upon these results, it has become a goal of cancer
immunotherapists to induce regressions of tumors. However,
historically, successful efforts have been sporadic and generally
minor in frequency and magnitude.
[0006] A fundamental problem in the effort to immunize cancer
patients is that the tumor-bearing state is associated with
immunosuppressive mechanisms derived from both the tumor and the
host's disturbed immune system (Kavanaugh D Y, et al, Hematol-Oncol
Clinics of North Amer 10(4):927-951, 1996), thereby making
immunization difficult and until now impossible on a consistent
basis. Immune suppression or depletion involves a reduced capacity
of the immune system to respond. Such suppression can be drug or
disease induced. The condition can be drug induced by treatment,
virus induced as in AIDS, or induced by a disease state such as
cancer. The immune system in this condition is effectively turned
off.
[0007] A variety of tumor immunization strategies have been
developed. However, all of these strategies are complex and deviate
significantly from the conventional immunization strategies used
for infectious diseases (Weber J. Tumor, Medscape Anthology 3:2,
2000). One such tumor immunization strategy involves
Theratope.RTM., a Sialyl T.sub.N polysaccharide mucin antigen
conjugated with keyhole limpet hemocyanine and administered with
Detox.RTM. mycobacterium adjuvant and low dose cyclophosphamide
(Maclean G D, et al, J Immunother Emphasis Tumor Immunol
19(4):309-316, 1996). However, use of this vaccine in patients with
metastatic breast and ovarian cancer has yielded major clinical
responses in a low percentage of patients. A major response means
greater than 50% tumor reduction.
[0008] Gene therapy has also been attempted using an adenovirus
construct as an expression vector for genes expressing Papilloma
virus peptide 16 has been used for immunization or patients with
cervical cancer and has yielded major clinical responses in a low
percentage of patients (Borysiewickz L K, et al, Lancet
347:1524-1527, 1996).
[0009] Dendritic cell mediated therapy has also been attempted,
wherein dendritic cells were pulsed with oligopeptide fragments of
prostate specific antigens (PSA). Prostate specific membrane
antigen (PSMA) has been used in patients with metastatic prostate
cancer with major clinical responses in a low percentage of
patients (Sanda M G, et al, Urology 52:2, 1999; Murphy G P, et al,
The prostate. 38:43-78, 1999)
[0010] Additionally, autologous tumors have been used with low dose
cyclophosphamide and BCG to immunize cancer patients with malignant
melanoma. However, few clinical responses were reported
(Mastrangelo M J, et al, Seminars in Oncology 23(6):773-781, 1996).
Another strategy attempted included using MAGE antigens with a
variety of vaccine adjuvants. Again, this has yielded few, if any,
responses in patients with malignant melanoma (personal
communication Thierry Boon).
[0011] Several patents to Doyle et al (U.S. Pat. Nos. 5,503,841;
5,800,810; 6,060,068; 5,643,565; 5,100,664) disclose methods of
enhancing the immune response in patients using Interleukin
2-(IL-2). This method is disclosed for use in response to
infectious diseases and primarily functions using antigens known to
be immunogenic. Limited applicability was demonstrated. As
disclosed above, the treatment of cancer is known to require
different approaches. To date, treatment with IL-2 has shown minor
effects in two cancers, renal cell and malignant melanoma (response
rates less than 20%). It is generally considered ineffective in
squamous cell head and neck and cervical cancer and in prostate
cancer. Hence, it is not approved for these uses. It would
therefore not be within the skill of one in the art to apply the
method of the Doyle et al patents to the use of small peptides in
the treatment of cancer.
[0012] It is important to contrast prevention with known "classic"
antigens of complex structure and high molecular weights in healthy
patients vs. treatment (generally unsuccessful) with tumor antigens
or peptides (general unsuccessful) in immunosupressed patients
(generally unsuccessful). The first is easy and our current viral
vaccines attest to their efficacy. The latter is nearly impossible
on a routine basis despite 30 years of intense effort.
[0013] It is important that this invention relates to, but not
exclusively to, immunizing with endogenous peptide processed and
presented by dendritic cells or endogenously administered to an
environment (lymph node) where dendritic cells have been prepared
and can present them to T-cells effectively. This goal is
considered by many immunologists to be insurmountable, Peptides are
much too small to be effective immunogens, their one half life is
short they are often nonmutated self antigens to which
[0014] In several of the above strategies, cellular and/or tumoral
immunity to tumor-associated antigens has been induced (Weber J.
Tumor, Medscape Anthology 3:2, 2000; Maclean G D, et al, J
Immunother Emphasis Tumor Immunol 19(4):309-316, 1996; Borysiewickz
L K, et al, Lancet 347:1524-1527, 1996; Sanda M G, et al, Urology
52:2, 1999). This is especially so in association with tumor
regression. Nevertheless, the success rate of such treatments is
negligible and inconsistent (<30%).
[0015] It would therefore be useful to develop a consistent and
effective method of immunizing cancer patients.
SUMMARY OF THE INVENTION
[0016] In accordance with the present invention there is provided a
method for overcoming immune depression by inducing production of
naive T cells and restoring T-cell immunity. That is the present
invention provides an immune restoration. The present invention
further provides a method of vaccine immunotherapy including the
steps of inducing production of naive T cells and exposing the
naive T cells to endogenous or exogenous antigens at an appropriate
site. Additionally, the present invention provides a method for
unblocking immunization at a regional lymph node by promoting
differentiation and maturation of immature dendritic cells at a
regional lymph node and allowing presentation of processed peptides
by resulting mature dendritic cells, thus exposing tumor peptides
to T cells to gain immunization of the T cells. Additionally, the
present invention provides a method of treating cancer and other
persistent lesions by administering an effective amount of a
natural cytokine mixture as an adjuvant to endogenous or
exogenously administered antigen of the cancer or other persistent
lesions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Other advantages of the present invention are readily
appreciated as the same becomes better understood by reference to
the following detailed description when considered in connection
with the accompanying drawings wherein:
[0018] FIG. 1 is a graph showing a comparison of NCM in different
media utilizing continuous versus pulsed exposure to PHA;
[0019] FIG. 2 is a graph showing the effect of cell concentration
with continuous exposure to PHA;
[0020] FIG. 3 is a bar graph similar to FIG. 1 with PHA at twice
the concentration (2 micrograms per ml);
[0021] FIG. 4 is a graph of thymidine uptake versus units per ml of
IL2 relating to splenocytes;
[0022] FIG. 5 is a graph similar to FIG. 2 related to
thymocytes;
[0023] FIG. 6 is a graph showing ratio to control versus in vivo
treatments for mice with involuted thymuses is treated with ILL IL2
or IL combinations, NCM, or saline;
[0024] FIG. 7 is a graph also showing a comparison of treatment
with recombinant IL1, IL2, IL1 plus IL2, and NCM;
[0025] FIG. 8 is a graph demonstrating the effect of NCM treatment
in vivo on splenocyte and thymocyte markers;
[0026] FIG. 9 is a bar graph also demonstrating the effect of NCM
treatment in vivo on splenocyte and thymocyte markers;
[0027] FIG. 10 is a graph demonstrating splenocyte and splenocyte
responses to in vitro media including various recombinant
interleukins or NCM after treatment in vivo with control media or
NCM;
[0028] FIG. 11 is a bar graph demonstrating the splenocyte and
thymocyte responses in vitro to media, various interleukins, or NCM
in vivo with control media or NCM;
[0029] FIG. 12 demonstrates responses in splenocyte and thymocyte
in vitro to ConA and PHA after treatment in vivo with control or
NCM;
[0030] FIG. 13 demonstrates responses in splenocyte and thymocyte
in vitro to ConA and PHA after treatment in vivo with control or
NCM;
[0031] FIG. 14 is a bar graph showing node size in controls, and
cancer controls or IRX-2(NCM) treated populations with squamous
cell head & neck cancer (H&NSCC);
[0032] FIG. 15A and FIG. 15 B shows two bar graphs, FIG. 15A
showing T-cell area and FIG. 15B showing density in controls and
head and neck squamous cancer controls and patients treated with
NCM(IRX-2);
[0033] FIG. 16A and FIG. 16B shows two bar graphs, FIG. 16A showing
B-cell area and FIG. 16B showing follicles in the three treatment
groups;
[0034] FIG. 17A shows a comparison of other cells in the three
treatment groups and FIG. 17B shows a comparison of sinus
histocytosis in the three treatment groups; and
[0035] FIG. 18 is a graph showing node B&T and Cancer B&T
fit plot.
DESCRIPTION OF THE INVENTION
[0036] Generally, the present invention provides methods for
treating patients utilizing vaccine immunotherapy wherein the
patients are immune suppressed. By immune suppressed, it is meant
that the patient has reduced cellular immunity and thus impaired
capacity to respond to new antigens. More specifically, in blood, T
lymphocyte counts are reduced and/or function of these cells is
impaired, as shown, e.g. by PHA proliferation assay.
[0037] T lymphocytopenia (low T cell levels in blood) is a
diagnostic characteristic of cellular immune deficiency; impaired
function of existing thymphocytes is the other characteristic.
There is no generally accepted (clinically approved) way to treat T
lymphocytopenia. Bone marrow transplants (.+-.thymus transplants)
have been used in cases of severe combined immunodeficiency
(SCID--congenital, irradiation or chemotherapy induced).
Recombinant IL2 has been tried in AIDS with some effect by much
toxicity.
[0038] There are two ways to make new T cells to attempt to correct
T lymphocytopenia. One way, as in rIL-2 therapy, expands T cells
already in the periphery, i.e., memory T cells (CD.sub.45RO)
(blood, lymph node and spleen). The other involves processing in
the thymus of new T cells from bone marrow--derived precursors.
This happens naturally in children but not in adults. These new
cells are called recent "thymic emigres" and have the surface
marker of "naive" T cells i.e., CD.sub.45RA. NCM therapy (plus
Thymosin .alpha..sub.1) results in the production of these new T
cells as well as expanding preexisting memory T cells.
[0039] More specifically, the present invention utilizes new
discoveries relating to immunization to provide an immune response
to antigens which is either endogenously or exogenously
administered. Such antigens in the past may have been believed to
be immunogenic while others used in the present invention may have
been thought previously to be non-immunogenic. Examples of such
antigens are EADPTGHSY (SEQ ID NO: 1) (melanoma) from MAGE-1
protein, EVDPIGHLY (SEQ ID NO: 2) (lung carcinoma) from MAGE-3, and
many others. (See Bellone, et al, Immunology Today, Vol 20, No. 10,
p 457-462, 1999.)
[0040] The present invention utilizes several general newly derived
method steps for obtaining immunization in subjects where such
immunization was previously thought to be impossible. More
specifically, the present invention provides a method for
overcoming immune depression by inducing production of naive T
cells. The term "naive" T cells is meant to mean newly produced T
cells, even in adults, wherein these T cells have not yet been
exposed to antigen. Such T cells at this stage are non-specific yet
capable of becoming specific upon presentation by a mature
dendritic cell having antigen, such as tumor peptides, exposed
thereon. Thus, the present invention replenishes or generates new T
cells. This is generally accomplished by administering a natural
cytokine mixture (NCM). The NCM includes ILL IL2, IL6, IL8, IL10,
IL12, IFN-.gamma., TNF.alpha. and G- and GM-CSF. The amount and
proportions of these constituents are detailed below. Preferably,
about 150-600 units of IL2 are contained in the NCM.
[0041] Preferably, the NCM is injected around lymphatics that drain
into lymph nodes regional to a lesion, such as a tumor or other
persistent lesions being treated. Perilymphatic administration into
the lymphatics which drain into the lymph nodes, regional to the
lesion, such as a cancer, is critical. Peritumoral injection has
been associated with little response, even progression and is thus
contraindicated. A ten (10) day injection scheme is optimal and a
twenty (20) day injection protocol, while effective clinically,
tends to reduce the TH1 response and shift towards a less desirable
TH2 response as measured by lymphoid infiltration into the cancer.
Bilateral injections are effective. Where radical neck dissection
has occurred, contralateral injection is effective.
[0042] It is preferable to block endogenous suppression of T cells,
such as caused by various cancer lesions. Blocking is effected by
the codelivery of low dose cyclophosphamide and a non-steroidal
anti-inflammatory drug (NSAID). The NSAID of choice is
indomethacin. While indomethacin is the most effective NSAID, it is
also arguably the most toxic. Celebrex.RTM. (celecoxib) and
Vioxx.RTM. (rofecoxib), Cox II NSAIDS, are less effective.
Vioxx.RTM. can be more toxic, causing gastritis in many patients.
Ibuprofen was effective but the histological responses were
characteristic of a TH2 rather than TH1 mediated response, this
being less desirable. Side effects of NSAIDS are to be aggressively
treated with proton inhibitors and a prostaglandin E analog. Zinc
and multi-vitamins are useful agents to help restore T cell
immunity. Applicants have found that treatment with
contrasuppression and zinc without the NCM is ineffective.
[0043] In summary, the minimum regimen is perilymphatic treatment
with the NCM combined with contrasuppression using cyclophosphamide
and an NSAID. The alternative regimen is the previously mentioned
regimen further including zinc and vitamins, possibly including the
addition of selenium. Preferable dosing of Zinc is 50 to 75 mg. A
standard multivitamin can be administered. The zinc can be an
available gluconate.
[0044] In order to maximize clinical response and for the greatest
increase in survival rate, the degree and type of lymphocyte
infiltration is important. Lymphocyte: granulocyte or macrophage
infiltration of a 90:10 ratio is optimal. T and/or B cell
infiltration preferably is diffuse and intense and not peripheral.
Light infiltration of less than 20% is not associate with a robust
clinical response. Tumor reduction and fragmentation in the
histological samples is preferred in reflecting a good
response.
[0045] Lymph node changes key to good response involve at least
five (5) aspects. Lymph node enlargement and not just reversal of
tumor induced reduction of size but overall increase in size
compared to normal is preferred. Increased T and B cell areas
indicate an immunization. Sinus histocytosis (SH) is believed to be
the accumulation of immature dendritic cells which have ingested
and processed tumor antigens but are unable to mature and present
these tumor peptides to naive T cells capable of stimulating TH1
and TH2 effective cells which lead to cytotoxin T cell and B cells.
Reversal of SH is preferred
[0046] Thus, the present invention provides for unblocking
immunization at a regional lymph node by promoting differentiation
and maturation of immature dendritic cells in a regional lymph node
and thus allowing presentation by resulting mature dendritic cells
of small peptides, generally nine amino acids in length to T cells
to gain immunization of the T cells. Additionally, induction of
mature dendritic cells is required. Finally, mobilization of
peripheral blood T-lymphocytes in T-lymphocytopoenic patients in
the presence of induction of naive T cells capable of responding to
dendritic cells presenting endogenous tumor peptides is desired.
(See Sprent, et al, Science, Vol 293, Jul. 13, 2001, pgs
245-248).
[0047] In view of the above, the key mechanistic features of the
present invention are the in vivo maturation of dendritic cells
resulting in effective peptide antigen presentation. Based on the
examples presented below, increases in CD45 RA positive naive
uncommitted T cells have been found. With antigen, this leads to T
and B cell clonal expansion, creating immunity in the patient. The
resulting infiltration into tumors by hematogenous spread leads to
robust tumor destruction. The result, as found in the data below,
is increased survival due to immunologic memory. (See Sprent et al,
cited above).
[0048] It is predicted logically that exogenously provided
synthetic or extracted tumor peptides (See Bellone, et al, cited
above) can be delivered into the pre-primed or co-primed regional
or distal lymph node and yield tumor antigen specific T cells, with
or without B cells. Three examples are set forth below. In view of
the above, it can be concluded that the action of NCM plus other
agents is useful as for any tumor antigens (synthetic and
endogenous, peptides and proteins). Many of these peptides are not
normally immunogenic and only when presented by a matured,
activated dendritic cell, will they be effective in immunizing
naive T cells. Thus, the appearance of an immune T cell means, de
facto, that a dendritic cell has been made or allowed to work
properly. Also de facto, dendritic cell activation and maturation
is to be considered a key factor in cancer immunodeficiency as well
as the well-known defects in T cells such as a decreased number and
function with anergy and presumed apoptosis.
[0049] Referring more specifically to the protocol and medicant
delivered in accordance with the present invention, the invention
utilizes the natural cytokine mixture (NCM) to immunize patients,
such as cancer patients, as well as patients with other lesions or
antigen producing disease conditions. More specifically, the
present invention utilizes a method of enhancing the immune
response of cancer patients to a cancer by administering an
effective amount of a composition containing therein the NCM and a
tumor-associated antigen, the NCM acting as an adjuvant to produce
the immune response. The tumor associated antigen can be either an
endogenously processed tumor peptide preparation resident in
regional nodes of patients with cancer or in conjunction with an
exogenously administered tumor antigen preparation in or near these
nodes. Tumor peptides, as well as antigens, are included herein
even though peptides are not expected to be immunogenic where tumor
associated protein antigens would more likely be more so since they
are complete.
[0050] In the preferred embodiment, the composition of the present
invention involves the administration of the NCM plus a tumor
associated or specific antigen, as defined below with low doses of
cyclophosphamide, a cyclooxygenase inhibitor, and other similar
compounds which have been shown to further increase the effects of
the composition of the present invention.
[0051] To clarify and further define the above, the following
definitions are provided. By "adjuvant" it is meant a composition
with the ability to enhance the immune response to a particular
antigen. To be effective, an adjuvant must be delivered at or near
the site of antigen. Such ability is manifested by a significant
increase in immune mediated protection. Enhancement of immunity is
typically manifested by a significant increase (usually greater
than 10 fold) in the titer of antibody raised to the antigen.
Enhancement of cellular immunity can be measured by a positive skin
test, cytotoxic T-cell assay, ELISPOT assay for IFN-.gamma. or
IL-2, or T-cell infiltration into the tumor (as described
below).
[0052] By "tumor associated antigen", it is meant an analogous
protein or peptide (which were previously shown to work by pulsing
of dendritic cell ex vivo) or other equivalent antigen. This can
include, but is not limited to PSMA peptides, MAGE peptides (Sahin
U, et al, Curr Opin Immunol 9:709-715, 1997; Wang R F, et al,
Immunologic Reviews 170:85-100, 1999), Papilloma virus peptides (E6
and E7), MAGE fragments, NY ESO-1 or other similar antigens.
Previously, these antigens were not considered to be effective in
treating patients based either on their size, i.e. they are too
small or that they were previously thought to not have the
immunogenic properties (i.e., self antigens).
[0053] NCM, a non-recombinant cytokine mixture, is defined as set
forth in U.S. Pat. Nos. 5,632,983 and 5,698,194. Briefly, NCM is
prepared in the continuous presence of a 4-aminoquinolone
antibiotic sand with the continuous or pulsed presence of a mitogen
which in the preferred embodiment is PHA.
[0054] According to the present invention, there is provided a
partially characterized NCM that has been previously shown to be
effective in promoting T cell development and function in aged,
immunosuppressed mice. Upon administering this NCM to
immunosuppressed patients with head and neck cancer, it is
demonstrated in this application for the first time that the
mobilization of T lymphocytes in the blood of cancer patients
treated with the NCM produces an increase in immature, naive T
cells bearing both CD2 and CD45 RA. This is one of the first
demonstrations that adult humans can generate naive T cells.
Previous references: Mackall et al, (New England Journal of
Medicine (1995), Vol. 332, pp. 143-149); and a review by Mackall
(Stem Cells 2000, Vol. 18. pp. 10-18) discusses the inability to
generate new T cells in adults but not children, and discusses the
problem of trying to replenish T cells following cancer
chemotherapy and/or radiotherapy. In general there is the dogma
that new T cells are not generated in the adult human. However,
following bone marrow transplantation for intense chemotherapy,
there has been evidence that new T cells can be generated in the
adult. No molecular therapy to date has been able to achieve this,
although increase in lymphocytes counts have been achieved with
prolonged and intense therapy with recombinant interleukin-2 in
patients infected by HIV. These have not been clearly demonstrated
to be thymus derived T cells and are presumably an expansion of
pre-existing peripheral T cells.
[0055] Previously, Cortesina et al. employed a natural IL-2,
perilymphatically in patients with head and neck cancer and induced
several tumor regressions (Cortesina G, et al, Cancer 62:2482-2485,
1988) with some tumor infiltration with leukocytes (Valente G, et
al, Modem Pathol 3(6):702-708, 1990). Untreatable recurrences
occurred and the response was termed non-specific and without
memory and thus nonimmunologic (Cortesina G, et al, Br J Cancer
69:572-577, 1994). The repeated attempts to confirm the initial
observations with recombinant IL-2 were substantially unsuccessful
(Hadden J W, Int'l J Immunopharmacol 11/12:629-644, 1997).
[0056] The method of the present invention involves using NCM with
local perilymphatic injections or other injections that are known
to those of skill in the art to provide sufficient localization of
the immunotherapy compound. In the preferred embodiment, the
injections take place in the neck, but can be applied in other
locations as required by the disease to be treated. This treatment
induced clinical regressions in a high percentage of patients who
also showed improved, recurrence free survival (Hadden J W, et al,
Arch Otolaryngol Head Neck Surg. 120:395-403, 1994; Meneses A, et
al, Arch Pathol Lab Med 122:447-454, 1998; Barrera J, et al, Arch
Otolaryngol Head Neck Surg 126:345-351, 2000; Whiteside, et al,
Cancer Res. 53:564-5662, 1993). Whiteside, et al (Cancer Res.
53:5654-5662, 1993) observed that in head and neck cancer, tumoral
injection of recombinant interleukin-2 produced a T cell lymphocyte
infiltrate, but without significant clinical responses. Peritumoral
injection of Multikine (Celsci Website) (in combination with
perilymphatic injection in up to 150 patients resulted in
significant tumor responses, i.e. greater than 50% tumor reduction
in only 11 patients, making their response rate less than 10% in
contrast to the high degree of response observed in the present
studies, 40%. In addition, they noted 50% non-responders where
Applicants have observed only 20%.
[0057] Applicants, have observed that peritumoral and intratumoral
injection can be associated with progression of disease even in
patients who initially have had a positive response to the NCM
protocol, thus undoing its benefit. Peritumoral injection is thus
contraindicated and is excluded as part of the present invention.
This has led Applicants to the interpretation that the tumor is not
the site of immunization and the present application presents
documentation that the regional lymph node is the site of
immunization. Then, unpublished analysis of regional lymph nodes
revealed data which indicated that the regional lymph node is the
site of immunization to postulated tumor antigens (FIGS. 14-18).
With the identification of a number of different tumor antigens, it
has been a conundrum over the last decade that given the presence
of such antigens, they have not been employed effectively in
immunization protocols. Sporadic positive examples have been
reported, but in the main, the data are negative. The problem of
antigen presentation has been focused on in the last decade and the
dendritic cell has emerged as a critical player in the presentation
of small peptides derived from tumors. See DeLaugh and Lotts,
Current Opinion In Immunology, 2000, Vol. 12, pp. 583-588;
Banchereau et al, Annual Reviews of Immunology, (2000), Vol. 18,
pp. 767-811; also Albert et al, Nature, Vol. 392, pp. 86-89
(1998).
[0058] In brief, in order for tumor antigens to be properly
antigenic, they must arrive from an apoptotic rather than a
necrotic tumor cell (Albert, Nature, 39 2:86-87, 1997). They need
to be captured by immature dendritic cells that have the morphology
of large histocytes. These immature dendritic cells process antigen
(endocytosis, phagocytosis and digestion) and evolve into mature
dendritic cells which display peptide fragments (generally nine
amino acids) of the digested antigen in the MHC groove for
presentation to T cells. T cells, in order to respond, must have
antigen presented to them in the MHC groove plus various
co-stimulatory signals. References: Banchereau and DeLaugh.
[0059] Investigators, such as Murphy et al, 1999, have utilized
dendritic cells generated in culture and then pulsed with tumor
antigens and have achieved a small degree of success in immunizing
patients against prostate specific membrane antigen peptides.
Unfortunately, this approach of pulsing dendritic cells is
cumbersome and has been rather inefficient. In the present
invention, Applicants have shown that the cells present in the
lymph node sinuses, which accumulate in cancer, are cells of the
lineage of dendritic cells and that following the in vivo treatment
with the NCM protocol, these cells disappear and antigen ultimately
then becomes immunogenic for T cells. They are able then to respond
to the tumor. So a critical aspect of this invention is being able
to generate a microenvironment in the regional lymph node which
allows effective antigen processing and presentation. The
immunization which derives results in T cells able to traffic to
the lesion and destroy tumors is de facto demonstration of adequate
antigen processing by dendritic cells. Additionally, none of the
patients treated with NCM developed distant metastasis which is
expected in up to 15% clinically and up to 50% pathologically. This
indicates that a systemic immunity rather than merely a local
immunity has been induced by the treatment. This is a drastic
improvement over the compositions in the prior art, because the
prior art compositions, at best, were inconsistently effective
against metastatic disease. The ability of the composition of the
present invention to create systemic immunity allows more effective
and efficient treatment of a patient. Further, the magnitude of
systemic response enables an individual to be administered smaller
doses without limiting the effectiveness of the treatment and
without toxicity.
[0060] The literature (Hadden J W, Int'l J Immunopharmacol
11/12:629-644, 1997; Hadden J W. Immunology and immunotherapy of
breast cancer: An update: Int'l J Immunopharmacol 21:79-101, 1999)
has indicated that for both SCC and adenocarcinomas, the two major
types of cancer, regional lymph nodes reflect abnormalities related
to the tumor, including sinus histocytosis, lymphoid depletion and
often the presence of anergic tumor associated lymphocytes (capable
of reacting to tumor cells with ex vivo expansion and recovery
using IL-2). Then, with metastases, lymphoid depletion and
depressed function occur. Additionally, uninvolved cervical lymph
nodes of such patients have shown a reduction in average size and
an increase in sinus histocytosis associated with head and neck
cancers. (See FIGS. 14-17).
[0061] Specifically relating to the composition, the composition of
the present invention involves the natural cytokine mixture plus
either endogenous or exogenous tumor associated antigen.
Additionally, low doses of cyclophosphamide, cyclooxygenase
inhibitors, zinc, and other similar compounds have been shown to
further increase the effects of the composition of the present
invention.
[0062] Immunization for treatment of patients with cellular immune
deficiencies associated with cancer, HIV infection, aging, renal
transplants and other such deficiencies can be achieved with the
composition of the present invention.
[0063] Administration and protocols for treatment as follows:
Delivery of Gene Products/Synthetic Antigens with:
[0064] The compounds of the present invention (including NCM), and
exogenous antigens are administered and dosed to achieve optimal
immunization, taking into account the clinical condition of the
individual patient, the site and method of administration,
scheduling of administration, patient age, sex, body weight. The
pharmaceutically "effective amount" for purposes herein is thus
determined by such considerations as are known in the art. The
amount must be effective to achieve immunization including but not
limited to improved tumor reduction, fragmentation and
infiltration, survival rate or more rapid recovery, or improvement
or elimination of symptoms.
[0065] In the method of the present invention, the compounds of the
present invention can be administered in various ways. It should be
noted that they can be administered as the compound or as
pharmaceutically acceptable salt and can be administered alone or
as an active ingredient in combination with pharmaceutically
acceptable carriers, diluents, adjuvants and vehicles. The
compounds can be administered intra or subcutaneously, or peri or
intralymphatically, intranodally or intrasplenically or
intramuscularly, intraperitoneally, and intrathorasically. Implants
of the compounds can also be useful. The patient being treated is a
warm-blooded animal and, in particular, mammals including man. The
pharmaceutically acceptable carriers, diluents, adjuvants and
vehicles as well as implant carriers generally refer to inert,
non-toxic solid or liquid fillers, diluents or encapsulating
material not reacting with the active ingredients of the
invention.
[0066] The doses can be single doses or multiple doses over a
period of several days.
[0067] When administering the compound of the present invention, it
is generally formulated in a unit dosage injectable form (solution,
suspension, emulsion). The pharmaceutical formulations suitable for
injection include sterile aqueous solutions or dispersions and
sterile powders for reconstitution into sterile injectable
solutions or dispersions. The carrier can be a solvent or
dispersing medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils.
[0068] Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, can also be used as solvent
systems for compound compositions. Additionally, various additives
which enhance the stability, sterility, and isotonicity of the
compositions, including antimicrobial preservatives, antioxidants,
chelating agents, and buffers, can be added. Prevention of the
action of microorganisms can be ensured by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, and the like. In many cases, it is desirable
to include isotonic agents, for example, sugars, sodium chloride,
and the like. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption,
for example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
would have to be compatible with the compounds.
[0069] Peptides may be polymerized or conjugated to carriers such
as human serum albumen as is well known in the art.
[0070] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various of the other ingredients, as desired.
[0071] A pharmacological formulation of the present invention can
be administered to the patient in an injectable formulation
containing any compatible carrier, such as various vehicle,
additives, and diluents; or the compounds utilized in the present
invention can be administered parenterally to the patient in the
form of slow-release subcutaneous implants or targeted delivery
systems such as monoclonal antibodies, vectored delivery,
iontophoretic, polymer matrices, liposomes, and microspheres.
Examples of delivery systems useful in the present invention
include: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217;
4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439; 196;
and 4,475,196. Many other such implants, delivery systems, and
modules are well known to those skilled in the art.
[0072] The foregoing provides a protocol for using NCM as an
adjuvant to immunize cancer patients against tumor antigens, either
autologous or as defined proteins or peptides.
TABLE-US-00001 The antigen preparations to be used: In Cancer: 1)
PSMA peptides (9) - obtained commercially Prostate 2) MAGE 1 &
3 & MAGE fragments & NY ESO-1 Melanoma, obtained from the
Ludwig Inst. Of Immunol. H&NSCC 3) Papilloma virus E6 & E7
obtained commercially Cervical SCC
[0073] The commercially route of antigen administration is
preferentially the neck because it is accessible and it contains
>30% of the bodies lymph nodes and systemic immunity can be
envisioned to result.
[0074] Low-dose cyclosphosphamide: Low dose CY has been used to
augment cellular immunity and decrease suppression by lymphocytes
in mice and patients with cancer (Berd D., Progress in Clin Biol
Res 288:449-458, 1989; Berd D, et al, Cancer Research 47:3317-3321,
1987) and it has been employed in effective immunotherapy of cancer
patients (Weber J., Medscape Anthology 3:2, 2000; Murphy G P, Tjoa
B A, Simmons S J. The prostate. 38:43-78, 1999; Hadden J W, et al,
Arch Otolaryngol Head Neck Surg. 120:395-403, 1994).
[0075] Zinc: Zinc deficiency is associated with improved cellular
immunity and treatment with zinc is immunorestorative in mice
(Hadden J W., Int'l J Immunopharmacol 17:696-701, 1995; Saha A., et
al. Int'l J Immunopharmacol 17:729-734, 1995).
[0076] A cyclooxygenase inhibitor (COXi) like indomethacin: Cancers
produce prostaglandins and induce host macrophage production of
prostaglandins (Hadden J W. The immunopharmacology of head and neck
cancer: An update. Int'l J Immunopharmacol 11/12:629-644, 1997).
Since prostaglandins are known to be immunosuppressive for T cells,
inhibition of PG synthesis with cyclooxygenase inhibitors is
appropriate.
Recombinant Protein Purification
[0077] Marshak et al, "Strategies for Protein Purification and
Characterization. A laboratory course manual." CSHL Press,
1996.
Dose and Frequency of Antigens 1-1000 .mu.g, preferably 10-500;
form--soluble (partially polymerized or conjugated to carrier, if
necessary)
Schedule: Day 1, Day 12, Day 21
(Pre-Rx). Day 12, Day 21, Day 31
[0078] Site of injection: local injection, ie., neck injections
Expected Responses Tumor reduction [0079] Tumor pathological
changes (reduction, fragmentation, lymphoid infiltration) [0080]
Humoral immunity to antigen (RAI or ELISA) [0081] Cellular immunity
to antigen (intracutaneous skin test in vitro lymphocyte
proliferation, of ELISPOT ASSAY)
[0082] Keep in mind that oligopeptides like PSMA, MAGE fragments,
E6, E7 peptides would be poorly immunogenic even pulsing on to
dendritic cells. Thus effective immunization would not be expected
to occur. Even with effective immunization, tumor regression would
be considered surprising by this method, particularly at a distance
as with prostate and cervix. Regression of metastatic disease is
always a surprising event with immunotherapy. Degree and frequency
of clinical responses are a factor in the effectiveness and thus
the novelty of this approach.
[0083] Diagnostic skin tests are another way to guide us to more
effective immunization. Patients can be pretreated with IRX-2 (NCM)
to induce better responses (increase NCM and PHA skin tests and
lymphocyte counts and reversal of lymph node abnormalities).
This creates an Adjuvant strategy
[0084] Combining immunorestoration and adjuvancy
[0085] Making peptides and proteins immunogenic
[0086] Getting the degree of immune response to effect tumor
regression at a distance.
[0087] It can extend to all forms of tumor antigens and haptens
including peptides and/or carbohydrates
[0088] It can extend to areas of applicability as in AIDS virus
vaccine in HIV+patients; other difficult to manage situations;
renal transplants, aged, etc.
[0089] Patients will be skin tested for one or more tumor peptide
prior to consideration of the protocol, 100 .mu.g of one or more
tumor peptides will be perilymphatically administered in the neck
with NCM using the NCM protocol as discussed below on day 1 and 10
of the NCM series. The combination will be repeated on day 21. In
addition to tumor response and histology, immune reaction to the
peptides will be monitored by repeat skin test or by other means
known in the art.
Example 1
[0090] All steps relating to cell culture are performed under
sterile conditions. General methods of cellular immunology not
described herein are performed as described in general references
for Cellular immunology techniques such as Mishell and Shiigi
(Selected Methods in Cellular Immunology, 1981) and as are known in
the art.
Preparation of Natural Cytokine Mixture (NCM)
[0091] The buffy coat white cells of human blood from multiple
HIV-negative hepatitis virus-negative donors is collected. In an
alternative embodiment, animals could be the cell source for
veterinary uses. The cells from the donors are pooled and layered
on ficoll hypaque gradients (Pharmacia) to yield lymphocytes free
of neutrophils and erythrocytes. Alternative methods could be used
that would result in the same starting lymphocyte population as are
known in the art.
[0092] The lymphocytes are washed and distributed in X vivo-10
media (Whittaker Bioproducts) to surface activated cell culture
flasks for selection of cell subsets MICROCELLECTOR.TM. T-25 Cell
Culture Flasks) in which are immobilized stimulants, i.e. mitogens
like PHA. In one set of experiments, X vivo-15 and X vivo-20 media
were used as indicated. The immobilization process for the
stimulants is as described by the manufacturer for immobilizing
various substances for panning procedures, i.e. separating cells,
in the flasks. Alternatively, the lymphocytes are exposed to
stimulants e.g. PHA for 2-4 hours then washed three times.
[0093] The cells are incubated for 24-48 hours in X vivo-10 media
with 80 .mu.g/ml ciprofloxacin (Miles Lab) at 37.degree. in a
CO.sub.2/air incubator. Alternatively, RPMI 1640 media could be
used (Webb et al. 1973). Generally the HSA is used at 0.1 to 0.5%
(weight by volume). Following incubation the supernatants are
poured off and collected. Human serum albumin (HSA) may be added to
stabilize further the interleukins if HSA-free media is used for
generations. The supernatants are stored at 4.degree. C. to
-70.degree.
Characterization of Supernatants
[0094] The pooled supernatants are characterized by measuring the
cytokine content by bioassay for IL-2 and ELISAs for the remaining
interleukins IL-1-IL-15, CSFs, TNFs, and IFNs. Sterility is tested
by culture in thioglycolate broth and endotoxin measured by limulus
lysate assay as is known in the art.
[0095] Standardization of supernatant for cytokine content:
[0096] Each supernatant is standardized either by concentration or
amount administered so that comparisons can be made.
[0097] Removal of contaminants from supernatant:
[0098] DNA and virus exclusion, if used, employ such techniques as
ultrafiltration, column chromatography, virus retentive filters,
ethanol fractionation, polyethylene glycol/bentonite precipitation,
gamma irradiation, and/or solvent/detergent treatment as has been
used for intravenous gamma globulin and monoclonal antibodies (e.g.
IGIV News Update brochure).
Model
[0099] The model of hydrocortisone induced thymic involution in
aged mice was used unless otherwise indicated (Hadden J W, et al,
Int'l J Immunopharmacol 17:821-828. 1995).
Laboratory Animals
[0100] Female BALB/c (Life Science, St. Petersburg, Fla.) aged
retired breeder mice (8-9 months) whose thymuses had begun to
involute were employed in in vivo tests. Mice were weight matched
and randomly pooled in groups of five. Animals were fed standard
laboratory diets with drinking water ad lib. All mice, with
exception of a control group, were treated intraperitoneally (i.p.)
with hydrocortisone (5 mg/mouse in 0.1 ml 0.9% sodium chloride) for
two consecutive days to induce a chemical thymectomy and reduction
of spleen weight.
[0101] Hydrocortisone-treated adult mice show acute thymic
involution (less than 30% of control) and reduction in spleen size
(less than 80% of control) at two days with progressive recovery to
10 days.
Experimental Design
[0102] Each treatment group had five (5) animals and each
experiment was repeated 2-5 times. Treatment was initiated
intraperitoneally (i.p.) on Day 3 and continued once per day for a
total of five (5) days. Treatment groups were injected with one of
the following in vivo treatments as indicated in the text: [0103]
1. pyrogen free saline (controls); [0104] 2. recombinant
interleukin-1 (rIL-1; 4 ng); [0105] 3. recombinant interleukin-2
(rIL-2; 50 units); [0106] 4. rIL-1+mL-2 (4 ng+50 units,
respectively) [0107] 5. natural cytokine mixture (NCM; 50 units
IL-2 equivalence)
[0108] On day 8, the mice were weighed, sacrificed by cervical
dislocation, and their spleens and thymuses removed and weighed.
The organs were minced, the residual erythrocytes were lysed using
ammonium chloride (Mishell and Shiigi 1981), and the cells
counted.
[0109] The proliferative response of the cells to various
substances was then determined. A sample of cells was prepared for
cell culture at 37.degree. C., 5% CO.sub.2 in RPMI 1640 medium with
5% fetal bovine serum, penicillin (100 U/ml), streptomycin (100
.mu.g/ml) and 2-mercaptoethanol (2.times.10.sup.-5 M). The cells
were plated in 0.2 ml microwell plates in quadruplicate at a
concentration of 1.5.times.10.sup.6/ml and incubated for 72 hours
with one of the following as indicated in the text: [0110] 1.
control diluent (complete RPMI 1640 medium); [0111] 2. rIL-1 (1
ng/ml); [0112] 3. rIL-2 (2 Units/ml); [0113] 4. NCM (2 Units/ml of
IL-2 equivalence) [0114] 5. concanavalin A (Con A; 1.5 .mu.g/ml)
[0115] 6. phytohemagglutinin (PHA; 0.5 .mu.g/ml)
[0116] The culture was terminated to measure DNA synthesis, thus
cell proliferation, with an 18 hours pulse of tritiated thymidine
(3H-Thymidine; New England Nuclear, Boston, Mass.; specific
activity 6.7 Ci/mM), harvested with a multiple automatic sample
harvester and processed for liquid scintillation counting. Marker
studies were also performed as described by, Hadden et al. (1992).
The results were expressed as arithmetic mean of cpm from three
samples for each animal. In order to simplify the representation of
data obtained with different animals, the results with the
different animals were pooled and calculated together and in some
cases are expressed as ratio to control and others as
means+brackets for standard error of the mean (SEM).
Statistical Analysis
[0117] Student's T test was used to analyze data as
appropriate.
[0118] Results:
[0119] The objective was to find a way to stimulate lymphocytes to
produce high levels of interleukin-2 in the absence of serum and in
a way which did not yield significant quantities of PHA in the
supernatant. To do this, the PHA was immobilized on surface
activated cell culture flasks for selection of cell subsets (AIS
MICROCELLECTOR.TM. T-25 plates) as described in the manufacturer's
instructions for "panning" cell separation or pulsed into the cells
followed by washing (pulse technique).
[0120] Media employed in these experiments was X vivo-10
(Whittaker) and is approved for administration to humans by the
U.S. Food and Drug Administration for interleukin-2-lymphokine
activated killer (LAK) cell protocols. Serum-free media capable of
supporting human lymphocyte proliferation like minimal essential
media (MEM) or RPMI-1640 (Sigma) could also be used.
[0121] Initial experiments indicated that PHA (HA-16, Murex
Diagnostics Ltd., Dartford, U.K.) could be immobilized by the
technique described by the manufacturer and that under appropriate
optimal conditions of cell number of 7.5-15.times.10.sup.6/ml, time
of exposure of 24 hours-48 hours, and PHA concentration of 25 or 50
.mu.g/ml a high yield of interleukin-2 in the serum-free
supernatant could be obtained. The yield was superior to the pulse
technique employing brief exposures to PHA (NI) followed by washing
and subsequent culture with ciprofloxacin (NIM) in serum-free media
(Table 1).
TABLE-US-00002 TABLE 1 IL content of supernatant/ml PHA brief
exposure(NI) 2-20 units PHA brief exposure & ciprofloxacin
(NIM) (80 .mu.g/ml) 8-140 units PHA flask immobilization &
ciprofloxacin (80 .mu.g/ml) 100-353 units IL-2 content was measured
in the supernatant using the CTLL IL-2 dependent cell line by the
methods described by Gillis et al. (1978). IL-2 was quantitated in
international units against a known standard containing 640 units
(Pharmacia AB).
[0122] The cell free supernatants from flasks incubated without
cells were tested on human lymphocytes to determine if residual PHA
was present in sufficient quantities to produce a proliferative
response. Any residual PHA greater than 0.01 .mu.g/ml would give
such a response. In the absence of cells, small amounts of PHA were
observed in the supernatant at 40-48 hours; however, when PHA (25
.mu.g/ml) was used for only 24 hours, these levels were negligible.
24 hours incubation was thus considered optimal. A comparison of X
vivo-10, X vivo-15 and X vivo-20 (Whittaker) and MEM in the present
invention was undertaken and shown in FIGS. 1-3. X vivo-10 and X
vivo-15; are approved for administration to humans by the U.S. Food
and Drug Administration for interleukin-2-lymphokine activated
killer (LAK) cell protocols. Generation of NCM was compared in
different media utilizing continuous vs. pulsed exposure to PHA at
1 .mu.g/ml (FIG. 1). The effect of cell concentration was explored
with continuous exposure to PHA at 1 .mu.g/ml (FIG. 2) and PHA at 2
.mu.g/ml (FIG. 3). The optimal combination of these factors was
found to be continuous exposure by immobilization in X-vivo-10 at
cell concentrations of 2.5 or 5.0.times.10.sup.6/ml with PHA at 2
.mu.g/ml or at 5.times.10.sup.6 cells/ml with PHA at 1 .mu.g/ml.
Because the per cell yield is most efficient at 2.5.times.10.sup.6
that concentration with PHA at 2 .mu.g/ml is chosen as the
optimal.
[0123] Preliminary experiments, in tubes rather than flasks, were
performed to determine the parameters for ciprofloxacin and two
other 4-aminoquinolone antibiotics (Norfloxacin and Ofloxacin) to
enhance cytokine production from human leukocytes following
exposure to PHA. Table III shows that 80 .mu.l/ml of each of these
4-aminoquinolone antibiotics enhanced production of IL-1, IL-2,
IL-6, IFN.gamma., TNF.alpha., and G-CSF. IL-8 production was
maximal. IL-3, IL-4, and IL-7 were undetectable under these
circumstances in all supernatants. These results indicate that
under these serum free conditions al 4-aminoquinolones tested at 80
.mu.g/ml enhanced PHA induced cytokine production under serum-free
conditions.
TABLE-US-00003 TABLE II PHA Ciprofloxacin Norfloxacin Ofloxacin
Alone & PHA & PHA & PHA IL-1-.beta. 81 1080 783 810
IL-2 ND 120 32 82 IL-6 1665 >3000 >3000 >3000 IL-8 18000
>18000 >18000 >18000 IFN.gamma. ND 750 210 380 TNF .alpha.
54 1935 1500 4000 GM-CSF 114 4.5 4.5 72 G-CSF 41 555 800 630 Units
for cytokines other than IL2 are pg/ml and for IL2 international
unit/ml.
[0124] It was also determined that a monoclonal antibody, OKT-3,
(Ortho) which induces T lymphocytes to proliferate and produce
interleukins could be employed as a stimulant under these
conditions. Table III shows that OKT-3 induced cytokines similar to
those induced by PHA plus ciprofloxacin with cells incubated in
flasks as set forth in Example 1. IL-3,4,5 and 7 were not detected
with either set of stimulants. OKT-3 produced a small additive
effect for several ILs when joined with PHA and ciprofloxacin
(CIPRO).
TABLE-US-00004 TABLE III CIPRO OKT-3 + CIPRO + PHA + PHA OKT-3
IL-1-.beta. 1080 1530 1125 IL-2 120 340 ND IFN gamma. 750 4660
11280 IL-6 >3000 >3000 1980 IL-8 >18000 >18000
>18000 TNF alpha 1935 2700 2500 GM-CSF 4.5 12 75 G-CSF 555 375
ND Units of interleukins other than IL2 are pg/ml and for IL2
international units/ml. ND not done.
[0125] In order to show the superiority of the NCM over rIL-2 in
vitro, mouse splenocytes and thymocytes were cultured with MEM and
rIL-2 at comparable levels of IL2 as determined by bioassay and DNA
synthesis measured by tritiated thymidine incorporation. NCM
induces greater proliferation of splenocytes (FIG. 4) and
thymocytes (FIG. 5) then rIL-2 based on IL2 content.
[0126] In a series of experiments as set forth in FIGS. 6 and 7,
mice with involuted thymuses were treated in vivo with rIL-1,
rIL-2, combinations of these factors, NCM or saline (controls). The
spleens and thymuses were removed, the cells tested for cell
proliferation responses against the interleukins (IL-1, IL-2), NCM
and the mitogen ConA. The results are expressed as ratio to the
saline treated control. In vivo treatment with rIL-1, rIL-2, and
their combination (rIL-1 and rIL-2) had no significant effect to
increase proliferative responses of splenocytes (FIG. 6) or of
thymocytes (FIG. 7) to in vitro stimulation with IL-1, IL-2, NCM or
ConA. NCM treatment in vivo augmented significantly both
splenocytes and thymocytes to all four stimuli. These results are
consistent with an enhanced sensitivity of these cells to
stimulation and/or an increase in the number of responsive
cells.
[0127] FIGS. 8 and 9 demonstrate the effect of NCM treatment in
vivo on splenocyte and thymocyte markers. Non-mature T-cells are
indicated by--and may represent T lymphocyte precursors
particularly in the thymus. NCM increased proportionately this
population in spleen and thymus Immature T-cells are indicated by
++ and this population is proportionately decreased in thymus by
NCM treatment. Mature T-cells are indicated by CD4+ and CD8+. NCM
increased the proportions of mature T-cells in thymus and their
number in spleen. These results are consistent with an effect of
NCM to increase T cell precursors and to promote their development
to mature T cells in thymus.
[0128] FIGS. 10 and 11 demonstrate the splenocyte and thymocyte
responses in vitro to media (RPMI), rIL-1 (IL1), rIL-2 (IL.sub.2),
or NCM after treatment in vivo with control media or NCM in the
hydrocortisone model. The mice were treated as described
hereinabove. These data demonstrate that NCM augments background
splenocyte responses, splenocyte responses to IL-1 and IL-2, but
not NCM and background thymocyte responses and thymocyte responses
to IL-1, IL-2, and NCM.
[0129] FIGS. 12 and 13 demonstrate the splenocyte and thymocyte
responses in vitro to ConA and PHA after treatment in vivo with
control media or NCM. The mice were treated as described
hereinabove.
[0130] The in vitro studies demonstrate the superiority of NCM over
rIL-2 at equivalent doses in sensitizing splenocytes and thymocytes
to proliferation signals. The effects on thymocytes reflect
promotion of differentiation as well. The NCM composition, but not
rIL-1, rIL-2, nor their combination, potently promotes in vivo T
lymphocyte function (IL responses) and development (mitogen
responses and cell markers) which is therapeutically relevant in
any therapeutic measures requiring stimulation of the immune system
or restoring even partial functioning of a damaged or defective
immune system. For example chemotherapeutic agents can damage
cells, including T lymphocytes, involved in the immune response.
The present invention by stimulating the T lymphocyte functioning
and development can restore, either partially or entirely, this
feature of the immune system if damaged.
Example 2
[0131] There is shown that local perilymphatic injections in the
neck having NCM plus low dose cyclophosphamide, indomethacin, and
zinc and induced clinical regressions in a high percentage of
patients with squamous cell head and neck cancer (H&NSCC)
(Hadden J W, et al., Arch Otolaryngol Head Neck Surg. 120:395-403,
1994; Meneses A, et al., Arch Pathol Lab Med 122:447-454, 1998;
Barrera J, et al., Arch Otolaryngol Head Neck Surg 126:345-351,
2000) with evidence of improved, recurrence-free survival. Overall,
including minor response (25%-50%) tumor shrinkage and reduction of
tumor in pathological specimens, over 90% responded and the
majority had greater than 50% tumor reduction.
[0132] These responses were speculated to be mediated by immune
regression since both B and T lymphocytes were observed
infiltrating the tumors. The therapy was not associated with
significant toxicity.
[0133] Several unpublished observations serve to document this
speculation and lead to the present invention.
[0134] 1) Treatment of lymphocytopenic cancer patients with the
combination of NCM has resulted in marked lymphocyte mobilization;
where analyzed, these patients showed increases in CD45RA positive
T-cells (i.e., naive T cells (Table IV).
[0135] 2) Intratumoral or peritumoral injection of NCM in patients
with H&NSCC resulted in either reversing immunotherapy-induced
tumor regression or in progression of the tumor. The tumor is thus
not the site of immunization.
[0136] 3) Analysis of regional lymph nodes revealed unpublished
data which indicate that the regional lymph node is the site of
immunization to postulated tumor antigens (see FIGS. 14-18).
[0137] 4) None of these patients treated with NCM developed
metastasis expected in 15% clinically and up to 50% pathologically,
indicating systemic immunity rather than merely local immunity had
been induced.
[0138] 5) Patients were pretested with a skin test to 0.1 ml of NCM
prior to treatment. More than 90% of those with a positive skin
test (>0.3 mm at 24 hours) had robust clinical and pathological
response. Patients with negative skin tests had weak or no
response. Thus skin testing appears to select good responders.
[0139] Major increases were observed in T lymphocyte counts
(CD.sub.2) 752.fwdarw.1020 in these T lymphocytopoenic patients (T
cell counts 752 vs. normal=1600). Importantly there was a
corresponding increase in "naive" CD45RA positive T cells
(532.fwdarw.782). As mentioned previously these increases are
generally not thought to occur in adults particularly with a
pharmacological therapy like NCM. These cells presumably are recent
thymic emigres and could be considered a major new capacity for
responding to new antigens like tumor antigens. The preexisting
CD45RA positive cells were not responding to the tumor antigens and
may well be incapable of doing so due to the tumor-induced immune
suppression (anergy).
[0140] The literature (Hadden J W, Int'l J Immunopharmacol
11/12:629-644, 1997; Hadden J W, Int'l J Immunopharmacol 21:79-101,
1999) indicates that for both SCC and adenocarcinomas, the two
major types of cancer, regional lymph nodes reflect abnormalities
related to the tumor, including sinus histocytosis, lymphoid
depletion and often the presence of tumor-associated lymphocytes
capable of reacting to tumor cells (with IL-2). With metastasis
lymphoid depletion and depressed function occur. An unpublished
analysis of uninvolved cervical lymph nodes 10H&NSCC and 10
controls showed reduction in average size and an increase in sinus
histocytosis associated with H&NSCC (FIGS. 14-17).
TABLE-US-00005 TABLE IV NA VE T CELL PAN T CELL PATIENT MARKER
MARKER # PRE POST INCREASE PRE POST INCREASE 1 479 778 +299 704
1171 +467 2 938 1309 +371 1364 1249 -115 3 98 139 +41 146 178 +32 4
341 438 +97 655 590 -65 5 567 652 +97 453 643 +190 6 658 1058 +400
1118 1714 +569 7 642 1101 +459 822 1601 +779 MEAN 532 782 +250 752
1020 +269
[0141] Following treatment with one cycle of the NCM (IRX-2)
protocol (Hadden J W, et al., Arch Otolaryngol Head Neck Surg.
120:395-403, 1994; Meneses A, et al., Arch Pathol Lab Med
122:447-454, 1998; Barrera J, et al., Arch Otolaryngol Head Neck
Surg 126:345-351, 2000), the uninvolved cervical lymph nodes showed
the changes indicated in FIGS. 14-17). Compared to the regional
lymph nodes of patients with H&NSCC not treated with NCM, these
nodes showed a significant increase in size, T cell area and
density, and decreases in number of germinal centers and sinus
histocytosis and congestion. The lymph nodes of treated patients
were all stimulated and were larger than control nodes with
increased T cell area and density. These nodes were thus not only
restored to normal but showed evidence of T cell predominance, a
known positive conelate with survival in H&NSCC (Hadden J W.
Int'l J Immunopharmacol 11/12:629-644, 1997).
[0142] Importantly, when the lymph node changes related to B and T
cell areas were correlated with the changes in their tumors
reflecting T and B cell infiltration, a high degree of correlation
was obtained for T cells (p.<0.01) and B cells (<0.01) and
overall lymphoid presence (p.<0.001). (FIG. 18) In turn, these
changes conelate with tumor reduction by pathological and clinical
criteria. These findings indicate that the tumor reactions are
directly and positively correlated with lymph node changes and that
the tumor reaction reflects the lymph node changes as the dependent
variable. These findings, taken into conjunction with knowledge
about how the immune system works in general (Roitt I, Brostoff J,
Male D. Immunology, JB Lippincott Co, Phila, Pa., 1989), and
following tumor transfection with a cytokine gene (Maass G, et al,
Proc Natl Acad Sci USA, 1995, 92:5540-5542), indicate that the NCM
protocol immunizes these patients to yet unidentified tumor
antigens at the level of the lymph nodes. No one has previously
presented evidence for lymph node changes reflecting immunization
with autologous tumor antigens. These data convince the applicant
that this constitutes a good starting point for trying to induce
immunization with previously ineffective or poorly effective tumor
antigens in an effect to yield regression of distant
metastases.
Example 3
[0143] Two patients were treated with lymphoma of the head and
neck.
[0144] The patients included were those with head and neck cancer
who agreed to participate in the protocol. The following scheme was
followed:
[0145] Before treatment, the patients were skin-tested with NCM 0.1
ml subcutaneously in the forearm, the region was marked, and 24
hrs. later the test was read. The test was considered positive if
the induction and erythema was equal or larger than 3 mm.
[0146] Each Cycle of NCM was for 21 Days as Follows: [0147] Day 1:
Low dose cyclophosphamide (300 mg/m.sup.21 [0148] Day 1-21:
Indomethacin 25 mg p.o. 3 times daily [0149] Zinc sulfate 50 mg
p.c. once daily [0150] Day 2: NCM 200 units five as 1 ml
subcutaneously perilymphatic in the neck.
Case #1
[0151] The patient was a 23-year-old male who presented on with a
prior history of three months of the presence of a tumor on the
left submaxillary region, with no other symptoms. In the emergency
room, he was found to have lymph adenopathy of the left
submaxillary triangle of approximately 6.5 cm in diameter of a hard
consistency, partially fixed at deep levels. The rest of the
physical exam was normal. The incisional biopsy showed Hodgkin's
lymphoma. The lesion was staged ECIIA. A one-cycle treatment of NCM
was given, obtaining a minor response, as the adenopathy reduced in
size by 1 cm in diameter. The biopsy report obtained after NCM
treatment showed 60% of the lesion showed normal lymphocytic
infiltration, and the rest of the neoplasia (40%) showed necrosis.
No viable tumor cells were found.
[0152] Following this, the patient received radiation treatment in
the neck of 3600 rads. The patient is currently free of
disease.
Case #2
[0153] The patient is an 82-year-old male, who presented with a
two-month history of a painful mid-neck tumor mass, as well as a 10
kg loss of weight. On physical exam, the patient presented with
tumor on the right palatine tonsil, which was enlarged to
approximately 4.times.3 cm, with an ulcer in the center of the
tonsil. On the neck, a right submaxillary lymph node measured
approximately 2.times.2 cm and a lymph node mass at level II and
III of approximately 5.times.5 cm. The rest of the exam was normal.
The incisional biopsy of the tonsil and one of the necks lymph
nodes demonstrated defined non-Hodgkin's lymphoma mixed, of
intermediate grade.
[0154] The patient was subjected to two cycles of NCM at the end of
which a 1 cm reduction in the diameter of the tonsil and neck
adenopathy was observed. The pathological report post-NCM treatment
showed live tumor 20%, fragmented and necrotic 30% and normal
lymphocyte infiltration 50%.
[0155] The patient was given chemotherapy (CHOP) for 6 cycles and
later external radiotherapy (RT) at a total dose of 4600 rads. He
recurred at eight months post RT with adenomegaly at the occipital
level. The patient died three months later with evidence of neck
disease.
Example 4
[0156] Ten patients with untreated early stage cervical cancer,
clinically staged IB1, IB2 and IIA were treated with local,
perilymphatic injections NCM as IRX-2 (10 daily injections)
followed by radical hysterectomy at day 21. One day before starting
IRX 2, patients received a single IV dose of cyclophosphamide at
300 mg/m.sup.2. oral indomethacin or ibuprofen and zinc sulfate
were administered from days 1 to 21. The clinical and pathological
response, toxicity and disease-free survival were evaluated.
[0157] All patients completed NCM treatment and were evaluated for
response and toxicity. Clinical response was seen in 50% of
patients (3 partial response (PR), 2 minor response (MR)
(>25%<50% reduction)). Seven patients underwent surgery,
Pathologically tumor reduction associated with tumor fragmentation
was found in five cases. There was a rather heterogeneous pattern
of cell types infiltrating the tumor which included lymphocytes,
plasma cells, neutrophils, macrophages and eosinophils. Treatment
was well-tolerated except for mild pain and minor bleeding during
injection and gastric intolerance to indomethacin. At a 24 months
of follow-up, nine patients are disease-free.
[0158] This previously unpublished study shows that peritumoral NCM
induces immune-mediated tumor response in early stage untreated
cervical carcinoma.
Example 5
[0159] Two patients with liver metastasis from primary
hepatocellular carcinoma were treated with intrasplenic NCM (1 or 3
injections). The protocol was otherwise as previously described for
the H&NSCC, cervical, or lymphoma cases. One patient with
advanced hepatocellular carcinoma had a partial response confirmed
by tomography, no histology is available. The other had a partial
response confirmed by surgery. Histological exam showed tumor
reduction, fragmentation, and lymphorid infiltration.
Example 6
[0160] Four patients with squamous cell carcinoma of the penis
(human papiloma virus associated) were treated with the NCM
protocol as described above; all four had partial responses
clinically and the surgical specimen showed tumor reduction and
fragmentation and lymphoid infiltration characteristic of the
H&NSCC cancer patients.
Example 7
[0161] Mice were immunized with PMSA peptides conjugated to
ovalbumen 100 .mu.g at 3 sites (day 1, 14, and 21) with alum (1:1
Vol) as adjuvant (5@) or NCM (20 units IL2 equivalence) (5@)
animals were skin tested at day 28 with ovalbumen (100 .mu.g) (2@)
or peptides (100 .mu.g) (3 @). Two animals treated with ovalbumen
plus NCM without peptides responded to ovalbumen with positive skin
tests. Two animals treated with ovalbumen plus alum did not
respond. 2 of 3 animals treated with ovalbumen plus peptides and
NCM responded. None of the animals treated with ovalbumen plus
peptides and alum responded. Thus NCM was a superior adjuvant to
alum for both tumor peptides and ovalbumen as antigens.
[0162] Throughout this application, various publications, including
U.S. patents, are referenced by author and year and patents by
number. Full citations for the publications are listed below. The
disclosures of these publications and patents in their entireties
are hereby incorporated by reference into this application in order
to more fully describe the state of the art to which this invention
pertains.
[0163] The invention has been described in an illustrative manner,
and it is to be understood that the terminology which has been used
is intended to be in the nature of words of description rather than
of limitation.
[0164] Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the described
invention, the invention can be practiced otherwise than as
specifically described.
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Sequence CWU 1
1
219PRTArtificial SequenceMAGE-1 protein 1Glu Ala Asp Pro Thr Gly
His Ser Tyr 1 5 29PRTArtificial SequenceMAGE-3 protein 2Glu Val Asp
Pro Ile Gly His Leu Tyr 1 5
* * * * *