U.S. patent application number 13/653152 was filed with the patent office on 2013-06-27 for immunotherapy for reversing immune suppression.
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 | 20130164255 13/653152 |
Document ID | / |
Family ID | 23350824 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164255 |
Kind Code |
A1 |
Hadden; John W. |
June 27, 2013 |
IMMUNOTHERAPY FOR REVERSING IMMUNE SUPPRESSION
Abstract
A method for overcoming 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; preferably the natural cytokine
mixture is administered in combination with thymosin
.alpha..sub.1.
Inventors: |
Hadden; John W.; (Cold
Spring Harbor, NY) |
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Applicant: |
Name |
City |
State |
Country |
Type |
IRX Therapeutics, Inc.; |
New York |
NY |
US |
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|
Assignee: |
IRX Therapeutics, Inc.
New York
NY
|
Family ID: |
23350824 |
Appl. No.: |
13/653152 |
Filed: |
October 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12748622 |
Mar 29, 2010 |
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13653152 |
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10280627 |
Oct 26, 2002 |
7731945 |
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12748622 |
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60344509 |
Oct 26, 2001 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 2039/5158 20130101;
A61K 2039/57 20130101; A61K 38/19 20130101; A61P 7/00 20180101;
A61P 37/04 20180101; A61K 2039/5154 20130101; A61P 41/00 20180101;
A61P 43/00 20180101; A61K 39/39 20130101; A61K 39/0011 20130101;
A61P 31/04 20180101; A61K 38/2013 20130101; A61K 2039/55522
20130101; A61P 31/18 20180101; A61P 37/06 20180101; A61K 38/2292
20130101; A61K 2039/55527 20130101; A61P 35/00 20180101; A61K
38/2292 20130101; A61K 2300/00 20130101; A61K 38/2013 20130101;
A61K 2300/00 20130101; A61K 38/19 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/00 20060101 A61K039/00 |
Claims
1-32. (canceled)
33. A method for inducing an immune response in a subject, the
method comprising: a. administering an effective amount of a
natural cytokine mixture (NCM) to the subject, wherein the NCM
comprises Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6
(IL-6), Interleukin-8 (IL-8), Interleukin-12 (IL-12),
Interferon-.gamma. (IFN-.gamma.), Tumor necrosis factor-.alpha.
(TNF-.alpha.), Granulocyte colony-stimulating factor (G-CSF), and
Granulocyte macrophage colony-stimulating factor (GM-CSF); b.
administering an effective amount of a thymic peptide to the
subject; and c. administering at least one exogenous antigen to the
subject, wherein an immune response to the at least one exogenous
antigen is induced in the patient.
34. The method of claim 33, wherein the thymic peptide is thymosin
.alpha.1.
35. The method of claim 33, wherein the IL-2 in the NCM is present
in an amount of 100-353 units/mL.
36. The method of claim 33, wherein the administering is via
injection.
37. The method of claim 36, wherein the injection is a
perilymphatic injection.
38. The method of claim 37, wherein the perilymphatic injection is
a bilateral or contralateral injection.
39. The method of claim 33, wherein the NCM and thymic peptide
antigen are administered at the same time.
40. The method of claim 33, wherein the method further comprises:
a. administering an effective amount of a cyclophosphamide to the
subject.
41. The method of claim 33, wherein the method further comprises:
a. administering an effective amount of a non-steroidal
anti-inflammatory drug (NSAID) to the subject.
42. The method of claim 41, wherein the NSAID is indomethacin.
43. The method of claim 33, wherein the method further comprises:
a. administering an effective amount of a cyclophosphamide to the
subject; and b. administering an effective amount of a
non-steroidal anti-inflammatory drug (NSAID) to the subject.
44. The method of claim 43, wherein the NSAID is indomethacin.
45. The method of claim 43, wherein the method further comprises:
a. administering an effective amount of zinc to the subject.
46. The method of claim 33, wherein the subject has a cellular
immune deficiency.
47. The method of claim 46, wherein the subject has cancer.
48. The method of claim 47, wherein the at least one exogenous
antigen is at least one exogenous tumor antigen.
49. The method of claim 48, wherein the at least one exogenous
tumor antigen is a prostate specific membrane antigen (PSMA)
peptide.
50. The method of claim 33, wherein the at least one exogenous
antigen is at least one exogenous tumor, viral, or fungal antigen.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.120 of U.S. application Ser. No. 12/748,622, entitled
"IMMUNOTHERAPY FOR REVERSING IMMUNE SUPPRESSION" filed on Mar. 29,
2010, which is herein incorporated by reference in its entirety.
Application Ser. No. 12/748,622 claims the benefit under 35 U.S.C.
.sctn.120 of U.S. application Ser. No. 10/280,627, entitled
"IMMUNOTHERAPY FOR REVERSING IMMUNE SUPPRESSION" filed on Oct. 26,
2002, which is herein incorporated by reference in its entirety.
Application Ser. No. 10/280,627 claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/344,509,
entitled "IMMUNOTHERAPY FOR REVERSING IMMUNE SUPPRESSION" filed on
Oct. 26, 2001, which is herein incorporated by reference in its
entirety.
BACKGROUND OF INVENTION
[0002] 1. Technical Field
[0003] The present invention relates to vaccine therapy for cancer
patients and patients having persistent lesions, such as
infections. More specifically, the present invention relates to a
vaccine immunotherapy that immunizes patients, having immune
suppression, to both endogenous and exogenous tumor peptides or
proteins, as well as those derived from other persistent
lesions.
[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 cancers has yielded major
clinical responses in a low percentage of patients. A major
response means greater than 50% tumor reduction.
[0008] Gene therapy also has been attempted using an adenovirus
construct as an expression vector for genes expressing Papilloma
virus. Peptide 16 has been used for immunization for 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 also has 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.
[0011] Several U.S. 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 cancer, 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 versus treatment (generally unsuccessful) with tumor
antigens or peptides (generally unsuccessful) in immunosuppressed
patients (generally unsuccessful). The first is easy and 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 half-life is
short, they are often nonmutated self-antigens to which the patient
is immunologically tolerant, and gaining a response is tantamount
to inducing autoimmunity.
[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 to the cancer or other persistent
lesions.
BRIEF DESCRIPTION OF DRAWINGS
[0017] Other advantages of the present invention are readily
appreciated as the same becomes better understood by reference to
the following detailed descriptions 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
IL-2 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 treated with IL-1, IL-2
or IL combinations, NCM, or saline;
[0024] FIG. 7 is a graph also showing a comparison of treatment
with recombinant IL-1, IL-2, IL-1 plus IL-2, 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 and neck cancer (H&NSCC);
[0032] FIG. 15A and FIG. 15B show 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 show two bar graphs, FIG. 16A showing
B cell area and FIG. 16B showing follicles in the three treatment
groups;
[0034] FIG. 17A and FIG. 17B show two bar graphs, FIG. 17A showing
other cells and FIG. 17B showing sinus histiocytosis in the three
treatment groups;
[0035] FIG. 18 is a graph showing node B and T and cancer B and T
fit plot:
[0036] FIG. 19 shows increases of lymphocyte populations in the
blood of IRX-3 treated patients induced by a 10-day treatment of
NCM plus thymosin .alpha.1;
[0037] FIG. 20 shows increases in naive T cells (CD45RA+) and
memory T cells in the blood of IRX-3 treated patients induced by a
10-day treatment of NCM plus thymosin .alpha.1;
[0038] FIG. 21 is a bar graph of thymocyte response in vitro to
media (open bar). rIL-1 (closed bar), rIL-2 (cross-hatched), and
NCM (diagonal lines) after treatment in vivo with saline, thymosin
.alpha.1 (5 .mu.g/animal/day), NCM (50 units IL-2 equivalence) and
thymosin .alpha.1 (5 .mu.g/animal/day)+NCM (50 units IL-2
equivalence); and
[0039] FIG. 22 is a bar graph of splenocyte responses in vitro to
media (open bar), rIL-1 (closed bar), rIL-2 (cross-hatched) and NCM
(diagonal lines) after treatment in vivo with saline, thymosin
.alpha.1, NCM and thymosin .alpha.1+NCM as in FIG. 21.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0040] 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.
[0041] T lymphocytopenia (low T cell levels in blood) is a
diagnostic characteristic of cellular immune deficiency; impaired
function of existing lymphocytes 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 IL-2 has been tried in AIDS with some effect by much
toxicity.
[0042] 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 (CD45RO) (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., CD45RA. NCM therapy (plus thymosin .alpha.1) results
in the production of these new T cells as well as expanding
pre-existing memory T cells.
[0043] More specifically, the present invention utilizes new
discoveries relating to immunization to provide an immune response
to antigens that is either endogenously or exogenously
administered. Such antigens in the past have been believed to be
immunogenic while others used in the present invention have been
previously thought to be nonimmunogenic. Examples of such antigens
are EADPTGHSY (melanoma) from MAGE-1 protein, EVDPIGHLY (lung
carcinoma) from MAGE-3, EVDPIGHLY (lung carcinoma) from MAGE-3, and
many others (see, Bellone, et al., Immunology Today, 20(10):457-462
(1999)).
[0044] 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 severe 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 nonspecific 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 IL-1, IL-2, IL-6, IL-8,
IL-10, IL-12, .delta.IFN, TNF.alpha., and both G- and GM-CSF plus
thymosin .alpha.1. The amount and proportions of these constituents
are detailed below. Preferably, about 150-600 units of IL-2 are
contained in the NCM and 1.6 mg of thymosin .alpha.1.
[0045] Preferably, the NCM with thymosin .alpha.1 is injected
around lymphatics that drain into lymph nodes regional to a lesion,
such as tumor or other persistent lesions being treated.
Perilymphatic administration into the lymphatics that 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 10-day
injection scheme is optimal and a 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.
[0046] 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 nonsteroidal
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. and Vioxx.RTM., 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 multivitamins are useful agents to
help restore T cell immunity. Treatment with contrasuppression and
zinc without the NCM is ineffective.
[0047] In summary, the minimum regimen is perilymphatic treatment
with the NCM plus thymosin .alpha.1 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 is
zinc 50 to 75 mg. A standard multivitamin can be administered. The
zinc can be an available gluconate.
[0048] In order to maximize clinical response, and for the greatest
increase in survival rate, the degree and type of lymphocyte
infiltration is important. 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 associated with a robust
clinical response. Tumor reduction and fragmentation in the
histological samples are preferred in reflecting a good
response.
[0049] Lymph node changes key to good response involve at least
five 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. The present data indicate that sinus
histiocytosis (SH) is an accumulation of nonactivated mature
dendritic cells (CD68+CD83+DC) that 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 that lead to cytotoxin T and B cells. Reversal of
SH and the activation of DC is a key to the invention.
[0050] Thus, the present invention provides for unblocking
immunization at a regional lymph node by promoting maturation and
activation of 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, as discussed in greater detail below.
Additionally, induction of mature dendritic cells is required.
Finally, mobilization of peripheral blood T lymphocytes in T
lymphocytopenic 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,
293:245-248 (2001)).
[0051] 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 herein, increases in CD45RA positive naive
uncommitted T cells have been found. 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 herein, is
increased survival due to immunologic memory (see, Sprent, et
al.).
[0052] It is predicted logically that exogenously provided
synthetic or extracted tumor peptides (see, Bellone, et al.) can be
delivered into the preprimed or coprimed regional or distal lymph
node and yield tumor antigen specific T cells, with or without B
cells. Examples are set forth below. In view of the above, it can
be concluded that the action of NCM and thymosin .alpha.1 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 matured, activated
dendritic cells, 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 are
considered key factors 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.
[0053] Referring more specifically to the protocol and medicant
delivered in accordance with the present invention, the invention
utilizes the natural cytokine mixture plus thymosin .alpha.1 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
plus thymosin .alpha.1 and a tumor-associated antigen, the NCM plus
thymosin .alpha.1 acting as an adjuvant to produce the immune
response.
[0054] 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 be more likely so since
they are complete.
[0055] In the preferred embodiment, the composition of the present
invention involves the administration of the NCM plus thymosin
.alpha.1 plus a tumor-associated or specific antigen, as defined
below with low doses of cyclophosphamide, a cyclooxygenase
inhibitor, and other similar compounds that have been shown to
further increase the effects of the composition of the present
invention. The NCM and thymosin .alpha.1 combination is an adjuvant
creating an immune response to antigens not otherwise found to be
effectively antigenic. Moreover, this adjuvant effect has been
accomplished in patients who are severely immune deficient.
[0056] 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 MFN or IL-2, or T
cell infiltration into the tumor as described herein.
[0057] By "tumor associated antigen," it is meant an analogous
protein or peptide (which were previously shown to work by pulsing
of dendritic cells 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, 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).
[0058] NCM, a nonrecombinant 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 and with the continuous or pulsed presence of a mitogen,
which in the preferred embodiment, is PHA.
[0059] Pooled lymphocytes, generally from the buffy coat, free of
neutrophils and erythrocytes from HIV-negative, hepatitis
virus-negative multiple donors are used to produce a mixed
lymphocyte response (MLR). Further, in a preferred embodiment, up
to 50 donors are used each time to produce the mixture to ensure
that the MLR response is constant for each preparation and to even
out variation.
[0060] In an alternative embodiment, autologous lymphocytes are
used to generate the NCM. In these cases, the patient does have to
be virus-free. Further, if autologous lymphocytes are used, they
can be returned to the patient as needed. In an alternative
embodiment, animals can be the cell source for veterinary uses.
[0061] The lymphocytes are cultured in the presence of immobilized
mitogens in a tissue culture vessel. In a preferred embodiment, the
mitogen is 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. However, other
methods of immobilizing mitogens on the surface of the culture
vessel such as methods incorporating other "panning" techniques or
coupling to sepharose 4B beads could be used as are well known in
the art of cell isolation. The use of immobilizing cells for
selection is well known in the art.
[0062] The mitogens are generally selected from lectins and
monoclonal antibodies that stimulate lymphocytes to produce
cytokines. In a preferred embodiment, phytohemagglutinin (PHA) or
OKT3 (Orthoclone.RTM., Ortho Pharmaceuticals) are used. Other
lectins such as concanavalin A (ConA) or pokeweed mitogen that
stimulate B cells can be used. Monoclonal antibodies to T cell
receptors such as CD2, CD28, CD45 can be used as mitogens.
Anti-CD28 and CD45 antibodies are reported to be hyperproducers of
IL-2 (Deans, et al., (1989); June, et al., (1989). Further,
antilymphocyte globulin (ALG) has mitogenic activity for T cells.
In addition, combinations of mitogens can be used to activate a
combination of lymphocyte subpopulations. PHA is used in the
preferred embodiment and is coated at a starting concentration of
about 25 .mu.g/ml.
[0063] The lymphocytes are incubated for 24 to 28 hours in a
serum-free media with continuous exposure to the mitogen, i.e., no
washings. In a preferred embodiment, the media is either X vivo-10
or X vivo-15 media (Whittaker). This is a serum-free and
FDA-approved media for IL-2/LAK infusions in patients as set forth
in the manufacturer's brochure. Serum-free media capable of
supporting human lymphocyte proliferation, such as RPMI-1640
(Sigma), can be also used.
[0064] The media also contains a 4-aminoquinolone antibiotic. In
the preferred embodiment, the antibiotic is Ciprofloxacin. The
antibiotic is used to maintain sterility and to hyperproduce
lymphokines. Ciprofloxacin and related antibiotics have been
reported to increase IL-2 and other cytokines in the presence of
the soluble mitogen and serum (Riesenbeck, et al. (1994)). They
have not been reported to be effective in the absence of serum.
Ciprofloxacin is used in the preferred embodiment at a
concentration of from about 20 to about 200 .mu.g/ml and more
preferably, at a concentration of about 80 .mu.g/ml.
[0065] The supernatant is removed and is the source of the NCM of
the present invention. The supernatant is free of the mitogen as
shown in Example 1. In animal and initial human studies. it does
not have to be concentrated.
[0066] Human serum albumin (HSA) can be added to stabilize the NCM
in the supernatant. HSA is used instead of serum albumin from a
nonhuman source because HSA has been approved by the FDA for human
use.
[0067] A cytokine profile of the supernatant is established
utilizing the following assays. The interleukin content of the
supernatant is confirmed by bioassay for IL-2 and by ELISA's for
other interleukins, CSFs, TNFs, and IFNs. Sterility is tested and
endotoxin measured by limulus lysate assay. Specifically, the
following assays and kits are used in a preferred embodiment:
INF-.gamma. ELISA (ENDOGEN), IL-1, IL-2, IL-3, IL-4, IL-6, IL-7,
IL-8, GM-CSF, G-CSF, and TNF-.alpha. ELISAs (R&D Systems). The
IL-2 bioassay of Gillis, et al., 1978, is expressed as units/ml
compared to a known standard of IL-2 (Schiapparelli Biosystems,
Inc., Fairfield, N.J.).
[0068] In the preferred embodiment, wherein PHA is used as the
mitogen, the cytokine profile for the supernatant has a profile
of:
TABLE-US-00001 CYTOKINE AMOUNT IL-1 10-2000 pg/ml IL-2 100-500
units/ml IL-6 250-10,000 pg/ml IL-8 12,000-100,000 pg/ml IL-12
100-10,000 pg/ml IFN-.gamma. 50-15,000 pg/ml TNF-.alpha. 50-15,000
pg/ml CSF-G 50-1,500 pg/ml CSF-GM 10-1,500 pg/ml IL-3/IL-4/IL-7
Trace Amounts
[0069] Immobilization of the mitogen produces a higher yield of NCM
than does pulse techniques. For example, production of interleukins
by a pulse technique with PHA in serum-free media yielded IL-2 at
0-20 units/ml media (U.S. Pat. Nos. 4,390,623 and 4,464,355).
However, the present method allows an increased production with a
pulse technique by adding a 4-aminoquinolone antibiotic to the
serum-free media to hyperinduce interleukin and yielded about 8-140
units/ml of IL-2. As predicted by the animal studies, this
preparation, characterized as a natural interleukin mixture (NIM),
at 200 units IL-2/dose, increased T lymphocyte counts in blood of
lymphopenic patients with head and neck cancer (Hadden, et al.
(1994)). This result has not been reported at doses greater than
5,000 times the amount of IL-2 in NCM. Thus, it is important to
note that the dose IL-2 equivalent for NCM is used as an index of
its potency and is not meant to imply that the total biological
activity of NCM is that of only IL-2.
[0070] In the preferred embodiment of the present invention,
utilizing continuous exposure to the mitogen by immobilization and
the presence of a 4-aminoquinolone antibiotic, the NCM that is
generated generally contains IL-2 at 100-353 units/ml (an index of
the potency of the preparation). In the less preferred embodiment,
the invention can be practiced with the continuous presence of
4-aminoquinolone antibiotic and a pulsed presence of the mitogen,
producing NIM. This combination produces a level of cytokines
greater than the other prior art methods with a pulsed mitogen
only, but does not produce the levels seen with the preferred
embodiment of the present invention.
[0071] The invention can be also practiced with a natural
nonrecombinant interleukin mixture (NIM) that is produced with the
continuous presence of 4-aminoquionlone antibiotic but with only a
pulsed presence of a mitogen such as PHA. Other immunomodulating
natural nonrecombinant cytokine preparations such as an NIM
preparation, also can be used in the present invention. The various
preparations are compared by IL-2 content, and the dosage is
referred to as IL-2 equivalents.
[0072] Thymic peptides are used in the present invention
coadministered with the immunomodulator-cytokine preparations.
Thymosin .alpha.1 (T-.alpha.1), or its analogs and fragments, is
used in the preferred embodiment of the present invention. In
addition, other thymic peptides, such as thymosin .alpha.11 and
prothymosin and their analogs can be used. Thymic peptides,
analogs, and fragments that contain the thymosin .alpha.1 sequence
can be also used.
[0073] An analog will be generally at least 70% homologous over any
portion that is functionally relevant. In more preferred
embodiments, the homology will be at least 80% and can approach 95%
homology to the thymic peptide, particularly the thymosin .alpha.1
sequence. The amino acid sequence of an analog may differ from that
of the thymic peptide when at least one residue is deleted,
inserted, or substituted. Differences in glycosylation can provide
analogs. Analogs as set forth in U.S. Pat. Nos. 4,116,951;
4,353,821; 4,466,918; 4,470,926; 4,612,365; and 4,910,296 are
examples of such analogs and can be used in the present
invention.
[0074] A partially characterized NCM has been previously shown to
be effective in promoting T cell development and function in aged,
immunosuppressed mice. Thymosin .alpha.1 also protected T cell
development and function in aged immunosuppressed mice and the
combination of NCM plus thymosin .alpha.1 was dramatic in its
action to produce new T cells in the spleen (U.S. Pat. No.
5,698,194). 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 CD45RA. This is one of the first demonstrations that adult
humans can generate naive T cells. It is described in this
application that NCM plus thymosin cd produces increased "naive" T
cells in irradiated patients resistant to NCM treatment. Previous
references (Mackall, et al., New Eng J Medicine 332:143-149 (1995);
and a review by Mackall, Stem Cells 18:10-18 (2000)) discuss the
inability to generate new T cells in adults but not children, and
discuss 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 an increase in lymphocyte 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.
[0075] 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., Modern Pathol 3(6):702-708 (1990)).
Untreatable recurrences occurred and the response was termed
nonspecific 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., J Immunopharmacol,
11/12:629-644 (1997)).
[0076] The method of the present invention involves using NCM plus
thymosin .alpha.1 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)), 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% nonresponders where
Applicant has observed only 20%.
[0077] Applicant has 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 Applicant 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 that 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 (DeLaugh, et al., Curr Opin
in Immunol 12:583-588 (2000); Buchereau, et al., Ann Rev of Immunol
18:767-811 (2000); Albert, et al., Nature 392:86-89 (1998)).
[0078] In brief, in order for tumor antigens to be properly
antigenic, they must arrive from an apoptotic rather than a
necrotic tumor cell (Albert reference in Nature). They need to be
captured by immature dendritic cells that have the morphology of
large histocytes. These immature dendritic cells process
(endocytosis, phagocytosis and digestion) and evolve into mature
dendritic cells that 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
costimulatory signals (Banchereau and DeLaugh).
[0079] 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. Herein, Applicant has
shown that the cells present in the lymph node sinuses, which
accumulate in cancer, are cells of the lineage of dendritic cells.
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. Therefore, a
critical aspect of this invention is being able to generate a
microenvironment in the regional lymph node that allows effective
antigen processing and presentation. The immunization derives T
cells able to traffic to the lesion and destroy tumors. This is de
facto demonstration of adequate antigen processing by dendritic
cells. Additionally, none of the patients treated with NCM
developed distant metastasis that 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.
[0080] The literature (Hadden, J W, J Immunopharmacol 11/12:629-544
(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 histiocytosis, 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 histiocytosis associated with head and neck
cancers. (See FIGS. 14 through 17).
[0081] 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.
[0082] 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.
[0083] Administration and protocols for treatment follow.
[0084] Delivery of Gene Products/Synthetic Antigens:
[0085] 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, and 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, tumor reduction, fragmentation and infiltration,
survival rate or more rapid recovery, or improvement or elimination
of symptoms.
[0086] 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 a
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 also can 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,
nontoxic solid or liquid fillers, diluents, or encapsulating
material not reacting with the active ingredients of the
invention.
[0087] The doses can be single doses or multiple doses over a
period of several days.
[0088] When administering the compound of the present invention
parenterally, 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 (PEG), and the like), suitable
mixtures thereof, and vegetable oils. It is notable that PEG
induces a chemically modified (NCE) cytokine preparation
(Pegylation).
[0089] 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 as cottonseed oil, sesame
oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil
and esters, such as isopropyl myristate, can be also used as
solvent systems for compound compositions. Additionally, various
additives that 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.
[0090] Peptides may be polymerized or conjugated to carriers such
as ovalbumen or human serum albumen as is well known in the
art.
[0091] 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.
[0092] A pharmacological formulation of the present invention can
be administered to the patient in an injectable formulation
containing any compatible carrier, such as various vehicles,
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.
[0093] Generally, the initial dose of NCM may be administered
either simultaneously with thymosin .alpha.1 or by administering
one drug followed by the other, generally, and preferably, on the
same day. The NCM is administered at low doses (200-500 units) of
IL-2 equivalence as it is important not to use high doses (>1000
units/dose) as effect is lost and toxicity increases.
[0094] More specifically, 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-00002 The antigen preparations to be used In Cancer 1)
PSMA peptides-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
[0095] The route of antigen plus NCM+thymosin .alpha.1
administration is preferentially the neck because it is accessible
and it contains greater than 30% of the body's lymph nodes and
systemic immunity can be envisioned to result.
[0096] Low dose cyclophosphamide has been used to augment cellular
immunity and decrease suppression by lymphocytes in mice and
patients with cancer (Berd, D, Prog in Clin Biol Res 288:449-458
(1989); Berd, D, et al., Canc Res 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, et al., The Prostate
38:43-78 (1999); Hadden, J W, et al., Arch Otolaryngol Head Neck
Surg 120:395-403 (1994)).
[0097] Zinc deficiency is associated with improved cellular
immunity and treatment with zinc is immunorestorative in mice
(Hadden, J W, J Immunopharmacol 17:696-701 (1995); Saha, A. et al.,
Int'l J Immunopharmacol 17:729-734 (1995)).
[0098] A cyclooxygenase inhibitor (COXi) such as indomethacin is
used. 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.
[0099] Recombinant Protein Purification [0100] Marshak, et al.,
Strategies for Protein Purification and Characterization, a
Laboratory Course Manual, CSHL Press (1996).
[0101] Dose and Frequency of Antigens
[0102] 1-1000 .mu.g, preferably 10-50 are used. The form of antigen
is soluble (partially polymerized or conjugated to carrier, only if
necessary).
[0103] Schedule: Day 1, Day 12, Day 21
[0104] (Pre-Rx) Day 12, Day 21, Day 31
[0105] Site of Injection: local injection, i.e., neck
injections
[0106] Expected responses are tumor reduction and tumor
pathological changes reduction, fragmentation, lymphoid
infiltration). Humoral immunity to antigen (RAI or ELISA) is
expected, as well as cellular immunity to antigen (intracutaneous
skin test in vitro lymphocyte proliferation or ELISPOT ASSAY).
[0107] Oligopeptides such as PSMA, MAGE fragments, E6 and E7
peptides would not normally be immunogenic without conjugation to
carrier or pulsed 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 factors in the effectiveness and thus the novelty of
this approach. Diagnostic skin tests are another guide to more
effective immunization. Patients can be pretreated with IRX-2 (NCM)
and thymosin .alpha.1 to induce better responses (increase NCM and
PHA skin tests and lymphocyte counts and reversal of lymph node
abnormalities).
[0108] This creates an adjuvant strategy:
[0109] 1) Combining immunorestoration and adjuvancy;
[0110] 2) Making peptides and proteins immunogenic;
[0111] 3) Obtaining the degree of immune response to effect tumor
regression at a distance; and
[0112] 4) Extending to all forms of tumor antigens and haptens
including peptides and/or carbohydrates.
[0113] It can extend to areas of applicability as in AIDS virus
vaccine in HIV positive patients; other difficult to manage
situations; renal transplants, aged, etc.
[0114] Patients are HLA matched for MHC restricted peptides and
skin tested for one or more tumor peptides prior to consideration
of the protocol. 100 .mu.g of one or more tumor peptides are
perilymphatically administered in the neck with NCM plus thymosin
.alpha.1 using the NCM protocol as discussed below on days 1 and 10
of the NCM series. The combination is repeated on day 21. In
addition to tumor response and histology, immune reaction to the
peptides is monitored by repeat skin test or by other means known
in the art.
[0115] The following examples demonstrate the utility of the
present invention to provide immune restoration and adjuvant effect
of NCM plus thymosin .alpha.1. As an introduction to the examples,
reference is made to U.S. Pat. No. 5,632,983 ('983 patent), the
patent having the same inventor as the inventor herein. The '983
patent presents data resulting from in vivo treatments on thymocyte
(FIG. 21) and splenocyte (FIG. 22). The data represent in vitro
response to stimulation with media control (open bars), rIL-1
(solid bars), rIL-2 (cross-hatched), and NCM (diagonal lines).
Thymosin .alpha.1 and NCM alone increase many of the responses in
both the central lymphoid organs. The combination produced dramatic
and highly significant increases for all four responses. This data
was only presented in vivo in mice.
Example 1
[0116] 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.
[0117] Preparation of Natural Cytokine Mixture (NCM)
[0118] The buffy coat white cells of human blood from multiple
HIV-negative hepatitis virus-negative donors is collected. In an
alternative embodiment, animals are 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.
[0119] 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), which contain 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.
[0120] The cells are incubated for 24 to 48 hours in X vivo-10
media with 80 .mu.g/ml Ciprofloxacin (Miles Lab) at 37.degree. in a
CO2/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 store at 4.degree. C. to
-70.degree..
[0121] Characterization of Supernatants
[0122] 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.
[0123] Standardization of Supernatant of Cytokine Content:
[0124] Each supernatant is standardized either by concentration or
amount administered so that comparisons can be made.
[0125] Removal of Contaminants from Supernatant:
[0126] 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).
[0127] Model
[0128] 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)).
[0129] Laboratory Animals
[0130] 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.
[0131] 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
ten days.
[0132] Experimental Design
[0133] Each treatment group had five animals and each experiment
was repeated two to five times. Treatment was initiated
intraperitoneally (i.p.) on day 3 and continued once per day for a
total of five days. Treatment groups were injected with one of the
following in vivo treatments as indicated in the text:
[0134] 1. pyrogen free saline (controls);
[0135] 2. recombinant interleukin-1 (rIL-1; 4 ng);
[0136] 3. recombinant interleukin-2 (ft-2; 50 units);
[0137] 4. rIL-1+rIL-2 (4 ng+50 units, respectively);
[0138] 5. natural cytokine mixture (NCM; 50 units IL-2
equivalence)
[0139] 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.
[0140] 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% CO2 in RPMI 1640 medium with 5%
fetal bovine serum, penicillin (100 .mu./ml), streptomycin (100
.mu.g/ml) and 2-mercaptoethanol (2.times.10-5 M). The cells were
plated in 0.2 ml microwell plates in quadruplicate at a
concentration of 1.5.times.106/ml and incubated for 72 hours with
one of the following as indicated in the text:
[0141] 1. control diluent (complete RPMI 1640 medium);
[0142] 2. rIL-1 (1 ng/ml);
[0143] 3. rIL-2 (2 Units/ml);
[0144] 4. NCM (2 Units/ml of IL-2 equivalence);
[0145] 5. concanavalin A (ConA; 1.5 .mu.g/ml);
[0146] 6. phytohemagglutinin (PHA; 0.5 .mu.g/ml)
[0147] The culture was terminated to measure DNA synthesis, and
thereby cell proliferation, with an 18-hour 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).
[0148] Statistical Analysis
[0149] Student's T test was used to analyze data as
appropriate.
[0150] Results
[0151] 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 that 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).
[0152] 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.
[0153] Initial experiments indicated that PHA (HA-16, Murex
Diagnostics Ltd., Dartford, UK) could be immobilized by the
technique described by the manufacturer and that under appropriate
optimal conditions of cell number of 7.5-15.times.106/ml, time of
exposure of 24 hours to 48 hours, and PHA concentration of 25 or 50
.mu.g/ml a high yield of IL-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 I). Therefore, this flask procedure is used to generate the
NCM mixture.
TABLE-US-00003 TABLE I IL content of supernatant/ml PHA brief
exposure (NI) 2-20 units PHA brief exposure 8-140 units &
Ciprofloxacin (NIM) (80 .mu.g/ml) PHA flask immobilization 100-353
units & Ciprofloxacin (80 .mu.g/ml) 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).
[0154] 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 to 48 hours; however, when PHA
(25 .mu.g/ml) was used for only 24 hours, these levels were
negligible. Twenty-four hours incubation was thus considered
optimal.
[0155] 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 IL-2 lymphokine activated killer (LAK) cell protocols.
Generation of NCM was compared in different media utilizing
continuous versus 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.106/ml with PHA at 2 .mu.g/ml or
at 5.times.106 cells/ml with PHA at 1 .mu.g/ml. Because the per
cell yield is most efficient at 2.5.times.106 cells/ml, that
concentration with PHA at 2 .mu.g/ml is chosen as the optimal.
[0156] 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 II 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, all 4-aminoquinolones tested at
80 .mu.g/ml enhanced PHA induced cytokine production under
serum-free conditions.
TABLE-US-00004 TABLE II PHA Alone & PHA & Ciprofloxacin
Norfloxacin Ofloxacin 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 IL-2 are pg/ml and for IL-2
international unit/ml.
[0157] 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-00005 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 IL-2 are pg/ml and for IL-2
international units/ml. ND not done.
[0158] In order to show the superiority of the NCM over rIL-1 in
vitro, mouse splenocytes and thymocytes were cultured with MEM and
rIL-2 at comparable levels of IL-2 as determined by bioassay and
DNA synthesis measured by tritiated thymidine incorporation. NCM
induces greater proliferation of splenocytes (FIG. 4) and
thymocytes (FIG. 5) than rIL-2 based on IL-2 content.
[0159] 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 ft-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 4 stimuli. These results are
consistent with an enhanced sensitivity of these cells to
stimulation and/or an increase in the number of responsive
cells.
[0160] FIGS. 8 and 9 demonstrate the effect of NCM treatment in
vivo on splenocyte and thymocyte markers. Nonmature 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.
[0161] FIGS. 10 and 11 demonstrate the splenocyte and thymocyte
responses in vitro to media (RPMI), rIL-1 (IL-1), ft-2 (IL-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.
[0162] FIGS. 12 and 13 demonstrate the splenocyte and thymocyte
response in vitro to ConA and PHA after treatment in vivo with
control media or NCM. The mice were treated as described
hereinabove.
[0163] The in vitro studies demonstrate the superiority of NCM over
ft-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, ft-2, nor their combination, potently promotes in vivo T
lymphocyte function (IL responses) and development (mitogen
responses and cell markers) that 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
[0164] There is shown that local perilymphatic injections in the
neck having NCM plus low dose cyclophosphamide, indomethacin, and
zinc 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.
[0165] 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.
[0166] Several unpublished observations serve to document this
speculation and lead to the present invention.
[0167] 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 I)).
[0168] 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.
[0169] Analysis of regional lymph nodes revealed unpublished data
that indicate that the regional lymph node is the site of
immunization to postulated tumor antigens (see FIGS. 14 to 18).
[0170] 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.
[0171] Patients were pre-tested with a skin test to 0.1 ml of NCM
prior to treatment. More than 90% of those with a positive skin
test (70.3 mm at 24 hours) had robust clinical and pathological
response. Patients with negative skin tests had weak or no
response. This skin testing appears to select good responders.
[0172] Major increases were observed in T lymphocyte counts (CD2)
752.fwdarw.1020 in these T lymphocytopenic patients (T cell counts
752 versus 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 pre-existing 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).
[0173] The literature (Hadden J W, Intl J Immunopharmacol
11/12:629-644 (1997); Hadden J W, Intl 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 histiocytosis, 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
histiocytosis associated with H&NSCC (FIGS. 14-17).
TABLE-US-00006 TABLE IV Treatment of Lymphocyte Phase Patients with
H&NSCC with NCM - Increases in Naive T Cells in Blood
(#/mm.sup.3) NA VE T CELL MARKER PAN T CELL MARKER Patient # 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
[0174] 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 to 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
histiocytosis 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 also showed evidence of T cell
predominance, a known positive correlate with survival in
H&NSCC (Hadden J W, Intl J Immunopharmacol 11/12:629-644
(1997)).
[0175] 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). These changes
correlate 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 (Roth, I, et al., 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 92:5540-5542 (1995)), 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. 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
[0176] Two patients were treated with lymphoma of the head and
neck. The patients included were those with head and neck cancer
who agreed to participate in the protocol. The following scheme was
followed:
[0177] Before treatment, the patients were skin-tested with NCM 0.1
ml subcutaneously in the forearm, the region was marked, and 24
hours later, the test was read. The test was considered positive if
the induction and erythema was equal or larger than 3 mm.
[0178] Each cycle of NCM was for 21 days, as follows:
TABLE-US-00007 Case #1 Day 1 Low dose cyclophosphamide (300
mg/m.sup.2 i.v.) Days 1-21 Indomethacin 25 mg p.o. 3 times daily
Zinc sulfate 50 mg p.o. once daily Days 3-12 NCM 200 units 5 as 1
ml subcutaneously perilymphatic in the neck
[0179] The patient was a 23-year-old male who presented with a
prior history of 3 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.
[0180] Following this, the patient received radiation treatment in
the neck of 3600 rads. The patient is currently free of
disease.
Case #2
[0181] 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 neck's lymph
nodes demonstrated defined non-Hodgkin's lymphoma mixed, of
intermediate grade.
[0182] 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%.
[0183] The patient was given chemotherapy (CHOP) for six 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
[0184] 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 i.v. dose of cyclophosphamide at
300 mg/m2. 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.
[0185] 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 that included lymphocytes,
plasma cells, neutrophils, macrophages and eosinophils. Treatment
was well-tolerated except for severe pain and minor bleeding during
injection and gastric intolerance to indomethacin. After 24 months
of follow-up, nine patients are disease-free.
[0186] This previously unpublished study shows that peritumoral NCM
induces immune-mediated tumor response in early stage untreated
cervical carcinoma.
Example 5
[0187] Two patients with liver metastasis from primary
hepatocellular carcinoma were treated with intrasplenic NCM (1 or
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 lymphoid infiltration.
Example 6
[0188] Four patients with squamous cell carcinoma of the penis
(human Papilloma 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
[0189] Mice were immunized with PMSA peptides conjugated to
ovalbumen 100 .mu.g at 3 sites (days 1, 14, and 21) with alum (1:1
Vol) as adjuvant (5@) or NCM (20 units IL-2 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. Two of three 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.
Example 8
Phase I/II Study
NCM an NCM+Thymosin .alpha.1 in Lymphocytopenic Patients
[0190] Following radiotherapy patients show marked decline of total
lymphocyte counts, CD3 and T lymphocytes including both CD4 and CD8
subsets: (CD4 drops more than CD8 so that CD4/CD8 ratio drops from
2 to close to 1). During 18 months follow-up these levels did not
recover (Wolf, et al. (1985)). Following NCM treatment of T
lymphocytopenic patients prior to surgery, the lymphocyte counts
increased significantly (Verastegui, et al. (1999)).
[0191] A series of post radiotherapy T lymphogenic patients were
treated with IRX-2 (NCM) (7 patients) or IRX-2 (NCM) and Thymosin
al (7 patients). At onset, both groups had mean lymphocyte counts
of 800. Patients were treated daily for 10 days with perilymphatic
injections in the neck or axilla (to avoid irradiated area) with 1
ml IRX-2 (approximately 150 units IL-2 by ELISA, 640 by bioassay)
or IRX-2 plus thymosin .alpha.1, 1.6 mg/l ml. Lymphocyte counts and
various mononuclear cell subsets (CD2, 3, 4, 8, 16, 19, 25 CD45 RO
RA and 56) were analyzed by FACS at day 0 and approximately at day
12. The patients treated with IRX-2 showed no change in mean
lymphocyte counts at day 12 (800.fwdarw.700) and no change in:
TABLE-US-00008 T cells and T cell subsets counts (not shown) B cell
counts (CD19 - not shown) macrophages (CD16 - not shown) non-T,
non-B lymphocyte counts: (259--->265) CD45 RA counts:
(279--->290)
[0192] Overall the seven patients treated with NCM+thymosin
.alpha.1 showed increases in:
TABLE-US-00009 total lymphocyte counts 800--->914 p = NS non-T,
non-B cells 261--->451 p < .05 CD45 RA 221--->443 p <
.05
[0193] Of the seven patients treated with NCM+thymosin .alpha.1,
four showed marked increases in mean lymphocyte counts, non-T,
non-B lymphocytes counts and CD45RA and CD45RO counts without
significant changes in T cells, B cells or macrophages (FIGS. 19
and 20). The non-T, non-B cells were CD56 negative (not NK cells)
and correlated well with CD45RA positive.
[0194] Four patients showed increases in TLC following treatment
and these patients were studied for CD2 and were for a prolonged
period. They give a clearer picture of maturation process.
[0195] At the onset, almost one-third of peripheral blood
lymphocytes are non-T, non-B. With treatment, these data show a
progressive increase early (10 to 12) days of 450 lymphocytes
comprising three populations of T cells: approximately 1/2 of these
are CD2+CD3CD45RA+CD4+ or CD8+, i.e., naive mature T cells;
approximately 1/4 of the remaining are non-T and non-B, but CD45RA+
and 3/4 of the remaining CD2++CD3-. These latter are immature T
cells. Between two weeks and three-plus months, the TLC increases
persist, CD3 and aBTcr (one patient only) progressively increase as
do both CD4 and CD8 signifying a progressive maturation to fully
mature T cells. Correspondingly, CD45RO+ cells increase indicating
that new memory cells have been produced, meaning they have seen
antigen and have been immunized.
[0196] CD2+CD3CD45RA+CD4+ or CD8+ Lymphocytes Over a 6-Week
Period
[0197] Three patients showed no net gain in total lymphocyte counts
(TLC) comparing early treatments with late results (1-3 months) TLC
967.fwdarw.933, however, several observations were of note:
TABLE-US-00010 All three showed increases in CD45RA +173 decrease
in Non-T Non-B -159 increases in CD3 +101 increase in B +75 No
increase in CD4 and CD8 -- .alpha. BTcr increase (1 patient)
+155
[0198] These changes signify an internal shift from almost half
null cells (meaning non-T, non-B markers) towards early T cells
CD3+, CD45RA+, .alpha. BTcrR+, CD4-CD8-. The shift involves
approximately 170 cells/mm.sup.2 or approximately 20% of the cells;
if B cells are included, it involves 25% of the cells. This major
internal shift has never been seen before in association with
immunotherapy. It signifies a major abnormal circulating population
of immature cells (committed at least, in part, to the T cell
lineage, perhaps to both, 170-T:75-B) present in these cancer
patients. The treatment of NCM+thymosin .alpha.1 induced a major
shift towards more mature T cells, yet they lack CD4 and CD8
characteristic of mature T cells. The presence of null cells has
been noted in cancer and other immunodeficiencies, yet the
induction of immature CD4-CD8- T cells in the circulation has not
been observed before.
[0199] These data document the induction and maturation of new T
cells as a result of treatment with NCM and thymosin .alpha.1.
Intravascular transitional T cells progressed to maturity, i.e.:
[0200] CD2+CD45RA+CD3CD2+CD45RA+CD3+TCR+CD4-CD8.fwdarw.CD2,3+CD4+
and CD2,3+CD8+ subsets.
[0201] Where these cells are maturing is a matter of speculation.
Normally these events are thought to occur in the thymus. In
general, these patients are thought to have involuted thymuses
incapable of making new T cells. The composition and method of the
present invention apparently induces an increase and mobilization
of bone marrow T cell precursors (perhaps to a lesser degree, early
B cells) that are either trafficking in and out of the thymus or
are differentiating extrathymically. The progressive appearance of
memory cells is important in indicating that these new CD45RA+naive
T cells are transitioning to CD45RO memory cells as a response to
antigen exposure.
[0202] According to the definition of adjuvant to be used in a
treatment of infectious pathogens or tumors, these features are
requisite:
[0203] the presence and the generation of "naive" cells capable of
reacting to antigen if T lymphocytopenia is present;
[0204] the presence of endogenous or exogenously administered
peptides capable of being presented to T cells by mature dendritic
cells; and
[0205] the action of adjuvant plus antigen in an environment
capable of generating immunity, such as the regional lymph node, to
yield a robust immunity particularly of the TH1 type. This cellular
immunity or T cell immunity is considered central in the resistance
to most pathogens and tumors.
[0206] NCM is capable of, in the strategy with low dose
cyclophosphamide and an NSAID such as indomethacin, creating lymph
node changes (including dendritic cell maturation) leading to an
immunization to cancer and immune rejection characterized by tumor
reduction and fragmentation and a heavy lymphoid infiltration. The
combination with NCM+thymosin .alpha.1 is expected to be even more
active.
[0207] In the examples below, NCM was employed in combination with
thymosin .alpha.1 plus low dose cyclophosphamide and indomethacin
to treat recurrent head and neck squamous cell cancer (H&NSCC).
It is notable that recurrent cancers of this type following intense
immunosuppression by X-irradiation would be considered by the
cancer immunotherapy community not amenable to any form of
immunotherapy. While NCM was effective to palliate recurrent
H&NSCC in several patients, a cure was not considered a
possibility.
[0208] The next set of examples describe relations to endogenous
antigens associated with tumor and/or chronic/latent
infections.
Example 9
[0209] Patient was a 68-year-old female smoker who was treated for
stage 11 SCC (T2N0M0) of the tongue with partial glossectomy. Ten
months later, the patient had a 1.times.1 cm local recurrence at
the base of the residual tongue. The patient was treated with the
NCM protocol as described above containing 250 units of IL-2 by
ELISA, however, in addition, 1.6 mg of Thymosin .alpha.1
(Zadaxin/Thymalfin) was administered with each of 10 perilymphatic
injections of NCM. The patient showed an increase of lymphocyte
count from 600 to 900/mm3 with the appearance of approximately 300
CD2+CD3CD8+CD45RA+naive T lymphocytes. No toxicity was observed.
The tumor underwent a complete clinical regression with no further
treatment. Histological examination of a locally respected specimen
showed no tumor cells and a marked lymphoid infiltration. This
immune regression exemplifies the antitumor adjuvant potential for
the NCM+thymosin .alpha.1 mixture in combination with endogenous
tumor peptides.
[0210] Three additional examples show the action of NCM+thymosin
.alpha.1 to act as an adjuvant of endogenous pathogen-related
antigens.
Example 10
[0211] Patient was a 30-year-old female with cervical cancer
treated with irradiation. The patient had a long history (>3
years) of condyloma accuminata (venereal warts) indicative of
Papilloma virus infection. The persistent lymphocytopenia following
x-ray therapy prompted use of NCM in combination with thymosin
.alpha.1 (250 units IL-2+1.6 mg respectively) for 10 daily
injections in the axilla (without low dose cyclophosphamide and
indomethacin). Three to four weeks following the initiation of
treatment, the condyloma accuminata regressed completely and did
not recur. Lymphocyte counts rose from a low 800 to 1300 mm3 over a
four-week period. No other treatment was given.
[0212] NCM+thymosin .alpha.1 is interpreted to have induced
immunity to HPV and thus regression of the venereal warts.
Example 11
[0213] Patient is a 56-year-old male with Stage 1V H&NSCC gum
cancer treated successfully with the NCM protocol plus surgery
(maxillectomy and radiotherapy). Due to persistent lymphocytopenia
(lymphocyte count of 275/mm3) and oral thrush, the patient was
treated with NCM+thymosin .alpha.1 (250 IL-2 equivalence+1.6 mg
respectively) for ten daily perilymphatic injections in the axilla.
His lymphocyte count rose to a high of 1500 mm3. Three weeks
following the initiation of therapy, he developed a maxillary
sialadenitis typical of mumps infection. This resolved
spontaneously without other treatment. The patient also had a
reduction in oral thrush (candida infection). In this circumstance,
NCM+thymosin .alpha.1 is interpreted to eradicate and thus to act
as an adjuvant for another viral antigen, as well as the fungal
antigen.
Example 12
[0214] A 66-year-old female with H&NSCC cancer of the tongue
was treated. As a result of the radiotherapy, the patient suffered
for 11/2 years persistent oral thrush (Candidiasis) and
lymphocytopenia. The patient was treated with 10 daily
perilymphatic injections in the neck with the combination of
NCM+thymosin .alpha.1 as in the above examples. Following the
treatment, the patient's lymphocyte count rose from 800 to
1,200/mm3 and the oral Candidiasis resolved completely without
other treatments and did not recur. The combination of NCM+thymosin
.alpha.1 is interpreted to be an adjuvant to induce immunity to
Candida albicans antigens leading to the resolution of a chronic
parasitization by this fungus.
[0215] The above four examples exemplify how NCM+thymosin .alpha.1
can be administered with exogenous tumor, viral, or fungal antigens
to induce immunity and resolution of the condition. In the case of
the tumor antigen, contrasuppression with low dose cyclophosphamide
was necessary to interfere with tumor-induced immune-suppression to
effect an immunization. These preceding examples predict that the
combination will similarly be an adjuvant with exogenously
administered antigens in a classical adjuvant protocol, i.e., mixed
with tumor or pathogen, antigen, or peptides in either the
prevention or treatment of cancer or infection.
[0216] The novelty of the foregoing is based on the following three
points:
[0217] Lymphocyte counts do not rise or only slightly rise
following radiotherapy over 18 months of observation (Wolf, et al.,
Arch Otolaryngol 111:716-725 (1985));
[0218] No one has observed a progressive increase in the
circulation of [0219]
CD2+CD3-CD45RA+.fwdarw.CD2+CD3-CD45RA+CD4-CD8.fwdarw.
[0220] CD2+CD3CD45RA+CD4+ or CD8+ lymphocytes over a 6-week period
following any 2-week treatment period including bone marrow with or
without thymus transplantation. This progression of events is
thought to occur in the thymus and not to occur in adults as seen
here; and
[0221] Complete resolution of cancer, two viral infections (one as
a benign tumor, venereal warts) and a fungal infection within three
weeks following initiation of treatment (as would be expected
timewise for an immune response under the circumstances) is
completely unexpected.
[0222] Throughout this application, various publications, including
United States 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.
[0223] The invention has been described in an illustrative manner,
and it is to be understood that the terminology that has been used
is intended to be in the nature of words of description rather than
of limitation.
[0224] 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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