U.S. patent application number 11/804182 was filed with the patent office on 2007-10-04 for immunological methods of treating cancer.
Invention is credited to Peter Leskovar.
Application Number | 20070231326 11/804182 |
Document ID | / |
Family ID | 27205254 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070231326 |
Kind Code |
A1 |
Leskovar; Peter |
October 4, 2007 |
Immunological Methods of Treating Cancer
Abstract
A drug, affecting the hyperactivated immunologic effector cells,
comprising (I) a Ca-antagonist and (II) an agent, reducing the
intracellular cAMP/cGMP-ratio, is being described. In addition, a
drug, affecting the hyperactivated immunologic effector cells,
consisting of (I) an agent, eliminating the hyperactivated effector
cells and (II) alloreactive cells with predetermined cell death, is
being described.
Inventors: |
Leskovar; Peter; (Rosenheim,
DE) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
27205254 |
Appl. No.: |
11/804182 |
Filed: |
May 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10406077 |
Apr 2, 2003 |
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11804182 |
May 17, 2007 |
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09694131 |
Oct 20, 2000 |
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10406077 |
Apr 2, 2003 |
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08564370 |
Jan 18, 1996 |
6156312 |
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PCT/EP94/01992 |
Jun 19, 1994 |
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09694131 |
Oct 20, 2000 |
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Current U.S.
Class: |
424/138.1 ;
424/93.21 |
Current CPC
Class: |
A61K 2039/5152 20130101;
A61K 45/06 20130101; A61K 31/495 20130101; A61K 31/495 20130101;
A61K 31/135 20130101; A61K 31/495 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/138.1 ;
424/093.21 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 35/12 20060101 A61K035/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 7, 1994 |
DE |
44 11 956.9 |
Jun 23, 1993 |
DE |
43 20 878.9 |
Jul 25, 1993 |
DE |
43 24 877.2 |
Claims
1-15. (canceled)
16. A method of treating cancer in a subject, comprising a)
administering to the subject an agent eliminating tumor-protecting
suppressor T cells; b) making fusion cells by fusing tumor cells of
a subject with MHC II-positive cells in vitro; c) pre-programming
the fusion cells to die; and d) administering the fusion cells to
the subject, whereby administering the agent and the fusion cells
to the subject treats cancer.
17. The method of claim 16, wherein the agent comprises an antibody
against CD8 and/or CD3.
18. The method of claim 16, wherein the MHC II-positive cells are
selected from the group consisting of autologous MHC II-positive
cells and allogeneic MHC II-positive cells.
19. The method of claim 16, further comprising administering to the
subject inactivated peripheral blood lymphocytes derived from a
donor of the MHC II-positive cells.
20. The method of claim 16, further comprising administering to the
subject a fusion cell derived from a tumor cell of the subject and
an allogeneic MHC II-positive cell of the same cell type as the
tumor cell.
21. The method of claim 20, wherein the subject has leukemia and
wherein the fusion cell consists of a lymphocyte from the subject
and an allogeneic lymphocyte.
22. The method of claim 20, wherein the allogeneic MHC II-positive
cell has a pre-programmed cell death and is selected from the group
consisting of peripheral blood mononuclear cells and T cells.
23. A method of treating cancer in a subject, comprising
administering to the subject an agent eliminating tumor-protecting
suppressor T cells and a fusion cell having a pre-determined cell
death comprising a tumor cell from the subject and a MHC
II-positive cell, whereby administering the agent and the fusion
cell to the subject treats cancer.
24. The method of claim 23, wherein the agent comprises an antibody
against CD8 and/or CD3.
25. The method of claim 23, wherein the MHC II-cell is homologous.
Description
BACKGROUND
[0001] The invention deals with an agent, able to affect the
hyperactivated immununological effector cells and with its use.
[0002] Different situations and disorders of humans are associated
with a hyperactivated state of the immunological-effector cells,
caused e.g. by cytokines; such effector cells lose their ability to
respond to new specific signals. The immune system is impaired or
even switched off in such situations. This occurs e.g. following a
persistent stimulation during a prolonged infection or in
situations of cell hyperactivation due to an excessive release of
endogenous cytokines. The term "immunological effector cells"
comprises e.g. T cells, macrophages/monocytes, NK cells and other
immunological cells;
[0003] A variety of immunological processes include on the cellular
level the cyclic adenosine phosphate (cAMP) which is produced by
the enzyme adenylate cyclase (AC) from adenosine triphosphate
(ATP). The cAMP plays as "second messenger" a central role in the
hormonal regulation as well as in the metabolism (through
activation of protein kineses, e.g. protein kinase A (PKA). The PKA
phosphorylates proteins which in turn depress the immune response.
In this way, the hyperactivation of effector cells results in the
down regulation of the immune function.
[0004] This reaction cascade is regulated by the production of
cyclic guanosine monophosphate (cGMP) which antagonizes the cAMP.
This reaction cascade is also influenced by the group of G-protein
coupled receptors, comprising receptors such as adrenergic,
muscarinic, histamine, serotonin and adenosine receptors. The
G-proteins (guanine nucleotide-binding proteins) are able to
stimulate (Gs) or to inhibit (Gi) the production of second
messengers. By affecting either the Gs- or the Gi-receptors, the
stimulation of AC and herewith the cAMP-production can be
regulated. Situations with a disturbed equilibrium are e.g. cancer,
viral diseases and autoimmune disorders, as well as the inducing
and disease-maintaining component of the atherosclerosis.
SUMMARY
[0005] The objective of the invention was to provide an agent, able
to fight with such disorders and to restore the susceptibility of
the hyperactivation-depressed immune system for signals and a
normal immune response. This objective can be achieved by an agent,
capable to affect the hyperactivated immunological effector cells
which comprises (I) a Ca-antagonist, and (II) an agent, able to
decrease the intracellular cAMP/cGMP-ratio. Surprisingly, it could
be found out that such a combination reduced or prevented the
hyperactivation of effector cells. In this way, these cells
re-acquire their susceptibility for specific signals and show a
normal immune reaction. It could be shown that hyperactivated cells
contain an excess of calcium ions and are characterized by an
increased cAMP/cGMP-ratio.
[0006] According to the invention, the principle of the agent is
the prevention of Ca.sup.2+-influx and reduction of cAMP/cGMP-ratio
in immune effector cells. This can be achieved by the combination
of component I and II. Surprisingly, in this way, diseases as
different as cancer, autoimmune disorders, arteriosclerosis which
seems to need an autoimmune promotor for its provocation, further
bacterial, viral, including retro viral infections, as well as some
"modern" diseases, based on immunological derailment or deviations,
e.g. the CFS (chronic fatigue syndrome) can be treated. All these
situations and disorders appear to contain--in the immunological
sense--common disregulation elements. These common elements are
e.g. a persistent or excessive activation of certain
leukocyte-subpopulations, mostly macrophages and helper T cells, as
well as suppressor cells. During a simultaneous hyperactivation of
macrophages and helper T cells, as observed in HIV-patients, a
mutual stimulation of both interdependent leukocyte-subpopulations
can occur.
[0007] A further common element is the deblockade of
blastogenically pretransformed T4 and plasma (B) cells as
consequence of preceding, persistent latent or manifest
immunosuppression; it is based on a "critical drop" of autoantigen
or pathogen-specific surveyor cells or suppressor T cells. From the
molecular-biologic point of view, the persistence of an increased
intracellular Ca.sup.2+-level, leading to an immunosuppressive
counter regulation and to an impaired sensitivity to new antigenic
signals, is a common element of a variety of disorders mentioned
above. This results not only in the impairment of the existent set
of immunocompetent cells but, in addition, in a disturbed
recruitment of new, intact immunocytes.
[0008] The combination of components I and II, according to the
invention, deblocks the misprogrammed immunocytes or effector
cells, primarily those in the hyperactivated state, and has
therefore impact on the "wound healing" or "tissue
repair"--function of misprogrammed macrophages and cytokine-hyper
secreting helper T cells. This hyperactivated or persistently
activated state of effector cells can be prevented through
component I by breaking the Ca.sup.2+-rigidity, i.e. by breaking
the Ca.sup.2+-overload of effector cells; on the other hand, the
Ca.sup.2+-level is regulated by the intracellular pHi. An
additional mechanism is the control of the electrolyte transport
through the cell membrane, i.e. the regulation of the Ca.sup.2+--,
Na.sup.+-- and K.sup.+-channels and of the Na.sup.+/K.sup.+--,
Na.sup.+/H.sup.+--, K.sup.+/H.sup.+--, HCO.sub.3--/CI-- and
Ca.sup.2+/Na.sup.+-antiport, symport and different ATPases,
respectively. According to the invention, the combination was able
to reduce the primary tumor and metastases in cancer patients by
1/3 to 2/3 within few weeks, without a simultaneous radio- or
chemotherapy.
[0009] The CFS-patients also showed surprising therapeutic results.
Here presented examples of component I and II can be combined as
well. Their effect in patients with neoplastic and autoimmune
diseases, atherosclerosis, amyloidosis, Alzheimer disease and CFS
can further be potentiated by combining them with known
immunostimulators and BRMs. In addition, these combinations can be
used therapeutically in bacterial and viral infections. Herewith,
the initial derailment, i.e. the hyperactivation of
leukocyte-subpopulations can be reversed. The restoration of the
original immune state then occurs spontaneously.
[0010] The therapeutic protocol can consist of 2 or 3 steps. So, in
the first phase a deblockade of immunocompetent, misprogrammed
effector cells, primarily macrophages and T cells, can occur, and
in the second phase, a controlled stimulation of immunocytes can
follow.
[0011] In a further modification, a "freezing-up" of the controlled
stimulated cell state, i.e. a prolongation of the second phase
(phase 3) is foreseen. In the first phase, the drug combination,
according to the invention, is used. In the second phase, well
known immunostimulators and in the third phase, the same
combination as in the first phase, but at reduced concentration,
e.g. 20 to 50% of the concentration, used in the first phase, are
foreseen.
DETAILED
[0012] Details about the deblocking of immunocompetent cells in
hypoxic and/or acidified milieu of chronically inflammated tissues,
including necrotic tumor tissue are described.
[0013] Hyper- or persistently activated macrophages consume up to
10-20 times more oxygen than resting macrophages; in the presence
of gamma-interferon, this O.sub.2-consumption increases
additionally.
[0014] The phagocytosis of opsonized particles (bacteria, latex
etc.) as well as of immune complexes via the Fc-gamma-receptor
switches on the Embden-Mayerhof-glycolysis pathway. Herewith, the
generation of the microbicidal oxygen radicals (ROI) from molecular
oxygen is stimulated. In addition, the synthesis of leukotriens C,
D and E (via reduced glutathione) is impaired due to a direct
electron and proton transfer on O.sub.2-radicals (mediated by
cytochrome P450). Leukotrien B which is synthesized instead of the
leukotriens C, D and E stimulates directly the cytochrome
P450-mediated ROI-generation.
[0015] The glycolysis leads via the excessive production and
secretion of lactic acid to a strong acidification of the
macrophage micro milieu. The pH-drop results in the suppression of
immunocyte function. The impaired synthesis of Leukotrien C, D and
E, additionally affected by ROI, results in an inhibited
glycosylation and secretion of cytokines and immunoglobulins.
[0016] The hypoxia inflammation tissue, e.g. necrotic tumor tissue,
caused by the impaired angiogenesis and hyperactivated macrophages
leads to pH-drop which results in cell depression (below pH 6,8)
and cell death at a continued pH-drop.
[0017] Both, the apoptotic and the autolytic cell death appear to
arise from the drop of intracellular pH which is associated with
the activation of lysosomal enzymes. A defect in the H.sup.+-pump
which maintains a lysosomal pH of 4,5-5,0 precedes the cell
death.
[0018] As known from the myocard cells, the cell death is
accelerated in the presence of oxygen as terminal electron acceptor
if the cell is activated and herewith the intracellular Cai-level
is increased. Therefore, under hypoxic or ischemic conditions, the
glucose catabolism via the TCC/citrate cycle should be inhibited by
special drugs. Herewith, the overproduction of NADH.sub.2 (and
NADPH.sub.2) which leads to an accelerated glycolytic lactate
synthesis and to pH drop, can be prevented. For this reason, BRMs
have to be combined with deblocking substances, or the deblocking
of immunocompetent cells has to precede other (immuno)therapeutic
steps. The component II comprises an agent which is able to affect
the cAMP/cGMP-ratio. One variant proposes the increase of
intracellular cGMP-level.
[0019] One of the main reasons for the blocked function of
immunocompetent cells (mononuclear and polymorphonuclear
phagocytes, NK cells, K cells, T cells) in situ/in vitro e.g. in
tumor lesions or in chronic inflammation tissues is the low
cGMP-level or the increased cAMP-level in the cytosol of
immunocompetent cells.
[0020] The increased cAMP-level arises primarily from the
activation of adenylate cyclase by hyperproduced prostaglandins
(PGE2/E1); these prostaglandins are secreted by hyperactivated
macrophages, fibroblasts and synovial cells. They activate,
together with catecholarnines, the enzyme adenylate cyclase which
results in cAMP-increase in cytosol.
[0021] The intracellular cAMP-rise inhibits different cell
functions of various immunocyte subclasses. Therefore the induction
of guanylate cyclase and herewith the cGMP-rise in immunocompetent
cells is recommended according the invention. In this way, the
blocked (preinactivated) effector cells switch their function from
the suppressor to the effector.
[0022] According to the invention, the following agents can be used
therapeutically (a) as single agents, or (b) combined with each
other, or (c) combined as single agents or as mutual combinations
(see (b)) with different BRMs: [0023] (1) alkalizing substances,
such as alkali-(bi)carbonate and alkali-salts of metabolizable
organic acids (e.g. Nalactate, Na-gluconate, Na--, K-citrate)
[0024] (2) reversible competitive inhibitors of citrate oxidation
in Krebs/citrate cycle, i.e. inhibitors of tricarboxylic acids in
citrate cyclus (e.g. tricarballylate, methyl/ethyl-succinate,
malonate etc.). The working mechanism is the prevention of cell
(e.g. macrophage) hyperactivation in situations, associated with
O.sub.2-deficit. [0025] (3) .beta.-blockers in general. The working
mechanism is the inhibition of intracellular cAMP-rise and of
A-kinase (PKA)-activity. [0026] (4) Ca-antagonists in general. The
working mechanism is the inhibition of Ca.sup.2+-influx into the
hyperactivated (hypoxic) cell which prevents the
Ca.sub.i-stimulated oxidation in Krebs cycle during O.sub.2-deficit
and herewith the cell death. In the case of hyperactivated
macrophages, the ROI generation can be inhibited in this way. These
ROIs block in turn the glycosylation and secretion of cytokines.
[0027] (5) Ca-agonists in general. The working mechanism is like in
the case of .beta.-blocker (point (3)), the Ca.sub.i-increase. The
following 2-step-protocol is recommended: first, the activity of
the "misprogrammed", i.e. hyperactivated macrophages has to be
blocked by Ca-antagonists (point (4)); the 2.sup.nd step is the
activation of immunocompetent cells by Ca-agonists and/or
.beta.-blockers. [0028] (6) Substances, preventing pHi-drop by
penetrating into the cell and binding the excessive H.sup.+-ions.
This class of substances comprises all representatives of alkaline
compounds, e.g. derivatives of TRIZMA, HEPES, mono-, di- and
triethanolamines, as well as chloroquine and other antimalaria
drugs, further monensin and compounds which are able to release NH3
intracellularly, such as glutamine and asparagine. Further
pHi-increasing substances are LI--, Cs-- and Rb-salts of carbonic
acid and organic acids. The weak bases have to be used as free
bases, as (bi)carbonate or as salts of metabolizable organic acids,
but not as chloride, sulfate, nitrate etc. These substances can be
combined with alkali-salts of metabolizable organic acids (see
point (1)) and/or with inhibitors of TCC/citrate cycle. [0029] (7)
Inhibitors of LDH (lactate dehydrogenase) and XOD (xanthin
oxydase). The working mechanism is the inhibition of lactate
production which prevents the excessive acidification of cytosol
and extracellular milieu. These LDH- and XOD-inhibitors can be
combined with inhibitors of TCC/Krebs-cycle. [0030] (8) Substances,
able to correct the intracellular redox-potential, e.g.
fumarate/maleinate, vitamin C, vitamin A, vitamin E, alkali
(K)-ferrocyanide, Se-compounds (e.g. Na-selenite),
Na--/K-thiosulfate, alkali-sulfate and compounds carrying reduced
forms of mercaptylt/thionyl (--SH)-groups, e.g. glutathione,
penicillamine, thiola(thiopronine), cysteine, methionine etc.
[0031] (9) Substances, correcting the intracellular
NAD(P).sup.+/NAD(P)H.sub.2-- or the GSSG/2GSH-ratio, e.g. N-acetyl
cysteine. [0032] (10) Substances, replacing the terminal
electron-acceptor (molecular O.sub.2) of the oxidative
phosphorylation, e.g. ascorbate, dehydroascorbate, insaturated
fatty acids. [0033] (11) ROI-scavengers and/or antioxidants, e.g.
(a) phenols such as tocopherols, flavonoids, phenolic acid plus
esters, benzodioxols, lignanes (NDGA), BHT, BRA, THBP (b) amines
such as tetramethyl-p-phenylenediamine (c) heterocyclic compounds
such as ethoxyquinine, barbiturates, carbazols, phenothiazines,
levamisole, nafazatron, naloxone and tinoridine (d) different
compounds such as vitamin C, glutathione (GSH), .beta.-carotine and
vitamine A-derivatives. [0034] (12) Cl.sup.--channel blockers
[0035] (13) Cyclooxygenase inhibitors/NSAIDs [0036] (14)
H.sub.2-specific antihistaminics (antagonists of the
H.sub.2-histamine receptor), e.g. cimetidine [0037] (15) inhibitors
of the cAMP- and cGMP-phosphodiesterase (methylxanthines; e.g.
theophylline or theobromine [0038] (16) Since the immune complexes
(IC) suppress--via special receptors, such as the Fc(gamma)-R
and/or CR1, CR3, CRq and other receptors--both the
macrophages/monocytes and NK cells and stimulate suppressor T cells
(T.sub.G/Ts), the IC interaction with immunocyte receptors has to
be blocked, according to the invention, (a) by the
Fc(gamma)-subunit of Ig (IgG), (b) by the biotechnologically
modified complement subunits (replacement of key amino acids), e.g.
c3b, c3bi or c1q and/or (c) by an IgG-excess. [0039] (17)
Substances, acting anti-denaturing and partially re-naturing on
biorelevant proteins, e.g. formamide, acetamide, anilide
(fomlanilide, acetanilide), and their alkyl- and dialkyl
derivatives, especially the non-toxic mono methyl-derivatives and
other water-miscible compounds. They act by weakening the hydration
and the dielectric constant (DK) of the medium. These substances
can be combined with PEG, PVP and/or DMSO. The same substances and
their combinations with PEG, PVP, glycerol and/or DMSO are at the
same time, according to the invention, highly efficient
cryoprotectants for cells and proteins. The PVP seems to be
non-toxic, as it has been clinically used for years as plasma
expander. [0040] Each class of the above described deblocking
substances can also be used as additive to conventional vaccines.
[0041] (18) Anti-gamma-interferon, anti-M-CSF, anti-GM-CSF and
anti-TNFalpha [0042] (19) Liposomes, containing (a) cytotoxic
agents (b) other cytotoxins, e.g. ricin, abrin [0043] (20)
Substances, preventing histamine release from mast cells, e.g.
cromoglycin and intal [0044] (21) Substances, reducing the
Cl-concentration of the extracellular fluid (ECP) [0045] (22)
Substances, increasing the ECF-concentration of HCO.sub.3-ions.
According to the invention, a Ca-overload blocker, especially
cinnarizin (component I) should be combined with an antagonist of
.beta.-adrenoceptor, histamine-H2-receptor and/or A2-purinergic
receptor, especially propranolol (component II). In addition, some
selected preparations should be listed which can be used in
combination with 1,2 or more other substances (A+B, A+C . . . ,
B+C, B+D . . . , A+B+C, ABD . . . ). They have a special advantage
to represent--as single substances--preparations which can be
directly used clinically. They can be combined--as single or as
complex preparations--with BRMs. [0046] 1 Ca-antagonists
(objective: prevention of the intracellular Ca-over-load which
results in the deblockade of guanylate cyclase and 5-lipcxygenase
and in the inhibition of CaM (calcium modulin)-mediated activation
of Ca-ATPase and adenylate cyclase). [0047] 1.1 based on nifedipin:
adalate (26.071) or aprical 5/-10/-retard (26.072), or
bayotensin/-mite (26.075) [0048] 1.2 based on verapamil: azuparnil
40/-80/-120 (26.073) or dignover 40/-80 (2.081) or drostreakard
40/-80/-120 (26.083) or verapamil-ratiopharm (26.108) [0049] 1.3
based on cinnarizin: cinnarizin-ratiopharm (36.035) or cinnarizin
Siegfried (36.036) or cinnarizin R.A.N. 36.034) or cinnacet
(36.033) or cerepae (36.032). [0050] 2 .beta.-blockers (Objective:
inhibition of the cAMP-increasing sub-receptors for catecholamin,
PGE1/PGE2 and histamine (H2-R) [0051] 2.1 based on acetolol: neptal
400 (26.036) or prent 400 (26.040) [0052] 2.2 based on metoprolol:
lopresor/-mite (26.035) [0053] 2.3 based on propranolol: indobloc
10/40/80 (26.032) or efectolol 10/40/80 (26.024) or elbrol 40
(elbrol 80) 26.027) [0054] 3 combination drugs: Ca-antagonists plus
.beta.-blockers: beloif (26.070) or tredalat (16.132) or nif-ten 50
(16.131) [0055] 4 non-steroidal antiflogistics/antirhumatics
(inhibitors of cyclooxygenase/prostaglandin-synthetase) [0056] 4.1
based on diclofenac: diclofenac-Wolff-25/50 (05.142) or diclo-OPT
50, 100 retard (05.143) or diclophlogent (05.144) [0057] 4.2 based
on ibuprofen: dolgit 200/400/SL (05.149) or ibuphlogent 200/-400
(05.161) [0058] 4.3 based on indometacin: indo Tablinen (05.170) or
indomat retard-rqatiopharm 75 (05.167) or amuno/retard (MSD)
(05.129) [0059] 4.4 based on ketoprofen: alrheumun (05.128) [0060]
4.5 based on acetylsalicylic acid: spelt (05.122) or solpyron
(05.121) or gepan/mite (05.117) [0061] 5 drugs, affecting the
intracellular pHi (objective: impact on intracellular pH, K-- and
Na-ion, as well as on the HCO.sub.3/CI.sup.--ratio and herewith on
the deblockade of hyper- or persistently activated immunocompetent
cells, primarily macrophages) [0062] (a) hanooxygen (03.007) or
gelum oral--rd (03.006(b)) acidovert (03.005) or acetolyt (03.001)
or nephrotrans (03.004) or NaHCO.sub.3 1 g (03.003) [0063] (c)
histinorm (05.210) [0064] 6 drugs, correcting the redox-potential
(NAD(P)H.sub.2/NAD(P) and GSH/GSSG) [0065] 6.1 drugs, improving the
mercaptyl-/thionyl-, sulflhydryl-/disulfide-ratio [0066] (a) acetyl
cysteine (23.119) or acetylcystein-ratiopharm 100/200 (23.120)
[0067] (b) metalcaptase 150/-300 (05.201) or trolovol (05.206)
[0068] 6.2 reducing drugs: cebion (83.099) or cedoxon Cassis
(83.100) or cetebe (83.102) or resochin (05.203) or quensyl
(05.202) or tauredon (10/20/50). The first two drugs are
recommended especially for their oral administration. [0069] 7
gold-based drugs (contribute--like chloroquine--to the
stabilization of hyperactivated macrophages): ridaura (05.204)
(oral) or aureostan 10/25/50/100 (05.199) [0070] 9 methylxanthines
(e.g. theophylline) (objective: stabilization of the increased
cGMP-level following the corrector of the cAMP/cGMP-ratio;
methyl-xanthines inhibit not only the cAMP-phosphodiesterase but
also the cGMP-PDE): theophyllin retard-ratiopharm 125/250/350/500
(27.082) or theospirex (27.084) [0071] 10 antidiabetics (objective:
mimicking of the insulin activity or antagonization of the
glycolysis- and TCC-inhibiting and gluconeogenase-stimulating
activity of glucagon) [0072] 10.1 based on biguanidine
(metformine): glucophage retard/-mite (11.070) [0073] 10.2 based on
tolbutamide: artosin 1,0 (11.037) or rastinon-Hoechst (11.065) or
tolbutamide 0,5 g/1 g (11.067) [0074] 10.3 based on glibenclamide:
azuglucon -3,5/-1,75 (11.038) or diamicron (11.041) or glucononn
1,75/3,5 (11.051) [0075] 10.4 based on glisoxepid: pro-diaban
(11.064) [0076] 11 guanylate cyclase (cGMP)-stimulating drugs
[0077] 11.1 based on isosorbid-dinitrate: coleb 20/-40 (54.044) or
dignonitrat 40/-60/-100 (54.049) [0078] 11.2 based on
isosorbid-mononitrate: conpin 20/-40 (54.054) or coragin 20/60
(54.046) [0079] 11.3 based on glycerol-trinitrate: nitroglycerin
retard-ratiopharm (54.023). An alternative is the
sydnonimin-derivative molsidomine (e.g. molsidomine 1/2/4 from
ct/Berlin). The working mechanism on the cellular level corresponds
to that of org. nitrates; In both cases, the activity of guanylate
cyclase and herewith the intracellular cGMP-level is increased.
[0080] 12 inhibitors of xanthin-oxidase (XOD), with the aim to
depress the generation of oxygen radicals (see point (5)):
allopurinol 300 Stada (43.007) or allopurinol Dorsch (43.008) or
allopurinol-retard Woelm (43.011) [0081] 13 some K-saving diuretics
(due to K-retard-effect or indirectly due to the stimulation of the
H.sup.+/K.sup.+-antiport and herewith the pHi-rise). [0082] 13.1
based on aldosteron-antagonist spironolacton:
spironolacton-ratiopharm 50/-100 (02.016) [0083] 13.2 based on
triemteren: jatropur (35.060) [0084] 13.3 based on amiloride:
amiloride per se or in combination with hydrochlorothiazide as
amilorid comp.-ratiopharm (35.063) [0085] 14
carboanhydrase-blocker, e.g. acetazolamide (due to the impact on
the pHi, on the glycolysis, gluconeogenesis and TCC/Krebs (citrate)
cycle, as well as due to the depression of the glycolysis
inhibiting FPK/fructose phosphate kinase ): diamox retard (67.151)
or diamox (39.006) [0086] 15 oxygen carriers (objective: an
improved O.sub.2-transport to the hypoxic inflammatory tissue):
oxoferin or TCDO (tetrachlorodekaoxid). The same or a better effect
can be achieved with an oxygen therapy. Oxoferin and TCDO can be
combined with ascorbate, succinate and/or fumarate. [0087] 16
parathormone-antagonizing drugs (PTH acts via adenylate cyclase and
cAMP immunosuppressive, like e.g. glucagon, histamine (via
H2-receptor), adenosine, PGE2, noradrenaline (via
.beta.1-adrenoceptor), adrenaline and isoproterenol (via
.beta.2-adrenoceptor). The macrophages express receptors for (a)
insulin (b) glucagon (c) histamine (d) serotonine (e) parathormone
(f) calcitonine (g) somatotropine (h) somatostatine (i) PGE2 (j)
cAMP (k) .beta.-adrenoceptor (l) neuropeptides (endorphine) (m)
arginin-vasopressin, and (n) transferrin. [0088] 16.1 based on
etidronacid:diphos (65.005) [0089] 16.2 based on clodronacid: ostac
(65.007) [0090] 17 serene-compounds (objective: ROI-neutralization,
stimulation of glutathione-peroxidase) [0091] 17.1 based on
Na-selenite-pentabydrat: selenase (GN-Pharm) [0092] 17.2 based on
ebselen [0093] 18 Li-compounds (objective: increase of cytoplasmic
pHi) [0094] 18.1 based on Li-aspartate: Lithium-aspartat-Dragees
120 (70.235) [0095] 18.2 based on Li-orotate: Lithium-orotat Tabl.
(70.237) [0096] 18.3 based on Li-carbonate: hypnorex retard
(70.234) [0097] 18.4 based on Li-sulfate: Lithium-duriles (70.236)
[0098] 19 some essential precursors, needed during switching from
catabolic to anabolic function: 19.1. L-glutarnine 19.2. ribose
19.3. creatin(in) 19.4. ATP(ADP,AMP) and/or GTP (GDP, GMP, guanin)
19.5. unsaturated fatty acids+ascorbic acid 19.6. EPL 19.7.
vanadin-compounds 19.8. glutathion 19.9. folic acid
[0099] The best combinations are 1+2; 1 or 3+5; 1 or 3+6; 1 or
3+5+6 and 5+6.
[0100] Especially recommendable are: in the group 1 1.3., in the
group 5 hanooxygen and in the group 6 6.1a and/or 6.2. These
preferred combinations can be further combined with 4,12, 17 and/or
18,7,8, and 10. An especially preferred combination comprises
cinnarizine as component I and propranol as component II. The
conventional stimulation e.g. by BRMs can be supported by sub-dosed
11 and/or low-dosed lymphokines (e.g. IL-2, gamma IFN).
[0101] The fact that immunocytes react--like other somatic
cells--on different, Ca.sub.i.sup.2+-increasing signals has not
been considered up to now.
[0102] Therefore, the activation of immunocytes, as well as the in
situ/in vivo deblockade of inactivated effector cells
(monocytes/macrophages, Ts, NK-cells, K-cells) by
alpha-sympathomimetics (e.g. phenylephrine), by Ca-agonists (e.g.
Bay K 8644 or CGP 28392) and by p-receptor blockers (beta-blockers,
.beta.-sympatholytics) is recommended according the invention.
These, Ca.sub.i.sup.2+-increasing compounds can be used alone or
combined with each other and/or with immunostimulators and BRMs.
They are suitable for the in vitro and in viva activation and
reactivation/deblockade of immunocompetent cells. .beta.-blockers
are able to focus the activity of the physiological agonists
noradrenaline (norepinephrine) and adrenaline (epinephrine) on the
alpha1- and alpha2-adrenoceptor which favours cell activation
(cGMP-rise and cAMP-drop).
[0103] According to the invention, a combination of agents with a
double impact on Ca.sub.i.sup.2+-level, i.e. by increasing
Ca.sup.2+-influx and by maintaining the so elevated
Ca.sub.i.sup.2+-level in cytosol through the later blockade of
Ca-channels, is of a special interest. Such drug combinations
comprise on the one hand alpha-sympathomimetes, Ca-agonists and
.beta.-blockers, and on the other hand Ca-channel blockers
(Ca-antagonists, such as nifedipin, verapamil and diltiazem).
[0104] Examples of cGMP-increasing compounds are organic nitrites
and nitrates, i.e. esters of the nitrous and nitric acid, such as
amylnitrite, nitroglycerol, isosorbitnitrate and
5-isosorbitmononitrate, further Na-nitroprusside and
parasympathomimetics; the latter can be subdivided in 3 groups: (a)
choline ester (e.g. carbachol, bethanechol, metacholin); (b)
alkaloids with parasympathomimetic activity (e.g. pilocarpin); (c)
inhibitors of choline esterase (reversible inhibitors:
physostigmin, neostigmine and pyridostigmin; irreversible
inhibitors: fluostigmin and tetrastigmin).
[0105] In accordance with the further aspect of the invention, a
preparation, consisting of (I) an agent, eliminating the
hyperactivated effector cells, and (II) of alloreactive cells with
preprogrammed cell death, is recommended. The working mechanism of
this preparation is based on the replacement of "handicapped",
misprogrammed immunocompetent cells of the patient by the
corresponding, in vitro pregenerated (specifically tailored)
autologous or homologous/allogeneic immunocompetent cells, a
procedure called "microimmunosurgery". This "microimmunosurgery"
can be used in patients (a) with cancer (especially solid tumors)
(b) with (retro)viral infections, including AIDS, (c) with
autoimmune disorders, and (d) with atherosclerosis-based disorders.
In other terms, according to the invention, this preparation
represents a combination (a) of breaking down (by impairing or
eliminating) the resistance of the pathological set of patient's
immunocytes, and (b) of reinfusion of the ex vivo pregenerated
(specifically tailored) immunocompetent cells. An additional effect
is the circumvention of the critical labile interphase by
allogeneic and/or autologous immunocompetent cells, pretreated in
vitro in a novel way.
[0106] The impairment or elimination of the "misprogrammed"
(pathologic) immunocompetent cells of the recipient (patient) has
been described in all details in the patent applications DE
3812605A1 and PCT/EP89/00403. Here, the novel in visor preparation
of immunocompetent cells (a) for the critical labile interphase,
and (b) for the reinfusion of a new (healthy) set of specifically
tailored immunocompetent cells, is dealt with in detail. The
problems, associated with bone marrow transplantations (BMT) are an
extreme susceptibility of patients for infections and the
unevitable "explosion" of residual tumor cells due to the
immunocompromized state of the patients. If the donor-bone marrow
cells are not completely depleted of immunocompetent T cells, so
called graft-versus-leukemia (GvL)-effect of the non-depleted donor
T cells helps to increase the resistance against the infection and
the tumor; this advantage is, however, associated with the
disadvantage of the graft-versus-host (GvH)-reaction which shows
similar fatal complications. With autologous BMT, this GvHR can be
prevented. A broader clinical use, e.g. in patients with solid
tumors, is however dampened by the extreme immunologic lability of
the BMT-conditioned patients and the herewith associated enormous
costs (ca. 160.000 dollars/patient).
[0107] Both problems, the GvHR in allogeneic system and the extreme
immunological instability, associated with extreme costs, both in
autologous and allogeneic BMT, can be solved by the 2.sup.nd
variant of the preparation according to the invention, implicating
a novel in vitro premanipulation of allogeneic immunocompetent
cells. In this way, both the infection and the
tolerance-reinduction against the inevitable residual tumor cells
in the critical phase, following the removal of primary tumor can
be prevented. In autologous BMT, the patient benefits from the
"inner immunological stability", which minimizes the outer,
extremely expensive sterility measures. This opens the ways to the
introduction of BMT in patients with solid tumors. A further
advantage is the replacement of the patient-compromising
whole-body-irradiation and/or high-dose chemotherapy by a selective
depletion of immunocompetent T cells by different Mabs and
Mab-based immunoconjugates. This improvement opens new ways for
BMT, both in patients with solid tumors and in those, suffering
from autoimmune disorders.
[0108] According to the invention, the preparation is suitable for
a therapeutic procedure, consisting of 3 phases: [0109] (a) In the
phase I, the "misprogrammed" immunocompetent cells are eliminated
by whole-body-irradiation, by high-dose chemotherapy, by specific
Mabs (or corresponding immunotoxins), directed against T cells or
their subpopulations (Ts in tumor patients, Tac-R.sup.+-cells in
autoimmune disorders). [0110] (b) In the phase II, the in vitro
premanipulated, specifically tailored immunocompetent cells are
injected into the patient, to confer on the patient
immunocompetence in the critical, labile interphase. [0111] (c) In
the phase III, the patient is injected by ex vivo pregenerated
effector cells (CTLS, TILs, LAKs in the case of tumor patients and
autoantigen-specific Ts in patients with autoimmune disorders).
[0112] Since the phase I is subject of the patent application
PCT/EP89/00403 (author: P. Leskovar), only phase II and III will be
described here.
[0113] The effector cells, needed for the phase II are generated in
vitro as follows: [0114] (a) Allogeneic (i.e. donor-) T
cells/lymphocytes are cultured first in an isoleucine- or serum
free medium to synchronize their cell cycle. Then, isoleucine or
serum, respectively, is added and following progression in cell
cycle (G.sub.1 . . . S . . . G.sub.2-phase), the cells are treated
by mitomycin C. The mitomycin-concentration is adjusted in the way
to allow a 2-5 cell division before cells die (e.g. 1-5 mg/10.sup.6
cells). Instead of isoleucin or serum, other essential cell
substrates can be used for the cell cycle arrest. Similarly,
mitomycin C can be replaced by different mitomycinc-homologs, such
as BMV 25282 and BMY 25067, as well as by other DNA-damaging,
RNA-sparing substances, such as inhibitors of DNA-polymerase, DNA
crosslinking cytotoxic agents and irradiation.
[0115] After 12-24 hrs, ideally 18 hrs of incubation with mitomycin
C (or other compounds, damaging DNA in a reversible or irreversible
way), the allogeneic (donor-)T cells are resuspended in a fresh
medium.
[0116] To improve the in vitro activation, donor lymphocytes of the
bone marrow and/or peripheral blood can be preincubated in a kind
of one-way-MLC/MLR with the recipient lymphocytes (favorably with
T-depleted or preselected MHC II-positive B cells and/or adherent
cells).
[0117] This preincubation occurs in isoleucin- or serum free
medium; the recipient MHC II-positive cells (B cells) have to be,
however, pretreated by mitomycin C or irradiation so that they stay
metabolically active but unable to proliferate. During the later
mitomycin C-treatment of allogeneic (donor-)lymphocytes (T cells),
they get an additional dose of cytotoxic agent, leading to their
selective death. The so premanipulated donor cells are activated
during the contact with patient's MHC II-positive cells (B cells,
monocytes/macrophages, activated T cells), following their infusion
into the patient; they secrete IL2 and other cytokines which are
reduced or absent in immunoincompetent recipients (due to the lack
of mature helper T cells.)
[0118] These donor cells can, however, not induce the fatal GvHR or
GvHD, as they are "preprogrammed" and die after few cell divisions.
[0119] (b) By an alternative procedure donor's mitomycin
C-pretreated PBMs(without bone marrow) are injected in the first
phase, and donor's T-depleted bone marrow is post-transfused in the
second phase. The donor T cells, pretreated in this way, can be
replaced or combined with allogeneic (donor-) LAK cells.
[0120] Normally, only autologous LAK cells are used
therapeutically.
[0121] According to the invention, allogeneic (donor-)instead of or
in addition to autologous LAK cells should be used therapeutically.
The GvHD-complication is not a problem, because the LAK cells
consist of up to 90% activated NK cells and of up to 10%
non-MHC-restricted CD3.sup.+ (T) cells. The T subpopulations which
are responsible for the alloreaction and GvHD, cannot survive
during the LAK-generation in vitro, due to the lack of antigen
(i.e. alloantigen of recipient MHC II-positive cells). T cells can,
however, be depleted in vitro by specific Mabs or immunotoxins for
reasons of an additional security. Alternatively, L AIC cells can
be pretreated in vitro by mitomycin C (or other DNA-damaging
substances), as described above. [0122] (c) If for unknown reasons
GvHR or GvHD are observed, so a novel strategy can prevent these
complications and the GvHD-establishment in general, according to
the invention. The principle is the in vitro generation of
alloreactive T cells (a) from recipient or (b) from a third person
(second donor), directed against donor cells by means of the
MLC/MLR-technique.
[0123] The donor (first donor) cells have to be pretreated by
proliferation-preventing mitomycin C-doses in order to be able to
act as stimulator cells in the MLC.
[0124] The responder cells (from recipient or 2.sup.nd donor) are
thereafter treated by mitomycin C (or other DNA-damaging
substances) in a way, allowing the cells to divide for 2-5 times
before they die.
[0125] To increase further the efficiency, the autoaggressive
subpopulation of patient T cells can be preeliminated by
immunotoxins, consisting of cytotoxin (e.g. abrin, ricin,
doxorubicin, .sup.131I-radionuclid) plus IL-2 or
anti-Tac/II-2-R-Mab.
[0126] According to the invention, the so pretreated effector cells
can be frozen, similar to the effector cells with restricted
lifespan, described under (a),(b) and (c), in a medium, containing
8-15% DMSO and PVP or PEG of different concentration and mol weight
(patent application DE 3812605 A1 and PCT/EP89/00403).
[0127] The addition of PVP and/or PEG improves the viability and
preserves-in contrast to the sole DMSO-addition-the preactivated
state of cryopreserved effector cells. In this way, a repeated
infusion of effector cells into the recipient became possible.
[0128] An additional improvement is the separation of donor
adherent cells (macrophages/monocytes) before the mitomycin
C-treatment, followed by their later readdition to the mitomycin
C-pretreated effector cells (T cells, lymphocytes) and infusion
into the patient.
[0129] Mitomycin C can be combined with interferon (alpha, beta,
gamma), TNFalpha, DMSO, vitamin A and E, as well as the
(re)differentiation substances, such as butyrate.
[0130] The following procedure is also especially preferred:
Patient's lymphocytes can be clonally expanded in vitro into
tumoricidal CTLs and plasma cells by mitogenic lectins (PHA,ConA)
and mitogenic antibodies (anti-CD3/Ti, anti-CD2/T11). This in vitro
postexpansion comprises only the blastogenically pretransformed T
and B clones, i.e. memory T and B cells. Since tumor patients have
a persistent contact with tumor cells and AIDS/ARC/LAS-patients
with HIV and opportunistic infections, respectively, their blood
contains corresponding memory cells. Depending on the cell
structure conditions, these memory cells can be directed toward
CTLs (CD8.sup.+, and CD4.sup.+) or Ts cells; this opens new ways
for their therapeutic use (a) in tumor- and AIDS-patients, and (b)
in patients with autoimmune disorders.
[0131] If the resistance (immunocompetence) of the patient is
temporarily down-regulated, the infusion of in vitro clonally
postexpanded patient's lymphocytes (T cells) can have an essential
impact on the disease development. The expansion of CTLs can be
achieved e.g. by the PHA-treatment for 3-6 days. The autotolerant
Ts cells, necessary for the therapy of autoimmune disorders, can be
generated e.g. by the treatment with PWM for 7 days or with the PHA
for 3-4 weeks. The CD8.sup.+-rise is accompanied by a
CD4.sup.+-drop. These Ts cells can be cultured for more than 6
months under following conditions: 2 times weekly, IL2 is added to
the medium and cells are restimulated by feeder cells and PHA every
2 weeks. These cells suppress the proliferation of autologous and
heterologous CD4.sup.+ T cells when stimulated by PWM, OKT3 or
tetanus toxoid.
[0132] Alternatively, Ts cells can be generated in vitro in the
presence of PGE2, anti-IL1, anti-IL2, anti-IL4, cyclosporinA,
rapamycin and/or FK506.
[0133] The Ts-depleted cytotoxic effector cells, needed in the
tumor and AIDS/ARC-therapy, can alternatively be enriched by the
elimination of CD8.sup.+ cells (by means of Mabs or immunotoxins).
The spared CD4.sup.+ T cells are able to induce in vivo new CTLs;
this process is accelerated by anti-PGE2,
anti-lipocortin/macrocortin and anti-TGF-beta. In
AIDS/ARC/LAS-patients, it is advantageous to preimmunize the
patient with the lysate of opportunistic infections, before the in
vitro expansion of memory cells; alternatively, a healthy donor can
be preimmunized (a) with a viral antigen (8p 120) and (b) with
mentioned lysate.
[0134] The so generated clonally postexpanded allogeneic memory
cells (T and B cells) can be treated by mitomycin C, washed and
injected into the AIDS-patient.
[0135] Atherosclerosis and other coronary diseases show an
autoimmune genesis (our own experiments, reports of others., e.g.
W. Hollander).; therefore, the agent(s), based on
"microimmunosurgery", are able to replace or support the
"conventional" treatment.
[0136] According to the invention, hyperactivated B or T or both B
and T cells of atherosclerotic patients are depleted/inactivated by
means of "microimmunosurgery". It is advantageous to replace them
by the in vitro pregenerated Ts fraction. These Ts cells can be
generated by a simple, 3-5 week incubation of patient's PBMs with
ConA or PHA. Alternatively or supportingly, the patient can be
treated by special preparations which inactivate the hyperactivated
macrophages; they consist of Fab/F(ab').sub.2-subunits (labeling)
or immunotoxins (depletion), directed (a) against the (acid-labile
and acid-resistant) Fc(gaTnma)-receptor, (b) against the complement
receptor (CRT, CR3), (c) against the scavenger/AcLDL-receptor (I
and II), (d) against the gamma-interferon-receptor and/or (e)
against the LPs/endotoxin-receptor. Alternatively, the
biotechnologically synthesized receptor per se or its subunits can
be used therapeutically.
[0137] In addition, denatured (e.g. heat-denatured) complement
subunits (c1q, c3b, c3d etc.) or biotechnologically produced defect
C-components can be used; they can be combined with antioxidants
(vitamin E,A, probucol). The in vivo neutralization of the
solubilized ApoB,E- and ApoE-receptor in the patient's plasma,
following their quantitative determination in vitro, is also
advantageous.
[0138] An alternative way comprises a combination (a) with
Ca-antagonists/Ca-channel blockers (verapamil, nifedipin,
dilthiatem), (b) with cGMP-increasing substances (e.g.
Na-nitroprusside, org. nitrates), and (c) with pHi-raising
substances. In this way, the replacement of "wrongly programmed"
(hyperactivated) macrophages by "fresh" monocytes should take
place.
[0139] The protracted or repeated microimmunosurgery includes the
prevention of neutralizing antibodies against xenogeneic (mostly
murine) Mabs and Mab-based immunoconjugates by substances,
specified in the patent application PCT/EP89/00403, as well as by
some procedures, described here: [0140] (1) Prevention of
neutralizing antibodies by the pretreatment of recipient with Mabs
which are coupled as a kind of hapten to a tolerogen as carrier.
Examples of such tolerogenic carriers are polyethyleneglycol (PEG),
polyvinylpyrrolidone (PVP) and different copolymers of D-amino
acids (e.g. D-glutamine-lysine, shortly D-GL). [0141] (2) The same
principle (like under (1)) can be applied to different
immunoconjugates, e.g. conjugates of Mabs (directed against tumor
cells, viral/bacterial infections, leukocyte-subpopulations) with
(a) cytotoxins, such as ricin or abrin, (b) with cytotoxic agents,
such as doxorubicin, (c) with radionuclids, such as 131I, and (d)
with target cells-starving enzymes, such as arginase, asparaginase
etc. A two-step-administration (first sub-immunogenic, then
immunogenic dose), can additionally potentiate the effect. [0142]
(3) Neutralizing antibodies against xenogeneic (murine) proteins
(e.g.Mabs) can be prevented also by strictly monomeric,
molecular-dispersed structure of these xenogeneic proteins, which
can be achieved by their pretreatment (a) by mercaptoethanol (b) by
glutathione (c) by N-acetyl-cysteine (d) by penicillamine D (e) by
other substances which support the
disulfide-to-thioVsulfhydril-interconversion, as well as by 6M-urea
and guanidine-hydrochloride. [0143] (4) Prevention of neutralizing
antibodies by a direct in vivo use of Mab-producing plasma cells,
which induce a low-zone tolerance against xenogeneic proteins by
secreting strictly monomeric Mabs; these are not xenogenized by in
vitro manipulation. [0144] (5) Prevention of neutralizing
antibodies by the direct in vivo use of Mab-secreting hybridoma
cells, which were pretreated in vitro by mitomycin C and/or other
DNA-crosslinking cytotoxic agents (restricted lifespan, 2-5 cell
divisions only). [0145] (6) Prevention of neutralizing antibodies
by aggregate-preventing substances, e.g. protein-solubilizing
tensids (e.g. salts of higher fatty acids, Iysolecithin) at
extremely low concentrations).
[0146] As next, some further details to this invention will be
dealt with:
[0147] The hyperactivated, suppressive macrophages can be
inactivated as follows: [0148] (a) by antioxydants (b) by
inhibitors of enzymes, involved in ROI-synthesis (c) by
PAF-blockers (d) by PLAZ/PLC-inhibitors and/or (e) by Ca-channel
blockers/ Ca-antagonists. In the 2.sup.nd phase, the recruitment of
new macrophages from monocytes is foreseen.
[0149] The in vitro generation of blastogenically pretransformed
(memory) cells can be accelerated if patient's PBMs are first
treated by anti-CD8- and/or anti-CD3-Mabs plus complement or by
corresponding immunotoxins. This leads to a partial Ts-depletion
and should be followed by the treatment with mitogens (lectins or
mitogenic Mabs).
[0150] In patients with autoimmune disorders, the addition of Ts
-stimulating substances (anti-HLA-DR-Mab, anti-LFA-1beta-Mab,
cyclosporinA, corticosteroids, FK 506, rapamycin, ConA) and in
patients, suffering of cancer or AIDS (and other viral diseases),
the addition of Tc(CTL)-promoting substances (anti-HLA-DQ-Mab,
anti-LFA-alpha-Mab, cyclooxygenase-inhibitors such as aspirin,
indomethacin, anti-PGE-Mab etc.) is recommended. The preferred
generation of Tc instead of Ts cells can be observed in vitro also
when adherent cells are removed. This technique is based on the
effect of ex vivo specifically tailored effector cells on the
disease regression; the patient's immunologic resistance has to be
temporarily reduced by anti-CD8-Mab and/or anti-CD3-Mab (in the
case of tumor- and AIDS-patients) and by anti-CD3-Mab (in patients
with autoimmune disorders): The therapeutic efficiency in tumor
patients is further increased if patient's tumor cells are
incubated in the presence of gamma-interferon, TNFalpha and/or
5-HETE in order to induce MHC I and/or MHC II-postexpression on the
cells and are then reinjected along with patient's specifically
tailored, in vitro postexpanded memory cells.
[0151] A further increase in efficiency can be achieved by the
confusion of patient's glutaraldehyde-pretreated macrophages which
have been preincubated with tumor antigen. An additional advantage
is the confusion of patient's inactivated (preirradiated or
glutaraldehyde-pretreated) leukocytes (PBMs) which results in the
induction of antiidiotypes, directed against patient's Ts cells.
The infusion of hybridoma cells, based on patient's tumor cells and
MHC II-positive autologous and homologous cells is advantageous, as
well.
[0152] The above discussed LAK/TIL-techniques can be further
improved by adding mitomycin C-pretreated allogeneic (donor) T
cells to LAK or TIL cells. This increases the number of
lymphokine-secreting cells which is especially important for the
TIL-technique.
[0153] These premanipulated allogeneic cells are able to play a
similar positive role in bone marrow recipients. The preexpanded
allogeneic cells can be used also in the treatment of autoimmune
disorders and GvHD, if they are pretreated by DNA-damaging agents.
The RES-elimination (99%) of LAK and TIL cells due to their
"xenogenization" during the ex vivo manipulation can be reduced,
according to the invention, by addition of alpha2-macroglobulin,
antitrypsin and/or cortisone to the medium.
[0154] A variant of the described procedure renders the externally
controlled in vivo production (a) of cytokines (e.g. TNFalpha,
IL-1, IL-2, IL-4, IL-6, IL-3, G-CSF, M-CSF,GM-CSF), (b) of hormones
(e.g. insulin, parathormone etc.), and (c) of other physiologically
important cell factors possible. The principle is the transfection
of donor specific recipient T cells by corresponding, these
cytokines or hormones encoding genes; the preselection of
alloreactive, donor-specific recipient T cells can be achieved by a
(repeated) one-way--MLC (stimulator cells: donor-PBMs; responder
cells: recipient-PBMs).
[0155] After the reinfusion of the transfected recipient T cells,
these can be repeatedly reactivated in vivo to secrete cytokines or
hormones by injecting i.v. inactivated (mitomycin C-pretreated or
preirradiated) donor-PBMs. After the in vitro pregeneration of
autologous, alloreactive T memory cells, directed against donor A,
donor B, donor C etc., different functions can be transfected
donor-dependently into these recipient memory cells and "recalled"
in vivo, following the reinjection of these manipulated autologous
cells into the recipient. By the intratumoral injection of these
cells, the effect can be localized to the tumor tissue. A similar
in vivo "switching on" of the desired function can be achieved by
the following procedure: the recipient T cells are first primed in
vivo by a model antigen and then clonally postexpanded in vitro in
the presence of the same antigen. In the next step, these cells are
transfected by the gene of interest, e.g. cytokine- or hormone
encoding gene(s) and reinjected into the recipient, favorably after
the temporary depletion of recipient's immunocompetent cells. Later
restimulation of the recipient by the same model antigen "turns on"
the desired ("transfected") cell function. Representatives of such
antigens are tuberculin and other antigens, used in cutaneous
tests, as well as haptens (DNCB, DNBB, DNBS, TNBS etc.).
[0156] Instead of the above described alloantigens and model
antigens, low-dose allergens can be used.
[0157] This externally controlled in vivo secretion of cytokines is
of special interest in the therapy of strongly immunocompromised
patients (AIDS/ARC/LAS-patients, patients with advanced cancer,
recipients of bone marrow grafts).
[0158] The above discussed transplantation (a) of organs (b) of
bone marrow and (c) of mitomycin C-pretreated, hormones (e.g.
insulin) and cytokines-secreting allogeneic or xenogeneic cells and
hybridomas can be essentially improved by the following procedure:
First, the immune resistance of the recipient must be temporarily
down-regulated ("broken") by specific antibodies (anti-panT- or
anti-CD8-Mabs) or Mab-based immunotoxins. Then, the in vitro
pregenerated suppressor T cells (Ts) (see above !) are reinjected,
immediately before the transplantation. The main advantage over the
"conventional" grafts is that here--in contrast to the
"conventional" grafts--the recipient is not confronted
"unexperienced" with the MHC II-positive donor cells (bone marrow
macrophages and B cells, as well as "passenger lymphocytes" in
organ grafts) but preinjected by allotolerant Ts cells; these
donor-specific Ts-memory cells direct the CD4/CD8-double positive
precursor cells (inducer/transducer suppressor cells) towards the
Ts-effector cells before the alloreactive Tc/CTL cells predominate.
This seemingly minimal deviation in the procedure can decide about
the survival of organ- and bone marrow recipients.
[0159] The tolerance against the organ or bone marrow graft, as
well as against the hormone (e.g. insulin) or cytokine-producing
allogeneic cells can be induced also in the following way:
Alloreactive donor-specific recipient T cells are selected and
expanded in vitro by means of MLC (responder cells: recipient PBMs,
stimulator cells: donor PBMs). The repeated MLC results in a 95%
enrichment of these alloreactive T cells. The next step is the
inactivation of recipient's donor-specific alloreactive PBMs or T
cells by irradiation or mitomycin C: Before they are injected into
the recipient, the latter must be temporarily rendered
immunoincompetent (by anti-CD3- or anti-CD1- or anti-CD8-Mab). The
principle here is the induction of anti-idiotypes in the recipient
(in vivo), before this encounters MHC II-positive donor cells (bone
marrow cells or "passenger lymphocytes") Therefore, this induction
of anti-idiotypes has to be carried out several days before the
real transplantation.
[0160] Problems, associated with the transplantation, such as
immune suppression or susceptibility for infection can be reduced
by the co-infusion of in vitro inactivated allogeneic (donor) MHC
II-positive cells (B cells, macrophages), along with the
allotolerant Ts cells. These MHC II-positive cells can be
pregenerated in vitro by a kind of MLC (responder cells:
recipient-PBMs, stimulator cells: donor-PBMs), frozen and during or
after the transplantation repeatedly reinjected into the
recipient.
[0161] The precursor cells can be directed toward Ts cells by the
addition of anti-HLA-DR- and/or anti-LFA-1beta-Mabs. In contrast,
the generation of Tc/CTLs can be induced in the presence of
anti-HLA-DQ- and/or anti-LFA-1alpha-Mabs. The rise of MHC
II-positive APCs ("passenger lymphocytes"), which is critical for
the graft failure, can be prevented by the addition of
anti-HLA-DR-Mab or the corresponding Fab/F(ab').sub.2-subunit
and/or Ca-channel blocker (verapamil,nifedipin, dilthiazem).
[0162] A further improvement of the above discussed
LAK/TIL-technique can be achieved (a) by co-infusion of mitomycin
C-- preinactivated allogeneic, MHC II-positive cells (B cells,
adherent cells), and (b) by the addition of sub-dosed
corticosteroids and/or serum proteinase inhibitors
(alpha2-macroglobulin or alpha1-antitrypsin) to the medium during
LAK or TIL generation. The so modified culture medium prevents the
in vitro xenogenization of LAK and TIL cells and herewith their
early RES-elimination in vivo.
[0163] The activation of tumoricidal/virucidal effector cells can
be achieved also through a controlled treatment of these effector
cells (NK cells, T cells, macrophages) (a) by fusogenic substances
in sub-fusogenic concentrations (e.g. PEG, PVP), further (b) by
electrofusion under sub-fusogenic conditions (1000-5000 kHz;
10-150V/cm.sup.2) and/or (c) by proteolytic enzymes and
lipases.
[0164] The above described hybridomas can be used, according to the
invention, also to stabilize and to establish herewith new cell
lines which normally wouldn't survive in vitro.
[0165] An improved localization of the above described,
transfected, cytokines or hormones secreting T cells can be
achieved by "arming" of these transfected cells with bifunctional
Mabs which recognize these both transfected cells and the tumor
cells.
[0166] In order to induce a kind of allergic reaction against tumor
cells, the "conventional" anti-tumor-Mabs which are normally of the
IgG-isotype, can be combined with anti-tumor-Mabs of the
IgE-isotype. The latter can be produced in vitro by the "isotype
switching" of plasma cells from the IgG- to the IgE-production; the
process of "isotype switching" can be induced by addition of
anti-gamma interferon and anti-IL2-Mab, as well as IL3, IL4 and IL5
to the medium. A simultaneous addition of anti-CD8-Mab is
advantageous. In the case of anti-tumor-IgGs the immortalization
(hybridoma formation) is carried out before and in the case of
anti-tumor-IgEs after the "isotype switching" of plasma cells.
Alternatively, anti-tumor-Mabs of the IgE-isotype can be
constructed by the conjugation of xenogeneic (murine)
anti-tumor-Mabs (more precisely: Fab/F(ab').sub.2-subunits) with
human Fc-subunits.
[0167] According to a further variant, a direct in vivo use of
hybridoma cells, produced before (IgG) or after (IgE) the "isotype
switching" is recommended; these hybridoma cells have to be
pretreated by DNA-damaging, RNA-sparing agents (e.g. MMS, mitomycin
C etc.)
[0168] In a further variant, anti-gamma-interferon plus IL4 (IL-3,
IL-5) are directly injected into the patient; herewith the activity
of T.sub.H2 cells is increased and that Of T.sub.H1 cells depressed
which leads to an early "isotype-switching".
[0169] The next strategy is the replacement of the immortalizing
(transformed) partner cell (e.g. NS-1) in the hybridoma and
quadroma cell by MHC II/HLA-DR-positive (allogeneic) cell. In this
way, the hybridoma and quadroma cells can be externally switched on
and off. The new procedure is based (a) on the in vitro
pregeneration of alloreactive T cells of the recipient, directed
against the donor A and further donors (donor B, donor C etc.) by
means of the (repeated) MLC, (b) on the fusion of preselected
alloreactive memory-T-cells with partner cells which are used for
"conventional" hybridizations (e.g. anti-tumor-Mab producing plasma
cells), as well as (c) on the reinfusion of the so generated
hybridoma cells into the temporarily immunocompetent recipient.
These hybridoma cells which are well tolerated, as they are fully
(in the case of human autologous plasma cells) or partially
autologous (in the case of murine plasma cells), can be later
reactivated repeatedly by the injection of donor-lymphocytes.
[0170] A kind of tumor-directed "autoreactivity" can be achieved as
follows: tumor cells of the patient are stimulated in vitro by
gamma-interferon (TNFalpha, 5HETE) to post-express MHC II (and MHC
I) on the cell surface. Alternatively, tumor cells can be fused (a)
with autologous or (b) allogeneic, MHC II-positive cells and washed
thoroughly.
[0171] A further xenogenization of patient's tumor cells can be
achieved by the fusion with LPS-containing gram-negative bacteria
and/or yeast cells. After their inactivation, these manipulated
tumor cells are injected into the patient who has to be rendered
immunoincompetent temporarily (by injection of anti-CD3-, anti-CD1,
anti-CD2- or anti-CD8-Mab). In this way, the autoaggression against
tumor cells can be induced.
[0172] Hybridoma cells, arising from patient's tumor cells and MHC
II-positive allogeneic cells (e.g. allogeneic B cells) are able to
accelerate the induction of the autoaggressive reaction against
tumor cells if they are combined with (repeated) injection of
inactivated PBMs from that donor whose MHC II-positive cells were
used as partner cells for the hybridization with patient's tumor
cells (see above).
[0173] The same techniques can also be used for the improvement of
conventional vaccines (e.g. against bacterial and (retro)viral
infections, including HIV). So, the efficiency of conventional
vaccines is essentially increased if they are combined with
anti-CD8-Mabs (or the corresponding immunotoxins) and/or with
complete and incomplete (Freud)-adjuvant. It is also advantageous
to combine the 1.sup.st vaccination ("priming") but not the 2nd
vaccination ("boosting") with anti-B-cell-Mabs (e.g. anti-CD19-,
anti-20-, anti-CD21-, anti-CD22-Mab). The in vitro preformed immune
complexes, composed of pathogen and anti-pathogen (IgM-isotype)
with or without bound complement or complement-subunits (c1q,
c3b/c3d etc) can also be promising. Alternatively, the efficacy of
the vaccine can be improved by IgE-inducing IL3, IL4, IL5 and
anti-gamma-interferon.
[0174] The above discussed GVHD can be prevented also by autologous
PBMs which have been also preactivated by a repeated MLC. In this
MLC, the responder cells are autologous (recipient) PBMs and
stimulator cells the allogeneic (donor) PBMs; the latter have to be
preirradiated or mitomycin C-pretreated. The repeated co-incubation
of donor- and recipient-PBMs leads to the generation of highly
efficient alloreactive memory cells which are able to eliminate the
GvHD-causing donor-T-cells.
[0175] The autologous BMT can be improved: (a) by preelimination of
suppressor-T-cells in vitro, e.g. by anti-CD8-Mabs or corresponding
immunotoxins, (b) by in vitro preactivation of effector cells, (c)
by addition of autologous and/or allogeneic LAK-cells (the
allogeneic LAK-cells can, but need not be predepleted of
CD3-positive cells), (d) by addition of autologous,
CD8.sup.+-depleted PBMs to the autologous bone marrow cells (the
PBMs should be preactivated in a tumor-specific way), as well as
(e) by the addition of allogeneic, favorably preactivated
allogeneic PBMs which have to be pretreated by mitomycin C or other
DNA-damaging and RNA-sparing substances in order to confer to the
alloreactive subpopulation a restricted lifespan.
[0176] According to the invention, the stepwise addition of
increasing doses of anti-CD8- or anti-CD3-Mab plus complement (or
of corresponding immunotoxins) to the patient's PBMs allows the
determination of the critical T cell number/ml which directs the
immune response from suppression to stimulation (deblockade) of T4
and B memory cells. The so premanipulated autologous PBMs mimic the
favorable clinical situation, when autologous marrow cells are
collected during remission (PR or CR) and not during relapse.
[0177] This technique is recommended especially for patients with
solid tumors, as well as for AIDS/ARC/LAS-patients.
[0178] The above mentioned LAK cells show the special advantage
that they cannot be contaminated with (tumor-specific) Ts cells; a
further advantage is the lymphokine-production by autologous T
cells, directed against allogeneic LAK-cells, which is especially
important for immunocompromized recipients. The LAK-cells of one or
more persons could be frozen and used for different patients,
especially after an anti-CD3-pretreatment ("standardized
LAK-cells").
[0179] As next, some additional improvements of the techniques
described above should be quoted: So, the restricted lifespan of
hybridoma cells can be achieved by the change of the ratio of
partner cells during hybridization, instead of the treatment of
hybridoma cells with DNA-damaging and RNA-sparing agents. It
concerns the chemical (PEG) and viral (SV-40) fusion, as well as
the electrofusion.
[0180] If the ratio of partner cells which contribute to the
immortalization of hybridoma cells in comparison to those which
contribute the desired function is reduced from "classical" 1:1 to
1:2; 1:3; 1:4; 1:51: . . . 1:10, a controlled longevity of
hybridoma constructs can be achieved, similar to that, achievable
by low-dose mitomycin C. By an early fusion of instable cell lines,
which don't grow ex vivo, with transformed cells, these cells can
be "stabilized" and are able to survive in vitro.
[0181] According to the invention, the fusion of lymphokine and
hormone-producing cells with transformed cells, followed by the
mitomycin C-treatment, opens for the first time the possibility of
a direct in vivo implantation of cytokine and hormone-producing
cells (e.g. insulin production). The so pretreated secretory cells
die after a limited number of cell divisions and can be substituted
by a new set of premanipulated cells; this allows an efficient
control of the cytokine and hormone level.
[0182] The tolerance against such secretory cells can be achieved
(a) by the simultaneous treatment with immunosuppressive substances
(cytotoxic agents, sub-dosed anti-CD4- or anti-MHC II-Mab), (b) by
cell implantation in thymus and/or (c) by "autologization", i.e. by
use of autologous partner cells for hybridization. Further
comments: In AIDS-patients, the in vitro presensibilization of
patient's lymphocytes (T cells) by preirradiated of mitomycin
C-pretreated donor-lymphocytes (T cells), followed by the
transfection of alloactivated patient's T cells with cytokine or
anti-HIV-Ab-producing genes, and reinfusion of the so transfected
autologous T cells into the patient is recommended. A part of
transfected cells can carry the gene for the IgG-isotype and the
other part that for the IgE-isotype. The later "switching on" of
the cytokine and antibody-production occurs by the injection of
inactivated donor lymphocytes.
[0183] According to a simplified version, the patient's T cells in
general, not only his donor-specific alloreactive T-subclones can
be transfected in vitro with cytokine and/or
anti-HIV-antibodies-encoding genes, and reinjected into the
patient. Again, the later "switching on" of the in vivo cytokine
and antibody production occurs by the injection of
donor-lymphocytes; the system works because patient's lymphocytes
always contain T cells which are alloreactive against the
donor.
[0184] In conventional vaccines, the ratio between "positive" (Th
andTc/CTL) and "negative" (Ts) memory cells can be increased as
follows: (a) In the 2.sup.nd phase, i.e. 4-6 days after the
vaccination, the couterregulation of persistently activated
macrophages can be delayed by Ca-channel blockers and
macrophages-inactivating measures (ROI-inactivating agents, such as
retinol, tocopherol or carotinoids, glutathione, ascorbate,
radical-scavengers). (b) With anti-HLA-DQ (not DR) and
anti-LFA-1alpha (not beta), the (Th+Tc) /Ts-ratio can be positively
influenced. (c) A similar effect (increase of the (Th+Tc)/Ts-ratio)
can be achieved by
anti-lipomodulin/anti-lipocortin/anti-macrocortin-Mabs and/or
mellitin. (d) Ba the conjugation of pathogen antigens (viral or
bacterial structures) to carriers which are used as components of
common vaccines, such as tetanus and diphterytoxoids, tuberculin,
inactivated M. tuberculosis and BCG; the efficiency of conventional
vaccines can be further increased. (e) an additional improvement is
the use of anti-thymosine-alpha7 (not alpha1). (f) The efficiency
of vaccines can be also increased by anti-PGE2(E1) and by
PGF2alpha. (g) The resistance against pathogenic microbes can be
increased as well, if first contact with antigen structures of the
pathogen (e.g. gp120/160 or gp 41 of HIV) occurs via alveolar
macrophages which prevents its early contact with Ts cells (as in
the case of the i.v. administration). Therefore, the
sensibilization by aerosol-spray as the only or at least first step
("priming") is recommended. (h) In the 2.sup.nd phase of
vaccination, i.e. 4-7 days after vaccination, the Ts generation
should be prevented by anti-PGE2- and anti-interferon-Mabs. (i) The
in vitro preformed IgE.antigen:IgE-complexes are able to activate
the IgE-R-expressing cells (macrophages, NCF-A-, ECF-A- and
kininprotease-secreting mast cells) in the critical early
phase.
[0185] Some additional techniques: (a) Tumor patients can be
treated by inactivated hybridoma cells, arising from patient's
tumor cells and allogeneic (donor) cells of the same origin, i.e.
the same organ like tumor. An example are leukemia cells, fusioned
by the same lymphocyte-subclass of the healthy donor. (b) The
Th(=T.sub.M) cells can be stimulated in vivo if the injection of
antibodies of the IgM-isotype is followed by the injection of the
corresponding antigen. Analogously, the Ts (T.sub.G) cells can be
stimulated in vivo by the successive infusion of IgG and the
corresponding antigen. (c) Similarly to the above described
"allogeneic switching", autologous lymphocytes can be primed in
vitro by a model antigen and the resulting memory cells transfected
by cytokine and Mab-encoding genes. After the reinfusion they
respond to the resensibilization by the same model antigen. The
strongest response show the cutaneous antigens. It is advantageous
to use the preexistent memory cells which stem e.g. from previous
vaccinations (e.g. tuberculin) or sensibilization (DNCB, DNBB,
TNBS). (d) By anti-CD3-, anti-TCR/Ti-, anti-CD2/T11- and other
mitogenic Mabs, the memory but not naive T cells can be stimulated
to proliferate; in naive/non-primed T cells, the mean distance
between membrane antigens of the same structure, e.g. CD3 or Ti is
too long to be "bridged" by both Fab-subunits. Therefore, novel
antibodies are recommended according to the invention, containing
two or more anti-CD3(Ti, CS2 . . . )-Mabs or their
Fab/F(ab').sub.2-subunits, conjugated to different
spacer-molecules; this renders a direct crosslinking of CD3-, Ti-,
CD2- and other membrane structures possible even on naive Th-cells.
In this way, an APC-independent activation of naive Th-cells is
possible which can be of relevance in immunosuppressed AIDS and
tumor patients.
[0186] In order to prevent at the same time the neutralizing
antibodies against xenogeneic (murine) Mabs, the use of strong
tolerogens, especially polymers/copolymers of D-amino acids, e.g.
D-GL, or PEG or heparin or sialic acid-based polymers, is
recommended.
[0187] The efficacy of the novel type of antibodies can be further
increased by their combination with interferon which increases the
surface density of structures to be crosslinked.
[0188] The tumoricidal and/or virucidal effector cells can be
activated in a novel way by conjugating Mabs or their
Fab/F(ab').sub.2-subunits, recognizing (a) cytokine receptors or
(b) growth factors (EGF, IGF, PDGF etc.) with spacer molecules,
e.g. the CH.sub.2)n-chain or D-GL. D-polylysine as spacer molecule
is of special interest, as it is a tolerogen and as it
facilitates--through the positive charge--the approach of the
Mab-construct to the target cell. The same is true with the
polymers or copolymers of other basic, D-aminoacids. The here
described new principle of introducing spacer molecules as carriers
of Mabs with the same or different specifity, is also applicable
for all simple or combined Mabs, specified in the patent
applications PCT/EP ( )/00403 and DE 3812605A1. (e) Since LAK cells
consist to 90% of NK cells and to 10% of non-MHC-restricted
CD3.sup.+-cells and are free of alloreactive, GvH-inducing T cells,
the allogeneic LAK cells can be used to circumvent the
immunoincompetent phase which is critical for later relapses (a) in
bone marrow recipients and (b) in patients, immediately following
tumor surgery. LAK cells can be depleted of T cells before their
infusion into the patient. LAK cells can be frozen by a special
procedure (with PEG/PVP-addition to DMSO), preserving their
preactivated state.
[0189] *(f) Atherosclerosis seems to be induced by an autoimmune
primer, including hyperactivated macrophages. The latter can be
depressed (a) by corticosteroids (b) by Ca-antagonists (c) by
masking the Fc and CR1/CR3-receptors with Fab/F(ab').sub.2-subunits
of the anti-receptor-Mabs (d) by ROI-reducing agents and
radical-scavengers, and (e) by anti-CD19(CD20, CD21, CS22)-Mabs
(temporary depletion of CIS-producing B-cells). Within the
inflammatory tissue, the immunocytes are exposed (a) to
hypoxic/anoxic and/or (b) acidic micromilieu. (a) The unfavorable
redox-potential in this tissue has to be normalized by
O.sub.2-carriers (e.g. oxoferin, TCDO=tetrachlorodekaoxid) and/or
by other electrone acceptors (e.g. ascorbate, dehydro-ascorbate,
(poly)unsaturated fatty acids). (b) The low pH in the inflammation
tissue inhibits immunocompetent cells. By the pH-rise, the
H.sup.+/Na.sup.+-pump of immunocytes must be relieved. Examples of
pH.sub.i,-raising agents are quaternary bases and their salts
(carbonate, citrate, maleate), further TRIZMA (as base or as salt),
THAM/tromethamine/trometanol, mono-, di- and triethanolamine, etc.
An intracellular pH-rise to 7,3-7,6 is the prerequisite for cell
proliferation. The O.sub.2-deficit is associated also with
prostaglandin instead of leukotrien-synthesis. In order to
down-regulate the immunosuppressive effect of hyperactivated
macrophages on immunocompetent cells in inflammatory tissue (tumor,
persistent infection etc.), the following steps are recommended:
(a) treatment by Mabs and immunotoxins, directed against the
surface antigens on end differentiated macrophages (x-4, x-11,
x-12, x-14, x-15) and against the differentiation antigens on
hyperactivated macrophages (b) treatment by cytotoxic substances,
encapsulated in liposomes (c) treatment by agents which stabilize
the lysosome-membrane in macrophages (e.g. gold preparations such
as aurothioglucose, aurothiopolypeptide or Na-aurothiomalate,
further antimalaria-agents such as chloroquine and
D-penicillamine).
[0190] The proteases are able to switch the cell from the
suppressor--to the effector function. The GEF (glycosylation
enhancing factor), a kallikreinlike kinin-protease prevents the GIF
(glycosylatibn inhibition factor)--induced immune suppression; a
membrane-associated serine-protease is directly involved in the
process of cell activation.
[0191] The lipases are able to activate--via
lysophosphatide--immunocytes (macrophages, NK--, K--, T-cells).
This activation results partly from the mimicking of
membrane-associated phospholipases (PLC, PLA2). The effect of
mucopolysaccharidase is similar to that of proteases. Therefore,
the deblockade of immunocytes by proteases, lipases and
mucopolysaccharidases is recommended, both per se or in combination
with the above described cell activators.
[0192] Interferons, TNFalpha and TGFB switch the cell from
proliferation to dedifferentiation. Their pathologically increased
levels exert an antiproliferative effect on precursor cells,
depressing in this way the recruitment of immunocompetent cells.
Such pathologically raised levels of cytokines can be measured in
AIDS- and tumor-patients, but also in autoimmune disorders and
persistent infections. Therefore, these antiproliferative cytokines
should be neutralized by specific Mabs. In tumor- and
AIDS-patients, the immune response can be improved by special
conjugates, consisting of the Fab/F(ab').sub.2-subunit of tumor- or
HIV-specific Mabs of murine origin plus the Fc epsilon-subunit of
human origin.
[0193] Some membrane-associated immunorelevant structures (e.g. MHC
II, MHC I, CD4, CD8, B2m) become immunosuppressive if they are
shedded into the plasma. The neutralization of such solubilized
membrane structures by specific Mabs helps to prevent their
immunosuppressive effect.
[0194] A further advantage is the use of anti-HLA-DQ- and
anti-LFA-1alpha Mabs.
[0195] The following procedure is of special interest:
[0196] (a) Tumor patients are first in vivo depleted of the
tumor-protecting suppressor T cells (see patent applications
DE3812605A1 and PCT 94/EP89/00403.
[0197] (b) Patient's tumor cells are treated in vitro by IFN gamma
and/or TNF allpha (postexpression of MHC II and MHC I), inactivated
by anti-HLA-DQ- and/or anti-LFA-1alpha or their Fab/F(ab')-subunit
and reinjected into the patient. Alternatively, the tumor cells can
be fusioned (in the presence of PEG) with autologous and/or
allogeneic MHC II-positive cells (B cells or macrophages), treated
by anti-HLA-DQ- and/or anti-LFA-1alpha-Mab and reinjected into the
patient. Both alternative procedures include the immune
reactivation of the patient as an important step.
[0198] The principle of the here discussed novel tumor therapy is
to combine (a) the temporary breaking of resistance of
immunocompetent cells with (b) the reinfusion of in vitro
pregenerated tumor-specific T cells. It is essential that these
cells are memory cells; according to laws of the so-called
"restricted" CML (CML to non-MHC-molecules), only the
blastogenically pretransformed T cells are able to induce in vitro
the response to the (soluble) antigen in question.
[0199] The tumor-specific T cells are capable to induce in vivo a
kind of autoimmune reaction against the tumor antigen if they are
generated as follows:
[0200] (a) Patient's lymphocytes (T cells) are postexpanded in
vitro polyclonally, e.g. by PHA, then the CD8-positive Ts cells are
eliminated e.g. by specific Mabs or immunotoxins, and the residual
CD4-positive T cells are reinjected into the patient, along with
the memory cells. In vivo, these T4 cells are able to induce the
CTLs with specificity for tumor cells. Instead of the
CD8.sup.+-postdepletion, the inducer-suppressor subpopulation can
be inactivated in advance by a 500 Rad-preirradiation.
[0201] (b) Alternatively, patient's lymphocytes (T cells) can be
co-incubated in vitro with his malignant cells, after these have
been induced to post-express the MHC II-antigen.
[0202] (c) An interesting further way comprises the reinfusion of
patient's lymphocytes (T cells), along with mitomycin C-pretreated
patient's macrophages.
[0203] The success of organ- and bone marrow transplantation can be
essentially improved if the donor organ, more precisely its
"passenger lymphocytes". or donor bone marrow cells, respectively,
are pretreated in vitro by anti-HLA-DR- and/or anti-LFA-1alpha-Mabs
(or their Fab/F(ab').sub.2-subunit).
[0204] Alternatively or additionally, the MHC-postexpression on
"passenger lymphocytes" can be depressed by Ca-antagonists (e.g.
verapamil, nifedipin, diltiazem) or by anti-mitogenic substances
(colchicin, domecolcin, gliotoxin). This MHC II--postexpression,
following the organ surgery seems to be critical for the later
organ rejection. As mentioned above, the bone marrow
cryopreservation can be essentially improved by the addition of PEG
and/or PVP of dofferent mol weight to DMSO.
[0205] An interesting novel approach in tumor therapy is the use of
hybridoma cells with restricted lifespan, constructed of
tumor-specific immunocytes and mitomycinc-pretreated immortalized
partner cells. One variant proposes (a) to fuse the pregenerated
immunocytes with myeloma cells which are routinely used for
hybridization purpose, and to treat the resulting hybridoma cells
with mitomycin C. (b) The other possibility is to fuse the
pregenerated immunocytes with mitomycinc-pretreated myeloma partner
cells, or(c) to use the commercially available non-transformed
fibro-blasts or embryonal cells instead of myeloma cells as partner
cells. Instead of mitomycin C, other cell proliferation-limiting
agents or a controlled cell irradiation can be foreseen.
[0206] A typical mitomycin-concentration is 10 ug/ml, a typical
incubation time 18 hours (for 5 ug/106 cells). In this way,
hybridoma cells, based on tumor-specific plasma cells, CTLs, NK--,
K/ADCC-cells and preactivated macrophages can be constructed. They
can be kept in culture or frozen (PEG and/or PVP addition to DMSO);
the preservation of the preactivated state by this improved
cryoprotective mixture is of special relevance for repeated
infusion. The use of the HAT- or HAs-medium guarantees a sufficient
selection of the hybridoma cells; a time-consuming cloning is not
necessary.
[0207] As next, some novel approaches in the treatment of cancer,
chronic infections and autoimmune disorders, respectively, should
be discussed briefly. [0208] (1) In tumor patients and patients
with(chronic) infections, especially those with (retro)viral
infections, the treatment with anti-B-cell-Mabs is recommended,
since the level of immunosuppressive ICs is reduced and
CTL-hindering, antigen-masking specific antibodies are prevented to
be synthesized. Examples of such Mabs are anti-CD19-, anti-CD20-,
anti-CD21- and anti-CD22-Mab, as well as polyclonal anti-B-cell
antibodies. [0209] (2) Due to the negative impact of persistently
activated macrophages on the progress of the disease, their
elimination by monoclonal and polyclonal antibodies, both in tumor
patients and in patients with chronic infections, including
(retro)viral (HIV)ones, is recommended.
[0210] Examples: anti-CD 15-, anti-CD 14-, anti-CD11c- and anti CD
11b-Mates. [0211] (3) The Mabs, described under points (1) and (2)
can be injected as coctail too. [0212] (4) The points (1), (2) and
(3) are valid also for the patients with autoimmune disorders.
[0213] (5) The Mabs can be replaced by their conjugates with
cytokines, radionuclids and/or cytotoxic agents to increase their
efficacy. [0214] (6) The Mabs can be also replaced by
Fab/Fab').sub.2-subunits, masking--not eliminating--B cells and
macrophages. [0215] (7) An essential progress in tumor therapy,
according to the invention, is the introduction of tumor cells with
post-expressed MHC II, induced by interferons and/or TNFalpha.
Before the reinjection, tumor cells are inactivated by mitomycin C,
by heat or formaldebyde and/or glutaraldehyde. [0216] (8)
Alternatively, to point (7), hybridoma cells, arising from the
fusion (in 20-40% PEG) of tumor cells with patient's MHC
II-positive cells (macrophages, B cells), can be used. This cell
fusion can be carried out with non-viable cells and doesn't include
cell cloning. [0217] (9) A further possibility is the presentation
of tumor antigen plus autologous MHC II-antigen on the surface of
liposomes. [0218] (10) A new principle in tumor therapy is the
mimicking of the strong immune reaction, observed (a) during graft
rejection and (b) during the autoimmune reaction. In both cases,
the immune system encounters the antigen (the allogen in organ
grafts and the autoantigen in autoimmune reaction), first in its
"processed" form, i.e. in context with the MHC II-complex. This
favours the generation of Th and Tc cells and depresses the Ts
cells being able to interact with soluble antigens directly, i.e.
without antigen processing. Therefore, the in vivo situation in
graft recipients and patients with autoimmune disorders should be
mimicked in tumor-excised cancer patients by the injection of
patient's tumor cells with the post-expressed MHC II-antigen. An
additional improvement of the therapy is the use of the
above-entioned constructs, consisting of the
Fab/F(ab').sub.2-subunit of tumor-specific IgG or IgM, plus
Fc-subunit of IgE, alone or combined with IL4. [0219] (11) As next,
the in vitro preprocessing of patient's tumor-antigen is
recommended; this occurs by the phagocytosis of the soluble (e.g.
3M-KCI-extract) of tumor antigen or inactivated tumor cells,
followed by the reinfusion of the involved macrophages into the
patient. [0220] (12) The procedure, based on the generation of
Th-memory cells by the in vitro incubation of patient's T cells
with his inactivated tumor cells, followed by the elimination of Ts
cells through anti-CD8-Mabs plus complement, is of special
interest. Both, the so pretreated macrophages and Th-cells mimic
the situation during organ transplantation and autoimmune disorders
which are characterized by a strong in vivo immune response. In
this case, the in vivo immune reaction is directed against tumor
cells. [0221] (13) By the titration, i.e. "neutralization" of the
soluble fraction of immunorelevant membrane structures in plasma by
specific Mabs, the immunosuppressive effect of these humoral
factors can be prevented. By the quantitative analysis, the
patient-dependent amount of the neutralizing Mabs has to be
determined in advance. Both the specific diagnostic tests and the
in vivo use of the neutralizing Mab are subjects of this patent
application. Examples of such molecules are the
immune-response-mediating or potentiating membrane receptors in
general, e.g. IL2/Tac/CD25- and T11/CD2/Leu5a/IL4-receptor, further
various receptor: ligand-systems of cooperating immunocytes
(integrins such as CD2: LFA3, ICAM-1: LFA1), or immunorelevant
structures in general, such as CD4, CD8 and MHC II. [0222] (14) The
procedures, described under points (11) and (12) are also valid for
HIV-infected persons. Classical vaccines can be essentially
improved as follows: (1) by the preformation of antigen(pathogen):
IgM-immunocomplexes which activates the macrophages before Ts cells
are stimulated (2) by the use of preformed complexes, consisting of
pathogen: IgM/IgG: complement (or C-subunit) (3) by the use of
pathogen-specific IgE and/or conjugates of pathogen-specific
Fab/F(ab').sub.2 subunit of IgG/IgM plus Fc-fragment of IgE (of any
specificity) (4) by combining the priming with anti-CD8-Mab plus
anti-B-cell-Mab and the boosting with anti-CD8-Mab alone (without
anti-B-cell-Mab) (5) by masking the macrophages/monocytes with the
Fab/F(ab').sub.2-subunit of the specific Mabs, after the first or
during the second vaccination (6) by injecting Mabs, neutralizing
gamma-interferon, TNFalpha, PGE2 and/or TGFbeta (.+-.IL4) 4-6-days
after the first or during the second vaccination.
[0223] The next point is the prevention of tumor relapses after BMT
by the potentiation of the GvL-effect, without the danger of the
parallelly increased GvH-reaction. After years of research, several
procedures, based on the circumvention of the critical, often fatal
immunosuppressive phase immediately after the tumor removal, have
been worked out. These procedures can be used per se or in
combination with other techniques, described in this patent
application.
[0224] One procedure comprises the admixing of donor lymphocytes,
whose lifespan has been predetermined (preprogrammed) by a special
3-step-procedure, to the donor bone marrow cells. In this way, the
patient gets immunocompetence, until he restores his own immune
response; he is continuously able to combat infections and to
prevent the reinduction of tolerance against residual tumor cells
and herewith the later relapses. Before the donor T cells can
provoke the GvH-disease, they die due to the preprogrammed cell
death. The cell death can be predetermined by a controlled in vitro
pretreatment of donor T cells with DNA-crosslinking agents. The
preprogrammed cell death of immunocompetent donor T cells can be
achieved also--via the intracellular irradiation--by the in vitro
incorporation of radiolabeled, i.e.
radionuclid/radioisotope-containing nucleosides, nucleotides, free
bases and their derivatives into the DNA. Examples of such
radiolabeled nucleosides are: 2'-deoxyuridine-2-.sup.14C, further
2'-deoxyuridine-5-.sup.3H, 2'-deoxycytidine-S-.sup.3H and
5-bromo-2'-deoxyuridine-2-.sup.14C. All nucleosides and
nucleotides, labeled by various redionuclides (e.g. from H,P,S and
other) and added to the culture medium, are subject of this patent
application.
[0225] If donoe's immunocompetent T cells are coincubated with the
recipient's mitimycinc- or irradiation-inactivated MHC II (and/or
MHC 1)-positive cells, the cell death-preprogramming radiolabeled
nucleosides are incorporated nearly selectively into the
alloreactive, recipient-recognizing donor T cells.
[0226] In the special case of the radiolabeled bromo-deoxyuridine,
the prelabeled immunocompetent donor's T cells, added to the
T-depleted donor-bone marrow, can be treated after the restoration
of patient's own immune system ex vivo by UV-irradiation to support
additionally the self-destruction of these cells by radio labeling.
A similar technique of radionuclide-incorporation into the cell
nucleus can be used, according to the invention, for killing of
tumor cells in situ. Here, the carriers of the radioactive,
tumor-destroying radiation are not tumor-specific Mabs but (a) the
tumor-recognizing TIL (tumor infiltrating lymphocytes), and (b) the
tumor-specific Tc/CTL(cytotoxic T cells).
[0227] A special advantage of the cellular over the molecular, i.e.
Mab-mediated transport of the tumor-destroying radionuclides into
the immediate neighborhood of tumor cells is the essentially
intensity (density) of the cellular irradiation source. The
selectivity (a) of TIL cells as carriers of radioactivity is
guaranteed by the tumor-specificity of TIL-cells (Rosenberg,
Anderson, Blaese), and (b)) that of tumor-specific Tc/CTL by their
selective recognition of tumor antigens (TATA, TSTA, TSA). Though
TIL cells belong both to the CD4-and to the CD8-positive T cells, a
clear cut-off between TIL- and Tc/CTL-cells is not possible.
[0228] The preparation of TIL- and Tc/CTL-cells as carriers of
tumoricidal irradiation includes radiolabeled amino acids and other
radiolabeled cellular components (precursors), in addition to the
radiolabeled nucleosides; they must be added to the culture medium
and are incorporated into TIL- and/or Tc/CTL-cells.
[0229] A further possibility of bridging the fatal
immunosuppressive phase after the removal of primary tumor or after
following the BMT is the in vitro pretreatment of donor's
immunocompetent cells by photosensitive dye-stuffs (e.g. psoralen),
followed by their addition to the donor's T-depleted bone marrow,
and their transfusion into the recipient. Later, after having
protected the patient (recipient) from infections and from the
clonal expansion of residual tumor cells, these photolabeled cells
are selectively eliminated ex vivo by UV-irradiation. The used
technique (photopheresis/PUVA) profits of predeveloped devices. For
efficiency reasons, the combination of this technique with other
here described techniques is recommended.
[0230] In analogous technique, photosensitive dye-stuffs are
combined with or replaced by the radiolabeled or non-labeled
bromo-deoxyuridine; again, the labeled cells are selectively killed
ex vivo by the UV-irradiation. The use of radioactive instead of
the cold isotope in BrdUr increases the efficacy of the technique.
It should be combined with a preincubation of donor immunocompetent
cells with inactivated MHC II (and MHC-1)-positive recipient cells
which allows a selective preprogramming of cell death in the
recipient-specific alloreactive subpopulation of the donor.
[0231] A further way is the in vitro transfection of donor's
immunocompetent T cells with strongly immunogenic surface antigens,
followed by their addition to the T cell-depleted donor bone marrow
and by later transfusion into the recipient (e.g. tumor patient).
The later in vivo elimination of the mature donor T cells which
helps to bridge the immunoincompetent phase is carried out by
antibodies, directed against the transfected membrane antigen.
These antibodies can be conjugated with cytotoxins in order to
increase their efficacy. A further modification of this technique
is based on the addition of T cells of a second MHC-incompatible
donor to the T depleted bone marrow of the first donor. After the
restoration of patient's own immunocompetence, mature
histoincompatible donor T cells are eliminated in viva by allotypic
antibodies or corresponding immunotoxins. All these techniques can
be used alone or in different combinations. The next procedure is
based on the cumulative (additive) effect of DNA-damaging by
cytotoxic agents and/or irradiation. The immunocompetent cells of
the donor are treated first in vitro by the sub-lethal dose of the
DNA damaging cytotoxic agent and/or irradiation and later in vivo
after the finished mission (bridging of the fatal immunosuppressive
interphase, before the restoration of patient's immune competence)
by a second (lethal) dose of cytotoxic agent(s) or irradiation. The
strong GvL-effect of the so pretreated donor bone marrow can be
further intensified if the donor's immunocompetent T cells to be
admixed to the donor bone marrow are predepleted of alloreactive
subclones and activated in a tumor-specific or non-specific way,
before they have been injected into the patient. In addition, the
technique, based on a novel type of immunocompetent T cells which
is characterized by a "frozen" activated functional state, should
be described briefly. Details about this novel cell type,
implicating a constitutively activated state without cell
proliferation, follow below.
[0232] Cells of this novel type can be added to T-depleted donor
bone marrow and must not be later on eliminated in vivo, because
their ability to proliferate is genetically switched off.
Characteristically for all these techniques is (a) a strongly
increased GvL-effect, without a simultaneous GvH-reaction, (b)
opening of new ways for histoincompatible BMT, (c) the option of an
additional tumor-specific and/or non-specific preactivation of
immunocompetent T cells, admixed to the T-depleted donor bone
marrow, and (d) the impact on both the GvH- and HvG-reaction which
minimizes the BMT-associated complications.
[0233] These facts support the inventor's idea to introduce BMT
obligately in the patients with solid tumors. According to the
invention, the classical BMT could be replaced by the simple
exchange of patient's (recipient's) T cells by T lymphocytes of the
donor in patients with solid tumors. This can be carried out by an
in vivo depletion of patient's T cells by specific Mabs or
immunotoxins and transfusion of healthy donor T cells; the
histoincompatibility problems can be overcome by the above
described special pretreatment of immunocompetent T cells.
[0234] According to a further therapeutic model, the bridging of
the fatal immunosuppressive phase, following the tumor excision or
BMT can be achieved by autologous or allogeneic LAK-cells plus
rIL-2. Since LAK-cells consist of ca. 90% NK-cells and of ca. 10%
non-MHC-restricted CD3.sup.+-T cells, the in vitro predepletion of
CD3.sup.+, cells is recommended in the case of allogeneic
LAK-cells. Alternatively, the complete LAK-population can be
treated by one of the above described cell death-preprogramming
procedures. The so pretreated allogeneic LAK cells are added to the
donor bone marrow before their injection into the recipient. An
important positive "side effect" is the in vivo killing of
recipient's HvG-inducing residual T cells. In this way, the
complications of a radical recipient conditioning could be
prevented. This HvGR-inhibiting effect is based on the property of
NK-- and LAK-cells to recognize and to inactivate--via the
4F2/TNKTar-antigen--fastly proliferating cells. The plasmapheresis
brings some additional advantages in the autologous or allogeneic
BMT. The removal of immunosuppressive factors, belonging primarily
to the immune complexes, to the solubilized cytokine- and growth
factor-receptors, as well as to the prostaglandins, has been
neglected in conventional BMT. A further improvement is the
addition of fibroblast to the donor BM. The situation following BMT
shows a common element with the situation of immunocytes in the
limiting dilution-test, in which the added fibroblasts, by
secretion of growth factors, make the growth and the survival of
immunocompetent cells possible.
[0235] Though there are trends to shorten the phase of
immunoincompetence in tumor patients and BM-recipients by cytokines
(G-CSF, GM-CSF), fibroblasts, admixed to the donor bone marrow are
expected to secrete a much broader spectrum of cytokines.
[0236] As next, the addition of donor-macrophages to donor-BM is
recommended; in this way, the cooperation of accessory cells (APC)
with T cells in the critical post-transplantation phase is
guaranteed. The temporary masking of recipient-macrophages (to
inhibit the preexistent suppressor monocytes) is also of
interest.
[0237] The next point is the selective in vivo depletion of the
recipient macrophages/monocytes (e.g. by specific Mabs or
immunotoxins) and/or the addition of donor-macrophages to donor
bone marrow. Finally, the use of neutralizing Mabs, directed
against all those cytokines which allow the mutual activation of
macrophages, T4 cells and NK cells in the critical phase of the
GvH- and HvG-reaction, is recommended. So, the clonal expansion of
alloreactive subpopulations, following allogeneic bone marrow and
organ transplantation, can be prevented by neutralization of TNF
alpha, IL-1, IL-6 and/or gamma IFN. As next, a novel cell type
should be described which is characterized by the "frozen", i.e.
constitutively activated functional state and by a parallel
switching off of the cell proliferation. This state is associated
with the cell arrest in the G.sub.1- or G.sub.2-phase and with a
permanently increased level of the intracellular
Ca.sup.2+(Ca.sub.1). In the case of ex vivo generated LAK- and
TlL-cells, the problem of a rapid activity drop of these cells in
vivo could be solved by the corresponding cell modification. This
in vivo inactivation of LAK- and TlL-cells stems from the induction
of lipocortin/lipomodulin by plasma corticosteriods (cortisol) and
can be prevented in two ways: (1) by the constitutive,
proliferation-free activation of LAK- and TlL-cells, and (2) by the
transfection of LAK- and TIL-cells with the CDNA, encoding the
cortisol-cleaving enzymes, such as
20alpha-hydroxysteroid-dehydrogenase and B-glucuronidase.
[0238] The T cell-activation (a) by different cytokines, such as
IL-2, IL-3 and CSF-1/M-CSF, and (b) by (processed) antigen occurs
in the same way, i.e. via the activation of the PI
(phosphoinositol)--dependent PLC (phospholipaseC) and results in
the increase of the intracellular Ca,.sup.2+-level.
[0239] The constitutively increased Ca,.sup.2+-level in the cell
cycle-arrested cells (subject of this patent application) opens a
novel way of cell activation. A special advantage is the cell
activation and the maintaining of this activated state even in the
absence of the specific signal, e.g. the processed antigen in the
case of helper T cells. In other terms, the constitutively
.sup.increased cai2+-level in the cell cycle-arrested cells confers
to these cells the genetically predetermined (highly specialized)
function, e.g. the production of specific antibodies or the CTL- or
ADCC-activity. A further advantage is the inner stability, i.e. the
resistance against the specific or non-specific suppression by
suppressor factors, such as prostaglandins (e.g. PGE1/E2) or
corticosteroids, without the danger of an uncontrolled cell
proliferation (because of the cell cycle-arrest).
[0240] This goal can be reached in two ways: (a) by hybridization
of cells with the desired function, e.g. plasma cells, CTL/Tc,
T4/Th etc. with immortalized cells (autologous tumor cells of the
patient or transformed cells of other origin), followed by the
treatment of formed hybridoma cells by cell death-preprogramming
techniques, as described above, and (b) by a double transfection of
the cells, showing the desired function (B, T4, T8), with the
sense-cDNA, encoding the constitutive cell activation plus
antisense-cDNA, encoding the switching off of cell
proliferation.
[0241] Ad (a): The cell death-preprogramming treatment can occur
either in hybridoma cells or in immortalized partner cells, before
these have fused to give hybridoma cells.
[0242] The rejection of hybridoma cells, based on non-allogeneic
immortalized cells, must be prevented (a) by their repeated
incubation in the presence of the alloantigen-specific antibody, or
(b) by transfection of the cells with the histospecific
antisense-cDNA.
[0243] Candidates for the immortalized partner cells are those
transformed cells which maintain an increased intracellular
Ca.sub.i.sup.2+-level by autocrine or paracrine mechanisms or such
transformed cells which are able to maintain--independently of
external signals--the activated state either by the constitutive,
ligand-dependent activity of tyrosine- or serine/threonine-kinases
or by the continuing PI-conversion to PIP2.
[0244] Ad (b): The double transfection of the target cells, showing
the desired function (B,T4,T8 . . . ) (b1) with sense-cDNA,
encoding the constitutive expression of cell-activating signals,
plus (b2) with antisense-cDNA, encoding the turning off of cell
proliferation, creates a permanently "turned on", non-proliferating
cell which doesn't need any specific signals (e.g. processed
antigen) for its activation.
[0245] These novel cell constructs have an enormous practical
relevance. Examples are (1) the intracranial injection of NGF
(nerve-growth-factor)--and/or DOPA-secreting, long-lasting,
fibroblast-based cells constructs which cannot be down-regulated by
plasma suppressor factors and are of special interest in the
treatment of M. Alzheimer and M. Parkinson, further (2) the
cytokines and growth factors-producing cell constructs which
support patient's immune system, following high-dose
(radio)chemotherapy and BMT, as well as (3) cell constructs, based
on tumor-specific plasma cells which are able to produce
anti-tumor-Mabs in situ, preventing in this way the induction of
neutralizing immunoglobulins.
[0246] As the sense-cDNA, encoding cell activating signals (1) the
cDNA, encoding various cytokines or (hematologic) growth factors
and/or their receptors, further (2) the PLC- and PLA2-encoding
cDNA, (3) the cDNA, encoding different cytoplasmic
serine/threonine-kinases, and (4) the cDNA, encoding various
protein-kinases, such as C-kinase, Ca/calmodulin-kinase,
casein-kinase II and G-kinase, are recommended.
[0247] The prevention of cell proliferation can be achieved by the
antisense-cDNA of all cell division-inducing factors. Examples are
(a) cyclinA, cyclinB 1, c-ras, c-raf, PSTAIRE, MPF, p34.sup.cdc2,
p13, further (b) the DNA-transcription factors like AP-1 (AP-I) and
AP-2(AP-II), and (c) DNA-polymerase-alpha, PCNA and (protein)
elongation factor (e1F-2/e1F-2p). PSTAIRE is a cdc2-subregion
(aminoacids 42-56) and belongs, like p34.sup.cdc2, to the family of
cell cycle-specific protein-kineses. The PCNA (proliferating cell
nuclear antigen) is a 36 kD-intra-nuclear-polypeptide and component
of polymerase-delta; the MPF stands for mitose- or
M-phase-promoting-factor.
[0248] Alternatively to these antisense-cDNAs, various sense-cDNAs,
encoding suppressor-oncogenes, such as p105RB and p53 can be
transfected to prevent uncontrolled cell proliferation. By
transfection of extra-copies of the p53 and/or RB-cDNAs,somatic
cells, including leukocytes, can be made more resistant against
different carcerogenic agents.
[0249] The uncontrolled proliferation can be switched off also by
cell fusion with normal cells, expressing the wild type or the
wild-type p53.
[0250] One of the points, according to the invention, is the
prevented expression of the MHC II or MHC I complex on the cell
construct, achieved by the transfection of the MHC II or MHC
I-encoding antisense-cDNA. The antisense-cDNA can be replaced (a)
by ribozymes, (b) by psoralent derivatives of the
antisense-oligonucleosides or -oligonucleosidemethylphosphonates,
and (c) by antigen- and
antisense-oligonucleotid-intercalator-conjugates.
[0251] (a) The advantage of ribozymes, called also "catalytic RNA",
over the corresponding antisense-cDNA is their irreversibility as
they cleave the sense-DNA. The smallest and simplest self-cleaving
domain of ribosome is the "hammerhead"-structure, e.g. the
"structure I", described by Uhlenbeck or the "form IV", reported by
Haselhoff and Gerlach.
[0252] (b) The psoralene-derivatives of
antisense-oligonucleosidmethylphosphonates are also irreversible in
their action; here, the desired sense-DNA is switched off by the
photoinduced DNA-crosslinking.
[0253] (c) When the technique, based on
oligonucleotid-intercalator-conjugates is used, the irreversibility
is achieved by the conjugation of antisense-cDNA or -RNA with the
chemically (Cu.sup.2+-phenanthroline) or photochemically
(ellipticin) inducible intercalator-molecules.
[0254] The immunotherapy of malignancies and autoimmune disorders,
as well as of bacterial and (retro)viral infections is based on the
same principles as the improvement of conventional vaccines and
prevention of graft rejection, namely (a) on the deblockade of the
hyperactivated state of immunocompetent cells and/or (b) on the
elimination or inactivation of hyperactivated effector cells
("microimmunosurgery"). (a) This deblockade of the hyperactivated
state of immunocytes can be achieved, according to the invention,
by combining agents which block the "voltage-operated" and/or
"receptor-operated" Ca.sup.2+-channels ("component I"), with agents
which reduce the intracellular cAMP or the cAMP/cGMP-ratio,
respectively ("component II").
[0255] The "component I" comprises "classical" Ca-antagonists
(Ca-channel-blockers) of all subtypes, e.g. phenylalkylamines,
dihydropyridines, benzothiazepines, piperazines, quinoxalines,
quinazolines (e.g. bepridil and perhexilin). Alternatively or
additionally, a parallel inhibition (blockade) of alpha- plus B
adrenoceptors, of H2-plus H1-histamine receptors, of A2- plus
A1-adenosine receptors, of 5-HT/serotonine) receptors and/or
receptors of various inflammation mediators (e.g. bradykinin,
kinin-cascade, complement-cascade, especially c5a, c4a, c3a, PAF
etc.) is recommended. Examples of preferred combinations are listed
below; they can be combined with sub-dosed nitro-compounds,
including molsidomine, Ca-overload blockers, e.g. cinnarizine,
Ca-antagonists, BRM and/or cytokines. The combination of
molsidomine and nicerogoline (an alpha-blocker), with or without
Ca-overload-blocker(s) (e.g.cinnarizine) is of a special
interest.
[0256] The "component II" comprises cAMP/cGMP- or cAMP-reducing
agents. Examples are antagonists of all cell receptors which are
coupled via the Gs-protein to the membrane-associated adenylate
cyclase (AC). This effect is increased if agonists of G.sub.p-,
G.sub.i- or G.sub.o-protein-coupled receptors and/or of
cGMP-increasing, Ca.sub.i, reducing nitro-compounds (e.g.
isosorbid-mono- and -dinitrate, glycerol-trinitrate/nitroglycerol,
erythrit-tetranitrate, pentaerathrit-tetranitrate, amylnitrite,
molsidomine) are given parallelly. The antagonists of those
Gs-coupled receptors whose normal, physiological agonists bind at
the same time P.sub.p-, G.sub.i- or G.sub.o-coupled receptors are
of a special interest. After having blocked the. Gs-coupled
receptors, the Gs-coupled receptors, the endogenous ligand bind the
G.sub.p-, G.sub.i- or G.sub.o-coupled receptors to a higher degree.
In this way, B-blocker achieve 2 effects, the cAMP-drop and the
cGMP-rise in cytosol of immunocompetent target cells. Examples of
such endogenous agonists are catecholamines (adrenaline and
nonadrenaline), histamine and adenosine. So, the antagonists of
B-adrenoceptor (on immunocytes, e.g. T cells and
monocytes/macrophages) cause catecholamines, primarily adrenaline,
to interact with the alpha--instead of B-adrenoceptor. Similarly,
the antagonists of H2-histamine receptor make the histamine ligate
the cAMP-lowering H1--instead of the cAMP-increasing H2-receptor.
In the presence of A2-antagonists, the endogenous agonist adenosine
binds to the cAMP-depressing A1--instead of to the cAMP-raising
A2-subtype P1-adenosine receptor.
[0257] It is also advantageous to use the agents which inhibit
reversibly both, the Ca-- and the Na-channels. The working
mechanism is the increase of the resting potential of
immunocompetent cells which is decreased during the hyperactivated
state. This class of substances comprises (a) some Ca-antagonists
like cinnarizine, flunarizine, fendiline, bepridil, tiapamil and
partly verapamil and gallopamil, further (b)sub-dosed
antiarrhytmics (class I to IV), especially the combination of class
IB with class III, due to the equilibrated K-efflux (class IB) and
K-influx (class III) and synergism in the inhibition of Na-channel
(among these agents are adrenoceptor-blockers sotalol and
propranolol). The combination of cinnarizine and propranolol is of
a special interest.
[0258] The central point of this invention is the combination of
agents which decrease the cAMP-level or the cAMP/cGMP-ratio with
those which block the "voltage-operated" and/or "receptor-operated"
Ca-channels. In special cases, the "component I" and "component II"
are identic. So, some special nitro-compounds, such as syndnonimine
derivatives (e.g. mosidomine), show both effects (cGMP-rise,
Ca.sub.i-decrease); they are also subject of this invention.
[0259] Both, the therapy of the diseases discussed above and the
efficiency of conventional vaccines can be further improved by
methylxanthines, by pHi-increasing substances, ba redox-potential,
GSH/GSSG and NADP)H/NAD(P).sup.+ correcting agents, as well as by
ionic homeostasis and K-balance influencing substances.
[0260] (b) Inactivation/elimination of hyper- or persistently
activated immunocompetent cells.
[0261] The principle of this in vivo inactivation or depletion of
hyperactivated effector cells is the "microimmunosurgery", a new
technique which would compete with or complement the gene-therapy
in future. The principle is the selective inactivation of
disease-inducing and disease-maintaining lymphocyte-subclones by
the combination of (b1) panT- or T subclass-specific Mabs or
Mab-derived immunotoxins, plus (b2) alloreactive T cells of a
healthy donor. This combination of humoral (Mab) and cellular
(allogeneic T cells) technique is able to increase essentially the
efficiency of the Mab-mediated depletion of pathogenic
immunocyte-subclones, as shown in animal model. Tumor patients as
well as patients with (chronic) infections (including HIV) and
those, suffering from CFS (chronic fatigue syndrome) show a
strongly increased number of CD8-positive (Ts) and/or HLA-DR(MHC
II)-positive T cells. By the combination of anti-CD3- or
anti-CD8-Mabs plus allogeneic donor-PBM/PBL or donor T cells whose
cell death is "preprogrammed"/"predetermined" by a special in vitro
treatment, the meotioned pathologic (hyperactivated)
CD8:HLA-DR-positive effector cells can be selectively eliminated in
vivo. In this way, a 94-100%-survival rate in tumor-bearing mice
could be achieved.
[0262] The "preprogramming" of cell death in allogeneic donor
PBM/PBL or T cells is a multistep in vitro procedure, comprising
the following steps: [0263] (1) Synchronization of "donor effector
cells" (a) by their incubation in serum-free medium, followed by
the incubation in serum-containing medium, or (b) by cell
incubation first in the absence and later in the presence of
essential aminoacids (e.g. isoleucin), or (c) by cell incubation in
the presence of synchronizing cytotoxic agents, such as
vinchristine, hydroxyurea or bleomycin. [0264] (2) Treatment of
"donor-effector cells" by (a) bifunctional alkylating agents, i.e.
DNA-cross-linking agents (e.g. mitomycin C) and/or (b) by
inhibitors of the enzyme ribonucletoid-reductase (e.g.
hydroxyurera) which block the DNA--but not the RNA--or
protein-synthesis. [0265] (3) Incubation of "donor effector cells"
in the presence of inhibitors of DNA-reparases (DNA repair system)
(e.g. hydroxyurea). [0266] (4) A thorough washing of cells, e.g. by
PBS or RPMI 1640. [0267] (5) Infusion of so pretreated "donor
effector cells" into the recipient (e.g. tumor patient).
[0268] An alternative procedure consists of the incubation of
"donor effector cells" (a) with radiolabeled DNA-constituents
(purine- and pyrimidine bases) and/or (b) with radiolabeled
aminoacids.
[0269] A further alternative is the irradiation of "donor effector
cells", followed by their incubation in the presence of inhibitors
of DNA-reparases (e.g. hydroxyurea).
[0270] In the special case of autoimmune disorders, the target
cells of "preprogrammed" alloreactive donor T cells are also the
HLA-DR(MHC II)-positive autoaggressive T cells (mostly T4, partly
T8 cells).
[0271] The procedure consists of the in vivo depletion of T cells
(by anti-panT/CD3-Mabs) or their T8 and/or T4 subclass. Because of
the high costs for the "pure" Mabs and Mab-derived immunotoxins,
their combination with cytotoxic agents, especially
cyclophosphamide is recommended.
[0272] A further improvement of the technique is the reinfusion of
patient's peripheral blood cells which have been preactivated ex
vivo against the donor PBMs/PBLs, into the patient, after the
premanipulated donor effector cells have eliminated the
pathologically activated recipient lymphocytes.
[0273] As animal experiments have shown, the efficiency of
"microimmunosurgery" is so high that the donor effector cells are
able to lyse patient's pathologic leukocyte-subclones even (a)
without a preceding in vivo Ts-depletion (by Ts- and/or
panT/CD3-specific Mabs), though the combined attack on the cellular
and humoral level remains the most efficient approach. All
experiments, however, show an enormous rise in their efficacy when
the in vivo used Mabs are combined with the cell
death-preprogrammed effector cells. This concerns the
therapeutically used anti-tumor-Mabs, the Mabs, utilized in the
recipients of organ grafts (e.g. anti-CD3-Mabs) and the Mabs, used
in the treatment of autoimmune disorders (e.g. anti-CD4-Mabs).
[0274] A further possibility (a) to bridge the critical
immunoincompetent phase, e.g. after the excision of primary tumor
or following the BMT, and (b) to prevent GVHR (but not GVLR), is as
follows:
[0275] (a) Patients, e.g. those with solid tumors, are first
"conditioned" by anti-CD3-Mab (or the corresponding immunotoxin),
or by the Mab-saving combination of cytotoxic agent (e.g.
cyclophosphamide=plus Mab
[0276] b) In addition, the CD8:HLA-DR/DQ-double positive suppressor
fraction is eliminated by means of "microimmunosurgery".
[0277] (c) The next step is the in vivo depletion of donor effector
cells by patient's (i.e. autologous) T cells or PBMs/PBLs. The
ratio between the autologous T cells or PBMs/PBLs (point (c)) on
the one hand and the corresponding leukocyte-subpopulations of the
healthy donor (point (b)) on the other hand should be 3:1 to 10:1;
in this case, the cell death-programming can be omitted. Both, the
donor effector cells (point(b)) and the autologous effector cells
(point (c)) can, but must not be prealloactivated. It is
advantageous if parental PBMs/PBLs are used as donor effector
cells.
[0278] The presensibilization of donor alloreactive effector cells
against the recipient lymphocytes brings an additional advantage:
The so presensibilized (primed) donor effector cells are
blastogenically pretransformed and can be activated in vivo, in
some analogy to the effector cells of the secondary MLC and those
of the PLT 1-2 days earlier as the non-primed clones. This fact
confers to the presensibilized (primed) donor cells the crucial
advantage, that they are able to eliminate--following their
infusion into the patient--his pathologically (hyper)activated, MHC
II-positive subpopulations before the patient's defense against
these therapeutically utilized donor-effectors can be organized.
For this reason, the number of donor effectors to be transfused can
be reduced and the cell death-preprogramming possibly omitted.
[0279] (c) Improvement of organ grafts (kidney-, heart/lung-,
liver-allografts)--The acute rejection, leading to the loss of the
graft organ can be essentially improved, according to the
invention, if the classical procedures, based (a) on a generalized
immunosuppression (azathioprine, prednison, (methyl)prednisolon,
cyclosporinA), and/or (b) on the in vivo T cell depletion by
anti-CD3-Mabs or ALG or ATG, are completed or partly replaced by
the "microimmunosurgery" or by the cell death-preprogrammed
effector cells.
[0280] (d) Improvement of conventional vaccines--The same principle
(deblockade and/or inactivation/elimination of hyperactivated
effector cells) can be used to improve the conventional
antibacterial and anti(retro)viral vaccines, including the
anti-HIV-vaccines; the target cells are here the CD8: MHC II-double
positive suppressor effector cells. The principle is the increase
of the absolute number of blastogenically pretransformed,
pathogen-specific Th-, Tc/CTL- and/or B(plasma) cells by (a)
deblockade and/or (b) inactivation/elimination of pathogen-specific
Ts cells. In an early phase, these Ts inhibit the generation of
pathogen-specific Th-, Tc/CTL- and B-memory cells. By (a)
deblocking and/or (b) inactivating/eliminating the Ts-cells, the
clonal expansion of "positive" memory cells (Th, Tc, B) is strongly
increased and the protection of infection significantly
improved.
[0281] (a) The deblocking of Ts-cells occurs by the combination of
"component I" and "component II"
[0282] (b) The inactivation/elimination of CD8:HLA-DR/DQ-positive
Ts-cells is performed by alloreactive, cell-death-preprogrammed
effector cells (see above)
[0283] (c) The "immunologic memory", induced in a pathogen-specific
way by vaccination, can be further improved by eliminating in vivo
the pathogen-specific Ts-cells of the vaccinated person with subset
(CD8/Ts)- and/or panT(CD3)-specific Mabs or corresponding
immunotoxins
[0284] (d) The number of pathogen-specific Th-, Tc- and B-cells can
be increased also by delaying the antibody-production through Mabs
(and immunotoxins), directed against (d1) B-cells, (d2)
Th(T4)-cells, (d3) B-plus Th(T4)-cells, (d4) monocytes/macrophages
("suppressor monocytes"), and/or (d5) B-cells plus
suppressor-monocytes.
[0285] Again, "pure" Mabs can be replaced by the Mabs-saving
mixture of cytotoxic agents (e.g. cyclophosphamide) plus Mab.
[0286] (e) Support and partial replacement of glucocorticoids (e.g.
cortison) by low-dosed antagonists of Gi(Gp,Go)-coupled receptors
and/or by low-dosed agonists of Gs-coupled receptors
[0287] Glucocortocoids, characterized by strong side-effects, can
be supported or partly replaced by sub-dosed blockers of Gi-, Gp-
or Go-coupled receptors and/or by low-dosed ligands of Gs-coupled
receptors. These agents can be combined with sub-dosed
Ca-antagonists.
[0288] The indication of these novel combination preparations
corresponds to that of "classical" glucocorticoids. Again, the
antagonists of those Gi(GP, Go)-coupled receptors which share the
endogenous ligand with a Gs-coupled receptor (e.g. catecholamines,
histamine, adenosine) are of a special interest. With such
combinations, other clinically used immunosuppressive agents (e.g.
cyclosporine, FK506, rapamycin, azathioprin, cyclophosphamide) can
be supported or partly replaced.
[0289] Since the hyperactivated state of effector cells (T cells,
macrophages etc.) is an important element in the pathogenesis (a)
of cancer (b) of autoimmune disorders (c) atherosclerosis (d)
infectious diseases (including HIV), the procedures, described
under the point (a) ("Deblocking of hyper- or persistently
activated immunocompetent cells") and under the point (b)
("Inactivation/elimination of hyper- or persistently activated
immunocompetent cells") are valid for all these diseases.
[0290] The reduction of background-signals, i.e. "filtering out" of
non-specific (non-productive) transmembranal signals increases the
susceptibility of immunocompetent cells for specific,
immunorelevant signals. [0291] 1 Combination of sub-dosed B-(B1-
plus b B2-) and sub-dosed alpha-(alpha1- plus alpha2) adrenoceptor
blockers (antagonists) (objective: prevention of hypoergic or
anergic state of hyperactivated immunocompetent cells by lowering
the level of non-specific background-signals). [0292] 1.1
Combination of preparations on pindolol-basis (e.g.
durapindol/-15/-retard (26.039) or pinbetol/forte (26.067) plus
preparations on phenoxybenzamine-basis (e.g. dibenzyran
1/5/10(81.091)). Pindolol is a B1- plus B2-sympatholytic,
phenoxybenzamin an alpha1- plus alpha2-blocker. Recommended dose:
1.times.15 mg/d or 3.times.5 mg/d or 2-3.times.1 mg/d dibenzyran.
[0293] 2 Combination of sub-dosed B-(B1- plus
B2-)-adrenoceptor-blockers with sub-dosed
alpha1-receptor-antagonists, blocking at the same time the
H1-histamine and the 5-HT.(serotonin) receptor (objective: see
point (1)). [0294] 2.1 Combination of drugs on pindolol-basis (e.g.
durapindol/-15/-retard (26.039), or pinbetol/forte (26.067) plus
preparations on indoramin-basis (e.g. wydora/50(16.039)). Indoramin
is the antagonist of alpha1-adrenoceptor, of H1-histamine receptor
and of 5-HT receptor. Recommended dose: durapindol (see above);
indoramin 1.times.25 mg/d.
[0295] Combination of sub-dosed B- (B1- plus B2-)
adrenoceptor-antagonists with sub-dosed alpha- (alpha1- or alpha2-)
receptor blockers (objective: see point (1) and (2)). [0296] 3
Combination of preparations, based on 3.1.1. alprenolol (e.g. aptin
(26.002)), 3.1.2. bupranolol (e.g. betadrenol 50/-100(26.017)),
3.1.3. penbutolol-sulfate (e.g. betapressin (26.019))., 3.1.4.
bisoprololfumarat (e.g. concor 5/10 (26.025)) or 3.1.5. carteolol
(e.g. endak 5/10 (26.046)) plus preparations, based on 3.1.1.
urapidil (e.g. ebrantil 30/60/90 (16.032)), [0297] 3.1.2.
doxazosinmesilate (e.g. cardular 1 mg/-2 mg/-4 mg (16.029) or
diblocin 1 mg/-2 mg/-4 mg (16.030)) or 3.1.3. terazosin (e.g.
heitrin 1/2/5 (16.035)). Recommended doses: aptin-duriles
1.times.200 mg/d; betadrenol 1-2.times.50 mg/d; betapresin
0,5-1.times.40 mg/d; concor 1.times.5 mg or 1.times.10 mg/d; endok
5 mg/d; ebrantil 30 mg/d, cardular 1 mg/d; diblocin 1 mg/d; heitrin
0,5-1 mg/d. [0298] 4 Combination of sub-dosed H2- and H1-histamine
receptor antagonists (objective: see above). [0299] 4.1 Combination
of preparations, based on cymetidine (e.g. sigacimet 200/-400/-800
(59.102) or tagamet 200/-400/-800 (59.105) or H2-blocker ratiopharm
(59.096) plus preparations, based on 4.1.1. oxatomid (e.g. barpet
(07004)), 4.1.2. bromopheniramine-hydrogenmaleate (e.g. dimegan
(07.005)), 4.1.3. dimetindenmaleate (e.g. fenistil (07.006)) or
4.1.4.terfenadine (e.g. hisfedin (07.009)). Recommended doses:
sigacimet 1.times.200 mg/d; tagamet 1.times.200 mg/d; H2-blocker
ratiopharm 1.times.200 mg/d; barpet 1.times.30 mg/d; dimegan
1.times.12 mg/d; fenistil 1.times.1 mg/d; hisfedin 0,5-1.times.60
mg/d. [0300] 5 Combination of sub-dosed antagonists of A2- plus
A1-P1-purinergic (adenosine) receptors (objective: see above.
[0301] 5.1 Combination of preparations, based on methylxanthines
(theophylline) (e.g. aerobin mite (27.102) or contiphyllin retard
(27.113) or euphyllin N (27.125)) plus preparations, based on
ipratropium-bromide (e.g. atrovent (27.048) or itrop (09.028)).
Recommended doses: aerobin 0,5-1.times.200 mg/d; contiphyllin
0,5-1.times.300 mg/d; euphyllin N 1.times.73 mg/d; atrovent
0,5-1.times.200 mg/d; itrop 0,5-1.times.10 mg/d. Combinations,
based on cromoglicinic acid (diNa-salt) and ketotifen-hydrofunarate
are also of an interest.
[0302] Sub-dosed antagonists under the points (1)(2)(3) and (4) can
be combined. In this case, the dose has to be reduced to 5-50% of
the dose, quoted under the points (1) (2) (3) and (4). The strongly
sub-dosed antagonist under the points (1)(2)(3) and (4) can be
combined with conventional Ca-antagonists and/or with sub-dosed
agonists of Gp- and Gi-coupled receptors.
(I) Malignancies
[0303] (1) The restoration of the immunocompetence following the
sublethal irradiation or high-dose chemotherapy can be
accelerated--according to the invention--if cytokines such as M-CSF
or GM-CSF are combined with (a) antagonists of Gs-coupled
receptors, (b) agonists of Gi(Gp/Go)-coupled receptors, and/or
(c) Ca-antagonists.
[0304] (2) Therapeutic approaches, based on tumor-specific Mabs
(and corresponding immunotoxins), can be improved essentially by
combining these Mabs (a) with anti-CD3- and/or anti-CD8-Mabs, and
(b) with "micro-immunosurgery", tumor patients are (a) first
treated by the combination of Mab-saving cyclophosphamide plus
anti-CD3- or anti-CD8-Mab, then (b) injected by alloreactive donor
effector cells (PBM/PBL or T cells) which can be primed against the
recipient and (c) treated again by cyclophosphamide plus anti-CD3-
or anti-CD8-Mabs in order to eliminate the alloreactive donor
cells, and (d) finally reinjected with the autologous PBM or T
cells, collected before the start of therapy, to restore the
recipient's immunocompetence. In a simplified version, the step (c)
can be omitted.
[0305] (4) The reinduction of tumor-specific Ts cells can be
prevented (a) by Mabs, directed against immature T cells (e.g.
anti-T6-, antiT9-, anti-T10-Mabs), (b) by antagonists of Gs-coupled
receptors, and/or (c) by agonists of Gi-coupled receptors.
[0306] (5) To prevent an early RES-elimination of allogeneic
effector cells during the "microimmunosurgery", a simultaneous
injection of allogeneic erythrocytes or inactivated autologous
erythrocytes is recommended (temporary "RES-blockade")
[0307] (6) The combination of (a) insuline or antidiabetes with (b)
glucose or di- and tricarbonic acids can amplify the above
described deblocking process of hyperactivated macrophages and T
cells. Instead of di- and tricarbonic acids, their alkali-salts are
recommended, which lead to the rise of intracellular pHi:
[0308] (7) The combination of (a) amiloride plus plasma-acidifying
agents (e.g. NH.sub.4Cl, Iysine.HCl, methionine HCl) with (b) TNF
(or TNF-inducers such as LPS) or interferon, augments their
ROI-mediated target cell damaging by inhibiting the synthesis of
ROI-cleaving enzymes (e.g. SOD).
[0309] (8) Since hyper- or persistently activated effector cells
(macrophages, T cells etc.) represent a common element (a) in
neiplastic, (b) in autoimmune diseases (c) in atherosclerosis, (d)
in bacterial and (retro)viral infections, including HIV, and (e) in
vaccines, the use of antagonists of Gs-coupled receptors and of
agonists of Gi(Gp)-coupled receptors, as well as of
Ca-antagonists/Ca-overload-blockers is recommended in all these
situations.
[0310] (9) The efficiency of "microimmunosurgery" can be further
increased by the in vivo or in vitro sensibilization of donor
effector cells against the recipient (patient). Blastogenic
pretransformation confers the donor effector cells a higher
efficacy and a time advantage of 24 days which allows these donor
cells to inactivate the patient's pathological subclone(s) before
the recipient can organize the anti-donor defense. In this way, the
pretreatment of the recipient (patient) by cyclophosphamide plus
Mab can be reduced to a minimum.
[0311] (10) Agents, increasing the intracellular cGMP, such as (a)
nitrocompounds (e.g. glycerolnitrates, isosorbit-mono- and
dinitrates), vasodilators (e.g. minoxidil, (di)hydralazine,
Na-nitroprusside) and (c) molsidomine can be used alone or in
combination with BRMs, lymphokines, growth factors, methylxanthines
(e.g. theophylline), Ca-antagonists or Ca-overload-blockers,
agonists of Gi(Gp)-coupled receptors, antagonists of Gs-coupled
receptors and/or pHi-increasing or redox potential-correcting
substances.
[0312] (11) Since vasodilators (e.g. nitrocompounds) and
molsidomine inhibit the IP3-mediated Ca.sup.2+-mobilization, they
can be used, alone or combined with Ca-antagonists, for a rapid
deblockade of those hyperactivated effector cells which show a
strongly increased Ca.sup.2+-level due to an excess of signals, as
observed e.g. in chronic inflammation and autoimmune disorders.
Favorable is the combination with pHi-increasing and/or redox
potential correcting agents.
[0313] (12) The combination of molsidomine plus nicergoline (with
or without cinnarizine) is of a special interest.
[0314] (13). Infection and inflammations can be treated by
combining NSAID with sub-dosed (a) antagonists of Gs-coupled
receptors (b) agonists of Gi(Gp)-coupled receptors and/or (c)
sub-dosed Ca-antagonists (optimally: Ca-- and
Na-channels-inhibiting Ca-overload-blockers, such as
cinnarizine).
[0315] (14) With the same classes of substances, the in vitro
generation of LAK- and TL-cells could be improved essentially.
[0316] (15) The suppressor-monocytes seem to play a crucial
negative role in the process of immunosuppression. Therefore, they
have to be depleted in vivo by anti-Macrophage-Mabs (or
immunotoxins), along with the neutralization of their secretory
products (monokins) by Mabs, directed against TNFalpha and IL-1. In
addition, the macrophage-stimulating gamma-IFN must be neutralized
by the specific Mab.
[0317] The effector cells, active in the "microimmunosurgery"
process, seem to inactivate both the hyperactivated T cells and the
pathologically activated macrophages.
[0318] (16) The cGMP-synthesizing guanylate cyclase (G.C.) can be
stimulated, according to the invention, (a) by nitro compounds
("organic nitrates"), such as glycerol-, erythrite- and
isosorbite-nitrate, (b) by further vasodilators, such as minoxidil,
(di)hydralazine, Na-nitroprusside, (c) by compounds such as
arginine, N-methyl-D-aspartate(NMDA), L-glutamate,
S-acetylthiocholin-iodide, paraquate, D- and L-ornithine, further
extreme low concentrations of NO and CO. The most act via
stimulation of NO-synthetase (NOS). The monokines such as TNFalpha
and L-1 and lymphokines, such as IFN-gamma (a) switch the cell
function from the cell proliferation to the cell differentiation,
and (b) induce the secretion of numerous cytokines via the
H.sub.2O.sub.2 -intermediate. Interestingly, the H.sub.2O.sub.2 per
se is able to induce, at very low concentrations, the
TNF-production. The No-synthesis goes parallel with the ROI
(H.sub.2O.sub.2) -formation and is dependent in its last step
(oxydation of the hydroxylamine-intermediate) on the cooperation
with the compound I (catalase: H.sub.2O.sub.2-complex). The NO is
oxydized to NO.sub.2 and NO.sub.3.
[0319] The cGMP-synthesis and mediately the secretion of cytokines
can be achieved also by extremely low concentrations of ROI (e.g.
H.sub.2O.sub.2 or Mg-peroxide or
(NH.sub.4).sub.2S.sub.2O.sub.4).
[0320] On the other hand, by the inhibition of NO-synthetase or by
ROI-scavengers and reducing agents (e.g. N-acetyl-cysteine,
glutathione, cysteine, ascorbate, penicillamine etc.), the
(hyper)production of cytokines can be inhibited.
[0321] (17) The efficacy of the treatment can be increased by the
intratumoral/intralesional application (a) of NOS-activators (b) of
agonists of Gi(Gp)-coupled receptors and/or (c) of antagonists of
Gs-coupled receptors.
[0322] (18) Since the cGMP-increasing agents (e.g. nitrocompounds,
molsidomine) decrease the Ca.sub.i-concentration, the combination
of these compounds with Ca.sub.i- and pHi-increasing agents is
recommended. By a fine mutual balancing of these substance classes,
first the energy of immunocompetent cells, caused by hyperactivity,
can be broken by nitrocompounds and subsequently the biosynthesis
of cytokines and growth factors can be induced by a parallel
Ca.sub.i- and pHi-elevation.
[0323] (19) On the other hand, the combination (a) of
Ca.sub.i-reducing stimulators of NOS (b) with agonists of
Gs-coupled receptors or with antagonists of Gi(Gp)-coupled
receptors and/or (c) with Ca-antagonists results in the
inactivation of pathologically hyperactivated subclones. This
effect can be strengthened by the TNF (or TNF-inducers, like LPS),
IFN-gamma and IL-I.
[0324] Of a special interest is also the combination of
Ca-overload-blockers, e.g. piperazines (cinnarizine, lidoflazine,
flunarizine) with Ca-antagonists (controlled Ca.sub.i- and
cGMP-rise).
[0325] (20) The use (a) of agonists of Gi(Gp)-coupled receptors,
(b) of antagonists of Gs-coupled receptors, and/or (c) of
NOS-stimulating compounds helps to save cytokines and growth
factors. By a fine mutual balancing, a parallel Ca.sub.i-, pHi- and
cGMP-rise and herewith a maximal cytokine production can be
achieved.
[0326] (21) The pretreatment ("conditioning") of recipient, i.e.
tumor patient with the cyclophospharnide plus anti-CD3- or
anti-CD8-Mab as part of the "microimmunosurgery" can be replaced by
the treatment with the cyclophosphamide plus anti-T6 (anti-T9;
anti-T10; anti-TdT)Mab.
[0327] The combination of cyclophosphamide with the mixture of
Mabs, directed against the mature cells (anti-CD3 Mabs) and T cell
precursors (e.g. anti-T6-Mabs) is also recommended.
[0328] (22) A combination (a) of antagonists of Gi(Gp)-coupled
receptors, (b) of Ca.sup.2+-influx inhibiting Ca-antagonists, (c)
of Ca.sup.2+-mobilization-inhibiting nitrocompounds, (d) of
agonists of Gs-coupled receptors (B-sympathomimetics) and/or (e) of
alkalizing agents could be lifesaving even during a fatal immune
over-reaction (septic or anaphylactic shock). The K-preparations,
O.sub.2-scavengers and tolbutamide or biguanine plus glucose can
support this treatment.
[0329] (23) The Ts-cells can be converted to the CTL/Tc-cells (and
vice versa). The intracellular ADP/ATP-ratio seems to be critical:
a n ATP-drop or an ADP-rise seems to signalize the
Tc:Ts-interconversion.
[0330] A key role seems to play also the ADP/ATP-dependent
cAMP/cGMP-ratio. The AMP, formed by the reaction: 2ADP=ATP +AMP, is
partly dephosphorylated to adenosine which raises, via A2, the
purinergic receptors, the intracellular cAMP-level. The
ADP/ATP-decrease (in different stress situations, such as
O.sub.2-deficit, physical work, cold, heat) is associated by
catecholamine (adrenaline)-rise. The adrenaline causes also the
rise of intracellular cAMP-level. In order to increase the
ADP/ATP-ratio, the following agents are recommended: [0331] (a)
O.sub.2 or O.sub.2-carriers (b) glucose, di- and tricarbonic acids
(as alkali-salts), along with antidiabetics (tolbutamide, biguanine
etc.), (c) ribose and/or (d) glutamine, glycine and other
ATP-precursors. The adenosine or adrenaline-induced cAMP-rise can
be inhibited by the corresponding receptor blockers.
[0332] (24) The reinduction of tolerance against the tumor cells
after removal of primary tumor (a) by surgery and/or (b) by
chemotherapy and/or (c) by radiotherapy can be inhibited by the
antagonists of Gs-coupled receptors and by ligands of
Gi(Gp)-coupled receptors. In the case of B-blockers, the
H.sub.2-antihistaminics and A2-purinergic blockers, the ligands are
directed from Gs- to Gi(Gp)-coupled receptors.
[0333] (25) The therapy of (a) lymphomas and (b) leukemias of the
T-type can be performed by the "microimmunosurgery" like in solid
tumors; again, the sublethal whole body irradiation or high-dose
chemotherapy is replaced by the T cell depletion (by Mab-saving
cyclophosphamide plus anti-CD3-Mabs). The transformed T cells are
eliminated by allogeneic donor effector cells. The mature
(post-thymus) malignant cells are MHC II-positive. The immature
(pre-thymus) cells are recognized and depleted via blast-specific
structures.
[0334] (26) Autologous BMT in patients with blood cell tumors and
those with solid tumors (e.g. lung cancer) can be improved by the
in vitro treatment of autologous BM with allogeneic effector cells
whose cell death is preprogrammed. This Ts-depletion on cellular
level can be combined with that on humoral level, i.e. with
anti-CD8-Mabs and/or with the depletion of contaminating tumor
cells ("purgic").
[0335] (27) A further elegant technique of deblockade of
hyperactivated effector cells and of their reactivation in the
short opening of the voltage-operating Ca.sup.2+-channels (a) by a
high frequency (HF)current, or (b) by magnetic induction.
[0336] The HF-currents must be significantly lower than those, used
in the electroporation or electrofusion (Zimmermann). The deblocked
or reactivated state can be "frozen" by Ca-antagonists.
[0337] (28) The efficiency of "microimmunosurgery" can be increased
by the combination of Gs- and Gi(Gp)-influencing agents.
[0338] (29) The disappointing efficacy of (a) LAK- and (b)TIL-cells
in vivo can be explained by the cortisol-depression in plasma.
Therefore, (a) the use of adrenostatics (e.g.metopiron), inhibiting
the 11-B-hydroxylation of glucocorticoids during the LAK- and
TIL-infusion, and (b) the admixing of LAK- or TIL-cells,
pretransfected in vitro by the cDNA, encoding either the
cortisone-cleaving enzyme 20alpha-hydroxysteroid-dehydrogenase (20
alpha SDH) or the cortisone-inactivating enzyme B-glucuronidase,
are recommended.
[0339] (30) The efficacy of "microimmunosurgery" can be increased
also by the pretreatment of the recipient (a) with the BRMs or (b)
with the lymphokines (e.g. IFNgamma or IL-4), to render target
cells, i.e. pathological subclones more vulnerable.
[0340] (31) The standardized "microimmunosurgery" comprises the
following steps: (a) Pooled PBs/PBLs or T cells (from donor A,B,C)
whose pathogen-specific subclones can be clonally postexpanded in
vitro, e.g. by lectins, are (b) "cell death-preprogrammed" and (c)
frozen in a medium (e.g. RPMI1640), containing special
cryoprotectants (based on PEG and/or PVP) which warrant the
preservation of the preactivated cell state, in addition to an
improved cell viability. (d) Treatment of patients, suffering e.g.
of cancer or autoimmune disorders is restricted to the T cell
depletion because the "microimmunosurgery" is based on the simple
replacement of the pathologic patient's T set by the healthy
donor's T set.
[0341] (32) According to a modified "microimmunosurgery"-procedure,
the immature T- and B-cell precursors, both of the donor (in vitro)
and of the recipient (patient) are eliminated temporarily by
specific Mabs or Mabs plus cyclophosphamide; then, the patient's
pathological subclones are eradicated and--as the last step--the
depleted precursors are replaced by autologous or allogeneic
(T-depleted) bone marrow. The special advantage is the early
exclusion of inducer and transducer suppressor T cells.
[0342] The effector cells can be supported by allogeneic LAK-cells;
these can be either premanipulated to express a preprogrammed cell
death or depleted of their T cells (10% of cells). In certain
situations, the so pretreated allogeneic LAK-cells can be used
alone to eliminate the pathologic subclones.
[0343] (33) Immunocompetent cells, e.g. macrophages express a
variety of additional, not typically "immunologic" receptors (e.g.
for the insuline, the glucagon, the parathormone etc.) which have
been neglected up to date though they contribute essentially to the
activity of immunocompetent cells. The therapeutic manipulation of
all these receptors by agonists and antagonists is subject of this
invention.
[0344] (34) Since the early induction of tolerance involves
inducer- and transducer suppressor cells which are both
CD4-positive, the use of anti-VD4-Mabs (or immunotoxins) is
recommended for the prevention of tolerance in general. An
established tolerance, on the other hand, is maintained by mature,
CD8-positive Ts cells. The same concerns the reinduction of
tolerance against the same antigen. In both cases, the use of
anti-CD8-Mabs is recommended. Since immature, CD4-positive inducer
and transducer Ts cells are involved in the reinduction of
tolerance, anti-CD4-Mab (alone or combined with anti-CD8-Mab) are
recommended.
[0345] (35) As the IL-2(Tac) receptor (=CD25) is expressed only on
(hyper)activated macrophages/monocytes and on the T cells, the
parallel depletion of both pathologically hyperactivated
subpopulations can be achieved by anti-CD25-Mabs (or immunotoxins).
This procedure can improve the "microimmunosurgery" (e.g. in the
bone marrow and organ transplantation).
[0346] (36) Fibroblasts support the immunosuppressive "tissue
repair"/"wound healing"-function of macrophages and increase e.g.
their PGE2 secretion. Therefore, the neutralization of specific
fibroblast growth factors, e.g. PDGF, bFGF, FAF, FGTB, PF4 and LTBS
is recommended.
[0347] (37) As pathologic processes, such as GvHR, autoreactivity
or sarcoidosis, arise from a mutual stimulation of T4 cells and
monocytes/macrophages, the interruption of this activation chain by
Mabs against IL-I, TNFalpha and/or IFNgamma is recommended.
[0348] (38) Similarly, in BMT the mutual activation cascade,
leading to GvH- and HvG-reactivity, has to be interrupted,
according to the invention, by neutralizing the involved
cytokine(s). The use of Mabs is favorable. It is directed against
the TNFalpha, IL-1, IFNgamma, GM-CSF, M-CSF, IL-6 and IL-4.
[0349] (39) The combating of residual tumor cells and (premicro)
metastases, following tumor excision has best chances if the
following 2 principles are combined: (a) 1.sup.st principle: MHC
II-expression on the patient tumor cells, both (a1) by the in vitro
MHC II-postexpression, induced by IFNgamma, TNFalpha and/or IL-4,
and (a2) by the fusion of tumor cells with autologous or allogeneic
MHC II-positive cells (b) 2.sup.nd principle: transfection of
patient tumor cells with cytokines, primarily GM-CSF and IL-4.
Before their reinfusion, such modified tumor cells can be
manipulated to express the preprogrammed cell death. The local
cytokine enrichment can be achieved by the addition of allogeneic B
cells.
[0350] (40) To increase the humoral response e.g. against HIV, the
combination of antibodies of the IgG- or IgM-isotype with those of
the IgE-isotype is recommended. The latter can be constructed by
the replacement of the Fc gamma or Fcu-subunit by the Fc
epsilon-subunit. Alternatively, the IgE-percentage in vivo can be
increased by the simultaneous injection of anti-IFNgamma and
IL-4(IL-5).
[0351] (41) A long-term incubation of PBMs with lectins (e.g. 1
week with ConA or 3 weeks with PHA) leads to a high enrichment of
CD8-positive Ts cells; therefore, the depletion of so selected Ts
cells (a) by anti-CD8-Mabs (plus complement) or (b) by alloreactive
effector cells, with preprogrammed cell death, is recommended. The
residual PBMs--released of the depression by the Ts cells--are able
to post-generate the Tc/CTLs and Th cells.
[0352] In a next procedure, the patient's PBMs are first incubated
in vitro with lectins (e.g.PHA) and thereafter treated by
increasing concentrations of anti-CD8-Mab (+complement), until the
onset of the Tc- and Th-deblockade. Among the deblocked Tc- and
Th-cells, tumor-specific clones can be expected; after a further
clonal expansion, these tumoricidal subclones can be reinjected
into the patient.
[0353] (42) During their maturation process, the erythrocytes lose
the nuclea. Therefore, the ennucleated precursors (reticulocytes)
are of a special interest as carriers of temporarily limited
functions (e.g. in vivo production of cytokines, Mabs and
suppressor factors or local source of irradiation). In these cells,
the cell death need not be preprogrammed.
[0354] (43) The controlled DNA-damaging described above ("cell
death preprogramming") can be carried out by DNA-selective
cytotoxic agents, primarily vinca alkaloids, bleomycin, ICRF-159,
busulfan, DDP, VP16231 (EPE) and EPT, in addition to the above
mentioned mitomycinc. The controlled DNA-cross-linking can be
achieved by so called "bifunctional alkylating agents".
[0355] According to a further aspect of the invention, the use of
the combination of a Ca-antagonist plus an agent, decreasing the
cAMP/cGMP-ratio is recommended as the drug for the treatment of
cancer, viral and bacterial infections, as well as autoimmune
disorders.
[0356] In addition, the use of a combination, consisting of an
agent, eliminating hyperactivated effector cells, and of
alloreactive cells with preprogrammed cell death is recommended as
the drug for the treatment of cancer, viral infections and
autoimmune diseases.
[0357] According to the invention, an additional approach in the
treatment of cancer, viral infections and autoimmune disorders,
based on the elimination or down-regulation of immunological
effector cells, enables the patient's own immune system to restore
the pre-disease state.
* * * * *