Date: March 29, 2011
URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&d=PALL&RefSrch=yes&Query=PN%2F7914785
B-cell depleting agents, like anti-CD20 antibodies or fragments
thereof for the treatment of chronic fatigue syndrome
---------------------------------------------------------------
Abstract
The present invention relates in a first aspect to a B-cell depleting
anti-CD20 antibody or a CD20-binding antibody fragment thereof for the
treatment of chronic fatigue syndrome and myalgic encephalomyelitis.
In particular, the present invention relates to the use of anti-CD20
monoclonal antibodies or fragments thereof which are preferably
humanized for the treatment of chronic fatigue syndrome/myalgic
encephalomyelitis in a subject afflicted with said disease.
Inventors: Mella; Olav (Olsvik, NO), Fluge; Oystein (Morvik, NO)
Assignee: Bergen Teknologieverforing AS (Bergen, NO)
Appl. No.: 12/348,024
Filed: January 2, 2009
Current U.S. Class: 424/130.1; 424/133.1; 424/135.1; 424/152.1;
424/155.1; 514/249
Current International Class: A01N 43/48 (20060101); A61K 39/395 (20060101)
(...)
Other References
Center for Disease Control and Prevention, "Chronic Fatigue Syndrome:
Causes" [online], last updated Oct. 15, 2010 [retrieved Nov. 1, 2010],
Retrieved from internet URL:
http://www.cdc.gov/cfs/general/causes/index.html#>. cited by examiner.
Nye, F., Letters to the editor: Chronic fatigue syndrome and myalgic
encephalomyelitis: The 2007 guildelines from the National Institute of
Clinical Excellence, J. Infection, 55:569-572, 2007. cited by examiner.
Lundell et al., Clinical activity of folinic acid in patients with
chronic fatigue syndrome, Arznelm.-Forsch./Drug Res. 56(6):399-404,
2006. cited by examiner.
Lyall et al., A systematic review and critical evaluation of the
immunology of chronic fatigue syndrome, J. Physchosomatic Res.
55:79-90, 2003. cited by examiner.
Prins et al., Chronic fatigue syndrome, The Lancet, 376:346-355, Jan.
28, 2006. cited by examiner.
Klimas, N., "Immunologic Abnormalities in Chronic Fatigue Syndrome",
Journal of Clinical Microbiolgy, vol. 28, No. 6, 1990, pp. 1403-1410.
cited by other.
Robertson, et al., "Lyphocyte Subset Differences in Patients with
Chronic Fatigue Syndrome, Multiple Sclerosis and Major Depression",
Clinical and Expermimental Immuology, vol. 141, 2005, pp. 326-332.
cited by other.
Eurpoean search report for EP 08000006.0, filed Jan. 2, 2008. cited by other.
Primary Examiner: Spector; Lorraine
Assistant Examiner: Kaufman; Claire
Attorney, Agent or Firm: Whitham Curtis Christofferson & Cook, PC
Claims
The invention claimed is:
1. A method of treating chronic fatigue syndrome and optionally
myalgic encephalomyelitis comprising the step of administering a
B-cell depleting agent to a subject afflicted therewith.
2. The method for treating chronic fatigue syndrome and myalgic
encephalomyelitis as recited in claim 1 wherein said step of
administering includes providing said B-cell depleting agent to said
subject afflicted therewith one or two infusions twice within two weeks.
3. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 1 wherein said B-cell depleting
agent is a B-cell depleting anti CD20 antibody or CD20-binding
antibody fragment thereof, and wherein Methotrexate is administered
simultaneously, separately or sequentially with said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof to said
subject.
4. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 2 wherein said B-cell depleting
agent is a B-cell depleting anti CD20 antibody or CD20-binding
antibody fragment thereof, and wherein Methotrexate is administered
simultaneously, separately or sequentially with said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof to said
subject.
5. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 1, wherein said B-cell depleting
agent is a B-cell depleting anti CD20 antibody or CD20-binding
antibody fragment thereof.
6. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 5, wherein said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof is a
monoclonal antibody or CD20-binding antibody fragment thereof.
7. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 5, wherein said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof is a
humanized anti CD20 antibody or CD20-binding antibody fragment thereof.
8. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 5, wherein said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof is a
B-cell depleting CD20 antibody fragment binding to CD20.
9. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 8, wherein said B-cell depleting
CD20 antibody fragment is selected from the group consisting of
F(ab').sub.2, F(ab'), Fab, Fv and sFv.
10. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 1, wherein said B-cell depleting
agent is selected from the group consisting of Rituximab. Ofatumumab,
Ocrelizumab, GA101, BCX-301, Veltuzumab, and DXL 625.
11. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 1, wherein said B-cell depleting
agent is Rituximab.
12. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 1, wherein said B-cell depleting
agent is Methotrexate TRU-015 or SBI-087.
13. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 5, wherein said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof is
administered in an amount in the range of 10 mg to 5000 mg per dosage.
14. The method of treating chronic fatigue syndrome and myalgic
encephalomyelitis according to claim 5, wherein said B-cell depleting
anti CD20 antibody or CD20-binding antibody fragment thereof is
administered to said subject in a single therapeutically effective
dosage of said antibody of 50 to 2000 mg/m.sup.2 or multiple of
therapeutically effective dosages of said anti CD20 antibody or anti
CD20-binding antibody fragment thereof of 50 to 2000 mg/m.sup.2.
Description
The present invention relates in a first aspect to a B-cell depleting
anti-CD20 antibody or a CD20-binding antibody fragment thereof for the
treatment of chronic fatigue syndrome and myalgic encephalomyelitis.
In particular, the present invention relates to the use of anti-CD20
monoclonal antibodies or fragments thereof which are preferably
humanized for the treatment of chronic fatigue syndrome/myalgic
encephalomyelitis in a subject afflicted with said disease.
In a further aspect, the present invention relates to B-cell depleting
agents in general for the treatment of chronic fatigue syndrome and
myalgic encephalomyelitis in a subject afflicted therewith.
TECHNICAL BACKGROUND
Chronic Fatigue Syndrome
Chronic Fatigue Syndrome (CFS) is characterized by an unexplained,
severe fatigue, persisting for at least six consecutive months, and
with a substantial reduction of previous levels in occupational,
social, or personal activities. Also, the patients often experience
persistent or recurrent symptoms such as impairment of short-term
memory or concentration, muscle pain, joint pain without evidence of
arthritis, headache, sleep disturbances, and post-exercise exhaustion
(Fukuda K, et al., 1994, Ann Intern Med 121:953-9 et al 1994).
Although many studies have shown subtle alterations in blood tests or
radiological investigations, no biomarker or diagnostic test exists.
The prevalence of CFS worldwide is thought to be at least 0.5%, and
the female:male ratio is 3:1 (Wyller V B. 2007, Acta Neurol Scand
Suppl 187:7-14).
The aetiology of CFS remains unclear. The various hypotheses include
immunological, virological, neuroendocrinological, and psychological
mechanisms. The pathogenesis of CFS is presumed to be multifactorial
and to involve both host and environmental factors (Devanur & Kerr
2006).
In a recent review of November 2007, describing current research
priorities in CFS, the urgent need to elucidate the pathogenesis is
highlighted (Kerr J R et al., 2007, J Clin Pathol 60:113-6).
Many patients suffering from CFS have a history of an acute viral
infection preceding the development of fatigue. Although research data
indicate evidence of immune system activation, the disease mechanisms
remain unknown. A collaborative study group was formed in 2001; to
elucidate the molecular mechanisms of CFS, with the aims to develop a
diagnostic test and also to guide the development of more specific
treatment (Devanur L D, Kerr J R. 2006, J Clin Virol 37:139-50).
Several gene expression studies have been performed in CFS, indicating
that there are specific but complex gene alterations in accordance
with a dysfunction in immune response and in defence mechanisms. One
microarray study showed differential expression of 16 genes in CFS,
suggesting T-cell activation and a disturbance of neuronal and
mitochondrial function (Kaushik N, et. Al., 2005, J Clin Pathol
58:826-32). Another microarray study using serial samples of
peripheral blood mononuclear cells total RNA, from patients developing
CFS after Epstein Barr virus (EBV) infection and also from subjects
with EBV infection without development of fatigue, concluded that
several genes affecting mitochondrial function and cell cycle were
deregulated (Vernon S D, et. Al., 2006, BMC Infect Dis 6:15). Another
gene expression study in CFS suggested disturbance of exercise
responsive genes including several involved in membrane transport and
ion channels (Whistler T, et. al., 2005, BMC Physiol 5:5). Recently,
an analysis of gene networks in CFS revealed seven distinct genomic
subtypes with differences in clinical presentation and severity (Kerr
J, et. al., 2007, J Clin Pathol). Several other studies have addressed
global gene expression in CFS (Fang H, et. al., 2006, Pharmacogenomics
7:429-40; Whistler T, et al., 2003, J Transl Med 1:10).
The gene expression data are not conclusive, but suggests that there
are gene expression disturbances in CFS representing various cellular
functions, and may indicate that the disease has a heterogeneous
pathogenesis.
A prevailing theme in CFS research has been a sustained immune
deregulation, following acute exogenous stimuli such as a viral
infection. Among the microbial pathogens reported to be associated
with CFS are Epstein-Barr virus (Lerner A M, et al., 2004, In Vivo
18:101-6), enterovirus (Chia J K, Chia A Y. 2007, J Clin Pathol),
parvovirus B19 (Matano S, et al., 2003, Intern Med 42:903-5),
cytomegalovirus (Lerner A M, et al., 2002, In Vivo 16:153-9), human
herpesvirus type 6 (Chapenko S, et al., 2006, J Clin Virol 37 Suppl
1:S47-51; Komaroff A L. 2006, J Clin Virol 37 Suppl 1:S39-46),
Chlamydia pneumoniae (Nicolson G L, et al., 2003, Apmis 111:557-66).
However, the data are not consistent (Soto N E, Straus S E., 2000,
Herpes 7:46-50).
A recent study of postinfective fatigue syndrome found no differences
in ex vivo cytokine production over a 12-month period, as compared to
controls recovering promptly after infection (Vollmer-Conna U, et al.,
2007, Clin Infect Dis 45:732-5). Others claim that despite evidence of
immune activation, as demonstrated by increased number of activated
T-cells and elevated levels of cytokines, the CFS patients may have a
reduced immune cell function with a low NK-cell cytotoxicity and
immunoglobulin deficiencies (Patarca R. 2001, Ann N Y Acad Sci
933:185-200).
Others reported a high number of circulating B-lymphocytes, altered
NK-cells subsets also with increased expression of adhesion molecules,
as compared to controls (Tirelli U, et al., 1994, Scand J Immunol
40:601-8), while another study showed reduced CD56+ NK-cells, and
reduced CD4+ and CD8+ T-lymphocytes in CFS patients (Racciatti D, et
al., 2004, Int J Immunopathol Pharmacol 17:57-62). Also, T- and
NK-cells from CFS patients were found to express lower levels of the
intracellular granule protein perforin, indicating a reduced ability
to mediate cytotoxicity.
One study showed several abnormalities in laboratory markers
associated with immune function in CFS patients (Klimas N G, et al.,
1990, J Clin Microbiol 28:1403-10). The most consistent result was a
low NK cell cytotoxicity, but also an increase in CD8+ T-cells,
elevated number of CD20+ B-cells, and increase in the B-cell subset
coexpressing CD20 and CD5 (Klimas et al 1990). These data were to some
extent supported by a study reporting expansion of activated CD8+
cytotoxic T lymphocytes, along with a marked decrease in NK cell
activity, in CFS patients (Barker E, et al., 1994. Clin Infect Dis 18
Suppl 1:S136-41).
A recent study comparing CFS patients and controls, reported decreased
expression of CD69 on T-cells and NK-cells after mitogenic stimulation
in vitro, indicating a disorder in the early activation of cellular
immunity mediated by these cells (Mihaylova I, et al., 2007, Neuro
Endocrinol Lett 28:477-83).
However, the data on immune deregulation in CFS are not consistent,
and a study comparing lymphocyte subsets in CFS patients to those of
patients with depression, multiple sclerosis and healthy controls,
found no difference in T-, B-, or NK-cell subsets (Robertson M J, et
al., 2005, Clin Exp Immunol 141:326-32). Similarly, a review of the
immunology in CFS concluded that the studies performed in the research
field had varying quality, and that no consistent pattern of
immunological abnormalities could be identified (Lyall M, et al.,
2003, J Psychosom Res 55:79-90).
Along with hypotheses of immune deregulation in CFS, autoimmunity to
endogenous vasoactive neuropeptides has been proposed as a mechanism
for the disease (Staines D R., 2005, Med Hypotheses 64:539-42),
however not supported by scientific data. The author has also
suggested a similar mechanisms in the aetiology of fibromyalgia,
multiple sclerosis and amyotrophic lateral sclerosis, Parkinson's
disease, and sudden infant death syndrome hypothesizing that
autoimmunity against vasoactive neuropeptides acting as hormones,
neurotransmitters, immunmodulators and neurotrophes may explain the
complex clinical pictures of these diseases. However, no
autoantibodies to these neuropeptides have been documented in CFS.
One study investigated the presence of circulating anti-muscle and
anti-CNS antibodies in CFS patients and controls, with no detected
pathogenic antibodies. Another report of antinuclear autoantibodies in
CFS concluded that there was no association (Skowera A, et al., 2002,
Clin Exp Immunol 129:354-8), while another investigating common
autoantibodies and antibodies to neuron specific antigens showed
higher rates of antibodies to microtubule-associated protein 2 and
ssDNA in CFS (Vernon S D, Reeves W C. 2005, J Autoimmune Dis 2:5). A
single study showed the presence of autoantibodies to muscarinic
cholinergic receptor in a subset of CFS patients (Tanaka S, et al.,
2003, Int J Mol Med 12:225-30), and higher levels of autoantibodies to
insoluble cellular antigens were reported in CFS as compared to
controls (von Mikeecz A., et al., 1997, Arthritis Rheum 40: 295-305).
However, there is no direct evidence with consistent data for the
presence of pathogenic autoantibodies, or for T-lymphocyte-mediated
autoimmunity. No indirect evidence has recreated the CFS disease in an
animal model by immunization with antigens analogous to (putative)
human autoantigens.
CFS is at present not defined as an autoimmune disease, and a recent
protocol for a Cochrane review of pharmacological treatment in CFS
states the aetiology as unknown. (Rawson K M, et al., 2007.
Pharmacological treatments for chronic fatigue syndrome in adults.
(Protocol) Cochrane Database of Systematic Reviews, Issue 4. Art. No.:
CD006813.)
Other hypotheses for CFS pathogenesis are blood platelet dysfunction
(Kennedy G, et al., 2006, Blood Coagul Fibrinolysis 17:89-92),
neurological (Natelson B H, et al., 2005, Clin Diagn Lab Immunol
12:52-5), neuroendocrine (Van Den Eede F, et al., 2007,
Neuropsychobiology 55:112-20), metabolic or autonomic disturbances,
ion channel dysfunction (Chaudhuri A, et al., 2000, Med Hypotheses
54:59-63), zinc deficiency (Maes M, et al., 2006, J Affect Disord
90:141-7), toxin exposure or prior vaccinations (Appel S, et al.,
2007, Autoimmunity 40:48-53). Others have focused on an abnormal
response to exercise with intracellular immune deregulation as a
possible mechanism in CFS pathogenesis (Nijs J, et al., 2004, Med
Hypotheses 62:759-65). Also, post-infective impairment of the ability
to synthesise n-3 and n-6 long-chain polyunsaturated fatty acids has
been proposed as important in the pathophysiology of CFS (Puri B K.
2007, J Clin Pathol 60:122-4).
Hence, recent reviews on CFS in renown journals state that the disease
at present has an unknown cause (Hampton T. 2006, Jama 296:2915;
Hooper M. 2007, J Clin Pathol 60:466-71; Prins J B, et al., 2006,
Lancet 367:346-55). Thus, no consistent picture has emerged for the
aetiology and pathogenesis of CFS.
Current Treatment of CFS
Due to the lack of knowledge of the exact pathogenesis, and with no
known causal mechanism, there is no current standard specific
treatment for CFS. A systematic review concluded that CFS should be
associated with a "biopsychosocial model" with emphasis on progressive
muscular rehabilitation, combined with behavioural and cognitive
treatment (Maquet et al, 2006, Ann Readapt Med Phys 49:337-47, 418-27).
The unknown aetiology of CFS is probably the reason for the remarkably
few studies performed, evaluating therapy based upon a biological
hypothesis.
As the majority of evidence suggests an immune system deregulation,
perhaps precipitated by an exogenous stimulus, two studies have
assessed use of intravenous gammaglobulin for CFS. One was a case
report in three patients with CFS following an acute parvovirus B19
infection, treated with 5-days intravenous immunoglobulin, with
improvement of clinical symptoms and resolution of cytokine
dysregulation (Kerr et al, 2003, Clin Infect Dis 36:e100-6). In a
double-blind, placebo-controlled, randomized study of 71 adolescents
with CFS, three infusions of gammaglobulin were given one month apart,
with functional improvement in the gammaglobulin-treated group at
six-month follow-up with average duration 18 months. In the first six
months of the trial, both the placebo group and the
gammaglobulin-treated group reported improvement (Rowe 1997, J
Psychiatr Res 31:133-47).
In a pilot study reported in abstract form (Lamprecht 2001, Meeting of
the American association of chronic fatigue syndrome (AACFS). Seattle)
six patients with CFS were given etanercept (Enbrel.TM., i.e. human
tumor necrosis factor receptor p75 Fc fusion protein, which is a
soluble competitive TNF-receptor acting to inhibit the TNF-mediated
cellular response) and a clinical benefit was reported.
Among other therapeutic strategies, valganciclovir was used to treat
12 patients with long-standing fatigue and elevated antibody-titres to
Epstein-Barr virus or human herpes virus-6, and nine had improvement
of the symptoms, however with uncertainty as to whether the effects
were mediated through anti-viral effect or through immunomodulation
(Kogelnik A M,. 2006, J Clin Virol 37 Suppl 1:S33-8). Treatment with
azithromycin, an antibiotic with immunomodulating properties, gave
improvement in 59% of 99 CFS patients (Vermeulen & Scholte 2006, J
Transl Med 4:34).
In a recent review of current research priorities in CFS (Kerr et al
2007, J Clin Pathol 60:113-6), new studies are encouraged to focus on
the understanding of the molecular pathogenesis of the disease, to
test useful biomarkers, and to aid in the development of specific
treatment. Various molecular techniques are available and have been
used for this purpose, including global gene expression analyses using
microarrays.
Rituximab as an Example of B-Cell Depleting Antibodies in B-Cell
Lymphoma and Autoimmunity
Rituximab (Mabthera, RITUXAN.RTM.) is a monoclonal antibody directed
against an epitope in the extracellular portion of the transmembrane
molecule CD20. The antibody is a chimeric human-mouse in which the
fragment antigen binding (Fab) part is mouse and the Fc-part is human.
The CD20 protein is expressed on B-lymphocytes, but not on stem cells
or on the mature plasma cells. CD20 is also expressed on the vast
majority of B-cell lymphomas. CD20 is implicated in regulation of
transmembrane calcium conductance and cell cycle progression, but the
precise function is unknown (Janas et al 2005, Biochem Soc
Symp:165-75). Upon binding of Rituximab to CD20, an immunological cell
killing is mediated through the binding of complement to the Fc part
with activation of the complement cascade, and also through activation
antibody-dependent cellular cytotoxicity (ADCC) (Glennie et al 2007,
Mol Immunol 44:3823-37).
The molecule does not internalize or shed from the plasma membrane
after Rituximab binding, which allows the monoclonal antibody to
persist on the cell surface to extend the immunological attack.
The role of Rituximab in treatment of B-cell lymphomas has emerged
rapidly. Immunochemotherapy using Rituximab in combination with
chemotherapy, or Rituximab monotherapy in indolent lymphomas, are now
current standards of treatment, and has improved overall survival in
the most common type of aggressive B-cell lymphomas (Diffuse large
B-cell lymphoma), both in elderly (Coiffier et al 2002, N Engl J Med
346:235-42) and in younger patients (Pfreundschuh et al 2006, Lancet
Oncol 7:379-91), and also in the most common indolent lymphoma
(follicular lymphoma) (Marcus et al 2005, Blood 105:1417-23). In
selected patients with follicular lymphoma, Rituximab is also used as
maintenance treatment after induction therapy, with infusions every
third month for two years, showing improved overall survival (van Oers
et al 2006, Blood 108:3295-301).
In recent years, Rituximab was proved to be an effective treatment
also in autoimmune diseases, where the B-cell depletion is often
associated with a clinical improvement, e.g. in rheumatoid arthritis
(Dass et al 2006, Expert Opin Pharmacother 7:2559-70). The list of
different autoimmune diseases in which Rituximab has a therapeutic
role is growing (Sanz et al 2007, Front Biosci 12:2546-67). For future
B-cell targeting and depletion, the development of antibodies to
specific B-cell subsets will be important (Dorner & Lipsky 2007,
Expert Opin Biol Ther 7:1287-99).
The rituximab antibody is a genetically engineered chimeric
murine/human monoclonal antibody directed against the CD20 antigen.
Rituximab is the antibody called "C2B8" in U.S. Pat. No. 5,736,137.
RITUXAN.RTM. is indicated for the treatment of patients with relapsed
or refractory low-grade or follicular, CD20-positive, B cell
non-Hodgkin's lymphoma. In vitro mechanism of action studies have
demonstrated that RITUXAN.RTM. binds human complement and lyses
lymphoid B cell lines through complement-dependent cytotoxicity (CDC)
(Reff et al. Blood 83(2):435-445 (1994)). Additionally, it has
significant activity in assays for antibody-dependent cellular
cytotoxicity (ADCC). More recently, RITUXAN.RTM. has been shown to
have antiproliferative effects in tritiated thymidine incorporation
assays and to induce apoptosis directly, while other anti-CD19 and
CD20 antibodies do not (Maloney et al Blood 88(10):637a (1996)).
Synergy between RITUXAN.RTM. and chemotherapies and toxins has also
been observed experimentally. In particular, RITUXAN.RTM. sensitizes
drug-resistant human B cell lymphoma cell lines to the cytotoxic
effects of doxorubicin, CDDP, VP-16, diphtheria toxin and ricin
(Demidem et al Cancer Chemotherapy & Radiopharmaceuticals
12(3):177-186 (1997)). In vivo preclinical studies have shown that
RITUXAN.RTM. depletes B cells from the peripheral blood, lymph nodes,
and bone marrow of cynomolgus monkeys, presumably through complement
and cell-mediated processes (Reff et al. Blood 83(2):435-445 (1994)).
Patents and patent publications concerning CD20 antibodies include
U.S. Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and
6,682,734, as well as US patent appln nos. US 2002/0197255A1, US
2003/0021781A1, US 2003/0082172 AI, US 2003/0095963 AI, US
2003/0147885 AI (Anderson et al); U.S. Pat. No. 6,455,043BI and
WO00/09160 (Grillo-Lopez, A.); WO00/27428 (Grillo-Lopez and White);
WO00/27433 (Grillo-Lopez and Leonard); WO00/44788 (Braslawsky et al);
WO01/10462 (Rastetter, W.); WO01/10461 (Rastetter and White);
WO01/10460 (White and Grillo-Lopez); US2001/0018041A1,
US2003/0180292A1, WO01/34194 (Hanna and Hariharan); US appln no.
US2002/0006404 and WO02/04021 (Hanna and Hariharan); US appln no.
US2002/0012665 AI and WO01/74388 (Hanna, N.); US appln no. US
2002/0058029 AI (Hanna, N.); US appln no. US 2003/0103971 AI
(Hariharan and Hanna); US appln no. US2002/0009444A1, and WO01/80884
(Grillo-Lopez, A); WO01/97858 (White, C); US appln no.
US2002/0128488A1 and WO02/34790 (Reff, M.); WO02/060955 (Braslawsky et
al.,); WO2/096948 (Braslawsky et al.); WO02/079255 (Reff and Davies);
U.S. Pat. No. 6,171,586BI, and WO98/56418 (Lam et al); WO98/58964
(Raju, S.); WO99/22764 (Raju, S.); WO99/51642, U.S. Pat. No.
6,194,551BI, U.S. Pat. No. 6,242,195BI, U.S. Pat. No. 6,528,624BI and
U.S. Pat. No. 6,538,124 (Idusogie et al); WO00/42072 (Presta, L.);
WO00/67796 (Curd et al.); WO01/03734 (Grillo-Lopez et al.); US appln
no. US 2002/0004587A1 and WO01/77342 (Miller and Presta); US appln no.
US2002/0197256 (Grewal, L); US Appln no. US 2003/0157108 AI (Presta,
L.); U.S. Pat. Nos. 6,565,827BI, 6,090,365BI, 6,287,537BI, 6,015,542,
5,843,398, and 5,595,721, (Kaminski et al); U.S. Pat. Nos. 5,500,362,
5,677,180, 5,721,108, 6,120,767, 6,652,852BI (Robinson et al); U.S.
Pat. No. 6,410,391BI (Raubitschek et al); U.S. Pat. No. 6,224,866BI
and WO00/20864 (Barbera-Guillem, E.); WO01/13945 (Barbera-Guillem,
E.); WO00/67795 (Goldenberg); US Appl No. US 2003/0133930 AI and
WO00/74718 (Goldenberg and Hansen); WO00/76542 (Golay et al.);
WO01/72333 (Wolin and Rosenblatt); U.S. Pat. No. 6,368,596BI (Ghetie
et al); U.S. Pat. No. 6,306,393 and US Appln no. US2002/0041847 AI,
(Goldenberg, D.); US Appln no. US2003/0026801A1 (Weiner and Hartmann);
WO02/102312 (Engleman, E.); US Patent Application No. 2003/0068664
(Albitar et al); WO03/002607 (Leung, S.); WO 03/049694,
US2002/0009427A1, and US 2003/0185796 AI (Wolin et al); WO03/061694
(Sing and Siegall); US 2003/0219818 AI (Bohen et al); US 2003/0219433
AI and WO 03/068821 (Hansen et al.); US2003/0219818AI (Bohen et al);
US2002/0136719A1 (Shenoy et al); WO2004/032828 (Wahl et al), each of
which is expressly incorporated herein by reference. See, also, U.S.
Pat. No. 5,849,898 and EP appln no. 330,191 (Seed et al); U.S. Pat.
No. 4,861,579 and EP332,865A2 (Meyer and Weiss); U.S. Pat. No.
4,861,579 (Meyer et al); WO95/03770 (Bhat et al); US 2003/0219433 AI
(Hansen et al).
Safety Profile of Rituximab
The safety profile of Rituximab in treatment of B-cell lymphomas is
well known, and based on experience from a database with 370 000
patients (Kavanaugh 2006, J Rheumatol Suppl 77:18-23). In lymphoma
treatment, mild-to moderate reaction during the first infusion is the
most common side-effect, caused by cytokine release primarily in
patients with a high initial tumor burden (Solal-Celigny 2006, Leuk
Res 30 Suppl 1:S16-21). Allergic reactions may be seen during the
infusion, due to the protein nature of the Rituximab molecule.
Concern with all B-cell directed therapy is the anticipated effects on
humoral immunity. With extended treatment, and in particular with
maintenance treatment, i.e. infusions every third month for two years
(after induction therapy with perhaps 6-8 Rituximab-infusions every
third week), the B-cell depletion is more pronounced and most patients
will have hypogammaglobulinemia. However, the low levels of
immunoglobulins and B-cell depletion do not seem to have a major
impact on the clinical risk of infections.
One potential serious side-effect from use of Rituximab is the
development of interstitial lung disease. This is a potentially
life-threatening complication, but very rare with only 16 cases
reported in the literature (Wagner et al 2007, Am J Hematol 82:916-9).
Safety issues related to Rituximab-treatment in chronic autoimmune
diseases are exploited in clinical studies (Edwards et al 2006, Best
Pract Res Clin Rheumatol 20:915-28), and with less time for follow-up
so far. The long-term safety therefore remains to be clarified,
especially when Rituximab is to be given once or twice yearly for many
years. For patients with autoimmune diseases, Rituximab infusions are
often given twice (a few weeks apart), and this sequence may be
repeated after 6-12 months, i.e. considerably less doses than in
lymphoma patients (in the short term).
Today a next generation of anti CD-20 antibodies able to deplete
B-cells is used in clinical trials and will presumably be used in the
clinical practice within the next years. For example, the fully
humanized anti CD-20 antibody Ofatumomab of Glaxo-Smith-Kline is
presently in clinical trials for B-cell lymphoma relapse. Fully
humanized anti CD-20 antibody of the next generation are presumed to
result in even more potent B-cell depletion, and should therefore be
even more effective in the treatment of B-cell lymphomas than
Rituximab. Further, it is presumed that they would display less site
effects than described for Rituximab.
Various regimens for the treatment of chronic fatigue syndrome have
been suggested. For example, in US 2007/025375 a complex treatment
scheme is provided for the treatment of patient suffering from chronic
fatigue syndrome. Said region comprises inter alia the administration
of milna cipran.
In view of the unknown aetiology of CFS/ME (myalgic encephalomyelitis)
there is a continued demand for compounds useful for an effective
treatment of CFS.
BRIEF DESCRIPTION OF THE PRESENT INVENTION
The present invention aims in providing new compounds applicable in
the treatment of chronic fatigue syndrome. In particular, the
inventors have found that B-cell depleting agents, like anti-CD20
antibodies, are useful in the treatment of chronic fatigue syndrome.
Preferably, the anti-CD20 antibodies or CD20-binding antibody
fragments thereof are monoclonal antibodies. Particular preferred,
said monoclonal antibodies are humanized antibodies when administered
to human subjects. It is also contemplated in the present invention
that the antibodies may be present as antibody fragments which may
e.g. be produced recombinantly by genetic engineering.
In a further aspect, the present invention relates to methods for the
treatment of chronic fatigue syndrome/myalgic encephalomyelitis
comprising the step of administering a therapeutically effective
amount of a B-cell depleting agent, e.g. a B-cell depleting anti-CD20
antibody or a CD20-binding to a subject afflicted with said disease or
disorder.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows graphically the development of CFS symptoms for three
patients over a one year period outlining the different interventions,
namely Rituximab.RTM. or Methotrexat (M). The symptom score of CFS
symptoms which is in a range of 0 to 10 wherein 0 means no symptoms
while 10 refers to very severe symptoms of CFS.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns B-cell depleting agents either B-cell
depleting biological entities, like anti-CD20 antibodies or
CD20-binding antibody fragments thereof or chemical entities, like
small molecules having a B-cell depleting activity for the treatment
of chronic fatigue syndrome/myalgic encephalomyelitis.
In the context of the present invention, the terms "chronic fatigue
syndrome (CFS" and "myalgic encephalomyelitis (ME)" are used
synonymously.
As used herein, the term "B-cell depletion" or "B-cell depleting
activity" refers to the ability of the entity, either a chemical or
biological entity, e.g. an antibody, to reduce circulating B-cell
levels in a subject. B-cell depletion may be achieved e.g. by inducing
cell death or reducing proliferation.
The "CD20" antigen, or "CD20," is an about 35-kDa, non-glycosylated
phosphoprotein found on the surface of greater than 90% of B cells
from peripheral blood or lymphoid organs in humans. CD20 is present on
both normal B cells as well as malignant B cells, but is not expressed
on stem cells. Other names for CD20 in the literature include
"B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is
described in Clark et al. Proc. Natl. Acad. Sd. (USA) 82:1766 (1985),
for example. The term CD20 includes the equivalent molecules of other
species than human. Recently, low level expression of CD20 on a subset
of T-cells and NK-cells has been reported.
A "B-cell" is a lymphocyte that matures within the bone marrow, and
includes a naive B cell, memory B cell, or effector B cell (plasma
cells).
In a broader sense, the present invention relates not only to the use
of antibodies or fragments thereof for the treatment of CFS but to the
use of antagonists of the CD20 molecule in general having a B-cell
depleting activity for the treatment of CFS.
An "antagonist" or "B-cell depleting agent" which is used herein
interchangeably is a molecule which, e.g. upon binding to a B cell
surface marker, like CD20 on B cells, destroys or depletes B cells in
a mammal and/or interferes with one or more B cell functions, e.g. by
reducing or preventing a humoral response elicited by the B cell. The
antagonist or B-cell depleting agent according to the present
invention is able to deplete B cells (i.e. reduce circulating B cell
levels) in a mammal treated therewith. Such depletion may be achieved
via various mechanisms such antibody-dependent cell-mediated
cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC),
inhibition of B cell proliferation and/or induction of B cell death
(e.g. via apoptosis). Antagonists included within the scope of the
present invention include antibodies, synthetic or native sequence
peptides and small molecule antagonists which bind to the B cell
surface marker, optionally conjugated with or fused to a cytotoxic
agent. A preferred antagonist is a CD20 antibody or CD20-binding
antibody fragment. Furthermore, small molecule antagonists are
preferred, like the known B-cell depleting agent Methotrexat.
Insofar that other cells than B-cells express the CD20 antigen like a
subset of T-cells or NK-cells, these cells are also depleted with the
B-cells depleting agent being an agent acting via CD20.
Antagonists which "induce apoptosis" are those which induce programmed
cell death, e.g. of a B cell, as determined by standard apoptosis
assays, such as binding of annexin V, fragmentation of DNA, cell
shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
and/or formation of membrane vesicles (called apoptotic bodies).
The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies {e.g. bispecific antibodies) formed from at
least two intact antibodies, and antibody fragments so long as they
exhibit the desired biological activity.
In a preferred embodiment, the antibody useful for the treatment of
CFS is a B-cell depleting CD20-binding antibody fragment.
"CD20-binding antibody fragments" comprise a portion of an intact
antibody which comprises the antigen binding region thereof. Examples
of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. For the
purposes herein, an "intact antibody" is one comprising heavy and
light variable domains as well as an Fc region.
"Native antibodies" are usually heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light (L) chains and
two identical heavy (H) chains. Each light chain is linked to a heavy
chain by one covalent disulfide bond, while the number of disulfide
linkages varies among the heavy chains of different immunoglobulin
isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a
variable domain (V11) followed by a number of constant domains. Each
light chain has a variable domain at one end (V1) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The term "variable" refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies and
are used in the binding and specificity of each particular antibody
for its particular antigen. However, the variability is not evenly
distributed throughout the variable domains of antibodies. It is
concentrated in three segments called hypervaiiable regions both in
the light chain and the heavy chain variable domains. The more highly
conserved portions of variable domains are called the framework
regions (FRs). The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet configuration,
connected by three hypervariable regions, which form loops connecting,
and in some cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding site
of antibodies (see Kabat et ah, Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in antibody
dependent cellular cytotoxicity (ADCC).
"Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and antigen-binding site. This region consists of
a dimer of one heavy chain and one light chain variable domain in
tight, non-covalent association. It is in this configuration that the
three hypervariable regions of each variable domain interact to define
an antigen-binding site on the surface of the VH-VL dimer.
Collectively, the six hypervariable regions confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three hypervariable regions specific
for an antigen) has the ability to recognize and bind antigen,
although at a lower affinity than the entire binding site. The Fab
fragment also contains the constant domain of the light chain and the
first constant domain (CH1) of the heavy chain. Fab' fragments differ
from Fab fragments by the addition of a few residues at the carboxy
terminus of the heavy chain CH1 domain including one or more cysteines
from the antibody hinge region. Fab'-SH is the designation herein for
Fab' in which the cysteine residue(s) of the constant domains bear at
least one free thiol group. F(ab')2 antibody fragments originally were
produced as pairs of Fab' fragments which have hinge cysteines between
them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate
species can be assigned to one of two clearly distinct types, called
kappa (K) and lambda (.lamda.), based on the amino acid sequences of
their constant domains. Depending on the amino acid sequence of the
constant domain of their heavy chains, antibodies can be assigned to
different classes. There are five major classes of intact antibodies:
IgA5 IgD, IgE, IgG, and IgM, and several of these maybe further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA,
and IgA2. The heavy chain constant domains that correspond to the
different classes of antibodies are called a, .delta., e, .gamma., and
.mu., respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well known.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of antibody, wherein these domains are present in a single
polypeptide chain. Preferably, the Fv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the
scFv to form the desired structure for antigen binding. For a review
of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies,
vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315 (1994).
The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy chain variable
domain (V11) connected to a light chain variable domain (V1) in the
same polypeptide chain (VH-V1). By using a linker that is too short to
allow pairing between the two domains on the same chain, the domains
are forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more fully
in, for example, EP 404097; WO 93/11161; and Hollinger et al, Proc.
Natl. Acad. Sd. USA, 90:6444-6448 (1993).
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical and/or bind the same epitope, except for possible variants
that may arise during production of the monoclonal antibody, such
variants generally being present in minor amounts. In contrast to
polyclonal antibody preparations which typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity, the monoclonal antibodies
are advantageous in that they are uncontaminated by other
immunoglobulins. The modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example, the
monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al, Nature, 256:495 (1975), or may be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries using
the techniques described in Clackson et al, Nature, 352:624-628 (1991)
and Marks et al, J. Mol Biol, 222:581-597 (1991), for example. The
monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding sequences
in antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences in
antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such antibodies,
so long as they exhibit the desired biological activity (U.S. Pat. No.
4,816,567; Morrison et al, Proc. Natl. Acad. Sd. USA, 81:6851-6855
(1984)). Chimeric antibodies of interest herein include "primatized"
antibodies comprising variable domain antigen-binding sequences
derived from a non-human primate (e.g. Old World Monkey, such as
baboon, rhesus or cynomolgus monkey) and human constant region
sequences (U.S. Pat. No. 5,693,780). "Humanized" forms of non-human
(e.g., murine) antibodies are chimeric antibodies that contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody) in
which residues from a hypervariable region of the recipient are
replaced by residues from a hypervariable region of a non-human
species (donor antibody) such as mouse, rat, rabbit or nonhuman
primate having the desired specificity, affinity, and capacity. In
some instances, framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are not
found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of a
non-human immunoglobulin and all or substantially all of the FRs are
those of a human immunoglobulin sequence, except for FR
substitution(s) as noted above. The humanized antibody optionally also
will comprise at least a portion of an immunoglobulin constant region,
typically that of a human immunoglobulin. For further details, see
Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992). The term "hypervariable region" when used herein refers to the
amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al, Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues from
a "hypervariable loop" {e.g. residues 26-32 (L1), 50-52 (L2) and 91-96
(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J.
Mol. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are those
variable domain residues other than the hypervariable region residues
as herein defined. A "naked antibody" is an antibody (as herein
defined) which is not conjugated to a heterologous molecule, such as a
cytotoxic moiety or radiolabel. Examples of antibodies which bind the
CD20 antigen include: "C2B8" which is now called "Rituximab"
("RITUXAN.RTM.") (U.S. Pat. No. 5,736,137, expressly incorporated
herein by reference); the yttrium-[90]-labeled 2B8 murine antibody
designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN.RTM. (U.S. Pat.
No. 5,736,137, expressly incorporated herein by reference); murine
IgG2a "BI," also called "Tositumomab," optionally labeled with 131I to
generate the "131I-BI" antibody (iodine 1131 tositumomab, BEXXAR.TM.)
(U.S. Pat. No. 5,595,721, expressly incorporated herein by reference);
murine monoclonal antibody "1F5" (Press et al. Blood 69(2):584-591
(1987) and variants thereof including "framework patched" or humanized
1F5 (WO03/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and
chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated
herein by reference); humanized 2H7; Ofatumumab, a fully humanized
IgG1 against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark;
WO2004/035607); AME-133 (Applied Molecular Evolution); A20 antibody or
variants thereof such as chimeric or humanized A20 antibody (cA20,
hA20, respectively) (US 2003/0219433, Immunomedics); and monoclonal
antibodies L27, G28-2, 93-1B3, B--Cl or NU-B2 available from the
International Leukocyte Typing Workshop (Valentine et ah, In:
Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press
(1987)). Further, suitable antibodies are e.g. Ocrelizumab, a fully
humanized anti-CD20 antibody of Biogen Idec/Genentech/Roche the
antibody GA101, a third generation humanized anti-CD20-antibody of
Biogen Idec/Genentech/Roche. Moreover, BLX-301 of Biolex Therapeutics,
a humanized anti CD20 with optimized glycosyylation or Veltuzumab
(hA20) of Immunomedics or DXL625 of Inexus Biotechnology both are
humanized anti-CD20 antibodies are suitable.
Moreover, it is assumed that other B-cell depleting agents, in
particular, anti-CD22 antibodies, like Epratuzumab or anti-CD19
humanized antibodies, like MDX-1342 can be used for the treatment of
CFS.
The terms "rituximab" or "RITUXAN.RTM." or "mabthera" herein refer to
the genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S. Pat.
No. 5,736,137, expressly incorporated herein by reference, including
fragments thereof which retain the ability to bind CD20. Purely for
the purposes herein and unless indicated otherwise, "humanized 2H7"
refers to a humanized antibody that binds human CD20, or an
antigen-binding fragment thereof, wherein the antibody is effective to
deplete primate B cells in vivo.
The expression "effective amount" of the B-cell depleting agent or
antagonist, in particular of the anti-CD20 antibody or CD20-binding
antibody fragment thereof, refers to an amount of the B-cell depleting
agent or antagonist which is effective for treating CFS. For example,
the anti-CD20 antibody for the treatment of chronic fatigue
syndrome/myalgic encephalomyelitis is administered in the range of 10
mg to 5000 mg per dosage. For example, the dosage may be in the range
of from 100 to 1000 mg/m2, in particular, 500 mg/m2 as a single
infusion for Rituximab. Typically, the dosage for Methotrexate is in
the range of 5 mg to 30 mg per week.
In one preferred embodiment, the B-cell depleting agent is a chemical
entity, e.g. a small molecule. A variety of B-cell depleting agents
are known in the art for example known B-cell depleting agents are
BAFF-antagonists. Furthermore, known B-cell depleting agents include
antagonist of BR3, agonists of alpha-4-integrins etc. For example,
Methotrexate is an analogue of folic acid displaying B-cell depleting
activity. Other useful B-cell depleting agent are small modular
immunopharmaceuticals (SMIP) against CD20. For example, SMIP acting as
B-cell depleting agents are TRU-015 or SBI-087 of Trubion
Pharmaceuticals. Also, SMIP can be single chain polypeptides, smaller
than antibodies, having a potent B-cell depletion activity.
In a preferred embodiment, a combination of an anti CD20 antibody and
representing a biological entity of a B-cell depleting agent and
Methotrexat, representing a chemical entity of a B-cell depleting
agent, are used for treating chronic fatigue syndrome of myalgic
encephalomyelitis. Administration of these entities may be effected
simultaneously, separately or sequentially. For example, in a first
regimen either the antibody or Methotrexat is administered to the
subject while in a second regimen the other agent is administered.
The composition comprising the B-cell depleting agent, the antagonist,
in particular, the anti CD20 antibody or the CD20-binding antibody
fragment thereof, will be formulated, dosed, and administered in a
fashion consistent with good medical practice. Factors for
consideration in this context include the stage of the particular
disease or disorder being treated, the particular mammal being
treated, the clinical condition of the individual subject, the site of
delivery of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners. The
effective amount of the B-cell depleting agent, like an antibody or
antibody fragment to be administered will be governed by such
considerations. As a general proposition, the effective amount of the
antagonist administered parenterally per dose will be in the range of
about 20 mg/m2 to about 10,000 mg/m2 of subject body, by one or more
dosages. Exemplary dosage regimens for intact antibodies include 375
mg/m2 weekly.times.4; 1000 mg.times.2 (e.g. on days 1 and 15); or 1
gram.times.3. The antibody for the administration to a subject in a
single therapeutically effective dosage of said antibody is of 50 to
2000 mg/m2 or multiple of therapeutically effective dosages of said
antibody or CD20-binding antibody fragment thereof of 50 to 2000
mg/m2. As noted above, however, these suggested amounts of antibody
are subject to a great deal of therapeutic discretion. The key factor
in selecting an appropriate dose and scheduling is the result
obtained, as indicated above. The B-cell depleting agent antagonist,
like the antibody, is administered by any suitable means, including
parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary,
intranasal, and/or intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. Intrathecal administration is also
contemplated. In addition, the B-cell depleting agent antagonist, like
the antibody may suitably be administered by pulse infusion, e.g.,
with declining doses of the antagonist. Preferably the dosing is given
by intravenous injections.
Methods for generating such B-cell depleting antagonists will be
described here. The antigen to be used for production of, or screening
for, antagonist(s) maybe, e.g., a soluble form of CD20 or a portion
thereof, containing the desired epitope. Alternatively, or
additionally, cells expressing CD20 at their cell surface can be used
to generate, or screen for, antagonist(s). Other forms of CD20 useful
for generating antagonists will be apparent to those skilled in the art.
While the preferred antagonist is an antibody, antagonists other than
antibodies are contemplated herein. For example, the antagonist may
comprise a small molecule antagonist. Libraries of small molecules may
be screened against CD20 in order to identify a small molecule which
binds to that antigen. Alternatively, the small molecules may be
screened on their B-cell depleting activity in general by known
techniques. The small molecule may further be screened for its
antagonistic properties. The antagonist may also be a peptide
generated by rational design or by phage display (see, e.g.,
WO98/35036 published 13 Aug. 1998). In one embodiment, the molecule of
choice maybe a "CDR mimic" or antibody analogue designed based on the
CDRs of an antibody. While such peptides may be antagonistic by
themselves, the peptide may optionally be fused to a cytotoxic agent
so as to add or enhance antagonistic properties of the peptide. A
description follows as to exemplary techniques for the production of
the antibody antagonists used in accordance with the present invention.
(i) Polyclonal Antibodies
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the relevant
antigen and an adjuvant. It may be useful to conjugate the relevant
antigen to a protein that is immunogenic in the species to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide
ester (conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride, SOCl2,
or R1N.dbd.C.dbd.NR, where R and R1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by combining, e.g., 100 .mu.g or 5 .mu.g of the protein or
conjugate (for rabbits or mice, respectively) with 3 volumes of
Freund's complete adjuvant and injecting the solution intradermally at
multiple sites. One month later the animals are boosted with 1/5 to
1/10 the original amount of peptide or conjugate in Freund's complete
adjuvant by subcutaneous injection at multiple sites. Seven to 14 days
later the animals are bled and the serum is assayed for antibody
titer. Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different
cross-linking reagent. Conjugates also can be made in recombinant cell
culture as protein fusions. Also, aggregating agents such as alum are
suitably used to enhance the immune response.
(ii) Monoclonal Antibodies
Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope except for
possible variants that arise during production of the monoclonal
antibody, such variants generally being present in minor amounts.
Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete or polyclonal antibodies.
For example, the monoclonal antibodies may be made using the hybridoma
method first described by Kohler et ah, Nature, 256:495 (1975), or may
be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
DNA encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies). The
hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not
otherwise produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review articles
on recombinant expression in bacteria of DNA encoding the antibody
include Skerra et ah, Curr. Opinion in Immunol., 5:256-262 (1993) and
Pluckthun, Immunol. Revs., 130:151-188 (1992). In a further
embodiment, antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described in
McCafferty et ah, Nature, 348:552-554 (1990). Clackson et ah, Nature,
352:624-628 (1991) and Marks et ah, J. Mol. Biol., 222:581-597 (1991)
describe the isolation of murine and human antibodies, respectively,
using phage libraries. Subsequent publications describe the production
of high affinity (nM range) human antibodies by chain shuffling (Marks
et ah, Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et ah, Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable alternatives
to traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies. The DNA also may be modified, for example,
by substituting the coding sequence for human heavy- and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, et ah, Proc. Natl. Acad. ScL USA,
81:6851 (1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site of
an antibody to create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and another
antigen-combining site having specificity for a different antigen.
(iii) Humanized Antibodies
Methods for humanizing non-human antibodies have been described in the
art. Preferably, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers (Jones et al, Nature, 321:522-525 (1986);
Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting hypervariable region
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies (U.S.
Pat. No. 4,816,567) wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species, hi practice, humanized antibodies are
typically human antibodies in which some hypervariable region residues
and possibly some FR residues are substituted by residues from
analogous sites in rodent antibodies. The choice of human variable
domains, both light and heavy, to be used in making the humanized
antibodies is very important to reduce antigenicity. According to the
so-called "best-fit" method, the sequence of the variable domain of a
rodent antibody is screened against the entire library of known human
variable-domain sequences. The human sequence which is closest to that
of the rodent is then accepted as the human framework region (FR) for
the humanized antibody (Sims et al, J. Immunol, 151:2296 (1993);
Chothia et al, J. Mol. Biol, 196:901 (1987)). Another method uses a
particular framework region derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chain
variable regions. The same framework may be used for several different
humanized antibodies (Carter et al, Proc. Natl. Acad. ScL USA, 89:4285
(1992); Presta et al, J. Immunol, 151:2623 (1993)). It is further
important that antibodies be humanized with retention of high affinity
for the antigen and other favorable biological properties. To achieve
this goal, according to a preferred method, humanized antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three-dimensional models
of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to those
skilled in the art. Computer programs are available which illustrate
and display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so that
the desired antibody characteristic, such as increased affinity for
the target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in
influencing antigen binding.
(iv) Human Antibodies
As an alternative to humanization, human antibodies can be generated.
For example, it is now possible to produce transgenic animals {e.g.,
mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that the
homozygous deletion of the antibody heavy chain joining region (J11)
gene in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice will
result in the production of human antibodies upon antigen challenge.
See, e.g., Jakobovits et al, Proc. Natl. Acad. ScL USA, 90:2551
(1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et
al, Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,
5,589,369 and 5,545,807. Alternatively, phage display technology
(McCafferty et al, Nature 348:552-553 (1990)) can be used to produce
human antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as MI 3 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA copy
of the phage genome, selections based on the functional properties of
the antibody also result in selection of the gene encoding the
antibody exhibiting those properties. Thus, the phage mimics some of
the properties of the B cell. Phage display can be performed in a
variety of formats; for their review see, e.g., Johnson, Kevin S, and
Chiswell, David J., Current Opinion in Structural Biology 3:564-571
(1993). Several sources of V-gene segments can be used for phage
display. Clackson et al, Nature, 352:624-628 (1991) isolated a diverse
array of anti-oxazolone antibodies from a small random combinatorial
library of V genes derived from the spleens of immunized mice. A
repertoire of V genes from unimmunized human donors can be constructed
and antibodies to a diverse array of antigens (including
self-antigens) can be isolated essentially following the techniques
described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or
Griffith et al, EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.
5,565,332 and 5,573,905. Human antibodies may also be generated by in
vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
(v) Antibody Fragments
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al, Journal of
Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et
al, Science, 229:81 (1985)). However, these fragments can now be
produced directly by recombinant host cells. For example, the antibody
fragments can be isolated from the antibody phage libraries discussed
above. Alternatively, Fab'-SH fragments can be directly recovered from
E. coli and chemically coupled to form F(ab')2 fragments (Carter et
al, Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner. In other embodiments,
the antibody of choice is a single chain Fv fragment (scFv). See WO
93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The
antibody fragment may also be a "linear antibody", e.g., as described
in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments
may be monospecific or bispecific.
Pharmaceutical Formulations
Therapeutic formulations of the B-cell depleting agents, like
antibodies or other antagonists used in accordance with the present
invention are prepared for storage by mixing an antibody or a fragment
thereof having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
{Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl
or benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins,
such as serum albumin, gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose, mannitol, trehalose or sorbitol; salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.TM.,
PLURONICS.TM. or polyethylene glycol (PEG).
Exemplary anti-CD20 antibody formulations are described in WO98/56418,
expressly incorporated herein by reference. This publication describes
a liquid multidose formulation comprising 40 mg/mL rituximab, 25 mM
acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20
at pH 5.0 that has a minimum shelf life of two years storage at
2-8.degree. C. Another anti-CD20 formulation of interest comprises 1
mg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium
citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for
Injection, pH 6.5. Lyophilized formulations adapted for subcutaneous
administration are described in U.S. Pat. No. 6,267,958 (Andya et ah).
Such lyophilized formulations may be reconstituted with a suitable
diluent to a high protein concentration and the reconstituted
formulation may be administered subcutaneously to the mammal to be
treated herein. Crystallized forms of the antibody or antagonist are
also contemplated. See, for example, US 2002/0136719AI.
The active ingredients may also be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed in
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the antagonist, which matrices are in
the form of shaped articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid
and ? ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydiOxybutyric acid. The formulations to be used for in
vivo administration must be sterile. This is readily accomplished by
filtration through sterile filtration membranes.
EXAMPLES
At the Department of Oncology and Medical Physics, Haukeland
University Hospital, a striking symptomatic improvement after
cytotoxic chemotherapy in a 43-year old female patient with stable CFS
(debut in 1997, preceded by Epstein-Barr infection), was observed. She
developed Hodgkin's disease in 2003, and was treated with chemotherapy
and radiation. She had a lymphoma relapse in 2004, and was treated
with chemotherapy. Contrary to the expected (CFS patients generally
tolerate all types of drugs and stress poorly), the patient
experienced a marked decrease in CFS symptoms during and after this
chemotherapy. The changes were not interpreted as related to lymphoma
activity, and the effects lasted for approximately 5 months after
chemotherapy initiation, with then gradual relapse of CFS-like
symptoms. In addition to the cytotoxic effects, the chemotherapeutics
given also had an immunomodulatory effect. It is assumed that the
effects on CFS symptoms are mediated mainly through the drug
Methotrexate administered during chemotherapy.
When reviewing the literature on CFS in an attempt to understand what
our patient encountered during and after cancer chemotherapy, the
conclusion was done that modification of the immune system seemed a
likely explanation of the marked, but transient symptom improvement
experienced. It may well be that chronic B-cell activation seen in CFS
patients is important for the symptoms and also physiological changes
reported, such as central nervous blood circulation alterations, and
reports of lymphocyte infiltration in brain tissue, spinal nerve roots
or cardiac muscle.
Possible modes of action of B-cell depletion could be at several
sites, such as interactions with the T-cell system, thus modifying
inflammatory processes, and in influencing the levels of important
pleiotropic players in the immune homeostasis, such as the vasoactive
neuropeptides. These have a wide range of activities in the central
nervous system.
Taking these existing data into account, together with the unexpected
improvement of fatigue and pain in the CFS patient after
immunomodulatory cytotoxic therapy, it is assumed that B-cell
depletion as a concept could allow treatment of CFS.
At present, achievement of B-cell depletion is most readily achieved
by the use of the monoclonal anti-CD20 antibody Rituximab. However,
also new generation anti-CD20 antibodies are supposed to have at least
a similar effect on CFS symptoms, due to a presumed more potent B-cell
depletion achieved.
Pilot Patient 1:
As a first pilot patient, the above-mentioned woman received Rituximab
500 mg/m.sup.2 as a single infusion after informing of the
experimental nature of the procedure and the risks involved. Prior to
treatment, she had a stable CFS with marked fatigue and she was not
able to work out-of-house or do house-keeping. She used an electrical
wheel-chair for outdoor movement. Starting between five and six weeks
after the infusion, she experienced a marked improvement in symptoms,
with profoundly less fatigue, decreasing muscle pain, decreasing
burning pain in the skin, and declining headaches, accompanied with a
diminishing need of opioid analgesics. Due to declined fatigue, she
could now go for long walks, resumed her hobbies and was able to do
house-keeping work and take care of her children. She also reported a
marked improvement in cognitive function, retaining the ability to
concentrate, and was again able to read and e.g. work with computers.
The effect after the first infusion lasted until 14 weeks after the
Rituximab infusion, then declined, with a gradual, but not complete
relapse of CFS symptoms.
Five months after the first Rituximab infusion, she again had stable
and disabling CFS symptoms. She received a new single infusion of
Rituximab in the same dosage. After 6 weeks, she again experienced a
gradual and major recovery from all CFS symptoms (fatigue, pain,
cognitive symptoms) with a major effect on the quality of life. After
the second infusion (also as a single infusion at a low dose 500
mg/m.sup.2), the therapeutic effect lasted until 16 weeks, with then
slowly and gradual symptom worsening thereafter.
It was then decided to start weekly oral low-dose Methotrexate from 18
weeks after the second Rituximab-infusion, starting at 7.5 mg per
week, and increasing the dose to 12.5 mg per week during the next two
months. From 12 weeks after onset of weekly Mtx, she has again
experienced gradual and moderate CFS symptom recovery. She has now
used Mtx for 22 weeks, and she interprets the improvement to be
moderate and significant, but at present not as pronounced and rapid
as after Rituximab treatment. However she still experiences a gradual
improvement in her condition. The development of CFS symptoms is shown
in FIG. 1.
Pilot Patient 2:
He is a 42-year old male, and developed CFS after an Epstein-Barr
infection 8 years ago. He had marked fatigue and was not able to do
any work since encountering the entity. He was constrained to sit in a
chair most of the days. After mild exercise he had major problems with
exhaustion, increasing muscle pain and headaches. He also had fever
sensations, sweating and diarrhoea. He had serious cognitive
disturbances. Although being a previous experienced computer engineer,
he was unable to use a computer or to read coherently more than 1-2
pages in a book.
He was given a single infusion of Rituximab 500 mg/m.sup.2. The first
symptom to improve (3 weeks after infusion) was the longstanding
diarrhea. Starting 6 weeks after infusion, he experiences a marked
response with a dramatic improvement in fatigue, pain, cognitive and
autonomic symptoms. He was then able to perform manual labour and
enjoy computer games and reading. After the relatively low dose
(single infusion 1000 mg) the effect was most prominent until 12
weeks, and thereafter gradually declined. He and his family described
the clinical improvement as significant, yielding a major impact on
the quality of life of the whole family.
Five months after the first Rituximab infusion, he has now been
retreated with two infusions of Rituximab 1000 mg two weeks apart. As
following the first treatment, he started to recover first from the
diarrhea (after 3 weeks). Then, after 6 weeks, he reported less
cognitive symptoms, and some days later the fatigue started to improve.
The double Rituximab infusion gave a clear CFS symptom improvement
most prominent 16 weeks after the infusion. Thereafter, he has
experienced a very slow and gradual increase in his symptoms. However,
5 months after the infusion, he still has a clinical response (he is
still better than his condition prior to treatment) (FIG. 1). He has
now started weekly oral low-dose Methotrexate treatment.
Pilot Patient 3:
She is a 22-year student, developing CFS after mononucleosis 7 years
ago. Initially, she had the full-blown clinical picture with marked
fatigue, with pain including headaches, cognitive disturbances and
autonomic symptoms. During the last four years, she had however
experienced some improvement, but still retained marked fatigue,
excess sleep requirement and loose bowels. She had moderate cognitive
disturbances and moderate muscle pain.
She was given a single infusion of Rituximab 500 mg/m.sup.2. This
patient also experienced improvement from loose bowels 3 weeks after
infusion. Six weeks after infusion, she noted some improvement in
muscle pain. The first five months, she also had slight improvement in
fatigue, but transient and of shorter duration than in the other
patients.
However, from 6 months after the infusion, she experienced a major
clinical response on all CFS symptoms, to a high level of functioning
not experienced the last 7 years. She started to study full-time, and
could read without problems, and also noted a marked improvement in
her short-time memory. This dramatic improvement has now lasted for
41/2 months. The following weeks she then experienced a gradual
relapse of CFS symptoms. She has now received new infusions of
Rituximab (two infusions 500 mg/m.sup.2, given two weeks apart).
Methotrexat (Mtx) is a therapeutic agent with known (but not well
understood) immunomodulatory properties. Given orally on a weekly
schedule for rheumatoid arthritis, one of the drug effects is a
moderate B-cell depletion, which mechanistically is similar to but not
as pronounced as the effect of Rituximab (Edwards et al. NEJM, 2004.
Efficacy of B-Cell Targeting Therapy with Rituximab in Patients with
Rheumatoid Arthritis). For one of the three pilot patients (patient
1), she was treated weekly with Methotrexat for the last 22 weeks,
also with a significant and moderate clinical response on CFS
symptoms, starting from 10 weeks after initiating Mtx.
In conclusion, a major clinical response after Rituximab treatment in
three out of three pilot patients, in two these with a repeated
clinical responses also after a second Rituximab treatment, has been
observed.
The third had a limited improvement in CFS symptoms from 6 weeks after
the Rituximab infusion. However, from 6 to 101/2 months after the
infusion she had a major clinical response on all CFS related symptoms
lasting until now (101/2 months after infusion). Then she had a
gradual relapse, and she has now received new Rituximab treatment (two
infusions, i.e. at weeks 47 and 49 after her first Rituximab treatment).
All five treatments of the three patients resulted in marked to
moderate main symptom improvement. For these three patients, the
kinetics in symptom improvement has been very similar, but with in
addition a major late and long-lasting response in patient 3, as shown
in FIG. 1. The interval is compatible with the known degradation and
half-time of certain proteins that can be produced by B-lymphocytes.
The reappearance of symptoms after Rituximab treatment is compatible
with the maturing of pre-plasma cell B-lymphocytes from stem cells
after the CD20 antigen directed B-cell lysis, which is mediated
through complement-directed cytotoxicity (CDC) and by antibody
dependent cellular cytotoxicity (ADCC). These immature B-cells have
been shown to be capable of protein production, among them the
production of antibodies.
--------
(c) 2011 United States Patent and Trademark Office
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