U.S. patent application number 13/127541 was filed with the patent office on 2013-12-05 for treatment of acute lymphoblastic leukemia.
This patent application is currently assigned to Micromet AG. The applicant listed for this patent is Evelyn Degenhard, Gerhard Zugmaier. Invention is credited to Evelyn Degenhard, Gerhard Zugmaier.
Application Number | 20130323247 13/127541 |
Document ID | / |
Family ID | 41692882 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323247 |
Kind Code |
A1 |
Zugmaier; Gerhard ; et
al. |
December 5, 2013 |
TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA
Abstract
The present invention relates to a method for the treatment,
amelioration or elimination of acute lymphoblastic leukemia (ALL),
the method comprising the administration of a pharmaceutical
composition comprising a CD19xCD3 bispecific single chain antibody
construct to an adult patient in the need thereof.
Inventors: |
Zugmaier; Gerhard; (Munchen,
DE) ; Degenhard; Evelyn; (Munchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zugmaier; Gerhard
Degenhard; Evelyn |
Munchen
Munchen |
|
DE
DE |
|
|
Assignee: |
Micromet AG
|
Family ID: |
41692882 |
Appl. No.: |
13/127541 |
Filed: |
November 6, 2009 |
PCT Filed: |
November 6, 2009 |
PCT NO: |
PCT/EP2009/007970 |
371 Date: |
May 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61112323 |
Nov 7, 2008 |
|
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61183291 |
Jun 2, 2009 |
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Current U.S.
Class: |
424/135.1 ;
530/387.3 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/31 20130101; C07K 16/2803 20130101; A61P 35/02 20180101;
C07K 16/2809 20130101; C07K 16/468 20130101 |
Class at
Publication: |
424/135.1 ;
530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/46 20060101 C07K016/46; A61P 35/02 20060101
A61P035/02 |
Claims
1. A method for the treatment, amelioration or elimination of acute
lymphoblastic leukemia (ALL), the method comprising the
administration of a pharmaceutical composition comprising a
CD19.times.CD3 bispecific single chain antibody construct to an
adult patient in the need thereof.
2. The method of claim 1, wherein said acute lymphoblastic leukemia
(ALL) is B-lineage acute lymphoblastic leukemia.
3. The method of claim 1, wherein said acute lymphoblastic leukemia
(ALL) is refractory to chemotherapy in patients non-eligible for
allogeneic hematopoietic stem cell transplantation.
4. The method of claim 1, followed by allogeneic hematopoietic stem
cell transplantation or wherein said method replaces allogeneic
hematopoietic stem cell transplantation in patients eligible for
allogeneic hematopoietic stem cell transplantation.
5. The method of claim 1, wherein the method is for the treatment,
amelioration or elimination of minimal residual disease (MRD) in a
patient with acute lymphoblastic leukemia (ALL).
6. The method of claim 5, wherein said patient is MRD-positive in
complete hematological remission.
7. The method of claim 5, wherein the administration of said
pharmaceutical composition results in stable disease or converts
MRD positive acute lymphoblastic leukemia (ALL) into an MRD
negative status.
8. The method of claim 5, wherein MRD is measured with quantitative
detection of individual rearrangements of immunoglobulin genes or
T-cell receptor (TCR) rearrangements, or by bcr/abl fusion
transcripts, or by t(4;11) translocations using PCR or FACS
analysis.
9. The method of claim 8, wherein the ALL patient shows a bcr/abl
or a t(4;11) translocation signal above detection limit and/or at
least one marker by rearrangement with a sensitivity
of>10.sup.-4.
10. The method of claim 8, wherein the time to molecular relapse
detectable by the method is more than 4 months.
11. The method of claim 1, wherein the corresponding variable heavy
chain regions (V.sub.H) and the corresponding variable light chain
regions (V.sub.L) regions in said CD19.times.CD3 bispecific single
chain antibody construct are arranged, from N-terminus to
C-terminus, in the order,
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.H(CD3)-V.sub.L(CD3).
12. The method of claim 11, wherein said CD19.times.CD3 bispecific
single chain antibody construct comprises an amino acid sequence as
set forth in SEQ ID NO. 1.
13. The method of claim 1, wherein one treatment cycle is a 4-week
continuous infusion.
14. The method of claim 13, wherein the treatment cycle is repeated
at least three times, after determination of a MRD negative status
(consolidation).
15. The method of claim 1, wherein the CD19.times.CD3 bispecific
single chain antibody construct is to be administered in a daily
dose of 5 .mu.g to 100 .mu.g per square meter patient body surface
area.
16. The method of claim 15, wherein the CD19.times.CD3 bispecific
single chain antibody construct is to be administered in a daily
dose of 15 .mu.g to 30 .mu.g per square meter patient body surface
area.
17. (canceled)
18. The method of claim 2, wherein said B-lineage acute
lymphoblastic leukemia is B-precursor acute lymphoblastic
leukemia.
19. The method of claim 11, wherein said CD19.times.CD3 bispecific
single chain antibody construct comprises an amino acid sequence at
least 90% identical to SEQ ID NO. 1.
20. The method of claim 11, wherein said CD19.times.CD3 bispecific
single chain antibody construct comprises an amino acid sequence at
least 95% identical to SEQ ID NO. 1.
21. The method of claim 13, wherein the at least four weeks of
continuous infusion is followed by at least a 2-week treatment-free
interval.
22. The method of claim 14, wherein each treatment cycle is
followed by at least a 2-week treatment-free interval.
Description
[0001] The present invention relates to a method for the treatment,
amelioration or elimination of acute lymphoblastic leukemia (ALL),
the method comprising the administration of a pharmaceutical
composition comprising a CD19xCD3 bispecific single chain antibody
construct to an adult patient in the need thereof.
[0002] Leukemias are clonal neoplastic proliferations of immature
hematopoietic cells that are characterized by aberrant or arrested
differentiation. Leukemia cells accumulate in the bone marrow,
ultimately replacing most of normal hematopoietic cells. This
results in bone marrow failure and its consequences of anemia,
hemorrhage and infection. Leukemia cells circulate into the blood
and other tissues throughout the body (DeVita, Hellmann, Rosenberg.
Cancer: principles and practice of oncology. Eight edition. Library
of Congress Cataloging-in-Publication Data, ISBN 0-781-72387-6).
The acute leukemias, which can be broadly grouped as either
lymphoblastic or myeloblastic can be identified phenotypically and
genetically and are characterized by a rapid clinical course
requiring immediate treatment. Acute leukemia's are derived from
early hematopoietic progenitor cells. In contrast chronic
leukemia's have the phenotype and biologic character of more mature
cells (DeVita et al., loc. cit.). Acute lymphoblastic leukemia
(ALL) is distinguished from the lymphomas because the latter
resemble more mature lymphoid cells and typically inhabit the lymph
nodes, spleen or other extramedullary sites before spreading to the
bone marrow. Certain lymphomas such as lymphoblastic lymphomas or
Burkitt's lymphomas retain features of both the leukemia's and
lymphomas but are derived from immature progenitor cells and
require therapy similar to that used for acute lymphoblastic
leukemia ALL). Other lymphomas however may spread widely into the
blood and bone marrow, and in such a phase can be described as
leukemic lymphomas but are not true leukemias (De Vita et al., loc.
cit.). Acute lymphoblastic leukemia is a relatively rare
malignancy. The total incidence of acute lymphoblastic leukemia
(ALL) is 1.1/100,000 per year. The incidence has its peak during
childhood, decreasing continuously with increasing age. From the
age of 35 years on the incidence rises again and a second peak is
observed starting from the age of 80 years (2.3/100,000 per year)
(Hoelzer and Gokbuget; Der Onkologe 12 (2006); 983-1002). Although
the etiology of acute lymphoblastic leukemia (ALL) is unclear, it
is one of the most carefully studied and best characterized
neoplasms. The acute lymphoblastic leukemia (ALL) subgroups are
defined mainly by immunophenotyping, cytogentics and molecular
genetics. B-lineage acute lymphoblastic leukemia (ALL) with 74% of
cases comprises the majority of ALL's. Seventy percent of all ALL's
are B-precursor ALL's and 4% are mature B-cell ALL's. T-lineage
ALL's covers 26% of all ALL's (Hoelzer and Gokbuget; Der Onkologe
12 (2006); 983-1002).
[0003] In the early 1980s, adult acute lymphoblastic leukemia (ALL)
was a rarely curable disease with an overall survival of less than
10%. After use of adapted regimens administered by pediatric groups
the outcome improved to 30-40%. A period of stagnation followed
with improvement only in distinct subgroups. However, in the last
five years, progress has been made in molecular diagnostics of
acute lymphoblastic leukemia (ALL). Stem cell transplantation (SCT)
has improved the outcome of acute lymphoblastic leukemia (ALL) and
has made treatment more feasible. Though various new targeted drugs
are under evaluation, effective targeted therapies for acute
lymphoblastic leukemia (ALL) are not yet available. Rapid diagnosis
and classification of acute lymphoblastic leukemia (ALL) is
increasingly important to identify prognostic and molecular genetic
subsets that will be the focus of targeted treatment (Hoelzer and
Gokbuget; Hematology (2006); 133-141). The Philadelphia chromosome
(Ph), the result of a reciprocal translocation fusing the abl
proto-oncogene from chromosome 9 with the breakpoint cluster region
sequences on chromosome 22, was the first neoplasm-specific
translocation to be identified. Translocation (9;22) is the most
frequent genetic aberration in adult acute lymphoblastic leukemia
(ALL). It is found in 20-30% of patients. The incidence increases
with age, approaching 50% in patients older than 50 years. In past
clinical studies, older patients were underrepresented due to the
perceived futility of treatment, but this pattern is changing with
the availability of promising novel treatment options. Notably, it
is found almost exclusively in CD10+ precursor B-cell acute
lymphoblastic leukemia (c-ALL and pre-B ALL); rare reports of its
presence in T-lineage ALL may represent chronic myeloid leukemia
(CML) in lymphoid blast crisis rather than bona fide Ph+ ALL.
Clinically, patients present with a variable white blood cell (WBC)
count, surface expression of CD19, CD10 and CD34 antigens, and
frequent co-expression of myeloid markers, e.g., CD13 and CD33,
have an increased risk of developing meningeal leukemia. The
prognosis of adult patients with Ph+ ALL treated only with
chemotherapy is poor, with a less than 10% probability of long-term
survival.
[0004] Because of the dismal outcome with chemotherapy, allogeneic
hematopoietic stem cell transplantation (HSCT) is currently
considered to be the treatment of choice in adult Ph+ ALL. 12% to
65% long-term survival rates have been reported for patients
undergoing SCT in first complete remission (CR), indicating that
this procedure is potentially curative. However, approximately 30%
of these patients experience relapses (Ottmann and Wassmann;
Hematology (2005), 118-122). The presence of leukemia cells below
the cytological detection limit (5% leukemic cells) is defined as
minimal residual disease (MRD). If no MRD is detectable
(<10.sup.-4, i.e. <1 leukemia cell per 10.sup.4 bone marrow
cells) a complete molecular remission is reached. In the last
years, a series of retrospective studies has shown that MRD in
adult acute lymphoblastic leukemia is an independent prognostic
factor as already demonstrated for childhood leukemia. Diagnostic
tools for MRD are polymerase chain reaction (PCR) and/or flow
cytometry. PCR analysis can detect fusion transcripts such as
bcr/abl and individual clonal rearrangements of immunoglobulins
(IgH) and/or T-cell receptor genes (TCR). About 25% of patients
with minimal residual disease (MRD) defined by rearrangement
comprise a high-risk group with a 94% relapse rate within 3 years.
In general, the decrease in MRD occurs more slowly in adults than
it does in children. Decision making about treatment
intensification by allogeneic peripheral blood stem cell
transplantation (PBSCT) is therefore too early after induction
treatment. However, after start of consolidation, minimal residual
disease (MRD) at any time point is associated with a high risk of
relapse (Bruggemann et al., Blood 107 (2006), 1116-1123; Raff et
al., Blood 109 (2007), 910-915).
[0005] Treatment of adult patients with acute lymphoblastic
leukemia (ALL) is becoming increasingly complex as diverse
treatment protocols are introduced for different subtypes of the
disease, reflecting the intention to optimally tailor therapy to
specific risk-adapted disease entities. Recent improvements have
been achieved by introducing new therapeutic principles, such as
the early addition of the tyrosine kinase inhibitor imatinib in
Ph-positive (Ph+) ALL (Lee et al., Blood 102 (2003), 3068-3070) or
the use of the anti-CD20 antibody rituximab in CD20.sup.+ cases of
B-lineage ALL (see e.g. Griffin et al., Pediatr Blood Cancer 2008).
Diagnostic improvements were achieved by assessing the level of
minimal residual disease (MRD) either by molecular genetic methods
or by flow cytometry, which has been shown to be predictive for
outcome in a number of studies in children (see e.g. Cave et al.,
N. Engl. J. Med. 339 (1998), 591-598) and adults (see e.g.
Bruggemann et al., Blood 107 (2006), 1116-1123). Survival rates
with modern treatment protocols for adult acute lymphoblastic
leukemia (ALL) patients have reached a plateau where the potential
benefit of more aggressive chemotherapeutic regimens is often
offset by an excess mortality due to complications, thus making
efforts to individualize treatment even more important. Whereas
standard-risk patients without conventional risk factors, who have
a greater than 50% chance of long-term survival with chemotherapy
alone (Hoelzer et al., Hematology Am. Soc. Hematol. Educ. Program 1
(2002), 162-192) are potentially put at unnecessary risk by
intensified and prolonged therapy, outcome in patients with
relapsed acute lymphoblastic leukemia (ALL) is extremely poor, even
if a second remission is achieved. In a recent study, minimal
residual disease (MRD) monitoring during the first year of
intensive chemotherapy led to an MRD-based risk stratification
(Bruggemann et al. (2006), loc. cit.). This classification allowed
the identification of an MRD low-risk group consisting of about 10%
of patients with a minimal chance of relapse at 3 years, an MRD
high-risk group of about 25% of patients with an almost 100% risk
of relapse, and an MRD intermediate-risk group. In the latter
group, about 30% of patients will eventually relapse despite
becoming MRD negative or reaching MRD levels below 10.sup.-4 at the
end of the first year of therapy.
[0006] These data show that acute lymphoblastic leukemia (ALL)
remains for most patients a fulminate and incurable disease. In
light of this, there is an urgent need for improved ALL
therapies.
[0007] The present invention provides for a method for the
treatment, amelioration or elimination of acute lymphoblastic
leukemia (ALL), the method comprising the administration of a
pharmaceutical composition comprising a CD19xCD3 bispecific single
chain antibody construct to an adult patient in the need thereof.
In a preferred embodiment of the pharmaceutical methods and means
of the invention, said acute lymphoblastic leukemia (ALL) is
B-lineage acute lymphoblastic leukemia (ALL), preferably
B-precursor acute lymphoblastic leukemia. B-lineage acute
lymphoblastic leukemia (ALL) comprises the majority of ALL's with
74% of cases. Seventy percent of all ALL's are B-precursor ALL's
and 4% are mature B-cell ALL's. Since the CD19xCD3 bispecific
single chain antibody described herein is directed against the B
cell-associated marker CD19, said antibody is particularly suitable
as a therapeutic agent for B-lineage acute lymphoblastic leukemia,
preferably for B-precursor ALL's which can be further subdivided
into pro-B ALL, pre-B ALL and common ALL (cALL).
[0008] The administration of the CD19xCD3 bispecific single chain
antibody (also termed blinatumomab or MT103) described in more
detail below provides for the first time a therapeutic approach
which allows the treatment of minimal residual disease in patients
with acute lymphoblastic leukemia (ALL). As shown in the following
examples and illustrated by FIG. 1, the CD19xCD3 bispecific single
chain antibody (the nucleic acid sequence and amino acid sequence
of which is depicted in SEQ ID NOs. 2 and 1, respectively) has been
designed to link T cells with CD19-expressing target cells
resulting in a non-restricted cytotoxic T-cell response and T-cell
activation. Recently, a phase I study has demonstrated significant
clinical activity of the CD19xCD3 bispecific single chain antibody
in relapsed B-cell non-Hodgkin's lymphoma (NHL) (Bargou et al.,
Science 321 (2008):974-7). Based on these results, a phase II study
was designed in collaboration with the German Multicenter Study
Group on Adult Acute Lymphoblastic Leukemia (GMALL) to investigate
efficacy, safety, and tolerability of the CD19xCD3 bispecific
single chain antibody in acute lymphoblastic leukemia (ALL)
patients who achieved a complete hematological remission, but still
had minimal residual disease (MRD). MRD is an independent
prognostic factor that reflects primary drug resistance and is
associated with a high relapse risk after start of consolidation.
MRD was measured with standardized methods either by quantitative
detection of individual rearrangements of immunoglobulin or T-cell
receptor (TCR) rearrangements, t(4;11) translocations or by bcr/abl
fusion transcripts (see e.g. Van der Velden et al., Leukemia 18
(2004), 1971-80). The study population includes adult patients with
acute B-precursor acute lymphoblastic leukemia (ALL) who show a
bcr/abl signal or t(4;11) signal above detection limit and/or at
least one marker by rearrangement with a sensitivity of
.gtoreq.10.sup.-4. Primary endpoint of the ongoing phase II study
is the conversion rate to minimal residual disease (MRD) negative
status as defined by a bcr/abl or a t(4;11) signal below detection
limit and/or by detection of individual rearrangements of
immunoglobulin or T-cell receptor (TCR) genes below 10.sup.-4. One
treatment cycle of the CD19xCD3 bispecific single chain antibody is
a 4-week continuous intravenous infusion, which can be followed by
allogeneic hematopoietic stem cell transplantation after the first
cycle, or by repeated cycles after a 2-week treatment-free
interval. The dosage of CD19xCD3 bispecific single chain antibody
is 15 microgram/m.sup.2/24 hr, whereby an intra-patient dose
escalation up to 30 microgram/m.sup.2/24 hr is allowed. Minimal
residual disease (MRD) status is controlled after each treatment
cycle. Patients who achieve MRD negativity might receive additional
treatment cycles.
[0009] To date, seventeen adult ALL patients have been treated, or
are still on treatment with the CD19xCD3 bispecific single chain
antibody. 14 patients received the dose level of 15
microgram/m.sup.2/24 hr of CD19xCD3 bispecific single chain
antibody, whereas in three patients the dose has been escalated
from 15 to 30 microgram/m.sup.2/24 hr after the first or further
treatment cycles. All of these ALL patients had minimal residual
disease (MRD): Eleven of them had MRD by immunoglobulin or TCR
rearrangements, two patients had t(4;11) translocations and four
patient had bcr/abl fusion transcripts.
[0010] As a result, MRD response was evaluable in 16 of 17
patients. 13 of 16 patients became MRD negative, which corresponds
to an extraordinary complete molecular response rate of 81%. More
specifically, in nine out of eleven patients with immunoglobulin or
TCR rearrangements, in one out of two patients with t(4;11)
translocations and in three out of four patients with bcr/abl
transcripts, MRD-negativity could be achieved. The longest duration
of MRD-negativity observed so far in a patient having not received
a transplantation after the antibody treatment is 41 weeks. Another
patient treated with the CD19xCD3 bispecific single chain antibody
with MRD-negativity from 23 Jun. 2008 to 27 Oct. 2008 and having
received a successful allogeneic stem cell transplantation
thereafter is relapse-free to date.
[0011] Remarkably, the bcr/abl patients who could successfully be
treated with the CD19xCD3 bispecific single chain antibody were
refractory or intolerant to tyrosine kinase inhibitors imatinib
and/or dasatinib in previous ALL treatment regimen. For example,
one of the bcr/abl responders to treatment with CD19xCD3 bispecific
single chain antibody had a T315I mutation which was refractory to
therapy by tyrosine kinase inhibitors. Thus, the administration of
the CD19xCD3 bispecific single chain antibody now provides for the
first time for a therapy for imatinib- and/or dasatinib-refractory
ALL patients with bcr/abl transcripts. Only three out of a total of
17 patients did not become MRD negative. However, in two of them
stable disease could be achieved. Only one patient had a testicular
relapse followed by a hematological relapse, after 19 weeks of
MRD-negativity. One patient was not evaluable due to a serious
adverse event (SAE) on study day 2.
[0012] In summary, an absolutely exceptional complete molecular
response rate of 81% could be achieved in adult patients with
B-precursor ALL upon treatment with the CD19xCD3 bispecific single
chain antibody. Activity of the mentioned antibody could be
observed in all ALL patient subsets treated, including tyrosine
kinase inhibitors-refractory (T315I) bcr/abl patients and patients
with t(4;11) translocations. These ALL patient subsets are
generally considered incurable by conventional ALL standard
therapy, except for the option of allogeneic HSCT. In addition,
treatment with CD19xCD3 bispecific single chain antibody shows a
favorable toxicity profile, in contrast to conventional ALL
therapies, such as chemotherapy. In light of this, the
administration of the CD19xCD3 bispecific single chain antibody
described herein provides a new and advantageous treatment option
for adult acute lymphoblastic leukemia (ALL), in particular for
cases in which the ALL is refractory to conventional ALL therapy,
such as chemotherapy and/or allogeneic HSCT. In addition, the
administration of the CD19xCD3 bispecific single chain antibody now
provides for the first time for a therapy for MRD-positive ALL.
[0013] The method of the present invention provides for the
following major advantages:
[0014] 1. Less adverse effects than conventional acute
lymphoblastic leukemia (ALL) therapies, including chemotherapy or
allogeneic HSCT. Conventional ALL therapies are associated with
considerable health risks for patients; see e.g. Schmoll, Hoffken,
Possinger: Kompendium Internistische Onkologie, S. 2660 ff.; 4.
Auflage, Springer Medizin Verlag Heidelberg).
[0015] 2. Though allogeneic HSCT is currently considered to be the
treatment of choice in adult Ph+ ALL, approximately one third of
the transplanted patients relapse. Ph+ ALL patients carry the
highest risk for a relapse among all patients within the ALL
subtypes. As shown in the following examples, the administration of
CD19xCD3 bispecific single chain antibody is especially appropriate
for adult ALL patients with minimal residual disease (MRD). This
accounts for minimal residual disease (MRD) defined by the
Philadelphia chromosome translocation as well as for MRD defined by
immunoglobulin or TCR rearrangement or t(4;11). Adult ALL patients,
non-eligible for bone marrow transplantation, carrying t(4;11) or
refractory Ph+ ALL patients have so far been considered incurable.
The pharmaceutical methods and means of the invention therefore
provide a therapeutic approach for the treatment, amelioration or
elimination of MRD in adult ALL, thereby reducing or even
abolishing the risk of a relapse for the patient. It is worth
noting that, curative treatment for MRD-positive ALL patients has
not yet been available until now.
[0016] 3. In particular, the CD19xCD3 bispecific single chain
antibody can be used for therapy of MRD-positive acute
lymphoblastic leukemia (ALL) refractory to conventional ALL
therapy, such as chemotherapy, administration of tyrosine kinase
inhibitors, and/or HSCT.
[0017] 4. Not only the CD19xCD3 bispecific single chain antibody
can replace conventional acute lymphoblastic leukemia (ALL)
therapies in patients non-eligible for allogeneic HSCT, it can also
be used to convert ALL patients eligible for said transplantation
into an MRD negative-status, as MRD-negative patients have a lower
risk of relapse after transplantation than MRD-positive
patients.
[0018] 5. The high cytotoxic activity of the CD19xCD3 bispecific
single chain antibody allows the elimination of leukemia cells in
the bone marrow.
[0019] Acute lymphoblastic leukemia (ALL), including B-precursor
acute lymphoblastic leukemia and other types of B (cell) lineage
ALL, and treatments thereof are reviewed e.g. in Pui and Evans, N.
Engl. J. Med. 354 (2006), 166-178; Hoelzer and Gokbuget; Hematology
(2006); 133-141; or Apostolidou et al., Drugs 67 (2007),
2153-2171.
[0020] Information with respect to ALL can also be found e.g. under
http://www.cancer.gov, http://www.wikipedia.org or
http://www.leukemia-lymphoma.org.
[0021] The term "bispecific single chain antibody" or "single chain
bispecific antibody" or related terms in accordance with the
present invention mean antibody constructs resulting from joining
at least two antibody variable regions in a single polypeptide
chain devoid of the constant and/or Fc portion(s) present in full
immunoglobulins. A "linker" as used herein connects V domains of
the same specificity, whereas a "spacer" as used herein connects V
domains of different specificities. For example, a bispecific
single chain antibody may be a construct with a total of two
antibody variable regions, for example two VH regions, each capable
of specifically binding to a separate antigen, and connected with
one another through a short (usually less than 10 amino acids)
synthetic polypeptide spacer such that the two antibody variable
regions with their interposed spacer exist as a single contiguous
polypeptide chain. Another example of a bispecific single chain
antibody may be a single polypeptide chain with three antibody
variable regions. Here, two antibody variable regions, for example
one VH and one VL, may make up an scFv, wherein the two antibody
variable regions are connected to one another via a synthetic
polypeptide linker, the latter often being genetically engineered
so as to be minimally immunogenic while remaining maximally
resistant to proteolysis. This scFv is capable of specifically
binding to a particular antigen, and is connected to a further
antibody variable region, for example a VH region, capable of
binding to a different antigen than that bound by the scFv. Yet
another example of a bispecific single chain antibody may be a
single polypeptide chain with four antibody variable regions. Here,
the first two antibody variable regions, for example a VH region
and a VL region, may form one scFv capable of binding to one
antigen, whereas the second VH region and VL region may form a
second scFv capable of binding to another antigen. Within a single
contiguous polypeptide chain, individual antibody variable regions
of one specificity may advantageously be separated by a synthetic
polypeptide linker as described above, whereas the respective scFvs
may advantageously be separated by a short polypeptide spacer as
described above. Non-limiting examples of bispecific single chain
antibodies as well as methods for producing them are shown in WO
99/54440, WO 2004/106381, WO 2007/068354, Mack, J. Immunol. (1997),
158, 3965-70; Mack, PNAS, (1995), 92, 7021-5; Kufer, Cancer
Immunol. Immunother., (1997), 45, 193-7; Loffler, Blood, (2000),
95, 6, 2098-103; Brahl, J. Immunol., (2001), 166, 2420-2426.
[0022] As used herein, "CD3" denotes an antigen that is expressed
on T cells, preferably human T cells as part of the multimolecular
T cell receptor complex, the CD3 consisting of five different
chains: CD3-epsilon, CD3-gamma, CD3-delta, CD3-eta and CD3 zeta.
Clustering of CD3 on T cells e.g. by anti-CD3 antibodies leads to T
cell activation similar to the binding of an antigen but
independent from the clonal specificity of the T cell subset. Thus,
the term "CD19xCD3 bispecific single chain antibody" as used herein
relates to a CD3-specific construct capable of binding to the human
CD3 complex expressed on human T cells and capable of inducing
elimination/lysis of target cells, wherein such target cells
carry/display an antigen which is bound by the other,
non-CD3-binding portion of the bispecific single chain antibody.
Binding of the CD3 complex by CD3-specific binders (e.g. a
bispecific single chain antibody as administered according to the
pharmaceutical means and methods of the invention) leads to
activation of T cells as known in the art; see e.g. WO 99/54440 or
WO 2007/068354. Accordingly, a construct appropriate for the
pharmaceutical means and methods of the invention is advantageously
able to eliminate/lyse target cells in vivo and/or in vitro.
Corresponding target cells comprise cells expressing a tumor
antigen, such as CD19, which is recognized by the second
specificity (i.e. the non-CD3-binding portion of the bispecific
single chain antibody) of the mentioned construct. Preferably, said
second specificity is for human CD19 which has already been
described in WO 99/54440, WO 2004/106381 or WO 2007/068354.
According to this embodiment, each antigen-specific portion of the
bispecific single chain antibody comprises an antibody VH region
and an antibody VL region. An advantageous variant of this
bispecific single chain antibody is from N terminus to C
terminus:
TABLE-US-00001
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.H(CD3)-V.sub.L(CD3). (SEQ ID NO.:
1)
[0023] Within the meaning of the invention, the term "specifically
binding" or related terms such as "specificity" is/are to be
understood as being characterized primarily by two parameters: a
qualitative parameter (the binding epitope, or where an antibody
binds) and a quantitative parameter (the binding affinity, or how
strongly this antibody binds where it does). Which epitope is bound
by an antibody can advantageously be determined by e.g. FACS
methodology, ELISA, peptide-spot epitope mapping, or mass
spectroscopy. The strength of antibody binding to a particular
epitope may advantageously be determined by e.g. known Biacore
and/or ELISA methodologies. A combination of such techniques allows
the calculation of a signal:noise ratio as a representative measure
of binding specificity. In such a signal:noise ratio, the signal
represents the strength of antibody binding to the epitope of
interest, whereas the noise represents the strength of antibody
binding to other, non-related epitopes differing from the epitope
of interest. A signal:noise ratio of, for example at least 50, but
preferably about 80 for a respective epitope of interest as
determined e.g. by Biacore, ELISA or FACS may be taken as an
indication that the antibody evaluated binds the epitope of
interest in a specific manner, i.e. is a "specific binder". The
term "binding to/interacting with" may also relate to a
conformational epitope, a structural epitope or a discountinuous
epitope consisting of two or even more regions of the human target
molecules or parts thereof. A conformational epitope is defined by
two or more discrete amino acid sequences separated in the primary
sequence which come together on the surface of the molecule when
the polypeptide folds to the native protein (Sela, (1969) Science
166, 1365 and Laver, (1990) Cell 61, 553-6). The term
"discontinuous epitope" means non-linear epitopes that are
assembled from residues from distant portions of the polypeptide
chain. These residues come together on the surface of the molecule
when the polypeptide chain folds into a three-dimensional structure
to constitute a conformational/structural epitope.
[0024] The term "treatment" as used herein means in the broadest
sense medical procedures or applications that are intended to
relieve illness. In the present case, the administration of the
CD19xCD3 bispecific single chain antibody (prepared for
administration to an adult ALL patient) as described herein is for
the treatment, amelioration or elimination of the ALL disease in
adult patients.
[0025] The term "patient" as used herein refers to a human adult
patient. The term "adult ALL" or "adult ALL patient" or "adult
patient" as referred to herein denotes adults aged more than 18
years, i.e. patients aged 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,
or 50 years or more. Even patients with 70, 75, 80, 85, 90, 100
years or older may be treated by the methods and means of the
invention. The indicated age is to be understood as the age of the
adult at diagnosis of the ALL disease.
[0026] The term "amelioration" as used herein is synonymous with
improvement. If an adult ALL patient's condition shows
amelioration, the patient is clearly better--there is some
improvement in her or his condition. For example, it may be an
improvement in the ALL patient's condition, if a stabilization of
the ALL disease can be achieved (also termed stable disease), i.e.
the ALL disease is no longer progressive. Even better, MRD positive
acute lymphoblastic leukemia (ALL) is converted into an MRD
negative status.
[0027] The term "elimination" as used herein means the removal of
leukemic cells from the body of an adult ALL patient. As shown in
the following example, administration of the CD19xCD3 bispecific
single chain antibody is able to convert MRD positive acute
lymphoblastic leukemia (ALL) into an MRD negative status in various
ALL subtypes.
[0028] The term "administration" as used herein means
administration of a therapeutically effective dose of the
aforementioned CD19xCD3 bispecific single chain antibody to an
individual, i.e. a human patient. Preferably, the ALL patient is an
adult patient, as defined herein.
[0029] By "therapeutically effective amount" is meant a dose that
produces the effects for which it is administered, preferably the
conversion of an minimal residual disease (MRD)-positive acute
lymphoblastic leukemia (ALL) status into an MRD-negative ALL
status. The exact dose will depend on the purpose of the treatment,
and will be ascertainable by one skilled in the art using known
techniques. As is known in the art and described above, adjustments
for systemic versus localized delivery, age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0030] The attending physician and clinical factors will determine
the dosage regimen. As is well known in the medical arts, dosages
for any one patient depends upon many factors, including the
patient's size, body surface area, age, the particular compound to
be administered, sex, time and route of administration, general
health status, and other drugs being administered concurrently.
[0031] As is well known in the medical arts, dosages for any one
patient depends upon many factors, including the adult patient's
size, body surface area, age, the particular compound to be
administered, sex, time and route of administration, general health
status, and other drugs being administered concurrently. A typical
dose can be, for example, in the ranges set forth in the
embodiments of the invention and the appended examples; however,
doses below or above this exemplary range are envisioned,
especially considering the aforementioned factors.
[0032] The term "continuous infusion" refers to an infusion which
is allowed to proceed permanently over a time period, i.e. without
interruption. "Continuous infusion" refers to a permanently
administered infusion. Accordingly, in the context of the
invention, the terms "permanent" and "continuous" are used as
synonyms. Within the meaning of the invention, e.g. the term "4
week continuous infusion" denote(s) a situation in which the
CD19xCD3 bispecific single chain antibody used in the
pharmaceutical means and methods according to the invention is
continuously administered to the body of an adult patient over a
period of 4 weeks in a sustained, constant fashion throughout the
entire duration required in the pharmaceutical means and methods of
the invention. Continuous administration schemes of the CD19xCD3
bispecific single chain antibody are described in more detail in WO
2007/068354. An interruption of the introduction of CD19xCD3
bispecific single chain antibody is avoided, that is to say a
transition from a state in which this antibody is being
administered to the body of the patient to a state in which this
antibody is no longer being administered to the body of the patient
does not, or does not significantly occur over the entire duration
of administration required by the pharmaceutical means and methods
of the invention for other reasons than replenishing the supply of
CD19xCD3 bispecific single chain antibody being administered or
medical interventions which become necessary and the like. In as
far as such necessary replenishing leads to a temporary
interruption of the introduction of the antibody administered, such
administration is still to be understood as being "uninterrupted"
or "permanent" in the sense of the pharmaceutical means and methods
according to the invention. In most cases, such replenishing will
generally be of such a short duration that the time during which
antibody is not being introduced into the body of the patient will
be vanishingly small when compared to the time planned for the
overall administration regimen according to the pharmaceutical
means and methods according to the invention. In accordance with
the invention, one treatment cycle is to be understood as a 4-week
continuous infusion of the CD19xCD3 bispecific single chain
antibody to the adult ALL patient, followed by a 2-week
treatment-free interval. It may be that upon MRD staging of the
treated patient(s) after a 4 week-continuous administration or one
treatment cycle, a minimal response or partial response to the
bispecific single chain antibody treatment may be diagnosed. In
this case, the continuous administration may be extended by
additional one, two, three, four, five or even up to ten treatment
cycles in order to achieve a better therapeutic result, e.g. stable
disease or even a complete response. Preferably, said complete
response is MRD-negativity. In an alternative embodiment, the
4-week continuous infusion of the CD19xCD3 bispecific single chain
antibody to the adult ALL patient may be followed by allogeneic
HSCT. It is also envisaged that a patient treated by one, two,
three, four or even more treatment cycles as set forth above may
receive an allogeneic HSCT transplantation thereafter.
[0033] As shown in the following example, 13 of 16 adult ALL
patients became MRD negative upon treatment with the CD19xCD3
bispecific single chain antibody, which corresponds to an
extraordinary complete molecular response rate of 81%. More
specifically, in nine out of eleven patients with immunoglobulin or
TCR rearrangements, one out of two patients with t(4;11)
translocations and three out of four patients with bcr/abl
transcripts MRD-negativity could be achieved. Preferably, the major
therapeutic goal of the administration of the CD19xCD3 bispecific
single chain antibody, either alone or in combination with
allogeneic HSCT, to an adult ALL patient is the conversion of an
MRD-positive status into an MRD-negative status, as defined
herein.
[0034] Continuing uninterrupted administration of the bispecific
single chain antibody in the manner of the pharmaceutical means and
methods according to the invention for longer periods of time
allows the advantageous T cell activation mentioned in the examples
to exert its effect for long enough to advantageously clear all
diseased cells from the body. Since the rate of uninterruptedly
administered bispecific single chain antibody is kept low,
application of therapeutic agent may be continued longer without
risk of deleterious side effects for the patient.
[0035] The CD19xCD3 bispecific single chain antibody as used herein
is advantageously in the form of a pharmaceutical composition for
administration to a human patient diagnosed with acute
lymphoblastic leukemia (ALL). The human patient is preferably an
adult as defined herein below. While the bispecific single chain
antibody as used herein may be administered per alone, preferred is
administration in a pharmaceutically acceptable carrier. Examples
of suitable pharmaceutical carriers are well known in the art and
include phosphate buffered saline solutions, water, liposomes,
various types of wetting agents, sterile solutions, etc.
Compositions comprising such carriers can be formulated by well
known conventional methods. These pharmaceutical compositions can
be administered to the subject at a suitable dose. The dosage
regimen will be determined by the attending physician and clinical
factors. As is well known in the medical arts, dosages for any one
patient depends upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and other
drugs being administered concurrently. Preparations for parenteral
administration include sterile aqueous or non-aqueous solutions, or
suspensions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, aqueous solutions, or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, or lactated Ringer's. Intravenous
vehicles include fluid and nutrient replenishes, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like. Preservatives and other additives may also be present such
as, for example, antimicrobials, anti-oxidants, chelating agents,
and inert gases and the like. In addition, the composition might
comprise proteinaceous carriers, like, e.g., serum albumine or
immunoglobuline, preferably of human origin. It is envisaged that
the composition might comprise, in addition to the proteinaceous
bispecific single chain antibody further biologically active
agents, depending on the intended use of the pharmaceutical
composition. Such agents might be agents acting as cytostatica,
agents preventing hyperurikemia, agents inhibiting immune reactions
(e.g. corticosteroids, FK506), drugs acting on the circulatory
system and/or agents such as T-cell co-stimulatory molecules or
cytokines known in the art.
[0036] Preferably, the CD19xCD3 bispecific single chain antibody as
defined herein is formulated in a buffer, a stabilizer and a
surfactant. The buffer may be a phosphate, citrate, succinate or
acetate buffer. The stabilizer may be (an) amino acid(s) and/or a
sugar. The surfactants may be detergents, PEGs, or the like. More
preferably, the CD19xCD3 bispecific single chain antibody as
defined herein is formulated in citrate, lysine, trehalose and
Tween 80. As a diluent for the pharmaceutical composition of the
invention, isotonic saline and Tween 80 is preferred.
[0037] Preferably, in the uses or methods of the invention, the
pharmaceutical composition is to be administered to a human adult
patient diagnosed with acute lymphoblastic leukemia (ALL).
[0038] The success of the CD19xCD3 bispecific single chain antibody
therapy may be monitored by established standard methods for the
respective disease entities:
[0039] For B cell ALL therapy, Fluorescence Activated Cell Sorting
(FACS), bone marrow aspiration and various leukemia specific
clinical chemistry parameters and other established standard
methods may be used. Methods and means for the determination of the
minimal residual disease (MRD) status have been described
above.
[0040] Cytotoxicity can be detected by methods known in the art and
methods as illustrated e.g. in WO 99/54440, WO 2004/106381, WO
2007/068354:
[0041] In a preferred embodiment, the acute lymphoblastic leukemia
(ALL) of the adult patient(s) is refractory to chemotherapy,
preferably refractory to chemotherapy with respect to MRD (i.e. the
MRD in these ALL patients is resistant to chemotherapy). Even more
preferred, the acute lymphoblastic leukemia (ALL) is refractory to
chemotherapy in patients non-eligible for allogeneic HSCT.
[0042] The term "chemotherapy" as used herein denotes chemotherapy
used for the treatment of acute lymphoblastic leukemia (ALL).
Chemotherapy is the initial treatment of choice for ALL. Most ALL
patients end up receiving a combination of different treatments. In
the treatment of ALL, there are no surgical options, due to the
body-wide distribution of the malignant cells. In general,
cytotoxic chemotherapy for ALL combines multiple anti-leukemic
drugs in various combinations. Chemotherapy for ALL consists of
three phases: remission induction, intensification, and maintenance
therapy. Chemotherapy is also indicated to protect the central
nervous system from leukemia. The aim of remission induction is to
rapidly kill most tumor cells and get the patient into remission.
This is defined as the presence of less than 5% leukemic blasts in
the bone marrow (as determined by light microscopy), normal blood
cells and absence of tumor cells from blood, and absence of other
signs and symptoms of the disease. For example a combination of
Prednisolone or dexamethasone (in children), vincristine,
asparaginase, and daunorubicin (used in Adult ALL) is used to
induce remission. Intensification uses high doses of intravenous
multidrug chemotherapy to further reduce tumor burden. Typical
intensification protocols use vincristine, cyclophosphamide,
cytarabine, daunorubicin, etoposide, thioguanine or mercaptopurine
given as blocks in different combinations. Since ALL cells
sometimes penetrate the Central Nervous System (CNS), most
protocols include delivery of chemotherapy into the CNS fluid
(termed intrathecal chemotherapy). Some centers deliver the drug
through Ommaya reservoir (a device surgically placed under the
scalp and used to deliver drugs to the CNS fluid and to extract CNS
fluid for various tests). Other centers perform multiple lumbar
punctures as needed for testing and treatment delivery. Intrathecal
methotrexate or cytarabine is usually used for this purpose. The
aim of maintenance therapy is to kill any residual cell that was
not killed by remission induction, and intensification regimens.
Although such cells are few, they will cause relapse if not
eradicated. For this purpose, daily oral mercaptopurine, once
weekly oral methotrexate, once monthly 5-day course of intravenous
vincristine and oral corticosteroids are usually used. The length
of maintenance therapy is 3 years for boys, 2 years for girls and
adults. Central nervous system relapse is treated with intrathecal
administration of hydrocortisone, methotrexate, and cytarabine
(Hoffbrand et al., Essential Hematology, Blackwell, 5th edition,
2006). As the chemotherapy regimens can be intensive and protracted
(often about 2 years in case of the GMALL UKALL, HyperCVAD or CALGB
protocols; about 3 years for males on COG protocols), many patients
have an intravenous catheter inserted into a large vein (termed a
central venous catheter or a Hickman line), or a Portacath (a
cone-shaped port with a silicone nose that is surgically planted
under the skin, usually near the collar bone).
[0043] Chemotherapy for ALL has been described e.g. in Schmoll,
Hoffken, Possinger (loc. cit.).
[0044] In light of the above, the term "refractory to chemotherapy"
as used herein denotes resistance of the acute lymphoblastic
leukemia cells to chemotherapy.
[0045] Patients can experience a recurrence of ALL after initial
therapy and/or become refractory to chemotherapy following
treatment. ALL patients who are refractory to chemotherapy have a
markedly poor prognosis. In particular, the prognosis of adult
patients with Ph+ ALL treated only with chemotherapy is poor, with
a less than 10% probability of long-term survival. Since the
pharmaceutical methods and means of the invention are capable of
rendering the adult ALL patients MRD-negative, they are
particularly useful for the treatment of ALL patients refractory to
chemotherapy.
[0046] The term "allogeneic hematopoietic stem cell
transplantation" as used herein means allogeneic hematopoietic stem
cell transplantation (HSCT) or bone marrow transplantation (BMT)
which is a medical procedure in the field of hematology and
oncology that involves transplantation of hematopoietic stem cells
(HSCs). It is most often conducted in patients with diseases of the
lymph nodes, blood or bone marrow, such as ALL. Allogeneic HSCT is
a procedure in which a person receives blood-forming stem cells
(cells from which all blood cells develop) from a genetically
similar, but not identical, donor. This is often a close relative,
such as a mother, father, sister or brother, but could also be an
unrelated donor. Most recipients of HSCTs are leukemia (e.g. ALL)
patients who would benefit from treatment with high doses of
chemotherapy or total body irradiation. However allogeneic HSCT
remains a risky and toxic treatment.
[0047] The term "non-eligible for HSCT" as used herein means those
adult patients for whom allogeneic HSCT is not the ALL treatment of
choice, for instance, due to medical reasons. For example, it can
be the case that no appropriate donor is available, or the patient
has exceeded the upper age limit. As shown in the following
example, all patients have been refractory to chemotherapy, or in
case of Ph+ ALL also refractory or intolerant to tyrosine kinase
before inclusion into the study. Eight patients treated with the
CD19xCD3 bispecific single chain antibody have been non-eligible
for allogeneic HSCT, such as for example patients 111-003, 108-002,
109-006 or 109-007.
[0048] So far, ALL meant the death sentence for patients refractory
to chemotherapy and non-eligible for allogeneic HSCT. The
pharmaceutical methods and means of the invention for the first
time provide a therapy for this patient population in that it
eliminates the minimal residual disease (MRD) which otherwise would
cause a relapse and kill said patients.
[0049] In an alternative embodiment of the pharmaceutical methods
and means of the invention, said method is followed by allogeneic
hematopoietic stem cell transplantation or said method replaces
allogeneic hematopoietic stem cell transplantation in adult
patients eligible for allogeneic HSCT.
[0050] The term "eligible for allogeneic HSCT " as used herein
means that allogeneic HSCT is the required therapy for the adult
ALL patient. In cases, where the ALL patient is eligible for
allogeneic HSCT, the following two scenarios may be envisaged.
First, in one embodiment of the pharmaceutical methods and means of
the invention, the administration of the CD19xCD3 bispecific single
chain antibody (alone or preferably as a pharmaceutical
composition) can be used to replace allogeneic HSCT used as a
conventional therapy for adult ALL patients eligible for
transplantation. So the pharmaceutical methods and means of the
invention can avoid the health risks for the ALL patients
associated with allogeneic hematopoietic stem cell transplantation.
In addition, 30% of the transplanted ALL patients usually relapse
after transplantation. So the pharmaceutical methods and means of
the invention can be used to treat these patients. In an
alternative embodiment, the continuous infusion of the CD19xCD3
bispecific single chain antibody to the adult ALL patient may be
followed by an allogeneic hematopoietic stem cell transplantation.
In this embodiment, the administration of a pharmaceutical
composition comprising the CD19xCD3 bispecific single chain
antibody construct can be used to convert ALL patients eligible for
transplantation into an MRD negative-status before they receive the
transplantation. So, the pharmaceutical methods and means of the
invention can be used in order to eliminate MRD, which leads to a
lower risk of relapse than the transplantation treatment of
MRD-positive patients. The example presents a patient who has first
been converted into an MRD-negative status upon treatment with the
CD19xCD3 bispecific single chain antibody, followed by an
allogeneic transplantation. So far, this patient is still MRD
negative, with duration of MRD-negativity of 47 weeks until to
date.
[0051] It is also within the scope of the pharmaceutical methods
and means of the invention, that the CD19xCD3 bispecific single
chain antibody construct be administered to adult ALL patients who
have received an allogeneic HSCT and relapse thereafter.
[0052] In another preferred embodiment, the pharmaceutical methods
and means of the invention are for the treatment, amelioration or
elimination of minimal residual disease (MRD) in an adult patient
with acute lymphoblastic leukemia (ALL).
[0053] The term "minimal residual disease (MRD)" as defined herein
denotes a disease status after treatment e.g. with
chemotherapeutics when leukemia cells cannot be found any longer in
the bone marrow by light microscopic methods. More sensitive tests
such as flow cytometry (FACS based methods) or polymerase chain
reaction (PCR) have to be used in order to find evidence that
leukemia cells remained in the bone marrow of the ALL patient. More
specifically, the presence of leukemia cells below the cytological
detection limit (5% leukemic cells) is defined as minimal residual
disease (MRD). If no MRD is detectable (<10.sup.-4, i.e. less
than 1 leukemia cell per 10.sup.4 bone marrow cells detectable), a
complete molecular remission is reached. A "MRD positive status" as
defined herein means a bcr/abl signal or t(4;11) signal above
detection limit and/or by individual rearrangements of
immunoglobulin or T-cell receptor (TCR) genes above 10.sup.-4. A
"MRD negative status" as defined herein means a bcr/abl signal or a
t(4;11) translocation signal below detection limit or by individual
rearrangements of immunoglobulin or T-cell receptor (TCR) genes
below 10.sup.-4. The MRD status can be measured by PCR or FACS
analysis in that the individual rearrangements of immunoglobulin
genes or T-cell receptor (TCR) rearrangements, or bcr/abl fusion
transcripts, or t(4;11) are quantitatively detected. For example,
PCR analysis can detect fusion transcripts such as bcr/abl, or
t(4;11) translocations and individual clonal rearrangements of
immunoglobulins (IgH) and/or T-cell receptor genes (TCR).
[0054] Recurrent chromosomal abnormalities in the malignant cells
of patients with acute lymphoblastic leukemia are hallmarks of the
disease (Harrison and Foroni, Rev. Clin. Exp. Hematol. 6 (2002),
91-113). Specific aberrations which are frequently indicative of
consistent underlying molecular lesions can assist or even
establish the diagnosis and determine optimal therapy. In childhood
ALL, numerous good and high-risk cytogenetic subgroups have been
identified which are regularly used to stratify patients to
particular therapies (Pui and Evans, N. Engl. J. Med. 354 (2006),
166-178). However, in adult ALL the role of cytogenetics in patient
management has largely been centered on the presence of the
Philadelphia (Ph) chromosome which usually arises from
t(9;22)(q34;q11.2) and results in BCR-ABL (bcr/abl) fusion (Faderl
et al., Blood 91 (1998), 3995-4019). Although the overall incidence
of Ph+ ALL in adults is approximately 25%, it is correlated with
age and rises to greater than 50% among patients older than the age
of 55 years (Appelbaum, American Society of Clinical Oncology 2005
education book. Alexandria: ASCO, 2005: 528-532). Other cytogenetic
translocations associated with specific molecular genetic
abnormalities in acute lymphoblastic leukemia (ALL) are shown in
Table 1.
TABLE-US-00002 TABLE 1 Cytogenetic translocation Molecular genetic
abnormality t(9; 22)(q34; q11) BCR-ABL fusion(P185) t(12;
21)CRYPTIC TEL-AML1fusion t(1; 19)(q23; p13) E2A-PBX fusion t(4;
11)(q21; q23) MLL-AF4 fusion t(8; 14)(q24; q32) IGH-MYC fusion
t(11; 14)(p13; q11) TCR-RBTN2 fusion
[0055] Cytogenetics, has been increasingly recognized as an
important predictor of outcome in ALL (Moormann et al., Blood 109
(2007), 3189-97).
[0056] Some cytogenetic subtypes have a worse prognosis than
others. These include e.g.:
[0057] (i) A translocation between chromosomes 9 and 22, the
Philadelphia chromosome (Ph+), occurs in about 20% of adult and 5%
in pediatric cases of ALL.
[0058] (ii) A translocation between chromosomes 4 and 11 occurs in
about 4% of cases and is most common in infants under 12
months.
[0059] Rearrangements of immunoglobulin genes or T-cell receptor
(TCR) rearrangements and their role in ALL have been described in
the art (see e.g. Szczepa ski et al., Leukemia 12 (1998),
1081-1088).
[0060] In another preferred embodiment of the pharmaceutical
methods and means of the invention, said adult patient is
MRD-positive in complete hematological remission.
[0061] The term "remission" or "hematological remission" as used
herein is to be understood as having no evidence of disease after
treatment, e.g. after chemotherapy or transplantation. This means
that the bone marrow contains fewer than 5% blast cells as
determined by light microscopy, the blood cell counts are within
normal limits, and there are no signs or symptoms of the ALL
disease. A molecular complete remission means there is no evidence
of leukemia cells in biopsies of the bone marrow, even when using
very sensitive tests such as PCR. Put in other words: If no MRD is
detectable (<10.sup.-4, i.e. <1 leukemia cell per 10.sup.4
bone marrow cells), a complete molecular remission is reached.
[0062] After complete remission of the leukemia lesion(s) in a
human adult ALL patient by chemotherapeutic treatment or allogeneic
hematopoietic stem cell transplantation it may be the case that not
all leukemia cells could be eliminated from the body. However,
these remaining tumor cells may give rise to recurrent leukemia.
The pharmaceutical means and methods of the invention can be used
to kill these remaining tumor cells in order to prevent recurrence
of the leukemia (originating from the occult leukemia cells
remaining in the body after primary therapy). In this way, the
pharmaceutical means and methods help to prevent disease relapse in
adult ALL patients.
[0063] In another preferred embodiment of the pharmaceutical
methods and means of the invention, the administration of said
pharmaceutical composition converts MRD positive acute
lymphoblastic leukemia (ALL) into an MRD negative status.
[0064] In another preferred embodiment of the pharmaceutical
methods and means of the invention, MRD is measured with
quantitative detection of individual rearrangements of
immunoglobulin genes or T-cell receptor (TCR) rearrangements, or by
bcr/abl fusion transcripts, or t(4;11) using PCR or FACS
analysis.
[0065] As shown in the following examples, the administration of
CD19xCD3 bispecific single chain antibody is especially appropriate
for adult patients with minimal residual disease (MRD). This
accounts for minimal residual disease (MRD) defined by the
Philadelphia chromosome translocation or t(4;11) as well as for MRD
defined by immunoglobulin or TCR rearrangements. The pharmaceutical
methods and means of the invention therefore provide a therapeutic
approach for the treatment, amelioration or elimination of MRD,
thereby reducing or even abolishing the risk of relapse for the
adult patient. Notably, curative treatment of MRD in ALL patients
has not yet been available so far.
[0066] In another preferred embodiment of the pharmaceutical
methods and means of the invention, said patient shows a bcr/abl
signal or a t(4;11) signal above detection limit and/or at least
one marker by rearrangement with a sensitivity of
.gtoreq.10.sup.-4.
[0067] The term "bcr/abl signal or t(4;11) translocation signal
above detection limit" as used herein means that PCR or FACS
analysis leads to a detectable bcr/abl signal or t(4;11)
signal.
[0068] In another preferred embodiment of the pharmaceutical
methods and means of the invention, the time to molecular relapse
(detectable by the assays described above) is more than 4
months.
[0069] The term "molecular relapse" as used herein means that said
patient shows a bcr/abl or t(4;11) translocation signal above
detection limit and/or at least one marker by rearrangement with a
sensitivity of .gtoreq.10.sup.-4.
[0070] The term "with a sensitivity of .gtoreq.10.sup.-4" as used
herein means one or more than one leukemia cell(s) can be detected
in 10.000 cells, in particular bone marrow cells.
[0071] In another preferred embodiment of the pharmaceutical
methods and means of the invention, the corresponding variable
heavy chain regions (V.sub.H) and the corresponding variable light
chain regions (V.sub.L) regions in said CD19xCD3 bispecific single
chain antibody construct are arranged, from N-terminus to
C-terminus, in the order,
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.H(CD3)-V.sub.L(CD3).
[0072] The corresponding variable heavy chain regions (V.sub.H) and
the corresponding variable light chain regions (V.sub.L) regions of
the CD3 and CD19 binding domains of the CD19xCD3 bispecific single
chain antibody are shown in SEQ ID NOs. 3 to 10, respectively. The
corresponding CDR regions of the respective VH and VL regions of
the mentioned CD19xCD3 bispecific single chain antibody are shown
in SEQ ID NOs. 11 to 22.
[0073] In another preferred embodiment of the pharmaceutical
methods and means of the invention, said CD19xCD3 bispecific single
chain antibody construct comprises an amino acid sequence as set
forth in SEQ ID NO. 1, or an amino acid sequence at least 90%,
preferably at least 95% identical to SEQ ID NO. 1.
[0074] The invention describes a bispecific single chain antibody
molecule comprising an amino acid sequence as depicted in SEQ ID
NO. 1, as well as an amino acid sequence at least 90% or preferably
95% identical, most preferred at least 96, 97, 98, or 99% identical
to the amino acid sequence of SEQ ID NO. 1. The invention describes
also the corresponding nucleic acid sequence as depicted in SEQ ID
NO. 2 as well as a nucleic acid sequence at least 90%, preferably
95% identical, most preferred at least 96, 97, 98, or 99% identical
to the nucleic acid sequence shown in SEQ ID NO. 2. It is to be
understood that the sequence identity is determined over the entire
nucleotide or amino acid sequence. Moreover, it is to be understood
that a bispecific single chain antibody molecule comprising an
amino acid sequence at least 90% or preferably 95% identical, most
preferred at least 96, 97, 98, or 99% identical to the amino acid
sequence of SEQ ID NO. 1 contains all of the CDR sequences shown in
SEQ ID NOs. 11 to 22. For sequence alignments, for example, the
programs Gap or BestFit can be used (Needleman and Wunsch J. Mol.
Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2
(1981), 482-489), which is contained in the GCG software package
(Genetics Computer Group, 575 Science Drive, Madison, Wis., USA
53711 (1991). It is a routine method for those skilled in the art
to determine and identify a nucleotide or amino acid sequence
having e.g. 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the
nucleotide or amino acid sequences of the CD19xCD3 bispecific
single single chain antibody described herein. For example,
according to Crick's Wobble hypothesis, the 5' base on the
anti-codon is not as spatially confined as the other two bases, and
could thus have non-standard base pairing. Put in other words: the
third position in a codon triplet may vary so that two triplets
which differ in this third position may encode the same amino acid
residue. Said hypothesis is well known to the person skilled in the
art (see e.g. http://en.wikipedia.org/wiki/Wobble_Hypothesis;
Crick, J Mol Biol 19 (1966): 548-55).
[0075] In another preferred embodiment of the pharmaceutical
methods and means of the invention, one treatment cycle is a 4-week
continuous infusion, followed by repeated cycles after a 2-week
treatment-free interval or by an allogeneic hematopoietic stem cell
transplantation.
[0076] In another preferred embodiment of the pharmaceutical
methods and means of the invention, the treatment cycle is repeated
at least three times, preferably four, five, six, seven or even up
to ten times after determination of a MRD negative status
(consolidation).
[0077] In another preferred embodiment of the pharmaceutical
methods and means of the invention, the bispecific single chain
antibody construct is to be administered in a daily dose of 10
.mu.g to 100 .mu.g per square meter patient body surface area.
[0078] As used herein, a dose range which is defined as "X to Y"
equates with a dose range which is defined as "between X and Y".
The range includes the upper limit and also the lower limit. This
means that for example a daily dose of 10 .mu.g to 100 .mu.g per
square meter patient body surface area includes "10 .mu.g" and "100
.mu.g".
[0079] In an even more preferred embodiment of the pharmaceutical
methods and means of the invention, the CD19xCD3 bispecific single
chain antibody construct is to be administered in a daily dose of
15 .mu.g, 30 .mu.g, 60 .mu.g or 90 .mu.g per square meter patient
body surface area. Even more preferred, said antibody is to be
administered in a daily dose of 15 to 30 .mu.g per square meter
patient body surface area, most preferred in a daily dose of 15 or
30 .mu.g per square meter patient body surface area.
[0080] The average body surface area of an adult patient is hereby
calculated in the context of the pharmaceutical method or use
according to the invention to be in a range of 1.7 to 2.2
m.sup.2.
[0081] Advantageously, the pharmaceutical composition comprising
the CD19xCD3 bispecific single chain antibody as described herein
further comprises, optionally (a) reaction buffer(s), storage
solutions and/or remaining reagents or materials required for the
recited method or use. Furthermore, said components can be packaged
individually in vials or bottles or in combination in containers or
multicontainer units.
[0082] In order to evaluate safety and tolerability of the CD19xCD3
bispecific single chain antibody as described herein, the compound
is to be administered by long-term continuous infusion.
[0083] It has been found that the beneficial and unexpected effects
of the pharmaceutical means and methods of the invention can be
obtained by administering the CD19xCD3 bispecific single chain
antibody in a daily dose of 10 microgram to 100 microgram per
square meter body surface area. The daily dose may be kept constant
over the administration period. However, it is also within the
ambit of this embodiment that for the initial day(s) of the
infusion period a lower dose of bispecific single chain antibody be
administered ("initial dose") prior to the pharmaceutical methods
described herein, whereas for the remaining infusion period a
higher dose ("maintenance dose") be applied. For example, 5
microgram of bispecific single chain antibody per square meter body
surface area may be administered at the first day(s) of the
infusion period followed by administration of 15 microgram per
square meter body surface as daily dose for the remaining treatment
period. Or 15 microgram of bispecific single chain antibody per
square meter body surface area may be administered at the first
day(s) of the infusion period followed by administration of 30 or
45 microgram per square meter body surface as daily dose for the
remaining treatment period. The initial dose may be administered
for one, two or more days or even for one week (seven days). It is
also envisaged that 5 microgram of bispecific single chain antibody
per square meter body surface area may be administered at the first
day(s) of the infusion period, followed by administration of 15
microgram of bispecific single chain antibody per square meter body
surface area at the following day(s) of the infusion period,
followed by administration of 45 microgram per square meter body
surface as daily (maintenance) dose for the remaining treatment
period. The average body surface area of an adult patient is hereby
calculated in the context of the pharmaceutical method or use
according to the invention to be in a range of 1.7 to 2.2
m.sup.2.
[0084] In another embodiment of the methods and uses of the
invention, the dose is escalated after the first or further
treatment cycles, for example from 15 to 30 or 60 or even 90
microgram/m.sup.2/24 hr.
[0085] The uninterrupted administration of the CD19xCD3 bispecific
single chain antibody may be intravenous, parenteral, subcutaneous,
transdermal, intraperitoneal, intramuscular or pulmonary. The
intravenous mode of administration will in most cases be the mode
of choice for uninterruptedly administering the CD19xCD3 bispecific
single chain antibody and, as the case may be, for
co-administration of a pharmaceutical agent as part of a regimen of
co-therapy. As such, intraveneous administration is especially
preferred. In this case, a suitable metering device such as the
multi-therapy infusion pump model 6060 manufactured by Baxter may
advantageously be chosen. Whatever metering device is chosen, it
should be of such design and construction as to minimize or,
better, preclude an interruption of administration of therapeutic
agent in the event of cartridge exchange and/or power cell
replacement or recharging. This may be accomplished, for example by
choosing a device with a secondary reservoir of CD19xCD3 bispecific
single chain antibody solution apart from the cartridge to be
exchanged so that continuous infusion from this secondary reservoir
into the patient may continue even while the empty or almost empty
cartridge is removed and replaced with a fresh one.
[0086] A mode of intravenous administration and, as the case may
be, of co-administration as part of a regimen of co-therapy
involves the implantation of a pump into the body of the patient
for metering such administration. One of ordinary skill in the art
is aware of such metering pumps, for example model 6060
manufactured by Baxter as set forth above.
[0087] As a non-limiting example, it may be that the uninterrupted,
i.e. continuous administration is to be realized by a small pump
system worn by or implanted into the patient for metering the
influx of therapeutic agent into the body of the patient. Such pump
systems are generally known in the art, and commonly rely on
periodic exchange of cartridges containing the therapeutic agent to
be infused. When exchanging the cartridge in such a pump system, a
temporary interruption of the otherwise uninterrupted flow of
therapeutic agent into the body of the patient may ensue. In such a
case, the phase of administration prior to cartridge replacement
and the phase of administration following cartridge replacement
would still be considered within the meaning of the pharmaceutical
means and methods of the invention to together make up one
"uninterrupted administration" of such therapeutic agent. The same
would apply for very long administrations in which the cartridge
would require replacement more than once, or in which the power
cells driving the pump would require replacement, leading to a
temporary offset of the flow of therapeutic solution into the body
of the patient.
[0088] Appropriate measures should also be taken to minimize the
danger of infection at the puncture site of administration into the
patient's body, as such long-term wounds are especially prone to
such infection. The above also applies for intramuscular
administration via a similar delivery system.
[0089] The continuous administration may be transdermal by way of a
patch worn on the skin and replaced at intervals. One of skill in
the art is aware of patch systems for drug delivery suitable for
this purpose. It is of note that transdermal administration is
especially amenable to uninterrupted administration, as exchange of
a first exhausted patch can advantageously be accomplished
simultaneously with the placement of a new, second patch, for
example on the surface of the skin immediately adjacent to the
first exhausted patch and immediately prior to removal of the first
exhausted patch. Issues of flow interruption or power cell failure
do not arise.
[0090] In a further preferred embodiment, the continuous
administration is accomplished via a pulmonary route, for example
via a tube worn in one or both nostrils of the nose, the tube being
connected to a pressurized tank, the content of which is precisely
metered.
[0091] Furthermore, the invention relates to a CD19xCD3 bispecific
single chain antibody construct for the treatment, amelioration or
elimination of adult acute lymphoblastic leukemia (ALL). The
invention further relates to the use of a CD19xCD3 bispecific
single chain antibody construct for the preparation of a
pharmaceutical composition for the treatment, amelioration or
elimination of adult acute lymphoblastic leukemia (ALL).
Preferably, said acute lymphoblastic leukemia (ALL) is B-lineage
acute lymphoblastic leukemia, more preferably B-precursor acute
lymphoblastic leukemia.
[0092] In a preferred embodiment of the mentioned medical uses,
said acute lymphoblastic leukemia (ALL) is refractory to
chemotherapy in patients non-eligible for allogeneic HSCT.
[0093] In an alternative embodiment of the mentioned medical uses,
the administration of the CD19xCD3 bispecific single chain antibody
construct is followed by allogeneic HSCT or said uses replace
allogeneic HSCT in patients eligible for allogeneic HSCT
[0094] In another preferred embodiment of the mentioned medical
uses, the CD19xCD3 bispecific single chain antibody construct is
for the treatment, amelioration or elimination of minimal residual
disease (MRD) in a patient with acute lymphoblastic leukemia (ALL).
Preferably, said patient is MRD-positive in complete hematological
remission.
[0095] In a further preferred embodiment of the mentioned medical
uses, the administration of said CD19xCD3 bispecific single chain
antibody results in stable disease or converts MRD positive acute
lymphoblastic leukemia (ALL) into an MRD negative status.
[0096] Preferably, MRD is measured with quantitative detection of
individual rearrangements of immunoglobulin genes or T-cell
receptor (TCR) rearrangements, or by bcr/abl fusion transcripts, or
t(4;11), using PCR or FACS analysis.
[0097] Even more preferred, the ALL patient shows a bcr/abl or a
t(4;11) signal above detection limit and/or at least one marker by
rearrangement with a sensitivity of .gtoreq.10.sup.-4.
[0098] In another preferred embodiment of the mentioned medical
uses, the time to molecular relapse detectable by the indicated
detection methods is more than 4 months.
[0099] In another preferred embodiment of the mentioned medical
uses, the corresponding variable heavy chain regions (V.sub.H) and
the corresponding variable light chain regions (V.sub.L) regions in
said CD19xCD3 bispecific single chain antibody construct are
arranged, from N-terminus to C-terminus, in the order,
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.H(CD3)-V.sub.L(CD3).
[0100] Preferably, said CD19xCD3 bispecific single chain antibody
construct comprises an amino acid sequence as set forth in SEQ ID
NO. 1, or an amino acid sequence at least 90%, preferably 95%
identical to SEQ ID NO. 1.
[0101] In a further preferred embodiment of the mentioned medical
uses, one treatment cycle is a 4-week continuous infusion, followed
by repeated cycles after a 2-week treatment-free interval.
[0102] Preferably, the treatment cycle is repeated at least three
times, after determination of a MRD negative status
(consolidation).
[0103] In another preferred embodiment of the mentioned medical
uses, the CD19xCD3 bispecific single chain antibody construct is to
be administered in a daily dose of 10 .mu.g to 100 .mu.g per square
meter patient body surface area.
[0104] Preferably, the CD19xCD3 bispecific single chain antibody
construct is to be administered in a daily dose of 15 .mu.g to 30
.mu.g per square meter patient body surface area.
[0105] The definitions and explanations provided with respect to
the pharmaceutical methods and means of the invention apply mutatis
mutandis to the medical uses of the CD19xCD3 bispecific single
chain antibody construct described herein.
[0106] The Figures show:
[0107] FIG. 1: CD19xCD3 bispecific single chain antibody mode of
action. CD19xCD3 bispecific single chain antibody (blinatumomab or
MT103) redirects CD3-positive cytotoxic T cells to eliminate human
acute lymphoblastic leukemia cells carrying the CD19 antigen.
[0108] FIG. 2: Example of minimal residual disease (MRD) course.
PCR based measurement of TCR rearrangement (MRD) in common acute
lymphoblastic leukemia (cALL) patient 109-002 shows an MRD positivy
before treatment with CD19xCD3 bispecific single chain antibody and
ongoing MRD negativity starting after the 1st cycle CD19xCD3
bispecific single chain antibody.
[0109] FIG. 3: T cell kinetics of CD4 and CD8 T cells of patient
109-002 during treatment cycle 1. Representative example of
pharmacodynamics, showing rapid redistribution of T cells and an
increase mainly in the number of cytotoxic CD8 T cells.
[0110] FIG. 4: T cell kinetics of T cell subsets of patient 109-002
during treatment cycle 1. Representative example of
pharmacodynamics, showing rapid redistribution of T cells and
expansion of T effector memory cells (TEM). Naive T cells are not
expanded.
[0111] FIG. 5: The first four patients who have been enrolled in
the phase II study. All patients had previously received standard
chemotherapy regimens for ALL according to GMALL protocols
including at least one consolidation treatment.
[0112] FIG. 6: Minimal residual disease (MRD) responses in the
indicated ALL patients (i.e. the first four patients enrolled in
the phase II study) after the first treatment cycle with CD19xCD3
bispecific single chain antibody.
[0113] FIG. 7: Update on minimal residual disease (MRD) responses.
In nine out of eleven patients with immunoglobulin or TCR
rearrangements, in one out of two patients with t(4;11)
translocations and in three out of four patients with bcr/abl
transcripts, MRD-negativity could be achieved. In sum, 13 of 16
evaluable patients (81%) became MRD negative.
[0114] FIG. 8: Duration of minimal residual disease
(MRD)-negativity (status as of 25 May 2009). The longest duration
of MRD-negativity observed so far in patient 108-001 having not
received a transplantation after the antibody treatment is 41
weeks. Patient 111-001 with MRD-negativity from 23 Jun. 2008 to 27
Oct. 2008 after CD19xCD3 bispecific single chain antibody-treatment
and having received a successful allogeneic hematopoietic stem cell
transplantation thereafter is relapse-free to date. The arrowhead
means that the response is still ongoing (status May 25, 2009).
Patient 109-002 (*) had a testicular relapse followed by
hematological relapse after 19 weeks of MRD-negativity.
[0115] The invention is further illustrated by the following
example:
EXAMPLE
[0116] 1. The generation, expression and cytotoxic activity of the
CD19xCD3 bispecific single chain antibody has been described in WO
99/54440. The corresponding amino and nucleic acid sequences of the
CD19xCD3 bispecific single chain antibody are shown in SEQ ID NOs.
1 and 2, respectively. The VH and VL regions of the CD3 binding
domain of the CD19xCD3 bispecific single chain antibody are shown
in SEQ ID NOs. 7 to 10, respectively, whereas the VH and VL regions
of the CD19 binding domain of the CD19xCD3 bispecific single chain
antibody are shown in SEQ ID NOs 3 to 6, respectively. The
corresponding CDR regions are shown in SEQ ID NOs. 11 to 22.
[0117] 2. An ongoing phase 1 trial in relapsed B-NHL patients shows
high response rate at 60 .mu.g/m.sup.2/day of CD19xCD3 bispecific
single chain antibody. Responses have a duration of up to more than
12 months (ongoing in several patients). Removal of bone marrow
infiltrating B-NHL cells started at 15 .mu.g/m.sup.2/day (Bargou et
al., Science 2008).
[0118] 3. Based on these results, a phase II dose-escalating study
was designed in collaboration with the German Multicenter Study
Group on Adult Acute Lymphoblastic Leukemia (GMALL) to investigate
efficacy, safety, and tolerability of the CD19xCD3 bispecific
single chain antibody in adult (non-transplanted) acute
lymphoblastic leukemia (ALL) patients who achieved a complete
hematological remission, but remained minimal residual disease
(MRD)-positive. MRD is an independent prognostic factor that
reflects primary drug resistance and is associated with a high
relapse risk after start of consolidation. This applies for
Ph+/BCR-ABL-positive and -negative ALL. MRD was measured with
standardized methods either by quantitative detection of individual
rearrangements of immunoglobulin or T-cell receptor (TCR)
rearrangements, or by bcr/abl fusion transcripts or t(4;11)
translocations. The study population includes adult patients with
acute B-precursor acute lymphoblastic leukemia (ALL) who show a
bcr/abl or t(4;11) translocation signal above detection limit
and/or at least one marker by rearrangement with a sensitivity of
.gtoreq.10.sup.-4. More specifically, the major inclusion criteria
included: [0119] B-precursor ALL patients in complete hematological
remission with molecular failure or molecular relapse starting at
any time after consolidation 1 of front-line therapy within
standard protocols. [0120] Patients must have a molecular marker
for evaluation of minimal residual disease which is either bcr/abl
or a t(4;11) translocation at any detection level or individual
rearrangements of immunoglobulin or TCR-genes measured by an assay
with a sensitivity of minimum 10.sup.-4 and quantitative range to
10.sup.-4 for at least one marker.
[0121] Primary endpoint of the (ongoing) phase II study is the
conversion rate to minimal residual disease (MRD) negative status
as defined by a bcr/abl or t(4;11) translocation signal below
detection limit and/or by detection of individual rearrangements of
immunoglobulin or T-cell receptor (TCR) genes below 10.sup.-4.
Secondary endpoints are time to hematological relapse, time to MRD
progression, and time to molecular relapse. One treatment cycle of
the CD19xCD3 bispecific single chain antibody is a 4-week
continuous intravenous infusion, which can be followed by
allogeneic hematopoietic stem cell transplantation after the first
cycle or further cycles, or by repeated cycles after a 2-week
treatment-free interval. Minimal residual disease (MRD) status is
controlled after each treatment cycle. The starting dose level is
15 microgram/m.sup.2/24 hr, which may be escalated to 30
microgram/m.sup.2/24 hr and higher dose levels (60
microgram/m.sup.2/24 hr or 90 microgram/m.sup.2/24 hr) based on
clinical activity and safety data. For statistical design, Simon's
MinMax two stage design (14 to 21 patients) is being used.
[0122] In the following, the data of the first four patients
enrolled in the study are presented exemplarily in more detail.
These four patients aged 31, 57, 62, and 65 years received the
initial dose level of 15 microgram/m.sup.2/24 hr. As shown in FIG.
5, patient nos. 111001, 109002 and 110002 have been diagnosed with
c-ALL, whereas patient no. 108001 is a pre-B-ALL patient. The four
patients had previously received standard chemotherapy regimens for
ALL according to GMALL protocols including at least one
consolidation treatment. All of them have been refractory to
chemotherapy as regards minimal residual disease (MRD). More
specifically, all patients have been MRD-positive in complete
hematological remission. Patients nos. 110002, 108001 and 109002
have been non-eligible for allogeneic hematopoietic stem cell
transplantation, whereas patient no. 111001 has been eligible for
said transplantation.
[0123] As shown in FIG. 6, three out of the first 4 patients
enrolled in the study had minimal residual disease (MRD) by
immunoglobulin or TCR rearrangements at levels of 10.sup.-4
(patient no. 111001), 10.sup.-3 (patient no. 108001) and 10.sup.-1
(patient no. 109002), and one patient (patient no. 110002) had MRD
by bcr/abl fusion transcripts at a level of 10.sup.-4. Three out of
the 3 patients, i.e. patient nos. 111001, 108001 and 109002 with
immunoglobulin or TCR rearrangements turned MRD negative after the
first treatment cycle, independently from the level of MRD
positivity at baseline. Patient no. 111001, the only one of the
four patients eligible for allogeneic hematopoietic stem cell
transplantation, received a transplantation after having been
converted into MRD negativity upon CD19xCD3 bispecific single chain
antibody treatment.
[0124] FIG. 2 provides an example of the minimal residual disease
(MRD) course in patient 109002. PCR based measurement of TCR
rearrangement (MRD) in common acute lymphoblastic leukemia (cALL)
patient 109002 shows an MRD positivy before treatment with CD19xCD3
bispecific single chain antibody (Blinatumomab) and MRD negativity
starting after the 1st cycle CD19xCD3 bispecific single chain
antibody and lasting until week 19. Thereafter, the patient had a
testicular relapse, followed by a haematological relapse.
[0125] The other patient having no. 110002 had stable bcr/abl level
without signs of hematological relapse after the initial treatment
cycle; see FIG. 6.
[0126] The treatment of the patients with CD19xCD3 bispecific
single chain antibody was well tolerated: Except for fever on the
first 3 days of treatment, no clinically significant toxicities
were recorded.
[0127] Meanwhile, seventeen adult patients have been treated, or
are still on treatment with the CD19xCD3 bispecific single chain
antibody, up to date. All patients have been refractory to
conventional ALL therapies, including chemotherapy, before the
antibody treatment. None of them has received an allogeneic
hematopoietic stem cell transplantation before the antibody
treatment. The median age of the patients was 48 years, ranging
from 20 to 77 years. Ten of the patients were female, seven were
male patients. 14 patients received the dose level of 15
microgram/m.sup.2/24 hr of CD19xCD3 bispecific single chain
antibody, whereas in three patients the dose has been escalated
from 15 to 30 microgram/m.sup.2/24 hr after the first or further
treatment cycles: in patient 109-004 the dose escalation was
carried out after the second treatment cycle (with a total of three
treatment cycles, followed by allogeneic hematopoietic stem cell
transplantation), in patient 109-003 after the third treatment
cycle (with a total of four treatment cycles), and in patient
110-002 after the sixth treatment cycle (with a total of seven
treatment cycles). Eleven of these patients had minimal residual
disease (MRD) by immunoglobulin or TCR rearrangements, two patients
had t(4;11) translocations and four patient had bcr/abl fusion
transcripts.
[0128] As a result, MRD response was evaluable in 16 of 17
patients. As shown in FIG. 7, 13 of 16 evaluable patients became
MRD negative, which corresponds to an extraordinary complete
molecular response rate of 81%. More specifically, in nine out of
eleven patients with immunoglobulin or TCR rearrangements, one out
of two patients with t(4;11) translocations and three out of four
patients with bcr/abl transcripts MRD-negativity could be achieved.
As shown in FIG. 8, the longest duration of MRD-negativity in
patient 108-001 having not received a transplantation after the
antibody treatment observed so far is 41 weeks. Another patient
with MRD-negativity from 23 Jun. 2008 to 27 Oct. 2008 and having
received a successful allogeneic hematopoietic stem cell
transplantation after the antibody treatment is relapse-free to
date; see patient 111-001 in FIG. 8. Remarkably, the bcr/abl
patients who could successfully be treated with the CD19xCD3
bispecific single chain antibody were refractory or intolerant to
tyrosine kinase inhibitors imatinib and/or dasatinib in previous
ALL treatment regimen. In particular, one of the bcr/abl responders
to treatment with CD19xCD3 bispecific single chain antibody had a
T315I mutation which is refractory to therapy by tyrosine kinase
inhibitors. Thus, the administration of the CD19xCD3 bispecific
single chain antibody now provides for the first time for a therapy
for dasatinib-refractory ALL patients with bcr/abl transcripts.
Only three out of a total of 17 patients did not become MRD
negative. However, in two of them stable disease could be achieved.
Only one patient with initial stable disease had a hematological
relapse in the third treatment cycle. One patient was not evaluable
due to an SAE on study day 2.
[0129] In summary, an absolutely exceptional complete molecular
response rate of 81% could be achieved in patients with B-precursor
ALL upon treatment with CD19xCD3 bispecific single chain antibody.
Activity of the mentioned antibody could be observed in all
patients subsets treated, including tyrosine kinase
inhibitors-refractory (T315I) bcr/abl patients and patients with
t(4;11) translocations. In addition, treatment with CD19xCD3
bispecific single chain antibody shows a favorable toxicity
profile, in contrast to conventional ALL therapies, such as
chemotherapy. In light of this, the administration of the CD19xCD3
bispecific single chain antibody described herein provides a new
and advantageous treatment option for acute lymphoblastic leukemia
(ALL), in particular for cases in which the ALL is refractory to
conventional ALL therapy, such as chemotherapy. In addition, the
administration of the CD19xCD3 bispecific single chain antibody now
provides for the first time for a therapy for MRD-positive ALL.
[0130] These updated results indicate that treatment of acute
lymphoblastic leukemia (ALL) patients with the CD19xCD3 bispecific
single chain antibody is able to convert minimal residual disease
(MRD) positive acute lymphoblastic leukemia (ALL) into an MRD
negative status (as exemplified by the ALL patients with
immunoglobulin or TCR rearrangements, bcr/abl transcripts or
t(4;11) translocations), and that this treatment is well tolerated.
In light of this, the administration of the CD19xCD3 bispecific
single chain antibody described herein provides an alternative
treatment option especially for adult acute lymphoblastic leukemia
(ALL), in particular to ALL refractory to conventional ALL therapy,
such as chemotherapy and/or HSCT. Treatment with the CD19xCD3
bispecific single chain antibody is especially advantageous for the
treatment of MRD-positive ALL.
Sequence CWU 1
1
221498PRTArtificial Sequence="Description of artificial sequence
CD19xCD3 bispecific single chain antibody" 1Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180
185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met
Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr
Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Asp 245 250 255 Ile Lys Leu Gln Gln Ser
Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 260 265 270 Val Lys Met Ser
Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr 275 280 285 Met His
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300
Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 305
310 315 320 Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala
Tyr Met 325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys Ala 340 345 350 Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp
Tyr Trp Gly Gln Gly Thr 355 360 365 Thr Leu Thr Val Ser Ser Val Glu
Gly Gly Ser Gly Gly Ser Gly Gly 370 375 380 Ser Gly Gly Ser Gly Gly
Val Asp Asp Ile Gln Leu Thr Gln Ser Pro 385 390 395 400 Ala Ile Met
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 405 410 415 Ala
Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly 420 425
430 Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly
435 440 445 Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr
Ser Leu 450 455 460 Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr
Tyr Tyr Cys Gln 465 470 475 480 Gln Trp Ser Ser Asn Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu 485 490 495 Leu Lys 21494DNAArtificial
Sequence="Description of artificial sequence CD19xCD3 bispecific
single chain antibody" 2gatatccagc tgacccagtc tccagcttct ttggctgtgt
ctctagggca gagggccacc 60atctcctgca aggccagcca aagtgttgat tatgatggtg
atagttattt gaactggtac 120caacagattc caggacagcc acccaaactc
ctcatctatg atgcatccaa tctagtttct 180gggatcccac ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggaga
aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg
300acgttcggtg gagggaccaa gctcgagatc aaaggtggtg gtggttctgg
cggcggcggc 360tccggtggtg gtggttctca ggtgcagctg cagcagtctg
gggctgagct ggtgaggcct 420gggtcctcag tgaagatttc ctgcaaggct
tctggctatg cattcagtag ctactggatg 480aactgggtga agcagaggcc
tggacagggt cttgagtgga ttggacagat ttggcctgga 540gatggtgata
ctaactacaa tggaaagttc aagggtaaag ccactctgac tgcagacgaa
600tcctccagca cagcctacat gcaactcagc agcctagcat ctgaggactc
tgcggtctat 660ttctgtgcaa gacgggagac tacgacggta ggccgttatt
actatgctat ggactactgg 720ggccaaggga ccacggtcac cgtctcctcc
ggaggtggtg gatccgatat caaactgcag 780cagtcagggg ctgaactggc
aagacctggg gcctcagtga agatgtcctg caagacttct 840ggctacacct
ttactaggta cacgatgcac tgggtaaaac agaggcctgg acagggtctg
900gaatggattg gatacattaa tcctagccgt ggttatacta attacaatca
gaagttcaag 960gacaaggcca cattgactac agacaaatcc tccagcacag
cctacatgca actgagcagc 1020ctgacatctg aggactctgc agtctattac
tgtgcaagat attatgatga tcattactgc 1080cttgactact ggggccaagg
caccactctc acagtctcct cagtcgaagg tggaagtgga 1140ggttctggtg
gaagtggagg ttcaggtgga gtcgacgaca ttcagctgac ccagtctcca
1200gcaatcatgt ctgcatctcc aggggagaag gtcaccatga cctgcagagc
cagttcaagt 1260gtaagttaca tgaactggta ccagcagaag tcaggcacct
cccccaaaag atggatttat 1320gacacatcca aagtggcttc tggagtccct
tatcgcttca gtggcagtgg gtctgggacc 1380tcatactctc tcacaatcag
cagcatggag gctgaagatg ctgccactta ttactgccaa 1440cagtggagta
gtaacccgct cacgttcggt gctgggacca agctggagct gaaa
14943124PRTArtificial Sequence="Description of artificial sequence
VH anti CD19" 3Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg
Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp
Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr
Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala
Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105
110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
4372DNAArtificial Sequence="Description of artificial sequence VH
anti CD19" 4caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc
agtgaagatt 60tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt
gaagcagagg 120cctggacagg gtcttgagtg gattggacag atttggcctg
gagatggtga tactaactac 180aatggaaagt tcaagggtaa agccactctg
actgcagacg aatcctccag cacagcctac 240atgcaactca gcagcctagc
atctgaggac tctgcggtct atttctgtgc aagacgggag 300actacgacgg
taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc
360accgtctcct cc 3725111PRTArtificial Sequence="Description of
artificial sequence VL anti CD19" 5Asp Ile Gln Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser
Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr
Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu
Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65
70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln
Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 6333DNAArtificial Sequence="Description of
artificial sequence VL anti CD19" 6gatatccagc tgacccagtc tccagcttct
ttggctgtgt ctctagggca gagggccacc 60atctcctgca aggccagcca aagtgttgat
tatgatggtg atagttattt gaactggtac 120caacagattc caggacagcc
acccaaactc ctcatctatg atgcatccaa tctagtttct 180gggatcccac
ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat
240cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga
ggatccgtgg 300acgttcggtg gagggaccaa gctcgagatc aaa
3337119PRTArtificial Sequence="Description of artificial sequence
VH anti CD3" 7Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg
Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr
Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg
Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr
Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Thr Leu Thr Val Ser Ser 115 8357DNAArtificial
Sequence="Description of artificial sequence VH anti CD3"
8gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg
60tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg
120cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta
tactaattac 180aatcagaagt tcaaggacaa ggccacattg actacagaca
aatcctccag cacagcctac 240atgcaactga gcagcctgac atctgaggac
tctgcagtct attactgtgc aagatattat 300gatgatcatt actgccttga
ctactggggc caaggcacca ctctcacagt ctcctca 3579106PRTArtificial
Sequence="Description of artificial sequence VL anti CD3" 9Asp Ile
Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile
Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys 100 105 10318DNAArtificial Sequence="Description of
artificial sequence VL anti CD3" 10gacattcagc tgacccagtc tccagcaatc
atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gagccagttc aagtgtaagt
tacatgaact ggtaccagca gaagtcaggc 120acctccccca aaagatggat
ttatgacaca tccaaagtgg cttctggagt cccttatcgc 180ttcagtggca
gtgggtctgg gacctcatac tctctcacaa tcagcagcat ggaggctgaa
240gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt
cggtgctggg 300accaagctgg agctgaaa 3181115PRTartificial
sequence="Description of artificial sequence anti-CD19 L1" 11Lys
Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn 1 5 10 15
127PRTartificial sequence="Description of artificial sequence
anti-CD19 L2" 12Asp Ala Ser Asn Leu Val Ser 1 5 139PRTartificial
sequence="Description of artificial sequence anti-CD19 L3" 13Gln
Gln Ser Thr Glu Asp Pro Trp Thr 1 5 145PRTartificial
sequence="Description of artificial sequence anti-CD19 H1" 14Ser
Tyr Trp Met Asn 1 5 1517PRTartificial sequence="Description of
artificial sequence anti-CD19 H2" 15Gln Ile Trp Pro Gly Asp Gly Asp
Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly 1615PRTartificial
sequence="Description of artificial sequence anti-CD19 H3" 16Arg
Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 15
175PRTartificial sequence="Description of artificial sequence
anti-CD3 H1" 17Arg Tyr Thr Met His 1 5 1817PRTartificial
sequence="Description of artificial sequence anti-CD3 H2" 18Tyr Ile
Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15
Asp 1910PRTartificial sequence="Description of artificial sequence
anti-CD3 H3" 19Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10
2010PRTartificial sequence="Description of artificial sequence
anti-CD3 L1" 20Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10
217PRTartificial sequence="Description of artificial sequence
anti-CD3 L2" 21Asp Thr Ser Lys Val Ala Ser 1 5 229PRTartificial
sequence="Description of artificial sequence anti-CD3 L3" 22Gln Gln
Trp Ser Ser Asn Pro Leu Thr 1 5
* * * * *
References