U.S. patent application number 14/813881 was filed with the patent office on 2016-01-21 for use of an active substance binding to cd28 for producing a pharmaceutical composition for the treatment of b-cll.
The applicant listed for this patent is TheraMAB LLC. Invention is credited to Thomas Hanke.
Application Number | 20160017039 14/813881 |
Document ID | / |
Family ID | 34585025 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160017039 |
Kind Code |
A1 |
Hanke; Thomas |
January 21, 2016 |
USE OF AN ACTIVE SUBSTANCE BINDING TO CD28 FOR PRODUCING A
PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF B-CLL
Abstract
The invention relates to the use of a superagonistic monoclonal
antibody (mAb), which is specific for a naturally costimulatory
receptor expressed on T cells, or a mimicry compound thereto, for
producing a pharmaceutical composition for the treatment of
diseases occurring with lacking costimulability of T cells, in
particular of the B-CLL.
Inventors: |
Hanke; Thomas; (Wurzburg,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TheraMAB LLC |
Moscow |
|
RU |
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|
Family ID: |
34585025 |
Appl. No.: |
14/813881 |
Filed: |
July 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13625521 |
Sep 24, 2012 |
9119840 |
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14813881 |
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11646986 |
Dec 28, 2006 |
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13625521 |
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10988207 |
Nov 12, 2004 |
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11646986 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
C07K 2317/515 20130101;
A61K 39/39558 20130101; C07K 2317/75 20130101; C07K 2317/74
20130101; C07K 16/2818 20130101; C07K 2317/73 20130101; C07K
2317/51 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 11, 2003 |
DE |
103 52 900.4 |
Claims
1-12. (canceled)
13. A method for stimulating the antigen presenting function of B
cells in a cell suspension, comprising reacting the cell suspension
with a superagonistic monoclonal antibody specific for CD28,
wherein: the cell suspension is prepared from a body fluid taken
from a B-cell chronic lymphocytic leukemia (B-CLL) patient.
14. The method of claim 13, wherein the B cells are induced to
express B7.1.
15. The method of claim 13, wherein the B cells are induced to
express B7.2.
16. The method of claim 13, wherein the method further comprises
activation and expansion of T cells in the cell suspension.
17. The method of claim 16, wherein the T cells are activated to
express CD40 ligand (CD40L).
18. The method of claim 16, wherein the number of T cells in the
cell suspension are increased.
19. The method of claim 13, wherein the body fluid is blood.
20. The method of claim 13, wherein the superagonistic monoclonal
antibody specific for CD28 comprises a heavy chain variable region
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
SEQ ID NO: 19.
21. The method of claim 13, wherein the superagonistic monoclonal
antibody specific for CD28 comprises a light chain variable region
comprising the amino acid sequence set forth in SEQ ID NO: 25.
22. The method of claim 13, wherein the superagonistic monoclonal
antibody specific for CD28 comprises a heavy chain constant region
comprising the amino acid sequence set forth in one of SEQ ID NO:
22, SEQ ID NO: 23, and SEQ ID NO: 24.
23. The method of claim 13, wherein the superagonistic monoclonal
antibody specific for CD28 comprises a light chain constant region
comprising the amino acid sequence set forth in SEQ ID NO: 21.
Description
STATEMENT OF RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/625,521, filed Sep. 24, 2012, now U.S. Pat.
No. 9,119,840, which is a continuation of U.S. patent application
Ser. No. 11/646,986, filed Dec. 28, 2006, which is a continuation
of U.S. patent application Ser. No. 10/988,207, filed Nov. 12,
2004, each of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the use of an active substance for
producing a pharmaceutical composition for the treatment of chronic
lymphocytic leukemia of the B-cell type (B-CLL).
BACKGROUND OF THE INVENTION AND PRIOR ART
[0003] For understanding the invention, firstly the following
terminological background is important. The activation of resting T
cells for proliferation and functional differentiation firstly
requires the occupation of two surface structures, so-called
receptors: 1. of the antigen receptor having a different
specificity from cell to cell and being necessary for detecting
antigens, for instance viral fission products; and 2. of the CD28
molecule equally expressed on all resting T cells with the
exception of a sub-group of the CD8 T cells of man, the CD28
molecule naturally binding to ligands on the surface of other cells
of the immune system. This is the "costimulation" of the
antigen-specific immune reaction by CD28. In a cell culture, these
processes can be simulated by occupation of the antigen receptor
and of the CD28 molecule with suitable monoclonal antibodies
(mAb's). In the classic system of costimulation, neither the
occupation of the antigen receptor nor that of the CD28 molecule
alone will lead to a T cell proliferation, the occupation of both
receptors is however effective. This observation was made in T
cells of humans, mouse and rat.
[0004] There are however also known CD28-specific mAb's that can
initiate the T cell proliferation without costimulation. Such a
superagonistic, i.e. independent from the occupation of the antigen
receptor, activation of resting T lymphocytes by CD28-specific
mAb's is known in the art from the document Tacke et al., Eur. J.
Immunol., 1997, 27:239-247. According thereto, two types of
CD28-specific monoclonal antibodies having different functional
properties are described: costimulatory mAb's costimulating the
activation of resting T cells only with simultaneous occupation of
the antigen receptor; and superagonistic mAb's, which can activate
T lymphocytes of all classes in vitro and in the test animal for
proliferation without occupation of the antigen receptor. From the
documents DE 197 22 888 and PCT/DE03/00890 are also known
CD28-specific mAb's, which efficiently activate T lymphocytes in
vitro as well as in vivo without TCR stimulation, i.e. which act
"superagonistically". The mAb's, at least those, which are directed
against human CD28, are also called SuperMAb's.
[0005] The chronic lymphatic leukemia of the B-cell series (chronic
lymphocytic leukemia of the B-cell type, B-CLL) is with an
incidence of 3 per 100,000 inhabitants the most frequent leukemia
of adults (Landis et al., 1999). The disease is characterized by a
progressive accumulation of malignant monoclonal B lymphocytes in
the blood, lymph nodes, liver and bone marrow. With progressing
disease, the lymphocyte count in the blood will be increased, lymph
nodes, liver and spleen will become enlarged, and an anemia and
thrombocytopenia will occur (Caligaris-Cappio, 2000; Rozman and
Montserrat, 1995). A curative therapy of the B-CLL is not possible
at present.
[0006] To the main complications of the B-CLL belong infections,
for which can be blamed, among other reasons, the T cell defects or
low T cell counts to be observed in many patients (Cantwell et al.,
1997; Scrivener et al., 2003).
[0007] A limited function can further also be found in the tumor
cells themselves. The property of the B-CLL B cells, to act as
antigen-presenting cells (APC's) and to thus be detected and
eliminated by tumor-specific T cells, is substantially limited. The
reason for this, among others, is that B-CLL B cells do not express
or only insufficiently express the natural CD28 ligands CD80
(=B7.1) and CD86 (=B7.2) at their cell surface. Thus the
potentially tumor-specific T cells will obtain a single signal only
for the interaction between the T cell receptor and MHC+tumor
antigen and not the second costimulatory signal necessary for the
full activation of a T cell. As a summary, this indicates that the
immune system of B-CLL patients obviously is not capable of
eliminating the tumor cells by itself.
[0008] One approach for therapy aims for improving the APC function
of the B-CLL B cells and thus reinforcing the anti-tumor immune
response of the patient. Various working groups have already tried
to specifically activate B-CLL B cells and to thereby induce the
expression of costimulatory ligands. They were successful in
expressing, by gene transfer, the B cell stimulating CD40 ligand
(CD40L) in the B-CLL B cells, and thus making possible the
interaction between CD40L and the protein CD40 being present on all
B cells (including the B-CLL B cells). The signal forwarded by CD40
into the cell interior led to the desired expression of the
costimulatory ligand CD80 and CD86 (Kato et al., 1998; Wendtner et
al., 2002). The leukemia cells thus modified could now in fact act
in a more efficient way than APC's, and were now also
eliminated--firstly in vitro--by autologous T cells (Kato et al.,
1998). The in vivo gene transfer of CD40L was also effective: it
led in the patients to an increase in the T cell count as well as
to a reduction of the leukemic cells (Wierda et al., 2000). This
therapeutic approach does not influence, however, the above
mentioned T cell defects of the B-CLL disease.
[0009] In the following is shown the bibliography of the above
scientific literature. Caligaris-Cappio, F., Rev Clin Exp Hematol
4, 5-21 (2000); Cantwell, M. et al., Nat Med 3, 984-989 (1997);
Kato, K. et al., J Clin Invest 101, 1133-1141 (1998); Landis, S. H.
et al., CA Cancer J Clin 49, 8-31 (1999); Rozman, C. et al., N Engl
J Med 333, 1052-1057 (1995); Scrivener, S. et al., Leuk Lymphona
44, 383-389 (2003); Wendtner, C. M. et al., Blood 100, 1655-1661
(2002); Wierda, W. G. et al., Blood 96, 2917-2924 (2000).
TECHNICAL OBJECT OF THE INVENTION
[0010] It is the technical object of the invention to provide a
pharmaceutical composition, by means of which B-CLL can be treated,
and whereby simultaneously the immune system weakened by the
disease, in particular the T cell-mediated cellular immunity, is
generally improved.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic representation of a dot-dot analysis
showing B-CLL T Cell expansion upon induction with SuperMAB.
[0012] FIG. 2 is a schematic representation of a histogram showing
the expression of CD40L on T cells as a function of cell count.
[0013] FIG. 3 are schematic representations of histograms showing
the expression of B7.1 and B7.2 on B-CLL B cells.
[0014] FIG. 4 is a graphical representation showing the
proliferation of SuperMAB B-CLL B cells and not-SuperMAB-pretreated
B-CLL B Cells.
[0015] FIGS. 5A, 5B and 5C are graphical representations showing
the effect of SuperMAB pretreatment on activation and proliferation
of autologous T cells.
[0016] FIG. 6. is a diagrammatic representation showing T cell
proliferation as measured by the incorporation of 3H thymidine.
[0017] FIG. 7 is a diagrammatic representation showing stimulation
cells cultivated with autologous cells in the presence of the
cytokine, Interleukin 2.
[0018] FIG. 8 is a diagrammatic representation of the mechanism for
the use of SuperMAB to activate tumor-specific autologous T cells
and to induce an elimination of tumor cells by an induction of
costimulatory proteins on the surface of the antigen-presenting
B-CLL B cells.
[0019] FIGS. 9A and 9B show the amino acid sequence of the heavy
and light chains, respectively, of the monoclonal antibody,
TGN1412.
[0020] FIGS. 9C and 9D are nucleotide sequences of cDNA's coding
for the heavy and light chain, respectively, of the monoclinal
antibody, TGN 1412, inserted into plasmids pLNOH and pLNOK.
BASICS OF THE INVENTION AND PREFERRED EMBODIMENTS
[0021] For achieving this technical object, the invention teaches
the use of a superagonistic monoclonal antibody (mAb), which is
specific for a naturally costimulatory receptor expressed on
resting T cells, in particular a CD28, CTLA-4, ICOS or PD1-specific
superagonistic mAb, or a mimicry compound thereto, for producing a
pharmaceutical composition for the treatment of diseases lacking
costimulability of T cells, in particular of chronic lymphocytic
leukemia of the B-cell type (B-CLL). Other diseases lacking
costimulability of T cells are for instance agammaglobulinemia,
selective immunoglobulin deficiencies, such as selective IgA
deficiency, common variable immunodeficiencies (CVID), diseases,
where compared to solid tumors such as lung carcinoma, the
capability of tumor-specific T cells is limited due to the lack of
expression of costimulatory ligands on tumor cells, to
cytotoxically react against the tumor cells, and where T cells are
made capable, by superagonistic CD28 stimulation, to cytotoxically
react.
[0022] The invention is based on the finding that with the use of
the mAb's according to the invention a double effect so to speak is
obtained. On the one hand, normal T cells, which are repressed
because of the disease, are activated or expanded. Thereby, often
highly critical infections can be prevented or reduced in the
course of the disease. Simultaneously, the body-own immune system
is excited to eliminate the B-CLL B cells normally not or only
weakly attacked by tumor-specific T cells. This is achieved in that
the B-CLL B cells are activated to present a costimulatory ligand
not normally expressed by them. Thus, the body-own tumor-specific T
cells reliant upon such a signal are able to effectively attack the
B-CLL B cells.
[0023] An mAb according to the invention can, for instance, be
produced by immunizing a non-human mammal with CD28 or a partial
sequence thereof, for instance the C'-D loop, wherein from the
non-human mammal cells are taken, and from the cells hybridoma
cells are produced, and wherein the thus obtained hybridoma cells
are selected such that there are contained mAb's superagonistically
binding to CD28 in their culture supernatant. However, other common
methods for obtaining corresponding mAb's can also be used, such as
phage display or human-Ig transgenic mice. Suitable mAb's can also
be produced by that B lymphocytes are selected, which
superagonistically bind to CD28 or its C'-D loop, and its expressed
immunoglobulin genes are cloned. For ICOS, CTLA-4 and PD1
corresponding considerations apply.
[0024] In principle, the mAb's used according to the invention may
also be antibodies derived from the described mAb's, such as
chimeric or genetically humanized antibodies.
[0025] An active substance according to the invention binds to
CD28, CTLA-4, ICOS or PD1, in particular CD28 or ICOS, or to a
partial sequence therefrom. The CD28 partial sequence may for
instance contain an amino acid sequence SEQ ID NO: 1, or 2-7, or
17, which is located at least in part in the region of the C'-D
loop of CD28. At one of the sequences with val at the 5' terminal,
one or several amino acids of the sequence 8 may be connected in
the order defined there. The loop is in the region with the
sequence GNYSQQLQVYSKTGF. The binding may however also take place
at other partial sequences of the region of the C'-D loop, and
reference is made to the definition of the region of the C'-D loop.
There are for instance also mentioned possible sequences from
CTLA-4, ICOS and PD1.
[0026] Mimicry compounds according to the invention can be
identified in a screening method, wherein a prospective mimicry
compound or a mixture of prospective mimicry compounds are
subjected to a binding assay with CD28, CTLA-4, ICOS or PD1 or to a
partial sequence therefrom, for instance, as mentioned above, and
wherein active substances binding to CD28, CTLA-4, ICOS, or PD1 or
to the partial sequence therefrom are selected, if applicable
followed by an assay for testing for superagonistic stimulation of
several to all sub-groups of the T lymphocytes. In the case of a
mixture, it will be suitable to perform a deconvolution. Among the
selected mimicry compounds, so to speak a ranking according to the
selectivity and/or affinity may take place, with highly affinitive
active substances being preferred.
[0027] The mAb may, for instance, be obtained from hybridoma cells,
as filed under the DSM numbers DSM ACC2531 (mAb: 9D7 or 9D7G3H11)
or DSM ACC2530 (mAb: 5.11A or 5.11A1C2H3). The mAb or the mimicry
compound preferably contains one or several sequences SEQ ID NOS:
9, 11, 13 and/or 15, or one or several sequences SEQ ID NOS: 10,
12, 14 and/or 16, or one or several sequences 18 and/or 19, or
sequences being homologous thereto.
[0028] Another mAb TGN1412 with improved properties suitable for
the purpose of the invention contains as the variable region of the
heavy chain (VHC) the amino acid sequence of FIG. 9a and as the
variable region of the heavy chain the amino acid sequence of FIG.
9b. The leader peptides cited in FIGS. 9a and 9b are irrelevant for
the function of the mAb and solely serve for making the passage of
the antibodies through the cell membrane easier. The variants of
the C terminus shown in the figures do not lead to a change in the
function of the antibodies.
[0029] The invention further relates (among other countries, for
the U.S.A.) to a method for the treatment of in particular B-CLL,
wherein either to a patient is administered a pharmaceutical
composition comprising a superagonistic monoclonal antibody used
according to the invention or a mimicry compound thereto and
galenically prepared for a defined and employed dosage form, for
instance IV injection, or from a patient is taken a body liquid
(=cell suspension), in particular blood, comprising T lymphocytes
and/or forerunner cells thereto, the body liquid, if applicable
after a preparation step, is reacted with a superagonistic
monoclonal antibody used according to the invention or a mimicry
compound thereto, and the thus treated body liquid is again
administered to the patient, for instance IV injected.
[0030] It may be advisable, prior to the treatment, to take a blood
sample from a patient diseased with B-CLL, to cultivate in vitro
PBMC's included therein, to incubate the culture with mAb's used
according to the invention, and to determine the activation of T
cells and/or the elimination or reduction of B-CLL B cells. With
regard to a possible determination method, reference, for instance,
is made to the embodiments, of course other usual determination
methods and the means used therefor are also suitable. In so far,
the invention also comprises a test kit for testing for the
therapeutic effectivity of a prospective treatment of B-CLL with a
pharmaceutical composition according to the invention with the
following components: i) means for PBMC cultivation in vitro; ii) a
preparation comprising mAb's according to the invention in a
physiologically effective dose; iii) means for determining the T
cell counts or the activation and/or means for determining the
B-CLL B cell counts.
[0031] The administered daily dose for the indication B-CLL may be
in the range of 0.1 to 50, preferably 1 to 10 mg/kg body
weight.
[0032] The invention finally relates to a method for reducing (in
vivo) B-CLL B cells and expanding T cells in a liquid containing
normal T cells, B cells and B-CLL B cells, wherein by interaction
of the normal T cells present in the liquid with a superagonistic
mAb used according to the invention or a mimicry compound thereto,
the normal T cells are activated or expanded, and wherein by
interaction of the B-CLL T cells present in the liquid with the
superagonistic CD28-specific mAb or the mimicry compound thereto or
a superagonistic mAb different therefrom or a mimicry compound
different therefrom are activated to the expression of a CD40
ligand and if applicable expanded, wherein the activated or
expanded B-CLL T cells carrying CD40 ligands induce the expression
of CD86 and/or CD80 in B-CLL B cells carrying CD40, wherein
autologous tumor-specific T cells are costimulatorily activated or
expanded by the B-CLL B cells carrying CD86 and/or CD80, and
wherein the activated tumor-specific T cells eliminate the B-CLL B
cells.
DEFINITIONS
[0033] Monoclonal antibodies (mAb's) are antibodies, which are
produced by hybrid cell lines (so-called hybridomas) typically
generated by fusion of a B cell of animal or human origin producing
antibodies with a suitable myeloma tumor cell.
[0034] The amino acid sequence of human CD28 is known under the
accession No. NM.sub.--006139. The amino acid sequence of human
CTLA-4 is known under the accession No. L15006. The amino acid
sequence of human ICOS is known under the accession No. AJ277832.
The amino acid sequence of human PD1 is known under the accession
No. U64863.
[0035] The C'-D loop of CD28 comprises the amino acids 43 to 70, in
particular 52 to 66 of the above CD28 sequence (for numbering see
also Ostrov, D. A., et al.; Science (2000), 290:816-819). The term
C'-D loop will in the following also include any partial sequences
therefrom. The term region of the C'-D loop comprises in addition
to or in lieu of the C'-D loop spatially adjacent regions of the
CD28. These are according to the above numbering system in
particular the amino acids 78 (Tyr) to 87 (Thr). With regard to
partial sequences in CTLA-4, ICOS and PD1 corresponding to the CD28
C'-D loop, reference is made to the document DE-101 60 516 A1, in
particular FIG. 7 and text belonging thereto and sequence
information given there.
[0036] A loop or a binding site arranged therein is freely
accessible, if for a defined binding partner for the binding site
in the loop there is no steric hindrance by the sequences or
molecules following to the loop.
[0037] Activation and/or expansion of T cells is the increase of
metabolic activity, enlargement of the cell volume, synthesis of
immunologically important molecules, such as CD40 ligand, and
initiation of the cell division (proliferation) upon an external
stimulus. As a result, there are more T cells after the activation
or expansion than before.
[0038] Homology is at least 70%, preferably at least 80%, most
preferably at least 90% sequence identity on a protein level, a
homologous protein or peptide binding a defined binding partner
with at least identical affinity. Deviations in the sequence may be
deletions, substitutions, insertions and elongations.
[0039] A mimicry compound is a natural or synthetic chemical
structure behaving in a defined binding assay as a defined mAb
mimicried by the mimicry compound.
[0040] The term "mAb's" comprises, in addition to structures of the
conventional Fab/Fc type, also structures exclusively consisting of
the Fab fragment. It is also possible to use the variable region
only, the fragment of the heavy chains being connected with the
fragment of the light chain in a suitable manner, for instance also
by means of synthetic bridge molecules. The term antibody also
comprises (possibly complete) chimeric and genetically humanized
antibodies.
[0041] Superagonistic stimulation of the proliferation of
CD28-specific T cells means that no costimulation, i.e. no further
binding event in addition to a binding of an mAb or of a mimicry
compound to CD28 is necessary for the stimulation or inhibition of
the proliferation.
[0042] In the following, the invention is explained in more detail
with reference to embodiments representing examples only.
Example 1
SuperMAB Induces the In Vitro Expansion of B-CLL T Cells
[0043] In a first experiment, peripheral mononuclear cells were
isolated from the blood (PBMC's) of a B-CLL patient, and 2.times.10
5 cells each were cultivated in 96-well microtiter plates at
37.degree. C. either in medium only or in the presence of SuperMAB
(0.5 .mu.g/ml in solution). After four days, the total cell count
was determined in the Neubauer counting chamber. Further, the cells
were incubated with fluorescence-marked monoclonal antibodies with
specificity for CD19 (B cell marker) and CD5 (expressed on B-CLL B
cells and T cells) and analyzed in the FACS. FIG. 1 shows a
so-called dot blot analysis. Every dot represents an analyzed cell
and is localized according to the expression of CD19 (x axis) and
CD5 (y axis). R1 marks the population of the T cells (CD19-CD5+),
R2 the population of the B-CLL B cells (CD19+CD5+). FIG. 1 shows
that SuperMAB is capable to induce a massive expansion of the B-CLL
T cells. Whilst in the processes "ex vivo" and "medium" the T cells
represent only 3% and 8%, resp., of the total population, this
portion increased in the SuperMAB-treated cells to 47%. The
analysis of the total cell counts showed that this is not only a
relative, but also an absolute increase of T cells. Starting from
6.times.10 3 T cells/well (ex vivo) or 1.1.times.10 4 T cells/well
(medium), resp., the addition of SuperMAB to the culture led to an
increase to 7.4.times.10 4 T cells/well. In the SuperMAB cultures,
not only a proliferation of the tumor cells could be avoided, but
also a reduction of these cells by approx. 50% could be
achieved.
[0044] In further experiments, it was investigated now whether the
expansion of the T cells induced by SuperMAB is also accompanied by
qualitative changes, and can at last strengthen the immune response
in B-CLL patients.
Example 2
SuperMAB Induces the Expression of the B Cell-Stimulating CD40
Ligand on the Surface of B-CLL T Cells
[0045] In earlier experiments it could already be shown that
SuperMAB is capable to induce the expression of the B
cell-stimulating CD40 ligand. It was now of interest whether this
is also possible for T cells of B-CLL patients, and whether thus
B-CLL B cells can be activated by a CD40L-CD40 interaction (see
above). As described above, PBMC's of a B-CLL patient were
cultivated in the presence or the absence of SuperMAB and stained
after one day with a fluorescence-marked antibody with specificity
for CD40L. FIG. 2 shows a so-called histogram, in which the
expression of CD40L on the T cells (x axis: since PBMC's are a
mixed culture, the T cells were limited before by a so-called
"electronic gate") is shown as a function of the cell count (y
axis). Compared to cells, which are only cultivated in medium, the
T cells of the SuperMAB-treated culture showed a clearly stronger
expression of CD40L. Thus, in principle the conditions are met that
the T cells now expressing CD40L interact with the CD40 on the
B-CLL B cells, and can thus induce the expression of costimulating
ligands on these cells.
Example 3
SuperMAB Induces the Expression of the Costimulating Ligand B7.2 on
B-CLL B Cells
[0046] PBMC's of a B-CLL patient were cultivated as described above
and stained after 4 days with fluorescence-marked monoclonal
antibodies with specificity for B7.1 and B7.2. The histograms in
FIG. 3 show the expression of these two proteins on the B-CLL B
cells: whilst SuperMAB only moderately induced B7.1 on these cells
(histogram a), it could drastically increase the expression of B7.2
(histogram b). In order to be able to show that the induction of
B7.2 is the consequence of the expression of CD40L on
SuperMAB-treated T cells shown in FIG. 2, in an additional process
a blocking antibody (10 .mu.g/ml) with specificity for CD40L was
given into the cultures. As a consequence of its ability to block
the interaction between CD40L and CD40, thus the induction of B7.2
on B-CLL B cells should also be prevented. The drastically reduced
expression of B7.2 in the thus treated cells (histogram d) in fact
shows that the blocking of CD40L prevents the induction of B7.2 on
B-CLL B cells. This result thus provided the indirect proof that
CD40L-expressing T cells activated by SuperMAB interact with CD40
on the surface of B-CLL B cells and can therefore induce the
expression of costimulating proteins.
Example 4
SuperMAB Improves the Antigen-Presenting Function of B-CLL B Cells
(Allogeneic Mixed Lymphocyte Culture)
[0047] The decisive question whether the expression of
costimulatory ligands on the B-CLL B cells also occurs with an
improved antigen-presenting function, can only be answered by
functional tests: in the mixed lymphocyte culture, T cells of a
person (=responder cells) are cultivated together with so-called
stimulator cells. The stimulator cells are mitomycin C-treated
cells of a second not related person (=allogeneic donor), which
differs in the expression pattern of the genes of the major
histocompatibility complex (MHC) from the first person. The T cells
detect the foreign MHC proteins together with the costimulating
ligands on the surface of the stimulator cells and are excited to
proliferation (because of mitomycin C treatment, the stimulator
cells cannot divide anymore). After four days, the T cell
proliferation is measured by the incorporation of 3H thymidine (see
FIG. 6). For answering the question, PBMC's of B-CLL patients were
isolated and cultivated in medium or with SuperMAB (see above).
After four days, the B-CLL B cells were separated from these
cultures by magnetic separation, incubated with mitomycin C (60
.mu.g/ml) for 1 h at 37.degree. C., and served then as stimulator
cells in the subsequent mixed lymphocyte culture: For this purpose,
1.times.10 4 stimulator cells were cultivated four five days with
4.times.10 4 T cells of an allogeneic donor (=responder cells), and
the proliferation of the T cells was measured by the incorporation
of 3H thymidine during the last 16 h (see FIG. 6). FIG. 4 shows
that not-SuperMAB-pretreated B-CLL B cells (processes medium/ex
vivo) can only cause a very weak proliferation of the indicator
cells, which could substantially, i.e. by at least the factor 2, be
increased by the addition of SuperMAB (process SuperMAB). By the
addition of blocking monoclonal antibodies with specificity for
B7.1 and B7.2 (5 .mu.g/ml each; process SuperMAB+anti CD80/86), the
proliferation could be reduced nearly to the level of the cells
pretreated in the medium. As a summary can be said that these
results make obvious that SuperMAB improves the antigen-preventing
function of the B-CLL B cells by induction of the costimulatory
proteins B7.1 and B7.2.
Example 5
SuperMAB Improves the Antigen-Presenting Function of B-CLL B Cells
(Autologous Mixed Lymphocyte Culture)
[0048] The aim of the immune therapy for B-CLL patients is to
activate autologous (i.e. originating within the body itself)
tumor-specific T cells by improving the antigen-presenting function
of the B-CLL B cells and to thus permit them to eliminate tumor
cells (i.e. B-CLL B cells). By means of the so-called autologous
mixed lymphocyte culture, the question was investigated whether
SuperMAB has such properties. For this purpose, again PBMC's of a
B-CLL patient were isolated and cultivated in medium or in presence
of SuperMAB. The FACS analysis showed in this experiment, too, an
induction of B7.1 and in particular B7.2 in the SuperMAB-treated
B-CLL B cells. After three days the B-CLL B cells were isolated
from these cultures by magnetic separation, treated with mitomycin
C and then used as antigen-presenting stimulator cells in the
following autologous mixed lymphocyte culture: 1.times.10 4
stimulator cells were cultivated with 1.times.10 5 autologous T
cells (=responder cells) in the presence of the cytokine
Interleukin 2, in order to answer the following questions (FIG.
7).
[0049] Are B-CLL B cells from the SuperMAB process capable to
activate autologous T cells and drive them to proliferation? The
test system was here the 3H thymidine incorporation. Result:
compared to the medium/ex vivo processes, the proliferation of the
autologous T cells could be increased by a factor of 4, because of
the pretreatment with SuperMAB (FIG. 5A).
[0050] Can the thus activated T cells produce the cytokine
interferon-gamma? Interferon-gamma is produced, among other
sources, by so-called effector T cells. To the effector T cells
belong the cytotoxic T lymphocytes, which are capable to eliminate
tumor cells. The concentration of interferon-gamma in the cell
culture supernatant was determined by means of ELISA. Result:
whereas in the processes medium/ex vivo practically no
interferon-gamma was produced, the responder T cells from the
SuperMAB process could obviously be excited to the synthesis of
this cytokine (FIG. 5B).
[0051] Are the activated T cells capable to eliminate autologous
tumor cells? For answering this question, 3.times.10 4 responder T
cells (=effector T cells in question) were isolated on day 8 of the
mixed lymphocyte culture and cultivated for 3 hours with
1.5.times.10 4 freshly isolated B-CLL B cells (=target cells). Then
the incubation with the protein annexin V and a monoclonal antibody
with specificity for CD19 took place. By annexin V, to which a
fluorescence dye is coupled, apoptotic cells (these are cells,
which have died because of a programmed cell death) can be analyzed
by flow cytometry. An induction by treatment with SuperMAB of
potent tumor-specific effector T cells and an increased death of
tumor cells resulting therefrom should therefore be accompanied by
an increase of the percentage of annexin V-positive cells in the
FACS analysis. Result: in this test, too, the SuperMAB process
showed the best result: in the processes medium/ex vivo, about 12%
of the target cells (=CD19+ tumor cells) could be stained with
annexin V, in the SuperMAB process however more than 25% (FIG.
5C).
[0052] Obviously, by the use of SuperMAB, it is possible to
activate tumor-specific autologous T cells and to induce an
efficient elimination of the tumor cells. Presumably, the reason
for this is an induction of costimulatory proteins on the surface
of the antigen-presenting B-CLL B cells. The possible mechanism is
diagrammatically shown in FIG. 8.
Example 6
Production of a Monoclonal Antibody TGN1412 According to the
Invention
[0053] In the following, the production of a monoclonal antibody
TGN1412 according to the invention is described, which consists of
the amino acid sequences of the light and heavy chains cited in
FIGS. 9a and 9b.
[0054] The DNA molecules cited in FIGS. 9c and 9d were synthesized
in a way usual in this technology. The obtained cDNA's,
respectively coding for the heavy and the light chain of the
monoclonal antibody as indicated, were inserted into plasmids pLNOH
and pLNOK. With regard to the method in detail, the plasmids and
the interfaces, reference is made to the document Norderhaug et
al., Journal of Immunological Methods, 204:77-87 (1997).
[0055] With the respective plasmids, E. coli cells were
transformed. The transformed E. coli cells were multiplied, and the
respective plasmids were obtained therefrom to a larger scale.
[0056] With the obtained respective plasmids, comprising the DNA
information for the complete heavy or light chain, COST cells were
transiently cotransfected, and CHO and NSO cells were stably
cotransfected. The cotransfection was made in a usual way with
Lipofectamine (Invitrogen) or Effectene (QIAGEN) according to the
respective manufacturer's instructions.
[0057] The obtained transfected cells were cultivated in standard
culture medium in CO2 incubators (5% CO2) at 37.degree. C. in T
flasks and roller bottles. Stably transfected cells, which express
the intact antibody, were selected by means of G418 and hygromycin
B selection. Stable transformants were then subcloned and further
cultivated.
[0058] Cell culture supernatants with secreted antibodies were
analyzed by means of activity assays, binding assays (FACS
analysis), SDS-PAGE and Western blotting.
[0059] For purification, cell culture supernatants were guided over
a protein A or protein G column, in order to specifically bind the
desired antibodies. Bound antibodies were eluted with acidic buffer
(0.1 M glycin, pH 3.0-3.4) and then neutralized with 1 M Tris (pH
8.0). The thus purified antibodies were dialyzed against PBS buffer
and concentrated up according to requirements by means of membrane
filtration, if applicable. The obtained antibodies were then used
for proliferation assays, activity assays, binding assays (FACS
analysis for binding to CD28-positive cells), ELISA, SDS-PAGE and
Western blotting.
Sequence CWU 1
1
2816PRTArtificial SequenceHuman CD28 partial sequence 1Val Tyr Ser
Lys Thr Gly 1 5 24PRTArtificial SequenceHuman CD28 partial sequence
2Tyr Ser Lys Thr 1 35PRTArtificial SequenceHuman CD28 partial
sequence 3Tyr Ser Lys Thr Gly 1 5 45PRTArtificial SequenceHuman
CD28 partial sequence 4Val Tyr Ser Lys Thr 1 5 56PRTArtificial
SequenceHuman CD28 partial sequence 5Tyr Ser Lys Thr Gly Phe 1 5
67PRTArtificial SequenceHuman CD28 partial sequence 6Val Tyr Ser
Lys Thr Gly Phe 1 5 75PRTArtificial SequenceHuman CD28 partial
sequence 7Ser Lys Thr Gly Phe 1 5 88PRTArtificial SequenceHuman
CD28 partial sequence 8Gly Asn Tyr Ser Gln Gln Leu Gln 1 5
9321DNAArtificial Sequencemab 9D7 partial sequence 9gatatccaga
cgacacagac tacatcctcc cgttctgcct ctctgggaga cagagtcacc 60atcagttgca
gggcaggtca ggacattagt aattatttaa actggtatca gcagaaacca
120gatggaactg ttaagctcct gatctactac acatcaagat tacactcagg
agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca
ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag
ggtcatacgc ttccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa a
32110107PRTArtificial Sequencemab 5.11A partial sequence 10Asp Ile
Gln Thr Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Gly Gln Asp Ile Ser Asn Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln
Gln Gly His Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 11363DNAArtificial Sequencemab 9D7 partial
sequence 11gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc
tctgtccctc 60acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg
gatccggcag 120tttccaggaa acaaactgga gtggatgggc tacataagat
acagtggtag tactagctac 180aatccatctc tcaaaagtcg aatctctatc
actcgagaca catccaagaa ccagttcttc 240ctgcagttga attctgtgac
tactgaggac acagccacat attactgtgc aagagattgg 300ccgcgaccga
gctactggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360tca
36312121PRTArtificial Sequencemab 5.11A partial sequence 12Asp Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20
25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu
Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Ser Gly Ser Thr Ser Tyr Asn
Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser
Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Pro Arg Pro
Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val
Thr Val Ser Ser 115 120 13360DNAArtificial Sequencemab 9D7 partial
sequence 13caggtccaac tgcagcagtc cggacctgag ctggtgaagc cggggacttc
agtgaggatt 60tcctgcgagg cttctggcta caccttcaca agctactata tacactgggt
gaaacagagg 120cctggacagg gacttgagtg gattggatgt atttatcctg
gaaatgtcaa tactaactat 180aatgagaagt tcaaggacaa ggccacactg
attgtagaca catcctccaa cactgcctac 240atgcagctca gcagaatgac
ctctgaggac tctgcggtct atttctgtac aagatcacac 300tacggcctcg
actggaactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca
36014120PRTArtificial Sequencemab 5.11A partial sequence 14Gln Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15
Ser Val Arg Ile Ser Cys Glu Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn
Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Ile Val Asp Thr Ser
Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Arg Met Thr Ser Glu
Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser His Tyr Gly Leu
Asp Trp Asn Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser 115 120 15321DNAArtificial Sequencemab 9D7 partial
sequence 15gacatccaga tgaaccagtc tccatccagt ctgtctgcat cccttggaga
cacaattacc 60atcacttgcc atgccagtca aaacatttat gtttggttaa actggtacca
gcagaaacca 120ggaaatattc ctaaactctt gatctataag gcttccaacc
tgcacacagg cgtcccatca 180aggtttagtg gcagtggatc tggaacaggc
ttcacattaa ccatcagcag cctgcagcct 240gaagacattg ccacttacta
ctgtcaacag ggtcaaactt atccgtacac gttcggaggg 300gggaccaagc
tggaaataaa a 32116107PRTArtificial Sequencemab 5.11A partial
sequence 16Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn
Ile Tyr Val Trp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Asn
Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 1715PRTArtificial
Sequencehuman CD28 partial sequence 17Gly Asn Tyr Ser Gln Gln Leu
Gln Val Tyr Ser Lys Thr Gly Phe 1 5 10 15 18107PRTArtificial
Sequencemab 9D7 partial sequence 18Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asn Ile Tyr Val Trp 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys
Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr
Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 19120PRTArtificial Sequencemab 5.11A partial sequence 19Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn
Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp
Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser His Tyr Gly Leu
Asp Trp Asn Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser 115 120 2019PRTArtificial Sequencemab TGN1412 leader
peptide 20Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly 1 5 10 15 Val His Ser 21107PRTArtificial Sequencemab
TGN1412 constant region light chain 21Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55
60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 22327PRTArtificial Sequencemab TGN 1412 constant region heavy
chain variant 1 22Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100
105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225
230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu
Ser Leu Gly Lys 325 23328PRTArtificial Sequencemab TGN1412 constant
region heavy chain variant 2 23Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys
Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195
200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315
320 Leu Ser Leu Ser Leu Gly Lys Lys 325 24326PRTArtificial
Sequencemab TGN1412 constant region heavy chain variant 3 24Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly 325
25107PRTArtificial Sequencemab TGN1412 varianble region light chain
25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val
Trp 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Gly Gln Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 26120PRTArtificial Sequencemab TGN1412
variable region heavy chain 26Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile
Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys
Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe
Cys 85 90 95 Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val
Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120
271721DNAArtificial Sequencemab TGN1412 light chain 27ggtaccgggc
cgacctcacc atgggatgga gctgtatcat cctcttcttg gtagcaacag 60ctacaggtaa
ggggctcaca gtagcaggct tgaggtctgg acatatatat gggtgacaat
120gacatccact ttgcctttct ctccacaggt gtgcattccg acatccagat
gacccagtct 180ccatcctccc tgtctgcatc tgtaggagac agagtcacca
tcacttgcca tgccagtcaa 240aacatttatg tttggttaaa ctggtatcag
cagaaaccag ggaaagcccc taagctcctg 300atctataagg cttccaacct
gcacacaggg gtcccatcaa ggttcagtgg cagtggatct 360gggacagatt
tcactctcac catcagcagt ctgcaacctg aagattttgc aacttactac
420tgtcaacagg gtcaaactta tccgtacacg ttcggcggag ggaccaaggt
ggagatcaaa 480cgtgagtcgt acgctagcaa gcttgatatc gaattctaaa
ctctgagggg gtcggatgac 540gtggccattc tttgcctaaa gcattgagtt
tactgcaagg tcagaaaagc atgcaaagcc 600ctcagaatgg ctgcaaagag
ctccaacaaa acaatttaga actttattaa ggaatagggg 660gaagctagga
agaaactcaa aacatcaaga ttttaaatac gcttcttggt ctccttgcta
720taattatctg ggataagcat gctgttttct gtctgtccct aacatgccct
gtgattatcc 780gcaaacaaca cacccaaggg cagaavtttg ttacttaaac
accatcctgt ttgcttcttt 840cctcaggaac tgtggctgca ccatctgtat
tcatcttccc gccatctgat gagcagttga 900aatctggaac tgcctctgtt
gtgtgcctgc tgaataactt ctatcccaga gaggccaaag 960tacagtggaa
ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc
1020aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc
aaagcagact 1080acgagaaaca caaagtctac gcctgcgaag tcacccatca
gggcctgagc tcgcccgtca 1140caaagagctt caacagggga gagtgttaga
gggagaagtg cccccacctg ctcctcagtt 1200ccagcctgac cccctcccat
cctttggcct ctgacccttt ttccacaggg gacctacccc 1260tattgcggtc
ctccagctca tctttcacct cacccccctc ctcctccttg gctttaatta
1320tgctaatgtt ggaggagaat gaataaataa agtgaatctt tgcacctgtg
gtttctctct 1380ttcctcattt aataattatt atctgttgtt ttaccaacta
ctcaatttct cttataaggg 1440actaaatatg tagtcatcat aaggcgcata
accatttata aaaatcatcc ttcattctat 1500tttaccctat catcctctgc
aagacagtcc tccctcaaac ccacaagcct tctgtcctca 1560cagtcccctg
ggccatggta ggagagactt gcttccttgt tttccccccc tcagcaagcc
1620ctcatagtcc tttttaaggg tgacaggtct tacagtcata tatcctttga
ttcaattccc 1680tgagaatcaa ccaaagcaaa ttcctgcagc ccgggggatc c
1721282551DNAArtificial Sequencemab TGN1412 heavy chain
28ggtaccgggc cgacctcacc atgggatgga gctgtatcat cctcttcttg gtagcaacag
60ctacaggtaa ggggctcaca gtagcaggct tgaggtctgg acatatatat gggtgacaat
120gacatccact ttgcctttct ctccacaggt gtgcattccg aggtgcagct
ggtgcagtct 180ggggctgagg tgaagaagcc tggggcctca gtgaaggtct
cctgcaaggc ttctggatac 240accttcacca gctactatat acactgggtg
cgacaggccc ctggacaagg gcttgagtgg 300attggatgta tttatcctgg
aaatgtcaat actaactata atgagaagtt caaggacagg 360gccaccctga
ccgtagacac gtccatcagc acagcctaca tggagctgag caggctgaga
420tctgacgaca cggccgtgta tttctgtaca agatcacact acggcctcga
ctggaacttc 480gatgtctggg gccaagggac cacggtcacc gtctcctcag
gtgagtcgta cgctagcaag 540ctttctgggg caggccgggc ctgactttgg
ctgggggcag ggagggggct aaggtgacgc 600aggtggcgcc agccaggtgc
acacccaatg cccatgagcc cagacactgg accctgcatg 660gaccatcgcg
gatagacaag aaccgagggg cctctgcgcc ctgggcccag ctctgtccca
720caccgcggtc acatggcacc acctctcttg cagcttccac caagggccca
tccgtcttcc 780ccctggcgcc ctgctccagg agcacctccg agagcacagc
cgccctgggc tgcctggtca 840aggactactt ccccgaaccg gtgacggtgt
cgtggaactc aggcgccctg accagcggcg 900tgcacacctt cccggctgtc
ctacagtcct caggactcta ctccctcagc agcgtggtga 960ccgtgccctc
cagcagcttg ggcacgaaga cctacacctg caacgtagat cacaagccca
1020gcaacaccaa ggtggacaag agagttggtg agaggccagc acagggaggg
agggtgtctg 1080ctggaagcca ggctcagccc tcctgcctgg acgcaccccg
gctgtgcagc cccagcccag 1140ggcagcaagg catgccccat ctgtctcctc
acccggaggc ctctgaccac cccactcatg 1200ctcagggaga gggtcttctg
gatttttcca ccaggctccg ggcagccaca ggctggatgc 1260ccctacccca
ggccctgcgc atacaggggc aggtgctgcg ctcagacctg ccaagagcca
1320tatccgggag gaccctgccc ctgacctaag cccaccccaa aggccaaact
ctccactccc 1380tcagctcaga caccttctct cctcccagat ctgagtaact
cccaatcttc tctctgcaga 1440gtccaaatat ggtcccccat gcccatcatg
cccaggtaag ccaacccagg cctcgccctc 1500cagctcaagg cgggacaggt
gccctagagt agcctgcatc cagggacagg ccccagccgg 1560gtgctgacgc
atccacctcc atctcttcct cagcacctga gttcctgggg ggaccatcag
1620tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc
cctgaggtca 1680cgtgcgtggt ggtggacgtg agccaggaag accccgaggt
ccagttcaac tggtacgtgg 1740atggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagttc aacagcacgt 1800accgtgtggt cagcgtcctc
accgtcctgc accaggactg gctgaacggc aaggagtaca 1860agtgcaaggt
ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc tccaaagcca
1920aaggtgggac ccacggggtg cgagggccac atggacagag gtcagctcgg
cccaccctct 1980gccctgggag tgaccgctgt gccaacctct gtccctacag
ggcagccccg agagccacag 2040gtgtacaccc tgcccccatc ccaggaggag
atgaccaaga accaggtcag cctgacctgc 2100ctggtcaaag gcttctaccc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg 2160gagaacaact
acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2220agcaggctaa ccgtggacaa gagcaggtgg caggagggga atgtcttctc
atgctccgtg 2280atgcatgagg ctctgcacaa ccactacaca cagaagagcc
tctccctgtc tctgggtaaa 2340tgagtgccag ggccggcaag cccccgctcc
ccgggctctc ggggtcgcgc gaggatgctt 2400ggcacgtacc ccgtctacat
acttcccagg cacccagcat ggaaataaag cacccaccac 2460tgccctgggc
ccctgtgaga ctgtgatggt tctttccacg ggtcaggccg agtctgaggc
2520ctgagtgaca tgagggaggc agagcggatc c 2551
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