U.S. patent application number 12/884563 was filed with the patent office on 2011-07-07 for anti cd37 antibodies.
This patent application is currently assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH. Invention is credited to Eric BORGES, Karl-Heinz HEIDER, Elinborg OSTERMANN.
Application Number | 20110165153 12/884563 |
Document ID | / |
Family ID | 38925567 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110165153 |
Kind Code |
A1 |
HEIDER; Karl-Heinz ; et
al. |
July 7, 2011 |
ANTI CD37 ANTIBODIES
Abstract
Chimeric and humanized anti-CD37 antibodies and pharmaceutical
compositions containing them are useful for the treatment of B cell
malignancies and autoimmune and inflammatory diseases that involve
B cells in their pathology.
Inventors: |
HEIDER; Karl-Heinz;
(Ingelheim am Rhein, DE) ; BORGES; Eric; (Maria
Enzersdorf, AT) ; OSTERMANN; Elinborg; (Vienna,
AT) |
Assignee: |
BOEHRINGER INGELHEIM INTERNATIONAL
GMBH
INGELHEIM AM RHEIN
DE
|
Family ID: |
38925567 |
Appl. No.: |
12/884563 |
Filed: |
September 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12672378 |
Apr 14, 2010 |
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PCT/EP2008/060464 |
Aug 8, 2008 |
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12884563 |
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Current U.S.
Class: |
424/133.1 ;
435/243; 435/320.1; 435/328; 435/375; 435/419; 435/69.6; 530/387.3;
530/387.7; 536/23.53 |
Current CPC
Class: |
A01K 2227/105 20130101;
A61K 45/06 20130101; C07K 2317/73 20130101; C07K 16/3061 20130101;
A01K 2207/15 20130101; C07K 2317/72 20130101; A01K 67/0275
20130101; A61K 2039/505 20130101; A61P 37/02 20180101; A61P 43/00
20180101; A61P 29/00 20180101; C07K 2317/92 20130101; A61P 35/02
20180101; C07K 16/28 20130101; A01K 2217/072 20130101; C07K 16/2896
20130101; C07K 2317/76 20130101; A61P 35/00 20180101; A61P 37/00
20180101; C07K 2317/24 20130101; C07K 2317/732 20130101; A61K
39/39558 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 530/387.7; 536/23.53; 435/320.1; 435/419; 435/243;
435/328; 435/69.6; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/32 20060101 C07K016/32; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12N 1/00 20060101 C12N001/00; C12P 21/02 20060101
C12P021/02; C12N 5/0781 20100101 C12N005/0781; A61P 35/00 20060101
A61P035/00; A61P 35/02 20060101 A61P035/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2007 |
EP |
07114128.7 |
Claims
1) A humanized antibody molecule that binds to human CD37 and that
is further defined by a) CDRs contained within the variable heavy
chain as shown in SEQ ID NO:2, b) CDRs contained within the
variable light chain as shown in SEQ ID NO:4, c) frameworks
supporting said CDRs that are derived from a human antibody, and d)
constant heavy and light chains that are from a human antibody.
2) The antibody molecule of claim 1 further defined by i) a
variable heavy chain comprising the amino acid sequence shown in
SEQ ID NO: 6; and ii) a variable light chain comprising the amino
acid sequence shown in SEQ ID NO: 12.
3) The antibody of claim 2, wherein i) the constant heavy chain is
a IgG1 chain, and ii) the constant light chain is a kappa
chain.
4) The antibody of claim 3, wherein said constant heavy chain i)
comprises the amino acid sequence shown in SEQ ID NO:24 and wherein
said constant light chain ii) comprises the amino acid sequence
shown in SEQ ID NO:26.
5-11. (canceled)
12) The antibody of claim 1, wherein said antibody has one or more
mutations in the Fc domain that modulate one or more effector
functions.
13) The antibody of claim 12, wherein said modulation of effector
function is an increase in antibody-dependent cell-mediated
cytotoxicity.
14) The antibody of claim 12 wherein said one or more mutations in
the Fc domain is a single substitution at position 332, numbered
according to the Kabat EU numbering index.
15) The antibody of claim 12, wherein said one or more mutations in
the Fc domain is a combination of substitutions at positions 239
and 332, numbered according to the Kabat EU numbering index.
16) The antibody of claim 12, wherein said one or more mutations in
the Fc domain is a combination of substitutions at positions 236
and 332, numbered according to the Kabat EU numbering index.
17) The antibody of claim 12, wherein said one or more mutations in
the Fc domain is a combination of substitutions at positions 236,
239 and 332, numbered according to the Kabat EU numbering
index.
18) The antibody of claim 14, wherein said substitutions are 1332E,
S239D and G236A.
19) The antibody of claim 15, wherein said substitutions are 1332E,
S239D and G236A.
20) The antibody of claim 16, wherein said substitutions are 1332E,
S239D and G236A.
21) An antibody that binds to human CD37 and has a heavy chain
comprising the amino acid sequence of SEQ ID NO:36.
22) The antibody of claim 21, which has a light chain comprising
the amino acid sequence of SEQ ID NO:38.
23-24. (canceled)
25) An antibody that binds to human CD37 and has a heavy chain
comprising the amino acid sequence of SEQ ID NO:40.
26) The antibody of claim 25, which has a light chain comprising
the amino acid sequence of SEQ ID NO:42.
27) A DNA molecule comprising a region encoding the variable heavy
chain of an antibody of claim 1.
28) The DNA molecule of claim 27, wherein said variable heavy chain
encoding region is fused to a region encoding a constant heavy
chain of human origin.
29) The DNA molecule of claim 28, wherein said human constant heavy
chain is IgG1.
30) The DNA molecule of claim 29, wherein said IgG1 is encoded by a
sequence shown in SEQ ID NOs:23, 35 or 39.
31) The DNA molecule of claim 28, wherein said human constant heavy
chain has one or more substitutions in the Fc region at position
332, 229 or 236, numbered according to the Kabat EU numbering
index.
32) A DNA molecule comprising a region encoding the variable light
chain of an antibody of claim 1.
33) The DNA molecule of claim 32, wherein said variable light chain
encoding region is fused to a region encoding a constant light
chain of human origin.
34) The DNA molecule of claim 33, wherein said constant light chain
is a kappa chain.
35) The DNA molecule of claim 34, wherein said kappa light chain is
encoded by a sequence shown in SEQ ID NOs:25, 37 or 41.
36) An expression vector comprising a DNA molecule comprising a
region encoding the variable heavy chain of claim 1 and/or a DNA
molecule comprising a region encoding the variable light chain of
an antibody of claim 1.
37) A host cell carrying one or more vectors of claim 36.
38) A host cell carrying an expression vector comprising a DNA
molecule comprising a region encoding the variable heavy chain of
an antibody of claim 1, and a second expression vector comprising a
DNA molecule comprising a region encoding the variable light chain
of an antibody of claim 1.
39) (canceled)
40) A method for producing an antibody comprising transfecting a
mammalian host cell with one or more vectors of claim 36, culturing
the host cell and recovering and purifying the antibody
molecule.
41) A pharmaceutical composition comprising, as the active
ingredient, one or more anti-CD37 antibody molecules of claim 1,
and a pharmaceutically acceptable carrier.
42) The pharmaceutical composition of claim 41, further comprising
one or more additional therapeutic agents.
43) The pharmaceutical composition of claim 42, wherein said one or
more additional therapeutic agents are selected from agents that
target a B cell antigen wherein said B cell antigen is CD20.
44-48. (canceled)
49) The pharmaceutical composition of claim 41 for the treatment of
B cell malignancies, wherein said B cell malignancy is selected
from B cell non-Hodgkins lymphoma, B cell chronic lymphocytic
leukemia and multiple myeloma.
50) (canceled)
51) A method of depleting CD37 expressing B cells from a population
of cells, comprising administering to said population of cells an
antibody molecule of claim 1 or a pharmaceutical composition
containing such antibody molecule.
52) (canceled)
53) A method for treating a patient suffering from a B cell
malignancy selected from B cell non-Hodgkins lymphoma, B cell
chronic lymphocytic leukemia and multiple myeloma, comprising
administering to said patient an effective amount of a
pharmaceutical composition of claim 41.
54) A DNA molecule comprising a region encoding the variable heavy
chain of an antibody of claim 21.
55) The DNA molecule of claim 54, wherein said human constant heavy
chain has one or more substitutions in the Fc region at position
332, 229 or 236, numbered according to the Kabat EU numbering
index.
56) A DNA molecule comprising a region encoding the variable light
chain of an antibody of claim 22.
57) An expression vector comprising a DNA molecule comprising a
region encoding the variable heavy chain of claim 21.
58) An expression vector comprising a DNA molecule comprising a
region encoding the variable heavy chain of claim 22 and the DNA
molecule comprising a region encoding the variable light chain of
an antibody of claim 22.
59) A host cell carrying one or more vectors of claim 57.
60) A pharmaceutical composition comprising, as the active
ingredient, the anti-CD37 antibody molecule of claim 22.
61) A method of depleting CD37 expressing B cells from a population
of cells, comprising administering to said population of cells an
antibody molecule of claim 22 or a pharmaceutical composition
containing such antibody molecule.
Description
INTRODUCTION
[0001] The present invention relates to immunotherapies that are
based on B cell depletion. In particular, the present invention
relates to anti-CD37 antibody molecules for use in such therapies,
e.g. in the treatment of B cell malignancies and autoimmune
conditions.
[0002] Immunotherapy using monoclonal antibodies (mAbs) has been
emerging as a safe and selective method for the treatment of cancer
and other diseases. In particular, the role of monoclonal
antibodies in therapies that are based on B cell depletion, e.g. in
the treatment of B cell malignancies, has expanded since the
introduction of rituximab (Rituxan.RTM.), an antibody that is
directed against the CD20 antigen on the B cell surface. Numerous
studies have confirmed the efficacy of rituximab as a single agent
and in combination therapy in low-grade NHL (Hiddemann et al.,
2005a; Hiddemann et al., 2005b; Hainsworth 2004; McLaughlin et al.,
1998), mantle cell lymphoma (Forstpointner et al., 2004; Kahl et
al., 2006; Foran et al., 2000; Howard et al., 2002; Romaguera et
al., 2005), diffuse large cell lymphoma (DLCL) (Coiffier et al.,
1998; Feugier et al., 2005), and Burkitt leukemia/lymphoma (Thomas
et al., 2006). However, only a subset of patients respond to
therapy and the majority of those eventually relapse following
rituximab treatment. Therefore, new therapeutic targets on B cells
have been sought that are potentially more effective than CD20 for
therapy of B cell malignancies (Zhao et al., 2007). The CD37
antigen is a cell surface antigen that has, to date, not been
considered as a target for B cell malignancies to the same extent
as the B cell antigen CD20.
[0003] CD37, a member of the tetraspanin superfamily, is a heavily
glycosylated cell surface molecule with four transmembrane domains
and two extracellular loops. CD37 is almost exclusively expressed
on mature B cells, with highest expression levels on peripheral
blood B cells, reduced levels on plasma cells and non-detectable
levels on CD10+ precursor B cells in the bone marrow. Low level
expression of CD37 has also been reported on resting and activated
T cells, granulocytes, and monocytes. In B cell neoplasm, CD37
expression is mainly observed in aggressive non-Hodgkin's lymphoma
(NHL) and chronic lymphoid leukemia (CLL). High level of CD37
expression is also found on mantle cell lymphoma (MCL). This
expression pattern makes CD37 an attractive target for
antibody-mediated cancer therapy.
[0004] CD37 was first described in 1986 and characterized by the
murine monoclonal antibody MB-1 (Link et al., 1986).
[0005] The physiological role of CD37 is unknown. Mice deficient
for CD37 display no changes in development and cellular composition
of lymphoid organs, but have reduced levels of IgG1 and attenuated
T cell mediated immune reactions (Knobeloch et al., 2000). Studies
with CD37.sup.-/- T cells suggest a role for CD37 in T cell
proliferation (van Spriel et al., 2004).
[0006] CD37 expression on malignant B cells of various diseases has
been reported. CD37 is expressed in the majority of mature B cell
malignancies like Burkitt lymphoma, follicular lymphoma and
lymphocytic lymphoma (Link et al., 1986). High levels of CD37
expression have been observed in hairy cell leukemia and in samples
of patients with chronic lymphocytic leukemia (CLL) and different
subtypes of non-Hodgkin's lymphoma (NHL) including mantle cell
lymphoma (MCL) (Schwartz-Albiez et al., 1988; Barrena et al.,
2005). One report utilizing antibody micro array for
immunophenotyping claims CD37 to be a good discriminator between
malignant CLL cells (high CD37 expression) versus normal peripheral
blood (PB) lymphocytes (low CD37 expression) (Belov et al.,
2001).
[0007] Binding of a CD37-specific mAb to cancer cells may trigger
various mechanisms of action: First, after the antibody binds to
the extracellular domain of the CD37 antigen, it may activate the
complement cascade and lyse the targeted cell.
[0008] Second, an anti-CD37 antibody may mediate antibody-dependent
cell-mediated cytotoxicity (ADCC) to the target cell, which occurs
after the Fc portion of the bound antibody is recognized by
appropriate receptors on cytotoxic cells of the immune system.
[0009] Third, the antibody may alter the ability of B cells to
respond to antigen or other stimuli. Finally, anti-CD37 antibody
may initiate programmed cell death (apoptosis).
[0010] Anti-CD37 mAb MB-1 was evaluated in two radio-immunotherapy
trials in B-NHL patients (B-cell non-Hodgkin's lymphoma; Press et
al., 1989; Kaminski et al., 1992). Therapeutic doses of
.sup.131I-MB-1 were administered to 6 relapsed NHL patients in one
trial and all 6 patients achieved a clinical complete remission
(CR) with a median duration of 7 months. Of note, two of the six
patients showed clinical regressions already after administration
of only the tracer dose of MB-1 suggesting a direct anti-tumor
effect of the antibody itself. In the second trial radio-labeled
MB-1 was applied for the treatment of refractory NHL patients and
resulted in 3 out of 9 evaluable patients with objective responses
of limited duration (Kaminski et al., 1992). In both trials a rapid
and transient depletion of peripheral B cells after injection of
the trace labeled MB-1 antibody dose was reported. These
observations support the conclusion that MB-1 exerts a cytotoxic
activity on its own. In summary, these clinical trials underscore
the feasibility of CD37-targeting for B-cell malignancies and point
to a potential clinical relevance of anti-CD37 therapy.
[0011] There is experimental evidence with a CD37 specific
antibody-like single chain molecule ("Small Modular
ImmunoPharmaceutical", SMIP) that treatment with that molecule
induces apoptosis in vitro and delays Burkitt lymphoma growth in a
xenograft model in vivo. Anti-apoptotic activity of the recombinant
anti-CD37 SMIP Tru16.4 from Trubion was described recently (Zhao et
al., 2004). Tru 16.4 induced caspase-independent apoptosis on
primary CLL cells from tumor patients. Induction of apoptosis on
these cells was greater than that of Rituximab and comparable to
that of Alemtuzumab, a CD52 antagonist. The degree of apoptosis
induction was directly proportionate to CD37 cell surface
expression and could be further enhanced by cross-linking with an
anti-human IgG antibody. A correlation of CD37 expression and ADCC
was demonstrated on cell lines in vitro. In a Burkitt lymphoma
mouse model (Raji) treatment with anti-CD37 scFv revealed
therapeutic efficacy (Zhao et al., 2007). These data provide first
evidence that CD37-targeting is a promising approach for targeted
anti-tumor therapy by induction of apoptosis and ADCC.
[0012] In conclusion, it has been shown that the CD37 antigen is
frequently expressed on tumor cells in several human B cell
malignancies and on mature normal
[0013] B lymphocytes and that anti-CD37-based therapy may be a
promising approach for treating B cell malignancies. The depletion
of CD37-positive normal B cells is not considered critical since
clinical data from numerous patients show that even prolonged
depletion of B cells up to 6 months with an anti-CD20 mAb does not
significantly reduce IgG serum levels or increases the risk of
infections (Van der Kolk et al., 2002).
[0014] Although the anti-CD37 antibodies or antibody-like molecules
described above (MB-1 and SMIP Tru16.4) have shown anti-tumor
efficacy in B-cell malignancies and the potential to target CD37,
there is a need for alternate anti-CD37 inhibitors to improve
therapies based on B-cell depletion.
SUMMARY OF INVENTION
[0015] It was an object of the invention to provide novel CD37
antagonists for the treatment of B cell malignancies and other
disorders which respond to the depletion of CD37 positive B
cells.
[0016] Furthermore, it was an object of the invention to provide
anti-CD37 antibodies with improved effector functions. In
particular, the inventors sought to provide anti-CD37 mABs with
antibody-dependent cell-mediated cytotoxicity (ADCC).
[0017] To solve the problem underlying the invention, a murine
monoclonal anti-CD37 antibody was used as a starting antibody for
generating chimeric and humanized anti-CD37 antibodies that are
useful in human therapy.
[0018] In a first aspect, the present invention provides an
antibody molecule that binds to human CD37 and that is derived from
[0019] a) a murine monoclonal antibody that is defined by [0020] i.
a variable heavy chain comprising the amino acid sequence shown in
SEQ ID NO: 2; and [0021] ii. a variable light chain comprising the
amino acid sequence shown in SEQ ID NO:4, or from [0022] b) a
non-human antibody recognizing the same epitope of human CD37 as
the antibody defined in a) or recognizing an epitope that is close
to or overlaps with said epitope;
[0023] wherein said antibody molecule is a chimeric or a humanized
antibody.
[0024] As will be understood from the following, an antibody that
is "derived" from another antibody, i.e. the starting antibody,
means that said antibody has been generated by modification of the
starting antibody as described below.
[0025] In a preferred embodiment, the antibody molecule is a
chimeric or humanized antibody molecule derived from the starting
antibody defined in a). An antibody with a related sequence was
designated G28.1 and described in WO 2005/017148.
[0026] The starting antibody of category b) may, for example, be
selected from the CD37-specific antibodies that characterized, like
G28.1, the CD37 antigen in the Third HLDA Workshop; these
antibodies were designated HD28, HH1, BI14, F97-3G6 (Ling and
MacLennan, 1987). Other CD37-specific antibodies that have been
described include RFB-7, Y29/55, MB-1, M-B371, M-B372 and IPO-24.
According to Moldenhauer, 2000, and Schwartz-Albiez et al., 1988,
all these antibodies (including G28.1) recognize the same or an
overlapping or close CD37 epitope. Schwartz-Albiez et al., 1988,
indicates that the epitope is situated in the carbohydrate moiety
of CD37. A number of the above antibodies is commercially
available, e.g. HH1 (SantaCruz), RFB-7 (Biodesign), Y29/55
(Biogenesis), M-B371 (BD Biosciences), M-B372 (SantaCruz) and
IPO-24 (AbCam).
[0027] Other CD37-specific antibodies are S-B3 (Biosys), NMN46
(Chemicon), and ICO-66 (Bioprobe). Whether an antibody recognizes
the same epitope as G28.1 can be determined by competitive binding
assays or by cross inhibition radioimmunoassays as described by
Moldenhauer et al., 1987, and Moldenhauer, 2000.
[0028] By way of example, competitive binding may be determined in
an ELISA, using plates coated with CD37 protein or CD37 peptides or
with CD37 positive cells (Cell ELISA) and measuring binding of
biotinylated antibody in the presence of a competitor candidate
antibody. In the presence of a competing antibody or
antibody-derived fragment, the binding of biotinylated G28.1 (or
another antibody known to recognize the same epitope) is reduced in
the case that the antibodies recognize a shared epitope. To
identify the G28.1 epitope peptide, fragments or short polypeptides
or recombinant proteins derived from the CD37 sequence can be
synthesized or produced and the binding of G28.1 to said
peptides/polypeptides measured in an ELISA assay. Competitive
binding can also be determined by FACS analysis, as described in
the Examples.
[0029] An antibody defined in b) may be used in an analogous manner
as G28.1 as a starting antibody for the generation of chimeric or
humanized antibody molecules.
[0030] A starting antibody of category b) may also be generated de
novo by using peptides or protein fragments containing the relevant
epitope, or DNA molecules encoding such peptides/fragments,
respectively for immunization to obtain antibodies reactive with
the same epitope as G28.1.
[0031] A starting antibody b) may also be obtained by immunization
with whole cells carrying the relevant epitope; the thus obtained
hybridoma cells are then screened for competitive binding of the
secreted antibodies.
[0032] The term "anti-CD37 antibody molecule" encompasses anti-CD37
antibodies and anti-CD37 antibody fragments as well as conjugates
with antibody molecules. Antibodies include, in the meaning of the
present invention, chimeric monoclonal and humanized monoclonal
antibodies. The term "antibody", which may interchangeably be used
with "antibody molecule", shall encompass complete immunoglobulins
(as they are produced by lymphocytes and for example present in
blood sera), monoclonal antibodies secreted by hybridoma cell
lines, polypeptides produced by recombinant expression in host
cells, which have the binding specificity of immunoglobulins or
monoclonal antibodies, and molecules which have been derived from
such antibodies by modification or further processing while
retaining their binding specificity.
[0033] In an embodiment of the invention, the anti-CD37 antibody
molecule is a chimeric antibody defined by [0034] i) a variable
heavy chain comprising the amino acid sequence shown in SEQ ID NO:
2; [0035] ii) a variable light chain comprising the amino acid
sequence shown in SEQ ID NO:4; [0036] iii) constant heavy and light
chains that are of human origin.
[0037] The construction and production of chimeric mouse/human
antibodies is well known in the art (Boulianne et al., 1984). The
variable regions of the non-human antibody are typically linked to
at least a portion (F.sub.C) of the immunoglobulin constant region
of a human immunoglobulin. Human constant region DNA sequences can
be isolated in accordance with well-known procedures from a variety
of human cells, preferably from immortalized B cells (see Kabat et
al., 1991; and WO 87/02671). The antibody molecules may contain all
or only a portion of the constant region as long as they exhibit
specific binding to the CD37 antigen and the Fc receptors. The
choice of the type and length of the constant region depends on
whether effector functions like complement fixation or
antibody-dependent cell-mediated toxicity are desired, and on the
desired pharmacological properties of the antibody molecule.
[0038] In certain embodiments, the antibody molecule of the
invention is a chimeric CD37-specific antibody that has the heavy
chain variable region of a non-human antibody defined in a) or b)
fused to the human heavy chain constant region IgG1 and the light
chain variable region of a non-human antibody defined in a) or b)
fused to the human light chain constant region kappa.
[0039] In yet another embodiment, the antibody molecule is a
chimeric CD37-specific antibody that has the heavy chain variable
region shown in SEQ ID NO:2 fused to the human heavy chain constant
region IgG1 which is an IgG1 molecule with the sequence shown in
SEQ ID NO:24 (coding DNA sequence: SEQ ID NO: 23) or a mutated IgG1
molecule derived therefrom and that has the light chain variable
region shown in SEQ ID NO:4 fused to the human light chain constant
region kappa shown in SEQ ID NO:26 (coding DNA sequence: SEQ ID NO:
25).
[0040] Other human constant regions for chimerizing a non-human
starting antibody defined in a) or b) are available to the person
skilled in the art, e.g. IgG2, IgG3, IgG4, IgA, IgE or IgM (instead
of IgG1) or lambda (instead of kappa). The constant regions may
also be chimeric, for example, a heavy chain IgG1/IgG2 or IgG1/IgG3
chimera.
[0041] In certain embodiments of the invention, the anti-CD37
antibody molecule is a humanized antibody that is defined by [0042]
i. CDRs contained within the variable heavy chain as shown in SEQ
ID NO:2 and by [0043] ii. CDRs contained within the variable light
chain as shown in SEQ ID NO:4, [0044] iii. frameworks supporting
said CDRs that are derived from a human antibody, [0045] iv.
constant heavy and light chains that are from a human antibody.
[0046] Humanized forms of non-human (e.g. murine, rat or rabbit
antbodies) antibodies are immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding molecules with subsequences of antibodies) that
contain minimal sequences derived from non-human
immunoglobulin.
[0047] Humanized antibodies include human immunoglobulins (from the
recipient antibody) in which residues from a complementarity
determining region (CDR) of the recipient antibody are replaced by
residues from a CDR of a non-human species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human
residues.
[0048] In the humanized antibodies of the invention, the sequences
encoding CDRs of a non-human starting antibody defined in a) or b)
have been grafted into the respective genes of human immunoglobulin
heavy and light chains.
[0049] "Complementarity determining regions" (CDRs) of a monoclonal
antibody are understood to be those amino acid sequences involved
in specific antigen binding according to Kabat et al., 1991, in
connection with Chothia and Lesk (1987). From the sequences of the
variable regions as shown in SEQ ID NO:2 and SEQ ID NO:4, the CDR
sequence can be routinely determined by searching the Kabat
sequence database for sequence features.
[0050] Techniques for obtaining humanized antibodies are routinely
available to the skilled person, they have been described, inter
alia, in U.S. Pat. No. 5,225,539; U.S. Pat. No. 6,548,640; and U.S.
Pat. No. 6,982,321.
[0051] Appropriate framework residues of the CDR-grafted antibody
may be reverted to murine residues to improve binding affinity. As
described above, from methods pertinent to the art, the expert
knows how to obtain the CDRs from a given non-human antibody, to
choose and obtain appropriate human immunoglobulin genes, to graft
the CDRs into these genes, to modify selected framework residues,
to express the CDR-grafted antibody in appropriate host cells, e.g.
Chinese hamster ovary (CHO) cells, and to test the resulting
recombinant antibodies for binding affinity and specificity.
[0052] To obtain a humanized antibody, the antigen binding sites,
which are formed by the CDRs of the heavy chain and CDRs of the
light chain, are excised from the DNA of cells secreting the rodent
(murine) monoclonal antibody and grafted into the DNA coding for
the framework of the human antibody.
[0053] Alternatively to CDR grafting, non-human, in particular
murine, anti-CD37 antibodies can be humanized by the so-called
"resurfacing" technology, whereby the rodent frameworks are left
unchanged with the exception of surface-exposed residues, as
described in U.S. Pat. No. 5,639,641.
[0054] In a further aspect, the invention relates to humanized
antibodies having a variable heavy chain with a sequence shown in
SEQ ID NO:6 and a variable light chain with a sequence selected
from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
[0055] In another aspect, the invention relates to humanized
antibodies having a variable heavy chain with a sequence shown in
SEQ ID NO:8 and a variable light chain with a sequence selected
from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
[0056] In another aspect, the invention relates to humanized
antibodies having a variable heavy chain with a sequence shown in
SEQ ID NO:10 and a variable light chain with a sequence selected
from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
[0057] The above defined humanized antibodies are shown in Table
1.
[0058] In certain embodiments, the humanized antibody has a human
heavy chain constant region IgG1 and a human light chain constant
region kappa. As described above for the chimeric antibodies, the
constant regions may be selected from other classes and
subclasses.
[0059] In certain embodiments, in humanized antibodies of the
invention, the human constant heavy chain IgG1 is an IgG1 molecule
with the sequence shown in SEQ ID NO:24 or a mutated IgG1 molecule
derived therefrom and the human light chain constant region kappa
has the sequence shown in SEQ ID NO:26.
[0060] Anti-CD37 antibody molecules of the invention may also be
variants of the antibodies that are defined by the amino acid
sequences shown in the sequence listing. Using routinely available
technologies, the person skilled in the art will be able to
prepare, test and utilize functional variants of the above-defined
antibodies. Examples are variant antibodies with at least one
position in a CDR and/or framework altered, variant antibodies with
single amino acid substitutions in the framework region where there
is a deviation from the germline sequence, antibodies with
conservative amino substitutions, antibodies that are encoded by
DNA molecules that hybridize, under stringent conditions, with the
DNA molecules presented in the sequence listing encoding antibody
variable chains.
[0061] Given the properties of individual amino acids, rational
substitutions can be performed to obtain antibody variants that
conserve the overall molecular structure of the starting antibody.
Amino acid substitutions, i.e., "conservative substitutions", may
be made, for instance, on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the respective amino acid. The skilled person
is familiar with commonly practiced amino acid substitutions, as
described e.g. in WO 2007/042309, and methods for obtaining thus
modified antibodies. Given the genetic code and recombinant and
synthetic DNA techniques, DNA molecules encoding variant antibodies
with one or more conservative amino acid exchanges can be routinely
designed and the respective antibodies readily obtained.
[0062] In comparison with an antibody as defined by its variable
chains shown in the sequence listing, antibody variants encompassed
by the invention have a sequence identity in the CDR regions of at
least 60%, more preferably, at least 70% or 80%, still more
preferably at least 90% and most preferably at least 95%. Preferred
antibodies also have a sequence similarity in the CDR regions of at
least 80%, more preferably 90% and most preferably 95%. Preferred
antibody variants have a sequence identity in the variable regions
of at least 60%, more preferably, at least 70% or 80%, still more
preferably at least 90% and most preferably at least 95%. Preferred
antibodies also have a sequence similarity in the variable regions
of at least 80%, more preferably 90% and most preferably 95%.
[0063] "Sequence identity" between two polypeptide sequences
indicates the percentage of amino acids that are identical between
the sequences. "Sequence similarity" indicates the percentage of
amino acids that either are identical or that represent
conservative amino acid substitutions.
[0064] A variant may also be obtained by using an antibody with a
defined sequence as shown in the sequence listing as a starting
point for optimization and diversifying one or more amino acid
residues, preferably amino acid residues in one or more CDRs, and
by screening the resulting collection of antibody variants for
variants with improved properties. Diversification of one or more
amino acid residues in CDR3 of the variable light chain, CDR3 of
the variable heavy chain, CDR1 of the variable light and/or CDR2 of
the variable heavy chain has been proven useful. Diversification
can be done by methods known in the art, e.g. the so-called TRIM
technology referred to in WO 2007/042309.
[0065] In a further embodiment, the anti-CD37 antibody molecule of
the invention is an "affinity matured" antibody.
[0066] An "affinity matured" anti-CD37 antibody is an anti-CD37
antibody derived from an antibody with the sequences shown in the
sequence listing, that has one or more alterations in one or more
CDRs which result in an improvement in the affinity for the
antigens, compared to the respective original non-matured antibody.
One of the procedures for generating such antibody mutants involves
phage display (Hawkins et al., 1992; and Lowman et al., 1991).
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino acid substitutions at each
site. The antibody mutants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed mutants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed.
[0067] Affinity matured antibodies may also be produced by methods
as described, for example, by Marks et al., 1992, (affinity
maturation by variable heavy chain (VH) and variable light chain
(VL) domain shuffling), or Barbas, et al., 1994; Shier et al.,
1995; Yelton et al., 1995; Jackson et al., 1995; and Hawkins et
al., 1992 (random mutagenesis of CDR and/or framework residues).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. In a further
embodiment, the anti-CD37 antibody molecule of the invention is a
"de-immunized" antibody.
[0068] A "de-immunized" anti-CD37 antibody is an antibody derived
from a humanized or chimeric antibody with a sequence shown in the
sequence listing, that has one or more alterations in its amino
acid sequence which result in a reduction of immunogenicity of the
antibody, compared to the respective original non-dehumanized
antibody. One of the procedures for generating such antibody
mutants involves the identification and removal of T-cell epitopes
of the antibody molecule (Baker and Jones, 2007). In a first step,
the immunogenicity of the antibody molecule can be determined by
several methods, e.g. by in vitro determination of T-cell epitopes
or in silico prediction of such epitopes, as has been described in
the literature (Jones et al., 2004; Jones et al., 2005; Reche et
al., 2004; Hertz et al., 2006). Once the critical residues for
T-cell epitope function have been identified, mutations can be made
to remove immunogenicity and retain antibody activity (Jones et
al., 2005; Tangri et al., 2005). Methods for introduction of
mutations in proteins are well-known in the art, e.g. by
overlapping PCR techniques.
[0069] Since the Fc region of an antibody interacts with a number
of Fc receptors, which results in a number of important functional
capabilities (which are referred to as "effector functions"), the
antibody is, in certain embodiments, a full length antibody or an
antibody that contains a portion of the Fc region, the latter as
long as the antibody exhibits specific binding both to the relevant
portion of the antigen and to Fc receptors. The choice of the type
and length of the constant region depends on whether effector
functions like complement fixation or antibody-dependent
cell-mediated cytotoxicity are desirable features, and on the
desired pharmacological properties of the antibody protein.
[0070] In an embodiment of the invention, the anti-CD37 antibody is
a chimeric or humanized antibody with an Fc region, or the relevant
section thereof, that has been engineered to modulate effector
functions, in particular to enhance binding of the antibody to one
or more Fc receptors, thereby enhancing the effector function ADDC.
Engineering of the Fc region mediates the antibody's effector
function in the presence of effector cells more effectively than
that of the non-Fc-engineered parent antibody. In one embodiment,
such antibody variant mediates ADCC that is greater than that
mediated by the parent antibody. (In the following, if not
otherwise stated, the term "parent" in the context of an antibody
molecule, or in the context of IgG or the Fc region, refers to the
non-engineered antibody molecule, Fc region or IgG, respectively,
from which the mutated (engineered) molecule is derived.)
[0071] A variety of modifications of the Fc region have been
suggested in the art, both in the scientific literature and in
patent documents, e.g. in EP 0307434, WO 9304173, WO 9734631, WO
9744362, WO 9805787, WO 9943713, WO 9951642, WO 9958572, WO
02060919, WO 03074679, WO 2004016750, WO 2004029207, WO 2004063351,
WO 2004074455, WO 2004035752, WO 2004099249, WO 2005077981, WO
2005092925, WO 2006019447, WO 2006031994, WO 2006047350, WO
2006053301, WO 2006088494 and WO 2007041635.
[0072] In preferred embodiments, the antibodies of the invention
are Fc variants with amino acid substitutions at positions 332
and/or 239 and/or 236. In preferred embodiments, the antibodies of
the invention have mutations in the Fc domain selected from the
group of [0073] i) a single substitution at position 332,
preferably 1332E; [0074] ii) a combination of substitutions at
positions 239 and 332, preferably S239D/I332E; [0075] iii) a
combination of substitutions at positions 236 and 332, preferably
G236A/1332E; [0076] iv) a combination of substitutions at positions
236, 239 and, 332, preferably G236A /S239D/I332E.
[0077] The above defined substitutions have, for example, been
described by Lazar et al., 2006, in WO 2004029207 and WO
2007041635.
[0078] The Fc variants in the antibodies of the present invention
are defined according to the amino acid modifications that compose
them. Thus, for example, I332E is an Fc variant with the
substitution I332E relative to the parent Fc polypeptide. Likewise,
S239D/I332E defines an Fc variant with the substitutions S239D and
I332E and S239D/I332E/G236A defines an Fc variant with the
substitutions S239D, I332E, and G236A relative to the parent Fc
polypeptide.
[0079] Numbering is according to the EU numbering scheme (Kabat et
al., 1991), which refers to the numbering of the EU antibody
(Edelman et al., 1969). The person skilled in the art will
appreciate that these conventions consist of nonsequential
numbering in specific regions of an immunoglobulin sequence,
enabling a normalized reference to conserved positions in
immunoglobulin families.
[0080] In the above defined antibodies, the substituted positions
236, 239 and 332 correspond to positions 119, 122 and 215,
respectively, of the IgG1 heavy chain depicted in SEQ ID NO:24. (In
the full-length sequences of the heavy chains of antibodies A2, A4,
B2 and B4 shown in SEQ ID NOs: 28, 32, 36 and 40, the substituted
amino acids are at positions 235, 238 and 331).
[0081] In certain embodiments, the Fc variants of the invention are
based on human IgG sequences, and thus human IgG sequences are used
as the "base" sequences against which other sequences are compared.
For the antibodies of the present invention, the engineered Fc
region is preferably IgG, in particular IgG1, but it may also be
IgG2 or variant sequences from other immunoglobulin classes such as
IgA, IgE, IgGD, IgM or chimeric versions of two or more
immunoglobulin classes (e.g. IgG2/IgG1) and the like. Although the
Fc variants of the present invention are engineered in the context
of one parent IgG, the variants may be engineered in or
"transferred" to the context of another, second parent IgG. This is
done by determining the "equivalent" or "corresponding" residues
and substitutions between the first and second IgG, typically based
on sequence or structural homology between the sequences of the
first and second IgGs. In order to establish homology, the amino
acid sequence of a first IgG outlined herein is directly compared
to the sequence of a second IgG. After aligning the sequences,
using one or more of the homology alignment programs known in the
art, allowing for necessary insertions and deletions in order to
maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of the
first Fc variant are defined. Regardless of how equivalent or
corresponding residues are determined, and regardless of the
identity of the parent IgG in which the IgGs are made, what is
meant to be conveyed is that the Fc variants used in the present
invention may be engineered into any second parent IgG that has
significant sequence or structural homology with the Fc variant.
Thus for example, if a variant antibody is generated wherein the
parent antibody is human IgG1, by using the methods described above
or other methods for determining equivalent residues, the variant
antibody may be engineered, for example, in a human 1gG2 parent
antibody, a human IgA parent antibody (see WO 2007041635).
[0082] The antibodies of the invention target the antigen CD37,
which may be advantageous over targeting CD20 in diseases in which
the level of CD37 expression is higher than that of CD20, as e.g.
in chronic lymphocytic leukemia, where samples have shown high
levels of CD37 mRNA expression compared to low level expression of
CD20 mRNA.
[0083] It has been shown that antibodies of the invention are
superior to rituximab, a registered anti-CD20 antibody, with
respect to ADCC activity on Ramos cells, normal B cell depletion in
whole blood and Ramos Burkitt's lymphoma cell depletion. As could
be shown in the experiments of the invention, the antibodies of the
invention (both the non-Fc engineered and the Fc-engineered ones)
have a B cell depleting activity that is superior to that of
rituximab. The antibodies with the mutated Fc region show a ca. 10
fold increase of B cell depletion activity as compared to rituximab
(FIG. 11B).
[0084] Representatives of CD37 antibodies of the invention show
potent pro-apoptotic activity without cross-linking; in this
respect, antibodies with this property are superior to the
anti-CD37 SMIP Tru16.4, which does not show apoptosis without
cross-linking (Zhao et al., 2007). Induction of apoptosis without
cross-linking, which could be shown for antibodies of the invention
both with and without Fc engineering, is advantageous in the
absence of a cross-linking agent in vivo (e.g. effector cells
harboring Fc.gamma. receptors) or at low density of the target
antigen CD37 (e.g. tumor cells with low level expression of CD37).
An antibody which induces apoptosis without cross-linking may still
cause cell death, whereas an antibody dependent on cross-linking
does not.
[0085] In a further aspect, an anti-CD37 antibody molecule of the
invention is an antibody fragment that is derived from a humanized
or chimeric CD37 specific antibody according to the present
invention. To obtain antibody fragments, e.g. Fab fragments,
digestion can be accomplished by means of routine techniques, e.g.
using papain. Examples of papain digestion are described in WO
94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of
antibodies typically produces two identical antigen binding
fragments, so-called Fab fragments, each with a single antigen
binding site, and a residual Fc fragment. Pepsin treatment yields
an F(ab').sub.2 fragment that has two antigen combining sites and
is still capable of cross-linking the antigen.
[0086] The Fab fragments obtained by digestion of the antibody also
contain the constant domains of the light chain and the first
constant domain (CH.sub.1) of the heavy chain. Fab' fragments
differ from Fab fragments in that they contain additional residues
at the carboxy terminus of the heavy chain CH.sub.1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Antibody
fragments can also be generated by molecular biology methods
producing the respective coding DNA fragments.
[0087] The antibody molecule will typically be a tetramer
consisting of two light chain/heavy chain pairs, but may also be
dimeric, i.e. consist of a light chain/heavy chain pair, e.g. a Fab
or Fv fragment, or it may be a monomeric single chain antibody
(scFv; Johnson and Bird, 1991), a minibody, or a diabody.
[0088] The anti-CD37 antibody molecule may also be in the form of a
conjugate, i.e. an antibody molecule that is chemically coupled to
a cytotoxic agent, particularly a cytotoxic agent that induces
cytotoxicity (e.g. apoptosis or mitotic arrest) of tumor cells. As
a result of normal pharmacologic clearance mechanisms, an antibody
employed in a drug conjugate (an "immunoconjugate") contacts and
binds to target cells only in limited amounts. Therefore, the
cytotoxic agent employed in the conjugate must be highly cytotoxic
such that sufficient cell killing occurs to elicit a therapeutic
effect. As described in US 2004/0241174, examples of such cytotoxic
agents include taxanes (see, e.g. WO 01/38318 and WO 03/097625),
DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines,
tubulysin analogs, duocarmycin analogs, doxorubicin, auristatin E,
ricin A toxin, and cytotoxic agents comprising a reactive
polyethylene glycol moiety (see, e.g., Sasse et al., 2000; Suzawa
et al., 2000; Ichimura et al., 1991; Francisco et al., 2003; U.S.
Pat. No. 5,475,092; U.S. Pat. No. 6,340,701; U.S. Pat. No.
6,372,738; and U.S. Pat. No. 6,436,931; US 2001/0036923; US
2004/0001838; US 2003/0199519; and WO 01/49698).
[0089] In a preferred embodiment, the cytotoxic agent is a
maytansinoid, i.e. a derivative of maytansine (CAS 35846538),
maytansinoids being known in the art to include maytansine,
maytansinol, C-3 esters of maytansinol, and other maytansinol
analogues and derivatives (see, e.g., U.S. Pat. No. 5,208,020; and
U.S. Pat. No. 6,441,163).
[0090] Anti-CD37 antibody immunoconjugates may be designed and
synthesized as described in WO 2007/077173 for anti-FAP
immunoconjugates.
[0091] In a further embodiment, the anti-CD37 molecule of the
invention may be radioactively labeled to form an
radioimmunoconjugate, an approach suggested for the anti-CD37
antibody MB-1 (Buchsbaum et al., 1992, see above). Radionuclides
with advantageous radiation properties are known in the art,
examples are Phosphorus-32, Strontium-89, Yttrium-90, Iodine-131,
Samarium-153, Erbium-169, Ytterbium-175, Rhenium-188, that have
been successfully and stably coupled to MAbs. The anti-CD37
antibody molecules of the invention may be labeled with various
radionuclides using direct labeling or indirect labeling methods
known in the art, as described in U.S. Pat. No. 6,241,961. A review
on technologies for generating and applying novel radiolabled
antibody conjugates that are useful in the present invention, is
given by Goldenberg and Sharkey, 2007.
[0092] An antibody molecule of the invention, whether Fc-engineered
or not, may also be bispecific, i.e. an antibody molecule that
binds to two different targets, one of them being CD37, the other
one being selected from e.g. surface antigens expressed by T cells,
e.g. CD3, CD16 and CD56.
[0093] The present invention also relates to DNA molecules that
encode the chimeric or humanized anti-CD37 antibody molecules of
the invention. The sequences encoding variable heavy chains of the
antibody molecules of the invention are shown in SEQ ID NO:1, SEQ
ID NO:5, SEQ ID NO:7 and SEQ ID NO:9. The sequences encoding
variable light chains of the antibody molecules of the invention
are shown in SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO: 21.
[0094] Nucleic acid molecules coding for the light chain and the
heavy chain may be synthesized chemically and enzymatically (PCR
amplification) by standard methods. First, suitable
oligonucleotides can be synthesized with methods known in the art
(e.g. Gait, 1984), which can be used to produce a synthetic gene.
Methods to generate synthetic genes from oligonucleotides are known
in the art (e.g. Stemmer et al., 1995; Ye et al., 1992; Hayden et
Mandecki, 1988; Frank et al., 1987).
[0095] The DNA molecules of the invention include, but are not
limited to, the DNA molecules shown in the sequence listing.
Accordingly, the present invention also relates to nucleic acid
molecules that hybridize to the DNA molecules set forth in the
sequence listing under high stringency binding and washing
conditions, as defined in WO 2007/042309, where such nucleic
molecules encode an antibody or functional fragment thereof that
has properties equivalent or superior to an antibody encoded by a
sequence shown in the sequence listing. Preferred molecules (from
an mRNA perspective) are those that have at least 75% or 80%
(preferably at least 85%, more preferably at least 90% and most
preferably at least 95%) homology or sequence identity with one of
the DNA molecules described herein.
[0096] Yet another class of DNA variants that are within the scope
of the invention may be defined with reference to the polypeptide
they encode. These DNA molecules deviate with respect to their
sequence from those depicted in the sequence listing, but encode,
due to the degeneracy of the genetic code, antibodies with the
identical amino acid sequences. By way of example, in view of
expressing the antibodies in eukaryotic cells, the DNA sequences
shown in the sequence listing have been designed to match codon
usage in eukaryotic cells. If it is desired to express the
antibodies in E. coli, these sequences can be changed to match E.
coli codon usage. Variants of DNA molecules of the invention can be
constructed in several different ways, as described e.g. in WO
2007/042309.
[0097] For producing the recombinant anti-CD37 antibody molecules
of the invention, the DNA molecules encoding full-length light and
heavy chains or fragments thereof are inserted into an expression
vector such that the sequences are operatively linked to
transcriptional and translational control sequences.
[0098] For manufacturing the antibodies of the invention, the
skilled artisan may choose from a great variety of expression
systems well known in the art, e.g. those reviewed by Kipriyanow
and Le Gall, 2004.
[0099] Expression vectors include plasmids, retroviruses, cosmids,
EBV-derived episomes, and the like. The expression vector and
expression control sequences are selected to be compatible with the
host cell. The antibody light chain gene and the antibody heavy
chain gene can be inserted into separate vectors. In certain
embodiments, both DNA sequences are inserted into the same
expression vector. Convenient vectors are those that encode a
functionally complete human CH or CL immunoglobulin sequence, with
appropriate restriction sites engineered so that any VH or VL
sequence can be easily inserted and expressed, as described above.
The constant chain is usually kappa or lambda for the antibody
light chain, for the antibody heavy chain, it can be, without
limitation, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other
immunoglobulins, including allelic variants.
[0100] The recombinant expression vector may also encode a signal
peptide that facilitates secretion of the antibody chain from a
host cell. The DNA encoding the antibody chain may be cloned into
the vector such that the signal peptide is linked in-frame to the
amino terminus of the mature antibody chain DNA. The signal peptide
may be an immunoglobulin signal peptide or a heterologous peptide
from a non-immunoglobulin protein. Alternatively, the DNA sequence
encoding the antibody chain may already contain a signal peptide
sequence.
[0101] In addition to the DNA sequences encoding the antibody
chains, the recombinant expression vectors carry regulatory
sequences including promoters, enhancers, termination and
polyadenylation signals and other expression control elements that
control the expression of the antibody chains in a host cell.
Examples for promoter sequences (exemplified for expression in
mammalian cells) are promoters and/or enhancers derived from (CMV)
(such as the CMV Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e. g., the adenovirus major late
promoter (AdMLP)), polyoma and strong mammalian promoters such as
native immunoglobulin and actin promoters. Examples for
polyadenylation signals are BGH polyA, SV40 late or early polyA;
alternatively, 3'UTRs of immunoglobulin genes etc. can be used.
[0102] The recombinant expression vectors may also carry sequences
that regulate replication of the vector in host cells (e. g.
origins of replication) and selectable marker genes. Nucleic acid
molecules encoding the heavy chain or an antigen-binding portion
thereof and/or the light chain or an antigen-binding portion
thereof of an anti-CD37 antibody, and vectors comprising these DNA
molecules can be introduced into host cells, e.g. bacterial cells
or higher eukaryotic cells, e.g. mammalian cells, according to
transfection methods well known in the art, including
liposome-mediated transfection, polycation-mediated transfection,
protoplast fusion, microinjections, calcium phosphate
precipitation, electroporation or transfer by viral vectors.
[0103] Preferably, the DNA molecules encoding the heavy chain and
the light chain are present on two vectors which are co-transfected
into the host cell, preferably a mammalian cell.
[0104] Mammalian cell lines available as hosts for expression are
well known in the art and include, inter alia, Chinese hamster
ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells or the
derivatives/progenies of any such cell line. Other mammalian cells,
including but not limited to human, mice, rat, monkey and rodent
cells lines, or other eukaryotic cells, including but not limited
to yeast, insect and plant cells, or prokaryotic cells such as
bacteria may be used. The anti-CD37 antibody molecules of the
invention are produced by culturing the host cells for a period of
time sufficient to allow for expression of the antibody molecule in
the host cells.
[0105] Antibody molecules are preferably recovered from the culture
medium as a secreted polypeptide or it can be recovered from host
cell lysates if for example expressed without a secretory signal.
It is necessary to purify the antibody molecules using standard
protein purification methods used for recombinant proteins and host
cell proteins in a way that substantially homogenous preparations
of the antibody are obtained. By way of example, state-of-the art
purification methods useful for obtaining the anti-CD37 antibody
molecule of the invention include, as a first step, removal of
cells and/or particulate cell debris from the culture medium or
lysate. The antibody is then purified from contaminant soluble
proteins, polypeptides and nucleic acids, for example, by
fractionation on immunoaffinity or ion-exchange columns, ethanol
precipitation, reverse phase HPLC, Sephadex chromatography,
chromatography on silica or on a cation exchange resin. As a final
step in the process for obtaining an anti-CD37 antibody molecule
preparation, the purified antibody molecule may be dried, e.g.
lyophilized, as described below for therapeutic applications. In a
further aspect, the present invention relates to a pharmaceutical
composition containing, as the active ingredient, the anti-CD37
antibody molecule of the invention.
[0106] To be used in therapy, the anti-CD37 antibody is included
into pharmaceutical compositions appropriate to facilitate
administration to animals or humans. Typical formulations of the
anti-CD37 antibody molecule can be prepared by mixing the anti-CD37
antibody molecule with physiologically acceptable carriers,
excipients or stabilizers, in the form of lyophilized or otherwise
dried formulations or aqueous solutions or aqueous or non-aqueous
suspensions. Carriers, excipients, modifiers or stabilizers are
nontoxic at the dosages and concentrations employed. They include
buffer systems such as phosphate, citrate, acetate and other
anorganic or organic acids and their salts; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone or polyethylene
glycol (PEG); amino acids such as glycine, glutamine, asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides,
oligosaccharides or polysaccharides and other carbohydrates
including glucose, mannose, sucrose, trehalose, dextrins or
dextrans; chelating agents such as EDTA; sugar alcohols such as,
mannitol or sorbitol; salt-forming counter-ions such as sodium;
metal complexes (e.g. Zn-protein complexes); and/or ionic or
non-ionic surfactants such as TWEEN.TM. (polysorbates),
PLURONICS.TM. or fatty acid esters, fatty acid ethers or sugar
esters. Also organic solvents can be contained in the antibody
formulation such as ethanol or isopropanol. The excipients may also
have a release-modifying or absorption-modifying function.
[0107] The anti-CD37 antibody molecules may also be dried
(freeze-dried, spray-dried, spray-freeze dried, dried by near or
supercritical gases, vacuum dried, air-dried), precipitated or
crystallized or entrapped in microcapsules that are prepared, for
example, by coacervation techniques or by interfacial
polymerization using, for example, hydroxymethylcellulose or
gelatin and poly-(methylmethacylate), respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), in
macroemulsions or precipitated or immobilized onto carriers or
surfaces, for example by pcmc technology (protein coated
microcrystals). Such techniques are disclosed in Remington: The
Science and Practice of Pharmacy, 21.sup.st edition, Hendrickson R.
Ed.
[0108] Naturally, the formulations to be used for in vivo
administration must be sterile; sterilization may be accomplished
be conventional techniques, e.g. by filtration through sterile
filtration membranes.
[0109] It may be useful to increase the concentration of the
anti-CD37 antibody to come to a so-called high concentration liquid
formulation (HCLF); various ways to generate such HCLFs have been
described.
[0110] The anti-CD37 antibody molecule may also be contained in a
sustained-release preparation. Such preparations include solid,
semi-solid or liquid matrices of hydrophobic or hydrophilic
polymers, and may be in the form of shaped articles, e.g. films,
sticks or microcapsules and may be applied via an application
device. Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or
sucrose acetate butyrate), or poly(vinylalcohol)), polylactides
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and
.gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S-S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilization (e.g. as described in
WO 89/011297) from acidic solutions, controlling moisture content,
using appropriate additives, and developing specific polymer matrix
compositions.
[0111] Formulations that may also be used for the anti-CD37
antibody molecule of the invention are described in U.S. Pat. No.
7,060,268 and U.S. Pat. No. 6,991,790.
[0112] The CD37 antibody molecule can be incorporated also in other
application forms, such as dispersions, suspensions or liposomes,
tablets, capsules, powders, sprays, transdermal or intradermal
patches or creams with or without permeation enhancing devices,
wafers, nasal, buccal or pulmonary formulations, or may be produced
by implanted cells or--after gene therapy--by the individual's own
cells.
[0113] An anti-CD37 antibody molecule may also be derivatized with
a chemical group such as polyethylene glycol (PEG), a methyl or
ethyl group, or a carbohydrate group. These groups may be useful to
improve the biological characteristics of the antibody, e.g. to
increase serum half-life or to increase tissue binding.
[0114] The preferred mode of application is parenteral, by infusion
or injection (intraveneous, intramuscular, subcutaneous,
intraperitoneal, intradermal), but other modes of application such
as by inhalation, transdermal, intranasal, buccal, oral, may also
be applicable.
[0115] For the prevention or treatment of disease, the appropriate
dosage of antibody will depend on the type of disease to be
treated, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments.
[0116] Depending on the type and severity of the disease, about
0.01 .mu.g/kg to 40 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. For repeated administrations over several
days or longer, depending on the condition, the treatment is
sustained until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and assays,
e.g. by determining the extent of B cell depletion (e.g. using flow
cytometry).
[0117] The "therapeutically effective amount" of the antibody to be
administered is the minimum amount necessary to prevent,
ameliorate, or treat a disease or disorder.
[0118] The anti-CD37 antibody molecule of the invention and
pharmaceutical compositions containing it are useful to deplete B
cells that express CD37 on their surface and that cause cancerous
or autoimmune/inflammatory disease.
[0119] In a first aspect, the pharmaceutical composition of the
invention is useful for the treatment of cancers, in particular any
CD37-positive malignancies.
[0120] B cell malignancies include, without limitation, B cell
lymphomas (e.g. various forms of Hodgkin's disease, B cell
non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g.
Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic
lymphoma or immunocytoma) or central nervous system lymphomas),
leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic
lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic
leukemia BCLL), hairy cell leukemia and chronic myoblastic
leukemia) and myelomas (e.g. multiple myeloma). Additional B cell
malignancies include small lymphocytic lymphoma, B cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma
of bone, extraosseous plasmacytoma, extra-nodal marginal zone B
cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal
marginal zone B cell lymphoma, follicular lymphoma, mantle cell
lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large
B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma,
B cell proliferations of uncertain malignant potential,
lymphomatoid granulomatosis, and post-transplant
lymphoproliferative disorder.
[0121] In a further aspect, a pharmaceutical composition containing
anti-CD37 antibodies is useful for the treatment of autoimmune and
inflammatory diseases that involve B cells in their pathology.
[0122] Such diseases include, but are not limited to: arthritis,
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, polychondritis, psoriatic arthritis, psoriasis,
dermatitis, polymyositis/dermatomyositis, inclusion body myositis,
inflammatory myositis,toxic epidermal necrolysis, systemic
scleroderma and sclerosis, CREST syndrome, responses associated
with inflammatory bowel disease, Crohn's disease, ulcerative
colitis, respiratory distress syndrome, adult respiratory distress
syndrome (ARDS), meningitis, encephalitis, uveitis, colitis,
glomerulonephritis, allergic conditions, eczema, asthma, conditions
involving infiltration of T cells and chronic inflammatory
responses, atherosclerosis, autoimmune myocarditis, leukocyte
adhesion deficiency, systemic lupus erythematosus (SLE), subacute
cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus
cerebritis, juvenile onset diabetes, multiple sclerosis, allergic
encephalomyelitis, neuromyelitis optica, rheumatic fever,
Sydenham's chorea, immune responses associated with acute and
delayed hypersensitivity mediated by cytokines and T-lymphocytes,
tuberculosis, sarcoidosis, granulomatosis including Wegener's
granulomatosis and Churg-Strauss disease, agranulocytosis,
vasculitis (including hypersensitivity vasculitis/angiitis, ANCA
and rheumatoid vasculitis), aplastic anemia, Diamond Blackfan
anemia, immune hemolytic anemia including autoimmune hemolytic
anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA),
Factor VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
central nervous system (CNS) inflammatory disorders, multiple organ
injury syndrome, myasthenia gravis, antigen-antibody complex
mediated diseases, anti-glomerular basement membrane disease,
anti-phospholipid antibody syndrome, allergic neuritis, Behcet
disease, Castleman's syndrome, Goodpasture's syndrome,
Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's
syndrome, Stevens-Johnson syndrome, solid organ transplant
rejection, graft versus host disease (GVHD), pemphigoid bullous,
pemphigus, autoimmune polyendocrinopathies, seronegative
spondyloarthropathies, Reiter's disease, stiff-man syndrome, giant
cell arteritis, immune complex nephritis, IgA nephropathy, IgM
polyneuropathies or IgM mediated neuropathy, idiopathic
thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura
(TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia,
autoimmune disease of the testis and ovary including autoimmune
orchitis and oophoritis, primary hypothyroidism; autoimmune
endocrine diseases including autoimmune thyroiditis, chronic
thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis,
idiopathic hypothyroidism, Addison's disease, Grave's disease,
autoimmune polyglandular syndromes (or polyglandular endocrinopathy
syndromes), Type I diabetes also referred to as insulin-dependent
diabetes mellitus (IDDM) and Sheehan's syndrome; autoimmune
hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre' Syndrome,
large vessel vasculitis (including polymyalgia rheumatica and giant
cell (Takayasu's) arteritis), medium vessel vasculitis (including
Kawasaki's disease and polyarteritis nodosa), polyarteritis nodosa
(PAN) ankylosing spondylitis, Berger's disease (IgA nephropathy),
rapidly progressive glomerulonephritis, primary biliary cirrhosis,
Celiac sprue (gluten enteropathy), cryoglobulinemia,
cryoglobulinemia associated with hepatitis, amyotrophic lateral
sclerosis (ALS), coronary artery disease, familial Mediterranean
fever, microscopic polyangiitis, Cogan's syndrome, Whiskott-Aldrich
syndrome and thromboangiitis obliterans (see WO 2007/014278).
[0123] Depending on the disorder to be treated, the anti-CD37
antibody molecule of the invention may be used on its own or in
combination with one or more additional therapeutic agents, in
particular selected from DNA damaging or tubulin binding agents or
therapeutically active compounds that inhibit angiogenesis, signal
transduction pathways or mitotic checkpoints in cancer cells.
[0124] The additional therapeutic agent may be administered
simultaneously with, optionally as a component of the same
pharmaceutical preparation, or before or after administration of
the anti-CD37 antibody molecule.
[0125] In certain embodiments, the additional therapeutic agent may
be, without limitation, one or more inhibitors selected from the
group of inhibitors of EGFR family, VEGFR family, IGF-1R, Insulin
receptors, AuroraA, AuroraB, PLK and PI3 kinase, FGFR, PDGFR, Raf,
KSP or PDK1.
[0126] Further examples of additional therapeutic agents are
inhibitors of CDKs, Akt, Src, Bcr-Abl, cKit, cMet/HGF, c-Myc, Flt3,
HSP90, hedgehog antagonists, inhibitors of JAK/STAT, Mek, mTor,
NFkappaB, the proteasome, Rho, an inhibitor of Wnt signaling or
Notch signaling or an ubiquitination pathway inhibitor.
[0127] Examples for Aurora inhibitors are, without limitation,
PHA-739358, AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045,
PF-3814735, SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626,
MLN-8237 and ENMD-2076.
[0128] An example for a PLK inhibitor is GSK-461364.
[0129] Examples for raf inhibitors are BAY-73-4506 (also a VEGFR
inhibitor), PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib
(also a VEGFR inhibitor), XL-281, and Nevavar (also an inhibitor of
the VEGFR).
[0130] Examples for KSP inhibitors are ispinesib, ARRY-520,
AZD-4877, CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921,
LY-2523355, and EMD-534085.
[0131] Examples for a src and/or bcr-abl inhibitors are dasatinib,
AZD-0530, bosutinib, XL-228 (also an IGF-1R inhibitor), nilotinib
(also a PDGFR and cKit inhibitor), imatinib (also a cKit
inhibitor), NS-187, KX2-391, AP-24534 (also an inhibitor of EGFR,
FGFR, Tie2, Flt3), KM-80 and LS-104 (also an inhibitor of Flt3,
Jak2).
[0132] An example for a PDK1 inhibitor is AR-12.
[0133] An example for a Rho inhibitor is BA-210.
[0134] Examples for P13 kinase inhibitors are PX-866, PX-867,
BEZ-235 (also an mTor inhibitor), XL-147, and XL-765 (also an mTor
inhibitor), BGT-226, CDC-0941.
[0135] Examples for inhibitors of cMet or HGF are XL-184 (also an
inhibitor of VEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also
an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron,
Tie2), SU-11274, PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb,
CGEN-241, BMS-777607, JNJ-38877605, PF-4217903, SGX-126, CEP-17940,
AMG-458, NCB-028060, and E-7050.
[0136] An example for a c-Myc inhibitor is CX-3543.
[0137] Examples for Flt3 inhibitors are AC-220 (also an inhibitor
of cKit and PDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl
and Jak2), MC-2002, SB-1317, lestaurtinib (also an inhibitor of
VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK2), XL-999
(also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also
an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an
inhibitor of PDGFR, and cKit).
[0138] Examples for HSP90 inhibitors are, tanespimycin,
alvespimycin, IPI-504, STA-9090, MEDI-561, AUY-922, CNF-2024, and
SNX-5422.
[0139] Examples for JAK/STAT inhibitors are CYT-997 (also
interacting with tubulin), TG-101348 (also an inhibitor of Flt3),
and XL-019.
[0140] Examples for Mek inhibitors are ARRY-142886, AS-703026,
PD-325901, AZD-8330, ARRY-704, RDEA-119, and XL-518.
[0141] Examples for mTor inhibitors are temsirolimus, deforolimus
(which also acts as a VEGF inhibitor), everolimus (a VEGF inhibitor
in addition). XL-765 (also a PI3 kinase inhibitor), and BEZ-235
(also a PI3 kinase inhibitor).
[0142] Examples for Akt inhibitors are perifosine, GSK-690693,
RX-0201, and triciribine.
[0143] Examples for cKit inhibitors are masitinib, OSI-930 (also
acts as a VEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and
PDGFR), tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib
(also an inhibitor of VEGFR and PDGFR), sunitinib (also an
inhibitor of Flt3, PDGFR, VEGFR), and XL-820 (also acts as a VEGFR-
and PDGFR inhibitor), imatinib (also a bcr-abl inhibitor),
nilotinib (also an inhibitor of bcr-abl and PDGFR).
[0144] Examples for hedgehog antagonists are IPI-609, CUR-61414,
GDC-0449, IPI-926, and XL-139.
[0145] Examples for CDK inhibitors are seliciclib, AT-7519, P-276,
ZK-CDK (also inhibiting VEGFR2 and PDGFR), PD-332991, R-547,
SNS-032, PHA-690509, PHA-848125, and SCH-727965.
[0146] Examples for proteasome inhibitors are bortezomib,
carfilzomib, and NPI-0052 (also an inhibitor of NFkappaB).
[0147] Examples for proteasome inhibitors/NFkappaB pathway
inhibitors are bortezomib, carfilzomib, NPI-0052, CEP-18770,
MLN-2238, PR-047, PR-957, AVE-8680, and SPC-839.
[0148] An example for an inhibitor of the ubiquitination pathway is
HBX-41108.
[0149] Examples for anti-angiogenic agents are inhibitors of the
FGFR, PDGFR and VEGF(R), and thalidomides, such agents being
selected from, without limitation, bevacizumab, motesanib, CDP-791,
SU-14813, telatinib, KRN-951, ZK-CDK (also an inhibitor of CDK),
ABT-869, BMS-690514, RAF-265, IMC-KDR, IMC-18F1, IMiDs,
thalidomide, CC-4047, lenalidomide, ENMD-0995, IMC-D11, Ki-23057,
brivanib, cediranib, 1B3, CP-868596, IMC-3G3, R-1530 (also an
inhibitor of Flt3), sunitinib (also an inhibitor of cKit and Flt3),
axitinib (also an inhibitor of cKit), lestaurtinib (also an
inhibitor of Flt3 and PKC), vatalanib, tandutinib (also an
inhibitor of Flt3 and cKit), pazopanib, PF-337210, aflibercept,
E-7080, CHIR-258, sorafenib tosylate (also an inhibitor of Raf),
vandetanib, CP-547632, OSI-930, AEE-788 (also an inhibitor of EGFR
and Her2), BAY-57-9352 (also an inhibitor of Raf), BAY-73-4506
(also an inhibitor of Raf), XL-880 (also an inhibitor of cMet),
XL-647 (also an inhibitor of EGFR and EphB4), XL-820 (also an
inhibitor of cKit), nilotinib (also an inhibitor of cKit and
brc-abl), CYT-116, PTC-299, BMS-584622, CEP-11981, dovitinib,
CY-2401401, and ENMD-2976.
[0150] The additional therapeutic agent may also be selected from
EGFR inhibitors, it may be a small molecule EGFR inhibitor or an
anti-EGFR antibody. Examples for anti-EGFR antibodies, without
limitation, are cetuximab, panitumumab, nimotuzumab, zalutumumab;
examples for small molecule EGFR inhibitors are gefitinib,
erlotinib and vandetanib (also an inhibitor of the VEGFR). Another
example for an EGFR modulator is the EGF fusion toxin.
[0151] Further EGFR and/or Her2 inhibitors useful for combination
with an anti-CD37 antibody molecule of the invention are lapatinib,
trastuzumab, pertuzumab, XL-647, neratinib, BMS-599626 ARRY-334543,
AV-412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also an
inhibitor of VEGFR), AZD-8931, ARRY-380 ARRY-333786, IMC-11F8,
Zemab, TAK-285, AZD-4769.
[0152] The additional drug may also be selected from agents that
target the IGF-1R and insulin receptor pathways. Such agents
include antibodies that bind to IGF-1R (e.g. CP-751871, AMG-479,
IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab
IGFR and novel chemical entities that target the kinase domain of
the IGF1-R (e.g. OSI-906 or BMS-554417, XL-228, BMS-754807).
[0153] Other agents that may be advantageously combined in a
therapy with the anti-CD37 antibody molecule of the invention are
molecules targeting CD20, including CD20 specific antibodies like
rituximab, LY-2469298, ocrelizumab, MEDI-552, IMMU-106, GA-101
(=R7159), XmAb-0367, ofatumumab, radiolabbeled CD20 antibodies,like
tositumumab and ibritumomab tiuxetan or other CD20 directed
proteins, like the SMIP Tru015, PRO-131921, FBT-A05, veltuzumab,
R-7159.
[0154] CD37 antibodies may be combined with inhibitors of other
surface antigens expressed on leukocytes, in particular antibodies
or antibody-like molecules, e.g. anti-CD2 (siplizumab), anti-CD4
(zanolimumab), anti-CD19 (MT-103, MDX-1342, SAR-3419, XmAb-5574),
anti-CD22 (epratuzumab), anti-CD23 (lumiliximab), anti-CD30
(iratmumub), anti-CD32B (MGA-321), anti-CD38 (HuMax-CD38),
anti-CD40 (SGN40), anti-CD52 (alemtuzumab), anti-CD80 (galiximab).
An antibody of the invention may also be combined with another CD37
antagonist, e.g. TRU-016.
[0155] Other agents to be combined with CD37 antibodies are
immunotoxins like BL-22 (an anti-CD22 immunotoxin), inotuzumab
ozogamicin (an anti-CD23 antibody-calicheamicin conjugate),
RFT5.dgA (anti-CD25 Ricin toxin A-chain), SGN-35 (an
anti-CD30-auristatin E conjugate), and gemtuzumab ozogamicin (an
anti-CD33 calicheamicin conjugate), MDX-1411 (anti-CD70 conjugate),
or radiolabelled antibodies like .sup.90Y-epratuzumab (anti-CD22
radioimmunoconjugate).
[0156] In addition, anti-CD37 antibodies may be combined with
immunomodulators, agents, e.g. antibodies, that induce apoptosis or
modify signal transduction pathways like the TRAIL receptor
modulators mapatumumab (a TRAIL-1 receptor agonist), lexatumumab (a
TRAIL-2 receptor agonist), tigatuzumab, Apomab, AMG-951 and
AMG-655; an anti-HLA-DR antibody (like 1D09C3), an anti-CD74, an
osteoclast differentiation factor ligand inhibitor (like
denosumab), a BAFF antagonist (like AMG-623a) or an agonist of a
Toll-like receptor (e.g. TLR-4 or TLR-9).
[0157] Other drugs that may be used in combination with the
anti-CD37 antibody molecules of the present invention are selected
from, but not limited to hormones, hormonal analogues and
antihormonals (e.g. tamoxifen, toremifene, raloxifene, fulvestrant,
megestrol acetate, flutamide, nilutamide, bicalutamide, cyproterone
acetate, finasteride, buserelin acetate, fludrocortinsone,
fluoxymesterone, medroxyprogesterone, hydroxyprogesterone caproate,
diethylstilbestrol, testosterone propionate,
fluoxymesterone/equivalents, octreotide, arzoxifene, pasireotide,
vapreotide, adrenocorticosteroids/antagonists, prednisone,
dexamethasone, ainoglutethimide), aromatase inhibitors (e.g.
anastrozole, letrozole, liarozole, exemestane, atamestane,
formestane), LHRH agonists and antagonists (e.g. goserelin acetate,
leuprolide, abarelix, cetrorelix, deslorelin, histrelin,
triptorelin), antimetabolites (e.g. antifolates like methotrexate,
trimetrexate, pemetrexed, pyrimidine analogues like 5-fluorouracil,
fluorodeoxyuridine, capecitabine, decitabine, nelarabine,
5-azacytidine, and gemcitabine, purine and adenosine analogues such
as mercaptopurine, thioguanine, azathioprine, cladribine and
pentostatin, cytarabine, fludarabine, clofarabine); antitumor
antibiotics (e.g. anthracyclines like doxorubicin, daunorubicin,
epirubicin and idarubicin, mitomycin-C, bleomycin dactinomycin,
plicamycin, splicamycin, actimomycin D, mitoxantrone,
mitoxantroneidarubicin, pixantrone, streptozocin, aphidicolin);
platinum derivatives (e.g. cisplatin, oxaliplatin, carboplatin,
lobaplatin, satraplatin); alkylating agents (e.g. estramustine,
semustine, mechlorethamine, melphalan, chlorambucil, busulphan,
dacarbazine, cyclophosphamide, ifosfamide, hydroxyurea,
temozolomide, nitrosoureas such as carmustine and lomustine,
thiotepa); antimitotic agents (e.g. vinca alkaloids like
vinblastine, vindesine, vinorelbine, vinflunine and vincristine;
and taxanes like paclitaxel, docetaxel and their formulations,
larotaxel; simotaxel, and epothilones like ixabepilone, patupilone,
ZK-EPO); topoisomerase inhibitors (e.g. epipodophyllotoxins like
etoposide and etopophos, teniposide, amsacrine, topotecan,
irinotecan, banoxantrone, camptothecin) and miscellaneous
chemotherapeutics such as retinoic acid derivatives, amifostine,
anagrelide, interferon alpha, interferon beta, interferon gamma,
interleukin-2, procarbazine, N-methylhydrazine, mitotane, and
porflmer, bexarotene, celecoxib, ethylenemine/methyl-melamine,
thriethyienemelamine, triethylene thiophosphoramide,
hexamethylmelamine, and enzymes L-asparaginase, L-arginase and
metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,
etanidazole, nimorazole, RSU 1069, E09, RB 6145, SR4233,
nicotinamide, 5-bromodeozyuridine, 5-iododeoxyuridine,
bromodeoxycytidine, erythrohydroxynonyl-adenine, anthracenedione,
GRN-163L (a competitive telomerase template antagonist), SDX-101 (a
PPAR agonist), talabostat (a DPP inhibitor), forodesine (a PNP
inhibitor), atacicept (a soluble receptor targeting TNF family
members BLyS and APRIL), TNF-alpha neutralizing agents (Enbrel,
Humira, Remicade), XL-844 (a CHK1/2 inhibitor), VNP-40101M (a DNA
alkylating agent), SPC-2996 (an antisense bcl2 inhibitor),
obatoclax (a bcl2 inhibitor), enzastaurin (a PKC beta modulator),
vorinistat (an HDAC inhibitor), romidepsin (an HDAC inhibitor),
AT-101 (a Bcl-2/Bcl-xL inhibitor), plitidepsin (a multi-actioned
depsipeptide), SL-11047 (a polyamine metabolism modulators).
[0158] In certain embodiments, the anti-CD37 antibody molecule is
applied together with "CHOP" (a combination of cyclophosphamide,
doxorubicin, vincristine and prednisone).
[0159] The anti-CD37 antibody molecule of the invention may also be
used in combination with other therapies including surgery,
radiotherapy, endocrine therapy, biologic response modifiers,
hyperthermia and cryotherapy and agents to attenuate any adverse
effect (e.g. antiemetics), G-CSF, GM-CSF, photosensitizers such as
hematoporphyrin derivatives, Photofrin.RTM., benzoporphyrin
derivatives, Npe6, tin etioporphyrin, pheoboride-a
bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc
phthalocyanines
[0160] Monoclonal antibodies show exquisite antigen specificity and
frequently only react with the human target antigen, but not with
homologue proteins from animal species. To support development of
therapeutic antibodies, appropriate animal models for assessment of
in vivo toxicity and pharmacodynamic behavior are desirable. One
possibility for an in vivo model is a transgenic mouse in which the
endogenous target antigen is replaced by its human homologue
("knock-out/knock-in mouse"). In particular, for developing
therapeutic anti-CD37 antibodies , the murine CD37 gene can be
replaced by the human CD37 gene. This can be achieved by
constructing a targeting vector which contains the coding genomic
sequence of the human CD37 gene flanked by non-translated
sequences. This targeting vector can be used for homologous
recombination using mouse ES cells. Transgenic animals homozygous
for human CD37 expression can be used to assess the pharmacodynamic
effect of antibodies directed against human CD37, e.g. by
monitoring the number of peripheral B cells after application of
the antibodies. Alternatively, those mice can be used to
investigate potential toxic effects of human CD37 specific
antibodies, after i.v. application.
[0161] Another possibility in the case of lack of animal
cross-reactivity of monoclonal antibodies is the generation of a
so-called surrogate antibody. A surrogate antibody is an antibody
which reacts with the homologous protein of an animal species which
is relevant and useful for investigation of pharmacodynamic and
toxic effects, e.g. the mouse or the cynomolgus monkey. In case of
CD37, monoclonal antibodies are developed which are specific for
macaque CD37 or mouse CD37, respectively Ideally, such a surrogate
antibody should have similar binding and functional properties as
the development antibody. This can be investigated by the use of
assay systems which utilize macaque or mouse CD37 expressing cells
as target cells, e.g. for binding, FACS Scatchard analysis, ADCC
and apoptosis assays. Ultimately, the surrogate antibody can be
selected by virtue of its B cell depleting activity in macaque or
mouse blood in vitro.
BRIEF DESCRIPTION OF THE FIGURES
[0162] FIG. 1: Chimeric Antibody A0 specifically recognizes the
CD37 antigen, determined by FACS competition assay
[0163] FIG. 2: Binding of humanized versions of A0 to cellular CD37
antigen, determined by FACS
[0164] FIG. 3: Binding of humanized versions of A0 to cellular CD37
antigen, determined by FACS
[0165] FIG. 4: Affinity of humanized versions of A0 to cellular
CD37 antigen, determined by FACS scatchard analysis
[0166] FIG. 5: ADCC activity of humanized versions of A0 on Ramos
cells
[0167] FIG. 6: Pro-apoptotic activity of humanized versions of A0
on Ramos cells
[0168] FIG. 7: ADCC activity of Fc-engineered versions of mAb A0 on
Ramos cells
[0169] FIG. 8: ADCC activity of Fc-engineered versions of mAb B0 on
Ramos cells
[0170] FIG. 9: Pro-apoptotic activity of mAb A0 and B0
[0171] FIG. 10: Pro-apoptotic activity of Fc-engineered versions of
mAb A0
[0172] FIG. 11A: Depletion of normal human B cells in a whole blood
assay by Fc-engineered antibodies A2 and B2 in comparison to
Rituximab
[0173] FIG. 11B: Superior B cell depleting activity of antibodies
after Fc-engineering in comparison to Rituximab
[0174] FIG. 11C: Antibodies A2 and B2 do not deplete T cells and
monocytes in whole blood assays
[0175] FIG. 12: Superior ADCC activity after Fc-engineering
compared to Rituximab
[0176] FIG. 13: Depletion of Ramos Burkitt's lymphoma cells in a
whole blood assay by Fc-engineered antibodies A2 and B2 in
comparison to Rituximab
[0177] FIG. 14: In vivo tumor growth inhibition of Ramos xenograft
tumors in nude mice by Fc-engineered antibodies A2 and B2
[0178] FIG. 15: Expression of CD37 on multiple myeloma cells
[0179] FIG. 16: ADCC activity of antibodies A2 and B2 on multiple
myeloma cells
[0180] FIG. 17: Pro-apoptotic activity of antibodies A2 and B2 on
patient-derived CLL cells
EXAMPLE 1
[0181] Generation of Chimeric and Humanized Anti-CD37
Antibodies
[0182] a) Generation of Chimeric Antibody A0
[0183] Based on the variable heavy and light chain amino acid
sequences shown in SEQ ID NO:2 and SEQ ID NO:4, the corresponding
DNA sequences are synthesized applying codon usage optimized for
mammalian cells (GeneArt, Regensburg, Germany), adding at the 5'
end a HindIII and at the 3' end a BamH1 cloning site. The
synthesized DNA molecules are digested with HindIII and BamHI and
the resulting DNA fragments (SEQ ID NO:1 and SEQ ID NO:3 plus
restriction sites) are cloned into pcDNA3.1 based expression
vectors coding for human IgG1 constant region and human kappa light
chain constant region, respectively (SEQ ID NO:24 and SEQ ID
NO:26). EndoFree plasmid preparations (Qiagen) are prepared and the
heavy and light chain plasmids are co-transfected into HEK293
freestyle cells (Invitrogen) at a concentration of 1 mg/L of each
plasmid according to the supplier's protocol. After 72 hours the
supernatant is harvested and the IgG concentration is determined by
ELISA. The resulting chimeric anti-CD37 antibody (designated A0) is
purified on a modified protein A column (GE Healthcare), eluted
into a citrate buffer then dialyzed in PBS.
[0184] b) Generation of Humanized Versions of Chimeric Antibody
A0
[0185] Humanization of chimeric mAb A0, as obtained in a), is
performed using a CDR grafting approach, as described e.g. in U.S.
Pat. No. 5,225,539; U.S. Pat. No. 6,548,640; U.S. Pat. No.
6,982,321.
[0186] To establish a structural model of the mAb A0 VL domain, a
structural template is chosen from the Protein Data Bank (PDB) of
Brookhaven National Laboratory. The VL domain from the murine
monoclonal antibody entry "1KB5" is chosen with 88% sequence
identity/81% similarity and 2.5 .ANG. resolution. For the mAb A0 VH
domain, the same mouse monoclonal antibody structure "1KB5" with
90% sequence identity and 91% similarity is chosen as the main
modeling template. The best fit for human consensus framework is
found to be of the type human Vkappa1 (hVK1) and human VH1 (hVH1).
As an alternative design, a graft to the most stable human
consensus domains hVK3 and hVH3 is chosen. For grafting, the mAb
A0_VL and mAb A0_VH models are combined with human consensus domain
models hVK1, hVK3, hVH1A and hVH3 and combined to produce to
Fv-models. Loop grafting is performed by embedding the murine mAb
A0 CDR regions into the human antibody frameworks and the DNA
molecules of the humanized chain constructs are synthesized.
[0187] The respective humanized variable regions are synthesized
and cloned into immunoglobulin expression vectors and transiently
expressed in the HEK 293 freestyle expression system (Invitrogen),
as described in a), in the combinations of heavy and light chain
sequences as shown in Table 1, and purified on protein A
columns.
TABLE-US-00001 TABLE 1 Sequences of heavy and light variable chains
of the chimeric and humanized anti-CD37 antibodies used in the
Examples. Heavy Chain Light Chain SEQ ID NO: SEQ ID NO: Antibody
aa/DNA aa/DNA A (=A0) seq 2/1 seq 4/3 B seq 6/5 seq 12/11 C seq 6/5
seq 14/13 D seq 6/5 seq 16/15 H seq 8/7 seq 18/17 I seq 8/7 seq
20/19 J seq 8/7 seq 22/21 K seq 10/9 seq 18/17 L seq 10/9 seq 20/19
M seq 10/9 seq 22/21
[0188] c) Generation of Fc-Engineered Chimeric and Humanized
Anti-CD37 Antibodies
[0189] The generation of Fc mutants is performed as described by
Lazar et al., 2006. The resulting Fc-engineered heavy chain
sequence is introduced into the expression vector pAD-CMV1
(described in EP 393 438) and co-transfected together with a
plasmid containing the light chain encoding sequence into CHO-DG44
cells. The antibody is harvested from cell culture media 5 to 7
days after transfection and purified via protein A chromatography,
eluted into a citrate buffer then dialyzed in PBS. The protein
content of the samples is determined via Protein A HPLC, the
endotoxin content is determined via Kinetic-QCL Kinetic Chromogenic
Assay (Lonza). The monomer content of the samples is determined by
HP-SEC, all samples used for functional testing show a monomer
content of >95%.
TABLE-US-00002 TABLE 2 Sequences of heavy and light variable chains
(columns III and IV) and Fc mutations (coulum II) of the chimeric
and humanized anti-CD37 antibodies (antibody A0, B0, C0 etc. is
identical with antibody A and B, C etc. in Table 1). V VI III IV
SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: complete complete II v
heavy v light heavy light I Fc substitution(s) chain chain chain
chain Ab (Kabat numbering) aa/DNA aa/DNA aa/DNA aa/DNA A0 -- seq
2/1 seq 4/3 2/1 fused 4/3 to 24/23 fused to 26/25 A1 I332E seq 2/1
seq 4/3 2/1 fused 4/3 to fused to 24*/23* 26/25 A2 S239D/I332E seq
2/1 seq 4/3 28/27 30/29 A3 I332E/G236A seq 2/1 seq 4/3 2/1 fused
4/3 to fused to 24*/23* 26/25 A4 S239D/I332E/G236A seq 2/1 seq 4/3
32/31 34/33 B0 -- seq 6/5 seq 12/11 6/5 fused 12/11 to 24/23 fused
to 26/25 B1 I332E seq 6/5 seq 12/11 6/5 fused 12/11 to fused to
24*/23* 26/25 B2 S239D/I332E seq 6/5 seq 12/11 36/35 38/37 B3
I332E/G236A seq 6/5 seq 12/11 6/5 fused 12/11 to fused to 24*/23*
26/25 B4 S239D/I332E/G236A seq 6/5 seq 12/11 40/39 42/41 C0 -- seq
6/5 seq 14/13 6/5 fused 14/13 to 24/23 fused to 26/25 C1 I332E seq
6/5 seq 14/13 6/5 fused 14/13 to fused to 24*/23* 26/25 C2
S239D/I332E seq 6/5 seq 14/13 6/5 fused 14/13 to fused to 24*/23*
26/25 C3 I332E/G236A seq 6/5 seq 14/13 6/5 fused 14/13 to fused to
24*/23* 26/25 C4 S239D/I332E/G236A seq 6/5 seq 14/13 6/5 fused
14/13 to fused to 24*/23* 26/25 D0 -- seq 6/5 seq 16/15 6/5 fused
16/15 to 24/23 fused to 26/25 D1 I332E seq 6/5 seq 16/15 6/5 fused
16/15 to fused to 24*/23* 26/25 D2 S239D/I332E seq 6/5 seq 16/15
6/5 fused 16/15 to fused to 24*/23* 26/25 D3 I332E/G236A seq 6/5
seq 16/15 6/5 fused 16/15 to fused to 24/*23* 26/25 D4
S239D/I332E/G236A seq 6/5 seq 16/15 6/5 fused 16/15 to fused to
24/*23* 26/25 H0 -- seq 8/7 seq 18/17 8/7 fused 18/17 to 24/23
fused to 26/25 H1 I332E seq 8/7 seq 18/17 8/7 fused 18/17 to fused
to 24*/23* 26/25 H2 S239D/I332E seq 8/7 seq 18/17 8/7 fused 18/17
to fused to 24*/23* 26/25 H3 I332E/G236A seq 8/7 seq 18/17 8/7
fused 18/17 to fused to 24*/23* 26/25 H4 S239D/I332E/G236A seq 8/7
seq 18/17 8/7 fused 18/17 to fused to 24*/23* 26/25 I-0 -- seq 8/7
seq 20/19 8/7 fused 20/19 to 24/23 fused to 26/25 I-1 I332E seq 8/7
seq 20/19 8/7 fused 20/19 to fused to 24*/23* 26/25 I-2 S239D/I332E
seq 8/7 seq 20/19 8/7 fused 20/19 to fused to 24*/23* 26/25 I-3
I332E/G236A seq 8/7 seq 20/19 8/7 fused 20/19 to fused to 24*/23*
26/25 I-4 S239D/I332E/G236A seq 8/7 seq 20/19 8/7 fused 20/19 to
fused to 24*/23* 26/25 J0 -- seq 8/7 seq 22/21 8/7 fused 22/21 to
24/23 fused to 26/25 J1 I332E seq 8/7 seq 22/21 8/7 fused 22/21 to
fused to 24*/23* 26/25 J2 S239D/I332E seq 8/7 seq 22/21 8/7 fused
22/21 to fused to 24*/23* 26/25 J3 I332E/G236A seq 8/7 seq 22/21
8/7 fused 22/21 to fused to 24*/23* 26/25 J4 S239D/I332E/G236A seq
8/7 seq 22/21 8/7 fused 22/21 to fused to 24*/23* 26/25 K0 -- seq
10/9 seq 18/17 10/9 fused 18/17 to 24/23 fused to 26/25 K1 I332E
seq 10/9 seq 18/17 10/9 fused 18/17 to fused to 24*/23* 26/25 K2
S239D/I332E seq 10/9 seq 18/17 10/9 fused 18/17 to fused to 24*/23*
26/25 K3 I332E/G236A seq 10/9 seq 18/17 10/9 fused 18/17 to fused
to 24*/23* 26/25 K4 S239D/I332E/G236A seq 10/9 seq 18/17 10/9 fused
18/17 to fused to 24*/23* 26/25 L0 -- seq 10/9 seq 20/19 10/9 fused
20/19 to 24/23 fused to 26/25 L1 I332E seq 10/9 seq 20/19 10/9
fused 20/19 to fused to 24*/23* 26/25 L2 S239D/I332E seq 10/9 seq
20/19 10/9 fused 20/19 to fused to 24*/23* 26/25 L3 I332E/G236A seq
10/9 seq 20/19 10/9 fused 20/19 to fused to 24*/23* 26/25 L4
S239D/I332E/G236A seq 10/9 seq 20/19 10/9 fused 20/19 to fused to
24*/23* 26/25 M0 -- seq 10/9 seq 22/21 10/9 fused 22/21 to 24/23
fused to 26/25 M1 I332E seq 10/9 seq 22/21 10/9 fused 22/21 to
fused to 24*/23* 26/25 M2 S239D/I332E seq 10/9 seq 22/21 10/9 fused
22/21 to fused to 24*/23* 26/25 M3 I332E/G236A seq 10/9 seq 22/21
10/9 fused 22/21 to fused to 24*/23* 26/25 M4 S239D/I332E/G236A seq
10/9 seq 22/21 10/9 fused 22/21 to fused to 24*/23* 26/25
Full-length sequences of heavy and light chains are listed in
colums V and VI. (The sequences in column V marked with * refer to
the IgG1 sequence of SEQ ID NOs 24 and 23 (wild-type sequences)
that have been modified to have the substitution(s) corresponding
to column II, and the respective mutation(s) in the coding
DNA).
EXAMPLE 2
[0190] Chimeric mAb A0 Specifically Recognizes the CD37 Antigen
[0191] The specificity of MAb A0 for cellular CD37 is tested in a
FACS competition assay on Ramos Burkitt lymphoma cells (ATCC
#CRL-1596). Cells are grown in tissue culture flasks (175 cm.sup.2)
using RPMI-1640+GlutaMAX supplemented with 10% heat-inactivated
fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM
non-essential amino acids as culture medium. Cells are cultivated
with an initial density of 3.times.10.sup.5 cells/ml at 37.degree.
C. and 5% CO.sub.2 in a humidified atmosphere for three days. The
cultures are maintained at a cell concentration between
3.times.10.sup.5 and 1.8.times.10.sup.6/ml by sub-cultivation in a
ratio of 1:6 with fresh culture medium 2-3 times a week. For FACS
competition analysis, the CD37-specific mAb HH1 (Santa Cruz)
directly labeled with phycoerythrin (PE) is used at a concentration
of 1 .mu.g/ml. The antibody is preincubated with the unlabelled
competitor antibody A0 for 10 min at 4.degree. C. at the indicated
molar ratio. Thereafter, 1.times.10.sup.5 Ramos cells are incubated
for 30 min with the antibody mixture on ice. Thereafter, cells are
washed twice in phosphate buffered saline (PBS), resuspended in
FACS buffer and measured on a BD FACS Canto. Results of such an
assay are shown in FIG. 1. Addition of a control human IgG1
antibody (Sigma IgG1 kappa) at 20-fold molar excess does not
significantly reduce the mean fluorescence intensity (MFI) of Ramos
cells. Addition of either unlabelled HH1 antibody or A0 antibody at
20-fold molar excess almost completely abrogated binding of the
directly labeled HH1 antibody. This indicates that A0 and HH1
antibodies recognize identical or similar epitopes on Ramos cells
and compete for binding to cellular CD37 antigen.
EXAMPLE 3
[0192] Binding of Humanized Versions of mAb A0 to Cellular CD37
Antigen
[0193] Humanized versions of A0 are tested for their binding to
cellular CD37 antigen by FACS analysis. Antibodies are added to
Ramos cells at the indicated concentrations and allowed for binding
for 30 min at 4.degree. C. Thereafter, bound antibody is detected
with PE-labelled goat-anti-human IgG antibody (Sigma), cells are
washed twice with PBS, and thereafter cells are resuspended in FACS
buffer and analyzed by FACS on a BD FACS Canto. Examples are shown
in FIGS. 2 and 3 (antibodies A, B, C, D, I or A, H, I, J, K, L and
M, respectively; see Table 1). Several of the humanized versions of
A0 show similar binding to Ramos cells as the parental antibody A0,
indicating that humanization does not reduce binding to cellular
CD37 antigen.
EXAMPLE 4
[0194] FACS Scatchard Analysis of Humanized Versions of Chimeric
mAb A0
[0195] The affinity of humanized versions of antibody A0
(designated B, C, D, H, I and K; see Table 1) to cellular CD37
antigen is determined by FACS scatchard analysis as described
elsewhere (Brockhoff et al., 1994). Briefly, antibody dilutions are
prepared in a 96 well plate starting with 100-400 nM in the first
well (80 .mu.l), followed by 11 dilution steps (1:2, 40+40 .mu.l).
50 .mu.l of mAb dilutions are added to FACS tubes, 150 .mu.l cells
(0.8.times.10.sup.6/ml=1.2.times.10.sup.5 cells/tube) are added to
each FACS tube. Cells are gently mixed and incubated for 1 h on
ice. Thereafter 50 .mu.l FITC conjugated secondary antibody
(conc.15 .mu.g/ml; mouse mAb anti-hu IgG all subclasses, Zymed
05-4211) is added, mixed, and incubated for 30 min on ice. 4 ml PBS
ph7.2 containing 0.02% acid are added thereafter, cells are
pelleted and resuspended in 300 .mu.l PBS pH 7.2 and subjected to
FACS analysis using a BD FACS Canto. All experimental steps are
performed on wet ice, all antibody dilutions are made in PBS/0,5%
BSA+0.02% acid. FACS calibration is performed using Quantum FITC
MESF (Premix) High Level Beads (Bangs Laboratories). All samples
are measured using the same FACS parameters. The ratio of bound IgG
versus free IgG is calculated from MFI values at different antibody
concentrations and displayed as scatchard blot. FIG. 4 shows the
MFI/antibody concentration relationship of several humanized
variants of A0. The results show similar binding to Ramos cells of
some of the humanized versions as the starting antibody, with
dissociation constants (K.sub.d) ranging from 2.15 to 4.90
nanomoles/liter.
EXAMPLE 5
[0196] ADCC Activity of Humanized Versions of the Chimeric mAb
A0
[0197] The ability of humanized versions of A0 (designated B, C, D,
H, J, K; see Table 1) to mediate antibody-dependent cell-mediated
cytotoxicity (ADCC) is assessed using Ramos cells as target cells
and IL2-stimulated human PBMCs as effector cells. Ramos cells
(Burkitt's lymphoma; ATCC #CRL-1596) were purchased from ATCC.
Cells are grown in tissue culture flasks (175 cm.sup.2) using
RPMI-1640+GlutaMAX supplemented with 10% heat-inactivated fetal
bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM
non-essential amino acids as culture medium. Cells are cultivated
with an initial density of 3.times.10.sup.5 cells/ml at 37.degree.
C. and 5% CO.sub.2 in a humidified atmosphere for three days. The
cultures are maintained at a cell concentration between
3.times.10.sup.5 and 1.8.times.10.sup.6/ml by sub-cultivation in a
ratio of 1:6 with fresh culture medium 2-3 times a week. An aliquot
of the cell culture at a cell density between 1.5.times.10.sup.6/ml
and 1.8.times.10.sup.6/ml and growing in the log-phase is
centrifuged (200.times.g, i.e. 1000 rpm) for 10 min. Cells are
washed once with washing medium (RPMI 1640 w/o L-glutamine) and
pelleted (200.times.g, i.e. 1000 rpm; 10 min). Cell pellet is
resuspended in assay medium [1% BSA in RPMI w/o L-glutamine] and
cell count is determined. Cell concentration is adjusted to
2.times.10.sup.5/ml.
[0198] Approximately 50-80 ml whole blood drawn from healthy donors
is used for the isolation of PBMC. 10 ml whole blood are diluted
1:3.6 with 26 ml HBSS (Hanks' Balanced Salt Solution w/o calcium
and magnesium) in a 50 ml tube. 18 ml diluted whole blood is
layered on top of 12 ml Lymphoprep (Nycomed Pharma) in a 50 ml tube
and centrifuged at 370.times.g (1400 rpm) for 35 min. The
mononuclear cells from the interface are aspirated and washed first
with HBSS (750.times.g, i.e. 1900 rpm; 10 min), then a second time
with HBSS (300.times.g, i.e. 1200 rpm; 10 min) and at last with
HBSS (160.times.g, i.e. 900 rpm; 10 min). The pelleted cells are
gently resuspended in culture medium/assay medium (10%
heat-inactivated human AB serum in RPMI 1640 w/o L-glutamine) using
a pipette and the cell count is determined in the cell counter. The
PBMC concentration is adjusted to 1.times.10.sup.7/ml. The freshly
isolated PBMC (5.times.10.sup.5/ml) are maintained in culture
medium (RPMI 1640 w/o L-glutamine supplemented with 10% human AB
serum) in a tissue culture flask (75 cm.sup.2) at 37.degree. C. in
CO.sub.2 incubator over night. On the following day cells are
stimulated with hIL-2 at a final concentration of 1 U/ml for 3
further days. IL-2 stimulated PBMC are separated from cell debris
on a Lymphoprep gradient. The purified IL-2 stimulated PBMC are
suspended in culture medium/assay medium at a concentration of
1.times.10.sup.7/ml.
[0199] The co-cultivation of effector cells with target cells in
presence of specific or unspecific antibody is performed in
duplicates or triplicates in 96-well round-bottom microtiter plates
in a final volume of 200 .mu.l assay medium per well consisting of
10% human AB serum and 1% BSA in RPMI in 1:1 ratio. First effector
cells (freshly isolated PBMC cells in 100 .mu.l 10% human AB serum
in RPMI per well) are plated, followed by target cells and antibody
solution diluted in 50 .mu.l 1% BSA in RPMI. As a control, effector
cells are cultivated in assay medium alone (effector cell control)
and target cells are cultivated either in assay medium alone
(spontaneous lysis) or in assay medium supplemented with 1% Triton
X-100 (maximal lysis). The co-culture is incubated at 37.degree. C.
in a humid CO.sub.2 incubator for 3 hours. At the end of the
incubation cells are removed from the culture medium by
centrifugation (200.times.g, i.e. 1000 rpm; 10 min) at room
temperature. Cell free supernatants (100 .mu.l/well) are
transferred into corresponding wells of a 96-well flat-bottom
plate. To determine the LDH activity in these supernatants 100
.mu.l reaction mixture (freshly mixed 250 .mu.l catalyst with 11.25
ml dye solution) are added to each well and incubated 30 min at
room temperature in the dark. Then the absorbance is measured as
described below.
[0200] Cytotoxicity Detection Kit (LDH; Roche) is used to measure
ADCC activity. The detection of cytotoxicity is based on the
measurement of LDH enzyme activity released from plasma
membrane-damaged cells. LDH released into the culture supernatants
reduces the tetrazolium salt from the kit to formazan. The
absorption maximum of formazan dye is measured at 490 nm against a
reference wavelength of 650 nm in an ELISA plate reader. To
calculate percent cell mediated cytotoxicity five controls are
performed in each experimental setup. [0201] Background control I
(1): LDH activity contained in the assay medium, which is
subtracted from values (3) and (5). [0202] Background control II
(2): LDH activity contained in 1% Triton-X100 in assay medium,
which is subtracted from maximal LDH release values (4). [0203]
Spontaneous LDH release (3): LDH activity released from target
cells alone. [0204] Maximal LDH release (4): Maximum releasable LDH
activity in the target cells. [0205] Effector cell control (5): LDH
activity released from effector cells only.
[0206] To determine the percentage cell mediated cytotoxicity, the
average absorbance of the triplicates or duplicates is calculated
and the background is subtracted according to the manufacturer's
instructions. In FIG. 5, the results from an ADCC assay using an
E:T ratio of 25:1 and Ramos target cells are shown. Antibodies are
added in a concentration of 30 ng/ml. Both the starting mAb and
humanized versions thereof display similar ADCC activity against
the Ramos cells. In conclusion, humanization of anti-CD37 mAb A
does not significantly alter its ADCC inducing capacity.
EXAMPLE 6
[0207] Pro-Apoptotic Activity of Humanized Versions of the Chimeric
mAb A0
[0208] The pro-apoptotic activity of mAb A0 (=A) and humanized
versions thereof (B, C, D and I; see Table 1) is assessed by
measurement of AnnexinV/PI positive cells after incubation of Ramos
cells with mAbs. Ramos cells (Burkitt's lymphoma; ATCC #CRL-1596)
are received from ATCC. Cells are grown in tissue culture flasks
(175 cm.sup.2) using RPMI-1640+GlutaMAX supplemented with 10%
heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium
pyruvat, 1% MEM non-essential amino acids as culture medium. Cells
are cultivated with an initial density of 3.times.10.sup.5 cells/ml
at 37.degree. C. and 5% CO.sub.2 in a humidified atmosphere for
three days. The cultures are maintained at a cell concentration
between 3.times.10.sup.5 and 1.8.times.10.sup.6/ml by
sub-cultivation in a ratio of 1:6 with fresh culture medium 2-3
times a week. An aliquot of the cell culture at a cell density
between 1.5.times.10.sup.6/ml and 1.8.times.10.sup.6/ml and growing
in the log-phase is centrifuged (200.times.g, i.e. 1000 rpm) for 10
min. Cells are washed once with washing medium (RPMI 1640 w/o
L-glutamine) and pelleted (200.times.g, i.e. 1000 rpm; 10 min).
Cell pellet is resuspended in culture medium and cell count is
determined. Cell concentration is adjusted to 1.times.10.sup.6/ml.
100 .mu.l cell suspension per well are plated into 96-well round
bottom plates. Antibodies are diluted in cell culture medium
containing 10% FBS and 100 .mu.l antibody solution are added per
well. Cells are incubated for 20 to 24 hrs at 37.degree. C. in
CO.sub.2 incubator and thereafter stained with Vybrant apoptosis
assay kit #2. Alexa Fluor 488 labelled Annexin V and propidium
iodide solution are added to the cells and incubated for 15 min in
the dark. Thereafter cells are resuspended in 400 .mu.l AnnexinV
binding buffer and subjected to FACS analysis using a BD FACS
Canto. The percentage of AnnexinV positive/PI negative cells and
AnnexinV/PI positive cells is determined in two-dimensional dot
blots using FL1/FL2 channels. An isotype matched non-binding
antibody (Sigma human IgG1) is used as negative control.
[0209] In FIG. 6, the pro-apoptotic effect of various humanized
versions of mAb A on Ramos cells are shown. Cells are incubated
with antibody at 10 .mu.g/ml for 24 hrs, the total percentage of
AnnexinV positive cells (PI positive and PI negative) is displayed.
Parental mAb A shows potent pro-apoptotic activity. Surprisingly,
humanized versions show a significantly reduced number of AnnexinV
positive cells compared to the parental mAb A, indicative for
altered pro-apoptotic activity of the humanized antibodies. In
conclusion, humanization of MAb A leads to a reduction of its
pro-apoptotic activity in this experimental setting.
EXAMPLE 7
[0210] ADCC Activity of Fc-Engineered Versions of Chimeric mAb
A0
[0211] ADCC activity of Fc-engineered versions of mAb A0
(designated A1, A2, A3, A4; see Table 2) is assessed using Ramos
cells as target cells. ADCC assay is performed as described above
(Example 5). The result of the experiment is shown in FIG. 7.
Fc-engineered versions of A0 show clearly improved potency and
efficacy compared to the parent mAb A0. Certain Fc variants show
improvement in maximal lysis of up to 100% compared to the parent
mAb and improvement in EC.sub.50 of up to 10-fold compared to the
parent mAb. In conclusion, introduction of specific Fc mutants
strongly increases the ADCC activity of chimeric mAb A0.
EXAMPLE 8
[0212] ADCC Activity of Fc-Engineered Versions of mAb B0
[0213] ADCC activity of Fc-engineered versions of mAb B0
(designated B1, B2, B3, B4; see Table 2) is assessed using Ramos
cells as target cells. ADCC assay is performed as described above
(Example 5). Fc-engineered versions of B0 show clearly improved
potency and efficacy compared to the parent mAb B0. Certain Fc
variants show improvement in maximal lysis of up to 80% compared to
the parent mAb and improvement in EC.sub.50 of up to 20-fold
compared to the parent mAb. In conclusion, introduction of specific
Fc mutants strongly increases the ADCC activity of humanized mAb
B0. The results of the experiments are shown in FIG. 8.
EXAMPLE 9
[0214] Pro-Apoptotic Activity of mAbs A0 and B0
[0215] The pro-apoptotic activity of mAbs A0 and B0 on Ramos cells
before and after cross-linking with anti-IgG mAb is displayed in
FIG. 9. The apoptotic assay is performed as described in Example 6,
for antibody cross-linking an anti-human IgG antibody
(.gamma.-chain specific; Sigma) is added to the antibodies in a
ratio of 1:1 and incubated for 15 min at 37.degree. C. prior to
adding to the target cells. In FIG. 9 the CD37-specific mAbs are
added at a concentration of 1 .mu.g/ml with and without
cross-linking Chimeric mAb A0 is a potent inducer of apoptosis even
without cross-linking, this effect is significantly enhanced after
cross-linking of the mAb. Surprisingly, without cross-linking, the
humanized mAb B0 is completely devoid of pro-apoptotic activity,
however shows potent pro-apoptotic activity after cross-linking
with anti-IgG Ab. In conclusion, this experiment shows that
pro-apoptotic activity of a humanized version of mAb A0 can be
restored after antibody cross-linking
EXAMPLE 10
[0216] Pro-Apoptotic Activity of Fc-Engineered Versions of mAb
A0
[0217] The pro-apoptotic activity of Fc-engineered versions of
chimeric mAb A0 on Ramos cells is assessed by AnnexinV/PI staining
as described in Example 6. Parent1 antibody A0 and Fc-engineered
variants A2 and A4 are titrated over a concentration range from 0.1
to 10.000 ng/ml. As can be seen in FIG. 10, all 3 antibodies show
similar pro-apoptotic activity. In conclusion, this experiment
shows that Fc-engineering of mAb A0 does not alter its
pro-apoptotic activity.
EXAMPLE 11
[0218] a) B Cell Depleting Activity of Fc-Engineered Antibodies A2
and B2 in a Whole Blood Assay
[0219] The efficacy and potency of depletion of normal B cells from
human blood is assessed using a whole blood assay. In this assay
format, the test antibody is added to EDTA-treated samples of human
blood from healthy individuals and subsequently, after 3 to 4 hrs
incubation at 37.degree. C., the number of B cells is
quantitatively measured by a 4-color FACS assay. By comparison to
buffer or IgG controls, the degree of B cell depletion by the test
agent can be calculated. Due to the presence of human IgG levels
and effector cells similar to the situation in human beings in
vivo, this assay type is considered of high relevance for
predicting the effect of the test antibodies in vivo.
[0220] A quantitative FACS assay is used to determine the number of
B cells and/or spiked Ramos cells in blood samples derived from
healthy individuals. Quantification is performed using BD Trucount
tubes which contain a known number of fluorescent beads which serve
as internal standard for quantification of the cell population of
interest. B cells are identified by 4-colour analysis using 4
different CD markers (CD3/CD14/CD19/CD45) in combination with
FSC/SSC analysis.
[0221] 270 .mu.l fresh blood per well is incubated in a 48 well
plate together with 30 .mu.l antibody dilution (in PBS) or PBS
(buffer control) in duplicates. Samples are incubated for 4h at
37.degree. C. and thereafter immediately placed on ice. 33 .mu.l of
CD marker master mix is added to Trucount tubes and 50 .mu.l of the
blood-antibody mixture is added. Samples are vortexed and incubated
for 15 min at room temperature. Thereafter 450 .mu.l of lysis
buffer is added, vortexed, and incubated for additional 15 min at
room temperature. Samples are placed on ice and immediately
subjected to FACS analysis using a BD FACS Canto.TM. Flow
Cytometer. Evaluation of data is performed using the BD FACSDiva
software (Version 5.0.2).
[0222] Fc-engineered chimeric and humanized mAbs A2 and B2 show
excellent potency on normal B cell depletion with EC.sub.50 values
ranging from 0.15 to 0.35 nM. The degree of normal B cell depletion
ranges from 57% to 65%. Rituximab, a registered antibody used for
the treatment of B-NHL, is tested in parallel and yields
significantly lower depletion of B-cells in this assay format (FIG.
11A).
[0223] b) Fc-Engineering Introduces Superior B Cell Depleting
Activity of A0 and B0 Compared to Rituximab
[0224] The effect of mAbs on B cell depletion in human blood
derived from healthy individuals is assessed as described in a).
The non Fc-engineered mAbs A0 and B0 show B cell depleting activity
in the range from 13% to 26%, similar to Rituximab. Fe-engineering
results in a dramatic increase of B cell depleting activity for
both mAbs, with a mean percentage of depletion of 75%. This clearly
demonstrates the superiority of A2 and B2 compared to Rituximab
(FIG. 11B).
[0225] c) Antibodies A2 and B2 Do Not Deplete T Cells and Monocytes
in Whole Blood Assays
[0226] The effect of A2 and B2 on T lymphocytes (CD3-positive) and
monocytes (CD14-positive) is assessed in parallel to the effect on
B lymphocytes. No significant change of either T cell numbers or
monocyte numbers is observed, whereas a significant reduction of
the number of B cells is seen (FIG. 11C). This indicates that A2
and B2 specifically deplete B cells from human blood.
EXAMPLE 12
[0227] Fc-Engineering Introduces Superior ADCC Activity Compared to
Rituximab
[0228] ADCC activity of the Fc-engineered version A2 of mAb A0 is
assessed using Ramos cells as target cells. ADCC assay is performed
as described above (Example 5). The non Fc-engineered antibody A0
shows a maximal lysis of Ramos target cells which is inferior to
Rituximab, an antibody specific for CD20 which is an approved
treatment for patients suffering from B cell lymphomas.
Surprisingly, Fc-engineering of A0 leads to a clearly improved
potency and efficacy of A2 over Rituximab. This indicates, that at
similar antigen densities of CD20 and CD37 on Ramos cells the
Fc-engineered anti-CD37 mAb A2 shows clearly improved ADCC activity
than Rituximab (FIG. 12).
EXAMPLE 13
[0229] Lymphoma Cell Depleting Activity of Fc-Engineered Antibodies
A2 and B2 in a Whole Blood Assay
[0230] The efficacy and potency of depletion of Ramos cells, a
Burkitt's lymphoma derived cell line from human blood is assessed
using a whole blood assay as described in Example 11. In a
modification of the assay, Ramos tumor cells are spiked in about a
tenfold excess compared to endogenous B cells into the whole blood
matrix, and their depletion is also monitored by FACS analysis.
Fc-engineered chimeric and humanized mAbs A2 and B2 show good
potency on Ramos cell depletion with EC.sub.50 values ranging from
0.35 to 0.54 nM. The degree of Ramos cell depletion ranges from 36%
to 55%. Rituximab, a registered antibody used for the treatment of
B-NHL, is tested in parallel and yields significantly lower
depletion of Ramos cells in this assay format (FIG. 13).
EXAMPLE 14
[0231] In vivo Efficacy of Fc-Engineered Antibodies A2 and B2 in
Disease-Related Model
[0232] The in vivo anti-tumor efficacy of mAbs A2 and B2 is
assessed using a Ramos Burkitt's lymphoma model in nude mice.
CD37-positive Ramos cells are injected subcutaneously into the
flank of the animals and i.v. treatment of the animals started when
tumors are established. A twice weekly treatment schedule is chosen
(q3/4d), two different doses (8 mg/kg and 25 mg/kg) are tested in
parallel. Both mAbs show significant anti-tumor efficacy with T/C
values ranging from 0.2% to 26%. No significant difference between
the two dose levels and between the two antibodies are observed.
However, there is a trend towards better efficacy in the high dose
A2 treated animals, with T/C of 0.2% and 5/10 complete tumor
regressions. All treatments are well tolerated with no apparent
weight loss. In conclusion, mAbs A2 and B2 showed significant
anti-tumor efficacy in the Ramos Burkitt's lymphoma model, with
maximum activity already obtained at the 8 mg/kg dose level. The
activity is comparable to that of rituximab which is tested in
parallel. It has to be noted that the in vivo activity observed
with the Fc-engineered antibodies A2 and B2 may be underestimated
since these mAbs are optimized for interaction with human but not
murine effector cells. This optimized interaction, which leads to
strongly improved ADCC activity in vitro when using human effector
cells (Example 8), is not reflected in the mouse model used.
However, the data obtained in this experiment (shown in FIG. 14)
provide in vivo proof of concept of CD37 targeting and thus can be
used for estimating the therapeutic dose in humans.
EXAMPLE 15
[0233] Correlation of Pharmacokinetic and Pharmacodynamic Effect of
A2 and B2 in Mice for Estimating the Therapeutic Dose in Humans
[0234] A correlation between the serum concentrations of A2 and B2
to their pharmacodynamic effect is established in mice using the
Ramos tumor xenograft model. These studies demonstrate that a dose
of 8 mg/kg A2 and B2 (formulated in a citrate buffer: 25 mM
Na-Citrate, 115 mM NaCl, 0.04% Tween 80, pH 6.0) causes significant
retardation of tumor growth in this aggressive s.c. (subcutaneous)
tumor model using a standard q3 or 4d antibody dosing schedule in
mice, thus indicating continued activity throughout the dosing
interval. Furthermore, pharmacokinetic data are established for the
same dose.
[0235] Using this PK/PD association in mice, an estimated human
dose can be calculated using published data for the clearance (CL)
of humanized antibodies in humans (Lobo et al., 2004).
[0236] Full calculation for A2: [0237] Mean AUC(0-.infin.) after
single dose of 8 mg/kg=6099 .mu.gh/mL. [0238] Given AUC(0-.infin.)
in mice=AUC(ss,.tau.) in mice, and AUC(ss,.tau.)/.tau.=C(ave,ss).
[0239] C(ave,ss) in mice (for .tau.=84 hours)=73 .mu.g/mL, which is
presumably equivalent to C(ave,ss) in men (for .tau.=168 h). [0240]
Since AUC(ss,.tau.) in men=D/CL, and using the humanized antibody
clearance (CL) range in humans reported by Lobo et al, 2004.: CL=7
mL/h/70 kg to 15 mL/h/70 kg. [0241] For 7 mL/h/70 kg: 168
hr.times.7=1176 mL.times.73 .mu.g=86 mg. [0242] For 15 mL/h/70 kg:
168 hr.times.15=2520 mL.times.73 .mu.g=184 mg.
[0243] Therefore, for A2, the estimated weekly dose for a 70 kg
human is in the range of 86 to 184 mg. Using the same assumptions
as described above, the calculated estimated human weekly dose for
B2 for a 70 kg human is 189 to 404 mg.
EXAMPLE 16
[0244] Antibodies A2 and B2 Show ADCC Activity on Multiple Myeloma
Cells
[0245] The expression of CD37 on a panel of multiple myeloma cell
lines is assessed by FACS analysis using antibodies specific for
CD37. Cells are either incubated with a directly fluorescently
labeled anti-CD37 antibody or an unlabeled CD37-specific antibody
followed by a second fluorescently labeled antibody directed
against the primary antibody. The fluorescence activity of the
labeled cells is measured with a FACS Canto Flow Cytometer (BD
Biosciences) and the fluorescence intensity is recorded as MFI
using the FACS Diva Software. 6 out of 11 tested multiple myeloma
demonstrate cell surface expression of CD37 (FIG. 15). One cell
line (RPMI 8226) is subsequently tested in an ADCC assay as
described in Example 5 using the CD37-specific antibodies A2 and
B2. Both antibodies demonstrate potent ADCC activity on RPMI 8226
cells with EC.sub.50 values in the range of 25 ng/ml and a maximal
cell lysis of about 20% (FIG. 16). This example demonstrates that
CD37-positive multiple myeloma cells are susceptible to ADCC
mediated cell lysis using the CD37-specific mAbs A2 and B2.
[0246] FIG. 15 shows the FACS analysis of six multiple myeloma
derived cell lines for CD37 expression. The open curves indicate
reactivity with the CD37-specific antibody, the filled curves
represent the negative control antibody.
EXAMPLE 17
[0247] Pro-Apoptotic Activity of Antibodies A2 and B2 on
Patient-Derived CLL Cells
[0248] The pro-apoptotic activity of A2 and B2 is assessed on
patient-derived chronic lymphocytic leukemia (CLL) cells.
Peripheral blood mononuclear cells (PBMCs) are prepared from a
patient with diagnosed CLL, after informed consent according to the
declaration of Helsinki has been obtained. The primary CLL cells
are purified from freshly collected blood according to
Ficoll-Paque.RTM. plus procedure (StemCell Technologies, Meylan,
France) and stored at 4.degree. C. in RPMI 1640 culture medium
containing 10% heat inactivated human AB serum (Sigma, France)
until use. The culture medium for primary CLL cells is RPMI 1640
supplemented with 2 mM L-glutamine and 10% of heat inactivated
human AB serum. For experimental use, primary CLL cells are counted
in a hemocytometer and their viability is assessed by 0.25% trypan
blue exclusion. The viability of CLL samples is more than 90%.
Cells are incubated at 37.degree. C. for 24 hours with the
antibodies at 30 .mu.g/ml and thereafter the percentage of AnnexinV
positive cells is determined as described in Example 6. As shown in
FIG. 17, Fc-engineered antibodies A2 and B2 show strong
pro-apoptotic activity on the primary CLL cells with about 90% (A2)
and 40% (B2) AnnexinV positive cells. Both mAbs are clearly
superior to rituximab, a B cell-specific antibody approved for the
treatment of B-NHL. Mab A2 demonstrates also clearly superior
activity compared to alemtuzumab, an antibody approved for the
treatment of B-CLL.
EXAMPLE 18
[0249] Generation of a Transgenic Mouse Model in which the
Endogenous CD37 Gene is Replaced by its Human Homologue
[0250] A targeting vector is constructed which contains the coding
sequence of human CD37 (BAC (bacterial artificial chromosome) IDs:
RP11-433N13, RP11-50111) flanked by non-translated sequences. This
targeting vector (which contains, in addition, loxP sites flanking
exons 3-4 and the neo selection marker flanked by frt sites) is
then used for homologous recombination, using mouse ES cells and
standard technology to replace exons 1-8 of the mouse genomic
sequence with the human counterpart sequences, To this end, the
C57BL/6N ES cell line is grown on a mitotically inactivated feeder
layer comprised of mouse embryonic fibroblasts (MEF) in DMEM High
Glucose medium containing 20% FBS (PAN) and 1200 u/mL Leukemia
Inhibitory Factor (Millipore ESG 1107). 1.times.10.sup.7 cells and
30 g of linearized DNA vector are electroporated (Biorad Gene
Pulser) at 240 V and 500 F. G418 selection (200 g/mL) started on
d2. Counterselection with Gancyclovir (2 M) starts on d5 after
electroporation. ES clones are isolated on d8 and analyzed by
Southern Blotting according to standard procedures, e.g. by the use
of radiolabelled DNA probes specific for the target gene after
expansion and freezing of clones in liquid nitrogen. Transgenic
animals are then generated by standard procedures known in the art,
e.g. by blastocyst injection and subsequent generation of chimeric
animals. Animals heterozygous and homozygous for human CD37 are
obtained by conventional breeding of chimeric and heterozygous
animals, respectively. The successful knock-out of the murine CD37
gene and the knock-in of the human CD37 gene is monitored at the
protein level using standard procedures, e.g. FACS analysis of
peripheral blood lymphocytes or immunohistochemical analysis of
tissue sections.
EXAMPLE 19
[0251] Generation of Surrogate Antibodies
[0252] Monoclonal antibodies specific for macaque CD37 are
generated by genetic immunization of mice and rabbits using the
complete coding sequence of the macaque CD37 antigen (Acc. No.
ENSMMUT00000020744). Specific antibodies are selected using
recombinant HEK293 or CHO cells expressing the macaque CD37
antigen, e.g. by standard ELISA or FACS techniques. The variable
heavy and light chain coding sequences of these antibodies are
retrieved by PCR cloning and used for generation of chimeric
antibodies (as described in Example 1) which harbor the VH and VL
region derived from the murine or rabbit starting antibody and an
Fc portion identical to that of an antibody of the invention, e.g.
A2 or B2. The binding and functional properties can be investigated
by the use of assay systems which utilize macaque CD37 expressing
cells as target cells, e.g. for binding, FACS, Scatchard analysis,
ADCC and apoptosis assays. Ultimately, the surrogate antibody is
selected by virtue of its B cell depleting activity in Cynomolgus
monkey blood in vitro.
EXAMPLE 20
[0253] Preparation of Clones for Producing the Antibodies
[0254] In order to prepare clones for producing antibodies of the
invention, e.g. antibodies A2, A4, B2 or B4, the DNA molecule
encoding the complete heavy chain, e.g. with the sequence shown in
SEQ ID NO: 27, 31, 35 or 39, respectively, is inserted into the
eukaryotic expression vector designated pBI-26, encoding in
addition the selection marker dihydrofolate reductase from
hamster.
[0255] The DNA molecule encoding the complete light chain, depicted
in SEQ ID NO: 29, 33, 37 and 41, respectively, is inserted into the
eukaryotic expression vector designated pBI-49, encoding in
addition the selection marker neomycin phosphotransferase. The DNA
sequences of the entire heavy and light chains are sequenced
completely.
[0256] The hamster cell line CHO-DG44, grown in suspension in
chemically defined media, is co-transfected with the eukaryotic
expression vectors for the heavy and for the light chain of the
antibodies, as described above. Transfected cells are selected in
medium without hypoxanthine and thymidine and in the presence of
the antibiotic G418. Subsequently, cells are subjected to stepwise
selection and amplification using increasing concentrations of
methotrexate (MTX). From the 800 nM MTX amplification step, a
single cell clone is selected based on growth performance and
antibody production in spinner runs, and is cryopreserved in a
Safety Cell Bank (SCB).
REFERENCES
[0257] American Cancer Society (Cancer Facts & FIGS. 2005).
[0258] Baker and Jones, Curr Opinion in Drug Discovery &
Development, 10, 219-227, 2007. [0259] Barbas, et al., Proc. Nat.
Acad. Sci, USA 91:3809-3813, 1994. [0260] Barrena et al., Leukemia
19: 1376-1383, 2005. [0261] Belov et al., Cancer Research 61:
4483-4489, 2001. [0262] Boulianne G. L., Hozumi N. and Shulman, M.
J., Production of functional chimeric mouse/human antibody. Nature
312: 643, 1984. [0263] Brockhoff G, Hofstaedter F, Knuechel R.,
Cytometry 1994, 17(1):75-83. [0264] Buchsbaum et al., Cancer
Research 52: 6476-6481, 1992. [0265] Chothia and Lesk., J. Mol.
Biol. 196: 901-917, 1987. [0266] Coiffier B, et al. Rituximab
(anti-CD20 monoclonal antibody) for the treatment of patients with
relapsing or refractory aggressive lymphoma: a multicenter phase II
study. Blood 1998; 92: 1927-1932. [0267] Coiffier, JCO, 23,
6387-93, 2005. [0268] Edelman et al. Proc Natl Acad Sci USA 63:
78-85, 1969. [0269] Feugier P, et al. Long-term results of the
R-CHOP study in the treatment of elderly patients with diffuse
large B-cell lymphoma: a study by the Groupe d'Etude des Lymphomes
de l'Adulte. J Clin Oncol 2005; 23: 4117-4126. [0270] Foran J M, et
al. European phase II study of rituximab (chimeric anti-CD20
monoclonal antibody) for patients with newly diagnosed mantle-cell
lymphoma and previously treated mantle-cell lymphoma, immunocytoma,
and small B-cell lymphocytic lymphoma. J Clin Oncol 2000; 18:
317-324. [0271] Forstpointner R, et al. The addition of rituximab
to a combination of fludarabine, cyclophosphamide, mitoxantrone
(FCM) significantly increases the response rate and prolongs
survival as compared with FCM alone in patients with relapsed and
refractory follicular and mantle cell lymphomas: results of a
prospective randomized study of the German Low-Grade Lymphoma Study
Group. Blood, 2004; 104: 3064-3071. [0272] Francisco et al., Blood,
2003 Aug. 15; 102(4):1458-65. [0273] Frank, et al., Methods
Enzymol. 154: 221-249, 1987. [0274] Gait, M. J., Oligonucleotide
Synthesis. A Practical Approach. IRL Press, Oxford, UK (1984).
[0275] Goldenberg D M and Sharkey R M, Oncogene (2007) 26,
3734-3744. Novel radiolabeled antibody conjugates. [0276]
Hainsworth J D. Prolonging remission with rituximab maintenance
therapy. Semin Oncol 2004; 31: 17-21. [0277] Hawkins et al., J.
Mol. Biol. 254:889-896, 1992. [0278] Hayden and Mandecki. Gene
synthesis by serial cloning of oligonucleotides. DNA 7(8): 571-7,
1988. [0279] Hertz T, Yanover C: PepDist: A new framework for
protein-peptide binding prediction based on learning peptide
distance functions. BMC Bioinform (2006) 7 (Suppl 1):S3-S17. [0280]
Hiddemann W, et al. Frontline therapy with rituximab added to the
combination of cyclophosphamide, doxorubicin, vincristine, and
prednisone (CHOP) significantly improves the outcome for patients
with advanced-stage follicular lymphoma compared with therapy with
CHOP alone: results of a prospective randomized study of the German
Low-Grade Lymphoma Study. Group. Blood 2005; 106: 3725-3732
(2005a). [0281] Hiddemann W, et al. Treatment strategies in
follicular lymphomas: current status and future perspectives. J
Clin Oncol 2005b: 23; 6394-6399. [0282] Howard O M, et al.
Rituximab and CHOP induction therapy for newly diagnosed
mantle-cell lymphoma: molecular complete responses are not
predictive of progression-free survival. J Clin Oncol 2002; 20:
1288-1294. [0283] Ichimura et al., J. Antibiot. (Tokyo), 44,
1045-53, 1991. [0284] Jackson et al., 1995, J. Immunol.
154(7):3310-9. [0285] Johnson S, Bird R E. Construction of
single-chain derivatives of monoclonal antibodies and their
production in Escherichia coli. Methods Enzymol. 203: 88-98, 1991.
[0286] Jones T D, Hanlon M, Smith B J, Heise C T, Nayee P D,
Sanders D A, Hamilton A, Sweet C, Unitt E, Alexander G, Lo K M et
al: The development of a modified human IFN-.alpha.2b linked to the
Fc portion of human IgG1 as a novel potential therapeutic for the
treatment of hepatitis C virus infection. J Interferon Cytokine Res
(2004) 24(9):560-572. [0287] Jones T D, Phillips W J, Smith B J,
Bamford C A, Nayee P D, Baglin T P, Gaston J S, Baker M P:
Identification and removal of a promiscuous CD4+ T cell epitope
from the C1 domain of Factor VIII. J Thromb Haemost (2005) 3(5):
991-1000. [0288] Kabat E. A., Wu T. T., Perry H. M., Gottesman K.
S. and Foeller C. Sequences of Proteins of Immunological Interest
(5th Ed.). NIH Publication No. 91-3242. U.S. Department of Health
and Human Services, Public Health Service, National Institutes of
Health, Bethesda, Md. 1991. [0289] Kahl B, et al. Maintenance
rituximab following induction chemoimmunotherapy may prolong
progression-free survival in mantle cell lymphoma: a pilot study
from the Wisconsin Oncology Network. Ann Oncol, 2006; 17:
1418-1423. [0290] Kaminski et al., JCO 10: 1696-1711, 1992. [0291]
Kipriyanow and Le Gall, Molecular Biotechnology 26: 39-60, 2004.
[0292] Knobeloch et al., Mol Cell Biol 20: 5363-5369, 2000. [0293]
van der Kolk L E, Baars J W, Prins M H, van Oers M H. Rituximab
treatment results in impaired secondary humoral immune
responsiveness. Blood, 2002, Sep. 15; 100(6):2257-9. [0294]
McLaughlin P, et al. Rituximab chimeric anti-CD20 monoclonal
antibody therapy for relapsed indolent lymphoma: half of patients
respond to a four-dose treatment program. J Clin Oncol 1998; 16:
2825-2833. [0295] Lazar et al., Proc Natl Acad Sci USA. 2006 Mar.
14; 103(11):4005-10, 2006. [0296] Ling and MacLennan, pp. 302-335
in Leucocyte Typing III. White Cell Differentiation Antigens,
Oxford University Press, 1987. [0297] Link et al., Journal Immunol
137: 3013-3018, 1986. [0298] Lobo et al., J Pharm Sci
2004;93(11):2645-2668. [0299] Lowman et al., Biochemistry 30(45):
10832-10837, 1991. [0300] Marks et al., Biotechnology 10:779-783,
1992. [0301] Moldenhauer G., et al., 1987. Biochemical
characterization and epitope analysis of B lymphocyte-specific
surface antigens defined by clustering workshop monoclonal
antibodies. In Leukocyte Typing 111. A. McMichael. ed. Oxford
University Press, Oxford. p. 378. [0302] Moldenhauer G.: CD37. J.
Biol Regul Homeost Agents 2000; 14: 281-83. [0303] Press et al.,
JCO 7, 1989. [0304] Press O W, et al. Radiolabeled-antibody therapy
of B-cell lymphoma with autologous bone marrow support. N Engl J
Med 1993; 329: 1219-1224. [0305] Reche P A, Glutting J P, Zhang H,
Reinherz E L: Enhancement to the RANKPEP resource for the
prediction of peptide binding to MHC molecules using profiles.
Immunogenetics (2004), 56(6):405-419. [0306] Remington: The Science
and Practice of Pharmacy, 21st edition, Hendrickson R. Ed. [0307]
Romaguera J E, et al. High rate of durable remissions after
treatment of newly diagnosed aggressive mantle-cell lymphoma with
rituximab plus hyper-CVAD alternating with rituximab plus high-dose
methotrexate and cytarabine. J Clin Oncol, 2005; 23: 7013-7023.
[0308] Sasse et al., J. Antibiot. (Tokyo), 53, 879-85, 2000. [0309]
Shier et al., 1995, Gene 169:147-155. [0310] Schwartz-Albiez et al,
Journal Immunol 140: 905-914, 1988. [0311] Stemmer et al.
Single-step assembly of a gene and entire plasmid from large
numbers of oligodeoxyribonucleotides, Gene 164(1): 49-53, 1995.
[0312] van Spriel et al., Journal Immunol 172: 2953-2961, 2004.
[0313] Suzawa et al., Bioorg. Med. Chem., 8, 2175-84, 2000. [0314]
Tangri S, Mothe B R, Eisenbraun J, Sidney J, Southwood S, Briggs K,
Zinckgraf J, Bilsel P, Newman M, Chesnut R, LiCalsi C, Sette A:
Rationally engineered therapeutic proteins with reduced
immunogenicity. J Immunol (2005) 174(6):3187-3196. [0315] Thomas D
A, et al. Chemoimmunotherapy with hyper-CVAD plus rituximab for the
treatment of adult Burkitt and Burkitt-type lymphoma or acute
lymphoblastic leukemia. Cancer 2006; 106: 1569-1580. [0316] Ye et
al. Gene synthesis and expression in E. coli for pump, a human
matrix metalloproteinase. Biochem Biophys Res Commun 186(1):143-9,
1992. [0317] Yelton et al., 1995, Immunol. 155:1994-2004. [0318]
Zhao et al., Blood 104: Abstract 2515, 2004. [0319] Zhao X B,
Lapalombella R, Joshi T, Cheney C, Gowda A, Hayden-Ledbetter M S,
Baum P R, Lin T S, Jarjoura D, Lehman A, Kussewitt D, Lee R J,
Caligiuri M A, Tridandapani S, Muthusamy N, Byrd J C. Targeting
CD37+ lymphoid malignancies with a novel engineered small modular
immunopharmaceutical 2007; BLOOD, 1 Oct. 2007, VOLUME 110, NUMBER 7
(Epub ahead of print. Blood. 2007 Apr. 17)
Sequence CWU 1
1
421348DNAMus musculusCDS(1)..(348) 1gcg gtc cag ctg cag cag tct gga
cct gag ctg gaa aag cct ggc gct 48Ala Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15tca gtg aag att tcc tgc aag
gct tct ggt tac tca ttc act ggc tac 96Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30aat atg aac tgg gtg aag
cag aat aat gga aag agc ctt gag tgg att 144Asn Met Asn Trp Val Lys
Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45gga aat att gat cct
tat tat ggt ggt act acc tac aac cgg aag ttc 192Gly Asn Ile Asp Pro
Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60aag ggc aag gcc
aca ttg act gta gac aaa tcc tcc agc aca gcc tac 240Lys Gly Lys Ala
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80atg cag
ctc aag agt ctg aca tct gag gac tct gca gtc tat tac tgt 288Met Gln
Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95gca
aga tcg gtc ggc cct atg gac tac tgg ggt caa gga acc tca gtc 336Ala
Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105
110acc gtc tct tct 348Thr Val Ser Ser 1152116PRTMus musculus 2Ala
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10
15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp
Ile 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg
Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr Trp
Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 1153324DNAMus
musculusCDS(1)..(324) 3gac atc cag atg act cag tct cca gcc tcc cta
tct gca tct gtg gga 48Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
Ser Ala Ser Val Gly1 5 10 15gag act gtc acc atc aca tgt cga aca agt
gaa aat gtt tac agt tat 96Glu Thr Val Thr Ile Thr Cys Arg Thr Ser
Glu Asn Val Tyr Ser Tyr 20 25 30ttg gct tgg tat cag cag aaa cag gga
aaa tct cct cag ctc ctg gtc 144Leu Ala Trp Tyr Gln Gln Lys Gln Gly
Lys Ser Pro Gln Leu Leu Val 35 40 45tct ttt gca aaa acc tta gca gaa
ggt gtg cca tca agg ttc agt ggc 192Ser Phe Ala Lys Thr Leu Ala Glu
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60agt gga tca ggc aca cag ttt
tct ctg aag atc agc agc ctg cag cct 240Ser Gly Ser Gly Thr Gln Phe
Ser Leu Lys Ile Ser Ser Leu Gln Pro65 70 75 80gaa gat tct gga agt
tat ttc tgt caa cat cat tcc gat aat ccg tgg 288Glu Asp Ser Gly Ser
Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acg ttc ggt gga
ggc acc gaa ctg gag atc aaa cga 324Thr Phe Gly Gly Gly Thr Glu Leu
Glu Ile Lys Arg 100 1054108PRTMus musculus 4Asp Ile Gln Met Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile
Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Ser Phe Ala
Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys Arg 100
1055348DNAArtificialHumanized murine immunoglobulin sequence 5gcc
gtg cag ctg gtg cag agc gga gcc gag gtg aag aag ccc ggc agc 48Ala
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10
15agc gtc aag gtg tcc tgc aag gcc agc ggc tac agc ttc acc ggc tac
96Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30aac atg aac tgg gtg cgg cag gcc cca ggc cag gga ctg gaa tgg
atg 144Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45ggc aac atc gac ccc tac tac ggc ggc acc acc tac aac cgg
aag ttc 192Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg
Lys Phe 50 55 60aag ggc cgg gtg acc ctg acc gtg gac aag agc agc agc
acc gcc tac 240Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80atg gaa ctg agc agc ctg cgg agc gag gac acc
gcc gtg tac tac tgc 288Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95gcc aga tcc gtg ggc ccc atg gac tac tgg
ggc cag ggc acc ctg gtc 336Ala Arg Ser Val Gly Pro Met Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110acc gtc tct tca 348Thr Val Ser
Ser 1156116PRTArtificialSynthetic Construct 6Ala Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asn
Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60Lys
Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser 1157348DNAArtificialHumanized murine
immunoglobulin sequence 7gcc gtg cag ctg gtc gag tct ggc ggc gga
ctg gtg cag cct ggc ggc 48Ala Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15agc ctg aga ctg agc tgc aag gcc agc
ggc tac agc ttc acc ggc tac 96Ser Leu Arg Leu Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Gly Tyr 20 25 30aac atg aac tgg gtg cgg cag gcc
cct ggc aag ggc ctg gaa tgg gtg 144Asn Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45ggc aac atc gac ccc tac tac
ggc ggc acc acc tac aac cgg aag ttc 192Gly Asn Ile Asp Pro Tyr Tyr
Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60aag ggc agg gcc acc ctg
agc gtg gac aag agc agc agc acc gcc tac 240Lys Gly Arg Ala Thr Leu
Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80ctg cag atg aac
agc ctg cgg gcc gag gac acc gcc gtg tac tac tgc 288Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcc aga tcc
gtg ggc ccc atg gac tac tgg ggc cag ggc acc ctg gtc 336Ala Arg Ser
Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110acc
gtc tct tca 348Thr Val Ser Ser 1158116PRTArtificialSynthetic
Construct 8Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr
Tyr Asn Arg Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Ser Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Val Gly Pro Met
Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
1159348DNAArtificialHumanized murine immunoglobulin sequence 9gcc
gtg cag ctg gtc gag tct ggc ggc gga ctg gtg cag cct ggc ggc 48Ala
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15agc ctg aga ctg agc tgc aag gcc agc ggc tac agc ttc acc ggc tac
96Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30aac atg aac tgg gtg cgg cag gcc cct ggc aag ggc ctg gaa tgg
gtg 144Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45gcc aac atc gac ccc tac tac ggc ggc acc acc tac aac cgg
aag gtc 192Ala Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg
Lys Val 50 55 60aag ggc agg ttc acc atc agc gtg gac aag agc agc agc
acc gcc tac 240Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80ctg cag atg aac agc ctg cgg gcc gag gac acc
gcc gtg tac tac tgc 288Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95gcc aga tcc gtg ggc ccc atg gac tac tgg
ggc cag ggc acc ctg gtc 336Ala Arg Ser Val Gly Pro Met Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110acc gtc tct tca 348Thr Val Ser
Ser 11510116PRTArtificialSynthetic Construct 10Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn
Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser 11511324DNAArtificialHumanized
murine immunoglobulin sequence 11gac atc cag atg acc cag agc ccc
agc agc ctg agc gcc agc gtg ggc 48Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15gac cgg gtg acc atc acc tgc
cgg acc agc gag aac gtg tac agc tac 96Asp Arg Val Thr Ile Thr Cys
Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag
aag ccc ggc aag gcc ccc aag ctg ctg gtg 144Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45tcc ttc gcc aag acc
ctg gcc gag ggc gtg ccc agc cgg ttt agc ggc 192Ser Phe Ala Lys Thr
Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60agc ggc tcc ggc
acc gac ttc acc ctg acc atc agc agc ctg cag ccc 240Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80gag gac
ttc gcc acc tac ttt tgc cag cac cac agc gac aac ccc tgg 288Glu Asp
Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc
ttc ggc cag ggc acc aag gtg gag atc aaa cgt 324Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 10512108PRTArtificialSynthetic
Construct 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val
Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Val 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys
Gln His His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 10513324DNAArtificialHumanized murine
immunoglobulin sequence 13gac atc gtg atg acc cag agc ccc agc agc
ctg agc gcc agc gtg ggc 48Asp Ile Val Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15gac cgg gtg acc atc acc tgc cgg gtc
agc gag aac gtg tac agc tac 96Asp Arg Val Thr Ile Thr Cys Arg Val
Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc
ggc aag gcc ccc aag ctg ctg atc 144Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45tcc ttc gcc aag acc ctg gcc
gag ggc gtg ccc agc cgg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala
Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc gac
ttc acc ctg acc atc agc agc ctg cag ccc 240Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80gag gac ttc gcc
acc tac ttt tgc cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala
Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc
cag ggc acc aag gtg gag atc aaa cgt 324Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 10514108PRTArtificialSynthetic Construct
14Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Val Ser Glu Asn Val Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys Gln His
His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10515324DNAArtificialHumanized murine
immunoglobulin sequence 15gac atc cag atg acc cag agc ccc agc agc
ctg agc gcc agc gtg ggc 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15gac cgg gtg acc atc acc tgc cgg acc
agc gag aac gtg tac agc tac 96Asp Arg Val Thr Ile Thr Cys Arg Thr
Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc
ggc aag gcc ccc aag ctg ctg gtg 144Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Val 35 40 45tcc ttc gcc aag acc ctg gcc
gag ggc gtg ccc agc cgg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala
Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc cag
ttc acc ctg acc atc agc agc ctg cag ccc 240Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80gag gac ttc gcc
acc tac ttt tgc cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala
Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc
gga ggc acc gag ctg gag atc aaa cgt 324Thr Phe Gly Gly Gly Thr Glu
Leu Glu Ile Lys Arg 100 10516108PRTArtificialSynthetic Construct
16Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Val 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys Gln His
His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Glu Leu Glu
Ile Lys Arg 100 10517324DNAArtificialHumanized murine
immunoglobulin sequence 17gac atc gtg atg acc cag agc ccc gcc acc
ctg agc ctg agc cct ggc 48Asp Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15gag cgg gcc acc ctg tcc tgc cgg acc
agc gag aac gtg tac agc tac 96Glu Arg Ala Thr Leu Ser Cys Arg Thr
Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc
ggc cag gcc ccc aga ctg ctg gtg 144Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Val 35 40 45tcc ttc gcc aag acc ctg gcc
gag ggc gtg ccc gcc agg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala
Glu Gly Val Pro Ala Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc gac
ttc acc ctg acc atc agc agc ctg gaa ccc 240Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80gag gac ttc gcc
gtg tac ttc tgc cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala
Val Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc
cag ggc acc aag gtg gag atc aaa cgt 324Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 10518108PRTArtificialSynthetic Construct
18Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Val 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Gln His
His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10519324DNAArtificialHumanized murine
immunoglobulin sequence 19gac atc gtg atg acc cag agc ccc gcc acc
ctg agc ctg agc cct ggc 48Asp Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15gag cgg gcc acc ctg tcc tgc cgg gtc
agc gag aac gtg tac agc tac 96Glu Arg Ala Thr Leu Ser Cys Arg Val
Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc
ggc cag gcc ccc aga ctg ctg atc 144Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45tcc ttc gcc aag acc ctg gcc
gag ggc gtg ccc gcc agg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala
Glu Gly Val Pro Ala Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc gac
ttc acc ctg acc atc agc agc ctg gaa ccc 240Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80gag gac ttc gcc
gtg tac ttc tgc cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala
Val Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc
cag ggc acc aag gtg gag atc aaa cgt 324Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 10520108PRTArtificialSynthetic Construct
20Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Val Ser Glu Asn Val Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Gln His
His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10521324DNAArtificialHumanized murine
immunoglobulin sequence 21gac atc gtg atg acc cag agc ccc gcc acc
ctg agc ctg agc cct ggc 48Asp Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15gag cgg gcc acc ctg tcc tgc cgg acc
agc gag aac gtg tac agc tac 96Glu Arg Ala Thr Leu Ser Cys Arg Thr
Ser Glu Asn Val Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc
ggc cag gcc ccc aga ctg ctg gtg 144Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Val 35 40 45tcc ttc gcc aag acc ctg gcc
gag ggc gtg ccc gcc agg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala
Glu Gly Val Pro Ala Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc cag
ttc acc ctg acc atc agc agc ctg gaa ccc 240Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80gag gac ttc gcc
gtg tac ttc tgc cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala
Val Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc
gga ggc acc gag ctg gag atc aaa cgt 324Thr Phe Gly Gly Gly Thr Glu
Leu Glu Ile Lys Arg 100 10522108PRTArtificialSynthetic Construct
22Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Val 35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Gln His
His Ser Asp Asn Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Glu Leu Glu
Ile Lys Arg 100 10523990DNAHomo sapiensCDS(1)..(990) 23gcc tcc acc
aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag 48Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15agc acc
tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac 96Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30ttc
ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc
192Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag
acc 240Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80tac atc tgc aac gtg aat cac aag ccc agc aac acc aag
gtg gac aag 288Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95aga gtt gag ccc aaa tct tgt gac aaa act cac aca
tgc cca ccg tgc 336Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110cca gca cct gaa ctc ctg ggg gga ccg tca
gtc ttc ctc ttc ccc cca 384Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125aaa ccc aag gac acc ctc atg atc
tcc cgg acc cct gag gtc aca tgc 432Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140gtg gtg gtg gac gtg agc
cac gaa gac cct gag gtc aag ttc aac tgg 480Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160tac gtg gac
ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 528Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175gag
cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 576Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac
624His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc
aaa ggg 672Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220cag ccc cga gaa cca cag gtg tac acc ctg ccc cca
tcc cgg gag gag 720Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu225 230 235 240atg acc aag aac cag gtc agc ctg acc
tgc ctg gtc aaa ggc ttc tat 768Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255ccc agc gac atc gcc gtg gag
tgg gag agc aat ggg cag ccg gag aac 816Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270aac tac aag acc acg
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 864Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285ctc tat agc
aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 912Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300gtc
ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg 960Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320cag aag agc ctc tcc ctg tcc ccg ggt aaa 990Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 33024330PRTHomo sapiens 24Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33025318DNAHomo
sapiensCDS(1)..(318) 25act gtg gct gca cca tct gtc ttc atc ttc ccg
cca tct gat gag cag 48Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln1 5 10 15ttg aaa tct gga act gcc tct gtt gtg tgc
ctg ctg aat aac ttc tat 96Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 20 25 30ccc aga gag gcc aaa gta cag tgg aag
gtg gat aac gcc ctc caa tcg 144Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser 35 40 45ggt aac tcc cag gag agt gtc aca
gag cag gac agc aag gac agc acc 192Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60tac agc ctc agc agc acc ctg
acg ctg agc aaa gca gac tac gag aaa 240Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80cac aaa gtc tac gcc
tgc gaa gtc acc cat cag ggc ctg agc tcg ccc 288His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95gtc aca aag agc
ttc aac agg gga gag tgt 318Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 10526106PRTHomo sapiens 26Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100
105271338DNAArtificialFc-engineered chimeric immunoglobulin
sequence 27gcg gtc cag ctg cag cag tct gga cct gag ctg gaa aag cct
ggc gct 48Ala Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro
Gly Ala1 5 10 15tca gtg aag att tcc tgc aag gct tct ggt tac tca ttc
act ggc tac 96Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30aat atg aac tgg gtg aag cag aat aat gga aag agc
ctt gag tgg att 144Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser
Leu Glu Trp Ile 35 40 45gga aat att gat cct tat tat ggt ggt act acc
tac aac cgg aag ttc 192Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr
Tyr Asn Arg Lys Phe 50 55 60aag ggc aag gcc aca ttg act gta gac aaa
tcc tcc agc aca gcc tac 240Lys Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80atg cag ctc aag agt ctg aca tct
gag gac tct gca gtc tat tac tgt 288Met Gln Leu Lys Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95gca aga tcg gtc ggc cct atg
gac tac tgg ggt caa gga acc tca gtc 336Ala Arg Ser Val Gly Pro Met
Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110acc gtc tct tca gcc
tcc acc aag ggc cca tcg gtc ttc ccc ctg gca 384Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125ccc tcc tcc
aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg 432Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140gtc
aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc 480Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc
tca 528Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc
agc agc ttg 576Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190ggc acc cag acc tac atc tgc aac gtg aat cac
aag ccc agc aac acc 624Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205aag gtg gac aag aga gtt gag ccc aaa
tct tgt gac aaa act cac aca 672Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220tgc cca ccg tgc cca gca cct
gaa ctc ctg ggg gga ccg gac gtc ttc 720Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Asp Val Phe225 230 235 240ctc ttc ccc cca
aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 768Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255gag gtc
aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc
816Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc
aag aca 864Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285aag ccg cgg gag gag cag tac aac agc acg tac cgt
gtg gtc agc gtc 912Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300ctc acc gtc ctg cac cag gac tgg ctg aat
ggc aag gag tac aag tgc 960Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320aag gtc tcc aac aaa gcc ctc
cca gcc ccc gaa gag aaa acc atc tcc 1008Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335aaa gcc aaa ggg cag
ccc cga gaa cca cag gtg tac acc ctg ccc cca 1056Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350tcc cgg gag
gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc 1104Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365aaa
ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1152Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac
1200Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400ggc tcc ttc ttc ctc tat agc aag ctc acc gtg gac
aag agc agg tgg 1248Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415cag cag ggg aac gtc ttc tca tgc tcc gtg
atg cat gag gct ctg cac 1296Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430aac cac tac acg cag aag agc ctc
tcc ctg tcc ccg ggt aaa 1338Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 44528446PRTArtificialSynthetic Construct
28Ala Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly
Tyr 20 25 30Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu
Trp Ile 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn
Arg Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Lys Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155
160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Asp Val Phe225 230 235 240Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 44529642DNAArtificialChimeric immunoglobulin
sequence 29gac atc cag atg act cag tct cca gcc tcc cta tct gca tct
gtg gga 48Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly1 5 10 15gag act gtc acc atc aca tgt cga aca agt gaa aat gtt
tac agt tat 96Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val
Tyr Ser Tyr 20 25 30ttg gct tgg tat cag cag aaa cag gga aaa tct cct
cag ctc ctg gtc 144Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val 35 40 45tct ttt gca aaa acc tta gca gaa ggt gtg cca
tca agg ttc agt ggc 192Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60agt gga tca ggc aca cag ttt tct ctg aag
atc agc agc ctg cag cct 240Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys
Ile Ser Ser Leu Gln Pro65 70 75 80gaa gat tct gga agt tat ttc tgt
caa cat cat tcc gat aat ccg tgg 288Glu Asp Ser Gly Ser Tyr Phe Cys
Gln His His Ser Asp Asn Pro Trp 85 90 95acg ttc ggt gga ggc acc gaa
ctg gag atc aaa cga act gtg gct gca 336Thr Phe Gly Gly Gly Thr Glu
Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110cca tct gtc ttc atc
ttc ccg cca tct gat gag cag ttg aaa tct gga 384Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125act gcc tct
gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140aaa
gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc
agc 528Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac
aaa gtc tac 576Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190gcc tgc gaa gtc acc cat cag ggc ctg agc tcg
ccc gtc aca aag agc 624Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205ttc aac agg gga gag tgt 642Phe Asn
Arg Gly Glu Cys 21030214PRTArtificialSynthetic Construct 30Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu
Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser
Asp Asn Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210311338DNAArtificialFc-engineered chimeric immunoglobulin
sequence 31gcg gtc cag ctg cag cag tct gga cct gag ctg gaa aag cct
ggc gct 48Ala Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro
Gly Ala1 5 10 15tca gtg aag att tcc tgc aag gct tct ggt tac tca ttc
act ggc tac 96Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30aat atg aac tgg gtg aag cag aat aat gga aag agc
ctt gag tgg att 144Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser
Leu Glu Trp Ile 35 40 45gga aat att gat cct tat tat ggt ggt act acc
tac aac cgg aag ttc 192Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr
Tyr Asn Arg Lys Phe 50 55 60aag ggc aag gcc aca ttg act gta gac aaa
tcc tcc agc aca gcc tac 240Lys Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80atg cag ctc aag agt ctg aca tct
gag gac tct gca gtc tat tac tgt 288Met Gln Leu Lys Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95gca aga tcg gtc ggc cct atg
gac tac tgg ggt caa gga acc tca gtc 336Ala Arg Ser Val Gly Pro Met
Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110acc gtc tct tca gcc
tcc acc aag ggc cca tcg gtc ttc ccc ctg gca 384Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125ccc tcc tcc
aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg 432Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140gtc
aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc 480Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc
tca 528Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc
agc agc ttg 576Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190ggc acc cag acc tac atc tgc aac gtg aat cac
aag ccc agc aac acc 624Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205aag gtg gac aag aga gtt gag ccc aaa
tct tgt gac aaa act cac aca 672Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220tgc cca ccg tgc cca gca cct
gaa ctc ctg gcg gga ccg gat gtc ttc 720Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Ala Gly Pro Asp Val Phe225 230 235 240ctc ttc ccc cca
aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 768Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255gag gtc
aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 816Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca
864Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc
agc gtc 912Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag
gag tac aag tgc 960Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320aag gtc tcc aac aaa gcc ctc cca gcc
ccc gaa gag aaa acc atc tcc 1008Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Glu Glu Lys Thr Ile Ser 325 330 335aaa gcc aaa ggg cag ccc cga
gaa cca cag gtg tac acc ctg ccc cca 1056Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350tcc cgg gag gag atg
acc aag aac cag gtc agc ctg acc tgc ctg gtc 1104Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365aaa ggc ttc
tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1152Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380cag
ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 1200Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400ggc tcc ttc ttc ctc tat agc aag ctc acc gtg gac aag agc agg
tgg 1248Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag
gct ctg cac 1296Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430aac cac tac acg cag aag agc ctc tcc ctg tcc
ccg ggt aaa 1338Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 44532446PRTArtificialSynthetic Construct 32Ala Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn
Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile 35 40
45Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln
Gly Thr Ser Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185
190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly
Pro Asp Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310
315 320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile
Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln
Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44533642DNAArtificialChimeric immunoglobulin sequence 33gac atc cag
atg act cag tct cca gcc tcc cta tct gca tct gtg gga 48Asp Ile Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15gag act
gtc acc atc aca tgt cga aca agt gaa aat gtt tac agt tat 96Glu Thr
Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30ttg
gct tgg tat cag cag aaa cag gga aaa tct cct cag ctc ctg gtc 144Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40
45tct ttt gca aaa acc tta gca gaa ggt gtg cca tca agg ttc agt ggc
192Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60agt gga tca ggc aca cag ttt tct ctg aag atc agc agc ctg cag
cct 240Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Ser Leu Gln
Pro65 70 75 80gaa gat tct gga agt tat ttc tgt caa cat cat tcc gat
aat ccg tgg 288Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser Asp
Asn Pro Trp 85 90 95acg ttc ggt gga ggc acc gaa ctg gag atc aaa cga
act gtg gct gca 336Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110cca tct gtc ttc atc ttc ccg cca tct gat
gag cag ttg aaa tct gga 384Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125act gcc tct gtt gtg tgc ctg ctg
aat aac ttc tat ccc aga gag gcc 432Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140aaa gta cag tgg aag gtg
gat aac gcc ctc caa tcg ggt aac tcc cag 480Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160gag agt gtc
aca gag cag gac agc aag gac agc acc tac agc ctc agc 528Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175agc
acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc
624Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205ttc aac agg gga gag tgt 642Phe Asn Arg Gly Glu Cys
21034214PRTArtificialSynthetic Construct 34Asp Ile Gln Met Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile
Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Ser Phe Ala
Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210351338DNAArtificialFc-engineered
humanized immunoglobulin sequence 35gcg gtc cag ctg gtg cag agc gga
gcc gag gtg aag aag ccc ggc agc 48Ala Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15agc gtc aag gtg tcc tgc aag
gcc agc ggc tac agc ttc acc ggc tac 96Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30aac atg aac tgg gtg cgg
cag gcc cca ggc cag gga ctg gaa tgg atg 144Asn Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45ggc aac atc gac ccc
tac tac ggc ggc acc acc tac aac cgg aag ttc 192Gly Asn Ile Asp Pro
Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60aag ggc cgg gtg
acc ctg acc gtg gac aag agc agc agc acc gcc tac 240Lys Gly Arg Val
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80atg gaa
ctg agc agc ctg cgg agc gag gac acc gcc gtg tac tac tgc 288Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcc
aga tcc gtg ggc ccc atg gac tac tgg ggc cag ggc acc ctg gtc 336Ala
Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110acc gtc tct tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca
384Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc
tgc ctg 432Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg
tgg aac tca ggc 480Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly145 150 155 160gcc ctg acc agc ggc gtg cac acc ttc
ccg gct gtc cta cag tcc tca 528Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175gga ctc tac tcc ctc agc agc
gtg gtg acc gtg ccc tcc agc agc ttg 576Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190ggc acc cag acc tac
atc tgc aac gtg aat cac aag ccc agc aac acc 624Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205aag gtg gac
aag aga gtt gag ccc aaa tct tgt gac aaa act cac aca 672Lys Val Asp
Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220tgc
cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg gac gtc ttc 720Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe225 230
235 240ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc
cct 768Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac
cct gag gtc 816Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270aag ttc aac tgg tac gtg gac ggc gtg gag gtg
cat aat gcc aag aca 864Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285aag ccg cgg gag gag cag tac aac agc
acg tac cgt gtg gtc agc gtc 912Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300ctc acc gtc ctg cac cag gac
tgg ctg aat ggc aag gag tac aag tgc 960Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320aag gtc tcc aac
aaa gcc ctc cca gcc ccc gaa gag aaa acc atc tcc 1008Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser 325 330 335aaa gcc
aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 1056Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc
1104Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc
aat ggg 1152Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380cag ccg gag aac aac tac aag acc acg cct ccc gtg
ctg gac tcc gac 1200Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp385 390 395 400ggc tcc ttc ttc ctc tat agc aag ctc
acc gtg gac aag agc agg tgg 1248Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415cag cag ggg aac gtc ttc tca
tgc tcc gtg atg cat gag gct ctg cac 1296Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430aac cac tac acg cag
aag agc ctc tcc ctg tcc ccg ggt aaa 1338Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 44536446PRTArtificialSynthetic
Construct 36Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr
Tyr Asn Arg Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Val Gly Pro Met
Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Asp Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Glu Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 44537642DNAArtificialHumanized immunoglobulin
sequence 37gac atc cag atg acc cag agc ccc agc agc ctg agc gcc agc
gtg ggc 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15gac cgg gtg acc atc acc tgc cgg acc agc gag aac gtg
tac agc tac 96Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val
Tyr Ser Tyr 20 25 30ctg gcc tgg tat cag cag aag ccc ggc aag gcc ccc
aag ctg ctg gtg 144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Val 35 40 45tcc ttc gcc aag acc ctg gcc gag ggc gtg ccc
agc cgg ttt agc ggc 192Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60agc ggc tcc ggc acc gac ttc acc ctg acc
atc agc agc ctg cag ccc 240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80gag gac ttc gcc acc tac ttt tgc
cag cac cac agc gac aac ccc tgg 288Glu Asp Phe Ala Thr Tyr Phe Cys
Gln His His Ser Asp Asn Pro Trp 85 90 95acc ttc ggc cag ggc acc aag
gtg gag atc aaa cga act gtg gct gca 336Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110cca tct gtc ttc atc
ttc ccg cca tct gat gag cag ttg aaa tct gga 384Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125act gcc tct
gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140aaa
gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc
agc 528Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac
aaa gtc tac 576Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190gcc tgc gaa gtc acc cat cag ggc ctg agc tcg
ccc gtc aca aag agc 624Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205ttc aac agg gga gag tgt 642Phe Asn
Arg Gly Glu Cys 21038214PRTArtificialSynthetic Construct 38Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val
35 40 45Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser
Asp Asn Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210391338DNAArtificialFc-engineered humanized immunoglobulin
sequence 39gcg gtc cag ctg gtg cag agc gga gcc gag gtg aag aag ccc
ggc agc 48Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15agc gtc aag gtg tcc tgc aag gcc agc ggc tac agc ttc
acc ggc tac 96Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30aac atg aac tgg gtg cgg cag gcc cca ggc cag gga
ctg gaa tgg atg 144Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45ggc aac atc gac ccc tac tac ggc ggc acc acc
tac aac cgg aag ttc 192Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr
Tyr Asn Arg Lys Phe 50 55 60aag ggc cgg gtg acc ctg acc gtg gac aag
agc agc
agc acc gcc tac 240Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80atg gaa ctg agc agc ctg cgg agc gag gac
acc gcc gtg tac tac tgc 288Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95gcc aga tcc gtg ggc ccc atg gac tac
tgg ggc cag ggc acc ctg gtc 336Ala Arg Ser Val Gly Pro Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110acc gtc tct tca gcc tcc acc
aag ggc cca tcg gtc ttc ccc ctg gca 384Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125ccc tcc tcc aag agc
acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg 432Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140gtc aag gac
tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc 480Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155
160gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca
528Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc
agc ttg 576Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190ggc acc cag acc tac atc tgc aac gtg aat cac aag
ccc agc aac acc 624Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205aag gtg gac aag aga gtt gag ccc aaa tct
tgt gac aaa act cac aca 672Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr 210 215 220tgc cca ccg tgc cca gca cct gaa
ctc ctg gcg gga ccg gat gtc ttc 720Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Ala Gly Pro Asp Val Phe225 230 235 240ctc ttc ccc cca aaa
ccc aag gac acc ctc atg atc tcc cgg acc cct 768Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255gag gtc aca
tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 816Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270aag
ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 864Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc
912Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac
aag tgc 960Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320aag gtc tcc aac aaa gcc ctc cca gcc ccc gaa
gag aaa acc atc tcc 1008Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu
Glu Lys Thr Ile Ser 325 330 335aaa gcc aaa ggg cag ccc cga gaa cca
cag gtg tac acc ctg ccc cca 1056Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350tcc cgg gag gag atg acc aag
aac cag gtc agc ctg acc tgc ctg gtc 1104Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365aaa ggc ttc tat ccc
agc gac atc gcc gtg gag tgg gag agc aat ggg 1152Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380cag ccg gag
aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 1200Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400ggc tcc ttc ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg
1248Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct
ctg cac 1296Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430aac cac tac acg cag aag agc ctc tcc ctg tcc ccg
ggt aaa 1338Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 44540446PRTArtificialSynthetic Construct 40Ala Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55
60Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr
Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44541642DNAArtificialHumanized immunoglobulin sequence 41gac atc
cag atg acc cag agc ccc agc agc ctg agc gcc agc gtg ggc 48Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15gac
cgg gtg acc atc acc tgc cgg acc agc gag aac gtg tac agc tac 96Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25
30ctg gcc tgg tat cag cag aag ccc ggc aag gcc ccc aag ctg ctg gtg
144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val
35 40 45tcc ttc gcc aag acc ctg gcc gag ggc gtg ccc agc cgg ttt agc
ggc 192Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60agc ggc tcc ggc acc gac ttc acc ctg acc atc agc agc ctg
cag ccc 240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80gag gac ttc gcc acc tac ttt tgc cag cac cac agc
gac aac ccc tgg 288Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser
Asp Asn Pro Trp 85 90 95acc ttc ggc cag ggc acc aag gtg gag atc aaa
cga act gtg gct gca 336Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110cca tct gtc ttc atc ttc ccg cca tct
gat gag cag ttg aaa tct gga 384Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125act gcc tct gtt gtg tgc ctg
ctg aat aac ttc tat ccc aga gag gcc 432Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140aaa gta cag tgg aag
gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160gag agt
gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac
576Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca
aag agc 624Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205ttc aac agg gga gag tgt 642Phe Asn Arg Gly Glu
Cys 21042214PRTArtificialSynthetic Construct 42Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45Ser Phe
Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210
* * * * *