U.S. patent application number 14/958959 was filed with the patent office on 2016-04-07 for anti-cd38 human antibodies and uses thereof.
The applicant listed for this patent is MorphoSys AG. Invention is credited to Ute Jaeger, Michael Tesar.
Application Number | 20160096901 14/958959 |
Document ID | / |
Family ID | 35197540 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160096901 |
Kind Code |
A1 |
Tesar; Michael ; et
al. |
April 7, 2016 |
ANTI-CD38 HUMAN ANTIBODIES AND USES THEREOF
Abstract
The present invention provides recombinant antigen-binding
regions and antibodies and functional fragments containing such
antigen-binding regions that are specific for CD38, which plays an
integral role in various disorders or conditions. These antibodies,
accordingly, can be used to treat, for example, hematological
malignancies such as multiple myeloma. Antibodies of the invention
also can be used in the diagnostics field, as well as for
investigating the role of CD38 in the progression of disorders
associated with malignancies. The invention also provides nucleic
acid sequences encoding the foregoing antibodies, vectors
containing the same, pharmaceutical compositions and kits with
instructions for use. The invention also provides isolated novel
epitopes of CD38 and methods of use therefore.
Inventors: |
Tesar; Michael; (Friedberg,
DE) ; Jaeger; Ute; (Munich, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MorphoSys AG |
Martinsried/Planegg |
|
DE |
|
|
Family ID: |
35197540 |
Appl. No.: |
14/958959 |
Filed: |
December 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14630042 |
Feb 24, 2015 |
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14958959 |
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13427305 |
Mar 22, 2012 |
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14630042 |
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10588568 |
Oct 14, 2009 |
8263746 |
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PCT/IB2005/002476 |
Oct 14, 2009 |
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13427305 |
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60614471 |
Oct 1, 2004 |
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60599014 |
Aug 6, 2004 |
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60553948 |
Mar 18, 2004 |
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60547584 |
Feb 26, 2004 |
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60541911 |
Feb 6, 2004 |
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Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
C07K 2317/732 20130101;
C07K 2317/567 20130101; A61P 29/00 20180101; C07K 16/3061 20130101;
C07K 2317/33 20130101; C07K 2317/565 20130101; C12N 9/2497
20130101; C07K 2317/92 20130101; C07K 2317/34 20130101; C07K
2319/30 20130101; C07K 16/2896 20130101; C12Y 302/02024 20130101;
A61P 35/04 20180101; A61P 37/00 20180101; C07K 2317/24 20130101;
C07K 2317/52 20130101; C07K 2317/56 20130101; C07K 2317/734
20130101; A61K 2039/505 20130101; C07K 2317/74 20130101; C07K
16/005 20130101; A61P 35/02 20180101; A61P 19/02 20180101; C07K
2317/21 20130101; A61P 7/00 20180101; C07K 2317/55 20130101; C07K
16/40 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1-114. (canceled)
115. An isolated monoclonal non-chimeric, non-CDR grafted antibody
based on a human sequence that binds human CD38 (SEQ ID NO: 22),
(a) wherein said antibody mediates killing of a CD38+ target cell
by antibody dependent cellular cytotoxicity with an EC50 of 1 nM or
less when a human PBMC cell is employed as the effector cell,
wherein said CD38+ target cell is selected from LP-1 (DSMZ: ACC41)
or RPMI-8226 (ATCC: CCL-155), and wherein the ratio of effector
cells to target cells is from 30:1 to 50:1, (b) wherein said
antibody mediates killing of a CD38-transfected CHO cell by
complement dependent cytotoxicity with an EC50 of 3.2 nM or less,
(c) wherein said antibody comprises a VH3 variable heavy chain and
kappa variable light chain, and (d) wherein the antibody comprises
an IgG1 constant region.
116. The antibody according to claim 115, wherein the variable
heavy chain comprises a framework 1 region comprising
SGGGLVQPGGSLRLSC, a framework 2 region comprising VRQAPGKGLEW, a
framework 3 region comprising FTISRDNSKNTLYLQMNSLRAEDTAV and a
framework 4 region comprising GQGTLVTV.
117. The antibody according to claim 116, wherein the variable
light chain comprises a framework 1 region comprising TQSP, a
framework 2 region comprising QKPG, a framework 3 region comprising
RFSGSGSGTDFTL and a framework 4 region comprising TFGQGTKVEIK.
118. The antibody according to claim 115, wherein the antibody
mediates killing of a CD38+ target cell by antibody dependent
cellular cytotoxicity with an EC50 of 0.60 nM or less.
119. The antibody according to claim 118, wherein the antibody
mediates killing of a CD38+ target cell by antibody dependent
cellular cytotoxicity with an EC50 of 0.28 nM or less.
120. The antibody according to claim 119, wherein the antibody
mediates killing of a CD38+ target cell by antibody dependent
cellular cytotoxicity with an EC50 of 0.17 nM or less.
121. The antibody according to claim 115, wherein the antibody
mediates killing of a CD38-transfected CHO cell by complement
dependent cytotoxicity with an EC50 of 0.94 nM or less.
122. The antibody according to claim 121, wherein the antibody
mediates killing of a CD38-transfected CHO cell by complement
dependent cytotoxicity with an EC50 of 0.41 nM or less.
123. The antibody according to claim 115, wherein the antibody
mediates killing of a CD38-transfected CHO cell by complement
dependent cytotoxicity with an EC50 in the range of 3.2 nM to 0.94
nM.
124. The antibody according to claim 115, wherein the antibody
mediates killing of a CD38-transfected CHO cell by complement
dependent cytotoxicity with an EC50 in the range of 3.2 nM to 0.41
nM.
125. The antibody according to claim 115, wherein the antibody has
a CDRH1 having a length about 5, CDRH2 having a length about 17,
CDRH3 having a length about 13, a CDRL1 having a length about 11,
CDRL2 having a length about 7, and CDRL3 having a length about
9.
126. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acid
residues within amino acids 44-66 of CD38 (SEQ ID NO: 22).
127. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 110-122 of CD38 (SEQ ID NO: 22).
128. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 148-164 of CD38 (SEQ ID NO: 22).
129. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 186-200 of CD38 (SEQ ID NO: 22).
130. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 202-224 of CD38 (SEQ ID NO: 22).
131. The antibody according to claim 115, wherein said antibody
competes in binding to CD38 with an antibody comprising an H-CDR1,
H-CDR2 and H-CDR3 depicted in SEQ ID NO: 5 and an L-CDR1, L-CDR2
and L-CDR3 depicted in SEQ ID NO:13.
132. The antibody according to claim 131, wherein said antibody
binds to the same epitope of CD38 of an antibody comprising an
H-CDR1, H-CDR2 and H-CDR3 depicted in SEQ ID NO: 5 and an L-CDR1,
L-CDR2 and L-CDR3 depicted in SEQ ID NO: 13.
133. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 192-206 of CD38 (SEQ ID NO: 22).
134. The antibody according to claim 115, wherein said antibody
competes in binding to CD38 with an antibody comprising an H-CDR1,
H-CDR2 and H-CDR3 depicted in SEQ ID NO: 6 and an L-CDR1, L-CDR2
and L-CDR3 depicted in SEQ ID NO: 14.
135. The antibody according to claim 134, wherein said antibody
binds to the same epitope of CD38 with an antibody comprising an
H-CDR1, H-CDR2 and H-CDR3 depicted in SEQ ID NO: 6 and an L-CDR1,
L-CDR2 and L-CDR3 depicted in SEQ ID NO: 14.
136. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 82-94 of CD38 (SEQ ID NO: 22).
137. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 158-170 of CD38 (SEQ ID NO: 22).
138. The antibody according to claim 115, wherein said antibody
competes in binding to CD38 with an antibody comprising an H-CDR1,
H-CDR2 and H-CDR3 depicted in SEQ ID NO: 7 and an L-CDR1, L-CDR2
and L-CDR3 depicted in SEQ ID NO: 15.
139. The antibody according to claim 138, wherein said antibody
binds to the same epitope of CD38 with an antibody comprising an
H-CDR1, H-CDR2 and H-CDR3 depicted in SEQ ID NO: 7 and an L-CDR1,
L-CDR2 and L-CDR3 depicted in SEQ ID NO: 15.
140. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 82-94 of CD38 (SEQ ID NO: 22).
141. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 142-154 of CD38 (SEQ ID NO: 22).
142. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 158-170 of CD38 (SEQ ID NO: 22).
143. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 188-200 of CD38 (SEQ ID NO: 22).
144. The antibody according to claim 115, wherein said antibody
binds an epitope of CD38 that contains one or more amino acids
residues within amino acids 280-296 of CD38 (SEQ ID NO: 22).
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 14/630,042 filed on Feb. 24, 2015, which is pending, which is a
Continuation of U.S. application Ser. No. 13/427,305, filed Mar.
22, 2012, which is abandoned, which is a Divisional of U.S.
application Ser. No. 10/588,568, which issued as U.S. Pat. No.
8,263,746, which is the US National Stage application of
PCT/IB05/002476, filed Feb. 7, 2005, which claims priority to U.S.
provisional application numbers 60/541,911 filed Feb. 6, 2004,
60/547,584 filed Feb. 26, 2004, 60/553,948 filed Mar. 18, 2004, and
60/599,014 filed Aug. 6, 2004, and 60/614,471, filed Oct. 1, 2004,
the contents of each of which are incorporated herein in their
entireties.
BACKGROUND OF THE INVENTION
[0002] CD38 is a type-II membrane glycoprotein and belongs to the
family of ectoenzymes, due to its enzymatic activity as ADP
ribosyl-cyclase and cADP-hydrolase. During ontogeny, CD38 appears
on CD34+ committed stem cells and lineage-committed progenitors of
lymphoid, erythroid and myeloid cells. It is understood that CD38
expression persists only in the lymphoid lineage, through the early
stages of T- and B-cell development.
[0003] The up-regulation of CD38 serves as a marker for lymphocyte
activation--in particular B-cell differentiation along the
plasmacytoid pathway. (Co-)receptor functions of CD38 leading to
intracellular signaling or intercellular communication via its
ligand, CD31, are postulated, as well as its role as an
intracellular regulator of a second messenger, cyclic ADPr, in a
variety of signaling cascades. However, its physiological
importance remains to be elucidated, since knock out of the murine
analogue or anti-CD38 auto-antibodies in humans do not appear to be
detrimental.
[0004] Apart from observing its expression in the hematopoetic
system, researchers have noted the up-regulation of CD38 on various
cell-lines derived from B-, T-, and myeloid/monocytic tumors,
including B- or T-cell acute lymphoblastic leukemia (ALL), acute
myeloid leukemia (AML), Non-Hodgkin's lymphoma (NHL) and multiple
myeloma (MM). In MM, for example, strong CD38 expression is
witnessed in the majority of all patient samples.
[0005] Hence, over-expression of CD38 on malignant cells provides
an attractive therapeutic target for immunotherapy. Of special
attraction is the fact that the most primitive pluripotent stem
cells of the hematopoietic system are CD38-negative and that the
extent of cytotoxic effects by ADCC or CDC correlates well with the
expression-levels of the respective target.
[0006] Current approaches of anti-CD38 therapies can be divided in
two groups: in vivo and ex vivo approaches. In in vivo approaches,
anti-CD38 antibodies are administered to a subject in need of
therapy in order to cause the antibody-mediated depletion of
CD38-overexpressing malignant cells. Depletion can either be
achieved by antibody-mediated ADCC and/or CDC by effector cells, or
by using the anti-CD38 antibodies as targeting moieties for the
transport of cytotoxic substances, e.g. saporin, to the target
cells, and subsequent internalization. In the ex vivo approach,
cell population, e.g. bone marrow cells, comprising CD38
overexpressing malignant cells are removed from an individual in
need of treatment and are contacted with anti-CD38 antibodies. The
target cells are either destroyed by cytotoxic substances, e.g.
saporin, as described for the in vivo approach, or are removed by
contacting the cell population with immobilized anti-CD38
antibodies, thus removing CD38 overexpressing target cells from the
mixture. Thereafter, the depleted cell population is reinserted
into the patient.
[0007] Antibodies specific for CD38 can be divided in different
groups, depending on various properties. Binding of some antibodies
to the CD38 molecule (predominantly aa 220-300) can trigger
activities within the target cell, such as Ca2+ release, cytokine
release, phosphorylation events and growth stimulation based on the
respective antibody specificity (Konopleva et al., 1998; Ausiello
et al., 2000), but no clear correlation between the binding site of
the various known antibodies and their (non-)agonistic properties
could be seen (Funaro et al., 1990).
[0008] Relatively little is known about the efficacy of published
anti-CD38 antibodies. What is known is that all known antibodies
seem to exclusively recognize epitopes (amino acid residues 220 to
300) located in the C-terminal part of CD38. No antibodies are
known so far that are specific for epitopes in the N-terminal part
of CD38 distant from the active site in the primary protein
sequence. However, we have found that OKT10, which has been in
clinical testing, has a relatively low affinity and efficacy when
analyzed as chimeric construct comprising a human Fc part.
Furthermore, OKT10 is a murine antibody rendering it unsuitable for
human administration. A human anti-CD38 scFv antibody fragment has
recently been described (WO 02/06347). However, that antibody is
specific for a selectively expressed CD38 epitope.
[0009] Correspondingly, in light of the great potential for
anti-CD38 antibody therapy, there is a high need for human
anti-CD38 antibodies with high affinity and with high efficacy in
mediating killing of CD38 overexpressing malignant cells by ADCC
and/or CDC.
[0010] The present invention satisfies these and other needs by
providing fully human and highly efficacious anti-CD38 antibodies,
which are described below.
SUMMARY OF THE INVENTION
[0011] It is an object of the invention to provide human and
humanized antibodies that can effectively mediate the killing of
CD38-overexpressing cells.
[0012] It is another object of the invention to provide antibodies
that are safe for human administration.
[0013] It is also an object of the present invention to provide
methods for treating disease or and/or conditions associated with
CD38 up-regulation by using one or more antibodies of the
invention. These and other objects of the invention are more fully
described herein.
[0014] In one aspect, the invention provides an isolated antibody
or functional antibody fragment that contains an antigen-binding
region that is specific for an epitope of CD38, where the antibody
or functional fragment thereof is able to mediate killing of a
CD38+ target cell (LP-1 (DSMZ: ACC41) and RPMI-8226 (ATCC:
CCL-155)) by antibody-dependent cellular cytotoxicity ("ADCC") with
an at least two- to five-fold better efficacy than the chimeric
OKT10 antibody having SEQ ID NOS: 23 and 24 (under the same or
substantially the same conditions), when a human PBMC cell is
employed as an effector cell, and when the ratio of effector cells
to target cells is between about 30:1 and about 50:1. Such an
antibody or functional fragment thereof may contain an
antigen-binding region that contains an H-CDR3 region depicted in
SEQ ID NO: 5, 6, 7, or 8; the antigen-binding region may further
include an H-CDR2 region depicted in SEQ ID NO: 5, 6, 7, or 8; and
the antigen-binding region also may contain an H-CDR1 region
depicted in SEQ ID NO: 5, 6, 7, or 8. Such a CD38-specific antibody
of the invention may contain an antigen-binding region that
contains an L-CDR3 region depicted in SEQ ID NO: 13, 14, 15, or 16;
the antigen-binding region may further include an L-CDR1 region
depicted in SEQ ID NO: 13, 14, 15, or 16; and the antigen-binding
region also may contain an L-CDR2 region depicted in SEQ ID NO: 13,
14, 15, or 16.
[0015] In another aspect, the invention provides an isolated
antibody or functional antibody fragment that contains an
antigen-binding region that is specific for an epitope of CD38,
where the antibody or functional fragment thereof is able to
mediate killing of a CD38-transfected CHO cell by CDC with an at
least two-fold better efficacy than chimeric OKT10 (SEQ ID NOS: 23
and 24) under the same or substantially the same conditions as in
the previous paragraph. An antibody satisfying these criteria may
contain an antigen-binding region that contains an H-CDR3 region
depicted in SEQ ID NO: 5, 6, or 7; the antigen-binding region may
further include an H-CDR2 region depicted in SEQ ID NO: 5, 6, or 7;
and the antigen-binding region also may contain an H-CDR1 region
depicted in SEQ ID NO: 5, 6, or 7. Such a CD38-specific antibody of
the invention may contain an antigen-binding region that contains
an L-CDR3 region depicted in SEQ ID NO: 13, 14, or 15; the
antigen-binding region may further include an L-CDR1 region
depicted in SEQ ID NO: 13, 14, or 15; and the antigen-binding
region also may contain an L-CDR2 region depicted in SEQ ID NO: 13,
14, or 15.
[0016] Antibodies (and functional fragments thereof) of the
invention may contain an antigen-binding region that is specific
for an epitope of CD38, which epitope contains one or more amino
acid residues of amino acid residues 43 to 215 of CD38, as depicted
by SEQ ID NO: 22. More specifically, an epitope to which the
antigen-binding region binds may contain one or more amino acid
residues found in one or more of the amino acid stretches taken
from the list of amino acid stretches 44-66, 82-94, 142-154,
148-164, 158-170, and 192-206. For certain antibodies, the epitope
may be linear, whereas for others, it may be conformational (i.e.,
discontinuous). An antibody or functional fragment thereof having
one or more of these properties may contain an antigen-binding
region that contains an H-CDR3 region depicted in SEQ ID NO: 5, 6,
7, or 8; the antigen-binding region may further include an H-CDR2
region depicted in SEQ ID NO: 5, 6, 7, or 8; and the
antigen-binding region also may contain an H-CDR1 region depicted
in SEQ ID NO: 5, 6, 7, or 8. Such a CD38-specific antibody of the
invention may contain an antigen-binding region that contains an
L-CDR3 region depicted in SEQ ID NO: 13, 14, 15, or 16; the
antigen-binding region may further include an L-CDR1 region
depicted in SEQ ID NO: 13, 14, 15, or 16; and the antigen-binding
region also may contain an L-CDR2 region depicted in SEQ ID NO: 13,
14, 15, or 16.
[0017] Peptide variants of the sequences disclosed herein are also
embraced by the present invention. Accordingly, the invention
includes anti-CD38 antibodies having a heavy chain amino acid
sequence with: at least 60 percent sequence identity in the CDR
regions with the CDR regions depicted in SEQ ID NO: 5, 6, 7, or 8;
and/or at least 80 percent sequence homology in the CDR regions
with the CDR regions depicted in SEQ ID NO: 5, 6, 7, or 8. Further
included are anti-CD38 antibodies having a light chain amino acid
sequence with: at least 60 percent sequence identity in the CDR
regions with the CDR regions depicted in SEQ ID NO: 13, 14, 15 or
16; and/or at least 80 percent sequence homology in the CDR regions
with the CDR regions depicted in SEQ ID NO: 13, 14, 15 or 16.
[0018] An antibody of the invention may be an IgG (e.g.,
IgG.sub.1), while an antibody fragment may be a Fab or scFv, for
example. An inventive antibody fragment, accordingly, may be, or
may contain, an antigen-binding region that behaves in one or more
ways as described herein.
[0019] The invention also is related to isolated nucleic acid
sequences, each of which can encode an antigen-binding region of a
human antibody or functional fragment thereof that is specific for
an epitope of CD38. Such a nucleic acid sequence may encode a
variable heavy chain of an antibody and include a sequence selected
from the group consisting of SEQ ID NOS: 1, 2, 3, or 4, or a
nucleic acid sequence that hybridizes under high stringency
conditions to the complementary strand of SEQ ID NO: 1, 2, 3, or 4.
The nucleic acid might encode a variable light chain of an isolated
antibody or functional fragment thereof, and may contain a sequence
selected from the group consisting of SEQ ID NOS: 9, 10, 11, or 12,
or a nucleic acid sequence that hybridizes under high stringency
conditions to the complementary strand of SEQ ID NO: 9, 10, 11, or
12.
[0020] Nucleic acids of the invention are suitable for recombinant
production. Thus, the invention also relates to vectors and host
cells containing a nucleic acid sequence of the invention.
[0021] Compositions of the invention may be used for therapeutic or
prophylactic applications. The invention, therefore, includes a
pharmaceutical composition containing an inventive antibody (or
functional antibody fragment) and a pharmaceutically acceptable
carrier or excipient therefor. In a related aspect, the invention
provides a method for treating a disorder or condition associated
with the undesired presence of CD38 or CD38 expressing cells. Such
method contains the steps of administering to a subject in need
thereof an effective amount of the pharmaceutical composition that
contains an inventive antibody as described or contemplated
herein.
[0022] The invention also relates to isolated epitopes of CD38,
either in linear or conformational form, and their use for the
isolation of an antibody or functional fragment thereof, which
antibody of antibody fragment comprises an antigen-binding region
that is specific for said epitope. In this regard, a linear epitope
may contain amino acid residues 192-206, while a conformational
epitope may contain one or more amino acid residues selected from
the group consisting of amino acids 44-66, 82-94, 142-154, 148-164,
158-170 and 202-224 of CD38. An epitope of CD38 can be used, for
example, for the isolation of antibodies or functional fragments
thereof (each of which antibodies or antibody fragments comprises
an antigen-binding region that is specific for such epitope),
comprising the steps of contacting said epitope of CD38 with an
antibody library and isolating the antibody(ies) or functional
fragment(s) thereof.
[0023] In another embodiment, the invention provides an isolated
epitope of CD38, which consists essentially of an amino acid
sequence selected from the group consisting of amino acids 44-66,
82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 of CD38. As
used herein, such an epitope "consists essentially of" one of the
immediately preceding amino acid sequences plus additional
features, provided that the additional features do not materially
affect the basic and novel characteristics of the epitope.
[0024] In yet another embodiment, the invention provides an
isolated epitope of CD38 that consists of an amino acid sequence
selected from the group consisting of amino acids 44-66, 82-94,
142-154, 148-164, 158-170, 192-206 and 202-224 of CD38.
[0025] The invention also provides a kit containing (i) an isolated
epitope of CD38 comprising one or more amino acid stretches taken
from the list of 44-66, 82-94, 142-154, 148-164, 158-170, 192-206
and 202-224; (ii) an antibody library; and (iii) instructions for
using the antibody library to isolate one or more members of such
library that binds specifically to such epitope.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1a provides nucleic acid sequences of various novel
antibody variable heavy regions.
[0027] FIG. 1b provides amino acid sequences of various novel
antibody variable heavy regions. CDR regions HCDR1, HCDR2 and HCDR3
are designated from N- to C-terminus in boldface.
[0028] FIG. 2a provides nucleic acid sequences of various novel
antibody variable light regions.
[0029] FIG. 2b provides amino acid sequences of various novel
antibody variable light regions. CDR regions LCDR1, LCDR2 and LCDR3
are designated from N- to C-terminus in boldface.
[0030] FIG. 3 provides amino acid sequences of variable heavy
regions of various consensus-based HuCAL antibody master gene
sequences. CDR regions HCDR1, HCDR2 and HCDR3 are designated from
N- to C-terminus in boldface.
[0031] FIG. 4 provides amino acid sequences of variable light
regions of various consensus-based HuCAL antibody master gene
sequences. CDR regions LCDR1, LCDR2 and LCDR3 are designated from
N- to C-terminus in boldface.
[0032] FIG. 5 provides the amino acid sequence of CD38 (SWISS-PROT
primary accession number P28907).
[0033] FIG. 6 provides the nucleotide sequences of the heavy and
light chains of chimeric OKT10.
[0034] FIG. 7 provides a schematic overview of epitopes of
representative antibodies of the present invention.
[0035] FIG. 8 provides the DNA sequence of pMORPHO h IgG1 1 (bp
601-2100) (SEQ ID NO: 32): The vector is based on the pcDNA3.1+
vectors (Invitrogen). The amino acid sequence encoded by the DNA
sequence is presented (SEQ ID NO: 35), and the amino acid sequence
of the NH-stuffer sequence is indicated in bold, whereas the final
reading frames of the VH-leader sequence and the constant region
gene are printed in non-bold. Restriction sites are indicated above
the sequence. The priming sites of the sequencing primers are
underlined. The antisense strand of the DNA sequence is presented
in SEQ ID NO. 44.
[0036] FIG. 9 provides the DNA sequence of Ig kappa light chain
expression vector pMORPHO h Igic 1 (bp 601-1400) (SEQ ID NO: 33):
The vector is based on the pcDNA3.1+ vectors (Invitrogen). The
amino acid sequences sequence encoded by the DNA sequence is
presented (SEQ ID NO: 36), and the amino acid sequence of the
Nx-stuffer sequence is indicated in bold, whereas the final reading
frames of the Vic-leader sequence and of the constant region gene
are printed in non-bold. Restriction sites are indicated above the
sequence. The priming sites of the sequencing primers are
underlined. The antisense strand of the DNA sequence is presented
in SEQ ID NO. 45.
[0037] FIG. 10 provides the DNA sequence of HUCAL.RTM. Ig lambda
light chain vector pMORPHO h Igk t (bp 601-1400) (SEQ ID NO: 34):
The amino acid sequence encoded by the DNA sequence is presented
(SEQ ID NO: 37), and the amino acid sequence of the VX-stuffer
sequence is indicated in bold, whereas the final reading frames of
the VX-leader sequence and of the constant region gene are printed
in non-bold. Restriction sites are indicated above the sequence.
The priming sites of the sequencing primers are underlined. The
antisense strand of the DNA sequence is presented in SEQ ID NO.
46.
[0038] FIG. 11 provides the results of the proliferation assay:
PBMCs from 6 different healthy donors (as indicated by individual
dots) were cultured for 3 days in the presence of HUCAL.RTM.
antibodies Mab#1 (=MOR03077), Mab#2 (=MOR03079), and Mab#3
(=MOR03080), the reference antibody chOKTl0, the agonistic (ag.)
control IB4, an irrelevant HUCAL.RTM. negative control IgG1 (NC)
and a murine IgG2a (Iso) as matched isotype control for IB4. A
standard labeling with BrdU was used to measure proliferation
activity and its incorporation (as RLU=relative light units)
analyzed via a chemiluminescence-based ELISA.
[0039] FIG. 12 provides the results of the IL-6 Release Assay:
PBMCs from 4-8 different healthy donors (as indicated by individual
dots) were cultured for 24 hrs in the presence of HUCAL.RTM.
antibodies Mab#1 (=MOR03077), Mab#2 (=MOR03079), and Mab#3
(=MOR03080), the reference antibody chOKTlO, the agonistic (ag.)
control IB4, an irrelevant HUCAL.RTM. negative control (NC) and
medium only (Medium). IL-6 content in relative light units (RLU)
was analyzed from culture supernatants via a chemiluminescence
based ELISA.
[0040] FIG. 13 provides data about the cytotoxicity towards
CD34+/CD38+ progenitor cells: PBMCs from healthy donors harboring
autologous CD34+/CD38+ progenitor cells were incubated with
HUCAL.RTM. Mab#1 (=MOR03077), Mab#2 (=MOR03079), and Mab#3
(=MOR03080), the positive control (PC=chOKTlO) and an irrelevant
HUCAL.RTM. negative control for 4 hours, respectively. Afterwards,
the cell suspension was mixed with conditioned methyl-cellulose
medium and incubated for 2 weeks. Colony forming units (CFU)
derived from erythroid burst forming units (BFU-E; panel B) and
granulocyte/erythroid/macrophage/megakaryocyte stem cells
(CFU-GEMM; panels B) and granulocyte/macrophage stem cells (CFU-GM;
panel C) were counted and normalized against the medium control
("none"=medium). Panel A represents the total number of CFU (Total
CFUc) for all progenitors. Mean values from at least 10 different
PBMC donors are given. Error bars represent standard error of the
mean.
[0041] FIG. 14 provides data about ADCC with different cell-lines:
[0042] a: Single measurements (except for RPMI8226: average from 4
indiv. Assays); E:T-ratio: 30:1 [0043] b: Namba et al., 1989 [0044]
c: 5 .mu.g/ml used for antibody conc. (except for Raji with 0.1
.mu.g/ml) [0045] d: addition of retinoic assay for stimulation of
CD38-expression specific killing [%]=[(exp. killing-medium
killing)/(1-medium killing)]*100 [0046] PC: Positive control
(=chOKT10) [0047] MM: Multiple myeloma [0048] CLL: Chronic B-cell
leukemia [0049] ALL: Acute lymphoblastic leukemia [0050] AML: Acute
myeloid leukemia [0051] DSMZ: Deutsche Sammlung fur Mikroorganismen
and Zellkulturen GmbH [0052] ATCC: American type culture collection
[0053] ECACC: European collection of cell cultures [0054] MFI: Mean
fluorescence intensities.
[0055] FIG. 15 provides data about ADCC with MM-samples: [0056]
.sup.a: 2-4 individual analyses
[0057] FIG. 16 provides the experimental results of mean tumor
volumes after treatment of human myeloma xenograft with MOR03080:
group 1: vehicle; group 2: MOR03080 as hIgG1 1 mg/kg 32-68 days
every second day; group 3: MOR03080 as hIgG1 5 mg/kg 32-68 days
every second day; group 4: MOR03080 as chIgG2a 5 mg/kg 32-68 days
every second day; group 5: MOR03080 as hIgG1 1 mg/kg, 14-36 days
every second day; group 6: untreated
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention is based on the discovery of novel
antibodies that are specific to or have a high affinity for CD38
and can deliver a therapeutic benefit to a subject. The antibodies
of the invention, which may be human or humanized, can be used in
many contexts, which are more fully described herein.
[0059] A "human" antibody or functional human antibody fragment is
hereby defined as one that is not chimeric (e.g., not "humanized")
and not from (either in whole or in part) a non-human species. A
human antibody or functional antibody fragment can be derived from
a human or can be a synthetic human antibody. A "synthetic human
antibody" is defined herein as an antibody having a sequence
derived, in whole or in part, in silico from synthetic sequences
that are based on the analysis of known human antibody sequences.
In silico design of a human antibody sequence or fragment thereof
can be achieved, for example, by analyzing a database of human
antibody or antibody fragment sequences and devising a polypeptide
sequence utilizing the data obtained therefrom. Another example of
a human antibody or functional antibody fragment, is one that is
encoded by a nucleic acid isolated from a library of antibody
sequences of human origin (i.e., such library being based on
antibodies taken from a human natural source).
[0060] A "humanized antibody" or functional humanized antibody
fragment is defined herein as one that is (i) derived from a
non-human source (e.g., a transgenic mouse which bears a
heterologous immune system), which antibody is based on a human
germline sequence; or (ii) chimeric, wherein the variable domain is
derived from a non-human origin and the constant domain is derived
from a human origin or (iii) CDR-grafted, wherein the CDRs of the
variable domain are from a non-human origin, while one or more
frameworks of the variable domain are of human origin and the
constant domain (if any) is of human origin.
[0061] As used herein, an antibody "binds specifically to," is
"specific to/for" or "specifically recognizes" an antigen (here,
CD38) if such antibody is able to discriminate between such antigen
and one or more reference antigen(s), since binding specificity is
not an absolute, but a relative property. In its most general form
(and when no defined reference is mentioned), "specific binding" is
referring to the ability of the antibody to discriminate between
the antigen of interest and an unrelated antigen, as determined,
for example, in accordance with one of the following methods. Such
methods comprise, but are not limited to Western blots, ELISA-,
RIA-, ECL-, IRMA-tests and peptide scans. For example, a standard
ELISA assay can be carried out. The scoring may be carried out by
standard color development (e.g. secondary antibody with
horseradish peroxide and tetramethyl benzidine with
hydrogenperoxide). The reaction in certain wells is scored by the
optical density, for example, at 450 nm. Typical background
(=negative reaction) may be 0.1 OD; typical positive reaction may
be 1 OD. This means the difference positive/negative can be more
than 10-fold. Typically, determination of binding specificity is
performed by using not a single reference antigen, but a set of
about three to five unrelated antigens, such as milk powder, BSA,
transferrin or the like.
[0062] However, "specific binding" also may refer to the ability of
an antibody to discriminate between the target antigen and one or
more closely related antigen(s), which are used as reference
points, e.g. between CD38 and CD157. Additionally, "specific
binding" may relate to the ability of an antibody to discriminate
between different parts of its target antigen, e.g. different
domains or regions of CD38, such as epitopes in the N-terminal or
in the C-terminal region of CD38, or between one or more key amino
acid residues or stretches of amino acid residues of CD38.
[0063] Also, as used herein, an "immunoglobulin" (Ig) hereby is
defined as a protein belonging to the class IgG, IgM, IgE, IgA, or
IgD (or any subclass thereof), and includes all conventionally
known antibodies and functional fragments thereof. A "functional
fragment" of an antibody/immunoglobulin hereby is defined as a
fragment of an antibody/immunoglobulin (e.g., a variable region of
an IgG) that retains the antigen-binding region. An
"antigen-binding region" of an antibody typically is found in one
or more hypervariable region(s) of an antibody, i.e., the CDR-1,
-2, and/or -3 regions; however, the variable "framework" regions
can also play an important role in antigen binding, such as by
providing a scaffold for the CDRs. Preferably, the "antigen-binding
region" comprises at least amino acid residues 4 to 103 of the
variable light (VL) chain and 5 to 109 of the variable heavy (VH)
chain, more preferably amino acid residues 3 to 107 of VL and 4 to
111 of VH, and particularly preferred are the complete VL and VH
chains (amino acid positions 1 to 109 of VL and 1 to 113 of VH;
numbering according to WO 97/08320). A preferred class of
immunoglobulins for use in the present invention is IgG.
"Functional fragments" of the invention include the domain of a
F(ab')2 fragment, a Fab fragment and scFv. The F(ab')2 or Fab may
be engineered to minimize or completely remove the intermolecular
disulphide interactions that occur between the Cm and CL
domains.
[0064] An antibody of the invention may be derived from a
recombinant antibody library that is based on amino acid sequences
that have been designed in silico and encoded by nucleic acids that
are synthetically created. In silico design of an antibody sequence
is achieved, for example, by analyzing a database of human
sequences and devising a polypeptide sequence utilizing the data
obtained therefrom. Methods for designing and obtaining in
silico-created sequences are described, for example, in Knappik et
al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol.
Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issued to
Knappik et al., which hereby are incorporated by reference in their
entirety.
Antibodies of the Invention
[0065] Throughout this document, reference is made to the following
representative antibodies of the invention: "antibody nos." or
"LACS" or "MOR" 3077, 3079, 3080 and 3100. LAC 3077 represents an
antibody having a variable heavy region corresponding to SEQ ID NO:
1 (DNA)/SEQ ID NO: 5 (protein) and a variable light region
corresponding to SEQ ID NO: 9 (DNA)/SEQ ID NO: 13 (protein). LAC
3079 represents an antibody having a variable heavy region
corresponding to SEQ ID NO: 2 (DNA)/SEQ ID NO: 6 (protein) and a
variable light region corresponding to SEQ ID NO: 10 (DNA)/SEQ ID
NO: 14 (protein). LAC 3080 represents an antibody having a variable
heavy region corresponding to SEQ ID NO: 3 (DNA)/SEQ ID NO: 7
(protein) and a variable light region corresponding to SEQ ID NO:
11 (DNA)/SEQ ID NO: 15 (protein). LAC 3100 represents an antibody
having a variable heavy region corresponding to SEQ ID NO: 4
(DNA)/SEQ ID NO: 8 (protein) and a variable light region
corresponding to SEQ ID NO: 12 (DNA)/SEQ ID NO: 16 (protein).
[0066] In one aspect, the invention provides antibodies having an
antigen-binding region that can bind specifically to or has a high
affinity for one or more regions of CD38, whose amino acid sequence
is depicted by SEQ ID NO: 22. An antibody is said to have a "high
affinity" for an antigen if the affinity measurement is at least
100 nM (monovalent affinity of Fab fragment). An inventive antibody
or antigen-binding region preferably can bind to CD38 with an
affinity of about less than 100 nM, more preferably less than about
60 nM, and still more preferably less than about 30 nM. Further
preferred are antibodies that bind to CD38 with an affinity of less
than about 10 nM, and more preferably less than 3 about nM. For
instance, the affinity of an antibody of the invention against CD38
may be about 10.0 nM or 2.4 nM (monovalent affinity of Fab
fragment).
[0067] Table 1 provides a summary of affinities of representative
antibodies of the invention, as determined by surface plasmon
resonance (Biacore) and FACS Scatchard analysis:
TABLE-US-00001 TABLE 1 Antibody Affinities BIACORE FACS Scatchard
Antibody (Fab) (IgG1).sup.b (Fab or IgG1) K.sub.D [nM].sup.a
K.sub.D [nM].sup.a MOR03077 56.0 0.89 MOR03079 2.4 0.60 MOR03080
27.5 0.47 MOR03100 10.0 6.31 Chimeric OKT10 not determined 8.28
.sup.amean from at least 2 different affinity determinations
.sup.bRPMI8226 MM cell-line used for FACS-Scatchards
[0068] With reference to Table 1, the affinity of LACs 3077, 3079,
3080 and 3100 was measured by surface plasmon resonance (Biacore)
on immobilized recombinant CD38 and by a flow cytometry procedure
utilizing the CD38-expressing human RPMI8226 cell line. The Biacore
studies were performed on directly immobilized antigen (CD38-Fc
fusion protein). The Fab format of LACs 3077, 3079, 3080 and 3100
exhibit an monovalent affinity range between about 2.4 and 56 nM on
immobilized CD38-Fc fusion protein with LAC 3079 showing the
highest affinity, followed by Fabs 3100, 3080 and 3077.
[0069] The IgG1 format was used for the cell-based affinity
determination (FACS Scatchard). The right column of Table 1 denotes
the binding strength of the LACS in this format. LAC 3080 showed
the strongest binding, which is slightly stronger than LACS 3079
and 3077.
[0070] Another preferred feature of preferred antibodies of the
invention is their specificity for an area within the N-terminal
region of CD38. For example, LACs 3077, 3079, 3080, and 3100 of the
invention can bind specifically to the N-terminal region of
CD38.
[0071] The type of epitope to which an antibody of the invention
binds may be linear (i.e. one consecutive stretch of amino acids)
or conformational (i.e. multiple stretches of amino acids). In
order to determine whether the epitope of a particular antibody is
linear or conformational, the skilled worker can analyze the
binding of antibodies to overlapping peptides (e.g., 13-mer
peptides with an overlap of 11 amino acids) covering different
domains of CD38. Using this analysis, the inventors have discovered
that LACS 3077, 3080, and 3100 recognize discontinuous epitopes in
the N-terminal region of CD38, whereas the epitope of LAC 3079 can
be described as linear (see FIG. 7). Combined with the knowledge
provided herein, the skilled worker in the art will know how to use
one or more isolated epitopes of CD38 for generating antibodies
having an antigen-binding region that is specific for said epitopes
(e.g. using synthetic peptides of epitopes of CD38 or cells
expressing epitopes of CD38).
[0072] An antibody of the invention preferably is species
cross-reactive with humans and at least one other species, which
may be a rodent species or a non-human primate. The non-human
primate can be rhesus, baboon and/or cynomolgus. The rodent species
can be mouse, rat and/or hamster. An antibody that is cross
reactive with at least one rodent species, for example, can provide
greater flexibility and benefits over known anti-CD38 antibodies,
for purposes of conducting in vivo studies in multiple species with
the same antibody.
[0073] Preferably, an antibody of the invention not only is able to
bind to CD38, but also is able to mediate killing of a cell
expressing CD38. More specifically, an antibody of the invention
can mediate its therapeutic effect by depleting CD38-positive
(e.g., malignant) cells via antibody-effector functions. These
functions include antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC).
[0074] Table 2 provides a summary of the determination of EC50
values of representative antibodies of the invention in both ADCC
and CDC:
TABLE-US-00002 TABLE 2 EC50 Values of Antibodies ADCC CDC Antibody
EC50 [nM] EC50 [nM] (IgG1) LP-1 RPMI8226 CHO-transfectants MOR03077
0.60a 0.08a 0.8c; 0.94d MOR03079 0.09.sup.a 0.04.sup.a 0.41.sup.c
MOR03080 0.17.sup.b 0.05.sup.a 3.2.sup.c; 2.93.sup.d MOR03100
1.00.sup.b 0.28.sup.a 10.9.sup.c; 13.61.sup.e Chimeric 5.23.sup.a
4.10.sup.a 9.30.sup.c OKT10 .sup.amean from at least 2 EC50
determinations .sup.bsingle determination .sup.cmean from 2 EC50
determinations .sup.dmean from 3 EC50 determinations .sup.emean
from 4 EC50 determinations
[0075] CD38-expression, however, is not only found on immune cells
within the myeloid (e.g. monocytes, granulocytes) and lymphoid
lineage (e.g. activated B and T-cells; plasma cells), but also on
the respective precursor cells. Since it is important that those
cells are not affected by antibody-mediated killing of malignant
cells, the antibodies of the present invention are preferably not
cytotoxic to precursor cells.
[0076] In addition to its catalytic activities as a cyclic
ADP-ribose cyclase and hydrolase, CD38 displays the ability to
transduce signals of biological relevance (Hoshino et al., 1997;
Ausiello et al., 2000). Those functions can be induced in vivo by,
e.g. receptor-ligand interactions or by cross-linking with
agonistic anti-CD38 antibodies, leading, e.g. to calcium
mobilization, lymphocyte proliferation and release of cytokines.
Preferably, the antibodies of the present invention are
non-agonistic antibodies.
Peptide Variants
[0077] Antibodies of the invention are not limited to the specific
peptide sequences provided herein. Rather, the invention also
embodies variants of these polypeptides. With reference to the
instant disclosure and conventionally available technologies and
references, the skilled worker will be able to prepare, test and
utilize functional variants of the antibodies disclosed herein,
while appreciating that variants having the ability to mediate
killing of a CD38+ target cell fall within the scope of the present
invention. As used in this context, "ability to mediate killing of
a CD38+ target cell" means a functional characteristic ascribed to
an anti-CD38 antibody of the invention. Ability to mediate killing
of a CD38+ target cell, thus, includes the ability to mediate
killing of a CD38+ target cell, e.g. by ADCC and/or CDC, or by
toxin constructs conjugated to an antibody of the invention.
[0078] A variant can include, for example, an antibody that has at
least one altered complementarity determining region (CDR)
(hyper-variable) and/or framework (FR) (variable) domain/position,
vis-a-vis a peptide sequence disclosed herein. To better illustrate
this concept, a brief description of antibody structure
follows.
[0079] An antibody is composed of two peptide chains, each
containing one (light chain) or three (heavy chain) constant
domains and a variable region (VL, VH), the latter of which is in
each case made up of four FR regions and three interspaced CDRs.
The antigen-binding site is formed by one or more CDRs, yet the FR
regions provide the structural framework for the CDRs and, hence,
play an important role in antigen binding. By altering one or more
amino acid residues in a CDR or FR region, the skilled worker
routinely can generate mutated or diversified antibody sequences,
which can be screened against the antigen, for new or improved
properties, for example.
[0080] Tables 3a (VH) and 3b (VL) delineate the CDR and FR regions
for certain antibodies of the invention and compare amino acids at
a given position to each other and to corresponding consensus or
"master gene" sequences (as described in U.S. Pat. No.
6,300,064):
TABLE-US-00003 TABLE 3a VH Sequences ##STR00001##
TABLE-US-00004 TABLE 3b VL Sequences ##STR00002## ##STR00003##
[0081] The skilled worker can use the data in Tables 3a and 3b to
design peptide variants that are within the scope of the present
invention. It is preferred that variants are constructed by
changing amino acids within one or more CDR regions; a variant
might also have one or more altered framework regions. With
reference to a comparison of the novel antibodies to each other,
candidate residues that can be changed include e.g. residues 4 or
37 of the variable light and e.g. residues 13 or 43 of the variable
heavy chains of LACs 3080 and 3077, since these are positions of
variance vis-a-vis each other. Alterations also may be made in the
framework regions. For example, a peptide FR domain might be
altered where there is a deviation in a residue compared to a
germline sequence.
[0082] With reference to a comparison of the novel antibodies to
the corresponding consensus or "master gene" sequence, candidate
residues that can be changed include e.g. residues 27, 50 or 90 of
the variable light chain of LAC 3080 compared to VL.lamda.3 and
e.g. residues 33, 52 and 97 of the variable heavy chain of LAC 3080
compared to VH3. Alternatively, the skilled worker could make the
same analysis by comparing the amino acid sequences disclosed
herein to known sequences of the same class of such antibodies,
using, for example, the procedure described by Knappik et al., 2000
and U.S. Pat. No. 6,300,064 issued to Knappik et al.
[0083] Furthermore, variants may be obtained by using one LAC as
starting point for optimization by diversifying one or more amino
acid residues in the LAC, preferably amino acid residues in one or
more CDRs, and by screening the resulting collection of antibody
variants for variants with improved properties. Particularly
preferred is diversification of one or more amino acid residues in
CDR-3 of VL, CDR-3 of VH, CDR-1 of VL and/or CDR-2 of VH.
Diversification can be done by synthesizing a collection of DNA
molecules using trinucleotide mutagenesis (TRIM) technology
(Virnekas, B., Ge, L., Pluckthun, A., Schneider, K. C., Wellnhofer,
G., and Moroney S. E. (1994) Trinucleotide phosphoramidites: ideal
reagents for the synthesis of mixed oligonucleotides for random
mutagenesis. Nucl. Acids Res. 22, 5600.).
Conservative Amino Acid Variants
[0084] Polypeptide variants may be made that conserve the overall
molecular structure of an antibody peptide sequence described
herein. Given the properties of the individual amino acids, some
rational substitutions will be recognized by the skilled worker.
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 residues involved.
[0085] For example, (a) nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; (b) polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; (c) positively charged (basic) amino acids include
arginine, lysine, and histidine; and (d) negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
Substitutions typically may be made within groups (a)-(d). In
addition, glycine and proline may be substituted for one another
based on their ability to disrupt .alpha.-helices. Similarly,
certain amino acids, such as alanine, cysteine, leucine,
methionine, glutamic acid, glutamine, histidine and lysine are more
commonly found in .alpha.-helices, while valine, isoleucine,
phenylalanine, tyrosine, tryptophan and threonine are more commonly
found in .beta.-pleated sheets. Glycine, serine, aspartic acid,
asparagine, and proline are commonly found in turns. Some preferred
substitutions may be made among the following groups: (i) S and T;
(ii) P and G; and (iii) A, V, L and I. Given the known genetic
code, and recombinant and synthetic DNA techniques, the skilled
scientist readily can construct DNAs encoding the conservative
amino acid variants. In one particular example, amino acid position
3 in SEQ ID NOS: 5, 6, 7, and/or 8 can be changed from a Q to an
E.
[0086] As used herein, "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. Preferred
polypeptide sequences of 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%.
DNA Molecules of the Invention
[0087] The present invention also relates to the DNA molecules that
encode an antibody of the invention. These sequences include, but
are not limited to, those DNA molecules set forth in FIGS. 1a and
2a.
[0088] DNA molecules of the invention are not limited to the
sequences disclosed herein, but also include variants thereof. DNA
variants within the invention may be described by reference to
their physical properties in hybridization. The skilled worker will
recognize that DNA can be used to identify its complement and,
since DNA is double stranded, its equivalent or homolog, using
nucleic acid hybridization techniques. It also will be recognized
that hybridization can occur with less than 100% complementarity.
However, given appropriate choice of conditions, hybridization
techniques can be used to differentiate among DNA sequences based
on their structural relatedness to a particular probe. For guidance
regarding such conditions see, Sambrook et al., 1989 (Sambrook, J.,
Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, USA) and Ausubel et al., 1995 (Ausubel, F. M., Brent, R.,
Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., &
Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New
York: John Wiley and Sons).
[0089] Structural similarity between two polynucleotide sequences
can be expressed as a function of "stringency" of the conditions
under which the two sequences will hybridize with one another. As
used herein, the term "stringency" refers to the extent that the
conditions disfavor hybridization. Stringent conditions strongly
disfavor hybridization, and only the most structurally related
molecules will hybridize to one another under such conditions.
Conversely, non-stringent conditions favor hybridization of
molecules displaying a lesser degree of structural relatedness.
Hybridization stringency, therefore, directly correlates with the
structural relationships of two nucleic acid sequences. The
following relationships are useful in correlating hybridization and
relatedness (where T. is the melting temperature of a nucleic acid
duplex): [0090] a. T.sub.m=69.3+0.41(G+C)% [0091] b. The T.sub.m of
a duplex DNA decreases by 1.degree. C. with every increase of 1% in
the number of mismatched base pairs. [0092] c.
(T.sub.m).sub..mu.2-(T.sub.m).sub..mu.1=18.5 log.sub.10 .mu.2/.mu.1
[0093] where .mu.1 and .mu.2 are the ionic strengths of two
solutions.
[0094] Hybridization stringency is a function of many factors,
including overall DNA concentration, ionic strength, temperature,
probe size and the presence of agents which disrupt hydrogen
bonding. Factors promoting hybridization include high DNA
concentrations, high ionic strengths, low temperatures, longer
probe size and the absence of agents that disrupt hydrogen bonding.
Hybridization typically is performed in two phases: the "binding"
phase and the "washing" phase.
[0095] First, in the binding phase, the probe is bound to the
target under conditions favoring hybridization. Stringency is
usually controlled at this stage by altering the temperature. For
high stringency, the temperature is usually between 65.degree. C.
and 70.degree. C., unless short (<20 nt) oligonucleotide probes
are used. A representative hybridization solution comprises
6.times.SSC, 0.5% SDS, 5.times.Denhardt's solution and 100 .mu.g of
nonspecific carrier DNA. See Ausubel et al., section 2.9,
supplement 27 (1994). Of course, many different, yet functionally
equivalent, buffer conditions are known. Where the degree of
relatedness is lower, a lower temperature may be chosen. Low
stringency binding temperatures are between about 25.degree. C. and
40.degree. C. Medium stringency is between at least about
40.degree. C. to less than about 65.degree. C. High stringency is
at least about 65.degree. C.
[0096] Second, the excess probe is removed by washing. It is at
this phase that more stringent conditions usually are applied.
Hence, it is this "washing" stage that is most important in
determining relatedness via hybridization. Washing solutions
typically contain lower salt concentrations. One exemplary medium
stringency solution contains 2.times.SSC and 0.1% SDS. A high
stringency wash solution contains the equivalent (in ionic
strength) of less than about 0.2.times.SSC, with a preferred
stringent solution containing about 0.1.times.SSC. The temperatures
associated with various stringencies are the same as discussed
above for "binding." The washing solution also typically is
replaced a number of times during washing. For example, typical
high stringency washing conditions comprise washing twice for 30
minutes at 55.degree. C. and three times for 15 minutes at
60.degree. C.
[0097] Accordingly, the present invention includes nucleic acid
molecules that hybridize to the molecules of set forth in FIGS. 1a
and 2a under high stringency binding and washing conditions, where
such nucleic molecules encode an antibody or functional fragment
thereof having properties as described herein. 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. In one particular example of a
variant of the invention, nucleic acid position 7 in SEQ ID NOS: 1,
2, 3 and/or 4 can be substituted from a C to a G, thereby changing
the codon from CAA to GAA.
Functionally Equivalent Variants
[0098] Yet another class of DNA variants within the scope of the
invention may be described with reference to the product they
encode (see the peptides listed in FIGS. 1b and 2b). These
functionally equivalent genes are characterized by the fact that
they encode the same peptide sequences found in FIGS. 1b and 2b due
to the degeneracy of the genetic code. SEQ ID NOS: 1 and 31 are an
example of functionally equivalent variants, as their nucleic acid
sequences are different, yet they encode the same polypeptide, i.e.
SEQ ID NO: 5.
[0099] It is recognized that variants of DNA molecules provided
herein can be constructed in several different ways. For example,
they may be constructed as completely synthetic DNAs. Methods of
efficiently synthesizing oligonucleotides in the range of 20 to
about 150 nucleotides are widely available. See Ausubel et al.,
section 2.11, Supplement 21 (1993). Overlapping oligonucleotides
may be synthesized and assembled in a fashion first reported by
Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel
et al., supra, Section 8.2. Synthetic DNAs preferably are designed
with convenient restriction sites engineered at the 5' and 3' ends
of the gene to facilitate cloning into an appropriate vector.
[0100] As indicated, a method of generating variants is to start
with one of the DNAs disclosed herein and then to conduct
site-directed mutagenesis. See Ausubel et al., supra, chapter 8,
Supplement 37 (1997). In a typical method, a target DNA is cloned
into a single-stranded DNA bacteriophage vehicle. Single-stranded
DNA is isolated and hybridized with an oligonucleotide containing
the desired nucleotide alteration(s). The complementary strand is
synthesized and the double stranded phage is introduced into a
host. Some of the resulting progeny will contain the desired
mutant, which can be confirmed using DNA sequencing. In addition,
various methods are available that increase the probability that
the progeny phage will be the desired mutant. These methods are
well known to those in the field and kits are commercially
available for generating such mutants.
Recombinant DNA Constructs and Expression
[0101] The present invention further provides recombinant DNA
constructs comprising one or more of the nucleotide sequences of
the present invention. The recombinant constructs of the present
invention are used in connection with a vector, such as a plasmid
or viral vector, into which a DNA molecule encoding an antibody of
the invention is inserted.
[0102] The encoded gene may be produced by techniques described in
Sambrook et al., 1989, and Ausubel et al., 1989. Alternatively, the
DNA sequences may be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
OLIGONUCLEOTIDE SYNTHESIS (1984, Gait, ed., IRL Press, Oxford),
which is incorporated by reference herein in its entirety.
Recombinant constructs of the invention are comprised with
expression vectors that are capable of expressing the RNA and/or
protein products of the encoded DNA(s). The vector may further
comprise regulatory sequences, including a promoter operably linked
to the open reading frame (ORF). The vector may further comprise a
selectable marker sequence. Specific initiation and bacterial
secretory signals also may be required for efficient translation of
inserted target gene coding sequences.
[0103] The present invention further provides host cells containing
at least one of the DNAs of the present invention. The host cell
can be virtually any cell for which expression vectors are
available. It may be, for example, a higher eukaryotic host cell,
such as a mammalian cell, a lower eukaryotic host cell, such as a
yeast cell, but preferably is a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran mediated transfection, electroporation or phage
infection.
Bacterial Expression
[0104] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and,
if desirable, to provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus.
[0105] Bacterial vectors may be, for example, bacteriophage-,
plasmid- or phagemid-based. These vectors can contain a selectable
marker and bacterial origin of replication derived from
commercially available plasmids typically containing elements of
the well known cloning vector pBR322 (ATCC 37017). Following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
de-repressed/induced by appropriate means (e.g., temperature shift
or chemical induction) and cells are cultured for an additional
period. Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0106] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
protein being expressed. For example, when a large quantity of such
a protein is to be produced, for the generation of antibodies or to
screen peptide libraries, for example, vectors which direct the
expression of high levels of fusion protein products that are
readily purified may be desirable.
Therapeutic Methods
[0107] Therapeutic methods involve administering to a subject in
need of treatment a therapeutically effective amount of an antibody
contemplated by the invention. A "therapeutically effective" amount
hereby is defined as the amount of an antibody that is of
sufficient quantity to deplete CD38-positive cells in a treated
area of a subject--either as a single dose or according to a
multiple dose regimen, alone or in combination with other agents,
which leads to the alleviation of an adverse condition, yet which
amount is toxicologically tolerable. The subject may be a human or
non-human animal (e.g., rabbit, rat, mouse, monkey or other
lower-order primate).
[0108] An antibody of the invention might be co-administered with
known medicaments, and in some instances the antibody might itself
be modified. For example, an antibody could be conjugated to an
immunotoxin or radioisotope to potentially further increase
efficacy.
[0109] The inventive antibodies can be used as a therapeutic or a
diagnostic tool in a variety of situations where CD38 is
undesirably expressed or found. Disorders and conditions
particularly suitable for treatment with an antibody of the
inventions are multiple myeloma (MM) and other haematological
diseases, such as chronic lymphocytic leukemia (CLL), chronic
myelogenous leukemia (CML), acute myelogenous leukemia (AML), and
acute lymphocytic leukemia (ALL). An antibody of the invention also
might be used to treat inflammatory disease such as rheumatoid
arthritis (RA) or systemic lupus erythematosus (SLE).
[0110] To treat any of the foregoing disorders, pharmaceutical
compositions for use in accordance with the present invention may
be formulated in a conventional manner using one or more
physiologically acceptable carriers or excipients. An antibody of
the invention can be administered by any suitable means, which can
vary, depending on the type of disorder being treated. Possible
administration routes include parenteral (e.g., intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous),
intrapulmonary and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration. In
addition, an antibody of the invention might be administered by
pulse infusion, with, e.g., declining doses of the antibody.
Preferably, the dosing is given by injections, most preferably
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. The amount to be
administered will depend on a variety of factors such as the
clinical symptoms, weight of the individual, whether other drugs
are administered. The skilled artisan will recognize that the route
of administration will vary depending on the disorder or condition
to be treated.
[0111] Determining a therapeutically effective amount of the novel
polypeptide, according to this invention, largely will depend on
particular patient characteristics, route of administration, and
the nature of the disorder being treated. General guidance can be
found, for example, in the publications of the International
Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL
SCIENCES, chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R.
Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically,
determining a therapeutically effective amount will depend on such
factors as toxicity and efficacy of the medicament. Toxicity may be
determined using methods well known in the art and found in the
foregoing references. Efficacy may be determined utilizing the same
guidance in conjunction with the methods described below in the
Examples.
Diagnostic Methods
[0112] CD38 is highly expressed on hematological cells in certain
malignancies; thus, an anti-CD38 antibody of the invention may be
employed in order to image or visualize a site of possible
accumulation of malignant cells in a patient. In this regard, an
antibody can be detectably labeled, through the use of
radioisotopes, affinity labels (such as biotin, avidin, etc.)
fluorescent labels, paramagnetic atoms, etc. Procedures for
accomplishing such labeling are well known to the art. Clinical
application of antibodies in diagnostic imaging are reviewed by
Grossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)), Unger,
E. C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B. A.
et al., Science 209:295-297 (1980)).
[0113] The detection of foci of such detectably labeled antibodies
might be indicative of a site of tumor development, for example. In
one embodiment, this examination is done by removing samples of
tissue or blood and incubating such samples in the presence of the
detectably labeled antibodies. In a preferred embodiment, this
technique is done in a non-invasive manner through the use of
magnetic imaging, fluorography, etc. Such a diagnostic test may be
employed in monitoring the success of treatment of diseases, where
presence or absence of CD38-positive cells is a relevant indicator.
The invention also contemplates the use of an anti-CD38 antibody,
as described herein for diagnostics in an ex vivo setting.
Therapeutic and Diagnostic Compositions
[0114] The antibodies of the present invention can be formulated
according to known methods to prepare pharmaceutically useful
compositions, wherein an antibody of the invention (including any
functional fragment thereof) is combined in a mixture with a
pharmaceutically acceptable carrier vehicle. Suitable vehicles and
their formulation are described, for example, in REMINGTON'S
PHARMACEUTICAL SCIENCES (18th ed., Alfonso R. Gennaro, Ed., Easton,
Pa.: Mack Pub. Co., 1990). In order to form a pharmaceutically
acceptable composition suitable for effective administration, such
compositions will contain an effective amount of one or more of the
antibodies of the present invention, together with a suitable
amount of carrier vehicle.
[0115] Preparations may be suitably formulated to give
controlled-release of the active compound. Controlled-release
preparations may be achieved through the use of polymers to complex
or absorb anti-CD38 antibody. The controlled delivery may be
exercised by selecting appropriate macromolecules (for example
polyesters, polyamino acids, polyvinyl, pyrrolidone,
ethylenevinyl-acetate, methylcellulose, carboxymethylcellulose, or
protamine, sulfate) and the concentration of macromolecules as well
as the methods of incorporation in order to control release.
Another possible method to control the duration of action by
controlled release preparations is to incorporate anti-CD38
antibody into particles of a polymeric material such as polyesters,
polyamino acids, hydrogels, poly(lactic acid) or ethylene
vinylacetate copolymers. Alternatively, instead of incorporating
these agents into polymeric particles, it is possible to entrap
these materials in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatine-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences (1980).
[0116] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules, or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0117] The compositions may, if desired, be presented in a pack or
dispenser device, which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0118] The invention further is understood by reference to the
following working examples, which are intended to illustrate and,
hence, not limit the invention.
EXAMPLES
Cell-Lines
[0119] The following cell-lines were obtained from the European
Collection of Cell Cultures (ECACC), the German Collection of
Microorganisms (DSMZ) or the American Type Culture collection
(ATCC): hybridoma cell line producing the CD38 mouse IgG1
monoclonal antibody OKT10 (ECACC, #87021903), Jurkat cells (DSMZ,
ACC282), LP-1 (DSMZ, ACC41), RPMI8226 (ATCC, CCL-155), HEK293
(ATCC, CRL-1573), CHO-K1 (ATCC, CRL-61) and Raji (ATCC, CCL-86)
Cells and Culture-Conditions
[0120] All cells were cultured under standardized conditions at
37.degree. C. and 5% CO.sub.2 in a humidified incubator. The
cell-lines LP-1, RPMI8226, Jurkat and Raji were cultured in
RPMI1640 (Pan biotech GmbH, #P04-16500) supplemented with 10% FCS
(PAN biotech GmbH, #P30-3302), 50 U/ml penicillin, 50 .mu.g/ml
streptomycin (Gibco, #15140-122) and 2 mM glutamine (Gibco,
#25030-024) and, in case of Jurkat- and Raji-cells, additionally 10
mM Hepes (Pan biotech GmbH, #P05-01100) and 1 mM sodium pyruvate
(Pan biotech GmbH, # P04-43100) had to be added.
[0121] CHO-K1 and HEK293 were grown in DMEM (Gibco, #10938-025)
supplemented with 2 mM glutamine and 10% FCS. Stable CD38 CHO-K1
transfectants were maintained in the presence of G418 (PAA GmbH,
P11-012) whereas for HEK293 the addition of 1 mM sodium-pyruvate
was essential. After transient transfection of HEK293 the 10% FCS
was replaced by Ultra low IgG FCS (Invitrogen, #16250-078). The
cell-line OKT10 was cultured in IDMEM (Gibco, #31980-022),
supplemented with 2 mM glutamine and 20% FCS.
Preparation of Single Cell Suspensions from Peripheral Blood
[0122] All blood samples were taken after informed consent.
Peripheral blood mononuclear cells (PBMC) were isolated by
Histopaque.RTM.-1077 (Sigma) according to the manufacturer's
instructions from healthy donors. Red blood cells were depleted
from these cell suspensions by incubation in ACK Lysis Buffer (0.15
M NH4Cl, 10 mM KHCO.sub.3, 0.1 M EDTA) for 5 min at RT or a
commercial derivative (Bioscience, #00-4333). Cells were washed
twice with PBS and then further processed for flow cytometry or
ADCC (see below).
Flow Cytometry ("FACS")
[0123] All stainings were performed in round bottom 96-well culture
plates (Nalge Nunc) with 2.times.10.sup.5 cells per well. Cells
were incubated with Fab or IgG antibodies at the indicated
concentrations in 50 .mu.l FACS buffer (PBS, 3% FCS, 0.02%
NaN.sub.3) for 40 min at 4.degree. C. Cells were washed twice and
then incubated with R-Phycoerythrin (PE) conjugated goat-anti-human
or goat-anti-mouse IgG (H+L) F(ab').sub.2 (Jackson Immuno
Research), diluted 1:200 in FACS buffer, for 30 min at 4.degree. C.
Cells were again washed, resuspended in 0.3 ml FACS buffer and then
analyzed by flow cytometry in a FACSCalibur (Becton Dickinson, San
Diego, Calif.).
[0124] For FACS based Scatchard analyses RPMI8226 cells were
stained with at 12 different dilutions (1:2.sup.n) starting at 12.5
.mu.g/ml (IgG) final concentration. At least two independent
measurements were used for each concentration and K.sub.D values
extrapolated from median fluorescence intensities according to
Chamow et al. (1994).
Surface Plasmon Resonance
[0125] The kinetic constants k.sub.on and k.sub.off were determined
with serial dilutions of the respective Fab binding to covalently
immobilized CD38-Fc fusion protein using the BIACORE 3000
instrument (BIACORE, Uppsala, Sweden). For covalent antigen
immobilization standard EDC-NHS amine coupling chemistry was used.
For direct coupling of CD38 Fc-fusion protein CM5 senor chips
(BIACORE) were coated with -600-700 RU in 10 mM acetate buffer, pH
4.5.
[0126] For the reference flow cell a respective amount of HSA
(human serum albumin) was used. Kinetic measurements were done in
PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO4, 1.76 mM
KH.sub.2PO.sub.4 pH 7.4) at a flow rate of 20 .mu.l/min using Fab
concentration range from 1.5-500 nM. Injection time for each
concentration was 1 min, followed by 2 min dissociation phase. For
regeneration 5 .mu.l 10 mM HCl was used. All sensograms were fitted
locally using BIA evaluation software 3.1 (BIACORE).
Example 1
Antibody Generation from HuCAL.RTM. Libraries
[0127] For the generation of therapeutic antibodies against CD38,
selections with the MorphoSys HUCAL GOLD.RTM. phage display library
were carried out. HUCAL GOLD.RTM. is a Fab library based on the
HUCAL.RTM. concept (Knappik et al., 2000; Krebs et al., 2001), in
which all six CDRs are diversified, and which employs the
CYSDISPLAY.TM. technology for linking Fab fragments to the phage
surface (Lohning, 2001).
A. Phagemid Rescue, Phage Amplification and Purification
[0128] HUCAL GOLD.RTM. phagemid library was amplified in 2.times.TY
medium containing 34 .mu.g/ml chloramphenicol and 1% glucose
(2.times.TY-CG). After helper phage infection (VCSM13) at an OD600
of 0.5 (30 min at 37.degree. C. without shaking; 30 min at
37.degree. C. shaking at 250 rpm), cells were spun down (4120 g; 5
min; 4.degree. C.), resuspended in 2.times.TY/34 .mu.g/ml
chloramphenicol/50 [tg/mlkanamycin and grown overnight at
22.degree. C. Phages were PEG-precipitated from the supernatant,
resuspended in PBS/20% glycerol and stored at -80.degree. C. Phage
amplification between two panning rounds was conducted as follows:
mid-log phase TGI cells were infected with eluted phages and plated
onto LB-agar supplemented with 1% of glucose and 34 .mu.g/ml of
chloramphenicol (LB-CG). After overnight incubation at 30.degree.
C., colonies were scraped off, adjusted to an OD600 of 0.5 and
helper phage added as described above.
B. Pannings with HUCAL GOLD.RTM.
[0129] For the selections HUCAL GOLD.RTM. antibody-phages were
divided into three pools corresponding to different VH master genes
VH1/5.lamda..kappa., pool 2: VH3 .lamda..kappa., pool 3:
VH2/4/6.lamda..kappa.). These pools were individually subjected to
3 rounds of whole cell panning on CD38-expressing CHO-K1 cells
followed by pH-elution and a post-adsorption step on CD38-negative
CHO-K1-cells for depletion of irrelevant antibody-phages. Finally,
the remaining antibody phages were used to infect E. coli TGI
cells. After centrifugation the bacterial pellet was resuspended in
2.times.TY medium, plated on agar plates and incubated overnight at
30.degree. C. The selected clones were then scraped from the
plates, phages were rescued and amplified. The second and the third
round of selections were performed as the initial one.
[0130] The Fab encoding inserts of the selected HUCAL GOLD.RTM.
phages were subcloned into the expression vector pMORPH0.times.9
Fab FS (Rauchenberger et al., 2003) to facilitate rapid expression
of soluble Fab. The DNA of the selected clones was digested with
Xbal and EcoRI thereby cutting out the Fab encoding insert
(ompA-VLCL and phoA-Fd), and cloned into the Xbal/EcoRI cut vector
pMORPH0.times.9 Fab FS. Fab expressed in this vector carry two
C-terminal tags (FLAG.TM. and STREP-TAG.RTM. II) for detection and
purification.
Example 2
Biological Assays
[0131] Antibody dependent cellular cytotoxicity (ADCC) and
complement-dependent cytotoxicity was measured according to a
published protocol based on flow-cytometry analysis (Naundorf et
al., 2002) as follows:
ADCC:
[0132] For ADCC measurements, target cells (T) were adjusted to
2.0E.+-.05 cells/ml and labeled with 100 ng/ml Calcein AM
(Molecular Probes, C-3099) in RPMI1640 medium (Pan biotech GmbH)
for 2 minutes at room temperature. Residual calcein was removed by
3 washing steps in RPMI1640 medium. In parallel PBMC were prepared
as source for (natural killer) effector cells (E), adjusted to
1.0E+07 and mixed with the labeled target cells to yield a final
E:T-ratio of 50:1 or less, depending on the assay conditions. Cells
were washed once and the cell-mix resuspended in 200 .mu.l RPMI1640
medium containing the respective antibody at different dilutions.
The plate was incubated for 4 hrs under standardized conditions at
37.degree. C. and 5% CO.sub.2 in a humidified incubator. Prior to
FACS analysis cells were labeled with propidium-iodide (PI) and
analyzed by flow-cytometry (Becton-Dickinson). Between 50.000 and
150.000 events were counted for each assay.
[0133] The following equation gave rise to the killing activity [in
%]:
ED A EL A + ED A .times. 100 ##EQU00001##
with ED.sup.A=events dead cells (calcein+PI stained cells), and
EL.sup.A=events living cells (calcein stained cells)
CDC:
[0134] For CDC measurements, 5.0E+04 CD38 CHO-K1 transfectants were
added to a microtiter well plate (Nunc) together with a 1:4
dilution of human serum (Sigma, #S-1764) and the respective
antibody. All reagents and cells were diluted in RPMI1640 medium
(Pan biotech GmbH) supplemented with 10% FCS. The reaction-mix was
incubated for 2 hrs under standardized conditions at 37.degree. C.
and 5% CO.sub.2 in a humidified incubator. As negative controls
served either heat-inactivated complement or CD38-transfectants
without antibody. Cells were labeled with PI and subjected to
FACS-analysis.
[0135] In total 5000 events were counted and the number of dead
cells at different antibody concentrations used for the
determination of EC50 values. The following equation gave rise to
the killing activity [in %]:
ED C EL C + ED C .times. 100 ##EQU00002##
with ED.sup.C=events dead cells (PI stained cells), and
EL.sup.C=events living cells (unstained)
[0136] Cytotoxicity values from a total of 12 different
antibody-dilutions (1:2.sup.n) in triplicates were used in ADCC and
duplicates in CDC for each antibody in order obtain EC-50 values
with a standard analysis software (PRISM.RTM., Graph Pad
Software).
Example 3
Generation of Stable CD38-Transfectants and CD38 Fc-Fusion
Proteins
[0137] In order to generate CD38 protein for panning and screening
two different expression systems had to be established. The first
strategy included the generation of CD38-Fc-fusion protein, which
was purified from supernatants after transient transfection of
HEK293 cells. The second strategy involved the generation of a
stable CHO-K1-cell line for high CD38 surface expression to be used
for selection of antibody-phages via whole cell panning.
[0138] As an initial step Jurkat cells (DSMZ ACC282) were used for
the generation of cDNA (Invitrogen) followed by amplification of
the entire CD38-coding sequence using primers complementary to the
first 7 and the last 9 codons of CD38, respectively (primer MTE001
& MTE002rev; Table 4). Sequence analysis of the CD38-insert
confirmed the published amino acid sequence by Jackson et al.
(1990) except for position 49 which revealed a glutamine instead of
a tyrosine as described by Nata et al. (1997). For introduction of
restriction endonuclease sites and cloning into different
derivatives of expression vector pcDNA3.1 (Stratagene), the
purified PCR-product served as a template for the re-amplification
of the entire gene (primers MTE006 & MTE007rev, Table 4) or a
part (primers MTE004 & MTE009rev, Table 4) of it. In the latter
case a fragment encoding for the extracellular domain (aa 45 to
300) was amplified and cloned in frame between a human Vkappa
leader sequence and a human Fc-gamma 1 sequence. This vector served
as expression vector for the generation of soluble CD38-Fc
fusion-protein. Another pcDNA3.1-derivative without leader-sequence
was used for insertion of the CD38 full-length gene. In this case a
stop codon in front of the Fc-coding region and the missing
leader-sequence gave rise to CD38-surface expression. HEK293 cells
were transiently transfected with the Fc-fusion protein vector for
generation of soluble CD38 Fc-fusion protein and, in case of the
full-length derivative, CHO-K1-cells were transfected for the
generation of a stable CD38-expressing cell line.
TABLE-US-00005 TABLE 4 Primer # Sequence (5'->3') MTE001 ATG GCC
AAC TGC GAG TTC AGC (SEQ ID NO: 25) MTE002rev TCA GAT CTC AGA TGT
GCA AGA TGA ATC (SEQ ID NO: 26) MTE004 TT GGT ACC AGG TGG CGC CAG
CAG TG (SEQ ID NO: 27) MTE006 TT GGT ACC ATG GCC AAC TGC GAG (SEQ
ID NO: 28) MTE007rev CCG ATA TCA* GAT CTC AGA TGT GCA AGA TG (SEQ
ID NO: 29) MTE009rev CCG ATA TC GAT CTC AGA TGT GCA AGA TG (SEQ ID
NO: 30) *leading to a stop codon (TGA) in the sense
orientation.
Example 4
Cloning, Expression and Purification of HuCAL.RTM. IgG1
[0139] In order to express full length IgG, variable domain
fragments of heavy (VH) and light chains (VL) were subcloned from
Fab expression vectors into appropriate pMORPH.RTM._hIg vectors
(see FIGS. 8 to 10). Restriction endonuclease pairs BlpI/MfeI
(insert-preparation) and BlpI/EcoRI (vector-preparation) were used
for subcloning of the VH domain fragment into pMORPH.RTM._hIgG1.
Enzyme-pairs EcoRV/HpaI (lambda-insert) and EcoRV/BsiWI
(kappa-insert) were used for subcloning of the VL domain fragment
into the respective pMORPH.RTM._hIg.kappa._1 or
pMORPH.RTM._h_Ig.lamda._1 vectors. Resulting IgG constructs were
expressed in HEK293 cells (ATCC CRL-1573) by transient transfection
using standard calcium phosphate-DNA coprecipitation technique.
[0140] IgGs were purified from cell culture supernatants by
affinity chromatography via Protein A Sepharose column. Further
down stream processing included a buffer exchange by gel filtration
and sterile filtration of purified IgG. Quality control revealed a
purity of >90% by reducing SDS-PAGE and >90% monomeric IgG as
determined by analytical size exclusion chromatography. The
endotoxin content of the material was determined by a kinetic LAL
based assay (Cambrex European Endotoxin Testing Service,
Belgium).
Example 5
Generation and Production of Chimeric OKT10 (chOKT10; SEQ ID NOS:
23 and 24)
[0141] For the construction of chOKT10 the mouse VH and VL regions
were amplified by PCR using cDNA prepared from the murine OKT10
hybridoma cell line (ECACC #87021903). A set of primers was used as
published (Dattamajumdar et al., 1996; Zhou et al., 1994). PCR
products were used for Topo-cloning (Invitrogen; pCRII-vector) and
single colonies subjected to sequence analysis (M13 reverse primer)
which revealed two different kappa light chain sequences and one
heavy chain sequence. According to sequence alignments
(EMBL-nucleotide sequence database) and literature (Krebber et al,
1997) one of the kappa-sequence belongs to the intrinsic repertoire
of the tumor cell fusion partner X63Ag8.653 and hence does not
belong to OKT10 antibody. Therefore, only the new kappa sequence
and the single VH-fragment was used for further cloning. Both
fragments were reamplified for the addition of restriction
endonuclease sites followed by cloning into the respective
pMORPH.RTM. IgG1-expression vectors. The sequences for the heavy
chain (SEQ ID NO: 23) and light chain (SEQ ID NO: 24) are given in
FIG. 6. HEK293 cells were transfected transiently and the
supernatant analyzed in FACS for the chimeric OKT10 antibody
binding to the CD38 overexpressing Raji cell line (ATCC).
Example 6
Epitope Mapping
1. Materials and Methods:
Antibodies:
[0142] The following anti-CD38 IgGs were sent for epitope
mappings:
TABLE-US-00006 Conc. [mg/ml]/ MOR# Lot # Format Vol. [.mu.l]
MOR03077 2CHE106_030602 human IgG1 0.44/1500 MOR03079 2APO31 human
IgG1 0.38/500 MOR03080 030116_4CUE16 human IgG1 2.28/200 MOR03100
030612_6SBA6 human IgG1 0.39/500 chim. 030603_2CHE111 human IgG1
0.83/500 OKT10* *chimeric OKT10 consisting of human Fc and mouse
variable regions.
CD38-Sequence:
[0143] The amino acid (aa) sequence (position 44-300) is based on
human CD38 taken from the published sequence under SWISS-PROT
primary accession number P28907. At position 49 the aa Q (instead
of T) has been used for the peptide-design.
PepSpot-Analysis:
[0144] The antigen peptides were synthesized on a cellulose
membrane in a stepwise manner resulting in a defined arrangement
(peptide array) and are covalently bound to the cellulose membrane.
Binding assays were performed directly on the peptide array.
[0145] In general an antigen peptide array is incubated with
blocking buffer for several hours to reduce non-specific binding of
the antibodies. The incubation with the primary (antigen
peptide-binding) antibody in blocking buffer occurs followed by the
incubation with the peroxidase (POD)-labelled secondary antibody,
which binds selectively the primary antibody. A short T
(Tween)-TBS-buffer washing directly after the incubation of the
antigen peptide array with the secondary antibody followed by the
first chemiluminescence experiment is made to get a first overview
which antigen peptides do bind the primary antibody. Several buffer
washing steps follow (T-TBS- and TBS-buffer) to reduce false
positive binding (unspecific antibody binding to the cellulose
membrane itself). After these washing steps the final
chemiluminescence analysis is performed. The data were analysed
with an imaging system showing the signal intensity (Boehringer
Light units, BLU) as single measurements for each peptide. In order
to evaluate non-specific binding of the secondary antibodies
(anti-human IgG), these antibodies were incubated with the peptide
array in the absence of primary antibodies as the first step. If
the primary antibody does not show any binding to the peptides it
can be directly labelled with POD, which increases the sensitivity
of the system (as performed for MOR3077). In this case a
conventional coupling chemistry via free amino-groups is
performed.
[0146] The antigen was scanned with 13-mer peptides (11 amino acids
overlap). This resulted in arrays of 123 peptides. Binding assays
were performed directly on the array. The peptide-bound antibodies
MOR03077, MOR03079, MOR03080, MOR03100 and chimeric OKT10 were
detected using a peroxidase-labelled secondary antibody (peroxidase
conjugate-goat anti-human IgG, gamma chain specific, affinity
isolated antibody; Sigma-Aldrich, A6029). The mappings were
performed with a chemiluminescence substrate in combination with an
imaging system. Additionally, a direct POD-labelling of MOR03077
was performed in order to increase the sensitivity of the
system.
2. Summary and Conclusions:
[0147] All five antibodies showed different profiles in the PepSpot
analysis. A schematic summary is given in FIG. 7, which illustrates
the different aa sequences of CD38 being recognized. The epitope
for MOR03079 and chimeric OKT10 can clearly be considered as
linear. The epitope for MOR03079 can be postulated within aa
192-206 (VSRRFAEAACDVVHV (SEQ ID NO:38)) of CD38 whereas for
chimeric OKT10 a sequence between aa 284 and 298 (FLQCVKNPEDSSCTS
(SEQ ID NO:39)) is recognized predominantly. The latter results
confirm the published data for the parental murine OKT10 (Hoshino
et al., 1997), which postulate its epitope between aa 280-298. Yet,
for a more precise epitope definition and determination of key
amino acids (main antigen-antibody interaction sites) a shortening
of peptides VSRRFAEAACDVVHV (SEQ ID NO: 38) and FLQCVKNPEDSSCTS
(SEQ ID NO:39) and an alanine-scan of both should be envisaged.
[0148] The epitopes for MOR03080 and MOR03100 can be clearly
considered as discontinuous since several peptides covering
different sites of the protein sites were recognized. Those
peptides comprise aa 82-94 and aa 158-170 for MOR03080 and aa
82-94, 142-154, 158-170, 188-200 and 280-296 for MOR03100. However,
some overlaps between both epitopes can be postulated since two
different sites residing within aa positions 82-94 (CQSVWDAFKGAFI
(SEQ ID NO:40); peptide #20) and 158-170 (TWCGEFNTSKINY (SEQ ID
NO:41); peptide #58) are recognized by both antibodies.
[0149] The epitope for MOR03077 can be considered as clearly
different from the latter two and can be described as
multisegmented discontinuous epitope. The epitope includes aa
44-66, 110-122, 148-164, 186-200 and 202-224.
Example 7
IL-6-Release/Proliferation Assay
1. Materials and Methods:
[0150] Proliferation- and a IL-6 release assays have been performed
according to Ausiello et al. (2000) with the following
modifications: PBMCs from different healthy donors (after obtaining
informed consent) were purified by density gradient centrifugation
using the Histopaque cell separation system according to the
instructions of the supplier (Sigma) and cultured under standard
conditions (5% CO2, 37.degree. C.) in RPMI1640 medium, supplemented
with 10% FCS and glutamine ("complete RPMI1640"). For both assays
the following antibodies were used: HuCAL.RTM. anti-CD38 IgG1s Mabs
MOR03077, MOR03079, and MOR03080, an agonistic murine IgG2a
monoclonal antibody (IB4; Malavasi et al., 1984), an irrelevant
HuCAL.RTM. IgG1 antibody, a matched isotype control (murine IgG2a:
anti-trinitrophenol, hapten-specific antibody; cat. #: 555571,
clone G155-178; Becton Dickinson) or a medium control. For the IL-6
release assay, 1.0 E+06 PBMCs in 0.5 ml complete RPMI1640 medium
were incubated for 24 hrs in a 15 ml culture tube (Falcon) in the
presence of 20 .mu.g/ml antibodies. Cell culture supernatants were
harvested and analysed for IL-6 release using the Quantikine kit
according to the manufacturer's protocol (R&D systems). For the
proliferation assay 2.0E+05 PBMCs were incubated for 3 days in a
96-well flat bottom plate (Nunc) in the presence of 20 .mu.g/ml
antibodies. Each assay was carried out in duplicates. After 4 days
BrdU was added to each well and cells incubated for an additional
24 hrs at 37.degree. C. prior to cell fixation and DNA denaturation
according to the protocol of the supplier (Roche). Incorporation of
BrdU was measured via an anti-BrdU peroxidase-coupled antibody in a
chemiluminescence-based setting.
2. Summary and Conclusions:
Proliferation Assay:
[0151] In addition to its catalytic activities as a cyclic
ADP-ribose cyclase and hydrolase, CD38 displays the ability to
transduce signals of biological relevance (Hoshino et al., 1997;
Ausiello et al., 2000). Those functions can be induced in vivo by
e.g. receptor-ligand interactions or by cross-linking with
anti-CD38 antibodies. Those signalling events lead e.g. to calcium
mobilization, lymphocyte proliferation and release of cytokines.
However, this signalling is not only dependent on the antigenic
epitope but might also vary from donor to donor (Ausiello et al.,
2000). In the view of immunotherapy non-agonistic antibodies are
preferable over agonistic antibodies. Therefore, HuCAL.RTM.
anti-CD38 antibodies (Mabs MOR03077; MOR03079, MOR03080) were
further characterized in a proliferation assay and IL-6-(important
MM growth-factor) release assay in comparison to the reference
antibody chOKT10 and the agonistic anti-CD38 monoclonal antibody
IB4.
[0152] As demonstrated in FIG. 11 and FIG. 12 the HUCAL.RTM.
anti-CD38 antibodies Mab#1, 2 and 3 as well as the reference
antibody chOKT10 and corresponding negative controls showed no or
only weak induction of proliferation and no IL-6-release as
compared to the agonistic antibody IB4.
Example 8
Clonogenic Assay
1. Materials and Methods:
[0153] PBMCs harbouring autologous CD34+/CD38+ precursor cells were
isolated from healthy individuals (after obtaining informed
consent) by density gradient centrifugation using the Histopaque
cell separation system according to the instructions of the
supplier (Sigma) and incubated with different HUCAL.RTM. IgG1
anti-CD38 antibodies (Mabs MOR03077, MOR03079, and MOR03080) and
the positive control (PC) chOKTlO at 10 .mu.g/ml. Medium and an
irrelevant HUCAL.RTM. IgG1 served as background control. Each
ADCC-assay consisted of 4.0E+05 PBMCs which were incubated for 4
hrs at 37.degree. C. in RPMI1640 medium supplemented with 10% FCS.
For the clonogenic assay 2.50 ml "complete" methylcellulose
(CellSystems) was inoculated with 2.5 E+05 cells from the
ADCC-assay and incubated for colony-development for at least 14
days in a controlled environment (37.degree. C.; 5% CO2). Colonies
were analyzed by two independent operators and grouped into
BFU-E+CFU-GEMM (erythroid burst forming units and
granulocyte/erythroid/macrophage/megakaryocyte stem cells) and
CFU-GM (granulocyte/macrophage stem cells).
2. Summary and Conclusions:
[0154] Since CD38-expression is not only found on immune cells
within the myeloid (e.g. monocytes, granulocytes) and lymphoid
lineage (e.g. activated B and T-cells; plasma cells) but also on
the respective precursor cells (CD34+/CD38+), it is important that
those cells are not affected by antibody-mediated killing.
Therefore, a clonogenic assay was applied in order to analyse those
effects on CD34+/CD38+ progenitors.
[0155] PBMCs from healthy donors were incubated with HUCAL.RTM.
anti-CD38 antibodies (Mab#1, Mab#2 and Mab#3) or several controls
(irrelevant HUCAL.RTM. antibody, medium and reference antibody
chOKT10 as positive control) according to a standard ADCC-protocol
followed by further incubation in conditioned methylcellulose for
colony-development. As shown in FIG. 13 no significant reduction of
colony-forming units are shown for all HUCAL.RTM. anti-CD38
antibodies as compared to an irrelevant antibody or the reference
antibody.
Example 9
ADCC Assays with Different Cell-Lines and Primary Multiple Myeloma
Cells
1. Materials and Methods:
[0156] Isolation and ADCC of MM-patient samples: Bone marrow
aspirates were obtained from multiple myeloma patients (after
obtaining informed consent). Malignant cells were purified via a
standard protocol using anti-CD138 magnetic beads (Milteny Biotec)
after density gradient centrifugation (Sigma). An ADCC-assay was
performed as described before.
2. Summary and Conclusions:
[0157] Several cell-lines derived from different malignancies were
used in ADCC in order to show the cytotoxic effect of the
HUCAL.RTM. anti-CD38 antibodies on a broader spectrum of cell-lines
including different origins and CD38 expression-levels. As shown in
FIG. 14, all cells were killed in ADCC at constant antibody
concentrations (5 .mu.g/ml) and E:T ratios at 30:1. Cytotoxicity
via ADCC was also shown for several multiple myeloma samples from
patients. All HUCAL.RTM. anti-CD38 antibodies were able to perform
a dose-dependent killing of MM-cells and the EC50-values varied
between 0.006 and 0.249 nM (FIG. 15).
Example 10
Cross-Reactivity Analysis by FACS and Immunohisto-Chemistry
(IHC)
1. Materials and Methods:
[0158] IHC with tonsils: For IHC HUCAL.RTM. anti-CD38 Mabs and an
irrelevant negative control antibody were converted into the
bivalent dHLX-format (Pliickthun & Pack, 1997). 5 .mu.m cryo
sections from lymph nodes derived from Cynomolgus monkey, Rhesus
monkey and humans (retrieved from the archives of the Institute of
Pathology of the University of Graz/Austria) were cut with a Leica
CM3050 cryostat. Sections were air-dried for 30 minutes to 1 hour
and fixed in ice-cold methanol for 10 minutes and washed with PBS.
For the detection of the dHLX-format a mouse anti-His antibody
(Dianova) in combination with the Envision Kit (DAKO) was used. For
the detection of the anti-CD38 mouse antibodies (e.g. reference
mouse monoclonal OKT10) the Envison kit was used only.
[0159] FACS-analysis of lymphocytes: EDTA-treated blood samples
were obtained from healthy humans (after obtaining informed
consent), from Rhesus and Cynomolgus monkeys and subjected to
density gradient centrifugation using the Histopaque cell
separation system according to the instructions of the supplier
(Sigma). For FACS-analysis cells from the interphase were incubated
with primary antibodies (HUCAL.RTM. anti-CD38 and negative control
Mabs as murine IgG2a or Fab-format, the positive control murine
antibody OKT10 and a matched isotype control) followed by
incubation with anti-M2 Flag FLAG (Sigma; only for Fab-format) and
a phycoerythrin (PE)-labeled anti-mouse conjugate (Jackson
Research). FACS analysis was performed on the gated lymphocyte
population.
2. Summary and Conclusions:
[0160] HUCAL.RTM. anti-CD38 were analyzed for inter-species CD38
cross-reactivity. Whereas all anti-CD38 Mabs were able to detect
human CD38 on lymphocytes in FACS and IHC, only MOR03080 together
with the positive control OKT10 showed an additional reactivity
with Cynomolgus and Rhesus monkey CD38 (see Table 5:
Cross-reactivity analysis).
TABLE-US-00007 TABLE 5 Lymphocytes (FACS) and lymph-nodes (IHC)
from: Cynomolgus Rhesus Antibody Human Monkey Monkey Mab#1 ++ - -
Mab#2 ++ - - Mab#3 ++ ++ ++ PC ++ ++ ++ NC - - - ++: strong
positive staining; -: no staining; NC: negative control; PC:
positive control (=reference cMAb)
Example 11
Treatment of Human Myeloma Xenografts in Mice (Using the RPMI8226
Cell Line) with MOR03080
1. Establishment of Subcutaneous Mouse Model:
[0161] A subcutaneous mouse model for the human myeloma-derived
tumor cell line RPMI8226 in female C.B-17-SCID mice was established
as follows by Aurigon Life Science GmbH (Tutzing, Germany): on day
-1, 0, and 1, anti-asialo GM1 polyclonal antibodies (ASGM)
(WAKO-Chemicals), which deplete the xenoreactive NK-cells in the
SCID mice were applied intravenously in order to deactivate any
residual specific immune reactivity in C.B-17-SCID mice. On day 0,
either 5.times.10.sup.6 or 1.times.10.sup.7 RPMI8226 tumor cells in
50 .mu.l PBS were inoculated subcutaneously into the right flank of
mice either treated with ASGM (as described above) or untreated
(each group consisting of five mice). Tumor development was similar
in all 4 inoculated groups with no significant difference being
found for treatment with or without anti-asialo GM1 antibodies or
by inoculation of different cell numbers. Tumors appear to be
slowly growing with the tendency of stagnation or oscillation in
size for some days. Two tumors oscillated in size during the whole
period of investigation, and one tumor even regarded and
disappeared totally from a peak volume of 321 mm.sup.3. A treatment
study with this tumor model should include a high number of
tumor-inoculated animals per group.
2. Treatment with MOR03080:
2.1 Study Objective
[0162] This study was performed by Aurigon Life Science GmbH
(Tutzing, Germany) to compare the anti-tumor efficacy of
intraperitoneally applied antibodies (HUCAL.RTM. anti-CD38) as
compared to the vehicle treatment (PBS). The human antibody
hMOR03080 (isotype IgG1) was tested in different amounts and
treatment schedules. In addition the chimeric antibody chMOR03080
(isotype IgG2a: a chimeric antibody comprising the variable regions
of MOR03080 and murine constant regions constructed in a similar
way as described in Example 5 for chimeric OKT10 (murine VH/VL and
human constant regions)) was tested. The RPMI8226 cancer cell line
had been chosen as a model and was inoculated subcutaneously in
female SCID mice as described above. The endpoints in the study
were body weight (b.w.), tumor volume and clinical signs.
2.2 Antibodies and Vehicle
[0163] The antibodies were provided ready to use to Aurigon at
concentrations of 2.13 mg/ml (MOR03080 hIgG1) and 1.73 mg/ml
(MOR03080 chIgG2a, and stored at -80.degree. C. until application.
The antibodies were thawed and diluted with PBS to the respective
end concentration. The vehicle (PBS) was provided ready to use to
Aurigon and stored at 4.degree. C. until application.
2.3 Animal Specification
[0164] Species: mouse
[0165] Strain: Fox chase C.B-17-scid (C.B-Igh-1b/IcrTac)
[0166] Number and sex: 75 females
[0167] Supplier: Taconic M&B, Bomholtvej 10, DK-8680 Ry
[0168] Health status: SPF
[0169] Weight ordered: appr. 18 g
[0170] Acclimatization: 9 days
2.4 Tumor Cell Line
[0171] The tumor cells (RPMI8226 cell line) were grown and
transported to Aurigon Life Science GmbH, where the cells were
splitted and grown for another cycle. Aurigon prepared the cells
for injection on the day of inoculation. The culture medium used
for cell propagation was RPMI 1640 supplemented with 5% FCS, 2 mM
L-Glutamin and PenStrep. The cells showed no unexpected growth rate
or behaviour.
[0172] For inoculation, tumor cells were suspended in PBS and
adjusted to a final concentration of 1.times.10.sup.7 cells/50
.mu.l in PBS. The tumor cell suspension was mixed thoroughly before
being injected.
2.5 Experimental Procedure
[0173] On day 0, 1.times.10.sup.7 RPMI8226 tumor cells were
inoculated subcutaneously into the right dorsal flank of 75 SCID
mice. A first group was built with 15 randomly chosen animals
(group 5) directly after inoculation. This group was treated with 1
mg/kg b.w. hIgG1-MOR03080 every second day between day 14 and 36.
From all other 60 animals 4 groups were built with ten animals in
each group on day 31 (tumor volume of about 92 mm.sup.3). Groups
1-4 were built with comparable means tumor sizes and standard
deviations. An additional group of 5 animals (group 6) was chosen
showing relatively small tumor volumes (tumor volume of about 50
mm.sup.3) for comparison with pre-treated group 5 (all but three
mice showing tumor volumes of less than 10 mm.sup.3, one with about
22 mm.sup.3, one with about 44 mm.sup.3 and one with about 119
mm.sup.3). Groups 1 to 4 were treated every second day from day 32
to day 68 with either PBS (Vehicle; group 1), 1 mg/kg b.w.
hIgG1-MOR03080 (group 2) or 5 mg/kg b.w.hIgG1-MOR03080 (group 3),
or with 5 mg/kg b.w. chIgG2a-MOR03080 (group 4). Group 6 did not
receive any treatment (see Table 6). Tumor volumes, body weight and
clinical signs were measured two times a week until end of
study.
TABLE-US-00008 TABLE 6 No. of Type of Treatment dose Appl. volume
Group animals application Substance Schedule [mg/kg] [.mu.l/kg] 1
10 i.p. vehicle every second day -- 10 (PBS) between day 32 and day
68 2 10 i.p. MOR03080 every second day 1 10 human IgG1 between day
32 and day 68 3 10 i.p. MOR03080 every second day 5 10 human IgG1
between day 32 and day 68 4 10 i.p. MOR03080 every second day 5 10
chimeric between day 32 and IgG2a day 68 5 15 i.p. MOR03080 every
second day 1 10 human IgG1 between day 14 and day 36 6 5 -- -- --
-- --
2.6 Results
Clinical Observations and Mortality
[0174] No specific tumor or substance related clinical findings or
mortality were observed. In group 3 (hIgG1 5 mg/kg) four animals
died during blood sampling (one on day 3, one on day 34; two on day
52). In group 4 (muIgG2a 1 mg/kg) a single animal died during blood
sampling (day 34). All other animals, that died during the study
have been euthanized because of the tumor size.
Body Weight Development
[0175] No drug related interference with weight development was
observed in comparison to group 1 (vehicle). Body weight was
markedly influenced by blood sampling in groups 3 (hIgG1 5 mg/kg)
and 4 (muIgG2a 5 mg/kg). Despite such interruptions the mean weight
gain of all groups was continuous.
Tumor Development (See FIG. 16)
[0176] In group 1 (vehicle) tumor growth was found in the expected
rate with a slow progression. As this cell line has a pronounced
standard deviation values for the largest and smallest tumor have
been excluded from further statistical analysis. The tumor growth
of animals in group 1 was comparable to the tumor growth in group 6
(untreated), although this group started with a lower mean tumor
volume on day 31. Treatment might therefore have a slight influence
on the tumor growth rate. In group 1, two mice had to be euthanized
before day 83 because of the tumor size, and a further one before
day 87, so that the mean value of tumor volume is no longer
representative after day 80. In group 6, one mouse had to be
euthanized before day 80 because of the tumor size, two mice before
day 83, and a further one before day 87, so that the mean value of
tumor volume is no longer representative after day 76.
[0177] In group 2, treated with 1 mg/kg b.w. of hIgG1, one animal
has been excluded from further analysis, because the tumor grew
into the muscular tissue and this usually enhances the speed of
tumor growth. Compared with the control group 1 (vehicle) the mean
tumor size started to differ significantly starting with day 45
until the end of the study. No enhanced tumor growth was observed
after end of treatment (day 68).
[0178] Animals of group 3 (5 mg/kg b.w. hIgG1) revealed a marked
decrease in tumor growth in comparison to group 1 (vehicle),
getting statistically significant with day 38 until day 83. The
mean tumor volume started to strongly regrow about two weeks after
the end of treatment. One out of ten tumors disappeared at day 45
and did not regrow up to 19 days after end of treatment.
[0179] The best performance of all treatment groups starting with
92 mm.sup.3 tumor volume was found in group 4 (5 mg/kg b.w.
muIgG2a), where the mean tumor volume showed clear regression and
tumors even disappeared in 4 animals until the end of the
observation period. The difference to the mean tumor volume of
group 1 (vehicle) was highly significant beginning from day 38
until the end of study.
[0180] The early treatment with 1 mg/kg b.w. hIgG1 between days 14
and 36 (group 5) revealed an early as well as long lasting effect
on tumor development. One animal has been excluded from further
analysis as the tumor grew into muscular tissue. On day 31, only
five animals had a measurable tumor at the site of inoculation, in
comparison to the rest of the inoculated animals, where only 2 out
of 60 did not respond to tumor inoculation. The tumor progression
was delayed of about 31 days (comparison of day 52 of control group
1 with day 83 of group 5). About 50% of the animals did not show
tumors at the site of inoculation at the end of the study.
2.7 Conclusion
[0181] No specific tumor or substance related clinical findings or
mortality were observed in comparison with group1 (control).
[0182] No drug related interference with weight development was
observed.
[0183] Tumor growth of RPMI8226 tumor cells after treatment was
reduced in the order of efficiency: hIgG1 1 mg/kg, 14-36 days every
second day (group 5)>muIgG2a 5 mg/kg 32-68 days every second day
(group 4)>hIgG1 5 mg/kg 32-68 days every second day (group
3)>hIgG1 1 mg/kg 32-68 days every second day (group 2). In
groups 2 to 4, mean tumor volumes were again increased after end of
treatment to varying extents.
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Sequence CWU 1
1
431363DNAHomo sapiens 1caggtgcaat tggttcagag cggcgcggaa gtgaaaaaac
cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact tcttattcta
ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg gatgggctat
atcgatccga atcgtggcaa tacgaattac 180gcgcagaagt ttcagggccg
ggtgaccatg acccgtgata ccagcattag caccgcgtat 240atggaactga
gcagcctgcg tagcgaagat acggccgtgt attattgcgc gcgtgagtat
300atttatttta ttcatggtat gcttgatttt tggggccaag gcaccctggt
gacggttagc 360tca 3632366DNAHomo sapiens 2caggtgcaat tggtggaaag
cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt
taccttttct aattatggta tgcattgggt gcgccaagcc 120cctgggaagg
gtctcgagtg ggtgagcaat atccgttctg atggtagctg gacctattat
180gcggatagcg tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa
caccctgtat 240ctgcaaatga acagcctgcg tgcggaagat acggccgtgt
attattgcgc gcgtcgttat 300tggtctaagt ctcatgcttc tgttactgat
tattggggcc aaggcaccct ggtgacggtt 360agctca 3663366DNAHomo sapiens
3caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taccttttct tcttatggta tgcattgggt gcgccaagcc
120cctgggaagg gtctcgagtg ggtgagcaat atctattctg atggtagcaa
taccttttat 180gcggatagcg tgaaaggccg ttttaccatt tcacgtgata
attcgaaaaa caccctgtat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgcgc gcgtaatatg 300tatcgttggc cttttcatta
tttttttgat tattggggcc aaggcaccct ggtgacggtt 360agctca
3664357DNAHomo sapiens 4caggtgcaat tggtggaaag cggcggcggc ctggtgcaac
cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt taccttttct tctaatggta
tgtcttgggt gcgccaagcc 120cctgggaagg gtctcgagtg ggtgagcaat
atctcttatc tttctagctc tacctattat 180gcggatagcg tgaaaggccg
ttttaccatt tcacgtgata attcgaaaaa caccctgtat 240ctgcaaatga
acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtttttat
300ggttatttta attatgctga tgtttggggc caaggcaccc tggtgacggt tagctca
3575121PRTHomo sapiens 5Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ser Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro
Asn Arg Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Tyr Ile Tyr Phe Ile His Gly Met Leu Asp Phe Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 6122PRTHomo
sapiens 6Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Asn Ile Arg Ser Asp Gly Ser
Trp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Arg Tyr Trp Ser Lys Ser His Ala Ser Val Thr Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 7122PRTHomo sapiens
7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Asn Ile Tyr Ser Asp Gly Ser Asn Thr Phe
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Met Tyr
Arg Trp Pro Phe His Tyr Phe Phe Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 8119PRTHomo sapiens 8Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20
25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Asn Ile Ser Tyr Leu Ser Ser Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Tyr Gly Tyr Phe
Asn Tyr Ala Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 9342DNAHomo sapiens 9gatatcgtga tgacccagag cccactgagc
ctgccagtga ctccgggcga gcctgcgagc 60attagctgca gaagcagcca aagcctgctt
tttattgatg gcaataatta tctgaattgg 120taccttcaaa aaccaggtca
aagcccgcag ctattaattt atcttggttc taatcgtgcc 180agtggggtcc
cggatcgttt tagcggctct ggatccggca ccgattttac cctgaaaatt
240agccgtgtgg aagctgaaga cgtgggcgtg tattattgcc agcagtattc
ttctaagtct 300gctacctttg gccagggtac gaaagttgaa attaaacgta cg
34210327DNAHomo sapiens 10gatatccaga tgacccagag cccgtctagc
ctgagcgcga gcgtgggtga tcgtgtgacc 60attacctgca gagcgagcca ggatatttct
gcttttctga attggtacca gcagaaacca 120ggtaaagcac cgaaactatt
aatttataag gtttctaatt tgcaaagcgg ggtcccgtcc 180cgttttagcg
gctctggatc cggcactgat tttaccctga ccattagcag cctgcaacct
240gaagactttg cgacttatta ttgccagcag gcttattctg gttctattac
ctttggccag 300ggtacgaaag ttgaaattaa acgtacg 32711324DNAHomo sapiens
11gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc
60tcgtgtagcg gcgataatat tggtaataag tatgtttctt ggtaccagca gaaacccggg
120caggcgccag ttgttgtgat ttatggtgat aataatcgtc cctcaggcat
cccggaacgc 180tttagcggat ccaacagcgg caacaccgcg accctgacca
ttagcggcac tcaggcggaa 240gacgaagcgg attattattg ctcttcttat
gattcttctt attttgtgtt tggcggcggc 300acgaagttaa ccgttcttgg ccag
32412327DNAHomo sapiens 12gatatcgaac tgacccagcc gccttcagtg
agcgttgcac caggtcagac cgcgcgtatc 60tcgtgtagcg gcgataatat tggtcattat
tatgcttctt ggtaccagca gaaacccggg 120caggcgccag ttcttgtgat
ttatcgtgat aatgatcgtc cctcaggcat cccggaacgc 180tttagcggat
ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa
240gacgaagcgg attattattg ccagtcttat gattatcttc atgattttgt
gtttggcggc 300ggcacgaagt taaccgttct tggccag 32713114PRTHomo sapiens
13Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Phe
Ile 20 25 30 Asp Gly Asn Asn Tyr Leu Asn Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg
Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85 90 95 Ser Ser Lys Ser Ala
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
14109PRTHomo sapiens 14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ala Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Val Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Gly Ser Ile 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
15108PRTHomo sapiens 15Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp
Asn Ile Gly Asn Lys Tyr Val 20 25 30 Ser Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Val Val Ile Tyr 35 40 45 Gly Asp Asn Asn Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80Asp
Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Asp Ser Ser Tyr Phe Val 85 90
95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
16109PRTHomo sapiens 16Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp
Asn Ile Gly His Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Arg Asp Asn Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Tyr Leu His Asp Phe 85 90
95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
17120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 17Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70
75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
12018120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 18Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
12019107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 19Ser Tyr Glu Leu Thr Gln Pro Pro Ser
Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser
Gly Asp Ala Leu Gly Asp Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asp Asp Ser
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70
75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
20108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 20Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro
Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 21113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 21Asp Ile Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln
His 85 90 95 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 110 Arg 22300PRTHomo sapiens 22Met Ala Asn Cys
Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu
Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30
Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35
40 45 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val
Leu 50 55 60 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met
Arg His Val 65 70 75 80Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly
Ala Phe Ile Ser Lys 85 90 95 His Pro Cys Asn Ile Thr Glu Glu Asp
Tyr Gln Pro Leu Met Lys Leu 100 105 110 Gly Thr Gln Thr Val Pro Cys
Asn Lys Ile Leu Leu Trp Ser Arg Ile 115 120 125 Lys Asp Leu Ala His
Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr 130 135 140 Leu Glu Asp
Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys 145 150 155
160Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp
165 170 175 Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys
Thr Val 180
185 190 Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met
Leu 195 200 205 Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr
Phe Gly Ser 210 215 220 Val Glu Val His Asn Leu Gln Pro Glu Lys Val
Gln Thr Leu Glu Ala 225 230 235 240Trp Val Ile His Gly Gly Arg Glu
Asp Ser Arg Asp Leu Cys Gln Asp 245 250 255 Pro Thr Ile Lys Glu Leu
Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270 Phe Ser Cys Lys
Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285 Lys Asn
Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 300231317DNAHomo
sapiens 23caggtggaat tggtggaatc tggaggatcc ctgaaactct cctgtgcagc
ctcaggattc 60gattttagta gatcctggat gaattgggtc cggcaggctc caggaaaagg
gctagaatgg 120attggagaaa ttaatccaga tagcagtacg ataaactata
cgacatctct aaaggataaa 180ttcatcatct ccagagacaa cgccaaaaat
acgctgtacc tgcaaatgac caaagtgaga 240tctgaggaca cagcccttta
ttactgtgca agatatggta actggtttcc ttattggggc 300caagggactc
tggtcactgt cagctcagcc tccaccaagg gtccatcggt cttccccctg
360gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct
ggtcaaggac 420tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg
ccctgaccag cggcgtgcac 480accttcccgg ctgtcctaca gtcctcagga
ctctactccc tcagcagcgt ggtgaccgtg 540ccctccagca gcttgggcac
ccagacctac atctgcaacg tgaatcacaa gcccagcaac 600accaaggtgg
acaagaaagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg
660tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc
aaaacccaag 720gacaccctca tgatctcccg gacccctgag gtcacatgcg
tggtggtgga cgtgagccac 780gaagaccctg aggtcaagtt caactggtac
gtggacggcg tggaggtgca taatgccaag 840acaaagccgc gggaggagca
gtacaacagc acgtaccggg tggtcagcgt cctcaccgtc 900ctgcaccagg
actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc
960ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga
accacaggtg 1020tacaccctgc ccccatcccg ggatgagctg accaagaacc
aggtcagcct gacctgcctg 1080gtcaaaggct tctatcccag cgacatcgcc
gtggagtggg agagcaatgg gcagccggag 1140aacaactaca agaccacgcc
tcccgtgctg gactccgacg gctccttctt cctctacagc 1200aagctcaccg
tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg
1260catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
131724642DNAHomo sapiens 24gatatcctga tgacccagtc tcaaaaaatc
atgcccacat cagtgggaga cagggtcagc 60gtcacctgca aggccagtca aaatgtggat
actaatgtag cctggtatca acagaaacca 120ggacagtctc ctaaagcact
gatttactcg gcatcctacc gatacagtgg agtccctgat 180cgcttcacag
gcagtggatc tgggacagat ttcactctca ccatcaccaa tgtgcagtct
240gaggacttgg cagagtattt ctgtcagcaa tatgacagct atcctctcac
gttcggtgct 300gggaccaagc tggacctgaa acgtacggtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt
6422521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25atggccaact gcgagttcag c 212627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26tcagatctca gatgtgcaag atgaatc 272725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27ttggtaccag gtggcgccag cagtg 252823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
28ttggtaccat ggccaactgc gag 232929DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 29ccgatatcag atctcagatg
tgcaagatg 293028DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 30ccgatatcga tctcagatgt gcaagatg
2831363DNAHomo sapiens 31caggtgcaat tagtccaaag tggtgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttattcta ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggctat atcgatccga atcgtggcaa tacgaattac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtgagtat 300atttatttta ttcatggtat gcttgatttt tggggccaag
gcaccctggt gacggttagc 360tca 363321500DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
construct 32tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 60actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
ttttggcacc 120aaaatcaacg ggactttcca aaatgtcgta acaactccgc
cccattgacg caaatgggcg 180gtaggcgtgt acggtgggag gtctatataa
gcagagctct ctggctaact agagaaccca 240ctgcttactg gcttatcgaa
attaatacga ctcactatag ggagacccaa gctggctagc 300gccacc atg aaa cac
ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc 348 Met Lys His Leu Trp
Phe Phe Leu Leu Leu Val Ala Ala Pro 1 5 10 aga tgg gtc ctg tcc cag
gtg gaa ttc tgc agg cgg tta gct cag 393Arg Trp Val Leu Ser Gln Val
Glu Phe Cys Arg Arg Leu Ala Gln15 20 25 cctccaccaa gggtccatcg
gtcttccccc tggcaccctc ctccaagagc acctctgggg 453gcacagcggc
cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt
513ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta
cagtcctcag 573gactctactc cctcagcagc gtggtgaccg tgccctccag
cagcttgggc acccagacct 633acatctgcaa cgtgaatcac aagcccagca
acaccaaggt ggacaagaaa gttgagccca 693aatcttgtga caaaactcac
acatgcccac cgtgcccagc acctgaactc ctggggggac 753cgtcagtctt
cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg
813aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
ttcaactggt 873acgtggacgg cgtggaggtg cataatgcca agacaaagcc
gcgggaggag cagtacaaca 933gcacgtaccg ggtggtcagc gtcctcaccg
tcctgcacca ggactggctg aatggcaagg 993agtacaagtg caaggtctcc
aacaaagccc tcccagcccc catcgagaaa accatctcca 1053aagccaaagg
gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc
1113tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc
agcgacatcg 1173ccgtggagtg ggagagcaat gggcagccgg agaacaacta
caagaccacg cctcccgtgc 1233tggactccga cggctccttc ttcctctaca
gcaagctcac cgtggacaag agcaggtggc 1293agcaggggaa cgtcttctca
tgctccgtga tgcatgaggc tctgcacaac cactacacgc 1353agaagagcct
ctccctgtct ccgggtaaat gagggcccgt ttaaacccgc tgatcagcct
1413cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
ccttccttga 1473ccctggaagg tgccactccc actgtcc 150033800DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
construct 33tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 60actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
ttttggcacc 120aaaatcaacg ggactttcca aaatgtcgta acaactccgc
cccattgacg caaatgggcg 180gtaggcgtgt acggtgggag gtctatataa
gcagagctct ctggctaact agagaaccca 240ctgcttactg gcttatcgaa
attaatacga ctcactatag ggagacccaa gctggctagc 300gccacc atg gtg ttg
cag acc cag gtc ttc att tct ctg ttg ctc tgg 348 Met Val Leu Gln Thr
Gln Val Phe Ile Ser Leu Leu Leu Trp 1 5 10 atc tct ggt gcc tac ggg
gat atc gtg atg att aaa cgt acg gtg gct 396Ile Ser Gly Ala Tyr Gly
Asp Ile Val Met Ile Lys Arg Thr Val Ala15 20 25 30gca cca tct gtc
ttc atc ttc ccg cca tct gat gag cag ttg aaa tct 444Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 35 40 45 gga act
gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag 492Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 50 55 60
gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc
540Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
65 70 75 cag gag agt gtc aca gag cag gac agc aag gac agc acc tac
agc ctc 588Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu 80 85 90 agc agc acc ctg acg ctg agc aaa gca gac tac gag
aaa cac aaa gtc 636Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val95 100 105 110tac gcc tgc gaa gtc acc cat cag ggc
ctg agc tcg ccc gtc aca aag 684Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys 115 120 125 agc ttc aac agg gga gag tgt
taggggcccg tttaaacccg ctgatcagcc 735Ser Phe Asn Arg Gly Glu Cys 130
tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
795accct 80034800DNAArtificial SequenceDescription of Artificial
Sequence Synthetic nucleotide construct 34tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 60actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 120aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
180gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 240ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa gctggctagc 300gccacc atg gcc tgg gct ctg ctg ctc ctc acc
ctc ctc act cag ggc 348 Met Ala Trp Ala Leu Leu Leu Leu Thr Leu Leu
Thr Gln Gly 1 5 10 aca gga tcc tgg gct gat atc gtg atg cac gaa gtt
a acc gtc cta ggt 397Thr Gly Ser Trp Ala Asp Ile Val Met His Glu
Val Thr Val Leu Gly15 20 25 30cag ccc aag gct gcc ccc tcg gtc act
ctg ttc ccg ccc tcc tct gag 445Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu 35 40 45 gag ctt caa gcc aac aag gcc
aca ctg gtg tgt ctc ata agt gac ttc 493Glu Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe 50 55 60 tac ccg gga gcc gtg
aca gtg gcc tgg aag gga gat agc agc ccc gtc 541Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Gly Asp Ser Ser Pro Val 65 70 75 aag gcg gga
gtg gag acc acc aca ccc tcc aaa caa agc aac aac aag 589Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 80 85 90 tac
gcg gcc agc agc tat ctg agc ctg acg cct gag cag tgg aag tcc 637Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser95 100
105 110cac aga agc tac agc tgc cag gtc acg cat gaa ggg agc acc gtg
gag 685His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu 115 120 125 aag aca gtg gcc cct aca gaa tgt tca taggggcccg
tttaaacccg 732Lys Thr Val Ala Pro Thr Glu Cys Ser 130 135
ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
792gccttcct 80035359PRTArtificial SequenceDescription of Artificial
Sequence Synthetic protein construct 35Met Lys His Leu Trp Phe Phe
Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val
Glu Phe Cys Arg Arg Leu Ala Gln Ala Ser Thr 20 25 30 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 35 40 45 Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 50 55
60 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
65 70 75 80Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 85 90 95 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys 100 105 110 Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu 115 120 125 Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185
190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 225 230 235 240Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys 260 265 270 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 305 310
315 320Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355
36133PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 36Met Val Leu Gln Thr Gln Val Phe Ile
Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val
Met Ile Lys Arg Thr Val Ala Ala Pro 20 25 30 Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 35 40 45 Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 50 55 60 Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 65 70
75 80 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser 85 90 95 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala 100 105 110 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe 115 120 125 Asn Arg Gly Glu Cys 130
37135PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 37Met Ala Trp Ala Leu Leu Leu Leu Thr
Leu Leu Thr Gln Gly Thr Gly 1 5 10 15 Ser Trp Ala Asp Ile Val Met
His Glu Val Thr Val Leu Gly Gln Pro 20 25 30Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 35 40 45 Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 50 55 60 Gly Ala
Val Thr Val Ala Trp Lys Gly Asp Ser Ser Pro Val Lys Ala65 70 75
80Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
85 90 95Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
Arg 100 105 110 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys Thr 115 120 125Val Ala Pro Thr Glu Cys Ser 130 135
3815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val
Val His Val 1 5 10 153915PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Phe Leu Gln Cys Val Lys Asn
Pro Glu Asp Ser Ser Cys Thr Ser 1 5 10 154013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Cys
Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile 1 5 10
4113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn
Tyr 1 5 10 42120PRTHomo sapiens 42Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 12043113PRTHomo sapiens 43Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Gln Gln His 85 90 95 Tyr Thr Thr Pro Pro Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg
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