U.S. patent application number 16/821895 was filed with the patent office on 2020-09-10 for monoclonal antibodies against bcma.
The applicant listed for this patent is ENGMAB SARL. Invention is credited to Oliver Ast, Marina Bacac, Camille Delon, Anne Freimoser-Grundschober, Lydia Jasmin Hanisch, Christian Klein, Ekkehard Moessner, Samuel Moser, Klaus Strein, Pablo Umana, Minh Diem Vu, Tina Weinzierl.
Application Number | 20200283545 16/821895 |
Document ID | / |
Family ID | 1000004845364 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283545 |
Kind Code |
A1 |
Vu; Minh Diem ; et
al. |
September 10, 2020 |
MONOCLONAL ANTIBODIES AGAINST BCMA
Abstract
The invention relates to new antibodies against BCMA, their
manufacture and use.
Inventors: |
Vu; Minh Diem; (Wollerau,
CH) ; Strein; Klaus; (Weinheim, DE) ; Ast;
Oliver; (Bassersdorf, CH) ; Bacac; Marina;
(Zuerich, CH) ; Delon; Camille; (Oberengstingen,
CH) ; Hanisch; Lydia Jasmin; (Birmensdorf, CH)
; Freimoser-Grundschober; Anne; (Zuerich, CH) ;
Klein; Christian; (Bonstetten, CH) ; Moessner;
Ekkehard; (Kreuzlingen, CH) ; Moser; Samuel;
(Rotkreuz, CH) ; Umana; Pablo; (Wollerau, CH)
; Weinzierl; Tina; (Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENGMAB SARL |
Boudry |
|
CH |
|
|
Family ID: |
1000004845364 |
Appl. No.: |
16/821895 |
Filed: |
March 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15747385 |
Jan 24, 2018 |
10683369 |
|
|
PCT/EP2016/068549 |
Aug 3, 2016 |
|
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16821895 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
C07K 16/468 20130101; C07K 2317/66 20130101; C07K 2317/31 20130101;
C07K 2319/70 20130101; C07K 2317/73 20130101; A61P 35/02 20180101;
C07K 16/2878 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; A61P 35/02 20060101 A61P035/02; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2015 |
EP |
15179549.9 |
Claims
1.-23. (canceled)
24. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof an antibody specifically
binding to BCMA, wherein the antibody is characterized in
comprising a VH region comprising a CDR1H region having SEQ ID
NO:21, a CDR2H region having SEQ ID NO:22 and a CDR3H region having
SEQ ID NO:17 and a VL region comprising a CDR3L region having SEQ
ID NO:20 and a CDR1L and CDR2L region combination selected from the
group of: a) CDR1L region having SEQ ID NO:23 and CDR2L region
having SEQ ID NO:24, b) CDR1L region having SEQ ID NO:25 and CDR2L
region having SEQ ID NO:26, and c) CDR1L region having SEQ ID NO:27
and CDR2L region having SEQ ID NO:28.
25. The method of claim 24, wherein the antibody is characterized
in comprising a VH region comprising a CDR1H region having SEQ ID
NO:21, a CDR2H region having SEQ ID NO:22 and a CDR3H region having
SEQ ID NO:17; and a VL region comprising a CDR1L region having SEQ
ID NO:27, a CDR2L region having SEQ ID NO:28 and a CDR3L region
having SEQ ID NO:20.
26. The method of claim 24, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
27. The method of claim 24, wherein the plasma cell disorder is
multiple myeloma.
28. The method of claim 24, wherein the antibody is administered at
a dose of 200 to 2000 mg/m.sup.2/week.
29. The method of claim 24, wherein the antibody is characterized
in comprising a VH region having SEQ ID NO:10 and a VL region
selected from the group consisting of VL regions SEQ ID NO:12, 13,
and 14.
30. The method of claim 25, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
31. The method of claim 25, wherein the plasma cell disorder is
multiple myeloma.
32. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof a bispecific antibody
specifically binding to BCMA and human CDR3.epsilon. (CD3), wherein
the antibody is characterized in comprising a CDR3H region having
SEQ ID NO:17 and a CDR3L region having SEQ ID NO:20 and a CDR1H,
CDR2H, CDR1L, and CDR2L region combination selected from the group
of: a) CDR1H region having SEQ ID NO:21 and CDR2H region having SEQ
ID NO:22, CDR1L region having SEQ ID NO:23, and CDR2L region having
SEQ ID NO:24, b) CDR1H region having SEQ ID NO:21 and CDR2H region
having SEQ ID NO:22, CDR1L region having SEQ ID NO:25, and CDR2L
region having SEQ ID NO:26, c) CDR1H region having SEQ ID NO:21 and
CDR2H region having SEQ ID NO:22, CDR1L region having SEQ ID NO:27,
and CDR2L region having SEQ ID NO:28, d) CDR1H region having SEQ ID
NO:29 and CDR2H region having SEQ ID NO:30, CDR1L region having SEQ
ID NO:31, and CDR2L region having SEQ ID NO:32, e) CDR1H region
having SEQ ID NO:34 and CDR2H region having SEQ ID NO:35, CDR1L
region having SEQ ID NO:31, and CDR2L region having SEQ ID NO:32,
and f) CDR1H region having SEQ ID NO:36 and CDR2H region having SEQ
ID NO:37, CDR1L region having SEQ ID NO:31, and CDR2L region having
SEQ ID NO:32.
33. The method of claim 32, characterized in comprising a light
chain and a heavy chain of an antibody specifically binding to CD3,
wherein the variable domains VL and VH or the constant domains CL
and CH1 are replaced by each other.
34. The method of claim 32, characterized in that a variable domain
VH of an anti-CD3 antibody portion of the bispecific antibody (CD3
VH) comprises heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as
respective heavy chain CDR1, CDR2 and CDR3 and the variable domain
VL of the anti-CD3 antibody portion (CD3 VL) comprises light chain
CDRs having SEQ ID NO: 4, 5 and 6 as respective light chain CDR1,
CDR2 and CDR3.
35. The method of claim 32, wherein the bispecific antibody is
characterized in comprising a VH region of an anti-BCMA antibody
portion of the bispecific antibody comprising a CDR1H region having
SEQ ID NO:21, a CDR2H region having SEQ ID NO:22 and a CDR3H region
having SEQ ID NO:17; and a VL region of an anti-BCMA antibody
portion of the bispecific antibody comprising a CDR1L region having
SEQ ID NO:27, a CDR2L region having SEQ ID NO:28 and a CDR3L region
having SEQ ID NO:20; and, wherein the bispecific antibody is
further characterized in comprising a VH region of an anti-CD3
antibody portion of the bispecific antibody comprising a CDR1H
region having SEQ ID NO:1, a CDR2H region having SEQ ID NO:2 and a
CDR3H region having SEQ ID NO:3; and a VL region of an anti-CD3
antibody portion of the bispecific antibody comprising a CDR1L
region having SEQ ID NO:4, a CDR2L region having SEQ ID NO:5 and a
CDR3L region having SEQ ID NO:6.
36. The method of claim 32, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
37. The method of claim 32, wherein the plasma cell disorder is
multiple myeloma.
38. The method of claim 32, wherein the bispecific antibody is
administered at a dose of 0.1 to 250 mg/m.sup.2/week.
39. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof a bispecific antibody
specifically binding to two targets which are the extracellular
domain of human BCMA (BCMA) and human CDR3.epsilon. (CD3),
characterized in comprising a VH region comprising a CDR1H region
having SEQ ID NO:21, a CDR2H region having SEQ ID NO:22 and a CDR3H
region having SEQ ID NO:17 and a VL region comprising a CDR3L
region having SEQ ID NO:20 and a CDR1L and CDR2L region combination
selected from the group of: a) CDR1L region having SEQ ID NO:23 and
CDR2L region having SEQ ID NO:24, b) CDR1L region having SEQ ID
NO:25 and CDR2L region having SEQ ID NO:26, and c) CDR1L region
having SEQ ID NO:27 and CDR2L region having SEQ ID NO:28.
40. The method of claim 39, characterized in comprising a BCMA VH
region having SEQ ID NO:10 and a VL region having SEQ ID NO:12, or
a BCMA VH region having SEQ ID NO:10 and a VL region having SEQ ID
NO:13, or a BCMA VH region having SEQ ID NO:10 and a VL region
having SEQ ID NO:14.
41. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof a bispecific antibody
specifically binding to BCMA and CD3, wherein the bispecific
antibody is characterized in comprising a) a first light chain and
a first heavy chain of a first anti-BCMA antibody characterized in
comprising a CDR3H region having SEQ ID NO:17 and a CDR3L region
having SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region
combination selected from the group of: i) CDR1H region having SEQ
ID NO:21 and CDR2H region having SEQ ID NO:22, CDR1L region having
SEQ ID NO:23, and CDR2L region having SEQ ID NO:24, ii) CDR1H
region having SEQ ID NO:21 and CDR2H region having SEQ ID NO:22,
CDR1L region having SEQ ID NO:25, and CDR2L region having SEQ ID
NO:26, iii) CDR1H region having SEQ ID NO:21 and CDR2H region
having SEQ ID NO:22, CDR1L region having SEQ ID NO:27, and CDR2L
region having SEQ ID NO:28, iv) CDR1H region having SEQ ID NO:29
and CDR2H region having SEQ ID NO:30, CDR1L region having SEQ ID
NO:31, and CDR2L region having SEQ ID NO:32, v) CDR1H region having
SEQ ID NO:34 and CDR2H region having SEQ ID NO:35, CDR1L region
having SEQ ID NO:31, and CDR2L region having SEQ ID NO:32, and vi)
CDR1H region having SEQ ID NO:36 and CDR2H region having SEQ ID
NO:37, CDR1L region having SEQ ID NO:31, and CDR2L region having
SEQ ID NO:32; and b) a second light chain and a second heavy chain
of a second antibody which specifically binds to CD3, and wherein
the variable domains VL and VH in the second light chain and second
heavy chain of the second antibody are replaced by each other; and
c) wherein in a constant domain CL of the first light chain a) an
amino acid at position 124 is substituted independently by lysine
(K), arginine (R) or histidine (H) (numbering according to Kabat),
and wherein in a constant domain CH1 of the first heavy chain a) an
amino acid at position 147 and an amino acid at position 213 are
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to EU index of Kabat).
42. The method of claim 41, wherein the bispecific antibody is
characterized in comprising in addition a Fab fragment of said
first antibody (BCMA-Fab) and wherein in the constant domain CL of
said BCMA-Fab the amino acid at position 124 is substituted
independently by lysine (K), arginine (R) or histidine (H)
(numbering according to Kabat), and wherein in the constant domain
CH1 of said BCMA-Fab the amino acid at positions 147 and the amino
acid at position 213 are substituted independently by glutamic acid
(E), or aspartic acid (D) (numbering according to EU index of
Kabat) (see e.g. FIGS. 2A, 2C).
43. The method of claim 41, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
44. The method of claim 41, wherein the plasma cell disorder is
multiple myeloma.
45. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof a bispecific antibody
specifically binding to BCMA and CD3, characterized in comprising
a) a first light chain and a first heavy chain of a first antibody
specifically binding to human B cell maturation antigen (BCMA),
characterized in comprising a CDR3H region having SEQ ID NO:17 and
a CDR3L region having SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and
CDR2L region combination selected from the group of: i) CDR1H
region having SEQ ID NO:21 and CDR2H region having SEQ ID NO:22,
CDR1L region having SEQ ID NO:23, and CDR2L region having SEQ ID
NO:24, ii) CDR1H region having SEQ ID NO:21 and CDR2H region having
SEQ ID NO:22, CDR1L region having SEQ ID NO:25, and CDR2L region
having SEQ ID NO:26, iii) CDR1H region having SEQ ID NO:21 and
CDR2H region having SEQ ID NO:22, CDR1L region having SEQ ID NO:27,
and CDR2L region having SEQ ID NO:28, iv) CDR1H region having SEQ
ID NO:29 and CDR2H region having SEQ ID NO:30, CDR1L region having
SEQ ID NO:31, and CDR2L region having SEQ ID NO:32, v) CDR1H region
having SEQ ID NO:34 and CDR2H region having SEQ ID NO:35, CDR1L
region having SEQ ID NO:31, and CDR2L region having SEQ ID NO:32,
and vi) CDR1H region having SEQ ID NO:36 and CDR2H region having
SEQ ID NO:37, CDR1L region having SEQ ID NO:31, and CDR2L region
having SEQ ID NO:32; and b) a second light chain and a second heavy
chain of a second antibody which specifically binds to CD3, and
wherein the variable domains VL and VH in the second light chain
and second heavy chain of the second antibody are replaced by each
other; and wherein c) in a constant domain CL of the second light
chain under b) an amino acid at position 124 is substituted
independently by lysine (K), arginine (R) or histidine (H)
(numbering according to Kabat), and wherein in a constant domain
CH1 of the second heavy chain under b) an amino acid at positions
147 and an amino acid at position 213 are substituted independently
by glutamic acid (E), or aspartic acid (D) (numbering according to
EU index of Kabat).
46. The method of claim 45, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
47. The method of claim 45, wherein the plasma cell disorder is
multiple myeloma.
48. A method of treatment of a plasma cell disorder comprising
administering to a subject in need thereof a bispecific antibody
specifically binding to BCMA and to CD3, characterized in
comprising a heavy and light chain set selected from the group
consisting of polypeptides: i) SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, and SEQ ID NO:51 (2.times.), ii) SEQ ID NO:48, SEQ ID NO:52,
SEQ ID NO:53, and SEQ ID NO:54 (2.times.), or iii) SEQ ID NO:48,
SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57 (2.times.).
49. The method of claim 48, wherein the plasma cell disorder is
selected from the group consisting of multiple myeloma, systemic
lupus erythematosus, plasma cell leukemia, and AL-amyloidosis.
50. The method of claim 48, wherein the plasma cell disorder is
multiple myeloma.
51. The method of claim 48, wherein the bispecific antibody is
administered at a dose of 0.1 to 250 mg/m.sup.2/week.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. application Ser. No. 15/747,385, filed Jan. 24, 2018, which is
a 35 U.S.C. .sctn. 371 national phase application of International
Application No. PCT/EP2016/068549, filed Aug. 3, 2016, and
published under PCT Article 21(2) in English, which designated the
U.S., and claims the benefit of priority from European Application
No. EP15179549.9, filed Aug. 3, 2015, and each of which prior
applications are incorporated by reference herein into this
application in their entirety including all tables, figures and
claims.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which is
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Mar. 15,
2020, is named 298068-00319_Sequence_Listing.txt and is 70,441
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to new antibodies against
BCMA, their manufacture and use.
BACKGROUND OF THE INVENTION
[0004] Human B cell maturation antigen, also known as BCMA;
TR17_HUMAN, TNFRSF17 (UniProt Q02223), is a member of the tumor
necrosis receptor superfamily that is preferentially expressed in
differentiated plasma cells (Laabi et al. 1992; Madry et al. 1998).
BCMA is a non-glycosylated type III transmembrane protein, which is
involved in B cell maturation, growth and survival. BCMA is a
receptor for two ligands of the TNF superfamily: APRIL (a
proliferation-inducing ligand), the high-affinity ligand to BCMA
and the B cell activation factor BAFF, the low-affinity ligand to
BCMA (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4).
APRIL and BAFF show structural similarity and overlapping yet
distinct receptor binding specificity. The negative regulator TACI
also binds to both BAFF and APRIL. The coordinate binding of APRIL
and BAFF to BCMA and/or TACI activates transcription factor
NF-.kappa.B and increases the expression of pro-survival Bcl-2
family members (e.g. Bcl-2, Bcl-xL, Bcl-w, Mc1-1, A1) and the
downregulation of pro-apoptotic factors (e.g. Bid, Bad, Bik, Bim,
etc.), thus inhibiting apoptosis and promoting survival. This
combined action promotes B cell differentiation, proliferation,
survival and antibody production (as reviewed in Rickert R C et
al., Immunol Rev (2011) 244 (1): 115-133).
[0005] Antibodies against BCMA are described e.g. in Gras M-P. et
al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812,
WO2010104949 and WO2012163805. Antibodies against BCMA and their
use for the treatment of lymphomas and multiple myeloma are
mentioned e.g. in WO2002066516 and WO2010104949. WO2013154760 and
WO2015052538 relate to chimeric antigen receptors (CAR) comprising
a BCMA recognition moiety and a T-cell activation moiety. Ryan, M C
et al., Mol. Cancer Ther.
[0006] 6 (2007) 3009-3018 relate to anti BCMA antibodies with
ligand blocking activity that could promote cytotoxicity of
multiple myeloma (MM) cell lines as naked antibodies or as
antibody-drug conjugates. Ryan showed that SG1, an inhibitory BCMA
antibody, blocks APRIL-dependent activation of nuclear factor-KB in
a dose-dependent manner in vitro. Ryan also mentioned antibody SG2
which inhibited APRIL binding to BCMA not significantly.
[0007] A wide variety of recombinant bispecific antibody formats
have been developed in the recent past, e.g. by fusion of, e.g. an
IgG antibody format and single chain domains (see e.g. Kontermann R
E, mAbs 4:2, (2012) 1-16). Bispecific antibodies wherein the
variable domains VL and VH or the constant domains CL and CH1 are
replaced by each other are described in WO2009080251 and
WO2009080252.
[0008] An approach to circumvent the problem of mispaired
byproducts, which is known as `knobs-into-holes`, aims at forcing
the pairing of two different antibody heavy chains by introducing
mutations into the CH3 domains to modify the contact interface. On
one chain bulky amino acids were replaced by amino acids with short
side chains to create a `hole`. Conversely, amino acids with large
side chains were introduced into the other CH3 domain, to create a
`knob`. By coexpressing these two heavy chains (and two identical
light chains, which have to be appropriate for both heavy chains),
high yields of heterodimer formation (`knob-hole`) versus homodimer
formation (`hole-hole` or `knob-knob`) was observed (Ridgway J B,
Presta L G, Carter P. Protein Eng. 9, 617-621 (1996); and
WO1996027011). The percentage of heterodimer could be further
increased by remodeling the interaction surfaces of the two CH3
domains using a phage display approach and the introduction of a
disulfide bridge to stabilize the heterodimers (Merchant A. M, et
al, Nature Biotech 16 (1998) 677-681; Atwell S, Ridgway J B, Wells
J A, Carter P., J Mol. Biol 270 (1997) 26-35). New approaches for
the knobs-into-holes technology are described in e.g. in EP
1870459A1. Although this format appears very attractive, no data
describing progression towards the clinic are currently available.
One important constraint of this strategy is that the light chains
of the two parent antibodies have to be identical to prevent
mispairing and formation of inactive molecules. Thus this technique
is not appropriate for easily developing recombinant, bispecific
antibodies against two targets starting from two antibodies against
the first and the second target, as either the heavy chains of
these antibodies and/or the identical light chains have to be
optimized. Xie, Z., et al, J Immunol. Methods 286 (2005) 95-101
refers to a format of bispecific antibody using scFvs in
combination with knobs-into-holes technology for the FC part.
[0009] The TCR/CD3 complex of T-lymphocytes consists of either a
TCR alpha (.alpha.)/beta (.beta.) or TCR gamma (.gamma.)/delta
(.delta.) heterodimer coexpressed at the cell surface with the
invariant subunits of CD3 labeled gamma (.gamma.), delta (.delta.),
epsilon (E), zeta (0, and eta (q). Human CD3E is described under
UniProt P07766 (CD3E_HUMAN).
[0010] An anti CD3E antibody described in the state of the art is
SP34 (Yang S J, The Journal of Immunology (1986) 137; 1097-1100).
SP34 reacts with both primate and human CD3. SP34 is available from
Pharmingen. A further anti CD3 antibody described in the state of
the art is UCHT-1 (see WO2000041474). A further anti CD3 antibody
described in the state of the art is BC-3 (Fred Hutchinson Cancer
Research Institute; used in Phase I/II trials of GvHD, Anasetti et
al., Transplantation 54: 844 (1992)). SP34 differs from UCHT-1 and
BC-3 in that SP-34 recognizes an epitope present on solely the
.epsilon. chain of CD3 (see Salmeron et al., (1991) J. Immunol.
147: 3047) whereas UCHT-1 and BC-3 recognize an epitope contributed
by both the .epsilon. and .gamma. chains. Further anti-CD3
antibodies are described in WO2008119565, WO2008119566,
WO2008119567, WO2010037836, WO2010037837, WO2010037838, and U.S.
Pat. No. 8,236,308 (WO2007042261). CDRs, VH and VL sequences of a
further anti-CD3 antibody are shown in SEQ ID NO:7 and 8.
[0011] Bispecific antibodies against CD3 and BCMA are mentioned in
WO2007117600, WO2009132058, WO2012066058, and WO2012143498. CAR
compounds of antibodies against BCMA are mentioned in WO2013154760,
WO2013154760, and WO2014140248.
[0012] Cell-mediated effector functions of monoclonal antibodies
(like antibody dependent cellular cytotoxicity (ADCC)) can be
enhanced by engineering their oligosaccharide composition at Asn297
as described in Umana, P., et al., Nature Biotechnol. 17 (1999)
176-180; and U.S. Pat. No. 6,602,684. WO1999054342, WO2004065540,
WO2007031875, and WO2007039818, Hristodorov D, Fischer R, Linden
L., Mol Biotechnol. 2012 Oct. 25. (Epub) also relate to the
glycosylation engineering of antibodies to enhance Fc-mediated
cellular cytotoxicity.
[0013] Also several amino acid residues in the hinge region and the
CH2 domain influence cell-mediated effector functions of monoclonal
antibodies (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319
(1995), Chemical Immunology, 65, 88 (1997)] Chemical Immunology,
65, 88 (1997)]. Therefore modification of such amino acids can
enhance cell-mediated effector functions. Such antibody
modifications to increase cell-mediated effector functions are
mentioned in EP1931709, WO200042072 and comprise in the Fc part
substitutions at amino acid position(s) 234, 235, 236, 239, 267,
268, 293, 295, 324, 327, 328, 330, and 332.
[0014] Further antibody modifications to increase cell-mediated
effector functions are mentioned in EP1697415 and comprise amino
acid replacement of EU amino acid positions 277, 289, 306, 344, or
378 with a charged amino acid, a polar amino acid, or a nonpolar
amino acid.
[0015] Antibody formats and formats of bispecific and multispecific
antibodies are also pepbodies (WO200244215), Novel Antigen Receptor
("NAR") (WO2003014161), diabody-diabody dimers "TandAbs"
(WO2003048209), polyalkylene oxide-modified scFv (U.S. Pat. No.
7,150,872), humanized rabbit antibodies (WO2005016950), synthetic
immunoglobulin domains (WO2006072620), covalent diabodies
(WO2006113665), flexibodies (WO2003025018), domain antibodies, dAb
(WO2004058822), vaccibody (WO2004076489), antibodies with new world
primate framework (WO2007019620), antibody-drug conjugate with
cleavable linkers (WO2009117531), IgG4 antibodies with hinge region
removed (WO2010063785), bispecific antibodies with IgG4 like CH3
domains (WO2008119353), camelid Antibodies (U.S. Pat. No.
6,838,254), nanobodies (U.S. Pat. No. 7,655,759), CAT diabodies
(U.S. Pat. No. 5,837,242), bispecific (scFv).sub.2 directed against
target antigen and CD3 (U.S. Pat. No. 7,235,641),), sIgA
plAntibodies (U.S. Pat. No. 6,303,341), minibodies (U.S. Pat. No.
5,837,821), IgNAR (US2009148438), antibodies with modified hinge
and Fc regions (US2008227958, US20080181890), trifunctional
antibodies (U.S. Pat. No. 5,273,743), triomabs (U.S. Pat. No.
6,551,592), troybodies (U.S. Pat. No. 6,294,654).
[0016] WO2014122143 disclose anti-human BCMA antibodies
characterized in that the binding of said antibody is not reduced
by 100 ng/ml APRIL for more than 20% measured in an ELISA assay as
OD at 405 nm compared to the binding of said antibody to human BCMA
without APRIL, said antibody does not alter APRIL-dependent
NF-.kappa.B activation for more than 20%, as compared to APRIL
alone, and said antibody does not alter NF-.kappa.B activation
without APRIL for more than 20%, as compared without said antibody.
WO2014122144 discloses bispecific antibodies specifically binding
to the two targets human CD3E and human BCMA, comprising anti-human
BCMA antibodies of WO2014122143. An anti-human BCMA antibody with
unique properties, especially in regard to its therapeutic use as a
bispecific T cell binder, is antibody 83A10, characterized by
comprising as CDR regions CDR1H of SEQ ID NO:15, CDR2H of SEQ ID
NO16, CDR3H of SEQ ID NO:17, CDR1L of SEQ ID NO:18, CDR3L of SEQ ID
NO:19, and CDR3L of SEQ ID NO:20, disclosed also in WO2014122143
and WO2014122144.
SUMMARY OF THE INVENTION
[0017] The invention comprises monoclonal antibodies specifically
binding to human B cell maturation antigen (BCMA). The antibodies
according to the invention comprise as CDR3H and CDR3L regions the
same CDR regions as antibody 83A10.
[0018] The antibodies according to the invention comprise in an
embodiment as CDR3H and CDR3L regions the same CDR regions as
antibody 83A10, but show especially potent and efficient advantages
in comparison to antibody 83A10 for killing of MM cells in patient
bone marrow aspirates.
[0019] The invention comprises a monoclonal antibody specifically
binding to BCMA, characterized in comprising a CDR3H region of SEQ
ID NO:17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H,
CDR1L, and CDR2L region combination selected from the group of
a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b)
CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c)
CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28, d)
CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, e)
CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and
f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,
[0020] The invention comprises a monoclonal antibody specifically
binding to BCMA, characterized in comprising a VH region comprising
a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and
a CDR3H region of SEQ ID NO:17 and a VL region comprising a CDR3L
region of SEQ ID NO:20 and a CDR1L and CDR2L region combination
selected from the group of
a) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,
b) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26,
or c) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID
NO:28.
[0021] The invention provides an antibody according to the
invention, characterized in comprising a VL region selected from
the group consisting of VL regions of SEQ ID NO:12, 13, and 14
wherein amino acid 49 is selected from the group of amino acids
tyrosine(Y), glutamic acid (E), serine (S), and histidine (H). In
one embodiment amino acid 49 is E within SEQ ID NO:12, S within SEQ
ID NO:13 or H within SEQ ID NO:14.
[0022] The invention provides an antibody according to the
invention, characterized in comprising a VL region selected from
the group consisting of VL regions of SEQ ID NO:12, 13, and 14
wherein amino acid 74 is threonine (T) or alanine (A). In one
embodiment amino acid 74 is A within SEQ ID NO:14.
[0023] The antibodies according to the invention comprise in an
embodiment as CDR3H, CDR1L, CDR2L, and CDR3L regions the same CDR
regions as antibody 83A10. The invention comprises a monoclonal
antibody specifically binding to BCMA, characterized in comprising
a VH region comprising a CDR3H region of SEQ ID NO:17 and a VL
region comprising a CDR1L region of SEQ ID NO:31, a CDR2L region of
SEQ ID NO:32 and a CDR3L region of SEQ ID NO:20 and a CDR1L and
CDR2L region combination selected from the group of
a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,
b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35,
or c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID
NO:37.
[0024] The invention provides in one embodiment an antibody
according to the invention, characterized in comprising a VL region
of SEQ ID NO:12 and a VH region selected from the group comprising
the VH regions of SEQ ID NO:38, 39, and 40. The invention provides
an antibody according to the invention, characterized in comprising
a VL region SEQ ID NO:12, wherein amino acid 49 is selected from
the group of amino acids tyrosine(Y), glutamic acid (E), serine
(S), and histidine (H). In one embodiment amino acid 49 is E.
[0025] The invention provides in one embodiment an antibody
according to the invention, characterized in comprising as VH
region a VH region of SEQ ID NO:10. The invention provides in one
embodiment an antibody according to the invention, characterized in
comprising as VL region a VL region selected from the group
consisting of VL regions of SEQ ID NO:12, 13, and 14. The invention
provides in one embodiment an antibody according to the invention,
characterized in that comprising as VH region a VH region of SEQ ID
NO:10 and as VL region a VL region of SEQ ID NO:12. The invention
provides in one embodiment an antibody according to the invention,
characterized in that comprising as VH region a VH region of SEQ ID
NO:10 and as VL region a VL region of SEQ ID NO:13. The invention
provides in one embodiment an antibody according to the invention,
characterized in that comprising as VH region a VH region of SEQ ID
NO:10 and as VL region a VL region of SEQ ID NO:14.
[0026] The invention provides in one embodiment an antibody
according to the invention, characterized in comprising as VH
region a VH region selected from the group consisting of SEQ ID
NO:38, 39, and 40. The invention provides in one embodiment an
antibody according to the invention, characterized in that
comprising as VH region a VH region of SEQ ID NO:38 and as VL
region a VL region of SEQ ID NO:12. The invention provides in one
embodiment an antibody according to the invention, characterized in
that comprising as VH region a VH region of SEQ ID NO:39 and as VL
region a VL region of SEQ ID NO:12.
[0027] The invention provides in one embodiment an antibody
according to the invention, characterized in that comprising as VH
region a VH region of SEQ ID NO:40 and as VL region a VL region of
SEQ ID NO:12.
[0028] In one embodiment the antibody according to the invention is
further characterized in that it binds also specifically to
cynomolgus BCMA. In one embodiment an antibody of the invention
shows regarding binding to BCMA a cyno/human affinity gap between
1.5 and 5 or 1.5 and 10 or 1.5 and 16 (table 5).
[0029] The bispecific antibody according to the invention is
therefore in one embodiment characterized in that it binds also
specifically to cynomolgus CD3. In one embodiment the bispecific
anti-BCMA/anti-CD3 antibody of the invention shows a cyno/human gap
of Mab CD3 between 1.25 and 5 or between 0.8 and 1.0.
[0030] In a further embodiment of the invention the antibody
according to the invention is an antibody with an Fc part or
without an Fc part including a multispecific antibody, bispecific
antibody, a single chain variable fragment (scFv) such as a
bispecific T cells engager, diabody, or tandem scFv, an antibody
mimetic such as DARPin, a naked monospecific antibody, or an
antibody drug conjugate. In one embodiment a multispecific
antibody, bispecific antibody, a bispecific T cells engager,
diabody, or tandem scFv is specifically binding to BCMA and
CD3.
[0031] Based on an antibody according to the invention it is
possible to generate antibody-drug conjugates against BCMA and
multispecific or bispecific antibodies against BCMA and one or more
further targets in different formats with or without an Fc portion
known in the state of the art (see e. g. above in "background of
the invention"), single chain variable fragments (scFv) such as
bispecific T cells engagers, diabodies, tandem scFvs, and antibody
mimetics such as DARPins, all of them are also embodiments of the
invention.
[0032] Bispecific antibody formats are well known in the state of
the art and e.g. also described in Kontermann R E, mAbs 4:2 1-16
(2012); Holliger P., Hudson P J, Nature Biotech. 23 (2005)
1126-1136 and Chan A C, Carter P J Nature Reviews Immunology 10,
301-316 (2010) and Cuesta A M et al., Trends Biotech 28 (2011)
355-362.
[0033] A further embodiment of the invention is a bispecific
antibody against the two targets human CD3.epsilon. (further named
also as "CD3") and the extracellular domain of human BCMA (further
named also as "BCMA"), characterized in comprising as BCMA binding
portion an anti-BCMA antibody according to the invention.
[0034] The invention relates in one embodiment to a bispecific
antibody against BCMA and CD3, characterized in comprising within
the BCMA binding portion a CDR3H region of SEQ ID NO:17 and a CDR3L
region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region
combination selected from the group of
a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b)
CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26, c)
CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22,
CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28, d)
CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, e)
CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and
f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37,
CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,
[0035] The invention relates in one embodiment to a bispecific
antibody against BCMA and CD3, characterized in comprising a VH
region of an antibody according to the invention (further named
"BCMA VH") comprising a CDR1H region of SEQ ID NO:21, a CDR2H
region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VL
region (further named "BCMA VL") comprising a CDR3L region of SEQ
ID NO:20 and a CDR1L and CDR2L region combination selected from the
group of
a) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,
b) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26,
or c) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID
NO:28.
[0036] The invention provides in one embodiment a bispecific
antibody according to the invention, characterized in comprising as
BCMA VH a VH region of SEQ ID NO:10.
[0037] The invention relates in one embodiment to a bispecific
antibody against BCMA and CD3, characterized in that the BCMA VL is
selected from the group consisting of VL regions of SEQ ID NO:12,
13, and 14. The invention provides in one embodiment an antibody
according to the invention, characterized in comprising as BCMA VH
region a VH region of SEQ ID NO:10 and as VL region a VL region of
SEQ ID NO:12. The invention provides in one embodiment an antibody
according to the invention, characterized in comprising as BCMA VH
a VH region of SEQ ID NO:10 and as VL region a VL region of SEQ ID
NO:13. The invention provides in one embodiment an antibody
according to the invention, characterized in comprising as BCMA VH
a VH region of SEQ ID NO:10 and as VL region a VL region of SEQ ID
NO:14.
[0038] The invention provides a bispecific antibody according to
the invention, characterized in comprising a VL region selected
from the group consisting of VL regions of SEQ ID NO:12, 13, and 14
wherein amino acid 49 is selected from the group of amino acids
tyrosine(Y), glutamic acid (E), serine (S), and histidine (H). In
one embodiment amino acid 49 is E (SEQ ID NO:12), S (SEQ ID NO:13)
or H (SEQ ID NO:14). The invention provides a bispecific antibody
according to the invention, characterized in comprising a VL region
selected from the group consisting of VL regions of SEQ ID NO:12,
13, and 14 wherein amino acid 74 is threonine (T) or alanine (A).
In one embodiment amino acid 74 is A within SEQ ID NO:14.
[0039] The invention relates to a bispecific antibody against BCMA
and CD3, characterized in comprising a BCMA VH comprising a CDR3H
region of SEQ ID NO:17 and a BCMA VL comprising a CDR1L region of
SEQ ID NO:31, a CDR2L region of SEQ ID NO:32 and a CDR3L region of
SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from
the group of
a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,
b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35,
or c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID
NO:37.
[0040] The bispecific antibody against BCMA and CD3 is
characterized in one embodiment in comprising an anti BCMA antibody
according to the invention and an anti CD3 antibody, wherein
a) the light chain and heavy chain of an antibody specifically
binding to one of said targets CD3 and BCMA; and b) the light chain
and heavy chain of an antibody specifically binding to the other
one of said targets, wherein the variable domains VL and VH or the
constant domains CL and CH1 are replaced by each other.
[0041] In one embodiment a VH domain of said anti-CD3 antibody
portion is linked to a CH1 or CL domain of said anti-BCMA antibody
portion. In one embodiment a VL domain of said anti-CD3 antibody
portion is linked to a CH1 or CL domain of said anti-BCMA antibody
portion.
[0042] In one embodiment the bispecific antibody comprises not more
than one Fab fragment of an anti-CD3 antibody portion, not more
than two Fab fragments of an anti-BCMA antibody portion and not
more than one Fc part, in one embodiment a human Fc part. In one
embodiment not more than one Fab fragment of the anti-CD3 antibody
portion and not more than one Fab fragment of the anti-BCMA
antibody portion are linked to the Fc part and linking is performed
via C-terminal binding of the Fab fragment(s) to the hinge region.
In one embodiment the second Fab fragment of the anti-BCMA antibody
portion is linked via its C-terminus either to the N-terminus of
the Fab fragment of the anti-CD3 antibody portion or to the hinge
region of the Fc part and therefore between the Fc part and the
anti-CD3 antibody portion. The preferred bispecific antibodies are
shown in FIGS. 1 to 3.
[0043] Especially preferred are the bispecific antibodies
comprising only the Fab fragments and the Fc part as specified,
with or without "aa substitution":
Fab BCMA-Fc-Fab CD3 (bispecific format FIG. 1A or 1B), Fab
BCMA-Fc-Fab CD3-Fab BCMA (bispecific format FIG. 2A or 2B), Fab
BCMA-Fc-Fab BCMA-Fab CD3 (bispecific format FIG. 2C or 2D), Fc-Fab
CD3-Fab BCMA (bispecific format FIG. 3A or 3B), Fc-Fab BCMA-Fab CD3
(bispecific format FIG. 3C or 3D).
[0044] As shown in FIGS. 1 to 3 "Fab BCMA-Fc, "Fab BCMA-Fc-Fab CD3"
and "Fab BCMA-Fc-Fab CD3" means that the Fab fragment(s) is (are)
bound via its (their) C-terminus to the N-terminus of the Fc
fragment. "Fab CD3-Fab BCMA" means that the Fab CD3fragment is
bound with its N-terminus to the C-terminus of the Fab BCMA
fragment. "Fab BCMA-Fab CD3" means that the Fab BCMA fragment is
bound with its N-terminus to the C-terminus of the Fab CD3
fragment.
[0045] In one embodiment the bispecific antibody comprises a second
Fab fragment of said anti-BCMA antibody linked with its C-terminus
to the N-terminus of the CD3 antibody portion of said bispecific
antibody. In one embodiment a VL domain of said first anti-CD3
antibody portion is linked to a CH1 or CL domain of said second
anti-BCMA antibody.
[0046] In one embodiment the bispecific antibody comprises a second
Fab fragment of said anti-BCMA antibody linked with its C-terminus
to the Fc part (like the first Fab fragment of said anti-BCMA
antibody) and linked with its N-terminus to the C-terminus of the
CD3 antibody portion. In one embodiment a CH1 domain of said
anti-CD3 antibody portion is linked to the VH domain of said second
anti-BCMA antibody portion.
[0047] In one embodiment the bispecific antibody comprises an Fc
part linked with its N-terminus to the C-terminus of said CD3
antibody Fab fragment. In one embodiment the bispecific antibody
comprises an Fc part linked with its first N-terminus to the
C-terminus of said CD3 antibody Fab fragment and a second Fab
fragment of said anti-BCMA antibody linked with its C-terminus to
the second N-terminus of the Fc part. In one embodiment the CL
domain of the CD3 antibody Fab fragment is linked to the hinge
region of the Fc part. In one embodiment the CH1 domain of the BCMA
antibody Fab fragment is linked to the hinge region of the Fc
part.
[0048] The Fab fragments are chemically linked together by the use
of an appropriate linker according to the state of the art. In one
embodiment a (Gly4-Ser1)3 linker is used (Desplancq D K et al.,
Protein Eng. 1994 August; 7(8):1027-33 and Mack M. et al., PNAS
Jul. 18, 1995 vol. 92 no. 15 7021-7025). "Chemically linked" (or
"linked") means according to the the invention that the fragments
are linked by covalent binding. As the linker is a peptidic linker,
such covalent binding is usually performed by biochemical
recombinant means, using a nucleic acid encoding the VL and/or VH
domains of the respective Fab fragments, the linker and if
appropriate the Fc part chain.
[0049] The invention relates in one embodiment to a bispecific
antibody against BCMA and CD3 according to the invention,
characterized in that the variable domain VH of the anti-CD3
antibody portion (further named as "CD3 VH") comprises the heavy
chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain
CDR1H, CDR2H and CDR3H and the variable domain VL of the anti-CD3
antibody portion (further named as "CD3 VL") comprises the light
chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain
CDR1L, CDR2L and CDR3L.
[0050] In one embodiment such a bispecific antibody according to
the invention is characterized in that the variable domains of the
anti CD3E antibody portion are of SEQ ID NO:7 and 8.
[0051] The invention relates to a bispecific antibody according to
the invention, characterized in that the anti-CD3 antibody portion
is linked at its N-terminus to the C-terminus of a of the anti-BCMA
antibody portion and the variable domains VL and VH of the anti-CD3
antibody portion or the constant domains CL and CH1 are replaced by
each other.
[0052] In one embodiment the VH domain of said anti-CD3 antibody
portion is linked to a CH1 or CL domain of said anti-BCMA antibody
portion. In one embodiment a VL domain of said anti-CD3 antibody
portion is linked to a CH1 or CL domain of said anti-BCMA antibody
portion.
[0053] An antibody portion according to the invention is in one
embodiment a Fab fragment of the respective antibody.
[0054] In a further embodiment of the invention the bispecific
antibody wherein the variable domains VL and VH in the light chain
and the respective heavy chain of the anti-CD3 antibody portion or
the anti-BCMA antibody portion are replaced by each other, is
characterized in comprising a constant domain CL of the anti-CD3
antibody portion or the anti-BCMA antibody portion wherein the
amino acid at position 124 is substituted independently by lysine
(K), arginine (R) or histidine (H) (numbering according to Kabat),
and in the respective constant domain CH1 the amino acid at
position 147 and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D). In one
embodiment the antibody is monovalent for CD3 binding. In one
embodiment in addition to the amino acid replacement at position
124 in the constant domain CL the amino acid at position 123 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (further called as "charge variant exchange"). In one
embodiment the antibody is monovalent for CD3 binding and amino
acid 124 is K, amino acid 147 is E, amino acid 213 is E, and amino
acid 123 is R. In one embodiment the bispecific antibody comprises
in addition the same anti-BCMA binding portion once more (in one
embodiment a Fab fragment). That means also, that if the first
anti-BCMA binding portion comprises the charge variant exchange,
then the second anti-BCMA binding portion comprise the same charge
variant exchange.
[0055] The invention relates to a bispecific antibody according to
the invention, characterized in comprising a) the first light chain
and the first heavy chain of a first antibody which specifically
binds to BCMA; and
b) the second light chain and the second heavy chain of a second
antibody which specifically binds to CD3, and wherein the variable
domains VL and VH in the second light chain and second heavy chain
of the second antibody are replaced by each other; and c) wherein
in the constant domain CL of the first light chain under a) the
amino acid at position 124 is substituted independently by lysine
(K), arginine (R) or histidine (H) (numbering according to Kabat),
and wherein in the constant domain CH1 of the first heavy chain
under a) the amino acid at position 147 and the amino acid at
position 213 is substituted independently by glutamic acid (E), or
aspartic acid (D) (numbering according to EU index of Kabat) (see
e.g. FIGS. 1A, 2A, 2C, 3A, 3C).
[0056] In one embodiment said bispecific antibody described in the
last preceding paragraph is further characterized in that said
bispecific antibody comprises in addition a Fab fragment of said
first antibody (further named also as "BCMA-Fab") and in the
constant domain CL said BCMA-Fab the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of said BCMA-Fab the amino acid at positions 147 and the
amino acid at position 213 is substituted independently by glutamic
acid (E), or aspartic acid (D) (numbering according to EU index of
Kabat) (see e.g. FIGS. 2A, 2C).
[0057] The invention further relates to a bispecific antibody
according to the invention, characterized in comprising
a) the first light chain and the first heavy chain of a first
antibody which specifically binds to BCMA; and b) the second light
chain and the second heavy chain of a second antibody which
specifically binds to CD3, and wherein the variable domains VL and
VH in the second light chain and second heavy chain of the second
antibody are replaced by each other; and wherein c) in the constant
domain CL of the second light chain under b) the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat), and wherein in
the constant domain CH1 of the second heavy chain under b) the
amino acid at positions 147 and the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to EU index of Kabat).
[0058] In one embodiment in addition to the amino acid replacement
at position 124 in the constant domain CL of the first or second
light chain the amino acid at position 123 is substituted
independently by lysine (K), arginine (R) or histidine (H).
[0059] In one embodiment in the constant domain CL the amino acid
at position 124 is substituted by lysine (K), in the constant
domain CH1 the amino acid at position 147 and the amino acid at
position 213 are substituted by glutamic acid (E). In one
embodiment in addition in the constant domain CL in the amino acid
at position 123 is substituted by arginine (R).
[0060] In a preferred embodiment of the invention the bispecific
antibody according to the invention consists of one Fab fragment of
an antibody specifically binding to CD3 (further named also as
"CD3-Fab"), and one Fab fragment of an anti-BCMA antibody according
to the invention (further named also as "BCMA-Fab(s)") and a Fc
part, wherein the CD3-Fab and the BCMA-Fab are linked via their
C-termini to the hinge region of said Fc part. Either the CD3-Fab
or the BCMA-Fab comprises aa substitution and the CD3-Fab comprises
crossover (FIGS. 1A and 1B).
[0061] In a preferred embodiment of the invention the bispecific
antibody according to the invention consists of one CD3-Fab, and
one BCMA-Fab and a Fc part, wherein the CD3-Fab and the BCMA-Fab
are linked via their C-termini to the hinge region of said Fc part
and a second BCMA-Fab, which is linked with its C-terminus to the
N-terminus of the CD3-Fab. The CD3-Fab comprises crossover and
either the CD3-Fab or both BCMA-Fabs comprise aa substitution
(FIGS. 2A and 2B). Especially preferred is a bispecific antibody
comprising BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs
comprise aa substitution and the CD3-Fab comprises VL/VH crossover
(FIG. 2A). Especially preferred is a bispecific antibody consisting
of BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs comprise aa
substitution Q124K, E123R, K147E and K213E and the CD3-Fab
comprises VL/VH crossover. Especially preferred is that both
BCMA-Fabs comprise as CDRs the CDRs of antibody 21, 22, or 42, or
as VH/VL the VH/VL of antibody 21, 22, or 42.
[0062] In a preferred embodiment of the invention the bispecific
antibody according to the invention consists of two BCMA-Fabs and
an Fc part, wherein one BCMA-Fab and the CD3 Fab are linked via
their C-termini to the hinge region of said Fc part and the second
BCMA-Fab is linked with its C-terminus to the N-terminus of the
CD3-Fab. The CD3-Fab comprises crossover and either the CD3-Fab or
both BCMA-Fabs comprise aa substitution (FIGS. 2A and 2B).
[0063] In a preferred embodiment of the invention the bispecific
antibody according to the invention consists of two BCMA-Fabs and
an Fc part, wherein the BCMA-Fabs are linked via their C-termini to
the hinge region of said Fc part and a CD3-Fab, which is linked
with its C-terminus to the N-terminus of one BCMA-Fab. The CD3-Fab
comprises crossover and either the CD3-Fab or both BCMA-Fabs
comprise aa substitution (FIGS. 2C and 2D).
[0064] In a preferred embodiment of the invention the antibody
according to the invention consists of one CD3-Fab, which is linked
via its C-terminus to the hinge region of said Fc part and a
BCMA-Fab, which is linked with its C-terminus to the N-terminus of
the CD3-Fab. The CD3-Fab comprises crossover and either the CD3-Fab
or the BCMA-Fab comprise aa substitution (FIGS. 1A and 1B).
[0065] In a preferred embodiment of the invention the antibody
according to the invention consists of one CD3-Fab, which is linked
via its C-terminus to the hinge region of said Fc part and a
BCMA-Fab, which is linked with its C-terminus to the N-terminus of
the CD3-Fab. The CD3-Fab comprises crossover and either the CD3-Fab
or the BCMA-Fab comprise aa substitution (FIGS. 3A and 3B).
[0066] In a preferred embodiment of the invention the antibody
according to the invention consists of one BCMA-Fab, which is
linked via its C-terminus to the hinge region of said Fc part and a
CD3-Fab, which is linked with its C-terminus to the N-terminus of
the BCMA-Fab. The CD3-Fab comprises crossover and either the
CD3-Fab or the BCMA-Fab comprise aa substitution (FIGS. 3C and
3D).
[0067] The Fab fragments are chemically linked together by the use
of an appropriate linker according to the state of the art. In one
embodiment a (Gly4-Ser1)3 linker is used (Desplancq D K et al.,
Protein Eng. 1994 August; 7(8):1027-33 and Mack M. et al., PNAS
Jul. 18, 1995 vol. 92 no. 15 7021-7025). Linkage between two Fab
fragments is performed between the heavy chains. Therefore the
C-terminus of CH1 of a first Fab fragment is linked to the
N-terminus of VH of the second Fab fragment (no crossover) or to VL
(crossover). Linkage between a Fab fragment and the Fc part is
performed according to the invention as linkage between CH1 and
CH2.
[0068] The first and a second Fab fragment of an antibody
specifically binding to BCMA are in one embodiment derived from the
same antibody and in one embodiment identical in the CDR sequences,
variable domain sequences VH and VL and/or the constant domain
sequences CH1 and CL. In one embodiment the amino acid sequences of
the first and a second Fab fragment of an antibody specifically
binding to BCMA are identical. In one embodiment the BCMA antibody
is an antibody comprising the CDR sequences of antibody 21, 22, or
42, an antibody comprising the VH and VL sequences of antibody 21,
22, or 42, or an antibody comprising the VH, VL, CH1, and CL
sequences of antibody 21, 22, or 42.
[0069] In one embodiment the bispecific antibody comprises as Fab
fragments and Fc part, not more than one Fab fragment of an
anti-CD3 antibody, not more than two Fab fragments of an anti-BCMA
antibody and not more than one Fc part, in one embodiment a human
Fc part. In one embodiment the second Fab fragment of an anti-BCMA
antibody is linked via its C-terminus either to the N-terminus of
the Fab fragment of an anti-CD3 antibody or to the hinge region of
the Fc part. In one embodiment linkage is performed between CH1 of
BCMA-Fab and VL of CD3-Fab (VL/VH crossover).
[0070] In one embodiment the antibody portion specifically binding
to human CD3, in one embodiment the Fab fragment, is characterized
in comprising a variable domain VH comprising the heavy chain CDRs
of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1, CDR2 and
CDR3 and a variable domain VL comprising the light chain CDRs of
SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1, CDR2 and
CDR3 of the anti-CD3E antibody (CDR MAB CD3). In one embodiment the
antibody portion specifically binding to human CD3 is characterized
in that the variable domains are of SEQ ID NO:7 and 8 (VHVL MAB
CD3).
[0071] The invention relates to a bispecific antibody specifically
binding to the extracellular domain of human BCMA and to human
CD3.epsilon., characterized in comprising a heavy and light chain
set selected from the group consisting of polypeptides
i) SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID
NO:51(2.times.); (set 1 TCB of antibody 21), ii) SEQ ID NO:48, SEQ
ID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2.times.) (set 2 TCB of
antibody 22), and iii) SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56,
and SEQ ID NO:57(2.times.) (set 3 TCB of antibody 42).
[0072] In one embodiment the bispecific antibody according to the
invention is characterized in that the CH3 domain of one heavy
chain and the CH3 domain of the other heavy chain each meet at an
interface which comprises an original interface between the
antibody CH3 domains; wherein said interface is altered to promote
the formation of the bispecific antibody, wherein the alteration is
characterized in that: [0073] a) the CH3 domain of one heavy chain
is altered, so that within the original interface the CH3 domain of
one heavy chain that meets the original interface of the CH3 domain
of the other heavy chain within the bispecific antibody, an amino
acid residue is replaced with an amino acid residue having a larger
side chain volume, thereby generating a protuberance within the
interface of the CH3 domain of one heavy chain which is
positionable in a cavity within the interface of the CH3 domain of
the other heavy chain and [0074] b) the CH3 domain of the other
heavy chain is altered, so that within the original interface of
the second CH3 domain that meets the original interface of the
first CH3 domain within the bispecific antibody an amino acid
residue is replaced with an amino acid residue having a smaller
side chain volume, thereby generating a cavity within the interface
of the second CH3 domain within which a protuberance within the
interface of the first CH3 domain is positionable.
[0075] In one embodiment such a bispecific antibody is
characterized in that said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0076] In one embodiment such a bispecific antibody is
characterized in that said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0077] In one embodiment such a bispecific antibody is
characterized in that both CH3 domains are further altered by the
introduction of cysteine (C) as amino acid in the corresponding
positions of each CH3 domain.
[0078] In one embodiment such a bispecific antibody is
characterized in that one of the constant heavy chain domains CH3
of both heavy chains is replaced by a constant heavy chain domain
CH1; and the other constant heavy chain domain CH3 is replaced by a
constant light chain domain CL.
[0079] The invention relates further to an antibody according to
the invention, comprising a modified Fc part inducing cell death of
20% or more cells of a preparation BCMA expressing cells after 24
hours at a concentration of said antibody of 100 nM by ADCC
relative to a control under identical conditions using the same
antibody with the parent Fc part as control. Such an antibody is in
one embodiment a naked antibody.
[0080] In one embodiment the antibody according to the invention is
an antibody with an amount of fucose of 60% or less of the total
amount of oligosaccharides (sugars) at Asn297 (see e.g.
US20120315268).
[0081] In one embodiment the Fc part comprises the amino acid
substitutions which are introduced in a human Fc part and disclosed
in SEQ ID NO:55 and 56.
[0082] A further embodiment of the invention is a chimeric antigen
receptor (CAR) of an anti-BCMA antibody according to the invention.
In such an embodiment the anti-BCMA antibody consists of a single
chain VH and VL domain of an antibody according to the invention
and a CD3-zeta transmembrane and endodomain. Preferably the CD3
zeta domain is linked via a spacer with the C-terminus of said VL
domain and the N terminus of the VL domain is linked via a spacer
to the C terminus of said VH domain. Chimeric antigen receptors of
BCMA antibodies, useful transmembrane domains and endodomains, and
methods for the production are described e.g. in Ramadoss N S. et
al., J. Am. Chem. Soc. J., DOI: 10.1021/jacs.5b01876 (2015),
Carpenter R O et al., Clin. Cancer. Res. DOI:
10.1158/1078-0432.CCR-12-2422 (2013), WO2015052538 and
WO2013154760.
[0083] Further embodiments of the invention are the antibodies
Mab21, Mab22, Mab42, Mab27, Mab33, and Mab39 as described herein by
their CDR sequences, and/or VH/VL sequences together with the
described CL and CH1 sequences, as antigen binding fragments,
especially Fab fragments, as bispecific antibodies binding to BCMA
and CD3, with and without Fc part, as bispecific antibodies in the
described formats, especially the 2+1 format, and the bispecific
antibodies with the heavy and light chains as described herein,
especially as described in table 1A.
[0084] A further embodiment of the invention is a method of
generation an anti-BCMA antibody which depletes, in the bispecific
format according to the invention, human malignant plasma cells in
Multiple Myeloma MM bone marrow aspirates to at least 80% after a
48 hour treatment in a concentration of between 10 nM and 1 fM
inclusively, characterized in panning a variable heavy chain (VH)
and a variable light chain (VL) phage-display library of antibody
83A10 (VH library, VL library) with 1-50 nM cyno BCMA in 1-3 rounds
and selecting a variable light chain and a variable heavy chain
which have such properties as such bispecific T cell binder.
Preferably panning is performed in 3 rounds, using 50 nM cynoBCMA
for round 1, 25 nM cyBCMA for round 2 and 10 nM cyBCMA for round 3.
Preferably the libraries are randomized in either the light chain
CDR1 and CDR2 or the heavy chain CDR1 and CDR2. Preferably a light
and heavy chain are identified which each bind as Fab fragment,
comprising in addition the corresponding VH or VL of antibody
83A10, to huBCMA with a Kd of 50 pM to 5 nM and to cyno BCMA with a
Kd of 0.1 nM to 20 nM. Preferably the bispecific format is the
format of FIG. 2A, comprising the respective constant domains VL
and VH of the CD3 Fab replacement by each other and within both
BCMA Fabs amino acid exchanges K213E and K147E in the CH1 domain
and amino acid exchanges E123R and Q124K in the CL domain.
[0085] A further embodiment of the invention is a method for the
preparation of an antibody according to the invention comprising
the steps of [0086] a) transforming a host cell with [0087] b)
vectors comprising nucleic acid molecules encoding the light chain
and heavy chain of an antibody according to the invention, [0088]
c) culturing the host cell under conditions that allow synthesis of
said antibody molecule; and [0089] d) recovering said antibody
molecule from said culture.
[0090] A further embodiment of the invention is a method for the
preparation of a bispecific antibody according to the invention
comprising the steps of [0091] e) transforming a host cell with
[0092] f) vectors comprising nucleic acid molecules encoding the
light chain and heavy chain of an antibody specifically binding to
the first target [0093] g) vectors comprising nucleic acid
molecules encoding the light chain and heavy chain of an antibody
specifically binding to the second target, wherein the variable
domains VL and VH or the constant domains CL and CH1 are replaced
by each other; [0094] h) culturing the host cell under conditions
that allow synthesis of said antibody molecule; and [0095] i)
recovering said antibody molecule from said culture.
[0096] A further embodiment of the invention is a host cell
comprising vectors comprising nucleic acid molecules encoding an
antibody according to the invention. A further embodiment of the
invention is a host cell comprising vectors comprising nucleic acid
molecules encoding the light chain and heavy chain of an antibody
specifically binding to the first target and vectors comprising
nucleic acid molecules encoding the light chain and heavy chain of
an antibody specifically binding to the second target, wherein the
variable domains VL and VH or the constant domains CL and CH1 are
replaced by each other.
[0097] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention and a
pharmaceutically acceptable excipient.
[0098] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention for
use as a medicament.
[0099] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention for
use as a medicament in the treatment of plasma cell disorders.
[0100] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention for
use as a medicament in the treatment of Multiple Myeloma.
[0101] A further embodiment of the invention is pharmaceutical
composition comprising an antibody according to the invention for
use as a medicament in the treatment of systemic lupus
erythematosus.
[0102] A further embodiment of the invention is pharmaceutical
composition comprising an antibody according to the invention,
including a monospecific antibody, an ADCC enhanced naked antibody,
an antibody-drug conjugate, a multispecific antibody or a
bispecific antibody for use as a medicament in the treatment of
antibody-mediated rejection.
[0103] In one embodiment an antibody according to the invention can
be used for the treatment of plasma cell disorders like Multiple
Myeloma MM or other plasma cell disorders expressing BCMA as
described below.
[0104] MM is a plasma cell malignancy characterized by a monoclonal
expansion and accumulation of abnormal plasma cells in the bone
marrow compartment. MM also involves circulating clonal plasma
cells cells with same IgG gene rearrangement and somatic
hypermutation. MM arises from an asymptomatic, premalignant
condition called monoclonal gammopathy of unknown significance
(MGUS), characterized by low levels of bone marrow plasma cells and
a monoclonal protein. MM cells proliferate at low rate. MM results
from a progressive occurrence of multiple structural chromosomal
changes (e.g. unbalanced translocations). MM involves the mutual
interaction of malignant plasma cells and bone marrow
microenvironment (e.g. normal bone marrow stromal cells). Clinical
signs of active MM include monoclonal antibody spike, plasma cells
overcrowding the bone marrow, lytic bone lesions and bone
destruction resulting from overstimulation of osteoclasts
(Dimopulos & Terpos, Ann Oncol 2010; 21 suppl 7: vii143-150).
Another plasma cell disorder involving plasma cells i.e. expressing
BCMA is systemic lupus erythematosus (SLE), also known as lupus.
SLE is a systemic, autoimmune disease that can affect any part of
the body and is represented with the immune system attacking the
body's own cells and tissue, resulting in chronic inflammation and
tissue damage. It is a Type III hypersensitivity reaction in which
antibody-immune complexes precipitate and cause a further immune
response (Inaki & Lee, Nat Rev Rheumatol 2010; 6: 326-337).
Further plasma cell disorders are plasma cell leukemia and
AL-Amyloidosis (see also Examples 19 and 20). In all these plasma
cell disorders depletion of plasma cells/malignant plasma cells by
antibodies according to this invention is expected to be beneficial
for the patients suffering from such a disease.
[0105] A further embodiment of this invention is an antibody
according to the invention for the treatment of antibody-mediated
allograft rejection involving plasma cells and alloantibodies
including acute and chronic antibody-mediated rejection (AMR).
Acute AMR is characterized by graft dysfunction that occurs over
days and is the result of either pre-formed or de novo donor
specific antibodies developed post-transplant. It occurs in about
5-7% of all kidney transplants and causes 20-48% of acute rejection
episodes among pre-sensitized positive crossmatch patients (Colvin
and Smith, Nature Rev Immunol 2005; 5 (10): 807-817).
Histopathology in patients with acute AMR often reveals endothelial
cell swelling, neutrophilic infiltration of glomeruli and
peritubular capillaries, fibrin thrombi, interstitial edema, and
hemorrhage (Trpkov et al. Transplantation 1996; 61 (11):
1586-1592). AMR can be identified with C4d-staining or other
improved methods of antibody detection in allograft biopsies.
Another form of AMR is also known as chronic allograft injury which
also involves donor specific antibodies but manifests within months
and even years after transplantation. It is seen as transplant
glomerulopathy (also known as chronic allograft glomerulopathy) on
kidney biopsies and is characterized by glomerular mesangial
expansion and capillary basement membrane duplication (Regele et
al. J Am Soc Nephrol 2002; 13 (9): 2371-2380). The clinical
manifestations vary from patients being asymptomatic in the early
stages to having nephrotic range proteinuria, hypertension, and
allograft dysfunction in the advanced stages. Disease progression
can be quite rapid, especially with ongoing acute AMR, resulting in
graft failure within months (Fotheringham et al. Nephron--Clin
Pract 2009; 113 (1): c1-c7). The prevalence of transplant
glomerulopathy in patient biopsies varies between 5% at 1 yr to 20%
at 5 years (Cosio et al. Am J Transplant 2008; 8: 292-296).
[0106] A further embodiment of the invention is an antibody
according to the invention for use as a medicament.
[0107] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention for
use as a medicament.
[0108] A further embodiment of the invention is a pharmaceutical
composition comprising a naked antibody or a bispecific antibody
according to the invention for use as a medicament.
[0109] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention with
increased effector function for use as a medicament.
[0110] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention with
decreased effector function for use as a medicament.
[0111] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention as
bispecific antibody for use as a medicament.
[0112] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention as
multispecific antibody for use as a medicament.
[0113] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention as
conjugate with a therapeutic agent (drug conjugate) e.g. with a
cytotoxic agent or radiolabel for use as a medicament.
[0114] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention as a
diabody for use as a medicament.
[0115] In one embodiment the antibody according to the invention,
especially when being a bispecific antibody against CD3 and BCMA,
is administered once or twice a week in one embodiment via
subcutaneous administration (e.g. in one embodiment in the dose
range of 0.1 to 2.5, preferably to 25 mg/m.sup.2/week, preferably
to 250 mg/m.sup.2/week). Due to superior cytotoxicity activities of
the antibody according to the invention it can be administered at
least at the same magnitude of clinical dose range (or even lower)
as compared to conventional monospecific antibodies or conventional
bispecific antibodies that are not T cell bispecifics (i.e. do not
bind to CD3 on one arm). It is envisaged that for an antibody
according to the invention subcutaneous administration is preferred
in the clinical settings (e.g. in the dose range of 0.1-250
mg/m.sup.2/week). In addition, in patients with high levels of
serum APRIL and BAFF (e.g. multiple myeloma patients) it may not be
required to increase the dose for an antibody according to this
invention as it may not be affected by ligand competition. In
contrast, the doses for other ligand-blocking/competing anti-BCMA
antibodies may need to be increased in those patients. Another
advantage of the antibody according to the invention is an
elimination half-life of about 4 to 12 days which allows at least
once or twice/week administration.
[0116] In one embodiment the antibody according to the invention in
the case of naked/unconjugated ADCC enhanced monospecific
antibodies is an antibody with properties allowing for once/twice a
week treatment by intravenous route but preferably via subcutaneous
administration (e.g. a dosage in the range of 200-2000 mg/m/week
for 4 weeks). It is envisaged that for an antibody according to the
invention subcutaneous administration is possible and preferred in
the clinical settings (e.g. in the dose range of 200-2000
mg/m.sup.2/week, depending on the disease indications). In
addition, in patients with high levels of serum APRIL and BAFF
(e.g. multiple myeloma patients) it may not be required to increase
the dose for an antibody according to this invention (e.g.
non-ligand blocking/competing antibody) as it may not be affected
by ligand competition. In contrast, the doses for other
ligand-blocking/competing anti-BCMA antibodies may need to be
increased in those patients, making subcutaneous administration
technically more challenging (e.g. pharmaceutical). Another
advantage of the antibody according to the invention is based on
the inclusion of an Fc portion, which is associated with an
elimination half-life of 4 to 12 days and allows at least once or
twice/week administration.
[0117] A further preferred embodiment of the invention is a
diagnostic composition comprising an antibody according to the
invention.
DESCRIPTION OF THE FIGURES
[0118] FIGS. 1A-1B. Bispecific bivalent antibodies comprising only
the Fab fragments (specific to CD3 and BCMA) and the Fc part as
specified: (FIG. 1A) Fab BCMA(RK/EE)-Fc-Fab CD3; (FIG. 1B) Fab
BCMA-Fc-Fab CD3(RK/EE). aa substitutions for RK/EE introduced in
CL-CH1 to reduce LC mispairing/side products in production. The Fab
CD3 includes a VL-VH crossover to reduce LC mispairing and
side-products.
[0119] FIGS. 2A-2D. Preferred bispecific trivalent antibodies
comprising only the Fab fragments (specific to CD3 and BCMA) and
the Fc part as specified: (FIG. 2A) Fab BCMA(RK/EE)-Fc-Fab CD3-Fab
BCMA(RK/EE); (FIG. 2B) Fab BCMA-Fc-Fab CD3(RK/EE)-Fab BCMA; (FIG.
2C) Fab BCMA(RK/EE)-Fc-Fab BCMA(RK/EE)-Fab CD3; (FIG. 2D) Fab
BCMA-Fc-Fab BCMA-Fab CD3(RK/EE). aa substitutions for RK/EE
introduced in CL-CH1 to reduce LC mispairing/side-products in
production. Preferably, the Fab CD3 includes a VL-VH crossover to
reduce LC mispairing and side-products. Preferably, Fab CD3 and Fab
BCMA are linked to each other with flexible linkers.
[0120] FIGS. 3A-3D. Bispecific bivalent antibodies comprising only
the Fab fragments (specific to CD3 and BCMA) and the Fc part as
specified: (FIG. 3A) Fc-Fab CD3-Fab BCMA(RK/EE); (FIG. 3B) Fc-Fab
CD3(RK/EE)-Fab BCMA; (FIG. 3C) Fc-Fab BCMA(RK/EE)-Fab CD3; (FIG.
3D) Fc-Fab BCMA-Fab CD3(RK/EE). Preferably, the Fabs CD3 include a
VL-VH crossover to reduce LC mispairing and side-products. Fab CD3
and Fab BCMA are linked to each other with flexible linkers.
[0121] FIGS. 4A-4D. Redirected T-cell lysis of H929 MM cells
induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies as
measured by LDH release. Concentration response curves for lysis of
H929 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closed
triangle), 42-TCBcv (closed square) in comparison with 83A10-TCBcv
(open circle, dotted line). There was a concentration-dependent
killing of H929 cells for all anti-BCMA/anti-CD3 T cell bispecific
antibodies while no killing was observed with the control-TCB.
Experiments were performed with PBMC donor 1 (FIG. 4A), donor 3
(FIG. 4B), donor 4 (FIG. 4C), donor 5 (FIG. 4D) using an effector
cell to tumor target cell (E:T) ratio of 10 PBMCs to 1 MM cell (see
example 8).
[0122] FIGS. 5A-5E. Redirected T-cell lysis of L363 MM cells
induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies as
measured by LDH release. Concentration response curves for lysis of
L363 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closed
triangle), 42-TCBcv (closed square) in comparison with 83A10-TCBcv
(open circle, dotted line). A concentration-dependent killing of
L363 cells was observed for all anti-BCMA/anti-CD3 T cell
bispecific antibodies while no killing was observed with the
control-TCB. Experiments were performed with PBMC donor 1 (FIG.
5A), donor 2 (FIG. 5B), donor 3 (FIG. 5C), donor 4 (FIG. 5D), donor
5 (FIG. 5E) using an E:T ratio of 10 PBMCs to 1 MM cell (see
example 9).
[0123] FIGS. 6A-6D. Redirected T-cell lysis of RPMI-8226 MM cells
induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies as
measured by LDH release. Concentration response curves for lysis of
RPMI-8226 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv
(closed triangle), 42-TCBcv (closed square) in comparison with
83A10-TCBcv (open circle, dotted line). A concentration-dependent
killing of RPMI-8226 cells was observed for all anti-BCMA/anti-CD3
T cell bispecific antibodies while no killing was observed with the
control-TCB. Experiments were performed with PBMC donor 2 (FIG.
6A), donor 3 (FIG. 6B), donor 4 (FIG. 6C), donor 5 (FIG. 6D) using
an E:T ratio of 10 PBMCs to 1 MM cell (see example 10).
[0124] FIGS. 7A-7D. Redirected T-cell lysis of JJN-3 MM cells
induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies as
measured by flow cytometry. Concentration-dependent killing of
JJN-3 MM cells by 22-TCBcv (closed triangle), 42-TCBcv (closed
square) in comparison with 83A10-TCBcv (open circle, dotted line).
Percentage of annexin-V positive JJN-3 cells (FIG. 7A, FIG. 7C) and
tumor cell lysis (FIG. 7B, FIG. 7D) were determined and plotted.
The percentage of lysis of JJN-3 cells induced by a specific
concentration of anti-BCMA/anti-CD3 T cell bispecific antibody
determined as the following: the absolute count of
annexin-V-negative JJN-3 cells at a given TCB concentration and
subtracting it from the absolute count of annexin-V-negative JJN-3
cells without TCB; divided by the absolute count of
annexin-V-negative JJN-3 cells without TCB. Experiments were
performed with 2 PBMC donors: donor 1 (FIG. 7A, FIG. 7B) and donor
2 (FIG. 7C, FIG. 7D) using an E:T ratio of 10 PBMCs to 1 MM cell
(see example 11).
[0125] FIGS. 8A-8C. Redirected T-cell lysis of multiple myeloma
patient bone marrow myeloma plasma cells in presence of autologous
bone marrow infiltrating T cells (patient's whole bone marrow
aspirates) induced by anti-BCMA/anti-CD3 T-cell bispecific
antibodies as measured by multiparameter flow cytometry. Percentage
of annexin-V positive myeloma plasma cells was determined and
plotted against TCB concentrations. Concentration-dependent and
specific lysis of patient myeloma plasma cells were observed while
lysis of T cells, B cells, and NK cells was not observed based on
an 8-color multiparameter panel. No induction of cell death of
myeloma plasma cells with control-TCB at the highest concentration
of TCB antibodies tested. As compared to 83A10-TCBcv (FIG. 8A),
42-TCBcv (FIG. 8B) and 22-TCBcv (FIG. 8C) were more potent to
induce killing of patient bone marrow myeloma plasma cells (see
example 13).
[0126] FIGS. 9A-9B. Redirected T-cell lysis of multiple myeloma
patient bone marrow myeloma plasma cells in presence of autologous
bone marrow infiltrating T cells (patient's whole bone marrow
aspirates) induced by anti-BCMA/anti-CD3 T-cell bispecific
antibodies as measured by flow cytometry. Percentage of annexin-V
negative myeloma plasma cells was determined and plotted against
TCB concentrations. Concentration-dependent and specific lysis of
patient myeloma plasma cells were observed while lysis of
non-malignant bone marrow cells was not observed (data not shown).
No induction of cell death of myeloma plasma cells observed with
control-TCB at the highest concentration of TCB antibodies tested
(data not shown). As compared to 83A10-TCBcv, 42-TCBcv and 22-TCBcv
were more potent to induce killing of patient bone marrow myeloma
plasma cells as reflected by the concentration-dependent reduction
of viable (annexin-V negative) myeloma plasma cells. Representative
experiments in patient 001 (FIG. 9A) and patient 007 (FIG. 9B) (see
example 13).
[0127] FIGS. 10A-10H. Redirected T-cell lysis of multiple myeloma
patient bone marrow myeloma plasma cells in presence of autologous
bone marrow infiltrating T cells induced by anti-BCMA/anti-CD3
T-cell bispecific antibodies as measured by flow cytometry.
Percentage of propidium iodide negative myeloma plasma cells was
determined and the percentage of viable bone marrow plasma cells
relative to the medium control (MC) was plotted against TCB
concentrations. Concentration-dependent and specific lysis of
patient myeloma plasma cells were observed (FIG. 10A-FIG. 10G)
while lysis of bone marrow microenvironment (BMME) was not observed
(FIG. 10H). No induction of cell death of myeloma plasma cells
observed with control-TCB at the highest concentration of TCB
antibodies tested. As compared to 83A10-TCBcv, 42-TCBcv and
22-TCBcv were more potent to induce killing of patient bone marrow
myeloma plasma cells as reflected by the concentration-dependent
reduction of viable (propidium iodide negative) myeloma plasma
cells. An effect was considered statistically significant if the
P-value of its corresponding statistical test was <5% (*),
<1% (**) or <0.1% (***). Experiments performed using bone
marrow aspirate samples collected from patient 1 (FIG. 10A),
patient 2 (FIG. 10B), patient 3 (FIG. 10C), patient 4 (FIG. 10D),
patient 5 (FIG. 10E), patient 6 (FIG. 10F), and patient 7 (FIG.
10G, FIG. 10H) (see example 13).
[0128] FIGS. 11A-11C. Activation of myeloma patient bone marrow T
cells in presence of bone marrow plasma cells (patient whole bone
marrow aspirates) induced by anti-BCMA/anti-CD3 T-cell bispecific
antibodies as measured by multiparameter flow cytometry (8-color
staining panel). FIG. 11A: Magnitude of T-cell activation (top
graph: CD4 T-Cell Activation; bottom graph: CD8 T-Cell Activation)
was shown for 83A10-TCBcv. FIG. 11B: Magnitude of T-cell activation
(top graph: CD4 T-Cell Activation; bottom graph: CD8 T-Cell
Activation) was shown for 42-TCBcv. FIG. 11C: Magnitude of T-cell
activation (top graph: CD4 T-Cell Activation; bottom graph: CD8
T-Cell Activation) was shown for 22-TCBcv. (see example 14).
[0129] FIG. 12. Concentrations of 83A10-TCBcv measured from serum
samples (closed symbols with full lines) and bone marrow samples
(open symbols with dotted lines) after single intravenous (IV)
injection in cynomolgus monkeys with 0.003, 0.03 and 0.1 mg/kg of
83A10-TCBcv. Serum samples collection was performed at pre-dose and
30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after dosing. Bone
marrow samples were collected at pre-dose, and 96 and 336 h after
dosing (see example 16).
[0130] FIG. 13. Peripheral T-cell redistribution observed in
cynomolgus monkeys following a single IV injection of 83A10-TCBcv
(0.003, 0.03 and 0.3 mg/kg) Animals A and B, C and D, and E and F
respectively received an IV injection of 0.003, 0.03 and 0.3 mg/kg
of 83A10-TCBcv. Absolute blood T-cell cell counts (CD2+ cells per
.mu.L of blood) were plotted against time post treatment (see
example 16).
[0131] FIGS. 14A-14B. Reduction of blood plasma cells observed in
cynomolgus monkeys following a single IV injection of 83A10-TCBcv
(0.3 mg/kg) as measured by multiparameter flow cytometry. FIG. 14A:
Plasma cells (PCs) were identified based on a 6-color staining
panel and percentages of PCs over lymphocytes were measured and
plotted in contour plots. FIG. 14B: Kinetic of blood plasma cell
depletion after treatment with 83A10-TCBcv 0.3 mg/kg in cynomolgus
monkeys was plotted (see example 16).
[0132] FIGS. 15A-15C. Antitumoral activity induced by 83A10-TCBcv
anti-BCMA/anti-CD3 T cell bispecific antibody in the H929 human
myeloma xenograft model using PBMC-humanized NOG mice.
Immunodeficient NOD/Shi-scid IL2rgamma (null) (NOG) received on day
0 (d0) human multiple myeloma H929 cells as a subcutaneous (SC)
injection into the right dorsal flank. On day 15 (d15), NOG mice
received a single intraperitoneal (IP) injection of human PBMCs.
Mice were then carefully randomized into the different treatment
and control groups (n=9/group) and a statistical test was performed
to test for homogeneity between groups. The experimental groups
were the control untreated group, control-TCB treated group,
83A10-TCBcv 2.6 nM/kg treated group and BCMA50-BiTE.RTM. (BCMAxCD3
(scFv).sub.2) 2.6 nM/kg treated group. Antibody treatment given by
tail vein injection started on day 19 (d19), i.e. 19 days after SC
injection of H929 tumor cells. The TCB antibody treatment schedule
consisted of a once a week IV administration for up to 3 weeks
(i.e. total of 3 injections of TCB antibody). Tumor volume (TV) was
measured by caliper during the study and progress evaluated by
intergroup comparison of TV. TV (mm3) plotted against day post
tumor injection. On d19, first day of treatment, the mean tumor
volume had reached 300.+-.161 mm3 for the vehicle treated control
group (FIG. 15A), 315.+-.148 mm3 for the 2.6 nM/kg control-TCB
treated group (FIG. 15A), 293.+-.135 mm3 for the 2.6 nM/kg
83A10-TCBcv group (FIG. 15B) and 307.+-.138 mm3 for the 2.6 nM/kg
BCMA50-BiTE.RTM. group (FIG. 15C). TV of each individual mouse per
experimental group were plotted against day post tumor injection:
(FIG. 15A) control groups including vehicle control (full line) and
control-TCB (dotted line), (FIG. 15B) 83A10-TCBcv (2.6 nM/kg)
group, and (FIG. 15C) BCMA50-BiTE.RTM. (2.6 nM/kg). Black arrows
show the TCB treatment given by IV injection. In the 83A10-TCBcv
(2.6 nM/kg) group, 6 out of 9 mice (67%) had their tumor regressed
even below TV recorded at d19 i.e. first TCB treatment and tumor
regression was maintained until termination of study. The 3 mice in
the 83A10-TCBcv (2.6 nM/kg) treated group which failed to show
tumor regression had their TV equal to 376, 402 and 522 mm3
respectively at d19. In contrast, none of the 9 mice (0%) treated
with an equimolar dose of BCMA50-BiTE.RTM. (2.6 nM/kg) at a once a
week schedule for 3 weeks had their tumor regressed at any
timepoint (see example 17).
[0133] FIG. 16. Percentage of tumor growth (TG) calculated for d19
to d43 and compared between 83A10-TCBcv (2.6 nM/kg) group and
BCMA50-BiTE.RTM. (2.6 nM/kg). The percentage of tumor growth
defined as TG (%) was determined by calculating TG (%)=100.times.
(median TV of analyzed group)/(median TV of control vehicle treated
group). For ethical reason, mice were euthanized when TV reached at
least 2000 mm3 TG (%) was consistently and significantly reduced in
the 83A10-TCBcv (2.6 nM/kg) group as well as the TG (%) was always
lower when compared to BCMA50-BiTE.RTM. (2.6 nM/kg) (see example
17).
[0134] FIGS. 17A-17B. Surface plasmon resonance (SPR) of 70 clones
selected from ELISA. All experiments were performed at 25.degree.
C. using PBST as running buffer (10 mM PBS, pH 7.4 and 0.005% (v/v)
Tween.RTM.20) with a ProteOn XPR36 biosensor equipped with GLC and
GLM sensor chips and coupling reagents. Immobilizations were
performed at 30 .mu.l/min on a GLM chip. pAb (goat) anti hu IgG,
F(ab)2 specific Ab (Jackson) was coupled in vertical direction
using a standard amine-coupling procedure: all six ligand channels
were activated for 5 min with a mixture of EDC (200 mM) and
sulfo-NHS (50 mM) Immediately after the surfaces were activated,
pAb (goat) anti hu IgG, F(ab)2 specific antibody (50 .mu.g/ml, 10
mM sodium acetate, pH 5) was injected across all six channels for 5
min. Finally, channels were blocked with a 5 min injection of 1 M
ethanolamine-HCl (pH 8.5). Final immobilization levels were similar
on all channels, ranging from 11000 to 11500 RU. The Fab variants
were captured from E. coli supernantants by simultaneous injection
along five of the separate whole horizontal channels (30 .mu.l/min)
for 5 min and resulted in levels, ranging from 200 to 900 RU,
depending on the concentration of Fab in supernatant; conditioned
medium was injected along the sixth channel to provide an `in-line`
blank for double referencing purposes. FIG. 17A: One-shot kinetic
measurements were performed by injection of a dilution series of
human (50, 10, 2, 0.4, 0.08, 0 nM, 50 .mu.l/min) for 3 min along
the vertical channels. Dissociation was monitored for 5 min.
Kinetic data were analyzed in ProteOn Manager v. 2.1. FIG. 17B:
One-shot kinetic measurements were performed by injection of a
dilution series of cyno BCMA (50, 10, 2, 0.4, 0.08, 0 nM, 50
.mu.l/min) for 3 min along the vertical channels. Dissociation was
monitored for 5 min. Kinetic data were analyzed in ProteOn Manager
v. 2.1. Processing of the reaction spot data involved applying an
interspot-reference and a double-reference step using an inline
buffer blank (Myszka, 1999). The processed data from replicate
one-shot injections were fit to a simple 1:1 Langmuir binding model
without mass transport (O'Shannessy et al., 1993).
[0135] FIGS. 18A-18B. FIG. 18A: Binding affinity of BCMA antibodies
on HEK-huBCMA cells as measured by flow cytometry. The anti-BCMA
antibodies were used as first antibody then a secondary PE-labeled
anti-human Fc was used as detection antibody. It was found that
binding of antibodies Mab 21, Mab 22, Mab 27, Mab 39 and Mab 42 to
huBCMA on HEK cells was not significantly better than the binding
of Mab 83A10 to huBCMA-HEK cells. FIG. 18B: Binding affinity of
BCMA antibodies on HEK-huBCMA cells as measured by flow cytometry.
The anti-BCMA antibodies were used as first antibody then a
secondary PE-labeled anti-human Fc was used as detection antibody.
It was found that binding of antibodies Mab 21, Mab 22, Mab 27, Mab
39 and Mab 42 to huBCMA on HEK cells was not significantly better
than the binding of Mab 83A10 to huBCMA-HEK cells.
[0136] FIGS. 19A-19B. Concentrations of 42-TCBcv measured in serum
and bone marrow after single IV or SC injection in cynomolgus
monkeys. Animals received a single IV (FIG. 19A) or SC (FIG. 19B)
injection of 42-TCBcv) and blood samples per timepoint were
collected via the peripheral vein for PK evaluations at Pre-dose,
30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after dosing. Blood
samples were allowed to clot in tubes for serum separation for 60
min at room temperature. The clot was spun down by centrifugation.
The resultant serum was directly stored at -80.degree. C. until
further analysis. Bone marrow samples for PK evaluations were also
collected at the femur under anesthesia/analgesic treatment at
Pre-dose, 96 and 336 h after dosing. Bone marrow samples were
allowed to clot in tubes for serum separation for 60 min at room
temperature. The clot was spun down by centrifugation. The
resultant bone marrow was directly stored at -80.degree. C. until
further analysis. The PK data analysis and evaluation were
performed. Standard non compartmental analysis was performed using
Watson package (v 7.4, Thermo Fisher Scientific Waltman, Mass.,
USA) or Phoenix WinNonlin system (v. 6.3, Certara Company, USA).
Effective concentration range of 42-TCBcv in multiple myeloma
patient bone marrow aspirates corresponding to 10 pm to 10 nM (grey
area). Concentrations in parenthesis are in nM.
[0137] FIGS. 20A-20B. Redirected T-cell lysis of plasma cell
leukemia patient bone marrow leukemic cells in presence of
autologous T cells or bone marrow infiltrating T cells induced by
anti-BCMA/anti-CD3 T-cell bispecific antibodies as measured by flow
cytometry. Percentage of propidium iodide negative myeloma plasma
cells was determined and the percentage of viable bone marrow
plasma cell leukemic cells relative to the medium control (MC) was
plotted against TCB concentrations. Concentration-dependent and
specific lysis of patient plasma cell leukemic cells were observed
(FIG. 20A, FIG. 20B) while lysis of bone marrow microenvironment
(BMME) was not observed (data not shown). No induction of cell
death of myeloma plasma cells observed with control-TCB at the
highest concentration of TCB antibodies tested. 42-TCBcv was very
potent to induce killing of patient bone marrow plasma cell
leukemic cells as reflected by the concentration-dependent
reduction of viable (propidium iodide negative) myeloma plasma
cells. An effect was considered statistically significant if the
P-value of its corresponding statistical test was <5% (*),
<1% (**) or <0.1% (***). The figure shows results obtained
from bone marrow samples of patient 1 (FIG. 20A) and patient 2
(FIG. 20B) (see also example 20).
DETAILED DESCRIPTION OF THE INVENTION
[0138] The term "BCMA, the target BCMA, human BCMA" as used herein
relates to human B cell maturation antigen, also known as BCMA;
TR17_HUMAN, TNFRSF17 (UniProt Q02223), which is a member of the
tumor necrosis receptor superfamily that is preferentially
expressed in differentiated plasma cells. The extracellular domain
of BCMA consists according to UniProt of amino acids 1-54 (or
5-51). The term "antibody against BCMA, anti-BCMA antibody" as used
herein relates to an antibody specifically binding to the
extracellular domain of BCMA.
[0139] "Specifically binding to BCMA or binding to BCMA" refer to
an antibody that is capable of binding to the target BCMA with
sufficient affinity such that the antibody is useful as a
therapeutic agent in targeting BCMA. In some embodiments, the
extent of binding of an anti-BCMA antibody to an unrelated,
non-BCMA protein is about 10-fold preferably >100-fold less than
the binding of the antibody to BCMA as measured, e.g., by surface
plasmon resonance (SPR) e.g. Biacore.RTM., enzyme-linked
immunosorbent (ELISA) or flow cytometry (FACS). In one embodiment
the antibody that binds to BCMA has a dissociation constant (Kd) of
10 M or less, preferably from 10 M to 10'' M, preferably from
10.sup.9 M to 10.sup.-13 M. In one embodiment the anti-BCMA
antibody binds to an epitope of BCMA that is conserved among BCMA
from different species, preferably among human and cynomolgus, and
in addition preferably also to mouse and rat BCMA. "Bispecific
antibody specifically binding to CD3 and BCMA, bispecific antibody
against CD3 and BCMA" refers to a respective definition for binding
to both targets. An antibody specifically binding to BCMA (or BCMA
and CD3) does not bind to other human antigens. Therefore in an
ELISA, OD values for such unrelated targets will be equal or lower
to that of the limit of detection of the specific assay, preferably
>0.3 ng/mL, or equal or lower to OD values of control samples
without plate-bound-BCMA or with untransfected HEK293 cells.
[0140] Preferably the anti-BCMA antibody is specifically binding to
a group of BCMA, consisting of human BCMA and BCMA of non-human
mammalian origin, preferably BCMA from cynomolgus, mouse and/or
rat. "cyno/human gap" refer to the affinity ratio KD cynomolgus
BCMA[M]/KD human BCMA[M] (details see example 3). "cyno/human gap
of Mab CD3" as used herein refer to affinity ratio KD cynomolgus
CD3[M]/KD human CD3[M]. In one embodiment the bispecific
anti-BCMA/anti-CD3 antibody of the invention shows a cyno/human gap
of Mab CD3 between 1.25 and 5 or between 0.8 and 1.0. The
bispecific antibody according to the invention is in one embodiment
characterized in that it binds also specifically to cynomolgus CD3.
In one embodiment the bispecific anti-BCMA/anti-CD3 antibody of the
invention shows a cyno/human gap of Mab CD3 between 1.25 and 5 or
between 0.8 and 1.0. Preferably the cyno/human gap is in the same
range for anti-BCMA- and the anti-CD3 antibody.
[0141] The term "APRIL" as used herein relates to recombinant,
truncated murine APRIL (amino acids 106-241; NP_076006). APRIL can
be produced as described in Ryan, 2007 (Mol Cancer Ther; 6 (11):
3009-18).
[0142] The term "BAFF" as used herein relates to recombinant,
truncated human BAFF (UniProt Q9Y275 (TN13B_HUMAN) which can be
produced as described in Gordon, 2003 (Biochemistry; 42 (20):
5977-5983). Preferably a His-tagged BAFF is used according to the
invention. Preferably the His-tagged BAFF is produced by cloning a
DNA fragment encoding BAFF residues 82-285 into an expression
vector, creating a fusion with an N-terminal His-tag followed by a
thrombin cleavage site, expressing said vector and cleaving the
recovered protein with thrombin.
[0143] Anti-BCMA antibodies are analyzed by ELISA for binding to
human BCMA using plate-bound BCMA. For this assay, an amount of
plate-bound BCMA preferably 1.5 .mu.g/mL and concentration(s)
ranging from 0.1 pM to 200 nM of anti-BCMA antibody are used.
[0144] The term "NF-.kappa.B" as used herein relates to recombinant
NF-.kappa.B p50 (accession number (P19838). NF-.kappa.B activity
can be measured by a DNA-binding ELISA of an extract of NCI-H929 MM
cells (CRL-9068.TM.). NCI-H929 MM cells, untreated or treated with
0.1 .mu.g/mL TNF-.alpha., 1000 ng/mL heat-treated
HT-truncated-BAFF, 1000 ng/mL truncated-BAFF, 0.1 pM to 200 nM
isotype control, and with or without 0.1 pM to 200 nM anti-BCMA
antibodies are incubated for 20 min. NF-.kappa.B activity can be
assayed using a functional ELISA that detects chemiluminescent
signal from p65 bound to the NF-.kappa.B consensus sequence (U.S.
Pat. No. 6,150,090).
[0145] The term "further target" as used herein means preferably
CD3.epsilon.. The term "first target and second target" means
either CD3 as first target and BCMA as second target or means BCMA
as first target and CD3 as second target.
[0146] The term "CD3.epsilon. or CD3" as used herein relates to
human CD3.epsilon. described under UniProt P07766 (CD3E_HUMAN). The
term "antibody against CD3E, anti CD3.epsilon. antibody" relates to
an antibody specifically binding to CD3E. In one embodiment the
antibody comprises a variable domain VH comprising the heavy chain
CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H,
CDR2H and CDR3H and a variable domain VL comprising the light chain
CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1L,
CDR2L and CDR3L. In one embodiment the antibody comprises the
variable domains of SEQ ID NO:7 (VH) and SEQ ID NO:8 (VL).
[0147] The term "antibody" as used herein refers to a monoclonal
antibody. An antibody consists of two pairs of a "light chain" (LC)
and a "heavy chain" (HC) (such light chain (LC)/heavy chain pairs
are abbreviated herein as LC/HC). The light chains and heavy chains
of such antibodies are polypeptides consisting of several domains.
Each heavy chain comprises a heavy chain variable region
(abbreviated herein as HCVR or VH) and a heavy chain constant
region. The heavy chain constant region comprises the heavy chain
constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD, and
IgG) and optionally the heavy chain constant domain CH4 (antibody
classes IgE and IgM). Each light chain comprises a light chain
variable domain VL and a light chain constant domain CL. The
variable domains VH and VL can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
"constant domains" of the heavy chain and of the light chain are
not involved directly in binding of an antibody to a target, but
exhibit various effector functions. The term "antibody" as used
herein refers comprises also the portion of an antibody which is
needed at least for specific binding to the antigen CD3 resp. BCMA.
Therefore such an antibody (or antibody portion) can be in one
embodiment a Fab fragment, if said antibody portion is comprised in
a bispecific antibody according to the invention. The antibody
according to the invention can also be a Fab', F(ab').sub.2, a
scFv, a di-scFv, or a bi-specific T-cell engager (BiTE).
[0148] The term "antibody" includes e.g. mouse antibodies, human
antibodies, chimeric antibodies, humanized antibodies and
genetically engineered antibodies (variant or mutant antibodies) as
long as their characteristic properties are retained. Especially
preferred are human or humanized antibodies, especially as
recombinant human or humanized antibodies. Further embodiments are
heterospecific antibodies (bispecific, trispecific etc.) and other
conjugates, e.g. with cytotoxic small molecules.
[0149] The term "bispecific antibody" as used herein refers in one
embodiment to an antibody in which one of the two pairs of heavy
chain and light chain (HC/LC) is specifically binding to CD3 and
the other one is specifically binding to BCMA. The term also refers
to other formats of bispecific antibodies according to the state of
the art, in one embodiment to bispecific single-chain
antibodies.
[0150] The term "TCB" as used herein refer to a bispecific antibody
specifically binding to BCMA and CD3. The term "83A10-TCBcv" as
used herein refer to a bispecific antibody specifically binding to
BCMA and CD3 as specified by its heavy and light chain combination
of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 (2.times.), and SEQ ID
NO:48, and as shown in FIG. 2A and described in EP14179705. The
terms "21-TCBcv, 22-TCBcv, 42-TCBcv" as used herein refer to the
respective bispecific antibodies of Mab21, as specified by its
heavy and light chain combination of SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50, and SEQ ID NO:51 (2.times.), Mab 22 as specified by
its heavy and light chain combinations of SEQ ID NO:48, SEQ ID
NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2.times.), and Mab42 as
specified by its heavy and light chain combination of SEQ ID NO:48
of SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57-(2.times.).
[0151] The term "naked antibody" as used herein refers to an
antibody which is specifically binding to BCMA, comprising an Fc
part and is not conjugated with a therapeutic agent e.g. with a
cytotoxic agent or radiolabel. The term "conjugated antibody, drug
conjugate" as used herein refers to an antibody which is
specifically binding to BCMA, and is conjugated with a therapeutic
agent e.g. with a cytotoxic agent or radiolabel.
[0152] The term "bispecific single-chain antibody" as used herein
refers to a single polypeptide chain comprising in one embodiment
two binding domains, one specifically binding to BCMA and the other
one in one embodiment specifically binding to CD3. Each binding
domain comprises one variable region from an antibody heavy chain
("VH region"), wherein the VH region of the first binding domain
specifically binds to the CD3 molecule, and the VH region of the
second binding domain specifically binds to BCMA. The two binding
domains are optionally linked to one another by a short polypeptide
spacer. A non-limiting example for a polypeptide spacer is
Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) and repeats thereof. Each binding
domain may additionally comprise one variable region from an
antibody light chain ("VL region"), the VH region and VL region
within each of the first and second binding domains being linked to
one another via a polypeptide linker, long enough to allow the VH
region and VL region of the first binding domain and the VH region
and VL region of the second binding domain to pair with one another
such that, together, they are able to specifically bind to the
respective first and second binding domains (see e.g. EP0623679).
Bispecific single-chain antibodies are also mentioned e.g. in Choi
B D et al., Expert Opin Biol Ther. 2011 July; 11(7):843-53 and Wolf
E. et al., Drug Discov Today. 2005 Sep. 15; 10(18):1237-44.
[0153] The term "diabody" as used herein refers to a small bivalent
and bispecific antibody fragment comprising a heavy (VH) chain
variable domain connected to a light chain variable domain (VL) on
the same polypeptide chain (VH-VL) connected by a peptide linker
that is too short to allow pairing between the two domains on the
same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This
forces pairing with the complementary domains of another chain and
promotes the assembly of a dimeric molecule with two functional
antigen binding sites. To construct bispecific diabodies of the
invention, the V-domains of an anti-CD3 antibody and an anti-BCMA
antibody are fused to create the two chains VH(CD3)-VL(BCMA),
VH(BCMA)-VL(CD3). Each chain by itself is not able to bind to the
respective antigen, but recreates the functional antigen binding
sites of anti-CD3 antibody and anti-BCMA antibody on pairing with
the other chain. The two scFv molecules, with a linker between
heavy chain variable domain and light chain variable domain that is
too short for intramolecular dimerization, are co-expressed and
self-assemble to form bi-specific molecules with the two binding
sites at opposite ends. By way of example, the variable regions
encoding the binding domains for BCMA and CD3, respectively, can be
amplified by PCR from DNA constructs obtained as described, such
that they can be cloned into a vector like pHOG, as described in
Kipiriyanov et al., J. Immunol, Methods, 200, 69-77 (1997a). The
two scFV constructs are then combined in one expression vector in
the desired orientation, whereby the VH-VL linker is shortened to
prevent backfolding of the chains onto themselves. The DNA segments
are separated by a STOP codon and a ribosome binding site (RBS).
The RBS allows for the transcription of the mRNA as a bi-cistronic
message, which is translated by ribosomes into two proteins which
non-covalently interact to form the diabody molecule. Diabodies,
like other antibody fragments, have the advantage that they can be
expressed in bacteria (E. coli) and yeast (Pichia pastoris) in
functional form and with high yields (up to Ig/1).
[0154] The term "tandem scFVs" as used herein refers to a single
chain Fv molecule (i.e. a molecule formed by association of the
immunoglobulin heavy and light chain variable domains, VH and VL,
respectively) as described e.g, in WO 03/025018 and WO 03/048209.
Such Fv molecules, which are known as TandAbs.RTM. comprise four
antibody variable domains, wherein (i) either the first two or the
last two of the four variable domains bind intramolecularly to one
another within the same chain by forming an antigen binding scFv in
the orientation VH/VL or VL/VH (ii) the other two domains bind
intermolecularly with the corresponding VH or VL domains of another
chain to form antigen binding VH/VL pairs. In a preferred
embodiment, as mentioned in WO 03/025018, the monomers of such Fv
molecule comprise at least four variable domains of which two
neighboring domains of one monomer form an antigen-binding VH-VL or
VL-VH scFv unit.
[0155] The term "DARPins" as used herein refers to a bispecific
ankyrin repeat molecule as described e.g. in US 2009082274. These
molecules are derived from natural ankyrin proteins, which can be
found in the human genome and are one of the most abundant types of
binding proteins. A DARPin library module is defined by natural
ankyrin repeat protein sequences, using 229 ankyrin repeats for the
initial design and another 2200 for subsequent refinement. The
modules serve as building blocks for the DARPin libraries. The
library modules resemble human genome sequences. A DARPin is
composed of 4 to 6 modules. Because each module is approx. 3.5 kDa,
the size of an average DARPin is 16-21 kDa. Selection of binders is
done by ribosome diplay, which is completely cell-free and is
described in He M and Taussig M J., Biochem Soc Trans. 2007,
November; 35(Pt 5):962-5.
[0156] The term "T cell bispecific engager" are fusion proteins
consisting of two single-chain variable fragments (scFvs) of
different antibodies, or amino acid sequences from four different
genes, on a single peptide chain of about 55 kilodaltons. One of
the scFvs binds to T cells via the CD3 receptor, and the other to a
BCMA.
[0157] There are five types of mammalian antibody heavy chains
denoted by the Greek letters: .alpha., .delta., .epsilon., .gamma.,
and .mu. (Janeway C A, Jr et al (2001). Immunobiology. 5th ed.,
Garland Publishing). The type of heavy chain present defines the
class of antibody; these chains are found in IgA, IgD, IgE, IgG,
and IgM antibodies, respectively (Rhoades R A, Pflanzer R G (2002).
Human Physiology, 4th ed., Thomson Learning). Distinct heavy chains
differ in size and composition; .alpha. and .gamma. contain
approximately 450 amino acids, while .mu. and .epsilon. have
approximately 550 amino acids.
[0158] Each heavy chain has two regions, the constant region and
the variable region. The constant region is identical in all
antibodies of the same isotype, but differs in antibodies of
different isotype. Heavy chains .gamma., .alpha. and .delta. have a
constant region composed of three constant domains CH1, CH2, and
CH3 (in a line), and a hinge region for added flexibility (Woof J,
Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains .mu. and
.epsilon. have a constant region composed of four constant domains
CH1, CH2, CH3, and CH4 (Janeway C A, Jr et al (2001).
Immunobiology. 5th ed., Garland Publishing). The variable region of
the heavy chain differs in antibodies produced by different B
cells, but is the same for all antibodies produced by a single B
cell or B cell clone. The variable region of each heavy chain is
approximately 110 amino acids long and is composed of a single
antibody domain.
[0159] In mammals there are only two types of light chain, which
are called lambda (20 and kappa (.kappa.). A light chain has two
successive domains: one constant domain CL and one variable domain
VL. The approximate length of a light chain is 211 to 217 amino
acids. In one embodiment the light chain is a kappa (.kappa.) light
chain, and the constant domain CL is in one embodiment derived from
a kappa (K) light chain (the constant domain CK).
[0160] "aa substitution" as used herein refer to independent amino
acid substitution in the constant domain CH1 at the amino acid at
positions 147 and 213 by glutamic acid (E), or aspartic acid (D)
and in the constant domain CL the amino acid at position 124 is
substituted by lysine (K), arginine (R) or histidine (H). In one
embodiment in addition in the constant domain CL the amino acid at
position 123 is independently substituted by lysine (K), arginine
(R) or histidine (H). In one embodiment amino acid 124 is K, amino
acid 147 is E, amino acid 213 is E, and amino acid 123 is R. The aa
substitutions are either in the CD3 Fab or in one or two BCMA Fabs.
Bispecific antibodies against BCMA and CD3 as charge variants are
described in EP14179705, disclosed by reference (further called as
"charge variants resp. charge variant exchange").
[0161] All amino acid numbering herein is according to Kabat
(Kabat, E. A. et al, Sequences of Proteins of Immunological
Interest, 5th ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), NIH Publication 91-3242).
[0162] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0163] The "antibodies" according to the invention can be of any
class (e.g. IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or
subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably
IgG1), whereby both antibodies, from which the bivalent bispecific
antibody according to the invention is derived, have an Fc part of
the same subclass (e.g. IgG1, IgG4 and the like, preferably IgG1),
preferably of the same allotype (e.g. Caucasian).
[0164] A "Fc part of an antibody" is a term well known to the
skilled artisan and defined on the basis of papain cleavage of
antibodies. The antibodies according to the invention contain as Fc
part, in one embodiment a Fc part derived from human origin and
preferably all other parts of the human constant regions. The Fc
part of an antibody is directly involved in complement activation,
C1q binding, C3 activation and Fc receptor binding. While the
influence of an antibody on the complement system is dependent on
certain conditions, binding to C1q is caused by defined binding
sites in the Fc part. Such binding sites are known in the state of
the art and described e.g. by Lukas, T J., et al., J. Immunol. 127
(1981) 2555-2560; Brunhouse, R., and Cebra, J. J., MoI. Immunol. 16
(1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;
Thommesen, J. E., et al., MoI. Immunol. 37 (2000) 995-1004;
Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh,
M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al,
Immunology 86 (1995) 319-324; and EP 0 307 434.
[0165] Such binding sites are e.g. L234, L235, D270, N297, E318,
K320, K322, P331 and P329 (numbering according to EU index of
Kabat). Antibodies of subclass IgG1, IgG2 and IgG3 usually show
complement activation, C1 q binding and C3 activation, whereas IgG4
do not activate the complement system, do not bind C1q and do not
activate C3. In one embodiment the Fc part is a human Fc part.
[0166] In one embodiment an antibody according to the invention
comprises an Fc variant of a wild-type human IgG Fc region, said Fc
variant comprising an amino acid substitution at position Pro329
and at least one further amino acid substitution, wherein the
residues are numbered according to the EU index of Kabat, and
wherein said antibody exhibits a reduced affinity to the human
Fc.gamma.RIIIA and/or Fc.gamma.RIIA and/or Fc.gamma.RI compared to
an antibody comprising the wildtype IgG Fc region, and wherein the
ADCC induced by said antibody is reduced to at least 20% of the
ADCC induced by the antibody comprising a wild-type human IgG Fc
region. In a specific embodiment Pro329 of a wild-type human Fc
region in the antibody according to the invention is substituted
with glycine or arginine or an amino acid residue large enough to
destroy the proline sandwich within the Fc/Fc.gamma. receptor
interface, that is formed between the proline329 of the Fc and
tryptophane residues Trp 87 and Tip 110 of Fc.gamma.RIII
(Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In a
further aspect of the invention the at least one further amino acid
substitution in the Fc variant is S228P, E233P, L234A, L235A,
L235E, N297A, N297D, or P331S and still in another embodiment said
at least one further amino acid substitution is L234A and L235A of
the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc
region. Such Fc variants are described in detail in
WO2012130831.
[0167] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC. By "effector cell" as used
herein is meant a cell of the immune system that expresses one or
more Fc receptors and mediates one or more effector functions.
Effector cells include but are not limited to monocytes,
macrophages, neutrophils, dendritic cells, eosinophils, mast cells,
platelets, B cells, large granular lymphocytes, Langerhans' cells,
natural killer (NK) cells, and .gamma..delta. T cells, and may be
from any organism including but not limited to humans, mice, rats,
rabbits, and monkeys. By "library" herein is meant a set of Fc
variants in any form, including but not limited to a list of
nucleic acid or amino acid sequences, a list of nucleic acid or
amino acid substitutions at variable positions, a physical library
comprising nucleic acids that encode the library sequences, or a
physical library comprising the Fc variant proteins, either in
purified or unpurified form.
[0168] By "Fc gamma receptor" or "Fc.gamma.R" as used herein is
meant any member of the family of proteins that bind the IgG
antibody Fc region and are substantially encoded by the Fc.gamma.R
genes. In humans this family includes but is not limited to
Fc.gamma.RI (CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms
Fc.gamma.R11a (including allotypes H131 and R131), Fc.gamma.R11b
(including Fc.gamma.R11b-1 and Fc.gamma.R11b-2), and Fc.gamma.R11c;
and Fc.gamma.RIII (CD16), including isoforms Fc.gamma.R111a
(including allotypes V158 and F158) and Fc.gamma.R111b (including
allotypes Fc.gamma.R111b-NA1 and Fc.gamma.R111b-NA2) (Jefferis et
al., 2002, Immunol Lett 82:57-65), as well as any undiscovered
human Fc.gamma.Rs or Fc.gamma.R isoforms or allotypes. An
Fc.gamma.R may be from any organism, including but not limited to
humans, mice, rats, rabbits, and monkeys. Mouse Fc.gamma.Rs include
but are not limited to Fc.gamma.RI (CD64), Fc.gamma.RII (CD32),
Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2 (CD16-2), as well as any
undiscovered mouse Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes.
[0169] "Fc variant with increased effector function" as used herein
is meant an Fc sequence that differs from that of a parent Fc
sequence by virtue of at least one amino acid modification or
relates to other modifications like amendment of glycosylation at
e.g. Asn279 that increase effector functions. Such modifications
are e.g. mentioned in Duncan et al., 1988, Nature 332:563-564; Lund
et al., 1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol
Immunol 29:53-59; Alegre et al., 1994, Transplantation
57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA
92:11980-11984; Jefferis et al., 1995, //77muno/Lett 44:111-117;
Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996,
Immunol Lett 54:101-104; Lund et al., 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000,
J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200: 16-26;
Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al.,
2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol
Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490;
U.S. Pat. Nos. 5,624,821; 5,885,573; 6,194,551; WO200042072;
WO199958572. Such Fc modifications also include according to the
invention engineered glycoforms of the Fc part. By "engineered
glycoform" as used herein is meant a carbohydrate composition that
is covalently attached to an Fc polypeptide, wherein said
carbohydrate composition differs chemically from that of a parent
Fc polypeptide. Engineered glycoforms may be generated by any
method, for example by using engineered or variant expression
strains, by co-expression with one or more enzymes, for example
D1-4-N-acetylglucosaminyltransferase III (GnTIII), by expressing an
Fc polypeptide in various organisms or cell lines from various
organisms, or by modifying carbohydrate(s) after the Fc polypeptide
has been expressed. Methods for generating engineered glycoforms
are known in the art and mentioned in Umana et al., 1999, Nat
Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740;
Shinkawa et at, 2003, J Biol Chem 278:3466-3473) U.S. Pat. No.
6,602,684; WO200061739; WO200129246; WO200231140; WO200230954;
Potelligent.TM. technology (Biowa, Inc., Princeton, N.J.);
GlycoMAb.TM. glycosylation engineering technology (GLYCART
biotechnology AG, Zurich, Switzerland)). Engineered glycoform
typically refers to the different carbohydrate or oligosaccharide
composition than the parent Fc polypeptide.
[0170] Antibodies according to the invention comprising a Fc
variant with increased effector function show high binding affinity
to the Fc gamma receptor III (Fc.gamma.RIII, CD 16a). High binding
affinity to Fc.gamma.RIII denotes that binding is enhanced for
CD16a/F158 at least 10-fold in relation to the parent antibody (95%
fucosylation) as reference expressed in CHO host cells, such as CHO
DG44 or CHO K1 cells, or/and binding is enhanced for CD16a/V158 at
least 20-fold in relation to the parent antibody measured by
Surface Plasmon Resonance (SPR) using immobilized CD 16a at an
antibody concentration of 100 nM. Fc.gamma.RIII binding can be
increased by methods according to the state of the art, e.g. by
modifying the amino acid sequence of the Fc part or the
glycosylation of the Fc part of the antibody (see e.g. EP2235061).
Mori, K et al., Cytotechnology 55 (2007)109 and Satoh M, et al.,
Expert Opin Biol Ther. 6 (2006) 1161-1173 relate to a FUT8
(.alpha.-1,6-fucosyltransferase) gene knockout CHO line for the
generation of afucosylated antibodies.
[0171] The term "chimeric antibody" refers to an antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are preferred. Other preferred forms of
"chimeric antibodies" encompassed by the present invention are
those in which the constant region has been modified or changed
from that of the original antibody to generate the properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding. Such chimeric antibodies are also
referred to as "class-switched antibodies". Chimeric antibodies are
the product of expressed immunoglobulin genes comprising DNA
segments encoding immunoglobulin variable regions and DNA segments
encoding immunoglobulin constant regions. Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0172] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Other forms of
"humanized antibodies" encompassed by the present invention are
those in which the constant region has been additionally modified
or changed from that of the original antibody to generate the
properties according to the invention, especially in regard to C1q
binding and/or Fc receptor (FcR) binding.
[0173] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies (see, e.g., Jakobovits, A.,
et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits,
A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year
Immunol. 7 (1993) 33-40). Human antibodies can also be produced in
phage display libraries (Hoogenboom, H. R., and Winter, G., J. MoI.
Biol. 227 (1992) 381-388; Marks, J. D., et al., J. MoI. Biol. 222
(1991) 581-597). The techniques of Cole et al. and Boerner et al.
are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol.
147 (1991) 86-95). As already mentioned for chimeric and humanized
antibodies according to the invention the term "human antibody" as
used herein also comprises such antibodies which are modified in
the constant region to generate the properties according to the
invention, especially in regard to C1q binding and/or FcR binding,
e.g. by "class switching" i.e. change or mutation of Fc parts (e.g.
from IgG1 to IgG4 and/or IgG1/IgG4 mutation.)
[0174] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NSO or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions in a rearranged
form. The recombinant human antibodies according to the invention
have been subjected to in vivo somatic hypermutation. Thus, the
amino acid sequences of the VH and VL regions of the recombinant
antibodies are sequences that, while derived from and related to
human germ line VH and VL sequences, may not naturally exist within
the human antibody germ line repertoire in vivo.
[0175] The "variable domain" (variable domain of a light chain
(VL), variable region of a heavy chain (VH)) as used herein denotes
each of the pair of light and heavy chains which is involved
directly in binding the antibody according to the invention. The
domains of variable human light and heavy chains have the same
general structure and each domain comprises four framework (FR)
regions whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the .beta.-sheet structure. The
CDRs in each chain are held in their three-dimensional structure by
the framework regions and form together with the CDRs from the
other chain the binding site. The antibody heavy and light chain
CDR3 regions play a particularly important role in the binding
specificity/affinity of the antibodies according to the invention
and therefore provide a further object of the invention.
[0176] The terms "hypervariable region" or "target-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for target-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to target binding. CDR and FR regions are determined according
to the standard definition of Kabat et al., Sequences of Proteins
of Immunological Interest, 5th ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). The terms "CDR1H, CDR2H
and CDR3H" as used herein refer to the respective CDRs of the heavy
chain located in the variable domain VH. The terms "CDR1L, CDR2L
and CDR3L" as used herein refer to the respective CDRs of the light
chain located in the variable domain VL.
[0177] The constant heavy chain domain CH1 by which the heavy chain
domain CH3 is replaced can be of any Ig class (e.g. IgA, IgD, IgE,
IgG, and IgM), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2). The constant light chain domain CL by which the heavy chain
domain CH3 is replaced can be of the lambda (.lamda.) or kappa
(.kappa.) type, preferably the kappa (.kappa.) type.
[0178] The term "target" or "target molecule" as used herein are
used interchangeable and refer to human BCMA. In regard to
bispecific antibodies the term refers to BCMA and the second
target. Preferably in regard to bispecific antibodies the term
refers to BCMA and CD3.
[0179] The term "epitope" includes any polypeptide determinant
capable of specific binding to an antibody. In certain embodiments,
epitope determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of a target that is bound
by an antibody.
[0180] In general there are two vectors encoding the light chain
and heavy chain of an antibody according to the invention. In
regard to a bispecific antibody there are two vectors encoding the
light chain and heavy chain of said antibody specifically binding
to the first target, and further two vectors encoding the light
chain and heavy chain of said antibody specifically binding to the
second target. One of the two vectors is encoding the respective
light chain and the other of the two vectors is encoding the
respective heavy chain. However in an alternative method for the
preparation of an antibody according to the invention, only one
first vector encoding the light chain and heavy chain of the
antibody specifically binding to the first target and only one
second vector encoding the light chain and heavy chain of the
antibody specifically binding to the second target can be used for
transforming the host cell.
[0181] The term "nucleic acid or nucleic acid molecule", as used
herein, is intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0182] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the
context.
[0183] The term "transformation" as used herein refers to process
of transfer of a vectors/nucleic acid into a host cell. If cells
without formidable cell wall barriers are used as host cells,
transfection is carried out e.g. by the calcium phosphate
precipitation method as described by Graham and Van der Eh,
Virology 52 (1978) 546ff. However, other methods for introducing
DNA into cells such as by nuclear injection or by protoplast fusion
may also be used. If prokaryotic cells or cells which contain
substantial cell wall constructions are used, e.g. one method of
transfection is calcium treatment using calcium chloride as
described by Cohen S N, et al, PNAS 1972, 69 (8): 2110-2114.
[0184] Recombinant production of antibodies using transformation is
well-known in the state of the art and described, for example, in
the review articles of Makrides, S. C, Protein Expr. Purif. 17
(1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)
271-282; Kaufman, R J., MoI. Biotechnol. 16 (2000) 151-161; Werner,
R. G., et al., Arzneimittelforschung 48 (1998) 870-880 as well as
in U.S. Pat. Nos. 6,331,415 and 4,816,567.
[0185] As used herein, "expression" refers to the process by which
a nucleic acid is transcribed into mRNA and/or to the process by
which the transcribed mRNA (also referred to as transcript) is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectively referred to as gene product. If the polynucleotide is
derived from genomic DNA, expression in a eukaryotic cell may
include splicing of the mRNA.
[0186] A "vector" is a nucleic acid molecule, in particular
self-replicating, which transfers an inserted nucleic acid molecule
into and/or between host cells. The term includes vectors that
function primarily for insertion of DNA or RNA into a cell (e.g.,
chromosomal integration), replication of vectors that function
primarily for the replication of DNA or RNA, and expression vectors
that function for transcription and/or translation of the DNA or
RNA. Also included are vectors that provide more than one of the
functions as described.
[0187] An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide. An "expression system" usually
refers to a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0188] The antibodies according to the invention are preferably
produced by recombinant means. Such methods are widely known in the
state of the art and comprise protein expression in prokaryotic and
eukaryotic cells with subsequent isolation of the antibody
polypeptide and usually purification to a pharmaceutically
acceptable purity. For the protein expression, nucleic acids
encoding light and heavy chains or fragments thereof are inserted
into expression vectors by standard methods. Expression is
performed in appropriate prokaryotic or eukaryotic host cells like
CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast,
or E. coli cells, and the antibody is recovered from the cells
(supernatant or cells after lysis). The bispecific antibodies may
be present in whole cells, in a cell lysate, or in a partially
purified or substantially pure form. Purification is performed in
order to eliminate other cellular components or other contaminants,
e.g. other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, column chromatography
and others well known in the art. See Ausubel, F., et al., ed.,
Current Protocols in Molecular Biology, Greene Publishing and Wiley
Interscience, New York (1987).
[0189] Expression in NS0 cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0190] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0191] The antibodies are suitably separated from the culture
medium by conventional immunoglobulin purification procedures such
as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography. DNA or RNA encoding the monoclonal antibodies is
readily isolated and sequenced using conventional procedures. The
hybridoma cells can serve as a source of such DNA and RNA. Once
isolated, the DNA may be inserted into expression vectors, which
are then transfected into host cells such as HEK293 cells, CHO
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of recombinant
monoclonal antibodies in the host cells.
[0192] Amino acid sequence variants (or mutants) of an antibody
according to the invention are prepared by introducing appropriate
nucleotide changes into the antibody DNA, or by nucleotide
synthesis. Such modifications can be performed, however, only in a
very limited range, e.g. as described above. For example, the
modifications do not alter the above mentioned antibody
characteristics such as the IgG isotype and target binding, but may
improve the yield of the recombinant production, protein stability
or facilitate the purification.
[0193] The invention provides in one embodiment an isolated or
purified nucleic acid sequence encoding a chimeric antigen receptor
(CAR), wherein the CAR comprises an antigen recognition moiety
directed against BCMA, a transmembrane moiety and a T-cell
activation moiety, characterized in that the antigen recognition
moiety is an antibody according to the invention (here not the
bispecific antibody). The encoded antibody can be also an antigen
binding fragment thereof as specified. Structures and generation of
such "BCMA CARs" are described e.g. in WO2013154760, WO2015052538,
WO2015090229, and WO2015092024.
[0194] In one embodiment the invention comprises a chimeric antigen
receptor (CAR) comprising: [0195] (i) a B cell maturation antigen
(BCMA) recognition moiety; [0196] (ii) a spacer domain; and [0197]
(ii) a transmembrane domain; and [0198] (iii) an intracellular T
cell signaling domain, characterized in that the BCMA recognition
moiety is a monoclonal antibody specifically binding to BCMA,
characterized in comprising a CDR3H region of SEQ ID NO:17 and a
CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L
region combination selected from the group of a) CDR1H region of
SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ
ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region of SEQ
ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID
NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID
NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID
NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID
NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, e) CDR1H region of SEQ ID
NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, and f) CDR1H region of SEQ
ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32.
[0199] The T-cell activation moiety can be any suitable moiety
derived or obtained from any suitable molecule. In one embodiment,
for example, the T-cell activation moiety comprises a transmembrane
domain. The transmembrane domain can be any transmembrane domain
derived or obtained from any molecule known in the art. For
example, the transmembrane domain can be obtained or derived from a
CD8a molecule or a CD28 molecule. CD8 is a transmembrane
glycoprotein that serves as a co-receptor for the T-cell receptor
(TCR), and is expressed primarily on the surface of cytotoxic
T-cells. The most common form of CD8 exists as a dimer composed of
a CD8 alpha and CD8 beta chain. CD28 is expressed on T-cells and
provides co-stimulatory signals required for T-cell activation.
CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In a
preferred embodiment, the CD8 alpha and CD28 are human. In addition
to the transmembrane domain, the T-cell activation moiety further
comprises an intracellular (i.e., cytoplasmic) T-cell signaling
domain. The intercellular T-cell signaling domain can be obtained
or derived from a CD28 molecule, a CD3 zeta molecule or modified
versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27
molecule, an OX40 molecule, a 4-IBB molecule, or other
intracellular signaling molecules known in the art. As discussed
above, CD28 is a T-cell marker important in T-cell co-stimulation.
CD3 zeta, associates with TCRs to produce a signal and contains
immunoreceptor tyrosine-based activation motifs (ITAMs). 4-1BB,
also known as CD137, transmits a potent costimulatory signal to
T-cells, promoting differentiation and enhancing long-term survival
of T lymphocytes. In one embodiment, the CD28, CD3 zeta, 4-1BB,
OX40, and CD27 are human.
[0200] The invention provides in one embodiment an isolated or
purified nucleic acid sequence encoding a chimeric antigen receptor
(CAR) as specified above.
[0201] T cell bispecific (TCB) binders have very high
concentration/tumor-cell-receptor-occupancy dependent potency in
cell killing (e.g. EC.sub.50 in in vitro cell killing assays in the
sub- or low picomolar range; Dreier et al. Int J Cancer 2002),
T-cell bispecific binder (TCB) are given at much lower doses than
conventional monospecific antibodies. For example, blinatumomab
(CD19.times.CD3) is given at a continuous intravenous dose of 5 to
15 .mu.g/m.sup.2/day (i.e. only 0.35 to 0.105 mg/m.sup.2/week) for
treatment of acute lymphocytic leukemia or 60 .mu.g/m.sup.2/day for
treatment of Non Hodgkin Lymphoma, and the serum concentrations at
these doses are in the range of 0.5 to 4 ng/ml (Klinger et al.,
Blood 2012; Topp et al., J Clin Oncol 2011; Goebeler et al. Ann
Oncol 2011). Because low doses of TCB can exert high efficacy in
patients, it is envisaged that for an antibody according to the
invention subcutaneous administration is possible and preferred in
the clinical settings (preferably in the dose range of 0.1 to 2.5,
preferably 25 mg/m.sup.2/week, preferably 250 mg/m2/week). Even at
these low concentrations/doses/receptor occupancies, TCB can cause
considerable adverse events (Klinger et al., Blood 2012). Therefore
it is critical to control tumor cell occupancy/coverage. In
patients with high and variable levels of serum APRIL and BAFF
(e.g. multiple myeloma patients, Moreaux et al. 2004; Blood 103(8):
3148-3157) number of TCB bound to the tumor cells resp. tumor cell
occupancy may be considerably influenced by APRIL/BAFF. But by
using said antibody of this invention, tumor cell occupancy
respectively efficacy/safety it may not be required to increase the
dose for an antibody according to this invention as said antibody
may not be affected by APRIL/BAFF ligand competition. Another
advantage of the antibody according to the invention is based on
the inclusion of an Fc portion, which increases the elimination
half-life to about 4 to 12 days and allows at least once or
twice/week administrations as compared to TCBs without an Fc
portion (e.g. blinatumomab) which are required to be given
intravenously and continuously with a pump carried by patients.
[0202] The biological properties of the antibodies according to the
invention respectively their anti-BCMA/anti-CD3 TCB antibodies have
been investigated in several studies in comparison to 83A10-TCBcv.
The potency to induce T-cell redirected cytotoxicity of e.g.
anti-BCMA/anti-CD3 TCB antibodies 21-TCBcv, 22-TCBcv, 42-TCBcv in
comparison to 83A10-TCBcv was measured on H929 MM cell line
(Example 8, Table 12, FIG. 4). The antibodies of this invention
were studied and analysis showed that concentration dependent
killing of H929 cells resp. the EC50 values were found to be higher
than EC50 values determined for 83A10-TCBcv; suggesting that the
anti-BCMA antibodies according to the invention as TCBs were less
potent to induce killing of H929 MM cells than Mab 83A10 as TCB.
Surprisingly a turnover was observed when T-cell redirected
cytotoxicity was measured on RPMI-8226 MM cell line and also JJN-3
cell line (respectively, examples 10 and 11, Tables 13, and 14 and
15, FIGS. 6 and 7): the antibodies according to the invention as
TCBs showed lower EC50 and therefore higher potency than
83A10-TCBcv. To the surprise of the inventors, the antibodies
according to the invention as TCBs showed several advantages in a
direct comparison with 83A10 TCBcv in bone marrow aspirates freshly
taken from MM patients (note: to get the best possible comparison,
in all bone marrow aspirates always all T-cell bispecific (TCB)
antibodies have been tested at same concentrations);
[0203] Higher killing potency of myeloma cells, i.e. same % of
killing already at lower concentrations than with 83A10-TCBcv
respectively concentration response curves for killing shifted to
the left (Example 13, Tables 18, 19 and 20, FIGS. 8, 9 and 10).
Already at a concentration of 1 nM of antibodies as TCBs according
to the invention in seven different patient bone marrow aspirates
reduction relative to control of propidium iodide negative viable
multiple myeloma cancer cells was between 77.1 and 100%. With 1 nM
83A10-TCBcv in same seven bone marrow aspirates reductions of only
37.1 to 98.3% have been achieved (Tables 20 and 21).
[0204] Higher maximal killing as compared to 83A10-TCBcv was
achieved at the highest concentration tested (10 nM) in the same
experiment with the seven (7) bone marrow aspirates for antibodies
as TCBs according to the invention (Tables 20 and 21).
[0205] Non responders to 83A10-TCBcv can be turned to responders if
22-TCBcv/42-TCBcv are used: In two (2) bone marrow patient samples
in which no killing response to 83A10-TCBcv was observed,
surprisingly killing could be found with antibodies as TCBs
according to the invention (FIGS. 9A and 9B).
[0206] The BCMAxCD3 TCB of this invention bind to human and
cynomolgus monkeys (cyno) BCMA and to BCMA of mice and rat,
appropriate for toxicological examination in cynomolgus monkeys if
the CD3 binder also binds to cynomolgus CD3 or in mouse/rat if the
CD3 binder also binds to mouse/rat BCMA. Surprisingly the binding
affinity to cyno BCMA is very close to the binding affinity to
human BCMA. SPR has been used to measure binding affinities to
human and cyno BCMA (Example 2, Table 4). Cyno/human gap (ratio of
affinity for cyno to human BCMA, KD) has been calculated from
measured affinity data by dividing affinity to cyno BCMA through
affinity to human BCMA (Example 3, Table 5). For 83A10 a cyno/human
gap of 15.3 was found (i.e. 15.3 times lower binding affinity to
cyno than to human BCMA). To the surprise of the inventors the
antibodies according to the invention showed cyno/human gaps
between 15.4 and 1.7, which is similar or in majority more
favorable cyno/human gap than that of 83A10 (Table 5). Because the
CD3 binder used in the BCMAxCD3 TCB according to the invention is
cross-reactive to cynomolgus monkey CD3, pharmacokinetics and
pharmacodynamics investigations can be obtained from cynomolgus
monkeys (see Example 16). Also toxicological investigations in
cynomolgus monkeys are predictive of the pharmacological and
toxicological effects in humans and the cross-reactivity to
cynomolgus monkeys feature is to the benefit of patients. The BCMA
antibodies of this invention also bind to murine BCMA (e.g. Kd of
clones 22 and 42 measured by SPR as 0.9 nM and 2.5 nM) see table 2D
in Example 1.1.1A.4). The CD3 binder of the BCMAxCD3 TCB is not
cross-reactive to murine CD3.
[0207] In summary the potency and efficacy advantages for killing
of low BCMA expressing MM cell lines like RPMI-8226 and JJN-3 and
especially for killing of MM cells in patient bone marrow aspirates
and in addition the very favorable cyno/human gap in binding
affinities to BCMA make the antibodies of this invention and
respective TCBs essentially promising agents for treatment of MM
patients. In addition the anti-BCMAxCD3 TCBcv of this invention
have, as 83A10-TCBcv, favorable properties like long elimination
half-life, efficacy at once a week administration (intravenously,
subcutaneously), low or no tendency to aggregation and can be
manufactured with high purity and good yield.
TABLE-US-00001 TABLE 1A Antibody sequences SEQ ID NO: Name(s) aa
sequences 1 CD3 CDR1H TYAMN 2 CD3 CDR2H RIRSKYNNYATYYADSVKG 3 CD3
CDR3H HGNFGNSYVSWFAY 4 CD3 CDR1L GSSTGAVTTSNYAN 5 CD3 CDR2L GTNKRAP
6 CD3 CDR3L ALWYSNLWV 7 CD3 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVR
QAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDD
SKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFA YWGQGTLVTVSS 8 CD3 VL
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWV
QEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALT
LSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 9 83A10 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 10 Mab21 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV Mab22 VH
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK Mab42 VH
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 11 83A10 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ
KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIK 12 Mab21 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSEYYLAWYQQ Mab27 VL
KPGQAPRLLIEHASTRATGIPDRFSGSGSGTDFTLTISRLE Mab33 VL
PEDFAVYYCQQYGYPPDFTFGQGTKVEIK Mab39 VL 13 Mab22 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSYYLAWYQQ
KPGQAPRLLISGAGSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIK 14 Mab42 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSDEYLSWYQQ
KPGQAPRLLIHSASTRATGIPDRFSGSGSGTDFTLAISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIK 15 83A10 CDR1H SYAMS 16 83A10 CDR2H
AISGSGGSTYYADSVKG 17 83A10 CDR3H VLGWFDY Mab21 CDR3H Mab22 CDR3H
Mab42 CDR3H Mab27 CDR3H Mab33 CDR3H Mab39 CDR3H 18 83A10 CDR1L
RASQSVSSSYLAW 19 83A10 CDR2L YGASSRAT 20 83A10 CDR3L QQYGYPPDFT
Mab21 CDR3L Mab22 CDR3L Mab42 CDR3L 21 Mab21 CDR1H DNAMG Mab22
CDR1H Mab42 CDR1H 22 Mab21 CDR2H AISGPGSSTYYADSVKG Mab22 CDR2H
Mab42 CDR2H 23 Mab21 CDR1L RASQSVSEYYLAW 24 Mab21 CDR2L EHASTRAT 25
Mab22 CDR1L RASQSVSSYYLAW 26 Mab22 CDR2L SGAGSRAT 27 Mab42 CDR1L
RASQSVSDEYLSW 28 Mab42 CDR2L HSASTRAT 29 Mab27 CDR1H SAPMG 30 Mab27
CDR2H AISYIGHTYYADSVKG 31 Mab27 CDR1L RASQSVSEYYLA Mab33 CDR1L
Mab39 CDR1L 32 Mab27 CDR2L HASTRAT Mab33 CDR2L Mab39 CDR2L 33 Mab27
CDR3L QQYGYPPDFT Mab33 CDR3L Mab39 CDR3L 34 Mab33 CDR1H TNAMG 35
Mab33 CDR2H AINRFGGSTYYADSVKG 36 Mab39 CDR1H QNAMG 37 Mab39 CDR2H
AISPTGFSTYYADSVKG 38 Mab27 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAPMGWVR
QAPGKGLEWVSAISYIGHTYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTV SS 39 Mab33 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFYTNAMGWV
RQAPGKGLEWVSAINRFGGSTYYADSVKGRFTISRDNS
KNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTL VTVSS 40 Mab39 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFTQNAMGWV
RQAPGKGLEWVSAISPTGFSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 41 83A10 BCMA CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Mab21 BCMA CH1
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT Mab22 BCMA CH1
QTYICNVNHKPSNTKVDEKVEPKSC Mab42 BCMA CH1 42 83A10 BCMA CL
RTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKV Mab21 BCMA CL
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA Mab22 BCMA CL
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC Mab42 BCMA CL 43 CD3 CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC
44 CD3 CL ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 45 83A10 knob HC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGG
GSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYAN
WVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 46
83A10 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCP
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
47 83A10 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ
KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFI
FPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 48
CD3 LC EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVR
QAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDD
SKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFA
YWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 49 Mab21 knob HC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGG
GSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYAN
WVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 50
Mab21 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCP
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
51 Mab21 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSEYYLAWYQQ
KPGQAPRLLIEHASTRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFI
FPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 52
Mab22 knob HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGG
GSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYAN
WVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 53
Mab22 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCP
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
54 Mab22 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSYYLAWYQQ
KPGQAPRLLISGAGSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFI
FPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 55
Mab42 knob HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGG
GSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYAN
WVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAA
LTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 56
Mab42 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV
RQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCP
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
57 Mab42 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSDEYLSWYQQ
KPGQAPRLLIHSASTRATGIPDRFSGSGSGTDFTLAISRLE
PEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFI
FPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC
Remark: SEQ ID NO: 20 and SEQ ID NO: 33 are identical
TABLE-US-00002 TABLE 1B Antibody sequences (short list) SEQ ID NO:
VH VL CDR1H CDR2H CDR3H CDR1L CDR2L CDR3L CD3 antibody 7 8 1 2 3 4
5 6 BCMA antibody 83A10 9 11 15 16 17 18 19 20 Mab21 10 12 21 22 17
23 24 20 Mab22 10 13 21 22 17 25 26 20 Mab42 10 14 21 22 17 27 28
20 Mab27 38 12 29 30 17 31 32 33 Mab33 39 12 34 35 17 31 32 33
Mab39 40 12 36 37 17 31 32 33
TABLE-US-00003 TABLE 2A Additional constructs SEQ ID NO:
Fragment/Construct 83A10 Mab21 Mab22 Mab42 BCMA CH1 41 41 41 41
BCMA CL 42 42 42 42 CD3 CH1 43 43 43 43 CD3 CL 44 44 44 44
TABLE-US-00004 TABLE 2B Additional constructs SEQ ID NO: Construct
83A10 Mab21 Mab22 Mab42 BCMA VH_CH1cv .times. CD3 45 49 52 55
VL_CH1 Fc knob LALA PG (knob HC) BCMAcv HC hole LALA PG 46 50 53 56
(hole HC) BCMAcv hum IgG1 LC (BCMA 47 51 54 57 LC) CD3 VH_CL (CD3
LC) 48 48 48 48
[0208] To make the following (2+1) Fc-containing anti-BCMA/anti-CD3
TCBs, the respective constructs/sequence IDs as mentioned in the
table 2B above were used:
83A10-TCBcv: 45, 46, 47 (.times.2), 48 (FIG. 2A)
21-TCBcv: 48, 49, 50, 51 (.times.2) (FIG. 2A)
22-TCBcv: 48, 52, 53, 54 (.times.2) (FIG. 2A)
42-TCBcv: 48, 55, 56, 57 (.times.2) (FIG. 2A)
[0209] In the following specific embodiments of the invention are
listed:
1. A monoclonal antibody specifically binding to BCMA,
characterized in comprising a CDR3H region of SEQ ID NO:17 and a
CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L
region combination selected from the group of a) CDR1H region of
SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ
ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region of SEQ
ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID
NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID
NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID
NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID
NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, e) CDR1H region of SEQ ID
NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, and f) CDR1H region of SEQ
ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32. 2. A monoclonal antibody
specifically binding to BCMA, characterized in comprising a VH
region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of
SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VL region
comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L
region combination selected from the group of a) CDR1L region of
SEQ ID NO:23 and CDR2L region of SEQ ID NO:24, b) CDR1L region of
SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or c) CDR1L region
of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28. 3. The antibody
according to embodiment for 2, characterized in comprising as VL
region a VL region selected from the group consisting of VL regions
of SEQ ID NO:12, 13, and 14. 4. The antibody according to any one
of embodiments 1 to 3, characterized in comprising as VH region a
VH region of SEQ ID NO:10 and as VL region a VL region of SEQ ID
NO:12. 5. The antibody according to any one of embodiment 1 to 3,
characterized in comprising as VH region a VH region of SEQ ID
NO:10 and as VL region a VL region of SEQ ID NO:13. 6. The antibody
according to any one of embodiment 1 to 3, characterized in
comprising as VH region a VH region of SEQ ID NO:10 and as VL
region a VL region of SEQ ID NO:14. 7. The antibody according to
embodiment 1 or 2, characterized in that amino acid 49 of the VL
region is selected from the group of amino acids tyrosine (Y),
glutamic acid (E), serine (S), and histidine (H). 8. The antibody
according to embodiment 7, characterized in that amino acid 74 of
the VL region is threonine (T) or alanine (A). 9. A monoclonal
antibody specifically binding to BCMA, characterized in comprising
a VH region comprising a CDR3H region of SEQ ID NO:17 and a VL
region comprising a CDR1L region of SEQ ID NO:31, a CDR2L region of
SEQ ID NO:32 and a CDR3L region of SEQ ID NO:20 and a CDR1L and
CDR2L region combination selected from the group of a) CDR1H region
of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, b) CDR1H region
of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or c) CDR1H
region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37. 10. The
antibody according to embodiment 9, characterized in comprising a
VL region of SEQ ID NO:12 and a VH region selected from the group
comprising the VH regions of SEQ ID NO:38, 39, and 40. 11. The
antibody according to embodiment 9 or 10, characterized in
comprising in in that amino acid 49 of the VL region is selected
from the group of amino acids tyrosine(Y), glutamic acid (E),
serine (S), and histidine (H). 12. The antibody according to
embodiment 9 or 10, characterized in that amino acid 74 of the VL
region is threonine (T) or alanine (A). 13. The antibody according
to any one of embodiments 1 to 12, characterized in that it binds
also specifically to cynomolgus BCMA and comprises an additional
Fab fragment specifically binding to CD3.epsilon.. 14. The antibody
according to any one of embodiments 1 to 13, characterized in being
an antibody with an Fc or without an Fc part. 15. A bispecific
antibody specifically binding to BCMA and CD3e, characterized in
comprising a CDR3H region of SEQ ID NO:17 and a CDR3L region of SEQ
ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination
selected from the group of a) CDR1H region of SEQ ID NO:21 and
CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and
CDR2L region of SEQ ID NO:24, b) CDR1H region of SEQ ID NO:21 and
CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and
CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ ID NO:21 and
CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and
CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID NO:29 and
CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID NO:31, and
CDR2L region of SEQ ID NO:32, e) CDR1H region of SEQ ID NO:34 and
CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31, and
CDR2L region of SEQ ID NO:32, and f) CDR1H region of SEQ ID NO:36
and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO:31, and
CDR2L region of SEQ ID NO:32. 16. A bispecific antibody
specifically binding to the two targets which are the extracellular
domain of human BCMA (further named also as "BCMA") and human
CD3.epsilon. (further named also as "CD3"), characterized in
comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a
CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and
a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L
and CDR2L region combination selected from the group of a) CDR1L
region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24, b) CDR1L
region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or c)
CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28. 17.
The bispecific antibody according to embodiment 15 or 16,
characterized in comprising as VH region a VH region of SEQ ID
NO:10. 18. The bispecific antibody according to any one of
embodiments 15 to 16, characterized in that the BCMA VL is selected
from the group consisting of VL regions of SEQ ID NO:12, 13, and
14. 19. The bispecific antibody according to any one of embodiments
14 to 18, characterized in comprising as BCMA VH region a VH region
of SEQ ID NO:10 and as VL region a VL region of SEQ ID NO:12, or as
BCMA VH a VH region of SEQ ID NO:10 and as VL region a VL region of
SEQ ID NO:13, or as BCMA VH a VH region of SEQ ID NO:10 and as VL
region a VL region of SEQ ID NO:14. 20. The bispecific antibody
according to any one of embodiments 15 or 19, characterized in
comprising in in that amino acid 49 of the VL region is selected
from the group of amino acids tyrosine(Y), glutamic acid (E),
serine (S), and histidine (H). 21. The bispecific antibody
according to any one of embodiments 15 to 20, characterized in that
amino acid 74) of the VL region is threonine (T) or alanine (A).
22. A bispecific antibody specifically binding to BCMA and CD3,
characterized in comprising a VH region comprising a CDR3H region
of SEQ ID NO:17 and a VL region comprising a CDR1L region of SEQ ID
NO:31, a CDR2L region of SEQ ID NO:32 and a CDR3L region of SEQ ID
NO:20 and a CDR1L and CDR2L region combination selected from the
group of a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID
NO:30, b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID
NO:35, or c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ
ID NO:37. 23. The bispecific antibody according to embodiment 22,
characterized in comprising a VL region of SEQ ID NO:12 and a VH
region selected from the group comprising the VH regions of SEQ ID
NO:38, 39, and 40. 24. The bispecific antibody according to
embodiment 22 or 23, characterized in comprising in in that amino
acid 49 of the VL region is selected from the group of amino acids
tyrosine(Y), glutamic acid (E), serine (S), and histidine (H). 25.
The bispecific antibody according to any one of embodiments 22 to
24, characterized in that amino acid 74 of the VL region is
threonine (T) or alanine (A). 26. The bispecific antibody according
to any one of embodiments 15 to 25, characterized in comprising an
anti BCMA antibody according to the invention and an anti CD3
antibody, wherein a) the light chain and heavy chain of an antibody
according to any one of embodiments 1 to 7; and b) the light chain
and heavy chain of an antibody specifically binding to CD3, wherein
the variable domains VL and VH or the constant domains CL and CH1
are replaced by each other. 27. The bispecific antibody according
to any one of embodiments 15 to 26, characterized in comprising not
more than one Fab fragment of an anti-CD3 antibody portion, not
more than two Fab fragments of an anti-BCMA antibody portion and
not more than one Fc part. 28. The bispecific antibody according to
any one of embodiments 15 to 27, characterized in comprising a Fc
part linked with its N-terminus to the C-terminus of said CD3
antibody Fab fragment and to the C-terminus of one of said BCMA
antibody Fab fragments. 29. The bispecific antibody according to
any one of embodiments 15-28, characterized in comprising a second
Fab fragment of said anti-BCMA antibody (BCMA antibody portion)
linked with its C-terminus to the N-terminus of said Fab fragment
of said anti-CD3 antibody (CD3 antibody portion) of said bispecific
antibody. 30. The bispecific antibody according to embodiment 29,
characterized in that the VL domain of said anti-CD3 antibody Fab
fragment is linked to the CH1 domain of said second anti-BCMA
antibody Fab fragment. 31. The bispecific antibody according to any
one of embodiments 15 to 30, characterized in that the variable
domain VH of the anti-CD3 antibody portion (further named as "CD3
VH") comprises the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as
respectively heavy chain CDR1, CDR2 and CDR3 and the variable
domain VL of the anti-CD3 antibody portion (further named as "CD3
VL") comprises the light chain CDRs of SEQ ID NO: 4, 5 and 6 as
respectively light chain CDR1, CDR2 and CDR3. 32. The bispecific
antibody according to any one of embodiments 15 to 31,
characterized in that the variable domains of the anti CD3.epsilon.
antibody portion are of SEQ ID NO:7 and 8. 33. A bispecific
antibody specifically binding to the two targets which are the
extracellular domain of human BCMA and human CD3E, characterized in
comprising a) the first light chain and the first heavy chain of a
first antibody according to any one of claims 1 to 7; and b) the
second light chain and the second heavy chain of a second antibody
which specifically binds to CD3, and wherein the variable domains
VL and VH in the second light chain and second heavy chain of the
second antibody are replaced by each other; and c) wherein in the
constant domain CL of the first light chain under a) the amino acid
at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat), and
wherein in the constant domain CH1 of the first heavy chain under
a) the amino acid at position 147 and the amino acid at position
213 is substituted independently by glutamic acid (E), or aspartic
acid (D) (numbering according to EU index of Kabat) (see e.g. FIGS.
1A, 2A, 2C, 3A, 3C). 34. A bispecific antibody specifically
according to claim 33, characterized in comprising in addition a
Fab fragment of said first antibody (further named also as
"BCMA-Fab") and in the constant domain CL said BCMA-Fab the amino
acid at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat), and
wherein in the constant domain CH1 of said BCMA-Fab the amino acid
at positions 147 and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to EU index of Kabat) (see e.g. FIGS. 2A, 2C). 35. A
bispecific antibody specifically binding to the two targets which
are the extracellular domain of human BCMA and human CD3E,
characterized in comprising a) the first light chain and the first
heavy chain of a first antibody according to any one of claims 1 to
7; and b) the second light chain and the second heavy chain of a
second antibody which specifically binds to CD3, and wherein the
variable domains VL and VH in the second light chain and second
heavy chain of the second antibody are replaced by each other; and
wherein c) in the constant domain CL of the second light chain
under b) the amino acid at position 124 is substituted
independently by lysine (K), arginine (R) or histidine (H)
(numbering according to Kabat), and wherein in the constant domain
CH1 of the second heavy chain under b) the amino acid at positions
147 and the amino acid at position 213 is substituted independently
by glutamic acid (E), or aspartic acid (D) (numbering according to
EU index of Kabat). 36. A bispecific antibody specifically binding
to the two targets which are the extracellular domain of human BCMA
and human CD3E, characterized in comprising a heavy and light chain
set selected from the group consisting of polypeptides i) SEQ ID
NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2.times.);
(set 1 TCB of antibody 21), ii) SEQ ID NO:48, SEQ ID NO:52, SEQ ID
NO:53, and SEQ ID NO:54 (2.times.) (set 2 TCB of antibody 22), and
iii) SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57
(2.times.) (set 3 TCB of antibody 42). 37. A method for the
preparation of an antibody according to any one of claims 1 to 36
comprising the steps of a) transforming a host cell with b) vectors
comprising nucleic acid molecules encoding the light chain and
heavy chains of an antibody according to any one of claims 1 to 36,
c) culturing the host cell under conditions that allow synthesis of
said antibody molecule; and d) recovering said antibody molecule
from said culture. 38. A host cell comprising vectors comprising
nucleic acid molecules encoding an antibody according to any one of
claims 1 to 36. 39. A pharmaceutical composition comprising an
antibody according to any one of claims 1 to 36 and a
pharmaceutically acceptable excipient. 40. A pharmaceutical
composition comprising an antibody according to any one of claims 1
to 36 for use as a medicament. 41. A pharmaceutical composition
comprising an antibody according to any one of claims 1 to 36 for
use as a medicament in the treatment of plasma cell disorders. 42.
A pharmaceutical composition comprising an antibody according to
any one of claims 1 to 36 for use as a medicament in the treatment
of Multiple Myeloma, Plasma Cell Leukemia and AL-Amyloidosis 43. A
pharmaceutical composition comprising an antibody according to any
one of claims 1 to 36 for use as a medicament in the treatment of
systemic lupus erythematosus. 44. A pharmaceutical composition
comprising an antibody according to any one of claims 1 to 36,
including a monospecific antibody, an ADCC enhanced naked antibody,
an antibody-drug conjugate or a bispecific antibody for use as a
medicament in the treatment of antibody-mediated rejection. 45. A
chimeric antigen receptor (CAR) comprising: an antigen recognition
moiety directed against BCMA and a T-cell activation moiety,
characterized in that the antigen recognition moiety is a
monoclonal antibody or antibody fragment according to any one of
embodiments 1 to 14.
46. A chimeric antigen receptor (CAR) according to embodiment 45,
characterized in comprising: (i) a B cell maturation antigen (BCMA)
recognition moiety; (ii) a spacer domain; and (ii) a transmembrane
domain; and (iii) an intracellular T cell signaling domain. 47. A
chimeric antigen receptor (CAR) according to embodiment 45 or 46,
characterized in that the antigen recognition moiety is a
monoclonal antibody or antibody fragment specifically binding to
BCMA, characterized in comprising a CDR3H region of SEQ ID NO:17
and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and
CDR2L region combination selected from the group of a) CDR1H region
of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of
SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region of
SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ
ID NO:25, and CDR2L region of SEQ ID NO:26, c) CDR1H region of SEQ
ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID
NO:27, and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID
NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, e) CDR1H region of SEQ ID
NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32, and f) CDR1H region of SEQ
ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID
NO:31, and CDR2L region of SEQ ID NO:32. 48. An isolated or
purified nucleic acid sequence encoding a chimeric antigen receptor
(CAR), according to any one of embodiments 45 to 47. 49. A method
of generation a monoclonal antibody specifically binding to BCMA,
which depletes as bispecific antibody according to any one of
embodiments 15 to 36 human malignant plasma cells in multiple
myeloma MM bone marrow aspirates in a manner to at least 80% after
a 48 hour treatment in a concentration of between 10 nM and 1 fM,
anti-BCMA antibody, characterized in a) panning a variable heavy
chain (VH) phage-display library of SEQ ID NO:9 with 1-50 nM
cynomolgus BCMA in 1-3 rounds and selecting a variable heavy chain,
which when combined with the variable light chain of SEQ ID NO:11
to a bispecific antibody according to any one of embodiments 15 to
36 which depletes such human malignant plasma cells in such manner,
c) panning a variable light chain (VL) phage-display library of SEQ
ID NO:11 with 1-50 nM cynomolgus BCMA in 1-3 rounds and b)
selecting a variable light chain, which when combined with the
variable heavy chain of SEQ ID NO:9 to to a bispecific antibody
according to any one of embodiments 15 to 36 which depletes such
human malignant plasma cells in such manner, and combining said
selected variable heavy chain and selected variable light chain to
to a bispecific antibody according to any one of embodiments 4 to
16 which depletes such human malignant plasma cells in such manner.
50. A pharmaceutical composition comprising an antibody according
to any one of claims 1 to 36 and 45 to 47 for use as a medicament
in the treatment of multiple myeloma or systemic lupus
erythematosus or plasma cell leukemia or AL-amyloidosis.
[0210] In one embodiment the binding of the antibody according to
the invention is not reduced by 100 ng/ml APRIL for more than 20%
measured in an ELISA assay as OD at 405 nm compared to the binding
of said antibody to human BCMA without APRIL, does not alter
APRIL-dependent NF-.kappa.B activation for more than 20%, as
compared to APRIL, and does not alter NF-.kappa.B activation
without APRIL for more than 20%, as compared without said
antibody.
[0211] In one embodiment the binding the antibody in a
concentration of 6.25 nM is not reduced by 140 ng/ml murine APRIL
for more than 10%, preferably not reduced by for more than 1%
measured in an ELISA assay as OD at 450 nm compared to the binding
of said antibody to human BCMA without APRIL. The binding of said
antibody in a concentration of 50 nM is not reduced by 140 ng/ml
murine APRIL for more than 10%, measured in an ELISA assay as OD at
450 nm, compared to the binding of said antibody to human BCMA
without APRIL.
[0212] In one embodiment the binding of said antibody is not
reduced by 100 ng/ml APRIL and not reduced by 100 ng/ml BAFF for
more than 20% measured in an ELISA assay as OD at 405 nm compared
to the binding of said antibody to human BCMA without APRIL or BAFF
respectively, the antibody does not alter APRIL-dependent
NF-.kappa.B activation for more than 20%, as compared to APRIL
alone, does not alter BAFF-dependent NF-.kappa.B activation for
more than 20%, as compared to BAFF alone, and does not alter
NF-.kappa.B activation without BAFF and APRIL for more than 20%, as
compared without said antibody.
[0213] In one embodiment the binding of said antibody to human BCMA
is not reduced by 100 ng/ml APRIL for more than 15%, measured in
said ELISA, not reduced by 1000 ng/ml APRIL, for more than 20%,
measured in said ELISA, and not reduced by 1000 ng/ml APRIL for
more than 15%, measured in said ELISA.
[0214] In one embodiment the binding of said antibody to human BCMA
is not reduced by 100 ng/ml APRIL and not reduced by 100 ng/ml BAFF
for more than 15%, measured in said ELISA, not reduced by 1000
ng/ml APRIL and not reduced by 1000 ng/ml BAFF, for more than 20%,
measured in said ELISA, not reduced by 1000 ng/ml APRIL and not
reduced by 1000 ng/ml BAFF for more than 15%, measured in said
ELISA.
[0215] In one embodiment the antibody according to the invention
does not alter APRIL-dependent NF-kB activation for more than 15%,
does not alter BAFF-dependent NF-kB activation for more than 15%,
and does not alter NF-.kappa.B activation without APRIL and BAFF
for more than 15%.
[0216] In one embodiment the binding of the antibody to BCMA is not
reduced by APRIL, not reduced by BAFF for more than 25%, not more
than 20%, and not more than 10%, measured as binding of said
antibody in a concentration of 5 nM, preferably 50 nM, and 140 nM
to NCI-H929 cells (ATCC.RTM. CRL9068.TM.) in presence or absence of
APRIL or respectively BAFF in a concentration of 2.5 .mu.g/ml
compared to the binding of said antibody to NCI-H929 cells without
APRIL or BAFF respectively.
[0217] In one embodiment the following examples, sequence listing
and figures are provided to aid the understanding of the present
invention, the true scope of which is set forth in the appended
claims. It is understood that modifications can be made in the
procedures set forth without departing from the spirit of the
invention.
[0218] Materials & General Methods
[0219] Recombinant DNA Techniques
[0220] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions. General information regarding the
nucleotide sequences of human immunoglobulins light and heavy
chains is given in: Kabat, E. A. et al., (1991) Sequences of
Proteins of Immunological Interest, 5.sup.th ed., NIH Publication
No. 91-3242. Amino acids of antibody chains were numbered and
referred to according to Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md., (1991).
[0221] Gene Synthesis
a) Desired gene segments were prepared from oligonucleotides made
by chemical synthesis. The 600-1800 bp long gene segments, which
were flanked by singular restriction endonuclease cleavage sites,
were assembled by annealing and ligation of oligonucleotides
including PCR amplification and subsequently cloned via the
indicated restriction sites e.g. Kpnl/Sad or Ascl/Pacl into a
pPCRScript (Stratagene) based pGA4 cloning vector. The DNA
sequences of the subcloned gene fragments were confirmed by DNA
sequencing. Gene synthesis fragments were ordered according to
given specifications at Geneart (Regensburg, Germany). b) Desired
gene segments where required were either generated by PCR using
appropriate templates or were synthesized by Geneart A G
(Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. The gene segments flanked by
singular restriction endonuclease cleavage sites were cloned into
standard expression vectors or into sequencing vectors for further
analysis. The plasmid DNA was purified from transformed bacteria
using commercially available plasmid purification kits. Plasmid
concentration was determined by UV spectroscopy. The DNA sequence
of the subcloned gene fragments was confirmed by DNA sequencing.
Gene segments were designed with suitable restriction sites to
allow sub-cloning into the respective expression vectors. If
required, protein coding genes were designed with a 5'-end DNA
sequence coding for a leader peptide which targets proteins for
secretion in eukaryotic cells.
[0222] DNA Sequence Determination
[0223] DNA sequences were determined by double strand
sequencing.
DNA and Protein Sequence Analysis and Sequence Data Management
[0224] The Clone Manager (Scientific & Educational Software)
software package version 9.2 was used for sequence mapping,
analysis, annotation and illustration.
[0225] Expression Vectors
a) The fusion genes comprising the described antibody chains as
described below were generated by PCR and/or gene synthesis and
assembled with known recombinant methods and techniques by
connection of the according nucleic acid segments e.g. using unique
restriction sites in the respective vectors. The subcloned nucleic
acid sequences were verified by DNA sequencing. For transient
transfections larger quantities of the plasmids are prepared by
plasmid preparation from transformed E. coli cultures (Nucleobond A
X, Macherey-Nagel). b) For the generation of anti-BCMA antibody
expression vectors, the variable regions of heavy and light chain
DNA sequences were subcloned in frame with either the human IgG1
constant heavy chain or the hum IgG1 constant light chain
pre-inserted into the respective generic recipient expression
vector optimized for expression in mammalian cell lines. The
antibody expression is driven by a chimeric MPSV promoter
comprising a CMV enhancer and a MPSV promoter followed by a 5' UTR,
an intron and a Ig kappa MAR element. The transcription is
terminated by a synthetic polyA signal sequence at the 3' end of
the CDS. All vectors carry a 5'-end DNA sequence coding for a
leader peptide which targets proteins for secretion in eukaryotic
cells. In addition each vector contains an EBV OriP sequence for
episomal plasmid replication in EBV EBNA expressing cells.
[0226] c) For the generation of BCMAxCD3 bispecific antibody
vectors, the IgG1 derived bispecific molecules consist at least of
two antigen binding moieties capable of binding specifically to two
distinct antigenic determinants CD3 and BCMA. The antigen binding
moieties are Fab fragments composed of a heavy and a light chain,
each comprising a variable and a constant region. At least one of
the Fab fragments was a "Crossfab" fragment, wherein VH and VL were
exchanged. The exchange of VH and VL within the Fab fragment
assures that Fab fragments of different specificity do not have
identical domain arrangements. The bispecific molecule design was
monovalent for CD3 and bivalent for BCMA where one Fab fragment was
fused to the N-terminus of the inner CrossFab (2+1). The bispecific
molecule contained an Fc part in order for the molecule to have a
long half-life. A schematic representation of the constructs is
given in FIG. 2; the preferred sequences of the constructs are
shown in SEQ ID NOs 39 to 52. The molecules were produced by
co-transfecting HEK293 EBNA cells growing in suspension with the
mammalian expression vectors using polymer-based solution. For
preparation of 2+1 CrossFab-IgG constructs, cells were transfected
with the corresponding expression vectors in a 1:2:1:1 ratio
("vector Fc(knob)": "vector light chain": "vector light chain
CrossFab": "vector heavy chain-CrossFab").
[0227] Cell Culture Techniques
[0228] Standard cell culture techniques are used as described in
Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso,
M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M.
(eds.), John Wiley & Sons, Inc.
[0229] Transient Expression in HEK293 Cells (HEK293-EBNA
System)
[0230] Bispecific antibodies were expressed by transient
co-transfection of the respective mammalian expression vectors in
HEK293-EBNA cells, which were cultivated in suspension, using
polymer-based solution. One day prior to transfection the
HEK293-EBNA cells were seeded at 1.5 Mio viable cells/mL in Ex-Cell
medium, supplemented with 6 mM of L-Glutamine. For every mL of
final production volume 2.0 Mio viable cells were centrifuged (5
minutes at 210.times.g). The supernatant was aspirated and the
cells resuspended in 100 .mu.L of CD CHO medium. The DNA for every
mL of final production volume was prepared by mixing 1 .mu.g of DNA
(Ratio heavy chain:modified heavy chain:light chain:modified light
chain=1:1:2:1) in 100 .mu.L of CD CHO medium. After addition of
0.27 .mu.L of polymer-based solution (1 mg/mL) the mixture was
vortexed for 15 seconds and left at room temperature for 10
minutes. After 10 minutes, the resuspended cells and
DNA/polymer-based solution mixture were put together and then
transferred into an appropriate container which was placed in a
shaking device (37.degree. C., 5% CO.sub.2). After a 3 hours
incubation time 800 .mu.L of Ex-Cell Medium, supplemented with 6 mM
L-Glutamine, 1.25 mM valproic acid and 12.5% Pepsoy (50 g/L), was
added for every mL of final Production volume. After 24 hours, 70
.mu.L of feed solution was added for every mL of final production
volume. After 7 days or when the cell viability was equal or lower
than 70%, the cells were separated from the supernatant by
centrifugation and sterile filtration. The antibodies were purified
by an affinity step and one or two polishing steps, being cation
exchange chromatography and size exclusion chromatography. When
required, an additional polishing step was used. The recombinant
anti-BCMA human antibody and bispecific antibodies were produced in
suspension by co-transfecting HEK293-EBNA cells with the mammalian
expression vectors using polymer-based solution. The cells were
transfected with two or four vectors, depending in the format. For
the human IgG1 one plasmid encoded the heavy chain and the other
plasmid the light chain. For the bispecific antibodies four
plasmids were co-transfected. Two of them encoded the two different
heavy chains and the other two encoded the two different light
chains. One day prior to transfection the HEK293-EBNA cells were
seeded at 1.5 Mio viable cells/mL in F17 Medium, supplemented with
6 mM of L-Glutamine.
[0231] Protein Determination
[0232] Determination of the antibody concentration was done by
measurement of the absorbance at 280 nm, using the theoretical
value of the absorbance of a 0.1% solution of the antibody. This
value was based on the amino acid sequence and calculated by GPMAW
software (Lighthouse data).
[0233] SDS-PAGE
[0234] The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) is used
according to the manufacturer's instruction. In particular, 10% or
4-12% NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels (pH 6.4) and a
NuPAGE.RTM. MES (reduced gels, with NuPAGE.RTM. Antioxidant running
buffer additive) or MOPS (non-reduced gels) running buffer is
used.
[0235] Protein Purification
[0236] By Protein a Affinity Chromatography
[0237] For the affinity step the supernatant was loaded on a
protein A column (HiTrap Protein A FF, 5 mL, GE Healthcare)
equilibrated with 6 CV 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. After a washing step with the same buffer the
antibody was eluted from the column by step elution with 20 mM
sodium phosphate, 100 mM sodium chloride, 100 mM Glycine, pH 3.0.
The fractions with the desired antibody were immediately
neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooled and
concentrated by centrifugation. The concentrate was sterile
filtered and processed further by cation exchange chromatography
and/or size exclusion chromatography.
[0238] By Cation Exchange Chromatography
[0239] For the cation exchange chromatography step the concentrated
protein was diluted 1:10 with the elution buffer used for the
affinity step and loaded onto a cation exchange colume (Poros 50
HS, Applied Biosystems). After two washing steps with the
equilibration buffer and a washing buffer resp. 20 mM sodium
phosphate, 20 mM sodium citrate, 20 mM TRIS, pH 5.0 and 20 mM
sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium
chloride pH 5.0 the protein was eluted with a gradient using 20 mM
sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium
chloride pH 8.5. The fractions containing the desired antibody were
pooled, concentrated by centrifugation, sterile filtered and
processed further a size exclusion step.
[0240] By Analytical Size Exclusion Chromatography
[0241] For the size exclusion step the concentrated protein was
injected in a XK16/60 HiLoad Superdex 200 column (GE Healthcare),
and 20 mM Histidine, 140 mM Sodium Chloride, pH 6.0 with or without
Tween20 as formulation buffer. The fractions containing the
monomers were pooled, concentrated by centrifugation and sterile
filtered into a sterile vial.
[0242] Measurement of Purity and Monomer Content
[0243] Purity and monomer content of the final protein preparation
was determined by CE-SDS (Caliper LabChip GXII system (Caliper Life
Sciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusion
column (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium
chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium
azide, pH 6.7 buffer.
[0244] Molecular Weight Confirmation by LC-MS Analyses
[0245] Deglycosylation
[0246] To confirm homogeneous preparation of the molecules final
protein solution of was analyzed by LC-MS analyses. To remove
heterogeneity introduced by carbohydrates the constructs are
treated with PNGaseF (ProZyme). Therefore the pH of the protein
solution was adjusted to pH7.0 by adding 2 .mu.l 2 M Tris to 20
.mu.g protein with a concentration of 0.5 mg/ml. 0.8 .mu.g PNGaseF
was added and incubated for 12 h at 37.degree. C.
[0247] LC-MS Analysis--On Line Detection
[0248] The LC-MS method was performed on an Agilent HPLC 1200
coupled to a TOF 6441 mass spectrometer (Agilent). The
chromatographic separation was performed on a Macherey Nagel
Polysterene column; RP1000-8 (8 .mu.m particle size, 4.6.times.250
mm; cat. No. 719510). Eluent A was 5% acetonitrile and 0.05% (v/v)
formic acid in water, eluent B was 95% acetonitrile, 5% water and
0.05% formic acid. The flow rate was 1 ml/min, the separation was
performed at 40.degree. C. and 6 .mu.g (15 .mu.l) of a protein
sample obtained with a treatment as described before.
TABLE-US-00005 Time (min.) % B 0.5 15 10 60 12.5 100 14.5 100 14.6
15 16 15 16.1 100
[0249] During the first 4 minutes the eluate was directed into the
waste to protect the mass spectrometer from salt contamination. The
ESI-source was running with a drying gas flow of 121/min, a
temperature of 350.degree. C. and a nebulizer pressure of 60 psi.
The MS spectra were acquired using a fragmentor voltage of 380 V
and a mass range 700 to 3200 m/z in positive ion mode using. MS
data were acquired by the instrument software from 4 to 17
minutes.
[0250] Isolation of Human PBMCs from Blood
[0251] Peripheral blood mononuclear cells (PBMCs) were prepared by
Histopaque density centrifugation from enriched lymphocyte
preparations (huffy coats) obtained from local blood banks or from
fresh blood from healthy human donors. Briefly, blood was diluted
with sterile PBS and carefully layered over a Histopaque gradient
(Sigma, H8889). After centrifugation for 30 minutes at 450.times.g
at room temperature (brake switched off), part of the plasma above
the PBMC containing interphase was discarded. The PBMCs were
transferred into new 50 ml Falcon tubes and tubes were filled up
with PBS to a total volume of 50 ml. The mixture was centrifuged at
room temperature for 10 minutes at 400.times.g (brake switched on),
The supernatant was discarded and the PBMC pellet washed twice with
sterile PBS (centrifugation steps at 4.degree. C. for 10 minutes at
350.times.g). The resulting PBMC population was counted
automatically (ViCell) and stored in RPMI1640 medium, containing
10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree.
C., 5% CO.sub.2 in the incubator until assay start.
[0252] Isolation of Primary Cynomolgus PBMCs from Heparinized
Blood
[0253] Peripheral blood mononuclear cells (PBMCs) were prepared by
density centrifugation from fresh blood from healthy cynomolgus
donors, as follows: Heparinized blood was diluted 1:3 with sterile
PBS, and Lymphoprep medium (Axon Lab #1114545) was diluted to 90%
with sterile PBS. Two volumes of the diluted blood were layered
over one volume of the diluted density gradient and the PBMC
fraction was separated by centrifugation for 30 min at 520.times.g,
without brake, at room temperature. The PBMC band was transferred
into a fresh 50 ml Falcon tube and washed with sterile PBS by
centrifugation for 10 min at 400.times.g at 4.degree. C. One
low-speed centrifugation was performed to remove the platelets (15
min at 150.times.g, 4.degree. C.), and the resulting PBMC
population was automatically counted. (ViCell) and immediately used
for further assays.
EXAMPLES
Example 1: Generation of Anti-BCMA Antibodies
Example 1.1: Production of Antigens and Tool Reagents
Example 1.1.1: Recombinant, Soluble, Human BCMA Extracellular
Domain
[0254] The extracellular domains of human, cynomolgus and murine
BCMA that were used as antigens for phage display selections were
transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA
cells and in vivo site-specifically biotinylated via co-expression
of BirA biotin ligase at the avi-tag recognition sequence located
at the C-terminus of the Fc portion carrying the receptor chain (Fc
knob chain). The extracellular domains of human and cynomolgus BCMA
comprised methionine 4 to asparagine 53, and methionine 4 to
asparagine 52, respectively. These were N-terminally fused to the
hinge of a human IgG1 enabling heterodimerization with an unfused
human IgG1 Fc portion (hole chain) by knobs-into-holes
technology.
Example 1.1.1A: Generation of Anti-BCMA Antibodies by
Maturation
1.1.1A.1 Libraries and Selections
[0255] Two libraries were constructed on basis of antibody 83A10.
These libraries are randomized in either CDR1 and CDR2 of the light
chain (83A10 L1/12) or CDR1 and CDR2 of the heavy chain (83A10
H1412), respectively. Each of these libraries was constructed by 2
subsequent steps of amplification and assembly. Final assembly
products have been digested NcoI/BsiWI for 83A10 L1/L2 library,
Muni and NheI for 83A10 library, alongside with similarly treated
acceptor vectors based on plasmid preparations of clone 83A10. The
following amounts of digested randomized (partial) V-domains and
digested acceptor vector(s) were ligated for the respective
libraries (.mu.g V-domain/.mu.g vector): a.m.83A10 L1/L2 library
(3/10), 83A10 H1/H2 library (3/10), Purified ligations of 83A10
L1/L2 and 83A10 H1/H2 libraries were pooled, respectively, and used
for 15 transformations of E. coli TG1 cells for each of the 2
libraries, to obtain final library sizes of 2.44.times.10.sup.10
for 83A10 L1/L2 library, and 1.4.times.10.sup.10 for a.m.83A10
H1/H2 library. Phagemid particles displaying these Fab libraries
were rescued and purified.
1.1.1A.2 Selections of Clones
[0256] Selections were carried out against the ectodomain of human
or cyno B-cell maturation antigen (BCMA) to which were cloned
upstream a Fc and an avi-tag. Prior to selections, the Fc depleter
was coated onto neutravidin plates at a concentration of 500 nM.
Selections were carried out according to the following pattern:
1) binding of .about.10.sup.12 phagemid particles of library.83A10
L1/L2 library or 83A10 H1/H2 library to immobilized Fc depleter for
1h, 2) transfer of unbound phagemid particles of library.83A10
L1/L2 library or 83.A10 H1/H2 library to 50 nM, 25 nM, 10 nM, or 2
nM human or cyno BCMA (depending on library and selection round)
for 20 min, 3) adding magnetic streptavidin beads for 10 min, 4)
washing of magnetic streptavidin beads using 10.times.1 ml
PBS/Tween.RTM. 20 and 10.times.1 ml PBS, 5) elution of phage
particles by addition of 1 ml 100 mM TEA (triethylamine) for 10 min
and neutralization by addition of 500 ul 1M Tris.RTM./HCl pH 7.4
and 6) re-infection of log-phase E. coli TG1 cells, infection with
helperphage VCSM13 and subsequent PEG/NaCl precipitation of
phagemid particles to be used in subsequent selection rounds.
[0257] Selections have been carried out over 3 rounds and
conditions were adjusted in 5 streamlines for each of the 2
libraries individually. In detail selection parameters were:
Streamline 1 (50 nM huBCMA for round 1, 25 nM cynoBCMA for round 2.
10 nM huBCMA for round 3). Streamline 2 (50 nM huBCMA for round 1,
10 nM huBCMA for round 2. 2 nM huBCMA for round 3), Streamline 3
(50 nM huBCMA for round 1, 25 nM huBCMA for round 2, 1 nM cynoBCMA
for round 3), Streamline 4 (50 nM huBCMA for round 1, 25 nM
cynoBCMA for round 2, 10 nM cynoBCMA for round 3), Streamline 5 (50
nM cynoBCMA for round 1, 25 nM cynoBCMA for round 2, 10 nM cynoBCMA
for round 3).
[0258] The heavy chains of Mab 21, Mab 22, Mab 33, and Mab 42 BCMA
antibodies were derived from Streamline 5 which used only
cynoBCMA.
[0259] 1.1.1A.3 Screening Method
[0260] Individual clones were bacterially expressed as 1 ml
cultures in 96-well format and supernatants were subjected to a
screening by ELISA. Specific binders were defined as signals higher
than 5.times.background for human and cyno BCMA and signals lower
than 3.times. background for Fc depleter. Neutravidin 96 well strip
plates were coated with 10 nM of huBCMA. 10 nM cyBCMA or 50 nM
Fc-depleter followed by addition of Fab-containing bacterial
supernatants and detection of specifically binding Fabs via their
Flag-tags by using an anti-Flag/HRP secondary antibody.
ELISA-positive clones were bacterially expressed as 1 ml cultures
in 96-well format and supernatants were subjected to a kinetic
screening experiment ProteOn. 500 positive clones were identified,
most of them having similar affinity.
[0261] 1.1.1A.4 Surface Plasmon Resonance Screen with Soluble Fabs
and IgGs
[0262] 70 clones were further tested by SPR. All experiments were
performed at 25.degree. C. using PBST as running buffer (10 mM PBS.
pH 7.4 and 0.005% (v/v) Tween.RTM.20). A ProteOn XPR36 biosensor
equipped with GLC and GLM sensor chips and coupling reagents (10 mM
sodium acetate, pH 4.5, sulfo-N-hydroxysuccinimide,
1-ethyl-3-(3-dimethylaminpropyl)-carbodiimide hydrochloride [EDC]
and ethanolamine) was purchased from BioRad Inc. (Hercules,
Calif.). Immobilizations were performed at 30 .mu.l/min on a GLM
chip. pAb (goat) anti hu IgG, F(ab)2 specific Ab (Jackson) was
coupled in vertical direction using a standard amine-coupling
procedure: all six ligand channels were activated for 5 min with a
mixture of EDC (200 mM) and sulfo-NHS (50 mM). Immediately after
the surfaces were activated, pAb (goat) anti hu IgG, F(ab)2
specific antibody (50 .mu.g/ml, 10 mM sodium acetate, pH 5) was
injected across all six channels for 5 min. Finally, channels were
blocked with a 5 min injection of 1 M ethanolamine-HCl (pH 8.5).
Final immobilization levels were similar on all channels, ranging
from 11000 to 11500 RU. The Fab variants were captured from E. coli
supernantants by simultaneous injection along five of the separate
whole horizontal channels (30 .mu.l/min) for 5 min and resulted in
levels, ranging from 200 to 900 RU, depending on the concentration
of Fab in supernatant; conditioned medium was injected along the
sixth channel to provide an `in-line` blank for double referencing
purposes. One-shot kinetic measurements were performed by injection
of a dilution series of human, cyno and mouse BCMA (50, 10, 2, 0.4,
0.08, 0 nM. 50 .mu.l/min) for 3 min along the vertical channels.
Dissociation was monitored for 5 min. Kinetic data were analyzed in
ProteOn Manager v. 2.1. Processing of the reaction spot data
involved applying an interspot-reference and a double-reference
step using an inline buffer blank (Myszka, 1999). The processed
data from replicate one-shot injections were fit to a simple 1:1
Langmuir binding model without mass transport (O'Shannessy et al.,
1993).
[0263] For measurements of IgG from supernatants of HEK productions
in 6-well format, the IgG variants were captured from HEK293
supernatants by simultaneous injection along five of the separate
whole horizontal channels (30 .mu.l/min) for 5 min and resulted in
levels, ranging from 200 to 400 RU: conditioned medium was injected
along the sixth channel to provide an `in-line` blank for double
referencing purposes. One-shot kinetic measurements were performed
by injection of a dilution series of human, cyno and mouse BCMA
(25, 5, 1, 0.2, 0.04, 0 nM, 50 .mu.l/min) for 3 min along the
vertical channels. Dissociation was monitored for 5 min. Kinetic
data were analyzed as described above. The OSK measurements are
summarized in Table 2D; i/m, inconclusive measurement. Affinity to
huBCMA was found to be between about 50 pm to 5 nM. Affinity to
cynoBCMA was found to be between about 2 nM to 20 nM (few clones
fall outside the range, see FIG. 17).
[0264] 1.1.1A5. Further Selection of HC and LC Clones
[0265] Due to their experience the inventors selected out of these
70 clones further 27 clones based on their binding properties to
huBCMA, cynoBCMA, murineBCMA, and ratio, measured in different
assays. Out of these clones 4VH and 9VL clones were selected, which
results in 34 VH/VL combinations. Binding affinity on HEK-huBCMA
cells was measured (FIG. 18 and Table 2E). It was found that
binding of antibodies Mab 21, Mab 22, Mab 27, Mab 39 and Mab 42 to
huBCMA on HEK cells was not significantly better than the binding
of Mab 83A10 to huBCMA-HEK cells. However Mab21, Mab 22, Mab27,
Mab33, Mab39, and Mab42 were selected due to their overall
properties, like affinity for huBCMA, cynoBCMA, binding as
bispecific antibody to BCMA-positive multiple myeloma cell lines
H929, L363 and RPMI-8226 by flow cytometry, killing potency of
myeloma cells H929, L363 and RPMI-8226, of viable myeloma plasma
cells from patient bone marrow aspirates, and pharmacokinetics
(PK)) and pharmacodynamics (killing of BCMA positive cells) data in
cynomolgus monkeys.
TABLE-US-00006 TABLE 2C Relationship of antibodies to streamlines
Derived from Derived from Mab No. library 2 (HC) Clone HC library 1
(LC) Clone LC Mab 21 Streamline 5 5F04 Streamline 1 1D04 Mab 22
Streamline 5 5F04 Streamline 1 1C05 Mab 27 Streamline 1 1A08
Streamline 1 1D04 Mab 33 Streamline 5 5D03 Streamline 1 1D04 Mab 39
Streamline 2 2E12 Streamline 1 1D04 Mab 42 Streamline 5 5F04
Streamline 5 5A11
TABLE-US-00007 TABLE 2D One-shot-kinetic affinity measurements to
human, cynomolgus and mouse BCMA Mab KD KD KD No. VH VL huBCMA
cyBCMA muBCMA 83A10 pCON1532 pCON1080 1.5E-09 1.4E-08 i/m Mab 21
pCON1531 pCON1522 2.8E-11 5.1E-11 7.3E-10 Mab 22 pCON1531 pCON1521
4.8E-11 i/m 9.0E-10 Mab 27 pCON1520 pCON1522 3.9E-13 1.0E-10
9.7E-10 Mab 33 pCON1530 pCON1522 1.7E-11 3.4E-11 4.9E-10 Mab 39
pCON1524 pCON1522 6.2E-11 2.7E-10 i/m Mab 42 pCON1531 pCON1527
2.3E-10 3.9E-10 2.5E-09
TABLE-US-00008 TABLE 2E Binding of IgG variants on HEK-huBCMA cells
Binding Binding EC50 Mab No VH VL EC50 [nM] [.mu.g/mL] 83A10
PCON1532 PCON1080 2.4 0.34 Mab 14 PCON1530 PCON1527 1.47 0.21 Mab
21 pCON1531 PCON1522 2.46 0.35 Mab 22 PCON1531 pCON1521 2.08 0.30
Mab 23 PCON1531 PCON1519 4.97 0.71 Mab 27 PCON1520 PCOM1522 10.57
1.52 Mab 28 PCON1520 PCOM1521 11.34 1.63 Mab 30 PCON1530 PCON1526
10.35 1.49 Mab 31 PCON1530 PCON1525 1.34 0.19 Mab 33 pCOM1530
PCON1522 1.18 0.17 Mab 34 PCON1530 PCON1521 1.24 0.18 Mab 35
PCON1530 PCON1519 1.63 0.23 Mab 39 PCON1524 PCON1522 1.73 0.25 Mab
42 PCON1531 pCON1527 2.10 0.30 Mab 44 PCON1520 PCON1527 1.55
0.22
Example 1.2: BCMA-Expressing Cells as Tools
Example 1.2.1: Human Myeloma Cell Lines Expressing BCMA on their
Surface and Quantification of BCMA Receptor Number on Cell
Surface
[0266] BCMA expression was assessed on five human myeloma cell
lines (NCI-H929, RPMI-8226, U266B1, L-363 and JJN-3) by flow
cytometry. NCI-H929 cells ((H929) ATCC.RTM. CRL-9068.TM.) were
cultured in 80-90% RPMI 1640 with 10-20% heat-inactivated FCS and
could contain 2 mM L-glutamine, 1 mM sodium pyruvate and 50 .mu.M
mercaptoethanol. RPMI-8226 cells ((RPMI) ATCC.RTM. CCL-155.TM.)
were cultured in a media containing 90% RPMI 1640 and 10%
heat-inactivated FCS. U266B1 ((U266) ATCC.RTM. TIB-196.TM.) cells
were cultured in RPMI-1640 medium modified to contain 2 mM
L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose,
and 1500 mg/L sodium bicarbonate and 15% heat-inactivated FCS.
L-363 cell line (Leibniz Institute DSMZ--German collection of
microorganisms and cell cultures; DSMZ No. ACC 49) was cultured in
85% RPMI 1640 and 15% heat-inactivated FCS. JJN-3 cell line (DSMZ
No. ACC 541) was cultured in 40% Dulbecco's MEM+40% Iscove's
MDM+20% heat-inactivated FBS. Briefly, cells were harvested,
washed, counted for viability, resuspended at 50,000 cells/well of
a 96-well round bottom plate and incubated with anti-human BCMA
antibody (Abcam, # ab54834, mouse IgG1) at 10 .mu.g/ml for 30 min
at 4.degree. C. (to prevent internalization). A mouse IgG1 was used
as isotype control (BD Biosciences, #554121). Cells were then
centrifuged (5 min at 350.times.g), washed twice and incubated with
the FITC-conjugated anti mouse secondary antibody for 30 min at
4.degree. C. At the end of incubation time, cells were centrifuged
(5 min at 350.times.g), washed twice with FACS buffer, resuspended
in 100 ul FACS buffer and analyzed on a Cantoll device running FACS
Diva software. The relative quantification of BCMA receptor number
on the surface membrane of H929, RPMI-8226 and U266B1 myeloma cell
lines was assessed by QIFIKIT analysis (Dako, # K0078, following
manufacturer's instructions). H929 cells expressed human BCMA with
the highest density, up to 5-6-fold higher more than other myeloma
cell lines. H929 is considered as a high BCMA-expressing myeloma
cell line as compared to U266 and L363 which are medium/low
BCMA-expressing myeloma cells, RPMI-8226 which are low
BCMA-expressing myeloma cells and JJN-3 which are very low
BCMA-expressing myeloma cells. Table 3 summarizes the relative BCMA
receptor number on the cell surface of human multiple myeloma cell
lines per each experiment (n=5).
TABLE-US-00009 TABLE 3 Quantification of BCMA receptor number on
membrane surface of H929, L363, RPMI-8226, U266B1 and JJN-3 human
myeloma cell lines Human Specific antigen binding capacity (SABC)
myeloma Exper- Exper- Exper- Exper- Exper- cell lines iment 1 iment
2 iment 3 iment 4 iment 5 H929 19357 54981 44800 100353 98050 L363
16,970 / 11300 11228 / U266(B1) / 12852 11757 / 9030 RPMI-8226 1165
5461 / 11361 2072 JJN-3 / / / / 650
Example 2: BCMA Binding Assays: Surface Plasmon Resonance
[0267] Assessment of binding of anti-BCMA antibodies to recombinant
BCMA by surface plasmon resonance (SPR) as follow. All SPR
experiments were performed on a Biacore T200 at 25.degree. C. with
HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM
EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). The
avidity of the interaction between anti-BCMA antibodies and
recombinant BCMA Fc(kih) (human and cynomolgus) was determined.
Biotinylated recombinant human and cynomolgus BCMA Fc(kih) were
directly coupled on a SA chip following instructions (Biacore,
Freiburg/Germany). The immobilization level ranged from 200 to 700
RU. The anti-BCMA antibodies were passed at a 2-fold concentration
range (1.95 to 500 nM) with a flow of 30 .mu.L/minutes through the
flow cells over 120 seconds. The dissociation was monitored for 180
seconds. Bulk refractive index differences were corrected for by
subtracting the response obtained on the reference flow cell. Here,
the anti-BCMA antibodies were flown over an empty surface
previously activated and deactivated as described in the standard
amine coupling kit. Apparent kinetic constants were derived using
the Biacore T200 Evaluation Software (vAA, Biacore AB,
Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding by
numerical integration, despite the bivalency of the interaction for
comparison purposes. The affinity of the interaction between
anti-BCMA antibodies and recombinant human BCMA Fc(kih) was also
determined. Anti-human Fab antibody (GE Healthcare) was directly
coupled on a CM5 chip at pH 5.0 using the standard amine coupling
kit (Biacore, Freiburg/Germany). The immobilization level was about
6500 RU. Anti-BCMA antibody was captured for 90 seconds at 25 nM.
Recombinant human BCMA Fc(kih) was passed at a 4-fold concentration
range (1.95 to 500 nM) with a flow of 30 .mu.J/minutes through the
flow cells over 120 seconds. The dissociation was monitored for 120
seconds. Bulk refractive index differences were corrected for by
subtracting the response obtained on reference flow cell. Here,
recombinant BCMA was flown over a surface with immobilized
anti-human Fab antibody but on which HBS-EP has been injected
rather than anti-BCMA antibody. Kinetic constants were derived
using the Biacore T100 Evaluation Software (vAA, Biacore AB,
Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding by
numerical integration (Table 4).
TABLE-US-00010 TABLE 4 Affinity constants determined by fitting
rate equations for 1:1 Langmuir binding Ligand Analyte Kon[1/Ms]
Koff[1/s] KD[M] 83A10 IgG huBCMA Fc(kih) 5.07E+05 2.92E-03 5.76E-09
cynoBCMA Fc(kih) 2.29E+05 2.03E-02 8.86E-08 Mab 21 IgG huBCMA
Fc(kih) 8.51E+05 4.39E-05 5.16E-11 cynoBCMA Fc(kih) 4.91E+05
2.35E-04 4.78E-10 Mab 22 IgG huBCMA Fc(kih) 8.14E+05 5.15E-05
6.33E-11 cynoBCMA Fc(kih) 4.54E+05 4.42E-04 9.74E-10 Mab 42 IgG
huBCMA Fc(kih) 8.03E+05 2.98E-04 3.71E-10 cynoBCMA Fc(kih) 7.07E+05
4.53E-04 6.41E-10 Mab 27 IgG huBCMA Fc(kih) 3.59E+05 5.93E-05
1.65E-10 cynoBCMA Fc(kih) 2.16E+05 4.55E-04 2.11E-09 Mab 33 IgG
huBCMA Fc(kih) 2.00E+05 3.55E-05 1.78E-10 cynoBCMA Fc(kih) 1.32E+05
9.76E-05 7.39E-10 Mab 39 IgG huBCMA Fc(kih) 3.61E+05 5.58E-05
1.55E-10 cynoBCMA Fc(kih) 2.15E+05 4.67E-04 2.17E-09
Example 3: Human/Cynomolgus (Hu/Cyno) Affinity Gap
[0268] Based on the affinity values described in Example 2, the
affinity of anti-BCMA antibodies to human BCMA vs. cynomolgus BCMA
were compared and cyno/hu affinity ratio (gap) values were
calculated (Table 5). Affinity cyno/hu gap was calculated as
affinity of antibody to cynomolgus BCMA divided by affinity to
human BCMA and means that BCMA antibody binds to human BCMA with x
fold binding affinity than to cynomolgus BCMA, where x=cyno/hu gap
value. Results are shown in Table 5.
TABLE-US-00011 TABLE 5 Affinity of anti-BCMA antibodies to human
BCMA vs. cynomolgus BCMA and hu/cyno gap values K.sub.D human KD
cynomolgus Affinity .alpha.-BCMA IgG BCMA[M] BCMA[M] cyno/hu gap
83A10 5.76E-09 8.86E-08 15.3 Mab 21 5.16E-11 4.78E-10 9.3 Mab 22
6.33E-11 9.74E-10 15.4 Mab 42 3.71E-10 6.41E-10 1.7 Mab 27 1.65E-10
2.11E-09 12.7 Mab 33 1.78E-10 7.39E-10 4.2 Mab 39 1.55E-10 2.17E-09
14
Example 4: Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific
Antibodies
[0269] Anti-BCMA/anti-CD3 T cell bispecific antibodies were
generated according to WO2014/122144, which is incorporated by
reference.
Example 4.1: Anti-CD3 Antibodies
[0270] The term "CD3.epsilon. or CD3" as used herein relates to
human CD3.epsilon. described under UniProt P07766
(CD3.epsilon._HUMAN). The term "antibody against CD3, anti CD3
antibody" relates to an antibody binding to CD3.epsilon..
Preferably the antibody comprises a variable domain VH comprising
the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy
chain CDR1. CDR2 and CDR3 and a variable domain VL comprising the
light chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light
chain CDR1. CDR2 and CDR3. Preferably the antibody comprises the
variable domains of SEQ ID NO:7 (VH) and SEQ ID NO:8 (VL). Anti-CD3
antibody as described above was used to generate the T cell
bispecific antibodies which were used in the following
examples.
Example 4.2: Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific
Antibodies of Fc-Containing 2+1 Format
[0271] cDNAs encoding the full heavy and light chains of the
corresponding anti-BCMA IgG1 antibodies as well as the anti-CD3 VH
and VL cDNAs were used as the starting materials. For each
bispecific antibody, four protein chains were involved comprising
the heavy and light chains of the corresponding anti-BCMA antibody
and the heavy and light chains of the anti-CD3 antibody described
above, respectively. In order to minimize the formation of
side-products with mispaired heavy chains, for example with two
heavy chains of the anti-CD3 antibody, a mutated heterodimeric Fc
region is used carrying "knob-into-hole mutations" and an
engineered disulphide bond, as described in WO2009080251 and in
WO2009080252. In order to minimize the formation of side-products
with mispaired light chains, for example with two light chains of
the anti-BCMA antibody, a CH1.times. constant kappa crossover is
applied to the heavy and light chains of the anti-CD3 antibody
using the methodology described in WO2009080251 and in
WO2009080252.
[0272] a) An anti-BCMA/anti-CD3 T cell bispecific antibody with a
2+1 format i.e. bispecific (Fab).sub.2.times.(Fab) antibody that is
bivalent for BCMA and monovalent for CD3 would have advantages on
potency, predictability for efficacy and safety because it would
preferentially bind to the tumor target BCMA and avoid CD3 antibody
sink, thus higher probability for drug exposure focused to the
tumor.
[0273] Anti-BCMA/anti-CD3 T cell bispecific of the 2+1 format (i.e.
bispecific (Fab).sub.2.times.(Fab) antibody bivalent for BCMA and
monovalent for CD3 with Fc were produced for the human BCMA
antibodies previously selected. cDNAs encoding the full Fabs (heavy
chain VH and CH1 domains plus light chain VL and CL domains) of the
corresponding anti-BCMA IgG1 antibodies as well as the anti-CD3 VH
and VL cDNAs, were used as the starting materials. For each
bispecific antibody, four protein chains were involved comprising
the heavy and light chains of the corresponding anti-BCMA antibody
and the heavy and light chains of the anti-CD3 antibody described
above, respectively, with Fc regions.
[0274] Briefly, each bispecific antibody is produced by
simultaneous cotransfection of four mammalian expression vectors
encoding, respectively: a) the full light chain cDNA of the
corresponding BCMA antibody, b) a fusion cDNA generated by standard
molecular biology methods, such as splice-overlap-extension PCR,
encoding a fusion protein made of (in N- to C-terminal order)
secretory leader sequence, Fab (VH followed by CH1 domains) of the
corresponding anti-BCMA antibody described above, a flexible
glycine(Gly)-serine(Ser) linker with the sequence
Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, Fab (VH followed by CH1
domains) of the corresponding anti-BCMA antibody described above, a
flexible glycine(Gly)-serine(Ser) linker with the sequence
Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, the VH of the anti-CD3
antibody described above and the constant kappa domain of a human
light chain cDNA, c) a fusion cDNA generated by standard molecular
biology methods, such as splice-overlap-extension PC, encoding a
fusion protein made of (in N- to C-terminal order) secretory leader
sequence, VL of the anti-CD3 antibody described above, constant CH1
domain of a human IgG1 cDNA. Co-transfection of mammalian cells and
antibody production and purification using the methods described
above for production of human or humanized IgG1 antibodies, with
one modification: for purification of antibodies, the first capture
step is not done using ProteinA, but instead is done using an
affinity chromatography column packed with a resin binding to human
kappa light chain constant region, such as KappaSelect (GE
Healthcare Life Sciences). In addition, a disulfide can be included
to increase the stability and yields as well as additional residues
forming ionic bridges and increasing the heterodimerization yields
(EP 1870459A1).
[0275] For the generation of BCMAxCD3 bispecific antibody vectors,
the IgG1 derived bispecific molecules consist at least of two
antigen binding moieties capable of binding specifically to two
distinct antigenic determinants CD3 and BCMA. The antigen binding
moieties were Fab fragments composed of a heavy and a light chain,
each comprising a variable and a constant region. At least one of
the Fab fragments was a "Crossfab" fragment, wherein the constant
domains of the Fab heavy and light chain were exchanged. The
exchange of heavy and light chain constant domains within the Fab
fragment assures that Fab fragments of different specificity do not
have identical domain arrangements and consequently do not
interchange light chains. The bispecific molecule design was
monovalent for CD3 and bivalent for BCMA where one Fab fragment is
fused to the N-terminus of the inner CrossFab (2+1). The bispecific
molecule contained an Fc part in order to have a longer half-life.
A schematic representation of the constructs is given in FIGS. 1-3;
the sequences of the preferred constructs are shown in Table 2A.
The molecules were produced by co-transfecting HEK293 EBNA cells
growing in suspension with the mammalian expression vectors using
polymer-based solution. For preparation of 2+1 CrossFab-IgG
constructs, cells were transfected with the corresponding
expression vectors in a 1:2:1:1 ratio ("vector Fc(knob)":"vector
light chain":"vector light chain CrossFab":"vector heavy
chain-CrossFab").
Example 4.3: Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific
Antibodies for Comparison
[0276] The generation of BCMA50-sc(Fv).sub.2 (also known as
BCMA50-BiTE.RTM.) anti-BCMA/anti-CD3 T cell bispecific antibody and
the amino acid sequences used were according to WO2013072406 and
WO2013072415.
Example 5: Production and Purification of Anti-BCMA/Anti-CD3
Fc-Containing (2+1) T Cell Bispecific Antibodies with Charge
Variants
[0277] Anti-BCMA/anti-CD3 T cell bispecific antibodies were
produced and purified according to WO2014/122144, which is
incorporated by reference.
[0278] For the production of the bispecific antibodies, bispecific
antibodies were expressed by transient co-transfection of the
respective mammalian expression vectors in HEK293-EBNA cells, which
were cultivated in suspension, using polymer-based solution. One
day prior to transfection the HEK293-EBNA cells were seeded at 1.5
Mio viable cells/mL in Ex-Cell medium, supplemented with 6 mM of
L-Glutamine. For every mL of final production volume 2.0 Mio viable
cells were centrifuged (5 minutes at 210.times.g). The supernatant
was aspirated and the cells resuspended in 100 .mu.L of CD CHO
medium. The DNA for every mL of final production volume was
prepared by mixing 1 .mu.g of DNA (Ratio heavy chain:modified heavy
chain:light chain:modified light chain=1:1:2:1) in 100 .mu.L of CD
CHO medium. After addition of 0.27 .mu.L of polymer-based solution
(1 mg/mL) the mixture was vortexed for 15 seconds and left at room
temperature for 10 minutes. After 10 minutes, the resuspended cells
and DNA/polymer-based solution mixture were put together and then
transferred into an appropriate container which was placed in a
shaking device (37.degree. C., 5% CO.sub.2). After a 3 hours
incubation time 800 .mu.L of Ex-Cell Medium, supplemented with 6 mM
L-Glutamine, 1.25 mM valproic acid and 12.5% Pepsoy (50 g/L), was
added for every mL of final Production volume. After 24 hours, 70
.mu.L of feed solution was added for every mL of final production
volume. After 7 days or when the cell viability was equal or lower
than 70%, the cells were separated from the supernatant by
centrifugation and sterile filtration. The antibodies were purified
by an affinity step and one or two polishing steps, being cation
exchange chromatography and size exclusion chromatography. When
required, an additional polishing step was used.
[0279] For the affinity step the supernatant was loaded on a
protein A column (HiTrap Protein A FF, 5 mL, GE Healthcare)
equilibrated with 6 CV 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. After a washing step with the same buffer the
antibody was eluted from the column by step elution with 20 mM
sodium phosphate, 100 mM sodium chloride, 100 mM Glycine, pH 3.0.
The fractions with the desired antibody were immediately
neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooled and
concentrated by centrifugation. The concentrate was sterile
filtered and processed further by cation exchange chromatography
and/or size exclusion chromatography.
[0280] For the cation exchange chromatography step the concentrated
protein was diluted 1:10 with the elution buffer used for the
affinity step and loaded onto a cation exchange colume (Poros 50
HS, Applied Biosystems). After two washing steps with the
equilibration buffer and a washing buffer resp. 20 mM sodium
phosphate, 20 mM sodium citrate, 20 mM TRIS, pH 5.0 and 20 mM
sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium
chloride pH 5.0 the protein was eluted with a gradient using 20 mM
sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium
chloride pH 8.5. The fractions containing the desired antibody were
pooled, concentrated by centrifugation, sterile filtered and
processed further a size exclusion step.
[0281] For the size exclusion step the concentrated protein was
injected in a XK16/60 HiLoad Superdex 200 column (GE Healthcare),
and 20 mM Histidine, 140 mM Sodium Chloride, pH 6.0 with or without
Tween20 as formulation buffer. The fractions containing the
monomers were pooled, concentrated by centrifugation and sterile
filtered into a sterile vial.
[0282] Determination of the antibody concentration was done by
measurement of the absorbance at 280 nm, using the theoretical
value of the absorbance of a 0.1% solution of the antibody. This
value was based on the amino acid sequence and calculated by GPMAW
software (Lighthouse data).
[0283] Purity and monomer content of the final protein preparation
was determined by CE-SDS (Caliper LabChip GXII system (Caliper Life
Sciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusion
column (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium
chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium
azide, pH 6.7 buffer.
[0284] To verify the molecular weight of the final protein
preparations and confirm the homogeneous preparation of the
molecules final protein solution, liquid chromatography-mass
spectometry (LC-MS) was used. A deglycosylation step was first
performed. To remove heterogeneity introduced by carbohydrates, the
constructs were treated with PNGaseF (ProZyme). Therefore, the pH
of the protein solution was adjusted to pH7.0 by adding 2 .mu.l 2 M
Tris to 20 .mu.g protein with a concentration of 0.5 mg/ml. 0.8
.mu.g PNGaseF was added and incubated for 12 h at 37.degree. C. The
LC-MS online detection was then performed. LC-MS method was
performed on an Agilent HPLC 1200 coupled to a TOF 6441 mass
spectrometer (Agilent). The chromatographic separation was
performed on a Macherey Nagel Polysterene column; RP1000-8 (8 .mu.m
particle size, 4.6.times.250 mm; cat. No. 719510). Eluent A was 5%
acetonitrile and 0.05% (v/v) formic acid in water, eluent B was 95%
acetonitrile, 5% water and 0.05% formic acid. The flow rate was 1
ml/min, the separation was performed at 40.degree. C. and 6 .mu.g
(15 .mu.l) of a protein sample obtained with a treatment as
described before.
[0285] During the first 4 minutes, the eluate was directed into the
waste to protect the mass spectrometer from salt contamination. The
ESI-source was running with a drying gas flow of 12 l/min, a
temperature of 350.degree. C. and a nebulizer pressure of 60 psi.
The MS spectra were acquired using a fragmentor voltage of 380 V
and a mass range 700 to 3200 m/z in positive ion mode using. MS
data were acquired by the instrument software from 4 to 17
minutes.
[0286] FIG. 10 of EP 14179705 (incorporated by reference) depicts
the CE-SDS (non-reduced) graphs of the final protein preparations
after different methods of purification for 83A10-TCB and
83A10-TCBcv antibodies. Protein A (PA) affinity chromatography and
size exclusion chromatographic (SEC) purification steps applied to
83A10-TCB antibody resulted in a purity of <30% and 82.8% of
monomer content (A). When additional purifications steps including
cation exchange chromatography (cIEX) and a final size exclusion
chromatographic (re-SEC) steps were applied to the final protein
preparations in (A), the purity was increased to 93.4% but the
monomer content remained the same and the yield was significantly
reduced to 0.42 mg/L. However, when specific charge modifications
were applied to 83A10 anti-BCMA Fab CL-CH1, namely 83A10-TCBcv
antibody, a superior production/purification profile of the TCB
molecule, as demonstrated by a purity of 95.3%, monomer content of
100% and yield of up to 3.3 mg/L, could already be observed even
when PA+cIEX+SEC purification steps were applied (C) in comparison
to (B) with a production/purification profile showing a 7.9-fold
lower yield and 17.2% lower monomer content despite including an
additional re-SEC purification step.
[0287] A head-to-head production run to compare the
production/purification profile of 83A10-TCB vs. 83A10-TCBcv
antibodies was then conducted to further evaluate the advantages of
the CL-CH1 charge modifications applied to the antibodies.
83A10-TCB and 83A10-TCBcv molecules are both of molecular format as
described in FIG. 2a. As depicted in FIG. 11, properties of
83A10-TCB and 83A10-TCBcv antibodies were measured side-by-side and
compared after each purification steps 1) PA affinity
chromatography only (A, B), 2) PA affinity chromatography then SEC
(C, D) and 3) PA affinity chromatography then SEC then cIEX and
re-SEC (E, F). The CE-SDS (non-reduced) graphs of the final protein
solutions after the respective methods of purification for
83A10-TCB and 83A10-TCBcv antibodies are demonstrated in FIG. 11 of
EP14179705 (incorporated by reference). As shown in FIGS. 11A and
11B of EP14179705 (incorporated by reference), improvements with
applying the charge variants to the TCB antibody were already
observed after purification by PA affinity chromatography only. In
this head-to-head study, PA affinity chromatography purification
step applied to 83A10-TCB antibody resulted in a purity of 61.3%, a
yield of 26.2 mg/L and 63.7% of monomer content (11A). In
comparison, when 83A10-TCBcv antibody was purified by PA affinity
chromatography all the properties were improved with a better
purity of 81.0%, a better yield of 51.5 mg/L and 68.2% of monomer
content (11B). When an additional SEC purification step was applied
to the final protein preparations as seen in FIGS. 12A and 12B of
EP14179705 (incorporated by reference), 83A10-TCB gained a purity
of 69.5%, a yield of 14.1 mg/L and 74.7% of monomer content (C) as
compared to 83A10-TCBcv with improved purity and monomer content of
up to 91.0% and 83.9% respectively, and a yield of 10.3 mg/L (D).
Even though the yield was slightly less (i.e. 27% less) for
83A10-TCBcv than for 83A10-TCB in this particular experiment, the
percentage of correct molecule was much better for 83A10-TCBcv than
for 83A10-TCB, respectively 90% vs. 40-60%, as measured by LC-MS.
In the third head-to-head comparison, 83A10-TCB and 83A10-TCBcv
final protein preparations from FIGS. 11C and 11D of EP14179705
(incorporated by reference) were pooled with approximately 1 L
(equivolume) of respective final protein preparations from another
purification batch (same production) following PA affinity
chromatography purification step only. The pooled protein
preparations were then being further purified by cIEX and SEC
purification methods. As depicted in FIGS. 11E and 11F of
EP14179705 (incorporated by reference), improvement of the
production/purification profile of the TCB antibody with the charge
variants was consistently observed when compared to TCB antibody
without charge variant. After several steps of purification methods
(i.e. PA+/-SEC+cIEX+SEC) were used to purify 83A10-TCB antibody,
only 43.1% purity was reached and 98.3% of monomer content could be
achieved but to the detriment of the yield which was reduced to
0.43 mg/L. The percentage of correct molecule as measured by LC-MS
was still poor with 60-70%. At the end, the quality of the final
protein preparation was not acceptable for in vitro use. In stark
contrast, when the same multiple purification steps with the same
chronology were applied to 83A10-TCBcv antibody, 96.2% purity and
98.9% of monomer content were reached as well as 95% of correct
molecule as measured by LC-MS. The yield however was also greatly
reduced to 0.64 mg/L after cIEX purification step. The results show
that better purity, higher monomer content, higher percentage of
correct molecule and better yield can be achieved with 83A10-TCBcv
antibody only after two standard purification steps i.e. PA
affinity chromatography and SEC (FIG. 11D of EP14179705) while such
properties could not be achieved with 83A10-TCB even when
additional purification steps were applied (FIG. 11E of
EP14179705). Table 12 of EP14179705 (incorporated by reference)
summarizes the properties of 83A10-TCB as compared to 83A10-TCVcv
following PA purification step. Table 13 of EP14179705
(incorporated by reference) summarizes the properties of 83A10-TCB
as compared to 83A10-TCVcv following PA and SEC purification steps.
Table 14 of EP14179705 (incorporated by reference) summarizes the
properties of 83A10-TCB as compared to 83A10-TCVcv following PA and
SEC plus PA alone then cIEX and re-SEC purification steps. For
Tables 12 to 14 of EP14179705 (incorporated by reference), the
values in bold highlight the superior property as compared between
83A10-TCB vs. 83A10-TCVcv. With one exception (i.e. yield
respectively amount, see Table 13 of EP14179705 (incorporated by
reference)) which may not be representative, all the
production/purification parameters and values resulting from the 3
head-to-head comparison experiments were superior for 83A10-TCBcv
as compared to 83A10-TCB. The overall results clearly demonstrate
that advantages in production/purification features could be
achieved with applying CL-CH1 charge modifications to TCB
antibodies and that only two purification steps (i.e PA affinity
chromatography and SEC) were required to achieve already high
quality protein preparations with very good developability
properties. Based on the improved production/purification
properties of 83A10-TCBcv, 21-TCBcv, 22-TCBcv, 27-TCBcv, 33-TCBcv,
39-TCBcv and 42-TCBcv were generated with charge variants, in a
similar way as 83A10-TCBcv.
TABLE-US-00012 TABLE 6 Production/purification profile of
anti-BCMA/anti- CD3 T cell bispecific antibodies following protein
A affinity chromatography purification step 83A10-TCB 83A10-TCBcv
Purity (%) 61.3 81.0 Yield (mg/L) 26.2 51.5 Amount (mg) 24.3 50.2
Monomer (%) 63.7 68.2 Correct molecule by n.d. n.d LC-MS (%)
TABLE-US-00013 TABLE 7 Production/purification profile of
anti-BCMA/anti-CD3 T cell bispecific antibodies following protein A
affinity chromatography and size exclusion chromatography
purification steps 83A10-TCB 83A10-TCBcv Purity (%) 69.5 91.0 Yield
(mg/L) 14.1 10.3 Amount (mg) 13.1 10.0 Monomer (%) 74.7 83.9
Correct molecule by 40-60 90 LC-MS (%)
TABLE-US-00014 TABLE 8 Production/purification profile of
anti-BCMA/anti-CD3 T cell bispecific antibodies following 1.a)
protein A affinity chromatography and size exclusion chromatography
and 1.b) protein A affinity chromatography only pooled together
then 2) cation exchange chromatography and 3) final size exclusion
chromatography purification steps 83A10-TCB 83A10-TCBcv Purity (%)
43.1 96.2 Yield (mg/L) 0.43 0.64 Amount (mg) 0.73 1.27 Monomer (%)
98.3 98.9 Correct molecule by 60-70% >95% LC-MS (%)
Example 6: Binding of Anti-BCMA/Anti-CD3 T-Cell Bispecific
Antibodies to BCMA-Positive Multiple Myeloma Cell Lines (Flow
Cytometry)
[0288] Anti-BCMA/anti-CD3 TCB antibodies (21-TCBcv, 22-TCBcv,
42-TCBcv, 83A10-TCBcv) were analyzed by flow cytometry for binding
to human BCMA on BCMA-expressing H929, L363 and RPMI-8226 cells.
MKN45 (human gastric adenocarcinoma cell line that does not express
BCMA) was used as negative control. Briefly, cultured cells are
harvested, counted and cell viability was evaluated using ViCell.
Viable cells are then adjusted to 2.times.10.sup.6 cells per ml in
BSA-containing FACS Stain Buffer (BD Biosciences). 100 .mu.l of
this cell suspension were further aliquoted per well into a
round-bottom 96-well plate and incubated with 30 .mu.l of the
anti-BCMA antibodies or corresponding IgG control for 30 min at
4.sup.0c. All Anti-BCMA/anti-CD3 TCB antibodies (and TCB controls)
were titrated and analyzed in final concentration range between
1-300 nM. Cells were then centrifuged (5 min, 350.times.g), washed
with 120 .mu.l/well FACS Stain Buffer (BD Biosciences), resuspended
and incubated for an additional 30 min at 4.degree. C. with
fluorochrome-conjugated PE-conjugated AffiniPure F(ab')2 Fragment
goat anti-human IgG Fc Fragment Specific (Jackson Immuno Research
Lab; 109-116-170). Cells were then washed twice with Stain Buffer
(BD Biosciences), fixed using 100 ul BD Fixation buffer per well (#
BD Biosciences, 554655) at 4.degree. C. for 20 min, resuspended in
120 .mu.l FACS buffer and analyzed using BD FACS CantoII. When
applicable, EC50 were calculated using Prism GraphPad (LaJolla,
Calif., USA) and EC50 values denoting the antibody concentration
required to reaching 50% of the maximal binding for the binding of
anti-BCMA/anti-CD3 TCB antibodies to H929 cells, L363 cells and
RPMI-8226 cells are summarized in Table 8, Table 9, and Table 10
respectively. Asterix denotes estimated EC50 values as extrapolated
and calculated by Prism software. EC50 values for binding of
21-TCBcv to L363 cells and binding of 22-TCBcv to RPMI-8226 cells
could not be estimated
TABLE-US-00015 TABLE 8 EC50 values for binding of
anti-BCMA/anti-CD3 T-cell bispecific antibodies to H929 multiple
myeloma cells Estimated EC50 83A10-TCBcv 21-TCBcv 22-TCBcv 42-TCBcv
nM 12.0 11.0 7.9 13.6 .mu.g/ml 1.725 1.589 1.142 1.956
TABLE-US-00016 TABLE 9 EC50 values for binding of
anti-BCMA/anti-CD3 T-cell bispecific antibodies to L363 multiple
myeloma cells Estimated EC50 83A10-TCBcv 21-TCBcv 22-TCBcv 42-TCBcv
nM 17.4 / 30.0 3.8 .mu.g/ml 2.507 / 4.328 0.5534
TABLE-US-00017 TABLE 10 EC50 values for binding of
anti-BCMA/anti-CD3 T-cell bispecific antibodies to RPMI-8226
multiple myeloma cells Estimated EC50 83A10-TCBcv 21-TCBcv 22-TCBcv
42-TCBcv nM ~188428* 6.8 / 13.2 .mu.g/ml ~27151* 0.9817 / 1.907
Example 7: Cytokine Production from Activated T Cells Upon Binding
of Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies to CD3-Positive
T Cells and BCMA-Positive Multiple Myeloma Cell Lines (Cytokine
Release Assay CBA Analysis)
[0289] Anti-BCMA/anti-CD3 T cell bispecific antibodies are analyzed
for their ability to induce T-cell mediated cytokine production de
novo in the presence or absence of human BCMA-expressing human
myeloma cells (RPMI-8226, JJN-3). Briefly, human PBMCs are isolated
from Buffy Coats and 0.3 million cells per well are plated into a
round-bottom 96-well plate. Alternatively, 280 .mu.l whole blood
from a healthy donor are plated per well of a deep-well 96-well
plate. BCMA-positive tumor target cells are added to obtain a final
E:T-ratio of 10:1. Anti-BCMA/anti-CD3 TCB antibodies and controls
are added for a final concentration of 0.1 pM-10 nM. After an
incubation of up to 24 h at 37.degree. C., 5% CO.sub.2, the assay
plate is centrifuged for 5 min at 350.times.g and the supernatant
is transferred into a new deep-well 96-well plate for the
subsequent analysis. The CBA analysis was performed on FACS Cantoll
according to manufactur'er's instructions, using either the Human
Th1/Th2 Cytokine Kit II (BD #551809) or the combination of the
following CBA Flex Sets: human granzyme B (BD #560304), human
IFN-.gamma. Flex Set (BD #558269), human TNF-.alpha. Flex Set (BD
#558273), human IL-10 Flex Set (BD #558274), human IL-6 Flex Set
(BD #558276), human IL-4 Flex Set (BD #558272), human IL-2 Flex Set
(BD #558270). Table 13 shows that 83A10-TCBcv induced a
concentration-dependent increase in cytokine production and serine
protease granzyme B, a marker of cytotoxic T-cell function. Table
11 shows the EC50 values and amount of secreted cytokines/proteases
per anti-BCMA/anti-CD3 T-cell bispecific antibody
concentrations.
TABLE-US-00018 TABLE 11 Secretion of cytokine and proteases induced
by anti-BCMA/anti- CD3 T-cell bispecific antibodies in presence of
RPMI-8226 cells Cytokines/ EC50 83A10-TCBcv concentration (nM)
proteases (nM) 0.00064 0.0032 0.016 0.08 0.4 2 10 TNF-.alpha. 0.52
-6.95 -6.49 -0.65 46.72 161.24 315.11 371.47 (pg/mL) IL-10 0.30
-9.21 1.95 25.17 125.82 401.42 602.64 680.05 (pg/mL) Granzyme B
0.34 220.54 331.55 889.13 5855.02 15862.84 21270.43 27120.52
(pg/mL)
Example 8: Redirected T-Cell Cytotoxicity of BCMA-High Expressing
H929 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific
Antibodies (Colorimetric LDH Release Assay)
[0290] Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their
potential to induce T cell-mediated apoptosis in BCMA
high-expressing MM cells upon crosslinking of the construct via
binding of the antigen binding moieties to BCMA on cells. Briefly,
human BCMA high-expressing H929 multiple myeloma target cells were
harvested with Cell Dissociation Buffer, washed and resuspended in
RPMI supplemented with 10% fetal bovine serum (Invitrogen).
Approximately, 30,000 cells per well were plated in a round-bottom
96-well plate and the respective dilution of the construct was
added for a desired final concentration (in triplicates); final
concentrations ranging from 0.1 pM to 10 nM. For an appropriate
comparison, all TCB constructs and controls were adjusted to the
same molarity. Human PBMCs (effector cells) were added into the
wells to obtain a final E:T ratio of 10:1, corresponding to a E:T
ratio of approximately 3 to 5 T cells for 1 tumor target cells.
Negative control groups were represented by effector or target
cells only. For normalization, maximal lysis of the H929 MM target
cells (=100%) was determined by incubation of the target cells with
a final concentration of 1% Triton X-100, inducing cell death.
Minimal lysis (=0%) was represented by target cells co-incubated
with effector cells only, i.e. without any T cell bispecific
antibody. After 20-24h or 48h incubation at 37.degree. C., 5%
CO.sub.2, LDH release from the apoptotic/necrotic MM target cells
into the supernatant was then measured with the LDH detection kit
(Roche Applied Science), following the manufacturer's instructions.
The percentage of LDH release was plotted against the
concentrations of anti-BCMA/anti-CD3 T cell bispecific antibodies
in concentration-response curves. The EC50 values were measured
using Prism software (GraphPad) and determined as the TCB antibody
concentration that results in 50% of maximum LDH release. As shown
in FIG. 4, all anti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-,
and 83A10-TCBcv) induced a concentration-dependent killing of
BCMA-positive H929 myeloma cells as measured by LDH release. The
lysis of H929 cells was specific since control-TCB antibody which
does not bind to BCMA-positive target cells but only to CD3 on T
cells did not induce LDH release, even at the highest concentration
tested. Table 12 summarizes the EC50 values for the redirected
T-cell killing of BCMA high-expressing H929 cells induced by
anti-BCMA/anti-CD3 TCB antibodies.
TABLE-US-00019 TABLE 12 EC50 values for redirected T-cell killing
of H929 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Anti-BCMA/ anti-CD3 EC50 (pM) TCB antibodies Donor 1 Donor 2 Donor
3 Donor 4 Donor 5 Donor 6 21-TCBcv 97.1 / 42.1 53.9 38.7 / 22-TCBcv
53.2 / 42.2 23.2 28.9 / 42-TCBcv 9.7 / 11.7 7.2 6.8 / 83A10-TCBcv
3.9 / 8.5 5.0 4.3 1.5
Example 9: Redirected T-Cell Cytotoxicity of BCMA-Medium/Low
Expressing L363 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell
Bispecific Antibodies (LDH Release Assay)
[0291] Anti-BCMA/anti-CD3 TCB antibodies were also analyzed for
their ability to induce T cell-mediated apoptosis in BCMA
medium/low-expressing MM cells upon crosslinking of the construct
via binding of the antigen binding moieties to BCMA on cells.
Briefly, human BCMA medium/low-expressing L363 multiple myeloma
target cells are harvested with Cell Dissociation Buffer, washed
and resuspended in RPMI supplemented with 10% fetal bovine serum
(Invitrogen). Approximately, 30,000 cells per well are plated in a
round-bottom 96-well plate and the respective dilution of the
construct is added for a desired final concentration (in
triplicates); final concentrations ranging from 0.1 pM to 10 nM.
For an appropriate comparison, all TCB constructs and controls are
adjusted to the same molarity. Human PBMCs (effector cells) were
added into the wells to obtain a final E:T ratio of 10:1,
corresponding to a E:T ratio of approximately 3 to 5 T cells for 1
tumor target cells. Negative control groups were represented by
effector or target cells only. For normalization, maximal lysis of
the MM target cells (=100%) is determined by incubation of the
target cells with a final concentration of 1% Triton X-100,
inducing cell death. Minimal lysis (=0%) was represented by target
cells co-incubated with effector cells only, i.e. without any T
cell bispecific antibody. After 20-24 h incubation at 37.degree.
C., 5% CO.sub.2, LDH release from the apoptotic/necrotic MM target
cells into the supernatant was then measured with the LDH detection
kit (Roche Applied Science), following the manufacturer's
instructions. The percentage of LDH release was plotted against the
concentrations of anti-BCMA/anti-CD3 T cell bispecific antibodies
in concentration-response curves. The EC50 values were measured
using Prism software (GraphPad) and determined as the TCB antibody
concentration that results in 50% of maximum LDH release. As shown
in FIG. 5, all anti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-,
and 83A10-TCBcv) induced a concentration-dependent killing of
BCMA-positive L363 myeloma cells as measured by LDH release. The
lysis of L363 cells was specific since control-TCB antibody which
does not bind to BCMA-positive target cells but only to CD3 on T
cells did not induce LDH release, even at the highest concentration
tested. Table 13 summarizes the EC50 values for the redirected
T-cell killing of BCMA medium/low-expressing L363cells induced by
anti-BCMA/anti-CD3 TCB antibodies.
TABLE-US-00020 TABLE 13 EC50 values for redirected T-cell killing
of L363 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Anti-BCMA/anti- CD3 EC50 (pM) TCB antibodies Donor 1 Donor 2 Donor
3 Donor 4 Donor 5 21-TCBcv 83.6 38.4 18.9 19.1 46.4 22-TCBcv 97.5
27.7 16.5 14.6 56.0 42-TCBcv 15.5 16.7 5.2 2.2 10.6 83A10-TCBcv
16.8 47.8 28.4 12.6 39.0
Example 10: Redirected T-Cell Cytotoxicity of BCMA-Medium/Low
Expressing RPMI-8226 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T
Cell Bispecific Antibodies (LDH Release Assay)
[0292] Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their
ability to induce T cell-mediated apoptosis in BCMA
medium/low-expressing MM cells upon crosslinking of the construct
via binding of the antigen binding moieties to BCMA on cells.
Briefly, human BCMA medium/low-expressing L363 multiple myeloma
target cells are harvested with Cell Dissociation Buffer, washed
and resuspended in RPMI supplemented with 10% fetal bovine serum
(Invitrogen). Approximately, 30,000 cells per well are plated in a
round-bottom 96-well plate and the respective dilution of the
construct is added for a desired final concentration (in
triplicates); final concentrations ranging from 0.1 pM to 10 nM.
For an appropriate comparison, all TCB constructs and controls are
adjusted to the same molarity. Human PBMCs (effector cells) were
added into the wells to obtain a final E:T ratio of 10:1,
corresponding to a E:T ratio of approximately 3 to 5 T cells for 1
tumor target cells. Negative control groups were represented by
effector or target cells only. For normalization, maximal lysis of
the MM target cells (=100%) was determined by incubation of the
target cells with a final concentration of 1% Triton X-100,
inducing cell death. Minimal lysis (=0%) was represented by target
cells co-incubated with effector cells only, i.e. without any T
cell bispecific antibody. After 20-24 h incubation at 37.degree.
C., 5% CO.sub.2, LDH release from the apoptotic/necrotic MM target
cells into the supernatant was then measured with the LDH detection
kit (Roche Applied Science), following the manufacturer's
instructions. The percentage of LDH release was plotted against the
concentrations of anti-BCMA/anti-CD3 T cell bispecific antibodies
in concentration-response curves. The EC50 values were measured
using Prism software (GraphPad) and determined as the TCB antibody
concentration that results in 50% of maximum LDH release. As shown
in FIG. 6, all anti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-,
and 83A10-TCBcv) induced a concentration-dependent killing of
BCMA-positive RPMI-8226 myeloma cells as measured by LDH release.
The lysis of RPMI-8226 cells was specific since control-TCB
antibody which does not bind to BCMA-positive target cells but only
to CD3 on T cells did not induce LDH release, even at the highest
concentration tested. Table 13 summarizes the EC50 values for the
redirected T-cell killing of BCMA medium/low-expressing RPMI-8226
cells induced by anti-BCMA/anti-CD3 TCB antibodies.
TABLE-US-00021 TABLE 13 EC50 values for redirected T-cell killing
of RPMI-8226 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Anti-BCMA/anti- CD3 EC50 (pM) TCB antibodies Donor 1 Donor 2 Donor
3 Donor 4 Donor 5 21-TCBcv / 41.3 8.8 4.0 8.4 22-TCBcv / 47.6 7.6
3.2 5.5 42-TCBcv / 382.8 18.7 3.5 1.5 83A10-TCBcv / 620.5 229.3
35.0 64.9
Example 11: Redirected T-Cell Cytotoxicity of BCMA-Low Expressing
JJN-3 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific
Antibodies (Flow Cytometry and LDH Release)
[0293] Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their
ability to induce T cell-mediated apoptosis in BCMA low-expressing
MM cells upon crosslinking of the construct via binding of the
antigen binding moieties to BCMA on cells. Briefly, human BCMA
low-expressing JJN-3 multiple myeloma target cells are harvested
with Cell Dissociation Buffer, washed and resuspended in RPMI
supplemented with 10% fetal bovine serum (Invitrogen).
Approximately, 30,000 cells per well are plated in a round-bottom
96-well plate and the respective dilution of the construct is added
for a desired final concentration (in triplicates); final
concentrations ranging from 0.1 pM to 10 nM. For an appropriate
comparison, all TCB constructs and controls are adjusted to the
same molarity. Human PBMCs (effector cells) were added into the
wells to obtain a final E:T ratio of 10:1, corresponding to a E:T
ratio of approximately 3 to 5 T cells for 1 tumor target cells.
Negative control groups were represented by effector or target
cells only. For normalization, maximal lysis of the MM target cells
(=100%) was determined by incubation of the target cells with a
final concentration of 1% Triton X-100, inducing cell death.
Minimal lysis (=0%) was represented by target cells co-incubated
with effector cells only, i.e. without any T cell bispecific
antibody. i) After 48 h incubation at 37.degree. C., 5% CO.sub.2,
the cultured myeloma cells were collected, washed and stained with
fluorochrome-conjugated antibodies and Annexin-V for determination
of apoptotic myeloma cells. The staining panel comprised
CD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V-
450/Annexin-V-PerCP-Cy5.5. Fluorochrome-labelled antibodies used
were purchased from BD Biosciences (San Jose, Calif.) and Caltag
Laboratories (San Francisco Calif.). Acquisition was performed
using a multicolor flow cytometer and installed software (e.g.
Cantoll device running FACS Diva software or FACSCalibur flow
cytometer using the CellQUEST software). The Paint-A-Gate PRO
program (BD Biosciences) was used for data analysis. Annexin-V was
measured on JJN-3 cells and the percentage of annexin-v-positive
JJN-3 cells was plotted against the concentration of
anti-BCMA/anti-CD3 T cell bispecific antibodies. The percentage of
lysis of JJN-3 cells induced by a specific concentration of
anti-BCMA/anti-CD3 T cell bispecific antibody was also determined
by measuring the absolute count of annexin-V-negative JJN-3 cells
at a given TCB concentration and subtracting it from the absolute
count of annexin-V-negative JJN-3 cells without TCB; divided by the
absolute count of annexin-V-negative JJN-3 cells without TCB. FIG.
7 shows that anti-BCMA/anti-CD3 TCB antibodies (22-, 42-, and
83A10-TCBcv) induced a concentration-dependent killing of BCMA
low-expressing JJN-3 myeloma cells as measured by flow cytometry.
The lysis of JJN-3 cells was specific since control-TCB antibody
which does not bind to BCMA-positive target cells but only to CD3
on T cells did not induce increase in annexin-v positive JJN-3
cells or JJN-3 cell lysis, even at the highest concentration
tested. Table 14 and Table 15 summarize respectively the
percentages of annexin-v positive JJN-3 cells and percentages of
lysis of JJN-3 cells induced by anti-BCMA/anti-CD3 TCB
antibodies.
[0294] Detection of LDH is also performed after 20-24 h or 48 h
incubation at 37.degree. C., 5% CO.sub.2. LDH release from the
apoptotic/necrotic JJN-3 MM target cells into the supernatant is
then measured with the LDH detection kit (Roche Applied Science),
following the manufacturer's instructions. The percentage of LDH
release is plotted against the concentrations of anti-BCMA/anti-CD3
T cell bispecific antibodies in concentration-response curves. The
EC50 values are measured using Prism software (GraphPad) and
determined as the TCB antibody concentration that results in 50% of
maximum LDH release.
TABLE-US-00022 TABLE 14 Redirected T-cell killing of BCMA
low-expressing JJN-3 cells induced by anti-BCMA/anti-CD3 TCB
antibodies: percentages of annexin-V positive cells Annexin-V
positive Anti-BCMA/anti-CD3 TCB concentration (pM) JJN-3 cells (%)
10000 1000 100 10 1 0.1 0 Experiment 1 83A10-TCBcv 16.78 10.21 9.12
11.11 11.36 8.14 9.6 42-TCBcv 24.83 16.84 8.62 12.3 11.9 / 9.6
22-TCBcv 22.95 26.15 12.48 13.29 9.3 12.48 9.6 Control-TCB 8.84 / /
/ / / / Experiment 2 83A10-TCBcv 22.86 17.53 16.5 15.94 14.32 13.07
10.74 42-TCBcv 26.88 21.68 14.42 13.6 13.47 12.75 10.74 22-TCBcv
29.72 26.97 18.35 15.94 15 14.8 10.74 Control-TCB 12.82 / / / / /
/
TABLE-US-00023 TABLE 15 Redirected T-cell killing of BCMA
low-expressing JJN-3 cells induced by anti-BCMA/anti-CD3 TCB
antibodies: percentages of lysis of JJN-3 cells Lysis of
Anti-BCMA/anti-CD3 TCB concentration (pM) JJN-3 cells (%) 10000
1000 100 10 1 0.1 0 Experiment 1 83A10-TCBcv 70.30 26.66 18.43
41.88 24.42 -14.45 0.00 42-TCBcv 92.92 84.02 41.87 38.96 40.29 /
0.00 22-TCBcv 88.02 90.54 56.26 73.56 -4.29 26.28 0.00 Control-TCB
-6.55 / / / / / / Experiment 2 83A10-TCBcv 51.18 25.30 20.12 39.58
-1.88 22.28 0.00 42-TCBcv 90.37 81.12 55.32 39.44 34.94 17.62 0.00
22-TCBcv 91.21 94.12 53.03 41.66 24.36 36.47 0.00 Control-TCB 4.18
/ / / / / /
Example 12: BCMA Expression on Bone Marrow Myeloma Plasma Cells
from Multiple Myeloma Patients
[0295] Human cell lines expressing the tumor target of interest are
very useful and practical tools for the measurement of TCB antibody
potency to induce tumor cell cytotoxicity in presence of T cells
and determination of EC50 values and for the ranking of TCB
molecules. However, despite being readily accessible and practical
human myeloma cell lines have the caveat of not representing the
heterogeneity of multiple myeloma, a very complex disease which is
characterized by a significant heterogeneity at the molecular
level. In addition, myeloma cell lines do not express BCMA receptor
with the same intensity and density as some cells express BCMA more
strongly than others (e.g. H929 cells vs. RPMI-8226 cells), and
such heterogeneity at the cellular level may also be observed among
different patients. Throughout academic collaborations with key
opinion leaders in multiple myeloma, determination of BCMA
expression and density in patient samples and evaluation of the
anti-BCMA/anti-CD3 TCB antibodies with clinical patient samples are
being investigated. Blood and bone marrow aspirates are collected
from multiple myeloma patients after informed consent is given, in
accordance with local ethical committee guidelines and the
Declaration of Helsinki.
[0296] a) BCMA Expression as Detected by Multiparameter Flow
Cytometry (Mean Fluorescence Intensity)
[0297] To determine the expression of BCMA receptor on bone marrow
myeloma cells, immunophenotypic analyses were performed using
freshly isolated whole bone marrow aspirates. Erythrocyte-lysed
K.sub.3-EDTA (ethylenediaminetetraacetic acid) anticoagulated whole
bone marrow samples were used for the immunophenotypic analyses. A
total of 2.times.10.sup.6 cells per tube were stained, lysed, and
then washed using a direct immunofluorescence technique and
multicolor staining, which was aimed at the specific identification
and immunophenotypic characterization of malignant plasma cells
identified as CD138.sup.+ CD38.sup.+ CD45.sup.+ CD19.sup.-
CD56.sup.+. The cells were then stained using a panel of
fluorochrome-conjugated antibodies including at least
CD38-FITC/CD56-PE/CD19-PerCP-Cy7/CD45-V450/BCMA-APC.
Fluorochrome-labelled antibodies used are purchased from BD
Biosciences (San Jose, Calif.) and Caltag Laboratories (San
Francisco Calif.). In-house APC-conjugated anti-human BCMA antibody
was used in the immunophenotypic analyses. Acquisition was
performed using a multicolor flow cytometer and installed software
(e.g. Cantoll device running FACS Diva software or FACSCalibur flow
cytometer using the CellQUEST software). The Paint-A-Gate PRO
program (BD Biosciences) was used for data analysis. BCMA
expression was measured gated on the malignant plasma cell
population and mean fluorescence intensity (MFI) values were
determined and compared among the myeloma patients.
TABLE-US-00024 TABLE 16 BCMA expression on patient bone marrow
myeloma plasma cells as detected by multiparameter flow cytometry
(mean fluorescence intensity) Patient No MFI.sub.BCMA P1 2863 P2
3528 P3 602 P4 389 P5 955 P6 1475 P7 282 P8 1621 P9 116 P10 125 P11
1495 P12 2451 P13 398 P14 2040 P15 678 P16 945 P17 1672 P18 1491
P19 2198 P20 1058 P21 3594 P22 615 P23 159
[0298] b) Determination of BCMA Specific Antigen Binding Capacity
(Quantitative Flow Cytometry Analysis)
[0299] The Qifikit (Dako) method was used to quantify BCMA specific
antigen binding capacity (SABC) on the cell surface of patient bone
marrow myeloma plasma cells. Myeloma plasma cells isolated from
whole bone marrow aspirates were stained with 50 .mu.l of mouse
anti-human BCMA IgG (BioLegend #357502) or a mouse IgG2a isotype
control (BioLegend #401501) diluted in FACS buffer (PBS, 0.1% BSA)
to a final concentration of 25 .mu.g/ml (or at saturation
concentrations) and staining was performed for 30 min at 4.degree.
C. in the dark. Next, 100 .mu.l of the Set-up or Calibration Beads
were added in separate wells and the cells, as well as the beads
were washed twice with FACS buffer. Cells and beads were
resuspended in 25 .mu.l FACS buffer, containing fluorescein
conjugated anti-mouse secondary antibody (at saturation
concentrations), provided by the Qifikit. Cells and beads were
stained for 45 min at 4.degree. C. in the dark. The cells were
washed once and all samples were resuspended in 100 .mu.l FACS
buffer. Samples were analyzed immediately on a multicolor flow
cytometer and installed software (e.g. Cantoll device running FACS
Diva software or FACSCalibur flow cytometer using the CellQUEST
software).
TABLE-US-00025 TABLE 17 BCMA specific antigen binding capacity on
patient bone marrow myeloma plasma cells as measured by
quantitative flow cytometry analysis Patient No SABC.sub.BCMA P1
n/a P2 n/a P3 679 P4 145 P5 957 P6 969 P7 554 P8 4479 P9 350 P10
414 P11 2756 P12 2911 P13 1267 P14 3453 P15 1006 P16 1097 P17 1622
P18 429 P19 1684 P20 383 P21 1602 P22 799 P23 204
Example 13: Redirected T-Cell Cytotoxicity of Bone Marrow Patient
Myeloma Plasma Cells in Presence of Autologous Bone Marrow
Infiltrating T Cells Induced by Anti-BCMA/Anti-CD3 T Cell
Bispecific Antibodies (Multiparameter Flow Cytometry)
[0300] One of the most meaningful and critical in vitro
characterization during preclinical evaluation of TCB antibody
candidates for multiple myeloma is whether the TCB molecule could
activate the patients' T cells and induce redirected T-cell killing
of primary myeloma plasma cells from the patients' bone marrow. To
evaluate the effect of anti-BCMA/anti-CD3 TCB antibodies to induce
redirected T-cell killing of bone marrow myeloma plasma cells,
whole bone marrow aspirates were collected from multiple myeloma
patients in EDTA-coated tubes and immediately used for the cell
culture assays. The ratio of effector cells to tumor cells (E:T
ratio) present in the whole bone marrow samples was determined and
measured by flow cytometry. Briefly, 200 .mu.l of bone marrow
samples were transferred into 96 deep-well plates.
Anti-BCMA/anti-CD3 TCB antibody and control antibody dilutions were
prepared in sterile medium and 10 .mu.l of the preparation were
added to the respective wells for final concentrations ranging from
0.1 pM to 30 nM. The bone marrow-antibody suspension is mixed by
gentle shaking and then incubated at 37.degree. C., 5% CO.sub.2 for
48 h, sealed with paraffin film. After the incubation period, 20
.mu.l of a corresponding FACS antibody solution prepared based on
an antibody-panel including
CD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V450/BCMA-A-
PC/Annexin-V-PerCP-Cy5.5 were added into a 96-U-bottom plate.
Fluorochrome-labelled antibodies were purchased from BD Biosciences
(San Jose, Calif.) and Caltag Laboratories (San Francisco Calif.)
and in-house APC-conjugated anti-human BCMA antibody was used. The
samples were then incubated for 15 minutes in the dark at room
temperature and acquired and analyzed using a multicolor flow
cytometer. Cell death of the myeloma cells was determined by
evaluating annexin-V positive expression gated on the myeloma cell
populations CD138.sup.+ CD38.sup.+ CD45.sup.+ CD19.sup.-
CD56.sup.+. Percentage of myeloma cell death was then determined.
The percentage of lysis of patient bone marrow myeloma plasma cells
induced by a specific concentration of anti-BCMA/anti-CD3 T cell
bispecific antibody was also determined by measuring the absolute
count of annexin-V-negative myeloma plasma cells at a given TCB
concentration and subtracting it from the absolute count of
annexin-V-negative myeloma plasma cells without TCB; divided by the
absolute count of annexin-V-negative myeloma plasma cells without
TCB. To verify the specificity of the anti-BCMA/anti-CD3 T cell
bispecific antibodies, annexin-V expression was also measured in
other bone marrow cell types such as T cells, B cells and NK cells.
As shown in FIG. 8, there was a concentration-dependent and
specific lysis of patient myeloma plasma cells while lysis of T
cells, B cells, and NK cells was not observed. In addition,
control-TCB which binds to CD3 only but not to BCMA did not induce
cell death of myeloma plasma cells at the highest concentrations of
TCB antibodies. As shown in Table 18, percentage of annexin-V
positive patient bone marrow myeloma cells at the highest
concentration (30 nM) reached up to 52.54% and 55.72% for 42-TCBcv
and 22-TCBcv respectively as compared to 29.31% for 83A10-TCBcv,
concluding that 42-TCBcv and 22-TCBcv are more potent than
83A10-TCBcv to induce killing of patient bone marrow myeloma plasma
cells.
TABLE-US-00026 TABLE 18 Percentage of annexin-V positive myeloma
plasma cells from patient bone marrow aspirates induced by anti-
BCMA/anti-CD3 T cell bispecific antibodies. Annexin-V positive
Anti-BCMA/anti-CD3 T cell bispecific myeloma plasma antibody
concentration (pM) cells (%) 30000 10000 1000 100 10 0 83A10-TCBcv
29.31 30.95 23.14 15.74 16.76 13.11 42-TCBcv 52.54 39.87 29.96
10.51 19.6 13.11 22-TCBcv 55.72 51.71 31.01 14.81 14.19 13.11
Control-TCB 15.18 10.93 / / / /
[0301] In another study in bone marrow aspirates from 5 different
MM patients, the percentage of viable myeloma plasma cells was
determined by gating on annexin-V negative cell population and
plotted against the concentration of anti-BCMA/anti-CD3 T cell
bispecific antibody. The EC50 values were measured and determined
as the TCB antibody concentration that results in 50% of maximum
viable myeloma plasma cells. EMAX (%) was determined as maximum of
viable myeloma plasma cells in presence of respective
anti-BCMA/anti-CD3 T cell bispecific antibody. 83A10-TCBcv was much
less potent in inducing lysis of myeloma plasma cells than 22-TCBcv
and 42-TCBcv in majority of the five myeloma patient bone marrow
aspirate samples (Table 26; FIG. 9 shows as example concentration
response curves for 2 of the 5 patients). Concentration-dependent
reduction of viable myeloma cells was observed in 5/5 patient
samples treated with 22-TCBcv or 42-TCBcv, as compared to only 1/5
patient samples for 83A10-TCBcv. Table 19 shows the comparison of
83A10-TCBcv with 22-TCBcv and 42-TCBcv and the effect of the
anti-BCMA/anti-CD3 T cell bispecific antibodies on viability of
bone marrow myeloma plasma cells. The results clearly show that
there were less viable bone marrow myeloma plasma cells with
22-TCBcv and 42-TCBcv (i.e. more lysis of the bone marrow myeloma
plasma cells) in 4/5 patient samples as demonstrated by lower EMAX
(%) values for 22-TCBcv and 42-TCBcv vs. 83A10-TCBcv in respective
patient samples. Concentration-dependent and specific lysis of
patient myeloma plasma cells were observed while lysis of
non-malignant bone marrow cells was not observed (data not
shown).
TABLE-US-00027 TABLE 19 EMAX (%) values in respect to annexin-V
negative viable myeloma plasma cells from patient bone marrow
aspirates in presence of by anti-BCMA/anti-CD3 T cell bispecific
antibodies. Bone marrow aspirate 83A10-TCBcv 22-TCBcv 42-TCBcv
patient sample (Study 2) EMAX (%) Patient 001 100 7.6 22.6 Patient
003 54.3 38.9 44.6 Patient 004 100 66.6 53.9 Patient 006 81.8 65.9
73.5 Patient 007 81.8 48.6 72.8
[0302] In a further investigations of the new anti-BCMA/anti-CD3 T
cell bispecific antibodies of this invention compared to
83A10-TCBcv, seven freshly taken patient whole bone marrow
samples/aspirates were stained with CD138 magnetic microbeads
(Miltenyi Biotec, Bergisch Gladbach, Germany), passed through an
autoMACS cell separation column and the collected fractions with
sufficient remaining number of MM plasma cells of usually >4%
myeloma plasma cells were used for further experiments. In 24-well
plates, 500,000 cells/well were incubated and cultured for 48
hours. Anti-BCMA/anti-CD3 TCB antibodies and control antibody
dilutions were added to the respective wells for a final TCB
concentration of 0.1. pM to 10 nM. Each dose point was done in
triplicates. Viability of the plasma cells and cells of the bone
marrow microenvironment was investigated by propidium
iodide/CD138-FITC double-staining using flow cytometry
(FACSCalibur; Becton Dickinson). Data analysis was performed using
FACSDiva Software (Becton Dickinson). As depicted in FIG. 10, bar
plots show mean values normalized on the mean over the triplicates
of the respective medium control (MC). For statistical analysis, a
one-sided t-test was used.
[0303] The maximum inhibition of MM plasma cell growth at a
concentration of 10 nM (IMAX10) and the inhibition measured at 1 nM
(IMAX1), respectively, were given in percent as referred to the
medium control. The maximum inhibition of the control-TCB antibody
(10 nM) compared to the medium control was also depicted.
Computations were performed using R 3.1.19, and Bioconductor
2.1310, but for calculation of the IMAX values (Microsoft
Excel.RTM.; Microsoft Office Professional 2013). An effect was
considered statistically significant if the P-value of its
corresponding statistical test was <5% (*), <1% (**) or
<0.1% (***). As shown in FIGS. 10A-10G, the results clearly show
that there were less viable bone marrow myeloma plasma cells with
22-TCBcv and 42-TCBcv (i.e. more lysis of the bone marrow myeloma
plasma cells) in 717 patient samples as compared to 83A10-TCBcv.
Table 20 demonstrates the percentage of viable myeloma plasma cells
from patient bone marrow aspirates induced by anti-BCMA/anti-CD3 T
cell bispecific antibodies relative to medium control. Table 21
shows the IMAX10 and IMAX1 values. The results demonstrate that
22-TCBcv and 42-TCBcv are clearly more potent than 83A10-TCBcv to
induce killing of patient bone marrow myeloma plasma cells. Despite
specific lysis of bone marrow plasma cells (BMPC) induced by the
anti-BCMA/anti-CD3 T cell bispecific antibodies and observed in all
bone marrow patient samples, the bone marrow microenvironment
(BMME) was unaffected in the respective samples (FIG. 10H,
representative of 7 patient samples).
TABLE-US-00028 TABLE 20 Relative percentage of propidium iodide
negative viable myeloma plasma cells from patient bone marrow
aspirates induced by anti-BCMA/anti-CD3 T cell bispecific
antibodies. Anti-BCMA/anti-CD3 T cell bispecific antibody
concentration (nM) 0.01 0.1 1 10 Patient sample No. 1/Viable
myeloma plasma cells (%) 83A10-TCBcv 181.3 106.3 31.3 9.4 42-TCVcv
81.3 15.6 9.4 9.4 22-TCVcv 37.5 6.3 6.3 9.4 Ctrl-TCB / / / 162.5
Patient sample No. 2/Viable myeloma plasma cells (%) 83A10-TCBcv
89.5 31.6 5.3 0 42-TCVcv 42.1 10.5 0 0 22-TCVcv 15.8 5.3 0 0
Ctrl-TCB / / / 94.7 Patient sample No. 3/Viable myeloma plasma
cells (%) 83A10-TCBcv 76.7 35.0 1.7 0 42-TCVcv 13.3 0 0 0 22-TCVcv
3.3 0 0 0 Ctrl-TCB / / / 86.7 Patient sample No. 4/Viable myeloma
plasma cells (%) 83A10-TCBcv 93.9 51.5 9.1 6.1 42-TCVcv 9.1 0 0 0
22-TCVcv 15.2 15.2 0 0 Ctrl-TCB / / / 127.3 Patient sample No.
5/Viable myeloma plasma cells (%) 83A10-TCBcv 100 91.4 62.9 20.0
42-TCVcv 71.4 34.3 22.9 11.4 22-TCVcv 20.0 22.9 14.3 11.4 Ctrl-TCB
/ / / 85.7 Patient sample No. 6/Viable myeloma plasma cells (%)
83A10-TCBcv 55.6 22.2 6.7 4.4 42-TCVcv 35.6 6.7 4.4 4.4 22-TCVcv
24.4 3.3 8.9 2.2 Ctrl-TCB / / / 117.8 Patient sample No. 7/Viable
myeloma plasma cells (%) 83A10-TCBcv 84.4 82.6 46.8 19.3 42-TCVcv
67.0 33.9 12.8 5.5 22-TCVcv 24.4 3.3 8.9 2.2 Ctrl-TCB / / /
106.4
TABLE-US-00029 TABLE 21 IMAX10 and IMAX1 values in respect to
maximal inhibition of MM plasma cell growth at 10 nM IMAX10 and
inhibition at 1 nM IMAX1 based on propidium iodide negative viable
myeloma plasma cells from patient bone marrow aspirates in presence
of by anti-BCMA/anti-CD3 T cell bispecific antibodies. Patient
83A10-TCBcv 42-TCBcv 22-TCBcv Ctrl-TCB Sample IMAX10 IMAX1 IMAX10
IMAX1 IMAX10 IMAX1 IMAX10 No. (%) (%) (%) (%) (%) (%) (%) 1 90.6
68.8 90.6 90.6 90.6 93.8 -62.5 3 100 94.7 100 100 100 100 5.3 4 100
98.3 100 100 100 100 13.3 5 93.9 90.9 100 100 100 100 -27.3 6 80.0
37.1 88.6 77.1 88.6 85.7 14.3 7 95.6 93.3 95.6 95.6 97.8 91.1 -17.8
8 80.7 53.2 94.5 87.2 97.2 97.2 -6.4
Example 14: T-Cell Activation of Patient Bone Marrow T Cells
Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies
(Multiparameter Flow Cytometry)
[0304] To evaluate whether anti-BCMA/anti-CD3 TCB antibodies induce
activation of myeloma patient CD4.sup.+ and CD8.sup.+ T cells (i.e.
bone marrow infiltrated T cells (MILs)), the samples from the
respective treated, untreated and control groups after 48 h of
incubation were also stain with a FACS antibody solution prepared
based on an antibody-panel including eight markers:
CD8/CD69/TIM-3/CD16/CD25/CD4/HLA-DR/PD-1. The samples were then
incubated for 15 minutes in the dark at room temperature and
acquired and analyzed using a multicolor flow cytometer. T-cell
activation was determined by evaluating CD25, CD69 and/or HLA-DR
positive expression gated on CD4.sup.+ and CD8.sup.+ T-cell
populations. Percentages of T-cell activation were then measured.
FIG. 11 shows a concentration-dependent upregulation of CD69 and
CD25 on bone marrow-infiltrated CD4.sup.+ and CD8.sup.+ T cells
from multiple myeloma patients. Table 22 summarizes the increase of
CD69 and CD25 expression on CD4.sup.+ and CD8.sup.+ T cells induced
by anti-BCMA/anti-CD3 TCB antibodies; data from one patient.
TABLE-US-00030 TABLE 22 T-cell activation of myeloma patient
autologous T cells induced by anti-BCMA/anti-CD3 T-cell bispecific
antibodies in presence of patient bone marrow myeloma plasma cells
Anti-BCMA/anti-CD3 T cell bispecific antibody concentration (pM)
30000 10000 1000 100 10 0 CD69+/CD4 T cells (%) 83A10-TCBcv 21.8
14.93 1.80 0.93 1.02 0.85 42-TCBcv 29.6 24.8 1.90 1.57 0.94 0.85
22-TCBcv 34.99 30.72 3.62 1.69 2.31 0.85 Control-TCB 0.7 0.62 / / /
/ CD69+/CD8 T cells (%) 83A10-TCBcv 25.50 22.07 8.330 5.60 5.14
5.30 42-TCBcv 23.61 24.22 11.125 9.26 6.28 5.30 22-TCBcv 25.48
28.14 11.460 6.64 14.08 5.30 Control-TCB 5.71 4.93 / / / /
CD25+/CD4 T cells (%) 83A10-TCBcv 17.47 12.86 5.18 4.58 4.07 7.5
42-TCBcv 8.65 7.42 3.51 2.71 2.81 7.5 22-TCBcv 12.34 11.52 5.23
4.89 4.90 7.5 Control-TCB 6.90 6.50 / / / / CD25+/CD8 T cells (%)
83A10-TCBcv 9.79 6.560 0.42 0.13 0.12 0.12 42-TCBcv 2.20 2.231 0.42
0.14 0.08 0.12 22-TCBcv 3.57 4.110 0.65 0.10 0.08 0.12 Control-TCB
0.09 0.100 / / / /
Example 15: Increased T-Cell Function (Cytokine Production) of
Patient Bone Marrow T Cells Induced by Anti-BCMA/Anti-CD3 T Cell
Bispecific Antibodies (Multiplexed-Bead Based Immunoassay/Flow
Cytometry)
[0305] To evaluate whether anti-BCMA/anti-CD3 TCB antibodies
(83A10-TCBcv, 22-TCBcv and 42-TCBcv) induce T-cell activation and
increased function of myeloma patient bone marrow infiltrating
CD4.sup.+ and CD8.sup.+ T cells, supernatant were collected from
the culture of the respective treated, untreated and control groups
after 48 h of incubation and the content of cytokines and serine
proteases were measured. The cytokine bead array (CBA) analysis is
performed on a multicolor flow cytometer according to
manufacturer's instructions, using either the Human Th1/Th2
Cytokine Kit II (BD #551809) or the combination of the following
CBA Flex Sets: human granzyme B (BD #560304), human IFN-.gamma.
Flex Set (BD #558269), human TNF-.alpha.c Flex Set (BD #558273),
human IL-10 Flex Set (BD #558274), human IL-6 Flex Set (BD
#558276), human IL-4 Flex Set (BD #558272), human IL-2 Flex Set (BD
#558270).
Example 16: Pharmacokinetic/Pharmacodynamic (PK/PD) Study in
Cynomolgus Monkeys
[0306] A clear advantage an anti-BCMA/anti-CD3 TCBcv antibody could
have over other bispecific antibodies such as (scFV).sub.2 (e.g.
BCMAxCD3 bispecific T-cell engager BiTE.RTM. as described in
WO2013072415 and WO2013072406) is the much longer elimination
half-life/lower clearance in vivo which could allow a twice or once
a week IV or SC administration as compared to the very short
elimination half-life of (scFV).sub.2 (e.g. 1 to 4 hours) requiring
treatment administered via a pump carried by the patients for weeks
to months (Topp et al. J Clin Oncol 2011; 29(18): 2493-8). A twice
or once a week administration would be much more convenient for the
patients and also much less risky (e.g. failure of pump, issues
with the catheter, etc.).
[0307] a) To verify the elimination half-life/clearance of
anti-BCMA/anti-CD3 83A10-TCBcv antibody in vivo, single dose
pharmacokinetic (PK) pharmacodynamic (PD) studies with
anti-BCMA/anti-CD3 T-cell bispecific antibodies (83A10-TCBcv,
22-TCBcv and 42-TCBcv) were conducted at experienced
AAALAC-accredited CRO. Biologically naive adult cynomolgus monkeys
of about two years old and weighing approximately 3 kg were
acclimatized for at least 40 days and selected on the basis of body
weight, clinical observations and clinical pathology examinations.
Animals were identified by Individual tattoos and color-coded cage
cards. All the animal procedures (including housing, health
monitoring, restrain, dosing, etc) and ethical revision was
performed according to the current country legislation enforcing
the Directive on the protection of animals used for biomedical
research. Animals were randomly assigned to the treatment group
based on the most recent pretest body weight. After excluding
animals with unacceptable pretest findings, a computer program
included in the Pristima.RTM. system designed to achieve balance
with respect to pretest body weights was used to exclude animals
from both body weight extremes and randomize the remaining animals
to the treatment group. Animals were assigned to three treatment
groups with 83A10-TCBcv (n=2 animals i.e. 1 female and 1 male per
group) at 0.003; 0.03; and 0.3 mg/kg. Animals received a single
i.v. injection of 83A10-TCBcv and at least 0.8 mL of blood samples
per timepoint were collected via the peripheral vein for PK
evaluations according to the following collection schedule and
procedures: Pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504
h after dosing. Blood samples were allowed to clot in tubes for
serum separation for 60 min at room temperature. The clot was spun
down by centrifugation (at least 10 min., 1200 g, +4.degree. C.).
The resultant serum (about 300 .mu.L) was directly stored at
-80.degree. C. until further analysis. Bone marrow samples for PK
evaluations were also collected at the femur under
anesthesia/analgesic treatment according to the following
collection schedule: Pre-dose, 96 and 336 h after dosing. Bone
marrow samples were allowed to clot in tubes for serum separation
for 60 min at room temperature. The clot was spun down by
centrifugation (at least 10 min, 1200 g, +4.degree. C.). The
resultant bone marrow (about 1 mL) was directly stored at
-80.degree. C. until further analysis. The PK data analysis and
evaluation are performed. Standard non compartmental analysis is
performed using Watson package (v 7.4, Thermo Fisher Scientific
Waltman, Mass., USA) or Phoenix WinNonlin system (v. 6.3, Certara
Company, USA). As shown in FIG. 12 and Table 23, serum
concentrations of 83A10-TCBcv were measured by ELISA from serum
samples collected at different timepoints after IV injection. Table
24 shows the concentrations of 83A10-TCBcv in bone marrow as
measured by ELISA for each treatment group (BLQ means below level
of quantification).
[0308] Several information relevant for potential clinical use of a
bispecific antibody according to the invention can be taken from
FIG. 12, Table 23 and Table 24: [0309] In bone marrow aspirates
from MM patients, concentrations of 1 nM or 10 nM of TCBs of this
invention induce significant or even total killing of MM plasma
cells; at the dose 0.03 mg/kg in the interval from injection to 168
hours (7 days) plasma concentrations between approx. 1 nM and 4 nM
have been achieved showing that once a week therapy with doses of
approx. 0.03 mg/kg may well be feasible (200 ng/ml corresponds to
approx. 1 nM) [0310] FIG. 12 shows that in the investigated dose
range PK is largely dose linear; that means concentrations are
proportional to dose; a useful property for clinical therapy MM is
a disease mainly located in the bone marrow; Concentrations of
83A10-TCBcv detected in bone marrow are close to serum
concentrations (Table 24), e.g. at 96 h after injection bone marrow
concentrations of approx. 1 and 2 nM have been measured; these are
concentrations of TCB of this invention at which significant
killing of MM plasma cells is observed in bone marrow aspirates
freshly taken from MM Patients; demonstrating again the opportunity
for convenient dosing intervals like once a week [0311] Between 24
and 504 hours post injection, the elimination is largely first
order with an elimination half-life of approx. 6 to 8 days showing
again the opportunity for e.g. once a week dosing
TABLE-US-00031 [0311] TABLE 23 Serum concentrations of 83A10-TCBcv
after IV treatment in cynomolgus monkeys 83A10-TCBcv 0.003 mg/ 0.03
mg/ 0.3 mg/ Conc. kg IV kg IV kg IV (ng/mL) A B C D E F Pre-dose
0.00 0.00 0.00 0.00 0.00 0.00 30 min 75.69 74.99 668.66 796.54
17207.20 14943.95 90 min 70.92 74.56 951.81 628.72 12831.54
16248.97 180 min 76.54 62.55 981.42 722.27 10653.28 6824.72 7 h
53.17 77.39 700.67 972.38 8204.77 4560.36 24 h 33.16 50.41 358.90
532.11 4609.28 4127.41 48 h 26.05 37.40 279.80 433.30 3546.09
2700.43 96 h 17.28 19.52 226.01 429.80 1959.96 2006.92 168 h 17.33
15.87 55.58 365.67 1918.06 1382.57 336 h 11.21 4.43 102.94 153.54
1102.96 773.55 504 h 4.33 BLQ 43.99 130.14 952.03 377.04
TABLE-US-00032 TABLE 24 Bone marrow concentrations of 83A10-TCBcv
after single IV treatment in cynomolgus monkeys Conc. 0.003 mg/kg
0.03 mg/kg 0.3 mg/kg (ng/mL) A B C D E F Pre-dose 0.00 0.00 0.00
0.00 0.00 0.00 96 h 25.07 37.15 179.87 469.08 3432.54 2674.70 336 h
9.92 6.90 59.39 47.22 1987.48 850.87
[0312] Pharmacodynamics (PD) measurements: Blood samples
(timepoints: pre-dose, 24, 48, 96, 168, 336, 504 h after dosing)
and bone marrow samples (timepoints: pre-dose, 96 and 336 hs after
dosing) were collected in tubes containing 7.5% K3 EDTA for PD
evaluation by flow cytometry to evaluate the effect of 83A10-TCBcv
give i.v. as single dose on blood and bone marrow plasma cells, B
cells, and T cells. A "lyse and wash" direct immunofluorescence
staining method of the surface markers was applied. Briefly, 100
.mu.L of blood or bone marrow was incubated with two antibody
mixtures including CD45/CD2/CD16/CD20/CD27/CD38 or
CD45/CD2/CD16/CD4/CD25/CD8 in the dark for 30 min at +4.degree. C.
To lyse red blood cells, 2 mL of lysing buffer solution was added
to the sample and incubated 15 min at room temperature in the dark.
Cells were collected by centrifugation and washed with staining
buffer (PBS 2% Fetal Bovine Serum). The stained samples were kept
refrigerated, protected from light, until acquisition with
cytometer on the same day. FACS data acquisition was performed with
a Becton Dickinson flow cytometer equipped with 488 and 635 laser
lines, BD FACS Canto II. BD FACSDiva software was used for data
collection and analysis. The absolute cell number enumeration was
performed with a double platform, based upon the WBC count obtained
by the hematology analyzer (ADVIA.TM. 120, Siemens). As shown in
FIG. 13, peripheral T-cell redistribution was observed in all
animals receiving a single dose IV treatment of 83A10-TCBcv as
shown by the decrease in circulating T cell counts. As shown in
FIG. 14A, already at 24h after treatment with 83A10-TCBcv 0.3 mg/kg
a decrease in blood plasma cells (BCMA-positive cells) was observed
in animals treated while there was no decrease in total B cells
(BCMA-negative cells). FIG. 14b shows the kinetic of plasma cell
reduction in blood after treatment with 83A10-TCBcv 0.3 mg/kg in
cynomolgus monkeys.
[0313] Blood samples were also processed for plasma collection for
cytokine analysis (IL-1b, IL-2, IL-6, IL-10, TNF-.alpha. and
IFN-.gamma.) in accordance with the following collection schedule:
Pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168 h after dosing. Blood
samples were put in plastic tubes kept in an ice-water bath, then
centrifuged (at least 10 min., 1200 g, +4.degree. C.). The
resultant plasma was directly stored at -80.degree. C. until
analysis. Cytokines analysis is performed with Multiplex bead-based
cytokine immunoassay (Luminex Technology). Data are analyzed using
Bio-Plex Manager 4.1 software (Bio-Rad): a five-parameter logistic
regression model (5PL) is used.
[0314] b) In a further study, cynomolgus monkeys were treated with
42-TCBcv or 22-TCBcv. Animals (n=2/group) received a single IV
(0.01; 0.1; and 1.0 mg/kg) or SC (0.01 and 0.1 mg/kg) injection of
42-TCBcv or single IV injection with 22-TCBCv (0.1 mg/kg). Blood
and bone marrow samples are collected at timepoints following a
defined collection schedule and processed accordingly for PK and PD
measurement (immunophenotyping and cytokine production).
[0315] Animals received a single IV or SC. injection of 42-TCBcv or
22-TCBcv (only IV) and blood samples per timepoint were collected
via the peripheral vein for PK evaluations according to the
following collection schedule and procedures: Pre-dose, 30, 90, 180
min, 7, 24, 48, 96, 168, 336, 504 h after dosing. Blood samples
were allowed to clot in tubes for serum separation for 60 min at
room temperature. The clot was spun down by centrifugation (at
least 10 min., 1200 g, +4.degree. C.). The resultant serum (about
300 .mu.L) was directly stored at -80.degree. C. until further
analysis. Bone marrow samples for PK evaluations were also
collected at the femur under anesthesia/analgesic treatment
according to the following collection schedule: Pre-dose, 96 and
336 h after dosing. Bone marrow samples were allowed to clot in
tubes for serum separation for 60 min at room temperature. The clot
was spun down by centrifugation (at least 10 min, 1200 g,
+4.degree. C.). The resultant bone marrow (about 1 mL) was directly
stored at -80.degree. C. until further analysis. The PK data
analysis and evaluation were performed. Standard non compartmental
analysis was performed using Watson package (v 7.4, Thermo Fisher
Scientific Waltman, Mass., USA) or Phoenix WinNonlin system (v.
6.3, Certara Company, USA). As shown in FIG. 19 and Table 24A-D,
concentrations of 42-TCBcv were measured by ELISA from serum and
bone marrow samples collected at different timepoints after IV or
SC injection. Effective concentration range of 42-TCBcv in multiple
myeloma patient bone marrow aspirates corresponding to 10 pm to 10
nM (grey area). Concentrations in parenthesis are in nM. BLQ, below
level of quantification; i/m, inconclusive measurement.
TABLE-US-00033 TABLE 24A Serum concentrations of 42-TCBcv after IV
treatment in cynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg IV 0.1
mg/kg IV 1.0 mg/kg IV (ng/mL) Male Female Male Female Male Female
Pre-dose BLQ BLQ i/m BLQ BLQ BLQ 30 min 468.57 613.44 4720.33
4506.64 41939.31 32677.23 90 min 333.09 427.16 4284.66 3214.61
30889.73 103925.73 180 min 392.37 422.36 4336.89 2865.36 29201.69
36157.78 7 h 421.96 356.34 4028.47 3070.84 25064.81 29962.62 24 h
242.64 305.74 2996.24 2321.66 19365.86 23656.65 48 h i/m 192.97
2595.62 1781.91 20539.59 13523.68 96 h 128.50 148.02 2153.34
1277.02 13147.09 12755.58 168 h 51.13 72.64 1388.24 948.31 6189.79
3952.05 336 h 27.68 13.03 195.51 190.87 5337.85 54.15 504 h 18.17
8.04 275.93 13.96 3678.69 37.88
TABLE-US-00034 TABLE 24B Bone marrow concentrations of 42-TCBcv
after single IV treatment in cynomolgus monkeys 42-TCBcv Conc. 0.01
mg/kg IV 0.1 mg/kg IV 1.0 mg/kg IV (ng/mL) Female Male Female
Female Male Female Pre-dose BLQ BLQ 406.99 BLQ BLQ BLQ 96 h 54.39
130.03 956.56 1022.87 4089.88 4339.33 336 h 27.23 18.49 227.20
170.34 3705.74 62.44
TABLE-US-00035 TABLE 24C Serum concentrations of 42-TCBcv after SC
treatment in cynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg SC 0.1
mg/kg SC (ng/mL) Male Female Male Female Pre-dose 4.76 12.41 BLQ
BLQ 30 min 8.25 12.51 25.11 14.62 90 min 16.38 22.71 140.73 145.39
180 min 23.75 48.51 334.95 269.66 7 h 37.46 63.48 836.86 565.10 24
h 68.15 115.31 2100.42 904.22 48 h 116.63 118.03 1956.60 1111.06 96
h 150.77 120.62 1810.13 1817.52 168 h 106.28 98.64 1192.65 1653.26
336 h 67.02 46.21 482.39 571.04 504 h 25.69 31.99 4.08 83.91
TABLE-US-00036 TABLE 24D Bone marrow concentrations of 42-TCBcv
after single SC treatment in cynomolgus monkeys 42-TCBcv Conc. 0.01
mg/kg SC 0.1 mg/kg SC (ng/mL) Female Male Female Female Pre-dose
5.59 10.70 BLQ BLQ 96 h 109.88 73.93 1064.66 1066.79 336 h 29.35
48.78 518.40 906.48
[0316] The results from Tables 24A and 24C show an attractive serum
concentration profile suitable for once a week or even once every
two weeks treatment with 42-TCBcv. Area under the curve AUC for
serum concentrations after IV and SC administration were
determined, comparison of the AUC values showed high
bioavailability of close to 100% with SC injection of 42-TCBcv. In
addition, the results show that concentration of 42-TCBcv in bone
marrow is very similar to 42-TCBcv serum concentrations. 42-TCBcv
concentrations in the serum could well represent the concentrations
of 42-TCBcv available in the bone marrow i.e. at the main location
where the myeloma tumor cells are enriched.
[0317] Pharmacodynamic (PD) measurements are valuable information
to corroborate with PK measurements. Further PD analyses were
performed. Cynomolgus CD20.sup.+ B cells from blood also express
BCMA on the cell surface and are significantly more frequent
(higher absolute count) than plasma cells in blood. Blood B-cell
depletion was used as a reliable pharmacodynamic effect of
anti-BCMA/anti-CD3 TCBcv antibodies and to compare the in vivo
efficacy between 83A10-TCBcv, 42-TCBcv and 22-TCBcv. Absolute
B-cell counts were calculated based on the double platform
consisting of flow cytometry and WBC count obtained with a
hematology analyser and measured at the following timepoints:
pre-dose, 24h, 48h, 96h and 196h after 10-min IV infusion. The
percentage of B-cell depletion was calculated as followed:
= [ absolute B - cell count at pre - dose ] - [ absolute B - cell
count at timepoint ] [ absolute B - cell count at pre - dose ] *
100 ##EQU00001##
TABLE-US-00037 TABLE 24E Pharmacodynamic effects of anti-BCMA/anti-
CD3 TCBcv antibodies: B-cell depletion B-cell depletion relative to
pre-dose (%) Time after 83A10-TCBcv 42-TCBcv 22-TCBcv IV injection
0.3 mg/kg 0.1 mg/kg 0.1 mg/kg (hours) (n = 2) (n = 2) (n = 2) 24 h
19.9 .+-. 0.21 91.4 .+-. 3.8 77.8 .+-. 3.7 48 h 11.9 .+-. 17.6 88.8
.+-. 3.9 61.5 .+-. 9.8 96 h 5.0 .+-. 10.8 93.0 .+-. 7.2 89.2 .+-.
4.8 168 h -0.23 .+-. 61.4 96.6 .+-. 3.5 91.9 .+-. 3.9
[0318] 42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv to
induce depletion of BCMA-expressing B cells in cynomolgus monkeys
following a single dose IV injection (see Table 24E). Since the
three molecules share the same molecular structure and CD3 binder,
the difference in efficacy in cynomolgus monkeys could be mainly
attributed to the respective BCMA antibody.
[0319] To confirm that depletion of BCMA-expressing B cells in
cynomolgus monkeys after IV injection is a result of the
mechanistic pharmacodynamic effects of anti-BCMA/anti-CD3 TCBcv
antibodies, the increase of activated CD8.sup.+ cytotoxic T cells
(i.e. effector cells) was measured in the bone marrow enriched of
BCMA-positive cells (i.e. target cells) 4 days (96 h) and 3 weeks
(336h) after IV injection. Absolute CD8.sup.+ CD25.sup.+ activated
T-cell counts were calculated based on the double platform
consisting of flow cytometry and WBC count obtained with a
hematology analyser
TABLE-US-00038 TABLE 24F Pharmacodynamic effects of
anti-BCMA/anti-CD3 TCBcv antibodies: Increase in
CD8.sup.+CD25.sup.+ activated T cells Increase in CD8.sup.+
CD25.sup.+ activated T cells relative to pre-dose (%) Time after
83A10-TCBcv 42-TCBcv 22-TCBcv IV injection 0.3 mg/kg 0.1 mg/kg 0.1
mg/kg (hours) (n = 2) (n = 2) (n = 2) 96 h 284 .+-. 244% 585 .+-.
496% 1449 .+-. 1715% 336 h -0.9 .+-. 1.3% 110 .+-. 187% -6.6 .+-.
45.3%
[0320] 42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv to
induce T-cell activation in cynomolgus monkeys following a single
dose IV injection (see Table 24F). Since the three molecules share
the same molecular structure and CD3 binder, the difference in
pharmacodynamic effects in cynomolgus monkeys could be mainly
attributed to the respective BCMA antibody. The results indicate
that depletion of BCMA-positive B cells in bone marrow and in blood
is most likely the result of activation of cytotoxic T cells
induced by anti-BCMA/anti-CD3 TCBcv antibodies.
Example 17: Antitumoral Activity Induced by Anti-BCMA/Anti-CD3 T
Cell Bispecific Antibody in the H929 Human Myeloma Xenograft Model
Using PBMC-Humanized NOG Mice
[0321] With a long elimination half-life, Fc-containing
anti-BCMA/anti-CD3 TCBcv antibodies could be more efficacious than
(scFv).sub.2-based bispecific antibodies such as BCMA50-BiTE.RTM.
given at equimolar doses, in a once a week schedule. The in vivo
effect of 83A10-TCBcv and BCMA50-BiTE.RTM. (as described in
WO2013072415 and WO2013072406) was compared and evaluated in the
H929 human myeloma xenograft model in PBMC-humanized NOG mice. NOG
mice are appropriate for humanized mouse models as they completely
lack of immune cells including resident NK cell population and are
therefore more permissive to tumor engraftment of human xenogeneic
cells (Ito et al. Curr Top Microbiol Immunol 2008; 324: 53-76).
Briefly, on day 0 (d0) of the study, 5.times.10.sup.6 human myeloma
cell line NCI-H929 (NCI-H929, ATCC.RTM. CRL-9068.TM.) in 100 .mu.L
RPMI 1640 medium containing 50:50 matrigel (BD Biosciences, France)
were subcutaneously (SC) injected into the right dorsal flank of
immunodeficient NOD/Shi-scid IL2rgamma (null) (NOG) female mice of
8-10 weeks of age (Taconic, Ry, Danemark). Twenty-four to 72 hours
prior to H929 tumor cell SC implantation, all mice received a whole
body irradiation with a .gamma.-source (1.44 Gy, .sup.60Co, BioMep,
Bretenieres, France). On day 15 (d15), NOG mice received a single
intraperitoneal (IP) injection of 2.times.10.sup.7 human PBMCs (in
500 .mu.L PBS 1.times.pH7.4). Characterization of the human PBMC
was performed by immunophenotyping (flow cytometry). Mice were then
carefully randomized into the different treatment and control
groups (n=9/group) using Vivo Manager.RTM. software (Biosystemes,
Couternon, France) and a statistical test (analysis of variance)
was performed to test for homogeneity between groups. Antibody
treatment started on day 19 (d19), i.e. 19 days after SC injection
of H929 tumor cells when the tumor volume had reached at least
100-150 mm.sup.3 in all mice, with a mean tumor volume of
300.+-.161 mm.sup.3 for the vehicle treated control group,
315.+-.148 mm.sup.3 for the 2.6 nM/kg control-TCB treated group,
293.+-.135 mm.sup.3 for the 2.6 nM/kg 83A10-TCBcv group and
307.+-.138 mm.sup.3 for the 2.6 nM/kg BCMA50-(scFv).sub.2
(BCMA50-BiTE.RTM.) group. The TCB antibody treatment schedule was
based on the pharmacokinetic results previously obtained with
83A10-TCBcv and consisted of a once a week IV administration for up
to 3 weeks (i.e. total of 3 injections of TCB antibody). Four days
after reconstitution of the host mice with human PBMCs (d19), a
first dose of the anti-BCMA/anti-CD3 83A10-TCBcv antibody (2.6
nM/kg respectively 0.5 mg/kg) was given via tail vein injection.
Blood samples were collected by jugular/mandibular vein puncture
(under anesthesia) 1 h before each treatment, 2 h before the second
treatment and at termination in mice from all groups treated with
83A10-TCBcv and control-TCBcv. Blood samples were immediately
transferred into clot activator containing tubes (T MG tubes,
cherry red top, Capiject.RTM., Terumo.RTM.). Tubes were left at
room temperature for 30 min to allow clotting. Then tubes were
centrifuged at 1,300 g for 5 min for clot/serum separation. Serum
aliquots were prepared, flash frozen in liquid nitrogen and stored
at -80.degree. C. until further analysis. Tumor volume (TV) was
measured by caliper during the study and progress evaluated by
intergroup comparison of TV. The percentage of tumor growth defined
as TG (%) was determined by calculating TG (%)=100.times. (median
TV of analysed group)/(median TV of control vehicle treated group).
For ethical reason, mice were euthanized when TV reached at least
2000 mm.sup.3. FIG. 15 shows the TV of each individual mouse per
experimental group: (A) control groups including vehicle control
(full line) and control-TCB (dotted line), (B) 83A10-TCBcv (2.6
nM/kg) group, and (C) BCMA50-BiTE.RTM. (2.6 nM/kg). In the
83A10-TCBcv (2.6 nM/kg) group, 6 out of 9 mice (67%) had their
tumor regressed even below TV recorded at d19 i.e. first TCB
treatment and tumor regression was maintained until termination of
study. The 3 mice in the 83A10-TCBcv (2.6 nM/kg) treated group
which failed to show tumor regression had their TV equal to 376,
402 and 522 mm.sup.3 respectively at d19. In contrast, none of the
9 mice (0%) treated with an equimolar dose of BCMA50-BiTE.RTM. (2.6
nM/kg) at a once a week schedule for 3 weeks had their tumor
regressed at any timepoints. Table 25 shows progression of tumor
volumes over time in all experimental groups. The percentage of
tumor growth was calculated for d19 to d43 and compared between
83A10-TCBcv (2.6 nM/kg) group and BCMA50-BiTE.RTM. (2.6 nM/kg)
(FIG. 16). The results demonstrate that TG (%) is consistently and
significantly reduced in the 83A10-TCBcv (2.6 nM/kg) group as well
as the TG (%) is always lower when compared to BCMA50-BiTE.RTM.
(2.6 nM/kg). Table 26 shows the median tumor volume (TV) and
percentage of tumor growth (TG (%)) at days 19 to 43. The overall
results clearly demonstrated that 83A10-TCBcv is superior to
BCMA50-BiTE.RTM. to induce antitumor activity in vivo when
treatment is given at equimolar dose in once a week schedule for 3
weeks.
TABLE-US-00039 TABLE 25 Progression of tumor volumes over time in
mice from control vehicle group and mice treated with equimolar
doses of control-TCB, 83A10-TCBcv and BCMA50-(scFv).sub.2
(BCMA50-BiTE .RTM.) Tumor volume Control vehicle Group A (mm.sup.3)
A1 A2 A3 A4 A5 A6 A7 A8 A9 Mean SD Day 5 95 58 63 71 63 68 67 65 36
65 15 Day 8 70 61 71 70 56 68 74 70 49 66 8 Day 12 66 65 53 50 57
58 60 59 56 58 5 Day 15 101 95 131 80 61 65 89 37 161 91 37 Day 19
333 327 566 123 197 191 444 92 427 300 161 Day 23 565 481 1105 470
310 309 517 281 581 513 249 Day 27 1071 877 1989 823 560 675 1089
530 870 943 440 Day 30 1870 1129 x 419.2 867 1060 1368 673 1331
1090 450 Day 34 x 1653 507 1056 1521 1805 1008 2042 1370 535 Day 37
2140 2043 1309 2017 2394 1267 x 1862 464 Day 40 x x 1592 x x 1346
1469 174 Day 43 1548 1994 1771 314 Day 47 x x Day 51 Tumor volume
2.6 nM/kg Control TCB Group B (mm.sup.3) B1 B2 B3 B4 B5 B6 B7 B8 B9
Mean SD Day 5 68 65 84 83 46 63 73 74 67 69 11 Day 8 55 64 54 73 60
103 56 55 76 66 16 Day 12 45 92 73 76 83 78 103 69 76 77 16 Day 15
72 169 64 99 69 150 223 115 88 117 54 Day 19 257 334 71 318 268 460
602 236 285 315 148 Day 23 430 773 95 444 553 738 808 381 461 520
227 Day 27 924 1252 232 780 768 1009 915 606 630 791 289 Day 30
1191 1714 326 867 1230 1349 1118 817 783 1044 398 Day 34 1684 x 592
1466 1660 1954 1765 1180 576 1359 529 Day 37 2522 597 1735 1105 x x
1402 861 1370 691 Day 40 x 978 2388 1952 2277 1365 1792 604 Day 43
1302 x x x 1895 1599 419 Day 47 2346 2373 2359 19 Day 51 x x Tumor
volume 2.6 nM/kg 83A10-TCBcv Group C (mm.sup.3) C1 C2 C3 C4 C5 C6
C7 C8 C9 Mean SD Day 5 78 79 55 77 53 47 39 53 60 60 15 Day 8 69 37
67 75 62 59 59 77 75 64 12 Day 12 58 61 60 69 48 59 46 63 87 61 12
Day 15 136 41 61 138 48 57 76 71 217 94 58 Day 19 376 151 238 522
154 133 377 287 402 293 135 Day 23 656 322 375 847 311 249 642 395
681 498 210 Day 27 1119 376 443 1400 253 253 678 371 1166 673 441
Day 30 1607 187 260 1975 88 113 219 191 1590 692 783 Day 34 2143 68
100 x 34 54 63 53 2429 618 1033 Day 37 x 41 44 43 34 34 35 x 38 5
Day 40 64 40 43 38 32 39 43 11 Day 43 40 43 33 24 32 25 33 8 Day 47
14 21 16 12 19 14 16 3 Day 51 15 30 20 20 15 18 20 6 Tumor volume
2.6 nM/kg BCMA50-(scFv).sub.2 (BCMA50-BiTE .RTM.) Group D
(mm.sup.3) D1 D2 D3 D4 D5 D6 D7 D8 D9 Mean SD Day 5 75 92 78 86 57
91 74 58 62 75 13 Day 8 51 87 61 99 70 88 90 73 71 77 15 Day 12 70
73 63 76 84 76 85 58 113 78 16 Day 15 142 72 61 128 87 77 121 60
188 104 44 Day 19 232 212 81 474 303 260 360 304 539 307 138 Day 23
560 483 121 811 665 408 654 457 1115 586 278 Day 27 827 879 216
1224 1092 732 886 908 1526 921 359 Day 30 1026 1414 227 1476 1373
1256 1210 1228 2433 1294 567 Day 34 1368 1855 418 2185 1734 1936
1465 1645 x 1576 535 Day 37 1691 2754 599 2542 2102 2062 1958 765
Day 40 2764 x 706 x x x 1735 1455 Day 43 x 807 807 n/a Day 47 x Day
51
TABLE-US-00040 TABLE 26 Median tumor volume (TV) and percentage of
tumor growth (TG (%)) at days 19 to 43: 83A10-TCBcv in comparison
to BCMA50-BiTE .RTM.. Tumor Vehicle treated 83A10-TCBcv BCMA50-BiTE
.RTM. Control-TCB growth Control 2.6 nM/kg 2.6 nM/kg 2.6 nM/kg
inhibition Median TG Median TG Median TG Median TG TG.sub.inh (%)
TV (%) TV (%) TV (%) TV (%) Day 19 327 100 287 87.8 303 92.7 285
87.2 Day 23 481 100 395 82.1 560 116.4 461 95.8 Day 27 870 100 443
50.9 886 101.8 780 89.7 Day 30 1094.5 100 219 20.0 1256 114.8 1118
102.1 Day 34 1521 100 65.5 4.3 1689.5 111.1 1563 102.8 Day 37 2030
100 38 1.9 2082 102.6 1253.5 61.7 Day 40 1469 100 39.5 2.7 1735
118.1 1952 132.9 Day 43 1771 100 32.5 1.8 807 45.6 1598.5 90.3 Day
47 / / 15 / / / 2359.5 / Day 51 / / 19 / / / / /
Example 18: Antitumoral Activity Induced by Anti-BCMA/Anti-CD3
T-Cell Bispecific Antibodies in RPMI-8226 Human Myeloma Xenograft
Model in PBMC-Humanized NOG Mice
[0322] Alternatively to H929 myeloma cell line, human myeloma
RPMI-8226 cell line for which the level of expression of surface
BCMA is lower than that of H929 and more representative of the
level detected on primary myeloma cells is used as tumor xenograft.
Briefly, on day 0 (d0) of the study,
10.times.10.sup.6-20.times.10.sup.6 human myeloma cell line
RPMI-8226 (ATCC.RTM. CCL-155.TM.) in 200 .mu.L 0.9% NaCl solution
containing 50:50 matrigel (BD Biosciences, France) are
subcutaneously (SC) injected into the right dorsal flank of
immunodeficient NOD/Shi-scid IL2rgamma (null) (NOG) female mice of
8-10 weeks of age (Taconic, Ry, Danemark). Twenty-four to 72 hours
prior to RPMI-8226 cell line SC implantation, all mice received a
whole body irradiation with a .gamma.-source (1.44 Gy, .sup.6Co,
BioMep, Bretenieres, France). NOG mice receive a single
intraperitoneal (IP) injection of 2.times.10.sup.7 human PBMCs (in
500 .mu.L PBS 1.times.pH7.4) once between day 9 (d9) and day 45
(d45) once the tumor volumes reach at least 100-150 mm.sup.3.
Characterization of the human PBMC is performed by
immunophenotyping (flow cytometry). Mice are then carefully
randomized into the different treatment and control groups
(n=9/group) using Vivo Manager.RTM. software (Biosystemes,
Couternon, France) and a statistical test (analysis of variance) is
performed to test for homogeneity between the groups. Antibody
treatment starts at least 24h to 48h after human PBMC IP injection
and when the tumor volume reaches at least 100-150 mm.sup.3 in all
mice. The TCB antibody treatment schedule is based on previous
pharmacokinetic results and consisted of a once or twice a week IV
administration via the tail vein for up to 3 weeks (i.e. total of 3
injections of TCB antibody). Blood samples are collected by
jugular/mandibular vein puncture (under anesthesia) 1 h before each
treatment, 2 h before the second treatment and at termination.
Blood samples are immediately transferred into clot activator
containing tubes (T MG tubes, cherry red top, Capiject.RTM.,
Terumo.RTM.). Tubes are left at room temperature for 30 min to
allow clotting. Then tubes are centrifuged at 1,300 g for 5 min for
clot/serum separation. Serum aliquots are prepared, flash frozen in
liquid nitrogen and stored at -80.degree. C. until further
analysis. Tumor volume (TV) is measured by caliper during the study
and progress evaluated by intergroup comparison of TV. The
percentage of tumor growth as defined as inhibition TG (%) is
determined by calculating TG (%)=100.times. (median TV of analysed
group)/(median TV of control vehicle treated group).
[0323] The model development of the RPMI-8226 human myeloma
xenograft in PBMC-humanized NOG mice was first performed to ensure
that the xenograft model was appropriate for testing
anti-BCMA/anti-CD3 T-cell bispecific antibodies.
BCMA.sup.low-expressing RPMI-8226 MM cells were injected SC to NOG
mice on day 0. At day 22, human PBMCs were injected IP and human T
cells could be detected in blood one week later (data not shown).
As depicted in FIG. 20, tumor growth and bodyweight were measured
until day 50. Unfortunately and unexpectedly, this xenograft model
turned out to be unsuitable to test the antitumor activity of
anti-BCMA/anti-CD3 T-cell bispecific antibodies for the following
reasons: 1) RPMI-8226 human myeloma xenograft failed to grow
consistently in the PBMC-humanized NOG mice; 2) the PBMC-humanized
NOG mice transplanted with RPMI-8226 xenograft started losing
bodyweight soon after IP injection of human PBMC, a sign of
graft-versus host disease. These mice were euthanized for ethical
reasons; 3) loss of BCMA expression observed in tumor xenograft
post SC injection at sacrifice of the host mice.
Example 19: Redirected T-Cell Cytotoxicity of Plasma Cells from
Peripheral Blood Mononuclear Cells or Bone Marrow Aspirates of
Patient with Plasma Cell Leukemia (PCL) in Presence of Autologous T
Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies as
Measured by Flow Cytometry
[0324] Plasma cell leukemia (PCL) is a leukemic variant of myeloma
arising either de novo or from clinically pre-existent multiple
myeloma (MM). The current available treatments are rather limited
and consist mainly of combinations of MM drugs and chemotherapy. To
date no therapy has ever been explicitly registered for this highly
aggressive and deadly disease. BCMA plays an essential role in the
survival of normal plasma cells and an anti-BCMA/anti-CD3 T cell
bispecific antibodies according to the invention can be used for
plasma cell leukemia treatment in a patient suffering from said
disease. Freshly taken peripheral blood mononuclear cells (PBMC)
from plasma cell leukemia patient samples containing >80% plasma
cells at high leucocyte counts are isolated by density gradient
using Ficoll or other comparable methods and incubated for 24h and
48h with anti-BCMA/anti-CD3 T cell bispecific antibody
concentrations or control antibodies of 0.1 pM to 30 nM at
37.degree. C. in a humidified air atmosphere. Whole bone marrow
aspirates from plasma cell leukemia patients can also be used as
samples. Each dose point is done in triplicates. Apoptosis is
determined by annexin/propidium iodide staining of the whole
population and of the CD138 positive cells on a FACSCalibur using
Diva software (BD). Viability of the plasma cells and PBMC whole
population are investigated by propidium iodide/CD138-FITC
double-staining using flow cytometry (FACSCalibur; Becton
Dickinson). Data analysis is performed using FACSDiva Software
(Becton Dickinson). Mean values are normalized on the mean over the
triplicates of the respective medium control (MC). For statistical
analysis, a one-sided t-test is used. The maximum inhibition of PCL
cell growth at a concentration of 10 nM (IMAX10) and the inhibition
measured at 1 nM (IMAX1), respectively, are given in percent as
referred to the medium control. The maximum inhibition of the
control-TCB antibody (10 or 30 nM) compared to the medium control
is also measured. Computations are performed using R 3.1.19, and
Bioconductor 2.1310, but for calculation of the IMAX values
(Microsoft Excel.RTM.; Microsoft Office Professional 2013). An
effect is considered statistically significant if the P-value of
its corresponding statistical test is <5% (*), <1% (**) or
<0.1% (***). BCMA expression is also measured on PBMC
CD138.sup.+ plasma cells from plasma cell leukemia patient samples
as well as effector cells to tumor cells (E:T) ratio is determined.
As shown in FIG. 20, the results clearly show that there was
significantly reduced viable bone marrow plasma cell leukemic cells
with 42-TCBcv (i.e. more lysis of the bone marrow plasma cell
leukemic cells) in two plasma cell leukemia patient samples as
compared to the medium control Table 27 demonstrates the percentage
of maximum inhibition of plasma cell leukemic cells from patient
bone marrow aspirates or peripheral blood induced by 10 nM (IMAX10)
and 1 nM (IMAX1) anti-BCMA/anti-CD3 T cell bispecific antibodies
relative to medium control. The results demonstrate that 42-TCBcv
is very potent to induce killing of patient bone marrow plasma cell
leukemic cells. Despite specific lysis of bone marrow plasma cell
leukemic cells induced by the anti-BCMA/anti-CD3 T cell bispecific
antibodies and observed bone marrow samples (PCL patient 1), the
bone marrow microenvironment (BMME) was unaffected in the
respective samples (data not shown).
TABLE-US-00041 TABLE 27 IMAX10 and IMAX1 values in respect to
maximal inhibition of plasma cell leukemia plasma cell growth at 10
nM (IMAX10) and inhibition at 1 nM (IMAX1) based on propidium
iodide negative viable plasma cell leukemic cells from patient bone
marrow aspirates in presence of by anti-BCMA/anti-CD3 T cell
bispecific antibodies. 42-TCBcv Ctrl-TCB Patient IMAX10 IMAX1
IMAX10 Sample No. (%) (%) (%) 1 99.6 88.2 -2.7 2 ~60.0 ~40.0
~8.0
Example 20: Redirected T-Cell Cytotoxicity of Bone Marrow Plasma
Cells from Patient with AL Amyloidosis in Presence of Autologous T
Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies as
Measured by Flow Cytometry
[0325] AL amyloidosis is a rare disease caused by a disorder of the
bone marrow which usually affects people from ages 50-80 and with
two-third of the patients being male. AL amyloidosis is reflected
by an abnormal production of antibody/immunoglobulin protein by the
plasma cells. In AL amyloidosis, the light chains (LC) of the
antibody are misfolded and the abnormal LC misfolded protein result
is the formation of amyloid. These misfolded amyloid proteins are
deposited in and around tissues, nerves and organs. As the amyloid
builds up in an organ, nerve or tissue, it gradually causes damage
and affects their function. Patients with AL amyloidosis are often
affected with more than one organ. Since BCMA plays an essential
role in the survival of normal plasma cells, it is highly justified
to evaluate the effect of anti-BCMA/anti-CD3 T cell bispecific
antibodies in killing plasma cells in AL amyloidosis. Freshly taken
AL amyloidosis patient whole bone marrow samples/aspirates are
either exposed directly to the anti-BCMA/anti-CD3 TCB antibodies or
stained with CD138 magnetic microbeads (Miltenyi Biotec, Bergisch
Gladbach, Germany), passed through an autoMACS cell separation
column and the collected fractions with sufficient remaining number
of AL amyloidosis plasma cells of usually >4% are used for
further experiments. In 24-well plates, 500,000 cells/well are
incubated and cultured for 48 hours. Anti-BCMA/anti-CD3 TCB
antibodies and control antibody dilutions are added to the
respective wells for a final TCB concentration of 0.1 pM to 30 nM.
Each dose point is done in triplicates. Viability of the plasma
cells and cells of the bone marrow microenvironment is investigated
by propidium iodide/CD138-FITC double-staining using flow cytometry
(FACSCalibur; Becton Dickinson). Data analysis is performed using
FACSDiva Software (Becton Dickinson). Mean values are normalized on
the mean over the triplicates of the respective medium control
(MC). For statistical analysis, a one-sided t-test is used. The
maximum inhibition of PCL cell growth at a concentration of 10 nM
(IMAX10) and the inhibition measured at 1 nM (IMAX1), respectively,
are given in percent as referred to the medium control. The maximum
inhibition of the control-TCB antibody (10 or 30 nM) compared to
the medium control is also measured. Computations are performed
using R 3.1.19, and Bioconductor 2.1310, but for calculation of the
IMAX values (Microsoft Excel.RTM.; Microsoft Office Professional
2013). An effect is considered statistically significant if the
P-value of its corresponding statistical test is <5% (*), <1%
(**) or <0.1% (***). BCMA expression is also measured on bone
marrow CD138.sup.+ plasma cells from AL amyloidosis patient samples
as well as effector cells to tumor cells (E:T) ratio is determined.
Sequence CWU 1
1
5715PRTMus musculus 1Thr Tyr Ala Met Asn1 5219PRTMus musculus 2Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1 5 10
15Val Lys Gly314PRTMus musculus 3His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe Ala Tyr1 5 10414PRTMus musculus 4Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5 1057PRTMus musculus 5Gly
Thr Asn Lys Arg Ala Pro1 569PRTMus musculus 6Ala Leu Trp Tyr Ser
Asn Leu Trp Val1 57125PRTMus musculus 7Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 1258109PRTMus musculus 8Gln Ala Val Val Thr Gln Glu Pro Ser Leu
Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu
Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys
Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp
Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 1059116PRTArtificial
SequenceVariable region VH 9Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val
Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 11510116PRTArtificial SequenceVariable region VH 10Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn
20 25 30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11511109PRTArtificial SequenceVariable region VL 83A10 11Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Gly Tyr Pro Pro 85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10512109PRTArtificial SequenceVariable region VL 12Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Glu Tyr
20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Glu His Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Tyr Pro Pro 85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 10513109PRTArtificial SequenceVariable region VL
Mab22 13Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Tyr 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45Ile Ser Gly Ala Gly Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Tyr Pro Pro 85 90 95Asp Phe Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10514109PRTArtificial SequenceVariable
region VL Mab42 14Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Asp Glu 20 25 30Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile His Ser Ala Ser Thr Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Ala Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95Asp Phe Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105155PRTHomo sapiens 15Ser Tyr
Ala Met Ser1 51617PRTHomo sapiens 16Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly177PRTHomo sapiens 17Val
Leu Gly Trp Phe Asp Tyr1 51813PRTHomo sapiens 18Arg Ala Ser Gln Ser
Val Ser Ser Ser Tyr Leu Ala Trp1 5 10198PRTHomo sapiens 19Tyr Gly
Ala Ser Ser Arg Ala Thr1 52010PRTHomo sapiens 20Gln Gln Tyr Gly Tyr
Pro Pro Asp Phe Thr1 5 10215PRTArtificial SequenceCDR1H 21Asp Asn
Ala Met Gly1 52217PRTArtificial SequenceCDR2H 22Ala Ile Ser Gly Pro
Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly2313PRTArtificial SequenceMab21 CDR1L 23Arg Ala Ser Gln Ser
Val Ser Glu Tyr Tyr Leu Ala Trp1 5 10248PRTArtificial SequenceMab21
CDR2L 24Glu His Ala Ser Thr Arg Ala Thr1 52513PRTArtificial
SequenceMab22 CDR1L 25Arg Ala Ser Gln Ser Val Ser Ser Tyr Tyr Leu
Ala Trp1 5 10268PRTArtificial SequenceMab22 CDR2L 26Ser Gly Ala Gly
Ser Arg Ala Thr1 52713PRTArtificial SequenceMab42 CDR1L 27Arg Ala
Ser Gln Ser Val Ser Asp Glu Tyr Leu Ser Trp1 5 10288PRTArtificial
SequenceMab42 CDR2L 28His Ser Ala Ser Thr Arg Ala Thr1
5295PRTArtificial SequenceMab27 CDR1H 29Ser Ala Pro Met Gly1
53016PRTArtificial SequenceMab27 CDR2H 30Ala Ile Ser Tyr Ile Gly
His Thr Tyr Tyr Ala Asp Ser Val Lys Gly1 5 10 153112PRTArtificial
SequenceCDR1L 31Arg Ala Ser Gln Ser Val Ser Glu Tyr Tyr Leu Ala1 5
10327PRTArtificial SequenceCDR2L 32His Ala Ser Thr Arg Ala Thr1
53310PRTArtificial SequenceCDR3L 33Gln Gln Tyr Gly Tyr Pro Pro Asp
Phe Thr1 5 10345PRTArtificial SequenceMab33 CDR1H 34Thr Asn Ala Met
Gly1 53517PRTArtificial SequenceMab33 CDR2H 35Ala Ile Asn Arg Phe
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly365PRTArtificial SequenceMab39 CDR1H 36Gln Asn Ala Met Gly1
53717PRTArtificial SequenceMab39 CDR2H 37Ala Ile Ser Pro Thr Gly
Phe Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly38115PRTArtificial SequenceMab27 VH 38Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala 20 25 30Pro Met Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Tyr Ile Gly His Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser 11539116PRTArtificial SequenceMab33 VH
39Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Tyr Thr
Asn 20 25 30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Asn Arg Phe Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11540116PRTArtificial SequenceMab39 VH 40Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Thr Gln Asn 20 25 30Ala Met Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Pro Thr Gly Phe Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser 11541103PRTArtificial SequenceCH1
domain 41Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Glu Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Glu 85 90 95Lys Val Glu Pro Lys Ser Cys
10042107PRTArtificial SequenceCL domain 42Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Arg1 5 10 15Lys Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10543103PRTArtificial SequenceCD3 CH1 43Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val Glu Pro Lys Ser Cys 10044107PRTArtificial SequenceCD3 CL
44Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10545671PRTArtificial Sequence83A10 knob HC 45Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Glu Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp
Gly Gly Gly Gly 210 215 220Ser Gly Gly Gly Gly Ser Gln Ala Val Val
Thr Gln Glu Pro Ser Leu225 230 235 240Thr Val Ser Pro Gly Gly Thr
Val Thr Leu Thr Cys Gly Ser Ser Thr 245 250 255Gly Ala Val Thr Thr
Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro 260 265 270Gly Gln Ala
Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 275 280 285Gly
Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 290 295
300Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr
Cys305 310 315
320Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu
325 330 335Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 340 345 350Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 355 360 365Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 370 375 380Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser385 390 395 400Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 405 410 415Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 420 425 430Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 435 440
445Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
450 455 460Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr465 470 475 480Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 485 490 495Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys 500 505 510Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520 525Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile545 550 555
560Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
565 570 575Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu 580 585 590Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 595 600 605Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 610 615 620Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg625 630 635 640Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665
67046446PRTArtificial Sequence83A10 hole HC 46Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu 130 135 140Val Glu Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
Pro Pro 340 345 350Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44547216PRTArtificial Sequence83A10 LC 47Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro
85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val 100 105 110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg
Lys Leu Lys 115 120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn145 150 155 160Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200
205Lys Ser Phe Asn Arg Gly Glu Cys 210 21548232PRTArtificial
SequenceCD3 LC 48Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His
Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val 115 120 125Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135
140Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg145 150 155 160Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn 165 170 175Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser 180 185 190Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys 195 200 205Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220Lys Ser Phe Asn
Arg Gly Glu Cys225 23049671PRTArtificial SequenceMab21 knob HC
49Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Asn 20 25 30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155
160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205Lys Val Asp Glu Lys Val Glu Pro Lys Ser
Cys Asp Gly Gly Gly Gly 210 215 220Ser Gly Gly Gly Gly Ser Gln Ala
Val Val Thr Gln Glu Pro Ser Leu225 230 235 240Thr Val Ser Pro Gly
Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr 245 250 255Gly Ala Val
Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro 260 265 270Gly
Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 275 280
285Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala
290 295 300Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr
Tyr Cys305 310 315 320Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly
Gly Gly Thr Lys Leu 325 330 335Thr Val Leu Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 340 345 350Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 355 360 365Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 370 375 380Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser385 390 395
400Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
405 410 415Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 420 425 430Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His 435 440 445Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val 450 455 460Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr465 470 475 480Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 485 490 495Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 500 505 510Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520
525Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
530 535 540Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
Thr Ile545 550 555 560Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 565 570 575Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu 580 585 590Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600 605Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 610 615 620Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg625 630 635
640Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
645 650 655His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 660 665 67050446PRTArtificial SequenceMab21 hole HC 50Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25
30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Glu Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro Pro 340 345 350Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 44551216PRTArtificial SequenceMab21 LC 51Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Glu Tyr 20 25 30Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile
Glu His Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro
85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val 100 105 110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg
Lys Leu Lys 115 120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn145 150 155 160Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200
205Lys Ser Phe Asn Arg Gly Glu Cys 210 21552671PRTArtificial
SequenceMab22 knob HC 52Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Asn 20 25 30Ala Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Pro Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu
Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120
125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Glu Lys
Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly 210 215 220Ser Gly Gly
Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu225 230 235
240Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
245 250 255Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu
Lys Pro 260 265 270Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn
Lys Arg Ala Pro 275 280 285Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
Leu Gly Gly Lys Ala Ala 290 295 300Leu Thr Leu Ser Gly Ala Gln Pro
Glu Asp Glu Ala Glu Tyr Tyr Cys305 310 315 320Ala Leu Trp Tyr Ser
Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 325 330 335Thr Val Leu
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 340 345 350Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 355 360
365Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
370 375 380Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser385 390 395 400Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser 405 410 415Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn 420 425 430Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His 435 440 445Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 450 455 460Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr465 470 475
480Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
485 490 495Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 500 505 510Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 515 520 525Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 530 535 540Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile Glu Lys Thr Ile545 550 555 560Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 565 570 575Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 580 585 590Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600
605Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
610 615 620Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg625 630 635 640Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 645 650 655His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 660 665 67053446PRTArtificial SequenceMab22
hole HC 53Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asp Asn 20 25 30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Gly Trp Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val
Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Glu Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 44554216PRTArtificial SequenceMab22 LC 54Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Ser Gly Ala Gly Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Tyr Pro Pro 85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Arg Lys Leu Lys 115 120 125Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170
175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr 195 200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
21555671PRTArtificial SequenceMab42 knob HC 55Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30Ala Met Gly
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu 130 135 140Val Glu Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly
210 215 220Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro
Ser Leu225 230 235 240Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr
Cys Gly Ser Ser Thr 245 250 255Gly Ala Val Thr Thr Ser Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro 260 265 270Gly Gln Ala Phe Arg Gly Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro 275 280 285Gly Thr Pro Ala Arg
Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 290 295 300Leu Thr Leu
Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys305 310 315
320Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu
325 330 335Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 340 345 350Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 355 360 365Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 370 375 380Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser385 390 395 400Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 405 410 415Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 420 425 430Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 435 440
445Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
450 455 460Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr465 470 475 480Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 485 490 495Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys 500 505 510Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520 525Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile545 550 555
560Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
565 570 575Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu 580 585 590Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 595 600 605Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 610 615 620Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg625 630 635 640Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665
67056446PRTArtificial SequenceMab42 hole HC 56Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30Ala Met Gly
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu 130 135 140Val Glu Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro Pro 340 345 350Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 44557216PRTArtificial SequenceMab42 LC 57Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Glu 20 25 30Tyr Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile
His Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro
Pro 85 90 95Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Arg Lys Leu Lys 115 120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200
205Lys Ser Phe Asn Arg Gly Glu Cys 210 215
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