U.S. patent application number 17/132989 was filed with the patent office on 2021-06-17 for anti-gd2 antibody for the treatment of neuroblastoma.
The applicant listed for this patent is UMC Utrecht Holding B.V.. Invention is credited to Johannes Gerardus Maria EVERS, Jeanette Henrica Wilhelmina LEUSEN.
Application Number | 20210179732 17/132989 |
Document ID | / |
Family ID | 1000005432563 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210179732 |
Kind Code |
A1 |
LEUSEN; Jeanette Henrica Wilhelmina
; et al. |
June 17, 2021 |
ANTI-GD2 ANTIBODY FOR THE TREATMENT OF NEUROBLASTOMA
Abstract
The invention relates to anti-ganglioside GD2 antibodies that
comprise an antibody variable domain and antibody constant domains,
wherein the variable domain comprises a heavy and light chain
variable region comprising respectively at least the CDR3 of the
heavy chain variable region of antibody ch14.18 and at least the
CDR3 of the light chain variable region of antibody ch14.18; and an
IgA hinge and C.sub.H2 domain and to methods of treatment of
subjects with a GD2 positive tumor, preferably neuroblastoma with
these antibodies.
Inventors: |
LEUSEN; Jeanette Henrica
Wilhelmina; (Utrecht, NL) ; EVERS; Johannes Gerardus
Maria; (Utrecht, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UMC Utrecht Holding B.V. |
Utrecht |
|
NL |
|
|
Family ID: |
1000005432563 |
Appl. No.: |
17/132989 |
Filed: |
December 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16825563 |
Mar 20, 2020 |
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17132989 |
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PCT/NL2018/050629 |
Sep 21, 2018 |
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16825563 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/565 20130101; C07K 2317/24 20130101; C07K 2317/732
20130101; C07K 2317/522 20130101; C07K 2317/21 20130101; C07K
2317/53 20130101; A61K 39/39558 20130101; C07K 16/3084 20130101;
C07K 2317/734 20130101; C07K 2317/526 20130101; A61K 31/203
20130101; A61K 2039/505 20130101; A61K 38/193 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61P 35/00 20060101 A61P035/00; A61K 31/203 20060101
A61K031/203; A61K 38/19 20060101 A61K038/19; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 2017 |
EP |
17192476.4 |
Claims
1-53. (canceled)
54. An engineered antibody that comprises: (a) an antigen binding
domain; and (b) a constant domain that comprises an immunoglobulin
A (IgA) heavy chain constant region that comprises a mutation in a
tailpiece region, wherein said mutation is a substitution, or a
deletion, wherein said mutation is not a deletion of amino acid
residue C471, or Y472, relative to a WT IgA heavy chain constant
region comprising an amino acid sequence of SEQ ID NO: 11,
numbering according to the myeloma IgA1 protein (Bur) scheme, and
wherein the antigen binding domain is from an immunoglobulin G
(IgG) antibody.
55. The engineered antibody of claim 54, wherein said IgA heavy
chain constant region further comprises; (i) an amino acid
substitution at N337, (ii) an amino acid substitution at 1338,
(iii) an amino acid substitution at T339, (iv) an amino acid
substitution at N166, (v) an amino acid substitution at P221, (vi)
an amino acid substitution at C311, or (vii) a combination thereof,
relative to the WT IgA heavy chain constant region comprising an
amino acid sequence of SEQ ID NO: 11, numbering according to the
myeloma IgA1 protein (Bur) scheme.
56. The engineered antibody of claim 54, wherein the IgA heavy
chain constant region further comprises; (i) a N337T amino acid
substitution, (ii) a I338L amino acid substitution, (iii) a T339S
amino acid substitution, (iv) a N166G amino acid substitution, (v)
a P221R amino acid substitution, (vi) a C311S amino acid
substitution, or (vii) a combination thereof, relative to the WT
IgA heavy chain constant region comprising an amino acid sequence
of SEQ ID NO: 11, numbering according to the myeloma IgA1 protein
(Bur) scheme.
57. The engineered antibody of claim 54, wherein the IgA heavy
chain constant region further comprises; (i) a N337T amino acid
substitution, (ii) a I338L amino acid substitution, (iii) a T339S
amino acid substitution, and (iv) a N166G amino acid substitution,
relative to a WT IgA heavy chain constant region comprising an
amino acid sequence of SEQ ID NO: 11, numbering according to the
myeloma IgA1 protein (Bur) scheme.
58. The engineered antibody of claim 54, wherein the IgA heavy
chain constant region further comprises; (i) a P221R amino acid
substitution, and (ii) a C311S amino acid substitution, relative to
the WT IgA heavy chain constant region comprising an amino acid
sequence of SEQ ID NO: 11, numbering according to the myeloma IgA1
protein (Bur) scheme.
59. The engineered antibody of claim 54, wherein the engineered
antibody exhibits antibody-dependent cell mediated cytotoxicity
(ADCC) as measured in a suitable in vitro ADCC assay.
60. The engineered antibody of claim 54, wherein the engineered
antibody is monomeric or dimeric.
61. A pharmaceutical composition comprising the engineered antibody
of claim 54.
62. A method of treating a subject in need thereof comprising;
administering to the subject a therapeutically effective amount of
the engineered antibody of claim 54.
63. An engineered antibody that comprises: (a) an antigen binding
domain; and (b) a constant domain that comprises an immunoglobulin
A (IgA) heavy chain constant region that comprises a mutation in a
tailpiece region, wherein said mutation comprises a deletion of at
least three amino acids, relative to a WT IgA heavy chain constant
region comprising an amino acid sequence of SEQ ID NO: 11,
numbering according to the myeloma IgA1 protein (Bur) scheme, and
wherein the antigen binding domain is from an immunoglobulin G
(IgG) antibody.
64. The engineered antibody of claim 63, wherein the at least three
amino acids comprise C471.
65. The engineered antibody of claim 63, wherein the at least three
amino acids comprise Y472.
66. The engineered antibody of claim 63, wherein said IgA heavy
chain constant region comprises; (i) an amino acid substitution at
N337, (ii) an amino acid substitution at 1338, (iii) an amino acid
substitution at T339, (iv) an amino acid substitution at N166, (v)
an amino acid substitution at P221, (vi) an amino acid substitution
at C311, or (vii) a combination thereof, relative to the WT IgA
heavy chain constant region comprising an amino acid sequence of
SEQ ID NO: 11, numbering according to the myeloma IgA1 protein
(Bur) scheme.
67. The engineered antibody of claim 63, wherein the IgA heavy
chain constant region further comprises; (i) a N337T amino acid
substitution, (ii) a I338L amino acid substitution, (iii) a T339S
amino acid substitution, (iv) a N166G amino acid substitution, (v)
a P221R amino acid substitution, (vi) a C311S amino acid
substitution, or (vii) a combination thereof, relative to the WT
IgA heavy chain constant region comprising an amino acid sequence
of SEQ ID NO: 11, numbering according to the myeloma IgA1 protein
(Bur) scheme.
68. The engineered antibody of claim 63, wherein the IgA heavy
chain constant region further comprises; (i) a N337T amino acid
substitution, (ii) a I338L amino acid substitution, (iii) a T339S
amino acid substitution, and (iv) a N166G amino acid substitution,
relative to the WT IgA heavy chain constant region comprising an
amino acid sequence of SEQ ID NO: 11, numbering according to the
myeloma IgA1 protein (Bur) scheme.
69. The engineered antibody of claim 63, wherein the IgA heavy
chain constant region further comprises; (i) a P221R amino acid
substitution, and (ii) a C311S amino acid substitution, relative to
the WT IgA heavy chain constant region comprising an amino acid
sequence of SEQ ID NO: 11, numbering according to the myeloma IgA1
protein (Bur) scheme.
70. The engineered antibody of claim 63, wherein the engineered
antibody exhibits antibody dependent cell mediated cytotoxicity
(ADCC) as measured in a suitable in vitro ADCC assay.
71. The engineered antibody of claim 63, wherein the engineered
antibody is monomeric or dimeric.
72. A pharmaceutical composition comprising the engineered antibody
of claim 63.
73. A method of treating a subject in need thereof comprising;
administering to the subject a therapeutically effective amount of
the engineered antibody of claim 63.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/825,563, filed Mar. 20, 2020, which is a
Continuation Application of International Patent Application
PCT/NL2018/050629, filed Sep. 21, 2018, which claims priority to EP
17192476.4, filed Sep. 21, 2017, each of which is entirely
incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 20, 2020, is named 55207_703_301_SequenceListing.txt and is
27,186 bytes in size.
TECHNICAL FIELD
[0003] The invention relates to the field of antibodies. In
particular it relates to antibodies that bind the ganglioside GD2.
It further relates to the use of GD2 antibodies in medical and
detection methods. The invention further relates to cells, nucleic
acid molecules and methods for the production of the
antibodies.
BACKGROUND
[0004] Approximately 12% of all pediatric cancer patients succumb
to neuroblastoma, the most common extracranial solid tumor of
childhood. The majority of patients is diagnosed with high-risk
neuroblastoma with a mortality of 50%. Neuroblastoma therapy
consists of intensive multimodality treatment with severe
toxicities. High-risk neuroblastoma therapy consists of intensive
multimodality treatment with severe short- and long-term
toxicities. Recurrence rates are high with limited further
treatment options. Residual disease is treated with intensive
chemotherapy, radiotherapy, high-dose chemotherapy followed by
autologous hematopoietic stem cell rescue and isotretinoin.
Complete eradication of the tumor is often not achieved (Matthay et
al., 1999; New Engl. J. of Med Vol 341: pp 1165-73).
Intensification of conventional therapy has not improved outcome
and is even associated with increased toxicity.
[0005] In 2015 the antibody dinutuximab (trade name Unituxin),
directed against ganglioside GD2, a carbohydrate antigen uniformly
expressed on neuroblastoma and neural tissue, was FDA-approved for
neuroblastoma treatment. Application of this antibody, combined
with cytokines and differentiation factors, has improved patient
prognosis and demonstrated that neuroblastoma is susceptible to
immunotherapy (Yu et al., 2010; New Engl. J. Med, Vol 363: pp
1324-34; and Suzuki and Cheung., 2015; Expert Opin Ther Targets Vol
19: p. 349-62). Dinutuximab significantly improved event-free
survival in comparison to standard treatment.
[0006] Dinutuximab is a chimeric monoclonal antibody composed of
the variable heavy- and light-chain regions of the murine anti-GD2
antibody 14.18 and the constant regions of human IgG1 heavy-chain
and kappa light-chain (Gillies et al., 1989; J Immunol Methods,
125: p. 191-202). It is directed against the end-terminal
penta-oligosaccharide of GD2, an extracellularly expressed
disialoganglioside on tissues of the central nervous system and
peripheral nerves, as well as on many tumors of neuroectodermal
origin, including neuroblastoma (Suzuki and Cheung, 2015; Expert
Opin Ther Targets Vol 19: p. 349-62). Antibody produced in SP2/0
mouse myeloma cells can result in aberrant glycosylation with
respect to natural human antibodies. In Europe the antibody is now
produced in CHO cells. The uniform expression of GD2 on
neuroblastoma together with the low expression on other tissues
makes this tumor associated antigen a promising target for antibody
therapy. Dinutuximab is used in combination with isotretinoin and
alternating administration of IL-2 and GM-CSF for the first-line
treatment against high-risk neuroblastoma in patients where a
response after induction therapy was shown (Suzuki and Cheung,
2015; Expert Opin Ther Targets Vol 19: p. 349-62).
[0007] Preclinical research has shown that dinutuximab mediates its
anti-tumor effects through antibody dependent cell mediated
cytotoxicity (ADCC) and complement mediated cytotoxicity (CDC)
(Barker et al., 1991; Cancer Res, Vol 51: p. 144-9). For ADCC
activity, most therapeutic antibodies depend on NK cells and
possibly macrophages for their action. Remarkably, the ADCC
activity of dinutuximab against neuroblastoma is for some reason
also dependent on granulocyte activation. This is shown in vitro
and in vivo, by showing that patient outcome depends in part on
granulocyte activation (Barker et al., 1991; Cancer Res, Vol 51: p.
144-9; Cheung et al., 2012; J Clin Oncol, Vol 30: p. 426-32; and
Batova et al., 1999; Clin Cancer Res Vol 5: p. 4259-63). These
observations have formed the basis for the inclusion of GM-CSF or
G-CSF in the current therapeutic regimen to further stimulate
granulocytes to enhance the anti-tumor response against
neuroblastoma (Cheung et al., 2012; J Clin Oncol, Vol 30: p.
426-32; and Batova et al., 1999; Clin Cancer Res Vol 5: p.
4259-63).
[0008] In spite of the good clinical effects of dinutuximab it is
associated with a number of toxicities. The most common toxicities
include tachycardia, hypertension, hypotension, difficult to treat
pain, fever and urticaria. Many of these toxicities are
dose-dependent and are rarely noted at low dosages. Other
toxicities include hyponatremia, hypokalemia, nausea, vomiting and
diarrhea (Mora J, 2016, Expert review of clinical pharmacology, pp
1-7; http://dx.doi.org/10.1586/17512433.2016.1160775).
[0009] The anti-GD2 antibodies and the methods of treatment of
present invention exhibit improved efficacy. In addition, toxicity
associated with dinutuximab treatment is reduced, in particular
pain associated toxicity is abrogated, as shown in pre-clinical
models.
SUMMARY OF THE INVENTION
[0010] The invention provides an anti-ganglioside GD2 antibody that
comprises an antibody variable domain and antibody constant
domains, wherein the variable domain comprises a heavy and light
chain variable region comprising respectively at least the CDR3 of
the heavy chain variable region of antibody ch14.18 and at least
the CDR3 of the light chain variable region of antibody ch14.18;
and an IgA hinge and C.sub.H2 domain. The variable domain
preferably comprises a heavy and light chain variable region
comprising respectively at least the CDR1, CDR2 and CDR3 of the
heavy chain variable region of antibody ch14.18 and at least the
CDR1, CDR2 and CDR3 of the light chain variable region of antibody
ch14.18. In a preferred embodiment the variable domain comprises
the heavy and light chain variable region of antibody ch14.18.
[0011] The invention further provides a method of treatment of a
subject that has a GD2 positive tumor or is at risk of having a GD2
positive tumor the method comprising administering a therapeutic
amount of an antibody of the invention to the subject in need
thereof. The method preferably further comprises administering
retinoic acid in an amount effective to upregulate the ganglioside
GD2 in said GD2 positive tumor, preferably neuroblastoma in said
subject. The method may further comprise administering
granulocyte-macrophage colony-stimulating factor (GM-CSF,
granulocyte colony-stimulating factor (G-CSF) or a combination
thereof to said subject. GM-CSF, G-CSF or a combination thereof can
increase the number of granulocytes in the subject but is also
administered to improve the induced cell killing.
[0012] The invention further provides an antibody of the invention
for use in the treatment of a subject that has a GD2 positive tumor
or is at risk of having a GD2 positive tumor. The invention further
provides an antibody of the invention in combination with retinoic
acid for use in the treatment of a subject that has a GD2 positive
tumor or is at risk of having a GD2 positive tumor. The invention
further provides an antibody of the invention in combination with
GM-CSF, G-CSF or a combination thereof for use in the treatment of
a subject that has a GD2 positive tumor or is at risk of having a
GD2 positive tumor. The invention further provides an antibody of
the invention in combination with GM-CSF, G-CSF or a combination
thereof and retinoic acid for use in the treatment of a subject
that has a GD2 positive tumor or is at risk of having a GD2
positive tumor.
[0013] The GD2 positive tumor is preferably a GD2 positive
neuroblastoma such as a neuroectoderm-derived tumor or a sarcoma.
In a preferred embodiment the GD2 positive tumor is a GD2 positive
neuroblastoma, retinoblastoma, melanoma, small cell lung cancer,
brain tumor such as glioblastoma, osteosarcoma, rhabdomyosarcoma,
Ewing's sarcoma in children and adolescents, or liposarcoma,
fibrosarcoma, leiomyosarcoma or another soft tissue sarcoma in
adults. In a preferred embodiment the GD2 positive tumor is a
neuroblastoma. The neuroblastoma treated with a method or use of
the invention is preferably a high risk neuroblastoma.
[0014] In a preferred embodiment an antibody of the invention
comprises a heavy chain variable region with the amino acid
sequence
TABLE-US-00001 EVQLLQSGPE LEKPGASVMI SCKASGSSFT GYNMNWVRQN
IGKSLEWIGA IDPYYGGTSY NQKFKGRATL TVDKSSSTAY MHLKSLTSED SAVYYCVSGM
EYWGQGTSVT VSS;
and a light chain variable region with the amino acid sequence
TABLE-US-00002 EIVMTQSPAT LSVSPGERAT LSCRSSQSLV HRNGNTYLHW
YLQKPGQSPK LLIHKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP
PLTFGAGTKL ELK; and
[0015] a heavy chain comprising an IgA hinge and C.sub.H2
domain.
[0016] Provided herein is an engineered antibody or fragment
thereof for use in improvement of patient compliance and/or a
reduction of a side effect associated with immunoglobulin G (IgG)
antibody therapy, wherein the engineered antibody or fragment
thereof comprises: (a) CDR1, CDR2, and CDR3 of an immunoglobulin G
(IgG); and (b) the constant regions or portion thereof of an
immunoglobulin A (IgA), wherein the engineered antibody or fragment
thereof results in reduction of at least one side effect upon
administration to the subject as compared to administration of a
comparable amount of a corresponding IgG antibody.
[0017] In an aspect, the engineered antibody or fragment thereof
further comprises a heavy chain and light chain variable region of
IgG. In an aspect, the constant regions or portion thereof of the
IgA comprises a hinge, a C.sub.H2 constant region, or a combination
thereof. In an aspect, the constant regions or portion thereof
comprises a hinge and a C.sub.H2 constant region of the IgA. In an
aspect, the constant regions comprise a constant heavy and constant
light chain of the IgA. In an aspect, the constant regions or
portion thereof of the IgA comprises a hinge and a constant heavy
chain domain. In an aspect, the antibody or fragment thereof is
chimerized, humanized, human, or non-human. In an aspect, the
constant regions or portion thereof is human.
[0018] In an aspect, the side effect comprises an innate immune
response. In some cases, the innate immune response comprises a
complement response. In some cases, the complement response
comprises IgG binding to Fc.gamma.Rs or C1q. In certain
embodiments, the side effect comprises one or more of pain,
visceral hypersensitivity, allodynia, hyperalgesia, allergic
reactions and flu-like symptoms. In some cases, the pain is acute
pain, migraine or intense visceral pain.
[0019] In some cases, determining the reduction of the side effect
is measured by performing flow cytometry, an in vitro assay, or a
combination thereof. In some cases, the flow cytometry comprises
determining cellular lysis, binding, cell death, receptor
expression, or a combination thereof. In some cases, the flow
cytometry comprises determining live/dead staining of a cell. In
some cases, the in vitro assay comprises ELISA, antibody-dependent
cell-mediated cytotoxicity (ADCC), complement-dependent
cytotoxicity (CDC), hemolytic assay, or a combination thereof.
[0020] In some cases, the engineered antibody or fragment thereof
when administered results in an increased antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or reduced
complement-dependent cytotoxicity (CDC) as compared to the
corresponding IgG antibody that comprises the same CDR1, CDR2, and
CDR3. In an aspect, the reduction comprises from 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, to 100% of the side effect as compared to administering a
comparable IgG antibody that comprises the same CDR1, CDR2, and
CDR3.
[0021] In some cases, the antibody or fragment thereof binds a
target present on a non-cancerous cell, a cancer cell, or a
combination thereof. In some cases, the antibody or fragment
thereof binds a target from a brain-metastasizing cancer. In some
cases, the antibody or fragment thereof binds one or more of GD2,
ALK, hNET, GD3, and CD20. In some cases, the GD2 positive tumor is
neuroblastoma, retinoblastoma, melanoma, small cell lung cancer,
glioblastoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma,
liposarcoma, fibrosarcoma, leiomyosarcoma, and any combinations
thereof. In an aspect, the antibody or fragment thereof binds a
neuroblastoma cell. In some cases, the ALK (anaplastic lymphoma
kinase) positive tumor is an anaplastic large-cell lymphoma, an
adenocarcinoma of the lung, a neuroblastoma, an inflammatory
myofibroblastic tumor, a renal cell carcinomas, esophageal squamous
cell carcinoma, breast cancer, a colonic adenocarcinoma, a
glioblastoma multiforme or an anaplastic thyroid cancer. In some
cases, the hNET (human norepinephrine transporter) positive tumor
is a bladder tumor, breast tumor, prostate tumor, carcinoma,
leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's lymphoma,
Non-Hodgkin's lymphoma, melanoma, neuroblastoma, ovarian tumor,
pancreatic tumor or a retinoblastoma. In some case, the GD3
positive tumor is a neuroectodermal tumor of the center nervous
system, glioma, neuroblastoma, retinoblastoma, ependymoma, sarcoma,
melanoma, breast cancer, ovarian cancer, glioblastoma, Ewing's
sarcoma, or small cell lung carcinoma. In some cases the CD20
positive tumor is a leukemia, a lymphoma or a neuroblastoma.
[0022] Provided herein is a pharmaceutical composition comprising
an engineered antibody or fragment thereof and a pharmaceutically
acceptable carrier.
[0023] Provided herein is a kit comprising an engineered antibody
or fragment thereof and instructions for use thereof.
[0024] Provided herein is a method comprising: administering to a
subject a pharmaceutical composition comprising an antibody or
fragment thereof comprising: (a) CDR1, CDR2, and CDR3 of an
immunoglobulin G (IgG); and (b) the constant regions or portion
thereof of an immunoglobulin A (IgA), wherein the administering
results in a reduction of a side effect in the subject as compared
to administering a comparable IgG antibody that comprises the same
CDR1, CDR2, and CDR3.
[0025] Provided herein is an engineered immunoglobulin G (IgG)
antibody fragment, wherein the engineered IgG antibody fragment
comprises at least a hinge domain and a C.sub.H2 domain of a human
IgA antibody. In some cases, the engineered IgG antibody fragment,
when administered to a subject, results in increased
antibody-dependent cell-mediated cytotoxicity (ADCC) and/or reduced
complement-dependent cytotoxicity (CDC) as compared to a comparable
antibody fragment absent the hinge domain and a C.sub.H2 domain of
a human IgA antibody
[0026] Provided herein is a method of treating pain or allodynia
associated with IgG administration comprising administering to a
subject an engineered IgG antibody or fragment thereof comprising
the constant regions of an IgA antibody. Provided herein is a
method of reducing complement activation resulting from IgG
administration comprising administering to a subject an engineered
IgG antibody or fragment thereof comprising the constant regions of
an IgA antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Gangliosides are sialic acid-containing glycosphingolipids
that play important roles in signal transduction as well as cell
adhesion and recognition. The ganglioside GD2 is a b-series
ganglioside that requires the enzymes GD3 synthase and GD2 synthase
to add sialic acid units onto its precursor G.sub.M2. Normal
tissues generally express a-series gangliosides, whereas b-series
gangliosides are expressed during fetal development and are
restricted primarily to the nervous system in healthy adults and at
low levels in peripheral nerves and skin melanocytes. A structure
of the ganglioside GD2 is depicted in FIG. 10.
[0028] The ganglioside GD2 is highly expressed on
neuroectoderm-derived tumors and sarcomas, including neuroblastoma,
retinoblastoma, melanoma, small cell lung cancer, brain tumors,
osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma in children and
adolescents, as well as liposarcoma, fibrosarcoma, leiomyosarcoma
and other soft tissue sarcomas in adults. GD2 expression in normal
individuals appears to be limited to the brain and certain
peripheral nerves and melanocytes. As the brain is typically not
accessible for normal circulating antibodies GD2 is considered an
attractive target for tumor-specific therapy (For review see
Mahiuddin et al., 2014; FEBS letters 588: 288-297). Mahiuddin et al
describe various GD2 targeted approaches among which there are the
GD2 specific antibodies. A number of targeted therapies has reached
the clinic and show promise in phase I, II and III trials.
[0029] Various GD2 antibodies are presently under development. The
antibodies are thought to induce ADCC and CDC, for which in
particular neuroblastoma appears to be relatively sensitive.
Various avenues are being pursued to improve the efficacy and
reduce the toxicity of the targeted approaches. The most frequently
used antibodies all originate from murine IgG3 antibodies. The
murine antibodies have been humanized in recent years. A number of
variants have been made. Murine antibody 14.18 has been chimerized
to ch14.18 and humanized into hu14.18 both have a human IgG1
background (Mahiuddin et al., 2014; FEBS letters 588: 288-297). The
murine IgG3 antibody 3F8 has been used in humans. To reduce human
anti-mouse responses and improve the efficacy of the antibody while
reducing toxicity 3F8 was chimerized (ch3F8) and humanized 3F8
(hu3F8-IgG1 and hu3F8-IgG4). In GD2 binding studies by SPR, ch3F8
and hu3F8 maintained KD comparable to m3F8. Unlike other anti-GD2
antibodies, m3F8, ch3F8 and hu3F8 had substantially slower koff.
Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth
in vitro, while cross-reactivity with other gangliosides was
comparable to that of m3F8. Both peripheral blood mononuclear cell
(PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8
and hu3F8-IgG1 were more potent than m3F8. Hu3F8-IgG4 had near
absent PBMC-ADCC and CDC. Hu3F8 and m3F8 had similar tumor-to-non
tumor ratios in biodistribution studies. Anti-tumor effect against
neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8 (see
Cheung et al., 2012; Oncoimmunology 1.4: 477-486).
[0030] An antibody (Ab), also known as an immunoglobulin (Ig), is a
large protein. An antibody interacts with various components of the
immune system. Some of the interactions are mediated by its Fc
region (located at the base), which contains site(s) involved in
these interactions.
[0031] Antibodies are proteins belonging to the immunoglobulin
superfamily. They typically have two heavy chains and two light
chains. There are several different types of antibody heavy chains
that define the five different types of crystallizable fragments
(Fc) that may be attached to the antigen-binding fragments. The
five different types of Fc regions allow antibodies to be grouped
into five isotypes. An Fc region of a particular antibody isotype
is able to bind to its specific Fc receptor (FcR) thus allowing the
antigen-antibody complex to mediate different roles depending on
which FcR it binds. The ability of an IgG antibody to bind to its
corresponding FcR is modulated by the presence/absence of
interaction sites and the structure of the glycan(s) (if any)
present at sites within its Fc region. The ability of antibodies to
bind to FcRs helps to direct the appropriate immune response for
each different type of foreign object they encounter.
[0032] Though the general structure of all antibodies is similar, a
region at the tip of the protein is extremely variable, allowing
millions of antibodies with slightly different tip structures, or
antigen-binding sites, to exist. This region is known as the
hypervariable region. The enormous diversity of antigen binding by
antibodies is largely defined by the hypervariable region and the
variable domain containing the hypervariable region.
[0033] An antibody of the invention is typically a full-length
antibody. The term `full length antibody` is defined as comprising
an essentially complete immunoglobulin molecule, which however does
not necessarily have all functions of an intact immunoglobulin. For
the avoidance of doubt, a full length antibody has two heavy and
two light chains. Each chain contains constant (C) and variable (V)
regions. A heavy chain of a full length antibody typically
comprises a C.sub.H1, a C.sub.H2, a C.sub.H3, a VH region and a
hinge region. A light chain of a full length antibody typically
comprises a CL region and a VL region.
[0034] An antibody binds to antigen via the variable region domains
contained in the Fab portion. An antibody variable domain comprises
a heavy chain variable region and a light chain variable region.
Full length antibodies according to the invention encompass heavy
and light chains wherein mutations may be present that provide
desired characteristics. Full length antibodies should not have
deletions of substantial portions of any of the regions. However,
IgG molecules wherein one or several amino acid residues are
substituted, inserted, deleted or a combination thereof, without
essentially altering the antigen binding characteristics of the
resulting antibody, are embraced within the term "full length"
antibody. For instance, a `full length" antibody can have a
substitution, insertion, deletion or a combination thereof, of
between 1 and 10 (inclusive) amino acid residues, preferably in
non-CDR regions, wherein the deleted amino acids are not essential
for the binding specificity of the antibody.
[0035] The four human IgG isotypes bind the activating Fc.gamma.
receptors (Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIIa), the
inhibitory Fc.gamma.RIIb receptor, and the first component of
complement (C1q) with different affinities, yielding very different
effector functions. Binding of IgG to the Fc.gamma.Rs or C1q
depends on residues located in the hinge region and the C.sub.H2
domain. Two regions of the C.sub.H2 domain are critical for
Fc.gamma.Rs and C1q binding, and have unique sequences in IgG2 and
IgG4. Substitutions into human IgG1 of IgG2 residues at positions
233-236 were shown to greatly reduce ADCC and CDC. Furthermore,
Idusogie et al. demonstrated that alanine substitution at different
positions, including K322A, significantly reduced complement
activation. Similarly, mutations in the CH2 domain of murine IgG2A
were shown to reduce the binding to Fc.gamma.RI, and C1q. Numerous
mutations have been made in the C.sub.H2 domain of human IgG1 and
their effect on ADCC and CDC tested in vitro. Notably, alanine
substitution at position 333 was reported to increase both ADCC and
CDC. Fc-receptor functions are among others reviewed in Bruhns et
al., 2009. Blood. 113(16):3716-25; Armour et al., 1999. Eur J
Immunol. 29(8):2613-24; Shields et al., 2001. J Biol Chem.
276(9):6591-604; Idusogie et al., 2000. J Immunol. 164(8):4178-84;
Steurer et al., 1995. J Immunol. 155(3):1165-74; and Idusogie. et
al., 2001. J Immunol. 166(4):2571-5. IgG1 antibodies have a C1q
binding site through which complement activation through the
classical pathway is achieved. IgA antibodies can also activate
complement. This can be through the alternative pathway, the
complement lectin pathway and the classical pathway (Jarvis et al.,
J Immunol. 1989; 143(5):1703-9; Hiemstra et al., European journal
of immunology. 1987; 17(3):321-6; Pfaffenbach et al., The Journal
of experimental medicine. 1982; 155(1):231-47; Roos et al., J
Immunol. 2001; 167(5):2861-8; Pascal et al., Haematologica DOI:
10.3324/haemato1.2011.061408; and Lohse et al., Britisch Journal of
Haematology. 2011doi: 10.1111/bjh.14624).
[0036] The present inventors found that ADCC activity of a murine,
murine chimerized or humanized IgG or human IgM GD2 antibody with
unmodified constant regions can be increased by replacing at least
the hinge domain and the C.sub.H2 domain thereof by a hinge domain
and C.sub.H2 domain of a human IgA antibody, preferably a human
IgA1 antibody. In a preferred embodiment also the C.sub.H3 domain
is of a human IgA antibody. In a preferred embodiment the antibody
comprises an essentially complete IgA constant region. The
invention therefore provides an antibody that can bind GD2 and that
comprises ADCC activity and that comprises a hinge domain and
C.sub.H2 domain of a human IgA antibody. A GD2 antibody of the
invention has a reduced capacity to induce CDC. A GD2 antibody of
the invention has a reduced capacity to induce pain when compared
to the murine, murine chimerized or humanized IgG or human IgM
original GD2 antibody.
[0037] An IgA constant domain or hinge region may have one or more
amino acid insertions, deletions, substitutions, additions or a
combination thereof at one or more positions with respect to a germ
line IgA domain or hinge region. An IgA domain or hinge region
preferably has at most 4 amino acid insertions, deletions,
substitutions, additions or a combination thereof, preferably at
most 3; 2 or preferably at most 1 amino acid insertions, deletions,
substitutions, additions or a combination thereof. Such a protein
is still an IgA constant domain or hinge region. An IgA constant
domain or hinge region may have one or more amino acid insertions,
deletions, substitutions, additions or a combination thereof at one
or more positions with respect to a germ line IgA. The IgA constant
part (including the three domains and the hinge region) can have 0;
1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; or 15 amino acid
insertions, deletions, substitutions, additions or a combination
thereof.
[0038] In one embodiment the invention provides an anti-ganglioside
GD2 antibody that comprises an antibody variable domain and
antibody constant domains, wherein the variable domain comprises a
heavy and light chain variable region comprising respectively at
least the CDR3 of the heavy and light chain variable regions of
antibody ch14.18 and an IgA hinge and C.sub.H2 domain. The variable
domain preferably comprises a heavy and light chain variable region
comprising respectively at least the CDR1, CDR2 and CDR3 of the
heavy and light chain variable regions of antibody ch14.18 as
depicted in FIG. 1A and FIG. 1B respectively. The variable domain
preferably comprises a heavy and light chain variable region of
antibody ch14.18 as depicted in FIG. 1A and FIG. 1B
respectively.
[0039] The anti-ganglioside GD2 antibody heavy chain variable
region is preferably a heavy chain variable region of antibody
ch14.18 with 0-5 amino acid insertions, deletions, substitutions,
additions or a combination thereof at one or more positions with
respect to the heavy chain variable region sequence of antibody
ch14.18 indicated in FIG. 1A. The one or more positions are
preferably not positions in the CDR1, CDR2 and CDR3 regions. The
sequence of the CDRs is thus as indicated for heavy chain variable
region of antibody ch14.18 of FIG. 1A. It is preferred that the
heavy chain variable region has 0-4 amino acid insertions,
deletions, substitutions, additions or a combination thereof at one
or more positions with respect to the heavy chain variable region
sequence of antibody ch14.18 indicated in FIG. 1A, wherein the one
or more positions are not positions in the CDR1, CDR2 and CDR3
regions. It is preferred that the heavy chain variable region has
0-3, more preferably 0-2, more preferably 0-1 amino acid
insertions, deletions, substitutions, additions or a combination
thereof at one or more positions with respect to the heavy chain
variable region sequence of antibody ch14.18 indicated in FIG. 1A,
wherein the one or more positions are not positions in the CDR1,
CDR2 and CDR3 regions. In a preferred embodiment a heavy chain
variable region in the antibody of the invention has 0 amino acid
insertions, deletions, substitutions, additions or a combination
thereof with respect to the heavy chain variable region sequence of
antibody ch14.18 in FIG. 1A.
[0040] The anti-ganglioside GD2 antibody light chain variable
region is preferably a light chain variable region of antibody
ch14.18 with 0-5 amino acid insertions, deletions, substitutions,
additions or a combination thereof at one or more positions with
respect to the light chain variable region sequence of antibody
ch14.18 indicated in FIG. 1B. The one or more positions are
preferably not positions in the CDR1, CDR2 and CDR3 regions. The
sequence of the CDRs is thus as indicated for light chain variable
region of antibody ch14.18 of FIG. 1B. It is preferred that the
light chain variable region has 0-4 amino acid insertions,
deletions, substitutions, additions or a combination thereof at one
or more positions with respect to the light chain variable region
sequence of antibody ch14.18 indicated in FIG. 1B, wherein the one
or more positions are not positions in the CDR1, CDR2 and CDR3
regions. It is preferred that the light chain variable region has
0-3, more preferably 0-2, more preferably 0-1 amino acid
insertions, deletions, substitutions, additions or a combination
thereof at one or more positions with respect to the light chain
variable region sequence of antibody ch14.18 indicated in FIG. 1B,
wherein the one or more positions are not positions in the CDR1,
CDR2 and CDR3 regions. In a preferred embodiment a light chain
variable region in the antibody of the invention has 0 amino acid
insertions, deletions, substitutions, additions or a combination
thereof with respect to the light chain variable region sequence of
antibody ch14.18 in FIG. 1B.
[0041] In another embodiment the invention provides an
anti-ganglioside GD2 antibody that comprises an antibody variable
domain and antibody constant domains, wherein the variable domain
comprises a heavy and light chain variable region comprising
respectively at least the CDR3 of the heavy and light chain
variable regions of antibody 3F8 and an IgA hinge and C112 domain.
The variable domain preferably comprises a heavy and light chain
variable region comprising respectively at least the CDR1, CDR2 and
CDR3 of the heavy and light chain variable regions of antibody 3F8
(as depicted in FIGS. 1E-1F). The variable domain preferably
comprises a heavy and light chain variable region of antibody 3F8
as depicted in FIGS. 1E-1F. The anti-ganglioside GD2 antibody heavy
chain variable region is preferably a heavy chain variable region
of antibody 3F8 with 0-5 amino acid insertions, deletions,
substitutions, additions or a combination thereof at one or more
positions with respect to the heavy chain variable region sequence
of antibody 3F8 indicated in FIG. 1E. The one or more positions are
preferably not positions in the CDR1, CDR2 and CDR3 regions. The
sequence of the CDRs is thus as indicated for heavy chain variable
region of antibody 3F8 of FIG. 1E. It is preferred that the heavy
chain variable region has 0-4 amino acid insertions, deletions,
substitutions, additions or a combination thereof at one or more
positions with respect to the heavy chain variable region sequence
of antibody 3F8 indicated in FIG. 1E, wherein the one or more
positions are not positions in the CDR1, CDR2 and CDR3 regions. It
is preferred that the heavy chain variable region has 0-3, more
preferably 0-2, more preferably 0-1 amino acid insertions,
deletions, substitutions, additions or a combination thereof at one
or more positions with respect to the heavy chain variable region
sequence of antibody 3F8 indicated in FIG. 1E, wherein the one or
more positions are not positions in the CDR1, CDR2 and CDR3
regions. In a preferred embodiment a heavy chain variable region in
the antibody of the invention has 0 amino acid insertions,
deletions, substitutions, additions or a combination thereof with
respect to the heavy chain variable region sequence of antibody 3F8
in FIG. 1E.
[0042] The anti-ganglioside GD2 antibody light chain variable
region is preferably a light chain variable region of antibody 3F8
with 0-5 amino acid insertions, deletions, substitutions, additions
or a combination thereof at one or more positions with respect to
the light chain variable region sequence of antibody 3F8 indicated
in FIG. 1F. The one or more positions are preferably not positions
in the CDR1, CDR2 and CDR3 regions. The sequence of the CDRs is
thus as indicated for light chain variable region of antibody 3F8
of FIG. 1F. It is preferred that the light chain variable region
has 0-4 amino acid insertions, deletions, substitutions, additions
or a combination thereof at one or more positions with respect to
the light chain variable region sequence of antibody 3F8 indicated
in FIG. 1F, wherein the one or more positions are not positions in
the CDR1, CDR2 and CDR3 regions. It is preferred that the light
chain variable region has 0-3, more preferably 0-2, more preferably
0-1 amino acid insertions, deletions, substitutions, additions or a
combination thereof at one or more positions with respect to the
light chain variable region sequence of antibody 3F8 indicated in
FIG. 1F, wherein the one or more positions are not positions in the
CDR1, CDR2 and CDR3 regions. In a preferred embodiment a light
chain variable region in the antibody of the invention has 0 amino
acid insertions, deletions, substitutions, additions or a
combination thereof with respect to the light chain variable region
sequence of antibody 3F8 in FIG. 1F.
[0043] IgA has two subclasses (IgA1 and IgA2) and can be produced
as a monomeric as well as a dimeric form. The antibody in the
present invention is preferably a monomeric antibody. The IgA
elements in an antibody of the invention are preferably human IgA
elements. An IgA element can be an IgA1 element or an IgA2 element.
IgA elements in an antibody of the invention can be all IgA1
elements or all IgA2 elements or a combination of IgA1 and IgA2
elements. An IgA element is preferably a human IgA element.
Preferably all IgA element in the antibody are human IgA elements.
The IgA elements can be IgA1 elements, preferably human IgA1
elements. The IgA elements can also be IgA2, preferably IgA2m(1)
elements, preferably human IgA1 elements. The C.sub.H1 domain
and/or C.sub.H3 domain of the antibody can be an IgG C.sub.H1
domain, an IgG C.sub.H3 domain or a combination thereof. It is
preferred that the C.sub.H1 domain, C.sub.H3 domain or combination
thereof is an IgA C.sub.H1 domain, an IgA C.sub.H3 domain or a
combination thereof. It is preferred that the IgA C.sub.H1 domain
and/or hinge region is a human IgA C.sub.H1 domain and/or human IgA
hinge region. Said human IgA C.sub.H1 domain and/or human IgA hinge
region is preferably an human IgA1 C.sub.H1 domain or human IgA1
hinge region. Said human IgA C.sub.H1 domain and/or human IgA hinge
region is preferably an human IgA2m(1) C.sub.H1 domain or human
IgA2m(1) hinge region. The constant domains and hinge region of the
antibody are preferably human constant regions and hinge region,
preferably of a human IgA antibody. The constant domains and hinge
region of the antibody are preferably human IgA1 or human IgA2m(1)
constant domains and hinge region.
[0044] A human constant region can have 0-15 amino acid changes
with respect to a human allele as found in nature. An amino acid
change may be introduced for various reasons. Non-limiting examples
include but are not limited to improving production or homogeneity
of the antibody, adapting half-life in the circulation, stability
of the HC/LC combination, optimizing glycosylation, adjusting
dimerization or complex formation, adjusting ADCC activity. A human
constant region can have 0; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12;
13; 14; and 15 amino acid changes with respect to a human allele as
found in nature. The changed amino acid is preferably one chosen
from an amino acid at a corresponding position of a different
isotype.
[0045] In one embodiment the constant regions of the heavy chain
are IgA2 constant regions, preferably human IgA2 constant regions,
preferably human IgA2m(1). In one aspect the human constant region
is a mutated IgA2m(1) sequence.
[0046] In one embodiment the antibody comprises the constant
regions of an IgA2m(1) sequence, preferably with at least one and
preferably at least 2; 3; 4; 5; and preferably at least 7 of the
following mutations: N166G; P221R; N337T; I338L; T339S; C331S; and
mutation of the C-terminal amino acid sequence which is a human
IgA2m(1) antibody is " . . . VDGTCY" into " . . . VDGT. FIG. 12A
shows the sequence of human IgA1; IgA2m(1) and a preferred mutated
IgA2m(1) sequence (hIgA2.0); see also FIG. 12A.
[0047] In one embodiment the GD2 antibody comprises a heavy chain
with the constant regions of FIG. 12A with 0; 1; 2; 3; 4; 5; 6; 7;
8; 9; 10; 11; 12; 13; 14; and 15 amino acid changes with respect to
the sequence provided in FIG. 12A, provided that amino acids at
positions 166; 221; 337; 338; 339; and 331 are 166G; 221R; 337T;
338L; 339S and 331S. Preferably the C-terminal amino acid sequence
which is a human IgA2m(1) antibody is " . . . VDGTCY" is " . . .
VDGT".
[0048] A human IgA sequence of whatever subtype relates to the
heavy chain of the antibody. The heavy chain is subject to isotype
switching.
[0049] The mention IgA antibody preferably comprises a ch14.18
variable domain or a 3F8 variable domain, preferably a ch14.18
variable domain.
[0050] An anti-ganglioside GD2 antibody comprising: [0051] a heavy
chain variable region of antibody ch14.18 with 0-5 amino acid
insertions, deletions, substitutions, additions or a combination
thereof at one or more positions with respect to the heavy chain
variable region sequence of antibody ch14.18 indicated in FIG. 1A.
The one or more positions are preferably not positions in the CDR1,
CDR2 and CDR3 regions. The sequence of the CDRs is thus as
indicated for heavy chain variable region of antibody ch14.18 of
FIG. 1A. It is preferred that the heavy chain variable region has
0-4 amino acid insertions, deletions, substitutions, additions or a
combination thereof at one or more positions with respect to the
heavy chain variable region sequence of antibody ch14.18 indicated
in FIG. 1A, wherein the one or more positions are not positions in
the CDR1, CDR2 and CDR3 regions. It is preferred that the heavy
chain variable region has 0-3, more preferably 0-2, more preferably
0-1 amino acid insertions, deletions, substitutions, additions or a
combination thereof at one or more positions with respect to the
heavy chain variable region sequence of antibody ch14.18 indicated
in FIG. 1A, wherein the one or more positions are not positions in
the CDR1, CDR2 and CDR3 regions. In a preferred embodiment a heavy
chain variable region in the antibody of the invention has 0 amino
acid insertions, deletions, substitutions, additions or a
combination thereof with respect to the heavy chain variable region
sequence of antibody ch14.18 in FIG. 1A; [0052] a light chain
variable region of antibody ch14.18 with 0-5 amino acid insertions,
deletions, substitutions, additions or a combination thereof at one
or more positions with respect to the light chain variable region
sequence of antibody ch14.18 indicated in FIG. 1B. The one or more
positions are preferably not positions in the CDR1, CDR2 and CDR3
regions. The sequence of the CDRs is thus as indicated for light
chain variable region of antibody ch14.18 of FIG. 1B. It is
preferred that the light chain variable region has 0-4 amino acid
insertions, deletions, substitutions, additions or a combination
thereof at one or more positions with respect to the light chain
variable region sequence of antibody ch14.18 indicated in FIG. 1B,
wherein the one or more positions are not positions in the CDR1,
CDR2 and CDR3 regions. It is preferred that the light chain
variable region has 0-3, more preferably 0-2, more preferably 0-1
amino acid insertions, deletions, substitutions, additions or a
combination thereof at one or more positions with respect to the
light chain variable region sequence of antibody ch14.18 indicated
in FIG. 1B, wherein the one or more positions are not positions in
the CDR1, CDR2 and CDR3 regions. In a preferred embodiment a light
chain variable region in the antibody of the invention has 0 amino
acid insertions, deletions, substitutions, additions or a
combination thereof with respect to the light chain variable region
sequence of antibody ch14.18 in FIG. 1B; [0053] an IgA heavy chain,
preferably comprising an amino acid sequence as depicted in FIG.
12A with 0; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; and 15
amino acid changes with respect to the sequence provided in FIG.
12A, provided that amino acids at positions 166; 221; 337; 338;
339; and 331 are 166G; 221R; 337T; 338L; 339S and 331S. Preferably
the C-terminal amino acid sequence which is a human IgA2m(1)
antibody is " . . . VDGTCY" is " . . . VDGT"; and [0054] a light
chain constant domain, preferably of antibody ch14.18.
[0055] The anti-ganglioside GD2 antibody of the invention
preferably exhibits more antibody-dependent cell-mediated
cytotoxicity (ADCC) than the antibody dinutuximab when measured in
a suitable in vitro ADCC assay. It preferably exhibits less
complement-dependent cytotoxicity (CDC) than the antibody
dinutuximab when measured in a suitable in vitro CDC assay. Various
ADCC and CDC assays are available to the person skilled in the art.
In the context of the present invention it is preferred that an
ADCC assay or a CDC assay as described in the examples is used.
ADCC function of an antibody as claimed is preferably measured in a
classical chromium release assay (as described for instance in
examples). CDC function of an antibody as claimed is preferably
measured in a method based on 7-AAD positivity in flow cytometry
(see for instance the examples). The antibody preferably exhibits
20% or less, more preferably 10% or less of the
complement-dependent cytotoxicity (CDC) of the antibody dinutuximab
when measured in a suitable in vitro CDC assay. In a preferred
embodiment the antibody comprises an albumin-binding domain (ABD)
attached to a heavy or light chain of the antibody. Reference is
made to Meyer, et al., 2016; MAbs. Vol. 8. No. 1; for details on
the linkage of the albumin-binding domain to a heavy or light
chain.
[0056] In another embodiment another domain is added to increase
the half-life of the antibody. In a preferred embodiment the domain
the DIII domain of human albumin. Such a domain is preferably
physically linked to a constant region of an IgA part of the
antibody.
[0057] In an aspect, an engineered antibody or fragment thereof, as
provided herein comprising the variable region of an IgG antibody
and the constant regions of an IgA antibody, can reduce a side
effect. In an aspect, an engineered antibody or fragment thereof,
as provided herein, can reduce a side effect associated with an IgG
antibody therapy. In an aspect, an engineered antibody or fragment
thereof, as provided herein, can reduce a side effect associated
with an innate immune response. Side effects associated with such
innate immune response can be inflammation, complement system
activation, white blood cell (mast cell, phagocyte, macrophage,
neutrophil, dendritic cell, basophil, eosinophil, natural killer
cell, and gamma delta cell activity. In some cases, an engineered
antibody or fragment thereof, as provided herein, can reduce a side
effect associated with complement system activation.
[0058] Complement system activation can be a biochemical cascade of
the immune system that complements the ability of antibodies or
fragments thereof to clear pathogens or mark them for destruction.
The complement cascade comprises a variety of plasma proteins,
which are synthesized in the liver, these proteins have various
functions comprising: triggering the recruitment of inflammatory
cells, tagging pathogens for destruction (opsonization),
perforating the plasma membrane of pathogens, eliminating
neutralized antigen-antibody complexes, and any combination
thereof. In some aspects, a reduction of a side effect can comprise
the reduction of complement system activation.
[0059] In an aspect, an IgG antibody or portion thereof can react
with a target resulting in binding of C1q to the Fc portion of
antigen-bound IgG after which C1r and C1s attach to form C1, an
enzyme associated with complement activation. C1 can proceed to
enzymatically cleave C4 into C4a and C4b. C4b binds to adjacent
proteins and carbohydrates on the surface of the target and then
binds C2. Activated C1 can cleave C2 into C2a and C2b forming
C4b2a, a C3 convertase. C3 convertase can cleave C3 into C3a and
C3b. In further steps of the complement activation pathway, C3b can
bind to C4b2a, a C3 convertase, to form C4b2a3b, a C5 convertase
that can cleave C5 into C5a and C5b. C5b can bind to the target and
also bind C6, C7, c8, and C9 to form C5b6789n, the membrane attack
complex (MAC). MAC can destroy gram-negative bacteria as well as
cells displaying foreign antigens (such as virus-infected cells,
tumor cells, to name a few) by causing their lysis. In an aspect, a
reduction in a complement response can be measured by the absence
or reduced level of any one of: C1q, C1r, C1s, C1, C4, C4a, C4b,
C2, C2a, C2b, C4b2a, C3, C3a, C3b, C4b2a3b, C5, C5a, C5b, C6, C7,
C8, C9, C5b6789m, MAC, and any combination thereof. In an aspect, a
reduction in a complement response can refer to a reduction of
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% of binding of a
complement factor selected from any one of: C1q, C1r, C1s, C1, C4,
C4a, C4b, C2, C2a, C2b, C4b2a, C3, C3a, C3b, C4b2a3b, C5, C5a, C5b,
C6, C7, C8, C9, C5b6789m, and MAC to the engineered antibody that
comprises an IgG variable region and the IgA constant regions or
portion thereof as described herein, as compared to a comparable
amount of an IgG antibody.
[0060] A reduction of a complement response can be determined by
performing an ELISA, flow cytometry, hematology, or an in vitro
assay to quantify an amount of a complement factor selected from
any one of: C1q, C1r, C1s, C1, C4, C4a, C4b, C2, C2a, C2b, C4b2a,
C3, C3a, C3b, C4b2a3b, C5, C5a, C5b, C6, C7, C8, C9, C5b6789m, MAC,
and any combination thereof. In some aspects, an antibody or
fragment thereof provided herein can result in a lower level of a
complement factor as compared to a comparable antibody or fragment
thereof comprising the provided CDR1, CDR2, and CDR3 regions. In
some aspects, an engineered IgG-IgA antibody or fragment thereof
provided herein can result in a lower level of a complement factor
as compared to a comparable IgG antibody or fragment thereof
comprising the same CDR1, CDR2, and CDR3. A reduction of a
complement response can also be measured by flow cytometric
quantification of complement dependent lysis of target cells as
well as described in the examples.
[0061] Additional side effects which can be mitigated or reduced by
administration of an engineered antibody or fragment thereof
described herein, as compared to administration of an IgG, can
refer to any side effects associated with IgG antibody therapy.
Side effects associated with IgG antibody therapy can include
inflammation, thromboembolic events, haemolytic events,
complement-system associated events, and a combination thereof. In
some cases, an IgG antibody therapy can result in side effects such
as: inflammation, hypertension, hypotension, pain, fever,
urticaria, allergy, chill, weakness, diarrhea, nausea, vomit, rash,
itch, cough, constipation, edema, headache, fever, shortness of
breath, muscle ache, pain, decreased appetite, insomnia, dizziness,
anaphylaxis, thrombosis, heart failure, bleeding, hepatitis,
enterocolitis, mucositis, cytokine syndrome, hypothyroidism,
hyponatremia, hypokalemia, capillary leak syndrome, and
allodynia.
[0062] In an aspect, reducing a complement response,
complement-associated side-effect, or a toxicity can improve
treatment compliance of a subject in need thereof. In some cases,
an antibody or fragment thereof provided herein, such as engineered
antibodies, can be administered at a higher dose, a longer
treatment period, or with more frequency as compared to a
comparable IgG antibody comprising the same CDR1, CDR2, and CDR3
regions. In some cases, an antibody or fragment thereof provided
herein, such as engineered antibodies, can be administered at a
higher dose from about 1.times., 2.times., 3.times., 4.times.,
5.times., 6.times., 7.times., 8.times., 9.times., 10.times., over
that of a comparable IgG antibody comprising the same CDR1, CDR2,
and CDR3 regions. In some cases, an antibody or fragment thereof
provided herein, such as engineered antibodies, can be administered
for a longer time period from about 1 hour, 5 hrs, 10 hrs, 1 day, 2
days, 3 days, 4 days, 5 days, 7 days, 14 days, 24 days, 30 days,
monthly, bimonthly, bi-yearly, yearly, and daily over that of a
comparable IgG antibody comprising the same CDR1, CDR2, and CDR3
regions. In some cases, an antibody or fragment thereof provided
herein, such as engineered antibodies, can be administered more
frequency such as hourly, daily, weekly, monthly, yearly as
compared to a comparable IgG antibody comprising the same CDR1,
CDR2, and CDR3 regions.
[0063] A reason for changing an amino acid at a certain position
can be immunogenicity. Other reasons include but are not limited to
improving production or homogeneity of the antibody. Antibodies of
the present invention have variable heavy and variable light chain
regions derived from a murine background. Antibodies with such
variable domains can be used in humans. Presently it is preferred
to de-immunize such variable domains. De-immunization typically
involves the modification of the murine sequence into a more human
sequence whenever possible. Typically such modifications are
directed towards removing one or more T-cell epitopes or one more
B-cell epitopes from the variable domain. In a preferred embodiment
one or more (human) T-cell epitopes have been removed by
replacement of at least one amino acid of the epitope with a
different amino acid.
[0064] Often it is sufficient to substitute the so-called "anchor"
amino acid. Suitable replacement amino acids can be obtained from
somatic cell hypermutants of the particular VH or VL. Replacement
with an amino acid that is naturally present at that position in a
human antibody is preferred. Also human B-cell epitopes can be
removed by replacement of at least one amino acid of the epitope
with a different amino acid. Often it is sufficient to substitute
only one amino acid of the epitope. Suitable replacement amino
acids can be obtained from somatic cell hypermutants of the
particular VH or VL. Replacement with an amino acid that is
naturally present at that position in a human antibody is
preferred. Preferably a variable domain of the invention is
modified with respect to one or more exterior residues. Such
residues are readily encountered by the immune system and are
preferably selectively replaced with human residues to provide a
hybrid molecule that comprises either a weakly immunogenic or
substantially non-immunogenic surface. Suitable replacement amino
acids can be obtained from somatic cell hypermutants of the
particular VH or VL. Replacement with an amino acid that is
naturally present at that position in a human antibody is
preferred. The invention thus further provides an antibody of the
invention that comprises a humanized heavy chain variable region, a
humanized light chain variable region or a combination thereof. A
light chain of an antibody as defined herein has a light chain
constant region. The light chain constant region is preferably the
light chain constant region of the corresponding antibody. A light
chain comprising a ch14.18 light chain variable region preferably
also comprises the light chain constant region of the ch14.18
antibody. A light chain comprising a 3F8 light chain variable
region preferably also comprises the light chain constant region of
the 3F8 antibody.
[0065] The invention further provides a method of treatment of a
subject that has a GD2 positive tumor or is at risk of having said
GD2 positive tumor the method comprising administering a
therapeutic amount of an antibody of the invention to the subject
in need thereof. Also provided is an antibody of the invention for
use in the treatment of a subject that has a GD2 positive tumor or
is at risk of having a GD2 positive tumor. The treatment preferably
further comprises administering retinoic acid in an amount
effective to upregulate the ganglioside GD2 in neuroblastoma cells
in said subject. The treatment preferably further comprises
administering granulocyte-macrophage colony-stimulating factor
(GM-CSF), G-CSF or a combination thereof in an amount effective to
increase the number of granulocytes in the subject and/or to
increase ADCC. The GM-CSF, G-CSF or a combination thereof is
preferably of a species that is to be treated. It is preferably
human GM-CSF, human G-CSF or a combination thereof.
[0066] The GD2 positive tumor is preferably a GD2 positive
neuroblastoma such as a neuroectoderm-derived tumor or a sarcoma.
In a preferred embodiment the GD2 positive tumor is a GD2 positive
neuroblastoma, retinoblastoma, melanoma, small cell lung cancer,
brain tumor, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma in
children and adolescents, or liposarcoma, fibrosarcoma,
leiomyosarcoma or another soft tissue sarcoma in adults. In a
preferred embodiment the GD2 positive tumor is a neuroblastoma. The
neuroblastoma treated with a method or use of the invention is
preferably a high risk neuroblastoma.
[0067] The retinoic acid is preferably 13-cis-retinoic acid
(isotretinoin).
[0068] The invention further provides an antibody of the invention
or a method or antibody for use of the invention, wherein the
antibody comprises a heavy chain variable region with the amino
acid sequence
TABLE-US-00003 EVQLLQSGPE LEKPGASVMI SCKASGSSFT GYNMNWVRQN
IGKSLEWIGA IDPYYGGTSY NQKFKGRATL TVDKSSSTAY MHLKSLTSED SAVYYCVSGM
EYWGQGTSVT VSS
[0069] and a light chain variable region with the amino acid
sequence
TABLE-US-00004 EIVMTQSPAT LSVSPGERAT LSCRSSQSLV HRNGNTYLHW
YLQKPGQSPK LLIHKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP
PLTFGAGTKL ELK.
[0070] The invention also provides a nucleic acid molecule or
combination of nucleic acid molecules that codes for a heavy chain,
a light or preferably both of an antibody as described herein. The
nucleic acid molecule or combination preferably further comprises
one or more sequences for the expression of an antibody as
described. Non-limiting examples of such expression sequences are a
promoter, a termination sequence, an enhancer, an intron etc. Such
sequences are not necessarily present on the nucleic acid molecule
as such sequences can be provided in cis by the integration site of
the nucleic acid molecule in, for instance, a chromosome of a cell,
or a vector comprising said nucleic acid molecule. Suitable
integration sites in a cellular chromosome can easily be determined
and targeted, for instance by means of homologous
recombination.
[0071] Further provided is a cell that comprises a nucleic acid
molecule or combination as described herein.
[0072] Further provided are means and methods for the production of
an antibody as described herein using a nucleic acid molecule or
combination of the invention or a cell comprising a nucleic acid
molecule or combination of nucleic acid molecules of the
invention.
[0073] A nucleic acid molecule or combination according to the
invention is for instance comprised in a cell. When said nucleic
acid is expressed in said cell the translated product of the
nucleic acid molecule can be, or be incorporated into, an antibody
of the invention. The invention thus also provides a cell
comprising a nucleic acid molecule or combination according to the
invention. The invention further provides a cell comprising a
nucleic acid molecule or combination of the invention and that is
capable of producing an antibody of the invention. Further provided
is a method for producing an antibody of the invention comprising
culturing a cell comprising expressing one or more nucleic acid
molecules that code for an antibody of the invention and harvesting
the antibody from the culture medium, the cell or a combination
thereof. Said cell is preferably an animal cell, more preferably a
mammalian cell. The cell is preferably a cell that is normally used
for the production of an antibody for use in humans. Non-limiting
examples of such cells are CHO, NSO, HEK cells preferably HEK293F
cells, and PER. C6 cells. Cells may specifically designed to suit
certain purposes, for instance, most cell lines used for the
production of antibodies have been adapted for growth in
suspension, in high densities and other properties. For the purpose
of the invention a suitable cell is any cell capable of comprising
and preferably of producing an antibody according to the
invention.
[0074] For the purpose of clarity and a concise description
features are described herein as part of the same or separate
embodiments, however, it will be appreciated that the scope of the
invention may include embodiments having combinations of all or
some of the features described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1A shows a Variable heavy and FIG. 1B shows a variable
light chain amino acid of the antibodies ch14.18 respectively; the
CDR regions are underlined. FIG. 1C shows DNA sequences coding for
the variable heavy and FIG. 1D shows DNA sequences coding for the
variable light chain of the antibody ch14.18 respectively.
[0076] FIG. 1E shows for 3F8 a chimeric gamma1 heavy chain (SEQ ID
NO: 1). FIG. 1F shows a chimeric kappa light chain (SEQ ID NO: 2).
The respective CDRs are underlined.
[0077] FIGS. 2A-2B shows quantification of IgA ch14.18 after
transfection with several heavy (HC) and light chain (LC) ratio's.
5 different ratios were used for IgG1 (FIG. 2A) and IgA1 (FIG. 2B)
production. The optimal ratios were used for larger scale
production.
[0078] FIG. 3A shows .kappa.-light chain specific affinity
chromatography of IgA ch14.18. FIG. 3B shows size exclusion
chromatography of IgA ch14.18 UV absorption, representing protein
concentration, is indicated by the trace.
[0079] FIGS. 4A-4B show binding of in-house produced and purified
IgG1 and IgA ch14.18 to the GD2 expressing neuroblastoma cell line
IMR32 (FIG. 4A) and SK-N-FI (FIG. 4B) in flow cytometry.
[0080] FIGS. 5A-5B show CDC assay of IgG1 and IgA after 1 hr (FIG.
5A) and 4 hrs (FIG. 5B) of incubation with IMR32 cell line. Cell
lysis through complement activation is analyzed by flow cytometric
analysis. 15% pooled human serum was added, and cells were
incubated for 60 min (FIG. 5A) and 4 hrs (FIG. 5B) at 37.degree. C.
The amount of cell lysis is measured by 7-AAD staining.
[0081] FIG. 6A shows Leukocyte and FIG. 6B shows PMN ADCC assay of
IgG1 and IgA after 4 hrs of incubation with the IMR32 cell lines.
FIG. 6C shows leukocyte and FIG. 6D shows PMN ADCC assay of IgG1
and IgA after 4 hrs of incubation with SK-N-F1 cell line. Red blood
cells were lysed and the remaining effector cells were added to
wells. After 4 hours of incubation at 37.degree. C., .sup.51Cr
release was measured in counts per minute (cpm) by a beta-gamma
counter. The percentage of specific lysis was calculated by
determining the maximal lysis in the presence of triton and basal
lysis in the absence of antibodies and effector cells.
[0082] FIGS. 7A-7D show Leukocyte ADCC assay of in-house produced
and purified IgG1 and IgA after 4 hrs of incubation with the IMR32
and SK-N-FI cell line. FIG. 7A shows leukocyte ADCC assay of
in-house produced and purified IgG1 and IgA after 4 hrs of
incubation with the IMR32 cell line. FIG. 7B shows leukocyte ADCC
assay of in-house produced and purified IgG1 and IgA after 4 hrs of
incubation with the IMR32 cell line. FIG. 7C shows leukocyte ADCC
assay of in-house produced and purified IgG1 and IgA after 4 hrs of
incubation with the SK-N-FI cell line. FIG. 7D shows leukocyte ADCC
assay of in-house produced and purified IgG1 and IgA after 4 hrs of
incubation with the SK-N-FI cell line. Red blood cells were lysed
and the remaining effector cells were added to wells together with
cytokines. After 4 hours of incubation at 37.degree. C., .sup.51Cr
release was measured in counts per minute (cpm) by a beta-gamma
counter. The percentage of specific lysis was calculated by
determining the maximal lysis in the presence of triton and basal
lysis in the absence of antibodies and effector cells.
[0083] FIGS. 8A-8D show PMN ADCC assay of IgG1 and IgA ch14.18
after 4 hrs of incubation with the IMR32 or SK-N-FI cell line. FIG.
8A shows PMN ADCC assay of IgG1 and IgA ch14.18 after 4 hrs of
incubation with the IMR32 cell line. FIG. 8B shows PMN ADCC assay
of IgG1 and IgA ch14.18 after 4 hrs of incubation with the IMR32
cell line. FIG. 8C shows PMN ADCC assay of IgG1 and IgA ch14.18
after 4 hrs of incubation with the SK-N-FI cell line. FIG. 8D shows
PMN ADCC assay of IgG1 and IgA ch14.18 after 4 hrs of incubation
with the SK-N-FI cell line. PMNs were isolated by Ficoll/Histopaque
separation. Subsequently, effector cells, cytokines and antibodies
at various concentrations were added to microtiter plates
containing target cells. E:T ratios were 40:1 (PMN). After 4 hours
of incubation at 37.degree. C., .sup.51Cr release was measured in
counts per minute (cpm) by a beta-gamma counter. The percentage of
specific lysis was calculated by determining the maximal lysis in
the presence of triton and basal lysis in the absence of antibodies
and effector cells.
[0084] FIG. 9A shows expression of GD2 and FIG. 9B shows expression
of MHC-I expression on IMR32 cells after exposure to different
concentrations of isotretinoin for 4 consecutive days. Cells were
stained with IgA ch14.18 and detected with a secondary anti-IgA PE
or anti-MHC-I-PE.
[0085] FIG. 10 shows schematic representation of the ganglioside
GD2.
[0086] FIG. 11 shows quantification of in vivo mechanical
thresholds. Experiments were conducted using female (aged 8-12
weeks) C57BL/6 mice (Harlan Laboratories). Mice received an
intravenous injection of 100 microgram of antibody. Mechanical
thresholds were determined using the von Frey test (Stoelting) with
the up-and-down method as is described in Eijkelkamp et al., 2010;
J. Neurosci. 30:2138-2149 and Chaplan et al., 1994; J. Neurosci.
Methods 53:55-63. All experiments were performed by experimenters
blinded to treatment.
[0087] FIGS. 12A-12D show primary sequence and modeling of the
IgA1/IgA2.0 hybrid antibody. FIG. 12A shows alignment of primary
sequences of the constant regions of hIgA1, IgA2m(1), and a
IgA1/IgA2m(1) hybrid (hIgA2.0). Residues are numbered according to
the myeloma IgA1 protein (Bur) scheme. Domain boundaries are
indicated by vertical lines above the sequences. The following
features are highlighted: light gray underlined residues are unique
for IgA1, dark gray underlined asparagines are conserved
N-glycosylation consensus sequences, and black underlined residues
are unique for IgA2.0. FIG. 12B shows the heavy chain of 225-IgA2.0
was modeled and illustrated in front and side view, with mutations
marked. FIG. 12C shows heavy chains of wild-type and mutant IgA2
were modeled. The resulting alignment indicates a different
orientation of C241 in the heavy chains of IgA2-wt compared with
IgA2.0, possibly due to the P221R mutation. FIG. 12D shows focus on
the tailpiece of 225-IgA2-wt (green, C471; red, Y 472) and IgA2.0
(red). Prediction and alignment of models were performed using
I-TASSER; models were modified in 3D-Mol Viewer.
[0088] FIGS. 13A-13B show HP-SEC analysis of IgA1 and IgG1 ch14.18.
Purified antibodies were subjected to HP-SEC analysis. Both IgA1
ch14.18 (FIG. 13A) and IgG1 ch14.18 (FIG. 13B) showed to be highly
monomeric. Traces show the UV absorbance at 280 nm.
[0089] FIGS. 14A-14B show binding of IgG1 and IgA1 ch14.18
antibodies to neuroblastoma cell lines. FIG. 14A shows antibody
binding of IgA1-FITC and IgG1-FITC ch14.18 to GD2 expressing
neuroblastoma cell line IMR32 and GD2-negative cell line GI-ME-N.
FIG. 14B shows real time cell-based affinity measurement of
neuroblastoma antibodies on IMR32 cells. IMR32 cell lines were
treated with 10 nM of antibody for 1 hour Subsequently,
concentration was increased to 20 nM of antibody for 1 hour).
Dissociation was followed from 130-250 minutes by replacing
antibody containing medium with medium without antibody. Calculated
affinities from cell-based affinity measurements are shown in FIG.
14B.
[0090] FIGS. 15A-15D show characterization of ADCC by IgA1 and IgG1
ch14.18 against a panel of neuroblastoma cell lines. FIG. 15A shows
ADCC assays with IgA1 and IgG1 ch14.18 on 3 different neuroblastoma
cell lines with leukocytes from peripheral blood as effector cells.
FIG. 15B shows ADCC assays with IgG1 ch14.18 on IMR-32 cell line
with isolated PBMC's (E:T ratio of 100:1) or h isolated neutrophils
(E:T ratio of 40:1) as effector cells. FIG. 15C shows ADCC assays
with IgG1 ch14.18 on IMR32 cell line with isolated PBMC's (E:T
ratio of 100:1) or isolated neutrophils (E:T ratio of 40:1) as
effector cells with co-treatment of 10 ng/ml GM-CSF, 6545 U/ml of
IL-2 and 24 hours of pre-incubation with 10 .mu.M of 11-cis
retinoic acid. FIG. 15D shows ADCC assays with leukocytes from
peripheral blood as effector cells in combination with 15% pooled
human complement active serum.
[0091] FIGS. 16A-16C show complement assays on a panel of
neuroblastoma cell lines by IgG1 and IgA1 ch14.18 antibodies. FIG.
16A shows lysis by IgG1 ch14.18 antibodies on 4 different
neuroblastoma cell lines. Cells were incubated with 4 different
concentrations of antibody and 15% serum for 15 minutes and 4
hours. FIG. 16B shows lysis by IgA1 ch14.18 antibodies on 4
different neuroblastoma cell lines. Cells were incubated with 4
different concentrations of antibody and 15% serum for 15 minutes
and 4 hours. FIG. 16C shows expression of complement regulatory
proteins CD46, CD55 and CD59 on neuroblastoma cell lines.
[0092] FIGS. 17A-17B show in vivo efficacy of IgG1 and IgA1 ch14.18
antibodies. FIG. 17A shows quantification of bioluminescent signal
of neuroblastoma cells after 24 hours of treatment with IgA1 or
IgG1 ch14.18. FIG. 17B shows quantification of bioluminescent
signal at 3 days of treatment after I.V. injection of tumor
cells.
[0093] FIGS. 18A-18D show nueronal exposure of to IgA1 does not
lead to decreases in mechanical withdrawal thresholds. FIG. 18A
shows plasma concentrations of IgA1 and IgG1 ch14.18 3 hours after
intravenous injection. FIG. 18B shows Von-Frey withdrawal
thresholds. FIG. 18C shows Von-Frey withdrawal thresholds after
I.V. injection of fluorescently labeled IgA1 ch14.18 or IgG1
ch14.18. FIG. 18D shows in left column: Visualization of
intravenously injected Alexa-488 labeled antibodies on sciatic
nerves. FIG. 18D shows in right column: Visualization of ex-vivo
staining of GD2 by incubation of neurons with Alexa-549 labeled
IgG1 ch14.18.
EXAMPLES
Example 1
Generation of Anti-GD2 Antibody Producing Vectors:
[0094] The amino acid sequences of the variable regions from
ch14.18 were found in the FDA application of dinutuximab
(application 125516; depicted in FIGS. 1A-1B). These amino acid
sequences were translated to the cDNA sequence representing the
most likely non-degenerate coding sequence. The cDNAs were
synthesized (Baseclear) and subcloned into the pEE14.4 expression
vectors which contain either the IgA1 or IgG1 backbone (described
in: Beyer, T., et al. "Serum-free production and purification of
chimeric IgA antibodies." Journal of immunological methods 346.1
(2009): 26-37). The optimal ratio of heavy to light chain DNA for
transfection was first determined by small scale test transfections
in HEK293F cells. Antibody production was then quantified by
anti-human IgG (FIG. 2A) or IgA ELISA (FIG. 2B).
[0095] Production was scaled up to allow for antibody
characterization and functional assays. Again, HEK293F cells were
transfected with the pEE14.4 IgA ch14.18 heavy chain and ch14.18
kappa light chain at the optimal heavy to light chain transfection
ratios. The produced IgA antibodies were purified by using a series
of two liquid chromatography steps. First, IgA ch14.18 was isolated
from the serum free supernatant using human .kappa.-light chain
specific affinity chromatography (FIG. 3A). Next, size exclusion
chromatography with a preparative prepacked Superdex200
26.times.600 column was used to separate intact IgA from free light
chain (FIG. 3B). Finally, IgG antibodies were purified by protein
affinity chromatography and were subsequently dialyzed to PBS. This
multistep procedure results in highly pure preparations of
recombinant monomeric IgA.
Characterization of Anti-GD2 Antibodies
[0096] Binding of anti-GD2 antibodies to the GD2 expressing cell
lines IMR32 and SK-N-FI was analyzed by staining cells on ice for
45 minutes with anti-GD2 antibody at several concentrations. Cells
were washed and a secondary goat-anti human IgA-PE or IgG-PE was
added to the cells for 45 minutes on ice in the dark. Afterwards,
antibody binding was quantified by flow cytometric analysis.
[0097] Complement activation of the produced antibodies was
assessed by incubation of IMR32 and SK-N-FI cells with 15% pooled
human serum and antibody (10-0.01 .mu.g/ml) for 1 and 4 hours at
37.degree. C. Afterwards, live/dead staining (7-AAD) of cells was
performed and cellular lysis was quantified via flow cytometric
analysis.
[0098] ADCC assays were performed to assess the efficacy of IgA and
IgG1 ch14.18 to recruit effector cells against neuroblastoma cells.
First, leukocyte ADCC's were performed. Hereto, peripheral blood
from healthy donors was treated with RBC Lysis buffer to remove red
blood cells. The remaining leukocytes were washed and added to
radioactively labeled neuroblastoma cells with or without IL-2 and
GM-CSF (both 10 ng/ml). After 4 hours of incubation at 37.degree.
C., cell death was quantified by .sup.51Cr release, measured by
liquid scintillation. The percentage of specific lysis was
calculated by determining the maximal lysis in the presence of 2.5%
triton X-100 and basal cell lysis in the absence of antibodies and
effector cells.
[0099] To evaluate whether PMNs are an important effector cell
population for IgA mediated killing, PMNs were isolated from
peripheral blood from healthy donors by ficoll-histopaque and added
in a 40:1 E:T ratio to radioactively labelled neuroblastoma cells
with or without IL-2 and GM-CSF (both 10 ng/ml).
Results and Discussion
[0100] The functional characterization of the antibodies was
performed by determining binding capacity to GD2, complement
activation and effector cell recruitment. Flow cytometric analysis
of binding to the GD2-positive cell lines IMR32 and SK-N-FI showed
similar binding patterns for both IgG1 and IgA (FIGS. 4A-4B).
Complement activation of the produced antibodies was assessed by
live/dead staining (7-AAD) after incubation with 15% pooled human
serum and several concentrations of antibody The IgA anti-GD2
antibody did not show activation of complement, while the IgG1
variant having the same variable regions did activate the
complement system after 1 hour of incubation (FIG. 5A). Lysis
increased further for IgG when incubation time increased to 4
hours, while for IgA no complement activation was observed (FIG.
5B)
[0101] ADCC assays were performed to assess the efficacy of IgA and
IgG1 ch14.18 to recruit effector cells against neuroblastoma cells.
First, leukocyte ADCC's were performed. Here, the IgA anti-GD2
antibody showed to be superior for both the IMR32 and SK-N-FI cell
line (FIGS. 6A and 6C respectively). To evaluate whether PMNs are
an important effector cell population for IgA mediated killing, PMN
were isolated from peripheral blood from healthy donors. It was
shown that the IgA anti-GD2 antibody stimulates PMN better than
IgG1 to kill IMR32 and SK-N-FI cells (FIGS. 6B and 6D
respectively).
[0102] When cells were incubated together with the clinically used
cytokine GM-CSF, maximal lysis induced by the IgA anti-GD2 antibody
strongly increased, while only minimal effects were seen for the
IgG1 variant (FIG. 7A and FIG. 7C). Furthermore, addition of IL-2
did not increase the observed lysis (FIG. 7B and FIG. 7D). Similar
effects were observed for PMN mediated ADCC (FIGS. 8A-8D).
[0103] We also show that treatment of neuroblastoma cell lines with
isotretinoin is able to influence GD2 expression. It was shown that
after 4 days of incubation with isotretinoin, binding of IgA
ch14.18 is increased, while MHC-I expression remained stable (FIGS.
9A-9B).
[0104] The examples show that an IgA isotype variant of dinutuximab
has higher ADCC capacity for killing of neuroblastoma's, and no
complement activation. The IgA anti-GD2 antibody enhances
destruction of neuroblastoma cells, whereas side effects such as
neuropathic pain are reduced.
[0105] In the present invention it is shown that isotype conversion
of dinutuximab to IgA provides a solution to one or more of the
toxicity problems of the dinutuximab antibody. The antibody of the
invention provides potent ADCC activity while simultaneously
reducing at least the pain problem associated with dinutuximab
administration. Antibodies of the invention efficiently activate
neutrophils presumably through the Fc.alpha.R (CD89). This
generates potent anti-tumor reactions. The susceptibility of
neuroblastoma cells to an antibody of the invention shows that an
IgA anti-GD2 exhibits improved efficacy against neuroblastoma when
compared to an IgG isotype variant with the same variable domains,
and simultaneously reduces treatment toxicity. This significantly
improves anti-neuroblastoma immunotherapy.
[0106] Antibody function is governed by a number of factors. The
target and the epitope that is recognized play an important role as
does the cell on which the target resides. Effector functions of
the antibody are often correlated with the isotype of the antibody.
Although this is useful as a general rule, many exceptions indeed
exist. This is exemplified for instance for ADCC activity by
Rajasekaran et al (Rajasekaran et al., 2015; ImmunoTargets and
Therapy Vol 4: 91) and for CDC activity by Lohse et al., 2017; Br J
Haematol. doi:10.1111/bjh.14624dgf; and Pascal, et al., (2012)
Haematologica 97.11: 1686-1694).
[0107] Conversion of IgG to IgA can alter the receptors which
interact with the antibodies. For IgG, the activating Fc gamma
receptors (Fc.gamma.R), bearing an ITAM motif, are the principal
mediators of antibody mediated activation of leukocytes. Inhibitory
Fc.gamma.Rs and polymorphisms in Fc.gamma.Rs interfere with these
effects. This can make IgG treatment less suitable for subgroups of
patients. We and others have shown that IgA also exerts anti-tumor
effects through an Fc receptor, the Fc.alpha.R, which is expressed
on neutrophils, monocytes and macrophages. Inhibitory receptors and
polymorphisms are not reported for Fc.alpha.RI. However, the
Fc.alpha.RI is not expressed in mice, making it for a long time not
possible to do the necessary preclinical in vivo studies with IgA
therapeutic antibodies. However, within our laboratory the human
Fc.alpha.RI transgenic mouse was generated. This mouse has a
similar expression pattern of human Fc.alpha.RI as in humans. These
mice were back-crossed in the relevant backgrounds, i.e. balb/c,
C57B/L6, and SCID for growing human tumors.
IgA as Therapeutic Antibody.
[0108] Although IgA is known as a mucosal antibody, in its
monomeric form it is the second class of antibody present in the
human serum. In previous studies we have shown that an anti-tumor
antibody IgA can be effective in vitro. The anti-tumor mechanism is
different and mainly through the recruitment of neutrophils, the
most abundant type of leucocytes. Also in vivo IgA can be
efficacious as a therapeutic antibody. A drawback of an IgA
molecule is its on average relatively short half-life, due to
different glycosylation and lack of binding to the neonatal Fc
receptor, FcRn. The present invention solves this problem for GD2
specific IgA antibodies by providing them with adapted
glycosylation and targeting of the IgA to FcRn indirectly (Meyer et
al., 2016 MAbs Vol 8: pp 87-98).
[0109] In the present invention we show that anti-GD2 IgA isotype
antibodies are targeting neuroblastoma, in vitro, in vivo and in
patient-derived models.
Example 2
[0110] The efficacy of the anti-GD2 antibodies to elicit ADCC, will
be assay against a further panel of GD2 expressing neuroblastoma
cell lines (e.g. IMR-32, SH-SY5Y, SK-N-FI, LAN-5), from ATCC) and
ex vivo neuroblastoma cells as targets. In addition, the panel will
include tumor-initiating cells that have been generated from
primary neuroblastoma samples and that contain stem cell-like cells
that may represent the chemotherapy-resistant cell population
responsible for resistance to conventional chemotherapy (such as
described in Bate-Eya, et al., 2014; European journal of cancer
50.3: 628-637). Both whole blood and isolated effector populations
from healthy volunteers and patients will be used as effector
cells. Practically, target cells will be labelled with .sup.51Cr
for 2 hours. PMNs and PBMCs will be isolated by Ficoll/Histopaque
separation. Subsequently, effector cells, antibodies at various
concentrations, and medium will be added to microtiter plates
containing target cells. E:T ratios will be 40:1 (PMN) and 50:1
(PBMC). After 4 hours of incubation at 37.degree. C., .sup.51Cr
release will be measured in counts per minute (cpm). The percentage
of specific lysis will be calculated by determining the maximal
lysis in the presence of triton and basal lysis in the absence of
antibodies and effector cells.
Testing on Organoids Derived from Neuroblastoma Patients
[0111] We have generated tumor-derived organoids from primary
neuroblastoma samples. Tumor-derived organoids reflect tumor
heterogeneity and allow performing these functional tests on
tissues representing primary tumor tissue. We will use the IgA
dinutuximab on these organoids. We will evaluate binding, ADCC
microscopically by death markers and complement depositions with
C1q and iC3b detecting antibodies.
In Vivo Experiments in Fc.alpha.R Transgenic Mice
[0112] For in vivo experiments, we will make use of syngeneic
models, xenograft models and patient derived xenograft (PDX)
models.
[0113] For syngeneic models, we will make use of the TH-MYCN
9464D-based syngeneic neuroblastoma mouse model, grown
orthotopically or subcutaneously.
[0114] For xenograft models we will make use of the HTLA-230 NB
cells implanted in SCID mice, as described in Bogenmann 1996 Int.
J. Cancer Vol 67: 379; and Raffaghello et al., 2003 Cancer Lett,
Vol 197(1-2): p. 205-9. Previously, the anti-GD2 antibody 14G2a was
tested in this model, and it was shown that it worked very
efficiently, but still in the absence of NK cells and macrophages
(Raffaghello et al., 2003 supra). That fits with the notion that
neutrophils are important effector cells for dinutuximab. When we
will test IgA in these mice, we will use the SCID/Fc.alpha.R
transgenic mice, available in our laboratory, since mice lack the
Fc.alpha.R. We will compare the efficacy of IgG and IgA in these
transgenic SCID mice.
[0115] For PDX models we will make use of the NSG mice
(NOD/SCID/gamma.sup.null mice). These models allows for orthotopic
positioning of patient tumors, outgrowth and subsequent treatment
(Braekeveldt et al., 2016 Cancer Lett. Vol 1; 375(2):384-9. doi:
10.1016/j.canlet.2016.02.046).
Example 3
[0116] Improvement in neuroblastoma patient survival came in 2015,
with the FDA approval of ch14.18, a chimeric antibody of the IgG1
isotype directed against the ganglioside GD2, expressed on
neuroblastoma cells, but also on peripheral and central nervous
tissue. Ch14.18 is given as second-line treatment in combination
with IL-2, GM-CSF and 11-cis retinoic acid for the treatment of
high-risk neuroblastoma after hematopoietic stem cell
transplantation. In a large phase III clinical trial (n=226) it was
shown that ch14.18 combination therapy resulted in 20% more
event-free survival than standard therapy and 10% more overall
survival, 2 years after treatment (Yu et al. 2010, N Engl J Med
363(14): 1324-1334). Although the inclusion of immunotherapy
improved the survival of neuroblastoma patients, there are
important side effects caused by the administration of ch14.18. Of
these, severe neuropathic pain is the most frequent (Yu et al.
2010, N Engl J Med 363(14): 1324-1334). This is believed to be
caused by ch14.18 binding to GD2 on A6 and C pain fibers and
activates the complement system locally, which is the cause for the
observed pain (Xiao et al 1997, Pain 69(1-2): 145-151). This pain
does not respond well to analgesics and can be dose limiting
(Gilman et al 2009, J Clin Oncol 27(1): 85-91).
[0117] Ch14.18-antibody-opsonized tumor cells can be killed by
leukocytes through antibody-dependent cell-mediated cytotoxicity
(ADCC), depending on antibody binding to Fc receptors on
leucocytes. Ch14.18 is also able to activate the complement system
on the tumor cell surface, causing lysis of the cell via
complement-dependent cytotoxicity (CDC). For ADCC mediated by IgG1
antibody therapy, natural killer (NK) cells are regarded as
important cells for mediating ADCC. For neuroblastoma, there is
evidence that granulocytes also play a role in mediating ADCC when
treated with an anti-GD2 IgG1 antibody (Bruchelt et al 1989,
Immunol Lett 22(3): 217-220; Gilman et al 2009, J Clin Oncol 27(1):
85-91; Cheung et al 2012, J Clin Oncol 30(4): 426-432).
Results
[0118] Production and Purification of IgG1 and IgA1 ch14.18
[0119] To compare IgA1 and IgG1 antibodies with the ch14.18
variable region, both antibodies were produced and purified
in-house. All antibodies were analyzed by HP-SEC to confirm their
purity. Both IgA1 ch14.18 (FIG. 13A) and IgG1 ch14.18 (FIG. 13B)
were shown to be monomeric, with a purity greater than 95%.
IgA1 and IgG1 ch14.18 Specifically Bind GD2 with Similar
Affinity
[0120] Next, we determined the binding of IgA1 and IgG1 ch14.18 to
a panel of neuroblastoma cell lines. Both antibodies recognized the
GD2 expressing neuroblastoma cell line IMR-32 similarly, while the
GI-ME-N cell line without GD2 expression was not bound (FIG. 14A).
To establish whether the affinity of the antibodies remained
unchanged after changing the isotype to IgA1, we performed real
time cell-based affinity measurements on IMR-32 cells with Ligand
Tracer. The antibodies closely follow the same association pattern
at 2 different concentrations (10 and 20 nM respectively) and also
overlap in the dissociation phase). The assays indicate that the
calculated affinity to IMR32 cells did not differ significantly
between IgA1 and IgG1 (3.8 nM vs 4.8 nM) (FIG. 14B).
Mechanism of Action of IgA1 and IgG1 ch14.18 Antibodies
[0121] Subsequently, we compared the in vitro mechanism of action
of these antibodies. Both ADCC and CDC are known to be induced by
ch14.18 against neuroblastoma in vivo. To compare killing with a
mix of effector cells, ADCC assays were performed on IMR-32,
SK-N-FI and LAN-1 neuroblastoma cell lines with leukocytes as
effector cells. Both IgA and IgG antibodies lysed IMR-32 cells to a
similar extent, while SK-N-FI and LAN-1 cells were killed better
with IgA1 ch14.18 (FIG. 15A). The GI-ME-N cell line, that has no
detectable GD2 expression on FACS could not be lysed by both
antibodies, showing that GD2 expression is a prerequisite for ADCC
(data not shown). To evaluate the relative importance of certain
leukocyte subsets in mediating ADCC, neutrophils and peripheral
blood mononuclear cells (PBMC) were separately used as effector
cells to determine their respective cytotoxic capacity against
neuroblastoma cell lines with these antibodies.
[0122] With PBMC's as effector cells, IgG1 ch14.18 effectively
lysed IMR32 cells, while lysis with the IgA antibody did not
perform as well (FIG. 15B). This was also seen for 2 other
neuroblastoma cell lines (data not shown) On the contrary, when
neutrophils were used as effector cells, IgA ch14.18 mediated
superior ADCC for all tested cell lines in comparison to IgG1 (FIG.
15B).
[0123] In the clinic, ch14.18 is administered as combination
therapy with GM-CSF, IL-2 and retinoic acid. Next, the impact of
these compounds on ADCC was assessed for both antibodies. When
PBMCs were used as effector cells, addition of GM-CSF did not
increase ADCC by IgG1. Combination of GM-CSF and IL-2 led to a
minor increase of cell death, even without the presence of
antibody, showing increases in PBMC-mediated killing regardless of
antibody. Finally, addition of pre-treatment of 11-cis retinoic
acid for 24 hours to the previous combination showed further
increases in killing for the IMR32 and SK-N-FI cell lines (FIG.
15C). With IgA, no antibody dependent killing could be induced by
PBMC's, but cells were lysed in presence of IL-2 and the
combination with cis-retinoic acid, indicating antibody independent
recognition of neuroblastoma cells by PBMC's (data not shown).
Different from IgG1, GM-CSF in combination with IgA1 ch14.18
boosted ADCC with neutrophils as effector cells (FIG. 15C).
Presence of IL-2 did not further increase neutrophil mediated
killing, while maximal lysis increased with pre-exposure to
11-cis-retinoic acid. For IgG1 antibodies, GM-CSF improved killing
with neutrophils slightly, while addition of IL-2 or retinoic acid
did not enhance killing further (data not shown).
[0124] Finally, ADCC assays were supplemented with human pooled
serum to investigate whether CDC could enhance neuroblastoma cell
lysis. For both IgG1 and IgA1 ch14.18 no significant differences in
lysis were observed after addition of serum (FIG. 15D).
IgA1 ch14.18 does not Activate Complement
[0125] A second effect mediated by ch14.18 is activation of the
complement system. Ch14.18 is known to lyse neuroblastoma target
cells via CDC in vitro. We assessed in vitro complement activation
by these antibodies on the same panel of neuroblastoma cell lines
as were used in ADCC assays. IgG1 ch14.18 lysed all tested
neuroblastoma cell lines except SK-N-FI via CDC after 15 minutes
(FIG. 16A). Contrary to this, no lysis could be observed for IgA1
ch14.18 (FIG. 16B). With longer incubation for 1 hour, the amount
of lysis further increased for IgG1, but cells remained negative
for 7-AAD with IgA1 (FIGS. 16A-16B). Since the amount of complement
regulatory proteins CD55 and CD59 on SK-N-FI is significantly
higher than on the other tested neuroblastoma cell lines, this cell
line seems to be less prone to complement mediated lysis (FIG.
16C).
[0126] IgA1 excels in tumor cell depletion in vivo in comparison to
IgG1 ch14.18 Finally, we analyzed the capacity of the antibodies to
kill GD2 expressing cells in vivo in 2 syngeneic mouse models. As a
model for a localized tumor, animals were intraperitoneally
injected with EL4 cells naturally expressing GD2. After 24 hours of
outgrowth, animals were treated with IgA1 or IgG1. Outgrowth of
tumor cells was evaluated 24 hours after injection of the antibody.
Although both antibodies reduced the average tumor burden treatment
with IgA which has only one of the two effector mechanisms, was
more effective (FIG. 17A).
[0127] In a second systemic mouse model, we assessed tumor cell
killing for a longer period of time. EL4 cells were intravenously
injected). Shortly after injection of cells, tumor cells localized
to the lungs, as observed with bioluminescent imaging (Data not
shown). After three days of treatment, both IgA1 and IgG1 cleared
the tumor cells, while cells were still present in mice treated
with PBS (FIG. 17B) Outgrowth was observed in the abdomen of the
mice by bioluminescent imaging (data not shown).
Anti GD2-IgA1 does not Induce Pain
[0128] A major limitation of IgG1 antibodies directed against GD2
is the increased sensitivity to light touch after treatment. To
test whether the lack of complement activation by IgA would
mitigate this problem, we conducted in vivo pain experiments in
mice. Paw retraction thresholds after stimulation with von Frey
hairs were determined as a measure for allodynia.
[0129] To correct for differences between the half-life of IgA1 and
IgG1, mice were intraperitoneally injected with either a low dose
of IgG1 (20 .mu.g), corresponding to a dose of 100 .mu.g of IgA at
24 hours or a high dose of IgG1 (100 .mu.g) (FIG. 18A). The
antibody concentration found back in the serum after 24 hours is in
line with the clinical phenotype after ch14.18 treatment. Treatment
with 20 .mu.g of IgG1 ch14.18 showed a significant decrease in
withdrawal threshold, which returned to baseline after 48 h. A
sub-therapeutic dose of IgG1 (4 .mu.g) did not lead to a
significant reduction in withdrawal threshold. Different from IgG1,
all tested doses of IgA1 did not decrease the paw retraction
threshold, indicating that IgA1 does not induce allodynia at levels
comparable with IgG (FIG. 18B). Similar results were obtained when
fluorescently labeled IgA1 and IgG1 ch14.18 antibodies were
injected intravenously. Here, IgG1 ch14.18 also reduced the
withdrawal threshold, while IgA1 ch14.18 did not (FIG. 18C.
Subsequently, binding of the antibodies was assessed on the sciatic
nerve. Antibody exposure was observed with 20 .mu.g of IgG1 and 100
.mu.g of IgA1 ch14.18 to a similar extent and corresponded to the
amount of antibody present in the serum (FIG. 18D). The anti-CD20
antibody rituximab was not detected on sciatic nerves. Ex vivo
staining of GD2 overlapped with the signal from directly labeled
antibodies.
Kits
[0130] Provided herein are kits comprising compositions of
engineered antibody or fragments thereof. Disclosed herein can also
be kits for the reduction of a complement response, such as
reducing complement activation, Disclosed herein can also be kits
for the treatment of a cancer, pathogen infection, immune disorder
or allogeneic transplant. In one embodiment, a kit can include a
therapeutic or prophylactic composition containing an effective
amount of an engineered antibody in unit dosage form. In some
embodiments, a kit comprises a sterile container which can contain
a therapeutic composition of engineered antibodies or fragments
thereof, such containers can be boxes, ampules, bottles, vials,
tubes, bags, pouches, blister-packs, or other suitable container
forms known in the art. Such containers can be made of plastic,
glass, laminated paper, metal foil, or other materials suitable for
holding medicaments. In some cases, engineered antibodies and
fragments thereof can be provided together with instructions for
administering the antibody or fragment thereof to a subject having
or at risk of developing a toxicity, complement-associated
toxicity, cancer, pathogen infection, immune disorder or allogeneic
transplant. Instructions can generally include information about
the use of the administration how to utilize the composition to
treat toxicity, a side effect, cancer, pathogen infection, immune
disorder or allogeneic transplant.
Discussion
[0131] The approval of ch14.18 for high-risk neuroblastoma led to
improvements in the survival of neuroblastoma patients.
Nevertheless, IgG1 antibody therapy for neuroblastoma comes with a
major limitation: presence of severe side effects such as
neuropathic pain and allodynia. We investigated whether changing
the isotype of ch14.18 from IgG1 to IgA1 would abrogate this
antibody-induced allodynia.
[0132] The present invention shows that IgA1 ch14.18 offers
surprisingly strong anti-tumoral effects through neutrophil
mediated ADCC and does not induce allodynia in vivo. Blocking the
C5a receptor with an antagonist completely stopped allodynia
(Sorkin et al 2010, Pain 149(1): 135-142). Thus far, several
approaches were undertaken to amend side-effects caused by ch14.18.
A first approach was to mutate the C1q-binding site of ch14.18
(K322A). This mutation is thought to abrogate complement activation
of ch14.18 (Sorkin et al 2010, Pain 149(1): 135-142). This approach
only reduced complement activation, and, as a result, residual pain
remained. Also in a phase I clinical trial with this antibody,
grade 3-4 toxicity in the form of pain occurred in 68% of patients
(Navid et al 2014, J Clin Oncol 32(14): 1445-1452).
[0133] Another approach to circumvent ch14.18 induced allodynia was
performed by targeting a differentially glycosylated variant of GD2
(O-acetyl-GD2), which is solely present on neuroblastoma cells and
not on peripheral nervous tissue by the antibody c.8B6 (Terme et al
2014, PLoS One 9(2): e87210). The antibody c.8B6 does not induce
complement activation around pain fibers, however, this antibody is
likely less effective.
[0134] During the first reports on 14.18, the parental
mouse-antibody where ch14.18 is derived from, it was noted that
granulocytes mediated effects against neuroblastoma (Bruchelt et al
(1989); Immunol Lett 22(3): 217-220). Later, it was shown that
their killing potential could be enhanced with the addition of
GM-CSF. In clinical studies, it was found that the activation of
granulocytes is an indicator for a good outcome of antibody therapy
against neuroblastoma (Cheung et al (2012); J Clin Oncol 30(4):
426-432). Besides co-treatment with GM-CSF, there has been a lack
of approaches to further engage this target cell population for
therapeutic purposes.
[0135] Next to an inherent vulnerability of neuroblastoma cells to
neutrophil mediated killing, the timing of the current treatment
protocol also favors an approach were neutrophils are used as
effector cells. Now, patients are prescribed with a myeloablative
regimen after which autologous stem cell transplantation follows
(Yu et al. (2010). N Engl J Med 363(14): 1324-1334). Shortly
thereafter, the immunotherapeutic protocol is started. Neutrophils
are often the first leukocyte population which is restored to
physiological levels and are therefore are an attractive leukocyte
subset to active in such an immune-compromised state. Less than 25%
of patients reached proper immune reconstitution for NK cells at
the moment of immunotherapy (Nassin et al 2018, Biol Blood Marrow
Transplant 24(3): 452-459). With an approach where neutrophils
instead of NK cells are addressed, therapeutic variability will be
reduced.
[0136] In the present invention it was found that IgA mediated
killing was improved by the addition of GM-CSF.
[0137] IL-2 does not seem to improve IgA or IgG mediated killing.
IL-2 was added to the clinical regimen after effects were seen in
GD2-expressing melanoma and sarcoma patients which were treated
with ch14.18. However, the effects of IL-2 on neuroblastoma therapy
remain unclear. This is further stressed by the clinical trial
which compared immunotherapy with and without IL-2 (Ladenstein et
al 2013 MAbs 5(5): 801-809). The authors show that the addition of
IL-2 did not have significant effects on EFS or OS and that early
termination because of toxicity was significantly higher in the
IL-2 arm.
[0138] Although IgA1 lacks a C1q binding site, complement
activation of IgA has been documented. The MBL pathway was shown to
be activated by polymeric IgA, while the classical complement
pathway was triggered for monomeric IgA directed against CD20 (Roos
et al; 2001; J Immunol 167(5): 2861-2868 and Lohse, Loew et al;
2018; Br J Haematol 181(3): 413-417). Nevertheless, IgA1 ch14.18
did not induce CDC of neuroblastoma cell lines and did not induce
complement-dependent allodynia in mice.
[0139] Although allodynia can be solved with IgA, complement as an
effector mechanism is also lost. In the present invention it was
surprisingly shown that the expected loss of therapeutic effect did
not occur. In the in vivo tumor models we indeed see that IgA is at
least as efficient as IgG in killing tumor cells. In vivo
complement consumption by anti-GD2 antibody has been demonstrated,
indicating that complement activation does take place (Cheung at al
2014, Int J Cancer 135(9): 2199-2205).
[0140] In our studies, we dosed mice with 5 times the amount of IgA
compared to IgG1 to adjust for the difference in half-life between
the two antibodies in mice. A similar neuronal exposure and serum
concentration could be achieved with the two antibodies. For longer
models, multiple doses of IgA1 were injected to account for this.
Although the in vivo half-life of IgA is approximately a week in
humans, several approaches can be undertaken to improve the
half-life of IgA in mice to improve future comparisons (Morell et
al, 1973, Clin Exp Immunol 13(4): 521-528). The glycosylation of
IgA can be reduced to decrease clearance by binding to the
asialoglycoprotein receptor. Silencing of multiple IgA
glycosylation sites was already accomplished before, leading to
improved pharmacokinetics (Lohse et al, 2016, Cancer Res 76(2):
403-417). Contrary to IgG, IgA does not interact with the neonatal
Fc receptor (FcRn). Therefore, antibodies pinocytosed by
endothelial cells are degraded via the lysosomal pathway. Both
albumin and IgG1 antibodies are rescued from degradation by binding
to this receptor. IgA was previously modified to facilitate FcRn
binding by introduction of a C-terminal albumin-binding site, which
improved its pharmacokinetic profile and anti-tumor effects (Meyer
et al, 2016 MAbs 8(1): 87-98).
[0141] The present invention shows that ch14.18 IgA offers both the
benefit of overcoming allodynia and improved neutrophil activation
in a single molecule. Our preclinical data shows that IgA can be
dosed higher than IgG without side effects.
Material and Methods
Antibody Production, Isolation and Quality Control
[0142] The variable heavy and light chain sequences of ch14.18 were
derived from Biologic License Application 125516. The variable
heavy chain sequences were cloned into Lonza expression vectors
(pEE14.4), coding for the IgA1 or IgG1 heavy chain while the
variable light chain sequences were cloned into Lonza expression
vectors (pEE14.4) coding for the kappa light chain. Monomeric
antibodies were produced by transient transfection of HEK293F cells
with vectors coding for the heavy chain, light chain and pAdvantage
(accession number U47294; promega), using 293Fectin transfection
reagent according to the manufacturer's instructions. IgG1
antibodies were purified using protein A columns (Hi-trap protein
A) coupled to an AKTAprime plus chromatography system (GE
lifesciences). Bound antibody was eluted with 0.1M sodium acetate
pH 2.5 and neutralized with 1M TRIS-HCl pH 8.8. The eluate was
dialyzed against PBS. IgA1 antibodies were purified using kappa
light chain affinity chromatography columns (Hi-trap kappaSelect)
and eluted with 0.1M glycine buffer pH 2.5. The eluate was applied
on a SEC column ran with PBS as mobile phase. The fractions
containing monomeric IgA were collected and concentrated with 100
KDa spin columns. All antibodies were filtered over 0.22 .mu.m
filters. Purity and stability of the antibodies was analyzed by
HP-SEC (Yarra 3u SEC-2000 column) with 100 mM sodium phosphate, 150
mM NaCl pH 6.8 as mobile phase with detection at 280 nm.
Fluorescent Labeling of Antibodies
[0143] Purified antibodies were labelled with fluorescein by
incubation at room temperature for 2 h with
N-hydroxy-succimidyl-FITC while stirring. Unbound NHS-fluorescein
was removed by using sephadex columns (NAP-5, GE-healthcare),
according to the manufacturer's instructions. Antibodies were
labelled with alexa fluor-488 antibody labeling kit (ThermoFisher)
according to the manufacturer's instructions.
Cell Lines
[0144] All neuroblastoma cell lines were cultured in DMEM culture
medium supplemented with HEPES, glutamax, 10% fetal calf serum, lx
penicillin and streptomycin at 37.degree. C. in a humidified
incubator containing 5% CO.sub.2. HEK293F cells were cultured in
FreeStyle 293 expression medium at 37.degree. C. in a humidified
incubator with orbital shaker platform containing 8% CO.sub.2.
Binding Assays
[0145] 100.000 neuroblastoma cells were plated out in 96 well
plates and centrifuged for 2 minutes at 1500 RPM. Cells were washed
and incubated with fluorescein-labelled antibody at several
concentrations for 45 minutes. Next, cells were centrifuged for 2
minutes at 1500 RPM, washed and resuspended in PBS. The amount of
bound antibody to the cells was quantified by flow cytometry (BD
FacsCanto II, BD).
Cell Based Affinity Measurements
[0146] 1.times.10.sup.6 IMR32 neuroblastoma cells were plated out
on the side of a 10 cm culture dish in an elliptical shape and
incubated overnight for attaching to the plate. Subsequently,
plates were washed with culture medium and transferred to the
LigandTracer apparatus (Ridgeview instruments). 10 nM of
fluorescein-labelled antibody was added to the cells and
association was measured for 1 hour. Afterwards, antibody
concentration was increased to 20 nM to assess association at
higher antibody concentrations for 1 hour. Finally, dissociation
was measured by replacing the antibody containing solution for
medium without antibody and dissociation was measured for 2 hours.
The affinity of the antibodies was calculated via TraceDrawer
software (Ridgeview instruments).
ADCC Assays
[0147] ADCC was quantified as described previously (Brandsma et al;
2015; Cancer Immunol Res 3(12): 1316-1324) In short, target cells
with or without pre-treatment of 10 .mu.M 11-cis-retinoic acid for
24 hours were labeled with 3.7 MBq .sup.51Cr for 2 hours.
Afterwards, cells were washed three times to remove excess
chromium. Blood for ADCC's was obtained from healthy donors at the
UMC Utrecht. For leukocyte isolation, blood was incubated with
demiwater for 30 seconds to lyse erythrocytes. Afterwards,
10.times.PBS was added to restore physiological osmolality. Cells
were washed in medium and resuspended in medium corresponding to
the original blood volume. The number of leukocytes used per well
corresponds to the number of leukocytes present in 501 of blood
before lysis. For PMN and PBMC isolation, blood was added on top of
Ficoll/Histopaque 1119 layers and centrifuged for 25 minutes at
1500 RPM without braking. Afterwards, PBMC's and PMN's were
collected from the interphase between serum and ficoll or in the
histopaque layer respectively. The effector-to-target (E:T) ratios
were 80:1 for PBMCs, 40:1 for PMNs. Effector cells, antibodies at
various concentration, GM-CSF and IL-2 and radioactively labeled
tumor cells were added to round-bottom microtiter plates (Corning
Incorporated) and incubated for 37.degree. C. in a humidified
incubator containing 5% CO.sub.2. Plates were centrifuged for 2
minutes at 1500 RPM and 50 .mu.l of the supernatant was transferred
to lumaplates. Radioactive signal (in cpm) was quantified in a
beta-gamma counter. Specific lysis was calculated using the
formula: ((Experimental cpm-basal cpm)/(maximal cpm-basal
cpm)).times.100, with maximal lysis determined by incubating
labelled cells with 2.5% triton and basal release was determined in
the absence of antibodies and effector cells.
CDC Assays
[0148] 10.sup.5 neuroblastoma cells were added to microtiter plates
and incubated for 30 minutes with antibodies at various
concentrations at room temperature. Afterwards, pooled human serum
(from 8 different healthy donors) was added to a concentration of
15% and incubated for 1 hour or 4 hours. Afterwards, cells were
washed and stained with 7-AAD for 15 minutes. 7-AAD uptake,
representing cell lysis was quantified by flow cytometry.
Antibody Concentration Determination in Mouse Serum
[0149] MaxiSorp 96 well ELISA plates were coated overnight with 0.5
.mu.g/ml goat IgG anti-human kappa diluted in PBS. Next, plates
were washed three times with 0.05% TWEEN 20 in PBS (PBST) and
blocked for 1 hour by incubating with 1% BSA in PBST. Serum samples
were diluted 1:2000 in 1% BSA in PBST and added to the wells and
incubated for 1.5 at room temperature. Next, plates were washed
three times with PBST. HRP-labeled-anti-human IgA or -IgG were used
to bind human IgA or human IgG respectively. Plates were developed
for 10 minutes with
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and
read out on a spectrophotometer at 415 nm.
Animals and Animal Experiments
[0150] Mice were maintained in the animal facility of the
University of Utrecht. Experiments were conducted using both male
and female C57BL/6 mice (Janvier) Mice were housed in groups under
a 12:12 light dark cycle, with food and water available ad libitum.
Mice were acclimatized for at least 1 week prior to the start of
experiment. Sample sizes were calculated with power analysis at the
time of the design of experiments.
[0151] Mechanical thresholds were determined using the von Frey
test in mice after intravenous injection with IgG1 ch14.18, IgA1
ch14.18 or fluorescently labeled variants thereof. (Stoelting, Wood
Dale, Ill., US) with the up-and-down method as was described before
previously (Eijkelkamp et al; 2016; J Neurosci 36(28): 7353-7363.
In brief, mechanical nociception was tested with a calibrated von
Frey hair monofilament (Stoelting). First, mice were acclimated for
15 to 20 min in a transparent box with a metal mesh floor. The von
Frey hair monofilament was applied through the mesh floor to the
plantar skin of the hindpaw. Mechanical nociception was measured as
the total number of paw withdrawals in response to a series of six
applications of a 0.02 mg von Frey hair, which does not elicit a
response in untreated animals (Alessandri-Haber et al., 2006; J
Neurosci. 2006 Apr. 5; 26(14):3864-74). In experiments, the average
of the left and right paw was considered as an independent measure.
To minimize bias, animals were randomly assigned to the different
groups prior to the start of experiment, and all experiments were
performed by experimenters blinded to treatment. At the end of the
experiments, mice were euthanized by cervical dislocation.
Mechanical thresholds were assessed for 48 hours.
[0152] For evaluating the in vivo efficacy of antibodies, mice were
injected intraperitoneally with 5.times.10.sup.6 GD2 expressing EL4
cells expressing (ATCC). After 1 day, mice were intraperitoneally
injected with 100 .mu.g of IgG1 ch14.18, or 100 .mu.g of IgA1
ch14.18. After 2 days, blood was taken and mice were injected with
luciferin and subjected to bioluminescence analysis. Afterwards
mice were euthanized by cervical dislocation.
[0153] GD2 antibody binding to neurons was visualized by i.v.
injection of 20 .mu.g or 100 .mu.g alexa-488 labeled IgG1 ch14.18
or 20 .mu.g of IgA1 ch14.18. Sciatic nerves were isolated and 10
.mu.m thick slices were prepared with a cryostat cryotome and
placed on slides. Slides were fixed for 10 minutes in 4% PFA and
washed. Finally, slides were counterstained with DAPI, washed and
treated with fluorsave. Slides were dried overnight at 4.degree. C.
and images were taken by fluorescence microscopy.
Sequence CWU 1
1
131444PRTArtificial Sequence3f8 heavy chain 1Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Val Thr Asn Tyr 20 25 30Gly Val His
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val
Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60Ser
Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75
80Lys Met Asn Ser Leu Gln Ile Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Asp Thr Leu
Met Ile Ser Arg Thr 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 Thr Lys 275 280 285Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315
320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Thr Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
4402210PRTArtificial Sequence3F8 light chain 2Ser Ile Val Met Thr
Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30Val Thr Trp
Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Asn Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Tyr Gly Thr Ala Phe Thr Phe Thr Ile Ser Thr Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly
85 90 95Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val 100 105 110Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser 115 120 125Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln 130 135 140Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val145 150 155 160Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 165 170 175Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Val Tyr Ala Cys Glu Val 180 185 190Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 195 200
205Glu Cys 2103113PRTArtificial Sequenceanti-GD2 antibody ch14.18
heavy chain 3Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys
Pro Gly Ala1 5 10 15Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser
Phe Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys
Ser Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met His Leu Lys Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Val Ser Gly Met Glu Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105
110Ser4113PRTArtificial Sequenceanti-GD2 antibody ch14.18 light
chain 4Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val
His Arg 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110Lys56PRTArtificial
Sequenceantibody C-terminus 5Val Asp Gly Thr Cys Tyr1
564PRTArtificial Sequencemutated antibody C-terminus 6Val Asp Gly
Thr17339DNAArtificial SequenceDNA sequence of antibody ch14.18
heavy chain 7gaagtgcagc tgctgcagag cggcccggaa ctggaaaaac cgggcgcgag
cgtgatgatt 60agctgcaaag cgagcggcag cagctttacc ggctataaca tgaactgggt
gcgccagaac 120attggcaaaa gcctggaatg gattggcgcg attgatccgt
attatggcgg caccagctat 180aaccagaaat ttaaaggccg cgcgaccctg
accgtggata aaagcagcag caccgcgtat 240atgcatctga aaagcctgac
cagcgaagat agcgcggtgt attattgcgt gagcggcatg 300gaatattggg
gccagggcac cagcgtgacc gtgagcagc 3398339DNAArtificial SequenceDNA
sequence of antibody ch14.18 light chain 8gaaattgtga tgacccagag
cccggcgacc ctgagcgtga gcccgggcga acgcgcgacc 60ctgagctgcc gcagcagcca
gagcctggtg catcgcaacg gcaacaccta tctgcattgg 120tatctgcaga
aaccgggcca gagcccgaaa ctgctgattc ataaagtgag caaccgcttt
180agcggcgtgc cggatcgctt tagcggcagc ggcagcggca ccgattttac
cctgaaaatt 240agccgcgtgg aagcggaaga tctgggcgtg tatttttgca
gccagagcac ccatgtgccg 300ccgctgacct ttggcgcggg caccaaactg gaactgaaa
33991017DNAArtificial SequenceIgA2.0 heavy chainCDS(1)..(1017) 9gca
tcc ccg acc agc ccc aag gtc ttc ccg ctg agc ctc gac agc acc 48Ala
Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr1 5 10
15ccc caa gat ggg aac gtg gtc gtc gca tgc ctg gtc cag ggc ttc ttc
96Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe
20 25 30ccc cag gag cca ctc agt gtg acc tgg agc gaa agc gga cag ggc
gtg 144Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly
Val 35 40 45acc gcc aga aac ttc cca cct agc cag gat gcc tcc ggg gac
ctg tac 192Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp
Leu Tyr 50 55 60acc acg agc agc cag ctg acc ctg ccg gcc aca cag tgc
cca gac ggc 240Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys
Pro Asp Gly65 70 75 80aag tcc gtg aca tgc cac gtg aag cac tac acg
aat ccc agc cag gat 288Lys Ser Val Thr Cys His Val Lys His Tyr Thr
Asn Pro Ser Gln Asp 85 90 95gtg act gtg ccc tgc cga gtt ccc cca cct
ccc cca tgc tgc cac ccc 336Val Thr Val Pro Cys Arg Val Pro Pro Pro
Pro Pro Cys Cys His Pro 100 105 110cga ctg tcg ctg cac cga ccg gcc
ctc gag gac ctg ctc tta ggt tca 384Arg Leu Ser Leu His Arg Pro Ala
Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125gaa gcg aac ctc acg tgc
aca ctg acc ggc ctg aga gat gcc tct ggt 432Glu Ala Asn Leu Thr Cys
Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140gcc acc ttc acc
tgg acg ccc tca agt ggg aag agc gct gtt caa gga 480Ala Thr Phe Thr
Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly145 150 155 160cca
cct gag cgt gac ctc tgt ggc tgc tac agc gtg tcc agt gtc ctg 528Pro
Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170
175cct ggc tct gcc cag cca tgg aac cat ggg gag acc ttc acc tgc act
576Pro Gly Ser Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr
180 185 190gct gcc cac ccc gag ttg aag acc cca cta acc gcc acc ctg
tca aaa 624Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Thr Leu
Ser Lys 195 200 205tcc gga aac aca ttc cgg ccc gag gtc cac ctg ctg
ccg ccg ccg tcg 672Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu
Pro Pro Pro Ser 210 215 220gag gag ctg gcc ctg aac gag ctg gtg acg
ctg acg tgc ctg gca cgt 720Glu Glu Leu Ala Leu Asn Glu Leu Val Thr
Leu Thr Cys Leu Ala Arg225 230 235 240ggc ttc agc ccc aag gat gtg
ctg gtt cgc tgg ctg cag ggg tca cag 768Gly Phe Ser Pro Lys Asp Val
Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255gag ctg ccc cgc gag
aag tac ctg act tgg gca tcc cgg cag gag ccc 816Glu Leu Pro Arg Glu
Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270agc cag ggc
acc acc acc ttc gct gtg acc agc ata ctg cgc gtg gca 864Ser Gln Gly
Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285gcc
gag gac tgg aag aag ggg gac acc ttc tcc tgc atg gtg ggc cac 912Ala
Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His 290 295
300gag gcc ctg ccg ctg gcc ttc aca cag aag acc atc gac cgc ttg gcg
960Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu
Ala305 310 315 320ggt aaa ccc acc cat gtc aat gtg tct gtt gtc atg
gcg gag gtg gac 1008Gly Lys Pro Thr His Val Asn Val Ser Val Val Met
Ala Glu Val Asp 325 330 335ggc acc tga 1017Gly
Thr10338PRTArtificial SequenceSynthetic Construct 10Ala Ser Pro Thr
Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr1 5 10 15Pro Gln Asp
Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30Pro Gln
Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45Thr
Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55
60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly65
70 75 80Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln
Asp 85 90 95Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys
His Pro 100 105 110Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu
Leu Leu Gly Ser 115 120 125Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly
Leu Arg Asp Ala Ser Gly 130 135 140Ala Thr Phe Thr Trp Thr Pro Ser
Ser Gly Lys Ser Ala Val Gln Gly145 150 155 160Pro Pro Glu Arg Asp
Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175Pro Gly Ser
Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190Ala
Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys 195 200
205Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser
210 215 220Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu
Ala Arg225 230 235 240Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp
Leu Gln Gly Ser Gln 245 250 255Glu Leu Pro Arg Glu Lys Tyr Leu Thr
Trp Ala Ser Arg Gln Glu Pro 260 265 270Ser Gln Gly Thr Thr Thr Phe
Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285Ala Glu Asp Trp Lys
Lys Gly Asp Thr Phe Ser Cys Met Val Gly His 290 295 300Glu Ala Leu
Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala305 310 315
320Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp
325 330 335Gly Thr11342PRTArtificial SequencehIgA2m(1) 11Ser Ser
Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp1 5 10 15Ser
Thr Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly 20 25
30Phe Phe Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln
35 40 45Asn Val Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly
Asp 50 55 60Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln
Cys Pro65 70 75 80Asp Gly Lys Ser Val Thr Cys His Val Lys His Tyr
Thr Asn Pro Ser 85 90 95Gln Asp Val Thr Val Pro Cys Pro Val Pro Pro
Pro Pro Pro Cys Cys 100 105 110His Pro Arg Leu Ser Leu His Arg Pro
Ala Leu Glu Asp Leu Leu Leu 115 120 125Gly Ser Glu Ala Asn Leu Thr
Cys Thr Leu Thr Gly Leu Arg Asp Ala 130 135 140Ser Gly Ala Thr Phe
Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val145 150 155 160Gln Gly
Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser 165 170
175Val Leu Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr
180 185 190Cys Thr Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala
Asn Ile 195 200 205Thr Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His
Leu Leu Pro Pro 210 215 220Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu
Val Thr Leu Thr Cys Leu225 230 235 240Ala Arg Gly Phe Ser Pro Lys
Asp Val Leu Val Arg Trp Leu Gln Gly 245 250 255Ser Gln Glu Leu Pro
Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln 260 265 270Glu Pro Ser
Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg 275 280 285Val
Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val 290 295
300Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp
Arg305 310 315 320Leu Ala Gly Lys Pro Thr His Val Asn Val Ser Val
Val Met Ala Glu 325 330 335Val Asp Gly Thr Cys Tyr
34012356PRTArtificial SequencehIgA1 12Ser Ser Ala Ser Pro Thr Ser
Pro Lys Val Phe Pro Leu Ser Leu Cys1 5 10 15Ser Thr Gln Pro Asp Gly
Asn Val Val Ile Ala Cys Leu Val Gln Gly 20 25 30Phe Phe Pro Gln Glu
Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln 35 40 45Gly Val Thr Ala
Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp 50 55 60Leu Tyr Thr
Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu65 70 75 80Ala
Gly Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser 85 90
95Gln Asp Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro
100 105 110Ser Pro Ser Thr Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys
His Pro 115 120 125Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu
Leu Leu Gly Ser 130 135 140Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly
Leu Arg Asp Ala Ser Gly145
150 155 160Val Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val
Gln Gly 165 170 175Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val
Ser Ser Val Leu 180 185 190Pro Gly Cys Ala Glu Pro Trp Asn His Gly
Lys Thr Phe Thr Cys Thr 195 200 205Ala Ala Tyr Pro Glu Ser Lys Thr
Pro Leu Thr Ala Thr Leu Ser Lys 210 215 220Ser Gly Asn Thr Phe Arg
Pro Glu Val His Leu Leu Pro Pro Pro Ser225 230 235 240Glu Glu Leu
Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 245 250 255Gly
Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 260 265
270Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro
275 280 285Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg
Val Ala 290 295 300Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys
Met Val Gly His305 310 315 320Glu Ala Leu Pro Leu Ala Phe Thr Gln
Lys Thr Ile Asp Arg Leu Ala 325 330 335Gly Lys Pro Thr His Val Asn
Val Ser Val Val Met Ala Glu Val Asp 340 345 350Gly Thr Cys Tyr
35513340PRTArtificial SequencehigA2.0 13Ser Ser Ala Ser Pro Thr Ser
Pro Lys Val Phe Pro Leu Ser Leu Asp1 5 10 15Ser Thr Pro Gln Asp Gly
Asn Val Val Val Ala Cys Leu Val Gln Gly 20 25 30Phe Phe Pro Gln Glu
Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln 35 40 45Gly Val Thr Ala
Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp 50 55 60Leu Tyr Thr
Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro65 70 75 80Asp
Gly Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser 85 90
95Gln Asp Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys
100 105 110His Pro Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu
Leu Leu 115 120 125Gly Ser Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly
Leu Arg Asp Ala 130 135 140Ser Gly Ala Thr Phe Thr Trp Thr Pro Ser
Ser Gly Lys Ser Ala Val145 150 155 160Gln Gly Pro Pro Glu Arg Asp
Leu Cys Gly Cys Tyr Ser Val Ser Ser 165 170 175Val Leu Pro Gly Ser
Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr 180 185 190Cys Thr Ala
Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Thr Leu 195 200 205Ser
Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro 210 215
220Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys
Leu225 230 235 240Ala Arg Gly Phe Ser Pro Lys Asp Val Leu Val Arg
Trp Leu Gln Gly 245 250 255Ser Gln Glu Leu Pro Arg Glu Lys Tyr Leu
Thr Trp Ala Ser Arg Gln 260 265 270Glu Pro Ser Gln Gly Thr Thr Thr
Phe Ala Val Thr Ser Ile Leu Arg 275 280 285Val Ala Ala Glu Asp Trp
Lys Lys Gly Asp Thr Phe Ser Cys Met Val 290 295 300Gly His Glu Ala
Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg305 310 315 320Leu
Ala Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu 325 330
335Val Asp Gly Thr 340
* * * * *
References