NZ744187B2 - Therapeutic anti-cd9 antibody - Google Patents
Therapeutic anti-cd9 antibody Download PDFInfo
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- NZ744187B2 NZ744187B2 NZ744187A NZ74418717A NZ744187B2 NZ 744187 B2 NZ744187 B2 NZ 744187B2 NZ 744187 A NZ744187 A NZ 744187A NZ 74418717 A NZ74418717 A NZ 74418717A NZ 744187 B2 NZ744187 B2 NZ 744187B2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
Abstract
antibody defined by CDRs that binds a CD9 epitope K169, D171, V172, L173, F176 and the use of the antibody in treating CD9 associated disorders.
Description
(12) Granted patent specificaon (19) NZ (11) 744187 (13) B2
(47) Publicaon date: 2021.12.24
(54) THERAPEUTIC ANTI-CD9 ANTIBODY
(51) Internaonal Patent Classificaon(s):
C07K 16/28 A61K 39/395 A61P 35/00
(22) Filing date: (73) Owner(s):
1.06 KLING BIOTHERAPEUTICS B.V.
(23) Complete specificaon filing date: (74) Contact:
2017.01.06 AJ PARK
(30) Internaonal Priority Data: (72) Inventor(s):
EP 16150698.5 2016.01.08 SPITS, Hergen
VAN HELDEN, Paula Maria Wilhelmina
(86) aonal Applicaon No.: POS, Wouter
SCHOTTE, Remko
FATMAWATI, Christien
(87) Internaonal Publicaon number: GO, Danïel l
WO/2017/119811 , Koen
VILLAUDY, Julien Christian
(57) Abstract:
An anbody defined by CDRs that binds a CD9 epitope K169, D171, V172, L173, F176 and the use
of the anbody in treang CD9 associated disorders.
NZ 744187 B2
Title: Therapeutic binding nds
The invention relates to the fields of biology, immunology and medicine.
Melanoma is caused by malignant melanocytes. It is primarily caused by
ultraviolet light exposure. Out of all different types of skin cancer malignant melanoma
has the highest rate of mortality. It is ted that world wide around 55,000 people
have died from metastatic melanoma in 2012, a number which is steadily increasing
every year. If spread has not yet occurred, most patients are cured by removing the
ma. Patients with spread melanoma are treated with chemotherapy, radiation
therapy and/or recently developed immunotherapies such as adoptive T-cell therapy or
so called checkpoint inhibitor antibodies. These new immunotherapies clearly show that
the immune system is able to recognize and attack the melanoma tumor cells. However,
the response rate to such therapies is less than 50% and s survival rates are
around 30%. Therefore, additional treatment s are highly needed. It is an object of
the present invention to provide means and methods for counteracting, preventing
and/or detecting melanoma and other diseases; and or to at least provide the public with
a useful choice.
Summary of the Invention
In a first aspect the present ion provides an dy or functional part or
functional equivalent thereof, that ses:
- a heavy chain CDR1 sequence DYAMH or DYAMY; and
- a heavy chain CDR2 sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence AVSGYYPYFDY or AVSGYFPYFDY or AVSGYYPYFHY
or AVSGYFPYFHY; and
- a light chain CDR1 sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence WASTRES or WASIRES; and
- a light chain CDR3 sequence QQYYTTP.
In a second aspect the present ion es an isolated, synthetic or
recombinant nucleic acid molecule with a length of at least 15 nucleotides, or a
functional equivalent thereof, encoding at least the heavy chain CDR 1-3 and the light
chain CDR 1-3 sequences of an antibody or functional part or functional lent
according to the first aspect.
In a third aspect the present invention provides a vector comprising a nucleic acid
molecule or functional equivalent according to the second aspect.
In a fourth aspect the present invention provides an isolated or inant cell,
or a non-human , comprising a nucleic acid molecule or functional equivalent
according to the second aspect or a vector ing to the third aspect.
In a fifth aspect the present invention es a composition comprising an
antibody or functional part or functional equivalent according to the first aspect, or a
nucleic acid molecule or functional equivalent according to the second aspect, or a vector
according to the third aspect, or a cell according to the fourth aspect.
In a sixth aspect the present invention provides a kit of parts comprising an
dy or functional part or functional equivalent according to the first aspect, a
nucleic acid molecule or functional equivalent ing to the second aspect, a vector
according to the third aspect or a cell according to the fourth aspect and a therapeutic
agent useful in the ent and/or prevention of a disorder associated with CD9-
sing cells.
In a seventh aspect the t invention provides a use of an antibody or
functional part or functional equivalent according to the first , or a nucleic acid
molecule or functional equivalent according to the second aspect, or a vector according to
the third aspect, or a cell according to the fourth aspect, in the manufacture of a
medicament or prophylactic agent.
In an eighth aspect the present invention provides a use of an antibody or
functional part or functional equivalent according to the first aspect in the manufacture
of a medicament for the diagnosis of a disorder associated with CD9-expressing cells.
In a ninth aspect the present ion provides a use of an antibody or functional
part or functional equivalent according to the first aspect for determining whether a
sample comprises CD9-expressing cells.
In a tenth aspect the present ion provides a method for determining
whether pressing cells are present in a sample comprising:
- contacting said sample with an antibody or functional part or functional equivalent
ing to athe first aspect, and
- allowing said antibody or functional part or functional equivalent to bind CD9-
expressing cells, if present, and
- determining whether or not CD9-expressing cells are bound to said antibody or
functional part or functional equivalent, thereby determining whether or not CD9-
expressing cells are present in said sample.
In an eleventh aspect the present ion provides a method for ing an
antibody or onal part or functional equivalent according to the first aspect, the
method comprising providing a cell with a nucleic acid le or functional equivalent
or a vector according to the second aspect, and allowing said cell to translate said nucleic
acid le or functional equivalent or vector, thereby producing said antibody or
onal part or functional equivalent according to the first aspect.
In a twelfth aspect the present invention es a use of an antibody or
functional part or functional equivalent ing to the first aspect, or a nucleic acid
molecule or functional equivalent according to the second aspect, or a vector according to
the third aspect, or a cell according to the fourth aspect, or a composition according to
the fifth asepct for the manufacture of a medicament for at least in part treating and/or
preventing a er associated with CD9-expressing cells.
In a thirteenth aspect the present invention es an ex vivo method for
determining whether an individual is suffering from a CD9-positive cancer, the method
comprising:
- contacting tumor cells in a sample from said individual with an antibody or functional
part or functional equivalent according to the first aspect,
- allowing said antibody or functional part or functional equivalent to bind CD9-
expressing cells, if present, and
- determining whether or not CD9-expressing cells are bound to said antibody or
functional part or functional equivalent, thereby determining whether or not said
dual is suffering from a CD9-positive cancer.
ption of the Invention
Some embodiments of the present disclosure be a patient-derived, human
antibody that is specific for CD9. Importantly, this antibody is derived from a late stage
IV melanoma patient who was in complete remission after immunotherapy and is still
alive and well 10 years after treatment. The human antibody, designated AT14-012, is
able to bind CD9-containing cells like melanoma, pancreas carcinoma, esophagus
carcinoma and colon carcinoma cells.
The transmembrane protein CD9, also referred to as, t other things,
MRP-1, MIC3, DRAP-27 and TSPAN-29, is a panin with a molecular weight of
about 23-27 kDa. It is ubiquitously present on the surface of many kinds of cells,
including melanocytes, endothelial cells, certain types of nervous cells, muscoskeletal
cells and certain types of immune cells. CD9 is also present on platelets. CD9 has four
transmembrane domains, a small intracellular loop and two extracellular loops, which
are referred to as the EC1 domain and the EC2 domain (Figure 1). CD9 interacts with
numerous other proteins, such as the most important integrins (Beta1 integrin), EWI
proteins (EWI-2 and EWI-F), CD81, CD63 and EGFR. CD9 plays amongst other things a
role in cell on, proliferation and migration, including tumor proliferation and
metastasis. In view of the presence of CD9 on the cell e of many kinds of cells,
including platelets, adverse side effects were feared for currently known CD9-specific
antibodies. Indeed, Kawakatsu et al. 1993 describes that platelet aggregation effects of
anti-CD9 antibodies led to lethal thrombosis in a primate model. Monkeys injected with
the anti-CD9 antibodies died within 5 minutes due to pulmonary thrombosis. This is
confirmed in the examples of the present application: the known anti-CD9 antibody
ALB6 s strong aggregation of platelets (Example 3). Although multiple CD9
antibodies have been ped and bed, due to this severe side-effect, none of
these known CD9 antibodies has proceeded to clinical trials so far.
Interestingly r, as shown in the Examples, antibody AT14-012 has a
higher g affinity for melanoma cells as compared to primary melanocytes. In
addition, AT14-012 has a higher binding activity for colon carcinoma as compared to
y colon epithelial cells. Moreover, AT14-012 binds several primary AML blasts
and multiple myeloma cell lines, s it exhibits only a weak reactivity against
primary human tonsil cells. The Examples show that antibody AT14-012 preferentially
binds to red CD9 over binding to monomeric CD9. It is known that formation of
homoclusters of CD9 is favored by palmitoylation of CD9 and that levels of CD9
homoclusters are elevated on primary tumor cells and in particular on atic tumor
cells (Yang et al., 2006). Hence, the preferred binding of AT14-012 may contribute to the
finding that antibody AT14-012 has a higher binding affinity for several tumor cells over
CD9-expressing non-tumor cells. In addition, multimerization of AT14-012 as a result of
binding to clustered CD9 may trigger a mechanism ically inhibiting tumor growth
or e spreading.
Importantly, whereas currently known CD9-specific antibodies such as for
instance ALB6 have the severe side-effect of platelet aggregation as described above,
hence ing the risk of thrombosis as a side effect, the present inventors have
demonstrated that antibody AT14-012, and several ts of AT14-012 that bind the
same unique epitope, do not induce any detectable platelet ation in vitro.
Although AT14-012 and such variants bind and even slightly activate platelets,
aggregation was not observed. This es the important advantage of AT14-012 and
these variants over currently known CD9-specific antibodies that the risk of thrombosis
is icantly reduced. Indeed, the melanoma patient from whom AT14-012 has been
derived did not show any sign of thrombosis. In fact, this melanoma patient did not
exhibit any sign of adverse side effects resulting from his immunotherapy treatment not
even vitiligo, which is a skin disease resulting in the loss of pigment.
In view of the above-mentioned teristics, antibody AT14-012, or a
functional part or functional equivalent thereof or variants thereof having the same
binding specificity, is an attractive choice for counteracting, preventing and/or detecting
disorders associated with CD9-expressing cells, like melanoma. The therapeutic
usefulness is already apparent from the fact that this antibody was isolated from a
melanoma patient who went into complete remission and is a long-time melanoma
survivor. Moreover, as shown in the Examples, AT14-012 binds melanoma, pancreas
carcinoma, esophagus carcinoma and colon carcinoma cells, several AML blasts and
some multiple myeloma cell lines. Moreover, the Examples have shown that 12
significantly counteracts tumor growth and outgrowth of metastases in an in vivo
ma mouse model. Antibody AT14-012, as well as functional parts and functional
lents f with the same binding specificity, and other binding compounds that
are specific for the same epitope as AT14-012 and/or compete with AT14-012 for binding
to the same e of CD9, are therefore particularly suitable for detecting and/or
counteracting diseases that are associated with CD9-containing cells, like for instance
CD9-positive tumors, osteoporosis, arthritis, lung inflammation, COPD, colitis,
Alzheimer’s disease and disorders associated with innate id cells. Moreover, since
CD9 is also expressed on extracellular vesicles, these vesicles are also interesting targets
of dy AT14-012 or the mentioned binding compounds.
As shown in the Examples, antibody AT14-012 binds a CD9 epitope that resides
in the m4 region. This epitope comprises at least CD9 amino acids corresponding to
K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in Figure 2. In
particular, it is demonstrated that AT14-012 binds to amino acids K169, D171, V172,
L173 and F176 of the CD9 sequence as ed in Figure 2.
ecific dies are known in the art. However, these antibodies are often
not human and recognize a different epitope. Human antibodies bed are derived
from artificial libraries where immunoglobulin heavy and light chains are randomly
paired. In contrast AT14-012 was derived from a human patient with naturally paired
heavy and light chains. For ce, international patent application
describes antibody 10E4, obtained from a human phage y library, that binds amino
acid positions 186-191 of the CD9 sequence as depicted in Figure 2.
describes murine CD9-specific monoclonal antibodies Z9.1 and
Z9.2 which show binding to amino acid positions 112-191 of the CD9 sequence as
depicted in Figure 2 and describes murine antibody A12, which
binds amino acid positions 112-194 of the CD9 sequence as depicted in Figure 2.
concerns antibody mAb7, which binds the amino acid sequence
PKKDV, which is present on amino acid positions 167-171 of the CD9 sequence as
depicted in Figure 2.
WO 823 concerns murine monoclonal antibody ES5.2D8 which binds the
CD9 sequence GLWLRFD. This ce is located between amino acid positions 31 and
37 of the CD9 sequence as depicted in Figure 2.
European patent EP 0508417 claims murine antibodies t amino acid
sequences of CD9, which sequences are selected from amino acid ons 35-60, 113-
142, 131-166 and 163-191 of the CD9 sequence as depicted in Figure 2.
Other ecific antibodies known in the art are murine antibodies ALB6 and
HI9a. As shown in the Examples, these murine antibodies also bind a different epitope
as compared to AT14-012. For instance, AT14-012 significantly binds CD9 amino acids
K169, D171, V172, L173 and F176, as depicted in Figure 2, whereas ALB6 does not
significantly bind these amino acid residues. Moreover, as shown in the Examples,
antibody AT14-012 is able to bind a CD9 mutant (mutant m1) wherein residues 112-134
of CD9 (numbering as depicted in Figure 2) were replaced by the corresponding region of
CD81, whereas antibodies ALB6 and HI9a are not able to bind this mutant.
Furthermore, HI9a is able to bind mutant m4 (wherein residues 168-180 of CD9 were
replaced by the corresponding region of CD81), whereas AT14-012 does not bind this
mutant m4. Finally, the Examples show that antibody ALB6 binds amino acids Q161 in
m3 and F176 in m4. Hence, ALB6 and HI9a bind a different epitope as ed to
AT14-012.
The Examples r show that AT14-012 reactivity is restricted to es,
binding to mouse and rabbit platelets expressing CD9 was not observed. This confirms
the uniqueness of the epitope in CD9 that is bound by AT14-012 as compared to that of
e.g. murine antibodies ALB6 and HI9a, which do bind mouse CD9.
In conclusion, a novel human ecific antibody is provided by the present
invention, which is specific for a novel CD9 epitope.
Some embodiments of the present disclosure therefore describe an isolated,
synthetic or recombinant antibody, or a functional part or a onal equivalent
thereof, that is specific for (i.e. able to specifically bind) at least 6 amino acids located
within amino acid positions 154-181 of the CD9 sequence as depicted in Figure 2.
Preferably, said antibody or functional part or functional equivalent is specific for at
least 6 amino acids located within amino acid positions 168-181 of the CD9 sequence as
ed in Figure 2. Also described is an isolated, synthetic or recombinant antibody, or
a functional part or a functional equivalent thereof, that is specific for (i.e. able to
specifically bind) at least 5 amino acids located within amino acid positions 168-181 of
the CD9 sequence as depicted in Figure 2.
Some embodiments describe an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, that is specific for an epitope of CD9,
wherein said e comprises at least one amino acid residue selected from the group
consisting of amino acids K169, D171, V172, L173 and T175 of the CD9 sequence as
depicted in Figure 2. Some embodiments describe an isolated, synthetic or recombinant
dy, or a functional part or a functional equivalent thereof, that is specific for an
epitope of CD9, n said epitope comprises at least one amino acid residue ed
from the group consisting of amino acids K169, D171, V172 and L173 of the CD9
sequence as depicted in Figure 2. Said antibody or functional part or functional
lent according to the disclosure is preferably also able to specifically bind amino
acid F176 of the CD9 sequence as depicted in Figure 2. Some embodiments describe an
antibody or onal part or functional equivalent that is specific for an epitope of CD9,
wherein said epitope comprises at least two amino acid residues selected from the group
consisting of amino acids K169, D171, V172, L173, T175 and F176 of the CD9 sequence
as depicted in Figure 2. In some embodiments, said dy or functional part or
functional equivalent is specific for an epitope of CD9, wherein said epitope comprises at
least three, or at least four or at least five, amino acid residues selected from the group
consisting of amino acids K169, D171, V172, L173, T175 and F176 of the CD9 sequence
as depicted in Figure 2. Said epitope preferably ses at least three, four or five
amino acid es selected from the group ting of amino acids K169, D171, V172,
L173 and F176 of the CD9 sequence as depicted in Figure 2. Preferred embodiments
be an isolated, synthetic or recombinant antibody, or a functional part or a
functional equivalent thereof according to the disclosure, that is specific for an epitope of
CD9 comprising amino acids corresponding to K169, D171, V172, L173 and F176 of the
CD9 sequence as depicted in Figure 2. Some embodiments describe an isolated, synthetic
or recombinant antibody, or a functional part or a functional lent thereof
according to the disclosure, that is specific for an epitope of CD9 comprising amino acids
corresponding to K169, D171, V172, L173 and F176 of the CD9 sequence as ed in
Figure 2.
As used herein, the term “CD9 sequence as depicted in Figure 2” means the
amino acid sequence of the human CD9 protein as depicted in Figure 2 (UniProt number
P21926; Genbank ion number NP-001760).
As used herein, the sions “located within CD9 amino acid ons X and Y
as depicted in Figure 2” and “located within amino acid positions X and Y of the CD9
sequence as depicted in Figure 2” encompass sequences that are d between the
recited positions and that include the amino acid(s) of position X and/or Y. In addition,
the terms embrace sequences that are located between the recited positions and that do
not contain the amino acid(s) of positions X and/or Y.
The term “antibody” as used herein, refers to an immunoglobulin protein
comprising at least a heavy chain variable region (VH), paired with a light chain
variable region (VL), that is specific for a target epitope.
A “functional part of an dy” is defined herein as a part that has at least one
shared property as said antibody in kind, not necessarily in amount. Said functional part
is capable of binding the same antigen as said antibody, albeit not necessarily to the
same extent. In one embodiment, a functional part of an antibody comprises at least a
heavy chain variable domain (VH). Non-limiting examples of a onal part of an
antibody are a single domain dy, a single chain antibody, a nanobody, an unibody,
a single chain variable fragment , a Fd fragment, a Fab fragment and a F(ab')2
fragment.
A “functional equivalent of an antibody” is defined herein as an artificial binding
compound, comprising at least one CDR sequence of an antibody, preferably a heavy
chain CDR3 sequence. Said functional equivalent preferably comprises the heavy chain
CDR3 sequence of an dy, as well as the light chain CDR3 sequence of said
antibody. More preferably, said functional equivalent comprises the heavy chain CDR1,
CDR2 and CDR3 sequences of an antibody, as well as the light chain CDR1, CDR2 and
CDR3 sequences of said antibody. A onal equivalent of an antibody is for instance
ed by altering an antibody such that at least an antigen-binding ty of the
resulting compound is essentially the same in kind, not necessarily in amount. This is
done in many ways, for instance through conservative amino acid substitution, whereby
an amino acid residue is substituted by another residue with generally similar ties
(size, hydrophobicity, etc.), such that the overall functioning of the antibody is
essentially not affected.
As is well known by the skilled person, a heavy chain of an antibody is the larger
of the two types of chains making up an immunoglobulin molecule. A heavy chain
comprises a constant domain and a variable domain, which le domain is involved
in antigen g. A light chain of an antibody is the smaller of the two types of chains
making up an immunoglobulin molecule. A light chain comprises a constant domain and
a variable domain. The variable domain is often, but not , together with the
variable domain of the heavy chain involved in antigen binding.
Complementary-determining regions (CDRs) are the hypervariable regions
present in heavy chain variable domains and light chain variable domains. In case of
whole antibodies, the CDRs 1-3 of a heavy chain and the CDRs 1-3 of the ted light
chain together form the antigen-binding site.
As used herein, the term “an antibody or functional part or onal equivalent
according to the invention” is also ed to as “a binding compound according to the
invention”.
The terms “specific for”, “able to specifically bind” and "capable of specifically
binding" are used herein interchangeably and refer to the interaction between an
antibody, or functional part or onal equivalent thereof, and its epitope. This means
that said antibody, or functional part or functional equivalent thereof, preferentially
binds to said epitope over other antigens or amino acid sequences. Thus, although the
antibody, functional part or equivalent may non-specifically bind to other antigens or
amino acid sequences, the binding affinity of said antibody or onal part or
functional equivalent for its epitope is significantly higher than the non-specific g
affinity of said antibody or functional part or functional equivalent for other antigens or
amino acid sequences.
An antibody or functional part or onal equivalent according to the invention
that is able to bind a particular epitope of CD9 can also be specific for other, non-CD9
cells if said epitope of CD9 also happens to be present in another protein. In that case an
antibody referred to herein as being specific for CD9 is also ic for such other
protein comprising the same epitope.
“Binding affinity” refers to the strength of the total sum of the noncovalent
interactions between a single binding site of an antibody or functional part or functional
equivalent and its binding partner (e.g., an antigen). Unless indicated otherwise, as used
herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and antigen). The affinity
can generally be represented by the equilibrium dissociation constant (KD), which is
calculated as the ka to kd ratio, see, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881.
Affinity can be measured by common methods known in the art, such as for instance a
Surface Plasmon Resonance (SPR) assay such as e (GE Healthcare) or IBIS-iSPR
instrument at IBIS Technologies BV (Hengelo, the Netherlands) or on phase
, such as Kinexa.
The percentage of identity of an amino acid or nucleic acid sequence, or the term
“% sequence identity”, is d herein as the percentage of residues in a candidate
amino acid or nucleic acid sequence that is identical with the residues in a reference
sequence after aligning the two sequences and introducing gaps, if necessary, to e
the maximum percent identity. Methods and computer programs for the alignment are
well known in the art, for example "Align 2".
An antibody or functional part or functional equivalent according to the disclosure
is preferably able to bind melanoma cells, colon carcinoma cells, pancreas carcinoma
cells and esophagus carcinoma cells. Such g nd is suitable for
counteracting various kinds of cancers and has, ore, a broad applicability.
icity for at least melanoma is preferred.
In some embodiments, an dy or functional part or functional equivalent
according to the disclosure is a human antibody or a onal part or functional
equivalent thereof. The presence of human amino acid sequences diminishes the chance
of adverse side effects during therapeutic use in human patients, as opposed to murine
or zed antibodies, wherein the non-human CDR or variable region sequences
lly result in an anti-murine immune response in human recipients.
In one particularly preferred embodiment, an antibody or functional part or
functional equivalent according to the disclosure is described wherein said antibody is of
the IgG isotype, preferably IgG1 or IgG3. This is beneficial for medical applications in
humans.
A preferred antibody according to the present disclosure is antibody AT14-012.
This antibody is preferred because it binds at least melanoma, as carcinoma,
esophagus carcinoma and colon carcinoma cells, several AML blasts and some multiple
myeloma cell lines. Moreover, it has been demonstrated that AT14-012 counteracts
metastases in vivo. This dy is, therefore, particularly suitable for counteracting
disorders associated with CD9-expressing cells, such as for instance a cancer involving
CD9-positive tumor cells, like melanoma. Interestingly, AT14-012 is of the IgG3 isotype
and s to the VH3-09 family. The heavy chain CDR1, CDR2 and CDR3 sequences,
and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT14-012 are
depicted in Table 1 and Figure 3.
As used herein, the term “AT14-012” encompasses all antibodies and onal
parts and onal equivalents thereof having at least the heavy chain and light chain
CDR1-3 sequences, preferably at least the heavy chain and light chain variable region
sequences, of antibody AT14-012. Such antibodies and functional parts and functional
equivalents for instance comprise isolated and/or purified antibodies, recombinant
antibodies, and/or antibodies obtained using an AT14-012 nucleic acid ce that has
been codon optimized for a producer host cell such as for instance a CHO cell.
Based on the AT14-012 sequences depicted in Table 1 and Figure 3, it is le
to produce an antibody or functional part or onal equivalent thereof comprising at
least one CDR ce of AT14-012, which is specific for CD9. Described is therefore an
isolated, recombinant and/or synthetic antibody or a functional part or functional
equivalent f comprising at least one CDR sequence of antibody 12, as
depicted in Table 1 and Figure 3. Said at least one CDR sequence preferably at least
comprises a CDR3 ce. Further described is therefore an isolated, synthetic or
recombinant antibody, or a functional part or a functional equivalent thereof, that
comprises at least a heavy chain CDR3 sequence having the sequence AVSGYYPYFDY
and a light chain CDR3 sequence having the sequence QQYYTTP. Preferably, binding
compounds are described that comprise at least two CDRs, more preferably at least three
CDRs, of antibody AT14-012. In some ments, at least two or three CDRs of the
heavy and light chains of antibody AT14-012 are jointly present in one binding
compound according to the disclosure. Preferably, a binding compound according to the
disclosure comprises all three heavy chain CDRs and all three light chain CDRs of
antibody AT14-012.
Optionally, at least one of said CDR sequences is optimized, y generating a
variant binding compound, preferably in order to improve binding efficacy, selectivity,
and/or ity. This is for instance done by mutagenesis procedures where after the
ity and/or binding cy of the resulting compounds are preferably tested and an
improved CD9-specific binding compound is selected. A skilled person is well capable of
generating variants comprising at least one altered CDR sequence according to the
disclosure. For instance, conservative amino acid substitution is d. Examples of
conservative amino acid substitution include the substitution of one hydrophobic residue
such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and
the substitution of one polar residue for another polar residue, such as the substitution
of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
Preferably, an antibody or functional part or functional equivalent is described
comprising a CDR sequence which is at least 80% identical to a CDR sequence of
antibody AT14-012, so that the ble CD9-binding characteristic is maintained or
even improved. Variant binding compounds comprising an amino acid sequence which is
at least 80% identical to a CDR sequence of antibody 12 are therefore also within
the scope of the present disclosure. Preferably, said binding compounds se heavy
chain and light chain CDR 1-3 sequences which are at least 80% identical to the heavy
and light chain CDR 1-3 sequences of antibody AT14-012. Preferably, the CDR
sequences of such variants differ in no more than three, preferably in no more than two,
preferably in no more than one amino acid from the al AT14-012 CDR sequences.
Besides optimizing CDR sequences in order to improve binding efficacy or
stability, at least one sequence in at least one of the framework regions can be optimized.
This is preferably done in order to improve g efficacy or ity. Framework
sequences are for instance zed by mutating a nucleic acid molecule encoding such
framework sequence where after the characteristics of the ing binding compound
are ably tested. This way, it is possible to obtain improved binding compounds. In
a preferred embodiment, human germline sequences are used for framework regions in
antibodies according to the disclosure. The use of human ne sequences minimizes
the risk of immunogenicity of said antibodies, because these sequences are less likely to
contain somatic alterations which are unique to individuals from which the framework
regions are derived, and may cause an immunogenic response when applied to another
human individual. Further bed is ore a synthetic or recombinant antibody or
functional part or functional equivalent according to the disclosure, comprising at least
one mutation in a framework region, as compared to the framework region of 12.
Additionally, or alternatively, a synthetic or inant antibody or functional part or
functional equivalent according to the disclosure is described that comprises at least one
mutation in a nt , as compared to the constant region of antibody AT14-012.
Such binding compound with at least one mutation as compared to AT14-012 does not
occur in nature. Instead, it has been artificially produced. In one embodiment, the IgG3
Fc region of antibody AT14-012 is at least partly replaced by an IgG1 Fc region. This
typically increases the stability and half life of the resulting immunoglobulin.
In some embodiments, a binding compound according to the present disclosure
comprises a human le region. In some embodiments, said binding compound
comprises a human constant region and a human variable region. In some preferred
embodiments, said binding nd is a human antibody. For therapeutic applications
in humans, the use of human CD9-specific antibodies is advantageous over the use of
non-human antibodies. The in vivo use of non-human antibodies for diagnosis and/or
treatment of human diseases is hampered by a number of factors. In particular, the
human body may ize non-human antibodies as foreign, which will result in an
immunogenic se against the non-human antibodies, resulting in e side
effects and/or rapid clearance of the antibodies from the ation. A human antibody
diminishes the chance of side-effects when administered to a human individual and often
results in a longer half-life in the ation e of reduced clearance when
compared to non-human antibodies.
In some embodiments, a binding compound according to the disclosure is a
chimeric antibody. In such chimeric antibody, sequences of st such as for instance
an additional binding site of interest are provided to a binding compound according to
the disclosure.
Binding compounds according to the disclosure are preferably monoclonal
antibodies. A monoclonal antibody is an antibody ting of a single molecular
s. Monoclonal antibodies can be produced in large quantities by monoclonal
antibody-producing cells or recombinant DNA technology.
Hence, variant binding compounds based on antibody AT14-012 can be generated,
using techniques known in the art such as for instance nesis. Typically, sequence
variations between 80 and 99% are tolerated while maintaining antigen specificity. One
embodiment therefore describes an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence that has at least 80% sequence identity with the
ce DYAMH; and
- a heavy chain CDR2 sequence that has at least 80% sequence identity with the
ce GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence that has at least 80% sequence identity with the
sequence AVSGYYPYFDY; and
- a light chain CDR1 ce that has at least 80% sequence identity with the sequence
KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence that has at least 80% sequence identity with the sequence
WASTRES; and
- a light chain CDR3 sequence that has at least 80% sequence identity with the sequence
QQYYTTP. Said dy or functional part or functional equivalent is preferably
specific for an epitope of CD9 comprising at least one amino acid ed from the group
consisting of K169, D171, V172 and L173 of the CD9 sequence as depicted in Figure 2,
more preferably specific for an epitope of CD9 comprising amino acids corresponding to
K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in Figure 2.
Preferably, said sequence identity is at least 85%, more preferably at least 86%,
more preferably at least 87%, more preferably at least 88%, more preferably at least
89%, more preferably at least 90%, more preferably at least 91%, more preferably at
least 92%, more preferably at least 93%, more preferably at least 94%, more preferably
at least 95%, more preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, more ably 100%.
The CDR ing and definition used herein is according to Kabat et al ,
unless indicted otherwise. Correspondence between different numbering system,
including the Kabat ing, the EU numbering and the IMGT numbering, is well
known to a person skilled in the art.
Some embodiments ore describe an ed, synthetic or recombinant
dy or a functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence ty with the sequence DYAMH; and
- a heavy chain CDR2 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence identity with the sequence GISWNSGSIVYADSVKG;
- a heavy chain CDR3 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence identity with the sequence PYFDY; and
- a light chain CDR1 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence identity with the ce KSSQSVLYSSNNKNYLG;
- a light chain CDR2 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence identity with the sequence WASTRES; and
- a light chain CDR3 sequence that has at least 85%, preferably at least 90%, more
preferably at least 95% sequence identity with the sequence QQYYTTP. Said antibody or
functional part or functional equivalent is ably specific for an epitope of CD9
comprising at least one amino acid selected from the group consisting of K169, D171,
V172 and L173 of the CD9 sequence as ed in Figure 2, more preferably specific for
an epitope of CD9 comprising amino acids corresponding to K169, D171, V172, L173 and
F176 of the CD9 sequence as depicted in Figure 2.
Some embodiments describe an isolated, synthetic or recombinant antibody or a
functional part or a functional lent thereof, that comprises:
- a heavy chain CDR1 sequence that has at least 97% sequence identity with the
sequence DYAMH; and
- a heavy chain CDR2 sequence that has at least 97% sequence identity with the
sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence that has at least 97% sequence identity with the
sequence AVSGYYPYFDY; and
- a light chain CDR1 sequence that has at least 97% sequence identity with the sequence
KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence that has at least 97% sequence identity with the sequence
S; and
- a light chain CDR3 sequence that has at least 97% sequence identity with the sequence
QQYYTTP. Said antibody or functional part or functional equivalent is preferably
specific for an epitope of CD9 comprising at least one amino acid selected from the group
consisting of K169, D171, V172 and L173 of the CD9 sequence as depicted in Figure 2,
more preferably specific for an epitope of CD9 comprising amino acids corresponding to
K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in Figure 2.
In the Examples, it is shown that a mutation in heavy chain CDR1 and/or one or
two mutations in CDR3 and/or a mutation in light chain CDR2 result in antibodies that
bind the same epitope as antibody AT14-012 and that have a binding affinity that is
equal to or higher than the g affinity of AT14-012. Importantly, as further shown
in the Examples, variants of antibody AT14-012 that have such ons in heavy
chain CDR1 and/or heavy chain CDR3 and/or light chain CDR2 also have the property
that they do not aggregate platelets, even if the variants have a higher affinity for CD9
as compared to AT14-012. Hence, the Examples show that antibodies sing heavy
chain CDR1 and/or heavy chain CDR3 sequences that are at least 80% identical to the
heavy chain CDR1, heavy chain CDR3 and light chain CDR2 sequences of antibody
AT14-012 have the same new and unique properties, including specificity for a novel
epitope of CD9 and absence of platelet aggregation, as antibody AT14-012. The affinity of
antibody AT14-012 is not linked to the e of platelet aggregation, but instead the
crucial characteristic that is associated with the absence of platelet aggregation is the
recognition of a unique epitope on CD9.
One embodiment therefore describe an isolated, synthetic or recombinant
antibody or a functional part or a functional equivalent thereof, that is specific for an
epitope of CD9 sing at least one amino acid ed from the group consisting of
K169, D171, V172 and L173 of the CD9 sequence as depicted in Figure 2, and that
comprises:
- a heavy chain CDR1 sequence that has at least 80% sequence ty with the
sequence DYAMH; and
- a heavy chain CDR2 sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence that has at least 80% ce identity with the
ce AVSGYYPYFDY; and
- a light chain CDR1 ce KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence that has at least 85% sequence identity with the sequence
WASTRES; and
- a light chain CDR3 sequence QQYYTTP. Said antibody or functional part or functional
equivalent is preferably specific for an epitope of CD9 comprising amino acids
corresponding to K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in
Figure 2.
Methods to determine r or not an antibody or functional part or functional
equivalent f is specific for an epitope of CD9 comprising at least one amino acid
ed from the group consisting of K169, D171, V172 and L173 of the CD9 sequence as
depicted in Figure 2 are well in the art and for instance described in the Examples
. Indeed, the Examples show how binding to CD9, the epitope of CD9 that is bound
by and antibody and the affinity of and antibody for CD9 can be determined.
The Examples also show that mutations in framework regions can be made
without affecting the binding specificity and affinity and/or t effecting the degree
of binding specificity and affinity. Indeed, the T29N mutation in heavy chain framework
region 1, the L94P mutation in the light chain framework region 3 and the L120V
mutation in light chain framework region 4 (IMGT numbering) did not have a major
impact on binding of AT14-012 or on improved binding of variants of AT14-012 that
show improved binding as ed to aT14-012.
As said before, typically at most 3 amino acid residues of a given CDR sequence
may vary while ing the same kind of binding activity (in kind, not necessarily in
amount). Hence, a binding compound according to the disclosure preferably contains
heavy chain and light chain CDR1-3 sequences wherein at most 3, preferably at most 2,
more preferably at most 1 amino acid deviate(s) from the heavy and light chain CDR1-3
sequences of antibody AT14-012. In some embodiments, the heavy and light chain
CDR1-3 sequences of a binding compound according to the disclosure are identical to the
heavy and light chain CDR1-3 sequences of antibody AT14-012. Further bed is
therefore an isolated, synthetic or recombinant antibody or a onal part or a
functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 ce having the sequence AVSGYYPYFDY; and
- a light chain CDR1 ce having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 ce having the sequence QQYYTTP.
In some embodiments, the heavy and light chain CDR1-3 sequences of a binding
compound according to the disclosure are identical to the heavy and light chain CDR1-3
sequences of variants of 12, which have an affinity that is comparable to or
higher than that of antibody AT14-012.
Further described is therefore an isolated, synthetic or recombinant antibody or a
functional part or a functional lent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the ce GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the ce AVSGYYPYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the ce WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is therefore an isolated, tic or recombinant antibody or a
functional part or a functional equivalent f, that ses:
- a heavy chain CDR1 sequence having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is therefore an isolated, synthetic or recombinant antibody or a
onal part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further bed is therefore an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 ce having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence S; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is therefore an ed, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 ce having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is therefore an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
r bed is therefore an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent f, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the ce AVSGYFPYFHY; and
- a light chain CDR1 sequence having the sequence LYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 ce having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the ce PYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is therefore an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 ce having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 ce having the sequence PYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 ce having the sequence QQYYTTP.
Further described is an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the ce DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and
- a light chain CDR1 sequence having the ce KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 ce having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is an isolated, synthetic or inant antibody or a
onal part or a onal equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 ce having the sequence QQYYTTP.
Further described is an isolated, synthetic or recombinant antibody or a
functional part or a functional equivalent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMH; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and
- a light chain CDR1 sequence having the ce KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
Further described is an isolated, tic or recombinant dy or a
functional part or a functional lent thereof, that comprises:
- a heavy chain CDR1 sequence having the sequence DYAMY; and
- a heavy chain CDR2 sequence having the sequence GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the ce AVSGYFPYFHY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASIRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP.
A particularly preferred antibody comprises:
- a heavy chain CDR1 sequence having the ce DYAMY; and
- a heavy chain CDR2 sequence having the ce GISWNSGSIVYADSVKG; and
- a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and
- a light chain CDR1 sequence having the sequence KSSQSVLYSSNNKNYLG; and
- a light chain CDR2 sequence having the sequence WASTRES; and
- a light chain CDR3 sequence having the sequence QQYYTTP. As demonstrated in the
Examples, such antibody has a particularly high affinity (see figure 19B).
Preferably, a binding compound according to the disclosure comprises a variable
heavy chain sequence and/or a variable light chain sequence of antibody AT14-012, or
heavy and light chain variable sequences that are at least 80% identical. The heavy and
light chain variable regions of antibody AT14-012 are also depicted in Table 1 and Figure
3. r described is ore an antibody or functional part or functional equivalent
according to the disclosure, comprising a heavy chain variable region sequence having at
least 80% sequence identity with the sequence
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGI
LVTVSS and/or a light chain le region sequence having at least 80% sequence
identity with the ce
DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS
TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK, or
sequences that are at least 85%, more preferably at least 86%, more preferably at least
87%, more preferably at least 88%, more preferably at least 89%, more preferably at
least 90%, more preferably at least 91%, more preferably at least 92%, more preferably
at least 93%, more preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more preferably at least 98%,
more preferably at least 99%, or even 100% identical to the above ned heavy chain
and/or light chain variable region sequences of AT14-012. Preferably, in the heavy chain
and light chain variable regions the heavy chain CDR1, CDR2 and CDR3 sequences have
at least 80% ce ty with the sequences DYAMH (CDR1),
GSIVYADSVKG (CDR2) and AVSGYYPYFDY (CDR3) and the light chain
CDR1, CDR2 and CDR3 sequences have at least 80% ce identity with the
sequences KSSQSVLYSSNNKNYLG , WASTRES (CDR2) and QQYYTTP
(CDR3). Said antibody or functional part or functional lent is preferably specific
for an epitope of CD9 comprising at least one amino acid selected from the group
consisting of K169, D171, V172 and L173 of the CD9 sequence as depicted in Figure 2,
more preferably specific for an epitope of CD9 sing amino acids corresponding to
K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in Figure 2. The
higher the identity, the more closely a binding compound resembles antibody AT14-012.
Preferably, a g compound according to the disclosure comprises both the heavy
chain variable region sequence and the light chain variable region sequence of antibody
AT14-012, as depicted in Table 1 and Figure 3, or heavy and light chain variable region
sequences that are at least 80%, preferably at least 85%, or at least 86%, or at least 87%,
or at least 88%, or at least 89%, or at least 90%, or 91%, or at least 92%, or at least 93%,
or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at
least 99%, identical thereto.
ularly preferred is an antibody or functional part or functional equivalent
thereof that is specific for an epitope of CD9 comprising at least one amino acid selected
from the group consisting of K169, D171, V172 and L173 of the CD9 sequence as
depicted in Figure 2, preferably specific for an epitope of CD9 comprising amino acids
corresponding to K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in
Figure 2 and that comprises:
- a heavy chain variable region sequence
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGI
LVTVSS, or
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI
LVTVSS or
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSG
SVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI
LVTVSS or
SGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHYWGQGI
LVTVSS or
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI
LVTVSS or
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSG
SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI
LVTVSS or
EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSG
SVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHYWGQGI
LVTVSS and/or
- a light chain variable region sequence having at least 80% sequence identity with the
sequence
DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS
TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK,
preferably whereby the CDR1, CDR2 and CDR3 sequences comprise at least 80%
sequence identity with the sequences KSSQSVLYSSNNKNYLG (CDR1), WASTRES
(CDR2) and P (CDR3), more ably a light chain variable region sequence
DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS
TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK or
DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS
IRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK.
As already mentioned before, an antibody or functional part or functional
equivalent according to the t disclosure is preferably of the IgG isotype, more
preferably IgG1 or IgG3, in view of the stability of such immunoglobulin in vivo. In
addition, as shown in the Examples, antibody AT14-012 is able to trigger complement
dependent cytotoxicity (CDC) in tumor cells when the antibody is in a IgG3 backbone.
Moreover, a mutation in the IgG Fc tail (E345R, EU numbering, as described in Kabat
1991) forces hexamerization of the dy upon target binding, C1q deposition on the
tumor cell surface and further s tration dependent cell death via CDC (De
Jong 2016). Hence, in a particularly preferred embodiment, an antibody, functional part
or functional lent according to the disclosure is of the IgG3 isotype. In a further
preferred embodiment, an antibody, functional part or functional equivalent according to
the disclosure is of the IgG isotype, preferably IgG1 or IgG3, more preferably IgG3, and
comprises an arginine at amino acid on 345 (EU numbering).
As shown in the Examples, antibody AT14-012 is able to specifically bind amino
acids K169, D171, V172, L173 and F176 of the CD9 sequence as ed in Figure 2.
Now that this is known, g compounds can be obtained or generated that compete
with AT14-012 for the same epitope. For instance, a CD9 peptide comprising at least 4,
preferably at least 5, or 6 of the above ned amino acid residues is described, or a
CD9 peptide consisting of at least 4, preferably at least 5, or 6 of the above mentioned
amino acid residues, where after a non-human animal is immunized with such CD9
peptide, or with an immunogenic compound comprising such CD9 peptide, or with a
nucleic acid molecule or functional equivalent thereof encoding such CD9 peptide,
preferably followed by one or more booster administrations. Subsequently, antibodies
that are specific for CD9 are harvested from said non-human animal. Alternatively, or
additionally, CD9-specific B cells are harvested from said non-human . Such CD9-
specific B-cells are particularly suitable for the production of CD9-specific antibodies.
CD9-specific B-cells harvested from said immunized animal are for ce used for the
production of hybridomas, from which CD9-specific antibodies are obtained. In other
embodiments, said CD9-specific B cells ted from said immunized animal are
transduced with a Bcl-6 nucleic acid and with an anti/apoptotic nucleic acid such as for
instance Bcl-xL or Mcl-1, and cultured in long term ex vivo B cell cultures, as for
instance described in European Patent No. 7 and US patent No. 9,127,251. This
way, long term replicating B cell cultures are generated, wherein the B cells both
replicate and produce antibody. In some ments, CD9-specific antibodies produced
by said hybridomas or by such B cell culture are harvested and for instance used for
anti-CD9 therapy, preferably after humanization of the antibodies in order to reduce
side-effects. In some embodiments, an antibody and/or B cell obtained from said nonhuman
animal is tested for competition with antibody AT14-012 for binding to CD9. This
is for instance done by incubating CD9-expressing cells with said dy or B cell
obtained from said non-human animal, and subsequently adding antibody AT14-012. As
a l, CD9-expressing cells are preferably incubated with antibody AT14-012 in the
e of any other antibody or B cell. If pre-incubation of CD9-expressing cells with an
antibody or B cell obtained from said non-human animal s to affect the binding of
AT14-012 to said cells, it is concluded that said antibody or b cell ed from said non-
human animal competes with antibody AT14-012 for binding to CD9.
In some embodiments, the variable domain-encoding nucleic acid sequences of
CD9-specific B cells obtained from said non-human animal are sequenced in order to
obtain the nucleic acid sequences of the CD9-specific variable domains, where after one
or more nucleic acid molecules comprising these sequences are introduced in producer
cells, such as for instance E. coli, Chinese hamster ovary (CHO) cells, NSO cells (a mouse
myeloma) or 293(T) cells, for the production of CD9-specific antibodies. Said one or more
nucleic acid sequences are preferably codon optimized for said producer cell. As used
herein, the term “codon” means a triplet of nucleotides (or functional equivalents thereof)
that encode a specific amino acid residue. The term “codon optimized” means that one or
more codons from the original, animal nucleic acid sequence are replaced by one or more
codons that are red by a n producer cell. These replacement codons
preferably encode the same amino acid residue as the original animal codon that has
been ed.
In some embodiments, CD9-specific antibodies obtained from said non-human
animal or from immune cells of said non-human animal are humanized, meaning that at
least part of the animal amino acid sequence, preferably at least part or the whole of the
framework sequences, is replaced by a human sequence in order to reduce adverse sideeffects
in humans.
Animal immunization ols, including suitable stration procedures and
adjuvants, procedures for obtaining and purifying antibodies and/or immune cells from
such immunized animals, competition experiments and humanization ures of nonhuman
antibodies are well known in the art. Reference is for instance made to Hanly et
al, 1995.
In some embodiments, a CD9 peptide comprising, or consisting of, at least 4,
preferably at least 5, or 6 of the CD9 amino acid residues selected from the group
consisting of K169, D171, V172, L173, T175 and F176 as depicted in Figure 2, preferably
of the CD9 amino acid residues ed from the group ting of K169, D171, V172,
L173 and F176, or a compound comprising such CD9 peptide, is used for screening a
phage display y in order to identify and/or isolate CD9-specific immunoglobulins
(typically Fab fragments). In some embodiments, a naïve phage display library is used.
In preferred embodiments, a phage display library derived from one or more melanoma
ts is used, so that the library will already be biased. In some embodiments, a CD9-
ic immunoglobulin obtained from said phage display library is tested for
competition with antibody AT14-012 for binding to CD9. This is for ce done using
a competition test described .
Antibodies that are obtained, produced or selected with a method as described
above will typically compete with antibody AT14-012 for at least part of the same CD9
e. Further described is, therefore, an isolated, synthetic or inant antibody
or functional part or functional equivalent thereof that competes with dy AT14-
012 for binding to CD9. Some embodiments describe an isolated, synthetic or
recombinant antibody or onal part or functional equivalent thereof that competes
with antibody AT14-012 for binding to at least 4 CD9 amino acids selected from the
group consisting of K169, D171, V172, L173 and F176, as depicted in Figure 2. In some
embodiments, said antibody or functional part or functional equivalent competes with
antibody AT14-012 for binding to at least 4, or at least 5, CD9 amino acids selected from
the group consisting of K169, D171, V172, L173 and F176, as ed in Figure 2. In
some embodiments, said antibody or functional part or functional equivalent competes
with antibody AT14-012 for binding to CD9 amino acids K169, D171, V172, L173 and
F176, as ed in Figure 2.
Also described is an antibody or functional part or functional equivalent according
to the disclosure, which is coupled to r compound. In one embodiment, a binding
compound according to the disclosure is d to another therapeutic moiety, such as a
chemotherapeutic drug or other toxic compound or radioactive compound, to form a so
called “antibody-drug conjugate”. In another embodiment, a moiety that is coupled to a
binding compound according to the disclosure is an immunomodulatory molecule such as
for instance a CD3-specific antibody. Such CD3-specific antibody is capable of binding T
cells and, if coupled to a binding compound according to the disclosure, it will target T
cells to CD9-containing cells such as melanoma cells, thereby enhancing an antimelanoma
T-cell response. This provides an even stronger anti-melanoma . Also
described is a bispecific or multispecific binding compound, comprising a CD9-specific
binding compound according to the present disclosure and an modulatory
molecule, preferably a CD3-specific binding compound. Another preferred embodiment
bes an anti-CD9 compound, said compound comprising a binding compound
according to the present disclosure, which is specific for CD9, and a toxic moiety. In some
other embodiments, a binding compound according to the present disclosure is d to
a label. This allows detection of CD9-containing cells, such as for instance melanoma
cells, using such labeled binding compound. Other embodiments describe a binding
nd according to the disclosure that is coupled to another CD9-specific g
compound. In some embodiments, such other CD9- specific binding compound is also a
binding nd according to the present disclosure. Described is ore a
compound comprising two binding compounds according to the disclosure that are
coupled to each other, such as for instance two coupled AT14-012 antibodies or
functional parts or functional equivalents thereof. In some embodiments, a binding
compound according to the disclosure is coupled to another CD9-specific binding
compound, such as for instance a currently known anti CD9 antibody, in order to
produce a bispecific compound. In some ments, a heavy chain of antibody
AT14-012 is paired with a heavy chain of another CD9-specific antibody, in order to
produce a bispecific antibody. Bispecific nds and bispecific antibodies according
to the disclosure allow, for ce, for increased binding of CD9-containing cells,
especially when the two coupled binding compounds are specific for different CD9
epitopes. Such bispecific compound and/or bispecific antibody is thus very suitable for
therapeutic or diagnostic applications. It is also possible to use bispecific compounds and
bispecific antibodies according to the disclosure in assays wherein different CD9-
containing cells are bound to the same bispecific binding nd.
In some embodiments, a synthetic or recombinant dy is described, or a
functional part or a functional equivalent thereof, which comprises one Fab nt of
an antibody according to the present disclosure, preferably an AT14-012 Fab fragment,
and one Fab fragment of another ecific antibody. The ing binding compound
is monospecific for CD9, but each Fab arm will typically bind its own CD9 epitope. In
some embodiments, the epitopes recognized by the Fab fragments are different from
each other. In another embodiment, the es are the same. The Fab arms may bind
the epitopes with different affinity. Alternatively, the Fab arms bind their epitopes with
essentially the same affinity, meaning that the KD of the Fab arms differ no more than
%, preferably no more than 20% or no more than 10% from each other.
In some embodiments, a synthetic or recombinant antibody is described, or a
functional part or a onal lent thereof, which comprises one Fab fragment of
an antibody according to the t disclosure, preferably an AT14-012 Fab fragment,
and one Fab fragment of r antibody. For instance, such antibody comprises one
Fab fragment of an antibody according to the disclosure and one Fab fragment of a
blocking antibody specific for a complement regulatory protein or a blocking antibody
specific for a co-inhibitory T cell molecule. Preferred examples of a ng antibody
specific for a complement regulatory protein from which a Fab fragment is t in
such antibody is a CD55 blocking antibody, a CD46 blocking antibody or a CD59
blocking antibody, more preferably a CD55 ng antibody. red es of a
blocking antibody against a ng antibody specific for a co-inhibitory T cell molecule
from which a Fab fragment is t in such antibody is an anti-CTLA4 antibody, an
anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα
antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD276 antibody, an
anti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, an anti-VISTA
antibody and an anti-IDO antibody, preferably an anti-PD-1 antibody or an anti-PD-L1
Further bed is therefore an antibody or functional part or functional
equivalent according to the disclosure, that is coupled to another compound. In some
embodiments, said other compound is a detectable label, a chemotherapeutic drug, a
toxic moiety, an immunomodulatory molecule, another CD9-specific binding compound,
or a radioactive compound. Some embodiments describe antibody AT14-012 that is
coupled to another compound, for instance any one of the compounds mentioned above.
Some embodiments describe a bispecific antibody, or a onal part or functional
equivalent thereof, comprising a Fab fragment of antibody AT14-012 and a Fab fragment
of another CD9-specific antibody. Some embodiments describe a bispecific antibody, or a
onal part or functional equivalent thereof, comprising a heavy chain of antibody
12 paired with a heavy chain of r CD9-specific antibody.
In some embodiments, a binding compound according to the sure is coupled
to another moiety, such as for example a chemotherapeutic agent or a CD3-specific
antibody, via a linker such as for instance an acid-labile hydrazone linker, or via a
peptide linker like line-valine, or through a thioether linkage, or by sortase A
catalyzed transamidation, which is described in detail in WO 87994.
Sortase catalyzed transamidation involves engineering of a sortase ition
site (LPETGG) on the heavy chain of an antibody, preferably on the C-terminal part of
the heavy chain, and on the moiety to be coupled to said antibody. The antibody and the
moiety further typically contain a GGGGS sequence and a tag for purification purposes,
such as a HIS tag. Subsequently sortase mediated transamidation is performed followed
by click try linkage. In a e catalyzed transamidation, "click chemistry
linkage" typically involves chemical coupling of, for instance, an alkyne-containing
t and, for instance, an azide-containing reagent which are added by e
h addition of glycines to the sortase motif on the heavy chain of the antibody and
to a sortase motif on the moiety (such as a protein, peptide or antibody) to be coupled to
the antibody. In one embodiment, described herein is an antibody according to the
disclosure wherein a sortase recognition site (LPETGG) is engineered on the heavy chain
of the antibody, preferably on the C-terminal part of the heavy chain, the antibody
preferably further containing a GGGGS sequence and a cation tag, such as a HIS
tag.
In another embodiment, a g compound according to the disclosure is
coupled to r moiety via a thioether linkage. In such case, one or more cysteines
are preferably orated into a binding compound according to the disclosure.
Cysteines contain a thiol group and, therefore, incorporation of one or more cysteines
into a binding compound according to the disclosure, or replacement of one or more
amino acids by one or more cysteines of a binding compound according to the disclosure,
enable coupling of said binding compound to another moiety. Said one or more cysteines
are preferably introduced into a binding compound according to the disclosure at a
position where it does not significantly influence g of said binding compound, and
does not significantly alter antigen binding or effector function. Also described is a
binding nd according to the disclosure that ses a heavy chain sequence of
antibody AT14-012, wherein at least one amino acid of said AT14-012 sequence (other
than cysteine) has been ed by a cysteine.
Also described is an antibody or functional part or functional equivalent ing
to the disclosure, which is combined with another therapeutic agent. For instance, an
antibody or functional part or functional equivalent according to the disclosure is
combined with another agent that is capable of at least in part treating or preventing a
disorder ated with CD9-expressing cells, ably a disorder selected from the
group consisting of CD9 positive cancer, osteoporosis, arthritis, lung inflammation,
COPD, colitis, and a disorder associated with innate lymphoid cells. An antibody or
functional part or functional equivalent according to the disclosure, which is ed
with another therapeutic agent useful in the treatment and/or prevention of a CD9
ve cancer. es of such agents are complement tory proteins, antibodies
specific for a co-inhibitory T cell molecule, small molecules against mutated BRAF (e.g.
vemurafenib or dabrafenib) and other chemotherapy agents. Described is therefore a use
or method for at least in part treating or ting a disorder associated with CD9-
expressing cells according to the disclosure whereby an antibody or functional part or
functional equivalent according to the disclosure is combined with a therapeutic agent
useful in the treatment and/or prevention of a disorder associated with CD9-expressing
cells, preferably a CD9 positive . Also described is a kit of parts comprising an
antibody or functional part or functional equivalent according to the disclosure and a
therapeutic agent useful in the treatment and/or prevention of a disorder associated with
CD9-expressing cells, preferably a CD9 positive cancer. Preferred, but non-limiting
es of such agents are complement regulatory proteins, antibodies specific for a coinhibitory
T cell molecule, small molecules against mutated BRAF (e.g. vemurafenib or
dabrafenib) and other chemotherapy agents. For instance, the Examples show that
dy AT14-012 E345R ently kills tumor cells by CDC in the presence of human
complement factors, which lack the expression of CD55, an inhibitor of C3 convertase
ion. Hence, when the antibody is combined with an agent capable of stimulating
C3 convertase formation or capable of racting inhibition of C3 convertase
formation, such as a CD55 blocking antibody, complement dependent cell death of tumor
cells may be induced. Antibodies against other complement regulatory proteins, blocking
of which enhances CDC, such as a CD46 blocking antibody or a CD59 blocking antibody,
may also be ageously combined with an antibody, functional part or functional
equivalent according to the disclosure.
Described is therefore a use or method for at least in part treating or preventing a
er associated with CD9-expressing cells according to the disclosure whereby an
dy or functional part or onal equivalent according to the disclosure is
combined with an agent capable of stimulating C3 tase formation or capable of
counteracting inhibition of C3 convertase formation. Said agent is preferably a CD55
blocking antibody, a CD46 blocking antibody or a CD59 blocking antibody, more
preferably a CD55 blocking antibody. Said disorder is preferably a CD9 positive cancer,
more preferably ed from the group consisting of melanoma, colorectal cancer,
pancreatic , esophageal cancer, lung , breast cancer, ovarian cancer,
stomach cancer, squamous cell carcinoma, AML, multiple myeloma, gastric cancer, liver
cancer, brain cancer, Kaposi sarcoma, carcinoma idermoid, choriocarcinoma,
fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lung arcinoma, ll-cell
lung carcinoma, bladder cancer and small cell lung cancer.
Also described is a kit of parts comprising an antibody or functional part or
functional lent ing to the disclosure and a eutic agent useful in the
treatment and/or prevention of a disorder associated with pressing cells,
preferably a CD9 positive . In a preferred embodiment, said agent is an agent
capable of stimulating C3 convertase formation or capable of counteracting inhibition of
C3 tase formation. Said agent is preferably a CD55 blocking antibody, a CD46
blocking antibody or a CD59 blocking antibody, more preferably a CD55 blocking
antibody. Also described is a kit of part comprising a c acid molecule or onal
equivalent, a vector or a cell according to the disclosure and an agent capable of
stimulating C3 convertase formation or capable of counteracting inhibition of C3
convertase formation. Said agent is preferably a CD55 blocking antibody, a CD46
blocking antibody or a CD59 blocking antibody, more preferably a CD55 blocking
antibody.
An antibody or functional part or functional equivalent according to the disclosure
is further optionally combined with an antibody specific for a co-inhibitory T cell
molecule, such as an antibody blocking the L1-axis. Antibodies blocking the
PD1-PDL1 axis, in particular those binding PD1, are now widely used to treat a wide
y of late stage cancer patients. The es show that when antibody AT14-012
is combined with nivolumab (Opdivo, Bristol-Myers Squibb), an anti-PD-1 dy, the
inhibition of tumor growth was strongly enhanced in comparison to treatment with
AT14-012 alone.
An antibody against a co-inhibitory T cell molecule is preferably a blocking
dy. A “blocking antibody” as used herein refers to an antibody or fragment whose
binding to it antigen reduces or blocks the interaction between the antigen and its
target. For instance, a blocking antibody against CTLA-4 refers to an antibody that
reduces or blocks the binding of soluble human CTLA-4 to cell-expressed CD80 and
CD86 (B7-1 and B7-2) and thereby inhibits the T cell tory activity of CTLA-4.
Suitable antibody against a co-inhibitory T cell molecule include, but are not limited to, a
blocking antibody ic for cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed
death-1 (PD-1), PD-ligand 1 (PD-L1), PD-L2, Signal-regulatory protein alpha (SIRPα), T-
cell immunoglobulin- and mucin domaincontaining molecule 3 (TIM3), lymphocyte-
activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), CD276, CD272,
A2AR, VISTA and indoleamine 2,3 dioxygenase (IDO).
Described is therefore a use or method for at least in part treating or preventing a
disorder associated with pressing cells according to the disclosure whereby an
antibody or functional part or functional equivalent according to the disclosure is
combined with a blocking antibody specific for a co-inhibitory T cell molecule. Said
antibody is preferably selected from the group consisting of an anti-CTLA4 antibody, an
anti-PD-1 antibody, an anti-PD-L1 antibody, an D-L2 dy, an anti-SIRPα
antibody, an IM3 dy, an anti-LAG3 antibody, an anti-CD276 antibody, an
anti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, an anti-VISTA
antibody and an anti-IDO antibody. Suitable antibodies used as a further
immunotherapy component are nivolumab, pembrolizumab, lambrolizumab, ipilimumab
and mab. In a particularly preferred embodiment, said antibody is an antibody
blocking the PD1-PDL1-axis, such as an anti-PD1 antibody or an DL1 antibody,
more preferably an anti-PD1 antibody. Said disorder is preferably a CD9 positive cancer,
more preferably selected from the group consisting of melanoma, colorectal cancer,
pancreatic cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer,
stomach cancer, squamous cell carcinoma, AML, multiple myeloma, gastric cancer, liver
cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma,
fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lung adenocarcinoma, nonsmall-cell
lung oma, bladder cancer and small cell lung cancer.
Also described is a kit of parts comprising an antibody or functional part or
onal equivalent according to the disclosure and a blocking antibody specific for a
co-inhibitory T cell molecule. Also described is a kit of part comprising a nucleic acid
molecule or onal equivalent, a vector or a cell ing to the disclosure and a
blocking antibody specific for a co-inhibitory T cell molecule. Said dy is preferably
ed from the group consisting of an anti-CTLA4 antibody, an D-1 antibody, an
anti-PD-L1 antibody, an anti-PD-L2 antibody, an IRPα antibody, an anti-TIM3
antibody, an anti-LAG3 dy, an anti-CD276 antibody, an anti-CD272 antibody, an
anti-KIR antibody, an anti-A2AR antibody, an anti-VISTA antibody and an anti-IDO
dy. Suitable antibodies used as a further immunotherapy component are
mab, pembrolizumab, lambrolizumab, ipilimumab and lirilumab. In a particularly
preferred embodiment, said antibody is an antibody blocking the L1-axis, such
as a PD1 blocking antibody or a PDL1 blocking antibody, more preferably a PD1
blocking antibody.
A kit of parts according to the disclosure may comprise one or more containers
filled with pharmaceutical composition sing an dy, functional part or
functional lent according to the disclosure and a pharmaceutical composition
sing the agent capable of stimulating C3 convertase formation or capable of
racting inhibition of C3 convertase formation, preferably a CD55 blocking
antibody, or the blocking dy specific for a co-inhibitory T cell molecule, preferably
a PD1 or PDL1 ng antibody. The kit of part or the one or more containers further
optionally comprises one or more pharmaceutically acceptable excipients. Associated
with such kit of parts or container(s) can be various written als such as
instructions for use, or a notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceuticals products, which notice
reflects approval by the agency of manufacture, use, or sale. Preferably, a kit of parts
comprises instructions for use.
Also described is a pharmaceutical composition comprising a an antibody or
functional part or functional equivalent according to any one of claims 1-15, a
therapeutic agent useful in the treatment and/or prevention of a disorder ated with
CD9-expressing cells, preferably a CD9 positive , and a pharmaceutically
acceptable carrier, diluent or excipient. In a preferred embodiment, said agent is an
agent capable of stimulating C3 convertase formation or capable of counteracting
inhibition of C3 convertase formation, preferably a CD55 ng antibody, a CD46
blocking antibody or a CD59 blocking antibody, more preferably a CD55 blocking
antibody. In a further preferred embodiment, said agent is a blocking antibody specific
for a co-inhibitory T cell molecule, preferably selected from the group consisting of an
TLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2
antibody, an anti-SIRPα antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an
anti-CD276 antibody, an anti-CD272 antibody, an anti-KIR antibody, an anti-A2AR
antibody, an anti-VISTA antibody and an anti-IDO antibody, more preferably a PD1
blocking antibody or a PDL1 blocking antibody.
Also, described herewith are c acid molecules and functional lents
thereof, and vectors, encoding at least one CDR region of an antibody, functional part or
functional equivalent or binding compound according to the disclosure. Preferably, at
least the heavy chain CDR1-3 regions and the light chain CDR 1-3 regions of such
binding compound are encoded by one or more nucleic acid molecules or functional
equivalents or vectors according to the present disclosure. In some embodiments, the
heavy and light chain variable regions of a binding compound ing to the disclosure
are encoded. Also described is an isolated, synthetic or recombinant c acid
molecule with a length of at least 15 nucleotides, or a onal lent thereof, or a
vector, encoding at least one CDR region of an antibody or functional part or functional
equivalent according to the disclosure. Preferably, said CDR region is a CDR region from
antibody AT14-012, or a variant thereof as bed herein that has the same or higher
binding affinity as antibody 12.
In some embodiments, a nucleic acid molecule according to the disclosure has a
length of at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably
at least 75 nucleotides. A nucleic acid molecule according to the disclosure is for instance
isolated from a B-cell which is capable of producing an antibody according to the
disclosure. Said B-cell preferably produces antibody AT14-012, or a variant thereof as
described herein that has the same or higher g affinity as antibody AT14-012.
Some embodiments describe one or more nucleic acid molecules, or functional
lents or vectors, encoding at least the heavy chain CDR3 sequence and the light
chain CDR3 sequence of AT14-012, or a variant thereof as bed herein that has the
same or higher binding affinity as antibody AT14-012.
As used herein the term “an isolated, synthetic or recombinant nucleic acid
molecule with a length of at least 15 nucleotides, or a functional equivalent thereof,
encoding at least one CDR region of an antibody or functional part or functional
equivalent according to the invention” is herein also referred to as “a nucleic acid
molecule or functional equivalent according to the invention”.
As used herein, a nucleic acid molecule or nucleic acid sequence of the disclosure
ably comprises a chain of nucleotides, more preferably DNA, cDNA or RNA. In
other ments, a nucleic acid le or nucleic acid sequence of the disclosure
ses other kinds of nucleic acid ures such as for ce a DNA/RNA helix,
peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other
nucleic acid structures are referred to as functional equivalents of a c acid
sequence. The term “functional equivalent of a nucleic acid molecule” thus encompasses
a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide
building blocks which exhibit the same function as natural nucleotides.
Nucleic acid sequences encoding the heavy chain and light chain CDR regions of
antibody AT14-012 are ed in Table 1 and Figure 3. Nucleic acid molecules having a
sequence that differs from any one of the CDR nucleic acid sequences depicted in Table 1
and Figure 3, but wherein nucleic acid codons are present which encode the same CDR
amino acid sequence(s) as depicted in Table 1 and Figure 3, are also encompassed by the
disclosure. Such nucleic acid molecules for instance comprise nucleic acid sequences that
have been codon zed for a producer cell, such as for instance E. coli or Chinese
hamster ovary (CHO) cells, NSO cells (a mouse myeloma) or 293(T) cells, enabling high
scale production of binding nds according to the sure having the same CDR
amino acid ce(s) as antibody AT14-012. It should be noted that antibody
production can be done by any recombinant antibody production system; the four
producer cell systems mentioned here are only a few examples of the many systems that
are available to date. As used herein, the term “codon” means a triplet of nucleotides (or
functional equivalents thereof) that encode a specific amino acid residue. The term
“codon optimized” means that one or more codons from the al, human nucleic acid
sequence is replaced by one or more codons that are red by a certain dy
production system. These replacement codons ably encode the same amino acid
residue as the original human codon that has been replaced. Alternatively, one or more
replacement codons encode a different amino acid residue. This preferably results in
conservative amino acid substitution, although this is not necessary. Typically, in
constant s and framework regions one or more amino acid substitutions are
generally allowed. In CDR regions, preferably codons are used that encode the same
amino acid residue as the al human codon that has been replaced.
Furthermore, nucleic acid molecules encoding a heavy or light chain CDR which
is not identical to, but based on, a CDR sequence of antibody 12 are also
encompassed by the disclosure, as long as the resulting CDR has at least 80% sequence
identity with a CDR sequence of dy AT14-012.
Further described is, therefore, a nucleic acid molecule or functional equivalent
thereof or a vector, comprising a sequence that has at least 80%, preferably at least 85%,
or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at
least 99%, sequence identity with a CDR sequence of antibody AT14-012. Preferably, the
resulting CDR s in no more than three, preferably in no more than two, preferably
in only one amino acid from the original CDR sequence of an antibody according to the
disclosure.
Some embodiments describe one or more nucleic acid molecules or functional
equivalents or s according to the disclosure, that encode at least the heavy chain
CDR 1-3 and the light chain CDR 1-3 regions of antibody AT14-012 or variants thereof
that have the same of higher binding affinity. Further described is therefore one or more
c acid molecules or functional equivalents or vectors ing to the disclosure
that comprise:
- a heavy chain CDR1 encoding nucleic acid sequence which encodes the sequence
DYAMH or DYAMY, and/or
- a heavy chain CDR2 encoding nucleic acid sequence which encodes the sequence
GISWNSGSIVYADSVKG, and/or
- a heavy chain CDR3 encoding nucleic acid sequence which encodes the sequence
AVSGYYPYFDY or AVSGYFPYFDY or AVSGYYPYFHY or AVSGYFPYFHY, and/or
- a light chain CDR1 encoding nucleic acid sequence which encodes the sequence
KSSQSVLYSSNNKNYLG, and/or
- a light chain CDR2 encoding nucleic acid sequence which encodes the sequence
WASTRES or WASIRES, and/or
- a light chain CDR3 encoding nucleic acid ce which encodes the sequence
Further described is one or more nucleic acid molecules or functional equivalents
or vectors, comprising a sequence that has at least 80% ce identity with one or
more sequences selected from the group consisting of:
- gat tat gcc atg cac; and
- ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc; and
- gcc gtg agt ggt tat tat ccc tac ttt gac tac; and
- aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt; and
- tgg gca tct acc cgg gaa tcc; and
. cag caa tat tat act act cct.
These are the heavy and light chain CDR1-3 c acid ces of antibody
AT14012, as depicted in Table 1 and Figure 3. In some embodiments, said sequence
identities are at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least
89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or
at least 96%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%.
Preferably, the above mentioned heavy and light chain CDR1-3 sequences of
AT14-012, or sequences that are at least 80% identical o, are all present. Further
described is therefore one or more nucleic acid molecules or functional equivalents or
vectors that comprise:
- a heavy chain CDR1 encoding nucleic acid sequence that has at least 80% sequence
identity with the sequence gat tat gcc atg cac, and
- a heavy chain CDR2 encoding sequence that has at least 80% sequence identity with
the sequence ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc, and
- a heavy chain CDR3 encoding sequence that has at least 80% ce identity with
the sequence gcc gtg agt ggt tat tat ccc tac ttt gac tac, and
- a light chain CDR1 encoding sequence that has at least 80% sequence identity with
the sequence aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt, and
- a light chain CDR2 ng sequence that has at least 80% sequence identity with
the sequence tgg gca tct acc cgg gaa tcc, and
- a light chain CDR3 encoding sequence that has at least 80% sequence ty with
the sequence cag caa tat tat act act cct.
In some ments, said sequence identities are at least 85%, or at least 86%, or at
least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least
92%, or at least 93%, or at least 94%, or at least 96%, or at least 96%, or at least 97%, or
at least 98%, or at least 99%, or 100%. Preferably, the encoded CDR amino acid
sequences differ in no more than three, preferably in no more than two, preferably in
only one amino acid from the heavy and light chain CDR1-3 amino acid sequences of
dy AT14-012.
Some embodiments describe nucleic acid molecules or functional lents or
vectors according to the disclosure that encode at least the heavy chain variable region
sequence and/or the light chain variable region sequence of an antibody or functional
part or functional equivalent according to the disclosure. Preferably, said at least one
nucleic acid molecule or functional equivalent or vector encodes at least the heavy chain
variable region sequence and/or the light chain variable region sequence of antibody
AT14-012, or a sequence that is at least 80% identical o.
Further described is therefore one or more nucleic acid molecules or onal
lents or vectors, comprising a sequence that has at least 80% sequence identity
with the sequence gaa gtg cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc agg tcc ctg
aga ctc tcc tgt gca gcc tct gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg
aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag
ggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa ctg aac agt ctg aga gct
gag gac acg gcc ttc tat tac tgt gca aaa gcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag
gga att ttg gtc acc gtc tcc tca, and/or comprising a sequence that has at least 80%
sequence identity with the sequence gac atc gtg atg acc cag tct cca gac tcc ctg tct gtg tct
ctg ggc gag agg gcc acc atc aac tgc aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac
tac tta ggt tgg tac cag cag aaa cca gga cag cct cct aag ctg ctc att tac tgg gca tct acc cgg gaa
tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc agc agc ctg cag
gct gaa gat gtg gca gtt tat tac tgt cag caa tat tat act act cct tcc acc ttc ggc caa ggg aca cga
ctg gag att aaa.
Preferably, one or more nucleic acid molecules or a functional equivalents or
vectors ing to the disclosure encode both a heavy chain variable region and a light
chain variable region that le the heavy chain variable region and the light chain
variable s of AT14-012 as depicted in Table 1 and Figure 3. Further described is
therefore one or more nucleic acid molecules or functional equivalents or vectors,
comprising a sequence that has at least 80% sequence identity with the sequence gaa gtg
cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct gga
ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg aag ggc ctg gag tgg gtc tca ggt
att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac
aac gcc aag aac tcc ctg tat ctg caa ctg aac agt ctg aga gct gag gac acg gcc ttc tat tac tgt gca
aaa gcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag gga att ttg gtc acc gtc tcc tca, and
comprising a sequence that has at least 80% sequence identity with the sequence gac atc
gtg atg acc cag tct cca gac tcc ctg tct gtg tct ctg ggc gag agg gcc acc atc aac tgc aag tcc agc
cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt tgg tac cag cag aaa cca gga cag cct
cct aag ctg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct
ggg aca gat ttc act ctc acc atc agc agc ctg cag gct gaa gat gtg gca gtt tat tac tgt cag caa tat
tat act act cct tcc acc ttc ggc caa ggg aca cga ctg gag att aaa.
In some embodiments, said sequence ties are at least 85%, or at least 86%,
or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at
least 92%, or at least 93%, or at least 94%, or at least 96%, or at least 96%, or at least
97%, or at least 98%, or at least 99%, or 100%.
In some ments, nucleic acid molecules and functional equivalents thereof
and vectors are described that encode an antibody or functional part or equivalent
according to the sure. In some embodiments, c acid molecules and functional
equivalents thereof and vectors are described that encode antibody AT14-012, or a
functional part or a functional lent thereof. In some embodiments, said nucleic
acid molecules or functional equivalents or vectors are codon optimized for a non-human
recombinant expression system, such as a non-human host cell like E. coli, CHO, NSO,
or 293 cells.
Some embodiments describe a vector comprising a c acid molecule or
functional equivalent according to the disclosure. As used herein “a vector comprising a
nucleic acid molecule or functional equivalent according to the invention” is also referred
to as “a vector according to the invention”. These terms ass one or more vector(s)
according to the disclosure, comprising one or more c acid molecule(s) or functional
equivalent(s) according to the disclosure. As used herein, the singular term “a”
encompasses the term “one or more”.
Methods for constructing vectors comprising one or more nucleic acid molecule(s)
or functional equivalent(s) according to the disclosure are well known in the art. Nonlimiting
examples of vectors suitable for generating a vector of the disclosure are
iral and lentiviral vectors. Such s are suitable for a variety of applications.
For instance, a vector of the disclosure comprising a therapeutically cial nucleic
acid sequence according to the disclosure is le for prophylactic or therapeutic
applications against melanoma. Administration of such vector(s) to an individual,
preferably a human, in need thereof results in expression of said prophylactic or
therapeutic nucleic acid sequence in vivo resulting in at least partial treatment or
prophylaxis against melanoma. Said vector can also be used in ations involving in
vitro sion of a nucleic acid molecule of interest, for instance for rcial)
production of antibodies or functional equivalents according to the sure. Hence,
nucleic acid molecules, functional equivalents and vectors according to the disclosure are
particularly useful for generating dies or functional parts or functional equivalents
according to the disclosure, which are specific for CD9. This is for instance done by
introducing such nucleic acid molecule(s) or functional equivalent(s) or vector(s) into a
cell so that the cell’s nucleic acid translation ery will produce the encoded
antibodies or functional parts or functional equivalents. In some embodiments, at least
one nucleic acid molecule or functional equivalent or vector encoding a heavy and light
chain variable region of a binding compound according to the disclosure is/are expressed
in so called producer cells, such as for instance E. coli, CHO, NSO or 293(T) cells, some of
which are adapted to commercial antibody production. Of note, any recombinant
antibody production system is suitable; these four producer cell systems mentioned are
only a few examples of the many systems that are available to date. As described herein
before, in such cases it is red to use nucleic acid molecules or onal
equivalents thereof wherein the original human AT14-012 sequences as described herein
are codon optimized for the producer cell. eration of said er cells results in a
producer cell line capable of producing g compounds according to the disclosure.
Preferably, said producer cell line is le for producing antibodies for use in humans.
Hence, said producer cell line is preferably free of pathogenic agents such as enic
micro-organisms. In some embodiments, antibody AT14-012 is produced in such
producer cell line.
Further described is therefore an isolated or recombinant cell, sing at least
one nucleic acid molecule and/or functional equivalent and/or vector according to the
disclosure. Such cell is preferably an antibody producing cell capable of producing a
binding compound according to the disclosure, such as for instance antibody AT14-012.
Further described is a method for producing an antibody or functional part or functional
equivalent according to the disclosure, the method comprising ing a cell with at
least one nucleic acid molecule or functional equivalent or vector according to the
disclosure, and ng said cell to translate said at least one nucleic acid molecule or
functional equivalent or vector, thereby producing said antibody or functional part or
functional equivalent according to the disclosure. In some ments, said antibody is
AT14-012 optionally having one or more of the heavy chain mutations H40Y, Y112F and
D116H and/or light chain mutation T66I (IMGT numbering), or a functional part or a
onal equivalent thereof. Said method according to the disclosure preferably further
comprises a step of harvesting, purifying and/or ing said antibody or functional
part or functional equivalent ing to the disclosure. Obtained binding nds
ing to the disclosure are for instance suitable for use in human therapy or
diagnostics, optionally after additional purifying, isolation or processing steps.
In some embodiments, at least one nucleic acid molecule or functional equivalent
or vector according to the disclosure is introduced into a non-human animal, for instance
for in vivo antibody production. Further described is therefore an isolated or
recombinant non-human animal, comprising at least one nucleic acid molecule or
functional equivalent or vector according to the disclosure. Methods for producing
transgenic non-human animals are known in the art. Reference is for instance made to
EC Lee, Nature Biotechnology, 2013.
Binding compounds according to the present disclosure are suitable for use
against melanoma. Furthermore, CD9 also has a role in other diseases, like for ce
other kinds of tumors that also express CD9. Other non-limiting es of diseases
that are associated with CD9-positive cells are osteoporosis and arthritis (Iwai et al. and
Hattori et al.), lung inflammation and COPD (Takeda et al. and Jin et al.), and colitis
(Wagner et al.). For instance, CD9 is ntly expressed in activated osteoclasts in
ovariectomy-induced osteoporosis and in bone erosions of collagen-induced arthritis (Iwai
et al. and Hattori et al.). CD9 is also sed in innate id cells. Other nonlimiting
examples of diseases that are associated with CD9-positive cells are virus
infections (for instance HIV or herpes or influenza), ial infections, CMV tis,
oral candidiasis, Glanzmann thrombasthenia and eria.
Since binding compounds according to the present disclosure are specific for CD9,
they are suitable for use against these ers as well. Binding compounds according
to the present disclosure are thus ularly suitable for use as a medicine or
prophylactic agent. Described is therefore an antibody or functional part or functional
equivalent according to the disclosure for use as a medicament and/or prophylactic
agent. In some embodiments, binding nds according to the disclosure are used
that consist of human sequences, in order to reduce the chance of e side effects
when human individuals are treated. Said antibody preferably comprises antibody AT14-
012. r described is therefore antibody AT14-012 for use as a medicament and/or
prophylactic agent. In some embodiments, human sequences are synthetically or
recombinantly produced based on the sequence of AT14-012, optionally using codon
optimized nucleic acid sequences that encode the same AT14-012 amino, or sequences
that are at least 80% identical thereto.
Also described is a nucleic acid molecule or functional equivalent thereof
according to the disclosure, or a vector according to the disclosure comprising such
nucleic acid molecule or functional equivalent, or a cell according to the disclosure, for
use as a medicament and/or prophylactic agent. When (a vector comprising) one or more
nucleic acid molecule(s) or functional lent(s) according to the disclosure is/are
administered, the nucleic acid molecule(s) or onal equivalent(s) will be translated
in situ into a binding compound according to the sure. The resulting binding
compounds ing to the disclosure will subsequently counteract or t disorders
associated with CD9-expressing cells, like for instance CD9-expressing tumors,
osteoporosis, arthritis, lung mation, COPD, colitis, or disorders associated with
innate lymphoid cells. Likewise, uction of a cell according to the disclosure into a
patient in need thereof will result in in vivo generation of therapeutic or lactic
anti-CD9 antibodies, or functional parts or functional equivalents, according to the
disclosure.
Some ments describe an antibody or functional part or functional
equivalent according to the disclosure, or a nucleic acid le or functional equivalent
or vector according to the disclosure, or a cell according to the disclosure, for use in a
method for at least in part treating or preventing a disorder associated with CD9-
sing cells. Some embodiments describe antibody AT14-012 for use in a method for
at least in part treating or preventing a disorder associated with CD9-expressing cells.
As used herein, the term “a disorder ated with CD9-expressing cells” means any
disease that involves the ce of CD9-expressing disease-specific cells. In some
embodiments, such cells are a causative factor of the disease, as is often the case for
CD9-expressing malignant cells. In some embodiments, the presence of such cells cause
adverse symptoms, such as for instance inflammation and/or pain. A non-limiting
example of a disorder associated with pressing cells is a cancer with
CD9-expressing tumor cells, like for instance melanoma, colorectal , pancreatic
cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer,
basal cell carcinoma, squamous cell carcinoma, AML, multiple myeloma, c cancer,
liver cancer, cervical cancer, renal cell carcinoma, prostate , brain cancer, Kaposi
sarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma, cervical carcinoma,
glioma, adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma, bladder
cancer and small cell lung cancer.
As used herein, a tumor cell that expresses CD9 is also referred to as a CD9-
positive tumor cell or a CD9-positive malignant cell. A cancer wherein at least part of the
tumor cells express CD9 is referred to as a “CD9-positive cancer”. Other non-limiting
examples of a disorder associated with CD9-expressing cells are osteoporosis, tis,
lung inflammation, COPD, colitis and disorders associated with innate lymphoid cells.
Further described is therefore an antibody or functional part or functional
equivalent ing to the disclosure, or a nucleic acid molecule or functional equivalent
or vector according to the disclosure, or a cell according to the disclosure, for use in a
method for at least in part treating or preventing a disorder associated with CD9-
expressing cells, wherein said disorder is selected from the group consisting of
CD9-positive cancer, osteoporosis, arthritis, lung inflammation, COPD, s, and a
disorder associated with innate lymphoid cells. Said CD9-positive cancer is preferably
selected from the group consisting of melanoma, colorectal cancer, pancreatic cancer,
esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer,
squamous cell carcinoma, AML, multiple myeloma, c cancer, liver cancer, brain
cancer, Kaposi sarcoma, oma mucoepidermoid, carcinoma, arcoma,
cervical carcinoma, glioma, arcinoma, lung adenocarcinoma, non-small-cell lung
oma, bladder cancer and small cell lung cancer.
The es show that AT14-012 is able to kill ma cells via antibody
dependent cytotoxicity (ADCC) while minimal cell death was ed when primary
Human Artery elial Cells (HAECs). In on, antibody AT14-012 has been
shown to be able to trigger complement dependent cytotoxicity (CDC) when the antibody
is in a IgG3 backbone. Hence, without wishing to be bound by theory, it is speculated
that the anti-tumor reactivity of AT1412 is at least in part mediated via ADCC. Hence,
in a preferred embodiment, an antibody, functional part or functional equivalent
according to the disclosure or for use ing to the disclosure is able to induced
antibody dependent cytotoxicity (ADCC) and/or complement ent cytotoxicity
(CDC) in CD9-expressing cells.
A preferred antibody for use in any of the recited methods is antibody 12,
or a variant of antibody AT14-012 described herein that the same binding specificity and
the same or higher affinity as antibody AT14-012.
In some embodiments, antibody AT14-012 optionally having one or more of the
mutations H40Y, Y112F and D116H and/or light chain mutation T66I (IMGT
numbering), or a functional part or functional equivalent thereof, or at least one nucleic
acid molecule or functional equivalent thereof encoding AT14-012 optionally having one
or more of the mutations H40Y, Y112F and D116H and/or light chain mutation T66I
(IMGT numbering), or a functional part or functional equivalent thereof, or at least one
vector or cell comprising said nucleic acid molecule or functional equivalent, is preferably
used for at least in part treating and/or preventing melanoma. As used herein the term
“at least in part treating and/or preventing ma” includes counteracting melanoma
tumor growth and/or alleviating symptoms resulting from the presence of melanoma
cells in a patient. Also described is therefore a use of antibody AT14-012 optionally
having one or more of the mutations H40Y, Y112F and D116H and/or light chain
mutation T66I (IMGT numbering), or a functional part or functional equivalent thereof,
or of at least one nucleic acid molecule or functional equivalent encoding AT14-012
optionally having one or more of the mutations H40Y, Y112F and D116H and/or light
chain mutation T66I (IMGT numbering), or a functional part or functional equivalent
thereof, or of at least one vector or cell comprising said nucleic acid molecule or
functional equivalent, for the preparation of a medicament and/or prophylactic agent for
at least in part treating and/or preventing melanoma. r described is dy
AT14-012 optionally having one or more of the mutations H40Y, Y112F and D116H
and/or light chain mutation T66I (IMGT numbering), or a onal part or onal
equivalent thereof, or at least one nucleic acid molecule or onal lent
encoding AT14-012 optionally having one or more of the mutations H40Y, Y112F and
D116H and/or light chain mutation T66I (IMGT numbering), or a onal part or
functional equivalent thereof, or at least one vector or cell comprising said nucleic acid
molecule or functional equivalent, for use in a method for at least in part treating and/or
preventing melanoma.
In some embodiments, a g nd according to the disclosure is coupled
to a therapeutic moiety, such as a chemotherapeutic drug or other toxic nd or a
radioactive compound or an immunomodulatory molecule such as for instance a CD3-
specific antibody, to form a so called ody-drug conjugate” or a “chimeric antigen
receptor (CAR) T cell”, respectively, which is able to counteract a myeloproliferative or
lymphoproliferative disorder.
Further embodiments describe a composition comprising an antibody or
functional part or functional equivalent according to the disclosure. A composition
comprising a nucleic acid molecule or onal equivalent according to the disclosure is
also described, as well as a composition sing a vector or a cell according to the
disclosure. In some embodiments, said antibody is AT14-012, optionally having one or
more of the mutations H40Y, Y112F and D116H and/or light chain mutation T66I
(IMGT numbering). In some embodiments, a composition according to the disclosure
ses antibody AT14-012 optionally having one or more of the mutations H40Y,
Y112F and D116H and/or light chain mutation T66I (IMGT numbering), or a onal
part or functional equivalent thereof, and another CD9-specific antibody. Said other
CD9-specific dy preferably binds a different CD9 epitope as compared to
AT14-012. Such ation of different CD9-specific binding compounds is ularly
suitable for binding and/or counteracting CD9-positve cells, such as melanoma cells or
other CD9-positve tumor cells.
In some embodiments, a composition according to the present disclosure is a
pharmaceutical composition. Such ceutical composition preferably also comprises
a ceutical able carrier, diluent and/or excipient. Non-limiting examples of
suitable carriers for instance comprise keyhole limpet haemocyanin (KLH), serum
albumin (e.g. BSA or RSA) and ovalbumin. In one red embodiment said suitable
carrier comprises a solution, like for example saline. A pharmaceutical composition
according to the disclosure is preferably suitable for human use.
Also described is a method for at least in part treating and/or preventing a
disorder associated with CD9-expressing cells, comprising administering to an individual
in need thereof a therapeutically effective amount of an antibody or functional part or
functional equivalent according to the disclosure, and/or a nucleic acid molecule or
functional equivalent thereof according to the disclosure, and/or a vector or cell according
to the disclosure, and/or a composition according to the disclosure. As used , an
“individual” or “subject” is a human or a non-human animal, preferably a human patient
suffering from a CD9-positive cancer, orosis, arthritis, lung inflammation, COPD,
colitis, or a disorder ated with innate lymphoid cells. In some embodiments, said
human individual is a melanoma patient. Said composition is ably a
pharmaceutical composition according to the disclosure. A binding compound or a nucleic
acid molecule or a functional equivalent or a vector or a pharmaceutical composition
ing to the disclosure is preferably administered via one or more injections. l
doses of administration of a binding compound according to the disclosure are between
0.1 and 10 mg per kg body weight.
A binding compound ing to the disclosure is also particularly useful for
detection of CD9 expressing cells. For ce, if an individual, preferably a human, is
suspected of suffering from a disorder associated with CD9-expressing cells, a sample
such as a blood or tissue sample from said individual can be tested for the presence of
CD9-expressing cells (also referred to as CD9-positive cells), using a binding compound
according to the disclosure. In some embodiments said sample is mixed with a binding
compound according to the disclosure, which will specifically bind CD9-positive cells.
CD9-positive cells, such as for instance melanoma cells, bound to a binding nd
according to the sure can be isolated from the sample and/or detected using any
method known in the art, for example, but not d to, isolation using ic beads,
streptavidin-coated beads, or isolation through the use of secondary antibodies
immobilized on a column. Alternatively, or additionally, a binding nd according
to the disclosure is d in order to be able to detect said binding nd. Such
binding compound is for instance fluorescently labeled, enzymatically labeled, or
radioactively labeled. Alternatively, a binding compound according to the disclosure is
detected using a labeled secondary antibody which is directed against said binding
If a binding compound according to the sure appears to be bound to a
ent of a patient’s sample, it is indicative for the presence of CD9-positive cells.
This way, disease-specific CD9 positive cells like melanoma cells can be detected. Some
embodiments ore describe a use of an antibody or functional part or functional
equivalent according to the disclosure for determining whether a sample comprises CD9-
expressing cells. In some embodiments said antibody or functional part or functional
equivalent ing to the disclosure is used for determining whether a sample
comprises CD9-expressing tumor cells. Also described is a method for determining
whether CD9-expressing cells, preferably CD9 positive tumor cells, are present in a
sample comprising:
- contacting said sample with an antibody or onal part or onal equivalent
according to the disclosure, and
- allowing said antibody or functional part or functional equivalent to bind CD9-
expressing cells, if present, and
- determining whether or not CD9-expressing cells, such as for instance CD9 positive
tumor cells, are bound to said dy or functional part or functional equivalent,
thereby determining whether or not CD9-expressing (tumor) cells are present in said
sample. In some embodiments, said CD9-expressing tumor cells are melanoma cells.
As shown in the Examples, antibody AT14-012 is particularly suitable for
detecting CD9-positive cells, like for instance CD9-positive tumor cells. r
described is therefore a use of antibody AT14-012 optionally having one or more of the
mutations H40Y, Y112F and D116H and/or light chain mutation T66I (IMGT
numbering), or a functional part or functional equivalent thereof, for determining
r a sample comprises CD9-expressing cells. Also described is a use of antibody
AT14-012 optionally having one or more of the mutations H40Y, Y112F and D116H
and/or light chain mutation T66I (IMGT numbering), or a functional part or functional
equivalent f, for determining r a sample comprises CD9-expressing tumor
cells, like for instance ma cells or colorectal cancer cells or pancreatic cancer cells
or esophageal cancer cells or lung cancer cells or breast cancer cells or ovarian cancer
cells or stomach cancer cells or squamous cell carcinoma cells or AML cells or multiple
myeloma cells or gastric cancer cells or liver cancer cells or brain cancer cells or Kaposi
sarcoma cells or carcinoma idermoid cells or choriocarcinoma cells or
fibrosarcoma cells or cervical carcinoma cells or glioma cells or adenocarcinoma cells or
lung adenocarcinoma cells or non-small-cell lung carcinoma cells or bladder cancer cells
or small cell lung cancer cells.
Also described is a method for determining whether CD9-expressing cells,
preferably CD9 positive tumor cells, are present in a sample comprising:
- contacting said sample with antibody AT14-012 optionally having one or more of the
mutations H40Y, Y112F and D116H and/or light chain mutation T66I (IMGT
ing), or with a functional part or functional equivalent thereof, and
- allowing antibody AT14-012 optionally having one or more of the ons H40Y,
Y112F and D116H (IMGT numbering), or said functional part or functional equivalent
thereof, to bind pressing cells, if t, and
- determining whether or not CD9-expressing cells, such as for instance CD9 positive
tumor cells, are bound to antibody AT14-012 optionally having one or more of the
mutations H40Y, Y112F and D116H and/or light chain mutation T66I (IMGT
numbering), or to said onal part or functional equivalent thereof, thereby
determining whether or not CD9-expressing cells are present in said sample.
Also described is a method according to the disclosure wherein said sample
comprises a blood sample, or a bone marrow sample, or a biopsy. In some embodiments,
said biopsy is from skin , in order to test for melanoma and/or squamous cell
carcinoma. In some embodiments, said biopsy is from the intestines, in order to test for
gastric cancer, colorectal cancer, esophageal cancer or stomach . In some
embodiments, said biopsy is from pancreatic tissue, to test for pancreatic cancer, or from
lung tissue, to test for lung cancer, or from breast tissue, to test for breast cancer, or
from ovarian tissue, to test for ovarian cancer, or from liver tissue, to test for liver
cancer, or from brain , to test for brain cancer or from mucoepidermoid tissue to
test for carcinoma mucoepidermoid, or from cervical tissue to test for cervical carcinoma,
or from bladder tissue to test for bladder cancer. In some embodiments, said sample is a
blood sample, which is for instance useful for testing for AML, multiple myeloma, cancer
related extracellular vesicles mes), or the presence of metastases of any of the
above mentioned solid tumors.
The test results with a binding compound according to the disclosure are useful
for typing of a sample. For instance, if a sample of an individual appears to contain
malignant CD9-positive cells, the sample is typed as containing disease-associated cells.
Such typing can subsequently be used for diagnosis of a disorder associated with CD9-
expressing cells. Some ments therefore describe an antibody or functional part or
functional equivalent according to the disclosure for use in diagnosis of a disorder
associated with CD9-expressing cells. Said disorder is preferably selected from the group
consisting of a CD9 positive cancer, arthritis, lung inflammation, COPD, colitis, and a
disorder ated with innate lymphoid cells. Said CD9 positive cancer is preferably
selected from the group consisting of melanoma, colorectal cancer, pancreatic cancer,
esophageal cancer, lung cancer, breast cancer, n cancer, stomach cancer,
squamous cell carcinoma, AML, multiple myeloma, gastric cancer, liver , brain
cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma,
cervical oma, glioma, adenocarcinoma, lung adenocarcinoma, non-small-cell lung
carcinoma, bladder cancer and small cell lung cancer. In some preferred embodiments,
antibody AT14-012 optionally having one or more of the mutations H40Y, Y112F and
D116H and/or light chain mutation T66I (IMGT numbering), or a functional part or
functional equivalent is used for the above-mentioned detection and diagnosis. Also
described is therefore dy AT14-012 ally having one or more of the mutations
H40Y, Y112F and D116H and/or light chain mutation T66I (IMGT numbering), or a
functional part or functional lent thereof, for use in diagnosis of a er
associated with CD9-expressing cells. Some embodiments describe dy AT14-012
optionally having one or more of the mutations H40Y, Y112F and D116H and/or light
chain mutation T66I (IMGT numbering), or a functional part or functional equivalent
thereof, for use in diagnosis of melanoma, colorectal , pancreatic cancer,
esophageal cancer, lung cancer, breast cancer, ovarian , stomach ,
squamous cell carcinoma, AML, le myeloma, gastric cancer, liver cancer, brain
cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma, arcoma,
cervical carcinoma, glioma, arcinoma, lung adenocarcinoma, non-small-cell lung
carcinoma, bladder cancer or small cell lung cancer.
Also described is an ex vivo method for ining whether an individual is
suffering from a CD9-positive cancer, the method comprising:
- contacting tumor cells from said individual with an antibody or functional part or
functional equivalent according to the disclosure,
- allowing said antibody or functional part or functional equivalent to bind CD9-
expressing cells, if present, and
- ining whether or not CD9-expressing cells are bound to said antibody or
functional part or functional equivalent, thereby determining whether or nor said
individual is suffering from a CD9-positive cancer.
Non-limiting examples of such CD9-positive cancer are listed above. Preferably,
antibody AT14-012 optionally having one or more of the mutations H40Y, Y112F and
D116H and/or light chain mutation T66I (IMGT numbering) or a functional part or
functional equivalent thereof is used for said method. Some ments therefore
describe an ex vivo method for determining whether an individual is suffering from a
CD9-positive cancer, the method comprising:
- contacting tumor cells from said individual with dy AT14-012 optionally having
one or more of the mutations H40Y, Y112F and D116H and/or light chain mutation T66I
(IMGT numbering), or with a onal part or functional equivalent thereof,
- allowing said antibody or functional part or functional equivalent to bind
CD9-expressing cells, if present, and
- determining whether or not CD9-expressing cells are bound to said dy or
functional part or functional equivalent, thereby determining whether or nor said
individual is suffering from a CD9-positive cancer.
As shown in the Examples, antibody AT14-012 binds at least 5 CD9 amino acids
located within ons 154-181, preferably 168-181, of the CD9 sequence as ed in
Figure 2. Antibody AT14-012 binds a CD9 epitope that comprises CD9 amino acids
corresponding to K169, D171, V172, L173 and F176 of the CD9 sequence as ed in
Figure 2. In particular, AT14-012 binds to amino acids K169, D171, V172, L173 and
F176 of this CD9 sequence. Now that this is known, it has become le to obtain or
generate further antibodies with specificity for CD9. As described herein before, this can
for instance be done by immunizing a non-human animal with a CD9 e comprising
at least 4, preferably at least 5, of the above mentioned amino acid residues, or with a
CD9 peptide ting of at least 4, preferably at least 5, of the above mentioned amino
acid residues, or with an immunogenic compound comprising such CD9 peptide, or with
a nucleic acid molecule or functional lent thereof encoding such CD9 peptide,
preferably followed by one or more booster administrations. Subsequently, antibodies
and/or B cells that are specific for CD9 can be harvested from said non-human animal.
In some embodiments, said antibody or B cell is tested for competition with antibody
12 for binding to CD9.
Alternatively, or additionally, said CD9 peptide is used to screen a phage y
library in order to fy and/or isolate CD9-specific immunoglobulins, typically Fab
fragments. Obtained antibodies, B cells or Fab fragments will typically e with
antibody AT14-012 for binding to CD9. In some embodiments, a competition assay is
performed.
The above mentioned CD9 peptides and uses thereof are also encompassed by the
t disclosure. Some embodiments therefore describe an isolated, inant or
purified CD9 peptide with a length of at most 60 amino acid residues, wherein said
peptide comprises at least 5 amino acid residues that are identical to at least 5 amino
acid residues located within CD9 amino acid positions 154-181, preferably amino acids
positions 168-181, as depicted in Figure 2. Some embodiments describe an isolated,
recombinant or purified CD9 e with a length of at most 60 amino acid residues,
n said peptide comprises at least 6 amino acid residues that are identical to at
least 6 amino acid residues located within CD9 amino acid positions 154-181, preferably
amino acids positions 168-181, as depicted in Figure 2. In some embodiments said CD9
e comprises 5 or 6 amino acid residues that are identical to 5 or 6 amino acid
residues located within CD9 amino acid positions 169-176 as depicted in Figure 2. In
some embodiments, said isolated, recombinant or purified CD9 peptide at least
comprises amino acids corresponding to K169, D171, V172, L173 and F176 of the CD9
ce as depicted in Figure 2. In some embodiments, said isolated, recombinant or
purified CD9 peptide at least comprises amino acids corresponding to K169, D171, V172,
L173 and T175 of the CD9 sequence as depicted in Figure 2. Preferably, said CD9
peptide further comprises an amino acid corresponding to F176 of the CD9 sequence as
depicted in Figure 2.
As used herein, any of the above-mentioned peptides are referred to as a “CD9
peptide according to the ion”.
In some embodiments, a CD9 peptide according to the disclosure has a length of
at most 55 amino acid residues. In some embodiments, a CD9 peptide according to the
present disclosure has a length of at most 50 amino acid residues or at most 45 amino
acid residues or at most 40 amino acid residues. In some embodiments, a CD9 peptide
according to the present disclosure has a length of at most 35 amino acid residues or at
most 30 amino acid residues or at most 25 amino acid residues or at most 20 amino acid
residues or at most 15 amino acid residues. In some embodiments, said CD9 peptide
according to the present disclosure has a length of 10 amino acid residues, or 9 amino
acid residues or 8 amino acid residues.
Besides the recited amino acid residues that are identical to at least 6 amino acid
residues located within positions 154-181 of the human CD9 protein as depicted in
Figure 2, preferably within positions 168-181 of said CD9 protein, a CD9 peptide
according to the t disclosure may further comprise other amino acid residues. In
some embodiments, said other amino acid residues are not derived from a human CD9
sequence. Said other amino acid residues, which are referred to as “non-CD9 amino acid
residues” may for instance function to enhance ity, and/or to enhance
immunogenicity, and/or to couple the CD9 peptide to another moiety such as for instance
a molecular scaffold or carrier. Non-limiting examples of such scaffold or rs are
e limpet hemocyanin and CLIPS scaffolds (such as for instance
bis(bromomethyl)benzene, tris(bromomethyl)benzene and tetra(bromomethyl)benzene,
bed in ). Some embodiments therefore describe an isolated,
recombinant or ed CD9 peptide with a length of at most 60 amino acid residues,
wherein said peptide comprises at least 5 amino acid residues that are identical to at
least 5 amino acid residues located within CD9 amino acid positions 154-181, preferably
amino acids positions 168-181, as depicted in Figure 2. ably, said peptide
comprises at least 6 amino acid residues that are cal to at least 6 amino acid
residues d within CD9 amino acid positions 154-181, preferably amino acids
positions 1, as depicted Figure 2, preferably selected from the group consisting of
K169, D171, V172, L173 and F176 of the CD9 sequence as depicted in Figure 2, more
preferably at least comprising K169, D171, V172, L173 and F176 of the CD9 sequence as
depicted in Figure 2, and wherein said peptide further comprises at least 1, or at least 2,
or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at
least 40, or at least 50, non-CD9 amino acid residues, wherein the full length sequence of
said non-CD9 amino acid residues is not present in the corresponding CD9 amino acid
position as depicted in Figure 2. Such peptide preferably comprises at least 6 amino acid
residues that are identical to at least 6 amino acid residues located within CD9 amino
acid positions 154-181, preferably amino acids positions 168-181, as depicted Figure 2,
preferably selected from the group consisting of K169, D171, V172, L173, T175 and F176
of the CD9 sequence as depicted in Figure 2, more preferably at least comprising K169,
D171, V172, L173, T175 and F176 of the CD9 sequence as depicted in Figure 2. Such
peptide is also embraced by the term “CD9 peptide according to the disclosure”. Some
ments describe an isolated, recombinant or purified CD9 peptide with a length of
at most 60 amino acid residues, wherein said peptide ses at least 5 amino acid
es that are identical to at least 6 amino acid residues located within CD9 amino
acid ons 154-181 as depicted Figure 2, preferably amino acid positions 168-181,
ably at least sing K169, D171, V172, L173 and F176 of the CD9 sequence as
ed in Figure 2, more preferably K169, D171, V172, L173, T715 and F176, that is
coupled to r peptide containing non-CD9 amino acid residues. Some embodiments
describe an isolated, recombinant or purified CD9 peptide with a length of at most 60
amino acid residues, wherein said e comprises at least K169, D171, V172, and
L173 and T175of the CD9 sequence as ed in Figure 2, and preferably also the F176
e of the CD9 sequence as depicted in Figure 2, wherein said peptide is coupled to
another peptide containing non-CD9 amino acid residues. In some embodiments, said
peptides are coupled to each other via a peptide bond. In other embodiments, said
peptides are coupled to each other via another, non-peptide bond, such as for instance a
linker.
As is known to the skilled , once an immunogenic sequence has been
described, it has become possible to alter the sequence to some extent, thereby preferably
zing the immunogenicity and/or stability of the resulting immunogen. This is for
instance done by mutagenesis procedures where after the stability and/or
immunogenicity of the resulting compounds are preferably tested and an improved CD9
antigenic compound is selected. A skilled person is well capable of generating antigen
variants starting from a certain amino acid sequence. In some ments, a
replacement net analysis is carried out, which involves replacement of one or more
amino acid residues by any other amino acid residue, and g the resulting
compounds. In some preferred embodiments, conservative amino acid substitution is
used. Examples of conservative amino acid substitutions include the substitution of one
hydrophobic residue such as isoleucine, , leucine or methionine for another
hydrophobic residue, and the tution of one polar residue for another polar residue,
such as the substitution of arginine for , glutamic acid for aspartic acid, or
glutamine for asparagine. Another example of conservative amino acid substitutions
includes the substitution of serine for threonine and tyrosine for phenylalanine.
r described is therefore an isolated, recombinant or purified CD9 peptide
according to the disclosure wherein at least one amino acid e selected from the
group consisting of K169, D171, V172, L173, T175 and F176 of the CD9 sequence as
depicted in Figure 2 is substituted by another amino acid residue, wherein said peptide
comprises an arginine at an amino acid position corresponding to K169 of the CD9
sequence depicted in Figure 2, and/or a glutamic acid at an amino acid on
corresponding to D171 of the CD9 sequence as depicted in Figure 2, and/or an amino acid
residue ed from the group consisting of isoleucine, leucine and methionine at an
amino acid position corresponding to V172 of the CD9 ce as depicted in Figure 2,
and/or an amino acid residue selected from the group consisting of isoleucine, valine and
methionine at an amino acid position corresponding to L173 of the CD9 sequence as
depicted in Figure 2, and/or a serine at an amino acid on corresponding to T175 of
the CD9 ce as depicted in Figure 2 , and/or a tyrosine at an amino acid position
corresponding to F176 of the CD9 sequence as depicted in Figure 2.
In other words, CD9 es according to the disclosure are described wherein
the lysine at position 169 has been replaced by an arginine, and/or n the aspartic
acid at position 171 has been replaced by a glutamic acid, and/or wherein the valine at
position 172 has been replaced by an isoleucine, leucine or methionine, and/or wherein
the leucine at position 173 has been replaced by isoleucine, valine or methionine, and/or
wherein the phenylalanine at position 176 has been replaced by a ne. These are
conservative amino acid substitutions, so that the resulting peptides will still be able to
bind antibody AT14-012. The resulting peptides will also be able to bind or generate
dies or functional parts or functional equivalents thereof that compete with
antibody AT14-012 for binding to CD9, preferably to the same epitope in CD9.
In some embodiments, the amino acid residues of a CD9 peptide according to the
disclosure are chosen from the 20 amino acid residues that naturally occur in
eukaryotes, which are also referred to as “standard” or “canonical” amino acids.
Alternatively, non-natural amino acid residues are included in a CD9 peptide according
to the disclosure, such as for instance D-amino acids (i.e. D-stereoisomers of amino acids)
or N-methyl amino acids.
Nucleic acid molecules, or functional equivalents thereof, encoding a CD9 peptide
according to the disclosure are also assed by the present disclosure. Further
described is therefore an isolated, synthetic or recombinant nucleic acid molecule, or a
functional equivalent thereof, encoding a CD9 peptide according to the disclosure. Said
nucleic acid molecule or functional equivalent ably comprises a chain of
nucleotides, more preferably DNA, cDNA or RNA. In other ments said nucleic
acid molecule or functional equivalent comprises other kinds of nucleic acid structures
such as for instance a DNA/RNA helix, e nucleic acid (PNA), locked nucleic acid
(LNA) and/or a ribozyme.
Said c acid les and functional equivalents are for instance useful for
the production of a CD9 peptide according to the present disclosure, using a nucleic acid
expression system such as for instance host cells like for instance E. coli, CHO, NSO or
293(T) cells. In some embodiments, said nucleic acid molecule or functional equivalent
according to the disclosure is present in a gene delivery vehicle, which facilitates
introduction of said nucleic acid molecule or functional equivalent into a cell of st.
r described is therefore a gene delivery vehicle, preferably a vector, comprising a
nucleic acid molecule or functional equivalent according to the disclosure. A host cell
comprising a nucleic acid le or functional lent according to the disclosure,
and/or a gene delivery vehicle according to the disclosure, is also described th.
As described above, a CD9 peptide according to the present disclosure, or a
nucleic acid molecule or functional equivalent encoding for a CD9 peptide according to
the sure is for instance useful for ing a ecific antibody according to
the disclosure, such as for instance an antibody that competes with antibody AT14-012
for binding to CD9. This is for instance done by immunizing a non-human animal with
said CD9 peptide or with a (vector comprising) a nucleic acid molecule or functional
equivalent encoding a CD9 e according to the disclosure. Alternatively, or
additionally, a phage display library is screened. Some embodiments therefore describe a
use of a CD9 peptide according to the disclosure, or a use of a nucleic acid molecule or
functional equivalent according to the disclosure, or a use of a vector according to the
disclosure, for producing, binding, detecting and/or obtaining an immune cell, such as for
instance a B cell, and/or an antibody or a functional part or functional equivalent
thereof, such as for instance a Fab nt, that is specific for CD9. Said immune cell
or antibody or functional part or functional equivalent thereof is preferably able to
specifically bind melanoma cells. A CD9 peptide according to the disclosure for use as an
immunogen is also herewith described, as well as a nucleic acid molecule or functional
equivalent encoding a CD9 peptide according to the disclosure for use as an immunogen.
Also bed is a method for producing a CD9-specific immune cell or a CD9-
specific antibody, the method comprising immunizing a non-human animal with a CD9
e ing to the disclosure or with a nucleic acid molecule or functional
equivalent or vector according to the sure. Said method preferably further
comprises harvesting an CD9-specific immune cell or antibody from said non-human
animal. As said , said immune cell or antibody or functional part or functional
equivalent thereof is preferably able to specifically bind melanoma cells.
A CD9-specific antibody or functional part or functional equivalent thereof
obtainable by a method according to the disclosure is also described herewith, as well as
an immune cell obtainable by a method according to the disclosure. Said CD9-specific
antibody, functional part, functional equivalent or immune cell preferably competes with
antibody AT14-012 for binding to CD9.
Said non-human animal ably comprises a mammal such as a rodent or
cattle. In some embodiments said non-human animal comprises a mouse, a rat, a ,
a llama, a camel, a pig, y, a cow, a goat, a horse, an ape, and/or a gorilla.
Some embodiments describe a composition, preferably an immunogenic
composition, comprising a CD9 e according to the present disclosure. In some
embodiments, said CD9 e is d to a pharmaceutically acceptable carrier or
scaffold. Some embodiments be a composition, preferably an immunogenic
composition, comprising a nucleic acid molecule or onal equivalent thereof
encoding a CD9 peptide according to the present disclosure. Some embodiments describe
a composition, preferably an immunogenic composition, sing a vector that
comprises said nucleic acid molecule or functional equivalent thereof. An immunogenic
composition according to the present disclosure preferably further comprises a
biocompatible additive, such as for instance a carrier, diluent, excipient or filler. Some
embodiments describe a vaccine comprising a CD9 peptide according to the disclosure, or
a vaccine comprising a compound that ses a CD9 peptide according to the
disclosure, or a e comprising a nucleic acid molecule or functional equivalent
thereof encoding a CD9 peptide ing to the disclosure. Some embodiments describe
a composition according to the disclosure, wherein said composition is a pharmaceutical
composition which further comprises a pharmaceutically acceptable carrier, t or
excipient.
CD9 peptides according to the present disclosure are also useful for testing for the
presence of CD9-specific binding compounds, such as for instance CD9-specific
antibodies or CD9-specific immune cells such as B cells or T cells, in a biological sample.
For instance, a sample from an individual, or a on of such sample that comprises
antibodies, B cells and/or T cells, is incubated with a CD9 peptide according to the
present disclosure, or with a compound that comprises a CD9 peptide according to the
disclosure, in order to screen for the presence of CD9-specific antibodies and/or CD9-
specific immune cells. If such antibodies or immune cells appear to be present in said
sample or in said sample fraction, and to bind said CD9 peptide according to the present
disclosure, said sample is typed as being positive for CD9-specific binding compounds
(i.e. antibodies and/or immune cells).
A CD9-specific antibody or CD9-specific immune cell is for instance detected
and/or fied using an immunoassay, such as for instance a n blot, a
(capture) ELISA or RIA. These assays are well known in the art. Labelled CD9 peptides
according to the disclosure (optionally in the context of an MHC x in order to
detect T cells) are for instance incubated with a blood sample or with a tissue sample
such as for instance a skin sample, or with a fraction of such sample that comprises
antibodies, B cells and/or T cells, where after unbound g compounds are washed
away. Subsequently, it is determined whether said labelled CD9 peptides according to
the disclosure are bound by ecific antibodies or immune cells. In some
embodiments, an led CD9 peptide ing to the disclosure, or an unlabeled
compound comprising a CD9 peptide ing to the disclosure (optionally in the
context of an MHC complex), is contacted with a sample that comprises dies
and/or immune cells, such as for instance a blood sample or tissue sample such as for
instance a skin sample, or with a fraction of such sample that comprises antibodies,
B cells and/or T cells. After incubation, one or more washing steps are preferably
performed in order to remove non-bound antibodies and unbound immune cells.
Subsequently, it is tested whether antibodies or immune cells have bound said CD9
e according to the disclosure, for instance using an antibody that is specifically
directed against human antibodies or human immune cells and that is coupled to a
marker, such as for instance a fluorescent compound or for instance horseradish
peroxidase or alkaline phosphatase. After a further washing step, it is preferably
ined whether the second antibody has bound, for instance by measuring light
emission or by adding a substrate of horseradish peroxidase or alkaline phosphatase.
These detection techniques are well known in the art.
In some embodiments, a CD9 peptide according to the disclosure, or a compound
or composition that comprises a CD9 peptide according to the disclosure (optionally in
the context of an MHC complex), is contacted with a fraction of a sample that has been
enriched for antibodies and/or immune cells. In some embodiments, said fraction is an in
vitro B cell culture or an in vitro T cell culture. In some embodiments, a CD9 peptide
according to the disclosure or a compound or composition that comprises a CD9 peptide
according to the disclosure is contacted with antibodies and/or immune cells that have
been essentially ed from a biological , such as for instance a purified B cell
fraction that has been obtained by selecting for CD19 ve cells and/or an
antibody/B cell fraction that has been purified using an anti Ig antibody or a protein A or
G purification method. Protein A or G purification s are well known in the art
and protocols and reagents are commercially available. As used herein, the term
“immune cells that have been essentially purified from a sample” means that at least
80%, preferably at least 85%, more preferably at least 90% or at least 95%, of the cells of
a resulting fraction consists of immune cells. The term “antibodies that have been
essentially purified from a sample” means that at least 80%, more preferably at least
85%, more preferably at least 90% or at least 95%, of the mass of a resulting fraction
consists of antibodies.
Further described is therefore a use of a CD9 e according to the disclosure,
or a use of a compound or ition that comprises a CD9 e according to the
disclosure, for binding and/or detecting a CD9-specific immune cell and/or a CD9-specific
antibody, or a functional part or functional equivalent thereof. Said immune cell and/or
antibody or functional part or functional equivalent thereof is ably able to
specifically bind CD9-positive tumor cells, such as for instance melanoma cells. A CD9
e according to the disclosure, or a nd that comprises a CD9 peptide
according to the disclosure, for use as a detection moiety for CD9-specific binding
nds such as dies and/or immune cells is also herewith described, as well as
a method for determining whether a sample comprises ecific antibodies and/or
CD9-specific immune cells, the method comprising incubating a CD9 peptide according
to the disclosure, or a compound or composition that comprises a CD9 peptide according
to the disclosure, with said sample, or with a fraction of said sample that comprises
antibodies and/or immune cells, and subsequently ining whether said CD9
peptide ing to the disclosure is bound by CD9-specific antibodies and/or by
CD9-specific immune cells, or whether said compound that comprises said CD9 peptide
according to the sure is bound by ecific antibodies and/or CD9-specific
immune cells. If such binding is detected, it is concluded that said sample ses
CD9-specific antibodies and/or CD9-specific immune cells, for instance antibodies and/or
immune cells that are able to specifically bind CD9-positive tumor cells like melanoma.
Also described is a method for determining whether a sample comprises
CD9-specific antibodies and/or CD9-specific immune cells, the method comprising
incubating a CD9 peptide ing to the disclosure, or a nd that comprises a
CD9 peptide according to the disclosure (optionally in the context of an MHC x),
with antibodies and/or immune cells that have been essentially purified from said
sample, and uently determining r said CD9 peptide according to the
disclosure is bound by CD9-specific antibodies and/or CD9-specific immune cells, or
whether said compound that comprises said CD9 peptide according to the disclosure is
bound by CD9-specific antibodies and/or ecific immune cells.
In some embodiments, the results of detection tests as described above are used
for determining whether an individual has a disorder associated with CD9-expressing
cells. For instance, if a sample from an individual that is tested for the presence of a
CD9-positive tumor appears to contain CD9-specific immune cells and/or CD9-specific
antibodies, it can be concluded that said individual is suffering from a CD9-positive
tumor, like for instance melanoma. Said sample preferably comprises tumor cells. For
instance, in order to test for the presence of melanoma cells, a biopsy of the skin area
with the suspected melanoma is preferably used. Alternatively, or additionally, a blood
sample or a lymph node sample is also useful for g for sitive tumor cells,
because metastases often circulate in the blood and lymphatic system.
A CD9 peptide according to the sure for use as a diagnostic agent is
therefore also described herewith, as well as a compound or composition that comprises a
CD9 peptide according to the disclosure for use as a diagnostic agent. Further described
is a use of a CD9 peptide according to the disclosure for diagnosing a disorder associated
with CD9-expressing cells, such as for instance a sitive tumor, or orosis, or
arthritis, or lung inflammation, or COPD, or colitis, or a disorder associated with innate
lymphoid cells, as well as a use of a compound or composition that comprises a CD9
peptide according to the disclosure for diagnosing a disorder associated with CD9-
expressing cells, such as for instance a CD9-positive tumor, or osteoporosis, or arthritis,
or lung inflammation, or COPD, or colitis, or a disorder associated with innate lymphoid
cells. In some embodiments, said CD9-positive cancer is melanoma. Some embodiments
therefore describe a CD9 peptide according to the disclosure for use in diagnosing
ma, as well as a use of a CD9 peptide according to the disclosure for the
preparation of a diagnostic kit for diagnosing melanoma.
Further described is a stic kit comprising:
- a CD9 peptide according to the sure, or a compound or composition that comprises
a CD9 peptide according to the disclosure, and
- means for detecting an antibody-bound CD9 peptide or an immune cell-bound CD9
peptide.
Such means for instance encompass labelled dies that are specifically directed
against human dies or human immune cells. In some embodiments, said labelled
antibodies are conjugated with horseradish peroxidase or alkaline phosphatase.
Some embodiments describe a method for determining whether an dual has
a CD9-positive tumor, the method comprising ting a CD9 peptide according to the
disclosure, or a compound or composition that comprises a CD9 peptide according to the
disclosure (optionally in the context of an MHC complex), with antibodies and/or immune
cells of said individual and determining whether said CD9 peptide according to the
disclosure, or said compound or composition comprising a CD9 peptide according to the
disclosure, is bound by at least one of said antibodies and/or immune cells of said
individual. If said CD9 peptide or said compound according to the disclosure is bound by
antibodies and/or immune cells of said individual, it is concluded that said individual has
a CD9-positive tumor. In some embodiments, said CD9-positve tumor is ma. In
some embodiments, a CD9 peptide according to the sure, or a compound that
comprises a CD9 e ing to the disclosure, is contacted with a sample that
comprises antibodies and/or immune cells of said individual, such as for ce a blood
sample or a bone marrow sample or a biopsy such as for ce a skin tissue. In other
embodiments, a CD9 peptide or compound according to the disclosure is contacted with a
fraction of a sample from said individual, wherein said fraction comprises immune cells
and/or antibodies. In some embodiments, a CD9 e or compound according to the
disclosure is contacted with antibodies and/or immune cells that have been essentially
ed from said sample, such as for instance a purified B cell fraction that has been
obtained by selecting for CD19 positive cells and/or an antibody/B cell fraction that has
been purified using an anti Ig antibody or a protein A or G purification method.
Another interesting application of the novel CD9 peptides according to the
present disclosure and nucleic acid molecules and functional equivalents encoding
therefore is therapy. For instance, a CD9 peptide according to the present
sure, or a nucleic acid molecule or functional equivalent encoding therefore, is used
for treatment of a CD9-positive tumor. As used herein, “treatment” encompasses
alleviation of at least one symptom, and/or delaying or even halting the progression of
disease, at least temporarily. In one red embodiment, a CD9 peptide according to
the disclosure, or a nucleic acid molecule or a functional equivalent encoding therefore,
or a compound or composition that comprises a CD9 peptide according to the disclosure,
is administered to a sitive cancer patient in order to boost his/her immune
system, resulting in an enhanced immune response. In some embodiments, naïve T cells
or B cells from a CD9-positive cancer t are cultured ex vivo and incubated with a
CD9 e or compound according to the disclosure, optionally in the context of an
MHC complex in case of a T cell culture, in order to obtain CD9-specific T cells or B cells
that are subsequently stered to the patient, optionally after ex vivo expansion. In
some embodiments, said CD9-positive cancer is melanoma.
In some embodiments, adoptive cell therapy is used. T cells from a CD9-positive
cancer t are preferably tested for binding or activation, using a CD9 peptide
according to the disclosure in the context of an MHC complex or using a compound or
composition that comprises a CD9 peptide according to the disclosure in the context of an
MHC complex. T cells recognizing said CD9 peptide are expanded ex vivo and
subsequently stered to the patient, which will result in an anti-CD9 T cell
se.
In some embodiments, adoptive cell therapy of donor lymphocytes is used. Donor
T cells ed from a CD9-positive cancer patient who received allogeneic HSCT or
isolated from the HSCT donor are preferably tested for CD9 binding or activation, using
a CD9 peptide in the context of an MHC complex, or a compound that comprises a CD9
e according to the disclosure in the context of an MHC complex, and donor T cells
recognizing said CD9 peptide are expanded ex vivo and subsequently administered to
the t, which will result in an anti-CD9 allogeneic T cell response.
In some embodiments, T cells are ed in order to provide them with a
CD9-specific binding moiety. Said T cells are preferably derived from a CD9-positive
cancer patient. In some embodiments, ic antigen receptor (CAR) T cells are
produced. These are T cells with modified T cell receptors, which have been provided
with a binding icity of interest, preferably d from an antibody. Typically,
CAR T cells are produced by fusing a single-chain variable domains (scFv) derived from
a monoclonal antibody to the ta embrane domain, so that a zeta signal will
be elicited upon target recognition by the scFv.
According to some embodiments, a CD9 peptide ing to the disclosure, or a
nucleic acid molecule or a functional equivalent encoding therefore, or a compound or
composition that comprises a CD9 peptide according to the disclosure, is used in order to
produce and/or isolate a CD9-specific antibody and/or B cell, which in turn is used for the
tion of a modified T cell. For instance, said CD9 peptide or compound or nucleic
acid molecule or functional equivalent is used in order to elicit, detect and/or isolate a
CD9-specific antibody or B cell. Subsequently, in some embodiments the heavy chain
and/or light chain variable domains of said CD9-specific antibody are provided to T cells,
thereby producing ed T cells with CD9 specificity. In some embodiments, these
modified T cells are subsequently stered to a CD9-positive cancer patient, which
will result in a tumor-specific T cell response. In some embodiments, said modified T
cells are CAR T cells. In some embodiments said CD9-specific antibodies or B cells are
tested for competition with antibody AT14-012 for binding to CD9 before the heavy chain
and/or light chain variable domains of said antibodies are provided to T cells. Such
competing dies are preferably selected for producing modified T cells with a CD9
specificity.
Further described is therefore a CD9 peptide according to the disclosure, or a
compound or composition that comprises a CD9 peptide according to the disclosure, or a
nucleic acid molecule or functional equivalent thereof ng a CD9 peptide according
to the disclosure, for use as a medicament. Also described is a use of a CD9 peptide
according to the disclosure nally in the context of an MHC complex), or use of a
compound or composition that comprises a CD9 peptide according to the disclosure
(optionally in the context of an MHC x), or use of a nucleic acid molecule or
functional lent thereof encoding said CD9 peptide according to the disclosure, for
the production of CD9-specific T cells. Some embodiments describe a method for
producing a modified T cell, the method comprising contacting an antibody-containing
sample or a B cell-containing sample from a CD9-positive cancer patient with a CD9
peptide or compound according to the disclosure, resulting in bound antibodies or B cells
against CD9, and subsequently obtaining one or more CD9-specific s from said
CD9-specific antibodies or B cells and providing said one or more domains to a T cell.
Some embodiments describe a method for producing a modified T cell, the method
comprising immunizing a non-human animal with a CD9 peptide or compound or nucleic
acid molecule or functional equivalent according to the disclosure, thereby eliciting an
immune response against CD9, and subsequently obtaining one or more ecific
domains from a CD9-specific antibody or CD9-specific B cell from said man
animal, or obtaining one or more c acid sequences encoding for said one or more
CD9-specific domains, and providing said one or more s, or said one or more
nucleic acid sequences, to a T cell.
A CD9 e according to the sure for use in immunotherapy is also
described herewith, as well as a nucleic acid molecule or functional equivalent thereof
encoding a CD9 peptide according to the disclosure for use in immunotherapy. A
compound or composition comprising a CD9 peptide ing to the disclosure for use in
immunotherapy is also described herewith. Some ments describe a use of a CD9
peptide according to the disclosure, or a use of a nd or composition that comprises
a CD9 e ing to the disclosure, or a use of a nucleic acid molecule or
functional equivalent thereof encoding a CD9 peptide according to the disclosure, for the
preparation of a medicament against a disorder associated with pressing cells,
such as for instance a CD9-positive tumor, or osteoporosis, or arthritis, or lung
inflammation, or COPD, or colitis, or a disorder associated with innate id cells. In
some embodiments, said CD9-positive tumor is selected from the group consisting of
melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, lung cancer, breast
cancer, n cancer, stomach cancer, squamous cell carcinoma, AML, multiple
myeloma, gastric cancer, liver cancer, brain cancer, Kaposi sarcoma, carcinoma
mucoepidermoid, choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma,
adenocarcinoma, lung adenocarcinoma, non-small-cell lung oma, bladder cancer
and small cell lung cancer.
In some embodiments, the results of detection tests according to the disclosure as
described hereinbefore are used for determining r an individual exhibits a
detectable immune response against a CD9-positive tumor like for instance melanoma.
This is for instance preferred for determining whether a t suffering from such
tumor who has received immunotherapy, has elicited an anti-tumor immune response.
Some embodiments therefore describe a method for determining r an individual
exhibits an immune response t a CD9-positve tumor, the method sing
contacting a CD9 peptide according to the disclosure (optionally in the context of an
MHC complex), or a compound or composition that comprises said CD9 peptide according
to the disclosure, with dies and/or immune cells of said individual and ining
whether said CD9 peptide according to the disclosure, or said nd or composition
that comprises said CD9 peptide according to the disclosure, is bound by at least one of
said antibodies and/or immune cells of said individual. If said CD9 peptide or said
compound appears to be bound, it indicates that said individual exhibits an immune
response against a CD9-positive tumor.
In some embodiments, an ed, recombinant or purified antibody, or a
onal part or a functional equivalent thereof, that competes with dy AT14-012
for binding to CD9 is used for treatment of melanoma. As described in the Examples,
antibody AT14-012 was obtained from a melanoma patient in complete remission,
demonstrating that AT14-012 is effective against ma. Antibodies that compete
with AT14-012 for CD9 will therefore also be effective. Hence, administration of such
antibodies to a melanoma patient will effectively counteract, and/or kill, melanoma cells.
Some embodiments therefore describe an ed, recombinant or purified antibody, or a
functional part or a functional equivalent thereof, that competes with antibody AT14-012
for binding to CD9, for use as a medicament. Some embodiments describe a use of an
isolated, recombinant or purified antibody, or a functional part or a functional equivalent
thereof, that competes with antibody AT14-012 for binding to Cd9, for the preparation of
a medicament.
Also described is an isolated, recombinant or purified antibody, or a functional
part or a functional lent thereof, that es with antibody AT14-012 for
binding to CD9, for use in a method for at least in part treating or preventing melanoma,
as well as a use of an isolated, recombinant or purified antibody, or a functional part or a
functional equivalent thereof, that competes with antibody AT14-012 for binding to CD9,
for the preparation of a ment against melanoma.
While the current application may describe features as part of the same
embodiment or as parts of separate embodiments, the scope of the present invention also
includes embodiments comprising any combination of all or some of the features
bed herein.
The invention is r explained in the following examples. These examples do
not limit the scope of the invention, but merely serve to clarify the invention.
Brief description of the drawings
Figure 1. Schematic structure of human CD9.
Figure (1a). Schematic representation of human tetraspanin CD9
Tetraspanins are terized by four spanning transmembrane regions and two
extracellular loops: small extracellular loop 1 (EC1) and large extracellular loop 2 (EC2).
The EC2 of CD9 is structurally and conformationally defined by two cysteine bonds (red)
(C152-C181 and C153-C167) and conserved adjacent residues (G154 and P168). Two
highly variable s among panin family members are located between amino
acid positions 154-167 and 168-181 (depicted in purple).
Figure (1b). Cartoon representation of the second extracellular loop.
A cartoon representation of the second extracellular loop divided in 5 different regions
and shown with corresponding amino acid numbers. These regions are chosen based on
overlap with other tetraspanin family members such as CD81. These regions are further
described in the epitope mapping studies (Figure 5).
Figure 2. Amino acid sequence of human CD9 (UniProt No. P21926)
Figure 3. Amino acid and nucleotide sequences of antibody AT14-012. The CDR
numbering is according to Kabat et al .
Figure 4. Target ID
Figure (4a). AT14-012 reacts with an antigen of ~25 kDa size.
Western blots were probed for binding of 12 towards lysates (50ug) of Caco2,
MelBLM and control HL-60 cell lines under ducing and reducing conditions (see
Materials&Methods of Example 2). AT14-012 shows reactivity of towards a ~25kDa size
antigen and is lost when the s are reduced, implying 12 reacts towards a
conformational epitope.
Figure (4b). Mass spectrometric is of immunoprecipitation with 12 on
cancer cell lines indicates CD9 as the target.
Sortase biotin labeled AT14-012 or control AT10-002 were incubated with lysates of
Caco2, MelBLM or control HL-60. Immunoprecipitated eluates were run on gel and
stained with coommassie blue to reveal a visible band of ~25 kDa in size overlapping
with western blot results. Mass ometric is revealed CD9 as the AT14-012
antigen. MS analysis identified precipitated CD9 for the MelBLM cell line as well
whereas no CD9 was found for HL-60 eluates.
Figure (4c). Confirmation of CD9 by commercial antibody ALB6.
IP eluates showed reactivity towards AT14-012 and anti-CD9 dy ALB6 confirming
CD9 to be the target antigen of AT14-012 whereas no reactivity was found for IP eluates
of HL-60 lysates.
Figure 5. Epitope mapping
Figure (5a). Epitope mapping by hybrid or d mutants reveals extracellular loop 2
(EC2) and more specific region 154 – 180 as the main epitope for 12.
Epitope mapping revealed the extracellular loop 2 to have epitopes of all anti-CD9
antibodies. AT14-012 showed loss of binding towards the variable CD9 loops m3 and m4.
Figure (5b). Epitope mapping by e scanning of region m4 of the EC2.
Reactivity of HI9a, ALB6 and AT14-012 (-Alexa647 labeled) towards alanine mutants of
region m4. HI9a was taken along as positive control. F176 was the only residue that
showed loss of binding towards both antibodies. The additional 5 residues showed loss of
binding towards AT14-012 (K169, D171, V172, L173 and T175).
Figure 6. Tumor binding.
Figure (6a). AT14-012 has broad binding reactivity against solid tumors. Flow cytometry
analysis of AT14-012 versus AT10-002 antibody g to a panel of solid tumor cell
lines.
Figure (6b). AT14-012 binds a selected number of Acute Myeloid Leukemia cell lines.
Flow cytometry analysis of AT14-012 versus CD30 dy binding to a panel of AML
cell lines.
Figure (6c). AT14-012 binds a selected number of Multiple Myeloma cell lines. Flow
try is of CD9 versus unstained and AT14-012 versus AT10-002 dy
binding to a panel of MM cell lines.
Figure 7. Binding to y cells
Figure 7a. AT14-012 binds stronger to melanoma as compared to primary melanocytes.
Analysis for 12 binding to melanoma cell lines and primary melanocytes. AT10-
002 (anti Influenza) was included as a negative control, Panitumumab (anti EGFR1) as a
positive control for binding to healthy cells. After staining with the primary antibody,
the cells were labelled with anti IgG-PE for ization by flow cytometry.
Figure 7b. AT14-012 binds stronger to colon carcinoma as compared to y colon
epithelial cells. Flow cytometry analysis for CD9 PE and AT14-012 A647 binding to colon
carcinoma cell lines and primary colon epithelial cells. AT10-002 A647 (anti Influenza)
was included as a negative control, anti CD81 PE was included to confirm absence of
CD9 expression on the Colo-320 cells.
Figure 7c. AT14-012 binds stronger to melanoma compared to primary tonsil
lymphocytes. Tonsillar lymphocytes were stained with antibodies t CD4, CD8 and,
CD9 to discriminate CD4 T, CD8 T and CD19 B cells respectively.
Figure 8. Platelets
Figure 8a AT14-012 binds human platelets. Fixed or xed y human platelets
were stained with CD41, CD9 or AT14-012 biotin/SA-PeCy7. Histograms are gated CD41
positive.
Figure 8b AT14-012 activates healthy platelets. Surface CD62P expression was
determined by flow cytometry on PRP incubated with TRAP ive control peptide),
ALB6 (positive control mouse IgG1 antibody), FLAG (negative control mouse IgG1
dy), AT10-002 or AT14-012 antibodies.
Figure 8c. AT14-012 does not induce platelet aggregation. Whole blood was incubated
with various stimuli and tested for the ion of platelet aggregation as measured
using a Multiplate reader.
Figure 9. In vivo experiments
Figure 9a AT14-012 inhibits lymph node metastasis in vivo. NSG mice subcutaneously
grafted with 500.000 MelBLM GFP/luciferase melanoma cells on both flanks are treated
with AT14-012 or AT10-002 control dy. Lymph nodes metastasis indicated by the
arrows were visualized using a Bioluminescence imager after luciferin injection and
subsequent exposure of internal organs.
Figure 9b AT14-012 impairs primary melanoma tumor growth in vivo. NSG mice
subcutaneously grafted with 200.000 MelBLM GFP/luciferase melanoma cells on both
flanks are treated with AT14-012 or AT10-002 control antibody from the start of the
experiment. Tumor growth is determined by caliper measurement.
Figure 9C AT14-012 recognizes melanoma tumors in vivo. MelBLM subcutaneous
tumors harvested from NSG mice treated with either 02 (anti nza) or AT14-
012 or we left untreated were embedded in paraffin. For immunohistochemistry sections
were stained with HRP labelled anti-lambda or anti-kappa antibodies.
Figure 9D Reduced surface CD9 levels after AT14-012 treatment. Subcutaneous
MelBLM tumors ted from NSG mice treated with 12 or AT10-002 control
antibody were digested and stained for flowcytometry with AT10biotin, AT14
biotin followed by fluorescent ed conjugated avidin, or direct labelled HI9a
CD9 or anti IgG antibodies. Samples were measured on a Fortessa X20 (Becton
Dickinson).
Figure 9E-G AT14-012 impairs growth of SK-MEL-5 melanoma. NSG mice carrying
subcutaneous SK-MEL-5 melanoma tumor are d with AT14-012 or the AT10-002
control dy. E. Tumor growth in time is determined by caliper measurement. Grey
area indicates period of antibody treatment. F. Ex vivo isolated tumors are weighed.
Weights are averaged per mouse. G. Tumors are digested by se ent and
stained for flowcytometry with CD9 HI9a-PE.
Figure 10. AT14-012 binding correlates with CD9 expression on recently established
tumor cell lines. Flowcytometry analysis for AT14-012 and CD9 HI9a binding to short
term cultured melanoma cells. A. Histograms for the CD9 HI9a, AT14-012 and AT10-002
anti-Influenza (control) antibody staining of melanoma cells. B. Mean scence
ities of the AT14-012 versus CD9 HI9a signals corrected for ound staining.
Arrows indicate melanoma samples Mel06.07 and Mel05.18 derived from the original
AT14-012 patient. C. Flowcytometry analysis for AT14-012 and CD9 HI9a binding to
ished cell lines 1 and CAPAN2) and recent patient derived pancreas
carcinoma tumor cells (53M and 193).
Figure 11. AT14-012 binding is restricted to (non)-human primates. Platelet rich plasma
isolated from A. NOD SCID γc-/- (NSG) mice; B. New Zealand White rabbits; C. crabeating
macaque (cynomolgus monkeys) and a human was electronically gated for CD41
expressing platelets. Binding of AT10-002, AT14-012 and anti CD9 to platelets of the
respective species was determined by flowcytometry.
Figure 12. Antibody dependent cytotoxicity (ADCC). um labeled A. MelBLM or
Human Artery Endothelial Cells or B. primary short term cultured melanoma cells
incubated with indicated antibodies were titrated against total human PBMCs. Cell
death was determined by the release of Chromium in the supernatant.
Figure 13. AT14-012 triggers ment mediated tumor cell death. A, B. ma
cell lines are incubated with ted antibodies and human serum and subsequently
tested for C1q deposition by flow cytometry using an anti C1q antibody. ment
mediated cytotoxicity was determined by incubating C. suspension or D. overnight
adhered ma cells with indicated antibodies and rabbit complement. E. Incubation
of CD55 positive (MelBLM) and CD55 negative 205) with indicated antibodies and
human serum. Percentage cell death of suspension of adhered cells is determined by
DAPI by flow cytometry or TO-PRO3 by microscopy respectively.
Figure 14. AT14-012 favors binding to homoclustered CD9. Two melanoma and one colon
carcinoma cell line were incubated with the inhibitor of palmitoylation 2-BP or DMSO
only for 36 hours. A. Cells were detached and stained for flow cytometry with the AT10-
002 influenza control antibody or different CD9 antibodies, AT14-012 or the
commercially available HI9a and ALB6 clones. B. Mean fluorescence intensity of the
histograms of the AT10-002 signals are deducted from the m.f.i. of the CD9 signals.
Ratio is the ∆m.f.i. values for the DMSO conditions divided over the corresponding
∆m.f.i. 2-BP values and plotted against the different cell lines. A ratio of 1 tes that
antibody binding is not affected by de-palmitoylation.
Figure 15. Alanine scanning of region m3 expressed on HL60 cells did not show any loss
of AT14-012 binding (n=2). Surprisingly, we determined that exchange of single amino
acids in the m3 region did not disrupt AT14-012 binding. As a control, the CD9-WT, the
CD9/CD81 EC2 hybrid mutant, the EC2 m3 81 hybrid mutant was included and
the binding data shows overlap with previous ed data (Figure 5).
Figure 16. AT14-012 has lower affinity for CD9 as compared to commercial mouse anti-
CD9 antibodies. A. CD9 binding of AT14-012 (+ control AT10-002) and commercial abs
HI9a and ALB6 (+ controls anti-FLAG for detection of the presence of CD9 protein on
the plate and OKT3) were compared. B. SPR curves showing binding of anti-CD9
antibodies AT14-012, ALB6 and HI9a for s injections of antibody for one single
spotted CD9-3xFLAG-rabbitFc-Sortase-biotin n. C. Affinity ement of the
different antibodies for human CD9 as determined by SPR. ka in 104 sec-1*M-1, kd in 10-5
sec-1, KD in pM. Shown are results on one CD9-spot coated with 7324 RU recombinant
CD9-3xFLAG-rabbitFc-Sortase-biotin /ml). To calculate binding kinetics, data
from three duplicate injections were fitted to a 1:1 binding model. Shown are averages
and standard deviations from measurements on three spots, coated with 0.5 or 1.0μg/ml
CD9-3xFLAG-rabbitFc-Sortase-biotin. *: When the apparent antibody dissociation rate
(kd) was too low for accurate fitting, a value of 0.1*10-5 is used. Shown are averages of
two ions on two spots, both coated with 0.5 μg/ml CD9-3xFLAG-rabbitFc-Sortasebiotin.
To ate the binding kinetics, data from three duplicate injections were used.
Figure 17. K169, D171, V172, L173 and F176 are part of the AT14-012 epitope. A.
ELISA data of AT14-012 (triangles, bottom line in all graphs), ALB6 les) and HI9a
(squares) binding on recombinantly expressed m4 e mutants. B. AT14-012 epitope
displayed onto a CD9 homology model (EC is extracellular and IC is ellular
orientation). Homology model was constructed using the I-Tasser server (Yang et al.,
2015) using the CD81 homology model (2AVZ; Seigneuret et al., 2006). As of November
2016, a crystal structure was published (Zimmerman et al., 2016) and a newly
constructed CD9 gy model based on this crystal ure did not show any
chances with respect to the orientation of the es related to the AT14-012 epitope.
C. AT14-012 FACS binding data to cells from different species (see Figure11) and
alignment of CD9 sequence (residues 23 – 228) from multiple species (here) strengthens
the AT14-012 epitope. Sequences were retrieved from the ensembl.org e around
June 2016. The first 22 residues were not well resolved for all found species and are
therefore omitted here. AT14-012 is known to react with cynomolgus cells, whereas
g is lost when assayed for binding to rabbit or mouse cells. The alignment of
region m4, flanked by C167 and C182 (green), is ghted (light grey) and more
specifically the 5 residues (dark grey) involved in AT14-012 binding. Apparently, the
F176L mutation does not induce a loss of binding of AT14-012 (see AT14-012 binding to
cynomolgus cells in Figure 11C).
Figure 18. Affinity ing AT14-012 using single cell sorted 2H15 B cells and SPR
(IBIS). A. SPR curves of 8 sequential injections of recombinant AT14-012 at increasing
concentrations (0 – 1.33 – 4.0 – 13.30 – 40.0 – 133.0 – and 0 nM of protein). The green
line is (no) g reactivity s CD81, orange binding to the anti-human IgG-Fc,
grey the binding to anti-CH1 nanobody and blue g to CD9. The ratio of the anti-
IgG-Fc and aCH1 can be used to observe the tration and integrity of the IgG in
the B cell sup (see B). B. After screening of 800 clones in SPR as described in the
materials and methods, we assayed newly grown B cell sups again for binding. Eight
clones were sed (1D5, 1F5, 2D12, 4H10, 6E10, 9E5, 10B9 and 10D1) whereas 4
doubtful clones (1C9, 2H10, 9A9, 9D12) were eventually not. At the beginning and end
there are injections of recombinant AT14-012 for reference. C. Sequence analysis of the
found clones including affinities shows overlap in mutations related to their
association/dissociation profiles. Group1 shows faster ation and slower
dissociation, group2 faster association and dissociation and group3 no clear difference
related to WT sequences (1G2, 1G3, 1G4, 1G5).
Figure 19. Design, expression and analysis of AT14-012 higher ty mutants binding
on cells and SPR. A. As only the major mutations were located in the heavy chain, we
only show the alignments of the heavy chain. Also, the combinations of group1 (red;
H40Y and Y112F) and group2 (blue-cyan; D116H and T29N) and both groups er
(H40Y, Y112F, D116H and T29N) were made. As a control, one mutation resulting in a
lack of CD9 binding was added to verify the results (dark blue; G110D). The original
AT14-012/2H15 hypermutations are highlighted in yellow. CDR numbering according to
the IMGT numbering system nc 1997, Lefranc 1999 and Lefranc et al. 2003). B-C.
MelWBO or short term cultured healthy melanocytes from two different donors’ cells
were incubated with CHO production supernatant of the different AT14-012 variants.
Binding was detected by flow cytometry using a goat-anti-human IgG-PE ary
dy. D-E. CHO production supernatant of the different AT14-012 variants was
assayed on CD9 coated chip by SPR (shown in duplicates). # = CHO production
supernatant. Affinities are in ka in 104 sec-1*M-1, kd in 10-5 sec-1, KD in pM. Shown are
averages of two titrations on two spots, both coated with 0.5 μg/ml CD9-3xFLAG-
rabbitFc-Sortase-biotin. Mutant samples were non-purified production sup, 1x diluted in
PBST. -: no g detected *: binding detected, but no good fit possible.
Figure 20. Platelet aggregation assay using the 12 high affinity s. As
controls, TRAP, ALB6 and previously examined recombinant purified full length AT14-
012 IgG1 and IgG3 including control AT10-002 IgG1 antibody were taken along.
Figure 21. B cell produced AT14-012/2H15 antibody is of allotype IGHG3*16. mRNA was
isolated from AT14-012/2H15 B cells. Figure is adapted from Vidarsson et al., 2014.
Figure 22. AT14-012 combined with anti PD1 inhibits tumor growth in vivo. NSG mice
carrying a human immune system are subcutaneous lanted with SK-MEL-5
luciferase expressing melanoma tumor cells. Mice are treated twice per week with
indicated dies. A. Tumor growth is determined by rase imaging. The grey
area indicates the period of antibody treatment. B. The percentage tumor growth
inhibition (TGI) is calculated based on the size of the tumors at the end of the
ment and the tumor size at the time of the first antibody injection.
The term “comprising” as used in this specification and claims means sting at least
in part of”. When interpreting statements in this specification, and claims which include
the term “comprising”, it is to be understood that other features that are additional to
the features prefaced by this term in each statement or claim may also be
present. Related terms such as “comprise” and “comprised” are to be interpreted in
similar manner.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically
stated otherwise, reference to such external nts is not to be construed as an
admission that such documents, or such sources of information, in any jurisdiction, are
prior art, or form part of the common general knowledge in the art.
In the description in this specification reference may be made to t matter that is
not within the scope of the claims of the t application. That subject matter should
be readily identifiable by a person skilled in the art and may assist in putting into
practice the ion as defined in the claims of this ation.
Examples
Example 1 – Isolation of AT14-012 B cell clone
ALS & METHODS
Melanoma cell cultures. ma cell lines MelBLM, Mel136.2 and MelWBO were
obtained via Rosalie Luiten (Academic Medical Centre, Dept. of Dermatology) and
maintained in IMDM (Life Technologies), 8% fetal calf serum using standard tissue
culture techniques. For minimal disruption of cell surface proteins tumor cells were
detached using Accutase (Life Technologies) or EDTA.
Melanoma donor PBMCs. Study protocols were approved by the Medical Ethical
Committee of the Leiden University Medical Center. A stage IV melanoma patient was
treated by adoptive transfer of autologous blood-derived tumor specific T cells in
ation with IFNα (Verdegaal Cancer Immunol Immunother 2011). After treatment
tumors regressed and the patient is the only long term survivor from its cohort.
Blood was collected five years after treatment, PBMCs were isolated from a ficoll
gradient and frozen to liquid nitrogen until B cell isolation.
B cell immortalization. Total IgG B cells were sorted from the thawed patient's PBMCs
using a FACSAria (Becton Dickinson) and immortalized as described in nbos
Nat Med 2010. Briefly, total IgG B cells were cultured and activated during 36 hours on
CD40L expressing L cells in the presence of recombinant mouse IL-21. By retroviral
transduction our proprietary construct expressing Bcl6 and Bcl-xL and the marker gene
GFP was introduced in the B cells rendering the B cell alized.
B cell culture. Immortalized B cells were maintained in IMDM (Gibco) supplemented
with 8% FCS (HyClone), llin/streptomycin (Roche) and recombinant mouse IL-21
(50 ng/ml, in house produced). Gamma-irradiated (50Gy) CD40L expressing mouse
fibroblasts were included as feeder cells. The es were routinely tested to be
negative for the presence of mycoplasma.
ion of melanoma binding B cell clones. Immortalized IgG B cells were seeded at 25
cells per well of a 384 well plate and expanded using L-cells and mIL21. After
approximately 2 weeks of culture antibody ning B cell supernatants were tested
for binding to a mixture of melanoma cell lines. Positive binding was visualized by flow
cytometry (FACS Canto and LSR Fortessa X20, Becton Dickinson) using an anti-human
IgG-PE antibody ern Biotech). The positive minicultures were expanded and the
procedure was repeated on single cell sorted B cells to retrieve the melanoma reactive B
cell clone from the 25 cell miniculture. Panitumumab (anti EGFR1) was included as a
positive control antibody.
Recombinant antibody production. To produce recombinant antibodies total RNA was
isolated with the ® mini kit (Qiagen), generated cDNA, performed PCR and
cloned the heavy and light chain variable regions into the pCR2.1 TA cloning vector
(Invitrogen). To rule out reverse transcriptase or DNA rase induced mutation
multiple clones were sequenced. The heavy and light variable regions of AT14-012 were
cloned in frame with human IgG1 or IgG3 and Kappa constant regions into a pcDNA3.1
(Invitrogen) based vector. The resulting vector was ently transfected 293T cells
and recombinant antibody was ed from the culture supernatant using an AKTA
purification system (General Electric Lifesciences). For control purposes an irrelevant
control antibody (AT10-002) recognizing the HA antigen on influenza virus was ed
in the experiments.
Identification and isolation of melanoma binding B cell clone. A patient with ous
melanoma and progressive metastatic disease stage IV was treated by adoptive transfer
of autologous tumor-reactive T cells (Verdegaal Cancer Immunol Immunotherapy 2011).
To this tumor tissue was obtained by surgery and used to establish an autologous
melanoma cell line. Peripheral blood mononuclear cells were isolated from blood and put
into ture with ly irradiated autologous melanoma cells in T-cell medium.
After 4 weeks of culture the tumor reactivity of the cultured T cells was confirmed in
onal assays (Verdegaal Cancer Immunol.Immunotherapy 2011). The patient
ed two rounds of expanded gous T cells and displayed a complete response
and is still tumor free over 9 years after therapy.
From PBMCs isolated five years after the adoptive T cell therapy the total IgG B cell
pool was retrovirally transduced with our proprietary Bcl6/Bcl-xL construct.
Immortalized GFP positive cells were tested for the presence of ma binding
antibodies by flow cytometry. An IgG3 B cell clone named AT14-012 displayed strong
reactivity against both melanoma lines initially tested (MelBLM and ). Variable
heavy and light chain sequences were determined (see Figure 3) and DNA cloned in both
an IgG1 and IgG3 backbone for recombinant dy production in 293 or CHO cells.
Example 2 – AT14-012 target antigen is CD9
MATERIALS & S
AT14-012 target identification and validation
Cells of the colon cancer cell line Caco2 (ATCC HTB-37), cells of the melanoma cell line
MelBLM and cells of the human promyelocytic leukemia cell line HL-60 (negative
control) were lysed (0,5% Triton X114 (Sigma), 150mM NaCl, 10mM CL pH7.4,
1,5mM MgCl2 supplemented with protease and phosphatase inhibitors (Roche)) and
precleared with an irrelevant antibody (in-house generated RSV antibody D25), Protein-
G and Streptavidin beads (Pierce) to remove non-specific binding proteins. For direct
n blotting with AT14-012, we incubated purified recombinant AT14-012 for at
least 1 hour at room temperature in TBS + 5% BSA (Thermo Fisher) and 0.1% Tween20
(Sigma) on SDS-Page and d lysates. Blots were washed 3 times for 5 minutes in
TBST and detected with a goat-anti-human-IgG (1:10.000 dilution HRP labeled; Jackson
Laboratories) in TBST + 5% BSA. Again, blots were washed 3 times for 5 minutes before
development by chemiluminescence treatment. Precleared s were then incubated
with bead-bound AT14-012 melanoma-specific antibody or with the influenza specific
antibody AT10-002 as a negative control (3 hrs. at 4°C). dy-incubated beads were
washed three times in lysis buffer and bound proteins were eluted from the beads (0,1M
Glycine pH10,5, 150mM NaCl, 1% Triton X100, 1mM EDTA) and neutralized with 1:10
volume of 2M Tris pH7.4. Again, samples were run on an SDS-PAGE gel. 85% of IP
samples was run on SDS-PAGE and stained with Imperial protein stain (Pierce) to stain
total proteins and excise specific bands for Mass Spectrometry. The rest of the
immunoprecipitation (IP) s were run on SDS-PAGE and transferred to PVDF
membrane (Bio-Rad) for immunoblotting. The blot was incubated with AT14-012 or
mouse-anti-CD9 (clone ALB6, Beckmann Coulter) for Western blot analysis to confirm
the identity of CD9 (data not shown).
Epitope mapping
Epitope mapping was done initially by ting hybrid mutants of CD9 (vs CD81).
There are two extracellular loops on CD9: the small EC1 (residues 34 – 58; or SEL) and
large EC2 (residues 112 – 195; or LEL) which possibly serve as the binding partners for
AT14-012 (figure 1a). In addition to the wildtype CD9 construct, we generated one swap
mutant replacing the first r loop, and one swap mutant replacing the larger loop
for the corresponding residues of CD81. To deconstruct the secondary loop even further,
we proposed smaller swap mutants replacing predicted alpha helical stretches for the
corresponding region of CD81 leading s 5 swap mutants: m1 ues 112-134 of
CD9 replaced by the corresponding CD81 residues), m2 (residues 135-151 of CD9
replaced by the corresponding CD81 residues), m3 ues 154-166 of CD9 ed by
the corresponding CD81 residues), m4 (residues 168-180 of CD9 replaced by the
ponding CD81 residues), and m5 (residues 5 of CD9 replaced by the
corresponding CD81 residues), see figure 1b. The cysteines are conserved among
tetraspanins (C152, C153, C167 and C181), and therefore the structural properties/fold
will likely be intact after swapping the designated s. Secondary structure
predictions show that CD9 folds in a similar manner as other tetraspanins, indicating
that this approach will work, as long as the cysteines are kept. All CD9 and swap
variants were constructed by GeneArt (Thermo Fisher Scientific) of the CD9 gene and
was C-terminal FLAG-tagged (3x FLAG: DYKDHDGDYKDHDIDYKDDDDK) for
possible detection on western blot. The CD9 cDNA was cloned into the pHEF-TIG third-
generation lentiviral vector containing an IRES-GFP 3’ of the CD9 cDNA; VSV-G
lentiviral particles were produced in HEK293T cells. The multiple myeloma CD9
negative cell line HL-60 (ATCC; CCL-240) was transduced with these viruses in the
presence of ectin (Takara, Clontech, Japan) and sorted for GFP to obtain a pure
population of CD9 overexpressing cells. Based on FACs binding results of 14-012
together with other cial anti-CD9 dies (ALB6; Beckmann Coulter and
HI9a; Biolegend), we generated alanine mutants of region m4 ues K169A, K170A,
D171A, V172A, L173A, E174A, T175A, F176A, T177A, V178A, K179A and S180A) to
examine which specific amino acids in this m4 region were attributing to the epitope.
Epitope mapping using alanine scanning in the m3 region of CD9
e scanning was performed as bed previously (see materials and methods
belonging to Figure 5B). The CD9 cDNA was cloned into the pHEF-TIG third generation
lentiviral vector containing an IRES-GFP 3’ of the CD9 cDNA; VSV-G lentiviral particles
were produced in T cells. The multiple myeloma CD9 negative cell line HL-60
(ATCC; CCL-240) was transduced with these viruses in the presence of retronectin
(Takara, Clontech, Japan) and sorted for GFP to obtain a pure population of CD9
overexpressing cells. Based on FACs binding results of AT14-012 together with other
cial anti-CD9 antibodies (ALB6 and HI9a), we generated alanine mutants of
region m3 (residues G154A, L155A, G157A, G158A, V159A, E160A, Q161A, F162A,
I163A, S164A, D165A, I166A) to e which specific amino acids in this m3 region
were attributing to the epitope.
The target of AT14-012 is CD9
We identified the target of AT14-012 using colon cancer cells Caco2 (ATCC HTB-37)
since the binding of AT14-012 was higher on FACs but confirmed in a similar manner on
lysates of the melanoma cell line MelBLM. Western blots of SDS-Page run lysates of
Caco2, MelBLM or HL-60 cells were probed for AT14-012 reactivity and detected with a
polyclonal goat anti-human-IgG (HRP labeled; Jackson laboratories). The blot showed
reactivity towards a ~25 kDa large protein (Figure 4a). Reactivity or signal was lost
when the lysates were run under reducing conditions, meaning that the antibody reacts
with a conformational epitope constrained by cysteines. precipitation (IP) of
Caco2 or MelBLM lysates incubated with biotin-labeled sortase-tagged AT14-012 yielded
also a ~25kDa band (Figure 4b). The band is specific as it was not seen in the AT10-002
IP of Caco2/MelBLM lysate nor in the HL-60 lysate IP. Mass-spectrometry (MS) is
of the immunoprecipitation band revealed CD9 as the target n. gh, no
coommassie band was visible by eye for the IP on MelBLM cells, MS analysis showed to
reveal the same precipitated CD9 antigen. Four extracellular peptides belonging to the
ellular loop 2 were identified, giving a 10% coverage of the protein.
Transmembrane peptides were not identified since these are difficult to detect due to
their hobic nature. CD9 binding by AT14-012 was confirmed by western blot
analysis (Figure 4c). Briefly, Caco2 or HL-60 lysates were immunoprecipitated with
AT14-012 or with the influenza-specific antibody AT10-002. Western blot analysis with
again AT14-012 and the mouse-anti-CD9 (clone ALB6) confirmed CD9 as the binding
target of AT14-012 (Figure 4c).
CD9 is widely expressed on healthy and ant cells. ecific antibodies have
been generated and are commercially ble, such as ALB6 and HI9a. With these
antibodies, we confirmed CD9 expression by Caco2 and BLM cells. Competition
experiments with AT14-012 showed that all commercial antibodies were able to e
for binding of AT14-012 as well as AT14-012 itself (data not shown).
To more specifically identify the binding epitope of AT14-012, hybrid mutants were
generated swapping protein regions of the CD9 homolog CD81 as described in the
Materials&Methods section. Binding of dies AT14-012, ALB6 and HI9a to these
mutants was . We identified that all anti-CD9 antibodies bound to the
extracellular loop 2 (EC2), g loss of binding when the EC2 of CD81 was swapped,
whereas binding was maintained when the first extracellular loop (EC1) was swapped.
No binding was observed for all antibodies when HL60 cells were transduced with empty
vector or non uced cells. The epitope on the EC2 loop was further examined by
hybrid mutants m1, m2, m3, m4 and m5, in which specific regions of CD9 were d
for the corresponding regions of CD81 (figure 1b). Of note, the cysteines were left
untouched in order to maintain the secondary structure. Swapping of the m2 and m5
region had no effect whatsoever on binding of any anti-CD9 antibody, showing that the
epitope did not reside in these regions. We could show that binding of all antibodies was
abrogated when region m3 was swapped, indicating that the epitope of all tested
antibodies resides in this . AT14-012 ined binding to the m1 mutant
whereas all commercial antibodies lost binding. ALB6 and AT14-012 showed loss of
binding towards the m4 mutant whereas HI9a retained binding. These results indicate
that the main epitope of AT14-012 resides in m3 and/or m4. This directed us to make
alanine mutants in m4 at first (as described in the Materials&Methods section). Herein,
HI9a was taken along as a positive control, controlling for expression of CD9 alanine
mutants on the surface of transduced HL60 cells. ALB6 was taken along as a
comparison, for examining whether this commercial widely used anti-CD9 antibody had
a similar epitope. After FACs analysis, we showed that F176A was the only alanine
mutant that showed loss of binding to ALB6. As for AT14-012, loss of g was
observed when residues K169, D171, V172, L173, T175 and F176 were substituted for an
alanine. We thus showed that the epitope of AT14-012 overlaps with the epitope of ALB6
but differs in at least 5 additional amino acids.
AT14-012 epitope is linear and s only in region m4. After the e scanning
binding experiment of region m4, we pursued to e the m3 region in more detail as
g was lost to the hybrid CD9/CD81 mutant (see Figure 15). We constructed
alanine mutants in region m3 spanning amino acids 154 to 166 with the exception of
A156. We transduced the CD9 negative HL60 cell line and GFP bulk sorted the cells
expressing CD9. AT14-012, ALB6 and HI9a binding to the cells was examined by FACS.
Surprisingly, no single alanine replacement in this region hed binding of AT14-012
or HI9a. The EC2 and m3 CD9/CD81 hybrid mutant displayed a lack of binding for all
dies and therefore, we have to postulate that this particular hybrid was probably
wrongly folded. ing to the crystal structure of CD81 and homology model of CD9
(see Figure 15) the m3 region is “locked” in a position between m1, m2 and, m4.
Therefore, it seems likely that the binding is lost for all anti-CD9 antibodies when any
large exchange of amino acids is brought to this region. On a side note, ALB6 lost
binding to the Q161A mutant and thereby, we solved the epitope for ALB6 as well (Q161
in m3 and F176 in m4). After analysis of the alanine scanning in region m3 and m4 we
can hypothesize that AT14-012 targets a linear folded epitope which is conformationally
held together or induced by other surrounding parts of CD9 (m2, m3 and m5 see
homology model in Figure 15).
AT14-012 favors binding to clustered CD9. The lab of Martin Hemler showed that the
formation of CD9 homoclusters is favored by palmitoylation of CD9 and that levels of
CD9 homoclusters are elevated on primary and in particular on metastatic tumor cells
(Yang JBC 2006). To determine the dependence of AT14-012 on the palmitoylation
status of CD9 tumor cells were cultured in presence of 2-bromo-palmitate (2-BP), a
known tor of palmitoylation. Melanoma BLM cells clearly show reduced binding of
AT14-012 to 2-BP treated cells whereas binding of the commercially available CD9 HI9a
antibody is not affected by depalmitoylation [Figure 14A, B]. Of interest the observed
effect was strongest on the highly sive MelBLM lomé AJP 2009) and not
seen on the non-metastatic colon carcinoma CaCo2 cells. This suggests that CD9
homoclusters are at higher levels in advanced disease, ting AT14-012 may be used
to monitor tumor progression.
Example 3 – Functional characterization of 12
MATERIALS & METHODS
Tumor cell lines. Melanoma (MelBLM, MelWBO, Mel136.2), Colon Carcinoma (CaCo2,
Colo320, HT29, LSTR), Pancreas Carcinoma (PANC-1, CAPAN-2, MiaPACA, ,
Esophagus Carcinoma (OE19, OE33) and, Acute Myeloid Leukemia (THP-1) cell lines
were ined under standard tissue culture conditions. For minimal disruption of
cell surface proteins tumor cells were detached using se (Life Technologies).
Flow cytometry and antibodies. Detached solid tumor cells and primary fibroblasts, non
adherent tumor cells and other primary cells were ed for flow cytometry analysis
at 50.000 cells in a 96 well plate. Cells were incubated with commercial antibodies
against CD4, CD8, CD9, CD19, CD41, CD62P, CD81 (Biolegend). In house generated
02, anti CD30, or AT14-012 were either led, biotin or Alexa 647 d.
Unlabeled antibodies and biotin labelled antibodies were secondary stained with anti
IgG-PE ern Biotech) or anti streptavidin PeCy7 (Becton Dickinson) respectively.
Panitumumab (anti EGFR1) was included as a positive control antibody in some
experiments. Samples were analyzed on a FACS Canto and LSR sa X20 (Becton
Dickinson).
Platelet activation. Blood collected from healthy eers in citrate containing blood
collection tubes (Becton Dickinson) was spun for 10 minutes at 800g. The top Platelet
Rich Plasma fraction (PRP) was collected and used to test for platelet activation. Briefly
µl PRP was incubated for 20 min at room temperature with 10 µg/ml antibody, Fab2-
fragments or the positive control Thrombin Receptor Activating Peptide (TRAMP).
Samples were analysed by flow cytometry (LSR Fortessa X20, BD) for surface expression
of CD41 and CD62P/ P-selectin using direct conjugated antibodies (Biolegend). CD9
HI9a expression was ined on unstimulated platelets.
et ation. Blood was collected from healthy volunteers in citrate containing
blood collection tubes (Becton Dickinson). 300 µl whole blood mixed with 300 µl assay
buffer was allowed to warm at 37 ⁰C for 2 minutes. Positive control peptide or antibody
(end concentration 10 µg/ml) was added and platelet aggregation was measured in time
using a Multiplate analyser (Cobas/Roche).
Xenograft mice. Immunodeficient mice were transplanted aneously with 200.000-
500.000 rase/GFP expressing MelBLM cells in High Concentration Matrigel
(Corning). AT14-012 or AT10-002 control antibody was given intravenously at 10mg/kg
mouse. Antibody treatment started at the day of tumor injection or tumors were allowed
to grow for 3 weeks to ine growth of the primary subcutaneous tumor or
outgrowth of metastasis respectively. Subcutaneous tumor growth was determined both
by caliper or luciferase imaging after luciferin (Promega) injection using a photon imager
(Biospace lab). The presence of metastasis was visualized by eye and luciferase imaging
at the end of the experiment.
Recently established tumor cell lines
Pieces of tumor tissue ally removed from melanoma patient were digested and put
into culture. Growing cells were maintained under standard tissue culture conditions.
Tumor tissue obtained from pancreas carcinoma ts are too small to directly
establish cell lines and are first grafted under the skin of NSG mice. Growing tumors are
harvested, digested and maintained under rd tissue culture conditions. Human
tumor cells and tumor infiltrating fibroblasts of mouse origin are ted by
tometry cell g based on EpCam expression.
RESULTS
AT14-012 has broad tumor reactivity. AT14-012 was identified by binding to Melanoma
cell lines MelBLM and MelWBO. Later it was found that AT14-012 displays binding
vity to all melanoma cell lines tested (Figure 6a and Figure 7a). A relative large
body of literature suggests that CD9 is broadly expressed and upregulated on a wide
variety of solid tumor cells. In line with this we found that AT14-012 reacts with a panel
of colon, esophagus and pancreas oma cell lines (Figure 6a and Figure 7b). In our
hands the only solid tumor cell line found thus far not to interact with AT14-012 is the
CD9 negative Colo-320 colon carcinoma line (Figure 7b). Although to a lesser extend
CD9 has also been found expressed on hematopoietic cells. In Figure 6b and Figure 6c it
is shown that AT14-012 is able to react to a ed number of Acute d Leukemia
and Myeloid Leukemia cells (AT14-012 appears to bind BL-007, BL-009, BL-037, BL-054
and BL-058, whereas it does not bind BL-014, BL-030 and BL-055). Altogether this
tes that AT14-012 is useful for a much broader therapeutic application than
melanoma only.
AT14-012 binds stronger to tumor than primary cells. None of the therapeutic antibodies
for use of solid cancer treatment currently used in the clinic recognize antigens that are
exclusively expressed on tumors. However, a therapeutic window for these antibodies
presents itself when the antigen is higher expressed on tumor cells as compared to
healthy cells. For example, Trastuzumab (Herceptin) is used to treat HER2
overexpressing breast cancer. Similar to this CD9 is known to be frequently upregulated
on a wide variety of solid cancers. If 12 reacts er to tumor cells as ed
to healthy cells AT14-012 could be used in a therapeutic setting r to tin.
Indeed, we found that AT14-012 reacts stronger to melanoma cells than to y
melanocytes (Figure 7a; all melanoma cell lines are bound by AT14-012, whereas
primary fibroblasts are not bound. Primary melanocytes are bound by AT14-012, but to a
lesser extent than most melanoma cell lines). Also, AT14-012 reacts stronger to colon
carcinoma cells than to primary colon epithelial cells (Figure 7b; AT14-012 binds CaCo2
(upper panel) er than primary colon epithelial cells (lower . Lastly,
AT14-012 was found to bind stronger to the melanoma MelBLM cells than to primary
tonsillar T and B lymphocytes (Figure 7c; the lower images in the middle and right
columns demonstrate stronger binding of AT14-012 to MelBLM as compared to the
binding of AT14-012 to total CD4 T cells, total CD8 T cells and total CD19 B cells (upper
images of the middle and right column)). These data indicate that AT14-012 is useful for
a clinical setting currently used for dy treatment of solid tumors.
AT14-012 binds and activates platelets but does not induce aggregation. It has been
previously published that CD9 is highly expressed on platelets and that antibodies
targeting CD9 can induce platelet activation and aggregation which potentially leads to
thrombosis in patients treated with such anti CD9 antibody. Although the melanoma
patient from which 12 was isolated did not display any signs of thrombosis we
needed to ensure that AT14-012 does not induce this serious side effect.
Firstly, the binding of AT14-012 to platelets was determined. Platelet rich plasma (PRP)
from a healthy volunteer was incubated with a commercial antibody against CD9 or
stained with AT14-012. Platelets were fixed to rule out any ence in cell surface
expression of CD9 due to auto activation of the platelets. As expected from literature the
commercial CD9 HI9a antibody strongly binds ets (lower images of Figure 8a). In
line with this AT14-012 also showed strong interaction with the platelets (Upper images
of Figure 8a).
Next, we assessed whether the platelets would be activated upon interaction with
AT14-012. Both Thrombin Receptor Activating e (TRAP) and the commercial CD9
antibody ALB6 are known to stimulate the activation of platelets as visualized by cell
e upregulation of P-selectin/CD62P. PRP from a healthy volunteer incubated with
TRAP or ALB6 indeed did show this surface induction of CD62P as compared to the
unstimulated condition and the irrelevant ALB6 isotype matched FLAG antibody
(Figure 8b). Also, AT14-012 in both recombinant IgG1 and IgG3 formats as well as the
antibody purified from the supernatant of the original B cell clone was able to activate
the platelets (Figure 8b).
Lastly it was ined whether AT14-012 induces the ation of platelets. For
this whole blood was incubated with the same stimulants as before with the addition of
Fab2 nts of the AT10-002 and 12 antibodies. As expected the TRAP
peptide and ALB6 antibody induced strong aggregation of platelets (Figure 8c). In
contrast to the commercial CD9 ALB6 antibody, AT14-012 did not trigger the
aggregation of platelets in any of the different s (IgG1, IgG3, purified from B cell
supernatant (2H15), or Fab2 fragment) (Figure 8c). Altogether this shows that although
AT14-012 binds and tes platelets, the interaction of AT14-012 with the platelets is
not inducing platelet aggregation. These findings are in line with the observation that
the AT14-012 donor did not y any signs of thrombosis. We therefore conclude that
AT14-012 can be clinically used without involving thrombosis as a serious side effect.
AT14-012 impairs wth of primary and secondary tumors. A tumor xenograft
mouse model was set up to determine an anti tumor effect of 12 in an in vivo
setting. Immunodeficient mice are a suitable model for tumor engraftment.
The mice received a subcutaneous transplant of 500.000 luciferase/GFP expressing
MelBLM cells in Matrigel on both flanks. Tumors were d to grow for 3 weeks
before the mice received intravenous injections of 10 mg/ml AT14-012 or our control anti
influenza antibody (AT10-002) twice weekly for one or two weeks (depending on the size
of the aneous tumor). At 4 or 5 weeks after tumor cell graft, mice were iced
and internal organs exposed. In 3 out of 4 mice in the AT10-002 treated group, large
luciferase positive lymph nodes were found suggesting that the MelBLM tumor cells are
able to metastasize and develop ary tumors in lymph nodes. In sharp contrast,
none of the five mice that received the AT14-012 antibody showed any signs of lymph
node metastasis (figure 9a). This demonstrates that AT14-012 is able to inhibit tumor
metastasis.
In a follow up experiment mice received 200.000 MelBLM GFP/luciferase tumor cells on
both flanks. This time antibody injection (twice weekly for 2 weeks 10 mg antibody per
kg mouse) was initiated at the same time as tumor grafting. The size of the
subcutaneous tumors was determined two times per week by caliper. As shown in figure
9b growth of tumors was reduced in the AT14 as ed to the control-treated
mice. This shows that 12 has a negative effect on tumor growth.
MelBLM subcutaneous tumors harvested from AT14-012 or AT10-002 control antibody
treated mice were tested for presence of bound AT14-012 antibody by
immunohistochemistry. Tumor tissue was imbedded in paraffin after which sections
were incubated with HRP labelled anti-lambda or anti-kappa recognizing the light chain
of the AT10-002 or AT14-012 dies respectively. As expected the AT10-002 anti-
Influenza control antibody does not bind tumor tissue whereas AT14-012 clearly binds
the outer layers of the tumor tissue and shows penetration to deeper layers (Figure 9C).
Single cell digests of MelBLM subcutaneous tumor cells harvested from AT14-012 or
AT10-002 control antibody treated mice are tested for binding of CD9 dies AT14-
012 and HI9a. Tumor cells harvested from AT14-012 treated mice show reduced binding
of both AT14-012 and CD9 HI9a as compared to tumor cells from AT10-002 treated mice
(Figure 9D). The observed effect is not due to pre-occupation of the AT14-012 epitope by
ed AT14-012 dy as tumor cells from both treatment groups stain ve
with an anti IgG antibody (Figure 9D).
Of interest, in a repeat ment with AT14-012 is also able to impair tumor growth of
subcutaneously growing SK-MEL-5 melanoma tumors (Figure 9E). The effect on tumor
growth inhibition is more apparent when the weight of the tumor is determined. AT14-
012 treated tumors y have a lower weight as their counterparts from AT10-002
treated mice (Figure 9F). Of note, the reduction of CD9 levels on the tumors cells as
observed on AT14-012 treated MelBLM tumors is confirmed when CD9 sion levels
are determined in ex vivo isolated and digested -5 tumors (Figure 9G).
AT14-012 binds recent patient derived melanoma and pancreas tumor cells. AT14-012 is
able to recognize a broad range of established solid tumor cell lines (Figure 6A). Next is
was tested if the AT14-012 binding reactivity also applies to tumor samples that were
recently isolated from cancer patients. Short term cultured patient derived ma
cells were tested for the binding of AT14-012. A positive signal with AT14-012 was
observed on all primary ma samples tested (Figure 10A-B). A strong correlation
of AT14-012 binding and CD9 expression was observed (Figure 10B). Of note, tumor cells
d from the melanoma patient from which AT14-012 was derived are the highest
AT14-012 binders in the panel. Likewise, patient derived pancreas carcinoma tumor
cells were subjected to binding of CD9 antibodies. In line with the efficient binding of
AT14-012 to established pancreas carcinoma cell lines (Figure 6A) AT14-012 display
strong reactivity towards both patient derived pancreas carcinoma lines tested (Figure
10C).
12 reactivity is restricted to es. Tetraspanins in general and CD9 in
particular are broadly expressed in a vast number of cells and tissues and have been
suggested to be evolutionary conserved h tly related species (Garcia-
Espana, Genomics, 2008). Platelets of mice, , cynomolgus monkeys and a human
are tested for binding of AT14-012. As expected CD9 is expressed on the platelets of all
species tested. r, AT14-012 only reacts with platelets of the Cynomolgus
monkeys and humans, binding to mice and rabbit was not ed (Figure 11 A-C).
Together this suggests that AT14-012 reactivity is restricted to primates.
Example 4 – Complement and antibody depended xicity
MATERIALS & S
Complement depended cytotoxicity (CDC) assay
Suspension or adhered melanoma cells were labelled for half an hour at room
temperature with antibody. uently cells were incubated with rabbit ment
(S7764, Sigma) for 45 minutes at 37 ⁰C. Percentage cell death is determined by DAPI
and flow cytometry (Fortessa X20, Becton Dickinson) or ToPRO3 and microscopy
(Operetta, Perkin Elmer) for suspension and adhered cells respectively.
dy dependent cellular cytoxicity (ADCC) assay
Chromium-51 labelled target cells are incubated with 10 ?g antibody for 30 min at 37
⁰C. CD3 depleted PBMCs are added in a serial dilution ed by an onal 4 hours
of incubation. The presence of Chromium-51 release in the supernatant is detected in
LumaPlates (Perkin Elmer) using a Wallac-counter. Plotted values for antibody induced
cell lysis are corrected for the spontaneous release of Chromium-51.
RESULTS
AT14-012 triggers antibody dependent cytotoxicity (ADCC). To determine whether
AT14-012 possess the ability to kill tumor cells via antibody dependent cytotoxicity
(ADCC) tumor cells were labelled with radioactive Chromium and subsequently
incubated with AT14-012, negative l AT10-002 (anti Influenza) or positive control
(Cetuximab, anti EGFR1) antibodies. PBMC effector/ melanoma target cell ratios were
varied. The percentage cell death was determined by the release of Chromium from the
dead cells in the medium. Less ent than Cetuximab AT14-012 was able to kill
MelBLM via ADCC while minimal cell death was observed when primary Human Artery
elial Cells (HAECs) were used as target cells (Figure 12A). In parallel primary
short termed cultured patient derived melanoma cells were tested for cell kill via ADCC
by AT14-012. Although some variation between the different melanoma cells was
observed AT14-012 was able to show ADCC ty over the AT10-002 control antibody
(Figure 12B). Altogether this suggests that the anti-tumor reactivity of AT14-012 is at
least in part mediated via ADCC.
AT14-012 triggers complement dependent cytotoxicity (CDC). Several variants of the
AT14-012 antibody were tested for their ability to trigger complement mediated
cytotoxicity (CDC). 2H15 is the antibody d from the original AT14-012
immortalized IgG3 B cell clone. The AT14-012 recombinant antibody based on 2H15 is
produced in both an IgG1 or IgG3 backbone. In addition, we constructed a variant of the
12 IgG1 antibody containing an E345R on in the Fc tail. This on has
been shown to force hexamerization of a particular antibody on its target y
efficiently triggering complement mediated xicity (CDC) (de Jong, PLOS Biology,
2016).
Melanoma lines in sion were incubated with ent AT14-012 variants in the
presence of human serum and subsequently tested for the presence of C1q on the cell
e. As expected C1q deposition was observed with the AT14-012 hexamerization
variant. In addition, C1q deposition was found with the 2H15 antibody purified from the
original B cell clone and the recombinant produced AT14-012 IgG3. Of note AT14-012
engineered as an IgG1 did not attract C1q to the cells [Figure 13A, B]. Suspension
MelBLM or SK-MEL5 were incubated with the AT10-002 control anti Influenza antibody
or any of the 12 variants in the presence of rabbit complement. The AT14-012
IgG1 antibody does not induce any cytotoxicity similar to the anti Influenza negative
control antibody [Figure 13C]. In sharp contrast and in line with published observations
[de Jong, PLOS Biology, 2016] introduction of the E345R mutation the dy induces
concentration dependent cell death via CDC [Figure 13C]. These observations are
comparable for sion and adhered melanoma cells [Figure 13 C, D]. Of interest
AT14-012 recombinant produced as an IgG3 (thus without E345R mutation) is also able
to trigger CDC [Figure 13D]. Surprisingly, the original B cell produced 2H15 antibody
does attract C1q, but does not induce complement mediated cell death e 13A, C].
AT14-012 E345R efficiently kills tumor cells by CDC in the presence of rabbit
complement. While AT14-012 E345R is able to attract human C1q to the cell surface
[Figure 13A, B] the antibody is not able to r CDC mediated cell death in the
presence of human ment factors [Figure 13E]. We igated whether the
discrepancy of cell kill between rabbit and human complement is related to the
expression of complement regulatory proteins (CRPs). Colo-205 which completely lack
the expression of CD55, an inhibitor of C3 convertase formation, did allow antibody
mediated CDC in the presence of human serum [Figure 13E]. This suggests that AT14-
012 may be able to induced complement dependent cell death of tumor cells when
combined with a CD55 blocking antibody.
Example 5 – Affinity ements
MATERIALS & METHODS
ELISA binding 12 compared to commercial anti-CD9 antibodies
Binding of AT14-012 (IgG1) and control human AT10-002 antibody was assayed in an
ELISA format to make a comparison to commercial antibodies ALB6, HI9a and mouse
dy controls anti-FLAG (for detection of CD9-3xFLAG-RabbitFc-Sort-biotin to the
plate) and anti-CD3 OKT3 onab). The ELISA setup is similar as described above
to assay the amount of biotinylation of the CD9 molecules. The commercial abs were
added in a serial dilution similar to that of AT14-012. The commercial abs were detected
with a goat anti-mouse HRP labeled dy (1:4000 from Jackson) whereas the human
abs were detected with the goat uman HRP d dy (1:4000 from
Jackson). To compare the affinity differences in a better manner, we applied the
antibodies in a surface plasmon resonance (SPR) assay on a CD9-3xFLAG-rabbitFc-
Sortase-biotin coated SPR chip. EC50 values were calculated using GraphPad 7.0
software.
Affinity measurement using Surface plasmon resonance (SPR)
The chip for binding of the anti-CD9 antibodies was made in a similar manner as
described for the ove detection (see below). Here, AT14-012 (+ controls) affinity
is measured in a 'classical' setup, regenerating the chip after each antibody injection.
Binding was analyzed on the IBIS MX96 ment by performing injections with
dilution series of recombinant antibody diluted in binding buffer (PBS + 0.05% Tween20
+ 0.05% sodium azide + 0.01% BSA) on the chip. In each injection, complexes were
injected and incubated for 8 min, followed by 12 min thorough washing with system
buffer (PBS + 0.05 % Tween20 + 0.05% sodium azide) to measure dissociation. Injections
were repeated at least three times for every tested antibody and injections with blank
binding buffer were used as reference. After each concatenated injection, the chip was
regenerated with 10 mM glycine-HCl, pH 2.0 + 150 mM NaCl. Experimental data were
processed with SPRintX software (IBIS Technologies) and kinetic constants were
determined using Scrubber2 software (BioLogic).
Cloning, expression, purification and Sortase A site specific biotinylation of 2-
-rabbit-Fc-Sortase-HIS (+ control CD81-EC2-3xFLAG-Rabbit-Fc-SortaseHIS)
Freestyle cells (Thermo) were adapted and taken in culture for one week in serum free
Freestyle medium (Gibco) in a 125ml vent capped g flask on a shaker platform
(140rpm) at 37 degrees with 8% CO2. Transient transfection was performed using the
pcDNA3.4 vector containing the CD9 sequence of the extracellular large loop 2 (amino
acids 112-195; t P21926) fused er with a 3xFLAG tag (-
DYKDHDGDYKDHDIDYKDDDDK-) and subsequently the Fc region (CH2-CH3) of a
rabbit IgG1 protein (amino acids 108-322; UniProt P01870). The CD9 was spaced from
the 3xFLAG tag by a -GGGT- , the 3xFLAG from the rabbit Fc by a -GSS- linker.
The SortaseHIS tag (-LPETGGHHHHHHstop) was spaced from the Fc part by a -GGGS-
linker. The insert was cloned into the pcDNA3.4 vector using the NcoI and PmeI
restriction s (NEB) and a large DNA preparation was isolated using a Qiagen
plasmid maxi kit. DNA (3ug of plasmid) and 6µl of ExtremeGene9 (Sigma) solution in
Optimem (Gibco) was incubated separately for 10 minutes in 100µl Optimem. The 100µl
Optimem-ExtremeGene9 on mix was added to the 100µl Optimem-DNA mix and
incubated for another 30 minutes before adding se to a 3ml culture having 0.5 x
10^6 cells/ml. Two days later, the medium was fed with another 2ml of fresh Freestyle
medium. The medium was ted after 5-7 days of culture and put into a -80°C
r for further use. Culture conditions were scaled up if necessary for larger
productions. Protein production was measured using a quantitative rabbit IgG ELISA
(Jackson). Medium was defrosted and filtered before being applied to a 5ml of protein G
column (GE Healthcare) at a flow rate of 1ml/min on an AKTA Explorer system (GE).
The column was pre-equilibrated with PBS until a stable UV280/215nm baseline was
achieved. After application of the sample, the column was again washed with at least 5
column volumes PBS and until a stable UV280/215nm baseline was kept. Bound protein
was eluted with 0.2M Glycine + 150mM NaCl pH2.5. Top protein fractions were
neutralized with 1:10 v/v 1M Tris pH9.0. The fractions were combined and applied onto a
Superdex 200 16/60 column (GE) which was equilibrated with PBS. The monomeric peak
was ted and quantified on a nanodrop 1000 system with the appropriate
extinction/size settings for this protein. The protein was aliquoted for further use and
stored at -20°C for short term storage. The enzyme Sortase A (see Wagner et al., 2014 for
preparation) was used in a 1:1 molar ratio together with a 10 times molar ratio of the
otin nucleophile to enzymatically attach a biotin moiety to the molecule in which
the HIS tag was removed by the tic e A reaction. The reaction ed in
25mM Tris, 150mM NaCl pH7.5 and 2mM CaCl2 for 4 hours at 37°C with occasional
gentle vortexing. The on was stopped by 1mM EDTA. The biotinylated CD9 protein
was separated from Sortase A and smaller components (free GGG-biotin nucleophile and
free HIS tag) on a PBS equilibrated Superdex200 16/60 column. Top fractions were
collected. The amount of ylation was checked via ELISA. In short, 5ug/ml
streptavidin was coated overnight in PBS onto a 96 well high binding ELISA plate
(Costar). The biotinylated CD9 was applied to the wells in a serial two-step dilution with
start concentration of 10ug/ml in PBS + 2.5% BSA for one hour. 12 was added in
a serial dilution as well to obtain a grid to e the optimal signal for one hour in
PBST + 2.5%BSA. AT14-012 was detected by incubation of a goat-anti-human HRP
labeled dy (1:4000 dilution from Jackson) in PBST + 2.5%BSA and developed
using a TMB/H2O2 acidic solution. The reaction was stopped using 1M H2S04 and
measured using 450nM on a Perkin Elmer Envision plate reader. The protein was
sufficiently biotinylated and optimal concentrations were between 2.5 and 5ug/ml. All
the steps above were repeated for the control protein CD81-EC2-3xFLAG-rabbit-Fc-
Sortase-HIS, as the coding region of CD9 was replaced for the EC2 coding region of
CD81 (amino acids 113-201; UniProt P60033). The integrity and biotinylation of this
protein was checked by ELISA using the anti-CD81 dy clone JS81 (BD) and
detected with a goat-anti-mouse HRP labeled antibody (Jackson).
e mapping of AT14-012 using soluble CD9-EC2-FLAG-rabbit-Fc protein in ELISA
Alanine mutants of region m4 (amino acids 169-180) were cloned into the pcDNA3.4
vector mentioned above. The proteins were expressed in small scale (3ml) and quantified
using the rabbit IgG ELISA. To examine AT14-012, ALB6 and HI9a binding, we coated
anti-FLAG antibody (Sigma) at 5ug/ml overnight in PBS. Unpurified serum free
supernatants were subjected to binding to the FLAG antibody at 1ug/ml for 1 hour in
PBST + 2.5% BSA. After washing the random biotinylated HI9a, ALB6 and AT14-012
were subjected to binding to the captured CD9-FLAG-rabbit-Fc-SortaseHIS molecules.
Bound antibody was detected with streptavidin-HRP (1:10.000 dilution from Thermo).
ELISA was developed as described earlier. Random ylation of the antibodies was
d out using the EZ-Link NHS-Biotin kit (Thermo). Purified antibodies in PBS were
subjected by an tion of a 10-fold molar ratio of biotin label for 30 minutes at room
temperature. The reaction was stopped by size exclusion. The biotinylated antibody was
separated from the free label by applying the sample (1ml at 1mg/ml) on a PBS preequilibrated
Superdex 200 16/60 column.
Construction of an m4 circular peptide.
The m4 region of CD9 (167-PKKDVLETFTVKS-180) was synthetically made by a
peptide synthesis lab, analyzed by LCMS and purified by RP-HPLC using an
Acetonitrile gradient. The peptide was lyophilized until completion. The peptide was
flanked by two additional serines to mimic the space that the cysteine knot creates (see
crystal structure of CD81) and ed by two cysteines that would make the peptide
circular. For detection or capture purposes, a biotin moiety was placed at the N-terminus
which was spaced by a single PEG2 group biotin-PEG(2)-CSPKKDVLETFTVKSSC
ines are linked).
RESULTS
AT14-012 is a medium affinity antibody. First, the amount of hypermutations brought to
the heavy (4 amino acid replacements) and light chain (3 amino acid replacements) of
AT14-012 might be an indication that the immune system was not adequately
challenged in the patient to bring additional hypermutations to the variable domain
sequences. Second, we could show that AT14-012 does not induce platelet aggregation
whereas commercial dies developed previous by others did induce platelet
aggregation. Also, the t did not p any thrombotic or ocytopenic
symptoms (low platelets counts due to antibody ed platelet destruction or
aggregation) and was not treated with any agents that could have resolved this
undiagnosed issue. The main question is whether the lower ty of AT14-012 is
cial to the properties of a “type” of anti-CD9 dy that causes the optimal
platelet phenotype or the usage of the unique epitope on CD9 (m4) targeted by AT14-012
results in an optimal non-aggregative et state. The binding of AT14-012 was tested
in an ELISA setup using the recombinant expressed second ellular loop of CD9
(EC2) to examine the differences in binding affinities with common used commercial
murine anti-CD9 antibodies. We determined before (Figure 5) that the epitope for all
tested D9 antibodies resides in the EC2 loop. Although the two ELISA setups are
different (detection with different secondary antibodies) between the human and mouse
(commercial) antibodies, we could estimate that the EC50 of AT14-012 was significantly
lower (EC50 ~250ng/ml) ed to the commercial HI9a (EC50 ~20ng/ml) and ALB6
(EC50 ~13 ng/ml). To make a better estimate in the affinity of AT14-012 compared to the
commercial dies, we employed a label free detection setup using surface plasmon
resonance (SPR). Three separate injections were employed over a CD9 layered SPR chip.
The averaged affinity of AT14-012 in this setup was in the nM range (~44nM) and the
commercial dies were ~145pM for ALB6 and HI9a ~2.33pM (Figure 16). The
dissociation rate for AT14-012 measured is 700 times higher compared to HI9a which
means that AT14-012 is able to detach from CD9 quite easily. HI9a is ~19,000 higher
affinity due to its low dissociation rate (kd). We did not examine longer dissociation times
for HI9a or ALB6 as the result for AT14-012 was obvious. ALB6 is still able to dissociate
in a slow fashion after binding (~22 higher dissociation compared to HI9a) and
butes to the at lower but still very high affinity when compared to HI9a.
e confirmation of AT14-012 using recombinantly expressed CD9-EC2 m4 alanine
mutants. The epitope was igated in r detail using an ELISA setup by
incubation of random biotinylated anti-CD9 antibodies onto FLAG-tag captured CD9-
EC2-3xFLAG-rabbitFc protein. The alanine mutants (amino acids K169A to S180A
described in Figure 5 were cloned and expressed as described in the als and
methods. Binding for AT14-012 is lost totally when alanine mutants are made at
positions K169A, D171A, L173A, F176A and a significant decreased binding can be
observed for V172A (Figure 17A) which is line with previous FACS data (Figure 5).
ALB6 showed decreased binding to the F176A mutant as observed previously. HI9a does
not lose any reactivity to any mutant and is an internal l for the presence of CD9
protein on the ELISA plate. Therefore, we can conclude that the epitope for HI9a does
not reside in m3 or m4 or that a single e mutation brought to these regions does
not abolish binding as the affinity of HI9a is of a significantly high value in this SPR
setup. The epitope was mapped (highlighted by red) on the constructed homology of CD9
(Figure 17B) and was located on the edge of the ellular part of the n. The
alanine scan of m3 on FACs did not reveal any loss of binding to AT14-012 and we could
not show any binding to a circular constructed m4 peptide in ELISA or SPR (data not
shown). Therefore, we hypothesize that the correct folding of the m4 region is strongly
influenced by other CD9 s (m3 and m5) which leads to an AT14-012
conformational linear epitope. A stal is required to confirm the epitope mapping
data in further detail and to examine the bution of every single amino acid on the
CD9 epitope as well as the AT14-012 paratope. The epitope mapping data was r
confirmed using the binding of AT14-012 to various s like lgus, mouse and
rabbit cells (Figure 11). AT14-012 was able to react with cynomolgus cells, whereas
binding was lost when assayed for binding to rabbit or mouse cells. The 5 residues
contributing to the epitope of AT14-012 are aligned in Figure 17C (highlighted in dark /
red). Apparently, AT14-012 binding is lost when too many residues are varied as
observed for the rabbit and mouse CD9 m4 sequences. Presumably, rabbit mutations
(V172I, T175S, F176I and T177Q) and mouse mutations (D172Q, V172L, T175S and
T177Q) in region m4 causes a major conformational change or shift of the AT14-012
epitope that might explain the lack of AT14-012 g. On a side note, the F176L
mutation alone does not induce a loss of binding of AT14-012 to cynomolgus cells (Figure
17C).
Example 6 – Affinity improving AT14-012
MATERIALS & METHODS
Affinity improving AT14-012 using single cell sorted 2H15 B cells and SPR
The original identified B cell clone of AT14-012 (2H15; IgG3) was single cell sorted using
a BD FACs ARIA III in ~20x 384 well plates with appropriate culturing ions. B
cell outgrowth (~70%) was monitored via an Operetta al machine (Perking Elmer)
observing Bcl6/Bcl-xL transduced GFP positive cells. Wells with positive signal were
transferred to a fresh 96 well plate (8 plates in total) and cultured up to 1 – 2 weeks
before the supernatant was harvested (100ul) in a 96 well PCR plate and diluted 1:1
with PBS + 0.05% Tween20 + 0.05% sodium azide and sealed frozen in a -80°C freezer
until further usage. Two wells were attributed to control supernatants of the original
2H15 clone and a control IgG3 B cell supernatant of an anti-HRV clone that does not
bind to CD9 or CD81. SPR was performed on an IBIS Mx96 instrument (IBIS
Technologies). Proteins are immobilized on an SPR chip pre-coated with streptavidin (G-
STREP H825-065 (Sens logies) using a CFM microfluidics r device
(Wasatch Microfluidics). Biotinylated anti-human CH1 nanobody (Thermo) and
biotinylated full length anti-human Fc antibody (Jackson) were spotted at various
concentrations for quantitation (examine the IgG concentration) as well as qualitative
measures (IgG integrity). CD9- and CD81-3xFLAG-rabbitFc-Sortase-Biotin was also
spotted at s concentrations to examine CD9 binding compared to a serial dilution
of inant 12 (IgG1) antibody. The binding was ed for all spots as
such that similar amounts of spotted CD9 and CD81 could be compared. Binding of IgGs
was monitored using the IBIS surface plasmon resonance imager described earlier and
after each concatenated injection, the chip was regenerated with 10mM glycine-HCl
pH2.0 + 150mM NaCl. The total amount of injections per plate in detail were as s:
(1) two injections with PBST to enforce a baseline, (2) one injection with anti-rabbit to
check whether CD9 and CD81 were still on the chip and did not degrade over time/usage
because of the extensive stripping, (3) one injection with PBST, (4) a serial dilution of
recombinant AT14-012 IgG1 to measure the RU’s for IgG concentration as well as CD9
g (CD81 as control binding) – 1.33 – 4.0 – 13.30 – 40.0 – M of protein, (5)
two rows of B cell sups (A1 to A12 and B1 to B12), (6) one injection of inant AT14-
012 IgG1, (7) two rows of B cell sups (C1 to C12 and D1 to D12), (8) one injection of
recombinant 12 IgG1 (9) one injection with PBST, (10) two rows of B cell sups (E1
to E12 and F1 to F12), (11) one injection of recombinant AT14-012 IgG1, (12) dependent
on the plate a well was sacrificed to include a control B cell sup (IgG3 HRV clone plate 1
= G1, plate 2 = G2 etc.), (13) one row of B cell sups (G X? to G X? depends on the plate
which numbers), (14) dependent on the plate a well was sacrificed to include a control B
cell sup (2H15; IgG3 original clone, plate 1 = G1, plate 2 = G2 etc.), (15) one row of B cell
sups (H X? to H X? depends on the plate which numbers), (16) one injection of
recombinant AT14-012 IgG1, (17) a serial dilution of recombinant AT14-012 IgG1 to
measure the RU’s for IgG concentration as well as CD9 binding CD81 as control binding)
– 1.33 – 4.0 – 13.30 – 40.0 – 133.0nM of protein to check for difference in RU’s in the
beginning of the run and after, (18) one injection with anti-rabbit to check whether CD9
and CD81 were still on the chip and did not degrade over time/usage because of
stripping, (19) one final injection with PBST. In total, there were 117 injections and
control checks (PBST and recombinant AT14-012 IgG1) with an association time of 8
minutes and dissociation time of 8 minutes which led to a total run time of ~50 hours per
plate. Data is processed with SPRintX re (IBIS Technologies). B cell sub clone
RNA isolation, cDNA amplification and sequencing were executed as described
previously (Kwakkenbos et al., 2010).
Expression and analysis of the AT14-012 high affinity mutants
CHO1-KSV cell line was taken up in e for one week in CD CHO medium and
refreshed every 2 to 3 days. Cells were transiently expressed (adapted from ra et
al., 2015) with the designated single mutants (H40Y, Y112F, D116H and T29N),
combination double mutants (H40Y/Y112F and T29N) and combined quadruple
mutant (H40Y/Y112F/D116H/T29N) including WT 12 and control mutant
(G110D), which does not show CD9 binding. In short, 4.0 x 10^6 cells/ml (10ml) were
taken up in CD CHO media supplemented with 0.25%DMA (Sigma). Cultures were
added with 3.2ug/ml DNA (pXC39 vector expressing both heavy and light chains)
subsequently with PEImax (Sigma). After two days, a feed of 10 ml of fresh medium was
added. Medium was harvested after 7 days and IgG expression was quantified using the
IgG quantification ELISA (Jackson). Cell culture supernatants were tested for binding to
MelWBO cultured cells (see elsewhere) in FACS and in a similar manner using the same
SPR chip setup used for the AIMMprove method. Alignments were made using Seaview
software (Gouy et al., 2010).
RESULTS
Development of high affinity AT14-012 variants using SPR. As mentioned before, the
main question for development of AT14-012 antibody would be whether a higher affinity
variant (as comparable affinity to ALB6) would lead to platelet ation.
Alternatively, does AT14-012 target such a unique e in which the affinity would
not make any difference? Activation-induced cytidine ase, also known as AID, is
still expressed and active in the immortalized B cell repertoire. Expression of AID did
not result in c instability leading to growth arrest and cell death, as 63% of wells
that were seeded showed robust expansion enbos et al., 2010). Thus, AID is still
able to induce mutations randomly or ably at a mutational hotspot. One approach
to identify mutations which lead to a higher affinity antibody/antigen binding, would be
to sort single B cells that would bind scently labeled soluble CD9 protein and
compare it with the IgG expression. In short, we were not able to find any setup to have
soluble CD9 either in a single, tetramer (using streptavidin-PE) or multimerized form
(using PE labeled dextramers) to bind the 2H15 B cells (data not . This is
probably due to the cis-type binding of the 2H15 B cell receptor to CD9 expressed on the
surface of the B cell itself. ore, we used a similar setup to single cells sort the
original 2H15 B cells but now test the binding of the produced IgG to a recombinant
CD9-EC2 protein in SPR (similar CD9 protein as used for the AT14-012 ty
determination). The 2H15 single B cells are able to produce sufficient amounts of IgGs to
be tested especially if a mutation induces increased binding. Initially, we examined
optimal SPR settings using the bulk 2H15 cultured IgGs in supernatant and compared it
to increasing trations of recombinant AT14-012 (Figure 18A) as a l. After
validation of a proper ation and iation phase of the 2H15 IgGs, we employed
a setup in which the observed concentration and ity of the IgG in the B cell
supernatant could be related to the binding of recombinant purified AT14-012 at
properly determined IgG concentrations (see Figure 18A). The ratio related to the
binding to the anti-human Fc and the anti-CH1 showed a good ation between
concentration and stably produced antibody. The SPR curves of the anti-human Fc
increase in a linear manner whereas the SPR curves for the H1 increase in an
exponential manner. We were able to identify 13 clones with enhanced or altered binding
pattern after examination of eight 96 well plates (~800 clones) and ~400 hours of SPR
run time (raw data not shown). These clones were taken into culture and examined
again on a newly generated SPR chip (Figure 18B). Out of the initial 13 clones only 8
still showed significantly enhanced binding. There were 3 different groups designated in
which group 1 showed faster association and slower dissociation (clones 1D5, 1F5, 4H10,
10B9 and 10D1). Group 2 clones showed a faster association but also a faster
dissociation (clones 2D12, 4D4, 6E10 and 9E5) and group 3 did not show any difference
(clones 1C9, 2H10, 9A9 and 9D12). Enhanced binding of the high affinity clones was also
ed and detected on two different melanoma cell lines (data not . Again,
group 1 clones showed the best cell population shift on FACs. One single mutation in the
heavy chain of the clone attributes to the enhanced binding pattern in SPR and on cells
(see Figure 18C). Surprisingly, one clone 4D4 was the only clone to have light chain
mutations and no heavy chain mutations. We also examined a few clones that did show
IgG expression but no CD9 binding (referred to as “disPROVE”, clones 1E3, 1E4, 1E5,
1F12, 2A3 and 5B1). And, we included a few clones (1G2, 1G3, 1G4 and 1G5) that
showed a consensus binding pattern among all 800 analyzed clones. It was established
that these clones were most germline (Figure 18C). The most important mutations were
the H40Y (4x) and Y112F (1x) (group 1) and the D116H (2x) and T29N (1x) (group 2).
The L120V mutation (clone 4H10) as well as the S28N (clone 9E5), both located in the
light chain, were omitted because of other clones having the same mutation in the heavy
chain and also equal CD9 affinities.
Examination of the high affinity mutants and combination thereof in recombinant AT14-
012 background. The high affinity mutations were transiently expressed as single,
double or quadruple variants in CHO cells (see Figure 19A) for the positions and
alignments). WT AT14-012 and the mutant G110D, which resulted in a lack of CD9
g, were taken along as controls. The antibodies were assayed for integrity and
break down products on SDS-page and western blot. No obvious abnormalities could be
detected when the antibodies were ed with a rabbit anti-human IgG heavy/light
chain dy (data not shown). Production supernatant was used at a serial dilution on
melanoma cells to examine CD9 cell surface binding (Figure 19B). The G110D mutant,
which ed in a lack of CD9 binding, was genuinely interfering with the binding to
CD9. The T29N did not have a major impact on improved binding as observed for the B
cell supernatant screening. The D116H does show an expected improved binding. The
ation of these two group 2 mutations did not attribute to an even higher signal
compared to the single D116H binding. Again, this result explains the lack of impact the
T29N mutation has in inant form. Fortunately, the binding of the group 1
mutations was increased significantly better with the H40Y to have the most impact of
all single mutations. As for the double group 1 mutant (H40Y/Y112F) the effect became
even more enhanced. The quadruple variant did not show any beneficial effect over the
double group 1 mutant. In line with the binding n observed for WT AT14-012
(Figure 7A) also the high affinity variants show enhanced binding to melanoma cells as
compared to short term cultured healthy melanocytes (Figure 19C). An exact binding
profile was achieved using the CD9 SPR setup (Figure 19D). The contribution to the
higher binding was nicely ned by the overview of the association and iation
constants (Figure 19E). All single mutants, except for T29N, contributed to an enhanced
g affinity. The H40Y mutant showed a 100 times enhancement, as for Y112F 50
times and D116H 10 times. The combination group 1 mutant contributed to a 250 times
higher ty compared to WT 12 (~220pM). Surprisingly, the quadruple mutant
twice less high (~455pM) compared to the group 1 double mutant. This leads to an
dy with a comparable affinity range as ALB6. This mouse anti-CD9 antibody is
known to induce platelet aggregation and is used as a positive control in the platelet
aggregation assay. Now, we have comparable binding affinities to answer the question
whether the et aggregation is affinity or epitope related.
Affinity improved AT14-012 mutants do not ate platelets. In line with literature
we observed that the commercially ble anti CD9 antibody ALB6 antibody induces
the ation of platelets. In sharp contrast incubation of whole blood with the anti
CD9 antibody 12 does not cause ets to aggregate. Importantly, when the
high affinity mutants were tested in our platelet aggregation assay none of the AT14-012
affinity improved variants was able to induce the aggregation of platelets (Figure 20).
The ALB6 antibody was included in the assay as a positive control for aggregation of
platelets. The affinity of the double group 1 mutant is approximately the same as the
affinity of the ALB6 antibody (Figure 16C). This indicates that the affinity of AT14-012
is not linked to the absence of platelet aggregation, but instead the l characteristic
is the recognition of a unique epitope on CD9.
e 7 – IgG iso- and allotype
MATERIALS & METHODS
cing of the CD9 open reading frame from patient d tumour material and
cancer cell lines
In short, mRNA was isolated using Trizol reagent and cDNA amplification was
performed using random primers. CD9 was amplified by PCR using CD9 specific primers
described in Huang et al., 1998. CD9 sequences was analyzed from frozen cell pellets of
two short term cultured primary tumor al sources (AT14-012 derived) designated
Mel05.18 from a skin lesion and Mel06.07 from a brain lesion. Two other short term
cultured primary tumor material, both AT14-012 binding positive from other melanoma
patients were taken along as ls. Furthermore, melanoma cell lines MelBLM,
MelWBO, A375 and Jurkat T cell line (negative for CD9 binding) as well as AML cell
line HL-60 were used to examine the CD9 sequence. The B cell clone 2H15 and IgG3
anti-HRV B cell clone were also ed. Sequencing was carried out on the PCR
products itself (CD9-fw 5’-TGCATCTGTATCCAGCGCCA-3’and CD9-rev 5’-
CTCAGGGATGTAAGCTGACT-3’).
Sequencing of the IgG3 2H15 allotype
The IgG3 constant region of the 2H15 B cell clone was determined by isolation of RNA
using the Trizol method (Kwakkenbos et al., 2010). cDNA was made using random
primers. A PCR reaction was performed using CH1 forward (5’-
CACCAAGGGCCCATCGGTCTTC-3’) and CH3 reverse primers (5’-
CCCGGAGACAGG-3’). Primers were constructed based on the human IgG3
sequences found at the IMGT website
(http://www.imgt.org/IMGTrepertoire/Proteins/alleles/index.php?species=Homo%20sapie
ns&group=IGHC&gene=IGHG3). Sequencing was d out on the PCR products itself
(fw and rev) to determine the allotype according to Vidarsson et al., 2014.
cing of AT14-012 IgG specific heavy and light chains from the patient B cell
repertoire
The variable heavy and light chains of AT14-012 were amplified separately from cDNA
that was constructed from the isolated RNA pool of the total B cell repertoire of the
t of the time of B cell screening. RNA was isolated by the Trizol method and cDNA
was made as previously described (Kwakkenbos et al., 2010). First, to amplify the heavy
chain, a pre-amplification step was performed using a mix of two VH3 family specific
forward primers VH3-9L (5’-CCATGGAGTTGGGACTGAGC-3’) and VH3LB (5’-
CACCATGGARYTKKGRCTBHGC-3’) and IgG ic reverse s OCG1 (5’-
GTCCACCTTGGTGTTGCTGGGCTT-3’, OCG2 (5’-CTGCTGAGGGAGTAGAGTCC-3’)
and OCG3 (5’-GGTGTGCACGCCGCTGGTCAG-3’). The PCR product was harvested
from the DNA gel by the Qiagen gel extraction kit. A secondary amplification step was
performed to amplify AT14-012 specific sequences. Four different PCR reactions were
med using one AT14-012 specific heavy chain forward primer (5’-
GTGTCCAGTGTGAAGTGCAGG-3’) and 4 different reverse s AT14-012Hrev A
(5’-GGGATAATAACCACTCACGGC-3’), AT14-012Hrev B (5’-
GTAGGGATAATAACCACTCAC-3’), AT14-012Hrev C (5’-
GTCAAAGTAGGGATAATAAC-3’) and AT14-012Hrev D (5’-
CCAGTAGTCAAAGTAGGG-3’) that recognize the rearranged HCDR3 region in a
stepwise manner to cover the widest VDJ rearranged sequence. Hereby, framework 4 is
not sequenced. The final PCR products from A, B, C and D PCRs were all combined and
one single DNA mix was ligated into the pCR2.1 TA g vector (Thermo). There was
no need to perform the is for all reverse reactions separately because of the
stepwise annealing on the HCDR3 region. Hereby, we could identify by cing
which product was amplified by which reverse primer. The inserts were sequenced using
the generic M13 reverse and M13 forward primers. To amplify the 12 light chain,
a similar protocol was executed. The forward primers to amplify the VK4 family step
were VK4L-Fw-leader-ATG: 5’-ACCATGGTGTTGCAGACCCAG-3’ and VK4L 5’-
TYYCTSYTSCTYTGGATCTCTG-3’ and the reverse primer OCK 5’-
ACACTCTCCCCTGTTGAAGCTCTT-3’. For the second AT14-012 specific amplification
step the forward primer was Fw1-1412L 5’-CAGTCTCCAGACTCCCTGT-3’ s the
three LCDR3 specific reverse primers were AT14-012Lrev A (5’-
GGCCGAAGGTGGAAGGAGTAG-3’) AT14-012Lrev B (5’-
GTCCCTTGGCCGAAGGTGGAAG-3’) and AT14-012rev C (5’-
TGTCCCTTGGCCGAAGGTGG-3’). Again, the framework 4 of the light chain is not
resolved by this PCR method.
RESULTS
AT14-012 recognizes non-mutated CD9. To confirm that the epitope on CD9 recognized
by 12 is non-mutated, sequence analysis was performed on a panel of different
cell types. Tumor cells including melanoma cells derived from the original t as well
as the AT14-012/2H15 original B cells were subjected to . None of the cells tested
showed mutations in the AT14-012 epitope confirming that AT14-012 recognizes a wild
type sequence on CD9. Also, these data show that tive CD9 domain on expressed
on Bcl6/xL immortalized B cells is the wild type sequence.
The B cell derived 2H15/ AT14-012 antibody is of allotype IGHG3*16. The original
patient derived 12/2H15 B cell is of the IgG3 isotype. To determine the allotype of
the produced dy mRNA of the B cells was isolated and subjected to RT-PCR using
primers specific to the Fc region. The obtained sequence together with published data
(Vidarsson et al., 2014) reveal that the AT14-012 patient derived B cell clone is of
pe IGHG3*16 (Figure 21). The impact this IgG3 allotype has on the antibody that
AT14-012 is and should be is tly unknown. No published work shows the side by
side comparison of all allotypes in an effector function assay such as CDC.
The AT14-012 heavy and light chain sequences are able to be retrieved from the total B
cell repertoire
Our aim here was to investigate r the AT14-012 ce was present in the
patients’ total B cell repertoire. Using a PCR approach applying a pre-amplification step
on the variable heavy (VH3) and light chain (VK4) IgG family, we succeeded to acquire
proper AT14-012 sequences with the introduced hypermutations during a secondary
AT14-012 specific PCR (see materials and methods). The framework region 4 for both
chains was not able to be resolved due to the limitations of this approach. There were no
obvious additional or less hypermutations found (data not shown) in the heavy chain but
at position T109 of the light chain (IMGT numbering), we found that not all sequences
had the introduced hypermutation. The original germline ce contains a serine and
this hypermutation is rather conserved in its properties and should not have a major
impact on the overall structure or CD9 binding (not tested).
Example 8 – Combination with anti-PD1 antibodies
MATERIALS & METHODS
Generation of human immune system mice (van Lent, Methods Mol Biol, 2010)
Sublethally ated (350 cGy) neonatal (<1 wk old) NSG mice were injected
intrahepatically with human CD34+ CD38− hematopoietic itor cells. Mice that are
reconstituted well and produce human immune cells are ined to be suitable for
aft experiments.
RESULTS
Strong inhibition of in vivo melanoma growth by AT14-012 in combination with anti
PD1. dies blocking the PD1-PDL1 axis, in particular those binding PD1, are now
widely used to treat a wide variety of late stage cancer patients. Response rates differ
per type of cancer, in general only a minor fraction of patients respond well to the
treatment. Many clinical trials are being med to test anti PD1 antibodies in
combination with new or registered compounds. We tested the efficacy of AT14-012 in
eradication of tumor cells in the presence of Nivolumab (Opdivo, Bristol-Myers Squibb)
in a humane immune system (HIS) mouse model. HIS mice are generated by grafting
human hematopoietic stem cells in NSG mice (van Lent, Methods Mol Biol, 2010). After
an immune system had formed, as characterized by the presence of human immune in
the ation, the mice received a subcutaneous graft of luciferase expressing
melanoma cells. Tumors were allowed to grow for 4 weeks to about 100 mm3 in size
before the start of ent. Mice are randomized over 4 different treatment groups
receiving intraperitoneal antibodies injections twice per week.
AT10-002 (15 mg/kg) + PBSAT10-002 (15mg/kg) + Nivolumab (2.5 mg/kg)
12 (15 mg/kg) + PBSAT14-012 (15mg/kg) + Nivolumab (2.5 mg/kg)
As determined by luciferase imaging mice receiving the AT14-012 antibody alone showed
delayed tumor growth as compared to the mice in the AT10-002 (anti Influenza) group
(Figure 22A, B). Interestingly, when the administration of AT14-012 was combined with
the anti-PD1 antibody the inhibition of tumor growth was strongly enhanced in
comparison the other antibody regimen (Figure 22A). Calculating the tumor size at the
day of sacrifice in relation to the size at the start of the ent revealed that the
ation AT14-012+Nivolumab reduced the size of the tumor with almost 70%
(Figure 22B). These data clearly show that combining the AT14-012 anti-CD9 dy
with a T-cell stimulating antibody holds great potential in eradication of tumor cells.
Table 1. Antibody AT14-012
Heavy chain CDR1 DYAMH
Heavy chain CDR2 GISWNSGSIVYADSVKG
Heavy chain CDR3 AVSGYYPYFDY
Light chain CDR1 KSSQSVLYSSNNKNYLG
Light chain CDR2 WASTRES
Light chain CDR3 P
Heavy chain EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ
APGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLY
LQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGILVTVSS
Light chain DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGW
YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK
Heavy chain CDR1 gat tat gcc atg cac
Heavy chain CDR2 ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag
Heavy chain CDR3 gcc gtg agt ggt tat tat ccc tac ttt gac tac
Light chain CDR1 aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta
Light chain CDR2 tgg gca tct acc cgg gaa tcc
Light chain CDR3 cag caa tat tat act act cct
Heavy chain gaa gtg cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc agg
tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt gat gat tat gcc
atg cac tgg gtc cgg caa gct cca ggg aag ggc ctg gag tgg gtc tca
ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag
ggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa
ctg aac agt ctg aga gct gag gac acg gcc ttc tat tac tgt gca aaa
gcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag gga att ttg
gtc acc gtc tcc tca
Light chain gac atc gtg atg acc cag tct cca gac tcc ctg tct gtg tct ctg ggc gag
agg gcc acc atc aac tgc aag tcc agc cag agt gtt tta tac agc tcc
aac aat aag aac tac tta ggt tgg tac cag cag aaa cca gga cag cct
cct aag ctg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc cct gac
cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc agc agc
ctg cag gct gaa gat gtg gca gtt tat tac tgt cag caa tat tat act act
cct tcc acc ttc ggc caa ggg aca cga ctg gag att aaa
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Claims (60)
1.
An antibody or functional part or functional equivalent thereof, that comprises: - a heavy chain CDR1 sequence DYAMH or DYAMY; and - a heavy chain CDR2 sequence GISWNSGSIVYADSVKG; and 5 - a heavy chain CDR3 sequence AVSGYYPYFDY or AVSGYFPYFDY or AVSGYYPYFHY or AVSGYFPYFHY; and - a light chain CDR1 sequence KSSQSVLYSSNNKNYLG; and - a light chain CDR2 ce WASTRES or WASIRES; and - a light chain CDR3 sequence QQYYTTP. 10 2. An dy or functional part or functional lent according to claim 1, that comprises: - a heavy chain CDR1 sequence DYAMH; and - a heavy chain CDR2 sequence GISWNSGSIVYADSVKG; and - a heavy chain CDR3 sequence AVSGYYPYFDY; and 15 - a light chain CDR1 ce KSSQSVLYSSNNKNYLG; and - a light chain CDR2 sequence WASTRES; and - a light chain CDR3 sequence QQYYTTP.
3. An antibody or functional part or functional equivalent ing to claim 1 or 2, comprising a heavy chain variable region sequence having at least 80% sequence identity 20 with the sequence EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSG SIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGI LVTVSS.
4. An antibody or functional part or functional equivalent according to any one of 25 claims 1-3, comprising a light chain le region sequence having at least 80% sequence identity with the sequence DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK.
5. An antibody or functional part or functional equivalent according to any one of 30 claims 1-4 comprising: a heavy chain variable region sequence EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISW YADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDY WGQGILVTVSS, or 35 EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWN SGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYW GQGILVTVSS or EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISW NSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDY 5 WGQGILVTVSS or EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISW NSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHY WGQGILVTVSS or EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWN 10 SGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYW GQGILVTVSS or EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWN SGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYW GQGILVTVSS or 15 EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWN SGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHY WGQGILVTVSS and/or a light chain variable region sequence SPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIY 20 WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLE
6. An antibody or functional part or functional equivalent according to any one of claims 1-5, that is a human antibody or functional part or functional equivalent f.
7. An antibody or functional part or functional equivalent according to any one of 25 claims 1-6, that is able to bind melanoma cells, colon carcinoma cells, pancreas carcinoma cells and esophagus carcinoma cells.
8. An dy or functional part or functional lent according to any one of claims 1-7, n said antibody is of the IgG isotype.
9. An antibody or functional part or functional equivalent according to claim 8, 30 wherein the antibody is of the IgG1 or IgG3 isotype.
10. An antibody or functional part or functional lent according to claim 8 or 9 comprising an arginine in the IgG Fc tail at amino acid position 345 (EU numbering).
11. An antibody or onal part or functional equivalent according to any one of claims 1-10, that is coupled to another compound.
12. An antibody or functional part or functional equivalent according to claim 11, n said other compound is a detectable label, a chemotherapeutic drug, a toxic moiety, an immunomodulatory molecule, another CD9-specific binding compound, or a ctive compound. 5
13. An isolated, synthetic or recombinant nucleic acid molecule with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least the heavy chain CDR 1-3 and the light chain CDR 1-3 sequences of an antibody or functional part or functional equivalent according to any one of claims 1-12.
14. A nucleic acid molecule or functional equivalent ing to claim 13, that 10 encodes at least the heavy chain variable region sequence and/or the light chain variable region sequence of an antibody or functional part or onal equivalent according to any one of claims 1-12.
15. A nucleic acid molecule or functional equivalent according to claim 13 or 14, comprising a sequence that has at least 80% sequence identity with a ce selected 15 from the group ting of: - gat tat gcc atg cac; and - ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc; and - gcc gtg agt ggt tat tat ccc tac ttt gac tac; and - aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt; and 20 - tgg gca tct acc cgg gaa tcc; and - cag caa tat tat act act cct.
16. A nucleic acid molecule or functional equivalent according to any one of claims 13- 15, comprising a sequence that has at least 80% sequence identity with the ce gaa gtg cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct 25 gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa gct cca ggg aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa ctg aac agt ctg aga gct gag gac acg gcc ttc tat tac tgt gca aaa gcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag gga att ttg gtc acc gtc tcc tca, and/or comprising a sequence that has at least 80% sequence identity with the sequence 30 gac atc gtg atg acc cag tct cca gac tcc ctg tct gtg tct ctg ggc gag agg gcc acc atc aac tgc aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt tgg tac cag cag aaa cca gga cag cct cct aag ctg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc agc agc ctg cag gct gaa gat gtg gca gtt tat tac tgt cag caa tat tat act act cct tcc acc ttc ggc caa ggg aca cga ctg gag att aaa.
17. A nucleic acid le or functional equivalent thereof, ng an antibody or functional part or onal equivalent according to any one of claims 1-12.
18. A nucleic acid molecule according to any one of claims 13-17, that comprises cDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), or a A helix. 5
19. A nucleic acid molecule according to any one of claims 13-18, that is codon zed for expression in a non-human host cell.
20. A vector comprising a nucleic acid molecule or functional equivalent according to any one of claims 13-19.
21. An isolated or recombinant cell, or a non-human animal, comprising a nucleic 10 acid molecule or onal equivalent according to any one of claims 13-19 or a vector according to claim 20.
22. A ition comprising an antibody or functional part or functional equivalent according to any one of claims 1-12, or a nucleic acid molecule or functional equivalent according to any one of claims 13-19, or a vector according to claim 20, or a cell according 15 to claim 21.
23. A composition according to claim 22, wherein said composition is a pharmaceutical composition that also ses a pharmaceutically able carrier, diluent or excipient.
24. A kit of parts comprising an antibody or functional part or functional equivalent 20 according to any one of claims 1-12, a c acid molecule or onal equivalent according to any one of claims 13-19, a vector according to claim 20 or a cell according to claim 21 and a therapeutic agent useful in the treatment and/or prevention of a disorder associated with CD9-expressing cells.
25. The kit of parts according to claim 24 wherein the disorder is a CD9 positive 25 .
26. The kit of parts according to claim 24 or 25, wherein said agent is an agent capable of stimulating C3 convertase formation or capable of counteracting inhibition of C3 convertase formation.
27. The kit of parts according to claim 26, wherein said agent is a CD55 blocking 30 antibody, a CD46 blocking antibody or a CD59 blocking antibody.
28. The kit of parts according to claim 27, wherein the agent is a CD55 blocking antibody.
29. The kit of parts according to claim 24 or 25, wherein said agent is a blocking antibody specific for a co-inhibitory T cell molecule.
30. The kit of parts according to claim 29, wherein said antibody is selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, an anti-TIM3 dy, an anti-LAG3 antibody, an anti-CD276 antibody, an anti-CD272 antibody, an anti-KIR 5 antibody, an anti-A2AR antibody, an anti-VISTA antibody and an anti-IDO antibody.
31. The kit of parts according to claim 30, wherein said antibody is a PD1 blocking antibody or a PDL1 blocking antibody.
32. A use of an antibody or functional part or functional equivalent according to any one of claims 1-12, or a c acid molecule or onal equivalent according to any 10 one of claims 13-19, or a vector according to claim 20, or a cell according to claim 21, in the manufacture of a medicament or lactic agent.
33. A use of an antibody or functional part or functional equivalent according to any one of claims 1-12 in the manufacture of a medicament for the diagnosis of a disorder associated with pressing cells. 15
34. A use according to claim 33, wherein said disorder is selected from the group consisting of CD9 positive cancer, osteoporosis, arthritis, lung inflammation, COPD, colitis, and a disorder associated with innate lymphoid cells.
35. A use according to claim 34, wherein said CD9 ve cancer is selected from the group consisting of melanoma, colorectal cancer, pancreatic cancer, esophageal cancer, 20 lung cancer, breast cancer, ovarian cancer, h cancer, us cell oma, AML, multiple myeloma, gastric cancer, liver cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma, bladder cancer and small cell lung cancer. 25
36. Use of an antibody or onal part or functional equivalent according to any one of claims 1-12 for determining whether a sample ses CD9-expressing cells.
37. Use ing to claim 36, for determining whether a sample comprises CD9- expressing tumor cells.
38. A method for ining whether pressing cells are present in a sample 30 comprising: - contacting said sample with an antibody or functional part or functional equivalent according to any one of claims 1-12, and - allowing said antibody or onal part or functional equivalent to bind CD9- expressing cells, if present, and 35 - determining whether or not CD9-expressing cells are bound to said antibody or functional part or functional equivalent, thereby determining whether or not CD9- expressing cells are present in said sample.
39. A method according to claim 38, wherein said CD9 expressing cells are CD9 positive tumor cells. 5
40. A method for producing an antibody or functional part or functional equivalent according to any one of claims 1-12, the method comprising ing a cell with a nucleic acid molecule or functional equivalent or a vector according to any one of claims 13-20, and allowing said cell to translate said nucleic acid le or functional equivalent or vector, thereby producing said antibody or functional part or functional 10 equivalent according to any one of claims 1-12.
41. The method according to claim 40, n the method further comprises harvesting, purifying and/or isolating said antibody or functional part or functional equivalent according to any one of claims 1-12.
42. Use of an antibody or functional part or functional equivalent according to any 15 one of claims 1-12, or a c acid molecule or functional equivalent according to any one of claims 13-19, or a vector according to claim 20, or a cell according to claim 21, or a composition according to claim 22 or 23, for the manufacture of a medicament for at least in part treating and/or preventing a disorder associated with CD9-expressing cells.
43. A use ing to claim 42, wherein said er is selected from the group 20 ting of CD9 positive cancer, orosis, tis, lung inflammation, COPD, colitis, and a disorder associated with innate lymphoid cells.
44. A use according to claim 43, wherein said CD9 positive cancer is selected from the group consisting of melanoma, colorectal cancer, atic cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, squamous cell carcinoma, 25 AML, multiple myeloma, gastric cancer, liver cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma, bladder cancer and small cell lung cancer.
45. A use according to any one of claims 42-44, whereby said antibody or functional 30 part or functional equivalent or c acid le or functional equivalent or vector or cell is ed with a therapeutic agent useful in the treatment and/or prevention of a disorder associated with pressing cells.
46. A use according to claim 45, wherein the disorder is a CD9 positive cancer.
47. A use according to claim 45 or 46, wherein said agent is an agent capable of stimulating C3 convertase formation or capable of counteracting inhibition of C3 convertase formation.
48. A use according to claim 47, wherein said agent is a CD55 blocking antibody, a 5 CD46 blocking antibody or a CD59 blocking antibody.
49. A use according to claim 45 or 46, wherein said agent is a blocking antibody specific for a co-inhibitory T cell molecule.
50. A use according to claim 49, wherein said antibody is ed from the group consisting of an anti-CTLA4 antibody, an D-1 antibody, an anti-PD-L1 antibody, 10 an anti-PD-L2 antibody, an IRPα antibody, an anti-TIM3 dy, an anti-LAG3 antibody, an anti-CD276 antibody, an anti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, an anti-VISTA antibody and an anti-IDO antibody.
51. A use according to claim 50, wherein said antibody is a PD1 blocking antibody or a PDL1 blocking antibody. 15
52. An ex vivo method for ining whether an individual is suffering from a CD9-positive , the method comprising: - contacting tumor cells in a sample from said individual with an antibody or functional part or functional lent according to any one of claims 1-12, - allowing said antibody or functional part or functional equivalent to bind CD9- 20 expressing cells, if present, and - determining whether or not pressing cells are bound to said antibody or functional part or functional equivalent, thereby determining r or not said individual is suffering from a CD9-positive cancer.
53. An antibody or functional part or functional equivalent according to any one of 25 claims 1-12 substantially as herein described and with reference to any example f.
54. A nucleic acid le or functional equivalent according to any one of claims 13- 19 substantially as herein described and with reference to any example thereof.
55. A vector as d in claim 20 substantially as herein described and with reference to any example thereof. 30
56. An isolated or recombinant cell, or a non-human animal as claimed in claim 21 substantially as herein described and with reference to any example thereof.
57. A composition as claimed in claim 22 or 23 substantially as herein bed and with reference to any example thereof.
58. A kit of parts as claimed in any one of claims 24-31 substantially as herein 35 described and with reference to any example thereof.
59. A use as claimed in any one of claims 32-37 and 42-51 substantially as herein described and with reference to any example thereof.
60. A method as d in any one of claims 38-41 and 52 substantially as herein described and with reference to any example thereof. wo
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16150698.5 | 2016-01-08 | ||
EP16150698 | 2016-01-08 | ||
PCT/NL2017/050003 WO2017119811A1 (en) | 2016-01-08 | 2017-01-06 | Therapeutic anti-cd9 antibody |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ744187A NZ744187A (en) | 2021-08-27 |
NZ744187B2 true NZ744187B2 (en) | 2021-11-30 |
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