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SENESCENT CELL-ASSOCIATED ANTIGEN-BINDING DOMAINS,
ANTCCBIES AND CHIMERIC ANTIGEN RECEPTORS COMPRISING THE
SAME, AND USES THEREOF
FIELD OF INVENTION
The invention relates to DEP1-binding domains and DPP4-binding domains, as
well as
antibodies and chimeric antigen receptors (CAR) comprising the same. Also
disclosed
are uses and methods for treating, preventing or alleviating senescence-
related diseases
or disorders, or for depleting and/or killing senescent cells.
BACKGROUND OF INVENTION
Cellular senescence is an evolutionarily conserved state of stable replicative
arrest
induced by several pro-ageing stressors, including telomere attrition,
oxidative stress,
DNA damage and oncogene activation. Cellular senescence is associated with
apoptosis
resistance, and results in secretion of a broad repertoire of cytokines,
chemokines, growth
factors, matrix remodeling proteases: the so-called senescence-associated
secretory
phenotype (SASP). This cellular state also promotes proliferation and tissue
deterioration.
Conversely, senescence is also anti-proliferative, and may be requisite for
optimal
cutaneous wound healing. Therefore, cellular senescence is an example of
antagonistic
pleiotropy in which natural selection favors processes that are beneficial
early in life, even
if they cause harmful effects later in post-reproduction life.
Since its discovery, senescence, once defined as the limited replicative
capacity of
primary human fibroblasts, now serves as a key player driving organismal aging
via
exhaustion of tissue repair capacity. Several human pathologies have been
associated with
detrimental effects of senescence such as lung fibrosis, type 2 diabetes,
obesity,
osteoarthritis, ocular diseases, Alzheimer's and Parkinson's disease (Munoz-
Espin and
Serrano, 2014. Nat Rev Mol Cell Biol. 2014 Jul; 15(7):482-96). Therapeutic
strategies so
far to balance these pathologies related to accumulation of senescent cells
are dependent
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on direct elimination of senescent cells based on their intrinsic properties
(e.g., their
apoptotic resistance or P53 dependence) (Yosef et al., 2016. Nat Commun.
7:11190;
Chang et al., 2016. Nat Med. 22(1):78-83; Baar et al., 2017. Cell. 169(1):132-
147).
Although these first-generation senolytic approaches serve as a proof-of-
principle for
drug discovery targeting senescence, they are limited by their observed toxic
side effects.
While the role of senescence and the contribution of senescent cells are
increasingly
recognized in the context of aging and a variety of disease states, relatively
little is known
regarding the influences of senescent cells in normal lung growth and agingper
se, or in
the induction or progression of lung diseases across the age spectrum, such as
bronchopulmonary dysplasia, asthma, chronic obstructive pulmonary disease, or
pulmonary fibrosis. However, crucial evidences have been recently provided by
several
groups that cellular senescence contributes to lung ageing (Hashimoto et al.,
2016. JCI
Insight. 1(12):e87732; Lehmann et al., 2017. Eur Respir J. 50(2):1602367;
Schafer et
al., 2017. Nat Commun. 8:14532).
Among lung diseases, idiopathic pulmonary fibrosis (IPF) is a typical example
of an
ageing disease characterized by a progressive destruction of lung parenchyma
and
interstitial remodeling, leading to IPF symptoms (i.e., chronic shortness of
breath, cough,
fatigue and weight loss) and resulting in dramatic truncation of healthspan
and lifespan.
The potential to blunt lung disease by targeting senescent cells using a novel
class of
drugs called "senolytics" is currently discussed. Indeed, two studies by
Lehmann et al.
and Schafer et al. suggest that cellular senescence is a salient feature of
lung fibrosis, and
that targeting/elimination of these cells could be beneficial. In particular,
they show that
cellular senescence markers such as SAPG, P21, P16INK4a and DNA damage
response
are detectable within IPF patients, as well as in experimental models of lung
fibrosis.
They further demonstrate that senescent cell elimination rejuvenates pulmonary
health in
aged mice. However, it is unclear whether and how senescent cells regulate IPF
in humans
or if their removal may be an efficacious intervention strategy.
Although promising, it cannot be excluded that senolytic drugs could be
detrimental in
IPF patients. Indeed, senolytic drug treatment may result in massive
epithelial cell
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depletion by apoptosis, which could trigger diffuse alveolar damage and acute
exacerbation, since the regenerative capacity of epithelial cells in IPF
patients is impaired.
There remains thus a need for alternative strategies for depleting senescent
cells and
improving health and lung functions of IPF patients.
The Inventors have developed such alternative strategy, by potentiating an
immune
response against senescent cells in a way that would lead to their clearance
from lung
tissue. They provide herein a new association of two cell surface markers,
DPP4 and
DEP1, which are targeted to detect and deplete senescent cells in the lung.
Senescent cells
are immunogenic in nature and are subject to immune surveillance mechanisms.
Dipeptidyl peptidase 4 (DPP4, also named CD26) is a cell surface protease with
a wide
range of biological functions. As a serine-type protease, DPP4 preferentially
cleaves off
substrates with proline and alanine at the penultimate position. Expression of
DPP4 is
widespread throughout the body. Interestingly, DPP4 has been identified as a
senescent
cell surface targetable protein, functionally required for fibroblast
activation and tissue
fibrosis (Kyoung et al., 2017. Genes Dev. 31(15):1529-1534).
Density Enhanced Protein Tyrosine Phosphatase (DEP1, also named CD148, HPTP-
eta,
or PTP receptor type J (PTPRJ)) is an enzyme that removes phosphate groups
covalently
attached to tyrosine residues in proteins. DEP1 is highly expressed on both
hematopoietic
and nonhematopoietic cells, including lung cells. It has been shown that DEP1
can
directly interact with and dephosphorylate the regulatory subunit of PI3K
(p85) (Tsuboi
et al., 2008. Biochem J. 413(1):193-200) and that hyperactivation of PI3K/Akt
plays an
important role in the profibrotic phenotype of IPF-derived lung fibroblasts by
promoting
cell proliferation and migration and myofibroblast differentiation (Kral et
al., 2016. Sci
Rep. 6:23034; Nho et al., 2014. PLoS One. 9(4):e94616).
The Inventors herein provide antibodies, bispecific antibodies, chimeric
antigen receptors
(CARs) and bispecific CARs, including immune cell populations expressing said
CARs
directed specifically against senescent cells for treatment and prophylaxis of
age-related
diseases and disorders, and other diseases and disorders associated or
exacerbated by the
presence of senescent cells, such as, for example, pulmonary fibrosis. The
antibodies and
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CARs described herein are specific for at least one senescent cell-associated
antigen (e.g.,
DEP1 and/or DPP4), and induce the clearance (i.e., removal, elimination,
destruction) of
senescent cells. Said clearance may, for example, be mediated by antibody-
dependent
cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) or both.
SUMMARY
The present invention relates to a DPP4-binding domain, comprising a
combination of
three heavy chain variable region (HCVR)'s complementary-determining regions
(CDRs) and three light chain variable region (LCVR)'s CDRs, said combination
being as
defined in Table 3.
In one embodiment, the DPP4-binding domain comprises:
a) a HCVR which comprises the following three CDRs:
VH-CDR1 selected from the group consisting of SEQ ID NO: 109 and 108;
VH-CDR2 selected from the group consisting of SEQ ID NO: 122 and 127;
VH-CDR3 selected from the group consisting of SEQ ID NO: 139 and 138;
b) a LCVR which comprises the following three CDRs:
VL-CDR1 selected from the group consisting of SEQ ID NO: 148 and 147;
VL-CDR2 selected from the group consisting of SEQ ID NO: 160 and 159;
VL-CDR3 selected from the group consisting of SEQ ID NO: 172 and 171.
In one embodiment, the DPP4-binding domain is selected from the group
consisting of:
i. a DPP4-binding domain comprising a VH-CDR1 with SEQ ID NO: 109, a VH-
CDR2 with SEQ ID NO: 122, a VH-CDR3 with SEQ ID NO: 139, a VL-CDR1 with
SEQ ID NO: 148, a VL-CDR2 with SEQ ID NO: 160 and a VL-CDR3 with
SEQ ID NO: 172; and
ii. a DPP4-binding domain comprising a VH-CDR1 with SEQ ID NO: 108, a VH-
CDR2 with SEQ ID NO: 127, a VH-CDR3 with SEQ ID NO: 138, a VL-CDR1 with
SEQ ID NO: 147, a VL-CDR2 with SEQ ID NO: 159 and a VL-CDR3 with
SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain is selected from the group
consisting of:
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i. a
DPP4-binding domain comprising a HCVR with a sequence sharing at least 80%
of sequence identity with the non-CDR regions of SEQ ID NO: 185 and a LCVR
with
a sequence sharing at least 80% of sequence identity with the non-CDR regions
of
SEQ ID NO: 213; and
5 ii. a DPP4-binding domain comprising a HCVR with a sequence sharing at
least 80%
of sequence identity with the non-CDR regions of SEQ ID NO: 197 and a LCVR
with
a sequence sharing at least 80% of sequence identity with the non-CDR regions
of
SEQ ID NO: 212.
The present invention also relates to a DEP1-binding domain, comprising a
combination
of three heavy chain variable region (HCVR)'s complementary-determining
regions
(CDRs) and three light chain variable region (LCVR)'s CDRs, said combination
being as
defined in Table 1.
In one embodiment, the DEP1-binding domain comprises:
a) a HCVR which comprises the following three CDRs:
VH-CDR1 selected from the group consisting of SEQ ID NO: 10, 11 and 5;
VH-CDR2 selected from the group consisting of SEQ ID NO: 21, 25 and 12;
VH-CDR3 selected from the group consisting of SEQ ID NO: 30, 32 and 29;
b) a LCVR which comprises the following three CDRs:
VL-CDR1 selected from the group consisting of SEQ ID NO: 37, 38 and 33;
VL-CDR2 selected from the group consisting of SEQ ID NO: 44, 46 and 40;
VL-CDR3 selected from the group consisting of SEQ ID NO: 53, 52 and 49.
In one embodiment, the DEP1-binding domain is selected from the group
consisting of:
i. a DEP1-binding domain comprising a VH-CDR1 with SEQ ID NO: 10, a VH-
CDR2 with SEQ ID NO: 21, a VH-CDR3 with SEQ ID NO: 30, a VL-CDR1 with
SEQ ID NO: 37, a VL-CDR2 with SEQ ID NO: 44 and a VL-CDR3 with
SEQ ID NO: 53;
ii. a DEP1-binding domain comprising a VH-CDR1 with SEQ ID NO: 11, a VH-
CDR2 with SEQ ID NO: 25, a VH-CDR3 with SEQ ID NO: 32, a VL-CDR1 with
SEQ ID NO: 38, a VL-CDR2 with SEQ ID NO: 46 and a VL-CDR3 with
SEQ ID NO: 52; and
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iii. a DEP1-binding domain comprising a VH-CDR1 with SEQ ID NO: 5, a VH-CDR2
with SEQ ID NO: 12, a VH-CDR3 with SEQ ID NO: 29, a VL-CDR1 with
SEQ ID NO: 33, a VL-CDR2 with SEQ ID NO: 40 and a VL-CDR3 with
SEQ ID NO: 49.
In one embodiment, the DEP1-binding domain is selected from the group
consisting of:
i. a DEP1-binding domain comprising a HCVR with a sequence sharing at
least 80%
of sequence identity with the non-CDR regions of SEQ ID NO: 68 and a LCVR with
a sequence sharing at least 80% of sequence identity with the non-CDR regions
of
SEQ ID NO: 89;
ii. a DEP1-binding domain comprising a HCVR with a sequence sharing at least
80%
of sequence identity with the non-CDR regions of SEQ ID NO: 72 and a LCVR with
a sequence sharing at least 80% of sequence identity with the non-CDR regions
of
SEQ ID NO: 93; and
iii. a DEP1-binding domain comprising a HCVR with a sequence sharing at least
80%
of sequence identity with the non-CDR regions of SEQ ID NO: 58 and a LCVR with
a sequence sharing at least 80% of sequence identity with the non-CDR regions
of
SEQ ID NO: 78.
The present invention also relates to an isolated antibody or antigen-binding
fragment
thereof comprising the DPP4-binding domain or the DEP1-binding domain of the
invention.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
is a
bispecific antibody comprising the DPP4-binding domain and the DEP1-binding
domain
of the invention.
The present invention also relates to a chimeric antigen receptor (CAR)
comprising:
a. at least one extracellular binding domain, comprising at least one DPP4-
binding
domain and/or at least one DEP1-binding domain of the invention,
b. an extracellular spacer domain,
c. a transmembrane domain,
d. optionally, at least one costimulatory domain, and
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e. at least one intracellular signaling domain.
The present invention also relates to an immune cell engineered to express the
CAR of
the invention at its surface.
The present invention also relates to a population of immune cells, comprising
a plurality
of immune cells of the invention.
In one embodiment, the population of immune cells of the invention comprises:
a) a plurality of immune cells of the invention, engineered to express a CAR
comprising at least one DPP4-binding domain of the invention at its surface;
and a
plurality of immune cells of the invention, engineered to express a CAR
comprising at
least one DEP1-binding domain of the invention at its surface; or
b) a plurality of immune cells of the invention, engineered to express a CAR
comprising at least one DPP4-binding domain of the invention and a CAR
comprising
at least one DEP1-binding domain of the invention, at its surface; or
c) a plurality of immune cells of the invention, engineered to express a CAR
comprising at least one DPP4-binding domain of the invention and at least one
DEP1-
binding domain of the invention at its surface.
The present invention also relates to a composition comprising:
- the isolated antibody or antigen-binding fragment thereof of the
invention,
- the immune cell of the invention, and/or
- the population of immune cells of the invention.
In one embodiment, the composition of the invention is a pharmaceutical
composition
and further comprising at least one pharmaceutically acceptable excipient.
In one embodiment, the composition of the invention is for use as a drug.
In one embodiment, the composition of the invention is for use in treating,
preventing or
alleviating a senescence-related disease or disorder, preferably selected from
the group
consisting of fibrotic diseases, premalignant disorders, inflammatory diseases
and
cancers.
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In one embodiment, the senescence-related disease or disorder is a fibrotic
disease,
preferably a pulmonary fibrotic disease.
In one embodiment, the composition of the invention is for use in depleting
and/or killing
senescent cells.
DEFINITIONS
"A", "an" and "the" are intended to include both singular and plural forms,
unless the
context clearly indicates otherwise.
"About", preceding a figure encompasses plus or minus 10%, or less, of the
value of said
figure. It is to be understood that the value to which the term "about" refers
is itself also
specifically, and preferably, disclosed.
"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-
mediated
cytotoxicity induced in an antibody-dependent manner when the Fc region of
said
antibody bound to its antigen binds to the Fc receptor on effector cells such
as natural
killer cells, macrophages, neutrophils, eosinophils and mononuclear cells
(e.g., peripheral
blood mononuclear cells), thereby leading to lysis of the target cell. ADCC
can be
measured using assays that are known and available in the art (e.g., Clynes et
al., 1998.
Proc Natl Acad Sci USA. 95(2):652-6).
"Antibody-dependent cell-mediated phagocytosis" or "ADCP" or "opsonisation"
refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g.,
phagocytes,
macrophages) that express Fc receptors (FcRs) recognize antibody bound on a
target cell
and induce phagocytosis of the target cell. ADCP can be measured using assays
that are
known and available in the art (e.g., Clynes et al., 1998. Proc Natl Acad Sci
U S A.
95(2):652-6).
"Adnectins", also known as monobodies, is well known in the art and refer to
proteins
designed to bind with high affinity and specificity to antigens. They belong
to the class
of molecules collectively called "antibody mimetics".
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"Allogeneic" refers to a graft derived from a different animal of the same
species.
"Alphabody" that may also be referred to as Cell-Penetrating Alphabodies,
refer to a type
of antibody mimetics consisting of small 10 kDa proteins engineered to bind to
a variety
of antigens. Alphabodies are able to reach and bind to intracellular protein
targets.
.. "Affibodies" are well-known in the art and refer to affinity proteins based
on a 58 amino
acid residue protein domain, derived from one of the IgG binding domain of
staphylococcal protein A (Frejd & Kim, 2017. Exp Mol Med. 49(3):e306; Patent
US5,831,012).
"Affilins" are well known in the art and refer to artificial proteins designed
to selectively
bind antigens. They resemble antibodies in their affinity and specificity to
antigens but
not in structure which makes them a type of antibody mimetic "Affinity" and
"avidity"
are well-known in the art and are used to defined the strength of an antibody-
antigen
complex. Affinity measures the strength of interaction between an epitope and
an antigen
binding site on an antibody. It may be expressed by an affinity constant KA or
by a
dissociation constant KD. Avidity (or functional affinity) gives a measure of
the overall
strength of an antibody-antigen complex. It may depend on different
parameters,
including in particular the affinity of the antibody or antigen-binding
fragment thereof for
an epitope, (ii) the valency of both the antibody and the antigen and (iii)
structural
arrangement of the parts that interact. Affinities of antibodies or antigen-
binding fragment
thereof can be readily determined using conventional techniques, for example,
those
described by Scatchard, 1949. Ann NY Acad Sci. 51:660-672. Binding properties
of an
antibody or antigen-binding fragment thereof to antigens, cells or tissues may
generally
be determined and assessed using immunodetection methods including, for
example,
ELISA, immunofluorescence-based assays, such as immuno-histochemistry (IHC)
and/or
fluorescence-activated cell sorting (FACS) or by surface plasmon resonance
(SPR, e.g.,
using BIAcore ).
"Antibody" and "immunoglobulin" may be used interchangeably and refer to a
protein
having a combination of two heavy and two light chains whether or not it
possesses any
relevant specific immunoreactivity. "Antibodies" refers to such assemblies
which have
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significant known specific immunoreactive activity to an antigen of interest
(e.g., human
DEP1 and/or DPP4). The term "anti-DEP1 antibodies or anti-DPP4 antibodies" is
used
herein to refer to antibodies which exhibit immunological specificity for
human DEP1
antigen or human DPP4 antigens, respectively. As explained elsewhere herein,
5 .. "specificity" for human DEP1 does not exclude cross-reaction with species
homologues
of human DEP1, such as, for example, with simian DEP1, and "specificity" for
human
DPP4 does not exclude cross-reaction with species homologues of human DPP4
such as,
for example, with simian DPP4.
Antibodies and immunoglobulins comprise light and heavy chains, with or
without an
10 interchain covalent linkage between them. Basic immunoglobulin
structures in vertebrate
systems are relatively well understood. The generic term "immunoglobulin"
comprises
five distinct classes of antibody that can be distinguished biochemically.
Although the
following discussion will generally be directed to the IgG class of
immunoglobulin
molecules, all five classes of antibodies are within the scope of the present
invention.
With regard to IgG, immunoglobulins comprise two identical light polypeptide
chains of
molecular weight of about 23 kDa, and two identical heavy chains of molecular
weight
of about 53-70 kDa. The four chains are joined by disulfide bonds in a "Y"
configuration
wherein the light chains bracket the heavy chains starting at the mouth of the
"Y" and
continuing through the variable region. The light chains of an antibody are
classified as
either kappa (K) or lambda (k). Each heavy chain class may be bonded with
either a K or
k light chain. In general, the light and heavy chains are covalently bonded to
each other,
and the "tail" regions of the two heavy chains are bonded to each other by
covalent
disulfide linkages or non-covalent linkages when the immunoglobulins are
generated
either by hybridomas, B cells or genetically engineered host cells. In the
heavy chain, the
amino acid sequences run from an N-terminus at the forked ends of the Y
configuration
to the C-terminus at the bottom of each chain. Those skilled in the art will
appreciate that
heavy chains are classified as gamma (y), mu (0, alpha (a), delta (6) or
epsilon (6) with
some subclasses among them (e.g., yl-y4). It is the nature of this chain that
determines
the "class" of the antibody as IgG, IgM, IgA IgD or IgE, respectively. The
immunoglobulin subclasses or "isotypes" (e.g., IgGl, IgG2, IgG3, IgG4, IgAl,
etc.) are
well characterized and are known to confer functional specialization. Modified
versions
of each of these classes and isotypes are readily discernable to the skilled
artisan in view
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of the instant disclosure and, accordingly, are within the scope of the
present invention.
As indicated above, the variable region of an antibody allows the antibody to
selectively
recognize and specifically bind epitopes on antigens. That is, the light chain
variable
domain (VL domain) and heavy chain variable domain (VH domain) of an antibody
combine to form the variable region that defines a three-dimensional antigen
binding site.
This quaternary antibody structure forms the antigen binding site presents at
the end of
each arm of the "Y". More specifically, the antigen binding site is defined by
three
complementarity determining regions (CDRs) on each of the VH and VL chains.
"Anticalins" are well known in the art and refer to an antibody mimetic
technology,
wherein the binding specificity is derived from lipocalins. Anticalins may
also be
formatted as dual targeting protein, called Duocalins.
"Antigen" refers any substance that is capable of stimulating an immune
response,
specifically activating immunes cells. In general, two main divisions of
antigens are
recognized: foreign antigens (or heteroantigens) and autoantigens (or self-
antigens).
Antigen molecules possess by definition, at least one epitope (or antigenic
sites) which
produce corresponding antibodies.
"Antigen-binding fragment", as used herein, refers to a part or region of an
antibody or
chimeric antigen receptor (CAR), which comprises fewer amino acid residues
than the
whole antibody or CAR. An "antigen-binding fragment" binds to an antigen
and/or
competes with the whole antibody and/or CAR from which it was derived for
antigen
binding (e.g., specific binding to human senescent associated-cell antigen).
Antigen-
binding fragments encompasses, without any limitation, single chain
antibodies, Fv, Fab,
Fab', Fab' -SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies
and
tetrabodies.
"Armadillo repeat protein-based scaffold", as used herein, refers to a type of
antibody
mimetics corresponding to artificial peptide binding scaffolds based on
armadillo repeat
proteins. Armadillo repeat proteins are characterized by an armadillo domain,
composed
of tandem armadillo repeats of approximately 42 amino acids, which mediates
interactions with peptides or proteins.
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"Atrimers" are well known in the art and refers to binding molecules for
target protein
that trimerize as a perquisite for their biological activity. They are
relatively large
compared to other antibody mimetic scaffolds.
"Autologous" is meant to refer to any material derived from the same
individual to which
it is later to be re-introduced into the individual.
"Avimers" are well known in the art and refer to an antibody mimetic
technology.
"Complement-dependent cytotoxicity" or "CDC" refers to the induction of the
lysis of
antigen-expressing cells recognized by an antibody or antigen-binding fragment
thereof
of the invention in the presence of complement. The complement activation
pathway is
.. initiated by the binding of the first component of the complement system
(Cl q) to a
molecule (e.g., an antibody) complexed with a cognate antigen. CDC can be
measured
using assays that are known and available in the art (e.g., Clynes et al,
1998. Proc Natl
Acad Sci U S A. 95(2):652-6; Gazzano-Santaro et al., 1997. J Immunol Methods.
202(2): 163-71).
"CDR" or "complementarity determining region" means the non-contiguous antigen
combining sites found within the variable region of both heavy and light chain
polypeptides. The precise amino acid sequence boundaries of a given CDR can be
determined using any of a number of well-known schemes, including those
described by
Kabat et al. (1991), "Sequences of Proteins of Immunological Interest" 5th Ed.
Public
Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering
scheme), Al-Lazikani et al., 1997. J Mol Biol. 273(4):927-48 ("Chothia"
numbering
scheme), or a combination thereof. More recently, a universal numbering system
has been
developed and widely adopted, ImMunoGeneTics (IMGT) Information System
(Lefranc et al., 1999. Nucleic Acids Res. 27(4209-12). IMGT is an integrated
information system specializing in immunoglobulins (IG), T cell receptors (TR)
and
major histocompatibility complex (MHC) of human and other vertebrates. Herein,
the
CDRs are referred to in terms of both the amino acid sequence and the location
within the
light or heavy chain. As the "location" of the CDRs within the structure of
the
immunoglobulin variable domain is conserved between species and present in
structures
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called loops, by using numbering systems that align variable domain sequences
according
to structural features, CDR and framework residues may be readily identified.
This
information can be used in grafting and replacement of CDR residues from
immunoglobulins of one species into an acceptor framework from, typically, a
human
antibody. Correspondence between the Kabat numbering and the IMGT unique
numbering system is also well known to one skilled in the art (e.g., Lefranc
et al., supra).
Thus, in one embodiment, by CDR regions or CDR, it is intended to indicate the
hypervariable regions of the heavy and light chains of the immunoglobulins as
defined
by IMGT numbering system (e.g. Lefranc et al., supra).
Heavy chain variable region (HCVR or VII)
VH-CDR1 VH-CDR2 VH-CDR3
Approx. at residue 26
(always 4 after a Cys) Always 33 residues after
Always 15 residues after
according to end of VH-CDR2
the end of VH-CDR1
Chothia/AbM's definition
eh' according to Kabat/AbM's
definition Always 2 residues after a
Kabat's definition starts 5 Cys
residues later
4, Always Cys-Xaa-Xaa-Xaa, Always Cys-Xaa-Xaa, with
t.) Typically, Leu-Glu-Trp-Ile-
-as t with Xaa being any amino Xaa being any amino acid
Gly, but a number of
acid according to
p4 variations
Chothia/AbM's definition Typically, Cys-Ala-Arg
4, Always Trp
Lys/Arg- Always Trp-Gly-Xaa-Gly,
Leu/IleNal/Phe/Thr/Ala- with Xaa being any amino
c)..) Typically, Trp-Val, but
Thr/Ser/Ile/Ala acid
also, Trp-Ile or Trp-Ala
to 12 residues according
to AbM's definition
16 to 19 residues according
to Kabat's definition
r)io Chothia's definition
3 to 25 residues
0.) excludes the last 4 residues
AbM's definition ends 7
residues earlier
5 to 7 residues according to
Kabat's definition
Light chain variable region (LCVR or VL)
VL-CDR1 VL-CDR2 VL-CDR3
Always 33 residues after
*)) end of VL-CDR2 (except
Always 16 residues after
at Approx. at residue 24 NEW (PDB ID: 7FAB)
eh' the end of VL-CDR1
which has the deletion at
the end of CDR-L2*)
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-0 0 Generally, Ile-Tyr, but also
Always Cys
Val-Tyr, Ile-Lys or Ile-Phe' Always Cys
1:4
Always Trp
Always Phe-Gly-Xaa-Gly,
MS a)
Typically, Trp-Tyr-Gln, but with Xaa being any amino
0.)
r:4 also, Trp-Leu-Gln, Trp- acid
Phe-Gln or Trp-Tyr-Leu
Always 7 residues (except
NEW (PDB ID: 7FAB)
c 10 to 17 residues . 7 to 11 residues
which has a deletion in this
region*)
* Saul & Poljak, 1992. Proteins. 14(3):363-71
"Cell", "cell line" and "cell culture" may be used interchangeably. All of
these terms
also include their progeny, which is any and all subsequent generations. It is
understood
that all progeny may not be identical due to deliberate or inadvertent
mutations. In the
context of expressing a heterologous nucleic acid sequence, "host cell" refers
to a
eukaryotic cell that is capable of replicating a vector and/or expressing a
heterologous
gene encoded by a vector. A host cell can, and has been, used as a recipient
for vectors.
A host cell may be "transfected" or "transformed," which refers to a process
by which
exogenous nucleic acid is transferred or introduced into the host cell. A
transformed cell
includes the primary subject cell and its progeny. As used herein, the terms
"engineered"
and "recombinant" cells or host cells are intended to refer to a cell into
which an
exogenous nucleic acid sequence, such as, for example, a vector, has been
introduced.
Therefore, recombinant cells are distinguishable from naturally occurring
cells which do
not contain a recombinantly introduced nucleic acid
"Chimeric antigen receptor" or "CAR refers to engineered receptors, which
graft an
antigen specificity onto cells with intracellular signal generation (such as,
for example,
T cells or phagocytic cells). CARs are also known as artificial T cell
receptors, chimeric
T cell receptors or chimeric immunoreceptors.
"Co-stimulatory domain" or "CSD", when used in a relationship with a chimeric
antigen receptor (CAR), refers to the portion of the CAR which enhances the
proliferation, survival and/or development of memory cells. The CARs of the
invention
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may comprise one or more co-stimulatory domains. Co-stimulatory domains are
apparent
to those of skill in the art and may be used in connection with alternate
embodiments of
the invention.
"DARPins" or "Designed Ankyrin Repeat Proteins" are well known in the art and
refer
5 to an antibody mimetic DRP (designed repeat protein) technology developed
to exploit
the binding abilities of non-antibody polypeptides.
"DEP1", also known as PTPRJ, SCC1, CD148, HPTPeta or R-PTP-ETA, refers to a
protein encoded by a gene which is a member of the protein tyrosine
phosphatase (PTP)
family. PTPs are known to be signaling molecules that regulate a variety of
cellular
10 processes, including cell growth, differentiation, mitotic cycle, and
oncogenic
transformation. This PTP possesses an extracellular region containing five
fibronectin
type III repeats, a single transmembrane region, and a single intracytoplasmic
catalytic
domain, and thus represents a receptor-type PTP. This protein is present in
all
hematopoietic lineages, and was shown to negatively regulate T cell receptor
signaling
15 possibly through interfering with the phosphorylation of Phospholipase C
Gamma 1 and
Linker for Activation of T Cells. This protein can also dephosphorylate the
PDGF beta
receptor, and may be involved in UV-induced signal transduction. In human,
multiple
transcript variants encoding different isoforms have been found for this gene.
In the sense of the present invention, human DEP1 (or hDEP1) is a protein with
an amino
acid sequence SEQ ID NO: 1.
SEQ ID NO: 1
MKPAAREARLPPR SP GLRWALPLLLLLLRL GQILCAGGTP SPIPDP S VATVAT GE
NGITQIS STAESFHICQNGTGTPQVETNT SEDGE S SGAND SLRTPEQ GSNGTD GA S
QKTP S ST GP SPVFDIKAVSI SP TNVILTWK SNDTAASEYKYVVKHKMENEKTITV
VHQPWCNITGLRPAT SYVF SITPGIGNETWGDPRVIKVITEPIPVSDLRVALTGVR
KAALSW SNGNGTASCRVLLE SIGSHEELTQD SRL QVNI S GLKP GVQYNINPYLL
Q SNKTKGDPLGTEGGLDASNTERSRAGSPTAPVHDE SLVGPVDP S SGQQ SRDTE
VLLVGLEPGTRYNATVYSQAANGTEGQPQAIEFRTNAIQVFDVTAVNISATSLT
LIWKVSDNES S SNYTYKIHVAGETD S SNLNVSEPRAVIPGLRS STFYNITVCPVL
GDIEGTPGFLQVHTPPVPVSDFRVTVVSTTEIGLAW S SHDAESFQMHITQEGAG
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NSRVEITTNQSIIIGGLFPGTKYCFEIVPKGPNGTEGASRTVCNRTVP SAVFDIHV
VYVT T TEMWLDWK SPD GA SEYVYHLVIE SKHGSNHT STYDKAITLQGLIPGTL
YNITISPEVDHVWGDPNSTAQYTRP SNVSNIDVSTNTTAATL SWQNFDDASPTY
SYCLLIEKAGNS SNATQVVTDIGITDATVTELIPGS SYTVEIFAQVGDGIKSLEPG
RIC SF C TDPASMASFDCEVVPKEP ALVLKW TCPP GANAGF ELEVS SGAWNNATH
LESC SSENGTEYRTEVTYLNF ST SYNISITTVSCGKMAAPTRNTCTTGITDPPPPD
GSPNITSVSHNSVKVKF SGFEASHGPIKAYAVILTTGEAGHP SADVLKYTYEDFK
KGASDTYVTYLIRTEEKGRSQSLSEVLKYEIDVGNESTTLGYYNGKLEPLGSYR
AC VAGF TNI TFHP QNK GL ID GAE S YV SF SRY SD AV SLP QDP GVIC GAVF GC IF GA
LVIVTVGGF IFWRICKRKDAKNNEV SF SQIKPKKSKLIRVENFEAYFKKQQADSN
C GF AEE YEDL KLVGI S QP K YAAELAENRGKNRYNNVLP YD I SRVKL SVQTHST
DDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVE
QGRTKCEEYWP SKQAQDYGDITVAMT SEIVLPEWTIRDFTVKNIQT SE SHPLRQ
FHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAI
DRLI Y Q IENENT VD VY GIVYD LRMHRPLMVQ T ED Q YVF LNQ C VLD IVR S Q KD S
KVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA
hDEP1 is composed of several domains, as follows:
- a signal peptide, comprising or consisting of amino acid residues 1 ¨
35 of
SEQ ID NO: 1;
- an extracellular domain, comprising or consisting of amino acid residues
36 ¨ 975
of SEQ ID NO: 1, itself comprising:
= a fibronectin type-III domain 1, comprising or consisting of amino acid
residues 121 ¨ 209 of SEQ ID NO: 1;
= a fibronectin type-III domain 2, comprising or consisting of amino acid
residues 207 ¨ 291 of SEQ ID NO: 1;
= a fibronectin type-III domain 3, comprising or consisting of amino acid
residues 271 ¨ 364 of SEQ ID NO: 1;
= a fibronectin type-III domain 4, comprising or consisting of amino acid
residues 368 ¨ 456 of SEQ ID NO: 1;
= a fibronectin type-III domain 5, comprising or consisting of amino acid
residues 457 ¨ 541 of SEQ ID NO: 1;
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= a fibronectin type-III domain 6, comprising or consisting of amino acid
residues 542 ¨ 623 of SEQ ID NO: 1;
= a fibronectin type-III domain 7, comprising or consisting of amino acid
residues 625 ¨ 720 of SEQ ID NO: 1;
= a
fibronectin type-III domain 8, comprising or consisting of amino acid
residues 721 ¨ 817 of SEQ ID NO: 1; and
= a fibronectin type-III domain 9, comprising or consisting of amino acid
residues 816¨ 902 of SEQ ID NO: 1;
a transmembrane domain, comprising or consisting of amino acid residues
976 ¨ 996 of SEQ ID NO: 1; and
a cytoplasmic domain, comprising or consisting of amino acid residues 997¨
1337
of SEQ ID NO: 1.
"Diabodies", as used herein, refers to small antibody fragments prepared by
constructing
scFv fragments with short linkers (about 5-10 residues) between the HCVR and
LCVR
such that inter-chain but not intra-chain pairing of the variable domains is
achieved,
resulting in a bivalent fragment, i.e., fragment having two antigen-binding
sites.
Bispecific diabodies are heterodimers of two "crossover" scFv fragments in
which the
HCVR and LCVR of the two antibodies are present on different polypeptide
chains.
Diabodies are described more fully in European patent EP0404097, International
patent
application W01993011161; and in Holliger et al., 1993. Proc Natl Acad Sci
USA.
90(14):6444-8.
"Domain antibodies" are well-known in the art and refer to the smallest
functional
binding units of antibodies, corresponding to the variable regions of either
the heavy or
light chains of antibodies.
"Domain kunitz peptide" refer to a type of antibody mimetics, and is based on
the active
domains of proteins inhibiting the function of proteases.
"DPP4" (also known as ADABP, adenosine deaminase complexing protein 2, ADCP-2,
dipeptidyl peptidase IV, DPP IV, CD26, or TP103) refers to an intrinsic
membrane
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glycoprotein and a serine exopeptidase that cleaves X-proline dipeptides from
the N-
terminus of polypeptides.
In the sense of the present invention, human DPP4 (or hDPP4) is a protein with
an amino
acid sequence SEQ ID NO: 101.
SEQ ID NO: 101
MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKTYTLTDYLKNTY
RLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSIS
PDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTW SPVG
HICLAYVWNNDIYVKIEPNLP SYRITWTGKEDIIYNGITDWVYEEEVF S AY S ALW
W SPNGTFLAYAQFND TEVPLIE Y SF Y SDE SL QYPKTVRVPYPKAGAVNP TVKFF
VVNTDSL S SVTNAT SIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYS
VMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRP SEPHFTLDGNSFYKIISN
EEGYRHICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLY
KIQL SDYTKVTCL SCELNPERCQYYSVSF SKEAKYYQLRCSGPGLPLYTLHS SV
NDKGLRVLEDNSALDKMLQNVQMP SKKLDFIILNETKFWYQMILPPHFDKSKK
YPLLLDVYAGPC SQKADTVFRLNWATYLASTENIIVASFDGRGSGYQGDKIMH
AINRRLGTFEVEDQIEAARQF SKMGFVDNKRIAIWGW SYGGYVT SMVL GS GSG
VFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQ
VEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIAS STAHQ
HIYTHMSHFIKQCF SLP
hDPP4 is composed of several domains, as follows:
- a cytoplasmic domain, comprising or consisting of amino acid residues
1 ¨ 6 of
SEQ ID NO: 101;
- a transmembrane domain, comprising or consisting of amino acid
residues 7 ¨ 28
of SEQ ID NO: 101; and
- an extracellular domain, comprising or consisting of amino acid
residues 29 ¨ 766
of SEQ ID NO: 101.
"Epitope", also known as "antigenic determinant", refers to a specific
arrangement of
amino acids located on a protein or proteins (or antigen(s)) to which an
antibody or
antigen-binding fragment thereof or chimeric antigen receptor (CAR) binds.
Epitopes
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often consist of a chemically active surface grouping of molecules such as
amino acids
or sugar side chains, and have specific three-dimensional structural
characteristics as well
as specific charge characteristics. Epitopes can be linear (or sequential) or
conformational, i.e., involving two or more sequences of amino acids in
various regions
of the antigen that may not necessarily be contiguous.
"Evasins" are well known in the art and refer to a class of chemokine-binding
proteins.
"Extracellular spacer domain" or "ESD" or "hinge domain", when used in a
relationship with a chimeric antigen receptor (CAR), refers to the hydrophilic
region
which is between the antigen-specific targeting region and the transmembrane
domain.
The extracellular spacer domains are apparent to those of skill in the art and
may be used
in connection with alternate embodiments of the invention.
"Framework region" or "FR region" includes the amino acid residues that are
part of
the variable region, but are not part of the CDRs (e.g., using the IMGT
numbering
definition of CDRs). The framework regions for the light chain are similarly
separated by
each of the LCVR's CDRs. In naturally occurring antibodies, the six CDRs
present on
each monomeric antibody are short, non-contiguous sequences of amino acids
that are
specifically positioned to form the antigen binding site as the antibody
assumes its three-
dimensional configuration in an aqueous environment. The remainders of the
heavy and
light variable domains show less inter-molecular variability in amino acid
sequence and
are termed the framework regions. The framework regions largely adopt a 13-
sheet
conformation and the CDRs form loops which connect, and in some cases form
part of,
the 13-sheet structure. Thus, these framework regions act to form a scaffold
that provides
for positioning the six CDRs in correct orientation by inter-chain, non-
covalent
interactions. The antigen binding site formed by the positioned CDRs defines a
surface
complementary to the epitope on the immunoreactive antigen. This complementary
surface promotes the non-covalent binding of the antibody to the
immunoreactive antigen
epitope. The position of CDRs can be readily identified by one of ordinary
skill in the art.
"Fc domain" "Fc portion" and "Fc region" refer to a C-terminal fragment of an
antibody
heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human
gamma heavy
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chain or its counterpart sequence in other types of antibody heavy chains
(e.g., a, (3, c and
for human antibodies), or a naturally occurring allotype thereof.
"Fynomers" are well known in the art and refer to proteins that belong to the
class of
antibody mimetic. They are attractive binding molecules due to their high
thermal
5 stability and reduced immunogenicity.
as used herein, refers to the minimum antibody fragment that contains a
complete
antigen-recognition and -binding site. This fragment consists of a dimer of
one HCVR
and one LCVR in tight, non-covalent association. From the folding of these two
domains
emanate six hypervariable loops (three loops each from the heavy and light
chain) that
10 contribute to antigen binding and confer antigen binding specificity to
the antibody.
However, even a single variable domain (or half of an Fv comprising only three
CDRs
specific for an antigen) has the ability to recognize and bind antigen,
although at a lower
affinity than the entire binding site.
"Heavy chain region" includes amino acid sequences derived from the constant
domains
15 of an immunoglobulin heavy chain. A protein comprising a heavy chain
region comprises
at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge
region)
domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof In an
embodiment,
the antibody or antigen-binding fragment thereof according to the present
invention may
comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge
portion, a CH2
20 domain, and a CH3 domain). In another embodiment, the antibody or
antigen-binding
fragment thereof according to the present invention lacks at least a region of
a constant
domain (e.g., all or part of a CH2 domain). In certain embodiments, at least
one, and
preferably all, of the constant domains are derived from a human
immunoglobulin heavy
chain. For example, in one preferred embodiment, the heavy chain region
comprises a
fully human hinge region. In other preferred embodiments, the heavy chain
region
comprising a fully human Fc region (e.g., hinge, CH2 and CH3 domain sequences
from a
human immunoglobulin). In certain embodiments, the constituent constant
domains of
the heavy chain region are from different immunoglobulin molecules. For
example, a
heavy chain region of a protein may comprise a CH2 domain derived from an IgG1
molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other
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embodiments, the constant domains are chimeric domains comprising regions of
different
immunoglobulin molecules. For example, a hinge may comprise a first region
from an
IgG1 molecule and a second region from an IgG3 or IgG4 molecule. As set forth
above,
it will be understood by one of ordinary skill in the art that the constant
domains of the
heavy chain region may be modified such that they vary in amino acid sequence
from the
naturally occurring (wild-type) immunoglobulin molecule. That is, the antibody
or
antigen-binding fragment thereof according to the present invention may
comprise
alterations or modifications to one or more of the heavy chain constant
domains (CH1,
hinge, CH2 or CH3) and/or to the light chain constant domain (CL). Exemplary
modifications include additions, deletions or substitutions of one or more
amino acids in
one or more domains.
"Hinge region", when used in a relationship with an antibody, includes the
region of a
heavy chain molecule that joins the CH1 domain to the CH2 domain in an
antibody. This
hinge region comprises approximately 25 residues and is flexible, thus
allowing the two
N-terminal antigen binding regions to move independently. Hinge regions can be
subdivided into three distinct domains: upper, middle, and lower domains (Roux
et al.,
1998. J Immunol. 161(8):4083-90).
"Hypervariable loop" is not strictly synonymous to complementarity determining
region
(CDR), since the hypervariable loops (HVs) are defined on the basis of
structure, whereas
CDRs are defined based on sequence variability (Kabat et al, 1991. Sequences
of proteins
of immunological interest (5th ed.). Bethesda, MD: U.S. Dep. of Health and
Human
Services) and the limits of the HVs and the CDRs may be different in some VH
and Vi.
domains. The CDRs of the Vi. and VH domains can typically be defined by the
Kabat/Chothia definition as already explained hereinabove.
"Identity" or "identical", when used in a relationship between the sequences
of two or
more amino acid sequences, or of two or more nucleic acid sequences, refers to
the degree
of sequence relatedness between amino acid sequences or nucleic acid
sequences, as
determined by the number of matches between strings of two or more amino acid
residues
or nucleic acid residues. "Identity" measures the percent of identical matches
between the
smaller of two or more sequences with gap alignments (if any) addressed by a
particular
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mathematical model or computer program (i.e., "algorithms"). Identity of
related amino
acid sequences or nucleic acid sequences can be readily calculated by known
methods.
Such methods include, but are not limited to, those described in Lesk A. M.
(1988).
Computational molecular biology: Sources and methods for sequence analysis.
New
York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing:
Informatics and
genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G.
(1994).
Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von
Heijne G.
(1987). Sequence analysis in molecular biology: treasure trove or trivial
pursuit. San
Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence
analysis
primer. New York, NY: Stockton Press; Carillo et at, 1988. SIAM J Appl Math.
48(5):1073-82. Preferred methods for determining identity are designed to give
the largest
match between the sequences tested. Methods of determining identity are
described in
publicly available computer programs. Preferred computer program methods for
determining identity between two sequences include the GCG program package,
including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI;
Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN,
and
FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program
is
publicly available from the National Center for Biotechnology Information
(NCBI) and
other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894).
The well-known Smith Waterman algorithm may also be used to determine
identity.
"Intracellular signaling domain" or "ISD" or "cytoplasmic domain", when used
in a
relationship with a chimeric antigen receptor (CAR), refers to the portion of
the CAR
which transduces the effector function signal and directs the cell to perform
its specialized
function. Intracellular signaling domains are be apparent to those of skill in
the art and
may be used in connection with alternate embodiments of the invention.
"Isolated" means altered or removed from the natural state. For example, a
nucleic acid
or a peptide naturally present in a living animal is not "isolated" but the
same nucleic acid
or peptide partially or completely separated from the coexisting materials of
its natural
state is "isolated". An isolated nucleic acid or protein can exist in
substantially purified
form, or can exist in a non-native environment such as, for example, a host
cell.
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"Knottin" or "inhibitor cystine knot" refer to an antibody mimetic comprising
a protein
structural motif containing three disulfide bridges.
"Monoclonal antibody" refers to an antibody obtained from a population of
substantially
homogeneous antibodies, i.e., the individual antibodies comprised in the
population are
identical except for possible naturally occurring mutations that may be
present in minor
amounts. Monoclonal antibodies are highly specific, being directed against a
single
antigenic site. Furthermore, in contrast to polyclonal antibody preparations
that include
different antibodies directed against different determinants (epitopes), each
monoclonal
antibody is directed against a single determinant on the antigen. In addition
to their
specificity, the monoclonal antibodies are advantageous in that they may be
synthesized
uncontaminated by other antibodies. The modifier "monoclonal" is not to be
construed as
requiring production of the antibody by any particular method. For example,
the
monoclonal antibodies or antigen-binding fragment thereof according to the
present
invention may be prepared by the hybridoma methodology first described by
Kohler et
al, 1975. Nature. 256(5517):495-7, or may be made using recombinant DNA
methods in
bacterial, eukaryotic animal or plant cells (Patent US4,816,567). The
"monoclonal
antibodies" may also be isolated from phage antibody libraries using the
techniques
described in Clackson et al, 1991. Nature. 352(6336):624-8 and Marks et al.,
1991. J
Mol Biol. 222(3):581-97, for example.
"Linker", when used in a relationship with a chimeric antigen receptor (CAR),
refers to
an oligo- or polypeptide region from about 1 to 100 amino acids in length,
which links
together any of the domains/regions of the CAR of the invention. Linkers may
be
composed of flexible residues like glycine and serine so that the adjacent
protein domains
are free to move relative to one another. Longer linkers may be used when it
is desirable
to ensure that two adjacent domains do not sterically interfere with one
another. Linkers
may be cleavable or non-cleavable. Linkers are apparent to those of skill in
the art and
may be used in connection with alternate embodiments of the invention.
"Nanobodies" are well-known in the art and refer to antibody-derived
therapeutic
proteins that contain the unique structural and functional properties of
naturally-occurring
heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These
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heavy chain antibodies may contain a single variable domain (VHH) and two
constant
domains (CH2 and CH3).
"Prevent", "preventing" and "prevention" refer to prophylactic and
preventative
measures, wherein the object is to reduce the chances that a subject will
develop the
pathologic condition or disorder over a given period of time. Such a reduction
may be
reflected, e.g., in a delayed onset of at least one symptom of the pathologic
condition or
disorder in the subject.
"Proliferating cell" refers to a cell that is undergoing cell division.
"Promoter" is used to define a control sequence, that is a region of a vector
at which
initiation and rate of transcription are controlled. It may contain genetic
elements to which
regulatory proteins and molecules may bind, such as RNA polymerase and other
transcription factors, to initiate the specific transcription a nucleic acid
sequence. The
phrases "operatively positioned" "operatively linked" "under control" and
"under
transcriptional control" mean that a promoter is in a correct functional
location and/or
orientation in relation to a nucleic acid to control transcriptional
initiation and/or
expression of that nucleic acid.
A promoter typically comprises a sequence that functions to position the start
site for
RNA synthesis. The best known example of this is the TATA box, but in some
promoters
lacking a TATA box, such as, for example, the promoter for the mammalian
terminal
deoxynucleotidyl transferase gene and the promoter for the 5V40 late genes, a
discrete
element overlying the start site itself helps to fix the place of initiation.
Additional
promoter elements regulate the frequency of transcriptional initiation.
Typically, these
are located in the region 110 bp upstream of the start site, although a number
of promoters
have been shown to contain functional elements downstream of the start site as
well. To
bring a coding sequence "under the control of' a promoter, one positions the
5' end of
the transcription initiation site of the transcriptional reading frame
"downstream" of (i.e.,
3' of) the chosen promoter. The "upstream" promoter stimulates transcription
of the DNA
and promotes expression of the encoded RNA.
The spacing between promoter elements frequently is flexible, so that promoter
function
is preserved when elements are inverted or moved relative to one another. In
the tk
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promoter, the spacing between promoter elements can be increased to 50 bp
apart before
activity begins to decline. Depending on the promoter, it appears that
individual elements
can function either cooperatively or independently to activate transcription.
A promoter
may or may not be used in conjunction with an "enhancer", which refers to a
cis-acting
5 regulatory sequence involved in the transcriptional activation of a
nucleic acid sequence.
A promoter may be one naturally associated with a nucleic acid, as may be
obtained by
isolating the 5' non-coding sequences located upstream of the coding segment
and/or
exon. Such a promoter can be referred to as "endogenous". Similarly, an
enhancer may
be one naturally associated with a nucleic acid, located either downstream or
upstream of
10 that sequence. Alternatively, certain advantages will be gained by
positioning the coding
nucleic acid segment under the control of a recombinant or heterologous
promoter, which
refers to a promoter that is not normally associated with a nucleic acid
sequence in its
natural environment. A recombinant or heterologous enhancer refers also to an
enhancer
not normally associated with a nucleic acid sequence in its natural
environment. Such
15 promoters or enhancers may include promoters or enhancers of other
genes, and
promoters or enhancers isolated from any other virus, or prokaryotic or
eukaryotic cell,
and promoters or enhancers not "naturally occurring", i.e., containing
different elements
of different transcriptional regulatory regions, and/or mutations that alter
expression. For
example, promoters that are most commonly used in recombinant DNA construction
20 include the lactamase (penicillinase), lactose and tryptophan (trp)
promoter systems. In
addition to producing nucleic acid sequences of promoters and enhancers
synthetically,
sequences may be produced using recombinant cloning and/or nucleic acid
amplification
technology, including PCRTM, in connection with the compositions disclosed
herein (see
US patents 4,683,202 and 5,928,906). Furthermore, it is contemplated the
control
25 sequences that direct transcription and/or expression of sequences
within non-nuclear
organelles such as mitochondria, chloroplasts, and the like, can be employed
as well.
Naturally, it will be important to employ a promoter and/or enhancer that
effectively
directs the expression of the DNA segment in the organelle, cell type, tissue,
organ, or
organism chosen for expression. Those of skill in the art of molecular biology
generally
know the use of promoters, enhancers, and cell type combinations for protein
expression,
(see, for example Sambrook et al. 1989). The promoters employed may be
constitutive,
tissue-specific, inducible, and/or useful under the appropriate conditions to
direct high-
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26
level expression of the introduced DNA segment, such as is advantageous in the
large-
scale production of recombinant proteins and/or peptides. The promoter may be
heterologous or endogenous.
Additionally, any promoter/enhancer combination could also be used to drive
expression.
Use of a T3, T7 or SP6 cytoplasmic expression system is another possible
embodiment.
Eukaryotic cells can support cytoplasmic transcription from certain bacterial
promoters
if the appropriate bacterial polymerase is provided, either as part of the
delivery complex
or as an additional genetic expression construct.
The identity of tissue-specific promoters or elements, as well as assays to
characterize a
specific initiation signal also may be required for efficient translation of
coding
sequences. These signals include the ATG initiation codon or adjacent
sequences.
Exogenous translational control signals, including the ATG initiation codon,
may need to
be provided. One of ordinary skill in the art would readily be capable of
determining this
and providing the necessary signals.
"Recombinant antibody" refers to antibodies which are produced, expressed,
generated
or isolated by recombinant means, such as antibodies which are expressed using
a
recombinant expression vector transfected into a host cell; antibodies
isolated from a
recombinant combinatorial antibody library; antibodies isolated from an animal
(e.g., a
mouse) which is transgenic due to human immunoglobulin genes; or antibodies
which are
produced, expressed, generated or isolated in any other way in which
particular
immunoglobulin gene sequences (such as human immunoglobulin gene sequences)
are
assembled with other DNA sequences. Recombinant antibodies include, for
example,
chimeric and humanized antibodies.
"Senescent cells" refers to cells that are in cell cycle arrest, generally
during the G1
transition of the cell cycle or in few cases in G2, elicited by replicative
exhaustion due to
telomere attrition or in response to stresses such as DNA damage,
chemotherapeutic
drugs, or aberrant expression of oncogenes. According to one embodiment, the
senescent
cells are generally characterized by at least one or more of the following
characteristics:
activation of the p53/p21CIP1 and/or pRb/p16INK4A tumor suppressor pathways,
cells
whose proliferation is irreversibly arrested, shortening of telomere size,
expression of
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senescent-associated beta-galactosidase activity, specific chromatin
modification,
specific secretome, increase in reactive oxygen species and altered overall
mitochondrial
activity. Senescent cells and senescent cell-associated antigens can be
detected by
techniques and procedures described in the art.
"Single chain antibody", as used herein, refers to any antibody or fragment
thereof that
is a protein having a primary structure comprising or consisting of one
uninterrupted
sequence of contiguous amino acid residues, including without limitation (1)
single-chain
Fv molecules (scFv); (2) single chain proteins containing only one light chain
variable
domain, or a fragment thereof that contains the three CDRs of the light chain
variable
domain, without an associated heavy chain moiety; and (3) single chain
proteins
containing only one heavy chain variable region, or a fragment thereof
containing the
three CDRs of the heavy chain variable region, without an associated light
chain moiety.
"Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to antibody
fragments that
comprise the VH and VL antibody domains connected into a single amino acid
chain.
Preferably, the scFv amino acid sequence further comprises a flexible peptidic
linker
between the VH and VL domains that enables the scFv to form the desired
structure for
antigen binding (Pluckthun, 1994. "Antibodies from Escherichia coil". In
Rosenberg &
Moore (Eds.), The pharmacology of monoclonal antibodies. Handbook of
Experimental
Pharmacology, 113:269-315. Springer: Berlin, Heidelberg). Flexible peptidic
linkers are
generally composed of small, non-polar (e.g., glycine, Gly, G) or polar (e.g.,
serine, Ser,
S; or threonine, Thr, T) amino acids, as suggested by Argos (1990. J Mol Biol.
211(4):943-958). The small size of these amino acids provides flexibility, and
allows for
mobility of the connecting functional domains, such as the VH and VL domains.
In one
embodiment, the flexible peptidic linker may be a short oligo- or polypeptide,
preferably
having a length ranging from 2 to 30 amino acids. In one embodiment, the
flexible
peptidic linker comprises glycine-serine repeats. In one embodiment, the
flexible peptidic
linker comprises one, or several repeats of, such as 2, 3, 4, 5 or more
repeats of, GS
linker(s) (i.e., a sequence of one Gly and one Ser), G25 linker(s) (i.e., a
sequence of two
Gly and one Ser), G35 linker(s) (i.e., a sequence of three Gly and one Ser),
G45 linker(s)
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(i.e., a sequence of four Gly and one Ser), or G5S linker(s) (i.e., a sequence
of five Gly
and one Ser).
"Subject" refers to a mammal, preferably a human. In one embodiment, a subject
may
be a "patient", i.e., a warm-blooded animal, more preferably a human,
who/which is
awaiting the receipt of, or is receiving medical care or was/is/will be the
object of a
medical procedure, or is monitored for the development of a disease. The term
"mammal"
refers here to any mammal, including humans, domestic and farm animals, and
zoo,
sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs,
goats, rabbits, etc.
Preferably, the mammal is a primate, more preferably a human.
"Therapeutically effective amount" refers to the level or amount of an
antibody as
described herein that is aimed at, without causing significant negative or
adverse side
effects to the target, (1) delaying or preventing the onset of a disease,
disorder, or
condition; (2) slowing down or stopping the progression, aggravation, or
deterioration of
one or more symptoms of the disease, disorder, or condition; (3) bringing
about
ameliorations of the symptoms of the disease, disorder, or condition; (4)
reducing the
severity or incidence of the disease, disorder, or condition; or (5) curing
the disease,
disorder, or condition. A therapeutically effective amount may be administered
prior to
the onset of the disease, disorder, or condition, for a prophylactic or
preventive action.
Alternatively or additionally, the therapeutically effective amount may be
administered
after initiation of the disease, disorder, or condition, for a therapeutic
action.
"Transfected" or "transformed" or "transduced" as used herein refers to a
process by
which exogenous nucleic acid is transferred or introduced into the host cell.
A
"transfected" or "transformed" or "transduced" cell is one which has been
transfected,
transformed or transduced with exogenous nucleic acid.
"Transmembrane domain" or "TMD", when used in a relationship with a chimeric
antigen receptor (CAR), refers to the region of the CAR which crosses the
plasma
membrane. The transmembrane domain of the CAR of the invention is the
transmembrane region of a transmembrane protein (for example Type I
transmembrane
proteins), an artificial hydrophobic sequence or a combination thereof Other
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transmembrane domains are apparent to those of skill in the art and may be
used in
connection with alternate embodiments of the invention.
"Treating" or "treatment" or "alleviation" refers to both therapeutic
treatment and
prophylactic or preventative measures; wherein the object is to prevent or
slow down
(lessen) the targeted pathologic condition or disorder. Those in need of
treatment include
those already with the disorder as well as those prone to have the disorder or
those in
whom the disorder is to be prevented. A subject or mammal is successfully
"treated" for
a cancer or an infection if, after receiving a therapeutic amount of an
antibody according
to the methods of the present invention, the patient shows observable and/or
measurable
reduction in or absence of one or more of the following: reduction in the
number of cancer
cells (or tumor size), or pathogenic cells; reduction in the percent of total
cells that are
cancerous or pathogenic; and/or relief to some extent, one or more of the
symptoms
associated with the specific disease or condition; reduced morbidity and
mortality, and
improvement in quality of life issues. The above parameters for assessing
successful
treatment and improvement in the disease are readily measurable by routine
procedures
familiar to a physician.
"Unibodies" are well known in the art and refer to an antibody fragment
lacking the hinge
region of IgG4 antibodies. The deletion of the hinge region results in a
molecule that is
essentially half the size of traditional IgG4 antibodies and has a univalent
binding region
rather than the bivalent biding region of IgG4 antibodies.
"Variable" refers to the fact that certain regions of the variable domains \Tx
and VL differ
extensively in sequence among antibodies and are used in the binding and
specificity of
each particular antibody for its target antigen. However, the variability is
not evenly
distributed throughout the variable domains of antibodies. It is concentrated
in three
segments called "hypervariable loops" in each of the VL domain and the \Tx
domain which
form part of the antigen binding site. The first, second and third
hypervariable loops of
the Vk light chain domain are referred to herein as Li (k), L2 (k) and L3 (k)
and may be
defined as comprising residues 24-33 (Li(), consisting of 9, 10 or 11 amino
acid
residues), 49-53 L2 (k), consisting of 3 residues) and 90-96 (L3(k),
consisting of
6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79). The
first,
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second and third hypervariable loops of the Vic light chain domain are
referred to herein
as Ll(x), L2(x) and L3(x) and may be defined as comprising residues 25-33
(L1(x),
consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(x), consisting of 3
residues) and
90-97 (L3(x), consisting of 6 residues) in the VL domain (Morea et al., 2000.
Methods.
5 20(3):267-79). The first, second and third hypervariable loops of the VH
domain are
referred to herein as H1, H2 and H3 and may be defined as comprising residues
25-33
(H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4
residues) and 91-
105 (H3, highly variable in length) in the VH domain (Morea et al., 2000.
Methods.
20(3):267-79). Unless otherwise indicated, the terms Li, L2 and L3
respectively refer to
10 the first, second and third hypervariable loops of a VL domain, and
encompass
hypervariable loops obtained from both Vic and Vk isotypes. The terms H1, H2
and H3
respectively refer to the first, second and third hypervariable loops of the
VH domain, and
encompass hypervariable loops obtained from any of the known heavy chain
isotypes,
including gamma (y), mu ( ), alpha (a), delta (6) or epsilon (6). The
hypervariable loops
15 Li, L2, L3, H1, H2 and H3 may each comprise part of a "complementarity
determining
region" or "CDR", as defined hereinabove.
"Vector" is used to refer to a carrier nucleic acid molecule into which a
nucleic acid
sequence can be inserted, for introduction into a cell where it can be
replicated. A nucleic
acid sequence can be "exogenous", which means that it is foreign to the cell
into which
20 the vector is being introduced or that the sequence is homologous to a
sequence in the
cell but in a position within the host cell nucleic acid in which the sequence
is ordinarily
not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal
viruses, and
plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the
art would be
well equipped to construct a vector through standard recombinant techniques
(see, e.g.,
25 Maniatis et al., 1988 and Ausubel et al, 1994).
In one embodiment, the vector can be an "expression vector". This term refers
to any
type of genetic construct comprising a nucleic acid coding for an RNA capable
of being
transcribed. In some cases, RNA molecules are then translated into a protein,
polypeptide,
or peptide. In other cases, these sequences are not translated, e.g., in the
production of
30 antisense molecules or ribozymes. Expression vectors can contain a
variety of control
sequences, which refer to nucleic acid sequences necessary for the
transcription and
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possibly translation of an operably linked coding sequence in a particular
host cell. In
addition to control sequences that govern transcription and translation,
vectors and
expression vectors may contain nucleic acid sequences that serve other
functions as well
and are described herein. In particular, expression vectors can contain one or
several
promoter(s), enhancer(s), internal ribosome entry site(s) (TRES), multiple
cloning site(s)
(MCS), splicing site(s), termination signal(s), origin(s) of replication,
and/or selectable
marker(s).
The vector may be a "plasmid vector". In general, plasmid vectors containing
replicon
and control sequences which are derived from species compatible with the host
cell are
used in connection with these hosts. The vector ordinarily carries a
replication site, as
well as marking sequences which are capable of providing phenotypic selection
in
transformed cells. In a non-limiting example, E. colt is often transformed
using
derivatives of pBR322, a plasmid derived from an E. colt species. pBR322
contains genes
for ampicillin and tetracycline resistance and thus provides easy means for
identifying
transformed cells. The pBR plasmid, or other microbial plasmid or phage must
also
contain, or be modified to contain, for example, promoters which can be used
by the
microbial organism for expression of its own proteins.
In addition, phage vectors containing replicon and control sequences that are
compatible
with the host microorganism can be used as transforming vectors in connection
with these
hosts. For example, the phage lambda GEMTm 11 may be utilized in making a
recombinant phage vector which can be used to transform host cells, such as,
for example,
E. colt LE392.
Further useful plasmid vectors include pIN vectors (Inouye et al., 1985); and
pGEX
vectors, for use in generating glutathione S transferase (GST) soluble fusion
proteins for
later purification and separation or cleavage. Other suitable fusion proteins
are those with
galactosidase, ubiquitin, and the like.
Bacterial host cells, for example, E. colt, comprising the expression vector,
are grown in
any of a number of suitable media, for example, LB. The expression of the
recombinant
protein in certain vectors may be induced, as would be understood by those of
skill in the
art, by contacting a host cell with an agent specific for certain promoters,
e.g., by adding
IPTG to the media or by switching incubation to a higher temperature. After
culturing the
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bacteria for a further period, generally of between 2 and 24 hours, the cells
are collected
by centrifugation and washed to remove residual media.
The vector may be a "viral vector". The ability of certain viruses to infect
cells or enter
cells via receptor mediated endocytosis, and to integrate into host cell
genome and express
viral genes stably and efficiently have made them attractive candidates for
the transfer of
foreign nucleic acids into cells (e.g., mammalian cells).
In one embodiment, the viral vector may be an adenoviral vector. Although
adenoviral
vectors are known to have a low capacity for integration into genomic DNA,
this feature
is counterbalanced by the high efficiency of gene transfer afforded by these
vectors.
In one embodiment, the viral vector may be an adeno-associated viral (AAV)
vector.
Increased transfection efficiencies have been reported in cell systems using
adenovirus
coupled systems (Kelleher & Vos, 1994. Biotechniques. 17(6)1110-7; Cotten et
al.,
1992. Proc Natl Acad Sci USA. 89(13):6094-8; Curiel, 1994. Nat Immun. 13(2-
3):141-
64). AAV is an attractive vector system as it has a high frequency of
integration and it
can infect non-dividing cells, thus making it useful for delivery of genes
into mammalian
cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a
broad host
range for infectivity (Tratschin et al., 1984; Laughlin et al, 1986; Lebkowski
et al, 1988;
McLaughlin et al., 1988). Details concerning the generation and use of rAAV
vectors are
described in US patents 5,139,941 and 4,797,368.
In one embodiment, the viral vector may be a retroviral vector. Retroviruses
are useful as
delivery vectors because of their ability to integrate their genes into the
host genome,
transferring a large amount of foreign genetic material, infecting a broad
spectrum of
species and cell types and of being packaged in special cell lines. In order
to construct a
retroviral vector, a nucleic acid (e.g., one encoding the desired sequence) is
inserted into
the viral genome in the place of certain viral sequences to produce a virus
that is
replication defective. In order to produce virions, a packaging cell line
containing the gag,
pol, and env genes but without the LTR and packaging components is
constructed. When
a recombinant plasmid containing a cDNA, together with the retroviral LTR and
packaging sequences is introduced into a special cell line (e.g., by calcium
phosphate
precipitation), the packaging sequence allows the RNA transcript of the
recombinant
plasmid to be packaged into viral particles, which are then secreted into the
culture media.
The media containing the recombinant retroviruses is then collected,
optionally
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concentrated, and used for gene transfer. Retroviral vectors are able to
infect a broad
variety of cell types. However, integration and stable expression require the
division of
host cells. Lentiviruses are complex retroviruses, which, in addition to the
common
retroviral genes gag, poi, and env, contain other genes with regulatory or
structural
function. Lentiviral vectors are well known in the art (see, e.g., US patents
6,013,516 and
5,994,136). Some examples of lentivirus include the human immunodeficiency
viruses
HIV-1 and HIV-2, and the simian immunodeficiency virus SW. Lentiviral vectors
have
been generated by attenuating the HIV virulence genes, for example, the genes
env, vif,
vpr, vpu and nef are deleted making the vector biologically safe. Recombinant
lentiviral
vectors are capable of infecting non-dividing cells and can be used for both
in vivo and
ex vivo gene transfer and expression of nucleic acid sequences. For example,
recombinant
lentivirus capable of infecting a non-dividing cell wherein a suitable host
cell is
transfected with two or more vectors carrying the packaging functions, namely
gag, pol
and env, as well as rev and tat is described in US patent 5,994,136. One may
target the
recombinant virus by linkage of the envelope protein with an antibody or a
particular
ligand for targeting to a receptor of a particular cell-type. By inserting a
sequence
(including a regulatory region) of interest into the viral vector, along with
another gene
which encodes the ligand for a receptor on a specific target cell, for
example, the vector
is now target-specific.
Other viral vectors may also be employed in the present invention. Vectors
derived from
viruses such as vaccinia virus, sindbis virus, cytomegalovirus and herpes
simplex virus
may be employed. They offer several attractive features for various mammalian
cells.
"Versabodies" are well known in the art and refer to another antibody mimetic
technology. They are small proteins of 3-5 kDa with >15% cysteines, which form
a high
disulfide density scaffold, replacing the hydrophobic core the typical
proteins have. The
replacement of a large number of hydrophobic amino acids, comprising the
hydrophobic
core, with a small number of disulfides results in a protein that is smaller,
more
hydrophilic (less aggregation and non-specific binding), more resistant to
proteases and
heat, and has a lower density of T cell epitopes, because the residues that
contribute most
to MHC presentation are hydrophobic. All four of these properties are well-
known to
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affect immunogenicity, and together they are expected to cause a large
decrease in
immunogenicity.
"Xenogeneic" refers to a graft derived from an animal of a different species.
DETAILED DESCRIPTION
A first object of the present invention is an antigen-binding domain directed
to a senescent
cell-associated antigen.
In one embodiment, the antigen-binding domain of the invention recognizes and
is
capable of binding to a senescent cell-associated antigen.
The presence of senescent cells can be determined by detection of senescent
cell-
associated molecules include growth factors, proteases, cytokines (e.g.,
inflammatory
cytokines), chemokines, cell-related metabolites, reactive oxygen species
(e.g., H202), and
other molecules that stimulate inflammation and/or other biological effects or
reactions
that may promote or exacerbate the underlying disease of the subject.
Senescent cell-
associated molecules include those that are described in the art as comprising
the
senescence-associated secretory phenotype (SASP, i.e., which includes secreted
factors
which may make up the pro-inflammatory phenotype of a senescent cell),
senescent-
messaging secretome, and DNA damage secretory program (DDSP). For example, the
presence of senescent cells in tissues can be analyzed by histochemistry or
immunohistochemistry techniques that detect the senescence marker, SA-beta gal
(SA-
Bgal) (see, for exemple, Dimri et al , 1995. Proc Natl Acad Sci USA.
92(20):9363-7).
Senescent cell-associated antigens include molecules that are overexpressed in
senescent
cells compared to their quiescent or non-senescent counterparts. Certain
senescent cell-
associated antigens are tissue specific while others are ubiquitously
overexpressed in
senescent cells. In particular embodiments of the immunogenic compositions
described
herein, a senescent cell-associated antigen is an antigen present on the cell
surface of a
senescent cell (e.g., receptor proteins, channel forming proteins, proteins
that facilitate
diffusion or active transport of molecules and ion across the membrane, cell
recognition
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proteins, and enzymes). These antigens may be present on the cell surface of a
cell
exclusively or at a greater level on senescent cells compared with non-
senescent cells and
are therefore useful as immunogens for evoking a specific immune response.
Examples
of senescent cell-associated antigens include polypeptides and proteins
(including
5 glycoproteins), lipids, glycolipids, and carbohydrate molecules that
contribute to or are
markers of a senescence cell.
In one embodiment, the senescent cell according to the present invention
expresses a
senescent cell-associated antigen or a combination of senescent cell-
associated antigens
that are characteristic of senescence. Such senescent cell-associated antigens
include, but
10 are not limited to, actin cytoplasmic 1 (ACTB), A disintegrin and
metalloproteinase with
thrombospondin motifs 7 (ADAMTS7), amyloid-like protein 2 (APLP2), armadillo
repeat-
containing X-linked protein 3 (ARMCX-3), ATP synthase subunit alpha
mitochondrial
(ATP5F1A), V-type proton ATPase subunit d 2 (ATP6V0D2), beta-2-microglobulin
(B2MG), cholinesterase (BCHE), uncharacterized protein C 1 1 orf87 (C1
lorf87),
15 membrane cofactor protein (CD46), CD57, cyclin-dependent kinase
inhibitor 2A
"p16INK4a" (CDKN2A), cathepsin B (CTSB), neuferricin (CYB5D2), dipeptidyl
peptidase 4 "DPP4" (DPP4), electron transfer flavoprotein beta subunit lysine
methyltransferase (ETFB), F-box/LRR-repeat protein 7 (FBXL 7), integral
membrane
protein GPR137B (GPR137B), interferon alpha-inducible protein 27-like protein
1
20 (IF127L1), interleukin-15 receptor subunit alpha (IL15RA), killer cell
lectin-like receptor
subfamily G member 1 (KLRG1), lysosome-associated membrane glycoprotein 2
(LAMP2), glutathione S-transferase LANCL1 (LANCL1), major vault protein (MVP),
unconventional myosin-X (MY010), sialidase-1 (NEW), NETS-like protein 2
(NHSL2),
neurogenic locus notch homolog protein 3 (NOTCH3), neuronal PAS domain-
containing
25 protein 2 (NPAS2), olfactory receptor 1F1 (OR1F1), prolyl 4-hydroxylase
beta subunit
precursor (P4HB), protein disulfide isomerase (PD]), astrocytic phosphoprotein
PEA-15
(PEA15), phospholipase D3 (PLD3), receptor-type tyrosine-protein phosphatase C
isoform RA "CD45RA" (PTPRC), receptor-type tyrosine-protein phosphatase eta
"DEP1" (PTPRI), Ras-related protein Rab-23 (RAB23), retinoic acid receptor
beta
30 (RARB), RNA-binding region-containing protein 3 (RNPC3), protein
adenylyltransferase
Sel0 mitochondrial (SELO), thioredoxin reductase-like selenoprotein T (SEL7),
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semaphorin-5B (SEMA5B), stress-associated endoplasmic reticulum protein 1
(SERPI),
plasminogen activator inhibitor 1 (SERPINEI), sodium/hydrogen exchanger 7
(SLC9A 7),
sorting nexin-3 (SNX3), syntaxin-4 (STX4), TBC1 domain family member 1
(TBCIDI),
transforming growth factor beta regulator 1 (TBRGI), transcription elongation
factor A
N-terminal and central domain-containing protein (TCEANC), tissue factor
pathway
inhibitor (TFP1), BTB/POZ domain-containing adapter for CUL3-mediated RhoA
degradation protein 2 (INFAIP1), tumor necrosis factor receptor superfamily
member
10D "DCR2" (TNFRSFIOD), tubulin gamma-2 chain (TUBG2), Ubl carboxyl-terminal
hydrolase 18 (USP 18), vesicle-associated membrane protein 3 (VAMP3), vacuolar
protein sorting-associated protein 26A (VPS26A), and zinc finger protein 419
(ZNF4I9).
In one embodiment, the senescent cell-associated antigen is selected from the
group
comprising or consisting of DEP1 and DPP4.
In one embodiment, the senescent cell according to the present invention
expresses the
DEP1 and/or DPP4 antigen.
The presence of the senescent cell-associated antigens, in particular of DEP1
and/or
DPP4, can be determined by any one of numerous immunochemistry methods
practiced
in the art, such as immunoblotting analysis.
In one embodiment, the senescent cell-associated antigen is DEP1, such as,
e.g., human
DEP1, or orthologs thereof, including murine and rat DEP1. In one embodiment,
the
senescent cell-associated antigen is human DEP1 (hDEP1) with SEQ ID NO: 1.
In one embodiment, the antigen-binding domain of the invention recognizes and
is
capable of binding to DEP1, such as, e.g., to human DEP1, or orthologs
thereof, including
murine and rat DEP1. Hence, the antigen-binding domain of the invention is a
"DEP1-
binding domain".
In one embodiment, the DEP1-binding domain of the invention recognizes and is
capable
of binding to human DEP1 (hDEP1) with SEQ ID NO: 1.
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In one embodiment, the DEP1-binding domain of the invention recognizes and is
capable
of binding to the extracellular domain of human DEP1 (hDEP1) comprising or
consisting
of amino acid residues 36 ¨ 975 of SEQ ID NO: 1.
The binding between the DEP1-binding domain of the invention and DEP1 implies
that
said DEP1-binding domain exhibits appreciable affinity for DEP1. In other
words, the
DEP1-binding domain of the invention is specific for, or is immunospecific
for, or
specifically bind to, DEP1.
The affinity between the DEP1-binding domain of the invention and DEP1 can be
determined by various methods well known from the one skilled in the art.
These methods
include, but are not limited to, biosensor analysis (including, e.g., Biacore
analysis), Blitz
analysis and Scatchard plot.
Alternatively or additionally, whether the DEP1-binding domain of the
invention binds
to DEP1 can be tested readily by, inter alia, comparing the reaction of said
DEP1-binding
domain with DEP1 or a fragment thereof (in particular, a fragment comprising
or
consisting of an epitope of DEP1) with the reaction of said DEP1-binding
domain with
proteins or antigens other than DEP1 or a fragment thereof
In one embodiment, the DEP1-binding domain of the invention recognizes and is
capable
of binding to DEP1 with a KD-affinity constant less than or equal to 10' M,
preferably
less than or equal to 10-7 M, 5.10-8 M, 10-8 M, 5.10-9 M, 10-9 M or less; as
may be
determined, e.g., by biosensor analysis, particularly by Biacore Analysis.
In one embodiment, the DEP1-binding domain of the invention comprises a heavy
chain
variable region (abbreviated herein as HCVR or VH) which comprises at least
one,
preferably at least two, more preferably the following three complementary-
determining
regions (CDRs):
V11-CDR1: any one of SEQ ID NO: 5 to 11;
V11-CDR2: any one of SEQ ID NO: 12 to 25;
V11-CDR3: any one of SEQ ID NO: 26 to 32.
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SEQ ID SEQ ID
SEQUENCE SEQUENCE
NO NO
SYYIS 19 TISYDDSRTYYRDSVKG
6 NIAMY 20 YITNSFGSAYYRDSVKG
7 NYTIS 21 TISYDDYRTYYRDSVKG
8 SDSIS 22 YITNSLGSAYYRDSVKG
9 NYSIS 23 YITNSFGSTYYRDSVKG
DYNMA 24 YITNGYGSTYYRDSVKG
11 NYYMA 25 YITNGFGSTYYRDSVKG
12 YINTGSGGTNYNEKFKG 26 YFDY
13 HIRTKPHNFATYYANSVKG 27 GFGDY
14 YIYAGTGDTNYNEKFKG 28 YFDH
HIRTKPHNYATYYADSVKG 29 DKWVD
16 YIHPGSGVTNYNEKFKG 30 QGGIIRGVWFPY
17 YIHPGSGVTNYNEKFRG 31 VPLGAFVY
18 YIYPGSGDTNYNEKFKG 32 VPLGAFVS
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 5;
5 VH-CDR2: SEQ ID NO: 12;
VH-CDR3: SEQ ID NO: 26.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
10 VH-CDR1: SEQ ID NO: 6;
VH-CDR2: SEQ ID NO: 13;
VH-CDR3: SEQ ID NO: 27.
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In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 7;
VH-CDR2: SEQ ID NO: 14;
VH-CDR3: SEQ ID NO: 28.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 5;
VH-CDR2: SEQ ID NO: 12;
VH-CDR3: SEQ ID NO: 29.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 6;
VH-CDR2: SEQ ID NO: 15;
VH-CDR3: SEQ ID NO: 27.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 5;
VH-CDR2: SEQ ID NO: 16;
VH-CDR3: SEQ ID NO: 26.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 8;
VH-CDR2: SEQ ID NO: 16;
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VH-CDR3: SEQ ID NO: 26.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
5 VH-CDR1: SEQ ID NO: 5;
VH-CDR2: SEQ ID NO: 16;
VH-CDR3: SEQ ID NO: 26.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
10 complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 8;
VH-CDR2: SEQ ID NO: 17;
VH-CDR3: SEQ ID NO: 26.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
15 .. comprises at least one, preferably at least two, more preferably the
following three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 9;
VH-CDR2: SEQ ID NO: 18;
VH-CDR3: SEQ ID NO: 28.
20 .. In one embodiment, the DEP1-binding domain of the invention comprises a
HCVR which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 10;
VH-CDR2: SEQ ID NO: 19;
25 VH-CDR3: SEQ ID NO: 30.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 11;
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V11-CDR2: SEQ ID NO: 20;
SEQ ID NO: 31.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
V11-CDR1: SEQ ID NO: 10;
V11-CDR2: SEQ ID NO: 21;
V11-CDR3: SEQ ID NO: 30.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
.. comprises at least one, preferably at least two, more preferably the
following three
complementary-determining regions (CDRs):
V11-CDR1: SEQ ID NO: 11;
V11-CDR2: SEQ ID NO: 22;
V11-CDR3: SEQ ID NO: 31.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
V11-CDR1: SEQ ID NO: 11;
V11-CDR2: SEQ ID NO: 23;
V11-CDR3: SEQ ID NO: 31.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
V11-CDR1: SEQ ID NO: 11;
V11-CDR2: SEQ ID NO: 24;
SEQ ID NO: 31.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
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VH-CDR1: SEQ ID NO: 11;
VH-CDR2: SEQ ID NO: 25;
VH-CDR3: SEQ ID NO: 32.
In one embodiment, the DEP1-binding domain of the invention comprises a light
chain
variable region (abbreviated herein as LCVR or VI) which comprises at least
one,
preferably at least two, more preferably the following three complementary-
determining
regions (CDRs):
VL-CDR1: any one of SEQ ID NO: 33 to 39;
VL-CDR2: any one of SEQ ID NO: 40 to 46;
VL-CDR3: any one of SEQ ID NO: 47 to 54.
SEQ ID NO SEQUENCE SEQ ID NO SEQUENCE
33 RASQDVGIYVN 44 GATTLAD
34 KSSQSLKHSDGKTYLN 45 HANPLHD
35 QASQDIGNNLI 46 YANPLHD
36 RSSQSLKHSDGKTYLN 47 LQYDEFPPT
37 QASQDIGNWLA 48 CQGSYSPYT
38 LASEGISNYLA 49 LQYDEWPYT
39 LASEDIYSYLA 50 LQYDEYPPT
40 RATNLAD 51 QQTSSTPWT
41 QVSKLDS 52 QQGYKFPYT
42 YATNLAN 53 QQASSAPWT
43 RATTLAD 54 QQGYKFPYS
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 33;
VL-CDR2: SEQ ID NO: 40;
VL-CDR3: SEQ ID NO: 47.
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In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 34;
VL-CDR2: SEQ ID NO: 41;
VL-CDR3: SEQ ID NO: 48.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 35;
VL-CDR2: SEQ ID NO: 42;
VL-CDR3: SEQ ID NO: 47.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 33;
VL-CDR2: SEQ ID NO: 40;
VL-CDR3: SEQ ID NO: 49.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 36;
VL-CDR2: SEQ ID NO: 41;
VL-CDR3: SEQ ID NO: 48.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 33;
VL-CDR2: SEQ ID NO: 40;
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VL-CDR3: SEQ ID NO: 50.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 33;
VL-CDR2: SEQ ID NO: 43;
VL-CDR3: SEQ ID NO: 50.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 37;
VL-CDR2: SEQ ID NO: 44;
VL-CDR3: SEQ ID NO: 51.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 38;
VL-CDR2: SEQ ID NO: 45;
VL-CDR3: SEQ ID NO: 52.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 37;
VL-CDR2: SEQ ID NO: 44;
VL-CDR3: SEQ ID NO: 53.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 38;
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VL-CDR2: SEQ ID NO: 45;
VL-CDR3: SEQ ID NO: 54.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
5 complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 39;
VL-CDR2: SEQ ID NO: 45;
VL-CDR3: SEQ ID NO: 52.
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
which
10 comprises at least one, preferably at least two, more preferably the
following three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 38;
VL-CDR2: SEQ ID NO: 46;
VL-CDR3: SEQ ID NO: 52.
15 In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) at least one, preferably at least two, more preferably three HCVR's
CDRs and (ii)
at least one, preferably at least two, more preferably three LCVR's CDRs, said
combination being as defined in Table 1.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
20 of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination
being as
defined in Table 1.
TABLE 1. Preferred combinations of HCVR's and LCVR's CDRs. The CDRs are
defined by their SEQ ID NOs. First column indicates the clone's name.
Vll- Vii- Vll- VL- VL- VL-
Clone's name
CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
5738-10-R3A-B2 5 12 26 33 40 47
5738-10-R3A-C6 6 13 27 34 41 48
5738-10-R3A-D1 7 14 28 35 42 47
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5738-10-R3A-D5 5 12 29 33 40 49
5738-10-R3A-D8 6 15 27 36 41 48
5738-10-R3A-D11 5 16 26 33 40 47
5738-10-R4A-E7 8 16 26 33 40 47
5738-10-R4A-E9 5 16 26 33 40 50
5738-10-R4A-F12 8 16 26 33 40 50
5738-10-R4A-G4 8 17 26 33 40 47
5738-10-R4A-G11 9 18 28 33 43 50
5738-10-R4A-G12 8 16 26 33 40 50
5738-13-R2A-C1 10 19 30 37 44 51
5738-13-R2A-D3 11 20 31 38 45 52
5738-13-R4A-D11 10 21 30 37 44 53
5738-13-R3A-F5 11 22 31 38 45 54
5738-13-R4A-F11 11 23 31 39 45 52
5738-13-R2A-H3 11 24 31 38 45 52
5738-13-R2A-H4 11 25 32 38 46 52
5738-13-R4A-H9 11 25 32 38 45 52
5738-13-R4A-H11 11 25 32 38 45 52
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
any one of the following clones as defined in Table 1: 5738-13-R2A-C1, 5738-13-
R2A-
D3, 5738-13-R4A-D11, 5738-13-R3A-F5, 5738-13-R4A-F11, 5738-13-R2A-H3, 5738-
13-R2A-H4, 5738-13-R4A-H9, and 5738-13-R4A-H11.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
any one of the following clones as defined in Table 1: 5738-10-R3A-C6, 5738-10-
R3A-
D5, 5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
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any one of the following clones as defined in Table 1: 5738-10-R4A-G12, 5738-
13-R4A-
D11, and 5738-13-R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
.. any one of the following clones as defined in Table 1: 5738-13-R4A-D11, and
5738-13-
R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
clone 5738-13-R2A-C1 as defined in Table 1. In one embodiment, the DEP1-
binding
domain of the invention comprises a combination of (i) three HCVR's CDRs and
(ii) three
LCVR's CDRs, said combination being that of clone 5738-13-R2A-D3 as defined in
Table 1. In one embodiment, the DEP1-binding domain of the invention comprises
a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5738-13-R4A-D11 as defined in Table 1. In one embodiment,
the
DEP1-binding domain of the invention comprises a combination of (i) three
HCVR's
CDRs and (ii) three LCVR's CDRs, said combination being that of clone 5738-13-
R3A-
F5 as defined in Table 1. In one embodiment, the DEP1-binding domain of the
invention
comprises a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5738-13-R4A-F11 as defined in Table 1. In one
embodiment, the DEP1-binding domain of the invention comprises a combination
of (i)
three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of
clone
5738-13-R2A-H3 as defined in Table 1. In one embodiment, the DEP1-binding
domain
of the invention comprises a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5738-13-R2A-H4 as defined in
Table 1. 5738-13-R4A-H9 as defined in Table 1. In one embodiment, the DEP1-
binding
domain of the invention comprises a combination of (i) three HCVR's CDRs and
(ii) three
LCVR's CDRs, said combination being that of clone 5738-13-R4A-H11 as defined
in
Table 1.
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 55; or a HCVR comprising
or
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consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 55.
SEQ ID NO: 55
QVQLKQSGAELAKPGSSVKISCKASGYTFTSYYISWIKQTTGQGLEYIGYINTGS
GGTNYNEKFKGKATLTVDKS S STAFMQL S SLTPDDSAVYYCARYFDYWGQGV
MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 56; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 56.
SEQ ID NO: 56
E VKLVES GGGLVQPKE SLKI SC AA S GF TF SNIAMYWVRQAPGKGLEWVAHIRT
KPHNF AT YYAN S VKGRF TISRDD SKNMVYLQMDNLKPEDTAMYYC SVGF GDY
WGQGVMVTVSS
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 57; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 57.
SEQ ID NO: 57
EVQLQQSGAELAKPGS SVKISCKASGYTFTNYTISWIKQTTGQGLEYIGYIYAGT
GDTNYNEKFKGKATLTVDKS SNTAFMQL S SLTPDD SAVYYCARYFDHWGQGV
MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 58; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 58.
SEQ ID NO: 58
EVQLQQSGAELAKPGS SVKISCKASGYTFTSYYISWIKQTTGQGLEYIGYINTGS
GGTNYNEKFKGKATLTVDKS S STAFMQL S SLTPDDSAVYYCARDKWVDWGQ
GVMVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 59; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 59.
SEQ ID NO: 59
EVQLEESGGGLVQPKESLKISCAVSGFTFSNIAMYWVRQAPGKGLEWVGHIRT
KPHNYATYYAD S VKGRFTISRDD SNNMVYLEMDNLKPEDTAMYYC SVGF GDY
WGQGVMVTVSS
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 60; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 60.
SEQ ID NO: 60
QVQLKQSGAELAKPGSSVKISCKASGYTFTSYYISWIKQTTGQGLEYIGYIHPGS
GVTNYNEKFKGKATLTVDKS S STAFMQL SSLTPDDSAIYYCARYFDYWGQGV
MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 61; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 61.
SEQ ID NO: 61
QVQLKQ SGVELAKPGS S VKISCKASGYTF T SD SISWIKQTTGQGLEYIGYIHPGS
5 GVTNYNEKFKGKATLTVDKS S STAFMQL SSLTPDDSAIYYCARYFDYWGQGV
MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 62; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
10 98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 62.
SEQ ID NO: 62
EVQLQQSGVELAKPGS SVKISCKASGYTFT SD SISWIKQTTGQGLEYIGYIHPGS
GVTNYNEKFKGKATLTVDKS S STAFMQL SSLTPDDSAIYYCARYFDYWGQGV
15 MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 63; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
20 .. regions) of SEQ ID NO: 63.
SEQ ID NO: 63
EVQLQQSGVELAKPGS SVKISCKASGYTFT SD SISWIKQTTGQGLEYIGYIHPGS
GVTNYNEKFRGKATLTVDKS S STAFMQL S SLTPDDSAIYYCARYFDYWGQGV
MVTVS S
25 In one embodiment, the DEP1-binding domain of the invention comprises a
HCVR
comprising or consisting of the sequence SEQ ID NO: 64; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 64.
SEQ ID NO: 64
QVQLQQPRAELAKPGSSVKISCKASGYTFTNYSISWIKQTTGQGLEYIGYIYPGS
GDTNYNEKFKGKATLTVDKS S STAFMQL S SL TPDD SAVYYC ARYFDHWGQ GT
LVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 65; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 65.
SEQ ID NO: 65
QVQLKESGAELAKPGS SVKISCKASGYTF T SD SISWIKQTTGQ GLEYIGYIHPGS
GVTNYNEKFKGKATLTVDKS S STAFMQL SSLTPDDSAIYYCARYFDYWGQGV
MVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 66; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 66.
SEQ ID NO: 66
QVQLKESGGGLVQPGRSLKL SC AA S GF TF SDYNMAWVRQAPKKGLEWVATIS
YDD SRTYYRDSVKGRFAISRDDAKGTLNLQMDSLRSEDTATYYCARQGGIIRG
VWFPYWGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 67; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 67.
SEQ ID NO: 67
E VKLVES GGGLVQP GGSLKL S CAA S GF TF SNYYMAWVRQ APTKGLEWVAYIT
NSFGSAYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCSTVPLGAFVY
WGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 68; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 68.
SEQ ID NO: 68
QVQLKESGGGLVQPGRSLKL SC AA S GF SF GDYNMAWVRQAPKKGLEWVATIS
YDDYRTYYRDSVKGRFTISRDDAKATLYLQMDSLRSEDTATYYCARQGGIIRG
VWFPYWGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 69; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 69.
SEQ ID NO: 69
E VKLVES GGGLVQP GGSLKL S CAA S GF TF SNYYMAWVRQ APTKGLEWVAYIT
NSLGSAYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYC STVPLGAFV
YWGQGTLVTVSS
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 70; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 70.
SEQ ID NO: 70
E VKLVES GGGLVQP GGSLKL S CAA S GF TF SNYYMAWVRQ APTKGLEWVAYIT
NSF GSTYYRD S VKGRF TISRDNAK STLYLQMD SLRSEDTATYYC STVPLGAFVY
WGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 71; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 71.
SEQ ID NO: 71
E VKLVES GGGLVQP GRSLKL S CAA S GF TF SNYYMAWVRQAPTKGLEWVAYIT
NGYGSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCSTVPLGAFV
YWGQGTLVTVSS
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 72; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 72.
SEQ ID NO: 72
E VKLVES GGGLVQP GRSLKL S CAA S GF TF SNYYMAWVRQAPTKGLEWVAYIT
NGF GSTYYRD S VKGRF TISRDNAK STLYLQMD SLRSEDTATYYC STVPLGAF VS
WGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 73; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 73.
SEQ ID NO: 73
E VKLVES GGGLVQP GRSLKL S CAA S GF TF SNYYMAWVRQAPTKGLEWVAYIT
NGFGSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDAATYYCSTVPLGAFVS
WGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 74; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 74.
SEQ ID NO: 74
E VKLVES GGGLVQP GGSLKL S CAA S GF TF SNYYMAWVRQ APTKGLEWVAYIT
NGF GSTYYRD S VKGRF TISRDNAK STLYLQMD SLRSEDTATYYC STVPLGAF VS
WGQGTLVTVS S
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 75; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 75.
SEQ ID NO: 75
DIVMTQ SP S SM SVSL GDTVTITCRA S QDVGIYVNWF QQKP GKPPRRMIYRATNL
ADGVPSRFSGTRSGSDYSLTIS SLESEDVADYHCLQYDEFPPTFGSGTKLDIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 76; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 76.
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SEQ ID NO: 76
DIVMTQAPLSL SVAIGQSASISCKSSQSLKHSDGKTYLNWIFQSPGQSPKRLIYQ
VSKLDSGVPDRF S GTGSETDF TLKI SRVEAEDL GVYYCC Q GS Y SP YTF GAGTKL
5 ELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 77; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
10 regions) of SEQ ID NO: 77.
SEQ ID NO: 77
DILMTQ SP S SM SA SL GDRVTITCQ A S QDIGNNLIWF QQKP GK SPRRMIYYATNL
ANGVPSRFSGSRSGSDYSL SIS SLESEDVADYHCLQYDEFPPTFGSGTKLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
15 comprising or consisting of the sequence SEQ ID NO: 78; or a LCVR
comprising or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 78.
SEQ ID NO: 78
20 DILMT Q SP S SMSVSLGDTVTITCRASQDVGIYVNWFQQKPGKPPRRMIYRATNL
ADGVP SRF SGSRSGSNYSLTIRSLE SEDVADYHCLQYDEWPYTF GAGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 79; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
25 98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 79.
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SEQ ID NO: 79
DIVMTQAPLSL SVDIGQSASISCRS SQ SLKHSDGKTYLNWVFQSPGQSPKRLIYQ
VSKLDSGVPDRF SGSGSEADFTLKISRVEAEDLGVYYCCQGSYSPYTFGAGTKL
ELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 80; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 80.
SEQ ID NO: 80
DIQL TQ SP S SMSVSQGDTVTITCRASQDVGIYVNWFQQKPGKSPRRMIYRATNL
ADGVPSRFSGSRSGSDYSLTIASLESEDVADYHCLQYDEFPPTFGSGTNLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 81; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 81.
SEQ ID NO: 81
DILMTQ SP S SMSVSLGDTVTITCRASQDVGIYVNWFQQIPGKSPRRLIYRATNLA
DGVPSRF SGSRSGSDYSLTIASLESEDVADYHCLQYDEFPPTFGSGTKLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 82; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 82.
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SEQ ID NO: 82
DILMTQ SP S SMSVSQGDTVTITCRASQDVGIYVNWFQQKPGKSPRRMIHRATNL
ADGVP SRF SGSRSGSDYSLTIT SLESEDVADYHCLQYDEYPPTFGSGTNLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 83; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 83.
SEQ ID NO: 83
DILMT Q SP S SMSVSLGDTVTITCRASQDVGIYVNWFQQKPGKSPRRMIHRATNL
ADGVPSRFSGSRSGSDYSLTIS SLESEDVADYHCLQYDEYPPTFGSGTKLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 84; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 84.
SEQ ID NO: 84
DIVMTQ SP SSMSVSLGDTVTITCRASQDVGIYVNWFQQKPGKSPRRMIYRATNL
ADGVPSRFSGSRSGSDYSLTIASLESEDVADYHCLQYDEFPPTFGSGTKLEIK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 85; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 85.
SEQ ID NO: 85
DILMT Q SP S SMSVSLGDTVTITCRASQDVGIYVNWFQQKPGKSPRRMIYRATTL
ADGVPSRFSGSRSGSDYSLTIS SLESEDVADYHCLQYDEYPPTFGSGTKLEIK
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In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 86; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 86.
SEQ ID NO: 86
DILMTQ SP S SM S V SLGD TVTIT CRA S QDVGIYVNWF Q QKP GK SPRRMIYRATNL
ADGVPSRF SGSRSGSDYSLTIS SLESEDVADYHCLQYDEYPPTF GGGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 87; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 87.
SEQ ID NO: 87
DIQLTQ SPASL SASLEEIVTITCQASQDIGNWLAWYQQKPGKSPHLLIYGATTLA
DGVPSRSGSRSGTQYSLKISRLQVEDVGMYYCQQTS STPWTFGGGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 88; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 88.
SEQ ID NO: 88
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLISHANPLH
DGVPSRFSGDGSGTQYSLKIRNMQPEDEGVYYCQQGYKFPYTFGAGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 89; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 89.
SEQ ID NO: 89
DIQMTQTPASL SASLEEIVTITCQASQDIGNWLAWYQQKPGKSPHLLIYGATTL
ADGVPSRF SGSRSGTQYSLKISRLQAEDIGIYYCQQASSAPWTFGGGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 90; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 90.
SEQ ID NO: 90
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLISHANPLH
DGVPSRF SGSGSGTQYSLKIRNMQPEDEGVYYCQQGYKFPYSFGAGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 91; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 91.
SEQ ID NO: 91
DIQLTQ SPASL SASLGETVSIECLASEDIYSYLAWYQQKPGKSPQLLISHANPLH
DGVPSRF SGSGSGTQYSLKIRNMQPEDEGVYYCQQGYKFPYTFGAGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 92; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 92.
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SEQ ID NO: 92
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLISHANPLH
DGVPSRF SGSGSGTQYSLKIRNMQPEDEGVYYCQQGYKFPYTFGAGTKLELK
5 .. In one embodiment, the DEP1-binding domain of the invention comprises a
LCVR
comprising or consisting of the sequence SEQ ID NO: 93; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 93.
10 SEQ ID NO: 93
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLISYANPLH
DGVPSRF SGSGSGTQF SLKIRNMQPEDEGVYYCQQGYKFPYTFGAGTKLELT
In one embodiment, the DEP1-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 94; or a LCVR comprising
or
15 consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 94.
SEQ ID NO: 94
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLISHANPLH
20 DGVPSRF S GS GSGT QF SLKIRNMQPEDEGVYYCQQGYKFPYTFGAGTKLELK
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, said combination being as defined in Table 2.
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TABLE 2. Preferred combinations of HCVR and LCVR. The HCVR and LCVR are
defined by their SEQ ID NOs. First column indicates the clone's name.
Clone's name HCVR LCVR
5738-10-R3A-B2 55 75
5738-10-R3A-C6 56 76
5738-10-R3A-D1 57 77
5738-10-R3A-D5 58 78
5738-10-R3A-D8 59 79
5738-10-R3A-D11 60 80
5738-10-R4A-E7 61 81
5738-10-R4A-E9 60 82
5738-10-R4A-F12 62 83
5738-10-R4A-G4 63 84
5738-10-R4A-G11 64 85
5738-10-R4A-G12 65 86
5738-13-R2A-C1 66 87
5738-13-R2A-D3 67 88
5738-13-R4A-D11 68 89
5738-13-R3A-F5 69 90
5738-13-R4A-F11 70 91
5738-13-R2A-H3 71 92
5738-13-R2A-H4 72 93
5738-13-R4A-H9 73 94
5738-13-R4A-H11 74 94
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 2: 5738-13-R2A-C1, 5738-13-R2A-D3, 5738-13-R4A-D11, 5738-
13-R3A-F5, 5738-13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-H4, 5738-13-R4A-H9,
and 5738-13-R4A-H11.
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In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 2: 5738-10-R3A-C6, 5738-10-R3A-D5, 5738-10-R4A-G12, 5738-
13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 2: 5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 2: 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the DEP1-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of clone 5738-13-R2A-C1
as
defined in Table 2. In one embodiment, the DEP1-binding domain of the
invention
comprises a combination of (i) a HCVR and (ii) a LCVR, combination being that
of clone
5738-13-R2A-D3 as defined in Table 2. In one embodiment, the DEP1-binding
domain
of the invention comprises a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5738-13-R4A-D11 as defined in Table 2. In one embodiment,
the
DEP1-binding domain of the invention comprises a combination of (i) a HCVR and
(ii)
a LCVR, combination being that of clone 5738-13-R3A-F5 as defined in Table 2.
In one
embodiment, the DEP1-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5738-13-R4A-F11 as
defined
in Table 2. In one embodiment, the DEP1-binding domain of the invention
comprises a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R2A-H3 as defined in Table 2. In one embodiment, the DEP1-binding domain of
the
invention comprises a combination of (i) a HCVR and (ii) a LCVR, combination
being
that of clone 5738-13-R2A-H4 as defined in Table 2. In one embodiment, the
DEP1-
binding domain of the invention comprises a combination of (i) a HCVR and (ii)
a LCVR,
combination being that of clone 5738-13-R4A-H9 as defined in Table 2. 5738-13-
R4A-
H11 as defined in Table 2.
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In one embodiment, the senescent cell-associated antigen is DPP4, such as,
e.g., human
DPP4 or orthologs thereof, including murine and rat DPP4. In one embodiment,
the
senescent cell-associated antigen is human DPP4 (hDPP4) with SEQ ID NO: 101.
In one embodiment, the antigen-binding domain of the invention recognizes and
is
capable of binding to DPP4, such as, e.g., to human DPP4, or orthologs
thereof, including
murine and rat DPP4. Hence, the antigen-binding domain of the invention is a
"DPP4-
binding domain".
In one embodiment, the DPP4-binding domain of the invention recognizes and is
capable
of binding to human DPP4 (hDPP4) with SEQ ID NO: 101.
In one embodiment, the DPP4-binding domain of the invention recognizes and is
capable
of binding to the extracellular domain of human DPP4 (hDPP4) comprising or
consisting
of amino acid residues 29- 766 of SEQ ID NO: 101.
The binding between the DPP4-binding domain of the invention and DPP4 implies
that
said DPP4-binding domain exhibits appreciable affinity for DPP4. In other
words, the
DPP4-binding domain of the invention is specific for, or is immunospecific
for, or
specifically bind to, DPP4.
The affinity between the DPP4-binding domain of the invention and DPP4 can be
determined by various methods well known from the one skilled in the art.
These methods
include, but are not limited to, biosensor analysis (including, e.g., Biacore
analysis), Blitz
analysis and Scatchard plot.
Alternatively or additionally, whether the DPP4-binding domain of the
invention binds
to DPP4 can be tested readily by, inter alia, comparing the reaction of said
DPP4-binding
domain with DPP4 or a fragment thereof (in particular, a fragment comprising
or
consisting of an epitope of DPP4) with the reaction of said DPP4-binding
domain with
proteins or antigens other than DPP4 or a fragment thereof
In one embodiment, the DPP4-binding domain of the invention recognizes and is
capable
of binding to DPP4 with a KD-affinity constant less than or equal to 10' M,
preferably
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less than or equal to 10-7 M, 5.10-8 M, 10-8 M, 5.10-9 M, 10-9 M or less; as
may be
determined, e.g., by biosensor analysis, particularly by Biacore Analysis.
In one embodiment, the DPP4-binding domain of the invention comprises a heavy
chain
variable region (abbreviated herein as HCVR or VH) which comprises at least
one,
preferably at least two, more preferably the following three complementary-
determining
regions (CDRs):
VH-CDR1: any one of SEQ ID NO: 104 to 112;
VH-CDR2: any one of SEQ ID NO: 113 to 129;
VH-CDR3: any one of SEQ ID NO: 130 to 142.
SEQ ID SEQ ID
SEQUENCE SEQUENCE
NO NO
104 NYGMA 124 YINPGGGGIGYNEKFKG
105 TSDRCVS 125 QISHSGSTSYNPSLKS
106 NFGMA 126 SINPGSGGIGYNEKFKG
107 DNYWG 127 SINPGGGGTGYNEKFKG
108 TYDIG 128 QISHSGSTSYNPSLIS
109 GNYLA 129 QISHTGSSTYNPSLKS
110 SNYWG 130 HRLIYTTDYYYEVMDV
111 TYDRG 131 HRLIYTTDYYYEVMDA
112 GNYWG 132 NSGDGRFAY
113 TISYDGNDTYYRDSVKG 133 HICLIYTTDYYYEVMDA
114 TTSYDGNDTYYRDSVKG 134 HRLMYTTDYYYEVMDD
115 TIC WDDSKGYNPSLKN 135 HRLIYTTDYYYEVLDA
116 TINYDGRNTYYRDSVKG 136 HICLIYTTDYYYEVMDV
117 TINYDGSNTYYRDSVKG 137 YGAGASFDY
118 TINYDGRDTYYRDSVKG 138 PLRRVLDY
119 HISHSGSSTYNPSLKS 139 HGHYVMDV
120 YINPGSGGIGYNEKFKG 140 YGAGSSFDY
121 SINPGSGGIAYSEKFKG 141 PLRRVLDN
122 HIKSSGTTTYNPSLKS 142 PLRVLDY
123 SINPGSGGIGYNERFKG
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In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 104;
5 VH-CDR2: SEQ ID NO: 113;
VH-CDR3: SEQ ID NO: 130.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
10 VH-CDR1: SEQ ID NO: 104;
VH-CDR2: SEQ ID NO: 114;
VH-CDR3: SEQ ID NO: 131.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
15 complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 105;
VH-CDR2: SEQ ID NO: 115;
VH-CDR3: SEQ ID NO: 132.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
20 comprises at least one, preferably at least two, more preferably the
following three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 104;
VH-CDR2: SEQ ID NO: 116;
VH-CDR3: SEQ ID NO: 133.
25 In one embodiment, the DPP4-binding domain of the invention comprises a
HCVR which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 104;
VH-CDR2: SEQ ID NO: 117;
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VH-CDR3: SEQ ID NO: 134.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 104;
VH-CDR2: SEQ ID NO: 113;
VH-CDR3: SEQ ID NO: 135.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 104;
VH-CDR2: SEQ ID NO: 116;
VH-CDR3: SEQ ID NO: 136.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 106;
VH-CDR2: SEQ ID NO: 118;
VH-CDR3: SEQ ID NO: 131.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 107;
VH-CDR2: SEQ ID NO: 119;
VH-CDR3: SEQ ID NO: 137.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
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VH-CDR2: SEQ ID NO: 120;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 121;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 109;
VH-CDR2: SEQ ID NO: 122;
VH-CDR3: SEQ ID NO: 139.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 123;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 124;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
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VH-CDR1: SEQ ID NO: 110;
VH-CDR2: SEQ ID NO: 125;
VH-CDR3: SEQ ID NO: 140.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 126;
VH-CDR3: SEQ ID NO: 141.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 126;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 111;
VH-CDR2: SEQ ID NO: 127;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 110;
VH-CDR2: SEQ ID NO: 128;
VH-CDR3: SEQ ID NO: 140.
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In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 112;
VH-CDR2: SEQ ID NO: 125;
VH-CDR3: SEQ ID NO: 140.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 127;
VH-CDR3: SEQ ID NO: 138.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VH-CDR1: SEQ ID NO: 108;
VH-CDR2: SEQ ID NO: 127;
VH-CDR3: SEQ ID NO: 142.
In one embodiment, the DPP4-binding domain of the invention comprises a light
chain
variable region (abbreviated herein as LCVR or VI) which comprises at least
one,
preferably at least two, more preferably the following three complementary-
determining
regions (CDRs):
VL-CDR1: any one of SEQ ID NO: 143 to 151;
VL-CDR2: any one of SEQ ID NO: 152 to 163;
VL-CDR3: any one of SEQ ID NO: 164 to 173.
SEQ ID NO SEQUENCE SEQ ID NO SEQUENCE
143 KSSQSLLYNENKKNYLA 159 YTSNLQS
144 KSSQSLLHSNGNTYLN 160 DASHLAS
145 RSSQSLLHSNGNTYLN 161 YTS SFQD
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146 LASEGISNYLA 162 YTISLQD
147 RASQGISNKLN 163 YASSLQD
148 RASQSVSTSTYNFMH 164 QEYYKFPWT
149 RA SQ GIGNKLN 165 QDYYHFPWT
150 RASQGISKKLN 166 MQATHAPFT
151 GA S Q GIGNKVN 167 QQYYKFPWP
152 WASTRES 168 QQYYKFPWT
153 SVSKLES 169 QQYYKFPYT
154 WASTREA 170 QQGYKYPWT
155 WASTRKS 171 QQDASFPPT
156 SVSNLES 172 QQSRELPLT
157 YTSSLQD 173 QQDTSFPPT
158 YTSRLQS
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 143;
5 VL-CDR2: SEQ ID NO: 152;
VL-CDR3: SEQ ID NO: 164.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
10 VL-CDR1: SEQ ID NO: 143;
VL-CDR2: SEQ ID NO: 152;
VL-CDR3: SEQ ID NO: 165.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
15 complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 144;
VL-CDR2: SEQ ID NO: 153;
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VL-CDR3: SEQ ID NO: 166.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 143;
VL-CDR2: SEQ ID NO: 154;
VL-CDR3: SEQ ID NO: 167.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 143;
VL-CDR2: SEQ ID NO: 155;
VL-CDR3: SEQ ID NO: 168.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 145;
VL-CDR2: SEQ ID NO: 156;
VL-CDR3: SEQ ID NO: 166.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 143;
VL-CDR2: SEQ ID NO: 154;
VL-CDR3: SEQ ID NO: 169.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 146;
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VL-CDR2: SEQ ID NO: 157;
VL-CDR3: SEQ ID NO: 170.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 147;
VL-CDR2: SEQ ID NO: 158;
VL-CDR3: SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 147;
VL-CDR2: SEQ ID NO: 159;
VL-CDR3: SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 148;
VL-CDR2: SEQ ID NO: 160;
VL-CDR3: SEQ ID NO: 172.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 149;
VL-CDR2: SEQ ID NO: 159;
VL-CDR3: SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
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VL-CDR1: SEQ ID NO: 146;
VL-CDR2: SEQ ID NO: 161;
VL-CDR3: SEQ ID NO: 170.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 150;
VL-CDR2: SEQ ID NO: 159;
VL-CDR3: SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 151;
VL-CDR2: SEQ ID NO: 159;
VL-CDR3: SEQ ID NO: 171.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 146;
VL-CDR2: SEQ ID NO: 162;
VL-CDR3: SEQ ID NO: 170.
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 149;
VL-CDR2: SEQ ID NO: 159;
VL-CDR3: SEQ ID NO: 173.
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In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
which
comprises at least one, preferably at least two, more preferably the following
three
complementary-determining regions (CDRs):
VL-CDR1: SEQ ID NO: 146;
VL-CDR2: SEQ ID NO: 163;
VL-CDR3: SEQ ID NO: 170.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) at least one, preferably at least two, more preferably three HCVR's
CDRs and (ii)
at least one, preferably at least two, more preferably three LCVR's CDRs, said
combination being as defined in Table 3.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being as
defined in Table 3.
TABLE 3. Preferred combinations of HCVR's and LCVR's CDRs. The CDRs are
defined by their SEQ ID NOs. First column indicates the clone's name.
Vii- Vii- Vii- VL- VL- VL-
Clone's name
CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
5826-8-R6A-A1 0 104 113 130 143 152 164
5826-8-R6A-B11 104 114 131 143 152 165
5826-8-R6A-D12 105 115 132 144 153 166
5826-8-R6A-E10 104 116 133 143 154 167
5826-8-R5A-G6 104 117 134 143 152 165
5826-8-R5A-G8 104 113 135 143 155 168
5826-8-R6A-H9 104 116 136 143 154 167
5826-8-R6A-H11 105 115 132 145 156 166
5826-8-R6A-H12 106 118 131 143 154 169
5826-13-R3A-A10 107 119 137 146 157 170
5826-13-R3A-B1 108 120 138 147 158 171
5826-13-R3A-B3 108 121 138 147 159 171
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5826-13-R3A-D5 109 122 139 148 160 172
5826-13-R3A-D6 108 123 138 149 159 171
5826-13-R4A-E2 108 124 138 147 159 171
5826-13-R4A-E6 110 125 140 146 161 170
5826-13-R4A-E9 108 126 141 149 159 171
5826-13-R4A-F10 108 126 138 150 159 171
5826-13-R4A-G11 108 126 138 150 159 171
5826-13-R4A-G12 111 127 138 151 159 171
5826-13-R4A-H1 110 128 140 146 162 170
5826-13-R4A-H2 108 126 141 149 159 171
5826-13-R4A-H3 108 126 138 149 159 173
5826-13-R4A-H4 112 125 140 146 162 170
5826-13-R4A-H5 108 127 138 147 159 171
5826-13-R4A-H6 108 127 142 147 159 171
5826-13-R4A-H9 108 123 138 149 159 171
5826-13-R4A-H10 110 125 140 146 162 170
5826-13-R4A-H11 110 128 140 146 162 170
5826-13-R4A-H12 110 125 140 146 163 170
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
any one of the following clones as defined in Table 3: 5826-13-R3A-A10, 5826-
13-R3A-
B1, 5826-13-R3A-B3, 5826-13-R3A-D5, 5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-
5 R4A-E6, 5826-13-R4A-E9, 5826-13-R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-
G12,
5826-13-R4A-H1, 5826-13-R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-
R4A-H5, 5826-13-R4A-H6, 5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11,
and 5826-13-R4A-H12.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
10 of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination
being that of
any one of the following clones as defined in Table 3: 5826-8-R6A-E10, 5826-8-
R5A-
G8, 5826-8-R6A-H11, 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
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In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
any one of the following clones as defined in Table 3: 5826-13-R3A-D5, 5826-13-
R4A-
H5, and 5826-13-R4A-H12.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being
that of
clone 5826-13-R3A-A10 as defined in Table 3. In one embodiment, the DPP4-
binding
domain of the invention comprises a combination of (i) three HCVR's CDRs and
(ii) three
LCVR's CDRs, said combination being that of clone 5826-13-R3A-B1 as defined in
Table 3. In one embodiment, the DPP4-binding domain of the invention comprises
a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R3A-B3 as defined in Table 3. In one embodiment,
the
DPP4-binding domain of the invention comprises a combination of (i) three
HCVR's
CDRs and (ii) three LCVR's CDRs, said combination being that of clone 5826-13-
R3A-
D5 as defined in Table 3. In one embodiment, the DPP4-binding domain of the
invention
comprises a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R3A-D6 as defined in Table 3. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of
clone
5826-13-R4A-E2 as defined in Table 3. In one embodiment, the DPP4-binding
domain
of the invention comprises a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-E6 as defined in
Table 3. In one embodiment, the DPP4-binding domain of the invention comprises
a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-E9 as defined in Table 3. In one embodiment,
the
DPP4-binding domain of the invention comprises a combination of (i) three
HCVR's
CDRs and (ii) three LCVR's CDRs, said combination being that of clone 5826-13-
R4A-
F10 as defined in Table 3. In one embodiment, the DPP4-binding domain of the
invention
comprises a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-G11 as defined in Table 3. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
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three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of
clone
5826-13-R4A-G12 as defined in Table 3. In one embodiment, the DPP4-binding
domain
of the invention comprises a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-H1 as defined in
Table 3. In one embodiment, the DPP4-binding domain of the invention comprises
a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-H2 as defined in Table 3. In one embodiment,
the
DPP4-binding domain of the invention comprises a combination of (i) three
HCVR's
CDRs and (ii) three LCVR's CDRs, said combination being that of clone 5826-13-
R4A-
H3 as defined in Table 3. In one embodiment, the DPP4-binding domain of the
invention
comprises a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H4 as defined in Table 3. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of
clone
5826-13-R4A-H5 as defined in Table 3. In one embodiment, the DPP4-binding
domain
of the invention comprises a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-H6 as defined in
Table 3. In one embodiment, the DPP4-binding domain of the invention comprises
a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-H9 as defined in Table 3. In one embodiment,
the
DPP4-binding domain of the invention comprises a combination of (i) three
HCVR's
CDRs and (ii) three LCVR's CDRs, said combination being that of clone 5826-13-
R4A-
H10 as defined in Table 3. In one embodiment, the DPP4-binding domain of the
invention comprises a combination of (i) three HCVR's CDRs and (ii) three
LCVR's
CDRs, said combination being that of clone 5826-13-R4A-H11 as defined in Table
3. In
one embodiment, the DPP4-binding domain of the invention comprises a
combination of
(i) three HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that
of
clone 5826-13-R4A-H12 as defined in Table 3.
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 174; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 174.
SEQ ID NO: 174
EVQLEESGGGLVQPGRSLKL S CAA S GF TFNNYGMAWVRQAP TKGLEWVATI S
YDGNDTYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCVRHRLIYTT
DYYYEVMDVWGQGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 175; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 175.
SEQ ID NO: 175
QVQLKESGGGLVQPGRSLKL SC AA S GF TF SNYGMAWVRQAPTKGLEWVATTS
YDGNDTYYRDSVKGRFTVSRDNAKNTLYLQMDSLRSEDTATYYCVRHRLIYT
TDYYYEVMDAWGQGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 176; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 176.
SEQ ID NO: 176
QVTLKESGPGILQPSQTL SLTC SF SGF SL S T SDRC VS WIRQP SGKGLEWLATICW
DD SKGYNPSLKNRLTISKDTSNNQAFLKITSVGTADIAKYYCARNSGDGRFAY
WGQGTLVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 177; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 177.
SEQ ID NO: 177
EVQLEESGGGLVQPGRSLKL SC AA S GF TF SNYGMAWVRQAPTKGLEWVATIN
YDGRNTYYRDSVKGRFTISRDNAKSTLYLQVDSLQSEDTATYYCTREIKLIYTT
DYYYEVMDAWGQGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 178; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 178.
SEQ ID NO: 178
EVKLVESGGGLVQPGRSLKLSCAASGF SF TNYGMAWVRQAPTKGLEWVATIN
YDGSNTYYRDSVKGRFTISRDNAKRTLDLQMDSLRSEDTATYYCARHRLMYT
TDYYYEVMDDWGQGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 179; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 179.
SEQ ID NO: 179
EVKLVESGGGLVQPGRSLKL SCAAS GF SFRNYGMAWVRQAPTKGQEWVATIS
YDGNDTYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCTRHRLIYTT
DYYYEVLDAWGQGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 180; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 180.
SEQ ID NO: 180
EVKLVESGGGLVQPGRSLKLSCTASGFTF SNYGMAWVRQAPTKGLEWVATIN
5 YDGRNTYYRDSVKGRFTISRDNAKSTLYLQVDSLQSEDTATYYCTREIKLIYTT
D YY YEVMDVWGQ GA S VAVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 181; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
10 98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 181.
SEQ ID NO: 181
EVKLVESGGALVQPGRSLKLSCAASGFTF SNFGMAWVRQAPTKGLEWVATIN
YDGRDTYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCTRHRLIYTT
15 DYYYEVMDAWGRGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 182; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
20 regions) of SEQ ID NO: 182.
SEQ ID NO: 182
EVKLVESGPGLVKPSQSL SLAC SITDYSITDNYWGWIRKFPGNKMEWIGHISHS
GS STYNPSLKSRISFTRDTSKNQFFLQLNSVTPEDTATYFCARYGAGASFDYWG
QGVMVTVSS
25 In one embodiment, the DPP4-binding domain of the invention comprises a
HCVR
comprising or consisting of the sequence SEQ ID NO: 183; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 183.
SEQ ID NO: 183
EVQLQQSGAELTKPGSSVKISCKASGFTFTTYDIGWLKQRPGQALEWIGYINPG
SGGIGYNEKFKGKATLTVDKSS STAFMQL S SLTPEDTAVYYCARPLRRVLDYW
GQGVMVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 184; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 184.
SEQ ID NO: 184
EVQLQQ SGAGLTKPGAS VKISCKASGYTF TTYDIGWIKQRPGQALEWIGSINPG
SGGIAYSEKFKGKATLTVDKS S STAFMQLS SLTPEDTAVYYCARPLRRVLDYW
GQGVLVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 185; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 185.
SEQ ID NO: 185
E VKL VE S GP GL VKP SQ SL SLTC S VTGYF IT GNYL AWIRKFPGNKMEW IGHIK S S
GTTTYNPSLKSRVSITRDTSKNQFFLQLNSVTSEDTATYYCARHGHYVMDVWG
QGASVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 186; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 186.
SEQ ID NO: 186
EVQLQQSGAELTKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNERFKGKATLTVDKS S STAFMQL S SLTPEDTAVYYCARPLRRVLDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 187; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 187.
SEQ ID NO: 187
QVQLKQSGAELTKPGS SVKISCKASGYTFTTYDIGWLKQRPGQALEWIGYINPG
GGGIGYNEKFKGKATLTVDKS S STAFMQL S SLTPEDTAVYYCARPLRRVLDYW
GQGVMVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 188; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 188.
SEQ ID NO: 188
DVKLQESGPGLVKPSQSLSLTC SVTGHSITSNYWGWIRKFPGNKMEWIGQISHS
GSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCGRYGAGSSFDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 189; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 189.
SEQ ID NO: 189
EVQLQQSGAELTKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNEKFKGKATLTVDKS SSTVFMQL S SLTPEDTAVYYCARPLRRVLDNWG
QGVLVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 190; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 190.
SEQ ID NO: 190
EVQLQQSGAELAKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNEKFKGKATLTVDKS SRTVFMQL SSLTPEDTAVYYCARPLRRVLDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 191; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 191.
SEQ ID NO: 191
EVQLQQSGPELAKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNEKFKGKATLTVDKS SSTAFMQL S SLTPEDTAVYYCARPLRRVLDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 192; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 192.
SEQ ID NO: 192
EVQL QQ S GAGL TKP GA S VKI SC TA S GYTF T TYDRGWLRQRPGQ ALEWIGSINP G
GGGTGYNEKFKGNATLTVDKS S STAFMQL S SL TPED TAD YYCARPLRRVLD Y
WGQGVLVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 193; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 193.
SEQ ID NO: 193
DVKLQESGPGLVKPSQSLSLTCSVTGHSITSNYWGWIRKLPGNKMEWIGQISHS
GST SYNP SLISRISITRDT SNQFFLQLNSVTTEDTATYYCGRYGAGS SFDYWGQG
VMVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 194; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 194.
SEQ ID NO: 194
EVQLQQSGAELTKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNEKFKGKATLTVDRS S STAFMQL S SLTPEDTAVYYCARPLRRVLDNWG
QGVLVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 195; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 195.
SEQ ID NO: 195
EVQLQQSGGELTKPGS SVKISCKASGYTF STYDIGWIKQRPGQALEWIGSINPGS
5 GGIGYNEKFKGKATLTVDKS SSTAFMQL S SLTPEDTAVYYCARPLRRVLDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 196; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
10 98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 196.
SEQ ID NO: 196
DVKLQESGPGLVKPSQSLSLTC SVTGHSITGNYWGWIRKFPGNKMEWIGQISHS
GSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCGRYGAGSSFDYWG
15 QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 197; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
20 regions) of SEQ ID NO: 197.
SEQ ID NO: 197
EVQLQQ SGAGLTKPGGS VKISCKVSGYTF TTYDIGWLKQRPGQALEWIGSINPG
GGGTGYNEKFKGKATLTVDKS S STAFMQL S SLTPEDTAVYYCARPLRRVLDY
WGQGVLVTVS S
25 In one embodiment, the DPP4-binding domain of the invention comprises a
HCVR
comprising or consisting of the sequence SEQ ID NO: 198; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 198.
SEQ ID NO: 198
EVQLQQ SGAGLTKPGAS VKISCKASGYTF TTYDIGWLKQRPGQALEWIGSINPG
GGGTGYNEKFKGKATLTVDKS SSTAFMQL SSLTPEDTAVYYCARPLRVLDYW
GQGVLVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 199; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 199.
SEQ ID NO: 199
EVQLQQSGAELTKPGS SVKISCKASGYTFTTYDIGWIKQRPGQALEWIGSINPGS
GGIGYNERFKGKATLTVDKS S STAFMQL S SLTPEDTAVYYCARPLRRVLDYWG
RGVMVTVS S
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 200; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 200.
SEQ ID NO: 200
EVQLEESGPGLVKPSQSLSLTC SVTGHSITSNYWGWIRKFPGNKMEWIGQISHS
GSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCGRYGAGSSFDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a HCVR
comprising or consisting of the sequence SEQ ID NO: 201; or a HCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 201.
SEQ ID NO: 201
QVQLKESGPGLVKPSHSLSLTC SVTGHSITSNYWGWIRKFPGNKMEWIGQISHT
GS S TYNP SLK SRISF TRDT SKNQFFLQLNS VTTED SATYYCGRYGAGS SFDYWG
QGVMVTVSS
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 202; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 202.
SEQ ID NO: 202
DVLMTQTPS SQAASAGEKVTMSCKS SQSLLYNENKKNYLAWFQQKPGQSPKL
LIYWASTRESGVPDRFIGGGSGTDFTLTIS SVQAEDLAVYYCQEYYKFPWTFGG
GTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 203; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 203.
SEQ ID NO: 203
DIVMTQ SP S SQAVSAGEKVTMSCK S SQSLLYNENKKNYLAWFQQKPGQSPKLL
IYWASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQDYYHFPWTFGGG
TKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 204; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 204.
SEQ ID NO: 204
DVLMTQTPPTLSATIGQSVSISCKS SQSLLHSNGNTYLNWLLQRPGQSPQLLIYS
VSKLESGVPNRF S GS GS QTDF TLKISEVEAEDMGVYYCMQATHAPF TF GSWTK
LEIK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 205; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 205.
SEQ ID NO: 205
DIVMTQAP S SQAVSPGEKVTMSCKS SQSLLYNENKKNYLAWYQQKPGQSPKLL
IYWASTREAGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQQYYKFPWPFGGG
TKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 206; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 206.
SEQ ID NO: 206
DIVMTQAP S SQAVSAGEKVTMSCK SSQ SLLYNENKKNYLAWFQQKPGQSPKLL
IYWAS TRK S GVPDRFIGS GS GTDF TLTI S SVQAEDLAVYYCQQYYKFPWTFGGG
TKLELR
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 207; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 207.
SEQ ID NO: 207
DIVMTQ SP S SQAVSPGEKVTMNCKS SQSLLYNENKKNYLAWYQQKPGQSPKLL
IYWASTREAGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQQYYKFPWPFGGG
TKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 208; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 208.
SEQ ID NO: 208
DVLMTQTPPTLSATIGQSVSISCRS SQ SLLHSNGNTYLNWLLQRPGQ SP QLLIY S
VSNLESGVPNRF S GS GSETDF TLKI SGVEAEDL GVYYCMQATHAPF TF GSGTKL
E1K
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 209; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 209.
SEQ ID NO: 209
DIVMTQ SP S SQAVSPGEKVTMNCKS SQSLLYNENKKNYLAWYQQKPGQSPKLL
IYWA S TREAGVPDRFIGS GS GTDF TL TI S S VQAEDLAVYYCQ Q YYKFP YTF GAG
TKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 210; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 210.
SEQ ID NO: 210
DIQLTQSPHSLSASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLIYYT SSLQD
5 GVPSRF SGSGSGTQYSLKISNMQPEDEGVYYCQQGYKYPWTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 211; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
10 regions) of SEQ ID NO: 211.
SEQ ID NO: 211
DIVMTQ SP S SLPASLGERVTISCRASQ GISNKLNWYQQKPDGTIKPLIYYT SRLQ S
GVPSRF SGSGSGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGAGTKVELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
15 comprising or consisting of the sequence SEQ ID NO: 212; or a LCVR
comprising or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 212.
SEQ ID NO: 212
20 DIQLTQ SP S SLPASLGERVTISCRASQGISNKLNWYQQKPDGTIKPLIYYT SNLQS
GVPSRF SGSGSGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 213; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
25 98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 213.
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SEQ ID NO: 213
DIVLTQ SPVLAVSLGQRATISCRASQ SVS T S TYNFMIHWYQQKPGQQPRLLIYDA
SHLASSVPARF SGSGSGTDFTLTINPVQADDIATYYCQQSRELPLTFGSGTKLEIK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 214; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 214.
SEQ ID NO: 214
DILMTQ SP S SLSASLGERVTISCRASQGIGNKLNWYQQKPDGTIKPLIYYTSNLQ
SGVP SRF SGSGSGTDYSLTISSLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 215; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 215.
SEQ ID NO: 215
DIQLTQ SP S SLPASLGERVTISCRASQGISNKLNWYQQKPDGTIKPLIYYT SNLQ S
GVPSRF S GS GS GTDY SLTI S SLEPEDFAMYFCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 216; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 216.
SEQ ID NO: 216
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLIYYTS SF Q
DGVPSRF SGSGSGTQYSLKISNMQPEDEGVYYCQQGYKYPWTFGGGTKLELK
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In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 217; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 217.
SEQ ID NO: 217
DILMTQ SP S SRPASLGERVTISCRASQGIGNKLNWYQQKPDGTIKPLIYYT SNLQ
SGVP SRF SGSGSGTDYSLTISSLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 218; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 218.
SEQ ID NO: 218
DIQMTQTP S SLPASLGERVTISCRASQ GISKKLNWYQQKPDGTIKPLIYYT SNLQ
SGVP SRF SGSGSGTDYSLTIS SLEPEDFAIYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 219; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 219.
SEQ ID NO: 219
DIQLTQ SP S SLPASLGERVTISCRASQGISKKLNWYQQKPDGTIKPLIYYT SNLQS
GVPSRF SGS SGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 220; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
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98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 220.
SEQ ID NO: 220
DILMTQ SP S SLPASLGERVTISCGASQGIGNKVNWYQQKPDGTIKPLIYYTSNLQ
SGVP SRF SGSGTGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 221; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 221.
SEQ ID NO: 221
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQRKPGK SPQLLIYYTISLQD
GVP SRF SGSGSGTQYSLKISNMQPEDEGVFYCQQGYKYPWTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 222; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 222.
SEQ ID NO: 222
DIQLTQ SP S SLPASLGERVTISCRASQGIGNKLNWYQQKPDGTIKPLIYYTSNLQS
GVPSRF SGSGSGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 223; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 223.
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SEQ ID NO: 223
DILMTQ SP S SLSASLGERVTISCRASQGIGNKLNWYQQKPDGTIKPLIYYTSNLQ
SGVP SRF SGSGSGTDYSLTIS SLEPEDFAMYYCQQDTSFPPTFGAGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 224; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 224.
SEQ ID NO: 224
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLIYYTISLQ
DGVPSRF SGSGSGTQYSLKISNMQPEDEGVFYCQQGYKYPWTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 225; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 225.
SEQ ID NO: 225
DIQMTQTP SSLPASLERVTISCRASQGISNKLNWYQKKPDGTIKPLIYYTSNLQS
GVPSRF SGSGSGTDYSLTIS SLEPEDFAMYFCQQDASFPPTFGGGTQLELK
In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 226; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
regions) of SEQ ID NO: 226.
SEQ ID NO: 226
DIQLTQ SP S SRPASLGERVTISCRASQGIGNKLNWYQQKPDGTIKPLIYYTSNLQS
GVPSRF SGSGSGTDYSLTIS SLEPEDFAMYYCQQDASFPPTFGGGTKLELK
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In one embodiment, the DPP4-binding domain of the invention comprises a LCVR
comprising or consisting of the sequence SEQ ID NO: 227; or a LCVR comprising
or
consisting of a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%,
98%, 99% or more sequence identity with the non-CDR regions (i.e., the
framework
5 regions) of SEQ ID NO: 227.
SEQ ID NO: 227
DIQMTQTPHSL SASLGETVSIECLASEGISNYLAWYQQKPGKSPQLLIYYAS SLQ
DGVPSRF SGSGSGTQYSLKISNMQPEDEGVYYCQQGYKYPWTFGGGTKLELK
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
10 of (i) a HCVR and (ii) a LCVR, said combination being as defined in
Table 4.
TABLE 4. Preferred combinations of HCVR and LCVR. The HCVR and LCVR are
defined by their SEQ ID NOs. First column indicates the clone's name.
Clone's name HCVR LCVR
5826-8-R6A-A10 174 202
5826-8-R6A-B11 175 203
5826-8-R6A-D12 176 204
5826-8-R6A-E10 177 205
5826-8-R5A-G6 178 203
5826-8-R5A-G8 179 206
5826-8-R6A-H9 180 207
5826-8-R6A-H11 176 208
5826-8-R6A-H12 181 209
5826-13-R3A-A10 182 210
5826-13-R3A-B1 183 211
5826-13-R3A-B3 184 212
5826-13-R3A-D5 185 213
5826-13-R3A-D6 186 214
5826-13-R4A-E2 187 215
5826-13-R4A-E6 188 216
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5826-13-R4A-E9 189 217
5826-13-R4A-F10 190 218
5826-13-R4A-G11 191 219
5826-13-R4A-G12 192 220
5826-13-R4A-H1 193 221
5826-13-R4A-H2 194 222
5826-13-R4A-H3 195 223
5826-13-R4A-H4 196 224
5826-13-R4A-H5 197 212
5826-13-R4A-H6 198 225
5826-13-R4A-H9 199 226
5826-13-R4A-H10 200 224
5826-13-R4A-H11 201 224
5826-13-R4A-H12 200 227
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 4: 5826-13-R3A-A10, 5826-13-R3A-B1, 5826-13-R3A-B3, 5826-
13-R3A-D5, 5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6, 5826-13-R4A-E9,
5826-13-R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12, 5826-13-R4A-H1, 5826-13-
R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-R4A-H5, 5826-13-R4A-H6,
5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11, and 5826-13-R4A-H12.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 4: 5826-8-R6A-E10, 5826-8-R5A-G8, 5826-8-R6A-H11, 5826-13-
R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of any one of the
following clones
as defined in Table 4: 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
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In one embodiment, the DPP4-binding domain of the invention comprises a
combination
of (i) a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-A10
as
defined in Table 4. In one embodiment, the DPP4-binding domain of the
invention
comprises a combination of (i) a HCVR and (ii) a LCVR, combination being that
of clone
5826-13-R3A-B1 as defined in Table 4. In one embodiment, the DPP4-binding
domain
of the invention comprises a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R3A-B3 as defined in Table 4. In one embodiment,
the
DPP4-binding domain of the invention comprises a combination of (i) a HCVR and
(ii) a
LCVR, combination being that of clone 5826-13-R3A-D5 as defined in Table 4. In
one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-D6 as
defined
in Table 4. In one embodiment, the DPP4-binding domain of the invention
comprises a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-E2 as defined in Table 4. In one embodiment, the DPP4-binding domain of
the
invention comprises a combination of (i) a HCVR and (ii) a LCVR, combination
being
that of clone 5826-13-R4A-E6 as defined in Table 4. In one embodiment, the
DPP4-
binding domain of the invention comprises a combination of (i) a HCVR and (ii)
a LCVR,
combination being that of clone 5826-13-R4A-E9 as defined in Table 4. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-F10 as
defined
in Table 4. In one embodiment, the DPP4-binding domain of the invention
comprises a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-G11 as defined in Table 4. In one embodiment, the DPP4-binding domain of
the
invention comprises a combination of (i) a HCVR and (ii) a LCVR, combination
being
that of clone 5826-13-R4A-G12 as defined in Table 4. In one embodiment, the
DPP4-
binding domain of the invention comprises a combination of (i) a HCVR and (ii)
a LCVR,
combination being that of clone 5826-13-R4A-H1 as defined in Table 4. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-H2 as
defined
in Table 4. In one embodiment, the DPP4-binding domain of the invention
comprises a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-H3 as defined in Table 4. In one embodiment, the DPP4-binding domain of
the
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invention comprises a combination of (i) a HCVR and (ii) a LCVR, combination
being
that of clone 5826-13-R4A-H4 as defined in Table 4. In one embodiment, the
DPP4-
binding domain of the invention comprises a combination of (i) a HCVR and (ii)
a LCVR,
combination being that of clone 5826-13-R4A-H5 as defined in Table 4. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-H6 as
defined
in Table 4. In one embodiment, the DPP4-binding domain of the invention
comprises a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-H9 as defined in Table 4. In one embodiment, the DPP4-binding domain of
the
invention comprises a combination of (i) a HCVR and (ii) a LCVR, combination
being
that of clone 5826-13-R4A-H10 as defined in Table 4. In one embodiment, the
DPP4-
binding domain of the invention comprises a combination of (i) a HCVR and (ii)
a LCVR,
combination being that of clone 5826-13-R4A-H11 as defined in Table 4. In one
embodiment, the DPP4-binding domain of the invention comprises a combination
of (i)
a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-H12 as
defined
in Table 4.
Another object of the present invention is an isolated antibody or antigen-
binding
fragment thereof, wherein said antibody or antigen-binding fragment thereof
recognizes
and is capable of binding to a senescent cell-associated antigen, as defined
hereinabove.
In one embodiment, the senescent cell-associated antigen is selected from the
group
comprising or consisting of DEP1 and DPP4.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention recognizes and is capable of binding to DEP1, such as, e.g., human
DEP1, or
orthologs thereof, including murine and rat DEP1. In one embodiment, the
isolated
antibody or antigen-binding fragment thereof of the invention recognizes and
is capable
of binding to human DEP1 (hDEP1) with SEQ ID NO: 1. In one embodiment, the
isolated
antibody or antigen-binding fragment thereof of the invention recognizes and
is capable
of binding to the extracellular domain of human DEP1 (hDEP1) comprising or
consisting
of amino acid residues 36 ¨975 of SEQ ID NO: 1. Hence, the isolated antibody
or
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antigen-binding fragment thereof of the invention is an isolated "anti-DEP1
antibody or
antigen-binding fragment thereof'.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention recognizes and is capable of binding to DPP4, such as, e.g., human
DPP4 or
orthologs thereof, including murine and rat DPP4. In one embodiment, the
isolated
antibody or antigen-binding fragment thereof of the invention recognizes and
is capable
of binding to human DPP4 (hDPP4) with SEQ ID NO: 101. In one embodiment, the
isolated antibody or antigen-binding fragment thereof of the invention
recognizes and is
capable of binding to the extracellular domain of human DPP4 (hDPP4)
comprising or
consisting of amino acid residues 29 - 766 of SEQ ID NO: 101. Hence, the
antigen-
binding fragment of the invention is an isolated "anti-DPP4 antibody or
antigen-
binding fragment thereof'.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention recognizes and is capable of binding to DEP1 and DPP4.
The binding between the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention and DEP1, or between the anti-DPP4 antibody or antigen-binding
fragment
thereof of the invention and DPP4, implies that said antibody or antigen-
binding fragment
thereof exhibits appreciable affinity for DEP1 or DPP4, respectively. In other
words, the
anti-DEP1 antibody or antigen-binding fragment thereof of the invention or the
anti-
DPP4 antibody or antigen-binding fragment thereof of the invention, is
specific for, or is
immunospecific for, or specifically binds to, DEP1 or DPP4, respectively.
An antibody or antigen-binding fragment thereof is said to be "specific for",
"immunospecific for" or to "specifically bind to" an antigen if it reacts with
said antigen
(e.g., DEP1 and/or DPP4). An antibody or antigen-binding fragment thereof is
said to be
"specific for", "immunospecific for" or to "specifically bind to" an antigen
if it recognizes
and is capable of binding to antigen with a KD-affinity constant less than or
equal to 10-
6 M, preferably less than or equal to 10-7 M, 5.10-8 M, 10-8 M, 5.10-9 M, 10-9
M or less; as
may be determined, e.g., by biosensor analysis, particularly by Biacore
Analysis.
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In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention comprises an antigen-binding domain, as described hereinabove.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain, as described hereinabove.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) at
least one,
preferably at least two, more preferably three HCVR's CDRs and (ii) at least
one,
preferably at least two, more preferably three LCVR's CDRs, said combination
being as
defined in Table 1.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being as defined in
Table 1.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
the following clones as defined in Table 1: 5738-13-R2A-C1, 5738-13-R2A-D3,
5738-
13-R4A-D11, 5738-13-R3A-F5, 5738-13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-
H4, 5738-13-R4A-H9, and 5738-13-R4A-H11.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
the following clones as defined in Table 1: 5738-10-R3A-C6, 5738-10-R3A-D5,
5738-
10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
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the following clones as defined in Table 1: 5738-10-R4A-G12, 5738-13-R4A-D11,
and
5738-13-R2A-H4.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
the following clones as defined in Table 1: 5738-13-R4A-D11, and 5738-13-R2A-
H4.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R2A-C1 as defined in Table 1. In one embodiment, the anti-DEP1 antibody or
antigen-binding fragment thereof of the invention comprises a DEP1-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5738-13-R2A-D3 as defined in Table 1. In one
embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R4A-D11 as defined in Table 1. In one embodiment, the anti-DEP1 antibody or
antigen-binding fragment thereof of the invention comprises a DEP1-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5738-13-R3A-F5 as defined in Table 1. In one
embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R4A-F11 as defined in Table 1. In one embodiment, the anti-DEP1 antibody or
antigen-binding fragment thereof of the invention comprises a DEP1-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5738-13-R2A-H3 as defined in Table 1. In one
embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
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13-R2A-H4 as defined in Table 1. In one embodiment, the anti-DEP1 antibody or
antigen-binding fragment thereof of the invention comprises a DEP1-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5738-13-R4A-H9 as defined in Table 1. In one
embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R4A-H11 as defined in Table 1.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, said combination being as defined in Table 2.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 2: 5738-13-R2A-C1, 5738-13-R2A-D3, 5738-13-R4A-D11, 5738-13-R3A-F5,
5738-13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-H4, 5738-13-R4A-H9, and 5738-
13-R4A-H11.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 2: 5738-10-R3A-C6, 5738-10-R3A-D5, 5738-10-R4A-G12, 5738-13-R4A-D11,
and 5738-13 -R2A-H4 .
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 2: 5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
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and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 2: 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R2A-C1 as defined in
Table 2.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R2A-D3 as defined in
Table 2.
In one embodiment, the anti-DEP1 antibody or antigen-binding fragment thereof
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R4A-D11 as defined in
Table 2. In one embodiment, the anti-DEP1 antibody or antigen-binding fragment
thereof
of the invention comprises a DEP1-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5738-13-R3A-F5 as
defined in
Table 2. In one embodiment, the anti-DEP1 antibody or antigen-binding fragment
thereof
of the invention comprises a DEP1-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5738-13-R4A-F11 as
defined
in Table 2. In one embodiment, the anti-DEP1 antibody or antigen-binding
fragment
thereof of the invention comprises a DEP1-binding domain comprising a
combination of
(i) a HCVR and (ii) a LCVR, combination being that of clone 5738-13-R2A-H3 as
defined in Table 2. In one embodiment, the anti-DEP1 antibody or antigen-
binding
fragment thereof of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R2A-H4 as defined in Table 2. In one embodiment, the anti-DEP1 antibody or
antigen-
binding fragment thereof of the invention comprises a DEP1-binding domain
comprising
a combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R4A-H9 as defined in Table 2. In one embodiment, the anti-DEP1 antibody or
antigen-
binding fragment thereof of the invention comprises a DEP1-binding domain
comprising
a combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R4A-H11 as defined in Table 2.
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In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain, as described hereinabove.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) at
least one,
preferably at least two, more preferably three HCVR's CDRs and (ii) at least
one,
preferably at least two, more preferably three LCVR's CDRs, said combination
being as
defined in Table 3.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being as defined in
Table 3.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
the following clones as defined in Table 3: 5826-13-R3A-A10, 5826-13-R3A-B1,
5826-
13-R3A-B3, 5826-13-R3A-D5, 5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6,
5826-13-R4A-E9, 5826-13-R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12, 5826-13-
R4A-H1, 5826-13-R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-R4A-H5,
5826-13-R4A-H6, 5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11, and 5826-
13-R4A-H12.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
the following clones as defined in Table 3: 5826-8-R6A-E10, 5826-8-R5A-G8,
5826-8-
R6A-H11, 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of any one
of
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the following clones as defined in Table 3: 5826-13-R3A-D5, 5826-13-R4A-H5,
and
5826-13-R4A-H12.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R3A-A10 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R3A-B1 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R3A-B3 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R3A-D5 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R3A-D6 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-E2 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-E6 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-E9 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
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invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-F10 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-G11 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-G12 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H1 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H2 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H3 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H4 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H5 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H6 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
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comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H9 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H10 as defined in Table 3. In one embodiment, the anti-DPP4 antibody or
antigen-binding fragment thereof of the invention comprises a DPP4-binding
domain
comprising a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs,
said
combination being that of clone 5826-13-R4A-H11 as defined in Table 3. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H12 as defined in Table 3.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, said combination being as defined in Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 4: 5826-13-R3A-A10, 5826-13-R3A-B1, 5826-13-R3A-B3, 5826-13-R3A-D5,
5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6, 5826-13-R4A-E9, 5826-13-
R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12, 5826-13-R4A-H1, 5826-13-R4A-H2,
5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-R4A-H5, 5826-13-R4A-H6, 5826-13-
R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11, and 5826-13-R4A-H12.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 4: 5826-8-R6A-E10, 5826-8-R5A-G8, 5826-8-R6A-H11, 5826-13-R3A-D5,
5826-13-R4A-H5, and 5826-13-R4A-H12.
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In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of any one of the following clones as
defined in
Table 4: 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
.. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R3A-A10 as defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-B1 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-B3 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-D5 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R3A-D6 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-E2 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-E6 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-E9 as
defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-F10 as
defined
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in Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding
fragment
thereof of the invention comprises a DPP4-binding domain comprising a
combination of
(i) a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-G11 as
defined in Table 4. In one embodiment, the anti-DPP4 antibody or antigen-
binding
fragment thereof of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-G12 as defined in Table 4. 5826-13-R4A-H1 as defined in Table 4. In one
embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H2 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H3 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H4 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H5 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H6 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H9 as defined in
Table 4.
In one embodiment, the anti-DPP4 antibody or antigen-binding fragment thereof
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H10 as defined in
Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding fragment
thereof
of the invention comprises a DPP4-binding domain comprising a combination of
(i) a
HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-H11 as
defined
in Table 4. In one embodiment, the anti-DPP4 antibody or antigen-binding
fragment
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thereof of the invention comprises a DPP4-binding domain comprising a
combination of
(i) a HCVR and (ii) a LCVR, combination being that of clone 5826-13-R4A-H12 as
defined in Table 4.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a molecule selected from the group comprising or consisting of a
whole
antibody, a single-chain variable fragment (scFv), a dimeric single-chain
variable
fragment (di-scFv, such as a tandem scFv or a diabody), a trimeric single-
chain variable
fragment (tri-scFv, such as a triabody), a tetrameric single-chain variable
fragment (tetra-
scFv, such as a tetrabody), a Fv, a Fab, a Fab', a Fab'-SH, a F(ab')2, a Fabc,
and a Fd.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a mimetic selected from the group comprising or consisting of an
affibody,
an alphabody, an armadillo repeat protein based scaffold, a knottin, a kunitz
domain
peptide, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin,
an anticalin,
an avimer, a fynomer, a versabody or a duocalin.
Antigen-binding fragment of antibodies comprising specific antigen-binding
domains
may be generated by known methods. Methods for producing such antigen-binding
fragments of antibodies are known in the art, for example as described in Lo
(Ed.), 2004.
Antibody Engineering: Methods and Protocols (1st ed., Vol. 248). Totowa, NJ:
Humana
Press; and McCafferty, Hoogenboom & Chiswell (Eds.), 1996. Antibody
Engineering: a
Practical Approach (1st ed., Vol. 169). Oxford: 1RL Press at Oxford University
Press. For
example, F(ab')2 fragments can be produced by pepsin digestion of the whole
antibody
molecule, and Fab fragments can be generated by reducing the disulfide bridges
of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed
to allow
rapid and easy identification of monoclonal Fab fragments with the desired
specificity, as
described for example in Huse et al., 1989. Science. 246(4935):1275-81.
Antibodies may be generated using known methods. For the production of
antibodies,
various hosts including goats, rabbits, rats, mice, humans, and others, may be
immunized
by injection with an appropriate antigen. Depending on the host species,
various adjuvants
may be used to increase an immunological response. Such adjuvants include
Freund's
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adjuvant, mineral gels such as aluminium hydroxide, and surface-active
substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
keyhole limpet
hemocyanin, and dinitrophenol. Adjuvants are commercially available.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is polyclonal. In another embodiment, the isolated antibody or
antigen-binding
fragment thereof of the invention is monoclonal.
Methods of producing polyclonal and monoclonal antibodies as well as fragments
thereof
are well known in the art (see, for example, Harlow and Lane, Antibodies: A
Laboratory
Manual, Cold Spring Harbor Laboratory, New York, 1988).
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a purified antibody or a purified antigen-binding fragment
thereof
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is purified to:
(1) greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more by weight of
antibody or antigen-binding fragment thereof, as may be determined, e.g., by
the
Lowry method; and most preferably more than 96%, 97%, 98% or 99% by weight
of antibody or antigen-binding fragment thereof;
(2) a degree sufficient to obtain at least 15 amino acid residues of the N-
terminal, or of
an internal, amino acid sequence, e.g., by use of a spinning cup sequenator;
and/or
(3) homogeneity as shown, e.g., by SDS-PAGE under reducing or non-reducing
conditions and using, e.g., Coomassie blue staining or more preferably silver
staining.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention has an isotype selected from the group comprising or consisting of
IgG
(including IgGl, IgG2, IgG3 and IgG4), IgM, IgA (including IgAl and IgA2), IgD
and
IgE. The immunoglobulin subclasses or "isotypes" (e.g.õ IgAl, etc.).
The constant region of an antibody is important in the ability of an antibody
to fix
complement and mediate cell-dependent cytotoxicity and phagocytosis. Thus, as
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discussed herein, the isotype of an antibody may be selected on the basis of
whether it is
desirable for the antibody to mediate cytotoxicity/phagocytosis. Determination
or
selection of the isotype of an antibody may be by known methods in the art.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a murine, a chimeric or a humanized antibody or antigen-binding
fragment
thereof.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a murine antibody or antigen-binding fragment thereof
A "murine antibody or antigen-binding fragment thereof' refers to those
antibodies
or antigen-binding fragments thereof in which the variable region (including
the CDRs
and FRs) and the constant region are derived from a mouse.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a chimeric antibody or antigen-binding fragment thereof
A "chimeric antibody or antigen-binding fragment thereof' broadly refers to an
antibody or antigen-binding fragment thereof comprising a first amino acid
sequence
linked to a second amino acid sequence with which it is not naturally linked
in nature.
The amino acid sequences may normally exist in separate proteins that are
brought
together in the fusion protein or they may normally exist in the same protein
but are placed
in a new arrangement in the fusion protein. A chimeric protein may be created,
for
example, by chemical synthesis, or by creating and translating a
polynucleotide in which
the peptide regions are encoded in the desired relationship. The term
"chimeric antibody
or antigen-binding fragment thereof' encompasses herein antibodies and antigen-
binding fragments thereof in which:
(a) the
constant region (Fc), or a portion thereof, is altered, replaced or exchanged
so
that the variable region is linked to a constant region of a different or
altered class,
effector function and/or species, or an entirely different molecule which
confers
new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone,
growth
factor, drug, etc.; or
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(b) the variable region (Fv), or a portion thereof, is altered, replaced
or exchanged with
a variable region, or portion thereof, having a different or altered antigen
specificity; or with corresponding sequences from another species or from
another
antibody class or subclass.
Method to produce chimeric antibodies are well known in the art. For example,
chimeric
antibodies may be produced as described in Morrison et al., 1984. Proc Natl
Acad Sci U
SA. 81(21):6851-5; Neuberger et al., 1984. Nature. 312(5995):604-608; and
Takeda et
al., 1985. Nature, 314(6010):452-454.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a humanized antibody or antigen-binding fragment thereof.
A "humanized antibody or antigen-binding fragment thereof' refers to a
chimeric
antibody or antigen-binding fragment thereof which contains only minimal
sequence
derived from a non-human immunoglobulin. It includes antibodies made by a non-
human
cell having variable and constant regions which have been altered to more
closely
resemble antibodies that would be made by a human cell, e.g., by altering the
non-human
antibody amino acid sequence to incorporate amino acids found in human
germline
immunoglobulin sequences. Humanized antibodies or antigen-binding fragment
thereof
of the invention may include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or site-
specific
mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
The term
"humanized antibody or antigen-binding fragment thereof' also includes
antibodies and
antigen-binding fragment thereof in which CDR sequences derived from the
germline of
another mammalian species, such as a mouse, have been grafted onto human
framework
sequences. In other words, the term "humanized antibody or antigen-binding
fragment
thereof' refers to an antibody or antigen-binding fragment thereof in which
the CDRs of
a recipient human antibody are replaced by CDRs from a donor non-human
antibody.
Humanized antibodies or antigen-binding fragments thereof may also comprise
residues
of donor origin in the framework sequences. The humanized antibody or antigen-
binding
fragment thereof can also comprise at least a portion of a human
immunoglobulin constant
region. Humanized antibodies and or antigen-binding fragments thereof may also
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comprise residues which are found neither in the recipient antibody nor in the
imported
CDR or framework sequences. Humanization can be performed using methods known
in
the art (e.g., Jones et al, 1986. Nature. 321(6069):522-5; Riechmann et al,
1988. Nature.
332(6162):323-7; Verhoeyen etal., 1988. Science. 239(4847):1534-6; Presta,
1992. Curr
Opin Biotechnol. 3(4):394-8; Patent US4,816,567), including techniques such as
"superhumanizing" antibodies (e.g., Tan et al., 2002. J Immunol. 169(2):1119-
25) and
"resurfacing" (e.g., Staelens etal., 2006. Mol Immunol. 43(8):1243-57; Roguska
et at,
1994. Proc Natl Acad Sci USA. 91(3):969-73). A "humanized antibody or antigen-
binding
fragment thereof' retains a similar antigenic specificity as the original
antibody.
However, using certain methods of humanization, the affinity and/or
specificity of
binding of the antibody may be increased.
Methods for humanizing the isolated antibody or antigen-binding fragment
thereof of the
invention are well-known in the art. The choice of human variable domains,
both light
and heavy, to be used in making the humanized antibody or antigen-binding
fragment
thereof is very important to reduce antigenicity. According to the so-called
"best-fit"
method, the sequence of the variable domain of an isolated antibody or antigen-
binding
fragment thereof of the invention is screened against the entire library of
known human
variable-domain sequences. The human sequence that is closest to the mouse
sequence is
then accepted as the human framework (FR) for the humanized antibody (Sims et
al,
1993. J Immunol. 151(4):2296-308; Chothia & Lesk, 1987. JMolBiol. 196(4):901-
17).
Another method for humanizing the isolated antibody or antigen-binding
fragment
thereof of the invention uses a particular framework from the consensus
sequence of all
human antibodies of a particular subgroup of light or heavy chains. The same
framework
can be used for several different humanized antibodies (Carter et al, 1992.
Proc Natl
Acad Sci USA. 89(144285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). It
is
further important that antibodies be humanized with retention of high affinity
for DEP1
or DPP4 and other favorable biological properties. To achieve this goal,
according to a
preferred method, humanized antibodies and antigen-binding fragments thereof
are
prepared by a process of analysis of the parental sequences and various
conceptual
humanized products using three-dimensional models of the parental and
humanized
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sequences. Three-dimensional immunoglobulin models are commonly available and
are
familiar to those skilled in the art. Computer programs are available which
illustrate and
display probable three-dimensional structures of selected candidate
immunoglobulin
sequences. Inspection of these displays permits analysis of the likely role of
the residues
in the functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues
that influence the ability of the candidate immunoglobulin to bind its
epitope. In this way,
CDR residues can be selected and combined from the consensus and import
sequences so
that the desired antibody characteristic, such as an increased affinity for
DEP1 or DPP4,
is achieved. In general, the CDR residues are directly and most substantially
involved in
influencing antigen binding.
Another method for humanizing the isolated antibody or antigen-binding
fragment
thereof of the invention is to use a transgenic or transchromosomic animal
carrying parts
of the human immune system for immunization. As a host, these animals have had
their
immunoglobulin genes replaced by functional human immunoglobulin genes. Thus,
antibodies produced by these animals or in hybridomas made from the B cells of
these
animals are already humanized. Examples of such transgenic or transchromosomic
animal include, without limitation:
- the XenoMouse (Abgenix, Fremont, CA), described in Patents US5,939,598,
US6,075,181, US6,114,598, US6,150,584 and US6,162,963;
- the HuMAb Mouse (Medarex, Inc.), described in Lonberg et al, 1994. Nature.
368(6474):856-859; Lonberg & Huszar, 1995. Int Rev Immunol. 13(1):65-93;
Harding & Lonberg, 1995. Ann N Y Acad Sci. 764:536-46; Taylor et al., 1992.
Nucleic Acids Res. 20(23):6287-95; Chen et al., 1993. Int Immunol. 5(6):647-
56;
Tuaillon et al., 1993. Proc Natl Acad Sci USA. 90(8):3720-4; Choi et al, 1993.
Nat Genet. 4(2): 117-23; Chen et al, 1993. EMBO J. 12(3):821-30; Tuaillon et
al.,
1994. J Immunol. 152(6):2912-20; Taylor et al., 1994. Int Immunol. 6(4):579-
91;
Fishwild et al, 1996. Nat Biotechnol. 14(7):845-51;
- the KM Mouse , described in Patent application W02002043478;
- the TC mice, described in Tomizuka et al., 2000. Proc Nati Acad Sci USA.
97(2):722-7; and
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- the OmniRatTM (OMT, Inc.), described in Patent application W02008151081;
Geurts et al, 2009. Science. 325(5939):433; Menoret et al., 2010. Eur J
Immunol.
40(10):2932-41.
Humanized antibodies and antigen-binding fragments thereof may also be
produced
according to various other techniques, such as by using, for immunization,
other
transgenic animals that have been engineered to express a human antibody
repertoire
(Jakobovitz et al, 1993. Nature. 362(6417):255-8), or by selection of antibody
repertoires
using phage display methods. Such techniques are known to the skilled person
and can
be implemented starting from monoclonal antibodies or antigen-binding
fragments
thereof as disclosed in the present application.
Whether chimeric or humanized, the isolated antibody or antigen-binding
fragment
thereof of the invention may comprise a constant region (Fc) of human origin.
In one embodiment, especially when the isolated antibody or antigen-binding
fragment
thereof of the invention is intended for human therapeutic uses, it is typical
for the entire
constant region (Fc), or at least a part thereof, to have a fully or
substantially human amino
acid sequence. Therefore, one or more of, or any combination of, the CH1
domain, hinge
region, CH2 domain, CH3 domain and CL domain and CH4 domain (when present) may
be
fully or substantially human with respect to its amino acid sequence.
Advantageously, the
CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain and CH4 domain
(when present) may all have a fully or substantially human amino acid
sequence.
The term "substantially human", in the context of the constant region (Fc) of
a chimeric
or humanized antibody or antigen-binding fragment thereof, refers to an amino
acid
sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%,
95%, 96%,
97%, 98%, 99% or more with a human constant region (Fc).
The term "human amino acid sequence", in this context, refers to an amino acid
sequence which is encoded by a human immunoglobulin gene, which includes
germline,
rearranged and somatically mutated genes. The present invention also
contemplates
proteins comprising constant domains of "human" sequence which have been
altered, by
one or more amino acid additions, deletions or substitutions with respect to
the human
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sequence, excepting those embodiments where the presence of a "fully human
hinge
region" is expressly required.
The presence of a "fully human hinge region" in the isolated antibody or
antigen-
binding fragment thereof of the invention may be beneficial both to minimize
immunogenicity and to optimize stability of the antibody. It is considered
that one or
more amino acid substitutions, insertions or deletions may be made within the
constant
region of the heavy and/or the light chain, particularly within the Fc region.
Amino acid
substitutions may result in replacement of the substituted amino acid with a
different
naturally occurring amino acid, or with a non-natural or modified amino acid.
Other
structural modifications are also permitted, such as for example changes in
glycosylation
pattern (e.g., by addition or deletion of N- or 0-linked glycosylation sites).
Depending on
the intended use of the antibody or antigen-binding fragment thereof, it may
be desirable
to modify the isolated antibody or antigen-binding fragment thereof of the
invention with
respect to its binding properties to Fc receptors, for example to modulate
effector
function. For example, cysteine residue(s) may be introduced in the Fc region,
thereby
allowing interchain disulfide bond formation in this region. The homodimeric
antibody
thus generated may have improved effector function (Caron et al, 1992. J Exp
Med.
176(4):1191-5; Shopes, 1992. J Immunol. 148(9):2918-22).
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention may be a bispecific antibody (BsAb) with antigen binding to at least
two
senescent cell-associated antigens. In one embodiment, the isolated antibody
or antigen-
binding fragment thereof of the invention may be a bispecific antibody (BsAb)
which
binds to both DEP1 and DPP4.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention may be a bispecific antibody (BsAb) with antigen binding to at one
senescent
cell-associated antigen and one non-senescent cell-associated antigen. In one
embodiment, the isolated antibody or antigen-binding fragment thereof of the
invention
may be a bispecific antibody (BsAb) which binds to either of DEP1 or DPP4 on
the one
hand, and to one non-senescent cell-associated antigen on the other hand.
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In one embodiment, the non-senescent cell-associated antigen is a T cell
receptor or part
thereof, such as, e.g., any one of the CD36, CD3y, CD3E or CD3 subunit.
Examples of bispecific antibodies (BsAb) include, but are not limited to,
quadromas,
knobs-in-holes, CrossMab Fab, CrossMab VH-VL, CrossMab CH1-CL, TriMab, one-arm
single-chain Fab-immunoglobulin gamma (0AscFab-IgG), disulfide stabilized Fv-
IgG
(dsFv-IgG), DuetMab, controlled Fab-arm exchanged-IgG1 (cFAE-IgG1), charged
pair
scFv-Fc, strand-exchange engineered domain body (SEEDbody), two-arm leucine
zipper
heterodimeric monoclonal antibodies (two-arm LUZ-Y), kappa lambda body (K-
body),
bi-specific T cell engagers (BiTEs), diabodies, .tandab, dual-affinity
retargeting
molecules (DARTs), bispecific killer cell engagers (BiKEs), trispecific killer
cell
engagers (TriKEs), monomeric Fc-VH (mFc-VH) and Fc antigen binding (Fcab), all
reviewed and described in Liu et al, 2017 (Front Immunol. 8:38).
Methods for producing bispecific antibodies (BsAb) are well known in the art.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is a recombinant isolated antibody or antigen-binding fragment
thereof
Thus accordingly, the isolated antibody or antigen-binding fragment thereof of
the
invention may be produced recombinantly by methods known in the art, such as,
e.g., by
expression in Escherichia coil expression systems (see, e.g., US patent
4,816,567).
Antigen binding fragment may also be produced by phage display technologies,
which
are known in the art.
It will also be appreciated that the isolated antibody or antigen-binding
fragment thereof
of the invention can be modified using methods well known in the art, e.g., to
improve
the properties of the isolated antibody or antigen-binding fragment thereof
For example,
to slow clearance in vivo and obtain a more desirable pharmacokinetic profile,
the isolated
antibody or antigen-binding fragment thereof may be modified with polyethylene
glycol
(PEG). Methods for coupling and site-specifically conjugating PEG to an
antibody or
antigen-binding fragment thereof are described in, e.g., Leong et al., 2001.
Cytokine.
16(3):106-19; Delgado et al., 1996. Br J Cancer. 73(2):175-82. Another non-
limiting
example of modification consist in the modification of the human Fc region of
the
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antibody in order to enhance their affinity for an Fcy receptor. Methods of
enhancing Fc
receptor binding include Fc amino acid modification and modification of Fc
carbohydrate
structures. For immunoglobulins, it has been demonstrated that the attachment
of an N-
linked oligosaccharide to Asn-297 of the CH2 domain is critical for ADCC
activity.
Removal of the N-linked oligosaccharide through mutation of the N-linked
consensus site
or by enzymatic means results in little or no ADCC activity. Removal of the
core a-1,6-
fucose moiety from IgG1 Fc oligosaccharides has been demonstrated to improve
FcyRIII
binding and ADCC activity (see, e.g., Carter, 2001. Nat Rev Cancer. 1(2):118-
29; Kanda
et at, 2007. Glycobiology. 17(1):104-18; Shields et al., 2002. J Biol Chem.
277(30):26733-40; Shinkawa et al., 2003. J Biol Chem. 278(5):3466-73; Niwa et
al.,
2004. Cancer Res. 64(6):2127-33). The level of another glycoform, bisected N-
linked
carbohydrate, has also been suggested to increase ADCC (see, e.g., Umalia et
al., 1999.
NatBiotechnol. 17(2):176-80; Hodoniczky eta, 2005. BiotechnolProg. 21(6):1644-
52).
A variety of Fc sequence variants with optimized binding affinity for FcyRs
and/or
enhanced ADCC have been described and are known in the art.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention targets, blocks, depletes and/or kills senescent cells to which it
is bound.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention targets, blocks, depletes and/or kills senescent cells expressing at
least one
senescent cell-associated antigen, as defined hereinabove.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention may comprise human HCCRs (heavy chain constant regions) and allows
to
target, block, deplete and/or kill DEP1-expressing cells to which it is bound.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention may comprise human HCCRs (heavy chain constant regions) and allow to
target, block, deplete and/or kill DPP4-expressing cells to which it is bound.
In one embodiment where the isolated antibody or antigen-binding fragment
thereof is a
bispecific antibody as described above, said bispecific antibody may comprise
human
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HCCRs (heavy chain constant regions) and allow to target, block, and/or
deplete DEP1-
and/or DPP4-expressing cells to which it is bound.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention depletes and/or kills DEP1-expressing cells to which it is bound.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention depletes and/or kills DPP4-expressing cells to which it is bound.
In one embodiment where the isolated antibody or antigen-binding fragment
thereof is a
bispecific antibody as described above, said bispecific antibody depletes
and/or kills
DEP1- and/or DPP4-expressing cells to which it is bound.
By "deplete" or "depleting", it is referred to the killing, elimination,
lysis, or induction
of such killing, elimination or lysis, so as to negatively affect the number
of cells to which
the isolated antibody or antigen-binding fragment thereof is bound (such as,
e.g., DEP1-
and/or DPP4-expressing cells) present in a sample or in a subject. In one
embodiment,
such depletion occurs via ADCC. In one embodiment, such depletion occurs via
ADCP.
In one embodiment, such depletion occurs via CDC.
Thus, in one embodiment, the isolated antibody or antigen-binding fragment
thereof of
the invention leads, directly or indirectly, to the depletion of senescent
cells, in particular
of DEP1- and/or DPP4-expressing cells.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention leads, directly or indirectly, to the depletion of 10%, 20%, 30%,
40%, 50%,
60%, 70%, 80%, 90% or more of senescent cells, in particular of DEP1- and/or
DPP4-
expressing cells.
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention leads, directly or indirectly, to a decrease by 10%, 20%, 30%, 40%,
50%, 60%,
70%, 80%, 90% or more elimination of the number of senescent cells, in
particular of
DEP1- and/or DPP4-expressing cells.
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In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention induces any one or several, such as two or three, of:
- antibody dependent cellular cytotoxicity (ADCC);
- antibody-dependent cell-mediated phagocytosis (ADCP);
- complement-dependent cytotoxicity (CDC).
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is from the IgG1 subclass and has any one or several, such as two or
three, of:
- antibody dependent cellular cytotoxicity (ADCC);
- antibody-dependent cell-mediated phagocytosis (ADCP);
- complement-dependent cytotoxicity (CDC).
In one embodiment, the isolated antibody or antigen-binding fragment thereof
of the
invention is linked/fused/conjugated to a payload, e.g., a therapeutic moiety.
Such
conjugates are referred to herein as an "antibody drug conjugates" or "ADCs".
In one embodiment, the payload is selected from chemotherapeutic agents,
targeted
therapy agents, cytotoxic agents, antibiotics, antivirals, cell cycle-
synchronizing agents,
ligands for cellular receptor(s), immunomodulatory agents, pro-apoptotic
agents, anti-
angiogenic agents, cytokines, growth factors, hormones, coding or non-coding
oligonucleotides, photodetectable labels, contrast agents, radiolabels, and
the like.
Another object of the present invention is a nucleic acid encoding the antigen-
binding
domain, the antibody or the antigen-binding fragment thereof of the invention.
Another object of the present invention is a vector comprising the nucleic
acid encoding
the antigen-binding domain, the antibody or the antigen-binding fragment
thereof of the
invention.
In one embodiment, the vector is an expression vector and further comprises
regulatory
elements allowing for expression of the antigen-binding domain, the antibody
or the
antigen-binding fragment thereof, in a cell.
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In one embodiment, the expression vector may be monocistronic. By
"monocistronic",
it is meant that a single nucleic acid encoding a single protein is expressed
in a single
expression vector.
In one embodiment, the expression vector comprises a sequence encoding the
HCVR of
the antigen-binding domain, the antibody or the antigen-binding fragment
thereof of the
invention, preferably operably linked to regulatory elements.
In one embodiment, the expression vector comprises a sequence encoding the
HCVR of
the DEP1-binding domain, the anti-DEP1 antibody or the antigen-binding
fragment
thereof of the invention, preferably operably linked to regulatory elements.
In one embodiment, the expression vector comprises a sequence encoding the
HCVR of
the DEP1-binding domain, the anti-DEP1 antibody or the antigen-binding
fragment of
the invention, preferably operably linked to regulatory elements, said
sequence being
selected from the group comprising or consisting of SEQ ID NOs: 55 to 74.
In one embodiment, the expression vector comprises a sequence encoding the
HCVR of
the DPP4-binding domain, the anti-DPP4 antibody or the antigen-binding
fragment of the
invention, preferably operably linked to regulatory elements.
In one embodiment, the expression vector comprises a sequence encoding the
HCVR of
the DPP4-binding domain, the anti-DPP4 antibody or the antigen-binding
fragment of the
invention, preferably operably linked to regulatory elements, said sequence
being selected
from the group comprising or consisting of SEQ ID NOs: 174 to 201.
In one embodiment, the expression vector comprises a sequence encoding the
LCVR of
the antigen-binding domain, the antibody or the antigen-binding fragment
thereof of the
invention, preferably operably linked to regulatory elements.
In one embodiment, the expression vector comprises a sequence encoding the
LCVR of
the DEP1-binding domain, the anti-DEP1 antibody or the antigen-binding
fragment of
the invention, preferably operably linked to regulatory elements.
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In one embodiment, the expression vector comprises a sequence encoding the
LCVR of
the DEP1-binding domain, the anti-DEP1 antibody or the antigen-binding
fragment of
the invention, preferably operably linked to regulatory elements, said
sequence being
selected from the group comprising or consisting of SEQ ID NOs: 75 to 94.
In one embodiment, the expression vector comprises a sequence encoding the
LCVR of
the DPP4-binding domain, the anti-DPP4 antibody or the antigen-binding
fragment of the
invention, preferably operably linked to regulatory elements.
In one embodiment, the expression vector comprises a sequence encoding the
LCVR of
the DPP4-binding domain, the anti-DPP4 antibody or the antigen-binding
fragment of the
invention, preferably operably linked to regulatory elements, said sequence
being selected
from the group comprising or consisting of SEQ ID NOs: 202 to 227.
In one embodiment, the expression vector may be polycistronic. By
"polycistronic", it is
meant that at least two or more nucleic acids, each encoding a single protein,
are
expressed in a single expression vector.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the antigen-binding domain, the antibody
or the
antigen-binding fragment thereof of the invention, preferably operably linked
to
regulatory elements, and
- a sequence encoding the LCVR of the antigen-binding domain, the antibody
or the
antigen-binding fragment thereof of the invention, preferably operably linked
to
regulatory elements.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, and
- a sequence encoding the LCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements.
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In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, said sequence being selected from the group
comprising or consisting of SEQ ID NOs: 55 to 74, and
- a sequence encoding the LCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, said sequence being selected from the group
comprising or consisting of SEQ ID NOs: 75 to 94.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, and
- a sequence encoding the LCVR of the DEP1-binding domain, the anti-DEP1
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements,
wherein said sequence encoding the HCVR and said sequence encoding the LCVR
are
selected from the group comprising or consisting of the combinations of HCVR
and
LCVR as defined in Table 2.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, and
- a sequence encoding the LCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
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linked to regulatory elements, said sequence being selected from the group
comprising or consisting of SEQ ID NOs: 174 to 201, and
- a sequence encoding the LCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, said sequence being selected from the group
comprising or consisting of SEQ ID NOs: 202 to 227.
In one embodiment, the expression vector comprises:
- a sequence encoding the HCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements, and
- a sequence encoding the LCVR of the DPP4-binding domain, the anti-DPP4
antibody or the antigen-binding fragment of the invention, preferably operably
linked to regulatory elements,
wherein said sequence encoding the HCVR and said sequence encoding the LCVR
are
selected from the group comprising or consisting of the combinations of HCVR
and
LCVR as defined in Table 4.
Another object of the invention is a method of producing and purifying the
isolated
antibody or antigen-binding fragment thereof of the invention.
In one embodiment, the method comprises:
- culturing host cells comprising the nucleic acid or expression vector of
the present
invention, under conditions suitable for expression of the antibody or antigen-
binding fragment thereof, and
- recovering the expressed antibody or antigen-binding fragment thereof.
This recombinant process can be used for large scale production of antibodies
or antigen-
binding fragments thereof, including monoclonal antibodies intended for in
vitro, ex vivo
and/or in vivo therapeutic and/or diagnostic uses.
The nucleic acid or expression vector encoding the antibody or antigen-binding
fragment,
as described herein, may be propagated and expressed according to any of a
variety of
routinely practiced procedures for nucleic acid excision, ligation,
transformation, and
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transfection. In certain embodiments, expression of the antibody or antigen-
binding
fragment thereof may be carried out in a prokaryotic host cell (i.e., the host
cell
comprising the nucleic acid or expression vector of the present invention is a
prokaryotic
host cell), such as Escherichia coli (see, e.g., Pluckthun et al, 1989.
Methods Enzymol.
178:497-515). In certain other embodiments, the antibody or antigen-binding
fragment
thereof may be expressed in a eukaryotic host cell (i.e., the host cell
comprising the
nucleic acid or expression vector of the present invention is an eukaryotic
host cell),
including animal cells (such as mammalian cells), yeast (e.g., Saccharomyces
cerevisiae,
Schizosaccharomyces porn be, or Pichia pastoris); and plant cells. Examples of
suitable
animal cells include, but are not limited to, myeloma, HEK293, COS, or CHO
cells.
Examples of plant cells include tobacco, corn, soybean, and rice cells. By
methods known
to those having ordinary skill in the art and based on the present disclosure,
a nucleic acid
vector may be designed for expressing foreign sequences in a particular host
system, and
then polynucleotide sequences encoding the cellular polypeptide may be
inserted. The
regulatory elements will vary as appropriate for the particular host.
All these processes are well known in the art (Subramanian (Ed.), 2004.
Antibodies (1st
ed., Vol. 1: Production and Purification). New York, NY: Springer US).
In an embodiment, the expressed antibody or antigen-binding fragment thereof
is further
purified.
Methods to purify the antibody or antigen-binding fragment thereof of the
invention are
well known in the art (Subramanian (Ed.), 2004. Antibodies (1st ed., Vol. 1:
Production
and Purification). New York, NY: Springer US), and include, without
limitation,
chromatography, preferably by affinity chromatography, more preferably by
affinity
chromatography on protein L agarose.
Another object of the present invention is a chimeric antigen receptor (CAR)
comprising:
(i) at least one extracellular binding domain, comprising or consisting of
at least one
antigen-binding domain directed to a senescent cell-associated antigen,
(ii) an extracellular spacer domain,
(iii) a transmembrane domain,
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(iv) optionally, at least one costimulatory domain, and
(v) at least one intracellular signaling domain.
In one embodiment, the CAR of the invention comprises at least one
extracellular binding
domain (also called ectodomain), wherein said at least one extracellular
binding domain
recognizes and is capable of binding to a senescent cell-associated antigen,
as defined
hereinabove. In one embodiment, the extracellular binding domain comprises or
consists
of an antigen-binding domain directed to a senescent cell-associated antigen,
which
recognizes and is capable of binding to a senescent cell-associated antigen,
as defined
hereinabove.
In one embodiment, the senescent cell-associated antigen is selected from the
group
comprising or consisting of DEP1 and DPP4. Hence, in one embodiment, the at
least one
extracellular binding domain of the CAR of the invention recognizes and is
capable of
binding to DEP1 and/or DPP4. In one embodiment, the extracellular binding
domain
comprises or consists of an antigen-binding domain directed to DEP1 and/or
DPP4, which
recognizes and is capable of binding to DEP1 and/or DPP4, as defined
hereinabove.
In one embodiment, the at least one extracellular binding domain in the CAR of
the
invention recognizes and is capable of binding to DEP1, such as, e.g., human
DEP1, or
orthologs thereof, including murine and rat DEP1. In one embodiment, the at
least one
extracellular binding domain in the CAR of the invention recognizes and is
capable of
binding to human DEP1 (hDEP1) with SEQ ID NO: 1. In one embodiment, the at
least
one extracellular binding domain in the CAR of the invention recognizes and is
capable
of binding to the extracellular domain of human DEP1 (hDEP1) comprising or
consisting
of amino acid residues 36 ¨ 975 of SEQ ID NO: 1. In one embodiment, the
extracellular
binding domain comprises or consists of a DEP1-binding domain, as defined
hereinabove. Hence, the CAR of the invention is an "anti-DEP1 chimeric antigen
receptor (CAR)".
In one embodiment, the at least one extracellular binding domain in the CAR of
the
invention recognizes and is capable of binding to DPP4, such as, e.g., human
DPP4 or
orthologs thereof, including murine and rat DPP4. In one embodiment, the at
least one
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extracellular binding domain in the CAR of the invention recognizes and is
capable of
binding to human DPP4 (hDPP4) with SEQ ID NO: 101. In one embodiment, the at
least
one extracellular binding domain in the CAR of the invention recognizes and is
capable
of binding to the extracellular domain of human DPP4 (hDPP4) comprising or
consisting
of amino acid residues 29 - 766 of SEQ ID NO: 101. In one embodiment, the
extracellular
binding domain comprises or consists of a DPP4-binding domain, as defined
hereinabove.
Hence, the CAR of the invention is an "anti-DDP4 chimeric antigen receptor
(CAR)".
In one embodiment, the at least one extracellular binding domain in the CAR of
the
invention comprises an antigen-binding domain, as described hereinabove.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain, as described
hereinabove.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) at least one, preferably at least two, more preferably
three HCVR's
CDRs and (ii) at least one, preferably at least two, more preferably three
LCVR's CDRs,
said combination being as defined in Table 1.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being as defined in Table 1.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 1: 5738-13-
R2A-C1,
5738-13-R2A-D3, 5738-13-R4A-D11, 5738-13-R3A-F5, 5738-13-R4A-F11, 5738-13-
R2A-H3, 5738-13-R2A-H4, 5738-13-R4A-H9, and 5738-13-R4A-H11.
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In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 1: 5738-10-
R3A-C6,
5738-10-R3A-D5, 5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 1: 5738-10-
R4A-G12,
5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 1: 5738-13-
R4A-D11,
.. and 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5738-13-R2A-C1 as defined in Table 1. In one embodiment,
the
extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R2A-D3 as defined in Table 1. In one embodiment, the extracellular binding
domain
of the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5738-13-R4A-D11 as defined
in
Table 1. In one embodiment, the extracellular binding domain of the anti-DEP1
chimeric
antigen receptor (CAR) of the invention comprises a DEP1-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5738-13-R3A-F5 as defined in Table 1. In one embodiment,
the
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extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R4A-F11 as defined in Table 1. In one embodiment, the extracellular binding
domain
of the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5738-13-R2A-H3 as defined in
Table 1. In one embodiment, the extracellular binding domain of the anti-DEP1
chimeric
antigen receptor (CAR) of the invention comprises a DEP1-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5738-13-R2A-H4 as defined in Table 1. In one embodiment,
the
extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5738-
13-R4A-H9 as defined in Table 1. In one embodiment, the extracellular binding
domain
of the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5738-13-R4A-H11 as defined
in
Table 1.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, said combination being as defined
in
Table 2.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 2: 5738-13-R2A-C1, 5738-13-R2A-D3, 5738-
13-
R4A-D11, 5738-13-R3A-F5, 5738-13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-H4,
5738-13-R4A-H9, and 5738-13-R4A-H11.
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In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 2: 5738-10-R3A-C6, 5738-10-R3A-D5, 5738-
10-
R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 2: 5738-10-R4A-G12, 5738-13-R4A-D11, and
.. 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 2: 5738-13-R4A-D11, and 5738-13-R2A-H4.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R2A-C1 as defined in Table 2. In one embodiment, the extracellular binding
domain of
the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5738-13-R2A-D3 as defined in Table 2. In one embodiment,
the
extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R4A-D11 as defined in
Table 2. In one embodiment, the extracellular binding domain of the anti-DEP1
chimeric
antigen receptor (CAR) of the invention comprises a DEP1-binding domain
comprising
a combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R3A-F5 as defined in Table 2. In one embodiment, the extracellular binding
domain of
the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
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being that of clone 5738-13-R4A-F11 as defined in Table 2. In one embodiment,
the
extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R2A-H3 as defined in
Table 2.
In one embodiment, the extracellular binding domain of the anti-DEP1 chimeric
antigen
receptor (CAR) of the invention comprises a DEP1-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5738-13-
R2A-H4 as defined in Table 2. In one embodiment, the extracellular binding
domain of
the anti-DEP1 chimeric antigen receptor (CAR) of the invention comprises a
DEP1-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5738-13-R4A-H9 as defined in Table 2. In one embodiment,
the
extracellular binding domain of the anti-DEP1 chimeric antigen receptor (CAR)
of the
invention comprises a DEP1-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5738-13-R4A-H11 as defined in
Table 2.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain, as described
hereinabove.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) at least one, preferably at least two, more preferably
three HCVR's
CDRs and (ii) at least one, preferably at least two, more preferably three
LCVR's CDRs,
said combination being as defined in Table 3.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being as defined in Table 3.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
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combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 3: 5826-13-
R3A-A10,
5826-13-R3A-B1, 5826-13-R3A-B3, 5826-13-R3A-D5, 5826-13-R3A-D6, 5826-13-
R4A-E2, 5826-13 -R4A-E6, 5826-13 -R4A-E9, 5826-13 -R4A-F10, 5826-13 -R4A-G11,
5826-13-R4A-G12, 5826-13-R4A-H1, 5826-13-R4A-H2, 5826-13-R4A-H3, 5826-13-
R4A-H4, 5826-13-R4A-H5, 5826-13-R4A-H6, 5826-13-R4A-H9, 5826-13-R4A-H10,
5826-13-R4A-H11, and 5826-13-R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 3: 5826-8-
R6A-E10,
5826-8-R5A-G8, 5826-8-R6A-H11, 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-
R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of any one of the following clones as defined in Table 3: 5826-13-
R3A-D5,
5826-13-R4A-H5, and 5826-13-R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R3A-A10 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R3A-B1 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R3A-B3 as defined in
Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
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antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R3A-D5 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
.. invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R3A-D6 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-E2 as defined in
Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-E6 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-E9 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-F10 as defined
in
Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
.. being that of clone 5826-13-R4A-G11 as defined in Table 3. In one
embodiment, the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-G12 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-H1 as defined in
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Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-H2 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H3 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-H4 as defined in
Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-H5 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H6 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
LCVR's CDRs, said combination being that of clone 5826-13-R4A-H9 as defined in
Table 3. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) three HCVR's CDRs and (ii) three LCVR's CDRs, said
combination
being that of clone 5826-13-R4A-H10 as defined in Table 3. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i)
three
HCVR's CDRs and (ii) three LCVR's CDRs, said combination being that of clone
5826-
13-R4A-H11 as defined in Table 3. In one embodiment, the extracellular binding
domain
of the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) three HCVR's CDRs and (ii)
three
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LCVR's CDRs, said combination being that of clone 5826-13-R4A-H12 as defined
in
Table 3.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, said combination being as defined
in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 4: 5826-13-R3A-A10, 5826-13-R3A-B1, 5826-
13-
R3A-B3, 5826-13-R3A-D5, 5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6,
5826-13-R4A-E9, 5826-13-R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12, 5826-13-
R4A-H1, 5826-13-R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-R4A-H5,
5826-13-R4A-H6, 5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11, and 5826-
13-R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 4: 5826-8-R6A-E10, 5826-8-R5A-G8, 5826-8-
R6A-H11, 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of any one
of the
following clones as defined in Table 4: 5826-13-R3A-D5, 5826-13-R4A-H5, and
5826-
13-R4A-H12.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R3A-A10 as defined in Table 4. In one embodiment, the extracellular binding
domain of
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the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R3A-B1 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
.. invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R3A-B3 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R3A-D5 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R3A-D6 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-E2 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-E6 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R4A-E9 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-F10 as defined in
Table 4. In one embodiment, the extracellular binding domain of the anti-DPP4
chimeric
antigen receptor (CAR) of the invention comprises a DPP4-binding domain
comprising
a combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-G11 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
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being that of clone 5826-13-R4A-G12 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H1 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-H2 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R4A-H3 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H4 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-H5 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R4A-H6 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H9 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the anti-DPP4 chimeric
antigen
receptor (CAR) of the invention comprises a DPP4-binding domain comprising a
combination of (i) a HCVR and (ii) a LCVR, combination being that of clone
5826-13-
R4A-H10 as defined in Table 4. In one embodiment, the extracellular binding
domain of
the anti-DPP4 chimeric antigen receptor (CAR) of the invention comprises a
DPP4-
binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination
being that of clone 5826-13-R4A-H11 as defined in Table 4. In one embodiment,
the
extracellular binding domain of the anti-DPP4 chimeric antigen receptor (CAR)
of the
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invention comprises a DPP4-binding domain comprising a combination of (i) a
HCVR
and (ii) a LCVR, combination being that of clone 5826-13-R4A-H12 as defined in
Table 4.
In one embodiment, the extracellular binding domain of the CAR of the
invention
comprises or consists a single chain variable region (scFv) or a Fab fragment,
preferably
a scFv, comprising an one antigen-binding domain as defined hereinabove.
In one embodiment, the CAR of the invention comprises more than one
extracellular
binding domain, such as 2 extracellular binding domains, 3 extracellular
binding domains
or more. Such CARs are called "tandem CARs" or "multispecific CARs", such as
bispecific, trispecific, etc.
In this embodiment, the CAR of the invention is multispecific and comprises
extracellular
binding domains, wherein at least one of the extracellular binding domains
recognizes
and is capable of binding to a senescent cell-associated antigen.
In one embodiment, the CAR of the invention is multispecific and comprises
extracellular
binding domains, each of which recognizes and is capable of binding to a
different
senescent cell-associated antigen.
In one embodiment, the CAR of the invention is multispecific and comprises
extracellular
binding domains, wherein one extracellular binding domain recognizes and is
capable of
binding to either of DEP1 or DPP4, and at least one other extracellular
binding domain
recognizes and is capable of binding to another senescent cell-associated
antigen.
In one embodiment, the CAR of the invention is multispecific and comprises
extracellular
binding domains, wherein one extracellular binding domain recognizes and is
capable of
binding to DEP1, and at least one other extracellular binding domain
recognizes and is
capable of binding to DPP4.
In one embodiment, the CAR of the invention is bispecific and comprises two
extracellular binding domains, wherein one extracellular binding domain
recognizes and
is capable of binding to DEP1, and the other extracellular binding domain
recognizes and
is capable of binding to DPP4.
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In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) at least one, preferably
at
least two, more preferably three HCVR's CDRs and (ii) at least one, preferably
at
least two, more preferably three LCVR's CDRs, said combination being as
defined
in Table 1; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) at least one, preferably
at
least two, more preferably three HCVR's CDRs and (ii) at least one, preferably
at
least two, more preferably three LCVR's CDRs, said combination being as
defined
in Table 3,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being as defined in Table 1; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being as defined in Table 3,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 1: 5738-13-R2A-C1, 5738-13-R2A-D3, 5738-13-R4A-
D11, 5738-13-R3A-F5, 5738-13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-H4,
5738-13-R4A-H9, and 5738-13-R4A-H11; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
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clones as defined in Table 3: 5826-13-R3A-A10, 5826-13-R3A-B1, 5826-13-R3A-
B3, 5826-13-R3A-D5, 5826-13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6,
5826-13-R4A-E9, 5826-13-R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12,
5826-13-R4A-H1, 5826-13-R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-
13-R4A-H5, 5826-13-R4A-H6, 5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-
R4A-H11, and 5826-13-R4A-H12,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 1: 5738-10-R3A-C6, 5738-10-R3A-D5, 5738-10-R4A-
G12, 5738-13-R4A-D11, and 5738-13-R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 3: 5826-8-R6A-E10, 5826-8-R5A-G8, 5826-8-R6A-
H11, 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 1: 5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-
R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 3: 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-
R4A-H12,
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as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 1: 5738-13-R4A-D11, and 5738-13-R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) three HCVR's CDRs and
(ii) three LCVR's CDRs, said combination being that of any one of the
following
clones as defined in Table 3: 5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-
R4A-H12,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
said combination being as defined in Table 2; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
said combination being as defined in Table 4,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
2:
5738-13-R2A-C1, 5738-13-R2A-D3, 5738-13-R4A-D11, 5738-13-R3A-F5, 5738-
13-R4A-F11, 5738-13-R2A-H3, 5738-13-R2A-H4, 5738-13-R4A-H9, and 5738-
13-R4A-H11; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
4:
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5826-13-R3A-A10, 5826-13-R3A-B1, 5826-13-R3A-B3, 5826-13-R3A-D5, 5826-
13-R3A-D6, 5826-13-R4A-E2, 5826-13-R4A-E6, 5826-13-R4A-E9, 5826-13-
R4A-F10, 5826-13-R4A-G11, 5826-13-R4A-G12, 5826-13-R4A-H1, 5826-13-
R4A-H2, 5826-13-R4A-H3, 5826-13-R4A-H4, 5826-13-R4A-H5, 5826-13-R4A-
H6, 5826-13-R4A-H9, 5826-13-R4A-H10, 5826-13-R4A-H11, and 5826-13-R4A-
H12,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
2:
5738-10-R3A-C6, 5738-10-R3A-D5, 5738-10-R4A-G12, 5738-13-R4A-D11, and
5738-13-R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
4:
5826-8-R6A-E10, 5826-8-R5A-G8, 5826-8-R6A-H11, 5826-13-R3A-D5, 5826-
13-R4A-H5, and 5826-13-R4A-H12,
as described and defined hereinabove.
In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the
invention comprises
a DEP1-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
2:
5738-10-R4A-G12, 5738-13-R4A-D11, and 5738-13-R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
4:
5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12,
as described and defined hereinabove.
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In this embodiment,
a) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DEP1-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
2:
5738-13-R4A-D11, and 5738-13-R2A-H4; and
b) one extracellular binding domain of the bispecific CAR of the invention
comprises
a DPP4-binding domain comprising a combination of (i) a HCVR and (ii) a LCVR,
combination being that of any one of the following clones as defined in Table
4:
5826-13-R3A-D5, 5826-13-R4A-H5, and 5826-13-R4A-H12,
as described and defined hereinabove.
Additionally or alternatively, the CAR of the invention is multispecific and
comprises
extracellular binding domains, wherein at least two extracellular binding
domains
recognize and are capable of binding to the same senescent cell-associated
antigen, but
on different epitopes of said senescent cell-associated antigen.
Such multispecific CARs typically comprise:
(i) two of more extracellular binding domains, as defined hereinabove,
(ii) one extracellular spacer domain,
(iii) one transmembrane domain,
(iv) optionally, at least one costimulatory domain, and
(v) at least one intracellular signaling domain.
In a multispecific CAR, each extracellular binding domain comprises or
consists of a
single chain variable region (scFv) or a Fab fragment, preferably a scFv,
comprising the
antigen-binding domain as defined hereinabove.
In one embodiment, the extracellular binding domains in the multispecific CAR
of the
invention are linked or fused together through a flexible peptidic linker,
enabling each
extracellular binding domain to form the desired structure for antigen binding
(Pluckthun,
1994. "Antibodies from Escherichia coli". In Rosenberg & Moore (Eds.), The
pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology,
113:269-315. Springer: Berlin, Heidelberg). Flexible peptidic linkers are
generally
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composed of small, non-polar (e.g., glycine, Gly, G) or polar (e.g., serine,
Ser, S; or
threonine, Thr, T) amino acids, as suggested by Argos (1990. J Mol Biol.
211(4):943-
958). The small size of these amino acids provides flexibility, and allows for
mobility of
the connecting functional domains, such as the extracellular binding domains.
In one
embodiment, the flexible peptidic linker may be a short oligo- or polypeptide,
preferably
having a length ranging from 2 to 30 amino acids. In one embodiment, the
flexible
peptidic linker comprises glycine-serine repeats. In one embodiment, the
flexible peptidic
linker comprises one, or several repeats of, such as 2, 3, 4, 5 or more
repeats of, GS
linker(s) (i.e., a sequence of one Gly and one Ser), G2S linker(s) (i.e., a
sequence of two
Gly and one Ser), G3S linker(s) (i.e., a sequence of three Gly and one Ser),
G4S linker(s)
(i.e., a sequence of four Gly and one Ser), or G5S linker(s) (i.e., a sequence
of five Gly
and one Ser).
In one embodiment, the CAR of the invention comprises an extracellular spacer
domain
(also called hinge domain).
In one embodiment, the at least one extracellular binding domain is connected
to one
transmembrane domain through one extracellular spacer domain.
The extracellular spacer domain typically facilitates proper protein folding,
provides
flexibility to the at least one extracellular binding domain and helps
avoiding steric
hindrance. It typically comprises a hydrophilic region linking the at least
one extracellular
binding domain and the transmembrane domain.
Extracellular spacer domains may include, but are not limited to, Fc fragments
of
antibodies or fragments or derivatives thereof, hinge regions of antibodies or
fragments
or derivatives thereof, CH2 regions of antibodies, CH3 regions antibodies,
artificial spacer
sequences or combinations thereof.
Examples of extracellular spacer domains include, but are not limited to, CD8a
hinge;
CD28 hinge; flexible peptidic linkers (such as, e.g., Gly3); or hinge region,
CH1, CH2
and/or CH3 domains of IgG' s (such as human IgG4).
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In one embodiment, the extracellular spacer domain is selected from the group
comprising or consisting of (i) a hinge region, CH2 domain and CH3 domain of
IgG4, (ii)
a hinge region of IgG4, (iii) a hinge region and CH2 domain of IgG4, (iv) a
hinge region
of CD8a, (v) a hinge region, CH2 domain and CH3 domain of IgGl, (vi) a hinge
region of
IgG1 (vii) a hinge region and CH2 domain of IgGl, and (viii) a hinge region of
CD28;
and combinations thereof
Additional extracellular spacer domains will be apparent to those skilled in
the art and
may be used in connection with alternate embodiments of the invention.
In one embodiment, the CAR of the invention comprises a transmembrane domain.
In one embodiment, the transmembrane domain comprises an amino acid sequence
derived from the transmembrane domain of any protein which has such
transmembrane
domain, including any of the type I, type II or type III transmembrane
proteins.
In one embodiment, the transmembrane domain may also comprise an artificial
hydrophobic sequence.
Examples of transmembrane domains that are suitable in the CAR of the
invention
include, but are not limited to, transmembrane domains of an a, 0 or chain of
a T cell
receptor, or of CD28, CD3y, CD36, CD3c, CD3c CD45, CD4, CD5, CD8, CD9, CD16,
CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40,
CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR (CD357),
.. CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19,
IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,
ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 1 1 a, LFA-1, ITGAM, CD1 lb, PD1,
ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1
(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM
(CD355), Ly9 (CD229), CD160 (BY55), PSGL1, CDIO0 (SEMA4D), SLAMF6 (NTB-
A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162),
LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D or NKG2C.
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In one embodiment, the transmembrane domain comprises an amino acid sequence
derived from the transmembrane domain of CD28 or CD3.
Additional transmembrane domains will be apparent to those skilled in the art
and may
be used in connection with alternate embodiments of the invention.
In one embodiment, the transmembrane domain may be recombinant, in which case
it
will comprise predominantly hydrophobic amino acids, such as, e.g., valine,
Val, V or
leucine, Leu, L.
In one embodiment, the CAR of the invention can optionally comprise one or
several
costimulatory domain(s).
Costimulatory domains enhance cell proliferation, cell survival and
development of
memory cells.
Examples of costimulatory domains that are suitable in the CAR of the
invention include,
but are not limited to, costimulatory domains of any of the members of the
TNFR super
family, CD28, CD137 (4-1BB), CD134 (0X40), Dap10, CD27, CD2, CD5, ICAM-1,
Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, CTLA-4, ICOS, PD-1, and combinations
thereof.
Costimulatory domains from other proteins may also be used with the CARs of
the
invention. Additional costimulatory domains will be apparent to those skilled
in the art
and may be used in connection with alternate embodiments of the invention.
If the CAR of the invention comprises more than one costimulatory domain,
these
domains may be arranged in tandem, optionally separated by a linker, such as a
flexible
peptidic linker as has been described above.
In one embodiment, the costimulatory domain comprises a T cell costimulatory
molecule,
or a sequence derived therefrom.
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In one embodiment, the CAR of the invention comprises at least one
costimulatory
domain selected from the group comprising or consisting of 4-1BB, ICOS, CD27,
0X40,
CD28, CTLA4 and PD-1.
In one embodiment, the CAR of the invention comprises at least one
intracellular
signaling domain (also called endodomain).
The intracellular signaling domain is cytoplasmic, and allows to transduce the
effector
function signal and direct the cell to perform its specialized function upon
binding of the
extracellular binding domain to its antigen.
Examples of intracellular signaling domains that are suitable in the CAR of
the invention
include, but are not limited to, chain of the T cell receptor or any of its
homologs (such
as, e.g., q chain, FccRly and 0 chains, 1V1B1 (Iga) chain, B29 (Igf3) chain,
etc.), CD3
polypeptides (such as, e.g., A, 6 and 6), syk family tyrosine kinases (such
as, e.g., Syk,
ZAP 70, etc.), src family tyrosine kinases (such as, e.g., Lck, Fyn, Lyn,
etc.) and other
molecules involved in T cell transduction, such as, e.g., CD2, CD5 and CD28.
In one embodiment, the intracellular signaling domain may be human CD3 chain,
FcyRIII, FccRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-
based
activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof
Additional intracellular signaling domains will be apparent to those skilled
in the art and
may be used in connection with alternate embodiments of the invention.
In one embodiment, the at least one intracellular signaling domain may
comprise the
entire intracellular portion, or the entire native intracellular signaling
domain, of the
molecule from which it is derived, or a functional fragment or derivative
thereof
In one embodiment, the at least one intracellular signaling domain comprises
or consists
of a T cell primary signaling domain (or a sequence derived therefrom).
In one embodiment, the T cell primary signaling domain comprises or consists
of a
signaling domain of a protein selected in the group of CD3, CD3y, CD3, CD3c,
common FcRy (FCER1G), FcRE (Fc Epsilon Rib), CD79a, CD79b, FcyRIIa, DAP10,
DAP12, and sequences derived therefrom.
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In one embodiment, the T cell primary signaling domain comprises or consists
of a
functional signaling domain of CD3.
T cell primary signaling domains that act in a stimulatory manner may comprise
signaling
motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS).
Examples of ITAM containing T cell primary intracellular signaling domains
that are of
particular use in the invention include, but are not limited to, those of (or
derived from)
CD3c common FcRy (FCER1G), FcyRIIa, FcRf3 (FccR1b), CD3y, CD36, CD3c, CD5,
CD22, CD66b, CD79a, CD79b, DAP10, and DAP12.
In one embodiment, the T cell primary signaling domain comprises a modified
ITAM
domain (e.g., a mutated ITAM domain which has altered e.g., increased or
decreased)
activity as compared to the native ITAM domain. In one embodiment, a primary
signaling
domain comprises a modified ITAM-containing primary intracellular signaling
domain,
e.g., an optimized and/or truncated ITAM-containing primary intracellular
signaling
domain. In one embodiment, a primary signaling domain comprises one, two,
three, four
or more ITAM motifs.
In one embodiment, the at least one intracellular signaling domain and the at
least one
costimulatory domain, if present, may be linked to each other in tandem, in a
random or
in a specified order.
Optionally, a linker, such as a flexible peptidic linker as has been described
above, may
form the linkage between distinct intracellular signaling domains, and/or
between a
costimulatory domain and an intracellular signaling domain. Besides flexible
peptidic
linkers described above, a single amino acid (such as, e.g., alanine, Ala, A
or glycine,
Gly, G) may also be a suitable linker.
In one embodiment, the CAR of the invention comprises more than one
intracellular
signaling domain, such as 2, 3, 4, 5, or more, intracellular signaling
domains.
In one embodiment, the CAR of the invention may further comprise a tag, such
as, e.g.,
a tag for quality control, enrichment, tracking in vivo and the like.
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In one embodiment, said tag is localized N-terminally, C-terminally and/or
internally.
Examples of tags that that are suitable in the CAR of the invention include,
but are not
limited to, hemagglutinin tag, poly-arginine tag, poly-histidine tag, Myc tag,
strep tag, S-
tag, HAT tag, 3xFlag tag, calmodulin-binding peptide (CBP) tag, SBP tag,
chitin binding
domain (CBD) tag, GST tag, maltose-binding protein (MBP) tag, fluorescent
protein tag,
T7 tag, V5 tag and Xpress tag.
The CAR of the invention may be a first-generation, second-generation or third-
generation CAR.
The first generation of CARs was developed more than 30 years ago (Kuwana et
al.,
1987. Biochem Biophys Res Commun. 149(3):960-968; Gross et al., 1989.
Transplant
Proc. 21(1 Pt 1):127-130; Gross et al., 1989. Proc Natl Acad Sci USA.
86(24):10024-
10028).
In one embodiment, the CAR of the invention is a first-generation CAR and
comprises:
(i) at least one extracellular binding domain,
(ii) an extracellular spacer domain,
(iii) a transmembrane domain, and
(iv) one or more intracellular signaling domain(s).
A first-generation CAR can be, for example, a CAR in which signaling is
provided by
CD3c i.e., the intracellular signaling domain is CD3.
Second-generation CARs add a co-stimulatory domain, such as, e.g., CD28 or 4-
1BB.
The involvement of these intracellular signaling domains improve T cell
proliferation,
cytokine secretion, resistance to apoptosis, and in vivo persistence.
In one embodiment, the CAR of the invention is a second-generation CAR and
comprises:
(i) at least one extracellular binding domain,
(ii) an extracellular spacer domain,
(iii) a transmembrane domain,
(iv) a costimulatory domain(s), and
(v) one or more intracellular signaling domain(s).
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Third-generation CARs combine multiple co-stimulatory domains, such as, e.g.,
CD28-
4-1BB or CD28-0X40, to increase T cell activity.
In one embodiment, the CAR of the invention is a third-generation CAR and
comprises:
(i) at least one extracellular binding domain,
(ii) an extracellular spacer domain,
(iii) a transmembrane domain,
(iv) at least two costimulatory domains, and
(v) one or more intracellular signaling domain(s).
Another object of the present invention is a nucleic acid encoding the CAR of
the
invention.
Another object of the present invention is a vector comprising the nucleic
acid encoding
the CAR of the invention.
In one embodiment, the nucleic acid or vector of the invention comprises a
nucleic acid
sequence of the extracellular binding domain(s) operably linked to the nucleic
acid
sequence of an extracellular spacer domain, operably linked to the nucleic
acid sequence
of a transmembrane domain, operably linked to the nucleic acid sequence of a
cytoplasmic domain (i.e., at least one intracellular signaling domain and
optionally, at
least one costimulatory domain).
The nucleic acid or the vector of the invention can be prepared in
conventional ways (e.g.,
recombinant methods), where the genes and regulatory regions may be isolated,
as
appropriate, ligated, and cloned in an appropriate cloning host, analyzed by
restriction or
sequencing, or other convenient means. Particularly, using PCR, individual
fragments
including all or portions of a functional unit may be isolated, where one or
more mutations
may be introduced using "primer repair", ligation, in vitro mutagenesis, etc.,
as
appropriate. Alternatively, the gene of interest can be produced
synthetically, rather than
cloned.
In one embodiment, the vector comprises a first nucleic acid encoding a CAR of
the
invention, and further comprises a second nucleic acid encoding an antibody or
antigen-
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binding fragment thereof, a bispecific antibody (e.g., a BiTE), a cytokine or
a
costimulatory ligand. In one embodiment, the first nucleic acid and the second
nucleic
acid are each operably linked to a promoter. In one embodiment, the first
nucleic acid is
operably linked to a first promoter and the second nucleic acid is operably
linked to a
second promoter. The promoter can be a constitutively-expressed promoter
(e.g., an EFla
promoter) or an inducibly-expressed promoter (e.g., a NFAT promoter).
In one embodiment, expression of the CAR and expression of the antibody or
antigen-
binding fragment thereof, bispecific antibody (e.g., a BiTE), cytokine or
costimulatory
ligand are driven by the same promoter, e.g., a constitutively expressed
promoter (e.g.,
an EF la promoter). In one embodiment, expression of the CAR and expression of
the
antibody or antigen-binding fragment thereof, bispecific antibody (e.g., a
BiTE), cytokine
or costimulatory ligand are driven by different promoters.
In one embodiment, the nucleic acid encoding the CAR can be located upstream
or
downstream of the second nucleic acid encoding the antibody or antigen-binding
fragment thereof, the bispecific antibody (e.g., a BiTE), the cytokine or the
costimulatory
ligand.
Another object of the present invention is an immune cell, preferably an
isolated immune
cell engineered to express the chimeric antigen receptor (CAR) of the
invention at its
surface.
In one embodiment, the immune cell of the invention expresses at its cell
surface a CAR
comprising an extracellular binding domain comprising or consisting of an
antigen-
binding domain directed to a senescent cell-associated antigen, which
recognizes and is
capable of binding to a senescent cell-associated antigen, as defined
hereinabove.
In one embodiment, the immune cell of the invention expresses at its cell
surface an anti-
DEP1 chimeric antigen receptor (CAR), as defined hereinabove.
In one embodiment, the immune cell of the invention expresses at its cell
surface an anti-
DPP4 chimeric antigen receptor (CAR), as defined hereinabove.
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In one embodiment, the immune cell of the invention expresses at its cell
surface at least
two CARs, wherein the first CAR and the second CAR recognize two different
senescent
cell-associated antigens.
In one embodiment, the immune cell of the invention expresses at its cell
surface at least
two CARs, wherein the first CAR is an anti-DEP1 chimeric antigen receptor, and
the
second CAR is an anti-DPP4 chimeric antigen receptor, as defined hereinabove.
In one embodiment, the immune cell of the invention expresses at its cell
surface a
multispecific CAR, as defined hereinabove.
In one embodiment, the immune cell of the invention expresses at its cell
surface a
bispecific CAR, comprising two extracellular binding domains, wherein one
extracellular
binding domain recognizes and is capable of binding to DEP1, and the other
extracellular
binding domain recognizes and is capable of binding to DPP4, as defined
hereinabove.
In one embodiment, the immune cell of the invention further expresses at its
cell surface
a bispecific T cell engager (BiTE).
In one embodiment, the BiTE binds to at least one senescent cell-associated
antigen and
at least one non-senescent cell-associated antigen, as defined hereinabove.
In one embodiment, the immune cell of the invention is a T cell, preferably an
isolated
T cell. In one embodiment, the immune cell is a CDS+ T cell, a CD4+ T cells, a
natural
killer (NK) cell or an NKT cell.
In one embodiment, the immune cell of the invention is a cytotoxic T cell
(also known as
TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CDS+ T cells
or killer
T cell). NK cells and NKT cells are also encompassed in the invention.
In one embodiment, the T cell is a CDS+ T cytotoxic lymphocyte cell selected
from the
group comprising or consisting of naive CDS+ T cells, CDS+ memory T cells,
central
memory CDS+ T cells, regulatory CDS+ T cells, IPS-derived CDS+ T cells,
effector
memory CDS+ T cells and bulk CDS+ T cells.
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In one embodiment, the T cell is a CD4+ T helper lymphocyte cell selected from
the group
comprising or consisting of naive CD4+ T cells, CD4+ memory T cells, central
memory
CD4+ T cells, regulatory CD4+ T cells, IPS-derived CD4+ T cells, effector
memory CD4+
T cells and bulk CD4+ T cells.
In one embodiment, the immune cell of the invention is cytotoxic for cells
expressing at
their surface the senescent cell-associated antigen recognized by the CAR
(such as, e.g.,
DEP1 and/or DPP4). In one embodiment, the immune cell of the invention is not
cytotoxic for cells expressing at their surface the senescent cell-associated
antigen
recognized by the CAR (such as, e.g., DEP1 and/or DPP4).
In one embodiment, the immune cell of the invention is a phagocytic cell.
In one embodiment, the immune cell of the invention is a phagocytic cell
selected from
the group comprising or consisting of macrophages, monocytes, histiocytes,
Kupffer
cells, alveolar macrophages, microglial cells and dendritic cells.
In one embodiment, the immune cell of the invention is a mammal immune cell,
preferably a human immune cell.
In one embodiment, the immune cells of the invention is an autologous cell, a
syngeneic
cell, an allogenic cell, or a xenogeneic cell.
Another object of the present invention is a population of immune cells,
comprising a
plurality of immune cells of the invention.
In one embodiment, the population of immune cells may be homogeneous, i.e.,
composed
at more than 50%, such as more than 60%, 70%, 80%, 90%, 95% or more of the
same
immune cells, i.e., immune cells of same nature, origin, and/or expressing the
same CAR
at their cell surface.
In one embodiment, the population of immune cells may be heterogeneous, i.e.,
composed of a mix of different immune cells, i.e., immune cells of different
nature, origin,
and/or expressing a different CAR at their cell surface.
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Another object of the present invention is a method of obtaining an immune
cell
expressing the CAR of the invention at its surface, or a population of such
immune cells.
Means and methods to obtain immune cells expressing the CAR of the invention
at its
surface, or a population of such immune cells, are well known in the art.
In one embodiment, the method for obtaining an immune cell expressing the CAR
of the
invention at its surface, or a population of such immune cells, comprises one
or several
of:
- a step of isolating an immune cell or population of immune cells (e.g.,
T cells or
phagocytic cells) from a sample obtained from a subject,
- optionally, a step of selecting a specific subpopulation of immune cells,
- a step of transducing the immune cell or population of immune cells
with a nucleic
acid encoding the CAR of the invention,
- optionally, a step of expanding the transduced immune cell or
population of immune
cells,
- optionally, a step of washing the immune cell or population of immune cells,
- optionally, a step of cryopreserving the immune cell or population of
immune cells.
Prior to transduction and expansion of the immune cell of the invention, a
source of
immune cells (e.g., T cells or phagocytic cells) is obtained from a subject.
Thus, in one
embodiment, the immune cell or the population of immune cells of the invention
is
isolated and/or substantially purified.
T cells and/or phagocytic cells can be obtained from a number of sources,
including
peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord
blood, thymus
tissue, tissue from a site of infection, ascites, pleural effusion, spleen
tissue, and tumors.
In one embodiment, any number of T cell and/or phagocytic cells lines
available in the
art may be used.
In one embodiment, T cells and/or phagocytic cells can be obtained from a unit
of blood
collected from a subject using any number of techniques known to those skilled
in the art,
such as FicollTM separation.
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In one embodiment, cells from the circulating blood of a subject are obtained
by
apheresis. The apheresis product typically contains lymphocytes, including T
cells,
monocytes, granulocytes, B cells, other nucleated white blood cells, red blood
cells, and
platelets.
.. In one embodiment, cells from the circulating blood of a subject are
obtained by
leukapheresis. In one embodiment, cells collected by leukapheresis may be
washed to
remove the plasma fraction and to place the cells in an appropriate buffer or
media for
subsequent processing steps. In one embodiment of the invention, the cells are
washed
with phosphate buffered saline (PBS). In one embodiment, the wash solution
lacks
.. calcium, and may lack magnesium or many if not all divalent cations. After
washing, the
cells may be resuspended in a variety of biocompatible buffers, such as, e.g.,
Ca2+-free,
Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
Alternatively, the undesirable components of the leukapheresis sample may be
removed
and the cells directly resuspended in culture media.
In one embodiment, T cells and/or phagocytic cells are isolated from
peripheral blood
lymphocytes by lysing the red blood cells and depleting the monocytes, e.g.,
by
centrifugation through a PERCOLLTM gradient or by counterflow centrifugal
elutriation.
A specific subpopulation of T cells can be further isolated by positive or
negative
selection techniques. Those skilled in the art would recognize that multiple
rounds of
selection can also be used in the context of this invention.
In one embodiment, it may be desirable to perform the selection procedure and
use the
"unselected" cells in the activation and expansion process. "Unselected" cells
can also be
subjected to further rounds of selection. Enrichment of a T cell population by
negative
selection can be accomplished with a combination of antibodies directed to
surface
markers unique to the negatively selected cells. One method is cell sorting
and/or
selection via negative magnetic immuno-adherence or flow cytometry that uses a
cocktail
of monoclonal antibodies directed to cell surface markers present on the cells
negatively
selected.
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For example, to enrich CD4+ cells by negative selection, a monoclonal antibody
cocktail
typically includes antibodies directed to CD14, CD20, CD1 lb, CD16, HLA-DR,
and
CD8. To deplete T regulatory cells, anti-CD25 conjugated beads or other
similar method
of selection can be used. To enrich a population of monocytes, macrophages
and/or
dendritic cells by negative selection, a monoclonal antibody cocktail
typically includes
antibodies to CD34, CD3, CD4, CD8, CD14, CD19 or CD20. Those skilled in the
art are
familiar with such means and methods.
In one embodiment, the immune cell or population of immune cells of the
invention is
transduced with a nucleic acid encoding the CAR of the invention, or with a
vector
comprising the nucleic acid encoding the CAR of the invention, and optionally
expanded.
Methods for transfecting eukaryotic cells and tissues removed from an organism
in an ex
vivo method are known to those skilled in the art. In one embodiment, the
method is an
ex vivo method. Thus, it is contemplated that cells or tissues may be removed
and
transfected ex vivo using nucleic acids or vectors of the invention. In one
embodiment,
the transplanted cells or tissues may be placed into an organism. In one
embodiment, a
nucleic acid is expressed in the transplanted immune cell population.
The nucleic acid encoding the CAR of the invention, once completed and
demonstrated
to have the appropriate sequences, may be introduced into the immune cell by
any
convenient means, including, but not limited to, by fusion, electroporation,
biolistics,
transfection, lipofection, or the like. Alternatively, the nucleic acid
encoding the CAR of
the invention may also be integrated and packaged into non-replicating,
defective viral
genomes like adenovirus, adeno-associated virus (AAV), or herpes simplex virus
(HSV)
or others, including retroviral vectors or lentiviral vectors, for infection
or transduction
into cells. The nucleic acid encoding the CAR of the invention may include
viral
sequences for transfection, if desired. The engineered cells may be grown and
expanded
in culture before introduction of the construct(s), followed by the
appropriate treatment
for introduction of the construct(s) and integration of the construct(s). The
engineered
cells are then expanded and screened by virtue of a marker present in the
construct.
Various markers that may be used successfully include hprt, neomycin
resistance,
thymidine kinase, hygromycin resistance, etc.
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In one embodiment, one may have a target site for homologous recombination,
where it
is desired that a nucleic acid be integrated at a particular locus. For
example, one can
knock-out an endogenous gene and replace it (at the same locus or elsewhere)
with the
gene encoded for by the construct using materials and methods as are known in
the art
for homologous recombination. For homologous recombination, one may use either
OMEGA or 0-vectors. See, e.g., Thomas & Capecchi, 1987. Cell. 51(3):503-12;
Mansour
et al., 1988. Nature. 336(6197):348-352; and Joyner et al., 1989. Nature.
338(6211):153-
156.
The nucleic acid encoding the CAR of the invention may be introduced as a
single DNA
molecule encoding at least the CAR of the invention and optionally another
gene, or
different DNA molecules having one or more genes. Other genes include genes
that
encode therapeutic molecules or suicide genes, for example. The constructs may
be
introduced simultaneously or consecutively, each with the same or different
markers.
In one embodiment, suicide gene technology may be used. Different suicide gene
technologies are described in the art depending on their mechanism of action
(Jones et
al, 2014. Front Pharmacol. 5:254). Examples of gene-directed enzyme prodrug
therapy
(GDEPT) converting a nontoxic drug to a toxic drug include herpes simplex
virus
thymidine kinase (HSV-TK) and cytosine deaminase (CD). Other examples are
chimeric
proteins composed of a drug binding domain linked to apoptotic components such
as for
example the inducible Fas (iFas) or the inducible Caspase 9 (iCasp9) systems.
Other
examples include systems mediated by therapeutic antibodies such as inducing
overexpression of c-myc at the surface of the engineered cell to induce their
deletion by
administration of an anti-c-myc antibody. The use of EGFR is described as a
similar
system compared to the c-myc system.
Vectors containing useful elements such as bacterial or yeast origins of
replication,
selectable and/or amplifiable markers, promoter/enhancer elements for
expression in
prokaryotes or eukaryotes, etc. that may be used to prepare stocks of
construct DNAs and
for carrying out transfections are well known in the art, and many are
commercially
available.
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In one embodiment, the step of transducing the immune cell or population of
immune
cells corresponds to a gene disruption step, a gene correction step or a gene
addition step,
preferably a gene addition step.
The immune cells that have been transduced with the nucleic acid encoding the
CAR of
the invention are then grown in culture under selective conditions, to retain
only those
cells which were successfully transduced.
In one embodiment, the immune cell or the population of immune cells of the
invention
is or comprises or consists of a genetically modified immune cell.
In one embodiment, the genetically modified immune cell or the population of
immune
cells of the invention can be or comprise or consist of an allogeneic immune
cell. For
example, the allogeneic immune cell can be an immune cell lacking expression
of a
functional human leukocyte antigen (HLA), e.g., HLA class I and/or HLA class
II or a
T cell receptor (TCR).
In one embodiment, the immune cell or the population of immune cells of the
invention
can be engineered such that the immune cell does not express a functional HLA
and/or
TCR on its surface. For example, an immune cell can be engineered such that
cell surface
expression HLA, e.g., HLA class 1 and/or HLA class II or non-classical HLA
molecules,
is downregulated.
Modified immune cells that lack expression of a functional HLA and/or TCR can
be
obtained by any suitable means, including a knock-out or knock-down of one or
more
subunit of HLA. For example, the immune cell can include a knock-down of HLA
using
siRNA, shRNA, clustered regularly interspaced short palindromic repeats
(CRISPR)
transcription-activator like effector nuclease (TALEN), zinc finger
endonuclease (ZFN),
meganuclease (mn, also known as homing endonuclease), or megaTAL (combining a
TAL effector with a mn cleavage domain). Such systems are well known in the
art.
In one embodiment, the nucleic acid encoding a CAR as described herein is
inserted at a
specific locus in the genome of an immune cell, such as, e.g., at the locus of
a gene to be
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deleted. In one embodiment, the nucleic acid encoding a CAR as described
herein is
inserted within an HLA locus, thereby resulting in the inhibition of HLA
expression.
In one embodiment, the CAR of the invention, when expressed by a T cell or
phagocytic
cell, confers to the T cell or phagocytic cell the ability to bind to cells
expressing DEP1
and/or DPP4 on their cell surface and be activated by DEP1 and/or DPP4,
differently
from the antigen that the T cells or phagocytic cell are or would have been
specific or
activated by.
The immune cell population of the invention may thus be defined as a
redirected immune
cell population. The term "redirected" refers to such immune cells, e.g., a T
cell or
phagocytic cell, which carries a CAR as described herein, conferring to the
immune cell
the ability to bind to and be activated by a ligand that is different from the
one the immune
cell is or would have been specific or be activated by.
In one embodiment, the immune cell or population of immune cell of the
invention can
express certain gene products that can kill the modified cells under
controlled conditions,
such as inducible suicide genes.
In one embodiment, the immune cell or the population of immune cells of the
invention
is cultured for expansion. In one embodiment, the immune cell or the
population of
immune cells of the invention comprises or consists of progenitor cells, which
are
cultured for differentiation and expansion of the immune cells or population
of immune
cells as described herein.
Whether prior to or after transduction of the immune cells (i.e., T cells or
phagocytic
cells) to express a desirable CAR as described herein, the immune cells (i.e.,
T cells and/or
phagocytic cells) can be activated and expanded generally using methods as
described,
e.g., in US6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;
6,905,681;
7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;
6,797,514; 6,867,041; and 7,572,631.
In one embodiment, immune cells may be cryopreserved, either after isolation
and
optionally, selection, and/or after transduction and expansion.
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In one embodiment, cryopreserved cells are thawed and washed as described
herein and
allowed to rest for one hour at room temperature prior to activation.
Also contemplated in the context of the invention is the collection of blood
samples or
leukapheresis product from a subject at a time period prior to when the
expanded cells as
described herein might be needed. As such, the source of the cells to be
expanded can be
collected at any time point necessary, and desired cells, such as T cells
and/or phagocytic
cells, isolated and frozen for later use in cell therapy for any number of
diseases or
conditions that would benefit from cell therapy, such as those described
herein.
In one embodiment, a blood sample or a leukapheresis product is taken from a
generally
healthy subject. In one embodiment, a blood sample or a leukapheresis product
is taken
from a healthy subject who is at risk of developing a disease, but who has not
yet
developed said disease, and the cells of interest are isolated and
cryopreserved for later
use. In one embodiment, the T cells and/or phagocytic cells may be expanded,
frozen,
and used at a later time.
Another object of the invention is a composition comprising, consisting of or
consisting
essentially of:
an antigen-binding domain, as described above;
an antibody or antigen-binding fragment thereof, as described above;
a nucleic acid encoding the antigen-binding domain, the antibody or the
antigen-
binding fragment thereof, as described above;
a vector comprising the nucleic acid encoding the antigen-binding domain, the
antibody or the antigen-binding fragment thereof, as described above;
a CAR, as described above;
a nucleic acid encoding the CAR, as described above;
- a vector comprising the nucleic acid encoding the CAR, as described
above;
an immune cell engineered to express the CAR at its cell surface, as described
above;
a population of immune cells engineered to express the CAR at their cell
surface,
as described above.
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In one embodiment, the composition comprises, consists of or consists
essentially of:
a DEP1-binding domain, as described above;
an anti-DEP1 antibody or antigen-binding fragment thereof, as described above;
a nucleic acid encoding the DEP1-binding domain, the anti-DEP1 antibody or the
antigen-binding fragment thereof, as described above;
a vector comprising the nucleic acid encoding the DEP1-binding domain, the
anti-
DEP1 antibody or the antigen-binding fragment thereof, as described above;
an anti-DEP1 CAR, as described above;
a nucleic acid encoding the anti-DEP1 CAR, as described above;
- a vector comprising the nucleic acid encoding the anti-DEP1 CAR, as
described
above;
an immune cell engineered to express the anti-DEP1 CAR at its cell surface, as
described above;
a population of immune cells engineered to express the anti-DEP1 CAR at their
cell
surface, as described above.
In one embodiment, the composition comprises, consists of or consists
essentially of:
a DPP4-binding domain, as described above;
an anti-DPP4 antibody or antigen-binding fragment thereof, as described above;
a nucleic acid encoding the DPP4-binding domain, the anti-DPP4 antibody or the
antigen-binding fragment thereof, as described above;
a vector comprising the nucleic acid encoding the DPP4-binding domain, the
anti-
DPP4 antibody or the antigen-binding fragment thereof, as described above;
an anti-DPP4 CAR, as described above;
a nucleic acid encoding the anti-DPP4 CAR, as described above;
- a vector comprising the nucleic acid encoding the anti-DPP4 CAR, as
described
above;
an immune cell engineered to express the anti-DPP4 CAR at its cell surface, as
described above;
a population of immune cells engineered to express the anti-DPP4 CAR at their
cell
surface, as described above.
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In one embodiment, the composition comprises, consists of or consists
essentially of:
a DEP1-binding domain and a DPP4-binding domain, as described above;
an anti-DEP1 antibody or antigen-binding fragment thereof and an anti-DPP4
antibody or antigen-binding fragment thereof, as described above;
- an anti-DEP1/anti-DPP4 bispecific antibody;
a nucleic acid encoding the DEP1-binding domain, the anti-DEP1 antibody or the
antigen-binding fragment thereof and a nucleic acid encoding the DPP4-binding
domain, the anti-DPP4 antibody or the antigen-binding fragment thereof, as
described above;
- a nucleic acid encoding the anti-DEP1/anti-DPP4 bispecific antibody, as
described
above;
a vector comprising the nucleic acid encoding the DEP1-binding domain, the
anti-
DEP1 antibody or the antigen-binding fragment thereof and a vector comprising
the
nucleic acid encoding the DPP4-binding domain, the anti-DPP4 antibody or the
antigen-binding fragment thereof, as described above;
a vector comprising the nucleic acid encoding the DEP1-binding domain, the
anti-
DEP1 antibody or the antigen-binding fragment thereof and the nucleic acid
encoding the DPP4-binding domain, the anti-DPP4 antibody or the antigen-
binding
fragment thereof, as described above;
- a vector comprising the nucleic acid the anti-DEP1/anti-DPP4 bispecific
antibody,
as described above;
an anti-DEP1 CAR and an anti-DPP4 CAR, as described above;
an anti-DEP1/anti-DPP4 bispecific CAR, as described above;
a nucleic acid encoding the anti-DEP1 CAR and the anti-DPP4 CAR, as described
above;
a nucleic acid encoding the anti-DEP1/anti-DPP4 bispecific CAR, as described
above;
a vector comprising a nucleic acid encoding the anti-DEP1 CAR and a vector
comprising the nucleic acid encoding the anti-DPP4 CAR, as described above;
- a vector comprising a nucleic acid encoding the anti-DEP1 CAR and the
nucleic
acid encoding the anti-DPP4 CAR, as described above;
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a vector comprising a nucleic acid encoding the anti-DEP1/anti-DPP4 bispecific
CAR, as described above;
an immune cell engineered to express the anti-DEP1 CAR and an immune cell
engineered to express the anti-DPP4 CAR, as described above;
- an immune cell engineered to express the anti-DEP1 CAR and the anti-DPP4
CAR,
as described above;
an immune cell engineered to express the anti-DEP1/anti-DPP4 bispecific CAR,
as
described above;
a population of immune cells engineered to express the anti-DEP1 CAR and a
population of immune cells engineered to express the anti-DPP4 CAR, as
described
above;
a population of immune cells engineered to express the anti-DEP1 CAR and the
anti-DPP4 CAR, as described above;
a population of immune cells engineered to express the anti-DEP1/anti-DPP4
bispecific CAR, as described above.
In one embodiment, the composition has been frozen and thawed. In one
embodiment,
the composition is lyophilized.
In one embodiment, the compositions of the invention are pharmaceutical
compositions
and further comprise at least one pharmaceutically acceptable excipient.
The term "pharmaceutically acceptable excipient" includes any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption
delaying agents and the like. Said excipient does not produce an adverse,
allergic or other
untoward reaction when administered to an animal, preferably a human. For
human
administration, preparations should meet sterility, pyrogenicity, and general
safety and
purity standards as required by regulatory offices, such as, for example, FDA
Office or
EMA.
Pharmaceutically acceptable excipients that may be used in the pharmaceutical
composition of the invention include, without being not limited to, ion
exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human serum
albumin,
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buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate,
partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or
electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,
sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone,
cellulose-based substances (for example sodium carboxymethylcellulose),
polyethylene
glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers,
polyethylene glycol and wool fat.
In one embodiment, the compositions according to the present invention are
medicaments.
As used herein, the term "consisting essentially of', with reference to a
composition,
pharmaceutical composition or medicament, means that the antigen-binding
domain,
antibody or antigen-binding fragment thereof, nucleic acid, vector, CAR,
immune cell or
population of immune cells of the invention is/are the only therapeutic agent,
or agent
with a biologic activity, within said composition, pharmaceutical composition
or
medicament.
Such compositions and medicaments may comprise buffers such as neutral
buffered
saline, phosphate buffered saline and the like; carbohydrates such as glucose,
mannose,
sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as
glycine;
antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g.,
aluminum
hydroxide); and preservatives.
Since the present invention selectively target senescent cells, it is
contemplated that the
compositions according to the present invention may be cosmetic compositions,
and
further comprise at least one dermatologically acceptable excipient.
The term "dermatologically acceptable excipient" refers to excipient which are
suitable
for external topical application. Examples of dermatologically acceptable
excipients
include, but are not limited to, sebum-regulating agents, antibacterial
agents, antifungal
agents, keratolytic agents, keratoregulating agents, astringents, anti-
inflammatory agents,
anti-irritants, antioxidants, free-radical scavengers, cicatrizing agents,
anti-aging agents
and moisturizing agents.
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The administration of the composition, pharmaceutical composition, medicament
or
cosmetic composition of the invention may be carried out in any convenient
manner,
including by injection, aerosol inhalation, topical delivery (such as, for
example, by
transdermal delivery), oral delivery, rectal delivery, nasal delivery, or
vaginal delivery.
In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition of the present invention is in a form adapted for
injection, such as,
e.g., for trans-arterial, intravenous (iv.), intramuscular, intraperitoneal
(i.p.), intrapleural,
intradermal, subcutaneous, transdermal injection or infusion.
Examples of forms suitable for injectable use include, but are not limited to,
sterile
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile
injectable solutions or dispersions. The prevention against contamination by
microorganisms can be brought about by adding in the composition preservatives
such
as, e.g., various antibacterial and antifungal agents (e.g., parabens,
chlorobutanol, phenol,
sorbic acid, thimerosal and the like). In one embodiment, it may be preferable
to include
isotonic agents, e.g., sugars or sodium chloride, to reduce pain during
injection. In one
embodiment, prolonged absorption of the injectable compositions can be brought
about
by the use in the compositions of agents delaying absorption, e.g., aluminum
monostearate and gelatin.
In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition of the present invention is in an adapted form for a
parenteral
administration. Thus, in one embodiment, the composition, pharmaceutical
composition,
medicament or cosmetic composition of the invention is to be administered
parenterally.
In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition of the invention is in an adapted form for an intravenous
administration. Thus, in one embodiment, the composition, pharmaceutical
composition,
medicament or cosmetic composition of the present invention is to be
administered
intravenously.
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In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition of the present invention may be injected directly into
the site of the
disease or disorder to be treated.
In one embodiment, a lyophilized composition, a lyophilized pharmaceutical
composition, a lyophilized medicament or a lyophilized cosmetic composition of
the
invention is solubilized in water for injection and gently mixed, the mixture
is gently
mixed and charged into a suitable syringe. This invention thus also relates to
a medical
device, including a syringe filled or prefilled with a composition,
pharmaceutical
composition, medicament or cosmetic composition of the invention.
In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition of the invention is formulated for topical
administration. Thus, in
one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic
composition is to be administered topically.
Examples of forms adapted for topical administration include, without being
limited to,
liquid, paste or solid compositions, and more particularly aqueous solutions,
drops,
dispersions, sprays, microcapsules, micro- or nanoparticles, polymeric patch,
or
controlled-release patch, and the like.
In particular embodiments where the compositions of the invention comprise a
CAR, an
immune cell or a population of immune cells as described above, such
compositions may
be administered by intradermal or subcutaneous injection, such as by i.v.
injection.
In one embodiment, the compositions comprising a CAR, an immune cell or a
population
of immune cells may be injected directly into a lymph node, site of infection,
site of
inflammation or site of tissue or organ rejection. In one embodiment, the
compositions
comprising a CAR, an immune cell or a population of immune cells may be
injected
directly into the site of the autoimmune and/or inflammatory disease.
In one embodiment, the subject is administered, or is to be administered, with
autologous
cells. In some embodiments, the subject is administered, or is to be
administered, with
allogenic cells.
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In one embodiment, the subject may be a mammal. In one embodiment, the subject
may
be a human.
Another object of the present invention is the use of the composition,
pharmaceutical
composition or medicament described herein, as a medicament.
Another object of the present invention is the use of the composition,
pharmaceutical
composition or medicament described herein, in treating, preventing or
alleviating
senescence-related diseases or disorders.
Another object of the present invention is a method of treating, preventing or
alleviating
senescence-related diseases or disorders in a subject in need thereof,
comprising
administering to said subject the composition, pharmaceutical composition or
medicament described herein.
As used herein, the term "senescence-associated", "senescence-related" or "age-
related" diseases, disorders, or conditions refers to a physiological
condition that presents
with one or more symptoms or signs, wherein a subject having the condition
needs or
would benefit from a lessening of such symptoms or signs. The condition is
senescence-
associated if it is caused or mediated in part by senescent cells, which may
be induced by
multiple etiologic factors including age, DNA damage, oxidative stress,
genetic defects,
etc. Lists of senescence-associated disorders that can potentially be treated
or managed
using the methods and products taught in this disclosure include those
discussed in this
disclosure and the previous disclosures to which this application claims
priority.
Non-limiting examples of senescence-related diseases include : fibrotic
diseases, chronic
inflammatory diseases (e.g., arthritis or arthrosis), cancer, premalignant
lesions,
atherosclerosis, osteoarthritis, diabetes, diabetic ulcers, kyphosis,
scoliosis, hepatic
insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS),
laminopathies,
osteoporosis, dementia, (cardio)vascular diseases (e.g., angina, arrhythmia,
atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery
disease (CAD),
carotid artery disease, endocarditis, heart attack, coronary thrombosis,
myocardial
infarction, high blood pressure/hypertension,
hypercholesterolemia/hyperlipidemia,
mitral valve prolapsed, peripheral artery disease (PAD) and stroke), obesity,
metabolic
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syndrome, acute myocardial infarction, emphysema, insulin sensitivity,
boutonneuse
fever, sarcopenia, neurodegenerative diseases (e.g., Alzheimer's, Huntington's
or
Parkinson's disease), cataract, anemia, hypertension, age-related macular
degeneration,
COPD, asthma, renal insufficiency, incontinence, hearing loss such as
deafness, vision
loss such as blindness, sleeping disturbances, pain such as joint pain or leg
pain,
imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle
loss, loss of
bone density, frailty and/or reduced fitness.
Another object of the present invention is the use of the composition,
pharmaceutical
composition or medicament described herein, in treating, preventing or
alleviating
fibrotic diseases, premalignant lesions, inflammatory diseases and cancers.
Another object of the present invention is a method of treating, preventing or
alleviating
fibrotic diseases, premalignant lesions, inflammatory diseases and cancers in
a subject in
need thereof, comprising administering to said subject the composition,
pharmaceutical
composition or medicament described herein.
Senescent cells are present in fibrosis of many tissues including, but not
limited, to skin,
liver, lung, pancreas and prostate.
Thus, in one embodiment, the senescence-related disease or disorder to be
treated is a
fibrotic disease. Exemplary fibrotic diseases which may be treated by the
invention
include but are not limited to eosinophilic esophagitis, hypereosinophilic
syndromes
(HES), Loeffler's endomyocarditis, endomyocardial fibrosis, idiopathic
pulmonary
fibrosis, and scleroderma.
In one embodiment, the pulmonary fibrotic disease to be treated, prevented or
alleviated
is selected from the group comprising idiopathic pulmonary fibrosis (IPF),
chronic
obstructive pulmonary disease (COPD) acute respiratory distress syndrome
(ARDS),
combined pulmonary fibrosis and emphysema (CPFR), pulmonary edema, Loffler's
syndrome, eosinophilic pneumonia, respiratory hypersensitivity, allergic
bronchopulmonary aspergillosis (ABPA), Hamman-Rich syndrome, sarcoidosis,
pneumoconiosis, and hypersensitivity pneumonitis (HP).
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In one embodiment, the pulmonary fibrotic disease to be treated, prevented or
alleviated
is chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary
fibrosis.
In one embodiment, the senescence-related disease or disorder to be treated is
a
premalignant lesion.
As used herein, the phrase "premalignant lesion" refers to a mass of cells
and/or tissue
having increased probability of transforming into a malignant tumor.
Examples of premalignant lesions include, but are not limited to, adenomatous
polyps,
Barrett's esophagus, pancreatic intraepithelial neoplasia (PanIN), IPMN
(intraductal
papillary mucinus neoplasia), DCIS (ductal carcinoma in situ) in the breast,
leukoplakia
and erythroplakia. Thus, the premalignant lesion to be treated by the
invention can
transform into a malignant solid or non-solid (e.g., hematological
malignancies) cancer
(or tumor).
In one embodiment, the premalignant lesion which is to be treated is an
adenomatous
polyp of the colon, an adenomatous polyp of the rectum, an adenomatous polyp
of the
small bowel or Barrett's esophagus.
As used herein, the term "inflammatory diseases" refers to any abnormality
associated
with inflammation, such as, for example, chronic inflammatory diseases, acute
inflammatory diseases.
Examples of inflammatory disorders include, but are not limited to, rheumatic
diseases,
neurological diseases, cardiovascular diseases, uro-gynecological diseases,
eye and ear
diseases, mucocutaneous diseases, infectious diseases, graft rejection
diseases and
allergic diseases.
Examples of rheumatic diseases include, but are not limited to, arthritis,
osteoarthritis,
rheumatoid arthritis, osteoporosis, fibromyalgia, lupus, systemic lupus
erythematosus and
scleroderma.
Examples of neurological diseases include, but are not limited to, multiple
sclerosis,
Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke,
traumatic brain
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injury, spinal cord injury, dystonia, chronic regional pain syndrome, motor
neuron
disease/amyotrophic lateral sclerosis, Guillain-Barre syndrome, muscular
dystrophy,
cerebral palsy, neuropathy and myositis.
Examples of cardiovascular diseases include, but are not limited to, coronary
heart
disease, stroke, hypertensive heart disease, inflammatory heart disease,
rheumatic heart
disease, aortic aneurysm and dissection, congenital heart disease, deep vein
thrombosis
and pulmonary embolism and atherosclerosis.
Examples of uro-gynecological diseases include, but are not limited to,
glomerulonephritis, urinary incontinence and prolapse.
Examples of eye and ear diseases include, but are not limited to, cataract,
glaucoma, age-
related macular degeneration (AMD), presbyopia, dry eyes, corneal diseases,
diabetic
retinopathy, vertigo, tinnitus and Meniere's disease.
Examples of mucocutaneous diseases include, but are not limited to, eczema,
xeroderma
pigmentosum, oral lichen planus, mucous membrane pemphigoid and pemphigus
vulgaris.
Examples of infectious diseases include, but are not limited to, chronic
infectious
diseases, subacute infectious diseases, acute infectious diseases, viral
diseases, bacterial
diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma
diseases
and prion diseases.
Examples of diseases associated with transplantation of a graft include, but
are not limited
to, graft rejection, chronic graft rejection, subacute graft rejection,
hyperacute graft
rejection, acute graft rejection and graft versus host disease.
Examples of allergic diseases include, but are not limited to, asthma, hives,
urticaria,
pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex
allergy, chemical
allergy, drug allergy, insect bite allergy, animal dander allergy, stinging
plant allergy,
poison ivy allergy and food allergy.
In one embodiment, the senescence-related disease or disorder to be treated is
cancer.
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Non-limiting examples of cancers which may be treated according to this aspect
of the
present invention include, but are not limited to, adenocarcinoma, adrenal
gland tumor,
ameloblastoma, anaplastic, anaplastic carcinoma of the thyroid, angiofibroma,
angioma,
angiosarcoma, apudoma, argentaffmoma, arrhenoblastoma, ascites tumor cell,
ascitic
tumor, astroblastoma, astrocytoma, ataxia-telangiectasia, atrial myxoma, a
basal cell
carcinoma cell, bone cancer, brainstem glioma, brain tumor, breast cancer,
Burkitt's
lymphoma, cerebellar astrocytoma, cervical cancer, cherry angioma,
cholangiocarcinoma, cholangioma, chondroblastoma, chondroma, chondrosarcoma,
chorioblastoma, choriocarcinoma, colon cancer, common acute lymphoblastic
leukemia,
craniopharyngioma, cystocarcinoma, cystofbroma, cystoma, ductal carcinoma in
situ,
ductal papilloma, dysgerminoma, encephaloma, endometrial carcinoma,
endothelioma,
ependymoma, epithelioma, erythroleukemia, Ewing's sarcoma, extra nodal
lymphoma,
feline sarcoma, fibro adenoma, fibro sarcoma, follicular cancer of the
thyroid,
ganglioglioma, gastrinoma cell, glioblastoma multiform, glioma,
gonadoblastoma,
haemangioblastoma, haemangioendothelioblastoma, haemangioendothelioma,
haemangiopericytoma, haematolymphangioma, haemocytoblastoma, haemocytoma,
hairy cell leukemia, hamartoma, hepatocarcinoma, hepatocellular carcinoma,
hepatoma,
histoma, Hodgkin's disease, hypernephroma, infiltrating cancer, infiltrating
ductal cell
carcinoma, insulinoma, juvenile angioforoma, Kaposi sarcoma, kidney tumor,
large cell
lymphoma, leukemia, a leukemia, acute leukemia, lipoma, liver cancer, liver
metastases,
Lucke carcinoma, lymphadenoma, lymphangioma, lymphocytic leukemia, lymphocytic
lymphoma, lymphoeytoma, lymphoedema, lymphoma, lung cancer, malignant
mesothelioma, malignant teratoma, mastocytoma, medulloblastome., melanoma,
meningioma, mesothelioma, Morton's neuroma, multiple myeloma, myeloblastoma,
myeloid leukemia, myelolipoma, myeloma, myoblastoma, myxoma, nasopharyngeal
carcinoma, neoplastic, nephroblastoma, neuroblastoma,
neurofibroma,
neurofibromatosis, neuroglioma, neuroma, non-Hodgkin's
lymphoma,
oligodendroglioma, optic glioma, osteochondroma, osteogenic sarcoma,
osteosarcoma,
ovarian cancer, Paget's disease of the nipple, pancoast tumor, pancreatic
cancer,
phaeochromocytoma, pheoehromocytoma, plasmacytoma, primary brain tumor,
progonoma, prolactinoma, renal cell carcinoma, retinoblastoma,
rhabdomyosarcoma,
rhabdo sarcoma, a solid tumor, sarcoma, a secondary tumor, seminoma, skin
cancer, small
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cell carcinoma, squamous cell carcinoma, strawberry haemangioma, T cell
lymphoma,
teratoma, testicular cancer, thymoma, trophoblastic tumor, and Wilm's tumor.
In one embodiment, the composition, pharmaceutical composition or medicament
described herein is used alone. In one embodiment, the composition ,
pharmaceutical
composition, or medicament described herein is used in combination with at
least one
anti-cancer agent.
Indeed, many of existing and potential anti-cancer agents induce senescence of
cancer
cells, therefore the present invention can be used in combination with these
agents to
increase the efficacy of an anti-cancer treatment. Treatment by these agents
can also
reduce side effects of radiotherapy or chemotherapy with DNA-damaging agents.
Thus, the composition, pharmaceutical composition or medicament described
herein can
be used as an adjuvant therapy along with other treatment modalities for
cancers, which
are selected based on cancer type, location, the cell type and the grade of
malignancy.
Conventional therapies include surgery, radiation therapy, and chemotherapy.
Exemplary anti-cancer drugs that can be co-administered with the composition,
pharmaceutical composition or medicament described herein include, but are not
limited
to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adriamycin,
adozelesin,
aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide,
amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine,
azetepa,
azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride,
bisnafide
dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine,
busulfan,
cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine,
carubicin
hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin,
cladribine,
crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
.. daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine,
dezaguanine
mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride,
droloxifene,
droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate,
eflornithine
hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin
hydrochloride, erbulozole, esorubicin hydrochloride, estramustine,
estramustine
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phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine,
fadrozole
hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate,
fluorouracil,
fluorocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine
hydrochloride,
hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interferon alfa-
2a,
interferon alfa-2b, interferon alfa-nl, interferon alfa-n3, interferon beta-
la, interferon
gamma-lb, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole,
leuprolide
acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone
hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride,
megestrol
acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine,
methotrexate,
methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin,
mitocromin,
mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone
hydrochloride,
mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel,
pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide,
pipobroman,
piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer
sodium,
porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin
hydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingol
hydrochloride,
semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium
hydrochloride,
spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur,
talisomycin, taxol,
tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide,
teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofuirin,
tirapazamine,
topotecan hydrochloride, toremifene citrate, trestolone acetate, triciribine
phosphate,
trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride,
uracil
mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine
sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,
vinleurosine sulfate,
vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole,
zeniplatin,
zinostatin, and zorubicin hydrochloride.
Additional antineoplastic agents include those disclosed in Chabner et al.,
2001.
"Antineoplastic agents". In Goodman et al. (Eds.), Goodman & Gilman' s The
pharmacological basis of therapeutics (le ed., pp. 1315-1404). New York, NY:
McGraw-Hill.
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Another object of the present invention is the use of the cosmetic composition
described
herein, as cosmetics.
Indeed, since the present invention selectively target senescent cells, it is
contemplated
that the present invention can be used for skin care, skin anti-aging and/or
skin
rejuvenation.
Another object of the present invention is thus a method for rejuvenating the
skin in a
subject in need thereof, comprising administering to the subject the cosmetic
composition
described herein.
Because senescent cells drive age-related pathologies, a selective elimination
of these
cells can prevent or delay age-related deterioration. Thus, senescent cells
may be
therapeutic targets in the treatment of aging and age-related disease. As
such, removal of
senescent cells may delay tissue dysfunction and extend health span. Clearance
of
senescent cells is expected to improve tissue milieu, thereby improving the
function of
the remaining non-senescent cells.
Another object of the present invention is thus the use of the composition,
pharmaceutical
composition or medicament described herein, for depleting and/or killing
senescent cells.
Another object of the present invention is thus a method for depleting and/or
killing
senescent cells in a subject, comprising administering to the subject the
composition,
pharmaceutical composition or medicament described herein.
In one embodiment, the composition, pharmaceutical composition, medicament or
cosmetic composition described herein is to be administered to a subject in
need thereof
in a therapeutically effective amount.
It will be however understood that the total daily usage of the composition,
pharmaceutical composition, medicament or cosmetic composition described
herein will
be decided by the attending physician within the scope of sound medical
judgment. The
specific therapeutically effective dose level for any particular patient will
depend upon a
variety of factors including the disease being treated and the severity of the
disease;
activity of the therapeutic agent in the composition, pharmaceutical
composition,
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medicament or cosmetic composition (antigen-binding domain, antibody or
antigen-
binding fragment thereof, nucleic acid, vector, CAR, immune cell or population
of
immune cells of the invention) employed; the age, body weight, general health,
sex and
diet of the subject; the time of administration, route of administration, and
rate of
excretion of the specific composition, pharmaceutical composition, medicament
or
cosmetic composition employed; the duration of the treatment; drugs used in
combination
or coincidental with the composition, pharmaceutical composition, medicament
or
cosmetic composition employed; and like factors well known in the medical
arts. For
example, it is well within the skill of the art to start doses of the compound
at levels lower
than those required to achieve the desired therapeutic effect and to gradually
increase the
dosage until the desired effect is achieved. The total dose required for each
treatment may
be administered by multiple doses or in a single dose.
Disclosed herein is a chimeric antigen receptor (CAR) comprising:
(i) at least one extracellular binding domain, comprising at least one
antigen-binding
domain directed to a senescent cell-associated antigen, preferably to DEP1
and/or
DPP4,
(ii) an extracellular spacer domain,
(iii) a transmembrane domain,
(iv) optionally at least one costimulatory domain, and
(v) at least one intracellular signaling domain.
In one embodiment, said at least one antigen-binding domain is directed to
DEP1.
In one embodiment, said at least one antigen-binding domain is directed to
DPP4.
In one embodiment, said CAR is a bispecific CAR comprising two antigen-binding
domains.
In one embodiment, each of the at least two antigen-binding domains binds to a
different
antigen, preferably to DEP1 and DPP4.
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Also disclosed herein is an isolated immune cell population expressing at
least one CAR
according to the present disclosure, preferably the isolated immune cell
population
expresses:
at least one CAR directed to DEP1 and at least one CAR directed to DPP4; or
- at least one bispecific CAR comprising two antigen-binding domains,
preferably
wherein each of the at least two antigen-binding domains binds to a different
antigen, preferably to DEP1 and DPP4.
In one embodiment, the isolated immune cell population according to the
present
disclosure comprises immune cells selected from the group comprising T cells,
natural
killer (NK) cells, or a combination thereof.
Also disclosed herein is an isolated bispecific antibody or a fragment
thereof, comprising
at least two antigen binding domains directed to at least two senescent cell-
associated
antigens, preferably the at least two senescent cell-associated antigens are
DEP1 and
DPP4.
In one embodiment, the isolated bispecific antibody or fragment thereof
according to the
present disclosure comprises:
(i) an antigen-binding domain of an anti-human DEP1 antibody or a fragment
thereof;
and
(ii) an antigen-binding domain of an anti-human DPP4 antibody or a fragment
thereof.
Also disclosed herein is a composition comprising:
the isolated immune cell population according to the present disclosure;
the isolated bispecific antibody or fragment thereof according to the present
disclosure; or
a mixture of an isolated anti-human DEP1 antibody or a fragment thereof and an
isolated anti-human DPP4 antibody or a fragment thereof.
In one embodiment, the composition according to the present disclosure is a
pharmaceutical composition and further comprises at least one pharmaceutically
acceptable excipient.
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In one embodiment, the composition or pharmaceutical composition according to
the
present disclosure is for use as a medicament.
In one embodiment, the composition or pharmaceutical composition according to
the
present disclosure is:
- for use in treating, preventing or alleviating senescence-related
diseases or
disorders; or
for depleting and/or killing senescent cells.
In one embodiment, senescence-related diseases or disorders are selected from
the group
comprising fibrotic diseases, premalignant lesions, inflammatory diseases and
cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 ¨ 'The structure of CAR vector' ¨ is a schema illustrating the
construction of the
full-length CAR expression cassette, subcloned into Lenti-EFla-rhuMAB 41-VH-
Linker-VL-CFPCART, pCDCAR1. The full length of chimeric antigen receptor was
synthesized and subcloned into lentivirus vector. The insert was confirmed by
Sanger
sequencing and is schematically illustrated in the figure. This CAR vector was
used in the
CAR-T assays for the proof-of-concept experiments.
Figure 2 ¨ 'The assessment of senescent state and DPP4 levels in target cells'
¨ is a graph
showing the senescent state of WI-38 and GM21808 cells, confirmed by
senescence
.. associated P-galactosidase activity. Representative results of control
versus senescent
fibroblasts are shown. DPP4 levels of the cells were monitored by quantitative
RT-PCR.
Relative quantities of DPP4 in young WI-38 (low passage number: PDL < 20) or
old WI-
38 (high passage number: PDL > 50) and in GM21808 cells, untreated or cultured
with
DNA-damaging agent etoposide (adding etoposide every two days for fourteen
days)
were calculated. Fold-change, or relative quantity, of DPP4 in each sample was
calculated
relative to young WI-38 or untreated GM21808.
Figure 3 ¨ 'The ADCC Assay' ¨ is a graph showing the relative toxicity in
young and old
fibroblast cells (WI-38 cells), using 0.05 p.g/mL or 0.5 p.g/m1 of anti-DPP4
antibody.
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Figure 4 ¨ 'The CAR-T assay' ¨ is a graph showing the results of selective
elimination
of senescent cells by engineered CAR-T against DPP4.
Figure 5¨ 'The quantification of experimental CAR-T assay results' ¨ is a
graph showing
the results of the CAR-T assay on control (WI-38/GM21808) versus senescent
cells. Cells
were incubated with increasing ratios of CAR-T to target cells (1:1, 5:1 and
10:1).
Figure 6 ¨ scEv library screening by phage display' ¨ is a graph showing the
panning
using hDEP1 Peptide #4/BSA as target and standard elution strategy. Four
rounds were
carried out in order to isolate specific binders. The reactivity of phages
eluted after each
round against the targeted peptide was assessed in phage-ELISA.
Figure 7 ¨ scEv library screening by phage display' ¨ is a graph showing the
panning
using hDEP1-ECD-His as target and standard elution strategy. Four rounds were
carried
out in order to isolate specific binders. The reactivity of phages eluted
after each round
against the targeted peptide was assessed in phage-ELISA.
Figure 8¨ 'DPP4 and DEP I mRNA levels in normal and diseased primary human
lung
fibroblasts' ¨ is a graph showing the relative quantity of DPP4 and DEP-1 in
normal and
diseased human lung fibroblasts. Expression levels in NHLF (normal human lung
fibroblast), idiopathic pulmonary fibroblast (IPF) and chronic obstructive
pulmonary
disease (COPD) human fibroblast cells were calculated relative to NHLF.
Figure 9 ¨ 'DPP4 staining in fibroblasts' is a set of three graphs showing the
staining of
various primary human fibroblasts and different senescence-stressors in terms
of DPP4
surface antigen presentation in target senescent cells versus normal/young
cells. The data
also include normal human cells versus human lung fibrosis disease cells. WI-
38
(PDL < 20), GM21808 ( etoposide), normal human lung fibroblast (NHLF),
idiopathic
pulmonary fibroblast (IPF) and chronic obstructive pulmonary disease (COPD)
human
fibroblast cells were stained with anti-DPP4 antibodies or scFv. Control
staining
comprising secondary antibody only was included for all cell types, but only
the control
staining for NHLF cells is shown for illustration. Percentage of positive
cells is indicated
on the right side, with MFI indicated in parenthesis.
Figure 9A: staining with monoclonal antibody(Creative Biolabs);
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Figure 9B: staining with 5826-13-R3A-D5;
Figure 9C: staining with 5826-13-R4A-H5.
Figure 10 ¨ 'DEP 1 staining in fibroblasts' is a set of three graphs showing
the staining
of various primary human fibroblasts and different senescence-stressors in
terms of DEP1
surface antigen presentation in target senescent cells versus normal/young
cells. The data
also include normal human cells versus human lung fibrosis disease cells. WI-
38
(PDL < 20), GM21808 ( etoposide), normal human lung fibroblast (NHLF),
idiopathic
pulmonary fibroblast (IPF) and chronic obstructive pulmonary disease (COPD)
human
fibroblast cells were stained with anti-DEP1 antibodies or scFv. Control
staining
comprising secondary antibody only was included for all cell types, but only
the control
staining for NHLF cells is shown for illustration. Percentage of positive
cells is indicated
on the right side, with MFI indicated in parenthesis.
Figure 10A: staining with monoclonal antibody(AbCam)
Figure 10B: staining with 5738-10-R3A-D5;
Figure 10C: staining with 5738-13 -R4A-D 11;
Figure 10D: staining with 5738-13-R2A-H4.
EXAMPLES
The present invention is further illustrated by the following examples.
Example 1 ¨ DPP4 is expressed on the surface of senescent cells
Materials and Methods
Expression of DPP 4 (rhuMAB 41) antibody and antigen
The full-length of the antigen was synthesized and subcloned into an
expression vector.
The insert was confirmed by Sanger sequencing. After the vector was verified
by
sequencing, the vector was expressed in CHO-S cell line with chemically
defined culture
media. After 9 days cultivation, the protein was purified by Nickel columns,
ultrafiltration
and then subjected to 0.2-micron sterile filtration to get the bulk of high
purity.
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The heavy chain and light chain of the rhuMAB 41 antibody (Creative Biolabs)
were
synthesized and subcloned into Creative Biolabs property expression vector,
respectively.
The insert was confirmed by Sanger sequencing. After the vectors were verified
by
sequencing, they were expressed in HEK293 cell line with chemically defined
culture
media. After 9 days of cultivation, the protein was purified by Protein A
affinity
chromatography, ultrafiltration and then subjected to 0.2-micron sterile
filtration to get
the bulk of high purity.
Expression of DPP4 (rhuMAB 41) scFv
The scFv consists of variable regions of heavy and light chains that are
joined together
.. by a flexible peptide linker. In the scFv, the order of the domains can be
either VH-linker-
VL or VL-linker-VH. The affinity of the two construction types to the target
might be
different. Hence, the two construction types can lead to secretory expression
in different
level.
The scFv(s) were expressed and tested by flow cytometry to evaluate the
binding affinity
to target cells. 5 x 105 WI-38 cells were co-cultured with rhuMAB 41 antibody
(humanized antibody), VL-Linker-VH antibody and VH-Linker-VL antibody
(1 [tg/tube), respectively, and then analyzed by using PE-anti-human IgG Fc as
secondary
antibody.
The results indicated that VH-linker-VL antibody has higher affinity for
target cell WI-
38 and was chosen for CAR development.
CAR-T cell preparation and construction
Primary human T cells were used for CAR-T generation. Human primary T cells
were
isolated from PBMCs of healthy donors by magnetic beads and stimulated in
growth
medium supplemented with IL-2. Activated T cells were then transduced with
lentivirus
expressing customized chimeric antigen receptor. After CAR-T cell expansion,
CAR-
transduction efficiency was examined by FACS and qPCR.
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The full-length of chimeric antigen receptor was synthesized and subcloned
into
lentivirus vector. The insert was confirmed by Sanger sequencing. The
structure of CAR
vector is schematically illustrated in Figure 1.
Lentiviral vectors, which were derived from immunodeficiency viruses, were
used for
their relatively high efficiency for T cell transduction and their ability of
infection of the
non-proliferating cells. A second generation of the packaging system was
utilized to
generate transduction-ready pseudoviral particles in HEK293T cells. The titer
of the
lentivirus particle was determined by qPCR and cell-based titration assay. The
results of
lentivirus titration showed that prepared virus stock was at a high titer of
3.27 x 108 TU/mL.
Primary human T lymphocytes preparation and CAR-T preparation
PBMC from a healthy human donor was stimulated with anti-CD3/CD28 magnetic
beads
and the T cells were isolated using magnetic cell separation system. To
generate CAR-T
cells, the lentivirus particles with DPP4-CAR coding gene were incubated with
the T cells
in the presence of polybrene. After T cell expansion, the CAR-T cells were
used for in
vitro cytotoxicity assays.
Target cell preparation
WI-38 target cells were obtained at population doubling 19 (PD19) and passaged
until
they stopped proliferating. They were further analyzed by FACS for the
detection of their
surface antigen DPP4 and by qRT-PCT for the detection of their relative DPP4
mRNA
expression level.
Cellular senescence induced by DNA damage was also induced in GM21808
fibroblasts
by adding etoposide, as previously described (Robles et al., 1999. Biochem
Pharmacol.
58(4):675-685; Gey & Seeger, 2013. Mech Ageing Dev. 134(3-4):130-138).
Untreated
(control) and treated cells were further analyzed by qRT-PCT for the detection
of their
relative DPP4 mRNA expression level.
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Natural killer (NK) cell preparation
Effector cells (NK cells) were freshly prepared before the ADCC assay. By
using
EasySepTM Human NK Cell Isolation Kit (STEMCELL, Catalog:17955), NK cells were
isolated from a healthy human donor and resuspended in RPMI 1640 medium at
.. 5 x 106/mL.
ADCC assay
The target cells (WI-38), i.e., the senescent cells and proliferating cells,
were plated into
a 24-well plate at 1 x 105 cell/well in 100 [tL RPMI-1640 supplied with 5% FBS
24 hour
and cultured overnight. On the day of the assay, anti-DPP4 antibody (rhuMAB
41) was
.. added into each well at a final concentration of 0 [tg/mL, 0.05 [tg/mL, and
0.5 [tg/mL.
After 30 minutes of incubation at 37 C with 5% CO2, 100 [tL NK cells (5 x 105
cell/well)
were added to each well at E/T=5:1. After incubation for 6 hours at 37 C with
5% CO2,
the cells were stained with eBioscienceTM Annexin V Apoptosis Detection Kit PE
(Invitrogen, Catalog: 88-8102-74) with 7-AAD and analyzed by flow cytometry.
In vitro CAR-T activity assay by FACS
In vitro analysis was used for evaluation of the targeting effect of DPP4-CAR-
T. The
target cells (WI-38 or GM21808), i.e., the senescent cells and proliferating
cells, were
plated into a 24-well plate at 1 x 105 cell/well in 100 [tL RPMI-1640 supplied
with 5%
FBS and cultured overnight. On the day of the assay, effector cells (5 x 105
cell/well)
.. were added to each well at E/T=5:1. After incubation for 6 hours at 37 C
with 5% CO2,
the cells were stained with eBioscienceTM Annexin V Apoptosis Detection Kit PE
(Invitrogen, Catalog: 88-8102-74) with 7-AAD and analyzed by flow cytometry.
The
relative percentage was quantified by normalizing the results to control CAR-
T.
Results
Assessment of senescent state and DPP4 levels in target cells
The senescent state of the cells was confirmed by senescence associated fl-
galactosidase
activity. DPP4 levels were monitored by quantitative RT-PCR. Figure 2 shows
that DPP4
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is expressed selectively in senescent cells, but not in proliferating human
diploid
fibroblasts or healthy foreskin fibroblasts.
ADCC Assay
In vitro Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) assays are
common
tools for immunotherapeutic drug discovery and biosimilar development. For
this
experiment, cytotoxicity is defined as apoptosis monitored by annexin-V
positive and 7-
ADD negative cells counted.
Results of a standard ADCC assay are shown in Figure 3. This experiment
depicts a
difference between young and old fibroblast cells at indicated antibody
concentration as
listed 1.5 and 1.65-fold, respectively.
Our results show that high abundance of DPP4 on the surface of senescent cells
lead to a
1.5-fold selective elimination of senescent cells using anti-DPP4 antibody
under optimal
conditions.
DPP4- CAR-T Assay
When we used the same cells with control and DPP4-CAR-T cells, we observe a 3-
fold
selective elimination of old senescent cells with respect to the young ones.
CAR-T cells
engineered against DPP4 show high specificity against DPP4-bearing senescent
cells
(Figure 4). The quantification of experimental results also shows that
increasing CAR-T
levels are only cytotoxic to senescent cells, but not to control cells (Figure
5). This
indicates that the CAR-T assay selectively eliminates target cells, i.e.,
senescent cells.
Conclusion
DPP4 is a novel senescent surface marker identified in human fibroblasts both
in
replicative and oncogene-induced (RAS) senescence. DPP4 is robustly
upregulated on
the plasma membrane of senescent cells.
DPP4 is expressed selectively in senescent cells, but not proliferating human
diploid
fibroblasts, making it a suitable target for senolytic therapy (Figure 2). We
showed that,
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upon two different senescent-inducing stimuli in two different types of
fibroblasts, CAR-
T cell therapy against DPP4 selectively eliminates DPP4-positive senescent
cells
(Figure 5).
Example 2 ¨ Development of scFv against human DEP1
Materials
Rat immunization and splenectomy
- "hDEP1 Peptide #1": amino acid residues 36-48 of SEQ ID NO: 1,
conjugated to
BSA
- "hDEP1 Peptide #2": amino acid residues 436-452 of SEQ ID NO: 1,
conjugated
to BSA
- "hDEP1 Peptide #3": amino acid residues 728-741 of SEQ ID NO: 1,
conjugated
to BSA
- "hDEP1 Peptide #4": amino acid residues 864-881 of SEQ ID NO: 1,
conjugated
to BSA
- "hDEP1-ECD-Fc": amino acid residues 621-969 of SEQ ID NO: 1 conjugated to
an IgG1 Fc domain (SEQ ID NO: 2), produced recombinantly in CHO cells and
purified on protein A resin
- "hDEP1-ECD-His": amino acid residues 621-969 of SEQ ID NO: 1
conjugated to
a 6xHis tag through a linker (SEQ ID NO: 3), produced recombinantly in CHO
cells and purified on Ni-NTA resin
- B SA (NEB)
- Freund's complete and incomplete adjuvants (Sigma, F5881 and F5506)
- Standard ELISA material
- Fc-specific polyclonal anti-rat immunoglobulin G (IgG) (Jackson 112-
036-071;
1/5000)
RNA extraction
- RNAse AWAY (Molecular Bio-products, Cat. n 7002)
- Tri reagent (Molecular research center Inc, Cat. n TR118)
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- BCP (Molecular research center Inc, Cat. n 13P-ISI)
- Isopropanol, ethanol 75% (molecular grade)
RT-PCR, library pre-cloning
- SuperScript Reverse Transcriptase (Invitrogen, Cat. n 18064-014)
- Murine Primers set
- Taq DNA polymerase, recombinant (Invitrogen, Cat. 10342-020)
- pGemT Vector System (Promega, Cat. A3600)
- Electroporation device and material
- Electrocompetent Sure bacteria (E. coli) (Stratagene, Cat. 200227)
- Nucleobond AX (Macherey-Nagel, Cat. 740 573-100)
Ampli6;ingVH and VL
- Red Taq (Sigma, Hamburg) + 10x buffer
- dNTP mix (10 mM each)
- Oligonucleotide primer
- Agarose (Serva, Heidelberg)
- TAE-buffer 50x (2 M TrisHC1, 1 M acetic acid, 0.05 M EDTA pH 8)
- Nucleospin Extract 2 Kit (Macherey-Nagel, DUren)
Cloning VH and VL
- NotI, MluI, NcoI, HindIII (NEB)
- Buffer 2, Buffer 3 (NEB)
- BSA (NEB)
- Calf intestine phosphatase (CIP) (MBI Fermentas, St. Leon-Rot)
- T4 ligase (Promega, Mannheim)
- 3 M sodium acetate pH 5.2
- E. coli XL1-Blue MRF (Stratagene, Amsterdam), genotype: A(mcrA)183
A(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-1 recAl gyrA96 relAl lac [F'
proAB lacIqZAM15 Tn10 (Tetr)]
- Electroporator MicroPulser (BIO-RAD, Munchen)
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- 2 M glucose (sterile filtered)
- 2 M magnesium solution (1 M MgCl, 1 M MgSO4)
- SOC medium pH 7.0 (2 w/v tryptone, 0.5 % w/v yeast extract, 0.05 %
w/v NaCl,
20 mM Mg solution, 20 mM glucose)
- 2xYT-medium pH 7.0 (1.6 % w/v tryptone, 1 % w/v yeast extract, 0.5 % w/v
NaCl)
- 2xYT-GAT (2xYT + 100 mM glucose + 100 p.g/mL ampiciline + 20 p.g/mL
tetracycline)
- Ampiciline (100 mg/mL stock)
- Tetracycline (10 mg/mL stock)
- 9 cm-Petrie dishes
- 25 cm-square Petri dishes ("pizza plates")
- 2xYT-GAT agar plates (2xYT-GAT, 1.5 % w/v agar-agar)
- Nucleobond Plasmid Midi Kit (Macherey-Nagel)
- Glycerol 99.5 % (Roth, Karlsruhe)
Library packaging
- 2xYT-medium pH 7.0 (1.6 % w/v tryptone, 1 % w/v yeast extract, 0.5 %
w/v NaCl)
- 2xTY-GA (2xTY, 100 mM glucose, 100 p.g/mL ampicillin)
- M13K07 Helperphage for monovalent display (Stratagene)
- 2x TY-AK (2x TY + 100 p.g/mL ampicillin + 50 p.g/mL kanamycin)
- Sorval Centrifuge RC5B Plus, rotor G53 and SS34 (Thermo Scientific,
Waltham)
- Polyethylenglycol (PEG) solution (20 % w/v PEG 6000, 2.5 M NaCl)
- Phage dilution buffer (10 mM Tris-HC1 pH 7.5, 20 mM NaCl, 2 mM EDTA)
Library screening
- Microtiter plate (Nunc Maxisorp)
- 3 % BSA in TBS (50 mM Tris; 137 mM NaCl; 2.7 mM KCl; pH adjusted to 8.0)
- TBS supplemented with 0.1 % Tween20
- E. coli (SURE strain, Stratagene)
- SB (super broth) supplemented with tetracycline (10 p.g/mL) and
carbenicillin
(50 lig/mL).
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SB supplemented with tetracycline (10 ug/mL), carbenicillin (50 ug/mL) and
kanamycin (70 ug/mL)
Polyethylenglycol (PEG) solution (20 % w/v PEG 6000, 2.5 M NaCl)
Sorval Centrifuge RC5B Plus, rotor G53 and SS34 (Thermo Scientific, Waltham)
scFv production and purification
E. coli strain HB2151
SB (super broth) supplemented with carbenicillin (50 ug/mL) and 1 % glucose
1 mM IPTG
Polymyxin B sulfate
- His-Trap column (GE Healthcare, Buckinghamshire, UK)
Methods
Rat immunization
12 rats were immunized with i.p. injections of 50 ug of hDEP1 Peptide #1,
Peptide #2,
Peptide #3 or Peptide #4 (3 rats/peptide). In parallel, 5 rats were immunized
with i.p.
injections of 50 ug of hDEP1-ECD-Fc.
The three first injections were administered at 14 days intervals (DO, D14 and
D28). A
fourth injection was administered after animal selection according to the
titer reached.
The first injection at DO was mixed with complete Freund's adjuvant. The
remaining
injections were carried out with incomplete Freund's adjuvant.
The rat immune response was evaluated by ELISA performed in 96-well microtiter
plates
using hDEP1 Peptide #1, Peptide #2, Peptide #3, Peptide #4, hDEP1-ECD-His, or
BSA.
Pre-immune serum was used as a negative control. For detection, Fc-specific
polyclonal
anti-rat IgG was used and the titer was measured as the reciprocal of the
highest dilution
of immune serum giving a signal at 50 % of the highest OD.
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RNA extraction, RT-PCR, library pre-cloning
After the fourth injection, RNA was isolated with Tri Reagent/BCP method from
the
spleen of the immunized rats and used for reverse transcription.
Oligonucleotides sets
were used for amplification of the DNA encoding the lc and k light chain and
to amplify
the DNA encoding Fd fragments of the heavy y chain. To obtain two sub-
libraries
encoding the Fd fragment and light chain, the corresponding PCR products were
pooled
and sub-cloned into the pGEMT vector.
Library construction
The final library was constructed starting from these two sub-libraries. The
DNA
encoding the Fd fragment and the light chain was re-amplified with two
individual
oligonucleotide sets introducing the required restrictions sites for library-
cloning using
the pTH1 vector. First the pTH1 vector and the VL-amplified repertoire were
digested
with MluI and NotI. Afterwards, the enzyme reaction was terminated and the
vector was
dephosphorylated with calf intestinal phosphatase. The vector and the VL-
repertoire were
purified using NucleoSpinII Gel and PCR Clean-up Kit (Macherey-Nagel) and 270
ng of
the VL-repertoire was cloned into 1 lig of dephosphorylated vector using T4
DNA-ligase.
The pellet was washed twice with 70 % ethanol and resuspended in 30 tL H20
before it
was used for electroporation (1.7 kV) with 25 tL of XL1-BlueMRF'. The
transformed
bacteria were cultured on 2x YT agar plates supplemented with 100 [tg/mL
ampicillin,
20 [tg/mL tetracycline and 100 mM glucose. The colonies were harvested by
resuspension in 40 mL of 2x YT medium with a Drigalsky spatula and plasmids
were
isolated with the Nucleobond Plasmid Midi Kit. Following this, the VL-library
and the
VH-repertoire were digested with SfiI and HindIII, ligated and electroporated
as
described for VL, but this time 250 ng of the digested and purified VH-
repertoire was
inserted into 1 lig of the VL-library. The harvested bacteria of the final
scFv antibody
gene library were pooled, aliquoted and stored at -80 C. The library was
packaged with
M13K07.
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Library packaging
To package the library, inoculate 400 mL 2 x TY-GA in a 1 L Erlenmeyer flask
with 1 mL
antibody gene library stock and then infect 25 mL bacteria culture (¨ 1.25 x
1010 cells)
with 2.5 x 1011 colony forming units (cfu) of the helper phage M13K07.
Produce scFv-phage overnight at 250 rpm and 30 C. Pellet the bacteria by
centrifugation
for 10 minutes at 10000 g. Precipitate the phage from the supernatant by
adding 1/5
volume PEG solution. Incubate for 1 hour at 4 C with gentle shaking, followed
by
centrifugation for 1 hour at 10000g. Discard the supernatant, resolve each
pellet in 10 mL
phage dilution buffer and add 1/5 volume PEG solution. Incubate on ice for 20
minutes
and pellet the phage by centrifugation for 30 minutes at 10000 g. Discard the
supernatant
and put the open tubes upside down on tissue paper. Let the viscous PEG
solution move
out completely. Resuspend the phage pellet in 1 mL phage dilution buffer.
Library screening
For isolation of anti-DEP I specific scFv, microtiter plates were coated
overnight with
hDEP1-ECD-His or Peptide #4 at 10 1.tg/mL in PBS at 4 C. The plates were then
blocked
with 3% BSA in TBS for 2 hours at 37 C. After a washing step, the antibody
phage
display library was added and incubated for an additional 2 hours at 37 C.
During the first round of panning, the plates were washed 5 times with TBS
supplemented
with 0.1 % Tween20. The plates were finally rinsed with sterile PBS and phage
were
eluted with trypsin (1 mg/mL in TBS) for 30 minutes at 37 C. The eluted phages
were
used to infect E. coli cultured in SB supplemented with tetracycline and
carbenicillin.
For the production of new phage particles, infected E. coli were co-infected
with M13K07
and cultured overnight at 37 C in SB supplemented with tetracycline,
carbenicillin and
kanamycin. Phage particles were precipitated in PEG/NaCl (4 % w/v PEG8000, 3 %
w/v
NaCl) and used for the next round of panning. This was performed as described
above,
with the exception that the plates were washed with increasing stringency.
The infected E. coli of the last round of panning were grown on SB media in
petri dishes
and used for screening by ELISA using hDEP1-ECD-His or Peptide #4.
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scFv production and purification
For expression of soluble scFv, the DNA encoding the selected scFv was used to
transform the non-suppressor E. coil strain HB2151. Transformed E. coil were
then used
to inoculate 500 mL of SB medium and cultivated at 30 C until OD600 1.5. Then,
1 mM
IPTG was added to induce the expression of scFv and the culture was incubated
overnight
at 22 C. After harvesting by centrifugation at 2500 g for 15 minutes at 4 C,
scFv were
extracted with polymyxin B sulfate and purified using a His-Trap column
according to
the manufacturer's instructions. Purified scFv were quantified by Dm and
controlled
by SDS-PAGE analysis.
Confirmation of selected scFv by ELISA against screening peptide
The soluble anti-DEP1 scFv were tested in indirect ELISA for reactivities
against the 4
peptides (Peptide #1, Peptide #2, Peptide #3, and Peptide #4), against hDEP1-
ECD-His
and its murine counterpart with SEQ ID NO: 4; and against BSA as negative
control.
Reactivity was assessed by OD450, in serial dilutions.
Results
Serum of the 17 rats was collected on D38 (after the third immunization, on
D28) and Fc-
specific polyclonal anti-rat IgG was used to measure the titer at 50 % of
maximum signal.
Results are given in Table 5.
Table 5
hDEP1 hDEP1 hDEP1 hDEP1
hDEP1-
Peptide Peptide Peptide Peptide BSA
ECD-His
#1 #2 #3 #4
hDEP1 Rat 1 380000 ND ND ND ND ND
Peptide Rat 2 55000 ND ND ND ND ND
#1 Rat 3 11000 ND ND ND ND ND
hDEP1 Rat 4 ND 27000 ND ND ND
ND
Peptide Rat 5 ND 23000 ND ND ND ND
#2 Rat 6 A7) 1'0000 Ni) :\7) NI)
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(demi)
hDEP1 Rat 7 ND ND 12000 ND 500 ND
Peptide Rat 8 ND ND 18000 ND 500 ND
#3 Rat 9 ND ND 22000 ND 500
ND
hDEP1 Rat 10 ND ND ND 113000 6000 ND
Peptide Rat 11 ND ND ND 110000 4500 ND
#4 Rat 12 ND ND ND
113000 5000 500
Rat 13 500 500 500 ND 321000 ND
Rat 14 ND ND 500 ND 64000 500
hDEP1-
Rat 15 ND ND 500 ND 180000 500
ECD-Fc
Rat 16 500 500 500 500 82000 ND
Rat 17 ND ND 500 ND 40000 ND
High titers were harvested against each immunogenic peptide.
Peptides #1 and #4 appeared to be more immunogenic than Peptides #2 and #3,
allowing
better immune responses. Rats immunized with Peptide #4 also presented a
positive
immune response for the recombinant hDEP1-ECD-His protein, while rats
immunized
with Peptides #1 and #2 did not present such positive immune response.
Overall, rat 10
seemed to be the best candidate among rats 1 to 12.
High titers were also harvested against the recombinant protein hDEP1-ECD-His,
ranging
from 1/40000 to 1/321000. Overall, rat 13 seemed to be the best candidate
among rats 13
to 17.
A splenectomy was carried out on rats 10 and 13. RNA were extracted from the
spleens,
and total RNA was quantified (Table 6) and controlled on agarose gel (not
shown).
Table 6
Sample Concentration (pg/mL) A260nm A280nm A260nmi A280nm A260nmi A230nm
Rat 10 2108.9 52.7 29.5 1.78 1.55
Rat 13 2370.77 59.3 33.4 1.77 1.42
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The first step of the library construction consisted of the VL fragments
cloning in a
phagemid vector, and then the VH fragments were inserted into the vector
containing the
VL repertoire. The vector format VH/VL-6xHis-Flag was selected for
constructions.
Rat 10
The final scFv library consisted of 1 x 107 independent clones with a full-
size insert rate
of 92 % (by colony-PCR) and was finally packaged in M13K07 phage.
The panning was performed using hDEP1 Peptide #4/BSA as target and standard
elution
strategy. Four rounds were carried out in order to isolate specific binders.
After each
round, only the phages having interacted with hDEP1 Peptide #4/BSA were
eluted.
Between the first and the last round of panning, the number of eluted phages
increased,
indicating that amplification/selection of reactive phages was expected from
this panning.
The reactivity of the phages eluted after each round against the targeted
peptide was
assessed in phage-ELISA and confirmed that potential binders were eluted in
rounds 3
and 4 (Figure 6).
96 isolated clones from the second, third and fourth rounds of panning were
hand-picked
and used to produce the respective soluble scFv in microtiter plate. Each
clone was then
tested in ELISA against hDEP1 Peptide #4/BSA.
48 isolated clones showing a specific signal against hDEP1 Peptide #4/BSA
compared to
negative control (BSA) were selected. Their DNA was extracted and sequenced.
Sequences with an early stop codon or with an incorrect size were considered
as
recombined. The non-recombined sequences were aligned for redundancy analysis
in
order to identify the identical sequences (referred to as "redundant"
sequences).
Twelve sequences corresponding to non-recombined and non-redundant scFv
sequences
were identified, grouped into two clusters (Table 7).
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Table 7. HCVR and LCVR column indicate the amino acid sequence of the heavy
chain
and light chain variable regions, respectively.
Cluster Clone's name Redundancy HCVR LC VR
5738-10-R3A-C6 2 56 76
Cluster 1
5738-10-R3A-D8 1 59 79
5738-10-R3A-B2 1 55 75
5738-10-R3A-D1 1 57 77
5738-10-R3A-D5 1 58 78
5738-10-R3A-D11 3 60 80
5738-10-R4A-E7 1 61 81
Cluster 2
5738-10-R4A-E9 1 60 82
5738-10-R4A-F12 5 62 83
5738-10-R4A-G4 2 63 84
5738-10-R4A-G11 7 64 85
5738-10-R4A-G12 18 65 86
According to sequence and redundancy analysis, the following clones were
selected:
- 5738-10-R3A-C6 (cluster 1)
- 5738-10-R3A-D5 (cluster 2)
- 5738-10-R4A-G12 (cluster 2)
After transformation of the 3 selected clones DNA in an E. coli strain
dedicated to
production, the clones were expressed as soluble scFv and purified using the
His tag on
an Ni-NTA column. These soluble anti-hDEP1 scFv were tested in indirect ELISA
for
reactivities against the 4 peptides and DEP1 proteins (human and murine) and
against the
negative control (BSA only).
All three scFv were reactive against hDEP1 Peptide #4, as expected, especially
the two
scFv of cluster 2. No reactivity could be observed against the 3 other
peptides. Only clone
5738-10-R4A-G12 showed a reactivity against hDEP1, with no response for the
mDEP1
or the negative control.
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Rat /3
The final scFy library consisted of 1 x 107 independent clones with a full-
size insert rate
of 88 % (by colony-PCR) and was finally packaged in M13K07 phage.
The panning was performed using hDEP1-ECD-His as target and standard elution
strategy. Four rounds were carried out in order to isolate specific binders.
After each
round, only the phages having interacted with hDEP1-ECD-His were eluted.
Between the
first and the last round of panning, the number of eluted phages increased,
indicating that
amplification/selection of reactive phages was expected from this panning. The
reactivity
of the phages eluted after each round against the targeted peptide was
assessed in phage-
ELISA and confirmed that potential binders were eluted in rounds 2, 3 and 4
(Figure 7).
96 isolated clones from the second, third and fourth rounds of panning were
hand-picked
and used to produce the respective soluble scFy in microtiter plate. Each
clone was then
tested in ELISA against hDEP1-ECD-His.
48 isolated clones showing a specific signal against hDEP1-ECD-His compared to
negative control (BSA) were selected. Their DNA was extracted and sequenced.
Sequences with an early stop codon or with an incorrect size were considered
as
recombined. The non-recombined sequences were aligned for redundancy analysis
in
order to identify the identical sequences (referred to as "redundant"
sequences).
Nine sequences corresponding to non-recombined and non-redundant scFy
sequences
were identified, grouped into two clusters (Table 8).
Table 8. HCVR and LCVR column indicate the amino acid sequence of the heavy
chain
and light chain variable regions, respectively.
Cluster Clone's name Redundancy HCVR LCVR
5738-13-R2A-C1 1 66 87
Cluster 1
5738-13-R4A-D11 1 68 89
5738-13 -R2A-D3 1 67 88
Cluster 2 573 8-13 -R3A-F5 1 69 90
5738-13 -R4A-F11 1 70 91
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5738-13 -R2A-H3 3 71 92
5738-13 -R2A-H4 21 72 93
5738-13 -R4A-H9 2 73 94
5738-13 -R4A-H11 15 74 94
According to sequence and redundancy analysis, the following clones were
selected:
- 5738-13-R4A-D11 (cluster 1)
- 5738-13-R2A-H4 (cluster 2)
After transformation of the 2 selected clones DNA in an E. coli strain
dedicated to
production, the clones were expressed as soluble scFv and purified using the
His tag on
an Ni-NTA column. These soluble anti-hDEP1 scFv were tested in indirect ELISA
for
reactivities against the 4 peptides and DEP1 proteins (human and murine) and
against the
negative control (BSA only).
The two scFv were reactive against hDEP1, as expected. No reactivity could be
observed
against any of the 4 peptides, the mDEP1 or the negative control.
Example 3 ¨ Development of scFv against human DPP4
Materials
Rat immunization and splenectomy
- "hDPP4 Peptide P2": amino acid residues 170-191 of SEQ ID NO: 101,
conjugated to BSA
- "hDPP4 Peptide P3": amino acid residues 235-254 of SEQ ID NO: 101,
conjugated to BSA
- "hDPP4 Peptide P5": amino acid residues 492-517 of SEQ ID NO: 101,
conjugated to BSA
- "hDPP4 Peptide P6": amino acid residues 533-551 of SEQ ID NO: 101,
conjugated to BSA
- "hDPP4-ECD-His": SEQ ID NO: 102
- "mDPP4": murine DDP with SEQ ID NO: 103
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- BSA (NEB)
- Freund's complete and incomplete adjuvants (Sigma, F5881 and F5506)
- Standard ELISA material
- Fc-specific polyclonal anti-rat immunoglobulin G (IgG) (Jackson 112-
036-071;
1/5000)
RNA extraction
- RNAse AWAY (Molecular Bio-products, Cat. n 7002)
- Tri reagent (Molecular research center Inc, Cat. n TR118)
- BCP (Molecular research center Inc, Cat. n'BP-ISI)
- Isopropanol, ethanol 75% (molecular grade)
RT-PCR, library pre-cloning
- SuperScript Reverse Transcriptase (Invitrogen, Cat. n 18064-014)
- Murine Primers set
- Tag DNA polymerase, recombinant (Invitrogen, Cat. 10342-020)
- pGemT Vector System (Promega, Cat. A3600)
- Electroporation device and material
- Electrocompetent Sure bacteria (E. coli) (Stratagene, Cat. 200227)
- Nucleobond AX (Macherey-Nagel, Cat. 740 573-100)
Amplifying VH and VL
- Red Tag (Sigma, Hamburg) + 10x buffer
- dNTP mix (10 mM each)
- Oligonucleotide primer
- Agarose (Serva, Heidelberg)
- TAE-buffer 50x (2 M TrisHC1, 1 M acetic acid, 0.05 M EDTA pH 8)
- Nucleospin Extract 2 Kit (Macherey-Nagel, Diiren)
CloningVH and VL
- NotI, MluI, NcoI, HindIII (NEB)
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- Buffer 2, Buffer 3 (NEB)
- BSA (NEB)
- Calf intestine phosphatase (CIP) (MBI Fermentas, St. Leon-Rot)
- T4 ligase (Promega, Mannheim)
- 3 M sodium acetate pH 5.2
- E. coli XL1-Blue MRF (Stratagene, Amsterdam), genotype: A(mcrA)183
A(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-1 recAl gyrA96 relAl lac [F'
proAB lacIqZAM15 Tn10 (Tetr)]
- Electroporator MicroPulser (BIO-RAD, Munchen)
- 2 M glucose (sterile filtered)
- 2 M magnesium solution (1 M MgCl, 1 M MgSO4)
- SOC medium pH 7.0 (2 w/v tryptone, 0.5 % w/v yeast extract, 0.05 %
w/v NaCl,
mM Mg solution, 20 mM glucose)
- 2 xYT-medium pH 7.0 (1.6 % w/v tryptone, 1 % w/v yeast extract, 0.5 %
w/v NaCl)
15 - 2xYT-GAT (2xYT + 100 mM glucose + 100 ug/mL ampiciline + 20 ug/mL
tetracycline)
- Ampiciline (100 mg/mL stock)
- Tetracycline (10 mg/mL stock)
- 9 cm-Petrie dishes
20 - 25 cm-square Petri dishes ("pizza plates")
- 2 xYT-GAT agar plates (2 xYT-GAT, 1.5 % w/v agar-agar)
- Nucleobond Plasmid Midi Kit (Macherey-Nagel)
- Glycerol 99.5 % (Roth, Karlsruhe)
Library packaging
- 2 xYT-medium pH 7.0 (1.6 % w/v tryptone, 1 % w/v yeast extract, 0.5 % w/v
NaCl)
- 2xTY-GA (2xTY, 100 mM glucose, 100 ug/mL ampicillin)
- M13K07 Helperphage for monovalent display (Stratagene)
- 2x TY-AK (2x TY + 100 ug/mL ampicillin + 50 ug/mL kanamycin)
- Sorval Centrifuge RC5B Plus, rotor G53 and SS34 (Thermo Scientific,
Waltham)
- Polyethylenglycol (PEG) solution (20 % w/v PEG 6000, 2.5 M NaCl)
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- Phage dilution buffer (10 mM Tris-HC1 pH 7.5, 20 mM NaCl, 2 mM EDTA)
Library screening
- Microtiter plate (Nunc Maxisorp)
- 3 % BSA in TBS (50 mM Tris; 137 mM NaCl; 2.7 mM KC1; pH adjusted to
8.0)
- TBS supplemented with 0.1 % Tween20
- E. coli (SURE strain, Stratagene)
- SB (super broth) supplemented with tetracycline (10 p.g/mL) and
carbenicillin
(50 lig/mL).
- SB supplemented with tetracycline (10 pg/mL), carbenicillin (50
p.g/mL) and
kanamycin (70 lig/mL)
- Polyethylenglycol (PEG) solution (20 % w/v PEG 6000, 2.5 M NaCl)
- Sorval Centrifuge RC5B Plus, rotor G53 and SS34 (Thermo Scientific,
Waltham)
scFv production and purification
- E. coli strain HB2151
- SB (super broth) supplemented with carbenicillin (50 lig/mL) and 1 %
glucose
- 1 mM IPTG
- Polymyxin B sulfate
- His-Trap column (GE Healthcare, Buckinghamshire, UK)
Methods
Rat immunization
12 rats were immunized with i.p. injections of 50 lig of hDDP4 Peptide P1,
Peptide P3,
Peptide P5 or Peptide P6 (3 rats/peptide). In parallel, 5 rats were immunized
with i.p.
injections of 50 lig of hDDP4-ECD-His.
The three first injections were administered at 14 days intervals (DO, D14 and
D28). A
fourth injection was administered after animal selection according to the
titer reached.
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The first injection at DO was mixed with complete Freund' s adjuvant. The
remaining
injections were carried out with incomplete Freund's adjuvant.
The rat immune response was evaluated by ELISA performed in 96-well microtiter
plates
using hDDP4 Peptide P1, Peptide P3, Peptide P5, Peptide P6, hDPP4-ECD-His,
mDPP4,
or B SA.
Pre-immune serum was used as a negative control. For detection, Fc-specific
polyclonal
anti-rat IgG was used and the titer was measured as the reciprocal of the
highest dilution
of immune serum giving a signal at 50 % of the highest OD.
RNA extraction, RT-PCR, library pre-cloning
After the fourth injection, RNA was isolated with Tri Reagent/BCP method from
the
spleen of the immunized rats and used for reverse transcription.
Oligonucleotides sets
were used for amplification of the DNA encoding the lc and k light chain and
to amplify
the DNA encoding Fd fragments of the heavy y chain. To obtain two sub-
libraries
encoding the Fd fragment and light chain, the corresponding PCR products were
pooled
and sub-cloned into the pGEMT vector.
Library construction
The final library was constructed starting from these two sub-libraries. The
DNA
encoding the Fd fragment and the light chain was re-amplified with two
individual
oligonucleotide sets introducing the required restrictions sites for library-
cloning using
the pTH1 vector. First the pTH1 vector and the VL-amplified repertoire were
digested
with MluI and NotI. Afterwards, the enzyme reaction was terminated and the
vector was
dephosphorylated with calf intestinal phosphatase. The vector and the VL-
repertoire were
purified using NucleoSpinII Gel and PCR Clean-up Kit (Macherey-Nagel) and 270
ng of
the VL-repertoire was cloned into 1 lig of dephosphorylated vector using T4
DNA-ligase.
The pellet was washed twice with 70 % ethanol and resuspended in 30 tL H20
before it
was used for electroporation (1.7 kV) with 25 tL of XL1-BlueMRF'. The
transformed
bacteria were cultured on 2x YT agar plates supplemented with 10011g/mL
ampicillin,
20 1.tg/mL tetracycline and 100 mM glucose. The colonies were harvested by
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