U.S. patent application number 16/392794 was filed with the patent office on 2019-08-15 for methods for selecting binders by phage display and masked selection.
The applicant listed for this patent is CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (ONRS), INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), INSTITUT JEAN PAOLI & IRENE CALMETTES, UNIVERSITE D'AIX MARSEILLE. Invention is credited to Daniel BATY, Patrick CHAMES.
Application Number | 20190249170 16/392794 |
Document ID | / |
Family ID | 48483098 |
Filed Date | 2019-08-15 |
United States Patent
Application |
20190249170 |
Kind Code |
A1 |
BATY; Daniel ; et
al. |
August 15, 2019 |
METHODS FOR SELECTING BINDERS BY PHAGE DISPLAY AND MASKED
SELECTION
Abstract
The present invention relates to methods for selecting binders
by phage display and masked selection. More particularly, the
present invention relates to a method for selecting a plurality of
binders specific for at least one relevant target comprising
screening a phage binder library of binders against the relevant
target in the presence of a plurality of binders obtained from a
library of binders directed against at least one irrelevant target
and positively selecting the binders that are specific for the at
least one relevant target.
Inventors: |
BATY; Daniel; (Marseille,
FR) ; CHAMES; Patrick; (Marseille, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
UNIVERSITE D'AIX MARSEILLE
INSTITUT JEAN PAOLI & IRENE CALMETTES
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (ONRS) |
Paris
Marseille Cedex 07
Paris
Paris |
|
FR
FR
FR
FR |
|
|
Family ID: |
48483098 |
Appl. No.: |
16/392794 |
Filed: |
April 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15427126 |
Feb 8, 2017 |
10316315 |
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16392794 |
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14403228 |
Nov 24, 2014 |
9605257 |
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PCT/EP2013/060786 |
May 24, 2013 |
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15427126 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1037 20130101;
C40B 30/04 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2012 |
EP |
12305585.7 |
Claims
1. (canceled)
2. A method for selecting a plurality of binders specific for a
cell surface protein comprising: i) providing a cell that was
genetically transformed so as to express at its surface the cell
surface protein; ii) building a binder library by using peripheral
blood mononuclear cells (PBMCs) obtained from at least one animal
immunized with the cell of step i); iii) producing a phage binder
library by infecting the binder library of step ii) with a helper
phage; iv) performing with the phage binder library of step ii) at
least one round of selection against the cell of step i) that was
not genetically transformed; v) producing soluble binders with
selected clones obtained at step iv). vi) performing with the phage
binder library of step iii) at least one round of selection against
the cell of step i) in the presence of an excess of binders
produced by step vi); and vii) cloning and recovering the binders
from the clones selected at step vi).
3. (canceled)
4. A method for selecting a plurality of binders with cancer
specificity comprising: i) building a binder library by using
peripheral blood mononuclear cells (PBMCs) obtained from at least
one animal immunized with a mixture of cancer biopsy lysates; ii)
producing a phage binder library by infecting the binder library of
step i) with a helper phage;. iii) performing with the phage binder
library of step ii) at least one round of selection against a
mixture of normal biopsy lysate; iv) producing soluble binders with
selected clones obtained at step iii); v) performing with the phage
binder library of step ii) at least one round of selection against
the mixture of cancer biopsy lysates in the presence of an excess
of binders produced by step iv); and vi) cloning and recovering the
binders from the clones selected at step v).
5. A method for selecting a plurality of binders with cancer
specificity comprising: i) building a binder library by using
peripheral blood mononuclear cells (PBMCs) obtained from at least
one animal immunized with a mixture of cancer cell lines.; ii)
producing a phage binder library by infecting the binder library of
step i) with a helper phage; iii) performing with the phage binder
library of step ii) at least one round of selection against a
normal cell line; iv) producing soluble binders with selected
clones obtained at step v) performing with the phage binder library
of step ii) at least one round of selection against the mixture of
cancer cell lines in the presence of an excess of binders produced
by step iv); and vi) cloning and recovering the binders from the
clones selected at step v).
6. The method of claim 2, further comprising sequencing the binders
from the clones selected at step vi).
7. The method of claim 4, further comprising sequencing the binders
from the clones selected at step vi).
8. The method of claim 5, further comprising sequencing the binders
from the clones selected at step vi).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for selecting
binders by phage display and masked selection.
BACKGROUND OF THE INVENTION
[0002] Hybridoma [1] and phage-display recombinant antibody systems
[2] are currently the predominant methods for isolating monoclonal
Abs. Display of recombinant antibodies (Ab) on the surface of
bacteriophage M13 has numerous advantages compared to conventional
hybridoma technology. When combined with the use of large
non-immune libraries, phage Ab selection represents a rich source
of binders that can be isolated in a fraction of the time needed
for hybridoma-based approaches. As an in vitro selection methods,
it permits the selection of binders against toxic or highly
conserved antigens, which is not easily performed using the
conventional hybridoma techniques and importantly, it can be used
to isolate fully human antibody fragments [3]. Consequently, phage
display rapidly became an established procedure for the isolation
of binders against a wide variety of antigens.
[0003] Phage display-based antibody isolation typically relies on
the use of recombinant proteins for several steps, including
immunizations (if needed), library enrichment by selection on
immobilized antigen, screening, and characterization of antibodies
specificity and affinity [4]. This procedure is efficient but
depends on the availability of purified recombinant proteins.
Unfortunately, some surface molecules, such as G-protein coupled
receptors, cannot be easily expressed and purified in a native
conformation. Some molecules with large extracellular domains may
adopt a specific conformation due to interaction with other cell
surface proteins, thereby forming complexes that are cumbersome to
produce by recombinant expression. Moreover, many standard
screening practices, such as the adsorption of recombinant proteins
on plastic, may significantly alter protein conformations [5]. For
these reasons, Abs selected on the basis of binding to a
recombinant protein may not bind the native conformation of this
protein. It is thus of high interest to develop procedures entirely
based on the use of intact cells expressing the receptor of choice.
However, in this case, an extra step is necessary to enrich for
phage-Abs binding to the receptor of interest rather than to other
cell surface proteins. Since selection steps are performed in
vitro, it is possible to influence the outcome of a selection by
perfonning some additional steps such as deletion steps (also named
negative selection) prior to positive selections to remove unwanted
specificities or cross-reactions [6], or competitive elution using
a ligand or an antibody to favor the selection of binders against a
precise epitope [7].
[0004] Along this line, it would be of very high interest to
establish a procedure able to reliably guide the selection toward
an unknown but relevant antigen within a complex mixture, such as a
tumor maker overexpressed at the surface of intact cells, or in a
cell lysate. Indeed, during the past two decades, there has been a
growing interest in approaches aiming at discovering new diagnosis
biomarkers and identifying new potential surface markers for
targeted therapy. Several studies have described the use of phage
display and libraries of recombinant antibodies for the isolation
of tumor specific binders [8-14], leading in some cases to the
identification of new tumor markers [15, 16]. Most of these
strategies are based on the use of depletion steps on normal
samples followed by a selection step on the tumor sample.
Unfortunately, this procedure often leads to inconsistent results
and its efficiency can be a limiting factor in complex situation
such as the selection of antibodies against unknown overexpressed
tumor antigens.
SUMMARY OF THE INVENTION
[0005] The present invention relates to methods for selecting
binders by phage display and masked selection.
DETAILED DESCRIPTION OF THE INVENTION
[0006] The inventors have designed a new selection method, named
masked selection that is relying on the blockade of unwanted
epitopes to favor the accessibility of relevant ones. They
demonstrate the efficiency of this method by selecting binders
against a specific portion of a fusion protein, by selecting
binders against a member of the seven transmembrane receptor family
and a tyrosine kinase receptor using intact transfected HEK cells,
or by selecting binders against unknown breast cancer markers not
expressed on normal samples, as shown by flow cytometry and
immunohistochemistry. The universality and efficiency of this
approach should ultimately lead to the rapid selection of specific
binders and the development of diagnostic and targeted therapies in
various settings.
[0007] The present invention thus relates to a method for selecting
a plurality of binders specific for at least one relevant target
comprising screening a phage binder library of binders against the
relevant target in presence of a plurality of binders obtained from
a library of binders directed against at least one irrelevant
target and positively selecting the binders that are specific for
the at least one relevant target.
[0008] As used herein, the term "binder" refers to any kind of
antibody fragments (scFv, Fab fragments), alternative scaffolds
(darpins, monobodies, affibodies, anticalins) or peptides.
[0009] In one embodiment, the binders are single domain antibodies
(sdAbs). The term "single domain antibody" (sdAb) or "VHH" refers
to the single heavy chain variable domain of antibodies of the type
that can be found in Camelid mammals which are naturally devoid of
light chains. Such VHH are also called "nanobody.RTM.". According
to the invention, sdAb can particularly be llama sdAb.
[0010] Typically, the libraries of binders (e.g. antibodies)
according to the invention are generated after immunization with
the relevant or irrelevant target(s) as described in the
EXAMPLE.
[0011] In one embodiment, the relevant target consists of a protein
of surface (e.g. a receptor protein) or a portion thereof (e.g.
ectodomain of the protein).
[0012] For example, the protein may be specific for an immune cell
regulatory molecule such as CD3, CD4, CD8, CD25, CD28, CD26,
CTLA-4, ICOS, or CD11a. Other suitable protein include but are not
limited to those associated with immune cells including T
cell-associated molecules, such as TCR/CD3 or CD2; NK
cell-associated targets such as NKG2D, Fc.gamma.RIIIa (CD16), CD38,
CD44, CD56, or CD69; granulocyte-associated targets such as
Fc.gamma.RI (CD64), FcaRI (CD89), and CR3 (CD11b/CD18);
monocyte/macrophage-associated targets (such as Fc.gamma.RI (CD64),
FcaRI (CD89), CD3 (CD11b/CD18), or mannose receptor; dendritic
cell-associated targets such as Fc.gamma.RI (CD64) or mannose
receptor; and erythrocyte-associated targets such as CRI
(CD35).
[0013] Alternatively, the protein of surface is a cancer antigen.
Known cancer antigens include, without limitation, c-erbB-2 (erbB-2
is also known as c-neu or HER-2), which is particularly associated
with breast, ovarian, and colon tumor cells, as well as
neuroblastoma, lung cancer, thyroid cancer, pancreatic cancer,
prostate cancer, renal cancer and cancers of the digestive tract.
Another class of cancer antigens is oncofetal proteins of
nonenzymatic function. These antigens are found in a variety of
neoplasms, and are often referred to as "tumor-associated
antigens." Carcinoembryonic antigen (CEA), and .alpha.-fetoprotein
(AFP) are two examples of such cancer antigens. AFP levels rise in
patients with hepatocellular carcinoma: 69% of patients with liver
cancer express high levels of AFP in their serum. CEA is a serum
glycoprotein of 200 kDa found in adenocarcinoma of colon, as well
as cancers of the lung and genitourinary tract. Yet another class
of cancer antigens is those antigens unique to a particular tumor,
referred to sometimes as "tumor specific antigens," such as heat
shock proteins (e.g., hsp70 or hsp90 proteins) from a particular
type of tumor. Other targets include the MICA/B ligands of NKG2D.
These molecules are expressed on many types of tumors, but not
normally on healthy cells. Additional specific examples of cancer
antigens include epithelial cell adhesion molecule
(Ep-CAM/TACSTD1), mesothelin, tumor-associated glycoprotein 72
(TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7,
cancer-associated viral vaccines (e.g., human papillomavirus
antigens), prostate specific antigen (PSA, PSMA), RAGE (renal
antigen), CAMEL (CTL-recognized antigen on melanoma), CT antigens
(such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1,
SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1,
mucin-CA125, etc.), cancer-associated ganglioside antigens,
tyrosinase, gp75, C-myc, Martl, MelanA, MUM-1, MUM-2, MUM-3,
HLA-B7, Ep-CAM, tumor-derived heat shock proteins, and the like
(see also, e.g., Acres et al., Curr Opin Mol Ther 2004 February,
6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999
Oct. 8; 1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003
July-August; 2(4 Suppl 1):S161-8; and Ohshima et al., Int J Cancer.
2001 Jul. 1; 93(1):91-6). Other exemplary cancer antigen targets
include CA 195 tumor-associated antigen-like antigen (see, e.g.,
U.S. Pat. No. 5,324,822) and female urine squamous cell
carcinoma-like antigens (see, e.g., U.S. Pat. No. 5,306,811), and
the breast cell cancer antigens described in U.S. Pat. No.
4,960,716.
[0014] The protein may also be a receptor protein such as receptors
associated with cancer progression (e.g., one of the HER1-HER4
receptors).
[0015] In one embodiment, the relevant target may be a carbohydrate
antigen present at the surface of a cell (e.g. a cancer cell). For
example the target may be selected from glycosylation groups of
antigens that are preferentially produced by transformed
(neoplastic or cancerous) cells, infected cells, and the like
(cells associated with other immune system-related disorders). In
one aspect, the antigen is a tumor-associated antigen. In an
exemplary aspect, the antigen is O-acetylated-GD2 or glypican-3. In
another particular aspect, the antigen is one of the
Thomsen-Friedenreich (TF) antigens (TFAs).
[0016] In a further aspect, the present invention relates to a
method for selecting a plurality of binders specific for a relevant
polypeptide comprising
[0017] i) building a binder library by using PBMCs obtained from at
least one animal immunized with a fusion protein consisting of the
relevant polypeptide fused to an irrelevant polypeptide
[0018] ii) producing a phage binder library by infecting the binder
library of step i) with a helper phage
[0019] iii) performing with the phage binder library of step ii) at
least one round of selection against the irrelevant polypeptide
[0020] iv) producing soluble binders with the selected clones
obtained at step iii)
[0021] v) performing with the phage binder library of step ii) at
least one round of selection against the fusion protein of step i)
in the presence of an excess of binders produced by step iv)
[0022] iv) cloning, recovering and optionally sequencing the
binders from the clones selected at step v).
[0023] Methods for producing a phage binder library (e.g. a phage
antibody library) are well known in the art and are typically
described in the EXAMPLE. Typically, the helper phage is selected
among any phage well known for phage display and include for
example KM13 or Hyperphage (Progen biotechnik) helper phage such as
described in the example.
[0024] In one embodiment, the fusion protein is built so as to
solubilise the relevant polypeptide (e.g. ectodomain of cell
surface protein of interest) and typically may consist of the
relevant polypeptide fused to the Fc domain of an immunoglobulin
(e.g. IgG1). Accordingly the irrelevant polypeptide is the Fc
domain og the immunoglobulin.
[0025] In a further aspect, the present invention relates to a
method for selecting a plurality of binders specific for a cell
surface protein comprising
[0026] i) providing a cell that was genetically transformed so as
to express at its surface the cell surface protein
[0027] ii) building a binder library by using PBMCs obtained from
at least one animal immunized with the cell of step i)
[0028] iii) producing a phage binder library by infecting the
binder library of step ii) with a helper phage
[0029] iv) performing with the phage binder library of step ii) at
least one round of selection against the cell of step i) that was
not genetically transformed
[0030] v) producing soluble binders with selected clones obtained
at step iv)
[0031] vi) perfoiming with the phage binder library of step iii) at
least one round of selection against the cell of step i) in the
presence of an excess of binders produced by step v)
[0032] vii) cloning, recovering and optionally sequencing the
binders from the clones selected at step vi).
[0033] According to the invention, the cell is an eukaryotic cell.
Preferably said cell is a mammalian cell. Typically said mammalian
cells include but are not limited to cells from humans, dogs, cats,
cattle, horses, sheep, pigs, goats, and rabbits. In a particular
embodiment the cell is a human cell. In another particular
embodiment said cell is a cell line. (e.g. HEK) In particular
embodiment, the cell is a tumor cell obtainable from a patient.
[0034] The term "transformation" means the introduction of a
"foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA
sequence to a host cell, so that the host cell will express the
introduced gene or sequence to produce the desired protein coded by
the introduced gene or sequence. A host cell that receives and
expresses introduced DNA or RNA bas been "transformed". Typically,
said transformation may be performed by using any vector well known
in the art. Examples of suitable vectors include replicating
plasmids comprising an origin of replication, or integrative
plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other
examples of viral vector include adenoviral, retroviral, herpes
virus and AAV vectors. Such recombinant viruses may be produced by
techniques known in the art, such as by transfecting packaging
cells or by transient transfection with helper plasmids or viruses.
Typical examples of virus packaging cells include PA317 cells,
PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for
producing such replication-defective recombinant viruses may be
found for instance in WO 95/14785, WO 96/2378, U.S. Pat. No.
5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S.
Pat. No. 5,278,056 and WO 94/19478.
[0035] According to the invention, any eukaryotic cell may be used.
Preferably said cell is a mammalian cell. Typically said mammalian
cells include but are not limited to cells from humans, dogs, cats,
cattle, horses, sheep, pigs, goats, and rabbits. In a particular
embodiment the cell is a human cell. In particular embodiment, the
cell is a tumor cell obtainable from a patient.
[0036] In another particular embodiment said cell is a cell
line.
[0037] In a further aspect, the present invention relates to a
method for selecting a plurality of binders specific for a cell
comprising
[0038] i) building a binder library by using PBMCs obtained from at
least one animal immunized with the cell or a cell lysate
[0039] ii) producing a phage binder library by infecting the binder
library of step i) with a helper phage
[0040] iii) performing with the phage binder library of step ii) at
least one round of selection against a second cell or second cell
lysate
[0041] iv) producing soluble binders with selected clones obtained
at step iii)
[0042] v) performing with the phage binder library of step iv) at
least one round of selection against the cell in the presence of an
excess of binders produced by step iv)
[0043] vi) cloning, recovering and optionally sequencing the
binders from the clones selected at step v).
[0044] In a particular embodiment, the cell is also a eukaryotic
cell as above described.
[0045] In one embodiment the cell is specific for a tissue and the
second cell is specific for another type of tissue. In this case
the method will be particularly suitable for providing a plurality
of antibodies that may be used for diagnosing purpose or imaging
purpose.
[0046] In one embodiment, the cell is a cancer cell and the second
cell according to step iii) is a normal cell (i.e. the same type of
cell that was not malignant transformed). The cancer cell may be
isolated from a biopsy. The cancer may be selected from any cancer
(e.g. breast cancer). In this case, the method of the invention
will be particularly suitable for providing a set of antibodies
specific for cancer antigens. Thereafter said antibodies may be
useful for screening and identifying relevant cancer antigens that
can represent relevant therapeutic targets or relevant diagnostic
markers.
[0047] Accordingly, the present invention also relates to a method
for selecting a plurality of binders with cancer specificity
comprising
[0048] i) building a binder library by using PBMCs obtained from at
least one animal immunized with a mixture of cancer biopsy
lysates
[0049] ii) producing a phage binder library by infecting the binder
library of step i) with a helper phage
[0050] iii) performing with the phage binder library of step ii) at
least one round of selection against a mixture of normal biopsy
lysate
[0051] iv) producing soluble binders with selected clones obtained
at step iii)
[0052] v) performing with the phage binder library of step ii) at
least one round of selection against the mixture of cancer biopsy
lysates in the presence of an excess of binders produced by step
iv)
[0053] vi) cloning, recovering and optionally sequencing the
binders from the clones selected at step v).
[0054] The present invention also relates to a method for selecting
a plurality of binders with cancer specificity comprising
[0055] i) building a binder library by using PBMCs obtained from at
least one animal immunized with a mixture of cancer cell lines
[0056] ii) producing a phage binder library by infecting the binder
library of step i) with a helper phage
[0057] iii) performing with the phage binder library of step ii) at
least one round of selection against a normal cell line
[0058] iv) producing soluble binders with selected clones obtained
at step iii)
[0059] v) performing with the phage binder library of step ii) at
least one round of selection against the mixture of cancer cell
lines in the presence of an excess of binders produced by step
iv)
[0060] vi) cloning, recovering and optionally sequencing the
binders from the clones selected at step v).
[0061] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0062] FIGS. 1A and B: A) phage ELISA assay of clones selected on
recombinant antigen. 94 clones randomly picked from each type of
selection were assayed by monoclonal phage ELISA for binding to
HER2-Fc, or Fc. Clones yielding a positive signal on HER2-Fc but
not on Fc were considered anti-HER2. Those positive on HER2-Fc and
Fc were considered anti-Fc and those negative on both were
considered non binders (negative). Dep: depletion. Fc Masked:
selection done in the presence of masking sdAbs B) Phage ELISA on
cells using clones selected on transfected cells. 94 clones
randomly picked from each type of selection were assayed by
monoclonal phage ELISA for binding to HEK cells, HEK cells
transfected with HER2 (HEK-HER2) or cells transfected with mGluR4.
Binders yielding signals on transfected cells (HEK-HER2 or
HEK-mGluR2) but not on HEK cells were considered receptor-specific
(anti-Receptor), those yielding signal on all type of HEK cells
were considered HEK-specific (anti-HEK) and those negative on all
cells were considered negative. Sel.: selection.
[0063] FIG. 2A-C: Characterization of anti-HER2 sdAbs by
homogeneous time resolved fluorescence (HTRF) technology. A) Cells
transfected by SNAP-tag HER2 and labeled with donor fluorophore
were incubated with sdAbs and acceptor-labeled anti-6his mAb. The
sdAb binding, detected by FRET, is expressed as HTRF ratio to
normalize results for the HER2 receptor density (see Materials and
Methods). B) Various concentrations of sdAbs were incubated on
transfected and labeled cells and binding was followed by FRET as
for A). C) Dissociation constants were calculated using a
non-linear curve fitting software (Prism, GraphPad).
[0064] FIG. 3A-C: Characterization of anti-mGluR4 sdAbs by
homogeneous time resolved fluorescence (HTRF) technology. SdAb
targeting mGluR4 were characterized as described in FIG. 2. A).
Cells transfected by SNAP-tag mGluR4 and labeled with donor
fluorophore were incubated with sdAbs and acceptor-labeled
anti-6his mAb. The sdAb binding, detected by FRET, is expressed as
HTRF ratio to normalize results for the mGluR4 receptor density
(see Materials and Methods). B) Various concentrations of sdAbs
were incubated on transfected and labeled cells and binding was
followed by FRET as for A). C) Dissociation constants were
calculated using a non-linear curve fitting software (Prism,
GraphPad).
[0065] FIG. 4A-C: Fine characterization of candidate sdAbs isolated
against biopsy lysates. A) Reverse phase phage-ELISA. Various
biopsy lysates, a mixture of breast cancer cell lysates, (BC cell
lines), a human PBMC lysate, and a healthy breast epithelial cell
line HME1 lysate were coated on maxisorp. Phage-sdAbs were
incubated and washed. Bound phage were detected using
HRP-conjugated anti-M13 mAb. B) Tissue micro array analysis.
Paraffin embedded slide containing 80 breast cancer samples or 14
healthy breast samples were incubated with purified and in vitro
biotinylated sdAb J.H6. Bound sdAbs were detected using
HRP-conjugated streptavidin. Representative examples are shown. C)
Staining results were classified according to their intensity.
[0066] FIG. 5A-C: Fine characterization of candidate sdAbs against
intact breast cancer cells. A) A phage cytometry assay was
performed on 6 breast cancer cell lines, 7 cancer cell lines of
various origins (cervical, pancreas, ovarian, colon, prostate,
lymphocyte), on human PBMC and normal breast epithelium cell line
HME1. In vitro biotinylated sdAb were added on cells. After
washing, bound sdAbs were detected with HRP-conjugated
streptavidin. B) Tissue micro array analysis. Paraffin embedded
tissue array containing 80 breast cancer samples and 14 healthy
breast samples were incubated with in vitro biotinylated sdAbs.
Bound sdAbs were detected by HRP-conjugated streptavidin. Shown are
representative examples. C) Staining results were classified
according to their intensity.
[0067] FIG. 6A and B: Transfection level. Expression HER2 or mGluR4
(fused to SNAP and Flag tag) on the surface of HEK cells was
followed by flow cytometry. A) HER2-transfected HEK cells
(HEK-HER2) and B mGluR4-transfected HEK cells (HEK-mGLuR4) were
incubated with anti-Flag mAb (black line) or not (gray line).
Captured antibodies were detected using PE-conjugated anti-mouse
antibodies. Cells were analyzed by flow cytometry assay on a
MACSQuant flow cytometer (Miltenyi).
[0068] FIG. 7: Characterization of anti-HER2 sdAbs by ELISA.
HER2-Fc recombinant fusion or human Fc portion were covalently
immobilized on epoxy magnetic beads. Soluble sdAbs were incubated.
After washing, bound sdAbs were detected using a HRP-labeled
anti-6his mAb. aFc: anti human Fc sdAb used as positive
control.
[0069] FIG. 8A-C: Characterization of anti-HER2 binders by flow
cytometry. A) All selected phage-sdAbs were incubated with HER2+
SKOv3 cells. After washing, bound phage were detected using
PE-conjugated anti-M13 mAb. Cells were analyzed by flow cytometry
assay on a MACSQuant flow cytometer (Miltenyi). B) Competition flow
cytometry assay. Cells were incubated with phage-sdAbs in the
presence of an excess of soluble anti-HER2 sdAbs C.E4, A.E4 or
A.H10. Bound phage were detected as in A). C) Phage-sdAbs and sdAbs
able to compete (sharing overlapping epitopes) were classified into
groups.
EXAMPLE
[0070] Material & Methods
[0071] Cells Lines, Biopsies
[0072] MCF7, SK-BR-3 and T47D are a kind gift of Daniel Olive.
MDA-MB-231 and HCC1937 are a kind gift of Marie Alix Poul (IRCM,
Montpellier). BrCa-Mz-01, HCC1806, HCC1954 and BT474 are a kind
gift of Jean Imbert (INSERM, U928, TAGC, Marseille, France). Cells
lines MC38, MDA-MB-231, MCF7, T47D, HCC1937, HEK293T were cultured
in DMEM complemented with 10% (v/v) fetal calf serum. Cells lines
SK-BR-3, HCC1954, BrCaMz01, BT474 and HCC1806 were cultured in RPMI
complemented with 10% (v/v) fetal calf serum. HME1 cell line was
purchased from ATCC and grown as recommended by the manufacturer.
All cell lines were grown at 37.degree. C. in a humidified
atmosphere and with 5% CO.sub.2. PBMC are patient's donor cells.
For transfection assay, HEK/293T were tranfected with Lipofectamine
(Invitrogen), following the recommendation of the manufacturer.
[0073] Breast cancer biopsies (5801, 5772e, 5766, 5586, 5572i,
5592, 5011, 5712, 5713, 5033, 5627 kind gift of S. Garcia, CRCM,
Marseille) or cells were lysed with a potter in lysis buffer : 150
mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH8 with protease
inhibitor cocktail (Complete, Roche). The lysate was centrifuged
for 10 min at 13000 g at 4.degree. C. Supernatant was the final
cell lysate. Total protein concentration (average between 2-5
mg/ml) was determined spectrophotometrically using a protein assay
kit (Bio-Rad Laboratories, Hercules, Calif., USA).
[0074] Production and Purification of sdAbs
[0075] For polyclonal production of sdAbs from, 10 .mu.l of output
from selection round 1 and 2 were used to inoculate 200 ml of
2YT/ampicillin (100 .mu.g/mL). Cells were grown at 37.degree. C.
(250 rpm) until the OD.sub.600 reached 0.5. sdAb expression was
induced by the addition of 0.1 mM IPTG
(isopropyl-h-D-thiogalactopyranoside) at 30.degree. C. (250 rpm)
for 20 h.
[0076] sdAbs were purified by metal affinity chromatography as
described [19].
[0077] In Vitro Biotinylation
[0078] The in vitro biotinylation of protein was performed using
Ez-link micro NMHS-PEO4-biotinylation kit (Perbio science)
following the recommendation of the manufacturer.
[0079] Llama Immunization and Library Construction
[0080] Three young adult llama (Lama glama) were immunized
subcutaneously at days 1, 30, 60, 90 and 120 with breast cancer
biopsy lysate (two llamas) or with healthy breast biopsy (one
llama). One llama was immunized with HER2-Fc protein and HEK-mGluR4
cells.
[0081] VHH library constructions were performed as described [13,
20]
[0082] Selection of Phage-sdAbs
[0083] To produce phage-sdAb library, 10 .mu.L of the library was
grown in 50 mL of 2YT/ampicillin (100 .mu.g/mL)/glucose (2%) at
37.degree. C. to an OD.sub.600 of 0.5. Then, the culture was
infected with KM13 or Hyperphage (Progen biotechnik) helper phage
with a ratio of 20 phage/cell for 30 min at 37.degree. C. without
shaking. The culture was centrifuged for 10 min at 3000 g. The
bacterial pellet was resuspended in 250 mL of 2YT/ampicillin (100
.mu.g/mL) / kanamycine (25 .mu.g/mL), and incubated overnight at
30.degree. C. with shaking (250 rpm). Twenty five mL were then
centrifuged for 20 min at 3000 g. Five mL of 20% PEG 6000, 2.5 M
NaCl were added to the supernatant and incubated for 1 h on ice to
precipitate phage particles. The solution was centrifuged for 15
min at 3000 g at 4.degree. C. and the phage-containing pellet was
re-suspended with 1 mL of PBS.
[0084] Different strategies of panning were performed. Some phages
were selected using magnetic epoxy beads (Dynabeads, invitrogen)
coated with antigen or lysates immobilized on epoxy beads during 48
h at 4.degree. C. following recommendations of the manufacturer.
Other phages were selected directly on cells (2.times.10.sup.6
cells). Beads or cells were washed three times in PBS (using a
magnetic particle concentrator for magnetic beads and
centrifugation step for cells) and phage-sdAb library (1 ml) and
beads or cells were saturated in 2% milk PBS. For selection
including a depletion step, phage-sdAb library were incubated with
depletion support with rotation during 2 h at room temperature or
at 4.degree. C. for cells. Phage-sdAb libraries (depleted or not)
were recovered and incubated with beads with rotation during 2 h at
room temperature or at 4.degree. C. for cells. For masked selection
in the presence of soluble sdAbs, 10 .mu.M of pure sdAbs were added
during this step. Beads, cells or plate were washed 10 times with 1
ml 0.1% Tween PBS (without Tween for cells) and two times with PBS.
Bound phage were eluted with tryspin solution (Sigma) at 1 mg/ml
during 30 min at room temperature with rotation. Eluted phage were
incubated without shaking with log-phase TG1 cells and plated on
2YT/ampicillin (100 .mu.g/mL)/glucose (2%) in 15 cm Petri dishes.
Some isolated colonies were grown overnight in microtiter plate
containing 200 .mu.L 2YT/ampicillin (100 .mu.g/mL)/ glucose (2%)
and stored at -80.degree. C. after the addition of 15% glycerol
(masterplates). The remaining colonies were harvested from the
plates, suspended in 2 mL 2YT/ampicillin (100 .mu.g/mL)/glucose
(2%) and used for phage production for the next round of
selection.
[0085] Phage-sdAb ELISA on Epoxy Beads
[0086] A 96-well plate replicator was used to replicate the
masterplates in 150 .mu.L of fresh broth. Colonies were grown for 2
h at 37.degree. C. under shaking (400 rpm) and 15 .mu.L 2YT/
ampicillin (100 .mu.g/mL)/glucose (2%) containing 2.times.10.sup.9
M13K07 helper phage were added to each well and incubated for 30
min at 37.degree. C. without shaking. The plate was centrifuged for
10 min at 1200 g and bacterial pellets were suspended in 150 .mu.L
2YT/ampicillin (100 .mu.g/mL)/kanamycine (25 .mu.g/mL)1) and grown
for 16 h at 30.degree. C. under shaking (400 rpm). Phage-containing
supernatants were tested for binding by ELISA.
[0087] Antigens HER2-Fc (R & D systems) or Fc were immobilized
on magnetic epoxy beads (Dynabeads, invitrogen) during 48 h at
4.degree. C. following recommendation of the manufacturer. For
ELISA, 2.mu.l of beads/well was used. After three washes, beads
were blocked with 5% milk-PBS (MPBS) for two hours at RT. Plates
were incubated for 1 h at RT with 50 .mu.l/well of phage-containing
supernatants diluted at 1/2 in 4% MPBS. After three washes with
0.1% Tween PBS and three washes in PBS, plates were incubated with
HRP-conjugated anti-M13 mAb (Pharmacia) diluted 1/5000 during 1 h
at RT. After three washes with 0.1% Tween PBS and three washes in
PBS, bound secondary antibodies were detected using ABTS.
Coloration was followed at 405 nm.
[0088] Phage-sdAb ELISA with Lysate Coated on Plate
[0089] Fifty .sub.IA/well of biopsy mixture, or breast cancer cell
lines (BT474, SK-BR-3, HCC1954, MCF7, MDA-MB-231, T47D, HCC1806,
BRCA-Mz-01, HCC1937) or control cells HME1 and human PBMC lysates
(200 .mu.g/ml of total proteins) were coated overnight at 4.degree.
C. on maxisorp 96-well plate (Nunc). After three washes with PBS,
plates were blocked with 5% MPBS for two hours at RT. For
competitive assay, plates were incubated with 50 .mu.l/well of
sdAbs at 10 .mu.g/ml during 1 h at RT. Plates were incubated for 1
h at RT with 50 .mu.l/well of phage-containing supernatants diluted
at 1/2 in 4% MPBS. After three washes with 0.1% Tween PBS and three
washes in PBS, plates were incubated with HRP-conjugated anti-M13
mAb (Pharmacia) at 1/5000 during 1 h at RT. After three washes with
0.1% Tween PBS and three washes in PBS, bound secondary antibodies
were detected using ABTS. Coloration was followed at 405 nm.
[0090] Phage-sdAb Flow Cytometry Assay
[0091] Experiments were performed on ice with rocking in 1% BSA
PBS. Typically, 2.times.10.sup.5 cells resuspended in 50 ttl were
distributed in 96-well microtiter plates. For competitive assay,
plates were incubated with 50 .mu.L well of sdAbs at 10 .mu.g/ml
during 1 h at 4.degree. C. Fifty .mu.l/well of phage-containing
supernatants diluted at 1/2 in 2% BSA PBS were added and plates
were incubated for 1 h at 4.degree. C. with. After three washes in
PBS, plates were incubated with PE-conjugated anti-M13 mAb at 1/200
during 1 h at 4.degree. C. After three washes in PBS, fluorescence
was measured using a MACSQuant (Miltenyi) and results were analyzed
with the MACSQuant software. Negative (secondary antibody only)
controls were carried out.
[0092] HTRF Assay
[0093] A 96-well plate replicator was used to replicate the
masterplates in 150 uL of fresh broth. Colonies were grown for 2 h
at 37.degree. C. under shaking (900 rpm) and 15 .mu.L 2YT/
ampicillin (100 1.1g/mL)/ containing 0.1 mM IPTG were added to each
well. The plate was incubated for 16 h at 30.degree. C. with
shaking (400 rpm). sdAb-containing supernatants were tested for
binding by HTRF. To measure HTR-FRET signals, HEK cells were
transfected plasmids coding for SNAP-tagged HER2 or mGluR4
receptors N-terminally fused to a SNAP tag 24 h prior to the assay
(plasmids were a kind gift of Cisbio Bioassays and Jean-Philippe
Pin (IGF, Montpellier), respectively). Cells were labeled with
Tag-lite Snap-Lumi4-Tb, according to the manufacturer's kit
protocol (Cisbio Bioassays). sdAb and D2 labeled anti-6his mAb
(Cisbio) were added simultaneously. After incubation for 1 hour at
RT, HTR-FRET signal (665 nm) and Lumi4-Tb donor signal (620 nm)
were measured using a Tecan infinite M1000. HTRF ratio (665 nm/620
nm.times.10.sup.4) was calculated to eliminate quenching and
dispensing errors.
[0094] Immunohistochemistry Assay
[0095] In vitro biotinylated sdAbs were assayed in
immunohistochemistry on 5 .mu.m sections of paraffin-embedded
cancer tissus. In addition, adjacent normal breast epithelium
served as specificity control. A breast cancer tissue micro array
containing 80 samples in duplicate (containing lobular and ductal
breast cancer biopsies from grade I to III tumors with local lymph
node invasion or not) and 14 samples healthy breast tissues in
duplicate was also used. After deparaffinization of
paraffin-embedded tissues, antigen retrieval of paraffin-embedded
tissues was performed in 95.degree. C. pre-warmed citrate buffer
during 20 min. Endogenous peroxidase activity was blocked by
incubation with 3% H.sub.2O.sub.2. Slides were incubated for lh
with in vitro biotinylated sdAbs at 10 .mu.g/ml at room temperature
and washed. Detection was performed by incubations at room
temperature 30 min streptavidin peroxidase. Finally, visualization
was performed by a DAB revelation (Dako) peroxidase reaction with
haematoxylin as counterstain.
[0096] Results
[0097] 1. Selection of phage antibodies against a specific part of
recombinant protein HER2-Fc.
[0098] The proof of principle of this new approach was first
established on a simple selection procedure using a purified
recombinant protein. A single-domain antibody (sdAb) library was
built using PBMCs of llamas immunized with various recombinant
proteins consisting of fusion between ectodomains of relevant tumor
markers and human IgG1 Fc portion including HER2-Fc and HER4-Fc.
The aim of this first part of the study was to favor the selection
of HER2 binders compared to Fc binders.
[0099] A basic strategy (i.e. direct selection of phage antibody
produced using helper phage KM13) was compared to conventional
depletion strategies on irrelevant Fc bearing molecules prior to
positive selection on HER2-Fc fusion. A third approach was
developed using Hyperphage to produce the phage-antibody particles
since it was demonstrated that this helper phage can significantly
increase the percentage of phage actually displaying an antibody
fragment and the number of antibody fragment displayed per
particles (valency). Theoretically, these two factors should
increase the efficiency of the depletion strategy. Finally a masked
selection strategy was performed. This approach consisted in a
first selection of the library against a human IgG Fc portion. The
selected clones were then polyclonally produced as soluble sdAbs
which were added in excess during a second selection against
recombinant fusion protein HER2-Fc to block their corresponding
epitopes on the Fc portion. FIG. 1A shows that the direct selection
strategy yielded a majority of Fc binders. The proportion of Fc
binders was significantly reduced using a depletion step (with both
helper phages). Interestingly, the vast majority of binders
obtained by masked selection were HER2 ectodomain binders.
Sequencing of the various outputs revealed 8 different clones out
of 24 binders for the classical selection and 14 different clones
out of 70 binders for the masked selection, suggesting that the
masked selection on purified antigen could greatly improve the
frequency of relevant binders without markedly impacting the output
diversity.
[0100] 2. Selection of phage-antibodies against specific receptors
on transfected cells
[0101] Some targets, including for example members of the seven
transmembrane receptors, cannot easily be recombinantly produced as
soluble protein. In such cases, phage display can be used to select
relevant binders by using a positive selection on transfected cells
following a depletion step on the untransfected cell line. We
decided to compare the efficiency of depletion vs. masked selection
for the selection of binders against HER2 and mGluR4, a member of
the metabotropic G-protein-coupled glutamate receptor family. For
these experiments, we used a sdAb library that had been generated
after immunization with transfected cells expressing HER2 and
mGluR2. The two receptors were differentially expressed at the
surface of HEK293T cells (HEK), as shown in FIG. 6A and B, using an
anti-Flag tag antibody for detection. Masking sdAbs were obtained
by two rounds of selection on untransfected HEK cells.
[0102] The output of depletion strategies were mainly constituted
by HEK binders, and yielded only 7 and 8% of HER2 binders for KM13
and Hyperphage based approaches respectively, demonstrating a
limited effect of the depletion strategy, even in high display
conditions due to the use of hyperphage (FIG. 1B). The masked
selection approach significantly improved the output since 26% of
this output was constituted by HER2 binders.
[0103] Sequencing of a subset of the positive clones revealed 6
different clones out of 15 HER2 binders obtained by the two
depletion strategies and 5 different clones out of 20 HER2 binders
selected by masked selections. The dominant clone A.B5 was found in
all different strategies. Several clones selected on purified
antigens were not retrieved by cell selection. Conversely, clone
H.H8 was only retrieved by masked selection on cells.
[0104] All 15 anti-HER2 binders obtained so far using recombinant
antigen or transfected cells were produced as soluble fragments to
be further characterized in terms of specificity and affinity.
[0105] Their specificity was first confirmed by ELISA. FIG. 6A and
B shows that all clones yielded a high signal on HER2-Fc and were
negative against Fc, including sdAb H.H8. Next, flow cytometry on
HER2 positive cells was used to demonstrate the ability of these
anti-HER2 sdAbs to bind their antigen in a native context. All
clones were found positive (FIG. 7), including 7 clones that were
selected on recombinant antigen but not through cell selections.
Flow cytometry competition experiments following the binding of
phage-sdAbs in the presence of soluble sdAbs (FIG. 8B) could
establish at least three groups of sdAbs binding to three
independent HER2 epitopes, representing 11 clones (FIG. 8C).
[0106] A third independent approach based on Homogeneous Time
Resolved Fluorescence (HTRF) was finally used to confirm the
specificity and determine the affinity of anti-HER2 sdAbs. HEK
cells were transfected with HER2 fused to the SNAP tag allowing a
site-directed labeling of the receptor at the cell surface with a
donor fluorophore. Bound sdAbs were detected using an anti-His tag
monoclonal antibody labeled with an acceptor fluorophore. In this
setting, the excitation of the donor fluorophore can lead to an
emission of the acceptor fluorophore (FRET effect) if these two
molecules are in close proximity, i.e. if the sdAb is binding to
HER2. As shown in FIG. 2A, all anti-HER2 sdAbs were found positive
in this assay whereas an irrelevant (anti-Fc) sdAb did not yield
any signal. Three clones, binding overlapping epitopes yielded
significantly lower FRET signal (A.E6, A.E4 and C.E10), suggesting
that these epitopes are more distant to the SNAP tag than the other
epitopes. H.F6 also yielded a lower signal, in agreement with flow
cytometry results.
[0107] Dose response curves of the 12 best binders were used to
determine the sdAb dissociation constants (FIG. 2B). The calculated
K.sub.D values using a non-linear curve fitting program ranged
between 0.5 and 7.3 nM (FIG. 2C).
[0108] To see if similar results could be obtained on a different
type of receptors, depletion and masked selections were applied to
the selection of sdAbs against the metabotropic glutamate receptor
4(mGluR4). This receptor belongs to the family of
seven-transmembrane G protein-coupled receptor family and is
difficult to produce recombinantly. Thus in this case,
immunization, selection and screening steps were all perfonned
using transfected HEK cells.
[0109] A conventional depletion step on HEK cells followed by a
selection on mGluR4-transfected cells yielded a vast majority of
HEK binders and less than 13% of mGluR4 binders (FIG. 1B), despite
the high expression of the transfected receptor (FIGS. 6A and B).
In sharp contrast, the masked selection strategy was highly
successful since 90% of the output was specific for the relevant
receptors and only 4% of the outputs were HEK binders (FIG.
1B).
[0110] The sequencing of 47 mGluR4 binders obtained by masked
selection revealed 19 different clones, suggesting again this
approach can lead to a good diversity of binders.
[0111] Soluble sdAbs corresponding to these 19 clones were produced
and purified from E. coli for further characterization. As for HER2
binders, HTRF experiments were performed to establish their
specificity for mG1uR4. FIG. 3A shows that all clones yielded high
HTRF signals demonstrating their specificity for mGluR4. In
contrast, an irrelevant anti-Fc sdAb was found negative. Dose
response curves and non-linear curve fitting analysis of seven best
binders (FIG. 3B) were used to determine their dissociation
constants. K.sub.D values of these monovalent single domain
antibodies ranged from 0.3 to 9.3 nM (FIG. 3C).
[0112] 3. Selection of phage antibodies with breast cancer
specificity
[0113] The efficiency of masked selection being established on
transfected HEK cells, we applied this approach to the selection of
binders against unknown antigens overexpressed in cancer samples.
Llamas were immunized with breast cancer biopsies and the resulting
sdAb libraries were selected against several samples.
[0114] Selection on breast cancer biopsy lysates: Selections were
performed on a mixture of breast cancer biopsy lysates using a
lysate of human peripheral blood mononuclear cells mixed to a
lysate of normal human mammary epithelium cell line
HME1(immortalized by hTERT expression) as normal sample. A basic
approach using only depletion was compared to an approach using
depletion plus masking using sdAbs selected from the same library
by panning on the normal sample, and using KM13 or hyperphage as
helper phage.
[0115] Ninety six clones were picked after two rounds of selection
for each approach and a phage ELISA screening procedure using
plastic-adsorbed lysates was performed to evaluate the specificity
of the selected binders. The depletion strategy yielded 13% and 9%
of clones showing specificity for the biopsy lysates, for KM13 or
Hyperphage respectively. The addition of the masking procedure
massively decreased the number of non cancer specific clones
increased the proportion of cancer-specific clones to 40% and 30%
for KM13 and hyperphage respectively. Sequencing of biopsy lysate
binders revealed 9 different sequences including a highly dominant
clone. Competition experiments performed by phage ELISA in the
presence of an excess of each purified sdAb indicated that 4 of
these clones were sharing a common epitope. A representative clone
of this family and the 5 other sdAbs targeting independent epitopes
were chosen for further characterization.
[0116] To confirm the specificity of these sdAbs for breast cancer,
phage ELISA was performed on a panel of immobilized lysates from 11
different breast cancer biopsies, and on a mixture of breast cancer
cell line lysates (see material and methods). Lysates of PBMCs and
HME1 cell line were used as normal samples. This experiment
confirmed the absence of signals on normal samples for all tested
clones. Various binding profiles were generated against the 11
breast cancer biopsy lysates (FIG. 4A). To further confirm the
cancer specificity of these antibody fragments, they were produced
as soluble fragments and tested by immunohistochemistry. Out of the
6 tested sdAbs, only sdAb J.H6 yielded a strong signal on paraffin
embedded tissue. This sdAb was thus further characterized on larger
scale using breast cancer tissue microarray including 80 breast
cancer samples (lobular and ductal breast cancer biopsies) and 14
healthy breast samples. None of the normal samples were stained by
the sdAb and 75% of breast cancer biopsies were positive (FIG. 4C).
FIG. 4B shows results on representative samples (cancer biopsies
and normal samples).
[0117] Selection for cell surface binders on breast cancer cell
lines: While intracellular cancer specific antigens can be useful
as biomarkers for diagnosis purposes, they are not compatible with
some therapeutic approaches such as therapeutic antibodies. For
these approaches, the identification of cancer-specific membrane
antigens is required. To evaluate the potential of masked selection
in this case, and using the same sdAb library, we compared the
output of two rounds of selection performed on a mixture of 4
different breast cancer cell lines (MDA-MB-231, MCF7, SKBr3,
HCC1954) using a simple depletion step on a mixture of human PBMCs
and normal breast epithelial cell line HME1 (PBMC+HME1), or a
combination of deletion and masking using sdAbs from the same
library selected on PBMC+HME1. Ninety six clones from each
selection were screened as phage-sdAb by flow cytometry on the
mixture of breast cancer cell lines and normal sample.
[0118] A simple depletion did not lead to any cancer specific
clones. 60% and 35% of tested clones were positive on PBMC+HME1 for
KM13 and hyperphage respectively. The addition of the masking step
very efficiently blocked the selection of such binders since only 1
or 2 clones out of 96 were positive on PBMC+HME1. Interestingly, 6
and 12 clones were found positive on the mixture of breast cancer
cell lines, for KM13 and hyperphage respectively. Sequencing of
these binders revealed 11 different sequences. Flow cytometry
competition experiments performed using purified sdAbs indicated
that six different epitopes were targeted by these 11 different
sdAbs. A representative binder of each epitope was chosen for
further studies.
[0119] The six purified sdAbs were tested by flow cytometry against
6 different breast cancer cell lines, 7 other cancer cell lines of
various origins, against normal breast epithelial cell line HME1
and against human PBMCs. As shown in FIG. 5A, none of the sdAbs
were positive on normal cells. Four sdAbs (M.C9, M.B9, M.H3, M.H12)
were strongly positive against most tested cancer cell lines. sdAbs
M.D5 was only positive on two breast cancer cell lines whereas sdAb
M.A4 was positive on one breast cancer cell line (MDA-MB-231) as
well as on PC3(prostate cancer) and HeLa (cervical cancer) cell
lines.
[0120] Immunohistochemistry characterization demonstrated that two
of these sdAbs (M.H3, M.H12) were functional on paraffin embedded
tissues. These sdAbs were then assayed on breast cancer tissue
micro array. FIG. 58 displays representative results obtained with
these two sdAbs. None of the 14 normal samples were stained by
these two sdAbs and 98% or 79% of breast cancer biopsies were
strongly stained by sdAb M.H3 and M.H12, respectively (FIG.
5C).
[0121] Discussion
[0122] We have designed a new procedure to focus the output of a
phage display toward specific epitopes of a chimeric protein,
toward transfected membrane receptors and toward specific or
overexpressed cancer markers using crude lysates or intact cells
when surface markers are preferred. In all cases, the principle of
masking non relevant epitopes was shown to be more efficient than
conventional depletion. The general concept underlying this
approach is merely to block unwanted epitopes using the addition of
an excess of binders previously selected from the very same library
by panning against the non relevant sample.
[0123] This simple procedure is very powerful because it takes
advantage of the potential biases of a library. Indeed, libraries
often contain a large number of binders directed against non
relevant but highly abundant or very immunoreactive epitopes. These
binders often outcompete relevant binders against low abundant
epitopes when selections are performed on complex samples. Masked
selection should be very efficient in this case because these
dominant binders targeting abundant proteins would be very
efficiently selected. By this process, the polyclonal population of
soluble binders selected on the non-relevant sample (the mask) will
be enriched for binders that have to block the most problematic
(abundant and/or immunoreactive) epitopes.
[0124] Our results demonstrate some efficiency of depletion
strategies but also highlight their shortcomings in difficult
selections on complex samples. The principle of depletion relies on
the capture of non relevant binders from the phage population
before positive selection. This approach can improve the
specificity of an output but cannot be totally efficient since, as
for any non covalent interaction, a fraction of the total
phage-antibody to be depleted will stay in solution at a ratio
dependent on the affinity of the antibody. Increasing the number of
pill fused to an antibody fragment using for example hyperphage as
helper phage should increase the apparent affinity of binders and
thus increase the proportion of bound phage during the depletion
step. Such an effect could be seen in this study on depletion
performed on Fc portion but was not visible on selections performed
on complex samples. Other techniques have been proposed to focus a
phage selection on a particular antigen including guided selection
[17, 18] and competitive elutions [7]. These techniques have been
successfully used in several studies but they are limited in
several aspects. Guided selection depends on the availability of a
cloned antibody and is labor-intensive. Competitive elution uses a
known ligand or monoclonal antibody to specifically elute
phage-antibodies binding to the same epitopes. Unfortunately this
strategy naturally favors the selection of low affinity binders and
cannot be used for target discovery.
[0125] On the other hand, the masked selection is based on a
competition between masking antibody fragments and phage particles.
The input of a selection procedure usually contains 10.sup.12 phage
particle in 1 mL, leading to a phage concentration of around 2 nM.
Single domain antibodies are easily produced in E. coli and we
routinely use 10 .mu.M of polyclonal sdAbs for the masking
procedures, ensuring a huge excess of sdAbs over phage-sdAbs to
guarantee an efficient blocking of non relevant epitopes. This
process does not influence the affinity of selected binders and as
shown in this study, it can efficiently leads to the selection by
binders against unknown but differentially expressed targets.
[0126] In principle, this technique is very flexible and can be
used with any kind of antibody fragments (scFv, Fab fragments),
alternative scaffolds (darpins, monobodies, affibodies,
anticalins
[0127] As described in this work, masked selection can be used to
select binders against unknown targets that can be subsequently
identified by immunoprecipitation and mass spectrometry analysis
for example and has a great potential for cancer therapy. It should
also be of high interest for many other purposes, such as the
identification of specific cell surface receptors of some cell
types, such as normal and cancer stem cells or regulatory T cells
for example.
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