U.S. patent application number 16/450186 was filed with the patent office on 2019-10-10 for novel biotin-specific monoclonal antibody and use thereof.
This patent application is currently assigned to Roche Diagnostics Operations, Inc.. The applicant listed for this patent is Roche Diagnostics Operations, Inc.. Invention is credited to Michael Gerg, Lars Hillringhaus, Klaus Hirzel, Caroline Dorothea Hojer, Hans-Peter Josel, Michael Schraeml, Christoph Seidel, Leopold Von Proff.
Application Number | 20190309091 16/450186 |
Document ID | / |
Family ID | 61005782 |
Filed Date | 2019-10-10 |
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United States Patent
Application |
20190309091 |
Kind Code |
A1 |
Gerg; Michael ; et
al. |
October 10, 2019 |
NOVEL BIOTIN-SPECIFIC MONOCLONAL ANTIBODY AND USE THEREOF
Abstract
The present invention relates to a monoclonal antibody capable
of binding to biotin. In one embodiment the monoclonal antibody
according to the invention also does not bind to a biotin moiety on
a biotinylated molecule, wherein the biotin moiety is attached to
the molecule via the carbon atom of the carboxyl function of the
valeric acid moiety of biotin. Also disclosed is a method for
generation of an antibody as disclosed herein. The monoclonal
antibody according to the invention is of specific use in a method
for measuring an analyte in a sample, wherein a
(strept)avidin/biotin pair is used to bind a biotinylated analyte
specific binding agent to a (strept)avidin coated solid phase.
Inventors: |
Gerg; Michael; (Muenchen,
DE) ; Hillringhaus; Lars; (Koenigsdorf-Schoenrain,
DE) ; Hirzel; Klaus; (Baierbrunn, DE) ; Hojer;
Caroline Dorothea; (Muenchen, DE) ; Josel;
Hans-Peter; (Weilheim, DE) ; Seidel; Christoph;
(Weilheim, DE) ; Schraeml; Michael; (Penzberg,
DE) ; Von Proff; Leopold; (Hohenpeissenberg,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc. |
Indianapolis |
IN |
US |
|
|
Assignee: |
Roche Diagnostics Operations,
Inc.
Indianapolis
IN
|
Family ID: |
61005782 |
Appl. No.: |
16/450186 |
Filed: |
June 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2017/084538 |
Dec 22, 2017 |
|
|
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16450186 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/82 20130101;
C07K 16/44 20130101; G01N 33/54306 20130101; C07K 16/00 20130101;
C07K 2317/92 20130101; C07D 495/04 20130101; C07K 2317/31 20130101;
C07K 2317/34 20130101; C07K 16/40 20130101 |
International
Class: |
C07K 16/44 20060101
C07K016/44; G01N 33/543 20060101 G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2016 |
EP |
16206944.7 |
Jul 31, 2017 |
EP |
17184142.2 |
Dec 21, 2017 |
EP |
17209229.8 |
Claims
1. A monoclonal antibody specifically binding the compound of
Formula I, ##STR00028## characterized in that it also binds to
biotin, wherein X is selected from the group consisting of
(CH.sub.2).sub.n with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, and with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5 and R is selected from the group
consisting of H, OH, COOH, NH.sub.2, an azide group, a maleimide
group, and Z, wherein Z is A or B, wherein A is M-L with M being
selected from (i) a hapten which does not contain a biotin moiety
and (ii) a polypeptide, and L being a linker, wherein B is
##STR00029## with M being selected from (i) a hapten which does not
contain a biotin moiety and (ii) a polypeptide, and L being a
linker, whereby the nitrogen atom marked with an asterisk is
covalently bound to an adjacent CH.sub.2 group of X.
2. The antibody of claim 1 further characterized in that it does
not bind to a compound of Formula II, also depicted in FIG. 3A.
##STR00030##
3. The monoclonal antibody according to claim 1, wherein the
binding affinity of the monoclonal antibody to any one compound of
the group of compounds selected from ##STR00031## is higher by a
factor of at least 50 than the binding affinity to the compound of
Formula II.
4. The monoclonal antibody according to claim 3, wherein the
binding affinity of the monoclonal antibody to any one compound of
the group of compounds selected from Formula III A, Formula III B,
and Formula III C, is higher by a factor of at least 500 than the
binding affinity to the compound of Formula II.
5. A method for measuring an analyte in a sample, wherein a
(strept)avidin/biotin binding pair is used to bind a biotinylated
analyte specific binding agent to a (strept)avidin coated solid
phase, the method comprising adding to the sample a) an antibody
according to claim 1, b) a biotinylated analyte specific binding
agent, c) a (strept)avidin coated solid phase, followed by
measuring the analyte bound to coated (strept)avidin on the
solidphase and biotinylated analyte specific binding agent.
6. The method of claim 5, wherein step 5 (a) and optionally also
step 5 (b) is performed before step 5 (c).
7. An immunoassay test kit comprising at least a) an antibody
according to claim 1, b) a biotinylated analyte specific binding
agent, and c) a (strept)avidin coated solid phase or a labeled
(strept)avidin.
8-13. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/EP2017/084538 filed Dec. 22, 2017, which claims
priority to European Application No. 17209229.8 filed Dec. 21,
2017, European Application No. 17184142.2 filed Jul. 31, 2017, and
European Application No. 16206944.7 filed Dec. 27, 2016, the
disclosures of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a monoclonal antibody
capable of binding to biotin. In one embodiment the monoclonal
antibody according to the invention also does not bind to a biotin
moiety on a biotinylated molecule, wherein the biotin moiety is
attached to the molecule via the carbon atom of the carboxyl
function of the valeric acid moiety of biotin. Also disclosed is a
method for generation of an antibody as disclosed herein. The
monoclonal antibody according to the invention is of specific use
in a method for measuring an analyte in a sample, wherein a
(strept)avidin/biotin pair is used to bind a biotinylated analyte
specific binding agent to a (strept)avidin coated solid phase.
[0003] In living organisms as well as in biochemistry in vitro, the
primary function of biotin is that of a co-substrate which is
required as a prosthetic group for enzymes with carboxytransferase
activity, e.g. pyruvate carboxylase and acetyl-CoA-carboxylase. In
bacteria, biotin is attached to biotin carboxyl carrier protein by
biotin protein ligase. Further, a number of chemical processes are
known with which biotin can be covalently attached to a suitable
group on almost any molecule of interest. As a common feature, the
carbon atom of the carboxyl function of the valeric acid side chain
is reacted in a coupling reaction to an appropriate (receiving)
group on the molecule of interest, or to a reactive group of a
linker, wherein the linker itself is either already connected to
the molecule of interest or the linker is coupled to the molecule
once biotin is attached to the linker. Generally, such attachment
of biotin to various chemical sites via the carbon atom of the
carboxyl function of the valeric acid side chain is referred to as
biotinylation.
[0004] The extraordinary affinity of avidin and/or streptavidin
(=(strept)avidin), respectively, for biotin (K.sub.a=10.sup.15
M.sup.-1) is one of the strongest known non-covalent interactions
of a protein and a ligand. It allows biotinylated molecules in a
complex mixture to be specifically bound by (strept)avidin. For
this reason avidin and/or streptavidin are used in a large number
of immunological detection assays.
[0005] Besides a strong affinity for (strept)avidin, two further
properties make biotin particularly suited for tagging proteins and
other macromolecules. Firstly, the biotin molecule is substantially
smaller than proteins. Its molecular size allows one or more biotin
molecules to be conjugated to a molecule of interest while
minimizing loss of biological function of such molecule. Secondly,
the terminal carbon atom of the valeric acid side chain of biotin
can be derivatized easily, thereby facilitating conjugation to
reactive moieties on a molecule of interest, particularly a
protein. Notably biotinylation does noch change the structure of
the heterocyclic moiety of biotin.
[0006] After biotinylation via the terminal carbon atom of the
valeric acid side chain the biotin moiety preserves the capability
to interact specifically with (strept)avidin, as the moiety of the
biotin molecule that is responsible for specific interaction with
the binding pocket of avidin-type proteins is the heterocyclic
structure represented by the ureido ring that is fused with the
tetrahydrothiophene ring is not affected.
[0007] The heterocyclic structure of biotin is also targeted by
monoclonal antibodies (mAbs) of the prior art against biotin. Kohen
F. et al. (Methods in Enzymology 279 (1997) 451-463) generated
monoclonal antibodies using as an immunogen biotinylated bovine
serum albumin (BSA conjugated with N-hydroxysuccinimidobiotin). The
document reports analysis of amino acid sequences of antigen
binding regions of antibodies capable of binding the biotin moiety
of a biotinylated protein. Sequence alignments were made with
homologous stretches of the polypeptide sequences of avidin and
streptavidin which were reported to interact with the bicyclic ring
system of biotin. Notably, similarities with the polypeptide
sequences of avidin and streptavidin were identified in the CDR2
and CDR3 of biotin-specific antibodies. The results were
interpreted in that in the amino acid sequences of biotin binding
pockets a common pattern is necessary for biotin binding.
[0008] Dakshinamurti, K et al. (Biochem. J. 237 (1986) 477-482)
report the generation and characterization of murine mAbs using as
an immunogen keyhole limpet hemocyanin (KLH) to which an activated
form of biotin (N-hydroxysuccinimidobiotin) was coupled. Several
hybridoma clones were obtained, and the respective mAbs were
effective in binding free biotin, hapten-conjugated biotin,
protein-conjugated biotin, and biocytin. Notably, biocytin is a
naturally occurring derivative of biotin, an amide formed from the
valeric acid carboxyl function and the amino acid L-lysine. The
fact that such derivatized biotin is bound by the mAbs of
Dakshinamurti, K et al. (supra) indicates a binding specificity
which targets the heterocyclic structure of biotin, i.e. the ureido
ring that is fused with the tetrahydrothiophene ring.
[0009] While the mAbs reported by Dakshinamurti, K et al. (supra)
do bind conjugated biotin and also free biotin, JP 2008-094721
discloses a mAb that specifically binds to protein-conjugated
biotin, but not to free biotin. Table 1 summarizes the discussed
properties of the prior art antibodies.
TABLE-US-00001 TABLE 1 Binding properties of monoclonal antibodies
raised against biotin binding to biotinylated binding to Report
target free biotin Dakshinamurti, K et al. (Biochem. J. 237 yes yes
(1986) 477-482) Kohen F. et al. (M. Enzymol. 279 (1997) yes not
451-463) determined JP 2008-094721 yes no no report, yet no yes
[0010] Similar to the report of Dakshinamurti, K et al. (supra), WO
00/50088 A2 deals with antibodies having comparable properties;
specifically, antibodies are reported which have an affinity for
conjugated biotin one to four orders of magnitude greater than the
respective affinity for free biotin. The document discloses
immunization with an antigen to which biotin is conjugated via the
carbon atom of the carboxyl function of the valeric acid moiety. In
a first screening step antibodies are identified which bind to the
conjugated biotin; a subsequent second screening step is disclosed
which aims at identifying clones secreting monoclonal antibodies
capable of binding to the conjugated biotin even in the presence of
a defined amount of free biotin. Notably, the document is silent
concerning monoclonal antibodies which on the one hand bind to free
biotin (biotin that is not covalently bound to another molecule and
which is in dissociated form in aqueous solution) but on the other
hand do not bind to the biotin moiety on a biotinylated molecule,
i.e. a biotin moiety which could also be bound by
(strept)avidin.
[0011] Indyk H. E. et al. International Dairy Journal 35 (2014)
25-31 report an optical biosensor assay for the detection of free
biotin in milk. A Biacore Q biosensor with a CM5 sensor chip was
used; on an amine-modified sensor surface biotin was immobilized by
covalent coupling of its NHS-activated valeric acid terminal
carboxylate group. Notably, the orientation of the biotin molecules
coupled to the sensor chip was the same as for biotin on
biotinylated molecules, i.e. corresponds to a biotin moiety which
could also be bound by (strept)avidin. Thus, primarily the
heterocyclic structure of biotin was exposed for antibody binding.
Binding properties of three different biotin-specific polyclonal
antibodies and two different monoclonal antibodies were
characterized using the biotin sensor chip, in the presence of
different concentrations of free biotin as competitor.
[0012] Notably, no disclosure has been found in the prior art, so
far, that describes a monoclonal antibody which specifically binds
to free biotin, but not to the conjugated biotin on a biotinylated
target molecule. Such an antibody would bind the biotin primarily
from its "tail", i.e. would importantly interact with the valeric
acid moiety of biotin. In particular, no antibody has been
described, so far, which on the one hand specifically binds to free
biotin, but on the other hand does not bind to conjugated biotin,
wherein conjugated biotin is attached to the target molecule via
the carbon atom of the carboxyl function of the valeric acid
moiety, whereby the heterocyclic "head" structure of biotin is
located distal from the target molecule. Further, no monoclonal
antibody is known, so far, which in aqueous solution is
characterized by an affinity for free biotin K.sub.s[free] that is
higher than the affinity of the same monoclonal antibody for
conjugated biotin K.sub.s[conj.], wherein the conjugated biotin is
conjugated via the carbon atom of the carboxyl function of the
valeric acid moiety (see above), and wherein K.sub.s[free] differs
from K.sub.s[conj.] by a factor of at least 50, 100, 500, 1,000,
5,000, 10,000, 50,000, or at least 100,000.
[0013] Johnson L. C. ("The synthesis of new biotin derivatives and
their bioactivity", Master of Science thesis dated December 2002,
submitted to the Graduate Faculty of the Louisiana State University
and Agricultural and Mechanical College (US) retrieved from the
internet on Feb. 16, 2017;
URL:http://etd.lsu.edu/docs/available/etd-0927102-135929/unrestricted/Joh-
nson_thesis .pdf) describes chemical reactions of derivatization of
the heterocyclic "head" structure of biotin. Embodiments are
disclosed wherein the 1'-N atom comprised in the heterocyclic
structure of biotin is targeted by derivatization.
[0014] Wu F.-B. et al. Clinica Chimica Acta 308 (2001) 117-126
disclose a method to counteract matrix interference in a
competitive immunoassay. An initial assay setup consisting of an
immobilized second antibody bound to a target (serum thyroxin)
specific antibody was replaced. The replacement was provided by
firstly immobilizing biotinylated bovine serum albumin (BSA),
followed by binding streptavidin to the biotinylated BSA, followed
by binding the target-specific antibody to the streptavidin,
wherein prior to this step the target-specific antibody was
biotinylated. It was found that such a setup provided increased
binding capacity for the target of the assay (serum thyroxin) and
thereby increased resistance to matrix interference.
[0015] The present invention provides chemical structures of
Formula I (see below) which specifically present the valeric acid
"tail" moiety of a derivatized diaminobiotin as the distal (most
terminal) part of the respective structure. By way of derivatizing
the biotin analog diaminobiotin, and by way of attaching its "head"
moiety to the rest of the structure of Formula I, the valeric acid
moiety is primarily exposed and presented for physical interaction,
including interaction with immune cell receptors and antibodies. It
was subject of the present investigation to find out whether an
antibody interacting with the derivatized biotin according to
Formula I would have binding properties that could target the
valeric acid "tail" moiety. In the present study it was further
tested whether the monoclonal antibodies that can be elicited using
a structure of Formula I, and which are capable of reacting with
such a structure would also have the properties of a desired
antibody according to the invention.
[0016] Accordingly, a desired antibody of the invention is a
monoclonal antibody which specifically binds biotin, wherein the
biotin is not covalently bound to another molecule. Also in line
with the above reasoning, a desired antibody of the invention is a
monoclonal antibody which specifically binds biotin, wherein the
biotin is in dissociated form in aqueous solution. At the same
time, a desired antibody of the invention does not bind to
conjugated biotin on a biotinylated target molecule, wherein the
biotin is attached to the target molecule via the carboxy group of
the valeric acid moiety. In such a biotinylated target molecule,
the conjugated form of the "head" structure of the biotin is
presented and can be bound, e.g. by a (strept)avidin.
[0017] Thus the inventors set out to find out whether antibodies
could be made and isolated to specifically bind unconjugated (free)
biotin. Such binding to biotin would have a completely different
structural basis compared to the already described antibodies of
earlier reports which bind to the "head" portion of biotin.
[0018] For the purpose of this disclosure, it was an objective of
the present invention to provide a monoclonal antibody specifically
binding the compound of Formula I,
##STR00001##
characterized in that it also binds to biotin, wherein X is
selected from the group consisting of (CH.sub.2).sub.n with n being
an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being selected from (i) a hapten which does not contain a biotin
moiety and (ii) a polypeptide, and L being a linker connecting X
and M, and wherein B is
##STR00002##
with M being selected from (i) a hapten which does not contain a
biotin moiety and (ii) a polypeptide, and L being a linker, whereby
the nitrogen atom marked with an asterisk is covalently bound to an
adjacent CH.sub.2 group of X.
[0019] It was a further objective to provide a monoclonal antibody
binding to a molecule of Formula I and to biotin, but not to
conjugated biotin which is attached to a macromolecule via the
carbon atom of the carboxyl function of the valeric acid moiety. As
a further objective the antibody of the present invention is
binding to biotin comprising a chemically unmodified valeric acid
moiety. It was hence an objective to isolate a monoclonal antibody
with an affinity for conjugated biotin on a biotinylated target
molecule which is lower than the affinity for free biotin by a
factor selected from the group consisting of at least 50, 100, 500,
1,000, 5,000, 10,000, or higher. In other words, an objective was
to isolate a monoclonal antibody with an affinity for free biotin
which is higher than the affinity for conjugated biotin on a
biotinylated target molecule by a factor selected from the group
consisting of at least 50, 100, 500, 1,000, 5,000, and at least
10,000.
[0020] It has now surprisingly been found that such a monoclonal
antibody can be generated and isolated.
[0021] Recently, high dosage biotin supplementation has become
"fashionable". Biotin is believed to be a key contributor to
keratin, and high dose biotin thus could improve quality and
quantity of hair, nails and skin. Biotin is water-soluble and
excreted rapidly. However, if high dose biotin supplementation is
taken, rather high levels of biotin in the circulation may be
present and the biotin in the circulation will also be present in a
sample used for in vitro anylysis for measurement of an analyte,
i.e. in a sample like serum or plasma. Biotin comprised in a
sample, if present at high levels might interfere in an assay for
measurement of an analyte, which is employing a (strept)avidin
coated solid phase and a biotinylated specific binding agent.
[0022] Therefore, with the increased use of high dose biotin
supplements, an increasing need exists to reduce the potential
interference by abnormally high biotin levels in a sample with the
measurement of an analyte from the same sample in assays which are
based on the (strept)avidin-biotin binding pair.
[0023] It was a further task to investigate whether the antibodies
as disclosed herein can be used to reduce the potential
interference of biotin. It has been surprisingly found that a
monoclonal antibody capable of binding biotin but not binding the
biotin moiety on a biotinylated target molecule is particularly
useful to counteract a potential interference caused by abnormally
high levels of biotin in a sample in a method for measuring an
analyte in such sample, in an assay wherein a (strept)avidin/biotin
pair is used to bind a biotinylated analyte specific binding agent
to a (strept)avidin coated solid phase.
SUMMARY OF THE INVENTION
[0024] Herein is reported a monoclonal antibody specifically
binding the compound of Formula I,
##STR00003##
characterized in that it also binds to biotin, wherein X is
selected from the group consisting of (CH.sub.2).sub.n with n being
an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being selected from (i) a hapten which does not contain a biotin
moiety and (ii) a polypeptide, and L being a linker connecting X
and M, and wherein B is
##STR00004##
with M being selected from (i) a hapten which does not contain a
biotin moiety and (ii) a polypeptide, and L being a linker, whereby
the nitrogen atom marked with an asterisk is covalently bound to an
adjacent CH.sub.2 group of X.
[0025] Further reported is a method for measuring an analyte in a
sample, wherein a (strept)avidin/biotin binding pair is used to
bind a biotinylated analyte specific binding agent to a
(strept)avidin coated solid phase, the method comprising adding to
the sample (a) an antibody as reported herein, (b) a biotinylated
analyte specific binding agent, (c) a (strept)avidin coated solid
phase, followed by measuring the analyte bound to the solid phase
via (strept)avidin and biotinylated analyte specific binding
agent.
[0026] Further reported is a method for measuring an analyte in a
sample, wherein a (strept)avidin/biotin binding pair is used to
bind a biotinylated analyte specific binding agent to a label, the
method comprising adding to the sample (a) an antibody as reported
herein, (b) a biotinylated analyte specific binding agent, (c) a
(strept)avidin bound to a label, followed by separating the complex
comprising the analyte, the biotinylated analyte specific binding
agent and the labeled streptavidin, and determining the amount of
label bound to the analyte.
[0027] Further reported is the use of an antibody as reported
herein, in a method for measuring an analyte in a sample, wherein a
(strept)avidin/biotin pair is used to bind a biotinylated analyte
specific binding agent to a (strept)avidin coated solid phase phase
or to a labeled (strept)avidin.
[0028] Further reported is an immunoassay test kit comprising at
least (a) an antibody as disclosed herein, (b) a biotinylated
analyte specific binding agent, and (c) a (strept)avidin coated
solid phase or a labeled (strept)avidin.
[0029] Further reported is an immunogen according to of Formula
I
##STR00005##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being a polypeptide, and L being a linker connecting X and M, and
wherein B is
##STR00006##
with M being a polypeptide, and L being a linker, whereby the
nitrogen atom marked with an asterisk is covalently bound to an
adjacent CH.sub.2 group of X.
[0030] Further reported is a method for producing an antibody as
disclosed herein, the method comprising the steps of (a) immunizing
an experimental animal with an immunogen as disclosed herein,
thereby inducing B-cells producing antibodies binding to the
immunogen, (b) obtaining a monoclonal antibody binding to the
immunogen produced by the B-cell of step (a), either via hybridoma
technology or by B-cell PCR technology, (c) further selecting the
antibody of step (b) for binding to biotin, thereby obtaining an
antibody as disclosed herein.
[0031] Further reported is a method for producing an antibody as
disclosed herein, the method comprising the steps of a) immunizing
an experimental animal with an immunogen as disclosed herein,
thereby inducing B-cells producing antibodies binding to the
immunogen, b) obtaining a monoclonal antibody binding to the
immunogen produced by the B-cell of step (a), either via hybridoma
technology or by B-cell PCR technology, c) selecting the antibody
of step (b) for binding to biotin, and d) selecting those
antibodies which do not bind to the compound of Formula II,
##STR00007##
thereby obtaining an antibody as disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1A D(+)-biotin
[0033] FIG. 1B D(+)-biotin
[0034] FIG. 2A Synthesis schemes related to Examples 1 and 2
[0035] FIG. 2B Synthesis schemes related to Examples 1 and 2
[0036] FIG. 2C Synthesis schemes related to Examples 1 and 2
[0037] FIG. 3A Compound of Formula II
[0038] FIG. 3B Compound of Formula III B;
[0039] FIG. 3C Compound of Formula III C
[0040] FIG. 4 Exemplary kinetic signatures from the antibody
kinetic screening assay. Dotted line: SPR binding signal of the
Dig-Biotin-conjugate-M-D.G-Fab' complex injection. Solid line: SPR
binding signal of the Dig-Biotin-conjugate-M-D.G-Fab' complex
supplemented with 300 nM d-biotin. Typically, three classes of free
d-biotin blocking kinetics were observed. A: complete d-biotin
competition. B: Intermediate d-biotin blocking. C: No signal
interference by d-biotin. Suitable antibody candidates were
selected for from the classes A and B for subsequent detailed
investigations.
[0041] FIG. 5 Exemplary SPR Sensorgram overlay plot of the lead
antibody candidate clone L SPR IC50 measurement. 270 nM
Dig-Biotin-conjugate was mixed with free d-biotin concentrations at
270 nM (not shown), 90 nM, 30 nM (twice), 10 nM, 3.3 nM and 1 nM.
The mixture containing 90 nM free d-biotin concentration produced
the lowest response signal and with 1 nM free d-biotin produced the
highest response signal (labelled). X indicates the positions of
the report points, which were used for the IC50 calculation.
[0042] FIG. 6A Kinetic Screening Assay experimental SPR setup. At
first, the preformed Dig-Biotin-conjugate-M-D.G-Fab' was used as
analyte in solution to monitor the binding for the molecular weight
enhanced, conjugated d-biotin. Secondly, the d-biotin was added to
the analyte mixture in order to compete with the conjugate
binding.
[0043] FIG. 6B SPR assay experimental setup. The
Dig-Biotin-conjugate interaction was measured in presence and
absence of a d-biotin concentration series. In absence of d-biotin,
concentration dependent series of the Dig-biotin-conjugate were
used to determine the rabbit antibody kinetics versus the
Dig-biotin-conjugate. IC50 measurements were performed with a
constant concentration of the Dig-biotin-conjugate and increasing
concentrations of free d-biotin in the analyte sample mixture.
[0044] FIG. 6C Alternative SPR assay experimental setup. The
Dig-Biotin-conjugate interaction was measured in presence and
absence of a d-biotin concentration series. In absence of d-biotin,
concentration dependent series of the Dig-biotin-conjugate were
used to determine the rabbit antibody kinetics versus the
Dig-biotin-conjugate. In this case the Dig-Biotin-conjugate was
captured by the surface displayed M-D.G antibody, whereas the
rabbit antibody or fragments thereof were used as concentration
dependent analyte in solution. IC50 measurements were performed
with a constant concentration of the rabbit antibody and increasing
concentrations of free d-biotin in the analyte sample mixture.
DETAILED DESCRIPTION OF THE INVENTION
[0045] In one embodiment the present invention relates to a
monoclonal antibody specifically binding the compound of Formula
I,
##STR00008##
characterized in that it also binds to biotin, wherein X is
selected from the group consisting of (CH.sub.2).sub.n with n being
an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, [0046] and R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being selected from (i) a hapten which does not contain a biotin
moiety and (ii) a polypeptide, and L being a linker connecting X
and M and wherein B is
##STR00009##
[0046] with M being selected from (i) a hapten which does not
contain a biotin moiety and (ii) a polypeptide, and L being a
linker, whereby the nitrogen atom marked with an asterisk is
covalently bound to an adjacent CH.sub.2 group of X.
[0047] Surprsingly it has been found that a monoclonal antibody
binding to both, the structure of Formula I (which is described in
more detail further below) and biotin can be reliably generated
based on the materials and methods disclosed herein which also are
illustrated in more detail further below.
[0048] For the purpose of the present disclosure, in all aspects
and embodiments mentioned herein, the term "(strept)avidin" and
avidin-type protein can be used interchangeably. An avidin-type
protein is generally understood as a protein with at least one
binding pocket capable of binding specifically to the heterocyclic
structure of biotin that is represented by the ureido ring that is
fused with the tetrahydrothiophene ring. By virtue of this
property, an avidin-type protein is capable of binding to a
biotinylated target molecule, wherein biotin is covalently bound to
the molecule via the carbon atom of the carboxyl function of the
valeric acid side chain of biotin. Several embodiments of
avidin-type proteins are known to the art. More specifically, an
avidin-type protein can be selected from the group including
avidin, neutravidin, streptavidin, bradavidin, traptavidin, a
biotin-binding variant thereof, a mixture thereof, a monomer,
dimer, trimer, tetramer or multimer thereof, a conjugated form
thereof and an antibody binding to a conventionally biotinylated
molecule of interest. It is known that in their naturally occurring
forms a number of avidin-type proteins (especially those which are
not antibodies), specifically avidin and streptavidin, are
homotetramers; i.e. they consist of four identical subunits. In an
embodiment of a variant of a monomeric avidin-type protein, the
naturally occurring form may be a di-tri-, or tetra-oligomer with
each monomer having a biotin binding pocket. In an embodiment the
avidin-type protein is selected from a monomer, a homodimer, a
homotrimer, and a homotetramer.
[0049] Also more specifically, an avidin-type protein can be an
antibody with an antigen binding pocket capable of binding
specifically to the heterocyclic structure of biotin that is
represented by the ureido ring that is fused with the
tetrahydrothiophene ring. Examples of antibodies with this property
are known in the prior art and cited above. In an even more
specific embodiment, an avidin-type protein can be identified if it
specifically binds to the biotin moiety of the structure in FIG. 3A
(Formula II).
[0050] While an avidin-type protein may be capable of binding free
biotin, an avidin-type protein importantly is not capable of
specifically binding to the structure in FIG. 2C (Formula III A)
depicting an immunogen suitable for generating monoclonal
antibodies of the invention. More specifically, an avidin-type
protein does not specifically bind to the structure in FIG. 3C
(Formula III C) depicting a hapten-coupled screening agent useful
in identifying monoclonal antibodies of the invention. Even more
specifically, an avidin-type protein does not specifically bind to
the structure in FIG. 3B (Formula III B) depicting another
hapten-coupled screening agent useful in identifying monoclonal
antibodies that specifically bind to the "tail" aspect of
biotin.
[0051] In one embodiment the (strept)avidin according to the
present disclosure is selected from the group including avidin,
neutravidin, streptavidin, bradavidin, traptavidin, a
biotin-binding variant thereof, and a mixture thereof.
[0052] When referring to "(strept)avidin" or an avidin-type protein
in the present disclosure, it is understood that these terms
equally incorporate any variant thereof with the proviso that the
variant is capable of binding biotin non-covalently with at least
one binding pocket capable of binding specifically to the
heterocyclic structure of biotin that is represented by the ureido
ring that is fused with the tetrahydrothiophene ring. In this
respect, a variant is a "functionally equivalent polypetide" in
that the amino acids forming the at least one binding pocket bear
similar electrostatic and sterochemical attributes of the amino
acid sequence of the original avidin-type protein under
consideration, wherein the variant comprises one or more
conservative amino acid substitutions, analog amino acids
substitutions and/or deletions and/or additions of amino acids that
do not significantly affect or alter the function of the amino
acids of the binding pocket. "Functionally equivalent" also
includes a homologous amino acid sequence with regards to the
respective referenced amino acid sequence.
[0053] "Conservative substitutions" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, "conservatively substituted" refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill in the
art will recognize that each codon in a nucleic acid (except AUG,
which is ordinarily the only codon for methionine, and TGG, which
is ordinarily the only codon for tryptophan) can be modified to
yield a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence.
[0054] As to amino acid sequences, one of ordinary skill in the art
will recognize that individual substitutions in a peptide,
polypeptide, or protein sequence which alter a single amino acid or
a small percentage of amino acids in the amino acid sequence is a
"conservative substitution" where the alteration results in the
substitution of an amino acid with a chemically similar amino
acid.
[0055] Conservative substitution tables providing functionally
similar amino acids are known to those of ordinary skill in the
art. Conservative substitution tables providing functionally
similar amino acids are known to those of ordinary skill in the
art. The following eight groups each contain amino acids that are
conservative substitutions for one another.
[0056] The term "conservative amino acid substitutions" refers to
all substitutions wherein the substituted amino acid has similar
structural or chemical properties with the corresponding amino acid
in the reference sequence. By way of example, conservative amino
acid substitutions involve substitution of one aliphatic or
hydrophobic amino acids, e.g., alanine, valine, leucine,
isoleucine, methionine, phenylalanine, or tryptophan with another;
substitution of one hydroxyl-containing amino acid, e.g., serine
and threonine, with another; substitution of one acidic residue,
e.g., glutamic acid or aspartic acid, with another; replacement of
one amide-containing residue, e.g., asparagine and glutamine, with
another; replacement of one aromatic residue, e.g., phenylalanine
and tyrosine, with another; replacement of one basic residue, e.g.,
lysine, arginine and histidine, with another; and replacement of
one small amino acid, e.g., alanine, serine, threonine, and
glycine, with another.
[0057] As used herein "deletions" and "additions" in reference to
amino acid sequence, means deletion or addition of one or more
amino acids to the amino terminus, the carboxy-terminus, the
interior of the amino acid sequence or a combination thereof, for
example the addition can be to one of the antibodies subject of the
present application.
[0058] As used herein, "homologous sequences" have amino acid
sequences which are at least 70%, at least 80%, at least 90%, at
least 95%, or at least 99% homologous to the corresponding
reference sequences. Sequences which are at least 90% identical
have no more than 1 alteration, i.e., any combination of deletions,
additions or substitutions, per 10 amino acids of the reference
sequence. Percent homology is determined by comparing the amino
acid sequence of the variant with the reference sequence using, for
example, MEGALIGN.TM. project in the DNA STAR.TM. program.
[0059] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. Sequences are
"substantially identical" if they have a percentage of amino acid
residues or nucleotides that are the same (i.e., about 60%
identity, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, or about 95% identity over a specified region), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms (or other algorithms available to
persons of ordinary skill in the art) or by manual alignment and
visual inspection. This definition also refers to the complement of
a test sequence. The identity can exist over a region that is at
least about 50 amino acids or nucleotides in length, or over a
region that is 75-100 amino acids or nucleotides in length, or,
where not specified, across the entire sequence of a polynucleotide
or polypeptide. A polynucleotide encoding a polypeptide of the
present disclosure, including homologs from species other than
human, may be obtained by a process comprising the steps of
screening a library under stringent hybridization conditions with a
labeled probe having a polynucleotide sequence of the present
disclosure or a fragment thereof, and isolating full-length cDNA
and genomic clones containing said polynucleotide sequence. Such
hybridization techniques are well known to the skilled artisan.
[0060] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0061] For the purpose of the present disclosure it is understood
that the terms "biotin" or "free biotin" are used interchangeably
and denote the naturally occurring compound, i.e. D(+)-biotin.
[0062] Biotin (D(+)-biotin; C.sub.10H.sub.16N.sub.2O.sub.3S;
MW=244.31 g/mol; IUPAC name:
5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]p-
entanoic acid), CAS Registry Number 58-85-5 comprises a ureido ring
fused with a tetrahydrothiophene ring, and a valeric acid
substituent which is attached to one of the carbon atoms of the
tetrahydrothiophene ring. The basic structure of biotin is known
since long and was reported e.g. by Melville D. B. et al. (J. Biol.
Chem. 146 (1942) 487-492). Biotin has three contiguous chiral
carbon atoms and therefore, four diastereomeric racemic forms are
possible. Of the diastereomeric racemic forms, only D(+)-biotin
occurs in nature whereas other isomers are of synthetic origin. The
biologically active form is the (3aS,4S,6aR) configuration shown in
FIG. 1A and FIG. 1B.
[0063] According to Marquet A. (Pure & Appl. Chem. 49 (1977)
183-196), in the crystal structure of D(+)-biotin the ureido ring
is planar while the thiophane ring has an envelope conformation, as
shown in FIG. 1B. The valeric acid side chain is not fully extended
but twisted, and there is interaction between the C.sub.6 atom of
the side chain and the N'.sub.3 atom of the ureido ring; reportedly
this interaction has an impact on the reactivity of biotin. The
envelope conformation of the thiophane ring was also reported in
solution, as shown by NMR studies reported by Glasel J. A.
(Biochemistry 5 (1966) 1851-1855) and by Lett R. & Marquet
A./Tetrahedron 30 (1974) 3365-3377).
[0064] The term "biotin moiety" is used to refer to the
biotin-related part or biotin-derived part of a molecule as e.g.
obtained by any kind of biotinylation or chemical coupling.
[0065] The attachment of biotin to an appropriate chemical group on
a molecule of interest via the carbon atom of the carboxyl function
of the valeric acid side chain is referred to as "biotinylation" or
"conventional biotinylation". Accordingly, the biotin residue of a
"biotinylated" molecule of interest has an outward-facing ring
structure (i.e. the ureido ring that is fused with a
tetrahydrothiophene ring), whereas the linear portion of the biotin
residue is inward-facing, towards the surface of the biotinylated
molecule. The outward-facing ring structure can be bound by an
avidin-type protein. Thus, importantly the heterocyclic structure
of biotin needs to be exposed for specific binding by an
avidin-type protein. Interaction of diaminobiotin with streptavidin
was reported in Torregiani A. & Fini G. Biospectroscopy 4
(1998) 197-208.
[0066] The term "(strept)avidin/biotin binding pair" is perfectly
known to the person skilled in the art. It points to the fact that
biotin (including the biotin moiety of a biotinylated molecule) on
the one hand and (strept)avidin on the other hand represent the two
members of this binding pair. As described above, this binding pair
is outstanding in having one of the highest binding affinities
known for non-covalent interactions.
[0067] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an antibody" means one antibody
or more than one antibody.
[0068] The term "antibody" encompasses the various forms of
antibody structures including, but not being limited to, whole
antibodies and antibody fragments. The antibody according to the
invention is preferably a goat, sheep, mouse, rabbit, or rat
antibody, a chimeric antibody, or further genetically engineered
antibody as long as the characteristic properties according to the
invention are retained.
[0069] "Antibody fragments" comprise a portion of a full length
antibody, preferably the variable domain thereof, or at least the
antigen binding site thereof. Examples of antibody fragments
include diabodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments. scFv
antibodies are, e.g., described in Huston, J. S., Methods in
Enzymol. 203 (1991) 46-88. In addition, antibody fragments comprise
single chain polypeptides having the characteristics of a V.sub.H
domain, namely being able to assemble together with a V.sub.L
domain, or of a V.sub.L domain binding to IGF-1, namely being able
to assemble together with a V.sub.H domain to a functional antigen
binding site and thereby providing the properties of an antibody
according to the invention.
[0070] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0071] The term "specific binding agent" is used to indicate that
an agent is used which is able to either specifically bind to or to
be specifically bound by an analyte of interest. Many different
assay set-ups for immunoassays are known in the art. Dependent on
the specific assay set-up, various biotinylated specific binding
agents can be used. In one embodiment the biotinylated specific
binding agent is selected from the group consisting of a
biotinylated analyte-specific binding agent, a biotinylated analyte
bound to solid phase, and a biotinylated antigen bound to solid
phase.
[0072] The term "analyte-specific binding agent" refers to a
molecule specifically binding to the analyte of interest. An
analyte-specific binding agent in the sense of the present
disclosure typically comprises binding or capture molecules capable
of binding to an analyte (other terms analyte of interest; target
molecule). In one embodiment the analyte-specific binding agent has
at least an affinity of 10.sup.7 l/mol for its corresponding target
molecule, i.e. the analyte. The analyte-specific binding agent in
other embodiments has an affinity of 10.sup.8 l/mol or even of
10.sup.9 l/mol for its target molecule. As the skilled artisan will
appreciate the term specific is used to indicate that other
biomolecules present in the sample do not significantly bind to the
binding agent specific for the analyte. In some embodiments, the
level of binding to a biomolecule other than the target molecule
results in a binding affinity which is only 10%, more preferably
only 5% of the affinity of the target molecule or less. In one
embodiment no binding affinity to other molecules than to the
analyte is measurable. In one embodiment the analyte-specific
binding agent will fulfill both the above minimum criteria for
affinity as well as for specificity.
[0073] The term "analyte-specific binding" as used in the context
of an antibody refers to the immunospecific interaction of the
antibody with its target epitope on the analyte, i.e. the binding
of the antibody to the epitope on the analyte. The concept of
analyte-specific binding of an antibody via its epitope on an
analyte is fully clear to the person skilled in the art.
[0074] The terms "polypeptide," "peptide" and "protein" refer to a
polymer of amino acid residues. The terms apply to naturally
occurring amino acid polymers as well as amino acid polymers in
which one or more amino acid residues are a non-naturally encoded
amino acid. As used herein, the terms encompass amino acid chains,
wherein the amino acid residues are linked by covalent peptide
bonds. The polypeptides, peptides and proteins are written using
standard sequence notation, with the nitrogen terminus being on the
left and the carboxy terminus on the right. Standard single letter
notations have been used as follows: A--alanine, C--cysteine,
D--aspartic acid, E--glutamic acid, F--phenylalanine, G--glycine,
H--histidine, S--Isoleucine, K--lysine, L--leucine, M--methionine,
N--asparagine, P--proline, Q--glutamine, R--arginine, S--serine,
T--threonine, V--valine, W--tryptophan, Y--tyrosine. The term
"peptide" as used herein refers to a polymer of amino acids that
has a length of up to 5 amino acids. The term "polypeptide" as used
herein refers to a polymer of amino acids that has a length of 6 or
more amino acids. The term "protein" either signifies a polypeptide
chain or a polypeptide chain with further modifications such as
glycosylation, phosphorylation, acetylation or other
post-translational modifications
[0075] "Haptens" are small molecules (e.g. pesticides, fungicides,
drugs, hormones, toxins, synthetic peptides, etc.) which do not
directly induce an immune response such as formation of antibodies.
Techniques have been established to raise antibodies against
haptens by conjugating them with immunogenic carriers, such as
antigenic macromolecules. For the purpose of the present
disclosure, a hapten is understood as being a low molecular weight
molecule, specifically having a molecular weight of 10,000 Da or
less, which does not elicit immune response until and unless
conjugated with an immunogenic carrier, such as protein. Once the
antibody is formed, it can bind to the hapten. Antibodies thus
generated are useful in many fields, specifically in the
development of immunodiagnostic kits or biosensors. Thus, the term
"hapten" denotes a small molecule of 10,000 Da or less that can
elicit an immune response only when attached to an immunogenic
carrier such as a polypeptide of at least 30 amino acids. In this
sense, and in an embodiment, a hapten is an incomplete antigen that
cannot, by itself, promote antibody formation but that can do so
when conjugated to a protein of at least 30 amino acids. Exemplary
haptens are aniline, o-, m-, and p-aminobenzoic acid, quinone,
histamine-succinyl-glycine (HSG), hydralazine, halothane,
indium-DTPA, fluorescein, digoxigenin, theophylline,
bromodeoxyuridine, steroid compounds and dinitrophenol. In a
specific embodiment, the hapten is not biotin and does not contain
a biotin moiety. In one specific embodiment the hapten is
digoxigenin or theophylline or fluorescein or bromodeoxyuridine. A
hapten in the context of the disclosure of the compound according
to Formula I, a hapten as a moiety of the compound is understood as
a which is covalently coupled to the remaining portion of the
compound, wherein the hapten moiety (i.e. the chemical structure of
10,000 Da or less) is capable of eliciting an immune response only
when attached to an immunogenic carrier such as a polypeptide of at
least 30 amino acids.
[0076] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
some embodiments, an antibody is purified to greater than 95% by
weight of antibody, and in some embodiments, to greater than 99% as
determined by SDS-PAGE under reducing or nonreducing conditions
using, for example, Coomassie blue or silver stain.
[0077] Antibodies of the immunoglobulin G class usually are
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light-chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light-chain and heavy-chain variable
domains.
[0078] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "VH." The variable domain of the light chain may be
referred to as "VL." These domains are generally the most variable
parts of an antibody and contain the antigen-binding sites.
[0079] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions (HVRs) both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework regions (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a beta-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0080] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (k), based on the amino
acid sequences of their constant domains.
[0081] The antibodies used in a method according to the present
invention may be from any animal origin. In one embodiment the
antibodies are human, murine (e. g., mouse and rat), donkey,
monkey, rabbit, goat, guinea pig, camel, horse, or chicken
antibodies.
[0082] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into subclasses (isotypes), e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known and described generally in, for
example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W. B.
Saunders, Co., 2000). An antibody may be part of a larger fusion
molecule, formed by covalent or non-covalent association of the
antibody with one or more other proteins or peptides.
[0083] The terms "full-length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0084] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; single-chain antibody molecules; scFv, sc(Fv).sub.2;
diabodies; and multispecific antibodies formed from antibody
fragments.
[0085] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab') 2 fragment that has two antigen-combining sites
and is still capable of cross-linking antigen.
[0086] The Fab fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody-hinge region.
[0087] Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0088] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species
(sc(Fv)2). It is in this configuration that the three HVRs of each
variable domain interact to define an antigen-binding site on the
surface of the VH-VL dimer. Collectively, the six HVRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three HVRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0089] The present disclosure includes monovalent Fab fragments and
single chain Fv that are derived from monoclonal antibodies capable
of specifically binding free biotin as disclosed in here. Compared
with naturally occurring antibody forms the monovalent species can
diffuse faster in aqueous solution, owing to their smaller
molecular weight. Another aspect is that under suitable conditions
particularly scFv antibodies can be recombinantly produced in
prokaryotic expression systems.
[0090] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404097; WO 1993/01161; Hudson et
al., Nat. Med. 9:129-134 (2003); and Holliger et al., PNAS USA 90:
6444-6448 (1993). Triabodies and tetrabodies are also described in
Hudson et al., Nat. Med. 9:129-134 (2003).
[0091] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target-binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal-antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal-antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0092] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein., Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2.sup.nd ed. 1988); Haemmerling et al., in:
Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier,
N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567), phage-display technologies (see, e.g., Clackson et al.,
Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222:
581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004);
Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, PNAS
USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132(2004), and technologies for producing
human or human-like antibodies in animals that have parts or all of
the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., PNAS USA 90: 2551
(1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann
et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et
al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368:
856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et
al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature
Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.
Immunol. 13: 65-93 (1995).
[0093] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(e.g., U.S. Pat. No. 4,816,567 and Morrison et al., PNAS USA
81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED.RTM.
antibodies wherein the antigen-binding region of the antibody is
derived from an antibody produced by, e.g., immunizing macaque
monkeys with the antigen of interest.
[0094] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody-variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.
Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993) and Sheriff et al., Nature Struct. Biol. 3:733-736
(1996).
[0095] A number of HVR delineations are in use and are encompassed
herein. The HVRs that are Kabat complementarity-determining regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The AbM HVRs represent a compromise between
the Kabat CDRs and Chothia structural loops, and are used by Oxford
Molecular's AbM antibody-modeling software. The "contact" HVRs are
based on an analysis of the available complex crystal structures.
The residues from each of these HVRs are noted below.
TABLE-US-00002 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0096] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and
26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable-domain residues are numbered according to
Kabat et al., supra, for each of these extended-HVR
definitions.
[0097] The expression "variable-domain residue-numbering as in
Kabat" or "amino-acid-position numbering as in Kabat," and
variations thereof, refers to the numbering system used for
heavy-chain variable domains or light-chain variable domains of the
compilation of antibodies in Kabat et al., supra. Using this
numbering system, the actual linear amino acid sequence may contain
fewer or additional amino acids corresponding to a shortening of,
or insertion into, a FR or HVR of the variable domain. For example,
a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy-chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0098] The term "experimental animal" denotes a non-human animal.
In one embodiment the experimental animal is selected from rat,
mouse, hamster, rabbit, camel, llama, non-human primates, sheep,
dog, cow, chicken, amphibians, sharks and reptiles. In one
embodiment the experimental animal is a rabbit.
[0099] The present disclosure is based on derivatized diaminobiotin
(CAS Registry Number 22342-46-7), synonymous with
(2S,3S,4R)-cis-5-(3,4-Diaminotetrahydro-2-thienyl)valeric acid,
cis-3,4-Diamino-2-tetrahydrothiophenevaleric acid. Diaminobiotin as
reported herein is derivatized at the two nitrogen atoms
corresponding to the N'1 and N'3 position the heterocyclic ring
structure of biotin. Particularly owing to the larger substituent
at the N'1 atom, but also because of the smaller substituent at the
N'3 nitrogen atom, any molecule according to Formula I as reported
herein is incompatible with a binding pocket of (strept)avidin. The
essential tight interaction that are known to the art for the
heterocyclic structure of biotin cannot take place in the case of
the dibiotin derivatives, for several reasons. These include that
the ureido ring is not present as in biotin, and that both amino
groups of the diaminobiotin carry substituents thereby sterically
hindering access of derivatized diaminobiotin according to Formula
I to biotin binding pockets of avidin-type proteins. It is
hypothesized that this structure also prevents the formation of
antibodies binding to a conventionally biotinylated molecule of
interest once a compound according to Formula I is used as an
immunogen. At the same time, the structure still preservers the
"tail" aspect of biotin, and it was hypothesized that comprised in
an immunogen the derivatized diaminobiotin might be suited to
generate desired monoclonal antibodies against biotin.
[0100] In fact, in one embodiment of all aspects disclosed herein
the monoclonal antibody according to the invention does not bind a
conventionally biotinylated molecule, i.e. a molecule conjugated
with biotin, wherein the carbon atom of the carboxyl function of
the valeric acid side chain of the biotin moiety is covalently
coupled to the molecule. In a specific embodiment, the antibody
according to the invention does not bind to a compound of Formula
II, depicted in FIG. 3A (Formula II).
[0101] Rather, the monoclonal antibody according to the invention
binds to a compound of Formula I, wherein X is selected from the
group consisting of (CH.sub.2).sub.n with n being an integer from 1
to 20, [(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2
or 3, with m being 2 or 3, with k being an integer from 1 to 30,
and [(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being
an integer from 1 to 5, with t being an integer from 0 to 5, and
with s being an integer from 1 to 5, and wherein R is selected from
the group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z comprises a hapten. In a specific
embodiment, the monoclonal antibody according to the invention
specifically binds to a compound selected from the compound
depicted in FIG. 2C (Formula III A), and the compound depicted in
FIG. 3C (Formula III C).
[0102] In a more specific embodiment, the binding affinity of the
monoclonal antibody to a compound selected from the compound
depicted in FIG. 2C (Formula III A) and the compound depicted in
FIG. 3C (Formula III C) is higher by a factor of at least 50 than
the binding affinity to the compound of Formula II depicted in FIG.
3A (Formula II). In yet another more specific embodiment, the
binding affinity of the monoclonal antibody to a compound selected
from the compound depicted in FIG. 2C (Formula III A) and the
compound depicted in FIG. 3C (Formula III C) is higher by a factor
of at least 500, at least 1,000, at least 5,000, at least 10,000,
at least 50,000, and at least 100,000 than the binding affinity to
the compound of Formula II depicted in FIG. 3A (Formula II).
[0103] Thus, in another specific embodiment of all aspects as
disclosed herein, the affinity of the monoclonal antibody for
conjugated biotin on a biotinylated target molecule, including but
not limited to the exemplary compound of Formula II depicted in
FIG. 3A, is lower than the affinity for free biotin by a factor
selected from the group consisting of at least 50, 100, 500, 1,000,
5,000, and at least 10,000. In other words, the monoclonal antibody
according to the invention has an affinity for unconjugated (free)
biotin which is higher than the affinity for conjugated biotin on a
biotinylated target molecule by a factor selected from the group
consisting of at least 50, 100, 500, 1,000, 5,000, and at least
10,000.
[0104] Surprisingly, monoclonal antibodies according to the
invention also bind to a compound of Formula III B depicted in FIG.
3B. As a key feature here, the N'1 atom of the heterocyclic moiety
of biotin is modified and carries a substituent. Thus, there is a
cross-reactivity of these monoclonal antibodies in that an antibody
of this group is capable of specifically binding (i) to biotin that
is substituted at the N'1 atom of the ureido ring (see FIG.
1A).
[0105] In a more specific embodiment, the binding affinity of the
monoclonal antibody to a compound of Formula III B depicted in FIG.
3B is higher by a factor of at least 50 than the binding affinity
to the compound of Formula II. In yet another more specific
embodiment, the binding affinity of the monoclonal antibody to the
compound of Formula III B is higher by a factor of at least 500, at
least 1,000, at least 5,000, at least 10,000, at least 50,000, and
at least 100,000 than the binding affinity to the compound of
Formula II depicted in FIG. 3A.
[0106] Numerous methods and systems have been developed for the
detection and quantitation of analytes of interest in biochemical
and biological samples. Methods and systems which are capable of
measuring trace amounts of microorganisms, pharmaceuticals,
hormones, viruses, antibodies, nucleic acids and other proteins are
of great value to researchers and clinicians.
[0107] Many assay methods make use of an analyte-specific binding
agent to capture a specific target molecule of interest from a
sample, and allow for determination of the target molecule. In
other assays an analyte of interest can be detected by competitive
binding of a solid phase bound analyte and the analyte in the
sample with a detectably labeled analyte-specific binding agent. In
serological assays an antibody to an antigen, e.g. an infectious
agent is detected directly or in a so-called double antigen
sandwich assay.
[0108] Typically, the existence of an analyte of interest is
indicated by the presence or absence of an observable "label"
attached to one or more of the analyte-specific binding agents.
[0109] The vast majority of immunoassays nowadays one way or the
other employs a solid phase. Usually at least one of the specific
binding agents used in the assay is directly or indirectly bound to
the solid phase. The (strept)avidin-biotin binding pair is
characterized by an extremely high binding affinity. For this
reason the (strept)avidin-biotin binding pair is broadly used for
indirect binding of any appropriate biotinylated specific binding
agents to a solid phase coated with (strept)avidin.
[0110] A "Sandwich assay" is an assay type which is among the most
useful and commonly used assays. A number of variations of the
sandwich assay technique exist, and all are intended to be
encompassed by the present invention. Briefly, in a typical forward
assay, an unlabeled antibody is immobilized on a "solid phase", and
the sample to be tested is brought into contact with the bound
molecule. Immobilization of this capture antibody can be by direct
adsorption to a solid phase or indirectly, e.g. via a specific
binding pair, e.g. via the (strept)avidin-biotin binding pair.
After a suitable period of incubation, for a period of time
sufficient to allow formation of an antibody-antigen complex, a
second antibody binding to the antigen, labeled with a reporter
molecule capable of producing a detectable signal is then added and
incubated, allowing time sufficient for the formation of a
sandwich-complex of antibody-antigen-labeled antibody. Any
unreacted material is washed away, and the presence of the analyte
is determined by observation of a signal produced by the reporter
molecule. The results may either be qualitative, by simple
observation of the visible signal, or may be quantitated by
comparing with a control sample containing known amounts of
analyte.
[0111] For the purpose of the present invention, in a typical
sandwich assay a first biotinylated analyte specific binding agent,
e.g. a biotinylated antibody is bound non-covalently to a solid
phase, which is coated with (strept)avidin. The solid phase is
typically glass or a polymer, the most commonly used polymers being
cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride,
or polypropylene. The solid phase may be in the form of tubes,
beads, discs of microplates, or any other surface suitable for
conducting an immunoassay. Coating processes are well-known in the
art and generally consist of cross-linking, covalent binding, or
physically adsorbing. The (strept)avidin-coated solid phase is
usually treated to block non-specific binding and washed in
preparation for its in the testing procedure. An aliquot of the
sample to be tested is contacted with the first or capture antibody
and a labeled second antibody, and incubated for a period of time
sufficient (e.g. 2-40 minutes or overnight if more convenient) and
under suitable conditions (e.g., from room temperature to
40.degree. C. such as between 25.degree. C. and 32.degree. C.
inclusive) to allow for binding between the first or capture
antibody and the corresponding antigen, and the antigen with the
second antibody binding to another epitope on the antigen, thereby
forming a sandwich complex. Subsequently, the (strept)avidin-coated
solid phase is added and incubated for a period of time sufficient
(e.g. 2-40 minutes or overnight if more convenient) and under
suitable conditions (e.g., from room temperature to 40.degree. C.
such as between 25.degree. C. and 32.degree. C. inclusive) to allow
for binding between the first or capture antibody and the solid
phase. The second antibody is linked to a reporter molecule which
is used to indicate the binding of the second antibody to the
complex of first antibody and the antigen of interest.
[0112] Variations on the assay include a simultaneous assay, in
which both sample and labeled antibody are added simultaneously to
the bound antibody or the antibody capable of being bound to the
solid phase by means of a (strept)avidin/biotin binding pair. These
techniques are well known to those skilled in the art, including
any minor variations as will be readily apparent.
[0113] In a further alternative setting, a sandwich complex is
formed, wherein a first biotinylated antibody is provided, a second
antibody labeled with a hapten is provided, and the two antibodies
are contacted with a sample containing the corresponding antigen.
Upon incubation under conditions permissive with formation of a
sandwich comprising the first and the second antibody, and the
antigen, (strept)avidin carrying a label is added, and the complex
is captured by a solid phase capable of specifically binding the
hapten that is attached to the second antibody. After a washing
step the amount of label bound by the solid phase indicates the
presence and amount of the antigen of interest.
[0114] Generally, relating to all aspects and embodiments herein,
the present disclosure provides a compound of Formula I,
##STR00010##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being selected from (i) a hapten which does not contain a biotin
moiety and (ii) a polypeptide, and L being a linker connecting X
and M, and wherein B is
##STR00011##
with M being selected from (i) a hapten which does not contain a
biotin moiety and (ii) a polypeptide, and L being a linker, whereby
the nitrogen atom marked with an asterisk is covalently bound to an
adjacent CH.sub.2 group of X.
[0115] A first key feature of the structure of Formula I is the
diaminobiotin moiety, a biotin analog in the sense that it
comprises an unmodified, unconjugated valeric acid side chain, as
it is the case in biotin. It was speculated that the conformation
of the side chain relative to the tetrahydrothiophene structure of
the diaminobiotin moiety is not totally random. Without being bound
to theory, this assumption was based on a possible analogy to
conclusions about biotin, NMR data with biotin and the previous
interpretation concluding an interaction between the C6 atom being
part of the valeric acid side chain on the one hand, and the N'3
atom on the other hand. Thus, an unanswered question, so far, was
whether the "tail" portion of the biotin molecule that is opposite
to (distal from) the heterocyclic "head" portion which can be bound
by (strept)avidin is actually suited for recognition by a
monoclonal antibody. More specifically, the present disclosure
addresses the question whether even the "tail" portion of
diaminobiotin is suited for recognition by a monoclonal antibody,
and whether it may mimick the "tail" portion of unconjugated (free)
biotin. For the purpose of the present report, both nitrogen atoms
of diaminobiotin carry substitutions. The N'3 atom in the
diaminobiotin moiety comprised in Formula I atom is therefore an
important feature since in addition to the N'1 it is also subject
of modification.
[0116] Thus, derivatized diaminobiotin as provided in here is
prepared as a building block which allows, among other things, the
formation of immunogens and of molecules useful for detecting and
screening desired antibodies. Specifically for this purpose, the
terminal group R can be selected to be Z, wherein Z is A or B,
wherein A is M-L with M being selected from (i) a hapten which does
not contain a biotin moiety and (ii) a polypeptide, and L being a
linker connecting X and M, and wherein B is
##STR00012##
with M being selected from (i) a hapten which does not contain a
biotin moiety and (ii) a polypeptide, and L being a linker, whereby
the nitrogen atom marked with an asterisk is covalently bound to an
adjacent CH.sub.2 group of X.
[0117] In a specific embodiment, R is an azide group which can be
used as a reactive partner in a type of reaction known as "click"
chemistry. Thus, a conjugate of the hapten and the biotin
derivative can be formed with reactive groups having bioorthogonal
functional groups. A specific embodiments of a reactive group is an
azide group with which a "click" reaction can be performed with an
alkynes or a phosphine as reaction partner (J. C. Jewett, C. R.
Bertozzi, Chem. Soc. Rev. 2010, 39, 1272). Azides with phosphines
perform a Staudinger reaction, azides with alkynes a [3+2]
cycloaddition. Especially cyclooctyne derivatives are known to the
person of skill for the modification of biomolecules under mild
conditions (WO 2006/050262). In the specific embodiment in which R
is Z, and Z is B, the diaminobiotin moiety via the --X substituent
at the 1'N atom is connected to M, wherein B is obtained from a
[3+2] cycloaddition of an alkyne with the azide group being an
embodiment of R.
[0118] The term "linker" denotes a bifunctional or multifunctional
moiety which can be used to conjugate (link) a first moiety with a
second moiety or more moieties. Conjugates comprising a first and a
second moiety bound to each other can be conveniently prepared
using a linker having two reactive functionalities. In such
conjugate the two moieties are bound "via" this linker. As obvious
to the skilled artisan in such conjugate the functional moieties of
the linker are present as part of a bond and not as an unreacted
functional moiety.
[0119] As defined herein the term "reactive group" or "reactive
functionality" means any group, which is suitable for reacting with
amine groups, preferably an N-hydroxysuccinimide group or a
maleimide group, in order to bind a linker to an amino group; or a
group, which is suitable for a second functionality binding, e.g.
for reacting with an SH-group, preferably a maleimide group in
order to bind a linker to an SH-group.
[0120] In a specific embodiment, a heterobifunctional linker is
selected from the group consisting of NHS-maleimide linkers, which
are based on N-hydroxysuccinimide and maleimide reactive groups;
succinimidyl-(PEG)n NHS-PEG-maleimide linkers, NHS-haloacetyl
linkers; and NHS-pyridyldithiol linkers. In a particular preferred
embodiment the heterobifunctional linker is a succinimidyl-(PEG)n
NHS-PEG-maleimide linker.
[0121] In a specific embodiment, L has a backbone length of between
1 and 200 atoms. In other words if the backbone length is between 1
and 200 atoms, the shortest connection between Z and R consists of
1 to 200 atoms.
[0122] In case a ring system is present the shortest number of
atoms in the ring system is taken when assessing the linker length.
As an example, a phenylen ring accounts for a length of four atoms
in a linker.
[0123] In one embodiment of Formula I, L is a linker having as a
backbone a straight or branched saturated, unsaturated,
unsubstituted or substituted C1-C20 alkyl chain, or a 1 to 200 atom
chain consisting of carbon atoms, substituted carbon atoms and/or
one or more atoms selected from O, N, P and S, or substituted N, P,
S atoms, or a chain as described before with the backbone
containing one or more cyclic or heterocyclic aromatic or
non-aromatic ring systems.
[0124] In one embodiment of Formula I, the linker L has as a
backbone a straight or branched saturated, unsaturated,
unsubstituted or substituted C1-C100 alkyl chain, or a 1 to 100
atom chain consisting of carbon atoms, substituted carbon atoms
and/or one or more atoms selected from O, N, P and S, or
substituted N, P, or S atoms, or a chain as described before with
the backbone containing one or more cyclic or heterocyclic aromatic
or non-aromatic ring systems.
[0125] In one embodiment of Formula I, the linker L has as a
backbone a straight or branched saturated, unsaturated,
unsubstituted or substituted C1-C50 alkyl chain, or a 1 to 50 atom
chain consisting of carbon atoms, substituted carbon atoms and/or
one or more atoms selected from O, N, P and S, or substituted N, P,
or S atoms, or a chain as described before with the backbone
containing one or more cyclic or heterocyclic aromatic or
non-aromatic ring systems.
[0126] In one further embodiment of Formula I, the linker L has as
backbone of a straight or branched saturated, unsaturated,
unsubstituted or substituted C1-C20 alkyl chain, or a 1 to 20 atom
chain consisting of carbon atoms, substituted carbon atoms and/or
one or more atoms selected from O, N, P and S, or substituted N, P,
or S atoms, or a chain as described before with the backbone
containing one or more cyclic or heterocyclic aromatic or
non-aromatic ring systems.
[0127] In another specific embodiment of Formula I, L is a linker,
which is obtained upon reaction of a (homo-)bifunctional
crosslinker with an appropriate chemical group on each of the two
moieties linked by the linker, the (homo-)bifunctional crosslinker
is exemplified by
##STR00013##
[0128] In another specific embodiment of Formula I, L is a linker,
which is obtained upon reaction of a (hetero-)bifunctional
crosslinker with an appropriate chemical group on each of the two
moieties linked by the linker, the (hetero-)bifunctional
crosslinker can be selected from the group consisting of
NHS-maleimide crosslinkers, e.g.
##STR00014## ##STR00015## ##STR00016## ##STR00017##
[0129] In an embodiment of Formula I R is A or B, and A is M-L, and
B comprises M-L, wherein M is a polypeptide. Thus, another general
aspect of the original disclosure herein, and of the invention,
which is also related to all other aspects and embodiments as
disclosed herein is an immunogen according to Formula I,
##STR00018##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein A is M-L with M being a
polypeptide of at least 30 amino acids, and preferably being
keyhole limpet hemocyanin, and L being a linker connecting X and M,
and wherein B is
##STR00019##
with M being a polypeptide of at least 30 amino acids, and
preferably being keyhole limpet hemocyanin, and L being a linker,
whereby the nitrogen atom marked with an asterisk is covalently
bound to an adjacent CH.sub.2 group of X.
[0130] Importantly, the polypeptide is capable of eliciting an
immune response to the derivatized diaminobiotin moiety, while the
immunogenicity of diaminobiotin alone is low. In an embodiment, the
polypeptide is selected from rat, rabbit, mouse, porcine or bovine
serum albumin, bovine or porcine thyroglobulin, ovalbumin, tetanus
toxoid, gelatin, soybean trypsin inhibitor, keyhole limpet
hemocyanin and similar substances. In a specific embodiment, the
polypeptide is keyhole limpet hemocyanin (KLH).
[0131] In one embodiment the present disclosure related to all
other aspects and embodiments also provides methods for making
polyclonal antibodies (including antibodies, antibody fragments
thereof, and antigen binding fragments thereof) that specifically
bind to the immunogen according to the present disclosure. The
method comprises: (a) providing an immunogen according to the
invention; (b) immunizing an experimental animal with the immunogen
under conditions such that the immune system of the animal makes
the antibodies; and (c) removing the antibodies that specifically
bind to biotin from the animal. The animal can be a sheep, goat,
rabbit, rat, mouse and the like.
[0132] Another general aspect of the original disclosure herein,
and of the invention, which is also related to all other aspects
and embodiments as disclosed herein is a method of producing an
antibody according to the invention, the method comprising the
steps of (a) immunizing an experimental animal with an immunogen
according to the invention, thereby inducing B-cells producing
antibodies binding to the immunogen, (b) obtaining a monoclonal
antibody binding to the immunogen produced by the B-cell of step
(a), either via hybridoma technology or by B-cell PCR technology,
(c) further selecting the antibody of step (b) for binding to
biotin, thereby obtaining an antibody according to the
invention.
[0133] In an embodiment, in Formula I, R comprises M, and a
specific embodiment of M is a hapten, wherein the hapten does not
contain a biotin moiety. Thus, another general aspect of the
original disclosure herein, and of the invention, which is also
related to all other aspects and embodiments as disclosed herein is
a compound according to Formula I,
##STR00020##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is A or B, wherein A
is M-L with M being a hapten which does not contain a biotin
moiety, and L being a linker connecting X and M, and wherein B
is
##STR00021##
with M being a hapten which does not contain a biotin moiety, and L
being a linker, whereby the nitrogen atom marked with an asterisk
is covalently bound to an adjacent CH.sub.2 group of X.
[0134] In a specific embodiment, R is A, and derivatized
diaminobiotin is comprised in the structure of FIG. 3C (Formula III
C) which is useful for detecting and screening desired antibodies
according to the invention.
[0135] A compound according to this aspect is particularly useful
in capturing desired antibodies, e.g. in a process of identifying
polyclonal or monoclonal antibodies generated using an immunogen
according to the invention. Such antibodies will specifically bind
to the biotin moiety of the compound, that is to say the compound
is capable of capturing the desired antibodies according to the
invention. In this regard, the hapten must not be and/or must not
contain a further biotin moiety, in order to maintain the capturing
specificity of the compound. Thus, a suitable hapten which is not
biotin and/or does not contain biotin is selected from the group
consisting of dinitrophenol, aniline, aminobenzoic acid,
hydralazine, fluorescein, and digoxigenin.
[0136] In a specific embodiment, M is digoxigenin. A specific
embodiment is the compound disclosed in FIG. 3C comprising
digoxigenin (Formula III C).
[0137] Further, a specific embodiment according to all aspects as
disclosed herein is a monoclonal antibody which firstly
specifically binds to a compound according to Formula I,
##STR00022##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and R is A or B, wherein A is M-L
with M being a hapten which does not contain a biotin moiety, and L
being a linker connecting X and M, and wherein B is
##STR00023##
with M being a hapten which does not contain a biotin moiety, and L
being a linker, whereby the nitrogen atom marked with an asterisk
is covalently bound to an adjacent CH.sub.2 group of X, and
secondly specifically binds to a compound depicted in FIG. 3C
(Formula III C). In a more specific embodiment, the monoclonal
antibody firstly specifically binds to a compound depicted in FIG.
2C (Formula III A) and secondly to the compound depicted in FIG. 3C
(Formula III C).
[0138] Surprisingly it was additionally found that monoclonal
antibodies described in the aspects and embodiments herein not only
bind to compounds of Formula I as specified and described above,
but they also bind derivatized biotin which is characterized in
that unlike in Formula I diaminobiotin is replaced by biotin, and
the N'i atom of the ureido ring of the biotin carries a substituent
by which the biotin moiety is covalently attached to a carrier.
Thus, a specific embodiment according to all aspects as disclosed
herein is a monoclonal antibody which firstly specifically binds to
a compound according to Formula I,
##STR00024##
wherein X is selected from the group consisting of (CH.sub.2).sub.n
with n being an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and R is A or B, wherein A is M-L
with M being a hapten which does not contain a biotin moiety, and L
being a linker connecting X and M, and wherein B is
##STR00025##
with M being a hapten which does not contain a biotin moiety, and L
being a linker, whereby the nitrogen atom marked with an asterisk
is covalently bound to an adjacent CH.sub.2 group of X, and
secondly specifically binds to a compound of Formula III B,
depicted in FIG. 3B.
[0139] Another general aspect of the original disclosure herein,
and of the invention, which is also related to all other aspects
and embodiments as disclosed herein is a method of producing an
antibody according to the invention, the method comprising the
steps of (a) immunizing an experimental animal with an immunogen
according to the invention, thereby inducing B-cells producing
antibodies binding to the immunogen, (b) obtaining a monoclonal
antibody binding to the immunogen produced by the B-cell of step
(a), either via hybridoma technology or by B-cell PCR technology,
(c) further selecting the antibody of step (b) for binding to
biotin, thereby obtaining an antibody according to the
invention.
[0140] The generation of cell lines which produce monoclonal
antibodies by way of hybridoma technology is well known to the
skilled person.
[0141] B-cell PCR technology known to the skilled person takes
advantage from the fact that from B-cells the total mRNA can be
isolated and transcribed to cDNA. With specific primers the cognate
VH- and VL-region encoding nucleic acid can be amplified. Almost no
identical sequences are obtained. The method provides for highly
diverse antibodies binding to the same antigen.
[0142] The primers used for the amplification of the VH-encoding
nucleic acid can be used for cDNA obtained from cells from the
NMRI-mouse, the Armenian Hamster, the Balb/c-mouse as well as the
Syrian hamster and the rabbit.
[0143] In one embodiment of all methods as reported herein the
amino acid sequence is derived from the amplified VH-encoding
nucleic acid and the exact start and end point is identified by
locating the amino acid sequences of EVQL/QVQL to VSS (VH-region)
and DIVM/DIQM to KLEIK (VL-region).
[0144] Also reported herein is a method using B-cell PCR for
producing an antibody comprising the following steps: (a) providing
a population of (mature) B-cells (obtained from the blood of an
experimental non-human animal), (b) staining the cells of the
population of B-cells with at least one fluorescence dye (in one
embodiment with one to three, or two to three fluorescence dyes),
(c) depositing single cells of the stained population of B-cells in
individual containers (in one embodiment is the container a well of
a multi well plate), (d) cultivating the deposited individual
B-cells in the presence of feeder cells, (e) determining the
binding specificity of the antibodies secreted in the cultivation
of the individual B-cells, (f) determining the amino acid sequence
of the variable light and heavy chain domain of specifically
binding antibodies by a reverse transcriptase PCR and nucleotide
sequencing, and thereby obtaining a monoclonal antibody variable
light and heavy chain domain encoding nucleic acid, (g) introducing
the monoclonal antibody light and heavy chain variable domain
encoding nucleic acid in an expression cassette for the expression
of an antibody, (h) introducing the nucleic acid in a cell, (i)
cultivating the cell and recovering the antibody from the cell or
the cell culture supernatant and thereby producing an antibody.
[0145] In one embodiment the non-human animal is selected from rat,
mouse, hamster, rabbit, non-human primates, sheep, dog, cow,
chicken, amphibians, and reptiles.
[0146] In a specific embodiment of the herein disclosed method for
producing a monoclonal antibody, in step (c) the selection is
performed in a competitive assay using biotin as a competitor for
binding of the antibody to a compound of the group of compounds
selected from Formula III A depicted in FIG. 2C, Formula III B
depicted in FIG. 3B, and Formula III C depicted in FIG. 3C. These
compound have been discussed above, already. The compounds are
particularly useful in capturing desired antibodies. Such
antibodies will specifically bind to the biotin moiety of one or
both compounds, that is to say one or both compounds are capable of
capturing the desired antibodies according to the invention. In
this regard, the hapten as exemplified in Formula III B and Formula
III C must not be and/or must not contain a further biotin moiety,
in order to maintain the capturing specificity of the respective
compound. Thus, the hapten is used to anchor the capture reagent on
a solid phase, e.g. as exemplified with digoxigenin as the
hapten.
[0147] Another important step is the selection and/or confirmation
that an antibody, specifically a monoclonal antibody generated
using an immunogen as disclosed herein, and by a method as
disclosed herein is actually capable of binding biotin. A
competitive binding assay thereby ensures that the antibody is
capable of binding biotin when contacted with free biotin in
aqueous solution. Accordingly, in an embodiment of the herein
disclosed method for producing a monoclonal antibody in step (c)
the selection is performed in a competitive assay using biotin as a
competitor for binding of the antibody to the immunogen as
disclosed herein.
[0148] In another embodiment of the herein disclosed method for
producing a monoclonal antibody in step (c) the selection is
performed in a competitive assay using biotin as a competitor for
binding of the antibody to the to a compound of Formula III B
(Formula III B).
[0149] In another embodiment of the herein disclosed method for
producing a monoclonal antibody in step (c) the selection is
performed in a competitive assay using biotin as a competitor for
binding of the antibody to the to a compound of Formula III C
(Formula III C).
[0150] In another embodiment of the herein disclosed method for
producing a monoclonal antibody in step (c) the selection is
performed in a competitive assay using biotin as a competitor for
binding of the antibody to the to a compound of Formula III A
(Formula II C).
[0151] More generally, in another embodiment of the herein
disclosed method for producing a monoclonal antibody in step (c)
the selection is performed in a competitive assay using biotin as a
competitor for binding of the antibody to the to a compound
according to Formula I. In a specific embodiment, R is a hapten,
wherein the hapten does not contain a biotin moiety.
[0152] As evidenced in the Examples of present disclosure, the
invention in its aspects and embodiments now provides a novel
monoclonal antibody specifically binding a compound of Formula
I,
##STR00026##
characterized in that it also binds to biotin, wherein X is
selected from the group consisting of (CH.sub.2).sub.n with n being
an integer from 1 to 20,
[(CH.sub.2).sub.p--O].sub.k--(CH.sub.2).sub.m with p being 2 or 3,
with m being 2 or 3, with k being an integer from 1 to 30, and
[(CH.sub.2).sub.r--CONH].sub.s--(CH.sub.2).sub.t with r being an
integer from 1 to 5, with t being an integer from 0 to 5, and with
s being an integer from 1 to 5, and wherein R is selected from the
group consisting of H, OH, COOH, NH.sub.2, an azide group, a
maleimide group, and Z, wherein Z is A or B, wherein A is M-L with
M being selected from (i) a hapten which does not contain a biotin
moiety and (ii) a polypeptide, and L being a linker connecting X
and M, and wherein B is and wherein B is
##STR00027##
[0153] with M being selected from (i) a hapten which does not
contain a biotin moiety and (ii) a polypeptide, and L being a
linker, whereby the nitrogen atom marked with an asterisk is
covalently bound to an adjacent CH.sub.2 group of X.
[0154] The monoclonal antibody of the invention can be obtained by
a method according to the invention and as disclosed herein.
[0155] As described above, the monoclonal antibody according to the
invention is characterized in that it does not bind a compound of
Formula II. In Formula II the biotin moiety is coupled via a linker
to a hapten via carbon atom of the carboxyl function of the valeric
acid side chain. Thus the molecule exemplifies a conventionally
biotinylated molecule to which the desired antibodies according to
the invention do not bind.
[0156] Thus, another general aspect of the original disclosure
herein, and of the invention, which is also related to all other
aspects and embodiments as disclosed herein is a method for
producing an antibody which does not bind to a biotinylated
molecule, wherein the biotin moiety is coupled via a linker to a
hapten via the carbon atom of the carboxyl function of the valeric
acid side chain, the method comprising the steps of (a) immunizing
an experimental animal with an immunogen according to the
invention, thereby inducing B-cells producing antibodies binding to
the immunogen, (b) obtaining a monoclonal antibody binding to the
immunogen produced by the B-cell of step (a), either via hybridoma
technology or by B-cell PCR technology, (c) selecting the antibody
of step (b) for binding to biotin, and (d) selecting those
antibodies which do not bind to the biotinylated molecule, thereby
obtaining an antibody according to the invention which does not
bind to a biotinylated molecule. In a specific embodiment, step (d)
is performed with a compound according to Formula II depicted in
FIG. 3A.
[0157] Thus, more specifically, there is disclosed a method for
producing an antibody which does not bind to a compound of Formula
II, the method comprising the steps of (a) immunizing an
experimental animal with an immunogen according to the invention,
thereby inducing B-cells producing antibodies binding to the
immunogen, (b) obtaining a monoclonal antibody binding to the
immunogen produced by the B-cell of step (a), either via hybridoma
technology or by B-cell PCR technology, (c) selecting the antibody
of step (b) for binding to biotin, and (d) selecting those
antibodies which do not bind to the compound of Formula II, thereby
obtaining an antibody according to the invention which does not
bind to a compound according to Formula II.
[0158] Particularly in a method for measuring an analyte in a
sample, wherein a (strept)avidin/biotin binding pair is used to
bind a biotinylated analyte specific binding agent to a
(strept)avidin coated solid phase, the measurement of the analyte
may become inaccurate if the sample contains extraordinary high
amounts of biotin. For this reason it is highly desired to scavenge
biotin. This technical problem can be solved by sample
pre-treatment, i.e. with a process of removing free biotin before
the sample is subjected to the method for measuring the analyte.
However, this would involve an undesired large number of discrete
working steps such as, e.g., mixing the sample with magnetic
particles that are coated with (strept)avidin, incubating the
mixture thereby binding the biotin to the particles, followed by
magnetically removing the particles. Not only is such an approach
of sample pre-treatment consuming resources such as raw materials
and time; in addition such an approach bears the risk of changing
the sample (e.g. its volume and composition) which in turn may lead
to undesired effects in analyte detection and measurement.
[0159] Surprisingly it was found that a monoclonal antibody
according to the invention capable of binding biotin and not
binding to conventionally biotinylated molecules of interest
provides for an elegant solution to reduce biotin interference.
While acting as a biotin scavenger, the biotin binding property of
the antibody is such that it does not interfere with the binding of
e.g. a biotinylated analyte specific binding agent to
(strept)avidin. Thus, the monoclonal antibody according to the
invention turns out to be a powerful tool in counteracting
interference caused by a high level of biotin that might be present
in the sample.
[0160] Another general aspect of the original disclosure herein,
and of the invention, which is also related to all other aspects
and embodiments as disclosed herein is the use of an antibody
according to the invention, in a method for measuring an analyte in
a sample, wherein a (strept)avidin/biotin pair is used to bind a
biotinylated analyte specific binding agent to a (strept)avidin
coated solid phase.
[0161] Another general aspect of the original disclosure herein,
and of the invention, which is also related to all other aspects
and embodiments as disclosed herein is a method for measuring an
analyte in a sample, wherein a (strept)avidin/biotin binding pair
is used to bind a biotinylated analyte specific binding agent to a
(strept)avidin coated solid phase, the method comprising adding to
the sample a) an antibody according to the invention, b) a
biotinylated analyte specific binding agent, c) a (strept)avidin
coated solid phase, followed by measuring the analyte bound to the
solid phase via (strept)avidin and biotinylated analyte specific
binding agent.
[0162] The present disclosure provides the means to scavenge
potentially interfering unconjugated (free) biotin comprised in a
sample from which sample an analyte shall be measured and which
measuring method makes use of the (strept)avidin/biotin binding
pair. Preferred the scavenging step is performed prior to the
formation of the (strept)avidin/biotin pair. Thus, in an embodiment
there is provided a method for measuring an analyte in a sample,
wherein a (strept)avidin/biotin binding pair is used to bind a
biotinylated analyte specific binding agent to a (strept)avidin
coated solid phase, the method comprising adding to the sample a)
an antibody according to the invention, b) a biotinylated analyte
specific binding agent, c) a (strept)avidin coated solid phase,
followed by measuring the analyte bound to the solid phase via
(strept)avidin and biotinylated analyte specific binding agent,
wherein step (a) and optionally also step (b) is performed before
step (c). In yet another embodiment, step (a) and step (b) are
performed simultaneously before step (c) is performed. In yet
another embodiment, step (b) and step (c) are performed
simultaneously after step (a) has been performed. In yet another
embodiment, all three steps (a), (b) and (c) are performed
simultaneously. Surprisingly, a monoclonal antibody of the
invention is even technically suited as an effective scavenger for
free biotin for the use in a method for measuring an analyte in a
sample, the sample containing free biotin, wherein a
(strept)avidin/biotin pair is used to bind a biotinylated analyte
specific binding agent to a (strept)avidin coated solid phase or to
a labelled (strept)avidin, wherein the sample, the monoclonal
antibody of the invention, a biotinylated binding agent and either
of a (strept)avidin coated solid phase or a labeled stereptavidin
are contacted with each other simultaneously.
[0163] Concerning all aspects and embodiments of analyte detection
and/or measurement as disclosed herein an aqueous liquid sample can
be used in a method for specific in vitro-detection of an analyte
in a method according to the present disclosure. The sample may be
known to comprise the analyte or it may be suspected of comprising
the analyte. In one embodiment a sample for in vitro diagnosis used
in a method according to the present disclosure is a body fluid
selected from whole blood, blood serum, blood plasma, liquor, urine
or saliva. In one embodiment the sample suspected of comprising or
comprising the analyte is serum, plasma or liquor. In one
embodiment the sample suspected of comprising or comprising the
analyte is serum or plasma.
[0164] Another general aspect of the original disclosure herein,
and of the invention, which is also related to all other aspects
and embodiments as disclosed herein is an immunoassay test kit
comprising in separate containers at least (a) an antibody
according to any of claims according to the invention, (b) a
biotinylated analyte specific binding agent, and (c) a
(strept)avidin coated solid phase.
[0165] The term single container unit relates to the fact that for
many automatic analyzers, like the Elecsys.RTM. analyzer series
from Roche diagnostics, the reagents required to measure a certain
analyte are provide in the form of a "reagent pack", i.e. as one
container unit fitting on the analyzer and containing in different
compartments all the key reagents required for measurement of the
analyte of interest.
[0166] In one embodiment the present invention relates to a kit
wherein said first partner of a binding pair is avidin or
streptavidin, and wherein said second partner of said binding pair
is selected from biotin or biotin analogues such as aminobiotin,
iminobiotin or desthiobiotin.
[0167] The following examples and figures are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Example 1
Derivatization of Diaminobiotin and Synthesis Steps to Obtain an
Immunogen
[0168] 1.1
Synthesis of
5-{3-Amino-4-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-tet-
rahydro-thiophen-2-yl}-pentanoic acid
[0169] The reaction was performed in an inert atmosphere (argon)
using dried solvents and dried starting materials. 220 mg
FMOC-beta-alanine (CAS Registry Number 35737-10-1, other name
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-.beta.-alanin) and 112 mg HOB
t (Benzotriazol-1-ol, N-Hydroxybenzotriazole, CAS Number 2592-95-2)
were dissolved in 7.5 mL dry DMF (Dimethylformamide). 105 mg
1,3-Diisopropyl-carbodiimide were added and stirred for 1 h at room
temperature. 140 mg diaminobiotin (CAS Registry Number 22342-46-7)
dissolved in 7.5 mL DMF was added and the mixture stirred
overnight. After evaporation of the solvent the reaction mixture
was further purified by preparative HPLC. The yield was 174 mg.
[0170] HPLC-ESI-MS: M+512.4
1.2
Synthesis of
5-{3-Acetylamino-4-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamin-
o]-tetrahydro-thiophen-2-yl}-pentanoic acid
[0171] The reaction was performed in an inert atmosphere (argon)
using dried solvents and dried starting materials. 174 mg of
5-{3-amino-4-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-tet-
rahydro-thiophen-2-yl}-pentanoic acid were dissolved in 10 mL DMF
and 416 mg acetic acid anhydride added. The reaction mixture was
stirred at room temperature overnight. The solvent was removed by
evaporation and the product dried under vacuum. The yield was 94
mg.
[0172] HPLC-ESI-MS: M+554.4
1.3
Synthesis of
5-[3-Acetylamino-4-(3-amino-propionylamino)-tetrahydro-thiophen-2-yl]-pen-
tanoic acid
[0173] 94 mg
5-{3-acetylamino-4-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamin-
o]-tetrahydro-thiophen-2-yl}-pentanoic acid were dissolved in 10 mL
DMF and 2.5 mL piperidine were added. The mixture was stirred at
room temperature for 3 h. The yield was 52 mg crude product.
1.4
Synthesis of
5-{3-Acetylamino-4-[3-(2-azido-acetylamino)-propionylamino]-tetrahydro-th-
iophen-2-yl}-pentanoic acid=Azido-beta-Ala-Diaminobiotin
[0174] The reaction was performed in an inert atmosphere (argon)
using dried solvents and dried starting materials. 7 mg of
5-[3-acetylamino-4-(3-amino-propionylamino)-tetrahydro-thiophen-2-yl]-pen-
tanoic acid and 8 mg of azidoacetic acid succinimidyl ester (CAS
Registry Number 824426-32-6) were dissolved in 10 mL DMF and 8 mg
triethylamine were added. After stirring overnight the solvent was
removed by evaporation and the crude product was purified by
preparative HPLC.
[0175] Yield 2 mg
[0176] HPLC-ESI-MS: M+415.4
1.5
KLH-SXA1028-Azido-Ala-Diaminobiotin (Formula III A, see FIG.
2C)
[0177] Keyhole limpet hemocyanin (30 mg, Sigma Aldirch #8283) in
0.1 M potassium phosphate buffer (pH 7.2, 10 mL) was reacted with
(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succinimidyl carbonate
(6.6 mg, 22.6 .mu.mol, Synaffix Product # SX-A-1028). The mixture
was stirred for 5 hours at room temperature. Unreacted cyclooctyne
was removed by dialysis against 0.1 M potassium phosphate buffer
(pH 7.2). Azido-Ala-Diaminobiotin (3.52 mg, 8.5 .mu.mol) was
dissolved in DMSO and added to cyclooctyne-derivatized KLH. The
mixture was stirred overnight at 4.degree. C. Unreacted
Azido-Ala-Diaminobiotin was removed by dialysis against 0.1 M
potassium phosphate buffer (pH 7.2). The protein content was
determined by measuring absorbance at 280 nm.
Example 2
Synthesis of a Reagent Including a Diaminobiotin Derivative for
Screening Monoclonal Antibodies In Vitro
[0178] Dig-3-CME-AMCAP-beta-ala-biotin
[0179]
5-[3-Acetylamino-4-(3-amino-propionylamino)-tetrahydro-thiophen-2-y-
l]-pentanoic acid was synthesized as described above. (Example
1).
[0180] Digoxigenin-3-CME-AMCAP-NHS ester (other names: Digoxigenin
NHS-Ester, Card-20(22)-enolide,
3-[2-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-2-oxoethoxy]-1-
2,14-dihydroxy-, (3.beta.,5.beta.,12.beta.)-)=CAS Nr. 129273-26-3
was synthesized as described in DE3836656A1, or was obtained as
commercially available product (Merck-Sigma).
[0181] 20 mg Digoxigenin-3-CME-AMCAP-NHS ester is dissolved in 3 mL
DMF and 6 .mu.l triethylamine is added. Afterwards 10 mg
5-[3-Acetylamino-4-(3-amino-propionylamino)-tetrahydro-thiophen-2-yl]-pen-
tanoic acid is added and the mixture is stirred at room temp. for
3.5 h. The solvent is removed by evaporation and the product is
isolated by prep. HPLC chromatography.
Example 3
Derivatization of Biotin at the N'1 Atom
[0182] (1) FMOC-beta-alanine-acid Chloride
[0183] To 4.7 g FMOC-beta-alanine (IRIS, FAA1300) in a flask under
dry conditions ca. 20 mL thionyl chloride were added slowly. The
mixture was stirred at room temperature for 50 min. and afterwards
heated to reflux for 10 min. After cooling, the mixture was
evaporated, the residue was dissolved in absolute toluene three
times and evaporated each time.
[0184] The yield was 5.1 g.
(2) FMOC-beta-alanyl-biotin
[0185] The carboxylic acid of biotin was protected by reacting
biotin (2.48 g) with t-butylchlorodiphenylsilane (6.5 ml) in 15 mL
dry pyridine in the presence of DMAP (0.63 g) overnight at room
temperature in an inert atmosphere of argon as described by Fang
and Bergstrom in Nucleic Acids Research, 2003, Vol. 31, No. 2,
708.
[0186] 4.9 g FMOC-beta-alanine-acid chloride dissolved in 10 mL
dichloromethane were added and stirred for 3.5 h at room temp. The
mixture was evaporated, DMF added and evaporated again. The residue
was dissolved in DMF-H.sub.2O (3:1) and 50 mmol potassium carbonate
was added and the mixture was stirred for 30 min. After
acidification with citric acid to pH 4 the product was extracted
with ethyl acetate and purified by column chromatography (silica,
eluent ethyl acetate/methanol).
[0187] HPLC-ESI-MS: M.sup.+=538.3 Da. The yield was 1.5 g.
(3)
Beta-alanyl-biotin=5-[3-(3-Amino-propionyl)-2-oxo-hexahydro-thieno[3,-
4-d]imidazol-6-yl]-pentanoic acid
[0188] The FMOC protecting group was cleaved by dissolving 1.67 g
FMOC-beta-alanyl-biotin in a mixture of 20% piperidine in DMF (60
ml). The mixture was evaporated and dried under vacuum. Afterwards
the product was isolated by prep HPLC using reverse phase silica
and a H.sub.2O-acetonitrile gradient.
[0189] HPLC-ESI-MS: M.sup.+=316.3 Da. The yield was 0.55 g.
Example 4
Synthesis of Further Reagents for the Screening of Monoclonal
Antibodies
(1) Dig-3-CME-AMCAP-DADOO-biotin
[0190] Digoxigenin-3-CME-AMCAP-NHS ester (other names: Digoxigenin
NHS-Ester, Card-20(22)-enolide,
3-[2-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-2-oxoethoxy]-1-
2,14-dihydroxy-, (3.beta.,5.beta.,12.beta.)-)=CAS Nr. 129273-26-3
was synthesized as described in DE3836656A1, or was obtained as
commercially available product (Merck-Sigma).
[0191] 21 mg Digoxigenin-3-CME-AMCAP-NHS ester were dissolved in 3
mL DMF and 6 .mu.l trimethylamine were added. Afterwards 14 mg
Biotin-DADOO were added and the mixture was stirred at room temp.
for 2 h. The solvent was removed by evaporation and the product was
isolated by preparative HPLC chromatography.
[0192] HPLC-ESI-MS: M+=918.7 Da. Yield: 23 mg.
(2) Dig-3-CME-AMCAP-beta-ala-biotin
[0193]
Beta-alanyl-biotin=beta-ala-biotin=5-[3-(3-Amino-propionyl)-2-oxo-h-
exahydro-thieno[3,4-d]imidazol-6-yl]-pentanoic acid was synthesized
as described above (Example 1).
[0194] Digoxigenin-3-CME-AMCAP-NHS ester (other names: Digoxigenin
NHS-Ester, Card-20(22)-enolide,
3-[2-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-2-oxoethoxy]-1-
2,14-dihydroxy-, (3.beta.,5.beta.,12.beta.)-)=CAS Nr. 129273-26-3
was synthesized as described in DE3836656A1, or was obtained as
commercially available product (Merck-Sigma).
[0195] 20 mg Digoxigenin-3-CME-AMCAP-NHS ester were dissolved in 3
mL DMF and 6 .mu.l triethylamine were added. Afterwards 10.5 mg
beta-ala-biotin were added and the mixture was stirred at room
temp. for 3.5 h. The solvent was removed by evaporation and the
product was isolated by prep. HPLC chromatography.
[0196] HPLC-ESI-MS: M+=859.6 Da. Yield: 16 mg.
Example 5
Immunization of Rabbits for Generation of Antibodies Binding
Free-Biotin
[0197] In the present disclosure there is reported the development
of antibodies with the ability to scavenge free biotin. To this end
we generated antibodies which bind to biotin only if the COOH-group
of the valeric acid moiety of biotin is accessible and not used for
conjugation. The antibodies according to the invention do not bind
to biotinylated molecules, i.e. conventional biotin-conjugates
where biotin is covalently coupled via the carbon atom of the
carboxyl group.
[0198] For the generation of such antibodies, 12-16-weeks old NZW
rabbits were immunized with KLH-Biotin conjugate (see above,
Example 1, KLH conjugates [nn]). All rabbits were subjected to
repeated immunizations. In the first month the animals were
immunized weekly. From the second month onward the animals were
immunized once per month. For the first immunization 500 .mu.g KLH
conjugate [nn] (Formula III A) was dissolved in 1 mL 140 mM NaCl
and was emulsified in 1 mL CFA (complete Freund's adjuvans). For
all following immunizations, CFA was replaced by IFA (incomplete
Freund's adjuvans). The titers of the animals were evaluated on day
35 after start of the immunization.
Example 6
Antibody Titer Analysis of the Immunized Animals
[0199] The experimental setup of serum titrations was designed to
determine the amount of polyclonal antibodies that can discriminate
between (i) a conjugated biotin of the conventional type, i.e. a
biotin that is conjugated to the carrier via the carbon atom of the
carboxyl function of the valeric acid moiety, and (ii) a conjugated
biotin of which the ring structure is covalently attached to a
carrier.
[0200] Provided herein are the compounds of Formula III A, Formula
III B and Formula III C (depicted in FIGS. 2C, 3 B and 3C,
respectively. Further provided is the compound of Formula II
(depicted in FIG. 3A).
[0201] In an emodiment 96 well plates were first coated with 5
.mu.g/mL of a polyclonal sheep anti-Dig antibody (Sigma). After a
washing step, the plates were blocked with 5% BSA (Roche) to reduce
background signals. To capture the different biotin-Dig conjugates
(CME-AMCAP-beta-ala-biotin (Formula III B) and
Dig-3-CME-AMCAP-DADOO-biotin (Formula II)) the anti-Dig coated
plates were incubated with 250 ng/mL of the conjugates, in separate
wells. After an additional washing step, the rabbit sera were
diluted in PBS with 1% BSA, and the dilutions were added to the
plates. The sera were tested at dilutions 1:300, 1:900, 1:2,700,
1:8,100, 1:24,300, 1:72,900, 1:218,700 and 1:656,100. Bound
antibody was detected with a HRP-labeled F(ab')2 goat anti-rabbit
Fc.gamma. (Dianova) and ABTS (Roche) as a substrate. The titer of
the analyzed animals was set by 50% signal decrease of the dilution
curve.
TABLE-US-00003 TABLE 2 Titration of the sera of experimental
animals immunized with the Diaminobiotin-KLH immunogen, wherein a
Biotin analog is coupled to KLH in the form of a diaminobiotin
derivative. Compound Compound Formula Formula animal III B II
1#E40432 3684 842 2#E40338 1704 1157 3#E40362 7021 804
[0202] It is shown, that the polyclonal sera from the 3 immunized
animals preferentially bind Dig-3-CME-AMCAP-beta-ala-biotin, which
is similar to the immunogen in that the side chain aspect common to
biotin and diaminobiotin is presented for antibody binding and
wherein particularly the COOH-group of the valeric acid side chain
is accessible. Dig-3-CME-AMCAP-DADOO-biotin was detected only
weakly.
Example 7
Development of Monoclonal Antibodies Binding Free-Biotin
[0203] For the development of antibodies capable of binding to
biotin in solution (i.e. free biotin) and without cross reactivity
conventionally biotinylated targets, B-cell cloning as described in
Seeber et al. (2014), PLoS One. 2014 Feb. 4; 9(2) was used. For the
enrichment of antigen reactive B-cells a biotinylated mouse
anti-Dig antibody (Roche) was bound to streptavidin coated magnetic
beads (Miltenyi). Afterwards, the PBMC pool of the immunized
animals was prepared and incubated with 250 ng/mL
Dig-3-CME-AMCAP-beta-ala-biotin. After 1 h of incubation the cells
were washed with PBS and incubated with the pre-coated anti-Dig
magnetic beads. For the enrichment of antigen-reactive B-cells MACS
columns (Miltenyi) were used. B-cell sorting and incubation was
done as described in Seeber et al. (2014), PLoS One. 2014 Feb. 4;
9(2). 24 h before performing the ELISA, to identify
antigen-reactive clones, 2 .mu.g/mL streptavidin was added to the
cell culture supernatants to neutralize free biotin. This was done
as a precaution as free biotin in the culture medium or of cellular
origin could block the interaction of the antibodies in the
supernatant with the screening reagent
Dig-3-CME-AMCAP-beta-ala-biotin (Formula III B). For the ELISA 96
well plates were coated with 5 .mu.g/mL polyclonal goat
anti-rabbit-IgG antibody. After a washing step, the plates were
blocked with 5% BSA to reduce background signals. The plates were
washed again, and 30 .mu.l of the rabbit B-cell cultures were
transferred to the 96 well plates and incubated for 1 h at room
temperature. After another washing step, 50 ng/mL of the positive
screening reagent, Dig-3-CME-AMCAP-beta-ala-biotin, or the negative
screening reagent, Dig-3-CME-AMCAP-DADOO-biotin (Formula II), were
added to the wells and incubated for 1 h at room temperature. For
the detection of antibodies bound to the screening reagents, 3
.mu.g/mL of a POD labeled polyclonal sheep anti-Dig-antibody were
added to the plates. After a final washing step, ABTS (Roche) was
added as a POD substrate, and positive clones were identified by
measuring OD at 405 nm. Results are given in Table 3.
TABLE-US-00004 TABLE 3 ELISA results of 6 identified clones
producing antibody characterized by specific binding to the
positive screening reagent according to Formula III B but not to
the negative screening reagent according to Formula II.
Dig-3-CME-AMCAP-beta-ala- Dig-3-CME-AMCAP-DADOO- clone biotin
(Formula III B) biotin (Formula II) D 2.417 0.089 K 2.897 0.099 F
1.172 0.080 G 3.172 0.093 H 0.685 0.066 L 3.094 0.120
Example 8
Binding of Biotin by Monoclonal Antibodies
[0204] To demonstrate that the newly generated desired antibodies
(including those represented by the clones shown in Table 3)
additionally bind to free biotin, a competition ELISA assay was
designed and performed.
[0205] For this purpose the antibodies were firstly cloned as
described in Seeber et al. (2014), PLoS One. 2014 Feb. 4; 9(2). To
generate antibody containing supernatants, HEK cells were
transiently transfected with the expression plasmids coding for the
relevant heavy and light chains of the anti-biotin antibodies.
After 1 week of culture time the concentration of the anti-biotin
antibodies in the transient transfections were determined and the
concentrations of each of the selected clones were adjusted to 5
.mu.g/ml.
[0206] For the ELISA 96 well plates were coated with 5 .mu.g/mL
polyclonal goat anti-rabbit-IgG antibody. After washing, the plates
were blocked with 5% BSA to reduce background signals. The plates
were washed again and 30 .mu.l of the antibody supernatants from
the transient transfections (5 .mu.g/ml) were transferred into each
well of the 96 well plates, and incubated for 1 h at room
temperature.
[0207] Each of the selected clones was added to 8 different wells
in a row to perform a biotin titration.
[0208] Following incubation, the plates were washed again 3 times
to eliminate the streptavidin/biotin complexes present in the clone
supernatants. For the positive control, 50 ng/mL of the positive
screening reagent, Dig-3-CME-AMCAP-beta-ala-biotin (Formula III B)
was added to one of the wells for each antibody, and incubated for
1 h at room temperature.
[0209] To test for the binding of free biotin a titration was made.
For this purpose, free biotin was added to
Dig-3-CME-AMCAP-beta-ala-biotin in increasing concentrations.
Biotin was added at concentration of 10 ng/ml, 20 ng/ml, 40 ng/ml,
80 ng/ml, 160 ng/ml, 320 ng/mL and 640 ng/mL to an amount of 50
ng/mL Dig-3-CME-AMCAP-beta-ala-biotin. Afterwards the plates were
washed again and for the detection of antibodies bound to
Dig-3-CME-AMCAP-beta-ala-biotin, 3 .mu.g/mL of a POD labeled
polyclonal sheep anti-Dig-antibody were added to the plates. After
a final washing step, ABTS (Roche) was added as a POD substrate and
signals were detected by measuring OD (optical density) at 405 nm.
Notably, signal reduction was observed was reduced as a result of
increasing biotin concentrations in the presence of the screening
reagent. This indicates that free-biotin solution and
Dig-3-CME-AMCAP-beta-ala-biotin are competing for binding to the
selected antibodies.
Example 9
Counteracting Biotin Interference in an Immunological Assay
[0210] The control sample for the Elecsys TSH assay, PreciControl
Universal 2 is spiked with rising concentrations of biotin (e.g. 0,
100 and 200 ng/mL final concentration of biotin).
[0211] Reagent 2 of the Elecsys TSH sales kit containing the anti
TSH detection-antibody is used in a unmodified version (control),
and a modified version, additionally containing 300 .mu.g/mL of an
monoclonal anti Biotin antibody according to the invention.
[0212] The sandwich assay is performed according to the routine
assay protocol for the Elecsys TSH assay: Shortly, 50 .mu.L, of
Sample (PreciControl Universal 2 with or without biotin) are
incubated with 60 .mu.L, of Reagent 1 of the Elecsys TSH kit
containing the biotinylated anti-TSH antibody and 50 .mu.L of
Reagent 2 containing the ruthenylated anti-TSH antibody with or
without further addition of 300 .mu.g/mL of the monoclonal anti
Biotin antibody. After incubation at 37.degree. C. for 9 min, 40
.mu.L, suspension of streptavidin coated magnetic beads of the
Elecsys TSH kit are added, the reaction is incubated for another 9
min and finally the reaction mixture is aspirated into the
measuring cell where the microparticles are magnetically captured
onto the surface of the electrode. Unbound substances are then
removed with ProCell. Application of a voltage to the electrode
then induces electrochemiluminescense-based emission of light which
is measured by a photomultiplier.
[0213] Using the control reagent, presence of 100 or 200 ng/mL
biotin in the sample results in a prominent signal drop to 53% or
16% of the reference signal (sample without biotin).
[0214] In contrast to this, in the modified version of the kit
containing the biotin binding antibody in reagent R2, the
interfering effect of biotin is markedly reduced.
Example 10
Kinetic Antibody Screening
[0215] The kinetic screening was performed at 37.degree. C. on a GE
Healthcare Biacore 4000 instrument. A Biacore CM5 series S sensor
was mounted into the instrument and was hydrodynamically addressed
and preconditioned according to the manufacturer's instructions.
The system buffer was HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1
mM EDTA, 0.05% (w/v) P20). The sample buffer was the system buffer
supplemented with 1 mg/ml CMD (Carboxymethyldextran, Fluka).
[0216] A rabbit antibody capture system was established on the
biosensor. A polyclonal goat anti-rabbit IgG Fc capture antibody
GARbFc.gamma. (Code Nr.: 111-005-046, lot#105332, Jackson Immuno
Research) was immobilized according to the manufacturer's
instructions using NHS/EDC chemistry. 30 .mu.g/ml GARbFc.gamma. in
10 mM sodium acetate buffer (pH 4.5) were preconcentrated to the
spots 1, 2, 4 and 5 in the flow cells 1, 2, 3 and 4 and immobilized
with 10.000 RU GARbFc.gamma.. The sensor was subsequently saturated
with 1 M ethanolamine pH 8.5. The Spots 1 and 5 were used for the
interaction measurements and spots 2 and 4 were served as
references. Each rabbit antibody clone suspension was diluted 1:2
in sample buffer and was injected at a flow rate of 30 .mu.l/min
for 1 min. The rabbit antibody Capture Level (CL) in response units
(RU) was monitored.
[0217] Since the molecular weight of the 0.9 kDa
Dig-Biotin-conjugate is too small for the sensitivity range of the
SPR screening instrument, 300 nM Dig-Biotin-conjugate
(Dig-3-cme-Amcap-.beta.-Ala-Biotin (BMO No. 15420318)) were
preincubated for 2 hours at room temperature with 900 nM
<Dig>M-D.G-Fab'. The mAb<Dig>M-D.G-Fab binds to
digoxigenin with KD 5 pM affinity. M-D.G-Fab' also binds with
picomolar affinity to the digoxigenin moiety in the
Dig-Biotin-conjugate and does not interfere with the GARbFc.gamma.
capture system. M-D.G-Fab' does not interfere with anti-biotin
rabbit clones and free d-biotin. Using the 50 kDa M-D.G-Fab', the
0.9 kDa Dig-Biotin-conjugate was loaded with an additional mass.
This results in a highly stable immunocomplex with 50.9 kDa
molecular weight, which is optimal for the sensitivity range of the
SPR screening instrument. M-D.G-Fab' is monomeric and no analyte
avidity effect was generated nor detectable. The preformed
Dig-Biotin-conjugate-M-D.G-Fab' complex was singly injected at 30
.mu.l/min for 5 min to monitor the association phase to the
respective surface displayed anti-biotin rabbit mAb. The
dissociation of the conjugate from the rabbit clone were monitored
for 5 min. After each cycle of kinetic rates determination the
rabbit clones were completely washed from the biosensorcapture
system by a 1 min injection of 10 mM Glycin pH 2.0 followed by a 2
min injection of 10 mM Glycin pH 2.25 at 20 .mu.l/min.
[0218] In a second screening setup, rabbit anti-biotin antibodies,
which were sensitive for d-biotin binding interference, were
identified. An analyte mixture was prepared by overnight incubation
at room temperature, consisting of 300 nM Dig-Biotin-conjugate, 900
nM<Dig>M-D.G-Fab' and 300 nM free d-biotin (d-Biotin, CAS
No.: 58-85-5, Catalog No.: 47868, Supelco). The kinetic screening
was performed as described before but with this analyte mixture in
solution. In another embodiment different Dig-Biotin-conjugate and
<Dig>M-D.G-Fab' concentrations were used.
[0219] FIG. 6 explains the experimental setup for the
screening.
[0220] The kinetic traces of both analyte single concentration
kinetics were monitored by the Biacore 4000 Evaluation Software.
Furthermore, the kinetic data was interpreted by report point
characterisations and kinetic determinations. Two report points,
the recorded signal shortly before the end of the analyte
injection, Binding Late (BL) and the signal shortly before the end
of the dissociation time, Stability Late (SL), were used to
characterize the analyte/antigen binding stability. Furthermore,
the dissociation rate constant k.sub.d (1/s) was calculated
according to a Langmuir model and the antibody/antigen complex
half-life was calculated in minutes according to the Formula
ln(2)/(60*kd). The Molar Ratio, the binding stoichiometry was
calculated with the formula: MW (antibody)/MW (antigen)*BL
(antigen)/CL (antibody).
[0221] Finally, the kinetic traces of each anti-biotin rabbit mAb
were overlayed in one analysis plot. Mainly by visual inspection of
these overlay plots, antibodies were selected, which showed an
effective Dig-Biotin-conjugate binding signal reduction in the
presence of 300 nM free d-biotin. Effective means, more than 90%
Dig-Biotin-conjugate binding signal reduction. In another
embodiment antibodies with 80%, 70%, 60%, and 50% signal reduction
were selected. Selected antibodies were transferred to detailed
IC50 analyses.
[0222] FIG. 4 exemplarily shows three antibody blocking signatures
measured by SPR based kinetic screening. Class A shows full
d-biotin blockage of the Dig-Biotin-conjugate binding. Class B
features an antibody with just moderate sensitivity for the
d-biotin signal interference and Class C is not susceptible to
d-biotin.
Detailed SPR Based Functional Analyses
[0223] Detailed kinetic investigations were performed at 37.degree.
C. on a GE Healthcare T200 instrument. A Biacore CM5 series S
sensor was mounted into the instrument and was hydrodynamically
addressed and preconditioned according to the manufacturer's
instructions. The system buffer was HBS-EP (10 mM HEPES (pH 7.4),
150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). The sample buffer was the
system buffer supplemented with 1 mg/ml CMD (Carboxymethyldextran,
Fluka).
[0224] In one embodiment a rabbit antibody capture system was
established on the CM5 biosensor. A polyclonal goat anti-rabbit IgG
Fc capture antibody GARbFc.gamma. (Code Nr.: 111-005-046,
lot#105332, Jackson Immuno Research) was immobilized according to
the manufacturer's instructions using NHS/EDC chemistry. 30
.mu.g/ml GARbFc.gamma. in 10 mM sodium acetate buffer (pH 4.5) were
preconcentrated to the flow cells 1, 2, 3 and 4 and were
immobilized with 10.000 RU GARbFc.gamma.. The sensor was
subsequently saturated with 1 M ethanolamine pH 8.5.
[0225] Selected rabbit antibody clones from the initial kinetic
screening step were diluted in sample buffer at 500 nM each and
were captured on the biosensor at a flow rate of 5 .mu.l/min for 2
min followed by a 2 min washing step with 10-fold concentrated
HBS-EP system buffer at 60 .mu.l/min. The rabbit antibody Capture
Level (CL) in response units (RU) was monitored. The higher
sensitivity of the T200 instrument circumvented the additional
molecular mass load by <Dig>M-D.G-Fab'. A series of analytes
was injected at 60 .mu.l/min for 5 min association phase and the
dissociation phase was monitored for 5 min. First, the
Dig-Biotin-conjugate analyte was injected at 300 nM omitting
d-biotin in solution. Then, increasing d biotin concentrations of 4
nM, 8 nM, 15 nM, 30 nM, 90 nM and 270 nM were added to the
Dig-Biotin-conjugate mixture. Sensorgram overlay plots were
produced to analyze the Dig-Biotin-conjugate binding signal
suppression by the presence of increasing d-biotin concentrations.
The Binding Late report points from the signal plateau of the
analyte association phase were plotted over the increasing d-biotin
concentrations and the d-biotin IC50 values were determined using
the point-to-point mode in Biaevaluation software. Furthermore, the
sensorgram overlay plots were visually investigated for the
competing performance of d-biotin and a % signal suppression was
estimated. In another embodiment the % signal blocking was
calculated by comparison of the 0 nM d-biotin and the 270 nM
d-biotin sample injections.
[0226] FIG. 6 shows the SPR experimental setup of the IC50
measurements. FIG. 6B shows the preferred embodiment.
[0227] FIG. 5 exemplary shows a result of this competition assay.
Shown is the antibody lead candidate 5D9. The higher the free
d-biotin concentration in the sample, the lower is the
Dig-Biotin-conjugate binding signal and thus it was estimated the
antibody's susceptibility for d-biotin binding. The IC50
determination of the clone L was IC50=60 nM d-biotin.
[0228] In another embodiment the kinetic parameters ka [1/Ms], kd
[1/s], t1/2 diss [min], KD [M] and the binding stoichiometry (Molar
Ratio) of the Dig-Biotin-conjugate binding antibody were
determined.
[0229] The 37.degree. C. kinetics of clone 5D9 were determined with
ka=2.2*10E5 1/Ms, kd=2.0*1-3 (1/s), t1/2 diss=6 min, MR=1.1. The
affinity for the Dig-Biotin-conjugate was KD=9 nM.
[0230] In one embodiment, the T200 instrument assay setup was as
described above. The Dig-Biotin-conjugate was injected in a
concentration serie at 0 nM, 4 nM, 8 nM, 15 nM, 30 nM, 90 nM and
270 nM. Kinetic parameters were determined using the Biacore
evaluation software.
[0231] In another embodiment the kinetic parameters ka [1/Ms], kd
[1/s], t1/2 diss [min], KD [M] and the binding stoichiometry (Molar
Ratio) of the Dig-Biotin-conjugate antibody binding were determined
by an alternative sensor surface setup. In one embodiment a murine
antibody capture system was established on the CM5 biosensor. A
polyclonal rabbit anti-mouse IgG capture antibody (RbAMIgG (rabbit
anti mouse IgG), pAb<M-IgG>Rb-IgG(IS), BR-1008-38, 2017-08,
GE Healthcare) was immobilized according to the manufacturer's
instructions using NHS/EDC chemistry. 30 .mu.g/ml GARbFc.gamma. in
10 mM sodium acetate buffer (pH 5.0) were preconcentrated to the
flow cells 1, 2, 3 and 4 and were immobilized with 10.000 RU. The
sensor was subsequently saturated with 1 M ethanolamine pH 8.5.
[0232] Approximately 300 RU of 30 nM mAb<Dig>M-19/11-IgG(Q)
(Roche, 28 Jan. 1999, entrance 19.5.2017, Id. 2157861) were
captured at 10 .mu.l/min for 1 min. The Dig-Biotin-conjugate was
injected at 150 nM for 2 min at 30 .mu.l/min to be stably captured
by M-19/11-IgG.
[0233] FIG. 6 is describing the assay setup. This assay was
preferably used for the kinetic determination of recombinantely
expressed rabbit anti-biotin Fab' fragments.
[0234] Then anti-biotin rabbit antibodies or fragments thereof were
injected in concentration dependent series as described above in
order to determine binding kinetics.
Example 11
ITC Experiments
[0235] ITC experiments directly determine the affinity of the
selected antibodies to free biotin in solution, and the
thermodynamic parameters and stoichiometry of binding reaction.
[0236] All ITC experiments were conducted on a VP-ITC
microcalorimeter (Malvern Instruments). The experiments were
performed at 25.degree. C. in phosphate buffer (25 mM potassium
phosphate pH 7.4, 150 mM potassium chloride). Protein
concentrations were 750 .mu.g/ml for the exemplary monoclonal
antibodies (see Table 5) resulting in a molar concentration of 10
.mu.M for the paratopes. Biotin concentrations were 100 .mu.M. The
titrations were carried out with a stirring speed of 310 rpm and
200 s time intervals between 10 .mu.l injections. The first
injection for each sample was excluded from the data fitting. The
experimental data were fitted to a theoretical curve using the
NanoAnalyze Data Analysis software package (Version 3.6.0) to
afford values for K.sub.d (dissociation constant in M), n
(stoichiometry of binding) and .DELTA.H (the change in enthalpy in
kcal/mol). The thermodynamic parameters (.DELTA.G and .DELTA.S)
were calculated from K.sub.d and .DELTA.H using the equation:
.DELTA.G=RT ln(K.sub.d)=.DELTA.H-T.DELTA.S
where R is the universal gas constant, T the temperature and
.DELTA.G, .DELTA.H and .DELTA.S are the changes in Gibbs free
energy, enthalpy and entropy.
TABLE-US-00005 TABLE 5 Exemplary monoclonal antibody K.sub.d
.DELTA.H .DELTA.G -T.DELTA.S nM n kcal/mol kcal/mol kcal/mol N 5.5
1.09 -13 -11.3 2 M 10.5 0.92 -21 -10.9 10 L 4.4 1.07 -17 -11.4 5 J
1.6 1.03 -20 -12.0 8 K 0.7 1.00 -16 -12.5 3 G 1.0 1.04 -15 -12.3 3
F 1.0 1.00 -20 -12.3 8
Example 12
[0237] Purification of Fab Fragments from Prokaryotic Cell
Fermentation
[0238] The expressed polypeptides of the present invention are
secreted into and recovered from the periplasm of the host cells.
The cells may be harvested by centrifugation or filtration. After
resuspension of the biomass in an appropriate buffer with a pH
between 2.0 and 8.0 or using the complete fermentation broth the
cells are disrupted by physical, chemical or enzymatic methods.
Once cells are disrupted, cell debris or whole cells may be removed
by centrifugation or filtration. The purification of the
polypeptides may involve several precipitation steps and
fractionation on cation, anion exchange chromatography, hydrophobic
interaction, mixed mode, affinity or gel filtration chromatography.
For the removal of trace amounts of bound biotin a treatment of the
polypeptide solution with a binding agent e.g. streptavidin may be
necessary. For the final formulation of the polypeptide dialysis,
ultrafiltration or ultradialysis steps may be conducted. The
polypeptide may be stored as liquid, a frozen liquid or as a spray
dried or freeze dried solid.
* * * * *
References