U.S. patent application number 15/188870 was filed with the patent office on 2017-04-20 for method for obtaining immunoglobulin encoding nucleic acid.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Hans-Willi Krell, Alexander Lifke, Valeria Lifke, Kairat Madin, Christian Weilke.
Application Number | 20170107550 15/188870 |
Document ID | / |
Family ID | 40790896 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170107550 |
Kind Code |
A1 |
Krell; Hans-Willi ; et
al. |
April 20, 2017 |
METHOD FOR OBTAINING IMMUNOGLOBULIN ENCODING NUCLEIC ACID
Abstract
The current invention is directed to a method for obtaining a
nucleic acid encoding an immunoglobulin variable domain from a
single cell comprising the following steps: performing a first
polymerase chain reaction with three to six 5'-primer and one
3'-primer, performing with the product of the first polymerase
chain reaction a second polymerase chain reaction with thirteen to
sixteen 5'-primer and one 3'-primer, whereby the distance of the
binding locations of the primer employed in the second polymerase
chain reaction is reduced compared to the first polymerase chain
reaction.
Inventors: |
Krell; Hans-Willi;
(Penzberg, DE) ; Lifke; Alexander; (Penzberg,
DE) ; Lifke; Valeria; (Penzberg, DE) ; Madin;
Kairat; (Penzberg, DE) ; Weilke; Christian;
(Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
40790896 |
Appl. No.: |
15/188870 |
Filed: |
June 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13202446 |
Aug 19, 2011 |
9399670 |
|
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PCT/EP2010/001008 |
Feb 18, 2010 |
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15188870 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Q 2600/16 20130101; C07K 2317/14 20130101; C12Q 1/6858 20130101;
C12Q 1/6881 20130101; C12Q 1/6858 20130101; C07K 2317/21 20130101;
C07K 2317/55 20130101; C12Q 1/6804 20130101; C07K 16/00 20130101;
C12Q 2537/143 20130101; C12Q 2549/119 20130101; C12P 21/005
20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12P 19/34 20060101 C12P019/34; C07K 16/00 20060101
C07K016/00; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2009 |
EP |
09002396.1 |
Claims
1. A method for producing an immunoglobulin comprising the
following steps: (a) providing a single immunoglobulin producing
cell, (b) obtaining from said cell a nucleic acid, wherein said
nucleic acid encodes an immunoglobulin variable domain selected
from a heavy chain variable domain, a kappa light chain variable
domain, and a lambda light chain variable domain, comprising: (a)
performing a first polymerase chain reaction (PCR) to obtain a
first PCR product; and (b) performing a second PCR with the first
PCR product, whereby the distance of the binding locations of the
primers employed in the second PCR is reduced compared to the
distance in the first PCR, wherein: i) for obtaining the nucleic
acid encoding the immunoglobulin heavy chain variable domain, the
first PCR is performed with 5' primers comprising the nucleic acids
of SEQ ID NO: 05 and/or 06, 07 and/or 08, 09, 10 and/or 11, 12, 13,
and with 3' primers comprising the nucleic acids of SEQ ID NO:104
or 105 or 106, and the second PCR is performed with 5' primers
comprising the nucleic acids of SEQ ID NO: 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, and/or 142 and with 3'
primers comprising the nucleic acids of SEQ ID NO: 104 or 105 or
106 or 143, and ii) for obtaining the nucleic acid encoding the
immunoglobulin kappa light chain variable domain, the first PCR is
performed with 5' primers comprising the nucleic acids of SEQ ID
NO: 16, 17, 18, 19, and with a 3' primer comprising the nucleic
acid of SEQ ID NO: 115, and the second PCR is performed with 5'
primers comprising the nucleic acids of SEQ ID NO: 53 and/or 54, 55
and/or 56, 57 and/or 58, 59, 60, 61 and/or 62, 63 and/or 64, 65,
66, 67, 68, 69, 70, 144, and with 3' primers comprising the nucleic
acids of SEQ ID NO: 115 and/or 145, and iii) for obtaining the
nucleic acid encoding the immunoglobulin lambda light chain
variable domain, the first PCR is performed with 5' primers
comprising the nucleic acids of SEQ ID NO: 21, 22, 23 and/or 24
and/or 25 and/or 26, and with 3' primers comprising the nucleic
acids of SEQ ID NO:120 and/or 121 and/or 122 and/or 123 and/or 124
and/or 125, and the second PCR is performed with 5' primers
comprising the nucleic acids of SEQ ID NO: 72, 73 and/or 74, 75,
76, 77 and/or 78, 79, 80, 81, 82 and/or 83, 84 and/or 85, 86, 87
and/or 88, 89, 90 and/or 91, 92, and 3' primers comprising the
nucleic acid of SEQ ID NO: 120 and/or 121 and/or 122 and/or 123
and/or 124 and/or 125; (c) combining the nucleic acid encoding the
light chain variable domain with a nucleic acid encoding the
immunoglobulin light chain constant domain and combining the
nucleic acid encoding the heavy chain variable domain with a
nucleic acid encoding an immunoglobulin heavy chain constant
region, (d) transfecting a eukaryotic cell with the combined
nucleic acids, (e) cultivating said transfected cell under
conditions suitable for the expression of the immunoglobulin, (f)
recovering the immunoglobulin from the cell or the cultivation
medium; and thereby producing the immunoglobulin.
2. The method of claim 1, wherein the 3'-primer in the second PCR
is the same as in the first PCR and at least one 5'-primer is
changed, and whereby in the second PCR the number of nucleotides
between the 5'-end of each of the 5'-primer and the 3'-end of the
3'-primer is reduced compared to the number of nucleotides between
the 5'-end of each of the 5'-primer and the 3'-end of the 3'-primer
in the first PCR, when bound to the nucleic acid to be
amplified.
3. The method of claim 1, wherein the primers employed in the
second PCR provide for overhangs encoding the translational start
codon ATG for the 5'-primers and/or the translational stop codon
TTA for the 3'-primer.
4. The method of claim 1, wherein said method comprises the first
step of: providing a single cell and obtaining the mRNA of said
cell.
5. The method of claim 4, wherein said method further comprises the
following second step: --obtaining cDNA from said mRNA with a
reverse transcriptase PCR.
6. The method of claim 1, wherein the single cell is a B-cell, a
plasmablast, or a plasma cell.
7. The method of claim 1, wherein said immunoglobulin is a human
immunoglobulin.
8. The method of claim 1, wherein said immunoglobulin is an
immunoglobulin of the class G (IgG).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/202,446, filed Aug. 19, 2011, which is a national stage
application of International Application No. PCT/EP2010/001008
having an international filing date of Feb. 18, 2010 and which
claims benefit under 35 U.S.C. .sctn.119 to European Patent
Application No. 09002396.1, filed Feb. 20, 2009, the contents of
which are hereby incorporated by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
Jun. 21, 2016, is named P04661_US_1_Sequence_Listing.txt and is
29,346 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to a method and means for
obtaining immunoglobulin encoding nucleic acid from a single
immunoglobulin producing cell with a multiplexed polymerase chain
reaction (PCR), and also to a method for producing an
immunoglobulin whereby the immunoglobulin encoding nucleic acid is
obtained from a single immunoglobulin producing cell in combination
with in vitro translation. Also encompassed by the current
invention is a method for characterization of recombinantly
produced human Fab-fragments.
BACKGROUND OF THE INVENTION
[0004] Since the establishment of hybridoma technology (Cole, S. P.
C., et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991)
86-95), monoclonal immunoglobulins have emerged to play a pivotal
role in scientific research, human healthcare and diagnostics.
Consequently, the generation of monoclonal, especially therapeutic,
immunoglobulins is a field undergoing intensive research. In this
respect, the hybridoma technology and phage display technology
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597)
are, amongst others, two commonly used technologies for the
generation of monoclonal immunoglobulins. In hybridoma technology
obtaining of stable clones is a hurdle, thus, diminishing diversity
of the antibodies, as only a limited number of B-cells are
successfully fused, propagated and thereafter characterized.
Similarly, a drawback of phage or yeast display-based combinatorial
library approaches is the random pairing of the immunoglobulin
heavy and light chains. The dissociation of the original heavy and
light chain pairing, and non-cognate pairing, necessitate the
screening of a large number of immunoglobulin producing cells in
order to identify heavy and light chain pairs of high affinity. In
addition, such non-cognate pairs may display unwanted
cross-reactivity to human antigens. Finally, the genetic diversity
of target-specific immunoglobulins identified by selection and
screening of combinatorial libraries is commonly limited due to
inherent selection biases.
[0005] Generation of immunoglobulins from immunoglobulin producing
cell can be performed according to methods known in the art. Such
methods are e.g. hybridoma technique. A different method is based
on the identification of the nucleic acid sequence of the
immunoglobulin. Usually it is sufficient to identify the sequence
of the variable regions or even only the CDR regions or only the
CDR3 region. For example, the mRNA is isolated from a pool of
immunoglobulin producing cells and is used for the construction of
a cDNA-library encoding the CDR regions of the immunoglobulin. The
cDNA-library is then transfected into a suitable host cell, such as
NS0 or CHO, and screened for specific immunoglobulin
production.
[0006] WO 2008/104184 reports a method for cloning cognate
antibodies. The efficient generation of monoclonal antibodies from
single human B cells is reported by Tiller et al. (Tiller, T., et
al., J. Immunol. Meth. 329 (2007) 112-124). Braeuninger et al.
(Braeuninger, A., et al., Blood 93 (1999) 2679-2687) report the
molecular analysis of single B cells from T-cell-rich B-cell
lymphoma. Systematic design and testing of nested (RT-) PCR primer
is reported by Rohatgi et al. (Rohatgi, S., et al, J. Immunol.
Meth. 339 (2008) 205-219). In WO 02/13862 a method and composition
for altering a B-cell mediated pathology are reported. Haurum et
al. (Meijer, P. J. and Haurum, J. S., J. Mol. Biol. 358 (2006)
764-772) report a one-step RT-multiplex overlap extension PCR.
Stollar et al. and Junghans et al. report the sequence analysis by
single cell PCR reaction (Wang, X. and Stollar, B. D., J. Immunol.
Meth. 244 (2000) 217-225; Coronella, J. A. and Junghans, R. P.,
Nucl. Acids Res. 28 (2000) E85). Jiang, X. and Nakano, H., et al.
(Biotechnol. Prog. 22 (2006) 979-988) report the construction of a
linear expression element for in vitro transcription and
translation.
SUMMARY OF THE INVENTION
[0007] The current invention is directed in specific embodiments of
an aspect to a method for providing a human monoclonal antibody
comprising the in vitro translation of a nucleic acid encoding
human immunoglobulin G fragments whereby the nucleic acid is
obtained by specific amplification of cDNA fragments obtained from
the mRNA of a single immunoglobulin producing human B-cell,
plasmablast or plasma cell or a B-cell of an animal comprising a
human immunoglobulin locus.
[0008] With this method it is possible to characterize each of a
number of provided B-cells with respect to the antigen binding
characteristics of the produced immunoglobulin. Thus, no loss of
immunoglobulin diversity occurs. As the analyzed B-cells are mature
B-cells obtained after the in vivo maturation process it is very
unlikely that their produced immunoglobulins show cross-reactivity
with other antigens.
[0009] The invention comprises a method for the multiplex
semi-nested PCR and multiplex one tube RT-GSP-PCR (RT-Gene Specific
Primer-PCR) for the amplification of cognate IgG HC and IgG LC
chains (human IgG isotype) from a single B-cell or plasmablast or
plasma cell. The Fab PCR product is subsequently transcribed to
mRNA and translated in vitro in E. coli lysate. The expression was
examined using ELISA and Western blot.
[0010] The current invention comprises as first aspect a method for
obtaining a nucleic acid encoding an immunoglobulin variable domain
from a single cell comprising the following steps: [0011] obtaining
a first nucleic acid composition by performing a first polymerase
chain reaction with three to six 5'-primer and one 3'-primer,
[0012] obtaining a nucleic acid encoding an immunoglobulin variable
domain by performing with the composition obtained in the first
polymerase chain reaction a second polymerase chain reaction with
thirteen to sixteen 5'-primer and one 3'-primer, [0013] whereby,
when bound to the nucleic acid to be amplified, the distance of the
binding locations of the 5'-primer to the binding location of the
3'-primer employed in the second polymerase chain reaction is
reduced compared to that of the first polymerase chain
reaction.
[0014] A second aspect of the current invention is a method for
obtaining a nucleic acid encoding an immunoglobulin variable domain
from a single cell comprising the following steps: [0015] obtaining
a nucleic acid composition by performing a first polymerase chain
reaction with four to six 5'-primer and one 3'-primer, [0016]
obtaining a nucleic acid encoding an immunoglobulin variable domain
by performing with the composition obtained in the first polymerase
chain reaction a second polymerase chain reaction with thirteen to
fifteen 5'-primer and one 3'-primer, [0017] whereby in the second
polymerase chain reaction either the 5'-primer are the same as that
in the first polymerase chain reaction and the 3'-primer is changed
or the 3'-primer is the same as in the first polymerase chain
reaction and at least one 5'-primer is changed, [0018] whereby,
when bound to the nucleic acid to be amplified, the number of
nucleotides between the 5'-end of each of the 5'-primer and the
3'-end of the 3'-primer in the second polymerase chain reaction is
reduced compared to the number of nucleotides between the 5'-end of
each of the 5'-primer and the 3'-end of the 3'-primer in the first
polymerase chain reaction.
[0019] A further aspect of the current invention is a method for
obtaining a nucleic acid encoding an immunoglobulin variable domain
from a single cell by a multiplex one tube RT-GSP-PCR comprising
the following step: [0020] performing a reverse transcription and
polymerase chain reaction in one step with one 5'-primer and one
3'-primer.
[0021] In one embodiment the methods according to the invention are
characterized in that the 5'-primer employed in the first
polymerase chain reaction bind in the coding region for the leader
peptide of the immunoglobulin. In another embodiment the methods
according to the invention are characterized in that the 5'-primer
employed in the second polymerase chain reaction or the 5'-primer
employed in a multiplex one tube RT-GSP-PCR bind in the coding
region of the first framework region of the immunoglobulin. In a
further embodiment the methods according to the invention are
characterized in that the primer employed in the second polymerase
chain reaction provide for overhangs encoding the translational
start codon ATG for 5'-primer and/or the translational stop codon
TTA for 3'-primer. In still a further embodiment the methods
according to the invention are characterized in comprising the
additional step of: [0022] providing a single cell and obtaining
the mRNA of this cell.
[0023] In a further embodiment the methods according to the
previous embodiment are characterized in comprising the following
second step: [0024] obtaining cDNA from the provided mRNA with a
reverse transcriptase polymerase chain reaction (RT-PCR).
[0025] In another embodiment the methods according to the invention
are characterized in that six 5'-primer and one 3'-primer are
employed in the first polymerase chain reaction. In still a further
embodiment the methods according to the invention are characterized
in that four 5'-primer and one 3'-primer are employed in the second
polymerase chain reaction. In a further embodiment of the current
invention the methods are characterized in that [0026] a) for
obtaining a nucleic acid encoding an immunoglobulin heavy chain
variable domain the primer in the first polymerase chain reaction
comprise the nucleic acids of SEQ ID NO: 05 and/or 06, 07 and/or
08, 09, 10 and/or 11, 12, 13, and 104 and/or 105 and/or 106, and
the primer in the second polymerase chain reaction comprise the
nucleic acids of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, and 104 and/or 105 and/or 106, and/or
142, and/or 143, [0027] b) for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain the primer in the
first polymerase chain reaction comprise the nucleic acids of SEQ
ID NO: 16, 17, 18, 19, and 115, and the primer in the second
polymerase chain reaction comprise the nucleic acids of SEQ ID NO:
53 and/or 54, 55 and/or 56, 57 and/or 58, 59, 60, 61 and/or 62, 63
and/or 64, 65, 66, 67, 68, 69, 70, and/or 115, and/or 144, and/or
145, [0028] c) for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain the primer in the
first polymerase chain reaction comprise the nucleic acids of SEQ
ID NO: 21, 22, 23 and/or 24 and/or 25 and/or 26, and 120 and/or 121
and/or 122 and/or 123 and/or 124 and/or 125, and the primer in the
second polymerase chain reaction comprise the nucleic acids of SEQ
ID NO: 72, 73 and/or 74, 75, 76, 77 and/or 78, 79, 80, 81, 82
and/or 83, 84 and/or 85, 86, 87 and/or 88, 89, 90 and/or 91, 92,
and 120 and/or 121 and/or 122 and/or 123 and/or 124 and/or 125.
[0029] In one embodiment of the methods according to the invention
the immunoglobulin variable domain is an immunoglobulin heavy chain
variable domain or an immunoglobulin kappa light chain variable
domain or an immunoglobulin lambda light chain variable domain.
[0030] A further aspect of the current invention is a method for
producing an immunoglobulin Fab-fragment comprising the following
steps: [0031] providing a single immunoglobulin producing cell,
[0032] obtaining from the cell the nucleic acid encoding the
immunoglobulin light and heavy chain variable domains, optionally
also encoding a part of the light chain constant domain and a part
of the heavy chain C.sub.H1 domain, [0033] generating a linear
expression matrix comprising the obtained nucleic acid, [0034]
translating in vitro the nucleic acid and thereby producing the
immunoglobulin Fab fragment.
[0035] Another aspect of the current invention is a method for
producing an immunoglobulin comprising the following steps: [0036]
providing a single immunoglobulin producing cell, [0037] obtaining
from the cell the nucleic acid encoding the immunoglobulin light
and heavy chain variable domains, [0038] linking the nucleic acid
encoding the light chain variable domain with a nucleic acid
encoding an immunoglobulin light chain constant domain in operable
form, and linking the nucleic acid encoding the heavy chain
variable domain with a nucleic acid encoding an immunoglobulin
heavy chain constant region in operable form, [0039] transfecting a
eukaryotic or a prokaryotic cell with the nucleic acids obtained in
the previous step, [0040] cultivating the transfected cell, in one
embodiment under conditions suitable for the expression of the
immunoglobulin, [0041] recovering the immunoglobulin from the cell
or the cultivation medium and thereby producing an
immunoglobulin.
[0042] In one embodiment of all methods according to the invention
is the immunoglobulin an immunoglobulin of class G (IgG). In one
embodiment of the methods for producing an immunoglobulin Fab
fragment or an immunoglobulin is the obtaining of the nucleic acid
by a method according to an aspect of the current invention.
DESCRIPTION OF THE INVENTION
[0043] One aspect of the current invention is a method for
obtaining a nucleic acid encoding an immunoglobulin variable domain
from a single cell comprising the following steps: [0044]
performing a first polymerase chain reaction with three to six
5'-primer and one 3'-primer, [0045] performing with the product of
the first polymerase chain reaction a second polymerase chain
reaction with thirteen to sixteen 5'-primer and one 3'-primer,
thereby obtaining a nucleic acid encoding an immunoglobulin
variable domain, [0046] whereby the distance of the binding
locations of the primer employed in the second polymerase chain
reaction is reduced compared to the first polymerase chain
reaction.
[0047] By employing magnetic micro-beads coated with the human pan
B-cell marker, CD19 (see e.g. Bertrand, F. E., III, et al., Blood
90 (1997) 736-744), B-cells were isolated from peripheral blood.
With the limited dilution approach, single cells were placed in a
96 well microtiter plate. The mRNA of these cells was
extracted.
[0048] It has been found that by performing the IgG-specific PCR
amplification according to the current invention for obtaining
nucleic acid encoding an immunoglobulin variable domain from a
single cell in a thereafter following production of the respective
immunoglobulin or Fab-fragment in one embodiment OD-values in the
range of from 0.5 to 2.0 were obtained, and concomitantly, e.g.,
Fab-fragment yields of from 180 to 310 ng/ml were obtained.
[0049] With the methods according to the current invention a
multiplex polymerase chain reaction is used for the amplification
of heavy and light chain variable domains simultaneously in the
same polymerase chain reaction. In contrast to the amplification of
the heavy chain variable domain and the light chain variable domain
in separate reactions the current approach provides for an
increased sensitivity and an increased amount of amplified
sequences. The use of gene-specific primer in both, i.e. all,
polymerase chain reactions enhances the specificity and accuracy of
the methods.
[0050] More complex gene structure in the case of human IgG
requires a different strategy for the primer design, placement and
polymerase chain reaction for the sensitivity and accuracy
required.
[0051] Thus, herein is employed a multiplex polymerase chain
reaction either without or with the linkage of the heavy and light
chain regions that are amplified. For the in vitro translation of
the obtained nucleic acids it is beneficial that the encoded
domains comprise cysteine residues suitable for the formation of
interchain disulfide bonds.
[0052] Methods and techniques known to a person skilled in the art,
which are useful for carrying out the current invention, are
described e.g. in Ausubel, F. M., ed., Current Protocols in
Molecular Biology, Volumes I to III (1997), Wiley and Sons;
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), Morrison, S. L., et al., Proc. Natl. Acad.
Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat.
No. 5,204,244.
[0053] The term "immunoglobulin" denotes a protein consisting of
one or more polypeptide(s) substantially encoded by immunoglobulin
genes. The recognized immunoglobulin genes include the different
constant region genes as well as the myriad immunoglobulin variable
region genes. Immunoglobulins may exist in a variety of formats,
including, for example, Fv, Fab, and F(ab).sub.2 as well as single
chains (scFv) or diabodies (e.g. Huston, J. S., et al., Proc. Natl.
Acad. Sci. USA 85 (1988) 5879-5883; Bird, R. E., et al., Science
242 (1988) 423-426; Hood, L. E. et al., Immunology, Benjamin N.Y.,
2nd edition (1984); Hunkapiller, T. and Hood, L., Nature 323 (1986)
15-16).
[0054] An immunoglobulin in general comprises two so called light
chain polypeptides (light chain) and two so called heavy chain
polypeptides (heavy chain). Each of the heavy and light chain
polypeptides contains a variable domain (variable region)
(generally the amino terminal portion of the polypeptide chain)
comprising binding regions that are able to interact with an
antigen. Each of the heavy and light chain polypeptides comprises a
constant region (generally the carboxyl terminal portion).
[0055] The constant region of the heavy chain mediates the binding
of the antibody i) to cells bearing a Fc gamma receptor
(Fc.gamma.R), such as phagocytic cells, or ii) to cells bearing the
neonatal Fc receptor (FcRn) also known as Brambell receptor. It
also mediates the binding to some factors including factors of the
classical complement system such as component (C1q). The variable
domain of an immunoglobulin's light or heavy chain in turn
comprises different segments, i.e. four framework regions (FR) and
three hypervariable regions (CDR).
[0056] Genetic engineering of immunoglobulins is e.g. described in
Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984)
6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244;
Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger, M. S.,
et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23
(2005) 1117-1125.
[0057] The term "chimeric immunoglobulin" denotes an
immunoglobulin, preferably a monoclonal immunoglobulin, comprising
a variable domain, i.e. binding region, from a first non-human
species and at least a portion of a constant region derived from a
second different source or species. Chimeric immunoglobulins are
generally prepared by recombinant DNA techniques. In one embodiment
chimeric immunoglobulins comprise a mouse, rat, hamster, rabbit, or
sheep variable domain and a human constant region. In one
embodiment the human heavy chain constant region is a human IgG
constant region. In another embodiment the human light chain
constant region is a kappa chain or a lambda chain.
[0058] Other forms of chimeric immunoglobulins encompassed by the
present invention are those in which the class or subclass of the
non-human immunoglobulin from which the variable domain is derived
has been changed. Such immunoglobulins are also referred to as
"class-switched immunoglobulins". Forms of "class-switched
immunoglobulins" encompassed by the present invention are also
those in which the constant region has differences from the
wild-type constant region sequence that result in an immunoglobulin
with different properties, e.g. in regard to C1q binding and/or Fc
receptor (FcR) binding. The "Fc part" of an immunoglobulin is not
directly involved in binding to the antigen, but exhibit various
effector functions. Depending on the amino acid sequence of the
constant region of the heavy chain, immunoglobulins are divided in
the classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are
further divided into subclasses, i.e. IgG in IgG1, IgG2, IgG3, and
IgG4, or IgA in IgA1 and IgA2. According to the immunoglobulin
class to which an immunoglobulin belongs the heavy chain constant
regions of immunoglobulins are called .alpha. (IgA), .delta. (IgD),
.epsilon. (IgE), .gamma. (IgG), and .mu. (IgM),
.quadrature.respectively. The immunoglobulins according to the
invention belong in one embodiment to the IgG class. An "Fc part of
an immunoglobulin" is a term well known to the skilled artisan and
defined on basis of the papain cleavage of immunoglobulins. In one
embodiment of the invention the immunoglobulin contains as Fc part
a human Fc part or an Fc part derived from human origin. In a
further embodiment of the invention is the Fc part either an Fc
part of a human immunoglobulin of the subclass IgG4 or IgG1 or is
an Fc part of a human antibody of the subclass IgG1, IgG2, or IgG3,
which is modified in such a way that no Fc.gamma. receptor (e.g.
Fc.gamma.RIIIa) binding and/or no C1q binding as defined below can
be detected. In one embodiment the Fc part is a human Fc part, in
another embodiment a human IgG4 or IgG1 subclass Fc part or a
mutated Fc part from human IgG1 subclass. In a further embodiment
the Fc part is from human IgG1 subclass with mutations L234A and
L235A. While IgG4 shows reduced Fc.gamma. receptor (Fc.gamma.RIIIa)
binding, immunoglobulins of other IgG subclasses show strong
binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc
carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253,
Ser254, Lys288, Thr307, Gln311, Asn434, or/and His435 are residues
which, if altered, provide also reduced Fc.gamma. receptor binding
(Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund,
J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al.,
Immunology 86 (1995) 319-324; EP 0 307 434). In one embodiment the
immunoglobulin is in regard to Fc.gamma. receptor binding of IgG4
or IgG1 subclass or of IgG1 or IgG2 subclass, with a mutation in
L234, L235, and/or D265, and/or contains the PVA236 mutation. In
another embodiment the mutations are S228P, L234A, L235A, L235E,
and/or PVA236 (PVA236 means that the amino acid sequence ELLG
(given in one letter amino acid code) from amino acid position 233
to 236 of IgG1 or EFLG of IgG4 is replaced by PVA). In a further
embodiment the mutations are S228P of IgG4, and L234A and L235A of
IgG1. The Fc part of an immunoglobulin is directly involved in ADCC
(antibody-dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity). An immunoglobulin which does
not bind Fc.gamma. receptor and/or complement factor C1q does not
elicit antibody-dependent cellular cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC). In one embodiment the
heavy chain constant region has an amino acid sequences of SEQ ID
NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 01 with mutations L234A and
L235A, or SEQ ID NO: 02 with mutation S228P, and the light chain
constant region has an amino acid sequence of SEQ ID NO: 03 or SEQ
ID NO: 04.
[0059] "Humanized" or "CDR-grafted" forms of non-human (e.g. rodent
or rabbit) immunoglobulins are immunoglobulins that contain partial
sequences derived from a non-human immunoglobulin and partial
sequences derived from a human immunoglobulin. For the most part,
humanized immunoglobulins are derived from a human immunoglobulin
(recipient or acceptor immunoglobulin), in which residues from a
hypervariable region are replaced by residues from a hypervariable
region of a non-human species (donor immunoglobulin), such as
mouse, rat, hamster, rabbit, or non-human primate, having the
desired specificity and affinity (see e.g. Morrison, S. L., et al.,
Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.
5,202,238; U.S. Pat. No. 5,204,244). In some instances, framework
region (FR) residues of the acceptor immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized
immunoglobulins may comprise further modifications, e.g. amino acid
residues that are not found in the acceptor immunoglobulin or in
the donor immunoglobulin. Such modifications result in variants of
such recipient or donor immunoglobulin, which are homologous but
not identical to the corresponding parent sequence.
[0060] Methods for humanizing non-human immunoglobulin have been
described in the art. Generally, a humanized immunoglobulin
comprises one or more amino acid residues introduced into it from a
source which is non-human. These non-human amino acid residues are
often referred to as "import" residues, which are typically taken
from an "import" variable domain. Humanization can be essentially
performed following the method of Winter and co-workers by
substituting hypervariable region sequences for the corresponding
sequences of a non-human immunoglobulin (see e.g. Winter, G. and
Harris, W. J., Immunol. Today 14 (1993) 243-246).
[0061] The term "human immunoglobulin" as used herein, denotes an
immunoglobulin having variable and constant regions (domains)
derived from human germ line immunoglobulin sequences and having
high sequence similarity or identity with these germ line
sequences. The variable heavy chain region is in one embodiment
derived from germline sequence DP-50 (GenBank L06618) and the
variable light chain region is derived from germline sequence L6
(GenBank X01668) or the variable heavy chain region is derived
DP-61 (GenBank M99682) and the variable light chain region is
derived from germline sequence L15 (GenBank 1(01323). The constant
regions of the antibody are constant regions of human IgG1 or IgG4
type or a variant thereof. Such regions can be allotypic and are
described by, e.g., Johnson, G. and Wu, T. T., Nucleic Acids Res.
28 (2000) 214-218, and the databases referenced therein.
[0062] The term "recombinant immunoglobulin" as used herein denotes
an immunoglobulin that is prepared, expressed, or created by
recombinant means, such as immunoglobulins isolated from host
cells, such as E. coli, NS0, BHK, or CHO cells, or from an animal
(e.g. a mouse or rabbit) that is transgenic for human
immunoglobulin genes. "Recombinant human immunoglobulins" according
to the invention have in one embodiment variable and constant
regions in a rearranged form. The recombinant human immunoglobulins
according to the invention have been subjected to in vivo somatic
hypermutation. Thus, the amino acid sequences of the VH and VL
regions of the recombinant human immunoglobulins are sequences that
can be assigned to defined human germ line VH and VL sequences, but
may not naturally exist within the human antibody germ line
repertoire in vivo.
[0063] The term "monoclonal immunoglobulin" as used herein refers
to an immunoglobulin obtained from a population of substantially
homogeneous immunoglobulins, i.e. the individual immunoglobulins of
the population are identical except for naturally occurring
mutations that may be present in minor amounts. Monoclonal
immunoglobulins are highly specific, being directed against a
single antigenic site. Furthermore, in contrast to polyclonal
immunoglobulin preparations, which include different
immunoglobulins directed against different antigenic sites
(determinants or epitopes), each monoclonal immunoglobulin is
directed against a single antigenic site. In addition to their
specificity, the monoclonal immunoglobulins are advantageous in
that they may be synthesized uncontaminated by other
immunoglobulins. The modifier "monoclonal" indicates the character
of the immunoglobulin as being obtained from a substantially
homogeneous population of immunoglobulins and is not to be
construed as requiring production of the immunoglobulin by any
particular method.
[0064] Immunoglobulins having "conservative sequence
modifications", which are amino acid sequence modifications which
do not affect or alter the characteristics of the immunoglobulin,
are denoted as "variant immunoglobulins". Modifications can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions include ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. These families include amino acids with basic
side chains (e.g. lysine, arginine, histidine), acidic side chains
(e.g. aspartic acid, glutamic acid), uncharged polar side chains
(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, tryptophan), non-polar side chains (e.g. alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g. threonine, valine, isoleucine), and
aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted amino acid residue not essential for
antigen binding in an immunoglobulin can be replaced with another
amino acid residue from the same side chain family.
[0065] The term "variable domain" (variable domain of a light chain
(V.sub.L), variable domain of a heavy chain (V.sub.H)) as used
herein denotes each of the individual domains of a pair of light
and heavy chains of an immunoglobulin which are directly involved
in the binding of the target antigen. The variable domains are
generally the N-terminal domains of light and heavy chains. The
variable domains of the light and heavy chain have the same general
structure, i.e. they possess an "immunoglobulin framework", and
each domain comprises four "framework regions" (FR), whose
sequences are widely conserved, connected by three "hypervariable
regions" (or "complementarity determining regions", CDRs). The
terms "complementary determining region" (CDR) or "hypervariable
region" (HVR), which are used interchangeably within the current
application, denote the amino acid residues of an antibody which
are mainly involved in antigen-binding. "Framework" regions (FR)
are those variable domain regions other than the hypervariable
regions. Therefore, the light and heavy chain variable domains of
an immunoglobulin comprise from N- to C-terminus the regions FR1,
CDR1, FR2, CDR2, FR3, CDR3, and FR4. The framework regions adopt a
n-sheet conformation and the CDRs form loops connecting the n-sheet
structure. The CDRs in each chain are held in their
three-dimensional structure by the framework regions and form
together with the CDRs from the other chain the antigen binding
site. The immunoglobulin heavy and light chain CDR3 region plays a
particularly important role in the binding specificity/affinity of
the immunoglobulin. CDR and FR regions are determined according to
the standard definition of Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991).
[0066] The term "amino acid" as used within this application
denotes the group of carboxy .alpha.-amino acids, which directly or
in form of a precursor can be encoded by nucleic acid. The
individual amino acids are encoded by nucleic acids consisting of
three nucleotides, so called codons or base-triplets. Each amino
acid is encoded by at least one codon. The encoding of the same
amino acid by different codons is known as "degeneration of the
genetic code". The term "amino acid" as used within this
application denotes the naturally occurring carboxy .alpha.-amino
acids and comprises alanine (three letter code: ala, one letter
code: A), arginine (arg, R), asparagine (asn, N), aspartic acid
(asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid
(glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile,
I), leucine (leu, L), lysine (lys, K), methionine (met, M),
phenylalanine (phe, F), proline (pro, P), serine (ser, S),
threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and
valine (val, V).
[0067] A "nucleic acid" or a "nucleic acid sequence", which terms
are used interchangeably within this application, refers to a
polymeric molecule consisting of the individual nucleotides (also
called bases) `a`, `c`, `g`, and `t` (or `u` in RNA), i.e. to DNA,
RNA, or modifications thereof. This polynucleotide molecule can be
a naturally occurring polynucleotide molecule or a synthetic
polynucleotide molecule or a combination of one or more naturally
occurring polynucleotide molecules with one or more synthetic
polynucleotide molecules. Also encompassed by this definition are
naturally occurring polynucleotide molecules in which one or more
nucleotides are changed (e.g. by mutagenesis), deleted, or added. A
nucleic acid can either be isolated, or integrated in another
nucleic acid, e.g. in an expression cassette, a plasmid, or the
chromosome of a host cell. A nucleic acid is characterized by its
nucleic acid sequence consisting of individual nucleotides.
[0068] To a person skilled in the art procedures and methods are
well known to convert an amino acid sequence, e.g. of a
polypeptide, into a corresponding nucleic acid sequence encoding
this amino acid sequence. Therefore, a nucleic acid is
characterized by its nucleic acid sequence consisting of individual
nucleotides and likewise by the amino acid sequence of a
polypeptide encoded thereby.
[0069] It has now been found that a nucleic acid encoding a
monoclonal immunoglobulin can be obtained from a single cell with a
method according to the invention comprising a polymerase chain
reaction (PCR). Further it has been found that with a combination
of the PCR method according to the invention and an in vitro
translation method the nucleic acid encoding a monoclonal
immunoglobulin can be obtained from a single cell and the encoded
immunoglobulin can be provided in quantities sufficient for the
characterization of the immunoglobulin's binding properties. In
order to amplify the very low amount of mRNA obtained from a single
cell, the individual PCR (polymerase chain reaction) has to be very
sensitive and a combination of more than one PCR has to be
performed.
[0070] Thus, it has been found that based on the amplification of
nucleic acid encoding cognate IgG HC (immunoglobulin G heavy chain)
and IgG LC (immunoglobulin G light chain) of an IgG isotype
immunoglobulin from a single cell with subsequent in vitro
translation of the obtained amplified nucleic acid Fab fragments or
complete immunoglobulins can be provided. With this method a high
sensitive method for obtaining information about an immunoglobulin
produced by a single cell is provided. This is possible even from
the minute amounts of mRNA of a single cell. The method according
to the invention allows for the biochemical characterization of the
binding characteristics of an immunoglobulin expressed by a single
B-cell. Thus, with this method characterization of a higher
diversity as opposed to the hybridoma technology is possible.
Furthermore, as the cognate immunoglobulin chains are obtained from
mature B-cells after antigen contact, selectively the nucleic acids
encoding high specific and correctly assembled immunoglobulins are
obtained.
[0071] The method according to the current invention for obtaining
the nucleic acid encoding an immunoglobulin form a single cell
comprises a multiplex semi-nested PCR for the amplification of
cognate IgG HC and IgG LC (human IgG isotype) from a single B-cell.
For characterization of the binding characteristics of the
immunoglobulin encoded by the obtained nucleic acid, the PCR
product was transformed to a nucleic acid encoding the
corresponding Fab-fragment. Thereafter the Fab-fragment was
translated in vitro in E. coli lysate. The expression was confirmed
using ELISA and Western blot methods.
[0072] In general one aspect of the current invention is a method
employing the following steps i) isolating with magnetic
micro-beads coated with human CD19 B-cells from peripheral blood,
ii) depositing single cells e.g. by limited dilution or FACS, iii)
extracting the mRNA of the individualized B-cells, iv) obtaining
one or more nucleic acids encoding at least the variable domains
(VH and VL) of the immunoglobulin produced by the individualized
B-cell, v) translating in vitro a linear RNA template, and
optionally vi) characterizing the binding properties of the
immunoglobulin or immunoglobulin fragment.
[0073] The IgG-specific PCR amplification according to the current
invention was optimized and modified resulting in an increase in
determined OD-values and, thus, obtained immunoglobulin or
immunoglobulin fragment after in vitro translation.
[0074] Three novel PCR-based approaches were established which are
highly sensitive and result in high recovery of the amplified
nucleic acids encoding the immunoglobulin's heavy and light chains
or fragments thereof. Also provided is a method for the expression
of functional and stable Fab fragments after in vitro translation
of nucleic acid obtained with the PCR-based method according to the
invention.
[0075] The terms "polymerase chain reaction" and "PCR", which are
used interchangeably in this application, denote a method for
specifically amplifying a region of nucleic acids, e.g. of DNA or
RNA. This method has been developed by K. Mullis (see e.g. Winkler,
M. E., et al., Proc. Natl. Acad. Sci. USA 79 (1982) 2181-2185). The
region can be a single gene, a part of a gene, a coding or a
non-coding sequence. Most PCR methods typically amplify DNA
fragments of hundreds of base pairs (bp), although some techniques
allow for amplification of fragments up to 40 kilo base pairs (kb)
in size. A basic PCR set up requires several components and
reagents. These components include a nucleic acid template that
contains the region to be amplified, two primer complementary to
the 5'- and 3'-end of the region to be amplified, a polymerase,
such as Taq polymerase or another thermostable polymerase,
deoxynucleotide triphosphates (dNTPs) from which the polymerase
synthesizes a new strand, a buffer solution providing a suitable
chemical environment for optimum activity and stability of the
polymerase, divalent cations, generally Mg.sup.2+, and finally,
monovalent cations like potassium ions.
[0076] The term "semi-nested PCR" denotes two successive polymerase
chain reactions each employing at least a pair of PCR primer,
wherein in the first polymerase chain reaction a first pair of
primer is employed and in the second polymerase chain reaction a
second pair of primer is employed. In the first and second pair of
primer one of the primer is the same and the other primer is
changed, whereby the distance, i.e. the number of nucleotides,
between the 3'-end of the first primer and the 5'-end of the second
primer is reduced in the pair of primer used in the second
polymerase chain reaction compared to the pair of primer used in
the first polymerase chain reaction. The changed primer is either
the sense primer or the anti-sense primer. The first PCR amplifies
a sequence as seen in any PCR experiment. One primer of the second
pair of primer, i.e. the nested primer, for the second PCR binds
within the first PCR product and produces a second PCR product that
is shorter than the first one. The technique, because it uses four
specific primer, rather than two, has greater specificity than
regular PCR. It can also yield detectable product in cases where
simple PCR fails to do so.
[0077] The terms "multiplex polymerase chain reaction" or
"multiplex PCR", which are used interchangeably within the current
application, denote a polymerase chain reaction employing multiple,
unique primer in a single PCR reaction/mixture to produce amplicons
of varying sizes specific to different DNA sequences. By targeting
multiple genes at once, additional information can be obtained from
a single test run that otherwise would require several times the
reagents and more time to perform. Annealing temperatures for each
primer sets must be optimized to work correctly within a single
reaction. Besides, amplicon sizes should be different enough to
form distinct bands when visualized by gel electrophoresis.
[0078] In the human genome the chromosomal loci containing the
immunoglobulin encoding genes are located on chromosomes 2, 14, and
22 (see FIGS. 1A-C). The human immunoglobulin G heavy chain locus
can be found on chromosome 14 (14q32.2) with the chromosomal
orientation in the locus:
telomere-5'-end-V.sub.H-D-J.sub.H-C.sub.H-3'-end-centromere. The
V.sub.H segments on the chromosome are classified as depicted in
the following Table 1.
TABLE-US-00001 TABLE 1 Grouping of the V.sub.H-genes into V.sub.H
families according to Matsuda, F., et al., J. Exp. Med. 188 (1998)
2151-2162 and Tomlinson, I. M., et al., V Base sequence directory
1999. V.sub.H Number of family Genes with open reading family
members frame V.sub.H1 14 9/11 V.sub.H2 4 3 V.sub.H3 65 22 V.sub.H4
32 7/11 V.sub.H5 2 2 V.sub.H6 1 1 V.sub.H7 5 1
[0079] The human immunoglobulin G heavy chain locus comprises
overall 123-129 V.sub.H-genes, of which 51 are functional, 23
functional D-genes (D=diversity), grouped in seven families, 6
functional J.sub.H-genes (J joining) and in the most frequent
haplotype 9 functional C.sub.H-genes (C=constant).
[0080] The locus for the human immunoglobulin G light chains of the
types kappa (.kappa.) and lambda (.lamda.) is located on two
different chromosomes, chromosomes 2 and 22. The kappa light chain
locus can be found on the short arm of chromosome 2 (2p11.2) and
comprises 40 functional V.sub..kappa.-gene segments. These are
grouped in seven families. The locus also comprises 5
J.sub..kappa.-genes and a single C.sub..kappa.-gene (Schable, K. F.
and Zachau, H. G., Biol. Chem. Hoppe Seyler 374 (1993) 1001-1022;
Lefranc, M. P., Exp. Clin. Immunogenet. 18 (2001) 161-174).
TABLE-US-00002 TABLE 2 Grouping of the V.sub..kappa.-genes into
V.sub..kappa. families according to Foster, S. J., et al., J. Clin.
Invest. 99 (1997) 1614-1627. V.sub..kappa. Number of family
functional genes V.sub..kappa.1 19 V.sub..kappa.2 9 V.sub..kappa.3
7 V.sub..kappa.4 1 V.sub..kappa.5 1 V.sub..kappa.6 3
[0081] The lambda light chain locus can be found on the long arm of
chromosome 22 (22p11.2) and comprises 73-74 V.sub..lamda.-gene of
which 30 are functional. These are grouped in ten families which in
addition are grouped in three clusters. The locus also comprises 7
J.sub..lamda.-genes, of which 5 are functional.
TABLE-US-00003 TABLE 3 Grouping of the V.sub..lamda.-genes into
V.sub..lamda. families according to Frippiat, J. P., et al., Hum.
Mol. Genet. 4 (1995) 983-991; Farner, N. L., et al., J. Immunol.
162 (1999) 2137-2145; Lefranc, M. P., Exp. Clin. Immunogenet. 18
(2001) 242-254. V.sub..lamda. Number of family functional genes
Cluster V.sub..lamda.1 5 B V.sub..lamda.2 5 A V.sub..lamda.3 8 A
V.sub..lamda.4 3 A-C V.sub..lamda.5 3 B V.sub..lamda.6 1 C
V.sub..lamda.7 2 B V.sub..lamda.8 1 C V.sub..lamda.9 1 B
V.sub..lamda.10 1 C
[0082] The PCR-based amplification of the nucleic acid encoding an
IgG HC and LC or at least the variable domain thereof from a single
immunoglobulin producing cell, e.g. from a single B-cell, is based
on the single cell deposition of B-lymphocytes followed by a PCR
based nucleic acid amplification with specific primer for the
variable domain of the heavy and light chain. The outcome of the
PCR is essentially depending on the employed PCR primer. At best
the employed primer should cover all V-genes, should not be prone
to dimer formation and should specifically bind to the cDNA
encoding the immunoglobulin. Thus, in one embodiment the nucleic
acid encoding an immunoglobulin variable domain is obtained from
cDNA.
[0083] Due to the large number of functional genes on the human
immunoglobulin G locus it is necessary to employ different primer
in the PCR reaction in order to cover as many known genes as
possible. Therefore, a set of degenerated primer has been
established which is also an aspect of the current invention. In
one embodiment the amplification of the nucleic acid encoding the
heavy and light chain is performed in one polymerase chain
reaction. In this embodiment the primer are chosen in order to
provide for the amplification of nucleic acids of approximately the
same length in order to allow for the same PCR conditions. In this
embodiment primer for the nucleic acid encoding the heavy chain are
employed whereof one is binding in the heavy chain C.sub.H1 region,
thus, providing for a nucleic acid fragment of comparable size to
that of the corresponding nucleic acid encoding the light
chain.
[0084] One aspect of the current invention is a method for
obtaining a nucleic acid encoding at least an immunoglobulin
variable domain from a single cell comprising the following steps:
[0085] obtaining a first nucleic acid composition by performing a
first polymerase chain reaction with three to six 5'-primer and one
3'-primer, [0086] obtaining a nucleic acid encoding an
immunoglobulin variable domain by performing with the composition
obtained in the first polymerase chain reaction a second polymerase
chain reaction with thirteen to sixteen 5'-primer and one
3'-primer, whereby the distance of the binding locations of the
primer employed in the second polymerase chain reaction is reduced
compared to the first polymerase chain reaction.
[0087] In one embodiment of this method the 5'-primer employed in
the first polymerase chain reaction bind in the coding region for
the leader peptide of the immunoglobulin. In another embodiment the
5'-primer employed in the second polymerase chain reaction bind in
the coding region for the first framework region of the
immunoglobulin. In another embodiment the primer employed in the
second polymerase chain reaction provide for overhangs encoding the
translational start codon ATG for 5'-primer and/or the
translational stop codon TTA for 3'-primer. This overhang is useful
in an optional following overlapping polymerase chain reaction for
the generation of nucleic acids for the in vitro translation of the
obtained nucleic acid. In one embodiment the immunoglobulin
variable domain is an immunoglobulin heavy chain variable domain or
an immunoglobulin kappa light chain variable domain or an
immunoglobulin lambda light chain variable domain.
[0088] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin heavy chain
variable domain have the nucleic acid sequence of SEQ ID NO: 05
and/or 06, and SEQ ID NO: 07 and/or 08, and SEQ ID NO: 09, and SEQ
ID NO: 10 and/or 11, and SEQ ID NO: 12, and SEQ ID NO: 13, and SEQ
ID NO: 14 and/or 15.
TABLE-US-00004 TABLE 4 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub.H Primer
TCACCATGGACTG(C/G)ACCTGGA V.sub.HL-1 05, 06 binding in
CCATGGACACACTTTG(C/T)TCCAC V.sub.HL-2 07, 08 the leader
TCACCATGGAGTTTGGGCTGAGC V.sub.HL-3 09 peptide
AGAACATGAAACA(C/T)CTGTGGTTC V.sub.HL-4 10, 11 coding TT region
ATGGGGTCAACCGCCATCCT V.sub.HL-5 12 ACAATGTCTGTCTCCTTCCTCAT
V.sub.HL-6 13 primer GCCAGGGGGAAGAC(C/G)GATG huC.sub.H-II 14, 15
binding in the constant region coding region
[0089] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin kappa light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
16 to 20.
TABLE-US-00005 TABLE 5 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. Primer SEQ
description Sequence Denotation ID NO: V.kappa. primer
GCTCAGCTCCTGGGGCTCCTG V.kappa.L-1 16 binding in the
CTGGGGCTGCTAATGCTCTGG V.kappa.L-2 17 leader peptide
TTCCTCCTGCTACTCTGGCTC V.kappa.L-3 18 coding region
CAGACCCAGGTCTTCATTTCT V.kappa.L-4 19 primer binding in
TTTCAACTGCTCATCAGATGGCGG huC.kappa.-II 20 the constant region
coding region
[0090] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin lambda light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
21, and SEQ ID NO: 22, and SEQ ID NO: 23 and/or 24 and/or 25 and/or
26, and SEQ ID NO: 27 and/or 28.
TABLE-US-00006 TABLE 6 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain. Primer SEQ
description sequence denotation ID NO: V.lamda. primer
CCTCTCCTCCTCACCCTCCT V.lamda.L-1 21 binding in the
CTCCTCACTCAGGGCACA V.lamda.L-2 22 leader peptide
ATGGCCTGGA(T/C)C(C/G)CTCTCC V.lamda.L-3 23, 24, coding region 25,
26 primer binding in AGCTCCTCAGAGGAGGG(C/T)GG C.lamda.II 27, 28 the
constant region coding region
[0091] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin heavy chain
variable domain have the nucleic acid sequence of SEQ ID NO: 29
and/or 30, and SEQ ID NO: 31, and SEQ ID NO: 32 and/or 33, and SEQ
ID NO: 34 and/or 35, and SEQ ID NO: 36, and SEQ ID NO: 37 and/or
38, and SEQ ID NO: 39 and/or 40, and SEQ ID NO: 41, and SEQ ID NO:
42, and SEQ ID NO: 43 and/or 44, and SEQ ID NO: 45, and SEQ ID NO:
46 and/or 47, and SEQ ID NO: 48, and SEQ ID NO: 49 and/or 50, and
SEQ ID NO: 51 and/or 52.
TABLE-US-00007 TABLE 7 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. SEQ Primer ID
description Sequence Denotation NO: V.sub.H primer
CTTTAAGAAGGAGATATACCATGGT V.sub.HL-1a 29, 30 binding in the
(G/T)CAGCTGGTGCAG FR1 coding CTTTAAGAAGGAGATATACCATGCA V.sub.HL-1b
31 region GGTCCAGCTTGTGCAG CTTTAAGAAGGAGATATACCATG V.sub.HL-1c 32,
33 (G/C)AGGTCCAGCTGGTACAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-1d 34,
35 (A/G)ATGCAGCTGGTGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-2a 36 G
ATCACCTTGAAGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-2b 37, 38 G
GTCACCTTGA(A/G)GGAG CTTTAAGAAGGAGATATACCATGGA V.sub.HL-3a 39, 40
(A/G)GTGCAGCTGGTGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-3b 41 G
GTGCAGCTGGTGGAG CTTTAAGAAGGAGATATACCATGGA V.sub.HL-3c 42 G
GTGCAGCTGTTGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-4a 43,44
G(C/G)TGCAGCTGCAGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-4b 45 G
GTGCAGCTACAGCAG CTTTAAGAAGGAGATATACCATGGA V.sub.HL-5a 46, 47
(A/G)GTGCAGCTGGTGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-6a 48
GGTACAGCTGCAGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.HL-7a 49, 50
GGT(C/G)CAGCTGGTGCAA primer ATCGTATGGGTAGCTGGTCCCTTAG huC.sub.H-III
51, 52 binding in the AC(C/G)GATGGGCCCTTGGTGGA constant region
coding region
[0092] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin kappa light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
53 and/or 54, and SEQ ID NO: 55 and/or 56, and SEQ ID NO: 57 and/or
58, and SEQ ID NO: 59, and SEQ ID NO: 60, and SEQ ID NO: 61 and/or
62, and SEQ ID NO: 63 and/or 64, and SEQ ID NO: 65, and SEQ ID NO:
66, and SEQ ID NO: 67, and SEQ ID NO: 68, and SEQ ID NO: 69, and
SEQ ID NO: 70, and SEQ ID NO: 71.
TABLE-US-00008 TABLE 8 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. Primer SEQ
description Sequence Denotation ID NO: V.sub..kappa. primer
CTTTAAGAAGGAGATATACCATG V.sub..kappa.L-1a 53, 54 binding in the
(A/G)ACATCCAGATGACCCAG FR1 coding CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-1b 55, 56 region (A/C)CATCCAGTTGACCCAG
CTTTAAGAAGGAGATATACCATGGC V.sub..kappa.L-1c 57, 58
CATCC(A/G)GATGACCCAG CTTTAAGAAGGAGATATACCATGGT V.sub..kappa.L-1d 59
CATCTGGATGACCCAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-2a 60
TATTGTGATGACCCAG CTTTAAGAAGGAGATATACCATGG V.sub..kappa.L-2b 61, 62
AT(A/G)TTGTGATGACTCAG CTTTAAGAAGGAGATATACCATGG V.sub..kappa.L-3a
63, 64 AAATTGTGTTGAC(A/G)CAG CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-3b 65 AAATAGTGATGACGCAG CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-3c 66 AAATTGTAATGACACAG CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-4a 67 ACATCGTGATGACCCAG CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-5a 68 AAACGACACTCACGCAG CTTTAAGAAGGAGATATACCATGG
V.sub..kappa.L-6a 69 AAATTGTGCTCACTCAG CTTTAAGAAGGAGATATACCATGGA
V.sub..kappa.L-6b 70 TGTTGTGATGACACAG primer binding
ATCGTATGGGTAGCTGGTCCCTTAA huC.sub..kappa.-III 71 in the constant
AGATGAAGACAGATGGTGC region coding region
[0093] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin lambda light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
72, and SEQ ID NO: 73 and/or 74, and SEQ ID NO: 75, and SEQ ID NO:
76, and SEQ ID NO: 77 and/or 78, and SEQ ID NO: 79, and SEQ ID NO:
80, and SEQ ID NO: 81, and SEQ ID NO: 82 and/or 83, and SEQ ID NO:
84 and/or 85, and SEQ ID NO: 86, and SEQ ID NO: 87 and/or 88, and
SEQ ID NO: 89, and SEQ ID NO: 90 and/or 91, and SEQ ID NO: 92, and
SEQ ID NO: 93.
TABLE-US-00009 TABLE 9 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain. Primer SEQ
description Sequence Denotation ID NO: V.sub..lamda. primer
CTTTAAGAAGGAGATATACCATGCA V.sub..lamda.L-1a 72 binding in the
GTCTGTGCTGACTCAG FR1 coding CTTTAAGAAGGAGATATACCATGCA
V.sub..lamda.L-1b 73, 74 region GTCTGTG(C/T)TGACGCAG
CTTTAAGAAGGAGATATACCATGCA V.sub..lamda.L-1c 75 GTCTGTCGTGACGCAG
CTTTAAGAAGGAGATATACCATGCA V.sub..lamda.L-2a 76 GTCTGCCCTGACTCAG
CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3a 77, 78
CTATG(A/T)GCTGACTCAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3b 79
CTATGAGCTGACACAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3c 80
TTCTGAGCTGACTCAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3d 81
CTATGAGCTGATGCAG CTTTAAGAAGGAGATATACCATGCA V.sub..lamda.L-4a 82, 83
GC(C/T)TGTGCTGACTCAA CTTTAAGAAGGAGATATACCATGCAG V.sub..lamda.L-5a
84, 85 (C/G)CTGTGCTGACTCAG CTTTAAGAAGGAGATATACCATGAA
V.sub..lamda.L-6a 86 TTTTATGCTGACTCAG CTTTAAGAAGGAGATATACCATGCAG
V.sub..lamda.L-7a 87, 88 (A/G)CTGTGGTGACTCAG
CTTTAAGAAGGAGATATACCATGCAG V.sub..lamda.L-8a 89 ACTGTGGTGACCCAG
CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-4/9a 90, 91
(A/T)GCCTGTGCTGACTCAG CTTTAAGAAGGAGATATACCATGCAG V.sub..lamda.L-10a
92 GCAGGGCTGACTCAG primer binding ATCGTATGGGTAGCTGGTCCCTTAG
huC.sub..lamda.-III 93 in the constant GGAACAGAGTGACCG region
coding region
[0094] A further aspect of the current invention is a method for
obtaining a nucleic acid encoding at least an immunoglobulin
variable domain from a single cell comprising the following step:
[0095] obtaining a first nucleic acid composition by performing a
first polymerase chain reaction with three or four 5'-primer and
one 3'-primer, [0096] obtaining a nucleic acid encoding at least an
immunoglobulin variable domain by performing with the composition
obtained in the first polymerase chain reaction a second polymerase
chain reaction with one 5'-primer and one 3'-primer, whereby primer
employed in the first and in the second polymerase chain reaction
can be the same.
[0097] In one embodiment the primer employed in the polymerase
chain reaction provide for overhangs encoding the translational
start codon ATG for 5'-primer and/or the translational stop codon
TTA for 3'-primer. This overhang is useful in an optional following
overlapping polymerase chain reaction for the generation of nucleic
acids for the in vitro translation of the obtained nucleic acid. In
one embodiment the immunoglobulin variable domain is an
immunoglobulin heavy chain variable domain or an immunoglobulin
kappa light chain variable domain or an immunoglobulin lambda light
chain variable domain.
[0098] In one embodiment of this method the primer employed in the
first two-step polymerase chain reaction for obtaining a nucleic
acid encoding an immunoglobulin heavy chain variable domain have
the nucleic acid sequence of SEQ ID NO: 94, and SEQ ID NO: 95, and
SEQ ID NO: 96 and/or 97 and/or 98 and/or 99, and SEQ ID NO: 100
and/or 101 and/or 102 and/or 103, and SEQ ID NO: 104 and/or 105
and/or 106.
TABLE-US-00010 TABLE 10 Primer employed in the first two-step
polymerase chain reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub.H primer
CTTTAAGAAGGAGATATACCATGCA huV.sub.H-1 94 binding in
GGTGCAGCTGGTGCAGTC the FR1 CTTTAAGAAGGAGATATACCATGCA huV.sub.H-2 95
coding GGTCAACTTAAGGGAGTCTGG region CTTTAAGAAGGAGATATACCATGAG
huV.sub.H-3 96, 97, GTGCAGCTG(C/G)TG(C/G)AGTC 98, 99
CTTTAAGAAGGAGATATACCATGCA huV.sub.H-4 100, 101,
GGT(A/G)CAGCTGCAG(C/G)AGTC 102, 103 primer ATCGTATGGGTAGCTGGTCCCTTA
huC.sub.H-2 104, 105, binding in GTGGTGGTGGTGGTGGTGAACT 106 the
constant (C/G/T)TCTTGTCCACCTTGGTGTTG region coding region
[0099] In one embodiment of this method the primer employed in the
first two-step polymerase chain reaction for obtaining a nucleic
acid encoding an immunoglobulin kappa light chain variable domain
have the nucleic acid sequence of SEQ ID NO: 107 and/or 108 and/or
109 and/or 110, and SEQ ID NO: 111 and/or 112, and SEQ ID NO: 113,
and SEQ ID NO: 114, and SEQ ID NO: 115.
TABLE-US-00011 TABLE 11 Primer employed in the first two-step
polymerase chain reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub..kappa. primer
CTTTAAGAAGGAGATATACCATGG huV.sub..kappa.-1 107, 108, binding in the
A CATC(C/G)(A/T)GATGACCCAGTCT 109, 110 FR1 coding
CTTTAAGAAGGAGATATACCATGG huV.sub..kappa.-2 111, 112 region A
TATTGTG(A/C)TGACTCAGTCTCC CTTTAAGAAGGAGATATACCATGG
huV.sub..kappa.-3 113 A AATTGTGTTGACGCAGTCTCC
CTTTAAGAAGGAGATATACCATGG huV.sub..kappa.-4 114 A
AACGACACTCACGCAGTCTC primer ATCGTATGGGTAGCTGGTCCCTTAA
huC.sub..kappa.-2 115 binding in the CACCTCTCCCCTGTTGAAGCTC
constant region coding region
[0100] In one embodiment of this method the primer employed in the
first two-step polymerase chain reaction for obtaining a nucleic
acid encoding an immunoglobulin lambda light chain variable domain
have the nucleic acid sequence of SEQ ID NO: 116, and SEQ ID NO:
117, and SEQ ID NO: 118 and/or 119, and SEQ ID NO: 120 and/or 121
and/or 122 and/or 123 and/or 124 and/or 125.
TABLE-US-00012 TABLE 12 Primer employed in the first two-step
polymerase chain reaction for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub..lamda. primer
CTTTAAGAAGGAGATATACCATG huV.sub..lamda.-1 116 binding in the
CAGTCTGTGCTGACTCAGCC FR1 coding CTTTAAGAAGGAGATATACCATG
huV.sub..lamda.-2 117 region CAGTCTGCCCTGACTCAGCC
CTTTAAGAAGGAGATATACCATG huV.sub..lamda.-3 118, 119
TCCTATGAGCTGAC(A/T)CAGCC primer ATCGTATGGGTAGCTGGTCCCTTA
huC.sub..lamda.-2 120, 121, binding in the
TGAACATTC(C/T)G(C/T)AGGGGC 122, 123, constant (A/T)ACT 124, 125
region coding region
[0101] In one embodiment of this method the primer employed in the
second two-step polymerase chain reaction have the nucleic acid
sequence of SEQ ID NO: 126 and SEQ ID NO: 127.
TABLE-US-00013 TABLE 13 Primer employed in the second two-step
polymerase chain reaction. Primer SEQ ID description Sequence
Denotation NO: first primer CTTTAAGAAGGAGATATACCATG LTGS-lfp 126
second primer ATCGTATGGGTAGCTGG LTGS-rfp 127
[0102] Another aspect of the current invention is a method for
obtaining a nucleic acid encoding at least an immunoglobulin
variable domain from a single cell comprising the following steps:
[0103] obtaining a first nucleic acid composition by performing a
first polymerase chain reaction with four to six 5'-primer and one
3'-primer, [0104] obtaining a nucleic acid encoding at least an
immunoglobulin variable domain by performing with the composition
obtained in the first polymerase chain reaction a second polymerase
chain reaction with thirteen to fifteen 5'-primer and one
3'-primer, whereby in the second polymerase chain reaction either
the 5'-primer are the same as in the first polymerase chain
reaction and the 3'-primer is different or the 3'-primer is the
same as in the first polymerase chain reaction and at least one
5'-primer is different, and whereby in the second polymerase chain
reaction the number of nucleotides between the 5'-end of each of
the 5'-primer and the 3'-end of the 3'-primer is smaller compared
to the number of nucleotides between the 5'-end of each of the
5'-primer and the 3'-end of the 3'-primer in the first polymerase
chain reaction.
[0105] In one embodiment of this method the 5'-primer employed in
the first polymerase chain reaction bind in the coding region for
the leader peptide of the immunoglobulin. In another embodiment the
5'-primer employed in the second polymerase chain reaction bind in
the coding region for the first framework region of the
immunoglobulin. In another embodiment the primer employed in the
second polymerase chain reaction provide for overhangs encoding the
translational start codon ATG for 5'-primer and/or the
translational stop codon TTA for 3'-primer. This overhang is useful
in an optional following overlapping polymerase chain reaction for
the generation of nucleic acids for the in vitro translation of the
obtained nucleic acid. In one embodiment of this method for
obtaining an immunoglobulin variable domain encoding nucleic acid
six 5'-primer and one 3'-primer are employed in the first
polymerase chain reaction. In another embodiment four 5'-primer and
one 3'-primer are employed in the second polymerase chain reaction.
In one embodiment the immunoglobulin variable domain is an
immunoglobulin heavy chain variable domain or an immunoglobulin
kappa light chain variable domain or an immunoglobulin lambda light
chain variable domain.
[0106] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin heavy chain
variable domain have the nucleic acid sequence of SEQ ID NO: 05
and/or 06, and SEQ ID NO: 07 and/or 08, and SEQ ID NO: 09, and SEQ
ID NO: 10 and/or 11, and SEQ ID NO: 12, and SEQ ID NO: 13, and SEQ
ID NO: 104 and/or 105 and/or 106.
TABLE-US-00014 TABLE 14 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. SEQ Primer ID
description Sequence Denotation NO: V.sub.H primer
TCACCATGGACTG(C/G)ACCTGGA V.sub.HL-1 05, 06 binding in the
CCATGGACACACTTTG(C/T)TCCAC V.sub.HL-2 07, 08 leader
TCACCATGGAGTTTGGGCTGAGC V.sub.HL-3 09 peptide
AGAACATGAAACA(C/T)CTGTGGTTCTT V.sub.HL-4 10, 11 coding region
ATGGGGTCAACCGCCATCCT V.sub.HL-5 12 ACAATGTCTGTCTCCTTCCTCAT
V.sub.HL-6 13 primer TCGTATGGGTAGCTGGTCCCTTAGTGGT huC.sub.H-2 104,
binding in the GGTGGTGGTGGTGAACT(C/G/T)TCTTGT 105, constant
CCACCTTGGTGTTG 106 region coding region
[0107] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin kappa light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
16, and SEQ ID NO: 17, and SEQ ID NO: 18, and SEQ ID NO: 19, and
SEQ ID NO: 115.
TABLE-US-00015 TABLE 15 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub..kappa. primer
GCTCAGCTCCTGGGGCTCCTG V.sub..kappa.L-1 16 binding in the
CTGGGGCTGCTAATGCTCTGG V.sub..kappa.L-2 17 leader peptide
TTCCTCCTGCTACTCTGGCTC V.sub..kappa.L-3 18 coding region
CAGACCCAGGTCTTCATTTCT V.sub..kappa.L-4 19 primer binding in
ATCGTATGGGTAGCTGGTCCC huC.sub..kappa.-2 115 the constant
TTAACACTCTCCCCTGTTGAA region coding GCTC region
[0108] In one embodiment of the methods according to the invention
the primer employed in the first polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin lambda light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
21, and SEQ ID NO: 22, and SEQ ID NO: 23 and/or 24 and/or 25 and/or
26, and SEQ ID NO: 120 and/or 121 and/or 122 and/or 123 and/or 124
and/or 125.
TABLE-US-00016 TABLE 16 Primer employed in the first polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub..lamda. primer
CCTCTCCTCCTCACCCTCCT V.sub..lamda.L-1 21 binding in the
CTCCTCACTCAGGGCACA V.sub..lamda.L-2 22 leader peptide
ATGGCCTGGA(T/C)C(C/G)CTCTCC V.sub..lamda.L-3 23, 24, 25, coding
region 26 primer binding ATCGTATGGGTAGCTGGTCCCTTA huC.sub..lamda.-2
120, 121, in the constant TGAACATTC(C/T)G(C/T)AGGGGC 122, 123,
region coding (A/T)ACT 124, 125 region
[0109] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin heavy chain
variable domain have the nucleic acid sequence of SEQ ID NO: 128,
and SEQ ID NO: 129, and SEQ ID NO: 130, and SEQ ID NO: 131, and SEQ
ID NO: 132, and SEQ ID NO: 133, and SEQ ID NO: 134, and SEQ ID NO:
135, and SEQ ID NO: 136, and SEQ ID NO: 137, and SEQ ID NO: 138,
and SEQ ID NO: 139, and SEQ ID NO: 140, SEQ and ID NO: 141, and SEQ
ID NO: 104 and/or 105 and/or 106.
TABLE-US-00017 TABLE 17 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. SEQ Primer ID
description Sequence Denotation NO: V.sub.H primer
CTTTAAGAAGGAGATATACCATGCA V.sub.H-1a 128 binding in the
GGTKCAGCTGGTGCAG FR1 coding CTTTAAGAAGGAGATATACCATGCA V.sub.H-1b
129 region GGTCCAGCTTGTGCAG CTTTAAGAAGGAGATATACCATGSAG V.sub.H-1c
130 GTCCAGCTGGTACAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-1d 131
RATGCAGCTGGTGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-2a 132
GATCACCTTGAAGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-2b 133
GGTCACCTTGARGGAG CTTTAAGAAGGAGATATACCATGGA V.sub.H-3a 134
RGTGCAGCTGGTGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-3b 135
GGTGCAGCTGGTGGAG CTTTAAGAAGGAGATATACCATGGA V.sub.H-3c 136
GGTGCAGCTGTTGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-4a 137
GSTGCAGCTGCAGGAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-4b 138
GGTGCAGCTACAGCAG CTTTAAGAAGGAGATATACCATGGA V.sub.H-5a 139
RGTGCAGCTGGTGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-6a 140
GGTACAGCTGCAGCAG CTTTAAGAAGGAGATATACCATGCA V.sub.H-7a 141
GGTACAGCTGGTGCAA primer ATCGTATGGGTAGCTGGTCCCTTAGT huC.sub.H-2 104,
binding in the GGTGGTGGTGGTGGTGAACT(C/G/T)T 105, constant
CTTGTCCACCTTGGTGTTG 106 region coding region
[0110] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin kappa light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
53 and/or 54, and SEQ ID NO: 55 and/or 56, and SEQ ID NO: 57 and/or
58, and SEQ ID NO: 59, and SEQ ID NO: 60, and SEQ ID NO: 61 and/or
62, SEQ ID NO: 63 and/or 64, and SEQ ID NO: 65, and SEQ ID NO: 66,
and SEQ ID NO: 67, and SEQ ID NO: 68, and SEQ ID NO: 69, and SEQ ID
NO: 70, and SEQ ID NO: 115.
TABLE-US-00018 TABLE 18 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. SEQ Primer ID
description Sequence Denotation NO: V.sub..kappa. primer
CTTTAAGAAGGAGATATACCATG(A/G) V.sub..kappa.L-1a 53, 54 binding in
the ACATCCAGATGACCCAG FR1 coding CTTTAAGAAGGAGATATACCATGG(A/
V.sub..kappa.L-1b 55, 56 region C)CATCCAGTTGACCCAG
CTTTAAGAAGGAGATATACCATGGCC V.sub..kappa.L-1c 57, 58
ATCC(A/G)GATGACCCAG CTTTAAGAAGGAGATATACCATGGTC V.sub..kappa.L-1d 59
ATCTGGATGACCCAG CTTTAAGAAGGAGATATACCATGGAT V.sub..kappa.L-2a 60
ATTGTGATGACCCAG CTTTAAGAAGGAGATATACCATGGAT V.sub..kappa.L-2b 61, 62
(A/G)TTGTGATGACTCAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-3a 63,
64 AATTGTGTTGAC(A/G)CAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-3b
65 AATAGTGATGACGCAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-3c 66
AATTGTAATGACACAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-4a 67
CATCGTGATGACCCAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-5a 68
AACGACACTCACGCAG CTTTAAGAAGGAGATATACCATGGA V.sub..kappa.L-6a 69
AATTGTGCTGACTCAG CTTTAAGAAGGAGATATACCATGGAT V.sub..kappa.L-6b 70
GTTGTGATGACACAG primer ATCGTATGGGTAGCTGGTCCCTTAAC huC.sub..kappa.-2
115 binding in the CATCTCCCCTGTTGAAGCTC constant region coding
region
[0111] In one embodiment of the methods according to the invention
the primer employed in the second polymerase chain reaction for
obtaining a nucleic acid encoding an immunoglobulin lambda light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
72, and SEQ ID NO: 73 and/or 74, and SEQ ID NO: 75, and SEQ ID NO:
76, and SEQ ID NO: 77 and/or 78, and SEQ ID NO: 79, and SEQ ID NO:
80, and SEQ ID NO: 81, and SEQ ID NO: 82 and/or 83, and SEQ ID NO:
84 and/or 85, and SEQ ID NO: 86, and SEQ ID NO: 87 and/or 88, and
SEQ ID NO: 89, and SEQ ID NO: 90 and/or 91, and SEQ ID NO: 92, and
SEQ ID NO: 120 or 121 or 122 or 123 or 124 or 125.
TABLE-US-00019 TABLE 19 Primer employed in the second polymerase
chain reaction for obtaining a nucleic acid encoding an
immunoglobulin lambda light chain variable domain. SEQ Primer ID
description Sequence Denotation NO: V.sub..lamda. primer
CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-1a 72 binding in the
AGTCTGTGCTGACTCAG FR1 coding CTTTAAGAAGGAGATATACCATGC
V.sub..lamda.L-1b 73, 74 region AGTCTGTG(C/T)TGACGCAG
CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-1c 75 AGTCTGTCGTGACGCAG
CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-2 76 AGTCTGCCCTGACTCAG
CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3a 77, 78
CTATG(A/T)GCTGACTCAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3b 79
CTATGAGCTGACACAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3c 80
TTCTGAGCTGACTCAG CTTTAAGAAGGAGATATACCATGTC V.sub..lamda.L-3d 81
CTATGAGCTGATGCAG CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-4 82, 83
AGC(C/T)TGTGCTGACTCAA CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-5 84,
85 AG(C/G)CTGTGCTGACTCAG CTTTAAGAAGGAGATATACCATGA V.sub..lamda.L-6
86 ATTTTATGCTGACTCAG CTTTAAGAAGGAGATATACCATGC V2.sub..lamda.L-7 87,
88 AG(A/G)CTGTGGTGACTCAG CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-8
89 AGACTGTGGTGACCCAG CTTTAAGAAGGAGATATACCATGC V.sub..lamda.L-4/9
90, 91 (A/T)GCCTGTGCTGACTCAG CTTTAAGAAGGAGATATACCATGC
V.sub..lamda.L-10 92 AGGCAGGGCTGACTCAG primer
ATCGTATGGGTAGCTGGTCCCTTAT huC.sub..lamda.-2 120, binding in the
GAACATTC(C/T)G(C/T)AGGGGC 121, constant (A/T)ACT 122, region coding
123, region 124, 125
[0112] In one embodiment of the methods according to the invention
the nucleic acid encoding the light chain variable domain and
nucleic acid encoding the heavy chain variable domain are obtained
in one polymerase chain reaction by a combination of the different
5'- and 3'-primer in a single multiplex polymerase chain
reaction.
[0113] Another aspect of the current invention is a method for
obtaining a nucleic acid encoding at least an immunoglobulin
variable domain from a single cell comprising the following step:
[0114] performing a reverse transcription and polymerase chain
reaction in one step with a set of primer comprising one 5'-primer
and one 3'-primer.
[0115] In one embodiment of this method the 5'-primer employed in
the multiplex one tube reverse transcription gene specific primer
polymerase chain reaction (RT-GSP-PCR) binds in the coding region
for the first framework region of the immunoglobulin. In another
embodiment the primer employed in the RT-GSP-PCR reaction provide
for overhangs encoding the translational start codon ATG for the
5'-primer and/or the translational stop codon TTA for the
3'-primer. This overhang is useful in an optional following
overlapping polymerase chain reaction for the generation of nucleic
acids for the in vitro translation of the obtained nucleic acid. In
one embodiment this method is for obtaining an immunoglobulin heavy
chain variable domain with a RT-GSP-PCR reaction. In one embodiment
the immunoglobulin variable domain is an immunoglobulin heavy chain
variable domain or an immunoglobulin kappa light chain variable
domain or an immunoglobulin lambda light chain variable domain.
[0116] In one embodiment the primer employed in the multiplex one
tube RT-GSP-PCR for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain have the nucleic acid
sequence of SEQ ID NO: 142 and 143.
TABLE-US-00020 TABLE 20 Primer employed in the multiplex one tube
RT-GSP-PCR reaction for obtaining a nucleic acid encoding an
immunoglobulin heavy chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub.H primer
CTTTAAGAAGGAGATATACCAT V.sub.H-lfp 142 binding in the
GAACTBTCTTGTCCACCTTGGT FR1 coding GTTG region primer binding in
ATCGTATGGGTAGCTGGTCCCTT V.sub.H-rfp 143 the constant
AAACTBTCTTGTCCACCTTGGTG region coding TTG region
[0117] In one embodiment of the methods according to the invention
the primer employed in the multiplex one tube RT-GSP-PCR for
obtaining a nucleic acid encoding an immunoglobulin kappa light
chain variable domain have the nucleic acid sequence of SEQ ID NO:
144 and 145.
TABLE-US-00021 TABLE 21 Primer employed in the multiplex one tube
RT-GSP-PCR reaction for obtaining a nucleic acid encoding an
immunoglobulin kappa light chain variable domain. Primer SEQ ID
description Sequence Denotation NO: V.sub..kappa. primer
CTTTAAGAAGGAGATATACCAT VL(k)-1fp 144 binding in the
GACACTCTCCCCTGTTGAAGCTC FR1 coding region primer binding
ATCGTATGGGTAGCTGGTCCCTT VL(k)-rfp 145 in the constant
AACACTCTCCCCTGTTGAAGCTC region coding region
[0118] Further it has been found that with a combination of the PCR
method according to the invention and a cell-free in vitro
translation system the nucleic acids encoding the cognate
immunoglobulin VH and VL domains can be obtained from a single cell
whereby the encoded immunoglobulin variable domain is provided as
Fab fragment in quantities sufficient for the characterization of
the immunoglobulin's binding properties. In order to amplify the
very low amount of mRNA obtained from a single cell, the individual
PCR (polymerase chain reaction) has to be very sensitive and a
combination of more than one PCR has to be performed.
[0119] A "cell-free in vitro translation system" according to the
invention denotes a cell-free lysate of a prokaryotic or
eukaryotic, preferably of a prokaryotic, cell containing ribosomes,
tRNA, ATP, CGTP, nucleotides, and amino acids. In one embodiment
the prokaryote is E. coli.
[0120] Cell-free in vitro translation is a method which has been
known in the state of the art for a long time. Spirin et al.
developed in 1988 a continuous-flow cell-free (CFCF) translation
and coupled transcription/translation system in which a relatively
high amount of protein synthesis occurs (Spirin, A. S., et al.,
Science 242 (1988) 1162-1164). For such cell-free in vitro
translation, cell lysates containing ribosomes were used for
translation or transcription/translation. Such cell-free extracts
from E. coli were developed by, for example, Zubay (Zubay, G., et
al., Ann. Rev. Genetics 7 (1973) 267-287) and were used by Pratt
(Pratt, J. M., et al., Nucleic Acids Research 9 (1981) 4459-4474;
and Pratt, J. M., et al., Transcription and Translation: A
Practical Approach, Hames and Higgins (eds.), 179-209, IRL Press,
1984). Further developments of the cell-free protein synthesis are
reported in U.S. Pat. No. 5,478,730, U.S. Pat. No. 5,571,690, EP 0
932 664, WO 99/50436, WO 00/58493, and WO 00/55353. Eukaryotic
cell-free expression systems are reported by, for example, Skup, D.
and Millward, S., Nucleic Acids Research 4 (1977) 3581-3587;
Fresno, M., et al., Eur. J. Biochem. 68 (1976) 355-364; Pelham, H.
R. and Jackson, R. J., Eur. J. Biochem. 67 (1976) 247-256 and in WO
98/31827.
[0121] It has been found that based on the amplification of nucleic
acid encoding cognate IgG HC (immunoglobulin G heavy chain) and IgG
LC (immunoglobulin G light chain) of an IgG isotype immunoglobulin
from a single cell and the subsequent in vitro translation of the
obtained nucleic acid to provide Fab fragments of said
immunoglobulin a high sensitive method for obtaining information
about an immunoglobulin produced by a single cell from the minute
amounts of mRNA obtainable is provided. The method according to the
invention permits the investigation of the expressed immunoglobulin
from a single B-cell, thus, providing higher diversity as opposed
to the hybridoma technology. Furthermore, since the cognate
immunoglobulin variable domains or immunoglobulin chains are
obtained from mature B-cells after antigen contact, selectively the
nucleic acid encoding high specific and correctly assembled
immunoglobulins can be obtained.
[0122] Therefore, one aspect of the current invention is a method
for producing an immunoglobulin Fab fragment comprising the
following steps: [0123] providing a single immunoglobulin producing
cell, [0124] obtaining from the cell the nucleic acid encoding the
immunoglobulin light and heavy chain variable domains, optionally
also encoding a part of the light chain constant domain and a part
of the heavy chain C.sub.H1 domain, [0125] optionally generating a
linear expression matrix comprising the obtained nucleic acid,
[0126] translating in vitro the nucleic acid and thereby producing
the immunoglobulin Fab fragment.
[0127] In one embodiment the nucleic acid encoding the
immunoglobulin variable domains is obtained with a method according
to the previous aspects of the current invention. In one embodiment
of method according to the invention the obtaining the nucleic acid
encoding the immunoglobulin light and heavy chain variable domain
form a single cell comprises a multiplex polymerase chain reaction
according to the invention for the amplification of cognate IgG HC
and IgG LC (human IgG isotype) from a single B-cell. For
characterization of the binding characteristics of the
immunoglobulin encoded by the obtained nucleic acid the nucleic
acid is subsequently translated in vitro in an E. coli lysate to an
immunoglobulin Fab fragment.
[0128] In general one aspect of the current invention is a method
employing the following steps i) isolating with magnetic
micro-beads coated with the human CD19 B-cells from peripheral
blood, ii) depositing single cells by limited dilution or FACS,
iii) extracting the mRNA of the individualized B-cells, iv)
obtaining the nucleic acid encoding at least the variable domains
of the immunoglobulin produced by the individualized B-cell, v) in
vitro translating the linear mRNA template, and optionally vi)
characterizing the binding properties of the immunoglobulin or
immunoglobulin fragment.
[0129] For the recombinant production of an immunoglobulin
comprising the variable domains obtained from a single cell with a
method according to the invention the obtained nucleic acids
encoding the variable domain of the light and heavy immunoglobulin
chain are further modified. At first the nucleic acid encoding the
variable domain is combined with a nucleic acid encoding an
immunoglobulin constant region. In one embodiment the nucleic acid
encoding the light chain variable domain is combined with a nucleic
acid encoding human kappa light chain constant domain of SEQ ID NO:
03 or with a nucleic acid encoding human lambda light chain
variable domain of SEQ ID NO: 04. In another embodiment the nucleic
acid encoding the heavy chain variable domain is combined with a
nucleic acid encoding human immunoglobulin G1 (IgG1) constant
region of SEQ ID NO: 01 or with a nucleic acid encoding human
immunoglobulin G4 (IgG4) constant region of SEQ ID NO: 02. In
another embodiment the nucleic acid encoding the heavy chain
variable domain is combined with a nucleic acid encoding human
immunoglobulin G1 (IgG1) constant region 1 (C.sub.H1).
[0130] The nucleic acid molecules encoding the complete
immunoglobulin heavy and light chain or a fragment thereof are in
the following referred to as structural genes. They can be located
on the same expression plasmid or can alternatively be located on
different expression plasmids. The assembly of the immunoglobulin
or Fab-fragment takes place before the secretion of the
immunoglobulin to the cultivation medium and, thus, within the
expressing cell. Therefore, the nucleic acid molecules encoding the
immunoglobulin chains are in one embodiment expressed in the same
host cell. If after recombinant expression a mixture of
immunoglobulins is obtained, these can be separated and purified by
methods known to a person skilled in the art. These methods are
well established and widespread used for immunoglobulin
purification and are employed either alone or in combination. Such
methods are, for example, affinity chromatography using
microbial-derived proteins (e.g. protein A or protein G affinity
chromatography), ion exchange chromatography (e.g. cation exchange
(carboxymethyl resins), anion exchange (amino ethyl resins) and
mixed-mode exchange chromatography), thiophilic adsorption (e.g.
with beta-mercaptoethanol and other SH ligands), hydrophobic
interaction or aromatic adsorption chromatography (e.g. with
phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic
acid), metal chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
preparative electrophoretic methods (such as gel electrophoresis,
capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem.
Biotech. 75 (1998) 93-102).
[0131] With recombinant engineering methods known to a person
skilled in the art the conjugates can be tailor-made on the nucleic
acid/gene level. The nucleic acid sequences encoding immunoglobulin
light and heavy chains are known and can be obtained for example
from genomic databases. The elements required for the construction
of an expression plasmid for the expression of the immunoglobulin
obtained with a method according to the invention are, for example,
an expression cassette for the immunoglobulin light chain, an
expression cassette for the immunoglobulin heavy chain
(alternatively the light chain and the heavy chain structural genes
can be contained in the same expression cassette, e.g. as
bicistronic expression element), a selection marker, and an E. coli
replication as well as selection unit. An expression cassette
comprises in general a promoter, a DNA segment encoding a secretion
signal sequence, the structural gene, and a
terminator/polyadenylation signal. The elements are assembled in an
operatively linked form either on one plasmid encoding all chains
of the immunoglobulin, or on two plasmids each encoding one chain
of the immunoglobulin. For the expression of the structural genes
the plasmid(s) is (are) introduced into a suitable host cell.
Proteins are produced in mammalian cells such as CHO cells, NS0
cells, Sp2/0 cells, COS cells, HEK cells, K562 cells, BHK cells,
PER.C6.RTM. cells, and the like. In one embodiment the conjugate is
expressed in a CHO cell, or a BHK cell, or a HEK cell, or NS0 cell.
The regulatory elements of the plasmid have to be selected in a way
that they are functional in the selected host cell. For expression
the host cell is cultivated under conditions suitable for the
expression of the immunoglobulin, which are known to a person of
skill in the art. The expressed immunoglobulin chains are
functionally assembled and the fully processed immunoglobulin is
secreted into the medium.
[0132] An "expression plasmid" is a nucleic acid providing all
required elements for the expression of the comprised structural
gene(s) in a host cell. Typically, an expression plasmid comprises
a prokaryotic plasmid propagation unit, e.g. for E. coli,
comprising an origin of replication, and a selectable marker, an
eukaryotic selection marker, and one or more expression cassettes
for the expression of the structural gene(s) of interest each
comprising a promoter, a structural gene, and a transcription
terminator including a polyadenylation signal. Gene expression is
usually placed under the control of a promoter, and such a
structural gene is said to be "operably linked to" the promoter.
Similarly, a regulatory element and a core promoter are operably
linked if the regulatory element modulates the activity of the core
promoter.
[0133] "Operably linked" refers to a juxtaposition of two or more
components, wherein the components so described are in a
relationship permitting them to function in their intended manner.
The term "linking . . . in operable form" denotes the combination
of two or more individual nucleic acids in a way that the
individual nucleic acids are operably linked in the final nucleic
acid. For example, a promoter and/or enhancer are operably linked
to a coding sequence, if it acts in cis to control or modulate the
transcription of the linked sequence. Generally, but not
necessarily, the DNA sequences that are "operably linked" are
contiguous and, where necessary to join two protein encoding
regions such as first domain and a second domain, e.g. an
immunoglobulin variable domain and an immunoglobbulin constant
domain or constant region, contiguous and in (reading) frame. A
polyadenylation site is operably linked to a coding sequence if it
is located at the downstream end of the coding sequence such that
transcription proceeds through the coding sequence into the
polyadenylation sequence. A translation stop codon is operably
linked to an exonic nucleic acid sequence if it is located at the
downstream end (3' end) of the coding sequence such that
translation proceeds through the coding sequence to the stop codon
and is terminated there. Linking is accomplished by recombinant
methods known in the art, e.g., using PCR methodology and/or by
ligation at convenient restriction sites. If convenient restriction
sites do not exist, then synthetic oligonucleotide adaptors or
linkers are used in accord with conventional practice.
[0134] Thus, one aspect of the current invention is a method for
producing an immunoglobulin comprising the following steps: [0135]
providing a single immunoglobulin producing cell, [0136] obtaining
from this cell the nucleic acid encoding the immunoglobulin light
and heavy chain variable domains, [0137] linking the nucleic acid
encoding the light chain variable domain with a nucleic acid
encoding an immunoglobulin light chain constant domain of SEQ ID
NO: 03 or SEQ ID NO: 04 in operable form and linking the nucleic
acid encoding the heavy chain variable domain with a nucleic acid
encoding an immunoglobulin heavy chain constant region of SEQ ID
NO: 01 or SEQ ID NO: 02 in operable form, [0138] transfecting a
eukaryotic or prokaryotic cell with the nucleic acids of the
previous step, [0139] cultivating the transfected cell under
conditions suitable for the expression of the immunoglobulin,
[0140] recovering the immunoglobulin from the cell or the
cultivation medium and thereby producing an immunoglobulin.
[0141] The term "under conditions suitable for the expression of"
denotes conditions which are used for the cultivation of a cell
capable of expressing a heterologous polypeptide and which are
known to or can easily be determined by a person skilled in the
art. It is known to a person skilled in the art that these
conditions may vary depending on the type of cell cultivated and
type of polypeptide expressed. In general the cell is cultivated at
a temperature, e.g. between 20.degree. C. and 40.degree. C., and
for a period of time sufficient to allow effective production of
the conjugate, e.g. for of from 4 days to 28 days, in a volume of
0.01 liter to 10.sup.7 liter.
[0142] The following examples, sequence listing 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.
DESCRIPTION OF THE SEQUENCE LISTING
[0143] SEQ ID NO: 01 human IgG1 heavy chain constant region
[0144] SEQ ID NO: 02 human IgG4 heavy chain constant region
[0145] SEQ ID NO: 03 human IgG kappa light chain constant
domain
[0146] SEQ ID NO: 04 human IgG lambda light chain constant
domain
[0147] SEQ ID NO: 05 primer V.sub.HL-1 variant 1
[0148] SEQ ID NO: 06 primer V.sub.HL-1 variant 2
[0149] SEQ ID NO: 07 primer V.sub.HL-2 variant 1
[0150] SEQ ID NO: 08 primer V.sub.HL-2 variant 2
[0151] SEQ ID NO: 09 primer V.sub.HL-3
[0152] SEQ ID NO: 10 primer V.sub.HL-4 variant 1
[0153] SEQ ID NO: 11 primer V.sub.HL-4 variant 2
[0154] SEQ ID NO: 12 primer V.sub.HL-5
[0155] SEQ ID NO: 13 primer V.sub.HL-6
[0156] SEQ ID NO: 14 primer huC.sub.H-II variant 1
[0157] SEQ ID NO: 15 primer huC.sub.H-II variant 2
[0158] SEQ ID NO: 16 primer V.sub.kL-1
[0159] SEQ ID NO: 17 primer V.sub.kL-2
[0160] SEQ ID NO: 18 primer V.sub.kL-3
[0161] SEQ ID NO: 19 primer V.sub.kL-4
[0162] SEQ ID NO: 20 primer huC.sub.k-II
[0163] SEQ ID NO: 21 primer V.sub.lL-1
[0164] SEQ ID NO: 22 primer V.sub.lL-2
[0165] SEQ ID NO: 23 primer V.sub.lL-3 variant 1
[0166] SEQ ID NO: 24 primer V.sub.lL-3 variant 2
[0167] SEQ ID NO: 25 primer V.sub.lL-3 variant 3
[0168] SEQ ID NO: 26 primer V.sub.lL-3 variant 4
[0169] SEQ ID NO: 27 primer huC.sub.1-II variant 1
[0170] SEQ ID NO: 28 primer huC.sub.1-II variant 2
[0171] SEQ ID NO: 29 primer V.sub.HL-1a variant 1
[0172] SEQ ID NO: 30 primer V.sub.HL-1a variant 2
[0173] SEQ ID NO: 31 primer V.sub.HL-1b
[0174] SEQ ID NO: 32 primer V.sub.HL-1c variant 1
[0175] SEQ ID NO: 33 primer V.sub.HL-1c variant 2
[0176] SEQ ID NO: 34 primer V.sub.HL-1d variant 1
[0177] SEQ ID NO: 35 primer V.sub.HL-1d variant 2
[0178] SEQ ID NO: 36 primer V.sub.HL-2a
[0179] SEQ ID NO: 37 primer V.sub.HL-2b variant 1
[0180] SEQ ID NO: 38 primer V.sub.HL-2b variant 2
[0181] SEQ ID NO: 39 primer V.sub.HL-3a variant 1
[0182] SEQ ID NO: 40 primer V.sub.HL-3a variant 2
[0183] SEQ ID NO: 41 primer V.sub.HL-3b
[0184] SEQ ID NO: 42 primer V.sub.HL-3c
[0185] SEQ ID NO: 43 primer V.sub.HL-4a variant 1
[0186] SEQ ID NO: 44 primer V.sub.HL-4a variant 2
[0187] SEQ ID NO: 45 primer V.sub.HL-4b
[0188] SEQ ID NO: 46 primer V.sub.HL-5a variant 1
[0189] SEQ ID NO: 48 primer V.sub.HL-6a
[0190] SEQ ID NO: 49 primer V.sub.HL-7a variant 1
[0191] SEQ ID NO: 50 primer V.sub.HL-7a variant 2
[0192] SEQ ID NO: 51 primer huC.sub.H-III variant 1
[0193] SEQ ID NO: 52 primer huC.sub.H-III variant 2
[0194] SEQ ID NO: 53 primer V.sub.kL-1a variant 1
[0195] SEQ ID NO: 54 primer V.sub.kL-1a variant 2
[0196] SEQ ID NO: 55 primer V.sub.kL-1b variant 1
[0197] SEQ ID NO: 56 primer V.sub.kL-1b variant 2
[0198] SEQ ID NO: 57 primer V.sub.kL-1c variant 1
[0199] SEQ ID NO: 58 primer V.sub.kL-1c variant 2
[0200] SEQ ID NO: 59 primer V.sub.kL-1d
[0201] SEQ ID NO: 60 primer V.sub.kL-2a
[0202] SEQ ID NO: 61 primer V.sub.kL-2b variant 1
[0203] SEQ ID NO: 62 primer V.sub.kL-2b variant 2
[0204] SEQ ID NO: 63 primer V.sub.kL-3a variant 1
[0205] SEQ ID NO: 64 primer V.sub.kL-3a variant 2
[0206] SEQ ID NO: 65 primer V.sub.kL-3b
[0207] SEQ ID NO: 66 primer V.sub.kL-3c
[0208] SEQ ID NO: 67 primer V.sub.kL-4a
[0209] SEQ ID NO: 68 primer V.sub.kL-5a
[0210] SEQ ID NO: 69 primer V.sub.kL-6a
[0211] SEQ ID NO: 70 primer V.sub.kL-6b
[0212] SEQ ID NO: 71 primer huC.sub.k-III
[0213] SEQ ID NO: 72 primer V.sub.lL-1a
[0214] SEQ ID NO: 73 primer V.sub.lL-1b variant 1
[0215] SEQ ID NO: 74 primer V.sub.lL-1b variant 2
[0216] SEQ ID NO: 75 primer V.sub.lL-1c
[0217] SEQ ID NO: 76 primer V.sub.lL-2a
[0218] SEQ ID NO: 77 primer V.sub.lL-3a variant 1
[0219] SEQ ID NO: 78 primer V.sub.lL-3a variant 2
[0220] SEQ ID NO: 79 primer V.sub.lL-3b
[0221] SEQ ID NO: 80 primer V.sub.lL-3c
[0222] SEQ ID NO: 81 primer V.sub.lL-3d
[0223] SEQ ID NO: 82 primer V.sub.lL-4 variant 1
[0224] SEQ ID NO: 83 primer V.sub.lL-4 variant 2
[0225] SEQ ID NO: 84 primer V.sub.lL-5 variant 1
[0226] SEQ ID NO: 85 primer V.sub.lL-5 variant 2
[0227] SEQ ID NO: 86 primer V.sub.lL-6
[0228] SEQ ID NO: 87 primer V.sub.lL-7 variant 1
[0229] SEQ ID NO: 88 primer V.sub.lL-7 variant 2
[0230] SEQ ID NO: 89 primer V.sub.lL-8
[0231] SEQ ID NO: 90 primer V.sub.lL-4/9 variant 1
[0232] SEQ ID NO: 91 primer V.sub.lL-4/9 variant 2
[0233] SEQ ID NO: 93 primer huC.sub.l-III
[0234] SEQ ID NO: 94 primer huV.sub.H-1
[0235] SEQ ID NO: 95 primer huV.sub.H-2
[0236] SEQ ID NO: 96 primer huV.sub.H-3 variant 1
[0237] SEQ ID NO: 97 primer huV.sub.H-3 variant 2
[0238] SEQ ID NO: 98 primer huV.sub.H-3 variant 3
[0239] SEQ ID NO: 99 primer huV.sub.H-3 variant 4
[0240] SEQ ID NO: 100 primer huV.sub.H-4 variant 1
[0241] SEQ ID NO: 101 primer huV.sub.H-4 variant 2
[0242] SEQ ID NO: 102 primer huV.sub.H-4 variant 3
[0243] SEQ ID NO: 103 primer huV.sub.H-4 variant 4
[0244] SEQ ID NO: 104 primer huC.sub.H-2 variant 1
[0245] SEQ ID NO: 105 primer huC.sub.H-2 variant 2
[0246] SEQ ID NO: 106 primer huC.sub.H-2 variant 3
[0247] SEQ ID NO: 107 primer huV.sub.k-1 variant 1
[0248] SEQ ID NO: 108 primer huV.sub.k-1 variant 2
[0249] SEQ ID NO: 109 primer huV.sub.k-1 variant 3
[0250] SEQ ID NO: 110 primer huV.sub.k-1 variant 4
[0251] SEQ ID NO: 111 primer huV.sub.k-2 variant 1
[0252] SEQ ID NO: 112 primer huV.sub.k-2 variant 2
[0253] SEQ ID NO: 113 primer huV.sub.k-3
[0254] SEQ ID NO: 114 primer huV.sub.k-4
[0255] SEQ ID NO: 115 primer huC.sub.k-2
[0256] SEQ ID NO: 116 primer huV.sub.l-1
[0257] SEQ ID NO: 117 primer huV.sub.l-2
[0258] SEQ ID NO: 118 primer huV.sub.l-3 variant 1
[0259] SEQ ID NO: 119 primer huV.sub.l-3 variant 2
[0260] SEQ ID NO: 120 primer huC.sub.l-2 variant 1
[0261] SEQ ID NO: 121 primer huC.sub.l-2 variant 2
[0262] SEQ ID NO: 122 primer huC.sub.l-2 variant 3
[0263] SEQ ID NO: 123 primer huC.sub.l-2 variant 4
[0264] SEQ ID NO: 124 primer huC.sub.l-2 variant 5
[0265] SEQ ID NO: 125 primer huC.sub.l-2 variant 6
[0266] SEQ ID NO: 126 primer LTGS-lfp
[0267] SEQ ID NO: 127 primer LTGS-rfp
[0268] SEQ ID NO: 128 primer V.sub.H-1a
[0269] SEQ ID NO: 129 primer V.sub.H-1b
[0270] SEQ ID NO: 130 primer V.sub.H-1c
[0271] SEQ ID NO: 131 primer V.sub.H-1d
[0272] SEQ ID NO: 132 primer V.sub.H-2a
[0273] SEQ ID NO: 133 primer V.sub.H-2b
[0274] SEQ ID NO: 134 primer V.sub.H-3a
[0275] SEQ ID NO: 135 primer V.sub.H-3b
[0276] SEQ ID NO: 136 primer V.sub.H-3c
[0277] SEQ ID NO: 137 primer V.sub.H-4a
[0278] SEQ ID NO: 138 primer V.sub.H-4b
[0279] SEQ ID NO: 139 primer V.sub.H-5a
[0280] SEQ ID NO: 140 primer V.sub.H-6a
[0281] SEQ ID NO: 141 primer V.sub.H-7a
[0282] SEQ ID NO: 142 primer V.sub.H-lfp
[0283] SEQ ID NO: 143 primer V.sub.H-rfp
[0284] SEQ ID NO: 144 primer VL(k)-lfp
[0285] SEQ ID NO: 145 primer VL(k)-rfp
DESCRIPTION OF THE FIGURES
[0286] FIGS. 1A-C Chromosomal localization of the human
immunoglobulin G heavy chain locus (FIG. 1A), the human
immunoglobulin kappa light chain locus (FIG. 1B) and of the human
immunoglobulin lambda light chain locus (FIG. 1C).
[0287] FIG. 2 Scheme for the polymerase chain reaction for the
immunoglobulin light chain with a first and a second primer
set.
[0288] FIGS. 3A-B Agarose gel analysis of the amplified nucleic
acid after the first (FIG. 3A) and the second (FIG. 3B) polymerase
chain reaction with different primer sets; (A) 1--IgG HC and IgG
LC(.kappa.) 55.degree. C.; 2--IgG LC(.kappa.) 55.degree. C.; 3--IgG
HC 55.degree. C.; 4--IgG HC and IgG LC(.kappa.) 50.degree. C.;
[0289] 5--IgG LC(.kappa.) 50.degree. C.; 6--IgG HC 50.degree. C.;
7--H.sub.2O PCRI; (B) 1--IgG LC(.kappa.) 55.degree. C.; 2--IgG HC
and IgG LC(.kappa.) 55.degree. C.; 3--IgG HC 55.degree. C.; 4--IgG
HC and IgG LC(.kappa.) 50.degree. C.; 5--IgG LC(.kappa.) 50.degree.
C.; 6--IgG HC 50.degree. C.; 7--H.sub.2O PCRII; 8--H.sub.2O
PCRI.
[0290] FIG. 4 Scheme for the polymerase chain reaction for the
immunoglobulin heavy chain with a different second primer set in
the two polymerase chain reactions.
[0291] FIGS. 5A-B Agarose gel analysis of the amplified nucleic
acid after the first (FIG. 5A) and the second (FIG. 5B) polymerase
chain reaction with different primer sets; (A) 1--IgG HC and IgG
LC(.kappa.) 55.degree. C.; 2--IgG LC(.kappa.) 55.degree. C.; 3--IgG
HC 55.degree. C.; 4--IgG HC and IgG LC(.kappa.) 50.degree. C.;
5--IgG LC(.kappa.) 50.degree. C.; 6--IgG HC 50.degree. C.;
7--H.sub.2O PCRI; (B) 1--IgG HC and IgG LC(.kappa.) 55.degree. C.;
2--IgG LC(.kappa.) 55.degree. C.; 3--IgG HC 55.degree. C.; 4--IgG
HC and IgG LC(.kappa.) 50.degree. C.; 5--IgG LC(.kappa.) 50.degree.
C.; 6--IgG HC 50.degree. C.; 7--H.sub.2O PCRII; 8--H.sub.2O
PCRI.
[0292] FIG. 6 Scheme for the polymerase chain reaction for the
immunoglobulin heavy chain with identical primer set in the two
polymerase chain reactions.
[0293] FIGS. 7A-B Agarose gel analysis of the amplified nucleic
acid after the first (FIG. 7A) and the second (FIG. 7B) polymerase
chain reaction with different primer sets; (A) 1--IgG HC and IgG
LC(.kappa.) 55.degree. C.; 2--IgG LC(.kappa.) 55.degree. C.; 3--IgG
HC 55.degree. C.; 4--IgG HC and IgG LC(.kappa.) 50.degree. C.;
5--IgG LC(.kappa.) 50.degree. C.; 6--IgG HC 50.degree. C.;
7--H.sub.2O PCRI; (B) 1--IgG LC(.kappa.) 55.degree. C.; 2--IgG HC
and IgG LC(.kappa.) 55.degree. C.; 3--IgG HC 55.degree. C.; 4--IgG
HC and IgG LC(.kappa.) 50.degree. C.; 5--IgG LC(.kappa.) 50.degree.
C.; 6--IgG HC 50.degree. C.; 7--H.sub.2O PCRII; 8--H.sub.2O
PCRI.
[0294] FIG. 8 Scheme of overlapping extension PCR exemplified with
a C-terminal HA-tag.
[0295] FIG. 9 Agarose gel analysis of the linear expression
constructs of the three different polymerase chain reactions of
examples 1 to 3.
[0296] FIG. 10 Comparison of the result of the three polymerase
chain reactions according to the invention after in vitro
translation (determination at 450 nm, reference wavelength at 620
nm, background subtracted).
[0297] FIGS. 11A-B Agarose gel analysis of the amplified nucleic
acid after the first (FIG. 11A) and the second (FIG. 11B)
polymerase chain reaction with two identical sets of primer; 1--no
RT, 2--37 single cells, 38--H.sub.2O cDNA, 39--control mRNA,
40--H.sub.2O control mRNA, 41--H.sub.2O PCRII, 42--IgG HC/IgG
LC(.kappa.), 43--IgG HC, 44--IgG LC(.kappa.), 45--H.sub.2O PCRI,
46--GFP, 46--H.sub.2O GFP.
[0298] FIGS. 12A-C Agarose gel analysis of the amplified nucleic
acid after the second polymerase chain reaction with one variable
and one fixed set of primer (FIG. 12A-C).
[0299] FIG. 13 Agarose gel analysis of the linear expression
constructs obtained from nucleic acid after the second polymerase
chain reaction with one variable and one fixed set of primer.
[0300] FIG. 14 Result of the in vitro translation of an IgG
specific two-step polymerase chain reaction of a single cell with
one fixed set of primer and one variable set of primer.
[0301] FIG. 15 Human Fab immunoglobulin fragments after single cell
polymerase chain reaction and in vitro translation; 1-8 human Fab
fragments after in vitro translation of IgG HC and IgG LC(.kappa.)
obtained from a single cell and addition of IgG HC control sample,
9-11 human Fab fragments after in vitro translation of IgG HC and
IgG LC(.kappa.) obtained from a single cell, 12 human Fab fragments
after in vitro translation of IgG HC and IgG LC(.lamda.) obtained
from a single cell.
[0302] FIGS. 16A-B Western blot analysis after a two-step
polymerase chain reaction with one fixed set of primer and one
variable set of primer and in vitro translation; (FIG. 16A) 1--IgG
HC/IgG LC from a single cell combined with IgG HC, 2--4 IgG HC/IgG
LC from a single cell, 5--7 IgG HC/IgG LC(.kappa.) control, 8--IgG
HC and IgG LC(.kappa.) control, 9--negative control, 10--standard
Fab 0.5 ng/ml, 11--standard Fab 50 ng/ml, 12--standard Fab 5
.mu.g/ml; (FIG. 16B) 1--standard Fab 5 .mu.g/ml, 2--standard Fab 50
ng/ml, 3--standard Fab 0.5 ng/ml, 3--negative control, 5--12 IgG
HC/IgG LC from a single cell combined with IgG HC.
EXAMPLES
Materials & Methods
B-Cells and Plasma Cells
[0303] Samples used in this approach are B-cells and plasma cells
isolated from the peripheral blood of healthy donor and tissue
(spleen, bone marrow) of transgenic mice for human IgG. Solid
tissue is first of all manually disaggregated in DMEM in separate
tubes. In the later steps, gentle handling and low temperature
minimize cell lysis, which is important for the future positive
isolation of the cells of interest and to keep the source of mRNA
intact. Disaggregated tissue is suspended by the delicate addition
of cell separation media for making of a different cell type
gradient (Leucosep-tubes (Greiner Bio-One) with Ficoll density
gradient). Suspended cells are purified by centrifugation on the
cold separation medium for 20 min. at 800.times.g and 22.degree. C.
in a centrifuge without breaking in order to enrich for plasma
cells (PBMC) and lymphocytes. Cells are washed in cold buffer (PBS
(phosphate buffered saline), 0.1% (w/v) BSA (bovine serum albumin),
2 mM EDTA (ethylene diamin tetra acetate)) and the supernatant is
carefully discarded to keep only the lymphocytes. Lymphocytes are
than resuspended in PBS and mixed by carefully pipetting.
Centrifugation is effectuated for 5 min at 800.times.g and
22.degree. C. to pellet the cells. B-cells and plasma cells are
pretreated with murine and human FC blocker to block unspecific
binding of Abs on their cells surface. Cells are washed once with
buffer (PBS, 0.1% (w/v) BSA, 2 mM EDTA), centrifuged and
resuspended in PBS. Only the CD19+ B-cells and CD138+ plasma cells
were used. To prevent mRNA degradation an RNAse Inhibitor is added.
The positive isolation of the CD19+ B-cells (Dynal Biotech
Dynabeads CD19 Pan B) from the mouse spleen has been carried out
according to the manufacturer's instructions. The selection of the
CD138+ plasma cells (StemCell Technologies EasySep Human CD138
Selection Kit) has been carried out following the manufacturer's
instructions.
Separation into Single Cell by the Principle of the
Limiting-Dilution Culture or FACS Sorting:
[0304] Cells are counted and, by the principle of the
limiting-dilution culture, deposited as single cell into the wells
of 96-well PCR plates or 384-well plates. Plates are sealed with
PCR Film and immediately placed on ice. Sorted cells can be used
immediately in RT-PCR (reverse transcriptase polymerase chain
reaction) or stored at -20.degree. C. for short-term use or
-80.degree. C. for long-term use. Single-cell sorting was performed
on a FACSAria cell-sorting system (Becton Dickinson). Cells that
stained positive for CD19, highly positive for CD38 and
intermediately positive for CD45 were collected and designated
plasma cells (PC). Additional gates on forward scatter/side scatter
and side scatter width/side scatter height were included to select
live lymphocytes and singlets, respectively. Single cells were
distributed directly into the wells of 96-well PCR plates
(Eppendorf), containing all the necessary PCR reagents in a volume
of 10 except for reverse transcriptase, DNA polymerase, buffer and
dNTPs and frozen at -80.degree. C. for later processing.
Cell Lysis and Reverse Transcription:
[0305] To be able to amplificate the mRNA in a polymerase chain
reaction, B-cells and plasma cells must be lysed before the reverse
transcription reaction.
TABLE-US-00022 TABLE 22 Components used for the classical lysis.
Component Volume (.mu.l) Final concentration Water, PCR grade 1.75
5xRT Reaction Buffer 1.00 RNAse Inhibitor (40 U/.mu.l) 0.25 5 U
Gene Specific antisense primer 1.00 0.02 .mu.M Igepal 0.01% 1.00
0.01% Final volume 5.00
TABLE-US-00023 TABLE 23 Block cycler program for the cell lysis.
Temperature (.degree. C.) Time (sec) 65 60 55 30 45 30 35 20 23 120
4 .infin.
[0306] Plates with lysed cells are spun briefly in the centrifuge
for 30 sec. to collect liquid and cells in the bottom of wells. The
RT (reverse transcriptase) reaction as well as all the PCR reaction
was made in a 96-well plate. To each well containing 5 .mu.l
template is added 2.5 .mu.l of water, 1 .mu.l cold RT reaction
buffer, 1 .mu.l dNTPs, 0.25 .mu.l RNAse Inhibitor (40 U/.mu.l),
0.25 .mu.l reverse transcriptase (20 U/.mu.l), all from the First
Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim,
Germany) for a total volume of 10 .mu.l. RT plates are briefly
centrifuged and placed from ice to 55.degree. C. for 60 min. (with
heated lid), heated to 85.degree. C. for 2 min. (to inactivate the
reverse transcriptase), in a Block cycler (LightCycler 480, Roche
Diagnostics GmbH, Mannheim, Germany). Single-stranded cDNA was
stored at -20.degree. C. shortly after the reverse transcription
reaction to avoid degradation of the cDNA. A control synthesis
reaction was simultaneously performed without cells to test for
contamination.
TABLE-US-00024 TABLE 24 Components used for the reverse
transcription reaction. Component Volume (.mu.l) Final
concentration Water, PCR grade 6.8 PCR Master 10 Semi-nested Primer
IgG HC 0.4 0.02 .mu.M Semi-nested Primer IgG LC (k) 0.4 0.02 .mu.M
Semi-nested Primer IgG LC (l) 0.4 0.02 .mu.M cDNA template from RT
reaction 2 Final volume 20
Cell Lysis and Reverse Transcription:
[0307] To be able to amplificate the mRNA in a polymerase chain
reaction, B-cells and plasma cells must be lysed before the reverse
transcription reaction.
First PCR:
TABLE-US-00025 [0308] TABLE 25 Block cycler program for the first
PCR. Temperature (.degree. C.) Time Number of cycles 95 .sup. 2
min. 1 94 15 sec. 35 55 30 sec. 72 .sup. 1 min. 72 10 min. 1 4
.infin.
TABLE-US-00026 TABLE 26 Primer used for the first PCR. Ig heavy
chain Ig light chain (.kappa.) Ig light chain (.lamda.) primer
primer primer V.sub.HL-1 V.sub..kappa.L-1 V.sub..lamda.L-1
V.sub.HL-2 V.sub..kappa.L-2 V.sub..lamda.L-2 V.sub.HL-3
V.sub..kappa.L-3 V.sub..lamda.L-3 V.sub.HL-4 V.sub..kappa.L-4
huC.sub..lamda.-II V.sub.HL-5 huC.sub..kappa.-II V.sub.HL-6
huC.sub.H-II
Second PCR:
[0309] The .kappa. and .lamda. light chains and the heavy chains
were subsequently amplified with a second-round PCR according to
the following protocol. Using semi-nested primer, the second PCR
was performed to increase the amount of cDNA copies and to amplify
only from the variable part of the light chain (LC) and heavy chain
(HC) to the C.sub.H1 region. The HC amplification was performed
using 16 primer, the kappa LC using 17 primer, and the lambda LC
using 14 primer. The genes were amplified in a total volume of 20
.mu.l using 2 .mu.l of the first PCR product, 10 .mu.l of High
Fidelity PCR Master (containing 0.4 .mu.M each dNTPs, double
concentrated reaction buffer (with 3 mM MgCl.sub.2), 0.02 .mu.M of
each primer, all from the High Fidelity PCR Master Kit (Roche
Diagnostics GmbH, Mannheim, Germany) using the following second PCR
program: 2 min. at 95.degree. C., 45 cycles of 94.degree. C. for 15
sec., 55.degree. C. for 30 sec., 72.degree. C. for 1 min.,
following 10 min. at 72.degree. C.
TABLE-US-00027 TABLE 27 Components used for the second PCR.
Component Volume (.mu.l) Final concentration Water, PCR grade 7.8
High Fidelity PCR Master 10 1.8 mM MgCl.sub.2 Two-step Primer IgG
HC 0.4 0.02 .mu.M Two-step Primer IgG LC(.kappa.) 0.4 0.02 .mu.M
Two-step Primer IgG LC(.lamda.) 0.4 0.02 .mu.M First PCR product 2
Final volume 20
TABLE-US-00028 TABLE 28 Block cycler program for the second PCR.
Temperature (.degree. C.) Time Number of cycles 95 .sup. 2 min. 1
94 15 sec. 45 55 30 sec. 72 .sup. 1 min. 72 10 min. 1 4 .infin.
TABLE-US-00029 TABLE 29 Primer used for the second PCR IgG heavy
chain IgG light chain (.kappa.) IgG light chain (.lamda.) primer
primer primer V.sub.H1a V.sub..kappa.1a V.sub..lamda.1a V.sub.H1b
V.sub..kappa.1b V.sub..lamda.1b V.sub.H1c V.sub..kappa.1c
V.sub..lamda.1c V.sub.H1d V.sub..kappa.1d V.sub..lamda.2 V.sub.H2a
V.sub..kappa.2a V.sub..lamda.3a V.sub.H2b V.sub..kappa.2b
V.sub..lamda.3b V.sub.H3a V.sub..kappa.3a V.sub..lamda.3c V.sub.H3b
V.sub..kappa.3b V.sub..lamda.3d V.sub.H3c V.sub..kappa.3c
V.sub..lamda.4 V.sub.H4a V.sub..kappa.4a V.sub..lamda.5 V.sub.H4b
V.sub..kappa.5a V.sub..lamda.6 V.sub.H5a V.sub..kappa.6a
V.sub..lamda.7 V.sub.H6a V.sub..kappa.6b V.sub..lamda.8 V.sub.H7a
C.sub..kappa.III V.sub..lamda.4/9 C.sub.HIII V.sub..lamda.10
C.sub..lamda.III
One Step Multiplex RT-GSP (Gene Specific Primer)-PCR Reaction:
[0310] To be able to amplificate the mRNA in a polymerase chain
reaction, B-cells and plasma cells must be distributed directly
into the wells of 96-well PCR plates (Eppendorf), containing all
the necessary PCR reagents in a volume of 10 .mu.l, except for
reverse transcriptase, DNA polymerase, buffer and dNTPs and frozen
at -80.degree. C. for later processing.
RT-Step:
[0311] Reverse transcription and PCR were performed in one step
(one step Multiplex RT-PCR). The isolated, sorted and stored cells
were used as raw material for the reverse transcription or RT-PCR.
All necessary reagents were thawed at room temperature. All primer
were synthesized in the MOLBIOL TIB GmbH laboratories. The plates
and all other reagents were kept on ice during the entire
procedure. For cDNA syntheses the gene specific primer with
extensions were used directly. The enzyme complex consists of two
Sensiscript reverse transcriptases and one Omniscript polymerase
(Qiagen OneStep RT PCR). The rewriting of the mRNA into cDNA was
performed by the Sensiscript complex (Qiagen OneStep RT PCR) and
the amplification of the cDNA was performed using the HotStarTaq
DNA Polymerase (Qiagen OneStep RT PCR), which is a chemically form
of a recombinant 94 kDa DNA polymerase
(deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC
2.7.7.7), originally isolated from Thermus aquaticus expressed in
E. coli. The cells were sorted in a 96-well PCR plate and stored in
a volume of 10 .mu.l, containing 5 .mu.l PCR H.sub.2O grade, 1
.mu.l 0.1 .mu.M primer for VH and VL, 1 .mu.l RNAse inhibitor 20
U/reaction and 3 .mu.l Tris 1.5 mM. Before adding the other 10
.mu.l for performing the PCR reaction, the cells stored at
-60.degree. C. were briefly centrifuged (20 sec. at 1400 rpm) to
collect the liquid and cells on the bottom of the wells.
TABLE-US-00030 TABLE 30 Master Mix 1 used for the RT-PCR. Final
volume/well Master Mix 1 concentration/well (.mu.l) H.sub.2O 5
primer V.sub.H/VL(k) 0.1 .mu.M 1 RNAse Inhibitor 20 U/reaction 1
Tris-buffer 1.5 mM 3 B/Plasma cells final volume 10
TABLE-US-00031 TABLE 31 Master Mix 2 used for the RT-PCR. Final
volume/well Master Mix 2 concentration/well (.mu.l) H.sub.2O 1x 2.2
5x Buffer 1x 4 dNTP 10 mM each 400 .mu.M each 0.8 5x Q-Solution
0.25x 1 One Step RT PCR Enzyme 1.2 mix RNAse Inhibitor 20U 1 final
volume 10
[0312] 10 .mu.l per well of Master Mix 2 were added to the cells.
The second Master Mix contained 2.2 .mu.l H.sub.2O PCR grade, 4
.mu.l of 1.times. buffer, 0.8 .mu.l of dNTPs 400 .mu.M each, 1
.mu.l of Q-solution 0.25.times., 1.2 .mu.l of the enzyme complex
and 1 .mu.l of RNAse inhibitor 20U.
TABLE-US-00032 TABLE 32 Primer used for the RT-PCR. Ig heavy chain
primer Ig light chain (.kappa.) primer V.sub.H-lfp SEQ ID NO: 142
VL(k)-lfp SEQ ID NO: 144 V.sub.H-rfp SEQ ID NO: 143 VL(k)-rfp SEQ
ID NO: 145
TABLE-US-00033 TABLE 33 Block cycler program for the RT-GSP-PCR.
Temperature Time Step Cycles 50.degree. C. 30 min. reverse
transcription 1 95.degree. C. 15 min. denaturation 1 94.degree. C.
40 sec. denaturation 11 52.degree. C. 1 min. annealing 72.degree.
C. 1 min. elongation 94.degree. C. 41 sec. denaturation 29
60.degree. C. 1 min. annealing 72.degree. C. 1 min. elongation
72.degree. C. 10 min. final elongation 1 4.degree. C. .infin.
cooling
Purification of PCR Products:
[0313] To improve the efficiency of the generation of linear
template for the in vitro translation in the next overlapping PCR
(third PCR) the purification of the previously amplified PCR
products was performed by removing unincorporated primer, dNTPs,
DNA polymerases and salts used during PCR amplification in order to
avoid interference in downstream applications. Agencourt AMPure was
used. The buffer is optimized to selectively bind PCR amplicons 100
bp and larger to paramagnetic beads. Excess oligonucleotides,
nucleotides, salts, and enzymes can be removed using a simple
washing procedure. The resulting purified PCR product is
essentially free of contaminants and can be used in the following
applications: Fluorescent DNA sequencing (including capillary
electrophoresis), microarray spotting, cloning and primer extension
genotyping. The work flow for 96-well format started with gently
shaking the beads stored in buffer to resuspend any magnetic
particle that may have settled. The correct volume of 36 .mu.l of
beads solution was added to the 20 .mu.l of sample and the mix was
pipetted 10 times up and down. The following step was incubating
for 10 minutes and afterwards the reaction plate was placed onto a
magnetic plate for 10 minutes to separate beads from solution. The
cleared solution (supernatant) was aspirated from the reaction
plate and discard. For the beads-cDNA washing 200 .mu.l of 70%
ethanol were dispersed per well and incubated at room temperature
for at least 30 seconds. The ethanol was aspirated out and
discarded. The washing step was performed two times and then the
reaction plate was left to air-dry for 20 minutes at room
temperature. It followed with the addition of 40 .mu.l of elution
buffer and the mix was again pipetted 10 times up and down. After
the cDNA dissociation from the magnetic beads, the purified DNA was
transferred into a new plate.
Third PCR:
[0314] The amplified DNA of the second PCR was afterwards linked by
an overlapping extension PCR method with the following components,
necessary for the transcription/translation step: a ribosome
binding site (RBS), a T7 promoter and a T7 terminator sequences.
For this PCR, 2 .mu.l of the second PCR were taken to a final
volume of 20 .mu.l containing: 10.7 .mu.l water, 2 .mu.l of
10.times. reaction buffer with MgCl.sub.2 (10 mM), 0.8 .mu.l of
DMSO, 0.5 .mu.l dNTPs (10 mM each), 1.6 .mu.l T7 promotor and
terminator primer (6 .mu.M each), 0.4 .mu.l C-terminal HA-Tag
primer and 0.4 .mu.l of enzyme blend, all from the RTS E. coli
Linear Template Generation Set, HA-Tag (Roche Diagnostics GmbH,
Mannheim, Germany). Finally, the overlapping PCR products were used
as template for in vitro transcription using Escherichia coli
lysate and the resulting functional Fab was screened against the
F(ab').sub.2 IgG by enzyme-linked immunoadsorbent assay
(ELISA).
TABLE-US-00034 TABLE 34 Components used for the third PCR. Volume
Component (.mu.l) Final concentration Water, PCR grade 10.7 10x
Reaction Buffer with MgCl.sub.2 (10 mM) 2 1x DMSO 0.8 PCR
Nucleotide mix (10 mM each) 0.5 250 .mu.M Working solution T7 Prom
Primer (6 .mu.M) 1.6 0.48 .mu.M Working solution T7 Term Primer (6
.mu.M) 1.6 0.48 .mu.M Working solution C-term HA-tag (6 .mu.M) 0.4
0.48 .mu.M Enzyme Blend 0.4 PCR 2 product 2 Final volume 20
TABLE-US-00035 TABLE 35 Block cycler program for the third PCR.
Temperature (.degree. C.) Time Number of cycles 95 4 min. 1 95 1
min. 45 60 1 min. 72 1 min. 30 sec. 72 7 min. 1 4 .infin.
Gel Electrophoresis:
[0315] The gel electrophoresis analysis (1% agarose gel, Invitrogen
Corp., USA) was performed to evaluate the amplification and the
specificity of the cDNA templates with the appropriate
controls.
TABLE-US-00036 TABLE 36 Gel analysis protocol. Component Volume
(.mu.l) Migration time H.sub.20 6 5x Orange G 3 PCR product 6 Final
volume 15 Volume for gel 10 20 min.
In Vitro Transcription and Translation:
[0316] The in vitro coupled transcription and translation was
carried out following the manufacturer's protocol RTS 100 E. coli
Disulfide Kit (Roche Diagnostics GmbH, Mannheim, Germany) with
components as reported (see Table 12). 4 .mu.l of each overlapping
PCR product was transcribed and translated in a total volume of 50
.mu.l, at 37.degree. C. for 20 hours in the RTS Proteo Master
Instrument (Roche Diagnostics GmbH, Mannheim, Germany). A control
reaction was performed under identical conditions without cDNA
template. GFP (green fluorescent protein) vectors were added to the
reaction system for autoradiography as positive control. After the
in vitro transcription/translation, the 50 .mu.l reaction mixture
was transferred in 75 .mu.l PBS (1:2.5 dilution) and incubated at
4.degree. C. overnight for the correct folding and maturation of
the protein.
TABLE-US-00037 TABLE 37 Components for the in vitro transcription
and translation. Mix Component Volume (.mu.l) Mix 1: E. coli lysate
25 Lysate activator 1 Final volume 26 incubate for 10-20 min. at RT
Mix 2: Feeding mix 640 Amino acid mix 140 Methionine 20 H.sub.2O
200 Final volume 1000 Mix 3: Reaction mix 7 Amino acid mix 7
Methionine 1 Mix 1 25 GroE Supplement 5 RNAse inhibitor 1 PCR 3
product 4 Final volume 50
ELISA:
[0317] A 384-well plate (Nunc GmbH & Co. KG, Thermo Fisher
Scientific, Langenselbold, Germany) was coated with 50 .mu.l
(1:1000 in PBS) goat anti-human IgG Fab fragment (produced by
Bethyl Laboratories Inc., obtained from Biomol GmbH, Hamburg,
Germany, 1 mg/l ml) incubated at 4.degree. C. overnight. The plate
was washed three times with washing solution (100 .mu.l PBST
(phosphate buffered saline Tween-20)) and 60 .mu.l of Blocking
solution (0.25% CroteinC (w/v)/0.5% Tween (w/v)/PBS) was added,
incubated for 1 h at room temperature. Another washing step
(3.times.100 .mu.l PBST) was performed and 37.5 .mu.l sample was
transferred, as well as 37.5 ml negative control (negative control
from the in vitro transcription/translation) and 37.5 .mu.l
positive control, containing 0.75 .mu.l of human recombinant Fab
fragment (Roche Diagnostics GmbH, Mannheim, Germany). The samples
were titrated to a 1:3 dilution. The plate was incubated for 1.5 h
at room temperature. After a washing step (3.times.100 .mu.l PBST),
25 .mu.l goat anti-human IgG F(ab).sub.2 (Dianova, Hamburg,
Germany; 0.8 mg/ml (1:2000 diluted in Blocking Solution)) was added
and incubated for 1 h at room temperature. The last washing step
(3.times.100 .mu.l PBST) was performed and 25 .mu.l of TMB (POD
Substrate, Roche Diagnostics GmbH, Mannheim, Germany, Art-No: 1 484
281) was pipetted into each well. After 2-3 minutes the absorption
signal was detected at 405 nm and 495 nm (Tecan, Safire 2; Tecan
Deutschland GmbH, Crailsheim, Germany).
Flow Cytometric Analysis and Cell Sorting:
[0318] For FACS analysis and cell sorting monoclonal antibodies,
either biotinylated or conjugated with either FITC (fluorescein
isothiocyanate), PE (Phycoerythrin), or APC (allophycocyanin)
against the following antigens were used: CD3 (UCHT1), CD4
(13B8.2), CD8 (B9.11), CD40 (MAB89), CD80 (MAB104), CD83 (HB15a),
CD86 (HA5.2B7) (all available from Imunotech/Beckman Coulter,
Marseille, France), CD19 (HIB19), CD20 (2H7), CD34(581),
IL-3Ra/CD123 (9F5), CD11c (B-ly6) CD14 (M5E2), CD24, CD22a, CD38,
CD138 (all available from BD Pharmingen, San Diego, Calif., USA),
CD45 (HI30), CD45RA (MEM56), HLA-DR (TU36) (all available from
Caltag, Burlingame, Calif., USA), TLR2 (TL2.1), TLRR4 (HTA125),
TCRab (IP26), (all available from Bioscience, San Diego, Calif.),
BDCA-1, BDCA-2, BDCA-4, CD25 (4E3) (all available from Miltenyi
Biotec, Bergisch Gladbach, Germany), IgM (Jackson Immunoresearch,
West Grove, Pa., USA), CCR7 (3D12, provided by M. Lipp, Berlin,
Germany). The IOTest Beta Mark was used for Vb analysis
(Imunotech/Beckman Coulter). Streptavidin conjugated FITC, PE, or
APC (all BD Pharmingen) were used for visualization of biotinylated
antibodies. Dead cells were excluded by propidium iodide staining.
Appropriate isotype-matched, irrelevant control mAbs were used to
determine the level of background staining. Cells were analyzed
using a FACS Calibur and sorted using a FACSAria (Becton Dickinson
Immunocytometry Systems, Mountain View, Calif., USA).
Example 1
[0319] Amplification of IgG Genes from Humanized Immunized Mice's
Single B Cell by a Polymerase Chain Reaction with One Fixed Primer
Set and One Changed Primer Set
[0320] Single B-cells of a mouse having a human immunoglobulin
locus have been obtained as outlined above. The 3'-primer of the
first and second primer set employed in the polymerase chain
reaction were identical. The 5'-primer of the first and second
primer set different insofar as the primer of the first 5'-primer
set bound in the region encoding the leader peptide and the primer
of the second 5'-primer set bound in the FR1 region. A scheme of
this polymerase chain reaction is given in FIG. 2.
[0321] The employed sets of primer for the first polymerase chain
reaction are denoted in the following list:
TABLE-US-00038 Ig heavy chain Ig light chain (.kappa.) primer
primer Ig light chain (.lamda.) primer VHL-1 V.sub..kappa.L-1
V.sub..lamda.L-1 V.sub.HL-2 V.sub..kappa.L-2 V.sub..lamda.L-2
V.sub.HL-3 V.sub..kappa.L-3 V.sub..lamda.L-3 V.sub.HL-4
V.sub..kappa.L-4 huC.sub..lamda.-II V.sub.HL-5 huC.sub..kappa.-II
V.sub.HL-6 huC.sub.H-II
[0322] The employed sets of primer for the second polymerase chain
reaction are denoted in the following list:
TABLE-US-00039 IgG heavy chain IgG light chain (.kappa.) IgG light
chain primer primer (.lamda.) primer V.sub.H-1a SEQ ID NO:
V.sub..kappa.L-1a SEQ ID NO: V.sub..lamda.L-1a SEQ ID NO: 128 53,
54 72 V.sub.H-1b SEQ ID NO: V.sub..kappa.L-1b SEQ ID NO:
V.sub..lamda.L-1b SEQ ID NO: 129 55, 56 73, 74 V.sub.H-1c SEQ ID
NO: V.sub..kappa.L-1c SEQ ID NO: V.sub..lamda.L-1c SEQ ID NO: 130
57, 58 75 V.sub.H-1d SEQ ID NO: V.sub..kappa.L-1d SEQ ID NO:
V.sub..lamda.L-2 SEQ ID NO: 131 59 76 V.sub.H-2a SEQ ID NO:
V.sub..kappa.L-2a SEQ ID NO: V.sub..lamda.L-3a SEQ ID NO: 132 60
77, 78 V.sub.H-2b SEQ ID NO: V.sub..kappa.L-2b SEQ ID NO:
V.sub..lamda.L-3b SEQ ID NO: 133 61, 62 79 V.sub.H-3a SEQ ID NO:
V.sub..kappa.L-3a SEQ ID NO: V.sub..lamda.L-3c SEQ ID NO: 134 63,
64 80 V.sub.H-3b SEQ ID NO: V.sub..kappa.L-3b SEQ ID NO:
V.sub..lamda.L-3d SEQ ID NO: 135 65 81 V.sub.H-3c SEQ ID NO:
V.sub..kappa.L-3c SEQ ID NO: V.sub..lamda.L-4 SEQ ID NO: 136 66 82,
83 V.sub.H-4a SEQ ID NO: V.sub..kappa.L-4a SEQ ID NO:
V.sub..lamda.L-5 SEQ ID NO: 137 67 84, 85 V.sub.H-4b SEQ ID NO:
V.sub..kappa.L-5a SEQ ID NO: V.sub..lamda.L-6 SEQ ID NO: 138 68 86
V.sub.H-5a SEQ ID NO: V.sub..kappa.L-6a SEQ ID NO: V.sub..lamda.L-7
SEQ ID NO: 139 69 87, 88 V.sub.H-6a SEQ ID NO: V.sub..kappa.L-6b
SEQ ID NO: V.sub..lamda.L-8 SEQ ID NO: 140 70 89 V.sub.H-7a SEQ ID
NO: huC.sub..kappa.-2 SEQ ID NO: V.sub..lamda.L-4/9 SEQ ID NO: 141
115 90, 91 huC.sub.H-2 SEQ ID NO: V.sub..lamda.L-10 SEQ ID NO: 104,
105, 92 106 huC.sub..lamda.-2 SEQ ID NO: 120, 121, 122, 123, 124,
125
[0323] In FIGS. 3A-B the agarose gel of the amplified nucleic acid
fragments obtained in this polymerase chain reaction is shown. The
samples were analyzed after 40 amplification cycles with an
annealing temperature of 50.degree. C. and 55.degree. C.,
respectively. The blanks (water) were negative and the size of the
fragments correlated well to the expected sizes of 750 bp (IgG HC)
and 665 bp (IgG LC), respectively, after the first polymerase chain
reaction and of 711 bp and 688 bp, respectively, after the second
polymerase chain reaction.
Example 2
[0324] Amplification of IgG Genes from Humanized Immunized Mice's
Single B Cell by a Polymerase Chain Reaction with Two Changed
Primer Sets
[0325] Single B-cells of a mouse having a human immunoglobulin
locus have been obtained as outlined above. The 5'-primer and the
3'-primer of the first and second primer set were different to each
other insofar as the primer of each of the second sets bound in a
more inward region of the nucleic acid. A scheme of this polymerase
chain reaction is given in FIG. 4.
[0326] The employed sets of primer for the first polymerase chain
reaction are denoted in the following list:
TABLE-US-00040 Ig light Ig heavy chain primer chain (.kappa.)
primer Ig light chain (.lamda.) primer V.sub.HL-1 SEQ ID NO:
V.sub..kappa.L-1 SEQ ID NO: V.sub..lamda.L-1 SEQ ID NO: 05, 06 16
21 V.sub.HL-2 SEQ ID NO: V.sub..kappa.L-2 SEQ ID NO:
V.sub..lamda.L-2 SEQ ID NO: 07, 08 17 22 V.sub.HL-3 SEQ ID NO:
V.sub..kappa.L-3 SEQ ID NO: V.sub..lamda.L-3 SEQ ID NO: 09 18 23,
24, 25, 26 V.sub.HL-4 SEQ ID NO: V.sub..kappa.L-4 SEQ ID NO:
huC.sub..lamda.-II SEQ ID NO: 10, 11 19 27, 28 V.sub.HL-5 SEQ ID
NO: huC.sub..kappa.-II SEQ ID NO: 12 20 V.sub.HL-6 SEQ ID NO: 13
huC.sub.H-II SEQ ID NO: 14, 15
[0327] The employed sets of primer for the second polymerase chain
reaction are denoted in the following list:
TABLE-US-00041 IgG heavy IgG light chain (.kappa.) IgG light chain
primer primer chain (.lamda.) primer V.sub.HL-1a SEQ ID NO:
V.sub..kappa.L-1a SEQ ID NO: V.sub..lamda.L-1a SEQ ID NO: 29, 30
53, 54 72 V.sub.HL-1b SEQ ID NO: V.sub..kappa.L-1b SEQ ID NO:
V.sub..lamda.L-1b SEQ ID NO: 31 ?55, 56 73, 74 V.sub.HL-1c SEQ ID
NO: V.sub..kappa.L-1c SEQ ID NO: V.sub..lamda.L-1c SEQ ID NO: 32,
33 57, 58 75 V.sub.HL-1d SEQ ID NO: V.sub..kappa.L-1d SEQ ID NO:
V.sub..lamda.L-2a SEQ ID NO: 34, 35 59 76 V.sub.HL-2a SEQ ID NO:
V.sub..kappa.L-2a SEQ ID NO: V.sub..lamda.L-3a SEQ ID NO: 36 60 77,
78 V.sub.HL-2b SEQ ID NO: V.sub..kappa.L-2b SEQ ID NO:
V.sub..lamda.L-3b SEQ ID NO: 37, 38 61, 62 79 V.sub.HL-3a SEQ ID
NO: V.sub..kappa.L-3a SEQ ID NO: V.sub..lamda.L-3c SEQ ID NO: 39,
40 63, 64 80 V.sub.HL-3b SEQ ID NO: V.sub..kappa.L-3b SEQ ID NO:
V.sub..lamda.L-3d SEQ ID NO: 41 65 81 V.sub.HL-3c SEQ ID NO:
V.sub..kappa.L-3c SEQ ID NO: V.sub..lamda.L-4a SEQ ID NO: 42 66 82,
83 V.sub.HL-4a SEQ ID NO: V.sub..kappa.L-4a SEQ ID NO:
V.sub..lamda.L-5a SEQ ID NO: 43, 44 67 84, 85 V.sub.HL-4b SEQ ID
NO: V.sub..kappa.L-5a SEQ ID NO: V.sub..lamda.L-6a SEQ ID NO: 45 68
86 V.sub.HL-5a SEQ ID NO: V.sub..kappa.L-6a SEQ ID NO:
V.sub..lamda.L-7a SEQ ID NO: 46, 47 69 87, 88 V.sub.HL-6a SEQ ID
NO: V.sub..kappa.L-6b SEQ ID NO: V.sub..lamda.L-8a SEQ ID NO: 48 70
89 V.sub.HL-7a SEQ ID NO: huC.sub..kappa.-III SEQ ID NO:
V.sub..lamda.L-4/9a SEQ ID NO: 49, 50 71 90, 91 huC.sub.H- SEQ ID
NO: V.sub..lamda.L-10a SEQ ID NO: III 51, 52 92 huC.sub..lamda.-III
SEQ ID NO: 93
[0328] In FIGS. 5A-B the agarose gel of the amplified nucleic acid
fragments obtained in this polymerase chain reaction is shown. The
samples were analyzed after 40 amplification cycles with an
annealing temperature of 50.degree. C. and 55.degree. C.,
respectively. The blanks (water) were negative and the size of the
fragments correlated well to the expected sizes of 471 bp (IgG HC)
and 413 bp (IgG LC), respectively, after the first polymerase chain
reaction and of 442 bp and 399 bp, respectively, after the second
polymerase chain reaction.
Example 3
[0329] Amplification of IgG Genes from Humanized Immunized Mice's
Single B Cell by a Polymerase Chain Reaction with Two Identical
Primer Sets
[0330] Single B-cells of a mouse having a human immunoglobulin
locus have been obtained as outlined above. The 5'-primer and the
3'-primer of the first and second primer set were identical. A
scheme of this polymerase chain reaction is given in FIG. 6.
[0331] The employed sets of primer for the first and second
polymerase chain reaction are denoted in the following list:
TABLE-US-00042 Ig light chain (.kappa.) Ig heavy chain primer
primer Ig light chain (.lamda.) primer huV.sub.H-1 SEQ ID NO:
huV.sub..kappa.-1 SEQ ID NO: huV.sub..lamda.-1 SEQ ID NO: 94 107,
108, 116 109, 110 huV.sub.H-2 SEQ ID NO: huV.sub..kappa.-2 SEQ ID
NO: huV.sub..lamda.-2 SEQ ID NO: 95 111, 112 117 huV.sub.H-3 SEQ ID
NO: huV.sub..kappa.-3 SEQ ID NO: huV.sub..lamda.-3 SEQ ID NO: 96,
97, 98, 113 118, 119 99 huV.sub.H-4 SEQ ID NO: huV.sub..kappa.-4
SEQ ID NO: huC.sub..lamda.-2 SEQ ID NO: 100, 101, 114 120, 121,
122, 102, 103 123, 124, 125 huC.sub.H-2 SEQ ID NO:
huC.sub..kappa.-2 SEQ ID NO: 104, 105, 115 106
[0332] In FIGS. 7A-B the agarose gel of the amplified nucleic acid
fragments obtained in this polymerase chain reaction is shown. The
samples were analyzed after 40 amplification cycles with an
annealing temperature of 50.degree. C. and 55.degree. C.,
respectively. The blanks (water) were negative and the size of the
fragments correlated well to the expected sizes of 711 bp (IgG HC)
and 688 bp (IgG LC), respectively, after the first and second
polymerase chain reaction.
Example 4
Generation of Linear Template for In Vitro Translation
[0333] For the first polymerase chain reaction gene specific primer
have been designed comprising the necessary overlapping sequences
to the regulatory DNA regions of the T7 phage. For the second
polymerase chain reaction the product of the first PCR was combined
with nucleic acid fragments comprising the regulatory sequences and
encoding the tag-sequence, respectively. A 3'-terminal extension
was achieved by hybridization with the nucleic acid fragments
comprising the regulatory elements. This linear expression
construct is further amplified with the help of two terminal
primer. These primer comprise the following sequence:
5'-CTTTAAGAAGGAGATATACC+ATG+15-20 bp of the gene-specific sequence
(5'-primer, SEQ ID NO: 126) or 5'-ATCGTATGGGTAGCTGGTCCC+TTA+15-20
bp of the gene-specific sequence (3'-primer, SEQ ID NO: 127).
[0334] In FIG. 9 lanes 1, 5 and 9 represent the blank water
controls. The heavy chain nucleic acid are contained in lanes 4, 8,
and 12, and the kappa light chains in lanes 3, 7, and 11. Lanes 2,
6, and 10 show combined samples of both chains. All nucleic acids
have the expected size (see Table 38).
TABLE-US-00043 TABLE 38 Size of the linear expression constructs.
immunoglobulin two fixed primer one fixed primer two variable chain
sets set primer sets IgG HC ~1110 bp ~1110 bp ~822 bp IgG
LC(.kappa.) ~1089 bp ~1089 bp ~799 bp
Example 5
[0335] In Vitro Translation and huFab Specific ELISA
[0336] In vitro translation is carried out as outlined above.
[0337] As can be seem from FIG. 10 nucleic acids obtained with a
two-step polymerase chain reaction with two variable primer sets
does not provide for a linear expression construct which allows the
in vitro production of the encoded Fab immunoglobulin fragment. In
contrast the two-step polymerase chain reaction with one fixed and
one variable set of primer employed in separated successive
polymerase chain reactions allows for the subsequent provision of a
linear expression construct and the in vitro translation of IgG HC
and IgG LC comprising immunoglobulin Fab fragment.
[0338] In contrast to this is the two-step polymerase chain
reaction comprising one fixed set of primer more efficient in the
multiplex format as the polymerase chain reaction employing two
fixed sets of primer. By employing only one fixed set of primer up
to 5-times higher optical densities can be achieved.
Example 6
[0339] In Vitro Translation and huFab Specific ELISA with a Nucleic
Acid Obtained from a from Single Cell Two-Step Polymerase Chain
Reaction with Identical Primer Sets
[0340] As can be seen from FIGS. 11A-B the control samples yielded
no signal in an agarose gel. Single deposited B-cells also showed
no signal. IgG HC and IgG LC(.kappa.) could be amplified from
control samples.
Two-Step Polymerase Chain Reaction with One Fixed Set of Primer and
One Variable Set of Primer
[0341] As can be seen from FIG. 13 all nucleic acids, except for
sample 2, obtained with a two-step polymerase chain reaction with
one fixed set of primer and one variable set of primer allowed for
providing a linear expression construct for the production of IgG
HC and IgG LC. Thus, these multiplex polymerase chain reaction are
well suited.
[0342] The concentrations of the obtained human kappa Fab
immunoglobulin fragments are between 100 and 550 ng/ml. Lanes 9 to
11 of FIGS. 16A-B show IgG HC and IgG LC(.kappa.) obtained from a
single cell without the addition of IgG HC positive control. Here
the obtained amount of human Fab immunoglobulin fragment was
between 180 and 330 ng/ml.
Sequence CWU 1
1
1451330PRTHomo sapiens 1Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 2327PRTHomo sapiens 2Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145
150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
3107PRTHomo sapiens 3Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 4104PRTHomo
sapiens 4Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu 1 5 10 15 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe Tyr 20 25 30 Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Ser Ser Pro Val Lys 35 40 45 Ala Gly Val Glu Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr 50 55 60 Ala Ala Ser Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His 65 70 75 80 Arg Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 85 90 95 Thr Val
Ala Pro Thr Glu Cys Ser 100 521DNAHomo sapiens 5tcaccatgga
ctgcacctgg a 21621DNAHomo sapiens 6tcaccatgga ctggacctgg a
21722DNAHomo sapiens 7ccatggacac actttgctcc ac 22822DNAHomo sapiens
8ccatggacac actttgttcc ac 22923DNAHomo sapiens 9tcaccatgga
gtttgggctg agc 231025DNAHomo sapiens 10agaacatgaa acacctgtgg ttctt
251125DNAHomo sapiens 11agaacatgaa acatctgtgg ttctt 251220DNAHomo
sapiens 12atggggtcaa ccgccatcct 201323DNAHomo sapiens 13acaatgtctg
tctccttcct cat 231419DNAArtificialhuCH-II 14gccaggggga agaccgatg
191519DNAHomo sapiens 15gccaggggga agacggatg 191621DNAHomo sapiens
16gctcagctcc tggggctcct g 211721DNAHomo sapiens 17ctggggctgc
taatgctctg g 211821DNAHomo sapiens 18ttcctcctgc tactctggct c
211921DNAHomo sapiens 19cagacccagg tcttcatttc t 212024DNAHomo
sapiens 20tttcaactgc tcatcagatg gcgg 242120DNAHomo sapiens
21cctctcctcc tcaccctcct 202218DNAHomo sapiens 22ctcctcactc agggcaca
182319DNAHomo sapiens 23atggcctgga tccctctcc 192419DNAHomo sapiens
24atggcctgga cccctctcc 192519DNAHomo sapiens 25atggcctgga tcgctctcc
192619DNAHomo sapiens 26atggcctgga ccgctctcc 192720DNAHomo sapiens
27agctcctcag aggagggcgg 202820DNAHomo sapiens 28agctcctcag
aggagggtgg 202938DNAHomo sapiens 29ctttaagaag gagatatacc atggtgcagc
tggtgcag 383038DNAHomo sapiens 30ctttaagaag gagatatacc atggttcagc
tggtgcag 383141DNAHomo sapiens 31ctttaagaag gagatatacc atgcaggtcc
agcttgtgca g 413241DNAHomo sapiens 32ctttaagaag gagatatacc
atggaggtcc agctggtaca g 413341DNAHomo sapiens 33ctttaagaag
gagatatacc atgcaggtcc agctggtaca g 413441DNAHomo sapiens
34ctttaagaag gagatatacc atgcaaatgc agctggtgca g 413541DNAHomo
sapiens 35ctttaagaag gagatatacc atgcagatgc agctggtgca g
413641DNAHomo sapiens 36ctttaagaag gagatatacc atgcagatca ccttgaagga
g 413741DNAHomo sapiens 37ctttaagaag gagatatacc atgcaggtca
ccttgaagga g 413841DNAHomo sapiens 38ctttaagaag gagatatacc
atgcaggtca ccttgaggga g 413941DNAHomo sapiens 39ctttaagaag
gagatatacc atggaagtgc agctggtgga g 414041DNAHomo sapiens
40ctttaagaag gagatatacc atggaggtgc agctggtgga g 414141DNAHomo
sapiens 41ctttaagaag gagatatacc atgcaggtgc agctggtgga g
414241DNAHomo sapiens 42ctttaagaag gagatatacc atggaggtgc agctgttgga
g 414341DNAHomo sapiens 43ctttaagaag gagatatacc atgcagctgc
agctgcagga g 414441DNAHomo sapiens 44ctttaagaag gagatatacc
atgcaggtgc agctgcagga g 414541DNAHomo sapiens 45ctttaagaag
gagatatacc atgcaggtgc agctacagca g 414641DNAHomo sapiens
46ctttaagaag gagatatacc atggaagtgc agctggtgca g 414741DNAHomo
sapiens 47ctttaagaag gagatatacc atggaggtgc agctggtgca g
414841DNAHomo sapiens 48ctttaagaag gagatatacc atgcaggtac agctgcagca
g 414941DNAHomo sapiens 49ctttaagaag gagatatacc atgcaggtcc
agctggtgca a 415041DNAHomo sapiens 50ctttaagaag gagatatacc
atgcaggtgc agctggtgca a 415145DNAHomo sapiens 51atcgtatggg
tagctggtcc cttagaccga tgggcccttg gtgga 455245DNAHomo sapiens
52atcgtatggg tagctggtcc cttagacgga tgggcccttg gtgga 455341DNAHomo
sapiens 53ctttaagaag gagatatacc atgaacatcc agatgaccca g
415441DNAHomo sapiens 54ctttaagaag gagatatacc atggacatcc agatgaccca
g 415541DNAHomo sapiens 55ctttaagaag gagatatacc atggacatcc
agttgaccca g 415641DNAHomo sapiens 56ctttaagaag gagatatacc
atggccatcc agttgaccca g 415741DNAHomo sapiens 57ctttaagaag
gagatatacc atggccatcc agatgaccca g 415841DNAHomo sapiens
58ctttaagaag gagatatacc atggccatcc ggatgaccca g 415941DNAHomo
sapiens 59ctttaagaag gagatatacc atggtcatct ggatgaccca g
416041DNAHomo sapiens 60ctttaagaag gagatatacc atggatattg tgatgaccca
g 416141DNAHomo sapiens 61ctttaagaag gagatatacc atggatattg
tgatgactca g 416241DNAHomo sapiens 62ctttaagaag gagatatacc
atggatgttg tgatgactca g 416341DNAHomo sapiens 63ctttaagaag
gagatatacc atggaaattg tgttgacaca g 416441DNAHomo sapiens
64ctttaagaag gagatatacc atggaaattg tgttgacgca g 416541DNAHomo
sapiens 65ctttaagaag gagatatacc atggaaatag tgatgacgca g
416641DNAHomo sapiens 66ctttaagaag gagatatacc atggaaattg taatgacaca
g 416741DNAHomo sapiens 67ctttaagaag gagatatacc atggacatcg
tgatgaccca g 416841DNAHomo sapiens 68ctttaagaag gagatatacc
atggaaacga cactcacgca g 416941DNAHomo sapiens 69ctttaagaag
gagatatacc atggaaattg tgctcactca g 417041DNAHomo sapiens
70ctttaagaag gagatatacc atggatgttg tgatgacaca g 417144DNAHomo
sapiens 71atcgtatggg tagctggtcc cttaaagatg aagacagatg gtgc
447241DNAHomo sapiens 72ctttaagaag gagatatacc atgcagtctg tgctgactca
g 417341DNAHomo sapiens 73ctttaagaag gagatatacc atgcagtctg
tgctgacgca g 417441DNAHomo sapiens 74ctttaagaag gagatatacc
atgcagtctg tgttgacgca g 417541DNAHomo sapiens 75ctttaagaag
gagatatacc atgcagtctg tcgtgacgca g 417641DNAHomo sapiens
76ctttaagaag gagatatacc atgcagtctg ccctgactca g 417741DNAHomo
sapiens 77ctttaagaag gagatatacc atgtcctatg agctgactca g
417841DNAHomo sapiens 78ctttaagaag gagatatacc atgtcctatg tgctgactca
g 417941DNAHomo sapiens 79ctttaagaag gagatatacc atgtcctatg
agctgacaca g 418041DNAHomo sapiens 80ctttaagaag gagatatacc
atgtcttctg agctgactca g 418141DNAHomo sapiens 81ctttaagaag
gagatatacc atgtcctatg agctgatgca g 418241DNAHomo sapiens
82ctttaagaag gagatatacc atgcagcctg tgctgactca a 418341DNAHomo
sapiens 83ctttaagaag gagatatacc atgcagcttg tgctgactca a
418441DNAHomo sapiens 84ctttaagaag gagatatacc atgcagcctg tgctgactca
g 418541DNAHomo sapiens 85ctttaagaag gagatatacc atgcaggctg
tgctgactca g 418641DNAHomo sapiens 86ctttaagaag gagatatacc
atgaatttta tgctgactca g 418741DNAHomo sapiens 87ctttaagaag
gagatatacc atgcagactg tggtgactca g 418841DNAHomo sapiens
88ctttaagaag gagatatacc atgcaggctg tggtgactca g 418941DNAHomo
sapiens 89ctttaagaag gagatatacc atgcagactg tggtgaccca g
419041DNAHomo sapiens 90ctttaagaag gagatatacc atgcagcctg tgctgactca
g 419141DNAHomo sapiens 91ctttaagaag gagatatacc atgctgcctg
tgctgactca g 419241DNAHomo sapiens 92ctttaagaag gagatatacc
atgcaggcag ggctgactca g 419340DNAHomo sapiens 93atcgtatggg
tagctggtcc cttagggaac agagtgaccg 409443DNAHomo sapiens 94ctttaagaag
gagatatacc atgcaggtgc agctggtgca gtc 439546DNAHomo sapiens
95ctttaagaag gagatatacc atgcaggtca acttaaggga gtctgg 469642DNAHomo
sapiens 96ctttaagaag gagatatacc atgaggtgca gctgctgcag tc
429742DNAHomo sapiens 97ctttaagaag gagatatacc atgaggtgca gctgctggag
tc 429842DNAHomo sapiens 98ctttaagaag gagatatacc atgaggtgca
gctggtgcag tc 429942DNAHomo sapiens 99ctttaagaag gagatatacc
atgaggtgca gctggtggag tc 4210043DNAHomo sapiens 100ctttaagaag
gagatatacc atgcaggtac agctgcagca gtc 4310143DNAHomo sapiens
101ctttaagaag gagatatacc atgcaggtac agctgcagga gtc 4310243DNAHomo
sapiens 102ctttaagaag gagatatacc atgcaggtgc agctgcagca gtc
4310343DNAHomo sapiens 103ctttaagaag gagatatacc atgcaggtgc
agctgcagga gtc 4310467DNAHomo sapiens 104atcgtatggg tagctggtcc
cttagtggtg gtggtggtgg tgaactctct tgtccacctt 60ggtgttg
6710567DNAHomo sapiens 105atcgtatggg tagctggtcc cttagtggtg
gtggtggtgg tgaactgtct tgtccacctt 60ggtgttg 6710667DNAHomo sapiens
106atcgtatggg tagctggtcc cttagtggtg gtggtggtgg tgaactttct
tgtccacctt 60ggtgttg 6710744DNAHomo sapiens 107ctttaagaag
gagatatacc atggacatcc agatgaccca gtct 4410844DNAHomo sapiens
108ctttaagaag gagatatacc atggacatcg agatgaccca gtct 4410944DNAHomo
sapiens 109ctttaagaag gagatatacc atggacatcg agatgaccca gtct
4411044DNAHomo sapiens 110ctttaagaag gagatatacc atggacatcg
tgatgaccca gtct 4411146DNAHomo sapiens 111ctttaagaag gagatatacc
atggatattg tgatgactca gtctcc 4611246DNAHomo sapiens 112ctttaagaag
gagatatacc atggatattg tgctgactca gtctcc 4611346DNAHomo sapiens
113ctttaagaag gagatatacc atggaaattg tgttgacgca gtctcc
4611445DNAHomo sapiens 114ctttaagaag gagatatacc atggaaacga
cactcacgca gtctc 4511546DNAHomo sapiens 115atcgtatggg tagctggtcc
cttaacactc tcccctgttg aagctc 4611643DNAHomo sapiens 116ctttaagaag
gagatatacc atgcagtctg tgctgactca gcc 4311743DNAHomo sapiens
117ctttaagaag gagatatacc atgcagtctg ccctgactca gcc 4311843DNAHomo
sapiens 118ctttaagaag gagatatacc atgtcctatg agctgacaca gcc
4311943DNAHomo sapiens 119ctttaagaag gagatatacc
atgtcctatg agctgactca gcc 4312046DNAHomo sapiens 120atcgtatggg
tagctggtcc cttatgaaca ttccgcaggg gcaact 4612146DNAHomo sapiens
121atcgtatggg tagctggtcc cttatgaaca ttctgcaggg gcaact
4612246DNAHomo sapiens 122atcgtatggg tagctggtcc cttatgaaca
ttccgtaggg gcaact 4612346DNAHomo sapiens 123atcgtatggg tagctggtcc
cttatgaaca ttccgcaggg gctact 4612446DNAHomo sapiens 124atcgtatggg
tagctggtcc cttatgaaca ttctgtaggg gcaact 4612546DNAHomo sapiens
125atcgtatggg tagctggtcc cttatgaaca ttctgtaggg gctact
4612623DNAHomo sapiens 126ctttaagaag gagatatacc atg 2312724DNAHomo
sapiens 127atcgtatggg tagctggtcc ctta 2412841DNAHomo sapiens
128ctttaagaag gagatatacc atgcaggtkc agctggtgca g 4112941DNAHomo
sapiens 129ctttaagaag gagatatacc atgcaggtcc agcttgtgca g
4113041DNAHomo sapiens 130ctttaagaag gagatatacc atgsaggtcc
agctggtaca g 4113141DNAHomo sapiens 131ctttaagaag gagatatacc
atgcaratgc agctggtgca g 4113241DNAHomo sapiens 132ctttaagaag
gagatatacc atgcagatca ccttgaagga g 4113341DNAHomo sapiens
133ctttaagaag gagatatacc atgcaggtca ccttgargga g 4113441DNAHomo
sapiens 134ctttaagaag gagatatacc atggargtgc agctggtgga g
4113541DNAHomo sapiens 135ctttaagaag gagatatacc atgcaggtgc
agctggtgga g 4113641DNAHomo sapiens 136ctttaagaag gagatatacc
atggaggtgc agctgttgga g 4113741DNAHomo sapiens 137ctttaagaag
gagatatacc atgcagstgc agctgcagga g 4113841DNAHomo sapiens
138ctttaagaag gagatatacc atgcaggtgc agctacagca g 4113941DNAHomo
sapiens 139ctttaagaag gagatatacc atggargtgc agctggtgca g
4114041DNAHomo sapiens 140ctttaagaag gagatatacc atgcaggtac
agctgcagca g 4114141DNAHomo sapiens 141ctttaagaag gagatatacc
atgcaggtac agctggtgca a 4114248DNAArtificialVH-lfp 142ctttaagaag
gagatatacc atgaactbtc ttgtccacct tggtgttg
4814349DNAArtificialVH-rfp 143atcgtatggg tagctggtcc cttaaactbt
cttgtccacc ttggtgttg 4914445DNAArtificialVL(k)-lfp 144ctttaagaag
gagatatacc atgacactct cccctgttga agctc
4514546DNAArtificialVL(k)-rfp 145atcgtatggg tagctggtcc cttaacactc
tcccctgttg aagctc 46
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