U.S. patent application number 15/870647 was filed with the patent office on 2018-05-24 for antibody producing non-human animals.
This patent application is currently assigned to Merus N.V.. The applicant listed for this patent is Merus N.V.. Invention is credited to Cornelis A. DE KRUIF, Erwin HOUTZAGER, Robert A. KRAMER, Ton LOGTENBERG, Rui D. PINTO, Mark THROSBY.
Application Number | 20180142005 15/870647 |
Document ID | / |
Family ID | 42007787 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180142005 |
Kind Code |
A1 |
LOGTENBERG; Ton ; et
al. |
May 24, 2018 |
ANTIBODY PRODUCING NON-HUMAN ANIMALS
Abstract
Described are transgenic, non-human animals comprising a nucleic
acid encoding an immunoglobulin light chain, whereby the
immunoglobulin light chain is human, human-like, or humanized. The
nucleic acid is provided with a means that renders it resistant to
DNA rearrangements and/or somatic hypermutations. In one
embodiment, the nucleic acid comprises an expression cassette for
the expression of a desired molecule in cells during a certain
stage of development in cells developing into mature B cells.
Further provided is methods for producing an immunoglobulin from
the transgenic, non-human animal.
Inventors: |
LOGTENBERG; Ton;
(Driebergen, NL) ; THROSBY; Mark; (Utrecht,
NL) ; KRAMER; Robert A.; (Utrecht, NL) ;
PINTO; Rui D.; (Utrecht, NL) ; DE KRUIF; Cornelis
A.; (De Bilt, NL) ; HOUTZAGER; Erwin; (Zeist,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merus N.V. |
Utrecht |
|
NL |
|
|
Assignee: |
Merus N.V.
Utrecht
NL
|
Family ID: |
42007787 |
Appl. No.: |
15/870647 |
Filed: |
January 12, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12589181 |
Oct 19, 2009 |
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15870647 |
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12459285 |
Jun 29, 2009 |
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12589181 |
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61133274 |
Jun 27, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 21/00 20130101;
A01K 2217/075 20130101; A01K 2207/15 20130101; C12N 15/8509
20130101; A01K 2217/15 20130101; A01K 2217/052 20130101; A01K
2267/01 20130101; A01K 67/0278 20130101; C07K 16/248 20130101; A01K
2217/206 20130101; C07K 2317/24 20130101; C07K 16/1282 20130101;
C07K 14/47 20130101; A01K 2227/105 20130101; A01K 67/027
20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; C12P 21/00 20060101 C12P021/00; A01K 67/027 20060101
A01K067/027; C12N 15/85 20060101 C12N015/85; C07K 16/24 20060101
C07K016/24; C07K 14/47 20060101 C07K014/47 |
Claims
1. A B cell that produces an antibody that binds to a desired
antigen, which B cell is isolated from a transgenic non-human
animal that has been immunized with the antigen, wherein the genome
of said animal comprises a transgene encoding a rearranged human
immunoglobulin variable region and wherein the transgene comprises
an immunoglobulin constant region or said rearranged human
immunoglobulin variable region is operatively linked to an
endogenous immunoglobulin constant region, and wherein the
transgenic animal has produced a repertoire of antibodies that bind
said antigen having the rearranged human immunoglobulin variable
region encoded by the rearranged human immunoglobulin variable
region comprised in said transgene.
2. The B cell of claim 1, wherein the transgene encoding a
rearranged human immunoglobulin variable region comprises a human
germline light chain V gene segment joined to a human germline
light chain J gene segment, wherein there is no mutation due to
said joining and wherein said immunoglobulin constant region is a
light chain constant region.
3. The B cell of claim 1, wherein the transgene encoding a
rearranged human immunoglobulin variable region comprises a human
germline heavy chain V gene segment joined to a human germline
heavy chain D gene segment joined to a human heavy chain J gene
segment, wherein there is no mutation due to said joining and
wherein said immunoglobulin constant region is a heavy chain
constant region.
4. The B cell of claim 1 wherein the transgenic animal is a
mammal.
5. The B cell of claim 1 wherein the transgenic animal is a
rodent.
6. The B cell of claim 5 wherein the transgenic animal is a
mouse.
7. The B cell of claim 1 wherein, in said antibodies, the
rearranged variable region is encoded by the transgene that has not
undergone somatic hypermutation.
8. A method to obtain an antibody that binds to an antigen, the
method comprising culturing the B cell of claim 1 to obtain a
population of said B cells and recovering antibodies secreted by
said B cells.
9. A method to obtain an antibody that binds to an antigen, the
method comprising culturing the B cell of claim 2 to obtain a
population of said B cells and recovering antibodies secreted by
said B cells.
10. A method to obtain an antibody that binds to an antigen, the
method comprising culturing the B cell of claim 3 to obtain a
population of said B cells and recovering antibodies secreted by
said B cells.
11. A method to obtain an antibody or fragment thereof that binds
to an antigen, the method comprising (a) isolating nucleic acid
comprising nucleotide sequences encoding the antibody secreted by
the B cell of claim 1; (b) transfecting recombinant host cells with
expression system(s) for production of the antibody or fragment
encoded by the nucleotide sequences contained in the nucleic acid
obtained in step (a); (c) culturing said host cells, and (d)
recovering the antibodies or fragments secreted by the host
cells.
12. Recombinant host cells that comprise nucleic acids that express
nucleotide sequences encoding the antibody secreted by the B cell
of claim 1 or an antigen-binding fragment thereof.
13. A method to obtain an antibody or fragment thereof that binds
to an antigen which method comprises culturing the cells of claim
12 under conditions wherein said nucleotide sequences are expressed
and recovering said antibody or fragment.
14. A method to obtain an antibody or fragment thereof that binds
to an antigen, the method comprising (a) isolating nucleic acid
comprising nucleotide sequences encoding the antibody secreted by
the B cell of claim 2; (b) transfecting recombinant host cells with
expression system(s) for production of the antibody or fragment
encoded by the nucleotide sequences contained in the nucleic acid
obtained in step (a); (c) culturing said host cells, and (d)
recovering the antibodies or fragments secreted by the host
cells.
15. Recombinant host cells that comprise nucleic acids that express
nucleotide sequences encoding the antibody secreted by the B cell
of claim 2 or an antigen-binding fragment thereof.
16. A method to obtain an antibody or fragment thereof that binds
to an antigen which method comprises culturing the cells of claim
15 under conditions wherein said nucleotide sequences are expressed
and recovering said antibody or fragment.
17. A method to obtain an antibody or fragment thereof that binds
to an antigen, the method comprising (a) isolating nucleic acid
comprising nucleotide sequences encoding the antibody secreted by
the B cell of claim 3; (b) transfecting recombinant host cells with
expression system(s) for production of the antibody or fragment
encoded by the nucleotide sequences contained in the nucleic acid
obtained in step (a); (c) culturing said host cells, and (d)
recovering the antibodies or fragments secreted by the host
cells.
18. Recombinant host cells that comprise nucleic acids that express
nucleotide sequences encoding the antibody secreted by the B cell
of claim 3 or an antigen-binding fragment thereof.
19. A method to obtain an antibody or fragment thereof that binds
to an antigen which method comprises culturing the cells of claim
18 under conditions wherein said nucleotide sequences are expressed
and recovering said antibody or fragment.
20. A recombinant expression system which system is a nucleic acid
comprising a nucleotide sequence encoding at least a variable
region of an antibody secreted by the B cell of claim 1,
operatively linked to control sequences for expression.
21. A recombinant expression system which system is a nucleic acid
comprising a nucleotide sequence encoding at least a variable
region of an antibody secreted by the B cell of claim 2,
operatively linked to control sequences for expression.
22. A recombinant expression system which system is a nucleic acid
comprising a nucleotide sequence encoding at least a variable
region of an antibody secreted by the B cell of claim 3,
operatively linked to control sequences for expression.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/589,181, filed Oct. 19, 2009, pending, which
application is a continuation of U.S. patent application Ser. No.
12/459,285, filed Jun. 29, 2009, abandoned, which applications
claim the benefit, under 35 U.S.C. .sctn. 119(e), to U.S.
Provisional Patent Application Ser. No. 61/133,274, filed Jun. 27,
2008, for "Antibody Producing Non-Human Mammals," the entire
contents of each of which are hereby incorporated herein by this
reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn. 1.821(c) or (e)--SEQUENCE
LISTING SUBMITTED AS ASCII TEXT FILE
[0002] Pursuant to 37 C.F.R. .sctn. 1.821(c) or (e), files
containing a TXT version and a PDF version of the Sequence Listing
have been submitted concomitant with this application, the contents
of which are hereby incorporated by reference.
TECHNICAL FIELD
[0003] The invention relates to the production and use of non-human
animals capable of producing antibodies or derivatives thereof,
which are expressed from at least partially exogenous nucleic acids
(transgenes). Transgenes to produce such transgenic animals and
methods to produce such heterologous antibodies; methods and
vectors for producing such transgenic animals are disclosed.
BACKGROUND
[0004] B cells mediate humoral immunity by producing specific
antibodies. The basic structural subunit of an antibody (Ab) is an
immunoglobulin (Ig) molecule. Ig molecules consist of a complex of
two identical heavy (H) and two identical light (L) polypeptide
chains. At the amino terminus of each H chain and L chain is a
region that varies in amino acid sequence named the variable (V)
region. The remaining portion of the H and L chains is relatively
constant in amino acid sequence and is named the constant (C)
region. In an Ig molecule, the H and L chain V regions (VH and VL)
are juxtaposed to form the potential antigen-binding site. The
genes that encode H and L chain V regions are assembled somatically
from segments of germline DNA during precursor B (pre-B) cell
differentiation: V, D and J gene segments for the H chain and V and
J gene segments for the L chain. Within Ig V regions are three
regions of greatest amino acid sequence variability that interact
to form the antigen-recognition site and are thus referred to as
complementarity determining regions (CDRs).
[0005] The V gene segment encodes the bulk of the V region domain,
including CDR1 and CDR2. Diversity in CDR1 and CDR2 derives from
sequence heterogeneity among multiple different germline-encoded V
segments. CDR3 is encoded by sequences that are formed by the
joining of H chain V, D, and J gene segments and L chain V and J
segments and by mechanisms that create nucleotide sequence
heterogeneity where these segments are combined. Additional
diversity may be derived from pairing of different H and L chain V
regions. Collectively these processes yield a primary repertoire of
antibodies encoded by germline gene segments and expressed by newly
formed B cells.
[0006] An additional source of antibody diversity is imposed on top
of the diversity generated by recombination of Ig gene segments. B
cells are able to introduce mutations into the antibody V regions
that they express, a process called somatic hypermutation. Thus,
when an animal first encounters an antigen, the antigen binds to a
specific B cell which happens to carry antibodies which have a V
domain which binds the antigen. This primary response may activate
this B cell to go on to secrete the cognate antibody. These
activated B cells can also now target a somatic mutation process to
their rearranged antibody gene segments and thus allow the
production of daughter cells which make variants of the antibodies
of the primary response. A selection process amplifies those
variant B cell descendants which make an antibody of improved
affinity of the antigen. In B cells, somatic hypermutations are
targeted to a restricted genomic region including both the
rearranged VH and VL genes. Thus somatic mutation allows affinity
maturation--the production and selection of high affinity
antibodies. Therefore, somatic mutation is important for the
generation of high affinity antibodies.
[0007] The exquisite specificity and high affinity of antibodies
and the discovery of hybridoma technology allowing the generation
of monoclonal antibodies (mAbs) has generated great expectations
for their utilization as targeted therapeutics for human diseases.
MAbs are identical because they are produced by a single B cell and
its progeny. MAbs are made by fusing the spleen cells from a mouse
that has been immunized with the desired antigen with myeloma cells
to generate immortalized hybridomas. One of the major impediments
facing the development of in vivo applications for mAbs in humans
is the intrinsic immunogenicity of non-human Igs. Patients respond
to therapeutic doses of mouse mAbs by making antibodies against the
mouse Ig sequences (Human Anti Mouse Antibodies; HAMA), causing
acute toxicity, alter their biodistribution and accelerate
clearance, thus reducing the efficacy of subsequent administrations
(Mirick et al. (2004), Q. Nucl. Med. Mol. Imaging 48:251-257).
[0008] To circumvent the generation of HAMA, antibody humanization
methods have been developed in an attempt to produce mAbs with
decreased immunogenicity when applied to humans. These endeavors
have yielded various recombinant DNA-based approaches aimed at
increasing the content of human amino acid sequences in mAbs while
retaining the specificity and affinity of the parental non-human
antibody. Humanization began with the construction of mouse-human
chimeric mAbs (S. L. Morrison et al. (1984), Proc. Natl. Acad. Sci.
USA 81:6851-5), in which the Ig C regions in murine mAbs were
replaced by human C regions. Chimeric mAbs contain 60-70% of human
amino acid sequences and are considerably less immunogenic than
their murine counterparts when injected into humans, albeit that a
human anti-chimeric antibody response was still observed (W. Y.
Hwang et al. (2005), Methods 36:3-10).
[0009] In attempts to further humanize murine mAbs, CDR grafting
was developed. In CDR grafting, murine antibodies are humanized by
grafting their CDRs onto the VL and VH frameworks of human Ig
molecules, while retaining those murine framework residues deemed
essential for specificity and affinity (P. T. Jones et al. (1986),
Nature 321:522). Overall, CDR-grafted antibodies consist of more
than 80% human amino acid sequences (C. Queen et al. (1989), Proc.
Natl. Acad. Sci. U.S.A. 86:10029; P. Carter et al. (1992), Proc.
Natl. Acad. Sci. U.S.A. 89:4285). Despite these efforts,
CDR-grafted, humanized antibodies were shown to still evoke an
antibody response against the grafted V region (W. Y. Hwang et al.
(2005), Methods 36:3).
[0010] Subsequently to CDR grafting, humanization methods based on
different paradigms such as resurfacing (E. A. Padlan et al.
(1991), Mol. Immunol. 28:489), superhumanization (P. Tan D. A. et
al. (2002), J. Immunol. 169:1119), human string content
optimization (G. A. Lazar et al. (2007), Mol. Immunol. 44:1986) and
humaneering have been developed in an attempt to further decrease
the content of non-human sequences in therapeutic mAbs (J. C.
Almagro et al. (2008), Frontiers in Bioscience 13:1619). As in CDR
grafting approaches, these methods rely on analyses of the antibody
structure and sequence comparison of the non-human and human mAbs
in order to evaluate the impact of the humanization process into
immunogenicity of the final product. When comparing the
immunogenicity of chimeric and humanized antibodies, humanization
of variable regions appears to decrease immunogenicity further (W.
Y. Hwang et al. (2005), Methods 36:3-10).
[0011] De-immunization is another approach developed to reduce the
immunogenicity of chimeric or mouse antibodies. It involves the
identification of linear T-cell epitopes in the antibody of
interest, using bioinformatics, and their subsequent replacement by
site-directed mutagenesis to human or non-immunogenic sequences (WO
9852976 A1, the contents of which are incorporated by this
reference). Although de-immunized antibodies exhibited reduced
immunogenicity in primates, compared with their chimeric
counterparts, some loss of binding affinity was observed (M. Jain
et al. (2007), Trends in Biotechnol. 25:307).
[0012] The development of phage display technology complemented and
extended humanization approaches in attempts to obtain less
immunogenic mAbs for therapy in humans. In phage display, large
collections ("libraries") of human antibody VH and VL regions are
expressed on the surface of filamentous bacteriophage particles.
From these libraries, rare phages are selected through binding
interaction with antigen; soluble antibody fragments are expressed
from infected bacteria and the affinity of binding of selected
antibodies is improved by mutation (G. Winter et al. (1994), Annu.
Rev. Immunol. 12:433). The process mimics immune selection, and
antibodies with many different bindings specificities have been
isolated using this approach (H. R. Hoogenboom et al. (2005), Nat.
Biotechnol. 23:1105). Various sources of H and L chain V regions
have been used to construct phage display libraries including those
isolated from non-immune or immune donors. In addition, phage
display libraries have been constructed of V regions that contain
artificially randomized synthetic CDR regions in order to create
additional diversity. Often, antibodies obtained from phage display
libraries are subjected to in vitro affinity maturation to obtain
high affinity antibodies (H. R. Hoogenboom et al. (2005), Nat.
Biotechnol. 23:1105).
[0013] The creation of transgenic mouse strains producing human
antibodies in the absence of mouse antibodies has provided another
technology platform for the generation of specific and high
affinity human mAbs for application in humans. In these transgenic
animals, the endogenous mouse antibody machinery is inactivated and
replaced by human Ig loci to substantially reproduce the human
humoral immune system in mice (A. Jakobovits et al. (2007), Nat.
Biotechnol. 25:1134; N. Lonberg (2005), Nat. Biotechnol. 23:1117).
B cell development as well as Ig diversification by recombination
of gene segments is faithfully reproduced in these mice, leading to
a diverse repertoire of murine B cells expressing human Igs. By
immunizing these mice with antigens, it was further demonstrated
that these transgenic animals accumulated somatic mutations in the
V regions of both heavy and light chains to produce a wide
diversity of high-affinity human mAbs (N. Lonberg (2005), Nat.
Biotechnol. 23:1117).
[0014] The question, whether "fully human" mAbs such as derived
from phage display libraries or transgenic mice are less
immunogenic than humanized mAbs cannot be answered yet, because
full immunogenicity data are available for just two human mAbs. An
anti-tumor necrosis factor mAb, developed from phage-displayed
human libraries induced antibody responses in 12% of patients--at
the higher end of the incidence of anti-antibody responses of the
humanized antibodies (W. Y. Hwang et al. (2005), Methods
36:3-10).
[0015] Evaluation of the immunogenicity of the first registered
human mAb generated by the transgenic approach demonstrated that
mAb treatment resulted in the generation of antibodies in
approximately 5.5% of treated cancer patients (A. Jakobovits et al.
(2007), Nat. Biotechnol. 25:1134; J. A. Lofgren et al. (2007), J.
Immunol. 178:7467).
DISCLOSURE OF THE INVENTION
[0016] Disclosed are a method and means for producing antibodies
that are specific for their targets, but are less immunogenic.
Described herein, the reduction of immunogenicity is at least
partially achieved by providing a transgenic non-human mammal
comprising, at least in its B cell lineage, a nucleic acid encoding
at least an immunoglobulin light chain or heavy chain, wherein the
heavy- or light chain encoding sequence is provided with a means
that renders it resistant to DNA rearrangements and/or somatic
hypermutations, preferably such a non-human animal is a rodent,
more specifically a mouse. In certain embodiments, the nucleic acid
encodes a human, human-like, or humanized immunoglobulin chain.
[0017] In the remainder of this specification, mice are typically
used as examples of the non-human mammals. The transgenic,
non-human, mammalian hosts are capable of mounting an immune
response to an antigen, where the response produces antibodies
having primate, particularly human, variable regions. Various
transgenic hosts may be employed, particularly murine, lagomorpha,
ovine, avine, porcine, equine, canine, feline, or the like. Mice
have been used for the production of B-lymphocytes for
immortalization for the production of antibodies. Since mice are
easy to handle, can be bred in large numbers, and are known to have
an extensive immune repertoire, mice will usually be the animal of
choice. Therefore, in the following discussion, the discussion will
refer to mice, but it should be understood that other animals,
particularly non-primate mammals, may be readily substituted for
the mice, following the same procedures.
[0018] The reason for preventing rearrangements and hypermutation
is that in this manner a non-immunogenic polypeptide can be chosen
beforehand knowing that this polypeptide chain will remain
non-immunogenic. At least one of the chains of the resulting
immunoglobulin is thus less immunogenic. The resulting antibody
needs to have (usually) both a light- and a heavy chain. The
non-immunogenic chain must therefore be capable of pairing with the
other chain. The other chain may be an endogenous chain, an
exogenous chain or a hybrid of both. For human therapy, the
non-immunogenic chain should be as close to human as possible.
[0019] A means for rendering a gene encoding an immunoglobulin
chain (or chains) resistant to DNA rearrangement and/or mutation is
of course removal of all genetic elements responsible for the
rearrangement and/or mutation. The drawback thereof is that the
variability of the two chains is eliminated, whereas the invention
preferably retains the variability in one chain (preferably the
heavy chain) and inhibits and/or prevents the
rearrangement-mutation of the other chain (preferably the light
chain).
[0020] The elements for rearrangement and/or hypermutation
characterized so far are located within the loci for
immunoglobulins. Therefore the means for rendering the
immunoglobulin encoding sequence resistant to DNA rearrangement
and/or mutation is inserting the gene in a locus outside the
immunoglobulin loci.
[0021] Thus, described herein, a transgenic non-human mammal is
provided wherein the light/heavy chain encoding sequence is
integrated in the genome of the non-human mammal in a locus outside
the immunoglobulin loci. Preferably the insertion is in a locus
that is resistant to gene silencing. Described herein, the
integration is in the Rosa-locus or a comparable locus.
[0022] In certain embodiments, provided is an expression cassette
that can be inserted into a Rosa locus or comparable locus with a
means that allows expression of the immunoglobulin chain(s)
essentially limited to cells of B cell lineage, preferably with a
means that allows expression of the light chain encoding nucleic
acid during a certain stage of the development of B cells. The term
"essentially limited expression" indicates that expression is
predominantly in cells of the B-cell lineage, but that lower levels
of expression in other cells, as compared to the level of
expression in B-cells, is possible. In certain embodiments, the
term "essentially limited expression" indicates that the expression
is exclusively present in cells of the B-cell lineage. Such means
typically and preferably include B cell (developmental stage)
specific promoters such as CD19, CD20, .mu.HC (all V-genes),
VpreB1, VpreB2, VpreB3, .lamda.5, Ig.alpha., Ig.beta., .kappa.LC
(all genes), .lamda.LC (all genes), BSAP (Pax5). Although it is
very well possible to direct the expression of the DNA
rearrangement and/or mutation resistant chain by such promoters,
they are relatively weak. A strong promoter will typically be
required to ensure adequate surface expression of the B cell
receptor (made up of the membrane attached Ig H and L chain) and to
compete with the expression and pairing of endogenous chains (if
present) through allelic exclusion. Such a promoter, however is
usually not tissue specific. To confer tissue specificity, an
indirect system employing Cre/lox or the like is preferred. The
desired chain is put under control of a strong promoter inhibited
by an element that can be removed by the action of a Cre-protein,
leading to activation of the desired immunoglobulin encoding gene.
This system is described in detail in F. T. Wunderlich (2004),
"Generation of inducible Cre systems for conditional gene
inactivation in mice," Inauguraldissertation zur Erlangung des
Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultat der
Universitat zu Koln; on the internet at deposit.
ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=d1&dok_ext=pdf&filename=9755-
72 30x. pdf.
[0023] Preferably the immunoglobulin chain produced in a manner
resistant to rearrangements and hypermutation is a light chain
capable of pairing with different heavy chains encoded by the
non-human mammal. The light chain will then be the same (and less
immunogenic) in all antibodies, but variety in specificity is
retained through rearrangements and hypermutations in the heavy
chains. It may in that case be preferable to silence at least one
of the endogenous loci encoding a light chain, although allelic
exclusion may render this unnecessary.
[0024] According to this embodiment, preferably the endogenous
kappa (.kappa.) light chain locus is functionally silenced.
[0025] If the endogenous .kappa. light chain locus is silenced, but
also for other reasons, it is preferred that the resistant light
chain is a .kappa. light chain, preferably a light chain that has a
germline-like sequence. Described herein such a light chain would
lead to an antibody with reduced immunogenicity. The preferred
germline sequence is based on the human IGKV1-39 (O12) as this
light chain is very frequently observed in the human repertoire (de
Wildt et al. 1999, J Mol. Biol. 285(3):895 and has superior
thermodynamic stability, yield and solubility (Ewert et al. 2003,
J. Mol. Biol. 325(3):531).
[0026] The following gives more specific embodiments of the
expression cassette with which the non-human animal can be provided
described herein. Although this is typically advantageous for
immunoglobulins, other genes of interest are also contemplated.
[0027] Thus, provided in a specific embodiment is a transgenic
non-human mammal wherein the light chain encoding nucleic acid
comprises in 5'-3' direction: a B cell specific promoter, a leader,
a rearranged human V gene, optionally a mouse .kappa.-intron
enhancer (MoE.kappa.i), a constant region (.kappa.) and optionally
a (truncated) mouse .kappa.-3' enhancer (MoE.kappa.3'). Neuberger
identified and examined a novel B-cell specific enhancer located
downstream of the kappa constant region (Neuberger, EP 00469025 B
1, the contents of which are incorporated herein by this
reference). This enhancer has been shown to play a crucial role in
the expression of kappa genes as removal of the 808 bp enhancer
strongly reduced expression. Deletion of the 3' kappa enhancer also
strongly reduced the level of somatic hypermutations (SHM). In
transgenic and cell expression studies. it has been revealed that
reduced, mutated or deleted 3' kappa enhancers not only lowered
expression levels but also decreased the level of somatic
hypermutations. Currently, it cannot be determined whether the 3'
kappa enhancer is involved in SHM processes, expression regulation
or both (review V. H. Odegard et al. (2006), Nat. Rev. Immunol.
6:573; M. Inlay et al. (2002), Nat. Immunol. 3:463).
[0028] Detailed expression studies using engineered variants of the
3' kappa enhancer indicated that a 50 nucleotide region is
sufficient to drive expression. However for proper expression a
reduced sequence of 145 nucleotides is preferred (EP04690251; K. B.
Meyer et al. (1990), Nucleic Acids Res. 18(19):5609-15).
[0029] Thus, the invention in one aspect provides a nucleic acid
for insertion into the genome of a non human animal that is an
expression cassette for the expression of a desired proteinaceous
molecule in cells developing into mature B cells during a certain
stage of development, the cassette comprising means for preventing
silencing of expression of the desired proteinaceous molecule after
introduction into a host cell, and means for timing expression of
the desired proteinaceous molecule with the desired developmental
stage of the host cell.
[0030] An expression cassette is defined as a nucleic acid that has
been provided with means for introduction into the genome of a host
cell, such as sequences which allow for homologous recombination
with a certain site in the genome. Usually the nucleic acid will be
DNA, typically double stranded. Typically the expression cassette
will be provided to the cell in a vector from which it is
transferred to the genome of the cell. The expression cassette
further comprises all elements necessary for expression of the gene
in a host cell, although in certain embodiments some of such
elements may be present on a second nucleic acid to be introduced,
whereby these elements act in trans. Elements necessary for
expression in a host cell include promoters, enhancers and other
regulatory elements. Only those elements are necessary that are not
provided by the host cell.
[0031] The expression of the gene of interest should not be
silenced in the genome of the host cell, especially not in the
development stage where expression is required. This can be done by
various means, such as insertion into the endogenous locus or by
providing the cassette with nucleic acid elements that prevent
silencing (Kwaks et al. (2006), Trends Biotechnol. 24(3):137-142,
which is incorporated herein by reference). It is preferred that
the expression cassette is inserted in a locus that is not silenced
in the host cells (EP 01439234; which is incorporated herein by
reference).
[0032] The means for prevention of silencing comprise STabilizing
Anti-Repression-sequences (STAR.RTM.-sequences) and Matrix
Attachment Regions (MARs). A STAR sequence is a nucleic acid
sequence that comprises a capacity to influence transcription of
genes in cis. Typically, although not necessarily, a STAR sequence
does not code by itself for a functional protein element. In one
embodiment one STAR element is used. Preferably, however, more than
one STAR element is used. In a particularly preferred embodiment an
expression cassette described herein is provided with two STAR
sequences; one STAR sequence at the 5' side of the coding sequence
of the immunoglobulin gene and one STAR sequence at the 3' side of
the coding sequence of the immunoglobulin gene. MARs are DNA
sequences that are involved in anchoring DNA/chromatin to the
nuclear matrix and they have been described in both mammalian and
plant species. MARs possess a number of features that facilitate
the opening and maintenance of euchromatin. MARs can increase
transgene expression and limit position-effects.
[0033] Expression from the cassette should only occur during a
certain period in the development of a cell, in particular a
developing B cell, more in particular a B cell in a transgenic
non-human animal, in particular a mouse. In this particular case
the developmental period is chosen such that the expression of the
gene from the cassette (typically a light- or heavy chain-like
polypeptide) does not significantly interfere with the normal
differentiation and/or maturation of the cell and when applicable,
allows for pairing of the polypeptide chain produced with its
counterpart.
[0034] This may, in one embodiment, be achieved by providing a
nucleic acid described herein, wherein the means for timing
expression is a promoter of which the activity is essentially
limited to the certain stage of development. In a developing B
cell, which, e.g., after immunization is maturing and/or
differentiating, the expression of the gene of interest, when it is
one of the polypeptide chains of an immunoglobulin, must not
interfere (significantly) with the maturation and/or
differentiation and it needs to be timed such that the resulting
polypeptide can pair with its counterparts. Therefore, provided is
a nucleic acid described herein wherein the certain stage starts at
a stage immediately preceding or coinciding with the onset of the
expression of light chain molecules by the cells at a certain stage
of development into a mature B cell. This may be achieved by
selecting a promoter which is active only during the suitable
period. Such a promoter may be a CD19 promoter, the Ig-.alpha.
promoter, the Ig-.beta. promoter, the .mu.hc (all genes) promoter,
the Vk promoter or analogues or homologues thereof.
[0035] In a specific embodiment, the promoter as disclosed above
does not drive the expression of the gene of interest directly.
Instead it drives the expression of a gene of which the product
activates in trans the expression of the gene of interest. Such an
activating gene may be a gene encoding a so-called Cre recombinase
or Cre-like protein. The expression cassette for the gene of
interest may, e.g., be provided with a sequence that inhibits
expression of the gene of interest. The sequence can be removed by
the action of the Cre recombinase, which is under control of the
desired promoter (active during the proper stage of development).
In this embodiment a set of expression cassettes is required.
[0036] Therefore, provided is a set of nucleic acids that are
expression cassettes, wherein one nucleic acid comprises an
expression cassette encoding a Cre-like protein under control of a
promoter active during the desired stage of development of the host
cell and the second nucleic acid comprises a sequence encoding a
desired proteinaceous molecule under control of a constitutive
promoter which can be activated by the action of a Cre-like
protein. The activation is preferably achieved by removal of a stop
sequence flanked by loxP sites. The Cre/lox system is described in
detail in Rajewsky et al. (1996), J. Clin. Invest. 98:600-603,
which is incorporated herein by reference. Such systems are
reviewed in F. T. Wunderlich (2004), "Generation of inducible Cre
systems for conditional gene inactivation in mice,"
Inauguraldissertation zur Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultat der Universitat zu
Koln; on the World Wide Web at
deposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=d1&dok_ext=pdf&filen-
ame=97557230x.pd, which is incorporated herein by reference.
[0037] Further provided is a transgenic non-human animal that has
been provided with an expression cassette hereof, wherein the
desired proteinaceous molecule is a polypeptide chain of an
immunoglobulin. A preferred polypeptide chain is a light chain. A
more preferred polypeptide is a germline or germline-like light
chain. A most preferred polypeptide is encoded by the
immunoglobulin kappa variable 1-39 (IGKV1-39, also known as O12)
gene segment, preferably the rearranged germline kappa light chain
IGKV1-39*01/IGKJ1*01 (nomenclature according to the IMGT database,
at [worldwideweb].imgt.org).
[0038] In certain embodiments, the polypeptide chain is rendered
essentially incapable of rearrangement and/or of excluded of any
sequence modification such as normally operating on Ig during the
process of B cell affinity maturation. Therefore, provided is a
transgenic non-human animal that has been provided with an
expression cassette described herein, wherein the rearrangement
and/or sequence modifications are prevented by the absence of
elements at least partially responsible for somatic hypermutation
such as, for example, the MoE.kappa.i enhancer.
[0039] A preferred expression cassette described herein comprises
means for prevention of silencing. In one embodiment, the means for
prevention of silencing are means for insertion into a locus in the
genome of the host cell that is resistant to silencing. The means
for insertion are preferably means for homologous recombination
into the site resistant to silencing. A preferred locus when the
non-human animal is a mouse is the rosa-locus.
[0040] A further preferred expression cassette described herein
comprises in 5'-3' direction: a V.kappa. promoter, a mouse leader,
a human V gene, optionally a MoE.kappa.i enhancer, a rat constant
region (C.kappa.) and optionally a (truncated) MoE.kappa.3'
enhancer.
[0041] Yet a further preferred expression cassette described herein
comprises in 5'-3' direction: a V.kappa. promoter, a human leader,
a human V gene, optionally a MoE.kappa.i enhancer, a rat constant
region (C.kappa.) and optionally a (truncated) MoE.kappa.3'
enhancer.
[0042] Certain antibodies produced as described herein may be used
in human therapeutics and diagnostics. Thus, provided is a method
for producing a desired antibody comprising exposing a non-human
mammal described herein to an antigen such that an antibody
response is induced and isolating the antibodies specific for the
antigen.
[0043] In certain embodiments, provided are methods for producing a
desired antibody comprising exposing a non-human mammal described
herein to an antigen such that an antibody response is induced and
isolating cells producing such antibodies, culturing and optionally
immortalizing the cells and harvesting the antibodies.
[0044] In certain embodiments, provided is a method for producing a
desired antibody comprising exposing a non-human mammal described
herein to an antigen such that an antibody response is induced and
isolating a nucleic acid encoding at least part of such an
antibody, inserting the nucleic acid or a copy or a derivative
thereof in an expression cassette and expressing the antibody in a
host cell.
[0045] The methods for producing antibodies from transgenic mice
are known to a person skilled in the art. Particularly preferred
are methods for production of mixtures of antibodies from one cell,
whereby the nucleic acids encoding these antibodies have been
derived from mice described herein.
[0046] These so-called oligoclonics are disclosed in WO04106375 and
WO05068622, which are incorporated herein by reference.
[0047] Described herein are transgenic non-human mammals,
preferably mice, capable of generating specific and high affinity
hybrid mouse-human antibodies with preferably human immunoglobulin
light chain variable (VL) regions in or near germline configuration
and preferably murine immunoglobulin heavy chain variable (VH)
regions that may have accumulated somatic mutations during the
process of antigen-driven affinity maturation. It is envisaged that
the murine VH regions of the hybrid antibodies may be subjected to
humanization procedures to yield mAbs that have reduced
immunogenicity when applied in humans based on germline or
near-germline VL regions and murine VH regions that have been
humanized.
[0048] In particular, we have shown that transgenic mice that
harbor a DNA expression construct that encodes a rearranged human
VL region under the control of cis-acting genetic elements that
provide timely and regulated expression of the transgene on a
significant proportion of B cells during B cell development, yet
lack elements that direct the somatic hypermutation machinery to
the transgene, are capable of generating specific and high affinity
mouse-human hybrid antibodies with essentially unmutated L chains.
It is shown that the rearranged human transgene is capable of
pairing with a diversity of endogenous murine immunoglobulin H
chains to form mouse-human hybrid immunoglobulins expressed on the
surface of B cells and to sufficiently facilitate murine B cell
development to obtain a sizeable and diverse peripheral B cell
compartment.
[0049] In certain embodiments, the transgene expression construct
harbors the coding sequences of a human rearranged L chain V region
under the control of a human VL promoter to direct B-cell specific
expression. In addition, the construct harbors the murine 3' Ck
enhancer sequence for B cell specific and inducible and high level
expression of the transgene. Furthermore, the construct is designed
to lack regulatory elements that facilitate the recruitment of the
somatic hypermutation machinery to the transgene, such as the
intron enhancer and the 3' C-kappa enhancer.
[0050] In a related embodiment, the rearranged human VL gene is
inserted in the murine Rosa26 locus by site-specific integration.
The Rosa26 locus is useful in the context of the "targeted
transgenesis" approach for efficient generation of transgenic
organisms (such as mice) with a predictable transgene expression
pattern.
[0051] In certain embodiments, the rearranged human VL region is
selected for its capacity to pair with many different murine VH
genes so as to ensure the generation of a population of B cells
with a diverse VH gene repertoire. A method of obtaining such VL
regions comprises amplifying a repertoire of rearranged VH genes
from the B cells of mice and a repertoire of human rearranged
germline VL regions from the B cells of humans and cloning them
into phagemid display vectors to prepare diverse libraries of
hybrid immunoglobulins in bacteria. By nucleotide sequence analysis
of collections of unselected and antigen-selected VH/VL pairs,
human germline VL genes that pair with many different murine VH
genes are identified. A collection of human germline VL genes with
this capacity is described.
[0052] In one embodiment, it is shown that upon immunization with
antigen, the B cells are capable of mounting an immune response,
leading to the generation of B cells that secrete hybrid antibodies
with high specificity and affinity. The V regions encoding these
antibodies are characterized by the human transgenic light chain
that harbors no or very few mutations and a murine heavy chain that
harbors a variable number of mutations introduced by the somatic
hypermutation machinery.
[0053] In a related embodiment, strategies to obtain high affinity
hybrid monoclonal antibodies from the transgenic mice by hybridoma
and display technologies are contemplated as well as procedures to
humanize the murine VH regions to obtain less immunogenic
antibodies for application in humans.
[0054] In one embodiment, provided is an immunoglobulin L chain
transgene construct comprising DNA sequences that encode a human
immunoglobulin VL region in combination with a light chain constant
region (CL) of an animal immunoglobulin protein, which sequences
are operably linked to transcription regulatory sequences that,
when integrated in a non-human transgenic animal, produce an Ig
VL-CL polypeptide with a human VL region that is not or marginally
subject to somatic hypermutation. The Ig VL is capable of pairing
with rearranged VH-CH polypeptides that are generated during B cell
development in the non-human transgenic animal, with the VH-CH
polypeptides retaining the capacity to undergo somatic
hypermutation upon stimulation. The CL region may be of any animal
species and is generally capable of pairing with the CH regions of
the non-human transgenic animal.
[0055] Also included is the use of a transgene construct as above
in producing a transgenic non-human animal capable of the
production of hybrid antibodies consisting of VL-CL polypeptides
and VH-CH polypeptides in which the VL region is of human origin
and the CL, VH and CH may be of any animal species, including
human. Upon immunization, these transgenic animals are capable of
generating high affinity antibodies encoded by somatically
hypermutated VH genes and essentially non-mutated VL genes encoded
by the transgene.
[0056] In another aspect, provided is a process for the production
of a transgenic non-human animal capable of the production of
hybrid antibodies in response to antigenic challenge, comprising
functionally disrupting the endogenous immunoglobulin light chain
locus and inserting into the animal genome a transgene construct of
the invention.
[0057] Included is the use of animals obtainable by this process in
the production of B cells that produce immunoglobulin having human
VL light chain. In another aspect of the invention there is
provided a process for the production of B cells that produce
immunoglobulin having human VL and binding to a selected antigen,
comprising challenging an animal obtainable by a process as above
with the antigen and screening for B cells from the animal that
bind the antigen. Further included is B cells obtainable by this
process and hybridomas obtainable by immortalizing such B cells,
e.g., hybridomas obtained by fusing B cells as above with myeloma
cells. Also included is a process for producing monoclonal antibody
comprising cultivating such a hybridoma. In yet a further aspect,
provided is the use of the above B cells in producing a hybridoma
or corresponding monoclonal antibody.
[0058] Described herein is a process for the production of
immunoglobulin having human VL chain and binding to a selected
antigen, comprising challenging an animal obtainable as above with
the antigen and obtaining immunoglobulin there from.
[0059] In one strategy, as an individual step, a rearranged VL
region encoded by human germline V and J gene segments and a light
chain constant region of any animal species but preferably a murine
constant region is introduced into the mouse germ line. The
transgene DNA may be introduced into the pronuclei of fertilized
oocytes or embryonic stem cells. The integration may be random or
homologous depending on the particular strategy to be employed. For
example, the VL transgene may be introduced by random insertion,
resulting in mice that bear one or multiple copies of the transgene
in the genome. Alternatively, the human VL transgene may be
targeted to a specific genomic locus using site-specific
recombination as described in the art.
[0060] In certain embodiments, the VL transgene is targeted to the
murine ROSA26 locus which is a suitable integration site allowing
strong and predictable expression of inserted transgenes (European
Patent Office document EP 1,439,234 A1, the contents of which are
incorporated herein by this reference). The targeting vector allows
insertion of a single copy of a gene expression cassette, thus
avoiding modulation of transgene expression by the arrangement of
multiple copies. By choosing the autosomal Rosa26 locus as
insertion site, the expression pattern of the inserted transgene in
the non-human animal is predictable. Furthermore, random X
inactivation and/or modulation by chromosomal position effects are
avoided. This also eliminates the need to generate and analyze
multiple transgenic strains for any given transgene. Finally, the
Rosa26 targeting vector for the site-specific integration can be
used for multiple gene expression cassettes. Thus, it may be
envisaged that two or more different rearranged germline human VL
regions are inserted into the Rosa26 locus to further increase the
diversity of the repertoire of hybrid or human antibodies.
[0061] In another embodiment, a rearranged human VL region may be
targeted to the murine Ig kappa or lambda light chain locus so as
to functionally inactivate the endogenous locus or mice containing
the rearranged human VL region may be bred with mice that lack
functional kappa or lambda Ig loci or both. Thus, by using
transformation, using repetitive steps or in combination with
breeding, transgenic animals may be obtained which are able to
produce antibodies harboring the human VL transgene in the
substantial absence of endogenous host immunoglobulin light
chains.
[0062] In one embodiment, a human VL transgene is selected for its
capacity to pair with a substantial portion of murine VH regions to
form a diverse repertoire of functional mouse-human hybrid
antibodies expressed on the surface of B cells. By a substantial
portion of murine VH regions is meant that the human VL pairs with
at least with 0.1% of the murine VH regions generated during B cell
development, more preferably with at least 1% and most preferably
with at least 10%. Methods to identify human VL genes with this
characteristic include randomly pairing a repertoire of human VL
regions with a repertoire of murine VH regions, co-expression of VH
and VL regions in appropriate eukaryotic or prokaryotic expression
vectors and screening for human VL regions that pair with a
substantial portion of murine VH regions. In one embodiment,
phagemid vectors may be used to direct expression of mouse-human
antibody fragments in bacterial cells or to the surface of
filamentous phage and analysis of binding capacity of antibody
fragments by methods known in the art.
[0063] In another embodiment, a human VL transgene is selected for
its capacity to pair with a substantial portion of human VH regions
to form a diverse repertoire of human antibodies expressed on the
surface of B cells. By a substantial portion of human VH regions is
meant that the human VL pairs with at least with 0.1% of the human
VH regions generated during B cell development, more preferably
with at least 1% and most preferably with at least 10%.
[0064] In the latter embodiment, the human VL transgenic mice are
crossed with mice that harbor functional rearranged or
non-rearranged human H chain immunoglobulin loci and functionally
inactivated endogenous H chain Ig loci as described in the art. The
functional inactivation of the two copies of each of the three host
Ig loci (heavy chain, kappa and lambda light chain), where the host
contains the human IgH and the rearranged human VL transgene would
allow for the production of purely human antibody molecules without
the production of host or host human chimeric antibodies. Such a
host strain, by immunization with specific antigens, would respond
by the production of mouse B-cells producing specific human
antibodies, which B-cells are subsequently fused with mouse myeloma
cells or are immortalized in any other manner for the continuous
stable production of human monoclonal antibodies. Alternatively,
the population of B cells is used as a source of VH regions that
can be obtained by constructing cDNA libraries or by PCR
amplification using primers for human VH regions as is known in the
art.
[0065] A human rearranged VL gene is reconstructed in an
appropriate eukaryotic or prokaryotic microorganism and the
resulting DNA fragments can be introduced into pronuclei of
fertilized mouse oocytes or embryonic stem cells. Various
constructs that direct B cell specific expression of VL transgenes
have been described in the art and have the following general
format: a leader sequence and relevant upstream sequences to direct
B cell specific expression of the transgene, a coding sequence of a
human VL transgene, an enhancer sequence that directs B cell
specific and high level expression of the transgene and a murine
constant region gene. In a preferred format, the enhancer is the
C-kappa 3' enhancer because it directs high level expression in
B-lineage cells, but does not recruit somatic hypermutation when
used in transgene constructs.
[0066] In one embodiment, animals, preferably mice, comprising one
or multiple copies of the transgene in the genome are isolated and
analyzed for stable expression. Animals are selected that show
stable expression of the transgene over longer periods of time,
preferably in B-cells. If required, different animal lines
comprising independent insertions of one or multiple copies of the
transgene, preferably on different chromosomes, are crossed to
obtain animals with different insertions of one or multiple copies
of the transgene to increase expression of the transgene in
animals, preferably in B-cells.
[0067] Further provided is progeny of a transgenic non-human animal
described herein, the progeny comprising, at least in its B-cell
lineage, a heavy- or light chain encoding sequence together with a
means that renders the sequence resistant to DNA rearrangements
and/or somatic hypermutations.
[0068] Further provided is progeny of a transgenic non-human animal
described herein, the progeny comprising an expression cassette for
the expression of a desired proteinaceous molecule in cells during
a certain stage of development in cells developing into mature B
cells.
[0069] In addition, provided is a cell that is isolated from a
transgenic non-human animal described herein, the cell comprising a
heavy- or light chain encoding sequence together with a means that
renders the sequence resistant to DNA rearrangements and/or somatic
hypermutations. In addition, provided is a cell that is isolated
from a transgenic non-human animal described herein, the cell
comprising an expression cassette for the expression of a desired
proteinaceous molecule in cells during a certain stage of
development in cells developing into mature B cells. A cell
described herein, preferably an antibody-producing B-cell or a cell
that is capable of differentiating or maturating into an
antibody-producing B-cell, can be used for in vitro production of
antibodies, as is known to the skilled person, for example, from
Gascan et al. 1991, J. Exp. Med. 173:747-750. Methods for
immortalization of a cell described herein are known in the art and
include the generation of hybridomas, for example, by fusion with a
myeloma cell, transformation with Epstein Barr Virus; expression of
the signal transducer of activation and transcription (STAT),
activation via CD40 and IL4 receptor signaling, and/or expression
of Bc16 (Shvarts et al. 2002, Genes Dev. 16: 681-686).
[0070] In a separate step, the mouse endogenous Kappa and Lambda
light chain loci are rendered essentially non-functional such that
at least the majority of B cells in the transgenic mice bear Ig
receptors that contain the transgenic human VL region. Inactivation
of the endogenous mouse immunoglobulin loci is achieved by targeted
disruption of the appropriate loci by homologous recombination in
mouse embryonic stem cells. The targeted disruption comprises
alteration of the genomic sequence such that substantially no
functional endogenous mouse immunoglobulin Kappa and/or Lambda
light chain is produced. The term "substantially no functional
endogenous mouse immunoglobulin" indicates that the endogenous
Kappa and/or Lambda light chain loci are functionally silenced such
that the level of functional protein expression of the endogenous
Kappa and/or Lambda light chain loci, preferably the endogenous
Kappa light chain locus, is reduced to about 20% of the level of
expression in a reference mouse, more preferred to about 10%, more
preferred to about 5%, more preferred to about 2% and more
preferred to about 1%. In a most preferred embodiment, the level of
functional protein expression of the endogenous Kappa and/or Lambda
light chain loci is reduced to 0%. The level of functional protein
expression can be determined by means known to the skilled person,
including western blotting and pairing with a mouse heavy chain.
The reference mouse is a mouse in which the endogenous Kappa and/or
Lambda light chain loci is not disrupted. The alteration comprises
mutation and/or deletion of gene sequences that are required for
functional expression of the endogenous immunoglobulin genes.
Alternatively, the alteration comprises insertion of a nucleic acid
into the endogenous mouse immunoglobulin Kappa and/or Lambda light
chain loci such that the functional expression of the endogenous
immunoglobulin genes is reduced. In one embodiment, the nucleic
acid comprises a silencing element resulting in transcriptional
silencing of the endogenous immunoglobulin gene. In a further
embodiment, or in addition, the nucleic acid comprises a sequence
that disrupts splicing and/or translation of the endogenous
immunoglobulin gene, for example, by introducing an exon that
renders a frame shift in the coding sequence, or that comprises a
premature stop codon. In each case chimeric animals are generated
which are derived in part from the modified embryonic stem cells
and are capable of transmitting the genetic modifications through
the germ line. The mating of mouse strains with human
immunoglobulin loci to strains with inactivated mouse loci yields
animals which produce antibodies comprising essentially only human
light chains.
[0071] A construct for homologous recombination is prepared by
means known in the art and any undesirable sequences are removed,
e.g., procaryotic sequences. Any convenient technique for
introducing a construct for homologous recombination into a target
cell may be employed. These techniques include spheroplast fusion,
lipofection, electroporation, calcium phosphate-mediated DNA
transfer or direct microinjection. After transformation or
transfection of the target cells, target cells are selected by
means of positive and/or negative markers, for example, by neomycin
resistance and/or acyclovir and/or gancyclovir resistance. Those
cells which show the desired phenotype may then be further analyzed
by restriction analysis, electrophoresis, Southern analysis, PCR,
or the like. By identifying fragments which show the presence of
the lesion(s) at the target locus, cells in which homologous
recombination has occurred to inactivate a copy of the target locus
are identified.
[0072] Furthermore, it is shown that upon immunization, the murine
and human VH regions in the afore-mentioned transgenic mice but not
the VL regions are capable of undergoing somatic hypermutations to
generate high affinity antibodies. Advantageously, these antibodies
encoded by germline VL regions are predicted to contribute to lower
immunogenicity when applied in humans and result in more stable
antibodies that are less prone to aggregation and thus safer for
therapeutic use in humans.
[0073] MAbs derived from the afore-mentioned non-human transgenic
animals or cells all share the same identical human VL regions. It
has been described that mAbs that share the same identical VL
region may be co-expressed in a single clonal cell for the
production of mixtures of recombinant antibodies with functional
binding sites (see the incorpoarated WO04106375 and WO05068622).
Thus, provided is a platform for the generation of specific and
high affinity mAbs that constitute the basis for mixtures of mAbs
produced by clonal cells.
[0074] It is preferred that mAbs derived from the afore-mentioned
non-human transgenic animals or cells are directed against cellular
targets. Preferred targets are human surface-expressed or soluble
proteins or carbohydrate molecules. Further preferred targets are
surface-expressed proteins or carbohydrate molecules that are
expressed on the surface of bacteria, viruses, and other pathogens,
especially of humans.
[0075] More specifically, preferred targets include cytokines and
chemokines, including but not limited to InterLeukin 1beta
(IL1beta), IL2, IL4, IL5, IL7, IL8, IL12, IL13, IL15, IL18, IL21,
IL23 and chemokines such as, for example, CXC chemokines, CC
chemokines, C chemokines (or y chemokines) such as XCL1
(lymphotactin-a) and XCL2 (lymphotactin-B), and CX3C chemokines.
Further included as preferred targets are receptor molecules of the
cytokines and chemokines, including type I cytokine receptors such
as, for example, the IL-2 receptor, type II cytokine receptors such
as, for example, interferon receptors, immunoglobulin (Ig)
superfamily receptors, tumor necrosis factor receptor family
including receptors for CD40, CD27 and CD30,
serine/threonine-protein kinase receptors such as TGF beta
receptors, G-protein coupled receptors such as CXCR1-CXCR7, and
tyrosine kinase receptors such as fibroblast growth factor receptor
(FGFR) family members, EGF receptor family members including erbB1
(EGF-R; HER1), erbB2, (HER2), erbB3 (HER3), and erbB4 (HER4),
insulin receptor family members including IGF-R1 and IGF-RII, PDGF
receptor family members, Hepatocyte growth factor receptor family
members including c-Met (HGF-R), Trk receptor family members, AXL
receptor family members, LTK receptor family members, TIE receptor
family members, ROR receptor family members, DDR receptor family
members, KLG receptor family members, RYK receptor family members,
MuSK receptor family members, and vascular endothelial growth
factor receptor (VEGFR) family members.
[0076] Further preferred targets are targets that are
over-expressed or selectively expressed in tumors such as, for
example, VEGF, CD20, CD38, CD33, CEA, EpCAM, PSMA, CD54, Lewis Y,
CD52, CD40, CD22, CD51/CD61, CD74, MUC-1, CD38, CD19, CD262
(TRAIL-R2), RANKL, CTLA4, and CD30; targets that are involved in
chronic inflammation such as, for example, CD25, CD11a, TNF, CD4,
CD80, CD23, CD3, CD14, IFNgamma, CD40L, CD50, CD122, TGFbeta and
TGFalpha.
[0077] Preferred surface-expressed proteins or carbohydrate
molecules that are expressed on the surface of bacteria, viruses,
and other parasitic pathogens, especially of humans, include
surface markers of influenza A and B viruses such as hemagglutinin
(HA) and neuraminidase (NA), filoviruses such as Ebola virus,
rabies, measles, rubella, mumps, flaviviruses such as Dengue virus
types 1-4, tick-borne encephalitis virus, West Nile virus, Japanese
encephalitis virus, and Yellow fever virus, Paramyxoviruses
including Paramyxovirus such as Parainfluenza 1, 3, Rubulavirus
such as Mumpsvirus and Parainfluenza 2, 4, Morbillivirus, and
Pneumovirus such as Respiratory syncytial virus, Vaccinia, small
pox, coronaviruses, including Severe Acute Respiratory Syndrome
(SARS) virus, hepatitis virus A, B and C, Human Immunodeficiency
Virus, Herpes viruses, including cytomegalovirus, Epstein Barr
virus, Herpes simplex virus, and Varicella zoster virus,
parvoviruses such as, for example, B19; Legionella pneumophila;
Listeria monocytogenes; Campylobacter jejuni; Staphylococcus
aureus; E. coli O157:H7; Borrelia burgdorferi; Helicobacter pylori;
Ehrlichia chaffeensis; Clostridium difficile; Vibrio cholera;
Salmonella enterica Serotype Typhimurium; Bartonella henselae;
Streptococcus pyogenes (Group A Strep); Streptococcus agalactiae
(Group B Strep); Multiple drug resistant S. aureus (e.g., MRSA);
Chlamydia pneumoniae; Clostridium botulinum; Vibrio vulnificus;
Parachlamydia pneumonia; Corynebacterium amycolatum; Klebsiella
pneumonia; Linezolid-resistant enterococci (E. faecalis and E.
faecium); and Multiple drug resistant Acinetobacter baumannii.
[0078] Most preferred targets are IL-6 and its receptor,
IL-6Ralpha, glycoprotein-denominated gp130, RSV, especially the
surface proteins F, G and SH and non-structural proteins such as N
and M, and receptor tyrosine kinases, in particular erbB1 (EGF-R;
HER1), erbB2, (HER2), erbB3 (HER3), erbB4 (HER4), IGF-R1 and
IGF-RII, c-Met (HGF-R).
[0079] Therefore, provided is a platform for the generation of
specific and high affinity mAbs against the above mentioned targets
that constitute the basis for mixtures of mAbs produced by clonal
cells. In certain embodiments, the specific and high affinity mAbs
comprise mAbs that are directed against different epitopes on at
least one of the targets. In a further preferred embodiment, the
specific and high affinity mAbs comprise mAbs that are directed
against different targets, such as, for example, one or more
members of the EGF-receptor family, including erbB1 (EGF-R; HER1),
erbB2, (HER2), erbB3 (HER3) and erbB4 (HER4).
[0080] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
foregoing techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (2001)), which is incorporated herein by reference. The
nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques are used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
DESCRIPTION OF THE DRAWINGS
[0081] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0082] FIG. 1: A topology map of the annealing locations of mouse
specific VH primers and the position of required restriction sites
that are introduced by overhanging sequences at the 3' end of
primers.
[0083] FIG. 2: PCR amplification steps (Amplification, Intermediate
and Site introduction). The location and names of the mouse VH
amplification primers (and mixtures of primers) are indicated per
step.
[0084] FIG. 3: Topology of the MV1043 vector. This vector is used
for the cloning of human or murine VH fragments. O12 (IGKV1-39) is
indicated as the VL gene. Products of this vector in combination
with helper phages in E. coli cells allow the generation of phages
that display Fab fragments on the surface of the phage particles as
a fusion product to the g3 protein and presence of the vector in
the phage as the genetic content (F1 ORI).
[0085] FIG. 4: The topology of the mouse Ckappa locus downstream of
the J-segments. Both enhancers and Ckappa region are indicated. The
lower arrow indicates the region that is removed in order to
silence the locus.
[0086] FIG. 5: The topology of the mouse C-lambda locus. All three
active V-regions are indicated (Igl-V1, V2 and V3) as are the
J-segments (Igl-J1, Igl-J2, Igl-J3, Igl-J4 and the pseudo segment
Igl-J3p) and constant regions (Igl-C1, Igl-C2, Igl-C3 and Igl-C4).
The regions that are deleted in order to silence the locus are
indicated by deletion markers. These deletions include all active V
genes (1, 2 and 3) and the intergenic segment between V2 and
V3.
[0087] FIG. 6: Construct topology of IGKV1-39/J-Ck with an intron
located in the leader open reading frame (ORF).
[0088] FIG. 7: Construct topology of IGLV2-14/J-Ck with an intron
located in the leader open reading frame (ORF).
[0089] FIG. 8: Construct topology of VkP-IGKV1-39/J-Ck (VkP-O12).
The promoter originates from the IGKV1-39 gene and is placed
directly in front of the required elements for efficient
transcription and translation. Intergenic sequences (including the
enhancers) are derived from mice and obtained from BAC clones. The
C-kappa sequence codes for the kappa constant region of rat.
[0090] FIG. 9: Construct topology of VkP-IGLV2-14/J-Ck (VkP-2a2).
The promoter originates from the IGKV1-39 gene and is placed
directly in front of the required elements for efficient
transcription and translation. Intergenic sequences (including the
enhancers) are derived from mice and obtained from BAC clones. The
C-kappa sequence codes for the kappa constant region of rat.
[0091] FIG. 10: Construct topology of VkP-IGKV1-39/J-Ck-A1
(VkP-O12-del1) is identical to VkP-IGKV1-39/J-Ck from FIG. 9 except
that the intron enhancer region is removed.
[0092] FIG. 11: Construct topology of VkP-IGKV1-39/J-Ck-A2
VkP-O12-de12) is identical to VkP-IGKV1-39/J-Ck-A1 from FIG. 10
except that a large piece of the intergenic region between the Ck
gene and 3' enhancer is deleted. In addition, the 3' enhancer is
reduced in size from 809 bp to 125 bp.
[0093] FIG. 12: Overview of the sequences used or referred to in
this application: Human germline IGKV1-39/J DNA (SEQ ID NO:84);
human germline IGKV1-39/J Protein (SEQ ID NO:85); human germline
IGLV2-14/J DNA (SEQ ID NO:86); human germline IGLV2-14/J Protein
(SEQ ID NO:87); Rat IGCK allele a DNA (SEQ ID NO:88); Rat IGCK
allele a protein (SEQ ID NO:89); IGKV1-39/J-Ck (SEQ ID NO:90);
IGLV2-14/J-Ck (SEQ ID NO:91); VkP-IGKV1-39/J-Ck (SEQ ID NO:92);
VkP-IGKV1-39/J-Ck-A1 (SEQ ID NO:93); VkP-IGKV1-39/J-Ck-A2 (SEQ ID
NO:94); VkP-IGLV2-14/J-Ck (SEQ ID NO:95); pSELECT-IGKV1-39/J-Ck
(SEQ ID NO:96); pSelect-IGLV2-14/J-Ck (SEQ ID NO:97); MV1043 (SEQ
ID NO:98); and MV1057 (SEQ ID NO:99).
[0094] FIGS. 13A-C: Generation of Rosa26-IgVk1-39 KI allele. FIG.
13A Schematic drawing of the pCAGGS-IgVK1-39 targeting vector. FIG.
13B Nucleotide sequence of the pCAGGS-IgVK1-39 targeting vector
(SEQ ID NO:100). FIG. 13C Targeting strategy.
[0095] FIGS. 14A-C: FIG. 14A Southern blot analysis of genomic DNA
of ES clones comprising an insertion of the pCAGGS-IgVK1-39
targeting vector. Genomic DNA of four independent clones was
digested with AseI and probed with 5e1 indicating the 5'-border of
the targeting vector. All clones comprise a correct insertion of
the targeting vector at the 5' end.
[0096] FIG. 14B Southern blot analysis of genomic DNA of ES clones
comprising an insertion of the pCAGGS-IgVK1-39 targeting vector.
Genomic DNA of four independent clones was digested with MscI and
probed with 3e1 indicating the 3'-border of the targeting vector.
All clones comprise a correct insertion of the targeting vector at
the 3' end. FIG. 14C Southern blot analysis of genomic DNA of ES
clones comprising an insertion of the pCAGGS-IgVK1-39 targeting
vector. Genomic DNA of four independent clones was digested with
BamHI and probed with an internal Neo probe indicating the
5'-border of the targeting vector. All clones comprise a correct,
single insertion of the targeting vector.
[0097] FIGS. 15A-C: Generation of Rosa26-IgV12-14 KI allele. FIG.
15A Schematic drawing of the pCAGGS-IgVL2-14 targeting vector. FIG.
15B Nucleotide sequence of the pCAGGS-IgVL2-14 targeting vector
containing the CAGGS expression insert (SEQ ID NO:101) based on the
rearranged germline IGLV2-14/J V lambda region (IGLV2-14/J-Ck).
FIG. 15C Targeting strategy.
[0098] FIGS. 16A-C: Epibase.RTM. profile of IGKV1-39 residues 1-107
(SEQ ID NO:85).
[0099] FIG. 16A displays the binding strength for DRB1 allotypes,
while FIG. 16C displays the binding strength for DRB3/4/5, DQ and
DP allotypes. The values in the figure represent dissociation
constants (Kds) and are plotted on a logarithmic scale in the range
0.01 .mu.M-0.1 .mu.M (very strong binders may have run off the
plot). For medium binding peptides, qualitative values are given
only, and weak and non-binders are not shown. Values are plotted on
the first residue of the peptide in the target sequence (the
peptide itself extends by another nine residues). Importantly, only
the strongest binding receptor for each peptide is shown:
cross-reacting allotypes with lower affinity are not visible in
this plot. The strongest binding receptor is indicated by its
serotypic name. Finally, any germline-filtered peptides are plotted
with a lighter color in the epitope map (in this case, no non-self
epitopes were found). FIG. 16B shows the HLA binding promiscuity
for every decameric peptide (Y-axis: the number of HLA allotypes
recognizing critical epitopes in each of the peptides starting at
the indicated residue shown on the X-axis). The promiscuity is
measured as the number of allotypes out of the total of 47 for
which the peptide is a critical binder. White columns refer to
self-peptides, and black columns (absent here) to non-self
peptides.
[0100] FIG. 17: Epitope map of IGKV1-39 showing the presence of
peptide binders predicted in the sequence of IGKV1-39 by serotype
in the 15-mer format. Each 15-mer is numbered as indicated in the
top of the figure. The full sequence of the corresponding 15-mer is
listed in Table 7. Black boxes indicate the presence of one or more
critical self-epitopes in the 15-mer for the serotype listed on the
left. Critical epitopes are operationally defined as strong or
medium DRB1 binders and strong DRB3/4/5 or DP or DQ binders.
[0101] FIGS. 18A-B: Constitutive knock-out (KO) of the Ig kappa
locus. FIG. 18A Targeting strategy. FIG. 18B Schematic drawing of
the pIgKappa targeting vector.
[0102] FIGS. 19A-B: Constitutive KO of the Ig lambda locus. FIG.
19A First step of the targeting strategy. FIG. 19B Second step of
the targeting strategy.
[0103] FIGS. 20A-C: Schematic drawing of targeting vectors. FIG.
20A pVkP-O12 (VkP-IGKV1-39/J-Ck); FIG. 20B pVkP-O12-del1
(VkP-IGKV1-39/J-Ck-A1); FIG. 20C pVkP-O12-de12
(VkP-IGKV1-39/J-Ck-A2).
[0104] FIGS. 21A-C: Targeting strategies for insertion of transgene
into the Rosa26 locus by targeted transgenesis using RMCE. FIG. 21A
VkP-O12 (VkP-IGKV1-39/J-Ck); FIG. 21B VkP-O12-del1
(VkP-IGKV1-39/J-Ck-A1); FIG. 21C VkP-O12-de12
(VkP-IGKV1-39/J-Ck-A2).
[0105] FIG. 22: Topology of the MV1057 vector. Replacing the
indicated stuffer fragment with a VH fragment yields an expression
vector that can be transfected to eukaryotic cells for the
production of IgG1 antibodies with light chains containing an O12
(IGKV1-39) VL gene.
[0106] FIG. 23: Lack of transgenic human Vk1 light chain expression
in non-B cell populations of the spleen.
[0107] FIG. 24: Transgenic human Vk1 light chain is expressed in
all B cell populations of the spleen.
[0108] FIG. 25: Transgenic human Vk1 light chain is expressed in B1
cells of the peritoneal cavity.
[0109] FIGS. 26A-B: Transgenic human Vk1 light chain is not
expressed in pro- and pre-B cells but in the immature and
recirculating populations B cells in the bone marrow. FIG. 26A
Gating of bone marrow cells. FIG. 26B Histograms of transgene
expression with overlay from one WT control.
[0110] FIG. 27: Transgenic human Vk1 light chain is directly
correlated with endogenous light chain and IgM expression in
circulating B cells in the blood.
[0111] FIG. 28: Parameters of stability for stable clones
containing the germline IGKV1-39 gene.
[0112] FIG. 29A-B: Antibody mixtures used for staining of
lymphocyte populations. BM=bone marrow, PC=peritoneal cavity,
PP=Peyer's patches.
DETAILED DESCRIPTION OF THE INVENTION
Examples
Example 1: Human Light Chain V-Gene Clones
[0113] This example describes the rationale behind the choice of
two human light chain V-genes, one gene of the kappa type and one
gene of the lambda type, that are used as a proof of concept for
light chain expressing transgenic mice. De Wildt et al. 1999 (de
Wildt et al. (1999), J. Mol. Biol. 285(3):895) analyzed the
expression of human light chains in peripheral IgG-positive
B-cells. Based on these data, IGKV1-39 (O12) and IGLV2-14 (2a2)
were chosen as light chains as they were well represented in the
B-cell repertoire. The J-segment sequence of the light chains has
been chosen based upon sequences as presented in GenBank ABA26122
for IGKV1-39 (B. J. Rabquer, S. L. Smithson, A. K. Shriner and M.
A. J. Westerink) and GenBank AAF20450 for IGLV2-14 (O. Ignatovich,
I. M. Tomlinson, A. V. Popov, M. Bruggemann and G. J. Winter, J.
Mol. Biol. 294 (2):457-465 (1999)).
[0114] All framework segments are converted into germline amino
acid sequences to provide the lowest immunogenicity possible in
potential clinical applications.
Example 2: Obtaining Mouse Heavy Chain V-Genes that Pair with Human
IGKV1-39 Gene Segment to Form Functional Antibody Binding Sites
[0115] This example describes the identification of mouse heavy
chain V-genes that are capable of pairing with a single, rearranged
human germline IGKV1-39/J region. A spleen VH repertoire from mice
that were immunized with tetanus toxoid was cloned in a phage
display Fab vector with a single human IGKV1-39-C kappa light chain
and subjected to panning against tetanus toxoid. Clones obtained
after a single round of panning were analyzed for their binding
specificity. The murine VH genes encoding tetanus toxoid-specific
Fab fragments were subjected to sequence analysis to identify
unique clones and assign VH, DH and JH utilization.
[0116] Many of the protocols described here are standard protocols
for the construction of phage display libraries and the panning of
phages for binding to an antigen of interest and described in
Antibody Phage Display: Methods and Protocols (editor(s): Philippa
M. O'Brien and Robert Aitken).
Immunizations
[0117] BALB/c mice received one immunization with tetanus toxoid
and were boosted after six weeks with tetanus toxoid.
Splenocyte Isolation
[0118] Preparation of spleen cell suspension. After dissection, the
spleen was washed with PBS and transferred to a 60 mm Petri dish
with 20 ml PBS. A syringe capped with 20 ml PBS and a G20 needle
was used to repeatedly flush the spleen. After washing the flushed
cells with PBS, the cells were carefully brought into suspension
using 20 ml PBS and left on a bench for five minutes to separate
the splenocytes from the debris and cell clusters. The splenocytes
suspension was transferred on top of a Ficoll-Paque.TM. PLUS-filled
tube and processed according to the manufacturer's procedures for
lymphocyte isolation (Amersham Biosciences).
RNA Isolation and cDNA Synthesis
[0119] After isolation and pelleting of lymphocytes, the cells were
suspended in TRIzol LS Reagent (Invitrogen) for the isolation of
total RNA according to the accompanying manufacturer's protocol and
subjected to reverse transcription reaction using 1 microgram of
RNA, Superscript III RT in combination with dT20 according to
manufacturer's procedures (Invitrogen).
PCR Amplification of cDNA
[0120] The cDNA was amplified in a PCR reaction using primer
combinations that allow the amplification of approximately 110
different murine V-genes belonging to 15 VH families (Table 1;
RefSeq NG 005838; Thiebe et al. 1999, European Journal of
Immunology 29:2072-2081). In the first round, primer combinations
that bind to the 5' end of the V-genes and 3' end of the J regions
were used. In the second round, PCR products that were generated
with the MJH-Rev2 primer were amplified in order to introduce
modifications in the 3' region to enable efficient cloning of the
products. In the last round of amplification, all PCR products were
amplified using primers that introduce a SfiI restriction site at
the 5' end and a BstEII restriction site at the 3' end (see FIGS. 1
and 2, and Table 1).
[0121] Reaction conditions for 1st round PCR: four different
reactions combining all 25 forward primers (MVH1 to MVH25, Table 1
and FIG. 2) and one reverse primer per reaction (MJH-Rev1,
MJH-Rev2, MJH-Rev3 or MJH-Rev4; see Table 1 and FIG. 2). Fifty
microliters PCR volumes were composed of 2 microliters cDNA (from
RT reactions), 10 microliters 5* Phusion polymerase HF buffer, 40
nM of each of the 25 forward primers (total concentration of 1
micromolar), 1 micromolar reverse primer, 1 microliter 10 mM dNTP
stock, 1.25 unit Phusion polymerase and sterile MQ water. The
thermocycler program consisted of a touch down program: one cycle
98.degree. C. for 30 seconds, 30 cycles 98.degree. C. for ten
seconds, 58.degree. C. decreasing 0.2.degree. C. per cycle ten
seconds, 72.degree. C. 20 seconds and one cycle 72.degree. C. for
three minutes. The second round PCR program was set up only for the
products of the first PCR that contain the MJH-Rev2 primer: two
different reactions combining either the ExtMVH-1 or ExtMVH-2
primers (Table 1 and FIG. 2) in combination with the reverse primer
ExtMJH-Rev2int (Table 1 and FIG. 2). Fifty microliters PCR volumes
were composed of 50 ng PCR product (from first PCR round), 10
microliters 5* Phusion polymerase HF buffer, 500 nM of each forward
primer, 1 micromolar reverse primer, 1 microliter 10 mM dNTP stock,
1.25 unit Phusion polymerase and sterile MQ water. The thermocycler
program consisted of a touch down program followed by a regular
amplification step: one cycle 98.degree. C. for 30 seconds, ten
cycles 98.degree. C. for ten seconds, 65.degree. C. decreasing
1.5.degree. C. per cycle ten seconds, 72.degree. C. 20 seconds, ten
cycles 98.degree. C. for ten seconds, 55.degree. C. ten seconds,
72.degree. C. 20 seconds and one cycle 72.degree. C. for three
minutes. The third round PCR program was setup as described in FIG.
2. Fifty microliters PCR volumes were composed of 50 ng PCR product
(from earlier PCR rounds, FIG. 2), 10 microliters 5* Phusion
polymerase HF buffer, 1 micromolar forward primer (Table 1 and FIG.
2), 1 micromolar reverse primer, 1 microliter 10 mM dNTP stock,
1.25 unit Phusion polymerase and sterile MQ water. The program
consists of a touch down program followed by a regular
amplification step: one cycle 98.degree. C. for 30 seconds, ten
cycles 98.degree. C. for ten seconds, 65.degree. C. decreasing
1.5.degree. C. per cycle ten seconds, 72.degree. C. 20 seconds, ten
cycles 98.degree. C. for ten seconds, 55.degree. C. ten seconds,
72.degree. C. 20 seconds and one cycle 72.degree. C. for three
minutes. After PCR amplifications, all PCR products were gel
purified using Qiaex II according to the manufacturer's
protocols.
Restriction Enzyme Digestions
[0122] Purified products were digested with BstEII and SfiI in two
steps. First 1 microgram of DNA was digested in 100 microliters
reactions consisting of 10 microliters of 10* NEB buffer 3 (New
England Biolabs), 1 microliter 100* BSA, 12.5 unit BstEII and
sterile water for six hours at 60.degree. C. in a stove. The
products were purified using Qiaquick PCR Purification kit from
Qiagen according to the manual instructions and eluted in 40
microliters water. Next all products were further digested with
SfiI in 100 microliters reactions consisting of 10 microliters of
10* NEB buffer 2 (New England Biolabs), 1 microliter 100* BSA, 12.5
unit SfiI and sterile water for 12 hours at 50.degree. C. in a
stove. The digested fragments were purified by Qiaquick Gel
Extraction kit following gel separation on a 20 cm 1.5% agarose TBE
plus ethidium bromide gel at 80 V. 100 micrograms of the acceptor
vector (MV1043, FIGS. 3 and 12) was digested with 50 units Eco91I
in 600 microliters under standard conditions (Tango buffer) and
next purified on a 0.9% agarose gel. After a second digestion step
under prescribed conditions with 400 units SfiI in 500 microliters
for 12 hours, 100 units BsrGI were added for three hours at
50.degree. C.
Ligations
[0123] Each PCR product was ligated separately according to the
following scheme: 70 ng digested PCR products, 300 ng digested
acceptor vector, 100 units T4 Ligase (NEB), 1* ligase buffer in 30
microliters for 16 hours at 12.degree. C. The ligation reactions
were purified with phenol/chloroform/isoamyl alcohol extractions
followed by glycogen precipitations (Sigma Aldrich #G1767)
according to the manufacturer's protocol and finally dissolved in
25 microliters sterile water.
Transformations and Library Storage
[0124] The purified ligation products were transformed by
electroporation using 1200 microliters TG1 electrocompetent
bacteria (Stratagene #200123) per ligation batch and plated on LB
carbenicillin plates containing 4% glucose. Libraries were
harvested by scraping the bacteria in 50 ml LB carbenicillin. After
centrifugation at 2000 g for 20 minutes at 4.degree. C., the
bacterial pellets were resuspended carefully in 2 ml ice cold
2*TY/30% glycerol on ice water and frozen on dry ice/ethanol before
storage at -80.degree. C.
Library Amplification
[0125] Libraries were grown and harvested according to procedures
as described by Kramer et al. 2003 (Kramer et al. (2003), Nucleic
Acids Res. 31(11):e59) using VCSM13 (Stratagene) as helper phage
strain.
Selection of Phages on Coated Immunotubes
[0126] Tetanus toxoid was dissolved in PBS in a concentration of 2
.mu.g/ml and coated to MAXISORP.TM. Nunc-Immuno Tube (Nunc 444474)
overnight at 4.degree. C. After discarding the coating solution,
the tubes were blocked with 2% skim milk (ELK) in PBS (blocking
buffer) for one hour at RT. In parallel, 0.5 ml of the phage
library was mixed with 1 ml blocking buffer and incubated for 20
minutes at room temperature. After blocking the phages, the phage
solution was added to the tetanus toxoid-coated tubes and incubated
for two hours at RT on a slowly rotating platform to allow binding.
Next, the tubes were washed ten times with PBS/0.05% TWEEN'-20
detergent followed by phage elution by an incubation with 1 ml 50
mM glycine-HCl pH 2.2 ten minutes at RT on rotating wheel and
directly followed by neutralization of the harvested eluent with
0.5 ml 1 M Tris-HCl pH 7.5.
Harvesting Phage Clones
[0127] Five ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 was
added to the harvested phage solution and incubated for 30 minutes
at 37.degree. C. without shaking to allow infection of the phages.
Bacteria were plated on Carbenicillin/Tetracycline 4% glucose 2*TY
plates and grown overnight at 37.degree. C.
Phage Production
[0128] Phages were grown and processed as described by Kramer et
al. 2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using
VCSM13 as helper phage strain.
Phage ELISA
[0129] ELISA plates were coated with 100 microliters tetanus toxoid
per well at a concentration of 2 micrograms/ml in PBS overnight at
4.degree. C. Plates coated with 100 microliters thyroglobulin at a
concentration of 2 micrograms/ml in PBS were used as a negative
control. Wells were emptied, dried by tapping on a paper towel,
filled completely with PBS-4% skimmed milk (ELK) and incubated for
one hour at room temperature to block the wells. After discarding
the block solution, phage minipreps pre-mixed with 50 .mu.l
blocking solution were added and incubated for one hour at RT. Next
five washing steps with PBS-0.05% Tween-20 removed unbound phages.
Bound phages were detected by incubating the wells with 100
microliters anti-M13-HRP antibody conjugate (diluted 1/5000 in
blocking buffer) for one hour at room temperature. Free antibody
was removed by repeating the washing steps as described above,
followed by TMB substrate incubation until color development was
visible. The reaction was stopped by adding 100 microliters of 2 M
H.sub.2SO.sub.4 per well and analyzed on an ELISA reader at 450 nm
emission wavelength (Table 2). Higher numbers indicate stronger
signals and thus higher incidence of specific binding of the
phage-Fab complex.
Sequencing
[0130] Clones that gave signals at least three times above the
background signal (Table 2) were propagated, used for DNA miniprep
procedures (see procedures Qiagen miniPrep manual) and subjected to
nucleotide sequence analysis. Sequencing was performed according to
the Big Dye 1.1 kit accompanying manual (Applied Biosystems) using
a reverse primer (CH1_Rev1, Table 1) recognizing a 5' sequence of
the CH1 region of the human IgG1 heavy chain (present in the Fab
display vector MV1043, FIGS. 3 and 12). Mouse VH sequences of 28
tetanus toxoid binding clones are depicted in Table 3. The results
show that the selected murine VH genes belong to different gene
families, and different individual members from these gene families
are able to pair with the rearranged human IGKV1-39/J VH region to
form functional tetanus toxoid-specific antibody binding sites.
From the sequence analyses, it was concluded that the murine VH
regions utilize a diversity of DH and JH gene segments.
Example 3: Silencing of the Mouse Kappa Light Chain Locus
[0131] This example describes the silencing of the mouse endogenous
kappa light chain locus. The endogenous kappa locus is modified by
homologous recombination in ES cells, followed by the introduction
of genetically modified ES cells in mouse embryos to obtain
genetically adapted offspring.
[0132] A vector that contains an assembled nucleotide sequence
consisting of a part comprising the J-region to 338 bp downstream
of the J5 gene segment fused to a sequence ending 3' of the 3' CK
enhancer is used for homologous recombination in ES cells. The
assembled sequence is used to delete a genomic DNA fragment
spanning from 3' of the JK region to just 3' of the 3' CK enhancer.
As a consequence of this procedure, the CK constant gene, the 3'
enhancer and some intergenic regions are removed (see FIGS. 4 and
18A-B).
Construction of the Targeting Vector
[0133] A vector that received 4.5-8 kb flanking arms on the 3' and
5' end fused to the deletion segment was used for targeted
homologous recombination in an ES cell line. Both arms were
obtained by PCR means ensuring maximum homology. The targeting
strategy allows generation of constitutive KO allele. The mouse
genomic sequence encompassing the Igk intronic enhancer, Igk
constant region and the Igk 3' enhancer was replaced with a PuroR
cassette, which was flanked by F3 sites and inserted downstream of
the Jk elements. Flp-mediated removal of the selection marker
resulted in a constitutive KO allele. The replacement of the Igk
MiEk-Igk C-Igk 3'E genomic region (approximately 10 kb) with a
F3-Puro cassette (approx. 3 kb) was likely to decrease the
efficiency of homologous recombination. Therefore, the arms of
homology were extended accordingly and more ES cell colonies were
analyzed after transfection in order to identify homologous
recombinant clones.
Generation of ES Cells Bearing the Deleted Kappa Fragment
[0134] The generation of genetically modified ES cells was
essentially performed as described (Seibler et al. (2003), Nucleic
Acids Res. February 15; 31(4):e12). See also Example 14 for a
detailed description.
Generation of ES Mice by Tetraploid Embryo Complementation
[0135] The production of mice by tetraploid embryo complementation
using genetically modified ES cells was essentially performed as
described (Eggan et al., PNAS 98:6209-6214; J. Seibler et al.
(2003), Nucleic Acids Res. February 15; 31(4):e12; Hogan et al.
(1994), Summary of mouse development, Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
N.Y., pp. 253-289).
Example 4: Silencing of the Mouse Lambda Light Chain Locus
[0136] This example describes the silencing of the mouse endogenous
lambda light chain locus. The endogenous lambda locus is modified
by homologous recombination in ES cells followed by the
introduction of genetically modified ES cells in mouse embryos to
obtain genetically adapted offspring.
[0137] Two regions of the murine lambda locus that together contain
all functional lambda V regions are subject to deletion.
[0138] The first region targeted for homologous recombination-based
deletion is a region that is located 408 bp upstream of the start
site of the IGLV2 gene segment and ends 215 bp downstream of IGLV3
gene segment, including the intergenic sequence stretch between
these IGLV gene segments. The second region that is subject to a
deletion involves the IGLV1 gene segment consisting of a fragment
spanning from 392 bp upstream to 171 bp downstream of the IGLV1
gene segment. As a consequence of these two deletion steps, all
functional V-lambda genes segments are deleted, rendering the locus
functionally inactive (FIGS. 5 and 19A-B).
Construction of the Targeting Vectors
[0139] Vectors that received 3-9.6 kb flanking arms on the 3' and
5' end fused to the deletion segment were used for targeted
homologous recombination in an ES cell line. Both arms were
obtained by PCR means ensuring maximum homology. In a first step,
the mouse genomic sequence encompassing the Igl V2-V3 regions were
replaced with a PuroR cassette flanked by F3 sites, which yields a
constitutive KO allele after Flp-mediated removal of selection
marker (see FIG. 19A). In a second step, the mouse genomic sequence
encompassing the Igl V1 region was replaced with a Neo cassette in
ES cell clones which already carried a deletion of the Igl V2-V3
regions (see FIG. 19B). The selection marker (NeoR) was flanked by
FRT sites. A constitutive KO allele was obtained after Flp-mediated
removal of selection markers.
Generation of ES Cells Bearing the Deleted Lambda Fragment
[0140] The generation of genetically modified ES cells was
essentially performed as described (J. Seibler, B. Zevnik, B.
KUter-Luks, S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N.
Faust, G. Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kuhn,
F. Schwenk (2003), Nucleic Acids Res., February 15; 31(4):e12). See
also, Example 14 for a detailed description. To show that both
targeting events occurred on the same chromosome several double
targeted clones were selected for the in vitro deletion with pCMV
C31deltaCpG. The clones were expanded under antibiotic pressure on
a mitotically inactivated feeder layer comprised of mouse embryonic
fibroblasts in DMEM High Glucose medium containing 20% FCS (PAN)
and 1200.mu./mL Leukemia Inhibitory Factor (Millipore ESG 1107).
1.times.10.sup.7 cells from each clone were electroporated with 20
.mu.g of circular pCMV C31deltaCpG at 240 V and 500 .mu.F and
plated on four 10 cm dishes each. Two to three days after
electroporation, cells were harvested and analyzed by PCR. Primers
used were:
TABLE-US-00001 (SEQ ID NO: 1) 20055: CCCTTTCCAATCTTTATGGG (SEQ ID
NO: 2) 20057: AGGTGGATTGGTGTCTTTTTCTC (SEQ ID NO: 3) 20059:
GTCATGTCGGCGACCCTACGCC
[0141] PCR reactions were performed in mixtures comprising 5 .mu.l
PCR Buffer 10x (Invitrogen), 2 .mu.l MgCl.sub.2 (50 mM), 1 .mu.l
dNTPs (10 mM), 1 .mu.l first primer (5 .mu.M), 1 .mu.l second
primer (5 .mu.M), 0.4 .mu.l Taq (5 U/ul, Invitrogen), 37.6 .mu.l
H.sub.2O, and 2 .mu.l DNA. The program used was 95.degree. C. for
five minutes; followed by 35 cycles of 95.degree. C. for 30
seconds; 60.degree. C. for 30 seconds; 72.degree. C. for 1 minute;
followed by 72.degree. C. for ten minutes.
Generation of ES Mice by Tetraploid Embryo Complementation
[0142] The production of mice by tetraploid embryo complementation
using genetically modified ES cells was essentially performed as
described (Eggan et al., PNAS 98:6209-6214; J. Seibler, B. Zevnik,
B. Kiiter-Luks, S. Andreas, H. Kern, T. Hennek, A. Rode, C.
Heimann, N. Faust, G. Kauselmann, M. Schoor, R. Jaenisch, K.
Rajewsky, R. Kuhn, and F. Schwenk (2003), Nucleic Acids Res.,
February 15; 31(4):e12; Hogan et al. (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor N.Y.), pp. 253-289).
Example 5: Construction of the CAGGS Expression Insert Based on a
Rearranged Human Germline IGKV1-39/J-Ck Gene (IGKV1-39/J-Ck)
[0143] This example describes the construction of a CAGGS
expression cassette incorporating the rearranged human germline
IGKV1-39/J region. This insert expression cassette encompasses
cloning sites, a Kozak sequence, a leader sequence containing an
intron, an open reading frame of the rearranged IGKV1-39 region, a
rat CK constant region from allele a and a translational stop
sequence (IGKV1-39/J-Ck; FIG. 6). The primary construct consists of
naturally occurring sequences and has been analyzed and optimized
by removing undesired cis acting elements like internal TATA-boxes,
poly adenylation signals, chi-sites, ribosomal entry sites, AT-rich
or GC-rich sequence stretches, ARE-, INS- and CRS sequence
elements, repeat sequences, RNA secondary structures, (cryptic)
splice donor and acceptor sites and splice branch points (GeneArt
GmbH). In addition, the codon usage in the open reading frame
regions is optimized for expression in mice. The intron sequence is
unchanged and thus represents the sequence identical to the coding
part of the human IGKV1-39 leader intron.
[0144] At the 5' end of the expression cassette, a NotI site was
introduced and on the 3' site a NheI site. Both sites are used for
cloning in the CAGGS expression module. After gene assembly
according to methods used by GeneArt, the insert is digested with
NotI-NheI and cloned into the expression module containing a CAGGS
promoter, a stopper sequence flanked by LoxP sites ("foxed"), a
polyadenylation signal sequence and, at the 5' and 3' end,
sequences to facilitate homologous recombination into the Rosa26
locus of mouse ES cell lines. Promoter and/or cDNA fragments were
amplified by PCR, confirmed by sequencing and/or cloned directly
from delivered plasmids into an RMCE exchange vector harboring the
indicated features. A schematic drawing and the confirmed sequence
of the final targeting vector pCAGGS-IgVK1-39 are shown in FIGS.
13A and 13B. The targeting strategy is depicted in FIG. 13C.
Example 6: CAGGS Expression Insert Based on the Rearranged Germline
IGLV2-14/J V Lambda Region (IGLV2-14/J-Ck)
[0145] This example describes the sequence and insertion of an
expression cassette incorporating the rearranged germline
IGLV2-14/J V lambda region. This insert encompasses cloning sites,
a Kozak sequence, a leader sequence containing an intron, an open
reading frame of the rearranged IGLV2-14/J region, a rat CK
constant region from allele a and a translational stop sequence
(IGLV2-14/J-Ck; FIG. 7). The primary construct consists of
naturally-occurring sequences and has been analyzed and optimized
by removing undesired cis acting elements like: internal
TATA-boxes, poly adenylation signals, chi-sites, ribosomal entry
sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRS
sequence elements, repeat sequences, RNA secondary structures,
(cryptic) splice donor and acceptor sites and splice branch points
(GeneArt GmbH). In addition, the codon usage in the open reading
frame regions was optimized for expression in mice. The intron
sequence is unchanged and thus represents the sequence identical to
the human IGKV1-39 leader intron.
[0146] At the 5' end of the expression cassette, a NotI site was
introduced and on the 3' site a NheI site. Both sites are used for
cloning in the CAGGS expression module as described by
TaconicArtemis. After gene assembly according to methods used by
GeneArt, the insert was digested with NotI-NheI and cloned into the
expression module containing a CAGGS promoter, a stopper sequence
flanked by LoxP sites ("foxed"), a polyadenylation signal sequence
and, at the 5' and 3' end, sequences to facilitate homologous
recombination into the Rosa26 locus of mouse ES cell lines. To
construct the final ROSA26 RMCE targeting vector, promoter and/or
cDNA fragments were amplified by PCR. Amplified products were
confirmed by sequencing and/or cloned directly from delivered
plasmids into an RMCE exchange vector harboring the indicated
features. A schematic drawing and the confirmed sequence of the
final targeting vector pCAGGS-IgVL2-14 is shown in FIGS. 15A and
15B. The targeting strategy is depicted in FIG. 15C.
Example 7: Expression of IGKV1-39/J-Ck in HEK293 Cell Lines
(pSELECT-IGKV1-39/J-Ck)
[0147] This example describes a method to verify that the
IGKV1-39/J-Ck constructs described in Example 5 enable expression
and detection of the IGKV1-39/J-Ck L chain in HEK293 cells. The
IGKV1-39/J insert (FIG. 6) was modified at the 5' end by changing
the NotI site into a SalI site. This change is required for cloning
of the product into the expression cassette plasmid pSELECT-hygro
(InvivoGen). The CAGGS expression insert IGKV1-39/J-Ck and
pSELECT-hygro were digested with SalI and NheI, ligated and used to
transform competent XL1-Blue cells using standard techniques.
Colonies were picked and DNA purified using Qiagen Midi-prep
columns according to the manufacturer's procedures. The resulting
light chain (LC) expressing vector named 0817676pSELECT 0815426 was
used to transfect HEK293 cells with Fugene6 (Roche) according to
the manufacturer's protocols. Supernatants were screened for the
presence of IGKV1-39/J-Ck light chains by ELISA and western blot
using anti-rat-Ck antibodies (Beckton Dickinson #550336 and 553871)
and protocols used in the art.
[0148] The VH of anti-tetanus toxoid (TT) IgG MG1494 was cloned
into IgG expression vector MV1056 using restriction sites SfiI and
BstEII. The resulting clone was sequence verified. HEK293T cells
were transfected with five different vector combinations as shown
in Table 4 (see Example 8 for details of vector
0817678_pSELECT_0815427). Supernatants were harvested and IgG
concentrations determined (see Table 4). No IgG could be detected
for supernatants A and B containing light chain only as expected
(detection antibody recognized Fc part of IgG). IgG concentration
in supernatants C and D was comparable to that of positive control
supernatant E, indicating correct expression of the light chain
constructs.
[0149] Binding to TT was analyzed by ELISA to check functionality
of the produced antibodies, using hemoglobin as negative control
antigen. No TT-specific binding could be detected for supernatants
A and B containing light chain only, as expected. TT-specific
binding for supernatants C and D was at least as good as for
positive control supernatant E, confirming correct expression of
the light chain constructs and functional assembly with heavy
chain. Antibodies were detected not only using an anti-human IgG
secondary antibody, but also an anti-rat Ckappa light chain
secondary antibody. The results confirm that the anti-rat Ckappa
antibody (BD Pharmingen #553871, clone MRK-1) recognizes the light
chain expressed by the pSELECT vectors.
[0150] Supernatants were analyzed by non-reducing SDS-PAGE and
Western blot (not shown). Detection using an anti-human IgG heavy
chain antibody did not show bands for supernatants A and B
containing light chain only, as expected. Results for supernatants
C and D were comparable to positive control supernatant E, with a
band close to the 170 kD marker as expected for intact IgG.
Additional lower molecular weight bands were observed as well for
supernatants C, D and E, which might represent degradation
products, IgG fragments resulting from (partial) reduction and/or
irrelevant protein bands due to non-specific binding of the
detection antibody.
[0151] Detection using an anti-rat Ckappa light chain antibody
showed a band close to the 26 kD marker for supernatants A and B,
as expected for light chain only. This band was much more intense
for A compared to B, indicating that the free IGKV1-39 light chain
may be better expressed and/or more stable than the free IGLV2-14
light chain. No bands were detected for control supernatant E as
expected, since the expressed IgG contains a human Ckappa light
chain. For supernatants C and D, expected bands close to the 170 kD
marker were observed; lower molecular weight bands were also
observed, but to a lesser extent than above using the anti-human
IgG antibody.
[0152] In conclusion, transfection of the light chain expression
constructs combined with the heavy chain of anti-tetanus toxoid
(TT) IgG MG1494 resulted in IgG production comparable to the
positive control construct for both the pSELECT kappa and lambda
light chain constructs. Both IgG productions yielded ELISA signals
in a TT ELISA that were better than or comparable to the control
IgG. SDS-PAGE and Western blot analysis confirmed the presence of
intact IgG. The tested anti-rat Ckappa antibody worked efficiently
in both ELISA and Western blot. Culture supernatant from cells
transfected with light chain constructs only did not result in
detectable IgG production nor in detectable TT-specific binding,
while free light chain was detected on Western blot.
Example 8: Expression of IGLV2-14/J-Ck in HEK293 Cell Lines
(pSELECT-IGLV2-14/J-Ck)
[0153] This example describes a method to verify that the
IGLV2-14/J constructs described in Example 6 enable expression and
detection of the IGLV2-14/J-Ck L chain in HEK293 cells. The
IGLV2-14/J-Ck insert (FIG. 7) was modified at the 5' end by
changing the NotI site into a SalI site. This change is required
for cloning of the product into the expression cassette plasmid
pSELECT-hygro (InvivoGen). The CAGGS expression insert
IGLV2-14/J-Ck and pSELECT-hygro were digested with SalI and NheI
ligated and used to transform competent XL1-Blue cells using
standard techniques. Colonies were picked and DNA purified using
Qiagen Midi-prep columns according to the manufacturer's
procedures. The resulting light chain (LC) expressing vector named
0817678pSELECT 0815427 was used to transfect HEK293 cells with
Fugene6 (Roche) according to the manufacturer's protocols.
Supernatants were screened for the presence of IGLV2-14/J-Ck light
chains by ELISA and western blot using anti-rat-Ck antibodies
(Becton Dickinson #550336 and 553871) and protocols used in the
art. See Example 7 for details and results.
Example 9: Construction of a VK Promoter-Driven Expression
Construct Containing an IGKV1-39/J Insert and Multiple Enhancer
Elements Derived from the Murine CK Locus (VkP-IGKV1-39/J-Ck;
VkP-O12)
[0154] This example describes the construction of an expression
cassette that contains relevant elements to enable B-cell and
developmental/differentiation stage-specific expression of the
rearranged human IGKV1-39 VK region, based on the IGKV1-39 VK
promoter region, leader containing an intron, germline V-gene,
CDR3, IGKJ segment, mouse intergenic region located between Jk and
CK, rat Ck allele a open reading frame, and a mouse intergenic
fragment from the 3' end of the mouse CK gene ending just 3' of the
3' CK enhancer.
[0155] Optimized open reading frames of the leader, IGKV1-39
rearranged gene, and rat CK allele a gene, as described in Example
5, was used for the construction of the expression cassette. The VK
promoter region was obtained by gene synthesis procedures (GeneArt,
GmbH) and is almost identical to the sequence of the human IGKV1-39
region between -500 bp and the ATG (start site) of the gene. The
only deviation from the natural sequence is the introduction of a
GCCACCATGG Kozak sequence (SEQ ID NO:102) at the ATG (start) site
in order to promote translation. A genomic fragment from a mouse
BAC clone (TaconicArtemis) is used as the basis for the
introduction of individual elements. This fragment is identical to
the sequence of the mouse VK locus starting with the intron donor
site located directly 3' of the JK5 region and ending just 3' of
the 3' CK enhancer and covers approximately 12.5 kb.
[0156] The final construct contains from 5' to 3' end the following
elements: human genomic IGKV1-39 promoter (500 bp), a Kozak
sequence, a human IGKV1-39 leader part 1 (optimized), a human
IGKV1-39 leader intron, a human IGKV1-39 leader part 2 (optimized),
a human IGKV1-39 germline gene (optimized), a human J-region
(optimized), a mouse intergenic region including the intron
enhancer element, a rat (Rattus norvegicus) kappa constant region
(optimized), and a mouse intergenic region including the 3' kappa
enhancer. The elements of this expression cassette are shown in
FIG. 8 and named VkP-IGKV1-39/J-Ck (VkP-O12). An outline of the
pVkP-O12 vector and the targeting strategy is depicted in FIGS. 20A
and 21A. The vector was introduced into ES cells following standard
procedures (see Example 14).
Example 10: Construction of a VK Promoter-Driven Expression
Construct Containing an IGLV2-14/J Clone and Multiple CK
Locus-Derived Enhancer Elements (VkP-IGLVL2-14/J-Ck; VkP-2a2)
[0157] This example describes the same construct as described in
Example 9, except that the IGKV1-39 gene and J-region are replaced
by the optimized human IGLV2-14 germline gene including a unique
V-J region (VkP-IGLV2-14/J-Ck; VkP-2a2; FIG. 9).
Example 11: Construction of a VK Promoter-Driven Expression
Construct Containing an IGKV1-39 Clone Lacking the CK Intron
Enhancer Element (VkP-IGKV1-39/J-Ck-.DELTA.1; VkP-O12-del1)
[0158] The construct described in Example 9 was modified by
removing the CK intron enhancer element, located in the intergenic
region between the human J region and the rat CK region by standard
PCR modification and DNA cloning methodologies (GeneArt, GmBH). The
resulting expression cassette is shown in FIG. 10 and named
VkP-IGKV1-39/J-Ck-.DELTA.1 (VkP-O12-del1).
[0159] An outline of the pVkP-O12-del1 vector and the targeting
strategy is depicted in FIGS. 20B and 21B. The vector was
introduced into ES cells following standard procedures (see Example
14).
Example 12: Construction of a VK Promoter-Driven Expression
Construct Containing an IGKV1-39 Clone Lacking the CK Intron
Enhancer Element and a Truncated 3' CK Enhancer Element
(VkP-IGKV1-39/J-Ck-.DELTA.2; VkP-O12-De12)
[0160] The construct described in Example 11 was modified by
truncating the 3' CK enhancer element and deleting part of the
intergenic region 3' of the rat Ck gene, to remove potential
inhibitory elements. This was achieved by removing the intergenic
sequence between an EcoRV site (located 3' of the rat Ck gene) and
the NcoI site present in the 3' enhancer (5993 bp) and further
removing the sequence between the 3' enhancer BstXI site and the
BstXI site 3' of the 3' enhancer (474 bp) using standard methods.
The resulting expression cassette is shown in FIG. 11 and named
VkP-IGKV1-39/J-Ck-.DELTA.2 (VkP-O12-de12).
[0161] An outline of the pVkP-O12-de12 vector and the targeting
strategy is depicted in FIGS. 20C and 21C. The vector was
introduced into ES cells following standard procedures (see Example
14).
Example 13: Expression of Vk Constructs in Cell Lines
[0162] The constructs described in Examples 9-12 are tested for
their ability to produce light chain proteins in the myeloma cell
lines MPC11 (ATCC CCL167), B-cell lymphoma WEHI231 (ATCC CRL-1702),
the T-cell lymphoma EL4 (ATCC TIB-39) and in HEK293 (ATCC CRL1573).
The enhancer and promoter elements in the construct enable
expression in the B-cell lines but not in cell lines derived from
other tissues. After transfection of the cell lines using purified
linearized DNA and Fugene6 (Roche) cells are cultured for transient
expression. Cells and supernatant are harvested and subjected to
SDS-PAGE analysis followed by western blotting using a specific
anti-rat-C-kappa antibody. Supernatants are analyzed in ELISA for
secreted L chains using the anti-rat CK antibody (Beckton Dickinson
#550336).
Example 14: Generation of Transgenic ES Lines
[0163] All constructs as described in Examples 3, 4, 5, 6, 9, 10,
11 and 12 were used to generate individual stable transgenic ES
lines by means of homologous recombination. The methods for
generation of transgenic ES lines via homologous recombination are
known in the field (e.g., Eggan et al., PNAS 98:6209-6214; J.
Seibler, B. Zevnik, B. KUter-Luks, S. Andreas, H. Kern, T. Hennek,
A. Rode, C. Heimann, N. Faust, G. Kauselmann, M. Schoor, R.
Jaenisch, K. Rajewsky, R. Kuhn, F. Schwenk (2003), Nucleic Acids
Res., February 15; 31(4):e12; Hogan et al. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).
[0164] For all constructs described in Examples 5 and 6, and
Examples 9-12, the RMCE ES cell line (derived from mouse strain
129S6B6F1-Gt(ROSA)26Sortml0Arte) was grown on a mitotically
inactivated feeder layer comprised of mouse embryonic fibroblasts
(MEF) in DMEM High Glucose medium containing 15% FBS (PAN
1302-P220821). Leukemia Inhibitory Factor (Chemicon ESG 1107) was
added to the medium at a concentration of 900 U/mL. For
manipulation, 2.times.10.sup.5 ES-cells were plated on 3.5 cm
dishes in 2 ml medium. Directly before transfection, 2 ml fresh
medium was added to the cells. Three .mu.l Fugene6 Reagent (Roche;
Catalog No. 1 814 443) was mixed with 100 .mu.l serum free medium
(OptiMEM I with Glutamax I; Invitrogen; Catalog No. 51985-035) and
incubated for five minutes. One hundred .mu.l of the Fugene/OptiMEM
solution was added to 2 .mu.g circular vector and 2 CAGGS-Flp and
incubated for 20 minutes. This transfection complex was added
dropwise to the cells and mixed. Fresh medium was added to the
cells the following day. From day 2 onwards, the medium was
replaced daily with medium containing 250 .mu.g/mL G418 (Geneticin;
Invitrogen; Catalog No. 10131-019). Seven days after transfection,
single clones were isolated, expanded, and molecular analyzed by
Southern blotting according to standard procedures.
[0165] For each construct, analysis of multiple clones by
restriction enzyme digestion of genomic DNA of single clones
followed by hybridization with 5' probes, 3' probes, and internal
probes resulted in clones that comprised a correct, single
insertion at the correct position in the Rosa26 locus. An example
is provided in FIGS. 14A-C.
Example 15: Generation of Transgenic Mouse Strains
[0166] All ES cell lines that were generated and verified for their
modifications as described in Example 14 were used to generate
stable transgenic mice by means of tetraploid recombination. The
methods are known in the field. In general, after administration of
hormones, superovulated Balb/c females were mated with Balb/c
males. Blastocysts were isolated from the uterus at dpc 3.5. For
microinjection, blastocysts were placed in a drop of DMEM with 15%
FCS under mineral oil. A flat tip, piezo actuated
microinjection-pipette with an internal diameter of 12-15
micrometers was used to inject 10-15 targeted C57BL/6 N.tac ES
cells into each blastocyst. After recovery, injected blastocysts
were transferred to each uterine horn of 2.5 days post coitum,
pseudopregnant NMRI females. Chimerism was measured in chimeras
(G0) by coat color contribution of ES cells to the Balb/c host
(black/white). Highly chimeric mice were bred to strain C57BL/6
females. Depending on the project requirements, the C57BL/6 mating
partners are non-mutant (W) or mutant for the presence of a
recombinase gene (Flp-Deleter or Cre-deleter or CreER inducible
deleter or combination of Flp-deleter/CreER). Germline transmission
was identified by the presence of black, strain C57BL/6, offspring
(G1).
[0167] For example, ESC clone IgVK1-39 2683 8 (see Examples 5 and
14) was injected in a total of 62 blastocysts in three independent
experiments. Three litters were obtained with a total of six pups.
All pups were chimeric. Three heterozygous offspring pups were
obtained that were used for further crossing.
[0168] ESC Clone Kappa 2692 A-C10 (see Examples 3 and 14) was
injected in a total of 54 blastocysts in three independent
experiments. Three litters were obtained with a total of eleven
pups, of which ten were chimeric. Eight heterozygous offspring pups
were obtained that were used for further crossing.
[0169] ESC Clone Kappa 2692 B-C1 (see Examples 3 and 14) was
injected in a total of 51 blastocysts in three independent
experiments. Two litters were obtained with a total of six pups, of
which four were chimeric. Three heterozygous offspring pups were
obtained that were used for further crossing.
Example 16: Breeding
[0170] This example describes the breeding for obtaining mice that
contain transgenic expression cassettes as described Example 14 and
knock-out mice in which the endogenous lambda and kappa loci have
been silenced. The localization of V-lambda on chromosome 16 and
CD19 on chromosome 7 allow standard breeding procedures. The
breeding of the co-localized Vk locus and Rosa26 locus on
chromosome 6 with a distance of about 24 cM requires special
attention during the screening as only a percentage of the
offspring shows crossover in a way that both modifications are
brought together on one chromosome.
[0171] All four loci have to be combined in a single mouse strain
that is homo- or heterozygous for CD19-cre (not described) and
modified Rosa26 transgene and homozygous for the other loci.
Breeding is performed by standard breeding and screening techniques
as appropriate and offered by commercial breeding companies (e.g.,
TaconicArtemis).
Example 17: Immunizations of Mice
[0172] Primary and booster immunization of mice are performed using
standard protocols.
[0173] To validate the transgenic expression of human rearranged
V.kappa. O12 (IGKV1-39)--rat C.kappa. light chains (see Examples 5,
14-16) in B cells from CD19-HuV.kappa.1 mice and to assess its
impact on VH repertoire size, diversity of VH family usage and
V(D)J recombination after immunization, the CD19-HuV.kappa.1
transgenic mice are immunized with tetanus toxin vaccine (TT
vaccine) and VH sequence diversity of randomly picked clones from
CD19-HuV.kappa.1 mice are compared with TT-immunized wt mice and
CD19-Cre HuVk1 negative littermates. Data on the SHM frequency of
the human V.kappa. O12 transgene in the immunized mice are
obtained. A diverse collection of at least 40 TT-specific,
clonally-unrelated mAbs containing the human V.kappa. O12 are
recovered from CD19-HuV.kappa.1 mice by phage display.
[0174] For this, three adult CD19-HuV.kappa.1 mice are vaccinated
with TT vaccine using standard immunization procedures. After
immunization, serum titers are measured using TT specific ELISA
(TT: Statens Serum Institute, Art. no. 2674) and spleen suspensions
subjected to cell sorting by the FACS procedure after staining with
a rat C.kappa.-specific monoclonal antibody to isolate transgenic B
cells (clone RG7/9.1; BD Pharmingen#553901, Lot#06548). RNA from
rat C.kappa.-positive B cells are extracted and the resulting cDNA
material used for library building and SHM analysis.
[0175] The standard monoclonal mouse anti-rat C.kappa. antibody
(clone RG7/9.1; BD Pharmingen#553901, Lot#06548) is used in FACS
analysis of transgene expressing B cells (Meyer et al. (1996), Int.
Immunol. 8:1561). The clone RG7/9.1 antibody reacts with a
monotypic (common) kappa chain determinant. This anti-rat C.kappa.
antibody (clone RG7/9.1 (BD Pharmingen#553901, Lot#06548) is
labeled with R-phycoerythrin (PE) using the LYNX rapid conjugation
kit according to the manufacturer's instructions for FACS analysis
and sorting. The labeled antibody is firstly tested by flow
cytometry for binding to rat C.kappa.-containing functional light
chain proteins produced into transiently transfected HEK-293T
cells; the un-conjugated antibody serves as a positive control. Two
other antibodies shown to bind to rat C.kappa. by ELISA and
Western-blot (see Example 7) are tested as well by flow
cytometry.
[0176] Fab-phage display library building is carried out with a set
of optimized degenerate PCR primers designed to amplify C57BL/6 VH
genes; the minimal library size is 10.sup.6 clones, and minimal
insert frequency is 80%. The vector used, MV1043 (FIGS. 3 and 12),
contains the human V.kappa. O12 fused to a human C.kappa. region.
The rat C.kappa. is therefore exchanged for the human counterpart
in the library generation process.
[0177] Before selection, VH sequencing of 96 randomly picked clones
is performed to validate VH repertoire diversity that is compared
to diversity obtained from an unselected library previously
generated using the same procedures from BALB/c mice immunized with
TT. A library from C57Bl/6 wt mice that are immunized in the same
way allows diversity comparison between two preselected libraries
sharing the same vaccine and the same genetic background.
[0178] Several independent selections are performed on TT coated in
immunotubes. Variables that may be included are selections using
biotinylated antigens in solution or selections on captured TT.
Based on the number and diversity of ELISA-positive clones obtained
in the first selections, decisions on additional rounds of
selection are made. Clones are considered positive when
>3.times. positive over a negative control clone. Positive
clones are analyzed by ELISA against a panel of negative control
antigens to verify antigen specificity. The aim is to identify at
least 40 unique VH regions, as based on unique CDR3 sequences and
V.sub.HDJ.sub.H rearrangements.
[0179] Amplification of the cDNA material from rat CK-positive
sorted B cells is performed with a PCR forward primer specific to
the human leader sequence and a PCR reverse primer specific to the
rat C.kappa. sequence, in a region not redundant with the mouse
C.kappa. sequence, as reported in a recent study (Brady et al.
(2006), JIM 315:61). Primer combinations and annealing temperatures
are firstly tested on cDNA from HEK-293T cells transfected with
0817676_pSELECT_0815426=pSELECT vector with IGKV1-39 DNA cassette
(see Example 7).
[0180] The amplification products is cloned in pJET-1 vector and
after XL1-blue transformation, 96 colonies are sequenced for
assessing VL SHM frequency by direct comparison to the V.kappa. O12
(IGKV1-39) germline sequence. The R/S ratio method, as described in
our study on human TT-specific antibodies (de Kruif et al. (2009),
J. Mol. Biol. 387:548) allows discrimination between random
mutations and antigen-driven mutations that occurred on VL
sequences.
Example 18: Immunofluorescent Analysis of B Cell Populations in
Transgenic Mouse Lines
[0181] This example describes the use of antibodies and flow
cytometry to analyze B cell populations in primary (bone marrow)
and secondary (spleen, peritoneal) lymphoid organs and blood.
Methods and reagents are described in Middendorp et al. (2002), J.
Immunol. 168:2695; and Middendorp et al. (2004), J. Immunol.
172:1371. For analysis of early B cell development in bone marrow,
cells were surface stained with combinations of antibodies (Becton
Dickinson) specific for B220, CD19, CD25, IgM, IgD, mouse Ckappa,
mouse Clambda and rat Ckappa to detect pro-B cells, pre-B cells,
large pre-B cells, early and late immature B cells and
recirculating B cell populations expressing the transgene on their
surface. DAPI staining (Invitrogen) was included to exclude dead
cells from the analysis and FC block (Becton Dickinson) to inhibit
antibody interaction with Fc receptors on myeloid cells. For
analysis of surface transgene expression on B cell populations in
peripheral lymphoid organs and blood, cells were stained with
combinations of antibodies (Becton Dickinson) specific for B220,
CD5, CD19, CD21, CD23, IgM, IgD, mouse Ckappa, mouse Clambda and
rat Ckappa. DAPI staining was included to exclude dead cells from
the analysis and FC block to inhibit antibody interaction with Fc
receptors on myeloid cells. In addition, combinations of antibodies
(Becton Dickinson) specific for CD3, CD4, CD11b, CD11c and NK1.1
were included to determine if transgene expression occurred in cell
types outside of the B cell compartment.
[0182] Three mice heterozygous for the human IGKV1-39/rat Ckappa
transgene and heterozygous for the CD19-Cre transgene on a C57BL6
background (HuVk1/CD19-Cre) were analyzed. As controls for the FACS
analysis, three littermate mice wild-type for the human
IGKV1-39/rat Ckappa transgene and heterozygous for the CD19-Cre
transgene on a C57BL6 background (CD19-Cre) and two C57BL6/NTac
mice (Wt) were included. All animals were allowed to acclimatize in
the animal facility for one week before analysis and all mice were
male and six weeks of age. Lymphocytes were isolated from the
femurs, spleens, peritoneal cavity and blood of mice using
conventional techniques as previously described (Middendorp et al.
(2002), J. Immunol. 168:2695; and Middendorp et al. (2004), J.
Immunol. 172:1371). Antibodies were pre-combined as shown in FIG.
29A-B and staining was carried out in 96-well plates. Incubation
with the PE-conjugated anti-rat C kappa (described above) was
carried out before staining with the rat anti-murine antibodies to
avoid non-specific binding. After completion of cell staining,
labeled cells were analyzed on a Becton Dickinson LSR II FACS
machine and the acquired data analyzed with FlowJo software
(v6.4.7).
[0183] Transgenic mice were similar in weight, appearance and
activity to wild-type mice. No gross anatomical alterations were
observed during the harvesting of tissues. No difference was
observed in the numbers of B cells in the bone marrow (BM) and
spleen (Table 9) or in the numbers of B cells, T cells and myeloid
cells in peripheral organs between transgenic and wild-type mice.
In addition, the frequency or proportion of the cells in the
different lymphocyte developmental pathways was not altered in
transgenic mice when compared to wild-type mice. Thus in the double
transgenic (HuVk1/CD19-Cre) and transgenic (CD19-Cre) mice lymphoid
and most importantly B cell development was indistinguishable from
wild-type mice.
[0184] In the peripheral lymphoid organs, staining with the
transgene specific antibody (anti-ratCkappa-PE) was only observed
in the B cell populations. T cell, myeloid cell and NK cell
populations were all negative for surface expression of the
transgene in the spleen (FIG. 23). In contrast, in cells stained
with the pan B cell markers B220 and CD19 all cells were shifted to
the right in the FACS plot indicating cell surface expression of
the transgene (FIG. 24). A similar transgene-specific staining was
measured in CD5.sup.+ B1 cells of the peritoneum, a developmentally
distinct population of B cells (FIG. 25).
[0185] Differentiation of B cells from multilineage precursors to
mature B cells occurs in the bone marrow. In the lymphocytes
analyzed from the bone marrow, extracellular and transgene
expression was not detectable in the earliest B cell progenitors
the pro- and pre-B cell consistent with the pattern of normal light
chain expression (FIGS. 26A-B). Transgene expression first becomes
detectable in immature B cells, the developmental stage at which
the germline murine light chain undergoes rearrangement and is
expressed at the cell surface in the context of the preselected
heavy chain (FIGS. 26A-B). Consistent with the staining in the
spleen transgenic light chain expression is also detected on mature
recirculating B cells (FIGS. 26A-B). Thus the CD19-Cre driven
expression of the transgene is consistent with the normal pattern
of light chain expression. The staining with the endogenous light
chain-specific antibody is more intense than that of the
transgene-specific light chain antibody. This may indicate a higher
expression level of the endogenous light chain, a more sensitive
staining with the endogenous light chain-specific antibody or a
combination of both. Importantly, the intensity of the surface
expression of the transgenic light chain is correlated with both
endogenous light chain and IgM surface expression as observed in
staining of circulating B cells in the blood (FIG. 27).
[0186] Thus, overall this analysis demonstrates that expression of
the human IGKV1-39/Ckappa transgene is restricted to the B cell
compartment and the temporal regulation of its expression is
similar to the endogenous kappa and lambda light chains resulting
in normal development of all B cell populations. The apparent lower
level of expression of the transgene could be explained by the
strength of the promoter in comparison to the promoter and
enhancers present on endogenous light chain genes or by a delay in
transgene expression that gives the endogenous light chains a
competitive advantage in pairing with the rearranged heavy chain.
This is consistent with the observation that as B cells mature the
relative intensity of transgene staining increases compared to the
endogenous light chains. In addition, the observation that B cells
numbers are normal and that every surface Ig+B cell co-expresses an
endogenous and transgenic light chain supports the conclusion that
the IGKV1-39 variable region is capable of pairing with a normal
repertoire of different murine heavy chain variable regions. We
conclude from this analysis that insertion of the IGKV1-39/rat
Ckappa transgene driven by the CD19-Cre activated CAGGS promoter in
the Rosa locus facilitates timely and B cell-specific expression of
the transgene and that the transgene is capable of pairing with a
normal repertoire of murine heavy chains.
Example 19: Epibase.RTM. T-Cell Epitope Profile for IGKV1-39
[0187] The protein sequence of IGKV1-39 (FIG. 12, human germline
IGKV1-39/J Protein) was scanned for the presence of putative HLA
class II restricted epitopes, also known as T.sub.H-epitopes. For
this, Algonomics' Epibase.RTM. platform was applied to IGKV1-39. In
short, the platform analyzes the HLA binding specificities of all
possible 10-mer peptides derived from a target sequence (Desmet et
al. (1992), Nature 356:539-542; Desmet et al. (1997), FASEB J.
11:164-172; Desmet et al. (2002), Proteins 48:31-43; Desmet et al.
(2005), Proteins 58:53-69). Profiling is done at the allotype level
for 20 DRB1, 7 DRB3/4/5, 13 DQ and 7 DP, i.e., 47 HLA class II
receptors in total (see Table 5). Epibase.RTM. calculates a
quantitative estimate of the free energy of binding
.DELTA.G.sub.bind of a peptide for each of the 47 HLA class II
receptors. These data were then further processed as follows:
[0188] Free energies were converted into Kd-values through
.DELTA.G.sub.bind=RT ln(Kd).
[0189] Peptides were classified as strong (S), medium (M), weak and
non (N) binders. The following cutoffs were applied:
[0190] S: strong binder: Kd<0.1 .mu.M.
[0191] M: medium binder: 0.1 .mu.M.ltoreq.Kd<0.8 .mu.M.
[0192] N: weak and non-binder: 0.8 .mu.M.ltoreq.Kd.
[0193] Peptides corresponding to self-peptides were treated
separately. The list of self-peptides was taken from 293 antibody
germline sequences. They are referred to as "germline-filtered"
peptides.
[0194] S- and M-peptides are mapped onto the target sequence in
so-called epitope maps; S-affinities are plotted quantitatively;
M-values are presented qualitatively. As a general overview of the
results, Table 6 lists the number of strong and medium binders in
the analyzed proteins, for the groups of HLA class II receptors
corresponding to the DRB1, DQ, DP and DRB3/4/5 genes. Counting was
done separately for strong and medium affinity binders. Peptides
binding to multiple allotypes of the same group were counted as
one. Values between brackets refer to germline-filtered peptides.
In Table 7, the sequence is shown in a format suitable for
experimental work. The sequence is broken down in consecutive
15-mers overlapping by 12 residues. For each 15-mer, the
promiscuity is listed (the number of allotypes out of a total of 47
for which the 15-mer contains a critical binder), as well as the
implied serotypes. The Epibase.RTM. profile and epitope maps are
shown in FIGS. 16A-C and 17.
[0195] It was concluded that IGKV1-39 contains no strong non-self
DRB1 binders. Typically, significantly more binders were found for
DRB1 than for other HLA genes. This is in agreement with
experimental evidence that allotypes belonging to the DRB1 group
are more potent peptide binders. Medium strength epitopes for DRB1
allotypes are expected to contribute to the population response,
and cannot be disregarded. Again, no non-self DRB1 binders were
found in IGKV1-39.
[0196] In the humoral response raised against an antigen, the
observed T.sub.H cell activation/proliferation is generally
interpreted in terms of the DRB1 specificity. However, one cannot
ignore the possible contribution of the DRB3/4/5, DQ and DP genes.
Given the lower expression levels of these genes as compared to
DRB1, the focus was on the class of strong epitopes for DRB3/4/5,
DQ and DP. "Critical epitopes" are those epitopes that are strong
binders for any DRB1, DRB3/4/5, DQ or DP allotype or are medium
binders for DRB1. IGKV1-39 contains no strong or medium non-self
binders for DRB3/4/5, DQ, or DP.
[0197] A number of peptides are also present in germline sequences
(values between brackets in Table 6). Such peptides may very well
bind to HLA but they are assumed to be self and, hence,
non-immunogenic. In total, six strong and 16 medium
germline-filtered DRB1 binders were found in IGKV1-39. Framework
region 1 up to framework region 3 is an exact match for germline
V-segment VKI 2-1-(1) O12 (VBase), a.k.a. IGKV1-39*01 (IMGT).
Framework region 4 is an exact match for germline J-segment JK1
(V-base) a.k.a. IGKJ1*01(IMGT). It is hardly surprising that these
segments do not contain any non-self epitopes.
Example 20: Production Characteristics of IGKV1-39
[0198] There is a great demand for antibody discovery platforms
that yield therapeutic antibodies that are thermodynamically stable
and give good expression yields. These characteristics are
important in ensuring the stability of the drug substance during
production and after injection of the drug product into the
patient. In addition good expression yields impact directly on the
cost of drug manufacture and thus pricing, patient access and
profitability. Virtually all therapeutic antibodies in clinical use
today are composed of human IgG1 and kappa constant regions but use
different heavy and light chain variable regions that confer
specificity. Human variable heavy and light chain domains can be
divided into families that have greater than 80% sequence
divergence. When rearranged examples of these families in germline
configuration are combined and compared for stability and yield it
is clear that the gene families are not equal in terms of
biophysical properties. In particular V.sub.H3, V.sub.H1 and
V.sub.H5 have favourable stability for the heavy chains and Vk1 and
Vk3 have the best stability and yield of light chains. In addition
when mutations are introduced as part of the somatic hypermutation
process they can interfere with V.sub.H/V.sub.L pairing. To assess
the effect that different light chain genes with different rates of
mutation have on the production characteristics of a fixed V.sub.H
chain, a Fab phage display library was built of light chains (kappa
and lambda) from six naive healthy donors combined with a panel of
44 TT binding heavy chains from immunized donors. After one round
of selection TT binding Fab clones were isolated. Several of these
shared the same V.sub.H gene as the TT clone PG1433 in combination
with different light chains. The Fab light chain fragments were
recloned into a kappa expression vector and transfected in
combination with DNA encoding the heavy chain of PG1433 into 293
cells and specific IgG production measured by ELISA. As
demonstrated in Table 8 the selected clones containing PG1433
V.sub.H combined with different light chains had between five- and
ten-fold lower protein expression PG1433 V.sub.H combined with
IGKV1-39. Note that all of the light chains contained amino acid
mutations within their coding regions that might disrupt V.sub.H
paring and reduce production stability. Thus, in addition to
reducing the chances of unwanted immunogenicity, it is expected
that the use of the light chain IGKV1-39 without mutations
contributes to improved production stability and yields of various
specificity-contributing V.sub.H genes. Indeed stable clones
generated by the transfection of different V.sub.H genes all paired
with IGKV1-39 are able to be passaged extensively and still retain
robust production characteristics as shown in Table 9 FIG. 28.
Example 21: Generation of Mice Expressing Fully Human VH and VL
Regions
[0199] Transgenic mice described herein are crossed with mice that
already contain a human VH locus. Examples of appropriate mice
comprising a human VH locus are disclosed in Taylor et al. (1992),
Nucleic Acids Res. 20:6287-95; Lonberg et al. (1994), Nature
368:856-9; Green et al. (1994), Nat. Genet. 7:13-21; Dechiara et
al. (2009), Methods Mol. Biol. 530:311-24.).
[0200] After crossing and selecting for mice that are at least
heterozygous for the IGKV1-39 transgene and the human VH locus,
selected mice are immunized with a target. VH genes are harvested
as described hereinabove. This method has the advantage that the VH
genes are already fully human and thus do not require
humanization.
Example 22: Isolation, Characterization, Oligoclonics Formatting
and Production of Antibodies Targeting Human IL6 for Treatment of
Chronic Inflammatory Diseases Such as Rheumatoid Arthritis
[0201] A spleen VH repertoire from transgenic mice that are
immunized with human recombinant IL6 is cloned in a phage display
Fab vector with a single human IGKV1-39-C kappa light chain
(identical to the mouse transgene) and subjected to panning against
the immunogen human IL6. Clones that are obtained after two to four
rounds of panning are analyzed for their binding specificity. VH
genes encoding IL6-specific Fab fragments are subjected to sequence
analysis to identify unique clones and assign VH, DH and JH
utilization. The Fab fragments are reformatted as IgG1 molecules
and transiently expressed. Unique clones are then grouped based on
non-competition in binding assays and subjected to affinity and
functional analysis. The most potent anti-IL6 IgG1 mAbs are
subsequently expressed as combinations of two, three, four or five
heavy chains comprising different VH-regions in the Oligoclonics
format, together with one IGKV1-39-C-based kappa light chain and
tested in vitro for complex formation with IL-6. The Oligoclonics
are also tested in vivo for clearance of human IL-6 from mice. An
Oligoclonic with the most potent clearance activity is chosen and
the murine VH genes humanized according to conventional methods.
The humanized IgG1 are transfected into a mammalian cell line to
generate a stable clone. An optimal subclone is selected for the
generation of a master cell bank and the generation of clinical
trial material.
[0202] Many of the protocols described here are standard protocols
for the construction of phage display libraries and the panning of
phages for binding to an antigen of interest and are described, for
example, in Antibody Phage Display: Methods and Protocols (2002),
Editor(s) Philippa M. O'Brien, Robert Aitken, Humana Press, Totowa,
N.J., USA.
Immunizations
[0203] Transgenic mice receive three immunizations with human IL6
every two weeks using the adjuvant Sigma titerMax according to
manufacturer's instructions.
RNA Isolation and cDNA Synthesis
[0204] Three days after the last immunization, spleens and
lymphnodes from the mice are removed and passed through a 70 micron
filter into a tube containing PBS pH 7.4 to generate a single cell
suspension. After washing and pelleting of lymphocytes, cells are
suspended in TRIzol LS Reagent (Invitrogen) for the isolation of
total RNA according to the manufacturer's protocol and subjected to
reverse transcription reaction using 1 microgram of RNA,
Superscript III RT in combination with dT20 according to
manufacturer's procedures (Invitrogen).
[0205] The generation of Fab phage display libraries is carried out
as described in Example 2.
Selection of Phages on Coated Immunotubes
[0206] Human recombinant IL6 is dissolved in PBS in a concentration
of 5 .mu.g/ml and coated to MAXISORP.TM. Nunc-Immuno Tube (Nunc
444474) overnight at 4.degree. C. After discarding the coating
solution, the tubes are blocked with 2% skim milk (ELK) in PBS
(blocking buffer) for one hour at Room Temperature (RT). In
parallel, 0.5 ml of the phage library is mixed with 1 ml blocking
buffer and incubated for 20 minutes at room temperature. After
blocking the phages, the phage solution is added to the IL6-coated
tubes and incubated for two hours at RT on a slowly rotating
platform to allow binding. Next, the tubes are washed ten times
with PBS/0.05% TWEEN.TM.-20 detergent followed by phage elution by
incubating with 1 ml 50 mM glycine-HCl pH 2.2 ten minutes at RT on
rotating wheel and directly followed by neutralization of the
harvested eluent with 0.5 ml 1 M Tris-HCl pH 7.5.
Harvesting Phage Clones
[0207] A 5 ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 is
added to the harvested phage solution and incubated for 30 minutes
at 37.degree. C. without shaking to allow infection of the phages.
Bacteria are plated on Carbenicillin/Tetracycline 4% glucose 2*TY
plates and grown overnight at 37.degree. C.
Phage Production
[0208] Phages are grown and processed as described by Kramer et al.
2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using
VCSM13 as helper phage strain.
Phage ELISA
[0209] ELISA plates are coated with 100 microliters human
recombinant IL6 per well at a concentration of 2.5 micrograms/ml in
PBS overnight at 4.degree. C. Plates coated with 100 microliters
thyroglobulin at a concentration of 2 micrograms/ml in PBS are used
as a negative control. Wells are emptied, dried by tapping on a
paper towel, filled completely with PBS-4% skimmed milk (ELK) and
incubated for one hour at room temperature to block the wells.
After discarding the block solution, phage minipreps pre-mixed with
50 .mu.l blocking solution are added and incubated for one hour at
RT. Unbound phages are subsequently removed by five washing steps
with PBS-0.05% Tween-20. Bound phages are detected by incubating
the wells with 100 microliters anti-M13-HRP antibody conjugate
(diluted 1/5000 in blocking buffer) for one hour at room
temperature. Free antibody is removed by repeating the washing
steps as described above, followed by TMB substrate incubation
until color development was visible. The reaction is stopped by
adding 100 microliters of 2 M H2SO4 per well and analyzed on an
ELISA reader at 450 nm emission wavelength.
Sequencing
[0210] Clones that give signals at least three times above the
background signal are propagated, used for DNA miniprep procedures
(see procedures Qiagen miniPrep manual) and subjected to nucleotide
sequence analysis. Sequencing is performed according to the Big Dye
1.1 kit accompanying manual (Applied Biosystems) using a reverse
primer (CH1_Rev1, Table 1) recognizing a 5' sequence of the CH1
region of the human IgG1 heavy chain (present in the Fab display
vector MV1043, FIGS. 3 and 12). The sequences of the murine VH
regions are analyzed for diversity of DH and JH gene segments.
Construction and Expression of Chimeric IgG1
[0211] Vector MV1057 (FIGS. 12 and 22) was generated by cloning the
transgene (IGKV1-39) L chain fragment into a derivative of vector
pcDNA3000Neo (Crucell, Leiden, The Netherlands) that contains the
human IgG1- and kappa constant regions. VH regions are cloned into
MV1057 and nucleotide sequences for all constructs are verified
according to standard techniques. The resulting constructs are
transiently expressed in HEK293T cells and supernatants containing
chimeric IgG1 are obtained and purified using standard procedures
as described before (M. Throsby 2006, J. Virol. 80:6982-92).
IgG1 Binding and Competition Analysis
[0212] IgG1 antibodies are titrated in ELISA using IL6-coated
plates as described above and an anti-human IgG peroxidase
conjugate. Competition ELISAs to group antibodies based on epitope
recognition are performed by incubating Fab phages together with
IgG1 or with commercial antibodies against IL6 (e.g., Abcam cat.
no. ab9324) in IL6-coated plates, followed by detection of bound
Fab phage using an anti-M13 peroxidase conjugate.
IgG1 Affinity Measurements
[0213] The affinities of the antibodies to IL6 are determined with
the Quantitative kinetic protocol on the Octet (ForteBio).
Antibodies are captured onto an Anti-Human IgG Fc Capture biosensor
and exposed to free IL6 and analyzed using proprietary software to
calculate the Kd of each antibody.
Functional Activity of IL6 Antibodies
[0214] To test the ability of the selected antibodies to inhibit
binding between IL6 and IL6 receptor (IL6R), an ELISA based assay
is used. Various concentrations of antibody are mixed with a fixed
concentration (10 ng/ml) of biotinylated IL6 as described by Naoko
et al. 2007, Can. Res. 67:817-875. The IL6-antibody immune complex
is added to immobilized IL6R. The binding of biotinylated IL6 to
IL6R is detected with horseradish peroxidase-conjugated
streptavidin. The reduction of ELISA signal is a measurement of
inhibition. As positive control for inhibition of binding between
IL6 and IL6R either anti-IL6R antibody (Abcam cat. no. ab34351;
clone B-R6) or anti IL6 antibody (Abcam cat. no. ab9324) is
used.
[0215] In vitro blocking activity of the selected anti-IL6
antibodies is measured in a proliferation assay using the
IL6-dependent cell line 7TD1. Briefly, cells are incubated with
different concentrations of human IL6 with or without the anti-IL6
antibody. The available amount of IL6 determines the degree of
proliferation. Thus if an added antibody blocks IL6 binding the
proliferation readout is reduced compared to a non binding antibody
control. Proliferation is measured by the incorporation of
5-bromo-2'-deoxy-uridine (BrdU) into the DNA using the BrdU
proliferation kit (Roche cat. no. 11444611001) according to the
manufacturer's instructions.
Generation of Anti-IL6 Oligoclonics
[0216] The most potent anti-IL6 antibodies are selected from each
epitope group. The expression constructs expressing these
antibodies are transfected into HEK293T cells in non-competing
groups of three in different ratios (1:1:1; 3:1:1; 1:3:1; 1:1:3;
3:3:1; 1:3:3; 3:1:3; 10:1:1; 1:10:1; 1:1:10; 10:10:1; 1:10:10;
10:1:10; 3:10:1; 10:3:1; 1:10:3; 3:1:10; 10:1:3; 1:3:10). Antibody
containing supernatants are harvested and purified and
characterized as above.
Complex Formation and In Vivo Clearance of Anti-IL6
Oligoclonics
[0217] To measure the ability of anti-IL6 Oligoclonics to form
immune complexes and to analyze these complexes Size Exclusion
Chromatography (SEC) is used according to the approach disclosed by
Min-Soo Kim et al. (2007), JMB 374:1374-1388, to characterize the
immune-complexes formed with different antibodies to TNF.alpha..
Different molar ratios of the anti-IL6 Oligoclonics are mixed with
human IL6 and incubated for 20 hours at 4.degree. C. or 25.degree.
C. The mixture is analyzed on an HPLC system fitted with a size
exclusion column; different elution times are correlated to
molecular weight using a molecular weight standards.
[0218] The ability of antibodies to form complexes with IL6 is
correlated with their ability to rapidly clear the cytokine from
the circulation in vivo. This is confirmed by measuring the
clearance of radiolabelled IL6 from mice. Briefly, female, six- to
eight-week-old Balb/c mice are obtained and 18 hours before the
experiment, the animals are injected intravenously (IV) via the
lateral tail vein with different doses of purified anti-IL6
Oligoclonics. On day 0, the mice are injected IV with 50
microliters of radiolabeled IL-6 (1.times.10E7 cpm/mL) under the
same conditions. Blood samples (approximately 50 microliters) are
collected at several time intervals and stored at 4.degree. C. The
samples are centrifuged for five minutes at 4000.times.g and the
radioactivity of the serum determined. All pharmacokinetic
experiments are performed simultaneously with three animals for
each treatment.
Generation of Anti-IL6 Oligoclonics Stable Clones and Preclinical
Development
[0219] A lead anti-IL6 Oligoclonic is selected based on the in
vitro and in vivo potency as determined above. The murine VH genes
are humanized according to standard methods and combined with the
fully human IGKV1-39 light chain in an expression vector as
described above. Examples of humanization methods include those
based on paradigms such as resurfacing (E. A. Padlan et al. (1991),
Mol. Immunol. 28:489), superhumanization (P. Tan, D. A., et al.
(2002), J. Immunol. 169:1119) and human string content optimization
(G. A. Lazar et al. (2007), Mol. Immunol. 44:1986). The three
constructs are transfected into PER.C6 cells at the predetermined
optimal ratio (described above) under the selective pressure of
G418 according to standard methods. A stable high producing
anti-IL6 Oligoclonic clone is selected and a working and qualified
master cell bank generated.
TABLE-US-00002 TABLE 1 List of primers DO- Primer Sequence 0012
CH1_Rev1 TGCCAGGGGGAAGACCGATG (SEQ ID NO: 4) 0656 MVH-1
GCCGGCCATGGCCGAGGTRMAGCTTCAGGAGTCAGGAC (SEQ ID NO: 5) 0657 MVH-2
GCCGGCCATGGCCGAGGTSCAGCTKCAGCAGTCAGGAC (SEQ ID NO: 6) 0658 MVH-3
GCCGGCCATGGCCCAGGTGCAGCTGAAGSASTCAGG (SEQ ID NO: 7) 0659 MVH-4
GCCGGCCATGGCCGAGGTGCAGCTTCAGGAGTCSGGAC (SEQ ID NO: 8) 0660 MVH-5
GCCGGCCATGGCCGARGTCCAGCTGCAACAGTCYGGAC (SEQ ID NO: 9) 0661 MVH-6
GCCGGCCATGGCCCAGGTCCAGCTKCAGCAATCTGG (SEQ ID NO: 10) 0662 MVH-7
GCCGGCCATGGCCCAGSTBCAGCTGCAGCAGTCTGG (SEQ ID NO: 11) 0663 MVH-8
GCCGGCCATGGCCCAGGTYCAGCTGCAGCAGTCTGGRC (SEQ ID NO: 12) 0664 MVH-9
GCCGGCCATGGCCCAGGTYCAGCTYCAGCAGTCTGG (SEQ ID NO: 13) 0665 MVH-10
GCCGGCCATGGCCGAGGTCCARCTGCAACAATCTGGACC (SEQ ID NO: 14) 0666 MVH-11
GCCGGCCATGGCCCAGGTCCACGTGAAGCAGTCTGGG (SEQ ID NO: 15) 0667 MVH-12
GCCGGCCATGGCCGAGGTGAASSTGGTGGAATCTG (SEQ ID NO: 16) 0668 MVH-13
GCCGGCCATGGCCGAVGTGAAGYTGGTGGAGTCTG (SEQ ID NO: 17) 0669 MVH-14
GCCGGCCATGGCCGAGGTGCAGSKGGTGGAGTCTGGGG (SEQ ID NO: 18) 0670 MVH-15
GCCGGCCATGGCCGAKGTGCAMCTGGTGGAGTCTGGG (SEQ ID NO: 19) 0671 MVH-16
GCCGGCCATGGCCGAGGTGAAGCTGATGGARTCTGG (SEQ ID NO: 20) 0672 MVH-17
GCCGGCCATGGCCGAGGTGCARCTTGTTGAGTCTGGTG (SEQ ID NO: 21) 0673 MVH-18
GCCGGCCATGGCCGARGTRAAGCTTCTCGAGTCTGGA (SEQ ID NO: 22) 0674 MVH-19
GCCGGCCATGGCCGAAGTGAARSTTGAGGAGTCTGG (SEQ ID NO: 23) 0675 MVH-20
GCCGGCCATGGCCGAAGTGATGCTGGTGGAGTCTGGG (SEQ ID NO: 24) 0676 MVH-21
GCCGGCCATGGCCCAGGTTACTCTRAAAGWGTSTGGCC (SEQ ID NO: 25) 0677 MVH-22
GCCGGCCATGGCCCAGGTCCAACTVCAGCARCCTGG (SEQ ID NO: 26) 0678 MVH-23
GCCGGCCATGGCCCAGGTYCARCTGCAGCAGTCTG (SEQ ID NO: 27) 0679 MVH-24
GCCGGCCATGGCCGATGTGAACTTGGAAGTGTCTGG (SEQ ID NO: 28) 0680 MVH-25
GCCGGCCATGGCCGAGGTGAAGGTCATCGAGTCTGG (SEQ ID NO: 29) 0681 ExtMVH-1
CAGTCACAGATCCTCGCGAATTGGCCCA ATGGCCSANG (SEQ ID NO: 30) 0682
ExtMVH-2 CAGTCACAGATCCTCGCGAATTGGCCCA ATGGCCSANC (SEQ ID NO: 31)
0683 MJH-Revl GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 32) 0684
MJH-Rev2 GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 33) 0685
MJH-Rev3 GGGGGTGTCGTTTTGGCTGCAGAGAC AGAG (SEQ ID NO: 34) 0686
MJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC GAGG (SEQ ID NO:3 5) 0687
ExtMJH-Rev1& GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 36)
0688 ExtMJH-Rev2in GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 37)
0690 ExtMJH-Rev3 GGGGGTGTCGTTTTGGCTGAGGAGAC AGAG (SEQ ID NO: 38)
0691 ExtMJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC GAGG (SEQ ID NO:
39)
TABLE-US-00003 TABLE 2 Phage ELISA signal levels as measured at 450
nm. TT-coated plates represent plates that were coated with tetanus
toxoid. Thyroglobulin-coated plates are used as negative controls.
10/10 and 15/15 indicate the number of wash steps with PBS-Tween
during panning procedures. The 10/10 tetanus toxoid and 10/10
thyroglobulin plates and the 15/15 tetanus toxoid and 15/15
thyroglobulin plates are duplicates from each other except for the
coating agent. OD values higher than three times the background are
assumed specific. 1 2 3 4 5 6 7 8 9 10 11 12 TT-coated plate 10/10
washings A 0.139 0.093 0.089 0.121 0.117 0.598 0.146 0.115 0.18
0.155 0.543 0.601 B 0.136 0.404 0.159 0.187 0.489 0.134 0.216 0.092
0.222 0.108 0.181 0.484 C 0.197 0.526 0.09 0.213 0.395 0.155 0.108
0.12 0.183 0.136 0.092 0.866 D 0.143 0.258 0.101 0.422 0.088 0.243
0.485 0.251 0.304 0.198 0.478 0.091 E 0.445 0.169 0.526 0.481 0.206
0.285 0.111 0.119 0.128 0.2 0.118 0.098 F 0.237 0.291 0.594 0.139
0.206 0.565 0.543 0.091 0.136 0.227 0.228 0.099 G 0.459 0.102 0.152
0.659 0.203 0.452 0.152 0.133 0.094 0.102 0.375 0.098 H 0.341 0.623
0.745 0.415 0.682 0.527 0.655 0.114 0.258 0.284 0.685 0.113
TT-coated plate 15/15 washings A 0.247 0.582 0.421 0.428 0.133
0.082 0.262 0.079 0.343 0.414 0.095 0.292 B 0.065 0.364 0.073 0.042
0.049 0.071 0.046 0.103 0.078 0.057 0.048 0.155 C 0.081 0.044 0.066
0.082 0.225 0.444 0.203 0.362 0.122 0.047 0.052 0.309 D 0.092 0.11
0.59 0.22 0.33 0.544 0.058 0.159 0.047 0.174 0.086 0.05 E 0.469
0.577 0.206 0.304 0.13 0.749 0.431 0.062 0.167 0.049 0.056 0.049 F
0.846 0.07 0.561 0.656 0.882 0.094 0.383 0.13 0.152 0.098 0.134
0.048 G 0.537 0.052 0.49 0.105 0.337 0.193 0.514 0.294 0.068 0.35
0.525 0.05 H 0.061 0.306 0.157 0.853 0.054 0.534 0.102 0.235 0.441
0.412 0.565 0.061 Thyroglobulin-coated plate 10/10 washings A 0.047
0.051 0.045 0.043 0.051 0.044 0.046 0.042 0.047 0.048 0.049 0.05 B
0.042 0.042 0.042 0.042 0.043 0.041 0.041 0.042 0.043 0.045 0.042
0.046 C 0.044 0.043 0.043 0.044 0.043 0.044 0.043 0.042 0.043 0.041
0.044 0.046 D 0.045 0.044 0.044 0.044 0.045 0.046 0.045 0.056 0.045
0.049 0.048 0.73 E 0.046 0.045 0.046 0.044 0.045 0.044 0.044 0.044
0.047 0.046 0.047 0.926 F 0.048 0.045 0.044 0.046 0.044 0.043 0.044
0.046 0.046 0.046 0.046 0.792 G 0.051 0.048 0.045 0.045 0.044 0.043
0.048 0.045 0.048 0.051 0.045 0.053 H 0.064 0.05 0.049 0.047 0.05
0.051 0.047 0.046 0.047 0.047 0.047 0.056 Thyroglobulin-coated
plate 15/15 washings A 0.036 0.049 0.045 0.044 0.046 0.047 0.046
0.042 0.042 0.043 0.042 0.041 B 0.045 0.042 0.041 0.043 0.043 0.043
0.045 0.045 0.047 0.048 0.044 0.045 C 0.049 0.047 0.047 0.046 0.046
0.046 0.045 0.047 0.046 0.045 0.045 0.052 D 0.047 0.049 0.048 0.048
0.048 0.048 0.047 0.052 0.048 0.046 0.048 0.456 E 0.049 0.047 0.047
0.047 0.047 0.049 0.047 0.048 0.047 0.046 0.048 0.412 F 0.05 0.047
0.046 0.046 0.046 0.046 0.046 0.046 0.046 0.047 0.048 0.528 G 0.05
0.048 0.045 0.045 0.046 0.049 0.048 0.046 0.053 0.049 0.05 0.057 H
0.057 0.05 0.046 0.045 0.047 0.049 0.047 0.047 0.046 0.047 0.053
0.048
TABLE-US-00004 TABLE 3 Protein sequence analysis of ELISA positive
tetanus toxoid binders. CDR3 V Gene CDR3/SEQ ID NO: length VH DH JH
family HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3
mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11
JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192
DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15
musIGHV192 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40)
15 musIGHV192 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO:
40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID
NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ
ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY
(SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183
HGAFYTYDEKPWFAY (SEQ ID NO: 41) 15 musIGHV192 IGHD2- JH3 mouse
VH7183 14*01 HISYYRYDEEVSFAY (SEQ ID NO: 42) 15 musIGHV192 IGHD2-
JH3 mouse VH7183 14*01 HISYYRYDEEVSFAY (SEQ ID NO: 42) 15
musIGHV192 IGHD2- JH3 mouse VH7183 14*01 GWRAFAY (SEQ ID NO: 43) 7
musIGHV131 DSP2.9 JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43) 7
musIGHV131 DSP2.9 JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43) 7
musIGHV131 DSP2.9 JH3 mouse VH7183 DRGNYYGMDY (SEQ ID NO: 44) 10
musIGHV178 DSP2.1 JH4 mouse VH7183 LGDYYVDWFFAV (SEQ ID NO: 45) 12
musIGHV165 DFL16.1 JH1 mouse VH7183 NFPAWFAF (SEQ ID NO: 46) 8
musIGHV547 DST4.3inv JH3 mouse VJH558 NFPAWFAY (SEQ ID NO: 46) 8
musIGHV547 DSP2.1 JH3 mouse VJH558 NFPAWFVY (SEQ ID NO: 46) 8
musIGHV547 DSP2.1 JH3 mouse VJH558 SFTPVPFYYGYDWYFDV (SEQ ID NO:
47) 17 musIGHV532 DSP2.3 JH1 mouse VJH558 SFTPVPFYYGYDWYFDV (SEQ ID
NO: 47) 17 musIGHV532 DSP2.3 JH1 mouse VJH558 SDYDWYFDV (SEQ ID NO:
48) 9 musIGHV286 DSP2.2 JH1 mouse VJH558 SDYDWYFDV (SEQ ID NO: 48)
9 musIGHV286 DSP2.2 JH1 mouse VJH558 DSKWAYYFDY (SEQ ID NO: 49) 10
musIGHV532 DST4.3 JH2 mouse VJH558 GDYTGYGMDY (SEQ ID NO: 50) 10
musIGHV125 DSP2.13 JH4 mouse VHSM7 GDYTGYGMDY (SEQ ID NO: 50) 10
musIGHV125 DSP2.13 JH4 mouse VHSM7 GGYDGYWFPY (SEQ ID NO: 51) 10
musIGHV125 DSP2.9 JH3 mouse VHSM7 CDR3 sequence, CDR3 length, VH
family members and specific name, JH origin and DH origin of the
clones is indicated.
TABLE-US-00005 TABLE 4 Vector combinations that were transfected to
HEK293T. Combined Conc. Code HC vector LC vector vector Prep name
(.mu.g/ml) A x 0817676_pSELECT_0815426 x PIGKV1-39/ -- (IGKV1-39)
P1 B x 0817678_pSELECT_0815427 x PIGLV2-14/ -- (IGLV2-14) P1 C
MV1110 0817676_pSELECT_0815426 x PMV1110/ 11.0 (IGKV1-39)
IGKV1-39/P1 D MV1110 0817678_pSELECT_0815427 x PMV1110/ 15.4
(IGLV2-14) IGLV2-14/P1 E x x MG1494 MG1494/P2 16.1
TABLE-US-00006 TABLE 5 HLA allotypes considered in T.sub.H-epitope
profiling. The corresponding serotypes are shown, as well as
allotype frequencies in the Caucasian population (Klitz et al.
(2003), Tissue Antigens 62: 296-307; Gjertson and Terasake (eds)
in: HLA 1997; Gjertson and Terasake (eds) in: HLA 1998; Castelli et
al. (2002), J. Immunol. 169: 6928-6934). Frequencies can add up to
more than 100% since each individual has two alleles for each gene.
If all allele frequencies of a single gene were known, they would
add up to slightly less than 200% due to homozygous individuals.
HLA type Serotype Population % DRB1*0101 DR1 17.4 DRB1*0102 DR1 4.9
DRB1*0301 DR17(3) 21.2 DRB1*0401 DR4 11.5 DRB1*0402 DR4 3.1
DRB1*0404 DR4 5.5 DRB1*0405 DR4 2.2 DRB1*0407 DR4 <2 DRB1*0701
DR7 23.4 DRB1*0801 DR8 3.3 DRB1*0802 DR8 <2 DRB1*0901 DR9 <2
DRB1*1101 DR11(5) 17 DRB1*1104 DR11(5) 5.7 DRB1*1201 DR12(5) 3.1
DRB1*1301 DR13(6) 15.4 DRB1*1302 DR13(6) 10.8 DRB1*1401 DR14(6) 4.2
DRB1*1501 DR15(2) 13.2 DRB1*1601 DR16(2) 5.5 DRB3*0101 DR52 24.6
DRB3*0202 DR52 43 DRB3*0301 DR52 10 DRB4*0101 DR53 25.5 DRB4*0103
DR53 21 DRB5*0101 DR51 15.8 DRB5*0202 DR51 5.7 DQA1*0101/DQB1*0501
DQ5(1) 20.5 DQA1*0102/DQB1*0502 DQ5(1) 2.6 DQA1*0102/DQB1*0602
DQ6(1) 26.5 DQA1*0102/DQB1*0604 DQ6(1) 6.7 DQA1*0103/DQB1*0603
DQ6(1) 11 DQA1*0104/DQB1*0503 DQ5(1) 4 DQA1*0201/DQB1*0202 DQ2 20.9
DQA1*0201/DQB1*0303 DQ9(3) 7.2 DQA1*0301/DQB1*0301 DQ7(3) 12.5
DQA1*0301/DQB1*0302 DQ8(3) 18.3 DQA1*0401/DQB1*0402 DQ4 4.5
DQA1*0501/DQB1*0201 DQ2 24.6 DQA1*0501/DQB1*0301 DQ7(3) 20.9
DPA1*0103/DPB1*0201 DPw2 19.9 DPA1*0103/DPB1*0401 DPw4 65.1
DPA1*0103/DPB1*0402 DPw4 24.3 DPA1*0201/DPB1*0101 DPw1 6.3
DPA1*0201/DPB1*0301 DPw3 <2 DPA1*0201/DPB1*0501 DPw5 <2
DPA1*0201/DPB1*0901 -- 2.4
TABLE-US-00007 TABLE 6 T.sub.H epitope counts for IGKV1-39.
Peptides binding to multiple HLAs of the same group (DRB1,
DRB3/4/5, DP, DQ) are counted as one. Values between brackets refer
to germline-filtered peptides. DRB1 DRB3/4/5 DQ DP Strong Medium
Strong Medium Strong Medium Strong Medium Merus IGKV1-39 0 (+6) 0
(+16) 0 (+0) 0 (+5) 0 (+3) 0 (+9) 0 (+0) 0 (+9)
TABLE-US-00008 TABLE 7 Mapping of Epibase .RTM. predictions for
Merus IGKV1-39 in the classical 15-mer peptide format. Start
Allotype 15 mer Position 15-mer sequence count Implicated serotypes
1 1 DIQMTQSPSSLSASV 6 DR1, DR4, DR7, DR9 2 4 MTQSPSSLSASVGDR 5 DR1,
DR4, DR9 3 7 SPSSLSASVGDRVTI 0 4 10 SLSASVGDRVTITCR 0 5 13
ASVGDRVTITCRASQ 0 6 16 GDRVTITCRASQSIS 2 DR11(5), DR7 7 19
VTITCRASQSISSYL 4 DQ2, DR11(5), DR4, DR7 8 22 TCRASQSISSYLNWY 2
DQ2, DR4 9 25 ASQSISSYLNWYQQK 5 DR13(6), DR15(2), DR4 10 28
SISSYLNWYQQKPGK 8 DR12(5), DR13(6), DR15(2), DR16(2), DR4, DR8 11
31 SYLNWYQQKPGKAPK 10 DR1, DR12(5), DR16(2), DR4, DR51, DR8 12 34
NWYQQKPGKAPKLLI 9 DR1, DR15(2), DR4, DR51, DR8 13 37
QQKPGKAPKLLIYAA 7 DQ4, DR1, DR11(5), DR15(2), DR51, DR8 14 40
PGKAPKLLIYAASSL 7 DQ4, DR1, DR11(5), DR4, DR8 15 43 APKLLIYAASSLQSG
15 DR1, DR11(5), DR12(5), DR13(6), DR14(6), DR15(2), DR4, DR51,
DR8, DR9 16 46 LLIYAASSLQSGVPS 15 DR1, DR11(5), DR12(5), DR13(6),
DR14(6), DR15(2), DR4, DR51, DR8, DR9 17 49 YAASSLQSGVPSRFS 1
DR15(2) 18 52 SSLQSGVPSRFSGSG 1 DR15(2) 19 55 QSGVPSRFSGSGSGT 0 20
58 VPSRFSGSGSGTDFT 0 21 61 RFSGSGSGTDFTLTI 0 22 64 GSGSGTDFTLTISSL
1 DR52 23 67 SGTDFTLTISSLQPE 4 DR4, DR52, DR7, DR9 24 70
DFTLTISSLQPEDFA 4 DQ2, DR4, DR7, DR9 25 73 LTISSLQPEDFATYY 1 DQ2 26
76 SSLQPEDFATYYCQQ 0 27 79 QPEDFATYYCQQSYS 1 DR4 28 82
DFATYYCQQSYSTPP 5 DR4, DR51, DR7 29 85 TYYCQQSYSTPPTFG 4 DR4, DR51,
DR7 30 88 CQQSYSTPPTFGQGT 0 31 91 SYSTPPTFGQGTKVE 0 32 94
TPPTFGQGTKVEIK 0 This table shows the allotype count of critical
epitopes (SEQ ID NOs: 52-83) and implicated serotypes for each of
the 15-mers spanning the Merus IGKV1-39 sequence.
TABLE-US-00009 TABLE 8 The V.sub.H gene from PG1433 paired with
various light chain genes with differing rates of amino acid
mutation were compared for production levels with the original
clone containing the IGKV1-39 gene. Light Number of amino acid
concentration IgG name chain gene mutations (.mu.g/ml) PG1433 1-39
0 63, 45.5, 38.6 (avg = 49) PG1631 1-12 4 10.5 PG1632 1-27 7 9.3
PG1634 1D-12 10 10.8 PG1635 1D-33 6 10.2 PG1642 1-5 8 7.1 PG1644
1-9 3 7.8 PG1650 1D-39 3 9.1 PG1652 2D-28 3 7.1 PG1653 3-15 14 7
PG1654 3-20 2 5.2 PG1674 1-40 7 8.2 PG1678 2-11 2 8.1 PG1680 2-14
15 10.8 PG1682 3-1 13 9.9 PG1683 6-57 6 13.9
TABLE-US-00010 TABLE 9 Numbers of lymphocytes harvested from the
bone marrow and spleen of wild-type and transgenic mice *10e6/ml
total vol total cells cells (ml) *10.sup.6 Bone Marrow Wt 18.82
5.05 95.0 Wt 19.24 4.96 95.4 CD19-Cre 23.42 5.08 119.0 CD19-Cre
20.58 4.82 99.2 CD19-Cre 25.77 5.15 132.7 CD19-Cre/HuVk1 17.71 5.06
89.6 CD19-Cre/HuVk1 12.60 5.33 67.2 CD19-Cre/HuVk1 18.13 5.27 95.5
Spleen Wt 41.70 5.36 223.5 Wt 37.85 4.71 178.3 CD19-Cre 60.19 3.77
226.9 CD19-Cre 35.06 3.66 128.3 CD19-Cre 80.69 4.60 371.2
CD19-Cre/HuVk1 51.67 4.48 231.5 CD19-Cre/HuVk1 58.80 6.24 366.9
CD19-Cre/HuVk1 24.37 6.25 152.3
Sequence CWU 1
1
102120DNAArtificial Sequenceprimer 1ccctttccaa tctttatggg
20223DNAArtificial Sequenceprimer 2aggtggattg gtgtcttttt ctc
23322DNAArtificial Sequenceprimer 3gtcatgtcgg cgaccctacg cc
22420DNAArtificial Sequenceprimer 4tgccaggggg aagaccgatg
20538DNAArtificial Sequenceprimer 5gccggccatg gccgaggtrm agcttcagga
gtcaggac 38638DNAArtificial Sequenceprimer 6gccggccatg gccgaggtsc
agctkcagca gtcaggac 38736DNAArtificial Sequenceprimer 7gccggccatg
gcccaggtgc agctgaagsa stcagg 36838DNAArtificial Sequenceprimer
8gccggccatg gccgaggtgc agcttcagga gtcsggac 38938DNAArtificial
Sequenceprimer 9gccggccatg gccgargtcc agctgcaaca gtcyggac
381036DNAArtificial Sequenceprimer 10gccggccatg gcccaggtcc
agctkcagca atctgg 361136DNAArtificial Sequenceprimer 11gccggccatg
gcccagstbc agctgcagca gtctgg 361238DNAArtificial Sequenceprimer
12gccggccatg gcccaggtyc agctgcagca gtctggrc 381336DNAArtificial
Sequenceprimer 13gccggccatg gcccaggtyc agctycagca gtctgg
361439DNAArtificial Sequenceprimer 14gccggccatg gccgaggtcc
arctgcaaca atctggacc 391537DNAArtificial Sequenceprimer
15gccggccatg gcccaggtcc acgtgaagca gtctggg 371635DNAArtificial
Sequenceprimer 16gccggccatg gccgaggtga asstggtgga atctg
351735DNAArtificial Sequenceprimer 17gccggccatg gccgavgtga
agytggtgga gtctg 351838DNAArtificial Sequenceprimer 18gccggccatg
gccgaggtgc agskggtgga gtctgggg 381937DNAArtificial Sequenceprimer
19gccggccatg gccgakgtgc amctggtgga gtctggg 372036DNAArtificial
Sequenceprimer 20gccggccatg gccgaggtga agctgatgga rtctgg
362138DNAArtificial Sequenceprimer 21gccggccatg gccgaggtgc
arcttgttga gtctggtg 382237DNAArtificial Sequenceprimer 22gccggccatg
gccgargtra agcttctcga gtctgga 372336DNAArtificial Sequenceprimer
23gccggccatg gccgaagtga arsttgagga gtctgg 362437DNAArtificial
Sequenceprimer 24gccggccatg gccgaagtga tgctggtgga gtctggg
372538DNAArtificial Sequenceprimer 25gccggccatg gcccaggtta
ctctraaagw gtstggcc 382636DNAArtificial Sequenceprimer 26gccggccatg
gcccaggtcc aactvcagca rcctgg 362735DNAArtificial Sequenceprimer
27gccggccatg gcccaggtyc arctgcagca gtctg 352836DNAArtificial
Sequenceprimer 28gccggccatg gccgatgtga acttggaagt gtctgg
362936DNAArtificial Sequenceprimer 29gccggccatg gccgaggtga
aggtcatcga gtctgg 363045DNAArtificial
Sequenceprimermisc_feature(44)..(44)n is a, c, g, or t 30cagtcacaga
tcctcgcgaa ttggcccagc cggccatggc csang 453145DNAArtificial
Sequenceprimermisc_feature(44)..(44)n is a, c, g, or t 31cagtcacaga
tcctcgcgaa ttggcccagc cggccatggc csanc 453237DNAArtificial
Sequenceprimer 32gggggtgtcg ttttggctga ggagacggtg accgtgg
373337DNAArtificial Sequenceprimer 33gggggtgtcg ttttggctga
ggagactgtg agagtgg 373437DNAArtificial Sequenceprimer 34gggggtgtcg
ttttggctgc agagacagtg accagag 373537DNAArtificial Sequenceprimer
35gggggtgtcg ttttggctga ggagacggtg actgagg 373637DNAArtificial
Sequenceprimer 36gggggtgtcg ttttggctga ggagacggtg accgtgg
373737DNAArtificial Sequenceprimer 37gggggtgtcg ttttggctga
ggagacggtg acagtgg 373837DNAArtificial Sequenceprimer 38gggggtgtcg
ttttggctga ggagacggtg accagag 373937DNAArtificial Sequenceprimer
39gggggtgtcg ttttggctga ggagacggtg accgagg 374015PRTArtificial
SequenceCDR3 40His Gly Ala Tyr Tyr Thr Tyr Asp Glu Lys Ala Trp Phe
Ala Tyr 1 5 10 15 4115PRTArtificial SequenceCDR3 41His Gly Ala Phe
Tyr Thr Tyr Asp Glu Lys Pro Trp Phe Ala Tyr 1 5 10 15
4215PRTArtificial SequenceCDR3 42His Ile Ser Tyr Tyr Arg Tyr Asp
Glu Glu Val Ser Phe Ala Tyr 1 5 10 15 437PRTArtificial SequenceCDR3
43Gly Trp Arg Ala Phe Ala Tyr 1 5 4410PRTArtificial SequenceCDR3
44Asp Arg Gly Asn Tyr Tyr Gly Met Asp Tyr 1 5 10 4512PRTArtificial
SequenceCDR3 45Leu Gly Asp Tyr Tyr Val Asp Trp Phe Phe Ala Val 1 5
10 468PRTArtificial SequenceCDR3 46Asn Phe Pro Ala Trp Phe Ala Phe
1 5 4717PRTArtificial SequenceCDR3 47Ser Phe Thr Pro Val Pro Phe
Tyr Tyr Gly Tyr Asp Trp Tyr Phe Asp 1 5 10 15 Val 489PRTArtificial
SequenceCDR3 48Ser Asp Tyr Asp Trp Tyr Phe Asp Val 1 5
4910PRTArtificial SequenceCDR3 49Asp Ser Lys Trp Ala Tyr Tyr Phe
Asp Tyr 1 5 10 5010PRTArtificial SequenceCDR3 50Gly Asp Tyr Thr Gly
Tyr Gly Met Asp Tyr 1 5 10 5110PRTArtificial SequenceCDR3 51Gly Gly
Tyr Asp Gly Tyr Trp Phe Pro Tyr 1 5 10 5215PRTArtificial
Sequenceepitope IGKV1-39 52Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val 1 5 10 15 5315PRTArtificial Sequenceepitope
IGKV1-39 53Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
Arg 1 5 10 15 5415PRTArtificial Sequenceepitope IGKV1-39 54Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 1 5 10 15
5515PRTArtificial Sequenceepitope IGKV1-39 55Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg 1 5 10 15 5615PRTArtificial
SequenceEpitope IGKV1-39 56Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln 1 5 10 15 5715PRTArtificial SequenceEpitope
IGKV1-39 57Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser 1 5 10 15 5815PRTArtificial SequenceEpitope IGKV1-39 58Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 1 5 10 15
5915PRTArtificial SequenceEpitope IGKV1-39 59Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 1 5 10 15 6015PRTArtificial
SequenceEpitope IGKV1-39 60Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
Trp Tyr Gln Gln Lys 1 5 10 15 6115PRTArtificial SequenceEpitope
IGKV1-39 61Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys 1 5 10 15 6215PRTArtificial SequenceEpitope IGKV1-39 62Ser Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 1 5 10 15
6315PRTArtificial SequenceEpitope IGKV1-39 63Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 6415PRTArtificial
SequenceEpitope IGKV1-39 64Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr Ala Ala 1 5 10 15 6515PRTArtificial SequenceEpitope
IGKV1-39 65Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
Leu 1 5 10 15 6615PRTArtificial SequenceEpitope IGKV1-39 66Ala Pro
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly 1 5 10 15
6715PRTArtificial SequenceEpitope IGKV1-39 67Leu Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 1 5 10 15 6815PRTArtificial
SequenceEpitope IGKV1-39 68Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser 1 5 10 15 6915PRTArtificial SequenceEpitope
IGKV1-39 69Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly 1 5 10 15 7015PRTArtificial SequenceEpitope IGKV1-39 70Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 1 5 10 15
7115PRTArtificial SequenceEpitope IGKV1-39 71Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 7215PRTArtificial
SequenceEpitope IGKV1-39 72Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile 1 5 10 15 7315PRTArtificial SequenceEpitope
IGKV1-39 73Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu 1 5 10 15 7415PRTArtificial SequenceEpitope IGKV1-39 74Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 1 5 10 15
7515PRTArtificial SequenceEpitope IGKV1-39 75Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 1 5 10 15 7615PRTArtificial
SequenceEpitope IGKV1-39 76Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr 1 5 10 15 7715PRTArtificial SequenceEpitope
IGKV1-39 77Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln 1 5 10 15 7815PRTArtificial SequenceEpitope IGKV1-39 78Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser 1 5 10 15
7915PRTArtificial SequenceEpitope IGKV1-39 79Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 1 5 10 15 8015PRTArtificial
SequenceEpitope IGKV1-39 80Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr
Pro Pro Thr Phe Gly 1 5 10 15 8115PRTArtificial SequenceEpitope
IGKV1-39 81Cys Gln Gln Ser Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly
Thr 1 5 10 15 8215PRTArtificial SequenceEpitope IGKV1-39 82Ser Tyr
Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 1 5 10 15
8314PRTArtificial SequenceEpitope IGKV1-39 83Thr Pro Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 84321DNAHomo
sapiensCDS(1)..(321) 84gac atc cag atg acc cag agc ccc agc agc ctg
agc gcc agc gtg ggc 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 gac aga gtg acc atc acc tgc aga gcc
agc cag agc atc agc agc tac 96Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30 ctg aac tgg tat cag cag aag
ccc ggc aag gcc ccc aag ctg ctg atc 144Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 tac gcc gcc agc tcc
ctg cag agc ggc gtg ccc agc aga ttc agc ggc 192Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agc ggc tcc
ggc acc gac ttc acc ctg acc atc agc agc ctg cag ccc 240Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gag
gac ttc gcc acc tac tac tgc cag cag agc tac agc acc ccc ccc 288Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90
95 acc ttc ggc cag ggc acc aag gtg gag atc aag 321Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 85107PRTHomo sapiens 85Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 86330DNAHomo sapiensCDS(1)..(330) 86cag tct
gcc ctg acc cag ccc gcc tct gtg tct ggc agc cct ggc cag 48Gln Ser
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15
agc atc acc atc agc tgc acc ggc acc agc agc gac gtg ggc ggc tac
96Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30 aac tac gtg tcc tgg tat cag cag cac ccc ggc aag gcc ccc
aag ctg 144Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 atg atc tac gag gtg tcc aac aga ccc agc ggc gtg
agc aac aga ttc 192Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 agc ggc agc aag agc ggc aac acc gcc agc
ctg acc atc agc ggc ctc 240Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 cag gct gag gac gag gcc gac tac
tac tgc agc agc tac acc agc agc 288Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 tcc acc ctg gtg ttt ggc
ggc gga aca aag ctg acc gtg ctg 330Ser Thr Leu Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 110 87110PRTHomo sapiens 87Gln Ser
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20
25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys
Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu
Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Leu Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 110 88321DNARattus
norvegicusCDS(1)..(321) 88aga gcc gac gcc gct ccc acc gtg tcc atc
ttc ccc ccc agc atg gaa 48Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Met Glu 1 5 10 15 cag ctg acc tct ggc gga gcc acc
gtg gtc tgc ttc gtg aac aac ttc 96Gln Leu Thr Ser Gly Gly Ala Thr
Val Val Cys Phe Val Asn Asn Phe 20 25 30 tac ccc aga gac atc agc
gtg aag tgg aag atc gac ggc agc gag cag 144Tyr Pro Arg Asp Ile Ser
Val Lys Trp Lys Ile Asp Gly Ser Glu Gln 35 40 45 agg gac ggc gtg
ctg gac agc gtg acc gac cag gac agc aag gac tcc 192Arg Asp Gly Val
Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 acc tac
agc atg agc agc acc ctg agc ctg acc aag gtg gag tac gag 240Thr Tyr
Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu Tyr Glu 65 70 75 80
agg cac aac ctg tac acc tgc gag gtg gtg cac aag acc agc tcc agc
288Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser Ser
85 90 95 ccc gtg gtc aag tcc ttc aac cgg aac gag tgt 321Pro Val Val
Lys Ser Phe Asn Arg Asn Glu Cys 100 105 89107PRTRattus norvegicus
89Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Met Glu 1
5 10 15 Gln Leu Thr Ser Gly Gly Ala Thr Val Val Cys Phe Val Asn Asn
Phe 20 25 30 Tyr Pro Arg Asp Ile Ser Val Lys Trp Lys Ile Asp Gly
Ser Glu Gln 35 40 45 Arg Asp Gly Val Leu Asp Ser Val Thr Asp Gln
Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser
Thr Leu Ser Leu Thr Lys Val Glu Tyr Glu 65 70 75 80 Arg His Asn Leu
Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser Ser 85 90 95 Pro Val
Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 90865DNAArtificial
SequenceIGKV1-39/J-Ck 90ggtaccgcgg ccgccaccat ggacatgaga gtgcccgccc
agctcctggg gctcctgcta 60ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt gtgctcagta 120ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa tatttgtttt 180tatgtttcca atctcaggtg
ccagatgtga catccagatg acccagagcc ccagcagcct 240gagcgccagc
gtgggcgaca gagtgaccat cacctgcaga gccagccaga gcatcagcag
300ctacctgaac tggtatcagc agaagcccgg caaggccccc aagctgctga
tctacgccgc 360 cagctccctg cagagcggcg tgcccagcag attcagcggc
agcggctccg gcaccgactt 420caccctgacc atcagcagcc tgcagcccga
ggacttcgcc acctactact gccagcagag 480 ctacagcacc ccccccacct
tcggccaggg caccaaggtg gagatcaaga gagccgacgc 540cgctcccacc
gtgtccatct tcccccccag catggaacag ctgacctctg gcggagccac
600cgtggtctgc ttcgtgaaca acttctaccc cagagacatc agcgtgaagt
ggaagatcga 660cggcagcgag cagagggacg gcgtgctgga cagcgtgacc
gaccaggaca gcaaggactc 720cacctacagc atgagcagca ccctgagcct
gaccaaggtg gagtacgaga ggcacaacct 780gtacacctgc gaggtggtgc
acaagaccag ctccagcccc gtggtcaagt ccttcaaccg 840 gaacgagtgt
tgagctagcg agctc 86591874DNAArtificial SequenceIGLV2-14/J-Ck
91ggtaccgcgg ccgccaccat ggacatgaga gtgcccgccc agctcctggg gctcctgcta
60ctctggctcc gaggtaagga tggagaacac taggaattta ctcagccagt gtgctcagta
120ctgactggaa cttcagggaa gttctctgat aacatgatta atagtaagaa
tatttgtttt 180tatgtttcca atctcaggtg ccagatgtca gtctgccctg
acccagcccg cctctgtgtc 240tggcagccct ggccagagca tcaccatcag
ctgcaccggc accagcagcg acgtgggcgg 300ctacaactac gtgtcctggt
atcagcagca ccccggcaag gcccccaagc tgatgatcta 360cgaggtgtcc
aacagaccca gcggcgtgag caacagattc agcggcagca agagcggcaa
420caccgccagc ctgaccatca gcggcctcca ggctgaggac gaggccgact
actactgcag 480cagctacacc agcagctcca ccctggtgtt tggcggcgga
acaaagctga ccgtgctgag 540agccgacgcc gctcccaccg tgtccatctt
cccccccagc atggaacagc tgacctctgg 600cggagccacc gtggtctgct
tcgtgaacaa cttctacccc agagacatca gcgtgaagtg 660gaagatcgac
ggcagcgagc agagggacgg cgtgctggac agcgtgaccg accaggacag
720caaggactcc acctacagca tgagcagcac cctgagcctg accaaggtgg
agtacgagag 780gcacaacctg tacacctgcg aggtggtgca caagaccagc
tccagccccg tggtcaagtc 840cttcaaccgg aacgagtgtt gagctagcga gctc
8749213373DNAArtificial SequenceVkP-IGKV1-39/J-Ck 92ggccggccca
catgaaacaa tgggaaccat gtgacaatca cagaggtgtt gttactatag 60caaaagggat
tgttactctc cacatccctt taagtaactt gaaggcctga tagacccacc
120ctctaagact tcattagaca ttccctacga atggttatac tctcctgtat
actcccaata 180caactctaaa atatattatt ccatatagtc cttaggtttg
tattaaagtt tgactttttt 240ccttcaaaat atctcttgtc acaacagcgg
ctctagagag aaatacattc cctccaggca 300aatctatgct gcgctggtct
gacctgggac cctggggaca ttgcccctgt gctgagttac 360taagatgagc
cagccctgca gctgtgctca gcctgcccca tgccctgctg attgatttgc
420atgttccaga gcacagcccc ctgccctgaa gactttttta tgggctggtc
gcaccctgtg 480caggagtcag tctcagtcag gagccaccat ggacatgaga
gtgcccgccc agctcctggg 540gctcctgcta ctctggctcc gaggtaagga
tggagaacac taggaattta ctcagccagt 600gtgctcagta ctgactggaa
cttcagggaa gttctctgat aacatgatta atagtaagaa 660tatttgtttt
tatgtttcca atctcaggtg ccagatgtga catccagatg acccagagcc
720ccagcagcct gagcgccagc gtgggcgaca gagtgaccat cacctgcaga
gccagccaga 780gcatcagcag ctacctgaac tggtatcagc agaagcccgg
caaggccccc aagctgctga 840tctacgccgc cagctccctg cagagcggcg
tgcccagcag attcagcggc agcggctccg 900gcaccgactt caccctgacc
atcagcagcc tgcagcccga ggacttcgcc acctactact 960gccagcagag
ctacagcacc ccccccacct tcggccaggg caccaaggtg gagatcaaac
1020gtaagtacac ttttctcatc tttttttatg tgtaagacac aggttttcat
gttaggagtt 1080aaagtcagtt cagaaaatct tgagaaaatg gagagggctc
attatcagtt gacgtggcat 1140acagtgtcag attttctgtt tatcaagcta
gtgagattag gggcaaaaag aggctttagt 1200tgagaggaaa gtaattaata
ctatggtcac catccaagag attggatcgg agaataagca 1260tgagtagtta
ttgagatctg ggtctgactg caggtagcgt ggtcttctag acgtttaagt
1320gggagatttg gaggggatga ggaatgaagg aacttcagga tagaaaaggg
ctgaagtcaa 1380gttcagctcc taaaatggat gtgggagcaa actttgaaga
taaactgaat gacccagagg 1440atgaaacagc gcagatcaaa gaggggcctg
gagctctgag aagagaagga gactcatccg 1500tgttgagttt ccacaagtac
tgtcttgagt tttgcaataa aagtgggata gcagagttga 1560gtgagccgta
ggctgagttc tctcttttgt ctcctaagtt tttatgacta caaaaatcag
1620tagtatgtcc tgaaataatc attaagctgt ttgaaagtat gactgcttgc
catgtagata 1680ccatggcttg ctgaataatc agaagaggtg tgactcttat
tctaaaattt gtcacaaaat 1740gtcaaaatga gagactctgt aggaacgagt
ccttgacaga cagctcaagg ggtttttttc 1800ctttgtctca tttctacatg
aaagtaaatt tgaaatgatc ttttttatta taagagtaga 1860aatacagttg
ggtttgaact atatgtttta atggccacgg ttttgtaaga catttggtcc
1920tttgttttcc cagttattac tcgattgtaa ttttatatcg ccagcaatgg
actgaaacgg 1980tccgcaacct cttctttaca actgggtgac ctcgcggctg
tgccagccat ttggcgttca 2040ccctgccgct aagggccatg tgaacccccg
cggtagcatc ccttgctccg cgtggaccac 2100tttcctgagg cacagtgata
ggaacagagc cactaatctg aagagaacag agatgtgaca 2160gactacacta
atgtgagaaa aacaaggaaa gggtgactta ttggagattt cagaaataaa
2220atgcatttat tattatattc ccttatttta attttctatt agggaattag
aaagggcata 2280aactgcttta tccagtgtta tattaaaagc ttaatgtata
taatctttta gaggtaaaat 2340ctacagccag caaaagtcat ggtaaatatt
ctttgactga actctcacta aactcctcta 2400aattatatgt catattaact
ggttaaatta atataaattt gtgacatgac cttaactggt 2460taggtaggat
atttttcttc atgcaaaaat atgactaata ataatttagc acaaaaatat
2520ttcccaatac tttaattctg tgatagaaaa atgtttaact cagctactat
aatcccataa 2580ttttgaaaac tatttattag cttttgtgtt tgacccttcc
ctagccaaag gcaactattt 2640aaggaccctt taaaactctt gaaactactt
tagagtcatt aagttattta accactttta 2700attactttaa aatgatgtca
attccctttt aactattaat ttattttaag gggggaaagg 2760ctgctcataa
ttctattgtt tttcttggta aagaactctc agttttcgtt tttactacct
2820ctgtcaccca agagttggca tctcaacaga ggggactttc cgagaggcca
tctggcagtt 2880gcttaagatc agaagtgaag tctgccagtt cctcccaggc
aggtggccca gattacagtt 2940gacctgttct ggtgtggcta aaaattgtcc
catgtggtta caaaccatta gaccagggtc 3000tgatgaattg ctcagaatat
ttctggacac ccaaatacag accctggctt aaggccctgt 3060ccatacagta
ggtttagctt ggctacacca aaggaagcca tacagaggct aatatcagag
3120tattcttgga agagacagga gaaaatgaaa gccagtttct gctcttacct
tatgtgcttg 3180tgttcagact cccaaacatc aggagtgtca gataaactgg
tctgaatctc tgtctgaagc 3240atggaactga aaagaatgta gtttcaggga
agaaaggcaa tagaaggaag cctgagaata 3300tcttcaaagg gtcagactca
atttactttc taaagaagta gctaggaact agggaataac 3360ttagaaacaa
caagattgta tatatgtgca tcctggcccc attgttcctt atctgtaggg
3420ataagcgtgc ttttttgtgt gtctgtatat aacataactg tttacacata
atacactgaa 3480atggagccct tccttgttac ttcataccat cctctgtgct
tccttcctca ggggccgacg 3540ccgctcccac cgtgtccatc ttccccccca
gcatggaaca gctgacctct ggcggagcca 3600ccgtggtctg cttcgtgaac
aacttctacc ccagagacat cagcgtgaag tggaagatcg 3660acggcagcga
gcagagggac ggcgtgctgg acagcgtgac cgaccaggac agcaaggact
3720ccacctacag catgagcagc accctgagcc tgaccaaggt ggagtacgag
aggcacaacc 3780tgtacacctg cgaggtggtg cacaagacca gctccagccc
cgtggtcaag tccttcaacc 3840ggaacgagtg ttgaagacaa aggtcctgag
acgccaccac cagctcccca gctccatcct 3900atcttccctt ctaaggtctt
ggaggcttcc ccacaagcga cctaccactg ttgcggtgct 3960ccaaacctcc
tccccacctc cttctcctcc tcctcccttt ccttggcttt tatcatgcta
4020atatttgcag aaaatattca ataaagtgag tctttgcact tgagatctct
gtctttctta 4080ctaaatggta gtaatcagtt gtttttccag ttacctgggt
ttctcttcta aagaagttaa 4140atgtttagtt gccctgaaat ccaccacact
taaaggataa ataaaaccct ccacttgccc 4200tggttggctg tccactacat
ggcagtcctt tctaaggttc acgagtacta ttcatggctt 4260atttctctgg
gccatggtag gtttgaggag gcatacttcc tagttttctt cccctaagtc
4320gtcaaagtcc tgaaggggga cagtctttac aagcacatgt tctgtaatct
gattcaacct 4380acccagtaaa cttggcgaag caaagtagaa tcattatcac
aggaagcaaa ggcaacctaa 4440atgtgcaagc aataggaaaa tgtggaagcc
catcatagta cttggacttc atctgctttt 4500gtgccttcac taagttttta
aacatgagct ggctcctatc tgccattggc aaggctgggc 4560actacccaca
acctacttca aggacctcta taccgtgaga ttacacacat acatcaaaat
4620ttgggaaaag ttctaccaag ctgagagctg atcaccccac tcttaggtgc
ttatctctgt 4680acaccagaaa ccttaagaag caaccagtat tgagagactc
atttatgaaa gtctaaaact 4740ggatacaacc aaaatgtcca ccaacagtta
aattatgaca tgttcacaat tgagctatta 4800cttaataagg agaattaata
aaataaaact taagagcata gtttaatctc ataaacaaga 4860taataagcaa
aacaaaacat tttttcatcc atgtaagttt aaaagcaggt aaaatttaaa
4920attaagagag acataagttt tgaggtagca agatggaaac tctggggctt
ggggaatgtt 4980ctgtctctct gtatgggatg tgaaagttac tattgtggaa
ttgggatcta tgttcttcct 5040gtatatattg tatacttcat aataacttca
cctaaagaaa tatctaatac ccagtgcata 5100cataaaagag gatacaagga
atgaatcata cgtcaaggcc agaaagacaa taaagtaggg 5160gatccaggat
caaatctccc acaaccttga gccttctact attctgcctt ccagagctca
5220aagtacaaaa cacataattc aaacacatga tccctccttg gggtctcttc
cttcatgcat 5280cgaattagaa atagccatgt ataaaatgag atagaagaga
ccttcatcaa caggtcaaag 5340aatataggta attttgtctg ggtatgaaga
gcccacgtat caaaggttac attagggaag 5400gaagaggaca ctaacagtga
ctttcattct ccccctcttc ctggaggccc ctgcatttag 5460tccctcgtgg
gctcatccac tcagcacaca tttactaagc atcttctcag cctacactct
5520gaaggcagtg cagaataatg ttagtgtccc ttcccccagt taatatgcag
tccagtttcc 5580ctgctccttc cctttctcag tccacataag gatgatggga
aaggacagtc accaaatagg 5640agagggcaac cctttgcctt cctacctctt
gagaatgtac attattatcc actttttgaa 5700acttctttta attgcttttt
tttaatttgt cttttcaaat agcataacct tgttcatcca 5760tttctgggaa
ccaaatttat caatcaacag tgcctctaat ctggctatta atacaaaaat
5820gcctcctcaa aatatatatg ttcgagtctt atctaaaaca gaacccacaa
taaaaaagaa 5880gaaagaatac atataagcat ttatataatt ctgagcaacc
ttgtgctttg tgaaaaaaat 5940ataatctaat gtcacatgct gtattctttt
tatttaacac tggtgaaatt ataccattag 6000agagaaagag gacagatcac
tgatcctagg atctagggat gttacagata agaaaacaaa 6060tgtgacaaag
agctgtcaca aggaggatct tcaaggtcac agaatcactg tcttgatttc
6120agtggtggtt acatacattt aaatatgtga taaaatgttg ttgaactata
ttcatatatt 6180gtaccaatgt caaatgctta attttggctc tatagtataa
ttatgcacta aataactatt 6240tggacaaaga aaatgatgtt tacatcaaag
gtgaggccat atttgttagg aacataactt 6300aaaaaccatt ttggataact
aatgaaaagc cattttgtgt gccttggcat atcatgccta 6360agctgtcacc
agatagatct aataagacct aagcctcaga agcaagcccc tgcccagcaa
6420gcaggcagca cagataagag ctaaacccag gacaggccat gatatgctaa
tgaactacct 6480tcaaggtggt gttgctgacc tagtgaacca gccccaagct
gtgagcccca atagcacaaa 6540gctactgccc aaagaaatta tacaaaaatt
ggaactttgg gaatggtgtg caggatcgct 6600ctgctgtatg cctggaacac
agcttctcta tgttttgtat tgataccagt ctagaagctt 6660ccaaaacttt
ctcactgaag aagattcccc atgtgggacc cctacagact cttttgccca
6720aacaactgct tccctcctgg tgtgatatct gttttgcttt tatgttagca
taatattata 6780aggaatgttt gtgtgaataa accaaacata ttttaaaagc
aaatattgta tgcacatcct 6840aattgctaaa aagtttacag ctaatagtcc
catgctctcc acaatactgg atccaaataa 6900gtcctaattt caatgttggg
catctttaca gagagaaaga cattaaaaat gaagagacat 6960gcagagagtg
caccatgcca tcgtggagac agactgaagt gacacaactg ttagtcaaag
7020aggattaagg acttccagaa gccaccaaag gaaggaggta tgaagtggtt
tctccctcag 7080agtatccaga ggagactaaa ccaaccaaca cctttttgct
taagacttct tgccttcagg 7140actgtgagaa ggtagcttcc tattgttcta
agccccagta tgtggcattt tgttaaggta 7200gagtcaagaa accaataaaa
tgcagacaga caaaaggata gctgagtttt ccaggccctt 7260ccttcttatt
tttggttttg ttggtggtgg tggtggtggt ggtgatggtg gtggttttgt
7320ttatgttttg tttggggagt tttttggggt ttttttgggt tttgtttttg
ttgttgtttt 7380gggggttttt gttgttgttg ttgtttgctt ttttgttttt
tgttttttgt ttttttgaga 7440cagtgtttct ctgtatagcc ctggctgtcc
tggagttcct tctatctcta atgtctacat 7500ctcagagggg atcctctaat
ttcaaatgag cagtagctct ccatttttag ctcttattta 7560ttcatttatt
tacttactta cttattgtct gtagatgaaa gaattttgga gtgggaaagg
7620gttcatgagc ccccagcaac taatgaggag ctacagacaa ttgatgtttc
tggggaaagg 7680agactcagtt tctttgagag tatagcttct gatgggtcaa
ccatgttcct gtggctgatg 7740tcacacccag gagtatgcag acaacagaaa
ctggagttaa tgagttgttt taaaaataaa 7800aaagggcatg aagcttggga
tagaaattaa ggataaatac aattaaatac aggaaattct 7860gaaagaatta
ataaaaacat ttcttttttt aaaaaaaaat ccagaattag ctatgcttct
7920tcaaaattgc ttctggagaa ctttacaagt taaataagtt atattgtaga
aaaggtagag 7980aggagaatag tggaagagag agataaggag acttcaaaag
gagtggaggg agatagagga 8040ggagaaagca gaagcaatgg ctgatagaca
caggataaga gggaacagaa aggagaaaga 8100ggaagccagg atgggtattt
ctttgcctat ctgtgacttg cacatggtct tggcaattat 8160tgatgagttc
aaggcttaat tcttcacttg tgccaactca acagagtctt tctttcttat
8220aaccaggccc ccagtatgct catgtatgta tcaggtcctc ttatctcctt
atagcaatcc 8280tgtttataac tgggtaactt tgtgaaggga aggaagtgca
cactgagatg tgctacaact 8340ttttaataca aaattttgaa gagtttgtac
aatgtatgta taattaataa ttaatattat 8400gcactttaga ttttgatttc
aactcaagat actaattcta tatatatggg ttaaatcaat 8460atattaataa
gtttaatttc acatgcttat ttttattgtg gttttcgaga cagggtttct
8520ctgtatagcc ctggctgtcc tggaacccac tttgtagacc aggctggcct
caaactcaga 8580aacctacctg cctctgcctc tgcctctgcc tctgcctctg
cctctgcctc tgcctctgcc 8640tctgcctctg cctctgcctc tgcctctgcc
tctgcctctg cctctgcctc tgcctctgcc 8700tctgcctctg cctctgcctc
tgcctctgcc tctgcctctg cctctgcctc tgcctctgcc 8760tctgcctagt
gctggaatta aaggtttgcg ccaccacgcc cggtgaaatt tttaaacttt
8820atatatgtct cattctattt ctatcagata ggactgtgta gactgtgcta
aactaataaa 8880tgtgccctca aaagtaatcg caagttgtat tgttgttgtt
ttgctttgct ttgctttgct 8940ttgctttgct ttgctttgct ttgctttgct
ttgctttgct ttgctttgct ttgctttgct 9000ttgctttgct ttgctttgct
ttgctttttt gttttgggtt tttttccggg ggagggaggg 9060tggagaaaga
atcttactat gaagctctga ctgtcctggg aactcactat atagatcagg
9120cttgattcaa ctcatagaga tctgccttct tctgcctccc aagtgctggg
aataaaggca 9180tacacctcca tgcccagata gtgatcccaa gttttagcaa
aagtttctag acttgacatt 9240aatcgatgga gatagacatg aattacacaa
agaactaatg tggagtttac ctgaatcata 9300ctctatactt tatcagagat
taaattaaca tttaataatc cagtgccagg ctagaggcac 9360cattcaatgg
cagtgtttgc catcatgcat aggcttagtc ttcagtgctg aaaggcattg
9420ggggcaatat tactcattat acagatgaga aactgggaaa gacttgcctc
agattctcta 9480ctgaaaggct gagtttgtgg cttctagaaa atcttttact
ttcaatattt ttaatgtata 9540atttttttat ttccactgat tttatttttt
atttttaaca tttataagaa ataaatgcaa 9600taaaccaaat acatggacaa
aaaaatacaa gaatcatatg atcacctcaa tggaaggaaa 9660aaaaaagaaa
gaaaaagtct ttgataagat tcaacattca ttcttttttt attagatatt
9720ttcttcattt acatttcaaa tgctatcccc aaagccccct ataccttccc
ctgccctgct 9780ccccaaccca cccactcctg ctttctggcc ctggcattcc
tctgtactga ggcatatgat 9840cttcaaaaaa ccaagggcct ctcctctcat
tggtggccga ctattaggcc atcttttgct 9900acatatgcaa ctagagacac
agctctgggg gttactggtt agttcatatt gttagtcctc 9960ctatagagtt
gcagacccct ttagctcctt ggatactttc tctagttcct tcattagggg
10020ccctgtgtcc catccaatag atgactgtga gcatccactt ctgtatttgc
caggcactgg 10080catagcctca cgagaaagag agagctatgt caggatcctg
tcagtaaaat ctttctggca 10140tatgcaatag tatctgggtt tggtggttgt
atatgggatg gatccccaag tggagcagtc 10200tctgaatggt ccttccttcc
atctcagctc caaactttgt ctctataact ccttccatgg 10260gtattttgtt
ccccattcta agaaggagtg aagaatccac actttggtct tccttcttct
10320tgagtttcat atgttgcatc ttggatattc taagtttctg ggttaatatc
cacgtatcag 10380tgagtgcata tcatgcgtgt tattttgtga ttagtttacc
tcactcagga tgatatcctc 10440cagatgcatc catttgccta agaatttcat
taattcactg tttttaattg ctgaatagta 10500ctccattgtg taaatgtacc
acattttctg tatccattcc tctgttgagg ggcatctggg 10560ttctttccag
cttctggcta ttataaataa ggctgctatg agcatagcgg agcatgtgtc
10620cttatcaagt tggaacatct tctaggtata tgcccaggag aggaattgct
ggatcttccg 10680gtagtaccat caacatgcat tcttaataaa agccctagaa
caaggaggac tgtaggaaac 10740atattccaac ataataaagg ttatgtatga
caaactcatg accaatatca tcctaaatga 10800atgaaaccat taataagctc
cattaaaatc agaggactgc ccactatccc tacttctcat 10860ccataatgag
attgaagcat tagctggagc aataaggcaa gagaagggat acaaatggga
10920aaatattaag tcaaattgtt ttcaattgaa gattatatta tcttataccc
aatgacctca 10980aattttgact agaaaaattg tagaaattat caataatttc
agcaaagtgt tatgatgcac 11040cacatcctta ttcttctccc cagcttctgc
ttgcttctct cttcttgctc ttcatccttt 11100ctgtccttcc atctgcctgc
actcttgtct caagactgag tgcagcgtgt aactctcctg 11160tgactgagta
tctcacaaaa cgttctacct gccaaacctg gatgagccct ttgtctttct
11220gaagctatga ggctctctac atagactcaa gaaggaaatg acagggagga
ggtaataatg 11280aagtggggaa ggctgacatt agcattgctc ctgtgtggct
ccttaatttc tcatacttca 11340cactgagatg ttattaactg tgactcatag
gtgaagaagc cagagctaag gttctcatat 11400ttgagtgtta tagaatgagt
agagcagtag ttctcaaact atgggtcatg actcctttat 11460gggtcaaact
accctttcac acaggttgca tatcagatat cctaatttta tatacatata
11520tatatgcata tgtatatata tatatttcac aacagtagga aaattattta
gtaatcattt 11580tatagttgtg ggtcatggca acatgaggaa ctgtattaaa
gggttgcagc attaggaatg 11640ttgagaccca ctgtaataga gaatgaggct
taaggcaggg ctataaagcc caatggacca 11700tgtgcctttt ccaacatttg
ccacatggta agctctgtat agacttttta aagaacattg 11760gtttgtaatt
ttaaatggat aagggtcttc actgtctatc acccatctat ataataaata
11820cataagtttt gattccacca tggattcaaa tgcaaaaatc ctcaacctaa
gacatagcag 11880tgaaacattg atgaccaaat aggaaatcca tgtagagacc
ttctatcttc tgatggctcc 11940acaggcacca tcttgcaaca gagttctact
ttgctaccag taatgaatac agtgtctcaa 12000ctcctgccat tgaatcttca
ggaagcccct gaaatgactt gtactacacc atttcttaaa 12060gacagaaaag
ctaagactta gagggaataa atgtcatgcc tgagatcatg caaccaatta
12120agtccaactt ggcctgatca agaggcacaa ttcaaaagca atgttgttcc
ttcactagct 12180cttgtgtatg gttgctgatt ccggaagcaa agtatcagtg
aatatcccta gtgggaaaag 12240acttggaaat caaatgtctc atttaacaga
ttaggagatg aaacggtaga ctctgtgtag 12300ttgtacaccc ctgtgatccc
atcgctagga agactgaggc aggaagtcct cgagctcaaa 12360ccagcttagg
ctacacagag aaactatcta aaaaataatt actaactact taataggaga
12420ttggatgtta agatctggtc actaagaggc agaattgaga ttcgaagcca
gtattttcta 12480cctggtatgt tttaaattgc agtaaggatc taagtgtaga
tatataataa taagattcta 12540ttgatctctg caacaacaga gagtgttaga
tttgtttgga aaaaaatatt atcagccaac 12600atcttctacc atttcagtat
agcacagagt acccacccat atctccccac ccatccccca 12660taccagactg
gttattgatt ttcatggtga ctggcctgag aagattaaaa aaagtaatgc
12720taccttattg ggagtgtccc atggaccaag atagcaactg tcatagctac
cgtcacactg 12780ctttgatcaa gaagaccctt tgaggaactg aaaacagaac
cttaggcaca tctgttgctt 12840tcgctcccat cctcctccaa cagcctgggt
ggtgcactcc
acaccctttc aagtttccaa 12900agcctcatac acctgctccc taccccagca
cctggccaag gctgtatcca gcactgggat 12960gaaaatgata ccccacctcc
atcttgtttg atattactct atctcaagcc ccaggttagt 13020ccccagtccc
aatgcttttg cacagtcaaa actcaacttg gaataatcag tatccttgaa
13080gagttctgat atggtcactg ggcccatata ccatgtaaga catgtggaaa
agatgtttca 13140tggggcccag acacgttcta gaagtacctg agagtggcaa
aaaatagttg tgctaaatag 13200tttggccatc tttaggctga gagactagga
aatacagcga tggactatat cagcattgca 13260ggatagttgt cagtaaacac
cccacaaccc ataacagaag tattctcttc tttctatatc 13320ccttttccat
ccatgtagat ggctgtcttc atatttgttc tagacggccg gcc
133739312892DNAArtificial SequenceVkP-IGKV1-39/J-Ck-delta1
93ggccggccca catgaaacaa tgggaaccat gtgacaatca cagaggtgtt gttactatag
60caaaagggat tgttactctc cacatccctt taagtaactt gaaggcctga tagacccacc
120ctctaagact tcattagaca ttccctacga atggttatac tctcctgtat
actcccaata 180caactctaaa atatattatt ccatatagtc cttaggtttg
tattaaagtt tgactttttt 240ccttcaaaat atctcttgtc acaacagcgg
ctctagagag aaatacattc cctccaggca 300aatctatgct gcgctggtct
gacctgggac cctggggaca ttgcccctgt gctgagttac 360taagatgagc
cagccctgca gctgtgctca gcctgcccca tgccctgctg attgatttgc
420atgttccaga gcacagcccc ctgccctgaa gactttttta tgggctggtc
gcaccctgtg 480caggagtcag tctcagtcag gagccaccat ggacatgaga
gtgcccgccc agctcctggg 540gctcctgcta ctctggctcc gaggtaagga
tggagaacac taggaattta ctcagccagt 600gtgctcagta ctgactggaa
cttcagggaa gttctctgat aacatgatta atagtaagaa 660tatttgtttt
tatgtttcca atctcaggtg ccagatgtga catccagatg acccagagcc
720ccagcagcct gagcgccagc gtgggcgaca gagtgaccat cacctgcaga
gccagccaga 780gcatcagcag ctacctgaac tggtatcagc agaagcccgg
caaggccccc aagctgctga 840tctacgccgc cagctccctg cagagcggcg
tgcccagcag attcagcggc agcggctccg 900gcaccgactt caccctgacc
atcagcagcc tgcagcccga ggacttcgcc acctactact 960gccagcagag
ctacagcacc ccccccacct tcggccaggg caccaaggtg gagatcaaac
1020gtaagtacac ttttctcatc tttttttatg tgtaagacac aggttttcat
gttaggagtt 1080aaagtcagtt cagaaaatct tgagaaaatg gagagggctc
attatcagtt gacgtggcat 1140acagtgtcag attttctgtt tatcaagcta
gtgagattag gggcaaaaag aggctttagt 1200tgagaggaaa gtaattaata
ctatggtcac catccaagag attggatcgg agaataagca 1260tgagtagtta
ttgagatctg ggtctgactg caggtagcgt ggtcttctag acgtttaagt
1320gggagatttg gaggggatga ggaatgaagg aacttcagga tagaaaaggg
ctgaagtcaa 1380gttcagctcc taaaatggat gtgggagcaa actttgaaga
taaactgaat gacccagagg 1440atgaaacagc gcagatcaaa gaggggcctg
gagctctgag aagagaagga gactcatccg 1500tgttgagttt ccacaagtac
tgtcttgagt tttgcaataa aagtgggata gcagagttga 1560gtgagccgta
ggctgagttc tctcttttgt ctcctaagtt tttatgacta caaaaatcag
1620tagtatgtcc tgaaataatc attaagctgt ttgaaagtat gactgcttgc
catgtagata 1680ccatggcttg ctgaataatc agaagaggtg tgactcttat
tctaaaattt gtcacaaaat 1740gtcaaaatga gagactctgt aggaacgagt
ccttgacaga cagctcaagg ggtttttttc 1800ctttgtctca tttctacatg
aaagtaaatt tgaaatgatc ttttttatta taagagtaga 1860aatacagttg
ggtttgaact atatgtttta atggccacgg ttttgtaaga catttggtcc
1920tttgttttcc cagttattac tcgattgtaa ttttatatcg ccagcaatgg
actgaaacgg 1980tccgcaacct cttctttaca actgggtgac ctcgcggctg
tgccagccat ttggcgttca 2040ccctgccgct aagggccatg tgaacccccg
cggtagcatc ccttgctccg cgtggaccac 2100tttcctgagg cacagtgata
ggaacagagc cactaatctg aagagaacag agatgtgaca 2160gactacacta
atgtgagaaa aacaaggaaa gggtgactta ttggagattt cagaaataaa
2220atgcatttat tattatattc ccttatttta attttctatt agggaattag
aaagggcata 2280aactgcttta tccagtgtta tattaaaagc ttaatgtata
taatctttta gaggtaaaat 2340ctacagccag caaaagtcat ggtaaatatt
ctttgactga actctcacta aactcctcta 2400aattatatgt catattaact
ggttaaatta atataaattt gtgacatgac cttaactggt 2460taggtaggat
atttttcttc atgcaaaaat atgactaata ataatttagc acaaaaatat
2520ttcccaatac tttaattctg tgatagaaaa atgtttaact cagctactat
aatcccataa 2580ttttgaaaac tatttatttg gctacaccaa aggaagccat
acagaggcta atatcagagt 2640attcttggaa gagacaggag aaaatgaaag
ccagtttctg ctcttacctt atgtgcttgt 2700gttcagactc ccaaacatca
ggagtgtcag ataaactggt ctgaatctct gtctgaagca 2760tggaactgaa
aagaatgtag tttcagggaa gaaaggcaat agaaggaagc ctgagaatat
2820cttcaaaggg tcagactcaa tttactttct aaagaagtag ctaggaacta
gggaataact 2880tagaaacaac aagattgtat atatgtgcat cctggcccca
ttgttcctta tctgtaggga 2940taagcgtgct tttttgtgtg tctgtatata
acataactgt ttacacataa tacactgaaa 3000tggagccctt ccttgttact
tcataccatc ctctgtgctt ccttcctcag gggccgacgc 3060cgctcccacc
gtgtccatct tcccccccag catggaacag ctgacctctg gcggagccac
3120cgtggtctgc ttcgtgaaca acttctaccc cagagacatc agcgtgaagt
ggaagatcga 3180cggcagcgag cagagggacg gcgtgctgga cagcgtgacc
gaccaggaca gcaaggactc 3240cacctacagc atgagcagca ccctgagcct
gaccaaggtg gagtacgaga ggcacaacct 3300gtacacctgc gaggtggtgc
acaagaccag ctccagcccc gtggtcaagt ccttcaaccg 3360gaacgagtgt
tgaagacaaa ggtcctgaga cgccaccacc agctccccag ctccatccta
3420tcttcccttc taaggtcttg gaggcttccc cacaagcgac ctaccactgt
tgcggtgctc 3480caaacctcct ccccacctcc ttctcctcct cctccctttc
cttggctttt atcatgctaa 3540tatttgcaga aaatattcaa taaagtgagt
ctttgcactt gagatctctg tctttcttac 3600taaatggtag taatcagttg
tttttccagt tacctgggtt tctcttctaa agaagttaaa 3660tgtttagttg
ccctgaaatc caccacactt aaaggataaa taaaaccctc cacttgccct
3720ggttggctgt ccactacatg gcagtccttt ctaaggttca cgagtactat
tcatggctta 3780tttctctggg ccatggtagg tttgaggagg catacttcct
agttttcttc ccctaagtcg 3840tcaaagtcct gaagggggac agtctttaca
agcacatgtt ctgtaatctg attcaaccta 3900cccagtaaac ttggcgaagc
aaagtagaat cattatcaca ggaagcaaag gcaacctaaa 3960tgtgcaagca
ataggaaaat gtggaagccc atcatagtac ttggacttca tctgcttttg
4020tgccttcact aagtttttaa acatgagctg gctcctatct gccattggca
aggctgggca 4080ctacccacaa cctacttcaa ggacctctat accgtgagat
tacacacata catcaaaatt 4140tgggaaaagt tctaccaagc tgagagctga
tcaccccact cttaggtgct tatctctgta 4200caccagaaac cttaagaagc
aaccagtatt gagagactca tttatgaaag tctaaaactg 4260gatacaacca
aaatgtccac caacagttaa attatgacat gttcacaatt gagctattac
4320ttaataagga gaattaataa aataaaactt aagagcatag tttaatctca
taaacaagat 4380aataagcaaa acaaaacatt ttttcatcca tgtaagttta
aaagcaggta aaatttaaaa 4440ttaagagaga cataagtttt gaggtagcaa
gatggaaact ctggggcttg gggaatgttc 4500tgtctctctg tatgggatgt
gaaagttact attgtggaat tgggatctat gttcttcctg 4560tatatattgt
atacttcata ataacttcac ctaaagaaat atctaatacc cagtgcatac
4620ataaaagagg atacaaggaa tgaatcatac gtcaaggcca gaaagacaat
aaagtagggg 4680atccaggatc aaatctccca caaccttgag ccttctacta
ttctgccttc cagagctcaa 4740agtacaaaac acataattca aacacatgat
ccctccttgg ggtctcttcc ttcatgcatc 4800gaattagaaa tagccatgta
taaaatgaga tagaagagac cttcatcaac aggtcaaaga 4860atataggtaa
ttttgtctgg gtatgaagag cccacgtatc aaaggttaca ttagggaagg
4920aagaggacac taacagtgac tttcattctc cccctcttcc tggaggcccc
tgcatttagt 4980ccctcgtggg ctcatccact cagcacacat ttactaagca
tcttctcagc ctacactctg 5040aaggcagtgc agaataatgt tagtgtccct
tcccccagtt aatatgcagt ccagtttccc 5100tgctccttcc ctttctcagt
ccacataagg atgatgggaa aggacagtca ccaaatagga 5160gagggcaacc
ctttgccttc ctacctcttg agaatgtaca ttattatcca ctttttgaaa
5220cttcttttaa ttgctttttt ttaatttgtc ttttcaaata gcataacctt
gttcatccat 5280ttctgggaac caaatttatc aatcaacagt gcctctaatc
tggctattaa tacaaaaatg 5340cctcctcaaa atatatatgt tcgagtctta
tctaaaacag aacccacaat aaaaaagaag 5400aaagaataca tataagcatt
tatataattc tgagcaacct tgtgctttgt gaaaaaaata 5460taatctaatg
tcacatgctg tattcttttt atttaacact ggtgaaatta taccattaga
5520gagaaagagg acagatcact gatcctagga tctagggatg ttacagataa
gaaaacaaat 5580gtgacaaaga gctgtcacaa ggaggatctt caaggtcaca
gaatcactgt cttgatttca 5640gtggtggtta catacattta aatatgtgat
aaaatgttgt tgaactatat tcatatattg 5700taccaatgtc aaatgcttaa
ttttggctct atagtataat tatgcactaa ataactattt 5760ggacaaagaa
aatgatgttt acatcaaagg tgaggccata tttgttagga acataactta
5820aaaaccattt tggataacta atgaaaagcc attttgtgtg ccttggcata
tcatgcctaa 5880gctgtcacca gatagatcta ataagaccta agcctcagaa
gcaagcccct gcccagcaag 5940caggcagcac agataagagc taaacccagg
acaggccatg atatgctaat gaactacctt 6000caaggtggtg ttgctgacct
agtgaaccag ccccaagctg tgagccccaa tagcacaaag 6060ctactgccca
aagaaattat acaaaaattg gaactttggg aatggtgtgc aggatcgctc
6120tgctgtatgc ctggaacaca gcttctctat gttttgtatt gataccagtc
tagaagcttc 6180caaaactttc tcactgaaga agattcccca tgtgggaccc
ctacagactc ttttgcccaa 6240acaactgctt ccctcctggt gtgatatctg
ttttgctttt atgttagcat aatattataa 6300ggaatgtttg tgtgaataaa
ccaaacatat tttaaaagca aatattgtat gcacatccta 6360attgctaaaa
agtttacagc taatagtccc atgctctcca caatactgga tccaaataag
6420tcctaatttc aatgttgggc atctttacag agagaaagac attaaaaatg
aagagacatg 6480cagagagtgc accatgccat cgtggagaca gactgaagtg
acacaactgt tagtcaaaga 6540ggattaagga cttccagaag ccaccaaagg
aaggaggtat gaagtggttt ctccctcaga 6600gtatccagag gagactaaac
caaccaacac ctttttgctt aagacttctt gccttcagga 6660ctgtgagaag
gtagcttcct attgttctaa gccccagtat gtggcatttt gttaaggtag
6720agtcaagaaa ccaataaaat gcagacagac aaaaggatag ctgagttttc
caggcccttc 6780cttcttattt ttggttttgt tggtggtggt ggtggtggtg
gtgatggtgg tggttttgtt 6840tatgttttgt ttggggagtt ttttggggtt
tttttgggtt ttgtttttgt tgttgttttg 6900ggggtttttg ttgttgttgt
tgtttgcttt tttgtttttt gttttttgtt tttttgagac 6960agtgtttctc
tgtatagccc tggctgtcct ggagttcctt ctatctctaa tgtctacatc
7020tcagagggga tcctctaatt tcaaatgagc agtagctctc catttttagc
tcttatttat 7080tcatttattt acttacttac ttattgtctg tagatgaaag
aattttggag tgggaaaggg 7140ttcatgagcc cccagcaact aatgaggagc
tacagacaat tgatgtttct ggggaaagga 7200gactcagttt ctttgagagt
atagcttctg atgggtcaac catgttcctg tggctgatgt 7260cacacccagg
agtatgcaga caacagaaac tggagttaat gagttgtttt aaaaataaaa
7320aagggcatga agcttgggat agaaattaag gataaataca attaaataca
ggaaattctg 7380aaagaattaa taaaaacatt tcttttttta aaaaaaaatc
cagaattagc tatgcttctt 7440caaaattgct tctggagaac tttacaagtt
aaataagtta tattgtagaa aaggtagaga 7500ggagaatagt ggaagagaga
gataaggaga cttcaaaagg agtggaggga gatagaggag 7560gagaaagcag
aagcaatggc tgatagacac aggataagag ggaacagaaa ggagaaagag
7620gaagccagga tgggtatttc tttgcctatc tgtgacttgc acatggtctt
ggcaattatt 7680gatgagttca aggcttaatt cttcacttgt gccaactcaa
cagagtcttt ctttcttata 7740accaggcccc cagtatgctc atgtatgtat
caggtcctct tatctcctta tagcaatcct 7800gtttataact gggtaacttt
gtgaagggaa ggaagtgcac actgagatgt gctacaactt 7860tttaatacaa
aattttgaag agtttgtaca atgtatgtat aattaataat taatattatg
7920cactttagat tttgatttca actcaagata ctaattctat atatatgggt
taaatcaata 7980tattaataag tttaatttca catgcttatt tttattgtgg
ttttcgagac agggtttctc 8040tgtatagccc tggctgtcct ggaacccact
ttgtagacca ggctggcctc aaactcagaa 8100acctacctgc ctctgcctct
gcctctgcct ctgcctctgc ctctgcctct gcctctgcct 8160ctgcctctgc
ctctgcctct gcctctgcct ctgcctctgc ctctgcctct gcctctgcct
8220ctgcctctgc ctctgcctct gcctctgcct ctgcctctgc ctctgcctct
gcctctgcct 8280ctgcctagtg ctggaattaa aggtttgcgc caccacgccc
ggtgaaattt ttaaacttta 8340tatatgtctc attctatttc tatcagatag
gactgtgtag actgtgctaa actaataaat 8400gtgccctcaa aagtaatcgc
aagttgtatt gttgttgttt tgctttgctt tgctttgctt 8460tgctttgctt
tgctttgctt tgctttgctt tgctttgctt tgctttgctt tgctttgctt
8520tgctttgctt tgctttgctt tgcttttttg ttttgggttt ttttccgggg
gagggagggt 8580ggagaaagaa tcttactatg aagctctgac tgtcctggga
actcactata tagatcaggc 8640ttgattcaac tcatagagat ctgccttctt
ctgcctccca agtgctggga ataaaggcat 8700acacctccat gcccagatag
tgatcccaag ttttagcaaa agtttctaga cttgacatta 8760atcgatggag
atagacatga attacacaaa gaactaatgt ggagtttacc tgaatcatac
8820tctatacttt atcagagatt aaattaacat ttaataatcc agtgccaggc
tagaggcacc 8880attcaatggc agtgtttgcc atcatgcata ggcttagtct
tcagtgctga aaggcattgg 8940gggcaatatt actcattata cagatgagaa
actgggaaag acttgcctca gattctctac 9000tgaaaggctg agtttgtggc
ttctagaaaa tcttttactt tcaatatttt taatgtataa 9060tttttttatt
tccactgatt ttatttttta tttttaacat ttataagaaa taaatgcaat
9120aaaccaaata catggacaaa aaaatacaag aatcatatga tcacctcaat
ggaaggaaaa 9180aaaaagaaag aaaaagtctt tgataagatt caacattcat
tcttttttta ttagatattt 9240tcttcattta catttcaaat gctatcccca
aagcccccta taccttcccc tgccctgctc 9300cccaacccac ccactcctgc
tttctggccc tggcattcct ctgtactgag gcatatgatc 9360ttcaaaaaac
caagggcctc tcctctcatt ggtggccgac tattaggcca tcttttgcta
9420catatgcaac tagagacaca gctctggggg ttactggtta gttcatattg
ttagtcctcc 9480tatagagttg cagacccctt tagctccttg gatactttct
ctagttcctt cattaggggc 9540cctgtgtccc atccaataga tgactgtgag
catccacttc tgtatttgcc aggcactggc 9600atagcctcac gagaaagaga
gagctatgtc aggatcctgt cagtaaaatc tttctggcat 9660atgcaatagt
atctgggttt ggtggttgta tatgggatgg atccccaagt ggagcagtct
9720ctgaatggtc cttccttcca tctcagctcc aaactttgtc tctataactc
cttccatggg 9780tattttgttc cccattctaa gaaggagtga agaatccaca
ctttggtctt ccttcttctt 9840gagtttcata tgttgcatct tggatattct
aagtttctgg gttaatatcc acgtatcagt 9900gagtgcatat catgcgtgtt
attttgtgat tagtttacct cactcaggat gatatcctcc 9960agatgcatcc
atttgcctaa gaatttcatt aattcactgt ttttaattgc tgaatagtac
10020tccattgtgt aaatgtacca cattttctgt atccattcct ctgttgaggg
gcatctgggt 10080tctttccagc ttctggctat tataaataag gctgctatga
gcatagcgga gcatgtgtcc 10140ttatcaagtt ggaacatctt ctaggtatat
gcccaggaga ggaattgctg gatcttccgg 10200tagtaccatc aacatgcatt
cttaataaaa gccctagaac aaggaggact gtaggaaaca 10260tattccaaca
taataaaggt tatgtatgac aaactcatga ccaatatcat cctaaatgaa
10320tgaaaccatt aataagctcc attaaaatca gaggactgcc cactatccct
acttctcatc 10380cataatgaga ttgaagcatt agctggagca ataaggcaag
agaagggata caaatgggaa 10440aatattaagt caaattgttt tcaattgaag
attatattat cttataccca atgacctcaa 10500attttgacta gaaaaattgt
agaaattatc aataatttca gcaaagtgtt atgatgcacc 10560acatccttat
tcttctcccc agcttctgct tgcttctctc ttcttgctct tcatcctttc
10620tgtccttcca tctgcctgca ctcttgtctc aagactgagt gcagcgtgta
actctcctgt 10680gactgagtat ctcacaaaac gttctacctg ccaaacctgg
atgagccctt tgtctttctg 10740aagctatgag gctctctaca tagactcaag
aaggaaatga cagggaggag gtaataatga 10800agtggggaag gctgacatta
gcattgctcc tgtgtggctc cttaatttct catacttcac 10860actgagatgt
tattaactgt gactcatagg tgaagaagcc agagctaagg ttctcatatt
10920tgagtgttat agaatgagta gagcagtagt tctcaaacta tgggtcatga
ctcctttatg 10980ggtcaaacta ccctttcaca caggttgcat atcagatatc
ctaattttat atacatatat 11040atatgcatat gtatatatat atatttcaca
acagtaggaa aattatttag taatcatttt 11100atagttgtgg gtcatggcaa
catgaggaac tgtattaaag ggttgcagca ttaggaatgt 11160tgagacccac
tgtaatagag aatgaggctt aaggcagggc tataaagccc aatggaccat
11220gtgccttttc caacatttgc cacatggtaa gctctgtata gactttttaa
agaacattgg 11280tttgtaattt taaatggata agggtcttca ctgtctatca
cccatctata taataaatac 11340ataagttttg attccaccat ggattcaaat
gcaaaaatcc tcaacctaag acatagcagt 11400gaaacattga tgaccaaata
ggaaatccat gtagagacct tctatcttct gatggctcca 11460caggcaccat
cttgcaacag agttctactt tgctaccagt aatgaataca gtgtctcaac
11520tcctgccatt gaatcttcag gaagcccctg aaatgacttg tactacacca
tttcttaaag 11580acagaaaagc taagacttag agggaataaa tgtcatgcct
gagatcatgc aaccaattaa 11640gtccaacttg gcctgatcaa gaggcacaat
tcaaaagcaa tgttgttcct tcactagctc 11700ttgtgtatgg ttgctgattc
cggaagcaaa gtatcagtga atatccctag tgggaaaaga 11760cttggaaatc
aaatgtctca tttaacagat taggagatga aacggtagac tctgtgtagt
11820tgtacacccc tgtgatccca tcgctaggaa gactgaggca ggaagtcctc
gagctcaaac 11880cagcttaggc tacacagaga aactatctaa aaaataatta
ctaactactt aataggagat 11940tggatgttaa gatctggtca ctaagaggca
gaattgagat tcgaagccag tattttctac 12000ctggtatgtt ttaaattgca
gtaaggatct aagtgtagat atataataat aagattctat 12060tgatctctgc
aacaacagag agtgttagat ttgtttggaa aaaaatatta tcagccaaca
12120tcttctacca tttcagtata gcacagagta cccacccata tctccccacc
catcccccat 12180accagactgg ttattgattt tcatggtgac tggcctgaga
agattaaaaa aagtaatgct 12240accttattgg gagtgtccca tggaccaaga
tagcaactgt catagctacc gtcacactgc 12300tttgatcaag aagacccttt
gaggaactga aaacagaacc ttaggcacat ctgttgcttt 12360cgctcccatc
ctcctccaac agcctgggtg gtgcactcca caccctttca agtttccaaa
12420gcctcataca cctgctccct accccagcac ctggccaagg ctgtatccag
cactgggatg 12480aaaatgatac cccacctcca tcttgtttga tattactcta
tctcaagccc caggttagtc 12540cccagtccca atgcttttgc acagtcaaaa
ctcaacttgg aataatcagt atccttgaag 12600agttctgata tggtcactgg
gcccatatac catgtaagac atgtggaaaa gatgtttcat 12660ggggcccaga
cacgttctag aagtacctga gagtggcaaa aaatagttgt gctaaatagt
12720ttggccatct ttaggctgag agactaggaa atacagcgat ggactatatc
agcattgcag 12780gatagttgtc agtaaacacc ccacaaccca taacagaagt
attctcttct ttctatatcc 12840cttttccatc catgtagatg gctgtcttca
tatttgttct agacggccgg cc 12892946425DNAArtificial
SequenceVkP-IGKV1-39/J-Ck-delta2 94ggccggccca catgaaacaa tgggaaccat
gtgacaatca cagaggtgtt gttactatag 60caaaagggat tgttactctc cacatccctt
taagtaactt gaaggcctga tagacccacc 120ctctaagact tcattagaca
ttccctacga atggttatac tctcctgtat actcccaata 180caactctaaa
atatattatt ccatatagtc cttaggtttg tattaaagtt tgactttttt
240ccttcaaaat atctcttgtc acaacagcgg ctctagagag aaatacattc
cctccaggca 300aatctatgct gcgctggtct gacctgggac cctggggaca
ttgcccctgt gctgagttac 360taagatgagc cagccctgca gctgtgctca
gcctgcccca tgccctgctg attgatttgc 420atgttccaga gcacagcccc
ctgccctgaa gactttttta tgggctggtc gcaccctgtg 480caggagtcag
tctcagtcag gagccaccat ggacatgaga gtgcccgccc agctcctggg
540gctcctgcta ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt 600gtgctcagta ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa 660tatttgtttt tatgtttcca atctcaggtg
ccagatgtga catccagatg acccagagcc 720ccagcagcct gagcgccagc
gtgggcgaca gagtgaccat cacctgcaga gccagccaga 780gcatcagcag
ctacctgaac tggtatcagc agaagcccgg caaggccccc aagctgctga
840tctacgccgc cagctccctg cagagcggcg tgcccagcag attcagcggc
agcggctccg 900gcaccgactt caccctgacc atcagcagcc tgcagcccga
ggacttcgcc acctactact 960gccagcagag ctacagcacc ccccccacct
tcggccaggg caccaaggtg gagatcaaac 1020gtaagtacac ttttctcatc
tttttttatg tgtaagacac aggttttcat gttaggagtt 1080aaagtcagtt
cagaaaatct tgagaaaatg gagagggctc attatcagtt gacgtggcat
1140acagtgtcag attttctgtt tatcaagcta gtgagattag gggcaaaaag
aggctttagt 1200tgagaggaaa gtaattaata ctatggtcac catccaagag
attggatcgg agaataagca 1260tgagtagtta ttgagatctg ggtctgactg
caggtagcgt ggtcttctag acgtttaagt 1320gggagatttg gaggggatga
ggaatgaagg aacttcagga tagaaaaggg ctgaagtcaa 1380gttcagctcc
taaaatggat gtgggagcaa actttgaaga taaactgaat gacccagagg
1440atgaaacagc gcagatcaaa gaggggcctg gagctctgag aagagaagga
gactcatccg 1500tgttgagttt ccacaagtac tgtcttgagt
tttgcaataa aagtgggata gcagagttga 1560gtgagccgta ggctgagttc
tctcttttgt ctcctaagtt tttatgacta caaaaatcag 1620tagtatgtcc
tgaaataatc attaagctgt ttgaaagtat gactgcttgc catgtagata
1680ccatggcttg ctgaataatc agaagaggtg tgactcttat tctaaaattt
gtcacaaaat 1740gtcaaaatga gagactctgt aggaacgagt ccttgacaga
cagctcaagg ggtttttttc 1800ctttgtctca tttctacatg aaagtaaatt
tgaaatgatc ttttttatta taagagtaga 1860aatacagttg ggtttgaact
atatgtttta atggccacgg ttttgtaaga catttggtcc 1920tttgttttcc
cagttattac tcgattgtaa ttttatatcg ccagcaatgg actgaaacgg
1980tccgcaacct cttctttaca actgggtgac ctcgcggctg tgccagccat
ttggcgttca 2040ccctgccgct aagggccatg tgaacccccg cggtagcatc
ccttgctccg cgtggaccac 2100tttcctgagg cacagtgata ggaacagagc
cactaatctg aagagaacag agatgtgaca 2160gactacacta atgtgagaaa
aacaaggaaa gggtgactta ttggagattt cagaaataaa 2220atgcatttat
tattatattc ccttatttta attttctatt agggaattag aaagggcata
2280aactgcttta tccagtgtta tattaaaagc ttaatgtata taatctttta
gaggtaaaat 2340ctacagccag caaaagtcat ggtaaatatt ctttgactga
actctcacta aactcctcta 2400aattatatgt catattaact ggttaaatta
atataaattt gtgacatgac cttaactggt 2460taggtaggat atttttcttc
atgcaaaaat atgactaata ataatttagc acaaaaatat 2520ttcccaatac
tttaattctg tgatagaaaa atgtttaact cagctactat aatcccataa
2580ttttgaaaac tatttatttg gctacaccaa aggaagccat acagaggcta
atatcagagt 2640attcttggaa gagacaggag aaaatgaaag ccagtttctg
ctcttacctt atgtgcttgt 2700gttcagactc ccaaacatca ggagtgtcag
ataaactggt ctgaatctct gtctgaagca 2760tggaactgaa aagaatgtag
tttcagggaa gaaaggcaat agaaggaagc ctgagaatat 2820cttcaaaggg
tcagactcaa tttactttct aaagaagtag ctaggaacta gggaataact
2880tagaaacaac aagattgtat atatgtgcat cctggcccca ttgttcctta
tctgtaggga 2940taagcgtgct tttttgtgtg tctgtatata acataactgt
ttacacataa tacactgaaa 3000tggagccctt ccttgttact tcataccatc
ctctgtgctt ccttcctcag gggccgacgc 3060cgctcccacc gtgtccatct
tcccccccag catggaacag ctgacctctg gcggagccac 3120cgtggtctgc
ttcgtgaaca acttctaccc cagagacatc agcgtgaagt ggaagatcga
3180cggcagcgag cagagggacg gcgtgctgga cagcgtgacc gaccaggaca
gcaaggactc 3240cacctacagc atgagcagca ccctgagcct gaccaaggtg
gagtacgaga ggcacaacct 3300gtacacctgc gaggtggtgc acaagaccag
ctccagcccc gtggtcaagt ccttcaaccg 3360gaacgagtgt tgaagacaaa
ggtcctgaga cgccaccacc agctccccag ctccatccta 3420tcttcccttc
taaggtcttg gaggcttccc cacaagcgac ctaccactgt tgcggtgctc
3480caaacctcct ccccacctcc ttctcctcct cctccctttc cttggctttt
atcatgctaa 3540tatttgcaga aaatattcaa taaagtgagt ctttgcactt
gagatctctg tctttcttac 3600taaatggtag taatcagttg tttttccagt
tacctgggtt tctcttctaa agaagttaaa 3660tgtttagttg ccctgaaatc
caccacactt aaaggataaa taaaaccctc cacttgccct 3720ggttggctgt
ccactacatg gcagtccttt ctaaggttca cgagtactat tcatggctta
3780tttctctggg ccatggtagg tttgaggagg catacttcct agttttcttc
ccctaagtcg 3840tcaaagtcct gaagggggac agtctttaca agcacatgtt
ctgtaatctg attcaaccta 3900cccagtaaac ttggcgaagc aaagtagaat
cattatcaca ggaagcaaag gcaacctaaa 3960tgtgcaagca ataggaaaat
gtggaagccc atcatagtac ttggacttca tctgcttttg 4020tgccttcact
aagtttttaa acatgagctg gctcctatct gccattggca aggctgggca
4080ctacccacaa cctacttcaa ggacctctat accgtgagat tacacacata
catcaaaatt 4140tgggaaaagt tctaccaagc tgagagctga tcaccccact
cttaggtgct tatctctgta 4200caccagaaac cttaagaagc aaccagtatt
gagagactca tttatgaaag tctaaaactg 4260gatacaacca aaatgtccac
caacagttaa attatgacat gttcacaatt gagctattac 4320ttaataagga
gaattaataa aataaaactt aagagcatag tttaatctca taaacaagat
4380aataagcaaa acaaaacatt ttttcatcca tgtaagttta aaagcaggta
aaatttaaaa 4440ttaagagaga cataagtttt gaggtagcaa gatggaaact
ctggggcttg gggaatgttc 4500tgtctctctg tatgggatgt gaaagttact
attgtggaat tgggatctat gttcttcctg 4560tatatattgt atacttcata
ataacttcac ctaaagaaat atctaatacc cagtgcatac 4620ataaaagagg
atacaaggaa tgaatcatac gtcaaggcca gaaagacaat aaagtagggg
4680atccaggatc aaatctccca caaccttgag ccttctacta ttctgccttc
cagagctcaa 4740agtacaaaac acataattca aacacatgat ccctccttgg
ggtctcttcc ttcatgcatc 4800gaattagaaa tagccatgta taaaatgaga
tagaagagac cttcatcaac aggtcaaaga 4860atataggtaa ttttgtctgg
gtatgaagag cccacgtatc aaaggttaca ttagggaagg 4920aagaggacac
taacagtgac tttcattctc cccctcttcc tggaggcccc tgcatttagt
4980ccctcgtggg ctcatccact cagcacacat ttactaagca tcttctcagc
ctacactctg 5040aaggcagtgc agaataatgt tagtgtccct tcccccagtt
aatatgcagt ccagtttccc 5100tgctccttcc ctttctcagt ccacataagg
atgatgggaa aggacagtca ccaaatagga 5160gagggcaacc ctttgccttc
ctacctcttg agaatgtaca ttattatcca ctttttgaaa 5220cttcttttaa
ttgctttttt ttaatttgtc ttttcaaata gcataacctt gttcatccat
5280ttctgggaac caaatttatc aatcaacagt gcctctaatc tggctattaa
tacaaaaatg 5340cctcctcaaa atatatatgt tcgagtctta tctaaaacag
aacccacaat aaaaaagaag 5400aaagaataca tataagcatt tatataattc
tgagcaacct tgtgctttgt gaaaaaaata 5460taatctaatg tcacatgctg
tattcttttt atttaacact ggtgaaatta taccattaga 5520gagaaagagg
acagatcact gatcctagga tctagggatg ttacagataa gaaaacaaat
5580gtgacaaaga gctgtcacaa ggaggatctt caaggtcaca gaatcactgt
cttgatttca 5640gtggtggtta catacattta aatatgtgat aaaatgttgt
tgaactatat tcatatattg 5700taccaatgtc aaatgcttaa ttttggctct
atagtataat tatgcactaa ataactattt 5760ggacaaagaa aatgatgttt
acatcaaagg tgaggccata tttgttagga acataactta 5820aaaaccattt
tggataacta atgaaaagcc attttgtgtg ccttggcata tcatgcctaa
5880gctgtcacca gatagatcta ataagaccta agcctcagaa gcaagcccct
gcccagcaag 5940caggcagcac agataagagc taaacccagg acaggccatg
atatgctaat gaactacctt 6000caaggtggtg ttgctgacct agtgaaccag
ccccaagctg tgagccccaa tagcacaaag 6060ctactgccca aagaaattat
acaaaaattg gaactttggg aatggtgtgc aggatcgctc 6120tgctgtatgc
ctggaacaca gcttctctat gttttgtatt gataccagtc tagaagcttc
6180caaaactttc tcactgaaga agattcccca tgtgggaccc ctacagactc
ttttgcccaa 6240acaactgctt ccctcctggt gtgatcatgg accaagatag
caactgtcat agctaccgtc 6300acactgcttt gatcaagaag accctttgag
gaactgaaaa cagaacctta ggcacatctg 6360ttgctttcgc tcccatcctc
ctccaacagc atggctgtct tcatatttgt tctagacggc 6420cggcc
64259513382DNAArtificial SequenceVkP-IGLV2-14/J-Ck 95ggccggccca
catgaaacaa tgggaaccat gtgacaatca cagaggtgtt gttactatag 60caaaagggat
tgttactctc cacatccctt taagtaactt gaaggcctga tagacccacc
120ctctaagact tcattagaca ttccctacga atggttatac tctcctgtat
actcccaata 180caactctaaa atatattatt ccatatagtc cttaggtttg
tattaaagtt tgactttttt 240ccttcaaaat atctcttgtc acaacagcgg
ctctagagag aaatacattc cctccaggca 300aatctatgct gcgctggtct
gacctgggac cctggggaca ttgcccctgt gctgagttac 360taagatgagc
cagccctgca gctgtgctca gcctgcccca tgccctgctg attgatttgc
420atgttccaga gcacagcccc ctgccctgaa gactttttta tgggctggtc
gcaccctgtg 480caggagtcag tctcagtcag gagccaccat ggacatgaga
gtgcccgccc agctcctggg 540gctcctgcta ctctggctcc gaggtaagga
tggagaacac taggaattta ctcagccagt 600gtgctcagta ctgactggaa
cttcagggaa gttctctgat aacatgatta atagtaagaa 660tatttgtttt
tatgtttcca atctcaggtg ccagatgtca gtctgccctg acccagcccg
720cctctgtgtc tggcagccct ggccagagca tcaccatcag ctgcaccggc
accagcagcg 780acgtgggcgg ctacaactac gtgtcctggt atcagcagca
ccccggcaag gcccccaagc 840tgatgatcta cgaggtgtcc aacagaccca
gcggcgtgag caacagattc agcggcagca 900agagcggcaa caccgccagc
ctgaccatca gcggcctcca ggctgaggac gaggccgact 960actactgcag
cagctacacc agcagctcca ccctggtgtt tggcggcgga acaaagctga
1020ccgtgctgcg taagtacact tttctcatct ttttttatgt gtaagacaca
ggttttcatg 1080ttaggagtta aagtcagttc agaaaatctt gagaaaatgg
agagggctca ttatcagttg 1140acgtggcata cagtgtcaga ttttctgttt
atcaagctag tgagattagg ggcaaaaaga 1200ggctttagtt gagaggaaag
taattaatac tatggtcacc atccaagaga ttggatcgga 1260gaataagcat
gagtagttat tgagatctgg gtctgactgc aggtagcgtg gtcttctaga
1320cgtttaagtg ggagatttgg aggggatgag gaatgaagga acttcaggat
agaaaagggc 1380tgaagtcaag ttcagctcct aaaatggatg tgggagcaaa
ctttgaagat aaactgaatg 1440acccagagga tgaaacagcg cagatcaaag
aggggcctgg agctctgaga agagaaggag 1500actcatccgt gttgagtttc
cacaagtact gtcttgagtt ttgcaataaa agtgggatag 1560cagagttgag
tgagccgtag gctgagttct ctcttttgtc tcctaagttt ttatgactac
1620aaaaatcagt agtatgtcct gaaataatca ttaagctgtt tgaaagtatg
actgcttgcc 1680atgtagatac catggcttgc tgaataatca gaagaggtgt
gactcttatt ctaaaatttg 1740tcacaaaatg tcaaaatgag agactctgta
ggaacgagtc cttgacagac agctcaaggg 1800gtttttttcc tttgtctcat
ttctacatga aagtaaattt gaaatgatct tttttattat 1860aagagtagaa
atacagttgg gtttgaacta tatgttttaa tggccacggt tttgtaagac
1920atttggtcct ttgttttccc agttattact cgattgtaat tttatatcgc
cagcaatgga 1980ctgaaacggt ccgcaacctc ttctttacaa ctgggtgacc
tcgcggctgt gccagccatt 2040tggcgttcac cctgccgcta agggccatgt
gaacccccgc ggtagcatcc cttgctccgc 2100gtggaccact ttcctgaggc
acagtgatag gaacagagcc actaatctga agagaacaga 2160gatgtgacag
actacactaa tgtgagaaaa acaaggaaag ggtgacttat tggagatttc
2220agaaataaaa tgcatttatt attatattcc cttattttaa ttttctatta
gggaattaga 2280aagggcataa actgctttat ccagtgttat attaaaagct
taatgtatat aatcttttag 2340aggtaaaatc tacagccagc aaaagtcatg
gtaaatattc tttgactgaa ctctcactaa 2400actcctctaa attatatgtc
atattaactg gttaaattaa tataaatttg tgacatgacc 2460ttaactggtt
aggtaggata tttttcttca tgcaaaaata tgactaataa taatttagca
2520caaaaatatt tcccaatact ttaattctgt gatagaaaaa tgtttaactc
agctactata 2580atcccataat tttgaaaact atttattagc ttttgtgttt
gacccttccc tagccaaagg 2640caactattta aggacccttt aaaactcttg
aaactacttt agagtcatta agttatttaa 2700ccacttttaa ttactttaaa
atgatgtcaa ttccctttta actattaatt tattttaagg 2760ggggaaaggc
tgctcataat tctattgttt ttcttggtaa agaactctca gttttcgttt
2820ttactacctc tgtcacccaa gagttggcat ctcaacagag gggactttcc
gagaggccat 2880ctggcagttg cttaagatca gaagtgaagt ctgccagttc
ctcccaggca ggtggcccag 2940attacagttg acctgttctg gtgtggctaa
aaattgtccc atgtggttac aaaccattag 3000accagggtct gatgaattgc
tcagaatatt tctggacacc caaatacaga ccctggctta 3060aggccctgtc
catacagtag gtttagcttg gctacaccaa aggaagccat acagaggcta
3120atatcagagt attcttggaa gagacaggag aaaatgaaag ccagtttctg
ctcttacctt 3180atgtgcttgt gttcagactc ccaaacatca ggagtgtcag
ataaactggt ctgaatctct 3240gtctgaagca tggaactgaa aagaatgtag
tttcagggaa gaaaggcaat agaaggaagc 3300ctgagaatat cttcaaaggg
tcagactcaa tttactttct aaagaagtag ctaggaacta 3360gggaataact
tagaaacaac aagattgtat atatgtgcat cctggcccca ttgttcctta
3420tctgtaggga taagcgtgct tttttgtgtg tctgtatata acataactgt
ttacacataa 3480tacactgaaa tggagccctt ccttgttact tcataccatc
ctctgtgctt ccttcctcag 3540gggccgacgc cgctcccacc gtgtccatct
tcccccccag catggaacag ctgacctctg 3600gcggagccac cgtggtctgc
ttcgtgaaca acttctaccc cagagacatc agcgtgaagt 3660ggaagatcga
cggcagcgag cagagggacg gcgtgctgga cagcgtgacc gaccaggaca
3720gcaaggactc cacctacagc atgagcagca ccctgagcct gaccaaggtg
gagtacgaga 3780ggcacaacct gtacacctgc gaggtggtgc acaagaccag
ctccagcccc gtggtcaagt 3840ccttcaaccg gaacgagtgt tgaagacaaa
ggtcctgaga cgccaccacc agctccccag 3900ctccatccta tcttcccttc
taaggtcttg gaggcttccc cacaagcgac ctaccactgt 3960tgcggtgctc
caaacctcct ccccacctcc ttctcctcct cctccctttc cttggctttt
4020atcatgctaa tatttgcaga aaatattcaa taaagtgagt ctttgcactt
gagatctctg 4080tctttcttac taaatggtag taatcagttg tttttccagt
tacctgggtt tctcttctaa 4140agaagttaaa tgtttagttg ccctgaaatc
caccacactt aaaggataaa taaaaccctc 4200cacttgccct ggttggctgt
ccactacatg gcagtccttt ctaaggttca cgagtactat 4260tcatggctta
tttctctggg ccatggtagg tttgaggagg catacttcct agttttcttc
4320ccctaagtcg tcaaagtcct gaagggggac agtctttaca agcacatgtt
ctgtaatctg 4380attcaaccta cccagtaaac ttggcgaagc aaagtagaat
cattatcaca ggaagcaaag 4440gcaacctaaa tgtgcaagca ataggaaaat
gtggaagccc atcatagtac ttggacttca 4500tctgcttttg tgccttcact
aagtttttaa acatgagctg gctcctatct gccattggca 4560aggctgggca
ctacccacaa cctacttcaa ggacctctat accgtgagat tacacacata
4620catcaaaatt tgggaaaagt tctaccaagc tgagagctga tcaccccact
cttaggtgct 4680tatctctgta caccagaaac cttaagaagc aaccagtatt
gagagactca tttatgaaag 4740tctaaaactg gatacaacca aaatgtccac
caacagttaa attatgacat gttcacaatt 4800gagctattac ttaataagga
gaattaataa aataaaactt aagagcatag tttaatctca 4860taaacaagat
aataagcaaa acaaaacatt ttttcatcca tgtaagttta aaagcaggta
4920aaatttaaaa ttaagagaga cataagtttt gaggtagcaa gatggaaact
ctggggcttg 4980gggaatgttc tgtctctctg tatgggatgt gaaagttact
attgtggaat tgggatctat 5040gttcttcctg tatatattgt atacttcata
ataacttcac ctaaagaaat atctaatacc 5100cagtgcatac ataaaagagg
atacaaggaa tgaatcatac gtcaaggcca gaaagacaat 5160aaagtagggg
atccaggatc aaatctccca caaccttgag ccttctacta ttctgccttc
5220cagagctcaa agtacaaaac acataattca aacacatgat ccctccttgg
ggtctcttcc 5280ttcatgcatc gaattagaaa tagccatgta taaaatgaga
tagaagagac cttcatcaac 5340aggtcaaaga atataggtaa ttttgtctgg
gtatgaagag cccacgtatc aaaggttaca 5400ttagggaagg aagaggacac
taacagtgac tttcattctc cccctcttcc tggaggcccc 5460tgcatttagt
ccctcgtggg ctcatccact cagcacacat ttactaagca tcttctcagc
5520ctacactctg aaggcagtgc agaataatgt tagtgtccct tcccccagtt
aatatgcagt 5580ccagtttccc tgctccttcc ctttctcagt ccacataagg
atgatgggaa aggacagtca 5640ccaaatagga gagggcaacc ctttgccttc
ctacctcttg agaatgtaca ttattatcca 5700ctttttgaaa cttcttttaa
ttgctttttt ttaatttgtc ttttcaaata gcataacctt 5760gttcatccat
ttctgggaac caaatttatc aatcaacagt gcctctaatc tggctattaa
5820tacaaaaatg cctcctcaaa atatatatgt tcgagtctta tctaaaacag
aacccacaat 5880aaaaaagaag aaagaataca tataagcatt tatataattc
tgagcaacct tgtgctttgt 5940gaaaaaaata taatctaatg tcacatgctg
tattcttttt atttaacact ggtgaaatta 6000taccattaga gagaaagagg
acagatcact gatcctagga tctagggatg ttacagataa 6060gaaaacaaat
gtgacaaaga gctgtcacaa ggaggatctt caaggtcaca gaatcactgt
6120cttgatttca gtggtggtta catacattta aatatgtgat aaaatgttgt
tgaactatat 6180tcatatattg taccaatgtc aaatgcttaa ttttggctct
atagtataat tatgcactaa 6240ataactattt ggacaaagaa aatgatgttt
acatcaaagg tgaggccata tttgttagga 6300acataactta aaaaccattt
tggataacta atgaaaagcc attttgtgtg ccttggcata 6360tcatgcctaa
gctgtcacca gatagatcta ataagaccta agcctcagaa gcaagcccct
6420gcccagcaag caggcagcac agataagagc taaacccagg acaggccatg
atatgctaat 6480gaactacctt caaggtggtg ttgctgacct agtgaaccag
ccccaagctg tgagccccaa 6540tagcacaaag ctactgccca aagaaattat
acaaaaattg gaactttggg aatggtgtgc 6600aggatcgctc tgctgtatgc
ctggaacaca gcttctctat gttttgtatt gataccagtc 6660tagaagcttc
caaaactttc tcactgaaga agattcccca tgtgggaccc ctacagactc
6720ttttgcccaa acaactgctt ccctcctggt gtgatatctg ttttgctttt
atgttagcat 6780aatattataa ggaatgtttg tgtgaataaa ccaaacatat
tttaaaagca aatattgtat 6840gcacatccta attgctaaaa agtttacagc
taatagtccc atgctctcca caatactgga 6900tccaaataag tcctaatttc
aatgttgggc atctttacag agagaaagac attaaaaatg 6960aagagacatg
cagagagtgc accatgccat cgtggagaca gactgaagtg acacaactgt
7020tagtcaaaga ggattaagga cttccagaag ccaccaaagg aaggaggtat
gaagtggttt 7080ctccctcaga gtatccagag gagactaaac caaccaacac
ctttttgctt aagacttctt 7140gccttcagga ctgtgagaag gtagcttcct
attgttctaa gccccagtat gtggcatttt 7200gttaaggtag agtcaagaaa
ccaataaaat gcagacagac aaaaggatag ctgagttttc 7260caggcccttc
cttcttattt ttggttttgt tggtggtggt ggtggtggtg gtgatggtgg
7320tggttttgtt tatgttttgt ttggggagtt ttttggggtt tttttgggtt
ttgtttttgt 7380tgttgttttg ggggtttttg ttgttgttgt tgtttgcttt
tttgtttttt gttttttgtt 7440tttttgagac agtgtttctc tgtatagccc
tggctgtcct ggagttcctt ctatctctaa 7500tgtctacatc tcagagggga
tcctctaatt tcaaatgagc agtagctctc catttttagc 7560tcttatttat
tcatttattt acttacttac ttattgtctg tagatgaaag aattttggag
7620tgggaaaggg ttcatgagcc cccagcaact aatgaggagc tacagacaat
tgatgtttct 7680ggggaaagga gactcagttt ctttgagagt atagcttctg
atgggtcaac catgttcctg 7740tggctgatgt cacacccagg agtatgcaga
caacagaaac tggagttaat gagttgtttt 7800aaaaataaaa aagggcatga
agcttgggat agaaattaag gataaataca attaaataca 7860ggaaattctg
aaagaattaa taaaaacatt tcttttttta aaaaaaaatc cagaattagc
7920tatgcttctt caaaattgct tctggagaac tttacaagtt aaataagtta
tattgtagaa 7980aaggtagaga ggagaatagt ggaagagaga gataaggaga
cttcaaaagg agtggaggga 8040gatagaggag gagaaagcag aagcaatggc
tgatagacac aggataagag ggaacagaaa 8100ggagaaagag gaagccagga
tgggtatttc tttgcctatc tgtgacttgc acatggtctt 8160ggcaattatt
gatgagttca aggcttaatt cttcacttgt gccaactcaa cagagtcttt
8220ctttcttata accaggcccc cagtatgctc atgtatgtat caggtcctct
tatctcctta 8280tagcaatcct gtttataact gggtaacttt gtgaagggaa
ggaagtgcac actgagatgt 8340gctacaactt tttaatacaa aattttgaag
agtttgtaca atgtatgtat aattaataat 8400taatattatg cactttagat
tttgatttca actcaagata ctaattctat atatatgggt 8460taaatcaata
tattaataag tttaatttca catgcttatt tttattgtgg ttttcgagac
8520agggtttctc tgtatagccc tggctgtcct ggaacccact ttgtagacca
ggctggcctc 8580aaactcagaa acctacctgc ctctgcctct gcctctgcct
ctgcctctgc ctctgcctct 8640gcctctgcct ctgcctctgc ctctgcctct
gcctctgcct ctgcctctgc ctctgcctct 8700gcctctgcct ctgcctctgc
ctctgcctct gcctctgcct ctgcctctgc ctctgcctct 8760gcctctgcct
ctgcctagtg ctggaattaa aggtttgcgc caccacgccc ggtgaaattt
8820ttaaacttta tatatgtctc attctatttc tatcagatag gactgtgtag
actgtgctaa 8880actaataaat gtgccctcaa aagtaatcgc aagttgtatt
gttgttgttt tgctttgctt 8940tgctttgctt tgctttgctt tgctttgctt
tgctttgctt tgctttgctt tgctttgctt 9000tgctttgctt tgctttgctt
tgctttgctt tgcttttttg ttttgggttt ttttccgggg 9060gagggagggt
ggagaaagaa tcttactatg aagctctgac tgtcctggga actcactata
9120tagatcaggc ttgattcaac tcatagagat ctgccttctt ctgcctccca
agtgctggga 9180ataaaggcat acacctccat gcccagatag tgatcccaag
ttttagcaaa agtttctaga 9240cttgacatta atcgatggag atagacatga
attacacaaa gaactaatgt ggagtttacc 9300tgaatcatac tctatacttt
atcagagatt aaattaacat ttaataatcc agtgccaggc 9360tagaggcacc
attcaatggc agtgtttgcc atcatgcata ggcttagtct tcagtgctga
9420aaggcattgg gggcaatatt actcattata cagatgagaa actgggaaag
acttgcctca 9480gattctctac tgaaaggctg agtttgtggc ttctagaaaa
tcttttactt tcaatatttt 9540taatgtataa tttttttatt tccactgatt
ttatttttta tttttaacat ttataagaaa 9600taaatgcaat aaaccaaata
catggacaaa aaaatacaag aatcatatga tcacctcaat 9660ggaaggaaaa
aaaaagaaag aaaaagtctt tgataagatt caacattcat tcttttttta
9720ttagatattt tcttcattta catttcaaat gctatcccca aagcccccta
taccttcccc 9780tgccctgctc cccaacccac ccactcctgc tttctggccc
tggcattcct ctgtactgag 9840gcatatgatc ttcaaaaaac caagggcctc
tcctctcatt ggtggccgac tattaggcca 9900tcttttgcta catatgcaac
tagagacaca gctctggggg ttactggtta gttcatattg 9960ttagtcctcc
tatagagttg cagacccctt tagctccttg gatactttct ctagttcctt
10020cattaggggc cctgtgtccc atccaataga tgactgtgag
catccacttc tgtatttgcc 10080aggcactggc atagcctcac gagaaagaga
gagctatgtc aggatcctgt cagtaaaatc 10140tttctggcat atgcaatagt
atctgggttt ggtggttgta tatgggatgg atccccaagt 10200ggagcagtct
ctgaatggtc cttccttcca tctcagctcc aaactttgtc tctataactc
10260cttccatggg tattttgttc cccattctaa gaaggagtga agaatccaca
ctttggtctt 10320ccttcttctt gagtttcata tgttgcatct tggatattct
aagtttctgg gttaatatcc 10380acgtatcagt gagtgcatat catgcgtgtt
attttgtgat tagtttacct cactcaggat 10440gatatcctcc agatgcatcc
atttgcctaa gaatttcatt aattcactgt ttttaattgc 10500tgaatagtac
tccattgtgt aaatgtacca cattttctgt atccattcct ctgttgaggg
10560gcatctgggt tctttccagc ttctggctat tataaataag gctgctatga
gcatagcgga 10620gcatgtgtcc ttatcaagtt ggaacatctt ctaggtatat
gcccaggaga ggaattgctg 10680gatcttccgg tagtaccatc aacatgcatt
cttaataaaa gccctagaac aaggaggact 10740gtaggaaaca tattccaaca
taataaaggt tatgtatgac aaactcatga ccaatatcat 10800cctaaatgaa
tgaaaccatt aataagctcc attaaaatca gaggactgcc cactatccct
10860acttctcatc cataatgaga ttgaagcatt agctggagca ataaggcaag
agaagggata 10920caaatgggaa aatattaagt caaattgttt tcaattgaag
attatattat cttataccca 10980atgacctcaa attttgacta gaaaaattgt
agaaattatc aataatttca gcaaagtgtt 11040atgatgcacc acatccttat
tcttctcccc agcttctgct tgcttctctc ttcttgctct 11100tcatcctttc
tgtccttcca tctgcctgca ctcttgtctc aagactgagt gcagcgtgta
11160actctcctgt gactgagtat ctcacaaaac gttctacctg ccaaacctgg
atgagccctt 11220tgtctttctg aagctatgag gctctctaca tagactcaag
aaggaaatga cagggaggag 11280gtaataatga agtggggaag gctgacatta
gcattgctcc tgtgtggctc cttaatttct 11340catacttcac actgagatgt
tattaactgt gactcatagg tgaagaagcc agagctaagg 11400ttctcatatt
tgagtgttat agaatgagta gagcagtagt tctcaaacta tgggtcatga
11460ctcctttatg ggtcaaacta ccctttcaca caggttgcat atcagatatc
ctaattttat 11520atacatatat atatgcatat gtatatatat atatttcaca
acagtaggaa aattatttag 11580taatcatttt atagttgtgg gtcatggcaa
catgaggaac tgtattaaag ggttgcagca 11640ttaggaatgt tgagacccac
tgtaatagag aatgaggctt aaggcagggc tataaagccc 11700aatggaccat
gtgccttttc caacatttgc cacatggtaa gctctgtata gactttttaa
11760agaacattgg tttgtaattt taaatggata agggtcttca ctgtctatca
cccatctata 11820taataaatac ataagttttg attccaccat ggattcaaat
gcaaaaatcc tcaacctaag 11880acatagcagt gaaacattga tgaccaaata
ggaaatccat gtagagacct tctatcttct 11940gatggctcca caggcaccat
cttgcaacag agttctactt tgctaccagt aatgaataca 12000gtgtctcaac
tcctgccatt gaatcttcag gaagcccctg aaatgacttg tactacacca
12060tttcttaaag acagaaaagc taagacttag agggaataaa tgtcatgcct
gagatcatgc 12120aaccaattaa gtccaacttg gcctgatcaa gaggcacaat
tcaaaagcaa tgttgttcct 12180tcactagctc ttgtgtatgg ttgctgattc
cggaagcaaa gtatcagtga atatccctag 12240tgggaaaaga cttggaaatc
aaatgtctca tttaacagat taggagatga aacggtagac 12300tctgtgtagt
tgtacacccc tgtgatccca tcgctaggaa gactgaggca ggaagtcctc
12360gagctcaaac cagcttaggc tacacagaga aactatctaa aaaataatta
ctaactactt 12420aataggagat tggatgttaa gatctggtca ctaagaggca
gaattgagat tcgaagccag 12480tattttctac ctggtatgtt ttaaattgca
gtaaggatct aagtgtagat atataataat 12540aagattctat tgatctctgc
aacaacagag agtgttagat ttgtttggaa aaaaatatta 12600tcagccaaca
tcttctacca tttcagtata gcacagagta cccacccata tctccccacc
12660catcccccat accagactgg ttattgattt tcatggtgac tggcctgaga
agattaaaaa 12720aagtaatgct accttattgg gagtgtccca tggaccaaga
tagcaactgt catagctacc 12780gtcacactgc tttgatcaag aagacccttt
gaggaactga aaacagaacc ttaggcacat 12840ctgttgcttt cgctcccatc
ctcctccaac agcctgggtg gtgcactcca caccctttca 12900agtttccaaa
gcctcataca cctgctccct accccagcac ctggccaagg ctgtatccag
12960cactgggatg aaaatgatac cccacctcca tcttgtttga tattactcta
tctcaagccc 13020caggttagtc cccagtccca atgcttttgc acagtcaaaa
ctcaacttgg aataatcagt 13080atccttgaag agttctgata tggtcactgg
gcccatatac catgtaagac atgtggaaaa 13140gatgtttcat ggggcccaga
cacgttctag aagtacctga gagtggcaaa aaatagttgt 13200gctaaatagt
ttggccatct ttaggctgag agactaggaa atacagcgat ggactatatc
13260agcattgcag gatagttgtc agtaaacacc ccacaaccca taacagaagt
attctcttct 13320ttctatatcc cttttccatc catgtagatg gctgtcttca
tatttgttct agacggccgg 13380cc 13382964638DNAArtificial
SequencepSELECT-IGKV1-39/J-Ck 96gcggccgcaa taaaatatct ttattttcat
tacatctgtg tgttggtttt ttgtgtgaat 60cgtaactaac atacgctctc catcaaaaca
aaacgaaaca aaacaaacta gcaaaatagg 120ctgtccccag tgcaagtgca
ggtgccagaa catttctcta tcgaaggatc tgcgatcgct 180ccggtgcccg
tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
240gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
gaaagtgatg 300tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
accgtatata agtgcagtag 360tcgccgtgaa cgttcttttt cgcaacgggt
ttgccgccag aacacagctg aagcttcgag 420gggctcgcat ctctccttca
cgcgcccgcc gccctacctg aggccgccat ccacgccggt 480tgagtcgcgt
tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg
540taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
agcctaccta 600gactcagccg gctctccacg ctttgcctga ccctgcttgc
tcaactctac gtctttgttt 660cgttttctgt tctgcgccgt tacagatcca
agctgtgacc ggcgcctacc tgagatcacc 720ggcgtgtcga cgccaccatg
gacatgagag tgcccgccca gctcctgggg ctcctgctac 780tctggctccg
aggtaaggat ggagaacact aggaatttac tcagccagtg tgctcagtac
840tgactggaac ttcagggaag ttctctgata acatgattaa tagtaagaat
atttgttttt 900atgtttccaa tctcaggtgc cagatgtgac atccagatga
cccagagccc cagcagcctg 960agcgccagcg tgggcgacag agtgaccatc
acctgcagag ccagccagag catcagcagc 1020tacctgaact ggtatcagca
gaagcccggc aaggccccca agctgctgat ctacgccgcc 1080agctccctgc
agagcggcgt gcccagcaga ttcagcggca gcggctccgg caccgacttc
1140accctgacca tcagcagcct gcagcccgag gacttcgcca cctactactg
ccagcagagc 1200tacagcaccc cccccacctt cggccagggc accaaggtgg
agatcaagag agccgacgcc 1260gctcccaccg tgtccatctt cccccccagc
atggaacagc tgacctctgg cggagccacc 1320gtggtctgct tcgtgaacaa
cttctacccc agagacatca gcgtgaagtg gaagatcgac 1380ggcagcgagc
agagggacgg cgtgctggac agcgtgaccg accaggacag caaggactcc
1440acctacagca tgagcagcac cctgagcctg accaaggtgg agtacgagag
gcacaacctg 1500tacacctgcg aggtggtgca caagaccagc tccagccccg
tggtcaagtc cttcaaccgg 1560aacgagtgtt gagctagctg gccagacatg
ataagataca ttgatgagtt tggacaaacc 1620acaactagaa tgcagtgaaa
aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 1680tttgtaacca
ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
1740tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
ctacaaatgt 1800ggtatggaat tctaaaatac agcatagcaa aactttaacc
tccaaatcaa gcctctactt 1860gaatcctttt ctgagggatg aataaggcat
aggcatcagg ggctgttgcc aatgtgcatt 1920agctgtttgc agcctcacct
tctttcatgg agtttaagat atagtgtatt ttcccaaggt 1980ttgaactagc
tcttcatttc tttatgtttt aaatgcactg acctcccaca ttcccttttt
2040agtaaaatat tcagaaataa tttaaataca tcattgcaat gaaaataaat
gttttttatt 2100aggcagaatc cagatgctca aggcccttca taatatcccc
cagtttagta gttggactta 2160gggaacaaag gaacctttaa tagaaattgg
acagcaagaa agcgagcttc tagcgaattc 2220tcgactcatt cctttgccct
cggacgagtg ctggggcgtc ggtttccact atcggcgagt 2280acttctacac
agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg
2340acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca
agctgcatca 2400tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa
gaccaatgcg gagcatatac 2460gcccggagcc gcggcgatcc tgcaagctcc
ggatgcctcc gctcgaagta gcgcgtctgc 2520tgctccatac aagccaacca
cggcctccag aagaagatgt tggcgacctc gtattgggaa 2580tccccgaaca
tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg
2640acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca
gtcctcggcc 2700caaagcatca gctcatcgag agcctgcgcg acggacgcac
tgacggtgtc gtccatcaca 2760gtttgccagt gatacacatg gggatcagca
atcgcgcata tgaaatcacg ccatgtagtg 2820tattgaccga ttccttgcgg
tccgaatggg ccgaacccgc tcgtctggct aagatcggcc 2880gcagcgatcg
catccatgag ctccgcgacg ggttgcagaa cagcgggcag ttcggtttca
2940ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt
caggctctcg 3000ctgaattccc caatgtcaag cacttccgga atcgggagcg
cggccgatgc aaagtgccga 3060taaacataac gatctttgta gaaaccatcg
gcgcagctat ttacccgcag gacatatcca 3120cgccctccta catcgaagct
gaaagcacga gattcttcgc cctccgagag ctgcatcagg 3180tcggagacgc
tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca
3240ggctttttca tgatggccct cctatagtga gtcgtattat actatgccga
tatactatgc 3300cgatgattaa ttgtcaaaac agcgtggatg gcgtctccag
cttatctgac ggttcactaa 3360acgagctctg cttatataga cctcccaccg
tacacgccta ccgcccattt gcgtcaatgg 3420ggcggagttg ttacgacatt
ttggaaagtc ccgttgattt actagtcaaa acaaactccc 3480attgacgtca
atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
3540attgatgtac tgccaaaacc gcatcatcat ggtaatagcg atgactaata
cgtagatgta 3600ctgccaagta ggaaagtccc ataaggtcat gtactgggca
taatgccagg cgggccattt 3660accgtcattg acgtcaatag ggggcgtact
tggcatatga tacacttgat gtactgccaa 3720gtgggcagtt taccgtaaat
actccaccca ttgacgtcaa tggaaagtcc ctattggcgt 3780tactatggga
acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
3840aggcgggcca tttaccgtaa gttatgtaac gcctgcaggt taattaagaa
catgtgagca 3900aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg 3960ctccgccccc ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg 4020acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4080ccgaccctgc
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
4140tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc 4200tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt 4260gagtccaacc cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt 4320agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 4380tacactagaa
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
4440agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 4500tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 4560acggggtctg acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catggctagt 4620taattaacat ttaaatca
4638975349DNAArtificial SequencepSelect-IGVL2-14/J-Ck 97gcggccgcaa
taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat 60cgtaactaac
atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg
120ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgaaggatc
tgcgatcgct 180ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
ccccgagaag ttggggggag 240gggtcggcaa ttgaacgggt gcctagagaa
ggtggcgcgg ggtaaactgg gaaagtgatg 300tcgtgtactg gctccgcctt
tttcccgagg gtgggggaga accgtatata agtgcagtag 360tcgccgtgaa
cgttcttttt cgcaacgggt ttgccgccag aacacagctg aagcttcgag
420gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
ccacgccggt 480tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg
aactgcgtcc gccgtctagg 540taagtttaaa gctcaggtcg agaccgggcc
tttgtccggc gctcccttgg agcctaccta 600gactcagccg gctctccacg
ctttgcctga ccctgcttgc tcaactctac gtctttgttt 660cgttttctgt
tctgcgccgt tacagatcca agctgtgacc ggcgcctacc tgagatcacc
720ggcgtgtcga cgccaccatg gacatgagag tgcccgccca gctcctgggg
ctcctgctac 780tctggctccg aggtaaggat ggagaacact aggaatttac
tcagccagtg tgctcagtac 840tgactggaac ttcagggaag ttctctgata
acatgattaa tagtaagaat atttgttttt 900atgtttccaa tctcaggtgc
cagatgtcag tctgccctga cccagcccgc ctctgtgtct 960ggcagccctg
gccagagcat caccatcagc tgcaccggca ccagcagcga cgtgggcggc
1020tacaactacg tgtcctggta tcagcagcac cccggcaagg cccccaagct
gatgatctac 1080gaggtgtcca acagacccag cggcgtgagc aacagattca
gcggcagcaa gagcggcaac 1140accgccagcc tgaccatcag cggcctccag
gctgaggacg aggccgacta ctactgcagc 1200agctacacca gcagctccac
cctggtgttt ggcggcggaa caaagctgac cgtgctgaga 1260gccgacgccg
ctcccaccgt gtccatcttc ccccccagca tggaacagct gacctctggc
1320ggagccaccg tggtctgctt cgtgaacaac ttctacccca gagacatcag
cgtgaagtgg 1380aagatcgacg gcagcgagca gagggacggc gtgctggaca
gcgtgaccga ccaggacagc 1440aaggactcca cctacagcat gagcagcacc
ctgagcctga ccaaggtgga gtacgagagg 1500cacaacctgt acacctgcga
ggtggtgcac aagaccagct ccagccccgt ggtcaagtcc 1560ttcaaccgga
acgagtgttg agctagctgg ccagacatga taagatacat tgatgagttt
1620ggacaaacca caactagact gactcagcct gcctccgtgt ctgggtctcc
tggacagtcg 1680atcaccatct cctgcactgg aaccagcagt gacgttggtg
gttataacta tgtctcctgg 1740taccaacagc acccaggcaa agcccccaaa
ctcatgattt atgaggtcag taatcggccc 1800tcaggggttt ctaatcgctt
ctctggctcc aagtctggca acacggcctc cctgaccatc 1860tctgggctcc
aggctgagga cgaggctgat tattactgca gctcatatac aagcagcagc
1920actctcgtat tcggcggagg gaccaagctg accgtcctac gggctgatgc
tgcaccaact 1980gtatccatct tcccaccatc catggaacag ttaacatctg
gaggtgccac agtcgtgtgc 2040ttcgtgaaca acttctatcc cagagacatc
agtgtcaagt ggaagattga tggcagtgaa 2100caacgagatg gtgtcctgga
cagtgttact gatcaggaca gcaaagacag cacgtacagc 2160atgagcagca
ccctctcgtt gaccaaggtt gaatatgaaa ggcataacct ctatacctgt
2220gaggttgttc ataagacatc atcctcaccc gtcgtcaaga gcttcaacag
gaatgagtgt 2280taggctagct ggccagacat gataagatac attgatgagt
ttggacaaac cacaactaga 2340atgcagtgaa aaaaatgctt tatttgtgaa
atttgtgatg ctattgcttt atttgtaacc 2400attataagct gcaataaaca
agttaacaac aacaattgca ttcattttat gtttcaggtt 2460cagggggagg
tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggaa
2520ttctaaaata cagcatagca aaactttaac ctccaaatca agcctctact
tgaatccttt 2580tctgagggat gaataaggca taggcatcag gggctgttgc
caatgtgcat tagctgtttg 2640cagcctcacc ttctttcatg gagtttaaga
tatagtgtat tttcccaagg tttgaactag 2700ctcttcattt ctttatgttt
taaatgcact gacctcccac attccctttt tagtaaaata 2760ttcagaaata
atttaaatac atcattgcaa tgaaaataaa tgttttttat taggcagaat
2820ccagatgctc aaggcccttc ataatatccc ccagtttagt agttggactt
agggaacaaa 2880ggaaccttta atagaaattg gacagcaaga aagcgagctt
ctagcgaatt ctcgactcat 2940tcctttgccc tcggacgagt gctggggcgt
cggtttccac tatcggcgag tacttctaca 3000cagccatcgg tccagacggc
cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg 3060gctccggatc
ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
3120ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata
cgcccggagc 3180cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
agcgcgtctg ctgctccata 3240caagccaacc acggcctcca gaagaagatg
ttggcgacct cgtattggga atccccgaac 3300atcgcctcgc tccagtcaat
gaccgctgtt atgcggccat tgtccgtcag gacattgttg 3360gagccgaaat
ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
3420agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac
agtttgccag 3480tgatacacat ggggatcagc aatcgcgcat atgaaatcac
gccatgtagt gtattgaccg 3540attccttgcg gtccgaatgg gccgaacccg
ctcgtctggc taagatcggc cgcagcgatc 3600gcatccatga gctccgcgac
gggttgcaga acagcgggca gttcggtttc aggcaggtct 3660tgcaacgtga
caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc
3720ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
ataaacataa 3780cgatctttgt agaaaccatc ggcgcagcta tttacccgca
ggacatatcc acgccctcct 3840acatcgaagc tgaaagcacg agattcttcg
ccctccgaga gctgcatcag gtcggagacg 3900ctgtcgaact tttcgatcag
aaacttctcg acagacgtcg cggtgagttc aggctttttc 3960atgatggccc
tcctatagtg agtcgtatta tactatgccg atatactatg ccgatgatta
4020attgtcaaaa cagcgtggat ggcgtctcca gcttatctga cggttcacta
aacgagctct 4080gcttatatag acctcccacc gtacacgcct accgcccatt
tgcgtcaatg gggcggagtt 4140gttacgacat tttggaaagt cccgttgatt
tactagtcaa aacaaactcc cattgacgtc 4200aatggggtgg agacttggaa
atccccgtga gtcaaaccgc tatccacgcc cattgatgta 4260ctgccaaaac
cgcatcatca tggtaatagc gatgactaat acgtagatgt actgccaagt
4320aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
taccgtcatt 4380gacgtcaata gggggcgtac ttggcatatg atacacttga
tgtactgcca agtgggcagt 4440ttaccgtaaa tactccaccc attgacgtca
atggaaagtc cctattggcg ttactatggg 4500aacatacgtc attattgacg
tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc 4560atttaccgta
agttatgtaa cgcctgcagg ttaattaaga acatgtgagc aaaaggccag
4620caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc 4680cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
ggcgaaaccc gacaggacta 4740taaagatacc aggcgtttcc ccctggaagc
tccctcgtgc gctctcctgt tccgaccctg 4800ccgcttaccg gatacctgtc
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4860tcacgctgta
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
4920gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
tgagtccaac 4980ccggtaagac acgacttatc gccactggca gcagccactg
gtaacaggat tagcagagcg 5040aggtatgtag gcggtgctac agagttcttg
aagtggtggc ctaactacgg ctacactaga 5100agaacagtat ttggtatctg
cgctctgctg aagccagtta ccttcggaaa aagagttggt 5160agctcttgat
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
5220cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
tacggggtct 5280gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
tcatggctag ttaattaaca 5340tttaaatca 5349986772DNAArtificial
SequenceMV1043 98cttgatttgg gtgatggttc acgtagtggg ccatcgccct
gatagacggt ttttcgccct 60ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
tccaaactgg aacaacactc 120aactctatct cgggctattc ttttgattta
taagggattt tgccgatttc ggtctattgg 180ttaaaaaatg agctgattta
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 240acaattttat
ggtgcagtct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
300cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
ggcatccgct 360tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
agaggttttc accgtcatca 420ccgaaacgcg cgagacgaaa gggcctcgtg
atacgcctat ttttataggt taatgtcatg 480ataataatgg tttcttagac
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 540atttgtttat
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
600taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
ccgtgtcgcc 660cttattccct tttttgcggc attttgcctt cctgtttttg
ctcacccaga aacgctggtg 720aaagtaaaag atgctgaaga tcagttgggt
gcccgagtgg gttacatcga actggatctc 780aacagcggta agatccttga
gagttttcgc cccgaagaac gttttccaat gatgagcact 840tttaaagttc
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
900ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
cacagaaaag 960catcttacgg atggcatgac agtaagagaa ttatgcagtg
ctgccataac catgagtgat 1020aacactgcgg ccaacttact tctgacaacg
atcggaggac cgaaggagct aaccgctttt 1080ttgcacaaca tgggggatca
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 1140gccataccaa
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
1200aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
agactggatg 1260gaggcggata aagttgcagg accacttctg cgctcggccc
ttccggctgg ctggtttatt 1320gctgataaat ctggagccgg tgagcgtggg
tctcgcggta tcattgcagc actggggcca 1380gatggtaagc cctcccgtat
cgtagttatc tacacgacgg ggagtcaggc aactatggat
1440gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
gtaactgtca 1500gaccaagttt actcatatat actttagatt gatttaaaac
ttcattttta atttaaaagg 1560atctaggtga agatcctttt tgataatctc
atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc
cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc
acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg
ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg
gttcgtgcat acagcccagc ttggagcgaa cgacctacac cgaactgaga
2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga
gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt
cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct
tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
2340gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
ccgaacgacc 2400gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
caatacgcaa accgcctctc 2460cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca ggtttcccga ctggaaagcg 2520ggcagtgagc gcaacgcaat
taatgtgagt tagctcactc attaggcacc ccaggcttta 2580cactttatgc
ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
2640ggaaacagct atgaccatga ttacgccaag ctttggagcc ttttttttgg
agattttcaa 2700cgtgaaaaaa ttattattcg caattccttt agttgttcct
ttctattctc acagtgcaca 2760gatccaaatg acccagtctc catcctccct
gtctgcatct gtaggagaca gagtcaccat 2820cacttgccgg gcaagtcaga
gcattagcag ctacttaaat tggtatcagc agaaaccagg 2880gaaagcccct
aagctcctga tctatgctgc atccagtttg caaagtgggg tcccatcaag
2940gttcagtggc agtggatctg ggacagattt cactctcacc atcagcagtc
tgcaacctga 3000agattttgca acttactact gtcaacagag ttacagtacc
cctccaacgt tcggccaagg 3060gaccaagctc gagatcaaac gtactgtggc
tgcaccatct gtcttcatct tcccgccatc 3120tgatgagcag ttgaaatctg
gaactgcctc tgttgtgtgc ctgctgaata acttctatcc 3180cagagaggcc
aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga
3240gagtgtcaca gagcaggaca gcaaggacag cacctacagc ctcagcagca
ccctgacgct 3300gagcaaagca gactacgaga aacacaaagt ctacgcctgc
gaagtcaccc atcagggcct 3360gagctcgccc gtcacaaaga gcttcaacag
gggagagtgt tagtaaggcg cgccaattct 3420atttcaagga gacagtcata
atgaaatacc tattgcctac ggcagccgct ggattgttat 3480tactcgcggc
ccagccggcc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
3540ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
atcaggacat 3600agcgttggct acccgtgata ttgctgaaga gcttggcggc
gaatgggctg accgcttcct 3660cgtgctttac ggtatcgccg ctcccgattc
gcagcgcatc gccttctatc gccttcttga 3720cgagttcttc tgagcgggac
tctggggttc ggtgctacga gatttcgatt ccaccgccgc 3780cttctatgaa
aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
3840gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
cagcttataa 3900tggttacaaa taaagcaata gcatcacaaa tttcacaaat
aaagcatttt tttcactgca 3960ttctagttgt ggtttgtcca aactcatcaa
tgtatcttat catgtctgta taccgtcgac 4020ctctagctag agcttggcgt
aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4080gctcacaatt
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
4140atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
agtcgggaaa 4200cctgtcgtgc cagaattgca tgaagaatct gcttagggtt
aggcgttttg cgctgcttcg 4260ctaggtggtc aatattggcc attagccata
ttattcattg gttatatagc ataaatcaat 4320attggctatt ggccattgca
tacgttgtat ccatatcata atatgtacat ttatattggc 4380tcatgtccaa
cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca
4440attacggggt cattagttca tagcccatat atggagttcc gcgttacata
acttacggta 4500aatggcccgc ctggctgacc gcccaacgac ccccgcccat
tgacgtcaat aatgacgtat 4560gttcccatag taacgccaat agggactttc
cattgacgtc aatgggtgga gtatttacgg 4620taaactgccc acttggcagt
acatcaagtg tatcatatgc caagtacgcc ccctattgac 4680gtcaatgacg
gtcaccgtct caagcgcctc caccaagggc ccatcggtct tccccctggc
4740accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg
tcaaggacta 4800cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
ctgaccagcg gcgtccacac 4860cttcccggct gtcctacagt cctcaggact
ctactccctc agcagcgtag tgaccgtgcc 4920ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg aatcacaagc ccagcaacac 4980caaggtggac
aagaaagttg agcccaaatc ttgtgcggcc gcacatcatc atcaccatca
5040cggggccgca gaacaaaaac tcatctcaga agaggatctg aatggggccg
catagactgt 5100tgaaagttgt ttagcaaaac ctcatacaga aaattcattt
actaacgtct ggaaagacga 5160caaaacttta gatcgttacg ctaactatga
gggctgtctg tggaatgcta caggcgttgt 5220ggtttgtact ggtgacgaaa
ctcagtgtta cggtacatgg gttcctattg ggcttgctat 5280ccctgaaaat
gagggtggtg gctctgaggg tggcggttct gagggtggcg gttctgaggg
5340tggcggtact aaacctcctg agtacggtga tacacctatt ccgggctata
cttatatcaa 5400ccctctcgac ggcacttatc cgcctggtac tgagcaaaac
cccgctaatc ctaatccttc 5460tcttgaggag tctcagcctc ttaatacttt
catgtttcag aataataggt tccgaaatag 5520gcagggtgca ttaactgttt
atacgggcac tgttactcaa ggcactgacc ccgttaaaac 5580ttattaccag
tacactcctg tatcatcaaa agccatgtat gacgcttact ggaacggtaa
5640attcagagac tgcgctttcc attctggctt taatgaggat ccattcgttt
gtgaatatca 5700aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct
ggcggcggct ctggtggtgg 5760ttctggtggc ggctctgagg gtggcggctc
tgagggtggc ggctctgagg gtggcggttc 5820tgagggtggc ggctctgagg
gtggcggttc cggtggcggc tccggttccg gtgattttga 5880ttatgaaaaa
atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc
5940gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg
gtgctgctat 6000cgatggtttc attggtgacg tttccggcct tgctaatggt
aatggtgcta ctggtgattt 6060tgctggctct aattcccaaa tggctcaagt
cggtgacggt gataattcac ctttaatgaa 6120taatttccgt caatatttac
cttctttgcc tcagtcggtt gaatgtcgcc cttatgtctt 6180tggcgctggt
aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg
6240tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcga
cgtttgctaa 6300catactgcgt aataaggagt cttaataaga attcactggc
cgtcgtttta caacgtcgtg 6360actgggaaaa ccctggcgtt acccaactta
atcgccttgc agcacatccc cctttcgcca 6420gctggcgtaa tagcgaagag
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 6480atggcgaatg
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
6540gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
cggcgggtgt 6600ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
ttagcgcccg ctcctttcgc 6660tttcttccct tcctttctcg ccacgttcgc
cggctttccc cgtcaagctc taaatcgggg 6720gctcccttta gggttccgat
ttagtgcttt acggcacctc gaccccaaaa aa 67729910293DNAArtificial
SequenceMV1057 99tactcttcct ttttcaatat tattgaagca tttatcaggg
ttattgtctc atgagcggat 60acatatttga atgtatttag aaaaataaac aaataggggt
tccgcgcaca tttccccgaa 120aagtgccacc tgacgtcgac ggatcgggag
atctcccgat cccctatggt gcactctcag 180tacaatctgc tctgatgccg
catagttaag ccagtatctg ctccctgctt gtgtgttgga 240ggtcgctgag
tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
300ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgctagg
tggtcaatat 360tggccattag ccatattatt cattggttat atagcataaa
tcaatattgg ctattggcca 420ttgcatacgt tgtatccata tcataatatg
tacatttata ttggctcatg tccaacatta 480ccgccatgtt gacattgatt
attgactagt tattaatagt aatcaattac ggggtcatta 540gttcatagcc
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
600tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
catagtaacg 660ccaataggga ctttccattg acgtcaatgg gtggagtatt
tacggtaaac tgcccacttg 720gcagtacatc aagtgtatca tatgccaagt
acgcccccta ttgacgtcaa tgacggtaaa 780tggcccgcct ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac 840atctacgtat
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
900cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
cgtcaatggg 960agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
gtcgtaacaa ctccgcccca 1020ttgacgcaaa tgggcggtag gcgtgtacgg
tgggaggtct atataagcag agctcgttta 1080gtgaaccgtc agatcgcctg
gagacgccat ccacgctgtt ttgacctcca tagaagacac 1140cgggaccgat
ccagcctccg cggccgggaa cggtgcattg gaagcttggt accggtgaat
1200tggccggccc gcgccgtcga ggttatcgat ccgaccgacg cgttcgcgag
aggccgcaat 1260tccctagcca ccatgggatg gagctgtatc atcctcttct
tggtactgct gctggcccag 1320ccggccatgg ggcggagaat gggcggaact
gggcggagtt aggggcggga tgggcggagt 1380taggggcggg actatggttg
ctgactaatt gagatgcgga tccgctggca cgacaggttt 1440cccgactgga
aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
1500gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
tgtgagcgga 1560taacaatttc acacaggaaa cagctatgac catgattacg
ccaagcttgg gctgcaggtt 1620ctttccgcct cagaagccat agagcccacc
gcatccccag catgcctgct attgtcttcc 1680caatcctccc ccttgctgtc
ctgccccacc ccacccccca gaatagaatg acacctactc 1740agacaatgcg
atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
1800agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact
agaaggcaca 1860gtcgaggctg atcagcgagc tctagatcat cgatgcatgg
ggtcgtgcgc tcctttcggt 1920cgggcgctgc gggtcgtggg gcgggcgtca
ggcaccgggc ttgcgggtca tgcaccaggt 1980gcgcggtcct tcgggcacct
cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa 2040ggggaggttg
cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc
2100ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt
cgggcgagac 2160gccgacggtg gccaggaacc acgcgggctc cttgggccgg
tgcggcgcca ggaggccttc 2220catctgttgc tgcgcggcca gccgggaacc
gctcaactcg gccatgcgcg ggccgatctc 2280ggcgaacacc gcccccgctt
cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc 2340gtcgtccgcg
acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg
2400cagctcggtc accgtctcca gtgctagcac caagggccca tcggtcttcc
ccctggcacc 2460ctcctccaag agcacctctg ggggcacagc ggccctgggc
tgcctggtca aggactactt 2520ccccgaaccg gtgacggtgt cgtggaactc
aggcgccctg accagcggcg tgcacacctt 2580cccggctgtc ctacagtcct
caggactcta ctccctcagc agcgtcgtga ccgtgccctc 2640cagcagcttg
ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa
2700ggtggacaag agagttggtg agaggccagc acagggaggg agggtgtctg
ctggaagcca 2760ggctcagcgc tcctgcctgg acgcatcccg gctatgcagt
cccagtccag ggcagcaagg 2820caggccccgt ctgcctcttc acccggaggc
ctctgcccgc cccactcatg ctcagggaga 2880gggtcttctg gctttttccc
caggctctgg gcaggcacag gctaggtgcc cctaacccag 2940gccctgcaca
caaaggggca ggtgctgggc tcagacctgc caagagccat atccgggagg
3000accctgcccc tgacctaagc ccaccccaaa ggccaaactc tccactccct
cagctcggac 3060accttctctc ctcccagatt ccagtaactc ccaatcttct
ctctgcagag cccaaatctt 3120gtgacaaaac tcacacatgc ccaccgtgcc
cagcacctga actcctgggg ggaccgtcag 3180tcttcctctt ccccccaaaa
cccaaggaca ccctcatgat ctcccggacc cctgaggtca 3240catgcgtggt
ggtggacgtg agccacgaag accctgaggt caagttcaac tggtacgtgg
3300acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
aacagcacgt 3360accgtgtggt cagcgtcctc accgtcctgc accaggactg
gctgaatggc aaggagtaca 3420agtgcaaggt ctccaacaaa gccctcccag
cccccatcga gaaaaccatc tccaaagcca 3480aagggcagcc ccgagaacca
caggtgtaca ccctgccccc atcccgggag gagatgacca 3540agaaccaggt
cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg
3600agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
gtgctggact 3660ccgacggctc cttcttcctc tatagcaagc tcaccgtgga
caagagcagg tggcagcagg 3720ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac acgcagaaga 3780gcctctccct gtctccgggt
aaatgagttt aacggatctt aattaatccg agctcggtac 3840caagcttaag
tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
3900ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
actgtccttt 3960cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
gtgtcattct attctggggg 4020gtggggtggg gcaggacagc aagggggagg
attgggaaga caatagcagg catgctgggg 4080atgcggtggg ctctatggct
tctgaggcgg aaagaaccag ctggggctct agggggtatc 4140cccacgcgcc
ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
4200ccgctacact tgccagcgcc tagcgcccgc tcctttcgct ttcttccctt
cctttctcgc 4260cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
ctccctttag ggttccgatt 4320tagtgcttta cggcacctcg accccaaaaa
acttgattag ggtgatggtt cacgtagtgg 4380gccatcgccc tgatagacgg
tttttcgccc tttgacgttg gagtccacgt tctttaatag 4440tggactcttg
ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
4500ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
aacaaaaatt 4560taacgcgaat taattctgtg gaatgtgtgt cagttagggt
gtggaaagtc cccaggctcc 4620ccagcaggca gaagtatgca aagcatgcat
ctcaattagt cagcaaccag gtgtggaaag 4680tccccaggct ccccagcagg
cagaagtatg caaagcatgc atctcaatta gtcagcaacc 4740atagtcccgc
ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
4800ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
ctctgcctct 4860gagctattcc agaagtagtg aggaggcttt tttggaggcc
taggcttttg caaaaagctc 4920ccgggagctt ggatatccat tttcggatct
gatcaagaga caggatgagg atcgtttcgc 4980atgattgaac aagatggatt
gcacgcaggt tctccggccg cttgggtgga gaggctattc 5040ggctatgact
gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
5100gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
gaatgaactg 5160caggacgagg cagcgcggct atcgtggctg gccacgacgg
gcgttccttg cgcagctgtg 5220ctcgacgttg tcactgaagc gggaagggac
tggctgctat tgggcgaagt gccggggcag 5280gatctcctgt catctcacct
tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 5340cggcggctgc
atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
5400atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
tctggacgaa 5460gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
tcaaggcgcg catgcccgac 5520ggcgaggatc tcgtcgtgac ccatggcgat
gcctgcttgc cgaatatcat ggtggaaaat 5580ggccgctttt ctggattcat
cgactgtggc cggctgggtg tggcggaccg ctatcaggac 5640atagcgttgg
ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
5700ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
tcgccttctt 5760gacgagttct tctgagcggg actctggggt tcggtgctac
gagatttcga ttccaccgcc 5820gccttctatg aaaggttggg cttcggaatc
gttttccggg acgccggctg gatgatcctc 5880cagcgcgggg atctcatgct
ggagttcttc gcccacccca acttgtttat tgcagcttat 5940aatggttaca
aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
6000cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
tataccgtcg 6060acctctagct agagcttggc gtaatcatgg tcatagctgt
ttcctgtgtg aaattgttat 6120ccgctcacaa ttccacacaa catacgagcc
ggaagcataa agtgtaaagc ctggggtgcc 6180taatgagtga gctaactcac
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6240aacctgtcgt
gccagaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
6300cgctaggtgg tcaatattgg ccattagcca tattattcat tggttatata
gcataaatca 6360atattggcta ttggccattg catacgttgt atccatatca
taatatgtac atttatattg 6420gctcatgtcc aacattaccg ccatgttgac
attgattatt gactagttat taatagtaat 6480caattacggg gtcattagtt
catagcccat atatggagtt ccgcgttaca taacttacgg 6540taaatggccc
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
6600atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
gagtatttac 6660ggtaaactgc ccacttggca gtacatcaag tgtatcatat
gccaagtacg ccccctattg 6720acgtcaatga cggtaaatgg cccgcctggc
attatgccca gtacatgacc ttatgggact 6780ttcctacttg gcagtacatc
tacgtattag tcatcgctat taccatggtg atgcggtttt 6840ggcagtacat
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
6900ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
ccaaaatgtc 6960gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
tgtacggtgg gaggtctata 7020taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag acgccatcca cgctgttttg 7080acctccatag aagacaccgg
gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa 7140gcttggtacc
ggtgaattag gcgcgccgtc gaggttatcg atccgaccga cgcgttcgcg
7200agaggccgca attccctagc caccatggca tgccctggct tcctgtgggc
acttgtgatc 7260tccacctgtc ttgaattctc catggctgac atccagatga
cccagtctcc atcctccctg 7320tctgcatctg taggagacag agtcaccatc
acttgccggg caagtcagag cattagcagc 7380tacttaaatt ggtatcagca
gaaaccaggg aaagccccta agctcctgat ctatgctgca 7440tccagtttgc
aaagtggggt cccatcaagg ttcagtggca gtggatctgg gacagatttc
7500actctcacca tcagcagtct gcaacctgaa gattttgcaa cttactactg
tcaacagagt 7560tacagtaccc ctccaacgtt cggccaaggg accaaggtgg
agatcaaacg taagtgcact 7620ttgcggccgc taggaagaaa ctcaaaacat
caagatttta aatacgcttc ttggtctcct 7680tgctataatt atctgggata
agcatgctgt tttctgtctg tccctaacat gccctgtgat 7740tatccgcaaa
caacacaccc aagggcagaa ctttgttact taaacaccat cctgtttgct
7800tctttcctca ggaactgtgg ctgcaccatc tgtcttcatc ttcccgccat
ctgatgagca 7860gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat
aacttctatc ccagagaggc 7920caaagtacag tggaaggtgg ataacgccct
ccaatcgggt aactcccagg agagtgtcac 7980agagcaggac agcaaggaca
gcacctacag cctcagcagc accctgacgc tgagcaaagc 8040agactacgag
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc
8100cgtcacaaag agcttcaaca ggggagagtg ttaggtttaa cggatccgag
ctcggtacca 8160agctcaagtt taaaccgctg atcagcctcg actgtgcctt
ctagttgcca gccatctgtt 8220gtttgcccct cccccgtgcc ttccttgacc
ctggaaggtg ccactcccac tgtcctttcc 8280taataaaatg aggaaattgc
atcgcattgt ctgagtaggt gtcattctat tctggggggt 8340ggggtggggc
aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
8400gcggtgggct ctatggcttc tgaggcggaa agaaccagct gcattaatga
atcggccaac 8460gcgcggggag aggcggtttg cgtattgggc gctcttccgc
ttcctcgctc actgactcgc 8520tgcgctcggt cgttcggctg cggcgagcgg
tatcagctca ctcaaaggcg gtaatacggt 8580tatccacaga atcaggggat
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 8640ccaggaaccg
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
8700agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
ctataaagat 8760accaggcgtt tccccctgga agctccctcg tgcgctctcc
tgttccgacc ctgccgctta 8820ccggatacct gtccgccttt ctcccttcgg
gaagcgtggc gctttctcat agctcacgct 8880gtaggtatct cagttcggtg
taggtcgttc gctccaagct gggctgtgtg cacgaacccc 8940ccgttcagcc
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
9000gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
gcgaggtatg 9060taggcggtgc tacagagttc ttgaagtggt ggcctaacta
cggctacact agaagaacag 9120tatttggtat ctgcgctctg ctgaagccag
ttaccttcgg aaaaagagtt ggtagctctt 9180gatccggcaa acaaaccacc
gctggtagcg gtggtttttt tgtttgcaag cagcagatta 9240cgcgcagaaa
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
9300agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
aggatcttca 9360cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
ctaaagtata tatgagtaaa 9420cttggtctga cagttaccaa tgcttaatca
gtgaggcacc tatctcagcg atctgtctat 9480ttcgttcatc catagttgcc
tgactccccg tcgtgtagat aactacgata cgggagggct 9540taccatctgg
ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
9600tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
gcaactttat 9660ccgcctccat ccagtctatt
aattgttgcc gggaagctag agtaagtagt tcgccagtta 9720atagtttgcg
caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
9780gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
tcccccatgt 9840tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
tgtcagaagt aagttggccg 9900cagtgttatc actcatggtt atggcagcac
tgcataattc tcttactgtc atgccatccg 9960taagatgctt ttctgtgact
ggtgagtact caaccaagtc attctgagaa tagtgtatgc 10020ggcgaccgag
ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
10080ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
aggatcttac 10140cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
caactgatct tcagcatctt 10200ttactttcac cagcgtttct gggtgagcaa
aaacaggaag gcaaaatgcc gcaaaaaagg 10260gaataagggc gacacggaaa
tgttgaatac tca 102931008179DNAArtificial SequencepCAGGS-IgVK1-39
targeting vector 100atccaggcgc ggatcaataa aagatcatta ttttcaatag
atctgtgtgt tggttttttg 60tgtgccttgg gggaggggga ggccagaatg aggcgcggcc
aagggggagg gggaggccag 120aatgaccttg ggggaggggg aggccagaat
gaccttgggg gagggggagg ccagaatgag 180gcgcggatcc ggagaagttc
ctattccgaa gttcctattc ttcaaatagt ataggaactt 240cgctcgaggg
atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
300tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
gatgccgccg 360tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
caagaccgac ctgtccggtg 420ccctgaatga actgcaggac gaggcagcgc
ggctatcgtg gctggccacg acgggcgttc 480cttgcgcagc tgtgctcgac
gttgtcactg aagcgggaag ggactggctg ctattgggcg 540aagtgccggg
gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
600tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
ttcgaccacc 660aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
agccggtctt gtcgatcagg 720atgatctgga cgaagagcat caggggctcg
cgccagccga actgttcgcc aggctcaagg 780cgcgcatgcc cgacggcgag
gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 840tcatggtgga
aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
900accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
ggcggcgaat 960gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
cgattcgcag cgcatcgcct 1020tctatcgcct tcttgacgag ttcttctgag
gggatcgatc cgctgtaagt ctgcagaaat 1080tgatgatcta ttaaacaata
aagatgtcca ctaaaatgga agtttttcct gtcatacttt 1140gttaagaagg
gtgagaacag agtacctaca ttttgaatgg aaggattgga gctacggggg
1200tgggggtggg gtgggattag ataaatgcct gctctttact gaaggctctt
tactattgct 1260ttatgataat gtttcatagt tggatatcat aatttaaaca
agcaaaacca aattaagggc 1320cagctcattc ctcccactca tgatctatag
atctatagat ctctcgtggg atcattgttt 1380ttctcttgat tcccactttg
tggttctaag tactgtggtt tccaaatgtg tcagtttcat 1440agcctgaaga
acgagatcag cagcctctgt tccacataca cttcattctc agtattgttt
1500tgccaagttc taattccatc agaagctgac tctagatggc gcgtatgcag
gttttcgaca 1560ttgattattg actagttatt aatagtaatc aattacgggg
tcattagttc atagcccata 1620tatggagttc cgcgttacat aacttacggt
aaatggcccg cctggctgac cgcccaacga 1680cccccgccca ttgacgtcaa
taatgacgta tgttcccata gtaacgccaa tagggacttt 1740ccattgacgt
caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
1800gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
ccgcctggca 1860ttatgcccag tacatgacct tatgggactt tcctacttgg
cagtacatct acgtattagt 1920catcgctatt accatggtcg aggtgagccc
cacgttctgc ttcactctcc ccatctcccc 1980cccctcccca cccccaattt
tgtatttatt tattttttaa ttattttgtg cagcgatggg 2040ggcggggggg
gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
2100cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
tttcctttta 2160tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
cgcgcggcgg gcgggagtcg 2220ctgcgttgcc ttcgccccgt gccccgctcc
gcgccgcctc gcgccgcccg ccccggctct 2280gactgaccgc gttactccca
caggtgagcg ggcgggacgg cccttctcct ccgggctgta 2340attagcgctt
ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa agccttaaag
2400ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg
cgtgtgtgtg 2460tgcgtgggga gcgccgcgtg cggcccgcgc tgcccggcgg
ctgtgagcgc tgcgggcgcg 2520gcgcggggct ttgtgcgctc cgcgtgtgcg
cgaggggagc gcggccgggg gcggtgcccc 2580gcggtgcggg ggggctgcga
ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg 2640tgagcagggg
gtgtgggcgc ggcggtcggg ctgtaacccc cccctgcacc cccctccccg
2700agttgctgag cacggcccgg cttcgggtgc ggggctccgt gcggggcgtg
gcgcggggct 2760cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
ggcggggcgg ggccgcctcg 2820ggccggggag ggctcggggg aggggcgcgg
cggccccgga gcgccggcgg ctgtcgaggc 2880gcggcgagcc gcagccattg
ccttttatgg taatcgtgcg agagggcgca gggacttcct 2940ttgtcccaaa
tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
3000cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
ccttcgtgcg 3060tcgccgcgcc gccgtcccct tctccctctc cagcctcggg
gctgtccgcg gggggacggc 3120tgccttcggg ggggacgggg cagggcgggg
ttcggcttct ggcgtgtgac cggcggctct 3180agaagcgttg gggtgagtac
tccctctcaa aagcgggcat gacttctgcg ctaagattgt 3240cagtttccaa
aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg
3300tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg
aggtgtggca 3360ggcttgagat ctggccatac acttgagtga cattgacatc
cactttgcct ttctctccac 3420aggtgtccac tcccagggcg gcctccggag
cgatcgccga tccgcctagg caattgttta 3480aatcggccgg ccataacttc
gtataatgta tgctatacga agttatggat cctcacagta 3540ggtggcatcg
ttcctttctg actgcccgcc ccccgcatgc cgtcccgcga tattgagctc
3600cgaacctctc gccctgccgc cgccggtgct ccgtcgccgc cgcgccgcca
tggaatcgaa 3660gccaccatgg atcttaccgg aaaactcgac gcaagaaaaa
tcagagagat cctcataaag 3720gtcaagaagg gcggaaagat cgccgtgtaa
ttctagaccg gttcgagatc caggcgcgga 3780tcaataaaag atcattattt
tcaatagatc tgtgtgttgg ttttttgtgt gccttggggg 3840agggggaggc
cagaatgagg cgcggccaag ggggaggggg aggccagaat gaccttgggg
3900gagggggagg ccagaatgac cttgggggag ggggaggcca gaatgaggcg
cgccctccgt 3960cgacctataa cttcgtataa tgtatgctat acgaagttat
ggcggccgcc accatggaca 4020tgagagtgcc cgcccagctc ctggggctcc
tgctactctg gctccgaggt aaggatggag 4080aacactagga atttactcag
ccagtgtgct cagtactgac tggaacttca gggaagttct 4140ctgataacat
gattaatagt aagaatattt gtttttatgt ttccaatctc aggtgccaga
4200tgtgacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg
cgacagagtg 4260accatcacct gcagagccag ccagagcatc agcagctacc
tgaactggta tcagcagaag 4320cccggcaagg cccccaagct gctgatctac
gccgccagct ccctgcagag cggcgtgccc 4380agcagattca gcggcagcgg
ctccggcacc gacttcaccc tgaccatcag cagcctgcag 4440cccgaggact
tcgccaccta ctactgccag cagagctaca gcaccccccc caccttcggc
4500cagggcacca aggtggagat caagagagcc gacgccgctc ccaccgtgtc
catcttcccc 4560cccagcatgg aacagctgac ctctggcgga gccaccgtgg
tctgcttcgt gaacaacttc 4620taccccagag acatcagcgt gaagtggaag
atcgacggca gcgagcagag ggacggcgtg 4680ctggacagcg tgaccgacca
ggacagcaag gactccacct acagcatgag cagcaccctg 4740agcctgacca
aggtggagta cgagaggcac aacctgtaca cctgcgaggt ggtgcacaag
4800accagctcca gccccgtggt caagtccttc aaccggaacg agtgttgagc
tagcttaaga 4860tttaaatagg ccggccgcgt cgacctcgag atccaggcgc
ggatcaataa aagatcatta 4920ttttcaatag atctgtgtgt tggttttttg
tgtgccttgg gggaggggga ggccagaatg 4980aggcgcggcc aagggggagg
gggaggccag aatgaccttg ggggaggggg aggccagaat 5040gaccttgggg
gagggggagg ccagaatgag gcgcgccccc gggtaccgag ctcgaattag
5100tggatcctca cagtaggtgg catcgttcct ttctgactgc ccgccccccg
catgccgtcc 5160cgcgatattg agctccgaac ctctcgccct gccgccgccg
gtgctccgtc gccgccgcgc 5220cgccatggaa tcgcgccggt aaccgaagtt
cctatacttt ctagagaata ggaacttcgg 5280aataggaact tcaagccggt
acccagcttt tgttcccttt agtgagggtt aatttcgagc 5340ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
5400cacaacatac gagccgggag cataaagtgt aaagcctggg gtgcctaatg
agtgagctaa 5460ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct gtcgtgccag 5520ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg gcgctcttcc 5580gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5640cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
5700tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 5760cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 5820aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 5880cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5940gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
6000ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 6060cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 6120aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 6180tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 6240ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
6300tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 6360ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 6420agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca 6480atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 6540cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
6600ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
accgcgagac 6660ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag ggccgagcgc 6720agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg ccgggaagct 6780agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6840gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
6900cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc 6960gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat 7020tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag 7080tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aatacgggat 7140aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
7200cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
cactcgtgca 7260cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc aaaaacagga 7320aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat actcatactc 7380ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag cggatacata 7440tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
7500ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
tgttaaatca 7560gctcattttt taaccaatag gccgaaatcg gcaaaatccc
ttataaatca aaagaataga 7620ccgagatagg gttgagtgtt gttccagttt
ggaacaagag tccactatta aagaacgtgg 7680actccaacgt caaagggcga
aaaaccgtct atcagggcga tggcccacta cgtgaaccat 7740caccctaatc
aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
7800ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
aaggaaggga 7860agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
agcggtcacg ctgcgcgtaa 7920ccaccacacc cgccgcgctt aatgcgccgc
tacagggcgc gtcccattcg ccattcaggc 7980tgcgcaactg ttgggaaggg
cgatcggtgc gggcctcttc gctattacgc cagctggcga 8040aagggggatg
tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
8100gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
gaattggggg 8160taactaagta aggatcgag 81791018188DNAArtificial
SequencepCAGGS-IgVL2-14 targeting vector 101atccaggcgc ggatcaataa
aagatcatta ttttcaatag atctgtgtgt tggttttttg 60tgtgccttgg gggaggggga
ggccagaatg aggcgcggcc aagggggagg gggaggccag 120aatgaccttg
ggggaggggg aggccagaat gaccttgggg gagggggagg ccagaatgag
180gcgcggatcc ggagaagttc ctattccgaa gttcctattc ttcaaatagt
ataggaactt 240cgctcgaggg atcggccatt gaacaagatg gattgcacgc
aggttctccg gccgcttggg 300tggagaggct attcggctat gactgggcac
aacagacaat cggctgctct gatgccgccg 360tgttccggct gtcagcgcag
gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 420ccctgaatga
actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
480cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
ctattgggcg 540aagtgccggg gcaggatctc ctgtcatctc accttgctcc
tgccgagaaa gtatccatca 600tggctgatgc aatgcggcgg ctgcatacgc
ttgatccggc tacctgccca ttcgaccacc 660aagcgaaaca tcgcatcgag
cgagcacgta ctcggatgga agccggtctt gtcgatcagg 720atgatctgga
cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
780cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
ttgccgaata 840tcatggtgga aaatggccgc ttttctggat tcatcgactg
tggccggctg ggtgtggcgg 900accgctatca ggacatagcg ttggctaccc
gtgatattgc tgaagagctt ggcggcgaat 960gggctgaccg cttcctcgtg
ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 1020tctatcgcct
tcttgacgag ttcttctgag gggatcgatc cgctgtaagt ctgcagaaat
1080tgatgatcta ttaaacaata aagatgtcca ctaaaatgga agtttttcct
gtcatacttt 1140gttaagaagg gtgagaacag agtacctaca ttttgaatgg
aaggattgga gctacggggg 1200tgggggtggg gtgggattag ataaatgcct
gctctttact gaaggctctt tactattgct 1260ttatgataat gtttcatagt
tggatatcat aatttaaaca agcaaaacca aattaagggc 1320cagctcattc
ctcccactca tgatctatag atctatagat ctctcgtggg atcattgttt
1380ttctcttgat tcccactttg tggttctaag tactgtggtt tccaaatgtg
tcagtttcat 1440agcctgaaga acgagatcag cagcctctgt tccacataca
cttcattctc agtattgttt 1500tgccaagttc taattccatc agaagctgac
tctagatggc gcgtatgcag gttttcgaca 1560ttgattattg actagttatt
aatagtaatc aattacgggg tcattagttc atagcccata 1620tatggagttc
cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
1680cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
tagggacttt 1740ccattgacgt caatgggtgg agtatttacg gtaaactgcc
cacttggcag tacatcaagt 1800gtatcatatg ccaagtacgc cccctattga
cgtcaatgac ggtaaatggc ccgcctggca 1860ttatgcccag tacatgacct
tatgggactt tcctacttgg cagtacatct acgtattagt 1920catcgctatt
accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc
1980cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
cagcgatggg 2040ggcggggggg gggggggcgc gcgccaggcg gggcggggcg
gggcgagggg cggggcgggg 2100cgaggcggag aggtgcggcg gcagccaatc
agagcggcgc gctccgaaag tttcctttta 2160tggcgaggcg gcggcggcgg
cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg 2220ctgcgttgcc
ttcgccccgt gccccgctcc gcgccgcctc gcgccgcccg ccccggctct
2280gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
ccgggctgta 2340attagcgctt ggtttaatga cggctcgttt cttttctgtg
gctgcgtgaa agccttaaag 2400ggctccggga gggccctttg tgcggggggg
agcggctcgg ggggtgcgtg cgtgtgtgtg 2460tgcgtgggga gcgccgcgtg
cggcccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg 2520gcgcggggct
ttgtgcgctc cgcgtgtgcg cgaggggagc gcggccgggg gcggtgcccc
2580gcggtgcggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg
cgtggggggg 2640tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc
cccctgcacc cccctccccg 2700agttgctgag cacggcccgg cttcgggtgc
ggggctccgt gcggggcgtg gcgcggggct 2760cgccgtgccg ggcggggggt
ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg 2820ggccggggag
ggctcggggg aggggcgcgg cggccccgga gcgccggcgg ctgtcgaggc
2880gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
gggacttcct 2940ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
gccgcacccc ctctagcggg 3000cgcggggcga agcggtgcgg cgccggcagg
aaggaaatgg gcggggaggg ccttcgtgcg 3060tcgccgcgcc gccgtcccct
tctccctctc cagcctcggg gctgtccgcg gggggacggc 3120tgccttcggg
ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
3180agaagcgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg
ctaagattgt 3240cagtttccaa aaacgaggag gatttgatat tcacctggcc
cgcggtgatg cctttgaggg 3300tggccgcgtc catctggtca gaaaagacaa
tctttttgtt gtcaagcttg aggtgtggca 3360ggcttgagat ctggccatac
acttgagtga cattgacatc cactttgcct ttctctccac 3420aggtgtccac
tcccagggcg gcctccggag cgatcgccga tccgcctagg caattgttta
3480aatcggccgg ccataacttc gtataatgta tgctatacga agttatggat
cctcacagta 3540ggtggcatcg ttcctttctg actgcccgcc ccccgcatgc
cgtcccgcga tattgagctc 3600cgaacctctc gccctgccgc cgccggtgct
ccgtcgccgc cgcgccgcca tggaatcgaa 3660gccaccatgg atcttaccgg
aaaactcgac gcaagaaaaa tcagagagat cctcataaag 3720gtcaagaagg
gcggaaagat cgccgtgtaa ttctagaccg gttcgagatc caggcgcgga
3780tcaataaaag atcattattt tcaatagatc tgtgtgttgg ttttttgtgt
gccttggggg 3840agggggaggc cagaatgagg cgcggccaag ggggaggggg
aggccagaat gaccttgggg 3900gagggggagg ccagaatgac cttgggggag
ggggaggcca gaatgaggcg cgccctccgt 3960cgacctataa cttcgtataa
tgtatgctat acgaagttat ggcggccgcc accatggaca 4020tgagagtgcc
cgcccagctc ctggggctcc tgctactctg gctccgaggt aaggatggag
4080aacactagga atttactcag ccagtgtgct cagtactgac tggaacttca
gggaagttct 4140ctgataacat gattaatagt aagaatattt gtttttatgt
ttccaatctc aggtgccaga 4200tgtcagtctg ccctgaccca gcccgcctct
gtgtctggca gccctggcca gagcatcacc 4260atcagctgca ccggcaccag
cagcgacgtg ggcggctaca actacgtgtc ctggtatcag 4320cagcaccccg
gcaaggcccc caagctgatg atctacgagg tgtccaacag acccagcggc
4380gtgagcaaca gattcagcgg cagcaagagc ggcaacaccg ccagcctgac
catcagcggc 4440ctccaggctg aggacgaggc cgactactac tgcagcagct
acaccagcag ctccaccctg 4500gtgtttggcg gcggaacaaa gctgaccgtg
ctgagagccg acgccgctcc caccgtgtcc 4560atcttccccc ccagcatgga
acagctgacc tctggcggag ccaccgtggt ctgcttcgtg 4620aacaacttct
accccagaga catcagcgtg aagtggaaga tcgacggcag cgagcagagg
4680gacggcgtgc tggacagcgt gaccgaccag gacagcaagg actccaccta
cagcatgagc 4740agcaccctga gcctgaccaa ggtggagtac gagaggcaca
acctgtacac ctgcgaggtg 4800gtgcacaaga ccagctccag ccccgtggtc
aagtccttca accggaacga gtgttgagct 4860agcttaagat ttaaataggc
cggccgcgtc gacctcgaga tccaggcgcg gatcaataaa 4920agatcattat
tttcaataga tctgtgtgtt ggttttttgt gtgccttggg ggagggggag
4980gccagaatga ggcgcggcca agggggaggg ggaggccaga atgaccttgg
gggaggggga 5040ggccagaatg accttggggg agggggaggc cagaatgagg
cgcgcccccg ggtaccgagc 5100tcgaattagt ggatcctcac agtaggtggc
atcgttcctt tctgactgcc cgccccccgc 5160atgccgtccc gcgatattga
gctccgaacc tctcgccctg ccgccgccgg tgctccgtcg 5220ccgccgcgcc
gccatggaat cgcgccggta accgaagttc ctatactttc tagagaatag
5280gaacttcgga ataggaactt caagccggta cccagctttt gttcccttta
gtgagggtta 5340atttcgagct tggcgtaatc atggtcatag ctgtttcctg
tgtgaaattg ttatccgctc 5400acaattccac acaacatacg agccgggagc
ataaagtgta aagcctgggg tgcctaatga 5460gtgagctaac tcacattaat
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 5520tcgtgccagc
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
5580cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
gcggcgagcg 5640gtatcagctc actcaaaggc ggtaatacgg ttatccacag
aatcagggga taacgcagga 5700aagaacatgt gagcaaaagg ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg 5760gcgtttttcc ataggctccg
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5820aggtggcgaa
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
5880gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
tctcccttcg 5940ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
tcagttcggt gtaggtcgtt 6000cgctccaagc tgggctgtgt gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc 6060ggtaactatc gtcttgagtc
caacccggta agacacgact tatcgccact ggcagcagcc 6120actggtaaca
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
6180tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
gctgaagcca 6240gttaccttcg gaaaaagagt tggtagctct tgatccggca
aacaaaccac cgctggtagc 6300ggtggttttt ttgtttgcaa gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat 6360cctttgatct tttctacggg
gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6420ttggtcatga
gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
6480tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
atgcttaatc 6540agtgaggcac ctatctcagc gatctgtcta tttcgttcat
ccatagttgc ctgactcccc 6600gtcgtgtaga taactacgat acgggagggc
ttaccatctg gccccagtgc tgcaatgata 6660ccgcgagacc cacgctcacc
ggctccagat ttatcagcaa taaaccagcc agccggaagg 6720gccgagcgca
gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
6780cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
tgccattgct 6840acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
cattcagctc cggttcccaa 6900cgatcaaggc gagttacatg atcccccatg
ttgtgcaaaa aagcggttag ctccttcggt 6960cctccgatcg ttgtcagaag
taagttggcc gcagtgttat cactcatggt tatggcagca 7020ctgcataatt
ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
7080tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
cccggcgtca 7140atacgggata ataccgcgcc acatagcaga actttaaaag
tgctcatcat tggaaaacgt 7200tcttcggggc gaaaactctc aaggatctta
ccgctgttga gatccagttc gatgtaaccc 7260actcgtgcac ccaactgatc
ttcagcatct tttactttca ccagcgtttc tgggtgagca 7320aaaacaggaa
ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
7380ctcatactct tcctttttca atattattga agcatttatc agggttattg
tctcatgagc 7440ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
gggttccgcg cacatttccc 7500cgaaaagtgc cacctaaatt gtaagcgtta
atattttgtt aaaattcgcg ttaaattttt 7560gttaaatcag ctcatttttt
aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 7620aagaatagac
cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
7680agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
ggcccactac 7740gtgaaccatc accctaatca agttttttgg ggtcgaggtg
ccgtaaagca ctaaatcgga 7800accctaaagg gagcccccga tttagagctt
gacggggaaa gccggcgaac gtggcgagaa 7860aggaagggaa gaaagcgaaa
ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 7920tgcgcgtaac
caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
7980cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
ctattacgcc 8040agctggcgaa agggggatgt gctgcaaggc gattaagttg
ggtaacgcca gggttttccc 8100agtcacgacg ttgtaaaacg acggccagtg
agcgcgcgta atacgactca ctatagggcg 8160aattgggggt aactaagtaa ggatcgag
818810210DNAArtificial SequenceKozak sequence 102gccaccatgg 10
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