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Cancer Patent Abstract
The present invention provides an anti-ovarian cancer bispecific
antibody. Said antibody includes two polypeptide domains connected
by a polypeptide linker, one is anti-ovarian cancer antibody, or
its Fab fragment, single complementarity determining region (CDR)
antibody or single chain Fv (scFv) and the other is anti-CD3 antibody,
or its Fab fragment, single CDR antibody or scFv. The present invention
also provides DNA sequences encoding said antibody, an expression
vector containing said DNA sequence, and a host cell containing
said expression vector.
Cancer Patent Claims
What is claimed is:
1. A bispecific antibody against ovarian cancer, comprising (i)
an anti-ovarian cancer single chain antibody comprising a heavy
chain variable region and a light chain variable region, said heavy
chain variable region of said anti-ovarian cancer single chain antibody
comprising the amino acid sequence as set forth in SEQ ID NO: 17,
and said light chain variable region of said anti-ovarian cancer
single chain antibody comprising the amino acid sequence as set
forth in SEQ ID NO: 18; (ii) an anti-human CD3 single chain antibody;
and (iii) an interlinker connecting said anti-ovarian cancer single
chain antibody to said anti-human CD3 single chain antibody.
2. The bispecific antibody of claim 1, wherein said anti-human
CD3 single chain antibody is a reshaped antibody.
3. The bispecific antibody of claim 1 or 2, wherein said heavy
chain variable region of said anti-ovarian cancer single chain antibody
and said light chain variable region of said anti-ovarian cancer
single chain antibody is connected by an intralinker comprising
the amino acid sequence set forth in SEQ ID NO: 15.
4. The bispecific antibody of claim 1, wherein said anti-human
CD3 single chain antibody comprises a heavy chain variable region
and a light chain variable region, said heavy chain variable region
of said anti-human CD3 single chain antibody comprising the amino
acid sequence set forth in SEQ ID NO: 19.
5. The bispecific antibody of claim 1, wherein said anti-human
CD3 single chain antibody comprises a heavy chain variable region
and a light chain variable region, said light chain variable region
of said anti-human CD3 single chain antibody comprising the amino
acid sequence set forth in SEQ ID NO: 20.
6. The bispecific antibody of claim 5, wherein said heavy chain
variable region of said anti-human CD3 single chain antibody comprises
the amino acid sequence set forth in SEQ ID NO: 19.
7. The bispecific antibody of claim 1, 2, or 6 wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO; 23.
8. The bispecific antibody of claim 1, 2, or 6, wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO: 25.
9. The bispecific antibody of claim 1, 2, or 6, wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO: 27.
10. The bispecific antibody of claim 6 wherein said heavy chain
variable region of said anti-ovarian cancer single chain antibody
and said light chain variable region of said anti-ovarian cancer
single chain antibody is connected by an intralinker comprising
the amino acid sequence set forth in SEQ ID NO: 15.
11. The bispecific antibody of claim 10, wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO: 23.
12. The bispecific antibody of claim 10, wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO: 25.
13. The bispecific antibody of claim 10, wherein said interlinker
comprises the amino acid sequence set forth in SEQ ID NO: 27.
14. The bispecific antibody of claim 10, wherein said bispecific
antibody further comprises a segment of six consecutive histidine
residues.
15. The bispecific antibody of claim 12, wherein said bispecific
antibody further comprises a segment of six consecutive histidine
residues.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an anti-cancer bispecific antibody
constructed by gene engineering, nucleotide sequences encoding the
said bispecific antibody, the expression vectors containing the
said nucleotide sequences and host cells containing the vectors.
2. Description of the Related Art
Different from natural antibodies, two antigen-binding sites of
bispecific antibody (BsAb) bear different specificities, therefore,
it is bivalent in chemical structure but monovalent in binding function.
BsAb directed to both tumor-associated antigens and trigger molecules
on effector cells can recruit the immunological effector cells to
tumor sites efficiently and activate them to kill tumor cells specifically.
BsAbs are hybrid proteins that can be generated by chemical cross-link,
hybridoma technology or genetic methods. In the chemical cross-link
method, two kinds of monoclonal antibodies and fragments thereof
were dissociated by reductants to generate monovalent antibodies
and fragments thereof. The resulting BsAb is constructed via chemical
cross-linking of two monovalent antibodies and fragments thereof
from different parental antibodies. This strategy can be used for
rapid production of BsAb in large scale but BsAb can be inactivated
sometimes during cross-link and it is difficult to guarantee the
homogeneity of products. Another strategy for production of BsAb
is hybridoma technology by which an established hybridoma cell line
secreting one monoclonal antibody was fused to spleen cells immunized
with the other antigen or two established hybridoma cell lines secreting
two different monoclonal antibodies were fused each other to create
hybrid hybridomas. The former resulting hybridoma is called dimeric
hybridoma and tetrameric hybridoma. Generally, BsAb produced by
hybridoma technology keeps high bioactivities. However, the procedures
are tedious and time-consuming and it is not easy to isolate BsAb
from other non-active and unwanted antibodies generated simultaneously.
These BsAb formats encountered another predictable problems: too
large size and murine components contained in BsAb are immunogenic
in patients and will induce the production of human anti-mouse antibodies
(HAMA), which may prevent reuse of these BsAbs in clinic. Furthermore,
production and purification of these formats of BsAb are expensive,
which limits the application of BsAbs in clinic. Replacement of
these traditional methods with gene recombination approaches has
accelerated progress in this area. Based on the technology of small
molecular antibodies, production of BsAb by gene engineering has
advantages over those described above, such as the stability of
process, large scale production, low cost and easy-to-use. Gene
engineering has led to the development of various small molecular
BsAb formats by connecting two different kinds of scFvs. There are
three kinds of BsAb formats classified by different links. (1) mini-antibodies
are heterodimers assembled by connecting two scFv fragments together
with an oligomerized domain (e.g. leucine zipper motifs derived
from Fos or Jun transcription factors). (2) Diabodies are non-covalently
associated dimmers which are assembled by two single chains VH1-VL2
and VH2-VL1, both connected by a short linker that is too short
to allow pairing between V-domains from the same chain. Thus, each
chain alone is not capable of binding antigen, but co-expression
of two chains (VH.sub.1-VL.sub.2 and VH.sub.2-VL.sub.1) leads to
assembly of heterodimeric diabodies which can bind to two kinds
of antigens. (3) ScBsAb: a interlinker was used for connecting two
different scFvs with different specificities and ScBsAb was expressed
in the host cells as a single polypeptide. The intralinker between
two domains within scFv is often (Gly.sub.4Ser).sub.3. As for the
interlinker between two scFvs, there are two strategies for designing
it. For the purpose of avoiding false paring between heterogenous
variable regions, the interlinker is often a short peptide linker
less than ten amino acid residues such as Gly.sub.4Ser. Another
strategy is to select a longer linker for the interlinker. In our
lab, an interlinker with 25 amino acids named 205c', devised by
Gruber in construction anti-TCR.times.anti-fluorescent scBsAb, was
cited for one of three interlinkers. Another two interlinkers named
Fc (26 residues) and HSA were devised, which both result in the
proper folding of two scFvs and the formation of BsAb with two antigen-binding
sites with high activities. In a word, the most important for designing
interlinkers is to ensure the proper pairing between variable domains
and folding of proteins, resulting in the formation of BsAb which
maintains biological activities and stability. Some novel properties
for facilitating purification and extending the plasma half-life
time should be introduced.
BsAb-mediated immunotherapy plays a promising role in the clinical
biotherapy for tumors. The following is two characteristics of BsAb.
First, tumor-killing effects mediated by BsAb are based on stimulating
the immune system, highly specific with tumors and free of MHC restriction.
Second, due to lacking Fc domain, BsAb is harmless to normal tissues.
Therefore, BsAb-mediated therapy is the complementarity of traditional
methods such as surgery, radiotherapy and chemotherapy. The major
effect of this approach is based on clearing up sub-clinical residuals
and preventing or eliminating the tumor from recurrence and metastasis.
BsAb can not only cure tumors but also stimulate the immune system
to provide and keep the immune protection for a long time. Based
on results of experiments in mouse and clinic, BsAb prepared for
trial use should have at least five characteristics as follows:
{circle around (1)} It targets to the relevant tumor antigens with
high specificity and affinity; {circle around (2)} It can bind monovalently
to trigger factors on effector cells-cytotoxic cells and result
in cross link only when BsAb binds to tumor antigens due to lack
of Fc domain; {circle around (3)} BsAb is able to promote the effective
cytotoxicity and inflammation selectively produced by the corresponding
group of leukocytes at tumor sites; {circle around (4)} BsAb must
be humanized to minimize induction of human anti-mouse response
following repeated uses; Finally, {circle around (5)} BsAb should
be not only small enough to penetrate into tumors but also large
enough to keep in the circulation for a sufficient time.
Based on these points described above, numerous BsAbs triggering
many kinds of immune effector cells and targeting different tumor
cells have been developed in the past few years, wherein the effector
cells include T lymphocytes, NK cells, monocytes, macropghages,
neutrophils, LAK cells (lymphokine-activated cytotoxic cells) and
TIL cells (tumor infiltrating lymphocytes) etc. T cells are commonly
recognized as the major specific cells for immune responses. CD3
expressed on the surface of all mature T cells is the common surface
marker for T cells. CD3 binds to TCR non-covalently, forming the
whole TCR-CD3 complex, and involves in immune responses against
antigen stimulus. Now CD3 is surface trigger molecule on immune
effector cells used most widely and successfully. Following anti-CD3
antibody within BsAb binds to CD3 molecule on the surface of T cells,
numerous effects as follows will be produced to kill tumor cells.
These effects include: (1) proliferation and differentiation of
T cells. Firstly, BsAb can activate the rest T cells, resulting
in Th cell and Tc cell derived from the premature effector T cells
with CD4.sup.+ or CD8.sup.+. Secondly, BsAb can activate numerous
memory cells to proliferate and differentiate into effector T cells
which will attack and kill tumor cells. The number of effector cells
is directly related to the rate of tumor elimination. (2) release
of cytokines: CD4.sup.+ Th cells activated by BsAb can secrete a
great deal of IL-2. IL-2 not only stimulates the proliferation of
Th cells in autocrine, but also activates naive CD8.sup.+ T cells
in paracrine to become Tc cells, resulting in enlargement of cytotoxicity
of Tc cells. In addition, IL-2 is a costimulating signal for activating
T cells. Therefore, IL-2 plays a vital role in BsAb-mediated immune
effects. Some other cytokines, such as TNF-.alpha. and IFN--Y are
produced in the process of T-cell activation and can produce `stander-by`
effect by inhibiting the growth of `stander-by` tumor cells through
the medium among cells. (3) cytotoxicity: In vitro experiments indicate
that mediated by BsAb, CD8.sup.+ Tc interacts with tumor cells directly,
releases cytotoxic materials through granule exocytosis and lyses
target cells, which takes place rapidly usually within 4-6 hours
following targeting tumor cells. The major components in the cytotoxic
materials are perforin and serine easterases or granzymes. Perforins
can attack the plasma membrane and form ion channels, thus causing
entry of plenty of ions and water, resulting in the lysis and necrosis
of cells while granzymes are similar to lymphotoxin, capable of
activating DNases in the cell, thus causing lysis of nucleic DNA,
resulting in the apoptosis of target cells.
Currently, Fv fragment is widely used for construction of BsAb,
since it is the minimal unit with the complete antigen-binding site,
small (about 1/6 of the whole antibody), absence of Fc domain, lower
immunogenicity, easily penetration into the wall of blood vessels
and solid tumors, easily expressed in E. coli and lower production
cost. However, Fv is unstable and easy to dissociate in vivo because
the covalent bond between VH and VL domains is unable to generate.
In order to improve the stability of Fv fragment, a polypeptide
intralinker between VH and VL domains is used to form so called
ScFv. The intralinker is commonly a short flexible peptide with
15 amino acid residues in length such as (Gly.sub.4Ser).sub.3. In
the present invention, the said intralinker was used in both ScFvs.
As mentioned above, there are several methods to construct BsAb.
In the present invention, we constructed the single-chain bispecific
antibody (ScBsAb) connected by an interlinker. The general principle
for designing interlinkers is to ensure the proper pairing and folding
of variable domains from two antibodies, furthermore keep the biological
activities and stability of the said antibody. In addition, the
said interlinker should endow BsAb some novel properties, such as
easy purification and prolonged half-life time in the plasma etc.
Two kinds of interlinkers, Fc and HSA originally designed in the
present invention as a useful provide a novel idea for designing
interlinkers. 205c' interlinker cited from literature was used to
compare and verify the efficacy of interlinkers designed in the
present invention and the value of the said design in construction
of anti-ovarian BsAb. (1) Design of Fc interlinker: in order to
minimize the immunogenicity and molecule size, small molecular antibodies
are absent of Fv domains resulting in lack of several biological
function, such as ADCC, CDC and the classic complement activating
pathway. To resolve this problem, we devised the interlinkers to
make up the said shortcoming of genetically engineered antibodies.
IgG1 is the most potent molecule in inducing ADCC and CDC among
four subtypes of IgGs. It can induce the classic complement activating
pathway by combining to C1q with its C-terminal sequence of CH.sub.2,
wherein Gly318, Lys320 and Lys322 sites locate in the surface of
Fc molecule to form a cluster in conformation and combine to C1q
directly. In addition, Asn297 of CH.sub.2 contains a glycosylation
site which is vital to the effect of ADCC and CDC induced by Fc.
Thus, a fragment from 297 to 322 of CH.sub.2 in human IgG was selected
to construct the interlinker of ScBsAb. It has 26 residues in length
and contains the glycosylation site Asn297, the C1q-binding site
Glu318, Lys320 and Lys322 etc as well as an EcoRI site at the 5'
end and a SacI site at the 3' end for the purpose of gene clone.
ScBsAb constructed by this strategy is expected to have the prolonged
half life time in vivo and the effect for inducing CDC similar to
Fc. (2) HSA interlinker: Because of the smaller size, small molecular
antibodies have fast renal clearance, which results in a short retention
time in immunotherapy thus causing curative effects unperfect although
the shorter half life time is benefit for immunoimaging diagnosis
of tumors. Therefore, we devised HSA interlinker which is expected
to prolong the half life time of ScBsAb in vivo, improve the stability
and solubility of ScBsAb. HSA (human serum album) is an important
component of human serum. It is widely used as a stable natural
vector because of its stability, several week half-life time, lack
of specific enzymatic and immunological activities and slow clearance
in liver. It was showed in research that the stability of proteins
fused with HSA increased 20 to 40 times in animals. HSA molecule
with 585 amino acids in length is composed of three domains, wherein
the third domain DIII alone possesses the vector function of the
whole molecule. Herein, a fragment with 25 residues from 403 to
427 of DIII domain, which is lack of Cys but rich in polar amino
acids in HSA was used as another interlinker in construction of
BsAb to improve the stability and prolong the half-life time in
vivo. (3) 205c' interlinker: This interlinker is 25 amino acids
in length devised by Gruber in construction of anti-TCRxanti-fluorescence
scBsAb. The purpose of utilizing 205c' interlinker was to compare
and verify the efficacy of interlinkers designed in the present
invention and the value of the said design in construction of anti-ovarian
BsAb.
Facing HAMA problem induced by murine antibodies in clinic that
strongly limits repeated use and dose, further causing the poor
curative efficacy, murine antibodies must be humanized to minimize
their heterology, which is the urgent affairs for preparation of
antibodies used in clinic. The scFv against CD3 molecule in ScBsAb
used in the present invention is a reshaped antibody through humanization.
The reshaped antibody, so-called CDR-grafted antibody or humanized
antibody, is constructed by grafting complementarity-determing regions
(CDRs) from the variable domains of rodent antibodies into the framemork
regions of human variable domains. The space structure of antigen-binding
sites of antibodies is mainly determined by six CDRs of variable
domains. The said CDRs form three loops, which have decisive effects
in antigen-antibody recognition, in the upper site of variable domains
supported by four .beta.-sheet domains. The said reshaped antibody
remains the antigen-binding ability as well as the most characteristics
of human antibody, therefore minimizing HAMA response effectively.
Ovarian cancer remains the leading cause of death from gynecologic
malignancies. The five-year survival rate maintains only 30%. Because
of lack of the effective diagnostic methods for ovarian cancer located
deep into pelvic cavity and the vague symptoms associated in the
earlier stage, most patients with ovarian cancer present with an
advanced stage of cancer. Although methods of surgical operation
advance, drugs of chemotherapy renew and treatments of radiotherapy
improve stepwise, the prognosis of ovarian cancer didn't improve
at all. The easy recurrence after surgical operation and the side
effects and drug tolerance after repeated use of chemotherapy strongly
influence the effects of treatments. Therefore, specific diagnostic
methods for earlier stage of cancer and the timely clearance of
residual focuses are the key step for improving prognosis. BsAb
against the related antigens of ovarian cells is regarded as powerful
tools in clinic.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a biological
preparation with low toxicity, high efficiency and cost-effectiveness
against ovarian cancers-anti-human ovarian cancer.times.anti-human
CD3 bispecific antibody developed by gene engineering technology.
Another object of the present invention is to provide a nucleotide
sequence encoding the said BsAb.
Another object of the present invention is to provide a vector
for the said nucleotide sequence.
Further object of the present invention is to provide a host cell
transformed by the expression vector used in the invention.
In addition, based on the context of the disclosure, another aspects
of the present invention will be apparent to those with skill in
the art.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic presentation of plasmid pFUW80;
FIG. 2 shows the nucleotide sequences of variable domains of heavy
chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 3) of anti-ovarian
monoclonal antibody and the amino acid sequences (SEQ ID NO: 17
and SEQ ID NO: 18 respectively) encoded by the said nucleotide sequences;
FIG. 3 is activity of anti-ovarian scFv tested by ELISA;
FIG. 4 is a schematic presentation of plasmid pROH80;
FIG. 5 shows the nucleotide sequences of variable domains of heavy
chain (SEQ ID NO: 5) and light chain (SEQ ID NO: 7) of anti-CD3
reshaped scFv and the amino acid sequences (SEQ ID NO: 19 and SEQ
ID NO: 20 respectively) encoded by the said nucleotide sequences;
FIG. 6 is antigen-binding activity of anti-CD3 reshaped scFv tested
by FACS;
FIG. 7 is a schematic presentation of plasmid pAL781 and the synthesized
multi-cloning site (SEQ ID NO: 21 and SEQ ID NO: 28);
FIG. 8 shows the nucleotide sequences of three kinds of interlinkers
(Fc interlinker: SEQ ID NO: 22; HSA interlinker: SEQ ID NO: 24:
205C' interlinker: SEQ ID NO: 26) and the amino acid sequences (Fc
interlinker: SEQ ID NO: 23; HSA interlinker: SEQ ID NO: 25; 205C'
interlinker: SEQ ID NO: 27) encoded by the said nucleotide sequences;
FIG. 9 is a schematic presentation of plasmid palm;
FIG. 10 is a schematic presentation of plasmid pETAE;
FIG. 11 is a schematic presentation of plasmid pTMF;
FIG. 12 is anti-ovarian activity of BsAb tested by ELISA; and
FIG. 13 is SDS-PAGE analysis of bispecific antibody expressed from
pTMF.
DETAILED DESCRIPTION OF THE INVENTION
One antibody molecule consists of two identical heavy chains and
light chains, each of which is composed of one variable region and
one or more constant region. The variable region is responsible
for binding with antigens and the constant region is mainly responsible
for binding with effect molecules. There are three flexible loops
with high variability in each variable region, termed complementarity-determining
regions (CDRs), which are mainly responsible for recognizing antigens.
The other parts of variable regions, are composed of the rigid .beta.-sheets
and support the so-called framework regions (FRs). CDRs and FRs
arrange alternatively forming the "Sandwich" structure.
In the present invention, the used terms have meanings as follows:
"Fab antibody" refers to a heterodimer formed by Fd fragment
(consisting of heavy chain V.sub.H and CH1) and the whole light
chain which is connected to the former by a interchain disulfide.
The size of "Fab antibody" is 1/3 of the whole antibody
and it contains only one antigen binding site.
"Single chain antibody (scFv)" refers to an antibody
fragment constructed by gene engineering and a recombinant protein
consisting of heavy chain variable region (V.sub.H) and light chain
variable region (V.sub.L) which is joined to the former by a linker.
The size of scFv is about 1/6 of the whole antibody.
"Single domain antibody" consists of the heavy chain
variable region (V.sub.H) or the light chain variable region (V.sub.L).
Since this antibody fragment consists of only one domain, it is
called single domain antibody. The size of this fragment is 1/12
of the whole antibody.
"Minimal recognizing unit (MRU)" consists of single CDR
and its size is about 1/70 or 1/80 of the whole antibody.
"Reshaping antibody" is also called "CDR-grafted
antibody". Using gene synthesis or site-directed mutation,
CDRs in human antibody are replaced by those from murine antibody,
therefore the antigen-binding specificity of murine antibody was
kept. However, it should be considered that some amino acid residues
in human FRs are capable of interfering the conformation of antigen-binding
site formed by murine CDRs. Therefore, individual amino acid residues
in FRs need be mutated to obtain antibodies humanized to the most
extent and with high affinity.
The present invention provides a genetically engineered bispecific
antibody against ovarian cancers, wherein the antibody is the whole
antibody molecule, Fab, single domain antibody or single-chain Fv
(ScFv).
Preferably, the genetically engineered bispecific antibody against
ovarian cancer consists of two different single-chain antibodies.
In the present invention, the genetically engineered bispecific
antibody against ovarian cancers is preferably a hybrid protein
consisting of the single-chain Fv against ovarian cancer and the
reshaped single-chain Fv against human CD3. The said proteins is
the expressed products, which are capable of activating T lymphocytes
to kill ovarian cancer cells specifically, of anti-ovarian scFv
and anti-CD3 reshaped scFv connected by three interlinkers.
In bispecific antibodies of the present invention, the said anti-ovarian
scFv can contain the amino acid sequence of heavy chain variable
domain as follows:
TABLE-US-00001 1 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu 11 Val
Lys Lys Pro Gly Glu Thr Val Arg Ile 21 Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr 31 Thr Ala Gly Met Gln Trp Val Gln Lys Met 41 Pro Gly
Lys Gly Leu Lys Trp Leu Gly Trp 51 Ile Asn Thr Asn Ser Glu Val Pro
Lys Tyr 61 Ala Glu Asp Phe Arg Gly Arg Phe Ala Phe 71 Ser Leu Glu
Thr Ser Ala Ser Thr Ala Tyr 81 Leu Gln Ile Ser Asn Leu Lys Asn Glu
Asp 91 Thr Ala Thr Phe Phe Cys Ala Arg Ser Phe 101 Thr Trp Gly Thr
Met Asp Tyr Trp Gly Gln 111 Gly Thr Thr Val Thr Val Ser Ser
In bispecific antibodies of the present invention, the said anti-ovarian
scFv can contain the amino acid sequence of light chain variable
domain as follows:
TABLE-US-00002 1 Asp Val Val Met Thr Gln Thr Pro Leu Ser 11 Leu
Pro Val Ser Leu Gly Asp Gln Ala Ser 21 Ile Ser Cys Arg Ser Ser Gln
Thr Leu Val 31 His Ser Ile Gly Asn Thr Tyr Leu His Trp 41 Tyr Leu
Gln Lys Pro Gly Gln Ser Pro Lys 51 Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe 61 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 71 Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 81 Ser Arg Val Glu Ala Glu Asp Leu Gly
Val 91 Tyr Phe Cys Ser Gln Ser Thr His Val Pro 101 Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu 111 Leu Lys
In bispecific antibodies of the present invention, the said anti-human
CD3 reshaped scFv can contain the amino acid sequence of heavy chain
variable domain as follows:
TABLE-US-00003 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu 11 Val
Arg Lys Pro Gly Ala Ser Val Arg Val 21 Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr 31 Arg Tyr Thr Met His Trp Val Arg Gln Ala 41 Pro Gly
His Gly Leu Glu Trp Ile Gly Tyr 51 Ile Asn Pro Ser Arg Gly Tyr Thr
Asn Tyr 61 Asn Gln Lys Phe Lys Asp Arg Val Thr Met 71 Thr Thr Asp
Lys Ser Phe Ser Thr Ala Ile 81 Met Asp Leu Arg Ser Leu Arg Ser Asp
Asp 91 Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr 101 Asp Asp His Tyr
Cys Leu Asp Tyr Trp Gly 111 Gln Gly Thr Thr Val Thr Val Ser Ser
In bispecific antibodies of the present invention, the said anti-human
CD3 reshaped scFv can contain the amino acid sequence of light chain
variable domain as follows:
TABLE-US-00004 1 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr 11 Leu
Ser Leu Ser Pro Gly Glu Arg Ala Thr 21 Leu Ser Cys Ser Ala Ser Ser
Ser Val Ser 31 Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly 41 Gln Ala
Pro Arg Arg Trp Ile Tyr Asp Thr 51 Ser Lys Leu Ala Ser Gly Ile Pro
Ala Arg 61 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 71 Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 81 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Trp 91 Ser Ser Asn Pro Phe Thr Phe Gly Gly Gly 101 Thr Lys Val Glu
Ile Lys Arg
In bispecific antibodies of the present invention, the interlinker
connecting two single-chain Fv antibodies can contain the amino
acid sequence as follows:
TABLE-US-00005 1 Asn Ser Thr Tyr Arg Val Val Ser Val Leu 11 Thr
Val Leu His Gln Asp Trp Leu Asn Gly 21 Lys Glu Tyr Lys Cys Lys
In bispecific antibodies of the present invention, the interlinker
connecting two single-chain Fv antibodies can contain the amino
acid sequence as follows:
TABLE-US-00006 1 Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 11 Lys
Lys Val Pro Gln Val Ser Thr Pro Thr 21 Leu Val Glu Val Ser
In bispecific antibodies of the present invention, the interlinker
connecting two single-chain Fv antibodies can contain the amino
acid sequence as follows:
TABLE-US-00007 1 Ala Ser Ala Asp Asp Ala Lys Lys Asp Ala 11 Ala
Lys Lys Asp Asp Ala Lys Lys Asp Asp 21 Ala Lys Lys Asp Leu
The present invention still provides a nucleotide sequence encoding
the said BsAb, herein containing the nucleotide sequences of two
scFvs and the interlinkers between two scFvs. The said interlinker
may be any kind of which are capable of ensuring the proper folding
of each of two antibodies, furthermore keeping the biological activities
of the said antibodies. In addition, the said interlinker should
endow BsAb some novel properties to the products. Fc interlinker
and HSA interlinker designed and constructed in the present invention
and 205c' interlinker cited in the present invention are preferred.
The present invention provides the universal E. coli plasmids for
construction and expression of BsAb and the expression plasmids
containing the nucleotide sequences encoding BsAb of the present
invention. In the preferred embodiment of the present invention,
one plasmid is pALM derived from plasmid pAL781, which is a universal
plasmid for expression of BsAb. The said plasmid has characteristics
as follows: Based on the constructed BsAb, different types of BsAbs
can be generated by replacing anyone kind of scFv genes. The plasmid
from pALM containing the nucleotide sequences encoding BsAb of the
present invention was named pALMB. Another plasmid is pETAE, a derivative
of pET16 by inactivating EcoRI site, contains lac operator and T7
promoter, which makes pETAE an vector for expression of target proteins
with efficiency. The plasmid from pETAE containing the nucleotide
sequences encoding BsAb of the present invention was named pEMAB.
Another plasmid is pTMF, which was constructed from pET28a according
to the restriction endonuclease sites of BsAb and for the sake of
the need of further research and is a plasmid for expression of
proteins not limited to BsAb with high efficiency. Besides the high
efficiency of expression, the plasmid has the characteristics as
follows: it has several rare restriction endonuclease sites to facilitate
insertion of various foreign genes in the manner of co- or fusion
expression; there is a thrombin site between two groups of restriction
endonuclease sites, which facilitates the isolation of the desired
protein from the fusion protein; there is six codons encoding six
His residue at 3' end of the multiclonig site, which facilitates
the purification of expressed products with metal chelating chromatography.
The plasmid from pTMF containing the nucleotide sequences encoding
BsAb of the present invention was named pTMAB.
The present invention still provides the host cells containing
the above described expression vectors. The said host cells are
preferably E. coli.
The steps for production of genetically engineered BsAbs of the
present invention are described as follows:
1. VH and VL genes of the monoclonal antibody were amplified from
the hybridoma cell line secreting monoclonal antibody against tumor,
respectively, by PCR technology using the designed primers.
2. The obtained VH and VL genes against tumors were inserted into
the universal scFv expression plasmid, which was transformed into
E. coli, induced, expressed and characterized.
3. The amino acid sequences of reshaped VH and VL were designed,
respectively, according to the antigen binding sites of mouse anti-human
CD3 monoclonal antibody OKT3 by using molecular modeling. The nucleotide
acid sequences were deduced with E. coli bias codons. The whole
genes of VH and VL were obtained by splicing the synthetic oligonucleotide
fragments using PCR.
4. The resulting VH and VL genes of reshaped anti-human CD3 antibody
were inserted into the universal single-chain antibody expression
vector, which was transformed into E. coli, expressed under induction
and characterized.
5. The plasmid with a strong promoter was selected as the starting
plasmid. A DNA fragment containing the restriction endonuclease
sites which are not present in the starting plasmid, two scFvs and
three interlinkers was designed, synthesized and used to replace
the multicloning site of the starting plasmid. The synthesized oligonucleotide
sequences of interlinkers are inserted into the corresponding sites
in the new multicloning site, respectively. Therefore, the universal
intermediate vectors containing the interlinkers for BsAbs were
constructed.
6. A pair of primers was designed and scFv gene with flanking suitable
digestion sites was amplified by PCR from the plasmid containing
anti-CD3 reshaped scFv. The gene fragment was inserted into the
intermediate vectors for bispecific antibody to yield anti-human
CD3-based universal expression vectors for ScBsAb. The vectors were
transformed into E. coli, expressed under induction and the effects
of different interlinkers on the expression of scFvs were characterized.
7. The gene fragment of anti-human ovarian carcinoma scFv was obtained
by digesting the expression vector containing the gene of anti-human
ovarian carcinoma scFv with double restriction endonucleases. The
expression vector for bispecific antibody was constructed by inserting
the said gene fragment into the corresponding restriction sites
of anti-human CD3-based universal expression vector for ScBsAb.
8. The said expression vector from 7 was transformed into E. coli.
BsAb was expressed under induction. The molecular mass, expression
amount and expression form were analyzed by SDS-PAGE. The anti-tumor
activity of the expressed products was tested by ELISA. The anti-human
CD3 activity of the expressed products was tested by FACS. The expressed
products mediated anti-tumor capability and specificity was assayed
using Jurkat cells or human peripheral blood lymphocytes.
9. The expression vector with high efficiency was constructed and
the gene of BsAb was inserted into the said vector, resulting in
the expression vector with high efficiency for BsAb.
10. The constructed vector with high efficiency was transformed
into the host cells.
11. The transformed host cells were cultured and induced for the
expression of the said BsAb.
12. The expressed BsAb was isolated.
In additon, the present invention still relates to the drug compositions
for the treatments or prevention of tumors containing anti-human
ovarian cancer.times.anti-human CD3 bispecific antibody and the
pharmaceutical vectors of the present invention and uses of anti-human
ovarian cancer.times.anti-human CD3 bispecific antibody in preparation
of drugs for the treatments or prevention of tumors and in the treatments
or prevention of tumors thereof.
The present invention will now be described further by way of the
following examples which are intended to be illustrative only and
not limited to the scope of the present invention.
An embodiment is disclosed in following paragraphs.
1. Construction of Anti-Ovarian Carcinoma scFv and Anti-Human CD3
scFv
(1) Construction of Anti-Ovarian Carcinoma scFv Antibody
VH and VL genes of monoclonal antibody COC183B2 against human ovarian
carcinoma were cloned, respectively, by RT-PCR and PCR technology
with primers hybridizing to FR1 and FR4 sequences of VH or VL region
of mouse immunoglobulin (shown in FIG. 2). VH and VL fragments were
cloned to plasmid pUC19 and verified by DNA sequencing. Plasmid
pFVB2 was created by inserting VH and VL genes to plasmid pFUW80
(shown in FIG. 1) constructed by our lab with the order of VH and
VL from 5' to 3' end and there is a (Gly.sub.4Ser).sub.3 linker
(SEQ ID NO: 15) between VH and VL. E. coli strain Top10 was used
for the propagation of plasmid and the positive plasmid was verified
by digestion with proper restriction endonucleases. E. coli strain
XL1-Blue was transformed with pFVB2 and infected by helper phage
M13KO7. The phage particles were rescued and the binding activity
of the phage antibody was assayed by indirect ELISA. The results
indicate that the binding activity of the antibody is 2.5 times
higher than that of the negative control, which demonstrates that
anti-ovarian carcinoma scFv antibody was constructed successfully.
(2) Construction of Anti-Human CD3 Reshaped scFv Antibody
The amino acid sequences of reshaped VH and VL were designed, respectively,
according to the antigen binding sites of mouse anti-human CD3 monoclonal
antibody OKT3 by using molecular modeling. The nucleotide acid sequences
were deduced with E. coli bias codons. The genes of VH and VL (shown
in FIG. 5) were obtained by splicing the synthetic oligonucleotide
fragments using PCR. The VH and VL genes of reshaped anti-human
CD3 antibody were inserted into the universal single-chain antibody
expression vector pROH80 (shown in FIG. 4) in the orientation of
VL-(Gly4Ser)3-VH from 5' to 3' end. E. coli strain Top10 was used
for propagation of the yielding plasmid pROCD3 and the positive
plasmids were verified by digestion with proper restriction endonucleases.
Single colony was picked up from the transformed Top10 plate and
incubated in LB medium with corresponding antibiotics at 37.degree.
C. overnight. An aliquot of the culture was transferred to the fresh
medium with the proportion of 1-5% and shaking at 37.degree. C.
until an OD600 of 0.5-1.0 was reached. IPTG was added to the final
concentration of 0.4 mM to induce expression of target proteins.
The antigen-binding activity of reshaped anti-CD3 scFv was assayed
by FACS. The results demonstrates that the competitive inhibition
rate of anti-CD3 reshaped scFv to anti-CD3 monoclonal antibody was
18% (shown in FIG. 6), which implies anti-human CD3 reshaped scFv
was constructed and expressed successfully.
2. Construction and Expression of Anti-Human Ovarian Carcinoma.times.Anti-Human
CD3 Bispecific Antibody
(1) Construction of Intermediate Vector for Bispecific Antibody
An expression vector with suitable restriction endonuclease sites
was constructed for the insertion of two above described scFvs and
interlinker. Plasmid pAL-781 (shown in FIG. 7) was chosen as the
starting vector to construct the vector for bispecific antibodies.
In the present invention, an oligonucleotide fragment with 55 bps
was designed and synthesized to replace the multiple clone sites
(MCS) in pAL-781. This intermediate vector was named pALM. The new
MCS contains the start codon ATG integrated in restriction endonucleases
site NdeI. The gene fragment of anti-ovarian carcinoma scFv was
inserted between XhoI and EcoRI, interlinker was inserted between
EcoRI and SacI and the gene fragment of anti-CD3 reshaped scFv was
inserted between SacI and BamHI. And the following was DNA fragment
(CATCAC).sub.3 (SEQ ID NO: 16) encoding 6 His and the stop codon
TAA. Anyone of the components mentioned above could be substituted
for another by digestion and insertion. 3 kinds of the synthetic
interlinkers fragments (shown in FIG. 8) were inserted into the
proper restriction sites on pALM. Three intermediate vectors for
ScBsAb: pALM-Fc; pALM-HSA and pALM-205c' (shown in FIG. 9) were
constructed successfully and verified by digestion and DNA sequencing.
(2) Construction of CD3-based Universal Expression Vector for ScBsAb
A pair of primers were designed for amplification of CD3 scFv gene
with flanking digestion sites SacI at 5' end and BamHI at 3' end
from the plasmid containing anti-CD3 reshaped scFv by PCR. The gene
fragment was inserted into the intermediate vector for bispecific
antibody to yield anti-human CD3-based universal expression vector
for ScBsAb. The resulting vector was transformed to E. coli strain
GI724 and the protein was expressed. The results indicate that all
of three kinds of interlinkers had no negative effects on the expression
of scFv.
(3) Construction of Bispecific Antibody
The gene fragment of anti-human ovarian carcinoma scFv was obtained
by digesting the expression vector containing the gene of anti-human
ovarian carcinoma scFv with restriction endonucleases XhoI and EcoRI.
The expression vector for bispecific antibody was constructed by
inserting the said gene fragment into anti-human CD3-based universal
expression vector for ScBsAb. The resulting plasmids were propagated
in E. coli strain GI724, verified by digestion with proper restriction
endonucleases and named pAMAB.
(4) Expression of Bispecific Antibody in E. coli
Single colony of E. coli strain GI724 harboring pAMAB was picked
up and incubated in RM medium overnight at 30.degree. C. An aliquot
of culture was transferred to fresh medium with the proportion of
20%. When an OD550 of the culture was reached 0.5-1.0, tryptophan
was added to the final concentration of 100 ug/ml to induce expression
of the target protein. After 3 hours of induction, culture was precipitated
by centrifugation at 3,000 rpm for 10 min at 4.degree. C. The cell
pellet was resuspended in 1/2 culture-volume of PBS and broken by
ultrasonic in ice bath for 20 s for 6-8 times with 1-min interval
followed by centrifugation at 4.degree. C. at 12,000 rpm for 20
min. The precipitate was resuspended in PBS with the same volume
to the supernatant. The sonicate, supernatant and precipitate of
sonication were analyzed by 12% SDS-PAGE with the empty vector pALM
as negative control. Protein with molecular weight of 52 kDa was
found in both supernatant and precipitate. The result indicated
the target protein was expressed partly in soluble form. The soluble
protein could be used directly to identify the biological activity
of bispecific antibody.
3. Biological Activity Assay of Anti-Human Ovarian Carcinoma.times.Anti-Human
CD3 Bispecific Antibody
(1) Antigen-Binding Activity of Anti-Human Ovarian Carcinoma scFv
The antigen-binding activity of anti-ovarian carcinoma scFv in
bispecific antibody was assayed by direct ELISA. ELISA plate was
coated with bispecific antibody at 4.degree. C. overnight. Wells
coated with anti-ovarian monoclonal antibody were set as positive
control. Plate was washed 3 times with PBST (PBS-0.05% Tween 20)
for 5 min. HRP-OC183B2 diluted in 3% goat serum was added and incubate
at 37.degree. C. for 1 h. After washing plate 3 times with PBST,
the substrate of HRP was added and incubated for 20 min at room
temperature in the dark. 2M H2SO4 was added to stop the reaction.
The plates were read at 492 nm (data shown in FIG. 12). All the
values of OD492 of bispecific antibody were 2.5 times higher than
those of negative control and changed with gradient among the different
dilution of bispecific antibody. The data indicates that anti-ovarian
scFv in bispecific antibody has the binding activity to antigens
associated with ovarian carcinoma.
(2) Antigen-Binding Activity of Anti-Human CD3 scFv
The antigen-binding activity of anti-CD3 reshaped scFv in bispecific
antibody was assayed by FACS according to the principle of competitive
inhibition. 1.times.106 fresh Jurkat cells in an FACS tube were
washed 3 times with PBS containing 2% fetal bovine serum and 0.1%
NaN3. Bispecific antibody was added before the cells were incubated
at 4.degree. C. for 45 min. Cells incubated with PBS were set as
positive control. After washed 3 times with PBS containing 2% fetal
bovine serum and 0.1% NaN3, the cells were incubated with diluted
murine anti-CD3 monoclonal antibody for 30 min at 4.degree. C. After
washed 3 times with PBS containing 2% fetal bovine serum and 0.1%
NaN3, the cells were incubated with goat anti-mouse IgG-FITC (with
the dilution of 1:50) for 45 min at 4.degree. C. After washed 2
times with PBS containing 2% fetal bovine serum and 0.1% NaN3, the
cells were resuspended in 500 ul PBS and assayed on FACSort. The
data indicate bispecific antibody can greatly inhibit the antigen-binding
activity of anti-CD3 mouse monoclonal antibody. The inhibition rate
is 18%, which demonstrates anti-CD3 reshaped scFv in bispecific
antibody has binding activity to CD3. The results indicate two scFvs
within bispecific antibody both keep their antigen-binding activities.
(3) Cytotoxicity of Anti-Ovarian Bispecific Antibody Against Ovarian
Carcinoma Cells
Human ovarian cell line SKOV3 cells (target cells) were seeded
in 96-well plate with approximate 1.times.104/well. Bispecific antibody
renatured from inclusion bodies were added at three different volumes
of 5 ul, 10 ul and 20 ul. Plates were incubated in the incubator
with CO2 overnight. Jurkat cells (effector cells) were added at
different effector cells: target cells ratios. Plates were incubated
in the incubator with CO2 at 37.degree. C. for 48 h. 25 ul MTT was
added to each well. After incubation at 37.degree. C. for 4 h, the
plates were emptied and 100 ul acidic SDS (0.1N HCl, 1% SDS) was
added to each well. After incubation at 37C overnight, plates were
read at 570 nm. The rate of cytotoxicity was calculated according
to the formula below.
.times..times..times..times. ##EQU00001##
As shown in Table 1, the cytotoxicity rate of effector cells against
target cells increased in the case of addition of bispecific antibody
(shown in FIG. 13). The rate cytotoxicity increases with the increase
of the concentration of bispecific antibody, which indicates bispecific
antibody triggers the direct killing effect of effector cells against
target cells.
TABLE-US-00008 TABLE 1 BsAb 140 .mu.g/ml 0 .mu.l 5 .mu.l 10 .mu.l
20 .mu.l E/T cytotoxicity % 12.5:1 97.39 100.00 110.76 144.64 6.2:1
69.56 55.09 113.27 104.29 3.1:1 33.48 46.30 52.02 64.38
4. Construction of High Performance Expression Vector
For production purpose, high performance expression vector was
constructed for overexpression of bispecific antibody. pET.DELTA.E
(shown in FIG. 10) derived from pET16 by inactivated EcoRI site
is a T7 promoter-based high performance expression vector. The bispecific
antibody gene fragment digested from pAMAB with XhoI and BamHI was
inserted into the same sites of pETAE digested with the same restriction
endonucleases, yielding pEMAB. Proteins were expressed after pEMAB
was transformed to E. coli strain BL21(DE3). However, expressed
from pETAE, the target protein was fused to (His)10 tag which could
not be purified by IMAC as effectively as proteins fused to (His)6.
Therefore, plasmid pTMF (shown in FIG. 11) derived from pET28a was
constructed. pTMF contains T7 promoter for overexpression of target
proteins and several unusual restriction sites which were used for
facilitating the insertion of foreign genes followed by fusion or
co-expression of target proteins. A thrombin site was designed between
two groups of restriction sites. Following purification, the target
proteins could be separate from the fusion proteins by proteolysis
on thrombin site. Flanking 3' end of MCS is the sequence coding
for His6 tag which could be used in IMAC.
The gene fragment of bispecific antibody digested from pAMAB with
XhoI and BamHI was inserted into pTMF to generate plasmid pTMAB.
E. coli strains BL21(DE3) was transformed with pTMAB, single colony
was picked and grown until an the value of OD550 reached 0.5. Protein
expression was induced by adding IPTG to a final concentration of
0.4 mM. After 3 hours of induction, the culture was precipitated
by centrifugation, cell pellets were broken by ultrasonic and the
sonicate was analyzed by SDS-PAGE (shown in FIG. 13). In the total
whole-cell proteins, 27% is bispecific antibody 16% of which is
soluble. The expression level is eligible in production area. |