NZ615477B2 - Tumor necrosis factor-? humanized antibody - Google Patents
Tumor necrosis factor-? humanized antibody Download PDFInfo
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- NZ615477B2 NZ615477B2 NZ615477A NZ61547712A NZ615477B2 NZ 615477 B2 NZ615477 B2 NZ 615477B2 NZ 615477 A NZ615477 A NZ 615477A NZ 61547712 A NZ61547712 A NZ 61547712A NZ 615477 B2 NZ615477 B2 NZ 615477B2
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
Disclosed is a humanised anti-TNF monoclonal antibody comprising the CDRs and FRs of the sequences as defined in the specification. Also disclosed is its use in treatment.
Description
TUMOR NECROSIS FACTOR - ! HUMANIZED ANTIBODY
TECHNICAL FIELD
The present invention relates to at least a humanized anti tumor necrosis
factor-! (TNF!) or fragments thereof, including specific parts or variants, and nucleic
acid encoding the humanized anti-TNF! antibody and its complementary nucleic acid,
vectors, host cells, and the preparation method thereof, and compositions and kits
comprising the humanized anti-TNF! antibody, and the use thereof.
BACKGROUND
Human tumor necrosis factor-! (TNF!) is a proinflammatory cytokine
produced by monocytes and macrophages, which is a 26 kDA precursor protein when
initially produced with N terminal inside the cells and C terminal outside the cells,
named transmembrane type TNF!. Pennica et al. cloned TNF! gene cDNA for the first
time in 1984, and deduced that human TNF! molecule is composed of 157 amino acid
residues, and weights about 17 KD (Pennica D, et al, Nature 1984!312:724)"Huamn
TNF has two molecular forms, TNF! and TNF". TNF! is produced by activated
macrophages or monocytes, and causes neoplastic tissues hengrrhagie necrosis, thus
it is also called Cachectin. TNF" is mainly secreted by active T lymphocytes. Both
have similar pyretogenesis. TNF! acts on receptors on the surface of oncocytes, and
breaks into lysosome by identifying the cell, binding and endocytosing, and then
activates lysosomes and proteases to cause cell death. TNF! plays an important role in
immune response, inflammation, and response to injury, majorly affects the regulation
of cell proliferation and cell apoptosis. Besides the effects on tumor cell such as
cytotoxicity, cytolysis, induction of apoptosis and cell proliferation suppression, TNF!
also can facilitate cell differentiation of myeloid leukemia cell to macrophage, and
improve the phagocytic activity of neutrophile granulocyte.
An appropriate amount of TNF! can activate immune system to enhance
immunity of the body, and play an important role in defense system of host resisting
microbial invasion and tumor inhibition. But when over expressed, TNF! may cause
several pathologic damages with other inflammatory factors. Therefore, the activities of
TNF! may be suppressed or neutralized at different levels to block it from approaching
receptors, in turn avoid the consequence of signal transduction.
For the purpose of overcoming relevant problems caused by using non-
human antibodies, it is a relatively effective strategy of treatment to construct human-
murine chimeric antibody to decrease organism immunogenicity initiated by HAMA.
Such kind of chimeric antibody is made by incorporating non-human antibody variable
region into human antibody constant region while retaining amino acid sequences of
original heavy chain, light chain variable regions of non-human antibody (see, Daddona,
P.E et al. PCT publication WO92/16553, Le, J. et al., U.S. Patent NO. 5,919,452, Kang,
Heui H et al. PCT publication WO2005/047329, Jin BOquan et al. Chinese patent
publication CN1544466A), Jin Yihui et al. Chinese patent publication CN101177453
provides a chimeric antibody which can bind to human tumor necrosis factor.
Compared to non-human antibody, the immunogenicity of the chimeric antibody
decreases, however, it may cause HAMA response in varied degrees since the murine
derived portion in the chimeric antibody is still high, specifically including skin mucosa
reaction, allergic reaction, arrhythmia and stenocardia, renal insufficiency, even coma
when severe. Therefore, the clinical applications of this kind of chimeric antibodies are
greatly limited.
Clinical trials demonstrate that this kind of chimeric antibodies as
heterogeneous protein may cause immunological rejecting response of the
heterogeneous protein by organism immune system (i.e., Human anti mouse antibody,
HAMA response) when administrated to human. The response leads to rapid clearance
of the murine antibody in human bodies, and short half life. Repeated administration
may even result in severe anaphylactic shock. Moreover, the “foreign” antibody may be
attacked by immune antibody, so that they may be neutralized before presenting
pharmaceutical effects.
The inventors develop a new technique to prepare humanized antibody
by utilizing genetic technology for the purpose of reducing the murine derived portion to
minimum in the chimeric antibody on the basis of aforesaid patents. The technique
comprises separately incorporating complementary determining regions (CDRs) of
murine antibody heavy chain variable region and light chain variable region into human
antibody framework region (FR). The obtained humanized antibody is similar to human
sequence in structure as possible, meantime, it also can maintain CDR conformation
similar to parent non-human antibody. Compared to parent non-human antibody and
chimeric antibody, the portion of parent non-human amino acid sequence in the
engineered humanized antibody decreases, one hand, the ability of antibody
recognizing antigen is remained; the other hand, the immunogenicity of murine antibody
has been greatly decreased. Therefore, safety of the antibody in clinical applications
has been improved.
Consequently, the present invention provides a humanized antibody,
which is safer, has longer half life more significant effects in human body, compared to
murine chimeric antibody in prior arts.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides at least one humanized
anti tumor necrosis factor monoclonal antibody, or specified complementary determining
regions, heavy chain or light chain variable region, heavy chain or light chain constant
region, framework region, or any other arbitrary parts, thereof. The antibody of the
present invention may be derived from any mammals, for example, but not limited to
human, mouse, rat, rodent, primate or any combination thereof.
The antibody of the present invention specifically binds to at least one
epitope of TNF proteins, subunit, fragment, part thereof, or any combination thereof.
The said at least one epitope may comprise at least on antibody binding area. The said
at least one epitope may optionally comprise at least one complementary determining
area (CDR) (for example, heavy chain variable region or light chain variable region)
and/or at least constant or variable framework region (FR) or any arbitrary part. The
amino acid sequence of the said at least antibody may further optionally comprise
insertion, deletion or conservatively substitution of at least one amino acid residue.
In another aspect, the present invention provides at least one nucleic
acid molecules, the nucleic acid molecule comprises polynucleotide encoding at least
one humanized anti tumor necrosis factor antibody of the present invention, or is
complementary to or hybridized to the polynucleotide encoding at least one humanized
anti tumor necrosis factor antibody of the present invention, wherein the antibody
comprises at least one specific sequence, domain, part or variant thereof.
In another aspect, the present invention provides a recombinant vector
comprising the nucleic acid molecule encoding the humanized anti tumor necrosis factor
antibody, a host cell comprising the nucleic acid and/or recombinant vector, and the
preparation method and/or use of the nucleic acid, vector and/or host cell.
At least one antibody of the present invention has at least one activity,
for example, but not limited to neutralizing the toxicity of rhTNF! to L929 target cell,
Suppressing and/or competing the binding of TNF with receptor and/or other
monoclonal antibody for example, but not limited to Humira.
In another aspect, the present invention provide the use of antibody
and/or composition of the present invention in inhibiting hTNF! activities, wherein the
hTNF! related disease is selected for the group consisting of pyaemia, autoimmune
diseases, malignant tumor, lung function disorder, transplant rejection, bacterial
meningitis, cerebral malaria, AIDS and AIDS related complex (ARC), secondary
cytomegalovirus infection after transplantation.
In another aspect, the present invention provides the use of in
preparation of a medicament in diagnostic analysis of hTNF!, wherein the humanized
anti tumor necrosis factor antibody also may be labeled by detecting molecule and/or
may not be labeled, and the label comprises radioactive isotope; fluorescence label;
various kinds of enzyme substrate mark.
In another aspect, the present provides the use of antibody and/or
composition of the present invention in analysis method, wherein the analysis method
includes competitive binding analysis, direct or indirect sandwich analysis, or
immunoprecipitation analysis.
In another aspect, the present invention provide at least a composition,
which comprises the humanized anti tumor necrosis antibody and/or its encoding
nucleic acid, one or more auxiliaries selected from but not limited to pharmaceutically
accepted vector, excipients, diluent, and additive. The composition may further
optionally comprise at least one other antibody, nucleic acid, auxiliaries or any
combination.
In another aspect, the present invention provides a kit comprising
predetermined amount of reagents and instruction, the reagents comprise the antibody,
nucleic acid and/or composition of the present invention. The kit further comprises
other additives for example, but not limited to stabilizer, buffers.
BRIEF DESCRIPTION OF FIGURES
Persons skilled in the art may understand that the following figures are
for the purpose of illustrating the present invention, which do not limit the scope of the
present invention in any manners.
Figure 1 shows the curve of the antibody neutralizing the effect of TNF!
killing U937.
Figure 2 shows the score of degree of rat joint swelling induced by type II
collagen.
Figure 3A shows the score of Tg197 mouse arthritis, figure 3B shows the
histology evaluation of treating group of Tg197 mouse, figure 3C shows the effect of
treating group on arthritis score (AS) and histology score (HS) of Tg197 mouse.
DETAILED DESCRIPTION OF THE INVENTION
Anti tumor necrosis factor antibody
The antibody of the present invention comprises antibody amino acid
sequence encoded by any suitable polynucleotide, or any separated or prepared
antibody. The humanized antibody or antigen binding fragment preferably binds to
human tumor necrosis factor, and as result, partially, substantially or completely
neutralizes at least one activity of human tumor necrosis factor, consequently inhibits
the signal transduction process and physiological process mediated by the binding of
TNF with TNF receptor.
The humanized antibody of the present invention may be any type (IgG,
IgA, IgM, IgE, IgD, et al.) or isotype, and may comprise # or $ light chains, and!, µ, %, &
or ' heavy chains.
At least one antibody of the present invention binds to at least one
epitope of TNF protein, subunit, fragment, part thereof, or any combination thereof.
At least one anti tumor necrosis factor monoclonal antibody comprises
following amino acid sequence, wherein the amino acid sequence of heavy variable
region of the antibody is shown as SEQ ID NO: 1, and the amino acid sequence of light
variable region of the antibody is shown as SEQ ID NO: 2.
SEQ ID NO: 1:
QVQLVQSGPELKKPGASVKISCKASGYTFTHYGMHWVKQTPGRGLK
WVGWINTYTGEPTYDADFQGRFTFSLETSVSTAFLQINSLKDEDLATYFCARYDFDGF
DYWGQGTTLTVSS
SEQ ID NO: 2:
ENVLTQSPPILSASPGERVTMTCRASSSITFNYLHWYQQKSGDSPKVW
IYSTSNLVSGVPSRFSGSGSGTSYSLTISSLEAEDAATYYCQQYSDYPYTFGGGTKLEI
wherein complementary determining region CDR-H1 of the heavy chain
variable region has a sequence of SEQ ID NO: 3; CDR-H2 has a sequence of SEQ ID
NO: 4; CDR-H3 has a sequence of SEQ ID NO: 5; wherein, within framework region
FR-H1, A can be substituted by E at amino acid residue position 16, S can be
substituted by T at position 17, I can be substituted by V at position 20 (e.g., as shown
in SEQ ID NO: 11); within FR-H2, K can be substituted by R at position 3, G can be
substituted by S at position 9 (e.g., as shown in SEQ ID NO: 12); within FR-H3, T can
be substituted by V at position 3, E can be substituted by D at position 7, V can be
substituted by T at position 10, F can be substituted by Y at position 14, S can be
substituted by T at position 19, T can be substituted by V at position 27 (e.g., as shown
in SEQ ID NO: 13); and complementary determining region CDR-L1 has a sequence of
SEQ ID NO: 6; CDR-L2 has a sequence of SEQ ID NO: 7; CDR-L3 has a sequence of
SEQ ID NO: 8; wherein, within framework region FR-L1, L can be substituted by M at
position 11, R can be substituted by E at position 18, M can be substituted by I at
position 21 (e.g., as shown in SEQ ID NO: 14); within FR-L2, W can be by L at position
13 (e.g., as shown in SEQ ID NO: 15); within FR-L3, S can be substituted by A at
position 4, L can be substituted by V at position 22, A can be substituted by F at position
27 (e.g., as shown in SEQ ID NO: 16).
SEQ ID NO.3:
HYGMH
SEQ ID NO: 4:
WINTYTGEPTYDADFQG
SEQ ID NO: 5:
YDFDGFDY
SEQ ID NO: 6:
RASSSITFNYLH
SEQ ID NO: 7:
STSNLVS
SEQ ID NO: 8:
QQYSDYPYT
SEQ ID NO: 9:
QVQLVQSGPELKKPG(E/A)(T/S)VK(I/V)SCKASGYTFTHYGMHWV(K/R)
QTPGR(S/G)LKWVGWINTYTGEPTYDADFQGRF(T/V)FSL(E/D)TS(T/V)STA(F/Y)LQIN
(T/S)LKDEDLA(T/V)YFCARYDFDGFDYWGQGTTLTVSS
SEQ ID NO: 10:
ENVLTQSPPI(M/L)SASPGE(E/R)VT(M/I)TCRASSSITFNYLHWYQQKS
GDSPKV(W/L)IYSTSNLVSGVP(A/S)RFSGSGSGTSYSLTISS(V/L)EAED(A/F)ATYYCQ
QYSDYPYTFGGGTKLEIK
SEQ ID NO: 11:
QVQLVQSGPELKKPG(E/A)(T/S)VK(I/V)SCKASGYTFT
SEQ ID NO: 12:
WV(K/R)QTPGR(S/G)LKWVG
SEQ ID NO: 13#
RF(T/V)FSL(E/D)TS(T/V)STA(F/Y)LQIN(T/S)LKDEDLA(T/V)YFCAR
SEQ ID NO: 14:
ENVLTQSPPI(M/L)SASPGE(E/R)VT(M/I)TC
SEQ ID NO: 15:
WYQQKSGDSPKV(W/L)IY
SEQ ID NO: 16:
GVP(S/A)RFSGSGSGTSYSLTISS(V/L)EAED(A/F)ATYYC
In an embodiment of the present invention, the heavy chain constant
region sequence of the humanized anti tumor necrosis factor antibody is the heavy
chain constant region of human IgG1.
In an embodiment of the present invention, the light chain constant
region sequence of the humanized anti tumor necrosis factor antibody is the light chain
constant region of human antibody.
In a preferred embodiment of the present invention, the amino acid
sequence of humanized anti tumor necrosis factor monoclonal antibody may be
modified by inserting, deleting or conservatively substituting one or more amino acid
residues, preferably 1-5 amino acid residues.
The monoclonal antibody modified or mutated by one or more insertion,
deletion or conservatively substitution in any combinational forms may have differences
in the amino acid sequences. In the preferred variant, the modification is obtained by
amino acid conservative substitution from the aforesaid monoclonal antibody of the
present invention. The conservative substation means a specific amino acid is
substituted by another amino acid with similar properties. The amino acids below listed
in non-limited manners are considered as conservatively exchangeable (with similar
properties): a) alanine, serine and threonine; b) glutamic acid and aspartic acid; c)
asparagine and glutamine; d) arginine and lysine; e) isoleucine, leucylacid, methionine
and valine; and f) phenylalanine, tyrosine and tryptophan.
The functionally equivalent monoclonal antibody of the present invention
is a variant, wherein one or more amino acid residues, preferably 1-5 amino acid
residues are conservatively substituted. The conservative substitution includes: any
one of aromatic amino acids Ala, Val, Leu and Ile can be substituted by another;
hydroxyl residues Ser and Thr are exchangeable; acid residues Asp and Glu are
exchangeable; amide residues Asn and Glun are exchangeable; basic residues Lys and
Arg are exchangeable; aromatic residues Phe and Tyr are exchangeable.
Furthermore, present invention discloses the amino acid sequences
which have at least 50% identity with the amino acid sequences of the present
invention, or fragment thereof, and amino acid sequences with equivalent functions. In
one embodiment, the amino acid sequences have at least 75% identity with the amino
acid sequence SEQ ID NO:1 or 2 according to present invention, more preferably at
least 85% identity, even more preferably at least 90% identity, even more preferably at
least 95% identity, even more preferably at least 97% identity, and most preferably at
least 99% identity.
Several kinds of antibodies are encompassed by present invention. For
example, anti hTNF! antibody may be full-length antibody (for example, comprising
complete human Fc region); or antibody fragment (for example, Fv, scFv, Fab, Fab’ and
(Fab’)2). Moreover, the antibody can be labeled with detectable labels, fixed on solid
phase carrier, and/or coupled with heterologous compounds (such as cytotoxin
materials).
Fab is produced by treating IgG antibody molecule by protease/papain. It
is an antibody fragment with molecular weight of about 50,000 and have antigen binding
activity, wherein, in the fragment obtained by papain treatment (cleaving H chain at
amino acid residue 224 ), about half of H chain from the N-terminal and the whole L
chain are bound together by disulfide bonds. The Fab of the present invention also may
be produced by inserting DNA encoding Fab of the antibody into prokaryotes
expression vectors and/or eukaryotes expression vectors.
Fab’ is an antibody fragment with antibody binding activity, produced by
cleaving disulfide bonds in (Fab’)2 hinge region, with molecular weight of about 50,000.
The Fab’ of the present invention also may be produced by inserting DNA encoding
Fab’ of the antibody into prokaryotes expression vectors and/or eukaryotes expression
vectors, and subsequently introducing the vectors into prokaryote and/or eukaryote to
express Fab’.
(Fab’)2 is an antibody fragment with antigen binding activity, with
molecular weight of about 50,000, wherein, in in the fragment obtained by
protease/pepsin treatment (cleaving H chain at amino acid residue 234) of IgG antibody,
the antibody fragment of Fab by bound together with disulfide bonds in hinge region is
slightly larger. The (Fab’)2 of the present invention may be produced by treating
antibody with pepsin. Besides, the (Fab’)2 of the present invention also may be
produced by linking Fab’ with thioether bonds or disulfide bonds.
ScFv is an antibody fragment with antigen binding activity, consisting of a
chain V and a chain V which are connected by appropriate peptide joints. The scFv of
the present invention may be produced by obtaining cDNAs encoding V and V of the
antibody, constituting DNA encoding scFv, inserting the DNA encoding scFv into
prokaryotes expression vectors and/or eukaryotes expression vectors, and
subsequently introducing the vectors into prokaryote and/or eukaryote to express scFv.
Nucleic Acid
The nucleic acids of the present invention are nucleotide sequences
encoding at least one of SEQ ID NO: 1-16, specific fragments, variants thereof, or at
least 70-100% continuous amino acid sequences of consensus sequences. The nucleic
acid molecules encoding at least one anti TNF antibody may be obtained by the
methods described in present invention or known in the art.
Nucleic acid molecules of the present invention can be in the form of
RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including,
but not limited to, cDNA and genomic DNA obtained by cloning or produced
synthetically, or any combinations thereof. The DNA can be triple-stranded, double-
stranded or single-stranded, or any combination thereof. Any portion of at least one
strand of the DNA or RNA can be the coding strand, also known as the sense strand, or
it can be the non-coding strand, also referred to as the anti-sense strand.
As indicated herein, nucleic acid molecules of the present invention
which comprise a nucleic acid encoding an anti-TNF antibody can include, but are not
limited to, those encoding the amino acid sequence of an antibody fragment, by itself;
the coding sequence for the entire antibody or a portion thereof; the coding sequence
for an antibody, fragment or portion, as well as additional sequences, such as the
coding sequence of at least one signal leader or fusion peptide, with or without the
aforementioned additional coding sequences, such as at least one intron, together with
additional, non-coding sequences, including but not limited to, non-coding 5' and 3'
sequences, such as the transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and polyadenylation signals (for
example - ribosome binding'and stability of mRNA); an additional coding sequence that
codes for additional amino acids, such as those that provide additional functionalities.
Thus, the sequence encoding an antibody can be fused to a marker sequence, such as
a sequence encoding a peptide that facilitates purification of the fused antibody
comprising an antibody fragment or portion.
The nucleic acids of the present invention can be made using (a)
recombinant methods, (b) synthetic techniques, (c) purification techniques, or
combinations thereof, as well-known in the art.
The nucleic acid compositions of the present invention, such as RNA,
cDNA, genomic DNA, or any combination thereof, can be obtained from biological
sources using any number of cloning methodologies known to persons skilled in the art.
In some embodiments, oligonucleotide probes that selectively hybridize, under stringent
conditions, to the polynucleotides of the present invention are used to identify the
desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and
construction of cDNA and genomic libraries, is well known to persons skilled in the art.
A cDNA or genomic library can be screened using a probe based upon
the sequence of a polynucleotide of the present invention, such as those disclosed
herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to
isolate homologous genes in the same or different organisms. Persons skilled in the art
will appreciate that various degrees of stringency of hybridization can be employed in
the assay; and either the hybridization or the wash medium can be stringent. As the
conditions for hybridization become more stringent, there must be a greater degree of
complementarity between the probe and the target for duplex formation to occur. The
degree of stringency can be controlled by one or more of temperature, ionic strength,
pH and the presence of a partially denaturing solvent such as formamide. For example,
the stringency of hybridization is conveniently varied by changing the polarity of the
reactant solution through, for example, manipulation of the concentration of formamide
within the range of 0% to 50%. The degree of complementarity (sequence identity)
required for detectable binding will vary in accordance with the stringency of the
hybridization medium and/or wash medium. The degree of complementarity will
optimally be 100%, or 70-100%, or any range or value therein. However, it should be
understood that minor sequence variations in the probes and primers can be
compensated for by reducing the stringency of the hybridization and/or wash medium.
Methods of amplification of RNA or DNA are well known in the art and
can be used according to the present invention without undue experimentation, based
on the teaching and guidance presented herein.
The isolated nucleic acids of the present invention can also be prepared
by direct chemical synthesis by known methods. Chemical synthesis generally
produces a single-stranded oligonucleotide, which can be converted into double-
stranded DNA by hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template.
Construction of Humanized Monoclonal Antibody Expression
Vector
5’ fragment is obtained by using a plasmid (pHu-V ) comprising
humanized antibody heavy chain variable region V gene fragment as template, 5'
primer FVHX (5’-CGCGCAAG-CTTCCTCGAG-3’ SEQ ID NO: 17) and 3' primer RVCG
(5’-CGATGGGCCCTTGGTGGA-3’ SEQ ID NO: 18), which comprises the gene for
humanized antibody heavy chain variable region (V ) and 7 amino acid at 5'-teminal of
human IgG heavy chain constant region (C ). Meanwhile, A gene comprising human
1 %1
IgG heavy chain constant region (C ) encoding sequence is obtained from RNA
1 %1
prepared from human leucocyte by using 5' primer HuCGF (5’-
ACCAAGGGCCCATCGGTCTTC-3’; SEQ ID NO: 19) and 3' primer HUCGE (5’-
CGGAATTCTCATTTACCCGGAGACAGGGA 3’, SEQ ID NO: 20) through reverse
transcription and PCR. Finally, the fragment of humanized antibody heavy chain
variable region and human C gene are linked by PCR using 5' primer (FVHX, SEQ ID
NO: 17) and 3' primer (HUCGE, SEQ ID NO: 20) to obtain a gene fragment of length
about 1400 bp comprising heavy chain encoding sequence. The gene fragment is
treated with endonuclease Hind III and EcoR1, and then inserted into vectors such as
PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103-119).
5’ fragment is obtained by using a plasmid (pHu-V ) comprising
humanized antibody light chain variable region V gene fragment as template, 5' primer
FVHX (SEQ ID NO: 17) and 3' primer VKCKO (5’ -AGA TGG TGC AGC CAC AGT TCG
CTT GAT CTC CAG CTT GGT GCC -3’ SEQ ID NO: 21), which comprises the gene for
humanized antibody light chain variable region (V ) and 7 amino acid at 5'-teminal of
human # light chain constant region (C#). Meanwhile, A gene comprising human # light
chain constant region (C#) encoding sequence is obtained from RNA prepared from
human leucocyte by using 5' primer HuCKF (5’ -GTG GCT GCA CCA TCT GTC TTC -3’
SEQ ID NO: 22) and 3' primer HUCKB (5’ -TGC GGA TCC CTA ACA CTC TCC CCT
GTT GAA -3’, SEQ ID NO: 23) through reverse transcription and PCR. Finally, the
fragment of humanized antibody light chain variable region and human C# gene are
linked by PCR using 5' primer (FVHX, SEQ ID NO: 17) and 3' primer (HUCKB, SEQ ID
NO: 23) to obtain a gene fragment of length about 700 bp comprising light chain
encoding sequence. The gene fragment is treated with endonuclease Hind III and Bam
H1, and then inserted into vectors such as PUC19 (ref: Yanisch-Perron, C., Vieira, J.
and Messing, J. (1985) Gene, 33, 103-119.).
The cDNA encoding the heavy chain or light chain or the cDNA encoding
their modified products which are obtained by aforesaid methods are inserted into
pcDNA3 (Invitrogen USA, Carlsbad, CA, U.S.A.) vector to construct Hu_anti-TNF!
humanized expression vector. The expression vector plasmid comprises
cytomegalovirus early gene promoter-enhancer required for high level expression in
mammal cells. Meanwhile, the vector plasmid also comprises optional maker gene, so
as to have amicillin resistance in bacteria, have G418 resistance in mammal cells.
Furthermore, the vector plasmid comprises DHFR gene. In suitable host cells, chimeric
antibody gene and DHFR gene can be co-amplified by Methotrexate (MTX, Sigma)
(see, for example, Axel, R., et al. U.S Patent No. 5,179,017; Kaufman,R. and Sharp,P.,
J.Mol. Biol. 159:601-621,1982).
Antibody Host Cell
The present invention also relates to produce at least one anti TNF
antibody by using recombinant vector gene engineered host cell, and recombinant
technique in the art.
Polynucleotides may be optionally linked to vector comprising optional
labels, for amplification in the host. generally, the plasmid vector is introduced into a
precipitate, such as calcium phosphate, or introduced into complex comprising charged
lipid.
Appropriate culture mediums and conditions for the above-described
host cells are known in the art. Suitable vectors will be readily apparent to persons
skilled in the art. Introduction of a vector construct into a host cell can be effected by
calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-
mediated transfection, electroporation, transduction, infection or other known methods.
The host cells for the humanized anti tumor necrosis factor monoclonal
antibody of the present invention are derived from Chinese hamster ovary cells, which
are obtained by transfection with plasmid comprising anti-TNF! gene code, and
followed by a series of stringent, specified screening, which including drug screening,
gene amplification, and single cell cloning to produce the fanal cell strain. The host cell
of the present invention, Chinese hamster ovary cell strain CHO HUAT 132 was
deposited at CCTCC at March 7, 2011, with Deposit NO. C201117.
The cell of the cell strain can be bred when suspended in serum free
medium; when cultivated in 2 L Fermenter, The level of secreted anti-TNF! in medium
will not lower than 1 g/L after the cultivation cycle of 16-20 days ends. The anti-TNF!
produced by the cell strains is humanized monoclonal antibody.
Activity of Binding to TNF!
The humanized anti tumor necrosis factor monoclonal antibody of the
present invention only has specific affinity with recombinant human tumor necrosis
factor (rhTNF!, target molelules), but does not crosslinked to any other protein
molecules. In competitive assay of affinity to their target molecules, the humanized anti
tumor necrosis factor monoclonal antibody of the present invention has been
determined to have a similar affinity as Humira.
The humanized anti tumor necrosis factor monoclonal antibody of the
present invention also has the activity to neutralize the toxicity of rhTNF! to L929 target
cell, and its EC50 is similar to that of Humira, which is between 20.4 ng/mL and 50
ng/mL.
Therapeutic Use
Anti hTNF! antibody can be used to treat and/or prevent hTNF! related
diseases, wherein the hTNF! related disease may be, for example, pyaemia,
autoimmune diseases, malignant tumor, lung function disorder, transplant rejection,
bacterial meningitis, cerebral malaria, AIDS and AIDS related complex (ARC),
secondary cytomegalovirus infection after transplantation. The use of the antibody and
antibody components of the present invention in treating hTNF! related diseases will be
further discussed as follows:
1) Sepsis
The role of tumor necrosis factor in sepsis pathology, and the biological
effects thereof includes hypotension, myocardial depression, vascular leak syndrome,
organ necrosis (see for example, U.S. patent 5,231,024). Therefore, the humanized
antibodies of the present invention and antibody components can be used for the
treatment of any sepsis with clinical background, including septic shock, endotoxic
shock, gram-negative sepsis and toxic shock syndrome.
2) Autoimmune diseases
It has been found that the tumor necrosis factor plays a role in the
pathophysiology of a variety of autoimmune diseases, for example, it has been found
that TNF! is involved in activating tissue inflammation and lead to joint destruction in
rheumatoid arthritis (see, for example, U.S. Patent 5,231,024; Moeller, A. et al. (1990)
Cytokine 2:162-169;). It also has been found that TNF! is involved in promoting islet
cell death in diabetes and mediates the cytotoxicity to oligodendrocyte and induces
inflammatory plaques.
Humanized antibodies and antibody components of the present invention
can be used to treat autoimmune diseases, especially those associated with
inflammation, including rheumatoid arthritis, rheumatoid myelitis, osteoarthritis and gout
arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune eye uveitis, and
nephrotic syndrome.
3) Malignant tumors
It has been found that tumor necrosis factor has been found in malignant
tumors is involved in inducing cachexia, stimulating tumor growth, enhancing metastatic
potential and mediating cytotoxicity. Therefore, the antibodies and the antibody
components of the present invention can be used to treat malignant tumors, inhibit
tumor growth or metastasis and/or reduce the malignant secondary cachexia. The
antibody or antibody component can be systemic or local administrated to the tumor
site.
4) Pulmonary function disorders
It has been known that the tumor necrosis factor is involved in
pathophysiology of adult respiratory distress syndrome (ARDS), including stimulating
white blood cell - endothelial cell activation, cytotoxicity-oriented to lung cells and
inducing vascular leak syndrome. Thus, the antibody and the antibody components of
the present invention can be used to treat lung function disorders, including adult
respiratory distress syndrome, chronic pneumonia, pulmonary sarcoidosis, pulmonary
fibrosis and silicosis.
5) Intestinal dysfunction
Human antibodies and antibody components of the present invention can
be used to treat intestinal disorders such as idiopathic inflammatory bowel disease,
which includes two syndrome, Crohn and ulcerative colitis.
6) Transplantation
It has been found that tumor necrosis factor may be the key mediators of
allograft rejection and transplant plants versus-host disease (GVHD) , and mediates the
side effects observed in the inhibition of renal transplant rejection by rat antibody OKT3
whhich is targeted to T-cell receptor CD3 complex (see for example, Suthanthiran, M.,
and Strom, TB (1994) New Engl J.Med.331 :365-375)
Therefore, the antibody and the antibody components of the present
invention can be used to suppress transplant rejection, including allograft and xenograft
rejection, and suppress GVHD.
7) Infectious diseases
The antibodies and the antibody components of the present invetion can
be used to treat infectious diseases, including bacterial meningitis, cerebral malaria,
AIDS and AIDS-related syndrome (ARC), and secondary cytomegalovirus infection after
transplantation. They can also be used to reduce infectious diseases related
symptoms, including fever and muscle pain caused by infection (eg influenza), and
infection secondary cachexia (such as AIDS or secondary ARC).
Analysis and Diagnostic Purposes
The antibody of the present invention can be used in any known analysis
method, such as competitive binding assays, direct or indirect sandwich analysis and
immunoprecipitation analysis. Zola, Monoclonal Antibodies: Technical Manual
"(Monoclone Antibodies; A Manual of Techniques), pp. 147-158 (CRC Press, Inc.,
1987).
Competitive binding analysis depends on the ability of marked standard
material competing with analyte in the measured sample to bind limit amount of
antibody. Amount of the standard material is inversely proportional to the amount of
antibody bound with hTNF! in the measured sample. In order to facilitate the
determination of the amount of the bound standard material, the antibodies usually are
insoluble before or after the competition, so that it will be convenient to separate the
bound standard material and the analyte from unbound standard material and analyte
separation.
Sandwich analysis involves the use of two antibodies, each bind to
different immunogenicity site or epitope on target proteins. In sandwich analysis, the
measured sample analyte is bound to the first antibody fixed on the solid phase carrier,
and then the second antibody binds to the analyte, thus forming insoluble three-
component complex. See U.S. Patent 4,376,110. The second antibody itself can
marked with detectable part (direct sandwich assay), or can be detected by using anti-
immunoglobulin antibodies labeled with detectable part (indirect sandwich assay). For
example, one of the sandwich analysis is ELISA, in which the detectable part is
enzyme.
Anti-hTNF! antibodies can also be used in the diagnostic analysis of
hTNF!, for example, to detect its expression in specific cells, tissues or serum. This
diagnostic method can be used for the diagnosis of causes for autoimmune diseases
The antibody usually can be labeled with detectable molecular. Many
Markers can be used, and generally they can be classified as follows:
(a) Radioisotopes, such as 111In, 99Tc, 14C, 131I, 125I, 3H, 32P or
35S. Antibodies can be labeled with radioactive isotopes in accordance with the
methods decribed in Current Protocols in Immunology, Volume 1 and 2, Coligen eds,
Wiley-Interscience, New York, New York, the Pubs (1991), in which the radioactivity
may be determined by scintillation counting method, and the diseased sites can be
located by using immune-flash photography.
(b) Fluorescent marker, such as rare earth chelating agent (europium
chelator), or luciferase and its derivatives, rhodamine and its derivatives, dansyl,
lissamine, phycoerythrin and Texas Red. Fluorescent marker may be coupled with
antibody using the methods described in for example, Current Protocols in Immunology
mentioned above. Fluorescence can be quantified by fluorometer.
(c) A variety of substrate markers are available, and U.S. Patent
4,275,149 disclosed some of them. The enzymes usually catalyze chemical changes of
a variety of chromogenic substrates which can be detected by many techniques. For
example, the enzymes catalyze the color changes of the substrates, which can be
measured by spectrophotometer, or the enzymes change the fluorescence or
chemiluminescence of the substrate. Previously, the technology of quantitative
determination of the fluorescence changes has been described. Chemiluminescent
substrates are electrically excited due to chemical reactions, and then illuminate. The
light emitted can be determined (such as the use of chemical photometer) or provides
energy to fluorescent receptors. The enzyme markers include such as luciferase (for
example, firefly luciferase and bacterial fluorescence luciferase; U.S. Patent 4,737,456),
luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase
such as horseradish peroxidase (HRPO), alkaline phosphatase, b-galactosidase,
glucoamylase, lysozyme, sugar oxidase (eg glucose oxidase enzyme, galactose
oxidase and glucosephosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, micro-peroxidase. O’Sullivan describes
the technology of conjugating enzymes to antibodies in Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay (Methods In Enzym.)
(by J. Langone, and H. the Van Vunakis ed.), Academic press, New York, ,73:147-166
(1981).
The compositions of enzyme - substrate include, for example:
(i) Horseradish peroxidase (HRP) and hydrogen peroxidase as substrate,
wherein, the hydrogen peroxidase oxidize the dye precursor (eg, o-phenylenediamine
(OPD) or hydrochloric acid to 3,3',5,5'- tetramethylbenzidine (TMB));
(ii) Alkaline phosphatase (AP) and p-nitrophenyl phosphate as
chromogenic substrate;
(iii) b-D-galactosidase anhydrase (b-D-Gal) and chromogenic substrate
(for example, nitrophenyl-b-D-galactosidase) or fluorogenic substrate and 4-methyl
umbelliferone-b-D-galactosidase.
Persons skilled in the art may know many other enzyme-substrate
combinations. Review of these combinations can be found in U.S. patents 4,275,149
and 4,318,980. Sometimes, markers and antibodies are indirectly coupled. Persons
skilled in the art also know all kinds of methods to obtain the said compositions. For
example, the antibodies may coupled with biotin, and any one of the above three
categories of markers can coupled with avidin, or vice versa. Biotin selectively binds to
avidin, and the markers can be coupled with the antibody indirectly. Or, to coupled the
marker with antibody indirectly, the antibody can be coupled with a small hapten (eg
digoxin), while one of the markers of different types can be couple with anti-hapten
antibody (eg anti-digoxin antibody). Therefore, the indirect coupling of markers with the
antibody is obtained.
In another embodiment of the present invention, it is not necessary to
mark the anti-hTNF! antibodies, and its existence can be detected by marked antibody
which binds to the hTNF! antibody.
Affinity Purification Kit
The antibodies of the present invention can be used as affinity
purification reagents. In this method, the antibody is fixed on solid-phase, for example,
Sephadex resin or filter paper by methods known in the art. The fixed antibody contact
with hTNF!-containing samples to be purified, and then the carrier is washed with
suitable solvents, and the solvent can substantially remove all the other materials
expect hTNF! bound with immobilized antibody.
Pharmaceutical composition and mode of administration
The antibody and antibody components of the present invention may be
added into pharmaceutical composition suitable for administration to subjects, wherein
the pharmaceutical compositions comprise the antibodies of the present invention and
pharmaceutically acceptable excipients, and the pharmaceutical excipients include any
physiological applicable solvents, dispersion media, antibacterial agents, antifungal
agents, isotonic agents, coating, absorption delay agent. The pharmaceutical
compositions of the present invention can take various forms, such as liquid semi-solid
and solid dosage forms.
The anti-hTNF antibody of the present invention in a pharmaceutically
acceptable dosage form can be administrated to human using known methods. The
method includes intravenous (for example, intravenous injection of concentrated drug
(bolus) or infusion within a period of time), intramuscular, intraperitoneal, cerebrospinal
cavity, subcutaneous, intra-arterial, synovial cavity, intrathecal injection, oral, local
injections, or inhalation.. The antibody can also be appropriately administrated via
intratumor, around tumor, inside injury sites, around injury side to obtain local and
systemic treatment. Intraperitoneal injection is expected to be particularly useful, for
example, for the treatment of ovarian cancer.
In order to prevent or treat diseases, the appropriate dose of the
antibody will depend on the type of the disease to be treated as defined above, disease
severity and duration of disease, antibody given for prevention or for treatment, previous
treatment, and patient history and antibody response, as well as the attending
physician's independent judgment. The antibody is suitable for one-off or series doses
to the patients.
According to the type and severity of the disease, no matter one dose or
several separated doses, or continuous infusion, 1 µg/kg to 50 mg/kg (eg 0.1-20 mg/kg)
of the antibody is the initial candidate dose to patients. The typical daily dose or weekly
dose is about 1 mg/kg -20 mg/kg or more, depending on the factors mentioned above.
As to repeated dose within a few days or more (depending on condition), the treatment
should be continued until the disease symptoms have been inhibited as desired.
However, the other regimen can also be used. The progress of treatment can easily
monitored using conventional techniques and analysis methods.
Product
1) Injection
Another embodiment of the present invention provides a product
containing the material used for the treatment of these diseases. The product includes
a container and a label. The suitable containers include such as ordinary bottles,
medicine bottles, syringes and test tubes. The container can be made of various
materials, such as glass or plastic. The container contains the effective composition for
the treatment of diseases, and with a sterile entrance (for example, the container can be
a plugged intravenous infusion bag or bottle, the stopper can be penetrated using a
hypodermic needle). The active ingredient in the composition is anti-hTNF! antibody.
The label on the container or associated with the container associated demonstrates the
specific conditions treated by the composition. The products can also have another
container, which contains a pharmaceutically acceptable buffer, such as phosphate
buffer, Ringer’s solution and glucose solution. According to business needs or the
needs of users, it can include other materials, such as other buffers, diluents, filters,
needles, syringes, and instructions.
2) Sustained Release Formulations
The humanized anti-tumor necrosis factor monoclonal antibodies of the
present invention can be used for the preparation of sustained-release formulations.
The suitable sustained-release formulations include such as semi-permeable matrix
body comprising solid hydrophobic polymers of the antibodies, and the matrix body is a
tangible object, such as film or microcapsules. The suitable sustained-release matrix
body include such as polyesters, hydrogels (for example, poly(methacrylic acid 2-
hydroxy ethyl) or polyvinyl alcohol), polylactide (U.S. Patent 3,773,919), L-glutamic acid
and L-glutamic acid ethyl ester copolymer, non-degradable ethylene vinyl acetate,
degradable lactic acid-ethanol acid copolymer such as Lupron DepotTM (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate),
and poly-D-(-)hydroxybutyric acid. Molecular polymers such as ethylene vinyl
acetate and lactic acid-glycolic acid enable the release of molecules lasting for 100 days
or more, while some hydrogels release proteins in a short period of time. When
encapsulated antibodies are retained in the body for a long time, they may become
denaturation or cohesion for contacting with water at 37°C, resulting in biological activity
decrease and may lead to changes in immunogenicity. Reasonably stable strategy can
be designed according to the mechanism. For example, if the condensation mechanism
is the formation of intermolecular S-S bond by sulfur-disulfide interchange reaction, the
stabilization can be achieved by modifying thiol residues, lyophilizing acidic solutions,
controlling moisture content, using suitable additives and designing special polymer
matrix composition.
For convenience, the antibody of the present invention can be provided
in the form of kits, that is to say, predetermined amounts of reagents and the instruction
for diagnostic analysis are packed together. If the antibody is enzymatic marked, the kit
will contain the substrate and cofactors (such as the substrate precursors providing
detectable chromophores and fluorophores) required for the enzyme. Besides, it may
also include other additives such as stabilizers, buffers (such as blocking buffer or lysis
buffer). In order that the provided reagent concentration can achieve the highest
sensitivity of the analysis, the relative amounts of various reagents significantly change.
Specifically, the reagent can be dry powder, usually in lyophilized powder form, and
excipients may be included. They will form a reagent solution with appropriate
concentration upon dissolution.
The present invention is further set forth, in connection with following
specific embodiments. It should be understood that these embodiments are for
illustrating the present invention only, but not be used to limit the scope of the present
invention. In the following embodiment, the experimental methods without indicating the
specific conditions are usually performed in conventional conditions, for example, the
conditions described in Sambrook et al, Molecular Cloning: Laboratory Manual (New
York; the Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended
by the manufacturer.
Examples
Example 1: production of anti-hTNF! mouse monoclonal antibody
1) Immune
Some 7-11 weeks old female Balb/c mice were IP or ID immunized with
the recombinant hTNF (rhTNF!, purchased from Pepro Tech Inc.), and the recombinant
human TNF! was emulsified using an equal volume of TITERMAX or Freund's
complete adjuvant with final volume of 100-400 µL. At 1-7, 5-12, 10-18, 17-25 and/or
21-34 days thereafter, each mouse were IP (1-400 µg) and SC (1400 µg(2) with TNF
which was emusified with equal volume of TITERMAX or incomplete Freund adjuvant..
At 12-25 and 25-40 days thereafter, blood samples were collected from the mouse by
hip puncture under non-anticoagulant conditions. And the blood coagulates in RT for 1
hour to collect the serum. Titers were determined using TNF! ELA according to known
methods. When repeated injections do not lead to increase in titers, fusion was
conducted. At this point, 1-400 µg of of TNF! diluted in 100 µL of saline can be injected
to the mice for last booster shots. Three days later, the mice were sacrificed by
breaking cervical vertebrae, and spleens were removed under sterile conditions, and
immersed into 10 mL of cold hydrochloric acid buffered saline (PBS) containing 1000
U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (PSA).
Spleen cells were harvested by sterile perfusing the spleen with PSA-PBS, and counted
by trypan blue dye exclusion method, and resuspended in RPMI 1640 medium
containing 25 mM Hepes.
2) Serum Test
A plate was coated with 2 µg/mL of TNF! in PBS overnight. After
washed with 0.15 M saline containing 0.02% (v / v) Tween 20, each well was blocked
for 1 hour using 1% (w / v) BSA in PBS, 200 µL/well. at RT. The plate was immediately
used or frozen at -20 for future use. 50 µL/well of mouse serum dilutions were
incubated at RT for 1 hour on the TNF! coated plate. The plate was washed, and then
monitored at RT for 1 hour with of 1:30000 specific diluted 50 µL/well of HRP labeled
IgG Fc probe in 1% BSA-PBS, washed again, added 100 µL/well of citrate-phosphate
substrate solution (0.1 M citric acid and 0.2 M sodium sulfate, 0.01% H O and 1 mg/mL
OPD) at RT for 15 minutes, then added 25 µL/well termination solution (4N sulfuric
acid). And OD values were read at 490 nm with automatic plate spectrophotometric
photometer.
3) Cell Fusion
The live spleen cells identified with a high level of anti-hTNF! antibody in
serum in the serum test were fused with mouse myeloma cells at the ratio of 1:1 to 10:1.
As a non-restrictive example, the spleen cells and myeloma cells were co-precipitation,
and resuspended for 30 seconds or more at 37 in 1 mL of 50% (w/v) of PEG/PBS
solution (PEG molecular weight 1450, sigma). In order to terminate the fusion, 10.5 mL
of RPMI 1640 medium containing 25 mM Hepes (37 °C) for 1 minutes or more. The
fused cells were centrifuged at 500-1500 rpm for 5 minutes, and then were
resuspended in HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal
the Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 µg/mL of
celebration Trappe factors, 2.5% Origen culture supplement (Fisher), 10% of 653
adjusted RPMI 1640/Hepes medium, 50 µM 2-mercaptoethanol, 100 µM hypoxanthine
and 16 µM thymidine), and were seeded on 15 pieces of 96-well flat-bottom plates with
200 µL/well. And then the plates were placed in a 37 °C incubator with 5% CO and
95% humility for 7-10 days.
Example 2: Qualitative Test of Anti-hTNF! Murine Antibodies
Two kinds of qualitative tests are available for anti-hTNF! antibody. In
one test, the antibody and Humira are competitive in binding hTNF!, and the
competition is determined. In the other test, the ability of the antibody to neutralize
hTNF! is determined in the assay of determining L929 cell toxicity. Following, these
two methods and experimental results are described, respectively.
1) Competitive binding assay against humira
The anti-hTNF! human antibody humira marked with horseradish
peroxidase (HRP) was used as a reagent. Elisa plates were coated with rhTNF (50 µl
of 0.05 g/mL), and left overnight at room temperature. The coating solution was
discarded, and each well was blocked with 1% skim milk in phosphate buffered saline
(PBS) for about 0.5 hours, and washed with PBS containing 0.05% Tween 20. And
then a mixture of 50 µl of growth medium and 50 µl of HRP labeled humira was added
to each well. Unlabeled humira and antibody-free medium were used as positive and
negative controls. The method can screen out mouse monoclonal antibody that can
highly inhibit the binding of HRP-labeled humira to rhTNF!. The wells in which the
binding of HRP-labeled humira and rhTNF! was inhibited Kong were amplified and
subcloned, followed by analysis of several mouse monoclonal antibodies which show
the inhibition effects, and finally hybridoma cells were screened out. The hybridoma
cells were intermediate cultured, and the supernatant was taken for purification, and the
mouse monoclonal antibody TM212 and TM23. The purified mouse monoclonal
antibodies TM212 and TM23 were tested in competition binding assays. Even at
the concentration up to 1 µg/mL, the mouse antibody TM23 only competed out about
50% of the binding of humira to hTNF!. Another mouse antibodies TM212 showed
as good competitiveness as unlabeled humira, since at the concentration of about 0.05
µg/mL (equivalent to 3 ( 10 M), it can compete out about 50% of the binding of
humira to hTNF!.
2) Qualitative test of anti-hTNF! murine antibodies: determination of in
vitro activity to neutralize hTNF!
The biological activities of both anti-hTNF! mouse antibody and chimeric
antibody to neutralize hTNF! biological activity can be measured using the L929
cytotoxicity assay as described below. Each well of a 96-well culture plate was injected
7.5(10 of L929 cells (ATCC) (10 /mL, 75 µl), and was placed in a 37 °C, 5% CO
incubator for 24 hours. The growth medium for L929 cell was RPMI-1640 containing
% fetal bovine serum (GIBCO). Using another 96-well culture plate, the solution
containing anti-hTNF! antibody was 1/2 serially diluted with RPMI growth medium, and
rhTNF! was added to a final concentration of rhTNF! of 5 ng/mL in each sample well.
After the plate containing mixtures was placed in a 37 °C, 5% CO incubator for 2 hours,
the mixture of antibody and rhTNF! was added to the L929 cells well. In each line, the
concentrations of antibody in each well were in the order of 0.001 to 2 µg/mL. The
culture plate was placed in a 37 °C, 5% CO incubator. When surviving cells were
measured 3 days later, 20 µl of PBS containing 2.5 mg/mL 3-(4,4-dimethyl-thiazole
yl)-2,5-diphenyl-tetrazole bromide salt (MTT; purchased from Sigma Biochemicals) was
added, and incubated at 37 °C for 4 hours, and then 100 µl of 0.01 N HCl containing
% sodium dodecyl sulfate (SDS) was added overnight. Subsequently, 540/690 nm
optical density was tested for each well. The curve for the degree of optical density and
antibody concentration was plotted. IC were obtained by analyzing the binding curve,
wherein, IC50 means a concentration at which 50% of the rhTNF! toxicity to L929 cells
can be neutralized. Therefore, IC50 values can be used to compare the abilities of
antibodies to inhibit hTNF! cell toxicity. The IC50 values of several anti-hTNF! murine
antibody (TM212, TM220, TM22) and of humira were all in the range of 0.01
to 0.04 µg mL, which means they all have similar capabilities to neutralize L929
cytotoxic caused by rhTNF!. The anti-hTNF! mouse antibody TM212 was selected
for further preparation of the chimeric antibody.
Example 3: Cloning of TM212 murine antibody heavy chain and
light chain
1) Cloning of TM212 murine antibody heavy chain variable region
To design the humanization of murine antibody, DNA fragments
containing the murine antibody TM212 heavy chain and light chain variable region
encoding sequences must be obtained at the beginning. RNA was isolated from TM2-
11-12 mouse hybridoma cells with RNA purification kit (Invitrogen Corp.), in order to
prepare cDNA (GeneRacer kit, Invitrogen, Corp.). By polymerase chain reaction (PCR),
using 5' primer (5'-CGACTGGAGCACGAGGACACTGA-3', SEQ ID NO: 24) and 3'
primer (5'-TCCAGGGGCCAGTGGATAGAGAGA-3', SEQ ID NO: 25), heavy chain
variable region DNA fragment was isolated from cDNA. 3' primer is homologous
antisense to the mouse IgG1 heavy chain constant region. Those obtained DNA
fragments were cloned into TOPO TA vector (Invitrogen) and sequenced. The heavy
chain variable region amino acid sequence is SEQ ID NO: 34:
QVQLVQSGPELKKPGETVKISCKASGYTFTHYGMHWVKQTPGRSLKWVGWINTYTG
EPTYDADFQGRFTFSLETSTSTAFLQINTLKDEDLATYFCARYDFDGFDYWGQGTTLT
VSS. The amino acid sequences in complementary determining regions are CDR-H1
(SEQ ID NO: 3), CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5). The definition
for complementary determining regions can be found in Kabat E. et al. Sequences of
Proteins of Immunological Interest, 5th Edition U.S. Department of Health and Human
Services, NIH Publication No.91-3242.
2) Cloning of TM212 murine antibody light chain variable region
Similar to the PCR method of cloning the heavy chain variable region,
using SEQ ID NO: 29 as 5' primer and another 3' primer which is homologous antisense
to the mouse immunoglobulin # light chain constant region (5'-
CACTGGATGGTGGGAAGATGGATA-3', SEQ ID NO: 26), light chain variable region
DNA fragment was isolated from the cDNA. The obtained DNA fragments were cloned
into TOPO TA vector and sequenced. Two types of clones were found. About 3/4 of
the clones show that part of the nucleotide sequence cannot be translated into readable
amino acid sequence (sequences not shown). Such clones were mutated light chain
messenger RNA, which cannot encode functional antibody light chain protein. The
remaining about 1/4 of clones shows the nucleotide sequence which can be completely
translated into readable amino acid sequence. Such clones are derived from the
functional light chain messenger RNA. Those obtained DNA fragments were cloned
into the TOPO TA vector (Invitrogen) and sequenced. The light chain variable region is
amino acid sequence SEQ ID NO: 35:
ENVLTQSPPIMSASPGEEVTMTCRASSSITFNYLHWYQQKSGDSPKVWIYSTSNLVSG
VPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSDYPYTFGGGTKLEIK. The amino
acid sequences in complementary determining region are CDR-L1 (SEQ ID NO: 6),
CDR-L2 (SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8). The amino acid sequence was
used for designing humanized light chain.
Example 4: humanization design of heavy chain and light chain
variable region
In order to retain the antigen binding activity, all the amino acid
sequences within light chain and heavy chain hypervariable regions are still the same
as those of TM212 murine antibody during the humanizing process. The
humanization design includes changing amino acid residues within framework regions
in accordance with the sequences of human antibody, designing heavy chain variable
region and light chain variable region of humanized antibody with a variety of
modification, and directed mutating oligonucleotides sites of the antibody heavy chain
and light chain variable region sequence by computer simulation technology, so as to
increase antibody binding affinity or reduce antibody immunogenicity.
For the humanized antibody heavy chain variable region (SEQ ID NO: 1),
within framework region FR-H1, A can be substituted by E at amino acid residue
position 16, S can be substituted by T at position 17, I can be substituted by V at
position 20; within FR-H2, K can be substituted by R at position 3, G can be substituted
by S at position 9; within FR-H3, T can be substituted by V at position 3, E can be
substituted by D at position 7, V can be substituted by T at position 10, F can be
substituted by Y at position 14, S can be substituted by T at position 19, T can be
substituted by V at position 27.
For humanized antibody light chain variable region (SEQ ID NO: 2),
within framework region FR-L1, L can be substituted by M at position 11, R can be
substituted by E at position 18, M can be substituted by I at position 21; within FR-L2, W
can be by L at position 13; within FR-L3, S can be substituted by A at position 4, L can
be substituted by V at position 22, A can be substituted by F at position 27.
By introducing at least one of the aforesaid amino acid modifications, a
variety of amino acid sequences of several humanized antibody heavy and light chain
variable regions are designed. Several amino acid sequences of humanized antibody
heavy chain variable region VH and VL are shown in Table 1:
Table 1: amino acid sequences of humanized antibody heavy chain variable region VH
and light chain variable region VL
No. Amino Acid Sequence SEQ ID NO:
E001V QVQLVQSGPELKKPGASVKISCKASGYTFTHYGM
HWVKQTPGRGLKWVGWINTYTGEPTYDADFQGR
FTFSLETSVSTAFLQINSLKDEDLATYFCARYDFDG
FDYWGQGTTLTVSS
E002V QVQLVQSGPELKKPGASVKISCKASGYTFTHYGM
HWVKQTPGRGLKWVGWINTYTGEPTYDADFQGR
FTFSLDTSVSTAFLQINSLKDEDLAVYFCARYDFD
GFDYWGQGTTLTVSS
E003V QVQLVQSGPELKKPGESVKISCKASGYTFTHYGM
HWVKQTPGRGLKWVGWINTYTGEPTYDADFQGR
FTFSLETSTSTAYLQINSLKDEDLATYFCARYDFDG
FDYWGQGTTLTVSS
E004V QVQLVQSGPELKKPGATVKVSCKASGYTFTHYGM
HWVKQTPGRSLKWVGWINTYTGEPTYDADFQGR
FTFSLETSVSTAFLQINTLKDEDLATYFCARYDFDG
FDYWGQGTTLTVSS
E005V QVQLVQSGPELKKPGASVKVSCKASGYTFTHYGM
HWVRQTPGRGLKWVGWINTYTGEPTYDADFQGR
FTFSLETSVSTAYLQINSLKDEDLATYFCARYDFDG
FDYWGQGTTLTVSS
F001V ENVLTQSPPILSASPGERVTMTCRASSSITFNYLH
WYQQKSGDSPKVWIYSTSNLVSGVPSRFSGSGS
GTSYSLTISSLEAEDAATYYCQQYSDYPYTFGGGT
KLEIK
F002V ENVLTQSPPILSASPGEEVTMTCRASSSITFNYLH
WYQQKSGDSPKVWIYSTSNLVSGVPARFSGSGS
GTSYSLTISSLEAEDFATYYCQQYSDYPYTFGGGT
KLEIK
F003V ENVLTQSPPIMSASPGERVTMTCRASSSITFNYLH
WYQQKSGDSPKVLIYSTSNLVSGVPSRFSGSGSG
TSYSLTISSVEAEDAATYYCQQYSDYPYTFGGGTK
LEIK
F004V ENVLTQSPPILSASPGERVTLTCRASSSITFNYLHW
YQQKSGDSPKVWIYSTSNLVSGVPARFSGSGSGT
SYSLTISSLEAEDAATYYCQQYSDYPYTFGGGTKL
Example 5: Construction of pHu_anti-H1L1-of TNF! humanized
antibody expression vector
1) Construction of gene for humanized antibody light chain
First, the gene fragment for humanized antibody light chain variable
region (F001VL) is prepared by synthetic method. And the preparation procedures
include obtaining the nucleotides sequences by reverse translation from the amino acid
sequence of the light chain variable region according to their genetic codons; adding a
Kozak sequence and light chain leader sequence at 5' terminal; preparing the gene
fragment for light chain variable region by synthetic method. The gene fragment was
cloned into appropriate vectors to obtain pHu-VL1 plasmid. Subsequently, 5’ fragment
is obtained by using a pHu-VL plasmid as template, 5' primer FVHX (SEQ ID NO: 17)
and 3' primer VKCKO (SEQ ID NO: 21), which comprises the gene for humanized
antibody light chain variable region (V ) and 7 amino acid at 5'-teminal of human # light
chain constant region (C ). Meanwhile, A gene comprising human # light chain constant
region (C ) encoding sequence is obtained from RNA prepared from human leucocyte
by using 5' primer HuCKF (SEQ ID NO: 22) and 3' primer HUCKB (SEQ ID NO: 23)
through reverse transcription and PCR. Finally, the fragment of humanized antibody
light chain variable region and human C gene are linked by PCR using 5' primer
(FVHX, SEQ ID NO: 17) and 3' primer (HUCKB, SEQ ID NO: 23) to obtain a gene
fragment of length about 700 bp comprising light chain encoding sequence. The gene
fragment is treated with endonuclease Hind III and Bam H1, and then inserted into
vectors such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985)
Gene, 33, 103-119.).
2) Construction of gene for humanized antibody heavy chain
First, the gene fragment for humanized antibody heavy chain variable
region (E001VH) is prepared by synthetic method. And the preparation procedures
include obtaining the nucleotides sequences by reverse translation from the amino acid
sequence of the heavy chain variable region according to their genetic codons; adding a
Kozak sequence and heavy chain leader sequence at 5' terminal; preparing the gene
fragment for heavy chain variable region by synthetic method. The gene fragment was
cloned into appropriate vectors to obtain pHu-VH1 plasmid. Subsequently, 5’ fragment
is obtained by using a plasmid (pHu-V ) comprising humanized antibody heavy chain
variable region V gene fragment as template, 5' primer FVHX (SEQ ID NO: 17) and 3'
primer RVCG (SEQ ID NO: 18), which comprises the gene for humanized antibody
heavy chain variable region (V ) and 7 amino acid at 5'-teminal of human IgG heavy
chain constant region (C ). Meanwhile, A gene comprising human IgG heavy chain
%1 1
constant region (C ) encoding sequence is obtained from RNA prepared from human
leucocyte by using 5' primer HuCGF (SEQ ID NO: 19) and 3' primer HUCGE (SEQ ID
NO: 20) through reverse transcription and PCR. Finally, the fragment of humanized
antibody heavy chain variable region and human C gene are linked by PCR using 5'
primer (FVHX, SEQ ID NO: 17) and 3' primer (HUCGE, SEQ ID NO: 20) to obtain a
gene fragment of length about 1400 bp comprising heavy chain encoding sequence.
The gene fragment is treated with endonuclease Hind III and EcoR1, and then inserted
into vectors such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985)
Gene, 33, 103-119) to express humanized antibody heavy chain protein. The sequence
of the gene fragment has been verified to be correct by DNA sequencing.
3) Humanized antibody single-chain expression vector
The cDNAs encoding the heavy chain or light chain which are obtained
by aforesaid methods are inserted into pcDNA3 (Purchased from Invitrogen USA,
Carlsbad, CA, U.S.A.) vector to construct pHu_anti-H1L1-TNF! humanized expression
vector. The expression vector plasmid comprises cytomegalovirus early gene
promoter-enhancer required for high level expression in mammal cells. Meanwhile, the
vector plasmid also comprises optional maker gene, so as to have amicillin resistance in
bacteria, have G418 resistance in mammal cells. Furthermore, the vector plasmid
comprises DHFR gene. In suitable host cells, chimeric antibody gene and DHFR gene
can be co-amplified by Methotrexate (MTX, Sigma) (see, for example, Axel, R., et al.
U.S Patent No. 5,179,017; Kaufman,R. and Sharp,P., J.Mol. Biol. 159:601-621,1982).
Example 6: The expression of humanized antibodies
The constructed recombinant expression plasmid was transfected into
mammalian host cells to express anti-hTNF! humanized antibody. In order to stabilize
high level of expression, the preferred host cells are dihydrofolate reductase (DHFR)
deficient Chinese hamster ovary (CHO) cells (see, for example, Chasind, L., et al, U.S.
Patent 4,818,679). The preferred method of transfection is electroporation, and other
methods also can be used including calcium phosphate coprecipitation, lipid
transfection and protoplast fusion. In electroporation, Gene Pulser (Bio-Rad
Laboratories) set at 250V electric field and 960 µFd capacitor is used, 2(10 cells
suspended in 0.8 mL of PBS is added into a cuvette, which also contains 10µg
expression plasmid DNA linearized by using PvuI (TakaRa). After transfection for 2
days, 0.2 mg/mL of G418 and 200 nM methotrexate (methotrexate or MTX) are added.
In order to achieve a high level of expression, the transfected humanized antibody gene
is co-amplified by using DHFR gene inhibited by MTX drugs. The subcloning
transfectants are diluted, and the secretion rates of various cell lines are determined to
screen out cell lines with high-level expression of humanized antibody.
Example 7: Study on the antibodies neutralizing TNF! killing effect
on L929 cell
L929 cells were trypsinized, centrifuged, resuspended in 1640 medium
supplemented with 10% FCS, counted, and added to column 1 to 11 of 96-well plated at
a certain concentration. And then, an appropriate concentration of TNF! was added to
column 1 to 10 in 96-well plates. Respectively, 2-fold gradient dilutions of adalimumab
(purchased from Abbott Laboratories), amino acid unmodified chimeric antibody
AT(CE)-1, AT132, AT135, AT143, AT151, AT164 prepared in accordance with
Examples 5 and 6 were added to row A, B, C, D, E, F, G and H, in a serial of
concentrations from low to high (column 1 to 9). And column 10 is TNF! control,
column 11 is cell control, and column 12 is medium control. After addition, the plate
was placed in a 37 °C carbon dioxide incubator to cultivate. After incubation was
completed, color reagent was added, and continued to incubate. And then absorbance
was detected by microplate reader. The results were shown in Table 2.
Table 2: the antibodies neutralize TNF! killing effect on L929 cell
Antibody Type Heavy Chain Light Chain EC (ng/mL)
AT132 E001V F001V 20.4
AT135 E002V F001V 50
AT143 E003V F003V 39.8
AT151 E001V F002V 42.1
AT164 E002V F003V 45.5
AT(CE)-1 -- -- 15.2
Humira -- -- 19.1
The results in Table 2 show that humanized antibodies obtained by
mutation in FR region still have good activities in neutralizing TNF!. The EC50 of
AT132, 20.4 ng/mL, is similar to that of Humira.
Example 8: Study on AT132 neutralizing TNF! killing effect on
human U937 cells (human lymphoma)
U937 cells in good conditions were counted and adjusted to cell
concentration of 3.75 ( 10 /well with 1640 medium with 10% FCS. And then were
added to a 96-well plate, 75 µl/well. The cell culture medium containing 120 ng/mL of
TNF! was used to respectively gradient dilute AT132 standard sample and test
samples. The concentration in first well was 600 ng/mL, and dilution gradient was 1.5
fold. After dilutions, the samples were added to 96-well plate with 25 µl/well. The plate
was incubated for 40 hours in a 37 °C carbon dioxide incubator. After completion of the
incubation, each well was added 10 µl of CCK8 color reagent, and incubated for 3h,
detected with microplate reader at 490nm/630nm dual-wavelength. A four-parameter
curve fitting was performed to obtain ED50 of the standard sample and test samples,
and calculate the specific activity (formula: 100% (ED50 of standard sample / ED50 of
test samples).
Figure 1 shows the curve of antibody neutralizing TNF! killing effects of
U937 cells
The analysis results of figure 1 shows that, at extremely low
concentrations of AT132, TNF! kills cells. With the concentration of AT132 increases,
TNF! killing effects are gradually antagonized. When the concentration of AT132
reaches about 80 ng/mL, TNF! killing effects are completely antagonized. Clearly, it is
dose-dependent according to the results of several experiments. The average median
effective concentration of AT132 to neutralize 30 ng/mL of TNF! is 24.1 ng/mL.
Example 9: Determination of AT132 affinity
AT132 affinity was determined by Biacore X100, and analyzed with
Biacore X100 kinetics/affinity analysis software. Using indirect capture method, goat
anti-human IgG Fc polyclonal antibody was coupled to the surface of CM5 chip as a
capture molecule by using Amine Coupling Kit. By calculation, AT132 and Humira as
control were respectively diluted to a a certain concentration to be lately used as ligand,
and TNF! as the analyte. Analytes were diluted to 5 concentrations, and each
concentration as a cycle. First, using HBS-EP buffer to run for three cycles, designing
an analyte concentration of 0 concentration to run for two cycles, and finally, designing
a repeat analyte concentration to run for a cycle. The whole process ran 11 cycles, and
each cycle can draw a curve. The dynamics/affinity data of the measured antibody and
humira were analyzed by Biacore X100 kinetics/affinity analysis software.
Results: AT132 dissociation constant (Kd) of 1.19 ( 10 M, that is, the
-1 -10
affinity constant (Ka) of 8.4 ( 10 M ; Humira dissociation constant of 1.08 ( 10 M,
9 -1
that is, the affinity constant 9.3 ( 10 M .
Example 10: AT132 binding activity to mouse TNF! and monkey
TNF!
On a plate, respectively coat recombinant human TNF! 5 ng/well, mouse
TNF! 25 ng/well, and monkey TNF! 5 ng/well at room temperature, and blocked for 1h,
washed. And then 1.8-fold gradient dilutions of AT132 was added with an initial
concentration of 250 ng/mL, and incubated at 25 °C for 2h, washed, HRP-labeled anti-
human Fc antibody added, and incubated at room temperature for 1h, washed, and the
substrate solution added as 100 µl/well, placed in dark at 37 °C for 30 min. In
accordance with the order of the added color reagents, 0.2 M H SO , 50 µl/well was
added to terminate the reaction. Within about 5 minutes after termination, OD values
were test at 450 nm/630 nm on a microplate reader, and median effective
concentrations were obtained by four-parameter fitting. Compare the binding activities
of AT132 to different sources of TNF!.
Mouse TNF! binding activity results show that AT132 does not bind
mouse TNF!. Calculation based on coating concentration and AT132 sample
concentration suggests that AT132 binding activity to mouse TNF! is at least 1,000
times lower than that to human TNF!.
Monkey TNF! binding activity results show AT132 binding activity to
monkey TNF! was about 50% of that to human TNF!.
Example 11: Preparation of injections
The preparation of AT132, AT135 injection preparation is as follows:
1) Preparation of 20-L buffer (equivalent to 20.180 kg solution with a
density: 1.009 g / mL)
Weighing out the ingredients in the following weight: 240.0 g mannitol,
26.1 g monohydrate citric acid, 6.1 g sodium citrate, 30.6 g dihydrated disodium
hydrogen phosphate, 17.2 g dihydrate phosphate monobasic sodium, 123.3 g sodium
chloride, 20.0 g sorbitan polyoxyethylene(20) ether oleate and 19715.7 to 19716.1 g
water.
Mixing 40.0 g sodium hydroxide and 1000.8 g water for injection to
prepare sodium hydroxide solution.
Then, dissolving following pre-weighted ingredients (as described above)
in about 90% water for injection to prepare buffer: mannitol monohydrate citric acid,
sodium citrate dihydrate disodium hydrogen phosphate, ether oleate, sodium
dihydrogen phosphate, sodium chloride and sorbitan polyethylene(20) ether oleate.
After adding all of the above buffer ingredients, adjusting pH of the
solution with 1 M sodium hydroxide prepared by the above method. After adding
sodium hydroxide, adding the final weight of water. And then, The buffer is filtered into
a sterile container through a filter membrane (hydrophilic poly(vinylidene fluoride), 0.22
µm pore size). The filter media used in the filtration is ammonia for disinfection.
2) Preparation of 40 L formulation (equivalent to 40.88 kg)
Filtered buffer solution was added to antibody concentrate (the active
ingredient of the pharmaceutical formulation), which has been thawed and merged
according to the following methods. Before the preparation of pharmaceutical
formulation, the antibody (concentrate) was placed in a water bath to thaw. 34.207 g
antibody concentrate was used, which was equivalent to 2.0 kg of protein, protein
concentrate with a concentration of 60 mg protein/mL of. The density of the concentrate
was 1.0262 g/mL. Any protein concentrate within 25.655-37.316 can be used, which is
equivalent to a concentration of 50-80 mg/mL protein in the protein concentrate. Under
stirring, the buffer was added until it reaches the final weight of the total solution.
Then, the formulation which comprising all its ingredients was filtered in
accordance with the above method except that the formulation was filter by two layers
of sterile 0.22 µm membrane filter. After disinfection, the formulation was packed for
use in a vial or pre-filled syringe.
Example 12: AT132 acute toxicity test
Test samples: AT132 lyophilized powder, 20 mg/bottle; adjuvant control:
AT132 buffer (containing histidine, trehalose); solvent: sterilized water for injection.
Test animal grouping and dose: amount of 60 Kunming (KM) mice, 4-6 weeks old, 18-
22g weight, half male and half female, the SPF level. The animals were randomly
divided into three groups, each group of 20, half male and half female. Single dosed
and observed for 14 days. Table 3 shows the dose and route of administration:
Table 3: dose and route of administration
Dose in weight Dose in volume
No. Group
(mg/kg) (mL/kg)
1 Adjuvant control — —
2 Test sample, subcutaneous 500 25
injections
Test sample, intravenous
3 500 25
injections
Outcome measures: body weight, food intake, mental state, behavior,
stool. After the end of the trial observation period, the animals were put euthanasia and
pathology gross anatomical observed. And abnormal tissues or organs were
histologically examined.
Results: During the trial, no animal died or was dying; all animals were
in goold mental state, normal behavior, diet, water drinking, and showed no abnormal
performance. The weights of each treatment group animals showed no regular change
associated with the administration of dose. Pathology gross anatomy observation
showed no abnormal changes related to the administration of dose.
Conclusion: under the present experimental condition, when AT132
powder was administrated to mice by intravenous and subcutaneous injection with a
single injection of 500 mg/kg dose, no significant toxicity was observed, and the
maximum tolerated dose (MTD) was greater than 500 mg/kg.
Example 13: AT132 protective effect on D-galactosamine sensitized
mouse from rhTNF!-induced death
An amount of 51 C57BL/6 mic, weight 20.0 ± 2.0 g, divided into 6 groups
(Table 4). For group 2 to 5, each mouse was intraperitoneally injected 0.25 mL of
AT132 solution, wherein, based on the amount of AT132, each mouse in group 2 was
given a dose of 5.2 µg/mouse; in group 3, 26 µg/mouse; in group 4, 52 µg/mouse; in
group 5, 26 µg/mouse. For group 1, each mouse was intraperitoneally injected 0.25 mL
pH 5.63 citrate buffer. For group 6, each mouse was intraperitoneally injected 0.25 mL
of human IgG1 (HuIgG1, negative control), 26 µg/mouse based on the amount of
HuIgG1. 30 minutes later, every mouse in each group (except group 5) was
intraperitoneally injected 0.25 mL mixture solution of rhTNF! (Primegene, batch number
1030109021) and D-galactosamine. For group 5, each mouse was intraperitoneally
injected 0.25 mL of buffer. Observed the number of died mice in 48 hours, and
calculated survival rates as shown in Table 4.
Table 4: The survival rates of mice in each group
NO. Group n Survival/Total Survival Rate (%)
1 Buffer 9 0/9 0
2 5.2 µg AT132 9 3/9 33.33
3 26 µg AT132 9 9/9 100
4 52 µg AT132 10 10/10 100
26 µg AT132 7 7/7 100
(No rhTNF)
6 26µg HuIgG 7 0/7 0
Results: mice survival rates of buffer and HuIgG1 group were 0. But
AT132 protected rhTNF! and D-amino-galactose sensitized mice in dose dependent
manner. Therefore, AT132 showed protective effects on D-galactosamine sensitized
mice from rhTNF!-induced death.
Example 14: Study on the effect of AT132 on type II collagen-
induced arthritis
An amount of 40 Wistar female rats were randomly divided into 4 groups,
and each group is 10 rats. The groups are blank control group, inflammatory control
group (model group), AT132 1 mg/kg group, and AT132 5 mg/kg group.
Except for the blank control group, the rats in the other groups were
respectively intradermally injected on the back with collagen II immune. And the
proinflammatory control group was injected with 0.1 mol/L acetic acid and complete or
incomplete Freund's adjuvant emulsion (Sigma, lot number 129K8701) .
AT132 1 mg/kg group and AT132 5 mg/kg group began to be
intraperitoneal injected with AT132 at the day of immunization. One week after the first
immunization, the mice were immunized again in the same way to strengthen the
immune, and were administrated for a total of 28 days. At different times before and
after administration, rat joint swelling values were measured by YLS-7B toe volume
measuring instrument. The joint was taken for pathological examination after the
experiments. The swelling rate and inhibition rate were calculated, and the differences
between groups were compared by t test. Calculated as follows:
E = swelling values at different times after inflammation,
E = swelling values before inflammation
The experimental data were represented in mean values and standard
deviation (s), and t test was used for statistical analysis. The results are shown in
Figure 2. AT132 1 mg/kg group showed significant inhibitory effects, with a good effect
starting from day 19, and the best effect is 63.10%. Since then, the inhibitory effect
gradually decreased. The result is still good at day 28 with an inhibition rate of 55.71%.
AT132 5 mg/kg group showed significant inhibitory effects with significant treating effect
starting from day 19. The rat paw joint swelling gradually subsided, and activities
became normal, and the effect lasted until day 28 days. The highest inhibition rate of
47.27% appeared at day 21, and the other were about 40%.
Example 15: Pharmacodynamic Study of AT132 on Tg197 mouse
arthritis model
The experiment employed Tg197 transgenic mice (purchased from
Cyagen Biosciences), and the mice were divided into 6 groups, each group of 10 mice,
half male and half female. Specifically as follows: Group 1: AT132 1 mg/kg; Group 2:
solvent group (a buffer containing citric acid and sodium chloride); Group 3: AT132 30
mg/kg,; Group 4: AT132 10 mg/kg); Group 5: Humira 10 mg/kg; Group 6: AT132 3
mg/kg. Another 4 Tg197 mice were selected as blank control group.
3 weeks old Tg197 mice were intraperitoneally injected AT132, twice a
week, until 10 weeks old. AT132 was diluted to the desired concentration prior to
administration, and each group was given a dose of 10 µl/g weight. Observe the degree
of mice arthritis, and evaluate pathological scores of mice bare joint, and calculate the
inhibition rate based on arthritis scores and pathological scores.
1) Study of AT132 on Tg197 mouse arthritis degree
Mice joint morphological changes were evaluated weekly to assess the
degree of arthritis, and the specific arthritis scoring criteria as follows:
0.0 = no arthritis (appearance normal, the mice are able to support the
body weight, overall flexibility/evade capability normal, maximum grip strength);
0.5 = onset of arthritis (joints and paws slight swelling, appearance
normal, the mice are able to support the body weight, overall flexibility/evade capability
normal, maximum grip strength);
1.0 = mild arthritis (joints swelling and deformation, paws swelling,
appearance normal, the mice are able to support the body weight, overall
flexibility/evade capability normal, maximum grip strength);
1.5 = mild to moderate arthritis (joints and paws swelling, deformation,
and the last fingers inward deformation, barely able to support the weight, overall
flexibility reduced, grip strength decreased);
2.0 = moderate arthritis (severe joints, paws and fingers swelling, feet
joints deformation, cannot support the upper body weight and fall, disappearance of the
overall flexibility, grip strength disappeared, crawling/eating affected);
2.5 = moderate to severe arthritis (severe joints, paws and fingers
swelling, feet joints deformation, cannot support the upper body weight and fall,
disappearance of the overall flexibility, grip strength disappeared, crawling/eating
affected, finger paws deformation, mouse activity impaired);
3.0 = severe arthritis (joint stiffness, bending detected and activity
severely impared, the mice are dying).
Tg197 mice arthritis scores are shown below in Figure 3A.
2) Tg197 mice ankle joint histopathology study
To monitor the disease status, four littermates of Tg197 mice of the trial
(Numbered as Con1 to Con4) were sacrificed when 3 weeks old, as joint samples at the
beginning of treatment. Experimental mice were sacrificed when 10 weeks old, and
sample slices were taken from ankle joints. After hematoxylin/eosin staining,
microscopic histopathological scores of arthritis were evaluated in a blinded manner,
and the score of 0-4 as follows:
0 = no detectable pathology
1 = synovial proliferation, polymorphonuclear leukocytes infiltration
2 = pannus and fibrous tissue formation and subchondral bone erosion at
focus site
3 = cartilage destruction and bone erosion
4 = expanded cartilage destruction and bone erosion
Tg197 mice histopathological analysis were shown in Figure 3B and 3C
3) Calculation of inhibition rates according to the arthritis and
pathological scores.
Inhibition rates were calculated according to the arthritis scores as
follows:
En = arthritis score of each group
E0 = arthritis score of solvent group
Inhibition rates were calculated according to the pathological score as
follows:
E = pathological score of each group
E = pathological scores of solvent group
The results are shown in Table 5 below.
Table 5: mice arthritis score inhibition rate and pathological score inhibition rate
No. Group n arthritis score pathological
inhibition rate (%) score
inhibition rate
Group 1 AT132 1 mg/kg 10 15 3
Group 2 Solvent 10 0 0
Group 3 AT132 30 mg/kg 10 77 84
Group 4 AT132 10 mg/kg 10 79 86
Group 5 Humira 10 mg/kg 10 65 76
Group 6 AT132 3 mg/kg 10 46 41
Results: Administrated with different AT132 doses (group 1, group 3,
group 4, group 6), it shows significant inhibition effects on rheumatoid arthritis, which is
typically dose dependent. Especially from group 1 (1 mg/kg) to group 6 (3 mg/kg), the
inhibitions can be significantly distinguished therebetween. From group 6 (3 mg/kg) to
group 4 (10 mg/kg), the inhibitions also can be significantly distinguished therebetween.
Only between group 4 (10 mg/kg) group and the highest dose group 3 (30 mg / kg), the
inhibitions have no difference. It is important that at the same dose level of 10 mg/kg,
AT132 treatment group has no significant difference from Humira treatment group 5,
and have significantly improvement effect on arthritis histopathology when compared to
solvent group 2
Example 16: AT132 monoclonal antibody tissue cross-reaction
1) AT132 monoclonal antibody human tissue cross-reaction
Nasal polyps paraffin slices and four normal human tissue (donor A, B,
C, D, provided by National Institutes for Food and Drug Control, and National Center for
Safety Evaluation of Drugs) paraffin slices were divided into three groups, namely the
experimental group (AT132 biotin marker), positive control group (biotin-labeled
Humira), the negative control (buffer PBS). Observe the staining of tissue slicers after
tissue cross-reaction.
The experimental results show that the negative control group of human
nasal polyps and normal human tissues were not stained. Biotin-Humira positive
control group, the human nasal polyps macrophages, the normal human lymph nodes
macrophages and the lung alveolar macrophages were weakly to moderately stained,
while the other tissues were not stained. Biotin-AT132 experimental group, the human
nasal polyps macrophages showed weak to moderate staining. Normal human tissue
cross-reactions were similar to Humira.
2) AT132 monoclonal antibody cynomolgus monkey tissue cross-
reaction
Nasal polyps paraffin slices and three sets of cynomolgus monkey
tissues (donor A, B, C, provided by National Institutes for Food and Drug Control, and
National Center for Safety Evaluation of Drugs) paraffin slices were divided into three
groups, namely the experimental group (AT132 biotin marker), positive control group
(biotin-labeled Humira), the negative control (buffer PBS). Observe the staining of
tissue slicers after tissue cross-reaction.
The experimental results show that the negative control group of human
nasal polyps and normal cynomolgus monkey tissues were not stained. Biotin-Humira
positive control group, the human nasal polyps macrophages were weakly to
moderately stained, while the normal cynomolgus monkey tissues were not stained.
Biotin-AT132 experimental group, the human nasal polyps macrophages showed weak
to moderate staining. Normal cynomolgus monkey tissue cross-reactions were similar
to Humira.
Claims (16)
1. A humanized anti-TNF monoclonal antibody comprising a heavy chain and a light chain, the heavy chain comprising complementary determining regions (CDRs) CDR-H1, CDR-H2 and CDR-H3, and heavy chain framework regions FR-H1, FR-H2, and FR-H3, the light chain comprising CDRs CDR-L1, CDR-L2, CDR-L3 and light framework regions FR-L1, FR-L2 and FR-L3, wherein CDR-H1 of the heavy chain variable region comprises a sequence of SEQ ID NO: 3; CDR-H2 comprises a sequence of SEQ ID NO: 4; CDR-H3 comprises a sequence of SEQ ID NO: 5; complementary determining region CDR-L1 comprises a sequence of SEQ ID NO: 6; CDR-L2 comprises a sequence of SEQ ID NO: 7; and CDR-L3 comprises a sequence of SEQ ID NO: 8; and wherein one or more of the heavy chain framework regions and light chain framework regions comprises at least one substitution selected from, within the heavy chain framework region FR-H1, I is optionally substituted by V at position 20 (SEQ ID NO: 11); within FR-H2, K is optionally substituted by R at position 3 (SEQ ID NO: 12); within FR-H3, T is optionally substituted by V at position 3, E is optionally substituted by D at position 7, F is optionally substituted by Y at position 14, and T is optionally substituted by V at position 27 (SEQ ID NO: 13); within framework region FR- L1, M is optionally substituted by I at position 21 (SEQ ID NO: 14); within FR-L2, W is optionally substituted by L at position 13 (SEQ ID NO: 15); and within FR-L3, A is optionally substituted by F at position 27 (SEQ ID NO: 16).
2. The humanized anti-TNF monoclonal antibody comprising a heavy chain and a light chain according to claim 1, wherein the heavy chain comprises a sequence of SEQ ID NO: 1, and the light chain comprises a sequence of SEQ ID NO: 2.
3. A nucleic acid sequence which encodes the humanized anti-TNF monoclonal antibody according to claim 1 or 2.
4. A vector comprising the nucleic acid sequence according to claim 3.
5. The vector according to claim 4, wherein the vector further comprises a promoter which is operational linked to the nucleic acid sequence to facilitate its expression.
6. A host cell comprising the vector according to claim 5 which is not present within a human.
7. A diagnostic agent or medicament comprising the humanized anti-TNF monoclonal antibody according to claim 1 or 2.
8. A method for preparing a medicament in diagnostic analysis of hTNFα, comprising including in the medicament an effective amount of the humanized anti-TNF monoclonal antibody according to claim 1 or 2.
9. Use of an effective amount of humanized anti-TNF monoclonal antibody according to claim 1 or 2 in the manufacture of a medicament for treating a hTNF related disease, wherein the hTNFα related disease is selected from pyaemia, autoimmune diseases, malignant tumor, lung function disorder, transplant rejection, bacterial meningitis, cerebral malaria, AIDS and AIDS related complex (ARC), and secondary cytomegalovirus infection after transplantation.
10. A pharmaceutical composition comprising the humanized anti-TNF monoclonal antibody according to claim 1 or 2 and at least one pharmaceutically accepted excipient.
11. The humanized anti-TNF monoclonal antibody according to claim 1 or 2, wherein the at least one substitution is within FR-H1, wherein I is substituted by V at position 20 (SEQ ID NO: 11).
12. The humanized anti-TNF monoclonal antibody according to any one of claim 1, 2 and 11, wherein the at least one substitution is within FR-H2, wherein K is substituted by R at position 3 (SEQ ID NO: 12).
13. The humanized anti-TNF monoclonal antibody according to any one of claim 1, 2, and11-12, wherein the at least one substitution is within FR-H3, wherein T is substituted by V at position 3, E is substituted by D at position 7, F is substituted by Y at position 14 and T is substituted by V at position 27 (SEQ ID NO: 13).
14. The humanized anti-TNF monoclonal antibody according to any one of claim 1, 2 and 11-13, wherein the at least one substitution is within FR-L1, wherein M is substituted by I at position 21 (SEQ ID NO: 14).
15. The humanized anti-TNF monoclonal antibody according to any one of claims 1, 2 and 11-14, wherein the at least one substitution is within FR-L2, wherein W is substituted by L at position 13 (SEQ ID NO: 15).
16. The humanized anti-TNF monoclonal antibody according to any one of claims 1, 2 and 11-15, wherein the at least one substitution is within FR-L3, wherein A is substituted by F at position 27 (SEQ ID NO: 16). 1 / 3
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201110048505 | 2011-02-28 | ||
CN201110048505.1 | 2011-02-28 | ||
CN201210026698.5A CN102675460B (en) | 2011-02-28 | 2012-02-07 | The humanized antibody of Tumor necrosis factorα |
CN201210026698.5 | 2012-02-07 | ||
PCT/CN2012/071079 WO2012116595A1 (en) | 2011-02-28 | 2012-02-13 | Tumor necrosis factor-α humanized antibody |
Publications (2)
Publication Number | Publication Date |
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NZ615477A NZ615477A (en) | 2015-11-27 |
NZ615477B2 true NZ615477B2 (en) | 2016-03-01 |
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