U.S. patent application number 16/059761 was filed with the patent office on 2018-12-20 for antibody-drug conjugate having improved stability and use thereof.
The applicant listed for this patent is ABLBIO. Invention is credited to Jae Hyun Eom, Jin Won Jung, Jae Yong Kim, Ju Hee Kim, Young Min Kim, Min Ji Ko, Kyung Duk Moon, Dae Hae Song.
Application Number | 20180360986 16/059761 |
Document ID | / |
Family ID | 52142250 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180360986 |
Kind Code |
A1 |
Kim; Young Min ; et
al. |
December 20, 2018 |
ANTIBODY-DRUG CONJUGATE HAVING IMPROVED STABILITY AND USE
THEREOF
Abstract
The present invention relates to an antibody-drug conjugate
comprising a drug conjugated to an antibody, a preparation method
thereof and the use thereof.
Inventors: |
Kim; Young Min;
(Gyeonggi-do, KR) ; Ko; Min Ji; (Daejeon, KR)
; Kim; Jae Yong; (Seoul, KR) ; Kim; Ju Hee;
(Daejeon, KR) ; Moon; Kyung Duk; (Daejeon, KR)
; Song; Dae Hae; (Daejeon, KR) ; Eom; Jae
Hyun; (Sejong, KR) ; Jung; Jin Won; (Daejeon,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABLBIO |
Seoul |
|
KR |
|
|
Family ID: |
52142250 |
Appl. No.: |
16/059761 |
Filed: |
August 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14898126 |
Dec 12, 2015 |
10071170 |
|
|
PCT/KR14/05589 |
Jun 24, 2014 |
|
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16059761 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 2317/56 20130101; A61K 47/6803 20170801; C07K 2317/94
20130101; C07K 2317/92 20130101; A61K 2039/505 20130101; C07K
2317/24 20130101; C07K 2317/73 20130101; A61P 37/06 20180101; A61K
47/6899 20170801; A61P 37/00 20180101; A61P 35/00 20180101; A61K
47/6851 20170801; C07K 16/2803 20130101; A61K 47/6849 20170801;
C07K 16/2878 20130101; A61K 47/6817 20170801 |
International
Class: |
A61K 47/68 20170101
A61K047/68; C07K 16/28 20060101 C07K016/28; C07K 16/32 20060101
C07K016/32 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2013 |
KR |
10-2013-0072686 |
Claims
1. An antibody-drug conjugate comprising an immunosuppressive agent
conjugated to the N-terminal amino acid residue of the heavy chain
or light chain of an antibody, wherein the immunosuppressive agent
is site-specifically conjugated to the N-terminal .alpha.-amine
group of native first amino acid of the heavy chain or light chain
of the antibody, and the immunosuppressive agent is conjugated to
the antibody by a linker having a reactive aldehyde group, and the
conjugate is produced through reductive alkylation by the aldehyde
group.
2. The antibody-drug conjugate of claim 1, wherein the antibody
includes full-length antibodies or antibody fragments containing
antigen binding domains.
3. The antibody-drug conjugate of claim 2, wherein the antibody is
selected from the group consisting of IgG, scFv, Fv, Fab, Fab', and
F(ab').sub.2.
4. The antibody-drug conjugate of claim 1, wherein the
immunosuppressive agent is a cytokine antagonist.
5. The antibody-drug conjugate of claim 4, wherein the cytokine
antagonist is an interleukin antagonist.
6. The antibody-drug conjugate of claim 1, wherein the
immunosuppressive agent is a cytokine inhibitor.
7. The antibody-drug conjugate of claim 4, wherein the cytokine
inhibitor is an interleukin inhibitor.
8. The antibody-drug conjugate of claim 1, wherein the antibody is
selected from the group consisting of Trastuzumab, Lorvotuzumab,
Brentuximab, and Glembatumumab.
9. A pharmaceutical composition for treating autoimmune disease,
which comprises the antibody-drug conjugate of claim 1.
10. A method for preparing the antibody-drug conjugate of claim 1,
the method comprising conjugating the immunosuppressive agent to
the N-terminal .alpha.-amine group of the heavy chain or light
chain of the antibody, by reacting an antibody with an
immunosuppressive agent containing a reactive group capable of
crosslinking with an .alpha.-amine group.
11. The method of claim 10, further comprising separating the
antibody-drug conjugate from a reaction product including the
antibody and the immunosuppressive agent.
12. The method of claim 10, wherein the reactive group capable of
crosslinking with the .alpha.-amine group is selected from the
group consisting of isothiocyanate, isocyanate, acyl azide, NHS
ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,
carbonate, aryl halide, imidoester, carbodiimide, anhydride, and
fluorophenyl ester.
13. A method for treating autoimmune disease, comprising
administering the antibody-drug conjugate of claim 1 to a subject
having autoimmune disease.
14. The method of claim 13, wherein the autoimmune disease is
rheumatoid arthritis, systemic scleroderma, systemic lupus
erythematosus, atomic dermatitis, psoriasis, alopecia areata,
asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome,
Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis,
polymyositis, ankylsoing spondylitis, fibrositis, or polyarteritis
nodosa.
15. The method of claim 13, wherein the antibody of the
antibody-drug conjugate includes full-length antibodies or antibody
fragments containing antigen binding domains.
16. The method of claim 13, wherein the antibody of the
antibody-drug conjugate is selected from the group consisting of
IgG, scFv, Fv, Fab, Fab', and F(ab').sub.2.
17. The method of claim 13, wherein the immunosuppressive agent of
the antibody-drug conjugate is a cytokine antagonist.
18. The method of claim 17, wherein the cytokine antagonist is an
interleukin antagonist.
19. The method of claim 13, wherein the immunosuppressive agent of
the antibody-drug conjugate is a cytokine inhibitor.
20. The method of claim 19, wherein the cytokine inhibitor is an
interleukin inhibitor.
21. The method of claim 13, wherein the antibody of the
antibody-drug conjugate is selected from the group consisting of
Trastuzumab, Lorvotuzumab, Brentuximab, and Glembatumumab.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional under the provisions of 35 U.S.C.
.sctn. 120 of U.S. patent application Ser. No. 14/898,126 filed
Dec. 12, 2015, which in turn is a U.S. national phase under the
provisions of 35 U.S.C. .sctn. 371 of International Patent
Application No. PCT/KR14/05589 filed Jun. 24, 2014, which in turn
claims priority of Korean Patent Application No. 10-2013-0072686
filed Jun. 24, 2013. The disclosures of U.S. patent application
Ser. No. 14/898,126, International Patent Application No.
PCT/KR14/05589, and Korean Patent Application No. 10-2013-0072686
are hereby incorporated herein by reference in their respective
entireties, for all purposes.
TECHNICAL FIELD
[0002] The present invention relates to an antibody-drug conjugate
comprising a drug conjugated to the N-terminal amino acid residue
of the heavy chain or light chain of an antibody, a preparation
method thereof, and the use thereof.
BACKGROUND ART
[0003] In recent years, methods of diagnosing or treating various
diseases using antibodies have been studied. Particularly, because
of the target specificity of antibodies, various therapeutic
methods using antibodies have been developed, and various types of
drugs containing antibodies, for example, antibody-drug conjugates
(ADCs), have been developed. Thus, studies have been continuously
conducted to increase the in vivo stability of antibodies or
antibody-drug conjugates and maximize the therapeutic effects
thereof.
[0004] Among them, antibody-drug conjugates generally have the
disadvantage of low in vivo stability compared to natural
antibodies, but have been developed in order to overcome the
disadvantages (low therapeutic effects) of natural antibodies by
conjugating them to drugs. Various antibody-drug conjugates in
which drugs having certain medical effects, such as cytotoxin, are
conjugated to target-specific antibodies, have been developed. In
particular, a method of inducing cancer cell death by cytotoxin
conjugated to a cancer cell-specific antibody is a method that is
actually currently used.
[0005] However, such antibody-drug conjugates generally have low in
vivo stability compared to natural antibodies. Furthermore, if drug
antibody ratio (DAR) is increased in order to increase therapeutic
effects, there will be various technical problems to be solved.
First, an increase in drug antibody ratio should not interfere with
the antigen-binding ability and Fc function of antibodies for
target-specific therapy, should lead to an increase in therapeutic
effects, and should not reduce the in vivo stability (i.e., blood
half-life) of antibody-drug conjugates. The object of the current
antibody-drug conjugate preparation field is to maintain the
highest possible antibody drug ratio in view of the above-described
technical problems. In particular, considering that the expression
level of cancer cell surface antigens is low, the highest possible
drug antibody ratio (DAR) should be maintained in order to maintain
high cytotoxicity. However, if DAR reaches 8, there is a problem in
that the blood half-life of the antibody-drug conjugate decreases
due to the effect of the hydrophobic drug conjugated to the
antibody so that the toxicity thereof can increase and in vivo
efficacy thereof can decrease.
[0006] Under this background, the present inventors have made
extensive efforts to develop a technology capable of preparing an
antibody-drug conjugate which maintains the antigen-binding
activity of a parent antibody, exhibits excellent anticancer
effects, and has low drug toxicity and excellent in vivo efficacy.
As a result, the present inventors have found that, when a drug is
conjugated to the N-terminus of the heavy chain or light chain of
an antibody, the antibody-drug conjugate has excellent blood
stability and anticancer activity while having low in vivo toxicity
compared to previously reported antibody-drug conjugates, thereby
completing the present invention.
DISCLOSURE OF INVENTION
[0007] It is an object of the present invention to provide an
antibody-drug conjugate comprising a drug conjugated to the
N-terminal amino acid residue of the heavy chain or light chain of
an antibody.
[0008] Another object of the present invention is to provide a
method for preparing the above antibody-drug conjugate.
[0009] Still another object of the present invention is to provide
a composition comprising the above antibody-drug conjugate.
[0010] Yet another object of the present invention is to provide a
method for treating cancer, comprising administering the above
antibody-drug conjugate to a subject suspected of having
cancer.
[0011] A further object of the present invention is to provide a
method for treating autoimmune disease, comprising administering
the above antibody-drug conjugate to a subject suspected of having
autoimmune disease.
[0012] A still further object of the present invention is to
provide a method for screening an antibody suitable for use in
preparation of the above antibody-drug conjugate.
Advantageous Effects
[0013] A method for preparing an antibody-drug conjugate according
to the present invention can prepare an antibody-drug conjugate
having higher in vivo efficacy, stability and lower toxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the structural formula of toxin monomethyl
auristatin F (MMAF) having an aldehyde linker connected to the
end.
[0015] FIG. 2 is a schematic diagram showing the structure of a
non-genetically modified monoclonal antibody-cytotoxin conjugate in
which the number and site of cytotoxin moieties conjugated to an
antibody are homogeneous.
[0016] FIG. 3 shows the LC/MS profile of T-N-MMAF.
[0017] FIG. 4 shows the results of peptide mapping performed to
determine the site of binding of a drug in a prepared
Trastuzumab-N-MMAF (T-N-MMAF conjugate).
[0018] FIG. 5 shows the results of SEC-HPLC analysis of a prepared
T-N-MMAF.
[0019] FIG. 6 shows a time-dependent change in the blood
concentration of total antibody in rats.
[0020] FIG. 7 shows a time-dependent change in the blood
concentration of conjugated antibody.
[0021] FIG. 8 shows a comparison of the PK profiles of total
antibody and conjugated antibody between antibody-drug conjugates
(ADCs).
[0022] FIG. 9 shows growth curves of tumors formed by the HCC1954
cell line in nude rat xenograft models.
[0023] FIG. 10 shows survival curves obtained in nude rat xenograft
model experiments performed to measure the tumor volume at the
endpoint.
[0024] FIG. 11 shows the change and relative change in weight by
administration of each antibody-drug conjugate (ADC).
[0025] FIG. 12 shows the results of examining whether the
administration of each ADC caused hepatotoxicity.
[0026] FIG. 13 shows the changes in neutrophils and platelets by
administration of each ADC.
[0027] FIG. 14 shows the results of LC/MS analysis of T-N-MMAE.
[0028] FIG. 15 shows the rat PK profile of T-N-MMAE.
[0029] FIG. 16 shows the LC/MS profile of Brentuximab-N-MMAF
(B-N-MMAF).
[0030] FIG. 17 shows the results of analyzing the antigen-binding
activity of B-N-MMAF.
[0031] FIG. 18 shows the conjugation profile of Lorvotuzumab-N-MMAF
(L-N-MMAF).
[0032] FIG. 19 shows the antigen-binding activity of L-N-MMAF.
BEST MODE FOR CARRYING OUT THE INVENTION
[0033] In one aspect, the present invention provides is directed to
an antibody-drug conjugate comprising a drug conjugated to the
N-terminal amino acid residue of the heavy chain or light chain of
an antibody.
[0034] As used herein, the term "antibody-drug conjugate (ADC)"
refers to the form in which a drug and an antibody are chemically
conjugated to each other without reducing the biological activities
of the antibody and the drugs. In the present invention, the term
"antibody-drug conjugate" refers to the form in which the drug is
conjugated to the N-terminal amino acid residue of the heavy chain
and/or light chain of the antibody, particularly, the form in which
the drug is conjugated to the N-terminal a-amine group of the heavy
chain and/or light chain of the antibody. In the present invention,
it was found that, when a drug was site-specifically conjugated to
the N-terminus of the heavy chain or light chain among various
regions of an antibody, the antibody-drug conjugate had excellent
in vivo efficacy and stability and low toxicity, compared to
previously reported antibody-drug conjugates, including
antibody-drug conjugates formed by cysteine conjugation,
antibody-drug conjugates formed by thiol conjugation, and
antibody-drug conjugates formed by lysine conjugation, indicating
that the N-terminus of the heavy chain or light chain of antibodies
can be a site advantageous in terms of efficacy, stability and low
toxicity. This schematic view of an antibody-drug conjugate
according to the present invention is schematically shown in FIG.
2.
[0035] As used herein, the term "N-terminus" refers to the amino
terminus (N-terminus) of the heavy chain or light chain of an
antibody, which is a site to which a drug can be conjugated for the
purpose of the present invention. Examples of the N-terminus
include, but are not limited to, not only amino acid residues at
the distal end of the N-terminus, but also amino acid residues near
the N-terminus. Specifically, the term "N-terminus" refers to the
first amino acid residue of the heavy chain or light chain of an
antibody, and more specifically, refers to the a-amine group of the
first amino acid of the heavy chain or light chain of an antibody,
but is not limited thereto.
[0036] The antibody-drug conjugate according to the present
invention can have the advantage of guaranteeing homogeneity
through the site-specific conjugation or number-specific
conjugation between an antibody and a drug. Particularly, through
the optimization procedure of the present invention, 1-8 drug
moieties corresponding to the optimal drug-antibody ratio (DAR) can
be conjugated to the N-terminal amino acid residue of each antibody
molecule.
[0037] As used herein, the term "homogeneity" refers to the case in
which the ratio and site of conjugation between two substances in a
conjugate of the two substances are homogeneous. However, the term
is intended to include not only the case in which the ratio and
site of conjugation are completely homogeneous, but also the case
in which a specific ratio and site of conjugation are predominant.
When a conjugate has homogeneity, it is entirely homogeneous, and
the dose-dependent efficacy thereof can be accurately measured, and
thus the dose and number of administration thereof can be
standardized.
[0038] As used herein, the term "antibody" means a protein molecule
which comprises an immunoglobulin molecule immunologically reactive
with a certain antigen, and which serves as a receptor that
specifically recognizes the antigen. The term is intended to
encompass polyclonal antibodies, monoclonal antibodies, full-length
antibodies and antibody fragments containing antigen binding
domains. A full-length antibody has two full-length light chains
and two full-length heavy chains, in which each of the light chains
is linked to the heavy chain by a disulfide bond. The full-length
antibody comprises IgA, IgD, IgE, IgM and IgG, and subtypes of IgG
include IgG1, IgG2, IgG3 and IgG4. The term "antibody fragment"
refers to a fragment having an antigen-binding function, and is
intended to include Fab, Fab', F(ab')2, scFv and Fv. Fab comprises
light-chain and heavy-chain variable regions, a light-chain
constant region, and a heavy-chain first constant domain (CH1), and
has one antigen-binding site. Fab' differs from Fab in that it has
a hinge region including one or more cysteine residues at the
C-terminus of the heavy-chain CH1 domain. An F(ab')2 antibody is
formed by a disulfide bond between the cysteine residues of the
hinge region of Fab'. Fv means a minimal antibody fragment having
only a heavy-chain variable region and a light-chain variable
region. dsFv is has a structure in which a heavy-chain variable
region and a light-chain variable region are linked to each other
by a disulfide bond, and scFV generally has a structure in which a
heavy-chain variable region and a light-chain variable region are
covalently linked to each other by a peptide linker. These antibody
fragments can be obtained using proteases (for example, Fab
fragments can be obtained by digesting a full-length antibody with
papain, and F(ab')2 fragments can be obtained by digesting a
full-length antibody with pepsin). Preferably, these antibody
fragments can be produced by a genetic recombinant technique. These
antibody fragments can be obtained using proteases (for example,
digestion of a whole antibody with papain or pepsin affords Fab or
F(ab')2, respectively), and preferably may be constructed by
genetic recombination techniques.
[0039] In addition, the antibody that is used in the present
invention may be a natural antibody or a recombinant antibody. As
used herein, the term "natural antibody" refers to an antibody that
has undergone no genetic modification. The natural antibody may
have a significantly low risk of immunogenicity, unlike antibodies
genetically modified in vivo. As used herein, the term "recombinant
antibody" means a genetically modified antibody which may have an
antigen-binding activity or desired characteristic imparted by
genetic modification.
[0040] As used herein, the term "genetic modification" refers to an
action of changing the amino acid sequence of interest and is
intended to include the modification of polypeptides having amino
acid sequences that somewhat differ from the amino acid sequence of
a native sequence polypeptide encoding the amino acid sequence of
interest. Amino acid sequence variants contain an amino acid
sequence having a substitution, deletion or insertion of one or
more amino acid residues at one or more specific positions in a
native amino acid sequence.
[0041] The antibody that is used in the present invention may be an
antibody recognizing a cell surface antigen which is internalized
into cells when binding to the antibody. For the purpose of the
present invention, when an antigen is internalized into cells by
binding to the antibody, a drug, particularly a cytotoxic drug,
conjugated to the antibody, can enter the cells due to the
characteristics of the antibody, and thus can exhibit high
efficacy, but is not limited thereto.
[0042] In addition, the antibody that is used in the present
invention may be an antibody that binds specifically to a cancer
cell surface antigen or a surface antigen of a tissue in which an
autoimmune disease has occurred.
[0043] As used herein, the term "cancer cell surface antigen"
refers to either a substance that is not produced in normal cells
or not exposed to the cell surface, or a substance that is exposed
to the cell surface specifically in cancer cells, or s substance
that is present more on the surface of cancer cells than on the
surface of normal cells. When the substance of interest is
recognized by the antibody, it is referred to as an antigen.
[0044] Specifically, the cancer cell surface antigen that is used I
the present invention may be any cancer cell surface antigen that
can be recognized specifically by the antibody of the present
invention. Examples of the cancer cell surface antigen may include
CD19, CD20, CD30, CD33, CD37, CD22, CD56, CD70, CD74, CD138,
Muc-16, mesothelin, HER2, HER3, GPNMB(glycoprotein NMB), IGF-1R,
BCMA(B cell maturation antigen), PSMA(prostate-specific membrane
antigen), EpCAM(Epithelial cell adhesion molecule), and EGFR
(epidermal growth factor receptor). More specifically, the cancer
cell surface antigen may be any one selected from the group
consisting of HER2, CD30, CD56, and GPNMB, but is not limited
thereto. In an example of the present invention, Trastuzumab, a
kind of anti-HER2 antibody, Lorvotuzumab, a kind of anti-CD56
antibody, Brentuximab, a kind of anti-CD30 antibody, and
Glembatumumab, a kind of anti-GPNMB antibody, which recognize Her2,
CD56 and GPNMB, were used as model antibodies.
[0045] As used herein, the term "drug" means any substance having
cell-specific biological activity, and is intended to include
compounds, DNA, RNA, peptides and the like. The term "drug" is
intended to include not only substances containing a reactive group
capable of crosslinking with an a-amine group, but also substances
having a linker containing a reactive group capable of crosslinking
with an a-amine group. In this case, the drug can bind
site-specifically to the N-terminal amino acid residue of the
antibody by the linker, but is not limited thereto.
[0046] The term "linker" refers to a chemical moiety comprising an
atomic chain that allows the drug to bind covalently to the
antibody. The linker is prepared in a state in which it is
connected to the drug, and the end of the linker has a reactive
group that can be linked to the antibody.
[0047] Examples of the reactive group capable of crosslinking with
an .alpha.-amine group include any reactive groups known in the
art, which can crosslink with the N-terminal .alpha.-amine group of
the heavy chain or light chain of the antibody. Examples of the
reactive group may include isothiocyanate, isocyanate, acyl azide,
NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,
carbonate, aryl halide, imidoester, carbodiimide, anhydride, and
fluorophenyl ester. More preferably, the reactive group is aldehyde
or NHS ester, but not specifically limited thereto. Such reactive
groups can be bound with the amine group by acylation or
alkylation, but are not specifically limited thereto.
[0048] In particular, the antibody-drug conjugate of the present
invention may be an immunoconjugate in which the drug connected
with a linker having a reactive aldehyde group is conjugated to the
N-terminal amino acid residue of the antibody in a site-specific
and number-specific way.
[0049] The reactive aldehyde group is effective in
site-specifically conjugating the drug to the N-terminal amino acid
residue (particularly .alpha.-amine) of the antibody while
minimizing non-specific reactions. The final product produced
through reductive alkylation by an aldehyde bond is much more
stable than that linked by an amide bond. The reactive aldehyde
group has the property of selectively reacting with the N-terminal
.alpha.-amine at low pH. Thus, the conjugate of the present
invention has homogeneous in that the drug is site-specifically
conjugated to the N-terminal .alpha.-amine of the antibody. Thus,
the present invention overcomes the problem of the prior art in
which the uniform efficacy and quality of a drug cannot be
guaranteed because of heterogeneity of the number and site of
conjugations in conventional antibody-drug conjugates, but is not
specifically limited thereto.
[0050] In an example of the present invention, the present
inventors have found that, when a conjugation reaction is carried
out at a pH of 6.0 or lower in order to conjugate a cytotoxic drug
site-specifically to the .alpha.-amine of an antibody, the
conjugation of the cytotoxic drug to the .epsilon.-amine of lysine
residues can be minimized.
[0051] The drug that is used in the present invention may be any
substance that can induce the activation or inhibition of certain
signaling pathways, including cell death, cell proliferation,
immune activation and immune suppression. In particular, the drug
may be a cytotoxic drug or an immunosuppressive agent.
[0052] As used herein, the term "cytotoxic drug" refers to any
substance, for example, a compound, which has a cytotoxic or cell
proliferation inhibitory effect. The term "cytotoxic effect" refers
to the effect of inhibiting or reducing the function of cells to
induce disruption of the cells, and the term "cell proliferation
inhibitory effect" refers to the effect of limiting cell growth
functions such as cell growth or cell proliferation.
[0053] Examples of the cytotoxic drug that is used in the present
invention include chemotherapeutic agents, including microtubule
structure formation inhibitors, meiosis inhibitors, RNA polymerase
inhibitors, topoisomerase inhibitors, DNA intercalators, DNA
alkylators and ribosome inhibitors, protein toxins that can
function as enzymes, and radioisotopes. Examples of the cytotoxic
drug may include maytansinoid, auristatin, dolastatin, tubulysin,
calicheamicin, pyrrolobenzodiazepines, doxorubicin, duocamycin,
carboplatin(paraplatin), cisplatin, cyclophosphamide, ifosfamide,
nidran, [nitrogen mustard(mechlorethamine HCL)], bleomycin,
mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate,
methotrexate, etoposide, vinblastine, vinorelbine, alimta,
altretamine, procarbazine, taxol, taxotere, topotecan, irinotecan,
trichothecene, CC1065, alpha-amanitin, other enediyne antibiotics,
exotoxin, and plant toxin. In addition, compounds include their
stereoisomers and derivatives. Furthermore, the auristatin that is
used in the present invention may be monomethyl auristatin E or
monomethyl auristatin F, but is not limited thereto.
[0054] The term "immunosuppressive agent" refers to any compound
having the effect of reducing immune responses. The term means a
substance that can antagonize immune causing substances or that can
inhibit substances (cytokines such as interleukins) which are
involved in immune responses.
[0055] In an example of the present invention, Trastuzumab,
Lorvotuzumab, Brentuximab and Glembatumumab were used as model
antibodies, and monomethyl auristatin E ((MMAE)) or monomethyl
auristatin F (MMAF) were used as cytotoxic drugs to be conjugated
to the N-terminus of the antibodies (Examples 1 and 2). Trastuzumab
was allowed to react with MMAF or MMAE at a pH of 6.0 to conjugate
the drug to the N-terminus of the antibody, thereby preparing an
antibody-drug conjugate. In the case of this antibody-drug
conjugate, it was shown that the antigen binding activity of the
antibody and the cytotoxic efficacy of the drug were maintained
even after conjugation of the drug (Examples 3 to 5). In
particular, this antibody-drug conjugate showed excellent stability
in human serum in vitro compared to another antibody-drug conjugate
(comparative conjugate) having a cysteine or lysine bond, and also
showed excellent pharmacokinetics in an excellent pharmacokinetic
experiment performed using rats (Example 6). In addition, this
antibody-drug conjugate showed excellent anticancer activity
compared to the comparative conjugate in anticancer animal models,
but showed low toxicity similar to a control group in terms of
weight, hepatotoxicity, blood and the like (Examples 7 and 8).
Furthermore, results similar to the above-described results in
terms of antigen binding activity, cytotoxicity and the like were
obtained even when other drugs such as MMAE were used or when other
antibodies such as Lorvotuzumab, Brentuximab and Glembatumumab were
used (Example 9), indicating that the technology according to the
present invention, in which a drug is conjugated to the N-terminus
of the heavy chain or light chain of an antibody, can become a
platform technology in the preparation of antibody-drug
conjugates.
[0056] In another aspect, the present invention is directed to a
method for preparing the antibody-drug conjugate.
[0057] The antibody-drug conjugate and its components are as
described above.
[0058] Specifically, the method for preparing the antibody-drug
conjugate comprises allowing an antibody to react with a drug
containing a reactive group capable of crosslinking with an
.alpha.-amine group, thereby conjugating the drug to the N-terminal
.alpha.-amine group of the heavy chain or light chain of the
antibody.
[0059] In addition, the method for preparing the antibody-drug
conjugate may further comprise separating the antibody-drug
conjugate from a reaction product including the antibody and the
drug, which did not form the conjugate.
[0060] Specifically, in the preparation method, the antibody and
the drug may be conjugated to each other at a pH of 4.0-6.5, more
specifically 5.5-6.5, even more specifically 6.0. As described
above, the present invention has an advantage in that specific
conjugation between an aldehyde group present in the drug or its
linker and the N-terminal .alpha.-amine of the antibody can occur
at low pH.
[0061] The process of separating the antibody-drug conjugate can be
performed by various methods known in that art. For example, it can
be performed by a chromatographic process including size exclusion
chromatography, but is not specifically limited thereto.
[0062] In still another aspect, the present invention is directed
to a composition comprising the antibody-drug conjugate.
[0063] The composition may be in the form of a pharmaceutical
composition for treating cancer or autoimmune disease, which
comprise the antibody-drug conjugate. In this case, the antibody
may be an antibody that binds specifically to a cancer cell surface
antigen or a surface antigen of a tissue in which autoimmune
disease has occurred. The pharmaceutical composition of the present
invention may further comprise a pharmaceutically acceptable
carrier.
[0064] The antibody, the drug, the cancer cell surface antigen and
the surface antigen of the tissue in which autoimmune disease has
occurred are as described above.
[0065] As used herein, the term "cancer" includes all the kinds of
cancers without limitations, but examples of the cancer may include
esophageal cancer, stomach cancer, large intestine cancer, rectal
cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer,
colon cancer, breast cancer, uterine cervical cancer, endometrial
cancer, ovarian cancer, prostate cancer, testis cancer, bladder
cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer,
connective tissue cancer, skin cancer, brain cancer, thyroid
cancer, leukemia, Hodgkin's disease, lymphoma, and multiple myeloid
blood cancer. A cancer that can be treated depending on the kind of
an antigen specific to the cancer cell surface antigen may be
selected.
[0066] The term "autoimmune disease", as used herein, refers to any
autoimmune disease that is targeted by the antibody-drug conjugate.
Examples of the autoimmune disease include rheumatoid arthritis,
systemic scleroderma, systemic lupus erythematosus, atomic
dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease,
Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome,
chronic thyroiditis, multiple sclerosis, polymyositis, ankylsoing
spondylitis, fibrositis, and polyarteritis nodosa.
[0067] As used herein, the term "pharmaceutically acceptable
carrier" refers to a carrier or diluent that does not impair the
biological activity and characteristics of an administered compound
without irritating an organism. As a pharmaceutically acceptable
carrier in a composition that is formulated as a liquid solution, a
sterile and biocompatible carrier is used. The pharmaceutically
acceptable carrier may be physiological saline, sterile water,
Ringer's solution, buffered saline, albumin injection solution,
dextrose solution, maltodextrin solution, glycerol, ethanol, or a
mixture of two or more thereof. In addition, the composition of the
present invention may, if necessary, comprise other conventional
additives, including antioxidants, buffers, and bacteriostatic
agents.
[0068] The carrier is not limited particularly, but for oral
administration, the composition of the present invention can
comprises binders, lubricants, disintegrants, excipients,
emulsifiers, dispersions, stabilizers, suspending agents, pigments,
perfumes, etc., for injection administration, the composition of
the present invention can comprises buffers, preservatives,
analgesics, emulsifiers, isotonic agents, stabilizers, etc., and
for local administration, the composition of the present invention
can comprises bases, excipients, lubricants, preservatives, etc.,
can be used.
[0069] The inventive composition can be formulated with a
pharmaceutically acceptable carrier as described above in various
manners. For example, for oral administration, the composition of
the present invention can be formulated in the form of tablet,
troche, capsule, elixir, suspension, syrup, wafer, etc., and for
injection administration, the composition can be formulated as a
unit dosage ampoule or a multiple dosage form. The composition can
also be formulated as solution, suspension, tablet, pill, capsule,
sustained-release formulation, etc.
[0070] In the meantime, examples of carrier, excipient or diluent
suitable for the formulation of the composition may include
lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinylpyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate
and mineral oils. In addition, the composition of the present
invention may additionally contain fillers, anti-aggregating
agents, lubricants, wetting agents, perfumes, and
preservatives.
[0071] In addition, the pharmaceutical composition of the present
invention may include any one formulation selected from the group
consisting of tablets, pills, powders, granules, capsules,
suspensions, internal solutions, emulsions, syrups, sterilized
aqueous solutions, non-aqueous solvents, suspensions, emulsions,
lyophilized agents, and suppositories according to a conventional
method.
[0072] In addition, the conjugate may be used in a mixture with
various pharmaceutically acceptable carriers such as physiological
saline or organic solvents. To increase the stability or absorption
property of the conjugate, the conjugate may be used in combination
with carbohydrates such as glucose, sucrose or dextran,
antioxidants such as ascorbic acid or glutathione, chelating
agents, low-molecular-weight proteins, or stabilizers.
[0073] In yet another aspect, the present invention is directed to
a method of treating cancer or autoimmune disease using the
antibody-drug conjugate or the composition. The antibody may be an
antibody that binds specifically to a cancer cell surface antigen,
and the drug may be a drug for treating cancer. In addition, the
antibody may be an antibody that binds specifically a surface
antigen of a tissue in which autoimmune disease has occurred, and
the drug may be a drug for treating autoimmune disease.
[0074] The antibody and the drug are as described above.
[0075] The method may be a method for treatment of cancer or
autoimmune disease, which comprises administering the
pharmaceutical composition to a subject in need of the treatment.
The antibody-drug conjugate and carriers that are used in the
method are as described above.
[0076] The composition may be administered as single or multiple
doses in a pharmaceutically effective amount. In this case, the
composition may be administered in the form of liquid, powder,
aerosol, capsule, enteric-coated tablet, or suppository. The
composition of the present invention can be administered
intraperitoneally, intravenously, intramuscularly, subcutaneously,
transdermally, orally, topically, intranasally, intrapulmonarily or
intrarectally, but is not limited thereto. However, because the
protein antibody is digested when administered orally, the active
ingredient in the composition for oral administration should be
coated or formulated so as to be protected from degradation in the
stomach. In addition, the pharmaceutical composition may be
administered by any device by which the active ingredient may be
delivered to target cells. Furthermore, the pharmaceutical
composition of the present invention may be administered
individually or in combination with other therapeutic agents, and
may be administered sequentially or simultaneously with
conventional therapeutic agents.
[0077] The composition comprising the antibody-drug conjugate of
the present invention is administered in a pharmaceutically
effective amount. As used herein, the term "pharmaceutically
effective amount" refers to an amount sufficient to treat or
prevent the disease at a reasonable benefit/risk ratio applicable
for medical treatment or prevention, and an effective dosage level
can be determined according to the severity of disease, the
activity of the drug, the patient's age, weight, health conditions,
gender, and sensitivity to the drug, time of administration, route
of administration and the discharge rate, duration of treatment,
combination with the composition of the present invention used, or
well-known elements and elements in other medical fields, including
drugs used simultaneously.
[0078] In a further aspect, the present invention is directed to a
method for screening an antibody suitable for use in preparation in
the antibody-drug conjugate.
[0079] In the preparation of the antibody-drug conjugate according
to the present invention, an antibody suitable for use in
effectively preparing the antibody-drug conjugate by conjugating a
drug to the N-terminus (particularly .alpha.-amine) of the antibody
can be screened and selected.
EXAMPLES
[0080] Hereinafter, the present invention will be described in
further detail with reference to examples. It will be obvious to a
person having ordinary skill in the art that these examples are
illustrative purposes only and are not to be construed to limit the
scope of the present invention. Thus, the substantial scope of the
present invention will be defined by the appended claims and
equivalents thereof.
Example 1: Selection of Model Antibodies
[0081] In the preparation of an antibody-cytotoxin conjugate
representative of the antibody-drug conjugate of the present
invention, the anti-HER2 antibody Trastuzumab, the anti-CD56
antibody Lorvotuzumab, the anti-CD30 antibody Brentuximab and the
anti-GPNMB (glycoprotein NMB) antibody Glembatumumab were used as
model antibodies in order to examine whether cytotoxin having a
linker would be site-selectively conjugated to the antibodies.
[0082] The above antibodies were constructed into expression
vectors using known amino acid sequence information, and a stable
cell line was constructed from the CHO cell line. Alternatively,
the antibodies were expressed transiently, incubated and
purified.
Example 2: Synthesis of Toxin
[0083] Monomethyl auristatin F (MMAF) toxin having an aldehyde
linker connected to the end was synthesized (LegoChem Biosciences
or XcessBioscience) (FIG. 1). In addition, in order to examine
whether the N-terminal conjugation method of the present invention
can also be applied to toxins other than MMAF, Monomethyl
auristatin E (MMAE) was synthesized (XcessBioscience, USA).
Example 3: Preparation of Monoclonal Antibody-Cytotoxin
Conjugate
[0084] 3-1: Preparation of Monoclonal Antibody-Drug Conjugate
According to the Present Invention
[0085] An antibody was diluted in 100 mM potassium phosphate buffer
(pH 5.49) and concentrated to about 7.1 mg/ml. Next, MMAF (LegoChem
Biosciences, Korea) connected with a linker having an aldehyde
reactive group was dissolved in 50% DMSO (dimethyl sulfoxide)
solvent to a concentration of 2.5 mg/ml. Thereafter, the prepared
antibody solution and MMAF solution were mixed with each other so
as to achieve the following conditions: final 70 mM potassium
phosphate (pH 6.0); antibody concentration: 5.0 mg/ml; 14% DMSO;
MMAF concentration: 0.3 mg/ml; and the molar ratio between the
.alpha.-amine of the antibody and MMAF: about 1:2.3 (or the molar
ratio between the antibody and MMAF is 1:9). NaCNBH.sub.3 (Sigma,
USA) was added to the reaction solution to a final concentration of
20 mM, and then reacted at 4.degree. C. for 12 hours with gentle
stirring. To separate unreacted antibody and unreacted MMAF
connected with the linker, a Sephadex G-25 column (GE Healthcare,
USA) or a resource phenyl column (Resource Phe, GE Healthcare, USA)
was used. According to this process, a conjugate was prepared in
which about three MMAF toxin molecules per antibody molecule were
selectively conjugated to the amino terminus of the antibody (FIG.
2).
[0086] 3-2: Preparation of Control Antibody-Drug Conjugate
[0087] According to a conventional technology, a control
antibody-drug conjugate was prepared by cysteine conjugation
(Thiomab (HC-A114C)+Mal-C6-MMAF), thiol conjugation (Mal-C6-MMAF)
or lysine conjugation (SMCC linker, SH-C6-MMAF).
[0088] To prepare a thiol-conjugated antibody, an antibody was
reduced with TCEP at a pH of 8.0, and then Mal-C4-MMAF was added
thereto and allowed to react at 0.degree. C. for 3 hours. After the
reaction, thiol was added to the reaction product which was then
further reacted. After termination of the reaction, replacement
with 1.times.PBS using a G25 desalting column (GE healthcare, USA)
was performed to complete the reaction.
[0089] To prepare a cysteine-conjugated antibody, cysteine in a
purified antibody was activated, and then Mal-C6-MMAF was added
thereto, and conjugation was performed according to a process
similar to that used in the preparation of the thiol-conjugated
antibody.
[0090] A conjugate comprising a lysine-conjugated antibody was
prepared with reference to International Patent Publication No.
WO2005037992 (Immunogen). First, an antibody was reacted with an
SMCC linker, and unreacted SMCC was removed by buffer exchange. The
antibody-SMCC conjugate was reacted with SH-C4-MMAF (Concortis
bioscience, USA) containing a thiol group, thereby preparing an
antibody-SMCC-MMAF conjugate.
[0091] The antibody-cytotoxin conjugates prepared by .alpha.-amine
conjugation according to the present invention are summarized in
Table 1 below.
TABLE-US-00001 TABLE 1 Antibody-drug conjugates Conjugate name
(when Conjugation Trastuzumab and MMAF type Conjugation conditions
are used) Cys Thiomab (HC A114C), Mal-C6- Thiomab-MMAF conjugation
MMAF Thiol Mal-C6-MMAF T-C-MMAF conjugation Lys SMCC linker,
SH-C6-MMAF T-K-MMAF conjugation Amine ALD-C6-MMAF (weakly acidic
T-N-MMAF conjugation pH)
[0092] The four conjugates prepared as described above were
analyzed to determine the DAR (drug antibody ratio) and the site of
conjugation. The analysis was performed by LC-MS and peptide
mapping.
Example 4: Physicochemical Properties and Biological Properties
[0093] 4-1: Analysis of Molecular Weight
[0094] The molecular weights of the antibody-drug conjugates
(T-N-MMAF) were determined by LC-MS analysis. The theoretical
molecular weight of the drug (MMAF) used is 824.54 Da, and the
molecular weight of Trastuzumab is 145 kDa. Thus, the conjugation
of the drug to the antibody and the number of drug moieties
conjugated to one antibody molecule could be simultaneously
determined by mass spectrometry.
[0095] To determine the DAR of T-N-MMAF prepared in Example 3, the
molecular weight of T-N-MMAF was analyzed by LC/MS. The prepared
sample was treated with PNGaseF to remove sugar chains, and then
separated through an ACQUITY UPLC BEH 200 SEC column, after which
the sample was injected into the Waters Synapt G2-S system to
analyze the mass. The results of the analysis are shown in FIG.
3.
[0096] As a result, as shown in FIG. 3, chemical species ranging
from a chemical species (D0) having no drug moiety conjugated
thereto to a chemical species (D7) having 7 drug moieties
conjugated thereto were detected, and the number of drug moieties
conjugated was determined based on whether the difference in
molecular weight between peaks was consistent with or similar to
the molecular weight of the drug. The relative intensities of the
drug moieties are shown in Table 2 below. The DAR was calculated as
the weighted average of the chemical species and was DAR=3.2.
TABLE-US-00002 TABLE 2 No. of Relative bound drug Mass (Da)
intensity(%) Da 0 145179.5 1.8 -- 1 146005.3 10.7 825.8 2 146833.2
21.2 827.9 3 147661.1 25.4 827.9 4 148488.9 21.0 827.8 5 149317.0
12.5 828.1 6 150145.5 5.0 828.5 7 150964.2 2.3 818.7 DAR 3.219 DAR
= Sum(Intensity(%) .times. No. of Drug/100) Drug moiety mass: 828
Da
[0097] 4-2: Site of Conjugation of Drug
[0098] The site of conjugation of the drug in the prepared T-N_MMAF
conjugates was determined by peptide mapping. T-N-MMAF ADC (having
a DAR of 3.2) prepared in Example 3 was treated with Rapigest
(Waters), and then treated with trypsin (Roche) to make fragments.
The reaction product was separated through an ACQUITY UPLC PST
(BEH) C18 column, and the separated peaks were subjected to mass
spectrometry through the Waters Synapt G2-S Q/TOF system to
determine the sequence of the reaction product. The results of the
analysis are shown in FIG. 4.
[0099] As a result, as shown in FIG. 4, peaks which were not found
in the non-conjugated parent antibody were detected in the
chromatogram. The results of mass spectrometry indicated that the
fragments were the N-terminus of the heavy chain, the N-terminus of
the light chain, a portion of the heavy chain, and other small
fragments. The ratios of the fragments are shown in Table 3 below.
Thus, it can be seen that 75% of the drug was conjugated to the
N-terminus and 92% of the drug was selectively conjugated to the
N-terminus and the heavy-chain CH2 region, which can be clearly
defined.
TABLE-US-00003 TABLE 3 Trypsin fragment Ratio Heavy
EVQLVESGGGLVQPGGSLR (SEQ ID NO: 1) 46% chain-N- terminus Light
DIQMTQSPSSLSASVGDR (SEQ ID NO: 2) 29% chain-N- terminus Heavy
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR 17% chain-CH2 (SEQ ID NO: 3)
Others -- 8%
[0100] 4-3: Analysis of Purity
[0101] To determine the aggregate content of the prepared T-N-MMAF
conjugate, purity analysis of the conjugate was performed by
SE-HPLC and SDS-PAGE analysis. Size exclusion chromatography was
used in a TSK-Ge13000SWXL column using PBS as a mobile phase, and
SDS-PAGE was performed using 4-12% Novex NuPAGE gel. The results of
the chromatography are shown in FIG. 5.
[0102] As a result, as shown in FIG. 5, the purity of the monomer
was 98.8%, which is suitable for an efficacy test, and
fragmentation or cross-linking was not detected.
[0103] 4-4: Antigen Binding Activity
[0104] In order to examine the antigen binding activity of the
antibody is maintained even after the drug was conjugated thereto,
the antigen binding activity of the drug-conjugated antibody was
measured by a method of measuring surface plasmon resonance using
Biacore.TM.. As a control antibody, a natural antibody was used.
The antigen (ErbB2) binding activity was analyzed using Biacore
T200, and each antibody was immobilized on a CM5 sensor chip (GE
healthcare, USA) using an amine coupling kit, after which kD (M)
was calculated by measuring and analyzing on/off rate while
injecting ErbB2 at concentrations of 50, 16.67, 5.56, 1.85, 0.62
and 0.21 nM and at a rate of 30 uL/min.
TABLE-US-00004 TABLE 4 Antigen binding activity Samples Test 1
(10.sup.-10 M) Test 2 (10.sup.-10 M) Average (10.sup.-10 M)
Trastuzumab 1.3 2.2 1.8 T-N-MMAF 1.2 0.9 1.1 (DAR 1.6) T-N-MMAF 1.1
0.7 0.9 (DAR 3.2)
[0105] As a result, as summarized in Table 4 above, it was found
that an antigen binding activity of about 0.1 nM similar to that of
the natural antibody was maintained even after drug conjugation
regardless of the DAR.
Example 5: In Vitro Cytotoxicity Analysis
[0106] In order to examine the in vitro efficacy of the prepared
antibody-cytotoxin conjugate, an anti-proliferation assay was
performed using BT474, HCC1954, SKOV-3, JIMT-1 cell lines which are
HER2-expressing tumor cell lines. Each of the cell lines was
cultured and suspended at a concentration of 1.times.10.sup.5
cells/ml, and 100 of the suspension was loaded into each well of a
96-well plate. The cells were incubated in an incubator for 3
hours, and then 100 of the antibody-cytotoxin conjugate diluted to
various concentrations was added to each well of the plate and
incubated in an incubator for 4 days. A 1:10 dilution of CCK-8
(Dojindo) was added to each well of the plate, which was then
covered with a foil and incubated in an incubator for 2-5 hours.
Next, the absorbance of each well at 450 nm was measured using a
SpectraMax 190 microplate reader (Molecular Device).
TABLE-US-00005 TABLE 5 Cytotoxicity (IC.sub.50 (pM)) T-N-MMAF
T-C-MMAF T-K-MMAF Cell line (DAR 3.2) (DAR 3.6) (DAR 3.9) HCC1954
40 22 45 SKOV-3 104 59 N.D. JIMT1 253 98 727 BT474 116 49 77
[0107] As a result, as shown in Table 5 below, T-N-MMAF showed
cytotoxicity slightly lower than T-C-MMAF in all the four cancer
cell lines, but a significant decrease in cytotoxicity, which can
influence in vivo efficacy, was not observed.
Example 6: Stability Test
[0108] 6-1: Stability in Human Serum In Vitro
[0109] Using T-N-MMAF prepared in Example 3 and control antibodies,
including a natural antibody, T-C-MMAF and Thiomab-MMAF, a
stability test in human serum in vitro was performed. The
antibody-cytotoxin conjugate was buffer-exchanged with 1.times.PBS
and concentrated to 3.33 mg/ml, and then mixed with human serum
(Sigma, USA) at a ratio of 1:9 (v/v) and allowed to stand at
37.degree. C. for 7 days. After 7 days, to remove proteins other
the stored sample was treated with MabSelectSure (GE healthcare,
USA) than the antibody-cytotoxin conjugate contained in the sample
in order to minimize interference in LC/MS analysis. The stability
of the conjugate in human serum in vitro was analyzed by LC/MS, and
the results of the analysis are shown in Table 6 below.
TABLE-US-00006 TABLE 6 Relative content of Relative monoclonal
content of Relative antibody conjugate content of DAR (7 days of (7
days of (7 days of Samples storage, %) storage, %) storage, %)
Trastuzumab 90.0 -- -- T-N-MMAF 89.3 90.5 101.4 T-C-MMAF 49.2 32.3
65.6 Thiomab-MMAF 69.9 59.5 85.1
[0110] As a result, as can be seen in Table 6 above, the changes in
the content and DAR of T-N-MMAF compared to the control natural
antibody after 7 days of storage were not observed. However, in the
case of T-C-MMAF and Thiomab-MMAF, which are the comparative
antibody-drug conjugates, decreases in the total antibody content
and the DAR could be observed.
[0111] 6-2: Rat Pharmacokinetics (PK)
[0112] In vivo stability was compared and analyzed through a rat
pharmacokinetic experiment. Each of three ADCs (T-K-MMAF, T-C-MMAF,
and T-N-MMAF) and Trastuzumab were injected intravenously once to
female Sprague-Dawley rats at a dose of 2.5 mg/kg. At 0.05, 0.5, 1,
6, 24, 72, 168, 240 and 336 hours after administration of the
substances, blood was collected. A total antibody assay of
analyzing all antibodies binding to ErbB2 in blood and a conjugated
antibody assay of analyzing an antibody maintaining drug
conjugation were performed by an ELISA method.
[0113] The total antibody content was analyzed by an ELISA method
as follows.
[0114] A 96-well microplate was coated with ErbB2 (R&D
systems), and then a sample was added to the plate and incubated at
a temperature of 37r for 1 hour. The plate was washed with PBST to
remove all non-fixed substances, and then the absorbance of the
plate at 450 nm was measured using HRP-conjugated anti-human kappa
light chain antibody and 3,3',5,5'-tetramethylbenzidine (TMB,
Sigma, 10440), thereby determining the total antibody content of
the sample.
[0115] The conjugated antibody assay was performed by a method
similar to the above-described method. Specifically, a 96-well
microplate was coated with anti-MMAF antibody (Young In Frontier),
and then a sample was added to the plate and incubated at a
temperature of 37r for 1 hour. Next, biotinylated ErbB2
(ACROBIOSYSTEMS, USA), streptavidin-HRP and TMB were sequentially
added to the plate to develop color, and then the absorbance of the
plate at 450 nm was measured to determine the concentration of the
conjugated antibody. The results of the measurement are shown in
FIGS. 6 to 8 and Table 7 below.
TABLE-US-00007 TABLE 7 PK parameters measured after administering
ADCs to rats at a dose of 2.5 mg/kg Treatment (n = 5, each) Total
Ab Conjugated Ab 2-compartment AUC T.sub.1/2 C.sub.max AUC
T.sub.1/2 C.sub.max modeling (hr*.mu.g/ml) (hr) (.mu.g/ml)
(hr*.mu.g/ml) (hr) (.mu.g/ml) Trastuzumab 6964.9 115.7 128.1 -- --
-- T-N-MMAF 6795.7 122.1 111.2 6813.2 118.3 112.4 T-C-MMAF 5933.5
111.6 94.3 4315.4 84.6 93.7 T-K-MMAF 4324.3 65.1 157.5 3781.7 53.2
190.0
Example 7: Test for Anticancer Effect in Anticancer Model
Animals
[0116] In order to examine the efficacy of three ADCs prepared by
different techniques and the difference in efficacy by
drug-antibody ratio (DAR), an in vivo efficacy test was performed
in breast cancer (HCC 1954) xenograft models using nude rats.
[0117] Each of four ADCs, that is, T-N-M (DAR: about 1.6 and 3.2),
T-C-M (DAR: about 3.7) and T-K-M (DAR: about 3.9), was administered
intravenously once to HCC1954 cell-transplanted rats at a dose of 1
mg/kg, and then the degree of inhibition of growth of the
transplanted tumor was compared between the test groups. The
results are shown in FIGS. 9 and 10.
[0118] As a result, as shown in FIGS. 9 and 10, the antibody
according to the present invention had an excellent anticancer
effect compared to the control and comparative antibodies.
Example 8: Toxicity Test
[0119] In order to examine whether stability varying depending on
the technique for preparation of ADCs influences toxicity, a
single-dose toxicity test was performed using SD rats. Each of
three ADCs was administered intravenously once at a high dose of
200 mpk. As comparative groups, an antibody alone and MMAF were
administered at a dose of 200 mpk. The weight was measured everyday
during a period ranging from the time point of administration of
the test substance to the end of the test (day 12). Biochemical
analysis of the blood was performed at 5 days after administration.
Measurement items were AST and ALT for determining hepatotoxicity
and typical hematological toxicity, neutrophils and platelets.
[0120] 8-1: Change in Weight
[0121] The results of measurement of changes in the weight are
shown in FIG. 11. As shown in FIG. 11, the T-C-MMAF and T-K-MMAF
groups showed a distinct decrease in the weight compared to the
T-N-MMAF group and other groups. In particular, in the case of the
group administered with T-C-MMAF, all animals excluding one animal
did die after day 8.
[0122] 8-2: Biochemical Analysis (Hepatotoxicity)
[0123] In order to examine whether the ADCs cause hepatotoxicity,
biochemical analysis of blood collected at day 5 after
administration of the ADCs was performed. The analysis was
performed using the Au480 clinical analyzer (Beckman Coulter, USA),
and the levels of AST (aspartate aminotransferase) and ALT (alanine
aminotransferase) indicative of hepatotoxicity were measured. The
results of the measurement are shown in FIG. 12.
[0124] As a result, as shown in FIG. 12, it could be observed that
the T-N-MMAF group according to the present invention showed no
significant difference from other control groups including PBS,
indicating that it did not caused abrupt or serious hepatotoxicity.
However, a significant increase in AST and ALT was observed in the
T-C-MMAF and T-K-MMAF groups, indicating that administration of the
drugs caused hepatotoxicity.
[0125] 8-3: Hematological Analysis (Neutropenia and
Thrombocytopenia)
[0126] Because the major clinical toxicities of currently approved
ADCs indicate the hematological properties, hematological analysis
of blood collected at day 5 after administration of the ADCs was
performed using the Hemavet 950 FS hematological analyzer (Drew
Scientific Inc., USA). The results of the analyzer are shown in
FIG. 13.
[0127] As a result, as shown in FIG. 13, the T-N-MMAF group showed
no significant change in the number of neutrophils compared to the
control groups including PBS, suggesting that T-N-MMAF did not
cause abrupt and serious hematological toxicity. However, the
T-C-MMAF group showed a significant decrease in the number of
neutrophils, and the T-K-MMAF group showed a significant increase
in the number of neutrophils, which decreased immediately after
administration and then increased. Thus, for these two groups, it
could be concluded that abrupt hematological toxicity was caused by
administration of the drugs.
[0128] The number of platelets was noticeably smaller than in the
T-N-MMAF group than in other control groups including PBS. However,
the T-C-MMAF and T-K-MMAF groups showed a significant decrease in
the number of platelets, indicating that abrupt toxicity was caused
by administration of the drugs.
Example 9: Examination of Platform Function
[0129] Whether the method for preparing the antibody-drug conjugate
according to the present invention can be applied to various
antibody-drug conjugates was examined. For this, the method was
applied to various drugs or antibodies and various antibody forms
in order to examine the function thereof.
[0130] 9-1: Examination of Function According to Type of Drug
[0131] In order to determine whether the method for preparing the
antibody-drug conjugate according to the present invention can be
applied to various drugs, N-terminal conjugation of various drugs
was performed using Trastuzumab as a model antibody. Specifically,
two drugs (MMAF and MMAE) were used, and the results obtained using
MMAF are as described in the Examples above. Antibody-drug
conjugates were prepared according to the method described in
Example 1, and the DAR analysis, in vitro stability and rat PK of
the prepared antibody-drug conjugates were performed according to
the methods described in the Examples above.
[0132] 9-1-1: Preparation of T-N-MMAE
[0133] According to a conjugate between MMAE (XcessBioscience, USA)
and an antibody was prepared. To determine the DAR of the
conjugate, the molecular weight of the conjugate was analyzed by
LC/MS, and the results of the analysis are shown in FIG. 14 and
Table 8 below.
TABLE-US-00008 TABLE 8 Chemical species distribution by DAR of
T-N-MMAE and average DAR No. of Relative Delta drug Mass (Da)
content (%) mass D0 N/D N/D D1 146061.05 6.3 D2 146863.77 14.3
802.72 D3 147672.80 23.3 809.03 D4 148484.33 24.2 811.53 D5
149297.30 16.5 812.97 D6 150112.25 8.6 814.95 D7 150922.84 6.8
810.59 DAR 3.83
[0134] 9-1-2: Analysis of Stability of T-N-MMAE in Human Serum
[0135] According to the method of Example 6, the stability of
T-N-MMAE ADC in serum was evaluated. The concentration of ADC in
each sample was measured by the total antibody assay using ELISA,
and a change in the DAR was measured by LC/MS.
TABLE-US-00009 TABLE 9 .mu.g/ml % DAR % Day 0 364.8 100% 3.17 100%
Day 3 346.9 95% 3.33 105% Day 7 294.8 81% 3.28 103%
[0136] 9-1-3: Rat PK of T-N-MMAE
[0137] In order to evaluate the in vivo stability of the prepared
MMAE conjugate, a PK study in SD rats was performed according to a
method similar to that of Example 6. Shortly, 2.5 mg/pk of the ADC
was administered to female SD rats. At 12 min, 30 min, 1 hour, 6
hour, 24 hours, 3 days, 7 days, 10 days, 14 days, 17 days and 21
days after administration of the ADC, blood was collected from the
rats, and the concentrations of total protein and conjugated
antibody in the blood were measured according to the
above-described methods using an ELISA technique.
TABLE-US-00010 TABLE 10 Rat PK parameters of T-N-MMAE AUC
Conjugate/ half-life Conjugate/ Group (hr*.mu.g/ml) Total ratio
(hr) Total ratio trastuzumab 5868.83 169.6 T-N-MMAF (T) 6688.95
191.8 T-N-MMAF (C) 6871.71 103% 203.3 106% T-N-MMAE (T) 5639.24
173.9 T-N-MMAE (C) 5690.96 101% 163.7 94% * Trastuzumab and
T-N-MMAF were included for comparison between tests.
[0138] As a result, as shown in FIG. 15 and Table 10 above,
T-N-MMAE showed the profiles of total antibody and conjugated
antibody, which did not significantly differ from that of the
parent antibody, suggesting that the antibody-drug conjugate
prepared using MMAE has stability similar to that of the
antibody-drug conjugate prepared using MMAF.
[0139] 9-1-4: Activity of T-N-MMAE
[0140] In order to determine the biological activity of the
prepared MMAE conjugate, the activity thereof was measured using
four different tumor cell lines. The results of the measurement are
shown in Table 11 below. The method used was similar to that used
in Example 5.
TABLE-US-00011 TABLE 11 Cytotoxicity of T-N-MMAE for
HER2-expressing tumor cell lines IC.sub.50[nM] #1 #2 Average
HCC1954 0.39 0.26 0.33 SKOV-3 3.04 2.62 2.83 JIMT1 4.00 3.51 3.76
BT474 0.56 0.70 0.63
[0141] As a result, the measured IC50 was in the range of 0.33-3.76
nM, which was similar to the activity (0.47 nM) of the BT474 cell
line against the Trastuzumab/MMAE thiol conjugate reported in the
literature. This suggests that the method for selective conjugation
to the N-terminal .alpha.-amine according to the present invention
can also be applied to other types of drugs.
[0142] 9-2: Examination of Function According to Type of
Antibody
[0143] In order to examine whether the method for preparing the
antibody-drug conjugate according to the present invention can be
applied to various antibodies, N-terminal conjugation to three
anticancer antibodies (Brentuximab, Lorvotuzumab, Glembatumumab)
was performed, and the DAR and in vitro stability of the conjugates
were measured.
[0144] 9-2-1: Brentuximab
[0145] 9-2-1-1: Preparation of Brentuximab-N-MMAF
[0146] Using Brentuximab expressed from the CHO cell line,
Brentuximab-N-MMAF (B-N-MMAF) was prepared according to the method
of Example 3. The prepared ADC showed the LC/MS profile shown in
FIG. 16 and Table 12 below. In the ADC, chemical species ranging
from D0 to D6 were detected, and the DAR was calculated to be
2.90.
TABLE-US-00012 TABLE 12 No. of Relative Delta mass bound drug Mass
(Da) content (%) (Da) D0 145208.6 3.1 D1 146034.9 14 826.3 D2
146863.3 24.4 828.4 D3 147692 26.4 828.7 D4 148520.7 18.2 828.7 D5
149349.7 9.3 829 D6 150177.5 4.7 827.8 DAR 2.90
[0147] 9-2-1-2: Ligand Binding Assay
[0148] In order to determine whether the properties of the antibody
are changed by conjugation, the activity of binding of the antibody
to an antigen was measured by an ELISA technique. Specifically, 100
.mu.g of the antigen CD30 (R&D Systems) was coated on a 96-well
microplate, and then blocked with 1% BSA at 37.degree. C. for 1
hour. After the blocking solution was removed, a sample was added
to the plate and incubated at 37.degree. C. for 1 hour. The plate
was washed five times with PBST (PBS+0.05% tween 20), and then a
1000-fold dilution of HRP-conjugated anti-human kappa light-chain
antibody was added to the plate and incubated at 37.degree. C. for
1 hour. The plate was washed five times with PBST, and then TMB
(Sigma) was added to the plate which was then subjected to color
development for 10 minutes. 1N H.sub.2SO.sub.4 was added to the
plate to stop the reaction, and then the absorbance of the plate at
450 nm was measured. The results of the measurement are shown in
FIG. 17. In FIG. 17, the line indicated by .largecircle. indicate
results for non-conjugated Brentuximab, the line indicated by
.diamond. indicates results for B-N-MMAF having a DAR of 2.90, and
the line indicated by .DELTA. indicates results for B-N-MMAF having
a DAR of 4.22. As can be seen from the results, the activity of
binding of the antibody to the antigen did not change even after
conjugation regardless of the DAR value.
[0149] 9-2-1-3: In Vitro Cytotoxicity
[0150] To determine the in vitro efficacy of the prepared
antibody-cytotoxin conjugate, an anti-proliferation assay was
performed using Karpas-299 and L-540 cell lines that are
CD30-expressing cell lines.
[0151] Specifically, each of the cell lines was cultured and
suspended at a concentration of 1.times.10.sup.5 cells/ml, and 100
of the suspension was loaded into each well of a 96-well plate. The
cells were incubated in an incubator for 3 hours, and then 100 of
the antibody-cytotoxin conjugate diluted to various concentrations
was added to each well of the plate which was then incubated in an
incubator for 4 days. A 1:10 dilution of CCK-8 (Dojindo) was added
to each well of the plate, which was then covered with a foil and
incubated in an incubator for 2-5 hours. Next, the absorbance of
each well at 450 nm was measured using a SpectraMax 190 microplate
reader. The results of the measurement are shown in Table 13
below.
TABLE-US-00013 TABLE 13 Cell line IC.sub.50(pM) Karpas-299 32.2
L-540 37.1
[0152] As a result, a cytotoxicity lower than 40 pM was observed in
all the two cell lines (Karpas-299 and L-540).
[0153] 9-2-2: Lorvotuzumab
[0154] 9-2-2-1: Preparation of Lorvotuzumab-N-MMAF
[0155] Using Lorvotuzumab expressed transiently from CHO cells,
Lorvotuzumab-N-MMAF (L-N-MMAF) was prepared according to the method
of Example 3. As a result, the prepared ADC showed the conjugation
profile shown in FIG. 18 and Table 14 below, and the DAR of the
conjugate was determined to be 3.33.
TABLE-US-00014 TABLE 14 No. of Relative Delta mass bound drugs Mass
(Da) content (%) (Da) D0 147001.5 3.3 D1 147830.8 10.8 829.3 D2
148657.9 18.6 827.1 D3 149486.6 22.7 828.7 D4 150315.4 20.1 828.8
D5 151144.7 13.7 829.3 D6 151973.5 7 828.8 D7 152803 3.7 829.5 DAR
3.329
[0156] 9-2-2-2: Ligand Binding Assay
[0157] In order to determine whether the properties of the antibody
are changed by conjugation, the activity of binding of the antibody
to an antigen before and after conjugation was measured by an ELISA
technique. Specifically, 100 .mu.g of the antigen CD30 (R&D
Systems, 2408-NC-050) was coated on a 96-well microplate at a
concentration of 1 pg/ml, and then blocked with 1% BSA at
37.degree. C. for 1 hour. After the blocking solution was removed,
a test sample was added to the plate and incubated at 37.degree. C.
for 1 hour. The plate was washed five times with PBST (PBS+0.05%
tween 20), and then a 1000-fold dilution of HRP-conjugated
anti-human kappa light-chain antibody was added to the plate and
incubated at 37.degree. C. for 1 hour. The plate was washed five
times with PBST, and then TMB (Sigma) was added to the plate which
was then subjected to color development for 10 minutes. 1N
H.sub.2SO.sub.4 was added to the plate to stop the reaction, and
then the absorbance of the plate at 450 nm was measured. The
results of the measurement are shown in FIG. 19. In FIG. 19, the
line indicated by .smallcircle. indicate results for the antibody
not conjugated to the drug, the line indicated by .DELTA. indicates
results for L-N-MMAF having a DAR of 2.5, and the line indicated by
.diamond. indicates results for L-N-MMAF having a DAR of 3.3. As
can be seen from the results, the activity of binding of the
antibody to the antigen was maintained regardless of the DAR
value.
[0158] 9-2-2-2: In Vitro Cytotoxicity
[0159] To determine the in vitro efficacy of the prepared
antibody-cytotoxin conjugate, an anti-proliferation assay was
performed using an OPM-2 cell line. Specifically, the cell line was
cultured and suspended at a concentration of 1.times.10.sup.5
cells/ml, and 100 of the suspension was loaded into each well of a
96-well plate. The cells were incubated in an incubator for 3
hours, and then 100 of the antibody-cytotoxin conjugate diluted to
various concentrations was added to each well of the plate which
was then incubated in an incubator for 4 days. A 1:10 dilution of
CCK-8 (Dojindo) was added to each well of the plate, which was then
covered with a foil and incubated in an incubator for 2-5 hours.
Next, the absorbance of each well at 450 nm was measured using a
SpectraMax 190 microplate reader. The results of the measurement
are shown in Table 15 below.
TABLE-US-00015 TABLE 15 IC.sub.50 [nM] OPM-2 L-N-MMAF DAR 2.5 52.9
L-N-MMAF DAR 3.3 41.9
[0160] As can be seen in Table 15 above, the L-N-MMAF antibody
according to the present invention showed a cytotoxicity of about
42-53 nM.
[0161] 9-2-3: Glembatumumab
[0162] 9-2-3-1: In Vitro Cytotoxicity
[0163] To determine the in vitro efficacy of the prepared
antibody-cytotoxin conjugate, an anti-proliferation assay was
performed using a SK-MEL-2 cell line that is a skin cancer cell
line. Specifically, the cell line was cultured and suspended at a
concentration of 1.times.10.sup.5 cells/ml, and 100 of the
suspension was loaded into each well of a 96-well plate. The cells
were incubated in an incubator for 3 hours, and then 100 of the
antibody-cytotoxin conjugate diluted to various concentrations was
added to each well of the plate which was then incubated in an
incubator for 4 days. A 1:10 dilution of CCK-8 (Dojindo) was added
to each well of the plate, which was then covered with a foil and
incubated in an incubator for 2-5 hours. Next, the absorbance of
each well at 450 nm was measured using a SpectraMax 190 microplate
reader. The results of the measurement are shown in Table 16
below.
TABLE-US-00016 TABLE 16 SK-MEL-2 IC.sub.50(nM) G-N-MMAF DAR 2.2
5.47 G-N-MMAF DAR 3.4 3.36
[0164] As can be seen in Table 16 above, the G-N-MMAF according to
the present invention showed a cytotoxicity of about 3-5 nM.
[0165] The above-described results suggest that a new platform of
the antibody-drug conjugate prepared by site-specific conjugation
of the drug to the N-terminal amino acid residue of the heavy chain
or light chain of the antibody shows no reduction in the target
specificity of the antibody while having high stability and also
that the therapeutic effect of the antibody can be doubled by the
drug conjugated thereto.
[0166] From the foregoing, it will be understood by those skilled
in the art to which the present invention pertains that the present
invention can be carried out in other concrete embodiments without
changing the technical spirit or essential feature thereof. In this
regard, it should be understood that the aforementioned examples
are of illustrative in all aspects but not is limited. The scope of
the present invention should be construed to include the meaning
and scope of the appended claims, and all the alterations and
modified forms which are derived from the equivalent concept
thereof, rather than the detailed description.
Sequence CWU 1
1
3119PRTArtificial SequenceTrypsin fragment (heavy chain N-term)
1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg 218PRTArtificial SequenceTrypsin fragment
(light chain N-term) 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg 333PRTArtificial
SequenceTrypsin fragment (heavy chain-CH2) 3Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg
* * * * *