Note: Descriptions are shown in the official language in which they were submitted.
ANTI-HUMAN NGF ANTIBODIES AND METHODS USING SAME
Field of the invention
The invention concerns anti-NGF antibodies (such as anti-NGF antagonist
antibodies). The
invention further concerns the usage and/or application of the anti-NGF
antibody and/or its antigen
binding fragments.
Back2round of the invention
Nerve growth factor (NGF) is the frst discovered neurotrophin, and its role
includes both neuron
nutrition and neurite growth prompting. NGF plays an important role in
regulating the development,
.. differentiation, growth, regeneration and expression of central and
peripheral neurons. There are
three subunits (a, (3 and y) in NGF molecules. (3 subunits are the active
subunits of NGF, and their
function are nerve regeneration and repair. By now, there have been two NGF
receptors discovered:
TrkA tyrosine kinase receptor (high affinity NGF receptor) and p75 common
neurotrophin receptor
(p75NTR, also called low affinity NGF receptor).
Although the primary function of NGF is to promote the survival and
differentiation of neurons,
more and more studies have shown that NGF is related to the persistent or
chronic pain. In 1993, it
was reported that exogenous NGF could induce pain in rats (Lewin GR et al., J
Neurosci, 1993,
13:2136-2148). After that, it was found that intravenous NGF in human could
induce systemic
muscle pain, and local administration also could induce hyperalgesia and
abnormal pain at the
.. injection site (petty BG et al., Ann Neurol, 1994, 36:244-246). Other
studies have shown that NGF
upregulates the expression of neuropeptides in sensory neurons. After binding
to TrkA and p75NTR
receptors, NGF can increase pain response by up regulating the expression of a
sensory neuron -
nociceptor, which makes neurons more sensitive to potential pain stimulation
(Holmes D, Nat Rev
drug discov, 2012, 11:337-338). At present, it has been confirmed the hyper-
expression of NGF /
TrkA in articular cartilage of degenerative arthritis patients, and the
increased expression level of
NGF in rheumatic arthritis and interstitial cystitis patients. Thus,
development of monoclonal
antibodies targetting NGF to inhibit its function is expected to play an
active role in the prevention,
diagnosis and treatment of various pain related diseases.
There are tens of millions of patients suffering from chronic pain worldwide,
and the number is
increasing with the population. At present, the drugs of treatment of chronic
pain include
non-steroidal anti-inflammatory drugs, anticonvulsant drugs, and opioids et
al. However, these drugs
have been found they have limited efficacy in clinical applications and severe
side effects to pateints
health. For example, besides limited pharmacological efficacy, non-steroidal
anti-inflammatory
Date Recue/Date Received 2020-06-17
drugs' side effects includ gastrointestinal hemorrhage and nephrotoxicity,
while the side effect of
opioids is addiction. Thus, there is an unmet clinical need for non-opioid,
non-toxic and
not-easy-abuse new drugs for treatment of acute and chronic pain. Antagonist
antibodies tatrgetting
NGF has regarded as of great importance for new generation of anti-pain drug
development. By now,
there have been several anti-human NGF antibodies in different stage of
research and development.
Tanezumab (from Pfizer/Lilly company) and Fasinumab (from Regeneron/Sanofi
company) are the
two most advanced antibody candidates. Tanezumab is the first anti-NGF
antibody developed for
non-opiods based pain treatment. Tanezumab has shown efficacy over a wide
range of clinical trials.
It has been shown that Tanezumab had highly potent efficacy and analgesic
effect on joint pain,
chronic low back pain and bladder pain associated with interstitial cystitis
(Lane ne et al., n Engl J
Med, 2010, 363: 1521-1531). At present, Tanezuma is undergoing phase III
clinical trials of
indications such as osteoarthritis, back pain, cancer pain, etc. According to
the data of phase II / III
clinical study on the treatment of osteoarthritis pain with fasinumab, the
patients with the four doses
of fasinumab treatment group achieved significant improvement in pain relief.
Aditionally, the
clinical trials data of several NGF antibodies also showed that the NGF
antibodies may have limited
efficacy in severe ill patients, and limited dosage and administration time-
period for other patients,
which suggests the need of more prescise safety exploration of NGF antibodies.
Since NGF is a very important factor in the development of neurons, the amount
of NGF in
neurons should be taken into careful consideration when developing NGF
antibodies. On the one
hand, the effective dosage of antibody drugs depends on both the
neutralization activity of antigens
and the amount of antigens in the body, as the increasment of neutralization
activity is related to the
decrease of dosage. NGF antibody research needs to discovery novel CDR regions
with different
affinity for different epitopes or the same epitope. The difference in the
immunogenicity of varies
CDRs result in different antibody tolerance speed and toxicity, which directly
affects the efficacy.
On the other hand, the immune response of the subjects to the antibody itself
will result in the
formation of immune complex, which will change the pharmacokinetics of the
antibody, produce
unwanted allergic reaction, and finally affect the efficacy of the antibody.
Compared with mouse
antibody and chimeric antibody, humanized antibody has the lowest immune
response to human
immune system.The humanized antibody has a similar half-life with natural
human antibody, thus
ensuring less drug delivery frequency and lower dosage. Therefore, it is of
high importance for
developing an NGF antibody with good efficacy and safety window to treat or
prevent various
diseases related to NGF.
2
Date Recue/Date Received 2020-06-17
Brief summary of the invention
The invention disclosed herein concerns a safe and reliable anti-human NGF
antibody and/or an
antigen-binding fragment targeting huaman NGF. The antibody and/or its antigen-
binding fragment
can antagonize the binding of NGF to its receptor (NGF receptor) with high
specificity. Therefore,
the anti-human NGF antibody and/or its antigen-binding fragment provided by
the invention has
higher specificity, which is expected to improve the safety profile of the NGF
antibody in clinical
applications. In addition, the anti-human NGF antibody has higher efficacy,
which may reduce the
clinical treatment cost.
In another aspect, the invention is a humanized and affinity matured antibody
and its antigen
binding fragment, which specifically binds to human and rodent nerve growth
factor (NGF). The
anti-NGF antibody and its antigen binding fragment comprises:
Heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 sequences;
and
Light chain variable region, which includes CDR-L1, CDR-L2 and CDR-L3
sequences, and is
selected from the following groups:
(1) The heavy chain variable region includes the CDR-H1 amino acid sequence
shown in SEQ ID
No. 1, CDR-H2 amino acid sequence shown in SEQ ID No. 3, CDR-H3 amino acid
sequence shown
in SEQ ID No. 5, CDR-L1 amino acid sequence shown in SEQ ID No. 7, CDR-L2
amino acid
sequence shown in SEQ ID NO. 9, and CDR-L3 amino acid sequence shown in SEQ ID
No. 11;
(2) The heavy chain variable region includes the CDR-H1 amino acid sequence
shown in SEQ ID
No. 2, CDR-H2 amino acid sequence shown in SEQ ID No. 4, CDR-H3 amino acid
sequence shown
in SEQ ID No. 6, CDR-L1 amino acid sequence shown in SEQ ID No. 8, CDR-L2
amino acid
sequence shown in SEQ ID NO. 10, and SEQ ID The amino acid sequence of CDR-L3
shown in No.
12;
(3) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID No.
37, CDR-H2 amino acid sequence shown in SEQ ID No. 39, CDR-H3 amino acid
sequence shown in
SEQ ID No. 41, CDR-L1 amino acid sequence shown in SEQ ID No. 43, CDR-L2 amino
acid
sequence shown in SEQ ID No. 45, and CDR-L3 amino acid sequence shown in SEQ
ID No. 11;
(4) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID No.
47, CDR-H2 amino acid sequence shown in SEQ ID No. 48, CDR-H3 amino acid
sequence shown in
SEQ ID No. 41, CDR-L1 amino acid sequence shown in SEQ ID No. 49, CDR-L2 amino
acid
sequence shown in SEQ ID No. 50, and CDR-L3 amino acid sequence shown in SEQ
ID No. 11;
(5) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID No.
47, CDR-H2 amino acid sequence shown in SEQ ID No. 48, CDR-H3 amino acid
sequence shown in
3
Date Recue/Date Received 2020-06-17
SEQ ID No. 41, CDR-L1 amino acid sequence shown in SEQ ID No. 49, CDR-L2 amino
acid
sequence shown in SEQ ID No. 51, and CDR-L3 amino acid sequence shown in SEQ
ID No. 11;
(6) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID No.
38, CDR-H2 amino acid sequence shown in SEQ ID No. 40, CDR-H3 amino acid
sequence shown in
SEQ ID No. 42, CDR-L1 amino acid sequence shown in SEQ ID No. 44, CDR-L2 amino
acid
sequence shown in SEQ ID No. 46, and CDR-L3 amino acid sequence shown in SEQ
ID No. 12;
(7) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID No.
52, CDR-H2 amino acid sequence shown in SEQ ID No. 53, CDR-H3 amino acid
sequence shown in
SEQ ID No. 42, CDR-L1 amino acid sequence shown in SEQ ID No. 55, CDR-L2 amino
acid
sequence shown in SEQ ID No. 56, and CDR-L3 amino acid sequence shown in SEQ
ID No. 12;
(8) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID
No. 52, CDR-H2 amino acid sequence shown in SEQ ID No. 53, CDR-H3 amino acid
sequence
shown in SEQ ID No. 42, CDR-L1 amino acid sequence shown in SEQ ID No. 55, CDR-
L2 amino
acid sequence shown in SEQ ID No. 57, and CDR-L3 amino acid sequence shown in
SEQ ID No.
12;
(9) The heavy chain variable region contains CDR-H1 amino acid sequence shown
in SEQ ID
No. 52, CDR-H2 amino acid sequence shown in SEQ ID No. 54, CDR-H3 amino acid
sequence
shown in SEQ ID No. 42, CDR-L1 amino acid sequence shown in SEQ ID No. 55, CDR-
L2 amino
acid sequence shown in SEQ ID No. 57, and CDR-L3 amino acid sequence shown in
SEQ ID No.
12.
Further, the antigen or its antigen binding fragment is mouse derived,
chimeric or humanized.
In some embodiments of the invention, the antibody is mouse derived or
chimeric, and its heavy
chain variable region further comprises the heavy chain fr region of mouse
IgGl, IgG2a, IgG2b,
IgG3 or variants thereof; and its light chain variable region comprises the
light chain fr region of
mouse K. k chain or variants thereof
Preferably, the mouse derived or chimeric antibody comprises a heavy chain
variable region
amino acid sequence as shown in SEQ ID No. 13 or 15 respectively, and a light
chain variable region
amino acid sequence as shown in SEQ ID No. 14 or 16 respectively.
More preferably, the mouse antibody # 56 and the chimeric antibody AB5C1 in
the invention
comprise a heavy chain variable region amino acid sequence as shown in SEQ ID
No. 13 and a light
chain variable region amino acid sequence as shown in SEQ ID No. 14.
More preferably, the mouse antibody # 2 and the chimeric antibody ab5d1 in the
invention
comprise a heavy chain variable region amino acid sequence as shown in SEQ ID
No. 15 and a light
chain variable region amino acid sequence as shown in SEQ ID No. 16.
4
Date Recue/Date Received 2020-06-17
In some embodiments of the invention, the antibody is humanized. The
preparation of
humanized antibody can be completed by CDR transplantation technology, surface
remodeling
technology, computer simulation technology or other existing technologies.
In some embodiments of the invention, the mouse antibody Chen 56 is humanized
by CDR
transplantation. The resulting humanized antibody, preferably, has a heavy
chain variable region
containing an amino acid sequence selected from SEQ ID NOS: 17, 19, 21 or 23,
and a light chain
variable region containing an amino acid sequence selected from SEQ ID NOS:
18, 20, 22 or 24.
More preferably, the resulting humanized antibodies AB5C2, ab5c3, ab5c4 and
ab5c5, wherein the
heavy chain variable region respectively comprises an amino acid sequence as
shown in SEQ ID No.
17, 19, 21 or 23, and the light chain variable region respectively comprises
an amino acid sequence
as shown in SEQ ID No. 18, 20, 22 or 24.
In some embodiments of the invention, the mouse antibody Chen 56 is humanized
by surface
remodeling technology. Preferably, the resulting humanized antibody AB5C6 has
a heavy chain
variable region containing an amino acid sequence as shown in SEQ ID No. 25,
and a light chain
variable region containing an amino acid sequence as shown in SEQ ID No. 26.
In some embodiments of the invention, the mouse antibody Chen 2 is humanized
by CDR
transplantation. The resulting humanized antibody preferably has a heavy chain
variable region
containing an amino acid sequence selected from SEQ ID NOS: 27, 29, 31 or 33,
and a light chain
variable region containing an amino acid sequence selected from SEQ ID NOS:
28, 30, 32 or 34.
More preferably, the resulting humanized antibodies ab5d2, ab5d3, ab5d4 and
ab5d5, wherein the
heavy chain variable region respectively comprises an amino acid sequence as
shown in SEQ ID No.
27, 29, 31 or 33, and the light chain variable region respectively comprises
an amino acid sequence
as shown in SEQ ID No. 28, 30, 32 or 34.
In some embodiments of the invention, the mouse antibody#2 is humanized by
surface
remodeling technology. Preferably, the resulting humanized antibody ab5d6 has
a heavy chain
variable region containing an amino acid sequence as shown in SEQ ID No. 35,
and a light chain
variable region containing an amino acid sequence as shown in SEQ ID No. 36.
Without substantially affecting the antibody activity, those skilled in the
art may replace, add or
redeuce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino
acids of the sequence of the
antibody of the invention to obtain a variant of the antibody sequence. They
are all considered to be
included in the scope of protection of the invention. For example, amino acids
with similar properties
will be substituted in the variable region. The sequence of the variant of the
invention can be at least
80% homologous with its source sequence; more preferably, the sequence of the
variant of the
invention can be at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous with
its source
5
Date Recue/Date Received 2020-06-17
sequence.
In some embodiments of the invention, the antibody and/or its antigen binding
fragment
provided by the present invention is a full-length antibody that further
comprises an antibody
constant region of a human or mouse; or an antigen binding fragment that only
contains fab, Fab', f
(ab') 2 or scFv.
In a preferred example of this aspect, the heavy chain constant region
sequence is selected from
human IgGl, IgG2, IgG3 or IgG4.
In a preferred example in this respect, the light chain constant region
sequence of the antibody or
its antigen binding fragment is a human lc antibody light chain constant
sequence.
In one embodiment of the invention, the antibody binds to a human NGF.
Specifically, the
antibody can block the interaction between human NGF and corresponding human
NGF receptor. Kd
value of the antibody or its antigen binding fragment binding to NGF is
5 X 10-11 m,
preferably 1 x 10-11 m or smaller KD.
In a second aspect of the invention, a polynucleotide seqeunce encoding the
antibody or the
antigen binding fragment thereof is provided. Preferably, the polynucleotide
seqeunce encoding the
heavy chain variable region of the antibody or its antigen binding fragment is
as shown in SEQ ID
No. 58, 60 and 62 respectively, and the polynucleotide seqeunce encoding the
light chain variable
region of the antibody or its antigen binding fragment is as shown in SEQ ID
No. 59, 61 and 63
respectively.
For example, the polynucleotide seqeunce encoding the heavy chain variable
region of the
chimeric antibody AB5C1 preferred by the present invention is shown in SEQ ID
No. 58, and the
DNA molecule encoding the light chain variable region is shown in SEQ ID No.
59.
For example, the polynucleotide seqeunce encoding the heavy chain variable
region of the
humanized antibody AB5C2 preferred by the invention is shown in SEQ ID No. 60,
and the DNA
molecule encoding the light chain variable region is shown in SEQ ID No. 61.
As another example, the polynucleotide seqeunce encoding the heavy chain
variable region of
the humanized antibody AB5C6 is shown in SEQ ID No. 62, and the DNA molecule
encoding the
light chain variable region is shown in SEQ ID No. 63.
In a third aspect of the present invention, there is provided an expression
vector comprising the
polynucleotide seqeunce.
In a fourth aspect of the invention, a host cell transfected with the
expression vector is provided.
Preferably, the host cell is CHO cell.
In a fifth aspect of the invention, a pharmaceutical composition is provided.
The pharmaceutical
6
Date Recue/Date Received 2020-06-17
composition comprises at least one pharmaceutically acceptable carrier,
excipient or diluent, and an
effective amount of the antibody or its antigen binding fragment.
The sixth aspect of the invention provides the use of the antibody or its
antigen binding
fragment or pharmaceutical composition, preferably for the treatment of any
disease related to NGF,
in particular pain disease related to NGF. These diseases are usually related
to the overexpression and
elevation of NGF. The diseases include but are not limited to degenerative
arthritis, rheumatic
arthritis, interstitial cystitis, osteonecrosis, low back pain or diabetic
peripheral neuropathy, etc.
Preferably, a chimeric, humanized anti NGF antibody and its antigen binding
fragment can be
used in the preparation of a drug for treating the disease; more preferably, a
humanized anti NGF
antibody and its antigen binding fragment can be used.
The invention discloses that the antibody provided by the invention or the
antigen binding
fragment thereof has the following advantages:
1. The antibody provided by the invention has high affinity, and the affinity
constant Kd value is
5 X 10-11M, which can effectively block the binding between NGF and its
receptor, and block
the response of pain;
2. The antibody provided by the invention has strong specificity of binding
with antigen, and it
can be expected that the clinical dosage will also be reduced;
3. The antibody provided by the invention is expressed by CHO cells and has
the advantages of
high yield, high activity, simple purification process and low production
cost.
Description of the invention
Abbreviation and difinition
hNGF human nuron growth factor
CDR complementary determination region in immunoglobin proteins
variable region which
is difinied by IMGT system
ELISA enzyme linked immunosorbent assay
FR framework region
HRP Horseradish peroxidase
IgG Immunoglobulin G
Kabat Immunoglobulin protein comparison and coding system advocated by Elvin a
Kabat
IMGT International immunogenetic information system proposed by LaFranc et al
mAb monoclonal antibody
PCR polymerase chain reaction
V region IgG chain fragment with variable sequence between different
antibodies. It extends
7
Date Recue/Date Received 2020-06-17
to 109 residues of light chain and 113 residues of heavy chain.
VH Immunoglobulin heavy chain variable region
VK Immunoglobulin kappa light chain variable region
KD equilibration constant
kd dissociation rate constant
kon association rate constant
DEFINITIONS
The term "antibody" as used in the present invention refers to the
immunoglobulin molecule and
the immunoactive part of the immunoglobulin molecule, that is, the molecule
containing the antigen
binding site of the specific binding antigen, covering the full-length
antibody (e.g., IgG1 or IgG4
antibody), various functional segments (e.g., may only contain the antigen
binding part, such as Fab,
f (ab ') 2 or scFv segments), and modified Antibodies (e.g. humanization,
glycosylation, etc.).
Examples of antibodies include, but are not limited to, polyclonal antibodies,
monoclonal antibodies,
chimeric antibodies, humanized antibodies, domain antibodies, single chain
antibodies, Fab, Fab', f
(ab') 2 fragments, etc. The invention also includes an anti NGF antibody with
glycosylation
modification. In some applications, modification can be carried out to remove
undesirable
glycosylation sites, such as fucose modification on oligosaccharide chain to
enhance the function of
antibody dependent cytotoxicity (ADCC); in other applications, galactosylation
can be carried out to
change complement dependent cytotoxicity (CDC)
The term "monoclonal antibody or mAb" refers to an antibody obtained from a
single clone cell
line, which is not limited to a eukaryotic, prokaryotic or phage clone cell
line. Monoclonal antibodies
or antigen binding fragments can be reconstructed by hybridoma technology,
recombinant
technology, phage display technology, synthetic technology (such as CDR
grafting), or other existing
.. technologies.
"Antibody fragment" and "antigen binding fragment" mean the antigen binding
fragment and
antibody analogue of an antibody, which generally includes the antigen binding
region or variable
region (e.g. one or more CDRs) of at least part of the parent antibody.
Antibody fragments retain at
least some binding specificity of the parent antibody. In general, when the
activity is expressed on a
molar basis, the antibody fragment retains at least 10% of the maternal
binding activity. Preferably,
the antibody fragment retains at least 20%, 50%, 70%, 80%, 85%, 90%, 95% or
100% or more of the
binding affinity of the parent antibody to the target. Examples of antibody
fragments include, but are
not limited to: fab, Fab', f (ab') 2 and Fv fragments; double antibodies;
linear antibodies; single chain
antibody molecules, such as scFv, single antibody (technology from genmab);
nano antibody
8
Date Recue/Date Received 2020-06-17
(technology from domanti s); domain antibody (technology from Ablynx); and
multi-specific
antibody formed by antibody fragments. Antibody variants of engineering
modification are
summarized in Holliger et al., NAT biotechnology, 2005, 23:1126-1136.
"Fab fragment" consists of CH1 and variable region of a light chain and a
heavy chain. The
heavy chain of Fab molecule cannot form disulfide bond with another heavy
chain molecule.
"The Fab 'fragment" contains VH domain and CH1 domain of one light chain and
one heavy
chain, and the constant region between CH1 and CH2 domain. Thus, the
intermolecular disulfide
bond can be formed between the two heavy chains of two Fab' fragments to form
f (ab ') 2 molecule.
The "F (ab ) 2 fragment" contains VH domain and chi domain of two light chains
and two
heavy chains, as well as the constant region between chi and CH2 domains, thus
forming an inter
chain disulfide bond between the two heavy chains. Therefore, the f (AB
) 2 fragment consists of
two Fab fragments which are held together by the disulfide bond between
two heavy chains.
"FV region" contains variable region from heavy chain and light chain, but it
lacks constant
region.
"ScFy antibody" or "scFy antibody" refers to the antibody fragments containing
VH and VL
domains of the antibody, where these domains exist in a single polypeptide
chain. For a review of
scFv, see pluckthun, 1994, the pharmacology of monoclonal antibodies, Vol.
113, edited by
Rosenburg and Moore, Springer Verlag, Berlin, Heidelberg, pp. 269-315. See
also international
patent application Publication No. wo88 / 01649 and U.S. Patent No. 4946778
and No. 5260203.
The "FC" region contains two heavy chain fragments of chi and CH2 domains
containing
antibodies. The two heavy chain segments are held together by two or more
disulfide bonds and
through the hydrophobic action of the CH3 domain.
"Antigen binding fragment" is an immunoglobulin fragment with immunological
function,
which only contains heavy chain variable region or light chain variable region
chain.
The term "hypervariable region" or "CDR region" or "complementary determining
region" as
used herein refers to the amino acid residues of antibodies responsible for
antigen binding. The CDR
region sequence can be defined by IMgt, Kabat, chothia and ABM methods or
identified by any CDR
region sequence determination method well known in the art Antibody CDR can be
identified as
hypervariable regions initially defined by Kabat et al., for example, 24-34
(L1), 50-56 (L2) and
89-97 (L3) residues in light chain variable domains and 31-35 (H1), 50-65 (H2)
and 95-102 (H3)
residues in heavy chain variable domains. See Kabat EA et al., 1991, sequences
of proteins of
immune interest, public health service, National Institutes of health,
Bethesda, MD.; the location of
CDR can also be identified as originally defined by the "hypervariable ring"
(HVL) structure
9
Date Recue/Date Received 2020-06-17
described by chothia et al. IMgt (immunogenetics) also provides a numbering
system for
immunoglobulin variable region including CDR. CDR region is defined according
to IMgt number,
for example, 27-32 (L1), 50-52 (L2) and 89-97 (L3) residues of light chain
variable domain and
26-35 (H1), 51-57 (H2) and 93-102 (H3) residues of heavy chain variable
domain. See e.g. Lefranc
MP, etc., dev comp Immunol, 2003, 27:55-77. Other methods for
CDR identification include "ABM definition", which is a compromise between
Kabat and chothia
and is obtained by Oxford molecular's ABM antibody model software; or "contact
definition" of
CDR, which is based on the observed antigen contact and described in MacCallum
RM et al., J. Mol
Biol, 1996, 262:732-745. In the "configuration definition" method of CDR, the
position of CDR can
be identified as the residue contributing to the cooperative enthalpy of the
original junction. For
example, makabe K et al., J Biol Chem, 2008, 283:1156-1166. The methods used
in the present
invention may be defined by or according to CDR defined by any of these
methods, including but not
limited to any of Kabat definition, IMgt definition, chothia definition, ABM
definition, contact
definition and / or configuration definition.
The term "chimeric antibody" refers to an antibody fused with the variable
region of mouse
antibody and the constant region of human antibody, which can reduce the
immune response induced
by mouse antibody. To establish chimeric antibody, we need to select hybridoma
secreting mouse
specific monoclonal antibody, clone variable region gene from mouse hybridoma
cell, clone constant
region gene of human antibody as required, connect variable region gene of
mouse and constant
region gene of human into chimeric gene, insert it into vector, and express
chimeric antibody
molecule in eukaryotic expression system or prokaryotic expression system
In a preferred
embodiment of the present invention, the antibody light chain variable region
of the NGF chimeric
antibody further comprises the light chain fr region of mouse source K. k
chain or variants thereof
The antibody heavy chain variable region of the NGF chimeric antibody further
comprises the heavy
chain fr region of mouse derived IgGl, IgG2a, IgG2b or IgG3 or variants
thereof. The constant
region of human antibody can be selected from the constant region of heavy
chain of human IgGl,
IgG2, IgG3 or IgG4 or their variants, preferably including the constant region
of heavy chain of
human IgGl.
"Humanized" non-human (such as mouse) antibodies are chimeric antibodies that
contain the
minimum sequence of non-human immunoglobulins. For the most part, humanized
antibodies are
human immunoglobulins (recipient antibodies) in which the recipient
hypervariable region residue is
replaced by the hypervariable region residue of non-human sources (donor
antibodies) (such as mice,
rats, rabbits or non-human primates antibodies that require specificity,
affinity and activity). In some
Date Recue/Date Received 2021-08-23
cases, the fr residues of human immunoglobulin can be replaced by the
corresponding non-human
residues. In addition, humanized antibodies may include residues that neither
exist in the recipient
antibody nor in the donor antibody. These modifications can further improve
the performance of the
antibody. Generally, humanized antibody contains all (at least one, usually
two) variable regions, in
which all or basically all high variable regions correspond to the high
variable regions of non-human
immunoglobulin, and all or basically all fr regions are fr regions of human
immunoglobulin sequence.
Humanized antibodies preferably also include at least a portion of the
constant region (FC) of the
immunoglobulin (usually human immunoglobulin). For more details, please refer
to references Jones
Pt et al., nature, 1986, 321: 522-525.
The terms "immunobinding" and "immunobinding properties" used in this paper
refer to a non
covalent interaction between immunoglobulin molecules and antigens for which
immunoglobulins
are specific. The intensity or affinity of immune binding interaction can be
expressed by the
equilibrium dissociation constant (KD). The smaller the Kd value is, the
higher the affinity is. The
immunobinding properties of the selected polypeptide can be quantified by a
method known in the
art. One method involves measuring the rate of formation and dissociation of
antigen binding sites /
antigen complexes. Both "binding rate constant" (KA or Kon) and "dissociation
rate constant" (KD
or koff) can be calculated by the concentration and the actual rate of
association and dissociation
(Malmqvist m, nature, 1993, 361:186-187). The ratio of KD / Kon is equal to
the dissociation
constant KD (usually see Davies et al., annual Rev Biochem, 1990, 59:439-473).
KD, Kon and KD
can be measured by any effective method. In a preferred embodiment, the
dissociation constant is
measured by a bioluminescent interferometry, such as the fortebio octet method
described in
example 3. In other preferred embodiments, the dissociation constant may be
measured using a
surface plasmon resonance technique such as Biacore or kinexa. When the
equilibrium binding
constant (KD) is ---õ 5 >< 1-11
u
m, preferably ---õ 1 X 10-11 m, the antibody of the invention is
considered to specifically bind to the NGF epitope.
The term "labeled" or "labeled" as used herein refers to the incorporation of
a detectable marker,
such as an amino acid by incorporation of a radioactive marker or the
attachment of a biotin module
to a polypeptide that can be detected by a labeled affinity, such as a
streptomycin containing a
fluorescent marker or an enzymatic activity that can be detected by optical or
calorimetric methods.
In some cases, markers or markers can also be therapeutic. Various methods for
labeling
polypeptides and glycoproteins are known and can be used in the art. Examples
of markers for
polypeptides include, but are not limited to, radioisotopes or radionuclides
(such as 3h, 14C, 15N,
35S, 90Y, 99Tc, 111In, 1251, 1311), fluorescent markers (such as FITC,
rhodamine, lanthanide
11
Date Recue/Date Received 2020-06-17
phosphors), enzyme markers (such as horseradish peroxidase, p-galactosidase,
fluorescent enzyme,
alkaline phosphatase), chemiluminescence , biotin group, predetermined peptide
epitope recognized
by secondary reporter (such as leucine zipper pair sequence, binding site of
secondary antibody,
metal binding domain, epitope label).
Homologous antibody
In another aspect, the amino acid sequence contained in the heavy chain and
light chain variable
region of the antibody of the invention is homologous with the amino acid
sequence of the preferred
antibody described herein, and the antibody retains the desired functional
characteristics of the anti
NGF antibody of the invention.
For example, the invention provides humanized antibody or antigen binding
fragment of NGF,
which includes heavy chain variable region and light chain variable region,
wherein: (a) the heavy
chain variable region includes amino acid sequence at least 80% homologous
with amino acid
sequence selected from SEQ ID NOS: 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35;
more preferably, the
heavy chain variable region includes amino acid sequence selected from SEQ ID
NOS: amino acid
sequence of 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 is at least 85%, 90%,
95%, 96%, 97%, 98% or
99% homologous; (b) the light chain variable region contains and is selected
from seq ID NOS:
amino acid sequences of 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 are at least
80% homologous; more
preferably, the light chain variable region comprises amino acid sequences of
at least 85%, 90%,
95%, 96%, 97%, 98% or 99% homologous with amino acid sequences selected from
SEQ ID NOS:
18, 20, 22, 24, 26, 28, 30, 32, 34 or 36.
Antibody with conservative modification
The term "conservative modification" is intended to mean that amino acid
modification does not
significantly affect or alter the binding characteristics of antibodies
containing the amino acid
sequence. These conserved modifications include substitution, addition and
deletion of amino acids.
The modification can be introduced into the antibody of the invention through
the advantages of
standard techniques known in the art, such as site directed mutagenesis and
PCR mediated.
Conservative amino acid substitution refers to the substitution of amino acid
residues with amino
acid residues with similar side chains. A family of amino acid residues having
similar side chains has
been described in detail in the art. These families include those with basic
side chains (e.g. lysine,
arginine, histidine), acid side chains (e.g. aspartic acid, glutamic acid),
polar side chains without
charge (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, tryptophan),
nonpolar side chains (e.g. alanine, valine, leucine, isoleucine Amino acids
of, proline, phenylalanine,
methionine, (3 - branched side chains (such as threonine, valine, isoleucine)
and aromatic side chains
(such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or
more amino acid residues
12
Date Recue/Date Received 2020-06-17
in the CDR region of the antibody of the invention can be replaced with other
amino acid residues
from the same chain family.
In some embodiments of the invention, the antibodies of the present invention
include heavy
chain variable regions containing CDR-H1, CDR-H2 and CDR-H3 sequences and
light chain
variable regions containing CDR-L1, CDR-L2 and CDR-L3 sequences, wherein one
or more of these
CDR sequences contain specific amino acid sequences or conservative
modifications based on the
preferred antibodies herein, and the antibodies retain the period of the anti
NGF antibodies of the
present invention Functional characteristics of hope. Therefore, the invention
provides a separated
binding NGF antibody or an antigen binding part thereof, which comprises a
heavy chain variable
region containing CDR-H1, CDR-H2 and CDR-H3 sequences and a light chain
variable region
containing CDR-L1, CDR-L2 and CDR-L3 sequences, wherein: (a) the heavy chain
variable region
CDR-H1 sequence comprises a light chain variable region selected from SEQ ID
The amino acid
sequence shown in No. 1 and 2 and its conservatively modified amino acid
sequence; and / or the
heavy chain variable region CDR-H2 sequence includes the amino acid sequence
selected from the
amino acid sequence shown in SEQ ID No. 3 and 4 and its conservatively
modified amino acid
sequence; and / or the heavy chain variable region CDR-H3 sequence includes
the amino acid
sequence selected from the SEQ ID Amino acid sequences shown in No. 5 and 6
and their
conservatively modified amino acid sequences; and / or (b) the light chain
variable region CDR-L1
sequence comprises amino acid sequences selected from the amino acid sequences
shown in SEQ ID
No. 7 and 8 and their conservatively modified amino acid sequences; and / or
the light chain variable
region CDR-L2 sequence comprises amino acid sequences selected from the SEQ ID
The amino acid
sequences shown in No. 9 and 10 and their conservatively modified amino acid
sequences; and / or
the light chain variable region CDR-L3 sequences comprise amino acid sequences
selected from
those shown in SEQ ID NOS: 11 and 12 and their conservatively modified amino
acid sequences.
McAb production
The monoclonal antibody of the invention can be prepared by a variety of
technologies,
including conventional monoclonal antibody methodology, such as standard
somatic hybridization
technology described in Kohler g and Milstein C, nature, 1975:256:495.
Although somatic
hybridization procedures are preferred, other methods for the preparation of
monoclonal antibodies,
such as virus or carcinogenic transformation of B lymphocytes, may also be
used in principle.
The preferred animal system for hybridoma preparation is mouse family. It is a
perfect
procedure to prepare hybridoma in mice. It is known in the art that the immune
scheme and
technology for separating the immune spleen cells for fusion. Fusion mates
(such as mouse myeloma
13
Date Recue/Date Received 2020-06-17
cells) and fusion procedures are also known.
In order to express antibodies and their antibody fragments, the
polynucleotide seqeunce
encoding partial or full-length light and heavy chains can be obtained by
standard molecular
biological techniques (such as PCR amplification or cDNA cloning of hybridoma
expressing the
target antibody), and the polynucleotide seqeunce can be inserted into the
expression vector, so that
the target gene can be operatively linked with transcription and translation
regulatory sequence, and
then transferred to the host cell for expression The expression host preferred
eukaryotic expression
vector, more preferred mammalian cells, such as CHO and its derived cell
lines.
Antibodies can be purified by well-known techniques, such as affinity
chromatography using
.. protein A or protein G. Subsequently or alternatively, the specific antigen
or its epitope may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins was discussed by Wilkinson D,
for example (the
scientist, 2000, 8:25-28, the scientist, Inc., Philadelphia PA).
The chimeric or humanized antibody of the invention can be prepared according
to the sequence
of the prepared mouse monoclonal antibody. Polynucleotide seqeunce encoding
heavy and light
chain immunoglobulins can be obtained from target mouse hybridomas and
engineered to contain
non mouse (e.g., human) immunoglobulin sequences using standard molecular
biology techniques.
For example, in order to create chimeric antibodies, a method known in the art
can be used to
connect a mouse variable region to a human constant region (see U.S. Patent
No. 4816567, for
.. example, cabilly et al.). The separated DNA encoding VH region can be
transformed into full-length
heavy chain gene by another polynucleotide seqeunce operatively connecting VH
coding DNA to
coding heavy chain constant region (chi, CH2 and CH3). The sequence of human
heavy chain
constant region gene is known in the field (see, for example, Kabat EA et al.,
1991, sequences of
proteins of immune interest, public health service, National Institutes of
health, Bethesda, MD).
DNA fragments containing these regions can be amplified by standard PCR. The
heavy chain
constant region can be IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant
region, but the most
preferred region is IgG1 or IgG4 constant region.
In order to create humanized antibodies, the method known in the art can be
used to insert the
mouse CDR region into the human frame sequence (see U.S. patent no.5225539 of
winter and U.S.
patent nos.5530101; 5585089; 5693762 and 6180370 of queen, etc.). It is also
possible to utilize
transgenic animals, such as humAb mice (medarex, Inc.) that contain human
immunoglobulin gene
microloci (miniloci) encoding non rearranged human heavy chain (II and y) and
lc light chain
immunoglobulin sequences, in addition to targeted mutations that inactivate
endogenous p. and lc
chain loci (see, for example, lonberg et al., nature, 1994, 368: 856-859); or
"KM mouse TM" (see
14
Date Recue/Date Received 2020-06-17
patent wo02 / 43478) carrying human heavy chain transgene and human light
chain chromosomal
transformation for antibody humanization. Other methods of humanization
include surface
remodeling and phage display.
The invention is further described by the following embodiments, which shall
not be construed
.. as further limiting. All drawings and all references, patents and published
patent applications quoted
in the whole application are hereby expressly included as references.
Figure legents
Figure 1. Indirect ELISA was used to determine the binding titer of different
monoclonal
antibodies against human NGF mice.
Fig. 2. Competitive ELISA was used to determine the ability of anti human NGF
mouse
monoclonal antibody Chen 56 to block the binding of hNGF and its receptor
TrkA.
Fig. 3-1. The parallel comparison of amino acid sequences of AB5C2, ab5c3,
ab5c4, ab5c5 and
AB5C6 with the heavy chain variable region of mouse antibody # 56. Where the
underline is the
CDR area sequence (as defined by the IMgt system).
Fig. 3-2. Parallel comparison of amino acid sequences of AB5C2, ab5c3, ab5c4,
ab5c5 and
AB5C6, which are humanized antibodies against hNGF, with those of mouse
antibody # 56 light
chain variable region. Where the underline is the CDR area sequence (as
defined by the IMgt
system).
Fig. 4-1. Parallel comparison of amino acid sequences of ab5d2, ab5d3, ab5d4,
ab5d5 and ab5d6
humanized antibodies against hNGF with that of # 2 heavy chain variable region
of mouse antibody.
Where the underline is the CDR area sequence (as defined by the IMgt system).
Fig. 4-2. The parallel comparison of the amino acid sequences of ab5d2, ab5d3,
ab5d4, ab5d5
and ab5d6, which are humanized antibodies against hNGF, with that of the light
chain variable
region of mouse antibody # 2. Where the underline is the CDR area sequence (as
defined by the IMgt
system).
Fig. 5. Detection of anti-hngf antibody inhibiting NGF dependent cell survival
signal.
Fig. 6. Effect of AB5C1 on wound model after operation. Note: compared with
the model group,
* * P < 0.01, * * * P < 0.001.
Fig. 7. The effect of AB5C1 on the sciatic nerve ligation model in some mice.
Note: compared
with the model group, * * P <0.01, * * * P <0.001.
Fig. 8. Effect of AB5C1 and AB5C2 on acute gouty arthritis model induced by
sodium urate.
Note: compared with the model group, * P < 0.01, * P < 0.05.
Date Recue/Date Received 2020-06-17
Fig. 9. Effects of AB5C I and AB5C6 on complete Freund's adjuvant induced
inflammatory
pain model. Note: compared with the model group, * P < 0.01, * P < 0.05.
Examples
Example 1. Preparation of monoclonal antibody against hNGF
Four week old BALB / c mice were immunized subcutaneously with recombinant
human NGF
(peprotech company) and 20 la g / mouse fully emulsified with Freund's
adjuvant. The immune cycle
was three weeks. On the 10th day after the third immunization, blood was
collected from the eye
socket, and the titer of anti human NGF antibody was tested by ELISA to
monitor the immune
response of mice. The mice with the highest titer of anti human NGF antibody
were immunized once
three days before fusion. After 3 days, the spleen of the mouse was taken out
and fused with the
myeloma SP2 / 0 cell line. Mixed 5 x 108 SP2 / 0 cells and 5 x 108 mouse
spleen cells were fused
in 50% PEG (molecular weight 1450) and 5% DMSO solution. The number of spleen
cells was
adjusted to 7.5 x 105 / ml with Iscove medium (containing 10% fetal bovine
serum, 100 U / ml
penicillin, 100 p, g / ml streptomycin, 0.1 mm hypoxanthine, 0.4 n m
aminopterin and 16 p, m
thymidine), and 0.2 ml was added into 96 well plate. Placed in an incubator at
37 C with 5% CO2.
After 10 days, the high-throughput ELISA method was used to detect the ability
of the antibody in
the supernatant to compete with the FC labeled human TrkA receptor to bind to
hNGF, so as to
screen out the positive clones competing with the human TrkA receptor (see
example 3 for the
method). After subcloning the hybridoma cells, three positive hybridoma cell
lines A2, A56 and A98
were screened out by competitive ELISA.
Clones of specific antibodies were cultured in RPMI 1640 supplemented with 10%
FCS. When
the cell density reached about 5 x 105 cells / ml, the medium was replaced by
serum-free medium.
After 2 to 4 days, the culture medium was centrifuged to collect the
supernatant. Protein G column
was used to purify the antibody. The monoclonal antibody eluate was dialyzed
with 150 mM NaCl.
The dialyzed solution was filtered through a 0.2 pm filter to get the purified
mouse monoclonal
antibodies #2, #56 and #98.
Example 2. Titer determination of anti hNGF mouse antibody
Indirect ELISA was used to determine the binding titers of purified mouse
antibody #2, #56 and
#98 to hNGF. Among them, each pore was coated with 100 1-1 1 0.2 1-1 g / ml
hNGF (Corning
company) and placed at 4 C for 16-20 h. The PBS buffer in 96 well plate was
sucked off, washed
with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer once, then 200 n L /
well PBST / 1%
16
Date Recue/Date Received 2020-06-17
skimmed milk powder was added, incubated at room temperature for 1 h and
sealed. Remove the
blocking solution, wash the plate with PBST buffer for 3 times, add PBST / 1%
skimmed milk
powder to dilute to the appropriate concentration of anti-hngf mouse antibody
to be tested, 100 p. L /
well, incubate at room temperature for 1.5 h. Remove the reaction system, wash
the plate with PBST
three times, and then use the HRP labeled Sheep anti mouse IgG polyclonal
antibody (Jackson
Laboratory) diluted with PBST / 1% skimmed milk powder (dilution ratio 1:5000)
at 50 t L / well as
the detection antibody, and incubate at room temperature for 1 h. After 3
times of PBST washing,
add 100 p. L / well TMB and incubate at room temperature for 10-30 min. Add 50
t L / pore 0.2 M
sulfuric acid to stop the reaction. The absorbance value was detected at 0D450
nm by enzyme
labeling instrument, and the result is shown in Fig. 1.
It can be seen from Figure 1 that mouse antibody #2, #56 and #98 can all bind
to hNGF, and the
binding potency of #56 is the best.
Example 3. Binding inhibition assay of anti -hNGF mouse antibody and NGF
receptor TrkA
The enzyme plate was coated with 100 p. 1 2.5 t g / ml hNGF at room
temperature overnight.
Discard the coated solution, seal the holes with skimmed milk dissolved in PBS
for 0.5h, and wash
the holes with PBST. Then add 50 pL2p,g/ ml of TrkA (Beijing Yiqiao Shenzhou
Biology)
labeled with human Fc and 50 p, 1 of mixed solution with different
concentrations of #56 antibody
(10-0.15 t g / ml). The human IgG Fc polyclonal antibody (Jackson Laboratory)
labeled with HRP
was used as the detection antibody, and the 0D450 / 690nm absorbance was
recorded by TMB. It
can be seen from Figure 2 that #56 antibody can specifically block the binding
of NGF to trkA.
Example 4. Affinity analysis assay of anti- hNGF mouse antibody
The binding affinity constants of purified mouse monoclonal antibody # 56 and
# 2 with
antigens were determined by the bio film interference technique (fortebio
octet Red & QK system,
Pall company). The concentration gradient of multichannel parallel
quantitative analysis was set as
3.125, 6.25, 12.5, 25, 50 and 100 nm, and human NGF (his tag) was affinity
coupled to Ni NTA
sensor. After the kinetic fitting curve of affinity analysis, the affinity
constant was calculated by
analyzing the above data. The binding constant Kon value of mouse monoclonal
antibody # 56 =
7.23 x 105/ MS, the dissociation constant Kd value = 8.89 x 10 I s, the
equilibrium dissociation
constant Kd value = KD / Kon = 1.23 x 10-"M (0.0123 nM); the binding constant
Kon value of
mouse monoclonal antibody # 2 = 1.83 x 106 / MS, the dissociation constant KB
Value = 2.92 x 10-5
/ s, equilibrium dissociation constant Kd = KB / Kon = 1.59>< 10-11 M. The
binding affinity of mouse
17
Date Recue/Date Received 2020-06-17
monoclonal antibody # 56 and # 2 against hNGF is very high, which can reach
the order of 10-"M.
Example 5. Subtype identification and variable region amplification of anti-
hNGF mouse
monoclonal antibody
Antibody subtype identification: take the culture supernatant of hybridoma
cells, and identify the
antibody subtype by using the isostriptm mouse monoclonal antibody subtype
identification Kit
(Santa Cruz Biotechnology). The subtype of monoclonal antibody #56 was
identified as IgG1
(kappa), and the subtype of monoclonal antibody # 2 was identified as IgG1
(kappa).
Antibody variable region amplification: the candidate hybridoma cells A56 and
A2 were
cultured to a total number of 10 cells. The cells were collected by
centrifugation at 1000 rpm for 10
minutes. The total RNA was extracted by Trizol Kit (Invitrogen), the first
strand cDNA was
synthesized by smart race, and the corresponding antibody variable region DNA
sequence of
hybridoma cells was amplified by using the first strand cDNA as the subsequent
template. According
to the results of subtype identification, the heavy chain and light chain
constant region sequences of
the antibody subtype were obtained, and specific nested PCR primers were
designed. The primer
sequences used in the amplification reaction complemented the first frame
region and constant
region of the antibody variable region. The target gene was amplified by
conventional PCR, and the
amplified product was sequenced. The heavy chain variable region sequence SEQ
ID No. 13 and
light chain variable region sequence SEQ ID No. 14 of A56 secreting antibody
were obtained. The
amino acid sequence of the heavy chain CDR (CDR-HI, CDR-H2 and CDR-H3) of the
antibody was
identified as SEQ ID The amino acid sequences of light chain CDR (CDR-L1, CDR-
L2 and CDR-L3)
are shown in SEQ ID No. 7, 9 and 11, respectively. Hybridoma clone A2 secretes
the heavy chain
variable region sequence SEQ ID No. 15 and light chain variable region
sequence SEQ ID No. 16 of
antibody Chen 2; the amino acid sequence of heavy chain CDR (CDR-HI, CDR-H2
and CDR-H3)
of the antibody is shown as SEQ ID No. 2, 4 and 6, and the amino acid sequence
of light chain CDR
(CDR-L1, CDR-L2 and CDR-L3) is shown as SEQ ID No. 8, 10 and 12, respectively.
The above
CDR region sequence is defined by IMgt method, and any other known CDR region
sequence
determination method in the art can also be used to identify the amino acid
residues of CDR region
in the variable region.
Example 6. Humanization of mouse antibody against hNGF
6.1 sequencing of antibody variable region and antibody typing
According to the variable region sequence of antibody secreted by hybridoma
cells, the
humanization of antibody was carried out by CDR transplantation. In short, the
process of
18
Date Recue/Date Received 2020-06-17
humanization involves the following steps: comparing the amino acid sequence
of antibody secreted
by hybridoma cells with that of antibody of human embryonic line to find out
the sequence with high
homology; analyzing and investigating HLA-DR After that, using computer
simulation technology,
we used molecular docking to analyze the amino acid sequence of the variable
region and its
surrounding framework, and investigated its three-dimensional binding mode. By
calculating the
electrostatic force, van der Waals force, hydrophobicity and entropy, the key
amino acids which can
interact with NGF and maintain the spatial framework in the amino acid
sequence of antibody
secreted by hybridoma cells were analyzed, and then grafted back to the
selected human embryonic
antibody framework.
Among them, mouse antibody # 56 was constructed with human IGHV4-38-2 * 02
heavy chain
variable region and human IGKV1-33 * 01 light chain variable region as
template sequences, and
four different humanized antibodies were constructed, namely AB5C2, ab5c3,
ab5c4 and ab5c5. At
the same time, a human mouse chimeric antibody AB5C1 was constructed by
grafting the heavy
chain variable region of mouse antibody into the heavy chain constant region
of human IgG1 and the
light chain variable region of mouse antibody into the light chain constant
region of human kappa.
The amino acid sequence of the variable region of the humanized antibody is
shown in Table 1.
Among them, mouse antibody # 2 was based on human IGHV1-46 * 01 heavy chain
variable
region and human IGKV1-n11 * 01 light chain variable region as template
sequences. Four different
humanized antibodies were constructed, ab5d2, ab5d3, ab5d4 and ab5d5,
respectively. At the same
time, a human mouse chimeric antibody ab5d1 was constructed, which was
obtained by grafting the
heavy chain variable region of mouse antibody into the heavy chain constant
region of human IgG1
and the light chain variable region of mouse antibody into the light chain
constant region of human
kappa. The amino acid sequence of the variable region of the humanized
antibody is shown in Table
1.
In addition, the surface remodeling method was used to humanize the variable
region sequence
of the antibody secreted by the hybridoma cells. The surface remodeling method
refers to the
humanization of the amino acid residues on the surface of heteroantibodies. It
only replaces the
regions with obvious differences from the surface amino acids of human
antibodies. On the basis of
maintaining the antibody activity and reducing the heterogeneity, it selects
the amino acid
replacement similar to the surface residues of human antibodies. Specifically,
the process of
humanization of surface remodeling involves the following steps: first, the
amino acid sequence of
antibody secreted by hybridoma cells is compared with that of antibody of
human embryonic line to
find out the sequence with high homology; then, the computer simulation
technology is used to select
solvent When the accessibility is more than 30%, the exposed surface amino
acids will be replaced
19
Date Recue/Date Received 2020-06-17
by the adult embryonic antibody amino acids. As far as possible, the residues
that affect the size,
charge and hydrophobicity of the side chain, or may form hydrogen bond and
thus affect the
conformation of the complementary determining region of the antibody should
not be replaced.
Among them, human antibody AB5C6 was constructed by using human IGHV4-38-2 *
02 heavy
chain variable region and human IGKV1-33 * 01 light chain variable region as
template sequences,
and human IGHV1-46 * 01 heavy chain variable region and human IGKV1-n11 * 01
light chain
variable region as template sequences. The amino acid sequence of the variable
region of the
humanized antibody is shown in Table 1.
The affinity of the humanized antibody obtained by the method of example 4 is
shown in Table
2.
TABLE 1 Amino acid sequence of variable region of the humanized antibody
Name variable region of HC variable region of LC
AB5C2 SEQ ID NO: 17 SEQ ID NO: 18
AB5C3 SEQ ID NO: 19 SEQ ID NO: 20
AB5C4 SEQ ID NO: 21 SEQ ID NO: 22
AB5C5 SEQ ID NO: 23 SEQ ID NO: 24
AB5C6 SEQ ID NO: 25 SEQ ID NO: 26
AB5D2 SEQ ID NO: 27 SEQ ID NO: 28
AB5D3 SEQ ID NO: 29 SEQ ID NO: 30
AB5D4 SEQ ID NO: 31 SEQ ID NO: 32
AB5D5 SEQ ID NO: 33 SEQ ID NO: 34
AB5D6 SEQ ID NO: 35 SEQ ID NO: 36
TABLE 2 Binding affinity of the humanized antibody
TA1* Ku (M) kon (1/Ms) kd (1/s)
AB5C1 2.99E-11 1.81E+06 5.40E-05
AB5C2 1.30E-12 1.66E+06 2.16E-06
AB5C3 7.55E-12 1.31E+06 9.87E-06
AB5C4 1.53E-11 1.46E+06 2.23E-05
AB5C5 8.90E-12 1.78E+06 1.59E-05
Date Recue/Date Received 2020-06-17
AB5C6 1.02E-11 1.59E+06 1.61E-05
AB5D1 1.06E-11 1.46E+06 1.55E-05
AB5D2 6.33E-12 1.25E+06 7.94E-06
AB5D3 1.29E-11 1.49E+06 1.92E-05
AB5D4 1.82E-11 1.78E+06 3.25E-05
AB5D5 1.54E-11 1.23E+06 1.89E-05
AB5D6 1.57E-11 1.41E+06 2.22E-05
The amino acid sequences of the CDR regions of mouse McAbs # 56 and # 2 and
their derived
humanized antibodies AB5C2, AB5C3, AB5C4, AB5C5, AB5C6, AB5D2, AB5D3, AB5D4,
AB5D5 and AB5D6 are shown in TABLE 3-1 to TABLE 3-2, wherein the amino acid
sequences of
5 CDR are defined by Kabat and IMGT methods respectively, and the amino
acid sequences of CDR
region mutations in the humanized antibodies shown in TABLE 3-land TABLE 3-2
are highlited
using underline.
TABLE 3-1. CDR amino acid sequences of the #56 NGF antibody and its derived
humanized
antibodies
#56 AB5C2 AB5C6 AB5C3 AB5C4 AB5C5
GYGVN GYGVN GYGWG GYGWG
CDR¨H1
(SEQ NO ID: 37) (SEQ NO ID: 37) (SEQ NO ID: 47) (SEQ NO ID:
47)
MIWADGDTDYNSAL MIWADGDTDYNSAL SIWADGDTYYNPSL SIWADGDTYYNPSL
CDR¨H2 KS KS KS KS
(SEQ NO ID: 39) (SEQ NO ID: 39) (SEQ NO ID: 48) (SEQ NO ID:
48)
DSYYYGYNFFDV DSYYYGYNFFDV DSYYYGYNFFDV DSYYYGYNFFDV
CDR¨H3
Kabat
(SEQ NO ID: 41) (SEQ NO ID: 41) (SEQ NO ID: 41) (SEQ NO ID: 41)
RASQDISNYLN RASQDISNYLN QASQDISNYLN QASQDISNYLN
CDR¨L1
(SEQ NO ID: 43) (SEQ NO ID: 43) (SEQ NO ID: 49) (SEQ NO ID:
49)
YTSRLHS YTSRLHS YTSNLET YTSNLES
CDR¨L2
(SEQ NO ID: 45) (SEQ NO ID: 45) (SEQ NO ID: 50) (SEQ NO ID:
51)
QQGNTLPRT QQGNTLPRT QQGNTLPRT QQGNTLPRT
CDR¨L3
(SEQ NO ID: 11) (SEQ NO ID: 11) (SEQ NO ID: 11) (SEQ NO ID: 11)
GFSLTGYG
CDR¨H1
(SEQ NO ID: 1)
IMGT
IWADGDT
CDR¨H2
(SEQ NO ID: 3)
21
Date Recue/Date Received 2020-06-17
ARDSYYYGYNFFDV
CDR¨H3
(SEQ NO ID: 5)
QDISNY
CDR¨L1
(SEQ NO ID: 7)
YTS
CDR¨L2
(SEQ NO ID: 9)
QQGNTLPRT
CDR¨L3
(SEQ NO ID: 11)
TABLE 3-2. CDR amino acid sequences of the #2 NGF antibody and its derived
humanized
antibodies
#2 AB5D2 AB5D 6 AB5D3 AB5D 4 AB5D5
DYWMQ DYWMQ DYWMH DYWMH DYWMH
CDR¨H1 (SEQ NO ID: ( SEQ NO ID: (SEQ NO ID: (SEQ NO ID:
(SEQ NO ID:
3 8 ) 38) 52) 52) 52)
TIYPGDGYTR TIYPGDGYTRYI IIYPGDGYTS IIYPGDGYTS IIYPGDGYTS
YIQKFKG QKFKG YAQKFQG YAQKFQG YIQKFQG
CDR¨H2
(SEQ NO ID: ( SEQ NO ID: (SEQ NO ID: (SEQ NO ID: (SEQ NO ID:
4 0 ) 4 0 ) 53) 53) 54)
RAAYYTMDY RAAYYTMDY RAAYYTMDY RAAYYTMDY RAAYYTMDY
CDR¨H3 (SEQ NO ID: ( SEQ NO ID: (SEQ NO ID: (SEQ NO ID:
(SEQ NO ID:
42) 42) 42) 42) 42)
Kabat
KASQDVNTAV RASQDVNTAL RASQDVNTAL RASQDVNTAL
_ _ _ _ _ _
KASQDVNTAVA
A A A A
CDR¨L1 ( SEQ NO ID:
(SEQ NO ID: (SEQ NO ID: (SEQ NO ID: (SEQ NO ID:
44)
44) 55) 55) 55)
WASTRHT WASTRHT WASRLES WASRLET WASRLET
CDR¨L2 (SEQ NO ID: ( SEQ NO ID: (SEQ NO ID:
(SEQ NO ID: (SEQ NO ID:
46) 46) 56) 57) 57)
QQHYSSPWT QQHYSSPWT QQHYSSPWT QQHYSSPWT QQHYSSPWT
CDR¨L3 (SEQ NO ID: ( SEQ NO ID: (SEQ NO ID: (SEQ NO ID:
(SEQ NO ID:
12) 12) 12) 12) 12)
GYTFTDYW
CDR¨H1
IMGT (SEQ NO ID: 2)
CDR¨H2 IYPGDGYT
22
Date Recue/Date Received 2020-06-17
( SEQ NO ID: 4)
ARRAAYYTMDY
CDR¨H3
( SEQ NO ID: 6)
QDVNTA
CDR¨L1
( SEQ NO ID: 8)
WAS
CDR¨L2
( SEQ NO ID: 10)
QQHYS SPWT
CDR¨L3
( SEQ NO ID: 12)
Figure. 3-1 shows the amino acid sequences of HC variable region of five
humanized NGF
antibodies and mouse NGF antibody # 56. Figure 3-2 shows the amino acid
sequences of the LC
variable region of 5 humanized antibodies and mouse antibody #56. In the
variable region, CDR and
.. framework region are labeled as listed above, and CDR of HC and LC variable
region is defined by
IMGT method.
Fig. 4-1 shows the amino acid sequences of HC variable region of five
humanized NGF
antibodies and mouse NGF antibody #2. Figure 4-2 shows the amino acid
sequences of the LC
variable region of five humanized NGF antibodies and mouse NGF antibody #2. In
the variable
region, CDR and framework region are labeled as listed above, and CDR of HC
and LC variable
region is defined by IMGT method.
6.2 Expression vector and protein expression of the humanized antibodies
The cDNA of HC and LC obtained by the above method was inserted into the
pCMAB2M
eukaryotic expression vector (constructed in our laboratory) to construct the
humanized expression
vector. The expression vector plasmid contains the early gene promoter
enhancer of cytomegalovirus
required for high level expression in mammalian cells. At the same time, the
vector plasmids contain
selection marker genes with ampicillin resistance in bacteria and G418
resistance in mammalian cells.
In addition, the vector plasmid contains dihydrofolate reductase (DHFR) gene,
which can co amplify
antibody gene and DHFR gene with methotrexate (MTX) in suitable host cells.
The constructed recombinant expression vector plasmid was transfected into
mammalian host
cell line to express humanized antibody. In order to stabilize the high level
of expression, the
preferred host cell line is DHFR-deficient Chinese hamster ovary (CHO) cells
(see U.S. Patent No.
4818679). The preferred transfection method is electroporation, while other
transfection methods can
23
Date Recue/Date Received 2020-06-17
also be used, including calcium phosphate co-deposition, lipid transfection
and protoplast fusion. In
electroporation experiement, gene pulser (bio-rad laboratories) with 250 V
electric field and 960 p,
FD capacitance was used. 2 x 10' cells were added into the cuvette and
suspended in 0.8 ml PBS,
and 10 p. g expression vector plasmid was linearized with pvui (Takara). 2
days after transfection, 0.2
mg / ml G418 and 200 nm MTX (sigma) were added into the cell culture medium.
In order to
achieve a high level of antibody expression, the transfected antibody gene was
co-transfected with
DHFR gene which can be inhibited by MTX. Clones were selected by limited
dilution method and
the activity of the clones were validated by ELISA method. After several
rounds of selection and
validation, the clone with high level of antibody expression was selected out.
The cell culture
medium of the clones were collected for determination of their biological
activity in vitro and in
vivo.
Example 7. Determination of neutralization activity of NGF antibody in vitro
The biological activity of NGF antibody was validated by measurement of the
capability of the
NGF antibody to inhibit the NGF-dependent proliferation of TF-1 cells. TF-1
cell proliferation
highly dependents on granulocyte macrophage colony stimulating factor (GM-
CSF). The binding of
NGF to its receptor on TF-1 cell surface will induce the proliferation of TF-1
cells. Firstly, sample
plates were set as 50 p.1 of RPMI 1640 medium containing 10% fetal bovine
serum (FBS) plus 50
p.1 of antibodies of different concentrations and 50 p.1 of 800 ng / ml hNGF
per well. Wells without
addition of hNGF were used as negative control and medium-only wells were used
as blank control.
After shaking at room temperature for 30 minutes, add 100 1-1 1 105 / ml TF-1
cells into each well of
the sample plates, and culture the plates in a 5% CO2 incubator at 37 C for 5-
6 days. After that, 20
1 of MTT (2.5 mg / ml) was added into each well and incubated for 4 hours, and
then 100 ti L
10% SDS was added into each well and incubated overnight. The proliferation of
the TF-1 cells was
measured by monitoring the fluorescence signal at Ex570 nm and Em620 nm. Three
replicates were
set for each treatment, and the experiments were repeat twice for
confirmation.
The results are shown in Figure 5. Among the test groups, the binding affinity
of the
anti-hNGF antibody to NGF is relatively low at the concentration range from
0.005 to 0.3 p, g / ml.
When the concentration is higher than 0.3 p. g / ml, NGF antibody showed
inbihition of the effect of
NGF on TF-1 cell proliferation, indicating that NGF antibody can antagonize
the function of NGF.
In addition, the binding affinity of NGF humanized antibody AB5C2, AB5C6 and
mouse
monoclonal antibody # 56 are better than that of chimeric antibody AB5C1.
Example 8. In vivo analgesic efficacy study of NGF antibodies
24
Date Recue/Date Received 2020-06-17
8.1 Efficacy study with post-surgical pain model
We used the mimic post-surgical pain mouse model to evaluate the efficacy of
treatment of NGF
antibodies. In this experiment, the flexor muscles of the paw of the mice were
used for surgical
operation with a scalpel. This surgical operation results the stimulation and
finally the thermal
hyperalgesia of the mice. Efficacy of NGF antibodies on the pain elimination.
SPF grade C57BL / 6J male mice (Shanghai slake experimental animal Co., Ltd.)
of weight
around 25g, were randomly divided into two groups with 10 mice in each group.
Before the
experiment, the animals were put into the Hargreaves device (type 336, IITC
Life Science) for
adaptation for a period of time. The envioroment of the adaptation is light
intensity 17%, and the
light cut-off time was 25 s. Before administration, the basic thermal pain
threshold (the time from the
beginning of strong light radiation to the occurrence of foot contraction
reaction, i.e., the time of
thermal pain reaction) of each mouse was measured at 0.5-2 h interval, three
times in total. the
average value was set as the baseline (t0 value). Before the surgical
operation, NGF antibody AB5C1
was injected subcutaneously at the dose of 10 mg / kg in the test group and
PBS buffer at the same
volume dose (10 ml! kg) in the model group. One hour later, the flexor muscle
of the right paw of
the mice was separated and cut vertically. Then the site of the surgical
operation was sutured. During
the recovery time period, the mice were used to validate their thermal pain
threshold after 1, 24, 48,
72 and 96 h of the surgical operation. The percentage of the increase of the
thermal pain threshold
was calculated according to the following formula: the percentage of the
increase of the thermal pain
threshold = (the average thermal pain threshold after administration - the
average thermal pain
threshold before administration) / the average thermal pain threshold before
administration x 100%.
Statistical analysis was carried out based on the data. The experimental data
was expressed by means
SD. If the data were in accordance with normal distribution, SPSS 18.0
software, one-way
ANOVA or t-test was used; paired t-test was used for different influencing
factors before and after
treatment; nonparametric test, Mann Whitney test or Kruskal Wallis test were
used for scoring
statistics, P 0.05 had significant difference, P 0 There is a very
significant difference.
It can be seen from Fig. 6 that 1 h after the surgical operation, the thermal
pain threshold of the
mice in each group decreased due to post-surgical pain, while with the
extension of time, the
difference of the thermal pain threshold between the tested group and the
model group is more and
more significant. According to the student's t-test test, the time period from
t24 h to 72 h is
statistically significant, which shows that compared with the model group, the
pre-treatment of the
tested NGF antibody AB5C1 significantly reduces the post-osurgical pain. At
t72 h, the threshold of
thermal pain in the tested group was restored to the initial value, while that
in the model group was
Date Recue/Date Received 2020-06-17
still at a lower value; at t96 h, both groups were restored to the initial
value.
8.2 Efficacy study of NGF antibodies with sciatic nerve ligation model
We used sciatic nerve ligation model to evaluate the effect of NGF antibody on
chronic pain.
SPF C57BL / 6J female mice with weight of about 25 g were randomly divided
into 3 groups, 5 mice
in each group. Before the experiment, the test animals were put into the
Hargreaves device
(parameter setting as 8.1) for environment adaptation. The foot base pain
threshold of each mouse of
rest time was measured at 0.5-2 h interval, three times in total, and set the
average value of the foot
base pain threshold as the baseline (d0 value).
1 / 3 ¨ 1 / 2 of the sciatic nerve in the fascia and muscle of the right femur
of the mice were
ligated with 7-0 silk thread. Sew layer by layer the wounds of the tested
mice. In the negative control
group, the sciatic nerve was not ligated. On the 10th day (d10) and 17th day
(d17) after operation,
NGF antibody AB5C1 was subcutaneously injected into the tested group at a dose
of 100 mg / kg,
and PBS buffer of equal volume (20 ml / kg) was injected into the negative
control group and the
model group, respectively. The thermal pain thresholds of d10, d17 before
administration and d12,
d14, d16, d18, d20, d21 and d23 after administration were measured, and the
percentage (%) of the
increase of thermal pain threshold was calculated, the formula was the same as
8.1. For statistical
analysis of data, the software and analysis method are the same as 8.1
From the data in Table 4, it can be seen that on the 10th day after ligation,
the thermal pain
threshold of each group showed a downward trend compared with the basic
thermal pain threshold
measured before the experiment, and the percentage of increase of the thermal
pain threshold of NGF
antibody AB5C1 group, model group and negative control group was - 56.31%
6.98%, - 50.99%
8.19% and - 20.42% 5.35%, respectively. The first two groups and negative
control group were
statistically analyzed by t-test, and P = 0.014 < 0.05. After subcutaneous
administration for 48
hours (d12) on the 10th day, it was found that the increase rate of NGF
antibody AB5C1 in the
administration group was - 23.65% 5.17%, which was equivalent to that in the
negative control
group (- 18.70% 5.98%), the mean value was much higher than that in the
model group (- 51.44%
1.28%), and the statistical significance was not very significant, indicating
that the tested antibody
showed better analgesic effect at 48h than that of the control group. While
the thermal pain threshold
showed a downward trend on the 4th-7th day (d14-d17) after administration, as
shown in Figure 7.
The percentage of the increase of the thermal pain threshold on the 7th day
was almost the same as
that of the model group, indicating that the NGF antibody was constantly
metabolized and degraded
26
Date Recue/Date Received 2020-06-17
in mice with the extension of time.
One week after first administration, the second administration was carried out
on d17. It was
observed that at 24 h (d18) and 72 h (d20) after the second administration,
the increase percentage of
the thermal pain threshold in NGF antibody AB5C1 group was significantly
higher than that of the
model group. The data are shown in Table 3, and the statistical differences
are significant. Based on
the data, it is speculated that there are statistical differences in 48 h
tratement group, and those
indirectly validated the data of the first administration. All of those during
0-72h after administration.
TABLE 4, thermal pain threshold of mice
Days after AB5C1 group Model group Negative control
group
surgical
operation (d) SD SEM SD+SEM SD SEM
dO 0.00% 0.00% 0.00% 0.00% 0.00% 0.00%
d10/before -56.31% 6.98% -50.99% 8.19% -20.42%
5.35%
administration
d12 -23.65% 5.17% -51.44% 1.28% -18.70%
5.98%
d14 -33.76% 7.43% -32.23% 7.93% -11.93%
5.13%
d16 -33.36% 5.58% -45.41% 3.46% -17.46%
6.53%
d17/before
-42.23% 2.14% -40.84% 4.14% -37.29% 4.86%
administration
d18 -16.18% 5.09% -37.35% 2.84% -35.19%
4.26%
d20 -27.62% 3.55% -38.76% 3.01% -20.69%
6.34%
d21 -31.58% 3.62% -37.25% 4.23% -17.91%
3.79%
d23 -40.86% 3.22% -39.25% 2.44% -25.32%
3.00%
8.3 Efficacy study of NGF antibodies with gouty arthritis mice model induced
by sodium urate
This experiment is to validate the efficacy of NGF antibody on mice acute
gouty arthritis models
which induced by sodium urate. SPF C57BL / 6J female mice with weight of about
25 g were
randomly divided into 3 groups, 8 mice in each group. Before the experiment,
the mice were put into
the Hargreaves device (parameter setting as 8.1) to adapt for a period of
time. The foot base pain
threshold of each mouse of rest time was measured at 0.5-2 h interval, three
times in total, and set the
average value of the foot base pain threshold as the baseline (d0 value). NGF
antibody AB5C1 and
AB5C2 were injected subcutaneously at the dose of 10 mg / kg in the tested
group, and PBS buffer
of equal volume (10 ml! kg) was injected in the model group. One hour after
administration, 2.5%
sodium urate (sigma) solution was injected into the ankle joint for 30 u. L;
the post foot thermal pain
27
Date Recue/Date Received 2020-06-17
threshold was measured at 4, 24 and 48 h after injection of sodium urate at
0.5-2 h interval, three
times in total. The average value was taken as the actual threshold value, and
the percentage (%) of
increase in the thermal pain threshold was calculated using the same formula
as 8.1. For statistical
analysis of data, the software and analysis method are the same as 8.1.
Figure 8 showed that at 4-6 h after administration, the NGF antibody AB5C1 and
AB5C2
significantly prolonged the thermal pain threshold of mice in gouty arthritis
mice model. At 4-6 h, 24
h and 48 h, the statistical difference of AB5C1 was very significant, while
the statistical difference of
NGF antibody AB5C2 group was only significant at the day of administration;
however, the mean
value of the two groups was still very high compared with the model group,
showing significantly
prolonged the thermal pain threshold of mice.
8.4 Efficacy study of NGF antibodies with inflammatory pain model induced by
Freund's
adjuvant
To validate if the NGF antibodies could alleviate the pain in chronic
peripheral inflammatory
mice model or not, C57BL / 6J mice after subcutaneous injection of Freund's
adjuvant were used as
test model. SPF C57BL / 6J female mice with weight of about 25 g were randomly
divided into 3
groups, 8 mice in each group. Before the experiment, the mice were put into
the Hargreaves device
(parameter setting as 8.1) to adapt for a period of time. The foot base pain
threshold of each mouse
of rest time was measured at 0.5-2 h interval, three times in total, and set
the average value of the
foot base pain threshold as the baseline (d0 value). NGF antibody AB5C1 and
AB5C6 were injected
subcutaneously at the dose of 10 mg / kg in the tested group, and PBS buffer
of equal volume (10 ml
/ kg) was injected into the model group. After one hour of administration, 50
ill of 0.5% complete
Freund's adjuvant (BD company) solution was injected into the ankle joint;
after 4, 24, 48, 72 and 96
hours of injection, the thermal pain threshold of the foot bottom of the hind
limbs of each mouse was
measured at 0.5-2 h interval, three times in total. The average value was
taken as the actual threshold
value, and the increase percentage (%) of the thermal pain threshold was
calculated according to the
formula listed in 8.1. For statistical analysis of data, the software and
analysis method are the same
as 8.1.
Figure 9 shows that 4-72 h after administration, the thermal pain threshold of
NGF antibody
AB5C6 group is higher than that of the model group and the NGF antibody AB5C1
group, and the
statistical difference is significant. The efficacy of NGF antibody AB5C1 is
slightly weaker than that
of NGF antibody AB5C6, and the statistical difference can only be significant
at 48 h after
administration. There was no significant difference between them after 96
hours of administration. In
conclusion, the tested NGF antibody can relieve pain cause by immune
inflammatory in the pain
28
Date Recue/Date Received 2020-06-17
mice model, and among them , NGF antibody AB5C6 showed better efficacy than
that of NGF
antibody AB5C1.
It is understood that the examples and embodiments described herein are for
illustrative purpose
only and that various modifications or changes in light thereof will be
suggested to persons skilled in
the art and are to be included within the spirit and purview of this
application. Although preferred
examples of the invention are described, it is to be understood that those
skilled in the art may make
various changes in accordance with the teachings herein, which are not
contrary to the scope of the
invention.
All references mentioned in the invention are cited as references in the
application, just as each
reference is cited as a reference separately. In addition, it should be
understood that after reading the
above teaching contents of the invention, those skilled in the art can make
various changes or
modifications to the invention, and these equivalent forms also fall within
the scope of the claims
attached to the application.
29
Date Recue/Date Received 2020-06-17
