U.S. patent application number 17/669800 was filed with the patent office on 2022-05-26 for compositions and methods for cancer therapy.
The applicant listed for this patent is Board of Regents of the University of Texas System, Exphenom, LLC. Invention is credited to Bruce Beutler, Evan Nair-Gill, Pingping Wang, Jinglei Zhang, Xue Zhong.
Application Number | 20220162303 17/669800 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220162303 |
Kind Code |
A1 |
Beutler; Bruce ; et
al. |
May 26, 2022 |
COMPOSITIONS AND METHODS FOR CANCER THERAPY
Abstract
The present disclosure generally relates to compositions and
methods for cancer immunotherapy, as well as hematopoietic recovery
following cancer treatment such as chemotherapy or irradiation.
Inventors: |
Beutler; Bruce; (Austin,
TX) ; Nair-Gill; Evan; (Austin, TX) ; Zhong;
Xue; (Austin, TX) ; Zhang; Jinglei; (Austin,
TX) ; Wang; Pingping; (Austin, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents of the University of Texas System
Exphenom, LLC |
Austin
Carlsbad |
TX
CA |
US
US |
|
|
Appl. No.: |
17/669800 |
Filed: |
February 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16767152 |
May 27, 2020 |
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PCT/US2018/063082 |
Nov 29, 2018 |
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17669800 |
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62591874 |
Nov 29, 2017 |
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International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1-17. (canceled)
18. A method for cancer immunotherapy, comprising administering to
a patient having cancer a therapeutically effective amount of
anti-LRP10 antibody or antigen-binding fragment thereof.
19. The method of claim 18, wherein the anti-LRP10 antibody or
antigen-binding fragment thereof is an anti-LRP10 antibody.
20. The method of claim 18, wherein the anti-LRP10 antibody or
antigen-binding fragment thereof is an LRP10 antigen-binding
fragment.
21. The method of claim 18, wherein said anti-LRP10 antibody or
antigen-binding fragment thereof enhances tumor infiltration of
lymphocytes, thereby providing cancer immunotherapy.
22. The method of claim 21, wherein the lymphocytes are CD8+ T
cells and cytotoxic T lymphocytes.
23. A method for hematopoietic recovery, comprising administering
to a patient in need of hematopoietic recovery a therapeutically
effective amount of anti-LRP10 antibody or antigen-binding fragment
thereof.
24. The method of claim 23, wherein the anti-LRP10 antibody or
antigen-binding fragment thereof is an anti-LRP10 antibody.
25. The method of claim 24, wherein the anti-LRP10 antibody or
antigen-binding fragment thereof is an LRP10 antigen-binding
fragment.
26. The method of any one of claim 23, wherein said anti-LRP10
antibody or antigen-binding fragment thereof enhances recovery of
all hematopoietic lineages, in a patient who has received
chemotherapy and/or radiotherapy.
27. The method of claim 26, wherein said anti-LRP10 antibody or
antigen-binding fragment thereof enhances recovery of lymphoid
lineages.
28. The method of claim 18, wherein the anti-LRP10 antibody or
antigen-binding fragment thereof is administered parenterally.
29. The method of claim 18, wherein the method further comprises
administering at least one additional anti-cancer agent.
30. The method of claim 29, wherein the at least one additional
anti-cancer agent is a chemotherapeutic drug, an EGFR inhibitor, a
VEGF inhibitor, an anti-ErbB2 antibody, or any combination
thereof.
31. A method for identifying an LRP10 inhibitor, comprising
contacting a cell with a test agent, wherein an increase in
migration toward a chemotactic stimulus compared to a control cell
that is not contacted with the test agent indicates that the test
agent is an LRP10 inhibitor, wherein the chemotactic stimulus is
selected from C-X-C motif chemokine 12 (CXCL12), C-X-C motif
chemokine 10 (CXCL10), sphingosine-1-phosphate (S1P), C-C motif
ligand 2 (CCL2), and/or C-C motif ligand 21 (CCL21).
33. The method of claim 31, wherein the test agent is an anti-LRP10
antibody or antigen-binding fragment thereof or a soluble receptor
for one or more LRP10 ligands.
34. The method of claim 33, wherein the test agent is a soluble
receptor for one or more LRP10 ligands and is an LRP10 Q-Fc
chimera.
35. A method for identifying an LRP10 inhibitor, comprising
contacting a cell with a test agent, wherein an increase in
expression of Frizzled and/or p21 compared to a control cell that
is not contacted with the test agent indicates that the test agent
is an LRP10 inhibitor.
36. The method of claim 35, wherein the test agent is an anti-LRP10
antibody or antigen-binding fragment thereof or a soluble receptor
for one or more LRP10 ligands.
37. The method of claim 36, wherein the test agent is a soluble
receptor for one or more LRP10 ligands and is an LRP10 Q-Fc
chimera.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 62/591,874 filed Nov. 29, 2017,
the disclosure of which is incorporated herein by reference in its
entirety.
FIELD
[0002] The present disclosure generally relates to compositions and
methods for cancer immunotherapy, as well as hematopoietic recovery
following cancer treatment such as chemotherapy or irradiation.
BACKGROUND
[0003] Despite multiple preventative and therapeutic approaches,
cancer is one of the major causes of death worldwide. Between 2010
and 2020, the number of new cancer cases in the United States is
expected to increase by about 24% in men to more than 1 million
cases per year, and by about 21% in women to more than 900,000
cases per year.
[0004] The types of cancer that are expected to increase the most
are melanoma in both men and women; prostate, kidney, liver, and
bladder cancers in men; and lung, breast, uterine, and thyroid
cancers in women. Cancer remains the second most common cause of
death in the US, accounting for nearly 1 of every 4 deaths. Many
cancers are difficult or impossible to treat with current
approaches. Many cancers evade current treatment regimens, become
resistant to treatment, or reoccur after treatment. For example,
cytotoxic chemotherapy, one of the most common systemic treatment
options for cancer, has limited efficacy, especially in the
treatment of solid tumors.
[0005] Hematopoietic recovery is one of the key factors affecting
the outcome of chemotherapy. Faster bone marrow recovery leads to
fewer adverse consequences and enables patients to proceed with
further courses of chemotherapy without delay. A delayed bone
marrow recovery can lead to an uncontrolled infection that results
in treatment failure. The hematopoietic toxicity of chemotherapy is
an important factor in determining the doses for treatment
regimens. Drugs such as NEULASTA.RTM. and NEUPOGEN.RTM. (G-CSF) are
currently used to assist in hematopoietic recovery after
chemotherapy. However, these drugs are neutrophil boosters. There
is currently no drug that encourages lymphoid recovery. As such, a
need exists for effective methods and compositions to promote
hematopoietic recovery, preferably all hematopoietic lineages
including lymphoid cells, after chemotherapy.
SUMMARY
[0006] Provided herein are compositions and methods for cancer
immunotherapy, as well as hematopoietic recovery following cancer
treatment such as chemotherapy.
[0007] In one aspect, a method of providing cancer immunotherapy is
provided, comprising inhibiting LRP10 in a subject in need thereof.
In some embodiments, said inhibiting enhances tumor infiltration of
lymphocytes, preferably CD8+ T cells and cytotoxic T lymphocytes,
thereby providing cancer immunotherapy.
[0008] Another aspect relates to a method of enhancing
hematopoietic recovery, comprising inhibiting LRP10 in a subject in
need thereof. In various embodiments, said inhibiting enhances
recovery of all hematopoietic lineages, preferably lymphoid
lineages. In some embodiments, the subject has received
chemotherapy and/or radiotherapy.
[0009] In various embodiments, methods and use disclosed herein can
include administering to the subject an effective amount of
anti-LRP10 antibody or antigen-binding fragment thereof, or a
soluble receptor for one or more LRP10 ligands (e.g., an LRP10-Fc
chimera).
[0010] Another aspect relates to use of an LRP10 inhibitor or
competitor for the manufacture of a medicament for cancer
immunotherapy. In some embodiments, said LRP10 inhibitor or
competitor enhances tumor infiltration of lymphocytes, preferably
CD8+ T cells and cytotoxic T lymphocytes, thereby providing cancer
immunotherapy.
[0011] A further aspect relates to use of an LRP10 inhibitor or
competitor for the manufacture of a medicament for hematopoietic
recovery. In some embodiments, said LRP10 inhibitor or competitor
enhances recovery of all hematopoietic lineages, preferably
lymphoid lineages, in a patient who has received chemotherapy
and/or radiotherapy.
[0012] In certain embodiments, said LRP10 inhibitor is anti-LRP10
antibody or antigen-binding fragment thereof, preferably binding to
LRP10 ectodomain. In some embodiments, said LRP10 competitor is an
engineered, soluble receptor for one or more LRP10 ligands, wherein
said receptor competes for binding with endogenous LRP10 (e.g., an
LRP10-Fc chimera).
[0013] Also disclosed herein is an anti-LRP10 antibody, or antigen
binding fragment thereof, optionally for use in cancer
immunotherapy and/or hematopoietic recovery, comprising one or more
of the following CDRs:
TABLE-US-00001 VL CDR1 (SEQ ID NO: 33): RASQSISSYLN VL CDR2 (SEQ ID
NO: 62): X1ASX2LQS (X1 = N, A, R, D; X2 = D, P, R, A, L, T) VL CDR3
(SEQ ID NO: 63): QQX3X4X5X6PX7T (X3 = S, V, P, A, I, T, N; X4 = S,
A, T, D, K; X5 = R, S, T, A, Y; X6 = T, L, Y, R, G; X7 = T, N, L,
G) VH CDR1 (SEQ ID NO: 64): SX8AMS (X8 = Q, Y) VH CDR2 (SEQ ID NO:
65): X9IX10X11X12GX13X14TX15YADSVKG (X9 = S, Q, V; X10 = P, G, S,
Q, A, Y; X11 = P, T, R, S; X12 = G, M, T, Q, R, S; X13 = P, R, N,
T, Q, A; X14 = N, P, S, G, A, T; X15 = K, T, Y, E) VH CDR3 (SEQ ID
NO: 66): X16X17X18X19FDY (X16 = S, D, N; X17 = Y, G, A, S, R, T;
X18 = P, K, T, R, H, A; X19 = S, K, T)
[0014] In some embodiments, the antibody or fragment thereof can
include one or more of the following CDRs:
TABLE-US-00002 VL CDR1 (SEQ ID NO: 33): RASQSISSYLN VL CDR2 (SEQ ID
NO: 34): NASDLQS VL CDR2 (SEQ ID NO: 35): AASPLQS VL CDR2 (SEQ ID
NO: 36): RASRLQS VL CDR2 (SEQ ID NO: 37): AASALQS VL CDR2 (SEQ ID
NO: 38): DASLLQS VL CDR2 (SEQ ID NO: 39): AASTLQS VL CDR3 (SEQ ID
NO: 40): QQSSRTPTT VL CDR3 (SEQ ID NO: 41): QQVARTPNT VL CDR3 (SEQ
ID NO: 42): QQPTSLPLT VL CDR3 (SEQ ID NO: 43): QQADSYPTT VL CDR3
(SEQ ID NO: 44): QQIKTRPTT VL CDR3 (SEQ ID NO: 45): QQTSAGPGT VL
CDR3 (SEQ ID NO: 46): QQNDYYPTT VH CDR1 (SEQ ID NO: 47): SQAMS VH
CDR1 (SEQ ID NO: 48): SYAMS VH CDR2 (SEQ ID NO: 49):
QIGTMGRPTTYADSVKG VH CDR2 (SEQ ID NO: 50): SISTTGNSTYYADSVKG VH
CDR2 (SEQ ID NO: 51): VIQRQGTGTEYADSVKG VH CDR2 (SEQ ID NO: 52):
SIPSRGQATKYADSVKG VH CDR2 (SEQ ID NO: 53): SIATTGNTTYYADSVKG VH
CDR2 (SEQ ID NO: 54): SIPPGGPNTKYADSVKG VH CDR2 (SEQ ID NO: 55):
SIYTSGAATTYADSVKG VH CDR3 (SEQ ID NO: 56): SYPSFDY VH CDR3 (SEQ ID
NO: 57): SGKKFDY VH CDR3 (SEQ ID NO: 58): DATSFDY VH CDR3 (SEQ ID
NO: 59): NSRTFDY VH CDR3 (SEQ ID NO: 60): SRHTFDY VH CDR3 (SEQ ID
NO: 61): NTATFDY
[0015] In some embodiments, the antibody or fragment thereof can
have a VL sequence selected from the group consisting of SEQ ID
NOS: 1, 5, 7, 11, 15, 19, and 23 and a VH sequence selected from
the group consisting of SEQ ID NOS: 2, 8, 12, 16, 20, 24, 27, and
29.
[0016] A further aspect relates to a soluble receptor for one or
more LRP10 ligands, wherein said soluble receptor competes with
endogenous LRP10 for binding with the one or more LRP10 ligands. In
some embodiments, the soluble receptor is an LRP10-Fc chimera.
[0017] In some embodiments, in a migration assay, a cell (e.g.,
hematopoietic stem cell or T cell) contacted with the antibody or
fragment thereof or the soluble receptor for one or more LRP10
ligands (e.g., an LRP10-Fc chimera) migrates toward a chemotactic
stimulus more rapidly than a control cell that is not contacted
with the antibody or fragment thereof or the soluble receptor,
wherein preferably the chemotactic stimulus is selected from C-X-C
motif chemokine 12 (CXCL12), C-X-C motif chemokine 10 (CXCL10),
sphingosine-1-phosphate (S1P), C-C motif ligand 2 (CCL2), and/or
C-C motif ligand 21 (CCL21).
[0018] In certain embodiments, a cell (e.g., hematopoietic stem
cell or T cell) contacted with the antibody or fragment thereof or
the soluble receptor for one or more LRP10 ligands (e.g., an
LRP10-Fc chimera) shows increased expression of Frizzled and/or P21
compared to a control cell that is not contacted with the antibody
or fragment thereof or the soluble receptor.
[0019] Also provided herein is a pharmaceutical composition for
cancer immunotherapy and/or hematopoietic recovery, comprising the
antibody or fragment thereof and/or the soluble receptor for one or
more LRP10 ligands (e.g., an LRP10-Fc chimera) disclosed herein and
a pharmaceutically acceptable carrier. Use of the antibody or
fragment thereof and/or the soluble receptor for one or more LRP10
ligands (e.g., an LRP10-Fc chimera) disclosed herein, for the
manufacture of a medicament for cancer immunotherapy and/or
hematopoietic recovery, is also provided.
[0020] A method for identifying an LRP10 inhibitor is also
provided, comprising contacting a cell (e.g., hematopoietic stem
cell or T cell) with a test agent, wherein an increase in migration
toward a chemotactic stimulus compared to a control cell that is
not contacted with the test agent indicates that the test agent is
an LRP10 inhibitor, wherein preferably the chemotactic stimulus is
selected from C-X-C motif chemokine 12 (CXCL12), C-X-C motif
chemokine 10 (CXCL10), sphingosine-1-phosphate (SIP), C-C motif
ligand 2 (CCL2), and/or C-C motif ligand 21 (CCL21). In some
embodiments, the test agent is an antibody or antigen-binding
fragment thereof, or a soluble receptor for one or more LRP10
ligands (e.g., an LRP10-Fc chimera).
[0021] Another method for identifying an LRP10 inhibitor is also
provided, which includes contacting a cell (e.g., hematopoietic
stem cell or T cell) with a test agent, wherein an increase in
expression of p21 compared to a control cell that is not contacted
with the test agent indicates that the test agent is an LRP10
inhibitor. In some embodiments, the test agent is an antibody or
antigen-binding fragment thereof, or a soluble receptor for one or
more LRP10 ligands (e.g., an LRP10-Fc chimera).
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a schematic showing transplantation of bone marrow
cells from C57BL/6 mice (CD45.1), CRISPR-Lrp10 KO mice (CD45.2), or
a 1:1 mixture into irradiated Rag2.sup.-/- recipient mice.
[0023] FIG. 2 shows flow cytometry results of T cells, CD4+ cells,
CD8+ cells, B cells, and NK cells in peripheral blood from donor
chimeras (1:1 mixture of CD45.1 and CD45.2) in irradiated
Rag2.sup.-/- mice post-transplantation.
[0024] FIG. 3 shows the percentage of different lymphocytes
populations (T cells, CD4+ cells, CD8+ cells, B cells, and NK
cells) in peripheral blood from donor chimeras (1:1 mixture of
CD45.1 and CD45.2) in irradiated Rag2.sup.1 mice
post-transplantation, suggesting that chowmein homozygotes display
an increase in the competitive potential of hematopoietic stem
cells.
[0025] FIG. 4 shows the percentage of different myeloid populations
(macrophages and neutrophils cells) in peripheral blood from donor
chimeras (1:1 mixture of CD45.1 and CD45.2) in irradiated Rag2'
mice post-transplantation, suggesting that chowmein homozygotes
display an increase in the competitive potential of hematopoietic
stem cells.
[0026] FIG. 5 shows that Lrp10.sup.-/- peripheral T lymphocytes
(clear circles) migrate at a higher rate than Lrp10.sup.+/+ (dark
circles) in a chemotaxis assay.
[0027] FIG. 6 shows significantly increased expression of Frizzled
and P21 in Lrp10.sup.-/- T cells and hematopoietic progenitors than
Lrp10.sup.+/+ cells.
[0028] FIG. 7 shows that Lrp10.sup.-/- homozygotes showed higher
proportion of CD4 and CD8 T cells.
[0029] FIG. 8 shows that melanoma growth is retarded or arrested in
mice lacking Lrp10.
[0030] FIG. 9 shows that Lrp10.sup.-/- homozygotes challenged with
melanoma B16 cells showed higher active CD8+ T cells in the
blood.
[0031] FIG. 10 shows expression and purification of 9
antibodies.
[0032] FIG. 11 shows ELISA (enzyme-linked immunosorbent assay)
results of antibody binding to LRP10.
[0033] FIG. 12 shows that human anti-Lrp10 monoclonal antibody can
activate p21 in mouse T cells.
[0034] FIG. 13 shows that both AB2 and AB7 enhance T cell migration
in response to SDF12.
DETAILED DESCRIPTION
[0035] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the compositions
and methods of the present disclosure.
[0036] Disclosed herein are compositions and methods related to
inhibiting LRP10, enhancing hematopoietic recovery in a subject
after chemotherapy or irradiation, and/or improving cancer
immunotherapy, as well as kits that can be used in such methods.
One aspect of the present disclosure relates to the surprising
discovery that a mutation in Lrp10, or knockout of Lrp10, causes a
phenotype characterized by increased speed of hematopoietic
recovery, and in the case of mixed chimeras, out-competition of the
wild-type (WT) cells by mutant cells. As such, an inhibitor of
LRP10, such as an antibody, can be used to promote hematopoietic
recovery in a subject after, e.g., chemotherapy or irradiation. In
addition, without wishing to be bound by theory, it is believed
that LRP10 blockade may be equivalent to a checkpoint blockade,
allowing CD8+ T cells to infiltrate tumors more effectively than
they otherwise would. As such, LRP10 inhibition can also be used to
augment immunotherapy treatment of a cancer (e.g., by increasing
the number of tumor infiltrating T lymphocytes).
[0037] LRP10 is a single-spanning plasma membrane receptor,
previously of unknown function. Unexpectedly, Lrp10 KO T cells
migrate toward a chemotactic stimulus more rapidly than WT cells.
Furthermore, Lrp10 KO T cells show high expression of Fizzled and
p21, p21 being an end-product of the non-canonical Wnt signaling
pathway. In some embodiments, in a chemotaxis migration assay,
Frizzled expression and/or p21 expression can be used as a marker
in screening for inhibitors of LRP10.
[0038] Therefore, LRP10 inhibitors such as the antibodies and
soluble receptors disclosed herein, can be used to treat a subject,
by improving hematopoietic recovery after chemotherapy or
irradiation, and/or improving cancer immunotherapy.
Definitions
[0039] For convenience, certain terms employed in the
specification, examples, and appended claims are collected here.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
[0040] As used herein, the following terms and phrases are intended
to have the following meanings: The articles "a" and "an" are used
herein to refer to one or to more than one (i.e., to at least one)
of the grammatical object of the article. By way of example, "an
element" means one element or more than one element.
[0041] As used herein, the term "about" means acceptable variations
within 20%, more preferably within 10% and most preferably within
5% of the stated value.
[0042] "Lrp10" and "LRP10," also known as LRP9, LRP-10, MST087, or
MSTP087 are used interchangeably and refer to LDL receptor related
protein 10, with "Lrp10" generally referring to the gene or mRNA
and "LRP10" the protein product unless otherwise noted. It should
be understood that the terms include the complete gene, the cDNA
sequence, the complete amino acid sequence, or any fragment or
variant thereof. In some embodiments, the LRP10 is human LRP10.
[0043] As used herein, the term "LRP10 inhibitor" is intended to
include therapeutic agents that inhibit, down-modulate, suppress or
down-regulate LRP10 activity. The term is intended to include
chemical compounds, such as small molecule inhibitors and biologic
agents (e.g., antibodies), interfering RNA (shRNA, siRNA), soluble
antagonists, gene editing/silencing tools (CRISPR/Cas9, TALENs) and
the like.
[0044] An "anti-LRP10 antibody" is an antibody that
immunospecifically binds to LRP10 (e.g., its extracellular domain).
The antibody may be an isolated antibody. Such binding to LRP10
exhibits a K.sub.d with a value of, e.g., no greater than 1 .mu.M,
no greater than 100 nM or no greater than 50 nM. Kd can be measured
by any methods known to one skilled in the art, such as a surface
plasmon resonance assay or a cell binding assay. An anti-LRP10
antibody may be a monoclonal antibody, or antigen-binding fragments
thereof. Exemplary anti-LRP10 antibodies may inhibit LRP10 binding
with its ligand(s) (e.g., an endogenous ligand).
[0045] An "antibody," as used herein is a protein consisting of one
or more polypeptides comprising binding domains that bind to a
target epitope. The term antibody includes monoclonal antibodies
comprising immunoglobulin heavy and light chain molecules, single
heavy chain variable domain antibodies, and variants and
derivatives thereof, including chimeric variants of monoclonal and
single heavy chain variable domain antibodies. Binding domains are
substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes, wherein the protein immunospecifically binds
to an antigen. The recognized immunoglobulin genes include the
kappa, lambda, alpha, gamma, delta, epsilon and mu constant region
genes, as well as myriad immunoglobulin variable region genes.
Light chains are classified as either kappa or lambda. Heavy chains
are classified as gamma, mu, alpha, delta, or epsilon, which in
turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. For most vertebrate organisms, including humans and
murine species, the typical immunoglobulin structural unit
comprises a tetramer that is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kD) and
one "heavy" chain (about 50-70 kD). "V.sub.L" and V.sub.H" refer to
the variable domains of these light and heavy chains respectively.
"C.sub.L" and C.sub.H" refer to the constant domains of the light
and heavy chains. Loops of .beta.-strands, three each on the
V.sub.L and V.sub.H are responsible for binding to the antigen, and
are referred to as the "complementarity determining regions" or
"CDRs". The "Fab" (fragment, antigen-binding) region includes one
constant and one variable domain from each heavy and light chain of
the antibody, i.e., V.sub.L, C.sub.L, V.sub.H and C.sub.H1.
[0046] Antibodies include intact immunoglobulins as well as
antigen-binding fragments thereof. The term "antigen-binding
fragment" refers to a polypeptide fragment of an antibody which
binds antigen or competes with intact antibody (i.e., with the
intact antibody from which they were derived) for antigen binding
(i.e., specific binding). Antigen binding fragments can be produced
by recombinant or biochemical methods that are well known in the
art. Exemplary antigen-binding fragments include Fv, Fab, Fab',
(Fab').sub.2, CDR, paratope and single chain Fv antibodies (scFv)
in which a V.sub.H and a V.sub.L chain are joined together
(directly or through a peptide linker) to form a continuous
polypeptide.
[0047] Another class of antibodies known as heavy chain antibodies
(HCA, also referred to as two-chain or two-chain heavy chain
antibodies) have been reported in camelids such as dromedary
camels, Bactrian camels, wild Bactrian camels, llamas, alpacas,
vicunas, and guanacos (Hamers-Casterman et al., Nature, 363,
446-448 (1993); Wesolowski et al., Med. Microbiol. Immunol (2009)
198:157-174; see also U.S. Pat. Nos. 5,759,808; 5,800,988;
5,840,526; and 5,874,541). Compared with conventional four-chain
immunoglobulins of IgG-type, which are also produced by camelids,
these antibodies lack the light chains and CH1 domains of
conventional immunoglobulins, and their variable domains are
sometimes designated "V.sub.HH". V.sub.HH can include four
framework regions or "FR", FR1, FR2, FR3 and FR4. The framework
regions are interrupted by three CDRs, CDR1, CDR2 and CDR3. One of
the salient features of these naturally occurring heavy chain
antibodies is the predominant presence of Glu, Arg and Gly at VL
interface positions 44, 45 and 47 (Kabat numbering), respectively,
of their V.sub.HH. The same positions in the VH of conventional
four-chain antibodies (are almost exclusively occupied by Gly, Leu
and Trp. These differences are thought to be responsible for the
high solubility and stability of camelid HCA variable domain
(V.sub.HH), as compared with the relative insolubility of VH domain
of the conventional four-chain antibodies. Two more salient
features of camelid V.sub.HH domains are their comparatively longer
CDR3 and high incidence of cysteine pairs in CDRs. It appears that
cysteine pairs mediate the formation of a disulfide bridge and are
therefore involved in modulating the surface topology of the
antibody combining site. In the crystal structure of a camel
sdAb-lysozyme complex, a rigid loop protruding from the sdAb and
partly stabilized by a CDR disulfide linkage extends out of the
combining site and penetrates deeply into the lysozyme active site
(Desmyter et al., Nature Struct. Biol., 3, 803-811 (1996)).
[0048] Antibodies also include variants, chimeric antibodies and
humanized antibodies. The term "antibody variant" as used herein
refers to an antibody with single or multiple mutations in the
heavy chains and/or light chains. In some embodiments, the
mutations exist in the variable region. In some embodiments, the
mutations exist in the constant region. "Chimeric antibodies"
refers to those antibodies wherein one portion of each of the amino
acid sequences of heavy and light chains is homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular class, while the remaining
segment of the chains is homologous to corresponding sequences in
another. Typically, in these chimeric antibodies, the variable
region of both light and heavy chains mimics the variable regions
of antibodies derived from one species of mammals, while the
constant portions are homologous to the sequences in antibodies
derived from another. One clear advantage to such chimeric forms is
that, for example, the variable regions can conveniently be derived
from presently known sources using readily available hybridomas or
B cells from non-human host organisms in combination with constant
regions derived from, for example, human cell preparations. While
the variable region has the advantage of ease of preparation, and
the specificity is not affected by its source, the constant region
being human, is less likely to elicit an immune response from a
human subject when the antibodies are injected than would the
constant region from a non-human source. However, the definition is
not limited to this particular example. "Humanized" antibodies
refer to a molecule having an antigen-binding site that is
substantially derived from an immunoglobulin from a non-human
species and the remaining immunoglobulin structure of the molecule
based upon the structure and/or sequence of a human immunoglobulin.
The antigen-binding site may comprise either complete variable
domains fused onto constant domains or only the complementarity
determining regions (CDRs) grafted onto appropriate framework
regions in the variable domains. Antigen binding sites may be wild
type or modified by one or more amino acid substitutions, e.g.,
modified to resemble human immunoglobulin more closely. Some forms
of humanized antibodies preserve all CDR sequences (for example, a
humanized mouse antibody which contains all six CDRs from the mouse
antibodies). Other forms of humanized antibodies have one or more
CDRs (one, two, three, four, five, or six) which are altered with
respect to the original antibody, which are also termed one or more
CDRs "derived from" one or more CDRs.
[0049] As described herein, the amino acid residues of an antibody,
including VHH, can be numbered according to the general numbering
of Kabat (Kabat, et al. (1991) Sequences of Proteins of
Immunological Interest, 5th edition. Public Health Service, NTH,
Bethesda, Md.).
[0050] The term "binding" as used herein in the context of binding
between an antibody, such as a VHH, and an epitope of LRP10 as a
target, refers to the process of a non-covalent interaction between
molecules. Preferably, said binding is specific. The specificity of
an antibody can be determined based on affinity. A specific
antibody can have a binding affinity or dissociation constant Kd
for its epitope of less than 10.sup.-7 M, preferably less than
10.sup.-8 M.
[0051] The term "affinity" refers to the strength of a binding
reaction between a binding domain of an antibody and an epitope. It
is the sum of the attractive and repulsive forces operating between
the binding domain and the epitope. The term affinity, as used
herein, refers to the dissociation constant, K.sub.d.
[0052] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0053] The term "cancer" broadly refers to an uncontrolled,
abnormal growth of a host's own cells leading to invasion of
surrounding tissue and potentially tissue distal to the initial
site of abnormal cell growth in the host. Major classes include
carcinomas which are cancers of the epithelial tissue (e.g., skin,
squamous cells); sarcomas which are cancers of the connective
tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.);
leukemias which are cancers of blood forming tissue (e.g., bone
marrow tissue); lymphomas and myelomas which are cancers of immune
cells; and central nervous system cancers which include cancers
from brain and spinal tissue. "Cancer(s)," "neoplasm(s)," and
"tumor(s)" are used herein interchangeably. As used herein,
"cancer" refers to all types of cancer or neoplasm or malignant
tumors including leukemias, carcinomas and sarcomas, whether new or
recurring. Specific examples of cancers are: carcinomas, sarcomas,
myelomas, leukemias, lymphomas and mixed type tumors. Non-limiting
examples of cancers are new or recurring cancers of the brain,
melanoma, bladder, breast, cervix, colon, head and neck, kidney,
lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma,
stomach, uterus and medulloblastoma.
[0054] The term "cellular augmentation" or "immunotherapy
augmentation" broadly refers to the influx of cells or expansion of
cells in an environment that are not substantially present in the
environment prior to administration of a composition and not
present in the composition itself. Cells that augment the
environment include immune cells, stromal cells, bacterial and
fungal cells. Environments of particular interest are the
microenvironments where cancer cells reside or locate. In some
instances, the microenvironment is a tumor microenvironment or a
tumor draining lymph node. In other instances, the microenvironment
is a pre-cancerous tissue site or the site of local administration
of a composition or a site where the composition will accumulate
after remote administration.
[0055] The term "epitope" includes any determinant, preferably a
polypeptide determinant, capable of specific binding to an
immunoglobulin or T-cell receptor. In certain embodiments, epitope
determinants include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific
three-dimensional structural characteristics, and/or specific
charge characteristics. An epitope is a region of an antigen that
is bound by an antibody. In certain embodiments, an antibody is
said to specifically bind an antigen when it preferentially
recognizes its target antigen in a complex mixture of proteins
and/or macromolecules. Methods for epitope mapping are well known
in the art, such as X-ray co-crystallography, array-based
oligo-peptide scanning, site-directed mutagenesis, high throughput
mutagenesis mapping and hydrogen-deuterium exchange.
[0056] The site on the antibody that binds the epitope is referred
to as "paratope," which typically include amino acid residues that
are in close proximity to the epitope once bound. See Sela-Culang
et al., Front Immunol. 2013; 4: 302.
[0057] "Immunohistochemistry" or "IHC" refers to the process of
detecting an antigen in cells of a tissue section allowing the
binding and subsequent detection of antibodies immunospecifically
recognizing the antigen of interest in a biological tissue. For a
review of the IHC technique, see, e.g., Ramos-Vara et al.,
Veterinary Pathology January 2014 vol. 51 no. 1, 42-87,
incorporated herein by reference in its entirety. To evaluate IHC
results, different qualitative and semi-quantitative scoring
systems have been developed. See, e.g., Fedchenko et al.,
Diagnostic Pathology, 2014; 9: 221, incorporated herein by
reference in its entirety. One example is the H-score, determined
by adding the results of multiplication of the percentage of cells
with staining intensity ordinal value (scored from 0 for "no
signal" to 3 for "strong signal") with 300 possible values.
[0058] "Immunospecific" or "immunospecifically" (sometimes used
interchangeably with "specifically") refer to antibodies that bind
via domains substantially encoded by immunoglobulin genes or
fragments of immunoglobulin genes to one or more epitopes of a
protein of interest, but which do not substantially recognize and
bind other molecules in a sample containing a mixed population of
antigenic molecules. Typically, an antibody binds
immunospecifically to a cognate antigen with a K.sub.d with a value
of no greater than 50 nM, as measured by a surface plasmon
resonance assay or a cell binding assay. The use of such assays is
well known in the art.
[0059] The term "immune response" includes T cell mediated and/or B
cell mediated immune responses. Exemplary immune responses include
T cell responses, e.g., cytokine production and cellular
cytotoxicity. In addition, the term immune response includes immune
responses that are indirectly effected by T cell activation, e.g.,
antibody production (humoral responses) and activation of cytokine
responsive cells, e.g., macrophages.
[0060] "Immunotherapy" is treatment that uses a subject's immune
system to treat cancer and includes, for example innate
immunotherapy, adoptive transfer of tumor infiltrating lymphocytes
("TTL"), active specific immunotherapy ("ASI"), adoptive cellular
immunotherapy ("ACI"), adoptive immunotherapy, cancer antigen
immunotherapy ("CAI"), cytokine-expressing cancer immunotherapy,
monoclonal antibodies, therapeutic cancer vaccines, oncolytic virus
immunotherapy, adoptive T cell transfer, cytokine immunotherapy,
adjuvant immunotherapy.
[0061] The term "immunotherapeutic agent" can include any molecule,
peptide, antibody or other agent which can stimulate a host immune
system to generate an immune response to a tumor or cancer in the
subject. Various immunotherapeutic agents are useful in the
compositions and methods described herein.
[0062] The terms "cross-compete", "cross-competition",
"cross-block", "cross-blocked" and "cross-blocking" are used
interchangeably herein to mean the ability of an antibody or
fragment thereof to interfere with the binding directly or
indirectly through allosteric modulation of the anti-LRP10
antibodies of the present disclosure to the target LRP10. The
extent to which an antibody or fragment thereof is able to
interfere with the binding of another to the target, and therefore
whether it can be said to cross-block or cross-compete according to
the present disclosure, can be determined using competition binding
assays. One particularly suitable quantitative cross-competition
assay uses a FACS- or an AlphaScreen-based approach to measure
competition between the labelled (e.g. His tagged, biotinylated or
radioactive labelled) an antibody or fragment thereof and the other
an antibody or fragment thereof in terms of their binding to the
target. In general, a cross-competing antibody or fragment thereof
is for example one which can bind to the target in the
cross-competition assay such that, during the assay and in the
presence of a second antibody or fragment thereof, the recorded
displacement of the immunoglobulin single variable domain or
polypeptide according to the disclosure is up to 100% (e.g., in
FACS based competition assay) of the maximum theoretical
displacement (e.g., displacement by cold (e.g., unlabeled) antibody
or fragment thereof that needs to be cross-blocked) by the to be
tested potentially cross-blocking antibody or fragment thereof that
is present in a given amount. Preferably, cross-competing
antibodies or fragments thereof have a recorded displacement that
is between 10% and 100%, more preferred between 50% to 100%.
[0063] The terms "suppress", "suppression", "inhibit",
"inhibition", "neutralize" and "neutralizing" as used
interchangeably herein, refer to any statistically significant
decrease in biological activity (e.g., LRP10 activity or tumor cell
growth), including full blocking of the activity. For example,
"inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100% in biological activity.
[0064] The term "subject" or "patient" includes a human or other
mammalian animal that receives either prophylactic or therapeutic
treatment.
[0065] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures such as those
described herein. The methods of "treatment" employ administration
to a patient a LRP10 inhibitor provided herein, for example, a
patient having a cancer, in order to prevent, cure, delay, reduce
the severity of, or ameliorate one or more symptoms of the cancer
or recurring cancer, or in order to prolong the survival of a
patient beyond that expected in the absence of such treatment. The
methods of "treatment" also employ administration to a patient a
LRP10 inhibitor provided herein (e.g., an antibody) to enhance
hematopoietic recovery after chemotherapy or irradiation, and/or
improving cancer immunotherapy in a patient beyond that expected in
the absence of such treatment.
[0066] The term "effective amount," as used herein, refers to that
amount of an agent, such as a LRP10 inhibitor, for example an
anti-LRP10 antibody, which is sufficient to promote hematopoietic
recovery in a subject after chemotherapy or irradiation, and/or
effect treatment (e.g., immunotherapy), prognosis or diagnosis of a
cancer, when administered to a patient. A therapeutically effective
amount will vary depending upon the patient and disease condition
being treated, the weight and age of the patient, the severity of
the disease condition, the manner of administration and the like,
which can readily be determined by one of ordinary skill in the
art. The dosages for administration can range from, for example,
about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about
10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng
to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to
about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to
about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to
about 4,500 mg, about 500 ng to about 4,000 mg, about 1 .mu.g to
about 3,500 mg, about 5 .mu.g to about 3,000 mg, about 10 .mu.g to
about 2,600 mg, about 20 .mu.g to about 2,575 mg, about 30 .mu.g to
about 2,550 mg, about 40 .mu.g to about 2,500 mg, about 50 .mu.g to
about 2,475 mg, about 100 .mu.g to about 2,450 mg, about 200 .mu.g
to about 2,425 mg, about 300 .mu.g to about 2,000, about 400 .mu.g
to about 1,175 mg, about 500 .mu.g to about 1,150 mg, about 0.5 mg
to about 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to
about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to
about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to
about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about
925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875 mg,
about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30
mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to
about 750 mg, about 100 mg to about 725 mg, about 200 mg to about
700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg,
about 500 mg, or about 525 mg to about 625 mg, of an antibody or
antigen binding portion thereof, as provided herein. Dosing may be,
e.g., every week, every 2 weeks, every three weeks, every 4 weeks,
every 5 weeks or every 6 weeks. Dosage regimens may be adjusted to
provide the optimum therapeutic response. An effective amount is
also one in which any toxic or detrimental effects (side effects)
of the agent are minimized and/or outweighed by the beneficial
effects. Administration may be intravenous at exactly or about 6
mg/kg or 12 mg/kg weekly, or 12 mg/kg or 24 mg/kg biweekly.
Additional dosing regimens are described below.
[0067] Other terms used in the fields of recombinant nucleic acid
technology, microbiology, immunology, antibody engineering, and
molecular and cell biology as used herein will be generally
understood by one of ordinary skill in the applicable arts. For
example, conventional techniques may be used for preparing
recombinant DNA, performing oligonucleotide synthesis, and
practicing tissue culture and transformation (e.g.,
electroporation, transfection or lipofection). Enzymatic reactions
and purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See, e.g., Sambrook et al., 2001, Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated
herein by reference for any purpose. Unless specific definitions
are provided, the nomenclature utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0068] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are present in a given embodiment, yet open to the
inclusion of unspecified elements.
[0069] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the disclosure.
[0070] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0071] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0072] Various aspects and embodiments are described in further
detail in the following subsections.
LRP10
[0073] The low-density lipoprotein receptor (LDLR) is a cell
surface receptor that mediates lipoprotein metabolism in the body.
In humans, the LDLR gene resides on chromosome 19 at the band
19p13.2 and is split into 18 exons. Genetic deficiencies of the
LDLR gene give rise to familial hypercholesterolemia, one of the
most common genetic diseases in humans (Goldstein, J. L. et al. The
Metabolic Basis of Inherited Disease, sixth ed., McGraw-Hill, New
York, 1989, pp. 1215-1250). The LDLR gene encodes a single
transmembrane protein that consists of five functional domains: a
ligand binding domain composed of multiple cysteine-rich repeats;
an epidermal growth factor (EGF) precursor homology domain with the
sequence Tyr-Trp-Thr-Asp (YWTD) that forms a .beta.-propeller
structure; an O-linked sugar domain; a transmembrane domain; and a
cytoplasmic domain with a coated pit targeting signal (H.
Tolleshaug, J. L. et al., Cell, 30 (1982), pp. 715-724). In
addition to LDLR, the following receptors are considered to be part
of the LDLR gene family in mammals: very low-density lipoprotein
receptor (VLDLR); apolipoprotein E receptor 2 (ApoER2 also known as
LRP8); LDLR-related protein-1 (LRP1 also known as CD91, or
.alpha.2macroglobulin receptor, .alpha.2MR); LRP2 (also known as
megalin, or glycoprotein 330, GP330); LRP3 (closely resembles ST7
and LRP9); LRP4 (also known as corin); LRP5 (also known as LRP7);
LRP6 (also known as ADCAD2 or STHAG7); LRP10 (also known as LRP9,
LRP-10, MST087, or MSTP087), and LR11 (also known as sorLA) (Jeong,
Y. H. Biochem Biophys Res Commun. 2010 Jan. 1; 391(1):1110-5). Most
of the LDLR gene family is structurally similar to LDLR, so it has
been thought that most members of this family play a primary role
in lipoprotein metabolism. Other roles for the LDLR gene family are
the following: (1) VLDLR and ApoER2 transmit the extracellular
Reelin signal to migrating neurons, which governs neuronal layering
of the brain during embryonic brain development (Trommsdorff, M. et
al., Cell, 97 (1999), pp. 689-701); (2) LRP1 regulates cellular
entry of viruses and toxins and protects from atherosclerosis by
modulating plate-derived growth factor receptor-O signaling in the
vascular wall (Herz, J. et al., Curr. Opin. Lipidol., 15 (2004),
pp. 175-181); (3) LRP2 acts as an endocytic receptor that mediates
the availability of several extracellular signaling molecules such
as vitamin D, vitamin A and sex steroids (Hammes, A. et al., Cell,
122 (2005), pp. 751-762); (4) LRP3 modulates cellular uptake of
.beta.-VLDL; (5) LRP4 serves as a type II transmembrane serine
protease, and as a pro-atrial natriuretic peptide-converting enzyme
that regulates blood pressure (Yan, W. et al., Proc. Natl. Acad.
Sci. USA, 97 (2000), pp. 8525-8529); (6) LRP5 and LRP6 bind Wnt and
Frizzled proteins, and activate the Wnt signaling pathway involved
in cell proliferation, cell polarity and cell fate determination
(Tami, K. et al., Nature, 407 (2000), pp. 530-535), and (7) LR11
may participate in the development of Alzheimer's disease by
modulating endocytosis of the amyloid precursor protein, which
generates the amyloid 0 peptide (Herz, J. et al., Curr. Opin.
Lipidol., 15 (2004), pp. 175-181).
[0074] LRP10 is a single-spanning plasma membrane receptor and
consists of five functional domains characteristic of the LDLR gene
family. The structural organization of LRP10 predicts that LRP10
may bind ligands similarly to the LDLR gene family, and that LRP10
likely acts as an endocytic receptor or a signal transducer (Jeong,
Y. H. Biochem Biophys Res Commun. 2010 Jan. 1; 391(1):1110-5).
Prior to the present disclosure, the function of LRP10 had not been
elucidated.
[0075] The full gene sequence of human Lrp10 is 9,968 bp in length
(GenBank ID No. NC_000014.9). The mature type I membrane protein
contains 696 amino acids and has a calculated molecular mass of
74.8 kD (Sugiyama, T. et al., Biochemistry 39: 15817-15825, 2000).
The human cDNA sequence of low-density lipoprotein receptor-related
protein 10 isoform 2 precursor is 4,785 bp in length (GenBank ID
No. NM_001329226), and the human cDNA sequence of low-density
lipoprotein receptor-related protein 10 isoform 1 precursor 6,930
bp in length (GenBank ID No. NM_014045). The full gene sequence of
mouse Lrp10 gene is 7,334 bp in length (GenBank ID No.
NC_000080.6).
[0076] Compositions for inhibiting LRP10 and thus, enhancing
hematopoietic recovery in a subject after chemotherapy or
irradiation, and/or improving cancer immunotherapy in a subject are
also provided. The composition can include one or more anti-LRP10
antibody disclosed herein, or antigen binding fragment thereof. In
some embodiments, the anti-LRP10 antibodies disclosed herein can
inhibit a LRP10 ligand (e.g., in circulation) from binding with
LRP10. In some embodiments, other LRP10 inhibitors such as small
molecule compounds can also be used to inhibit one or more
activities of LRP10, including binding between LRP10 and its
ligand. In certain embodiments, a soluble version of LRP10 can be
engineered which can act to compete with the endogenous, membrane
protein LRP10 to bind to LRP10 ligand, thereby indirectly inhibit
binding between endogenous LRP10 and its ligand.
LRP10 Inhibitor
[0077] Inhibition of LRP10 can enhance hematopoietic recovery in a
subject, e.g., after chemotherapy or irradiation, and/or provide
cancer immunotherapy in a subject. As such, LRP10 inhibitors can be
used as an effective agent in cancer therapeutics.
[0078] Various LRP10 inhibitors are included in the present
disclosure. Examples include chemical compounds, such as small
molecule inhibitors and biologic agents (e.g., antibodies) that can
bind LRP10 and inhibit or decrease its activity, e.g., in a
chemotaxis migration assay or Frizzled and/or p21 expression assay.
Another exemplary LRP10 inhibitor is a soluble, decoy receptor that
binds to one or more LRP10 ligands. Agents that regulate Lrp10 gene
expression level are also included, such as interfering RNA (shRNA,
siRNA) and gene editing/silencing tools (CRISPR/Cas9, TALENs, zinc
finger nucleases) that are designed specifically to target the
Lrp10 gene or a regulatory sequence thereto.
[0079] In some embodiments, a method for identifying an LRP10
inhibitor is provided, which can include contacting a cell with a
test agent, wherein an increase in migration toward a chemotactic
stimulus compared to a control cell that is not contacted with the
antibody or fragment thereof indicates that the test agent is an
LRP10 inhibitor, wherein preferably the chemotactic stimulus is
selected from C-X-C motif chemokine 12 (CXCL12), C-X-C motif
chemokine 10 (CXCL10), sphingosine-1-phosphate (SIP), C-C motif
ligand 2 (CCL2), and/or C-C motif ligand 21 (CCL21).
[0080] Another method for identifying an LRP10 inhibitor can
include contacting a cell with a test agent, wherein an increase in
expression of Frizzled and/or P21 compared to a control cell that
is not contacted with the antibody or fragment thereof indicates
that the test agent is an LRP10 inhibitor.
[0081] The LRP10 inhibitor can be characterized by at least partial
inhibition of proliferation (e.g., by at least 10% relative to
control) of cancer cells or by at least partial inhibition of tumor
growth (e.g., volume and/or metastasis) in vivo in the patient.
Without wishing to be bound by theory, it is believed that in
cancer, one of the major problems is a failure of CD8 T cells and
their descendants, cytotoxic T lymphocytes, to infiltrate tumor
masses in order to kill tumor cells. The failure to do so may
depend on signaling via LRP10. Thus, functional inhibition of LRP10
using, e.g., an antibody or antigen-binding fragment thereof that
binds to LRP10 can allow such infiltration to occur.
[0082] In certain embodiments, the LRP10 inhibitor is an anti-LRP10
antibody, e.g., a monoclonal antibody, or an antigen-binding
fragment thereof. In certain embodiments, the anti-LRP10 antibody
can be a modified, e.g., chimeric or humanized antibody derived
from a mouse anti-LRP10 antibody. Methods for making modified
antibodies are known in the art. In some embodiments, the
anti-LRP10 antibody is an antibody or antigen binding fragment
thereof which binds to an epitope present on the human LRP10
protein, e.g., the extracellular ectodomain, or a portion
thereof.
[0083] In some embodiment, the anti-LRP10 antibody or
antigen-binding fragment thereof can comprise one or more of the
following VL (SEQ ID NOS: 1, 5, 7, 11, 15, 19, 23) and VH sequences
(SEQ ID NOS: 2, 8, 12, 16, 20, 24, 27, 29). Note that for the full
antibody sequence, each VL can be linked to a light chain constant
region such as Ck (SEQ ID NO: 31) to form a full light chain, and
each VH can be linked to a heavy chain constant region such as
human IgG1 CH123 (SEQ ID NO: 32) to form a full heavy chain.
TABLE-US-00003 VL (SEQ ID NO: 1):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YNASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSRTPTT FGQGTKVEIK VL
(SEQ ID NO: 5): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YAASPLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVARTPNT FGQGTKVEIK VL
(SEQ ID NO: 7): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPTSLPLT FGQGTKVEIK VL
(SEQ ID NO: 11): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADSYPTT FGQGTKVEIK VL
(SEQ ID NO: 15): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQIKTRPTT FGQGTKVEIK VL
(SEQ ID NO: 19): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYD
ASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSAGPGTFG QGTKVEIK VL (SEQ
ID NO: 23): TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
YAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNDYYPTT FGQGTKVEIK VH
(SEQ ID NO: 2): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEW
VSSIPPGGPNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKSYPSFDYWGQGTLVTVSS VH (SEQ ID NO: 8):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW
VSQIGTMGRPTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKSGKKFDYWGQGTLVTVSS VH (SEQ ID NO: 12):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRLAPGKGLEW
VSSISTTGNSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKDATSFDYWGQGTLVTVSS VH (SEQ ID NO: 16):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW
VSVIQRQGTGTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKNSRTFDYWGQGTLVTVSS VH (SEQ ID NO: 20):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW
VSSIPSRGQATKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKSRHTFDYWGQGTLVTVSS VH (SEQ ID NO: 24):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW
VSSIATTGNTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKNTATFDYWGQGTLVTVSS VH (SEQ ID NO: 27):
MAEVQLLESGGGLVQLGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEW
VSSIPPGGPNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKSYPSFDYWGQGTLVTVSS VH (SEQ ID NO: 29):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW
VSSIYTSGAATTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKSYPSFDYWGQGTLVTVSS Ck amino acid sequence (SEQ ID NO: 31):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC IgG1
CH123 amino acid sequence (SEQ ID NO: 32):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0084] In some embodiments, the anti-LRP10 antibody or
antigen-binding fragment thereof can comprise one or more of the
following CDRs:
TABLE-US-00004 VL CDR1 (SEQ ID NO: 33): RASQSISSYLN VL CDR2 (SEQ ID
NO: 34): NASDLQS VL CDR2 (SEQ ID NO: 35): AASPLQS VL CDR2 (SEQ ID
NO: 36): RASRLQS VL CDR2 (SEQ ID NO: 37): AASALQS VL CDR2 (SEQ ID
NO: 38): DASLLQS VL CDR2 (SEQ ID NO: 39): AASTLQS VL CDR3 (SEQ ID
NO: 40): QQSSRTPTT VL CDR3 (SEQ ID NO: 41): QQVARTPNT VL CDR3 (SEQ
ID NO: 42): QQPTSLPLT VL CDR3 (SEQ ID NO: 43): QQADSYPTT VL CDR3
(SEQ ID NO: 44): QQIKTRPTT VL CDR3 (SEQ ID NO: 45): QQTSAGPGT VL
CDR3 (SEQ ID NO: 46): QQNDYYPTT VH CDR1 (SEQ ID NO: 47): SQAMS VH
CDR1 (SEQ ID NO: 48): SYAMS VH CDR2 (SEQ ID NO: 49):
QIGTMGRPTTYADSVKG VH CDR2 (SEQ ID NO: 50): SISTTGNSTYYADSVKG VH
CDR2 (SEQ ID NO: 51): VIQRQGTGTEYADSVKG VH CDR2 (SEQ ID NO: 52):
SIPSRGQATKYADSVKG VH CDR2 (SEQ ID NO: 53): SIATTGNTTYYADSVKG VH
CDR2 (SEQ ID NO: 54): SIPPGGPNTKYADSVKG VH CDR2 (SEQ ID NO: 55):
SIYTSGAATTYADSVKG VH CDR3 (SEQ ID NO: 56): SYPSFDY VH CDR3 (SEQ ID
NO: 57): SGKKFDY VH CDR3 (SEQ ID NO: 58): DATSFDY VH CDR3 (SEQ ID
NO: 59): NSRTFDY VH CDR3 (SEQ ID NO: 60): SRHTFDY VH CDR3 (SEQ ID
NO: 61): NTATFDY
[0085] Alignments of the CDR sequences are shown in Tables 1-6.
TABLE-US-00005 TABLE 1 Alignment of VL CDR1 sequences Amino acid
position (Kabat numbering) Clone No. L24 L25 L26 L27 L27A L28 L29
L30 L31 L32 L33 L34 AB1: L1 + H1 R A S Q -- S I S S Y L N AB2: L2 +
H1 R A S Q -- S I S S Y L N AB3: L3 + H2 R A S Q -- S I S S Y L N
AB4: L4 + H3 R A S Q -- S I S S Y L N AB5: L5 + H4 R A S Q -- S I S
S Y L N AB6: L6 + H5 R A S Q -- S I S S Y L N AB7: L7 + H6 R A S Q
-- S I S S Y L N AB8: L1 + H7 R A S Q -- S I S S Y L N AB9: L1 + H8
R A S Q -- S I S S Y L N Conserved R A S Q -- S I S S Y L N
sequence
TABLE-US-00006 TABLE 2 Alignment of VL CDR2 sequences Amino acid
position (Kabat numbering) Clone No. L50 L51 L52 L53 L54 L55 L56
AB1: L1 + H1 N A S D L Q S AB2: L2 + H1 A A S P L Q S AB3: L3 + H2
R A S R L Q S AB4: L4 + H3 A A S A L Q S AB5: L5 + H4 R A S R L Q S
AB6: L6 + H5 D A S L L Q S AB7: L7 + H6 A A S T L Q S AB8: L1 + H7
N A S D L Q S AB9: L1 + H8 N A S D L Q S Conserved X1 A S X2 L Q S
sequence (X1 = N, A, R, D; X2 = D, P, R, A, L, T)
TABLE-US-00007 TABLE 3 Alignment of VL CDR3 sequences Amino acid
position (Kabat numbering) Clone No. L89 L90 L91 L92 L93 L94 L95
L95A L96 L97 AB1 :L1 + H1 Q Q S S R T P -- T T AB2: L2 + H1 Q Q V A
R T P -- N T AB3: L3 + H2 Q Q P T S L P -- L T AB4: L4 + H3 Q Q A D
S Y P -- T T AB5: L5 + H4 Q Q I K T R P -- T T AB6: L6 + H5 Q Q T S
A G P -- G T AB7: L7 + H6 Q Q N D Y Y P -- T T AB8: L1 + H7 Q Q S S
R T P -- T T AB9: L1 + H8 Q Q S S R T P -- T T Conserved Q Q X3 X4
X5 X6 P -- X7 T sequence (X3 = S, V, P, A, I, T, N; X4 = S, A, T,
D, K; X5 = R, S, T, A, Y; X6 = T, L, Y, R, G; X7 = T, N, L, G)
TABLE-US-00008 TABLE 4 Alignment of VH CDR1 sequences Amino acid
position (Kabat numbering) Clone No. H31 H32 H33 H34 H35 H35A AB1:
L1 + H1 S Q A M S -- AB2: L2 + H1 S Q A M S -- AB3: L3 + H2 S Y A M
S -- AB4: L4 + H3 S Y A M S -- AB5: L5 + H4 S Y A M S -- AB6: L6 +
H5 S Y A M S -- AB7: L7 + H6 S Y A M S -- AB8: L1 + H7 S Q A M S --
AB9: L1 + H8 S Y A M S -- Conserved S X8 A M S -- sequence (X8 = Q,
Y)
TABLE-US-00009 TABLE 5 Alignment of VH CDR2 sequences Amino acid
position (Kabat numbering) Clone No. H50 H51 H52 H52A H53 H54 H55
H56 H57 H58 H59 H60 H61 H62 H63 H64 H65 AB1: L1 + H1 S I P P G G P
N T K Y A D S V K G AB2: L2 + H1 S I P P G G P N T K Y A D S V K G
AB3: L3 + H2 Q I G T M G R P T T Y A D S V K G AB4: L4 + H3 S I S T
T G N S T Y Y A D S V K G AB5: L5 + H4 V I Q R Q G T G T E Y A D S
V K G AB6: L6 + H5 S I P S R G Q A T K Y A D S V K G AB7: L7 + H6 S
T A T T G N T T Y Y A D S V K G AB8: L1 + H7 S I P P G G P N T K Y
A D S V K G AB9: L1 + H8 S I Y T S G A A T T Y A D S V K G
Conserved X9 I X10 X11 X12 G X13 X14 T X15 Y A D S V K G sequence
(X9 = S, Q, V; X10 = P, G, S, Q, A, Y; X11 = P, T, R, S; X12 = G,
M, T, Q, R, S; X13 = P, R, N, T, Q, A; X14 = N, P, S, G, A, T; X15
= K, T, Y, E)
TABLE-US-00010 TABLE 6 Alignment of VH CDR3 sequences Amino acid
position (Kabat numbering) Clone No. H95 H96 H97 H98 H99 H100 H100A
H101 H102 AB1: L1 + H1 S Y P S F -- -- D Y AB2: L2 + H1 S Y P S F
-- -- D Y AB3: L3 + H2 S G K K F -- -- D Y AB4: L4 + H3 D A T S F
-- -- D Y AB5: L5 + H4 N S R T F -- -- D Y AB6: L6 + H5 S R H T F
-- -- D Y AB7: L7 + H6 N T A T F -- -- D Y AB8: L1 + H7 S Y P S F
-- -- D Y AB9: L1 + H8 S Y P S F -- -- D Y Conserved sequence X16
X17 X18 X19 F -- -- D Y (X16 = S, D, N; X17 = Y, G, A, S, R, T; X18
= P, K, T, R, H, A; X19 = S, K, T)
[0086] In some embodiments, the anti-LRP10 antibody or
antigen-binding fragment thereof can comprise one or more of the
following CDRs:
TABLE-US-00011 VL CDR1 (SEQ ID NO: 33): RASQSISSYLN VL CDR2 (SEQ ID
NO: 62): X1ASX2LQS (X1 = N, A, R, D; X2 = D, P, R, A, L, T) VL CDR3
(SEQ ID NO: 63): QQX3X4X5X6PX7T (X3 = S, V, P, A, I, T, N; X4 = S,
A, T, D, K; X5 = R, S, T, A, Y; X6 = T, L, Y, R, G; X7 = T, N, L,
G) VH CDR1 (SEQ ID NO: 64): SX8AMS (X8 = Q, Y) VH CDR2 (SEQ ID NO:
65): X9IX10X11X12GX13X14TX15YADSVKG (X9 = S, Q, V; X10 = P, G, S,
Q, A, Y; X11 = P, T, R, S; X12 = G, M, T, Q, R, S; X13 = P, R, N,
T, Q, A; X14 = N, P, S, G, A, T; X15 = K, T, Y, E) VH CDR3 (SEQ ID
NO: 66): X16X17X18X19FDY (X16 = S, D, N; X17 = Y, G, A, S, R, T;
X18 = P, K, T, R, H, A; X19 = S, K, T)
[0087] Antibodies or fragments thereof that cross-compete with any
of the anti-LRP10 antibody or antigen-binding fragment thereof
disclosed herein in a competition binding assay are also included
in the present disclosure. In some embodiments, such
cross-competing antibodies can bind to the same epitope as the
anti-LRP10 antibody or antigen-binding fragment thereof disclosed
herein.
[0088] In certain embodiment, the anti-LRP10 antibody can comprise
a mixture, or cocktail, of two or more anti-LRP10 antibodies, each
of which binds to the same or a different epitope on LRP10. In one
embodiment, the mixture, or cocktail, comprises three anti-LRP10
antibodies, each of which binds to the same or a different epitope
on LRP10.
[0089] In another embodiment, the LRP10 inhibitor can include a
nucleic acid molecule, such as an RNA molecule, that inhibits the
expression or activity of LRP10. Interfering RNAs specific for
Lrp10, such as shRNAs or siRNAs that specifically inhibits the
expression and/or activity of Lrp10, can be designed in accordance
with methods known in the art.
LRP10 Competitor
[0090] In some embodiments, the LRP10 competitor can be a soluble,
decoy receptor that can bind to one or more LRP10 ligands, thereby
competing with and inhibiting or decreasing the binding of the
ligand to endogenous LRP10. For example, the decoy receptor may
contain an amino acid sequence corresponding to all or a part of
the ectodomain of LRP10 and devoid of a transmembrane region. The
amino acid sequence can contain one or more substitutions,
deletions, and/or additions compared to the wild-type sequence,
while retaining or enhancing its binding activity with one or more
LRP10 ligands. The amino acid sequence can be fused or grafted to
the constant end of an antibody (e.g., IgG1) and thus, become
soluble in circulation. In some embodiments, the decoy receptor can
be a human LRP10-antibody Fc fragment (LRP10-Fc) chimera.
[0091] In some embodiments, the Fc domain is an IgG domain, e.g.,
an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc
domain comprises a CH2 domain and a CH3 domain. In some
embodiments, the Fc domain has dimerization activity.
[0092] In some embodiments, the Fc domain comprises an IgG1 Fc
domain of SEQ ID NO: 67 or an amino acid sequence having at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
TABLE-US-00012 (SEQ ID NO: 67)
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0093] In embodiments, the Fc domain is an effector-attenuated Fc
domain. In embodiments, the effector-attenuated Fc domain has
reduced effector activity, e.g., compared to a wild-type IgG1 Fc
domain, e.g., compared to a wild-type IgG1 Fc domain of SEQ ID NO:
67. In some embodiments, effector activity comprises
antibody-dependent cellular toxicity (ADCC). In embodiments, the
effector activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% in an ADCC assay, e.g., compared to a
wild-type IgG1 Fc domain of SEQ ID NO: 67. In some embodiments,
effector activity comprises complement dependent cytotoxicity
(CDC). In embodiments, the effector activity is reduced by 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a CDC assay
such as a CDC assay described in Armour et al., "Recombinant human
IgG molecules lacking Fc gamma receptor I binding and monocyte
triggering activities." Eur J Immunol (1999) 29:2613-24" e.g.,
compared to a wild-type IgG1 Fc domain of SEQ ID NO: 67.
[0094] In some embodiments, the Fc domain comprises an IgG2
constant region of SEQ ID NO: 68 or fragment thereof, or an amino
acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identity thereto.
TABLE-US-00013 (SEQ ID NO: 68)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK
[0095] In some embodiments, the Fc domain comprises a human IgG2 Fc
domain, e.g., a human IgG2 domain of SEQ ID NO: 69 or an amino acid
sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identity thereto.
TABLE-US-00014 (SEQ ID NO: 69)
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
[0096] In some embodiments, the Fc domain comprises an IgG2 Da Fc
domain of SEQ ID NO: 80 or an amino acid sequence having at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In
embodiments, the Fc domain comprises one or both of A330S and P331S
mutations using Kabat numbering system. In embodiments, the Fc
domain is one described in Armour et al. "Recombinant human IgG
molecules lacking Fc gamma receptor I binding and monocyte
triggering activities." Eur J Immunol (1999) 29:2613-24.
TABLE-US-00015 (SEQ ID NO: 70)
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
[0097] In various embodiments, the decoy receptor can be prepared
such that it is stable in circulation. In some embodiments, the
decoy receptor neutralizes and/or inhibits one or more LRP10
ligands, e.g., by competing with, thereby decreasing or preventing
binding of the LRP10 ligands with endogenous LRP10. In certain
embodiments, the decoy receptor may act more effectively than the
LRP10 ectodomain alone.
[0098] The soluble receptor can be prepared by methods known in the
art such as producing a fusion protein by recombinant DNA
technology. The LRP10 ectodomain or a functional portion thereof
(e.g., the ligand-binding fragment) can be fused to the constant
end of an antibody (e.g., IgG1). For example, the DNA sequence that
codes the human LRP10 ectodomain or a functional portion thereof,
can be engineered to link to, optionally through a linker, the DNA
sequence that codes the human gene for the Fc end of IgG1. The
engineered DNA can then be expressed to produce a protein that
links the LRP10 ectodomain to IgG1 Fc.
Preparation of Anti-LRP10 Antibodies
[0099] Anti-LRP10 antibodies can be made using various methods
generally known in the art. For example, phage display technology
can be used to screen a human antibody library, to produce a fully
human monoclonal antibody for therapy. High affinity binders can be
considered candidates for neutralization studies. Alternatively, a
conventional monoclonal approach can be used, in which mice or
rabbits can be immunized with the human protein, candidate binders
identified and tested, and a humanized antibody ultimately produced
by engrafting the combining sites of heavy and light chains into a
human antibody encoding sequence.
[0100] Antibodies typically comprise two identical pairs of
polypeptide chains, each pair having one full-length "light" chain
(typically having a molecular weight of about 25 kDa) and one
full-length "heavy" chain (typically having a molecular weight of
about 50-70 kDa). The amino-terminal portion of each chain
typically includes a variable region of about 100 to 110 or more
amino acids that typically is responsible for antigen recognition.
The carboxy-terminal portion of each chain typically defines a
constant region responsible for effector function. The variable
regions of each of the heavy chains and light chains typically
exhibit the same general structure comprising four relatively
conserved framework regions (FR) joined by three hyper variable
regions, also called complementarity determining regions or CDRs.
The CDRs from the two chains of each pair typically are aligned by
the framework regions, which alignment may enable binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chain variable regions typically comprise the domains FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to each domain is typically in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (1987 and
1991, National Institutes of Health, Bethesda, Md.), Chothia &
Lesk, 1987, J. Mol. Biol. 196:901-917, or Chothia et al., 1989,
Nature 342:878-883).
[0101] Antibodies became useful and of interest as pharmaceutical
agents with the development of monoclonal antibodies. Monoclonal
antibodies are produced using any method that produces antibody
molecules by continuous cell lines in culture. Examples of suitable
methods for preparing monoclonal antibodies include the hybridoma
methods of Kohler et al. (1975, Nature 256:495-497) and the human
B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and
Brodeur et al., 1987, Monoclonal Antibody Production Techniques and
Applications, Marcel Dekker, Inc., New York, pp. 51-63).
[0102] Monoclonal antibodies may be modified for use as
therapeutics. One example is a "chimeric" antibody in which a
portion of the heavy chain and/or light chain is identical with or
homologous to a corresponding sequence in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. Other examples are fragments of such antibodies, so long
as they exhibit the desired biological activity. See, U.S. Pat. No.
4,816,567; and Morrison et al. (1985), Proc. Natd. Acad. Sci. USA
81:6851-6855. A related development is the "CDR-grafted" antibody,
in which the antibody comprises one or more complementarity
determining regions (CDRs) from a particular species or belonging
to a particular antibody class or subclass, while the remainder of
the antibody chain(s) is/are identical with or homologous to a
corresponding sequence in antibodies derived from another species
or belonging to another antibody class or subclass.
[0103] Another development is the "humanized" antibody. Methods for
humanizing non-human antibodies are well known in the art (see U.S.
Pat. Nos. 5,585,089, and 5,693,762; see also Cecile Vincke et al.
J. Biol. Chem. 2009; 284:3273-3284 for humanization of llama
antibodies). Generally, a humanized antibody is produced by a
non-human animal, and then certain amino acid residues, typically
from non-antigen recognizing portions of the antibody, are modified
to be homologous to said residues in a human antibody of
corresponding isotype. Humanization can be performed, for example,
using methods described in the art (Jones et al., 1986, Nature
321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen
et al., 1988, Science 239:1534-1536), by substituting at least a
portion of a rodent variable region for the corresponding regions
of a human antibody.
[0104] More recent is the development of human antibodies without
exposure of antigen to human beings ("fully human antibodies").
Using transgenic animals (e.g., mice) that are capable of producing
a repertoire of human antibodies in the absence of endogenous mouse
immunoglobulin production, such antibodies are produced by
immunization with an antigen (typically having at least 6
contiguous amino acids), optionally conjugated to a carrier. See,
for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA
90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; and
Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of
these methods, transgenic animals are produced by incapacitating
the endogenous mouse immunoglobulin loci encoding the mouse heavy
and light immunoglobulin chains therein, and inserting loci
encoding human heavy and light chain proteins into the genome
thereof. Partially modified animals, which have less than the full
complement of modifications, are then cross-bred to obtain an
animal having all of the desired immune system modifications. When
administered an immunogen, these transgenic animals produce
antibodies that are immunospecific for these antigens having human
(rather than murine) amino acid sequences, including variable
regions. See PCT Publication Nos. WO96/33735 and WO94/02602,
incorporated by reference. Additional methods are described in U.S.
Pat. No. 5,545,807, PCT Publication Nos. WO91/10741, WO90/04036,
and in EP 546073B1 and EP 546073A1, incorporated by reference.
Human antibodies may also be produced by the expression of
recombinant DNA in host cells or by expression in hybridoma cells
as described herein.
[0105] In some embodiments, phage display technology may be used to
screen for therapeutic antibodies. In phage display, antibody
repertoires can be displayed on the surface of filamentous
bacteriophage, and the constructed library may be screened for
phages that bind to the immunogen. Antibody phage is based on
genetic engineering of bacteriophages and repeated rounds of
antigen-guided selection and phage propagation. This technique
allows in vitro selection of LRP10 monoclonal antibodies. The phage
display process begins with antibody-library preparation followed
by ligation of the variable heavy (VH) and variable light (VL) PCR
products into a phage display vector, culminating in analysis of
clones of monoclonal antibodies. The VH and VL PCR products,
representing the antibody repertoire, are ligated into a phage
display vector (e.g., the phagemid pComb3X) that is engineered to
express the VH and VL as an scFv fused to the pIII minor capsid
protein of a filamentous bacteriophage of Escherichia coli that was
originally derived from the M13 bacteriophage. However, the phage
display vector pComb3X does not have all the other genes necessary
to encode a full bacteriophage in E. coli. For those genes, a
helper phage is added to the E. coli that are transformed with the
phage display vector library. The result is a library of phages,
each expressing on its surface a LRP10 monoclonal antibody and
harboring the vector with the respective nucleotide sequence
within. The phage display can also be used to produce the LRP10
monoclonal antibody itself (not attached to phage capsid proteins)
in certain strains of E. Coli. Additional cDNA is engineered, in
the phage display vector, after the VL and VH sequences to allow
characterization and purification of the mAb produced.
Specifically, the recombinant antibody may have a hemagglutinin
(HA) epitope tag and a polyhistidine to allow easy purification
from solution.
[0106] Diverse antibody phage libraries are produced from
.about.10.sup.8 independent E. coli transformants infected with
helper phage. Using bio-panning, a library can screened for phage
binding to the immunogen sequence listed above, or a fragment
thereof, through the expressed surface of the monoclonal antibody.
Cyclic panning allows for pulling out potentially very rare
antigen-binding clones and consists of multiple rounds of phage
binding to antigen (immobilized on ELISA plates or in solution on
cell surfaces), washing, elution, and reamplification of the phage
binders in E. coli. During each round, specific binders are
selected out from the pool by washing away non-binders and
selectively eluting binding phage clones. After three or four
rounds, highly specific binding of phage clones through their
surface LRP10 monoclonal antibody is characteristic for directed
selection on the immobilized immunogen.
[0107] Another method is to add a C-terminal His tag, suitable for
purification by affinity chromatography, to the immunogen sequence
listed above. Purified protein can be inoculated into mice together
with a suitable adjuvant. Monoclonal antibodies produced in
hybridomas can be tested for binding to the immunogen, and positive
binders can be screened as described in the assays herein.
[0108] Fully human antibodies can also be produced from
phage-display libraries (as disclosed in Hoogenboom et al., 1991,
J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol.
222:581). These processes mimic immune selection through the
display of antibody repertoires on the surface of filamentous
bacteriophage, and subsequent selection of phage by their binding
to an antigen of choice. One such technique is described in PCT
Publication No. WO99/10494, incorporated by reference, which
describes the isolation of high affinity and functional agonistic
antibodies for MPL- and msk-receptors using such an approach.
[0109] In some embodiments, the human LRP10 ectodomain, consisting
of amino acids 18-713 of the 713 amino acid human LRP10 protein
sequence, can be used as the immunogen. A fragment or portion of
the LRP10 ectodomain can also be used as the immunogen. Monoclonal
antibodies can be raised using one or more immunogens. Potential
therapeutic anti-LRP10 antibodies can be generated.
[0110] In one example, using a mouse model having the human LRP10
ectodomain knocked into the mouse Lrp10 gene, and human T cells
(Jurkat or primary T cells from human donors), monoclonal
antibodies that phenocopy the knockout mutation could be tested and
identified as potential anti-LRP10 antibody candidates. Monoclonal
antibodies that phenocopy the knockout mutation include those that
have elevated number of T cells (e.g., CD4+ and CD8+) in blood, low
B:T cell ratio, and/or low NK cell count. Such tests include
screening endpoint(s), such as the augmentation of Frizzled and/or
P21 protein expression detected on, e.g., Western blot, and
secondarily, augmentation of T cell migration and enhanced
hematopoietic reconstitution after irradiation or chemotherapy.
After the screening, fully human monoclonal antibodies can be
developed for preclinical testing and then tested in clinical human
trials for safety and efficacy. Such antibodies can be clinical
candidates that can enhance the hematopoietic recovery in a subject
after chemotherapy or irradiation, and/or improve cancer
immunotherapy (e.g., by increasing the number of tumor infiltrating
lymphocytes).
[0111] Nucleotide sequences encoding the above antibodies can be
determined. Thereafter, chimeric, CDR-grafted, humanized, and fully
human antibodies also may be produced by recombinant methods.
Nucleic acids encoding the antibodies can be introduced into host
cells and expressed using materials and procedures generally known
in the art.
[0112] The disclosure provides one or more monoclonal antibodies
against LRP10. Preferably, the antibodies bind LRP10 ectodomain. In
preferred embodiments, the disclosure provides nucleotide sequences
encoding, and amino acid sequences comprising, heavy and light
chain immunoglobulin molecules, particularly sequences
corresponding to the variable regions thereof. In preferred
embodiments, sequences corresponding to CDRs, specifically from
CDR1 through CDR3, are provided. In additional embodiments, the
disclosure provides hybridoma cell lines expressing such
immunoglobulin molecules and monoclonal antibodies produced
therefrom, preferably purified human monoclonal antibodies against
human LRP10.
[0113] The CDRs of the light and heavy chain variable regions of
anti-LRP10 antibodies of the disclosure can be grafted to framework
regions (FRs) from the same, or another, species. In certain
embodiments, the CDRs of the light and heavy chain variable regions
of anti-LRP10 antibody may be grafted to consensus human FRs. To
create consensus human FRs, FRs from several human heavy chain or
light chain amino acid sequences are aligned to identify a
consensus amino acid sequence. The FRs of the anti-LRP10 antibody
heavy chain or light chain can be replaced with the FRs from a
different heavy chain or light chain. Rare amino acids in the FRs
of the heavy and light chains of anti-LRP10 antibody typically are
not replaced, while the rest of the FR amino acids can be replaced.
Rare amino acids are specific amino acids that are in positions in
which they are not usually found in FRs. The grafted variable
regions from anti-LRP10 antibodies of the disclosure can be used
with a constant region that is different from the constant region
of anti-LRP10 antibody. Alternatively, the grafted variable regions
are part of a single chain Fv antibody. CDR grafting is described,
e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089,
and 5,530,101, which are hereby incorporated by reference for any
purpose.
[0114] In some embodiments, antibodies of the disclosure can be
produced by hybridoma lines. In these embodiments, the antibodies
of the disclosure bind to LRP10 with a dissociation constant
(K.sub.d) of between approximately 4 pM and 1 .mu.M. In certain
embodiments of the disclosure, the antibodies bind to LRP10 with a
K.sub.d of less than about 100 nM, less than about 50 nM or less
than about 10 nM.
[0115] In preferred embodiments, the antibodies of the disclosure
are of the IgG1, IgG2, or IgG4 isotype, with the IgG1 isotype most
preferred. In preferred embodiments of the disclosure, the
antibodies comprise a human kappa light chain and a human IgG1,
IgG2, or IgG4 heavy chain. In particular embodiments, the variable
regions of the antibodies are ligated to a constant region other
than the constant region for the IgG1, IgG2, or IgG4 isotype. In
certain embodiments, the antibodies of the disclosure have been
cloned for expression in mammalian cells.
[0116] In alternative embodiments, antibodies of the disclosure can
be expressed in cell lines other than hybridoma cell lines. In
these embodiments, sequences encoding particular antibodies can be
used for transformation of a suitable mammalian host cell.
According to these embodiments, transformation can be achieved
using any known method for introducing polynucleotides into a host
cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus (or vector) or by transfection procedures known in the art.
Such procedures are exemplified by U.S. Pat. Nos. 4,399,216,
4,912,040, 4,740,461, and 4,959,455 (all of which are hereby
incorporated herein by reference for any purpose). Generally, the
transformation procedure used may depend upon the host to be
transformed. Methods for introducing heterologous polynucleotides
into mammalian cells are well known in the art and include, but are
not limited to, dextran-mediated transfection, calcium phosphate
precipitation, polybrene-mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0117] According to certain embodiments of the methods of the
disclosure, a nucleic acid molecule encoding the amino acid
sequence of a heavy chain constant region, a heavy chain variable
region, a light chain constant region, or a light chain variable
region of a LRP10 antibody of the disclosure is inserted into an
appropriate expression vector using standard ligation techniques.
In a preferred embodiment, the LRP10 heavy or light chain constant
region is appended to the C-terminus of the appropriate variable
region and is ligated into an expression vector. The vector is
typically selected to be functional in the particular host cell
employed (i.e., the vector is compatible with the host cell
machinery such that amplification of the gene and/or expression of
the gene can occur). For a review of expression vectors, see,
Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press.
N.Y.
[0118] Typically, expression vectors used in any of the host cells
can contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences
typically include one or more of the following nucleotide
sequences: a promoter, one or more enhancer sequences, an origin of
replication, a transcriptional termination sequence, a complete
intron sequence containing a donor and acceptor splice site, a
sequence encoding a leader sequence for polypeptide secretion, a
ribosome binding site, a polyadenylation sequence, a polylinker
region for inserting the nucleic acid encoding the polypeptide to
be expressed, and a selectable marker element. These sequences are
well known in the art.
[0119] Expression vectors of the disclosure may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the flanking sequences described
herein are not already present in the vector, they may be
individually obtained and ligated into the vector. Methods used for
obtaining each of the flanking sequences are well known to one
skilled in the art.
[0120] After the vector has been constructed and a nucleic acid
molecule encoding light chain or heavy chain or light chain and
heavy chain comprising an anti-LRP10 antibody has been inserted
into the proper site of the vector, the completed vector may be
inserted into a suitable host cell for amplification and/or
polypeptide expression. The transformation of an expression vector
for an anti-LRP10 antibody into a selected host cell may be
accomplished by well-known methods including transfection,
infection, calcium phosphate co-precipitation, electroporation,
microinjection, lipofection, DEAE-dextran mediated transfection, or
other known techniques. The method selected will in part be a
function of the type of host cell to be used. These methods and
other suitable methods are well known to the skilled artisan, and
are set forth, for example, in Sambrook et al., supra.
[0121] The host cell, when cultured under appropriate conditions,
synthesizes an anti-LRP10 antibody that can subsequently be
collected from the culture medium (if the host cell secretes it
into the medium) or directly from the host cell producing it (if it
is not secreted). The selection of an appropriate host cell will
depend upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
[0122] Mammalian cell lines available as hosts for expression are
well known in the art and include, but are not limited to, many
immortalized cell lines available from the American Type Culture
Collection (ATCC), including but not limited to Chinese hamster
ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), and a number of other cell lines. In certain
embodiments, one may select cell lines by determining which cell
lines have high expression levels and produce antibodies with
constitutive LRP10 binding properties. In another embodiment, one
may select a cell line from the B cell lineage that does not make
its own antibody but has a capacity to make and secrete a
heterologous antibody (e.g., mouse myeloma cell lines NS0 and
SP2/0).
Pharmaceutical Compositions and Use Thereof
[0123] In one aspect, use of LRP10 inhibitor or competitor for the
manufacture of a medicament for cancer immunotherapy and/or
hematopoietic recovery is provided. In another aspect, a method of
suppressing tumor growth in a patient is provided, the method
comprising administering to the patient an effective amount of an
LRP10 inhibitor or competitor.
[0124] In another aspect, pharmaceutical compositions are provided
that can be used in the methods disclosed herein, i.e.,
pharmaceutical compositions for enhancing hematopoietic recovery in
a subject, e.g., after chemotherapy or irradiation, and/or
providing cancer immunotherapy.
[0125] In some embodiments, the pharmaceutical composition
comprises an LRP10 inhibitor or competitor and a pharmaceutically
acceptable carrier. The LRP10 inhibitor or competitor can be
formulated with the pharmaceutically acceptable carrier into a
pharmaceutical composition. Additionally, the pharmaceutical
composition can include, for example, instructions for use of the
composition for the treatment of patients to enhance hematopoietic
recovery in a subject after chemotherapy or irradiation, and/or
provide cancer immunotherapy.
[0126] In one embodiment, the LRP10 inhibitor can be an anti-LRP10
antibody or antigen-binding fragment thereof.
[0127] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, buffers, and other excipients that are
physiologically compatible. Preferably, the carrier is suitable for
parenteral, oral, or topical administration. Depending on the route
of administration, the active compound, e.g., small molecule or
biologic agent, may be coated in a material to protect the compound
from the action of acids and other natural conditions that may
inactivate the compound.
[0128] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion, as well
as conventional excipients for the preparation of tablets, pills,
capsules and the like. The use of such media and agents for the
formulation of pharmaceutically active substances is known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions provided herein is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0129] A pharmaceutically acceptable carrier can include a
pharmaceutically acceptable antioxidant. Examples of
pharmaceutically-acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0130] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions provided herein
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, and injectable organic esters, such as ethyl oleate. When
required, proper fluidity can be maintained, for example, by the
use of coating materials, such as lecithin, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. In many cases, it may be useful to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0131] These compositions may also contain functional excipients
such as preservatives, wetting agents, emulsifying agents and
dispersing agents.
[0132] Therapeutic compositions typically must be sterile,
non-phylogenic, and stable under the conditions of manufacture and
storage. The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable to
high drug concentration.
[0133] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization, e.g., by
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
methods of preparation include vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The active agent(s) may be mixed
under sterile conditions with additional pharmaceutically
acceptable carrier(s), and with any preservatives, buffers, or
propellants which may be required.
[0134] Prevention of presence of microorganisms may be ensured both
by sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0135] Pharmaceutical compositions comprising an LRP10 inhibitor
can be administered alone or in combination therapy. For example,
the combination therapy can include a composition provided herein
comprising an LRP10 inhibitor and at least one or more additional
therapeutic agents, such as one or more chemotherapeutic agents
known in the art, discussed in further detail below. Pharmaceutical
compositions can also be administered in conjunction with radiation
therapy and/or surgery.
[0136] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation.
[0137] Exemplary dosage ranges for administration of an antibody
include: 10-1000 mg (antibody)/kg (body weight of the patient),
10-800 mg/kg, 10-600 mg/kg, 10-400 mg/kg, 10-200 mg/kg, 30-1000
mg/kg, 30-800 mg/kg, 30-600 mg/kg, 30-400 mg/kg, 30-200 mg/kg,
50-1000 mg/kg, 50-800 mg/kg, 50-600 mg/kg, 50-400 mg/kg, 50-200
mg/kg, 100-1000 mg/kg, 100-900 mg/kg, 100-800 mg/kg, 100-700 mg/kg,
100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100-300 mg/kg and
100-200 mg/kg. Exemplary dosage schedules include once every three
days, once every five days, once every seven days (i.e., once a
week), once every 10 days, once every 14 days (i.e., once every two
weeks), once every 21 days (i.e., once every three weeks), once
every 28 days (i.e., once every four weeks) and once a month.
[0138] It may be advantageous to formulate parenteral compositions
in unit dosage form for ease of administration and uniformity of
dosage. Unit dosage form as used herein refers to physically
discrete units suited as unitary dosages for the patients to be
treated; each unit contains a predetermined quantity of active
agent calculated to produce the desired therapeutic effect in
association with any required pharmaceutical carrier. The
specification for unit dosage forms are dictated by and directly
dependent on (a) the unique characteristics of the active compound
and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0139] Actual dosage levels of the active ingredients in the
pharmaceutical compositions disclosed herein may be varied so as to
obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. "Parenteral" as used herein in the context of
administration means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0140] The phrases "parenteral administration" and "administered
parenterally" as used herein refer to modes of administration other
than enteral (i.e., via the digestive tract) and topical
administration, usually by injection or infusion, and includes,
without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal, epidural and intrasternal injection and infusion.
Intravenous injection and infusion are often (but not exclusively)
used for antibody administration.
[0141] When agents provided herein are administered as
pharmaceuticals, to humans or animals, they can be given alone or
as a pharmaceutical composition containing, for example, 0.001 to
90% (e.g., 0.005 to 70%, e.g., 0.01 to 30%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0142] In certain embodiments, the methods and uses provided herein
for enhancing hematopoietic recovery in a subject after
chemotherapy or irradiation, and/or improving cancer immunotherapy,
can comprise administration of an LRP10 inhibitor and at least one
additional anti-cancer agent that is not an LRP10 inhibitor.
[0143] In one embodiment, the at least one additional anti-cancer
agent comprises at least one chemotherapeutic drug. Non-limiting
examples of such chemotherapeutic drugs include platinum-based
chemotherapy drugs (e.g., cisplatin, carboplatin), taxanes (e.g.,
paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.), EndoTAG-1.TM.
(a formulation of paclitaxel encapsulated in positively charged
lipid-based complexes; MediGene), Abraxane.RTM. (a formulation of
paclitaxel bound to albumin)), tyrosine kinase inhibitors (e.g.,
imatinib/Gleevec.RTM., sunitinib/Sutent.RTM.,
dasatinib/Sprycel.RTM.), and combinations thereof.
[0144] In another embodiment, the at least one additional
anti-cancer agent comprises an EGFR inhibitor, such as an anti-EGFR
antibody or a small molecule inhibitor of EGFR signaling. An
exemplary anti-EGFR antibody is cetuximab (Erbitux.RTM.). Cetuximab
is commercially available from ImClone Systems Incorporated. Other
examples of anti-EGFR antibodies include matuzumab (EMD72000),
panitumumab (Vectibix.RTM.; Amgen); nimotuzumab (TheraCIM.TM.) and
mAb 806. An exemplary small molecule inhibitor of the EGFR
signaling pathway is gefitinib (Iressa.RTM.), which is commercially
available from AstraZeneca and Teva. Other examples of small
molecule inhibitors of the EGFR signaling pathway include erlotinib
HCL (OSI-774; Tarceva.RTM., OSI Pharma); lapatinib (Tykerb.RTM.,
GlaxoSmithKline); canertinib (canertinib dihydrochloride, Pfizer);
pelitinib (Pfizer); PKI-166 (Novartis); PD158780; and AG 1478
(4-(3-Chloroanillino)-6,7-dimethoxyquinazoline).
[0145] In yet another embodiment, the at least one additional
anti-cancer agent comprises a VEGF inhibitor. An exemplary VEGF
inhibitor comprises an anti-VEGF antibody, such as bevacizumab
(Avastatin.RTM.; Genentech).
[0146] In still another embodiment, the at least one additional
anti-cancer agent comprises an anti-ErbB2 antibody. Suitable
anti-ErbB2 antibodies include trastuzumab and pertuzumab.
[0147] In one aspect, the improved effectiveness of a combination
according to the disclosure can be demonstrated by achieving
therapeutic synergy.
[0148] The term "therapeutic synergy" is used when the combination
of two products at given doses is more efficacious than the best of
each of the two products alone at the same doses. In one example,
therapeutic synergy can be evaluated by comparing a combination to
the best single agent using estimates obtained from a two-way
analysis of variance with repeated measurements (e.g., time factor)
on parameter tumor volume.
[0149] The term "additive" refers to when the combination of two or
more products at given doses is equally efficacious than the sum of
the efficacies obtained with of each of the two or more products,
whilst the term "superadditive" refers to when the combination is
more efficacious than the sum of the efficacies obtained with of
each of the two or more products.
[0150] Another measure by which effectiveness (including
effectiveness of combinations) can be quantified is by calculating
the log.sub.10 cell kill, which is determined according to the
following equation: log.sub.10 cell
kill=T-C(days)/3.32.times.T.sub.d in which T-C represents the delay
in growth of the cells, which is the average time, in days, for the
tumors of the treated group (T) and the tumors of the control group
(C) to have reached a predetermined value (1 g, or 10 mL, for
example), and Td represents the time, in days necessary for the
volume of the tumor to double in the control animals. When applying
this measure, a product is considered to be active if log.sub.10
cell kill is greater than or equal to 0.7 and a product is
considered to be very active if log.sub.10 cell kill is greater
than 2.8.
[0151] Using this measure, a combination, used at its own maximum
tolerated dose, in which each of the constituents is present at a
dose generally less than or equal to its maximum tolerated dose,
exhibits therapeutic synergy when the log.sub.10 cell kill is
greater than the value of the log.sub.10 cell kill of the best
constituent when it is administered alone. In an exemplary case,
the log.sub.10 cell kill of the combination exceeds the value of
the log.sub.10 cell kill of the best constituent of the combination
by at least one log cell kill.
[0152] Disclosed herein are compositions and methods for providing
cancer immunotherapy. The method can include inhibiting LRP10 or
competing for binding with endogenous LRP10 in a subject in need
thereof. In certain embodiments, inhibiting LRP10 or competing for
binding with endogenous LRP10 can enhance tumor infiltration of
lymphocytes, preferably CD8+ T cells and cytotoxic T lymphocytes,
thereby providing cancer immunotherapy. LRP10 inhibition (e.g., an
anti-LRP10 antibody) can be used as a stand-alone cancer
immunotherapy by, e.g., enhancing cytotoxic T lymphocytes tumor
infiltration. In some embodiments, LRP10 inhibition can be used in
conjunction with other cancer immunotherapy.
[0153] Also provided herein is a method of enhancing hematopoietic
recovery, comprising inhibiting LRP10 or competing for binding with
endogenous LRP10 in a subject in need thereof. In certain
embodiments, said inhibiting enhances recovery of all hematopoietic
lineages, preferably lymphoid lineages. In some embodiments, the
subject has received chemotherapy and/or radiotherapy.
[0154] In various embodiments, the methods disclosed herein can
include administering to the subject an effective amount of
anti-LRP10 antibody or antigen-binding fragment thereof, or a
decoy, soluble LRP10. In general, the effective amount can be
administered therapeutically and/or prophylactically.
[0155] Treatment can be suitably administered to subjects,
particularly humans, suffering from, having, susceptible to, or at
risk of developing such cancer. Determination of those subjects "at
risk" can be made by any objective or subjective determination by a
diagnostic test or opinion of a subject or health care provider
(e.g., genetic test, enzyme or protein marker, family history, and
the like). Identifying a subject in need of such treatment can be
in the judgment of a subject or a health care professional and can
be subjective (e.g. opinion) or objective (e.g. measurable by a
test or diagnostic method).
Administration of the Formulation
[0156] The formulations of the present disclosure, including but
not limited to reconstituted and liquid formulations, are
administered to a mammal in need of treatment with the anti-LRP10
antibodies, preferably a human, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes.
[0157] In preferred embodiments, the formulations are administered
to the mammal by subcutaneous (i.e., beneath the skin)
administration. For such purposes, the formulation may be injected
using a syringe. However, other devices for administration of the
formulation are available such as injection devices (e.g., the
INJECT-EASE.TM. and GENJECT.TM. devices); injector pens (such as
the GENPEN.TM.); auto-injector devices, needleless devices (e.g.,
MEDUECTOR.TM. and BIOJECTOR.TM.); and subcutaneous patch delivery
systems.
[0158] In a specific embodiment, the present disclosure is directed
to kits for a single dose-administration unit. Such kits comprise a
container of an aqueous formulation of therapeutic protein or
antibody, including both single or multi-chambered pre-filled
syringes. Exemplary pre-filled syringes are available from Vetter
GmbH, Ravensburg, Germany.
[0159] The appropriate dosage ("therapeutically effective amount")
of the protein will depend, for example, on the condition to be
treated, the severity and course of the condition, whether the
protein is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
anti-LRP10 antibody, the format of the formulation used, and the
discretion of the attending physician. The anti-LRP10 antibody is
suitably administered to the patient at one time or over a series
of treatments and may be administered to the patient at any time
from diagnosis onwards. The anti-LRP10 antibody may be administered
as the sole treatment or in conjunction with other drugs or
therapies useful in treating the condition in question.
[0160] For anti-LRP10 antibodies, an initial candidate dosage can
range from about 0.1-20 mg/kg for administration to the patient,
which can take the form of one or more separate administrations.
However, other dosage regimens may be useful. The progress of such
therapy is easily monitored by conventional techniques.
[0161] According to certain embodiments of the present disclosure,
multiple doses of an anti-LRP10 antibody (or a pharmaceutical
composition comprising a combination of an anti-LRP10 antibody and
any of the additional therapeutically active agents mentioned
herein) may be administered to a subject over a defined time
course. The methods according to this aspect of the disclosure
comprise sequentially administering to a subject multiple doses of
an anti-LRP10 antibody of the disclosure. As used herein,
"sequentially administering" means that each dose of anti-LRP10
antibody is administered to the subject at a different point in
time, e.g., on different days separated by a predetermined interval
(e.g., hours, days, weeks or months). The present disclosure
includes methods which comprise sequentially administering to the
patient a single initial dose of an anti-LRP10 antibody, followed
by one or more secondary doses of the anti-LRP10 antibody, and
optionally followed by one or more tertiary doses of the anti-LRP10
antibody. The anti-LRP10 antibody may be administered at a dose of
between 0.1 mg/kg to about 100 mg/kg.
[0162] The terms "initial dose," "secondary doses," and "tertiary
doses," refer to the temporal sequence of administration of the
anti-LRP10 antibody of the disclosure. Thus, the "initial dose" is
the dose which is administered at the beginning of the treatment
regimen (also referred to as the "baseline dose"); the "secondary
doses" are the doses which are administered after the initial dose;
and the "tertiary doses" are the doses which are administered after
the secondary doses. The initial, secondary, and tertiary doses may
all contain the same amount of anti-LRP10 antibody, but generally
may differ from one another in terms of frequency of
administration. In certain embodiments, however, the amount of
anti-LRP10 antibody contained in the initial, secondary and/or
tertiary doses varies from one another (e.g., adjusted up or down
as appropriate) during the course of treatment. In certain
embodiments, two or more (e.g., 2, 3, 4, or 5) doses are
administered at the beginning of the treatment regimen as "loading
doses" followed by subsequent doses that are administered on a less
frequent basis (e.g., "maintenance doses").
[0163] In certain exemplary embodiments of the present disclosure,
each secondary and/or tertiary dose is administered 1 to 26 (e.g.,
1, 11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8,
81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14,
141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20,
201/2, 21, 211/2, 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26,
261/2, or more) weeks after the immediately preceding dose. The
phrase "the immediately preceding dose," as used herein, means, in
a sequence of multiple administrations, the dose of anti-LRP10
antibody which is administered to a patient prior to the
administration of the very next dose in the sequence with no
intervening doses.
[0164] The methods according to this aspect of the disclosure may
comprise administering to a patient any number of secondary and/or
tertiary doses of an anti-LRP10 antibody. For example, in certain
embodiments, only a single secondary dose is administered to the
patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7,
8, or more) secondary doses are administered to the patient.
Likewise, in certain embodiments, only a single tertiary dose is
administered to the patient. In other embodiments, two or more
(e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are
administered to the patient.
[0165] In embodiments involving multiple secondary doses, each
secondary dose may be administered at the same frequency as the
other secondary doses. For example, each secondary dose may be
administered to the patient 1 to 2 weeks or 1 to 2 months after the
immediately preceding dose. Similarly, in embodiments involving
multiple tertiary doses, each tertiary dose may be administered at
the same frequency as the other tertiary doses. For example, each
tertiary dose may be administered to the patient 2 to 12 weeks
after the immediately preceding dose. In certain embodiments of the
disclosure, the frequency at which the secondary and/or tertiary
doses are administered to a patient can vary over the course of the
treatment regimen. The frequency of administration may also be
adjusted during the course of treatment by a physician depending on
the needs of the individual patient following clinical
examination.
[0166] The present disclosure includes administration regimens in
which 2 to 6 loading doses are administered to a patient at a first
frequency (e.g., once a week, once every two weeks, once every
three weeks, once a month, once every two months, etc.), followed
by administration of two or more maintenance doses to the patient
on a less frequent basis. For example, according to this aspect of
the disclosure, if the loading doses are administered at a
frequency of, e.g., once a month (e.g., two, three, four, or more
loading doses administered once a month), then the maintenance
doses may be administered to the patient once every five weeks,
once every six weeks, once every seven weeks, once every eight
weeks, once every ten weeks, once every twelve weeks, etc.).
EXAMPLES
[0167] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting the disclosure.
Example 1. Discovery of LRP10 as an Antibody Target for Use in
Humans
Summary
[0168] Using ENU mutagenesis we have created random germline
mutations and to date, have screened a total of 65,130 G3 mutant
mice from 2,289 pedigrees for unusual flow cytometry phenotypes in
peripheral blood. These mice have been carriers of a total of
126,220 point mutations affecting coding or splicing across the
proteome. One pedigree (R0765) showed a phenotype in which elevated
numbers of T cells (both CD4 and CD8 cells) were present in blood.
Other abnormalities included a low B:T cell ratio, and a low NK
cell count. The causative mutation was identified in Lrp10 (aka
Lrp9), a gene encoding a member of the LDL receptor family. Similar
findings were observed subsequently with other mutant alleles of
the gene, and CRISPR/Cas9 targeting verified the effect with an 8
bp frameshifting deletion mutation.
[0169] Using the CRISPR knockout mice, homozygous mutant T cells
were examined for migratory activity in response to stimulation
with a chemokine, CXCL12. The cells showed greatly enhanced
migration toward this stimulus.
[0170] When mixed with WT bone marrow at a 50:50 ratio and
transplanted into irradiated WT mice, it was observed that mutant
cells greatly outgrew the WT cells, suggesting much stronger
proliferative activity.
[0171] We propose that neutralizing LRP10 with a suitable
monoclonal antibody would assist in hematopoietic recovery after
chemotherapy or irradiation, as required during cancer
chemotherapy. We further propose that LRP10 blockade might be
equivalent to a checkpoint blockade, allowing CD8 T cells to
infiltrate tumors much more effectively than they otherwise
would.
Mutant Mice and Screening
[0172] Eight- to ten-week old pure C57BL/6J background males
purchased from The Jackson Laboratory were mutagenized with
N-ethyl-N-nitrosourea (ENU) to create random germline mutations, as
described previously (Wang T. et al., Proc Natl Acad Sci USA. 2015
Feb. 3; 112(5), PMID: 25605905). Mutagenized G0 males were bred to
C57BL/6J females, and the resulting G1 males were crossed to
C57BL/6J females to produce G2 mice. G2 females were backcrossed to
their G1 sires to yield G3 mice. The G3 mice were screened for
unusual flow cytometry phenotypes in the peripheral blood. To date,
a total of 65,130 G3 mutant mice have been screened from 2,289
pedigrees. These mice have been carriers of a total of 126,220
point mutations affecting coding or splicing across the
proteome.
FACS Analysis
[0173] Mutant G3 mice were generated through
(N-ethyl-N-nitrosourea) ENU-mutagenesis and strategic breeding as
described above. Peripheral blood was collected from G3 mice >6
weeks old by cheek bleeding. RBCs were lysed with hypotonic buffer
(ebiosceince, #00-4300-54). Samples were washed with FACs staining
buffer (PBS with 1% (w/v) BSA) one time at 500.times.g for 6
minutes. The RBC-depleted samples were stained for 1 hour at
4.degree. C., in 100 .mu.l 1:200 cocktail of
fluorescence-conjugated antibodies to 15 cell surface markers
encompassing the major immune lineages: B220 (BD Pharmingen,
#557957); CD19 (BD Biosceince, #563557); IgM (BD Pharmingen,
#550881); IgD, CD3e (BD Horizon, #553062); CD4 (BD Horizon,
#562464); CD8a (Biolegend, #100752); CD11b (Biolegend, #101237);
CD11c (BD Horizon, #563048); F4/80 (Tonbo Bioscience,
#50-4801-U100); CD44 (BD Horizon, #562464); CD62L (Tonbo
Bioscience, #60-0621-U100); CD5 (BD Horizon, #562739); CD43 (BD
Pharmingen, #560663); NK 1.1 (Biolegend, #564143), and 1:200 Fc
block (Fisher Scientific, #352235). Flow cytometry data was
collected on a BD LSR Fortessa cell analyzer and the proportions of
immune cell populations in each G3 mouse were analyzed with FlowJo
software. The resulting screening data were uploaded to Mutagenetix
for automated mapping of causative alleles.
Discovery of LRP10 as a Modulator of Immunity
[0174] Using the above screening method, multiple G3 mice from
pedigree R0765 of ENU-mutagenized mice showed an increased
proportion of peripheral blood CD4.sup.+ and CD8.sup.+ T cells.
Other abnormalities included a low B:T cell ratio, and a low NK
cell count. This phenotype was named chowmein and mapped to a
likely damaging missense mutation in the coding sequence of the
gene encoding low-density lipoprotein related receptor 10 (Lrp10),
a gene encoding a member of the LDL receptor family. Similar
findings were observed subsequently with other mutant alleles of
the gene, and CRISPR/Cas9 targeting verified the effect with an 8
bp frameshifting deletion mutation. The ENU allele resulted in a
change of aspartic acid to tyrosine at position 246 of the
polypeptide chain (D246Y), within the ectodomain of the single
spanning receptor protein.
[0175] To confirm cause and effect, female C57BL/6J mice were
superovulated by injection with 6.5 U pregnant mare serum
gonadotropin (PMSG; Millipore, #367222), then 6.5 U human chorionic
gonadotropin (hCG; Sigma-Aldrich, #C1063) 48 hours later. The
superovulated mice were subsequently mated with C57BL/6J male mice
overnight. The following day, fertilized eggs were collected from
the oviducts and in vitro transcribed Cas9 mRNA (50 ng/.mu.l) and
Lrp10 small base-pairing guide RNA (50 ng/.mu.l;
5'-ACAGCCCTGGACTTGAGTTA-3') were injected into the cytoplasm or
pronucleus of the embryos. The PCR primers were:
TABLE-US-00016 (SEQ ID NO: 71) (Forward) AGTCCCCCAGGAAGAGGCAA (SEQ
ID NO: 72) (Reverse) TCACCTAGGTTCTCACTAGCCCCGT
[0176] The sequencing primer was tgggagttctggcacttccctg. The
injected embryos were cultured in M16 medium (Sigma-Aldrich,
#M7292) at 37.degree. C. and 95% air/5% CO2. For the production of
mutant mice, 2-cell stage embryos were transferred into the ampulla
of the oviduct (10.sup.-20 embryos per oviduct) of pseudopregnant
Hsd:ICR (CD-1) (Harlan Laboratories, #030) females. The founder
Lrp10.sup.-/- mice have a 8-bp deletion in the fifth exon
(CCTGGACTTGAG(TTACGGAG)ATGCAGTGCA) (SEQ ID NO: 73) with 8-bp
deletion denoted in parenthesis), resulting in a frameshift
predicted to cause premature termination after 117 amino acids
compared to the 713 amino acid wild type LRP10 protein. The CRISPR
Lrp10 KO strain was subjected to the FACS screen and recapitulated
the increased peripheral CD4.sup.+ and CD8.sup.+ T cells observed
in chowmein.
Bone Marrow Chimeras
[0177] Recipient mice were given a 6 Gy exposure by X-ray
irradiator twice at a 5-hour interval. Antibiotic water was
provided to the recipients after irradiation.
[0178] Femurs from donor C57BL/6 mice (CD45.1), CRISPR-Lrp10 KO
(CD45.2) homozygotes were placed in a small dish (on ice)
containing medium [RPMI-1640 medium (Life Technologies, #72400-120)
supplemented with 10% (v/v) FBS (Life Technologies, #10082-147), 10
units/ml penicillin, and streptomycin (Life Technologies,
#15140-122). The femurs were flushed with this medium using a 25 G
needle. To remove bits of bone, the marrow was homogenized and the
solution was put through a sterile 40-.mu.m nylon cell strainer (BD
Biosciences, #352340) and collected in a 50-ml tube. The volume was
brought to 50 ml with medium and then centrifuged at 700.times.g
for 5 min at 4.degree. C. The cells were then resuspended in 5 ml
of red blood cell lysing buffer (Sigma-Aldrich, #R7757) and
incubated for 1 to 2 min. 5 ml of sterile PBS was added to the
tube, and 10 .mu.l cells and 10 .mu.l Trypan Blue were mixed to
count the total number of cells. Next, the cells were centrifuged
at 700.times.g for 5 min, and the cells were then resuspended in 1
ml PBS and transferred into 1.5 ml Eppendorf tubes and kept on ice.
Bone marrow cells from C57BL/6 mice (CD45.1), CRISPR-Lrp10 KO mice
(CD45.2), or a 1:1 mixture were transferred into the indicated
recipient mice through retro-orbital injection (FIG. 1). Peripheral
blood was sampled at time points post-transplantation and the
fraction of donor chimeras within the major immune cell populations
was assessed with flow cytometry using fluorescence-conjugated
antibodies against the CD45 congenic markers (CD45.1 Biolegend,
#110732, CD45.2 Biolegend, #109814) and CD3e (BD Horizon, #553062),
CD4 (BD Horizon, #562464), CD8a (Biolegend, #100752), B220 (BD
Pharmingen, #557957), CD19 (BD Biosceince, #563557), CD1 lb
(Biolegend, #101237), and NK 1.1 (Biolegend, #564143). Overall, in
each experiment, it was observed that mutant cells from the
CRISPR-Lrp10 KO mice (CD45.2) significantly outgrew the wild-type
C57BL/6 mice (CD45.1) cells, which suggested a much stronger
proliferative activity (FIG. 2, FIG. 3, and FIG. 4).
T Cell and HSC Migration Assay
[0179] Using the CRISPR knockout mice, homozygous mutant T cells
were examined for migratory activity in response to stimulation
with C-X-C motif chemokine 12 (CXCL12) (FIG. 5). To perform this
experiment, total T cells were isolated from the spleens and lymph
nodes of C57BL/6 mice and CRISPR-Lrp10 KO homozygotes using a
negative selection kit (StemCell Technologies, #19851).
Hematopoietic stem cells (HSCs) were then isolated from the bone
marrow of these mice using an HSC enrichment kit (StemCell
Technologies, #19756). Next, 10 .mu.l of cells and 10 .mu.l of
Trypan Blue were mixed together to count the total number of cells.
Cells were then resuspended in RPMI containing (w/v) 1% BSA at a
density of 10.sup.7/ml. 100 .mu.l of this suspension (10.sup.6
cells) was added to the upper chamber of a 24-well Transwell plate
containing 5-micron polycarbonate membrane inserts (Corning,
#CLS3421-48EA). 600 .mu.l of RPMI/(w/v)1% BSA containing 0, 20, or
200 ng/ml of stromal cell-derived factor 1 (SDF-1) (Peprotech,
#250-20A), also known as CXCL12, was added to the lower chamber.
The Transwell plates were incubated at 37 degrees for 3.5 hours.
After incubation, the upper chamber was discarded and the cells
that had migrated to the lower chamber were stained with
fluorescence-conjugated antibodies to CD4 (BD Horizon, #562464) and
CD8a (Biolegend, #100752). Cell counts were assessed by measuring
the total events collected in 30 s on an LSR Fortessa. The
percentage of migrating cells was calculated by dividing the number
of events collected at each chemokine concentration by the number
of events collected in 30 s from a 1:6 dilution of the input cell
population. Overall, the mutant cells from the CRISPR-Lrp10 KO mice
showed greatly enhanced migration toward the CXCL12 chemokine
stimulus compared to the wild-type C57BL/6 mouse cells (FIG.
5).
Western Blot Assays for p21 and Frizzled-6:
[0180] Wnt signaling pathways play fundamental roles in the
differentiation, proliferation and functions of many cells as well
as developmental, growth, and homeostatic processes in animals.
Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and
LRP6 are co-receptors of Wnt proteins together with Frizzled
receptors, triggering activation of canonical Wnt/beta-catenin
signaling. To understand how LRP10 regulates Wnt/.beta.-catenin
signaling, T cells and HSCs were enriched as described above.
Unstimulated cells (.about.10.sup.6) were then lysed in buffer
containing 1% SDS (ThermoFisher, #AM9820), 1:10,000 Benzonase
(Sigma, #E1014), and 1:100 Protease Inhibitor Cocktail (Cell
Signaling Technology, #5871S) in buffer A (50 mM HEPES, 2 mM
MgCl.sub.2, 10 mM KCl). Protein concentration was measured using
the BCA assay (Pierce). The cell extracts (20 .mu.l each;
equivalent to .about.-1.times.10.sup.6 cells) were separated on
NuPAGE.TM. Novex.TM. 4-12% Bis-Tris protein gels (Life
Technologies, #NP0336BOX) and proteins were transferred to
nitrocellulose membranes (Bio-Rad, #162-0115) for 1 hour at 12
voltage. After blocking in Tris-buffered saline containing 0.05%
(v/v) Tween-20 (TBS-T) with 5% (w/v) BSA at room temperature for 2
hours, the membrane was incubated overnight with primary antibody
anti-P21 (Santa Cruz Bio Tech, #SC-6246), anti-Frizzled (Santa Cruz
Bio Tech, #SC-393113), and anti-.beta.Actin (Cell Signaling,
#3700S) at 4.degree. C. in 5% (w/v) BSA in TBS-T with gentle
rocking. The membrane was then incubated with secondary antibody
goat anti-mouse IgM-HRP (Southern Biotech, #1021-05), goat
anti-rabbit IgG-HRP (Thermo fisher, #A16096), or goat anti-rabbit
IgG-HRP (Thermo fisher, #A16096) for 1 hour at room temperature.
The Chemiluminescence signal was developed by using SuperSignal
West Dura Extended Duration Substrate kit (Fisher Scientific,
#PIA34075) and detected by a G:Box Chemi XX6 system (Syngene). As
shown in FIG. 6, the Lrp10.sup.-/- T cells and the Lrp10.sup.-/-
hematopoietic cells have higher expression of p21, which is an end
product of Wnt signaling. Also, both the Lrp10.sup.-/- T cells and
the Lrp10.sup.-/- hematopoietic cells express the transmembrane
spanning receptor, Fizzled 6. Together, this data demonstrates that
the increase competitive potential of Lrp10.sup.-/- T cells and
Lrp10.sup.-/- hematopoietic cells is dependent on a non-canonical
Wnt/.beta.-catenin signaling pathway.
Flow Cytometry:
[0181] Peripheral blood cells were isolated, and red blood cell
(RBC) lysis buffer was added to remove the RBCs. Cells were stained
at a 1:200 dilution with 15 mouse fluorochrome-conjugated
monoclonal antibodies specific for the following murine cell
surface markers encompassing the major immune lineages: B220, CD19,
IgM, IgD, CD3R, CD4, CD5, CD11c, CD44, CD43, CD25, CD21, CD23, BP-1
(BD Pharmingen), CD8.alpha., CD11b, NK1.1 (Biolegend), F4/80, CD62L
(Tonbo Biosciences) and in the presence of anti-mouse CD16/32
antibody (Tonbo Biosciences) for 1 h at 4.degree. C. After
staining, cells were washed twice in PBS and analyzed by flow
cytometry.
[0182] As shown in FIG. 7, a point mutation in the extracellular
domain of Lrp10 (D246Y) was created during ENU mutagenesis. Mice
homozygous for this mutation showed an increased proportion of
CD3+, CD4+, and CD8+ T cells in their peripheral blood and were
named chowmein (Lrp10.sup.ch/ch). This mutation had a recessive
mode of inheritance as heterozyogotes (Lrp10.sup.+/ch) and
wild-type mice (Lrp10.sup.+/+) were not affected.
Tumor Inoculation, Anti-PD-1 Treatment and Tumor Measurement:
[0183] B16F10 melanoma cells were grown in DMEM containing 10%
vol/vol FBS. A total of 2.times.10.sup.5 B16F10 cells in 100 .mu.L
DPBS were injected s.c. into the right flank of C57BL/6J mice, wild
type, heterozygous and homozygous LRP10 CRISPR KO mice to establish
tumors. For anti-PD-1 treatment, 300 .mu.g anti-PD-1 in 200 .mu.L
DPBS was injected i.p. into mice on day 5, 8 and 11 after tumor
inoculation. Tumors were measured with a digital caliper (Fisher),
and the tumor sizes were calculated using the following formula:
volume=0.5.times.length.times.width.sup.2. Mice were killed when
the tumor length or width reached 2 cm.
[0184] As shown in FIG. 8, Lrp10 knockout mice (Lrp10.sup.-/-) were
made using CRISPR-Cas9. Lrp10.sup.+/+ and Lrp10.sup.-/- mice were
injected subcutaneously with the syngeneic B16 melanoma tumor cell
line. 7 days after tumor cell injection, mice were given an
intraperitoneal injection with anti-PD1 for checkpoint blockade.
Tumor volume was measured on days 17, 19, and 24 post-tumor
injection. Lrp10.sup.-/- showed significantly lower average tumor
volume at these time points.
[0185] As shown in FIG. 9, on day 19 post-tumor injection,
peripheral blood was harvested from B16-bearing Lrp10.sup.+/+ and
Lrp10.sup.-/- mice. Tumor-bearing Lrp10.sup.-/- mice had a higher
proportion of total T cells in the peripheral blood. In contrast to
tumor naive Lrp10.sup.-/-, only CD8+ T cells were elevated in
tumor-bearing Lrp10.sup.-/- mice and these had an activated
effector phenotype as measured by CD44 expression.
Example 2: Generation of LRP10 Monoclonal Antibody
[0186] Based on analysis of the NCBI Mus musculus
(www.ncbi.nlm.nih.gov/gene/65107) LRP10, it was determined that two
fragments (XP_006519421.1 and NP_075369.2) from Mus musculus LRP10
were predicted as alternatively spliced transcripts from Lrp10 EST
libraries. Also, based on analysis of Homo sapiens
(www.ncbi.nlm.nih.gov/gene/26020) LRP10 (also known as LRP9 in Homo
sapiens), it was determined that three fragments (NP_001316155.1,
NP_054764.2, XP_005267567) from Homo sapiens LRP10 were predicted
as alternatively spliced transcripts from Lrp10 EST libraries. Both
human NP_054764.2 and murine NP_075369.2, as canonical LRP10
proteins, are 713-residue long with 17-residue signal peptides, as
analyzed by SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/). Human
LRP10 protein has 88% identity to the murine LRP10 protein. Three
Homo sapiens LRP10 protein variants, including the 516-, 713-, or
721-residue variants, were aligned by ClustalW. Comparing to the
canonical protein, two isoforms lack residues 135-143, and human
LRP10 isoform2 lacks the last 157 residues. Canonical human LRP10
protein has an ectodomain consisting of the first 440 residues, a
transmembrane domain consisting of residues 441-463, and an
intracellular domain consisting of residues 464-713. In order to
target all the isoforms of human LRP10 protein, isoform2 was
analyzed by Lasergene/Protean. Based on this analysis, residues
159.sup.th-192.sup.th, 294.sup.th-360.sup.th, and
378.sup.th-434.sup.th polypeptides are candidates for the
anti-human Lrp10 antibodies.
[0187] The amino-acid sequence of the first 420 amino acids of the
protein, which can be expressed for use as an immunogen, is as
follows:
TABLE-US-00017 >sp|Q7Z4F1|LRP10_HUMAN Low-density lipoprotein
receptor-related protein 10 OS = Homo sapiens GN = LRP10 PE = 1 SV
= 2 (SEQ ID NO: 74)
MLLATLLLLLLGGALAHPDRIIFPNHACEDPPAVLLEVQGTLQRPLVRDS
RTSPANCTWLILGSKEQTVTIRFQKLHLACGSERLTLRSPLQPLISLCEA
PPSPLQLPGGNVTITYSYAGARAPMGQGFLLSYSQDWLMCLQEEFQCLNH
RCVSAVQRCDGVDACGDGSDEAGCSSDPFPGLTPRPVPSLPCNVTLEDFY
GVFSSPGYTHLASVSHPQSCHWLLDPHDGRRLAVRFTALDLGFGDAVHVY
DGPGPPESSRLLRSLTHFSNGKAVTVETLSGQAVVSYHTVAWSNGRGFNA
TYHVRGYCLPWDRPCGLGSGLGAGEGLGERCYSEAQRCDGSWDCADGTDE
EDCPGCPPGHFPCGAAGTSGATACYLPADRCNYQTFCADGADERRCRHCQ
PGNFRCRDEKCVYETWVCDGQPDCADGSDEWDCSYVLPRK
[0188] A portion of the above ectodomain, as opposed to the full
length, can also be used as an immunogen. Different methods known
in the art, and those that have been disclosed herein, may be used
to generate fully human or humanized anti-LRP10 antibodies. For
example, as described above, fully human LRP10 antibodies can also
be produced from phage-display libraries. Humanized anti-LRP10
antibodies can be prepared by humanizing monoclonal antibodies
obtained from hybridomas.
[0189] For example, a C-terminal His tag, suitable for purification
by affinity chromatography, can be added to the immunogen. Purified
protein can be inoculated into mice together with a suitable
adjuvant. Monoclonal antibodies produced in hybridomas can be
tested for binding to the immunogen, and positive binders can be
screened for ability to neutralize T cell migration or to affect
Frizzled and/or p21 expression in human lymphoid cells in the
assays described above. Thereafter, antibodies can be humanized for
preclinical and clinical studies.
Biopanning and Screening:
[0190] A typical biopanning includes 3-5 rounds to achieve an
acceptable enrichment.
1. Library Biopanning
[0191] 1) Coat each well of an ELISA plate with 2 .mu.g target of
interest in 50 .mu.L Coating Buffer (0.1 M NaHCO.sub.3, pH 9.6) and
incubate overnight at 4.degree. C. Discard the coating solution and
wash the wells with washing buffer of 0.1% PBST [PBS with 0.1%
(v/v) Tween 20] for 3 times and block the wells by adding 300 .mu.L
Blocking Buffer [2% PBSM (PBS containing 2% skim milk)]. Seal and
incubate overnight at 4.degree. C. [0192] 2) Discard the Blocking
Buffer and add 50-100 .mu.L phage library (.about.10.sup.11 phage
particles) to each well (4 wells). Seal and incubate for 2 h at
37.degree. C. [0193] 3) Remove the spent phage solution. Wash the
coated surface 9 times using an ELISA plate washer machine [0194]
4) After removing the final washing solution, add 200 .mu.L Elution
Buffer, incubate for 10 min at 37.degree. C. Pipet vigorously up
and down for 10 times. Transfer the eluate to a microfuge tube
containing 100 .mu.L Neutralizing Buffer.
2. Phage Titration
[0194] [0195] 1) Make serial dilutions of phages in 2.times.YT
Medium (For the phage input, expect titers of 10.sup.12-13
phage/mL. Make dilutions between 10.sup.-8 and 10.sup.-10. For the
phage output, make dilutions between 10.sup.-1 and 10.sup.1 for
Round 1, while make dilutions between 10.sup.-2 and 10.sup.-6 for
Round 2 and 3). [0196] 2) Add 500 .mu.L log phase E. coli TG1s
(OD600.apprxeq.0.5) (30 min 37.degree. C.). [0197] 3) Plate out 100
.mu.L infected cells onto 2.times.YT agar containing 2% glucose and
100 .mu.g/mL ampicillin and grow at 37.degree. C. O/N.
3. Rescue of the Selected Phages
[0197] [0198] 1) Infect E. coli TG1 with eluted phage output by
adding the total output to 6 mL TG1 (OD.sub.600.apprxeq.0.5).
[0199] 2) Allow infection to incubate in 37.degree. C. water bath
for 30 min. [0200] 3) Add 9 mL 2.times.YT-AG Medium. Grow overnight
at 37.degree. C. [0201] 4) Centrifuge at 5,000 rpm for 15 min at
4.degree. C. Resuspend the pellet in 2.times.YT Medium containing
15% glycerol and store at -80.degree. C. Master stock to be rescued
for future selections.
4. Phage Amplification for Next Round of Biopanning
[0201] [0202] 1) Inoculate part of the rescued glycerol stock into
25 mL 2.times.YT-AG Medium. [0203] 2) Grow to exponential phase
(OD.sub.600.apprxeq.0.5) by shaking at 37.degree. C. [0204] 3) Add
.about.2.times.10.sup.11 pfu M13KO7 Helper Phage. [0205] 4) Allow
infection to incubate in 37.degree. C. water bath for 30 min.
[0206] 5) Centrifuge at 5,000 rpm for 15 min. [0207] 6) Resuspend
the pellet in 25 mL 2.times.YT-AK Medium. [0208] 7) Transfer to a
250 mL flask. Grow shaking (225 rpm) overnight at 30.degree. C.
5. Phage Purification for Next Round of Biopanning
[0208] [0209] 1) Spin the culture in a 50 mL centrifuge tube at
4,000 g for 20 min to pellet the bacteria. [0210] 2) To the
supernatant, add 1/5.sup.th of the volume of 5.times.PEG/NaCl and
leave on ice for at least 1 h. [0211] 3) Pellet phages by spinning
at 12,000 g for 15 min at 4.degree. C. [0212] 4) Discard the
supernatant. [0213] 5) Resuspend the pellet in 1 mL sterile PBS.
[0214] 6) Transfer to a 1.5 mL microcentrifuge tube. [0215] 7) Spin
in microcentrifuge (2 min, maximum g) to remove the remaining
bacteria. [0216] 8) Transfer supernatant to a new tube. The phages
are now ready for use in the further screening rounds or selection
assays. Phage can be stored at 4.degree. C. without much decrease
in titer. For long-term storage, add glycerol to 20% and store at
-80.degree. C.
Antibody Expression and Purification:
[0216] [0217] 1. The cDNAs of light chain and heavy chain of the
antibody were chemically synthesized with optimization for
mammalian expression. The cDNAs were cloned in the expression
vector. [0218] 2. The encoding genes of light chain and heavy chain
were co-transfected with 30 mL 293F cells. The supernatant was
harvested on day 6 after transfection. [0219] 3. The antibodies
were purified by affinity chromatography using immobilized protein
A. [0220] 1) Transfer 1 ml of Protein A pure resin to a new
disposable plastic gravity flow column. Wash the resin with 10
column volumes (CVs) of ultrapure water, and then equilibrate with
10 CVs of TBS, pH 7.5/8.0 buffer. [0221] 2) Apply the filtered
antibody supernatant to the column and allow it to flow through by
gravity. Wash the resin twice with 10 CVs of PBS. Collect the
flow-throughs from both the supernatant and the washes, and keep
them on ice or at 4.degree. C. for further analysis. [0222] 3) For
elution of bound antibody, add 4 ml of glycine-HCl elution buffer
(pH 2.7) and collect the flow-through into tubes prefilled with 1
ml of Tris-HCl solution (pH 7.6). Collect additional fractions as
above. Stop elution when the OD280 nm of the fractions is zero
(this is usually after three to five fractions). [0223] 4) After
elution, subject the resin to sequential washes with 10 CVs of
glycine-HCl (pH 2.7), 10 CVs of PBS, 10 CVs of ultrapure water and
10 CVs of 20% (vol/vol) ethanol. Store the resin in 20% (vol/vol)
ethanol at 4.degree. C. for future use. [0224] 5) Analyze all
fractions by SDS-PAGE. [0225] 6) Combine the fractions with visible
protein bands and dialyze against >30 volumes of low-endotoxin
PBS buffer for at least 2 h at 4.degree. C. using dialysis tubing.
[0226] 7) Concentrate the dialyzed protein using a centrifugal
concentrator (<10 kDa cut-off) prewashed with PBS. [0227] 8)
Filter the concentrated sample through a syringe-driven 0.22-.mu.m
filter and calculate the protein concentration by BCA method.
ELISA Test:
[0227] [0228] 1) Coat the target protein LR10 (1 .mu.g/ml, 0.1
.mu.g/well) in a certain coating buffer (0.1 M NaHCO.sub.3, pH
9.6). Incubate overnight at 4.degree. C. [0229] 2) Wash the wells 3
times with 300 .mu.L washing buffer (0.1% PBST) per well. [0230] 3)
Block the wells with 300 .mu.L blocking buffer (3% BSA) for 1 h at
37.degree. C. [0231] 4) Discard the blocking buffer and wash the
plate 3 times with the washing buffer, slapping the plate face-down
onto a clean section of paper towel each time. [0232] 5) Add 100
.mu.L of a serial dilution of antibodies per well. Incubate at
37.degree. C. for 1 h. [0233] 6) Wash 3 times with the washing
buffer. Dilute HRP-goat anti-human IgG (1:4,000) in the blocking
buffer (3% BSA). Add 100 .mu.L of diluted conjugate per well and
incubate at 37.degree. C. for 1 h. [0234] 7) Wash 3 times with the
washing buffer. Dissolve Tetramethylbenzidine (TMB) substrate
(Sigma, USA) in 0.1 mmol/L citrate-phosphate buffer and add 1
.mu.L/mL H.sub.2O.sub.2 to the solution. Add 100 .mu.L of substrate
solution per well and incubate at room temperature for 15 minutes.
[0235] 8) Add 100 .mu.L of 2 mol/L H.sub.2SO.sub.4 solution to stop
the reaction. [0236] 9) Read plates using a microplate reader set
at 490 nm.
Results:
[0237] The Phage Display HuScl-2.TM. Library from Creative Biolabs
Inc. was used for 3 rounds of biopinning. His-tagged recombinant
human LRP10 protein, LRP10.sup.-516H was used as antigen.
Sequencing of the sublibrary from the 3.sup.rd round of biopanning
identified 9 unique clones having the following VL and VH sequences
(both amino acid and nucleotide sequences). Note that for the full
antibody sequence, each VL can be linked to a light chain constant
region such as Ck (SEQ ID NO: 31) to form a full light chain, and
each VH can be linked to a heavy chain constant region such as
human IgG1 CH123 (SEQ ID NO: 32) to form a full heavy chain.
TABLE-US-00018 Clone 1 (AB1) Protein sequence VL (SEQ ID NO: 1):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYNA
SDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSRTPTTFGQG TKVEIK VH (SEQ
ID NO: 2): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEWV
SSIPPGGPNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SYPSFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 3):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
AATGCATCCGATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGTCGTCGCGGACGCCTACGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 4):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
AGGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTCCTCCGGGTGGTCCTAATACAAAGTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AGTTATCCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 2
(AB2) Protein sequence VL (SEQ ID NO: 5):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASPLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVARTPNTFG QGTKVEIK VH (SEQ
ID NO: 2): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEWV
SSIPPGGPNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SYPSFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 6):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
GCGGCATCCCCGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGGTGGCTCGTACGCCTAATACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 4):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
AGGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTCCTCCGGGTGGTCCTAATACAAAGTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AGTTATCCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 3
(AB3) Protein sequence VL (SEQ ID NO: 7):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
RASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPTSLPLTFG QGTKVEIK VH (SEQ
ID NO: 8): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SQIGTMGRPTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SGKKFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 9):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
AGGGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGCCGACTTCGTTGCCTCTGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 10):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATAGATTGGGACGATGGGTCGGCCGACAACTTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AGTGGGAAGAAGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 4
(AB4) Protein sequence VL (SEQ ID NO: 11):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADSYPTTFG QGTKVEIK VH (SEQ
ID NO: 12): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRLAPGKGLEWV
SSISTTGNSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
DATSFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 13):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
GCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGGCTGATTCTTATCCTACTACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 14):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCTGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTTCTACTACTGGTAATAGTACATATTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
GATGCTACTAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 5
(AB5) Protein sequence VL (SEQ ID NO: 15):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
RASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQIKTRPTTFG QGTKVEIK VH (SEQ
ID NO: 16): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SVIQRQGTGTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
NSRTFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 17):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
CGTGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGATTAAGACAAGGCCTACGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 18):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCAGTTATTCAGCGTTAGGGTACTGGTACAGAGTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAAA
AATTCGCGGACGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 6
(AB6) Protein sequence VL (SEQ ID NO: 19):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
DASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSAGPGTFG QGTKVEIK VH (SEQ
ID NO: 20): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SSIPSRGQATKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SRHTFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 21):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
GATGCATCCCTTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGACTTCTGCGGGTCCTGGTACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 22):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCAAGTATTCCTAGTCGTGGTTAGGCAACAAAGTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGCGCGAAA
TCGCGTCATACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 7
(AB7) Protein sequence VL (SEQ ID NO: 23):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNDYYPTTFG QGTKVEIK VH (SEQ
ID NO: 24): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SSIATTGNTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
NTATFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 25):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
GCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGAATGATTATTATCCTACTACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 26):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTGCTACTACTGGTAATACTACATATTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AATACTGCTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 8
(AB8) Protein sequence VL (SEQ ID NO: 1):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
NASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSRTPTTFG QGTKVEIK VH (SEQ
ID NO: 27): MAEVQLLESGGGLVQLGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEWV
SSIPPGGPNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SYPSFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 3):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
AATGCATCCGATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGTCGTCGCGGACGCCTACGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 28):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCTTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
AGGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTCCTCCGGGTGGTCCTAATACAAAGTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AGTTATCCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Clone 9
(AB9) Protein sequence VL (SEQ ID NO: 1):
TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
NASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSRTPTTFG QGTKVEIK VH (SEQ
ID NO: 29): MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SSIYTSGAATTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SYPSFDYWGQGTLVTVSS Nucleotide sequence VL (SEQ ID NO: 3):
ACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATT
TAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
AATGCATCCGATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGG
ATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCAACAGTCGTCGCGGACGCCTACGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA VH (SEQ ID NO: 30):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG
GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT
ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCTATTTATACTTCTGGTGCTGCTACAACTTACGCAGACTCCGTGAA
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA
AGTTATCCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC GAGC Ck amino
acid sequence (SEQ ID NO: 31):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC IgG1
CH123 amino acid sequence (SEQ ID NO: 32):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0238] We chemically synthesized the cDNAs of these antibodies for
mammalian cell expression. The genes of light chain and heavy chain
were cloned into the expression vector, respectively, and
co-transfected into 293F cells. We first used an 80 mL culture for
each clone for the antibody expression test. As shown in FIG. 10, 8
antibodies were expressed successfully, while the expression of AB5
was too low to be detected. After purification by Protein A-based
affinity chromatography, all 8 antibodies achieved high purity.
[0239] For clone AB5, we also tried the CHO expression system but
its expression level was still quite low. This indicates that the
poor expressabilty of this clone is due to the antibody sequence
itself.
[0240] For the 8 well-expressed antibodies, we tested their binding
activities to the target antigen by ELISA (Table 7 and FIG. 11).
Among the 8 clones, 5 [AB1 (L1+H1), AB2 (L2+H1), AB3 (L3+H2),
AB7(L7+H6), and AB8 (L1+H7)] of them showed high affinity to the
antigen, whereas the other 3 antibodies did not exhibit binding
activities.
[0241] For the 5 high-affinity antibodies, AB1 and AB2 showed the
best binding activity, which was consistent with the sequencing
results that these two sequences had the highest frequencies.
TABLE-US-00019 TABLE 7 ELISA results Con. (ng/ml) ABI (L1 + H1) AB2
(L2 + H1) AB3 (L3 + H2) AB4 (L4 + H3) 1000 2.4919 2.5264 2.6404
2.6633 2.6678 2.6894 0.1135 0.108 250 2.4027 2.3947 2.3273 2.5826
2.4178 2.4264 0.0754 0.0744 62.5 2.1695 2.2884 2.1059 2.3035 1.878
1.7456 0.0768 0.0789 15.63 1.3403 1.4688 1.3149 1.4561 0.8578
0.8585 0.0612 0.0662 3.91 0.527 0.5902 0.5337 0.5752 0.318 0.3198
0.0591 0.0582 0.98 0.1853 0.1948 0.1855 0.2099 0.1201 0.1158 0.057
0.0598 0.24 0.0871 0.0916 0.0904 0.0906 0.0625 0.0596 0.0533 0.0578
0 0.0508 0.0592 0.0592 0.0697 0.0393 0.0575 0.0609 0.0664 EC50
12.97 14.78 34.87 Con.(ng/ml) AB6 (L6 + H5) AB7(L7 + H6) AB8 (L1 +
H7) AB9 (L1 + H8) 1000 0.2887 0.3495 3.1821 3.1195 1.872 1.8043
0.0561 0.052 250 0.1284 0.1332 2.7589 2.915 1.7051 1.7107 0.0665
0.0619 62.5 0.09 0.0989 2.168 2.0191 1.6008 1.6468 0.066 0.0548
15.63 0.0659 0.0656 0.9558 1.072 1.0632 1.1305 0.0619 0.0519 3.91
0.0615 0.0582 0.3916 0.3886 0.3891 0.4484 0.0523 0.0586 0.98 0.0588
0.0556 0.1293 0.0523 0.1585 0.1749 0.057 0.0464 0.24 0.0606 0.0573
0.0723 0.0801 0.0851 0.0859 0.0619 0.053 0 0.0595 0.0594 0.073
0.0579 0.0546 0.0515 0.0512 0.0452 EC50 35.68 11.71
Example 3. Functional Test of Monoclonal Antibodies
Western Blot
[0242] The expression of P21 was examined for the blocking of Lrp10
functions by human antibodies. For direct Western blot analysis in
purified splenic pan T cells isolated using a negative selection
method (StemCell Technologies, #19851) were cultured with or
without 100 ug/ml human anti-Lrp10 antibodies AB1, AB2, AB3, AB4,
AB6, AB7, AB8, or AB9 for 4 h, and lysed in buffer (1% (w/v) SDS
(Thermo), 0.01% (w/v) Benzonase (Sigma), protease inhibitor
cocktail (Cell Signaling Technology) in buffer A (50 mM HEPES, 2 mM
MgCl2, 10 mM KCl). Protein concentration was measured using a BCA
assay (Pierce). Ten micrograms of protein were separated on 4-12%
Bris-Tris protein gels (Life Technologies) and proteins were
transferred to nitrocellulose membranes (Bio-Rad) for 45 minutes at
13 volts. After blocking in Tris-buffered saline containing 0.05%
(v/v) Tween-20 (TBS-T) with 5% (w/v) non-fat dry milk (NFDM) at
room temperature for 1 hour, the membrane was incubated overnight
with primary antibodies anti-P21 (Santa Cruz) and anti-PActin (Cell
Signaling Technology), at 4.degree. C. in 5% (w/v) BSA in TBS-T
with gentle rocking. The membrane was then incubated with secondary
antibody goat anti-rabbit or mouse IgG-HRP (Thermo fisher) for 1
hour at room temperature with gentle rocking. The chemiluminescence
signal was developed by using SuperSignal West Dura Extended
Duration Substrate kit (Thermo Fisher) and detected by a G:Box
Chemi XX6 system (Syngene). Overall, AB2 showed greatly enhanced
P21 expression in in wild-type C57BL/6 mouse cells.
[0243] We had previously observed that T cells purified from
Lrp10.sup.-/- mice had increased expression of the cell cycle
inhibitor p21 compared to Lrp10.sup.+/+ T cells. T cells were
purified from the spleens of Lrp10.sup.+/+ mice and incubated with
different anti-human Lrp10 clones. P21 expression levels were
measured with Western blot. As shown in FIG. 12, here was no
difference in p21 expression between untreated T cells and T cells
treated with anti-Lrp10 antibodies. T cells treated with antibody 2
showed an intense immunoreactive band at .about.35 kD which may
reflect induction of a post-translational modification (e.g.
glycosylation or sumoylation). The left and right panel show two
separate experiments.
T Cells Migration Assay
[0244] Using the C57BL/6 T cells were examined for migratory
activity in response to stimulation with C-X-C motif chemokine 12
(CXCL12). To perform this experiment, total T cells were isolated
from the spleens and lymph nodes of C57BL/6 mice using a negative
selection kit (StemCell Technologies, #19851). Next, 10 .mu.l of
cells and 10 .mu.l of Trypan Blue were mixed together to count the
total number of cells. Cells were then resuspended in RPMI
containing (w/v) 1% BSA at a density of 10.sup.7/ml. 100 .mu.l of
this suspension (10.sup.6 cells) was pre-treated with or without 50
ug/ml Lrp10 antibody for half hours, and then cells were added to
the upper chamber of a 24-well Transwell plate containing 5-micron
polycarbonate membrane inserts (Corning, #CLS3421-48EA). 600 .mu.l
of RPMI/(w/v)1% BSA containing 200 ng/ml of stromal cell-derived
factor 1 (SDF-1) (Peprotech, #250-20A), also known as CXCL12, with
or without 50 ug/ml Lrp10 antibody, was added to the lower chamber.
The Transwell plates were incubated at 37 degrees for 3.5 hours.
After incubation, the upper chamber was discarded and the cells
that had migrated to the lower chamber were assessed by measuring
the total events collected in 60 s on an LSR Fortessa. The
percentage of migrating cells was calculated by dividing the number
of events collected at each chemokine concentration by the number
of events collected in 60 s from a 1:6 dilution of the input cell
population. Overall, AB2 and AB7 showed greatly enhanced migration
toward the CXCL12 chemokine stimulus in wild-type C57BL/6 mouse
cells.
[0245] The statistical significance of differences between groups
was analyzed using GraphPad by performing the indicated statistical
tests. Differences in the raw values among groups were considered
statistically significant when P<0.05. P values are denoted by *
P<0.05; ** P<0.01; *** P<0.001; ns, not significant with
P>0.05.
[0246] We had observed previously that Lrp10.sup.-/- T cells showed
an increased migration rate in response to SDF-1 (Cxcl12, ligand
for CXCR4) in a transwell assay. Lrp10.sup.+/+ splenocytes were
incubated with different anti-Lrp10 antibody clones or with vehicle
and placed in the upper well of a transwell chamber. The lower
chamber contained SDF-1 at 200 ng/ml. Cell were allowed to migrate
for 3.5 h. Afterward, the number of migrated T cells in the lower
chamber was counted on a flow cytometer. Migration rate is
presented as the number of T cells that migrated relative to the
input number. As shown in FIG. 13, all anti-Lrp10 antibodies
induced a higher migration rate compared to vehicle alone.
Antibodies 2, 7, and 8 induced the highest migration rate, about 2
fold higher than vehicle, in response to SDF-1.
[0247] Various aspects of the present disclosure may be used alone,
in combination, or in a variety of arrangements not specifically
discussed in the embodiments described in the foregoing and is
therefore not limited in its application to the details and
arrangement of components set forth in the foregoing description or
illustrated in the drawings. For example, aspects described in one
embodiment may be combined in any manner with aspects described in
other embodiments.
[0248] While specific embodiments of the subject disclosure have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the disclosure will become apparent
to those skilled in the art upon review of this specification. The
full scope of the disclosure should be determined by reference to
the claims, along with their full scope of equivalents, and the
specification, along with such variations.
INCORPORATION BY REFERENCE
[0249] All publications, patents and patent applications referenced
in this specification are incorporated herein by reference in their
entirety for all purposes to the same extent as if each individual
publication, patent or patent application were specifically
indicated to be so incorporated by reference.
Sequence CWU 1
1
741108PRTUnknownSynthetic 1Thr Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Asn Ala Ser Asp Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Arg Thr Pro 85 90 95Thr Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 1052118PRTUnknownSynthetic
2Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro1 5
10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30Ser Gln Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40 45Trp Val Ser Ser Ile Pro Pro Gly Gly Pro Asn Thr Lys
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Lys Ser Tyr Pro Ser Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
1153324DNAUnknownSynthetic 3acggacatcc agatgaccca gtctccatcc
tccctgtctg catctgtagg agacagagtc 60accatcactt gccgggcaag tcagagcatt
agcagctatt taaattggta tcagcagaaa 120ccagggaaag cccctaagct
cctgatctat aatgcatccg atttgcaaag tggggtccca 180tcaaggttca
gtggcagtgg atctgggaca gatttcactc tcaccatcag cagtctgcaa
240cctgaagatt ttgcaactta ctactgtcaa cagtcgtcgc ggacgcctac
gacgttcggc 300caagggacca aggtggaaat caaa 3244354DNAUnknownSynthetic
4atggccgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg
60agactctcct gtgcagcctc tggattcacc tttagcagct aggccatgag ctgggtccgc
120caggctccag ggaaggggct ggagtgggtc tcatctattc ctccgggtgg
tcctaataca 180aagtacgcag actccgtgaa gggccggttc accatctcca
gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc
gaggacacgg ccgtatatta ctgtgcgaaa 300agttatcctt cttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagc 3545108PRTUnknownSynthetic
5Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5
10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Ala Ala Ser Pro Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Val Ala Arg Thr Pro 85 90 95Asn Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 1056324DNAUnknownSynthetic 6acggacatcc agatgaccca
gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt gccgggcaag
tcagagcatt agcagctatt taaattggta tcagcagaaa 120ccagggaaag
cccctaagct cctgatctat gcggcatccc cgttgcaaag tggggtccca
180tcaaggttca gtggcagtgg atctgggaca gatttcactc tcaccatcag
cagtctgcaa 240cctgaagatt ttgcaactta ctactgtcaa caggtggctc
gtacgcctaa tacgttcggc 300caagggacca aggtggaaat caaa
3247108PRTUnknownSynthetic 7Thr Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Ala Ser Arg Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Pro Thr Ser Leu Pro 85 90 95Leu Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 1058118PRTUnknownSynthetic
8Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro1 5
10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40 45Trp Val Ser Gln Ile Gly Thr Met Gly Arg Pro Thr Thr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Lys Ser Gly Lys Lys Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
1159324DNAUnknownSynthetic 9acggacatcc agatgaccca gtctccatcc
tccctgtctg catctgtagg agacagagtc 60accatcactt gccgggcaag tcagagcatt
agcagctatt taaattggta tcagcagaaa 120ccagggaaag cccctaagct
cctgatctat agggcatccc gtttgcaaag tggggtccca 180tcaaggttca
gtggcagtgg atctgggaca gatttcactc tcaccatcag cagtctgcaa
240cctgaagatt ttgcaactta ctactgtcaa cagccgactt cgttgcctct
gacgttcggc 300caagggacca aggtggaaat caaa
32410354DNAUnknownSynthetic 10atggccgagg tgcagctgtt ggagtctggg
ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc
tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct
ggagtgggtc tcatagattg ggacgatggg tcggccgaca 180acttacgcag
actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg
240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta
ctgtgcgaaa 300agtgggaaga agtttgacta ctggggccag ggaaccctgg
tcaccgtctc gagc 35411108PRTUnknownSynthetic 11Thr Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr
Ala Ala Ser Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Tyr Pro
85 90 95Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10512118PRTUnknownSynthetic 12Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val
Arg Leu Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Ser Ile Ser
Thr Thr Gly Asn Ser Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Lys Asp Ala Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11513324DNAUnknownSynthetic 13acggacatcc
agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt
gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa
120ccagggaaag cccctaagct cctgatctat gctgcatccg ctttgcaaag
tggggtccca 180tcaaggttca gtggcagtgg atctgggaca gatttcactc
tcaccatcag cagtctgcaa 240cctgaagatt ttgcaactta ctactgtcaa
caggctgatt cttatcctac tacgttcggc 300caagggacca aggtggaaat caaa
32414354DNAUnknownSynthetic 14atggccgagg tgcagctgtt ggagtctggg
ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc
tttagcagct atgccatgag ctgggtccgc 120ctggctccag ggaaggggct
ggagtgggtc tcatctattt ctactactgg taatagtaca 180tattacgcag
actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg
240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta
ctgtgcgaaa 300gatgctacta gttttgacta ctggggccag ggaaccctgg
tcaccgtctc gagc 35415108PRTUNKNOWNSYNTHETIC 15Thr Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr
Arg Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Lys Thr Arg Pro
85 90 95Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10516118PRTUNKNOWNSYNTHETIC 16Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Val Ile Gln
Arg Gln Gly Thr Gly Thr Glu Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Lys Asn Ser Arg Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11517324DNAUNKNOWNSYNTHETIC 17acggacatcc
agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt
gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa
120ccagggaaag cccctaagct cctgatctat cgtgcatccc gtttgcaaag
tggggtccca 180tcaaggttca gtggcagtgg atctgggaca gatttcactc
tcaccatcag cagtctgcaa 240cctgaagatt ttgcaactta ctactgtcaa
cagattaaga caaggcctac gacgttcggc 300caagggacca aggtggaaat caaa
32418354DNAUNKNOWNSYNTHETIC 18atggccgagg tgcagctgtt ggagtctggg
ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc
tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct
ggagtgggtc tcagttattc agcgttaggg tactggtaca 180gagtacgcag
actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg
240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta
ctgtgcaaaa 300aattcgcgga cgtttgacta ctggggccag ggaaccctgg
tcaccgtctc gagc 35419108PRTUNKNOWNSYNTHETIC 19Thr Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr
Asp Ala Ser Leu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Ser Ala Gly Pro
85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10520118PRTUNKNOWNSYNTHETIC 20Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Ser Ile Pro
Ser Arg Gly Gln Ala Thr Lys Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Lys Ser Arg His Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11521324DNAUNKNOWNSYNTHETIC 21acggacatcc
agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt
gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa
120ccagggaaag cccctaagct cctgatctat gatgcatccc ttttgcaaag
tggggtccca 180tcaaggttca gtggcagtgg atctgggaca gatttcactc
tcaccatcag cagtctgcaa 240cctgaagatt ttgcaactta ctactgtcaa
cagacttctg cgggtcctgg tacgttcggc 300caagggacca aggtggaaat caaa
32422354DNAUNKNOWNSYNTHETIC 22atggccgagg tgcagctgtt ggagtctggg
ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc
tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct
ggagtgggtc tcaagtattc ctagtcgtgg ttaggcaaca 180aagtacgcag
actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg
240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta
ctgcgcgaaa 300tcgcgtcata cttttgacta ctggggccag ggaaccctgg
tcaccgtctc gagc 35423108PRTUNKNOWNSYNTHETIC 23Thr Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asp Tyr Tyr Pro
85 90 95Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10524118PRTUNKNOWNSYNTHETIC 24Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Ser Ile Ala
Thr Thr Gly Asn Thr Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Lys Asn Thr Ala Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11525324DNAUNKNOWNSYNTHETIC 25acggacatcc
agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt
gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa
120ccagggaaag cccctaagct cctgatctat gctgcatcca ctttgcaaag
tggggtccca 180tcaaggttca gtggcagtgg atctgggaca gatttcactc
tcaccatcag cagtctgcaa 240cctgaagatt ttgcaactta ctactgtcaa
cagaatgatt attatcctac tacgttcggc 300caagggacca aggtggaaat caaa
32426354DNAUNKNOWNSYNTHETIC 26atggccgagg tgcagctgtt ggagtctggg
ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc
tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct
ggagtgggtc tcatctattg ctactactgg taatactaca 180tattacgcag
actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg
240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta
ctgtgcgaaa 300aatactgcta cttttgacta ctggggccag ggaaccctgg
tcaccgtctc gagc 35427118PRTUNKNOWNSYNTHETIC 27Met Ala Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Leu1 5 10 15Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Gln Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val
Ser Ser Ile Pro Pro Gly Gly Pro Asn Thr Lys Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Ala Lys Ser Tyr Pro Ser Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11528354DNAUNKNOWNSYNTHETIC
28atggccgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcttgg ggggtccctg
60agactctcct gtgcagcctc tggattcacc tttagcagct aggccatgag ctgggtccgc
120caggctccag ggaaggggct ggagtgggtc
tcatctattc ctccgggtgg tcctaataca 180aagtacgcag actccgtgaa
gggccggttc accatctcca gagacaattc caagaacacg 240ctgtatctgc
aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa
300agttatcctt cttttgacta ctggggccag ggaaccctgg tcaccgtctc gagc
35429118PRTUNKNOWNSYNTHETIC 29Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Ser Ile Tyr
Thr Ser Gly Ala Ala Thr Thr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Lys Ser Tyr Pro Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11530354DNAUnknownSynthetic 30atggccgagg
tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60agactctcct
gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc
120caggctccag ggaaggggct ggagtgggtc tcatctattt atacttctgg
tgctgctaca 180acttacgcag actccgtgaa gggccggttc accatctcca
gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc
gaggacacgg ccgtatatta ctgtgcgaaa 300agttatcctt cttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagc 35431107PRTHomo sapiens 31Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10532330PRTHomo sapiens 32Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230
235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 3303311PRTUnknownSynthetic 33Arg Ala
Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 10347PRTUnknownSynthetic
34Asn Ala Ser Asp Leu Gln Ser1 5357PRTUnknownSynthetic 35Ala Ala
Ser Pro Leu Gln Ser1 5367PRTUnknownSynthetic 36Arg Ala Ser Arg Leu
Gln Ser1 5377PRTUnknownSynthetic 37Ala Ala Ser Ala Leu Gln Ser1
5387PRTUnknownSynthetic 38Asp Ala Ser Leu Leu Gln Ser1
5397PRTUnknownSynthetic 39Ala Ala Ser Thr Leu Gln Ser1
5409PRTUnknownSynthetic 40Gln Gln Ser Ser Arg Thr Pro Thr Thr1
5419PRTUnknownSynthetic 41Gln Gln Val Ala Arg Thr Pro Asn Thr1
5429PRTUnknownSynthetic 42Gln Gln Pro Thr Ser Leu Pro Leu Thr1
5439PRTunknownsynthetic 43Gln Gln Ala Asp Ser Tyr Pro Thr Thr1
5449PRTUnknownsynthetic 44Gln Gln Ile Lys Thr Arg Pro Thr Thr1
5459PRTunknownsynthetic 45Gln Gln Thr Ser Ala Gly Pro Gly Thr1
5469PRTunknownsynthetic 46Gln Gln Asn Asp Tyr Tyr Pro Thr Thr1
5475PRTUnknownsynthetic 47Ser Gln Ala Met Ser1
5485PRTunknownsynthetic 48Ser Tyr Ala Met Ser1
54917PRTunknownsynthetic 49Gln Ile Gly Thr Met Gly Arg Pro Thr Thr
Tyr Ala Asp Ser Val Lys1 5 10 15Gly5017PRTunknownsynthetic 50Ser
Ile Ser Thr Thr Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly5117PRTunknownsynthetic 51Val Ile Gln Arg Gln Gly Thr Gly Thr
Glu Tyr Ala Asp Ser Val Lys1 5 10 15Gly5217PRTUNKNOWNSYNTHETIC
52Ser Ile Pro Ser Arg Gly Gln Ala Thr Lys Tyr Ala Asp Ser Val Lys1
5 10 15Gly5317PRTUNKNOWNSYNTHETIC 53Ser Ile Ala Thr Thr Gly Asn Thr
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly5417PRTUNKNOWNSYNTHETIC
54Ser Ile Pro Pro Gly Gly Pro Asn Thr Lys Tyr Ala Asp Ser Val Lys1
5 10 15Gly5517PRTUNKNOWNSYNTHETIC 55Ser Ile Tyr Thr Ser Gly Ala Ala
Thr Thr Tyr Ala Asp Ser Val Lys1 5 10 15Gly567PRTUNKNOWNSYNTHETIC
56Ser Tyr Pro Ser Phe Asp Tyr1 5577PRTUNKNOWNSYNTHETIC 57Ser Gly
Lys Lys Phe Asp Tyr1 5587PRTUNKNOWNSYNTHETIC 58Asp Ala Thr Ser Phe
Asp Tyr1 5597PRTUNKNOWNSYNTHETIC 59Asn Ser Arg Thr Phe Asp Tyr1
5607PRTUNKNOWNSYNTHETIC 60Ser Arg His Thr Phe Asp Tyr1
5617PRTUNKNOWNSYNTHETIC 61Asn Thr Ala Thr Phe Asp Tyr1
5627PRTUNKNOWNSYNTHETICMISC_FEATURE(1)..(1)X=N, A, R,
DMISC_FEATURE(4)..(4)X=D, P, R, A, L, T 62Xaa Ala Ser Xaa Leu Gln
Ser1 5639PRTUNKNOWNSYNTHETICMISC_FEATURE(3)..(3)X=S, V, P, A, I, T,
NMISC_FEATURE(4)..(4)X=S, A, T, D, KMISC_FEATURE(5)..(5)X=R, S, T,
A, YMISC_FEATURE(6)..(6)X=T, L, Y, R, GMISC_FEATURE(8)..(8)X=T, N,
L, G 63Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1
5645PRTUNKNOWNSYNTHETICMISC_FEATURE(2)..(2)X=Q, Y 64Ser Xaa Ala Met
Ser1 56517PRTUNKNOWNSYNTHETICMISC_FEATURE(1)..(1)X=S, Q,
VMISC_FEATURE(3)..(3)X=P, G, S, Q, A, YMISC_FEATURE(4)..(4)X=P, T,
R, SMISC_FEATURE(5)..(5)X=G, M, T, Q, R, SMISC_FEATURE(7)..(7)X=P,
R, N, T, Q, AMISC_FEATURE(8)..(8)X=N, P, S, G, A,
TMISC_FEATURE(10)..(10)X=K, T, Y, E 65Xaa Ile Xaa Xaa Xaa Gly Xaa
Xaa Thr Xaa Tyr Ala Asp Ser Val Lys1 5 10
15Gly667PRTUNKNOWNSYNTHETICMISC_FEATURE(1)..(1)X=S, D,
NMISC_FEATURE(2)..(2)X=Y, G, A, S, R, TMISC_FEATURE(3)..(3)X=P, K,
T, R, H, AMISC_FEATURE(4)..(4)X=S, K, T 66Xaa Xaa Xaa Xaa Phe Asp
Tyr1 567232PRTHomo sapiens 67Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105
110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser
Leu Ser Leu Ser Pro Gly Lys225 23068326PRTHomo sapiens 68Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170
175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295
300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu305 310 315 320Ser Leu Ser Pro Gly Lys 32569228PRTHomo sapiens
69Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val1
5 10 15Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 35 40 45His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr65 70 75 80Phe Arg Val Val Ser Val Leu Thr Val Val
His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155
160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 210 215 220Ser Pro Gly Lys22570228PRTHomo
sapiens 70Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
Pro Val1 5 10 15Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 35 40 45His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr65 70 75 80Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150
155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 165 170 175Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser Pro Gly
Lys2257120DNAunknownsynthetic 71agtcccccag gaagaggcaa
207225DNAunknownsynthetic 72tcacctaggt tctcactagc cccgt
257330DNAunknownsynthetic 73cctggacttg agttacggag atgcagtgca
3074440PRTHomo sapiens 74Met Leu Leu Ala Thr Leu Leu Leu Leu Leu
Leu Gly Gly Ala Leu Ala1 5 10 15His Pro Asp Arg Ile Ile Phe Pro Asn
His Ala Cys Glu Asp Pro Pro 20 25 30Ala Val Leu Leu Glu Val Gln Gly
Thr Leu Gln Arg Pro Leu Val Arg 35 40 45Asp Ser Arg Thr Ser Pro Ala
Asn Cys Thr Trp Leu Ile Leu Gly Ser 50 55 60Lys Glu Gln Thr Val Thr
Ile Arg Phe Gln Lys Leu His Leu Ala Cys65 70 75 80Gly Ser Glu Arg
Leu Thr Leu Arg Ser Pro Leu Gln Pro Leu Ile Ser 85 90 95Leu Cys Glu
Ala Pro Pro Ser Pro Leu Gln Leu Pro Gly Gly Asn Val 100 105 110Thr
Ile Thr Tyr Ser Tyr Ala Gly Ala Arg Ala Pro Met Gly Gln Gly 115 120
125Phe Leu Leu Ser Tyr Ser Gln Asp Trp Leu Met Cys Leu Gln Glu Glu
130 135 140Phe Gln Cys Leu Asn His Arg Cys Val Ser Ala Val Gln Arg
Cys Asp145 150 155 160Gly Val Asp Ala Cys Gly Asp Gly Ser Asp Glu
Ala Gly Cys Ser Ser 165 170 175Asp Pro Phe Pro Gly Leu Thr Pro Arg
Pro Val Pro Ser Leu Pro Cys 180 185 190Asn Val Thr Leu Glu Asp Phe
Tyr Gly Val Phe Ser Ser Pro Gly Tyr 195 200 205Thr His Leu Ala Ser
Val Ser His Pro Gln Ser Cys His Trp Leu Leu 210 215 220Asp Pro His
Asp Gly Arg Arg Leu Ala Val Arg Phe
Thr Ala Leu Asp225 230 235 240Leu Gly Phe Gly Asp Ala Val His Val
Tyr Asp Gly Pro Gly Pro Pro 245 250 255Glu Ser Ser Arg Leu Leu Arg
Ser Leu Thr His Phe Ser Asn Gly Lys 260 265 270Ala Val Thr Val Glu
Thr Leu Ser Gly Gln Ala Val Val Ser Tyr His 275 280 285Thr Val Ala
Trp Ser Asn Gly Arg Gly Phe Asn Ala Thr Tyr His Val 290 295 300Arg
Gly Tyr Cys Leu Pro Trp Asp Arg Pro Cys Gly Leu Gly Ser Gly305 310
315 320Leu Gly Ala Gly Glu Gly Leu Gly Glu Arg Cys Tyr Ser Glu Ala
Gln 325 330 335Arg Cys Asp Gly Ser Trp Asp Cys Ala Asp Gly Thr Asp
Glu Glu Asp 340 345 350Cys Pro Gly Cys Pro Pro Gly His Phe Pro Cys
Gly Ala Ala Gly Thr 355 360 365Ser Gly Ala Thr Ala Cys Tyr Leu Pro
Ala Asp Arg Cys Asn Tyr Gln 370 375 380Thr Phe Cys Ala Asp Gly Ala
Asp Glu Arg Arg Cys Arg His Cys Gln385 390 395 400Pro Gly Asn Phe
Arg Cys Arg Asp Glu Lys Cys Val Tyr Glu Thr Trp 405 410 415Val Cys
Asp Gly Gln Pro Asp Cys Ala Asp Gly Ser Asp Glu Trp Asp 420 425
430Cys Ser Tyr Val Leu Pro Arg Lys 435 440
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