U.S. patent application number 17/327363 was filed with the patent office on 2021-09-09 for targeted therapy for small cell lung cancer.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Julien Sage, Kipp Andrew Weiskopf, Irving L. Weissman.
Application Number | 20210277115 17/327363 |
Document ID | / |
Family ID | 1000005600646 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210277115 |
Kind Code |
A1 |
Weiskopf; Kipp Andrew ; et
al. |
September 9, 2021 |
TARGETED THERAPY FOR SMALL CELL LUNG CANCER
Abstract
Methods are provided for treatment of lung cancers, particularly
small cell lung cancer with targeted therapy, which optionally
includes an agent that selectively blocks CD47 binding to
SIRP.alpha..
Inventors: |
Weiskopf; Kipp Andrew;
(Menlo Park, CA) ; Sage; Julien; (Stanford,
CA) ; Weissman; Irving L.; (Stanford, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
1000005600646 |
Appl. No.: |
17/327363 |
Filed: |
May 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15107852 |
Jun 23, 2016 |
11046763 |
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PCT/US15/10650 |
Jan 8, 2015 |
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17327363 |
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61925143 |
Jan 8, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6803 20170801;
A61K 39/395 20130101; A61K 47/6857 20170801; A61K 47/6869 20170801;
A61K 38/1709 20130101; C07K 2317/33 20130101; C07K 16/2896
20130101; C07K 16/2842 20130101; C07K 16/2803 20130101; A61K
2039/505 20130101; C07K 2317/76 20130101; C07K 16/3023 20130101;
A61K 2039/507 20130101; A61K 38/177 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 38/17 20060101 A61K038/17; A61K 39/395 20060101
A61K039/395; A61K 47/68 20060101 A61K047/68; C07K 16/30 20060101
C07K016/30 |
Claims
1-7. (canceled)
8. A method of treating an individual with lung cancer, the method
comprising: administering to a human subject in need thereof a
combination of (i) an agent that selectively blocks CD47 binding to
SIRP.alpha. and (ii) a targeted therapeutic agent that specifically
binds to one or more cell-surface antigens on lung cancer cells, in
a dose effective to increase depletion of the lung cancer
cells.
9. The method of claim 8, wherein the agent that selectively blocks
CD47 binding to SIRP.alpha. is an antibody.
10. The method of claim 9, wherein the antibody specifically binds
to CD47.
11. The method of claim 9, wherein the antibody specifically binds
to SIRP.alpha..
12. The method of claim 8, wherein the agent that selectively
blocks CD47 binding to SIRP.alpha. is a soluble SIRP.alpha.
polypeptide.
13. The method of claim 8, wherein the agent that selectively
blocks CD47 binding to SIRP.alpha. is a soluble CD47
polypeptide.
14. The method of claim 8, wherein the one or more cell-surface
antigens on lung cancer cells are selected from CD24, CD166, CD326,
CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, and
EpCAM.
15. The method of claim 8, wherein the combination of agents is
administered simultaneously.
16. The method of claim 8, wherein the combination of agents is
administered sequentially.
17. The method of claim 8, wherein the combination of agents is
administered in overlapping dosing regimens
18. The method of claim 8, wherein the individual is a human.
19. The method of claim 18, wherein the lung cancer is small cell
lung cancer.
20. The method of claim 19, wherein said marker is selected from
CD99, CD44 and EpCam.
21. The method of claim 8, wherein the combination of agents
provides for a synergistic effect.
22. The method of claim 10, wherein the anti-CD47 antibody
comprises an IgG4 Fc region.
23. The method of claim 22 wherein the antibody is 5F9-G4.
24. A method for the treatment of a lung cancer in a patient, the
method comprising: administering to said patient an effective dose
of a targeted therapeutic agent that specifically binds to a cell
surface antigen selected from CD99, CD44, EpCam, CD24, CD166, CD56,
CD326, CD298, CD29, CD63, CD9, CD164, CD46, CD59, CD57, and CD165.
Description
CROSS REFERENCE
[0001] This application is a Divisional and claims the benefit of
U.S. application Ser. No. 15/107,852, filed Jun. 23, 2016, which
claims the benefit of 371 Application No. PCT/US2015/010650, filed
Jan. 8, 2015, which claims the benefit of U.S. Provisional
Application No. 61/925,143, filed Jan. 8, 2014, which are
incorporated herein in their entirety for all purposes.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT
FILE
[0002] A Sequence Listing is provided herewith a text file,
(S13-499_STAN-1089DIV_Sequence_listinglist.txt), created on (Nov.
18, 2020) and having a size of (2 KB) The contents of the text file
are incorporated by reference herein in their entirety.
BACKGROUND
[0003] Targeted therapies, such as antibodies and specific ligands
have proven effective at fighting cancer, especially in cases where
conventional therapy fails. Even more encouraging is that
antibodies for cancer generally operate in a distinct mechanism
from traditional chemotherapy or radiotherapy, so they can often be
combined with traditional therapies to generate an additive or
synergistic effect.
[0004] Antibodies can achieve their therapeutic effect through
various mechanisms. They can have direct effects in producing
apoptosis or programmed cell death. They can block growth factor
receptors, effectively arresting proliferation of tumor cells. In
cells that express monoclonal antibodies, they can bring about
anti-idiotype antibody formation. Indirect effects include
recruiting cells that have cytotoxicity, such as monocytes and
macrophages. This type of antibody-mediated cell kill is called
antibody-dependent cell mediated cytotoxicity (ADCC). Monoclonal
antibodies also bind complement, leading to direct cell toxicity,
known as complement dependent cytotoxicity (CDC).
[0005] CD47 is a valuable target for anticancer therapy due to its
function as an inhibitor of macrophage phagocytosis as well as its
broad expression on a variety of human neoplasms. By binding to
signal-regulatory protein .alpha. (SIRP.alpha.), a receptor
expressed on the surface of macrophages, CD47 is able to transduce
inhibitory signals that prevent phagocytosis. Blocking the
interaction between CD47 and SIRP.alpha. with antibodies not only
stimulates macrophages to engulf cancer cells in vitro but also
exerts robust anticancer effects in vivo. Other CD47 blocking
agents include "next-generation" CD47 antagonists that bind and
block human CD47 with extraordinarily high affinity.
[0006] By disabling the inhibitory signals transduced by
SIRP.alpha., high-affinity SIRP.alpha. variants can reduce the
threshold for macrophage activation and promote phagocytic response
driven by tumor-specific antibodies. The degree to which the
anticancer activity of a given therapeutic antibody is enhanced by
CD47 blockade likely depends on multiple factors, including the
levels of antigen expression on the surface of malignant cells, the
isotype of its heavy chain, and the orientation assumed by the
antibody upon antigen binding, which affects its ability to engage
Fc receptors on immune effectors. High-affinity SIRP.alpha.
monomers represent therefore a rapid, safe and effective
alternative to several other approaches, including drug/toxin
conjugation strategies, that have been pursued in this
direction.
[0007] Identification of effective targets and combinations of
targeted therapies remain of high interest. The present invention
addresses this need.
SUMMARY OF THE INVENTION
[0008] Methods and compositions are provided for the treatment of
lung cancer with a targeted therapy. In some embodiments, the lung
cancer is small cell lung cancer. In some embodiments, the therapy
is targeted at one or more cell-surface antigens, including CD24,
CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46,
CD59, CD57, CD165, EpCAM, etc. In some embodiments the targeted
therapy comprises administering to an individual suffering from
lung cancer a therapeutic dose of an antibody that specifically
binds to a cell surface marker selected from CD24, CD166, CD56,
CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165
and EpCAM.
[0009] In some embodiments the targeted therapy is combined with a
CD47 blocking agent. Cancer cells evade macrophage surveillance by
upregulation of CD47 expression. Administration of agents that mask
the CD47 protein, e.g. antibodies or small molecules that bind to
CD47 or SIRP.alpha. and prevent interaction between CD47 and
SIRP.alpha., are administered to a patient, which increases the
clearance of cancer cells via phagocytosis. The agent that blocks
CD47 is combined with monoclonal antibodies directed against one or
more lung cancer cell markers, which compositions can be
synergistic in enhancing phagocytosis and elimination of cancer
cells as compared to the use of single agents.
[0010] Specific reagent combinations of interest for therapy
include anti-CD47 and anti-CD56; anti-CD47 and anti-CD44, anti-CD47
and anti-CD99, anti-CD47 and anti-EpCam. In some such embodiments
the anti-CD47 reagent is a high affinity SIRP.alpha. polypeptide,
which can be provided in the form of a monomer or a multimer, e.g.
as a fusion protein with an IgG Fc polypeptide.
[0011] In other embodiments, the therapy provides for a
multispecific antibody that targets CD47 and a second cancer cell
marker, including multispecific antibodies that target CD47 and
CD56; CD47 and CD44, CD47 and EpCam, etc. Compositions of such
multispecific antibodies are also provided, where the multispecific
antibody is desirably human or humanized; and may be modified to
extend the blood half-life, e.g. by pegylation, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1: CD47-blocking therapies stimulate macrophage
phagocytosis of small cell lung cancer. Human monocytes from
anonymous blood donors were purified by magnetic activated cell
sorting (MACS) using CD14+ selection. Monocytes were cultured in
the presence of 10% AB human serum for one week, at which point the
exhibited morphological changes characteristic of differentiation
to macrophages. Macrophages were co-cultured with primary human
small cell lung cancer cells (SCLC sample "H29") labeled with a
green fluorescent dye. Cells were treated with either a vehicle
control (phosphate buffered saling, PBS), anti-CD56 antibody (clone
MEM-188), or humanized anti-CD47 antibody (clone 5F9-G4).
Phagocytosis was evaluated by high-throughput flow cytometry as the
percentage of macrophages that had engulfed green fluorescent SCLC
cells. Treatment with anti-CD47 antibody was able to induce
elevated levels of phagocytosis as a single agent.
[0013] FIG. 2: CD47-blockade produces a therapeutic response
against small cell lung cancer cells in vivo using mouse
xenotransplantation models. Primary small cell lung cancer cells
(sample H29) were engrafted into immunodeficient NSG mice. After
approximately three weeks of growth, mice were randomized into two
treatment cohorts. The first cohort was treated with a vehicle
control (phosphate buffered saline, PBS; red), and the second
cohort was treated with daily injections of 250 .mu.g anti-CD47
antibody (clone 5F9-G4, blue). Tumor growth was monitored over
time. Each point represents a tumor growing in an individual mouse.
Black bars represent median tumor volume. Left Tumor volume
measurements over entire time course of study. Right Tumor volume
measurements on day 89 of study. Note logarithmic scale.
[0014] FIG. 3: Novel therapeutic targets highly expressed on the
surface of small cell lung cancer cells. Primary human small cell
lung cancer cells (sample H29) were subjected to comprehensive flow
cytometric immunophenotyping using a LEGENDScreen assay
(Biolegend). Surface antigens were ranked based on their geometric
mean fluorescence intensity (Geo. MFI). These antigens are
therapeutic targets for antibodies in combination with
CD47-blocking agents.
[0015] FIG. 4: The ability of SCLC-targeting antibodies to induce
macrophage phagocytosis can be enhanced by combination with
CD47-blocking therapies. Two human small cell lung cancer cell
lines (H82 and H69) were labeled with a green fluorescent dye, and
then were co-cultured with primary NSG mouse macrophages in the
presence of the indicated antibodies either in combination with
vehicle control (phosphate buffered saline, PBS; gray) or with
high-affinity SIRPalpha variant CV1 monomer (black). Phagocytosis
was evaluated by high-throughput flow cytometry as the percentage
of macrophages that had engulfed green fluorescent SCLC cells.
Anti-CD47 reagents 5F9-G4 and CV1-G4 were not tested in combination
with CV1 monomer due to direct competition.
[0016] FIG. 5. CD47-blockade induces macrophage phagocytosis of
SCLC cells in vitro. (A) Expression of CD47 on the surface of a
panel of human SCLC cell lines as evaluated by flow cytometry.
Black dotted line represents unstained NCI-H82 cells. (B)
Expression of CD47 on the surface of the primary human SCLC sample
H29. (C) Diagram depicting in vitro phagocytosis assays using human
macrophages and fluorescent tumor cells. (D) Representative flow
cytometry plots of phagocytosis assays performed with human
macrophages and calcein AM-labeled SCLC cells. (E) Representative
images of cell populations after fluorescence activated cell
sorting. The sorted double-positive population contained
macrophages with engulfed tumor cells. Scale bar represents 20
.mu.m. (F) Summary of phagocytosis assays using human macrophages
and calcein AM-labeled SCLC cells as analyzed by flow cytometry.
SCLC cells were treated with vehicle control (PBS) or anti-CD47
antibodies (clone Hu5F9-G4). The percentage of calcein AM+
macrophages was normalized to the maximal response by each
macrophage donor. (G) Phagocytosis of primary H29 SCLC cells by
human macrophages after treatment with vehicle control (PBS) or
anti-CD47 antibodies (clone Hu5F9-G4). (F-G) Phagocytosis assays
were performed with macrophages derived from four independent blood
donors. Data represent mean.+-.SD. ns, not significant;
**P<0.01; ****P<0.0001 for the indicated comparisons by
two-way analysis of variance with Sidak correction (F) or
two-tailed t test (G).
[0017] FIG. 6. CD47-blocking antibodies inhibit growth of human
SCLC tumors in vivo. (A) Growth of NCI-H82 cells in the
subcutaneously tissue of NSG mice. Mice were randomized into groups
treated with vehicle control (PBS) or anti-CD47 antibodies (clone
Hu5F9-G4). Growth was evaluated by tumor volume measurements. Seven
to eight mice were treated per cohort, and each point represents
tumor volume of independent animals. (B) Growth of GFP-luciferase+
patient-derived xenograft H29 tumors in the subcutaneous tissue of
NSG mice as evaluated by bioluminescence imaging. Mice were
randomized into groups treated with vehicle control (PBS) or
anti-CD47 antibodies (clone Hu5F9-G4). (C) Representative
bioluminescence images of H29 tumors on day 85 post-engraftment.
(D) Growth of H29 tumors as evaluated by tumor volume measurements.
(E) Survival of mice bearing patient-derived xenograft H29 tumors
that were treated with the indicated therapies. P=0.0004 by
Mantel-Cox test. A-E Black arrows indicate the start of treatment.
Points indicate measurements from independent animals, bars
indicate median values. Cohorts consisted of a minimum of 7-8 mice.
Measurements at each time point are staggered for clarity. ns, not
significant; *P<0.05; **P<0.01; ***P<0.001 for the
indicated comparisons by Mann-Whitney test.
[0018] FIG. 7. MCP-3 is a serum biomarker that predicts response to
CD47-blocking therapies. (A) Untreated NSG mice (No tumor) or NSG
mice bearing subcutaneous NCI-H82 cells were injected with a single
dose of anti-CD47 antibodies (clone Hu5F9-G4). Serum samples were
collected pre-treatment or 24 hours post-treatment. MCP-3 levels
were measured by Luminex multiplex array. (B) MCP-3 levels in mice
bearing patient-derived xenograft H29 tumors were evaluated as in
A. Points represent measurements from individual mice, bars
represent mean.+-.SD. Five mice were evaluated per condition.
ns=not significant; ****P<0.0001 by two-way analysis of variance
with Sidak correction.
[0019] FIG. 8. Comprehensive FACS-based antibody screening
identifies new and established therapeutic targets on SCLC. Antigen
expression on the surface of four SCLC cell lines and primary
patient sample H29 was assessed using LEGENDScreen Human Cell
Screening Kits (BioLegend), a collection of 332 antibodies
targeting cell surface antigens. Antibody binding was detected by
fluorescence-activated cell sorting (FACS) analysis. (A) Histogram
depicting geometric mean fluorescence intensity (MFI) of all
antibodies screened for SCLC surface binding. Data represent median
values for each antibody across all five SCLC samples. Data were
fit to Gaussian distribution (black curve), and negative antigens
(gray) were defined by median MFI less than two standard deviations
above the mean. Low antigens (red) defined as MFI less than one
order of magnitude above the negative threshold. High antigens
(blue) defined as one order of magnitude greater than negative
threshold. (B) Ranked list of the 39 antigens identified as `high`
based on median MFI across all five SCLC samples.
[0020] FIG. 9. High-affinity SIRP.alpha. variants enhance
macrophage phagocytosis of SCLC in response to tumor-binding
antibodies. Phagocytosis of NCI-H82 cells (A) and NCI-H524 cells
(B) in response to tumor-binding antibodies alone (red) or in
combination with the high-affinity SIRP.alpha. variant CV1 monomer
(blue). Points represent measurements from individual donors, bars
represent median values. Three clones of anti-CD56 (NCAM)
antibodies were tested, as well as antibodies to CD24, CD29, CD99,
and CD47 (clone Hu5F9-G4). (C) Phagocytosis of NCI-H82 SCLC cells
in response to varying concentrations of the anti-CD56 antibody
lorvotuzumab alone (red) or in combination with the high-affinity
SIRP.alpha. variant CV1 monomer (blue). Data represent mean.+-.SD.
(A-C) Phagocytosis assays were performed with human macrophages
derived from a minimum of four independent blood donors.
Measurements were normalized to the maximal response by each
macrophage donor. ns, not significant; *P<0.05; **P<0.01;
***P<0.001; ****P<0.0001 for the indicated comparisons by
two-way analysis of variance with Sidak correction.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0021] Methods and compositions are provided for the treatment of
lung cancer with a therapeutic agent, e.g. an antibody, targeted to
a marker of lung cancer, e.g. targeted to one or more cell-surface
antigens, including CD24, CD166, CD56, CD326, CD298, CD29, CD63,
CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc. In some
embodiments, a combination, e.g. a synergistic combination, of
agents is provided, wherein one agent is an anti-CD47 blocking
agent, and the second agent is targeted to a lung cancer marker,
e.g. CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99,
CD46, CD59, CD57, CD165, EpCAM, etc.
[0022] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0023] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0024] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events.
[0025] 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 invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0026] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0027] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0028] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Definitions
[0029] Synergistic combination. Synergistic combinations may
provide for a therapeutic effect that is comparable to the
effectiveness of a monotherapy, i.e. the individual components of
the combination, while reducing adverse side effects, e.g. damage
to non-targeted tissues, immune status, and other clinical indicia.
Alternatively synergistic combinations may provide for an improved
effectiveness when compared to the effectiveness of a monotherapy,
i.e. the individual components of the combination, which effect may
be measured by total tumor cell number; length of time to relapse;
and other indicia of patient health.
[0030] Synergistic combinations of the present invention combine an
agent that is targeted to inhibit or block CD47 function; and an
agent that is targeted to inhibit or block a second lung cancer
cell marker, usually a cell surface marker. The combination may be
provided with a combination of agents, e.g. two distinct proteins,
each of which is specific for a different marker; or may be
provided as a multispecific agent, e.g. antibody, that combines
specificity for two or more different markers.
[0031] Combination Therapy: As used herein, the term "combination
therapy" refers to those situations in which a subject is
simultaneously exposed to two or more therapeutic regimens (e.g.,
two or more therapeutic agents). In some embodiments, two or more
agents may be administered simultaneously; in some embodiments,
such agents may be administered sequentially; in some embodiments,
such agents are administered in overlapping dosing regimens.
[0032] Dosage Form: As used herein, the term "dosage form" refers
to a physically discrete unit of an active agent (e.g., a
therapeutic or diagnostic agent) for administration to a subject.
Each unit contains a predetermined quantity of active agent. In
some embodiments, such quantity is a unit dosage amount (or a whole
fraction thereof) appropriate for administration in accordance with
a dosing regimen that has been determined to correlate with a
desired or beneficial outcome when administered to a relevant
population (i.e., with a therapeutic dosing regimen). Those of
ordinary skill in the art appreciate that the total amount of a
therapeutic composition or agent administered to a particular
subject is determined by one or more attending physicians and may
involve administration of multiple dosage forms.
[0033] Dosing Regimen: As used herein, the term "dosing regimen"
refers to a set of unit doses (typically more than one) that are
administered individually to a subject, typically separated by
periods of time. In some embodiments, a given therapeutic agent has
a recommended dosing regimen, which may involve one or more doses.
In some embodiments, a dosing regimen comprises a plurality of
doses each of which are separated from one another by a time period
of the same length; in some embodiments, a dosing regimen comprises
a plurality of doses and at least two different time periods
separating individual doses. In some embodiments, all doses within
a dosing regimen are of the same unit dose amount. In some
embodiments, different doses within a dosing regimen are of
different amounts. In some embodiments, a dosing regimen comprises
a first dose in a first dose amount, followed by one or more
additional doses in a second dose amount different from the first
dose amount. In some embodiments, a dosing regimen comprises a
first dose in a first dose amount, followed by one or more
additional doses in a second dose amount same as the first dose
amount. In some embodiments, a dosing regimen is correlated with a
desired or beneficial outcome when administered across a relevant
population (i.e., is a therapeutic dosing regimen).
[0034] CD47 polypeptides. The three transcript variants of human CD
47 (variant 1, NM 001777; variant 2, NM 198793; and variant 3, NM
001025079) encode three isoforms of CD47 polypeptide. CD47 isoform
1 (NP 001768), the longest of the three isoforms, is 323 amino
acids long. CD47 isoform 2 (NP 942088) is 305 amino acid long. CD47
isoform 3 is 312 amino acids long. The three isoforms are identical
in sequence in the first 303 amino acids. Amino acids 1-8 comprise
the signal sequence, amino acids 9-142 comprise the CD47
immunoglobulin like domain, which is the soluble fragment, and
amino acids 143-300 is the transmembrane domain.
[0035] A "functional derivative" of a native sequence polypeptide
is a compound having a qualitative biological property in common
with a native sequence polypeptide. "Functional derivatives"
include, but are not limited to, fragments of a native sequence and
derivatives of a native sequence polypeptide and its fragments,
provided that they have a biological activity in common with a
corresponding native sequence polypeptide. The term "derivative"
encompasses both amino acid sequence variants of polypeptide and
covalent modifications thereof. Derivatives and fusion of soluble
CD47 find use as CD47 mimetic molecules.
[0036] The first 142 amino acids of CD47 polypeptide comprise the
extracellular region of CD47 (SEQ ID NO: 1). The three isoforms
have identical amino acid sequence in the extracellular region, and
thus any of the isoforms are can be used to generate soluble CD47.
"Soluble CD47" is a CD47 protein that lacks the transmembrane
domain. Soluble CD47 is secreted out of the cell expressing it
instead of being localized at the cell surface.
[0037] In vitro assays for CD47 biological activity include, e.g.
inhibition of phagocytosis of porcine cells by human macrophages,
binding to SIRP .alpha. receptor, SIRP .alpha. tyrosine
phosphorylation, etc. An exemplary assay for CD47 biological
activity contacts a human macrophage composition in the presence of
a candidate agent. The cells are incubated with the candidate agent
for about 30 minutes and lysed. The cell lysate is mixed with
anti-human SIRP .alpha. antibodies to immunoprecipitate SIRP
.alpha.. Precipitated proteins are resolved by SDS PAGE, then
transferred to nitrocellulose and probed with antibodies specific
for phosphotyrosine. A candidate agent useful as a CD47 mimetic
increases SIRP .alpha. tyrosine phosphorylation by at least 10%, or
up to 20%, or 50%, or 70% or 80% or up to about 90% compared to the
level of phosphorylation observed in the absence of candidate
agent. Another exemplary assay for CD47 biological activity
measures phagocytosis of hematopoietic cells by human macrophages.
A candidate agent useful as a CD47 mimetic results in the down
regulation of phagocytosis by at least about 10%, at least about
20%, at least about 50%, at least about 70%, at least about 80%, or
up to about 90% compared to level of phagocytosis observed in
absence of candidate agent.
[0038] By "manipulating phagocytosis" is meant an up-regulation or
a down-regulation in phagocytosis by at least about 10%, or up to
20%, or 50%, or 70% or 80% or up to about 90% compared to level of
phagocytosis observed in absence of intervention. Thus in the
context of decreasing phagocytosis of circulating hematopoietic
cells, particularly in a transplantation context, manipulating
phagocytosis means a down-regulation in phagocytosis by at least
about 10%, or up to 20%, or 50%, or 70% or 80% or up to about 90%
compared to level of phagocytosis observed in absence of
intervention.
[0039] Anti-CD47 agent. As used herein, the term "anti-CD47 agent"
refers to any agent that reduces the binding of CD47 (e.g., on a
target cell) to SIRP.alpha. (e.g., on a phagocytic cell).
Non-limiting examples of suitable anti-CD47 reagents include
SIRP.alpha. reagents, including without limitation high affinity
SIRP.alpha. polypeptides, anti-SIRP.alpha. antibodies, soluble CD47
polypeptides, and anti-CD47 antibodies or antibody fragments. In
some embodiments, a suitable anti-CD47 agent (e.g. an anti-CD47
antibody, a SIRP.alpha. reagent, etc.) specifically binds CD47 to
reduce the binding of CD47 to SIRP.alpha.. In some embodiments, a
suitable anti-CD47 agent (e.g., an anti-SIRP.alpha. antibody, a
soluble CD47 polypeptide, etc.) specifically binds SIRP.alpha. to
reduce the binding of CD47 to SIRP.alpha.. A suitable anti-CD47
agent that binds SIRP.alpha. does not activate SIRP.alpha. (e.g.,
in the SIRP.alpha.-expressing phagocytic cell).
[0040] The efficacy of a suitable anti-CD47 agent can be assessed
by assaying the agent (further described below). In an exemplary
assay, target cells are incubated in the presence or absence of the
candidate agent. An agent for use in the methods of the invention
will up-regulate phagocytosis by at least 10% (e.g., at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 100%, at least 120%, at
least 140%, at least 160%, at least 180%, or at least 200%)
compared to phagocytosis in the absence of the agent. Similarly, an
in vitro assay for levels of tyrosine phosphorylation of
SIRP.alpha. will show a decrease in phosphorylation by at least 5%
(e.g., at least 10%, at least 15%, at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, or 100%) compared to phosphorylation observed in
absence of the candidate agent.
[0041] In some embodiments, the anti-CD47 agent does not activate
CD47 upon binding. When CD47 is activated, a process akin to
apoptosis (i.e., programmed cell death) may occur (Manna and
Frazier, Cancer Research, 64, 1026-1036, Feb. 1 2004). Thus, in
some embodiments, the anti-CD47 agent does not directly induce cell
death of a CD47-expressing cell by apoptosis.
[0042] SIRP.alpha. reagent. A SIRP.alpha. reagent comprises the
portion of SIRP.alpha. that is sufficient to bind CD47 at a
recognizable affinity, which normally lies between the signal
sequence and the transmembrane domain, or a fragment thereof that
retains the binding activity. A suitable SIRP.alpha. reagent
reduces (e.g., blocks, prevents, etc.) the interaction between the
native proteins SIRP.alpha. and CD47. The SIRP.alpha. reagent will
usually comprise at least the dl domain of SIRP.alpha.. In some
embodiments, a SIRP.alpha. reagent is a fusion protein, e.g., fused
in frame with a second polypeptide. In some embodiments, the second
polypeptide is capable of increasing the size of the fusion
protein, e.g., so that the fusion protein will not be cleared from
the circulation rapidly. In some embodiments, the second
polypeptide is part or whole of an immunoglobulin Fc region. The Fc
region aids in phagocytosis by providing an "eat me" signal, which
enhances the block of the "don't eat me" signal provided by the
high affinity SIRP.alpha. reagent. In other embodiments, the second
polypeptide is any suitable polypeptide that is substantially
similar to Fc, e.g., providing increased size, multimerization
domains, and/or additional binding or interaction with Ig
molecules.
[0043] In some embodiments, a subject anti-CD47 agent is a "high
affinity SIRP.alpha. reagent", which includes SIRP.alpha.-derived
polypeptides and analogs thereof. High affinity SIRP.alpha.
reagents are described in international application PCT/US13/21937,
which is hereby specifically incorporated by reference. High
affinity SIRP.alpha. reagents are variants of the native
SIRP.alpha. protein. In some embodiments, a high affinity
SIRP.alpha. reagent is soluble, where the polypeptide lacks the
SIRP.alpha. transmembrane domain and comprises at least one amino
acid change relative to the wild-type SIRP.alpha. sequence, and
wherein the amino acid change increases the affinity of the
SIRP.alpha. polypeptide binding to CD47, for example by decreasing
the off-rate by at least 10-fold, at least 20-fold, at least
50-fold, at least 100-fold, at least 500-fold, or more.
[0044] A high affinity SIRP.alpha. reagent comprises the portion of
SIRP.alpha. that is sufficient to bind CD47 at a recognizable
affinity, e.g., high affinity, which normally lies between the
signal sequence and the transmembrane domain, or a fragment thereof
that retains the binding activity. The high affinity SIRP.alpha.
reagent will usually comprise at least the dl domain of SIRP.alpha.
with modified amino acid residues to increase affinity.
[0045] A SIRP.alpha. reagent can be used as a "monomer", in which
the binding domain of SIRP.alpha. is used, but where the binding
domain is provided as a soluble monomeric protein. In other
embodiments, a SIRP.alpha. variant of the present invention is a
fusion protein, e.g., fused in frame with a second polypeptide,
particularly where the second polypeptide provides for
multimerization. In some embodiments, the second polypeptide is
part or whole of an immunoglobulin Fc region. The Fc region aids in
phagocytosis by providing an "eat me" signal, which enhances the
block of the "don't eat me" signal provided by the high affinity
SIRP.alpha. reagent. In other embodiments, the second polypeptide
is any suitable polypeptide that is substantially similar to Fc,
e.g., providing increased size, multimerization domains, and/or
additional binding or interaction with Ig molecules.
[0046] The amino acid changes that provide for increased affinity
are localized in the dl domain, and thus high affinity SIRP.alpha.
reagents comprise a dl domain of human SIRP.alpha., with at least
one amino acid change relative to the wild-type sequence within the
dl domain. Such a high affinity SIRP.alpha. reagent optionally
comprises additional amino acid sequences, for example antibody Fc
sequences; portions of the wild-type human SIRP.alpha. protein
other than the dl domain, including without limitation residues 150
to 374 of the native protein or fragments thereof, usually
fragments contiguous with the dl domain; and the like. High
affinity SIRP.alpha. reagents may be monomeric or multimeric, i.e.
dimer, trimer, tetramer, etc.
[0047] Anti-CD47 antibodies. In some embodiments, a subject
anti-CD47 agent is an antibody that specifically binds CD47 (i.e.,
an anti-CD47 antibody) and reduces the interaction between CD47 on
one cell (e.g., an infected cell) and SIRP.alpha. on another cell
(e.g., a phagocytic cell). In some embodiments, a suitable
anti-CD47 antibody does not activate CD47 upon binding, for example
an antibody that does not induce apoptosis upon binding.
Non-limiting examples of suitable antibodies include clones B6H12,
5F9, 8B6, and C3 (for example as described in International Patent
Publication WO 2011/143624, herein specifically incorporated by
reference). Suitable anti-CD47 antibodies include fully human,
humanized or chimeric versions of such antibodies. Humanized
antibodies (e.g., hu5F9-G4) are especially useful for in vivo
applications in humans due to their low antigenicity. Similarly
caninized, felinized, etc. antibodies are especially useful for
applications in dogs, cats, and other species respectively.
Antibodies of interest include humanized antibodies, or caninized,
felinized, equinized, bovinized, porcinized, etc., antibodies, and
variants thereof.
[0048] Anti-CD47 antibodies. In some embodiments, a subject
anti-CD47 agent is an antibody that specifically binds CD47 (i.e.,
an anti-CD47 antibody) and reduces the interaction between CD47 on
one cell (e.g., an infected cell) and SIRP.alpha. on another cell
(e.g., a phagocytic cell). In some embodiments, a suitable
anti-CD47 antibody does not activate CD47 upon binding, for example
an antibody that does not induce apoptosis upon binding.
Non-limiting examples of suitable antibodies include clones B6H12,
5F9, 8B6, and C3 (for example as described in International Patent
Publication WO 2011/143624, herein specifically incorporated by
reference). The 5F9 antibody comprises CDR sequences (SEQ ID NO:1)
5F9 heavy chain CDR1: NYNMH; (SEQ ID NO:2) 5F9 heavy chain CDR2:
TIYPGNDDTSYNQKFKD; (SEQ ID NO:3) 5F9 heavy chain CDR3: GGYRAMDY;
(SEQ ID NO:4) 5F9 light chain CDR1: RSSQSIVYSNGNTYLG; (SEQ ID NO:5)
5F9 light chain CDR2: KVSNRFS; (SEQ ID NO:6) 5F9 light chain CDR3:
FQGSHVPYT. Suitable anti-CD47 antibodies include fully human,
humanized or chimeric versions of such antibodies. Humanized
antibodies (e.g., hu5F9-G4) are especially useful for in vivo
applications in humans due to their low antigenicity. Similarly
caninized, felinized, etc. antibodies are especially useful for
applications in dogs, cats, and other species respectively.
Antibodies of interest include humanized antibodies, or caninized,
felinized, equinized, bovinized, porcinized, etc., antibodies, and
variants thereof.
[0049] Soluble CD47 polypeptides. In some embodiments, a subject
anti-CD47 agent is a soluble CD47 polypeptide that specifically
binds SIRP.alpha. and reduces the interaction between CD47 on one
cell (e.g., an infected cell) and SIRP.alpha. on another cell
(e.g., a phagocytic cell). A suitable soluble CD47 polypeptide can
bind SIRP.alpha. without activating or stimulating signaling
through SIRP.alpha. because activation of SIRP.alpha. would inhibit
phagocytosis. Instead, suitable soluble CD47 polypeptides
facilitate the preferential phagocytosis of infected cells over
non-infected cells. Those cells that express higher levels of CD47
(e.g., infected cells) relative to normal, non-target cells (normal
cells) will be preferentially phagocytosed. Thus, a suitable
soluble CD47 polypeptide specifically binds SIRP.alpha. without
activating/stimulating enough of a signaling response to inhibit
phagocytosis.
[0050] In some cases, a suitable soluble CD47 polypeptide can be a
fusion protein (for example as structurally described in US Patent
Publication US20100239579, herein specifically incorporated by
reference). However, only fusion proteins that do not
activate/stimulate SIRP.alpha. are suitable for the methods
provided herein. Suitable soluble CD47 polypeptides also include
any peptide or peptide fragment comprising variant or naturally
existing CD47 sequences (e.g., extracellular domain sequences or
extracellular domain variants) that can specifically bind
SIRP.alpha. and inhibit the interaction between CD47 and
SIRP.alpha. without stimulating enough SIRP.alpha. activity to
inhibit phagocytosis.
[0051] In certain embodiments, soluble CD47 polypeptide comprises
the extracellular domain of CD47, including the signal peptide,
such that the extracellular portion of CD47 is typically 142 amino
acids in length, and has the amino acid sequence set forth in SEQ
ID NO:3. The soluble CD47 polypeptides described herein also
include CD47 extracellular domain variants that comprise an amino
acid sequence at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or
95%-99% (or any percent identity not specifically enumerated
between 65% to 100%), which variants retain the capability to bind
to SIRP.alpha. without stimulating SIRP.alpha. signaling.
[0052] In certain embodiments, the signal peptide amino acid
sequence may be substituted with a signal peptide amino acid
sequence that is derived from another polypeptide (e.g., for
example, an immunoglobulin or CTLA4). For example, unlike
full-length CD47, which is a cell surface polypeptide that
traverses the outer cell membrane, the soluble CD47 polypeptides
are secreted; accordingly, a polynucleotide encoding a soluble CD47
polypeptide may include a nucleotide sequence encoding a signal
peptide that is associated with a polypeptide that is normally
secreted from a cell.
[0053] In other embodiments, the soluble CD47 polypeptide comprises
an extracellular domain of CD47 that lacks the signal peptide. In
an exemplary embodiment, the CD47 extracellular domain lacking the
signal peptide has the amino acid sequence set forth in SEQ ID NO:1
(124 amino acids). As described herein, signal peptides are not
exposed on the cell surface of a secreted or transmembrane protein
because either the signal peptide is cleaved during translocation
of the protein or the signal peptide remains anchored in the outer
cell membrane (such a peptide is also called a signal anchor). The
signal peptide sequence of CD47 is believed to be cleaved from the
precursor CD47 polypeptide in vivo.
[0054] In other embodiments, a soluble CD47 polypeptide comprises a
CD47 extracellular domain variant. Such a soluble CD47 polypeptide
retains the capability to bind to SIRP.alpha. without stimulating
SIRP.alpha. signaling. The CD47 extracellular domain variant may
have an amino acid sequence that is at least 65%-75%, 75%-80%,
80-85%, 85%-90%, or 95%-99% identical (which includes any percent
identity between any one of the described ranges) to a reference
human CD47 sequence.
[0055] The term "antibody" or "antibody moiety" is intended to
include any polypeptide chain-containing molecular structure with a
specific shape that fits to and recognizes an epitope, where one or
more non-covalent binding interactions stabilize the complex
between the molecular structure and the epitope. Antibodies
utilized in the present invention may be polyclonal antibodies,
although monoclonal antibodies are preferred because they may be
reproduced by cell culture or recombinantly, and can be modified to
reduce their antigenicity.
[0056] Polyclonal antibodies can be raised by a standard protocol
by injecting a production animal with an antigenic composition.
See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988. When utilizing an entire protein,
or a larger section of the protein, antibodies may be raised by
immunizing the production animal with the protein and a suitable
adjuvant (e.g., Freund's, Freund's complete, oil-in-water
emulsions, etc.) When a smaller peptide is utilized, it is
advantageous to conjugate the peptide with a larger molecule to
make an immunostimulatory conjugate. Commonly utilized conjugate
proteins that are commercially available for such use include
bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In
order to raise antibodies to particular epitopes, peptides derived
from the full sequence may be utilized. Alternatively, in order to
generate antibodies to relatively short peptide portions of the
protein target, a superior immune response may be elicited if the
polypeptide is joined to a carrier protein, such as ovalbumin, BSA
or KLH. Alternatively, for monoclonal antibodies, hybridomas may be
formed by isolating the stimulated immune cells, such as those from
the spleen of the inoculated animal. These cells are then fused to
immortalized cells, such as myeloma cells or transformed cells,
which are capable of replicating indefinitely in cell culture,
thereby producing an immortal, immunoglobulin-secreting cell line.
In addition, the antibodies or antigen binding fragments may be
produced by genetic engineering. Humanized, chimeric, or xenogeneic
human antibodies, which produce less of an immune response when
administered to humans, are preferred for use in the present
invention.
[0057] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab')2, or other fragments) are useful
as antibody moieties in the present invention. Such antibody
fragments may be generated from whole immunoglobulins by ricin,
pepsin, papain, or other protease cleavage. "Fragment," or minimal
immunoglobulins may be designed utilizing recombinant
immunoglobulin techniques. For instance "Fv" immunoglobulins for
use in the present invention may be produced by linking a variable
light chain region to a variable heavy chain region via a peptide
linker (e.g., poly-glycine or another sequence which does not form
an alpha helix or beta sheet motif).
[0058] Antibodies include free antibodies and antigen binding
fragments derived therefrom, and conjugates, e.g. pegylated
antibodies, drug, radioisotope, or toxin conjugates, and the like.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the targeting and/or
depletion of cellular populations expressing the marker. Various
techniques can be utilized using monoclonal antibodies to screen
for cellular populations expressing the marker(s), and include
magnetic separation using antibody-coated magnetic beads, "panning"
with antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.
Cell, 96:737-49 (1999)). These techniques allow for the screening
of particular populations of cells; in immunohistochemistry of
biopsy samples; in detecting the presence of markers shed by cancer
cells into the blood and other biologic fluids, and the like.
[0059] Humanized versions of such antibodies are also within the
scope of this invention. Humanized antibodies are especially useful
for in vivo applications in humans due to their low
antigenicity.
[0060] The phrase "multispecific or bispecific antibody" refers to
a synthetic or recombinant antibody that recognizes more than one
protein. Bispecific antibodies directed against a combination of
epitopes, will allow for the targeting and/or depletion of cellular
populations expressing the combination of epitopes. Exemplary
bi-specific antibodies include those targeting a combination of
CD47 and an SCLC cancer cell marker. Generation of bi-specific
antibodies are described in the literature, for example, in U.S.
Pat. Nos. 5,989,830, 5,798,229, which are incorporated herein by
reference. Higher order specificities, e.g. trispecific antibodies,
are described by Holliger and Hudson (2005) Nature Biotechnology
23:1126-1136.
[0061] The efficacy of a CD47 inhibitor can be assessed by assaying
CD47 activity. The above-mentioned assays or modified versions
thereof are used. In an exemplary assay, SCLC are incubated with
bone marrow derived macrophages, in the presence or absence of the
candidate agent. An inhibitor of the cell surface CD47 will
up-regulate phagocytosis by at least about 10%, or up to 20%, or
50%, or 70% or 80% or up to about 90% compared to the phagocytosis
in absence of the candidate agent. Similarly, an in vitro assay for
levels of tyrosine phosphorylation of SIRP.alpha. will show a
decrease in phosphorylation by at least about 10%, or up to 20%, or
50%, or 70% or 80% or up to about 90% compared to phosphorylation
observed in absence of the candidate agent.
[0062] In one embodiment of the invention, the agent, or a
pharmaceutical composition comprising the agent, is provided in an
amount effective to detectably inhibit the binding of CD47 to
SIRP.alpha. receptor present on the surface of phagocytic cells.
The effective amount is determined via empirical testing routine in
the art. The effective amount may vary depending on the number of
cells being transplanted, site of transplantation and factors
specific to the transplant recipient.
[0063] The terms "phagocytic cells" and "phagocytes" are used
interchangeably herein to refer to a cell that is capable of
phagocytosis. There are four main categories of phagocytes:
macrophages, mononuclear cells (histiocytes and monocytes);
polymorphonuclear leukocytes (neutrophils) and dendritic cells.
[0064] The term "biological sample" encompasses a variety of sample
types obtained from an organism and can be used in a diagnostic or
monitoring assay. The term encompasses blood and other liquid
samples of biological origin, solid tissue samples, such as a
biopsy specimen or tissue cultures or cells derived therefrom and
the progeny thereof. The term encompasses samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components. The term encompasses a clinical sample, and also
includes cells in cell culture, cell supernatants, cell lysates,
serum, plasma, biological fluids, and tissue samples.
[0065] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, e.g. mouse, rat, rabbit, pig,
primate, including humans and other apes, particularly a human, and
includes: (a) preventing the disease or symptom from occurring in a
subject which may be predisposed to the disease or symptom but has
not yet been diagnosed as having it; (b) inhibiting the disease
symptom, i.e., arresting its development; or (c) relieving the
disease symptom, i.e., causing regression of the disease or
symptom.
[0066] The terms "recipient", "individual", "subject", "host", and
"patient", used interchangeably herein and refer to any mammalian
subject for whom diagnosis, treatment, or therapy is desired,
particularly humans.
[0067] A "host cell", as used herein, refers to a microorganism or
a eukaryotic cell or cell line cultured as a unicellular entity
which can be, or has been, used as a recipient for a recombinant
vector or other transfer polynucleotides, and include the progeny
of the original cell which has been transfected. It is understood
that the progeny of a single cell may not necessarily be completely
identical in morphology or in genomic or total DNA complement as
the original parent, due to natural, accidental, or deliberate
mutation.
[0068] The terms "cancer", "neoplasm", "tumor", and "carcinoma",
are used interchangeably herein to refer to cells which exhibit
relatively autonomous growth, so that they exhibit an aberrant
growth phenotype characterized by a significant loss of control of
cell proliferation. In general, cells of interest for detection or
treatment in the present application include precancerous (e.g.,
benign), malignant, pre-metastatic, metastatic, and non-metastatic
cells. Detection of cancerous cells is of particular interest. The
term "normal" as used in the context of "normal cell," is meant to
refer to a cell of an untransformed phenotype or exhibiting a
morphology of a non-transformed cell of the tissue type being
examined. "Cancerous phenotype" generally refers to any of a
variety of biological phenomena that are characteristic of a
cancerous cell, which phenomena can vary with the type of cancer.
The cancerous phenotype is generally identified by abnormalities
in, for example, cell growth or proliferation (e.g., uncontrolled
growth or proliferation), regulation of the cell cycle, cell
mobility, cell-cell interaction, or metastasis, etc.
[0069] "Therapeutic target" refers to a gene or gene product that,
upon modulation of its activity (e.g., by modulation of expression,
biological activity, and the like), can provide for modulation of
the cancerous phenotype. As used throughout, "modulation" is meant
to refer to an increase or a decrease in the indicated phenomenon
(e.g., modulation of a biological activity refers to an increase in
a biological activity or a decrease in a biological activity).
Lung Cancer
[0070] Lung carcinoma is the leading cause of cancer-related death
worldwide. About 85% of cases are related to cigarette smoking.
Symptoms can include cough, chest discomfort or pain, weight loss,
and, less commonly, hemoptysis; however, many patients present with
metastatic disease without any clinical symptoms. The diagnosis is
typically made by chest x-ray or CT and confirmed by biopsy.
Depending on the stage of the disease, treatment includes surgery,
chemotherapy, radiation therapy, or a combination. For the past
several decades, the prognosis for a lung cancer patient has been
poor, particularly for patients with stage IV (metastatic)
disease.
[0071] Respiratory epithelial cells require prolonged exposure to
cancer-promoting agents and accumulation of multiple genetic
mutations before becoming neoplastic (an effect called field
carcinogenesis). In some patients with lung cancer, secondary or
additional mutations in genes that stimulate cell growth (K-ras,
MYC), cause abnormalities in growth factor receptor signaling
(EGFR, HER2/neu), and inhibit apoptosis contribute to proliferation
of abnormal cells. In addition, mutations that inhibit
tumor-suppressor genes (p53, APC) can lead to cancer. Other
mutations that may be responsible include the EML-4-ALK
translocation and mutations in ROS-1, BRAF, and PI3KCA. Genes such
as these that are primarily responsible for lung cancer are called
driver mutations. Although driver mutations can cause or contribute
to lung cancer among smokers, these mutations are particularly
likely to be a cause of lung cancer among nonsmokers.
[0072] Chest x-ray is often the initial imaging test. It may show
clearly defined abnormalities, such as a single mass or multifocal
masses or a solitary pulmonary nodule, an enlarged hilum, widened
mediastinum, tracheobronchial narrowing, atelectasis, non-resolving
parenchymal infiltrates, cavitary lesions, or unexplained pleural
thickening or effusion. These findings are suggestive but not
diagnostic of lung cancer and require follow-up with CT scans or
combined PET-CT scans and cytopathologic confirmation.
[0073] CT shows many characteristic anatomic patterns and
appearances that may strongly suggest the diagnosis. CT also can
guide core needle biopsy of accessible lesions and is useful for
staging. If a lesion found on a plain x-ray is highly likely to be
lung cancer, PET-CT may be done. This study combines anatomic
imaging from CT with functional imaging from PET. The PET images
can help differentiate inflammatory and malignant processes.
[0074] SCLC has 2 stages: limited and extensive. Limited-stage SCLC
disease is cancer confined to one hemithorax (including ipsilateral
lymph nodes) that can be encompassed within one tolerable radiation
therapy port, unless there is a pleural or pericardial effusion.
Extensive-stage disease is cancer outside a single hemithorax or
the presence of malignant cells detected in pleural or pericardial
effusions. Less than one third of patients with SCLC will present
with limited-stage disease; the remainder of patients often have
extensive distant metastases. The overall prognosis for SCLC is
poor. The median survival time for limited-stage SCLC is 20 mo,
with a 5-yr survival rate of 20%. Patients with extensive-stage
SCLC do especially poorly, with a 5-yr survival rate of <1%.
[0075] NSCLC has 4 stages, I through IV (using the TNM system). TNM
staging is based on tumor size, tumor and lymph node location, and
the presence or absence of distant metastases. The 5-yr survival
rate of patients with NSCLC varies by stage, from 60 to 70% for
patients with stage I disease to <1% for patients with stage IV
disease.
[0076] Conventional treatment varies by cell type and by stage of
disease. Many patient factors not related to the tumor affect
treatment choice. Poor cardiopulmonary reserve, undernutrition,
frailty or poor physical performance status, comorbidities,
including cytopenias, and psychiatric or cognitive illness all may
lead to a decision for palliative over curative treatment or for no
treatment at all, even though a cure with aggressive therapy might
technically be possible.
[0077] SCLC of any stage is typically initially responsive to
treatment, but responses are usually short-lived. Chemotherapy,
with or without radiation therapy, is given depending on the stage
of disease. In many patients, chemotherapy prolongs survival and
improves quality of life enough to warrant its use. Surgery
generally plays no role in treatment of SCLC, although it may be
curative in the rare patient who has a small focal tumor without
spread (such as a solitary pulmonary nodule) who underwent surgical
resection before the tumor was identified as SCLC. Chemotherapy
regimens of etoposide and a platinum compound (either cisplatin or
carboplatin) are commonly used, as are other drugs, such as
irinotecan, topotecan, vinca alkaloids (vinblastine, vincristine,
vinorelbine), alkylating agents (cyclophosphamide, ifosfamide),
doxorubicin, taxanes (docetaxel, paclitaxel), and gemcitabine. When
disease is confined to a hemithorax, radiation therapy further
improves clinical outcomes; such response to radiation therapy was
the basis for the definition of limited-stage disease. The use of
cranial radiation to prevent brain metastases is also advocated in
certain cases; micrometastases are common in SCLC, and chemotherapy
has less ability to cross the blood-brain barrier.
[0078] In extensive-stage disease, treatment is based on
chemotherapy rather than radiation therapy, although radiation
therapy is often used as palliative treatment for metastases to
bone or brain. In patients with an excellent response to
chemotherapy, prophylactic brain irradiation is sometimes used as
in limited-stage SCLC to prevent growth of SCLC in the brain.
[0079] Treatment for NSCLC typically involves assessment of
eligibility for surgery followed by choice of surgery,
chemotherapy, radiation therapy, or a combination of modalities as
appropriate, depending on tumor type and stage.
Treatment of Cancer
[0080] The invention provides methods for reducing growth of lung
cancer cells through the introduction of an effective dose of a
targeted therapeutic agent directed to a lung cancer cell surface
marker, including without limitation CD24, CD166, CD56, CD326,
CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165,
EpCAM, etc. In some embodiments the marker is one of CD56, CD44,
CD99 and EpCam. In preferred embodiments the targeted therapeutic
agent is combined with a CD47 blocking agent, e.g. soluble
SIRP.alpha. monomer or multimer, an anti-CD47 antibody, small
molecule, etc. In certain embodiments the cancer is SCLC. By
blocking the activity of CD47, the downregulation of phagocytosis
that is found with certain tumor cells is prevented.
[0081] "Reducing growth of cancer cells" includes, but is not
limited to, reducing proliferation of cancer cells, and reducing
the incidence of a non-cancerous cell becoming a cancerous cell.
Whether a reduction in cancer cell growth has been achieved can be
readily determined using any known assay, including, but not
limited to, [.sup.3H]-thymidine incorporation; counting cell number
over a period of time; detecting and/or measuring a marker
associated with SCLC, etc.
[0082] Whether a substance, or a specific amount of the substance,
is effective in treating cancer can be assessed using any of a
variety of known diagnostic assays for cancer, including, but not
limited to biopsy, contrast radiographic studies, CAT scan, and
detection of a tumor marker associated with cancer in the blood of
the individual. The substance can be administered systemically or
locally, usually systemically.
[0083] As an alternative embodiment, an agent, e.g. a
chemotherapeutic drug that reduces cancer cell growth, can be
targeted to a cancer cell by conjugation to a CD47 specific
antibody. Thus, in some embodiments, the invention provides a
method of delivering a drug to a cancer cell, comprising
administering a drug-antibody complex to a subject, wherein the
antibody is specific for a cancer-associated polypeptide, and the
drug is one that reduces cancer cell growth, a variety of which are
known in the art. Targeting can be accomplished by coupling (e.g.,
linking, directly or via a linker molecule, either covalently or
non-covalently, so as to form a drug-antibody complex) a drug to an
antibody specific for a cancer-associated polypeptide. Methods of
coupling a drug to an antibody are well known in the art and need
not be elaborated upon herein.
[0084] In certain embodiments, a bi-specific antibody may be used.
For example a bi-specific antibody in which one antigen binding
domain is directed against CD47 and the other antigen binding
domain is directed against a cancer cell marker, such as CD24,
CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46,
CD59, CD57, CD165, EpCAM, etc. may be used.
[0085] Generally, as the term is utilized in the specification,
"antibody" or "antibody moiety" is intended to include any
polypeptide chain-containing molecular structure that has a
specific shape which fits to and recognizes an epitope, where one
or more non-covalent binding interactions stabilize the complex
between the molecular structure and the epitope. For monoclonal
antibodies, hybridomas may be formed by isolating the stimulated
immune cells, such as those from the spleen of the inoculated
animal. These cells are then fused to immortalized cells, such as
myeloma cells or transformed cells, which are capable of
replicating indefinitely in cell culture, thereby producing an
immortal, immunoglobulin-secreting cell line. The immortal cell
line utilized is preferably selected to be deficient in enzymes
necessary for the utilization of certain nutrients. Many such cell
lines (such as myelomas) are known to those skilled in the art, and
include, for example: thymidine kinase (TK) or hypoxanthine-guanine
phosphoriboxyl transferase (HGPRT). These deficiencies allow
selection for fused cells according to their ability to grow on,
for example, hypoxanthine aminopterinthymidine medium (HAT).
[0086] Antibodies which have a reduced propensity to induce a
violent or detrimental immune response in humans (such as
anaphylactic shock), and which also exhibit a reduced propensity
for priming an immune response which would prevent repeated dosage
with the antibody therapeutic or imaging agent (e.g., the
human-anti-murine-antibody "HAMA" response), are preferred for use
in the invention. These antibodies are preferred for all
administrative routes. Thus, humanized, chimeric, or xenogenic
human antibodies, which produce less of an immune response when
administered to humans, are preferred for use in the present
invention.
[0087] Chimeric antibodies may be made by recombinant means by
combining the murine variable light and heavy chain regions (VK and
VH), obtained from a murine (or other animal-derived) hybridoma
clone, with the human constant light and heavy chain regions, in
order to produce an antibody with predominantly human domains. The
production of such chimeric antibodies is well known in the art,
and may be achieved by standard means (as described, e.g., in U.S.
Pat. No. 5,624,659, incorporated fully herein by reference).
Humanized antibodies are engineered to contain even more human-like
immunoglobulin domains, and incorporate only the
complementarity-determining regions of the animal-derived antibody.
This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody, and fitting them to the structure of the human antibody
chains. Although facially complex, the process is straightforward
in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully
herein by reference.
[0088] Alternatively, polyclonal or monoclonal antibodies may be
produced from animals which have been genetically altered to
produce human immunoglobulins. The transgenic animal may be
produced by initially producing a "knock-out" animal which does not
produce the animal's natural antibodies, and stably transforming
the animal with a human antibody locus (e.g., by the use of a human
artificial chromosome). Only human antibodies are then made by the
animal. Techniques for generating such animals, and deriving
antibodies therefrom, are described in U.S. Pat. Nos. 6,162,963 and
6,150,584, incorporated fully herein by reference. Such fully human
xenogenic antibodies are a preferred antibody for use in the
methods and compositions of the present invention. Alternatively,
single chain antibodies can be produced from phage libraries
containing human variable regions. See U.S. Pat. No. 6,174,708,
incorporated fully herein by reference.
[0089] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) are
useful as antibody moieties in the present invention. Such antibody
fragments may be generated from whole immunoglobulins by ficin,
pepsin, papain, or other protease cleavage. "Fragment," or minimal
immunoglobulins may be designed utilizing recombinant
immunoglobulin techniques. For instance "Fv" immunoglobulins for
use in the present invention may be produced by linking a variable
light chain region to a variable heavy chain region via a peptide
linker (e.g., poly-glycine or another sequence which does not form
an alpha helix or beta sheet motif).
[0090] Fv fragments are heterodimers of the variable heavy chain
domain (VH) and the variable light chain domain (VL). The
heterodimers of heavy and light chain domains that occur in whole
IgG, for example, are connected by a disulfide bond. Recombinant
Fvs in which VH and VL are connected by a peptide linker are
typically stable, see, for example, Huston et al., Proc. Natl.
Acad, Sci. USA 85:5879 5883 (1988) and Bird et al., Science 242:423
426 (1988), both fully incorporated herein, by reference. These are
single chain Fvs which have been found to retain specificity and
affinity and have been shown to be useful for imaging tumors and to
make recombinant immunotoxins for tumor therapy. However,
researchers have found that some of the single chain Fvs have a
reduced affinity for antigen and the peptide linker can interfere
with binding. Improved Fv's have been also been made which comprise
stabilizing disulfide bonds between the VH and VL regions, as
described in U.S. Pat. No. 6,147,203, incorporated fully herein by
reference. Any of these minimal antibodies may be utilized in the
present invention, and those which are humanized to avoid HAMA
reactions are preferred for use in embodiments of the
invention.
[0091] Derivatized polypeptides with added chemical linkers,
detectable moieties such as fluorescent dyes, enzymes, substrates,
chemiluminescent moieties, specific binding moieties such as
streptavidin, avidin, or biotin, or drug conjugates may be utilized
in the methods and compositions of the present invention.
[0092] In some embodiments of the invention, the polypeptide
reagents of the invention are coupled or conjugated to one or more
therapeutic, cytotoxic, or imaging moieties. As used herein,
"cytotoxic moiety" (C) simply means a moiety which inhibits cell
growth or promotes cell death when proximate to or absorbed by the
cell. Suitable cytotoxic moieties in this regard include
radioactive isotopes (radionuclides), chemotoxic agents such as
differentiation inducers and small chemotoxic drugs, toxin
proteins, and derivatives thereof. Agents may be conjugated to a
polypeptide reagent of the invention by any suitable technique,
with appropriate consideration of the need for pharmokinetic
stability and reduced overall toxicity to the patient. A
therapeutic agent may be coupled to a suitable moiety either
directly or indirectly (e.g. via a linker group). A direct reaction
is possible when each possesses a functional group capable of
reacting with the other. For example, a nucleophilic group, such as
an amino or sulfhydryl group, may be capable of reacting with a
carbonyl-containing group, such as an anhydride or an acid halide,
or with an alkyl group containing a good leaving group (e.g., a
halide). Alternatively, a suitable chemical linker group may be
used. A linker group can function as a spacer to distance a
polypeptide reagent of the invention from an agent in order to
avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
a moiety or a polypeptide reagent of the invention, and thus
increase the coupling efficiency. An increase in chemical
reactivity may also facilitate the use of moieties, or functional
groups on moieties, which otherwise would not be possible.
[0093] Suitable linkage chemistries include maleimidyl linkers and
alkyl halide linkers (which react with a sulfhydryl on the antibody
moiety) and succinimidyl linkers (which react with a primary amine
on the antibody moiety). Several primary amine and sulfhydryl
groups are present on immunoglobulins, and additional groups may be
designed into recombinant immunoglobulin molecules. It will be
evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such
as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as a linker group. Coupling may be
effected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues. There are
numerous references describing such methodology, e.g., U.S. Pat.
No. 4,671,958. As an alternative coupling method, cytotoxic
moieties may be coupled to a polypeptide reagent of the invention
through a an oxidized carbohydrate group at a glycosylation site,
as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet another
alternative method of coupling a polypeptide reagent of the
invention to a cytotoxic or therapeutic moiety is by the use of a
non-covalent binding pair, such as streptavidin/biotin, or
avidin/biotin. In these embodiments, one member of the pair is
covalently coupled to the anti-CD47, CV1, etc. moiety and the other
member of the binding pair is covalently coupled to the
therapeutic, cytotoxic, or imaging moiety.
[0094] Where a cytotoxic moiety is more potent when free from the
binding portion of a polypeptide reagent of the invention, it may
be desirable to use a linker group which is cleavable during or
upon internalization into a cell, or which is gradually cleavable
over time in the extracellular environment. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of a cytotoxic moiety agent from these linker
groups include cleavage by reduction of a disulfide bond (e.g.,
U.S. Pat. No. 4,489,710), by irradiation of a photolabile bond
(e.g., U.S. Pat. No. 4,625,014), by hydrolysis of derivatized amino
acid side chains (e.g., U.S. Pat. No. 4,638,045), by serum
complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958), and
acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).
[0095] It may be desirable to couple more than one moiety to a
polypeptide reagent of the invention. By poly-derivatizing the
reagent, several strategies may be simultaneously implemented, e.g.
a therapeutic antibody may be labeled for tracking by a
visualization technique. Regardless of the particular embodiment,
conjugates with more than one moiety may be prepared in a variety
of ways. For example, more than one moiety may be coupled directly
to a polypeptide molecule, or linkers which provide multiple sites
for attachment (e.g., dendrimers) can be used. Alternatively, a
carrier with the capacity to hold more than one cytotoxic or
imaging moiety can be used.
[0096] A carrier may bear the agents in a variety of ways,
including covalent bonding either directly or via a linker group,
and non-covalent associations. Suitable covalent-bond carriers
include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234),
peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat.
No. 4,699,784), each of which have multiple sites for the
attachment of moieties. A carrier may also bear an agent by
non-covalent associations, such as non-covalent bonding or by
encapsulation, such as within a liposome vesicle (e.g., U.S. Pat.
Nos. 4,429,008 and 4,873,088). Encapsulation carriers are
especially useful for imaging moiety conjugation to antibody
moieties for use in the invention, as a sufficient amount of the
imaging moiety (dye, magnetic resonance contrast reagent, etc.) for
detection may be more easily associated with the antibody moiety.
In addition, encapsulation carriers are also useful in chemotoxic
therapeutic embodiments, as they can allow the therapeutic
compositions to gradually release a chemotoxic moiety over time
while concentrating it in the vicinity of the tumor cells.
[0097] Preferred radionuclides for use as cytotoxic moieties are
radionuclides which are suitable for pharmacological
administration. Such radionuclides include .sup.123I, .sup.125I,
.sup.131I, .sup.90Y, .sup.211At, .sup.67Cu, .sup.186Re, .sup.188Re,
.sup.212Pb, and .sup.212Bi. Iodine and astatine isotopes are more
preferred radionuclides for use in the therapeutic compositions of
the present invention, as a large body of literature has been
accumulated regarding their use.
[0098] Preferred chemotoxic agents include small-molecule drugs
such as carboplatin, cisplatin, vincristine, taxanes such as
paclitaxel and doceltaxel, hydroxyurea, gemcitabine, vinorelbine,
irinotecan, tirapazamine, matrilysin, methotrexate, pyrimidine and
purine analogs, and other suitable small toxins known in the art.
Preferred chemotoxin differentiation inducers include phorbol
esters and butyric acid. Chemotoxic moieties may be directly
conjugated to the antibody moiety via a chemical linker, or may be
encapsulated in a carrier, which is in turn coupled to the
antibody. Preferred toxin proteins for use as cytotoxic moieties
include ricins A and B, abrin, diphtheria toxin, bryodin 1 and 2,
momordin, trichokirin, cholera toxin, gelonin, Pseudomonas
exotoxin, Shigella toxin, pokeweed antiviral protein, and other
toxin proteins known in the medicinal biochemistry arts. As these
toxin agents may elicit undesirable immune responses in the
patient, especially if injected intravascularly, it is preferred
that they be encapsulated in a carrier for coupling to the
antibody.
[0099] For administration, a targeted therapeutic agent, or
combination of targeted therapeutic agents may be administered
separately or together; and will generally be administered within
the same general time frame, e.g. within a week, within 3-4 days,
within 1 day or simultaneously with each other.
[0100] The agent or agents are mixed, prior to administration, with
a non-toxic, pharmaceutically acceptable carrier substance.
Usually, this will be an aqueous solution, such as normal saline or
phosphate-buffered saline (PBS), Ringer's solution,
lactate-Ringer's solution, or any isotonic physiologically
acceptable solution for administration by the chosen means.
Preferably, the solution is sterile and pyrogen-free, and is
manufactured and packaged under current Good Manufacturing
Processes (GMPs), as approved by the FDA. The clinician of ordinary
skill is familiar with appropriate ranges for pH, tonicity, and
additives or preservatives when formulating pharmaceutical
compositions for administration by intravascular injection, direct
injection into the lymph nodes, intraperitoneal, or by other
routes. In addition to additives for adjusting pH or tonicity, the
agents may be stabilized against aggregation and polymerization
with amino acids and non-ionic detergents, polysorbate, and
polyethylene glycol. Optionally, additional stabilizers may include
various physiologically-acceptable carbohydrates and salts. Also,
polyvinylpyrrolidone may be added in addition to the amino acid.
Suitable therapeutic immunoglobulin solutions which are stabilized
for storage and administration to humans are described in U.S. Pat.
No. 5,945,098, incorporated fully herein by reference. Other
agents, such as human serum albumin (HSA), may be added to the
therapeutic or imaging composition to stabilize the antibody
conjugates.
[0101] The compositions of the invention may be administered using
any medically appropriate procedure, e.g., intravascular
(intravenous, intraarterial, intracapillary) administration,
injection into the tumor, etc. Intravascular injection may be by
intravenous or intraarterial injection. The effective amount of the
therapeutic compositions to be given to a particular patient will
depend on a variety of factors, several of which will be different
from patient to patient. A competent clinician will be able to
determine an effective amount of a therapeutic composition to
administer to a patient to retard the growth and promote the death
of tumor cells. Dosage of the agents will depend on the treatment
of the tumor, route of administration, the nature of the
therapeutics, sensitivity of the tumor to the therapeutics, etc.
Utilizing LD.sub.50 animal data, and other information available
for the conjugated cytotoxic or imaging moiety, a clinician can
determine the maximum safe dose for an individual, depending on the
route of administration. For instance, an intravenously
administered dose may be more than an locally administered dose,
given the greater body of fluid into which the therapeutic
composition is being administered. Similarly, compositions which
are rapidly cleared from the body may be administered at higher
doses, or in repeated doses, in order to maintain a therapeutic
concentration. Utilizing ordinary skill, the competent clinician
will be able to optimize the dosage of a particular therapeutic or
imaging composition in the course of routine clinical trials.
[0102] Typically an effective dosage will be 0.001 to 100
milligrams of antibody per kilogram subject body weight. The ratio
of anti-CD47 to the second agent may range from 1:100; 1:50; 1:10;
1:5; 1:2; 1:1; 2:1; 5:1; 10:1; 50:1; 100:1. The agents can be
administered to the subject in a series of more than one
administration. For therapeutic compositions, regular periodic
administration (e.g., every 2-3 days) will sometimes be required,
or may be desirable to reduce toxicity. For therapeutic
compositions which will be utilized in repeated-dose regimens,
antibody moieties which do not provoke HAMA or other immune
responses are preferred.
Example 1
High-Affinity SIRP.alpha. Variants Enhance Macrophage Destruction
of Small Cell Lung Cancer
[0103] CD47 allows cancer cells to evade the immune system by
signaling through SIRP.alpha., an inhibitory receptor on
macrophages. We recently developed next-generation CD47 antagonists
by engineering the N-terminal immunoglobulin domain of SIRP.alpha..
These "high-affinity SIRP.alpha. variants" have an affinity for
human CD47 (K.sub.D) as low as 11.1 pM, approximately 50,000-fold
improved over wild-type SIRP.alpha.. When combined with
tumor-specific antibodies, the high-affinity SIRP.alpha. variants
act as immunotherapeutic adjuvants to maximize macrophage
destruction of cancer cells.
[0104] We have now applied these reagents to small cell lung cancer
(SCLC), a cancer with poor prognosis for which no clinically
approved antibodies exist. We found SCLC cell lines and primary
samples expressed high levels of CD47 on their surface. Using human
macrophages, we found that CD47-blocking therapies were able to
induce macrophage phagocytosis of SCLC cells. Treatment of mice
bearing primary human SCLC tumors with CD47-blocking antibodies was
able to inhibit tumor growth and significantly prolong survival. To
identify novel SCLC antigens that can be targeted in combination
with high-affinity SIRP.alpha. variants, SCLC samples were screened
by flow cytometry using comprehensive antibody arrays.
[0105] We validated tumor-specific antigens on the surface of SCLC
cells, and identified antibodies to these antigens that could
stimulate phagocytosis in vitro. When combined with high-affinity
SIRP.alpha. monomers, the ability of these antibodies to stimulate
phagocytosis was dramatically enhanced.
Example 2
CD47-Blocking Therapies Stimulate Macrophage Destruction of Small
Cell Lung Cancer
[0106] Small cell lung cancer (SCLC) is a highly aggressive subtype
of lung cancer with dismal prognosis. There are no clinically
approved antibodies, targeted therapies, or immunotherapies for the
disease. We found that SCLC samples expressed high levels of CD47,
a cell-surface molecule that allows cancer cells to evade the
immune system. In particular, CD47 promotes immune evasion by
signaling through SIRP.alpha., an inhibitory receptor on
macrophages. We hypothesized that CD47-blocking therapies could be
applied to the treatment of SCLC. We found that CD47-blocking
therapies were able to induce macrophage phagocytosis of SCLC
samples in vitro. CD47-blocking therapies also inhibited tumor
growth and significantly prolonged survival of mice bearing SCLC
tumors. Furthermore, using comprehensive antibody arrays, we
identified several new and established therapeutic targets on the
surface of SCLC cells. Antibodies to these targets could elicit
macrophage phagocytosis and were enhanced when combined with
CD47-blocking therapies. These findings suggest that therapies that
disrupt the CD47-SIRP.alpha. axis could benefit patients with SCLC,
particularly when combined with tumor-specific antibodies.
[0107] Small cell lung cancer (SCLC), which derives from
neuroendocrine cells of the lung, is one of the most lethal
subtypes of cancer in humans. Each year, more than 25,000 patients
are diagnosed with SCLC in the United States alone, and patients
typically live only 6-12 months after diagnosis. The 5-year
survival rate has remained dismal, hovering around 5% since the
1970s. Except for the combination of radiation and chemotherapy,
there have been no new therapeutic approaches implemented in the
past 30 years. Despite a plethora of clinical trials, no targeted
therapies have been approved for SCLC. SCLC is strongly linked to
heavy cigarette smoking, and increased smoking rates in developing
countries will continue to increase the worldwide prevalence of
SCLC in the future. For these reasons, there is a need to identify
novel therapeutic targets and generate new treatments for patients
with SCLC.
[0108] One of the most promising advances in the field of oncology
is immunotherapy, which aims to stimulate a patient's own immune
system to attack and eliminate cancer. As tumors develop, they
acquire mechanisms to avoid destruction by the immune system. By
understanding these mechanisms, we can develop new strategies to
coax the immune system to recognize cancer as foreign. Previous
studies have identified CD47, a cell-surface molecule, as a "marker
of self" that prevents cells of the innate immune system from
attacking hematologic malignancies and certain types of solid
tumors. CD47 acts by sending inhibitory signals through
SIRP.alpha., a receptor expressed on the surface of macrophages and
other myeloid cells. In this sense, the CD47-SIRP.alpha.
interaction represents a myeloid-specific immune checkpoint. A
number of reagents have been generated to disrupt signaling by the
CD47-SIRP.alpha. axis, including anti-CD47 antibodies and
engineered variants of its receptor, SIRP.alpha.. Recent studies
have shown that blockade of CD47 lowers the threshold for
macrophage phagocytosis of cancer. We hypothesized that SCLC cells
also express CD47 and that CD47-blocking therapies could be used to
stimulate macrophage phagocytosis of SCLC cells and inhibit growth
of SCLC tumors in vivo.
[0109] Furthermore, CD47-blocking therapies have been shown to
enhance the response of macrophages to monoclonal antibodies.
Monoclonal antibodies--such as rituximab for lymphoma or
trastuzumab for Her2.sup.+ breast cancer--have demonstrated immense
success for the treatment of cancer. No monoclonal antibodies are
clinically approved for the treatment of SCLC, thus, we aimed to
identify new SCLC surface antigens that could be targeted with
monoclonal antibodies. While treatment with monoclonal antibodies
can produce robust anti-tumor effects, they often fail to elicit
cures when used as single agents, highlighting the need to improve
the efficacy of these approaches. Therefore, we aimed to combine
CD47-blocking therapies with other antibodies to achieve maximal
anti-tumor responses against SCLC.
[0110] As a first step in our approach, we investigated whether
CD47 was expressed on the surface of SCLC samples. Next, we
examined whether CD47-blocking therapies could stimulate macrophage
phagocytosis of SCLC in vitro. Mouse models of human cancer were
used to evaluate the response of SCLC samples to CD47-blocking
therapies in vivo. To identify new therapeutic targets on the
surface of SCLC samples, we performed high-throughput flow
cytometry using comprehensive antibody arrays. Last, we aimed to
demonstrate that antibodies towards the identified antigens could
be combined with CD47-blocking therapies to further increase
phagocytosis. The overall objectives of this study were to validate
CD47-blocking therapies for SCLC and identify additional antibodies
that could be used to target SCLC. In this manner, we aim to
identify new immunotherapeutic combinations that could be used for
the benefit of patients with SCLC.
Results
[0111] CD47 is Expressed on the Surface of SCLC. To evaluate
whether CD47-blocking therapies could be applied to SCLC, we first
examined expression of CD47 on the surface of SCLC cells. We
obtained six SCLC cell lines and subjected them to flow cytometry
to evaluate CD47 expression on the cell surface. All six cell lines
exhibited high CD47 expression (FIG. 5A). We also evaluated CD47
surface expression on a SCLC patient-derived xenograft obtained
from a primary SCLC patient sample. Similar to the cell lines, the
H29 patient sample also expressed high levels of CD47 on its
surface (FIG. 5B). These findings suggested that CD47 is an
immunotherapeutic target on SCLC.
[0112] CD47-blocking Antibodies Induce Phagocytosis of SCLC by
Human Macrophages. To validate CD47 as a genuine therapeutic target
on SCLC, we performed in vitro phagocytosis assays using human
macrophages and SCLC samples. Macrophages were co-cultured with
SCLC cells in the presence of a vehicle control or anti-CD47
antibodies. We tested anti-CD47 antibody clone Hu5F9-G4, a
humanized anti-CD47 antibody that blocks the interaction between
CD47 and SIRP.alpha. and is under investigation in a Phase I
clinical trial for solid tumors (ClinicalTrials.gov identifier:
NCT02216409). High-throughput flow cytometry was used to measure
phagocytosis, which was evaluated by the percentage of macrophages
engulfing calcein AM-labeled SCLC cells (FIGS. 5C and D).
Fluorescence-activated cell sorting was used to confirm the double
positive population contained macrophages with engulfed tumor cells
(FIG. 5E). Four SCLC samples were subjected to evaluation in
phagocytosis assays. Three cell lines (NCI-H524, NCI-1688, and
NCI-H82) exhibited significant increases in phagocytosis when
treated with the CD47-blocking antibody (FIG. 5F). One cell line,
NCI-H196, appeared to be resistant to phagocytosis, suggesting
additional mechanisms modify the susceptibility of this cell line
to macrophage attack. The patient-derived xenograft H29 was also
subjected to phagocytosis assays with human macrophages. Treatment
of this sample with anti-CD47 antibodies also resulted in a
significant increase in phagocytosis (FIG. 5G).
[0113] CD47-blocking Antibodies Inhibit Growth of SCLC Tumors in
vivo. To evaluate the potential of CD47-blocking agents when
administered as therapies for human SCLC, we established xenograft
models of human SCLC. We engrafted NCI-H82 cells into the lower
left flanks of NSG mice, which lack functional T cells, B cells,
and NK cells but retain functional macrophages. Approximately one
week after engraftment, mice were randomized into treatment with
vehicle control or 250 .mu.g anti-CD47 antibody clone Hu5F9-G4
administered every other day. Tumor volume measurements were used
to evaluate mice for a response to therapy. After two weeks of
treatment, a significant difference in median tumor volume was
observed that persisted through the remainder of the experiment
(FIG. 6A). After approximately one month of treatment, the median
tumor volume for the vehicle control cohort was 837.8 mm.sup.3
versus 160.2 mm.sup.3 for the cohort treated with the anti-CD47
antibody (P=0.0281). Therefore, the CD47-blocking antibody was able
to produce a significant inhibition of tumor growth.
[0114] We created a GFP-luciferase+ NCI-H82 cell line to monitor
growth and dissemination in vivo. As an orthotopic model of human
SCLC, we engrafted GFP-luciferase+ NCI-H82 cells into the left
intrathoracic space. Four days after injections, engraftment was
confirmed by bioluminescence imaging. We then randomized mice into
two cohorts treated with either vehicle control or 250 .mu.g
anti-CD47 antibody clone Hu5F9-G4 administered every other day. We
monitored tumor growth over time by bioluminescence imaging. Again,
the CD47-blocking antibody produced a significant inhibition of
tumor growth. Additionally, we observed a significant benefit in
survival for the cohort treated with the CD47-blocking antibody.
Post-mortem analysis revealed tumors formed within the thoracic
cavity or in the parathoracic region. Mice in the vehicle control
group also exhibited substantial metastases to the liver, which
were not observed in the cohort treated with the anti-CD47
antibody.
[0115] Since cell lines typically represent clonal populations of
cells, we next tested the in vivo efficacy of CD47-blocking
antibodies on a patient-derived xenograft, which more closely
models treatment in patients since it maintains the heterogeneity
of cancer cell populations within a tumor. Primary SCLC sample H29
was transduced to express GFP-luciferase to allow for dynamic
measurements of tumor growth in vivo. Tumors were then engrafted
into the lower left flanks of mice and allowed to establish for
approximately 2 weeks. Mice were then randomized into two treatment
cohorts with vehicle control or 250 .mu.g anti-CD47 antibody clone
Hu5F9-G4 administered every other day. We found the anti-CD47
antibody significantly inhibited tumor growth, as assessed by tumor
volume measurements and bioluminescence imaging (FIG. 6B-D).
Treatment with the CD47-blocking therapy also produced significant
benefits in survival. By day 125 post-engraftment, all mice in the
control group had died whereas the majority of mice in the
anti-CD47 antibody group had only small tumors that failed to
progress even after 225 days post-engraftment (FIG. 6E). These
models demonstrate that CD47-blocking therapies could be effective
for patients with SCLC.
[0116] Serum MCP-3 is a Biomarker of Response to CD47-blocking
Therapies. To identify potential biomarkers of a response to
CD47-blocking therapies, we again engrafted mice with NCI-H82
cells. We allowed tumors to grow to approximately 1.5 cm in
diameter and then we treated the mice with a single dose of vehicle
control or anti-CD47 antibody clone Hu5F9-G4. We collected serum
samples immediately before treatment and 24 hours post-treatment.
We subjected the serum samples to multiplex analysis of 38
cytokines. From this analysis, we found that macrophage chemotactic
protein 3 (MCP-3) was systemically increased following treatment
with anti-CD47 antibody clone Hu5F9-G4 (FIG. 7A). No significant
increase in MCP-3 was observed in mice without tumors that were
treated with anti-CD47 antibody clone Hu5F9-G4 (FIG. 7A). We also
performed a similar experiment using the patient-derived xenograft
H29. Again, mice bearing tumors were subjected to a single dose of
anti-CD47 antibody clone Hu5F9-G4. Serum cytokine analysis again
revealed that MCP-3 was significantly increased following treatment
with the CD47-blocking antibody (FIG. 7B). Therefore, MCP-3 may
serve as a biomarker of response to CD47-blocking therapies in
patients. Secretion of MCP-3 may be a positive feedback mechanism
that recruits more macrophages to the tumor and could in part
explain the robust effects of CD47-blocking therapies in vivo.
[0117] Comprehensive Antibody Arrays Identify Therapeutic Targets
on SCLC. Monoclonal antibodies have proven to be some of the most
effective treatments for cancer. However, there are few known
antibody targets on the surface of SCLC. For this reason, we aimed
to characterize the surface antigen profile of SCLC cells using
comprehensive antibody arrays. We subjected four SCLC cell lines
and the primary SCLC sample H29 to analysis using the BioLegend
LEGENDScreen array, a comprehensive collection of 332 antibodies to
human cell surface antigens. ***Discussion of histogram to define
negative, low, and high antigens (FIG. 8A). We identified 39
antigens that were highly expressed on the surface of the SCLC
samples, making them possible targets of therapeutic antibodies.
When we ranked these antigens by their median staining intensity,
we found that CD47 was the most intensely staining surface antigen
(FIG. 8B). Another highly expressed antigen across all samples was
CD56 (NCAM), a known marker of neuroendocrine tumors and a
therapeutic target currently under evaluation for SCLC, thus
validating our approach. A number of other highly expressed surface
antigens were also identified that could potentially be targeted by
monoclonal antibody therapies, including CD24, CD29, and CD99 (FIG.
8B). Interestingly, other immune checkpoint ligands such as CD80,
CD86, PD-L1, or PD-L2 were not appreciably expressed on the surface
of the SCLC samples.
[0118] Combining Antibodies with CD47-blockade Enhances
Phagocytosis of SCLC. To evaluate the therapeutic potential of the
antigens identified by the LEGENDScreen arrays, we next evaluated
their ability to be targeted by antibodies and induce phagocytosis
in vitro. We obtained antibodies to a number of highly expressed
surface antigens, including CD56 (clones HCD56 and MEM-188), CD24,
CD29, and CD99. Additionally, we obtained the sequence for
lorvotuzumab, an anti-CD56 antibody being evaluated in clinical
trials as an antibody-drug conjugate, and we produced it
recombinantly as a naked antibody. We tested these antibodies alone
and in combination with the high-affinity CD47 antagonist CV1,
which blocks CD47 but does not contribute an additional Fc stimulus
(FIGS. 9A and B). We tested the ability of these antibodies to
induce phagocytosis by human macrophages of two different SCLC cell
lines, NCI-H82 (FIG. 9A) and NCI-H524 (FIG. 9B). Of the three
anti-CD56 antibodies tested, we found that lorvotuzumab was able to
produce the greatest increase in phagocytosis, and this effect was
significantly enhanced by combination with CV1. Antibodies to CD24
or CD99 were also able to induce phagocytosis that was comparable
or exceeded that of treatment with anti-CD47 clone Hu5F9-G4. As
expected, phagocytosis with Hu5F9-G4 was entirely blocked when
combined with CV1, since CV1 competes for the same binding surface
and binds with extremely high affinity. Interestingly, the
anti-CD29 antibody was not able to induce phagocytosis even in
combination with CV1, an important demonstration that additional
factors such as surface binding geometry or the ability to engage
Fc receptors may modify the response of macrophages to therapeutic
antibodies.
[0119] Since lorvotuzumab is under evaluation as a therapeutic
agent for SCLC, we investigated its ability to induce phagocytosis
over a varying range of concentrations. Treatment with lorvotuzumab
alone produced a dose-response relationship for inducing macrophage
phagocytosis. Importantly, we found that over each lorvotuzumab
concentration tested, the addition of CV1 produced a greater degree
of phagocytosis (FIG. 9C). These findings demonstrate that CV1
could increase both the maximal efficacy and the potency of
lorvotuzumab, as previously observed when CV1 was combined with
rituximab, trastuzumab, and cetuximab.
[0120] Due to its poor prognosis and dearth of effective treatment
options, there is an imminent need to identify novel treatments for
SCLC. Immunotherapies are emerging as some of the most promising
new therapies for cancer, and here we show that CD47, the
myeloid-specific immune checkpoint, is a genuine immunotherapeutic
target for SCLC. CD47 was highly expressed on the surface of all
SCLC samples tested, and we found blocking CD47 enabled macrophage
phagocytosis of SCLC samples in vitro. Using multiple xenograft
models, the CD47-blocking antibody Hu5F9-G4 was able to inhibit
tumor growth and prolong survival of mice bearing SCLC tumors.
Importantly, we observed anti-tumor efficacy in a patient-derived
xenograft model of SCLC, which maintains the complexity of the
tumor-initiating cell population and thus serves as a more accurate
model for treatment in humans. Additionally we identified MCP-3 as
a serum biomarker that correlates with response to CD47-blocking
therapies. Since the anti-CD47 antibody Hu5F9-G4 is under
investigation in a Phase I clinical trial for human solid
malignancies (ClinicalTrials.gov identifier: NCT02216409), our
findings provide scientific justification for further evaluation of
anti-CD47 antibodies in subsets of patients with SCLC.
[0121] Furthermore, using comprehensive antibody arrays, we
identified several antigens on the surface of SCLC samples that
could be targeted with monoclonal antibodies therapies. Using the
high-affinity SIRP.alpha. variant CV1, a next-generation CD47
antagonist, we found that CD47-blockade augmented the efficacy of
anti-tumor antibodies for SCLC, as has been demonstrated for other
cancers. The combination of high-affinity SIRP.alpha. variants with
independent tumor-binding antibodies provided an optimal strategy
for targeting CD47 in SCLC. Blockade of CD47 on the surface of SCLC
was not sufficient to induce macrophage phagocytosis, but instead
it augmented macrophage phagocytosis when SCLC-binding antibodies
are present. Antibodies to CD56, CD24, and CD99 proved to be
effective at inducing phagocytosis of SCLC, particularly when
combined with CV1.
[0122] Additionally, we found that CD47-blockade was able to
enhance the efficacy of lorvotuzumab, an antibody proceeding
through clinical trials for SCLC as an antibody-drug conjugate
(ADC) with the cytotoxic agent mertansine. Combining therapeutic
antibodies with CD47-blocking therapies represents an alternative
method to enhance the efficacy of therapeutic antibodies. One
benefit of CV1 over ADCs is that it can be combined with any
antibody without further engineering. ADCs often rely on
internalization to deliver their cytotoxic payload, and this
dependency can limit efficacy and increase side effects. Since CD47
blockade stimulates macrophages to identify cells for removal,
there may be an added layer of specificity conferred by cell-cell
interactions than that achieved by ADCs. Nonetheless, it is likely
that even lorvotuzumab-mertansine could benefit from combination
with CV1 if the ability to engage Fc receptors is preserved.
[0123] Our approach to identifying novel SCLC surface antigens can
be applied to other types of cancer, and in the future could be
used to assemble oligoclonal cocktails of antibodies that could be
used to simulate the natural humoral immune response against
foreign pathogens or cells. These cocktails could be combined with
CD47-blocking therapies and other immunotherapies to mount an
effective immune response against SCLC cells. These studies show
that SCLC is responsive to CD47-blocking therapies.
Materials and Methods
[0124] Cell lines and culture: NCI-H82, NCI-524, NCI-H69, and
NCI-1688 were obtained from ATCC. Cells were cultured in RPMI-1640
supplemented with 10% fetal bovine serum (Hyclone), 1.times.
Glutamax (Invitrogen), and 100 U/mL penicillin and 100 ug/mL
streptomycin (Invitrogen). Cell lines were grown in suspension
(NCI-H82, NCI-524, NCI-H69) and dissociated by gentle pipetting or
brief incubation with 1.times. TrypLE (Invitrogen). NCI-1688 cells
were grown in adherent monolayers and or removed by brief
incubation with 1.times.TrypLE. Cell lines were cultured in
humidified incubators at 37.degree. C. with 5% carbon dioxide.
[0125] Human macrophage differentiation: Leukocyte reduction system
chambers were obtained from anonymous blood donors at the Stanford
Blood Center. Monocytes were purified on an AutoMACS (Miltenyi)
using CD14+ microbeads or CD14+ whole blood microbeads (Miltenyi)
according to the manufacturer's instructions. Purified CD14+
monocytes were plated on 15 cm tissue culture dishes at a density
of 10 million monocytes per plate. Monocytes were differentiated to
macrophages by culture in IMDM supplemented with 10% Human AB serum
(Invitrogen), 1.times. GlutaMax (Invitrogen), and 100 U/mL
penicillin and 100 ug/mL streptomycin for approximately 7-10
days.
[0126] In vitro phagocytosis assays: In vitro phagocytosis assays
were performed as previously described. Briefly, SCLC cancer cells
were removed from plates and washed with serum-free IMDM.
GFP-luciferase+ cells or cells labeled with calcein AM (Invitrogen)
were used as target cells. Macrophages were washed twice with HBSS,
then incubated with 1.times.TrypLE for approximately 20 minutes in
humidified incubators at 37.degree. C. Macrophages were removed
from plates using cell lifters (Corning), then washed twice with
serum-free IMDM. Phagocytosis reactions were carried out using
50,000 macrophages and 100,000 tumor cells. Cells were co-cultured
for two hours at 37.degree. C. in the presence of antibody
therapies. After co-culture, cells were washed with autoMACS
Running Buffer (Miltenyi) and prepared for analysis by flow
cytometry. Macrophages were stained using fluorophore-conjugated
antibodies to CD45 (BioLegend) in the presence of 100 .mu.g/mL
mouse IgG (Lampire). Dead cells were excluded from the analysis by
staining with DAPI (Sigma). Samples were analyzed by flow cytometry
using a LSRFortessa (BD Biosciences) equipped with a
high-throughput sampler. Phagocytosis was evaluated as the
percentage of calcein-AM.sup.+ macrophages using FlowJo v9.4.10
(Tree Star) and was normalized to the maximal response by each
independent donor where indicated. Statistical significance was
determined and data were fit to sigmoidal dose-response curves
using Prism 5 (Graphpad).
[0127] Additional reagents used in phagocytosis include the
high-affinity SIRP.alpha. variant CV1 monomer, which was produced
as previously described and used at a concentration of 1 .mu.M for
blocking. Antibodies to identified SCLC antigens were used in
phagocytosis assays at a concentration of 10 .mu.g/mL, including
anti-CD56 (NCAM) clone HCD56 (BioLegend), anti-CD56 (NCAM) clone
MEM-188 (BioLegend), anti-CD24 clone ML5 (Biolegend), anti-CD29
clone TS2/16 (BioLegend), anti-CD99 clone 12E7 (Abcam).
Additionally, lorvotuzumab was made recombinantly using the heavy
and light chain variable region sequences available in the KEGG
database (Drug: D09927). Lorvotuzumab variable regions were cloned
into pFUSE-CHIg-hG1 and pFUSE2-CLIg-hK (Invivogen) for expression.
Lorvotuzumab was produced recombinantly by transient transfection
of 293F cells (Invitrogen) using 293fectin (Invitrogen), followed
by purification over a HiTrap Protein A column (GE Healthcare).
Purified antibody was eluted with 100 mM citrate buffer (pH 3.0)
and neutralized with 1/10th volume of Tris buffer (pH 8.0).
Antibody was desalted using a PD-10 column (GE Healthcare).
[0128] Sorting of macrophage populations after phagocytosis: 2.5
million human macrophages were combined with 5 million GFP+ NCI-H82
cells and 10 .mu.g/mL anti-CD47 antibody (clone Hu5F9-G4) in
serum-free medium and incubated for two hours. Macrophages were
identified by staining with anti-CD45, and macrophages populations
were sorted on a FACSAria II cell sorter (BD Biosciences). Cells
from sorted populations were centrifuged onto microscope slides
then stained with Modified Wright-Giemsa stain (Sigma-Aldrich)
according to the manufacturer's instructions and imaged on a DM5500
B upright light microscope (Leica).
[0129] Mice: Nod.Cg-Prkdc.sup.scid IL2rg.sup.tmWj1/SzJ (NSG) mice
were used for all in vivo experiments. Mice were engrafted with
tumors at approximately 6-10 weeks of age, and experiments were
performed with age and sex-matched cohorts. Mice were maintained in
a barrier facility under the care of the Stanford Veterinary
Services Center and handled according to protocols approved by the
Stanford University Administrative Panel on Laboratory Animal
Care.
[0130] In vivo SCLC treatment models: 1.25.times.10.sup.6 NCI-H82
cells were subcutaneously engrafted into the flanks of NSG mice.
Tumors were allowed to grow for 8 days, then mice were randomized
into treatment groups with PBS or 250 .mu.g anti-CD47 antibody
(clone Hu5F9-G4). Treatment was administered every other day by
intraperitoneal injection. Tumor growth was monitored by tumor
dimension measurements that were used to calculate tumor volumes
according to the ellipsoid formula
(.pi./6.times.length.times.width.sup.2). For a patient-derived
xenograft model of SCLC, 3.times.10.sup.6 GFP-luciferase.sup.+ H29
cells were subcutaneously engrafted with 25% Matrigel (BD
Biosciences) into the flanks of NSG mice. Tumors were allowed to
grow for 15 days, then mice were randomized into treatment with
into treatment groups with PBS or 250 .mu.g anti-CD47 antibody
(clone Hu5F9-G4). Treatment was administered every other day by
intraperitoneal injection. Tumor growth was monitored by
bioluminescence imaging and tumor volume measurements as described
above. Statistical significance of tumor growth was determined by
Mann-Whitney test. Survival was analyzed by Mantel-Cox test. Pilot
in vivo experiments with H82 cells and H29 cells were performed
with smaller cohorts of mice with similar results.
[0131] GFP-fluorescence from tumor nodules was visualized on an
M205 FA fluorescent dissecting microscope (Leica) fitted with a DFC
500 camera (Leica).
[0132] Bioluminescence imaging: Mice bearing GFP-luciferase+ tumors
were imaged as previously described. Briefly, anesthetized mice
were injected with 200 .mu.L D-luciferin (firefly) potassium salt
(Biosynth) reconstituted at 16.67 mg/mL in sterile PBS.
Bioluminescence imaging was performed using an IVIS Spectrum
(Caliper Life Sciences) over 20 minutes to record maximal radiance.
Peak total flux values were assessed from the anatomical region of
interest using Living Image 4.0 (Caliper Life Sciences) and were
used for analysis.
[0133] Comprehensive FACS-based antibody screening: Antigens on the
surface of SCLC samples were analyzed using LEGENDScreen Human Cell
Screening Kits (BioLegend), according to the manufacturer's
protocol with the following modifications. Briefly, lyophilized
antibodies were reconstituted in molecular biology grade water and
added to cell samples at a 1:8 dilution. Approximately
20-40.times.10.sup.6 total cells were used for the analysis per
SCLC sample. NCI-H82 was labeled with calcein-AM and analyzed
simultaneously with NCI-H524. NCI-H69 was labeled with calcein-AM
and analyzed simultaneously with NCI-H1688. The primary patient
sample H69 was analyzed independently. It was freshly dissociated
from a low-passage xenograft and mouse lineage cells were excluded
from the analysis by staining with Pacific Blue anti-mouse
H-2k.sup.d (BioLegend). Samples were incubated with antibodies for
30 minutes on ice protected from light. For all samples, dead cells
were excluded from the analysis by staining with DAPI. [0134]
Jaiswal S, Jamieson C H, Pang W W, Park C Y, Chao M P, Majeti R, et
al. CD47 is upregulated on circulating hematopoietic stem cells and
leukemia cells to avoid phagocytosis. Cell. 2009; 138:271-85.
[0135] Majeti R, Chao M P, Alizadeh A A, Pang W W, Jaiswal S, Gibbs
K D, Jr., et al. CD47 is an adverse prognostic factor and
therapeutic antibody target on human acute myeloid leukemia stem
cells. Cell. 2009; 138:286-99. [0136] Willingham S B, Volkmer J P,
Gentles A J, Sahoo D, Dalerba P, Mitra S S, et al. The CD47-signal
regulatory protein alpha (SIRP.alpha.) interaction is a therapeutic
target for human solid tumors. Proceedings of the National Academy
of Sciences of the United States of America. 2012; 109:6662-7.
[0137] Weiskopf K, Ring A M, Ho C C, Volkmer J P, Levin A M,
Volkmer A K, et al. Engineered SIRPalpha Variants as
Immunotherapeutic Adjuvants to Anticancer Antibodies. Science.
2013. Chao M P, Alizadeh A A, Tang C, Myklebust J H, Varghese B,
Gill S, et al. Anti-CD47 antibody synergizes with rituximab to
promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell.
142:699-713. [0138] Maloney D G, Grillo-Lopez A J, White C A,
Bodkin D, Schilder R J, Neidhart J A, et al. IDEC-C2B8 (Rituximab)
anti-CD20 monoclonal antibody therapy in patients with relapsed
low-grade non-Hodgkin's lymphoma. Blood. 1997; 90:2188-95. [0139]
Vogel C L, Cobleigh M A, Tripathy D, Gutheil J C, Harris L N,
Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a
single agent in first-line treatment of HER2-overexpressing
metastatic breast cancer. J Clin Oncol. 2002; 20:719-26. [0140] Van
Cutsem E, Kohne C H, Hitre E, Zaluski J, Chang Chien C R, Makhson
A, et al. Cetuximab and chemotherapy as initial treatment for
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[0141] Willingham S B, Volkmer J P, Gentles A J, Sahoo D, Dalerba
P, Mitra S S, et al. The CD47-signal regulatory protein alpha
(SIRPa) interaction is a therapeutic target for human solid tumors.
Proc Natl Acad Sci USA. 109:6662-7. [0142] Shultz L D, Lyons B L,
Burzenski L M, Gott B, Chen X, Chaleff S, et al. Human lymphoid and
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Sequence CWU 1
1
615PRTMus musculus 1Asn Tyr Asn Met His1 5217PRTMus musculus 2Thr
Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10
15Asp38PRTMus musculus 3Gly Gly Tyr Arg Ala Met Asp Tyr1 5416PRTMus
musculus 4Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Asn Thr Tyr
Leu Gly1 5 10 1557PRTMus musculus 5Lys Val Ser Asn Arg Phe Ser1
569PRTMus musculus 6Phe Gln Gly Ser His Val Pro Tyr Thr1 5
* * * * *