U.S. patent application number 15/734961 was filed with the patent office on 2021-07-29 for methods of use of cd24 for the prevention and treatment of graft versus host disease and mucositis.
This patent application is currently assigned to ONCOIMMUNE, INC. The applicant listed for this patent is ONCOIMMUNE, INC.. Invention is credited to Martin Devenport, Yang Liu, Pan Zheng.
Application Number | 20210228680 15/734961 |
Document ID | / |
Family ID | 1000005549964 |
Filed Date | 2021-07-29 |
United States Patent
Application |
20210228680 |
Kind Code |
A1 |
Liu; Yang ; et al. |
July 29, 2021 |
METHODS OF USE OF CD24 FOR THE PREVENTION AND TREATMENT OF GRAFT
VERSUS HOST DISEASE AND MUCOSITIS
Abstract
The present invention relates to the use of a CD24 protein for
preventing or treating Graft versus Host Disease and mucositis.
Inventors: |
Liu; Yang; (Baltimore,
MD) ; Zheng; Pan; (Baltimore, MD) ; Devenport;
Martin; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONCOIMMUNE, INC. |
Rockville |
MD |
US |
|
|
Assignee: |
ONCOIMMUNE, INC
Rockville
MD
|
Family ID: |
1000005549964 |
Appl. No.: |
15/734961 |
Filed: |
June 3, 2019 |
PCT Filed: |
June 3, 2019 |
PCT NO: |
PCT/US2019/035200 |
371 Date: |
December 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62739742 |
Oct 1, 2018 |
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62739719 |
Oct 1, 2018 |
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62680218 |
Jun 4, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
37/06 20180101; A61K 38/177 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 37/06 20060101 A61P037/06; A61P 1/04 20060101
A61P001/04 |
Claims
1. (canceled)
2. (canceled)
3. A method of treating or preventing mucositis in a subject in
need thereof, comprising administering a CD24 protein to the
subject.
4. The method of claim 3, wherein the subject has a cancer.
5. The method of claim 4, wherein the cancer is Acute Myeloid
Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic
Myelogenous Leukemia (CML), Myelodysplastic syndrome (MDS), or
Chronic Myelomonocytic Leukemia (CMML).
6. The method of claim 3 wherein the CD24 protein is administered
at a dose of 240 mg or 480 mg.
7. The method of claim 3, wherein the subject will undergo or has
undergone a hematopoietic stem cell transplantation (HCT).
8. The method of claim 7, wherein the CD24 protein is administered
before or after the HCT.
9. The method of claim 8, wherein the CD24 protein is administered
one day before the HCT.
10. The method of claim 8, wherein the CD24 protein is administered
more than once.
11. The method of claim 10, wherein the CD24 protein is
administered in three biweekly doses, comprising a dose on the day
before the HCT, a dose on day 14 after the HCT, and a dose on day
28 after the HCT
12. The method of claim 11, wherein the doses of the CD24 protein
are 480 mg, 240 mg, and 240 mg, respectively.
13. The method of claim 3, wherein the CD24 protein comprises a
mature human CD24 polypeptide fused at its N-terminus or C-terminus
to a Fc region of a mammalian immunoglobulin (Ig) protein.
14. The method of claim 13, wherein the mature human CD24
polypeptide comprises the sequence set forth in SEQ ID NO: 1 or
2.
15. The method of claim 14, wherein the Ig protein is human, and
wherein the Fc region comprises a hinge region and CH2 and CH3
domains of IgG1, IgG2, IgG3, IgG4, or IgA.
16. The method of claim 14, wherein the Ig protein is human, and
wherein the Fc region comprises a hinge region and CH2, CH3 and CH4
domains of IgM.
17. The method of claim 15, wherein the CD24 protein comprises the
sequence set forth in SEQ ID NO: 6, 11, or 12.
18. The method of claim 17, wherein the amino acid sequence of the
CD24 protein consists of the sequence set forth in SEQ ID NO: 6,
11, or 12.
19. The method of claim 3, wherein the CD24 protein is soluble
and/or glycosylated.
20. (canceled)
21. The method of claim 3, wherein the CD24 protein is prepared
using a eukaryotic expression system.
22. The method of claim 21, wherein the eukaryotic expression
system comprises expression from a vector in mammalian cells.
23. The method of claim 22, wherein the mammalian cells are Chinese
Hamster Ovary cells.
24.-46. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for preventing and treating Graft versus Host Disease and
mucositis.
BACKGROUND OF THE INVENTION
[0002] Allogeneic hematopoietic stem cell transplantation (HCT) is
the only established curative therapy for a broad spectrum of high
risk leukemia and myelodysplasia in adults. An important function
of allogeneic transplantation is to use donor T cells to eliminate
allogeneic leukemia cells, this effect is called GVL. However,
Graft vs host disease (GVHD) is a life threatening complication
that occurs when the immune competent cells from the donor stem
cell graft mount an immune attack against the host. Activated donor
T cells damage host epithelial cells following an inflammatory
cascade that begins with the preparative regimen. The exact risk is
dependent on the stem cell source, age of the patient,
conditioning, and GVHD prophylaxis used. The incidence is directly
related to the degree of human leukocyte antigens (HLA) disparity.
The median onset of acute GVHD is typically 21 to 25 days after
transplantation. The incidence ranges from 30-65% in recipients of
fully histocompatible related donor transplants to 60% to 80% in
recipients of mismatched hematopoietic cells or hematopoietic cells
from an unrelated donor. Umbilical cord-blood transplantation has
been associated with slower neutrophil recovery with lower
incidence and later onset of acute GVHD. Factors that increase the
incidence include use of peripheral blood rather than bone marrow
as the source of hematopoietic cells and older recipient age. The
median time of diagnosis of chronic GVHD is 4.5 months after
HLA-identical sibling transplantation and 4 months after unrelated
donor transplantation. De novo chronic GVHD almost never occurs
after 2 years following allogeneic HCT.
[0003] For over 20 years, the combination of a calcineurin
inhibitor (e.g. cyclosporine and tacrolimus) with methotrexate has
remained the standard of care for the prevention of GVHD. Despite
routine administration of immune prophylaxis, clinically
significant GVHD (Grade II-IV) occurs in approximately 30 to 65% of
patients undergoing HLA matched related HCT and 60 to 80% of
patients receiving unrelated donor HCT. Acute GVHD is an early
event after HCT, with a median time to onset of approximately 25 to
30 days. In patients with very severe GVHD, mortality rates exceed
90%. One explanation for this is that, once established,
ineffective responses occur to front-line therapy with high dose
corticosteroids in greater than 50% of patients. Survival is
significantly diminished for patients who demonstrate steroid
refractoriness or who require prolonged treatment. Even when
successful, high doses of corticosteroids are a major source of
morbidity due to increased infections and deconditioning that
places patients at significant risk for TRM.
[0004] Host tissue injuries caused by the HCT conditioning
regimens, including high-dose chemotherapy and/or total body
irradiation (TBI), are considered to be the first step in the
development of acute GVHD. Host tissue injuries caused by the
conditioning regimen lead to the release of proinflammatory
cytokines (such as TNF-.alpha., IL-1.beta. and IL-6), and also the
release of damage-associated molecular patterns (DAMPs) and
pathogen-associated molecular patterns (PAMPs). Both DAMPs and
PAMPs can activate antigen-presenting cells (APCs), such as
dendritic cells (DCs), by binding to pattern recognition receptors
(PRRs). The host APCs subsequently activate donor T cells and an
immunologic cascade that results in the release of pro-inflammatory
cytokines and expansion of the antigen specific allo-reactive T
cells that target host tissues, resulting in GVHD. It is therefore
of great interest to explore whether GVHD can be attenuated by
targeting host response to tissue injuries and preventing
activation of APCs, the key processes in the initiation of
GVHD.
[0005] To date, treatment and prevention of GVHD has predominantly
focused on either pharmacologic inhibition or depletion of T cells
through in vivo or ex vivo approaches to limit expansion of
alloreactive T cells that mediate tissue injury. While
non-selective T-cell depleting strategies (e.g. antithymocyte
globulin) are efficacious in preventing GVHD, they do not improve
survival due to offsetting risks for relapse, infection and graft
rejection. Conversely, more selective inhibition by targeting
single pro-inflammatory cytokines has not demonstrated clinical
benefit in treating GVHD. As a result, apart from antibodies that
deplete T cells, no biologics have been approved for GVHD and the
combination of tacrolimus with methotrexate has remained the
standard of care for the prevention of GVHD. The significant unmet
medical needs call for more selective biological products for both
prophylaxis and treatment of GVHD.
[0006] Mucositis is a common and painful side effect of
chemotherapy and radiotherapy treatment for cancer that results in
inflammation and ulceration of the mucous membranes lining the
digestive tract. It is a result of tissue injury caused by the
radiation/radiotherapy (RT) or chemotherapy. Mucositis can occur
anywhere along the gastrointestinal (GI) tract, but oral mucositis
refers to the particular inflammation and ulceration that occurs in
the mouth. Oral and gastrointestinal (GI) mucositis affects almost
all patients undergoing high-dose chemotherapy with cytarabine and
high-dose 5-fluorouracil, alkylating agents and platinum based
compounds and the majority of patients with malignancies of the
head and neck receiving radiotherapy. Radiation induced oral
mucositis (RIOM) occurs in 100% of altered fractionation
radiotherapy head and neck cancer patients. Patients suffering from
oral mucositis experience severe pain, inflammation, ulceration,
and bleeding that can significantly impede the patient's ability to
swallow, which can also limit tumor control due to interruption of
cancer treatment. Accordingly, oral mucositis is an important
adverse effect seen in cancer patients on chemotherapy and/or
radiation therapy for the head and neck. Alimentary tract mucositis
increases mortality and morbidity and contributes to rising health
care costs. In the United Sates, the economic cost of RIOM was
estimated to reach 17,000.00 USD per patient with head and neck
cancers.
[0007] The most profound mucositis burden is experienced as a
result of the conditioning regimens used for hematopoietic stem
cell transplant (HCT). The treatment regimen for HCT includes a
pre-conditioning regimen, including highly mucotoxic chemotherapy
with or without total body irradiation (TBI), which is required to
kill off the recipient's cancerous hematopoietic cells prior to
transplant. This preconditioning treatment leads to serious damage
throughout the alimentary tract. Low grade mucositis includes
erythema of the mucosa and patchy ulcerations or pseudomembranes.
Severe mucositis (grade.gtoreq.3) is associated with confluent
ulcerations or pseudomembranes and bleeding with minor trauma,
which can progress to tissue necrosis, significant spontaneous
bleeding and life-threatening consequences. Another indication
related to mucositis in patients receiving HCT is oral
graft-versus-host disease (GVHD), which is a form of chronic GVHD.
As with chemotherapy and radiation induced mucositis, oral GVHD
includes mucosal erythema, ulcerations, and painful desquamative
oral lesions. However, a true clinical case definition of oral
acute GVHD is lacking, as several factors, particularly the
conditioning chemotherapy with or without concurrent radiation,
contribute to oral lesion development during the first 28 days
following HCT.
[0008] As indicated, GVHD, mucositis and related indications all
involve an element of tissue damage. To combat these challenges,
there is a need in the art for methods of effectively treating and
preventing GVHD and mucositis.
SUMMARY OF THE INVENTION
[0009] Provided herein is a method of preventing or treating Graft
versus Host Disease (GvHD) or mucositis in a subject in need
thereof, which may comprise administering a CD24 protein to the
subject. Also provided herein is use of a CD24 protein in the
manufacture of a medicament for preventing or treating GvHD or
mucositis in a subject. The method or use may reduce the subject's
risk of Grade III-IV acute GvHD. The subject may be human. The
subject may undergo or may have undergone a hematopoietic stem cell
transplantation (HCT). The subject may have cancer, which may be
Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL),
Chronic Myelogenous Leukemia (CML), Myelodysplastic syndrome (MDS),
or Chronic Myelomonocytic Leukemia (CMML).
[0010] The CD24 protein may be administered at a dose of 240 mg or
480 mg. The CD24 protein may be administered before or after the
HCT, and may be administered one day before the HCT. The CD24
protein may be administered more than once, and may be administered
in biweekly doses. The doses may comprise a dose on the day before
the HCT, a dose on day 14 after the HTC, and a dose on day 28 after
the HCT, and the doses may be, respectively, 480 mg, 240 mg, and
240 mg.
[0011] The CD24 protein may comprise a mature human CD24
polypeptide fused at its N-terminus or C-terminus to a Fc region of
a mammalian immunoglobulin (Ig) protein. The mature human CD24
polypeptide may comprise the sequence set forth in SEQ ID NO: 1 or
2. The Ig protein may be human. The Fc region may comprise a hinge
region and CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, or IgA.
The Fc region may comprise a hinge region and CH2, CH3 and CH4
domains of IgM. The CD24 protein may comprise the sequence set
forth in SEQ ID NO: 6, 11, or 12. The amino acid sequence of the
CD24 protein may consist of the sequence set forth in SEQ ID NO: 6,
11, or 12. The CD24 protein may be soluble, and may be
glycosylated. The CD24 protein may be prepared using a eukaryotic
expression system, which may comprise expression from a vector in
mammalian cells. The cells may be Chinese Hamster Ovary cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A shows the amino acid composition of the full length
CD24 fusion protein, CD24Fc (also referred to herein as CD24Ig)
(SEQ ID NO: 5). The underlined 26 amino acids are the signal
peptide of CD24 (SEQ ID NO: 4), which are cleaved off during
secretion from a cell expressing the protein and thus missing from
the processed version of the protein (SEQ ID NO: 6). The bold
portion of the sequence is the extracellular domain of the mature
CD24 protein used in the fusion protein (SEQ ID NO: 2). The last
amino acid (A or V) that is ordinarily present in the mature CD24
protein has been deleted from the construct to avoid
immunogenicity. The non-underlined, non-bold letters are the
sequence of IgG1 Fc, including the hinge region and CH1 and CH2
domains (SEQ ID NO: 7). FIG. 1B shows the sequence of CD24.sup.vFc
(SEQ ID NO: 8), in which the mature human CD24 protein (bold) is
the valine polymorphic variant of SEQ ID NO: 1. FIG. 1C shows the
sequence of CD24.sup.AFc (SEQ ID NO: 9), in which the mature human
CD24 protein (bold) is the alanine polymorphic variant of SEQ ID
NO: 1. The various parts of the fusion protein in FIGS. 1B and 1C
are marked as in FIG. 1A and the variant valine/alanine amino acid
is double underlined.
[0013] FIG. 2 shows amino acid sequence variations between mature
CD24 proteins from mouse (SEQ ID NO: 3) and human (SEQ ID NO: 2).
The potential O-glycosylation sites are bolded, and the
N-glycosylation sites are underlined.
[0014] FIGS. 3A-C. WinNonlin compartmental modeling analysis of
pharmacokenitics of CD24IgG1 (CD24Fc). The opened circles represent
the average of 3 mice, and the line is the predicted
pharmacokinetic curve. FIG. 3A. i.v. injection of 1 mg CD24IgG1.
FIG. 3B. s.c. injection of 1 mg CD24IgG1 (CD24Fc). FIG. 3C.
Comparison of the total amounts of antibody in the blood as
measured by areas under curve (AUC), half-life and maximal blood
concentration. Note that overall, the AUC and Cmax of the s.c.
injection is about 80% of i.v. injection, although the difference
is not statistically significant.
[0015] FIGS. 4A-B. CD24-Siglec G (10) interaction discriminates
between PAMP and DAMP. FIG. 4A. Host response to PAMP was
unaffected by CD24-Siglec G (10) interaction. FIG. 4B. CD24-Siglec
G (10) interaction represses host response to DAMP, possibly
through the Siglec G/10-associated SHP-1.
[0016] FIGS. 5A-C. CD24 Fc binds to Siglec 10 and HMGB1 and
activates Siglec G, the mouse homologue of human Siglec 10. FIG.
5A. Affinity measurement of the CD24Fc-Siglec 10 interaction. FIG.
5B. CD24Fc specifically interacts with HMGB-1 in a cation-dependent
manner CD24Fc was incubated with HMGB1 in 0.1 mM of CaCl.sub.2 and
MgCl.sub.2, in the presence or absence of the cation chelator EDTA.
CD24Fc is pulled down with protein G-beads, and the amounts of
HMGB1, CD24Fc or control Fc is determined by Western blot. FIG. 5C.
CD24Fc activates mouse Siglec G by inducing Tyrosine
phosphorylation (middle panel) and association with SHP-1 (upper
panel). The amounts of Siglec G are shown in the lower panel.
CD24.sup.-/- spleen cells were stimulated with 1 .mu.g/ml of
CD24Fc, control Fc or vehicle (PBS) control for 30 minutes. Siglec
G was then immunoprecipitated and probed with anti-phospho-tyrosine
or anti-SHP-1.
[0017] FIGS. 6A-B. CD24Fc inhibits production of TNF-.alpha. and
IFN-.gamma. by anti-CD3 activated human T cells. The human PBML
were stimulated with anti-CD3 for 4 days in the presence or absence
of CD24Fc and the amounts of IFN-.gamma. and TNF-.alpha. released
in the supernatant of cell culture were measured by ELISA. Data
shown are means of triplicate. Error bar, SEM.
[0018] FIGS. 7A-B. CD24 inhibits inflammatory cytokine production
by human macrophages. FIG. 7A. ShRNA silencing of CD24 leads to
spontaneous production of TNF-.alpha., IL-1.beta., and IL-6. THP1
cells were transduced with lentiviral vectors encoding either
scrambled or two independent CD24 shRNA molecules. The transduced
cells were differentiated into macrophages by culturing for 4 days
with PMA (15 ng/ml). After washing away PMA and non-adherent cells,
the cells were cultured for another 24 hours for measurement of
inflammatory cytokines, by cytokine beads array. FIG. 7B. As in
FIG. 7A, except that the given concentration of CD24Fc or control
IgG Fc was added to macrophages in the last 24 hours. Data shown in
FIG. 4A are means and S.D. from three independent experiments,
while those in FIG. 4B are representative of at least 3 independent
experiments.
[0019] FIG. 8 shows a plot of mean plasma CD24Fc concentration
(.+-.SD) by treatment for a PK Evaluable Population in human
subjects. PK=pharmacokinetic; SD=standard deviation.
[0020] FIG. 9 shows a dose proportionality plot of CD24Fc C.sub.max
versus dose for a PK Evaluable Population.
[0021] FIG. 10 shows a dose proportionality plot of CD24Fc
AUC.sub.0-42 d versus dose for a PK Evaluable Population.
[0022] FIG. 11 shows a dose proportionality plot of CD24Fc
AUC.sub.0-inf versus dose for a PK Evaluable Population.
[0023] FIG. 12 shows the trial design for the randomized,
placebo-controlled Phase IIa dose escalation trial was performed to
evaluate the addition of CD24Fc to standard of care acute GVHD
prophylaxis in cancer patients undergoing allogeneic myeloablative
hematopoietic stem cell transplantation (HCT).
[0024] FIG. 13 shows the dosing scheme for the single dose and
multi-dose cohorts in the Phase IIa trial.
[0025] FIG. 14 shows the median time to engraftment for patients
enrolled in the trial.
[0026] FIG. 15 shows the myeloid donor chimerism for patients
enrolled in the trial.
[0027] FIG. 16 shows the incidence of Grade II-IV and Grade III-IV
acute GVHD in the treatment (CD24Fc) cohort.
[0028] FIG. 17 shows the cumulative incidence of grade III-IV aGVHD
180 days post HCT in patients receiving
methotrexate/tacrolimus+CD24Fc as compared to contemporary control
patients receiving methotrexate/tacrolimus.
[0029] FIG. 18 shows the Kaplan-Meier survival analysis comparing
180 days Grade III-IV aGVHD, relapse-free survival in patients
receiving either CD24Fc or placebo control.
[0030] FIG. 19 shows the Kaplan-Meier survival analysis comparing
180 days Grade III-IV aGVHD, relapse-free survival in patients
receiving CD24Fc with contemporary control.
[0031] FIG. 20 shows the Kaplan-Meier survival analysis comparing
overall survival of patients receiving either CD24Fc or placebo
control at 800 days post transplant.
[0032] FIG. 21 shows the Kaplan-Meier survival analysis comparing
overall survival of patients receiving CD24Fc or contemporary
control at 800 days post transplant.
[0033] FIGS. 22A-B show the PK data from the 240 and 480 mg single
dose cohorts. FIG. 22A Plot of Mean (.+-.Standard Deviation) Plasma
CD24Fc Concentration (ng/mL) Versus Time on a Linear Scale. FIG.
22B. Plot of Mean (.+-.Standard Deviation) Plasma CD24Fc
Concentration (ng/mL) Versus Time on a Semi-Logarithmic Scale.
[0034] FIGS. 23A-B show the PK data from the multi-dose cohort.
FIG. 23A Plot of Mean (.+-.Standard Deviation) Plasma CD24Fc
Concentration (ng/mL) Versus Time on a Linear Scale. FIG. 23B. Plot
of Mean (.+-.Standard Deviation) Plasma CD24Fc Concentration
(ng/mL) Versus Time on a Semi-Logarithmic Scale.
[0035] FIGS. 24A-B show the effect of CD24Fc treatment on oral
mucositis. FIG. 24A. Combined mucositis scores. As a measure of the
effect on mucositis, we generated a multiple of the number of days
patients spent with serious (Grade 3 or 4) mucositis and this is
provided in the bars, along with the number of pts that had the
mucositis in parentheses. FIG. 24B. Dose-dependent reduction of
mucositis score by CD24Fc. Means and standard errors of individual
mucositis score are shown. Correlation coefficient and P-value
between drug dose and mucositis score of patients were determined
by using Pearson method. R=-0.9983, P=0.0009.
[0036] FIGS. 25A-B show the 180 Day Grade III-IV GVHD Free Survival
in the CD24Fc group compared to the placebo control group (FIG.
25A) and the contemporary control group (FIG. 25B).
[0037] FIGS. 26A-B show the Relapse Free Survival in the CD24Fc
group compared to the placebo control group (FIG. 26A) and the
contemporary control group (FIG. 26B).
DETAILED DESCRIPTION
[0038] Tissue damage can lead to the release of proinflammatory
cytokines (such as TNF-.alpha., IL-1.beta. and IL-6), and also the
release of damage-associated molecular patterns (DAMPs) and
pathogen-associated molecular patterns (PAMPs). Both DAMPs and
PAMPs can activate antigen-presenting cells (APCs), such as
dendritic cells (DCs), by binding to pattern recognition receptors
(PRRs). The host APCs subsequently activate donor T cells and an
immunologic cascade that results in the release of pro-inflammatory
cytokines and expansion of the antigen specific allo-reactive T
cells that target host tissues. It is these events that lead to the
development of GVHD and exacerbate the effects of mucositis. For
example, RIOM starts as an acute inflammation of oral mucosa,
tongue and pharynx following radiotherapy, which coincides with
recruitment of various inflammatory cells and release of
inflammatory cytokines, chemotactic mediators, and growth
factors.
[0039] The involvement of tissue damage in mucositis and GVHD
raised the prospect that negatively regulating host response to
DAMPs by CD24Fc can be explored for GVHD therapy. The inventors'
preclinical studies have demonstrated that CD24Fc specifically
targets DAMP-mediated inflammation and prevents GVHD in mouse
models, including a humanized mouse model. Importantly, the drug
has advantages over conventional immunosuppressant as it does not
cause general immune suppression and use of high doses of CD24Fc
does not block antibody response in non-human primates. The data
also demonstrate that CD24Fc prevents GVHD but preserves the graft
versus leukemia (GVL) effect, making it an ideal drug for
prophylaxis of GVHD in leukemia patients. Finally, the inventors'
studies in non-human primate demonstrate that CD24Fc does not
suppress antigen-specific immune responses, which suggest that
CD24Fc will not likely increase risk of infection.
[0040] The inventors have discovered that a soluble form of CD24 is
highly effective for preventing Graft versus Host Disease (GVHD)
and associated conditions such as mucositis, as well as for
preventing leukemia relapse following HCT. The inventors have also
discovered that CD24Fc produced a dose-dependent reduction in
severe mucositis (grade.gtoreq.3) among patients receiving HCT
therapy. These effects may be mediated through DAMPs. Pattern
recognition is involved in inflammatory response triggered by both
PAMPs and DAMPs. The inventors have realized that recent studies
have demonstrated that an exacerbated host response to DAMPs may
play a part in the pathogenesis of inflammatory and autoimmune
disease. DAMPs were found to promote the production of inflammatory
cytokines and autoimmune diseases and in animal models, and
inhibitors of DAMPs such as HMGB1 and HSP90 were consequently found
to ameliorate rheumatoid arthritis (RA). TLRs, RAGE-R, DNGR
(encoded by Clec9A), and Mincle have been shown to be receptors
responsible for mediating inflammation initiated by a variety of
DAMPs.
[0041] The inventors' recent work demonstrated that CD24-Siglec G
interactions discriminate innate immunity to DAMPs from PAMPs.
Siglec proteins are membrane-associated immunoglobulin (Ig)
superfamily members that recognize a variety of sialic
acid-containing structures. Most Siglecs have an intra-cellular
immune-tyrosine inhibitory motif (ITIM) that associates with SHP-1,
-2 and Cbl-b to control key regulators of inflammatory responses.
The inventors have reported CD24 as the first natural ligand for a
Siglec, Siglec G in mouse and Siglec 10 in human. Siglec G
interacts with sialylated CD24 to suppress the TLR-mediated host
response to DAMPs, such as HMGB1, via a SHP-1/2 signaling
mechanism.
[0042] Human CD24 is a small GPI-anchored molecule encoded by an
open-reading frame of 240 base pairs in the CD24 gene. Of the 80
amino acids, the first 26 constitute the signal peptide, while the
last 23 serve as a signal for cleavage to allow for the attachment
of the GPI tail. As a result, the mature human CD24 molecule has
only 31 amino acids. One of the 31 amino acids is polymorphic among
the human population. A C to T transition at nucleotide 170 of the
open-reading frame results in the substitution of Alanine (A) with
Valine (V) at residue 31 of the mature protein. Since this residue
is immediately N-terminal to the cleavage site, and since the
replacement is nonconservative, these two alleles may be expressed
at different efficiencies on the cell surface. Indeed, transfection
studies with cDNA demonstrated that the CD24.sup.v allele is more
efficiently expressed on the cell surface. Consistent with this,
CD24.sup.v/v PBL expressed higher levels of CD24, especially on T
cells.
[0043] The inventors have demonstrated that CD24 negatively
regulates host response to cellular DAMPs that are released as a
result of tissue or organ damage, and at least two overlapping
mechanisms may explain this activity. First, CD24 binds to several
DAMPs, including HSP70, HSP90, HMGB1 and nucleolin and represses
host response to these DAMPs. To do this, it is presumed that CD24
may trap the inflammatory stimuli to prevent interaction with their
receptors, TLR or RAGE. Second, using an acetaminophen-induced
mouse model of liver necrosis and ensuring inflammation, the
inventors demonstrated that through interaction with its receptor,
Siglec G, CD24 provides a powerful negative regulation for host
response to tissue injuries. To achieve this activity, CD24 may
bind and stimulate signaling by Siglec G wherein Siglec
G-associated SHP1 triggers the negative regulation. Both mechanisms
may act in concert as mice with targeted mutation of either gene
mounted much stronger inflammatory response. In fact, DC cultured
from bone marrow from either CD24.sup.-/- or Siglec G.sup.-/- mice
produced higher levels of inflammatory cytokines when stimulated
with either HMGB1, HSP70, or HSP90. To the inventors' knowledge,
CD24 is the only inhibitory DAMP receptor capable of shutting down
inflammation triggered by DAMPs and no drug is currently available
that specifically targets host inflammatory response to tissue
injuries. Furthermore, the inventors have demonstrated the ability
of exogenous soluble CD24 protein to alleviate DAMP-mediated
autoimmune disease using mouse models of RA, MS and GvHD.
1. Definitions
[0044] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise.
[0045] For recitation of numeric ranges herein, each intervening
number there between with the same degree of precision is
explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0046] A "peptide" or "polypeptide" is a linked sequence of amino
acids and may be natural, synthetic, or a modification or
combination of natural and synthetic.
[0047] "Substantially identical" may mean that a first and second
amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, or 300 amino acids.
[0048] "Treatment" or "treating," when referring to protection of
an animal from a disease, means preventing, suppressing,
repressing, or completely eliminating the disease. Preventing the
disease involves administering a composition of the present
invention to an animal prior to onset of the disease. Suppressing
the disease involves administering a composition of the present
invention to an animal after induction of the disease but before
its clinical appearance. Repressing the disease involves
administering a composition of the present invention to an animal
after clinical appearance of the disease.
[0049] A "variant" may mean a peptide or polypeptide that differs
in amino acid sequence by the insertion, deletion, or conservative
substitution of amino acids, but retain at least one biological
activity. Representative examples of "biological activity" include
the ability to bind to a toll-like receptor and to be bound by a
specific antibody. Variant may also mean a protein with an amino
acid sequence that is substantially identical to a referenced
protein with an amino acid sequence that retains at least one
biological activity. A conservative substitution of an amino acid,
i.e., replacing an amino acid with a different amino acid of
similar properties (e.g., hydrophilicity, degree and distribution
of charged regions) is recognized in the art as typically involving
a minor change. These minor changes can be identified, in part, by
considering the hydropathic index of amino acids, as understood in
the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The
hydropathic index of an amino acid is based on a consideration of
its hydrophobicity and charge. It is known in the art that amino
acids of similar hydropathic indexes can be substituted and still
retain protein function. In one aspect, amino acids having
hydropathic indexes of .+-.2 are substituted. The hydrophilicity of
amino acids can also be used to reveal substitutions that would
result in proteins retaining biological function. A consideration
of the hydrophilicity of amino acids in the context of a peptide
permits calculation of the greatest local average hydrophilicity of
that peptide, a useful measure that has been reported to correlate
well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,
incorporated fully herein by reference. Substitution of amino acids
having similar hydrophilicity values can result in peptides
retaining biological activity, for example immunogenicity, as is
understood in the art. Substitutions may be performed with amino
acids having hydrophilicity values within .+-.2 of each other. Both
the hydrophobicity index and the hydrophilicity value of amino
acids are influenced by the particular side chain of that amino
acid. Consistent with that observation, amino acid substitutions
that are compatible with biological function are understood to
depend on the relative similarity of the amino acids, and
particularly the side chains of those amino acids, as revealed by
the hydrophobicity, hydrophilicity, charge, size, and other
properties.
2. CD24
[0050] Provided herein is a CD24 protein, which may comprise a
mature CD24 or a variant thereof. Mature CD24 corresponds to the
extracellular domain (ECD) of CD24. The mature CD24 may be from a
human or another mammal As described above, mature human CD24
protein is 31 amino acids long and has a variable alanine (A) or
valine (V) residue at its C-terminal end. The mature CD24 protein
may comprise the following sequence:
TABLE-US-00001 (SEQ ID NO: 1)
SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A)
[0051] The C-terminal valine or alanine may be immunogenic and may
be omitted from the CD24 protein, which may reduce its
immunogenicity. Therefore, the CD24 protein may comprise the amino
acid sequence of mature human CD24 lacking the C-terminal amino
acid:
TABLE-US-00002 (SEQ ID NO: 2) SETTTGTSSNSSQSTSNSGLAPNPTNATTK
[0052] Despite considerable sequence variations in the amino acid
sequence of the mature CD24 proteins from mouse and human, they are
functionally equivalent, as human CD24Fc has been shown to be
active in the mouse. The amino acid sequence of the human CD24 ECD
shows some sequence conservation with the mouse protein (39%
identity; Genbank accession number NP_033976). However, it is not
that surprising that the percent identity is not higher as the CD24
ECD is only 27-31 amino acids in length, depending on the species,
and binding to some of its receptor(s), such as Siglec 10/G, is
mediated by its sialic acid and/or galactose sugars of the
glycoprotein. The amino acid sequence identity between the
extracellular domains of the human Siglec-10 (GenBank accession
number AF310233) and its murine homolog Siglec-G (GenBank accession
number NP_766488) receptor proteins is 63% (FIG. 2). As a result of
sequence conservation between mouse and human CD24 primarily in the
C-terminus and in the abundance of glycosylation sites, significant
variations in the mature CD24 proteins may be tolerated in using
the CD24 protein, especially if those variations do not affect the
conserved residues in the C-terminus or do not affect the
glycosylation sites from either mouse or human CD24. Therefore, the
CD24 protein may comprise the amino acid sequence of mature murine
CD24:
TABLE-US-00003 (SEQ ID NO: 3) NQTSVAPFPGNQNISASPNPTNATTRG.
[0053] The amino acid sequence of the human CD24 ECD shows more
sequence conservation with the cynomolgus monkey protein (52%
identity; UniProt accession number UniProtKB-I7GKK1) than with
mouse. Again, this is not surprising given that the percent
identity is not higher as the ECD is only 29-31 amino acids in
length in these species, and the role of sugar residues in binding
to its receptor(s). The amino acid sequence of cynomolgous
Siglec-10 receptor has not been determined but the amino acid
sequence identity between the human and rhesus monkey Siglec-10
(GenBank accession number XP_001116352) proteins is 89%. Therefore,
the CD24 protein may also comprise the amino acid sequence of
mature cynomolgous (or rhesus) monkey CD24:
TABLE-US-00004 (SEQ ID NO: 10) TVTTSAPLSSNSPQNTSTTPNPANTTTKA
[0054] The CD24 protein may be soluble. The CD24 protein may
further comprise an N-terminal signal peptide, which may allow
secretion of the protein from a cell expressing the protein. The
signal peptide sequence may comprise the amino acid sequence
MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). Alternatively, the
signal sequence may comprise any of those that are found on other
transmembrane or secreted proteins, or those modified from the
existing signal peptides known in the art.
a. Fusion
[0055] The CD24 protein may be fused at its N- or C-terminal end to
a protein tag, which may comprise a portion of a mammalian Ig
protein, which may be human or mouse or from another species. The
portion may comprise an Fc region of the Ig protein. The Fc region
may comprise at least one of the hinge region, CH2, CH3, and CH4
domains of the Ig protein. The Ig protein may be human IgG1, IgG2,
IgG3, IgG4, or IgA, and the Fc region may comprise the hinge
region, and CH2 and CH3 domains of the Ig. The Fc region may
comprise the human immunoglobulin G1 (IgG1) isotype SEQ ID NO: 7.
The Ig protein may also be IgM, and the Fc region may comprise the
hinge region and CH2, CH3, and CH4 domains of IgM. The protein tag
may be an affinity tag that aids in the purification of the
protein, and/or a solubility-enhancing tag that enhances the
solubility and recovery of functional proteins. The protein tag may
also increase the valency of the CD24 protein. The protein tag may
also comprise GST, His, FLAG, Myc, MBP, NusA, thioredoxin (TRX),
small ubiquitin-like modifier (SUMO), ubiquitin (Ub), albumin, or a
Camelid Ig. Methods for making fusion proteins and purifying fusion
proteins are well known in the art.
[0056] Based on preclinical research, for the construction of the
fusion protein CD24Fc identified in the examples, the truncated
form of native CD24 molecule of 30 amino acids, which lacks the
final polymorphic amino acid before the GPI signal cleavage site
(that is, a mature CD24 protein having SEQ ID NO: 2), has been
used. The mature human CD24 sequence is fused to a human IgG1 Fc
domain (SEQ ID NO: 7). The sequence of the full length CD24Fc
fusion protein is provided in SEQ ID NO: 5 (FIG. 1A), and the
sequence of the processed version of CD24Fc fusion protein that is
secreted from the cell (i.e. lacking the signal sequence which is
cleaved off) is provided in SEQ ID NO: 6. Processed polymorphic
variants of mature CD24 (that is, mature CD24 protein having SEQ ID
NO: 1) fused to IgG1 Fc may comprise the amino acid sequence set
forth in SEQ ID NO: 11 or 12.
b. Production
[0057] The CD24 protein may be heavily glycosylated, and may be
involved in functions of CD24 such as costimulation of immune cells
and interaction with a damage-associated molecular pattern molecule
(DAMP). The CD24 protein may be prepared using a eukaryotic
expression system. The expression system may entail expression from
a vector in mammalian cells, such as Chinese Hamster Ovary (CHO)
cells. The system may also be a viral vector, such as a
replication-defective retroviral vector that may be used to infect
eukaryotic cells. The CD24 protein may also be produced from a
stable cell line that expresses the CD24 protein from a vector or a
portion of a vector that has been integrated into the cellular
genome. The stable cell line may express the CD24 protein from an
integrated replication-defective retroviral vector. The expression
system may be GPEx.TM..
c. Pharmaceutical Composition
[0058] The CD24 protein may be contained in a pharmaceutical
composition, which may comprise a pharmaceutically acceptable
amount of the CD24 protein. The pharmaceutical composition may
comprise a pharmaceutically acceptable carrier. The pharmaceutical
composition may comprise a solvent, which may keep the CD24 protein
stable over an extended period. The solvent may be PBS, which may
keep the CD24 protein stable for at least 66 months at -20.degree.
C. (-15.about.-25.degree. C.). The solvent may be capable of
accommodating the CD24 protein in combination with another
drug.
[0059] The pharmaceutical composition may be formulated for
parenteral administration including, but not limited to, by
injection or continuous infusion. Formulations for injection may be
in the form of suspensions, solutions, or emulsions in oily or
aqueous vehicles, and may contain formulation agents including, but
not limited to, suspending, stabilizing, and dispersing agents. The
composition may also be provided in a powder form for
reconstitution with a suitable vehicle including, but not limited
to, sterile, pyrogen-free water.
[0060] The pharmaceutical composition may also be formulated as a
depot preparation, which may be administered by implantation or by
intramuscular injection. The composition may be formulated with
suitable polymeric or hydrophobic materials (as an emulsion in an
acceptable oil, for example), ion exchange resins, or as sparingly
soluble derivatives (as a sparingly soluble salt, for example). A
formulation for subcutaneous injection may be particularly relevant
for an indication like lupus and its associated manifestations and
complications.
3. Methods of Treatment
[0061] a. GVHD
[0062] Provided herein is a method of preventing, mitigating or
treating Graft versus Host Disease (GVHD) in a subject in need
thereof by administering the CD24 protein to the subject. The
subject may have or be at risk of developing GVHD. The subject may
undergo or may be undergoing hematopoietic stem cell
transplantation (HCT). The CD24 protein may be used
prophylactically to prevent GVHD in a subject undergoing HCT. The
GVHD may be acute GVHD. The CD24 protein may reduce the subject's
risk of grade III-IV acute GVHD. The GVHD may be chronic GVHD,
including oral GVHD.
[0063] The subject may have a cancer. The cancer may be Acute
Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic
Myelogenous Leukemia (CML), Myelodysplastic syndrome (MDS), or
Chronic Myelomonocytic Leukemia (CMML).
b. Mucositis
[0064] Provided herein is a method of preventing, mitigating or
treating mucositis in a subject in need thereof by administering
the CD24 protein to the subject. Also described herein is a method
of preventing, mitigating or treating other conditions associated
with GVHD or the HCT in a subject in need thereof by administering
the CD24 protein to the subject. The associated condition may be
mucositis, which may be oral mucositis. Mucositis is a painful
inflammation and ulceration of the mucous membranes lining the
digestive tract, usually as an adverse effect of the
preconditioning chemotherapy and/or radiotherapy regimen for HCT.
Mucositis can occur anywhere along the gastrointestinal (GI) tract.
Oral mucositis refers to the particular inflammation and ulceration
that occurs in the mouth. Oral mucositis is a common and often
debilitating complication of cancer treatment. Many hematopoietic
stem cell transplantation recipients experience mucositis, of which
oral mucositis is the most common and most debilitating.
[0065] The subject may have or be at risk of developing mucositis,
which may be oral mucositis. The subject may undergo or may be
undergoing at least one of chemotherapy and radiation therapy. The
CD24 protein may be used prophylactically to prevent mucositis in a
subject. The CD24 protein may reduce the subject's risk of severe
mucositis.
c. Medicaments
[0066] Also provided are uses of the CD24 protein in the
manufacture of a medicament for uses as described herein.
d. Dose Regimen
[0067] The dose of the CD24 protein administered may be 0.01 mg/kg
to 1000 mg/kg, and may be 1 to 500 mg/kg, depending on the desired
effect on GVHD or mucositis and the route of administration. The
CD24 protein may be administered by intravenous (IV) infusion or by
subcutaneous, intramural (that is, within the wall of a cavity or
organ), or intraperitoneal injection. The dose may be 10-1000 mg,
10-500 mg, 240 mg, or 480 mg, which in particular may be suitable
where the subject is a human.
[0068] The CD24 protein may be administered before or after the
stem cell transplant. The CD24 protein may be administered 1-4
days, particularly 1 day, before the stem cell transplant. The CD24
protein may also be administered in multiple doses before or after
stem cell transplant. The CD24 protein may be administered in 2, 3,
4, 5 or 6 bi-weekly doses. Each dose of the CD24 protein may be 240
mg or 480 mg. A first dose may be administered on day -4 to day 0
relative to the day of stem cell transplant (day 0), and may be
administered on day -1 in particular. Each subsequent dose may be
administered every 9-19 or 11-17 days thereafter. A second dose may
be administered on day +9 to +19 or day +11 to +17, particularly
day +14, relative to the day of stem cell transplant. A third dose
may be administered on day +18 to +38, day +23 to +33, or day +22
to +34, particularly day +28, relative to the day of stem cell
transplant. In particular, the CD24 protein may be administered in
three biweekly administrations of 480 mg, 240 mg, and 240 mg,
respectively on day -1, day +14 and day +28 relative to the day of
stem cell transplant. The CD24 protein may in particular be
CD24Fc.
[0069] The CD24 protein may be administered before or during at
least one of chemotherapy and radiation therapy is administered to
the subject. The CD24 protein may be administered so that it is
present when DAMPs are released.
e. Combination Treatment
[0070] The CD24 protein may be administered to the subject in
combination with standard of care GVHD prophylaxis. The standard of
care GVHD prophylaxis may comprise administration of methotrexate
plus calcineurin inhibitor, such as tacrolimus (Prograf, FK506) or
cyclosporine (Sandimmune, Neoral). Tacrolimus may be administered
on day -3 relative to the day of stem cell transplant, and may be
administered by IV or PO (orally). For IV dosing as a continuous
infusion the starting dose may be 0.03 mg/kg/day based on adjusted
body weight. For oral dosing the starting dose may be 0.045
mg/kg/dose twice daily. If the subject cannot tolerate tacrolimus,
then cyclosporine may be administered to the subject by IV at a
dose of 100.times. the IV tacrolimus dose (e.g., 3 mg/kg/day
starting dose). The cyclosporine may also be administered orally at
a dose of 3.times. the IV dose. When Neoral brand is used, because
of greater bioavailability, the cyclosporine may be administered
orally at 2.times. the IV dose.
[0071] In the absence of GVHD, tacrolimus levels may be monitored
for therapeutic dosing only during the first 100 days
post-transplant. The therapeutic target trough level for tacrolimus
may be 5-15 ng/mL. Tacrolimus levels may be monitored at a minimum
of three times (e.g. every 48-72 hours) for the first week post
CD24 protein infusion (day 0 to day 7). In the absence of GVHD or
relapse, tacrolimus tapering may begin on day +100 post-transplant.
In the presence of GVHD, tacrolimus may be continued at the
therapeutic dosing.
[0072] Methotrexate may be used in combination with tacrolimus for
standard GVHD prophylaxis. Methotrexate may be administered
intravenously at a dose of 15 mg/m.sup.2/dose once daily on Day 1
after HCT, and at a dose of 10 mg/m.sup.2/dose on days 3, 6, and 11
after HCT.
[0073] For mucositis, the CD24 protein may be administered with at
least one immunomodulatory agent (that is, other than a CD24
protein) to minimize the exacerbating inflammatory component. The
CD24 protein may be administered in combination with allopurinol
for subjects treated with 5-fluorouracil.
[0074] The CD24 protein may be administered in combination with one
or more of the following myeloablative conditioning regimens, and
may be administered before or during the conditioning regimens. The
CD24 protein may be administered so that it is present when DAMPs
are released in response to at least one of a myeloablative
conditioning regimen and the HCT.
Busulfan and Fludarabine
[0075] Busulfan may be administered on days -5 to -2 relative to
the day of stem cell transplant. The dose may be 3.2 mg/kg/day or
130 mg/m.sup.2/day, and may be administered intravenously. The
total dose may be 12.8 mg/kg or 520 mg/m.sup.2. Fludarabine may be
administered on days -5 to -2 relative to the day of stem cell
transplant. The dose may be 30-45 mg/m.sup.2/day, and the total
dose may be 120-180 mg/m.sup.2. The sequence and timing of busulfan
and fludarabine may be done according to institutional standards
for myeoablative conditioning that are known in the art.
Busulfan and Cyclophosphamide
[0076] Bulsulfan may be administered on days -7 to -4 relative to
the day of stem cell transplant. The dose may be 3.2 mg/kg/day or
130 mg/m.sup.2/day, and may be administered intravenously. The
total dose may be 12.8 mg/kg or 520 mg/m.sup.2. Cyclophosphamide
may be administered on days -3 to -2 relative to the day of stem
cell transplant. The dose may be 60 mg/kg/day, and the total dose
may be 120 mg/kg.
Cyclophosphamide and Total Body Irradiation
[0077] Total body irradiation (TBI) may be administered on days -7
to -4 relative to stem cell transplantation. Cyclophosphamide may
be administered on days -3 to -2 relative to the day of stem cell
transplant. The dose may be 60 mg/kg/day, and the total dose may be
120 mg/kg. The sequence of cyclophosphamide, TBI and TBI
administration practices for myeloablative regimens may be done
according to institutional standards for myeoablative conditioning
that are known in the art.
[0078] The CD24 protein may also be administered with individual
myeloablative treatments described above or combinations
thereof.
EXAMPLE 1
CD24 Pharmacokinetics in Mice
[0079] 1 mg of CD24Fc (CD24Fc) was injected into naive C57BL/6 mice
and collected blood samples at different timepoints (5 min, 1 hr, 4
hrs, 24 hrs, 48 hrs, 7 days, 14 days and 21 days) with 3 mice in
each timepoint. The sera were diluted 1:100 and the levels of
CD24Fc was detected using a sandwich ELISA using purified
anti-human CD24 (3.3 .mu.g/ml) as the capturing antibody and
peroxidase conjugated goat anti-human IgG Fc (5 .mu.g/ml) as the
detecting antibodies. As shown in FIG. 3a. The decay curve of
CD24Fc revealed a typical biphase decay of the protein. The first
biodistribution phase had a half-life of 12.4 hours. The second
phase follows a model of first-order elimination from the central
compartment. The half-life for the second phase was 9.54 days,
which is similar to that of antibodies in vivo. These data suggest
that the fusion protein is very stable in the blood stream. In
another study in which the fusion protein was injected
subcutaneously, an almost identical half-life of 9.52 days was
observed (FIG. 3b). More importantly, while it took approximately
48 hours for the CD24Fc to reach peak levels in the blood, the
total amount of the fusion protein in the blood, as measured by
AUC, was substantially the same by either route of injection. Thus,
from a therapeutic point of view, using a different route of
injection should not affect the therapeutic effect of the drug.
This observation greatly simplified the experimental design for
primate toxicity and clinical trials.
EXAMPLE 2
CD24-Siglec 10 Interaction in Host Response to Tissue Injuries
[0080] Nearly two decades ago, Matzinger proposed what was
popularly called danger theory. In essence, she argued that the
immune system is turned on when it senses the dangers in the host.
Although the nature of danger was not well defined at the time, it
has been determined that necrosis is associated with the release of
intracellular components such as HMGB1 and Heat-shock proteins,
which were called DAMP, for danger-associated molecular patterns.
DAMP were found to promote production of inflammatory cytokines and
autoimmune diseases. In animal models, inhibitors of HMGB1 and
HSP90 were found to ameliorate RA. The involvement of DAMP raised
the prospect that negative regulation for host response to DAMP can
be explored for RA therapy.
[0081] Using acetaminophen-induced liver necrosis and ensuring
inflammation, it was observed that through interaction Siglec G,
CD24 provides a powerful negative regulation for host response to
tissue injuries. CD24 is a GPI anchored molecules that is broadly
expressed in hematopoietic cells and other tissue stem cells.
Genetic analysis of a variety of autoimmune disease in human,
including multiple sclerosis, systemic lupus erythromatosus, RA,
and giant cell arthritis, showed significant association between
CD24 polymorphism and risk of autoimmune diseases. Siglec G is a
member of I-lectin family, defined by their ability to recognize
sialic acid containing structure. Siglec G recognized sialic acid
containing structure on CD24 and negatively regulates production of
inflammatory cytokines by dendritic cells. In terms of its ability
to interact with CD24, human Siglec 10 and mouse Siglec G are
functionally equivalent. However, it is unclear if there is a
one-to-one correlation between mouse and human homologues. Although
the mechanism remains to be fully elucidated, it is plausible that
Siglec G-associated SHP1 may be involved in the negative
regulation. These data lead to a new model in which CD24-Siglec
G/10 interaction may play a critical in discrimination
pathogen-associated molecular pattern (PAMP) from DAMP (FIG.
4).
[0082] At least two overlapping mechanisms may explain the function
of CD24. First, by binding to a variety of DAMP, CD24 may trap the
inflammatory stimuli to prevent their interaction with TLR or RAGE.
This notion is supported by observations that CD24 is associated
with several DAMP molecules, including HSP70, 90, HMGB1 and
nucleolin. Second, perhaps after associated with DAMP, CD24 may
stimulate signaling by Siglec G. Both mechanisms may act in concert
as mice with targeted mutation of either gene mounted much stronger
inflammatory response. In fact, DC cultured from bone marrow from
either CD24-/- or Siglec G-/- mice produced much higher
inflammatory cytokines when stimulated with either HMGB1, HSP70, or
HSP90. In contrast, no effect were found in their response to PAMP,
such as LPS and PolyI:C. These data not only provided a mechanism
for the innate immune system to distinguish pathogen from tissue
injury, but also suggest that CD24 and Siglec G as potential
therapeutic targets for diseases associated with tissue
injuries.
EXAMPLE 3
[0083] CD24Fc Interacts With HMGB1, Siglec 10 and Induces
Association Between Siglec G and SHP-1
[0084] To measure the interaction between CD24Fc and Siglec 10, we
immobilized CD24Fc onto a CHIP and used Biacore to measure the
binding of different concentrations of Siglec-10Fc. As shown in
FIG. 5a, CD24Fc binds with Siglec 10 with a Kd of
1.6.times.10.sup.-7M. This is 100-fold higher affinity than the
control Fc. The interaction between CD24Fc and HMGB1 was confirmed
by pull down experiments using CD24Fc-bound protein G beads
followed by Western blot with either anti-IgG or anti-HMGBE These
data demonstrate that CD24Fc, but not Fc, binds to HMGB1 and that
this binding is cation-dependent (FIG. 5b). To determine whether
CD24Fc is an agonist of Siglec G, the mouse counterpart of human
Siglec 10, we stimulated CD24-/- spleen cells with CD24Fc, control
Fc or vehicle (PBS) control for 30 minutes. Siglec G was then
immunoprecipitated and probed with anti-phospho-tyrosine or
anti-SHP-1. As shown in FIG. 5c, CD24Fc induced substantial
phosphorylation of Siglec G and association of SHP-1, a well-known
inhibitor for both adaptive and innate immunity.
[0085] In vitro efficacy studies of CD24Fc.
[0086] To study the impact of CD24Fc on the production of
inflammatory cytokines by human T cells, the mature T cells in
human PBML were activated by anti-CD3 antibody (OKT3), a commonly
used agonist of the T cell receptor in the presence of different
concentrations of CD24Fc or human IgG1 Fc. Four days later, the
supernatants were collected and the production of IFN-.gamma. and
TNF-.alpha. were measured by Enzyme-linked immunosorbent assay
(ELISA) to confirm activation. The results in FIG. 6 demonstrated
that CD24Fc from two different manufacturing lots significantly
reduced IFN-.gamma. and TNF-.alpha. production from the activated
human PBML compared with control IgG Fc control. In addition, when
CD24Fc was added, cytokine production was inhibited in a
dose-dependent manner Therefore, CD24Fc can inhibit anti-CD3
induced human PBML activation in vitro. This study not only
indicated the mechanism of action of CD24Fc might be through the
inhibition of T cell activation, but also established a reliable
bioassay for drug potency and stability testing.
[0087] To determine whether CD24Fc regulates production of
inflammatory cytokines in a human cell line, we first silenced CD24
in the human acute monocytic leukemia THP1 cell line using RNAi,
and then induced differentiation into macrophages by treating them
with PMA. As shown in FIG. 7a, CD24 silencing substantially
increased the production of TNF.alpha., IL-1.beta. and IL-6. These
data demonstrate an essential role for endogenous human CD24 in
limiting the production of inflammatory cytokines. Importantly,
CD24Fc restored inhibition of TNF.alpha. in the CD24-silenced cell
line (FIG. 7b), as well as IL-1.beta. and IL-6. These data not only
demonstrate the relevance of CD24 in inflammatory response of human
cells, but also provides a simple assay to assess biological
activity of CD24Fc.
[0088] Taken together, these data demonstrate that CD24Fc is
capable of inhibiting cytokine production triggered by adaptive and
innate stimuli. However, since the drug is much more effective in
reducing cytokine production by innate effectors, we consider that
the primary mechanism for its prophylactic function is to prevent
inflammation triggered by tissue injuries at the early phase of
transplantation.
EXAMPLE 4
CD24 Pharmacokinetics in Humans
[0089] This example shows an analysis of the pharmacokinetics of a
CD24 protein in humans. This was derived from a Phase I,
randomized, double-blind, placebo-controlled, single ascending dose
study to assess the safety, tolerability, and PK of CD24Fc in
healthy male and female adult subjects. A total of 40 subjects in 5
cohorts of 8 subjects each were enrolled in this study. Six of the
8 subjects in each cohort received study drug and 2 subjects
received placebo (0.9% sodium chloride, saline). The first cohort
was dosed with 10 mg. Succeeding cohorts received 30 mg, 60 mg, 120
mg, and 240 mg of CD24Fc or matching placebo and were dosed at
least 3 weeks apart to allow for review of safety and tolerability
data for each prior cohort. Administration of the next higher dose
to a new cohort of subjects was permitted only if adequate safety
and tolerability had been demonstrated.
[0090] In each cohort, the initial 2 subjects were 1 study drug
recipient and 1 placebo recipient on Day 1. The 3rd to 5th and 6th
to 8th subjects were dosed after Day 7 (a minimum of 24 hours apart
between the subgroups). Each subject was dosed at least 1 hour
apart in the same subgroup. If necessary, dosing of the rest of
subjects was delayed pending review of any significant safety
issues that may have arisen during the post-dose period involving
the first or second subgroups in that cohort. The subsequent cohort
was dosed at least 3 weeks after the prior cohort.
Screening Period:
[0091] The Screening Visit (Visit 1) occurred up to 21 days prior
to the beginning of the active treatment period. After providing
informed consent, subjects underwent screening procedures for
eligibility.
Treatment Period:
[0092] Subjects were admitted to the Clinical Pharmacology Unit
(CPU) on Day -1 (Visit 2), and the randomized treatment period
began on Day 1 following a 10-hour minimum overnight fast. Subjects
were randomly assigned to treatment with CD24Fc or placebo as a
single dose. Subjects remained confined until the morning of Day
4.
Follow-Up:
[0093] All subjects returned to the CPU on Day 7, Day 14, Day 21,
Day 28, and Day 42 (.+-.1 day) for follow-up visits (Visit 3, Visit
4, Visit 5, Visit 6, and Visit 7). Visit 7 was the final visit for
all subjects.
Duration of Treatment:
[0094] The total study duration for each subject was up to 63 days.
Single-dose administration occurred on Day 1.
[0095] Number of Subjects:
[0096] Planned: 40 subjects
[0097] Screened: 224 subjects
[0098] Randomized: 40 subjects
[0099] Completed: 39 subjects
[0100] Discontinued: 1 subject
Diagnosis and Main Criteria for Inclusion:
[0101] The population for this study was healthy males and females
between the ages of 18 and 55 years, inclusive, with a body mass
index between 18 kg/m.sup.2 and 30 kg/m.sup.2, inclusive.
Investigational Product and Comparator Information:
[0102] CD24Fc: single dose of 10 mg, 30 mg, 60 mg, 120 mg, or 240
mg administered via IV infusion; lot number: 09MM-036. CD24Fc was a
fully humanized fusion protein consisting of the mature sequence of
human CD24 and the fragment crystallizable region of human
immunoglobulin G1 (IgG1Fc). CD24Fc was supplied as a sterile,
clear, colorless, preservative-free, aqueous solution for IV
administration. CD24Fc was formulated as single dose injection
solution, at a concentration of 10 mg/mL and a pH of 7.2. Each
CD24Fc vial contained 160 mg of CD24Fc, 5.3 mg of sodium chloride,
32.6 mg of sodium phosphate dibasic heptahydrate, and 140 mg of
sodium phosphate monobasic monohydrate in 16 mL.+-.0.2 mL of
CD24Fc. CD24Fc was supplied in clear borosilicate glass vials with
chlorobutyl rubber stoppers and aluminum flip-off seals.
[0103] Matching placebo (0.9% sodium chloride, saline) administered
via IV infusion; lot numbers: P296855, P311852, P300715,
P315952.
[0104] The intent-to-treat (ITT) Population consisted of all
subjects who received at least 1 dose of the study drug. The ITT
Population was the primary analysis population for subject
information and safety evaluation.
[0105] Clinical laboratory evaluations (chemistry, hematology, and
urinalysis) were summarized by treatment and visit. Change from
baseline was also summarized. Vital signs (blood pressure, heart
rate, respiratory rate, and temperature) were summarized by
treatment and time point. Change from baseline was also summarized.
All physical examination data were listed. Electrocardiogram
parameters and the change from baseline were summarized. Overall
interpretations were listed.
Plasma CD24Fc Concentration
[0106] As shown in FIG. 8, the mean plasma concentration of CD24Fc
increased proportionally to the dose of CD24Fc administered. For
all dose groups except 120 mg, the maximum mean plasma
concentration of CD24Fc was reached at 1 hour post-dose. The
maximum mean plasma concentration of CD24Fc for the 120 mg group
was reached at 2 hours post-dose. By Day 42 (984 hours), the mean
plasma concentration of CD24Fc for all groups had decreased to
between 2% and 4% of the maximum mean plasma concentration.
[0107] Table 1 summarizes the plasma CD24Fc PK parameters by
treatment for the PK Evaluable Population.
TABLE-US-00005 TABLE 1 Summary of Plasma CD24Fc Pharmacokinetic
Parameters by Treatment - PK Evaluable Population CD24Fc CD24Fc
CD24Fc CD24Fc CD24Fc 10 mg 30 mg 60 mg 120 mg 240 mg Parameter
Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) C.sub.max (ng/mL)
n 6 6 6 6 6 Mean (SD) 2495 (576) 9735 (1715) 30 083 (7179) 52 435
(9910) 95 865 (10 734) CV % 23.1 17.6 23.9 18.9 11.2 Median 2371
9218 29 026 50 401 93 206 Min, Max 1,967, 3,390 8,583, 13,086
22,557, 42,628 40,434, 65,704 81,296, 110,110 Geometric mean 2,442
9,625 29,424 51,666 95,365 Geometric CV % 22.8 16.1 23.0 19.0 11.2
AUC.sub.0-42 d (ng*hr/mL) n 6 6 6 6 6 Mean (SD) 423,061 (99,615)
1,282,430 (88,798) 3,226,255 (702,862) 6,541,501 (2,190,944)
12,704,705 (1,918,596) CV % 23.5 6.9 21.8 33.5 15.1 Median 434,043
1,302,719 3,124,933 5,785,142 12,563,426 Min, Max 291,020, 528,079
1,175,733, 1,403,024 2,487,550, 4,139,748 4,485,193, 9,415,266
10,466,635, 15,693,606 Geometric mean 412,795 1,279,851 3,163,252
6,249,552 12,586,731 Geometric CV % 25.0 7.0 22.0 33.8 15.0
AUC.sub.0-inf (ng*hr/mL) n 6 6 6 6 6 Mean (SD) 462,260 (116,040)
1,434,464 (131,316) 3,497,196 (705,653) 7,198,196 (2,458,320)
13,861,796 (1,962,780) CV % 25.1 9.2 20.2 34.2 14.2 Median 470,426
1,422,205 3,519,732 6,463,665 13,713,034 Min, Max 310,956, 596,599
1,281,715, 1,650,503 2,703,655, 4,309,023 4,910,640, 10,479,940
11,822,988, 17,175,236 Geometric mean 449,583 1,429,578 3,437,036
6,862,129 13,750,972 Geometric CV % 26.7 9.0 20.7 34.6 13.8
T.sub.max (hr) n 6 6 6 6 6 Mean (SD) 1.15 (0.42) 1.17 (0.41) 1.01
(0.01) 1.34 (0.51) 1.33 (0.52) CV % 36.1 35.0 1.2 38.0 38.7 Median
1.00 1.00 1.00 1.03 1.00 Min, Max 0.92, 2.00 1.00, 2.00 1.00, 1.03
1.00, 2.00 1.00, 2.00 t.sub.1/2 (hr) n 6 6 6 6 6 Mean (SD) 280.83
(22.37) 327.10 (41.32) 279.82 (65.59) 286.45 (23.38) 285.33 (24.33)
CV % 8.0 12.6 23.4 8.2 8.5 Median 279.61 317.23 264.69 290.76
287.74 Min, Max 258.87, 321.26 289.82, 394.24 210.18, 362.46
243.89, 309.26 249.24, 322.26 AUCextr (%) n 6 6 6 6 6 Mean (SD)
7.61 (2.14) 10.44 (2.94) 7.88 (4.26) 8.92 (1.94) 8.46 (1.99) CV %
28.1 28.2 54.0 21.8 23.5 Median 7.16 10.01 6.35 9.27 8.45 Min, Max
5.46, 11.47 7.10, 15.05 3.92, 14.48 5.49, 10.99 5.56, 11.50 CL
(L/hr) n 6 6 6 6 6 Mean (SD) 0.0229 (0.0061) 0.0211 (0.0019) 0.0178
(0.0036) 0.0183 (0.0058) 0.0176 (0.0023) CV % 26.7 8.8 20.5 31.7
13.3 Median 0.0216 0.0211 0.0173 0.0191 0.0175 Min, Max 0.0168,
0.0322 0.0182, 0.0234 0.0139, 0.0222 0.0115, 0.0244 0.0140, 0.0203
Vd (L) n 6 6 6 6 6 Mean (SD) 9.153 (1.943) 9.867 (0.804) 7.289
(2.592) 7.491 (2.202) 7.276 (1.426) CV % 21.2 8.1 35.6 29.4 19.6
Median 8.507 10.007 7.486 7.691 7.151 Min, Max 7.326, 12.010 8.771,
10.958 4.222, 11.139 4.933, 9.974 5.814, 9.438 AUC.sub.0-42 d =
area under the concentration-time curve from time 0 to 42 days;
AUC.sub.0-inf = area under the concentration-time curve
extrapolated from time 0 to infinity; AUC.sub.extr = percentage of
AUC.sub.0-inf that was due to extrapolation from the time of the
last measurable concentration, per subject, to infinity; CL = total
body clearance; C.sub.max = maximum observed plasma drug
concentration; CV % = coefficient of variation; Min = minimum; Max
= maximum; SD = standard deviation; t.sub.1/2 = terminal
elimination half-life; T.sub.max = time of maximum observed plasma
drug concentration; V.sub.d = volume of distribution.
Plasma CD24Fc Dose Proportionality Analysis
[0108] FIG. 9 shows a dose proportionality plot of CD24Fc C.sub.max
versus dose for the PK Evaluable Population. FIG. 10 shows a dose
proportionality plot of CD24Fc AUC.sub.0-42 d versus dose for the
PK Evaluable Population. FIG. 11 shows a dose proportionality plot
of CD24Fc AUC.sub.0-inf versus dose for the PK Evaluable
Population. Table 2 shows a power analysis of dose
proportionality.
TABLE-US-00006 TABLE 2 Power Analysis of Dose Proportionality:
Plasma CD24Fc Pharmacokinetic Parameters - PK Evaluable Population
CD24Fc CD24Fc CD24Fc CD24Fc CD24Fc Dose Proportionality Parameter
10 mg 30 mg 60 mg 120 mg 240 mg Slope Standard Statistic (N = 6) (N
= 6) (N = 6) (N = 6) (N = 6) Estimate Error 90% CI C.sub.max
(ng/mL) 1.172 0.040 (1.105, 1.240) Geometric mean 2,441.8 9,624.9
29,424.4 51,666.4 95,364.9 Geometric CV % 22.8 16.1 23.0 19.0 11.2
AUC.sub.0-42 d (ng*hr/mL) 1.088 0.036 (1.027, 1.148) Geometric mean
412,794.8 1,279,850.8 3,163,251.7 6,249,551.9 12,586,731.3
Geometric CV % 25.0 7.0 22.0 33.8 15.0 AUC.sub.0-inf (ng*hr/mL)
1.087 0.036 (1.026, 1.148) Geometric mean 449,583.5 1,429,577.5
3,437,035.6 6,862,128.7 13,750,972.4 Geometric CV % 26.7 9.0 20.7
34.6 13.8 Geometric CV % = 100*sqrt(exp(SD.sup.2) - 1), where SD
was the standard deviation of the log-transformed data. The power
model was fitted by restricted maximum likelihood, regressing the
log-transformed PK parameter on log transformed dose. Both the
intercept and slope were fitted as fixed effects. Dose
proportionality was not rejected if the 90% CI lies within (0.8,
1.25). AUC.sub.0-42 d = area under the concentration-time curve
from time 0 to 42 days; AUC.sub.0-inf = area under the
concentration-time curve extrapolated from time 0 to infinity; CI =
confidence interval; C.sub.max = maximum observed plasma drug
concentration; CV % = coefficient of variation; PK =
pharmacokinetic; SD = standard deviation.
[0109] The C.sub.max slope estimate was 1.172 with a 90% CI of
1.105 to 1.240. The AUC.sub.0-42 d slope estimate was 1.088 with a
90% CI of 1.027 to 1.148. The AUC.sub.0-inf slope estimate was
1.087 with a 90% CI of 1.026 to 1.1.
Pharmacokinetic Conclusions
[0110] The C.sub.max and AUCs of plasma CD24Fc increased
proportionally to the doses administered in mouse, monkey and
human. The plasma CD24Fc reached T.sub.max between 1.01 and 1.34
hours. The t.sub.1/2 of plasma CD24Fc ranged between 280.83 and
327.10 hours.
EXAMPLE 5
CD24 can be Used to Treat Graft Versus Host Disease in Human
Subjects
[0111] A multicenter, prospective, double-blind, randomized,
placebo-controlled Phase IIa dose escalation trial was performed to
evaluate the addition of a CD24 protein, CD24Fc, to standard of
care acute GVHD prophylaxis in cancer patients undergoing
allogeneic myeloablative hematopoietic stem cell transplantation
(HCT). The trial design is shown in FIG. 12.
[0112] The primary objectives of the phase IIa study include
assessing the safety and tolerability of CD24Fc in combination with
methotrexate and tacrolimus prophylaxis in patients undergoing
matched unrelated donor HCT following myeloablative conditioning,
and to define the recommended phase 2 dose (RP2D) or maximum
tolerated dose (MTD). In addition, secondary efficacy objectives in
the phase IIa study include: [0113] determining if the addition of
CD24Fc to standard GVHD prophylaxis methotrexate and tacrolimus
reduces the cumulative incidence of grade II-IV aGVHD at day 100
after HCT [0114] estimating grade II-IV aGVHD free survival (GFS)
at day 180 after HCT, [0115] describing the incidence of cGVHD
(cGVHD) at 1 year [0116] describing the incidence of relapse one
year following HCT [0117] describing the incidence of
transplant-related mortality (TRM) one year following HCT [0118]
describing infection rates at day 100 following HCT [0119]
evaluating overall survival (OS), absence of grade III-IV GVHD, and
relapse-free survival one year following HCT [0120] evaluating
conditioning toxicity including oral mucositis and organ
failure
[0121] Other objectives include assessing the pharmacokinetic (PK)
profile of CD24Fc, examining the immune cell profile and functional
responses of APCs and T cells after HCT in the CD24Fc and placebo
groups, and assessing pharmacodynamics (PD) biomarkers such as the
plasma concentrations of pro-inflammatory cytokines, DAMPs, lipids,
and GVHD biomarkers in the CD24Fc and placebo groups.
[0122] The trial enrolled patients receiving transplants from
matched unrelated donors undergoing allogeneic HCT according to
institutional practice. Patients between the ages of 18-70 years
old undergoing matched unrelated donor allogeneic HCT for a
malignant hematologic condition with a Karnofsky performance
score.gtoreq.70% were eligible for the study. An 8/8 HLA allelic
match between the unrelated donor and the recipient at HLA-A,
HLA-B, HLA-C, and HLA-DRB1 was required. Restricting the study to
patients receiving HCT from unrelated donors is expected to limit
heterogeneity and facilitate statistical estimates of aGVHD
incidence for subsequent efficacy assessments, given the greater
incidence of grade II-IV aGVHD (60-80%) and grade III-IV aGVHD
(20-35%) in this population.
[0123] This trial exclusively utilized myeloablative conditioning
regimens and standard of care (SOC) prophylaxis comprising
tacrolimus and methotrexate since these patients experience the
most severe tissue injury and drug will likely have the strongest
biological effect in this setting. All patients received
myeloablative conditioning and standard of care GVHD prophylaxis
with methotrexate and tacrolimus per the phase IIa protocol.
Patients received a myeloablative conditioning regimen consisting
of either fludarabine and busulfan (Flu/Bu 4) or cyclophosphamide
and total body irradiation (Cy/TBI), as decided by the treating
physician, followed by an infusion of stem cells on day 0. GVHD
prophylaxis was administered to all patients and consisted of
tacrolimus (initiated Day -3 before transplant) and methotrexate
(initiated Day +1 after transplant) in combination with CD24Fc in
the treatment arm or saline in the placebo arm. In the absence of
GVHD, tacrolimus tapering started on day +100. The source of donor
stem cells was either peripheral blood stem cells (PBSC) or bone
marrow (BM).
[0124] The Phase IIa trial comprised two single ascending dose
cohorts (240 mg and 480 mg) and a single multi-dose cohort of
CD24Fc in addition to SOC GVHD prophylaxis as outlined in Table 3
below. As shown in FIG. 13, in the single dose cohort, the study
agent, CD24Fc, was administered intravenously on day -1 relative to
the day of stem cell transplant. In the multi-dosing cohort,
patients received 3 biweekly administrations of CD24Fc at 480 mg
(day -1), 240 mg (day +14) and 240 mg (day +28). Based upon PK data
for CD24Fc this biweekly dosing period will allow for passage of
greater than two half-lives. Dosing is based on a fixed amount and
not based on weight or BSA. Each dosing cohort enrolled 8 subjects
using a randomized 3:1 ratio (6 CD24Fc subjects and 2 placebo)
design for a total enrollment of 24 patients.
[0125] Table 4 lists demography information and clinical
characteristics for patients in the CD24Fc and placebo cohorts,
which were relatively balanced across risk factors such as age,
malignancy, and comorbidity. The most common malignancy in both the
CD24Fc and placebo cohorts was AML/MDS (66.7% and 83.3%). 72% of
the patients in the CD24Fc cohort and 50% in the placebo group had
a comorbidity index of intermediate or high. PBSCs were more
frequently used as the graft source as compared to bone marrow in
both cohorts, and Flu/Bu 4 was the most common conditioning regimen
across both cohorts. Four patients, all in the CD24Fc cohorts,
underwent Cy/TBI conditioning.
TABLE-US-00007 TABLE 4 Phase 2a Patient Characteristics CD24Fc +
Placebo + TAC/MTX TAC/MTX (N = 18) (N = 6) Age (years) Median
(range) 62 (23-68) 57 (36-66) Gender (N, %) Female 7 (38.8) 2
(33.3) Male 11 (61.1) 4 (66.7) Graft Source PBSC 15 (83.3) 4 (66.7)
(N, %) BM 3 (16.7) 2 (33.3) Malignancy AML/MDS 12 (66.7) 5 (83.3)
(N, %) CML 2 (11.1) 0 (0) CMML 1 (5.6) 1 (16.7) ALL 3 (16.7) 0 (0)
Comorbidity Low (0) 5 3 index (score) Intermediate (1-2) 9 2 High
(3-4) 4 1 Cytomegalovirus D+, R+ 5 1 status D+, R- 1 0 D-, R+ 3 1
D-, R- 8 4 Conditioning Flu/Bu 4 14 (77.8) 6 (100) regimen (N)
Cy/TBI 4 (22.2) 0 (0) Engraftment Neutrophil 13.0 (12, 23) 15.5
(12, 18) Day (Day) (min, max) Platelets 13.0 (9, 23) 15.0 (11,
>48) (min, max) BM = Bone marrow; Cy/TBI =
cyclophosphamide/total body irradiation; D = donor; Flu/Bu 4 =
fludarabine/busulfan; R = recipient
[0126] The primary objectives of the study are: to evaluate the
safety and tolerability of CD24Fc in subjects undergoing
myeloablative allogeneic hematopoietic cell transplantation (HCT);
and to determine the recommended Phase II dose (RP2D) or maximum
tolerable dose (MTD) of CD24Fc in patients undergoing HCT.
[0127] All patients enrolled in the study have completed the
Treatment period, which is the first day of treatment with CD24Fc
until 30 days after HCT for the single-dosing cohorts or 60 days
after HCT for the multi-dosing cohort (the exact days may vary
depending on the last day of administration of study drug without
constituting a deviation) and is the assessment and reporting
period for adverse events (AE) including dose limiting toxicities
potentially related to the study drug. Table 5 provides a summary
of toxicities observed in the Phase 2a trial. Overall this study
demonstrated that IV administration of CD24Fc up to 480 mg is
generally well tolerated in the intent-to-treat (ITT) population.
No infusion toxicities, dose-limiting toxicities (DLTs) or SAEs
attributable or likely attributable to the study drug have been
observed and no patients have been removed from the study.
[0128] All 24 subjects enrolled engrafted following transplant as
shown in FIG. 14. Neutrophils engrafted a median of 13.0 and 15.5
days after HCT in CD24Fc exposed and placebo patients,
respectively. Platelets engrafted a median of 13.0 and 15.0 days
after HCT in CD24Fc exposed and placebo patients, respectively,
with the exception of one patient in the placebo group in whom
platelets did not engraft and who died on Day 49. There were no
cases of graft failure. The median CD3 chimerism at day +30 was
82.5% (range 38-100%) in the CD24Fc exposed patients and 82.0%
(range, 62%-91%) in the placebo group (FIG. 15). The median donor
CD3 chimerism increased to 86% (range, 42%-100%) at day 100 in the
CD24Fc exposed patients and 84% (range, 17%-100%) in the placebo
group. Donor CD33 chimerism at day 30 and 100 was 100% in both the
CD24Fc and placebo groups.
TABLE-US-00008 TABLE 5 Summary of Toxicities Infusion Cohort
Treatment # of Pts Rxn SAE DLT Single CD24Fc 6 0 1 0 240 mg Placebo
2 0 2 0 Single CD24Fc 6 0 0 0 480 mg Placebo 2 0 1 0 Multidose
CD24Fc 6 0 4 0 Placebo 2 0 2 1
[0129] Efficacy analyses for the Phase 2a study are considered
secondary and include the following: to describe grade III-IV acute
GVHD free survival (GFS) at day 180 following HCT; to describe the
cumulative incidence of grade II-IV acute GVHD at day 100 after
HCT; to describe grade III-IV GVHD, Relapse Free Survival at day
180 after HCT; to describe grade II-IV acute GFS at day 180
following HCT; to describe incidence of chronic GVHD at one year
following HCT; to describe incidence of relapse at one year
following HCT; to describe incidence of transplant-related
mortality (TRM) at one year following HCT; to describe rates of
infection at day 100 following HCT; to evaluate overall survival
(OS) and disease free survival (DFS) at one year following HCT.
[0130] In addition to inclusion of the placebo arm in the phase IIa
study, data on contemporary controls (N=92) were collected from the
same institutions undergoing matched unrelated donor HCT following
the same myeloablative conditioning and GVHD prophylaxis regimens
(minus the experimental therapy CD24Fc) from the period of January
2012 to November 2017. A contemporary control cohort was included
given the small number of patients in the placebo control arm. The
demography data of the 92 adult patients in the contemporary
control cohort is summarized in Table 6.
TABLE-US-00009 TABLE 6 Characteristics of Patients Enrolled in the
Contemporary Control Cohort TAC/MTX (N = 92) Age (years) Median
(range) 49 (21-69) Gender Female 41 (44.5) (N, %) Male 51 (55.5)
Malignancy AML/MDS 63 (68.5) (N, %) CML 3 (3) CMML 2 (2) ALL 24
(26.5)
[0131] Tables 7 and 8 provide an overview of the clinical outcomes
of the Ph 2a study. Acute GVHD was graded according to consensus
guidelines utilized by the international CIBMTR registry and Blood
and Marrow Transplant Clinical Trials Network and recorded weekly.
Patients were evaluated for aGVHD following receipt of HCT on day 0
until day 100 after HCT.
TABLE-US-00010 TABLE 7 Overview of Clinical Outcomes, including the
cumulative incidence of grade II-IV and grade III-IV aGVHD. aGVHD
Relapse Death Cohort (Day 180) (Day 180) (Day 180) 1) Single Dose:
Gr II: 2 (skin) 0 0 240 mg Gr III: 0 (N = 6) Gr IV: 0 Gr II-IV:
33.3% (95% CI, 3.2, 70.4) Gr III-IV: 0% 2) Single Dose: Gr II: 2
(Skin and Upper GI) 1 0 480 mg Gr III: 1 (Lower GI) CMML (d 161)
.sup. (N = 6) Gr IV: 0 Gr II-IV: 50.0% (95% CI, 7.7, 82.9) Gr
III-IV: 16.7% 3) Multi-Dose: Gr II: 2 1 (N = 6) (Skin and Upper GI)
ALL (d 103) 0 Gr III: 0 Gr IV: 0 Gr II-IV: 33.3% (95% CI, 3.2,
70.4) Gr III-IV: 0% CD24Fc Total: Gr II: 6 2/18 (11.1%) 0/18* Gr
III: 1 Gr IV: 0 Gr II-IV: 38.9% (95% CI, 16.8, 60.7) Gr III-IV:
5.6% Placebo Gr II: 0 2/6 (33.3%) 1/6 (16.7%) (N = 6) Gr III: 1
(Lower GI) Gr IV: 0 Gr II-IV: 16.7% (95% CI, 0.5, 54.9) Gr III-IV:
16.7% Contemporary Control Gr II: 22 19/92 (23.1%) 22/92 (23.9%) (N
= 92) Grill: 16 (Death as Gr IV: 3 competing Gr II-IV: 50% factor)
(death as competing factor) (95% CI, 39.8, 60.2) Gr III-IV: 24%
(death as competing factor) (95% CI, 17.31) *Deaths (n = 2) after
day 180: Cohort 1 Pneumonitis 2/2 infections (d 210) and Cohort 2
relapse of CMML (d 196)
TABLE-US-00011 TABLE 8 Summary of Clinical Outcomes Statistical
CD24Fc Placebo Significance Number 18 6 aGVHD III-IV D 100 6% 17% D
180 6% 17% 1 yr Relapse 11% 33% 1 yr NRM 6% 17% 1.5 yr RFS 83% 50%
1.5 yr OS 89% 50% P = 0.046 D 180 Gr III-IV, Relapse 83% 33% P =
0.01 Free Survival
Incidence of Grade II to IV Acute Graft-Versus-Host Disease by Day
100
[0132] Table 9 summarizes the cumulative incidence of Grade II to
IV acute GVHD by Day 100 for the mITT Population. In total, 7
(38.9%) patients who received CD24Fc (2 [33.3%] patients in the 240
mg CD24Fc single dose cohort, 3 [50.0%] patients in the 480 mg
CD24Fc single dose cohort, and 2 [33.3%] patients in the 960 mg
CD24Fc multiple dose cohort) and 1 (16.7%) patient who received
placebo had Grade II to IV acute GVHD by Day 100. Additionally, 1
(16.7%) patient who received placebo died without Grade II to IV
acute GVHD by Day 100. Patients who were alive with no occurrence
of Grade II to IV acute GVHD through Day 100 were censored at their
last assessment for acute GVHD on or prior to Day 100. At least
50.0% of patients in each treatment group were censored.
TABLE-US-00012 TABLE 9 Cumulative Incidence of Grade II to IV Acute
Graft- Versus-Host Disease by Day 100 - mITT Population CD24Fc
CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc
Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N
= 6) (N = 18) No. of patients with Grade II-IV 1 (16.7) 2 (33.3) 3
(50.0) 2 (33.3) 7 (38.9) acute GVHD by Day 100 (n, %) No. of
patients who died without 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Grade II-IV acute GVHD by Day 100 (n, %) No. of patients censored
(n, %) 4 (66.7) 4 (66.7) 3 (50.0) 4 (66.7) 11 (61.1) Cumulative
incidence (%) of 16.7 33.3 50.0 33.3 38.9 Grade II-IV acute GVHD by
Day 100 [1] 95% CI (0.5, 54.9) (3.2, 70.4) (7.7, 82.9) (3.2, 70.4)
(16.8, 60.7) Treatment comparison; CD24Fc versus placebo [2] Hazard
ratio (90% CI) 2.6 (0.5, 14.7) Note: Day 100 = Day 100 (+7 days)
post-transplant (ie, Study Day 108). Percentage was calculated
using the number of patients in the column heading as the
denominator. [1] Grades were based on the CIBMTR grading scale. The
cumulative incidence (%) of acute GVHD by Day 100 and the 95% CI
were estimated using the cumulative incidence function with death
without Grade II to IV acute GVHD as a competing risk. [2] Hazard
ratio and 90% CI were based on a Fine and Gray model with treatment
as a covariate and death without Grade II to IV acute GVHD as a
competing risk. CI = confidence interval; CIBMTR = Center for
International Blood and Marrow Transplant Research; GVHD =
graft-versus-host disease; No. = number.
[0133] Overall, the cumulative incidence of Grade II to IV acute
GVHD by Day 100 (with 95% CI) was 38.9% (16.8%, 60.7%) for the
CD24Fc treatment group and 16.7% (0.5%, 54.9%) for the placebo
group. The hazard ratio (with 90% CI) for CD24Fc versus placebo was
2.6 (0.5, 14.7). The cumulative incidence of grade II-IV aGVHD was
50% in the contemporary control. In the CD24Fc treated group, four
cases of grade II aGVHD involved skin only and two cases involved
skin and the upper gastrointestinal (GI) tract. There were no cases
grade II aGVHD in the placebo group.
Grade II-IV Acute Graft-Versus-Host Disease-Free Survival Through
Day 180
[0134] Table 10 summarizes Grade II to IV acute GFS through Day 180
for the mITT Population. The median Grade II to IV acute GFS
Kaplan-Meier estimate was not reached in any treatment group.
Overall, the Grade II to IV acute GFS rate at Day 180 (with 95% CI)
was 61.1% (35.3%, 79.2%) for the CD24Fc treatment group and 50.0%
(11.1%, 80.4%) for the placebo group. The hazard ratio (with 90%
CI) for CD24Fc versus placebo was 0.8 (0.3, 2.5). Patients who were
alive and had no documented occurrence of Grade II to IV acute GVHD
at the data cutoff date were censored at the last date of acute
GVHD assessment on or prior to Day 180. In addition to the small
sample size, at least 50.0% of patients in each treatment group
were censored.
TABLE-US-00013 TABLE 10 Grade II to IV Acute Graft-Versus-Host
Disease-Free Survival Through Day 180 - mITT Population CD24Fc
CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc
Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N
= 6) (N = 18) No. of patients with events (n, %) 3 (50.0) 2 (33.3)
3 (50.0) 2 (33.3) 7 (38.9) Earliest contributing event Acute GVHD
(Grade II-IV) 2 (33.3) 2 (33.3) 3 (50.0) 2 (33.3) 7 (38.9) Death 1
(16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. of patients censored (n,
%) 3 (50.0) 4 (66.7) 3 (50.0) 4 (66.7) 11 (61.1) Acute GVHD (Grade
II-IV)-free survival (days) Kaplan-Meier estimate [1] Median (95%
CI) NE NE NE NE NE Mean (SD) [2] 144.2 (74.98) 145.7 (77.01) 116.2
(86.69) 150.5 (68.96) 137.4 (74.81) Median [2] 190.0 195.0 121.0
195.0 195.0 Min, max [2] 46, 195+ 32, 195+ 24, 195+ 59, 195+ 24,
195+ Treatment comparison: CD24Fc versus placebo [3] Hazard ratio
(90% CI) 0.8 (0.3, 2.5) Rate (%) of being alive without acute 50.0
(11.1, 80.4) 66.7 (19.5, 90.4) 50.0 (11.1, 80.4) 66.7 (19.5, 90.4)
61.1 (35.3, 79.2) GVHD (Grade II-IV) at Day 180 (95% CI) [4] Note:
Day 180 = Day 180 (+14 days) post-transplant (ie, Study Day 195).
Percentage was calculated using the number of patients in the
column heading as the denominator. [1] The 95% CI for median was
computed using the Brookmeyer and Crowley method with log-log
transformation. [2] Censoring was ignored in the calculation for
mean (SD) and median. A "+" after the min or max indicates a
censored observation. [3] Hazard ratio and 90% CI were based on a
Cox proportional hazards model with treatment as a covariate. [4]
Kaplan-Meier estimate. CI = confidence interval; GVHD =
graft-versus-host disease; log = logarithm; max = maximum; min =
minimum; NE = not estimable; No. = number; SD = standard
deviation.
Grade III to IV Acute GFS Through Day 180 for the mITT
Population
[0135] As shown in Table 11, in total, 1 (5.6%) patient who
received CD24Fc (1 [16.7%] patient in the 480 mg CD24Fc single dose
cohort) and 2 (33.3%) patients who received placebo had Grade III
to IV acute GVHD by Day 180. Overall, the Grade III to IV acute GFS
rate at Day 180 (with 95% CI) was 94.4% (66.6%, 99.2%) for the
CD24Fc treatment group and 50.0% (11.1%, 80.4%) for the placebo
group. The hazard ratio (with 90% CI) for CD24Fc versus placebo was
0.1 (0.0, 0.7). Patients who were alive and had no documented
occurrence of Grade III to IV acute GVHD at the data cutoff date
were censored at the last date of acute GVHD assessment on or prior
to Day 180. At least 50.0% of patients in each treatment group were
censored. Grade III to IV acute GFS rate at Day 180 was 24% in the
contemporary control cohort. FIG. 25 shows the 180 Day Grade III-IV
GVHD Free Survival in the CD24Fc group compared to the placebo
control group (FIG. 25A) and the contemporary control group (FIG.
25B).
TABLE-US-00014 TABLE 11 Grade III to IV Acute Graft-Versus-Host
Disease-Free Survival Through Day 180 - mITT Population CD24Fc
CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc
Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N
= 6) (N = 18) No. of patients with events (n, %) 3 (50.0) 0 (0.0) 1
(16.7) 0 (0.0) 1 (5.6) Earliest contributing event Acute GVHD
(Grade III-IV) 2 (33.3) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6) Death 1
(16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. of patients censored (n,
%) 3 (50.0) 6 (100.0) 5 (83.3) 6 (100.0) 17 (94.4) Acute GVHD
(Grade III-IV)-free survival (days) Kaplan-Meier estimate [1]
Median (95% CI) NE NE NE NE NE Mean (SD) [2] 144.2 (74.98) 195.0
(0.00) 166.5 (69.81) 195.0 (0.00) 185.5 (40.31) Median [2] 190.0
195.0 195.0 195.0 195.0 Min, max [2] 46, 195+ 195+, 195+ 24, 195+
195+, 195+ 24, 195+ Treatment comparison: CD24Fc versus placebo [3]
Hazard ratio (90% CI) 0.1 (0.0, 0.7) Rate (%) of being alive
without acute 50.0 (11.1, 80.4) 100.0 (NE) 83.3 (27.3,97.5) 100.0
(NE) 94.4 (66.6, 99.2) GVHD (Grade III-IV) at Day 180 (95% CI) [4]
Note: Day 180 = Day 180 (+14 days) post-transplant (ie, Study Day
195). Percentage was calculated using the number of patients in the
column heading as the denominator. [1] The 95% CI for median was
computed using the Brookmeyer and Crowley method with log-log
transformation. [2] Censoring was ignored in the calculation for
mean (SD) and median. A "+" after the min or max indicates a
censored observation. [3] Hazard ratio and 90% CI were based on a
Cox proportional hazards model with treatment as a covariate. [4]
Kaplan-Meier estimate. CI = confidence interval; GVHD =
graft-versus-host disease; log = logarithm; max = maximum; min =
minimum; NE = not estimable; No. = number; SD = standard
deviation.
[0136] All patients who developed aGVHD in the study at the time of
the data cutoff have responded to steroid treatment, as compared to
the 50% response rate observed in the contemporary cohort control.
After the first one hundred days post HCT, patients were evaluated
quarterly for late onset aGVHD (defined as acute GVHD onset after
day 100) or cGVHD until one year after HCT. No additional aGVHD
events were observed in the CD24Fc cohorts after Day 100
post-transplant.
[0137] FIG. 16 shows the cumulative incidence of Grade II-IV and
Grade III-IV acute GVHD in the treatment (CD24Fc) cohort. In
particular, only one patient had Grade III GVHD that involved the
lower GI and no liver GVHD was observed. The cumulative incidence
of grade III-IV aGVHD at Day 180 after HCT in the CD24Fc cohorts
trended lower than the grade III-IV aGVHD at Day 180 in the
contemporary control (P=0.097). There was an additional case of
Grade III aGVHD at Day 182, which resulted in death at day 184, in
a patient in the placebo group. This patient had a leukemia relapse
at Day 145. These results suggest that CD24Fc in addition to
methotrexate and tacrolimus prophylaxis reduces the risk of
developing more serious grade III and IV aGVHD in patients
undergoing HCT after receiving myeloablative conditioning.
Disease-Free Survival 1 Year Following Hematopoietic Stem Cell
Transplantation
[0138] Table 12 summarizes disease-free survival (DFS) 1 year
post-HCT for the mITT Population. The median DFS Kaplan-Meier
estimate was not reached for any treatment group. Overall, the DFS
rate at 1 year post-HCT (with 95% CI) was 83.3% (56.8%, 94.3%) for
the CD24Fc treatment group and 50.0% (11.1%, 80.4%) for the placebo
group. The hazard ratio (with 90% CI) for CD24Fc versus placebo was
0.2 (0.1, 0.9). Patients who were alive and did not experience
disease relapse at the end of the follow-up period were censored at
the last date of evaluation. At least 50.0% of patients in each
treatment group were censored. FIG. 26 shows the Relapse Free
Survival in the CD24Fc group compared to the placebo control group
(FIG. 26A) and the contemporary control group (FIG. 26B).
TABLE-US-00015 TABLE 12 Disease-Free Survival 1 Year Following
Hematopoietic Stem Cell Transplantation - mITT Population CD24Fc
CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc
Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N
= 6) (N = 18) No. of patients with events (n, %) 3 (50.0) 1 (16.7)
1 (16.7) 1 (16.7) 3 (16.7) Earliest contributing event Relapse 2
(33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11-1) Death 1 (16.7) 1 (16.7) 0
(0.0) 0 (0.0) 1 (5.6) No. of patients censored (n, %) 3 (50.0) 5
(83.3) 5 (83.3) 5 (83.3) 15 (83.3) Disease-free survival (days)
Kaplan-Meier estimate [1] Median (95% CI) NE NE NE NE NE Mean (SD)
[2] 232.7 (152.48) 342.5 (64.11) 335.2 (92.82) 324.2 (109.89) 333.9
(85.76) Median [2] 256.0 366.0 370.5 366.0 366.0 Min, max [2] 49,
371+ 212, 378+ 146, 380+ 100, 376+ 100, 380+ Treatment comparison:
CD24Fc versus placebo [3] Hazard ratio (90% CI) 0.2 (0.1, 0.9) Rate
(%) of being alive without 50.0 (11.1, 80.4) 83.3 (27.3, 97.5) 83.3
(27.3, 97.5) 83.3 (27.3, 97.5) 83.3 (56.8, 94.3) relapse at 1 year
post-HCT (95% CI) [4] Note: One year = Day 365 (+14 days)
post-transplant (ie, Study Day 380). Percentage was calculated
using the number of patients in the column heading as the
denominator. [1] The 95% CI for median was computed using the
Brookmeyer and Crowley method with log-log transformation. [2]
Censoring was ignored in the calculation for mean (SD) and median.
A "+" after the min or max indicates a censored observation. [3]
Hazard ratio and 90% CI were based on a Cox proportional hazards
model with treatment as a covariate. [4] Kaplan-Meier estimate. For
Day 365, if the maximum observed time was < Study Day 380, the
Kaplan-Meier estimate at the maximum observed time is presented for
a treatment group. CI = confidence interval; HCT = hematopoietic
stem cell transplantation; log = logarithm; max = maximum; min =
minimum; NE = not estimable; No. = number; SD = standard
deviation.
Overall Survival 1 Year Following Hematopoietic Stem Cell
Transplantation
[0139] Table 13 summarizes overall survival (OS) 1 year post-HCT
for the mITT Population. The median OS time Kaplan-Meier estimate
was not reached for any treatment group. Overall, the OS rate at 1
year (with 95% CI) was 83.3% (56.8%, 94.3%) for the CD24Fc
treatment group and 50.0% (11.1%, 80.4%) for the placebo group. The
hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.2 (0.1,
1.0). Patients who were alive at the end of the follow-up period
were censored at the last date that they were known to be alive. At
least 50.0% of patients in each treatment group were censored.
TABLE-US-00016 TABLE 13 Disease-Free Survival 1 Year Following
Hematopoietic Stem Cell Transplantation - mITT Population CD24Fc
CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc
Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N
= 6) (N = 18) No. of patients who died (n, %) 3 (50.0) 1 (16.7) 1
(16.7) 1 (16.7) 3 (16.7) No. of patients censored (n, %) 3 (50.0) 5
(83.3) 5 (83.3) 5 (83.3) 15 (83.3) Overall survival (days)
Kaplan-Meier estimate [1] Median (95% CI) NE NE NE NE NE Mean (SD)
[2] 276.3 (132.25) 343.0 (64.34) 343.5 (72.45) 367.2 (6.01) 351.2
(53.91) Median [2] 341.5 366.5 370.5 366.0 366.5 Min, max [2] 49,
371+ 212, 378+ 196, 380+ 358+, 376+ 196, 380+ Treatment comparison;
CD24Fc versus placebo [3] Hazard ratio (90% CI) 0.2 (0.1, 1.0) Rate
(%) of being alive at 1 year 50.0 (11.1, 80.4) 83.3 (27.3, 97.5)
83.3 (27.3, 97.5) 83.3 (27.3, 97.5) 83.3 (56.8, 94.3) post-HCT (95%
CI) [4] Note: One year = Day 365 (+14 days) post-transplant (ie,
Study Day 380). Percentage was calculated using the number of
patients in the column heading as the denominator. [1] The 95% CI
for median was computed using the Brookmeyer and Crowley method
with log-log transformation. [2] Censoring was ignored in the
calculation for mean (SD) and median. A "+" after the min or max
indicates a censored observation. [3] Hazard ratio and 90% CI were
based on a Cox proportional hazards model with treatment as a
covariate. [4] Kaplan-Meier estimate. For Day 365, if the maximum
observed time was < Study Day 380, the Kaplan-Meier estimate at
the maximum observed time is presented for a treatment group. CI =
confidence interval; HCT = hematopoietic stem cell transplantation;
log = logarithm; max = maximum; min = minimum; NE = not estimable;
No. = number; SD = standard deviation.
[0140] Estimates of overall survival (OS) at about 800 days post
HCT for patients in the phase IIa study are also encouraging.
Overall survival (OS) was about 80% for patients in the CD24Fc
cohorts, 50% for patients in the placebo cohort (p=0.06) (FIG. 20),
and 50% for patients in the contemporary control (p=0.05) (FIG.
21). The improved OS in the CD24Fc exposed patients as compared to
the placebo and contemporary control patients supports the other
findings described above which show that administration of CD24Fc
in combination with methotrexate and tacrolimus may yield a
substantial improvement on the outcome in patients undergoing HCT
following myeloablative conditioning.
Acute Graft-Versus-Host Disease-Free Survival and Relapse Free
Survival (aGRFS) Through Day 180
[0141] Therapeutic strategies designed to prevent GVHD may result
in an increase in leukemia relapse due to a reduction in the Graft
Versus Leukemia (GVL) effect. As shown in Table 7, the incidence of
leukemia relapse in patients exposed to CD24Fc at Day 180 post HCT
(11%) is lower as compared to patients in the placebo group (33%)
and the contemporary control (23%). One subject in the 480 mg
CD24Fc cohort experienced relapse of CMML on Day 146 and one
subject in the multi-dose 960 mg CD24Fc cohort experienced relapse
of ALL on Day 100 post HCT. The patient with CMML passed away on
Day 196 due to leukemia. The patient with ALL relapse was treated
with blinatumomab, achieved complete remission, and was alive as of
the data cutoff of Aug. 8, 2018. In the placebo cohort, one patient
experienced relapse of CMML on Day 94 and one patient with MDS
relapsed on Day 146 (the patient with CMML passed away on Day 316
and the patient with MDS passed away on Day 184). These results
suggest CD24Fc does not interfere with the beneficial
graft-versus-tumor (GVT) process, and may even reduce the risk of
leukemia relapse.
[0142] The number of deaths in the CD24Fc cohorts at Day 180 post
transplant is lower than in the placebo and contemporary control
cohorts (Table 7). At Day 180 post HCT, there were no deaths in any
of the CD24Fc cohorts, one death due to pneumonia in the placebo
cohort (16.7%), and 22 deaths in the contemporary control (23.9%).
Statistically significant improvements in the composite endpoint of
aGVHD grade III-IV, relapse-free survival (RFS) are observed in the
CD24Fc cohorts (83%) as compared to the placebo group (33%) at Day
180 post HCT (P=0.011, see FIG. 18) and the contemporary control
(53%) at Day 180 post HCT (P=0.017, see FIG. 19). This sort of
endpoint has become increasingly popular because it in theory
encapsulates the effect of an intervention not only on GVHD
suppression but potential impact toxicity, infection and relapse.
In support of the observations above, improvements in grade III-IV
aGVHD, RFS show that administering CD24Fc in combination with a
standard or care methotrexate and tacrolimus following a
myeloablative conditioning regimen is beneficial to patients in
preventing the occurrence of aGVHD while not affecting the GVL
effects of the graft.
[0143] The aGRFS through Day 180 post-HCT is a post hoc composite
endpoint in which events included Grade III to IV acute GVHD,
relapse, or death from any cause. Table 14 summarizes the Grade III
to IV acute GRFS through Day 180 for the mITT Population.
[0144] The Kaplan-Meier estimate of the median Grade III to IV
acute GRFS was not reached for the CD24Fc treatment groups. For the
placebo group, the Kaplan-Meier estimate of the median Grade III to
IV acute GRFS (with 95% CI) was 120.0 (46.0, not estimable).
Overall, the Grade III to IV acute GRFS rate at Day 180 (with 95%
CI) was 83.3% (56.8%, 94.3%) for the CD24Fc treatment group and
33.3% (4.6%, 67.6%) for the placebo group. The hazard ratio (with
90% CI) for CD24Fc versus placebo was 0.2 (0.0, 0.6). Patients who
were alive and had no documented occurrence of Grade III to IV
acute GVHD, chronic GVHD requiring systemic immunosuppressive
therapy, or relapse at the data cutoff date were censored at the
last assessment date.
TABLE-US-00017 TABLE 14 Grade III to IV Acute Graft-Versus-Host
Disease-Free Survival and Relapse Free Survival Through Day 180 -
mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single
Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic (N =
6) (N = 6) (N = 6) (N = 6) (N =1 8) No. of patients with events (n,
%) 4 (66.7) 0 (0.0) 2 (33.3) 1 (16.7) 3 (16.7) Earliest
contributing event Acute GVHD (Grade III-IV) 1 (16.7) 0 (0.0) 1
(16.7) 0 (0.0) 1 (5.6) Relapse 2 (33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2
(11.1) Death 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. of
patients censored (n, %) 2 (33.3) 6 (100.0) 4 (66.7) 5 (83.3) 15
(83.3) Grade III-IV acute GVHD-free survival and relapse-free
survival (days) Kaplan-Meier estimate [1] Median (95% CI) 120.0
(46.0, NE) NE NE NE NE Mean (SD) [2] 120.8 (67.99) 195.0 (0.00)
158.3 (68.67) 179.2 (38.78) 177.5 (45.47) Median [2] 120.0 195.0
195.0 195.0 195.0 Min, max [2] 46, 195+ 195+, 195+ 24, 195+ 100,
195+ 24, 195+ Treatment comparison: CD24Fc versus placebo [3]
Hazard ratio (90% CI) 0.2 (0.0, 0.6) Rate (%) of being alive
without 33.3 (4.6, 67.6) 100.0 (NE) 66.7 (19.5, 90.4) 83.3 (27.3,
97.5) 83.3 (56.8, 94.3) Grade III-IV acute GVHD or relapse at Day
180 (95% CI) [4] Note: Day 180 = Day 180 (+14 days) post-transplant
(ie, Study Day 195). Percentage was calculated using the number of
patients in the column heading as the denominator. [1] The 95% CI
for median was computed using the Brookmeyer and Crowley method
with log-log transformation. [2] Censoring was ignored in the
calculation for mean (SD) and median. A "+" after the min or max
indicates a censored observation. [3] Hazard ratio and 90% CI were
based on a Cox proportional hazards model with treatment as a
covariate. [4] Kaplan-Meier estimate. CI = confidence interval;
GVHD = graft-versus-host disease; log = logarithm; max = maximum;
min = minimum; NE = not estimable; No. = number; SD = standard
deviation.
Incidence of Relapse 1 Year Following Hematopoietic Stem Cell
Transplantation
[0145] Table 15 summarizes the cumulative incidence of relapse 1
year post-HCT for the mITT Population. Overall, the cumulative
incidence rate of relapse at 1 year post-HCT (with 95% CI) was
11.1% (1.7%, 30.4%) for the CD24Fc treatment group and 33.3% (2.9%,
71.1%) for the placebo group. The hazard ratio (with 90% CI) for
CD24Fc versus placebo was 0.3 (0.1, 1.4). Patients who were alive
and did not experience relapse at the end of the follow-up period
(Day 365 [1 year]) were censored at the last date of evaluation. At
least 50.0% of patients in each treatment group were censored.
TABLE-US-00018 TABLE 15 Cumulative Incidence of Relapse 1 Year
Following Hematopoietic Stem Cell Transplantation - mITT Population
CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple
CD24Fc Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N =
6) (N = 6) (N = 18) No. of patients with relapse by 1 year 2 (33.3)
0 (0.0) 1 (16.7) 1 (16.7) 2 (11.1) post-HCT (n, %) No. of patients
who died without 1 (16.7) 1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6) relapse
by 1 year post-HCT (n, %) No. of patients censored (n, %) 3 (50.0)
5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3) Cumulative incidence (%) of
relapse 33.3 0.0 16.7 16.7 11.1 at 1 year post-HCT (95% CI) [1]
(2.9, 71.1) (NE) (0.5, 54.9) (0.5, 54.9) (1.7, 30.4) Treatment
comparison: CD24Fc versus placebo [2] Hazard ratio (90% CI) 0.3
(0.1, 1.4) Note: One year = Day 365 (+14 days) post-transplant (ie,
Study Day 380). Percentage was calculated using the number of
patients in the column heading as the denominator. [1] The
cumulative incidence (%) of relapse 1 year post-HCT and the 95% CI
were estimated using the cumulative incidence function with death
without relapse as a competing risk. For Day 365, if the maximum
observed time was < Study Day 380, the cumulative incidence at
the maximum observed time is presented for a treatment group. [2]
Hazard ratio and 90% CI were based on a Fine and Gray model with
treatment as a covariate and death without relapse as a competing
risk. CI = confidence interval; HCT = hematopoietic stem cell
transplantation; NE = not estimable; No. = number.
Graft-Versus-Host Disease-Free Survival and Relapse-Free Survival
(GRFS) 1 Year Following Hematopoietic Stem Cell Transplantation
[0146] This GRFS through 1 year post-HCT is a composite endpoint in
which events included Grade III to IV acute GVHD, chronic GVHD
requiring systemic immunosuppressive therapy, relapse, or death
from any cause. Table 16 summarizes Grade III to IV acute GRFS 1
year post-HCT for the mITT Population.
[0147] The Kaplan-Meier estimate of the median GRFS (with 95% CI)
was 229.0 days (141.0, not estimable) for the overall CD24Fc
treatment group: 247.0 days (129.0, not estimable) for the 240 mg
CD24Fc single dose cohort, 287.0 (24.0, not estimable) for the 480
mg CD24Fc single dose cohort, and 193.5 (100.0, not estimable) for
the 960 mg CD24Fc multiple dose cohort. The Kaplan-Meier estimate
of the median GRFS (with 95% CI) was 120.0 days (46.0, not
estimable) for the placebo group. Overall, the GRFS rate at 1 year
post-HCT (with 95% CI) was 32.4% (12.7%, 54.0%) for the CD24Fc
treatment group and 33.3% (4.6%, 67.6%) for the placebo group. The
hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.7 (0.3,
1.7). Patients who were alive and had no documented occurrence of
Grade III to IV acute GVHD, chronic GVHD requiring systemic
immunosuppressive therapy, or relapse at the data cutoff date were
censored at the last assessment date.
TABLE-US-00019 TABLE 16 Graft-Versus-Host Disease-Free Survival and
Relapse-Free Survival 1 Year Following Hematopoietic Stem Cell
Transplantation - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480
mg 960 mg Single Single Multiple CD24Fc Placebo Dose Dose Doses
Total Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of
patients with events (n, %) 4 (66.7) 4 (66.7) 4 (66.7) 4 (66.7) 12
(66.7) Earliest contributing event Acute GVHD (Grade III-IV) 1
(16.7) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6) Chronic GVHD requiring 0
(0.0) 3 (50.0) 2 (33.3) 3 (50.0) 8 (44.4) systemic
immunosuppressive therapy Relapse 2 (33.3) 0 (0.0) 1 (16.7) 1
(16.7) 2 (11-1) Death 1 (16.7) 1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6) No.
of patients censored (n, %) 2 (33.3) 2 (33.3) 2 (33.3) 2 (33.3) 6
(33.3) GVHD-free survival and relapse-free survival (days)
Kaplan-Meier estimate [1] Median (95% CI) 120.0 (46.0, NE) 247.0
(129.0, NE) 287.0 (24.0, NE) 193.5 (100.0, NE) 229.0 (141.0, NE)
Mean (SD) [2] 178.7 (151.48) 260.5 (99.54) 250.3 (149.68) 227.2
(123.63) 246.0 (119.19) Median [2] 120.0 247.0 287.0 193.5 229.0
Min, max [2] 46, 371+ 129, 378+ 24, 380+ 100, 376+ 24, 380+
Treatment comparison: CD24Fc versus placebo [3] Hazard ratio (90%
CI) 0.7 (0.3, 1.7) Rate (%) of being alive without 33.3 (4.6, 67.6)
33.3 (4.6, 67.6) 33.3 (4.6, 67.6) 33.3 (4.6, 67.6) 32.4 (12.7,
54.0) GVHD or relapse at 1 year post-HCT (95% CI) [4] Note: One
year = Day 365 (+14 days) post-transplant (ie, Study Day 380).
Percentage was calculated using the number of patients in the
column heading as the denominator. [1] The 95% CI for median was
computed using the Brookmeyer and Crowley method with log-log
transformation. [2] Censoring was ignored in the calculation for
mean (SD) and median. A "+" after the min or max indicates a
censored observation. [3] Hazard ratio and 90% CI were based on a
Cox proportional hazards model with treatment as a covariate. [4]
Kaplan-Meier estimate. CI = confidence interval; GVHD =
graft-versus-host disease; HCT = hematopoietic stem cell
transplantation; log = logarithm; max = maximum; min = minimum; NE
= not estimable; No. = number; SD = standard deviation.
[0148] Incidence of Non-Relapse Mortality 1 Year Following
Hematopoietic Stem Cell Transplantation
[0149] Table 17 summarizes the cumulative incidence of NRM 1 year
post-HCT for the mITT Population. Overall, the cumulative incidence
rate of NRM at 1 year (with 95% CI) was 5.6% (0.3%, 23.1%) for the
CD24Fc treatment group and 16.7% (0.5%, 54.9%) for the placebo
group. The hazard ratio (with 90% CI) for CD24Fc versus placebo was
0.3 (0.0, 2.8). Patients who were alive at the end of the follow-up
period (Day 365 [1 year]) without relapse were censored at the last
date they were known to be alive. At least 50.0% of patients in
each treatment group were censored. The cumulative incidence rate
of NRM at Day 180 (with 95% CI) was 0.0% for the CD24Fc treatment
group and 16.7% (0.5%, 54.9%) for the placebo group.
TABLE-US-00020 TABLE 17 Cumulative Incidence of Non-Relapse
Mortality 1 Year Following Hematopoietic Stem Cell Transplantation
- mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single
Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic (N =
6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients who died
without 1 (16.7) 1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6) relapse by 1 year
post-HCT (n, %) No. of patients with relapse by 2 (33.3) 0 (0.0) 1
(16.7) 1 (16.7) 2 (11-1) 1 year post-HCT (n, %) No. of patients
censored (n, %) 3 (50.0) 5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3)
Cumulative incidence (%) of 16.7 (0.5, 54.9) 16.7 (0.5, 54.9) 0.0
(NE) 0.0 (NE) 5.6 (0.3, 23.1) NRM at 1 year post-HCT (95% CI) [1]
Treatment comparison: CD24Fc versus placebo [2] Hazard ratio (90%
CI) 0.3 (0.0, 2.8) Note: One year = Day 365 (+14 days)
post-transplant (ie, Study Day 380). Percentage was calculated
using the number of patients in the column heading as the
denominator. [1] The cumulative incidence (%) of NRM at 1 year
post-HCT and the 95% CI were estimated using the cumulative
incidence function with relapse as a competing risk. For Day 365,
if the maximum observed time was < Study Day 380, the cumulative
incidence at the maximum observed time is presented for a treatment
group. [2] Hazard ratio and 90% CI were based on a Fine and Gray
model with treatment as a covariate and relapse as a competing
risk. CI = confidence interval; HCT = hematopoietic stem cell
transplantation; NE = not estimable; No. = number; NRM =
non-relapse mortality.
Incidence of Chronic Graft-Versus-Host Disease 1 Year Following
Hematopoietic Stem Cell
[0150] Table 18 summarizes the cumulative incidence of chronic GVHD
1 year post-HCT for the mITT Population. Overall, the cumulative
incidence rate of chronic GVHD at 1 year post-HCT (with 95% CI) was
63.3% (34.1%, 82.4%) for the CD24Fc treatment group and 33.3%
(2.5%, 72.0%) for the placebo group. The hazard ratio (with 90% CI)
for CD24Fc versus placebo was 2.1 (0.6, 7.4). There were 3 moderate
chronic GVHD in the 240 mg CD24Fc single dose cohort, 3 mild and 1
moderate chronic GVHD in the 480 mg CD24Fc single dose cohort, and
2 mild and 3 moderate chronic GVHD in the 960 mg CD24Fc multiple
doses cohort. Two patients had mild chronic GVHD in the placebo
group. Overall, there were no instances of severe chronic GVHD.
Patients who were alive and did not experience chronic GVHD at the
end of the follow-up period (Day 365 [1 year]) were censored at the
last date of evaluation.
TABLE-US-00021 TABLE 18 Cumulative Incidence of Chronic
Graft-Versus-Host Disease 1 Year Following Hematopoietic Stem Cell
Transplantation - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480
mg 960 mg Single Single Multiple CD24Fc Placebo Dose Dose Doses
Total Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of
patients with chronic GVHD 2 (33.3) 3 (50.0) 3 (50.0) 5 (83.3) 11
(61.1) by 1 year post-HCT (n, %) No. of patients who died without 3
(50.0) 1 (16.7) 1 (16.7) 1 (16.7) 3 (16.7) chronic GVHD by 1 year
post-HCT (n, %) No. of patients censored (n, %) 1 (16.7) 2 (33.3) 2
(33.3) 0 (0.0) 4 (22.2) Cumulative incidence (%) of 33.3 50.0 50.0
83.3 63.3 chronic GVHD at 1 year post-HCT [1] 95% CI (2.5, 72.0)
(7.0, 83.5) (6.9, 83.6) (0.5, 99.4) (34.1, 82.4) Treatment
comparison: CD24Fc versus placebo [2] Hazard ratio (90% CI) 2.1
(0.6, 7.41 Note: One year = Day 365 (+14 days) post-transplant (ie,
Study Day 380). Percentage was calculated using the number of
patients in the column heading as the denominator. [1] The
cumulative incidence (%) of chronic GVHD at 1 year post-HCT and the
95% CI were estimated using the cumulative incidence function with
death without chronic GVHD as a competing risk. If the maximum
observed time was < Study Day 380, the cumulative incidence at
the maximum observed time is presented for a treatment group. [2]
Hazard ratio and 90% CI were based on a Fine and Gray model with
treatment as a covariate and death without chronic GVHD as a
competing risk. CI = confidence interval; GVHD = graft-versus-host
disease; HCT = hematopoietic stem cell transplantation; No. =
number.
Rate of Infection at Day 100
[0151] As with the effect on GVL, therapeutic strategies designed
to prevent GVHD through global immune suppression may result in an
increase in infection rates, including bacterial infections and CMV
reactivation.
[0152] Table 19 summarizes the incidence of infections through Day
100 for the mITT Population. In total, 13 (72.2%) patients who
received CD24Fc (5 [83.3%] patients in the 240 mg CD24Fc single
dose cohort, 2 [33.3%] patients in the 480 mg CD24Fc single dose
cohort, and 6 [100.0%] patients in the 960 mg CD24Fc multiple dose
cohort) and 2 (33.3%) patients who received placebo had an
infection through Day 100.
[0153] Most infections were considered to be controlled and
resolved. Patient 103-001 in the placebo group died from pneumonia.
Patient 102-002 in the placebo group had conjunctivitis that was
reported as recovering/resolving. Patient 101-010 in the 480 mg
CD24Fc single dose cohort and Patient 101-011 in the 480 mg CD24Fc
single dose cohort both had rash pustular that was reported as not
recovered/not resolved. Patient 102-006 in the 960 mg CD24Fc
multiple dose cohort had upper respiratory tract infection and
Clostridium difficile colitis that were reported as intervention
continued.
[0154] The majority of the infections were bacterial (9 [50.0%]
patients who received CD24Fc and 2 [33.3%] patients who received
placebo) or viral (7 [38.9%] patients who received CD24Fc and 1
[16.7%] patient who received placebo). The majority of infections
occurred in the blood (8 [44.4%] patients who received CD24Fc and 1
[16.7%] patient who received placebo), urine (4 [22.2%] patients
who received CD24Fc and no patients who received placebo), or feces
(2 [11.1%] patients who received CD24Fc and 2 [33.3%] patients who
received placebo). The majority of the bacteria recovered from
blood culture were common skin inhabitants and low virulence
pathogens (ie, coagulase negative staphylococci).
TABLE-US-00022 TABLE 19 Summary of Incidence of Infections Through
Day 100 - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg
Single Single Multiple CD24Fc Placebo Dose Dose Doses Total (N = 6)
(N = 6) (N = 6) (N = 6) (N = 18) Statistic n (%) n (%) n (%) n (%)
n (%) No. of patients with any infections 2 (33.3) 5 (83.3) 2
(33.3) 6 (100.0) 13 (72.2) through Day 100 Type of infection
Bacterial 2 (33.3) 2 (33.3) 2 (33.3) 5 (83.3) 9 (50.0) Fungal 0
(0.0) 0 (0.0) 2 (33.3) 0 (0.0) 2 (11.1) Viral 1 (16.7) 3 (50.0) 1
(16.7) 3 (50.0) 7 (38.9) Site of infection Blood 1 (16.7) 2 (33.3)
2 (33.3) 4 (66.7) 8 (44.4) Disseminated (2 or more sites) 0 (0.0) 0
(0.0) 0 (0.0) 1 (16.7) 1 (5.6) Feces 2 (33.3) 1 (16.7) 0 (0.0) 1
(16.7) 2 (11.1) Gastrointestinal system 0 (0.0) 0 (0.0) 0 (0.0) 1
(16.7) 1 (5.6) Genitalia 0 (0.0) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6)
Lower respiratory system 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Skin 0 (0.0) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11.1) Upper respiratory
system 0 (0.0) 1 (16.7) 0 (0.0) 2 (33.3) 3 (16.7) Urine 0 (0.0) 1
(16.7) 1 (16.7) 2 (33.3) 4 (22.2) Percentage was calculated using
the number of patients in the column heading as the denominator.
No. = number.
[0155] As shown in Table 20, there were 9 patients in the CD24Fc
group that had high risk of CMV reactivation (Donor/Recipient CMV
status before HCT: D+/R+, 5; D-/R+, 3; unknown D/R+, 1). One
patient in the CD24Fc group with D+/R- had intermediate risk for
CMV reactivation. Eight patients in the CD24Fc group had status of
D-/R-, which was considered to be low risk. Two D-/R+ patients had
CMV reactivation at Day 42 and Day 48, representing 22.2%
cumulative incidence of CMV reactivation at Day 100 in the high
risk group. Both patients had systemic steroid treatment prior to
the detection of CMV reactivation. In comparison, 2 patients in the
placebo group were high risk of CMV reactivation (D+/R+, 1; D-/R+,
1). One patient in the placebo group had CMV reactivation at Day 47
before systemic steroid treatment for acute GVHD (50.0% in high
risk group).
[0156] CMV infection rates in HCT patients stratified by donor and
recipient CMV status before transplant. D=donor, R=recipient, +is
positive, - is negative, U is unknown.
TABLE-US-00023 Cytomegalovirus status CD24Fc Group Placebo Group
D+, R+ 5 1 D+, R- 1 0 D-, R+ 3 1 D-, R- 8 4 DU, R+ 1 0
[0157] Overall, CD24Fc was well tolerated in the phase IIa study.
There were no infusion-related toxicities. There was one possible
drug related TEAE.gtoreq.grade III in patients exposed to CD24Fc in
the 480 mg group of hyperglycemia, which was managed with insulin.
One dose-limiting toxicity (DLT) was observed in the placebo group,
and no DLTs were observed in the CD24Fc groups. There were no
adverse events leading to death in patients administered CD24Fc
within the 180 days (at least 150 days after the last dosing of
CD24Fc). There was one adverse event of pneumonia that led to the
death of a subject at Day 48 in the placebo group. One patient in
CD24Fc group died 7 months after HCT, though the death was
determined to be unlikely related to study drug. Anti-drug
antibodies (ADA) were not detected in any of the 24 patients at any
point out to day 100 after HCT.
[0158] The most common TEAEs.gtoreq.grade III (>10%) included a
decrease in platelet counts (83.3% placebo and 94.4% CD24Fc),
decrease in WBC counts (66.7% placebo and 88.9% CD24Fc), decrease
in neutrophil counts (50% placebo and 83.3% CD24Fc), decrease in
lymphocyte counts (50% placebo and 77.8% CD24Fc), anemia (50%
placebo and 66.7% CD24Fc), stomatitis (83.3% placebo and 50%
CD24Fc), and nausea (0% placebo and 11.1% CD24Fc). These SAEs are
consistent with the known safety profile of myeloablative
conditioning regimens used in HCT.
[0159] Myeloablative conditioning for HCT is often associated with
severe regimen related toxicity including organ failure. Organ
failure is the most frequent cause of early onset transplantation
related mortality (TRM) or non-relapse mortality (NRM). In the
CD24Fc group of 18 patients, none of the patients died within the
first 100 days post HCT, while 1 out of 6 in the placebo group died
on Day 48 due to respiratory failure.
Pharmacokinetic Results
[0160] FIGS. 22-23 show the PK data from the three escalation
cohorts from the Phase 2a trial. The half-life from the 240 and 480
mg single dose cohorts (FIG. 22) was around about 14 days, which is
consistent with the data seen in healthy subjects. At 480 mg there
was higher cmx but no real increase in exposure after 14 days, the
time of peek GVHD incidence and engraftment. In the final
multi-dose cohort there was increased exposure through day 60 as
expected (FIG. 23), the period during which patients are most
susceptible to develop GVHD.
[0161] Table 21 summarizes the plasma PK parameters of CD24Fc for
the PK Population in the single dose cohorts. The geometric mean
C.sub.max,-1 d values were 52,145.41 and 84,155.08 ng/mL, the
geometric mean AUC.sub.0-last,-1 d values were 10,156,549.9 and
15,522,686.2 ng h/mL, the geometric mean AUC.sub.0-42 d values were
9,275,562.3 and 13,903,718.4 ng h/mL, and the geometric mean
AUC.sub.0-inf values were 10,383,503.9 and 15,716,616.4 ng h/mL for
the 240 and 480 mg CD24Fc single dose cohorts, respectively. Median
t.sub.max,-1 d was 2.10 h for both the 240 and 480 mg CD24Fc single
dose cohorts. The mean values of t1/2 were 414.739 and 406.648 h
and the mean values of .lamda.z were 0.0018 and 0.0017 h-1 for the
240 and 480 mg CD24Fc single dose cohorts, respectively. The mean
Vz values were 13.83 and 18.18 L, and the mean CL values were 0.024
and 0.031 L/h for the 240 and 480 mg CD24Fc single dose cohorts,
respectively.
TABLE-US-00024 TABLE 20 Summary of Plasma Pharmacokinetic
Parameters of CD24Fc - PK Population - Single Dose Cohorts CD24Fc
CD24Fc 240 mg 480 mg Single Dose Single Dose PK Parameter (Unit) N
Statistic N Statistic C.sub.max, -1 d (ng/mL) [1] 6 52,145.41
(22.3) 6 84,155.08 (24.6) t.sub.max, -1 d (h) [2] 6 2.10 (2.1, 2.4)
6 2.10 (2.0, 2.2) AUC.sub.0-42 d (ng h/mL) [1] 6 9,275, 562.3
(23.2) 6 13,903, 718.4 (19.7) AUC.sub.0-last, -1 d (ng h/mL) [1] 6
10,156, 549.9 (26.2) 6 15,522,686.2 (21.3) AUC.sub.0-inf (ng h/mL)
[1] 6 10,383, 503.9 (25.2) 6 15,716, 616.4 (21.5) AUC.sub.extrap
(%) [3] 6 2.17 (1.669) 6 1.23 (0.519) .lamda..sub.2 (h.sup.-1) [3]
6 0.0018 (0.0005) 6 0.0017 (0.0002) t.sub.1/2 (h) [3] 6 414.739
(110.4483) 6 406.648 (62.3044) V.sub.z (L) [3] 6 13.83 (3.586) 6
18.18 (4.529) CL (L/h) [3] 6 0.024 (0.0059) 6 0.031 (0.0071)
Geometric CV % = 100*(exp(SD.sup.2) - 1).sup.0.5, where SD is the
standard deviation of the logarithm-transformed data. [1] Geometric
mean (geometric CV %) [2] Median (minimum, maximum) [3] Mean (SD)
.lamda..sub.2 = apparent terminal elimination rate constant;
AUC.sub.0-42 d = the area under the plasma concentration versus
time curve from time 0 to Day 42; AUC.sub.0-inf = the area under
the concentration versus time curve from time 0 extrapolated to
infinity; AUC.sub.0-last, -1 d = the area under the plasma
concentration versus time curve from time 0 to the last measurable
plasma drug concentration for Day -1 dosing; AUC.sub.extrap =
percentage of AUC.sub.0-inf that was due to extrapolation from the
last measurable plasma drug concentration to infinity; CL = total
body clearance after intravenous administration; C.sub.max, -1 d =
maximum observed plasma concentration for Day -1 dosing; CV % =
coefficient of variation; PK = pharmacokinetic; SD = standard
deviation; t.sub.1/2 = apparent terminal elimination half-life;
t.sub.max, -1 d = the time of maximum observed plasma concentration
for Day -1 dosing; V.sub.z = volume of distribution based on the
terminal elimination phase.
[0162] Table 22 summarizes the plasma PK parameters of CD24 Fc for
the PK population in the multiple dose cohort on Day -1, Day 28,
and Day -1 to Day 100. The geometric mean C.sub.max,-1 d and
C.sub.max,28 d values were 96,942.71 ng/mL and 62,563.05 ng/mL,
respectively, for the 960 mg CD24Fc multiple dose cohort. The
geometric mean AUC.sub.0-last,-1 d, AUC.sub.0-14 d, AUC.sub.0-100
d, and AUC.sub.0-last, overall values were 12,317,971.2 ng h/mL,
9,688,933.9 ng h/mL, 37,736,555.1 ng h/mL, and 37,363,953.5 ng
h/mL, respectively, for the 960 mg CD24Fc multiple dose cohorts.
The median t.sub.max,-1 d and t.sub.max,28 d were 2.13 h and 2.52
h, respectively, for the 960 mg CD24Fc multiple dose cohort.
TABLE-US-00025 TABLE 21 Summary of Plasma Pharmacokinetic
Parameters of CD24Fc - PK Population - Multiple Dose Cohort - Day
-1, Day 28, and Day -1 to Day 100 CD24Fc CD24Fc CD24Fc 960 mg 960
mg 960 mg Multiple Doses Multiple Doses Multiple Doses Day -1 Day
28 Day -1 to Day 100 PK Parameter (Unit) N Statistic N Statistic N
Statistic C.sub.max, -1 d (ng/mL) [1] 6 96,942.71 (41.5) -- -- --
-- t.sub.max, -1 d (h) [2] 6 2.13 (2.0, 3.2) -- -- -- --
AUC.sub.0-last, -1 d (ng h/mL) [1] 6 12,317,971.2 (24.9) -- -- --
-- C.sub.max, 28 d (ng/mL) [1] -- -- 6 62,563.05 (34.1) -- --
t.sub.max, 28 d (h) [2] -- -- 6 2.52 (2.0, 4.0) -- -- C.sub.min
(ng/mL) [1] -- -- 6 13,233.79 (33.6) -- -- T.sub.min (h) [2] -- --
6 0.00 (0.0, 308.2) -- -- AUC.sub.0-14 d (ng h/mL) [1] -- -- 6
9,688,933.9 (30.9) -- -- C.sub.avg (ng/mL) [1] -- -- 6 28,836.11
(30.9) -- -- Cl.sub.ss (L/h) [3] -- -- 6 0.026 (0.0078) -- --
AUC.sub.0-last, overall (ng h/mL) [1] -- -- -- -- 6 37,363,953.5
(27.6) AUC.sub.0-100 d (ng h/mL) [1] -- -- -- -- 6 37,736,555.1
(29.3) Geometric CV % = 100*(exp(SD.sup.2) - 1).sup.0.5, where SD
is the standard deviation of the logarithm-transformed data. [1]
Geometric mean (geometric CV %) [2] Median (minimum, maximum) [3]
Mean(SD) AUC.sub.0-14 d = the area under the plasma concentration
versus time curve from time 0 to Tau; AUC.sub.0-100 d = the area
under the concentration versus time curve from time 0 on Day -1 to
Day 100; AUC.sub.0-last, -1 d = the area under the plasma
concentration versus time curve from time 0 to the last measurable
plasma drug concentration for Day -1 dosing; AUC.sub.0-last,
overall = the area under the plasma concentration versus time curve
from time 0 on Day -1 to the last measurable plasma drug
concentration after the last dose on Day 28; C.sub.avg = calculated
as AUC.sub.0-14 d divided by Tau; Cl.sub.ss = calculated as
Dose/AUC.sub.0-14 d; C.sub.max, 1 d = maximum observed plasma
concentration for Day -1 dosing; C.sub.max, 28 d = maximum observed
plasma concentration between dose time and dose time + Tau for Day
28 dosing; C.sub.min = minimum concentration between dose time and
dose time + Tau (at the time of minimum concentration sampled
during a dosing interval); CV % = coefficient of variation; PK =
pharmacokinetic; SD = standard deviation; t.sub.max, -1 d = the
time of maximum observed plasma concentration for Day -1 dosing;
t.sub.max, 28 d = the time of maximum observed plasma concentration
during a dosing interval for Day 28 dosing; T.sub.min = time of
minimum concentration sampled during a dosing interval.
[0163] The clinical evidence from the phase IIa study strongly
suggests that CD24Fc, administered in combination with methotrexate
and tacrolimus, greatly improves outcomes in leukemia patients
undergoing myeloablative allo-HCT by reducing both the likelihood
of severe aGVHD (grades III-IV) and the likelihood of leukemia
relapse. As described above, the cumulative incidence of grade
III-IV aGVHD is 5.6% in CD24Fc exposed patients as compared to
16.7% in the placebo cohort (saline plus methotrexate and
tacrolimus) and 24% in the contemporary control cohort
(methotrexate and tacrolimus alone). These data suggest that
administration of CD24Fc in combination with methotrexate and
tacrolimus as prophylaxis reduces the risk of grade III-IV aGVHD in
HCT patients, the most serious grades of aGVHD which are associated
with increased risk of non-relapse mortality. A trend of reduction
is observed in the incidence of relapse in patients who received
CD24Fc (11.1%) as compared to patients who did not, both as
compared to the placebo arm (33.3%) and the contemporary control
(23%), demonstrating that CD24Fc does not affect the GVT effects of
the graft and may even reduce the risk of leukemia relapse. The
benefit of including CD24Fc in standard GVHD prophylaxis regimens
is further supported by the better NRM in CD24Fc exposed patients
(5.6%) as compared to placebo (16.7%), better 1.5-year overall
survival (89% versus 50%, CD24Fc versus placebo control), a
statistically significant improvement in grade III-IV aGVHD RFS
(83% versus 33%, CD24Fc versus placebo control, respectively), a
dose-dependent reduction in severe mucositis, and a good safety
profile with only one drug-related TEAE (grade III) observed in the
study.
[0164] A prophylaxis agent that reduces the risk of both aGVHD and
leukemia relapse would be novel and extremely beneficial to
leukemia patients undergoing allo-HCT following myeloablative
conditioning. As described above, the early clinical data in this
application strongly suggests that administration of CD24Fc in
combination with methotrexate and tacrolimus provides a substantial
improvement over existing prophylaxis regimens on the clinically
significant endpoints of grade III-IV aGVHD prevention and leukemia
relapse, and thus should be eligible for Breakthrough Designation.
The effects of CD24Fc observed in the phase IIa portion of the
clinical study will be further investigated in the phase IIb
portion, which has been designed to confirm the efficacy of
prophylactic CD24Fc administration in reducing Grade III-IV aGVHD
and leukemia relapse in leukemia patients undergoing allo-HCT
following myeloablative conditioning.
EXAMPLE 6
CD24 can be Used to Reduce Mucositis in Subjects Undergoing HCT
[0165] Myeloablative conditioning for HCT is often associated with
severe regimen related toxicity including grade 3-4 mucositis.
Severe oral mucositis has been reported by HCT patients as the most
distressing symptom they experienced. As a measure of the effect of
CD24Fc treatment on mucositis in subjects in the Phase IIa GVHD
prophylaxis trial described in Example 5, we generated a combined
mucositis scoring system to study the outcome. This data is shown
in FIG. 24A and comprises the multiple of the number of days
patients spent with serious (Grade 3 or 4) mucositis. The score is
provided in the bars, along with the number of pts that had the
mucositis in parentheses. Oral mucositis was scored according to
CTCAE 4.01. As shown in FIG. 24B, dose-dependent reductions in the
mucositis grade-day scores were observed across all CD24Fc cohorts
as compared to placebo (R=-0.9983; P=0.0009) and a statistically
significant reduction was observed in the multi-dose cohort
(placebo vs. 960 mg multi-dose: P=0.035, Student's t-test).
Sequence CWU 1
1
12131PRTHomo sapiensVARIANT(31)...(31)Valine or Alanine 1Ser Glu
Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn
Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr Lys Xaa 20 25
30230PRTHomo sapiens 2Ser Glu Thr Thr Thr Gly Thr Ser Ser Asn Ser
Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala Pro Asn Pro Thr Asn
Ala Thr Thr Lys 20 25 30327PRTMus musculus 3Asn Gln Thr Ser Val Ala
Pro Phe Pro Gly Asn Gln Asn Ile Ser Ala1 5 10 15Ser Pro Asn Pro Thr
Asn Ala Thr Thr Arg Gly 20 25426PRTHomo sapiens 4Met Gly Arg Ala
Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu
Leu Pro Thr Gln Ile Tyr Ser 20 255287PRTArtificialFusion protein
5Met Gly Arg Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5
10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr
Gly 20 25 30Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu
Ala Pro 35 40 45Asn Pro Thr Asn Ala Thr Thr Lys Pro Lys Ser Cys Asp
Lys Thr His 50 55 60Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val65 70 75 80Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 85 90 95Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 100 105 110Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 115 120 125Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 130 135 140Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys145 150 155
160Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
165 170 175Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 180 185 190Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 195 200 205Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 210 215 220Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser225 230 235 240Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 245 250 255Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280
2856261PRTArtificialFusion protein 6Ser Glu Thr Thr Thr Gly Thr Ser
Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala Pro Asn
Pro Thr Asn Ala Thr Thr Lys Pro Lys 20 25 30Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35 40 45Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 50 55 60Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val65 70 75 80Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 85 90 95Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 100 105
110Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
115 120 125Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 130 135 140Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro145 150 155 160Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln 165 170 175Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala 180 185 190Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 195 200 205Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 210 215 220Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser225 230
235 240Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser 245 250 255Leu Ser Pro Gly Lys 2607231PRTHomo sapiens 7Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 5 10 15Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25
30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
35 40 45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170
175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
2308288PRTArtificialFusion protein 8Met Gly Arg Ala Met Val Ala Arg
Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln
Ile Tyr Ser Ser Glu Thr Thr Thr Gly 20 25 30Thr Ser Ser Asn Ser Ser
Gln Ser Thr Ser Asn Ser Gly Leu Ala Pro 35 40 45Asn Pro Thr Asn Ala
Thr Thr Lys Val Pro Lys Ser Cys Asp Lys Thr 50 55 60His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser65 70 75 80Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 85 90 95Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 100 105
110Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
115 120 125Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val 130 135 140Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr145 150 155 160Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 165 170 175Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu 180 185 190Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 195 200 205Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 210 215 220Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp225 230
235 240Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 245 250 255Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala 260 265 270Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 275 280 2859288PRTArtificialFusion protein 9Met
Gly Arg Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5 10
15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr Gly
20 25 30Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu Ala
Pro 35 40 45Asn Pro Thr Asn Ala Thr Thr Lys Ala Pro Lys Ser Cys Asp
Lys Thr 50 55 60His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser65 70 75 80Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 85 90 95Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 100 105 110Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 115 120 125Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 130 135 140Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr145 150 155 160Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 165 170
175Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
180 185 190Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 195 200 205Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 210 215 220Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp225 230 235 240Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 245 250 255Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280
2851029PRTMacaca fascicularis 10Thr Val Thr Thr Ser Ala Pro Leu Ser
Ser Asn Ser Pro Gln Asn Thr1 5 10 15Ser Thr Thr Pro Asn Pro Ala Asn
Thr Thr Thr Lys Ala 20 2511262PRTArtificialFusion protein 11Ser Glu
Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn
Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr Lys Val Pro 20 25
30Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
35 40 45Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 50 55 60Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp65 70 75 80Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 85 90 95Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 100 105 110Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp 115 120 125Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 130 135 140Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu145 150 155 160Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 165 170
175Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
180 185 190Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 195 200 205Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 210 215 220Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys225 230 235 240Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 245 250 255Ser Leu Ser Pro Gly
Lys 26012262PRTArtificialFusion protein 12Ser Glu Thr Thr Thr Gly
Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser1 5 10 15Asn Ser Gly Leu Ala
Pro Asn Pro Thr Asn Ala Thr Thr Lys Ala Pro 20 25 30Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 35 40 45Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 50 55 60Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp65 70 75
80Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
85 90 95Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 100 105 110Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 115 120 125Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 130 135 140Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu145 150 155 160Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 165 170 175Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 180 185 190Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 195 200
205Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
210 215 220Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys225 230 235 240Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 245 250 255Ser Leu Ser Pro Gly Lys 260
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