Note: Descriptions are shown in the official language in which they were submitted.
WO 2023/086929
PCT/US2022/079702
SIRP1A - and CD4OL-BASED CHIMERIC PROTEINS
TECHNICAL FIELD
The present technology relates to, inter alia, compositions and methods,
including chimeric proteins that find
use in the treatment of disease, such as immunotherapies for cancer comprising
doses, dosing regimens
that including biphasic dosing or dosing regimens comprising three cycles.
PRIORITY
This application claims the benefit of, and priority to, U.S. Provisional
Application No. 63/278,567, filed
November 12, 2021; and U.S. Provisional Application No. 63/371,083, filed
August 11, 2022, the contents of
each of which are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
The instant application contains a sequence listing, which has been submitted
in XML format via EFS-Web.
The contents of the XML copy named "SHK-055PC_116981-5055_Sequence_Listing,"
which was created
on November 9, 2022, and is 82,547 bytes in size, and the contents of which
are incorporated herein by
reference in their entirety.
BACKGROUND
The field of cancer immunotherapy has grown tremendously over the past several
years. This has been
largely driven by the clinical efficacy of antibodies targeting the family of
checkpoint molecules (e.g., CTLA-
4 and PD-1/L1) in many tumor types. However, despite this success, clinical
response to these agents as
monotherapy occurs in a minority of patients (10-45% in various solid tumors),
and these therapies are
hindered by side effects.
Rational dose selection and optimization of dosing regimens are of clinical
importance and are prerequisites
for enhancing patients' medication compliance and obtaining maximum clinical
benefits. Developing dosing
regimen typically relies on pharmacokinetic/pharmacodynamic studies performed
in animal models.
However, since immunotherapy does not exert direct anti-proliferative activity
on cancer cells, but are instead
expected to harness tumor immunity, typically nnurine surrogates are used for
some animal studies, making
the pharmacokinetic/pharmacodynamic studies not available. In addition,
physicians may vary the dosing
regimen of immunotherapy based on the immunogenicity of a tumor, disease
stage, and physical status of
patients. Therefore, novel strategies to develop dosing and regimens are
required.
1
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
SUMMARY
Accordingly, in various aspects, the present disclosure provides compositions
and methods that are useful
for developing strategies for developing doses and dosing regimen of cancer
immunotherapy. The present
disclosure is based, in part, on the discoveries that CD8O-F cells, CD8-F
cells, Granzyme B+ cells, CD68+
cells, Ki67+ cells, and PD-L1+ immune cells increase in the tumor
microenvironment (TME) following the
administration of the SIRPa-Fc-CD4OL chimeric protein; B cells and/or 0040+
cells marginate from
peripheral blood within hours of dosing and return to peripheral blood within
days; and that the levels of
certain serum cytokines increase within hours of dosing and decrease within
days following the administration
of the SIRPa-Fc-CD4OL chimeric protein.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level of a cell has
zo been measured in a pre-dose biological sample obtained from the subject
before the administration of the
first dose, wherein the marker is selected from one or more of a CD8O-F cell,
a CD8-F cell, a Granzyme B+
cell, a C068+ cell, a Ki67+ cell, and a PD-L1+ immune cell. In embodiments,
the method further comprises
administering to the human subject a second dose of the chimeric protein if a
post-dosing level of the cell is
greater than the background level of the cell. In embodiments, the second dose
is administered at least about
48 hours after the administration of the first dose. In embodiments, the
biological sample is a biopsy sample
or a surgical specimen. In embodiments, the biological sample is a tumor
biopsy sample or a tumor surgical
specimen. In embodiments, the level of the cell is measured by RNA sequencing,
immunohistochemical
staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
2
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
of B cells and/or CD4O-F cells has been measured in a first biological sample
obtained from the subject before
the administration of the first dose. In embodiments, an N-hr post-dose level
and/or activity of B cells and/or
CD40+ cells has been measured in a second biological sample obtained from the
subject after the
administration of the first dose. In embodiments, N is a number between 1 and
24. In embodiments, the N-
hr post-dose level and/or activity of B cells and/or CD40+ cells is less than
the background level and/or
activity of B cells and/or CD40+ cells. In embodiments, an M-day post-dose
level and/or activity of B cells
and/or CD40+ cells has been measured in a third biological sample obtained
from the subject after the
administration of the first dose. In embodiments, M is a number between 1 and
28. In embodiments, the
method further comprises (ii) administering to the human subject a second dose
of the chimeric protein if the
M-day post-dose level and/or activity of B cells and/or CD40+ cells is at
least about 50% higher than the N-
hour post-dose level and/or activity of B cells and/or CD4O-F cells. In
embodiments, the second dose is
administered at least about 48 hours after the administration of the first
dose. In embodiments, the first
biological sample, the second biological sample and the third biological
sample are blood. In embodiments,
the level and/or activity of B cells and/or CD40+ cells is measured by RNA
sequencing, immunohistochemical
zo staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background amount and/or
activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNa, ILI 5, IL23,
IL-12, MCP-1, MIP-1[3, MIP-
1a, and MDC has been measured in a first biological sample obtained from the
subject before the
administration of the first dose. In embodiments, a N-hr post-dose amount
and/or activity of the cytokine has
been measured in a second biological sample obtained from the subject after
the administration of the first
dose. In embodiments, N is a number between 1 and 24. In embodiments, the N-hr
post-dose amount and/or
activity of the cytokine is greater than the background amount and/or activity
of the cytokine. In embodiments,
the M-day post-dose amount and/or activity of the cytokine has been measured
in a third biological sample
obtained from the subject after the administration of the first dose. In
embodiments, M is a number between
3
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
1 and 28. In embodiments, the method further comprises (ii) administering to
the human subject a second
dose of the chimeric protein if the M-day post-dose amount and/or activity of
the cytokine is at least about
30% lower than the N-hr post-dose amount and/or activity of the cytokine. In
embodiments, the second dose
is administered at least about 48 hours after the administration of the first
dose. In embodiments, the first
biological sample, the second biological sample and the third biological
sample are blood. In embodiments,
the amount and/or activity of the cytokine is measured by RNA sequencing,
immunohistochemical staining,
western blotting, in cell western, immunofluorescent staining, [LISA, and
fluorescent activating cell sorting
(FACS) or a combination thereof.
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
is obtaining a biological sample obtained from the subject that has
received a first dose of a chimeric protein.
In embodiments, the chimeric protein has a general structure of: N terminus ¨
(a) ¨ (b) ¨ (c) ¨ C terminus,
wherein: (a) is a first domain comprising an extracellular domain of human
signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
zo ligand (CD4OL). In embodiments, a background level and/or activity of a
cell has been measured in a
biological sample obtained from the subject before the administration of the
first dose. In embodiments, the
cell is selected from one or more of a CD8O-F cell, a CD8+ cell, a Granzyme B+
cell, a CD68+ cell, a Ki67+
cell, and a PD-L1+ immune cell. In embodiments, the method further comprises
(ii) determining a post-dose
level and/or activity of the cell in the biological sample. In embodiments,
the method further comprises (iii)
25 determining that the chimeric protein is efficacious if the post-dose
level and/or activity of the cell is greater
than the background level and/or activity of the cell. In embodiments, the
biological sample is a tumor biopsy
sample or a tumor surgical specimen. In embodiments, the level and/or activity
of the cell is measured by
RNA sequencing, immunohistochemical staining, western blotting, in cell
western, immunofluorescent
staining, ELISA, and fluorescent activating cell sorting (FACS) or a
combination thereof.
30 An aspect of the present disclosure is a method of selecting a subject
for treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a biological
sample obtained from the subject
that has received a first dose of a chimeric protein. In embodiments, the
chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
35 domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c)
is a second domain comprising
4
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
of a cell has been measured in a biological sample obtained from the subject
before the administration of the
first dose. In embodiments, the cell is selected from one or more of a CD80+
cell, a CD8+ cell, a Granzyme
B+ cell, a CD68+ cell, a Ki67+ cell, and a PD-L1+ immune cell. In embodiments,
the method further
comprises (ii) determining a post-dose level and/or activity of the cell in
the biological sample. In
embodiments, the method further comprises (iii) selecting the subject for
treatment with the chimeric protein
if the post-dose level and/or activity of the cell is greater than the
background level and/or activity of the cell.
In embodiments, the biological sample is a tumor biopsy sample or a tumor
surgical specimen. In
embodiments, the level and/or activity of the cell is measured by RNA
sequencing, immunohistochemical
staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof.
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
obtaining a first biological sample obtained from the subject that has
received a first dose of a chimeric
protein. In embodiments, the chimeric protein has a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
zo terminus, wherein: (a) is a first domain comprising an extracellular
domain of human signal regulatory protein
a (CD172a (SI RPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
ligand (CD4OL). In embodiments, the method further comprises determining a
background level and/or
activity of B cells and/or CD40+ cells in the first biological sample . In
embodiments, the method further
comprises (iii) obtaining a second biological sample obtained from the subject
at N hr post-dose, wherein N
is a number between 1 and 24. In embodiments, the method further comprises
determining an N-hr post-
dose level and/or activity of B cells and/or CD40+ cells in the second
biological sample . In embodiments,
the method further comprises (iv) obtaining a third biological sample obtained
from the subject at M days
post-dose, wherein M is a number between 1 and 28. In embodiments, the method
further comprises
determining an M-day post-dose level and/or activity of B cells and/or 0D40-F
cells in the third biological
sample . In embodiments, the method further comprises (v) determining that the
chimeric protein is
efficacious if the N hr post-dose level and/or activity of B cells and/or
CD40+ cells is less than the background
level and/or activity of B cells and/or CD40+ cells, and/or if the M day post-
dose level and/or activity of B cells
and/or CD40+ cells is at least about 50% higher than the N-hour post-dose
level and/or activity of B cells
and/or CD40+ cells. In embodiments, the first biological sample, the second
biological sample and the third
5
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
biological sample are blood. In embodiments, the level and/or activity of B
cells and/or CD40+ cells is
measured by RNA sequencing, immunohistochemical staining, western blotting, in
cell western,
immunofluorescent staining, ELISA, and fluorescent activating cell sorting
(FAGS) or a combination thereof.
An aspect of the present disclosure is a method of selecting a subject for
treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a first biological
sample obtained from the
subject that has received a first dose of a chimeric protein. In embodiments,
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human 0040 ligand (CD4OL). In embodiments, the
method further comprises
is determining a background level and/or activity of B cells and/or CD4O-F
cells in the first biological sample. In
embodiments, the method further comprises (iii) obtaining a second biological
sample obtained from the
subject at N hr post-dose, wherein N is a number between 1 and 24. In
embodiments, the method further
comprises determining an N-hr post-dose level and/or activity of B cells
and/or CD4O-F cells in the second
biological sample . In embodiments, the method further comprises (iv)
obtaining a third biological sample
zo obtained from the subject at M days post-dose, wherein M is a number
between 1 and 28. In embodiments,
the method further comprises determining an M-day post-dose level and/or
activity of B cells and/or CD40+
cells in the third biological sample . In embodiments, the method further
comprises (v) selecting the subject
for treatment with the chimeric protein if the N hr post-dose level and/or
activity of B cells and/or CD40+ cells
is less than the background level and/or activity of B cells and/or 0D40+
cells, and/or if the M day post-dose
25 level and/or activity of B cells and/or 0040+ cells is at least about
50% higher than the N-hour post-dose
level and/or activity of B cells and/or 0D40+ cells. In embodiments, the first
biological sample, the second
biological sample and the third biological sample are blood. In embodiments,
the level and/or activity of B
cells and/or 0040+ cells is measured by RNA sequencing, immunohistochemical
staining, western blotting,
in cell western, immunofluorescent staining, ELISA, and fluorescent activating
cell sorting (FACS) or a
30 combination thereof.
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
obtaining a first biological sample obtained from the subject that has
received a first dose of a chimeric
protein. In embodiments, the chimeric protein has a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
35 terminus, wherein: (a) is a first domain comprising an extracellular
domain of human signal regulatory protein
6
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
a (CD172a (SI RPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
ligand (CD4OL). In embodiments, the method further comprises (ii) determining
in the first biological sample
a background amount and/or activity of a cytokine. In embodiments, the
cytokine is selected from CCL2,
CXCL9, CXCL10, IFNa, IL15, IL23, IL-12, MCP-1, MIP-113, MIP-la, and MDC In
embodiments, the method
further comprises (iii) obtaining a second biological sample obtained from the
subject at N hr post-dose,
wherein N is a number between 1 and 24. In embodiments, the method further
comprises determining an N-
hr post-dose amount and/or activity of the cytokine in the second biological
sample. In embodiments, the
method further comprises (iv) obtaining a third biological sample obtained
from the subject at M days post-
dose, wherein M is a number between 1 and 28. In embodiments, the method
further comprises determining
an M-day post-dose amount and/or activity of the cytokine in the third
biological sample. In embodiments,
the method further comprises (v) determining that the chimeric protein is
efficacious if the N-hr post-dose
amount and/or activity of the cytokine is greater than the background amount
and/or activity of the cytokine,
and/or if the M-day post-dose amount and/or activity of the cytokine is at
least about 50% lower than the N-
hour post-dose amount and/or activity of the cytokine. In embodiments, the
first biological sample, the second
zo biological sample and the third biological sample are blood. In
embodiments, the amount and/or activity of
the cytokine is measured by RNA sequencing, immunohistochemical staining,
western blotting, in cell
western, immunofluorescent staining, ELISA, and fluorescent activating cell
sorting (FACS) or a combination
thereof.
An aspect of the present disclosure is a method of selecting a subject for
treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a first biological
sample obtained from the
subject that has received a first dose of a chimeric protein. In embodiments,
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, the
method further comprises (ii)
determining in the first biological sample a background amount and/or activity
of a cytokine. In embodiments,
the cytokine is selected from CCL2, CXCL9, CXCL10, IFNa, 1L15, 11_23, IL-12,
MCP-1, MIP-1 13, MIP-la, and
MDC In embodiments, the method further comprises (iii) obtaining a second
biological sample obtained from
the subject at N hr post-dose, wherein N is a number between 1 and 24. In
embodiments, the method further
comprises determining an N-hr post-dose amount and/or activity of the cytokine
in the second biological
7
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
sample. In embodiments, the method further comprises (iv) obtaining a third
biological sample obtained from
the subject at M days post-dose, wherein M is a number between 1 and 28. In
embodiments, the method
further comprises determining an M-day post-dose amount and/or activity of the
cytokine in the third biological
sample. In embodiments, the method further comprises (v) selecting the subject
for treatment with the
chimeric protein if the N-hr post-dose amount and/or activity of the cytokine
is greater than the background
amount and/or activity of the cytokine, and/or if the M-day post-dose amount
and/or activity of the cytokine is
at least about 50% lower than the N-hour post-dose amount and/or activity of
the cytokine. In embodiments,
the first biological sample, the second biological sample and the third
biological sample are blood. In
embodiments, the amount and/or activity of the cytokine is measured by RNA
sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
fluorescent activating cell sorting (FACS) or a combination thereof.
In embodiments, the first domain is capable of binding a CD172a (SIRPa)
ligand. In embodiments, the first
domain comprises substantially all of the extracellular domain of CD172a
(SIRPa). In embodiments, the first
domain comprises an amino acid sequence that is at least about 90%, or at
least about 95%, or at least about
96%, or at least about 97%, or at least about 98%, or at least about 99%
identical to the amino acid sequence
zo of SEQ ID NO: 57.
In embodiments, the second domain is capable of binding a 0040 receptor. In
embodiments, the second
domain comprises substantially all of the extracellular domain of CD4OL. In
embodiments, the second domain
comprises an amino acid sequence that is at least about 90%, or at least about
95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about 99% identical
to the amino acid sequence of
SEQ ID NO: 58.
In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from
IgG4. In embodiments, the
linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. In
embodiments, the linker
comprises an amino acid sequence that is at least about 90%, or at least about
95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about 99% identical
to the amino acid sequence of
SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
In embodiments, (a) the first domain comprises the amino acid sequence of SEQ
ID NO: 57, (b) the second
domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the linker
comprises an amino acid
sequence that is at least about 90%, or at least about 95%, or at least about
96%, or at least about 97%, or
at least about 98%, or at least about 99% identical to the amino acid sequence
of SEQ ID NO: 1, SEQ ID
8
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
s NO: 2, or SEQ ID NO: 3. In embodiments, the chimeric protein further
comprises the amino acid sequence
of SEQ ID NO: 5 or SEQ ID NO: 7. In embodiments, the chimeric protein further
comprises the amino acid
sequence of SEQ ID NO: 5 and SEQ ID NO: 7.
In embodiments, the chimeric protein comprises an amino acid sequence that is
at least about 90%, or at
least about 95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%
identical to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61.
In embodiments, the first dose of the chimeric protein is in the range of from
about 0.03 mg/kg to 10 mg/kg.
In embodiments, the first dose is about 0.003, or about 0.01, or about 0.03,
or about 0.1, or about 0.3, or
about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or about
10 mg/kg.
In embodiments, the method of any of the aspects disclosed herein further
comprises administration of a
second dose of the chimeric protein. In embodiments, the second dose is
administered at least about 3 days,
or at least about 4 days, or at least about 5 days, or at least about 6 days,
or at least about 7 days, or at least
about 8 days, or at least about 9 days, or at least about 10 days, or at least
about 14 days, or at least about
21 days, or at least about 28 days after the administration of the first dose.
In embodiments, the second dose
of the chimeric protein is in the range of from about 0.03 mg/kg to 10 mg/kg.
In embodiments, the second
zo dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or
about 0.3, or about 1, or about 2, or about
3, or about 4, or about 6, or about 8, or about 10 mg/kg.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in
color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon request
and payment of the necessary fee.
FIG. 1 shows the diagrammatic representation of the SIRPa-Fc-CD4OL chimeric
protein (SL-172154). SL-
172154 (SIRPa-Fc-CD4OL) is a hexameric, bi-functional fusion protein
consisting of SIRPa (binding affinity
to CD47 is 0.628 nM) linked to CD4OL (binding affinity to CD40 is 4.74 nM)
through an Fc linker protein
FIG. 2 shows the Dose Escalation per modified Toxicity Probability (mTPI-2)
Design (N=15). The planned
dose escalation is in half-log increments. At least 3 subjects were enrolled
into sequential dose levels (DL)
and evaluated for dose limiting toxicity (DLT) in the first cycle of
treatment. Subjects receive intravenous (IV)
administration of SL-172154 on Schedule 1 or Schedule 2 until disease
progression, unacceptable toxicity,
or withdrawal of consent.
9
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
FIG. 3 shows the tumor response and duration of treatment iUPD = unconfirmed
progressive disease
(iRECIST) NE = non-evaluable; PD = progressive disease; SD = stable disease
FIG. 4A to FIG. 4D show the reproducible increases in serum cytokines
following repeated dosing of SL-
172154. The changes in the levels of CCL2 (MCP-1) (FIG. 4A), CCL4 (MIP-113)
(FIG. 4B), CCL3 (MIP-1a)
(FIG. 4C), and CCL22 (MDC) (FIG. 4D) are shown.
FIG. 5A and FIG. 5B show the dose-dependent and reproducible increases in
Serum IL-12. FIG. 5A shows
the subject level interleukin 12 (IL-12), a mediator of TH1 proinflammatory
responses over time typify the
cyclic effector cytokine responses observed in study subjects. FIG. 5B shows
median responses at the first
infusion (horizontal bars), preliminarily appear to be dose dependent.
FIG. 6A and FIG. 6B demonstrate the SL-172154 preferentially binds CD47 on
leukocytes but not RBCs.
FIG. 6A shows the CD47 receptor occupancy (RO) as evaluated by fluorescence
activated cell sorting
(FACS) analysis using whole blood on both red blood cells (RBC) and white
blood cells (WBC, leukocytes).
At one-hour post infusion on Cycle 1 Day 1 (C1 D1), median CD47 RO on
leukocytes (horizontal bars) is
-80%. FIG. 6B shows the CD47 RO on RBC is <5% for all dose levels.
FIG. 7A to FIG. 7C demonstrate that SL-172154 stimulates dose-dependent B cell
margination and
zo activation. FIG. 7A shows the median frequency of marginating cells
increases in a dose-dependent manner
(horizontal bars). Receptor engagement is -100% at all dose levels (data not
shown). FIG. 7B shows the
median B cell frequencies return to pre-infusion levels by the next infusion,
maintaining a cyclic pattern of
egress and return with each infusion cycle. FIG. 7C shows the returning B
cells exhibit increases in the co-
stimulatory marker 0D86, as well as the maturation marker 0D95, suggesting
that SL-172154 can induce
phenotypic changes.
FIG. 8A and FIG. 8B show the distinct profile of TNFa and Interleukin-6 (IL-6)
relative to CD40 mAbs. FIG.
8A shows the induction of TNFa at various doses of CP-870,893 (left panel) or
SL-172154 (right panel). FIG.
8B shows the induction of IL-6 at various doses of CP-870,893 (left panel) or
SL-172154 (right panel). The
CP-870,893 data are from Vonderheide etal., J Clin Once! 25:876-883 (2007).
FIG. 9A and FIG. 9B demonstrate that SL-172154 induces innate immune response
in tumor
microenvironment (TME). FIG. 9A shows the immunohistochemistry analysis of
biopsy sample from patient
A before and after the administration of SL-172154. Monocytes were detected by
staining for CD68 (a protein
highly expressed by cells in the monocyte lineage). FIG. 9B demonstrates the
upregulation of activation
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
markers, CD40 and MHC Class II in TME in tumor biopsy sample after the
treatment with SL-172154,
compared to pretreatment biopsy samples.
FIG. 10A and FIG. 10B demonstrate that SL-172154 induces adaptive immune
response in tumor
microenvironment (TME). FIG. 10A shows the CD8+ cells, Granzyme B + cells,
CD68-F cells, and Ki67+ cells
in biopsy sample from patient A before and after the administration of SL-
172154. The CD8+ cells, Granzyme
B+ cells, 0D68+ cells, and Ki67+ cells are increased in post-treatment biopsy
sample, compared to pre-
treatment biopsy sample. FIG. 10B is a plot comparing the tumor proportion
score (TPS) and combined
positive score (CPS).
FIG. 11 shows the planned clinical development strategy for SL-172154. The
strategy includes a trial for SL-
172154 monotherapy in ovarian cancer, combination therapy of SL-172154 +
liposomal doxorubicin in
ovarian cancer, combination therapy of SL-172154 azaciti di ne venetoclax in
AML, combination therapy
of SL-172154 + azacitidine in HR-MDS, and combination therapy of SL-172154 +
azaciti di ne in TP53 mutant
AML.
FIG. 12 shows increase in the abundance of CD80+ cells and/or CD80 expression
in tumor following
administration of SL-172154.
DETAILED DESCRIPTION
The present disclosure is based, in part, on the discovery that CD8O-F cells,
CD8-F cells, Granzyme B+ cells,
CD68+ cells, Ki67+ cells, and PD-L1+ immune cells increase in the tumor
microenvironment (TME) following
the administration of the SIRPa-Fc-CD4OL chimeric protein, which is well-
tolerated with no DLTs or evidence
of anemia, thrombocytopenia, liver dysfunction, cytokine release syndrome or
pneumonitis. The present
disclosure is also based, in part, on the discoveries that B cells and/or
CD40+ cells marginate from peripheral
blood and the levels of innate and adaptive serum cytokines such as CCL2,
CXCL9, CXCL10, IFNa, IL15,
IL23, IL-12, MCP-1, MIP-113, MIP-la, and MDC increase within hours of
following the administration of the
SI RPa-Fc-CD4OL chimeric protein, and that the B cells and/or CD40+ cells
return to peripheral blood as well
as the levels of innate and adaptive serum cytokines such as CCL2, CXCL9,
CXCL10, IFNa, 1L15, 1L23, IL-
12, MCP-1, MIP-13, MI P-1a, and MDC decrease within about a day following the
administration of the SIR Pa-
Fc-CD4OL chimeric protein. In some embodiments, the levels of B cells and/or
CD40+ cells, as well as the
levels of innate and adaptive serum cytokines such as CCL2, CXCL9, CXCL10,
IFNa, IL15, IL23, IL-12,
MCP-1, MIP-13, MI P-1a, and MDC get close to the background levels within a
day or two of the administration
of the SIRPa-Fc-CD4OL chimeric protein.
11
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Importantly, since a chimeric protein of the present disclosure (via binding
of the extracellular domain of
CD172a (SIRPa) to its receptor/ligand on a cancer cell) disrupts, blocks,
reduces, inhibits, and/or sequesters
the transmission of immune inhibitory signals, e.g., originating from a cancer
cell that is attempting to avoid
its phagocytosis and/or destruction, and (via binding of CD4OL to its
receptor) enhances, increases, and/or
stimulates the transmission of an immune stimulatory signal to an anti-cancer
immune cell, it can provide an
io anti-tumor effect by two distinct pathways; this dual-action is more
likely to provide any anti-tumor effect in a
patient and/or to provide an enhanced anti-tumor effect in a patient.
Furthermore, since such chimeric
proteins can act via two distinct pathways, they can be efficacious, at least,
in patients who respond poorly
to treatments that target one of the two pathways. Thus, a patient who is a
poor responder to treatments
acting via one of the two pathway, can receive a therapeutic benefit by
targeting the other pathway.
is Chimeric Proteins
The chimeric proteins of the present disclosure comprise an extracellular
domain of CD172a (SIRPa) and an
extracellular domain of CD4OL which together can simultaneously block immune
inhibitory signals and
stimulate immune activating signals.
Aspects of the present disclosure provide a chimeric protein comprising a
general structure of: N terminus ¨
20 (a) ¨ (b) ¨ (c) ¨ C terminus, where (a) is a first domain comprising an
extracellular domain of CD172a (SIRPa),
(b) is a linker adjoining the first domain and the second domain, e.g., the
linker comprising at least one
cysteine residue capable of forming a disulfide bond and/or comprising a hinge-
CH2-CH3 Fc domain, and
(c) is a second domain comprising an extracellular domain of CD4OL; wherein
the linker connects the first
domain and the second domain.
25 In embodiments, the first domain comprises substantially all of the
extracellular domain of CD172a (SIRPa).
In embodiments, the first domain is capable of binding a CD172a (SIRPa)
ligand. In embodiments, the first
domain is capable of binding a CD172a (SIRPa) ligand (e.g. 0D47) expressed on
cancer cell surface. In
embodiments, the first domain is capable of inhibiting the binding of a CD172a
(SIRPa) ligand (e.g. CD47)
to the CD172a (SIRPa) protein located on myeloid and hematopoietic stem cells
and neurons. In
30 embodiments, the first domain is capable of inhibiting an
immunosuppressive signal. In embodiments, the
first domain is capable of inhibiting an immunosuppressive signal. In
embodiments, the first domain is capable
of inhibiting a macrophage checkpoint or "do not eat me" signal. In
embodiments the therapy with the SIRPa-
Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61) stimulates
macrophages to phagocytize
tumor cells and effectively present the tumor antigens of phagocytized tumor
cells to T cells.
12
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the second domain is capable of binding a CD40 receptor. In
embodiments, the second
domain comprises substantially all of the extracellular domain of CD4OL. In
embodiments, the second domain
is capable of activating an immune stimulatory signal.
In embodiments, the chimeric protein is a recombinant fusion protein, e.g., a
single polypeptide having the
extracellular domains disclosed herein. For example, in embodiments, the
chimeric protein is translated as a
single unit in a prokaryotic cell, a eukaryotic cell, or a cell-free
expression system.
In embodiments, the present chimeric protein is producible in a mammalian host
cell as a secretable and fully
functional single polypeptide chain.
In embodiments, chimeric protein refers to a recombinant protein of multiple
polypeptides, e.g., multiple
extracellular domains disclosed herein, that are combined (via covalent or non-
covalent bonding) to yield a
single unit, e.g., in vitro (e.g., with one or more synthetic linkers
disclosed herein).
In embodiments, the chimeric protein is chemically synthesized as one
polypeptide or each domain is
chemically synthesized separately and then combined. In embodiments, a portion
of the chimeric protein is
translated and a portion is chemically synthesized.
In embodiments, an extracellular domain refers to a portion of a transmembrane
protein which is capable of
zo interacting with the extracellular environment. In embodiments, an
extracellular domain refers to a portion of
a transmembrane protein which is sufficient for binding to a ligand or
receptor and is effective in transmitting
a signal to a cell. In embodiments, an extracellular domain is the entire
amino acid sequence of a
transmembrane protein which is normally present at the exterior of a cell or
of the cell membrane. In
embodiments, an extracellular domain is that portion of an amino acid sequence
of a transmembrane protein
which is external of a cell or of the cell membrane and is needed for signal
transduction and/or ligand binding
as may be assayed using methods know in the art (e.g., in vitro ligand binding
and/or cellular activation
assays).
Transmembrane proteins typically consist of an extracellular domain, one or a
series of transmembrane
domains, and an intracellular domain. Without wishing to be bound by theory,
the extracellular domain of a
transmembrane protein is responsible for interacting with a soluble receptor
or ligand or membrane-bound
receptor or ligand (i.e., a membrane of an adjacent cell). Without wishing to
be bound by theory, the trans-
membrane domain(s) is responsible for localizing the transmembrane protein to
the plasma membrane.
Without wishing to be bound by theory, the intracellular domain of a
transmembrane protein is responsible
13
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
for coordinating interactions with cellular signaling molecules to coordinate
intracellular responses with the
extracellular environment (or visa-versa).
There are generally two types of single-pass transmembrane proteins: Type I
transmembrane proteins which
have an extracellular amino terminus and an intracellular carboxy terminus and
Type II transmembrane
proteins which have an extracellular carboxy terminus and an intracellular
amino terminus. Type I and Type
II transmembrane proteins can be either receptors or ligands. For Type I
transmembrane proteins (e.g.,
CD172a (SIRPa)), the amino terminus of the protein faces outside the cell, and
therefore contains the
functional domains that are responsible for interacting with other binding
partners (either ligands or receptors)
in the extracellular environment. For Type II transmembrane proteins (e.g.,
CD4OL), the carboxy terminus of
the protein faces outside the cell, and therefore contains the functional
domains that are responsible for
interacting with other binding partners (either ligands or receptors) in the
extracellular environment. Thus,
these two types of transmembrane proteins have opposite orientations to each
other relative to the cell
membrane.
The description of CD47 as a "do not eat me" signal in a broad range of
cancers stimulated exploration of
what combinations of "eat me" signals may enhance antitumor immunity in the
setting of CD47 blockade.
zo Willingham et al., The 0D47-signal regulatory protein alpha (SIRPa)
interaction is a therapeutic target for
human solid tumors. Proc Nat! Aced Sci U S A 109: 6662-6667 (2012); Jaiswal
etal., C047 is upregulated
on circulating hematopoietic stem cells and leukemia cells to avoid
phagocytosis. Cell 138:271-285 (2009);
Weiskopf et al., Engineered SIRPalpha variants as immunotherapeutic adjuvants
to anticancer antibodies.
Science 341:88-91 (2013). Preclinical combinations of CD47 blockade and ADCP-
competent antibodies,
including rituximab and trastuzumab, enhance tumor phagocytosis. Kauder etal.,
ALX148 blocks CD47 and
enhances innate and adaptive antitumor immunity with a favorable safety
profile. PLoS One 13: e0201832
(2018); Chao et al., Anti- CD47 antibody synergizes with rituximab to promote
phagocytosis and eradicate
non-Hodgkin lymphoma. Cell 142:699-713 (2010); Chao etal., Calreticulin is the
dominant pro-phagocytic
signal on multiple human cancers and is counterbalanced by CD47. Sci Transl
Med 2:63ra94 (2010); Advani
etal., CD47 Blockade by Hu5F9-G4 and rituximab in non-Hodgkin's lymphoma. N
Engl J Med 379:1711-
1721 (2018); Zhao et al., CD47-signal regulatory protein-alpha (SIRPalpha)
interactions form a barrier for
antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A 108:18342-
18347 (2011). At least 50%
of patients with relapsed or refractory diffuse large B-cell lymphoma or
follicular lymphoma treated with
Hu5F9-G4, a humanized, IgG4 isotype, CD47-blocking mAb, in combination with
rituximab demonstrate
objective responses. Advani etal., CD47 Blockade by Hu5F9-G4 and rituximab in
non-Hodgkin's lymphoma.
14
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
N Engl J Med 379:1711-1721 (2018). CD47 blockade enhances antigen presentation
in immune-neglected
tumors (Tseng et al., Anti-CD47 antibody-mediated phagocytosis of cancer by
macrophages primes an
effective antitumor 1-cell response. Proc Natl Acad Sci U S A 110:11103-11108
(2013)), yet only sporadic
clinical responses have been observed using CD47/SIRPa blocking therapeutics
as monotherapy or in
combination with PD-1/L1¨blocking antibodies.
Disrupting the binding of 0D47 to SIRPa has emerged as a promising
immunotherapeutic strategy for
advanced cancers by potentiating antibody-dependent cellular phagocytosis
(ADCP) of targeted antibodies.
Preclinically, CD47/SIRPa blockade induces antitumor activity by increasing
the phagocytosis of tumor cells
by macrophages and enhancing the cross-presentation of tumor antigens to CD8+T
cells by dendritic cells;
both of these processes are potentiated by CD40 signaling. Here a novel, two-
sided fusion protein
incorporating the extracellular domains of SIRPa and CD4OL, adjoined by a
central Fc domain, termed
SIRPa-Fc-CD4OL was generated. As shown herein, the SIRPa-Fc-CD4OL chimeric
protein bound 0D47 and
CD40 with high affinity and activated CD40 signaling in the absence of Fc
receptor cross-linking. No evidence
of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or
in cynomolgus macaques.
Further, as shown herein, the SIRPa- Fc-CD4OL chimeric protein outperformed
0D47 blocking and CD40
zo agonist antibodies in murine 0T26 tumor models and synergized with
immune checkpoint blockade of PD-1
and CTLA4. SIRPa-Fc-CD4OL activated a type I interferon response in
macrophages and potentiated the
activity of ADCP-competent targeted antibodies both in vitro and in vivo.
These data illustrated that whereas
0D47/SIRPa inhibition could potentiate tumor cell phagocytosis, CD40-mediated
activation of a type I
interferon response provided a bridge between macrophage- and T-cell¨mediated
immunity that significantly
enhanced durable tumor control and rejection.
Chimeric proteins of the present disclosure comprise an extracellular domain
of CD172a (SIRPa) and an
extracellular domain of CD4OL. Thus, a chimeric protein of the present
disclosure comprises, at least, a first
domain comprising the extracellular domain of CD172a (SIRPa), which is
connected ¨ directly or via a linker
¨ to a second domain comprising the extracellular domain of CD4OL. When the
domains are linked in an
amino-terminal to carboxy-terminal orientation, the first domain is located on
the "left" side of the chimeric
protein and is "outward facing" and the second domain is located on "right"
side of the chimeric protein and
is "outward facing".
Other configurations of first and second domains are envisioned, e.g., the
first domain is outward facing and
the second domain is inward facing, the first domain is inward facing and the
second domain is outward
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
facing, and the first and second domains are both inward facing. When both
domains are "inward facing", the
chimeric protein would have an amino-terminal to carboxy-terminal
configuration comprising an extracellular
domain of CD4OL, a linker, and an extracellular domain of CD172a (SIRPa). In
such configurations, it may
be necessary for the chimeric protein to include extra "slack", as described
elsewhere herein, to permit
binding domains of the chimeric protein to one or both of its
receptors/ligands.
io Constructs could be produced by cloning of the nucleic acids encoding
the three fragments (the extracellular
domain of CD172a (SIRPa), followed by a linker sequence, followed by the
extracellular domain of CD4OL)
into a vector (plasmid, viral or other) wherein the amino terminus of the
complete sequence corresponded to
the 'left' side of the molecule containing the extracellular domain of CD172a
(SIRPa) and the carboxy
terminus of the complete sequence corresponded to the 'right' side of the
molecule containing the
is extracellular domain of CD4OL. In some embodiments of chimeric proteins
having one of the other
configurations, as described above, a construct would comprise three nucleic
acids such that the translated
chimeric protein produced would have the desired configuration, e.g., a dual
inward-facing chimeric protein.
Accordingly, In embodiments, the present chimeric proteins are engineered as
such.
CD172a (SIRPa)-Fc-CD4OL Chimeric Protein
zo In embodiments, the chimeric protein is capable of contemporaneously
binding the human CD172a (SIRPa)
ligand and the human CD40 receptor, wherein the CD172a (SIRPa) ligand is CD47
and the CD4OL receptor
is CD40.
The chimeric protein has a general structure of: N terminus ¨ (a) ¨ (b) ¨ (c)
¨ C terminus, in which (a) is a
first domain comprising an extracellular domain of human signal regulatory
protein a (CD172a (SIRPa)), (b)
25 is a linker adjoining the first and second domains, wherein the linker
comprises a hinge-CH2-CH3 Fc domain,
and (c) is a second domain comprising an extracellular domain of human CD40
ligand (CD4OL). In
embodiments, the linker comprises at least one cysteine residue capable of
forming a disulfide bond.
Chimeric proteins of the present disclosure have a first domain which is
sterically capable of binding its
ligand/receptor and/or a second domain which is sterically capable of binding
its ligand/receptor. This means
30 that there is sufficient overall flexibility in the chimeric protein
and/or physical distance between an
extracellular domain (or portion thereof) and the rest of the chimeric protein
such that the ligand/receptor
binding domain of the extracellular domain is not sterically hindered from
binding its ligand/receptor. This
flexibility and/or physical distance (which is herein referred to as "slack")
may be normally present in the
extracellular domain(s), normally present in the linker, and/or normally
present in the chimeric protein (as a
16
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
whole). Alternately, or additionally, the chimeric protein may be modified by
including one or more additional
amino acid sequences (e.g., the joining linkers described below) or synthetic
linkers (e.g., a polyethylene
glycol (PEG) linker) which provide additional slack needed to avoid steric
hindrance.
In embodiments, the chimeric proteins of the present disclosure comprise
variants of the extracellular domain
of CD172a (SIRPa). As examples, the variant may have at least about 60%, or at
least about 61%, or at least
about 62%, or at least about 63%, or at least about 64%, or at least about
65%, or at least about 66%, or at
least about 67%, or at least about 68%, or at least about 69%, or at least
about 70%, or at least about 71%,
or at least about 72%, or at least about 73%, or at least about 74%, or at
least about 75%, or at least about
76%, or at least about 77%, or at least about 78%, or at least about 79%, or
at least about 80%, or at least
about 81%, or at least about 82%, or at least about 83%, or at least about
84%, or at least about 85%, or at
is least about 86%, or at least about 87%, or at least about 88%, or at
least about 89%, or at least about 90%,
or at least about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about
95%, or at least about 96%, or at least about 97%, or at least about 98%, or
at least about 99% sequence
identity with the known amino acid sequence of CD172a (SIRPa), e.g., human
CD172a (SIRPa).
In embodiments, the extracellular domain of CD172a (SIRPa) has the following
amino acid sequence:
EEELQVIQPDKSVLVAAGETATLRCTATSLI PVGPIQVVFRGAGPGRELIYNQKEGHFPRVTTVSDLT
KRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQ
HTVSFTCESHGFSPRDITL KVVF KNGNELSDFQTNVDPVGESVSYSI HSTAKWLTREDVHSQVICEV
AHVTLQGDP LRGTANLSETI RVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSR
TETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSN
TAAENTGSNERNIY (SEQ ID NO: 57).
In embodiments, a chimeric protein comprises a variant of the extracellular
domain of CD172a (SIRPa). As
examples, the variant may have at least about 60%, or at least about 61%, or
at least about 62%, or at least
about 63%, or at least about 64%, or at least about 65%, or at least about
66%, or at least about 67%, or at
least about 68%, or at least about 69%, or at least about 70%, or at least
about 71%, or at least about 72%,
or at least about 73%, or at least about 74%, or at least about 75%, or at
least about 76%, or at least about
77%, or at least about 78%, or at least about 79%, or at least about 80%, or
at least about 81%, or at least
about 82%, or at least about 83%, or at least about 84%, or at least about
85%, or at least about 86%, or at
least about 87%, or at least about 88%, or at least about 89%, or at least
about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about
17
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
96%, or at least about 97%, or at least about 98%, or at least about 99%
sequence identity with SEQ ID NO:
57.
In embodiments, the first domain of a chimeric protein comprises an amino acid
sequence that is at least
90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 57. In
embodiments, the first domain of a chimeric protein comprises an amino acid
sequence that is at least 95%
identical to the amino acid sequence of SEQ ID NO: 57. In embodiments, the
first domain of a chimeric
protein comprises an amino acid sequence that is at least 97% identical to the
amino acid sequence of SEQ
ID NO: 57. In embodiments, the first domain of a chimeric protein comprises an
amino acid sequence that is
at least 98% identical to the amino acid sequence of SEQ ID NO: 57. In
embodiments, the first domain of a
chimeric protein comprises an amino acid sequence that is at least 99%
identical to the amino acid sequence
is of SEQ ID NO: 57. In embodiments, the first domain of a chimeric protein
comprises an amino acid sequence
that is identical to the amino acid sequence of SEQ ID NO: 57.
One of ordinary skill may select variants of the known amino acid sequence of
CD172a (SIRPa) by consulting
the literature, e.g. Hatherley et al., "Paired receptor specificity explained
by structures of signal regulatory
proteins alone and complexed with CD47." MN Cell 31: 266-277 (2008); Hatherley
et al., "The Structure of
zo the Macrophage Signal Regulatory Protein Alpha (Sirpalpha) Inhibitory
Receptor Reveals a Binding Face
Reminiscent of that Used by T Cell Receptors." J Biol Chem 282: 14567 (2007);
Hatherley etal., "Structure
of Signal-Regulatory Protein Alpha: A Link to Antigen Receptor Evolution." J
Biol Chem 284: 26613 (2009);
Hatherley et al., "Polymorphisms in the Human Inhibitory Signal-Regulatory
Protein Alpha Do not Affect
Binding to its Ligand Cd47." J Biol Chem 289: 10024 (2014); Ring et al., "Anti-
SIRP alpha antibody
25 immunotherapy enhances neutrophil and macrophage antitumor activity."
Proc Natl Acad Sci U S A 114:
E10578-E10585 (2017), each of which is incorporated by reference in its
entirety.
In embodiments, the chimeric proteins of the present disclosure comprise
variants of the extracellular domain
of CD4OL. As examples, the variant may have at least about 60%, or at least
about 61%, or at least about
62%, or at least about 63%, or at least about 64%, or at least about 65%, or
at least about 66%, or at least
30 about 67%, or at least about 68%, or at least about 69%, or at least
about 70%, or at least about 71%, or at
least about 72%, or at least about 73%, or at least about 74%, or at least
about 75%, or at least about 76%,
or at least about 77%, or at least about 78%, or at least about 79%, or at
least about 80%, or at least about
81%, or at least about 82%, or at least about 83%, or at least about 84%, or
at least about 85%, or at least
about 86%, or at least about 87%, or at least about 88%, or at least about
89%, or at least about 90%, or at
18
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
least about 91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about 98%, or at
least about 99% sequence identity
with the known amino acid sequence of CD4OL, e.g., human CD4OL.
In embodiments, the extracellular domain of CD4OL has the following amino acid
sequence:
HRRLDKIEDERNLHEDFVFM KTIQRCNTGERSLSLLNCEE I KSQFEGFVK DIMLNKEETK KENSFEM
QKGDONPQIAAHVI SEASS KTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCS
NREASSQAPFIASLCLKSPGRFERILLRAANTHSSAK PCGQQSIHLGGVF ELQPGASVFVNVTDPSQ
VSHGTGFTSFGLLKL (SEQ ID NO: 58).
In embodiments, a chimeric protein comprises a variant of the extracellular
domain of CD4OL. As examples,
the variant may have at least about 60%, or at least about 61(3/0, or at least
about 62%, or at least about 63%,
or at least about 64%, or at least about 65%, or at least about 66%, or at
least about 67%, or at least about
68%, or at least about 69%, or at least about 70%, or at least about 71%, or
at least about 72%, or at least
about 73%, or at least about 74%, or at least about 75%, or at least about
76%, or at least about 77%, or at
least about 78%, or at least about 79%, or at least about 80%, or at least
about 81%, or at least about 82%,
or at least about 83%, or at least about 84%, or at least about 85%, or at
least about 86%, or at least about
zo 87%, or at least about 88%, or at least about 89%, or at least about
90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at least about
95%, or at least about 96%, or at
least about 97%, or at least about 98%, or at least about 99% sequence
identity with SEQ ID NO: 58.
In embodiments, the second domain of a chimeric protein comprises an amino
acid sequence that is at least
90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 58. In
embodiments, the second domain of a chimeric protein comprises an amino acid
sequence that is at least
95% identical to the amino acid sequence of SEQ ID NO: 58. In embodiments, the
second domain of a
chimeric protein comprises an amino acid sequence that is at least 97%
identical to the amino acid sequence
of SEQ ID NO: 58. In embodiments, the second domain of a chimeric protein
comprises an amino acid
sequence that is at least 98% identical to the amino acid sequence of SEQ ID
NO: 58. In embodiments, the
second domain of a chimeric protein comprises an amino acid sequence that is
at least 99% identical to the
amino acid sequence of SEQ ID NO: 58. In embodiments, the second domain of a
chimeric protein comprises
an amino acid sequence that is identical to the amino acid sequence of SEQ ID
NO: 58.
One of ordinary skill may select variants of the known amino acid sequence of
CD4OL by consulting the
literature, e.g. Karpusas et al., "2 A crystal structure of an extracellular
fragment of human CD40 ligand."
19
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Structure 3: 1031-1039 (1995); Karpusas eta!, "Structure of CD40 ligand in
complex with the Fab fragment
of a neutralizing humanized antibody." Structure 9: 321-329 (2001); Silvian
etal., "Small Molecule Inhibition
of the TNF Family Cytokine CD40 Ligand through a Subunit Fracture Mechanism."
ACS Chem Biol 6: 636-
647 (2011); An et aL, "Crystallographic and mutational analysis of the CD4O-
CD154 complex and its
implications for receptor activation." J Biol Chem 286: 11226-11235 (2011);
Karnell etal., "A CD4OL-targeting
protein reduces autoantibodies and improves disease activity in patients with
autoimmunity." Sci Trans! Med
11 (2019), each of which is incorporated by reference in its entirety.
In embodiments, the linker of a chimeric protein comprises an amino acid
sequence that is at least 90%, or
93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of
SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3. In embodiments, the linker of a chimeric protein comprises
an amino acid sequence that
is is at least 95% identical to the amino acid sequence of SEQ ID NO: 1,
SEQ ID NO: 2, or SEQ ID NO: 3. In
embodiments, the linker of a chimeric protein comprises an amino acid sequence
that is at least 97% identical
to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In
embodiments, the linker
of a chimeric protein comprises an amino acid sequence that is at least 98%
identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the
linker of a chimeric protein
zo comprises an amino acid sequence that is at least 99% identical to the
amino acid sequence of SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the linker of a chimeric
protein comprises an amino
acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1,
SEQ ID NO: 2, or SEQ ID NO:
3.
In embodiments, a chimeric protein of the present disclosure comprises: (1) a
first domain comprising the
25 amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or
98%, or 99% identical to SEQ ID NO:
57, (b) a second domain comprises the amino acid sequence that is at least
90%, or 93%, or 95%, or 97%,
or 98%, or 99% identical to SEQ ID NO: 58, and (c) a linker comprises an amino
acid sequence that is that
is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino
acid sequence of SEQ ID
NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
30 In embodiments, a chimeric protein of the present disclosure comprises:
(1) a first domain comprising the
amino acid sequence that is at least 95% identical to SEQ ID NO: 57, (b) a
second domain comprises the
amino acid sequence that is at least 95% identical to SEQ ID NO: 58, and (c) a
linker comprises an amino
acid sequence that is that is at least 95% identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3. In embodiments, a chimeric protein of the present
disclosure comprises: (1) a first domain
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
comprising the amino acid sequence that is at least 97% identical to SEQ ID
NO: 57, (b) a second domain
comprises the amino acid sequence that is at least 97% identical to SEQ ID NO:
58, and (c) a linker comprises
an amino acid sequence that is that is at least 97% identical to the amino
acid sequence of SEQ ID NO: 1,
SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, a chimeric protein of the
present disclosure comprises:
(1) a first domain comprising the amino acid sequence that is at least 98%
identical to SEQ ID NO: 57, (b) a
second domain comprises the amino acid sequence that is at least 98% identical
to SEQ ID NO: 58, and (c)
a linker comprises an amino acid sequence that is that is at least 98%
identical to the amino acid sequence
of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, a chimeric
protein of the present
disclosure comprises: (1) a first domain comprising the amino acid sequence
that is at least 99% identical to
SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is
at least 99% identical to
SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that
is at least 99% identical to
the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In
embodiments, a chimeric
protein of the present disclosure comprises: (1) a first domain comprising the
amino acid sequence of SEQ
ID NO: 57, (b) a second domain comprises the amino acid sequence of SEQ ID NO:
58, and (c) a linker
comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID
zo NO: 3. In embodiments, a chimeric protein of the present disclosure
comprises: (1) a first domain comprising
the amino acid sequence identical to SEQ ID NO: 57, (b) a second domain
comprises the amino acid
sequence that is to SEQ ID NO: 58, and (c) a linker comprises an amino acid
sequence that is that is identical
to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
In embodiments, a CD172a (SIRPa)-Fc-CD4OL chimeric protein of the present
disclosure has the following
amino acid sequence (the extracellular domain of CD172a (SIRPa) is shown in a
boldface font, the
extracellular domain of CD4OL is indicated by underline, Fc domain is shown in
italic:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSD
LTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAAR
ATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHS
QVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWL
ENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS
AHPKEQGSNTAAENTGSNERNIYSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTC
VVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVS
SKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMDH
21
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
RRLDKIEDERNLHEDFVFM KTIQRCNTGERSLSLLNCEEI KSQFEGFVKDIMLN KEETK KENSFEM
QKGDQ NPQIAAHVI SEASS KTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC
SNREASSQAPFIASLCLKSPGRFERI LLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDP
SQVSHGTGFTSFGLLKL (SEQ ID NO: 59).
The 792 amino acid sequence of the CD172a (SIRPa)-Fc-CD4OL chimeric protein
(SL-172154) (not including
the leader sequence) is shown above. The CD172a (SI RPa)-Fc-CD4OL chimeric
protein exists as a profile
of oligomeric forms. There are 17 cysteines in the amino acid sequence with 8
likely disulfide pairs. Both N
and 0-linked glycosylation have been identified.
In embodiments, the chimeric protein of the present disclosure comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 potential N glycosylation sites. In embodiments, the chimeric protein of
the present disclosure comprises
at least two potential N glycosylation sites. In embodiments, the chimeric
protein of the present disclosure
comprises at least four potential N glycosylation sites. In embodiments, the
chimeric protein of the present
disclosure comprises at least six potential N glycosylation sites. In
embodiments, the chimeric protein of the
present disclosure comprises at least eight potential N glycosylation sites.
In embodiments, the chimeric
protein of the present disclosure comprises at least ten potential N
glycosylation sites. In embodiments, the
zo chimeric protein of the present disclosure comprises at least 1, 2, 3,
4, 5, 6, 7, or 8 potential 0 glycosylation
sites. In embodiments, the chimeric protein of the present disclosure
comprises at least two potential 0
glycosylation sites. In embodiments, the chimeric protein of the present
disclosure comprises at least four
potential 0 glycosylation sites. In embodiments, the chimeric protein of the
present disclosure comprises at
least six potential 0 glycosylation sites. In embodiments, the chimeric
protein of the present disclosure
comprises at least eight potential 0 glycosylation sites. In embodiments, the
chimeric protein of the present
disclosure comprises at least two potential N glycosylation sites, and at
least two potential 0 glycosylation
sites. In embodiments, the chimeric protein of the present disclosure
comprises at least four potential N
glycosylation sites, and at least four potential 0 glycosylation sites. In
embodiments, the chimeric protein of
the present disclosure comprises at least six potential N glycosylation sites,
and at least six potential 0
glycosylation sites. In embodiments, the chimeric protein of the present
disclosure comprises at least eight
potential N glycosylation sites, and at least eight potential 0 glycosylation
sites. In embodiments, the chimeric
protein of the present disclosure comprises at least ten potential N
glycosylation sites, and at least eight
potential 0 glycosylation sites. In embodiments, the chimeric protein
expressed in Chinese Hamster Ovary
(CHO) cells is glycosylated.
22
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
There are 17 cysteines present in the SL-172154 chimeric protein. In some
embodiments, the SL-172154
chimeric protein has no disulfide bonds. In some embodiments, the SL-172154
chimeric protein has at least
one, or at least two, or at least 3, or at least 4, or at least 5, or at least
6, or at least 7, or at least 8, or at least
9, or at least 10 disulfide bonds. In some embodiments, the SL-172154 chimeric
protein has at least one, or
at least two interchain disulfide bonds. In some embodiments, the SL-172154
chimeric protein has at least
one, or at least two, or at least 3õ or at least 4, or at least 5, or at least
6, or at least 7, or 8 intrachain disulfide
bonds. In some embodiments, the SL-172154 chimeric protein has a C350=C350
interchain disulfide bond.
In some embodiments, the SL-172154 chimeric protein has a C353=C353 interchain
disulfide bond. In some
embodiments, the SL-172154 chimeric protein has a C153=C153 interchain
disulfide bond. In some
embodiments, the SL-172154 chimeric protein has a C25 = C91disulfide bond. In
some embodiments, the
SL-172154 chimeric protein has a C140 = C198 disulfide bond. In some
embodiments, the SL-172154
chimeric protein has a C243 = C301 disulfide bond. In some embodiments, the SL-
172154 chimeric protein
has a C385 = C445 disulfide bond. In some embodiments, the SL-172154 chimeric
protein has a C491 =
C549 disulfide bond. In some embodiments, the SL-172154 chimeric protein has a
C603 = C615 disulfide
bond. In some embodiments, the SL-172154 chimeric protein has a C709 = C725
disulfide bond. In some
zo embodiments, the SL-172154 chimeric protein has a C140 = C243 =
C709/C725 scrambled disulfide bond.
In some embodiments, the SL-172154 chimeric protein has a C615 (chain1) = C615
(chain2) scrambled
disulfide bond.
In some embodiments, the CD172a (SIRPa)-Fc-CD40L chimeric protein of the
present disclosure is encoded
by the following nucleotide sequence (leader sequence is shown by a bold-
underlined font):
ATGGAATGGAGCTGGGTGTTCTTGTTCTTCCTGTCCGTGACCACCGGCGTGCACTCGGAGGAGGAG
CTCCAGGTCATCCAGCCGGACAAGTCGGTGCTCGTGGCCGCCGGAGAAACTGCCACCCTGAGGTGC
ACCGCGACCTCGCTGATTCCCGTGGGCCCGATTCAGTGGTTCCGGGGGGCCGGGCCTGGCAGAGAA
CTGATCTACAACCAGAAGGAAGGCCATTTCCCTCGCGTGACTACTGTGTCCGATCTTACTAAGCGGAA
CAACATGGACTTCAGCATTAGGATCGGCAACATCACCCCTGCTGACGCGGGAACCTACTACTGCGTCA
AGTTCAGGAAAGGAAGCCCGGACGACGTGGAGTTCAAGAGCGGGGCGGGCACCGAACTGTCCGTGC
GCGCCAAGCCATCCGCGCCCGTGGTGTCCGGACCCGCAGCCAGAGCAACTCCGCAGCACACCGTGT
CGTTCACTTGCGAATCACACGGATTCTCCCCGCGCGATATCACGCTTAAGTGGTTCAAGAACGGGAAC
GAACTGAGCGACTTCCAGACCAACGTGGACCCCGTCGGAGAAAGCGTCAGCTACTCCATTCACTCGA
CCGCCAAAGTGGTGCTGACCAGGGAGGACGTGCATAGCCAAGTGATCTGCGAGGTCGCCCACGTCA
CTCTGCAAGGAGATCCGCTGCGGGGAACAGCCAACCTGTCCGAAACTATCCGCGTGCCTCCCACCCT
23
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
GGAAGTGACCCAGCAGCCCGTCCGAGCGGAGAATCAAGTCAATGTGACCTGTCAAGTCCGGAAATTC
TACCCTCAACGGCTCCAGCTGACCTGGCTGGAAAACGGAAACGTGTCCCGCACGGAAACCGCCTCGA
CCGTGACCGAGAACAAGGACGGCACCTACAACTGGATGTCCTGGCTCTTGGTGAACGTGTCAGCCCA
CCGGGACGATGTCAAGCTGACTTGCCAAGTGGAACATGATGGGCAGCCAGCTGTCAGCAAGAGCCAC
GACCTGAAGGTGTCCGCGCACCCGAAGGAACAGGGTTCGAATACTGCCGCCGAAAACACTGGTAGCA
ACGAACGGAACATCTACTCTAAGTACGGCCCACCTTGCCCTCCCTGCCCGGCACCTGAATTTCTGGGT
GGACCCTCCGTGTTTCTTTTCCCGCCCAAGCCAAAGGACCAGTTGATGATCTCCCGCACTCCGGAAGT
GACATGCGTGGTGGTGGACGTGTCCCAGGAAGATCCGGAAGTGCAGTTCAATTGGTACGTGGATGGC
GTGGAGGTCCATAACGCCAAGACTAAGCCGCGCGAGGAACAGTTCAATTCCACCTACCGGGTGGTGT
CCGTGCTGACCGTGCTGCATCAGGACTGGCTCTCCGGCAAAGAGTACAAGTGCAAGGTGTCATCCAA
GGGTCTGCCGTCGTCAATCGAAAAGACCATTTCCAATGCCACTGGGCAGCCCAGAGAACCTCAAGTCT
ACACCCTCCCACCGTCCCAAGAGGAAATGACCAAGAACCAAGTCTCGCTGACGTGTCTCGTGAAGGG
ATTCTACCCATCCGACATTGCTGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACTA
CCCCTCCCGTCCTGGACTCCGACGGTTCCTTCTTCCTTTACTCTCGCCTCACCGTGGATAAGTCGCGG
TGGCAGGAGGGGAACGTGTTCTCCTGCTCCGTCCTGCACGAAGCATTGCACAACCACTACACCCAGA
AGTCCCTGTCACTGTCCCTGGGAAAGATTGAGGGTCGGATGGATCATCGGCGCCTGGACAAGATCGA
GGACGAGCGGAACCTCCACGAGGATTTCGTGTTCATGAAAACCATCCAGAGATGCAACACCGGAGAG
AGAAGCCTGTCCCTGCTCAACTGCGAGGAAATCAAGTCCCAGTTTGAAGGATTTGTGAAGGACATTAT
GCTGAACAAGGAAGAGACTAAGAAGGAAAACTCCTTCGAGATGCAGAAGGGCGATCAGAACCCACAG
ATCGCGGCCCACGTGATCTCCGAGGCCTCGTCAAAGACCACTTCAGTGCTCCAATGGGCCGAGAAGG
GTTACTATACCATGAGCAACAACCTTGTGACCCTGGAGAACGGAAAGCAGCTCACCGTGAAAAGACAG
GGACTGTACTATATCTATGCCCAAGTCACCTTCTGTTCGAACCGCGAGGCTAGCAGCCAGGCCCCGTT
CATCGCCTCCCTCTGTTTGAAGTCGCCGGGGCGGTTTGAAAGGATTCTGCTGAGAGCTGCGAATACC
CATTCGTCCGCCAAGCCTTGCGGACAGCAGTCAATCCACCTGGGGGGAGTGTTCGAGCTGCAGCCTG
GCGCGAGCGTGTTCGTCAACGTGACCGACCCCTCCCAAGTGTCTCACGGCACCGGATTCACTTCGTT
TGGCCTGCTGAAGCTGTAA (SEQ ID NO: 60)
In some embodiments, the SEQ ID NO: 60 encodes for a precursor of the CD172a
(SIRPa)-Fc-CD4OL
chimeric protein of the present disclosure having following amino acid
sequence (leader sequence is shown
by an italic font):
MEWSINVFLFF LSVTIGVHSEEELQVIQP DK SVLVAAGETATL RCTATSL I PVGP IQVVF
RGAGPGRELIYN Q K
EGH FP RVTTVSD LT K RN NM D FSI RI GN IT PADAGTYYCVK F RKGSPDDVEF
KSGAGTELSVRAKPSAPVVS
24
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
GPAARATPQHTVSFICESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKWLTREDVHSQ
VI CEVAHVTLQGDPLRGTANLSETI RVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVS RT
ETASTVTENKDGTYNVVMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENT
GSNERNIYSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDOLMISRTPEVICVVVDVSQEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLP
PSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVLHEALHNHYTQKSLSLSLGKI EGRMDHRRLDKI EDERNLHEDFVFM KTIQRCNTGERSLSLLNCEEI
KSQFEGFVKDIMLNKEETK K ENSFEMQKGDQNPQIAAHVI SEASSKTTSVLQWAEKGYYTMSNNLVTLENG
KQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFEL
QPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 61)
is The chimeric protein of SEQ ID NO: 59 (also referred to herein as SL-
172154) is a recombinant fusion
glycoprotein comprising the extracellular domain of human CD172a (SIRPa)
(PDCD1, CD272a), a central
domain including the hinge-CH2-CH3 region from human immunoglobulin constant
gamma 4 (Inhibitory
receptor SHPS-1, IgG4), and the extracellular domain of human CD4OL (TNFSF5,
TRAP, 00154). The linear
configuration of SL-172154 is CD172a (SIRPa)-Fc-CD4OL.
zo The predicted molecular weight for the monomeric chimeric protein of
SEQ ID NO: 59 is 88.1 kDa. The
predicted molecular weight for the glycosylated monomeric chimeric protein of
SEQ ID NO: 59 is about 115
kDa.
The dual-sided nature of the chimeric proteins disclosed herein, such as the
00172a (SIRPa)-Fc-CD4OL
chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61), is designed to
intercept one of the key
25 immunosuppressive pathways within the tumor microenvironment (TME):
the CD172a (SIRPa) ¨ CD47
macrophage checkpoint.
Tumor cells may express 0047 on their cell surface, which can bind to CD172a
(SIRPa) expressed by a
macrophage to suppress phagocytosis of the tumor cells. Thus, the CD172a
(SIRPa)-Fc-CD4OL chimeric
protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61) can bind to 0047 expressed on
the surface of tumor, with
30 the CD172a (SIRPa) domain of the 00172a (SIRPa)-Fc-CD4OL chimeric
proteins disclosed herein intended
to provide competitive inhibition of 0047, and to replace the 0047 inhibitory
signal with functionally
trimerized/hexamerized CD4OL, resulting in an incoming T cell experiencing co-
stimulation via engagement
through its 0040 receptor instead of suppression through CD172a (SIRPa)
interactions. In other words,
because the extracellular domains (ECDs) of CD172a (SIRPa) and CD4OL are
physically linked to one
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
another and localized to the TME, tumor infiltrating T cells will receive co-
stimulation at the same time they
recognize a tumor antigen via its T cell receptor (TCR). Importantly, because
the ECDs of CD172a (SIRPa)
and CD4OL are physically linked to one another, and localized to the TME,
tumor infiltrating T cells will receive
costimulation at the same time they recognize a tumor antigen via the T cell
receptor. Together, these would
result in replacement of an inhibitory CD47 signal with a co-stimulatory CD4OL
signal to enhance the anti-
tumor activity of T cells.
The three constituent components of the chimeric proteins disclosed herein,
including the CD172a (SIRPa)-
Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61), have unique
attributes that facilitate
dimerization or oligomerization. The extracellular domain of CD172a (SIRPa)
normally exists as a monomer
and is not known to form higher-order homomeric complexes. The central Fc
domain contains cysteine
is residues that are capable of disulfide bonding to form a dimeric
structure. In embodiments, the chimeric
proteins disclosed herein, including the CD172a (SIRPa)-Fc-CD4OL chimeric
protein (e.g. SEQ ID NO: 59 or
SEQ ID NO: 61), contains an S228P mutation in the hinge region of the Fc
domain to prevent Fab arm
exchange. The CD4OL domain is known to form homotrimeric complexes, which are
stabilized through
noncovalent, electrostatic interactions. Although the chimeric proteins
disclosed herein, including the CD172a
zo (SIRPa)-Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61),
are expressed as a continuous
monomeric protein by production cell lines, the resulting monomeric proteins
self-assemble into higher-order
species based on these disulfide and charge-based interactions of CD4OL
(creating a trimer) and the
combined influence of these attractive forces, resulting in a hexamer (dimer
of trimers). The majority (>80%)
of the CD172a (SIRPa)-Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID
NO: 61) comprises the
zs hexamer and trimer forms, which have similar activity. Importantly,
because the CD172a (SIRPa)-Fc-CD4OL
chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61), are comprised of
hexamers and trimers, they
stimulate CD40 signaling in the absence of cross-linking by Fc receptors or
any other cross-linking agent.
The predicted tertiary structures of the CD172a (SIRPa)-Fc-CD4OL chimeric
protein (e.g. SEQ ID NO: 59 or
SEQ ID NO: 61) as a monomer and in various oligomeric states, based on
disulfide (Fc) and charge-based
30 (CD4OL) interactions shows visualization by electron microscopy of the
CD172a (SIRPa)-Fc-CD4OL chimeric
protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61) hexamers (top two images) and
the CD172a (SIRPa)-Fc-
CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61) trimers (bottom
two images). Accordingly,
the CD172a (SIRPa)-Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO:
61) forms
trimers/hexamers and activates CD40 without the need for cross-linking. It is
noteworthy that, unlike
26
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
monoclonal antibodies, Fc receptor cross-linking is not required for
functional activity of the CD172a (SIRPa)-
Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61).
In embodiments, a chimeric protein comprises a variant of the CD172a (SIRPa)-
Fc-CD4OL chimeric protein
(e.g. SEQ ID NO: 59 or SEQ ID NO: 61). As examples, the variant may have at
least about 60%, or at least
about 61%, or at least about 62%, or at least about 63%, or at least about
64%, or at least about 65%, or at
least about 66%, or at least about 67%, or at least about 68%, or at least
about 69%, or at least about 70%,
or at least about 71%, or at least about 72%, or at least about 73%, or at
least about 74%, or at least about
75%, or at least about 76%, or at least about 77%, or at least about 78%, or
at least about 79%, or at least
about 80%, or at least about 81%, or at least about 82%, or at least about
83%, or at least about 84%, or at
least about 85%, or at least about 86%, or at least about 87%, or at least
about 88%, or at least about 89%,
is or at least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least
about 99%, or at least about 99.2%, or at least about 99.4%, or at least about
99.6%, or at least about 99.8%
sequence identity with SEQ ID NO: 59 or SEQ ID NO: 61.
In embodiments, the first domain of a chimeric protein comprises an amino acid
sequence that is at least
zo 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 59 or
SEQ ID NO: 61. In embodiments, the first domain of a chimeric protein
comprises an amino acid sequence
that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59 or
SEQ ID NO: 61. In
embodiments, the first domain of a chimeric protein comprises an amino acid
sequence that is at least 97%
identical to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the first domain
25 of a chimeric protein comprises an amino acid sequence that is at least
98% identical to the amino acid
sequence of SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the first domain
of a chimeric protein
comprises an amino acid sequence that is at least 99% identical to the amino
acid sequence of SEQ ID NO:
59 or SEQ ID NO: 61. In embodiments, the first domain of a chimeric protein
comprises an amino acid
sequence that is identical to the amino acid sequence of SEQ ID NO: 59 or SEQ
ID NO: 61.
30 In embodiments, the first domain is capable of binding a CD172a (SIRPa)
ligand.
In embodiments, the first domain comprises substantially all of the
extracellular domain of CD172a (SIRPa).
In embodiments, the second domain is capable of binding a CD40 receptor.
In embodiments, the second domain comprises substantially all of the
extracellular domain of CD4OL.
27
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from
IgG4, e.g., human IgG4.
In embodiments, the linker comprises an amino acid sequence that is at least
95% identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the
linker comprises an amino
acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99%
identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the
linker comprises an amino
acid sequence that is at least 95% identical to the amino acid sequence of SEQ
ID NO: 1, SEQ ID NO: 2, or
SEQ ID NO: 3. In embodiments, the linker comprises an amino acid sequence that
is at least 97% identical
to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In
embodiments, the linker
comprises an amino acid sequence that is at least 98% identical to the amino
acid sequence of SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the linker comprises an
amino acid sequence that is at
is least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3. In
embodiments, the linker comprises an amino acid sequence that is identical to
the amino acid sequence of
SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
In embodiments, the first domain comprises an amino acid sequence that is at
least 95% identical to the
amino acid sequence of SEQ ID NO: 57. In embodiments, the first domain of a
chimeric protein comprises
zo an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or
98%, or 99% identical to the amino
acid sequence of SEQ ID NO: 57. In embodiments, the first domain of a chimeric
protein comprises an amino
acid sequence that is at least 95% identical to the amino acid sequence of SEQ
ID NO: 57. In embodiments,
the first domain of a chimeric protein comprises an amino acid sequence that
is at least 97% identical to the
amino acid sequence of SEQ ID NO: 57. In embodiments, the first domain of a
chimeric protein comprises
25 an amino acid sequence that is at least 98% identical to the amino acid
sequence of SEQ ID NO: 57. In
embodiments, the first domain of a chimeric protein comprises an amino acid
sequence that is at least 99%
identical to the amino acid sequence of SEQ ID NO: 57. In embodiments, the
first domain of a chimeric
protein comprises an amino acid sequence that is identical to the amino acid
sequence of SEQ ID NO: 57.
In embodiments, the second domain comprises an amino acid sequence that is at
least 95% identical to the
30 amino acid sequence of SEQ ID NO: 58. In embodiments, the second domain
of a chimeric protein comprises
an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%,
or 99% identical to the amino
acid sequence of SEQ ID NO: 58. In embodiments, the second domain of a
chimeric protein comprises an
amino acid sequence that is at least 95% identical to the amino acid sequence
of SEQ ID NO: 58. In
embodiments, the second domain of a chimeric protein comprises an amino acid
sequence that is at least
28
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
97% identical to the amino acid sequence of SEQ ID NO: 58. In embodiments, the
second domain of a
chimeric protein comprises an amino acid sequence that is at least 98%
identical to the amino acid sequence
of SEQ ID NO: 58. In embodiments, the second domain of a chimeric protein
comprises an amino acid
sequence that is at least 99% identical to the amino acid sequence of SEQ ID
NO: 58. In embodiments, the
second domain of a chimeric protein comprises an amino acid sequence that is
identical to the amino acid
sequence of SEQ ID NO: 58.
In embodiments, the (a) the first domain comprises the amino acid sequence of
SEQ ID NO: 57, (b) the
second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the
linker comprises an amino
acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or
SEQ ID NO: 3.
In embodiments, the chimeric protein further comprises an amino acid sequence
that is at least 90%, or 93%,
or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID
NO: 5 and/or SEQ ID NO:
7. In embodiments, the chimeric protein further comprises an amino acid
sequence that is at least 95%
identical to the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7. In
embodiments, the chimeric
protein further comprises an amino acid sequence that is at least 98%
identical to the amino acid sequence
of SEQ ID NO: 5 and/or SEQ ID NO: 7. In embodiments, the chimeric protein
further comprises the amino
zo acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7.
In embodiments, the chimeric protein comprises an amino acid sequence that is
at least about 95% identical
to SEQ ID NO: 59 or SEQ ID NO: 61, e.g., at least about 98% identical to SEQ
ID NO: 59 or SEQ ID NO: 61,
at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61, at least about
99.2% identical to SEQ ID
NO: 59 or SEQ ID NO: 61, at least about 99.4% identical to SEQ ID NO: 59 or
SEQ ID NO: 61, at least about
zs 99.6% identical to SEQ ID NO: 59 or SEQ ID NO: 61, or at least about
99.8% identical to SEQ ID NO: 59 or
SEQ ID NO: 61. In embodiments, the chimeric protein comprises the amino acid
sequence of SEQ ID NO:
59 or SEQ ID NO: 61.
In any herein-disclosed aspect and embodiment, the chimeric protein may
comprise an amino acid sequence
having one or more amino acid mutations relative to any of the protein
sequences disclosed herein. In
30 embodiments, the one or more amino acid mutations may be independently
selected from substitutions,
insertions, deletions, and truncations.
In embodiments, the amino acid mutations are amino acid substitutions, and may
include conservative and/or
non-conservative substitutions. "Conservative substitutions" may be made, for
instance, on the basis of
similarity in polarity, charge, size, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of
29
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
the amino acid residues involved. The 20 naturally occurring amino acids can
be grouped into the following
six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2)
neutral hydrophilic: Cys, Ser,
Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues
that influence chain orientation: Gly,
Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative
substitutions" are defined as exchanges
of an amino acid by another amino acid listed within the same group of the six
standard amino acid groups
shown above. For example, the exchange of Asp by Glu retains one negative
charge in the so modified
polypeptide. In addition, glycine and proline may be substituted for one
another based on their ability to
disrupt a-helices. As used herein, "non-conservative substitutions" are
defined as exchanges of an amino
acid by another amino acid listed in a different group of the six standard
amino acid groups (1) to (6) shown
above.
is In embodiments, the substitutions may also include non-classical amino
acids (e.g., selenocysteine,
pyrrolysine, N-formylmethionine p-alanine, GABA and 6-Aminolevulinic acid, 4-
aminobenzoic acid (PABA),
D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino
isobutyric acid, 4-aminobutyric acid,
Abu, 2-amino butyric acid, y-Abu, E-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme,
citrulline, homocitrulline, cysteic
zo acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
p-alanine, fluoro-amino acids, designer
amino acids such as 13 methyl amino acids, C a-methyl amino acids, N a-methyl
amino acids, and amino acid
analogs in general).
Mutations may also be made to the nucleotide sequences of the chimeric
proteins by reference to the genetic
code, including taking into account codon degeneracy.
zs In embodiments, a chimeric protein is capable of binding human
ligand(s)/receptor(s).
In embodiments, each extracellular domain (or variant thereof) of the chimeric
protein binds to its cognate
receptor or ligand with a KD of about 1 nM to about 5 nM, for example, about 1
nM, about 1.5 nM, about 2
nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about
5 nM. In embodiments, the
chimeric protein binds to a cognate receptor or ligand with a KD of about 5 nM
to about 15 nM, for example,
30 about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about
7.5 nM, about 8 nM, about 8.5 nM,
about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5
nM, about 12 nM, about
12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15
nM.
In embodiments, each extracellular domain (or variant thereof) of the chimeric
protein binds to its cognate
receptor or ligand with a KD of less than about 1 pM, about 900 nM, about 800
nM, about 700 nM, about 600
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 150 nM,
about 130 nM, about 100
nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50
nM, about 45 nM, about
40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about
10 nM, or about 5 nM, or
about 1 nM (as measured, for example, by surface plasmon resonance or biolayer
interferometry).
In embodiments, the chimeric protein binds to human CD47 with a KD of about 1
nM to about 5 nM, for
io example, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3
nM, about 3.5 nM, about 4 nM, about
4.5 nM, or about 5 nM. In embodiments, the chimeric protein binds to human
0D47 with a KID of less than
about 3 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM,
about 600 pM, about 500
pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about
80 pM, about 70 pM,
about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM,
about 30 pM, about 25 pM,
is about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured,
for example, by surface plasmon
resonance or biolayer interferometry).
In embodiments, the chimeric protein binds to human CD40 with a KD of less
than about 1 nM, about 900
pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM,
about 300 pM, about 200
pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55
pM about 50 pM about
20 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20
pM, about 15 pM, or about 10 pM,
or about 1 pM (as measured, for example, by surface plasmon resonance or
biolayer interferometry).
As used herein, a variant of an extracellular domain is capable of binding the
receptor/ligand of a native
extracellular domain. For example, a variant may include one or more mutations
in an extracellular domain
which do not affect its binding affinity to its receptor/ligand; alternately,
the one or more mutations in an
zs extracellular domain may improve binding affinity for the
receptor/ligand; or the one or more mutations in an
extracellular domain may reduce binding affinity for the receptor/ligand, yet
not eliminate binding altogether.
In embodiments, the one or more mutations are located outside the binding
pocket where the extracellular
domain interacts with its receptor/ligand. In embodiments, the one or more
mutations are located inside the
binding pocket where the extracellular domain interacts with its
receptor/ligand, as long as the mutations do
30 not eliminate binding altogether. Based on the skilled artisan's
knowledge and the knowledge in the art
regarding receptor-ligand binding, s/he would know which mutations would
permit binding and which would
eliminate binding.
In embodiments, the chimeric protein exhibits enhanced stability and protein
half-life.
31
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
A chimeric protein of the present disclosure may comprise more than two
extracellular domains. For example,
the chimeric protein may comprise three, four, five, six, seven, eight, nine,
ten, or more extracellular domains.
A second extracellular domain may be separated from a third extracellular
domain via a linker, as disclosed
herein. Alternately, a second extracellular domain may be directly linked
(e.g., via a peptide bond) to a third
extracellular domain. In embodiments, a chimeric protein includes
extracellular domains that are directly
linked and extracellular domains that are indirectly linked via a linker, as
disclosed herein.
Linkers
In embodiments, the chimeric protein comprises a linker.
In embodiments, the linker comprising at least one cysteine residue capable of
forming a disulfide bond. The
at least one cysteine residue is capable of forming a disulfide bond between a
pair (or more) of chimeric
proteins. Without wishing to be bound by theory, such disulfide bond forming
is responsible for maintaining
a useful nnultinneric state of chimeric proteins. This allows for efficient
production of the chimeric proteins; it
allows for desired activity in vitro and in vivo.
In a chimeric protein of the present disclosure, the linker is a polypeptide
selected from a flexible amino acid
sequence, an IgG hinge region, or an antibody sequence.
zo In embodiments, the linker is derived from naturally-occurring multi-
domain proteins or is an empirical linker
as described, for example, in Chichili etal., Protein Sci. 22(2):153-167
(2013); Chen etal., Adv Drug Deliv
Rev. 65(10):1357-1369 (2013), the entire contents of which are hereby
incorporated by reference. In
embodiments, the linker may be designed using linker designing databases and
computer programs such as
those described in Chen etal., Adv Drug Deliv Rev. 65(10):1357-1369 (2013);
and Crasto etal., Protein Eng.
13(5):309-312 (2000), the entire contents of which are hereby incorporated by
reference.
In embodiments, the linker comprises a polypeptide. In embodiments, the
polypeptide is less than about 500
amino acids long, about 450 amino acids long, about 400 amino acids long,
about 350 amino acids long,
about 300 amino acids long, about 250 amino acids long, about 200 amino acids
long, about 150 amino acids
long, or about 100 amino acids long. For example, the linker may be less than
about 100, about 95, about
90, about 85, about 80, about 75, about 70, about 65, about 60, about 55,
about 50, about 45, about 40,
about 35, about 30, about 25, about 20, about 19, about 18, about 17, about
16, about 15, about 14, about
13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5,
about 4, about 3, or about 2
amino acids long.
32
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the linker is flexible.
In embodiments, the linker is rigid.
In embodiments, the linker is substantially comprised of glycine and serine
residues (e.g., about 30%, or
about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about
90%, or about 95%, or about
97%, or about 98%, or about 99%, or about 100% glycines and seri nes).
In embodiments, the linker comprises a hinge region of an antibody (e.g., of I
gG, IgA, IgD, and IgE, inclusive
of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1, and IgA2)). The
hinge region, found in IgG, IgA,
IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab
portion to move freely in space. In
contrast to the constant regions, the hinge domains are structurally diverse,
varying in both sequence and
length among immunoglobulin classes and subclasses. For example, the length
and flexibility of the hinge
region varies among the IgG subclasses. The hinge region of IgG1 encompasses
amino acids 216-231 and,
because it is freely flexible, the Fab fragments can rotate about their axes
of symmetry and move within a
sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2
has a shorter hinge than IgG1,
with 12 amino acid residues and four disulfide bridges. The hinge region of
IgG2 lacks a glycine residue, is
relatively short, and contains a rigid poly-proline double helix, stabilized
by extra inter-heavy chain disulfide
zo bridges. These properties restrict the flexibility of the IgG2 molecule.
IgG3 differs from the other subclasses
by its unique extended hinge region (about four times as long as the IgG1
hinge), containing 62 amino acids
(including 21 prolines and 11 cysteines), forming an inflexible poly-proline
double helix. In IgG3, the Fab
fragments are relatively far away from the Fc fragment, giving the molecule a
greater flexibility. The elongated
hinge in IgG3 is also responsible for its higher molecular weight compared to
the other subclasses. The hinge
zs region of IgG4 is shorter than that of IgG1 and its flexibility is
intermediate between that of IgG1 and IgG2.
The flexibility of the hinge regions reportedly decreases in the order
IgG3>IgG1>IgG4>IgG2. In embodiments,
the linker may be derived from human IgG4 and contain one or more mutations to
enhance dimerization
(including S228P) or FcRn binding.
According to crystallographic studies, the immunoglobulin hinge region can be
further subdivided functionally
30 into three regions: the upper hinge region, the core region, and the
lower hinge region. See Shin et al.,
Immunological Reviews 130:87 (1992). The upper hinge region includes amino
acids from the carboxyl end
of CHI to the first residue in the hinge that restricts motion, generally the
first cysteine residue that forms an
interchain disulfide bond between the two heavy chains. The length of the
upper hinge region correlates with
the segmental flexibility of the antibody. The core hinge region contains the
inter-heavy chain disulfide
33
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
bridges, and the lower hinge region joins the amino terminal end of the CH2
domain and includes residues in
CH2. Id. The core hinge region of wild-type human IgG1 contains the sequence
CPPC (SEQ ID NO: 24) which,
when dimerized by disulfide bond formation, results in a cyclic octapeptide
believed to act as a pivot, thus
conferring flexibility. In embodiments, the present linker comprises, one, or
two, or three of the upper hinge
region, the core region, and the lower hinge region of any antibody (e.g., of
IgG, IgA, IgD, and I gE, inclusive
of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The
hinge region may also contain
one or more glycosylation sites, which include a number of structurally
distinct types of sites for carbohydrate
attachment. For example, IgA1 contains five glycosylation sites within a 17-
amino-acid segment of the hinge
region, conferring resistance of the hinge region polypeptide to intestinal
proteases, considered an
advantageous property for a secretory immunoglobulin. In embodiments, the
linker of the present disclosure
comprises one or more glycosylation sites.
In embodiments, the linker comprises an Fc domain of an antibody (e.g., of
IgG, IgA, I gD, and IgE, inclusive
of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)).
In a chimeric protein of the present disclosure, the linker comprises a hinge-
CH2-CH3 Fc domain derived
from IgG4. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain
derived from a human IgG4.
zo In embodiments, the linker comprises an amino acid sequence that is at
least 90%, or 93%, or 95%, or 97%,
or 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1
to SEQ ID NO: 3. In
embodiments, the linker comprises an amino acid sequence that is at least 95%
identical to the amino acid
sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 (e.g., at least 95%
identical to the amino acid
sequence of SEQ ID NO: 2.). In embodiments, the linker comprises one or more
joining linkers, such joining
linkers independently selected from SEQ ID NOs: 4-50 (or a variant thereof).
In embodiments, the linker
comprises two or more joining linkers each joining linker independently
selected from SEQ ID NOs: 4-50 (or
a variant thereof); wherein one joining linker is N terminal to the hinge-CH2-
CH3 Fc domain and another
joining linker is C terminal to the hinge-CH2-CH3 Fc domain.
In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a
human IgG1 antibody. In
embodiments, the Fc domain exhibits increased affinity for and enhanced
binding to the neonatal Fc receptor
(FcRn). In embodiments, the Fc domain includes one or more mutations that
increases the affinity and
enhances binding to FcRn. Without wishing to be bound by theory, it is
believed that increased affinity and
enhanced binding to FcRn increases the in vivo half-life of the present
chimeric proteins.
34
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the Fc domain in a linker contains one or more amino acid
substitutions at amino acid
residue 250, 252, 254, 256, 308, 309, 311, 416, 428, 433, or 434 (in
accordance with Kabat numbering, as
in as in Kabat, etal., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by
reference), or equivalents
thereof. In embodiments, the amino acid substitution at amino acid residue 250
is a substitution with
glutamine. In embodiments, the amino acid substitution at amino acid residue
252 is a substitution with
tyrosine, phenylalanine, tryptophan or threonine. In embodiments, the amino
acid substitution at amino acid
residue 254 is a substitution with threonine. In embodiments, the amino acid
substitution at amino acid
residue 256 is a substitution with serine, arginine, glutamine, glutamic acid,
aspartic acid, or threonine. In
embodiments, the amino acid substitution at amino acid residue 308 is a
substitution with threonine. In
embodiments, the amino acid substitution at amino acid residue 309 is a
substitution with proline. In
embodiments, the amino acid substitution at amino acid residue 311 is a
substitution with serine. In
embodiments, the amino acid substitution at amino acid residue 385 is a
substitution with arginine, aspartic
acid, serine, threonine, histidine, lysine, alanine or glycine. In
embodiments, the amino acid substitution at
amino acid residue 386 is a substitution with threonine, proline, aspartic
acid, serine, lysine, arginine,
zo isoleucine, or methionine. In embodiments, the amino acid substitution
at amino acid residue 387 is a
substitution with arginine, proline, histidine, serine, threonine, or alanine.
In embodiments, the amino acid
substitution at amino acid residue 389 is a substitution with proline, serine
or asparagine. In embodiments,
the amino acid substitution at amino acid residue 416 is a substitution with
serine. In embodiments, the amino
acid substitution at amino acid residue 428 is a substitution with leucine. In
embodiments, the amino acid
substitution at amino acid residue 433 is a substitution with arginine,
serine, isoleucine, proline, or glutamine.
In embodiments, the amino acid substitution at amino acid residue 434 is a
substitution with histidine,
phenylalanine, or tyrosine.
In embodiments, the Fc domain linker (e.g., comprising an IgG constant region)
comprises one or more
mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434,
or 436 (in accordance with
Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly
incorporated herein by
reference). In embodiments, the IgG constant region includes a triple
M252Y/52541/1-256E mutation or YTE
mutation. In embodiments, the IgG constant region includes a triple
H433K/N434FN436H mutation or KFH
mutation. In embodiments, the IgG constant region includes an YTE and KFH
mutation in combination.
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the linker comprises an IgG constant region that contains one
or more mutations at amino
acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance
with Kabat numbering, as in
as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by
reference). Illustrative mutations
include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F,
N434S, and H435A.
In embodiments, the IgG constant region comprises a M428L/N4345 mutation or LS
mutation. In
embodiments, the IgG constant region comprises a T2500/M428L mutation or QL
mutation. In embodiments,
the IgG constant region comprises an N434A mutation. In embodiments, the IgG
constant region comprises
a T307A/E380A/N434A mutation or AAA mutation. In embodiments, the IgG constant
region comprises an
1253A/H310A/H435A mutation or IHH mutation. In embodiments, the IgG constant
region comprises a
H433K/N434F mutation. In embodiments, the IgG constant region comprises a
M252Y/S254T/T256E and a
H433K/N434F mutation in combination.
Additional exemplary mutations in the IgG constant region are described, for
example, in Robbie, et al.,
Antimicrobial Agents and Chemotherapy 57(12):6147-6153 (2013); Dall'Acqua et
al., Journal Biol Chem
281(33):23514-24 (2006); Dall'Acqua et al., Journal of Immunology 169:5171-80
(2002); Ko et al. Nature
zo 514:642-645 (2014); Grevys etal. Journal of Immunology 194(11):5497-508
(2015); and U.S. Patent No.
7,083,784, the entire contents of which are hereby incorporated by reference.
An illustrative Fc stabilizing mutant is 8228P. Illustrative Fc half-life
extending mutants are 12500, M428L,
V308T, L309P, and Q311S and the present linkers may comprise 1, or 2, or 3, or
4, or 5 of these mutants.
In embodiments, the chimeric protein binds to FcRn with high affinity. In
embodiments, the chimeric protein
zs may bind to FcRn with a KD of about 1 nM to about 80 nM. For example,
the chimeric protein may bind to
FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM,
about 6 nM, about 7 nM,
about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM,
about 30 nM, about 35 nM,
about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM,
about 70 nM, about 71
nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77
nM, about 78 nM, about
30 79 nM, or about 80 nM. In embodiments, the chimeric protein may bind to
FcRn with a KD of about 9 nM. In
embodiments, the chimeric protein does not substantially bind to other Fc
receptors (i.e. other than FcRn)
with effector function.
In embodiments, the Fc domain in a linker has the amino acid sequence of SEQ
ID NO: 1 (see Table 1,
below), or at least 90%, or 93%, or 95%, 01 97%, or 98%, or 99% identity
thereto. In embodiments, mutations
36
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
are made to SEQ ID NO: 1 to increase stability and/or half-life. For instance,
in embodiments, the Fc domain
in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1,
below), or at least 90%, or
93%, or 95%, or 97%, or 98%, or 99% identity thereto. For instance, in
embodiments, the Fc domain in a
linker comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1, below),
or at least 90%, or 93%,
or 95%, or 97%, or 98%, or 99% identity thereto.
Further, one or more joining linkers may be employed to connect an Fc domain
in a linker (e.g., one of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or at least 90%, or 93%, or 95%, or 97%,
or 98%, or 99% identity
thereto) and the extracellular domains. For example, any one of SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may connect
an extracellular domain as
disclosed herein and an Fc domain in a linker as disclosed herein. Optionally,
any one of SEQ ID NOs: 4 to
is 50, or variants thereof are located between an extracellular domain as
disclosed herein and an Fc domain
as disclosed herein.
In embodiments, the present chimeric proteins may comprise variants of the
joining linkers disclosed in Table
1, below. For instance, a linker may have at least about 60%, or at least
about 61%, or at least about 62%,
or at least about 63%, or at least about 64%, or at least about 65%, or at
least about 66%, or at least about
zo 67%, or at least about 68%, or at least about 69%, or at least about
70%, or at least about 71%, or at least
about 72%, or at least about 73%, or at least about 74%, or at least about
75%, or at least about 76%, or at
least about 77%, or at least about 78%, or at least about 79%, or at least
about 80%, or at least about 81%,
or at least about 82%, or at least about 83%, or at least about 84%, or at
least about 85%, or at least about
86%, or at least about 87%, or at least about 88%, or at least about 89%, or
at least about 90%, or at least
25 about 91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or at least
about 99% sequence identity with
the amino acid sequence of any one of SEQ ID NOs: 4 to 50.
In embodiments, the first and second joining linkers may be different or they
may be the same.
Without wishing to be bound by theory, including a linker comprising at least
a part of an Fc domain in a
30 chimeric protein, helps avoid formation of insoluble and, likely, non-
functional protein concatemers and/or
aggregates. This is in part due to the presence of cysteines in the Fc domain
which are capable of forming
disulfide bonds between chimeric proteins.
In embodiments, a chimeric protein may comprise one or more joining linkers,
as disclosed herein, and lack
a Fc domain linker, as disclosed herein.
37
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the first and/or second joining linkers are independently
selected from the amino acid
sequences of SEQ ID NOs: 4 to 50 and are provided in Table 1 below:
Table 1: Illustrative linkers (Fc domain linkers and joining linkers)
SEQ ID Sequence
NO.
1 AP EFLGGPSVFLFPPK PKDTLMISRTP
EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK
PREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTI SNATGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT
VD KSSWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGK
2 AP EFLGGPSVFLFPPK PKDQLMI SRTP
EVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTK
PREEQFNSTYRVVSVLTTPHSDWLSGKEYKCKVSSKGLPSSI EKTISNATGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT
VDKSSWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK
3 AP EFLGGPSVFLFPPK PKDQLMI SRTP
EVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTK
PREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTI SNATGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT
VDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK
4 SKYGPPCPSCP
5 SKYGPPCPPCP
6 SKYGPP
7 IEGRMD
8 GGGVPRDCG
9 IEGRMDGGGGAGGGG
GGGSGGGS
11 GGGSGGGGSGGG
12 EGKSSGSGSESKST
13 GGSG
14 GGSGGGSGGGSG
EAAAK EAAAKEAAAK
16 EAAAREAAAREAAAREAAAR
38
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
17 GGGGSGGGGSGGGGSAS
18 GGGGAGGGG
19 GS or GGS or LE
20 GSGSGS
21 GSGSGSGSGS
22 GGGGSAS
23 APAPAPAPAPAPAPAPAPAP
24 CPPC
25 GGGGS
26 GGGGSGGGGS
27 GGGGSGGGGSGGGGS
28 GGGGSGGGGSGGGGSGGGGS
29 GGGGSGGGGSGGGGSGGGGSGGGGS
30 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
31 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
32 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
33 GGSGGSGGGGSGGGGS
34 GGGGGGGG
35 GGGGGG
36 EAAAK
37 EAAAKEAAAK
38 EAAAKEAAAKEAAAK
39 AEAAAKEAAAKA
40 AEAAAKEAAAKEAAAKA
41 AEAAAKEAAAKEAAAKEAAAKA
42 AEAAAKEAAAKEAAAKEAAAKEAAAKA
43 AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA
44 PAPAP
45 KESGSVSSEQLAQFRSLD
46 GSAGSAAGSGEF
39
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
47 GGGSE
48 GSESG
49 GSEGS
50 GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS
In embodiments, the joining linker substantially comprises glycine and serine
residues (e.g., about 30%, or
about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about
90%, or about 95%, or about
97%, or about 98%, or about 99%, or about 100% glycines and serines). For
example, in embodiments, the
joining linker is (Gly4Ser)n, where n is from about 1 to about 8, e.g., 1, 2,
3, 4, 5, 6, 7, or 8 (SEQ ID NO: 25 to
SEQ ID NO: 9, respectively). In embodiments, the joining linker sequence is
GGSGGSGGGGSGGGGS
(SEQ ID NO: 33). Additional illustrative joining linkers include, but are not
limited to, linkers having the
sequence LE, (EAAAK)n (n=1-3) (SEQ ID NO: 36 to SEQ ID NO: 38), A(EAAAK)nA (n
= 2-5) (SEQ ID NO: 39
to SEQ ID NO: 42), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 43), PAPAP (SEQ ID NO:
44),
KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46), and (XP)n,
with X
designating any amino acid, e.g., Ala, Lys, or Glu. In embodiments, the
joining linker is GGS. In embodiments,
a joining linker has the sequence (Gly)n where n is any number from 1 to 100,
for example: (Gly)8 (SEQ ID
NO: 34) and (Gly)8 (SEQ ID NO: 35).
In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO: 47),
GSESG (SEQ ID NO: 48),
GSEGS (SEQ ID NO: 49), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50),
and
a joining linker of randomly placed G, S, and E every 4 amino acid intervals.
In embodiments, the chimeric protein comprises a joining linker comprising the
amino acid sequence of SEQ
ID NO: 5 and/or SEQ ID NO: 7.
In embodiments, where a chimeric protein comprises an extracellular domain
(ECD) of CD172a (SIRPa), one
joining linker preceding an Fc domain, a second joining linker following the
Fc domain, and an ECD of CD4OL,
the chimeric protein may comprise the following structure:
ECD of human CD172a (SIRPa) - Joining Linker 1 - Fc Domain - Joining Linker 2-
ECD of human
CD4OL
The combination of a first joining linker, an Fc Domain linker, and a second
joining linker is referend to herein
as a "modular linker". In embodiments, a chimeric protein comprises a modular
linker as shown in Table 2:
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
TABLE 2: Illustrative modular linkers
Joining Linker Fc Joining
Modular Linker = Joining Linker
1 Linker 2 1 + Fc + Joining
Linker 2
SKYGPPCPSCP APEFLGGPSVFLFPPKPKDTL IEGRMD SKYGPPCPSCPAPEFLGGPSVFL
(SEQ ID NO: 4) MISRTPEVTCVVVDVSQEDPE (SEQ ID FPPKPKDTLMISRTPEVTCVWDV
VQFNWYVDGVEVHNAKTKPR NO: 7)
SQEDPEVQFNWYVDGVEVHNAK
EEQFNSTYRVVSVLTVLHQDVV
TKPREEQFNSTYRWSVLTVLHQ
LSGKEYKCKVSSKGLPSSIEKT
DWLSGKEYKCKVSSKGLPSSIEK
ISNATGQPREPQVYTLPPSQE
TISNATGQPREPQVYTLPPSQEE
EMTKNQVSLTCLVKGFYPSDIA
MTKNQVSLTCLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSSWQE
SFFLYSRLTVDKSSWQEGNVFSC
GNVFSCSVMHEALHNHYTQK
SVMHEALHNHYTQKSLSLSLGKIE
SLSLSLGK (SEQ ID NO: 1) GRMD (SEQ ID NO: 51)
SKYGPPCPSCP APEFLGGPSVFLFPPKPKDQL IEGRMD SKYGPPCPSCPAPEFLGGPSVFL
(SEQ ID NO: 4) MISRTPEVTCVVVDVSQEDPE (SEQ ID FPPKPKDQLMISRTPEVTCVWD
VQFNWYVDGVEVHNAKTKPR NO: 7)
VSQEDPEVQFNINYVDGVEVHNA
EEQFNSTYRVVSVLTTPHSDW
KTKPREEQFNSTYRVVSVLTTPH
LSGKEYKCKVSSKGLPSSIEKT
SDWLSGKEYKCKVSSKGLPSSIE
ISNATGQPREPQVYTLPPSQE
KTISNATGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIA
EMTKNQVSLTCLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSSVVQE
SFFLYSRLTVDKSSWQEGNVFSC
GNVFSCSVLHEALHNHYTQKS
SVLHEALHNHYTQKSLSLSLGKIE
LSLSLGK (SEQ ID NO: 2) GRMD (SEQ ID NO: 52)
SKYGPPCPSCP APEFLGGPSVFLFPPKPKDQL IEGRMD SKYGPPCPSCPAPEFLGGPSVFL
(SEQ ID NO: 4) MISRTPEVTCVVVDVSQEDPE (SEQ ID FPPKPKDQLMISRTPEVTCVWD
VQFNWYVDGVEVHNAKTKPR NO: 7)
VSQEDPEVQFNVVYVDGVEVHNA
EEQFNSTYRVVSVLTVLHQDVV
KTKPREEQFNSTYRVVSVLTVLH
LSGKEYKCKVSSKGLPSSIEKT
QDWLSGKEYKCKVSSKGLPSSIE
41
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
ISNATGQPREPQVYTLPPSQE
KTISNATGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIA
EMTKNQVSLTCLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSRVVQE
SFFLYSRLTVDKSRWQEGNVFSC
GNVFSCSVLHEALHNHYTQKS
SVLHEALHNHYTQKSLSLSLGKI E
LSLSLGK (SEQ ID NO: 3) GRMD (SEQ ID NO: 53)
SKYGPPCPPCP APEFLGGPSVFLFPPKPKDTL IEGRMD SKYGPPCPPCPAPEFLGGPSVFL
(SEQ ID NO: 5) MISRTPEVTCWVDVSQEDPE (SEQ ID FPPKPKDTLMISRTPEVTCVWDV
VQFNWYVDGVEVHNAKTKPR NO: 7)
SQEDPEVQFNWYVDGVEVHNAK
EEQFNSTYRVVSVLTVLHQDVV TK
PREEQFNSTYRWSVLTVLHQ
LSGKEYKCKVSSKGLPSSIEKT
DWLSGKEYKCKVSSKGLPSSI EK
ISNATGQPREPQVYTLPPSQE
TISNATGQPREPQVYTLPPSQEE
EMTKNQVSLTCLVKGFYPSDIA
MTKNOVSLICLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSSWQE
SFFLYSRLTVDKSSWQEGNVFSC
GNVFSCSVMHEALHNHYTQK
SVMHEALHNHYTQKSLSLSLGKIE
SLSLSLGK (SEQ ID NO: 1) GRMD (SEQ ID NO: 54)
SKYGPPCPPCP APEFLGGPSVFLFPPKPKDQL IEGRMD SKYGPPCPPCPAPEFLGGPSVFL
(SEQ ID NO: 5) MISRTPEVTCWVDVSQEDPE (SEQ ID FPPKPKDQLMISRTPEVTCVWD
VQFNWYVDGVEVHNAKTKPR NO: 7)
VSQEDPEVQFNVVYVDGVEVHNA
EEQFNSTYRVVSVLTTPHSDVV
KTKPREEQFNSTYRVVSVLTTPH
LSGKEYKCKVSSKGLPSSIEKT
SDWLSGKEYKCKVSSKGLPSSIE
ISNATGQPREPQVYTLPPSQE
KTISNATGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIA
EMTKNQVSLTCLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSSWQE
SFFLYSRLTVDKSSWQEGNVFSC
GNVFSCSVLHEALHNHYTQKS
SVLHEALHNHYTQKSLSLSLGKI E
LSLSLGK (SEQ ID NO: 2) GRMD (SEQ ID NO: 55)
SKYGPPCPPCP APEFLGGPSVFLFPPKPKDQL IEGRMD SKYGPPCPPCPAPEFLGGPSVFL
(SEQ ID NO: 5) MISRTPEVTCWVDVSQEDPE (SEQ ID FPPKPKDQLMISRTPEVTCVWD
VQFNWYVDGVEVHNAKTKPR NO: 7)
VSQEDPEVQFNVVYVDGVEVHNA
42
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
EEQFNSTYRVVSVLTVLHQDVV
KTKPREEQFNSTYRVVSVLTVLH
LSGKEYKCKVSSKGLPSSIEKT
QDWLSGKEYKCKVSSKGLPSSIE
ISNATGQPREPQVYTLPPSQE
KTISNATGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIA
EMTKNOVSLICLVKGFYPSDIAVE
VEWESNGQPENNYKTTPPVL
WESNGQPENNYKTTPPVLDSDG
DSDGSFFLYSRLTVDKSRVVQE
SFFLYSRLTVDKSRWQEGNVFSC
GNVFSCSVLHEALHNHYTQKS
SVLHEALHNHYTQKSLSLSLGKIE
LSLSLGK (SEQ ID NO: 3) GRMD (SEQ ID NO: 56)
In embodiments, the present chimeric proteins may comprise variants of the
modular linkers disclosed in
Table 2, above. For instance, a linker may have at least about 60%, or at
least about 61%, or at least about
62%, or at least about 63%, or at least about 64%, or at least about 65%, or
at least about 66%, or at least
about 67%, or at least about 68%, or at least about 69%, or at least about
70%, or at least about 71%, or at
least about 72%, or at least about 73%, or at least about 74%, or at least
about 75%, or at least about 76%,
io or at least about 77%, or at least about 78%, or at least about 79%, or
at least about 80%, or at least about
81%, or at least about 82%, or at least about 83%, or at least about 84%, or
at least about 85%, or at least
about 86%, or at least about 87%, or at least about 88%, or at least about
89%, or at least about 90%, or at
least about 91%, or at least about 92%, or at least about 93%, or at least
about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about 98%, or at
least about 99% sequence identity
is with the amino acid sequence of any one of SEQ ID NOs: 51 to 56.
In embodiments, the linker may be flexible, including without limitation
highly flexible. In embodiments, the
linker may be rigid, including without limitation a rigid alpha helix.
Characteristics of illustrative joining linkers
is shown below in Table 3:
TABLE 3: Characteristics of illustrative joining linkers
Joining Linker Sequence Characteristics
SKYGPPCPPCP (SEQ ID NO: 5) IgG4 Hinge Region
IEGRMD (SEQ ID NO: 7) Linker
GGGVPRDCG (SEQ ID NO: 8) Flexible
GGGSGGGS (SEQ ID NO: 10) Flexible
GGGSGGGGSGGG (SEQ ID NO: 11) Flexible
43
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Joining Linker Sequence Characteristics
EGKSSGSGSESKST (SEQ ID NO: 12) Flexible + soluble
GGSG (SEQ ID NO: 13) Flexible
GGSGGGSGGGSG (SEQ ID NO: 14) Flexible
EAAAKEAAAKEAAAK (SEQ ID NO: 15) Rigid Alpha Helix
EAAAREAAAREAAAREAAAR (SEQ ID NO: 16) Rigid Alpha Helix
GGGGSGGGGSGGGGSAS (SEQ ID NO: 17) Flexible
GGGGAGGGG (SEQ ID NO: 18) Flexible
GS (SEQ ID NO: 19) Highly flexible
GSGSGS (SEQ ID NO: 20) Highly flexible
GSGSGSGSGS (SEQ ID NO: 21) Highly flexible
GGGGSAS (SEQ ID NO: 22) Flexible
APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 23) Rigid
In embodiments, the linker may be functional. For example, without limitation,
the linker may function to
improve the folding and/or stability, improve the expression, improve the
pharmacokinetics, and/or improve
the bioactivity of the present chimeric protein. In another example, the
linker may function to target the
chimeric protein to a particular cell type or location.
In embodiments, a chimeric protein comprises only one joining linkers.
In embodiments, a chimeric protein lacks joining linkers.
In embodiments, the linker is a synthetic linker such as polyethylene glycol
(PEG).
In embodiments, a chimeric protein has a first domain which is sterically
capable of binding its ligand/receptor
and/or the second domain which is sterically capable of binding its
ligand/receptor. Thus, there is enough
overall flexibility in the chimeric protein and/or physical distance between
an extracellular domain (or portion
thereof) and the rest of the chimeric protein such that the ligand/receptor
binding domain of the extracellular
domain is not sterically hindered from binding its ligand/receptor. This
flexibility and/or physical distance
(which is referred to as "slack") may be normally present in the extracellular
domain(s), normally present in
the linker, and/or normally present in the chimeric protein (as a whole).
Alternately, or additionally, an amino
acid sequence (for example) may be added to one or more extracellular domains
and/or to the linker to
zo provide the slack needed to avoid steric hindrance. Any amino acid
sequence that provides slack may be
44
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
added. In embodiments, the added amino acid sequence comprises the sequence
(Gly)n where n is any
number from 1 to 100. Additional examples of addable amino acid sequence
include the joining linkers
described in Table '1 and Table 3. In embodiments, a polyethylene glycol (PEG)
linker may be added between
an extracellular domain and a linker to provide the slack needed to avoid
steric hindrance. Such PEG linkers
are well known in the art.
io In embodiments, a chimeric protein of the present disclosure comprises
the extracellular domain of human
CD172a (SIRPa) (or a variant thereof), a linker, and the extracellular domain
of human CD4OL (or a variant
thereof). In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain,
e.g., from an IgG1 or from
IgG4, including human IgG1 or IgG4. Thus, in embodiments, a chimeric protein
of the present disclosure
comprises the extracellular domain of human CD172a (SIRPa) (or a variant
thereof), linker comprising a
is hinge-CH2-CH3 Fc domain, and the extracellular domain of human CD4OL (or
a variant thereof). Such a
chimeric protein may be referred to herein as "hCD172a (SIRPa)-Fc-CD4OL" or
"SL-172154".
Diseases, Methods of Treatment, and Mechanisms of Action
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises (i) administering to the human subject a first dose of
chimeric protein having a general
zo structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is
a first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
of a cell has been measured in a biological sample obtained from the subject
before the administration of the
zs first dose, wherein the marker is selected from one or more of a CD8O-F
cell, a CD8-F cell, a Granzyme B+
cell, a CD68+ cell, a Ki67+ cell, and a PD-L1+ immune cell. In embodiments,
the method further comprises
(ii) administering to the human subject a second dose of the chimeric protein
if a post-dosing level and/or
activity of the cell is greater than the background level and/or activity of
the cell. In embodiments, the second
dose is administered at least about 48 hours after the administration of the
first dose.
30 In embodiments, the biological sample is selected from blood, plasma,
serum, blood cells, lacrimal fluid,
tears, bone marrow, ascites, tissue or fine needle biopsy sample, cell-
containing body fluid, free floating
nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid,
pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing
or lavage, aspirate, scraping,
bone marrow specimen, a biopsy specimen and a surgical specimen. In
embodiments, the biological sample
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
is a biopsy sample or a surgical specimen. In embodiments, the biological
sample is a tumor biopsy sample
or a tumor surgical specimen. In embodiments, the a tumor biopsy sample
derived from a tumor selected
ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous
cell carcinoma (CSCC), and
squamous cell carcinoma of the head and neck (SCCHN).
In embodiments, the level and/or activity of the cell is measured by RNA
sequencing, immunohistochemical
io staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof. In embodiments, the level and/or
activity of the cell is measured by
contacting the sample with an agent that specifically binds to one or more
molecules selected from CD80,
CD8, Granzyme B, CD68, Ki67, and PD-L1. In embodiments, the agent that
specifically binds to the one or
more molecules is an antibody or fragment thereof. In embodiments, the
antibody is a recombinant antibody,
is a monoclonal antibody, a polyclonal antibody, or fragment thereof.
In embodiments, the level and/or activity of the cell is measured by
contacting the sample with an agent that
specifically binds to one or more nucleic acids encoding one or more of CD80,
008, Granzyme B, CD68,
Ki67, and PD-L1. In embodiments, the agent that specifically binds to one or
more of the nucleic acids is a
nucleic acid primer or probe.
zo An aspect of the present disclosure is a method for treating a cancer in
a human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
chimeric protein having a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
25 an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
of B cells and/or CD40+ cells has been measured in a first biological sample
obtained from the subject before
the administration of the first dose. In embodiments, an N-hr post-dose level
and/or activity of B cells and/or
CD40+ cells has been measured in a second biological sample obtained from the
subject after the
administration of the first dose. In embodiments, N is a number between 1 and
24. In embodiments, the N-
30 hr post-dose level and/or activity of B cells and/or CD40+ cells is less
than the background level and/or
activity of B cells and/or CD40+ cells. In embodiments, an M-day post-dose
level and/or activity of B cells
and/or CD40+ cells has been measured in a third biological sample obtained
from the subject after the
administration of the first dose. In embodiments, M is a number between 1 and
28. In embodiments, the
method further comprises (ii) administering to the human subject a second dose
of the chimeric protein if the
46
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
M-day post-dose level and/or activity of B cells and/or C040+ cells is at
least about 50% higher than the N-
hour post-dose level and/or activity of B cells and/or CD40+ cells. In
embodiments, the second dose is
administered at least about 48 hours after the administration of the first
dose.
In embodiments, N is less than 12, or less than 8, or less than 6, or less
than 4, or less than 3, or less than
2, or less than 1. In embodiments, M is less than 21, or less than 14, or less
than 12, or less than 10, or less
than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is
less than 12, or less than 8, or less
than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M
is less than 21, or less than 14, or
less than 12, or less than 10, or less than 8, or less than 6, or less than 4,
or less than 2.
In embodiments, the N-hr post-dose level and/or activity of B cells and/or
CD40+ cells is greater than the
background level and/or activity of B cells and/or CD40+ cells. In
embodiments, the N-hr post-dose level
and/or activity of B cells and/or CD40+ cells is less than the background
level and/or activity of B cells and/or
CD40+ cells. In embodiments, the N-hr post-dose level and/or activity of B
cells and/or CD40+ cells is within
about 10%, or about 20%, or about 30%, or about 40% of the background level
and/or activity of B cells
and/or CD40+ cells.
In embodiments, the first biological sample, the second biological sample and
the third biological sample are
zo independently selected from blood, plasma, serum, blood cells, lacrimal
fluid, tears, bone marrow, ascites,
tissue or fine needle biopsy sample, cell-containing body fluid, free floating
nucleic acids, sputum, saliva,
urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal
swab, oral swab, nasal swab, washing or lavage, aspirate, scraping, bone
marrow specimen, a biopsy
specimen and a surgical specimen. In embodiments, the first biological sample,
the second biological sample
zs and the third biological sample are blood.
In embodiments, level and/or activity of B cells and/or CD40+ cells is
measured by RNA sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
fluorescent activating cell sorting (FACS) or a combination thereof. In
embodiments, level and/or activity of B
cells and/or CD40+ cells is measured by contacting the sample with an agent
that specifically binds to a
30 CD40 and/or a B-cell marker. In embodiments, the B-cell marker is
selected from CD19, CD20, 0D24, 0D27,
CD34, CD38, CD45R, 0D86, 0D95, IgM, IgD, and CD40. In embodiments, the agent
that specifically binds
to the one or more molecules is an antibody or fragment thereof. In
embodiments, the antibody is a
recombinant antibody, a monoclonal antibody, a polyclonal antibody, or
fragment thereof. In embodiments,
level and/or activity of B cells and/or CD40+ cells is measured by contacting
the sample with an agent that
47
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
specifically binds to one or more nucleic acids encoding a CD40 and/or a B-
cell marker. In embodiments, the
B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R,
CD86, CD95, I gM, IgD, and
CD40. In embodiments, the agent that specifically binds to one or more of the
nucleic acids is a nucleic acid
primer or probe.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
io the method comprises: (i) administering to the human subject a first
dose of the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background amount and/or
is activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNa, IL15,
IL23, IL-12, MCP-1, MIP-113, MIP-
1 a, and MDC has been measured in a first biological sample obtained from the
subject before the
administration of the first dose In embodiments, a N-hr post-dose amount
and/or activity of the cytokine has
been measured in a second biological sample obtained from the subject after
the administration of the first
dose, In embodiments, N is a number between 1 and 24. In embodiments, the N-hr
post-dose amount and/or
zo activity of the cytokine is greater than the background amount and/or
activity of the cytokine. In embodiments,
the M-day post-dose amount and/or activity of the cytokine has been measured
in a third biological sample
obtained from the subject after the administration of the first dose, wherein
M is a number between 1 and 28.
In embodiments, the method further comprises (ii) administering to the human
subject a second dose of the
chimeric protein if the M-day post-dose amount and/or activity of the cytokine
is at least about 30% lower
25 than the N-hr post-dose amount and/or activity of the cytokine. In
embodiments, the second dose is
administered at least about 48 hours after the administration of the first
dose.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
30 domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a
linker adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level of a cell has
been measured in a pre-dose biological sample obtained from the subject before
the administration of the
first dose, wherein the marker is selected from one or more of a CD8O-F cell,
a CD8+ cell, a Granzyme B+
35 cell, a CD68+ cell, a K167+ cell, and a PD-L1+ immune cell. In
embodiments, the method further comprises
48
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
administering to the human subject a second dose of the chimeric protein if a
post-dosing level of the cell is
greater than the background level of the cell. In embodiments, the second dose
is administered at least about
48 hours after the administration of the first dose.
In embodiments, the biological sample is selected from blood, plasma, serum,
blood cells, lacrimal fluid,
tears, bone marrow, ascites, tissue or fine needle biopsy sample, cell-
containing body fluid, free floating
io nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal
fluid, pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing
or lavage, aspirate, scraping,
bone marrow specimen, a biopsy specimen and a surgical specimen. In
embodiments, the biological sample
is a biopsy sample or a surgical specimen. In embodiments, the biological
sample is a tumor biopsy sample
or a tumor surgical specimen. In embodiments, the tumor biopsy sample or the
tumor surgical specimen
is derived from a tumor selected ovarian cancer, fallopian tube cancer,
peritoneal cancer, cutaneous squamous
cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck
(SCCHN).
In embodiments, the level and/or activity of the cell is measured by RNA
sequencing, immunohistochemical
staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof. In embodiments, the level and/or
activity of the cell is measured by
zo contacting the sample with an agent that specifically binds to one or
more molecules selected from CD80,
CD8, Granzyme B, CD68, Ki67, and PD-L1. In embodiments, the agent that
specifically binds to the one or
more molecules is an antibody or fragment thereof. In embodiments, the
antibody is a recombinant antibody,
a monoclonal antibody, a polyclonal antibody, or fragment thereof. In
embodiments, the level and/or activity
of the cell is measured by contacting the sample with an agent that
specifically binds to one or more nucleic
25 acids encoding one or more of 0D80, 008, Granzyme B, CD68, Ki67, and PD-
L1. In embodiments, the agent
that specifically binds to one or more of the nucleic acids is a nucleic acid
primer or probe.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
30 domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a
linker adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
of B cells and/or CD40+ cells has been measured in a first biological sample
obtained from the subject before
the administration of the first dose. In embodiments, an N-hr post-dose level
and/or activity of B cells and/or
49
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
CD40+ cells has been measured in a second biological sample obtained from the
subject after the
administration of the first dose. In embodiments, N is a number between 1 and
24. In embodiments, the N-
hr post-dose level and/or activity of B cells and/or CD40+ cells is less than
the background level and/or
activity of B cells and/or CD40+ cells. In embodiments, an M-day post-dose
level and/or activity of B cells
and/or CD40+ cells has been measured in a third biological sample obtained
from the subject after the
io administration of the first dose. In embodiments, M is a number between
1 and 28. In embodiments, the
method further comprises (ii) administering to the human subject a second dose
of the chimeric protein if the
M-day post-dose level and/or activity of B cells and/or CD40+ cells is at
least about 50% higher than the N-
hour post-dose level and/or activity of B cells and/or CD4O-F cells. In
embodiments, the second dose is
administered at least about 48 hours after the administration of the first
dose.
is In embodiments, N is less than 12, or less than 8, or less than 6, or
less than 4, or less than 3, or less than
2, or less than 1. In embodiments, M is less than 21, or less than 14, or less
than 12, or less than 10, or less
than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is
less than 12, or less than 8, or less
than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M
is less than 21, or less than 14, or
less than 12, or less than 10, or less than 8, or less than 6, or less than 4,
or less than 2.
zo In embodiments, the N-hr post-dose level and/or activity of B cells
and/or CD40+ cells is greater than the
background level and/or activity of B cells and/or CD4O-F cells. In
embodiments, the N-hr post-dose level
and/or activity of B cells and/or CD4O-F cells is less than the background
level and/or activity of B cells and/or
CD40+ cells. In embodiments, the N-hr post-dose level and/or activity of B
cells and/or CD40+ cells is within
about 10%, or about 20%, or about 30%, or about 40% of the background level
and/or activity of B cells
25 and/or C040+ cells.
In embodiments, the first biological sample, the second biological sample and
the third biological sample are
independently selected from blood, plasma, serum, blood cells, lacrimal fluid,
tears, bone marrow, ascites,
tissue or fine needle biopsy sample, cell-containing body fluid, free floating
nucleic acids, sputum, saliva,
urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal
30 swab, oral swab, nasal swab, washing or lavage, aspirate, scraping, bone
marrow specimen, a biopsy
specimen and a surgical specimen. In embodiments, the first biological sample,
the second biological sample
and the third biological sample are blood.
In embodiments, the level and/or activity of B cells and/or CD40+ cells is
measured by RNA sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
fluorescent activating cell sorting (FACS) or a combination thereof. In
embodiments, the level and/or activity
of B cells and/or CD4O-F cells is measured by contacting the sample with an
agent that specifically binds to a
CD40 and/or a B-cell marker. In embodiments, the B-cell marker is selected
from CD19, CD20, CD24, CD27,
CD34, C038, CD45R, CD86, CD95, IgM, IgD, and CD40. In embodiments, the agent
that specifically binds
to the one or more molecules is an antibody or fragment thereof. In
embodiments, the antibody is a
recombinant antibody, a monoclonal antibody, a polyclonal antibody, or
fragment thereof. In embodiments,
the level and/or activity of B cells and/or CD40+ cells is measured by
contacting the sample with an agent
that specifically binds to one or more nucleic acids encoding a CD40 and/or a
B-cell marker. In embodiments,
the B-cell marker is selected from CD19, CD20, CD24, CD 27, CD34, CD38, CD45R,
CD86, CD95, IgM, IgD,
and CD40. In embodiments, the agent that specifically binds to one or more of
the nucleic acids is a nucleic
acid primer or probe.
An aspect of the present disclosure is a method for treating a cancer in a
human subject. In embodiments,
the method comprises: (i) administering to the human subject a first dose of
the chimeric protein has a general
structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
zo domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c)
is a second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background amount and/or
activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNa, ILI 5, IL23,
IL-12, MCP-1, MIP-1 13, MIP-
1a, and MDC has been measured in a first biological sample obtained from the
subject before the
administration of the first dose. In embodiments, a N-hr post-dose amount
and/or activity of the cytokine has
been measured in a second biological sample obtained from the subject after
the administration of the first
dose. In embodiments, N is a number between 1 and 24. In embodiments, the N-hr
post-dose amount and/or
activity of the cytokine is greater than the background amount and/or activity
of the cytokine. In embodiments,
the M-day post-dose amount and/or activity of the cytokine has been measured
in a third biological sample
obtained from the subject after the administration of the first dose. In
embodiments, M is a number between
1 and 28. In embodiments, the method further comprises (ii) administering to
the human subject a second
dose of the chimeric protein if the M-day post-dose amount and/or activity of
the cytokine is at least about
30% lower than the N-hr post-dose amount and/or activity of the cytokine. In
embodiments, the second dose
is administered at least about 48 hours after the administration of the first
dose.
In embodiments, N is less than 12, or less than 8, or less than 6, or less
than 4, or less than 3, or less than
2, or less than 1. In embodiments, M is less than 21, or less than 14, or less
than 12, or less than 10, or less
51
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is
less than 12, or less than 8, or less
than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M
is less than 21, or less than 14, or
less than 12, or less than 10, or less than 8, or less than 6, or less than 4,
or less than 2.
In embodiments, the N-hr post-dose amount and/or activity of the cytokine is
greater than the background
amount and/or activity of the cytokine. In embodiments, the N-hr post-dose
amount and/or activity of the
cytokine is less than the background amount and/or activity of the cytokine.
In embodiments, the N-hr post-
dose amount and/or activity of the cytokine is within about 10%, or about 20%,
or about 30%, or about 40%
of the background amount and/or activity of the cytokine.
In embodiments, the first biological sample, the second biological sample and
the third biological sample are
independently selected from blood, plasma, serum, blood cells, lacrimal fluid,
tears, bone marrow, ascites,
tissue or fine needle biopsy sample, cell-containing body fluid, free floating
nucleic acids, sputum, saliva,
urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal
swab, oral swab, nasal swab, washing or lavage, aspirate, scraping, bone
marrow specimen, a biopsy
specimen and a surgical specimen. In embodiments, the first biological sample,
the second biological sample
and the third biological sample are blood.
zo In embodiments, the amount and/or activity of the cytokine is measured
by RNA sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
fluorescent activating cell sorting (FACS) or a combination thereof. In
embodiments, the amount and/or
activity of the cytokine is measured by contacting the sample with an agent
that specifically binds to the
cytokine. In embodiments, the agent that specifically binds to the one or more
molecules is an antibody or
zs fragment thereof. In embodiments, the antibody is a recombinant
antibody, a monoclonal antibody, a
polyclonal antibody, or fragment thereof. In embodiments, the amount and/or
activity of the cytokine is
measured by contacting the sample with an agent that specifically binds to one
or more nucleic acids
encoding the cytokine. In embodiments, the agent that specifically binds to
one or more of the nucleic acids
is a nucleic acid primer or probe.
30 The chimeric proteins disclosed herein, including the CD172a (SIRPa)-Fc-
CD4OL chimeric protein (e.g. SEQ
ID NO: 59 or SEQ ID NO: 61), finds use in methods for treating both advanced
solid tumors and advanced
lymphomas. These tumor types include: melanoma, non-small cell lung cancer
(squamous, adeno, adeno-
squamous), urothelial cancer, renal cell cancer, squamous cell cervical
cancer, gastric or gastro-esophageal
junction adenocarcinoma, squamous cell carcinoma of the anus, squamous cell
carcinoma of the head and
52
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
neck, squamous cell carcinoma of the skin, and microsatellite instability high
or mismatch repair deficient
solid tumors excluding central nervous system (CNS) tumors. Other tumors of
interest include Hodgkin's
lymphoma (HL), diffuse large B cell lymphoma, acute myeloid leukemia (AML) and
high-risk myelodysplastic
syndromes (HR-M DS).
In embodiments, the cancer comprises an advanced solid tumor (local and/or
metastatic). In embodiments,
the human subject has a cancer, wherein the cancer being treated is
characterized by having macrophages
in the tumor microenvironment and/or having tumor cells that are CD47+ cells
in the tumor. In embodiments,
the administration of the SIRPa- Fc-CD4OL chimeric protein blocks the "don't
eat me" signal of a tumor cell
and/or stimulates an "eat me" signal. In embodiments the therapy with the
SIRPa-Fc-CD4OL chimeric protein
(e.g. SEQ ID NO: 59 or SEQ ID NO: 61) stimulates macrophages to phagocytize
tumor cells and effectively
is present the tumor antigens of phagocytized tumor cells to T cells.
In embodiments, the cancer is a solid cancer. In embodiments, the cancer is a
solid tumor. In embodiments,
the cancer is a metastatic cancer. In embodiments, the cancer is a
hematological cancer. In embodiments,
the cancer expresses CD47.
In embodiments, the cancer comprises an advanced lymphoma. In embodiments, the
cancer comprises
zo acute myeloid leukemia (AML). In embodiments, the cancer comprises p53
mutant AML. In embodiments,
the cancer comprises a high-risk myelodysplastic syndrome (HR-MDS).
Aspects of the present disclosure provide methods of treating cancer. The
methods comprise a step of
administering to a subject in need thereof an effective amount of a chimeric
protein, e.g., in a pharmaceutical
composition, as disclosed herein.
25 It is often desirable to enhance immune stimulatory signal transmission
to boost an immune response, for
instance to enhance a patient's anti-tumor immune response.
In embodiments, the chimeric protein of the present disclosure comprises an
extracellular domain of human
CD172a (SIRPa), which disrupts, blocks, reduces, inhibits, and/or sequesters
the transmission of immune
inhibitory signals, e.g., originating from a cancer cell that is attempting to
avoid its detection and/or
30 destruction, and an extracellular domain of human CD4OL, which enhances,
increases, and/or stimulates the
transmission of an immune stimulatory signal to an anti-cancer immune cell.
Thus, the simultaneous binding
of the extracellular domain of CD172a (SIRPa) to its ligand/receptor and the
binding of the extracellular
domain of CD4OL with its receptor will prevent the transmission of an
immunosuppressive signal from the
53
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
cancer cell and will have stimulate immune activity in an immune system cell.
In other words, chimeric
proteins of the present disclosure are capable of treating cancer via two
distinct mechanisms.
In embodiments, the present disclosure pertains to cancers and/or tumors; for
example, the treatment or
prevention of cancers and/or tumors. As disclosed elsewhere herein, the
treatment of cancer involves, in
embodiments, modulating the immune system with the present chimeric proteins
to favor of increasing or
activating immune stimulatory signals. In embodiments, the method reduces the
amount or activity of
regulatory T cells (Tregs) as compared to untreated subjects or subjects
treated with antibodies directed to
CD172a (SIRPa), CD4OL, and/or their respective ligands or receptors. In
embodiments, the method
increases priming of effector T cells in draining lymph nodes of the subject
as compared to untreated subjects
or subjects treated with antibodies directed to CD172a (SIRPa), CD4OL, and/or
their respective ligands or
is receptors. In embodiments, the method causes an overall decrease in
immunosuppressive cells and a shift
toward a more inflammatory tumor environment as compared to untreated subjects
or subjects treated with
antibodies directed to the CD172a (SIRPa), CD4OL, and/or their respective
ligands or receptors.
In embodiments, the present chimeric proteins are capable of, or can be used
in methods comprising,
modulating the amplitude of an immune response, e.g. modulating the level of
effector output. In
zo embodiments, e.g. when used for the treatment of cancer, the present
chimeric proteins alter the extent of
immune stimulation as compared to immune inhibition to increase the amplitude
of a T cell response,
including, without limitation, stimulating increased levels of cytokine
production, proliferation or target killing
potential. In embodiments, the patient's T cells are activated and/or
stimulated by the chimeric protein, with
the activated T cells being capable of dividing and/or secreting cytokines.
25 Cancers or tumors refer to an uncontrolled growth of cells and/or
abnormal increased cell survival and/or
inhibition of apoptosis which interferes with the normal functioning of the
bodily organs and systems. Included
are benign and malignant cancers, polyps, hyperplasia, as well as dormant
tumors or micrometastases. Also,
included are cells having abnormal proliferation that is not impeded by the
immune system (e.g., virus infected
cells). The cancer may be a primary cancer or a metastatic cancer. The primary
cancer may be an area of
30 cancer cells at an originating site that becomes clinically detectable,
and may be a primary tumor. In contrast,
the metastatic cancer may be the spread of a disease from one organ or part to
another non-adjacent organ
or part. The metastatic cancer may be caused by a cancer cell that acquires
the ability to penetrate and
infiltrate surrounding normal tissues in a local area, forming a new tumor,
which may be a local metastasis.
The cancer may also be caused by a cancer cell that acquires the ability to
penetrate the walls of lymphatic
54
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
and/or blood vessels, after which the cancer cell is able to circulate through
the bloodstream (thereby being
a circulating tumor cell) to other sites and tissues in the body. The cancer
may be due to a process such as
lymphatic or hematogenous spread. The cancer may also be caused by a tumor
cell that comes to rest at
another site, re-penetrates through the vessel or walls, continues to
multiply, and eventually forms another
clinically detectable tumor. The cancer may be this new tumor, which may be a
metastatic (or secondary)
tumor.
The cancer may be caused by tumor cells that have metastasized, which may be a
secondary or metastatic
tumor. The cells of the tumor may be like those in the original tumor. As an
example, if a breast cancer or
colon cancer metastasizes to the liver, the secondary tumor, while present in
the liver, is made up of abnormal
breast or colon cells, not of abnormal liver cells. The tumor in the liver may
thus be a metastatic breast cancer
is or a metastatic colon cancer, not liver cancer.
The cancer may have an origin from any tissue. The cancer may originate from
melanoma, colon, breast, or
prostate, and thus may be made up of cells that were originally skin, colon,
breast, or prostate, respectively.
The cancer may also be a hematological malignancy, which may be leukemia or
lymphoma. The cancer may
invade a tissue such as liver, lung, bladder, or intestinal.
zo In embodiments, the chimeric protein is used to treat a subject that has
a treatment-refractory cancer. In
embodiments, the chimeric protein is used to treat a subject that is
refractory to one or more immune-
modulating agents. For example, in embodiments, the chimeric protein is used
to treat a subject that presents
no response to treatment, or whose disease progresses, after 12 weeks or so of
treatment. For instance, in
embodiments, the subject is refractory to one or more CD172a (SIRPa) and/or
CD47 agents, including, for
zs example, Magrolimab (5F9), Hu5F9-G4, CC-90002, Ti-061, SRF231, TTI-621,
TTI-622, or ALX148 refractory
patients. For instance, in embodiments, the subject is refractory to an anti-
CTLA-4 agent, e.g., ipilimumab
(YERVOY)-refractory patients (e.g., melanoma patients). Accordingly, in
embodiments the present disclosure
provides methods of cancer treatment that rescue patients that are non-
responsive to various therapies,
including monotherapy of one or more immune-modulating agents.
30 In embodiments, the present disclosure provides chimeric proteins which
target a cell or tissue within the
tumor microenvironment. In embodiments, the cell or tissue within the tumor
microenvironment expresses
one or more targets or binding partners of the chimeric protein. The tumor
microenvironment refers to the
cellular milieu, including cells, secreted proteins, physiological small
molecules, and blood vessels in which
the tumor exists. In embodiments, the cells or tissue within the tumor
microenvironment are one or more of:
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells;
endothelial progenitor cells (EPC);
cancer-associated fibroblasts; pericytes; other stromal cells; components of
the extracellular matrix (ECM);
dendritic cells; antigen presenting cells; T-cells; regulatory T cells;
macrophages; neutrophils; and other
immune cells located proximal to a tumor. In embodiments, the present chimeric
protein targets a cancer cell.
In embodiments, the cancer cell expresses one or more of targets or binding
partners of the chimeric protein.
The activation of regulatory T cells is critically influenced by costimulatory
and co-inhibitory signals. Two
major families of costimulatory molecules include the B7 and the tumor
necrosis factor (TNF) families. These
molecules bind to receptors on T cells belonging to the CD28 or TNF receptor
families, respectively. Many
well-defined co-inhibitors and their receptors belong to the B7 and 0D28
families.
In embodiments, an immune stimulatory signal refers to a signal that enhances
an immune response. For
example, in the context of oncology, such signals may enhance antitumor
immunity. For instance, without
limitation, immune stimulatory signal may be identified by directly
stimulating proliferation, cytokine
production, killing activity, or phagocytic activity of leukocytes. For
example, a chimeric protein may directly
stimulate the proliferation and cytokine production of individual T cell
subsets. Another example includes
direct stimulation of an immune inhibitory cell with through a receptor that
inhibits the activity of such an
zo immune suppressor cell. This would include, for example, stimulation of
CD4+FoxP3+ regulatory T cells,
which would reduce the ability of those regulatory T cells to suppress the
proliferation of conventional CD4+
or CD8+ T cells. In another example, this would include stimulation of CD40 on
the surface of an antigen
presenting cell, causing activation of antigen presenting cells including
enhanced ability of those cells to
present antigen in the context of appropriate native costimulatory molecules,
including those in the B7 or TNF
superfamily. In another example, the chimeric protein causes activation of the
lymphoid cell and/or production
of pro-inflammatory cytokines or chemokines to further stimulate an immune
response, optionally within a
tumor.
In embodiments, the present chimeric proteins are capable of, or find use in
methods involving, enhancing,
restoring, promoting and/or stimulating immune modulation. In embodiments, the
present chimeric proteins
described herein, restore, promote and/or stimulate the activity or activation
of one or more immune cells
against tumor cells including, but not limited to: T cells, cytotoxic T
lymphocytes, T helper cells, natural killer
(NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1
macrophages), B cells, and dendritic
cells. In embodiments, the present chimeric proteins enhance, restore, promote
and/or stimulate the activity
and/or activation of T cells, including, by way of a non-limiting example,
activating and/or stimulating one or
56
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
more T- cell intrinsic signals, including a pro-survival signal; an autocrine
or paracrine growth signal; a p38
MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; an anti-apoptotic
signal; and/or a signal
promoting and/or necessary for one or more of: pro-inflammatory cytokine
production or T cell migration or T
cell tumor infiltration.
In embodiments, the present chimeric proteins are capable of, or find use in
methods involving, causing an
io increase of one or more of T cells (including without limitation
cytotoxic T lymphocytes, T helper cells, natural
killer T (NKT) cells), B cells, natural killer (NK) cells, natural killer T
(NKT) cells, dendritic cells, monocytes,
and macrophages (e.g., one or more of M1 and M2) into a tumor or the tumor
microenvironment. In
embodiments, the chimeric protein enhances recognition of tumor antigens by
CD8-F T cells, particularly
those T cells that have infiltrated into the tumor microenvironment. In
embodiments, the present chimeric
is protein induces CD19 expression and/or increases the number of 0D19
positive cells (e.g., CD19 positive B
cells). In embodiments, the present chimeric protein induces IL-15Ra
expression and/or increases the
number of IL-15Ra positive cells (e.g., IL-15Ra positive dendritic cells).
In embodiments, the present chimeric proteins are capable of, or find use in
methods involving, inhibiting
and/or causing a decrease in immunosuppressive cells (e.g., myeloid-derived
suppressor cells (MDSCs),
zo regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2
macrophages, and tumor associated
macrophages (TAMs)), and particularly within the tumor and/or tumor
microenvironment (TME). In
embodiments, the present therapies may alter the ratio of M1 versus M2
macrophages in the tumor site
and/or TME to favor M1 macrophages. In embodiments, the SIRPa- Fc-CD4OL
chimeric protein
suppresses/reduces/eliminates a "don't eat me" signal via Sipri a/0D47 from
being transmitted on tumor
25 cells. In embodiments, the SIRPa- Fc-CD4OL chimeric protein makes a
tumor more likely to be attacked by
the immune system of the subject. In embodiments, the SIRPa- Fc-CD4OL chimeric
protein makes a tumor
more likely to be attacked by the innate immune system of the subject. In
embodiments, the SIRPa- Fc-
CD4OL chimeric protein makes a tumor more likely to be attacked by the
adaptive immune system of the
subject. S In embodiments, the SIRPa- Fc-CD4OL chimeric protein can
suppress/reduce/eliminate binding of
30 tumor-overexpressed 0D47 with phagocyte-expressed SIRPa to permit
phagocytic removal of cancer cells
and/or immunogenic processing of tumor antigens by macrophages and/or
dendritic cells. In embodiments,
the administration of the SIRPa- Fc-CD4OL chimeric protein blocks the "don't
eat me" signal of a tumor cell
and/or stimulates an "eat me" signal. In embodiments the therapy with the
SIRPa-Fc-CD4OL chimeric protein
57
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
(e.g. SEQ ID NO: 59 or SEQ ID NO: 61) stimulates macrophages to phagocytize
tumor cells and effectively
present the tumor antigens of phagocytized tumor cells to T cells.
In embodiments, the present chimeric proteins are able to increase the serum
levels of various cytokines
including, but not limited to, one or more of IFNy, TNFa, IL-2, IL-4, IL-5, IL-
9, IL-10, IL-13, IL-17A, IL-17F,
and IL-22. In embodiments, the present chimeric proteins are capable of
enhancing IL-2, IL-4, IL-5, IL-10, IL-
io 13, IL-17A, IL-22, or IFNy in the serum of a treated subject In
embodiments, the present chimeric proteins
do not increase the serum levels of certain cytokines. In embodiments, the
present chimeric proteins do not
increase the serum levels of IL-6 and/ or TNFa. In embodiments, the present
chimeric proteins do not
increase the serum levels of f IL-6 and/ or TNFa in the serum of a treated
subject. In embodiments, the
present chimeric proteins do not increase the serum levels off IL-6 and/ or
TNFa in the serum of a treated
is subject, while increasing the levels of other cytokines, including but
not limited to, CCL2, IL-8 and CXCL9 in
serum of a treated subject. Detection of such a cytokine response may provide
a method to determine the
optimal dosing regimen for the indicated chimeric protein.
In a chimeric protein of the present disclosure, the chimeric protein is
capable of increasing or preventing a
decrease in a sub-population of CD4+ and/or CD8+T cells.
zo In a chimeric protein of the present disclosure, the chimeric protein is
capable of enhancing tumor killing
activity by T cells.
In embodiments, the chimeric protein activates the human subject's T cells
when bound by the CD4OL domain
of the chimeric protein and (a) one or more tumor cells are prevented from
transmitting an
immunosuppressive signal when bound by the first domain of the chimeric
protein, (b) a quantifiable cytokine
25 response in the peripheral blood of the subject is achieved, and/or (c)
tumor growth is reduced in the subject
in need thereof as compared to a subject treated with CD40 agonist antibodies
and/or 0047 blocking
anti bodies.
In embodiments, the present chimeric proteins inhibit, block and/or reduce
cell death of an anti-tumor CD8+
and/or CD4+T cell; or stimulate, induce, and/or increase cell death of a pro-
tumor T cell. T cell exhaustion is
30 a state of T cell dysfunction characterized by progressive loss of
proliferative and effector functions,
culminating in clonal deletion. Accordingly, a pro-tumor T cell refers to a
state of T cell dysfunction that arises
during many chronic infections, inflammatory diseases, and cancer. This
dysfunction is defined by poor
proliferative and/or effector functions, sustained expression of inhibitory
receptors and a transcriptional state
distinct from that of functional effector or memory T cells. Exhaustion
prevents optimal control of infection
58
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
and tumors. Illustrative pro-tumor T cells include, but are not limited to,
Tregs, CD4+ and/or CD8+ T cells
expressing one or more checkpoint inhibitory receptors, Th2 cells and Th17
cells. Checkpoint inhibitory
receptors refer to receptors expressed on immune cells that prevent or inhibit
uncontrolled immune
responses. In contrast, an anti-tumor CD8+ and/or CD4+ T cell refers to T
cells that can mount an immune
response to a tumor.
io In embodiments, the present chimeric proteins are capable of, and can be
used in methods comprising,
increasing a ratio of effector T cells to regulatory T cells. Illustrative
effector T cells include ICOS+ effector T
cells; cytotoxic T cells (e.g., al3 TCR, CD3+, CD8+, CD45R0+); CD4+ effector T
cells (e.g., a13 TCR, CD3+,
CD4+, CCR7+, CD62Lhi, I L-7R/CD127+); CD8+ effector T cells (e.g., a13 TCR,
CD3+, CD8+, CCR7+, CD62Lhi,
IL-7R/CD127+); effector memory T cells (e.g., CD62Llow, CD44-F, TCR, CD3+, IL-
7R/CD127+, IL-15R+,
CCR7low); central memory T cells (e.g., CCR7+, CD62L+, CD27+; or CCR7hi,
CD44+, CD62Lhi, TCR, CD3+,
IL-7R/CD127+, IL-15R+); CD62L+ effector T cells; 008+ effector memory T cells
(TEM) including early effector
memory T cells (CD27-F CD62L-) and late effector memory T cells (0D27- CD62L-)
(TemE and TemL,
respectively); CD127( F)CD25(low/-) effector T cells; CD127(-)CD250 effector T
cells; CD8 F stem cell memory
effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca(+)); TH1
effector T-cells (e.g., CXCR3+,
zo CXCR6+ and CCR5+; or ap TCR, CD3+, CD4+, IL-12R+, IFNyR+, CXCR3+), TH2
effector T cells (e.g., CCR3+,
CCR4+ and CCR8+; or a13 TCR, CD3+, CD4+, IL-4R+, IL-33R+, CCR4+, IL-17RB+,
CRTH2+); TH9 effector T
cells (e.g., a13 TCR, CD3+, CD4+); TH17 effector T cells (e.g., up TCR, CD3+,
CD4+, IL-23R+, CCR6+, I L-1R+);
CD4+CD45RO+CCR7+ effector T cells, CD4+CD45RO+CCR7(-) effector T cells; and
effector T cells secreting
IL-2, IL-4 and/or IFN-y. Illustrative regulatory T cells include ICOS+
regulatory T cells, CD4+CD25+FOXP3+
regulatory T cells, CD4+CD25+ regulatory T cells, CD4+CD25- regulatory T
cells, CD4+CD25high regulatory
T cells, TIM-3+CD172a (SI RPa)+ regulatory T cells, lymphocyte activation gene-
3 (LAG-3)+ regulatory T cells,
CTLA-4/CD1524- regulatory T cells, neuropilin-1 (Nrp-1)+ regulatory T cells,
CCR44-CCR8+ regulatory T cells,
CD62L (L-selectin)+ regulatory T cells, CD45RBlow regulatory T cells, CD127low
regulatory T cells,
LRRC32/GARP+ regulatory T cells, CD39+ regulatory T cells, GITR+ regulatory T
cells, LAP + regulatory T
cells, 1611+ regulatory T cells, BTLA+ regulatory T cells, type 1 regulatory T
cells (Tr cells),T helper type 3
(Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs),
CD8+ regulatory T cells, CD8+CD28-
regulatory T cells and/or regulatory 1-cells secreting IL-10, IL-35, TGF-13,
TNF-a, Galectin-1, IFN-y and/or
MCP1.
In embodiments, the chimeric protein of the invention causes an increase in
effector T cells (e.g., CD4+0D25-
T cells).
59
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the chimeric protein causes a decrease in regulatory T cells
(e.g., CD41-CD25 T cells).
In embodiments, the chimeric protein generates a memory response which may,
e.g., be capable of
preventing relapse or protecting the animal from a recurrence and/or
preventing, or reducing the likelihood
of, metastasis. Thus, an animal treated with the chimeric protein is later
able to attack tumor cells and/or
prevent development of tumors when rechallenged after an initial treatment
with the chimeric protein.
io Accordingly, a chimeric protein of the present disclosure stimulates
both active tumor destruction and also
immune recognition of tumor antigens, which are essential in programming a
memory response capable of
preventing relapse.
In embodiments, the chimeric protein is capable of causing activation of
antigen presenting cells. In
embodiments, the chimeric protein is capable enhancing the ability of antigen
presenting cells to present
antigen.
In embodiments, the present chimeric proteins are capable of, and can be used
in methods comprising,
transiently stimulating effector T cells for longer than about 12 hours, about
24 hours, about 48 hours, about
72 hours or about 96 hours or about 1 week or about 2 weeks. In embodiments,
the transient stimulation of
effector T cells occurs substantially in a patient's bloodstream or in a
particular tissue/location including
zo lymphoid tissues such as for example, the bone marrow, lymph-node,
spleen, thymus, mucosa-associated
lymphoid tissue (MALT), non-lymphoid tissues, or in the tumor
microenvironment.
In a chimeric protein of the present disclosure, the present chimeric protein
unexpectedly provides binding of
the extracellular domain components to their respective binding partners with
slow off rates (Kd or Koff). In
embodiments, this provides an unexpectedly long interaction of the receptor to
ligand and vice versa. Such
an effect allows for a longer positive signal effect, e.g., increase in or
activation of immune stimulatory signals.
For example, the present chimeric protein, e.g., via the long off rate binding
allows sufficient signal
transmission to provide immune cell proliferation, allow for anti-tumor
attack, allows sufficient signal
transmission to provide release of stimulatory signals, e.g., cytokines.
In a chimeric protein of the present disclosure, the chimeric protein is
capable of forming a stable synapse
between cells. The stable synapse of cells promoted by the chimeric proteins
(e.g., between cells bearing
negative signals) provides spatial orientation to favor tumor reduction - such
as positioning the T cells to
attack tumor cells and/or sterically preventing the tumor cell from delivering
negative signals, including
negative signals beyond those masked by the chimeric protein of the invention.
In embodiments, this provides
longer on-target (e.g., intratumoral) half-life (t1/2) as compared to serum
ti/2 of the chimeric proteins. Such
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
properties could have the combined advantage of reducing off-target toxicities
associated with systemic
distribution of the chimeric proteins.
In embodiments, the chimeric protein is capable of providing a sustained
immunomodulatory effect.
The present chimeric proteins provide synergistic therapeutic effects (e.g.,
anti-tumor effects) as it allows for
improved site-specific interplay of two immunotherapy agents. In embodiments,
the present chimeric proteins
provide the potential for reducing off-site and/or systemic toxicity.
In embodiments, the present chimeric protein exhibit enhanced safety profiles.
In embodiments, the present
chimeric protein exhibit reduced toxicity profiles. For example,
administration of the present chimeric proteins
may result in reduced side effects such as one or more of diarrhea,
inflammation (e.g., of the gut), or weight
loss, which occur following administration of antibodies directed to the
ligand(s)/receptor(s) targeted by the
extracellular domains of the present chimeric proteins. In embodiments, the
present chimeric protein provides
improved safety, as compared to antibodies directed to the
ligand(s)/receptor(s) targeted by the extracellular
domains of the present chimeric proteins, yet, without sacrificing efficacy.
In embodiments, the present chimeric proteins provide reduced side-effects,
e.g., GI complications, relative
to current immunotherapies, e.g., antibodies directed to ligand(s)/receptor(s)
targeted by the extracellular
zo domains of the present chimeric proteins. Illustrative GI complications
include abdominal pain, appetite loss,
autoimmune effects, constipation, cramping, dehydration, diarrhea, eating
problems, fatigue, flatulence, fluid
in the abdomen or ascites, gastrointestinal (GI) dysbiosis, GI mucositis,
inflammatory bowel disease, irritable
bowel syndrome (I BS-D and IBS-C), nausea, pain, stool or urine changes,
ulcerative colitis, vomiting, weight
gain from retaining fluid, and/or weakness.
Pharmaceutical composition
Aspects of the present disclosure include a pharmaceutical composition
comprising a therapeutically effective
amount of a chimeric protein as disclosed herein.
Any chimeric protein disclosed herein may be used in a pharmaceutical
composition.
In embodiments, a chimeric protein disclosed herein is provided as a sterile
frozen solution in a vial or as a
sterile liquid solution in a vial. A drug product comprising a chimeric
protein disclosed herein comprises a
sterile-filtered, formulated chimeric protein disclosed herein solution filled
into a 10 mL single use glass vial
stoppered with a Flurotec rubber stopper and sealed with an aluminum flip off
seal. In embodiments, a
chimeric protein disclosed herein is formulated at between about 10mg/mL to
about 30 mg/mL, e.g., about
61
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
20 mg/mL in between about 30 mM to about 70 mM L-histidine, e.g., about 50 mM
L-histidine and between
about 125 mM and about 400 mM sucrose, e.g., about 250 mM sucrose in water for
injection. In
embodiments, each vial contains about 1 mL of drug product or about 20 mg of a
chimeric protein disclosed
herein.
The chimeric proteins disclosed herein, including the CD172a (SIRPa)-Fc-CD4OL
chimeric protein (e.g. SEQ
ID NO: 59 or SEQ ID NO: 61), can possess a sufficiently basic functional
group, which can react with an
inorganic or organic acid, or a carboxyl group, which can react with an
inorganic or organic base, to form a
pharmaceutically acceptable salt. A pharmaceutically-acceptable acid addition
salt is formed from a
pharmaceutically acceptable acid, as is well known in the art. Such salts
include the pharmaceutically
acceptable salts listed in, for example, Journal of Pharmaceutical Science,
66, 2-19 (1977) and The
is Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.
Stahl and C. G. VVermuth (eds.),
Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference
in their entirety.
In embodiments, the compositions disclosed herein are in the form of a
pharmaceutically acceptable salt.
Further, any chimeric protein disclosed herein can be administered to a
subject as a component of a
composition, e.g., pharmaceutical composition, that comprises a
pharmaceutically acceptable carrier or
zo vehicle. Such pharmaceutical compositions can optionally comprise a
suitable amount of a pharmaceutically
acceptable excipient so as to provide the form for proper administration.
Pharmaceutical excipients can be
liquids, such as water and oils, including those of petroleum, animal,
vegetable, or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. The
pharmaceutical excipients can be, for
example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal
silica, urea and the like. In addition,
zs auxiliary, stabilizing, thickening, lubricating, and coloring agents can
be used. In embodiments, the
pharmaceutically acceptable excipients are sterile when administered to a
subject. Water is a useful excipient
when any agent disclosed herein is administered intravenously. Saline
solutions and aqueous dextrose and
glycerol solutions can also be employed as liquid excipients, specifically for
injectable solutions. Suitable
pharmaceutical excipients also include starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica
30 gel, sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like. Any agent disclosed herein, if desired,
can also comprise minor amounts
of wetting or emulsifying agents, or pH buffering agents.
In embodiments, the compositions, e.g., pharmaceutical compositions, disclosed
herein are resuspended in
a saline buffer (including, without limitation TBS, PBS, and the like).
62
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the chimeric proteins may by conjugated and/or fused with
another agent to extend half-life
or otherwise improve pharmacodynamic and pharmacokinetic properties. In
embodiments, the chimeric
proteins may be fused or conjugated with one or more of PEG, XTEN (e.g., as
rPEG), polysialic acid
(POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein
(ELP), PAS, HAP, GLK, CTP,
transferrin, and the like. In embodiments, each of the individual chimeric
proteins is fused to one or more of
io the agents described in Stroh!, BioDrugs 29(4):215-239 (2015), the
entire contents of which are hereby
incorporated by reference.
The present disclosure includes the disclosed chimeric protein in various
formulations of pharmaceutical
composition. Any chimeric protein disclosed herein can take the form of
solutions, suspensions, emulsion,
drops, tablets, pills, pellets, capsules, capsules containing liquids,
powders, sustained-release formulations,
is suppositories, emulsions, aerosols, sprays, suspensions, or any other
form suitable for use. DNA or RNA
constructs encoding the protein sequences may also be used. In embodiments,
the composition is in the
form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of
suitable pharmaceutical
excipients are described in Remington's Pharmaceutical Sciences 1447-1676
(Alfonso R. Gen naro eds., 19th
ed. 1995), incorporated herein by reference.
zo Where necessary, the pharmaceutical compositions comprising the chimeric
protein (can also include a
solubilizing agent. Also, the agents can be delivered with a suitable vehicle
or delivery device as known in
the art. Compositions for administration can optionally include a local
anesthetic such as, for example,
lignocaine to lessen pain at the site of the injection.
The pharmaceutical compositions comprising the chimeric protein of the present
disclosure may conveniently
zs be presented in unit dosage forms and may be prepared by any of the
methods well known in the art of
pharmacy. Such methods generally include the step of bringing therapeutic
agents into association with a
carrier, which constitutes one or more accessory ingredients. Typically, the
pharmaceutical compositions are
prepared by uniformly and intimately bringing therapeutic agent into
association with a liquid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the product
into dosage forms of the desired
30 formulation (e.g., wet or dry granulation, powder blends, etc., followed
by tableting using conventional
methods known in the art)
In embodiments, any chimeric protein disclosed herein is formulated in
accordance with routine procedures
as a pharmaceutical composition adapted for a mode of administration disclosed
herein.
63
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Administration, Dosing, and Treatment Regimens
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
obtaining a biological sample obtained from the subject that has received a
first dose of a chimeric protein.
In embodiments, the chimeric protein has a general structure of: N terminus ¨
(a) ¨ (b) ¨ (c) ¨ C terminus,
io wherein: (a) is a first domain comprising an extracellular domain of
human signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
ligand (CD4OL). In embodiments, a background level and/or activity of a cell
has been measured in a
biological sample obtained from the subject before the administration of the
first dose. In embodiments, the
is cell is selected from one or more of a CD8O-F cell, a CD8+ cell, a
Granzyme B+ cell, a 0D68+ cell, a Ki67+
cell, and a PD-L1-F immune cell. In embodiments, the method further comprises
(ii) determining a post-dose
level and/or activity of the cell in the biological sample. In embodiments,
the method further comprises (iii)
determining that the chimeric protein is efficacious if the post-dose level
and/or activity of the cell is greater
than the background level and/or activity of the cell. In embodiments, the
biological sample is a tumor biopsy
zo sample or a tumor surgical specimen. In embodiments, the level and/or
activity of the cell is measured by
RNA sequencing, immunohistochemical staining, western blotting, in cell
western, immunofluorescent
staining, ELISA, and fluorescent activating cell sorting (FACS) or a
combination thereof.
An aspect of the present disclosure is a method of selecting a subject for
treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a biological
sample obtained from the subject
25 that has received a first dose of a chimeric protein. In embodiments,
the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, a
background level and/or activity
30 of a cell has been measured in a biological sample obtained from the
subject before the administration of the
first dose. In embodiments, the cell is selected from one or more of a CD8O-F
cell, a CD8-F cell, a Granzyme
B+ cell, a CD68+ cell, a Ki67+ cell, and a PD-L1+ immune cell. In embodiments,
the method further
comprises (ii) determining a post-dose level and/or activity of the cell in
the biological sample. In
embodiments, the method further comprises (iii) selecting the subject for
treatment with the chimeric protein
35 if the post-dose level and/or activity of the cell is greater than the
background level and/or activity of the cell.
64
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the biological sample is a tumor biopsy sample or a tumor
surgical specimen. In
embodiments, the level and/or activity of the cell is measured by RNA
sequencing, immunohistochemical
staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
sorting (FACS) or a combination thereof.
In embodiments, the biological sample is selected from blood, plasma, serum,
blood cells, lacrimal fluid,
io tears, bone marrow, ascites, tissue or fine needle biopsy sample, cell-
containing body fluid, free floating
nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid,
pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing
or lavage, aspirate, scraping,
bone marrow specimen, a biopsy specimen and a surgical specimen. In
embodiments, the biological sample
is a biopsy sample or a surgical specimen. In embodiments, the biological
sample is a tumor biopsy sample
is or a tumor surgical specimen. In embodiments, the tumor biopsy sample or
the tumor surgical specimen
derived from a tumor selected ovarian cancer, fallopian tube cancer,
peritoneal cancer, cutaneous squamous
cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck
(SCCHN).
In embodiments, the level and/or activity of the cell is measured by RNA
sequencing, immunohistochemical
staining, western blotting, in cell western, immunofluorescent staining,
ELISA, and fluorescent activating cell
zo sorting (FACS) or a combination thereof. In embodiments, the level
and/or activity of the cell is measured by
contacting the sample with an agent that specifically binds to one or more
molecules selected from CD80,
CD8, Granzyme B, CD68, Ki67, and PD-L1. In embodiments, the agent that
specifically binds to the one or
more molecules is an antibody or fragment thereof. In embodiments, the
antibody is a recombinant antibody,
a monoclonal antibody, a polyclonal antibody, or fragment thereof. In
embodiments, the level and/or activity
25 of the cell is measured by contacting the sample with an agent that
specifically binds to one or more nucleic
acids encoding one or more of CD80, C08, Granzyme B, CD68, Ki67, and PD-L1. In
embodiments, the agent
that specifically binds to one or more of the nucleic acids is a nucleic acid
primer or probe.
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
30 obtaining a first biological sample obtained from the subject that has
received a first dose of a chimeric
protein. In embodiments, the chimeric protein has a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
terminus, wherein: (a) is a first domain comprising an extracellular domain of
human signal regulatory protein
a (CD172a (SI RPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
ligand (CD4OL). In embodiments, the method further comprises determining a
background level and/or
activity of B cells and/or CD4O-F cells in the first biological sample . In
embodiments, the method further
comprises (iii) obtaining a second biological sample obtained from the subject
at N hr post-dose, wherein N
is a number between 1 and 24. In embodiments, the method further comprises
determining an N-hr post-
dose level and/or activity of B cells and/or CD40+ cells in the second
biological sample . In embodiments,
the method further comprises (iv) obtaining a third biological sample obtained
from the subject at M days
post-dose, wherein M is a number between 1 and 28. In embodiments, the method
further comprises
determining an M-day post-dose level and/or activity of B cells and/or CD40-F
cells in the third biological
sample . In embodiments, the method further comprises (v) determining that the
chimeric protein is
efficacious if the N hr post-dose level and/or activity of B cells and/or
CD40+ cells is less than the background
level and/or activity of B cells and/or CD40+ cells, and/or if the M day post-
dose level and/or activity of B cells
and/or CD40+ cells is at least about 50% higher than the N-hour post-dose
level and/or activity of B cells
and/or C040+ cells.
An aspect of the present disclosure is a method of selecting a subject for
treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a first biological
sample obtained from the
zo subject that has received a first dose of a chimeric protein. In
embodiments, the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SI RPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human 0040 ligand (CD4OL). In embodiments, the
method further comprises
determining a background level and/or activity of B cells and/or CD40+ cells
in the first biological sample. In
embodiments, the method further comprises (iii) obtaining a second biological
sample obtained from the
subject at N hr post-dose, wherein N is a number between 1 and 24. In
embodiments, the method further
comprises determining an N-hr post-dose level and/or activity of B cells
and/or CD40+ cells in the second
biological sample . In embodiments, the method further comprises (iv)
obtaining a third biological sample
obtained from the subject at M days post-dose, wherein M is a number between 1
and 28. In embodiments,
the method further comprises determining an M-day post-dose level and/or
activity of B cells and/or 0040+
cells in the third biological sample . In embodiments, the method further
comprises (v) selecting the subject
for treatment with the chimeric protein if the N hr post-dose level and/or
activity of B cells and/or CD4O-F cells
is less than the background level and/or activity of B cells and/or 0040+
cells, and/or if the M day post-dose
66
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
level and/or activity of B cells and/or CD40+ cells is at least about 50%
higher than the N-hour post-dose
level and/or activity of B cells and/or CD40+ cells.
In embodiments, N is less than 12, or less than 8, or less than 6, or less
than 4, or less than 3, or less than
2, or less than 1. In embodiments, M is less than 21, or less than 14, or less
than 12, or less than 10, or less
than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is
less than 12, or less than 8, or less
than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M
is less than 21, or less than 14, or
less than 12, or less than 10, or less than 8, or less than 6, or less than 4,
or less than 2.
In embodiments, the N-hr post-dose level and/or activity of B cells and/or
CD40+ cells is greater than the
background level and/or activity of B cells and/or CD40+ cells. In
embodiments, the N-hr post-dose level
and/or activity of B cells and/or CD40+ cells is less than the background
level and/or activity of B cells and/or
CD40+ cells. In embodiments, the N-hr post-dose level and/or activity of B
cells and/or CD40+ cells is within
about 10%, or about 20%, or about 30%, or about 40% of the background level
and/or activity of B cells
and/or CD40+ cells.
In embodiments, the first biological sample, the second biological sample and
the third biological sample are
independently selected from blood, plasma, serum, blood cells, lacrimal fluid,
tears, bone marrow, ascites,
zo tissue or fine needle biopsy sample, cell-containing body fluid, free
floating nucleic acids, sputum, saliva,
urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph,
gynecological fluid, skin swab, vaginal
swab, oral swab, nasal swab, washing or lavage, aspirate, scraping, bone
marrow specimen, a biopsy
specimen and a surgical specimen. In embodiments, the first biological sample,
the second biological sample
and the third biological sample are blood.
In embodiments, the level and/or activity of B cells and/or CD40+ cells is
measured by RNA sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
fluorescent activating cell sorting (FACS) or a combination thereof. In
embodiments, the level and/or activity
of B cells and/or CD40+ cells is measured by contacting the sample with an
agent that specifically binds to a
CD40 and/or a B-cell marker. In embodiments, the B-cell marker is selected
from CD19, CD20, 0D24, 0D27,
CD34, CD38, CD45R, 0D86, 0D95, IgM, IgD, and CD40. In embodiments, the agent
that specifically binds
to the one or more molecules is an antibody or fragment thereof. In
embodiments, the antibody is a
recombinant antibody, a monoclonal antibody, a polyclonal antibody, or
fragment thereof. In embodiments,
the level and/or activity of B cells and/or CD40+ cells is measured by
contacting the sample with an agent
that specifically binds to one or more nucleic acids encoding a CD40 and/or a
B-cell marker. In embodiments,
67
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
the B-cell marker is selected from CD19, CD20, CD24, CD 27, CD34, CD38, CD45R,
CD86, CD95, IgM, IgD,
and CD40. In embodiments, the agent that specifically binds to one or more of
the nucleic acids is a nucleic
acid primer or probe.
An aspect of the present disclosure is a method for evaluating the efficacy of
cancer treatment in a subject
in need thereof, wherein the subject is suffering from a cancer. In
embodiments, the method comprises: (i)
io obtaining a first biological sample obtained from the subject that has
received a first dose of a chimeric
protein. In embodiments, the chimeric protein has a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
terminus, wherein: (a) is a first domain comprising an extracellular domain of
human signal regulatory protein
a (CD172a (SI RPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
is ligand (CD4OL). In embodiments, the method further comprises (ii)
determining in the first biological sample
a background amount and/or activity of a cytokine. In embodiments, the
cytokine is selected from CCL2,
CXCL9, CXCL10, IFNa, IL15, IL23, IL-12, MCP-1, MIP-113, MIP-la, and MDC In
embodiments, the method
further comprises (iii) obtaining a second biological sample obtained from the
subject at N hr post-dose,
wherein N is a number between 1 and 24. In embodiments, the method further
comprises determining an N-
20 hr post-dose amount and/or activity of the cytokine in the second
biological sample. In embodiments, the
method further comprises (iv) obtaining a third biological sample obtained
from the subject at M days post-
dose, wherein M is a number between 1 and 28. In embodiments, the method
further comprises determining
an M-day post-dose amount and/or activity of the cytokine in the third
biological sample. In embodiments,
the method further comprises (v) determining that the chimeric protein is
efficacious if the N-hr post-dose
25 amount and/or activity of the cytokine is greater than the background
amount and/or activity of the cytokine,
and/or if the M-day post-dose amount and/or activity of the cytokine is at
least about 50% lower than the N-
hour post-dose amount and/or activity of the cytokine.
An aspect of the present disclosure is a method of selecting a subject for
treatment with a therapy for a
cancer. In embodiments, the method comprises: (i) obtaining a first biological
sample obtained from the
30 subject that has received a first dose of a chimeric protein. In
embodiments, the chimeric protein has a general
structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus, wherein: (a) is a
first domain comprising an extracellular
domain of human signal regulatory protein a (CD172a (SIRPa)), (b) is a linker
adjoining the first and second
domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a
second domain comprising
an extracellular domain of human CD40 ligand (CD4OL). In embodiments, the
method further comprises (ii)
35 determining in the first biological sample a background amount and/or
activity of a cytokine. In embodiments,
68
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
the cytokine is selected from CCL2, CXCL9, CXCL10, IFNa, IL15, IL23, IL-12,
MCP-1, MIP-1 (3, MIP-la, and
MDC In embodiments, the method further comprises (iii) obtaining a second
biological sample obtained from
the subject at N hr post-dose, wherein N is a number between 1 and 24. In
embodiments, the method further
comprises determining an N-hr post-dose amount and/or activity of the cytokine
in the second biological
sample. In embodiments, the method further comprises (iv) obtaining a third
biological sample obtained from
the subject at M days post-dose, wherein M is a number between 1 and 28. In
embodiments, the method
further comprises determining an M-day post-dose amount and/or activity of the
cytokine in the third biological
sample. In embodiments, the method further comprises (v) selecting the subject
for treatment with the
chimeric protein if the N-hr post-dose amount and/or activity of the cytokine
is greater than the background
amount and/or activity of the cytokine, and/or if the M-day post-dose amount
and/or activity of the cytokine is
at least about 50% lower than the N-hour post-dose amount and/or activity of
the cytokine.
In embodiments, N is less than 12, or less than 8, or less than 6, or less
than 4, or less than 3, or less than
2, or less than 1. In embodiments, M is less than 21, or less than 14, or less
than 12, or less than 10, or less
than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is
less than 12, or less than 8, or less
than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M
is less than 21, or less than 14, or
zo less than 12, or less than 10, or less than 8, or less than 6, or less
than 4, or less than 2.
In embodiments, the N-hr post-dose amount and/or activity of the cytokine is
greater than the background
amount and/or activity of the cytokine. In embodiments, the N-hr post-dose
amount and/or activity of the
cytokine is less than the background amount and/or activity of the cytokine.
In embodiments, the N-hr post-
dose amount and/or activity of the cytokine is within about 10%, or about 20%,
or about 30%, or about 40%
of the background amount and/or activity of the cytokine. In embodiments, the
first biological sample, the
second biological sample and the third biological sample are independently
selected from blood, plasma,
serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or
fine needle biopsy sample, cell-
containing body fluid, free floating nucleic acids, sputum, saliva, urine,
cerebrospinal fluid, peritoneal fluid,
pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab,
oral swab, nasal swab, washing or
lavage, aspirate, scraping, bone marrow specimen, a biopsy specimen and a
surgical specimen. In
embodiments, the first biological sample, the second biological sample and the
third biological sample are
blood.
In embodiments, the amount and/or activity of the cytokine is measured by RNA
sequencing,
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and
69
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
fluorescent activating cell sorting (FACS) or a combination thereof. In
embodiments, the amount and/or
activity of the cytokine is measured by contacting the sample with an agent
that specifically binds to the
cytokine. In embodiments, the agent that specifically binds to the one or more
molecules is an antibody or
fragment thereof. In embodiments, the antibody is a recombinant antibody, a
monoclonal antibody, a
polyclonal antibody, or fragment thereof. In embodiments, the amount and/or
activity of the cytokine is
measured by contacting the sample with an agent that specifically binds to one
or more nucleic acids
encoding the cytokine. In embodiments, the agent that specifically binds to
one or more of the nucleic acids
is a nucleic acid primer or probe.
In embodiments, the first dose of the chimeric protein is in the range of from
about 0.03 mg/kg to 10 mg/kg.
In embodiments, the first dose is about 0.003, or about 0.01, or about 0.03,
or about 0.1, or about 0.3, or
is about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or
about 10 mg/kg.
In embodiments, the method of any of the aspects disclosed herein further
comprises administration of a
second dose of the chimeric protein. In embodiments, the second dose is
administered at least about 3 days,
or at least about 4 days, or at least about 5 days, or at least about 6 days,
or at least about 7 days, or at least
about 8 days, or at least about 9 days, or at least about 10 days, or at least
about 14 days, or at least about
zo 21 days, or at least about 28 days after the administration of the first
dose. In embodiments, the second dose
of the chimeric protein is in the range of from about 0.03 mg/kg to 10 mg/kg.
In embodiments, the second
dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or about 0.3,
or about 1, or about 2, or about
3, or about 4, or about 6, or about 8, or about 10 mg/kg.
In embodiments, the method of any of the aspects disclosed herein further
comprises administration of a
25 second dose of the chimeric protein. In embodiments, the second dose is
administered at least about 3 days,
or at least about 4 days, or at least about 5 days, or at least about 6 days,
or at least about 7 days, or at least
about 8 days, or at least about 9 days, or at least about 10 days, or at least
about 14 days, or at least about
21 days, or at least about 28 days after the administration of the first dose.
In embodiments, the second dose
of the chimeric protein is in the range of from about 0.03 mg/kg to 10 mg/kg.
In embodiments, the second
30 dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or
about 0.3, or about 1, or about 2, or about
3, or about 4, or about 6, or about 8, or about 10 mg/kg.
In embodiments, the first domain is capable of binding a CD172a (SI RPa)
ligand. In embodiments, the first
domain comprises substantially all of the extracellular domain of CD172a (SI
RPa). In embodiments, the first
domain comprises an amino acid sequence that is at least about 90%, or at
least about 95%, or at least about
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
96%, or at least about 97%, or at least about 98%, or at least about 99%
identical to the amino acid sequence
of SEQ ID NO: 57.
In embodiments, the second domain is capable of binding a CD40 receptor. In
embodiments, the second
domain comprises substantially all of the extracellular domain of CD4OL. In
embodiments, the second domain
comprises an amino acid sequence that is at least about 90%, or at least about
95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about 99% identical
to the amino acid sequence of
SEQ ID NO: 58.
In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from
IgG4. In embodiments, the
linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. In
embodiments, the linker
comprises an amino acid sequence that is at least about 90%, or at least about
95%, or at least about 96%,
or at least about 97%, or at least about 98%, or at least about 99% identical
to the amino acid sequence of
SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
In embodiments, (a) the first domain comprises the amino acid sequence of SEQ
ID NO: 57, (b) the second
domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the linker
comprises an amino acid
sequence that is at least about 90%, or at least about 95%, or at least about
96%, or at least about 97%, or
zo at least about 98%, or at least about 99% identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3. In embodiments, the chimeric protein further comprises
the amino acid sequence
of SEQ ID NO: 5 or SEQ ID NO: 7. In embodiments, the chimeric protein further
comprises the amino acid
sequence of SEQ ID NO: 5 and SEQ ID NO: 7.
In embodiments, the chimeric protein comprises an amino acid sequence that is
at least about 90%, or at
least about 95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99%
identical to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the chimeric
protein comprises an amino acid sequence that is at least about 99% identical
to the amino acid sequence
of SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the chimeric protein
comprises an amino acid
sequence that is at least about 99.2% identical to the amino acid sequence of
SEQ ID NO: 59 or SEQ ID NO:
61. In embodiments, the chimeric protein comprises an amino acid sequence that
is at least about 99.4%
identical to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the chimeric
protein comprises an amino acid sequence that is at least about 99.6%
identical to the amino acid sequence
of SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the chimeric protein
comprises an amino acid
sequence that is at least about 99.8% identical to the amino acid sequence of
SEQ ID NO: 59 or SEQ ID NO:
71
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
61. In embodiments, the chimeric protein comprises the amino acid sequence of
SEQ ID NO: 59 or SEQ ID
NO: 61.
In embodiments, the human subject suffers from or is suspected to suffer from
an advanced solid tumor or a
lymphoma. In embodiments, the human subject suffers from or is suspected to
suffer from a cancer is
selected from ovarian cancer, fallopian tube cancer, peritoneal cancer,
cutaneous squamous cell carcinoma
io (CSCC), and squamous cell carcinoma of the head and neck (SCCHN). In
embodiments, the human subject
has failed one or more platinum-based therapies. In embodiments, the human
subject is ineligible for a
platinum therapy. In embodiments, the human subject is not receiving a
concurrent chemotherapy,
immunotherapy, biologic or hormonal therapy, and/or wherein the human subject
has received, been tolerant
to, or is ineligible for standard therapy and/or the cancer has no approved
therapy considered to be standard
is of care.
In embodiments, a chimeric protein disclosed herein is presented as a sterile
frozen solution at a
concentration of about 20 mg/mL and a total volume of about 1 mL, optionally
in a 10 mL glass vial. In
embodiments, a chimeric protein disclosed herein is administered by
intravenous (IV) infusion following
dilution with normal saline. Starting dose, dose escalation schema and dose
schedules of certain
zo embodiments are presented below.
In embodiments, the dose of the chimeric protein administered is at least
0.0001 mg/kg, e.g., between about
0.0001mg/kg and about 10 mg/kg. In embodiments, the dose of the chimeric
protein administered is at least
about 0.3 mg/kg, e.g., at least about 0.3 mg/kg, or about 1.0 mg/kg, or about
2 mg/kg, or about 3, about 4
mg/kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In embodiments,
the dose of the chimeric
25 protein administered is at least about 1 mg/kg, e.g., at least about 1.0
mg/kg, or about 2 mg/kg, or about 3,
about 4 mg/kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In
embodiments, the doses of the
SIRPa-Fc-CD4OL chimeric protein are not limited by anemia or another cytopenia
effects and are therefore
higher than doses are allowed compared to certain other therapeutics (e.g.
anti-CD47 antibodies or
SIRPalphaFc fusion protein). Further, in embodiments, a low dose priming is
not needed.
30 In embodiments, the administration is intravenous. In embodiments, the
administration is intratumoral. In
embodiments, the administration is by injection. In embodiments, the
administration is by infusion. In
embodiments, the administration is performed by an intravenous infusion. In
embodiments, the administration
is performed by an intratumoral injection.
72
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, about the chimeric protein is administered at an initial dose
(e.g., about one of about 0.0001,
about 0.001, about 0.003, about 0.01, about 0.03, about 0.1, about 0.3, about
1, about 2, about 3, about 4,
about 6, about 8 or about 10 mg/kg) and the chimeric protein is administered
in one or more subsequent
administrations. In embodiments, about the one or more subsequent
administrations has a dose of one or
more of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03, about
0.1, about 0.3, about 1, about
2, about 3, about 4, about 6, about 8, about and about 10 mg/kg.
In embodiments, the starting dose and/or the subsequent doses is the maximum
tolerated dose or less than
the maximum tolerated dose.
In embodiments, the dose escalates between one or more subsequent dose in log
increments, e.g., 0.0001
mg/kg to 0.001 mg/kg, 0.001 mg/kg to 0.01 mg/kg, and 0.01 mg/kg to 0.1 mg/kg.
is In embodiments, the dose escalates between one or more subsequent dose
in about half log increments, e.g.,
0.001 mg/kg to 0.003 mg/kg and 0.003 mg/kg to 0.01 mg/kg.
In embodiments, the human subject has failed one or more platinum-based
therapies, and optionally is
ineligible for a platinum therapy. In embodiments, the human subject is not
receiving a concurrent
chemotherapy, immunotherapy, biologic or hormonal therapy, and/or wherein the
human subject has
zo received, been tolerant to, or is ineligible for standard therapy and/or
the cancer has no approved therapy
considered to be standard of care.
In embodiments, the initial dose is less than the dose for at least one of the
subsequent administrations., e.g.,
each of the subsequent administrations.
In embodiments, the initial dose is the same as the dose for at least one of
the subsequent administrations,
25 e.g., each of the subsequent administrations.
In embodiments, the chimeric protein is administered at least about one time a
month.
In embodiments, the chimeric protein is administered at least about two times
a month.
In embodiments, the chimeric protein is administered at least about three
times a month.
In embodiments, the chimeric protein is first administered once a week for
three weeks and the chimeric
30 protein is then administered about once every three weeks or once every
four weeks.
73
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the chimeric protein is first administered once a week for
three weeks and the chimeric
protein is then administered about two times per month. For example, the
chimeric protein is first administered
once a week for three weeks and the chimeric protein is then administered
about once every two weeks.
In embodiments, the chimeric protein is administered at least about four times
a month. For example, the
chimeric protein is administered about once a week. In embodiments, the
chimeric protein is administered
once every week (once every seven days). in embodiments, the chimeric protein
is administered once every
two weeks.
In embodiments, the administration of the SI RPa-Fc-CD4OL chimeric protein
does not cause an anemia or
another cytopenia in the patient. In embodiments, the administration of the
does not cause lysis of RBCs. In
embodiments, the administration of the SIRPa-Fc-CD4OL chimeric protein is less
likely to cause anemia or
another cytopenia in than, e.g. an anti-CD47 Ab. In embodiments, the doses of
the SIRPa-Fc-CD4OL chimeric
protein are not limited by anemia or another cytopenia effects and are
therefore higher than doses are allowed
compared to certain other therapeutics (e.g. anti-CD47 antibodies or
SIRPalphaFc fusion protein). Further,
in embodiments, a low dose priming is not needed.
Another advantage the SIRPa-Fc-CD4OL chimeric protein (e.g. SEQ ID NO: 59 or
SEQ ID NO: 61) offers is
zo that despite targeting does not cause an anemia or another cytopenia in
the patient. This is because although
the CD47/SIRPa interaction plays a key role in the lysis of RBCs, as shown
herein, the SIRPa-Fc-CD4OL
chimeric protein does not cause lysis of RBCs. Accordingly, the present
methods are less likely to cause
anemia or another cytopenia in than, e.g. an anti-0D47 Ab.
A chimeric protein may be administered intravenously by intravenous infusion
or bolus injection into the
bloodstream. A chimeric protein may be administered intravenously by
intravenous infusion for patients
suffering from advanced ovarian, fallopian tube and primary peritoneal
cancers.
A chimeric protein may be administered an intratumoral injection. In
embodiments, the therapeutic dose for
intra-tumoral administration is equal or less than that of intravenous
infusion. In embodiments, the therapeutic
dose for intra-tumoral administration is equal to that of intravenous
infusion. In embodiments, the therapeutic
dose for intra-tumoral administration is less than that of intravenous
infusion. In embodiments, the therapeutic
dose for intra-tumoral administration for patients suffering from advanced or
metastatic CSCC and HNSCC.
In embodiments, the present chimeric protein allows for a dual effect that
provides less side effects than are
seen in conventional immunotherapy (e.g., treatments with one or more of
OPDIVO, KEYTRUDA, YERVOY,
74
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
and TECENTRIQ). For example, the present chimeric proteins reduce or prevent
commonly observed
immune-related adverse events that affect various tissues and organs including
the skin, the gastrointestinal
tract, the kidneys, peripheral and central nervous system, liver, lymph nodes,
eyes, pancreas, and the
endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash,
and rheumatic disease.
Dosage forms suitable for intravenous administration include, for example,
solutions, suspensions,
dispersions, emulsions, and the like. They may also be manufactured in the
form of sterile solid compositions
(e.g., lyophilized composition), which can be dissolved or suspended in
sterile injectable medium immediately
before use. They may contain, for example, suspending or dispersing agents
known in the art.
The dosage of any chimeric protein disclosed herein as well as the dosing
schedule can depend on various
parameters, including, but not limited to, the disease being treated, the
subject's general health, and the
administering physician's discretion.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject in need
thereof, the method comprising a step of administering to the human subject an
effective amount of a chimeric
protein having a general structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
terminus, wherein: (a) is a first domain
comprising an extracellular domain of human signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker
zo adjoining the first and second domains, wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c)
is a second domain comprising an extracellular domain of human CD40 ligand
(CD4OL), wherein the step of
administering comprises biphasic dosing. In embodiments, the first phase, and
the second phase each
independently comprise a dosing frequency of from about twice a week to about
once every two months. In
embodiments, the linker comprises at least one cysteine residue capable of
forming a disulfide bond. In
embodiments, the chimeric protein exhibits a linear dose response in the dose
range of e.g., about 0.3 mg/kg
to about 3 mg/kg, or about 0.5 mg/kg to about 3 mg/kg, or about 0.7 mg/kg to
about 3 mg/kg, or about 1
mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg
to about 3 mg/kg, or about
2.5 mg/kg to about 3 mg/kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about
0.3 mg/kg to about 2 mg/kg, or
about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 mg/kg, or
about 0.3 mg/kg to about 0.5
mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped
dose response.
In embodiments, the dosing frequency of the first phase, and the dosing
frequency of the second phase are
the same. In other embodiments, the dosing frequency of the first phase, and
the dosing frequency of the
second phase are different.
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the dosing frequency of the first phase is selected from about
every three days, about twice
a week, about every week, about every 10 days, about twice every 3 weeks,
about every 2 weeks, about
every 3 weeks, about every 4 weeks, about every month, about every 5 weeks,
about every 6 weeks, about
7 seven weeks, about every 8 weeks and about every 2 months. In embodiments,
the dosing frequency of
the first phase is selected from about every 3 days to about every 10 days,
about every week to about every
2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to
about every 4 weeks, about
every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6
weeks, about every 5 weeks
to about every 7 weeks, about every 6 weeks to about every 8 weeks, and ,
about every 6 weeks to about
every 2 months.
In embodiments, the dosing frequency of the second phase is selected from
about every three days, about
is twice a week, about every week, about every 10 days, about twice every 3
weeks, about every 2 weeks,
about every 3 weeks, about every 4 weeks, about every month, about every 5
weeks, about every 6 weeks,
about 7 seven weeks, about every 8 weeks and about every 2 months. In
embodiments, the dosing frequency
of the second phase is selected from about every 3 days to about every 10
days, about every week to about
every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks
to about every 4 weeks,
zo about every 3 weeks to about every 5 weeks, about every 4 weeks to about
every 6 weeks, about every 5
weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and,
about every 6 weeks to
about every 2 months.
In embodiments, the dosing frequency of the first phase is selected from from
about every 3 days to about
every 10 days, about every week to about every 2 weeks, about every 10 days to
about every 3 weeks; and
25 the frequency of the second phase is selected from from about every week
to about every 2 weeks, about
every 10 days to about every 3 weeks, about every 2 weeks to about every 4
weeks, about every 3 weeks to
about every 5 weeks, about every 4 weeks to about every 6 weeks.
Additionally, or alternatively, in embodiments, the first phase, and the
second phase each independently last
from about two days to about 12 months. In embodiments, the first phase lasts
from about two weeks to
30 about 2 months; and the second phase lasts from about 2 weeks to about
12 months. In embodiments, the
first phase lasts from about two weeks to about 1 month; and the second phase
lasts from about 2 weeks to
about 12 months. In embodiments, the first phase lasts from about two weeks to
about 1 month; and the
second phase lasts from about 4 weeks to about 12 months.
76
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Additionally, or alternatively, in embodiments, the effective amount for the
first phase, the second phase and
the third phase each independently comprise about 0.01 mg/kg to about 10
mg/ml. In embodiments, the
effective amount for the first phase, the second phase and the third phase
each independently selected from
about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3
mg/kg, about 10 mg/kg, and
any range including and/or in between any two of the preceding values. In
embodiments, the effective amount
for the first phase, the second phase and the third phase each independently
selected from from about 0.01
mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg
to about 1 mg/kg, about
0.3 mg/kg to about 3 mg/kg, and about 1 mg/kg to about 10 mg/kg. In
embodiments, the effective amount for
the first phase, the second phase and the third phase are same. In
embodiments, the effective amount for
the first phase, the second phase and the third phase are different. In
embodiments, the effective amount for
the first phase is greater than the effective amount for the second phase. In
embodiments, the effective
amount for the first phase is from about 0.03 mg/kg to about 0.3 mg/kg, about
0.1 mg/kg to about 1 mg/kg,
about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 10 mg/kg; and the
effective amount for the
second phase is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to
about 0.3 mg/kg, about 0.1
mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg to
about 10 mg/kg.
zo In embodiments, the chimeric proteins disclosed herein is the human
CD172a (SIRPa)-Fc-CD4OL chimeric
protein.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject in need
thereof the method comprising a step of administering to the human subject an
effective amount of a chimeric
protein having a general structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨ C
terminus, wherein: (a) is a first domain
comprising an extracellular domain of human signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker
adjoining the first and second domains, wherein the linker comprises a hinge-
CH2-CH3 Fc domain, and (c)
is a second domain comprising an extracellular domain of human CD40 ligand
(CD4OL), wherein the step of
administration comprises a first cycle, a second cycle and a third cycle. In
embodiments, the linker comprises
at least one cysteine residue capable of forming a disulfide bond. In
embodiments, the first cycle, the second
cycle and the third cycle each independently comprise a dosing frequency of
from about twice a week to
about once every two months. In embodiments, the dosing frequency of the first
cycle, the dosing frequency
of the second cycle and the dosing frequency of the third cycle are the same.
In embodiments, the dosing
frequency of the first cycle, the dosing frequency of the second cycle and the
dosing frequency of the third
cycle are different. In embodiments, the dosing frequency of the first cycle
is selected from about every three
days, about twice a week, about every week, about every 10 days, about twice
every 3 weeks, about every
77
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
2 weeks, about every 3 weeks, about every 4 weeks, about every month, about
every 5 weeks, about every
6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In
embodiments, the
chimeric protein exhibits a linear dose response in the dose range of e.g.,
about 0.3 mg/kg to about 3 mg/kg,
or about 0.5 mg/kg to about 3 mg/kg, or about 0.7 mg/kg to about 3 mg/kg, or
about 1 mg/kg to about 3
mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg,
or about 2.5 mg/kg to about
io 3 mg/kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to
about 2 mg/kg, or about 0.3 mg/kg to
about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 mg/kg, or about 0.3 mg/kg to
about 0.5 mg/kg. In
embodiments, the chimeric protein does not exhibit a bell-shaped dose
response.
In embodiments, the dosing frequency of the first cycle is selected from about
every 3 days to about every
days, about every week to about every 2 weeks, about every 10 days to about
every 3 weeks, about every
is 2 weeks to about every 4 weeks, about every 3 weeks to about every 5
weeks, about every 4 weeks to about
every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks
to about every 8 weeks,
and , about every 6 weeks to about every 2 months. In embodiments, the dosing
frequency of the second
cycle is selected from about every three days, about twice a week, about every
week, about every 10 days,
about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about
every 4 weeks, about every
zo month, about every 5 weeks, about every 6 weeks, about 7 seven weeks,
about every 8 weeks and about
every 2 months. In embodiments, the dosing frequency of the second cycle is
selected from about every 3
days to about every 10 days, about every week to about every 2 weeks, about
every 10 days to about every
3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to
about every 5 weeks, about
every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7
weeks, about every 6 weeks
25 to about every 8 weeks, and , about every 6 weeks to about every 2
months. In embodiments, the dosing
frequency of the third cycle is selected from about every three days, about
twice a week, about every week,
about every 10 days, about twice every 3 weeks, about every 2 weeks, about
every 3 weeks, about every 4
weeks, about every month, about every 5 weeks, about every 6 weeks, about 7
seven weeks, about every 8
weeks and about every 2 months. In embodiments, the dosing frequency of the
third cycle is selected from
30 about every 3 days to about every 10 days, about every week to about
every 2 weeks, about every 10 days
to about every 3 weeks, about every 2 weeks to about every 4 weeks, about
every 3 weeks to about every 5
weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to
about every 7 weeks, about
every 6 weeks to about every 8 weeks, and, about every 6 weeks to about every
2 months. In embodiments,
the dosing frequency of the first cycle is selected from from about every 3
days to about every 10 days, about
35 every week to about every 2 weeks, about every 10 days to about every 3
weeks; and the frequency of the
78
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
second cycle is selected from from about every week to about every 2 weeks,
about every 10 days to about
every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks
to about every 5 weeks,
about every 4 weeks to about every 6 weeks.
Additionally, or alternatively, in embodiments, the first cycle, the second
cycle and the third cycle each
independently last from about two days to about 12 months. In embodiments, the
first cycle lasts from about
two weeks to about 2 months; and the second cycle lasts from about 2 weeks to
about 12 months. In
embodiments, the first cycle lasts from about two weeks to about 2 months; the
second cycle lasts from about
2 weeks to about 12 months and the third cycle lasts from about 2 weeks to
about 6 months.
Additionally, or alternatively, in embodiments, the effective amount for the
first cycle, the second cycle and
the third cycle each independently comprise about 0.01 mg/kg to about 10
mg/ml. In embodiments, the
effective amount for the first cycle, the second cycle and the third cycle
each independently selected from
about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3
mg/kg, about 10 mg/kg, and
any range including and/or in between any two of the preceding values. In
embodiments, the effective amount
for the first cycle, the second cycle and the third cycle each independently
selected from from about 0.01
mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg
to about 1 mg/kg, about
zo 0.3 mg/kg to about 3 mg/kg, and about 1 mg/kg to about 10 mg/kg.
In embodiments, the effective amount for the first cycle, the second cycle and
the third cycle are same. In
other embodiments, the effective amount for the first cycle, the second cycle
and the third cycle are different.
In embodiments, the effective amount for the first cycle is greater than the
effective amount for the second
cycle. In other embodiments, the effective amount for the first cycle is
lesser than the effective amount for
the second cycle. In yet other embodiments, the effective amount for the first
cycle and the effective amount
for the second cycle are the same.
In embodiments, the effective amount for the first cycle is from about 0.03
mg/kg to about 0.3 mg/kg, about
0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg
to about 10 mg/kg; and the
effective amount for the second cycle is from about 0.01 mg/kg to about 0.1
mg/kg, about 0.03 mg/kg to
about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3
mg/kg, or about 1 mg/kg to
about 10 mg/kg.
In embodiments, the chimeric proteins disclosed herein is the human CD172a
(SIRPa)-Fc-CD4OL chimeric
protein.
79
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject in need
thereof the method comprising a step of administering to the human subject an
effective amount of an
effective amount of a chimeric protein having a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus,
wherein: (a) is a first domain comprising an extracellular domain of human
signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
ligand (CD4OL) with a dosing regimen, wherein the dosing regimen comprises
dosing with a frequency in the
range of about every three days to about every 2 months. In embodiments, the
linker comprises at least one
cysteine residue capable of forming a disulfide bond. In embodiments, the
dosing regimen is selected from
about every three days, about twice a week, about every week, about every 10
days, about twice every 3
weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about
every month, about every 5
weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about
every 2 months. In
embodiments, the dosing regimen is selected from about every week, about every
10 days, about every 2
weeks, about every 3 weeks, about every 4 weeks, about every month, about
every 5 weeks, about every 6
weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In
embodiments, the dosing
zo regimen is about every 2 weeks, about every 3 weeks, or about every 4
weeks.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject in need
thereof the method comprising a step of administering to the human subject an
effective amount of an
effective amount of a chimeric protein having a general structure of: N
terminus ¨ (a) ¨ (b) ¨ (c) ¨ C terminus,
wherein: (a) is a first domain comprising an extracellular domain of human
signal regulatory protein a
(CD172a (SIRPa)), (b) is a linker adjoining the first and second domains,
wherein the linker comprises a
hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an
extracellular domain of human CD40
ligand (CD4OL) with a dosing regimen selected from about every 3 days to about
every 10 days, about every
week to about every 2 weeks, about every 10 days to about every 3 weeks, about
every 2 weeks to about
every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks
to about every 6 weeks,
about every 5 weeks to about every 7 weeks, about every 6 weeks to about every
8 weeks, and , about every
6 weeks to about every 2 months. In embodiments, the linker comprises at least
one cysteine residue capable
of forming a disulfide bond. In embodiments, the dosing regimen is about every
week to about every 2 weeks,
about every 10 days to about every 3 weeks, or about every 2 weeks to about
every 4 weeks. In
embodiments, the chimeric protein exhibits a linear dose response in the dose
range of e.g., about 0.3 mg/kg
to about 3 mg/kg, or about 0.5 mg/kg to about 3 mg/kg, or about 0.7 mg/kg to
about 3 mg/kg, or about 1
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg
to about 3 mg/kg, or about
2.5 mg/kg to about 3 mg/kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about
0.3 mg/kg to about 2 mg/kg, or
about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 mg/kg, or
about 0.3 mg/kg to about 0.5
mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped
dose response.
In some embodiments of any of the aspects disclosed herein, the first domain
is capable of binding a CD172a
(SIRPa) ligand. In embodiments, the first domain comprises substantially all
of the extracellular domain of
CD172a (SIRPa). In embodiments, the second domain is capable of binding a CD40
receptor. In
embodiments, the second domain comprises substantially all of the
extracellular domain of CD4OL. In
embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4,
e.g., human IgG4. In
embodiments, the linker comprises an amino acid sequence that is at least 90%,
or 93%, or 95%, or 97%, or
98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3. In
embodiments, the linker comprises an amino acid sequence that is at least 95%
identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the
linker comprises an amino
acid sequence that is at least 98% identical to the amino acid sequence of SEQ
ID NO: 1, SEQ ID NO: 2,01
SEQ ID NO: 3. In embodiments, the first domain comprises an amino acid
sequence that is at least 90%, or
zo 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence
of SEQ ID NO: 57. In
embodiments, the first domain comprises an amino acid sequence that is at
least 95% identical to the amino
acid sequence of SEQ ID NO: 57. In embodiments, the first domain comprises an
amino acid sequence that
is at least 96% identical to the amino acid sequence of SEQ ID NO: 57. In
embodiments, the first domain
comprises an amino acid sequence that is at least 98% identical to the amino
acid sequence of SEQ ID NO:
57. In embodiments, the first domain comprises an amino acid sequence that is
at least 99% identical to the
amino acid sequence of SEQ ID NO: 57. In embodiments, the first domain
comprises an amino acid sequence
that is identical to the amino acid sequence of SEQ ID NO: 57.
In some embodiments of any of the aspects disclosed herein, the second domain
comprises an amino acid
sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99%
identical to the amino acid sequence
of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein,
the second domain
comprises an amino acid sequence that is at least 95% identical to the amino
acid sequence of SEQ ID NO:
58. In some embodiments of any of the aspects disclosed herein, the second
domain comprises an amino
acid sequence that is at least 96% identical to the amino acid sequence of SEQ
ID NO: 58. In some
embodiments of any of the aspects disclosed herein, the second domain
comprises an amino acid sequence
that is at least 97% identical to the amino acid sequence of SEQ ID NO: 58. In
some embodiments of any of
81
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
the aspects disclosed herein, the second domain comprises an amino acid
sequence that is at least 98%
identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of
any of the aspects disclosed
herein, the second domain comprises an amino acid sequence that is at least
99% identical to the amino
acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects
disclosed herein, the second
domain comprises an amino acid sequence that is identical to the amino acid
sequence of SEQ ID NO: 58.
io In embodiments, (a) the first domain comprises the amino acid sequence
of SEQ ID NO: 57, (b) the second
domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the linker
comprises an amino acid
sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ
ID NO: 3.
In embodiments, the chimeric protein further comprises the amino acid sequence
of SEQ ID NO: 5 or SEQ
ID NO: 7. In embodiments, the chimeric protein further comprises the amino
acid sequence of SEQ ID NO:
is 5 and SEQ ID NO: 7. In embodiments, the chimeric protein comprises an
amino acid sequence that is at
least 90%, or 93%, 01 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 59
or SEQ ID NO: 61. In
embodiments, the chimeric protein comprises an amino acid sequence that is at
least 95% identical to SEQ
ID NO: 59 or SEQ ID NO: 61. In embodiments, the chimeric protein comprises an
amino acid sequence that
is at least 96% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments,
the chimeric protein comprises
zo an amino acid sequence that is at least 97% identical to SEQ ID NO: 59
or SEQ ID NO: 61. In embodiments,
the chimeric protein comprises an amino acid sequence that is at least 98%
identical to SEQ ID NO: 59 or
SEQ ID NO: 61. In embodiments, the chimeric protein comprises an amino acid
sequence that is at least
99% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the chimeric
protein comprises an
amino acid sequence that is identical to SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the chimeric
25 protein comprises an amino acid sequence that is at least about 98%
identical to SEQ ID NO: 59 or SEQ ID
NO: 61. In embodiments, the chimeric protein comprises an amino acid sequence
that is at least about 99%
identical to SEQ ID NO: 59 or SEQ ID NO: 61.
Additionally or alternatively, in embodiments, the chimeric protein comprises
an amino acid sequence that is
at least about 99.2% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the chimeric protein
30 comprises an amino acid sequence that is at least about 99.4% identical
to SEQ ID NO: 59 or SEQ ID NO:
61. In embodiments, the chimeric protein comprises an amino acid sequence that
is at least about 99.6%
identical to SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the chimeric
protein comprises an amino
acid sequence that is at least about 99.8% identical to SEQ ID NO: 59 or SEQ
ID NO: 61. In embodiments,
the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 or SEQ
ID NO: 61. In
82
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
embodiments, the human subject has received, been tolerant to, or is
ineligible for standard therapy and/or
the cancer has no approved therapy considered to be standard of care.
In one aspect, the present disclosure relates to a method for promoting the
migration of lymphocytes from
peripheral blood into secondary lymphoid organs (e.g. the lymph nodes and
spleen in a human subject in
need thereof, the method comprising a step of administering to the human
subject an effective amount of a
chimeric protein having a general structure of: N terminus ¨ (a) ¨ (b) ¨ (c) ¨
C terminus, wherein: (a) is a first
domain comprising an extracellular domain of human signal regulatory protein a
(CD172a (SIRPa)), (b) is a
linker adjoining the first and second domains, wherein the linker comprises a
hinge-CH2-CH3 Fc domain,
and (c) is a second domain comprising an extracellular domain of human CD40
ligand (CD4OL).
In some embodiments of any of the aspects disclosed herein, the human subject
is not receiving a concurrent
chemotherapy, immunotherapy, biologic or hormonal therapy.
In one aspect, the present disclosure relates to a chimeric protein for use in
the method of any of the
embodiments disclosed herein.
In one aspect, the present disclosure relates to a chimeric protein comprising
an amino acid sequence that
is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the chimeric protein
comprises an amino acid sequence that is at least about 99% identical to SEQ
ID NO: 59 or SEQ ID NO: 61.
In embodiments, the chimeric protein comprises an amino acid sequence that is
identical to SEQ ID NO: 59
or SEQ ID NO: 61.
The dosing frequency of the first phase, and the dosing frequency of the
second phase may be same or
different. In embodiments, the dosing frequency of the first phase and the
dosing frequency of the second
phase are each independently selected from about every three days, about twice
a week, about every week,
about every 10 days, about twice every 3 weeks, about every 2 weeks, about
every 3 weeks, about every 4
weeks, about every month, about every 5 weeks, about every 6 weeks, about 7
seven weeks, about every 8
weeks and about every 2 months. In embodiments, the dosing frequency of the
first phase is selected from
about every 3 days to about every 10 days, about every week to about every 2
weeks, about every 10 days
to about every 3 weeks, about every 2 weeks to about every 4 weeks, about
every 3 weeks to about every 5
weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to
about every 7 weeks, about
every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every
2 months.
83
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the first phase, and the second phase each independently last
from about two days to about
12 months. For example, In embodiments, the first phase lasts from about two
weeks to about 2 months; and
the second phase lasts from about 2 weeks to about 12 months. In embodiments,
the first phase lasts from
about two weeks to about 1 month; and the second phase lasts from about 2
weeks to about 12 months. In
embodiments, the first phase lasts from about two weeks to about 1 month; and
the second phase lasts from
about 4 weeks to about 12 months.
The effective amount for the first phase, the second phase and the third phase
may be same or different. In
embodiments, the effective amount for the first phase, the second phase and
the third phase each
independently comprise about 0.01 mg/kg to about 10 mg/ml. In embodiments, the
effective amount for the
first phase is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to
about 1 mg/kg, about 0.3 mg/kg
to about 3 mg/kg, or about 1 mg/kg to about 10 mg/kg; and the effective amount
for the second phase is from
about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg,
about 0.1 mg/kg to about 1
mg/kg, about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 10 mg/kg.
In embodiments, the chimeric
proteins disclosed herein is the human CD172a (SIRPa)-Fc-CD4OL chimeric
protein.
In embodiments, the human CD172a (SIRPa)-Fc-CD4OL chimeric protein is capable
of providing a sustained
zo immunomodulatory effect.
In embodiments, the linker comprises an amino acid sequence that is at least
90%, or 93%, or 95%, or 97%,
or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3. In
embodiments, the linker comprises an amino acid sequence that is at least 95%
identical to the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the
linker comprises an amino
acid sequence that is at least 96% identical to the amino acid sequence of SEQ
ID NO: 1, SEQ ID NO: 2, or
SEQ ID NO: 3. In embodiments, the linker comprises an amino acid sequence that
is at least 98% identical
to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In
embodiments, the linker
comprises an amino acid sequence that is at least 99% identical to the amino
acid sequence of SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the linker comprises an
amino acid sequence that is
identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3.
In embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from IgG.
In embodiments, the
linker comprises hinge-CH2-CH3 Fc domain derived from an IgG selected from
IgG1 and IgG4. In
embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from human
IgG1 or human I gG4. In
84
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from IgG4.
In embodiments, the
hinge-CH2-CH3 Fc domain is derived from human IgG4.
Additionally, or alternatively, in embodiments, the extracellular domain of
human signal regulatory protein a
(CD172a (SIRPa)) comprises an amino acid sequence that is at least 90%, or
93%, or 95%, or 97%, or 98%,
or 99% identical to the amino acid sequence of SEQ ID NO: 57. In embodiments,
the extracellular domain of
human signal regulatory protein a (CD172a (SIRPa)) comprises an amino acid
sequence that is at least 95%
identical to the amino acid sequence of SEQ ID NO: 57. In embodiments, the
extracellular domain of human
signal regulatory protein a (CD172a (SIRPa)) comprises an amino acid sequence
that is at least 96%
identical to the amino acid sequence of SEQ ID NO: 57. In embodiments, the
extracellular domain of human
signal regulatory protein a (CD172a (SIRPa)) comprises an amino acid sequence
that is at least 98%
is identical to the amino acid sequence of SEQ ID NO: 57. In embodiments,
the extracellular domain of human
signal regulatory protein a (CD172a (SIRPa)) comprises an amino acid sequence
that is at least 99%
identical to the amino acid sequence of SEQ ID NO: 57. In embodiments, the
extracellular domain of human
signal regulatory protein a (CD172a (SIRPa)) comprises an amino acid sequence
that is identical to the
amino acid sequence of SEQ ID NO: 57.
zo Additionally, or alternatively, in embodiments, the extracellular domain
of human CD40 ligand (CD4OL)
comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%,
or 98%, or 99% identical
to the amino acid sequence of SEQ ID NO: 58. In embodiments, the extracellular
domain of human CD40
ligand (CD4OL) comprises an amino acid sequence that is at least 95% identical
to the amino acid sequence
of SEQ ID NO: 58. In embodiments, the extracellular domain of human CD40
ligand (CD4OL) comprises an
25 amino acid sequence that is at least 96% identical to the amino acid
sequence of SEQ ID NO: 58. In
embodiments, the extracellular domain of human CD40 ligand (CD4OL) comprises
an amino acid sequence
that is at least 98% identical to the amino acid sequence of SEQ ID NO: 58. In
embodiments, the extracellular
domain of human CD40 ligand (CD4OL) comprises an amino acid sequence that is
at least 99% identical to
the amino acid sequence of SEQ ID NO: 58. In embodiments, the extracellular
domain of human CD40 ligand
30 (CD4OL) comprises an amino acid sequence that is identical to the amino
acid sequence of SEQ ID NO: 58.
Additionally, or alternatively, in embodiments, the human CD172a (SIRPa)-Fc-
CD4OL chimeric protein
comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%,
or 98%, or 99% identical
to SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the human CD172a (SIRPa)-Fc-
CD4OL chimeric
protein comprises an amino acid sequence that is at least 95% identical to SEQ
ID NO: 59 or SEQ ID NO:
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
61. In embodiments, the human CD172a (SIRPa)-Fc-CD4OL chimeric protein
comprises an amino acid
sequence that is at least 96% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the human
CD172a (SIRPa)-Fc-CD4OL chimeric protein comprises an amino acid sequence that
is at least 97% identical
to SEQ ID NO: 59 or SEQ ID NO: 61. In embodiments, the human CD172a (SIRPa)-Fc-
CD4OL chimeric
protein comprises an amino acid sequence that is at least 98% identical to SEQ
ID NO: 59 or SEQ ID NO:
61. In embodiments, the human CD172a (SIRPa)-Fc-CD4OL chimeric protein
comprises an amino acid
sequence that is at least 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61. In
embodiments, the human
CD172a (SIRPa)-Fc-CD4OL chimeric protein comprises an amino acid sequence that
is identical to SEQ ID
NO: 59 or SEQ ID NO: 61.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject comprising:
is (i) administering to the human subject a chimeric protein having a
general structure of: N terminus ¨ (a) ¨ (b)
¨ (c) ¨ C terminus, wherein: (a) is a first domain comprising an extracellular
domain of human signal
regulatory protein a (CD172a (SIRPa)), (b) is a linker adjoining the first and
second domains, wherein the
linker comprises at least one cysteine residue capable of forming a disulfide
bond and/or comprises a hinge-
CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular
domain of human CD40 ligand
zo (CD4OL); and (ii) administering a second therapeutic agent. In
embodiments, the chimeric protein is
administered at a dose between about 0.0001 mg/kg and about 10 mg/kg. In
embodiments, the dose of the
chimeric protein administered is at least about 0.3 mg/kg, e.g., at least
about 0.3 ring/kg, or about 1.0 mg/kg,
or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg/kg, or about 8
mg/kg, or about 10 mg/kg. In
embodiments, the dose of the chimeric protein administered is at least about 1
mg/kg, e.g., at least about 1.0
25 mg/kg, or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg/kg, or
about 8 mg/kg, or about 10 mg/kg.
In embodiments, the chimeric protein exhibits a linear dose response in the
dose range of e.g., about 0.3
mg/kg to about 3 mg/kg, or about 0.5 mg/kg to about 3 mg/kg, or about 0.7
mg/kg to about 3 mg/kg, or about
1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2
mg/kg to about 3 mg/kg, or about
2.5 mg/kg to about 3 mg/kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about
0.3 mg/kg to about 2 mg/kg, or
30 about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0
mg/kg, or about 0.3 mg/kg to about 0.5
mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped
dose response.
In embodiments, the administration of the chimeric protein causes a CD47
receptor occupancy (RO) on
leukocytes that is at least about 30%, or at least about 40%, or at least
about 50%, or at least about 60%, or
at least about 65%, or at least about 70%, or at least about 75%, or at least
about 80%, or at least about
35 85%, or at least about 90%, or at least about 95% compared to the RO
prior to administration of the chimeric
86
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
protein, a second subject that is not administered the chimeric protein and/or
an external control. In
embodiments, the administration of the chimeric protein causes a CD47 receptor
occupancy (RO) on B cells
that is at least about 30%, or at least about 40%, or at least about 50%, or
at least about 60%, or at least
about 65%, or at least about 70%, or at least about 75%, or at least about
80%, or at least about 85%, or at
least about 90%, or at least about 95% compared to the RO prior to
administration of the chimeric protein, a
second subject that is not administered the chimeric protein and/or an
external control. In embodiments, the
administration of the chimeric protein causes an increase in the amount or
activity of one or more of IL-12,
MCP-1, MIP-1[3, MIP-1a, and MDC % compared to the RO prior to administration
of the chimeric protein, a
second subject that is not administered the chimeric protein and/or an
external control.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject comprising
is administering to a subject in need thereof: a chimeric protein of a
general structure of N terminus ¨ (a) ¨ (b)
¨ (c) ¨ C terminus, wherein: (a) is a first domain comprising an extracellular
domain of human signal
regulatory protein a (CD172a (SIRPa)), (b) is a linker adjoining the first and
second domains, wherein the
linker comprises at least one cysteine residue capable of forming a disulfide
bond and/or comprises a hinge-
CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular
domain of human CD40 ligand
zo (CD4OL); wherein: the subject is undergoing or has undergone treatment
with a second therapeutic agent. In
embodiments, the chimeric protein is administered at a dose between about
0.0001 mg/kg and about 10
mg/kg. In embodiments, the dose of the chimeric protein administered is at
least about 0.3 mg/kg, e.g., at
least about 0.3 ring/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3,
about 4 mg/kg, or about 6 mg/kg, or
about 8 mg/kg, or about 10 mg/kg. In embodiments, the dose of the chimeric
protein administered is at least
25 about 1 mg/kg, e.g., at least about 1.0 mg/kg, or about 2 mg/kg, or
about 3, about 4 mg/kg, or about 6 mg/kg,
or about 8 mg/kg, or about 10 mg/kg. In embodiments, the chimeric protein
exhibits a linear dose response
in the dose range of e.g., about 0.3 mg/kg to about 3 mg/kg, or about 0.5
mg/kg to about 3 mg/kg, or about
0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5
mg/kg to about 3 mg/kg, or
about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg, or about
0.3 mg/kg to about 2.5 mg/kg,
30 or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5
mg/kg, or about 0.3 mg/kg to about 1.0
mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In embodiments, the chimeric
protein does not exhibit a bell-
shaped dose response.
In one aspect, the present disclosure relates to a method for treating a
cancer in a human subject comprising
administering to a subject in need thereof a second anticancer therapeutic
agent, wherein the subject is
35 undergoing or has undergone treatment with a chimeric protein of a
general structure of N terminus ¨ (a) -
87
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
(b) ¨ (c) ¨ C terminus, wherein: (a) is a first domain comprising an
extracellular domain of human Signal
regulatory protein a (CD172a (SIRPa)), (b) is a linker adjoining the first and
second domains, wherein the
linker comprises at least one cysteine residue capable of forming a disulfide
bond and/or comprises a hinge-
CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular
domain of human CD40 ligand
(CD4OL). In embodiments, the chimeric protein is administered at a dose
between about 0.0001 mg/kg and
about 10 mg/kg. In embodiments, the chimeric protein exhibits a linear dose
response in the dose range of
e.g., about 0.3 mg/kg to about 3 mg/kg, or about 0.5 mg/kg to about 3 mg/kg,
or about 0.7 mg/kg to about 3
mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg,
or about 2 mg/kg to about
3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg, or about 0.3 mg/kg to about 2.5
mg/kg, or about 0.3 mg/kg to
about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to
about 1.0 mg/kg, or about 0.3
mg/kg to about 0.5 mg/kg. In embodiments, the chimeric protein does not
exhibit a bell-shaped dose
response.
In embodiments, the chimeric protein is administered before the second
therapeutic agent. In embodiments,
the second therapeutic agent is administered before the chimeric protein. In
embodiments, the second
therapeutic agent and the chimeric protein are administered substantially
together.
zo In embodiments, the second therapeutic agent is selected from an
antibody, and a chemotherapeutic agent.
In embodiments, the antibody is capable of antibody-dependent cellular
cytotoxicity (ADCC). In
embodiments, the antibody is selected from cetuximab, rituximab, obinutuzumab,
Hu14.18K322A, Hu3F8,
dinituximab, and trastuzumab. In embodiments, the antibody is capable of
antibody-dependent cellular
phagocytosis (ADCP). In embodiments, the antibody is selected from cetuximab,
daratumumab, rituximab,
and trastuzumab. In embodiments, the antibody is capable of binding a molecule
selected from
carcinoembryonic antigen (CEA), EGFR, HER-2, epithelial cell adhesion molecule
(EpCAM), and human
epithelial mucin-1, CD20, CD30, CD38, 0040, and 0D52. In embodiments, the
antibody is capable of binding
EGFR. In embodiments, the antibody is selected from Mab A13, AMG595, cetuximab
(Erbitux, 0225),
panitumumab (ABX-EGF, Vectibix), depatuxizumab (ABT 806), depatuxizumab,
mafodotin, duligotuzumab
(MEHD7945A, RG7597), Futuximab (Sym004), GC1118, imgatuzumab (GA201),
matuzumab (EMD 72000),
necitumumab (Portrazza), nimotuzumab (h-R3), anitumumab (Vectibix, ABX-EGF),
zalutumumab, humMR1,
and tomuzotuximab. In embodiments, the antibody is cetuximab.
88
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
In embodiments, the chemotherapeutic agent is an anthracycline. In
embodiments, the anthacycline is
selected from doxorubicin, daunorubicin, epirubicin and idarubicin, and
pharmaceutically acceptable salts,
acids or derivatives thereof. In embodiments, the chemotherapeutic agent is
doxorubicin.
In embodiments, the dose of the chimeric protein administered is at least
about 0.0001 mg/kg, e.g., between
about 0.0001 mg/kg and about 10.0 mg/kg. The chimeric protein may be
administered at an initial dose (e.g.,
at one of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03,
about 0.1, about 0.3, about 1, about
2, about 3, about 4, about 6 or about 10.0 mg/kg) and the chimeric protein is
administered in one or more
subsequent administrations (e.g., at one or more of about 0.0001, about 0.001,
about 0.003, about 0.01, about
0.03, about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 6, about
8, and about 10 mg/kg). In
embodiments, the dose of the chimeric protein administered is at least about
0.3 mg/kg, e.g., at least about
is 0.3 mg/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4
mg/kg, or about 6 mg/kg, or about 8
mg/kg, or about 10 mg/kg. In embodiments, the dose of the chimeric protein
administered is at least about 1
mg/kg, e.g., at least about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4
mg/kg, or about 6 mg/kg, or about
8 mg/kg, or about 10 mg/kg. In embodiments, the initial dose is less than the
dose for at least one of the
subsequent administrations (e.g. each of the subsequent administrations) or
the initial dose is the same as
zo the dose for at least one of the subsequent administrations (e.g., each
of the subsequent administrations). In
embodiments, the starting dose and/or the subsequent doses is the maximum
tolerated dose or less than the
maximum tolerated dose. In embodiments, the chimeric protein is administered
at least about one time a
month, e.g., at least about two times a month, at least about three times a
month, and at least about four
times a month. In embodiments, the chimeric protein is first administered once
a week for three weeks and
25 the chimeric protein is then administered about once every three weeks
or once every four weeks; alternately,
the chimeric protein is first administered once a week for three weeks and the
chimeric protein is then
administered about two times per month, e.g., once a week for three weeks and
the chimeric protein is then
administered about once every two weeks.
In embodiments, the cancer comprises an advanced solid tumor (local and/or
metastatic) or a lymphoma. In
30 embodiments, the cancer is selected from ovarian cancer, fallopian tube
cancer, peritoneal cancer, cutaneous
squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and
neck (SCCHN). In
embodiments, the cancer comprises an advanced solid tumor (local and/or
metastatic) or advanced
lymphoma.
89
CA 03237449 2024- 5-6
WO 2023/086929 PCT/US2022/079702
EXAMPLES
The examples herein are provided to illustrate advantages and benefits of the
present technology and to
further assist a person of ordinary skill in the art with preparing or using
the chimeric proteins of the present
technology. The examples herein are also presented in order to more fully
illustrate the preferred aspects of
the present technology. The examples should in no way be construed as limiting
the scope of the present
technology, as defined by the appended claims. The examples can include or
incorporate any of the
variations, aspects or embodiments of the present technology described above.
The variations, aspects or
embodiments described above may also further each include or incorporate the
variations of any or all other
variations, aspects or embodiments of the present technology.
Example 1: Phase 1 Clinical Trial of the SIRPa-Fc-CD4OL Chimeric Protein (SL-
172154)
This first-in-human Phase 1 dose escalation study is currently evaluating SL-
172154 as monotherapy in
subjects with platinum resistant ovarian cancer. Primary Objectives of the
study are to evaluate safety;
identify the maximum tolerated dose or maximum administered dose of SL-172154.
Secondary Objectives
of the study are to identify a dose and schedule (i.e., a recommended phase 2
dose [RP2D]), to characterize
the PK and immunogenicity, and to evaluate anti-tumor activity per RECISTv1.1
for solid tumors. Exploratory
zo Objectives are: to assess receptor occupancy of SIRPa and CD40 on PBMCs,
and to investigate
pharmacodynamic (PD) effects in blood and tumor.
The planned dose escalation is in half-log increments (FIG. 1). At least 3
subjects were enrolled into
sequential dose levels (DL) and evaluated for dose limiting toxicity (DLT) in
the first cycle of treatment.
Subjects receive intravenous (IV) administration of SL-172154 on Schedule 1 or
Schedule 2 until disease
progression, unacceptable toxicity, or withdrawal of consent. Currently
enrolling 10mg/kg.
Key Inclusion Criteria are:
= Locally advanced or metastatic ovarian cancer, primary peritoneal cancer
or fallopian tube cancer.
= Refractory to existing therapy(ies) and ineligible for a platinum
therapy. Subjects with homologous
recombination deficiency positive disease must have received prior PARPi with
or without
bevacizumab.
= Age 18 years or older
= ECOG performance status of 0 or 1
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
= Measurable disease per RECIST v1.1
Key Exclusion Criteria are:
= Primary platinum refractory as defined by progressing during or within 1
month of upfront platinum
therapy
= Prior treatment with an anti-CD47 or anti-SIRPa targeting agent or a CD40
agonist.
= Documented history of autoimmune disease or active pneumonitis
= Concurrent use of systemic corticosteroids or other immunosuppressive
medication
Subject Characteristic are shown in Table 4:
Table 4. Tumor Characteristics
Total number of subjects, n (Y0) N
15
t"::a:cer twe, OV3riilt3
i,60)
Pnnuvy ;),-.N-$zon,<,;C, 4
t2T:
204
AGO stage :.!i5ge 11;
, :...:..
GF;A:19 Hip 9Fi3Cie i
{73)
Gr000.*
f7)
3 (20)
Hixookt:34;7: ir3notri
Aderiocaminorna 2
;13
15 subjects of median age 67 years (range 33-79), having received a median of
5 prior lines of systemic
therapy (range 2-7) were treated with IV SL-172154 across 4 dose levels on 2
schedules. 13 of 15 subjects
had an ECOG PS of 1 at baseline. 6 subjects were treated on schedule 1 (days
1, 8, 15, 29, q2wks) at 0.1
mg/kg (n=3) and 0.3 mg/kg (n=3). 9 subjects treated on schedule 2 (once
weekly) at 0.3 mg/kg (n=3), 1.0
mg/kg (n=3), and 3.0 mg/kg (n=3). Most subjects had primary ovarian cancer
(n=9; 60%), FIGO stage IV
(n=9; 60%), high grade disease (n=11; 73%) and serous carcinoma histology
(n=11; 73%) as shown in Table
1. Adverse events are listed in Table 5.
Table 5. Most Common AEs in All Treated Subjects
91
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Total number of subjects, n (%) N= 15
Any AE 15
(WO)
(50)
:
Nausea 633)
D1::51if?;i1 4 (27)
apptite c20)
Prin!tus 3 (20)
Atiorninni:#04;w:t!im r19)
Back pain 2(3)
46*
2:03)::
Corislipation 2 (13)
:
3h00Øk : : =:
:
Hypomagnesonm 2 (13)
to*
Vomiting 2 (13)
No DLTs have been observed with SL-172154 on either schedule at doses ranging
from 0.1 mg/kg to 3.0
mg/kg. 15 subjects (100%) experienced an adverse event (AE) on treatment. The
most common AEs,
occurring in 3 subjects (all causality) were fatigue, infusion-related
reaction (IRR), nausea, diarrhea,
decreased appetite, dehydration, and pruritus (Table 2). 14 subjects (93%) had
treatment-related AEs
(TRAEs). The most common TRAEs, occurring in 2 subjects, were IRR (n=8; 53%)
fatigue (n=7; 47%),
nausea (n=4; 27%), decreased appetite (n=3; 20%), chills (n=2; 13%), diarrhea
(n=2; 13%) and dyspnea
(n=2, 13%). No G3 TRAEs have been reported. 8 subjects (53%) had infusion-
related reactions (IRRs)
dosed at 0.3 mg/kg (n=3), 1.0 mg/kg (n=2) and 3.0 mg/kg (n=3); 1 IRR event was
G3 and deemed secondary
to iron infusion, 14 IRRs were G2 in severity and 2 I RRs were G1 in severity.
IRRs were manageable with
pre-medications, did not prevent completion of IV dosing or lead to
discontinuation of SL- 172154. 13 subjects
(87%) subjects have discontinued SL-172154; 12 discontinued due to
radiological or clinical progression and
1 subject elected to stop treatment.
FIG.3 shows the tumor response and duration of treatment. Best response among
14 efficacy evaluable
subjects with post-baseline scan were:
¨ SD, n=4
¨ PD, n=9
¨ NE, n=1 (Subject had SD but did not meet protocol-specified minimum
interval for SD)
¨ 1 subject (3 mg/kg) had not reached the first on-treatment disease
assessment at week 8
92
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
Pharmacokinetics:
= Maximum concentration (Cmax) and Area under the curve, time 0
extrapolated to infinity
(AUCirif) increased disproportionately with increasing dose.
= Clearance decreased with increasing dose.
= PK appeared to be biphasic at 1.0 and 3.0 mg/kg.
= Elimination phase of the curve was not fully characterized at the doses
studied.
FIG. 4A to FIG. 4D show the reproducible increases in serum cytokines
following repeated dosing of SL-
172154. Plasma was prepared from subject blood draws at the indicated times.
Cytokine levels were assayed
using a multiplexed ECL ELISA method and select dose level cytokines are
shown. Proinflammatory
chemokines, CCL2 (MCP-1) (FIG. 4A), CCL4 (MIP-1[3) (FIG. 4B), CCL3 (MIP-1a)
(FIG. 4C), and CCL22
(MDC) (FIG. 4D) exhibited dose-dependent increases in plasma levels at all
post infusion measurements.
These increases were of equal duration and magnitude across all infusion
intervals.
FIG. 5A and FIG. 5B show the dose-dependent and reproducible increases in
Serum IL-12. FIG. 5A shows
the subject level interleukin 12 (IL-12), a mediator of TH1 proinflammatory
responses over time typify the
cyclic effector cytokine responses observed in study subjects. FIG. 5B shows
median responses at the first
zo infusion (horizontal bars), preliminarily appear to be dose dependent.
FIG. 6A and FIG. 6B demonstrate the SL-172154 preferentially binds 0D47 on
leukocytes but not RBCs.
FIG. 6A shows the CD47 receptor occupancy (RO) as evaluated by fluorescence
activated cell sorting
(FACS) analysis using whole blood on both red blood cells (RBC) and white
blood cells (WBC, leukocytes).
At one-hour post infusion on Cycle 1 Day 1 (Cl Dl), median CD47 RO on
leukocytes (horizontal bars) is
zs -80%. FIG. 6B shows the CD47 RO on RBC is <5% for all dose levels.
FIG. 7A to FIG. 7C demonstrate that SL-172154 stimulates dose-dependent B cell
margination and
activation. B cells represent a large pool of circulating immune cells
expressing high levels of CD40.
Fluorescence activated cell sorting (FACS) panels were designed to interrogate
CD40 receptor occupancy
and states of activation and maturation. On C1D1, nearly all (-80%) CD4O-F B
cells marginate, or exit the
30 circulation, within one-hour post-infusion. FIG. 7A shows the median
frequency of marginating cells increases
in a dose-dependent manner (horizontal bars). Receptor engagement is -100% at
all dose levels (data not
shown). FIG. 7B shows the median B cell frequencies return to pre-infusion
levels by the next infusion,
maintaining a cyclic pattern of egress and return with each infusion cycle.
FIG. 7C shows the returning B
93
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
cells exhibit increases in the co-stimulatory marker CD86, as well as the
maturation marker CD95, suggesting
that SL-172154 can induce phenotypic changes. Similarly, CD14-F monocytes
marginate, or exit the
circulation, within one hour of infusion; returning to pre-infusion levels by
the next infusion, maintaining a
cyclic pattern of egress and return for each infusion cycle. The observed
pattern of margination is driven
largely by CD86-F classical and non-classical monocytes (data not shown).
io FIG. 8A and FIG. 8B show the distinct profile of TNFa and Interleukin-6
(IL-6) relative to CD40 mAbs. FIG.
8A shows the induction of TNFa at various doses of CP-870,893 (left panel) or
SL-172154 (right panel). FIG.
8B shows the induction of IL-6 at various doses of CP-870,893 (left panel) or
SL-172154 (right panel). The
CP-870,893 data are from Vonderheide et al., J Clin Onco/ 25:876-883 (2007).
These results demonstrate,
inter alia, that SL-172154 does not induce TNFa and I nterleukin-6 (IL-6)
relative to CD40 mAbs. Dose limiting
is toxicities (DLTs) have been attributed to cytokine release syndrome
(CRS) have limited dose escalation of
CD40 agonist mAbs. Interestingly, no notable increases in TNFa and IL-6 have
been observed with SL-
172154. As a consequence, SL-172154 is currently dosing at 10x the dose of CP-
870,893
FIG. 9A and FIG. 9B demonstrate that SL-172154 induces innate immune response
in tumor
microenvironment (TME). FIG. 9A shows the immunohistochemistry analysis of
biopsy sample from patient
zo A before and after the administration of SL-172154. Monocytes were
detected by staining for CD68 (a protein
highly expressed by cells in the monocyte lineage). FIG. 9B demonstrates the
upregulation of activation
markers, CD40 and MHC Class II in TME in tumor biopsy sample after the
treatment with SL-172154,
compared to pretreatment biopsy samples.
FIG. 10A and FIG. 10B demonstrate that SL-172154 induces adaptive immune
response in tumor
zs microenvironment (TME). FIG. 9A shows the CD8-F cells, Granzyme B+
cells, 0D68+ cells, and Ki67-F cells
in biopsy sample from patient A before and after the administration of SL-
172154. CD8-F cells, Granzyme B-F
cells, CD68+ cells, and Ki67+ cells increased in post-treatment biopsy sample,
compared to pre-treatment
biopsy sample. FIG. 9B is a plot comparing the tumor proportion score (TPS)
and combined positive score
(CPS). Induction of PD-L1 on immune cells is a consequence of CD8-F T cell
activation.
30 These results suggest that SL-172154 was well-tolerated with no DLTs or
evidence of anemia,
thrombocytopenia, liver dysfunction, cytokine release syndrome or pneumonitis.
Dose escalation continues
at 10 mg/kg. Preliminary PK parameters for SL-172154 suggest target-mediated
drug disposition via receptor
binding. High receptor occupancy was observed for SL-172154 on CD47-F
leukocytes at the doses studied,
with minimal binding to RBCs. Binding of SL-172154 to CD40+ B cells and
monocytes led to rapid activation
94
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
and margination post infusion. Cyclical increases in innate and adaptive serum
cytokines were consistent
with CD40 receptor engagement and activation. There were no increases in IL-6
or TNFa, nor evidence of
bell-shaped dose responses. SL-172154 has been well-tolerated at doses which
saturate both CD40 and
CD47, with evidence of on-target PD activity which has not yet plateaued,
warranting further dose escalation.
These results further demonstrate, without wishing to be bound by theory, that
SL-172154 induces innate as
io well as adaptive immune response in tumor microenvironment (TME).
FIG. 11 shows the planned clinical development strategy for SL-172154. The
strategy includes a trial for SL-
172154 monotherapy in ovarian cancer, combination therapy of SL-172154 +
liposomal doxorubicin in
ovarian cancer, combination therapy of SL-172154 + azacitidine + venetoclax in
AML, combination therapy
of SL-172154 + azacitidine in HR-MDS, and combination therapy of SL-172154 +
azaciti dine in TP53 mutant
is AML.
Example 2: Increased Expression of CD80 in Tumor Following the Administration
of the SIRPa-Fc-CD4OL
Chimeric Protein (SL-172154)
SL-172154 was administered intratumorally to an ovarian cancer patient. A
tumor biopsy sample was
obtained from the patient prior to and after the administration of SL-172154.
The biopsy samples were
zo analyzed for the expression of CD80 in the tumor. As shown in FIG. 12,
compared to the biopsy sample
obtained prior to the administration of SL-172154, the sample obtained after
the administration of SL-172154
showed an increase in the abundance of CD80+ cells and/or CD80 expression in
tumor following
administration of SL-172154.
INCORPORATION BY REFERENCE
25 All patents and publications referenced herein are hereby incorporated
by reference in their entireties.
The publications discussed herein are provided solely for their disclosure
prior to the filing date of the present
application. Nothing herein is to be construed as an admission that the
present disclosure is not entitled to
antedate such publication by virtue of prior invention.
As used herein, all headings are simply for organization and are not intended
to limit the disclosure in any
30 manner. The content of any individual section may be equally applicable
to all sections.
EQUIVALENTS
While the invention has been disclosed in connection with specific embodiments
thereof, it will be understood
that it is capable of further modifications and this application is intended
to cover any variations, uses, or
CA 03237449 2024- 5-6
WO 2023/086929
PCT/US2022/079702
adaptations of the invention following, in general, the principles of the
invention and including such departures
from the present disclosure as come within known or customary practice within
the art to which the invention
pertains and as may be applied to the essential features hereinbefore set
forth and as follows in the scope
of the appended claims.
Those skilled in the art will recognize, or be able to ascertain, using no
more than routine experimentation,
numerous equivalents to the specific embodiments disclosed specifically
herein. Such equivalents are
intended to be encompassed in the scope of the following claims.
96
CA 03237449 2024- 5-6
