PROTEINES DE LIAISON A MOG ET LEURS UTILISATIONS

MOG-BINDING PROTEINS AND USES THEREOF

Abrégé français

La présente invention concerne le développement d'une nouvelle protéine (par exemple, un anticorps ou un fragment de celui-ci ) capable de se lier à MOG et l'inclusion de la partie de liaison à l'antigène de ladite protéine dans des récepteurs antigéniques chimériques (CAR) exprimés sur la surface cellulaire de cellules immunitaires régulatrices. Ces composés fournissent des outils thérapeutiques utiles pour réduire ou prévenir une démyélinisation, pour induire une remyélinisation, et pour traiter des maladies/troubles inflammatoires du SNC, comme la sclérose en plaques.

Abrégé anglais

The present invention relates to the development of a novel protein (e.g., antibody or fragment thereof) capable of binding to MOG and inclusion of the antigen-binding portion of said protein in chimeric antigen receptors (CAR) expressed on the cell surface of regulatory immune cells. These provide valuable therapeutic tools for reducing or preventing demyelination, for inducing remyelination, and for treating inflammatory CNS diseases/disorders, such as multiple sclerosis.

Revendications

WO 2023/035002
PCT/US2022/075956
CLAIMS
1. A myelin oligodendrocyte glycoprotein (MOG)-binding protein comprising:
(i) a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) 1-3 comprising SEQ ID NOs: 3-5, respectively; or any HCDR having an
amino acid sequence that shares at least about 90% of identity with one of SEQ
ID
NOs: 3-5; and
(ii)a light chain variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID
NOs:
6-8, respectively; or any LCDR having an amino acid sequence that shares at
least
about 90% of identity with one of SEQ ID NOs: 6-8.
2. A myelin oligodendrocyte glycoprotein (MOG)-binding protein comprising:
(i) a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) 1-3 having SEQ ID NOs: 3-5, respectively; and
(ii) a light chain variable domain (VL) comprising LCDRs 1-3 having SEQ ID
NOs: 6-8,
re sp ectiv el y.
3. The MOG-binding protein according to claim 1 or claim 2, wherein
said VH comprises SEQ ID NO: 11 or an amino acid sequence at least about 90%
identical
thereto, and
said VL comprises SEQ ID NO: 9 or any amino acid sequence at least about 90%
of
identical thereto.
4. The MOG-binding protein according to any one of claims 1 to 3 wherein
said VI-1 comprises SEQ ID NO: 11, and
said VL comprises SEQ ID NO: 9.
5. The MOG-binding protein of any one of claims 1 to 4, wherein the
protein is a single-chain
variable fragment (anti-MOG scF v).
6. The MOG-binding protein of claim 5, wherein the protein is a single-chain
variable
fragment (anti-MOG scFv) comprising SEQ ID NO: 12 or any amino acid sequence
at least
about 95% identical thereto.
7. The MOG-binding protein of claim 5 or claim 6, wherein the protein is a
single-chain
variable fragment (anti-MOG scFv) comprising SEQ ID NO: 12.
99
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
8. The MOG-binding protein of claim 5, wherein the protein is a single-chain
variable
fragment (anti-MOG scFv) comprising SEQ ID NO: 51 or any amino acid sequence
at least
about 95% identical thereto.
9. The MOG-binding protein of claim 5 or claim 8, wherein the protein is a
single-chain
variable fragment (anti-MOG scFv) comprising SEQ ID NO: 51.
10. The MOG-binding protein of any one of the preceding claims, wherein the
protein is
capable of binding mouse, cynomolgous and human MOG.
11. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising a MOG-binding protein according to any
one of
claims 1 to 10;
(ii)a transmembrane domain; and
(iii) a cytoplasmic domain comprising an intracellular signaling domain.
12. The CAR according to claim 11, wherein the intracellular signaling domain
comprises
a human CD28 costimulatory signaling domain, optionally comprising SEQ ID NO:
15 or
an amino acid sequence at least about 90% identical thereto, and/or
a human CD3 zeta domain, optionally comprising SEQ ID NO: 16 or an amino acid
sequence at least about 90% identical thereto.
13. The CAR according to any one of claims 11 or claim 12, wherein the
transmembrane
domain is derived from human CD8, optionally comprising SEQ ID NO: 14 or an
amino
acid sequence at least about 90% identical thereto.
14. A chimeric antigen receptor (CAR) comprising
(i) an anti-MOG scEv as defined in any one of claims 5 to 10,
(ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO:
13,
(iii)a transmembrane domain derived from human CD8, optionally comprising SEQ
ID
NO: 14,
(iv)an intracellular signaling domain comprising a human CD28 costimulatory
signaling
domain, optionally comprising SEQ ID NO: 15, and a human CD3 zeta domain,
optionally comprising SEQ ID NO: 16,
100
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
(v) optionally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
(vi)optionally a leader sequence, wherein the leader sequence optionally
comprises SEQ
ID NO: 1.
15. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising an anti-MOG scFv, the extracellular
domain
optionally comprising SEQ ID NO: 12;
(ii)a hinge domain derived from human CD8, the hinge domain optionally
comprising SEQ
ID NO: 13;
(iii)a transmembrane domain derived from human CD8, the transmembrane domain
optionally comprising SEQ ID NO: 14; and
(iv) a cytoplasmic domain comprising an intracellular signaling domain, the
intracellular
signaling domain comprising a human CD28 costimulatory signaling domain, the
human CD28 costimulatory signaling domain optionally comprising SEQ ID NO: 15,
and a human CD3 zeta domain, the human CD3 zeta domain optionally comprising
SEQ ID NO: 16;
(v) optionally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
(vi)optionally a leader sequence, wherein the leader sequence optionally
comprises SEQ
ID NO: 1.
16. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain compri sing an anti-MOG scFv, the extracellular
domain
optionally comprising SEQ ID NO: 51;
(ii)a hinge domain derived from human CD8, the hinge domain optionally
comprising SEQ
ID NO: 13;
(iii)a transmembrane domain derived from human CD8, the transmembrane domain
optionally comprising SEQ ID NO: 14; and
(iv) a cytoplasmic domain comprising an intracellular signaling domain, the
intracellular
signaling domain comprising a human CD28 costimulatory signaling domain, the
human CD28 costimulatory signaling domain optionally comprising SEQ ID NO: 15,
and a human CD3 zeta domain, the human CD3 zeta domain optionally comprising
SEQ ID NO: 16;
(v) opti onally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
101
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
(vi)optionally a leader sequence, wherein the leader sequence optionally
comprises SEQ
ID NO: 1.
17. The CAR according to any one of claims 11 to 15, wherein the extracellular
domain or anti-
MOG scFv comprises the sequence of SEQ ID NO: 12.
18. The CAR according to any one of claims 11 to 14 and 16, wherein the
extracellular domain
or anti-MOG scFy comprises the sequence of SEQ ID NO: 51.
19. A nucleic acid molecule encoding the MOG-binding protein according to any
one of claims
1 to 10, or the CAR according to any one of claims 1 1 to 18.
20. A vector comprising nucleic acid molecule according to claim 19.
21. A regulatory immune cell expressing the CAR according to any one of claims
11 to 18, or
comprising the nucleic acid molecule according to claim 19 or the vector of
claim 20.
22. The regulatory immune cell according to claim 21, wherein the regulatory
immune cell is
a regulatory T cell.
23. An isolated human T cell, wherein the T cell comprises a nucleic acid
molecule according
to claim 19 or the vector of claim 20.
24. A population of regulatory immune cells, wherein the population comprises
a plurality of
cells as defined in claim 21 or claim 22.
25. A composition comprising a regulatory immune cell according to claim 21 or
claim 22 or
a population of regulatory immune cells according to claim 24.
26. A regulatory immune cell according to 21 or claim 22, a population of
regulatory immune
cells according to claim 24, or a composition according to claim 25, for use
as a
medicament.
27. A regulatory immune cell according to claim 21 or claim 22, a population
of regulatory
immune cells according to claim 24, or a composition according to claim 25,
for use in
treating demyelinating diseases/disorders, particularly those associated with
the presence
of autoantibodies or self-reactive immune cells.
1 02
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
28. A regulatory immune cell according to claim 21 or claim 22, a population
of regulatory
immune cells according to claim 24, or a composition according to claim 25,
for use in
treating MOG-associated diseases/disorders (MOGAD), particularly MOG-
associated
inflammatory diseases/disorders, more particularly those associated with the
presence of
autoantibodies or self-reactive immune cells.
29. A regulatory immune cell according to claim 21 or claim 22, a population
of regulatory
immune cells according to claim 24, or a composition according to claim 25,
for use in
treating inflammatory CNS diseases/disorders, preferably wherein the
inflammatory CNS
disease/disorder is multiple sclerosis, more preferably wherein the multiple
sclerosis is:
(i) relapsing-remitting MS (RR1VIS),
(ii) primary-progressive MS (PPMS), or
(iii)secondary-progressive MS (SPMS).
30. A regulatory immune cell according to claim 21 or claim 22, a population
of regulatory
immune cells according to claim 24, or a composition according to claim 25,
for use treating
a demyelinating disorder caused or aggravated by auto-antigens and/or
autoantibodies.
31. A regulatory immune cell according to claim 21 or claim 22, a population
of regulatory
immune cells according to claim 24, or a composition according to claim 25,
for use in
reducing or preventing inflammation and/or damage including demyelination of
the CNS.
32. A method for treating a disorder or disease in a subject in need thereof,
wherein the method
comprises administering to said patient a regulatory immune cell according to
claim 21 or
claim 22, a population of regulatory immune cells according to claim 24, or a
composition
according to claim 25.
33. The method according to claim 32, wherein the disease or disorder is a MOG-
associated
disease/disorder (MOGAD), particularly MOG-associated
inflammatory
diseases/disorders, more particularly those associated with the presence of
autoantibodies
or self-reactive immune cells.
34. The method according to claim 32, wherein the disease or disorder is a
demyelinating
disorder caused or aggravated by auto-antigens and/or autoantibodies.
1 03
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
35. The method according to claim 32, wherein the method is for reducing or
preventing
inflammation and/or damage including demyelination of the CNS.
36. The method according to claim 32, wherein the method is for treating
inflammatory CNS
diseases/disorders, preferably wherein the inflammatory CNS disease/disorder
is multiple
sclerosis, more preferably wherein the multiple sclerosis is:
(i) relapsing-remitting MS (RRIV1S),
(ii) primary-progressive MS (PPMS), or
(iii) secondary-progressive MS (SPMS).
1 04
CA 03230229 2024- 2- 27

Description

WO 2023/035002
PCT/US2022/075956
MOG-BINDING PROTEINS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims priority from U.S. Patent Application
63/240,626, filed on
September 3, 2021. The disclosure of this priority application is incorporated
by reference
herein in its entirety.
SEQUENCE LISTING
100021 The instant application contains a Sequence Listing that has been
submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said
ASCII copy, created on September 2, 2022, is named 025297 W0040 SL.xml and is
96,546
bytes in size.
BACKGROUND OF THE INVENTION
100031 Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein found
primarily in the
central nervous system (CNS). MOG is found on the surface of myelinating
oligodendrocytes
and is a component of myelin sheaths; the protective coverings that surrounds
nerve fibres in
the brain, optic nerves and spinal cord.
100041 A demyelinating disease is any condition that results in damage or
destruction to the
myelin sheath (demyelination). When the myelin sheath is damaged, nerve
impulses slow or
even stop, causing neurological symptoms. Such symptoms, when isolated, are
defined as a
clinically isolated syndrome (CIS). Individuals who experience CIS may or may
not go on to
develop multiple sclerosis (MS).
100051 MS is the most common demyelinating disease of the central nervous
system and is
characterized by multiple areas of inflammation, demyelination and
neurodegeneration. There
are three main types of MS: relapsing-remitting MS (RRMS), primary-progressive
MS (PPMS)
and secondary-progressive MS (SPMS). In the early stage of MS, neural damage
is considered
to arise due to autoreactive T cells of the body's immune system recognising
myelin epitopes
and attacking the myelin sheath resulting in destruction of myelin expressing
cells. The
majority of patients will initially present with a relatively benign relapsing-
remitting disease
course (RRMS). Some will ultimately convert to a second progressive form
(SPMS). A smaller
1
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
percentage of patients will develop primary progression (PPMS) and will from
onset slowly
deteriorate in disease progression without any recovering phases of remission.
[0006] T regulatory cells (Tregs) play an active part in establishing and
maintaining
immunological tolerance and restraining various immune responses post-
inflammation.
Depletion of Tregs can inhibit natural recovery from an immune response-
induced disease and
transfer of these cells can reduce disease severity. Human Tregs play a key
role in maintenance
of immune homeostasis and thus may be used as therapeutic means in diverse
clinical
conditions. They also have potent immunosuppressive properties that can be
harnessed to
confer antigen-specific immunomodulation in a therapeutic setting.
[0007] An engineered, CNS-targeting regulatory T cell which expressed a
chimeric antigen
receptor (CAR) targeting mouse MUG was evaluated in vitro and in EAE mice in
Fransson et
al., Journal of Neuroinfl amm ati on, 2012, 9: 1 12.
[0008] There remains a need for effective treatments for inflammatory
diseases/disorders of
the central nervous system, particularly demyelinating disorders caused or
aggravated by auto-
antigens and/or anti- antibodies such as MS.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the development of a novel tool to
activate regulatory
immune cells at inflammation sites, based on the binding of MUG at
inflammation sites. More
specifically, the inventors disclose herein a novel protein (e.g., antibody or
fragment thereof)
capable of binding to MUG. Inclusion of the antigen-binding portion of said
protein (e.g., an
scFv) in chimeric antigen receptors (CAR) expressed on the cell surface of
regulatory immune
cells allows activation of these engineered cells in the CNS upon binding to
MUG. These
engineered regulatory immune cells would thus be a valuable therapeutic tool
for reducing or
preventing demyelination, for inducing remyelination, and for treating
inflammatory CNS
diseases/disorders, such as MS.
[0010] The present invention thus relates to a new protein (e.g., antibody or
fragment thereof),
which has a combination of unexpected and advantageous characteristics. The
present
invention further thus relates to a new chimeric antigen receptor (CAR)
targeting MUG (herein
referred to as an 'anti-MUG CAR'), which has a combination of unexpected and
advantageous
characteristics. Further, new Tregs are provided which express the new
chimeric antigen
receptor (CAR) targeting MUG (herein referred to as a 'CAR-MUG Tregs'). The
invention
2
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
further provides therapeutic agents and methods for reducing or preventing
demyelination, for
inducing remyelination, and for treatment of inflammatory CNS
diseases/disorders, such as
MS.
10011] In a first aspect, the present invention provides a MOG-binding protein
comprising a
heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs)
1-3 comprising SEQ ID NOs: 3-5, respectively; or any HCDR having an amino acid
sequence
that shares at least about 90% of identity with one of SEQ ID NOs: 3-5; and a
light chain
variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID NOs: 6-8,
respectively; or
any LCDR having an amino acid sequence that shares at least about 90% of
identity with one
of SEQ ID NOs: 6-8.
[0012] In an embodiment, the MOG-binding protein comprises a heavy chain
variable domain
(VH) comprising complementary-determining regions (HCDRs) 1-3 having SEQ ID
NOs: 3-
5, respectively; and a light chain variable domain (VL) comprising LCDRs 1-3
having SEQ ID
NOs: 6-8, respectively. In an embodiment, the VH comprises SEQ ID NO: 11 or an
amino acid
sequence at least about 90% identical thereto, and the VL comprises SEQ ID NO:
9 or any
amino acid sequence at least about 90% of identical thereto. Preferably, the
VH comprises SEQ
ID NO: 11, and the VL comprises SEQ ID NO: 9.
[0013] In a further embodiment, the MOG-binding protein is a single-chain
variable fragment
(anti-MOG scFv). More preferably, the MOG-binding protein is a single-chain
variable
fragment (anti-MOG scFv) comprising SEQ ID NO: 12 or any amino acid sequence
at least
about 95% identical thereto. More preferably, the MOG-binding protein is a
single-chain
variable fragment (anti-MOG scFv) comprising SEQ ID NO: 12. More preferably,
the MOG-
binding protein is a single-chain variable fragment (anti-MOG scFv) having the
amino acid
sequence of SEQ ID NO: 12. More preferably, the MOG-binding protein is a
single-chain
variable fragment (anti-MOG scFv) comprising SEQ ID NO: 51 or any amino acid
sequence
at least about 95% identical thereto. More preferably, the MOG-binding protein
is a single-
chain variable fragment (anti-MOG scFv) comprising SEQ ID NO: 51. More
preferably, the
MOG-binding protein is a single-chain variable fragment (anti-MOG scFv) having
the amino
acid sequence of SEQ ID NO. 51.
[0014] In an embodiment, the MOG-binding protein is capable of binding mouse
and human
myelin oligodendrocyte glycoprotein (MOG). Preferably, the MOG-binding protein
is also
capable of binding a cynomolgus myelin oligodendrocyte glycoprotein (MOG).
3
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0015] In a second aspect, the present invention provides a chimeric antigen
receptor (CAR)
comprising an extracellular domain comprising a MOG scFy or an antigen binding
fragment
of an anti-MOG antibody as described herein; a transmembrane domain; and a
cytoplasmic
domain comprising an intracellular signaling domain.
[0016] In an embodiment, the intracellular signaling domain comprises a human
CD28
costimulatory signaling domain, optionally comprising SEQ ID NO: 15 or an
amino acid
sequence at least about 90% identical thereto, and/or a human CD3 zeta domain,
optionally
comprising SEQ ID NO: 16 or an amino acid sequence at least about 90%
identical thereto.
Preferably, the transmembrane domain is derived from human CD8, optionally
comprising
SEQ ID NO: 14 or an amino acid sequence at least about 90% identical thereto.
[0017] In a preferred embodiment, the CAR comprises an anti-MOG scFv according
to the
invention; a hinge domain derived from human CD8, optionally comprising SEQ ID
NO: 13;
a transmembrane domain derived from human CD8, optionally comprising SEQ ID
NO: 14;
an intracellular signaling domain comprising a human CD28 costimulatory
signaling domain,
optionally comprising SEQ ID NO: 15, and a human CD3 zeta domain, optionally
comprising
SEQ ID NO: 16; and optionally a tag and/or a leader sequence.
[0018] In a preferred embodiment, the CAR comprises an extracellular domain
comprising an
anti-MOG scFy according to the invention, optionally comprising SEQ ID NO: 12;
a hinge
domain derived from human CD8, optionally comprising SEQ ID NO: 13; a
transmembrane
domain derived from human CD8, optionally comprising SEQ ID NO: 14; a
cytoplasmic
domain comprising an intracellular signaling domain comprising a human CD28
costimulatory
signaling domain, optionally comprising SEQ ID NO: 15, a human CD3 zeta
domain,
optionally comprising SEQ ID NO: 16; and optionally a tag wherein the tag
optionally
comprises SEQ ID NO: 2, and optionally a leader sequence, wherein the leader
sequence
optionally comprises SEQ ID NO: 1.
[0019] In a preferred embodiment, the CAR comprises an extracellular domain
comprising an
anti-MOG scFy according to the invention, optionally comprising SEQ ID NO: 51;
a hinge
domain derived from human CD8, optionally comprising SEQ ID NO: 13; a
transmembrane
domain derived from human CD8, optionally comprising SEQ ID NO: 14; a
cytoplasmic
domain comprising an intracellular signaling domain comprising a human CD28
costimulatory
signaling domain, optionally comprising SEQ ID NO: 15, a human CD3 zeta
domain,
optionally comprising SEQ ID NO: 16; and optionally a tag wherein the tag
optionally
4
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
comprises SEQ ID NO: 2, and optionally a leader sequence, wherein the leader
sequence
optionally comprises SEQ ID NO: 1.
[0020] In a third aspect, the present invention provides a nucleic acid
molecule encoding the
MOG-binding protein according to the invention, or the CAR according to the
invention.
[0021] In a fourth aspect, the present invention provides a vector comprising
nucleic acid
molecule according to the invention.
[0022] In a fifth aspect, the present invention provides a regulatory immune
cell expressing the
CAR according to the invention, or comprising the nucleic acid molecule
according to the
invention. In an embodiment, the regulatory immune cell is a regulatory T
cell.
[0023] The present invention also provides an isolated human T cell, wherein
the T cell
comprises a nucleic acid molecule according to the invention.
[0024] The present invention further provides a population of regulatory
immune cells,
wherein the population comprises a plurality of cells as defined herein.
[0025] In a sixth aspect, the present invention provides a composition
comprising a regulatory
immune cell according to the invention or a population of regulatory immune
cells according
to the invention.
[0026] In a seventh aspect, the present invention provides a regulatory immune
cell according
to the invention or a population of regulatory immune cells according to the
invention, for use
as a medicament.
[0027] The present invention also provides a regulatory immune cell according
to the
invention, a population of regulatory immune cells according to the invention,
or a composition
according to the invention, for use in treating demyelinating
diseases/disorders, particularly
those associated with the presence of autoantibodies or self-reactive immune
cells.
[0028] The present invention further provides a regulatory immune cell
according to the
invention, a population of regulatory immune cells according to the invention,
or a composition
according to the invention, for use in treating MOG-associated
diseases/disorders (MOGAD),
particularly MOG-associated inflammatory diseases/disorders, more particularly
those
associated with the presence of autoantibodies or self-reactive immune cells.
[0029] The present invention further provides a regulatory immune cell
according to the
invention, a population of regulatory immune cells according to the invention,
or a composition
according to the invention, for use in treating inflammatory CNS
diseases/disorders. In an
embodiment, the inflammatory CNS disease/disorder is multiple sclerosis. In an
embodiment,
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
multiple sclerosis is relapsing-remitting MS (RRMS). In another embodiment,
multiple
sclerosis is primary-progressive MS (PPMS). In another embodiment, multiple
sclerosis is
secondary-progressive MS (SPMS).
[0030] The present invention further provides a regulatory immune cell
according to the
invention, a population of regulatory immune cells according to the invention,
or a composition
according to the invention, for use treating a demyelinating disorder caused
or aggravated by
auto-antigens and/or autoantibodies.
[0031] The present invention further provides a regulatory immune cell
according to the
invention, a population of regulatory immune cells according to the invention,
or a composition
according to the invention, for use in reducing or preventing inflammation
and/or damage
including demyelination of the CNS.
[0032] The present invention further provides a method for treating a disorder
or disease in a
subject in need thereof, wherein the method comprises administering to said
patient a
regulatory immune cell as described herein, a population of regulatory immune
cells as
described herein, or a composition comprising a regulatory immune cell or a
population of
regulatory immune cells as described herein. In some embodiments, the disease
or disorder is
a MOG-associated disease/disorder (MOGAD), particularly MOG-associated
inflammatory
diseases/disorders, more particularly those associated with the presence of
autoantibodies or
self-reactive immune cells. In some embodiments, the disease or disorder is a
demyelinating
disorder caused or aggravated by auto-antigens and/or autoantibodies. In some
embodiments,
the method is for reducing or preventing inflammation and/or damage including
demyelination
of the CNS. In some embodiments, the method is for treating inflammatory CNS
diseases/disorders, preferably wherein the inflammatory CNS disease/disorder
is multiple
sclerosis, more preferably wherein the multiple sclerosis is relapsing-
remitting MS (RRMS),
primary-progressive MS (PPMS), or secondary-progressive MS (SPMS).
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 represents a schematic view of a human anti-MUG Chimeric
Antigen Receptor
(CAR) construct ("CAR 1"). The anti-MUG CAR construct of Figure 1 comprises,
an scFy
directed against the human/mouse MUG, a CD8 hinge (CD8 linker), a
transmembrane domain
derived from the human CD28 (CD28 TM), and CD3 zeta (CD3Z).
6
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0034] Figure 2 represents a schematic view of a mouse anti-MOG Chimeric
Antigen Receptor
(CAR) construct. The anti-MOG CAR construct of Figure 2 comprises an scFy
directed against
the human/mouse MUG, a CD8 hinge (CD8 linker), a transmembrane domain derived
from
the mouse CD28 (CD28 TM), and CD3 zeta (CD3Z).
[0035] Figure 3 is a dot plot of flow cytometry showing the transduction
efficiency assessed
by GFP expression and the CAR expression at human cell surface assessed by
protein-L.
[0036] Figure 4 is a graph monitoring the human Treg phenotype transduced or
not (NT for
Non-Transduced), with CAR-MUG of the present invention at the end of the first
cycle of
expansion. Treg cells were labeled with antibodies directed against human CD4,
CD25,
CD127, and CTLA-4. For detection of FOXP3 and Helios transcription factors, an
intra-
nuclear labeling was performed (A). Error bars represent mean SEM from 4
independent
experiments including 10 Treg donors in total.
[0037] Figure 5 is a graph showing human Treg activation status (measured by
CD69
expression, gated on GFP expression) either in absence of activation in media
only (NoAct) or
following 24h stimulation through the CAR (via addition of MUG coated beads)
or through
the TCR (via beads coated with anti-CD3 and anti-CD28; 3/28) from 3
independent
experiments including 8 Treg donors in total. Binding on MUG beads shows CAR-
mediated
activation. Binding with CD3/CD28 shows TCR mediated activation. Ctrl cells do
not have a
CAR and instead express GFP. Ctrl cells show no binding and activation in
presence of MUG.
CD3-CD28 control is performed to ensure that the cells can be activated
through their TCR.
[0038] Figure 6 is a combination of graphs showing that Treg cells expressing
the CAR-MUG
of the invention exhibit efficient CAR-mediated suppressive activity. Ctrl
cells do not have a
CAR and instead express GFP. Contact-dependent suppression mediated by CAR-MOG
in the
absence of any activation (grey curve) or after MUG-induced CAR activation
(red curve) or
after TCR-induced activation (blue curve) was evaluated by measuring the
proliferation of
conventional T cells (Tconv) using flow cytometry. Error bars represent mean
SEM from 4
independent experiments including 7 Treg donors in total.
[0039] Figure 7 is a dot plot of flow cytometry showing the transduction
efficiency assessed
by NGFR expression at mouse cell surface.
[0040] Figure 8 is a dot plot of flow cytometry showing the phenotype CD25+
FoxP3+ of
mouse CAR Tregs cells compared to NT cells after 7 days of expansion.
[0041] Figure 9 is a graph showing mouse Treg cells activation status
(measured by CD69
expression, gated on NGFR positive cells) either in absence of activation in
media only (No
7
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
Act) or following 24h stimulation through the TCR (beads coated with anti-CD3
and anti-
CD28) or through the CAR (via addition of MOG coated beads) from 4 independent
experiments. Binding on MOG beads shows CAR-mediated activation. Binding with
CD3/CD28 shows TCR mediated activation. CAR Ctrl cells have a truncated CAR
comprising
the anti-MOG scFy for binding, but no intracellular signaling domain. CAR Ctrl
cells show no
activation in presence of MOG. CD3-CD28 control is performed to ensure that
the cells can be
activated through their TCR.
[0042] Figure 10A is a combination of graphs showing mouse CAR Tregs
activation (% of
CD69) and proliferation (% of Ki67) in the CNS as compared to CAR Ctrl cells.
Cells with the
CAR MOG are NGFR-F cells and Controls are NGFR- cells. Figure 10B is a
combination of
graphs showing mouse CAR Tregs activation (% of CD69) and proliferation (% of
Ki67) in
the CNS as compared to spleen cells.
[0043] Figure 11A is a graph showing Treg cells activation status (measured by
CD69
expression, gated on NGFR positive cells) for a number of anti-MOG CAR,
including "CAR
1" which is an anti-MOG CAR in accordance with the invention. Figure 11B shows
the fold
increase in vivo of activation markers CD69, CD71, LAP and proliferation
marker Ki67 in the
CNS of animals injected with MOG CAR after 5 days ("short EAE model") versus
the level of
activation of CAR Tregs transduced with a truncated control CAR (MOG ScFy and
non-
signaling endodomain).
[0044] Figure 12 shows the level of activation in vitro of human MOG CAR Tregs
in response
of different doses of human (black line) or mouse (grey dotted line) coated
MOG. The
activation is measured by flow cytometry by looking at the level of expression
of the early
activation marker CD69.
[0045] Figure 13 shows the reactivity to MOG-protein from different species
(human, mouse
and cynomolgous) assessed by a cell based binding assay using flow cytometry.
MOG-CAR
expressing yeast cells were co-incubated with fluorescently labelled anti-myc
antibody to
detect scFV expression, and with the indicated concentration of the respective
biotinylated
MOG protein (left). Binding was quantified by measuring seFV/MOG double
positive cells
(right).
[0046] Figures 14A and 14B shows an exemplary study in which the CAR MOG Tregs
of the
present disclosure are administered to mice in which EAE has been induced by
pathogenic
cells.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0047] Figure 15 shows an exemplary study in which IFN-gamma positive cells
are determined
following MUG peptide stimulation of cells derived from the EAE mice treated
with MUG
CAR of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0048] In the present disclosure, the following terms have the following
meanings:
[0049] "About" when referring to a measurable value such as an amount, a
temporal duration,
and the like, is meant to encompass variations of +20% or in some instances
+10%, or in some
instances 5%, or in some instances +1%, or in some instances +0.1% from the
specified value,
as such variations are appropriate to perform the disclosed methods.
Preferably, as used herein,
the term "approximately" or "about" as applied to one or more values of
interest refers to a
value that is similar to a stated reference value. In certain embodiments, the
term refers to a
range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or
less in either
direction (greater than or less than) of the stated reference value unless
otherwise stated or
otherwise evident from the context
[0050] "Affinity" is used to define the strength of a protein (e.g., an
antibody)-antigen
complex. Affinity measures the strength of interaction between an epitope and
an antigen
binding site on a protein (e.g., an antibody). It may be expressed by an
affinity constant Ka or
by a dissociation constant Ku. A protein (e.g., an antibody) is said to
specifically bind to an
antigen when the KD is < 1 1.IM, preferably < 100 nM or < 10 nM. Ku can be
measured, e.g.,
by surface plasmon resonance (SPR) (BIAcore') or Bio-Layer Interferometry, for
example,
using the IBIS MX96 SPR system from IBIS Technologies, the ProteOnTM XPR36 SPR
system
from Bio-Rad, or the OctetTM system from F orteB i o.
10051] "Antibody" or "immunoglobulin" as used herein, refers to a tetramer
comprising two
heavy chains and two light chains interconnected by di sulfide bonds. Each
light chain is
composed of a light chain variable domain (VL) and a light chain constant
region (CL) and can
be a kappa (lc) light chain or a lambda (X) light chain. Each heavy chain is
comprised of a heavy
chain variable domain (VH) and a heavy chain constant region (CH). Based on
the amino acid
sequence of the CH, antibodies can be assigned to different isotypes: IgA,
IgD, IgE, IgG, or
IgM. The IgG and IgA isotypes are further divided into subclasses: IgGl, IgG2,
IgG3, IgG4,
IgAl, and IgA2. The pairing of a VH and a VL forms a single antigen-binding
site. In one
9
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
embodiment, the anti-MOG antibody of the present invention is an IgG antibody,
such as an
IgGl, IgG2, or IgG4 antibody.
[0052] "Antigen-binding fragment", as used herein, refers to a part or region,
or a derivative
of an antibody that comprises fewer amino acid residues than the whole
antibody and yet
remains capable of binding to the antigen (e.g., MOG) of the whole antibody.
Antigen-binding
fragments encompasses, without any limitation, single chain antibodies, Fy
(e.g., say), Fab,
Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies
and tetrabodies.
[0053] "Chimeric antigen receptor" or "CAR" refers to a protein, which when
expressed in
an immune cell (e.g., a regulatory immune cell), provides the cell with
specificity for a target
ligand and with intracellular signal generation. In some embodiments, the CAR
comprises a
set of polypeptides that include a dimerization switch that, upon the presence
of a dimerization
molecule, can couple the polypeptides to one another, e.g., can couple a
ligand-binding domain
to an intracellular signaling domain. In one embodiment, the CAR comprises an
optional leader
sequence at the N-terminus, wherein the leader sequence is cleaved during
cellular processing
and localization of the chimeric antigen receptor to the cellular membrane.
[0054] Complementarity-determining region" or "CDR" means the non-contiguous
antigen
combining sites found within the heavy chain variable domain (VH) and the
light chain variable
domain (VL). The precise amino acid sequence boundaries of a given CDR can be
determined
using any of a number of well-known schemes, including those described by
Kabat et al.,
"Sequences of Proteins of Immunological Interest,"5th Ed. (1991) Public Health
Service,
National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-
Lazikani et al.,
JN1B (1997) 273:927-948 ("Chothia" numbering scheme), or a combination
thereof. More
recently, a universal numbering system has been developed and widely adopted,
ImMunoGeneTics (IMGT) Information System (Lefranc et al., Nucleic Acids Res.
(1999)
27:209-212). In one embodiment, the CDR boundaries herein are defined in
accordance with
Kabat et al. (1991). The CDR of a VH domain may be labeled herein as a HCDR
domain. The
CDR of a VL domain may be labeled as a LCDR domain. The CDRs 1-3 of a VH
domain may
be labeled as CDR1-VH, CDR2-VH, and CDR3-VH, respectively, or as HCDR1, HCDR2,
and
HCDR3, respectively. The CDRs 1-3 of a VL domain may be labeled as CDR1-VL,
CDR2-
VL, and CDR3-VL, respectively, or as LCDR1, LCDR2, and LCDR3, respectively.
[0055] "Costimulatory molecule- refers to a cognate binding partner on a T
cell that
specifically binds with a costimulatory ligand, thereby mediating a
costimulatory response by
the T cell, such as, but not limited to, proliferation. Costimulatory
molecules are cell surface
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
molecules other than antigen receptors or their ligands that contribute to an
efficient immune
response. A costimulatory signaling domain can be the intracellular portion of
a costimulatory
molecule. A costimulatory molecule can be represented in the following protein
families: TNF
receptor proteins, immunoglobulin-like proteins, cytokine receptors,
integrins, signaling
lymphocytic activation molecules (SLAM proteins), and activating NK cell
receptors.
100561 -Epitope" refers to a specific arrangement of amino acids located on a
protein or
proteins to which an antibody or antigen-binding fragment thereof binds.
Epitopes often consist
of a chemically active surface grouping of molecules such as amino acids or
sugar side chains,
and have specific three-dimensional structural characteristics as well as
specific charge
characteristics. Epitopes can be linear (or sequential) or conformational,
i.e., involving two or
more sequences of amino acids in various regions of the antigen that may not
necessarily be
contiguous.
[0057] "Expression vector" refers to a vector comprising a recombinant
polynucleoti de
comprising expression control sequences operatively linked to a nucleotide
sequence to be
expressed. An expression vector comprises sufficient cis-acting elements for
expression; other
elements for expression can be supplied by the host cell or in an in vitro
expression system.
Expression vectors include all those known in the art, including cosmids,
plasmids (e.g., naked
or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses,
adenoviruses, and
adeno-associated viruses) that incorporate the recombinant polynucleotide
[0058] "Fe domain," "Fe portion," and "Fe region" refer to a C-terminal
fragment of an
antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of
human gamma
heavy chain or its counterpart sequence in other types of antibody heavy
chains (e.g., a, 5, e
and u for human antibodies), or a naturally occurring allotype thereof.
[0059] "Fv" is the minimum antibody fragment that contains a complete antigen-
recognition
and -binding site. This fragment consists of a dimer of one heavy-chain and
one light-chain
variable domain in tight, non-covalent association. From the folding of these
two domains
emanate six hypervariable loops (three loops each from the H and L chain) that
contribute to
the amino acid residues for antigen binding and confer antigen binding
specificity to the
antibody. However, even a single variable domain (or half of an Fv comprising
only three
CDRs specific for an antigen) has the ability to recognize and bind antigen,
although at a lower
affinity than the entire binding site.
[0060] "Identity" or "identical," when used herein to describe the
relationship between two
or more amino acid sequences, or between two or more nucleic acid sequences,
refers to the
11
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
degree of sequence relatedness between the compared sequences. "Identity"
measures the
percentage of identical matches between the smaller of two or more sequences
with gap
alignments (if any) addressed by a particular mathematical model or computer
program (i.e.,
"algorithms"). Identity of related amino acid sequences or nucleic acid
sequences can be
readily calculated by known methods. Such methods include, but are not limited
to, those
described in Lesk A. M. (1988). Computational molecular biology: Sources and
methods for
sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993).
Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press;
Griffin A.
M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa,
NJ: Humana
Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure
tro e or tri ial
pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991).
Sequence
analysis primer. New York, NY: Stockton Press; Carrillo et al., SIAM Appl
Math. (1988)
48(5).1073-82. Preferred methods for determining identity are designed to give
the largest
match between the sequences tested. Methods of determining identity are
described in publicly
available computer programs. Preferred computer program methods for
determining identity
between two sequences include the GCG program package, including GAP (Genetics
Computer Group, University of Wisconsin, Madison, WI; Devereux et at., Nucleic
Acids Res.
(1984) 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et at, .1 Mol
Biol. (1990)
215(3):403-10). The BLASTX program is publicly available from the National
Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et
al.
NCB/NLM/NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may
also
be used to determine identity.
[0061] "Intracellular signaling domain" as used herein, refers to an
intracellular portion of a
molecule. The intracellular signaling domain generates a signal that promotes
an immune
effector function of the chimeric receptor containing cell. Examples of immune
effector
function in a chimeric receptor-T cell may include cytolytic activity,
suppressive activity,
regulatory activity, and helper activity, including the secretion of
cytokincs.
[0062] An "isolated antibody", as used herein, is intended to refer to an
antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated
antibody that specifically binds MOG is substantially free of antibodies that
specifically bind
antigens other than MUG). An isolated antibody that specifically binds MUG
may, however,
have cross-reactivity to other antigens, such as MUG molecules from other
species. Moreover,
an isolated antibody may be substantially free of other cellular material
and/or chemicals, in
12
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
particular those that would interfere with therapeutic uses of the antibody,
including without
limitation, enzymes, hormones, and other proteinaceous or non-proteinaceous
components.
The isolated antibody herein may be an IgG antibody, such as an IgGl, IgG2, or
IgG4 antibody.
[0063] An "isolated nucleic acid", as used herein, is intended to refer to a
nucleic acid that is
substantially separated from other genome DNA sequences as well as proteins or
complexes
such as ribosomes and polymerases, which naturally accompany a native
sequence. the term
embraces a nucleic acid sequence that has been removed from its naturally
occurring
environment, and includes recombinant or cloned DNA isolates and chemically
synthesized
analogues or analogues biologically synthesized by heterologous systems. A
substantially pure
nucleic acid includes isolated forms of the nucleic acid.
[0064] This refers to the nucleic acid as originally isolated and does not
exclude genes or
sequences later added to the isolated nucleic acid by the hand of man.
[0065] "Subject" is intended to include living organisms in which an immune
response can be
elicited (e.g., mammals, human). In one embodiment, a subject may be a
"patient", i.e., a warm-
blooded animal, more preferably a human, who/which is awaiting the receipt of,
or is receiving
medical care or was/is/will be the object of a medical procedure, or is
monitored for the
development of the targeted disease or condition, such as, for example, an
inflammatory or
autoimmune condition. In one embodiment, the subject is an adult (for example
a subject above
the age of 18). In another embodiment, the subject is a child (for example a
subject below the
age of 18). In one embodiment, the subject is a male. In another embodiment,
the subject is a
female. In one embodiment, the subject is affected, preferably is diagnosed,
with an
autoimmune and/or inflammatory disease or disorder. In one embodiment, the
subject is at risk
of developing an autoimmune and/or inflammatory disease or disorder. Examples
of risks
factor include, but are not limited to, genetic predisposition, or familial
history of an
autoimmune and/or inflammatory disease or disorder.
[0066] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a
protein comprising
a variable domain of an antibody light chain (VL) and a variable domain of an
antibody heavy
chain, (Vii) wherein the light and heavy chain variable domains are
contiguously linked, e.g.,
via a synthetic linker, e.g., a short flexible polypeptide linker, and capable
of being expressed
as a single chain polypepti de, and wherein the scFv retains the specificity
of the intact antibody
from which it is derived. Unless specified, as used herein an scFv may have
the VL and VET
variable domains in either order, e.g., with respect to the N-terminal and C-
terminal ends of
13
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-
VL. In one
embodiment, the present antigen-binding fragment is a single chain Fv (scFv).
[0067] "Therapeutically effective amount" refers to the level or amount of an
antibody as
described herein that is aimed at, without causing significant negative or
adverse side effects
to the target, (1) delaying or preventing the onset of a disease, disorder, or
condition; (2)
slowing down or stopping the progression, aggravation, or deterioration of one
or more
symptoms of the disease, disorder, or condition; (3) bringing about
ameliorations of the
symptoms of the disease, disorder, or condition; (4) reducing the severity or
incidence of the
disease, disorder, or condition; or (5) curing the disease, disorder, or
condition. A
therapeutically effective amount may be administered prior to the onset of the
disease, disorder,
or condition, for a prophylactic or preventive action. Alternatively or
additionally, the
therapeutically effective amount may be administered after initiation of the
disease, disorder,
or condition, for a therapeutic action.
[0068] "Treating" or "treatment" or "alleviation" refers to both therapeutic
treatment and
prophylactic or preventative measures, wherein the objective is to prevent or
slow
down (lessen) the targeted pathologic condition or disorder. Those in need of
treatment include
those already with the disorder as well as those prone to have the disorder or
those in whom
the disorder is to be prevented. In one embodiment, a subject is successfully
"treated" for a
disease or disorder if, after receiving a therapeutic amount of an antibody or
of a cell according
to the present disclosure, the subject shows at least one of the following:
reduction in the
number or percentage of pathogenic cells; relief to some extent of one or more
of the symptoms
associated with the disease or disorder to be treated; reduced morbidity and
mortality; and
improvement in quality-of-life issues. The above parameters for assessing
successful treatment
and improvement in the disease are readily measurable by routine procedures
familiar to a
physician.
[0069] "Zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is
defined as the
protein provided as GenBank Ace. No. BAG36664.1, or the equivalent residues
from a non-
human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta
stimulatory domain"
or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory
domain" is
defined as the amino acid residues from the cytoplasmic domain of the zeta
chain, or functional
derivatives thereof, that are sufficient to functionally transmit an initial
signal necessary for T
cell activation. In one embodiment, the cytoplasmic domain of zeta comprises
residues 52
14
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a
non-human
species, e.g., mouse, rodent, monkey, ape and the like, that are functional
orthologs thereof.
I. Antigen-binding protein
[0070] The MOG-binding protein of the invention is an antigen-binding protein
capable of binding to
MOG (hereinafter referred to as a MOG-binding protein). The MOG-binding
protein of the
invention may be an antibody or an antigen-binding fragment thereof, in
particular an scFv.
[0071] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where the heavy chain variable domain comprises at least one, preferably at
least two, more
preferably all three, of the following heavy chain HCDRs.
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5).
[0072] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising a heavy chain variable domain (VH) comprising complementary-
determining
regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH, where the heavy chain variable
domain comprises at least one, preferably at least two, more preferably all
three, of the
following heavy chain HCDRs:
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5).
[0073] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising a heavy chain variable domain (VH) comprising complementary-
determining
regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; where the heavy chain variable
domain comprises all three of the following heavy chain HCDRs:
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5).
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0074] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where the light chain variable domain comprises at least one, preferably at
least two, more
preferably all three, of the following light chain LCDRs:
CDR1-VL: RASQSVSSNYLA (SEQ ID NO: 6)
CDR2-VL: GASSRAT (SEQ ID NO: 7)
CDR3-VL: QQYGTSPGLT (SEQ ID NO: 8).
[0075] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where the heavy chain variable domain comprises at least one, preferably at
least two, more
preferably all three, of the following heavy chain HCDRs:
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5);
and where the light chain variable domain comprises at least one, preferably
at least two, more
preferably all three, of the following light chain LCDRs:
CDR1-VL: RASQSVSSNYLA (SEQ ID NO: 6)
CDR2-VL: GAS SRAT (SEQ ID NO: 7)
CDR3-VL: QQYGTSPGLT (SEQ ID NO: 8).
[0076] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
16
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
where
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5).
[0077] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL.
where CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5).
[0078] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where
CDR1-VH comprises the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH comprises the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID
NO: 4);
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL comprises the amino acid sequence GAS SRAT (SEQ ID NO. 7); and
CDR3-VL comprises the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).
[0079] In one embodiment, the present invention provides a MOG-binding protein
(e.g, scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where
CDR1-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);
17
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7); and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).
10080] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-V1-1; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where
CDR1-VH comprises the amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino
acid sequence with at least about 90% identity thereto;
CDR2-VH comprises the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID
NO: 4) or an amino acid sequence with at least about 90% identity thereto;
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an
amino acid sequence with at least about 90% identity thereto;
CDR1-VL comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or
an amino acid sequence with at least about 90% identity thereto;
CDR2-VL comprises the amino acid sequence GAS SRAT (SEQ ID NO: 7) or an amino
acid sequence with at least about 90% identity thereto; and
CDR3-VL comprises the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an
amino acid sequence with at least about 90% identity thereto.
[0081] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where
CDR1-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino acid
sequence with at least about 90% identity thereto;
18
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
or an amino acid sequence with at least about 90% identity thereto;
CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an amino
acid sequence with at least about 90% identity thereto;
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or an
amino acid sequence with at least about 90% identity thereto;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7) or an amino acid
sequence with at least about 90% identity thereto; and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an amino
acid sequence with at least about 90% identity thereto.
[0082] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where
CDRI-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);
CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7), and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8);
and where the VH comprises SEQ ID NO: 11 or an amino acid sequence having at
least
about 90% identity thereto, and the VL comprises SEQ ID NO: 9 or an amino acid
sequence
having at least about 90% identity thereto.
[0083] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
19
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
where the VH comprises SEQ ID NO:11, and the VL comprises SEQ ID NO: 9.
[0084] In one embodiment, the present invention provides a MUG-binding protein
(e.g., scFv)
comprising:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and
- a light chain variable domain (VL) comprising complementary-determining
regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL;
where the VII has the amino acid sequence of SEQ ID NO: 11, and the VL has the
amino
acid sequence of SEQ ID NO: 9.
[0085] In an embodiment, the MUG-binding protein is an scFv, sdAb, or DARPin.
[0086] In an embodiment, the MUG-binding protein is an anti-MUG antibody or an
antigen-
binding fragment thereof, in particular a single-chain variable fragment
(scFv).
[0087] In an embodiment, the MUG-binding protein is a single-chain variable
fragment (scFv)
having a heavy chain variable domain (VH) and a light chain variable domain
(VL) where the
VH comprises SEQ ID NO: 11 or an amino acid sequence having at least about 90%
identity
thereto, and the VL comprises SEQ ID NO: 9 or an amino acid sequence having at
least about
90% identity thereto.
[0088] In an embodiment, the MOG-binding protein is a single-chain variable
fragment (scFv)
having a heavy chain variable domain (VH) and a light chain variable domain
(VL) where the
VH comprises SEQ ID NO: 11, and the VL comprises SEQ ID NO: 9.
[0089] In an embodiment, the MUG-binding protein is a single-chain variable
fragment (scFv)
comprising SEQ ID NO: 12 or any amino acid sequence at least about 90%
identical thereto.
[0090] In an embodiment, the MUG-binding protein is a single-chain variable
fragment (scFv)
comprising SEQ ID NO: 12.
[0091] In an embodiment, the MOG-binding protein is a single-chain variable
fragment (scFv)
comprising SEQ ID NO: 51 or any amino acid sequence at least about 90%
identical thereto.
[0092] In an embodiment, the MOG-binding protein is a single-chain variable
fragment (scFv)
comprising SEQ ID NO: 51.
[0093] The antibody or an antigen-binding fragment thereof (e.g., scFv) may
itself be fused to
another protein as described herein, thereby forming a fusion protein.
[0094] The present invention further provides a MOG-binding protein that binds
to the same
epitope on MOG as a protein of the invention (e.g., an antibody or an antigen-
binding fragment
thereof, in particular an scFv) as described anywhere herein.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0095] The present invention further provides a MOG-binding protein that
competes for
binding to MOG in a competition binding assay with a MOG-binding protein of
the invention
(e.g., an antibody or an antigen-binding fragment thereof, in particular an
scFv) as described
anywhere herein.
[0096] The protein of the invention, e.g., an scFv of the invention, as
described anywhere
herein may be comprised in the extracellular binding domain of a CAR,
particularly a CAR as
described herein
A. MOG-binding protein Junction: Antigen binding specificity and affinity
[0097] The MOG-binding protein of the invention is capable of binding to MOG
expressed on
cell surface. The MOG-binding protein of the invention is also capable of
binding to a soluble
MOG (i.e., not membrane bound).
100981 Advantageously, MOG-binding proteins of the invention have been found
to be capable
of binding to both a human and a mouse MOG. The MOG-binding proteins of the
invention
are also capable of binding to a cynomolgous MOG. This cross-reactivity is
beneficial for
extrapolating results from preclinical studies in mice and cynomolgous, and to
human clinical
studies for the drug approval process. In particular, MOG-binding proteins of
the invention
have been found to be capable of binding to both a human and a mouse MOG.
[0099] The MOG-binding protein of the invention recognizes and binds to a
human MOG.
Human MOG is a protein encoded by a 1775 bp long mRNA comprising 8 exons
(UniProtKB
identifier Q16653 and Genbank accession number: NM 002544.5).
[0100] In one embodiment, the present protein recognizes and is capable of
binding to a MOG
variant, such as a variant of a human MOG A variant of MOG refers to a
modified MOG
wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are deleted, added
or substituted as
compared to the original (wildtype) MOG.
[0101] Splice variants of human MOG have been previously identified. In
particular, 13
different isoforms of MOG have been described: isoform 1 (also known as alpha
1) (encoded
by a mRNA having the having the UniProtKB identifier Q16653-1), isoform 2
(also known as
alpha 2) (encoded by a mRNA having the UniProtKB identifier Q16653-2), isoform
3 (also
known as alpha 3) (encoded by a mRNA having the UniProtKB identifier Q16653-
3), isoform
4 (also known as alpha 4) (encoded by a mRNA having the UniProtKB identifier
Q16653-4),
isoform 5 (also known as beta 1) (encoded by a mRNA having the UniProtKB
number Q16653 -
5), isoform 6 (also known as beta 2) (encoded by a mRNA having the UniProtKB
identifier
21
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
Q16653-6), isoform 7 (also known as beta 3) (encoded by a mRNA having the
UniProtKB
identifier Q16653-7), isoform 8 (also known as beta 4) (encoded by a mRNA
having the
UniProtKB identifier Q16653-8), isoform 9 (encoded by a mRNA having the
UniProtKB
identifier Q16653-9), isoform 10 (encoded by a mRNA having the UniProtKB
identifier
Q16653-10), isoform 11 (encoded by a mRNA having the UniProtKB identifier
Q16653-11),
isoform 12 (encoded by a mRNA having the UniProtKB identifier Q16653-12) and
isoform 13
(encoded by a mRNA having the UniProtKB identifier Q16653-13).
[0102] Therefore, in one embodiment, the present protein recognizes and is
capable of binding
to one or more splice variant of human MOG selected from the group comprising
isoform 1,
isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, isoform 8,
isoform 9, isoform
10, isoform 11, isoform 12 and isoform 13.
[0103] In an embodiment, the protein of the invention, e.g., an scFy of the
invention, is able to
bind both mouse and human myelin oligodendrocyte glycoprotein (MOG). Thus,
cross-
reactivity of the scFy with mouse and human MOG is an advantageous feature.
[0104] Accordingly, in an embodiment, the protein also recognizes and binds to
a mouse MOG
(UniProtKB identifier Q61885).
[0105] In one embodiment, the present protein recognizes and is capable of
binding to a MOG
variant, such as a variant of a mouse MOG. A variant of MOG refers to a
modified MOG
wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are deleted, added
or substituted as
compared to the original (wildtype) MOG.
[0106] Advantageously, the present protein recognizes and is capable of
binding to a human
MOG (i.e. human MOG or a human MOG variant) and to a mouse MOG (i.e. mouse MOG
or
a mouse MOG variant). In an embodiment, the present protein recognizes and is
capable of
binding to human MOG and to a mouse MOG variant. In an embodiment, the present
protein
recognizes and is capable of binding to a human MOG variant and to mouse MOG.
In an
embodiment, the present protein recognizes and is capable of binding to a
human MOG variant
and to a mouse MOG variant.
[0107] In an embodiment, the protein of the invention, e.g., an scFy of the
invention, is also
able to bind cynomolgous myelin oligodendrocyte glycoprotein (MOG). Thus,
cross-reactivity
of the scFv with mouse, cynomolgous and human MOG is an advantageous feature.
[0108] Accordingly, in an embodiment, the protein also recognizes and binds to
a
cynomolgous MOG (UniProtKB identifier Q9BGS7).
22
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0109] In one embodiment, the present protein recognizes and is capable of
binding to a
cynomolgous variant, such as a variant of a cynomolgous MOG. A variant of MOG
refers to a
modified MOG wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are
deleted, added or
substituted as compared to the original (wildtype) MOG.
[0110] Advantageously, the present protein recognizes and is capable of
binding to a human
MOG (i.e. human MOO or a human MOO variant), to a mouse MOG (i.e. mouse MOG or
a
mouse MOG variant), and to a cynomolgous MOG (i.e. mouse MOG or a mouse MOG
variant).
B. Protein sequences
I. CDR sequences
[0111] In one embodiment, the MOG-binding protein of the invention (e.g.,
antibody or an
antigen-binding fragment thereof, in particular an scFv) comprises a heavy
chain variable
domain (VH) and a light chain variable domain (VL) each comprising
complementarity-
determining regions (CDRs). The CDRs are determined in accordance with the
Kabat CDR
definition system.
[0112] In one embodiment, the heavy chain of the present protein comprises at
least one,
preferably at least two, more preferably all three, of the following heavy
chain CDRs (HCDRs):
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VII: RERLYAGYY (SEQ ID NO: 5)
[0113] In a preferred embodiment, the MOG-binding protein of the invention
comprises all of
SEQ ID NOs: 3-5. In one embodiment, any of CDR-VH1, CDR2-VH and/or CDR3-VH may
comprise 1, 2, 3, or more amino acid modifications (e.g., substitutions) as
compared to SEQ
ID NOs: 3-5, respectively. In one embodiment, any of CDR1-VH, CDR2-VH and/or
CDR3-
VH has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%,
99% or more
of identity with SEQ ID NOs: 3-5, respectively.
[0114] In one embodiment, the light chain of the MOG-binding protein of the
invention
comprises at least one, preferably at least two, more preferably all three, of
the following light
chain CDRs (LCDRs):
CDR1-VL: RASQSVSSNYLA (SEQ ID NO: 6)
23
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CDR2-VL: GASSRAT (SEQ ID NO: 7)
CDR3-VL: QQYGTSPGLT (SEQ ID NO: 8)
[0115] In a preferred embodiment, the MOG-binding protein of the invention
comprises all of
SEQ ID NOs: 6-8. In one embodiment, any of CDR1-VL, CDR2-VL and/or CDR3-VL may
comprise 1, 2, 3, 4, 5, or more amino acid modifications (e.g., substitutions)
as compared to
SEQ ID NOs: 6-8, respectively. In one embodiment, any of CDR1-VL, CDR2-VL
and/or
CDR3-VL has an amino acid sequence that shares at least 90%, 95%, 96%, 97%,
98%, 99% or
more of identity with SEQ ID NOs: 6-8, respectively.
[0116] In one embodiment of the protein of the invention, at least one,
preferably at least two,
more preferably all three, of its heavy chain HCDRs 1-3 comprise SEQ ID NOs. 3-
5,
respectively; and at least one, preferably at least two, more preferably all
three, of its light chain
LCDRs 1-3 comprise SEQ ID NOs: 6-8, respectively.
[0117] In a preferred embodiment, the MOG-binding protein of the invention
comprises heavy
chain HCDRs 1-3 and light chain LCDRs 1-3 having the sequences of SEQ ID NOs:
3-8,
respectively.
[0118] In one embodiment of the protein of the invention, any of CDR1-VH, CDR2-
VH and/or
CDR3-VH has an amino acid sequence that shares at least 90%, 95%, 96%, 97%,
98%, 99%
or more of identity with SEQ ID NOs: 3-5, respectively; and any of CDR1-VL,
CDR2-VL
and/or CDR3-VL has an amino acid sequence that shares at least 90%, 95%, 96%,
97%, 98%,
99% or more of identity with SEQ ID NOs: 6-8, respectively.
2. VII and VL sequences
[0119] In one embodiment, the MOG-binding protein of the invention (e.g.,
antibody or an
antigen-binding fragment thereof, in particular an scFv) comprises a heavy
chain and a light
chain.
[0120] In one embodiment, the MOG-binding protein of the invention has a VH
amino acid
sequence comprising SEQ ID NO: 11.
[0121] In one embodiment, the MOG-binding protein of the invention has a VH
amino acid
sequence consisting of SEQ ID NO: 11.
[0122] In one embodiment, the VH amino acid sequence comprises SEQ ID NO: 11
having 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
amino acid modifications
(e.g., substitutions).
24
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0123] In one embodiment, the VH amino acid sequence consists of SEQ ID NO: 11
having 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
amino acid modifications
(e.g., substitutions).
[0124] In one embodiment, the VH amino acid sequence comprises the heavy chain
HCDRs
(e.g., SEQ ID NOs: 3-5) described above and shares at least 70%, 75%, 80%,
85%, 90%, 95%,
96%, 97%, 98%, 99% or more identity with SEQ ID NO: 11.
[0125] In one embodiment, the MOG-binding protein of the invention has a VL
amino acid
sequence comprising SEQ ID NO: 9
[0126] In one embodiment, the MOG-binding protein of the invention has a VL
amino acid
sequence consisting of SEQ ID NO: 9.
[0127] In one embodiment, the VL amino acid sequence comprises SEQ ID NO: 9
having 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
amino acid modifications
(e.g , sub sti tuti on s).
[0128] In one embodiment, the VL amino acid sequence consists of SEQ ID NO: 9
having 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
amino acid modifications
(e.g., substitutions).
[0129] In one embodiment, the VL amino acid sequence comprises the light chain
LCDRs
(e.g., SEQ ID NOs: 6-8) described above and shares at least 70%, 75%, 80%,
85%, 90%, 95%,
96%, 97%, 98%, 99% or more identity with SEQ ID NO: 9.
[0130] The present invention explicitly envisages combinations of any of the
VH described
herein with any of the VL described herein.
[0131] In one embodiment, the VH comprises SEQ ID NO: 11 the VL comprises SEQ
ID NO:
9, each optionally with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more
amino acid modifications (e.g., substitutions).
[0132] In one embodiment, the amino acid modification may be an insertion, a
deletion, or a
substitution. In one embodiment, the amino acid modification does not
significantly affect the
binding characteristics of the antibody or antigen-binding fragment thereof
containing the
modification. Specified variable domain and CDR sequences may comprise 1, 2,
3, 4, 5, 6, 7,
8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid
insertions, deletions, and/or
substitutions.
[0133] In one embodiment, the amino acid modification is a substitution made
preferably with
a conservative amino acid. A conservative amino acid is an amino acid having a
side chain
with similar physicochemical properties to those of the original amino acid.
Families of amino
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
acid residues having similar side chains have been defined in the art. These
families include
amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic
side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine,
serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains
(e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine), 0-branched side
chains (e.g.,
threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine,
phenylalanine,
tryptophan, histidine). Thus, one or more amino acid residues within the CDRs
and/or variable
domains of the present antibody or antigen-binding fragment can be replaced
with other amino
acid residues from the same side chain family, and the modified antibody or
antigen-binding
fragment can be tested for retained function (e.g., binding to MUG) using the
assays described
herein. In another embodiment, a string of amino acids within the CDRs and/or
variable
domains of the present antibody or antigen-binding fragment can be replaced
with a structurally
similar string that differs in order and/or composition of side chain family
members.
[0134] In one embodiment, the VH comprises at least one (preferably three)
heavy chain
HCDR as defined herein, and comprising or consisting of SEQ ID NO: 11 or an
amino acid
sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
more identity
thereto; and the VL comprises at least one (preferably three) light chain LCDR
as defined
herein, and comprising or consisting of SEQ ID NO: 9, or an amino acid
sequence with at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
[0135] In one embodiment, the VH and the VL comprise the CDRs (e.g., SEQ ID
NOs: 3-8)
as described above and share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%
or more identity with SEQ ID NOs: 11 and 9, respectively.
[0136] In one embodiment, the VH comprises SEQ ID NO: 11 and the VL comprises
SEQ ID
NO: 9.
[0137] In one embodiment, the VH consists of SEQ ID NO: 11 and the VL consists
of SEQ ID
NO: 9.
[0138] In one embodiment, the VH consists of SEQ ID NO: Ill and the VL
comprises SEQ ID
NO: 9.
[0139] In one embodiment, the VH comprises SEQ ID NO. 11 and the VL consists
of SEQ ID
NO: 9.
26
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
3. Linkers
[0140] In one embodiment, the MUG-binding protein of the invention (e.g.,
antibody or an
antigen-binding fragment thereof, in particular an scFv) comprises a linker
that links its VH
and VL (herein referred to as a VH-VL linker).
[0141] In one embodiment, the MOG-binding protein of the invention comprises,
from N-
terminus to C-terminus, the VL, a VH-VL linker, and the VH. In another
embodiment, the
MOG-binding protein of the invention comprises, from N-terminus to C-terminus,
the VH, a
VH-VL linker, and the VL.
[0142] In one embodiment, the VH-VL linker is a peptide linker, having a
length ranging from,
e.g., 2 to 20 or 2 to 15 amino acids.
[0143] For example, a glycine-serine doublet provides a particularly suitable
linker
(GS linker). In one embodiment, the VH-VL linker is a GS linker. Examples of
GS linkers
include, but are not limited to, GS linkers, G2S linkers (e.g., GGS and
(GGS)2), G3S linkers,
and Cr4S linkers
[0144] G3S linkers comprise the amino acid sequence (Gly-Gly-Gly-Ser)n or
(GGGS)n, where
n is a positive integer equal to or greater than 1 (such as, example, n-1,
n-2, n-3, n-4, n-5,
n=6, n=7, n=8, n=9, or n=10). Examples of G3S linkers include, but are not
limited to, GGGS
(SEQ ID NO: 71) corresponding to (GGGS)1 when n=1 and GGGSGGGSGGGSGGGS (SEQ
ID NO: 72) corresponding to (GGGS)4.
[0145] Examples of G4S linkers include, but are not limited to, (Gly4-Ser)
corresponding to
GGGGS (SEQ ID NO: 73); (Gly4-Ser)2 corresponding to GGGGSGGGGS (SEQ ID NO:
74);
(Gly4-Ser)3 corresponding to GGGGSGGGGSGGGGS (SEQ ID NO: 10); and (Gly4-Ser)4
corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75). In one embodiment, the
linker is SEQ ID NO: 10.
[0146] The present invention explicitly envisages the combination of any VH-VL
linker
described herein with any VH and VL domains described herein.
4. Types of proteins
[0147] The MOG-binding protein of the invention may be an antibody or antigen-
binding
fragment thereof. The MUG-binding protein of the invention may be a humanized
antibody or
antigen-hi n ding fragment thereof
[0148] In one embodiment, the antibody or antigen-binding fragment thereof is
an antigen-
binding fragment of an antibody, such as, for example, a single chain
antibody, a Fv (e.g.,
27
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
scFv), a Fab, a Fab', a Fab'-SH, a F(ab)'2, a Fd, a defucosylated antibody, a
diabody, a triabody
or a tetrabody.
[0149] In a preferred embodiment, the MOG-binding protein of the invention is
an scFv.
5. scFv sequence
[0150] The scFv of the invention advantageously can bind mouse and human
myelin
oligodendrocyte glycoprotein (MOG).
[0151] In one embodiment, the scFv of the invention comprises:
- -a heavy chain variable domain (VH) comprising at least one (preferably
three) heavy
chain CDR (HCDR) as defined herein, and comprising or consisting of SEQ ID NO:
11 or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or more identity thereto; and
- a light chain variable domain (VL) comprising at least one (preferably
three) light chain
CDR (HCDR) as defined herein, and comprising or consisting of SEQ ID NO: 9, or
an
amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or more identity thereto.
[0152] Preferably, the scFv of the invention comprises:
- a heavy chain variable domain (VH) comprising complementary-determining
regions
(HCDRs) 1-3 comprising SEQ ID NOs: 3-5, respectively; or any CDR having an
amino
acid sequence that shares at least about 90% of identity with one of SEQ ID
NOs: 3-5;
and
- a light chain variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID
NOs:
6-8, respectively; or any CDR having an amino acid sequence that shares at
least about
90% of identity with one of SEQ ID NOs: 6-8.
[0153] In an embodiment, the scFv of the invention comprises a VH comprising
SEQ ID NO:
11 or an amino acid sequence at least about 90% identical thereto, and a VL
comprising SEQ
ID NO: 9 or any amino acid sequence at least about 90% of identical thereto.
Preferably, the
scFv of the invention comprises a VH comprising SEQ ID NO: 11; and a VL
comprising SEQ
ID NO: 9. More preferably, the scFv of the invention comprises a VH consisting
of SEQ ID
NO: 11; and a VL consisting of SEQ ID NO: 9.
[0154] In one embodiment, the scFv of the invention comprises CDRs as defined
herein and
comprises or consists of SEQ ID NO: 12 or an amino acid sequence with at least
70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of identity thereto.
Preferably, the scFv
28
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
of the invention comprises SEQ ID NO: 12 or any amino acid sequence at least
about 95%
identical thereto. More preferably, the scFv of the invention comprises SEQ ID
NO: 12. More
preferably, the scFv of the invention consists of SEQ ID NO: 12.
[0155] In one embodiment, the scFv of the invention comprises CDRs as defined
herein and
comprises or consists of SEQ ID NO: 51 or an amino acid sequence with at least
70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of identity thereto.
Preferably, the scFv
of the invention comprises SEQ ID NO: 51 or any amino acid sequence at least
about 95%
identical thereto. More preferably, the say of the invention comprises SEQ ID
NO: 51. More
preferably, the scFv of the invention consists of SEQ ID NO: 51.
[0156] In one embodiment, the scFv of the invention is encoded by SEQ ID NO:
23 or a
nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO: 23.
[0157] In one embodiment, the scFv of the invention is encoded by SEQ ID NO:
23.
[0158] In an embodiment, the scFv of the invention further comprises a VH-VL
linker as
defined herein that links the VH and VL. Preferably, the linker is SEQ ID NO:
10.
[0159] In one embodiment, the scFv of the invention is encoded by SEQ ID NO:
76 or a
nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO: 76.
[0160] In one embodiment, the scFv of the invention is encoded by SEQ ID NO:
76.
C. Nucleic acid
[0161] An isolated nucleic acid encoding the present protein is disclosed
below.
[0162] In one embodiment, the nucleic acid encodes at least a VH or a VL of
the MUG-binding
protein of the invention. In one embodiment, the nucleic acid encodes the
variable domain (VL)
and the constant region of the light chain of the MUG-binding protein of the
invention. In one
embodiment, the nucleic acid encodes the variable domain (VH) and the constant
region of the
heavy chain of the MOG-binding protein of the invention. In one embodiment,
the nucleic acid
encodes both the heavy and light chains of the MUG-binding protein of the
invention.
[0163] In one embodiment, the nucleic acid herein comprises or consists of a
nucleotide
sequence encoding the VH of the protein of the invention, wherein said
nucleotide sequence is
SEQ ID NO: 22 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%,
95%, 96%,
97%, 98%, 99%, or more identity thereto.
29
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0164] In one embodiment, the nucleic acid herein comprises or consists of a
sequence
encoding the VL of the MOG-binding protein of the invention, wherein said
nucleotide
sequence is SEQ ID NO: 20 or a nucleotide sequence with at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or more identity thereto.
[0165] In one embodiment, the nucleic acid herein comprises nucleotide
sequences encoding
the VH and VL of the MOG-binding protein of the invention. In a further
embodiment, the
nucleic acid herein comprises SEQ ID NO s: 22 and 20.
[0166] In one embodiment, the nucleic acid herein further comprises a linker
nucleotide
sequence between the VL and VH coding sequences. In a further embodiment, the
linker
nucleotide sequence comprises or consists of SEQ ID NO: 21.
[0167] In one embodiment, the nucleic acid herein comprises or consists of SEQ
ID NO: 23 or
a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%,
or more identity thereto.
[0168] In one embodiment, the nucleic acid herein comprises or consists of SEQ
ID NO: 76 or
a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%,
or more identity thereto.
D. Production of proteins of the invention
[0169] The present invention also provides a vector for expression the MOG-
binding protein
of the invention and a method of using the vector for producing the protein.
[0170] In general, a suitable vector contains an origin of replication
functional in at least one
host organism, a promoter sequence, convenient restriction endonuclease sites,
one or more
selectable markers, and optionally an enhancer.
[0171] Examples of promoters and enhancers used in an expression vector for
mammalian cells
include, but are not limited to, early promoter and enhancer of SV40, LTR
promoter, and
enhancer of Moloney mouse leukemia virus, and the promoter and enhancer of
immunoglobulin H chain. See also below for additional examples of
transcriptional regulatory
sequences.
[0172] The present invention further provides a method of producing and
purifying the MOG-
binding protein of the invention as described herein. In one embodiment, the
method
comprises:
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
- introducing in vitro or ex vivo an expression vector comprising an
expression cassette
for the protein into a competent host cell (e.g., mammalian cells such as CHO
cells and
NSO cells);
- culturing in vitro or ex vivo the transformed host cells under conditions
suitable for
expression of the protein;
- optionally selecting the cells which express and/or secrete said protein;
and
- recovering the expressed protein from the cell culture, and
- optionally purifying the recovered protein.
[0173] Methods to purify a protein, in particular an antibody or antigen-
binding fragment (e.g,
an scFv), are well-known in the art and include, without limitation, protein A-
Sepharose, gel
electrophoresis, and chromatography (e.g., affinity chromatography such as
affinity
chromatography on protein L agarose).
II. Chimeric antigen receptor (CAR)
[0174] Proteins of the invention of particular interest are suitable for use
in a CAR. When used
in a CAR expressed by a regulatory immune cell, the protein of the invention
is expressed at
the cell surface.
[0175] Accordingly, the present invention further relates to a CAR comprising
a protein of the
invention. The CAR comprises an extracellular binding domain comprising a MOG-
binding
protein of the invention as described anywhere herein, e.g., an anti-MOG scFv,
sdAb or
DARPin as described herein.
[0176] A protein of the invention suitable for use in a CAR of the invention
comprises the
complementary-determining regions (HCDRs) 1-3 of the heavy chain comprising
SEQ ID
NOs: 3-5, respectively; or any CDR having an amino acid sequence that shares
at least about
90% of identity with one of SEQ ID NOs: 3-5. Preferably, the protein of the
invention for use
in a CAR of the invention further comprises the complementary-determining
regions (LCDRs)
1-3 of the light chain comprising SEQ ID NOs: 6-8, respectively; or any CDR
having an amino
acid sequence that shares at least about 90% of identity with one of SEQ ID
NOs: 6-8.
[0177] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
for use in a CAR of the invention, wherein the protein comprises a heavy chain
variable domain
(VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and
CDR3-VH; where the heavy chain variable domain comprises at least one,
preferably at least
two, more preferably all three, of the following heavy chain CDRs:
31
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: R1VPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5).
[0178] In one embodiment, the present invention provides a MOG-binding protein
(e.g., scFv)
for use in a CAR of the invention, wherein the protein comprises a heavy chain
variable domain
(VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and
CDR3-VH; where the heavy chain variable domain comprises all three of the
following heavy
chain CDR s:
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: R1VPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5).
[0179] In an embodiment, the extracellular binding domain of the CAR of the
invention
comprises a target binding domain comprising an antigen binding domain such as
an scFv,
sdAb, or DARPin.
A. Importantly, the protein of the invention suitable for use in a CAR of the
invention is
stable and has low immunogenicity. Preferably, the protein of the invention
suitable for
use in a CAR of the invention does not have any unintended secondary effects.
CAR
[0180] In one aspect of the invention, a CAR specific for MOG is provided. The
CAR may
comprise (i) an extracellular binding domain comprising a MOG-binding protein
of the
invention as described anywhere herein, e.g., an scFv as described herein,
(ii) optionally an
extracellular hinge domain, (iii) a transmembrane domain, (iv) an
intracellular signaling
domain, and (v) optionally a tag and/or a leader sequence. In one embodiment,
the CAR
comprises one or more polypeptides, such as, for example, two polypeptides.
[0181] The present invention explicitly envisages any and all combinations of
(i) extracellular
binding domain (ii) transmembrane domain, and (iii) intracellular signaling
domain as
disclosed herein.
[0182] The present invention explicitly envisages any and all combinations of
(i) extracellular
binding domain (ii) extracellular hinge domain, (iii) transmembrane domain,
(iv) intracellular
signaling domain, and (v) tag and/or a leader sequence as disclosed herein.
32
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
1. Extracelhdar binding domain
[0183] In one embodiment, the extracellular binding domain of the CAR
comprises a MOG-
binding protein of the invention, e.g., an scFv, sdAb, or DARPin of the
invention, as described
anywhere herein. In one embodiment, the extracellular binding domain of the
CAR comprises
a MOG-binding scFv of the invention, as described anywhere herein.
[0184] In one embodiment, the extracellular binding domain of the CAR consists
of a MOG-
binding protein of the invention, e.g., an scFv, sdAb, or DARPin of the
invention, as described
anywhere herein. In one embodiment, the extracellular binding domain of the
CAR consists of
a MOG-binding scFv of the invention, as described anywhere herein.
[0185] In one embodiment, the extracellular binding domain of the CAR
comprises a heavy
chain variable domain (VH) and a light chain variable domain (VL) each
comprising 3
complementarity-determining regions (LCDRs) where at least one of the heavy
chain HCDRs
1-3 has SEQ ID NOs: 3-5, respectively; and/or at least one of light chain
LCDRs 1-3 has SEQ
TD NOs. 6-8, respectively.
[0186] In a further embodiment, the extracellular binding domain of the CAR
comprises heavy
chain HCDRs 1-3 and light chain LCDRs 1-3 having the sequences of SEQ ID NOs:
3-8,
respectively. In one embodiment, the extracellular binding domain of the CAR
comprises a VH
having the sequence of SEQ ID NO: 11, or a sequence having at least about 70%,
preferably
at least about 75%, 80%, 85%, 90%, 95%, or more identity to SEQ ID NO: 11, and
a VL having
the sequence of SEQ ID NO: 9, or a sequence having at least about 70%,
preferably at least
about 75%, 80%, 85%, 90%, 95%, or more identity to SEQ ID NO: 9.
[0187] In one embodiment, the extracellular binding domain of the CAR
comprises an anti-
MOG scFv with a peptide linker between the VH and VL, wherein the peptide
linker comprises
SEQ ID NO: 10 or a sequence having at least about 90%, 95%, or more identity
thereto.
[0188] In one embodiment, the extracellular binding domain of the CAR consists
of an anti-
MOG scFv with a peptide linker between the VH and VL, wherein the peptide
linker comprises
SEQ ID NO: 10 or a sequence having at least about 90%, 95%, or more identity
thereto.
[0189] In one embodiment, the extracellular binding domain of the CAR
comprises an anti-
MOG scFv comprising SEQ ID NO: 12 or a or sequence having at least about 90%,
95%, or
more identity thereto.
[0190] In one embodiment, the extracellular binding domain of the CAR consists
of an anti-
MOG scFv comprising SEQ ID NO: 12 or a or sequence having at least about 90%,
95%, or
more identity thereto.
33
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0191] In one embodiment, the extracellular binding domain of the CAR
comprises an anti-
MOG scFy comprising SEQ ID NO: 51 or a or sequence having at least about 90%,
95%, or
more identity thereto.
[0192] In one embodiment, the extracellular binding domain of the CAR consists
of an anti-
MOG scFv comprising SEQ ID NO: 51 or a or sequence having at least about 90%,
95%, or
more identity thereto.
2. Hinge domain
[0193] In one embodiment, the extracellular MOG-binding domain is connected to
a
transmembrane domain by a hinge domain.
[0194] In one embodiment, the hinge domain is a peptide having a length of
about 2 to about
100 amino acids.
101951 In one embodiment, the hinge domain is a peptide having a length in the
range of from
about 2 to about 75 amino acids_
[0196] In one embodiment, the hinge domain is a peptide having a length in the
range of from
about 2 to about 20 amino acids.
[0197] In one embodiment, the hinge domain is a peptide having a length in the
range of from
about 2 to about 15 amino acids.
[0198] In one embodiment, the hinge domain comprises an amino acid sequence
derived from
a CD8 hinge (e.g., SEQ ID NO: 13) or an amino acid sequence with at least
about 95% (e.g.,
about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.
[0199] In one embodiment, the hinge domain consists of an amino acid sequence
derived from
a CD8 hinge (e.g., SEQ ID NO: 13) or an amino acid sequence with at least
about 95% (e.g.,
about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.
[0200] In one embodiment, the hinge domain comprises an amino acid sequence
having SEQ
ID NO: 13.
[0201] In one embodiment, the hinge domain consists of an amino acid sequence
having SEQ
ID NO: 13.
[0202] In one embodiment, the hinge domain is a CD8 hinge encoded by SEQ ID
NO: 24 or a
nucleotide sequence with at least about 95% (e.g, about 96%, 97%, 98%, or 99%)
identity to
SEQ ID NO: 24.
[0203] The present invention explicitly envisages any and all combinations of
a hinge domain
described anywhere herein with an extracellular binding domain described
anywhere herein.
34
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
3. Transmembrane domain
[0204] Examples of transmembrane domains that may be used in the present CAR
include, but
are not limited to, transmembrane domains of an alpha or beta chain of a T
cell receptor (TCR);
or of CD28, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8,
CD9,
CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40,
CD2, CD27, LFA-1 (CD! la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40,
BAFFR,
HVEM (LIGHTR), SLAM_F7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma,
IL7R
a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld,
ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 lb, PD1, ITGAX, CD11c, ITGB1,
CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4),
CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIO0
(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME
(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and
NKG2C
[0205] In one embodiment, the transmembrane domain comprises an amino acid
sequence
derived from a CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 14.
[0206] In one embodiment, the transmembrane domain consists of an amino acid
sequence
derived from a CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 14.
[0207] In one embodiment, the transmembrane domain comprises an amino acid
sequence
haying SEQ ID NO: 14.
[0208] In one embodiment, the transmembrane domain consists of an amino acid
sequence
having SEQ ID NO: 14.
[0209] In one embodiment, the transmembrane domain is a CD8 transmembrane
domain
encoded by SEQ ID NO: 25 or a nucleotide sequence with at least about 95%
(e.g., about 96%,
97%, 98% or 99%) identity to SEQ ID NO: 25.
[0210] In another embodiment, the transmembrane domain comprises or consists
of an amino
acid sequence derived from a CD28 transmembrane domain (e.g., SEQ ID NO: 29)
or an amino
acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to SEQ ID
NO: 29. In one embodiment, the transmembrane domain is a CD28 transmembrane
domain
encoded by SEQ ID NO: 30 or a nucleotide sequence with at least about 95%
(e.g., about 96%,
97%, 98% or 99%) identity to SEQ ID NO: 30.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0211] In another embodiment, the transmembrane domain comprises or consists
of an amino
acid sequence derived from a 4-1BB (CD137) transmembrane domain (e.g., SEQ ID
NO: 31)
or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98%
or 99%) identity
to SEQ ID NO: 31. In one embodiment, the transmembrane domain is a 4-1BB
transmembrane
domain encoded by SEQ ID NO: 32 or a nucleotide sequence with at least about
95 (e.g., 96%,
97%, 98% or 99%) identity to SEQ 113 NO: 32.
[0212] In another embodiment, the transmembrane domain comprises or consists
of an amino
acid sequence derived from a TNFR2 transmembrane domain (e.g., SEQ ID NO. 33)
or an
amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO. 33. In one embodiment, the transmembrane domain is a TNFR2
transmembrane
domain encoded by SEQ ID NO: 34 or a nucleotide sequence with at least about
95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 34.
[0213] In one embodiment, the transmembrane domain may be completely
artificial and may
comprise, for example, predominantly hydrophobic amino acids such as valine
and leucine.
[0214] The present invention explicitly envisages any and all combinations of
a
transmembrane domain described anywhere herein with an extracellular binding
domain
described anywhere herein.
4. Intracellular signaling domain
[0215] In one embodiment, the intracellular signaling domain of the present
CAR may
comprise the entire intracellular portion, or the entire native intracellular
signaling domain, of
the molecule from which it is derived, or a functional fragment or derivative
thereof.
[0216] In one embodiment, the intracellular signaling domain comprises a T
cell primary
signaling domain.
[0217] In one embodiment, the intracellular signaling domain comprises one or
more T cell
costimulatory domains.
[0218] In one embodiment, the intracellular signaling domain comprises at
least one T cell
costimulatory domain and a T cell primary signaling domain.
[0219] In one embodiment, the intracellular signaling domain consists of at
least one T cell
costimulatory domain and a T cell primary signaling domain.
[0220] In another embodiment, the intracellular signaling domain comprises two
T cell
costimulatory domains and a T cell primary signaling domain.
36
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0221] In another embodiment, the intracellular signaling domain consists of
two T cell
costimulatory domains and a T cell primary signaling domain.
[0222] In one embodiment, the T cell primary signaling domain comprises a
functional
signaling domain of CD3 zeta.
[0223] In one embodiment, the T cell primary signaling domain comprises the
amino acid
sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16, or an amino
acid sequence
with at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO:
16.
[0224] In one embodiment, the T cell primary signaling domain consists of the
amino acid
sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16, or an amino
acid sequence
with at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO:
16.
[0225] In one embodiment, the T cell primary signaling domain comprises the
amino acid
sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16.
[0226] In one embodiment, the T cell primary signaling domain consists of the
amino acid
sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16.
[0227] In one embodiment, the CD3 zeta primary signaling domain comprises an
amino acid
sequence having at least one, two, or three modifications ¨ but not more than
20, 10 or 5
modifications ¨ of SEQ ID NO: 16.
[0228] In one embodiment, the CD3 zeta primary signaling domain consists of an
amino acid
sequence having at least one, two, or three modifications ¨ but not more than
20, 10 or 5
modifications ¨ of SEQ ID NO: 16.
[0229] In one embodiment, the CD3 zeta primary signaling domain is encoded by
SEQ ID NO:
27 or a nucleotide sequence with at least about 95 (e.g., about 96%, 97%, 98%
or 99%) identity
to SEQ ID NO: 27.
[0230] T cell primary signaling domains that act in a stimulatory manner may
comprise
signaling motifs known as immunoreceptor tyrosine-based activation motifs
(ITAMS). In one
embodiment, the T cell primary signaling domain comprises a modified ITAM
domain, e.g., a
mutated ITAM domain which has altered (e.g., increased or decreased) activity
as compared
to the native ITAM domain. In one embodiment, a primary signaling domain
comprises a
modified ITAM-containing primary intracellular signaling domain, e.g., an
optimized and/or
truncated ITAM-containing primary intracellular signaling domain. In an
embodiment, a
primary signaling domain comprises one, two, three, four, or more ITAM motifs.
37
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0231] In one embodiment, the intracellular signaling domain of the present
CAR comprises a
T cell primary signaling domain (e.g., a CD3 zeta signaling domain) combined
with one or
more costimulatory signaling domains.
[0232] The costimulatory signaling domains may be derived from the
intracellular domains of
T cell costimulatory molecules or other cell surface molecules expressed on
immune cells.
Examples of costimulatory signal domains may be those derived from the
intracellular domains
of CD28, CD27, 4-1BB (CD137), an MHC class I molecule, BTLA, a Toll ligand
receptor,
0X40, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen-1
(LFA-
1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,
CDS,
ICAM-1, GITR, ARHR, BAFFR, HVEM (LIGHTR), SLA1V1F7, NKp80 (KLRF1), NKp44,
NKp30, NKp46, CD160 (BY55), CD19, CD19a, CD4, CD8alpha, CD8beta, IL2ra, IL6Ra,
IL2R beta, IL2R gamma, IL7R alpha, IL-13RA1/RA2, IL-33R(IL1RL1), IL-10RA/RB,
IL-4R,
IL-5R (CSF2RB), IL-21R, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,
CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 a/CD18, ITGAM, CD1 lb, ITGAX,
CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, CTLA-4 (CD152), CD95,
TNFR1 (CD120a/TNFRSF I A), TNFR2 (CD120b/TNFRSF IB),
TGFbR1/2/3,
TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),
CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAIV1F6 (NTB-A,
Ly108), SLAM (SLA_MF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR,
LAT, GADS, SLP-76, PAG/Cbp, common gamma chain, a ligand that specifically
binds with
CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.
[0233] In one embodiment of the present invention, the present CAR comprises
at least one
intracellular domain of a T cell costimulatory molecule selected from the
group comprising
CD28, TNFR2, 4-1BB, ICOS, CD27, 0X40, CTLA4, and PD-1.
[0234] In one embodiment, the T cell costimulatory signaling domain comprises
an amino acid
sequence derived from a CD28 intracellular domain (e.g., SEQ ID NO: 15) or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
15.
[0235] In one embodiment, the T cell costimulatory signaling domain consists
of an amino
acid sequence derived from a CD28 intracellular domain (e.g., SEQ ID NO: 15)
or an amino
acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to SEQ ID
NO: 15.
38
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0236] In one embodiment, the T cell costimulatory signaling domain comprises
an amino acid
sequence having at least one, two or three modifications¨but not more than 20,
10 or 5
modifications¨of an amino acid sequence of SEQ ID NO: 15.
[0237] In one embodiment, the T cell costimulatory signaling domain consists
of an amino
acid sequence having at least one, two or three modifications¨but not more
than 20, 10 or 5
modifications¨of an amino acid sequence of SEQ 113 NO: 15.
[0238] In one embodiment, the T cell costimulatory signaling domain comprises
an amino acid
having SEQ ID NO: 15.
[0239] In one embodiment, the T cell costimulatory signaling domain consists
of an amino
acid having SEQ ID NO: 15.
[0240] In one embodiment, the T cell costimulatory signaling domain is encoded
by SEQ ID
NO: 26 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%,
98% or 99%)
identity to SEQ ID NO: 26.
[0241] In one embodiment, the T cell costimulatory signaling domain comprises
or consists of
an amino acid sequence derived from a 4-1BB intracellular domain (e.g., SEQ ID
NO: 35) or
an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or
99%) identity
to SEQ ID NO: 35. In one embodiment, the T cell costimulatory signaling domain
comprises
or consists of an amino acid sequence having at least one, two or three
modifications ¨ but not
more than 20, 10 or 5 modifications ¨ of an amino acid sequence of SEQ ID NO:
35. In one
embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:
36 or a
nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO: 36.
[0242] In one embodiment, the T cell costimulatory signaling domain comprises
or consists of
an amino acid sequence derived from a CD27 intracellular domain (e.g., SEQ ID
NO: 37) or
an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or
99%) identity
to SEQ ID NO: 37. In one embodiment, the T cell costimulatory signaling domain
comprises
or consists of an amino acid sequence having at least one, two or three
modifications ¨ but not
more than 20, 10 or 5 modifications ¨ of an amino acid sequence of SEQ ID NO:
37. In one
embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:
38 or a
nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO: 38.
[0243] In one embodiment, the T cell costimulatory signaling domain comprises
or consists of
an amino acid sequence derived from a TNFR2 intracellular domain (e.g., SEQ ID
NO: 39) or
39
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or
99%) identity
to SEQ ID NO: 39. In one embodiment, the T cell costimulatory signaling domain
comprises
or consists of an amino acid sequence having at least one, two or three
modifications ¨ but not
more than 20, 10 or 5 modifications ¨ of an amino acid sequence of SEQ ID NO:
39. In one
embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:
40 or a
nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to
SEQ ID NO: 40.
[0244] In one embodiment, the intracellular signaling domain of the present
CAR comprises:
- the amino acid sequence of a CD28 intracellular domain of SEQ ID NO: 15
or an amino
acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%)
identity to SEQ ID
NO: 15; and
- the amino acid sequence of a CD3 zeta intracellular domain of SEQ ID NO:
16 or an amino
acid sequence with at least about 95% (e.g, about 96%, 97%, 98% or 99%)
identity to SEQ ID
NO: 16.
[0245] In one embodiment, the intracellular signaling domain of the present
CAR comprises
at least two different domains (e.g., a primary signaling domain and at least
one intracellular
domain of a T cell costimulatory molecule) that may be linked to each other in
a random order
or in a specified order.
[0246] Optionally, a peptide linker may be used to connect distinct signaling
domains In one
embodiment, a glycine-serine doublet (GS) is used as a suitable linker. In one
embodiment, a
single amino acid, e.g., an alanine (A) or a glycine (G), is used as a linker.
Other examples of
peptide linkers are described in Section I above.
[0247] In one embodiment, the intracellular signaling domain of the present
CAR comprises
two or more (e.g., 2, 3, 4, 5, or more) costimulatory signaling domains. In
one embodiment,
the two or more costimulatory signaling domains are separated by a peptide
linker such as those
described herein.
[0248] In one embodiment, the intracellular signaling domain of the present
CAR comprises
the primary signaling domain of CD3 zeta (e.g., SEQ ID NO: 16) and the co-
stimulatory
signaling domain of CD28 (e.g., SEQ ID NO: 15).
[0249] The present invention explicitly envisages any and all combinations of
an intracellular
signaling domain described anywhere herein with an extracellular binding
domain described
anywhere herein.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
5. Leader sequence
[0250] In one embodiment, the CAR of the present invention further comprises a
leader
sequence located N-terminal to the MOG-specific extracellular binding domain.
A leader
sequence may allow cell surface expression of the CAR protein after the
protein is secreted
from the Golgi complex. A non-limiting example of leader sequence is a leader
sequence of
CD8 that may comprise or consists of SEQ ID NO: 1. Preferably, the of leader
sequence is a
leader sequence of CD8 that consists of SEQ ID NO: 1.
[0251] In one embodiment, the nucleotide sequence encoding the leader sequence
comprises
or consists of a nucleotide sequence coding for a CD8 leader sequence (e.g.,
SEQ ID NO: 18)
or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or
99%) identity
to SEQ ID NO: 18.
[0252] The present invention explicitly envisages any and all combinations of
a leader
sequence described anywhere herein with an extracellular binding domain
described anywhere
herein
6. Tag
[0253] In one embodiment, the CAR further comprises a tag for, e.g., quality
control,
enrichment, and tracking in vivo. Said a tag may be localized at the N-
terminus or the C-
terminus of the CAR, or internally within the CAR polypeptide. Examples of
tags include, but
are not limited to, Hemagglutinin Tag, Poly Arginine Tag, Poly Hi sti dine
Tag, Myc Tag, Strep
Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin-binding peptide Tag, SBP Tag,
Chitin binding
domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7
Tag, V5
Tag, and Xpress Tag.
[0254] In one embodiment, the CAR of the present invention comprises a HA tag
(SEQ ID
NO: 2). In one embodiment the tag is encoded by SEQ ID NO: 19 or a nucleotide
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 19.
[0255] The present invention explicitly envisages any and all combinations of
a tag described
anywhere herein with an extracellular binding domain described anywhere
herein.
7. Exemplary CAR
[0256] The present invention provides a CAR comprising an extracellular domain
comprising
a MOG-binding protein according to the invention (e.g., an scFv according to
the invention), a
41
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
transmembrane domain, and a cytoplasmic domain comprising an intracellular
signaling
domain.
[0257] In an aspect, the invention provides a CAR comprising a MOG-binding
domain (e.g.,
a domain comprising or consisting of SEQ ID NO: 12), optionally an
extracellular hinge
domain, a transmembrane domain, a single intracellular domain of a T cell
costimulatory
molecule and a T cell primary signaling domain.
[0258] In an embodiment, the intracellular signaling domain comprises a human
CD28
costimulatory signaling domain, optionally comprising SEQ ID NO: 15 or an
amino acid
sequence at least about 90% identical thereto; and/or a human CD3 zeta domain,
optionally
comprising SEQ ID NO: 16 or an amino acid sequence at least about 90%
identical thereto.
[0259] In an embodiment, the transmembrane domain is derived from human CD8,
optionally
comprising SEQ ID NO: 14 or an amino acid sequence at least about 90%
identical thereto.
[0260] In one embodiment, the CAR of the invention comprises a MOG-binding
domain (e.g.,
SEQ ID NO: 12); a transmembrane domain of CD8 (e.g., SEQ ID NO: 14); an
intracellular
domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain
(e.g., SEQ
ID NO: 16).
[0261] In one embodiment, the CAR of the invention comprises a MOG-binding
domain (e.g.,
SEQ ID NO: 12); a hinge domain of CD8 (e.g., SEQ ID NO: 13); a transmembrane
domain of
CD8 (e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g., SEQ ID NO:
15); and a
CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).
[0262] In one embodiment, the CAR of the invention comprises an anti-MOG scFy
(e.g., an
scFy comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 56 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
56. Preferably, said CAR comprises SEQ ID NO: 56.
[0263] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFy comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 56 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
56. Preferably, said CAR consists of SEQ ID NO: 56.
42
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0264] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 57 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
57. Preferably, said CAR comprises SEQ Ill NO: 57.
[0265] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 57 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
57. Preferably, said CAR consists of SEQ ID NO: 57.
[0266] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 58 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
58. Preferably, said CAR comprises SEQ ID NO: 58.
[0267] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 58 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
58. Preferably, said CAR consists of SEQ ID NO: 58.
[0268] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 59 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
59. Preferably, said CAR comprises SEQ ID NO: 59.
[0269] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
43
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 59 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
59. Preferably, said CAR consists of SEQ ID NO: 59.
[0270] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 60 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
60. Preferably, said CAR comprises SEQ ID NO: 60.
[0271] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 60 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
60. Preferably, said CAR consists of SEQ ID NO: 60.
[0272] In an embodiment, the CAR comprises:
(i) an anti-MOG scFv, optionally comprising SEQ ID NO: 12,
(ii) a hinge domain derived from human CD8, optionally comprising SEQ ID
NO:
13,
(iii) a transmembrane domain derived from human CD8, optionally comprising SEQ
ID NO: 14,
(iv) an intracellular signaling domain comprising a human CD28
costimulatory
signaling domain, optionally comprising SEQ ID NO: 15, and a human CD3 zeta
domain, optionally comprising SEQ ID NO: 16, and
(v) optionally a tag and/or a leader sequence.
[0273] In another aspect, the invention provides a CAR comprising a MOG-
binding domain
(e.g, a domain comprising or consisting of SEQ ID NO: 51), optionally an
extracellular hinge
domain, a transmembrane domain, a single intracellular domain of a T cell
costimulatory
molecule and a T cell primary signaling domain.
[0274] In an embodiment, the intracellular signaling domain comprises a human
CD28
costimulatory signaling domain, optionally comprising SEQ ID NO: 15 or an
amino acid
44
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
sequence at least about 90% identical thereto; and/or a human CD3 zeta domain,
optionally
comprising SEQ ID NO: 16 or an amino acid sequence at least about 90%
identical thereto.
[0275] In an embodiment, the transmembrane domain is derived from human CD8,
optionally
comprising SEQ ID NO: 14 or an amino acid sequence at least about 90%
identical thereto.
[0276] In one embodiment, the CAR of the invention comprises a MOG-binding
domain (e.g.,
SEQ Ill NO: 51); a transmembrane domain of CD8 (e.g., SEQ ID NO: 14); an
intracellular
domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain
(e.g., SEQ
ID NO: 16).
[0277] In one embodiment, the CAR of the invention comprises a MOG-binding
domain (e.g.,
SEQ ID NO: 51); a hinge domain of CD8
SEQ ID NO: 13); a transmembrane domain of
CD8 (e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g., SEQ ID NO:
15); and a
CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).
[0278] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 17 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
17. Preferably, said CAR comprises SEQ ID NO: 17.
[0279] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 17 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
17. Preferably, said CAR consists of SEQ ID NO:17.
[0280] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 52 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
52. Preferably, said CAR comprises SEQ ID NO: 52.
[0281] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 52 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
52. Preferably, said CAR consists of SEQ ID NO: 52.
[0282] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 53 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
53. Preferably, said CAR comprises SEQ ID NO: 53.
[0283] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 53 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
53. Preferably, said CAR consists of SEQ ID NO: 53.
[0284] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 54 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
54. Preferably, said CAR comprises SEQ ID NO: 54.
[0285] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 54 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
54. Preferably, said CAR consists of SEQ ID NO: 54.
[0286] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 55 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
55. Preferably, said CAR comprises SEQ ID NO: 55.
46
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0287] In one embodiment, the CAR of the invention comprises an anti-MOG scFv
(e.g., an
scFy comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a
transmembrane
domain of human CD8, an intracellular domain of human CD28 and an
intracellular domain of
human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 55 or an
amino acid
sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity
to SEQ ID NO:
55. Preferably, said CAR consists of SEQ ID NO: 55.
[0288] In an embodiment, the CAR comprises:
(i) an anti-MOG say, optionally comprising SEQ ID NO: 51,
(ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO:
13,
(iii)a transmembrane domain derived from human CD8, optionally comprising SEQ
ID
NO: 14,
(iv)an intracellular signaling domain comprising a human CD28 costimulatory
signaling domain, optionally comprising SEQ ID NO: 15, and a human CD3 zeta
domain, optionally comprising SEQ ID NO: 16, and
(v) optionally a tag and/or a leader sequence.
8. Mouse CARs
[0289] In an aspect, the invention provides a mouse CARs that comprise a MOG-
binding
domain (e.g., a domain comprising or consisting of SEQ ID NO: 12), optionally
an extracellular
hinge domain, a transmembrane domain, a single intracellular domain of a T
cell costimulatory
molecule and a T cell primary signaling domain.
[0290] In one embodiment, the mouse CAR of the present invention comprises a
MOG-binding
domain (e.g., SEQ ID NO: 12); a transmembrane domain of mouse CD8 (e.g., SEQ
ID NO:
42); an intracellular domain of mouse CD28 (e.g., SEQ ID NO: 43); and a mouse
CD3 zeta
primary signaling domain (e.g., SEQ ID NO: 44). In certain embodiments, the
mouse CAR
may also comprise a hinge domain of mouse CD8 (e.g., SEQ ID NO: 41). A mouse
CAR with
any combination of the above domains is contemplated.
[0291] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFy
(e.g., an scFy comprising or consisting of SEQ ID NO: 12), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 65 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 65. Preferably, said CAR comprises SEQ ID NO: 65.
47
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0292] In one embodiment, said CAR consists of SEQ ID NO: 65 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 65.
Preferably, said CAR consists of SEQ ID NO: 65.
[0293] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 66 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 66. Preferably, said CAR comprises SEQ ID NO: 66.
[0294] In one embodiment, said CAR consists of SEQ ID NO: 66 or an amino acid
sequence
with at least about 95% (e.g-., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 66.
Preferably, said CAR consists of SEQ ID NO: 66.
[0295] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 67 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 67. Preferably, said CAR comprises SEQ ID NO: 67.
[0296] In one embodiment, said CAR consists of SEQ ID NO: 67 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 67.
Preferably, said CAR consists of SEQ ID NO: 67.
[0297] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g, an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 68 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 68. Preferably, said CAR comprises SEQ ID NO: 68.
[0298] In one embodiment, said CAR consists of SEQ ID NO: 68 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 68.
Preferably, said CAR consists of SEQ ID NO: 68.
[0299] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
48
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 69 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 69. Preferably, said CAR comprises SEQ ID NO: 69.
[0300] In one embodiment, said CAR consists of SEQ ID NO: 69 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 69.
Preferably, said CAR consists of SEQ ID NO: 69.
[0301] In another aspect, the invention provides a mouse CARs that comprise a
MOG-binding
domain (e.g., a domain comprising or consisting of SEQ ID NO: 51), optionally
an extracellular
hinge domain, a transmembrane domain, a single intracellular domain of a T
cell costimulatory
molecule and a T cell primary signaling domain.
[0302] In one embodiment, the mouse CAR of the present invention comprises a
MOG-binding
domain (e.g., SEQ ID NO: 51); a transmembrane domain of mouse CD8 (e.g., SEQ
ID NO:
42); an intracellular domain of mouse CD28 (e.g., SEQ ID NO: 43); and a mouse
CD3 zeta
primary signaling domain (e.g., SEQ ID NO: 44). In certain embodiments, the
mouse CAR
may also comprise a hinge domain of mouse CD8 (e.g., SEQ ID NO: 41). A mouse
CAR with
any combination of the above domains is contemplated.
[0303] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFy
(e.g., an scFy comprising or consisting of SEQ ID NO: 51), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO:45 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%,
98% or 99%)
identity to SEQ ID NO: 45. Preferably, said CAR comprises SEQ ID NO:45.
[0304] In one embodiment, said CAR consists of SEQ ID NO: 45 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 45.
Preferably, said CAR consists of SEQ ID NO:45.
[0305] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFy
(e.g., an scFy comprising or consisting of SEQ ID NO: 51), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 61 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 61. Preferably, said CAR comprises SEQ ID NO: 61.
49
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0306] In one embodiment, said CAR consists of SEQ ID NO: 61 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 61.
Preferably, said CAR consists of SEQ ID NO: 61.
[0307] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g., an scFv comprising or consisting of SEQ ID NO: 51), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 62 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 62. Preferably, said CAR comprises SEQ ID NO: 62.
[0308] In one embodiment, said CAR consists of SEQ ID NO: 62 or an amino acid
sequence
with at least about 95% (e.g-., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 62.
Preferably, said CAR consists of SEQ ID NO: 62.
[0309] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g., an scFv comprising or consisting of SEQ ID NO: 51), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 63 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 63. Preferably, said CAR comprises SEQ ID NO: 63.
[0310] In one embodiment, said CAR consists of SEQ ID NO: 63 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 63.
Preferably, said CAR consists of SEQ ID NO: 63.
[0311] In one embodiment, the mouse CAR of the invention comprises an anti-MOG
scFv
(e.g, an scFv comprising or consisting of SEQ ID NO: 51), a hinge region of
CD8, a
transmembrane domain of mouse CD8, an intracellular domain of mouse CD28 and
an
intracellular domain of mouse CD3 zeta. In one embodiment, said CAR comprises
SEQ ID
NO: 64 or an amino acid sequence with at least about 95% (e.g., about 96%,
97%, 98% or
99%) identity to SEQ ID NO: 64. Preferably, said CAR comprises SEQ ID NO: 64.
[0312] In one embodiment, said CAR consists of SEQ ID NO: 64 or an amino acid
sequence
with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID
NO: 64.
Preferably, said CAR consists of SEQ ID NO: 64.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
B. Nucleic acid encoding a CAR
[0313] The present invention also relates to a nucleic acid sequence encoding
a CAR as
described herein. An example of such a nucleic acid sequence is SEQ ID NO: 28
or a
degenerate or codon-optimized version thereof
[0314] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 28 or
a degenerate or codon-optimized version thereof. In one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 28 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 28.
[0315] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 77 or
a degenerate or codon-optimized version thereof. In one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 77 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 77.
[0316] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 78 or
a degenerate or codon-optimized version thereof. Tn one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 78 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 78.
[0317] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 79 or
a degenerate or codon-optimized version thereof. In one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 79 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 79.
[0318] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 80 or
a degenerate or codon-optimized version thereof. In one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 80 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 80.
[0319] In one embodiment, the human CAR of the invention is encoded by SEQ ID
NO: 81 or
a degenerate or codon-optimized version thereof. In one embodiment, the human
CAR of the
invention is encoded by SEQ ID NO: 81 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 81.
[0320] In one embodiment, the mouse CAR of the invention is encoded by SEQ ID
NO: 50 or
a degenerate or codon-optimized version thereof. In one embodiment, the mouse
CAR of the
invention is encoded by SEQ ID NO: 50 or a nucleotide sequence with at least
about 95% (e.g.,
about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 50.
51
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
C. Vector for expressing a CAR
[0321] The present invention further provides an expression vector comprising
a nucleic acid
encoding the CAR herein.
[0322] In one embodiment, the nucleic acid encoding the CAR is a DNA. In one
embodiment,
the nucleic acid encoding the CAR is an RNA. Examples of vectors that may be
used in the
present invention include, but are not limited to, a DNA vector, an RNA
vector, a plasmid, an
episome, a viral vector (e.g., an animal virus).
[0323] In one embodiment, the expression vector may comprise regulatory
elements, such as
a promoter, an enhancer, and a transcription terminator, to cause or direct
expression of the
transgene (e.g., CAR) thereon in host cells. The vector may also comprise one
or more
selectable markers.
[0324] Examples of promoters and enhancers used in the expression vector for
animal cell
include, but are not limited to, early promoter and enhancer of SV40, LTR
promoter and
enhancer of Moloney mouse leukemia virus, promoter, and enhancer of
immunoglobulin H
chain and the like. Other examples of suitable constitutive promoters include,
but are not
limited to, the immediate early cytomegalovirus (CMV) promoter sequence,
elongation factor
la (EF-1a) promoter, phosphoglycerate kinase (PGK) promoter, FOXP3 derived
promoter,
simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV)
promoter,
human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV
promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate
early promoter,
a Rous sarcoma virus promoter, as well as human gene promoters such as the
actin promoter,
the myosin promoter, the hemoglobin promoter, and the creatine kinase
promoter.
[0325] Examples of suitable inducible promoters include, but are not limited
to, a
m etall othi onin e promoter, a glucocorticoid promoter, a progesterone
promoter, a cum ate
promoter and a tetracycline promoter.
[0326] Examples of suitable bi-directional promoters include, but are not
limited to, the
promoters described by Luigi Naldini U.S. Pat. 8,501,464, incorporated herein
by reference,
disclosing a bi-directional promoter comprising i) a first minimal promoter
sequence derived
from cytomegalovirus (CMV) or mouse mammary tumor virus (MMTV) genomes and ii)
a
full efficient promoter sequence derived from an animal gene.
10327] Examples of suitable vectors include, but are not limited to, pAGE107,
pAGE103,
pHSG274, pKCR, pSG1 beta d2-4, and the like.
52
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0328] Examples of plasmids include, but are not limited to, replicating
plasmids comprising
an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR,
and the like.
[0329] A number of viral-based systems have been developed for gene transfer
into
mammalian cells. Examples of viral vectors include, but are not limited to
adenoviral vectors,
retroviral vectors, lentiviral vectors, herpes virus vectors and adeno-
associated viral (AAV)
vectors.
[0330] Retroviruses may provide a convenient platform for gene delivery
systems. A selected
gene can be inserted into a vector and packaged in retroviral particles using
techniques known
in the art. The recombinant virus can then be isolated and delivered to cells
of the subject either
lii 3)13)0 or ex i o. A number of retroviral systems are known in the art.
[0331] In some embodiments, adenovirus vectors are used. A number of
adenovirus vectors
are known in the art.
[0332] In one embodiment, lentivirus vectors are used.
[0333] In one embodiment, AAV vectors are used. As used herein, the term "AAV"
covers all
serotypes and variants, both naturally occurring and engineered forms. For
example, the term
encompasses AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV
type
4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV
type 8 (AAV-8), and AAV type 9 (AAV-9). In one embodiment, the vector is an
AAV6 vector.
In one embodiment, the AAV is a pseudotype AAV, such as an AAV having an AAV6
capsid
and a recombinant genome derived from another AAV serotype (e.g., having ITRs
from
AAV2).
[0334] The recombinant viruses may be produced by techniques known in the art,
such as by
transfecting packaging cells or by transient transfection with helper plasmids
or viruses.
Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells,
GPenv+ cells,
293 cells, 293T cells etc. Detailed protocols for producing such replication-
defective
recombinant viruses may be found in the art. Insect cells may also be used to
produce
recombinant viruses such as recombinant AAV.
III. Cell expressing a CAR
A. Regulatory immune cells
[0335] The present invention further relates to a regulatory immune cell and
to a regulatory
immune cell population engineered to express on the cell surface a CAR as
described herein.
53
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0336] The present invention provides a regulatory immune cell expressing the
CAR according
to the invention, or comprising the nucleic acid molecule according to the
invention or the
vector according to the invention.
[0337] In one embodiment, the regulatory immune cell is a T cell, such as, a
regulatory T cell
(Treg), a CD8+ T cell, a CD4+ T cell, or a NK T cell. In one embodiment, the
regulatory immune
cell is a CD4+CD25+CD1271' T reg. Foxp3 and Helios are transcription factors
expressed by
Treg cells and indicate maintenance of the desired phenotype of the cells.
Accordingly,
preferably, the Treg expresses high levels of Foxp3 and/or IIelios (see, for
example, Figure 4).
In an embodiment, the Treg expresses high levels of Foxp3. In an embodiment,
the Treg
expresses high levels of Helios.
[0338] The present invention also provides an isolated human T cell, wherein
the T cell
comprises a nucleic acid molecule according to the invention or the vector of
the invention. In
one embodiment, the regulatory immune cell is a T cell, such as, a regulatory
T cell (Treg), a
CD8+ T cell, a CD4+ T cell, or a NK T cell. In an embodiment, the isolated
human T cell is a
CD4+CD25+CD1271' T reg. Preferably, the Treg expresses high levels of Foxp3
and/or Helios.
In an embodiment, the Treg expresses high levels of Foxp3. In an embodiment,
the Treg
expresses high levels of Helios.
[0339] The present invention also relates to an isolated and/or substantially
purified regulatory
immune cell population, preferably a T cell population, comprising or
consisting of regulatory
immune cells engineered to express on the cell surface a CAR as described
herein.
[0340] The regulatory immune cells expressing the CAR of the invention are
directed to cells
in the central nervous system expressing MUG on their surface. The regulatory
immune cells
localize and bind to MUG via the CAR and then are activated. This process
allows the immune
cells to suppress autoimmune activity and inflammation causing demyelination,
thereby
treating the autoimmune or inflammatory disease. In this sense, the regulatory
immune cells
can be considered to provide a protective shield for the MUG expressing cells
against immune
attacks. The regulatory immune cells are also capable of improving
remyelination of neural
lesions.
[0341] Advantageously, in addition to demonstrating good activation (high
signal to
background ratio) and good suppressive activity, the regulatory immune cells
expressing the
CAR of the invention have been found to have a low tonic signaling. The term
"tonic signaling"
as used herein refers to an antigen-independent background of activation.
Methods for
measuring tonic signaling are well known to the person skilled in the art, and
include, without
54
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
limitation, measuring metabolic activity of the CAR-expressing cells,
measuring one or more
indicators of cell activation in the absence of stimulation by an antigen
recognized by the
receptor, measuring one or more phenotypical changes related to cell aging or
cell senescence,
determining cell cycle progression in the absence of antigenic stimulation;
and measuring the
size of cells expressing the receptor compared to the size of unmodified
cells.
[0342] The monitoring of CD69 spontaneous expression by CAR Treg cells as
compared to
untransduced Treg cells allows determination of tonic signaling intensity.
[0343] As demonstrated herein, engineered T cells and engineered Treg cells
expressing said
CAR constructs of the invention present a low tonic signaling and following
CAR engagement,
the engineered Treg cells showed highly efficient suppressive activity on T
effector cell
proliferation, thereby demonstrating the advantage of these Treg cells for
cell therapy.
[0344] In one embodiment, the regulatory immune cell population, preferably T
cell
population, comprises Treg cells, CDS+ T cells, OW T cells, and/or NK T cells.
[0345] In one embodiment, the regulatory immune cell population, preferably T
cell
population, consists of Treg cells, CD8+ T cells, CD4 T cells, and/or NK T
cells.
[0346] In one embodiment, the T cells of the present invention are Treg cells.
[0347] In one embodiment, the Treg cells in a cell population of the present
invention all
express a CAR described herein and may thus be defined as CAR-monospecific
(i.e., all the
Treg cells recognize the same antigen (MOG)). In one embodiment, the Treg cell
population
is TCR-monospecific (i.e., all the Treg cells recognize the same antigen with
their TCR). In
another embodiment, the Treg cell population is TCR-polyspecific (i.e., the
Tres cells may
recognize different antigens with their TCRs).
[0348] In one embodiment, the CAR of the present invention, when expressed by
a T (e.g.,
Treg) cell, confers to the T cell the ability to bind to cells expressing MOG
on their cell surface
and to be activated by binding to the MOG.
[0349] MOG is primarily expressed by oligodendrocytes in the CNS.
[0350] The regulatory immune cell population of the present invention (e.g.,
the T cell (e.g.,
Treg) population of the present invention) may thus be defined as a redirected
regulatory
immune cell population. As used herein, the term -redirected" refers to a
regulatory immune
cell carrying a CAR as described herein, which confers to the regulatory
immune cell the ability
to bind to and be activated by a ligand that is different from the one the
regulatory immune cell
would have been specific for or be activated by.
[0351] In one embodiment, Treg cells of the present invention are not
cytotoxic.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0352] In one embodiment, Treg cells of the present invention are cytotoxic.
[0353] In one embodiment, Treg cells of the present invention may be selected
from the group
comprising CD4+CD25+CD12710w FOXP3+ Treg cells, CD4 CD25 FOXP3+ Treg cells, In
cells, TGF-13-secreting Th3 cells, regulatory NK T cells, regulatory 76 T
cells, regulatory CD8-
T cells, and double negative regulatory T cells.
10354] In one embodiment, the regulatory immune cell is a CD4 rfreg cell. In
one
embodiment, the Treg is a thymus-derived Treg or an adaptive or induced Treg.
In one
embodiment, the Treg cell is a CD4+FOXP3+ Treg cell, or a CD4-TOXP3-
regulatory T cell
(Tr cell).
[0355] In one embodiment, the regulatory immune cell is a CD8+ Treg cell. In
one
embodiment, the CDS+ Treg cell is selected from the group consisting of a
CD8+CD28- Treg
cell, a CD8+CD103 Treg cell, a CD8'FOXP3-' Treg cell, a CD8'CD122-' Treg cell,
and any
combination thereof. In one embodiment, the regulatory cell is an
INF71L10+IL34+CD8+CD45RC1 ' Treg cell.
[0356] In one embodiment, the regulatory immune cells of the present invention
are human
Treg cells.
[0357] In one embodiment, the regulatory immune cells (e.g, the T cells or
Treg cells) are
derived from stem cells, such as induced pluripotent stem cells (iPSC).
[0358] As used herein, the term "induced pluripotent stem cells" or "iPSC"
refers to pluripotent
stem cells derived from non-pluripotent cells (e.g., adult somatic cells) by
de-differentiation or
reprogramming. In particular, iPSCs may be obtained by introducing a specific
set of
pluripotency-associated genes (reprogramming factors) into a cell.
Reprogramming factors
may be, for example, the transcription factors 0ct4 (Pou5f1), Sox2, c-Myc, and
Klf4.
[0359] In one embodiment, the Treg cell has the following phenotype:
CD4+CD25+, such as
CD4+CD25+CD127- and CD4'CD25'CD127-CD45RA'. In one embodiment, the Treg cell
has
the following phenotype: CD4+CD25+, such as CD4+CD25+CD12710' and
CD4 CD25 CD12710"CD45RAt In one embodiment, the Treg cell has the following
phenotype: CD4 CD25 , such as CD4 CD25 CD12710i- and CD4 CD25 CD12710"/-
CD45RA+. In one embodiment, the Treg cell has the following phenotype:
FOXP3 CD4+CD25+, such as FOXP3+CD4 CD25 CD127- and FOXP3+CD4+CD25+CD127-
CD45RA . In one embodiment, the Treg cell has the following phenotype:
FOXP3'CD4+CD25+, such as F OXP3 CD4'CD25'CD12710w
and
FOXP3 CD4+CD25 CD12710CD45RA . In one embodiment, the Treg cell has the
following
56
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
phenotype: FOXP3+CD4+CD25+, such as FOXP3+CD4+CD25+CD12710"/- and
FOXP3'CIN CD25+CD1271"i-CD45RAt
[0360] In one embodiment, the Treg cell has the following phenotype: CD4
CD25higli, such as
CD4+CD25highCD127- and CD4+CD25highCD127-CD45RA+. In one embodiment, the Treg
cell
has the following phenotype: CD4 CD25high, such as CD4 CD25h1ghCD1271" and
CD4+CD25highCD1271"CD45RA-'. In one embodiment, the Treg cell has the
following
phenotype: CD4+CD25ffigh, such as CD4 CD25highCD12710 and
CD4+CD25ffighCD12710"1-
CD45RA+. In one embodiment, the Treg cell has the following phenotype:
FOXP3'CD4+CD25h1gh, such as FOXP3'CD4VCD25h1gl1CD127-
and
FOXP3 CD4 CD25highCD127-CD45RA . In one embodiment, the Treg cell has the
following
phenotype: FOXP3+CD4+CD25 high-, such as FOXP3+CD4+CD25111ghCD1271' and
FOXP3'CD4+CD25highCD1271"CD45RA'. In one embodiment, the Treg cell has the
following phenotype: FOXP3 CD4 CD25high, such as FOXP3 CD4 CD25h1gl'CD1271"/-
and
FOXP3+CD4+CD25h1ghCD127101-CD45RA+.
[0361] In one embodiment, the regulatory immune cells (e.g, the T or Treg
cells) are
autologous cells. Autologous therapies are "custom" products for each patient.
In another
embodiment, the regulatory immune cells (e.g, the T or Treg cells) are
allogenic cells.
Allogenic cells can be used in allogenic therapies to provide "off-the-shelf'
products, used to
treat many patients. In such instances, the cell may be engineered to reduce
host rejection to
the cell (graft rejection) and/or the cell's potential attack on the host
(graft-versus-host disease).
By way of example, the cell may be engineered to have a null genotype for one
or more of the
following: (i) T cell receptor (TCR alpha chain or beta chain); (ii) a
polymorphic major
histocompatibility complex (MHC) class I or II molecule (e.g., HLA-A, HLA-B,
or HLA-C;
HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR; or 132-microglobu1in
(B2M)); (iii) a transporter associated with antigen processing (e.g., TAP-1 or
TAP-2); (iv)
Class II MHC transactivator (CIITA); (v) a minor histocompatibility antigen
(MiHA; e.g., HA-
1/A2, HA-2, HA-3, HA-8, HB-1H, or HB-1Y); and (vi) any combination thereof
[0362] The expression level of molecules may be determined by flow cytometry,
immunofluorescence, or image analysis. To detect intracellular proteins, cells
may be fixed and
permeabilized prior to flow cytometry analysis.
[0363] In one embodiment, the expression level of a molecule in a cell
population is indicated
by the percentage of cells of the cell population expressing the molecule
(i.e., cells "+" for the
molecule). The percentage of cells expressing the molecule may be measured by
FACS. The
57
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
expression level of the cell marker of interest may be determined by comparing
the Median
Fluorescence Intensity (MEI) of the cells from the cell population stained
with fluorescently
labeled antibody specific for this marker to the fluorescence intensity (II)
of the cells from the
same cell population stained with fluorescently labeled antibody with an
irrelevant specificity
but with the same isotype, the same fluorescent probe and originated from the
same specie
(referred as isotype control). 'The cells from the population stained with
fluorescently labeled
antibody specific for this marker and that show equivalent MI1 or a lower MF1
than the cells
stained with the isotype controls are not expressing this marker and then are
designated (-) or
negative. The cells from the population stained with fluorescently labeled
antibody specific for
this marker and that show a MFI value superior to the cells stained with the
isotype controls
are expressing this marker and then are designated (+) or positive.
[0364] The terms "expressing" (i.e., "positive" or "+") and "not expressing"
(i.e., "negative"
or "-") refer to the expression level of the cell marker of interest, in that
the expression level of
the cell marker corresponding to "+" is high or intermediate, also referred as
"+/-," and the
expression level of the cell marker corresponding to "-" is null. The term
"low" or "lo" or
"low/-" refers to the expression level of the cell marker of interest, in that
the expression level
of the cell marker is low by comparison with the expression level of that cell
marker in the
population of cells being analyzed as a whole. More particularly, the term
"lo" refers to a
distinct population of cells that express the cell marker at a lower level
than one or more other
distinct population of cells. The term "high" or "hi" or "bright" refers to
the expression level
of the cell marker of interest, in that the expression level of the cell
marker is high by
comparison with the expression level of that cell marker in the population of
cells being
analyzed as a whole. Generally, cells in the top 2, 3,4, or 5% of staining
intensity are designated
"hi," with those falling in the top half of the population categorized as
being "+." Those cells
falling below 50%, of fluorescence intensity are designated as "lo" cells and
below 5% as "-"
cells.
[0365] In one embodiment, the CAR of the present invention, when expressed by
a Treg cell,
allows for a reduction of the activation background of said Treg cells as
compared to other
CAR constructs directed to MOG.
B. Activation of regulatory immune cell
[0366] Importantly, once the CAR binds to its target, activation of the
regulatory immune cell,
preferably the T cell, more preferably the Treg cell, e.g., CD4 CD25 CD1271'
Treg cell, is
5g
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
further required such that the cell releases cytokines (e.g., IL-10, TGF-B)
and other soluble
mediators that suppress the activity of effector T cells (Teff cells) and
establish peripheral
tolerance. The cell also acts by cell contact via, for example, CTLA4 ¨
CD80/86 and LAG3. A
signal transduction in the cell is required for activation to occur in a
physiological manner.
Obtaining such signal transduction is challenging because it is reliant on how
(e,g. in which
position or configuration) the CAR binds to its target. In other words, mere
binding to MOG+
oligodendrocyte is not sufficient to induce a functional regulatory immune
cell and its retention
in the CNS.
[0367] The present inventors have demonstrated that the regulatory immune
cells expressing
the CAR of the invention, in particular Treg cells, e.g., CD4 CD25.+CD1271' T
reg cells,
present a MOG-binding CAR on their surface which is capable of binding MOG-
presenting
cells.
[0368] The CAR of the invention is a new CAR, which has a combination of
advantageous
characteristics as demonstrated in the Examples, including low CD69 expression
without
activation (low tonic/background activation) and good CAR-mediated activation
(high signal
to background ratio), e.g., activation increase by at least 2-fold. See Figure
11.
[0369] The CAR of the invention is a new and cross-reactive CAR, which is able
to bind mouse
and human MOG protein. See Figure 12.
[0370] CAR-specific activation of Treg cells and low background of activation
for the Treg
cells is shown in Figures 5 and 9. Tres cells expressing the CAR-MOG of the
invention exhibit
efficient CAR-mediated suppressive activity as shown in Figure 6. Importantly,
activation and
proliferation of the CAR was shown to take place in the CNS, see Figures 10A
and 10B.
IV. Composition, pharmaceutical composition, medicament
[0371] In an aspect, the present invention also provides a composition
comprising (including
consisting essentially of and consisting of) a MOG-binding protein of the
invention as
described herein (e.g., an antibody or fragment thereof, in particular an
scFv).
[0372] In an aspect, the present invention also provides a composition
comprising (including
consisting essentially of and consisting of) a nucleic acid or vector encoding
a protein of the
invention.
[0373] In another aspect, the present invention provides a composition
comprising (including
consisting essentially of and consisting of) a regulatory immune cell or
regulatory immune cell
population comprising the CAR according to the present invention. In an
embodiment, the
59
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
composition comprises a regulatory immune cell according to the invention or a
population of
regulatory immune cells invention.
[0374] In one embodiment, said composition is a pharmaceutical composition and
further
comprises a pharmaceutically acceptable excipient.
[0375] Consequently, the present invention further relates to a pharmaceutical
composition
comprising a regulatory immune cell or regulatory immune cell population
comprising the
CAR according to the present invention, and a pharmaceutically acceptable
excipient. In an
embodiment, the pharmaceutical composition consists of a regulatory immune
cell or
regulatory immune cell population comprising the CAR according to the present
invention, and
a pharmaceutically acceptable excipient.
[0376] In one embodiment, the MUG-binding protein of the invention (e.g., an
antibody or
fragment thereof, in particular an scFv), nucleic acid or expression vector,
or the regulatory
immune cell or regulatory immune cell population is the only therapeutic agent
or agent with
a biologic activity within said composition.
[0377] The term "pharmaceutically acceptable excipient" refers to solvents,
dispersion media,
coatings, antibacterial and antifungal agents, buffering agents, isotonic
agents, stabilizing
agents, preservatives, absorption-delaying agents, and the like. Said
excipient does not produce
an adverse, allergic, or other untoward reaction when administered to a
subject, such as a
human.
[0378] Examples of pharmaceutically acceptable excipients that may be used in
the
compositions of the present invention include, but are not limited to, ion
exchangers, alumina,
aluminum stearate, lecithin, serum proteins (e.g., human serum albumin),
buffer agents (e.g.,
phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride
mixtures of saturated
vegetable fatty acids, water, salts or electrolytes (e.g., sodium chloride,
protamine sulfate,
disodium hydrogen phosphate, potassium hydrogen phosphate, and zinc salts),
and
polyethylene glycol.
[0379] In one embodiment, the pharmaceutical compositions according to the
present
invention comprise vehicles which are pharmaceutically suitable for injection.
These may be,
for example, isotonic, sterile saline solutions (comprising, e.g., monosodium
or disodium
phosphate; sodium, potassium, calcium, or magnesium chloride; or mixtures of
such salts); or
dry (e.g., freeze-dried) compositions which, upon addition of a suitable
carrier such as sterilized
water or physiological saline, permit the constitution of injectable
solutions.
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0380] In another aspect, the invention provides a medicament comprising
(including
consisting essentially of and consisting of) a MUG-binding protein of the
invention.
[0381] The present invention further provides a medicament comprising
(including consisting
essentially of and consisting of) a population of regulatory immune cells
expressing a CAR of
the present invention.
10382] The present invention further provides a medicament comprising a
nucleic acid
encoding a MOG-binding protein of the invention.
[0383] The present invention further provides a medicament comprising a vector
of the
invention.
V. Administration route
[0384] Exemplary forms of administration include parenteral, by inhalation
spray, rectal, nasal,
or via an implanted reservoir.
[0385] Exemplary forms of administration include injection, including, without
limitation,
subcutaneous, intravenous, intramuscular, i ntra-arti cul ar, intra-synovi al,
i ntra-stern al,
intrathecal, intraperitoneal, intrahepatic, intralesional and intracranial
injection or infusion
techniques; preferably intravenous, intrathecal, or intraperitoneal injection,
more preferably
intravenous inj ection.
[0386] Exemplary forms adapted for injection include, but are not limited to,
solutions, such
as, for example, sterile aqueous solutions, gels, dispersions, emulsions,
suspensions, solid
forms suitable for using to prepare solutions or suspensions upon the addition
of a liquid prior
to use, such as, for example, powder, liposomal forms and the like.
VI. Dosage
[0387] It will be however understood that a therapeutically effective amount
and dosing
frequency will be decided by the attending physician within the scope of sound
medical
judgment. The specific therapeutically effective dose level for any particular
patient will
depend upon a variety of factors including the disease being treated and the
severity of the
disease; activity of the isolated MOG-binding protein, nucleic acid,
expression vector, or
regulatory immune cell employed; the age, body weight, general health, sex and
diet of the
subject; the time of administration, route of administration, and rate of
excretion of the specific
therapeutic agent employed; the duration of the treatment; drugs used in
combination or
coincidental with the specific therapeutic agent employed; and like factors
well known in the
61
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
medical arts. For example, it is well within the skill of the art to start
doses of the compound at
levels lower than those required to achieve the desired therapeutic effect and
to gradually
increase the dosage until the desired effect is achieved. The total dose
required for each
treatment may be administered by multiple doses or in a single dose.
[0388] In one embodiment, the subject (e.g., human) receives a single
administration of the
therapeutic agent (e.g., regulatory immune cell or regulatory immune cell
population) of the
present invention.
[0389] In one embodiment, the subject (e.g., human) receives at least two
administrations of
the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell
population) of
the present invention.
[0390] In one embodiment, the therapeutic agent (e.g., regulatory immune cell
or regulatory
immune cell population) of the present invention is administered once a week,
once a month,
or once a year to the subject.
[0391] In one embodiment, the number of regulatory immune cells administered
to the subject
ranges from about 102 to about 109, from about 103 to about 108, from about
104 to about 107,
or from about 105 to about 106.
[0392] In one embodiment, the therapeutic agent (e.g., regulatory immune cell
or regulatory
immune cell population) of the present invention is administered to the
subject in need thereof
in combination with another active agent. In one embodiment, said other active
agent is an
agent that may be used for treating an inflammatory CNS disease/disorder,
e.g., multiple
sclerosis. Examples of other active agents include, but are not limited to,
glucocorticoids
(including, without limitation, dexamethasone, prednisone, prednisolone,
methylprednisolone,
betamethasone, bedomethasone, tixocortol, triamcinolone, hydrocortisone,
budesonide or
fludrocortisone), antibodies or antagonists of human cytokines, molecules
(such as for
example, anti-CD20 such as ofatumumab, ocrelizumab, rituximab, tositumomab,
obinutuzumab; anti-CD52 such as alemtuzumab; anti-a401 integrin such as
natalizumab; anti-
CD25 such as daclizumab; anti-LINGO-1 such as opicinumab); Interferon-beta-la,
Peginterferon-beta-1a, Interferon-beta-lb, glatiramer acetate, mitoxantrone,
ibudilast,
simvastatin, biotin, laquinimod, ozanimod, fingolimod, siponimod, ponesimod,
evobrutinib
teriflunomi de, m on om ethyl fum arate, dim ethyl fumarate, di roxi m el
fumarate,
immunomodulators (such as, for example, tacrolimus, cyclosporine,
methotrexate,
thalidomide, leflunomide, and analogs of purines such as cladribine,
azathioprine, 6-
mercaptopurine), and plasma exchange.
62
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0393] In one embodiment, the administration of therapeutic agent (e.g.,
regulatory immune
cell or regulatory immune cell population) of the present invention allows a
reduction in the
amount of said other active agent received by the subject.
[0394] According to one embodiment, the therapeutic agent (e.g., regulatory
immune cell or
regulatory immune cell population) of the present invention is administered
before, at the same
time or after the administration of the other active agent.
VII. Therapeutic use
[0395] The present invention further relates to a cell expressing a CAR as
described anywhere
herein (e.g., the regulatory immune cells described herein, such as the Treg
cells described
herein) for use as a medicament.
[0396] The present invention further relates to a cell expressing a CAR as
described anywhere
herein (e.g., the regulatory immune cells described herein, such as the Treg
cells described
herein) for use in inflammatory CNS diseases/disorders, such as MS.
[0397] The present invention further relates to a cell expressing a CAR as
described anywhere
herein (e.g., the regulatory immune cells described herein, such as the Treg
cells described
herein) for use in treating demyelinating diseases/disorders, particularly
those associated with
the presence of autoantibodies or self-reactive immune cells.
[0398] The present invention further relates to a cell expressing a CAR as
described anywhere
herein (e.g., the regulatory immune cells described herein, such as the Treg
cells described
herein) for use in treating MOG-associated diseases/disorders (MOGAD),
particularly MOG-
associated inflammatory diseases/disorders, more particularly those associated
with the
presence of autoantibodies or self-reactive immune cells.
[0399] The present invention further relates to a cell expressing a CAR as
described anywhere
herein (e.g., the regulatory immune cells described herein, such as the Treg
cells described
herein) as described anywhere herein for reducing or preventing inflammation
of the CNS. The
present invention also relates to a cell expressing a CAR as described herein
(e.g., the
regulatory immune cells described herein, such as the Treg cells described
herein) for reducing
or preventing damage including demyelination of the CNS. The present invention
also relates
to a cell expressing a CAR as described herein (e.g., the regulatory immune
cells described
herein, such as the Treg cells described herein) for inducing remyelination of
neural lesions the
CNS.
63
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0400] The present invention further relates to a composition comprising a
regulatory immune
cell according to the invention or a population of regulatory immune cells
invention as
described anywhere herein for use as a medicament.
[0401] The present invention further relates to a composition a regulatory
immune cell
according to the invention or a population of regulatory immune cells
invention as described
anywhere herein for use in treating inflammatory CN S diseases/disorders, such
as MS.
[0402] The present invention further relates to a composition a regulatory
immune cell
according to the invention or a population of regulatory immune cells
invention as described
anywhere herein for use in treating demyelinating diseases/disorders,
particularly those
associated with the presence of autoantibodies or self-reactive immune cells.
[0403] The present invention further relates to a composition a regulatory
immune cell
according to the invention or a population of regulatory immune cells
invention as described
anywhere herein for use in treating MUG-associated diseases/disorders (MOGAD),
particularly MOG-associated inflammatory diseases/disorders, more particularly
those
associated with the presence of autoantibodies or self-reactive immune cells.
[0404] The present invention further relates to a composition a regulatory
immune cell
according to the invention or a population of regulatory immune cells
invention as described
anywhere herein for reducing or preventing inflammation and/or damage
including
demyelination of the CNS.
[0405] The present invention further relates to a method for treating a
disease, disorder, or
symptom of a disease or disorder in a subject in need thereof, comprising
administering to the
subject a cell population expressing a CAR as described anywhere herein (e.g.,
the regulatory
immune cells described herein, such as the Treg cells described herein). In
one embodiment,
the method is a method for treating inflammatory CNS diseases/disorders. In
one embodiment,
the method is a method for treating demyelinating diseases/disorders,
particularly those
associated with the presence of autoantibodies or self-reactive immune cells.
In one
embodiment, the method is a method for treating MOG-associated
diseases/disorders
(MOGAD), particularly MOG-associated inflammatory diseases/disorders, more
particularly
those associated with the presence of autoantibodies or self-reactive immune
cells.
[0406] The present invention further relates to a method for treating a
disease, disorder, or
symptom of a disease or disorder in a subject in need thereof, comprising
administering to the
subject a composition comprising a regulatory immune cell according to the
invention or a
population of regulatory immune cells invention as described anywhere herein.
In one
64
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
embodiment, the method is a method for treating inflammatory CNS
diseases/disorders. In one
embodiment, the method is a method for treating demyelinating
diseases/disorders, particularly
those associated with the presence of autoantibodies or self-reactive immune
cells. In one
embodiment, the method is a method for treating MOG-associated
diseases/disorders
(MOGAD), particularly MOG-associated inflammatory diseases/disorders, more
particularly
those associated with the presence of autoantibodies or self-reactive immune
cells.
[0407] The present invention further relates to a method for reducing or
preventing
inflammation and/or damage including demyelination of the CNS in a subject in
need thereof.
[0408] The present invention further relates to a cell therapy method for
treating in a subject
in need thereof an inflammatory CNS disease/disorder, e.g., multiple
sclerosis, wherein said
method comprises administering to the subject the regulatory immune cells
described herein,
e.g., the Treg cells described herein.
[0409] In one embodiment, the regulatory immune cells to be administered are
autologous
cells; in other words, the cell therapy is an autologous cell therapy. As used
herein, the term
"autologous" refers to any material derived from the same individual to whom
it is later to be
re-introduced.
[0410] In one embodiment, the cell therapy is a heterologous cell therapy. As
used herein, the
term "heterologous" refers to any material that is not derived from the
subject to be treated but
from an external source, e.g., induced pluripotent stem cells (iPSCs) or cells
of cadaveric
origin.
[0411] In one embodiment, the cell therapy is xenogeneic. As used herein, the
term
"xenogeneic" refers to any material derived from a subject of a different
species as the subject
to whom the material is introduced.
[0412] In another embodiment, the regulatory immune cells to be administered
are allogenic
cells; in other words, the cell therapy is an allogenic cell therapy. As used
herein, the term
"allogeneic" refers to any material derived from a different subject of the
same species as the
subject to whom the material is introduced. Two or more subjects are said to
be allogeneic to
one another when the genes at one or more loci are not identical. In a further
embodiment, the
regulatory immune cells are derived from a healthy human donor.
[0413] Examples of inflammatory CNS diseases/disorders include, but are not
limited to,
progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Parkinson's
disease (PD),
amyotrophic lateral sclerosis (AILS), autism spectrum disorders, Rasmussen
encephalitis,
chronic traumatic encephalopathy (CTE).
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0414] Preferred inflammatory CNS diseases/disorders are demyelinating
disorders caused or
aggravated by auto-antigens and/or autoantibodies such as Multiple Sclerosis
(MS), Clinically
Isolated Syndrome (CIS), Neuromyelitis Optica (N1VIO), Optic Neuritis, Acute
Disseminated
Encephalomyelitis, Transverse Myelitis, Adrenoleukodystrophy, Vanishing White
Matter
Disease, and Rubella induced mental retardation. More preferred demyelinating
disorders are
Multiple Sclerosis (MS) and Neuromyelitis Optica (NMO). In certain
embodiments, said MS
is selected from relapse-remitting MS (RRMS), secondary progressive MS (SPMS),
primary
progressive MS (PPMS), Acute fulminant Multiple Sclerosis and MS-suspected
radiology
isolated syndrome (RIS).
[0415] In one embodiment, the inflammatory CNS disease/disorder is Clinically
Isolated
Syndrome (CIS).
[0416] In one embodiment, the inflammatory CNS disease/disorder is multiple
sclerosis (MS).
[0417] In one embodiment, the multiple sclerosis is relapsing-remitting MS
(RRMS).
[0418] In one embodiment, the multiple sclerosis is primary-progressive MS
(PPMS).
[0419] In one embodiment, the multiple sclerosis is secondary-progressive MS
(SPMS).
VIII. Article of manufacture
[0420] The present invention also relates to an article of manufacture
containing materials
useful for the treatment of an inflammatory CNS disease/disorder, e.g.,
multiple sclerosis,
according to the invention.
[0421] The article of manufacture may comprise a container and a label or
package insert on
or associated with the container. Suitable containers include, for example,
bags, bottles, vials,
syringes, pouch, etc. The containers may be formed from a variety of materials
such as glass
or plastic
[0422] The article of manufacture, label or package insert may further
comprise instructional
material for administering the Treg cell population of the present invention
to the patient.
[0423] The present invention provides a kit comprising a regulatory immune
cell population
of the present invention. By "kit" is intended to mean any article of
manufacture (e.g., a
package or a container) comprising a Treg cell population of the present
invention. The kit may
also contain instructions for use.
[0424] Unless otherwise defined herein, scientific and technical terms used in
connection with
the present disclosure shall have the meanings that are commonly understood by
those of
ordinary skill in the art. Exemplary methods and materials are described
below, although
66
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
methods and materials similar or equivalent to those described herein can also
be used in the
practice or testing of the present disclosure. In case of conflict, the
present specification,
including definitions, will control. Generally, nomenclature used in
connection with, and
techniques of, medicine, medicinal and pharmaceutical chemistry, cell biology,
molecular
described herein are those well-known and commonly used in the art. Further,
unless otherwise
required by context, singular terms shall include pluralities and plural terms
shall include the
singular. Throughout this specification and embodiments, the words "have" and
"comprise,"
or variations such as "has," "having," "comprises," or "comprising," will be
understood to
imply the inclusion of a stated integer or group of integers but not the
exclusion of any other
integer or group of integers.
[0425] In order that this disclosure may be better understood, the following
examples are set
forth. These examples are for purposes of illustration only and are not to be
construed as
limiting the scope of the present invention in any manner.
EXAMPLES
[0426] The present invention if further illustrated by the following examples.
Material and methods
Determination of MUG-binding to different MUG variants
[0427] Yeast cells expressing the MOG-CAR of the present invention were
incubated with
biotinylated Human MUG-His (Uniprot Q16653), mouse MUG-His (Uniprot Q61885),
both
RND Systems; or cynomolgus monkey MOG-FC protein (Q9BGS7) mammalian cell
expressed at the indicated concentrations. MUG-protein bound to the scFV
expressing yeast
cells was then detected by flow cytometry using a fluorophore-tagged
streptavidin, while scF V
expression was detected by staining of a genetically encoded, N-terminally
fused MYC-tag.
See Figure 13.
A. Experiences with human cells
1. Human PBMC isolation
[0428] The blood of healthy donors is collected by the Etablissement Francais
du Sang (EFS).
The day after blood collection, peripheral blood mononuclear cells (PBMC) were
isolated from
buffy coats by Ficoll gradient centrifugation, which enables removal of
unwanted fractions of
67
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
blood product such as granulocytes, platelets and remaining red blood cell
contaminants. Then,
cells population of interest were isolated as follow.
2. FoxP3 Treg and CD4 CD25- conventional human T cells isolation
[0429] CD4+CD25+CD1271' Tregs were isolated using the Human CD4+CD12710wCD25-
Regulatory T Cell Isolation Kit (#18063; StemCell) following manufacturer's
instructions.
Briefly, CD25-' cells were first isolated from 400-500 x 106 PBMC by column-
free,
immunomagnetic positive selection using EasySepTm Releasable RapidSpheresTM.
Then,
bound magnetic particles were removed from the EasySepTm-isolated CD25+ cells,
and
unwanted non-Tregs were targeted for depletion. The final isolated fraction
contains highly
purified CD4 CD12710CD25. cells that express high levels of Foxp3 and were
immediately
used for downstream applications. CD4+CD25- conventional T cells were isolated
by choosing
the optional protocol for the isolation of CD4'CD25- responder T cells from
the kit #18063
(stemcell); for use in functional studies in parallel to Treg.
3. Activation and culture of isolated human Tregs
[0430] Isolated Treg cells were activated and cultured for 9 days. Briefly, at
day 0, Treg cells
(0.5 x 106) were cultured into 24 wells plate (Costar) with Xvivo 15 serum-
free medium
containing human transferrin (OZYME) and supplemented with 1000 U/ml IL-2
(Euromedex)
plus 100nM rapamycin (Sigma-Aldrich). Then, CD3/CD28 activation was performed
with
Dynabeads from Life Technology (0.5 x 106 beads per well). At day 2, 4 and 7
cells were feed
with fresh culture medium supplemented with 1000 U/ml IL-2. Finally, at day 9,
cells were
recovered, counted and reactivated.
4. Lentiviral vector production and titration
[0431] CAR-expressing lentiviral vectors (LVs) were produced using the
classical 4-plasmid
lentiviral system. Briefly, FIEK293T cells (Lenti-X, Ozyme) were transfected
with the CAR-
expressing transfer vector, the plasmid expressing HIV-1 Gag/pol (pMDLg/pRRE),
HIV-1 Rev
(pRSV.Rev) and for the viral envelope, the VSV-G glycoprotein (pMD2.G) (Didier
Trono,
EPFL, Switzerland) for transduction of human Tregs and the Ecotropic MLV
envelope
glycoprotein (pCMV-Eco, Cell Biolabs Inc.) for the transduction of mice Tregs.
24-hours post-
transfecti on, viral supernatants were harvested, concentrated by
centrifugation, ali quoted and
frozen at -80 C for long term storage. The infectious titers expressed in
transducing units per
milliliter (TU/ml) were obtained after transduction of the Jurkat T cell line
(for VSV-G
pseudotypes) or NIH-3T3 (for EcoMILV pseudotypes) with a serial dilution of
viral
supernatants and transduction efficiency evaluated after 4 days by monitoring
GFP expression.
68
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
5. Human Treg transduction protocol
[0432] Tregs were transduced 2 days after their activation with a chimeric
receptor (see below).
Briefly transduction was carried out by loading between 2 and 5 x106
Transducing Unit (TU)
per ml to each well. After 6 hours at 37 C, viral particles were removed by
washout. The plates
were then incubated at 37 C with 5% CO2. Five days after the transduction, the
transduction
efficiency was analyzed: the gene-transfer efficacy was measured by the
analysis of the
percentage of CEP positive cells in flow cytometry.
6. Human CAR construct used for transduction.
[0433] MOG CARs composed of the CD8 transmembrane (TM) and intracellular
domain of
CD28 in tandem with CD3C and associated with ScFv directed against MOG are
designed. The
construct used in this study is listed and described in FIG. 1.
7. Phenotype analysis of transduced human Treg
[0434] At day 9 of the culture, Treg phenotype was analyzed in flow cytometry
using the
markers listed in the Table 1.
Table 1: Material and reagents
Reagents Manufacturer
Reference
CD4 VioGreen Miltenyi
130-096-900
Helios eF450 (HamIgG) eBioscience 48-
9883-42
Treg CD25 PE Miltenyi
130-109-020
immuno-
phenotyping CD152 (CTLA-4) PECy7 (mIgG2a) Biolegend
369614
FoxP3 AF647 (mIgGl; 2 pi) BD
560045
CD127-APC-Vio770 Miltenyi
130-109-438
8. Activation assay of human CARs
[0435] Activation assay was performed at day 9 of the culture. Briefly, 0.05 x
106 Treg were
seeded in 96 U bottom plate alone or in presence of anti CD28/antiCD3 coated
beads (in a 1 to
69
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
1 Treg to beads ratio), or in MOG coated beads (in a 1 to 1 Tres to beads
ratio) in a 200 l.tl final
volume. After 24h at 37 C, 5% CO2, cells were stained for CD4 and CD69 and
then analyzed
using flow cytometry. The monitoring of the CD69 spontaneous expression in CAR
Treg cells,
compared to untransduced Treg cells, allows to determine the tonic signaling
intensity.
9. Suppression assay of human T cell proliferation
104361 The suppressive assays were performed at day 9 of the culture. Briefly,
rfreg were
recovered, counted and activated either through the TCR using anti
CD28/antiCD3 coated
beads (in a 1 to 1 Treg to beads ratio), or through the CAR using MOG coated
beads (in a 1 to
1 Treg to beads ratio) or kept without activation to evaluate their
spontaneous suppressive
activity. In parallel, allogeneic Tconv were thawed, stained with Cell Trace
Violet (CTV) and
activated with anti CD28/anti CD3 coated beads (in a 3 to 1 Tconv to beads
ratio). The day
after, beads were removed from Tconv before their coculture with un-activated
or activated
Treg (untransduced or transduced). At day 3, cells were harvested, and
proliferation of Tconv
was assessed by flow cytometry through the determination of CTV dilution. The
percentage of
inhibition of Tconv proliferation was calculated as followed:
% of Tconv proliferation in presence of CAR-Treg x 100
100
% of Tconv proliferation in absence of CAR-Treg
B. Experiences with mouse cells
1. Isolation of mouse Tregs
[0437] Spleen from C57/B16 are harvested and mashed up through cell strainers
to obtain a
single cell suspension. CD4+ CD25+ Tregs were isolated using Easy SepTm Mouse
CD4+CD25+ Regulatory T Cell Isolation Kit II (#18783; StemCell) following
manufacturer's
instructions. Briefly, CD4+ cells were first pre-enriched from splenocytes by
column-free,
immunomagnetic negative selection. Then, CD25+ are selected using CD25
positive selection
cocktail, which contains antibodies recognizing CD25 that link to magnetic
particles. Isolated
cells are immediately available for cell culture.
2. Activation and culture of isolated mouse Tregs
10438] Isolated Treg cells were activated and cultured for maximum 8 days.
Briefly, at day 0,
Treg cells (0,5.106) were cultured into 24 wells plate (Costar) with RPMI
containing with 10%
FBS, 2mM L-Glutamine, 1mM Sodium Pyruvate, 0,1mM Non-essential amino acids, 1%
Penicillin-Streptavidine and 5 laM 2-Betamercapto-ethanol (RPMI10)
supplemented with
1000U/m1 Rec huIL2 and 50nM Rapamycin. Then, CD3/CD28 activation was performed
with
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
Dynabeads mouse T activator from Life Technologies (2:1, Beads:Cell ratio). At
day 2, 4 and
6 cells were counted, fed with fresh culture medium supplemented with 1000
U/ml IL-2 and
Rapa (only at day 4).
3. Mouse Treg transduction protocol
[0439] Tregs were transduced 2 days after their activation with a chimeric
receptor. Briefly
transduction was carried out by loading 2 x107 rfransducing Unit (TU) per ml
of CAR vectors
to each well plus 15 g/m1 of protransducin B (PTDB). PTDB and vectors are
mixed for 5 min
at 37 C before to be added to the Tregs. A spinoculation is performed at 32 C,
1000g for 90
minutes. After 4 hours at 37 C, viral particles and PTDB were removed by
washout and fresh
media containing IL-2 (1000 U/ml) is added. The plates were then incubated at
37 C with 5%
CO2. Four to five days after transduction, the transduction efficiency was
analyzed: the gene-
transfer efficacy was measured by the analysis of the percentage of NGFR
positive cells in
fl ow cytom etry.
4. Mouse CAR construct used for transduction
[0440] MOG CARs composed of the CD8 transmembrane (TM) and intracellular
domain of
CD28 in tandem with CD3C and associated with ScFy directed against MOG are
designed. The
construction used in this study is listed and described in FIG. 2.
5. Phenotype analysis of transduced mouse Treg
[0441] At day 6-7 of the culture, Treg phenotype was analyzed in flow
cytometry using the
markers listed in the Table 2.
Table 2: Mouse antibodies
Reagents Manufacturer
Reference
CD4 V450 BD Biosciences 560468
CD8 BV510 Biolegend 100752
Tres
immuno- CD25 PE BD Biosciences 553866
phenotyping
FoxP3 APC Miltenyi 130-111-
601
NGFR Vio Bright 515 Miltenyi 130-112-
599
71
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
6. Activation assay of mouse CARs
[0442] Activation assay was performed at day 7 of the culture. Briefly, 0.05 x
106 Treg were
seeded in 96 U bottom plate alone or in presence of anti CD28/antiCD3 coated
beads (in a 1 to
1 Treg to beads ratio), or in MOG coated beads (in a 1 to 1 Treg to beads
ratio) in a 200 j.tl final
volume. After 24h at 37 C, 5% CO2, cells were stained for CD4 and CD69 and
then analyzed
using flow cytometry. The monitoring of the CD69 spontaneous expression in CAR
lreg cells,
compared to control Treg cells, allows the determination of the tonic
signaling intensity.
7. In vivo Activation assay of mouse CARs: short model
[0443] To determine if our MOG CAR can go in the target organ (Central Nervous
System:
CNS) and be activated and proliferate there, a short model using EAE was
developed. Briefly,
female mice were immunized with an emulsion containing MOG peptide + CFA to
induce the
disease. i.p injection of Pertusis toxin (PTX) were performed at day 0 and day
2 to help the
opening of the blood brain barrier (BBB). At the onset of disease (9-11 days
after
immunization), MOG CAR and CAR Ctrl Tregs were injected i.v. After 5 days,
mice were
sacrificed, draining lymph nodes (dLN) and CNS were harvested and analyzed by
flow
cytometry to assess activation (CD69, LAP and CD71) and proliferation (Ki67)
of the cells.
8. In vivo efficacy assay of mouse CARs in a mouse EAE model
[0444] Donor mice were immunized with CFA/ MOG emulsion using a standard
protocol.
Each mouse was subcutaneously immunized on the base of tail and on flanks with
a total of
100 pi CFA emulsion containing MOG peptide and Mycobacterium tuberculosis
(H37Ra).
After 14 days, mice were sacrificed, and spleen and LN were harvested then
smashed. Spleen
cells were cultured with polarization cocktail to enhance MOG specific
pathogenic cells. After
3 days, these cells were harvested and injected in females CD45.1 C57BL/6 mice
to induce
EAE (25x106 cells/mouse). Mouse CAR MOG Tregs of the present disclosure, CAR
control
Tregs, or saline were injected i.v 24h after pathogenic cells. Disease
progression was evaluated
based on the EAE scoring using the following criteria: 0 (normal), 1
(partially limb tail), 2
(paralyzed tail), 3 (hind limb weakness or loss of coordination), 4 (hind
limbs paralysis), 4.5
(hind limbs paralysis with forelimb weakness), and 5 (moribund or dead).
Clinical score and
bodyweight (BW) are measured every other day from Day 6 to Day 15. To avoid
biased results,
the EAE scoring was performed as part of a double-blind study.
[0445] After 15 days, mice were sacrificed, and CNS cells were harvested and
smashed.
Collagenase digestion was performed 30 min at 37 C followed by a Percoll
gradient to isolate
immune cells that infiltrated the CNS. These cells were incubated overnight in
complete RPMI
72
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
media containing MOG peptide (10 g/m1) to stimulate MOG specific cells. 16
hours later,
supernatants are harvested, and Brefeldin A is added to the media to stop
cytokine secretion.
Cells are then rinsed in cell staining buffer and stained for intracellular
cytokines before
acquisition in an Attune NxT.
Results
A. Experiences with human cells
I. Transduction efficiency and CAR expression at cell surface
[0443] Transduction efficiency was assessed by the percentage of GFP positive
cells
expression and CAR expression was monitored using recombinant protein L, an
immunoglobulin kappa light chain-binding protein. Results for the percentage
of transduction
efficiency and the percentage of transduced cells which expressed the CAR at
cell surface
compared to not transduced cells (NT) are given in the FIG. 3, as an example
of raw data.
2. New scFv highlighted a good Treg phenotype stability
[0444] A major issue with engineered T cells in general is to ensure the
maintenance of the
desired phenotype especially since it has been shown that high expression of
CARs has been
linked to undesired antigen-independent CAR activation (Frigault, 2015). A
panel of markers
related to Treg identity were analyzed to check if the Treg phenotype is
altered during
expansion and CAR engagement. Here the maintenance of Helios and FoxP3
expression as
well as other makers associated to Treg phenotype was checked on FoxP3 Treg
(FIG. 4). MOG
CAR-Tregs maintained high expression of FOXP3 and Helios after expansion, at
Day 9.
3. New scFv-derived CARs maintain CAR-specific activation
[0446] Low background of activation is observed with both constructs and the
CAR-MOG of
the invention is specifically activated by MOG beads (FIG. 5).
4. MUG CAR comprising new scFV exhibits efficient CAR-mediated suppressive
activity
[0447] For the CAR-MOG construct harboring a new scFV, a CAR-specific
triggering of the
suppressive activity compared to the CAR Control was observed (FIG. 6).
5. 1110G CAR Tregs show activation in response of mouse or human MOG
[0448] By analyzing the level of CD69 expression in MOG CAR-Tregs after
activation with
human and mouse MOG, cross reactivity of the scFy of the invention was
demonstrated. The
same activation curve in response of both human and mouse MOG targets was
observed (FIG.
12).
73
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
B. Experiences with mouse cells
I. Transduction efficiency
[0449] Transduction efficiency was assessed by the percentage of NGFR positive
cells
expression. Results for the percentage of transduction efficiency compared to
not transduced
cells (NT) are given in the FIG. 7, as an example of raw data.
2. New scFv highlighted a good Treg phenotype
[0450] A panel of markers related to Treg identity (Table 2) was analyzed to
check if the Treg
phenotype is altered during expansion and CAR engagement. here the maintenance
of FoxP3
at day 7 after isolation was checked on Tregs (FIG. 8). MOG CAR-Tregs
maintained high
expression of FOXP3 after expansion, between 60 to 70% of cells are CD25+
FoxP3+.
3. New scFv-derived CARs maintain CAR-specific activation
[0451] Low background of activation was observed with both constructs and MOG
CAR was
specifically activated by MOG beads (FIG. 9).
[0452] FIG. 11 A shows the level of activation in vitro of mouse Tregs
transduced with a
number of different MOG-CAR (NGFR+ cells) by MOG-coated beads (black bars)
versus
control beads (white bars) as measured by the level of expression of early
activation marker
CD69 by flow cytometry. CAR 1 is the same MOG CAR construct used in FIG. 9 and
is an
exemplary construct of the invention. CAR 2-6 are comparative MOG CAR
constructs.
[0453] The dotted line represents the threshold of CD69 expression of non-
activated reference
MOG-CAR (tonic signaling). The box highlights the two best candidates showing
low level of
tonic signaling and high level of activation by the target antigen MOG (best
signal to noise
ratio). The table in FIG. 11 A shows the fold increase of CD69 expression on
MOG CAR Tregs
after activation. The HLA-A2 CAR was used as a positive control in the assay.
[0454] FIG. 11B shows the fold increase in vivo of activation markers CD69,
CD71, LAP and
proliferation marker Ki67 in the CNS of animals injected with MOG CAR after 5
days ("short
EAE model") versus the level of activation of CAR Tregs transduced with a
truncated control
CAR (MOG ScFy and non-signaling endodomain).
4. In vivo Activation assay of mouse CARs: short model - MOG CAR Tregs are
going into
the CNS and are activated and proliferate there.
[0455] Six days after i v injection of mouse MOG CAR Tregs, activation and
proliferation of
NGFR+ and NGFR- Tregs were analysed by flow cytometry in CNS and spleen
(Figs.10A and
10B). CAR-MOG Tregs (NGFR+ cells) were more activated in the CNS compared to
Ctrl
Tregs (NGFR- cells) and, with respect to proliferation in the CNS, CAR-MOG
Tregs were
74
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
showing higher expression of Ki67 compared to Ctrl Tregs (Fig. 10A). Looking
at activation
and proliferation of the CAR-MOG Tregs in the CNS as compared to the spleen,
the CAR-
MOG Tregs showed a higher level of CD69 (activation) and Ki67 (proliferation)
in the CNS
than in the spleen (Fig. 10B).
5. Exengdary In vivo Efficacy of a MOG CAR Treg of the Present Disclosure in a
Mouse
EAE Model
[0456] In this exemplary study to demonstrate the functional in vivo efficacy
of our CAR MOG
Treg cells of the present disclosure, we used the adoptive transfer EAE in
C57B16 mice. As an
alternative to direct induction with MOG, EAE can also be induced in C57BL/6
mice by
adoptive transfer of in ino CNS antigen activated lymphocytes from mice
immunized with
these antigens. As shown in Figs. 14A and 14B, mice injected with pathogenic
cells are
developing signs of severe paralysis. Mice treated with CAR MOG Tregs of the
present
disclosure 24h after pathogenic cells injection are showing lower clinical
score (Fig. 14A) and
a delay in the disease incidence (Fig. 14B) compared to Saline or CAR control
(Ctrl) groups.
[0457] As shown in Fig. 15, the percentage of IFNg positive CNS cells from
mice that were
treated with MOG CAR of the present disclosure, a control CAR, or saline were
measured after
ex vivo stimulation with MOG peptide. After mice sacrifice, cells from CNS
were incubated
overnight with MOG peptide (10 g/m1). 16h later BFA was added to the media and
intracellular staining was performed. Error bars represent mean SEM from 2
independent
experiments including 15 mice/group. In this exemplary study, CNS cells from
mice treated
with MOG CAR of the present disclosure showed a lower percentage of IFN-gamma
positive
cells after MOG peptide incubation as compared to cells derived from mice
treated with saline
or a CAR control.
TABLES OF SEQUENCES
[0458] Table 3: Human aMOG CAR¨ Protein sequences (SEQ: SEQ ID NO)
SEQ Sequence function Sequence
1 hCD8 Leader MALPVTALLLPLALLLHAARP
2 Spacer HA-Tag- S YPYDVPDYA S
Spacer
3 CDR1-VH SSYAFS
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
4 CDR2-VH RIVPVVGTPNYAQKFQG
CDR3-VH RERLYAGYY
6 CDR1-VL RASQSVSSNYLA
7 CDR2-VL GASSRAT
8 CDR3-VL QQYGTSPGLT
ScFV-VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
9 AVVYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
QGTKVEIK
(G4S)3-Linker GGGGSGGGGSGGGGS
ScFV-VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
11 FSWVRQAPGQGLEVVMGRIVPVVGTPNYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
RLYAGYYFDHWGQGTLVTVSS
ScFV QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
FSVVVRQAPGQGLEVVMGRIVPVVGTPNYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
12 RLYAGYYFDHWGQGTLVTVSSGGGGSGGGGS
GGGGSEIVLTQSPGTLSLSPGERATLSCRASQS
VSSNYLAVVYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
PGLTFGQGTKVEIK
ScFV EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
51 QGTKVEIKGGGGSGGGGSGGGGSQVQLVQSG
AEVKKPGSSVKVSCKASGGTFSSYAFSVVVRQAP
GQGLEVVMGRIVPVVGTPNYAQKFQGRVTITADE
STSTAYMELSSLRSEDTAVYYCARERLYAGYYF
DHWGQGTLVTVSS
hCD8-Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
13
VHTRGLDFACD
14 hCD8- IYIWAPLAGTCGVLLLSLVITLYC
Transmembrane
hCD28
Costim- RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP
domain PRDFAAYRS
76
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
hCD3z
RVKFSRSADAPAYQQGQNQLYNELNLGRREEY
16
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
CAR
MALPVTALLLPLALLLHAARPSYPYDVPDYASEIV
LTQSPGTLSLSPGERATLSCRASQSVSSNYLAW
YOQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGTSPGLTFGQG
TKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEV
KKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQ
GLEVVMGRIVPVVGTPNYAQKFQGRVTITADEST
17 STAYMELSSLRSEDTAVYYCARERLYAGYYFDH
WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLR
PEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC
GVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPG
PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY
QQGQNQLYNELNLGRREEYDVLDKRRGRDPEM
GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQAL
PPR
CAR
SYPYDVPDYASEIVLTQSPGTLSLSPGERATLSC
RASQSVSSNYLAWYQQKPGQAPRLLIYGASSRA
TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ
QYGTSPGLTFGQGTKVEIKGGGGSGGGGSGGG
GSQVQLVQSGAEVKKPGSSVKVSCKASGGTFS
SYAFSVVVRQAPGQGLEVVMGRIVPVVGTPNYAQ
KFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
52 ARERLYAGYYFDHWGQGTLVTVSSTTTPAPRPP
TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA
CDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH
SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
RVKFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
CAR
MALPVTALLLPLALLLHAARPSEIVLTQSPGTLSL
SPGERATLSCRASQSVSSNYLAWYQQKPGQAP
RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGTSPGLTFGQGTKVEIKGGGG
SGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS
53 CKASGGTFSSYAFSWVRQAPGQGLEVVMGRIVP
VVGTPNYAQKFOGRVTITADESTSTAYMELSSLR
SEDTAVYYCARERLYAGYYFDHWGQGTLVTVSS
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP
77
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
GLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPR
CAR SEIVLTQSPGTLSLSPGERATLSCRASQSVSSNY
LAVVYQQKPGQAPRLLIYGASSRATGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTF
GQGTKVEIKGGGGSGGGGSGGGGSQVQLVQS
GAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQ
APGQGLEWMGRIVPVVGTPNYAQKFQGRVTITA
DESTSTAYMELSSLRSEDTAVYYCARERLYAGY
54 YFDHWGQGTLVTVSSTTTPAPRPPTPAPTIASQP
LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA
GTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPR
RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA
PAYQQGQNQLYNELNLGRREEYDVLDKRRGRD
PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALP PR
CAR EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
AVVYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
QGTKVEIKGGGGSGGGGSGGGGSQVQLVQSG
AEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAP
GQGLEWMGRIVPVVGTPNYAQKFQGRVTITADE
STSTAYMELSSLRSEDTAVYYCARERLYAGYYF
55 DHWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLS
LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRP
GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM
KGERRRGKGHDGLYQGLSTATKDTYDALHMQA
LPPR
CAR MALPVTALLLPLALLLHAARPSYPYDVPDYASQV
QLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFS
WVROAPGQGLEWMGRIVP\NGTPNYAQKFOGR
VTITADESTSTAYMELSSLRSEDTAVYYCARERL
YAGYYFDHWGQGTLVIVSSGGGGSGGGGSGG
56 GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSS
NYLAVVYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGTSPGL
TFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRP
EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG
VLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGP
TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ
7g
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMG
GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPP
R
CAR SYPYDVPDYASQVQLVQSGAEVKKPGSSVKVSC
KASGGTFSSYAFSWVRQAPGQGLEWMGRIVPV
VGTPNYAQKFOGRVTITADESTSTAYMELSSLRS
EDTAVYYCARERLYAGYYFDHWGQGTLVTVSS
GGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGE
RATLSCRASQSVSSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
57 AVYYCQQYGTSPGLTFGQGTKVEIKTTTPAPRPP
TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA
CDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH
SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
RVKFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
CAR MALPVTALLLPLALLLHAARPSQVQLVQSGAEVK
KPGSSVKVSCKASGGTFSSYAFSVVVRQAPGQG
LEWMGRIVPVVGTPNYAQKFQGRVTITADESTST
AYMELSSLRSEDTAVYYCARERLYAGYYFDHWG
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSP
GTLSLSPGERATLSCRASQSVSSNYLAVVYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI
58 SRLEPEDFAVYYCQQYGTSPGLTFGQGTKVEIKT
TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCR
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP
RDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
GLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPR
CAR SQVQLVQSGAEVKKPGSSVKVSCKASGGIFSSY
AFSVVVRQAPGQGLEWMGRIVPVVGTPNYAQKF
QGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
ERLYAGYYFDHWGQGTLVTVSSGGGGSGGGG
SGGGGSEIVLTQSPGTLSLSPGERATLSCRASQ
59 SVSSNYLAVVYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
PGLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLS
LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRP
GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
79
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM
KGERRRGKGHDGLYQGLSTATKDTYDALHMQA
LPPR
CAR QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
FSWVRQAPGQGLEWMGRIVPVVGTPNYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
RLYAGYYFDHWGQGTLVTVSSGGGGSGGGGS
GGGGSEIVLTQSPGTLSLSPGERATLSCRASQS
VSSNYLAVVYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
60 PGLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLS
LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRP
GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM
KGERRRGKGHDGLYQGLSTATKDTYDALHMQA
LPPR
[0459] Table 4: Human aMOG CAR¨ DNA sequences
SEQ Sequence function Sequence
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
18 hCD8 Leader
TCTGGCTCTGCTTCTTCATGCCGCCAGACCA
19 Spacer HA-Tag- TCT
TACCCCTACGATGTGCCTGATTATGCA
Spacer TCT
GAGATTGTGCTGACCCAGAGCCCTGGCACCCT
GTCTCTGAGCCCCGGAGAGAGAGCCACCCTG
AGCTGTCGGGCCAGCCAGAGCGTGAGCAGCA
ACTACCTGGCCTGGTATCAGCAGAAGCCCGGA
CAGGCCCCCAGACTGCTGATCTACGGCGCCTC
20 ScFV-VL CAGCAGAGCCACCGGAATCCCCGACAGATTTT
CTGGCAGCGGCTCTGGCACCGACTTCACCCTG
ACCATCTCCAGACTGGAGCCCGAGGACTTCGC
CGTGTACTACTGCCAGCAATACGGCACCAGCC
CAGGCCTGACCTTTGGTCAGGGCACAAAGGTG
GAAATCAAG
GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTG
21 (G4S)3-Linker
GTGGCGGAGGCTCA
CAGGTCCAGCTAGTACAAAGCGGCGCCGAAGT
22 ScFV-VH AAAGAAACCTGGTAGCTCTGTGAAGGTGAGCT
GCAAGGCCAGCGGCGGCACCTTCAGCAGCTA
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CGCCTTCAGCTGGGTTCGACAGGCCCCTGGAC
AGGGCCTAGAGTGGATGGGCAGAATCGTGCCT
GTGGTGGGCACCCCCAACTACGCCCAGAAGTT
CCAGGGCAGAGTTACCATCACCGCCGACGAGA
GCACCAGCACAGCCTATATGGAGCTGAGCAGC
CTGCGAAGCGAGGACACAGCTGTTTACTATTG
TGCCAGAGAGAGACTGTACGCCGGCTACTACT
TCGATCACTGGGGCCAGGGGACCCTGGTGAC
CGTTTCTTCT
CAGGTCCAGCTAGTACAAAGCGGCGCCGAAGT
AAAGAAACCTGGTAGCTCTGTGAAGGTGAGCT
GCAAGGCCAGCGGCGGCACCTTCAGCAGCTA
CGCCTTCAGCTGGGTTCGACAGGCCCCTGGAC
AGGGCCTAGAGTGGATGGGCAGAATCGTGCCT
GTGGTGGGCACCCCCAACTACGCCCAGAAGTT
CCAGGGCAGAGTTACCATCACCGCCGACGAGA
GCACCAGCACAGCCTATATGGAGCTGAGCAGC
CTGCGAAGCGAGGACACAGCTGTTTACTATTG
TGCCAGAGAGAGACTGTACGCCGGCTACTACT
TCGATCACTGGGGCCAGGGGACCCTGGTGAC
23 S cFV CGTTTCTTCTGGAGGCGGAGGTTCTGGCGGCG
GAGGAAGTGGTGGCGGAGGCTCAGAGATTGT
GCTGACCCAGAGCCCTGGCACCCTGTCTCTGA
GCCCCGGAGAGAGAGCCACCCTGAGCTGTCG
GGCCAGCCAGAGCGTGAGCAGCAACTACCTG
GCCTGGTATCAGCAGAAGCCCGGACAGGCCC
CCAGACTGCTGATCTACGGCGCCTCCAGCAGA
GCCACCGGAATCCCCGACAGATTTTCTGGCAG
CGGCTCTGGCACCGACTTCACCCTGACCATCT
CCAGACTGGAGCCCGAGGACTTCGCCGTGTAC
TACTGCCAGCAATACGGCACCAGCCCAGGCCT
GACCTTTGGTCAGGGCACAAAGGTGGAAATCA
AG
GAGATTGTGCTGACCCAGAGCCCTGGCACCCT
GTCTCTGAGCCCCGGAGAGAGAGCCACCCTG
AGCTGTCGGGCCAGCCAGAGCGTGAGCAGCA
ACTACCTGGCCTGGTATCAGCAGAAGCCCGGA
CAGGCCCCCAGACTGCTGATCTACGGCGCCTC
CAGCAGAGCCACCGGAATCCCCGACAGATTTT
76 ScFV CTGGCAGCGGCTCTGGCACCGACTTCACCCTG
ACCATCTCCAGACTGGAGCCCGAGGACTTCGC
CGTGTACTACTGCCAGCAATACGGCACCAGCC
CAGGCCTGACCTTTGGTCAGGGCACAAAGGTG
GAAATCAAGGGAGGCGGAGGTTCTGGCGGCG
GAGGAAGTGGTGGCGGAGGCTCACAGGTCCA
GCTAGTACAAAGCGGCGCCGAAGTAAAGAAAC
CTGGTAGCTCTGTGAAGGTGAGCTGCAAGGCC
81
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
AGCGGCGGCACCTTCAGCAGCTACGCCTTCAG
CTGGGTTCGACAGGCCCCTGGACAGGGCCTA
GAGTGGATGGGCAGAATCGTGCCTGTGGTGG
GCACCCCCAACTACGCCCAGAAGTTCCAGGGC
AGAGTTACCATCACCGCCGACGAGAGCACCAG
CACAGCCTATATGGAGCTGAGCAGCCTGCGAA
GCGAGGACACAGCTGTTTACTATTGTGCCAGA
GAGAGACTGTACGCCGGCTACTACTTCGATCA
CTGGGGCCAGGGGACCCTGGTGACCGTTTCTT
CT
ACAACAACACCTGCTCCTCGGCCTCCTACACC
AGCTCCTACAATTGCCAGCCAGCCACTGTCTC
24 hCD8- Hinge TGAGGCCCGAAGCTTGCAGGCCTGCTGCTGG
CGGAGCCGTGCATACAAGAGGACTGGATTTCG
CCTGCGAC
ATCTACATCTGGGCACCTCTGGCTGGAACCTG
hCD8-
25 TGGCGTGCTGCTGCTGAGCCTGGTCATCACCC
Transmembrane
TGTATTGC
CGGAGCAAGAGAAGCAGACTGCTGCACAGCG
hCD28 Costim- ACTACATGAACATGACCCCTAGACGGCCCGGA
26
domain CCTACCAGAAAGCACTACCAGCCTTACGCTCC
TCCTAGAGACTTCGCCGCCTACAGATCC
AGAGTGAAGTTCAGCAGATCCGCCGACGCTCC
TGCCTATCAGCAGGGCCAAAACCAGCTCTACA
ACGAGCTGAACCTGGGGAGAAGAGAAGAGTAC
GACGTGCTGGACAAGCGGAGAGGCAGAGATC
CTGAAATGGGCGGCAAGCCCAGACGGAAGAAT
27 hCD3z CCTCAAGAGGGCCTGTATAATGAGCTACAGAA
AGACAAGATGGCAGAGGCCTACAGCGAGATCG
GAATGAAGGGCGAGCGCAGAAGAGGCAAGGG
ACACGATGGACTGTACCAGGGCCTGAGCACCG
CCACCAAGGATACCTATGATGCCCTGCACATG
CAGGCCCTGCCTCCAAGA
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
TCTGGCTCTGCTTCTTCATGCCGCCAGACCAT
CTTACCCCTACGATGTGCCTGATTATGCATCTG
AGATTGTGCTGACCCAGAGCCCTGGCACCCTG
TCTCTGAGCCCCGGAGAGAGAGCCACCCTGA
28 CAR GCTGTCGGGCCAGCCAGAGCGTGAGCAGCAA
CTACCTGGCCTGGTATCAGCAGAAGCCCGGAC
AGGCCCCCAGACTGCTGATCTACGGCGCCTC
CAGCAGAGCCACCGGAATCCCCGACAGATTTT
CTGGCAGCGGCTCTGGCACCGACTTCACCCT
GACCATCTCCAGACTGGAGCCCGAGGACTTCG
CCGTGTACTACTGCCAGCAATACGGCACCAGC
82
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CCAGGCCTGACCTTTGGTCAGGGCACAAAGGT
GGAAATCAAGGGAGGCGGAGGTTCTGGCGGC
GGAGGAAGTGGTGGCGGAGGCTCACAGGTCC
AGCTAGTACAAAGCGGCGCCGAAGTAAAGAAA
CCTGGTAGCTCTGTGAAGGTGAGCTGCAAGGC
CAGCGGCGGCACCTTCAGCAGCTACGCCTTCA
GCTGGGTTCGACAGGCCCCTGGACAGGGCCT
AGAGTGGATGGGCAGAATCGTGCCTGTGGTG
GGCACCCCCAACTACGCCCAGAAGTTCCAGG
GCAGAGTTACCATCACCGCCGACGAGAGCAC
CAGCACAGCCTATATGGAGCTGAGCAGCCTGC
GAAGCGAGGACACAGCTGTTTACTATTGTGCC
AGAGAGAGACTGTACGCCGGCTACTACTTCGA
TCACTGGGGCCAGGGGACCCTGGTGACCGTT
TCTTCTACAACAACACCTGCTCCTCGGCCTCCT
ACACCAGCTCCTACAATTGCCAGCCAGCCACT
GTCTCTGAGGCCCGAAGCTTGCAGGCCTGCT
GCTGGCGGAGCCGTGCATACAAGAGGACTGG
ATTTCGCCTGCGACATCTACATCTGGGCACCT
CTGGCTGGAACCTGTGGCGTGCTGCTGCTGA
GCCTGGTCATCACCCTGTATTGCCGGAGCAAG
AGAAGCAGACTGCTGCACAGCGACTACATGAA
CATGACCCCTAGACGGCCCGGACCTACCAGAA
AGCACTACCAGCCTTACGCTCCTCCTAGAGAC
TTCGCCGCCTACAGATCCAGAGTGAAGTTCAG
CAGATCCGCCGACGCTCCTGCCTATCAGCAGG
GCCAAAACCAGCTCTACAACGAGCTGAACCTG
GGGAGAAGAGAAGAGTACGACGTGCTGGACA
AGCGGAGAGGCAGAGATCCTGAAATGGGCGG
CAAGCCCAGACGGAAGAATCCTCAAGAGGGC
CTGTATAATGAGCTACAGAAAGACAAGATGGC
AGAGGCCTACAGCGAGATCGGAATGAAGGGC
GAGCGCAGAAGAGGCAAGGGACACGATGGAC
TGTACCAGGGCCTGAGCACCGCCACCAAGGA
TACCTATGATGCCCTGCACATGCAGGCCGTGC
CTCCAAGA
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
TCTGGCTCTGCTTCTTCATGCCGCCAGACCAT
CTGAGATTGTGCTGACCCAGAGCCCTGGCACC
CTGTCTCTGAGCCCCGGAGAGAGAGCCACCC
TGAGCTGTCGGGCCAGCCAGAGCGTGAGCAG
77 CAR CAACTACCTGGCCTGGTATCAGCAGAAGCCCG
GACAGGCCCCCAGACTGCTGATCTACGGCGC
CTCCAGCAGAGCCACCGGAATCCCCGACAGAT
TTTCTGGCAGCGGCTCTGGCACCGACTTCACC
CTGACCATCTCCAGACTGGAGCCCGAGGACTT
CGCCGTGTACTACTGCCAGCAATACGGCACCA
GCCCAGGCCTGACCTTTGGTCAGGGCACAAA
83
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
GGTGGAAATCAAGGGAGGCGGAGGTTCTGGC
GGCGGAGGAAGTGGTGGCGGAGGCTCACAGG
TCCAGCTAGTACAAAGCGGCGCCGAAGTAAAG
AAACCTGGTAGCTCTGTGAAGGTGAGCTGCAA
GGCCAGCGGCGGCACCTTCAGCAGCTACGCC
TTCAGCTGGGTTCGACAGGCCCCTGGACAGG
GCCTAGAGTGGATGGGCAGAATCGTGCCTGT
GGTGGGCACCCCCAACTACGCCCAGAAGTTC
CAGGGCAGAGTTACCATCACCGCCGACGAGA
GCACCAGCACAGCCTATATGGAGCTGAGCAGC
CTGCGAAGCGAGGACACAGCTGTTTACTATTG
TGCCAGAGAGAGACTGTACGCCGGCTACTACT
TCGATCACTGGGGCCAGGGGACCCTGGTGAC
CGTTTCTTCTACAACAACACCTGCTCCTCGGC
CTCCTACACCAGCTCCTACAATTGCCAGCCAG
CCACTGTCTCTGAGGCCCGAAGCTTGCAGGCC
TGCTGCTGGCGGAGCCGTGCATACAAGAGGA
CTGGATTTCGCCTGCGACATCTACATCTGGGC
ACCTCTGGCTGGAACCTGTGGCGTGCTGCTGC
TGAGCCTGGTCATCACCCTGTATTGCCGGAGC
AAGAGAAGCAGACTGCTGCACAGCGACTACAT
GAACATGACCCCTAGACGGCCCGGACCTACCA
GAAAGCACTACCAGCCTTACGCTCCTCCTAGA
GACTTCGCCGCCTACAGATCCAGAGTGAAGTT
CAGCAGATCCGCCGACGCTCCTGCCTATCAGC
AGGGCCAAAACCAGCTCTACAACGAGCTGAAC
CTGGGGAGAAGAGAAGAGTACGACGTGCTGG
ACAAGCGGAGAGGCAGAGATCCTGAAATGGG
CGGCAAGCCCAGACGGAAGAATCCTCAAGAG
GGCCTGTATAATGAGCTACAGAAAGACAAGAT
GGCAGAGGCCTACAGCGAGATCGGAATGAAG
GGCGAGCGCAGAAGAGGCAAGGGACACGATG
GACTGTACCAGGGCCTGAGCACCGCCACCAA
GGATACCTATGATGCCCTGCACATGCAGGCCC
TGCCTCCAAGATAG
GAGATTGTGCTGACCCAGAGCCCTGGCACCCT
GTCTCTGAGCCCCGGAGAGAGAGCCACCCTG
AGCTGTCGGGCCAGCCAGAGCGTGAGCAGCA
ACTACCTGGCCTGGTATCAGCAGAAGCCCGGA
CAGGCCCCCAGACTGCTGATCTACGGCGCCT
CCAGCAGAGCCACCGGAATCCCCGACAGATTT
78 CAR TCTGGCAGCGGCTCTGGCACCGACTTCACCCT
GACCATCTCCAGACTGGAGCCCGAGGACTTCG
CCGTGTACTACTGCCAGCAATACGGCACCAGC
CCAGGCCTGACCTTTGGTCAGGGCACAAAGGT
GGAAATCAAGGGAGGCGGAGGTTCTGGCGGC
GGAGGAAGTGGTGGCGGAGGCTCACAGGTCC
AGCTAGTACAAAGCGGCGCCGAAGTAAAGAAA
84
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CCTGGTAGCTCTGTGAAGGTGAGCTGCAAGGC
CAGCGGCGGCACCTTCAGCAGCTACGCCTTCA
GCTGGGTTCGACAGGCCCCTGGACAGGGCCT
AGAGTGGATGGGCAGAATCGTGCCTGTGGTG
GGCACCCCCAACTACGCCCAGAAGTTCCAGG
GCAGAGTTACCATCACCGCCGACGAGAGCAC
CAGCACAGCCTATATGGAGCTGAGCAGCCTGC
GAAGCGAGGACACAGCTGTTTACTATTGTGCC
AGAGAGAGACTGTACGCCGGCTACTACTTCGA
TCACTGGGGCCAGGGGACCCTGGTGACCGTT
TCTTCTACAACAACACCTGCTCCTCGGCCTCCT
ACACCAGCTCCTACAATTGCCAGCCAGCCACT
GTCTCTGAGGCCCGAAGCTTGCAGGCCTGCT
GCTGGCGGAGCCGTGCATACAAGAGGACTGG
ATTTCGCCTGCGACATCTACATCTGGGCACCT
CTGGCTGGAACCTGTGGCGTGCTGCTGCTGA
GCCTGGTCATCACCCTGTATTGCCGGAGCAAG
AGAAGCAGACTGCTGCACAGCGACTACATGAA
CATGACCCCTAGACGGCCCGGACCTACCAGAA
AGCACTACCAGCCTTACGCTCCTCCTAGAGAC
TTCGCCGCCTACAGATCCAGAGTGAAGTTCAG
CAGATCCGCCGACGCTCCTGCCTATCAGCAGG
GCCAAAACCAGCTCTACAACGAGCTGAACCTG
GGGAGAAGAGAAGAGTACGACGTGCTGGACA
AGCGGAGAGGCAGAGATCCTGAAATGGGCGG
CAAGCCCAGACGGAAGAATCCTCAAGAGGGC
CTGTATAATGAGCTACAGAAAGACAAGATGGC
AGAGGCCTACAGCGAGATCGGAATGAAGGGC
GAGCGCAGAAGAGGCAAGGGACACGATGGAC
TGTACCAGGGCCTGAGCACCGCCACCAAGGA
TACCTATGATGCCCTGCACATGCAGGCCCTGC
CTCCAAGATAG
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
TCTGGCTCTGCTTCTTCATGCCGCCAGACCAT
CTTACCCCTACGATGTGCCTGATTATGCATCTC
AGGTCCAGCTAGTACAAAGCGGCGCCGAAGTA
AAGAAACCTGGTAGCTCTGTGAAGGTGAGCTG
CAAGGCCAGCGGCGGCACCTTCAGCAGCTAC
GCCTTCAGCTGGGTTCGACAGGCCCCTGGAC
79 CAR AGGGCCTAGAGTGGATGGGCAGAATCGTGCC
TGTGGTGGGCACCCCCAACTACGCCCAGAAGT
TCCAGGGCAGAGTTACCATCACCGCCGACGA
GAGCACCAGCACAGCCTATATGGAGCTGAGCA
GCCTGCGAAGCGAGGACACAGCTGTTTACTAT
TGTGCCAGAGAGAGACTGTACGCCGGCTACTA
CTTCGATCACTGGGGCCAGGGGACCCTGGTG
ACCGTTTCTTCTGGAGGCGGAGGTTCTGGCGG
CGGAGGAAGTGGTGGCGGAGGCTCAGAGATT
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
GTGCTGACCCAGAGCCCTGGCACCCTGTCTCT
GAGCCCCGGAGAGAGAGCCACCCTGAGCTGT
CGGGCCAGCCAGAGCGTGAGCAGCAACTACC
TGGCCTGGTATCAGCAGAAGCCCGGACAGGC
CCCCAGACTGCTGATCTACGGCGCCTCCAGCA
GAGCCACCGGAATCCCCGACAGATTTTCTGGC
AGCGGCTCTGGCACCGACTTCACCCTGACCAT
CTCCAGACTGGAGCCCGAGGACTTCGCCGTG
TACTACTGCCAGCAATACGGCACCAGCCCAGG
CCTGACCTTTGGTCAGGGCACAAAGGTGGAAA
TCAAGACAACAACACCTGCTCCTCGGCCTCCT
ACACCAGCTCCTACAATTGCCAGCCAGCCACT
GTCTCTGAGGCCCGAAGCTTGCAGGCCTGCT
GCTGGCGGAGCCGTGCATACAAGAGGACTGG
ATTTCGCCTGCGACATCTACATCTGGGCACCT
CTGGCTGGAACCTGTGGCGTGCTGCTGCTGA
GCCTGGTCATCACCCTGTATTGCCGGAGCAAG
AGAAGCAGACTGCTGCACAGCGACTACATGAA
CATGACCCCTAGACGGCCCGGACCTACCAGAA
AGCACTACCAGCCTTACGCTCCTCCTAGAGAC
TTCGCCGCCTACAGATCCAGAGTGAAGTTCAG
CAGATCCGCCGACGCTCCTGCCTATCAGCAGG
GCCAAAACCAGCTCTACAACGAGCTGAACCTG
GGGAGAAGAGAAGAGTACGACGTGCTGGACA
AGCGGAGAGGCAGAGATCCTGAAATGGGCGG
CAAGCCCAGACGGAAGAATCCTCAAGAGGGC
CTGTATAATGAGCTACAGAAAGACAAGATGGC
AGAGGCCTACAGCGAGATCGGAATGAAGGGC
GAGCGCAGAAGAGGCAAGGGACACGATGGAC
TGTACCAGGGCCTGAGCACCGCCACCAAGGA
TACCTATGATGCCCTGCACATGCAGGCCCTGC
CTCCAAGATAG
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
TCTGGCTCTGCTTCTTCATGCCGCCAGACCAT
CTCAGGTCCAGCTAGTACAAAGCGGCGCCGAA
GTAAAGAAACCTGGTAGCTCTGTGAAGGTGAG
CTGCAAGGCCAGCGGCGGCACCTTCAGCAGC
TACGCCTTCAGCTGGGTTCGACAGGCCCCTGG
ACAGGGCCTAGAGTGGATGGGCAGAATCGTG
80 CAR CCTGTGGTGGGCACCCCCAACTACGCCCAGA
AGTTCCAGGGCAGAGTTACCATCACCGCCGAC
GAGAGCACCAGCACAGCCTATATGGAGCTGAG
CAGCCTGCGAAGCGAGGACACAGCTGTTTACT
ATTGTGCCAGAGAGAGACTGTACGCCGGCTAC
TACTTCGATCACTGGGGCCAGGGGACCCTGGT
GACCGTTTCTTCTGGAGGCGGAGGTTCTGGCG
GCGGAGGAAGTGGTGGCGGAGGCTCAGAGAT
TGTGCTGACCCAGAGCCCTGGCACCCTGTCTC
86
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
TGAGCCCCGGAGAGAGAGCCACCCTGAGCTG
TCGGGCCAGCCAGAGCGTGAGCAGCAACTAC
CTGGCCTGGTATCAGCAGAAGCCCGGACAGG
CCCCCAGACTGCTGATCTACGGCGCCTCCAGC
AGAGCCACCGGAATCCCCGACAGATTTTCTGG
CAGCGGCTCTGGCACCGACTTCACCCTGACCA
TCTCCAGACTGGAGCCCGAGGACTTCGCCGT
GTACTACTGCCAGCAATACGGCACCAGCCCAG
GCCTGACCTTTGGTCAGGGCACAAAGGTGGAA
ATCAAGACAACAACACCTGCTCCTCGGCCTCC
TACACCAGCTCCTACAATTGCCAGCCAGCCAC
TGTCTCTGAGGCCCGAAGCTTGCAGGCCTGCT
GCTGGCGGAGCCGTGCATACAAGAGGACTGG
ATTTCGCCTGCGACATCTACATCTGGGCACCT
CTGGCTGGAACCTGTGGCGTGCTGCTGCTGA
GCCTGGTCATCACCCTGTATTGCCGGAGCAAG
AGAAGCAGACTGCTGCACAGCGACTACATGAA
CATGACCCCTAGACGGCCCGGACCTACCAGAA
AGCACTACCAGCCTTACGCTCCTCCTAGAGAC
TTCGCCGCCTACAGATCCAGAGTGAAGTTCAG
CAGATCCGCCGACGCTCCTGCCTATCAGCAGG
GCCAAAACCAGCTCTACAACGAGCTGAACCTG
GGGAGAAGAGAAGAGTACGACGTGCTGGACA
AGCGGAGAGGCAGAGATCCTGAAATGGGCGG
CAAGCCCAGACGGAAGAATCCTCAAGAGGGC
CTGTATAATGAGCTACAGAAAGACAAGATGGC
AGAGGCCTACAGCGAGATCGGAATGAAGGGC
GAGCGCAGAAGAGGCAAGGGACACGATGGAC
TGTACCAGGGCCTGAGCACCGCCACCAAGGA
TACCTATGATGCCCTGCACATGCAGGCCGTGC
CTCCAAGATAG
CAGGTCCAGCTAGTACAAAGCGGCGCCGAAG
TAAAGAAACCTGGTAGCTCTGTGAAGGTGAGC
TGCAAGGCCAGCGGCGGCACCTTCAGCAGCT
ACGCCTTCAGCTGGGTTCGACAGGCCCCTGG
ACAGGGCCTAGAGTGGATGGGCAGAATCGTG
CCTGTGGTGGGCACCCCCAACTACGCCCAGA
AGTTCCAGGGCAGAGTTACCATCACCGCCGAC
GAGAGCACCAGCACAGCCTATATGGAGCTGAG
81 CAR CAGCCTGCGAAGCGAGGACACAGCTGTTTACT
ATTGTGCCAGAGAGAGACTGTACGCCGGCTAC
TACTTCGATCACTGGGGCCAGGGGACCCTGGT
GACCGTTTCTTCTGGAGGCGGAGGTTCTGGCG
GCGGAGGAAGTGGTGGCGGAGGCTCAGAGAT
TGTGCTGACCCAGAGCCCTGGCACCCTGTCTC
TGAGCCCCGGAGAGAGAGCCACCCTGAGCTG
TCGGGCCAGCCAGAGCGTGAGCAGCAACTAC
CTGGCCTGGTATCAGCAGAAGCCCGGACAGG
87
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
CCCCCAGACTGCTGATCTACGGCGCCTCCAGC
AGAGCCACCGGAATCCCCGACAGATTTTCTGG
CAGCGGCTCTGGCACCGACTTCACCCTGACCA
TCTCCAGACTGGAGCCCGAGGACTTCGCCGT
GTACTACTGCCAGCAATACGGCACCAGCCCAG
GCCTGACCTTTGGTCAGGGCACAAAGGTGGAA
ATCAAGACAACAACACCTGCTCCTCGGCCTCC
TACACCAGCTCCTACAATTGCCAGCCAGCCAC
TGTCTCTGAGGCCCGAAGCTTGCAGGCCTGCT
GCTGGCGGAGCCGTGCATACAAGAGGACTGG
ATTTCGCCTGCGACATCTACATCTGGGCACCT
CTGGCTGGAACCTGTGGCGTGCTGCTGCTGA
GCCTGGTCATCACCCTGTATTGCCGGAGCAAG
AGAAGCAGACTGCTGCACAGCGACTACATGAA
CATGACCCCTAGACGGCCCGGACCTACCAGAA
AGCACTACCAGCCTTACGCTCCTCCTAGAGAC
TTCGCCGCCTACAGATCCAGAGTGAAGTTCAG
CAGATCCGCCGACGCTCCTGCCTATCAGCAGG
GCCAAAACCAGCTCTACAACGAGCTGAACCTG
GGGAGAAGAGAAGAGTACGACGTGCTGGACA
AGCGGAGAGGCAGAGATCCTGAAATGGGCGG
CAAGCCCAGACGGAAGAATCCTCAAGAGGGC
CTGTATAATGAGCTACAGAAAGACAAGATGGC
AGAGGCCTACAGCGAGATCGGAATGAAGGGC
GAGCGCAGAAGAGGCAAGGGACACGATGGAC
TGTACCAGGGCCTGAGCACCGCCACCAAGGA
TACCTATGATGCCCTGCACATGCAGGCCCTGC
CTCCAAGATAG
104601 Table 5: Other human transmembrane domains - Protein sequence and DNA
sequence
SEQ Sequence function Sequence
29 CD28 FVVVLVVVGGVLACYSLLVTVAF I I FVVV
transmembrane
domain
30 CD28 TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT
transmembrane GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT
domain coding TATTATTTTCTGGGT G
sequence
31 4-1 BB IISFFLALTSTALLFLLFFLTLRFSVV
transmembrane
domain
gg
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
32 4-1 BB ATCATCTCCTTCTTTCTTGCGCTGACcTCGACT
transmembrane GCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTC
domain coding CGTTTCTCTGTTGTT
sequence
33 TNFR2 FALPVGLIVGVTALGLLIIGVVNCVIMTQV
transmembrane
domain
34 TNFR2 TTCGCTCTTCCAGTTGGACTGATTGTGGGTGT
transmembrane GACAGCCTTGGGTCTACTAATAATAGGAGTGG
domain coding TGAACTGTGTCATCATGACCCAGGTG
sequence
104611 Table 6: Other human costimulatory signaling domains - Protein sequence
and DNA
sequence
SEQ Sequence function Sequence
35 4-1 BB costimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP
signaling domain EEEEGGCEL
AAACGGGGCAGAAAGAAACTCCTGTATATATTC
4-1BB costimulatory
AAACAACCATTTATGAGACCAGTACAAACTACT
36 signaling domain
CAAGAGGAAGATGGCTGTAGCTGCCGATTTCC
coding sequence
AGAAGAAGAAGAAGGAGGATGTGAACTG
37 CD27costimulatory QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTI
signaling domain PIQEDYRKPEPACSP
CAACGAAGGAAATATAGATCAAACAAAGGAGA
CD27 costimulatory AAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTT
38 signaling domain ACAGCTGCCCCAGGGAGGAGGAGGGCAGCAC
coding sequence CATCCCCATCCAGGAGGATTACCGAAAACCGG
AGCCTGCCTGCTCCCCC
KKKPLCLQREAKVPHLPADKARGTQGPEQQHLLI
TAPSSSSSSLESSASALDRRAPTRNQPQAPGVE
TNFR2
ASGAGEARASTGSSDSSPGGHGTQVNVTCIVNV
39 costimulatory
CSSSDHSSQCSSQASSTMGDTDSSPSESPKDE
signaling domain
QVPFSKEECAFRSQLETPETLLGSTEEKPLPLGV
PDAGMKPS
TNFR2 AAAAAGAAGCCCTTGTGCCTGCAGAGAGAAGC
costimulatory CAAGGTGCCTCACTTGCCTGCAGATAAGGCCA
signaling domain GGGGTACACAGGGTCCCGAGCAGCAGCACCT
coding sequence CCTGATCACAGCGCCGAGCTCCAGCAGCAGCT
CCCTGGAGAGCTCGGCCAGTGCGCTAGACAG
89
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
AAGGGCCCCCACTCGGAACCAGCCACAGGCA
CCAGGCGTGGAGGCGAGTGGGGCTGGGGAG
GCGAGGGCAAGCACCGGGAGCTCAGATTCTTC
CCCTGGTGGTCATGGCACCCAGGTCAATGTCA
CCTGTATCGTGAACGTCTGTAGCAGCTCTGAC
CACAGCTCACAGTGCTCCTCCCAAGCCTCGTC
CACAATGGGAGACACAGATTCCAGCCCCTCGG
AGTCCCCGAAGGACGAGCAAGTACCCTTCTCC
AAGGAGGAATGTGCCTTTCGGTCACAGCTGGA
GACGCCAGAGACCCTGCTGGGGAGCACCGAA
GAGAAGCCCCTGCCCCTTGGAGTGCCTGATGC
TGGGATGAAGCCCAGT
[0462] Table 7: Mouse aMOG CAR¨ Protein sequence
SEQ Sequence function Sequence
1 hCD8 Leader MALPVTALLLPLALLLHAARP
2 Spacer HA-Tag- S YPYDVPDYA S
Spacer
3 CDR1-VH SSYAFS
4 CDR2-VH RIVPVVGTPNYAQKFQG
CDR3-VH RERLYAGYY
6 CDR1-VL RASQSVSSNYLA
7 CDR2-VL GASSRAT
8 CDR3-VL QQYGTSPG LT
9 EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
AVVYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
ScFV-VL
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
QGTKVEIK
(G4S)3-Linker GGGGSGGGGSGGGGS
11 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
FV VH FSWVRQAPGQGLEWMGRIVPVVGTPNYAQKFQ
-
Sc
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
RLYAGYYFDHWGQGTLVTVSS
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
12 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
FSVVVRQAPGQGLEVVMGRIVPVVGTPNYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
RLYAGYYFDHWGQGTLVTVSSGGGGSGGGGS
ScFV
GGGGSEIVLTQSPGTLSLSPGERATLSCRASQS
VSSNYLAWYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
PGLTFGQGTKVEIK
EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
51 FV
QGTKVEIKGGGGSGGGGSGGGGSQVQLVQSG
Sc
AEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAP
GQGLEVVMGRIVPVVGTPNYAQKFQGRVTITADE
STSTAYMELSSLRSEDTAVYYCARERLYAGYYF
DHWGQGTLVTVSS
41 TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSV
mCD8-Hinge
KGTGLDFACD
42 mCD8- IYIWAPLAGICVALLLSLIITLI
Transmembrane
43 mCD28 Costim- NSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAP
domain ARDFAAYRP
44 RAKFSRSAETAANLQDPNQLYNELNLGRREEYD
VLEKKRARDPEMGGKQQRRRNPQEGVYNALQK
mCD3z
DKMAEAYSEIGTKGERRRGKGHDGLYQGLSTAT
KDTYDALHMQTLAPR
45 MALPVTALLLPLALLLHAARPSYPYDVPDYASEIV
LTQSPGTLSLSPGERATLSCRASQSVSSNYLAW
YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGTSPGLTFGQG
TKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEV
KKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQ
GLEVVMGRIVPVVGTPNYAQKFQGRVTITADEST
CAR STAYMELSSLRSEDTAVYYCARERLYAGYYFDH
WGQGTLVTVSSMHTTTKPVLRTPSPVHPTGTSQ
PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGIC
VALLLSLIITLICYTNSRRNRLLQSDYMNMTPRRP
GLTRKPYQPYAPARDFAAYRPRAKFSRSAETAA
NLQDPNQLYNELNLGRREEYDVLEKKRARDPEM
GGKQQRRRNPQEGVYNALQKDKMAEAYSEIGT
KGERRRGKGHDGLYQGLSTATKDTYDALHMQT
LAPR*
91
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
61 SYPYDVPDYASEIVLTQSPGTLSLSPGERATLSC
RASQSVSSNYLAVVYQQKPGQAPRLLIYGASSRA
TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ
QYGTSPGLTFGQGTKVEIKGGGGSGGGGSGGG
GSQVQLVQSGAEVKKPGSSVKVSCKASGGTFS
SYAFSVVVRQAPGQGLEWMGRIVPVVGTPNYAQ
KFOGRVTITADESTSTAYMELSSLRSEDTAVYYG
CAR ARERLYAGYYFDHWGQGTLVTVSSMHTTTKPVL
RTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLD
FACDIYIWAPLAGICVALLLSLIITLICYTNSRRNRL
LQSDYMNMTPRRPGLTRKPYQPYAPARDFAAY
RPRAKFSRSAETAANLQDPNQLYNELNLGRREE
YDVLEKKRARDPEMGGKQQRRRNPQEGVYNAL
QKDKMAEAYSEIGTKGERRRGKGHDGLYQGLST
ATKDTYDALHMQTLAPR
62 MALPVTALLLPLALLLHAARPSEIVLTQSPGTLSL
SPGERATLSCRASQSVSSNYLAWYQQKPGQAP
RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGTSPGLTFGQGTKVEIKGGGG
SGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS
CKASGGTFSSYAFSVVVRQAPGQGLEVVMGRIVP
VVGTPNYAQKFOGRVTITADESTSTAYMELSSLR
CAR SEDTAVYYCARERLYAGYYFDHVVGQGTLVTVSS
MHTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRG
SVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY
TNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYA
PARDFAAYRPRAKFSRSAETAANLQDPNQLYNE
LNLGRREEYDVLEKKRARDPEMGGKQQRRRNP
QEGVYNALQKDKMAEAYSEIGTKGERRRGKGH
DGLYQGLSTATKDTYDALHMQTLAPR
63 SEIVLTQSPGTLSLSPGERATLSCRASQSVSSNY
LAVVYQQKPGQAPRLLIYGASSRATGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTF
GQGTKVEIKGGGGSGGGGSGGGGSQVQLVQS
GAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQ
APGQGLEWMGRIVPVVGTPNYAQKFQGRVTITA
DESTSTAYMELSSLRSEDTAVYYCARERLYAGY
CAR YFDHWGQGTLVTVSSMHTTTKPVLRTPSPVHPT
GTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAP
LAG ICVALLLSLIITLICYTNSRRNRLLQSDYMNMT
PRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSA
ETAANLQDPNQLYNELNLGRREEYDVLEKKRAR
DPEMGGKQQRRRNPQEGVYNALQKDKMAEAY
SEIGTKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQTLAPR
92
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
64 EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYL
AVVYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGTSPGLTFG
QGTKVEIKGGGGSGGGGSGGGGSQVQLVQSG
AEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAP
GQGLEVVMGRIVPVVGTPNYAQKFQGRVTITADE
STSTAYMELSSLRSEDTAVYYCARERLYAGYYF
CAR DHWGQGTLVTVSSMHTTTKPVLRTPSPVHPTGT
SQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA
GICVALLLSLIITLICYTNSRRNRLLQSDYMNMTPR
RPGLTRKPYQPYAPARDFAAYRPRAKFSRSAET
AANLQDPNQLYNELNLGRREEYDVLEKKRARDP
EMGGKQQRRRNPQEGVYNALQKDKMAEAYSEI
GTKGERRRGKGHDGLYQGLSTATKDTYDALHM
QTLAPR
65 MALPVTALLLPLALLLHAARPSYPYDVPDYASQV
QLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFS
VVVRQAPGQGLEWMGRIVPVVGTPNYAQKFQGR
VTITADESTSTAYMELSSLRSEDTAVYYCARERL
YAGYYFDHWGQGTLVTVSSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSS
NYLAVVYQQKPGQAPRLLIYGASSRATGIPDRFS
CAR GSGSGTDFTLTISRLEPEDFAVYYCQQYGTSPGL
TFGQGTKVEIKMHTTTKPVLRTPSPVHPTGTSQP
QRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICV
ALLLSLIITLICYTNSRRNRLLQSDYMNMTPRRPG
LTRKPYQPYAPARDFAAYRPRAKFSRSAETAAN
LQDPNQLYNELNLGRREEYDVLEKKRARDPEMG
GKQQRRRNPQEGVYNALQKDKMAEAYSEIGTK
GERRRGKGHDGLYQGLSTATKDTYDALHMQTL
APR
66 SYPYDVPDYASQVQLVQSGAEVKKPGSSVKVSC
KASGGTFSSYAFSWVRQAPGQGLEWMGRIVPV
VGTPNYAQKFQGRVTITADESTSTAYMELSSLRS
EDTAVYYCARERLYAGYYFDHWGQGTLVTVSS
GGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGE
RATLSCRASQSVSSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
CAR AVYYCQQYGTSPGLTFGQGTKVEIKMHTTTKPV
LRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLD
FACDIYIWAPLAGICVALLLSLIITLICYTNSRRNRL
LQSDYMNMTPRRPGLTRKPYQPYAPARDFAAY
RPRAKFSRSAETAANLQDPNQLYNELNLGRREE
YDVLEKKRARDPEMGGKQQRRRNPQEGVYNAL
QKDKMAEAYSEIGTKGERRRGKGHDGLYQGLST
ATKDTYDALHMQTLAPR
93
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
67 MALPVTALLLPLALLLHAARPSQVQLVQSGAEVK
KPGSSVKVSCKASGGTFSSYAFSVVVRQAPGQG
LEWMGRIVPVVGTPNYAQKFQGRVTITADESTST
AYMELSSLRSEDTAVYYCARERLYAGYYFDHWG
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSP
GTLSLSPGERATLSCRASQSVSSNYLAVVYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI
CAR SRLEPEDFAVYYCQQYGTSPGLTFGQGTKVEIK
MHTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRG
SVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY
TNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYA
PARDFAAYRPRAKFSRSAETAANLQDPNQLYNE
LNLGRREEYDVLEKKRARDPEMGGKQQRRRNP
QEGVYNALQKDKMAEAYSEIGTKGERRRGKGH
DGLYQGLSTATKDTYDALHMQTLAPR
68 SQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY
AFSVVVRQAPGQGLEVVMGRIVPVVGTPNYAQKF
QGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
ERLYAGYYFDHWGQGTLVTVSSGGGGSGGGG
SGGGGSEIVLTQSPGTLSLSPGERATLSCRASQ
SVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
CAR PGLTFGQGTKVEIKMHTTTKPVLRTPSPVHPTGT
SQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA
GICVALLLSLIITLICYTNSRRNRLLQSDYMNMTPR
RPGLTRKPYQPYAPARDFAAYRPRAKFSRSAET
AANLQDPNQLYNELNLGRREEYDVLEKKRARDP
EMGGKQQRRRNPQEGVYNALQKDKMAEAYSEI
GTKGERRRGKGHDGLYQGLSTATKDTYDALHM
QTLAPR
69 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
FSWVRQAPGQGLEWMGRIVPVVGTPNYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYYCARE
RLYAGYYFDHWGQGTLVTVSSGGGGSGGGGS
GGGGSEIVLTQSPGTLSLSPGERATLSCRASQS
VSSNYLAWYQQKPGQAPRLLIYGASSRATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTS
CAR PGLTFGQGTKVEIKMHTTTKPVLRTPSPVHPTGT
SQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA
GICVALLLSLIITLICYTNSRRNRLLQSDYMNMTPR
RPGLTRKPYQPYAPARDFAAYRPRAKFSRSAET
AANLQDPNQLYNELNLGRREEYDVLEKKRARDP
EMGGKQQRRRNPQEGVYNALQKDKMAEAYSEI
GTKGERRRGKGHDGLYQGLSTATKDTYDALHM
QTLAPR
94
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
[0463] Table 8: Mouse aMOG CAR¨ DNA sequence
SEQ Sequence function Sequence
18 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
hCD8 Leader
TCTGGCTCTGCTTCTTCATGCCGCCAGACCA
19 Spacer HA-Tag- TCT
TACCCCTACGATGTGCCTGATTATGCA
Spacer TCT
20 GAGATTGTGCTGACCCAGAGCCCTGGCACCCT
GTCTCTGAGCCCCGGAGAGAGAGCCACCCTG
AGCTGTCGGGCCAGCCAGAGCGTGAGCAGCA
ACTACCTGGCCTGGTATCAGCAGAAGCCCGGA
CAGGCCCCCAGACTGCTGATCTACGGCGCCTC
ScFV-VL CAGCAGAGCCACCGGAATCCCCGACAGATTTT
CTGGCAGCGGCTCTGGCACCGACTTCACCCTG
ACCATCTCCAGACTGGAGCCCGAGGACTTCGC
CGTGTACTACTGCCAGCAATACGGCACCAGCC
CAGGCCTGACCTTTGGTCAGGGCACAAAGGTG
GAAATCAAG
21 GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTG
(G4S)3-Linker
GTGGCGGAGGCTCA
22 CAGGTCCAGCTAGTACAAAGCGGCGCCGAAGT
AAAGAAACCTGGTAGCTCTGTGAAGGTGAGCT
GCAAGGCCAGCGGCGGCACCTTCAGCAGCTA
CGCCTTCAGCTGGGTTCGACAGGCCCCTGGA
CAGGGCCTAGAGTGGATGGGCAGAATCGTGC
CTGTGGTGGGCACCCCCAACTACGCCCAGAA
-
ScFV VH
GTTCCAGGGCAGAGTTACCATCACCGCCGACG
AGAGCACCAGCACAGCCTATATGGAGCTGAGC
AGCCTGCGAAGCGAGGACACAGCTGTTTACTA
TTGTGCCAGAGAGAGACTGTACGCCGGCTACT
ACTTCGATCACTGGGGCCAGGGGACCCTGGT
GACCGTTTCTTCT
23 CAGGTCCAGCTAGTACAAAGCGGCGCCGAAGT
AAAGAAACCTGGTAGCTCTGTGAAGGTGAGCT
GCAAGGCCAGCGGCGGCACCTTCAGCAGCTA
CGCCTTCAGCTGGGTTCGACAGGCCCCTGGA
ScFV CAGGGCCTAGAGTGGATGGGCAGAATCGTGC
CTGTGGTGGGCACCCCCAACTACGCCCAGAA
GTTCCAGGGCAGAGTTACCATCACCGCCGACG
AGAGCACCAGCACAGCCTATATGGAGCTGAGC
AGCCTGCGAAGCGAGGACACAGCTGTTTACTA
TTGTGCCAGAGAGAGACTGTACGCCGGCTACT
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
ACTTCGATCACTGGGGCCAGGGGACCCTGGT
GACCGTTTCTTCTGGAGGCGGAGGTTCTGGCG
GCGGAGGAAGTGGTGGCGGAGGCTCAGAGAT
TGTGCTGACCCAGAGCCCTGGCACCCTGTCTC
TGAGCCCCGGAGAGAGAGCCACCCTGAGCTG
TCGGGCCAGCCAGAGCGTGAGCAGCAACTAC
CTGGCCTGGTATCAGCAGAAGCCCGGACAGG
CCCCCAGACTGCTGATCTACGGCGCCTCCAGC
AGAGCCACCGGAATCCCCGACAGATTTTCTGG
CAGCGGCTCTGGCACCGACTTCACCCTGACCA
TCTCCAGACTGGAGCCCGAGGACTTCGCCGTG
TACTACTGCCAGCAATACGGCACCAGCCCAGG
CCTGACCTTTGGTCAGGGCACAAAGGTGGAAA
TCAAG
46 ACTACTACCAAGCCAGTGCTGCGAACTCCCTC
ACCTGTGCACCCTACCGGGACATCTCAGCCCC
mCD8-Hinge AGAGACCAGAAGATTGTCGGCCCCGTGGCTCA
GTGAAGGGGACCGGATTGGACTTCGCCTGTGA
T
47 ATTTACATCTGGGCACCCTTGGCCGGAATCTG
mCD8-
CGTGGCCCTTCTGCTGTCCTTGATCATCACTCT
Transmembrane
CATCTTGATCATCACTCTCATC
48 AATAGTAGAAGGAACAGACTCCTTCAAAGTGA
mCD28 Costim- CTACATGAACATGACTCCCCGGAGGCCTGGGC
domain TCACTCGAAAGCCTTACCAGCCCTACGCCCCT
GCCAGAGACTTTGCAGCGTACCGCCCC
49 AGAGCCAAGTTCAGCAGATCCGCCGAAACAGC
CGCCAACTTGCAaGATCCTAACCAGCTGTACAA
CGAGCTGAACCTGGGGAGAAGAGAAGAGTAC
GACGTGCTGGAAAAGAAGAGGGCCAGAGATC
CAGAGATGGGCGGCAAGCAGCAGAGAAGAAG
mCD3z AAACCCTCAAGAGGGCGTGTACAACGCTCTGC
AaAAAGACAAGATGGCCGAGGCCTACAGCGAG
ATCGGAACTAAGGGCGAACGCAGAAGAGGCA
AGGGCCACGATGGACTGTACCAGGGCCTGAG
CACAGCCACCAAGGACACATACGATGCCCTGC
ACATGCAGACACTGGCCCCTAGATAG
50 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCC
TCTGGCTCTGCTTCTTCATGCCGCCAGACCAT
CTTACCCCTACGATGTGCCTGATTATGCATCTG
CAR AGATTGTGCTGACCCAGAGCCCTGGCACCCTG
TCTCTGAGCCCCGGAGAGAGAGCCACCCTGA
GCTGTCGGGCCAGCCAGAGCGTGAGCAGCAA
CTACCTGGCCTGGTATCAGCAGAAGCCCGGAC
AGGCCCCCAGACTGCTGATCTACGGCGCCTCC
96
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
AGCAGAGCCACCGGAATCCCCGACAGATTTTC
TGGCAGCGGCTCTGGCACCGACTTCACCCTGA
CCATCTCCAGACTGGAGCCCGAGGACTTCGCC
GTGTACTACTGCCAGCAATACGGCACCAGCCC
AGGCCTGACCTTTGGTCAGGGCACAAAGGTGG
AAATCAAGGGAGGCGGAGGTTCTGGCGGCGG
AGGAAGTGGTGGCGGAGGCTCACAGGTCCAG
CTAGTACAAAGCGGCGCCGAAGTAAAGAAACC
TGGTAGCTCTGTGAAGGTGAGCTGCAAGGCCA
GCGGCGGCACCTTCAGCAGCTACGCCTTCAG
CTGGGTTCGACAGGCCCCTGGACAGGGCCTA
GAGTGGATGGGCAGAATCGTGCCTGTGGTGG
GCACCCCCAACTACGCCCAGAAGTTCCAGGGC
AGAGTTACCATCACCGCCGACGAGAGCACCAG
CACAGCCTATATGGAGCTGAGCAGCCTGCGAA
GCGAGGACACAGCTGTTTACTATTGTGCCAGA
GAGAGACTGTACGCCGGCTACTACTTCGATCA
CTGGGGCCAGGGGACCCTGGTGACCGTTTCTT
CTATGCATACTACTACCAAGCCAGTGCTGCGA
ACTCCCTCACCTGTGCACCCTACCGGGACATC
TCAGCCCCAGAGACCAGAAGATTGTCGGCCCC
GTGGCTCAGTGAAGGGGACCGGATTGGACTTC
GCCTGTGATATTTACATCTGGGCACCCTTGGC
CGGAATCTGCGTGGCCCTTCTGCTGTCCTTGA
TCATCACTCTCATCTGCTACACAAATAGTAGAA
GGAACAGACTCCTTCAAAGTGACTACATGAAC
ATGACTCCCCGGAGGCCTGGGCTCACTCGAAA
GCCTTACCAGCCCTACGCCCCTGCCAGAGACT
TTGCAGCGTACCGCCCCAGAGCCAAGTTCAGC
AGATCCGCCGAAACAGCCGCCAACtTGCAaGA
TCCTAACCAGCTGTACAACGAGCTGAACCTGG
GGAGAAGAGAAGAGTACGACGTGCTGGAAAA
GAAGAGGGCCAGAGATCCAGAGATGGGCGGC
AAGCAGCAGAGAAGAAGAAACCCTCAAGAGG
GCGTGTACAACGCTCTGCAaAAAGACAAGATG
GCCGAGGCCTACAGCGAGATCGGAACTAAGG
GCGAACGCAGAAGAGGCAAGGGCCACGATGG
ACTGTACCAGGGCCTGAGCACAGCCACCAAG
GACACATACGATGCCCTGCACATGCAGACACT
GGCCCCTAGATAG
[0464] Table 9:0ther linkers - Protein sequence
SE Sequence function Sequence
Q
97
CA 03230229 2024- 2- 27

WO 2023/035002
PCT/US2022/075956
70 (GGS)2 -Linker GGSGGS
71 (G3S)-Linker GGGS
72 (G3S)4-Linker GGGSGGGSGGGSGGGS
73 (G4S)-Linker GGGGS
74 (G4S)2-Linker GGGGSGGGGS
75 (G4S)4-Linker GGGGSGGGGSGGGGSGGGGS
9g
CA 03230229 2024- 2- 27