U.S. patent application number 17/475189 was filed with the patent office on 2022-03-03 for ror1-specific chimeric antigen receptors (car) with humanized targeting domains.
The applicant listed for this patent is JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG. Invention is credited to Michael HUDECEK, Andreas MADES.
Application Number | 20220064258 17/475189 |
Document ID | / |
Family ID | 1000005960310 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064258 |
Kind Code |
A1 |
HUDECEK; Michael ; et
al. |
March 3, 2022 |
ROR1-SPECIFIC CHIMERIC ANTIGEN RECEPTORS (CAR) WITH HUMANIZED
TARGETING DOMAINS
Abstract
The invention relates to chimeric antigen receptors (CAR) with a
humanized targeting domain specific to the antigen ROR1. The
invention encompasses the polynucleotides, vectors encoding said
CARs and the isolated cells expressing them at their surface, in
particularly for their use in immunotherapy.
Inventors: |
HUDECEK; Michael; (Hochberg,
DE) ; MADES; Andreas; (Wiesbaden Biebrich,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG |
Wurzburg |
|
DE |
|
|
Family ID: |
1000005960310 |
Appl. No.: |
17/475189 |
Filed: |
September 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16607069 |
Oct 21, 2019 |
11149073 |
|
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PCT/EP2018/060887 |
Apr 27, 2018 |
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17475189 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70517 20130101;
C07K 14/7051 20130101; A61P 35/00 20180101; C07K 14/70578 20130101;
A61K 35/17 20130101; C07K 16/2803 20130101; C12N 2740/15043
20130101; C07K 2317/24 20130101; C07K 14/70521 20130101; C12N 15/86
20130101; C07K 2319/03 20130101; C12N 5/0636 20130101 |
International
Class: |
C07K 14/725 20060101
C07K014/725; A61P 35/00 20060101 A61P035/00; A61K 35/17 20060101
A61K035/17; C07K 14/705 20060101 C07K014/705; C07K 16/28 20060101
C07K016/28; C12N 5/0783 20060101 C12N005/0783; C12N 15/86 20060101
C12N015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 2017 |
EP |
17168805.4 |
Claims
1. A ROR1-specific CAR comprising: a humanized targeting domain
obtainable by humanization of a ROR1-binding fragment of a
monoclonal antibody capable of binding to ROR1, wherein said
monoclonal antibody is selected from the group consisting of the
monoclonal antibodies R11, R12 and 2A2.
2. The ROR1-specific CAR according to claim 1, wherein said
monoclonal antibody is selected from the group consisting of the
monoclonal antibodies R12 and 2A2.
3. A ROR1-specific CAR comprising a humanized targeting domain
capable of binding to ROR1, wherein the humanized targeting domain
comprises, preferably in an N- to C-terminal order: a) an antibody
heavy chain variable domain amino acid sequence selected from the
group consisting of: a1) the amino acid sequence of SEQ ID No: 1 or
an amino acid sequence at least 90% identical thereto; a2) the
amino acid sequence of SEQ ID No: 3 or an amino acid sequence at
least 90% identical thereto; or a3) the amino acid sequence of SEQ
ID No: 5 or an amino acid sequence at least 90% identical thereto;
and b) an antibody light chain variable domain amino acid sequence
selected from the group consisting of: b1) the amino acid sequence
of SEQ ID No: 2 or an amino acid sequence at least 90% identical
thereto; b2) the amino acid sequence of SEQ ID No: 4 or an amino
acid sequence at least 90% identical thereto; or b3) the amino acid
sequence of SEQ ID No: 6 or an amino acid sequence at least 90%
identical thereto.
4. The ROR1-specific CAR according to claim 3, wherein the
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 1 or an amino
acid sequence at least 90% identical thereto and the antibody light
chain variable domain amino acid sequence of SEQ ID No: 2 or an
amino acid sequence at least 90% identical thereto.
5. The ROR1-specific CAR according to claim 4, wherein the
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 1 and the
antibody light chain variable domain amino acid sequence of SEQ ID
No: 2.
6-12. (canceled)
13. A combination of CARs comprising at least a first and a second
CAR, the combination being ROR1-specific, wherein said first and
said second CAR are present on different polypeptide chains, and
wherein: c) said first CAR comprises a first humanized targeting
domain comprising an antibody heavy chain variable domain amino
acid sequence selected from the group consisting of: c1) the amino
acid sequence of SEQ ID No: 1 or an amino acid sequence at least
90% identical thereto; c2) the amino acid sequence of SEQ ID No: 3
or an amino acid sequence at least 90% identical thereto; or c3)
the amino acid sequence of SEQ ID No: 5 or an amino acid sequence
at least 90% identical thereto; and d) said second CAR comprises a
second humanized targeting domain comprising an antibody light
chain variable domain amino acid sequence selected from the group
consisting of: d1) the amino acid sequence of SEQ ID No: 2 or an
amino acid sequence at least 90% identical thereto; d2) the amino
acid sequence of SEQ ID No: 4 or an amino acid sequence at least
90% identical thereto; or d3) the amino acid sequence of SEQ ID No:
6 or an amino acid sequence at least 90% identical thereto.
14. The combination according to claim 13, wherein the first
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 1 or an amino
acid sequence at least 90% identical thereto and the second
humanized targeting domain comprises the antibody light chain
variable domain amino acid sequence of SEQ ID No: 2 or an amino
acid sequence at least 90% identical thereto.
15. The combination according to claim 14, wherein the first
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 1 and the second
humanized targeting domain comprises the antibody light chain
variable domain amino acid sequence of SEQ ID No: 2.
16. The combination according to claim 13, wherein the first
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 3 or an amino
acid sequence at least 90% identical thereto and the second
humanized targeting domain comprises the antibody light chain
variable domain amino acid sequence of SEQ ID No: 4 or an amino
acid sequence at least 90% identical thereto.
17. The combination according to claim 16, wherein the first
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 3 and the second
humanized targeting domain comprises the antibody light chain
variable domain amino acid sequence of SEQ ID No: 4.
18-32. (canceled)
33. A polynucleotide encoding the ROR1-specific CAR according to
claim 1.
34. A recombinant mammalian cell expressing at least one
ROR1-specific CAR according to claim 1.
35. The recombinant mammalian cell according to claim 34, wherein
said cell is an immune cell.
36-45. (canceled)
46. A method for producing a recombinant mammalian cell according
to claim 34, the method comprising the steps of: (a) providing a
mammalian cell; (b) introducing into said mammalian cell of step
(a) at least one polynucleotide encoding said at least one
ROR1-specific CAR or said combination of CARs; and (c) expressing
said at least one ROR1-specific CAR or said combination of CARs
from said at least one polynucleotide in said cell; thereby
obtaining said recombinant mammalian cell.
47. The method of claim 46, further comprising coculturing the
recombinant mammalian cell with lethally irradiated ROR1-expressing
target cells at a ratio of 4:1 for 72 h in the absence of exogenous
cytokines.
48. The method of claim 47, wherein at least 1%, preferably at
least 2%, more preferably at least 3%, and still more preferably at
least 4% of the recombinant mammalian cells undergo at least 5 cell
divisions during the coculturing.
49. The method according to claim 46, wherein said method is an in
vitro method.
50. The method according to claim 46, wherein said mammalian cells
of step (a) are cells obtained from donors.
51. The method according to claim 46, wherein said mammalian cells
of step (a) are cells obtained from patients.
52. A recombinant mammalian cell according to claim 46 for use in
medicine.
53-57. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to chimeric antigen receptors (CAR)
with a humanized targeting domain specific to the antigen ROR1. The
invention encompasses the polynucleotides, vectors encoding said
CARs and the isolated cells expressing them at their surface, in
particularly for their use in immunotherapy.
BACKGROUND OF THE INVENTION
[0002] The adoptive transfer of genetically modified T cells that
express a T cell receptor or a chimeric antigen receptor (CAR)
specific for a tumor-associated antigen is emerging as an effective
modality for cancer therapy [1-5]. CARs are synthetic receptors
most often constructed by linking a single chain variable fragment
(scFV) of a monoclonal antibody (mAb) specific for a tumor cell
surface molecule to a transmembrane domain, one or more
intracellular costimulatory signaling modules, and CD3.zeta. [6-8].
CAR-modified T-cells confer non-MHC restricted recognition of tumor
cells, and durable responses have been reported in patients with
B-cell malignancies after treatment with autologous T-cells
modified with CARs specific for the B-cell lineage restricted CD19
molecule. The major toxicities in these patients were related to
tumor lysis, cytokine release, and prolonged depletion of normal
B-lymphocytes [1-3, 5, 9]. A challenge in the field is to identify
and validate receptor constructs specific for molecules that are
expressed on a greater number of malignancies including common
epithelial tumors, and that are restricted in their expression to
malignant and not normal cells.
[0003] We have been investigating the receptor tyrosine kinase-like
orphan receptor 1 (ROR1) as a candidate for immunotherapy with
CAR-modified T-cells. ROR1 is a 120-kDa glycoprotein containing
extracellular immunoglobulin (Ig)-like, Frizzled, and Kringle
domains. ROR1 is expressed during embryogenesis but absent from
normal adult tissues, apart from a subset of immature B-cell
precursors, and low-level expression on adipocytes [10, 11]. ROR1
was first shown to be expressed in B-cell chronic lymphocytic
leukemia (B-CLL) by transcriptional profiling [12, 13], and was
subsequently identified on the surface of many cancers including
mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) with
a t(1;19) chromosome translocation, and a subset of lung, breast,
colon, pancreas, renal, and ovarian cancers [14-21]. In both lung
adenocarcinoma and t(1;19) ALL, ROR1 cooperates in oncogenic
signaling, and knockdown of ROR1 with siRNA exposed a critical role
for this molecule in maintaining tumor cell survival [15, 18, 22,
23]. WO 2016/115559 relates to antibodies and chimeric antigen
receptors specific for ROR1.
[0004] Clinical trials with CD19 CARs have demonstrated that a
paramount requirement for therapeutic efficacy is engraftment, in
vivo proliferation and persistence of CAR T cells after adoptive
transfer [24-26] (Clinical trial IDs: NCT02167360, NCT02030847
NCT01865617). In responding patients, CAR T cells undergo
substantial proliferation, to a point where they comprise a
substantial proportion of the patient's T-cell repertoire.
Non-responding patients by contrast have an insufficient CAR T cell
engraftment or the CAR T cell graft is rejected early after
adoptive transfer. CAR T cell proliferation is an `advanced`
effector function, requiring optimal binding of the CAR targeting
domain to the respective antigen. Therefore, to maximize the
potential of CAR T cell therapy it is important to chose a CAR that
exhibits strong antigen binding properties and also mediates high
CAR T cell expansion and long-term survival.
[0005] All CARs that are currently used in the clinic contain scFv
targeting domains that are derived from murine antibodies and the
majority of CARs that are in preclinical development also use
targeting domains of `foreign` origin. It has been noted that these
`foreign` targeting domains contain immunogenic epitopes that are
recognized by the patient's immune system and elicit cellular or
humoral immune responses that eventually mediate CAR-T cell
rejection; see [27], [28] and [29]. Reference [30] describes the
use of targeting domains from human antibodies.
[0006] In summary, there is a need in the art for improved CAR T
cell constructs and related products and methods that can be used
for therapies such as immunotherapies.
DESCRIPTION OF THE INVENTION
[0007] The present invention relates to humanized binding domains
based on the known anti-ROR1 antibodies R11, R12 and 2A2 and to
uses thereof for the construction of CARs and CAR engineered T
cells. See, for instance, reference [14] for a description of the
2A2 antibody, which is hereby incorporated by reference in its
entirety for all purposes; and reference [37] for a description of
the R11/R12 antibodies, which is hereby incorporated by reference
in its entirety for all purposes. The CARs according to the present
invention have a higher degree of "human-ness" of the humanized
R11, R12 and 2A2 binding domains, as opposed to the rabbit (R11
& R12) and mouse (2A2) binding domains. According to the
invention, these CARs and CAR engineered T cells are expected to
exhibit lower immunogenicity in clinical use in patients, as
opposed to the rabbit (R11 & R12) and mouse (2A2) binding
domains.
[0008] According to the invention, humanization can be carried out
by any method that is known in the art. As a non-limiting example,
humanization of the VH and VL domains of R11, R12 and 2A2 anti-ROR1
monoclonal antibodies has been performed by CDR grafting and
selection of best binders for the ROR-1 target by Transpo-mAb
Display. This method has been described for the R11 and 2A2
antibodies in reference [31], which is hereby incorporated by
reference in its entirety for all purposes. According to the
invention, recombinant mammalian cells expressing CARs such as CAR
T cells can be produced to express CARs with the humanized
anti-ROR1 binding domains of monoclonal anti-ROR-1 antibodies R11,
R12 and 2A2.
[0009] Humanization of ROR1-specific CARs is different from the
known clinical approaches which rely on non-humanized ROR1-specific
CARs. The present inventors have now surprisingly shown that
humanized ROR1-specific CARs have higher functionality than their
non-humanized counterparts. This advantage was unexpected, because
humanization is thought to decrease rather than increase
affinity.
[0010] Furthermore, the inventors have surprisingly shown that
humanized CARs with advantageous functional properties can be
produced according to the invention. For instance, CAR T cells
according to the invention are capable of target cell lysis and
exhibit strong proliferation upon stimulation with lethally
irradiated ROR1-expressing target cells.
[0011] Accordingly, the present invention provides the following
preferred embodiments: [0012] 1. A ROR1-specific CAR comprising:
[0013] a humanized targeting domain obtainable by humanization of a
ROR1-binding fragment of a monoclonal antibody capable of binding
to ROR1, wherein said monoclonal antibody is selected from the
group consisting of the monoclonal antibodies R11, R12 and 2A2.
[0014] 2. The ROR1-specific CAR according to item 1, wherein said
monoclonal antibody is selected from the group consisting of the
monoclonal antibodies R12 and 2A2. [0015] 3. A ROR1-specific CAR
comprising a humanized targeting domain capable of binding to ROR1,
wherein the humanized targeting domain comprises, preferably in an
N- to C-terminal order: [0016] a) an antibody heavy chain variable
domain amino acid sequence selected from the group consisting of:
[0017] a1) the amino acid sequence of SEQ ID No: 1 or an amino acid
sequence at least 90% identical thereto; [0018] a2) the amino acid
sequence of SEQ ID No: 3 or an amino acid sequence at least 90%
identical thereto; or [0019] a3) the amino acid sequence of SEQ ID
No: 5 or an amino acid sequence at least 90% identical thereto;
[0020] and [0021] b) an antibody light chain variable domain amino
acid sequence selected from the group consisting of: [0022] b1) the
amino acid sequence of SEQ ID No: 2 or an amino acid sequence at
least 90% identical thereto; [0023] b2) the amino acid sequence of
SEQ ID No: 4 or an amino acid sequence at least 90% identical
thereto; or [0024] b3) the amino acid sequence of SEQ ID No: 6 or
an amino acid sequence at least 90% identical thereto. [0025] 4.
The ROR1-specific CAR according to item 3, wherein the humanized
targeting domain comprises the antibody heavy chain variable domain
amino acid sequence of SEQ ID No: 1 or an amino acid sequence at
least 90% identical thereto and the antibody light chain variable
domain amino acid sequence of SEQ ID No: 2 or an amino acid
sequence at least 90% identical thereto. [0026] 5. The
ROR1-specific CAR according to item 4, wherein the humanized
targeting domain comprises the antibody heavy chain variable domain
amino acid sequence of SEQ ID No: 1 and the antibody light chain
variable domain amino acid sequence of SEQ ID No: 2. [0027] 6. The
ROR1-specific CAR according to item 5, wherein the humanized
targeting domain consists of the following sequences in an N- to
C-terminal order: the antibody heavy chain variable domain amino
acid sequence of SEQ ID No: 1, an amino acid linker sequence which
is preferably the amino acid sequence of SEQ ID No: 8, and the
antibody light chain variable domain amino acid sequence of SEQ ID
No: 2. [0028] 7. The ROR1-specific CAR according to item 3, wherein
the humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 3 or an amino
acid sequence at least 90% identical thereto and the antibody light
chain variable domain amino acid sequence of SEQ ID No: 4 or an
amino acid sequence at least 90% identical thereto. [0029] 8. The
ROR1-specific CAR according to item 7, wherein the humanized
targeting domain comprises the antibody heavy chain variable domain
amino acid sequence of SEQ ID No: 3 and the antibody light chain
variable domain amino acid sequence of SEQ ID No: 4. [0030] 9. The
ROR1-specific CAR according to item 8, wherein the humanized
targeting domain consists of the following sequences in an N- to
C-terminal order: the antibody heavy chain variable domain amino
acid sequence of SEQ ID No: 3, an amino acid linker sequence which
is preferably the amino acid sequence of SEQ ID No: 8, and the
antibody light chain variable domain amino acid sequence of SEQ ID
No: 4. [0031] 10. The ROR1-specific CAR according to item 3,
wherein the humanized targeting domain comprises the antibody heavy
chain variable domain amino acid sequence of SEQ ID No: 5 or an
amino acid sequence at least 90% identical thereto and the antibody
light chain variable domain amino acid sequence of SEQ ID No: 6 or
an amino acid sequence at least 90% identical thereto. [0032] 11.
The ROR1-specific CAR according to item 10, wherein the humanized
targeting domain comprises the antibody heavy chain variable domain
amino acid sequence of SEQ ID No: 5 and the antibody light chain
variable domain amino acid sequence of SEQ ID No: 6. [0033] 12. The
ROR1-specific CAR according to item 11, wherein the humanized
targeting domain consists of the following sequences in an N- to
C-terminal order: the antibody heavy chain variable domain amino
acid sequence of SEQ ID No: 5, an amino acid linker sequence which
is preferably the amino acid sequence of SEQ ID No: 8, and the
antibody light chain variable domain amino acid sequence of SEQ ID
No: 6. [0034] 13. A combination of CARs comprising at least a first
and a second CAR, the combination being ROR1-specific, wherein said
first and said second CAR are present on different polypeptide
chains, and wherein: [0035] c) said first CAR comprises a first
humanized targeting domain comprising an antibody heavy chain
variable domain amino acid sequence selected from the group
consisting of: [0036] c1) the amino acid sequence of SEQ ID No: 1
or an amino acid sequence at least 90% identical thereto; [0037]
c2) the amino acid sequence of SEQ ID No: 3 or an amino acid
sequence at least 90% identical thereto; or [0038] c3) the amino
acid sequence of SEQ ID No: 5 or an amino acid sequence at least
90% identical thereto; [0039] and [0040] d) said second CAR
comprises a second humanized targeting domain comprising an
antibody light chain variable domain amino acid sequence selected
from the group consisting of: [0041] d1) the amino acid sequence of
SEQ ID No: 2 or an amino acid sequence at least 90% identical
thereto; [0042] d2) the amino acid sequence of SEQ ID No: 4 or an
amino acid sequence at least 90% identical thereto; or [0043] d3)
the amino acid sequence of SEQ ID No: 6 or an amino acid sequence
at least 90% identical thereto. [0044] 14. The combination
according to item 13, wherein the first humanized targeting domain
comprises the antibody heavy chain variable domain amino acid
sequence of SEQ ID No: 1 or an amino acid sequence at least 90%
identical thereto and the second humanized targeting domain
comprises the antibody light chain variable domain amino acid
sequence of SEQ ID No: 2 or an amino acid sequence at least 90%
identical thereto. [0045] 15. The combination according to item 14,
wherein the first humanized targeting domain comprises the antibody
heavy chain variable domain amino acid sequence of SEQ ID No: 1 and
the second humanized targeting domain comprises the antibody light
chain variable domain amino acid sequence of SEQ ID No: 2. [0046]
16. The combination according to item 13, wherein the first
humanized targeting domain comprises the antibody heavy chain
variable domain amino acid sequence of SEQ ID No: 3 or an amino
acid sequence at least 90% identical thereto and the second
humanized targeting domain comprises the antibody light chain
variable domain amino acid sequence of SEQ ID No: 4 or an amino
acid sequence at least 90% identical thereto. [0047] 17. The
combination according to item 16, wherein the first humanized
targeting domain comprises the antibody heavy chain variable domain
amino acid sequence of SEQ ID No: 3 and the second humanized
targeting domain comprises the antibody light chain variable domain
amino acid sequence of SEQ ID No: 4. [0048] 18. The combination
according to item 13, wherein the first humanized targeting domain
comprises the antibody heavy chain variable domain amino acid
sequence of SEQ ID No: 5 or an amino acid sequence at least 90%
identical thereto and the second humanized targeting domain
comprises the antibody light chain variable domain amino acid
sequence of SEQ ID No: 6 or an amino acid sequence at least 90%
identical thereto. [0049] 19. The combination according to item 18,
wherein the first humanized targeting domain comprises the antibody
heavy chain variable domain amino acid sequence of SEQ ID No: 5 and
the second humanized targeting domain comprises the antibody light
chain variable domain amino acid sequence of SEQ ID No: 6. [0050]
20. The ROR1-specific CAR according to any one of the preceding
items, wherein the CAR further comprises a costimulatory domain
capable of mediating costimulation to immune cells; or the
combination according to any one of the preceding items, wherein at
least the first or the second CAR of the combination, preferably at
least the first and the second CAR of the combination, most
preferably all of the CARs of the combination, further comprise a
costimulatory domain capable of mediating costimulation to immune
cells. [0051] 21. The ROR1-specific CAR or the combination
according to item 20, wherein said costimulatory domain is from
4-1BB, CD28, Ox40, ICOS or DAP10. [0052] 22. The ROR1-specific CAR
or the combination according to item 21, wherein said costimulatory
domain has the amino acid sequence of SEQ ID No: 12. [0053] 23. The
ROR1-specific CAR according to any one of the preceding items,
further comprising a transmembrane polypeptide; or the combination
according to any one of the preceding items, wherein at least the
first or the second CAR of the combination, preferably at least the
first and the second CAR of the combination, most preferably all of
the CARs of the combination, further comprise a transmembrane
polypeptide. [0054] 24. The ROR1-specific CAR or the combination
according to item 23, wherein said transmembrane polypeptide is a
transmembrane domain from CD4, CD8 or CD28. [0055] 25. The
ROR1-specific CAR or the combination according to item 24, wherein
said transmembrane domain has the amino acid sequence of SEQ ID No:
11. [0056] 26. The ROR1-specific CAR according to any one of the
preceding items, further comprising a CAR spacer domain; or the
combination according to any one of the preceding items, wherein at
least the first or the second CAR of the combination, preferably at
least the first and the second CAR of the combination, most
preferably all of the CARs of the combination, further comprise a
CAR spacer domain. [0057] 27. The ROR1-specific CAR or the
combination according to item 26, wherein said CAR spacer domain is
from CD4, CD8, an FC-receptor, an immunoglobulin, or an antibody.
[0058] 28. The ROR1-specific CAR or the combination according to
item 27, wherein said CAR spacer domain has the amino acid sequence
of SEQ ID No: 9 or SEQ ID No: 10, preferably the amino acid
sequence of SEQ ID No: 9. [0059] 29. The ROR1-specific CAR
according to any one of the preceding items, further comprising a
suicide gene product that allows the selective killing of CAR T
cells; or the combination according to any one of the preceding
items, wherein at least the first or the second CAR of the
combination further comprises a suicide gene product that allows
the selective killing of CAR T cells. [0060] 30. The ROR1-specific
CAR or the combination according to item 29, wherein said suicide
gene product is iCasp9 or HSV-TK. [0061] 31. The ROR1-specific CAR
according to any one of the preceding items, comprising, in an N-
to C-terminal order: i) a signal peptide for direction into the
endoplasmic reticulum, the signal peptide preferably having the
amino acid sequence of SEQ ID No: 7; ii) said humanized targeting
domain; iii) the CAR spacer domain according to any one of items 26
to 28; iv) the transmembrane polypeptide according to any one of
items 23-25; v) the costimulatory domain according to any one of
items 20-22; vi) a CD3z signaling domain preferably having the
amino acid sequence of SEQ ID No: 13; and optionally further
comprising: vii) a T2A ribosomal skipping sequence preferably
having the amino acid sequence of SEQ ID No: 14; viii) a signal
peptide for direction into the endoplasmic reticulum, the signal
peptide preferably having the amino acid sequence of SEQ ID No: 7;
and ix) a detectable marker protein sequence preferably having the
EGFRt amino acid sequence of SEQ ID No: 15. [0062] 32. The
ROR1-specific CAR according to item 31, consisting of said
components i) to ix). [0063] 33. A polynucleotide encoding the
ROR1-specific CAR according to any one of the preceding items.
[0064] 34. A recombinant mammalian cell expressing at least one
ROR1-specific CAR according to any one of the preceding items or
expressing the combination of CARs according to any one of the
preceding items. [0065] 35. The recombinant mammalian cell
according to item 34, wherein said cell is an immune cell. [0066]
36. The recombinant mammalian cell according to any one of the
preceding items, wherein said cell is a lymphocyte. [0067] 37. The
recombinant mammalian cell according to any one of the preceding
items, wherein said lymphocyte is a B lymphocyte or T lymphocyte.
[0068] 38. The recombinant mammalian cell according to item 34,
wherein said cell is a CD8.sup.+ killer T cell, a CD4.sup.+ helper
T cell, a naive T cell, a memory T cell, a central memory T cell,
an effector memory T cell, a memory stem T cell, an invariant T
cell, an NKT cell, a cytokine induced killer T cell, a g/d T cell,
a natural killer cell, a monocyte, a macrophage, a dendritic cell,
or a granulocyte. [0069] 39. The recombinant mammalian cell
according to any one of the preceding items, wherein the cell is a
human cell. [0070] 40. The recombinant mammalian cell according to
any one of the preceding items, wherein the recombinant mammalian
cell is capable of at least one cell division when cocultured with
lethally irradiated ROR1-expressing target cells at an E:T ratio of
4:1 for 72 h in the absence of exogenous cytokines. [0071] 41. The
recombinant mammalian cell according to any one of the preceding
items, wherein the recombinant mammalian cell is capable of at
least two cell divisions when cocultured with lethally irradiated
ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the
absence of exogenous cytokines. [0072] 42. The recombinant
mammalian cell according to any one of the preceding items, wherein
the recombinant mammalian cell is capable of at least three cell
divisions when cocultured with lethally irradiated ROR1-expressing
target cells at an E:T ratio of 4:1 for 72 h in the absence of
exogenous cytokines. [0073] 43. The recombinant mammalian cell
according to any one of the preceding items, wherein the
recombinant mammalian cell is capable of at least four cell
divisions when cocultured with lethally irradiated ROR1-expressing
target cells at an E:T ratio of 4:1 for 72 h in the absence of
exogenous cytokines. [0074] 44. The recombinant mammalian cell
according to any one of the preceding items, wherein the
recombinant mammalian cell is capable of at least five cell
divisions when cocultured with lethally irradiated ROR1-expressing
target cells at an E:T ratio of 4:1 for 72 h in the absence of
exogenous cytokines. [0075] 45. The recombinant mammalian cell
according to any one of the preceding items, wherein the
ROR1-specific CAR or the ROR1-specific combination of CARs is
capable of binding to ROR1 with higher binding affinity than a
respective recombinant mammalian control cell expressing a
respective ROR1-specific CAR or a respective ROR1-specific
combination of CARs where none of the targeting domains is
humanized, and wherein said binding affinity is binding affinity to
fluorescently labelled recombinant aggregated ROR1 as assessed by
flow cytometry analysis.
[0076] 46. A method for producing a recombinant mammalian cell
according to any one of the preceding items, the method comprising
the steps of: [0077] (a) providing a mammalian cell; [0078] (b)
introducing into said mammalian cell of step (a) at least one
polynucleotide encoding said at least one ROR1-specific CAR or said
combination of CARs; and [0079] (c) expressing said at least one
ROR1-specific CAR or said combination of CARs from said at least
one polynucleotide in said cell; [0080] thereby obtaining said
recombinant mammalian cell. [0081] 47. The method of item 46,
further comprising coculturing the recombinant mammalian cell with
lethally irradiated ROR1-expressing target cells at a ratio of 4:1
for 72 h in the absence of exogenous cytokines. [0082] 48. The
method of item 47, wherein at least 1%, preferably at least 2%,
more preferably at least 3%, and still more preferably at least 4%
of the recombinant mammalian cells undergo at least 5 cell
divisions during the coculturing. [0083] 49. The method according
to any one of the preceding items, wherein said method is an in
vitro method. [0084] 50. The method according to any one of the
preceding items, wherein said mammalian cells of step (a) are cells
obtained from donors. [0085] 51. The method according to any one of
the preceding items, wherein said mammalian cells of step (a) are
cells obtained from patients. [0086] 52. A recombinant mammalian
cell according to any one of the preceding items for use in
medicine. [0087] 53. A recombinant mammalian cell according to any
one of the preceding items for use in a method for treating a
cancer that expresses ROR1 in a patient. [0088] 54. The recombinant
mammalian cell according to item 53 for the use according to item
53, wherein the cancer is ROR1-positive leukemia, mantle cell
lymphoma, breast-cancer or lung cancer. [0089] 55. A method for
treating a patient in need thereof, the method comprising
administering a recombinant mammalian cell according to any one of
the preceding items to said patient. [0090] 56. The method
according to item 55, wherein the method is for treating a cancer
that expresses ROR1. [0091] 57. The method according to item 56,
wherein the cancer is ROR1-positive leukemia, mantle cell lymphoma,
breast-cancer or lung cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0092] FIGS. 1A-1B: Amino acid sequences of the humanized 2A2 VH
and VL sequences and the complete h2A2 CAR coding sequence
[0093] FIG. 1A Humanized 2A2 VH and VL amino acid sequences
[0094] FIG. 1B Complete amino acid sequence of the h2A2 CAR,
represented in an N- to C-terminal order. Note that the asterisk
denotes the end of the amino acid sequence.
[0095] FIGS. 2A-2B: Amino acid sequences of the humanized R11 VH
and VL sequences and the complete R11 CAR coding sequence
[0096] FIG. 2A Humanized R11 VH and VL amino acid sequences
[0097] FIG. 2B Complete amino acid sequence of the R11 CAR,
represented in an N- to C-terminal order. Note that the asterisk
denotes the end of the amino acid sequence.
[0098] FIGS. 3A-3B: Amino acid sequences of the humanized R12 VH
and VL sequences and the complete R12 CAR coding sequence
[0099] FIG. 3A Humanized R12 VH and VL amino acid sequences
[0100] FIG. 3B Complete amino acid sequence of the R12 CAR,
represented in an N- to C-terminal order. Note that the asterisk
denotes the end of the amino acid sequence.
[0101] FIGS. 4A-4B: Enrichment and detection of CAR by EGFRt
transduction marker
[0102] Human CD4 or CD8 positive T cells were transduced with
lentiviral vector encoding a humanized or non-humanized ROR1 CAR
and subsequently enriched for CAR expressing cells by magnetic
activated cell sorting (MACS) making use of the truncated epidermal
growth factor receptor (EGFRt) transduction marker. The coding
sequence (CDS) for EGFRt is linked to the CAR CDS by a 2A ribosomal
skipping sequence and expression of EGFRt can be used as a
surrogate marker for CAR expression.
[0103] FIG. 4A Flow cytometry plots demonstrating the frequency of
EGFRt-positive CD4.sup.+ T cells after EGFRt enrichment by
MACS.
[0104] FIG. 4B Flow cytometry plots demonstrating the frequency of
EGFRt-positive CD8.sup.+ T cells after EGFRt enrichment by
MACS.
[0105] FIGS. 5A-5B: Cytolytic activity of hROR1 CAR-expressing T
cells
[0106] FIG. 5A Cytolytic activity of primary human CD8.sup.+ T
cells expressing the indicated non-humanized or humanized
ROR1-specific CARs against ROR1-positive target cells. K562 is a
ROR1-negative human leukemia cell line that was used as negative
control. K562-ROR1 originates from the same cell line but has been
engineered to stably express ROR1. MDA-MB-231 and A549 are human
breast and lung cancer cell lines that endogenously express ROR1.
All target cell lines were engineered to stably express a firefly
(P. pyralis) luciferase. The specific lysis of the target cells was
calculated based on the intensity of the luminescence signal after
addition of Luciferin to a final concentration of 150 .mu.g/ml.
[0107] FIG. 5B Summary of the cytolytic activity of human T cells
expressing a non-humanized or humanized ROR1-specific CAR against
two ROR1 positive target cells lines K562-ROR1 and MDA-MB-231. The
data was collected from n=3 independent experiments. The specific
lysis was calculated based on the luminesce intensity of
ffluc-positive target cells after 6 h incubation with an E:T ratio
of 10:1.
[0108] FIGS. 6A-6C: Cytokine secretion of hROR1 CAR-expressing T
cells
[0109] CD4.sup.+ or CD8.sup.+ CAR T cells expressing a
non-humanized or humanized ROR-specific CAR were co-cultured with
lethally irradiated ROR1-positive target cells at an E:T ratio of
4:1. Concentrations of the effector cytokines IL-2 and IFN-.gamma.
were measured by ELISA in the cell culture supernatants after 24 h
co-culture.
[0110] FIG. 6A Comparison of cytokine secretion from 2A2 and h2A2
CAR T cells
[0111] FIG. 6B Comparison of cytokine secretion from R11 and hR11
CAR T cells
[0112] FIG. 6C Comparison of cytokine secretion from R12 and hR12
CAR T cells
[0113] FIGS. 7A-7C: Proliferation of hROR1 CAR-expressing T cells
Proliferation of CD4.sup.+ ROR1-specific CAR T cells after
stimulation with ROR1-positive target cells at an E:T ratio of 4:1.
No exogenous cytokines were added to the culture media and the T
cell proliferation was assessed by CFSE dye dilution 72 h after
stimulation. For analysis, triplicate wells were pooled and the
proliferation of live 7AAD.sup.-, CD4.sub.+ T cells was
analyzed.
[0114] FIG. 7A CFSE flow cytometry histograms of ROR1 CAR T cells
with humanized (solid line) or non-humanized (dashed line). Grey
filled curves are from vector control T cells (EGFRt).
[0115] FIG. 7B Division indices of indicated ROR1 CAR T cells
[0116] FIG. 7C Table that summarized the percentages of T cells
that went through 0, 1, 2, 3, 4, and 5 cell division cycles.
[0117] FIG. 8: Binding of ROR1 protein by hROR1 CAR T cells
[0118] Human CD8.sup.+ T cells expressing non-humanized or
humanized ROR1 CARs were collected, washed with PBS, 0.25% FCS and
then incubated 10 min in the same buffer containing a final
concentration of 5.3 .mu./ml AlexaFluor 647-labeled aggregated ROR1
protein and monoclonal .alpha.EGFR antibody. Afterwards the cells
were washed with PBS, 0.25% FCS and analyzed by flow cytometry.
[0119] FIGS. 9A-9B: In vivo activity of hROR1 CAR T cells
[0120] NSG mice were inoculated with ROR1-expressing Jeko-1 mantle
cell lymphoma lines and received a treatment of humanized R12 or
2A2 CAR T cells 21 days later.
[0121] FIG. 9A Mice were injected with luciferin at day 28 and
radiance signals, which are emitted by ffluc-positive tumor cell,
were detected. Displayed is the change of average radiance per
mouse for the four best responding mice of each group compared to
the baseline signals, which were measured on day 21.
[0122] FIG. 9B Flow cytometry plots showing the frequency of
EGFRt-positive CD4.sup.+ and CD8.sup.+ T cells after EGFRt in the
bone marrow of mice that received hR12 or h2A2 ROR1 CAR T cells at
day 56.
DETAILED DESCRIPTION OF THE INVENTION
[0123] Unless specifically defined herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by a skilled artisan in the fields of gene therapy,
immunology, biochemistry, genetics, and molecular biology.
[0124] All methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, with suitable methods and materials being
described herein. All publications, patents and patent applications
cited herein are hereby incorporated by reference in their entirety
for all purposes. References referred to herein are indicated by a
reference number in square brackets (e.g. as "[31]" or as
"reference [31]"), which refers to the respective reference in the
list of references at the end of the description. In case of
conflict, the present specification, including definitions, will
prevail over the cited references. Further, the materials, methods,
and examples are illustrative only and are not intended to be
limiting, unless otherwise specified.
[0125] Antibodies of non-human origin can be humanized by CDR
grafting by methods known in the art. The humanization increases
the homology of the binding domains to human antibody binding
domains (i.e. the humanness), and reduces the immunogenic potential
of the humanized antibody in human beings, which in turn is
expected to increase the safety and therapeutic application profile
in human patients. On the other hand, antibody humanization is
often accompanied by a reduction of the binding affinity of the
humanized antibody to its antigen, often requiring tedious affinity
maturation, see reference [32], which is hereby incorporated by
reference in its entirety for all purposes. It has also been
experienced that the use of humanized antibody fragments for the
generation of a targeting domain of a CAR can result in a lower
performance of the CAR in respect to binding to the target antigen
and triggering effector functions of the CAR expressing cell.
[0126] In the present invention the inventors have fused humanized
VH and VL domains, as exemplified in FIGS. 1A-3B, of antibodies
originating from the anti-ROR1-specific antibody clones 2A2 (Mus
musculus, WO2010/124188, which is hereby incorporated by reference
in its entirety for all purposes), R11 and R12 (Oryctolagus
cuniculus, WO2012/075158, which is hereby incorporated by reference
in its entirety for all purposes). The humanized VH and VL domains
of these antibodies were then used to design single chain variable
fragments (scFvs) that were further used for the design of CARs
targeting the ROR1 antigen. Gene transfer vectors were created that
allow the transduction or transfection of primary human T cells
with the humanized CARs and allow the production of a CAR T cell
product. The effector functions of the CAR T cells expressing ROR1
CARs with humanized ROR1 targeting domains were compared to their
non-humanized counterparts. The humanized ROR1 CARs h2A2 and hR12
have conferred unexpected superior effector functions compared to
their non-humanized counterparts.
[0127] The present invention describes for the first time the
generation of humanized ROR1 CARs and their usage to redirect
immune cells for the killing of ROR1 expressing target cells.
Unexpectedly, the observed activity of two of the humanized CARs,
namely h2A2 and hR12, was higher than the non-humanized forms in
functional T cells assays with ROR1-expressing target cells. In
contrast, the CAR that was constructed with an scFv targeting
domain that originated from the R11 monoclonal antibody showed a
comparatively strong reduction of effector functions, thus
demonstrating the commonly expectable decline in therapeutic
potential.
[0128] The finding of the present invention, that humanized ROR1
CARs can mediate antigen-specific effector functions to immune
cells that are superior to those of the non-humanized counterparts
is unexpected and has, to the inventors' knowledge, not been
disclosed in the prior art. This finding is also unexpected,
because antibody humanization is often accompanied by a loss of
affinity, and/or a reduction of affinity to the target antigen.
CARs whose targeting domain originates from such humanized
antibodies oftentimes also exhibit lower effector functions and
less therapeutic potential. It could thus neither be anticipated
nor expected that our humanized ROR1 CARs demonstrate effector
functions that are superior to their non-humanized
counterparts.
[0129] The significantly higher function in combination with the
anticipated lower immunogenicity of our novel humanized ROR1 CARs
provides a substantial advantage for the clinical application of
these CARs, especially, but not limited to, their usage in the
context of immunotherapy against cancer.
[0130] A "recombinant mammalian cell" according to the invention
can be any cell as defined herein. Preferably, a recombinant
mammalian cell is an isolated cell. Recombinant mammalian cells
according to the invention can be produced in accordance with known
pharmaceutical standards. For instance, they can be formulated for
administration to humans.
[0131] A ROR1-specific CAR or a combination of CARs according to
the invention can be any possible form. In a preferred embodiment,
the ROR1-specific CAR or combination of CARs is present in an
isolated form. In another preferred embodiment the ROR1-specific
CAR or combination of CARs according to the invention can be
present in a composition, The composition may be a pharmaceutical
composition.
[0132] Sequence alignments of sequences according to the invention
are performed by suitable algorithms, and preferably by using the
BLAST algorithm, see references [33, 34], using suitable alignment
parameters as known in the art.
[0133] As used herein, each occurrence of terms such as
"comprising" or "comprises" may optionally be substituted with
"consisting of" or "consists of".
[0134] The sequences corresponding to the SEQ IDs referred to
herein are indicated in FIGS. 1A-3B and in the following
tables:
TABLE-US-00001 GMCSF signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID
No: 7) h2A2 heavy chain variable
EVQLVQSGAEVKKPGASVKVSCKASGYTFSDYEMHWVRQAPGQGLEWLGAIDPETGGTAYN
domain (VH)
QKFKGRVTMTGDTSISTAYMELSRLTSDDTAVYYCTGYYDYDSFTYWGQGTLVSVSS (SEQ ID
No: 1) 4(GS)x3 linker GGGGSGGGGSGGGGS (SEQ ID No: 8) h2A2 light
chain variable
DIQMTQSPSSLSTSVGDRVTITCKASQNVDAAVAWYQQKPGKAPKLLIYSASNRYTGVASR
domain (VL) FSGSGSGTDFTFTISSLQSEDLADYFCQQYDIYPYTFGQGTKLEIK (SEQ ID
No: 2) IgG4 hinge domain ESKYGPPCPPCP (SEQ ID No: 9) CD28
transmembrane domain MFWLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID No: 11)
4-1BB constimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
(SEQ ID No: 12) domain CD3z signaling domain
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE
LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No: 13)
T2A ribosomal skipping LEGGGEGRGSLLTCGDVEENPGPR (SEQ ID No: 14)
sequence GMCSF signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID No: 7)
EGFRt RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDI
LKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKE
ISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCW
GPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPD
NCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPT
NGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID No: 15) GMCSF signal peptide
MLLLVTSLLLCELPHPAFLLIP (SEQ ID No: 7) hR11 heavy chain variable
EVQLVQSGGGLVQPGGSLRLSCAASGSDINDYPISWVRQAPGKGLEWVSFINSGGSTWYAS
domain (VH)
WVKGRFTISRDNAKNSLYLQMNSLRDDDTATYFCARGYSTYYGDFNIWGQGTLVTVSS (SEQ ID
No: 3) 4(GS)x3 linker GGGGSGGGGSGGGGS (SEQ ID No: 8) hR11 light
chain variable
DIVMTQSPSSLSASVGDRVTITCQASQSIDSNLAWFQQKPGKAPKSLIYRASNLASGVPSK
domain (VL) FSGSGSGTDFTLTISSLQREDAATYYCLGGVGNVSYRTSFGGGTKVEIK (SEQ
ID No: 4) IgG4 CH2CH3 4/2NQ
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG
QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID No: 10) CD28
transmembrane domain MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID No: 11)
4-1BB constimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
(SEQ ID No: 12) domain CD3z signaling domain
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE
LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No: 13)
T2A ribosomal skipping LEGGGEGRGSLLTCGDVEENPGPR (SEQ ID No: 14)
sequence GMCSF signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID No: 7)
EGRFt RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDI
LKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKE
ISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCW
GPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPD
NCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPT
NGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID No: 15) GMCSF signal peptide
MLLLVTSLLLCELPHPAFLLIP (SEQ ID No: 7) hR12 heavy chain variable
QVQLVESGGALVQPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYAAS
domain (VH)
VQGRFTISADNAKNTVYLQMNSLTAADTATYFCARDSYADDGALFNIWGQGTLVTVSS (SEQ ID
No: 5) 4(GS)x3 linker GGGGSGGGGSGGGGS (SEQ ID NO: 8) hR12 light
chain variable
QLVLTQSPSVSAALGSSAKITCTLSSAHKTDTIDWYQQLAGQAPRYLMYVQSDGSYEKRSGVP
domain (VL) DRFSGSSSGADRYLIISSVQADDEADYYCGADYIGGYVFGGGTQLTVG (SEQ
ID No: 6) IgG4 hinge domain ESKYGPPCPPCP (SEQ ID No: 9) CD28
transmembrane domain MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID No: 11)
4-1BB costimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ
ID No: 12) domain CD3z signaling domain
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No: 13)
T2A ribosomal skipping LEGGGEGRGSLLTCGDVEENPGPR (SEQ ID No: 14)
sequence GMCSF signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID No: 7)
EGFRt
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILK
TVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDG
DVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
CVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHY
IDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIAT
GMVGALLLLLVVALGIGLFM (SEQ ID No: 15)
[0135] Additionally, preferred amino acid sequences of ROR1-binding
fragments of the non-humanized monoclonal antibodies R11, R12 and
2A2 that can be used as starting sequences for humanization in
accordance with the invention are as indicated below:
TABLE-US-00002 scFV of the non-humanized 2A2 antibody (SEQ ID No:
16): QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEW
IGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVY
YCTGYYDYDSFTYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSQ
KIMSTTVGDRVSITCKASQNVDAAVAWYQQKPGQSPKLLIYSASNRY
TGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIYPYTFGGGT KLEIK scFV of the
non-humanized R11 antibody (SEQ ID No: 17):
QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWI
GFINSGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCAR
GYSTYYGDFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVMTQTPSS
TSGAVGGTVTINCQASQSIDSNLAWFQQKPGQPPTLLIYRASNLASG
VPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNVSYRTSFGGG TEVVVK scFV of the
non-humanized R12 antibody (SEQ ID No: 18):
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEW
IATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATY
FCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLT
QSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQS
DGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGG YVFGGGTQLTVTG VH of
the non-humanized 2A2 antibody (SEQ ID No: 19):
QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEW
IGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVY
YCTGYYDYDSFTYWGQGTLVTVSA VL of the non-humanized 2A2 antibody (SEQ
ID No: 20): DIVMTQSQKIMSTTVGDRVSITCKASQNVDAAVAWYQQKPGQSPKLL
IYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIY PYTFGGGTKLEIK VH of
the non-humanized R11 antibody (SEQ ID No: 21):
QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWI
GFINSGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCAR
GYSTYYGDFNIWGPGTLVTISS VL of the non-humanized R11 antibody (SEQ ID
No: 22): ELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQKPGQPPTLL
IYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGN VSYRTSFGGGTEVVVK VH
of the non-humanized R12 antibody (SEQ ID No: 23):
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEW
IATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATY
FCARDSYADDGALFNIWGPGTLVTISS VL of the non-humanized R12 antibody
(SEQ ID No: 24): ELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYL
MQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGA
DYIGGYVFGGGTQLTVTG
Further Preferred Embodiments
[0136] A preferred embodiment of the humanized CARs of the
invention is their application in cellular immunotherapy against
malignancies that are associated with the aberrant occurrence of
ROR1-expressing cells. Preferably, the CAR modified cell is a
CD8.sub.+ killer T cell, a CD4.sub.+ helper T cell, a naive T cell,
a memory T cell, a central memory T cells, an effector memory T
cell, a memory stem T cell, an invariant T cell, an NKT cell, a
cytokine induced killer T cell, a gamma/delta T cell, a B
lymphocyte, a natural killer cell, a monocyte, a macrophage, a
dendritic cell, a granulocyte, or any other mammalian cell type
desirable to be used for genetic modification.
[0137] A particularly preferred embodiment is the usage use of CARs
of the invention with humanized targeting domains originating from
the 2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents
against ROR1-positive leukemia, mantle cell lymphoma,
breast-cancer, lung cancer or any other cancer that expresses
ROR1.
[0138] Another preferred embodiment is the usage use of CARs of the
invention with humanized targeting domains originating from the
2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents for
the treatment of obesity.
[0139] Another preferred embodiment is the usage use of CARs of the
invention with humanized targeting domains originating from the
2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents
against ROR1-positive autoimmune or infectious diseases.
[0140] Another preferred embodiment is the use of the humanized
targeting domains of the invention as component of CARs containing
a single costimulatory domain, including but not limited to 4-1BB,
CD28, Ox40, ICOS, DAP10 or any other domain that provides
costimulation to immune cells.
[0141] In another embodiment the humanized targeting domains of the
invention may be used as components of a CAR that mediates an
inhibitory signal due to the usage of a co-inhibitory signaling
domain. Such signaling domains can originate from the co-inhibitory
receptors CTLA-4, PD-1, BTLA, LAGS, TIM3 or any other receptor that
inhibits immune cell functions.
[0142] Another preferred embodiment is the usage of the humanized
targeting domains of the invention in a CAR that encompasses a
combination of two or more costimulatory or co-inhibitory
domains.
[0143] In another embodiment, the humanized targeting domains of
the invention may be used in a format that is different from the
presented scFv format to be included into a CAR construct. As a
non-limiting example such CARs may be composed of two different
polypeptide chains from which one chain encompasses the variable
heavy chain (VH) and one chain encompasses the variable light (VL)
chain of the disclosed humanized anti-ROR1 antibodies.
[0144] In another preferred embodiment the CAR gene, containing a
humanized targeting domain of the invention, is transferred into
the desired cells by non-viral transfection methods like
electroporation, nucleofection or together with a transposase like
Sleeping Beauty, PiggyBac, Frog Prince, Himar1, Passport, Minos,
hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmar1,
or any of their respective derivatives with equal, lower and/or
higher transposition activity.
[0145] In another preferred embodiment the CAR gene encompassing a
humanized targeting domain originating of the invention is
delivered as a part of a RNA or DNA polynucleotide molecule.
EXAMPLES
[0146] The present invention is exemplified by the following
non-limiting examples:
Example 1: Preparation and Functional Testing of ROR1-Specific
CAR-Modified Human CD8.sub.+ and CD4.sub.+ T Cells with Humanized
Targeting Domains
Materials and Methods:
Human Subjects
[0147] Blood samples were obtained from healthy donors who provided
written informed consent to participate in research protocols
approved by the Institutional Review Board of the University of
Wurzburg (Universitatsklinikum Wurzburg, UKW). Peripheral blood
mononuclear cells (PBMC) were isolated by centrifugation over
Ficoll-Hypaque (Sigma, St. Louis, Mo.).
Cell Lines
[0148] The 293T, K562, MDA-MB-231 and A549 cell lines were obtained
from the American Type Culture Collection. K562-ROR1 were generated
by lentiviral transduction with the full-length ROR1-gene.
Luciferase expressing lines were derived by lentiviral transduction
of the above mentioned cell lines with the firefly (P. pyralis)
luciferase (ffluc)-gene. The cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
100 U/ml penicillin/streptomycin.
Immunophenotyping
[0149] PBMC and T cell lines were stained with one or more of the
following conjugated mAb: CD3, CD4, CD8 and matched isotype
controls (BD Biosciences, San Jose, Calif.). Transduced T cell
lines were stained with biotin-conjugated anti-EGFR antibody
(ImClone Systems Incorporated, Branchburg, N.J.) and
streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with
7-AAD (BD Biosciences) was performed for live/dead cell
discrimination as directed by the manufacturer. Flow analyses were
done on a FACSCanto and data analyzed using FlowJo software
(Treestar, Ashland, Oreg.).
Lentiviral Vector Construction, Preparation of Lentivirus, and
Generation of CAR-T Cells
[0150] The construction of epHIV7 lentiviral vectors containing
ROR1-specific CARs with a short or long spacer and a 4-1BB
costimulatory domain has been described, see reference [35], which
is hereby incorporated by reference in its entirety for all
purposes. All CAR constructs encoded a truncated epidermal growth
factor receptor (EGFRt; also known as tEGFR), see reference [36],
which is hereby incorporated by reference in its entirety for all
purposes, downstream of the CAR. The genes were linked by a T2A
ribosomal skip element.
[0151] CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants
were produced in 293T cells co-transfected with each of the
lentiviral vector plasmids and the packaging vectors pCHGP-2,
pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech,
Mountain View, Calif.). Medium was changed 16 h after transfection,
and lentivirus collected after 72 h. CAR-T cells were generated as
described [35]. In brief, CD8.sup.+ or CD4.sup.+ bulk T cells were
sorted from PBMC of healthy donors, activated with anti-CD3/CD28
beads (Life Technologies), and transduced with lentiviral
supernatant. Lentiviral transduction was performed on day 2 by
spinoculation, and T cells propagated in RPMI-1640 with 10% human
serum, GlutaminMAX (Life technologies), 100 U/mL
penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was
performed to quantify viable T cells. After expansion, EGFRt.sup.+
T cells were enriched and expanded by polyclonal stimulation with
the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and
EBV-LCL feeder cells.
Cytotoxicity, Cytokine Secretion, and CFSE Proliferation Assays
[0152] Target cells stably expressing firefly luciferase were
incubated in triplicate at 5.times.10.sup.3 cells/well with
effector T cells at various effector to target (E:T) ratios. After
a four-hour incubation luciferin substrate was added to the
co-culture and the decrease in luminescence signal in wells that
contained target cells and T cells, compared to target cells alone,
measured using a luminometer (Tecan). Specific lysis was calculated
using the standard formula. For analysis of cytokine secretion,
5.times.10.sup.4 T cells were plated in triplicate wells with
target cells at a ratio of 4:1 and IFN-.gamma., TNF-.alpha., and
IL-2 measured by ELISA (Biolegend) in supernatant removed after a
24-hour incubation. For analysis of proliferation, 5.times.10.sup.4
T cells were labeled with 0.2 .mu.M carboxyfluorescein succinimidyl
ester (CFSE, Invitrogen), washed and plated in triplicate wells
with target cells at a ratio of 4:1 in CTL medium without exogenous
cytokines. After 72 h of incubation, cells were labeled with
anti-CD3 or anti-CD4 or anti-CD8 mAb and 7-AAD to exclude dead
cells from analysis. Samples were analyzed by flow cytometry and
cell division of live T cells assessed by CFSE dilution. The
division index was calculated using FlowJo software.
Results
Generation, Detection and Enrichment of Humanized ROR1 CAR T
Cells
[0153] PBMCs from healthy donors were isolated by Ficoll-Hypaque
density gradient centrifugation and bulk CD4.sup.+ or CD8.sup.+
human T cells were extracted from this cell population using MACS.
Directly after isolation the T cells were activated with CD3/28
Dynabeads for two days and then transduced by spinoculation with
lentiviral vectors encoding non-humanized or humanized versions of
the ROR1-specific CARs at a multiplicity of infection (MOI) of 5.
The Dynabeads were removed 4 days after transduction and at day 10
the T cells were enriched for EGFRt-positive cells by labeling with
a biotinylated monoclonal .alpha.EGFR antibody and MACS with
anti-biotin microbeads. After the enrichment, the EGFRt-positive
fraction reproducibly accounted for over 90% of total cells except
for the hR11 CAR that usually showed a slightly lower percentage of
EGFRt-positive cells (FIGS. 4A and 4B).
Cytolytic Activity of Humanized ROR1 CAR T Cells
[0154] CAR T cells were generated as described above and their
cytolytic activity was assessed in 6 h cytotoxicity assays against
the ROR1-positive and ffluc-expressing target cell lines K562-ROR1,
MDA-MB-231 and A549 (FIG. 5A). No specific lysis was detected
against ROR1-negative K562 controls. The T cells expressing the
h2A2 and hR12 CARs exhibited a very potent anti-tumor effect with a
high degree of target cell lysis that was dose dependent, with
higher E:T ratios causing higher percentages of target cell lysis.
A T cell line transduced with a vector control encoding for the
EGFRt transduction marker but not for a CAR caused no lysis of any
of the target cells. This demonstrates that the CAR itself induced
the target cell lysis and also that CAR-independent target cells
lysis, that could in principle occur due to allo-recognition by
endogenous TCRs, was not detectable in our experiments. In contrast
to the h2A2 and hR12 CARs the hR11 CAR was markedly impaired in its
cytolytic activity and showed no detectable lysis in the 6 h assay.
Notably it caused detectable and specific target cell lysis if the
incubation time was increased to e.g. 24 h.
[0155] The cytotoxicity assay was repeated under the same
conditions with CAR T cells that were generated from n=3 unrelated
healthy donors and the ROR1-positive target cells K562-ROR1 and
MDA-MB-231 (FIG. 5B). For all donors the lysis observed for the
h2A2 and hR12 CARs was consistently strong while the lysis mediated
by the hR11 CAR was barely detectable after 6 h incubation
time.
Effector Cytokine Secretion Following ROR1-Specific Activation of
hROR1 CAR T Cells
[0156] CD4.sup.+ or CD8.sup.+ CAR T cells were generated as
described above and co-cultured with lethally irradiated
ROR1-expressing target cell lines at an E:T ratio of 4:1 for 24 h.
After the incubation the cell culture supernatant was collected and
analyzed for the presence of the effector cytokines IL-2 and
IFN-.gamma. by ELISA. As controls, the cells were co-cultured with
ROR1-negative K562 cells or in absence of any target cell (media
control). For controlling the general ability of the CAR T cells to
produce the effector cytokines of interest the cells were
polyclonally stimulated with a combination of the protein kinase C
(PKC)/NF-.kappa.B-activator phorbol 12-Myristate 13-Acetate (PMA)
and the Ca2.sup.+ ionopohre ionomycin. The assay procedure was
repeated for up to n=3 unrelated healthy T cells donors and the
measured cytokine concentrations were used for group analysis.
[0157] The humanized 2A2 CAR showed a cytokine profile of that was
comparable to the non-humanized 2A2 CAR (FIG. 6A). IFN-.gamma. was
detected exclusively in samples that included ROR1-positive targets
or PMA/Iono and the average concentrations were in the range of
1000-1500 pg/ml for both CD4.sup.+ and CD8.sup.+ CAR T cells. IL-2
was also exclusively detected in samples that included
ROR1-positive target cells and the average concentrations were in
the range of 500-1000 pg/ml for CD4.sup.+ and CD8.sup.+ CAR T
cells. Surprisingly, the IL-2 secretion of the h2A2 CAR T cells was
elevated in comparison to the non-humanized variant for K562-ROR1
targets.
[0158] The humanized R11 CAR showed an impaired cytokine secretion
as compared to the non-humanized R11 CAR (FIG. 6B). The
concentrations of IFN-.gamma. and IL-2 were at background level for
the hR11 CAR even in the presence of ROR1-positive target cells
while for the non-humanized R11 CAR average concentrations of
IFN-.gamma. in the range of 1000 pg/ml for CD4.sup.+ T cells and
1800 pg/ml for CD8.sup.+ T cells as well as average IL-2
concentrations ranging from 500-1000 pg/ml were detected. Both CAR
T cells lines, either expressing the non-humanized or the humanized
R11 CAR, retained the general ability to produce IFN-.gamma. and
IL-2 in response to the antigen-unspecific stimulation with
PMA/Iono.
[0159] The humanized R12 CAR showed a cytokine profile of that was
comparable to the non-humanized R12 CAR (FIG. 6C). IFN-.gamma. was
detected exclusively in samples that included ROR1-spositive
targets or PMA/Iono and the average concentrations were in the
range of 1000-1500 pg/ml for both CD4.sup.+ and CD8.sup.+ CAR T
cells. IL-2 was also exclusively detected in samples that included
ROR1-positive target cells and the average concentrations were in
the range of 400 pg/ml for CD4.sup.+ and 500-800 pg/ml for
CD8.sup.+ CAR T cells.
[0160] Taken together these results demonstrate that the
humanization of the targeting domains of the h2A2 and the hR12 CAR
did not diminish the potential of CD4.sup.+ and CD8.sup.+ human T
cells expressing these CARs to secrete the effector cytokines
IFN-.gamma. and IL-2 after encounter of ROR1-positive target cells.
The cytokine levels detected were comparable to and in one instance
higher than for the non-humanized CARs. The hR11 CAR, in contrast,
mediated no detectable secretion of effector cytokines as response
to ROR1-positive target cells even though the T cells retained the
general ability for the secretion of both cytokines suggesting that
the humanization of the hR11 targeting domain was causative for the
observed loss of function. These data are evidence for the fact
that the use of humanized binding domains in CARs generated by CDR
grafting and only marginally decreasing the affinity of the
humanized anti-ROR1 antibodies, see reference [31], cannot predict
the functionality of CAR T cells comprising said humanized binding
domains in comparison to CAR T cells comprising the non-humanized
parental binding domains.
Proliferation of Humanized ROR1 CAR T Cells
[0161] CD4.sup.+ or CD8.sup.+ CAR T cells were generated as
described, labeled with CFSE and co-cultured with lethally
irradiated ROR1-expressing target cell lines at an E:T ratio of 4:1
for 72 h in the absence of exogenous cytokines. After the
incubation time the T cells were collected and analyzed for CFSE
dilution by flow cytometry. As a negative control, the CAR T cells
were cocultured with ROR1-negative K562 cells and as a positive
control in the presence of 50 UI/ml IL-2.
[0162] ROR1-negative K562 caused no proliferation of T cells
expressing any of the CAR constructs. T cells expressing a vector
construct encoding the EGFRt transduction marker but lacking a CAR
sequence showed no proliferation in response to any of the target
cells above background proliferation (FIGS. 7A-7C). This
demonstrates that the detected proliferation of the ROR1 CAR-T
cells was mediated specifically by the CAR as a response to
stimulation by ROR1-expressing cells.
[0163] Surprisingly, despite similar cytokine secretion profiles,
as determined above, T cells expressing the humanized 2A2 CAR
proliferated significantly stronger than T cells expressing the
non-humanized 2A2 CAR in response to ROR1-positive target cells
(FIG. 7A). Depending on the target cell line, the division indices
of the CD4.sup.+ h2A2 CAR T cells were consistently 2-3 fold higher
as compared to the non-humanized 2A2 ROR1 CAR (FIG. 7B). T cells
expressing the humanized 2A2 CAR went through a higher number of
cell divisions than T cells expressing the non-humanized 2A2 CAR
(FIG. 7C). 60% of the h2A2 CAR T cells with MDA-MB-231 target cells
went through three or more cell division cycles, for the
non-humanized 2A2 CAR this fraction was 20%. Similarly, 51% of the
h2A2 CAR T cells with K562-ROR1 target cells went through three or
more cell division cycles, for the non-humanized 2A2 CAR this
fraction was 18%. In line with the previous observations, 18% of
the h2A2 CAR T cells with A549 target cells went through three or
more cell division cycles, for the non-humanized 2A2 CAR this
fraction was 5%.
[0164] T cells expressing the humanized R11 showed a weaker
proliferation than T cells expressing the non-humanized R11 CAR but
the proliferation was clearly above background level (FIG. 7A).
Depending on the target cells, the division indices were reduced by
a factor of 1.5-3.5.
[0165] The proliferation of T cells expressing the humanized R12
CAR was specific and overall comparable to T cells expressing the
non-humanized variant. Significantly higher proliferation levels
were detected for T cells expressing the humanized R12 CAR as
compared to the non-humanized variant response to MDA-MB-231. The
proliferation index was about 2-fold increased as compared to the
non-humanized R12 CAR variant. The percentage of T cells that went
through 3 or more cell divisions was 37% for the humanized R12 CAR
and 20% for the non-humanized R12 CAR.
[0166] Taken together these results demonstrate that the humanized
variants of the 2A2 and R12 CARs are capable of activating T cell
proliferation in response to antigen encounter at a substantially
higher level than the non-humanized variants. The humanized R11 on
the other hand had a pronounced decrease in proliferative capacity
and the proliferation levels were markedly lowered as compared to
the non-humanized R11 CAR.
Example 2: Binding of ROR1-Protein by Humanized ROR1 CARs
Materials and Methods:
Human Subjects
[0167] Blood samples were obtained from healthy donors who provided
written informed consent to participate in research protocols
approved by the Institutional Review Board of the University of
Wurzburg (Universitatsklinikum Wurzburg, UKW). Peripheral blood
mononuclear cells (PBMC) were isolated by centrifugation over
Ficoll-Hypaque (Sigma, St. Louis, Mo.).
Immunophenotyping
[0168] PBMC and T cell lines were stained with one or more of the
following conjugated mAb: CD3, CD4, CD8 and matched isotype
controls (BD Biosciences, San Jose, Calif.). Transduced T cell
lines were stained with biotin-conjugated anti-EGFR antibody
(ImClone Systems Incorporated, Branchburg, N.J.) and
streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with
7-AAD (BD Biosciences) was performed for live/dead cell
discrimination as directed by the manufacturer. Flow analyses were
done on a FACSCanto and data analyzed using FlowJo software
(Treestar, Ashland, Oreg.).
Lentiviral Vector Construction, Preparation of Lentivirus, and
Generation of CAR-T Cells
[0169] The construction of epHIV7 lentiviral vectors containing
ROR1-specific CARs with a short or long spacer and a 4-1BB
costimulatory domain has been described, see reference [35], which
is hereby incorporated by reference in its entirety for all
purposes. All CAR constructs encoded a truncated epidermal growth
factor receptor (EGFRt; also known as tEGFR), see reference [36],
which is hereby incorporated by reference in its entirety for all
purposes, downstream of the CAR. The genes were linked by a T2A
ribosomal skip element.
[0170] CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants
were produced in 293T cells co-transfected with each of the
lentiviral vector plasmids and the packaging vectors pCHGP-2,
pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech,
Mountain View, Calif.). Medium was changed 16 h after transfection,
and lentivirus collected after 72 h. CAR-T cells were generated as
described [35]. In brief, CD8.sup.+ or CD4.sup.+ bulk T cells were
sorted from PBMC of healthy donors, activated with anti-CD3/CD28
beads (Life Technologies), and transduced with lentiviral
supernatant. Lentiviral transduction was performed on day 2 by
spinoculation, and T cells propagated in RPMI-1640 with 10% human
serum, GlutaminMAX (ThermoFisher Scientific, MA), 100 U/mL
penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was
performed to quantify viable T cells. After expansion, EGFRt.sup.+
T cells were enriched and expanded by polyclonal stimulation with
the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and
EBV-LCL feeder cells.
Binding of ROR1
[0171] Recombinant aggregated ROR1 protein was labeled with the
AlexaFluor647 labeling kit (ThermoFisher Scientific, MA) and used
to stain T cells expressing ROR1 CARs. The T cells were washed once
in PBS, 0.25% FCS and then resuspended in the same buffer
containing a final concentration of 5.3 .mu.g/ml labeled ROR1
protein and monoclonal .alpha.EGFRt antibody. After an incubation
time of 15 min the cells were washed with an excess of PBS, 0.25%
FCS and analyzed by flow cytometry.
Results:
[0172] Compared to their non-humanized counterparts the humanized
2A2 and R12 ROR1 CARs showed significantly stronger binding to the
ROR1 protein (FIG. 8). A higher overall percentage of ROR1 protein
binding was detected, suggesting a better surface availability
and/or binding capability of the humanized CARs. The percentage of
T cells with a distinct AlexaFluor647 signal was 62.7% for the
non-humanized and 92.1% for the humanized 2A2 CAR. Similarly, the
percentage of T cells with a distinct AlexaFluor647 signal was
47.6% for the non-humanized and 79.0% for the humanized R12
CAR.
[0173] Further, the percentage of CAR T cells that showed strong
ROR1 binding was increased for the humanized 2A2 and R12 CARs and
consequently a lesser frequency of weak ROR1-binding was detected
for these samples. The percentage of T cells with high
AlexaFluor647 signal was 2.61% for the non-humanized and 20.0% for
the humanized 2A2 CAR. Similarly, the percentage of T cells with
high AlexaFluor647 signal was 0.7% for the non-humanized and 7.68%
for the humanized R12 CAR. This accounts for a roughly 10-fold
increase in the number of T cells that strongly bind to the ROR1
protein.
[0174] The humanized R11 showed low overall ROR1 protein binding
with 4.98% of AlexaFluor647-positive T cells as compared to 97.9%
for the non-humanized R11 CAR. Similarly, the frequency of T cells
with high AlexaFluor647 signal was 0.019% for the humanized R11 and
66.5% for the non-humanized variant.
[0175] In summary these data demonstrate that the humanized
versions of the 2A2 and the R12 CAR have a stronger binding to the
ROR1 antigen than the non-humanized versions. That was unexpected
and may provide an explanation for the elevated activity in parts
of the assays that were performed for example 1.
Example 3: Regression of Human Jeko-1 Mantle Cell Lymphoma in
NOD/SCID/.gamma.c-/- (NSG) Mice after Adoptive Immunotherapy with
CAR-T Cells Expressing Humanized ROR1 CARs
Materials and Methods:
Human Subjects
[0176] Blood samples were obtained from healthy donors who provided
written informed consent to participate in research protocols
approved by the Institutional Review Board of the University of
Wurzburg (Universitatsklinikum Wurzburg, UKW). Peripheral blood
mononuclear cells (PBMC) were isolated by centrifugation over
Ficoll-Hypaque (Sigma, St. Louis, Mo.).
Cell Lines
[0177] The JeKo-1 (wild type), 293T, K562, MDA-MB-231 and A549 cell
lines were obtained from the American Type Culture Collection.
K562-ROR1 were generated by lentiviral transduction with the
full-length ROR1-gene. Luciferase expressing lines were derived by
lentiviral transduction of the above mentioned cell lines with the
firefly (P. pyralis) luciferase (ffluc)-gene. The cells were
cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum and 100 U/ml penicillin/streptomycin.
Immunophenotyping
[0178] PBMC and T cell lines were stained with one or more of the
following conjugated mAb: CD3, CD4, CD8 and matched isotype
controls (BD Biosciences, San Jose, Calif.). Transduced T cell
lines were stained with biotin-conjugated anti-EGFR antibody
(ImClone Systems Incorporated, Branchburg, N.J.) and
streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with
7-AAD (BD Biosciences) was performed for live/dead cell
discrimination as directed by the manufacturer. Flow analyses were
done on a FACSCanto and data analyzed using FlowJo software
(Treestar, Ashland, Oreg.).
Lentiviral Vector Construction, Preparation of Lentivirus, and
Generation of CAR-T Cells and JeKo-1/ffluc Cells
[0179] The construction of epHIV7 lentiviral vectors containing
ROR1-specific CARs with a short or long spacer and a 4-1BB
costimulatory domain has been described, see reference [35], which
is hereby incorporated by reference in its entirety for all
purposes. All CAR constructs encoded a truncated epidermal growth
factor receptor (EGFRt; also known as tEGFR), see reference [36],
which is hereby incorporated by reference in its entirety for all
purposes, downstream of the CAR. The genes were linked by a T2A
ribosomal skip element.
[0180] CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants
were produced in 293T cells co-transfected with each of the
lentiviral vector plasmids and the packaging vectors pCHGP-2,
pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech,
Mountain View, Calif.). Medium was changed 16 h after transfection,
and lentivirus collected after 72 h. CAR-T cells were generated as
described [35]. In brief, CD8.sup.+ or CD4.sup.+ bulk T cells were
sorted from PBMC of healthy donors, activated with anti-CD3/CD28
beads (Life Technologies), and transduced with lentiviral
supernatant. Lentiviral transduction was performed on day 2 by
spinoculation, and T cells propagated in RPMI-1640 with 10% human
serum, GlutaminMAX (Life technologies), 100 U/mL
penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was
performed to quantify viable T cells. After expansion, EGFRt.sup.+
T cells were enriched and expanded by polyclonal stimulation with
the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and
EBV-LCL feeder cells. JeKo-1/ffluc cells were generated by
lentiviral transduction with the ffluc/eGFP-encoding
lentivirus.
Cytotoxicity, Cytokine Secretion, and CFSE Proliferation Assays
[0181] Target cells stably expressing firefly luciferase were
incubated in triplicate at 5.times.10.sup.3 cells/well with
effector T cells at various effector to target (E:T) ratios. After
a four-hour incubation luciferin substrate was added to the
co-culture and the decrease in luminescence signal in wells that
contained target cells and T cells, compared to target cells alone,
measured using a luminometer (Tecan). Specific lysis was calculated
using the standard formula. For analysis of cytokine secretion,
5.times.10.sup.4 T cells were plated in triplicate wells with
target cells at a ratio of 4:1 and IFN-.gamma., TNF-.alpha., and
IL-2 measured by ELISA (Biolegend) in supernatant removed after a
24-hour incubation. For analysis of proliferation, 5.times.10.sup.4
T cells were labeled with 0.2 .mu.M carboxyfluorescein succinimidyl
ester (CFSE, Invitrogen), washed and plated in triplicate wells
with target cells at a ratio of 4:1 in CTL medium without exogenous
cytokines. After 72 h of incubation, cells were labeled with
anti-CD3 or anti-CD4 or anti-CD8 mAb and 7-AAD to exclude dead
cells from analysis. Samples were analyzed by flow cytometry and
cell division of live T cells assessed by CFSE dilution. The
division index was calculated using FlowJo software.
Experiments in NOD/SCID/.gamma.c-/- (NSG) Mice
[0182] The Institutional Animal Care and Use Committee approved all
mouse experiments. Six to 8-week old female NOD.Cg-Prkdcscid
II2rgtm1Wjl/SzJ (NSG) mice were obtained from the Jackson
Laboratory or bred in-house. Mice were injected with
0.5.times.10.sup.6 JeKo-1/ffluc tumor cells via tail vein and
received a subsequent tail vein injection of h2A2 or hR12 ROR1 CAR
cells. Both ROR1 CAR T cell lines (h2A2 and hR12) expressed the
transduction marker EGFRt. Control T cells expressed only the EGFRt
transduction marker.
[0183] For bioluminescence imaging of tumor growth, mice received
intraperitoneal injections of luciferin substrate (Caliper Life
Sciences) resuspended in PBS (15 .mu.g/g body weight). Mice were
anesthetized with isoflurane and imaged using an Xenogen IVIS
Imaging System (Caliper) 10 minutes after luciferin injection in
small binning mode at an acquisition time of 1 s to 1 min to obtain
unsaturated images. Luciferase activity was analyzed using Living
Image Software (Caliper). The photon flux (radiance) was measured
within regions of interest that encompassed the entire body of each
individual mouse.
Results:
[0184] To assess the in vivo activity of humanized ROR1 CARs, we
inoculated cohorts (n=5) of immunodeficient NSG mice with the
human, ROR1-expressing mantle cell lymphoma line JeKo-1/ffluc by
tail vein injection. 21 days later, when the tumor was
disseminated, the mice were treated with a single intravenous dose
of hR12 or h2A2 ROR1 CAR T cells. Control mice were treated with T
cells only expressing an EGFRt control vector or were left
untreated.
[0185] We observed rapid tumor regression and improved survival in
all of the mice treated with hR12 and h2A2 ROR1 CAR-T cells
(response rate 100%). FIG. 9A displays the tumor regression 7 days
after T cell transfer as percentage change from baseline radiance.
The baseline was measured for each mouse before the treatment on
day 21. In contrast to humanized ROR1 CAR-treated mice, the control
groups, which were either left untreated or were treated with T
cells expressing the EGFRt control vector, showed continued tumor
growth in all of the mice (response rate 0%). The average radiance
7 days after CAR T cell treatment was 2*10.sup.4 p/sec/cm.sup.2/sr
for hR12 and 1*10.sup.5 for h2A2. The average radiance of the
control groups was 6*10.sup.6 for untreated mice and 1*10.sup.6 for
mice treated with the EGFRt-only vector control.
[0186] Humanized ROR1 CAR T cells engrafted and persisted in the
mice and were detectable in spleen and bone marrow of the mice
until the end of the experiment. FIG. 9B shows representative flow
cytometry data from bone marrow samples of one mouse from the hR12
cohort and one mouse of the h2A2 cohort at day 56. CD4 and CD8 T
cells were detectable, and expressed the humanized ROR1 CAR. We
confirmed the presence of humanized ROR1 CAR T cells in organ
samples; the proportion of humanized ROR1 CAR T cells in the total
cells varied between individual mice but was generally in the range
of 1-10%.
[0187] In summary, these data demonstrate that the h2A2 and the
hR12 CARs conferred a strong and specific anti-tumor activity in
vivo.
INDUSTRIAL APPLICABILITY
[0188] The CARs, combinations of CARs, the recombinant mammalian
cells and the methods and medical uses according to the invention
are industrially applicable. For example, they can be used as, or
for the production of, pharmaceutical products.
REFERENCES
[0189] 1. Kalos, M., et al., T cells with chimeric antigen
receptors have potent antitumor effects and can establish memory in
patients with advanced leukemia. Sci Transl Med, 2011. 3(95): p.
95ra73. [0190] 2. Kochenderfer, J. N., et al., B-cell depletion and
remissions of malignancy along with cytokine-associated toxicity in
a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced
T cells. Blood, 2012. 119(12): p. 2709-20. [0191] 3. Kochenderfer,
J. N., et al., Eradication of 8-lineage cells and regression of
lymphoma in a patient treated with autologous T cells genetically
engineered to recognize CD19. Blood, 2010. 116(20): p. 4099-102.
[0192] 4. Morgan, R. A., et al., Cancer regression in patients
after transfer of genetically engineered lymphocytes. Science,
2006. 314(5796): p. 126-9. [0193] 5. Porter, D. L., et al.,
Chimeric antigen receptor-modified T cells in chronic lymphoid
leukemia. N Engl J Med, 2011. 365(8): p. 725-33. [0194] 6. Eshhar,
Z., et al., Specific activation and targeting of cytotoxic
lymphocytes through chimeric single chains consisting of
antibody-binding domains and the gamma or zeta subunits of the
immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA, 1993.
90(2): p. 720-4. [0195] 7. Kershaw, M. H., et al., Supernatural T
cells: genetic modification of T cells for cancer therapy. Nat Rev
Immunol, 2005. 5(12): p. 928-40. [0196] 8. Sadelain, M., I.
Riviere, and R. Brentjens, Targeting tumours with genetically
enhanced T lymphocytes. Nat Rev Cancer, 2003. 3(1): p. 35-45.
[0197] 9. Brentjens, R. J., et al., Safety and persistence of
adoptively transferred autologous CD19-targeted T cells in patients
with relapsed or chemotherapy refractory B-cell leukemias. Blood,
2011. 118(18): p. 4817-28. [0198] 10. Hudecek, M., et al., The
B-cell tumor-associated antigen ROR1 can be targeted with T cells
modified to express a ROR1-specific chimeric antigen receptor.
Blood, 2010. 116(22): p. 4532-41. [0199] 11. Matsuda, T., et al.,
Expression of the receptor tyrosine kinase genes, Ror1 and Ror2,
during mouse development. Mech Dev, 2001. 105(1-2): p. 153-6.
[0200] 12. Rosenwald, A., et al., Relation of gene expression
phenotype to immunoglobulin mutation genotype in B cell chronic
lymphocytic leukemia. J Exp Med, 2001. 194(11): p. 1639-47. [0201]
13. Klein, U., et al., Gene expression profiling of B cell chronic
lymphocytic leukemia reveals a homogeneous phenotype related to
memory B cells. J Exp Med, 2001. 194(11): p. 1625-38. [0202] 14.
Baskar, S., et al., Unique cell surface expression of receptor
tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.
Clin Cancer Res, 2008. 14(2): p. 396-404. [0203] 15. Bicocca, V.
T., et al., Crosstalk between ROR1 and the Pre-8 cell receptor
promotes survival of t(1;19) acute lymphoblastic leukemia. Cancer
Cell, 2012. 22(5): p. 656-67. [0204] 16. Daneshmanesh, A. H., et
al., Ror1, a cell surface receptor tyrosine kinase is expressed in
chronic lymphocytic leukemia and may serve as a putative target for
therapy. Int J Cancer, 2008. 123(5): p. 1190-5. [0205] 17. Fukuda,
T., et al., Antisera induced by infusions of autologous
Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and
receptor for Wnt5.alpha.. Proc Natl Acad Sci USA, 2008. 105(8): p.
3047-52. [0206] 18. Yamaguchi, T., et al.,
NKX2-1/TITF1/TFT-1-Induced ROR1 is required to sustain EGFR
survival signaling in lung adenocarcinoma. Cancer Cell, 2012.
21(3): p. 348-61. [0207] 19. Zhang, S., et al., ROR1 is expressed
in human breast cancer and associated with enhanced tumor-cell
growth. PLoS One, 2012. 7(3): p. e31127. [0208] 20. Zhang, S., et
al., The once-embryonic antigen ROR1 is expressed by a variety of
human cancers. Am J Pathol, 2012. 181(6): p. 1903-10. [0209] 21.
Dave, H., et al., Restricted cell surface expression of receptor
tyrosine kinase ROR1 in pediatric 8-lineage acute lymphoblastic
leukemia suggests targetability with therapeutic monoclonal
antibodies. PLoS One, 2012. 7(12): p. e52655. [0210] 22. Gentile,
A., et al., Ror1 is a pseudokinase that is crucial for Met-driven
tumorigenesis. Cancer Res, 2011. 71(8): p. 3132-41. [0211] 23.
Choudhury, A., et al., Silencing of ROR1 and FMOD with siRNA
results in apoptosis of CLL cells. Br J Haematol, 2010. 151(4): p.
327-35. [0212] 24. Maude, S. L., et al., Chimeric antigen receptor
T cells for sustained remissions in leukemia. N Engl J Med, 2014.
371(16): p. 1507-17. [0213] 25. Grupp, S. A., et al., Chimeric
antigen receptor-modified T cells for acute lymphoid leukemia. N
Engl J Med, 2013. 368(16): p. 1509-18. [0214] 26. Davila, M. L., et
al., Efficacy and toxicity management of 19-28z CAR T cell therapy
in B cell acute lymphoblastic leukemia. Sci Transl Med, 2014.
6(224): p. 224ra25. [0215] 27. Lamers, C. H., et al., Treatment of
metastatic renal cell carcinoma with CAIX CAR-engineered T cells:
clinical evaluation and management of on-target toxicity. Mol Ther,
2013. 21(4): p. 904-12. [0216] 28. Turtle, C. J., et al., CD19
CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL
patients. J Clin Invest, 2016. 126(6): p. 2123-38. [0217] 29. Maus,
M. V., et al., T cells expressing chimeric antigen receptors can
cause anaphylaxis in humans. Cancer Immunol Res, 2013. 1(1): p.
26-31. [0218] 30. Sommermeyer, D., et al., Fully human
CD19-specific chimeric antigen receptors for T-cell therapy.
Leukemia, 2017. [0219] 31. Waldmeier, L., et al., Transpo-mAb
display: Transposition-mediated B cell display and functional
screening of full-length IgG antibody libraries. MAbs, 2016. 8(4):
p. 726-40. [0220] 32. Baca, M., et al., Antibody humanization using
monovalent phage display. J Biol Chem, 1997. 272(16): p. 10678-84.
[0221] 33. Altschul, S. F., et al., Basic local alignment search
tool. J Mol Biol, 1990. 215(3): p. 403-10. [0222] 34. Altschul, S.
F., et al., Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res, 1997. 25(17): p.
3389-402. [0223] 35. Hudecek, M., et al., Receptor affinity and
extracellular domain modifications affect tumor recognition by
ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res,
2013. 19(12): p. 3153-64. [0224] 36. Wang, X., et al., A
transgene-encoded cell surface polypeptide for selection, in vivo
tracking, and ablation of engineered cells. Blood, 2011. 118(5): p.
1255-63. [0225] 37. Yang, J. et al., Therapeutic potential and
challenges of targeting receptor tyrosine kinase ROR1 with
monoclonal antibodies in B-cell malignancies. PLoS One. 2011;
6(6):e21018.
Sequence CWU 1
1
241118PRTArtificial Sequenceh2A2 VH sequence 1Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Glu Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45Gly Ala
Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Val Thr Met Thr Gly Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr Gly Tyr Tyr Asp Tyr Asp Ser Phe Thr Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Ser Val Ser Ser 1152107PRTArtificial
Sequenceh2A2 VL sequence 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asn Val Asp Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Asn Arg Tyr
Thr Gly Val Ala Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Leu Ala
Asp Tyr Phe Cys Gln Gln Tyr Asp Ile Tyr Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 1053119PRTArtificial
SequencehR11 VH sequence 3Glu Val Gln Leu Val Gln Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Asp Ile Asn Asp Tyr 20 25 30Pro Ile Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Phe Ile Asn Ser Gly Gly
Ser Thr Trp Tyr Ala Ser Trp Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Asp Asp Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95Arg Gly Tyr
Ser Thr Tyr Tyr Gly Asp Phe Asn Ile Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1154110PRTArtificial SequencehR11 VL
sequence 4Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile
Asp Ser Asn 20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro
Ser Lys Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Arg65 70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys
Leu Gly Gly Val Gly Asn Val Ser 85 90 95Tyr Arg Thr Ser Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 1105121PRTArtificial
SequencehR12 VH sequence 5Gln Val Gln Leu Val Glu Ser Gly Gly Ala
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Lys Ala Ser
Gly Phe Asp Phe Ser Ala Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Thr Ile Tyr Pro Ser Ser
Gly Lys Thr Tyr Tyr Ala Ala Ser Val 50 55 60Gln Gly Arg Phe Thr Ile
Ser Ala Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Asp
Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1206111PRTArtificial
SequencehR12 VL sequence 6Gln Leu Val Leu Thr Gln Ser Pro Ser Val
Ser Ala Ala Leu Gly Ser1 5 10 15Ser Ala Lys Ile Thr Cys Thr Leu Ser
Ser Ala His Lys Thr Asp Thr 20 25 30Ile Asp Trp Tyr Gln Gln Leu Ala
Gly Gln Ala Pro Arg Tyr Leu Met 35 40 45Tyr Val Gln Ser Asp Gly Ser
Tyr Glu Lys Arg Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Ser
Ser Gly Ala Asp Arg Tyr Leu Ile Ile Ser65 70 75 80Ser Val Gln Ala
Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr 85 90 95Ile Gly Gly
Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Gly 100 105
110722PRTArtificial SequenceGMCSF signal peptide 7Met Leu Leu Leu
Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu
Leu Ile Pro 20815PRTArtificial Sequence4(GS)x3 linker 8Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
15912PRTArtificial SequenceIgG4 hinge domain 9Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro1 5 1010228PRTArtificial SequenceIgG4
CH2CH3 4/2NQ 10Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Pro Val1 5 10 15Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 35 40 45Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Gln Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135
140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val 195 200 205Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser Leu Gly
Lys2251128PRTArtificial SequenceCD28 transmembrane domain 11Met Phe
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser1 5 10 15Leu
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 251242PRTArtificial
Sequence4-1BB costimulatory domain 12Lys Arg Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu
Gly Gly Cys Glu Leu 35 4013112PRTArtificial SequenceCD3z signaling
domain 13Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala Leu Pro Pro Arg 100 105 1101424PRTArtificial
SequenceT2A ribosomal skipping sequence 14Leu Glu Gly Gly Gly Glu
Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp1 5 10 15Val Glu Glu Asn Pro
Gly Pro Arg 2015335PRTArtificial SequenceEGFRt 15Arg Lys Val Cys
Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu1 5 10 15Ser Ile Asn
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30Ser Gly
Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45Thr
His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55
60Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn65
70 75 80Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly
Arg 85 90 95Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
Asn Ile 100 105 110Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser
Asp Gly Asp Val 115 120 125Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
Ala Asn Thr Ile Asn Trp 130 135 140Lys Lys Leu Phe Gly Thr Ser Gly
Gln Lys Thr Lys Ile Ile Ser Asn145 150 155 160Arg Gly Glu Asn Ser
Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175Cys Ser Pro
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190Cys
Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200
205Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
210 215 220Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys
Thr Gly225 230 235 240Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His
Tyr Ile Asp Gly Pro 245 250 255His Cys Val Lys Thr Cys Pro Ala Gly
Val Met Gly Glu Asn Asn Thr 260 265 270Leu Val Trp Lys Tyr Ala Asp
Ala Gly His Val Cys His Leu Cys His 275 280 285Pro Asn Cys Thr Tyr
Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300Thr Asn Gly
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala305 310 315
320Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325
330 33516240PRTArtificial SequencescFV of the non-humanized 2A2
antibody 16Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Ala1 5 10 15Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ser Asp Tyr 20 25 30Glu Met His Trp Val Ile Gln Thr Pro Val His Gly
Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala
Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Gly Tyr Tyr Asp Tyr Asp
Ser Phe Thr Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly
Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Ile Met 130 135 140Ser
Thr Thr Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln145 150
155 160Asn Val Asp Ala Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser 165 170 175Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr
Gly Val Pro 180 185 190Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile 195 200 205Ser Asn Met Gln Ser Glu Asp Leu Ala
Asp Tyr Phe Cys Gln Gln Tyr 210 215 220Asp Ile Tyr Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys225 230 235
24017241PRTArtificial SequencescFV of the non-humanized R11
antibody 17Gln Ser Val Lys Glu Ser Glu Gly Asp Leu Val Thr Pro Ala
Gly Asn1 5 10 15Leu Thr Leu Thr Cys Thr Ala Ser Gly Ser Asp Ile Asn
Asp Tyr Pro 20 25 30Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Phe Ile Asn Ser Gly Gly Ser Thr Trp Tyr Ala
Ser Trp Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr
Val Asp Leu Lys Met Thr65 70 75 80Ser Leu Thr Thr Asp Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Gly Tyr 85 90 95Ser Thr Tyr Tyr Gly Asp Phe
Asn Ile Trp Gly Pro Gly Thr Leu Val 100 105 110Thr Ile Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser
Glu Leu Val Met Thr Gln Thr Pro Ser Ser Thr Ser Gly 130 135 140Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile145 150
155 160Asp Ser Asn Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
Thr 165 170 175Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val
Pro Ser Arg 180 185 190Phe Ser Gly Ser Arg Ser Gly Thr Glu Tyr Thr
Leu Thr Ile Ser Gly 195 200 205Val Gln Arg Glu Asp Ala Ala Thr Tyr
Tyr Cys Leu Gly Gly Val Gly 210 215 220Asn Val Ser Tyr Arg Thr Ser
Phe Gly Gly Gly Thr Glu Val Val Val225 230 235
240Lys18248PRTArtificial SequencescFV of the non-humanized R12
antibody 18Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro
Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe
Ser Ala Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr
Tyr Ala Thr Trp Val 50 55 60Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
Ala Gln Asn Thr Val Asp65 70 75 80Leu Gln Met Asn Ser Leu Thr Ala
Ala Asp Arg Ala Thr Tyr Phe Cys 85 90 95Ala Arg Asp Ser Tyr Ala Asp
Asp Gly Ala Leu Phe Asn Ile Trp Gly 100 105 110Pro Gly Thr Leu Val
Thr Ile Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser
Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro 130 135 140Ser
Val Ser Ala Ala Leu Gly Ser Pro Ala Lys Ile Thr Cys Thr Leu145 150
155 160Ser Ser Ala His Lys Thr Asp Thr Ile Asp Trp Tyr Gln Gln Leu
Gln 165 170 175Gly Glu Ala Pro Arg Tyr Leu Met Gln Val Gln Ser Asp
Gly Ser Tyr 180 185 190Thr Lys Arg Pro Gly Val Pro Asp Arg Phe Ser
Gly Ser Ser Ser Gly 195 200 205Ala Asp Arg Tyr Leu Ile Ile Pro Ser
Val Gln Ala Asp Asp Glu Ala 210 215 220Asp Tyr Tyr Cys Gly Ala Asp
Tyr Ile Gly Gly Tyr Val Phe Gly Gly225 230 235 240Gly Thr Gln Leu
Thr Val Thr Gly 24519118PRTArtificial SequenceVH of the
non-humanized 2A2 antibody 19Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Thr Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Glu Met His Trp Val Ile Gln
Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro Glu
Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Ile
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Gly
Tyr Tyr Asp Tyr Asp Ser Phe Thr Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ala
11520107PRTArtificial SequenceVL of the non-humanized 2A2 antibody
20Asp Ile Val Met Thr Gln Ser Gln Lys Ile Met Ser Thr Thr Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Ala
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Met Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Asp Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 10521116PRTArtificial SequenceVH of the non-humanized
R11 antibody 21Gln Ser Val Lys Glu Ser Glu Gly Asp Leu Val Thr Pro
Ala Gly Asn1 5 10 15Leu Thr Leu Thr Cys Thr Ala Ser Gly Ser Asp Ile
Asn Asp Tyr Pro 20 25 30Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile Gly 35 40 45Phe Ile Asn Ser Gly Gly Ser Thr Trp Tyr
Ala Ser Trp Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Thr Ser Thr
Thr Val Asp Leu Lys Met Thr65 70 75 80Ser Leu Thr Thr Asp Asp Thr
Ala Thr Tyr Phe Cys Ala Arg Gly Tyr 85 90 95Ser Thr Tyr Tyr Gly Asp
Phe Asn Ile Trp Gly Pro Gly Thr Leu Val 100 105 110Thr Ile Ser Ser
11522110PRTArtificial SequenceVL of the non-humanized R11 antibody
22Glu Leu Val Met Thr Gln Thr Pro Ser Ser Thr Ser Gly Ala Val Gly1
5 10 15Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Asp Ser
Asn 20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Thr Leu
Leu Ile 35 40 45Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser
Gly Val Gln Arg65 70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly
Gly Val Gly Asn Val Ser 85 90 95Tyr Arg Thr Ser Phe Gly Gly Gly Thr
Glu Val Val Val Lys 100 105 11023121PRTArtificial SequenceVH of the
non-humanized R12 antibody 23Gln Glu Gln Leu Val Glu Ser Gly Gly
Arg Leu Val Thr Pro Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Lys Ala
Ser Gly Phe Asp Phe Ser Ala Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Thr Ile Tyr Pro Ser
Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val 50 55 60Asn Gly Arg Phe Thr
Ile Ser Ser Asp Asn Ala Gln Asn Thr Val Asp65 70 75 80Leu Gln Met
Asn Ser Leu Thr Ala Ala Asp Arg Ala Thr Tyr Phe Cys 85 90 95Ala Arg
Asp Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile Trp Gly 100 105
110Pro Gly Thr Leu Val Thr Ile Ser Ser 115 12024112PRTArtificial
SequenceVL of the non-humanized R12 antibody 24Glu Leu Val Leu Thr
Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser1 5 10 15Pro Ala Lys Ile
Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr 20 25 30Ile Asp Trp
Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met 35 40 45Gln Val
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp 50 55 60Arg
Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro65 70 75
80Ser Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr
85 90 95Ile Gly Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr
Gly 100 105 110
* * * * *