U.S. patent application number 16/079202 was filed with the patent office on 2019-02-14 for modified cells for immunotherapy.
The applicant listed for this patent is Cell Medica Switzerland AG, The University of North Carolina at Chapel Hill. Invention is credited to Gianpietro Dotti, Miriam Droste, Titus Kretzschmar, Elisa Landoni, Douglas Phillips, Abdijapar Shamshiev.
Application Number | 20190048085 16/079202 |
Document ID | / |
Family ID | 55446573 |
Filed Date | 2019-02-14 |
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United States Patent
Application |
20190048085 |
Kind Code |
A1 |
Dotti; Gianpietro ; et
al. |
February 14, 2019 |
MODIFIED CELLS FOR IMMUNOTHERAPY
Abstract
The present invention relates to engineered immune cells
expressing antigen receptors, such as a T cell receptor (TCR) or a
chimeric antigen receptor (CAR), as well as antibody targeting
PD-L1. Also provided are related nucleic acids, vectors,
compositions and method for enhancing the immune response towards
cancers and pathogens.
Inventors: |
Dotti; Gianpietro; (Chapel
Hill, NC) ; Landoni; Elisa; (Chapel Hill, NC)
; Shamshiev; Abdijapar; (Zurich, CH) ;
Kretzschmar; Titus; (Hunenberg, CH) ; Droste;
Miriam; (Dietikon, CH) ; Phillips; Douglas;
(Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cell Medica Switzerland AG
The University of North Carolina at Chapel Hill |
Schlieren
Chapel Hill |
NC |
CH
US |
|
|
Family ID: |
55446573 |
Appl. No.: |
16/079202 |
Filed: |
February 24, 2017 |
PCT Filed: |
February 24, 2017 |
PCT NO: |
PCT/US2017/019301 |
371 Date: |
August 23, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
A61K 39/39591 20130101; C07K 16/2827 20130101; G01N 2333/70532
20130101; A61K 2039/5156 20130101; C07K 2317/76 20130101; C07K
2319/03 20130101; A61K 45/06 20130101; C07K 14/705 20130101; A61P
35/00 20180101; C07K 2317/52 20130101; G01N 33/6872 20130101; C07K
2317/622 20130101; A61K 2039/505 20130101; C07K 2317/33 20130101;
A61K 39/39 20130101; C07K 2317/94 20130101; C07K 2317/24 20130101;
A61K 39/0011 20130101; A61K 35/17 20130101; C07K 2317/73 20130101;
A61K 2039/545 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/705 20060101 C07K014/705; A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 2016 |
EP |
16020057.2 |
Claims
1. An engineered immune cell expressing: i) an antigen receptor,
and ii) an antibody blocking PD-L1.
2. The engineered immune cell of claim 1, wherein the antibody
inhibits PD-L1 interaction with both CD80 and PD-1.
3. The engineered immune cell of claim 1 or 2, wherein the antibody
is humanized.
4. The engineered immune cell of any one of the preceding claims,
wherein said immune cell is a T cell, a Natural Killer T (NKT)
cell, a Natural Killer (NK) cell a human embryonic stem cell, a
hematopoietic stem cell (HSC) or an induced pluripotent stem cell
(iPS).
5. The engineered immune cell of any one of the preceding claims,
wherein said T cell is a cytotoxic T lymphocyte (CTL), a regulatory
T lymphocyte, an inflammatory T-lymphocytes, a helper T-lymphocyte
or a gamma-delta T cell.
6. The engineered immune cell of claim 4 or 5, wherein said T cell
is a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.
7. The engineered immune cell of any one of the preceding claims,
wherein said antigen receptor is a CAR, said CAR comprising a
cytoplasmic domain, a transmembrane domain and an extracellular
domain.
8. The engineered immune cell of claim 7, wherein said
transmembrane domain is a CD3 zeta, CD4, a CD28, a CD8 alpha or a
4-1BB transmembrane domain.
9. The engineered immune cell of any one of the preceding claims,
wherein the CAR further comprises one or more costimulatory
domains,
10. The engineered immune cell of any of claims 1-6, wherein said
antigen receptor is a TCR, such as an endogenous TCR or an
engineered TCR.
11. The engineered immune cell of any one of the preceding claims,
wherein said antigen receptor is recombinantly expressed.
12. The engineered immune cell of any one of the preceding claims,
wherein said antibody is a full-length immunoglobulin or an
antibody derivative.
13. The engineered immune cell of any one of the preceding claims,
wherein said antibody comprises a functional Fc domain.
14. The engineered immune cell of any one of the preceding claims,
wherein said antibody comprises a Fc domain which is modified such
that it does not induce cytotoxic immune responses or complement
activation.
15. The engineered immune cell of any one of the preceding claims,
wherein said antibody does not comprise a Fc domain.
16. The engineered immune cell of claim 15, wherein said antibody
is an antibody fragment selected from the group consisting of Fab,
Fab', scFab, scFv, Fv fragment, nanobody, VHH, dAb, minimal
recognition unit, diabody, single-chain diabody (scDb), BiTE or
DART.
17. The engineered immune cell of any one of the preceding claims,
wherein said antibody binds human PD-L1 with a KD of lower than 100
pM,
18. The engineered immune cell of any one of the preceding claims,
wherein said antibody comprises i) at least one of the variable
heavy chain (VH) CDR sequences CDR-H1, CDR-H2 or CDR-H3 as set
forth in SEQ ID NOs.: 6, 7, and 8, respectively, or variants
thereof, ii) at least one of the variable light chain (VL) CDR
sequences CDR-L1, CDR-L2 or CDR-L3 as set forth in SEQ ID NOs.: 3,
4, and 5, respectively, or variants thereof.
19. The engineered immune cell of any one of the preceding claims,
wherein said antibody comprises i) at least one VH sequence of SEQ
ID NO.: 2, and/or ii) at least one VL sequence of SEQ ID NO.:
1.
20. The engineered immune cell of any one of the preceding claims,
wherein said antibody comprises SEQ ID NO.: 9.
21. The engineered immune cell of any one of the preceding claims,
comprising i) at least one VH framework sequence of SEQ ID NO.: 2,
and/or ii) at least one VL framework sequence of SEQ ID NO.: 1.
22. The engineered immune cell of any one of claims 1 to 11,
wherein the antibody competes with the antibody of claims 17 to 19
for binding to PD-L1.
23. The engineered immune cell of any one of the preceding claims,
wherein said antibody is secreted by the cell and/or expressed on
its surface, for example is secreted.
24. The engineered immune cell of any one of the preceding claims,
wherein the cell further expresses one or more cytokine, preferably
human cytokine, such as IL-2, IL-4, IL-7, IL-12, IL-15, IL-21
and/or MIP-lalpha, and/or further expresses one or more antibodies
targeting an immune inhibitory molecule, such as transforming
growth factor-beta (TGF-.beta.), IL-10, Fas, CD47, CTLA-4, Tim-3,
LAG-3, and ligands thereof.
25. The engineered immune cell of any one of the preceding claims,
wherein the antigen receptor binds to an antigen that is expressed
by or derived from a tumor or a pathogen.
26. The engineered immune cell of any one of the preceding claims,
wherein the antigen is selected from the group consisting of GD2,
WT-1, 5T4, GPC3, CSPG4, MUC16, MUC1, CA1X, CEA, CDS, CD7, CD 10,
CD19, CD20, CD22, CD23, CD30, CD33, CD34, CD38, CD41, CD44, CD49f,
CD56, CD70, CD74, CD133, CD138, CD123, cytomegalovirus (CMV)
proteins such as pp65 or IE-1, human papillomavirus (HPV) proteins
such as E6 or E7, Epstein-Barr virus (EBV) proteins such as EBNA-1,
LMP-1, LMP-2, or BARF-1, ADV proteins such as hexon, EGP-2, EGP-40,
EpCAM, erb-B2, erb-B3, erb-B4, FBP, Fetal acetylcholine receptor,
folate receptor-a, GD3, Her-1, HER-2, HER2-HER3 in combination or
HER1-HER2 in combination, hTERT, IL-13R-a2, K-light chain, DR, LeY,
LI cell adhesion molecule, MAGE-AL MAGE-A4, MAGE-A10, Mesothelin,
NKG2D ligands, NY-ESO-1, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, EGFR,
EGFRvIII, mutated p53, mutated ras, mutated raf, mutated RAC1,
bcr/abl fusions, c-Met, alphafetoprotein, CA-125, MUC-1, epithelial
tumor antigen, prostate-specific antigen, melanoma-associated
antigen, folate binding protein, HIV-1 envelope glycoprotein gp120,
HIV-1 envelope glycoprotein gp41, meothelin, HERV-K, or ERBB2.
27. A nucleic acid encoding the antigen receptor and the antibody
according to any one of the preceding claims.
28. An expression vector comprising a nucleic acid encoding the
antigen receptor and/or the antibody of any one of claims 1 to
26.
29. The expression vector of claim 28, being a lentiviral, a
retroviral, an adenoviral, an Adeno-Associated Virus (AAV), a
plasmid, a transposon, and insertion sequence, or an artificial
chromosomal vector.
30. The expression vector of claim 28 or 29, being a multicistronic
vector, such as a bicistronic vector.
31. The expression vector of any one of claims 28 to 30, comprising
at least one IRES sequence and/or at least one self-cleaving
sequence, such as a 2A sequence.
32. The expression vector of any one of claims 28 to 31, further
comprising a safety switch, for example an inducible suicide
switch.
33. A cloning vector comprising the nucleic acid of claim 27.
34. A method of generating an immune cell according to any one of
claims 1 to 26, comprising the steps of: (a) Providing an immune
cell, (b) Introducing into said cell at least one nucleic acid
encoding said antigen receptor and at least one nucleic acid
encoding said antibody; and (c) Expressing said nucleic acids by
said cell.
35. The method of claim 34, wherein step (b) comprises introducing
the expression vector of any one of claims 28 to 32 into said
cell.
36. The method of claim 34 or 35, further comprising the step of:
(i) Introducing into said cell at least one other antigen receptor
having different antigen specificity than the antigen receptor of
claim 34; and/or introducing into said cell at least one other
antibody having a different antigen specificity than the antibody
of claim 34.
37. A pharmaceutical composition comprising i) an effective amount
of the engineered immune cell of any one of claims 1 to 26 or of
the expression vector of any one of claims 28 to 32, and ii) a
pharmaceutically acceptable excipient.
38. The engineered immune cell of any one of claims 1 to 26, the
expression vector of any one of claims 28 to 32 or the
pharmaceutical composition of claim 37 for use in therapy.
39. The engineered immune cell, the expression vector or the
pharmaceutical composition of claim 38, wherein therapy is in
combination with one or more therapies selected from the group of
antibodies therapy, chemotherapy, cytokines therapy, dendritic cell
therapy, gene therapy, hormone therapy, laser light therapy and
radiation therapy.
40. A method of treating a subject in need thereof comprising: (a)
Providing the engineered immune cell according to any one of claims
1 to 26; (b) Administrating said immune cells to said subject.
41. The method of claim 40, wherein said immune cell are autologous
or allogeneic.
42. The method of any one of claim 40 or 41, wherein cells are
administered one or more times to said subject.
43. A method of treating a subject in need thereof comprising: (a)
Providing the expression vector according to any one of claims 28
to 32 or the pharmaceutical composition of claim 37; (b)
Administrating said expression vector or said pharmaceutical
composition to said subject.
44. The method of any one of claims 40 to 43, in combination with
one or more therapies selected from the group of antibody therapy,
chemotherapy, cytokine therapy, dendritic cell therapy, gene
therapy, hormone therapy, laser light therapy and radiation
therapy.
45. The cell or the vector of claim 38 or 39, or the method of any
one of claims 40 to 44, wherein the condition to be treated is a
pre-malignant or malignant cancer condition, such as NSCLC (non
small cell lung carcinoma), urothelial cancer, melanoma, renal cell
carcinoma, Hodgkin's lymphoma, head and neck squamous cell
carcinoma, ovarian cancer, gastrointestinal cancer, hepatocellular
cancer, glioma, breast cancer, lymphoma, small cell lung carcinoma,
myelodysplastic syndromes, prostate cancer, bladder cancer,
cervical cancer, non clear cell kidney cancer, colorectal cancer,
sarcomas, colon cancer, kidney cancer, lung cancer, pancreatic
cancer or gastric cancer, skin cancer, uterine cancer,
glioblastoma, neuroblastoma, sarcoma, head and neck cancer,
leukemia, carcinoma, Merkel cell carcinoma or renal cell carcinoma
(RCC), blood cancer, multiple myeloma, lymphoblastic leukemia
(ALL), B cell leukemia, chronic lymphocytic leukemia, non-Hodgkin's
lymphoma, and ovarian cancer; pathogen infection, an autoimmune
disorder.
46. A kit for treatment of cancer, pathogen infection, an
autoimmune disorder comprising the engineered immune cell of any
one of claims 1 to 26 or the expression vector of any one of claims
28 to 32, and written instructions for use.
47. The kit of claim 46, further comprising an inducer of a safety
switch, such as an inducible suicide switch.
48. The engineered immune cell of claim 9, wherein the CAR further
comprises one or more costimulatory domains, for example CD28,
4-1BB (CD137), ICOS, or OX40 (CD134), or functional fragments
thereof, respectively.
Description
RELATED APPLICATIONS
[0001] This application is a US National stage entry of
International Application No. PCT/US2017/019301, which designated
the United States and was filed on Feb. 24, 2017, published in
English. This application claims priority under 35 U.S.C. .sctn.
119 or 365 to EP Application No. 16020057.2, filed Feb. 25, 2016.
The entire teachings of the above applications are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to engineered cells
expressing an antigen receptor and an antibody blocking PD-L1 as
well as methods of using the same for the treatment cancer and
other disorders.
BACKGROUND
[0003] Immunotherapy with antigen-specific immune cells, such as T,
NK cells or NKT cells offers a promising approach for the treatment
of different types of diseases, e.g., cancers.
[0004] One therapeutic strategy is by engineering immune cells to
express chimeric antigen receptors (CARs) which specifically target
tumor cells and typically comprise an extracellular antigen
recognition domain, a transmembrane domain, and an intracellular
signaling domain derived from, for example, the T-cell receptor
CD3-zeta chain. The signaling domain may be linked to one or more
costimulatory molecule endodomains.
[0005] Still another approach is the transfer of antigen-specific T
cell receptors (TCRs) with defined specificity into immune
cells.
[0006] Cell-based immunotherapy has a major advantage over the
currently available immunotherapies based on monoclonal antibodies,
due to the potential of such genetically modified immune cells to
traffic to sites of disease, to expand and to persist even after a
single dose of administration.
[0007] Cancer cells utilize numerous pathways to escape the immune
system. PD-L1 is often expressed on tumor cells and protects tumor
cells from T cell-mediated destruction by binding PD-1.
Up-regulated levels of PD-L1 correlate with increased tumor
aggressiveness and an increased risk of death.
[0008] Animal studies demonstrated that blocking of the PD-L1:PD-1
interaction via monoclonal antibodies improves T cell activation
and reduces tumor progression. In a clinical setting, monoclonal
antibodies that block either PD-1 or PD-L1 have demonstrated
impressive activity across a broad set of cancer subtypes, even at
advanced and metastatic stages of disease (Maute et al. (2015),
PNAS, 24; 112(47): E6506-E6514).
[0009] Therefore, a therapeutic approach using antigen-specific
immune cells in combination with anti-PD-L1 antibodies is
promising. Particularly attractive is the generation of immune
cells expressing both, an antigen receptor such as a TCR and/or a
CAR and an antibody against PD-L1, as secretion of the antibody at
the site of action of the immune cell would protect it from
inactivation. Moreover, such localized antibody delivery treatment
approach has advantages over a systemic approach as to amount of
drug to be administered as well as potential side-effects.
[0010] WO2014134165 describes the co-expression of a chimeric
antigen receptor (CAR) and an anti-mouse PD-1 scFv in mouse T
cells. The scFv was derived from the Armenian hamster anti-PD-1
antibody clone J43 as described in U.S. Pat. No. 7,858,746. The
scFv construct was cloned into a retroviral backbone expressing a
CAR targeting human CD19 or human MUC-CD. Primary murine T cells
were transduced with the respective constructs.
[0011] Both Suarez E. et al, Oncotarget. 2016 Jun 7; 7(23):
34341-34355 and WO2016100985 describe armored CAR T cells targeting
human anti-carbonic anhydrase IX (CAIX) and secreting human
anti-PD-L1 antibodies. Local antibody delivery at the tumor site
led to marked inhibition of immune checkpoint pathways. In a murine
model, tumor growth diminished 5 times and tumor weight reduced
50-80% when compared with the anti-CAIX CAR T cells alone. The
antibody was of IgG1 isotype and therefore capable of mediating
ADCC, which led to human NK cells recruitment to the tumor site in
the vivo model.
[0012] However, as a therapeutic has yet to be commercialized,
there is still a significant unmet medical need to provide
effective combination therapies involving immune cells expressing
both antigen receptors and antibodies against immune checkpoint
inhibitors.
SUMMARY OF THE INVENTION
[0013] Thus, in one aspect, the invention provides for an
engineered cell expressing:
[0014] i) an antigen receptor, and
[0015] ii) an antibody that binds and blocks PD-L1. Such antibody
may be a humanized antibody or a fully human antibody. In some
embodiments, the antibody may comprise at least one CDR sequence as
set forth in SEQ ID NOs.: 3, 4, 5, 6, 7 and 8 or variants
thereof.
[0016] Said antibody preferably binds to an epitope on PD-L1 such
that PD-L1 interaction with both CD80 and PD-1 is inhibited.
Further to PD-1, PD-L1 binds to CD80, a membrane receptor expressed
on T cells and B cells, although the binding affinity of PD-L1 for
CD80 is much lower than for PD-1. PD-L1 binding to either PD-1 or
CD80 transmits inhibitory signals to T-lymphocytes, suppressing
T-cell migration, proliferation and secretion of cytotoxic
mediators, and reducing tumor cell killing. However, while
PD-1/PD-L1 interaction drives T cell exhaustion, PD-L1/CD80
interaction drives T cell anergy. These are distinct processes as
exhaustion is progressive over a period of weeks or months and
depends on the chronic antigen stimulus, while anergy is induced
rapidly after antigen stimulation in the absence of appropriate
costimulation.
[0017] The cell is preferably a therapeutic cell. Suitable cells
may e.g. be a T cell, a Natural Killer T (NKT) cell, a natural
killer (NK) cell, a human embryonic stem cell, or a hematopoietic
stem cell (HSC) or induced pluripotent stem cells (iPS). Provided
herein are engineered immune cells expressing an antigen receptor
and an antibody.
[0018] In one embodiment, the cell is T cell. Such T cell may be a
cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an
inflammatory T- lymphocytes, a helper T-lymphocyte, or a
gamma-delta T cell.
[0019] In one embodiment, the cell is a NKT cell.
[0020] In one embodiment, the cell is of human in origin. In one
embodiment, the cell is autologous; in one embodiment, the cell is
allogenic.
[0021] In one embodiment, the antigen receptor is specific to a
cancer antigen, for example a antigen which is only expressed on a
cancer cell or is upregulated on a cancer cell.
[0022] In one embodiment, the antigen receptor is T cell receptor,
for example a native T cell receptor (for example stimulated to be
specific to antigen or selected for its specificity to antigen) or
an engineered T cell receptor.
[0023] In one embodiment, the antigen receptor is a chimeric
antigen receptor (CAR).
[0024] In one embodiment, the antigen receptor is membrane
anchored.
[0025] In one embodiment, an immune cell according to the present
disclosure may comprise a TCR and a CAR, for example a native TCR
and a CAR (in particular specific to cancer antigen).
Alternatively, the immune cell of the present disclosure (such as
an NK cell) may comprise an engineered TCR and a CAR.
[0026] Exemplary engineered immune cells described herein show
enhanced tumor-killing activity compared to engineered immune cells
expressing the antigen receptor only. For example, immune cells
encoding both an antigen receptor and an anti-PD-L1 antibody can be
effective in killing cancer cells for longer periods of time than
corresponding cells encoding only an antigen receptor. Thus, cells
according to the present disclosure are less susceptible to
exhaustion, especially in the tumor microenvironment.
[0027] Further provided are a nucleic acid encoding the antigen
receptor and the antibody as described herein, as well as vectors
comprising such nucleic acid.
[0028] Also provided are methods of generating an immune cell as
described herein, comprising the steps of:
[0029] (a) Providing an immune cell,
[0030] (b) Introducing into said cell at least one nucleic acid
encoding said antigen receptor and at least one nucleic acid
encoding said antibody; and
[0031] (c) Expressing said nucleic acids by said cell.
[0032] Further provided are pharmaceutical compositions
comprising
[0033] i) an effective amount of the engineered immune cell or of
the expression vector described herein, and
[0034] ii) a pharmaceutically acceptable excipient.
[0035] Also provided are the engineered immune cells, the
expression vector or the pharmaceutical composition as described
herein for use in therapy. Further provided are methods of treating
a subject in need thereof comprising:
[0036] (a) Providing the recombinant immune cell as described
herein;
[0037] (b) Administrating said immune cells to said subject.
[0038] The present invention provides substantial benefits over
alternatives such as the combined use of an engineered immune cell
and a purified checkpoint inhibitor antibody. In attempting to use
an engineered immune cell in combination with a checkpoint
inhibitor antibody, the skilled practitioner will recognize that
the timing of antibody delivery is a crucial factor. Furthermore,
the local concentration of the antibody is also important. The
antibody offering protective function should be present when cell
contact is established between the antigen receptor expressing
cells (effector cells) and their target cells, e.g., at the tumor
site. For the medical practitioner, it is practically impossible to
determine the precise timing of systemic antibody administration in
a traditional antibody formulation, for example by infusion, to
provide the antibody at the requisite cancer location. Furthermore,
full-length antibodies are generally not able to cross the blood
brain barrier. Thus, systemic delivery of a traditional (i.e.,
full-length) antibody formulation will not usually reach cancers in
the brain. Thus, the antibody is administered systemically in high
doses, thereby increasing the possibility of side-effects. In
contrast, the instant therapeutic approach offers a controlled
release of the antibody at the site of action, thereby improving
anti-tumor efficacy therapy and decreasing the likelihood of side
effects for the subject.
[0039] The therapy or method of treatment of the invention may be
in combination with one or more therapies selected from the group
of antibody therapy, chemotherapy, cytokine therapy, dendritic cell
therapy, gene therapy, hormone therapy, laser light therapy and
radiation therapy.
[0040] Further provided are kits for treatment of cancer, pathogen
infection, and/or an autoimmune disorder comprising the engineered
immune cell or the expression vector as described herein, and
written instructions for use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows the design of the scFv anti-PD-L1 retroviral
construct SFG.scFv.anti-PD-L1(I)eGFP having 8510 bp. The Ncol
restriction site at 5' end, the Sphl restriction site at 3' end and
the TGA stop codon were added to the scFv anti-PD-L1 DNA by PCR.
PCR product was cloned into the retroviral vector SFG(I)eGFP to
obtain the final vector SFG.scFv.anti-PD-L1(I)eGFP. The reporter
gene eGFP expressed upon IRES is used to assess the transduction
efficiency. LTR: long terminal repeat; SD: splicing donor; PS:
packaging signal; TGG: truncated gagpol; SA: splicing acceptor; SP:
signal peptide; VL: variable light chain of the scFv; L: linker;
VH: variable heavy chain of the scFv; IRES: internal ribosomal
entry site; eGFP: enhanced green fluorescent protein.
[0042] FIG. 2 shows that CD4 and CD8 T cells could be transduced
with the scFv anti-PD-L1 retroviral construct without alterations
of the CD4/CD8 ratio. Transduction efficiency of the
SFG.scFv.anti-PD-L1(I)eGFP vector is shown as percentage of CD4
(FIG. 2A) or CD8 (FIG. 2B) T cells expressing eGFP. FIG. 2C:
CD4/CD8 ratio of T cells. Mean and s.d are shown (n=4 independent
experiments)*P<0.1 by paired t test. NT: Non Transduced T
cells-PD-L1: T cells transduced only with
SFG.scFv.anti-PD-L1(I)eGFP vector; CAR.28 PD-L1: T cells
co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR
encoding the CD28 endodomain; CAR.BB PD-L1: T cells co-transduced
with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the
4-IBB endodomain.
[0043] FIG. 3 shows that T cells co-expressed GD2.CAR and eGFP
anti-PD-L1 upon double retroviral transduction. FIG. 3A:
Transduction efficiency of the SFG.scFv.anti-PD-L1(I)eGFP vector.
Shown is the percentage of eGFP positive T cells. FIG. 3B:
Transduction efficiency of GD2.CAR encoding either the CD28 or the
4-1BB endodomains.
[0044] FIG. 3C: Expression of GD2.CAR shown as RFI (Relative
Fluorescence Intensity). FIG. 3D: Percentage of T cells
co-transduced with the GD2.CAR and the scFv anti-PD-L1 retroviral
vectors. FIG. 3E: Representative plots of T cells at day 10 after
the T cell initiation. Mean and s.d are shown (n=6 independent
experiments)*P<0.1, **P<0.01 by paired t test. NT: Non
Transduced T cells, CAR.28: T cells transduced only with GD2.CAR
encoding the CD28 endodomain; CAR.BB: T cells transduced only with
GD2.CAR encoding the 4-1BB endodomain; -PD-L1: T cells transduced
only with SFG.scFv.anti-PD-L1(I)eGFP vector; CAR.28 PD-L1: T cells
co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR
encoding the CD28 endodomain; CAR.BB PD-L1: T cells co-transduced
with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the
4-1BB endodomain;. Taken together these results indicate that T
cells can be co-transduced with SFG.scFv.anti-PD-L1(I)eGFP and
GD2.CAR vectors and that the transduction with
SFG.scFv.anti-PD-L1(I)eGFP vector does not alter the expression
level of GD2.CAR.
[0045] FIG. 4 shows that transduction with the scFv anti-PD-L1 did
not affect T cell proliferation. Fold increase of T cells
calculated as number of T cells at day 6 (FIG. 4A) and at day 12
(FIG. 4B) after activation divided by number of T cells at 2 days
before transduction. Mean and s.d are shown (n=6 independent
experiments). NT: Non Transduced T cells, CAR.28: T cells
transduced only with GD2.CAR encoding the CD28 endodomain; CAR.BB:
T cells transduced only with GD2.CAR encoding the 4-1BB endodomain;
-PD-L1: T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP
vector; CAR.28 PD-L1: T cells co-transduced with
SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the CD28
endodomain; CAR.BB PD-L1: T cells co-transduced with
SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-1BB
endodomain;
[0046] FIG. 5 shows that expression of the anti-PD-L1 scFv did not
affect T cell subset compositions. FIG. 5A: Scheme of T lymphocyte
subpopulations determined by the expression of CD62L, CD45RA and
CD95 [Naive (TN) CD62L+CD45RA+CD95-, Stem Cell Memory (TSCM)
CD62L+CD45RA+CD95+, Central Memory (TCM) CD62L+CD45RA-, Effector
Memory (TEM) CD62L-CD45RA-, Effector T cell (TEFF) CD62L-CD45RA+].
T cell subset compositions (FIG. 5B), percentage of CD27 (FIG. 5C),
CD28 (FIG. 5D) and PD-1 (FIG. 5E) positive cells 10 days after
stimulation with immobilized anti-CD3/CD28. Mean and s.d. are shown
(n=4 independent experiments).
[0047] FIG. 6 shows that the anti-PD-L1 scFv is released by
transduced T cells. FIG. 6A: Quantification of anti-PD-L1 scFv
released from Non-transduced (NT) and anti-PD-L1 scFv transduced T
cells (scFv PD-L1) in T cell medium with 10% FBS. T cells were
seeded and activated with immobilized anti-CD3/CD28 antibodies. The
supernatant was collected after 18 hours and anti-PD-L1 scFv
quantified by a specific sandwich ELISA. FIG. 6B: The anti-PD-L1
scFv secreted by transduced T cells binds PD-L. The cell culture
supernatants of non-transduced (NT) and anti-PD-L1 transduced T
cells (scFv PD-L1) were tested for their binding to recombinant
human PD-L1. Anti-PD-L1 scFv produced in E. coli was used as a
reference control (scFv control).
[0048] FIG. 7 shows that GD2.CAR T cells with the 4-1BB endodomain
transduced with the anti-PD-L1 scFv show better killing of tumor
cells in the second cycle of co-culture compared to GD2.CAR T cells
with the 4-1BB endodomain that are not secreting the scFV
anti-PD-L1. FIG. 7A shows that in the first cycle (7 days) of
antigen stimulation GD2.CAR-Ts encoding 4-1BB efficiently
eliminated tumor cells and the presence of the secreted anti-PD-L1
scFv does not show any impairment of the GD2.CAR-T cytotoxic
function. During the second cycle (14 days) GD2.CAR T cells with
the 4-1BB endodomain transduced with the anti-PD-L1 scFv showed
enhanced killing of tumor cells than the GD2.CAR T cells without
the anti-PD-L1 scFv. Representative plots of T cells at the end of
the first (7 days of culture; FIG. 7B) and second cycle of
co-culture (14 days of culture; FIG. 7C) (T cells identifies as
CD3+ and tumor cells CHLA-255 identified as GD2+ cells,
respectively). E:T ratio 1:5. CHLA: tumor cells, T cells: T cells
alone with no tumor cells, Tc NT: Non Transduced T cells, Tc PD-L1:
T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP vector, Tc
CAR-41BB: T cells transduced only with GD2.CAR encoding the 4-1BB
endodomain, Tc CAR-41BB PD-L1: T cells co-transduced with
SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-1BB
endodomain.
[0049] FIG. 8 shows that in the first cycle (7 days) of antigen
stimulation GD2.CAR-Ts encoding 4-1BB efficiently release IFNgamma
and the presence of the secreted anti-PD-L1 scFv does not show any
impairment of the GD2. CAR-T function (IFNgamma release). During
the second cycle (14 days) GD2.CAR T cells with the 4-1BB
endodomain transduced with the anti-PD-L1 scFv showed enhanced
release of IFNgamma compared to GD2.CAR T cells without the
anti-PD-L1 scFv IFNgamma ELISA assay to quantify the IFNgamma
produced in the first 24 hours after the first (7 days of culture;
FIG. 8A) and second (14 days of culture; FIG. 8B) tumor specific
stimulation.
[0050] As used throughout the Figures "CAR.BB" refers to "CAR.41BB"
as well as "CAR.4-1BB".
DETAILED DESCRIPTION
[0051] Unless otherwise defined, all other scientific and technical
terms used in the description, figures and claims have their
ordinary meaning as commonly understood by one of ordinary skill in
the art. Although similar or equivalent methods and materials to
those described herein can be used in the practice or testing of
the engineered cells, antibodies, antigen receptors, nucleic acids,
vectors, compositions, methods and uses disclosed herein, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will prevail. The
materials, methods, and examples are illustrative only and not
intended to be limiting.
[0052] The term "engineered immune cell" as used herein refers to
an immune cell which was genetically modified to express the
proteins described herein.
[0053] As used herein, the term "antigen receptor" refers to a
receptor that is capable of activating an immune cell in response
to antigen binding. Exemplary antigen receptors may be endogenous
(i.e., native) or recombinant T cell receptors (TCRs) or chimeric
antigen receptors (CARs). TCRs are membrane-anchored heterodimeric
proteins expressed on immune cells. Upon binding to antigenic
molecules presented by antigen presenting cells, the immune cell is
activated. Whereas some TCRs comprise variable alpha and beta
chains, others comprise gamma and delta chains, the chains being
expressed as part of a complex with the invariant CD3 chain
molecules. Each of the alpha and beta chain may comprise a variable
region and a constant region, both being located extracellulary,
wherein each variable domain has three complementarity determining
regions (CDRs) which enable binding of the TCR to the peptide/MHC
complex. The variable region of the beta chain has an additional
hypervariable region HV4 which typically does not contact antigen
and is therefore not considered a CDR (see. e.g. Richman, S. A. et
al, Mol Immunol. 2009; 46(5): 902-916).
[0054] CARs typically comprise an extracellular domain
(ectodomain), a transmembrane domain and a cytoplasmic domain
(endodomain). The ectodomain provides antigen recognition and is
most commonly a scFv but other antibody formats may also be used.
The scFv is connected via a spacer to the transmembrane domain,
which is then connected to an endodomain. First generation CARs had
a simple structured endodomain comprising CD3-zeta. Upon antigen
binding, receptors cluster and an activation signal is transmitted
to the cell. To increase the activation signal, second generation
CARs further include a co-stimulatory domain, such as CD28, OX40
and/or 4-1BB, and third generation CARs include two or more
co-stimulatory domains (Maus MV et al (2014) Blood, 123:
2625-2635). Apart from CD3-zeta, other ITAM-containing domains have
been explored including the Fc receptor for IgE-.gamma. domain.
[0055] In some embodiments, binding of the antigen to the antigen
receptor activates the immune cell through induction of signal
transduction or changes in protein expression in the immune cell
which results in initiation of an immune response.
[0056] The term "endogenous" refers to a nucleic acid or a
polypeptide that is normally expressed in a cell or tissue, absent
recombinant engineering.
[0057] As used herein, "PD-L1" refers to the protein also known as
"programmed cell death ligand 1," "cluster of differentiation 274
(i.e., CD274)" or "B7 homolog 1 (i.e., B7-H1)". The native protein
comprises two extracellular domains, a transmembrane domain, and a
cytoplasmic domain. The term encompasses full-length and/or
unprocessed PD-L1 as well as any intermediate resulting from
processing in the cell. PD-L1 can exist as a transmembrane protein
or as a soluble protein; thus, the term as used herein may refer to
the full length or the extracellular domain of the protein. The
term also encompasses naturally occurring variants of PD-L1, e.g.,
splice variants or allelic variants. The protein may additionally
contain a tag, such as a his tag or Fc tag. The amino acid sequence
of exemplary human full-length PD-L1 protein can e.g. be found
under NCBI protein database accession number NP_054862.The term
"hPD-L1" refers to human PD-L1 and comprises natural hPD-L1 and
recombinant human rhPD-L1. "rPD-L1" refers to recombinant PD-L1.
Recombinant PD-L1 may or may not have an amino terminal methionine
residue, depending upon the method by which it is prepared.
"rhPD-L1" refers to recombinant human PD-L1. Likewise, PD-L1 may
also be obtained by isolation from biological samples of human or
non-human origin rhPD-L1 may, e.g., be obtained from RnD Systems,
USA, cat. no. 156-B7, or from Peprotech, USA, cat. no. 310-35.
"Monkey PD-L1" refers to PD-L1 of Rhesus macacque (Macaca mulatta).
The amino acid sequence of exemplary monkey PD-L1 protein can e.g.
be found under NCBI protein database accession number NP 001077358.
Monkey PD-L1 may, e.g., be obtained from Sino Biological, China,
cat. no. 90251-CO2H. "Rat PD-L1" refers to PD-L1 of Rattus
norvegicus (Norway rat). The amino acid sequence of exemplary rat
PD-L1 protein can e.g. be found under NCBI protein database
accession number NP_001178883 Rat PD-L1 may, e.g., be obtained from
Sino Biological, China, cat. no. 80450-RO2H. "Mouse PD-L1" refers
to PD-L1 of Mus musculus. The amino acid sequence of exemplary
mouse PD-L1 protein can e.g. be found under NCBI protein database
accession number NP_068693Mouse PD-L1 may, e.g., be obtained from
Sino Biological, China, cat. no. 50010-MO3H or from RnD Systems,
USA, cat. no. 1019-B7-100. "PD-1" is the programmed cell death
protein 1, also known as CD279 is a cell surface receptor for
PD-L1. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 is a
transmembrane protein including an extracellular domain followed by
a transmembrane region and an intracellular domain. The term
encompasses full-length and/or unprocessed PD-1 as well as any
intermediate resulting from processing in the cell. PD-1 can exist
as a transmembrane protein or as a soluble protein; thus, the term
as used herein may refer to the full length or the extracellular
domain of the protein. The term also encompasses naturally
occurring variants of PD-1, e.g., splice variants or allelic
variants. The protein may additionally contain a tag, such as a his
tag or Fc tag. The amino acid sequence of exemplary human PD-1
protein can e.g. be found under NCBI protein database accession
number NP_005009 The term "hPD-1" refers to human PD-1 and
comprises its natural form (hPD-1) as well as the recombinant human
form (rhPD-1). "rPD-1" refers to recombinant PD-1.
[0058] "CD80" refers to the cluster of differentiation 80, also
known as B7-1, B7.1, BB1, CD28LG, CD28LG1, LAB7. It is a membrane
receptor for CD28 and CTLA-4 as well as PD-L1 and comprises
extracellular domain followed by a transmembrane region and an
intracellular domain. The term encompasses full-length and/or
unprocessed CD80 as well as any intermediate resulting from
processing in the cell. CD80 can exist as a transmembrane protein
or as a soluble protein; thus, the term as used herein may refer to
the full length or the extracellular domain of the protein. The
term also encompasses naturally occurring variants of CD80, e.g.,
splice variants or allelic variants. The protein may additionally
contain a tag, such as a his tag or Fc tag. The amino acid sequence
of exemplary human CD80 protein can e.g. be found under NCBI
protein database accession number NP 005182, CD80 may, e.g., be
obtained from RnD Systems, USA, cat. no. 9050-B1-100. The term
"hCD80" refers to human CD80 and comprises its natural form (hCD80)
as well as the recombinant human form (rhCD80). "rCD80" refers to
recombinant CD80.
[0059] "PD-L2" refers to the protein also known as "Programmed cell
death 1 ligand 2", "B7-DC", or "CD273" (cluster of differentiation
273). The term as used herein encompasses full-length and/or
unprocessed PD-L2 as well as any intermediate resulting from
processing in the cell. PD-L2 can exist as a transmembrane protein
or as a soluble protein; thus, the term as used herein may refer to
the full length or the extracellular domain of the protein. The
term also encompasses naturally occurring variants of PD-L2, e.g.,
splice variants or allelic variants. The protein may additionally
contain a tag, such as a his tag or Fc tag. The amino acid sequence
of exemplary human full-length PD-L2 protein can e.g. be found
under NCBI protein database accession number NP 079515. PD-L2 may,
e.g., be obtained from RnD Systems, USA, cat. no. 1224-PL. The term
"rhPD-L2" refers to recombinant human PD-L2. "B7-H3" refers to the
protein also known as CD276 (Cluster of Differentiation 276). The
term as used herein encompasses full-length and/or unprocessed
B7-H3 as well as any intermediate resulting from processing in the
cell. B7-H3 can exist as a transmembrane protein or as a soluble
protein; thus, the term as used herein may refer to the full length
or the extracellular domain of the protein. The term also
encompasses naturally occurring variants of B7-H3, e.g., splice
variants or allelic variants. The protein may additionally contain
a tag, such as a his tag or Fc tag. The amino acid sequence of
exemplary human full-length B7-H3 protein can e.g. be found under
NCBI protein database accession number NP_079516. B7-H3 may, e.g.,
be obtained from RnD Systems, USA, cat. no. 1027-B3. The term
"rhB7-H3" refers to recombinant human B7-H3.
[0060] T cell exhaustion as employed herein is a state of T cell
dysfunction that arises during many chronic viral infections,
autoimmunity and cancer. It is characterized by poor effector
function, sustained expression of inhibitory receptors and a
transcriptional state which is distinct from that of functional
effector or memory T cells. Exhaustion prevents optimal control of
infectious conditions and tumors, i.e., in particular in chronic
environment.
[0061] Because anti-tumor T cells are persistently exposed to
antigen in the tumor microenvironment, they are particularly
susceptible to exhaustion. Exhaustion is a likely mechanism
contributing to T cell dysfunction in cancer patients. Accordingly,
exhausted T cells have been reported for melanoma patients as well
as patients with ovarian cancer and hepatocellular carcinoma.
Exhausted T cells express multiple inhibitory receptors including
PD-1 and LAG-3, and progressively lose cytotoxic and proliferative
potential. Ultimately, they may be driven to apoptosis. Expression
of high levels of inhibitory receptors, includes programmed cell
death protein 1 (PD-1), lymphocyte activation gene 3 protein
(LAG-3), T-cell immunoglobulin domain and mucin domain protein 3
(TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T
lymphocyte attenuator (BTLA) and T-cell immunoglobulin and
immunoreceptor tyrosine-based inhibitory motif domain (TIGIT). The
other principal characteristic of exhausted T cells is the
progressive loss of their ability to express effector cytokines.
Typically, interleukin-2 (IL-2) production and ex vivo killing
capacity are lost at the early stage of exhaustion. At the
intermediate stage, tumor necrosis factor-alpha (TNF-alpha)
production is lost. Finally, at the advanced stage of exhaustion,
interferon-gamma (IFN-gamma) and granzyme B (GzmB) production are
lost. The first evidence connecting exhausted T cells with tumor
microenvironment was that programmed cell death ligand 1 was
overexpressed. See for example the review T-cell exhaustion in the
tumor microenvironment, Jiang et al., Cell Death and Disease (2015)
6, e1792.
[0062] Whereas T cell exhaustion is a result of chronic
over-stimulation, T cell anergy typically refers to a
hyporesponsive state which is induced by triggering the TCR either
(i) without adequate concomitant co-stimulation through CD28 or
(ii) in the presence of high co-inhibitory molecule signaling. As a
result thereof, IL-2 is not effectively transcribed, but
anergy-associated genes such as GRAIL are expressed instead which
contribute to impaired TCR signaling via negative feedback.
[0063] The term "isolated" indicates that matter such as a peptide,
a nucleic acid molecule or a cell has been removed from its normal
physiological environment, e.g. a natural source, or that a peptide
or nucleic acid is synthesized. Use of the term "isolated"
indicates that a naturally occurring sequence has been removed from
its normal cellular (e.g., the chromosomal or cellular)
environment. Thus, the sequence may be in a cell-free solution or
placed in a different cellular environment. "Isolated" in reference
to a polypeptide or nucleic acid molecule means a polymer of amino
acids (2 or more amino acids) or nucleotides coupled to each other,
including a polypeptide or nucleic acid molecule that is isolated
from a natural source or that is synthesized. The term "isolated"
does not imply that the sequence is the only amino acid chain or
nucleotide chain present, but that it is essentially free of e.g.
non-amino acid material and/or non-nucleic acid material,
respectively, naturally associated with it. An "isolated cell"
refers to a cell that is separated from the molecular and/or
cellular components that naturally accompany the cell.
[0064] A "variant" refers to an amino acid or nucleic acid sequence
which differs from the parental sequence by virtue of addition
(including insertions), deletion, modification and/or substitution
of one or more amino acid residues or nucleobases while retaining
at least one desired activity of the parent sequence disclosed
herein. In the case of antibodies such desired activity may include
specific antigen binding. Similarly, a variant nucleic acid
sequence may be modified when compared to the parent sequence by
virtue of addition, deletion and/or substitution of one or more
nucleobases, but the encoded antibody retains the desired activity
as described above. Variants may be naturally occurring, such as
allelic or splice variants, or may be artificially constructed. The
term "identity" as used herein refers to the sequence match between
two proteins or nucleic acids. The protein or nucleic acid
sequences to be compared are aligned for maximum correspondence
over a comparison window, for example using bioinformatics tools
such as EMBOSS Needle (pair wise alignment; available at
www.ebi.ac.uk) or by manual alignment and visual inspection. When
the same position in the sequences to be compared is occupied by
the same nucleobase or amino acid residue, then the respective
molecules are identical at that very position. Accordingly, the
"percent identity" is a function of the number of matching
positions divided by the number of positions compared and
multiplied by 100%. For instance, if 6 out of 10 sequence positions
are identical, then the identity is 60%. Aligning sequences for
maximum correspondence may require introducing gaps. The percent
identity between two protein sequences can, e.g., be determined
using the Needleman and Wunsch algorithm (Needlemann S. B. and
Wunsch C. D. A general method applicable to the search for
similarities in the amino acid sequence of two proteins. J. Mol.
Biol. 1970, vol. 48, p.443) which has been incorporated into EMBOSS
Needle, using a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap
extend penalty" of 0.5, a false "end gap penalty", an "end gap open
penalty" of 10 and an "end gap extend penalty" of 0.5, or a method
of aligning sequences manually introducing gaps in a manner which
maximises identity can be used. Two molecules having the same
primary amino acid or nucleic acid sequence are identical
irrespective of any chemical and/or biological modification. For
example, two antibodies having the same primary amino acid sequence
but different glycosylation patterns are identical by this
definition. In case of nucleic acids, for example, two molecules
having the same sequence but different linkage components such as
thiophosphate instead of phosphate are identical by this
definition. Similarly, nucleobases that differ only because of
exocyclic modifications, for example cytosine and
5-methyl-cytosine, are identical by this definition.
[0065] A sequence being longer than any of the sequences provided
herein, for example because it comprises several variable domains
or one or more constant domains, shall nevertheless be identical to
the reference sequence disclosed herein if sequence identity over a
comparison window is given. A comparison window as used herein
includes the entire sequence as claimed.
[0066] The term "CDR" refers to the hypervariable regions of the
antibody which mainly contribute to antigen binding. Typically, an
antigen binding site includes six CDRs, embedded into a framework
scaffold. Herein, the CDRs of the VL are referred to as CDR-L1,
CDR-L2 and CDR-L3 whereas the CDRs of the VH are referred to as
CDR-H1, CDR-H2 and CDR-H3. These can be identified as described in
KABAT, E. A., et al. Sequences of Proteins of Immunological
Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND
HUMAN SERVICES. NIH Publications, 1991. p. 91-3242. CDR-H1 as used
herein, however, differs from the Kabat definition in that it
starts with position 27 and ends prior to position 36 (AHo
positions 28 to 42, inclusive).
[0067] As used herein, the numbering system to identify amino acid
residue positions in the VH and VL of the antibody corresponds to
the "AHo"-system described by Honegger A. and Pluckthun A. Yet
another numbering scheme for immunoglobulin variable domains: An
automatic modelling and analysis tool. J. Mol. Biol. 2001, vol.
309, p.657. The publication further provides conversion tables
between the AHo and the Kabat system (Kabat E. A. et al., Sequences
of Proteins of Immunological Interest. 5th edition. Edited by U.S.
Department of Health and Human Services. NIH Publications, 1991.
No. 91-3242). The term "framework" (FR) refers to the scaffold of
the variable antibody domain, either the variable light chain (VL)
or variable heavy chain (VH), embedding the respective CDRs. A VL
and/or VH framework typically includes four framework sections,
FR1, FR2, FR3 and FR4, flanking the CDR regions. Thus, as known in
the art, a VL has the general structure:
(FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), whereas
a VH has the general structure:
(FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H3)-(FR-H4). Various
aspects of the disclosure are described in further detail in the
following subsections. It is understood that the various
embodiments, preferences and ranges may be combined at will.
Further, depending of the specific embodiment, selected
definitions, embodiments or ranges may not apply.
[0068] In a first aspect, the invention provides an engineered
immune cell expressing:
[0069] i) an antigen receptor, and
[0070] ii) an antibody that blocks PD-L1.
[0071] Such immune cell may e.g. be a T cell, a Natural Killer T
(NKT) cell, a natural killer (NK) cell, a human embryonic stem
cell, a hematopoietic stem cell (HSC) or a induced pluripotent stem
cell (iPS).
[0072] Said T cell may be a cytotoxic T lymphocyte (CTL), a
regulatory T lymphocyte, an inflammatory T-lymphocytes, or a helper
T-lymphocyte or a gamma-delta T cell. Additionally or
alternatively, said T cell is a CD4+ or CD8+ or a mixed population
of CD4+ and CD8+ cells.
[0073] In some embodiments, the antigen receptor is a chimeric
antigen receptor (CAR). As explained above, CARs comprise a
cytoplasmic domain acting as intracellular signaling domain, a
transmembrane domain and an extracellular domain serving antigen
recognition. The extracellular domain may be connected to a signal
peptide, to direct the transport of the domain to the cell surface.
Said signal peptide may be cleavable.
[0074] Typically, a spacer or hinge region is present between the
transmembrane domain and the extracellular domain. Such hinge
region may e.g. be selected from the group consisting of a CD8a
hinge, an IgG1 hinge or a FcyRll hinge.
[0075] In some embodiments, the CAR comprises a CD3 zeta, a CD4, a
CD28, a CD8 alpha or a 4-1BB transmembrane domain.
[0076] Additionally, the CAR may comprise one or more costimulatory
domains, e.g., selected from the group consisting of CD28, 4-1BB
(CD137), ICOS, or OX40 (CD134) costimulatory domains, or functional
fragments thereof, respectively. In preferred embodiments, the CAR
comprises 4-1BB costimulatory domain or a functional fragment
thereof. Exemplary sequences of CD28 and 4-1BB co-stimulatory
domains are provided in SEQ ID NOs: 49 and 47, respectively.
[0077] Typically, the cytoplasmic domain comprises a CD3 zeta
signaling domain. An exemplary sequence of a CD3 zeta signaling
domain is given in SEQ ID NO: 48.
[0078] Corresponding sequences are well-known and available in the
art. Exemplary sequences of signal peptides, hinge regions,
transmembrane domains are provided in FIG. 1 of WO2016/034666,
which is herein incorporated by reference. Variants of said
sequences may also be used. Other signal peptides, hinge regions,
transmembrane domains, costimulatory domains and/or signaling
domains could also be used within the scope of the invention.
[0079] In some embodiments, the CAR architecture is as shown in
FIG. 1 of Heczey A. et al, Blood. 2014 Oct. 30; 124(18):2824-33,
incorporated herein by reference. In some embodiments, the CAR
comprises an extracellular domain targeting GD2, such as the 14g2a
scFv or an antibody comprising the 14g2a variable domains or an
antibody comprising the CDRs of the 14g2a scFv, or variants thereof
respectively, a CD3 zeta signaling domain and a CD28 costimulatory
domain. In some embodiments, the CAR comprises an extracellular
domain targeting GD2, such as the 14g2a scFv, an antibody
comprising the 14g2a variable domains or an antibody comprising the
CDRs of the 14g2a scFv, or variants thereof, respectively, a CD3
zeta signaling domain and a 4-1BB costimulatory domain. The VH and
VL sequences of the 14g2a scFv can e.g. be found in the PDB
database under accession number 4TUO_A and 4TUO_B, respectively
(see also SEQ ID NOs: 13 and 14). Also contemplated is the use of
variants of the 14g2a derived sequences, in particular variants
having framework mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
mutations in the variable light and/or heavy chain. Preferred is an
anti-GD2-CAR comprising at least one, preferably all of the CDR
sequences as shown in SEQ ID NOs: 13 and 14, i.e. at least one,
preferably all CDR sequences of SEQ ID NOs. 16 to 21. Exemplary
GD2-specific CAR constructs are described in Heczey A. et al,
Blood. 2014 Oct. 30; 124(18):2824-33, in Pule, M. A. et al, Mol
Ther, 12(5), November 2005, 933-941 (see FIG. 1 for the amino acid
sequence of transmembrane and endodomains of different receptors),
as well as in WO2012033885, all three incorporated herein by
reference.
[0080] In some embodiments, the CAR comprises an extracellular
domain targeting CSPG4. Preferably, such CAR comprises at least
one, preferably all CDRs of SEQ ID NOs.: 22 to 27. In some
embodiments, such CAR comprises a VL sequence of SEQ ID NO.: 28
and/or a VH sequence of SEQ ID NO.: 29. Exemplary CSPG4-specific
CAR constructs are described in WO2015/080981, incorporated herein
by reference.
[0081] In some embodiments, the CAR comprises an extracellular
domain targeting GPC3. Preferably, such CAR comprises at least one,
preferably all CDRs of SEQ ID NOs.: 30 to 35. In some embodiments,
such CAR comprises a VL sequence of SEQ ID NO.: 36 and/or a VH
sequence selected from the group consisting of SEQ ID NO.: 37 and
SEQ ID NO.: 38. Exemplary GPC3-specific CAR constructs are
described in WO2016/049459, incorporated herein by reference.
[0082] In some embodiments, the CAR comprises an extracellular
domain targeting 5T4. Preferably, such CAR comprises at least one,
preferably all CDRs of SEQ ID NOs.: 39 to 44. In some embodiments,
such CAR comprises a VL sequence of SEQ ID NO.: 45 and/or a VH
sequence of SEQ ID NO.: 46. Exemplary 5T4-specific CAR constructs
are described in WO2016/034666, incorporated herein by reference.
Additionally or alternatively, the antigen receptor is a T cell
receptor (TCR). The TCR may be an endogenous (or native) TCR or an
engineered TCR. The endogenous TCR may for example be selected for
its specificity to antigen.
[0083] In one embodiment, the engineered TCR is a native TCR the
sequence of which is recombinantly expressed in the immune cell. In
some embodiments, the engineered TCR is derived from a native TCR,
but comprises point mutations. In one embodiment, the engineered
TCR comprises a disulfide bond in the constant region, for example
as disclosed in WO2006/000830, incorporated herein by reference. In
particular the specific locations of the disulfide bonds in the
constant regions are incorporated.
[0084] In one embodiment, the engineered TCR is modified to
increase the surface expression as described in WO2016/170320,
incorporated herein by reference. For example, the TCR comprises at
least one of the following amino acid residues:
[0085] L96 of the alpha chain; R9 of the beta chain; Y10 of the
beta chain; T24 of the alpha chain; V19 of the alpha chain; T20 of
the alpha chain; M50 of the alpha chain; T5 of the alpha chain; Q8
of the alpha chain; S86 of the alpha chain; F39 of the alpha chain;
D55 of the alpha chain; R43 of the beta chain; A66 of the alpha
chain; V19 of the beta chain; L21 of the beta chain; L103 of the
beta chain; T3 of the alpha chain; S7 of the alpha chain; P9 of the
alpha chain; M11 of the alpha chain; A16 of the alpha chain; T18 of
the alpha chain; L21 of the alpha chain; S22 of the alpha chain;
D26 of the alpha chain; F40 of the alpha chain; S47 of the alpha
chain; R48 of the alpha chain; Q49 of the alpha chain; 151 of the
alpha chain; L52 of the alpha chain; V53 of the alpha chain; T67 of
the alpha chain; E68 of the alpha chain; N74 of the alpha chain;
F76 of the alpha chain; N79 of the alpha chain; Q81 of the alpha
chain; A83 of the alpha chain; K90 of the alpha chain; S92 of the
alpha chain; D93 of the alpha chain; and M101 of the alpha chain.
Preferably, said at least one amino acid residue is not present in
the corresponding germline framework TCR amino acid sequence.
[0086] In one embodiment, the engineered TCR comprises non-human
constant regions, for example murine constant regions.
[0087] In one embodiment, the TCR, such as an endogenous TCR of a
NKT cell, is capable of binding tumor associated macrophages.
[0088] In one embodiment, the TCR is specific for survivin.
Exemplary TCRs that are specific for the survivin tumor antigen but
do not have "on-target off tumor" toxicity are disclosed in
WO2016/070119. Such survivin specific TCR preferably comprises the
CDRs of SEQ ID NOs: 50 and 51. Preferably, the TCR comprises a beta
chain of SEQ ID NOs: 50 and/or an alpha chain of 51.
[0089] In one embodiment, the TCR is specific for WT-1. Exemplary
TCRs specific for WT-1 are disclosed in WO2005056595. WT-1 specific
TCRs preferably comprise at least one CDR from the group consisting
of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In one
embodiment, the alpha chain comprises SEQ ID NOs: 52, 53 and 54. In
one embodiment, the alpha chain comprises SEQ ID NOs: 52, 53 and
55. In one embodiment, the beta chain comprises SEQ ID NOs: 56, 57
and 58. In one embodiment, the beta chain comprises SEQ ID NOs: 56,
57 and 59. Such TCR may comprise the alpha chain of SEQ ID NOs: 60
or 62.
[0090] Additionally or alternatively, such TCR may comprise the
beta chain of SEQ ID NOs: 61 or 63. Thus, in one embodiment, the
TCR comprises SEQ ID NOs: 60 and 61. In one embodiment, the TCR
comprises SEQ ID NOs: 60 and 63. In one embodiment, the TCR
comprises SEQ ID NOs: 62 and 61. In one embodiment, the TCR
comprises SEQ ID NOs: 62 and 63.
[0091] In a preferred embodiment, said antigen receptor is
recombinantly expressed. Accordingly, the immune cell is transduced
or transfected with a vector encoding said antigen receptor.
[0092] The antigen to which the antigen receptor binds, is
preferably expressed by or derived from a tumor or a pathogen. In
some embodiments, in particular when using a CAR, the antigen
receptor may bind more than one target. Also contemplated are
immune cells expressing two or more, such as three, four or five,
different recombinant antigen receptors. Exemplary antigens to
which the antigen receptor binds may include, without being limited
to, GD2, WT-1, 5T4, GPC3, CSPG4, MUC16, MUC1, CA1X, CEA, CDS, CD7,
CD 10, CD19, CD20, CD22, CD23, CD30, CD33, CD34, CD38, CD41, CD44,
CD49f, CD56, CD70, CD74, CD133, CD138, CD123, cytomegalovirus (CMV)
proteins such as pp65 or IE-1, human papillomavirus (HPV) proteins
such as E6 or E7, Epstein-Barr virus (EBV) proteins such as EBNA-1,
LMP-1, LMP-2, or BARF-1, ADV proteins such as hexon, EGP-2, EGP-40,
EpCAM, erb-B2, erb-B3, erb-B4, FBP, Fetal acetylcholine receptor,
folate receptor-a, GD3, Her-1, HER-2, HER2-HER3 in combination or
HER1-HER2 in combination, hTERT, IL- 13R-a2, K-light chain, DR,
LeY, LI cell adhesion molecule, MAGE-AL MAGE-A4, MAGE-A10,
Mesothelin, NKG2D ligands, NY-ESO-1, PSCA, PSMA, ROR1, TAG-72,
VEGF-R2, EGFR, EGFRvIII, mutated p53, mutated ras, mutated raf,
mutated RAC1, bcr/abl fusions, c-Met, alphafetoprotein, CA-125,
MUC-1, epithelial tumor antigen, prostate-specific antigen,
melanoma-associated antigen, folate binding protein, HIV-1 envelope
glycoprotein gp120, HIV-1 envelope glycoprotein gp41, meothelin,
HERV-K, or ERBB2.
[0093] The antibody may be a full-length immunoglobulin or an
antibody derivative. Over the past decades, full-length
immunoglobulins have been dissected and the modules have been used
to create monovalent, bivalent or multivalent derivatives as well
as monospecific, bispecific or multispecific derivatives.
Initially, smaller antigen binding fragments were produced by
proteolysis and later, artificial constructs have been generated by
genetic engineering.
[0094] Antibody derivatives are thus recombinant molecules
including functional parts or the entirety of a full-length
immunoglobulin, possibly in multiple copies. Exemplary antibody
derivatives include, without being limited to, Fab, Fab', scFab,
scFv, Fv fragment, nanobody, VHH, minimal recognition unit,
diabody, single-chain diabody (scDb), tandem scDb (Tandab), a
linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb),
BiTE (or tandem di-scFv or tandem scFv), DART, tandem tri-scFv,
tri(a)body, bispecific Fab2, di-miniantibody, tetrabody,
scFv-Fc-scFv fusion, scFv-Fc fusion, di-diabody, DVD-Ig, CrossMab,
Duobody, scFab-Fc, scFab-Fc-scFab, IgG-scFab, scFab-dsscFv, Fv2-Fc,
IgG-scFv fusion (such as e.g., bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab,
Ts2A)b, Knob-into-Holes (KiHs), DuoBody, (see e.g., Holliger P and
Hudson J. Engineered antibody fragments and the rise of single
domains. Nature Biotechnol. 2005, vol. 23, 9, p.1126; Dimasi N. et
al (2009), JMB 393, 672-692)).
[0095] A subgroup of antibody derivatives are antibody fragments.
As used herein, the term "antibody fragments" refers to (i)
monovalent and monospecific antibody derivatives which comprise the
variable heavy and/or light chains or functional fragments of an
antibody and lack an Fc part; and (ii) BiTE (tandem scFv), DARTs,
diabodies and single-chain diabodies (scDB). Thus, an antibody
fragment is e.g. selected from the group consisting of: Fab, Fab',
scFab, scFv, Fv fragment, nanobody, VHH, dAb, minimal recognition
unit, single-chain diabody (scDb), BiTE and DART. The recited
antibody fragments have a molecular weight below 60 kDa. In one
embodiment, the antibody derivative is an antibody fragment,
preferably a humanized antibody fragment.
[0096] In one embodiment, the antibody comprises an Fc domain which
is capable of mediating cytotoxic immune responses. Non-limiting
examples of antibodies including an Fc domain are full-length
immunoglobulins, DVD-Ig, scFv-Fc and scFv-Fc.scFv fusions,
IgG-scFab, scFab-Fc, scFab-Fc-scFab,Fv2-Fc, IgG-scFv fusions (such
as e.g., bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab), DuoBody and
CrossMab.
[0097] In one embodiment, the antibody comprises an Fc domain which
is modified such that it does not induce cytotoxic immune responses
and/or or does not activate complement. In one embodiment, the
antibody derivative lacks an Fc domain. Exemplary antibody
derivatives lacking an Fc domain are Fab, Fab', scFab, scFv, Fv
fragment, nanobody, VHH, minimal recognition unit, diabody,
single-chain diabody (scDb), tandem scDb (Tandab), a linear dimeric
scDb (LD-scDb), circular dimeric scDb (CD-scDb), BiTE (also called
tandem di-scFv or tandem scFv), tandem tri-scFv, tri(a)body,
bispecific Fab2, di-miniantibody, di-diabody, scFab-dsscFv or DART.
In one embodiment, the antibody comprises a Fc domain engineered
using Knob into Holes (KiHs) technology.
[0098] The Fc part mediates cytotoxic immune responses such as
ADCC, ADCP and/or CDC; however, such Fc mediated effects are not
required or are even undesired when targeting the PD-1: PD-L1 axis
as both proteins are expressed on the surface of antitumor
cytotoxic T cells. Hence, administering full-length monoclonal
antibodies with functional Fc parts may result in the depletion of
the very lymphocytes they are intended to activate. Treatment with
anti-PD-1 antibodies was found to correlate with lower circulating
T-cell numbers in patients. PD-L1 is expressed on non-tumor cells
as well and it is not desirable to target these cells and mediate
ADCC, ADCP and/or CDC.
[0099] In some embodiments, the antibody derivative has a molecular
weight of about 60 kDa or lower, such as about 55 kDa, 50 kDa, 45
kDa, 40 kDa, 35 kDa, 30 kDa or 27 kDa or lower. Solid tumors have
substantial physical barriers that often prevent full-length
immunoglobulins to penetrate to the center which results in reduced
therapeutic effects (Christiansen, J., and Rajasekaran, A. K.
(2004), Mol. Cancer Ther. 3, 1493-1501). Smaller antibody
derivatives may in contrast penetrate deeper into the tumor.
Exemplary antibody derivates having a molecular weight of about 60
kDa or lower are antibody fragments, including, without being
limited to, Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH,
dAb, minimal recognition unit, single-chain diabody (scDb), or
DART.
[0100] The size and/or architecture of the antibody has
implications on its half-life. To decrease side-effects in a
therapeutic setting, it may be advantageous to use antibodies with
a short half-life. This may e.g. be achieved by using an antibody
derivative lacking an Fc part, more preferably an antibody
derivative having a low molecular weight, such as about 60 kDa or
lower, such as about 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa
or 27 kDa or lower.
[0101] Preferred antibody derivatives having these characteristics
are e.g. Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, dAb,
minimal recognition unit, single-chain diabody (scDb), BiTE or
DART.
[0102] The antibody can thus be monovalent or multivalent, i.e.
having one or more antigen binding sites. Non-limiting examples of
monovalent antibodies, in particular antibody derivatives, include,
without being limited to, scFv, Fv fragments, Fab, scFab, dAb, VHH,
nanobody or minimal recognition unit. A multivalent antibody can
have two, three, four or more antigen binding sites. Full-length
immunoglobulins, F(ab')2 fragments, single-chain diabody (scDb),
tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), circular
dimeric scDb (CD-scDb), BiTE (or tandem di-scFv or tandem scFv),
DART, tandem tri-scFv, tri(a)body, bispecific Fab2,
di-miniantibody, tetrabody, scFv-Fc-scFv fusion, scFv-Fc fusion,
di-diabody, DVD-Ig, CrossMab, Duobody, scFab-Fc, scFab-Fc-scFab,
IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, diabodies,
triabodies and tetrabodies are non-limiting examples of multivalent
antibodies; an exemplary multivalent antibody comprises two binding
sites, i.e. the antibody is bivalent.
[0103] In some embodiments, the antibody, in particular the
antibody derivative, is bispecific, i.e. the antibody derivative is
directed against two different targets or two different epitopes on
one target molecule. In some embodiments, the antibody derivative
is multivalent and comprises more than two, e.g., three or four
different binding sites for three or four, respectively, different
antigens. Such antibody is multivalent and multispecific, in
particular tri- or tetra-specific, respectively.
[0104] Preferably, the antibody derivative above is an scFv (a
"single chain variable fragment" or a "single chain antibody"). An
scFv is a fusion protein that includes the VH and VL domains of an
antibody connected by a linker. It thus lacks the constant Fc
region which is present in a full-length antibody. The VH and VL
domains can be connected in either orientation, VL-linker-VH or
VH-linker-VL, by a flexible linker. In a preferred embodiment, the
orientation is VL-linker-VH, i.e. the light chain variable region
being at the N-terminal end and the heavy chain variable region
being at the C-terminal end of the polypeptide. The linker may have
the sequence of SEQ ID NO: 10, however, shorter or longer linkers
or variants of SEQ ID NO: 10 may also be used.
[0105] Thus, in one embodiment, the cell provided herein is a T
cell expressing a scFv blocking PD-L1 and at least one CAR
according to the present disclosure. In one embodiment, the cell
provided herein is a T cell expressing a scFv blocking PD-L1 and at
least one TCR according to the present disclosure. In one
embodiment, the cell provided herein is a T cell expressing a scFv
blocking PD-L1 and at least one CAR as well as at least one TCR
according to the present disclosure.
[0106] Thus, in one embodiment, the cell provided herein is a
Natural Killer T (NKT) cell, expressing a scFv blocking PD-L1 and
at least one CAR according to the present disclosure. In one
embodiment, the cell provided herein is a NKT cell expressing a
scFv blocking PD-L1 and at least one TCR according to the present
disclosure. In one embodiment, the cell provided herein is a NKT
cell expressing a scFv blocking PD-L1 and at least one CAR as well
as at least one TCR according to the present disclosure.
[0107] Thus, in one embodiment, the cell provided herein is a
Natural Killer (NK) cell expressing a scFv blocking PD-L1 and at
least one CAR according to the present disclosure. In one
embodiment, the cell provided herein is a NK cell expressing a scFv
blocking PD-L1 and at least one TCR according to the present
disclosure. In one embodiment, the cell provided herein is a NK
cell expressing a scFv blocking PD-L1 and at least one CAR as well
as at least one TCR according to the present disclosure.
[0108] Thus, in one embodiment, the cell provided herein is a human
embryonic stem cell, expressing a scFv blocking PD-L1 and at least
one CAR according to the present disclosure. In one embodiment, the
cell provided herein is a human embryonic stem cell expressing a
scFv blocking PD-L1 and at least one TCR according to the present
disclosure. In one embodiment, the cell provided herein is a human
embryonic stem cell expressing a scFv blocking PD-L1 and at least
one CAR as well as at least one TCR according to the present
disclosure.
[0109] Thus, in one embodiment, the cell provided herein is a
hematopoietic stem cell (HSC) expressing a scFv blocking PD-L1 and
at least one CAR according to the present disclosure. In one
embodiment, the cell provided herein is a hematopoietic stem cell
expressing a scFv blocking PD-L1 and at least one TCR according to
the present disclosure. In one embodiment, the cell provided herein
is a hematopoietic stem cell expressing a scFv blocking PD-L1 and
at least one CAR as well as at least one TCR according to the
present disclosure.
[0110] Thus, in one embodiment, the cell provided herein is an
induced pluripotent stem cell (iPS) expressing a scFv blocking
PD-L1 and at least one CAR according to the present disclosure.
[0111] In one embodiment, the cell provided herein is an induced
pluripotent stem cell expressing a scFv blocking PD-L1 and at least
one TCR according to the present disclosure. In one embodiment, the
cell provided herein is an induced pluripotent stem cell expressing
a scFv blocking PD-L1 and at least one CAR as well as at least one
TCR according to the present disclosure.
[0112] Antibodies having the frameworks as used herein have been
described as being surprisingly stable in the scFv format (see,
e.g., WO/2009/155726 or Borras et al., JBC, Vol. 285, no. 12, 9
Mar. 2010, pages 9054-9066). Thus, the antibody preferably
comprises the framework sequences as comprised in SEQ ID Nos: 1
and/or 2 or variants thereof. Variants may e.g. include
modifications as described in WO2014/206561, in particular
including VL framework sequences SEQ ID NOs. 15 to 22 of
WO2014/206561.
[0113] The antibody is preferably humanized, to avoid an immune
response against the protein. "Humanized" antibodies refer to
antibodies that include one or more, typically all six CDR regions
of a non-human parent antibody or variants thereof or synthetic
CDRs, and of which the framework is, e.g., (i) a human framework,
potentially including one or more framework residues of the
non-human parent antibody, or (ii) a framework from a non-human
antibody modified to increase similarity to naturally produced
human frameworks. Methods of humanizing antibodies are known in the
art, see e.g. Leger O., and Saldanha J. Antibody Drug Discovery.
Edited by Wood C. London: Imperial College Press, 2011. ISBN
1848166281. p.1-23.
[0114] In some embodiments, the antibody is fully human.
[0115] In preferred embodiments, the antibody binds to an epitope
on PD-L1 such that PD-L1 interaction with both CD80 and PD-1 is
blocked. As PD-L1 binding to PD-1 drives T cell exhaustion and
PD-L1 binding to CD80 drives T cell anergy, simultaneously blocking
the binding of PD-L1 to CD80 and PD-1 prevents anergy and reverts
exhaustion.
[0116] In preferred embodiments, the antibody comprises
[0117] i) at least one of the variable heavy chain (VH) CDR
sequences CDR-H1, CDR-H2 or CDR-H3 as set forth in SEQ ID NOs.: 6,
7 and 8, respectively, or variants thereof,
[0118] ii) at least one of the variable light chain (VL) CDR
sequences CDR-L1 , CDR-L2 or CDR-L3 as set forth in SEQ ID NOs.: 3,
4, and 5, respectively, or variants thereof.
[0119] Preferably, the antibody comprises
[0120] i) at least one of VH sequence of SEQ ID NO: 2, and/or
[0121] ii) at least one VL sequence of SEQ ID NO: 1, or variants
thereof, respectively.
[0122] In some embodiments, said antibody is a scFv comprising SEQ
ID NO.: 9 or a variant thereof.
[0123] A variant may in some embodiments be an antibody that
differs from a given antibody, in one, two, three, four, five or
more positions of its amino acid sequence. Such difference may
e.g., be a substitution, addition, modification or deletion. In one
embodiment, the variant has at least 85%, more preferably 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100%
sequence identity to the sequences disclosed herein, in particular
SEQ ID NO: 1, 2 or 9.
[0124] Variants of the antibodies provided herein may be prepared
by introducing appropriate modifications into the nucleic acid
sequence encoding the antibody. Any combination(s) of deletions,
substitutions, additions, modifications and insertions can be made
to the framework or to the CDRs, provided that the generated
antibody possesses the desired characteristics for which it can be
screened using appropriate methods. Of particular interest are
substitutions, preferably conservative substitutions.
[0125] As used herein, "conservative substitution" refers to a
modification and a substitution, that maintains physically,
biologically, chemically and/or functionally the properties with
regard to the corresponding reference. A molecule that includes a
sequence with conservative substitution for instance may have a
similar size, shape, electric charge, chemical properties,
including a comparable ability to form covalent or hydrogen bonds,
and/or comparable polarity. Such conservative modifications
include, but are not limited to, one or more nucleobases and amino
acid substitutions, additions and deletions.
[0126] For example, conservative amino acid substitutions include
those in which the amino acid residue is replaced with an amino
acid residue having a similar side chain. For example, amino acid
residues being non-essential with regard to binding to an antigen
can be replaced with another amino acid residue from the same side
chain family, e.g. serine may be substituted for threonine. Amino
acid residues are usually divided into families based on common,
similar side-chain properties, such as:
[0127] 1. nonpolar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, methionine),
[0128] 2. uncharged polar side chains (e.g., asparagine, glutamine,
serine, threonine, tyrosine, proline, cysteine, tryptophan),
[0129] 3. basic side chains (e.g., lysine, arginine, histidine,
proline),
[0130] 4. acidic side chains (e.g., aspartic acid, glutamic
acid),
[0131] 5. beta-branched side chains (e.g., threonine, valine,
isoleucine) and
[0132] 6. aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). A conservative substitution can be taken to
be a substitution of a first amino acid within one of the six
groups above by a further amino acid within the same group of the
six groups. Preferred conservative substitutions include:
[0133] 1. Substituting alanine (A) by valine (V);
[0134] 2. Substituting arginine (R) by lysine (K);
[0135] 3. Substituting asparagine (N) by glutamine (Q);
[0136] 4. Substituting aspartic acid (D) by glutamic acid (E);
[0137] 5. Substituting cysteine (C) by serine (S);
[0138] 6. Substituting glutamic acid (E) by aspartic acid (D);
[0139] 7. Substituting glycine (G) by alanine (A);
[0140] 8. Substituting histidine (H) by arginine (R) or lysine
(K);
[0141] 9. Substituting isoleucine (I) by leucine (L);
[0142] 10. Substituting methionine (M) by leucine (L);
[0143] 11. Substituting phenylalanine (F) by tyrosine (Y);
[0144] 12. Substituting serine (S) by threonine (T);
[0145] 13. Substituting tryptophan (W) by tyrosine (Y);
[0146] 14. Substituting phenylalanine (F) by tryptophan (W);
and/or
[0147] 15. Substituting valine (V) by leucine (L) and vice versa.
Other substitutions such as substituting proline (P) by alanine (A)
are also permissible and can be determined empirically or in accord
with other known conservative or non-conservative substitutions. A
conservative substitution may also involve the use of a non-natural
amino acid.
[0148] The antibody described herein may comprise one or more, such
as two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve or more of such conservative substitutions.
[0149] In another embodiment, non-conservative substitutions are
introduced into any sequence disclosed herein to produce a variant.
In one embodiment, the antibody comprises one or more, such as two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve or
more of such non-conservative substitutions.
[0150] A particularly preferred type of variant is one where one or
more entire CDRs are replaced. Typically, the CDR-H3 and CDR-L3
contribute most significantly to antigen binding. For example, the
entire CDR-L1, CDR-L2, CDR-H1 and/or CDR-H2 may be replaced by a
different CDR of natural or artificial origin. In some embodiments,
one or more CDRs are replaced by an alanine-cassette.
[0151] In some embodiments, the variant does not show any
improvement over the parent antibody.
[0152] In some embodiments, a variant antibody as described
herein
[0153] (i) retains specific binding to PD-L1, in particular to
hPD-L1, preferably blocking the interaction between PD-L1 and PD-1;
and/or
[0154] (ii) has a KD to human PD-L1 of lower than 500 pM,
preferably lower than 250 pM, 100 pM, 75 pM, 50 pM, 40 pM, 30 pM,
20 pM, more preferably of lower than 10 pM as measured by
KinExA.RTM.; and/or
[0155] (iv) competes with the antibody disclosed herein for binding
to PD-L1; and/or
[0156] (v) has at least 80%, preferably at least 85%, 90%, 95% or
97% sequence identity to the sequences disclosed herein.
[0157] In some embodiments, the antibody has high affinity for
PD-L1 and binds hPD-L1 with a KD of lower than 100 pM, such as
lower than about 75 pM, 50 pM, 25 pM or 10 pM. For example, the
antibody is bivalent and binds PD-L1 with a KD of lower than 10 pM
as measured by KinExA.RTM., preferably lower than 5 pM, more
preferably about 3 pM, e.g., 2.9 pM, 2.8 pM or 2.7 pM. In some
embodiments, such bivalent antibody is a full-length
immunoglobulin.
[0158] In some embodiments, the antibody provided herein is
monovalent and binds human PD-L1 with a KD of lower than 50 pM as
measured by KinExA.RTM.. Said KD is preferably lower than about 10
pM, such as about 9 pM, e.g., 9.0 pM, 8.9 pM, 8.8 pM or 8.7 pM. In
some embodiments, said monovalent antibody is a scFv.
[0159] In some embodiments, the antibody provided herein is
monovalent and binds monkey PD-L1 with a KD of lower than 50 pM as
measured by KinExA.RTM.. Said KD is preferably lower than about 10
pM, more preferably lower than about 5 pM, such as e.g., about 3.4
pM, 3.3 pM or 3.2 pM as measured by KinExA.RTM..
[0160] Such KinExA.RTM..measurements are preferably done at room
temperature, more preferably under the conditions as described in
example 1.
[0161] High affinity antibodies may be advantageous to provide a
protective effect even if a small amount of engineered immune cells
is present at its target site, accordingly expressing a limited
amount of antibody.
[0162] Also provided are engineered immune cells as described above
expressing antibodies that bind to human PD-L1 as well as to monkey
PD-L1. Preferably, the affinity to monkey PD-L1 is at least twice
as tight as the affinity to human PD-L1. In some embodiments, the
affinity KD of a monovalent antibody, preferably a scFv, to monkey
PD-L1 is about 3.3 pM as measured by KinExA.RTM., for example as
measured at room temperature, preferably under the conditions
indicated in Example 1.
[0163] Further provided are antibodies that have no
cross-reactivity to other member of the B7 family, such as PD-L2
and B7-H3.
[0164] Further contemplated are engineered immune cells as
described above expressing antibodies that compete with the
antibodies disclosed herein for binding to PD-L1.
[0165] The engineered immune cell described herein may secreted the
antibody and/or express the antibody on its surface. In preferred
embodiments, the antibody is secreted.
[0166] In some embodiments, the cell may additionally recombinantly
express at least one further protein compound such as a second
antibody or a cytokine. Cytokines are e.g. selected from the group
consisting of IL-2, IL-4, IL-7, IL-12, IL-15, IL-21 or MIP-lalpha
and are preferably of human origin, i.e. hIL-2, hIL-4, hIL-7,
hIL-15, hIL-21, or hMIP-1 alpha. In some embodiments, such cell
expresses hIL-15. IL-15 has been described to improve in vivo
persistence and anti-tumor activity of CAR NKT cells (see
WO2013/040371). Such second antibody may e.g., target an immune
inhibitory molecule, such as transforming growth factor-beta
(TGF-.beta.), IL-10, Fas, CD47, CTLA-4, Tim-3, LAG-3, or ligands
thereof.
[0167] In some embodiments, the engineered immune cell expresses an
antigen receptor being a CAR comprising a 4-1BB costimulatory
domain, an antibody as described herein and further IL-15. In some
embodiments, the engineered immune cell expresses an antigen
receptor being a CAR comprising a CD28 costimulatory domain, an
antibody as described herein and further IL-15.
[0168] Thus there is provided an NKT cell with a native TCR, a
chimeric antigen receptor (in particular specific for a tumor
antigen), and an antibody to PD-L1 according to the present
disclosure, in particular a scFv. The NKT cell further encodes a
cytokine, such as IL-15.
[0169] Further contemplated is a nucleic acid encoding the antigen
receptor and/or the antibody described herein. The proteins may be
encoded by a plurality of nucleic acid sequences. In some
embodiments, the proteins are encoded by a single nucleic acid
sequence. Typically, the nucleic acid is an isolated nucleic
acid.
[0170] Knowing the sequence of the antibody and/or the antigen
receptor, cDNAs encoding the respective polypeptide sequence can be
generated by methods well known in the art, e.g. by gene synthesis.
These cDNAs can be cloned by standard cloning and mutagenesis
techniques into a suitable vector such as an expression vector or a
cloning vector. Thus, further contemplated is cDNA encoding the
antigen receptor and/or the antibody as described herein.
[0171] Based on the cloning strategy chosen, genetic constructs may
generate an antibody and/or an antigen receptor having one or more
additional residues at the N-terminal or C-terminal end. It is
therefore to be understood that the antibodies disclosed herein
include the disclosed sequences rather than consist of them.
[0172] Basic protocols of standard cloning, mutagenesis and
molecular biology techniques are described in, e.g., Molecular
Cloning, A Laboratory Manual (Green M. and Sambrook, J. Molecular
Cloning: a Laboratory Manual. 4th edition. Cold Spring Harbor
Laboratory, 2012. ISBN 1936113422).
[0173] Further contemplated are isolated nucleic acids hybridizing
with the nucleic acids described herein under stringent
conditions.
[0174] Also provided are vectors comprising the nucleic acid
provided herein, such as an expression vector or a cloning vector.
One, two or more nucleic acids encoding the antigen receptor and
the antibody described herein may be comprised in a vector, which
may be the same vector (bicistronic or multicistronic) or separate
vectors. The expression vector may be a multicistronic vector, such
as a bicistronic vector, e.g., using internal ribosome entry sites
(IRES), 2A-like sequences or dual promoters.
[0175] The expression vector may e.g. be a lentiviral, a
retroviral, an adenoviral or an Adeno-Associated Virus (AAV)
vector. The expression vector may also be a non-viral vector,
including a plasmid, a transposon, an inserting sequence, or an
artificial chromosome. A nucleic acid molecule may in some
embodiments define an expression cassette. An expression cassette
is a nucleic acid molecule capable of directing expression of a
particular nucleotide sequence in an appropriate host cell. An
expression cassette includes a promoter operatively linked to the
nucleotide sequence of interest, which is operatively linked to one
or more termination signals. It may also include sequences required
for proper translation of the nucleotide sequence. The coding
region can encode a polypeptide of interest. The expression of the
nucleotide sequence in the expression cassette can be under the
control of a constitutive promoter or of an inducible promoter that
initiates transcription only when the host cell is exposed to some
particular external stimulus. In some embodiments, the antibody is
under the control of the 5'' end LTR of the retrovirus. The nucleic
acid encoding the antigen receptor and/or the nucleic acid encoding
the antibody may each be operably linked to a promoter which may be
the same or different promoters.
[0176] The nucleic acid and/or vectors may further comprise a
signal peptide. Typically, a signal peptide is a 5-30 amino acid
peptide attached to the N-terminus of the protein to be secreted
and is attached to increase protein secretion. In preferred
embodiments, the signal peptide is a human signal peptide. In some
embodiments, the signal peptide is hIgGl. In some embodiments, the
signal peptide comprises SEQ ID NO: 15.
[0177] Additionally or alternatively, the antibody is membrane
anchored. Such membrane anchored antibody may comprise a
transmembrane domain. In some embodiments, the membrane anchored
antibody does not comprise no signaling domain. In one embodiment,
the antibody is secreted and in also provided as membrane anchored
form.
[0178] A genetically engineered immune cell may comprise a safety
switch, such as a suicide switch. Such switches suppress the cell's
activity if serious side effects emerge, or make the cells
self-destruct if they attack healthy tissue. Typically, such
switches are controllable and therefore require an additional
receptor or other target on the cell. Such safety switches are
controlled by administering a second medication to the subject.
Thus, in some embodiments, the vector comprises a nucleic acid
sequence encoding a safety switch, preferably a suicide switch.
[0179] The invention also provides a method of generating an immune
cell as described herein, comprising the steps of:
[0180] (a) Providing an immune cell,
[0181] (b) Introducing into said cell at least one nucleic acid
encoding said antigen receptor and at least one nucleic acid
encoding said antibody; and
[0182] (c) Expressing said nucleic acids by said cell.
[0183] In some embodiments, step (b) comprises introducing the
expression vector as described above into said cell.
[0184] The method may comprise the additional step of:
[0185] (i) Introducing into said cell at least one other antigen
receptor having a different antigen specificity than the antigen
receptor of step (b); and/or introducing into said cell at least
one other antibody having a different antigen specificity than the
antibody of step (b).
[0186] The invention also relates to a pharmaceutical composition
comprising
[0187] i) an effective amount of the engineered immune cell
described herein or of the expression vector described herein,
and
[0188] ii) a pharmaceutically acceptable excipient.
[0189] Suitable "excipients" include, without being limited to: (i)
buffers such as phosphate, citrate, or other, organic acids; (ii)
antioxidants such as ascorbic acid and tocopherol; (iii)
preservatives such as 3-pentanol, hexamethonium chloride,
benzalkonium chloride, benzyl alcohol, alkyl paraben, catechol, or
cyclohexanol; (iv) amino acids, such as e.g. histidine, arginine;
(v) peptides, preferably up to 10 residues such as polylysine; (vi)
proteins, such as bovine or human serum albumin; (vii) hydrophilic
polymers such as polyvinylpyrrolidone; (viii) monosaccharides,
disaccharides, polysaccharides and/or other carbohydrates including
glucose, mannose, sucrose, mannitol, trehalose, sorbitol,
aminodextran or polyamidoamines; (ix) chelating agents, e.g. EDTA;
(x) salt-forming ions such as sodium, potassium, and/or chloride;
(xi) metal complexes (e.g. Zn-protein complexes); (xii) ionic and
non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene
glycol (PEG), (xiii) cryopreservatives such as dimethyl sulfoxide
(DMSO).
[0190] The engineered immune cell described herein, the expression
vector described herein and/or the pharmaceutical composition
described herein are useful for therapy. Thus, further provided is
the engineered immune cell described herein, the expression vector
described herein and/or the pharmaceutical composition described
herein for use in therapy.
[0191] Also provided is a method of treating a subject in need
thereof comprising:
[0192] (a) Providing the engineered immune cell described herein;
and
[0193] (b) Administrating said immune cells to said subject.
[0194] Additionally or alternatively, the method of treatment
involves the provision and administration of the expression vector
described herein and/or the pharmaceutical composition described
herein.
[0195] The terms "treatment" and "treating" as used herein, include
a prophylactic or preventative measure having a therapeutic effect
and/or preventing, slowing down (lessening), or at least partially
alleviating or abrogating an abnormal, including pathologic,
condition in the organism of a subject. Treatment according to the
present disclosure involves the administration of a
pharmaceutically effective amount of a molecule, nucleic acid,
vector, pharmaceutical composition, and/or an engineered immune
cell as described herein, i.e. inter alia, the cell, the vector or
the composition disclosed herein, to a subject in need thereof to
prevent, cure, delay the onset and/or progression, reduce the
severity of, stabilize, modulate, cure or ameliorate one or more
symptoms of the condition to be treated. Those in need of treatment
include those already with the disorder as well as those prone to
having the disorder or those in whom the disorder is to be
prevented (prophylaxis). Generally, a treatment reduces,
stabilizes, or inhibits progression of a symptom that is associated
with the presence and/or progression of a disease or pathological
condition.
[0196] The subject in need of a treatment can be a human or a
non-human animal. Typically, the subject is a mammal, e.g., a
mouse, a rat, rabbit, a hamster, a dog, a cat, a monkey, an ape, a
goat, a sheep, a horse, a chicken, a guinea pig or a pig. In
typical embodiments, the subject is diagnosed with a cancer and/or
a PD-L1-related disorder or may acquire such a disorder. In case of
an animal model, the animal might be genetically engineered to
develop such disorder.
[0197] Typically, an effective amount of the cell, the vector or
the composition disclosed herein is administered to the subject. An
"effective amount" is an amount--either as a single dose or as part
of a series of doses--which at the dosage regimen applied yields
the desired therapeutic effect, i.e., to reach a certain treatment
goal. A therapeutically effective amount is generally an amount
sufficient to provide a therapeutic benefit in the treatment or
management of the relevant pathological condition, or to delay or
minimize one or more symptoms associated with the presence of the
condition. The dosage will depend on various factors including
patient and clinical factors (e.g., age, weight, gender, clinical
history of the patient, severity of the disorder and/or response to
the treatment), the nature of the disorder being treated, the
particular composition to be administered, the route of
administration, and other factors.
[0198] The term "administering", as used herein, refers to any mode
of transferring, delivering, introducing, or transporting matter
such as the cell, the vector or the composition described herein,
to a subject. Administration may be administered locally or
systemically. Preferred modes of administration include, without
being limited to, parenteral, e.g., intravenous, or systemic,
administration. Administration "in combination with" further matter
such as one or more therapeutic agents includes simultaneous
(concurrent) and consecutive administration in any order.
[0199] The cells, the vector or the composition are administered
one or more times to said subject.
[0200] The cells, the vector or the composition may be administered
in combination with one or more therapies selected from the group
of antibody therapy, chemotherapy, cytokine therapy, dendritic cell
therapy, gene therapy, hormone therapy, laser light therapy and
radiation therapy. The therapies of the present invention may
precede or follow the other agent treatment by intervals ranging
from minutes to weeks.
[0201] The cells may have originated from the subject or from
another individual of the same species, i.e. they are autologous or
allogeneic. Autologous adoptive transfer requires extraction of the
patient's cells, their genetic modification, e.g. as described
above and culturing said cells in vitro before returning them to
the same patient. Such individual preparation for each new patient
limits application of cellular immunotherapies in treating cancer.
As an off-the-shelf product, however, allogeneic T cells derived
from healthy donors carry the risk of recognizing the patient's
body as foreign, which can cause a serious side effect called graft
versus host disease (GvHD). Off-the-shelf therapies based on
CAR-modified NKT cells generated in large volumes from healthy
donors offer great promise. While endowed with powerful
cancer-killing properties like conventional T cells, invariant NKT
cells express special T cell receptors that are not associated with
GvHD. Hence allogeneic NKT cells can be used to treat multiple
cancer patients with minimal risk of GvHD.
[0202] The subject in need of treatment may have a condition,
without being limited to, a pre-malignant or malignant cancer
condition, such as NSCLC (non small cell lung carcinoma),
urothelial cancer, melanoma, renal cell carcinoma, Hodgkin's
lymphoma, head and neck squamous cell carcinoma, ovarian cancer,
gastrointestinal cancer, hepatocellular cancer, glioma, breast
cancer, lymphoma, small cell lung carcinoma, myelodysplastic
syndromes, prostate cancer, bladder cancer, cervical cancer, non
clear cell kidney cancer, colorectal cancer, sarcomas, colon
cancer, kidney cancer, lung cancer, pancreatic cancer or gastric
cancer, skin cancer, uterine cancer, glioblastoma, neuroblastoma,
sarcoma, head and neck cancer, leukemia, carcinoma, Merkel cell
carcinoma or renal cell carcinoma (RCC), multiple myeloma,
lymphoblastic leukemia (ALL), B cell leukemia, chronic lymphocytic
leukemia, non-Hodgkin's lymphoma; pathogen infection, an autoimmune
disorder.
[0203] The invention further provides a kit for treatment of
cancer, pathogen infection, an autoimmune disorder comprising the
engineered immune cell described herein, the expression vector
described herein or the pharmaceutical composition described
herein, and written instructions for use.
[0204] In some embodiments, the kit may further comprise an inducer
of a safety switch.
SEQUENCES
[0205] The sequences disclosed herein are
TABLE-US-00001 -VL of scFv SEQ ID NO: 1
EIVMTQSPSTLSASVGDRVIITCQASEDIYSLLAWYQQKPGKAPKLLIYDASDLASG
VPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNYGSSSSSSYGAVFGQGTKLTVLG -VH of
scFv SEQ ID NO: 2
EVQLVESGGGLVQPGGSLRLSCTVSGIDLSSYTMGWVRQAPGKGLEWVGIISSGG
RTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARGRYTGYPYYFAL WGQGTLVTVSS
-CDR-L1 of scFv SEQ ID NO: 3 QASEDIYSLLA -CDR-L2 of scFv SEQ ID NO:
4 DASDLAS -CDR-L3 of scFv SEQ ID NO: 5 QGNYGSSSSSSYGAV -CDR-H1 of
scFv SEQ ID NO: 6 IDLSSYTMG -CDR-H2 of scFv SEQ ID NO: 7
IISSGGRTYYASWAKG -CDR-H3 of scFv SEQ ID NO: 8 GRYTGYPYYFAL -scFv
SEQ ID NO: 9
EIVMTQSPSTLSASVGDRVIITCQASEDIYSLLAWYQQKPGKAPKLLIYDASDLASG
VPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGNYGSSSSSSYGAVFGQGTKLTVLG
GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGIDLSSYTMG
WVRQAPGKGLEWVGIISSGGRTYYASWAKGRFTISRDTSKNTVYLQMNSLRAED
TAVYYCARGRYTGYPYYFALWGQGTLVTVSS -linker SEQ ID NO: 10
GGGGSGGGGSGGGGSGGGGS -forward primer SEQ ID NO: 11
TAACCATGGAGTTTGGGCTGAG -reverse primer SEQ ID NO: 12
GACGCATGCTCAGCTCGACACGGTGACC -VL sequence of 14g2a scFv SEQ ID NO:
13 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHK
VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLE LKR (CDR
sequences highlighted in bold) -VH sequence of 14g2a scFv SEQ ID
NO: 14 EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYY
GGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTS VTVSS (CDR
sequences highlighted in bold) -hIgG1 signal peptide SEQ ID NO: 15
MEFGLSWLFLVAILKGVQ -CDR-L1 of anti-GD2-CAR SEQ ID NO: 16
RSSQSLVHRNGNTYLH -CDR-L2 of anti-GD2-CAR SEQ ID NO: 17 KVSNRFS
-CDR-L3 of anti-GD2-CAR SEQ ID NO: 18 SQSTHVPPLT -CDR-H1 of
anti-GD2-CAR SEQ ID NO: 19 SSFTGYNMN -CDR-H2 of anti-GD2-CAR SEQ ID
NO: 20 AIDPYYGGTSYNQKFKG -CDR-H3 of anti-GD2-CAR SEQ ID NO: 21 GMEY
-CDR-L1 of anti-CSPG4 CAR SEQ ID NO: 22 RASQTIYKNLH -CDR-L2 of
anti-CSPG4 CAR SEQ ID NO: 23 YGSDSIS -CDR-L3 of anti-CSPG4 CAR SEQ
ID NO: 24 LQGYSTPWT -CDR-H1 of anti-CSPG4 CAR SEQ ID NO: 25
YTFTDYSMH -CDR-H2 of anti-CSPG4 CAR SEQ ID NO: 26 WINTATGEPTYADDFKG
-CDR-H3 of anti-CSPG4 CAR SEQ ID NO: 27 YYDY -VL sequence of
anti-CSPG4 CAR SEQ ID NO: 28
LDIKLTQSPSILSVTPGETVSLSCRASQTIYKNLHWYQQKSHRSPRLLIKYGSDSISG
IPSRFTGSGSGTDYTLNINSVKPEDEGIYYCLQGYSTPWTFGGGTKLEIKR -VH sequence of
anti-CSPG4 CAR SEQ ID NO: 29
QVKLKESGPELKKPGETVKISCKASGYTFTDYSMHWVKKTPGKGLKWLGWINTA
TGEPTYADDFKGRFAISLETSARTVYLQINNLRNEDTATYFCFSYYDYWGQGTTV TVSS
-CDR-L1 of anti-GPC3 CAR SEQ ID NO: 30 RSSQSLVHSNRNTYLH -CDR-L2 of
anti-GPC3 CAR SEQ ID NO: 31 KVSNRFS -CDR-L3 of anti-GPC3 CAR SEQ ID
NO: 32 SQNTHVPPT -CDR-H1 of anti-GPC3 CAR SEQ ID NO: 33 YTFTDYEMH
-CDR-H2 of anti-GPC3 CAR SEQ ID NO: 34 ALDPKTGDTAYSQKFKG -CDR-H3 of
anti-GPC3 CAR SEQ ID NO: 35 FYSYTY -VL of anti-GPC3 CAR SEQ ID NO:
36 DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK R -VH of
anti-GPC3 CAR SEQ ID NO: 37
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALD
PKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWG QGTLVTVSS
-VH of anti-GPC3 CAR SEQ ID NO.: 38
QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDP
KTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQ GTLVTVSA
-CDR-L1 of anti-5T4 CAR SEQ ID NO: 39 YSFTGYYMH -CDR-L2 of anti-5T4
CAR SEQ ID NO: 40 RINPNNGVTLYNQKFKD -CDR-L3 of anti-5T4 CAR SEQ ID
NO: 41 STMITNYVMDY -CDR-H1 of anti-5T4 CAR SEQ ID NO: 42
KASQSVSNDVA -CDR-H2 of anti-5T4 CAR SEQ ID NO: 43 YTSSRYA -CDR-H3
of anti-5T4 CAR SEQ ID NO: 44 QQDYNSPPT -VL of anti-5T4 CAR SEQ ID
NO: 45 SIVMTQTPTFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPTLLISYTSSRYA
GVPDRFIGSGYGTDFTFTISTLQAEDLAVYFCQQDYNSPPTFGGGTKLEIKR -VH of
anti-5T4 CAR SEQ ID NO: 46
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGRINPNN
GVTLYNQKFKDKAILTVDKSSTTAYMELRSLTSEDSAVYYCARSTMITNYVMDY WGQVTSVTVSS
-41BB costimulatory domain SEQ ID NO: 47
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL -CD3 zeta intracellular
domain SEQ ID NO: 48
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN
PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR -CD28
costimulatory domain SEQ ID NO: 49
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS -Survivin specific TCR,
beta chain SEQ ID NO: 50
DAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKD
FNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSRGDSTAEPQHFGDGT RLSIL
-Survivin specific TCR, alpha chain SEQ ID NO: 51
GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKR
QGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAETVTDSWGKLQFGAGTQVVV TPD -WT-1
specific TCR. CDR1 alpha SEQ ID NO: 52 SSYSPS -WT-1 specific TCR.
CDR2 alpha
SEQ ID NO: 53 YTSAATL -WT-1 specific TCR. CDR3 alpha SEQ ID NO: 54
WSPFSGGGADGLT -WT-1 specific TCR. CDR3 alpha SEQ ID NO: 55
SPFSGGGADGLT -WT-1 specific TCR. CDR1 beta SEQ ID NO: 56 DFQATT
-WT-1 specific TCR. CDR2 beta SEQ ID NO: 57 SNEGSKA -WT-1 specific
TCR. CDR3 beta SEQ ID NO: 58 SARDGGEG -WT-1 specific TCR. CDR3 beta
SEQ ID NO: 59 RDGGEGSETQY -WT specific TCR, alpha chain SEQ ID NO:
60 MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYV
QHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCV
VSPFSGGGADGLT -WT-1 specific TCR, beta chain SEQ ID NO: 61
MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTXVKIECRSLDFQATTMFWYRQFPK
QSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYIC SARDGGEG
-WT-1 specific TCR, alpha chain SEQ ID NO: 62
MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYV
QHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCV
VSPFSGGGADGLT -WT-1 specific TCR, beta chain SEQ ID NO: 63
MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPK
QSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAR
DGGEGSETQY
[0206] The following are examples, illustrating the methods and
compositions disclosed herein. It is understood that various other
embodiments may be practiced, given the general description
provided above.
EXAMPLES
[0207] Cell lines
[0208] The 293T cells were obtained from ATCC while neuroblastoma
tumor cell line CHLA-255 was kindly provided by Dr Leonid Metelitsa
Baylor College of Medicine (Houston, Tex.). Cells were maintained
in culture with IMDM (Gibco) for 293T or RPMI 1640 (Gibco) for
CHLA-255, containing 10% FBS (Corning), 1% GlutaMAX and 1%
penicillin/streptavidin (Gibco) in a humidified atmosphere
containing 5% CO.sub.2 at 37.degree. C.
[0209] Example 1-Characterization of the scFv
[0210] Neutralization of Human PD-L1
[0211] The anti-PD-L1 scFv (see SEQ ID NO: 9) is a humanized
protein comprising rabbit CDRs. Its ability to inhibit the binding
of PD-L1 to PD-1 was tested by competition ELISA. Briefly, rhPD-L1
Fc fusion was coated onto 96-well microplates. After blocking,
serial dilutions of scFv were added to plates and incubated for one
hour at room temperature. Half of the scFv dilution was replaced
with biotinylated PD-1 Fc fusion and bound PD-1 was detected with
streptavidin-HRP.
[0212] Similarly, the ability to inhibit the binding of PD-L1 to
CD80 was tested by competition ELISA using rhCD80-His. After
blocking, serial dilution of scFv was prepared with a constant
concentration of 50 nM rhPD-L1 Fc fusion. This mixture was
incubated with the CD80 coated plates for 2 hours at room
temperature. The background level corresponding to no binding of
PD-L1 to CD80 was determined by including a dilution series of
scFv1 in the absence of any PD-L1-Fc. Bound PD-L1 Fc fusion was
detected with goat anti-human IgG Fc-HRP. In this assay, the
ability of PD-L1 to interact with CD80 generates an absorbance
signal, which is effectively neutralized to background level by the
scFv. Taken together, these results indicate that scFv blocks the
interaction of PD-L1 with both PD-1 and CD80.
[0213] Stability
[0214] Two different processes can be observed that may affect the
stability of scFvs. Firstly, scFvs could be prone to dimerization,
often followed by oligomerization and further aggregation and
precipitation. Secondly, scFv degradation, leading to smaller
fragments, can occur over time.
[0215] The stability of the scFv formulated in PBS pH 7.2 at 10
mg/mL concentration upon storage at different temperature
conditions (4.degree. C., 22.degree. C., 37.degree. C. and
-20.degree. C.) was investigated. Only a minor amount of
dimerization of the scFv or formation of high molecule weight
molecules was observed upon storage for 2 weeks at 4.degree. C.,
22.degree. C. and 37.degree. C. The scFv formed up to 1.8% and 2.7%
of dimers after 1 or 2 weeks of storage at 37.degree. C.,
respectively.
[0216] The thermal stability was assessed by differential scanning
fluorimetry (DSF) in a real-time PCR device (Corbett, Rotor-Gene).
The midpoint melting temperatures (Tm) of the scFv calculated using
Rotor-Gene 6000 Series Software 1.7. was 81.5.degree. C.
[0217] Proteinaceous biologics may become exposed to freeze/thaw
stress during manufacturing, storing and shipping which may cause
aggregation and degradation. In order to assess stability of the
anti-PD-L1 scFv during freeze/thaw cycles, it was formulated in PBS
pH 7.2 at 10 mg/mL in 1.5 mL polypropylene tubes. The vials were
submerged into liquid nitrogen, followed by incubation in a water
bath at room temperature. After centrifugation, supernatants were
analyzed by SE-HPLC. Virtually 100% of the scFv remained monomeric
after 10 freeze/thaw cycles and no protein loss or precipitation
was observed. The stability of the anti-PD-L1 scFv in human serum
(Sigma-Aldrich, cat no H4522) was assessed by ELISA. There was no
loss of binding activity of the scFv after up to 20 hours of
incubation with human serum at 37.degree. C.
[0218] Kinetic Exclusion Assay of the scFv and the full-length
antibody
[0219] The affinity of a monovalent and a bivalent antibody to
PD-L1 was determined by Kinetic Exclusion Assay (KinExA.RTM.) with
a KinExA 3200 (Sapidyne Instruments, USA, cat. no. 5001). The
KinExA.RTM. measures the equilibrium binding affinity and kinetics
between unmodified molecules in solution. The measurement requires
the immobilization of one interaction partner on a solid phase
solely to act as a probe to determine the concentration of the
corresponding binding partner in solution.
[0220] a) Monovalent antibody
[0221] The affinity of the scFv to PD-L1-Fc fusion was determined
at room temperature in PBS with 0.02% sodium azide, pH7.4. Two
curves were measured, one using 20 pM scFv1 with an incubation time
of 5 hours and the other at 10 pM scFv1 with an incubation time of
9 hours. The KD value for the scFv was 8.8 pM, calculated using the
"n-curve analysis" of the KinExA.RTM. Pro software version 4.1.9 or
4.2.10.
[0222] b) Bivalent antibody
[0223] The scFv was reformatted into IgG format and expressed in
suspension-adapted CHO K1 cells originally received from ATCC and
adapted to serum-free growth in suspension culture. IgG antibodies
were purified by Protein A chromatography followed by size
exclusion chromatography. The KD for binding to PD-L1-His was
calculated at room temperature in PBS with 0.02% sodium azide,
pH7.4, using two curves. One curve used 100 pM of IgG with an
incubation time of 5 hours, and the other used 10 pM of IgG with an
incubation time of 10 hours. The KD value calculated for the
binding of the IgG to human PD-L1 was 2.77 pM. The results
demonstrate that the IgG binds PD-L1 with an affinity around three
times tighter than the affinity of the scFv to PD-L1.
[0224] Selectivity
[0225] Cross-reactivity of the scFv to PD-L1 from other species was
determined by ELISA, using PD-L1 Fc fusions from human (RnD
Systems, USA, cat. no. 156-B7), rat (Sino Biological, China, cat.
no. 80450-RO2H) or monkey (Sino Biological, China, cat. no.
90251-C02H). The results indicated that the scFv specifically bound
to human and monkey PD-L1, but not to rat PD-L1. Crossreactivity of
the anti-PD-L1 scFv to monkey PD-L1 was further investigated using
KinExA.RTM.. The KD value was calculated at room temperature in PBS
with 0.02% sodium azide, pH7.4 using two curves. One curve used 50
pM of scFv with an incubation time of 6 hours, and the other used
10 pM of scFv with an incubation time of 16 hours. The KD value
calculated for the scFv was 3.3 pM. The results demonstrate that
the scFv binds to monkey PD-L1 with an affinity around 2.7 times
tighter than binding to human PD-L1.
[0226] Cross-reactivity of the scFv to recombinant human proteins
sharing sequence similarity to PD-L1 was determined by ELISA using
rhPD-L1 Fc fusion (RnD Systems, USA, cat. no. 156-B7), rhPD-L2 Fc
fusion (RnD Systems, USA, cat. no. 1224-PL) or rhB7-H3 Fc fusion
(RnD Systems, USA, cat. no. 1027-B3). The results indicated that
scFv specifically bound to human PD-L1, with no cross-reactivity to
human PD-L2 or B7-H3.
[0227] Binding to Cell Surface PD-L1
[0228] The ability of the scFv to bind to PD-L1 on the surface of
cells was determined by extracellular FACS staining of ES-2 cells
(ATCC, USA, cat. no. CRL-1978). The results demonstrate that the
scFv is able to specifically recognize the natural form of PD-L1
expressed on the surface of cells.
[0229] The binding of the scFv to cell surface PD-L1 was further
investigated at room temperature in PBS with 0.02% sodium azide,
pH7.4 using KinExA.RTM.. One curve was constructed, using 50 pM
scFv and an incubation time of 5 hours. The calculated KD value for
the scFv binding to cell surface expressed PD-L1 on ES-2 cells is
12.8 pM.
[0230] Example 2 - Generation of the retroviral construct encoding
the anti-PD-L1 scFv and retrovirus production
[0231] DNA encoding the anti-PD-L1 scFv was amplified by PCR with
the following primers: Forward: 5'-TAACCATGG AGTTTGGGCTGAG-3' (SEQ
ID NO: 11) and Reverse: 5'-GACGCATGCTCAGCTCGACACGGTGACC-3' (SEQ ID
NO: 12) in order to add the NcoI restriction site at 5' end
(forward primer) and the stop codon TGA and the restriction site
Sphl at the 3' end (reverse primer). The PCR product and the
retroviral backbone SFG(I)eGFP were digested with Ncol and Sphl and
ligated. The insert was sequenced to confirm that no mutations
occurred during the cloning. The final vector was named
SFG.scFv.anti-PD-L1(I)eGFP (see FIG. 1). The reporter gene GFP
expressed upon IRES is used to assess the transduction efficiency.
Transient retroviral supernatant was prepared by transfection of
293T cells with the retroviral vector and two plasmids encoding
gag-pol and RDF envelop, respectively. The supernatant collected at
48 hrs was used to transduce activated T cells isolated from
healthy donors. The retroviral vectors encoding the CAR that
targets the GD2 antigen (GD2.CAR) including either the CD28
(CAR.CD28 or CAR.28) or the 4-1BB (CAR.41BB or CAR.BB) endodomains
were previously described (Heczey A. et al, Blood. 2014 Oct. 30;
124(18):2824-33). Said CAR comprises the CDR sequences of SEQ ID
NOs: 16 to 21.
[0232] Example 3 - Generation and expansion of CAR T cells
producing the anti-PD-L1 scFv
[0233] Peripheral blood mononuclear cells (PBMCs) from healthy
human donors were isolated by Lymphoprep (Fresenius) density
gradient centrifugation. Primary T cells were cultured in complete
T cell media containing 44% Click's medium (Irvine Scientific), 44%
RPMI 1640 (Hyclone), 10% FBS (Hyclone), 1% Glutamax and 1%
penicillin/streptavidin in the presence of IL-7 (10 ng/mL) and
IL-15 (5 ng/mL) (from PeproTech). T cells were activated with
immobilized anti-CD3 (1 mcg/mL) (Miltenyi, Catalog Number:
130-093-387) and anti-CD28 (1 mcg/mL) (BD Biociences, Catalog
Number: 555725) in 24-well plate at the concentration of
0.5.times.10.sup.6 cell/mL in T cells media without cytokines.
Twenty four hours after the stimulation, IL-7 and IL-15 were added
to the medium. By day 2, T cells were transduced with the
retroviral supernatant SFG.scFv.anti-PD-L1(I)eGFP and/or for the
GD2.CAR (1 mL/well of retroviral supernatant in a
retronectin-coated 24-well plate). In order to generate T cells
expressing the GD2.CAR and releasing the anti-PD-L1 scFv a
co-transduction with both retroviral constructs has been performed
(1 mL/well of GD2. CAR supernatant plus 1 mL/well of anti-PD-L1
scFv). Non-Transduced (NT) T cells were plated at the same
concentration (0.25.times.10.sup.6 cells/mL) in a non-tissue
culture plate coated with retronectin. Seventy two hours after the
transduction T cells were washed, counted and suspended in complete
T cell media with IL-7/IL-15 at 1.times.10.sup.6 cells/mL. T cells
were expanded in vitro for 5 days and then analyzed by flow
cytometry to assess transduction efficiency and T cell composition.
Eleven to twelve days after initiation, T cells were tested in
functional assays.
[0234] Example 4-T cells co-transduced with the GD2.CAR and
SFG.scFv.anti-PD-L1 vectors express both GD2.CAR and anti-PD-L1
scFvs
[0235] The phenotype of T cells was assessed using mAbs for CD3,
CD4, CD8, CD60L, CD45RA, CD95, CD27, CD2, PD-L1 (BD Bioscience or
Biolegend). GD2.CAR expression was detected using the anti-idiotype
1A7 mAb followed by staining with a secondary rat
anti-mouse-IgG1-PE mAb (BD Bioscience). The transduction efficiency
of the SFG.scFv.anti-PD-L1(I)eGFP vector was assessed by measuring
GFP expression. GD2.CAR Relative Fluorescence Intensity (RFI) was
calculated as the Mean Fluorescence Intensity (MFI) of CAR T cells
divided by the MFI of non-transduced T cells. As shown in FIG. 2,
CD4 and CD8 T cells were successfully transduced with the
anti-PD-L1 scFv retroviral construct of Example 2 without
alteration of the CD4/CD8 ratio. T cells co-expressed GD2.CAR and
eGFP anti-PD-L1 upon double retroviral transduction (FIG. 3).
Transduction with the anti-PD-L1 scFv did not affect T cell
proliferation (FIG. 4). Moreover, expression of the anti-PD-L1 scFv
did not affect T cell subset compositions (FIG. 5). This example
demonstrates the generation of CART cells co-expressing anti-GD2
CARs and anti-PD-L1 scFvs.
[0236] Example 5-T cells transduced with the SFG.scFv.anti-PD-L1
vector release functionally active anti-PD-L1 scFvs upon activation
through the endogenous TCR/CD3 complexes
[0237] To test whether T cells are capable of secreting anti-PD-L1
scFvs the cells were stimulated through the endogenous TCR.
Non-transduced T cells or anti-PD-L1 scFv transduced T cells were
plated in tissue culture treated 24-well plates uncoated or coated
with immobilized anti-CD3 (1 .mu.g/ml, Miltenyi) and anti-CD28 (1
.mu.g/ml, BD Bioscences) anti-CD3/CD28 anti-CD3/CD28 antibodies at
the concentration of 0.5.times.10.sup.6 cells/mL in 2 mL/well of T
cells medium with 10% FBS. After 18 hours 1 mL of supernatant was
collected for the quantification of the anti-PD-L1 scFv released by
T cells using a specific sandwich ELISA assay. A matched pair of
anti-scFv monoclonal antibodies of mouse origin were used for this
sandwich ELISA. As shown in FIG. 6A, the anti-PD-L1 scFv was
released by transduced T cells after stimulation with immobilized
anti-CD.sup.3/anti-CD28 antibodies. These T cells express their
natural endogenous TCR/CD3 complexes. The results suggest that
activation of transduced T cells through the endogenous TCR/CD3
complexes is sufficient to induce synthesis and extracellular
secretion of anti-PD-L1 scFvs. The quantitative ELISA showed large
amounts of secreted anti-PD-L1 scFv (FIG. 6A). Non-transduced cells
did not secrete scFvs. Since the anti-PD-L1 scFv is under the
control of the constitutively active 5' LTR of a retroviral vector,
transduced T cells plated on uncoated wells released basal levels
of anti-PD-L1 scFvs which however significantly increased upon T
cell activation.
[0238] The anti-PD-L1 scFv produced by activated T cells is capable
of binding to immobilized PD-L1 (FIG. 6 B). Briefly, recombinant
human PD-L1-Fc (R&D Systems) was immobilized at a concentration
of 2 .mu.g/mL onto microplates in PBS. After, blocking with 5%
non-fat dry milk, increasing concentrations of scFv was added and
detected by Protein L-HRP (Sigma-Aldrich). FIG. 6B shows that T
cell-produced anti-PD-L1 scFv binds to PD-L1 equally well as a
reference control, the anti-PD-L1 scFv produced in E. coli. These
data demonstrate that the transduced T cells release functionally
active anti-PD-L1 scFvs in large amounts upon activation through
the endogenous TCR. These data also suggest that transduced T cells
will readily secrete anti-PD-L1 scFvs when they are activated
through genetically modified TCR and through CARs.
[0239] Example 6-T cells co-transduced with the GD2.CAR-4-1BB and
SFG.scFv.anti-PD-L1 vectors have enhanced tumor-killing
activity
[0240] Transduced and non-transduced T cells (0.5.times.10.sup.5
cells/well) were co-cultured with the tumor cell line CHLA-255
(2.5.times.10.sup.5 cells/well) at an effector:target (E:T) ratio
of 1:5 in 24-well plates, in the absence of exogenous cytokines.
After 7 days of co-culture, T cells were harvested and counted. If
the percentage of residual tumor cells was <5% (as assessed by
flow cytometry), T cells were re-plated with fresh tumor cells at
the same 1:5 E:T ratio for a second cycle of co-culture. After an
additional 7-8 days, cells were then collected and analyzed by flow
cytometry to enumerate T cells and residual tumor cells.
Specifically, CD3 and GD2 antibodies were used to stain T cells and
tumor cells, respectively. CountBright beads (Invitrogen) were used
for cell counting by flow cytometry. Supernatant was also collected
after 24 hours of culture to measure IFNgamma release by ELISA
(R&D System) according to the manufacturer's instruction.
[0241] GD2.CAR T cells with the 4-1BB endodomain transduced with
the anti-PD-L1 scFv showed better killing of tumor cells in the
second cycle of co-culture (14 days of culture) (FIG. 7) and
produced higher levels of IFNgamma in the second cycle of
co-culture (14 days of culture) with tumor cells (FIG. 8) than the
GD2.CAR T cells without the anti-PD-L1 svFv. Thus exhaustion of
GD2.CAR T cells engaging repetitively tumor cells occurs with 4-1BB
costimulation, but the presence of the anti PD-L1 scFv protect CART
cells from exhaustion and may provide prolonged anti tumor activity
in vivo and prevent tumor recurrence.
[0242] While there are shown and described presently preferred
embodiments of the invention, it is to be understood that the
invention is not limited thereto but may be otherwise variously
embodied and practiced within the scope of the following claims.
Since numerous modifications and alternative embodiments of the
present invention will be readily apparent to those skilled in the
art, this description is to be construed as illustrative only and
is for the purpose of teaching those skilled in the art the best
mode for carrying out the present invention. Accordingly, all
suitable modifications and equivalents may be considered to fall
within the scope of the following claims.
Sequence CWU 1
1
631114PRTArtificial SequenceVL of scFv 1Glu Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile
Thr Cys Gln Ala Ser Glu Asp Ile Tyr Ser Leu 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Asn Tyr Gly Ser
Ser Ser 85 90 95 Ser Ser Ser Tyr Gly Ala Val Phe Gly Gln Gly Thr
Lys Leu Thr Val 100 105 110 Leu Gly 2120PRTArtificial SequenceVH of
scFv 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Ser Ser Gly Gly Arg Thr
Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg
Tyr Thr Gly Tyr Pro Tyr Tyr Phe Ala Leu Trp Gly Gln 100 105 110 Gly
Thr Leu Val Thr Val Ser Ser 115 120 311PRTOryctolagus cuniculus
3Gln Ala Ser Glu Asp Ile Tyr Ser Leu Leu Ala 1 5 10
47PRTOryctolagus cuniculus 4Asp Ala Ser Asp Leu Ala Ser 1 5
515PRTOryctolagus cuniculus 5Gln Gly Asn Tyr Gly Ser Ser Ser Ser
Ser Ser Tyr Gly Ala Val 1 5 10 15 69PRTOryctolagus cuniculus 6Ile
Asp Leu Ser Ser Tyr Thr Met Gly 1 5 716PRTOryctolagus cuniculus
7Ile Ile Ser Ser Gly Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1
5 10 15 812PRTOryctolagus cuniculus 8Gly Arg Tyr Thr Gly Tyr Pro
Tyr Tyr Phe Ala Leu 1 5 10 9254PRTArtificial SequencescFv 9Glu Ile
Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Asp Ile Tyr Ser Leu 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln
Gly Asn Tyr Gly Ser Ser Ser 85 90 95 Ser Ser Ser Tyr Gly Ala Val
Phe Gly Gln Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 130 135 140 Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly 145 150
155 160 Ile Asp Leu Ser Ser Tyr Thr Met Gly Trp Val Arg Gln Ala Pro
Gly 165 170 175 Lys Gly Leu Glu Trp Val Gly Ile Ile Ser Ser Gly Gly
Arg Thr Tyr 180 185 190 Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ser 195 200 205 Lys Asn Thr Val Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg
Gly Arg Tyr Thr Gly Tyr Pro Tyr Tyr 225 230 235 240 Phe Ala Leu Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 1020PRTArtificial
Sequencelinker 10Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 1122DNAArtificial
Sequenceforward primer 11taaccatgga gtttgggctg ag
221228DNAArtificial Sequencereverse primer 12gacgcatgct cagctcgaca
cggtgacc 2813114PRTArtificial SequenceVL sequence of 14g2a scFv
13Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1
5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg
14113PRTArtificial SequenceVH sequence of 14g2a scFv 14Glu Val Gln
Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser
Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr 20 25
30 Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile
35 40 45 Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln
Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met His Leu Lys Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gly Met Glu Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser 1518PRTArtificial
SequencehIgG1 signal peptide 15Met Glu Phe Gly Leu Ser Trp Leu Phe
Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln 1616PRTArtificial
SequenceCDR-L1 of anti-GD2-CAR 16Arg Ser Ser Gln Ser Leu Val His
Arg Asn Gly Asn Thr Tyr Leu His 1 5 10 15 177PRTArtificial
SequenceCDR-L2 of anti-GD2-CAR 17Lys Val Ser Asn Arg Phe Ser 1 5
1810PRTArtificial SequenceCDR-L3 of anti-GD2-CAR 18Ser Gln Ser Thr
His Val Pro Pro Leu Thr 1 5 10 199PRTArtificial SequenceCDR-H1 of
anti-GD2-CAR 19Ser Ser Phe Thr Gly Tyr Asn Met Asn 1 5
2017PRTArtificial SequenceCDR-H2 of anti-GD2-CAR 20Ala Ile Asp Pro
Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
214PRTArtificial SequenceCDR-H3 of anti-GD2-CAR 21Gly Met Glu Tyr 1
2211PRTArtificial SequenceCDR-L1 of anti-CSPG4 CAR 22Arg Ala Ser
Gln Thr Ile Tyr Lys Asn Leu His 1 5 10 237PRTArtificial
SequenceCDR-L2 of anti-CSPG4 CAR 23Tyr Gly Ser Asp Ser Ile Ser 1 5
249PRTArtificial SequenceCDR-L3 of anti-CSPG4 CAR 24Leu Gln Gly Tyr
Ser Thr Pro Trp Thr 1 5 259PRTArtificial SequenceCDR-H1 of
anti-CSPG4 CAR 25Tyr Thr Phe Thr Asp Tyr Ser Met His 1 5
2617PRTArtificial SequenceCDR-H2 of anti-CSPG4 CAR 26Trp Ile Asn
Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly
274PRTArtificial SequenceCDR-H3 of anti-CSPG4 CAR 27Tyr Tyr Asp Tyr
1 28109PRTArtificial SequenceVL sequence of anti-CSPG4 CAR 28Leu
Asp Ile Lys Leu Thr Gln Ser Pro Ser Ile Leu Ser Val Thr Pro 1 5 10
15 Gly Glu Thr Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys
20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser His Arg Ser Pro Arg
Leu Leu 35 40 45 Ile Lys Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro
Ser Arg Phe Thr 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Asn Ile Asn Ser Val Lys 65 70 75 80 Pro Glu Asp Glu Gly Ile Tyr Tyr
Cys Leu Gln Gly Tyr Ser Thr Pro 85 90 95 Trp Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 29113PRTArtificial SequenceVH
sequence of anti-CSPG4 CAR 29Gln Val Lys Leu Lys Glu Ser Gly Pro
Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val
Lys Lys Thr Pro Gly Lys Gly Leu Lys Trp Leu 35 40 45 Gly Trp Ile
Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys
Gly Arg Phe Ala Ile Ser Leu Glu Thr Ser Ala Arg Thr Val Tyr 65 70
75 80 Leu Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr Tyr Phe
Cys 85 90 95 Phe Ser Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 100 105 110 Ser 3016PRTArtificial SequenceCDR-L1 of
anti-GPC3 CAR 30Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr
Tyr Leu His 1 5 10 15 317PRTArtificial SequenceCDR-L2 of anti-GPC3
CAR 31Lys Val Ser Asn Arg Phe Ser 1 5 329PRTArtificial
SequenceCDR-L3 of anti-GPC3 CAR 32Ser Gln Asn Thr His Val Pro Pro
Thr 1 5 339PRTArtificial SequenceCDR-H1 of anti-GPC3 CAR 33Tyr Thr
Phe Thr Asp Tyr Glu Met His 1 5 3417PRTArtificial SequenceCDR-H2 of
anti-GPC3 CAR 34Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln
Lys Phe Lys 1 5 10 15 Gly 356PRTArtificial SequenceCDR-H3 of
anti-GPC3 CAR 35Phe Tyr Ser Tyr Thr Tyr 1 5 36113PRTArtificial
SequenceVL of anti-GPC3 CAR 36Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr
Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln
Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg 37115PRTArtificial SequenceVH of
anti-GPC3 CAR v 37Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys
Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val
Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110 Val Ser Ser 115 38115PRTArtificial SequenceVH of anti-GPC3
CAR 38Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His
Gly Leu Lys Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp
Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe
Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val
Ser Ala 115 399PRTArtificial SequenceCDR-L1 of anti-5T4 CAR 39Tyr
Ser Phe Thr Gly Tyr Tyr Met His 1 5 4017PRTArtificial
SequenceCDR-L2 of anti-5T4 CAR 40Arg Ile Asn Pro Asn Asn Gly Val
Thr Leu Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp 4111PRTArtificial
SequenceCDR-L3 of anti-5T4 CAR 41Ser Thr Met Ile Thr Asn Tyr Val
Met Asp Tyr 1 5 10 4211PRTArtificial SequenceCDR-H1 of anti-5T4 CAR
42Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala 1 5 10
437PRTArtificial SequenceCDR-H2 of anti-5T4 CAR 43Tyr Thr Ser Ser
Arg Tyr Ala 1 5 449PRTArtificial SequenceCDR-H3 of anti-5T4 CAR
44Gln Gln Asp Tyr Asn Ser Pro Pro Thr 1 5 45108PRTArtificial
SequenceVL of anti-5T4 CAR 45Ser Ile Val Met Thr Gln Thr Pro Thr
Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile 35 40 45 Ser Tyr Thr
Ser Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly 50 55 60 Ser
Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln Ala 65 70
75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro
Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
105 46120PRTArtificial SequenceVH of anti-5T4 CAR 46Glu Val Gln Leu
Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asn Pro Asn Asn Gly Val Thr Leu Tyr Asn Gln Lys
Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Thr
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met Ile Thr Asn Tyr
Val Met Asp Tyr Trp Gly Gln 100 105 110 Val Thr Ser Val Thr Val Ser
Ser 115 120 4742PRTArtificial Sequence41BB costimulatory domain
47Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1
5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40
48112PRTArtificial SequenceCD3 zeta intracellular domain 48Arg Val
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20
25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg 65 70 75
80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
Pro Arg 100 105 110 4941PRTArtificial SequenceCD28 costimulatory
domain 49Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40
50114PRTArtificial SequenceSurvivin specific TCR, beta chain 50Asp
Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr Gln Phe Gly 1 5 10
15 Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His Asn Val Met
20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu Leu
Phe His 35 40 45 Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr
Pro Asp Asn Phe 50 55 60 Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys
Phe Leu Asp Ile Arg Ser 65 70 75 80 Pro Gly Leu Gly Asp Ala Ala Met
Tyr Leu Cys Ala Thr Ser Arg Gly 85 90 95 Asp Ser Thr Ala Glu Pro
Gln His Phe Gly Asp Gly Thr Arg Leu Ser 100 105 110 Ile Leu
51114PRTArtificial SequenceSurvivin specific TCR, alpha chain 51Gly
Glu Ser Val Gly Leu His Leu Pro Thr Leu Ser Val Gln Glu Gly 1 5 10
15 Asp Asn Ser Ile Ile Asn Cys Ala Tyr Ser Asn Ser Ala Ser Asp Tyr
20 25 30 Phe Ile Trp Tyr Lys Gln Glu Ser Gly Lys Gly Pro Gln Phe
Ile Ile 35 40 45 Asp Ile Arg Ser Asn Met Asp Lys Arg Gln Gly Gln
Arg Val Thr Val 50 55 60 Leu Leu Asn Lys Thr Val Lys His Leu Ser
Leu Gln Ile Ala Ala Thr 65 70 75 80 Gln Pro Gly Asp Ser Ala Val Tyr
Phe Cys Ala Glu Thr Val Thr Asp 85 90 95 Ser Trp Gly Lys Leu Gln
Phe Gly Ala Gly Thr Gln Val Val Val Thr 100 105 110 Pro Asp
526PRTArtificial SequenceWT-1 specific TCR, CDR1 alpha 52Ser Ser
Tyr Ser Pro Ser 1 5 537PRTArtificial SequenceWT-1 specific TCR,
CDR2 alpha 53Tyr Thr Ser Ala Ala Thr Leu 1 5 5413PRTArtificial
SequenceWT-1 specific TCR, CDR3 alpha 54Trp Ser Pro Phe Ser Gly Gly
Gly Ala Asp Gly Leu Thr 1 5 10 5512PRTArtificial SequenceWT-1
specific TCR, CDR3 alpha v 55Ser Pro Phe Ser Gly Gly Gly Ala Asp
Gly Leu Thr 1 5 10 566PRTArtificial SequenceWT-1 specific TCR, CDR1
beta 56Asp Phe Gln Ala Thr Thr 1 5 577PRTArtificial SequenceWT-1
specific TCR, CDR2 beta 57Ser Asn Glu Gly Ser Lys Ala 1 5
588PRTArtificial SequenceWT-1 specific TCR, CDR3 beta 58Ser Ala Arg
Asp Gly Gly Glu Gly 1 5 5911PRTArtificial SequenceWT-1 specific
TCR, CDR3 beta v 59Arg Asp Gly Gly Glu Gly Ser Glu Thr Gln Tyr 1 5
10 60124PRTArtificial SequenceWT-1 specific TCR, alpha chain 60Met
Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10
15 Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val
20 25 30 Ser Glu Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser
Ser Tyr 35 40 45 Ser Pro Ser Leu Phe Trp Tyr Val Gln His Pro Asn
Lys Gly Leu Gln 50 55 60 Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr
Leu Val Lys Gly Ile Asn 65 70 75 80 Gly Phe Glu Ala Glu Phe Lys Lys
Ser Glu Thr Ser Phe His Leu Thr 85 90 95 Lys Pro Ser Ala His Met
Ser Asp Ala Ala Glu Tyr Phe Cys Val Val 100 105 110 Ser Pro Phe Ser
Gly Gly Gly Ala Asp Gly Leu Thr 115 120 61116PRTArtificial
SequenceWT-1 specific TCR, beta chainmisc_feature(32)..(32)Xaa can
be any naturally occurring amino acid 61Met Leu Leu Leu Leu Leu Leu
Leu Gly Pro Gly Ser Gly Leu Gly Ala 1 5 10 15 Val Val Ser Gln His
Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Xaa 20 25 30 Val Lys Ile
Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe 35 40 45 Trp
Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser 50 55
60 Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys
65 70 75 80 Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
Val Thr 85 90 95 Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys
Ser Ala Arg Asp 100 105 110 Gly Gly Glu Gly 115 62124PRTArtificial
SequenceWT-1 specific TCR, alpha chain v 62Met Leu Leu Leu Leu Val
Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10 15 Gly Thr Arg Ala
Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val 20 25 30 Ser Glu
Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr 35 40 45
Ser Pro Ser Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln 50
55 60 Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile
Asn 65 70 75 80 Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe
His Leu Thr 85 90 95 Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu
Tyr Phe Cys Val Val 100 105 110 Ser Pro Phe Ser Gly Gly Gly Ala Asp
Gly Leu Thr 115 120 63121PRTArtificial SequenceWT-1 specific TCR,
beta chain v 63Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly
Leu Gly Ala 1 5 10 15 Val Val Ser Gln His Pro Ser Trp Val Ile Cys
Lys Ser Gly Thr Ser 20 25 30 Val Lys Ile Glu Cys Arg Ser Leu Asp
Phe Gln Ala Thr Thr Met Phe 35 40 45 Trp Tyr Arg Gln Phe Pro Lys
Gln Ser Leu Met Leu Met Ala Thr Ser 50 55 60 Asn Glu Gly Ser Lys
Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys 65 70 75 80 Phe Leu Ile
Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr 85 90 95 Ser
Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Asp 100 105
110 Gly Gly Glu Gly Ser Glu Thr Gln Tyr 115 120
* * * * *
References