U.S. patent application number 15/537779 was filed with the patent office on 2017-12-21 for chimeric antigen receptors and methods of use thereof.
The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Wayne A. MARASCO.
Application Number | 20170362297 15/537779 |
Document ID | / |
Family ID | 55229842 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170362297 |
Kind Code |
A1 |
MARASCO; Wayne A. |
December 21, 2017 |
CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE THEREOF
Abstract
The present invention provides chimeric antigen receptors, cells
expressing same and methods of using same for treatment various
disorders such as cancer.
Inventors: |
MARASCO; Wayne A.;
(Wellesley, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Brookline |
MA |
US |
|
|
Family ID: |
55229842 |
Appl. No.: |
15/537779 |
Filed: |
December 21, 2015 |
PCT Filed: |
December 21, 2015 |
PCT NO: |
PCT/US2015/067225 |
371 Date: |
June 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62094625 |
Dec 19, 2014 |
|
|
|
62252083 |
Nov 6, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/70578 20130101; C07K 14/70535 20130101; C07K 2319/03
20130101; C07K 14/70521 20130101; C07K 2319/02 20130101; C07K
14/7051 20130101; C07K 19/00 20130101 |
International
Class: |
C07K 14/725 20060101
C07K014/725; C07K 14/735 20060101 C07K014/735; C07K 14/705 20060101
C07K014/705; C07K 19/00 20060101 C07K019/00 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under [ ]
awarded by the [ ]. The government has certain rights in the
invention.
Claims
1. A chimeric antigen receptor (CAR) comprising an intracellular
signaling domain, a transmembrane domain and an extracellular
domain
2. The CAR of claim 1, wherein the transmembrane domain further
comprises a stalk region positioned between the extracellular
domain and the transmembrane domain.
3. The CAR or claim 1, wherein the transmembrane domain comprises
CD28.
4. The CAR of claim 1, further comprising one or more additional
costimulatory molecules positioned between the transmembrane domain
and the intracellular signaling domain
5. The CAR of claim 4, wherein the costimulatory molecules is CD28,
4-1BB, ICOS, or OX40.
6. The CAR of claim 1, wherein the intracellular signaling domain
comprises a CD3 zeta chain.
7. The CAR of claim 1, wherein the extracellular domain is an
antibody.
8. The CAR of claim 7, wherein the antibody is a Fab or a scFV.
9. The CAR of claim 7, wherein the antibody is specific for BCMA,
CD138, CCR4, PD-1, PDL-1, PD-L2, CXCR4, GITR, SARS virus,
flavivirus, MERS virus, influenza virus, or CAIX.
10. A nucleic acid encoding the CAR of any one of the preceding
claims further comprising a nucleic acid encoding a polypeptide
positioned after the intracellular signaling domain.
11. The nucleic acid of claim 10, wherein the polypeptide is an
antibody
12. The nucleic acid of claim 11, wherein the antibody is a
scFV.
13. The nucleic acid of claim 12, wherein the antibody is specific
for BCMA, CD138, CCR4, PD-1, PDL-1, PD-L2, CXCR4, GITR, SARS virus,
flavivirus, MERS virus, influenza virus, or CAIX
14. A vector comprising the nucleic acid of claim 11.
15. A cell comprising the vector of claim 14.
16. A genetically engineered cell which express and bear on the
cell surface membrane the chimeric antigen receptor of claim 1.
17. The genetically engineered cell of claim 16, wherein the cell
is a T-cell or an NK cell.
18. The genetically engineered cell of claim 17, wherein the T cell
is CD4.sup.+ or CD8.sup.+.
19. The genetically engineered cell of claim 18, which comprises a
mixed population of CD4.sup.+ and CD8 cells.sup.+.
20. The cell of claim 16, wherein the cell is further engineered to
express and secrete a polypeptide
21. The cell of claim 20, wherein the expressed and secreted
polypeptide is anti-PDL-1.
Description
RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of U.S.
Provisional Application No. 62/094,625 filed on Dec. 19, 2014, and
U.S. Provisional Application No. 62/252,083 filed on Nov. 6, 2015,
the contents of each of which are hereby incorporated in their
entireties.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0003] The contents of the text file named, "Omnibus Sequence
Listing for filing with DFCI-102_001WO_ST25", which was created on
Dec. 21, 2015, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0004] The present invention relates generally to chimeric antigen
receptor cells for and methods of using same for treatment cancer
and other disorders.
BACKGROUND OF THE INVENTION
[0005] T lymphocytes recognize specific antigens through
interaction of the T cell receptor (TCR) with short peptides
presented by major histocompatibility complex (MHC) class I or II
molecules. For initial activation and clonal expansion, naive T
cells are dependent on professional antigen-presenting cells (APCs)
that provide additional co-stimulatory signals. TCR activation in
the absence of co-stimulation can result in unresponsiveness and
clonal anergy. To bypass immunization, different approaches for the
derivation of cytotoxic effector cells with grafted recognition
specificity have been developed. Chimeric antigen receptors (CARs)
have been constructed that consist of binding domains derived from
natural ligands or antibodies specific for cell-surface components
of the TCR-associated CD3 complex. Upon antigen binding, such
chimeric antigen receptors link to endogenous signaling pathways in
the effector cell and generate activating signals similar to those
initiated by the TCR complex. Since the first reports on chimeric
antigen receptors, this concept has steadily been refined and the
molecular design of chimeric receptors has been optimized. Aided by
advances in recombinant antibody technology, chimeric antigen
receptors targeted to a wide variety of antigens on the surface of
cancer cells and of cells infected by human immunodeficiency virus
(HIV) have been generated.
SUMMARY OF THE INVENTION
[0006] In various aspects the invention provides a chimeric antigen
receptor (CAR) having an intracellular signaling domain, a
transmembrane domain and an extracellular domain.
[0007] In some aspects the transmembrane domain further includes a
stalk region positioned between the extracellular domain and the
transmembrane domain. The transmembrane domain includes CD28. In
another aspect the CAR further includes one or more additional
costimulatory molecules positioned between the transmembrane domain
and the intracellular signaling domain. The costimulatory molecules
is CD28, 4-1BB, ICOS, or OX40. The intracellular signaling domain
is for example a CD3 zeta chain. The extracellular domain is an
antibody such as a Fab or a scFV. Preferably the antibody is
specific for BCMA, CA-9, CD138, CCR4, or the influenza virus.
[0008] Also included in the invention are nucleic acids encoding
the CAR of the invention further including a nucleic acid encoding
a polypeptide positioned after the intracellular signaling domain.
The polypeptide is an antibody such as scFV. Preferably, the
antibody is specific for CCR4, PD-1, PDL-1, PD-L2, CXCR4, or GITR.
Also provided by the invention are vector including the nucleic
acid according to the invention and cells including the
vectors.
[0009] In yet a further aspect the invention provides a genetically
engineered cell which express and bear on the cell surface membrane
the chimeric antigen receptor of the invention. The cell is a
T-cell or an NK cell. The T cell is CD4.sup.+ or CD8.sup.+. The
cell is a mixed population of CD4.sup.+ and CD8 cells.sup.+. The
cell is further engineered to express and secrete a polypeptide
such as for example an antibody.
[0010] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are expressly incorporated by reference in their
entirety. In cases of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting.
[0011] Other features and advantages of the invention will be
apparent from and encompassed by the following detailed description
and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. ADCC of CAIX-specific Abs. 1 .mu.g/ml CAIX-specific
scFv-Fc minibodies were added to the target tumor cells in the
presence of human PBMC (E:T 25:1). Similar results were obtained in
2 experiments. Irrelevant anti-SARS scFv-Fc (11A) and anti-CCR4
scFv-Fc (48) minibodies were used as negative controls. A, CAIX+
sk-rc-09 cells; B, CAIX+ sk-rc-52 cells; C, CAIX- sk-rc-59
cells.
[0013] FIG. 2. Construction and expression of CAIX-specific CARs.
A.
[0014] Construction: The 1.sup.st generation CAR, scFv-CD8-TCR (CD8
CAR), is composed of a specific anti-CAIX scFv that is coupled to
truncated human CD8.alpha. extracellular domain, hinge (H),
transmembrane (TM) and intracellular regions, then to the signaling
domain of human TCR.zeta.. The 2.sup.nd generation CAR,
scFv-CD28-TCR.zeta. (CD28 CAR), contains anti-CAIX scFv fused with
human CD28 extracellular, TM and intracellular signaling domain to
TCR.zeta.. Both anti-CAIX CARs were cloned into a bicistronic
self-inactivating (SIN) lentiviral vector with expression driven by
an internal eF1-.alpha. promoter. The CAR control construct
contains an irrelevant anti-HIV CCRS specific A8 scFv substitution.
B. FACS analysis: Reporter gene ZsGreen was used to quantitate
primary T cell transduction efficiency by the lentiviral CAR
constructs. In addition, anti-CAIX scFv CARs were stained with
CAIX-Fc fusion protein and C9-tag (TETSQVAPA) was stained with 1D4
antibody. Untransduced activated T-cells, LAK only were served as
unstained cell control (i) or stained with 2.sup.nd antibody (ii.
PE-anti-human IgG and iii. APC-anti-mouse IgG) were used as
staining controls. C. Western blot: Molecular sizes of
monomer/dimer structures of anti-CAIX (clone G36) CD28 and
anti-CCRS (clone A8) CD28 CARs, as well as endogenous TCR.zeta.
chain of untransduced T cells were indicated.
[0015] FIG. 3. Effector functions of CAIX-specific CARTs. A.
Cytokine secretion. Anti-CAIX CART, irrelevant CART or activated
control T cells (LAK) were cocultivated overnight with kidney
cancer cell lines sk-rc-52 (CAIX+) and sk-rc-59 (CAIX-) for
cytokine production. One representative out of 2-3 results is
shown. B. ELISPOT. G36 CART or control A8 CART cells were added to
tumor cells overnight. IFN-.gamma. or granzyme B secreting T cells
detected by ELISPOT. Similar results were obtained in 2-3
experiments. C. Specific anti-tumor cytotoxicity of CAIX-specific
CART cells, control A8 CART cells or LAK cells were incubated in a
4-hour cytotoxicity assay at different amounts of target tumor cell
at the ratios as indicated. One out of two experiments is shown.
Clone 4-1 is a in vivo passaged subclone of sk-rc-52.
[0016] FIG. 4. Clonal expansion of CART cells after tumor contact.
A. Proliferation. CAR-transduced T cells or untransduced T cells
(LAK) were plated with irradiated tumor cells (CAIX+ sk-rc-52 &
CAIX- sk-rc-59) weekly at three different ratios of tumor to T
cells as indicated. Number of T cells was counted every 3-4 days in
triplicate from two separate wells. Similar results were obtained
in two experiments. B. Clonal enrichment. In tumor stimulation
experiments, cultures from CART- and LAK cells were assayed on one
week and two weeks by flow cytometry for expression of CART and
T-cell subset. One representative of two results is shown.
[0017] FIG. 5. Regression of established human RCC xenografts by
CART cells. Athymic null mice were inoculated subcutaneously with
7.5.times.10.sup.6 sk-rc-52 and 5.times.10.sup.6 sk-rc-59 RCC tumor
cells at left and right flank respectively. After 6 days of tumor
implantation, mice were injected i.v. with 50.times.10.sup.6 G36
CD28 CART cells, A8 CD28 CART cells (.gtoreq.20% CAR+), LAK, or PBS
alone. High dose of IL-2 (1.times.10.sup.5 U/ml) was injected every
2-3 days. Tumor size was measured by caliper every 2-3 days.
Experiment 1, n=7 & Experiment 2, n=8. Tumor size of these two
experiments was shown separately. +, p<0.05; *, p<0.01; **,
p<0.001 in groups of G36 Tandem treated mice versus control no T
cell treated mice in these two trials. Other statistic calculations
are reported in the text.
[0018] FIG. 6. In vivo anti-tumor activity of CAR+ T-cells. A.
Expression of ZsGreen by CART cells is shown in upper panel. CART
cells were pre-stained with Far Red dye, cytospun and examined by
fluorescent microscopy (lower panel). B. In situ staining of G36
CD28 CART cells in regressing tumor. CART-cells were i.v. injected
into RCC established mice and tumor tissue was collected on day
1-3. Confocal microscopy was used to measure apoptosis of tumor
cells by TUNNEL assay with PE-Cy5 dye (shown as red). Transduced T
cells were shown by ZsGreen. Nuclei were counterstained with DPAI.
Two representative slides were shown to indicate the apoptosis of
tumor cells at the edge of tumor (upper panel) and inside the tumor
bed (middle panel), respectively. The magnified image (lower panel)
demonstrates CART cells interacted with multiple tumors while a few
surrounding tumor cells were dying. C. Granzyme B+ T cells and
tumor necrosis. After the treatment with CART cells, the regressing
CAIX+ sk-rc-52 tumors were stained by granzyme B antibody (brown)
and H&E. The higher magnification view (middle and lower panels
of sections a and b in upper panel) shows the locations of granzyme
B+ T cells (shown by arrows) and the corresponding H&E slide
shows the tumor necrosis (shown by n). Granzyme B+ T cells are
distributed at the edge of tumor (middle panel) and inside the
tumor (lower panel).
[0019] FIG. 7. CAIX- sk-rc-52 tumors treated with control LAK cells
showed negative granzyme B staining (left) (upper panel) and the
corresponding histology was shown in H&E (right).
[0020] FIG. 8. Low background staining of granzyme B in CAIX-
sk-rc-59 tumors treated with G36 CD28z CART cells
[0021] FIG. 9. Low background staining of granzyme B in CAIX-
sk-rc-59 tumors treated with LAK cells.
[0022] FIG. 10. Positive control of granzyme B staining was
performed on sk-rc-52 tumors which was local injected with G36
CD28z CART cells (left) and tumor morphology was shown in H&E
(right).
[0023] FIG. 11 Expression of .alpha.CAIX CAR and .alpha.PD-L1
scFv-Fc transiently transfected 293T cells. 10.sup.6 of 293T cells
were transfected with or without lentiviral
pHAGE-EF1.alpha.G36-C9tag-CD28-CD3zeta-IRES-anti-PDL1 scFv-Fc
(IgG4)-WPRE or the control
pHAGE-EF1.alpha.-A716-C9tag-CD28-CD3zeta-IRES-ZsGreen-WPRE
plasmids. Cells and supernatant were harvested at 24 and 48 hours
post transfection. Left, purified CAIX(ECD)-Fc-Biotin and
Streptavidin-APC were used for cell staining and flow cytometry
analysis. Right, total human IgG in supernatant was quantitated
with human IgG quantification kit.
[0024] FIG. 12 (A) Total PBMCs from 4 donors were stimulated with
50 ng/ml SEB and treated with 10 ug/ml anti-PDL1 (#42) sIgG4 or
anti-influenza sIgG4. Restoration of IFN.gamma. production (upper)
or TNFa production (lower) was measured. (B) Parental anti-CCR4
antibody, c1567IgG, inhibited chemotaxis of Tregs to CCL22. (C)
CD4.sup.+CD25.sup.- T cells were CFSE-labeled, incubated with Tregs
at 10:1 ratio, and then stimulated with anti-CCR4 mAb and control
mAb in the presence of anti-CD3/28 co-stimulation. After 3 and 7
days cells were harvested and analyzed by flow cytometry. The
percentage of cells was calculated among the fluorescence positive
CD4.sup.+CD25.sup.- T and counting beads. The percent proliferation
was normalized to CD4.sup.+CD25.sup.- T effector cells at Day 0.
The data shown were calculated from two independent experiments.
Bars represent mean.+-.S.D. (D) IFN.gamma. ELISpot analysis of
mAb2-3 and control antibody treated CD4.sup.+CD25.sup.- Teff cells.
Data represents the quantification from three separate experiments
using three individual donor bloods. (E) Left, CFSE-labeled Teffs
(5.times.10.sup.4) and unlabeled Tregs (5.times.10.sup.3) were
co-incubated with 20 .mu.g/ml PHA in 96-well plates for 5 days. The
CFSE-labeled Teffs were harvested and CFSE intensity was analyzed
by flow cytometry. The proliferation of Teffs were only observed
when the coculture incubated with anti-GITR mAb. Right, IFN.gamma.
production in the same cultures was further measured by MSD.
CFSE-labeled Teffs (5.times.10.sup.4) and unlabeled Tregs
(5.times.10.sup.3) were co-incubated with 20 .mu.g/ml PHA in
96-well plates for 5 days. The CFSE-labeled Teffs were harvested
and CFSE intensity was analyzed by flow cytometry. Teffs were
proliferated after 5-day incubation with PHA, but not in the
Teff/Treg coculture. Left, IFN.gamma. production in the same
cultures was measured by MSD.
[0025] FIG. 13 is an illustration showing different CART
configurations according to the invention.
[0026] FIG. 14 is an illustration showing different armed CART
configurations according to the invention.
[0027] FIG. 15 is a further illustration showing different armed
CART configurations according to the invention.
[0028] FIG. 16 is a series of illustrations and graphs indicating
chimeric antigen receptors (CAR) constructs for CD8+ T cell
transduction. (A) Schematic representation of second generation
pHAGE lentiviral vectors containing CD28 as a co-stimulatory
domain. The anti-carbonic anhydrase IX (CAIX) or the Anti-B-cell
maturation antigen (BCMA) scFv (as a negative control) were
inserted after the eIF.alpha. promoter, in order to express the CAR
binding domain. The second cassette, after the Internal Ribosome
Entry Site (IRES) sequence, is responsible by coding the soluble
Anti-PD-L1 IgG1 or IgG4 isotypes or the Anti-Severe Acute
respiratory syndrome (SARS) coronavirus IgG1. To produce the
lentiviruses, the pHAGE vectors were transfected together with the
packaging plasmids (Gag, Rev, Tat and VSVG) into 293T LentiX cells
using Polyethyleneimine. The viruses were harvested two days after
transfection, purified and concentrated using LentiX Concentrator
(Clontech.TM.), according to the manufacturer instructions. LTR:
long terminal repeat, eIF.alpha.: eukaryotic initiation factor
alpha, scFv: single-chain variable fragment, C9 TAG: C9 peptide
TETSQVAPA, IRES: Internal Ribosome Entry Site, WPRE: Woodchuck
Hepatitis Virus Posttranscriptional Regulatory Element. (B) T cells
were transduced with the lentiviruses to generate Anti-CAIX CART
Cells, which are able to recognize CAIX positive RCC, and also
release Anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to
block PD-1/PD-L1-induced T cell exhaustion. (C) Percentage of CART
cells 14 days after transduction, representing the stable long-term
expression of CAR by the integrated lentiviruses in CD8+ T cells.
The CD8+ T cells were selected using Dynabeads.TM. CD8 Positive
isolation Kit (Life Technologies) and activated with Dynabeads.TM.
Human T Cell Activator CD3/CD28 (Life Technologies) in the presence
of IL-21 50 U/mL. IL-21 was added to the medium every 2 days. After
14 days, the CART cells were incubated with human CAIX-Fc or
BCMA-Fc, followed by incubation with an APC conjugated anti-human
Fc IgG and analyzed by FACS. (D) Concentration of IgG secreted to
the medium of transduced T cells evaluated by Human IgG ELISA
Quantitation Set (Bethyl Laboratories). (E) Concentration of
Anti-PD-L1 antibodies in the supernatant of 293T Cells transduced
with lentiviruses containing Anti-CAIX or Anti-BCMA CAR and
Anti-PD-L1 IgG1, Anti-PD-L1 IgG4 or unspecific Anti-SARS IgG1
sequences. The antibodies in the supernatant were purified with
Protein A sepharose beads (GE Healthcare) and biotinylated using
the EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific.TM.). These
antibodies were incubated with 5 .mu.g/mL of human PD-L1
pre-immobilized in the 96 wells MaxiSorp plate (Nunc.TM.). The
biotinylated antibodies were detected by incubation with
streptavidin-HRP for 1 h and developed with TMB. The absorbance was
read at .lamda.=450 nm. *P<0.001 compared to Anti BCMA/Anti SARS
IgG1 and Anti-CAIX/Anti-SARS IgG1. **P<0.05 compared to
Anti-CAIX/Anti-PD-L1 IgG1. (F and G) Clonal Expansion of CD8+ CART
cells. (F) Concentration of CART cells versus days in the presence
of skrc-52 CAIX+/PD-L1- cells. *P<0.05 comparing all CARS to
Anti-BCMA/Anti-SARS IgG1. (G) Percentage of transduced T cells
versus days in the presence of skrc-52 CAIX positive
cells.*P<0.05 comparing all CARS to Anti-BCMA/Anti-SARS IgG1.
The CD8+ CART cells were previously cultivated for five days after
transduction with Anti CAIX CAR able to express anti-PD-L1 IgG1
(Anti-CAIX/Anti-PD-L1 IgG1), IgG4 (Anti-CAIX/Anti-PD-L1 IgG4) or a
unspecific Anti-SARS Ab (Anti-CAIX/Anti SARS IgG1) or Anti-BCMA CAR
(negative control) able to express an unspecific anti-SARS Ab
(Anti-BCMA/Anti SARS IgG1) and activated with Dynabeads Human T
Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50
U/mL. The beads were removed and the CART cells were cultured with
skrc52 CAIX+PD-L1- and IL-21 (50 U/mL), which was added to the
medium every 2 days for 21 days. The results represent the
average.+-.SD of three donors in duplicate.
[0029] FIG. 17 is a series of graphs that illustrate CART cell
effector function. (A) Viability of Skrc59 CAIX+/PD-L1+ cells or
(B) Skrc52 CAIX-/PD-L1- cells incubated overnight (O.N.) with CART
cells Anti-CAIX/Anti-PD-L1 IgG1, Anti-CAIX/Anti-PD-L1 IgG4,
Anti-CAIX/Anti SARS IgG1 or Anti-BCMA/Anti SARS IgG1. These CART
cells were used 4 days after lentiviral transduction. The cell
viability was evaluated by MTT (Molecular Probes.TM.). *P<0.05
for all CART cells compared to Anti BCMA/Anti SARS IgG1 (C) IL2
released by CART cells after overnight contact with Skrc59
CAIX+/PD-L1+ cells or (D) Skrc52 CAIX-/PD-L1- cells. The IL-2
secretion was evaluated using the Human IL-2 ELISA Ready-SET-Go Kit
(eBioscience.TM.). *P<0.001 for all CART cells compared to Anti
BCMA/Anti SARS IgG1. (E) IFN.gamma. released by CART cells after
overnight contact with Skrc59 CAIX+/PD-L1+ or (F) Skrc52
CAIX-/PD-L1- cells. The IFN.gamma. secretion was evaluated using
the Human IFN.gamma. ELISA Ready-SET-Go Kit (eBiosciences.TM.).
*P<0.05 for all CART cells compared to Anti-BCMA/Anti-SARS IgG1.
(G) Ab-dependent cell-mediated cytotoxicity of skrc59 CAIX+/PD-L1+
or (H) skrc52 CAIX-/PD-L1- after incubation with the supernatant
(SN) of CART cells containing 500 ng/mL of the Anti-PD-L1 IgG1,
Anti-PD-L1 IgG4 or the Anti-SARS IgG1. To obtain the antibodies,
CART cells were incubated for six days with Dynabeads Human T
Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50
U/mL. After 7 days, the medium containing the mAbs was harvested NK
cells were purified using an EasySep.TM. Human NK Cell Enrichment
Kit (StemCell Technologies). RCC cell lines Skrc59 CAIX+PD-L1+ and
Skrc52 CAIX- PD-L1- were used as target cells and plated at
1.5.times.10.sup.3/well in a 96-well plate. RCC cells were
incubated for 1 hour, 37.degree. C., with 50 .mu.L of the CART
cells supernatant adjusted for 500 ng/mL of the respective Ab
Anti-PD-L1 IgG1, Anti-PD-L1 IgG4 or Anti-SARS IgG1. After the
incubation, the cells were washed with medium and incubated with
12.5:1, 25:1 or 50:1 NK cells for 4 h, 37.degree. C. Culture
supernatants were harvested by centrifugation and LDH measured in
the supernatant by CytoTox 96.RTM. Non-Radioactive Cytotoxicity
Assay (Promega.TM.) at 490 nm. These results represent the
average.+-.SD of three donors in duplicate.
[0030] FIG. 18 is a series of graphs that depict CART cells
expression of exhaustion markers. (A) Lag3, (B) Tim3 and (C) PD-1
expression. *P<0.05 compared to Anti-CAIX/Anti-SARS IgG1 and
Anti-BCMA/Anti-SARS IgG1. The CD8+ CART cells were selected using
Dynabeads.TM. CD8 Positive isolation Kit (Life Technologies),
activated with Dynabeads.TM. Human T Activator CD3/CD28 (Life
Technologies) and transduced with the following CARs:
Anti-CAIX/Anti-PD-L1 IgG1, Anti-CAIX/Anti-PD-L1 IgG4,
Anti-CAIX/Anti SARS IgG1 or Anti-BCMA/Anti SARS IgG1. These cells
were cultured in the presence of IL-21 50 U/mL and Dynabeads Human
T Activator CD3/CD28 for five days. After this period the CART
cells were co-cultured with Skrc-59 CAIX+PD-L1+ for 2 days in order
to stimulate exhaustion. CART cells were stained with
FITC-conjugated anti-human PD-1, PE-conjugated anti-human Tim3 and
PerCP/Cy5.5 anti-human Lag3 and analyzed by FACS. (D) Viability of
Skrc59 CAIX positive/PD-L1 positive cells after incubation with
exhausted CART cells. The cell viability was evaluated by MTT
(Molecular Probes). *P<0.05 compared to both Anti-CAIX/Anti-SARS
IgG1 and Anti-BCMA/Anti-SARS IgG1. **P<0.05 compared to
Anti-CAIX/Anti-PD-L1 IgG1. These results represent the
average.+-.SD of three donors.
[0031] FIG. 19 is a series of images and graphs that depict the
effect that CART cells have in an orthotopic model of human RCC.
(A) NSG Mice (N=35) were injected with 5.0.times.104 skrc-59 CAIX
positive, PD-L1 positive and luciferase positive RCC cells. After a
week, the mice were injected with 1.0.times.107 CART or
untransduced T cells IV (Day 0). The CART cells were previously
transduced with the following lentiviral sequences: Anti-BCMA
CAR/Anti-SARS IgG1, Anti-CAIX CAR/Anti SARS IgG1, Anti-CAIX
CAR/Anti-PD-L1 IgG1 and Anti-CAIX CAR/Anti-PD-L1 IgG4 (N=6 mice per
group). The tumor bioluminescence was quantified after 5 minutes of
luciferin IP injection using IVIS. A. Imaging of the tumors before
CART injection (Day 0), and after 7, 14, 23 and 30 days after first
CART cells injection. A second injection of 2.5.times.106 cells was
made on day 17. (B) Imaging of the tumors after excision at day 30.
Scale bar=1 cm. (C) Tumor growth curve. *P<0.05 when Anti-PD-L1
IgG1 and IgG4 groups were compared to Anti-BCMA/Anti-SARS IgG1 and
**P<0.05 when Anti-PD-L1 IgG1 and IgG4 groups were compared to
Anti-CAIX/Anti-SARS IgG1. (D) Tumor weight after 30 days of
treatment. *P<0.05 compared with Anti-BCMA/Anti SARS IgG1 CAR,
**P<0.05 compared with Anti-CAIX/Anti-SARS IgG1.
[0032] FIG. 20 is a series of graphs and histological images that
depict antitumor activity from CART cells. (A) Expression of
exhaustion markers in tumor infiltrating lymphocytes (TIL). The
kidney tumors from all mice were divided in two parts and one of
them was fragmented in small pieces and digested with collagenase
and DNAse to extraction of TIL. The CART cells were analyzed for
the exhaustion markers PD-1, Tim-3 and Lag3. *P<0.05 compared
with untransduced, Anti-BCMA/Anti SARS IgG1 CAR and
Anti-CAIX/Anti-SARS IgG1. (B) Detection of Ki67 as a tumor cell
proliferation marker and granzyme B to analyze CART cells activity
in the tissue. Four-micrometer sections of formalin-fixed,
paraffin-embedded tissues were dewaxed and rehydrated in a
decreasing ethanol series. Endogenous peroxidase activity was
quenched using 3% hydrogen peroxide. The antigen retrieval was
performed using pressure cooker in citrate buffer (pH=6.0) for 45
seconds at 123.degree. C., 15 PSI. The tissue sections were
incubated for 45 minutes with the rabbit anti human Ki67 polyclonal
Ab 1:2000 (Vector, VP-K451), mouse anti-human PD-L1 mAb 10.4
.mu.g/mL (Clone 405.9A11) developed by Dr. Gordon Freeman (Boston,
Mass.), biotinylated CAIX-Fc protein 17 .mu.g/mL (produced in our
lab), rabbit anti human granzyme B polyclonal Ab (Abcam, ab4059)
1:100 or rabbit anti-human NCAM (CD56) mAb 1:100 (Abcam, ab133345),
followed by secondary HRP conjugated anti-rabbit Ab or HRP-Avidin.
The slides were developed using 3,3'-diaminobenzidine (DAB) and
counterstained with hematoxylin. The images were obtained in an
Olympus BX51 microscopy using a DP71 digital camera (Olympus) and
analyzed in the DP Controller Software (Olympus.TM.). The scale
bars represent the magnification of the images of each column [500
.mu.m (40.times.), 100 .mu.m (200.times.) or 50 .mu.m
(400.times.)].(C) A series of graphs present percentage
quantification of TIL staining positive for Granzyme B, PD-L1-IHC,
and Ki67. Also presented in (C) is a Ki67-DAB Pixel count of
TILs.
[0033] FIG. 21 is a series of graphs that depict the evaluation of
IL-2 versus IL-21 to CD8+ CART cells proliferation. A and B.
Proliferation of CART transduced cells in the presence of IL-2 or
IL-21 evaluated 48 h, 72 h and 120 h after transduction with
lentiviruses. (A) Anti-CAIX CART cells or (B) Unspecific Anti-BCMA
CART cells (both with ZsGreen in the second cassette). The CD8+ T
cells were selected using Dynabeads.TM. CD8 Positive isolation Kit
(Life Technologies) and activated with Dynabeads.TM. Human T
Activator CD3/CD28 (Life Technologies) in the presence of IL2 or
IL-21 50 U/mL (Peprotech.TM.) The CART cells transduction was
evaluated by ZsGreen expression using FACS. The data represents the
average.+-.SD of two donors. *p<0.05 comparing IL-21 with non
treated control (Ctr); **p<0.05 comparing IL21 with IL-2. C and
D. Viability of RCC cells treated with CD8+ CART cells cultivated
in the presence of IL-2 or IL-21. The viability was evaluated by
MTT after an overnight incubation of CART cells Anti-BCMA,
Anti-CAIX or Untransduced T cells with (C) Skrc-59 CAIX+/PD-L1+ and
(F) Skrc-52 CAIX-/PD-L1- RCC cells. The CART cells were previously
cultivated in the presence of IL2 or IL-21 50 U/mL for 120 hours.
These results represent the average.+-.SD of two donors in
triplicate. *P<0.05 comparing Anti-CAIX CAR with Anti-BCMA CAR
or untransduced T cells.
[0034] FIG. 22 is a series of flow cytometry graphs that depict the
expression of PD-1 and CAIX in the renal cell carcinoma (RCC)
lines. (A) Negative control, (B) Skrc52 CAIX-PD-L1-, (C) Skrc52
CAIX+PD-L1-, (D) Skrc59 CAIX+PD-L1+. The cells were stained with
Anti-human CAIX antibody developed with APC-Anti-human Fc IgG and
Biotinylated Anti human PD-L1 antibody developed with PE-Avidin.
The analysis was performed by FACS.
[0035] FIG. 23 is a series of graphs that depict the
characterization of CART cells. (A) Proliferation of total CD8+ T
cells two or four days after transduction Anti CAIX CAR/Anti-PD-L1
IgG1), (Anti-CAIX CAR/Anti-PD-L1 IgG4), Anti-CAIX CAR/Anti SARS
IgG1) or Anti-BCMA CAR/Anti SARS IgG1). The CD8+ T cells were
selected using Dynabeads.TM. CD8 Positive isolation Kit (Life
Technologies) and activated with Dynabeads.TM. Human T Activator
CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL.
IL-21 was added to the medium every 2 days. The proliferation was
evaluated by FACS using Counting Beads (Molecular Probes). (B)
Concentration of CAR-transduced T cells two and four days after
transduction. The CART cells were incubated with human CAIX-Fc or
BCMA-Fc, followed by incubation with an APC conjugated anti-human
Fc IgG and analyzed by FACS. (C) Percentage of CART cells 2 and 4
days after transduction. The results represent the average.+-.SD of
three donors in duplicate.
[0036] FIG. 24 is a series of graphs that depict the effect that
CART cells have in an orthotopic model of human RCC. (A) Comparison
of tumor size detected by bioluminescence versus days among the
CART cells groups. NSG Mice (N=35) were injected in the renal
capsule with 5.0.times.104 skrc-59 CAIX+, PD-L1+ and luciferase+
RCC cells. After a week, the mice were injected with 1.0.times.107
CART or untransduced T cells IV. The CART cells were previously
transduced with the following lentiviral sequences: Anti-BCMA
CAR/Anti-SARS IgG1, Anti-CAIX CAR/Anti SARS IgG1, Anti-CAIX
CAR/Anti-PD-L1 IgG1 and Anti-CAIX CAR/Anti-PD-L1 IgG4 (N=6 mice per
group). The tumor bioluminescence was quantified after 5 minutes of
luciferin IP injection using IVIS. In the Day 17, more
2.5.times.106 CART cells were injected. *P<0.05 compared to
anti-BCMA CART cells. **P<0.05 compared to untransduced T cells,
***P<0.05 compared to Anti-CAIX/Anti-SARS IgG1. (B) Percentage
of T cells in the mice blood after 8 days of treatment. *P<0.05
compared to untransduced T cells. **P<0.05 compared to all
Anti-CAIX CARs. The red blood cells were lysed with ACK Lysing
Buffer (Lonza.TM.) and the remaining cells were stained with
Pacific Blue conjugated Anti-human CD45 and analyzed by FACS. (C)
Total tumor infiltrating lymphocytes (TIL) after 30 days of
treatment with CART cells. The tumors and kidney from all mice were
divided in two parts and one of them was fragmented in small pieces
and digested with collagenase and DNAse to extraction of TIL. The
cells were stained with Pacific Blue conjugated Anti-human CD45 and
analyzed by FACS.
[0037] FIGS. 25A and 25B are a series of graphs and histological
images that depict Human natural killer (NK) cells in the tumors
treated with Anti-CAIX CART cells releasing anti-PD-L1 IgG1 Ab. (A)
Percentage of CD56+ cells (NK marker) in the tumors. Two mice of
each group were injected with 4.5.times.106 NK cells one day before
the euthanasia. The kidney tumors from all mice were divided in two
parts and one of them was fragmented in small pieces and digested
with collagenase and DNAse to extraction of NK. NK cells present in
the tumor were stained with APC-Anti-CD56 Ab and analyzed by FACS.
*P<0.05. (B) CD56+ cells into the excised tumors detected by IHC
and quantified using IHC Profiler Plugin of ImageJ Software.
Four-micrometer sections of formalin-fixed, paraffin-embedded
tissues were dewaxed and rehydrated in a decreasing ethanol series.
Endogenous peroxidase activity was quenched using 3% hydrogen
peroxide. The antigen retrieval was performed using pressure cooker
in citrate buffer (pH=6.0) for 45 seconds at 123.degree. C., 15
PSI. The tissue sections were incubated for 45 minutes with the
rabbit anti-human CD56 mAb 1:100 (Abcam, ab133345), followed by
secondary HRP conjugated anti-rabbit or anti-mouse Ab. The slides
were developed using 3,3'-diaminobenzidine (DAB) and counterstained
with hematoxylin. The images were obtained in an Olympus BX51
microscopy using a DP71 digital camera (Olympus) and analyzed in
the DP Controller Software (Olympus). The quantification was
performed using the IHC Profiler Plugin of ImageJ Software (23).
The scale bars represent the magnification of the images
(400.times.). *P<0.05 compared with untransduced, **P<0.05
compared with untransduced and Anti-BCMA/Anti-SARS IgG1.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention relates to a chimeric antigen receptor
(CAR) particularly adapted to immune cells used in
immunotherapy.
[0039] In one embodiment, a double immunotherapeutic strategy is
described based on the blocking of T cell exhaustion using
anti-PD-L1 antibodies secreted by targeted anti-CAIX CAR T cells
combined in a single lentivirus construct for improving cancer
treatment.
[0040] An emerging mechanism associated with the progression of RCC
and other tumors is the immune checkpoint pathway, which consists
in cellular interactions that prevent excessive activation of T
cells under normal conditions, allowing T cell function in a
self-limited manner. As an evasion mechanism, many tumors are able
to stimulate the expression of immune checkpoint molecules,
resulting in an anergic phenotype of T cells that cannot restrain
tumor progression. Emerging clinical data highlight the importance
of one inhibitory ligand and receptor pair as an immune checkpoint:
the programmed death-ligand 1 (PD-L1; B7-H1 and CD274) and
programmed death receptor-1 (PD-1; CD279), in preventing killing of
cancer cells by cytotoxic T-lymphocytes. PD1 receptor is expressed
by many cell types like T cells, B cells, Natural Killer cells (NK)
and host tissues. Tumors and Antigen-presenting cells (APC)
expressing PD-L1 can block T cell receptor (TCR) signaling of
cytotoxic T-lymphocytes through binding to receptor PD-1,
decreasing the production of cytokines and T cell proliferation.
PD-L1 overexpression can be found in many tumor types and may also
mediate an immunosuppressive function through its interaction with
other proteins, including CD80 (B7.1), blocking its ability to
activate T cells through binding to CD28.
[0041] Genetic engineering of human lymphocytes to express
tumor-directed chimeric antigen receptors (CAR) can produce
antitumor effector cells that bypass tumor immune escape mechanisms
that are due to abnormalities in protein-antigen processing and
presentation. Moreover, these transgenic receptors can be directed
to tumor-associated antigens that are not protein-derived. In
certain embodiments of the invention there are lymphocytes (CARTS)
that are modified to comprise at least a CAR, and in particular
embodiments of the invention a single CAR targets two or more
antigens. In preferred embodiments, the CARTS are further modified
to express and secrete one or more polypeptides, such as for
example an antibody or a cytokine. Such CARTS are referred to
herein as armed CARTS. Armed CARTS allow for simultaneous secretion
of the polypeptide locally at the targeted site (i.e., tumor
site).
[0042] A modified TCR called chimeric antigen receptor (CAR)
containing single chain variable antibody fragment (scFv)
previously selected by high affinity against a specific tumor
associated antigen is a powerful new approach against cancer. The
scFv presented in the CAR is linked to an intracellular signaling
block that includes CD3 to induce T cell activation followed by
antigen binding. This structure is characteristic for
first-generation CARs, which were improved to second- and
generation CARs that link the signaling co-stimulatory endodomains
of CD28, 4-1BB, or OX40 to CD3 or 3.sup.rd-generation CARs that
links two elements to CD3.zeta. in tandem. These endodomains are
required for complete T cell activation during TCR recognition by
antigen-presenting cells (APCs), improving cytokine production and
proliferation of CAR-T cells. The effect of CART cells has
heretofore been modest for the treatment of solid tumors, due to
difficulty in finding unique tumor associated antigens, inefficient
homing of T cells to tumor locations, low persistence of T cells in
the body and the immunosuppressive microenvironment of solid
tumors.
[0043] In particular cases, the lymphocytes include a receptor that
is chimeric, non-natural and engineered at least in part by the
hand of man. In particular cases, the engineered chimeric antigen
receptor (CAR) has one, two, three, four, or more components, and
in some embodiments the one or more components facilitate targeting
or binding of the lymphocyte to one or more tumor
antigen-comprising cancer cells.
[0044] The CAR according to the invention generally comprises at
least one transmembrane polypeptide comprising at least one
extracellular ligand-biding domain and; one transmembrane
polypeptide comprising at least one intracellular signaling domain;
such that the polypeptides assemble together to form a Chimeric
Antigen Receptor.
[0045] The term "extracellular ligand-binding domain" as used
herein is defined as an oligo- or polypeptide that is capable of
binding a ligand. Preferably, the domain will be capable of
interacting with a cell surface molecule. For example, the
extracellular ligand-binding domain may be chosen to recognize a
ligand that acts as a cell surface marker on target cells
associated with a particular disease state.
[0046] In particular, the extracellular ligand-binding domain can
comprise an antigen binding domain derived from an antibody against
an antigen of the target.
[0047] As non limiting examples, the antigen of the target can be a
tumor-associated surface antigen, such as ErbB2 (HER2/neu),
carcinoembryonic antigen (CEA), epithelial cell adhesion molecule
(EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III
(EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2,
ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids,
glioma-associated antigen, .beta.-human chorionic gonadotropin,
alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,
MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),
intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,
prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53,
prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor
antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2,
CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor,
mesothelia, a major histocompatibility complex (MHC) molecule
presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30,
NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra
domain B (EDB) of fibronectin and the A1 domain of tenascin-C(TnC
A1) and fibroblast associated protein (fap); a lineage-specific or
tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33,
CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, a
major histocompatibility complex (MHC) molecule, BCMA (CD269,
TNFRSF 17), or a virus-specific surface antigen such as an
HIV-specific antigen (such as HIV gp120); an EBV-specific antigen,
a CMV-specific antigen, a HPV-specific antigen, a Lasse
Virus-specific antigen, an Influenza Virus-specific antigen as well
as any derivate or variant of these surface markers.
[0048] Preferably, the CAR is specific for BMCA, CAIX, CCR4, PD-L1,
PD-L2, PD1, Glucocorticoid-Induced Tumor Necrosis Factor Receptors
(GITR), Severe acute respiratory syndrome (SARS), influenza,
flavivirus or Middle East Respiratory Syndrome (MERS).
[0049] In a preferred embodiment, said extracellular ligand-binding
domain is a single chain antibody fragment (scFv) comprising the
light (V.sub.L) and the heavy (V.sub.H) variable fragment of a
target antigen specific monoclonal antibody joined by a flexible
linker.
[0050] In a more preferred embodiment, said scFv antibody is
specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, GITR, SARS,
influenza, flavivirus or MERS.
[0051] Exemplary antibodies useful in constructing the CAR
according to the invention includes antibodies disclosed in for
example: WO/2005/060520, WO/2006/089141, WO/2007/065027,
WO/2009/086514, WO/2009/079259, WO/2011/153380, WO/2014/055897, WO
2015/143194, WO 2015/164865, WO 2013/166500, and WO 2014/144061;
PCT/US2015/054202, PCT/US2015/054010 and 62/144,729 the contents of
which are hereby incorporated by reference in their entireties.
[0052] PDLL (68)
[0053] Exemplary anti-PDL1 antibodies include antibodies having
containing a .sub.VH nucleotide sequence having SEQ ID NO: 1485 and
a .sub.VL nucleotide sequence having SEQ ID NO: 1487; a VH
nucleotide sequence having SEQ ID NO: 1485 and a VL nucleotide
sequence having SEQ ID NO: 1487; a VH nucleotide sequence having
SEQ ID NO: 1489 and a VL nucleotide sequence having SEQ ID NO:
1491; a VH nucleotide sequence having SEQ ID NO: 1493 and a VL
nucleotide sequence having SEQ ID NO: 1495; a VH nucleotide
sequence having SEQ ID NO: 1497 and a VL nucleotide sequence having
SEQ ID NO: 1499; a VH nucleotide sequence having SEQ ID NO: 1501
and a VL nucleotide sequence having SEQ ID NO: 1503; a VH
nucleotide sequence having SEQ ID NO: 1505 and a VL nucleotide
sequence having SEQ ID NO: 1507; a VH nucleotide sequence having
SEQ ID NO: 1509 and a VL nucleotide sequence having SEQ ID NO:
1511; a VH nucleotide sequence having SEQ ID NO: 1513 and a VL
nucleotide sequence having SEQ ID NO: 1515; a VH nucleotide
sequence having SEQ ID NO: 1517 and a VL nucleotide sequence having
SEQ ID NO: 1519; a VH nucleotide sequence having SEQ ID NO: 1521
and a VL nucleotide sequence having SEQ ID NO: 1523; a VH
nucleotide sequence having SEQ ID NO: 1525 and a VL nucleotide
sequence having SEQ ID NO: 1527; a VH nucleotide sequence having
SEQ ID NO: 1529 and a VL nucleotide sequence having SEQ ID NO:
1531; a VH nucleotide sequence having SEQ ID NO: 1533 and a VL
nucleotide sequence having SEQ ID NO: 1535; a VH nucleotide
sequence having SEQ ID NO: 1537 and a VL nucleotide sequence having
SEQ ID NO: 1539.
[0054] Exemplary anti-PDL1 antibodies include antibodies having
containing a .sub.VH amino acid sequence having SEQ ID NO: 970 and
a .sub.VL amino acid sequence having SEQ ID NO: 971; a VH amino
acid having SEQ ID NO: 1486 and a VL polypeptide sequence having
SEQ ID NO: 1488 a VH amino acid having SEQ ID NO: 1490 and a VL
polypeptide sequence having SEQ ID NO: 1492 a VH amino acid having
SEQ ID NO: 1494 and a VL polypeptide sequence having SEQ ID NO:
1496 a VH amino acid having SEQ ID NO: 1498 and a VL polypeptide
sequence having SEQ ID NO: 1500 a VH amino acid having SEQ ID NO:
1502 and a VL polypeptide sequence having SEQ ID NO: 1504 a VH
amino acid having SEQ ID NO: 1506 and a VL polypeptide sequence
having SEQ ID NO: 1508 a VH amino acid having SEQ ID NO: 1510 and a
VL polypeptide sequence having SEQ ID NO: 1512 a VH amino acid
having SEQ ID NO: 1514 and a VL polypeptide sequence having SEQ ID
NO: 1516 a VH amino acid having SEQ ID NO: 1518 and a VL
polypeptide sequence having SEQ ID NO: 1520 a VH amino acid having
SEQ ID NO: 1522 and a VL polypeptide sequence having SEQ ID NO:
1524 a VH amino acid having SEQ ID NO: 1526 and a VL polypeptide
sequence having SEQ ID NO: 1528 a VH amino acid having SEQ ID NO:
1530 and a VL polypeptide sequence having SEQ ID NO: 1532 a VH
amino acid having SEQ ID NO: 1534 and a VL polypeptide sequence
having SEQ ID NO: 1536 a VH amino acid having SEQ ID NO: 1538 and a
VL polypeptide sequence having SEQ ID NO: 1540.
[0055] In other embodiments the anti-PDL1 antibodies have a heavy
chain with three CDRs including the amino acid sequences SEQ ID NO:
1541, 1554, 1569 respectively and a light chain with three CDRs
including the amino acid sequences 1584, 1599, 1610 respectively;
or a heavy chain with three CDRs comprising the amino acid
sequences 1543, 1556, 1571 and a light chain with three CDRs
comprising the amino acid sequences 1586, 1600, 1612; or a heavy
chain with three CDRs comprising the amino acid sequences 1544,
1557, 1572 and a light chain with three CDRs comprising the amino
acid sequences 1587, 1601, 1613; or a heavy chain with three CDRs
comprising the amino acid sequences 1545, 1558, 1573 and a light
chain with three CDRs comprising the amino acid sequences 1588,
1602, 1614; or a heavy chain with three CDRs comprising the amino
acid sequences 1546, 1559, 1574 and a light chain with three CDRs
comprising the amino acid sequences 1589, 1603, 1615; or a heavy
chain with three CDRs comprising the amino acid sequences 1547,
1560, 1575 and a light chain with three CDRs comprising the amino
acid sequences 1590, 1604, 1616; or a heavy chain with three CDRs
comprising the amino acid sequences 1548, 1561, 1576 and a light
chain with three CDRs comprising the amino acid sequences 1591,
1605, 1617; or a heavy chain with three CDRs comprising the amino
acid sequences 1541, 1562, 1577 and a light chain with three CDRs
comprising the amino acid sequences 1592, 1599, 1618; or a heavy
chain with three CDRs comprising the amino acid sequences 1549,
1563, 1578 and a light chain with three CDRs comprising the amino
acid sequences 1593, 1606, 1619; or a heavy chain with three CDRs
comprising the amino acid sequences 1550, 1564, 1579 and a light
chain with three CDRs comprising the amino acid sequences 1594,
1607, 1620; or a heavy chain with three CDRs comprising the amino
acid sequences 1551, 1565, 1580 and a light chain with three CDRs
comprising the amino acid sequences 1595, 1599, 1621; or a heavy
chain with three CDRs comprising the amino acid sequences 1542,
1566, 1581 and a light chain with three CDRs comprising the amino
acid sequences 1596, 1599, 1622; or a heavy chain with three CDRs
comprising the amino acid sequences 1552, 1567, 1582 and a light
chain with three CDRs comprising the amino acid sequences 1597,
1608, 1623; or a heavy chain with three CDRs comprising the amino
acid sequences 1553, 1568, 1583 and a light chain with three CDRs
comprising the amino acid sequences 1598, 1609 1624.
[0056] SARS (26)
[0057] Exemplary SARS neutralizing antibodies include antibodies
having containing a .sub.VH nucleotide sequence having SEQ ID NO:
1626 and a .sub.VL nucleotide sequence having SEQ ID NO: 1628; a VH
nucleotide sequence having SEQ ID NO: 1630 and a VL nucleotide
sequence having SEQ ID NO: 1639; a VH nucleotide sequence having
SEQ ID NO: 1634 and a VL nucleotide sequence having SEQ ID NO:
1640; a VH nucleotide sequence having SEQ ID NO: 1632 and a VL
nucleotide sequence having SEQ ID NO: 1641; a VH nucleotide
sequence having SEQ ID NO: 1633 and a VL nucleotide sequence having
SEQ ID NO: 1642; a VH nucleotide sequence having SEQ ID NO: 1634
and a VL nucleotide sequence having SEQ ID NO: 1643; a VH
nucleotide sequence having SEQ ID NO: 1635 and a VL nucleotide
sequence having SEQ ID NO: 1644; a VH nucleotide sequence having
SEQ ID NO: 1636 and a VL nucleotide sequence having SEQ ID NO:
1645; a VH nucleotide sequence having SEQ ID NO: 1637 and a VL
nucleotide sequence having SEQ ID NO: 1646
[0058] CXCR4 (33)
[0059] Exemplary anti-CXCR4 antibody include antibodies having a VH
amino acid sequence having SEQ ID NO: 771 and a VL amino acid
sequence having SEQ ID NO: 779; a VH amino acid sequence having SEQ
ID NO: 772 and a VL amino acid sequence having SEQ ID NO: 780; a VH
amino acid sequence having SEQ ID NO: 773 and a VL amino acid
sequence having SEQ ID NO: 781; a VH amino acid sequence having SEQ
ID NO: 774 and a VL amino acid sequence having SEQ ID NO: 782; a VH
amino acid sequence having SEQ ID NO: 775 and a VL amino acid
sequence having SEQ ID NO: 783; a VH amino acid sequence having SEQ
ID NO: 776 and a VL amino acid sequence having SEQ ID NO: 784; a VH
amino acid sequence having SEQ ID NO: 777 and a VL amino acid
sequence having SEQ ID NO: 785; or a VH amino acid sequence having
SEQ ID NO: 778 and a VL amino acid sequence having SEQ ID NO:
786.
[0060] In other embodiments the anti-CXCR4 antibodies have a heavy
chain with three CDRs including the amino acid sequences SEQ ID NO:
803, 804, 805 respectively and a light chain with three CDRs
including the amino acid sequences 806, 807, 808 respectively; or a
heavy chain with three CDRs comprising the amino acid sequences
809, 810, 811, respectively and a light chain with three CDRs
comprising the amino acid sequences 812, 813, 814, respectively; or
a heavy chain with three CDRs comprising the amino acid sequences
815, 816, 817 respectively and a light chain with three CDRs
comprising the amino acid sequences 818, 819, 820 respectively; or
a heavy chain with three CDRs comprising the amino acid sequences
827, 828, 829 respectively and a light chain with three CDRs
comprising the amino acid sequences 830, 831, 832 respectively; or
a heavy chain with three CDRs comprising the amino acid sequences
833, 834, 835, respectively and a light chain with three CDRs
comprising the amino acid sequences 836, 837, 838, respectively; or
a heavy chain with three CDRs comprising the amino acid sequences
839, 840, 841 respectively and a light chain with three CDRs
comprising the amino acid sequences 842, 843, 844 respectively.
[0061] Carbonic Anhydrase IX (40)
[0062] Exemplary anti-CA IX antibodies include antibodies having
containing a .sub.VH amino acid sequence having SEQ ID NO: 845 and
a .sub.VL amino acid sequence having SEQ ID NO: 846; a VH amino
acid sequence having SEQ ID NO: 847 and a VL amino acid sequence
having SEQ ID NO: 868; a VH amino acid sequence having SEQ ID NO:
848 and a VL amino acid sequence having SEQ ID NO: 869; a VH amino
acid sequence having SEQ ID NO: 849 and a VL amino acid sequence
having SEQ ID NO: 870; a VH amino acid sequence having SEQ ID NO:
850 and a VL amino acid sequence having SEQ ID NO: 871; a VH amino
acid sequence having SEQ ID NO: 851 and a VL amino acid sequence
having SEQ ID NO: 872; a VH amino acid sequence having SEQ ID NO:
852 and a VL amino acid sequence having SEQ ID NO: 873; a VH amino
acid sequence having SEQ ID NO: 853 and a VL amino acid sequence
having SEQ ID NO: 874; a VH amino acid sequence having SEQ ID NO:
854 and a VL amino acid sequence having SEQ ID NO: 875; a VH amino
acid sequence having SEQ ID NO: 855 and a VL amino acid sequence
having SEQ ID NO: 876; a VH amino acid sequence having SEQ ID NO:
856 and a VL amino acid sequence having SEQ ID NO: 877; a VH amino
acid sequence having SEQ ID NO: 857 and a VL amino acid sequence
having SEQ ID NO: 878; a VH amino acid sequence having SEQ ID NO:
858 and a VL amino acid sequence having SEQ ID NO: 879; a VH amino
acid sequence having SEQ ID NO: 859 and a VL amino acid sequence
having SEQ ID NO: 880; a VH amino acid sequence having SEQ ID NO:
860 and a VL amino acid sequence having SEQ ID NO: 881; a VH amino
acid sequence having SEQ ID NO: 861 and a VL amino acid sequence
having SEQ ID NO: 882 a VH amino acid sequence having SEQ ID NO:
862 and a VL amino acid sequence having SEQ ID NO: 883; a VH amino
acid sequence having SEQ ID NO: 863 and a VL amino acid sequence
having SEQ ID NO: 884; a VH amino acid sequence having SEQ ID NO:
864 and a VL amino acid sequence having SEQ ID NO: 885; a VH amino
acid sequence having SEQ ID NO: 865 and a VL amino acid sequence
having SEQ ID NO: 886; a VH amino acid sequence having SEQ ID NO:
866 and a VL amino acid sequence having SEQ ID NO: 887; a VH amino
acid sequence having SEQ ID NO: 867 and a VL amino acid sequence
having SEQ ID NO: 888.
[0063] In other embodiments the anti-CA IX antibodies have a heavy
chain with three CDRs including the amino acid sequences SEQ ID NO:
803, 804, 805 respectively and a light chain with three CDRs
including the amino acid sequences 806, 807, 808 respectively; or a
heavy chain with three CDRs comprising the amino acid sequences
899, 915, 909 and a light chain with three CDRs comprising the
amino acid sequences 905, 906, 952 or a heavy chain with three CDRs
comprising the amino acid sequences 899, 915, 909 and a light chain
with three CDRs comprising the amino acid sequences 935, 943, 953
or a heavy chain with three CDRs comprising the amino acid
sequences 899, 915, 909 and a light chain with three CDRs
comprising the amino acid sequences 935, 906, 954 or a heavy chain
with three CDRs comprising the amino acid sequences 910, 916, 923
and a light chain with three CDRs comprising the amino acid
sequences 936, 944, 955 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 915, 909 and a light chain with three
CDRs comprising the amino acid sequences 936, 944, 956 or a heavy
chain with three CDRs comprising the amino acid sequences 911, 917,
924 and a light chain with three CDRs comprising the amino acid
sequences 937, 945, 957 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 915, 909 and a light chain with three
CDRs comprising the amino acid sequences 935, 946, 958 or a heavy
chain with three CDRs comprising the amino acid sequences 899, 915,
909 and a light chain with three CDRs comprising the amino acid
sequences 938, 946, 959 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 915, 909 and a light chain with three
CDRs comprising the amino acid sequences 905, 946, 960 or a heavy
chain with three CDRs comprising the amino acid sequences 899, 918,
925 and a light chain with three CDRs comprising the amino acid
sequences 937, 947, 955 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 918, 926 and a light chain with three
CDRs comprising the amino acid sequences 937, 945, 957 or a heavy
chain with three CDRs comprising the amino acid sequences 912, 919,
927 and a light chain with three CDRs comprising the amino acid
sequences 937, 943, 961 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 918, 928 and a light chain with three
CDRs comprising the amino acid sequences 937, 906, 960 or a heavy
chain with three CDRs comprising the amino acid sequences 899, 918,
928 and a light chain with three CDRs comprising the amino acid
sequences 937, 906, 960 or a heavy chain with three CDRs comprising
the amino acid sequences 913, 920, 929 and a light chain with three
CDRs comprising the amino acid sequences 939, 948, 962 or a heavy
chain with three CDRs comprising the amino acid sequences 899, 918,
930 and a light chain with three CDRs comprising the amino acid
sequences 935, 944, 955 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 921, 931 and a light chain with three
CDRs comprising the amino acid sequences 935, 944, 955 or a heavy
chain with three CDRs comprising the amino acid sequences 912, 919,
932 and a light chain with three CDRs comprising the amino acid
sequences 940, 949, 963 or a heavy chain with three CDRs comprising
the amino acid sequences 899, 915, 909 and a light chain with three
CDRs comprising the amino acid sequences 935, 943, 960 or a heavy
chain with three CDRs comprising the amino acid sequences 914, 922,
933 and a light chain with three CDRs comprising the amino acid
sequences 941, 950, 964 or a heavy chain with three CDRs comprising
the amino acid sequences 912, 918, 934 and a light chain with three
CDRs comprising the amino acid sequences 942, 951, 965.
[0064] Cc-Chemokine Receptor 4 (Ccr4) (048)
[0065] Exemplary CC-chemokine receptor 4 (CCR4) antibodies include
antibodies having containing a .sub.VH nucleotide sequence having
SEQ ID NO: 969 and a .sub.VL nucleotide sequence having SEQ ID NO:
971; a .sub.VH nucleotide sequence having SEQ ID NO: 969 and a
V.sub.L nucleotide sequence having SEQ ID NO: 972.
[0066] Exemplary CCR4 antibodies include antibodies having
containing a .sub.VH amino acid sequence having SEQ ID NO: 970 and
a V.sub.L amino acid sequence having SEQ ID NO: 971.
[0067] In other embodiments the CCR4 antibodies have a heavy chain
with three CDRs including the amino acid sequences SEQ ID NO: 973,
974, 975 respectively and a light chain with three CDRs including
the amino acid sequences 976, 977, 978 respectively.
[0068] Middle East Respiratory Syndrome Coronavirus (MERS-CoV).
(85)
[0069] Exemplary anti-Middle East Respiratory Syndrome coronavirus
(MERS-CoV) antibody include antibodies having a VH nucleotide
sequence having SEQ ID NO: 677and a VL nucleotide sequence having
SEQ ID NO:679; a VH nucleotide sequence having SEQ ID NO: 681and a
VL nucleotide sequence having SEQ ID NO:683; a VH nucleotide
sequence having SEQ ID NO: 685and a VL nucleotide sequence having
SEQ ID NO:687; a VH nucleotide sequence having SEQ ID NO: 689and a
VL nucleotide sequence having SEQ ID NO:692; a VH nucleotide
sequence having SEQ ID NO: 693and a VL nucleotide sequence having
SEQ ID NO:695; a VH nucleotide sequence having SEQ ID NO: 697and a
VL nucleotide sequence having SEQ ID NO:699; and a VH nucleotide
sequence having SEQ ID NO: 701and a VL nucleotide sequence having
SEQ ID NO:703.
[0070] Exemplary anti-Middle East Respiratory Syndrome coronavirus
(MERS-CoV) antibody include antibodies having a VH amino acid
sequence SEQ ID NO: 678 and a VL amino acid sequence having SEQ ID
NO: 680; a VH amino acid sequence SEQ ID NO: 682 and a VL amino
acid sequence having SEQ ID NO: 684; a VH amino acid sequence SEQ
ID NO: 686 and a VL amino acid sequence having SEQ ID NO: 688; a VH
amino acid sequence SEQ ID NO: 690 and a VL amino acid sequence
having SEQ ID NO: 692; a VH amino acid sequence SEQ ID NO: 694 and
a VL amino acid sequence having SEQ ID NO: 696; a VH amino acid
sequence SEQ ID NO: 698 and a VL amino acid sequence having SEQ ID
NO: 700; and a VH amino acid sequence SEQ ID NO: 702 and a VL amino
acid sequence having SEQ ID NO: 704.
[0071] In other embodiments the anti-Middle East Respiratory
Syndrome coronavirus (MERS-CoV) antibody has a heavy chain with
three CDRs including the amino acid sequences of 705, 706, and 707
and a light chain with three CDRs including the amino acid
sequences 722, 723, and 724; a heavy chain with three CDRs
including the amino acid sequences of 708, 709, and 710 and a light
chain with three CDRs including the amino acid sequences 725, 726,
and 727; a heavy chain with three CDRs including the amino acid
sequences of 711, 712, and 713 and a light chain with three CDRs
including the amino acid sequences 728, 729, and 730; a heavy chain
with three CDRs including the amino acid sequences of 711, 735, and
715 and a light chain with three CDRs including the amino acid
sequences 731, 732, and 733; a heavy chain with three CDRs
including the amino acid sequences of 711, 735, and 716 and a light
chain with three CDRs including the amino acid sequences 737, 738,
and 739; a heavy chain with three CDRs including the amino acid
sequences of 717, 718, and 719 and a light chain with three CDRs
including the amino acid sequences 736, 742, and 743; and a heavy
chain with three CDRs including the amino acid sequences of 714,
720, and 721 and a light chain with three CDRs including the amino
acid sequences 740, 729, and 741.
[0072] GITR (93)
[0073] Exemplary anti-human GITR antibody include antibodies having
a VH nucleotide sequence having SEQ ID NO: 1361 and a VL nucleotide
sequence having SEQ ID NO: 1363; a VH nucleotide sequence having
SEQ ID NO: 1365 and a VL nucleotide sequence having SEQ ID NO:1367;
a VH nucleotide sequence having SEQ ID NO: 1369 and a VL nucleotide
sequence having SEQ ID NO: 1371; a VH nucleotide sequence having
SEQ ID NO: 1381 and a VL nucleotide sequence having SEQ ID NO:
1375; a VH nucleotide sequence having SEQ ID NO: 1377 and a VL
nucleotide sequence having SEQ ID NO: 1379; a VH nucleotide
sequence having SEQ ID NO: 1381 and a VL nucleotide sequence having
SEQ ID NO: 1383; a VH nucleotide sequence having SEQ ID NO: 1385
and a VL nucleotide sequence having SEQ ID NO: 1387; a VH
nucleotide sequence having SEQ ID NO: 1389 and a VL nucleotide
sequence having SEQ ID NO:1391; a VH nucleotide sequence having SEQ
ID NO: 1393 and a VL nucleotide sequence having SEQ ID NO: 1395; a
VH nucleotide sequence having SEQ ID NO: 1397 and a VL nucleotide
sequence having SEQ ID NO: 1398; or a VH nucleotide sequence having
SEQ ID NO: 1401and a VL nucleotide sequence having SEQ ID NO:
1403.
[0074] Exemplary anti-human GITR antibody include antibodies having
a VH amino acid sequence having SEQ ID NO: 1362 and a VL amino acid
sequence having SEQ ID NO: 1364; a VH amino acid having SEQ ID NO:
1366 and a VL polypeptide sequence having SEQ ID NO:1368; a VH
amino acid sequence having SEQ ID NO: 1371 and a VL amino acid
sequence having SEQ ID NO: 1372; a VH amino acid sequence having
SEQ ID NO: 1382 and a VL amino acid sequence having SEQ ID NO:
1376; a VH nucleotide sequence having SEQ ID NO: 1378 and a VL
nucleotide sequence having SEQ ID NO: 1380; a VH amino acid having
SEQ ID NO: 1382 and a VL polypeptide sequence having SEQ ID NO:
1384; a VH amino acid sequence having SEQ ID NO: 1386 and a VL
amino acid sequence having SEQ ID NO: 1388; a VH amino acid
sequence having SEQ ID NO: 1390 and a VL amino acid sequence having
SEQ ID NO: 1392; a VH amino acid having SEQ ID NO: 1394 and a VL
polypeptide sequence having SEQ ID NO: 1396; a VH amino acid
sequence having SEQ ID NO: 1399 and a VL amino acid sequence having
SEQ ID NO: 1400; or a VH amino acid sequence having SEQ ID NO: 1402
and a VL amino acid sequence having SEQ ID NO: 1404.
[0075] In other embodiments the anti-human GITR antibody has a
heavy chain with three CDRs including the amino acid sequences
1405, 1406, and 1407 and a light chain with three CDRs including
the amino acid sequences1408, 1409, and 1410 respectively; a heavy
chain with three CDRs including the amino acid sequences 1411,
1412, and 1413 and a light chain with three CDRs including the
amino acid sequences1414, 1415, and 1416 respectively; a heavy
chain with three CDRs including the amino acid sequences 1417,
1418, and 1419 and a light chain with three CDRs including the
amino acid sequences1420, 1421, and 1422 respectively; a heavy
chain with three CDRs including the amino acid sequences 1423,
1424, and 1425 and a light chain with three CDRs including the
amino acid sequences1426, 1427, and 1428 respectively; a heavy
chain with three CDRs including the amino acid sequences 1429,
1430, and 1431 and a light chain with three CDRs including the
amino acid sequences1432, 1433, and 1434 respectively; a heavy
chain with three CDRs including the amino acid sequences 1435,
1436, and 1437 and a light chain with three CDRs including the
amino acid sequences1438, 1439, and 1440 respectively; a heavy
chain with three CDRs including the amino acid sequences 1441,
1442, and 1443 and a light chain with three CDRs including the
amino acid sequences1444, 1445, and 1446 respectively; a heavy
chain with three CDRs including the amino acid sequences 1447,
1448, and 1449 and a light chain with three CDRs including the
amino acid sequences1450, 1451, and 1452 respectively; a heavy
chain with three CDRs including the amino acid sequences 1453,
1454, and 1455 and a light chain with three CDRs including the
amino acid sequences1456, 1457, and 1458 respectively; a heavy
chain with three CDRs including the amino acid sequences 1459,
1460, and 1461 and a light chain with three CDRs including the
amino acid sequences1462, 1463, and 1464 respectively; or a heavy
chain with three CDRs including the amino acid sequences 1465,
1466, and 1467 and a light chain with three CDRs including the
amino acid sequences1468, 1469, and 1470 respectively.
[0076] Flavivirus (73)
[0077] Exemplary anti-West Nile virus envelope protein E (WINE)
antibody include antibodies having a VH nucleotide sequence having
a VH amino acid sequence having SEQ ID NO: 1224 and a VL amino acid
sequence having SEQ ID NO: 1226.
[0078] Exemplary anti-West Nile virus envelope protein E (WINE)
antibody include antibodies having a VH nucleotide sequence having
SEQ ID NO: 1225 and a VL nucleotide sequence having SEQ ID NO:
1227.
[0079] In other embodiments the anti-West Nile virus envelope
protein E (WNE) antibody has a heavy chain with three CDRs
including the amino acid sequences 1244, 1245, and 1246 and a light
chain with three CDRs including the amino acid sequences 1247,
1248, and 1249 respectively.
[0080] CCR4 (65)
[0081] Exemplary anti-CC-chemokine receptor 4 (CCR4) antibody
include antibodies having a .sub.VH nucleotide sequence having SEQ
ID NO: 1329 and a .sub.VL nucleotide sequence having SEQ ID NO:
1331; a .sub.VH nucleotide sequence having SEQ ID NO: 1333 and a
V.sub.L nucleotide sequence having SEQ ID NO:1335; a .sub.VH
nucleotide sequence having SEQ ID NO: 1337 and a V.sub.L nucleotide
sequence having SEQ ID NO: 1192; a .sub.VH nucleotide sequence
having SEQ ID NO: 1341 and a V.sub.L nucleotide sequence having SEQ
ID NO: 1343; or a .sub.VH nucleotide sequence having SEQ ID NO:
1357 and a V.sub.L nucleotide sequence having SEQ ID NO:1359.
[0082] Exemplary anti-CC-chemokine receptor 4 (CCR4) antibody
include antibodies having a V.sub.H amino acid sequence having SEQ
ID NO: 1330 and a V.sub.L amino acid sequence having SEQ ID NO:
1332; a V.sub.H amino acid sequence having SEQ ID NO: 1334 and a
V.sub.L amino acid sequence having SEQ ID NO: 1336; a V.sub.H amino
acid sequence having SEQ ID NO: 1338 and a V.sub.L amino acid
sequence having SEQ ID NO: 1340; a V.sub.H amino acid sequence
having SEQ ID NO: 1342 and a V.sub.L amino acid sequence having SEQ
ID NO: 1344; or a V.sub.H amino acid sequence having SEQ ID NO:
1358 and a V.sub.L amino acid sequence having SEQ ID NO: 1360.
[0083] In other embodiments the anti-CC-chemokine receptor 4 (CCR4)
antibody has a heavy chain with three CDRs including the amino acid
sequences 1203, 1208, and 1211 and a light chain with three CDRs
including the amino acid sequences 1207, 1209, and 1216
respectively; or a heavy chain with three CDRs including the amino
acid sequences 1204, 1208, and 1212 and a light chain with three
CDRs including the amino acid sequences 1207, 1209, and 1217
respectively; or a heavy chain with three CDRs including the amino
acid sequences 1204, 1208, and 1213 and a light chain with three
CDRs including the amino acid sequences 1207, 1209, and 1217
respectively; or a heavy chain with three CDRs including the amino
acid sequences 1205, 1208, and 1214 and a light chain with three
CDRs including the amino acid sequences 1207, 1209, and 1218
respectively; or a heavy chain with three CDRs including the amino
acid sequences 1206, 1208, and 1210 and a light chain with three
CDRs including the amino acid sequences 1207, 1209, and 1220
respectively; or a heavy chain with three CDRs including the amino
acid sequences 1202, 1208, and 1210 and a light chain with three
CDRs including the amino acid sequences 1207, 1209, and 1219
respectively.
[0084] Human Immunoglobulin Heavy Chain Variable Region Germline
Gene VH1-69 (57)
[0085] Exemplary anti-human immunoglobulin heavy chain variable
region germline gene VH1-69 antibody include antibodies having a VH
nucleotide sequence having SEQ ID NO: 1153 and a VL nucleotide
sequence having SEQ ID NO: 1155; or a VH nucleotide sequence having
SEQ ID NO: 1163 and a VL nucleotide sequence having SEQ ID
NO:1155.
[0086] Exemplary anti-human immunoglobulin heavy chain variable
region germline gene VH1-69 antibody include antibodies having a
V.sub.H amino acid sequence having SEQ ID NO: 1154 and a V.sub.L
amino acid sequence having SEQ ID NO: 1156; or a V.sub.H amino acid
sequence having SEQ ID NO: 1164 and a V.sub.L amino acid sequence
having SEQ ID NO: 1156.
[0087] In other embodiments the anti-human immunoglobulin heavy
chain variable region germline gene VH1-69 antibody has a heavy
chain with three CDRs including the amino acid sequences 1157,
1158, and 1159 and a light chain with three CDRs including the
amino acid sequences 1160, 1161, and 1162 respectively.
[0088] Influenza (49)
[0089] Exemplary anti-influenza antibody include antibodies having
a VH nucleotide sequence having SEQ ID NO: 981 and a VL nucleotide
sequence having SEQ ID NO: 983; a VH nucleotide sequence having SEQ
ID NO: 985 and a VL nucleotide sequence having SEQ ID NO: 989; a VH
nucleotide sequence having SEQ ID NO: 987 and a VL nucleotide
sequence having SEQ ID NO: 991; a VH nucleotide sequence having SEQ
ID NO: 993 and a VL nucleotide sequence having SEQ ID NO: 997; a VH
nucleotide sequence having SEQ ID NO: 995 and a VK nucleotide
sequence having SEQ ID NO: 999; a VH nucleotide sequence having SEQ
ID NO: 1001 and a VL nucleotide sequence having SEQ ID NO: 1005; a
VH nucleotide sequence having SEQ ID NO: 1003 and a VL nucleotide
sequence having SEQ ID NO: 1007; a VH nucleotide sequence having
SEQ ID NO: 1009 and a VL nucleotide sequence having SEQ ID NO:
1011; a VH nucleotide sequence having SEQ ID NO: 1013 and a VL
nucleotide sequence having SEQ ID NO: 1015; and a VH nucleotide
sequence having SEQ ID NO: 1017 and a VK nucleotide sequence having
SEQ ID NO: 1019; a VH nucleotide sequence having SEQ ID NO: 1020
and a VL nucleotide sequence having SEQ ID NO: 1022.
[0090] Exemplary anti-influenza antibody include antibodies having
a VH amino acid sequence having SEQ ID NO: 982 and a VL amino acid
sequence having SEQ ID NO: 984; a VH amino acid sequence having SEQ
ID NO: 986 and a VL amino acid sequence having SEQ ID NO: 988; a VH
amino acid sequence having SEQ ID NO: 986 and a VL amino acid
sequence having SEQ ID NO: 990; a VH amino acid sequence having SEQ
ID NO: 992 and a VL amino acid sequence having SEQ ID NO: 994; a VH
amino acid sequence having SEQ ID NO: 992 and a VK amino acid
sequence having SEQ ID NO: 996; a VH amino acid sequence having SEQ
ID NO: 998 and a VL amino acid sequence having SEQ ID NO: 1000; a
VH amino acid sequence having SEQ ID NO: 998 and a VL amino acid
sequence having SEQ ID NO: 1002; a VH amino acid sequence having
SEQ ID NO: 1004 and a VL amino acid sequence having SEQ ID NO:
1006; a VH amino acid sequence having SEQ ID NO: 1008 and a VL
amino acid sequence having SEQ ID NO: 1010; a VH amino acid
sequence having SEQ ID NO: 1012 and a VK amino acid sequence having
SEQ ID NO: 1014; and a VH amino acid sequence having SEQ ID NO:
1016 and a VL amino acid sequence having SEQ ID NO: 1018.
[0091] In other embodiments the anti-influenza antibody has a heavy
chain with three CDRs including the amino acid sequences of 1023,
1031, and 1039 and a light chain with three CDRs including the
amino acid sequences 1047, 1059, and 1071; a heavy chain with three
CDRs including the amino acid sequences of 1023, 1032, and 1040 and
a light chain with three CDRs including the amino acid sequences
1048, 1060, and 1072; a heavy chain with three CDRs including the
amino acid sequences of 1025, 1032, and 1040 and a light chain with
three CDRs including the amino acid sequences 1057, 1069, and 1081;
a heavy chain with three CDRs including the amino acid sequences of
1026, 1033, and 1041 and a light chain with three CDRs including
the amino acid sequences 1049, 1061, and 1073; a heavy chain with
three CDRs including the amino acid sequences of 1026, 1033, and
1041 and a light chain with three CDRs including the amino acid
sequences 1054, 1066, and 1078; a heavy chain with three CDRs
including the amino acid sequences of 1027, 1034, and 1042 and a
light chain with three CDRs including the amino acid sequences
1050, 1062, and 1074; a heavy chain with three CDRs including the
amino acid sequences of 1027, 1034, and 1042 and a light chain with
three CDRs including the amino acid sequences 1056, 1068, and 1080;
a heavy chain with three CDRs including the amino acid sequences of
1028, 1035, and 1043 and a light chain with three CDRs including
the amino acid sequences 1051, 1063, and 1065; a heavy chain with
three CDRs including the amino acid sequences of 1028, 1036, and
1044 and a light chain with three CDRs including the amino acid
sequences 1052, 1064, and 1076; a heavy chain with three CDRs
including the amino acid sequences of 1029, 1037, and 1045 and a
light chain with three CDRs including the amino acid sequences
1053, 1065, and 1077; or a heavy chain with three CDRs including
the amino acid sequences of 1030, 1038, and 1046 and a light chain
with three CDRs including the amino acid sequences 1058, 1070, and
1082.
[0092] Influenza (78)
[0093] Exemplary anti-influenza antibodies include antibodies
having containing a .sub.VH nucleotide sequence having SEQ ID NO:
397 and a nucleotide sequence having SEQ ID NO: 398; a .sub.VH
nucleotide sequence having SEQ ID NO: 399 and a V.sub.L nucleotide
sequence having SEQ ID NO:400; a .sub.VH nucleotide sequence having
SEQ ID NO: 401 and a V.sub.L nucleotide sequence having SEQ ID NO:
402; a .sub.VH nucleotide sequence having SEQ ID NO: 403 and a
.sub.VL nucleotide sequence having SEQ ID NO: 404; or a .sub.VH
nucleotide sequence having SEQ ID NO: 405 and a .sub.VL nucleotide
sequence having SEQ ID NO:406; or a .sub.VH nucleotide sequence
having SEQ ID NO: 407 and a .sub.VL nucleotide sequence having SEQ
ID NO:408; or a .sub.VH nucleotide sequence having SEQ ID NO: 409
and a .sub.VL nucleotide sequence having SEQ ID NO:410; or a
.sub.VH nucleotide sequence having SEQ ID NO: 411 and a .sub.VH
nucleotide sequence having SEQ ID NO:412; or a .sub.VH nucleotide
sequence having SEQ ID NO: 413 and a .sub.VL nucleotide sequence
having SEQ ID NO:414; or a .sub.VH nucleotide sequence having SEQ
ID NO: 415 and a .sub.VL nucleotide sequence having SEQ ID NO:416;
or a .sub.VH nucleotide sequence having SEQ ID NO: 417 and a
.sub.VL nucleotide sequence having SEQ ID NO:418; or a .sub.VH
nucleotide sequence having SEQ ID NO: 419 and a .sub.VL nucleotide
sequence having SEQ ID NO:420; or a .sub.VH nucleotide sequence
having SEQ ID NO: 421 and a .sub.VL nucleotide sequence having SEQ
ID NO:422; or a .sub.VH nucleotide sequence having SEQ ID NO: 423
and a .sub.VL nucleotide sequence having SEQ ID NO:424; or a
.sub.VH nucleotide sequence having SEQ ID NO: 425 and a .sub.VL
nucleotide sequence having SEQ ID NO:426; or a .sub.VH nucleotide
sequence having SEQ ID NO: 427 and a .sub.VL nucleotide sequence
having SEQ ID NO:428; or a .sub.VH nucleotide sequence having SEQ
ID NO: 429 and a .sub.VL nucleotide sequence having SEQ ID NO:430;
or a .sub.VH nucleotide sequence having SEQ ID NO: 431 and a
.sub.VL nucleotide sequence having SEQ ID NO:432; or a .sub.VH
nucleotide sequence having SEQ ID NO: 433 and a .sub.VL nucleotide
sequence having SEQ ID NO:434; or a .sub.VH nucleotide sequence
having SEQ ID NO: 435 and a .sub.VL nucleotide sequence having SEQ
ID NO:436; or a .sub.VH nucleotide sequence having SEQ ID NO: 437
and a .sub.VL nucleotide sequence having SEQ ID NO:438; or a
.sub.VH nucleotide sequence having SEQ ID NO: 439 and a .sub.VL
nucleotide sequence having SEQ ID NO:440; or a .sub.VH nucleotide
sequence having SEQ ID NO: 441 and a .sub.VL nucleotide sequence
having SEQ ID NO:442; ; or a VH nucleotide sequence having SEQ ID
NO: 541 and a VL nucleotide sequence having SEQ ID NO: 542; or a VH
nucleotide sequence having SEQ ID NO: 543 and a VL nucleotide
sequence having SEQ ID NO: 544; or a VH nucleotide sequence having
SEQ ID NO: 545 and a VL nucleotide sequence having SEQ ID NO: 546;
or a VH nucleotide sequence having SEQ ID NO: 547 and a VL
nucleotide sequence having SEQ ID NO: 548; or a VH nucleotide
sequence having SEQ ID NO: 549 and a VL nucleotide sequence having
SEQ ID NO: 550; or a VH nucleotide sequence having SEQ ID NO: 551
and a VL nucleotide sequence having SEQ ID NO: 552; or a VH
nucleotide sequence having SEQ ID NO: 553 and a VL nucleotide
sequence having SEQ ID NO: 554; or a VH nucleotide sequence having
SEQ ID NO: 555 and a VL nucleotide sequence having SEQ ID NO: 556;
or a VH nucleotide sequence having SEQ ID NO: 557 and a VL
nucleotide sequence having SEQ ID NO: 558; or a VH nucleotide
sequence having SEQ ID NO: 559 and a VL nucleotide sequence having
SEQ ID NO: 560; or a VH nucleotide sequence having SEQ ID NO: 561
and a VL nucleotide sequence having SEQ ID NO: 562; or a VH
nucleotide sequence having SEQ ID NO: 563 and a VL nucleotide
sequence having SEQ ID NO: 564; or a VH nucleotide sequence having
SEQ ID NO: 565 and a VL nucleotide sequence having SEQ ID NO: 566;
or a VH nucleotide sequence having SEQ ID NO: 567 and a VL
nucleotide sequence having SEQ ID NO: 568; or a VH nucleotide
sequence having SEQ ID NO: 569 and a VL nucleotide sequence having
SEQ ID NO: 570; or a VH nucleotide sequence having SEQ ID NO: 571
and a VL nucleotide sequence having SEQ ID NO: 572; or a VH
nucleotide sequence having SEQ ID NO: 573 and a VL nucleotide
sequence having SEQ ID NO: 574; or a VH nucleotide sequence having
SEQ ID NO: 575 and a VL nucleotide sequence having SEQ ID NO: 576;
or a VH nucleotide sequence having SEQ ID NO: 577 and a VL
nucleotide sequence having SEQ ID NO: 578; or a VH nucleotide
sequence having SEQ ID NO: 579 and a VL nucleotide sequence having
SEQ ID NO: 580; or a VH nucleotide sequence having SEQ ID NO: 581
and a VL nucleotide sequence having SEQ ID NO: 582; or a VH
nucleotide sequence having SEQ ID NO: 583 and a VL nucleotide
sequence having SEQ ID NO: 584; or a VH nucleotide sequence having
SEQ ID NO: 585 and a VL nucleotide sequence having SEQ ID NO: 586;
or a VH nucleotide sequence having SEQ ID NO: 587 and a VL
nucleotide sequence having SEQ ID NO: 588; or a VH nucleotide
sequence having SEQ ID NO: 589 and a VL nucleotide sequence having
SEQ ID NO: 590; or a VH nucleotide sequence having SEQ ID NO: 591
and a VL nucleotide sequence having SEQ ID NO: 592; or a VH
nucleotide sequence having SEQ ID NO: 593 and a VL nucleotide
sequence having SEQ ID NO: 594; or a VH nucleotide sequence having
SEQ ID NO: 595 and a VL nucleotide sequence having SEQ ID NO: 596;
or a VH nucleotide sequence having SEQ ID NO: 597 and a VL
nucleotide sequence having SEQ ID NO: 598; or a VH nucleotide
sequence having SEQ ID NO: 599 and a VL nucleotide sequence having
SEQ ID NO: 600.
[0094] Exemplary anti-influenza antibodies antibody include
antibodies having containing a .sub.VH amino acid sequence having
SEQ ID NO: 469 and a .sub.VL amino acid sequence having SEQ ID NO:
470; a .sub.VH amino acid having SEQ ID NO: 471 and a V.sub.L
polypeptide sequence having SEQ ID NO:472; a .sub.VH amino acid
sequence having SEQ ID NO: 473 and a V.sub.L amino acid sequence
having SEQ ID NO: 474; a .sub.VH amino acid sequence having SEQ ID
NO: 475 and a .sub.VL amino acid sequence having SEQ ID NO: 476; or
a .sub.VH nucleotide sequence having SEQ ID NO: 477 and a .sub.VL
nucleotide sequence having SEQ ID NO:478; a .sub.VH amino acid
sequence having SEQ ID NO: 479 and a .sub.VL amino acid sequence
having SEQ ID NO: 480; a .sub.VH amino acid sequence having SEQ ID
NO: 481 and a .sub.VL amino acid sequence having SEQ ID NO: 482; a
.sub.VH amino acid sequence having SEQ ID NO: 483 and a .sub.VL
amino acid sequence having SEQ ID NO: 484; a .sub.VH amino acid
sequence having SEQ ID NO: 485 and a .sub.VL amino acid sequence
having SEQ ID NO: 486; a .sub.VH amino acid sequence having SEQ ID
NO: 487 and a .sub.VL amino acid sequence having SEQ ID NO: 488; a
.sub.VH amino acid sequence having SEQ ID NO: 489 and a .sub.VL
amino acid sequence having SEQ ID NO: 490; a .sub.VH amino acid
sequence having SEQ ID NO: 491 and a .sub.VL amino acid sequence
having SEQ ID NO: 492; a .sub.VH amino acid sequence having SEQ ID
NO: 493 and a .sub.VL amino acid sequence having SEQ ID NO: 494; a
.sub.VH amino acid sequence having SEQ ID NO: 495 and a .sub.VL
amino acid sequence having SEQ ID NO: 496; a .sub.VH amino acid
sequence having SEQ ID NO: 497 and a .sub.VL amino acid sequence
having SEQ ID NO: 498; a .sub.VH amino acid sequence having SEQ ID
NO: 499 and a .sub.VL amino acid sequence having SEQ ID NO: 500; a
.sub.VH amino acid sequence having SEQ ID NO: 501 and a amino acid
sequence having SEQ ID NO: 502; a .sub.VH amino acid sequence
having SEQ ID NO: 503 and a .sub.VL amino acid sequence having SEQ
ID NO: 504; a .sub.VH amino acid sequence having SEQ ID NO: 505 and
a .sub.VL amino acid sequence having SEQ ID NO: 506; a .sub.VH
amino acid sequence having SEQ ID NO: 507 and a .sub.VL amino acid
sequence having SEQ ID NO: 508; a .sub.VH amino acid sequence
having SEQ ID NO: 509 and a amino acid sequence having SEQ ID NO:
510; a .sub.VH amino acid sequence having SEQ ID NO: 511 and a
.sub.VL amino acid sequence having SEQ ID NO: 512; a .sub.VH amino
acid sequence having SEQ ID NO: 513 and a .sub.VL amino acid
sequence having SEQ ID NO: 514; a .sub.VH amino acid sequence
having SEQ ID NO: 515 and a .sub.VL amino acid sequence having SEQ
ID NO: 516; a .sub.VH amino acid sequence having SEQ ID NO: 517 and
a .sub.VL amino acid sequence having SEQ ID NO: 518; a .sub.VH
amino acid sequence having SEQ ID NO: 519 and a .sub.VL amino acid
sequence having SEQ ID NO: 520; a .sub.VH amino acid sequence
having SEQ ID NO: 521 and a amino acid sequence having SEQ ID NO:
522; a .sub.VH amino acid sequence having SEQ ID NO: 523 and a
.sub.VL amino acid sequence having SEQ ID NO: 524; a .sub.VH amino
acid sequence having SEQ ID NO: 525 and a .sub.VL amino acid
sequence having SEQ ID NO: 526; a .sub.VH amino acid sequence
having SEQ ID NO: 527 and a .sub.VL amino acid sequence having SEQ
ID NO: 528; a .sub.VH amino acid sequence having SEQ ID NO: 529 and
a amino acid sequence having SEQ ID NO: 530; a .sub.VH amino acid
sequence having SEQ ID NO: 531 and a .sub.VL amino acid sequence
having SEQ ID NO: 532; a .sub.VH amino acid sequence having SEQ ID
NO: 533 and a .sub.VL amino acid sequence having SEQ ID NO: 534; a
.sub.VH amino acid sequence having SEQ ID NO: 535 and a .sub.VL
amino acid sequence having SEQ ID NO: 536; a .sub.VH amino acid
sequence having SEQ ID NO: 537 and a .sub.VL amino acid sequence
having SEQ ID NO: 538; a .sub.VH amino acid sequence having SEQ ID
NO: 539 and a amino acid sequence having SEQ ID NO: 540 a VH amino
acid sequence having SEQ ID NO: 601 and a VL amino acid sequence
having SEQ ID NO: 602 a VH amino acid sequence having SEQ ID NO:
603 and a VL amino acid sequence having SEQ ID NO: 604 a VH amino
acid sequence having SEQ ID NO: 605 and a VL amino acid sequence
having SEQ ID NO: 606 a VH amino acid sequence having SEQ ID NO:
607 and a VL amino acid sequence having SEQ ID NO: 608 a VH amino
acid sequence having SEQ ID NO: 609 and a VL amino acid sequence
having SEQ ID NO: 610 a VH amino acid sequence having SEQ ID NO:
611 and a VL amino acid sequence having SEQ ID NO: 612 a VH amino
acid sequence having SEQ ID NO: 613 and a VL amino acid sequence
having SEQ ID NO: 614 a VH amino acid sequence having SEQ ID NO:
615 and a VL amino acid sequence having SEQ ID NO: 616 a VH amino
acid sequence having SEQ ID NO: 617 and a VL amino acid sequence
having SEQ ID NO: 618 a VH amino acid sequence having SEQ ID NO:
619 and a VL amino acid sequence having SEQ ID NO: 620 a VH amino
acid sequence having SEQ ID NO: 621 and a VL amino acid sequence
having SEQ ID NO: 622 a VH amino acid sequence having SEQ ID NO:
623 and a VL amino acid sequence having SEQ ID NO: 624 a VH amino
acid sequence having SEQ ID NO: 625 and a VL amino acid sequence
having SEQ ID NO: 626 a VH amino acid sequence having SEQ ID NO:
627 and a VL amino acid sequence having SEQ ID NO: 628 a VH amino
acid sequence having SEQ ID NO: 629 and a VL amino acid sequence
having SEQ ID NO: 630 a VH amino acid sequence having SEQ ID NO:
631 and a VL amino acid sequence having SEQ ID NO: 632 a VH amino
acid sequence having SEQ ID NO: 633 and a VL amino acid sequence
having SEQ ID NO: 634 a VH amino acid sequence having SEQ ID NO:
635 and a VL amino acid sequence having SEQ ID NO: 636 a VH amino
acid sequence having SEQ ID NO: 637 and a VL amino acid sequence
having SEQ ID NO: 638 a VH amino acid sequence having SEQ ID NO:
639 and a VL amino acid sequence having SEQ ID NO: 640 a VH amino
acid sequence having SEQ ID NO: 641 and a VL amino acid sequence
having SEQ ID NO: 642 a VH amino acid sequence having SEQ ID NO:
643 and a VL amino acid sequence having SEQ ID NO: 644 a VH amino
acid sequence having SEQ ID NO: 645 and a VL amino acid sequence
having SEQ ID NO: 646 a VH amino acid sequence having SEQ ID NO:
647 and a VL amino acid sequence having SEQ ID NO: 648 a VH amino
acid sequence having SEQ ID NO: 649 and a VL amino acid sequence
having SEQ ID NO: 650 a VH amino acid sequence having SEQ ID NO:
651 and a VL amino acid sequence having SEQ ID NO: 652 a VH amino
acid sequence having SEQ ID NO: 653 and a VL amino acid sequence
having SEQ ID NO: 654 a VH amino acid sequence having SEQ ID NO:
655 and a VL amino acid sequence having SEQ ID NO: 656 a VH amino
acid sequence having SEQ ID NO: 657 and a VL amino acid sequence
having SEQ ID NO: 658 a VH amino acid sequence having SEQ ID NO:
659 and a VL amino acid sequence having SEQ ID NO: 660.
[0095] In other embodiments the anti-influenza antibodies antibody
has a heavy chain with three CDRs including the amino acid
sequences SEQ ID NO: 1, 37, 73 respectively and a light chain with
three CDRs including the amino acid sequences 109, 145, 181
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 2, 38, 74 respectively and a light chain with three
CDRs comprising the amino acid sequences 110, 146, 182,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 3, 39, 75 respectively and a light chain with three
CDRs comprising the amino acid sequences 111, 147, 183,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 4, 40, 76 respectively and a light chain with three
CDRs comprising the amino acid sequences 112, 148, 184,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 5, 41, 77 respectively and a light chain with three
CDRs comprising the amino acid sequences 113, 149, 185,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 6, 42, 78 respectively and a light chain with three
CDRs comprising the amino acid sequences 114, 150, 186,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 7, 43, 79 respectively and a light chain with three
CDRs comprising the amino acid sequences 115, 151, 187,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 8, 44, 80 respectively and a light chain with three
CDRs comprising the amino acid sequences 116, 152, 188,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 9, 45, 81 respectively and a light chain with three
CDRs comprising the amino acid sequences 117, 153, 189,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 10, 46, 82 respectively and a light chain with three
CDRs comprising the amino acid sequences 118, 154, 190,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 11, 47, 83 respectively and a light chain with three
CDRs comprising the amino acid sequences 119, 155, 191,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 12, 48, 84 respectively and a light chain with three
CDRs comprising the amino acid sequences 120, 156, 192,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 13, 49, 85 respectively and a light chain with three
CDRs comprising the amino acid sequences 121, 157, 193,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 14, 50, 86 respectively and a light chain with three
CDRs comprising the amino acid sequences 122, 158, 194,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 15, 51, 87 respectively and a light chain with three
CDRs comprising the amino acid sequences 123, 159, 195,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 16, 52, 88 respectively and a light chain with three
CDRs comprising the amino acid sequences 124, 160, 196,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 17, 53, 89 respectively and a light chain with three
CDRs comprising the amino acid sequences 125, 161, 197,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 18, 54, 90 respectively and a light chain with three
CDRs comprising the amino acid sequences 126, 162, 198,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 19, 55, 91 respectively and a light chain with three
CDRs comprising the amino acid sequences 127, 163, 199,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 20, 56, 92 respectively and a light chain with three
CDRs comprising the amino acid sequences 128, 164, 200,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 21, 57, 93 respectively and a light chain with three
CDRs comprising the amino acid sequences 129, 165, 201,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 22, 58, 94 respectively and a light chain with three
CDRs comprising the amino acid sequences 130, 166, 202,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 23, 59, 95 respectively and a light chain with three
CDRs comprising the amino acid sequences 131, 167, 203,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 24, 60, 96 respectively and a light chain with three
CDRs comprising the amino acid sequences 132, 168, 204,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 25, 61, 95 respectively and a light chain with three
CDRs comprising the amino acid sequences 133, 169, 205,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 26, 62, 96 respectively and a light chain with three
CDRs comprising the amino acid sequences 134, 170, 206,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 27, 63, 97 respectively and a light chain with three
CDRs comprising the amino acid sequences 135, 171, 207,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 28, 64, 98 respectively and a light chain with three
CDRs comprising the amino acid sequences 136, 172, 208,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 29, 65, 99 respectively and a light chain with three
CDRs comprising the amino acid sequences 137, 173, 209,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 30, 66, 100 respectively and a light chain with
three CDRs comprising the amino acid sequences 138, 174, 210,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 31, 67, 101 respectively and a light chain with
three CDRs comprising the amino acid sequences 139, 175, 211,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 32, 68, 102 respectively and a light chain with
three CDRs comprising the amino acid sequences 140, 176, 212,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 33, 69, 103 respectively and a light chain with
three CDRs comprising the amino acid sequences 141, 177, 213,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 34, 70, 104 respectively and a light chain with
three CDRs comprising the amino acid sequences 142, 178, 214,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 35, 71, 105 respectively and a light chain with
three CDRs comprising the amino acid sequences 143, 179, 215,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 36, 72, 106 respectively and a light chain with
three CDRs comprising the amino acid sequences 144, 180, 216,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 217, 247, 277 respectively and a light chain with
three CDRs comprising the amino acid sequences 307, 337, 367,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 218, 248, 278 respectively and a light chain with
three CDRs comprising the amino acid sequences 308, 338, 368,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 219, 249, 279 respectively and a light chain with
three CDRs comprising the amino acid sequences 309, 339, 369,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 220, 250, 280 respectively and a light chain with
three CDRs comprising the amino acid sequences 310, 340, 370,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 221, 251, 281 respectively and a light chain with
three CDRs comprising the amino acid sequences 311, 341, 371,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 222, 252, 282 respectively and a light chain with
three CDRs comprising the amino acid sequences 312, 342, 372,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 223, 253, 283 respectively and a light chain with
three CDRs comprising the amino acid sequences 313, 343, 373,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 224, 254, 284 respectively and a light chain with
three CDRs comprising the amino acid sequences 314, 344, 374,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 225, 255, 285 respectively and a light chain with
three CDRs comprising the amino acid sequences 315, 345, 375,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 226, 256, 286 respectively and a light chain with
three CDRs comprising the amino acid sequences 316, 346, 376,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 227, 257, 287 respectively and a light chain with
three CDRs comprising the amino acid sequences 317, 347, 377,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 228, 258, 288 respectively and a light chain with
three CDRs comprising the amino acid sequences 318, 348, 378,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 229, 259, 289 respectively and a light chain with
three CDRs comprising the amino acid sequences 319, 349, 379,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 230, 260, 290 respectively and a light chain with
three CDRs comprising the amino acid sequences 320, 350, 380,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 231, 261, 291 respectively and a light chain with
three CDRs comprising the amino acid sequences 321, 351, 381,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 232, 262, 292 respectively and a light chain with
three CDRs comprising the amino acid sequences 322, 352, 382,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 233, 263, 293 respectively and a light chain with
three CDRs comprising the amino acid sequences 323, 353, 383,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 234, 273, 294 respectively and a light chain with
three CDRs comprising the amino acid sequences 324, 354, 384,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 235, 274, 295 respectively and a light chain with
three CDRs comprising the amino acid sequences 325, 355, 385,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 236, 275, 296 respectively and a light chain with
three CDRs comprising the amino acid sequences 326, 356, 386,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 237, 276, 297 respectively and a light chain with
three CDRs comprising the amino acid sequences 327, 357, 387,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 237, 277, 298 respectively and a light chain with
three CDRs comprising the amino acid sequences 328, 358, 388,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 238, 278, 299 respectively and a light chain with
three CDRs comprising the amino acid sequences 329, 359, 389,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 239, 279, 300 respectively and a light chain with
three CDRs comprising the amino acid sequences 330, 360, 390,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 240, 280, 301 respectively and a light chain with
three CDRs comprising the amino acid sequences 331, 361, 391,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 241, 281, 302 respectively and a light chain with
three CDRs comprising the amino acid sequences 332, 362, 392,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 242, 282, 303 respectively and a light chain with
three CDRs comprising the amino acid sequences 333, 363, 393,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 243, 283, 304 respectively and a light chain with
three CDRs comprising the amino acid sequences 334, 364, 394,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 244, 284, 305 respectively and a light chain with
three CDRs comprising the amino acid sequences 335, 365, 395,
respectively; or a heavy chain with three CDRs comprising the amino
acid sequences 245, 285, 306 respectively and a light chain with
three CDRs comprising the amino acid sequences 336, 366, 396,
respectively.
[0096] Other anti-influenza antibodies include those having the
amino acid or nucleic acid sequences shown in the below Table
1.
TABLE-US-00001 TABLE 1A Antibody 3I14 Variable Region nucleic acid
sequences V.sub.H chain of 3I14 (SEQ ID NO: 1665)
CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGGCA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATT
ATATCATTTGATGGAAGTAAAAAATATTATGCAAACTCCGTGAAGGGCCG
ATCCACCATCTCCAGAGACAATTCCAAGAACACGCTGTCTCTGCAAATGA
ACAGCCTGGGACCTGAGGACACGGCTCTATATTACTGTGCGAAACTGCCC
TCCCCGTATTACTTTGATAGTCGGTTCGTGTGGGTCGCCGCCAGCGCATT
TCACTTCTGGGGCCAGGGAATCCTGGTCACCGTCTCTTCA V.sub.L chain of 3I14 (SEQ
ID NO: 1667) AATTTTATGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGCTCTGGAAGCAGCTCCAACATCGGAGGTAATACTG
TACACTGGTTCCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT
ACTAATAGTCTGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAA
GTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG
AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTAAATGGTCAGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA
TABLE-US-00002 TABLE 1B Antibody 3I14 Variable Region amino acid
sequences V.sub.H chain of 3I14 (SEQ ID NO: 1666)
QVQLLESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAI
ISFDGSKKYYANSVKGRSTISRDNSKNTLSLQMNSLGPEDTALYYCAKLP
SPYYFDSRFVWVAASAFHFWGQGILVTVSS V.sub.L chain of 3I14 (SEQ ID NO:
1668) NFMLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWFQQLPGTAPKLLIY
TNSLRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGQV FGGGTKLTVL
TABLE-US-00003 TABLE 1C Antibody 3I14V.sub.LD94N Variable Region
nucleic acid sequence V.sub.L chain of 3I14V.sub.LD94N (SEQ ID NO:
1669) AATTTTATGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGG
GTCACCATCTCTTGCTCTGGAAGCAGCTCCAACATCGGAGGTAATACTGTA
CACTGGTTCCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATACT
AATAGTCTGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCT
GATTATTACTGTGCAGCATGGGATAACAGCCTAAATGGTCAGGTGTTCGGC
GGAGGGACCAAGCTGACCGTCCTA
TABLE-US-00004 TABLE 1C Antibody 3I14V.sub.LD94N Variable Region
amino acid sequence V.sub.L chain of 3I14V.sub.LD94N (SEQ ID NO:
1670) NFMLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWFQQLPGTAPKLLIYT
NSLRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDNSLNGQVFG GGTKLTVL
[0097] The amino acid sequences of the heavy and light chain
complementary determining regions of the 3I14 and 3I14V.sub.LD94N
neutralizing influenza antibodies are shown in the below Table
2
TABLE-US-00005 TABLE 2 HCDR1 GFTFSNYG (SEQ ID NO: 1671) HCDR2
ISFDGSKK (SEQ ID NO: 1672) HCDR3 CAKLPSPYYFDSRFVWVA (SEQ ID NO:
1673) ASAFHFW LCDR1 SSNIGGNT (SEQ ID NO: 1674) LCDR2 TNS (SEQ ID
NO: 1675) LCDR3 CAAWDDSLNGQVF (SEQ ID NO: 1676) 3I14V.sub.LD94N
CAAWDNSLNGQVF (SEQ ID NO: 1677) LCDR3
[0098] Other binding domain than scFv can also be used for
predefined targeting of lymphocytes, such as camelid single-domain
antibody fragments or receptor ligands, antibody binding domains,
antibody hypervariable loops or CDRs as non limiting examples.
[0099] In a preferred embodiment said transmembrane domain further
comprises a stalk region between said extracellular ligand-binding
domain and said transmembrane domain. The term "stalk region" used
herein generally means any oligo- or polypeptide that functions to
link the transmembrane domain to the extracellular ligand-binding
domain. In particular, stalk region are used to provide more
flexibility and accessibility for the extracellular ligand-binding
domain. A stalk region may comprise up to 300 amino acids,
preferably 10 to 100 amino acids and most preferably 25 to 50 amino
acids. Stalk region may be derived from all or part of naturally
occurring molecules, such as from all or part of the extracellular
region of CD8, CD4 or CD28, or from all or part of an antibody
constant region. Alternatively the stalk region may be a synthetic
sequence that corresponds to a naturally occurring stalk sequence,
or may be an entirely synthetic stalk sequence. In a preferred
embodiment said stalk region is a part of human CD8 alpha chain
[0100] The signal transducing domain or intracellular signaling
domain of the CAR of the invention is responsible for intracellular
signaling following the binding of extracellular ligand binding
domain to the target resulting in the activation of the immune cell
and immune response. In other words, the signal transducing domain
is responsible for the activation of at least one of the normal
effector functions of the immune cell in which the CAR is
expressed. For example, the effector function of a T cell can be a
cytolytic activity or helper activity including the secretion of
cytokines. Thus, the term "signal transducing domain" refers to the
portion of a protein which transduces the effector signal function
signal and directs the cell to perform a specialized function.
[0101] Signal transduction domain comprises two distinct classes of
cytoplasmic signaling sequence, those that initiate
antigen-dependent primary activation, and those that act in an
antigen-independent manner to provide a secondary or co-stimulatory
signal. Primary cytoplasmic signaling sequence can comprise
signaling motifs which are known as immunoreceptor tyrosine-based
activation motifs of ITAMs. ITAMs are well defined signaling motifs
found in the intracytoplasmic tail of a variety of receptors that
serve as binding sites for syk/zap70 class tyrosine kinases.
Examples of ITAM used in the invention can include as non limiting
examples those derived from TCR zeta, FcR gamma, FcR beta, FcR
epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b
and CD66d. In a preferred embodiment, the signaling transducing
domain of the CAR can comprise the CD3 zeta signaling domain, or
the intracytoplasmic domain of the Fc epsilon RI beta or gamma
chains. In another preferred embodiment, the signaling is provided
by CD3 zeta together with co-stimulation provided by CD28 and a
tumor necrosis factor receptor (TNFr), such as 4-1BB or OX40), for
example.
[0102] In particular embodiment the intracellular signaling domain
of the CAR of the present invention comprises a co-stimulatory
signal molecule. In some embodiments the intracellular signaling
domain contains 2, 3, 4 or more co-stimulatory molecules in tandem.
A co-stimulatory molecule is a cell surface molecule other than an
antigen receptor or their ligands that is required for an efficient
immune response.
[0103] "Co-stimulatory ligand" refers to a molecule on an antigen
presenting cell that specifically binds a cognate co-stimulatory
molecule on a T-cell, thereby providing a signal which, in addition
to the primary signal provided by, for instance, binding of a
TCR/CD3 complex with an MHC molecule loaded with peptide, mediates
a T cell response, including, but not limited to, proliferation
activation, differentiation and the like. A co-stimulatory ligand
can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86),
PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand
(ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70,
CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6,
ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor
and a ligand that specifically binds with B7-H3. A co-stimulatory
ligand also encompasses, inter alia, an antibody that specifically
binds with a co-stimulatory molecule present on a T cell, such as
but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1,
ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,
LTGHT, NKG2C, B7-H3, a ligand that specifically binds with
CD83.
[0104] A "co-stimulatory molecule" refers to the cognate binding
partner on a T-cell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the cell,
such as, but not limited to proliferation. Co-stimulatory molecules
include, but are not limited to an MHC class 1 molecule, BTLA and
Toll ligand receptor. Examples of costimulatory molecules include
CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the
like. The In another particular embodiment, said signal transducing
domain is a TNFR-associated Factor 2 (TRAF2) binding motifs,
intracytoplasmic tail of costimulatory TNFR member family.
Cytoplasmic tail of costimulatory TNFR family member contains TRAF2
binding motifs consisting of the major conserved motif
(P/S/A)X(Q/E)E) or the minor motif (PXQXXD), wherein X is any amino
acid. TRAF proteins are recruited to the intracellular tails of
many TNFRs in response to receptor trimerization.
[0105] The distinguishing features of appropriate transmembrane
polypeptides comprise the ability to be expressed at the surface of
an immune cell, in particular lymphocyte cells or Natural killer
(NK) cells, and to interact together for directing cellular
response of immune cell against a predefined target cell. The
different transmembrane polypeptides of the CAR of the present
invention comprising an extracellular ligand-biding domain and/or a
signal transducing domain interact together to take part in signal
transduction following the binding with a target ligand and induce
an immune response. The transmembrane domain can be derived either
from a natural or from a synthetic source. The transmembrane domain
can be derived from any membrane-bound or transmembrane
protein.
[0106] The term "a part of" used herein refers to any subset of the
molecule, that is a shorter peptide. Alternatively, amino acid
sequence functional variants of the polypeptide can be prepared by
mutations in the DNA which encodes the polypeptide. Such variants
or functional variants include, for example, deletions from, or
insertions or substitutions of, residues within the amino acid
sequence. Any combination of deletion, insertion, and substitution
may also be made to arrive at the final construct, provided that
the final construct possesses the desired activity, especially to
exhibit a specific anti-target cellular immune activity. The
functionality of the CAR of the invention within a host cell is
detectable in an assay suitable for demonstrating the signaling
potential of said CAR upon binding of a particular target. Such
assays are available to the skilled person in the art. For example,
this assay allows the detection of a signaling pathway, triggered
upon binding of the target, such as an assay involving measurement
of the increase of calcium ion release, intracellular tyrosine
phosphorylation, inositol phosphate turnover, or interleukin (IL)
2, interferon .gamma., GM-CSF, IL-3, IL-4 production thus
effected.
[0107] Cells
[0108] Embodiments of the invention include cells that express a
CAR (i.e, CARTS). The cell may be of any kind, including an immune
cell capable of expressing the CAR for cancer therapy or a cell,
such as a bacterial cell, that harbors an expression vector that
encodes the CAR. As used herein, the terms "cell," "cell line," and
"cell culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid sequence, "host cell" refers to a
eukaryotic cell that is capable of replicating a vector and/or
expressing a heterologous gene encoded by a vector. A host cell
can, and has been, used as a recipient for vectors. A host cell may
be "transfected" or "transformed," which refers to a process by
which exogenous nucleic acid is transferred or introduced into the
host cell. A transformed cell includes the primary subject cell and
its progeny. As used herein, the terms "engineered" and
"recombinant" cells or host cells are intended to refer to a cell
into which an exogenous nucleic acid sequence, such as, for
example, a vector, has been introduced. Therefore, recombinant
cells are distinguishable from naturally occurring cells which do
not contain a recombinantly introduced nucleic acid. In embodiments
of the invention, a host cell is a T cell, including a cytotoxic T
cell (also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell,
cytolytic T cell, CD8+ T-cells or killer T cell); NK cells and NKT
cells are also encompassed in the invention.
[0109] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0110] The cells can be autologous cells, syngeneic cells,
allogenic cells and even in some cases, xenogeneic cells.
[0111] In many situations one may wish to be able to kill the
modified CTLs, where one wishes to terminate the treatment, the
cells become neoplastic, in research where the absence of the cells
after their presence is of interest, or other event. For this
purpose one can provide for the expression of certain gene products
in which one can kill the modified cells under controlled
conditions, such as inducible suicide genes.
[0112] Armed CARTS
[0113] The invention further includes CARTS that are modified to
secrete one or more polypeptides. The polypeptide can be for
example an antibody or cytokine. Preferably, the antibody is
specific for CAIX, GITR, PD-L1, PD-L2. PD-1, or CCR4
[0114] Armed CARTS have the advantage of simultaneously secreting a
polypeptide at the targeted site, e.g. tumor site.
[0115] Armed CART can be constructed by including a nucleic acid
encoding the polypeptide of interest after the intracellular
signaling domain. Preferably, there is an internal ribosome entry
site, (IRES), positioned between the intracellular signaling domain
and the polypeptide of interest. One skilled in the art can
appreciate that more than one polypeptide can be expressed by
employing multiple IRES sequences in tandem.
[0116] In one embodiment, the methods and compositions presented
herein provide a target-specific Anti-CAIX CAR T cell of second
generation armed with the power to secrete anti-PD-L1 IgGs in the
RCC milieu to combat T cell exhaustion. The human Anti-CAIX CAR
containing the CD28 co-stimulatory domain was chosen based in its
killing activity and low immunogenicity in human cRCC xenografts in
mice. The Anti-CAIX CART cells-secreting Anti-PD-L1 IgG1 or IgG4
was extensively compared with an unrelated Anti-BCMA CAR or with an
Anti-CAIX CAR, both secreting an irrelevant Anti-SARS IgG1. We have
demonstrated stable expression of the CARs and high proliferation
of all CART cells. Anti-CAIX CART cells have capacity to undergo
clonal expansion when in contact with CAIX+ RCC cells, and were
also activated, releasing high levels of IFN.quadrature. and IL-2,
which could improve tumor suppression. These Anti-CAIX CART cells
are also able to secrete high levels of Anti-PD-L1 IgG1 or IgG4 and
these antibodies can interact specifically with PD-L1, inducing
downregulation of the exhaustion markers PD-1, Tim-3 and Lag-3 in
vitro and in vivo. These results have shown that the Anti-PD-L1
antibodies secreted in the tumor microenvironment are able to
revert T cell exhaustion, facilitating the Anti-CAIX CART cells
antitumor activity. These Anti-CAIX CART cells, mainly the ones
secreting anti-PD-L1, are also able to diminish the proliferation
of CAIX+ RCC cells, resulting in a slow tumor growth and small
tumor size and weight in an orthotopic NSG mice model of RCC. In
addition, Anti-CAIX CART cell secreting the IgG1 isotype of
Anti-PD-L1 was also able to induce ADCC in vitro and increased the
number of human NK cells infiltrating the tumor site in vivo. The
human NK cells were injected only in 2 mice of each group, two days
before their euthanasia to determine the NK cells capacity to
recognize the IgG1 isotype of the Anti-PD-L1 in vivo, which was
attested. The injection of human NK cells was not made in the
beginning of the treatment once our previous experience with this
mice model showed that these cells last only for a few days in
their blood
[0117] In one embodiment, the injection of CART cells into mice was
performed without the addition of interleukins to avoid their
influence in the therapeutic effect of CART cells alone.
[0118] In another embodiment, CART cells can be maintained with the
use of cytokines such as, for example, IL-2, IL-4, IL-7, IL-9,
IL-15 and IL-21.
[0119] Cytokines sharing the .gamma.c receptor, like IL-2, IL-4,
IL-7, IL-9, IL-15 and IL-21 are important for the development and
maintenance of memory T cells. Among them, IL-21 promote a less
differentiated phenotype, associated with an enrichment of
tumor-specific CD8 T cells, with increased anti-tumor effect in a
mouse melanoma model when compared to IL-2 or IL-15.
[0120] In certain embodiments, CART cells are maintained with
IL-21.
[0121] CAIX is a consistent marker for development of cancer
targeted systemic therapies due to it overexpression in many
tumors, remarkably in cRCC, but it is also expressed
physiologically in a few tissues.
[0122] In one embodiment, the anti-CAIX scFv in the CAR recognizes
the catalytic domain of CAIX, located in the central portion of the
protein, which could increase its specificity to sites of higher
expression of CAIX.
[0123] The choice of CD28 as a co-stimulatory domain for the CARs
presented herein was based in the fact that CD28 CARs direct an
active proliferative response and enhance effector functions,
whereas 4-1BB-based CARs induce a more progressive T cell
accumulation that may counterweigh for less immediate
effectiveness. In one embodiment, the CD28 is replaced by 41BB in
the CAR constructs.
[0124] T cell exhaustion is common in cancer and these T cells
present low capacities of proliferation and cytokine production
associated with high apoptosis rate and expression of inhibitory
receptors like PD-1, Tim-3 and Lag3. New strategies for preventing
T cell exhaustion include of PD-1/PD-L1 axis.
[0125] The methods and compositions presented herein provide
Anti-CAIX CART cells-secreting Anti-PD-L1 IgG1 or IgG4 that can
diminish T cell exhaustion, improving CART cell efficiency in the
cRCC treatment in vitro and in vivo.
[0126] Introduction of Constructs into CTLs
[0127] Expression vectors that encode the CARs can be introduced as
one or more DNA molecules or constructs, where there may be at
least one marker that will allow for selection of host cells that
contain the construct(s).
[0128] The constructs can be prepared in conventional ways, where
the genes and regulatory regions may be isolated, as appropriate,
ligated, cloned in an appropriate cloning host, analyzed by
restriction or sequencing, or other convenient means. Particularly,
using PCR, individual fragments including all or portions of a
functional unit may be isolated, where one or more mutations may be
introduced using "primer repair", ligation, in vitro mutagenesis,
etc., as appropriate. The construct(s) once completed and
demonstrated to have the appropriate sequences may then be
introduced into the CTL by any convenient means. The constructs may
be integrated and packaged into non-replicating, defective viral
genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes
simplex virus (HSV) or others, including retroviral vectors or
lentiviral vectors, for infection or transduction into cells. The
constructs may include viral sequences for transfection, if
desired. Alternatively, the construct may be introduced by fusion,
electroporation, biolistics, transfection, lipofection, or the
like. The host cells may be grown and expanded in culture before
introduction of the construct(s), followed by the appropriate
treatment for introduction of the construct(s) and integration of
the construct(s). The cells are then expanded and screened by
virtue of a marker present in the construct. Various markers that
may be used successfully include hprt, neomycin resistance,
thymidine kinase, hygromycin resistance, etc.
[0129] In some instances, one may have a target site for homologous
recombination, where it is desired that a construct be integrated
at a particular locus. For example,) can knock-out an endogenous
gene and replace it (at the same locus or elsewhere) with the gene
encoded for by the construct using materials and methods as are
known in the art for homologous recombination. For homologous
recombination, one may use either .OMEGA. or O-vectors. See, for
example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et
al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989)
338, 153-156.
[0130] The constructs may be introduced as a single DNA molecule
encoding at least the CAR and optionally another gene, or different
DNA molecules having one or more genes. Other genes include genes
that encode therapeutic molecules or suicide genes, for example.
The constructs may be introduced simultaneously or consecutively,
each with the same or different markers.
[0131] Vectors containing useful elements such as bacterial or
yeast origins of replication, selectable and/or amplifiable
markers, promoter/enhancer elements for expression in prokaryotes
or eukaryotes, etc. that may be used to prepare stocks of construct
DNAs and for carrying out transfections are well known in the art,
and many are commercially available.
[0132] Methods of Use
[0133] The cells according to the invention can be used for
treating cancer, viral infections or autoimmune disorders in a
patient in need thereof. In another embodiment, said isolated cell
according to the invention can be used in the manufacture of a
medicament for treatment of a cancer, viral infections of
autoimmune disorders, in a patient in need thereof.
[0134] The present invention relies on methods for treating
patients in need thereof, said method comprising at least one of
the following steps: (a) providing a chimeric antigen receptor
cells according to the invention and (b) administrating the cells
to said patient.
[0135] Said treatment can be ameliorating, curative or
prophylactic. It may be either part of an autologous immunotherapy
or part of an allogenic immunotherapy treatment. By autologous, it
is meant that cells, cell line or population of cells used for
treating patients are originating from said patient or from a Human
Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant
that the cells or population of cells used for treating patients
are not originating from said patient but from a donor.
[0136] The invention is particularly suited for allogenic
immunotherapy, insofar as it enables the transformation of T-cells,
typically obtained from donors, into non-alloreactive cells. This
may be done under standard protocols and reproduced as many times
as needed. The resulted modified T cells may be pooled and
administrated to one or several patients, being made available as
an "off the shelf" therapeutic product.
[0137] Cells that can be used with the disclosed methods are
described in the previous section. Said treatment can be used to
treat patients diagnosed with cancer, viral infection, autoimmune
disorders or Graft versus Host Disease (GvHD). Cancers that may be
treated include tumors that are not vascularized, or not yet
substantially vascularized, as well as vascularized tumors. The
cancers may comprise nonsolid tumors (such as hematological tumors,
for example, leukemias and lymphomas) or may comprise solid tumors.
Types of cancers to be treated with the CARs of the invention
include, but are not limited to, carcinoma, blastoma, and sarcoma,
and certain leukemia or lymphoid malignancies, benign and malignant
tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
Adult tumors/cancers and pediatric tumors/cancers are also
included.
[0138] It can be a treatment in combination with one or more
therapies against cancer selected from the group of antibodies
therapy, chemotherapy, cytokines therapy, dendritic cell therapy,
gene therapy, hormone therapy, laser light therapy and radiation
therapy.
[0139] According to a preferred embodiment of the invention, said
treatment can be administrated into patients undergoing an
immunosuppressive treatment. Indeed, the present invention
preferably relies on cells or population of cells, which have been
made resistant to at least one immunosuppressive agent due to the
inactivation of a gene encoding a receptor for such
immunosuppressive agent. In this aspect, the immunosuppressive
treatment should help the selection and expansion of the T-cells
according to the invention within the patient.
[0140] In a further embodiment, the cell compositions of the
present invention are administered to a patient in conjunction with
(e.g., before, simultaneously or following) bone marrow
transplantation, T cell ablative therapy using either chemotherapy
agents such as, fludarabine, external-beam radiation therapy (XRT),
cyclophosphamide, or antibodies such as OKT3 or CAM PATH. In
another embodiment, the cell compositions of the present invention
are administered following B-cell ablative therapy such as agents
that react with CD20, e.g., Rittman. For example, in one
embodiment, subjects may undergo standard treatment with high dose
chemotherapy followed by peripheral blood stem cell
transplantation. In certain embodiments, following the transplant,
subjects receive an infusion of the expanded immune cells of the
present invention. In an additional embodiment, expanded cells are
administered before or following surgery. Said modified cells
obtained by any one of the methods described here can be used in a
particular aspect of the invention for treating patients in need
thereof against Host versus Graft (HvG) rejection and Graft versus
Host Disease (GvHD); therefore in the scope of the present
invention is a method of treating patients in need thereof against
Host versus Graft (HvG) rejection and Graft versus Host Disease
(GvHD) comprising treating said patient by administering to said
patient an effective amount of modified cells comprising
inactivated TCR alpha and/or TCR beta genes.
[0141] Administration of Cells
[0142] The invention is particularly suited for allogenic
immunotherapy, insofar as it enables the transformation of T-cells,
typically obtained from donors, into non-alloreactive cells. This
may be done under standard protocols and reproduced as many times
as needed. The resulted modified T cells may be pooled and
administrated to one or several patients, being made available as
an "off the shelf" therapeutic product.
[0143] Depending upon the nature of the cells, the cells may be
introduced into a host organism, e.g. a mammal, in a wide variety
of ways. The cells may be introduced at the site of the tumor, in
specific embodiments, although in alternative embodiments the cells
hone to the cancer or are modified to hone to the cancer. The
number of cells that are employed will depend upon a number of
circumstances, the purpose for the introduction, the lifetime of
the cells, the protocol to be used, for example, the number of
administrations, the ability of the cells to multiply, the
stability of the recombinant construct, and the like. The cells may
be applied as a dispersion, generally being injected at or near the
site of interest. The cells may be in a physiologically-acceptable
medium.
[0144] In some embodiments, the cells are encapsulated to inhibit
immune recognition and placed at the site of the tumor.
[0145] The cells may be administered as desired. Depending upon the
response desired, the manner of administration, the life of the
cells, the number of cells present, various protocols may be
employed. The number of administrations will depend upon the
factors described above at least in part.
[0146] The administration of the cells or population of cells
according to the present invention may be carried out in any
convenient manner, including by aerosol inhalation, injection,
ingestion, transfusion, implantation or transplantation. The
compositions described herein may be administered to a patient
subcutaneously, intradermaly, intratumorally, intranodally,
intramedullary, intramuscularly, by intravenous or intralymphatic
injection, or intraperitoneally. In one embodiment, the cell
compositions of the present invention are preferably administered
by intravenous injection.
[0147] The administration of the cells or population of cells can
consist of the administration of 10.sup.4-10.sup.9 cells per kg
body weight, preferably 10.sup.5 to 10.sup.6 cells/kg body weight
including all integer values of cell numbers within those ranges.
The cells or population of cells can be administrated in one or
more doses. In another embodiment, said effective amount of cells
are administrated as a single dose. In another embodiment, said
effective amount of cells are administrated as more than one dose
over a period time. Timing of administration is within the judgment
of managing physician and depends on the clinical condition of the
patient. The cells or population of cells may be obtained from any
source, such as a blood bank or a donor. While individual needs
vary, determination of optimal ranges of effective amounts of a
given cell type for a particular disease or conditions within the
skill of the art. An effective amount means an amount which
provides a therapeutic or prophylactic benefit. The dosage
administrated will be dependent upon the age, health and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment and the nature of the effect desired.
[0148] It should be appreciated that the system is subject to many
variables, such as the cellular response to the ligand, the
efficiency of expression and, as appropriate, the level of
secretion, the activity of the expression product, the particular
need of the patient, which may vary with time and circumstances,
the rate of loss of the cellular activity as a result of loss of
cells or expression activity of individual cells, and the like.
Therefore, it is expected that for each individual patient, even if
there were universal cells which could be administered to the
population at large, each patient would be monitored for the proper
dosage for the individual, and such practices of monitoring a
patient are routine in the art.
[0149] Nucleic Acid-Based Expression Systems
[0150] The CARs of the present invention may be expressed from an
expression vector. Recombinant techniques to generate such
expression vectors are well known in the art.
[0151] Vectors
[0152] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques (see,
for example, Maniatis et al., 1988 and Ausubel et al., 1994, both
incorporated herein by reference).
[0153] The term "expression vector" refers to any type of genetic
construct comprising a nucleic acid coding for a RNA capable of
being transcribed. In some cases, RNA molecules are then translated
into a protein, polypeptide, or peptide. In other cases, these
sequences are not translated, for example, in the production of
antisense molecules or ribozymes. Expression vectors can contain a
variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host cell. In
addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well and are described
infra.
[0154] Promoters and Enhancers
[0155] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements at which regulatory
proteins and molecules may bind, such as RNA polymerase and other
transcription factors, to initiate the specific transcription a
nucleic acid sequence. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to
control transcriptional initiation and/or expression of that
sequence.
[0156] A promoter generally comprises a sequence that functions to
position the start site for RNA synthesis. The best known example
of this is the TATA box, but in some promoters lacking a TATA box,
such as, for example, the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40
late genes, a discrete element overlying the start site itself
helps to fix the place of initiation. Additional promoter elements
regulate the frequency of transcriptional initiation. Typically,
these are located in the region 30 110 bp upstream of the start
site, although a number of promoters have been shown to contain
functional elements downstream of the start site as well. To bring
a coding sequence "under the control of" a promoter, one positions
the 5' end of the transcription initiation site of the
transcriptional reading frame "downstream" of (i.e., 3' of) the
chosen promoter. The "upstream" promoter stimulates transcription
of the DNA and promotes expression of the encoded RNA.
[0157] The spacing between promoter elements frequently is
flexible, so that promoter function is preserved when elements are
inverted or moved relative to one another. In the tk promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription. A promoter may or may
not be used in conjunction with an "enhancer," which refers to a
cis-acting regulatory sequence involved in the transcriptional
activation of a nucleic acid sequence.
[0158] A promoter may be one naturally associated with a nucleic
acid sequence, as may be obtained by isolating the 5 prime'
non-coding sequences located upstream of the coding segment and/or
exon. Such a promoter can be referred to as "endogenous."
Similarly, an enhancer may be one naturally associated with a
nucleic acid sequence, located either downstream or upstream of
that sequence. Alternatively, certain advantages will be gained by
positioning the coding nucleic acid segment under the control of a
recombinant or heterologous promoter, which refers to a promoter
that is not normally associated with a nucleic acid sequence in its
natural environment. A recombinant or heterologous enhancer refers
also to an enhancer not normally associated with a nucleic acid
sequence in its natural environment. Such promoters or enhancers
may include promoters or enhancers of other genes, and promoters or
enhancers isolated from any other virus, or prokaryotic or
eukaryotic cell, and promoters or enhancers not "naturally
occurring," i.e., containing different elements of different
transcriptional regulatory regions, and/or mutations that alter
expression. For example, promoters that are most commonly used in
recombinant DNA construction include the lactamase (penicillinase),
lactose and tryptophan (trp) promoter systems. In addition to
producing nucleic acid sequences of promoters and enhancers
synthetically, sequences may be produced using recombinant cloning
and/or nucleic acid amplification technology, including PCR.TM., in
connection with the compositions disclosed herein (see U.S. Pat.
Nos. 4,683,202 and 5,928,906, each incorporated herein by
reference). Furthermore, it is contemplated the control sequences
that direct transcription and/or expression of sequences within
non-nuclear organelles such as mitochondria, chloroplasts, and the
like, can be employed as well.
[0159] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the organelle, cell type, tissue, organ, or organism chosen for
expression. Those of skill in the art of molecular biology
generally know the use of promoters, enhancers, and cell type
combinations for protein expression, (see, for example Sambrook et
al. 1989, incorporated herein by reference). The promoters employed
may be constitutive, tissue-specific, inducible, and/or useful
under the appropriate conditions to direct high level expression of
the introduced DNA segment, such as is advantageous in the
large-scale production of recombinant proteins and/or peptides. The
promoter may be heterologous or endogenous.
[0160] Additionally any promoter/enhancer combination could also be
used to drive expression. Use of a T3, T7 or SP6 cytoplasmic
expression system is another possible embodiment. Eukaryotic cells
can support cytoplasmic transcription from certain bacterial
promoters if the appropriate bacterial polymerase is provided,
either as part of the delivery complex or as an additional genetic
expression construct.
[0161] The identity of tissue-specific promoters or elements, as
well as assays to characterize their activity, is well known to
those of skill in the art.
[0162] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals
[0163] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages, and these may be used in the
invention.
[0164] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector. "Restriction enzyme
digestion" refers to catalytic cleavage of a nucleic acid molecule
with an enzyme that functions only at specific locations in a
nucleic acid molecule. Many of these restriction enzymes are
commercially available. Use of such enzymes is widely understood by
those of skill in the art. Frequently, a vector is linearized or
fragmented using a restriction enzyme that cuts within the MCS to
enable exogenous sequences to be ligated to the vector. "Ligation"
refers to the process of forming phosphodiester bonds between two
nucleic acid fragments, which may or may not be contiguous with
each other. Techniques involving restriction enzymes and ligation
reactions are well known to those of skill in the art of
recombinant technology.
[0165] Splicing sites, termination signals, origins of replication,
and selectable markers may also be employed.
[0166] Plasmid Vectors
[0167] In certain embodiments, a plasmid vector is contemplated for
use to transform a host cell. In general, plasmid vectors
containing replicon and control sequences which are derived from
species compatible with the host cell are used in connection with
these hosts. The vector ordinarily carries a replication site, as
well as marking sequences which are capable of providing phenotypic
selection in transformed cells. In a non-limiting example, E. coli
is often transformed using derivatives of pBR322, a plasmid derived
from an E. coli species. pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR plasmid, or other microbial
plasmid or phage must also contain, or be modified to contain, for
example, promoters which can be used by the microbial organism for
expression of its own proteins.
[0168] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM. 11 may be utilized in making a
recombinant phage vector which can be used to transform host cells,
such as, for example, E. coli LE392.
[0169] Further useful plasmid vectors include pIN vectors (Inouye
et al., 1985); and pGEX vectors, for use in generating glutathione
S transferase (GST) soluble fusion proteins for later purification
and separation or cleavage. Other suitable fusion proteins are
those with galactosidase, ubiquitin, and the like.
[0170] Bacterial host cells, for example, E. coli, comprising the
expression vector, are grown in any of a number of suitable media,
for example, LB. The expression of the recombinant protein in
certain vectors may be induced, as would be understood by those of
skill in the art, by contacting a host cell with an agent specific
for certain promoters, e.g., by adding IPTG to the media or by
switching incubation to a higher temperature. After culturing the
bacteria for a further period, generally of between 2 and 24 h, the
cells are collected by centrifugation and washed to remove residual
media.
[0171] Viral Vectors
[0172] The ability of certain viruses to infect cells or enter
cells via receptor mediated endocytosis, and to integrate into host
cell genome and express viral genes stably and efficiently have
made them attractive candidates for the transfer of foreign nucleic
acids into cells (e.g., mammalian cells). Components of the present
invention may be a viral vector that encodes one or more CARs of
the invention. Non-limiting examples of virus vectors that may be
used to deliver a nucleic acid of the present invention are
described below.
[0173] Adenoviral Vectors
[0174] A particular method for delivery of the nucleic acid
involves the use of an adenovirus expression vector. Although
adenovirus vectors are known to have a low capacity for integration
into genomic DNA, this feature is counterbalanced by the high
efficiency of gene transfer afforded by these vectors. "Adenovirus
expression vector" is meant to include those constructs containing
adenovirus sequences sufficient to (a) support packaging of the
construct and (b) to ultimately express a tissue or cell specific
construct that has been cloned therein. Knowledge of the genetic
organization or adenovirus, a 36 kb, linear, double stranded DNA
virus, allows substitution of large pieces of adenoviral DNA with
foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
[0175] AAV Vectors
[0176] The nucleic acid may be introduced into the cell using
adenovirus assisted transfection. Increased transfection
efficiencies have been reported in cell systems using adenovirus
coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992;
Curiel, 1994). Adeno associated virus (AAV) is an attractive vector
system for use in the cells of the present invention as it has a
high frequency of integration and it can infect nondividing cells,
thus making it useful for delivery of genes into mammalian cells,
for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has
a broad host range for infectivity (Tratschin et al., 1984;
Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,
1988). Details concerning the generation and use of rAAV vectors
are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each
incorporated herein by reference.
[0177] Retroviral Vectors
[0178] Retroviruses are useful as delivery vectors because of their
ability to integrate their genes into the host genome, transferring
a large amount of foreign genetic material, infecting a broad
spectrum of species and cell types and of being packaged in special
cell lines (Miller, 1992).
[0179] In order to construct a retroviral vector, a nucleic acid
(e.g., one encoding the desired sequence) is inserted into the
viral genome in the place of certain viral sequences to produce a
virus that is replication defective. In order to produce virions, a
packaging cell line containing the gag, pol, and env genes but
without the LTR and packaging components is constructed (Mann et
al., 1983). When a recombinant plasmid containing a cDNA, together
with the retroviral LTR and packaging sequences is introduced into
a special cell line (e.g., by calcium phosphate precipitation for
example), the packaging sequence allows the RNA transcript of the
recombinant plasmid to be packaged into viral particles, which are
then secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and stable expression require the division of host cells (Paskind
et al., 1975).
[0180] Lentiviruses are complex retroviruses, which, in addition to
the common retroviral genes gag, pol, and env, contain other genes
with regulatory or structural function. Lentiviral vectors are well
known in the art (see, for example, Naldini et al., 1996; Zufferey
et al., 1997; Blomer et al., 1997; U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentivirus include the Human
Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian
Immunodeficiency Virus: SIV. Lentiviral vectors have been generated
by multiply attenuating the HIV virulence genes, for example, the
genes env, vif, vpr, vpu and nef are deleted making the vector
biologically safe.
[0181] Recombinant lentiviral vectors are capable of infecting
non-dividing cells and can be used for both in vivo and ex vivo
gene transfer and expression of nucleic acid sequences. For
example, recombinant lentivirus capable of infecting a non-dividing
cell wherein a suitable host cell is transfected with two or more
vectors carrying the packaging functions, namely gag, pol and env,
as well as rev and tat is described in U.S. Pat. No. 5,994,136,
incorporated herein by reference. One may target the recombinant
virus by linkage of the envelope protein with an antibody or a
particular ligand for targeting to a receptor of a particular
cell-type. By inserting a sequence (including a regulatory region)
of interest into the viral vector, along with another gene which
encodes the ligand for a receptor on a specific target cell, for
example, the vector is now target-specific.
[0182] Other Viral Vectors
[0183] Other viral vectors may be employed as vaccine constructs in
the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988), sindbis virus, cytomegalovirus and herpes simplex
virus may be employed. They offer several attractive features for
various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal
and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0184] Delivery Using Modified Viruses
[0185] A nucleic acid to be delivered may be housed within an
infective virus that has been engineered to express a specific
binding ligand. The virus particle will thus bind specifically to
the cognate receptors of the target cell and deliver the contents
to the cell. A novel approach designed to allow specific targeting
of retrovirus vectors was developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification can permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0186] Another approach to targeting of recombinant retroviruses
was designed in which biotinylated antibodies against a retroviral
envelope protein and against a specific cell receptor were used.
The antibodies were coupled via the biotin components by using
streptavidin (Roux et al., 1989). Using antibodies against major
histocompatibility complex class I and class II antigens, they
demonstrated the infection of a variety of human cells that bore
those surface antigens with an ecotropic virus in vitro (Roux et
al., 1989).
[0187] Vector Delivery and Cell Transformation
[0188] Suitable methods for nucleic acid delivery for transfection
or transformation of cells are known to one of ordinary skill in
the art. Such methods include, but are not limited to, direct
delivery of DNA such as by ex vivo transfection, by injection, and
so forth. Through the application of techniques known in the art,
cells may be stably or transiently transformed.
[0189] Ex Vivo Transformation
[0190] Methods for transfecting eukaryotic cells and tissues
removed from an organism in an ex vivo setting are known to those
of skill in the art. Thus, it is contemplated that cells or tissues
may be removed and transfected ex vivo using nucleic acids of the
present invention. In particular aspects, the transplanted cells or
tissues may be placed into an organism. In preferred facets, a
nucleic acid is expressed in the transplanted cells.
Kits of the Invention
[0191] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, one or more cells for use in cell
therapy and/or the reagents to generate one or more cells for use
in cell therapy that harbors recombinant expression vectors may be
comprised in a kit. The kit components are provided in suitable
container means.
[0192] Some components of the kits may be packaged either in
aqueous media or in lyophilized form. The container means of the
kits will generally include at least one vial, test tube, flask,
bottle, syringe or other container means, into which a component
may be placed, and preferably, suitably aliquoted. Where there are
more than one component in the kit, the kit also will generally
contain a second, third or other additional container into which
the additional components may be separately placed. However,
various combinations of components may be comprised in a vial. The
kits of the present invention also will typically include a means
for containing the components in close confinement for commercial
sale. Such containers may include injection or blow molded plastic
containers into which the desired vials are retained.
[0193] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being particularly ueful. In some
cases, the container means may itself be a syringe, pipette, and/or
other such like apparatus, from which the formulation may be
applied to an infected area of the body, injected into an animal,
and/or even applied to and/or mixed with the other components of
the kit.
[0194] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. The kits may also comprise a
second container means for containing a sterile, pharmaceutically
acceptable buffer and/or other diluent.
[0195] In particular embodiments of the invention, cells that are
to be used for cell therapy are provided in a kit, and in some
cases the cells are essentially the sole component of the kit. The
kit may comprise reagents and materials to make the desired cell.
In specific embodiments, the reagents and materials include primers
for amplifying desired sequences, nucleotides, suitable buffers or
buffer reagents, salt, and so forth, and in some cases the reagents
include vectors and/or DNA that encodes a CAR as described herein
and/or regulatory elements therefor.
[0196] In particular embodiments, there are one or more apparatuses
in the kit suitable for extracting one or more samples from an
individual. The apparatus may be a syringe, scalpel, and so
forth.
[0197] In some cases of the invention, the kit, in addition to cell
therapy embodiments, also includes a second cancer therapy, such as
chemotherapy, hormone therapy, and/or immunotherapy, for example.
The kit(s) may be tailored to a particular cancer for an individual
and comprise respective second cancer therapies for the
individual.
[0198] Combination Therapy
[0199] In certain embodiments of the invention, methods of the
present invention for clinical aspects are combined with other
agents effective in the treatment of hyperproliferative disease,
such as anti-cancer agents. An "anti-cancer" agent is capable of
negatively affecting cancer in a subject, for example, by killing
cancer cells, inducing apoptosis in cancer cells, reducing the
growth rate of cancer cells, reducing the incidence or number of
metastases, reducing tumor size, inhibiting tumor growth, reducing
the blood supply to a tumor or cancer cells, promoting an immune
response against cancer cells or a tumor, preventing or inhibiting
the progression of cancer, or increasing the lifespan of a subject
with cancer. More generally, these other compositions would be
provided in a combined amount effective to kill or inhibit
proliferation of the cell. This process may involve contacting the
cancer cells with the expression construct and the agent(s) or
multiple factor(s) at the same time. This may be achieved by
contacting the cell with a single composition or pharmacological
formulation that includes both agents, or by contacting the cell
with two distinct compositions or formulations, at the same time,
wherein one composition includes the expression construct and the
other includes the second agent(s).
[0200] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with other therapies. In
the context of the present invention, it is contemplated that cell
therapy could be used similarly in conjunction with
chemotherapeutic, radiotherapeutic, or immunotherapeutic
intervention, as well as pro-apoptotic or cell cycle regulating
agents.
[0201] Alternatively, the present inventive therapy may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks. In embodiments where the other agent and present
invention are applied separately to the individual, one would
generally ensure that a significant period of time did not expire
between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several d
(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
[0202] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the inventive cell therapy.
[0203] Chemotherapy
[0204] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, abraxane,
altretamine, docetaxel, herceptin, methotrexate, novantrone,
zoladex, cisplatin (CDDP), carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the
foregoing and also combinations thereof.
[0205] In specific embodiments, chemotherapy for the individual is
employed in conjunction with the invention, for example before,
during and/or after administration of the invention
[0206] Radiotherapy
[0207] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0208] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0209] Immunotherapy
[0210] Immunotherapeutics generally rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0211] Immunotherapy other than the inventive therapy described
herein could thus be used as part of a combined therapy, in
conjunction with the present cell therapy. The general approach for
combined therapy is discussed below. Generally, the tumor cell must
bear some marker that is amenable to targeting, i.e., is not
present on the majority of other cells. Many tumor markers exist
and any of these may be suitable for targeting in the context of
the present invention. Common tumor markers include PD-1, PD-L1,
CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary
tumor associated antigen, fetal antigen, tyrosinase (p97), gp68,
TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen
receptor, laminin receptor, erb B and p155.
[0212] Genes
[0213] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as the present invention
clinical embodiments. A variety of expression products are
encompassed within the invention, including inducers of cellular
proliferation, inhibitors of cellular proliferation, or regulators
of programmed cell death.
[0214] Surgery
[0215] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0216] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0217] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0218] Other Agents
[0219] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abililties of the present invention by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
EXAMPLES
Example 1: Materials and Methods
[0220] Cells, Culture Media and Reagents.
[0221] Human CAIX+ renal cell carcinoma cell lines sk-rc-52,
sk-rc-09 and CAIX- sk-rc-59 were obtained from Dr. Gerd Ritter,
Memorial Sloan-Kettering Cancer Center, New York. They were
cultured at 37.degree. C. with 5% CO.sub.2 in R-10 complete medium
containing RPMI 1640 medium (Life Technologies) supplemented with
10% FCS, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100
.mu.g/ml streptomycin (Sigma). Primary human T cells were
maintained in R-10 with 10% human serum and 100 IU/ml recombinant
human interleukin 2 (IL-2) (Chiron). Human embryonic kidney cell
line 293T (ATCC) and mouse fibroblast NIH3T3 cells (ATCC) were
grown in D-10 complete medium (Life Technologies) containing DMEM
medium with 10% FCS, 100 U/ml penicillin, and 100 .mu.g/ml
streptomycin (Sigma). Leukopacks obtained from the blood bank of
the Children's Hospital Boston were collected from healthy
volunteers with written informed consent.
[0222] In one embodiment, Human ccRCC cell lines, Skrc52,
originally CAIX+/PD-L1-, and Skrc59, originally CAIX-/PD-L1+, were
obtained from Dr. Gerd Ritter (Memorial Sloan-Kettering Cancer
Center, New York). These cells were cultivated in RPMI 1640 Medium
(Life Technologies) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100
.mu.g/ml streptomycin. 293T (CRL-11268, ATCC) and Lenti-X 293T
(Clontech) cells were grown in DMEM Medium (Life Technologies)
supplemented with 10% FBS, 100 IU/ml penicillin and 100 .mu.g/ml
streptomycin. All cell lines used in this project were transduced
with luciferase through lentiviral transduction and maintained at
37.degree. C. with 5% CO2. The Skrc52 cells were selected for
CAIX-/PD-L1- and CAIX+/PD-L1- cell populations by Fluorescence
activated cell sorting (FACS) sorting. Skrc59 cells were engineered
to express high levels of human CAIX and CAIX+/PD-L1+ were selected
by FACS sorting.
[0223] Cloning of Anti-PD-L1 scFv-Fc IgG1 and IgG4 into a
Bicistronic Lentiviral Vector Encoding an Anti-CAIX 2.sup.nd
Generation CAR
[0224] The anti-PD-L1 antibody (Ab) used to construct the CART
cells was previously selected using a 27-billion-member human scFv
phage display library against a full length PD-L1 in the form of
paramagnetic proteoliposomes (manuscript in preparation). The DNA
sequences that encoded the anti-PD-L1 scFv (Clone 42)-Fc IgG1 or
IgG4 were codon optimized and synthesized (Genewiz) containing the
restriction sites for 5' NdeI and 3' ClaI to allow further scFv-Fc
cloning to replace ZsGreen in the lentiviral vector
pHAGE-eIF.alpha. signal-scFvG36
(anti-CAIX)-C9TAG-linker-CD28-CD3.zeta.-IRES-ZsGreen (e.g. CD28z).
This anti-CAIX original vector was previously constructed and
published (21). For the cloning of the negative controls, firstly
we replaced anti-PD-L1 scFv with an anti-Severe Acute Respiratory
Syndrome (SARS) scFv (Clone 11A) in the IgG1-Fc lentiviral vector
containing the anti-CAIX G36 scFv CAR. The DNA sequence that coded
for the anti-SARS scFv was amplified by PCR from the plasmid pHAGE
CMV-Anti-SARS (11A) scFv-Fc-CD28-gp41-IRES-ZsGreen to insert the
restriction sites for 5' MluI and 3' XbaI for further cloning using
the MluI forward primer 5' TCG ACG CGT GAG GTG CAG CTG GTG CAG T 3'
and XbaI reverse primer 5' TCC TCT AGA CAG GAC GGT GAC CTT GGT CC
3'. The final pHAGE-eIF.alpha.-scFv G36
(anti-CAIX)-C9TAG-linker-CD28-CD3.zeta.-IRES-anti-SARS (11A) IgG1
was used as one of the negative controls. We also replaced the
anti-CAIX scFv in the CAR structure containing the anti-SARS IgG1
for the anti-B cell maturation antigen (BCMA) scFv by double
digestion of the pHAGE-eIF.alpha.-A716scFv
(anti-BCMA)-C9TAG-linker-CD28-CD3.zeta.-IRES-ZsGreen and the
pHAGE-eIF.alpha.-G36scFv(anti-CAIX)-C9TAG-linker-CD28-CD3.zeta.-IRES-anti-
-SARS(11A) IgG1 with NcoI and NotI After the cloning processes we
obtained four main plasmids to start the Lentiviruses production:
Anti-CAIX CAR able to express anti-PD-L1 IgG1 (anti-CAIX/anti-PD-L1
IgG1), anti-CAIX CAR able to express anti-PD-L1 IgG4
(anti-CAIX/anti-PD-L1 IgG4), anti-CAIX CAR able to express an
irrelevant anti-SARS Ab (anti-CAIX/anti-SARS IgG1) and anti-BCMA
CAR able to express an irrelevant anti-SARS Ab (anti-BCMA/anti-SARS
IgG1).
[0225] scFv Isolation and Conversion of scFv to scFv-Fc.
[0226] CAIX-specific scFv antibodies were isolated from a
non-immune human scFv phage library as previously reported and
submitted to GenBank with accession numbers of
GQ903548-GQ903561.sup.23. scFv-coding DNA fragments from the
pFarber phagemid were digested with SfiI/NotI sites and subcloned
into the mammalian expression vector pcDNA3.1-F105L-hinge-stuffer
which has a human IgG1 F105 leader sequence and the human IgG1
hinge-CH2-CH3 Fc portion to express scFv-Fc antibodies. Plasmids of
scFv-Fc were transiently transfected into 293T cells by
lipofectamine 2000 (Invitrogen), and expressed antibodies were
purified using Sepharose protein A beads (Amersham Bioscience).
Specific binding to CAIX was tested by staining with phage scFv
antibodies or scFv converted into scFv-Fc format antibodies by
incubation with CAIX-expressing 293T and sk-rc-52 cell lines, and
with CAIX negative 293T and sk-rc-59 cell lines. In these
experiments, irrelevant anti-HIV CCRS antibody (clone A8).sup.25 or
anti-SARS antibody (11A).sup.24 and fluorescently conjugated
secondary antibodies alone were used as negative controls.
[0227] Construction of scFv-CD8-TCR.zeta. and scFv-CD28-TCR.zeta.
Constructs.
[0228] Pz1, scFv-CD8-TCR.zeta., and P28z, scFv-CD28-TCR.zeta., DNA
constructs in phagemid vector pSL1180 were obtained from Dr. Michel
Sadelain, Memorial Sloan-Kettering Cancer Center, New York. In Pz1,
the scFv and TCR.zeta. intracellular domain are appended to N- and
C-terminus of human CD8a chain, respectively. Similarly, in P28z,
the scFv and TCR.zeta. sequences are appended to the N- and
C-terminus of human CD28, respectively. The amino acid sequence of
human CD8a is 71 residues in length, consisting of 47 (aa 137-183),
23 (aa 184-206), and 2 (aa 207-208) residues of the CD8a
extracellular and hinge, transmembrane, and cytoplasmic domains,
respectively. The CD28 sequence in P28z is 107 residues in length,
consisting of 40 (aa 114-153), 23 (aa 154-176), and 44 (aa 177-220)
residues of the CD28 extracellular, transmembrane, and cytoplasmic
domains respectively. The human CD3.zeta. intracellular domain
common to both CARs consists of 112 amino acids (aa 52-163).
[0229] The nucleic acid sequence encoding an internal C9-tag (a
nine-amino acid peptide of human rhodopsin, TETSQVAPA) with a GGGGS
linker was amplified by PCR and was fused upstream with
CD8-TCR.zeta. and CD28-TCR.zeta. sequences with 5' NotI site and 3'
PacI sites. The primers used for cloning chimeric TCR.zeta.
constructs are
TABLE-US-00006 5' TAG GGC GCG GCC GCa acc gag acc agc cag gtg gcg
ccc gcc GGG GGA GGA GGC AGC CCC ACC ACG ACG CCA GCG CCG CGA 3'
(forward primer for CD8 construct where italic is the NotI site,
upper case is the C9 tag sequence, and underlining indicates the
GGGGS linker),
TABLE-US-00007 5' TAG GGC GCG GCC GCa acc gag acc agc cag gtg gcg
ccc gcc GGC GGA GGA GGC AGC ATT GAA GTT ATG TAT CCT CCT CCT 3'
(forward primer for CD28 construct) and reverse primer for both
constructs CTA GCC TT AAT TAA, TTA GCG AGG AGG GGG CAG GGC CTG CAT,
italic is Pac I site. These DNA fragments encoded functional
features which are arranged in accordance with the following
sequence: NotI-C9tag (TETSQVQPQ)-GGGGS-CD8 or CD28-TCR.zeta.-PacI.
The chimeric TCR constructs tagged with internal C9 peptide were
cloned into the pcDNA3.1-F105L-hinge stuffer vector containing
anti-CXCR4 scFv-Fc, clone 48, using NotI and PacI restriction
sites. This design allowed us to insert chimeric TCR receptor
constructs to replace Fc portion fragment. Later, anti-CAIX scFv
(clone G36) and anti-CCRS scFv (clone A8, as irrelevant scFv
control) antibody fragments were cloned to replace anti-CXCR4 scFv
at SfiI/NotI sites to create CAIX-specific chimeric TCR
constructs.
[0230] The lentivirus vector pHAGE-CMV-DsRed-IRES-ZsGreen, and four
HIV helper plasmids pHDM-Hgpm2 (HIV gag-pol), pMD-tat, pRC/CMV-rev,
and an Env VSV-G pseudotype were obtained from Dr. Richard
Mulligan, of the Virus Production Core at The Harvard Gene Therapy
Initiative in Boston. The CMV promoter in pHAGE-CMV-IRES-ZsGreen
was replaced by an EF1.alpha. promoter derived from the pSIN
lentivirus vector at SpeI/NotI sites. One of the 5 scFv-Fc
antibodies, G36, which possess high affinity to CAIX+ cells and
high ADCC only against CAIX+ tumor cells, was cloned into
pHAGE-EF1.alpha. lentivirus vector at AscI/BamHI to replace the
first cassette of the DsRed protein.
[0231] Production of Lentivirus and Transduction of Human Primary T
Cells.
[0232] Lentivirus was produced by five plasmid transient
transfection into 293T cells using lipofectamine 2000 as per the
manufacturer's instructions (Invitrogen). Cells were prepared for
80% confluence in 15 cm Petri dishes (Nalge Nunc) and transfected
with 30 .mu.g of total plasmid DNA. The ratio of vector plasmids
(pHDM-Hgpm2 (HIV gag-pol): pMD-tat: pRC/CMV-rev: Env VSV-G
pseudotype) was 20:1:1:1:2. After changing to D-10 medium, virus
supernatant was harvested on day 3, filtrated through a 0.45 .mu.m
filter, and concentrated by ultracentrifugation (Beckman Coulter,
Fullerton, Calif.) for 90 minutes at 16,500 rpm (48,960.times.g,
Beckman SW28 rotor) and 4.degree. C. The virus pellets were
resuspended in R-10 medium and kept frozen at -80.degree. C.
[0233] In one embodiment, Lentiviruses were produced by transient
transfection of five plasmids into 293T cells using
Polyethyleneimine (PEI). Briefly, each 80% confluent 293T cells in
15 cm plate (Nalge Nunc) was transfected with 30 .mu.g of total
five plasmids, being 5 .mu.g of each structural plasmid pHDH-Hgpm2
(HIV gag-pol), pMD-tat; pRC/CMV-rev and Env VSV-G, and 10 .mu.g of
the main plasmid codifying the CAR (anti-CAIX/anti-PD-L1 IgG1,
anti-CAIX/anti-PD-L1 IgG4, anti-CAIX/anti SARS IgG1 or
anti-BCMA/anti SARS IgG1). The virus supernatant was concentrated
using Lenti-X Concentrator (Clontech), following the manufacturer
instructions, and kept frozen at -80.degree. C.
[0234] Human PBMCs were isolated by ficoll density gradient
separation and were activated with 2 .mu.g/ml PHA (Sigma) plus 100
IU/ml human IL-2 for 4 days. The cells were infected with two or
three rounds of lentivirus transduction at multiplicity of
infection (MOI) of 10-20 in the presence of 10 .mu.g/ml DEAE. Three
days after transduction, transduced T cells were collected for
phenotypic and functional analyses in vitro, or were expanded for
in vivo experiments.
[0235] Selection, Activation and Lentivector Trasduction of CD8+ T
Cells.
[0236] Blood collars collected from healthy volunteers with written
informed consent were obtained from the blood bank of the Brigham
and Woman's Hospital (Boston, Mass.). The isolation of human
peripheral blood mononuclear cells (PBMCs) was performed using
Ficoll-Paque PLUS (GE Healthcare, NJ). The Dynabeads for CD8
Positive Isolation (Life Technologies) were used to isolate CD8
positive cells from PBMCs, which were cultured in RPMI 1640 medium
(Life Technologies) with 10% heat inactivated fetal bovine serum,
20 mM HEPES, 100 IU/ml penicillin and 100 .mu.g/ml streptomycin and
50 IU/mL of IL-21 (Peprotech) was added in the medium every 2 days.
The assays performed to determine if IL-2 or IL-21 were the best
cytokine to induce anti-CAIX CART cells proliferation were
performed with 50 IU/mL of each cytokine. The CD8+ T cells were
activated with Dynabeads Human T-Activator CD3/CD28 (Life
Technologies) using a ratio of 1:1. The cells were transduced with
the Lentiviruses at a multiplicity of infection of 20 and 10
.mu.g/mL of Diethylaminoethyl. All the assays were performed in
triplicate and using T cells from three different healthy
donors.
[0237] Flow Cytometric Analysis.
[0238] Transduction efficiency of human primary T cells was
assessed by expression of a reporter gene (ZsGreen). The CAIX-Fc
protein was expressed from a pcDNA3.1 plasmid that encoded amino
acids 38-397 of CAIX followed by human IgG1 hinge, CH2 and CH3
domains, the CAIX signal peptide (aa 1-37) was replaced with Ig
leader sequence. Expression of scFv(G250) on transduced T cells was
tested by staining the cells with 1 .mu.g CAIX-Fc protein, and then
APC-conjugated mouse anti-human IgG antibody (Jackson
ImmunoResearch). Additionally, expression of the internal rhodopsin
nonapeptide (TETSQVAPA) C9 tag of the scFv domain of TCR constructs
on transduced T cells was detected by staining with 5 .mu.g mouse
1D4 antibody followed by APC-conjugated goat anti-mouse IgG
antibody (Jackson ImmunoResearch). For analysis, the subsets of
human cells in culture during clonal expansion experiment were
stained with fluorescence conjugated mouse anti-human antibodies
(Invitrogen) against CD3 (clone S4.1), CD4 (clone S3.5) or CD8
(clone 3B5). In all cell staining, five hundred thousand cells were
stained with antibodies at recommended concentration according to
company's instruction. The matched isotype control antibodies for
each sample were used and the cells were analyzed using a
FACSCalibur cytometer (Becton-Dickinson).
[0239] In one embodiment, transduction of 293T cells or CD8+ T
cells was confirmed by FACS analysis of the anti-CAIX or anti-BCMA
expression. The cells were stained with 10 .mu.g/mL of human
CAIX-Fc produced in our lab or human BCMA-mouse-Fc (AB Bioscience)
and then developed with 1:250 APC-conjugated mouse anti-human IgG
Ab (Southern Biotech) or goat-anti mouse IgG Ab (Biolegend),
respectively. CountBright.TM. Absolute Counting Beads (Molecular
Probes) was used for the proliferation and clonal expansion assays.
All samples were analyzed with an LSR Fortessa or with a
FACSCalibur (BD Bioscience) and data were analyzed using FlowJo
software. To analyze the status of T cell exhaustion of the CART
cells they were cultured in the presence of IL-21 50 U/mL
(Peprotech) and Dynabeads Human T Activator CD3/CD28 for five days.
After this period the CART cells were co-cultured with Skrc-59
CAIX+ PD-L1+ cells for 2 days in order to stimulate exhaustion.
1.times.106 CART cells from this assay and Tumor-infiltrating
Lymphocytes (TIL) collected from the in vivo assay were stained
with FITC-conjugated anti-human PD-1, PE-conjugated anti-human
Tim3, PerCP/Cy5.5-conjugated anti-human Lag3 antibodies (Biolegend)
and Pacific Blue-conjugated anti-human CD45 and analyzed by FACS.
To verify the expression levels of CAIX and PD-L1 in the different
RCC cell lineages used in this project, we used 10 .mu.g/mL of the
anti-human CAIX mAb (Clone G36), produced in our laboratory, and 10
.mu.g/mL of the biotinylated mouse anti-human PD-L1 (Biolegend).
The primary antibodies were detected using 1:250 APC-conjugated
anti-human Ab and PE-conjugated avidin, respectively, and analyzed
by FACS.
[0240] ADCC and Cytotoxicity Assay of Lentivirus Transduced T
Cells.
[0241] Cytotoxicity assays were performed using the DELFIA EuTDA
Cytotoxicity kit (Perkin Elmer, Boston, Mass.) in accordance with
the manufacturer's instructions. Briefly, target tumor cells were
labeled with a fluorescent ligand (BATDA) for 30 minutes at
37.degree. C. and 1.times.10.sup.4 labeled cells were loaded per
well in 96-well U-bottom plate. For antibody-dependent cellular
cytotoxicity (ADCC) assay, a panel of anti-CAIX scFv-Fc antibodies
or irrevelant scFv-Fc antibody at a concentration of 1 .mu.g/ml or
5 .mu.g/ml was added separately. The assay was set up with ratios
of effector cells (human PBMC) to target cells (E:T) at 50:1, 25:1
and 12.5:1. For the T cell cytoxicity assay, different ratios of
effector cells (nontransduced or transduced T cells) to target
cells (E:T) were prepared (100:1, 50:1 and 25:1). The culture was
incubated for 4 hours in humidified 5% CO.sub.2 at 37.degree. C.
After the plate was spun for 5 minutes at 500.times. g, 20 .mu.l of
supernatant was transferred to a flat-bottom plate. 200 .mu.l of
Europium solution was added and the fluorescence released from the
cells was read by fluorometer (Victor.TM., PerkinElmer). The
control for spontaneous release was prepared by culturing the
labeling cells only and the control for maximum release was made by
adding lysis buffer (kit provided) to the labeling cells.
[0242] ELISA, ELISPOT Assays and Western Blot.
[0243] For cytokine secretion, RCC cell lines sk-rc-52 (CAIX+) or
sk-rc-59 (CAIX-) were seeded overnight at 1.times.10.sup.6 per well
in a 24-well plate, followed by 1.times.10.sup.6 untransduced or
transduced T cells. Before co-culture with tumor cells, T cells
were washed with PBS twice to remove human IL-2. After overnight
incubation, the supernatant was harvested and analyzed for IL-2 and
IFN-.gamma. by ELISA (e-Bioscience). In detecting T cells for the
IFN-.gamma. ELISPOT assay (e-Bioscience), a membrane was developed
using AEC substrate solution and the number of spots was counted by
ELISPOT plate reader (C.T.L. Cellular Technology).
[0244] For Western blot, preparation of untransduced and transduced
T cells was described.sup.50. One million cells were prepared in
non-reducing and reducing buffer (0.1 M dithiothreitol) and run on
a 10-20% polyacrylamide gradient gel (Invitrogen). Proteins were
transferred to polyvinylidence fluoride transfer membrane (NEN Life
Science Products, Boston, Mass.) at 100 V, 4.degree. C. overnight.
The membrane was incubated with 1:2000 primary antibody, anti-human
.zeta.-chain monoclonal antibody 8D3 (BD Pharmingen, San Diego,
Calif.) and then with 1:3000 secondary antibody horseradish
peroxidase (Caltag). Immunodetection was performed using the ECL
Plus Western blotting detection system (GE Healthcare, Piscataway,
N.J.) and x-ray film exposure.
[0245] Detection of IgG Secreted by CART Cells Using ELISA.
[0246] The total level of IgG secreted to the medium of transduced
cells was detected using Human IgG ELISA Quantitation Set (Bethyl
Laboratories). The Anti-PD-L1 Abs secreted by transduced CD8+ CART
Cells were purified with Protein A sepharose beads (GE Healthcare)
and biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin (Thermo
Scientific). These antibodies were incubated with 5 .mu.g/mL of
human PD-L1 produced in the lab, which was pre-immobilized in
MaxiSorp plates (Nunc) by 2 hours, RT. The biotinylated antibodies
were detected by incubation with streptavidin-HRP for 1 h and
developed with SureBlue.TM. TMB Peroxidase Substrate and TMB Stop
Solution (KPL). The absorbance was read at .lamda.=450 nm.
[0247] Proliferation, Clonal Expansion and Cytokine Secretion after
Tumor Cell Contact.
[0248] Tumor cells were irradiated (3,000 rads) and seeded at
2.5.times.10.sup.5 per well. T cells were added at 1.times.10.sup.6
in culture medium containing R-10 plus 100 IU/ml human IL-2 for a
week culture. T cells were split to maintain suitable density and
re-stimulated with tumor cells weekly. The number of T cells was
counted every 3 or 4 days for 2 weeks. The percentage expression of
ZsGreen by transduced T cells and T cell subsets were determined
weekly by fluorescence-activated cell sorting (FACS). For cytokine
secretion studies after tumor cell contact, T cells that were in
contact with irradiated tumor cells for one or two weeks were
washed, incubated with fresh tumor cells overnight and culture
supernatants were collected after 24 hrs for analysis.
[0249] Clonal Expansion of CAIX+ CART Cells.
[0250] In one embodiment, Skrc52 CAIX+/PD-L1- and Skrc52
CAIX-/PD-L1- cells were irradiated with 3,000 rads and seeded at
2.5.times.10.sup.5 per well. 1.times.10.sup.6 T cells were added at
the culture medium containing 50 IU/ml human IL-21 every two days.
T cells were split to maintain suitable density and re-stimulated
with tumor cells weekly. T cell number was counted once a week for
3 weeks by FACS.
[0251] Effect of Anti-CAIX CART Cells Secreting Anti-PD-L1
Antibodies on RCC Cells Viability and Antibody-Dependent Cellular
Cytotoxicity (ADCC).
[0252] 2.5.times.103 Skrc59 CAIX+/PD-L1+ and Skrc52 CAIX-/PD-L1-
were plated in 96 wells plate ON. Four days after the CART cells
transduction they were added to the RCC cells in the 25:1, 50:1 and
100:1 ratio Effector cells: Tumor cells (E:T) and incubated ON.
CART cells were removed and the viability of the tumor cells was
assayed for MTT (Life Technologies). For the ADCC assay RCC cells
were incubated for 1 hour, 37.degree. C. with 50 .mu.L of the CART
cells supernatant adjusted for 500 ng/mL of the respective Ab
Anti-PD-L1 IgG1, Anti-PD-L1 IgG4 or Anti-SARS IgG1. The cells were
then incubated with 12.5:1, 25:1 or 50:1 NK cells for 4 h,
37.degree. C., Lactate dehydrogenase (LDH) was measured in the
supernatant by CytoTox 96.RTM. Non-Radioactive Cytotoxicity Assay
(Promega).
[0253] ELISA Assays to Detect IL-2 and IFN.gamma. Released by
Functional CART Cells.
[0254] For analysis of cytokine secretion, 2.5.times.103 RCC cells
Skrc59 CAIX+/PD-L1+ or Skrc52 CAIX-/PD-L1- were seeded in 96 wells
plates ON, followed by 5:1, 25:1 and 50:1 CAR transduced T cells
addition and incubation ON. The supernatant was removed and
analyzed for IL-2 and IFN.gamma. secretion using the Human
IFN.gamma. or Human IL-2 ELISA Ready-SET-Go Kit (eBioscience).
[0255] Orthotopic Renal Cell Carcinoma Model Establishment and
CART-Cells Therapy.
[0256] 5.times.10.sup.4 passaged Skrc59 CAIX+/PD-L1+ cells were
suspended in 10 .mu.L of culture medium and diluted 1:1 in Matrigel
(Life Technologies) and injected into the left subrenal capsule of
6-8 week-old male NSG mice (N=35). After a week, tumor implantation
was confirmed by bioluminescence (BLI) image using a Xenogen IVIS
imaging system (Life Technologies) and 1.0.times.107 of each CART
cell (anti-BCMA CAR/anti-SARS IgG1, anti-CAIX CAR/anti SARS IgG1,
anti-CAIX CAR/anti-PD-L1 IgG1 and anti-CAIX CAR/anti-PD-L1 IgG4) or
untransduced T cells were injected intravenously in the tail vein
(Day 0); N=6 mice per group. The tumor BLI was quantified after 7,
14, 23 and 30 days of CART cells injection. A second injection of
2.5.times.10.sup.6 CAR or untransduced T cells was made on day 17.
Mice were sacrificed at 30 days post tumor engraftment by standard
CO2 inhalation, and tumors were harvested and weighted. The kidney
tumors from all mice were divided in two equal parts and one of
them was fragmented in small pieces and digested with collagenase
0.5 U/mL and DNAse 1.0 mg/mL to TIL extraction, which were analyzed
for the expression of the exhaustion markers and the percentage of
CART cells by FACS. The other part was fixed in 10% buffered
formaldehyde, and submitted to immunohistochemistry for different
markers. Two mice of each group were injected with 4.5.times.106 NK
cells 2 days before the euthanasia. NK cells present in the tumor
were stained with APC-Anti-CD56 Ab and analyzed by FACS. Animal
experiments were performed in accordance with the guidelines of the
DFCI Animal Care Committee.
[0257] Tumor Establishment and T Cell Therapy.
[0258] In one embodiment, due to immune-rejection of sk-rc-52 in
6-8 week-old female BALB/c nude mice and to accelerate in vivo
growth properties, five million cells were subcutaneously
inoculated into the mice, harvested, and expanded in vitro. The
cell line was then passaged two more times in nude mice and the
passaged cells were expanded for further experiments (subclone
4-1). For the therapeutic experiments, 5 million sk-rc-59 and 7.5
million passaged sk-rc-52 cells were subcutaneously inoculated on
opposing flanks into nude mice to yield comparable tumor growth
rates. After 7 days, tumors grew to the size of .about.6 mm, and 50
million nontransduced or transduced T cells were injected
intravenously. The mice were also treated with 20,000 IU human IL-2
by peritoneal injection every two days. Tumor size was measured by
caliper in two dimensions and the mean of two tumor diameter was
reported here. Animal experiments were performed in accordance with
the guidelines of the Dana Farber Cancer Institute Animal Care
Committee. Mice were sacrificed when tumors reached 15-mm diameter
or 2,000 mm.sup.3 and tumors were harvested.
[0259] Immunohistochemistry and Immunofluorescence Staining.
[0260] For in vitro examination of transduced T cells, the cultured
T cells were washed twice using PBS and resupended in 2 .mu.M Far
Red DDAO-SE CellTrace dye (Molecular Probe) in PBS for 15 minutes
at 37.degree. C. Then the cells were washed with culture medium
twice and cytospun on the glass slide. Far red pre-stained CART
cells with ZsGreen coexpression were visualized using confocal
microscopy (Zeiss) at the Optical Imaging Core facility, Harvard
NeuroDiscovery Center.
[0261] To examine the killing effect of transduced T cells in tumor
bed in situ, tumors were prepared for frozen sections for ApopTag
Peroxidase In Situ Apoptosis Detection kit (Millipore).
Cryosections were incubated with TdT enzyme (Millipore) for 1 hour.
Rabbit anti-DIG (Dako) was added and incubated for 30 minutes and
then Cy3-conjugated anti-rabbit antibody (Invitrogen) was added and
incubated for 30 minutes. Sections were mounted with DAPI antifade
mounting medium and fluorescent images were examined using confocal
microscopy.
[0262] Xenograft tumors and mouse spleens were harvested, fixed in
10% formalin/PBS solution, and submitted to the Harvard Medical
School, Rodent Histopathology Core Facility. Paraffin-embedded
sections were dewaxed with xylene and rehydrated through graded
alcohols before staining. Immunohistochemistry staining was
performed by incubating with anti-human granzyme B antibody (Dako,
clone GrB-7 (1:200)) as a primary antibody for 1 hour followed by
secondary anti-rabbit antibody (Pierce) or anti-mouse antibody
(Dako) for 30 minutes. Sections were developed using DAB substrate
and counterstained with hematoxylin.
[0263] In one embodiment, the fixed tumors were paraffin-embedded,
sectioned at four-micrometer, placed on slides and prepared for
IHQ. The tissues were stained with the anti human: Ki67 (Vector,
VP-K451), PD-L1 (Clone 405.9A11, produced in Dr. Gordon Freeman's
lab), granzyme B (Abcam, ab4059) or NCAM (CD56) (Abcam, ab133345)
antibodies, followed by secondary HRP conjugated anti-rabbit Ab or
HRP-Avidin. The slides were developed using DAB and counterstained
with hematoxylin. The images were obtained in an Olympus BX51
microscopy using a DP71 digital camera (Olympus) and analyzed in
the DP Controller Software (Olympus). The image quantification was
performed using the IHC Profiler Plugin of ImageJ Software as
described in Varghese F, Bukhari A B, Malhotra R, De A. IHC
Profiler: an open source plugin for the quantitative evaluation and
automated scoring of immunohistochemistry images of human tissue
samples. PloS one. 2014; 9:e96801.
[0264] Statistical Analyses.
[0265] Statistical significance was determined using the two-tailed
Student's t-test.
[0266] The statistical analysis associated with FIGS. 16-23 are
representative of at least three experiments unless otherwise
noted. The statistical significance of the data was evaluated using
ANOVA and Tukey posttest. P<0.05 was considered significant. The
statistical analysis was performed using the IBM SPSS Statistics
software version 20.
Example 2: ADCC Mediated Killing of Anti-Cam Antibodies and Choice
of Car Targeting Moiety
[0267] We have previously reported on a panel of high affinity
human anti-CAIX antibodies that differed in their epitope mapping,
expression levels and ability to internalize CAIX.sup.23. Our first
aim was to investigate the anti-tumor activity of five of these
anti-CAIX single-chain antibodies as candidates for CAR
construction. To test for anti-CAIX mAb mediated ADCC, the scFvs
were converted to scFv-Fc (hIgG1) minibodies.sup.23. We found that
all scFv-Fcs exhibited antigen-specific tumor lysis. For tumor cell
line sk-rc-09 with high CAIX+ expression, specific lysis ranged
from 40-57% and for sk-rc-52 with moderate CAIX+ expression,
specific lysis ranged from 46-60%, with background of lysis of
<5% for the CAIX- tumor cell line sk-rc-59. For negative control
scFv-Fcs such as anti-CXCR4 48-Fc.sup.23 and anti-SARS
11A-Fc.sup.24, only background levels of cell lysis were seen (FIG.
1). Based on ADCC killing and other published analyses, scFvG36 was
chosen for further evaluation as the CAR targeting moiety.
[0268] Construction and Expression of CAIX-Specific Chimeric
Receptors.
[0269] Two generations of anti-CAIX CARs were constructed: 1.sup.st
generation G36 CD8 CAR, with scFvG36 linked to CD8, truncated
extracellular, hinge, and transmembrane domains plus signaling
domain of TCR.zeta. (G36-CD8z). To deliver costimulatory signals,
2.sup.nd generation CD28 CAR was generated, consisting of scFvG36
fused to truncated extracellular, transmembrane and intracellular
domains of CD28 plus signaling domain of TCR.zeta. (G36-CD28z)
(FIG. 2A). Irrelevant 2.sup.nd generation CD28 CAR was made by
using anti-HIV CCRS (clone A8) scFv instead.sup.25. In order to
detect the expression of these constructs, human rhodopsin C9 tag
were inserted between the scFv and CD8 or CD28 domains,
respectively and ZsGreen was expressed after the IRES sequence.
High concentrations of viral stocks were obtained at comparable
levels among the different constructs that were tested by
cotransfection of vector plasmids into 293T cells (data not
shown).
[0270] For transduction, PHA mitogen was used to stimulate
peripheral blood lymphocytes for 3 days. Concentrated lentivirus
supernatants were used to infect human primary T cells in the
presence of cationic reagent DEAE as it increased the transduction
rate of 1.5-2.times. fold as compared with polybrene (data not
shown). The transduction rate of primary T cells ranged from 17% to
45% by ZsGreen expression in FACS analysis. A representative
experiment showing ZsGreen expression in circa 25% by primary CART
cells following lentivirus transduction is shown in FIG. 2B, left
column CAIX-Fc fusion protein can bind to the G36-CD8z and -CD28z
CART cells but not to control A8-CD28z CART cells (FIG. 2B, middle
column). C9-tag expression was only detected at circa one-third the
level of the CAIX-Fc protein (FIG. 2B, right column) which is
likely related to the finding that mAb 1D4 preferentially
recognizes the rhodopsin nonapeptide C9 when presented as a
carboxy-terminal verses internal polypeptide sequence (data not
shown). Transduced cells that were cultured in vitro for 6 weeks
maintained their expression of ZsGreen.
[0271] On Western blot under reducing conditions, G36 and A8 CD28z
CARs migrated with a mol wt of circa 53 kD whereas endogenous
TCR.zeta. was 16 kDa. G36-CD8z CAR migrated with a mol wt of circa
48 kD. Under nonreducing conditions, these two CD28z CARs formed
homodimers (FIG. 2C, data of CD8z CAR not shown).
Example 3: Enhanced Cytokine Secretion by Transduced T Cells on
Contact with CAIX+ Tumor
[0272] A study was performed to compare the reported superior
effects of using 2.sup.nd generation G36-CD28z CART cells that
incorporate signaling components of the costimulatory molecule CD28
to bypass MHC presentation and enhance T cell effector functions
verses 1.sup.st generation G36-CD8z CART cells. As seen in FIG. 3A,
after incubation with CAIX+ sk-rc-52 cells overnight, only low
levels of type I cytokines IL-2, IFN.gamma. and IL-17 secretion
were seen with control A8 CD28z CART cells or LAK cells alone. In
contrast, both 1.sup.st and 2.sup.nd generation G36 expressing CART
cells showed elevated levels of cytokine secretion with 2.sup.nd
generation G36-CD28z CART cells secreting higher amounts of type I
cytokines which reflects their higher activation status compared to
1.sup.st generation G36-CD8z CART cells. Specifically, G36-CD28z
CART cells secreted 6.5.times., 2.3.times. and 4.times. more IL-2,
IFN.gamma. and IL-17, respectively than G36-CD8z CART cells.
Specificity of cytokine secretion induction by the two G36 CART
cells is seen by their minimal stimulation with CAIX- sk-rc-59
cells.
[0273] In an Elispot study, after interaction with CAIX+ sk-rc-52
tumors, G36-CD28z CART cells became high capacity IFN-.gamma.
producing cells (FIG. 3B). G36-CD28z CART cells produced 6 times
more spots than seen for G36-CD8z CART cells upon interaction with
CAIX+ sk-rc 52 tumor cells and 12 times more spots than seen after
interaction with CAIX- sk-rc-59 tumor cells. Similarly, G36-CD28z
CART cells had a higher amount of granzyme B-secreting spots after
contact with CAIX+ tumors as compared with G36-CD8z CART cells and
control T cells. PMA and ionomycin stimulated T cells yielded the
highest amount of IFN-.gamma. and granzyme B secreting T cells.
These studies demonstrate both specificity and high capacity of
G36-CD28z CART cells to be activated by contact with CAIX+ tumor
cells.
Example 4: Specific Cytotoxicity Via Car Signaling in Transduced T
Cells
[0274] An in vitro cytotoxicity assay was established to further
evaluate the killing activity of the different G36 CART cells.
Using different ratios of effector-to-target, G36-CD28z CART cells
and its' twice in vivo passaged subclone 4-1 exhibited the highest
amount of cytolysis of CAIX+ tumor sk-rc-52 (FIG. 3C). With high
ratio of more than 25:1, G36-CD28z CART cells showed 2-3 fold
higher cytotoxicity than G36-CD8z CART cells and with low ratio of
5:1, G36-CD28z CART cells showed 8-9 fold higher lysis than
G36-CD8z CART cells. However, G36-CD8z CART cells still exhibited
good cytotoxicity with up to more than 60% tumor lysis using 100:1
of E:T ratio. Irrelevant A8-CD28z CART cells and control T cell LAK
showed the background non-specific tumor lysis with around 20%
lysis when using the highest 100:1 of E:T ratio. In all cases of
using CAIX- tumor sk-rc-59, transduced and untransduced T cells
showed background lysis.
Example 5: Improved In Vitro Proliferation in Cart Cells with
Prolonged Caix+ Tumor
[0275] Besides enhanced cytokine secretion and cytotoxicity on
short term CAIX+ tumor cell contact, incorporation of the CD28
costimulatory molecule into the CAR construct demonstrated improved
proliferation upon prolonged contact with antigen-specific tumor
cells. Untransduced and transduced (around 20%) T cells were mixed
with freshly irradiated tumor cells weekly in the presence of 100
units/ml human IL-2. To test the different levels of antigen
stimulation to a fixed amount of T cells, we used tumor cell to T
cell ratios of 1:8, 1:4 and 1:2. T cell numbers were counted by
trypan exclusion and CART cell fractions were examined by flow
cytometry. Under culture with CAIX- sk-rc-59 tumor cells, the
number of transduced and untransduced T cells was maintained (FIG.
4A bottom). The lack of basal level of proliferation of control T
cells might be due to the high amount of suppressive cytokines
secreted by the tumor cell line. In contrast, after two weeks of
culture with CAIX+ sk-rc-52 tumor cells, at ratio 1:8, the
population of G36-CD28z CART cells increased to 30-fold and
G36-CD8z CART cells proliferated up to 17-fold whereas at a ratio
of 1:4, the number of G36-CD28z CART cells increased 19-fold and
G36-CD8z CART cells proliferated 4-fold. With higher amounts of
tumor cells, neither G36-CD28z or G36-CD8z CART cells could
proliferate. Irrelevant A8-CD28z CART cells and control T cell LAK
showed no proliferation with tumor cells (FIG. 4A top).
[0276] Proliferating T cells were also harvested to examine their
enrichment on CAIX+ tumor cell contact. On CAIX- tumor contact,
there was no change in the percentage of any CART cells within the
population. However on contact with CAIX+ sk-rc-52 tumor cells,
there was enrichment in both populations of G36 CART cells. For
G36-CD28z CART cells, the positive population was enriched from 18%
on day 0 to 52% on day 8 to 88% on day 16. Expression of G36-CD8z
CART cells was enriched from 19% on day 0 (same levels at T cells
only) to 32% on day 8, and to 72% on day 16. No expansion of
A8-CD28z CART cells was seen over the two week study (FIG. 4B). The
percentage of CD8 cells remained constant throughout the 16 day
study under all conditions (FIG. 4C).
Example 6: Persistent Effector Function of Cart Cells after
Re-Contact with Tumor
[0277] Transduced T cells that were in contact with irradiated
tumor cells for one or two weeks were also tested for cytokine
secretion after 24 hours of contact with fresh non-irradiated tumor
cells. Upon contact with CAIX+ tumor (sk-rc-52) for one or two
weeks, G36-CD28z and G36-CD8z CART cells showed similar IFN-.gamma.
secretion levels although costimulatory signaling through G36-CD28z
CAR yielding 2.times. to 2.5.times. more IFN-.gamma. secretion than
seen for G36-CD8z CAR (Table 1). For IL-2 secretion, two weeks of
tumor contact for G36-CD28z and G36-CD8z CART cells exhibited more
IL-2 secretion than one week of contact. G36-CD28z CART cells
yielding 5.times. more IL2 than G36-CD8z CART cell on one week of
contact and 2.5.times. more on contact for two weeks. In addition,
G36-CD28z CART cells in contact with tumor cells for two weeks
secreted 3.3.times. more IL-2 than one time tumor contact whereas
G36-CD8z CART gave 6.8.times. more IL-2 secretion after two weeks
compared to after one week of tumor contact. These results indicate
that the transduced CART cells did not become exhausted and
maintained functional activity after a second tumor stimulation.
Only background levels of INF-.gamma. and IL-2 secretion were seen
with A8-CD28z, LAK and G36 CART cell treatments on contact with
CAIX- sk-rc59 cells.
Example 7: Suppression of Established Tumor by Cart Cells
[0278] We next tested CART cells to inhibit established tumor cell
growth in nude mice that were inoculated with sk-rc-52 tumor cells
on left flank and sk-rc-59 tumor cells on right flank that had been
established to yield similar tumor curves. On day 7 after tumor
implantation, with typical tumor size of .about.6.times.6 mm, 50
million G36-CD28z CART cells, A8-CD28z CART cell or untransduced T
cells (LAK) were injected intravenously. Adoptive T-cell therapy
was performed in two separate experiments with group sizes of n=7
in the first trial and n=8 in the second trial, in the presence of
high dose IL-2 (2.times.10.sup.5 IU) via intraperitoneal injection.
No T-cell treatment was included in order to compare the growth of
tumor and the effect of cell-therapy.
[0279] In trial one, treated and untreated CAIX- sk-rc-59 tumors
had average size of 6.09.+-.0.02 mm on day 4 and 9.29.+-.0.12 mm on
day 25 (within four tested groups). They exhibited the same tumor
growth rate in control groups and T-cell treated groups. Untreated
CAIX+ tumors that received no T cells showed similar tumor size as
CAIX- tumors, with an average size of 6.09.+-.0.13 mm on day 4 and
9.15.+-.0.11 mm on day 25. However, the tumor size of G36-CD28z
CART cell treated mice showed statistically significant reduction
in size compared to no T-cell treated mice at every time point that
was examined over the 25 day study (FIG. 5). G36-CD28z CART
treatment also led to a greater reduction in tumor size than seen
with A8-CD28z CART cell and LAK treated mice on day 7 (p<0.05)
and on day 25 (p<0.001), as calculated by two-tailed t test. In
trial two, tumor size of G36-CD28z CART cell treated mice was
significant smaller than that of no T-cell treated mice through the
29 day experiment. G36-CD28z CART cell treated mice also had
smaller tumors than were seen with A8 CD28z CART cell and LAK
treated mice on day 8 to day 26 with p<0.01 and on day 29 with
p<0.001 (FIG. 5).
[0280] Partial regression of CAIX+ tumor was considered when the
tumor size was smaller than 30% volume of control CAIX- tumor in a
same mouse receiving the same T-cell. Partial tumor regression was
observed in a high percentage of cases using G36-CD28z CART cells
(10 out of 15, (67%)), but only infrequently in irrelevant target
A8-CD28z CART cells (1 out of 15, (7%)) and in activated T cell
LAKs (2 out of 15, (13%)) (Table 2). Frequency of partial
regression response was found to be statistically significant for
mice treated with G36-CD28z CART cells versus control A8-CD28z CART
cells and LAKs at p<0.001 and p<0.005, respectively by Fisher
test.
Example 8: In Situ Cytotoxicity by Cart Cells
[0281] A sample of the whole population of transduced T cells used
for the in vivo study were pre-stained with Far red dye and the
CART cells expressing ZsGreen protein within the population were
analyzed by confocal microscopy. These results demonstrated circa
30% transduction efficiency which is in agreement with our FACS
analysis (FIG. 6A).
[0282] To provide evidence that G36-CD28z CART cell treatment of
CAIX+ sk-rc-52 tumor cells in vivo resulted in killing by
apoptosis, tumor sections were stained by Tunnel assay. On day 3
after adoptive T cell treatment, Tunnel staining identified
apoptotic tumor cells (red) at the edge of tumor (FIG. 6B upper
row) and inside the tumor bed (FIG. 6B middle row). The apoptoic
tumor cells lost the DAPI nuclear staining. Shown in the enlarged
graph (FIG. 6B bottom row) is a ZsGreen expressing CART cell
interacting with two tumor cells that were going apoptosis.
[0283] Due to the limitation of fluorescent signal, ZsGreen
expressing CART cells could not be observed from the whole tissue
section. Therefore on day 3 after G36-CD28z CART cell or LAK
treatment, the tumors were harvested and sections were also stained
with granzyme B antibody to locate the activated T cells. In FIG.
6C, the dark brown areas of staining show granzyme B+ T cells that
are seen infiltrating into the CAIX+ sk-rc-52 tumor sections (FIG.
6C upper left). These granzyme B+ T cells were seen surrounding the
tumor (FIG. 6C upper left (a) and middle) and inside the tumor
(FIG. 6C upper left (b) and lower). Tumors with necrotic areas were
shown in H&E stained slides (labeled as n inside FIG. 6C right
middle and lower) and lie at locations near to the granzyme B+ T
cells. In contrast, the CAIX+ sk-rc-52 tumors treated with control
activated T cells (LAK) (FIG. 7) did not show any granzyme B+ T
cells. Similarly, CAIX- sk-rc-59 treated with G36-CD28z CART cells
(FIG. 8) or treated with LAK (FIG. 9) showed a low background
staining while tumor was proliferating. For positive control of
granzyme B staining, CART cells was locally injected into the
established sk-rc-52 tumor in mice. After one day, the mice was
sacrificed and tumor tissue was sectioned for this staining (FIG.
10).
TABLE-US-00008 TABLE 1 Cytokine Secretion After One or Two Weeks of
Contact with Tumor Cells* IFN-.gamma. (pg/ml) IL-2 (pg/ml) CART
cells One week Two weeks One week Two weeks RC-SK-52 (CAIX+) Cells
G36-CD28z 25,788 28,192 7,524 24,937 G36-CD8z 13,096 10,961 1,470
10,029 A8-CD28z 55 55 9 13 LAK 68 58 9 13 RC-SK-59 (CAIX-) Cells
G36-CD28z 31 29 5 4 G36-CD8z 27 38 8 10 A8CD28z 56 55 7 8 LAK 49 56
10 8 *Transduced T cells were incubated with irradiated tumor cells
for one or two weeks then harvested, washed and incubated with
fresh non-irradiated tumor cells overnight and supernatants
collected after 24 hrs for cytokine analysis. For T cell cultures
that did not interact with tumor cells, only background level of
cytokines were detected at levels <50 pg/ml IFN-.gamma. and
<10 pg/ml IL-2.
TABLE-US-00009 TABLE 2 Frequency of Partial Regression of CAIX+
Tumors by G36-CD28z CART cells Gene construct LAK A8-CD28z
G36-CD28z Statistics Target antigen none irrelevant specific
Co-stimulatory none 2 signals 2 signals Partial 2 1 10 p <
0.005*; response p > 0.001** Non-partial 13 14 5 N.S. response
Mice from experiments reported in FIG. 5 (experiment 1, n = 7 &
experiment 2, n = 8) were scored for response at day 10. Partial
response is defined as the regression of tumor to smaller than 30%
volume of control tumor (same T-cell treatment in the same mouse
bearing left flank of sk-rc-52 and right flank of control tumor
sk-rc-59). Fisher test results - *G36-CD28z verses LAK; **G36-CD28z
verses A8-CD28z; N.S.--no statistically significant relationship
between number of tumors and partial response between T cells
transduced with LAK and with A8-CD28z.
Example 9: Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T
Cells Releasing Anti-PD-L1 Antibodies Revert T Cell Exhaustion and
Regress Renal Cell Carcinoma in a Humanized Mouse Model
[0284] Characterization of Anti-CAIX CAR T Cells Secreting
Anti-PDL1 IgG1 or IgG4
[0285] In order to develop a new CAR therapy for CAIX+ RCC, we
engineered a bicistronic lentiviral vector to express the anti-CAIX
(G36) scFv linked to CD28 and CD3-signaling domains (G36-CD28z CAR
in the first cassette and anti-PD-L1 IgG1 or IgG4 in a second
expression cassette after an IRES site (FIG. 16A). The anti-PD-L1
IgG sequence was inserted into the lentiviral vector with the aim
to block T-cell exhaustion, which we propose may improve the
efficacy of G36-CD28z CART cells (FIG. 16B). As controls, we used
an anti-CAIX CAR or anti-BCMA CAR containing an irrelevant
anti-SARS IgG1 mAb in the second cassette. The lentiviruses
generated from these constructs were transduced into CD8 T cells
and cultivated in the presence of IL-21, which yielded CART cells
with modestly improved proliferation than was seen with IL-2 (FIGS.
21A and 21B), while maintaining the same specific killing activity
for CAIX+ RCC (FIGS. 21C and 21D). The percentage of CAIX and PD-L1
expression in all RCC lineages used in our experiments is shown in
the FIG. 22.
[0286] The CART cell functionality is demonstrated in FIG. 23,
where CD8 T cells transduced with all CARS were able to proliferate
in the presence of IL-21 and anti-CD8/CD28 beads (FIGS. 23A and
23B), achieving transduction levels of 65-90% after four days (FIG.
23C). Fourteen days after transduction, we evaluated the stable
long-term expression of CAR by the integrated lentiviruses (FIG.
16C), which was maintained around 25-50% for all CARs. Total IgG
levels secreted by CD8 T cells was also determined, ranging around
300-650 ng/mL after 4 days (FIG. 16D). The binding specificity of
the anti-PD-L1 IgG1 and IgG4 antibodies for human PD-L1 was also
confirmed (FIG. 16E). The levels of biotinylated anti-PDL1 IgG1 and
4 were significantly lower than total IgG, which could be explained
by the fact that a portion of the IgG was wasted during the
purification process. The ability of anti-CAIX CART cells to
undergo clonal expansion in the presence of CAIX+ RCC cells was
established (FIGS. 16F and 16G). Anti-CAIX CART cells cannot expand
significantly in the presence of CAIX- RCC cells.
[0287] Effector Activity of Anti-CAIX CART Cells.
[0288] All anti-CAIX CART cells were able to induce around 50-70%
decrease in the viability of Skrc59 CAIX+/PD-L1+, indicating that
the Anti-PD-L1 IgG1 and IgG4, secreted by some CART cells, did not
augment cell death under these assay conditions (FIG. 17A).
Anti-CAIX CART cells induced IL-2 and IFN.gamma. release in the
presence of CAIX+/PD-L1+ cells, indicating the specific activation
of anti-CAIX CART cells (FIGS. 17C and 17E, respectively).
Regarding the anti-CAIX CART cells secreting anti-PD-L1 IgGs, the
unique differential effect was seen for the IgG1 isoform, which was
able to induce around 60% of ADCC in CAIX+/PD-L1+ RCC cells when
incubated with natural killer cells (NK) (FIG. 17G). No effect in
cell viability, cytokines secretion or ADCC was detected for any of
the CART cells in the presence of CAIX-/PD-L1- cells (FIGS. 17B,
17D, 17F and 17H).
[0289] Anti-CAIX CART Cells-Secreting Anti-PD-L1 Antibodies can
Diminish T Cell Exhaustion In Vitro.
[0290] A circa 50% decrease in the exhaustion markers Lag-3, Tim3
and PD-1 was found in the anti-PD-L1 IgG1 and IgG4 anti-CAIX CART
groups (FIGS. 18A, 18B and 18C, respectively) after induction of
exhaustion compared to the parental anti-CAIX or irrelevant
anti-BCMA CART cells. At this point, the killing activity of
anti-CAIX CART cells without anti-PD-L1 was retested over Skrc59
CAIX+/PD-L1+ cells. As can be seen in FIG. 18D, the anti-CAIX CART
cells had lost their killing activity against CAIX+/PD-L1+ RCC in
vitro to a level that was similar to the irrelavent CAR group,
establishing that the anti-CAIX CART cells became anergic. In
contract, the RCC viability diminished 25-50% for the anti-CAIX CAR
anti-PD-L1 IgG1 and IgG4 CART groups, thereby providing evidence
that the checkpoint blockade elicited by the presence of secreted
anti-PD-L1 IgGs can lead to diminished T cell exhaustion.
[0291] Anti-CAIX CART Cells Secreting Anti-PD-L1 Antibodies can
Further Decrease Tumor Growth in an Orthotopic Mouse Model of Human
RCC.
[0292] NSG mice were used to establish an orthotopic RCC model by
injecting Skrc-59 CAIX+PD-L1+luciferase+ positive RCC cells under
the kidney capsule followed by i.v. injection of 1.0.times.107 CART
or untransduced T cells (Day 0) and repeat treatment on Day 17 with
a lower dose (2.5.times.10.sup.6) of the same cells. We did not
treat the mice with systemic IL-2 to maintain the proliferation of
CART cells to avoid the bias that this molecule could exert over
the tumor growth. The data in FIGS. 19A-19C demonstrate that all
three anti-CAIX CART cell groups showed decreased RCC growth
compared to irrelevant anti-BCMA CART cells or untransduced cells
over the course of the experiment with the marked anti-tumor
effects of the anti-CAEX CART cells secreting anti-PD-L1 IgG1 or
IgG4 becoming evident at day 23 and 30 (FIGS. 19A and 19B).
However, even one week after i.v. treatment with CART cells, we
observed that the tumors were 2-3 times smaller in the
anti-PD-L1-secreting CART cells when compared with parental
anti-CAIX CART cells and the two control groups (FIG. 24A). We also
analyzed CD45+ T cell survival in the mouse blood to gauge their
survival in this passive transfer model. At Day 8 we observed that
the amount of human T cells within the PBMCs was only 10-15% (FIG.
24B) and for this reason we decided to expand the CART cells in
vitro to perform a second injection into the mail blood at Day 17.
One week after the second injection (Day 23) the anti-PD-L1 IgG1
and IgG4 groups had tumors 15 times smaller than the control groups
and 5 times smaller than the anti-CAIX CAR T cells without
anti-PD-L1 secretion (FIG. 19C and FIG. 24A). At day 30, the group
of mice treated with CART cells-secreting anti-PD-L1 antibody had
tumors 5 times smaller than the control groups (FIG. 19C and FIG.
24A). The excised tumors weights were also lower from the mice
treated CART cells-secreting anti-PD-L1 antibody and that was
particularly evident for the anti-PDLL IgG4 antibody group (FIGS.
19B and 19D).
[0293] Analysis of CART Cell Tumor Infiltrates and Evidence that
Anti-CAIX CART Cells Secreting Anti-PD-L1 Antibodies can Lead to
Reversal of T Cell Exhaustion.
[0294] Analysis in the excised tumors showed around 10% of TIL in
all groups (FIG. 24C)
[0295] One of the most important effects observed with the
anti-CAIX CART cells secreting anti-PD-L1 IgG1 or IgG4 antibodies
in vivo was their ability to decrease the expression of the
exhaustion markers PD-1, Tim-3 and Lag-3. As shown in FIG. 20A, we
observed a circa 40-50% decrease in the expression of these markers
in the TIL's when compared with the treatment using untransduced
control cells at the Day 30 There was also a circa 30-70% decrease
compared to anti-CAIX CAR cells That were not secreting anti-PDL-1
antibodies thereby providing evidence that the locally secreted
antibodies had an effect on decreasing T cell exhaustion.
[0296] The effector activity of CART cells and their influence over
RCC proliferation in vivo was evaluated by the immunohistochemical
detection of granzyme B in the TIL, the tumor proliferation marker
Ki67 and the tumor immunosuppressive protein PD-L1 (FIGS. 20B and
20C). The granzyme B staining showed the effector activity of CD8+
cells, especially in the RCC tumors treated with the anti-CAIX CART
cells secreting anti-PD-L1 IgG4, which presented an augmented
percentage of high positive stained cells (FIGS. 20B and 20C).
PD-L1 expression decreased dramatically in the tumors treated with
Antianti-CAIX CART cells, being more expressive in the
Anti-CAIX/Anti-PD-L1 IgG-secreting groups (FIGS. 20B and 20C). Ki67
expression decreased significantly in the Anti-CAIX/Anti-PD-L1
IgG-secreting groups, as we can visualize in the total DAB pixel
count graph, however when the intensity among the positive nuclei
staining (Ki67 IHC quantification graph) was evaluated, we can note
that the Anti-CAIX/Anti-PD-L1 IgG4 presented the lowest intensity
of Ki67 expression (FIGS. 20B and 20C). For the quantification of
nuclear proteins, like Ki67, DAB staining pattern is confined to
the nuclei, and the threshold feature of ImageJ should be used to
select the positive stained areas for quantification. For this
reason, there are no negative cells in the Ki67 IHC quantification
graph (FIG. 20C) and DAB pixel count were also shown for evaluation
of total Ki67 staining, including negative cells (FIG. 20C).
[0297] Anti-CAIX CART Cells Secreting Anti-PD-L1 IgG1 Antibodies
can Recruit NK Cells to the Tumor.
[0298] The tumors of the mice treated with Anti-CAIX/Anti-PD-L1
IgG1 that received an injection of NK cells 2 days before the
euthanasia showed the presence of 40% more NK cells inside the
tumor, when compared with Anti-BCMA/Anti-SARS IgG1, as detected by
Anti-CD56 staining by FACS (FIG. 25A). The increase in NK cells in
the Anti-CAIX/Anti-PD-L1 IgG1 group was also detected and
quantified by IHC (FIG. 25B). Very few NK cells were also detected
in the groups treated with CART cells secreting an unspecific IgG1
Ab.
Other Embodiments
[0299] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
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Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170362297A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170362297A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References