U.S. patent application number 16/923387 was filed with the patent office on 2021-05-20 for lym-1 and lym-2 targeted car cell immunotherapy.
The applicant listed for this patent is University of Southern California. Invention is credited to Alan L. Epstein.
Application Number | 20210147551 16/923387 |
Document ID | / |
Family ID | 1000005358513 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210147551 |
Kind Code |
A1 |
Epstein; Alan L. |
May 20, 2021 |
LYM-1 AND LYM-2 TARGETED CAR CELL IMMUNOTHERAPY
Abstract
CAR cells targeting and antibodies human HLA-DR are described as
a new method of cancer treatment. It is proposed that HLA-DR CAR
cells are safe and effective in patients and can be used to treat
human tumors expressing the HLA-DR.
Inventors: |
Epstein; Alan L.; (Pasadena,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Southern California |
Los Angeles |
CA |
US |
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|
Family ID: |
1000005358513 |
Appl. No.: |
16/923387 |
Filed: |
July 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15173534 |
Jun 3, 2016 |
10711064 |
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16923387 |
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62171004 |
Jun 4, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70517 20130101;
G01N 2333/70539 20130101; C07K 14/70521 20130101; C07K 2317/92
20130101; C07K 2317/73 20130101; C07K 2319/33 20130101; G01N
2800/52 20130101; C07K 16/2833 20130101; A61K 39/39558 20130101;
C07K 2317/622 20130101; G01N 33/57492 20130101; C07K 2319/03
20130101; A61K 2039/505 20130101; C07K 14/70578 20130101; C07K
14/7051 20130101; C07K 2317/565 20130101; C07K 2319/02
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/705 20060101 C07K014/705; C07K 14/725 20060101
C07K014/725; A61K 39/395 20060101 A61K039/395 |
Claims
1.-51. (canceled)
52. An isolated anti-Lym-2 antibody or an antigen binding fragment
thereof comprising a heavy chain variable region comprising
complementarity-determining regions (CDRs) comprising SEQ ID NOs:
2, 4, and 6; and a light chain variable region comprising CDRs
comprising SEQ ID NOs: 12, 14, and 16.
53. The isolated anti-Lym-2 antibody or the antigen binding
fragment thereof of claim 52, wherein the heavy chain variable
region comprises a polypeptide comprising at least 90% sequence
identity to SEQ ID NO: 10 and comprising the CDRs comprising of SEQ
ID NOs: 2, 4, 6, 12, 14 and 16.
54. The isolated anti-Lym-2 antibody or the antigen binding
fragment thereof of claim 52, wherein the heavy chain variable
region comprises SEQ ID NO: 10.
55. The isolated anti-Lym-2 antibody or the antigen binding
fragment thereof of claim 52, wherein the light chain variable
region comprises a polypeptide comprising at least 90% sequence
identity to SEQ ID NO: 20 and comprising the CDRs comprising of SEQ
ID NOs: 2, 4, 6, 12, 14 and 16.
56. The isolated anti-Lym-2 antibody or the antigen binding
fragment thereof of claim 52, wherein the light chain variable
region comprises SEQ ID NO: 20.
57. The isolated anti-Lym-2 antibody or the antigen binding
fragment thereof of claim 52, wherein the fragment of the isolated
anti-Lym-2 antibody is a scFv.
58. The isolated anti-Lym-2 antibody of claim 52, wherein the
isolated anti-Lym-2 antibody is a monoclonal antibody.
59. An isolated nucleic acid sequence encoding the isolated
anti-Lym-2 antibody or the antigen binding fragment thereof of
claim 52 or its complement or an equivalent of each thereof.
60. A vector comprising the isolated nucleic acid sequence of claim
59.
61. An isolated cell comprising the isolated anti-Lym-2 antibody or
the antigen binding fragment thereof of claim 52.
62. A complex comprising the isolated anti-Lym-2 antibody or the
antigen binding fragment thereof of claim 52 bound to a cell
expressing HLA-DR.
63. A method of inhibiting the growth of a tumor or treating a
cancer that overexpresses HLA-DR as compared to a normal,
non-cancerous counterpart cell in a subject in need thereof,
comprising administering to the subject an effective amount of the
isolated anti-Lym-2 antibody or the antigen binding fragment
thereof of claim 52.
64. The method of claim 63, further comprising administering to the
subject an anti-tumor therapy other than an HLA-DR CAR therapy.
65. The method of claim 63, wherein the tumor is cancerous.
66. The method of claim 63, wherein the cancer is a B-cell lymphoma
or a leukemia.
67. The method of claim 63, wherein the subject is a mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application No. 62/171,004, filed Jun.
4, 2015, the content of which is hereby incorporated by reference
in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 1, 2016, is named 064189-7201_SL.txt and is 51,805 bytes in
size.
BACKGROUND
[0003] The present disclosure relates generally to the field of
human immunology, specifically cancer immunotherapy.
[0004] The following discussion of the background is merely
provided to aid the reader in the understanding the disclosure and
is not admitted to describe or constitute prior art to the present
disclosure.
[0005] Lym-1 and Lym-2 are directed against MHC class II HLA-DR
molecules which are primarily expressed on the surface of human B
cells, dendritic cells, and B-cell derived lymphomas and
leukemias.
SUMMARY
[0006] Provided are methods and compositions relating to new cancer
immunotherapeutic chimeric antigen receptors (CARs). Aspects of the
disclosure relate to a chimeric antigen receptor (CAR) comprising,
or alternatively consisting essentially of, or yet further
consisting of: (a) an antigen binding domain of a Lym-1 and/or
Lym-2 antibody; (b) a hinge domain; (c) a transmembrane domain; and
(d) an intracellular domain. Further aspects of the disclosure
relate to a chimeric antigen receptor (CAR) comprising, or
alternatively consisting essentially of, or yet further consisting
of: (a) an antigen binding domain of a Lym-1 and/or Lym-2 antibody;
(b) a CD8 .alpha. hinge domain; (c) a CD8 .alpha. transmembrane
domain; (d) a CD28 costimulatory signaling region and/or a 4-1BB
costimulatory signaling region; and (e) a CD3 zeta signaling
domain.
[0007] Aspects of the disclosure relate to Lym-1 and Lym-2
antibodies.
[0008] Some aspects of the disclosure relate to a chimeric antigen
receptor (CAR) comprising an antigen binding domain specific to
human HLA-DR antigens--for example, the antigen binding domain of
Lym-1 and Lym-2 antibodies.
[0009] Further aspects of the disclosure relate to an isolated
nucleic acid sequence encoding a Lym1 or Lym-2 CARs and vectors
comprising the isolated nucleic acid sequences.
[0010] Other aspects of the disclosure relate to an isolated cell
comprising a Lym-1 or Lym-2 directed CAR and methods of producing
such cells. Still other method aspects of the disclosure relate to
methods for inhibiting the growth of a tumor and treating a cancer
patient comprising, or alternatively consisting essentially of, or
yet further consisting of, administering an effective amount of the
isolated cell to a tissue or subject in need of such.
[0011] Further method aspects of the disclosure relate to methods
for determining if a patient is likely or unlikely to respond to
Lym-1 CAR or Lym-2 CAR therapy through use of one or more of the
Lym-1 or Lym-2 antibodies and/or the Lym-1 CAR or Lym-2 CAR
cells.
[0012] Additional aspects of the disclosure relate to compositions
comprising a carrier and one or more of the products described in
the embodiments disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-1F show flow cytometric analysis of (FIG. 1A)
negative control; (FIG. 1B) Lym-1; (FIG. 1C) Lym-1 and B1; (FIG.
1D) B1 only; (FIG. 1E) Lym-2; and (FIG. 1F) Lym-2 and B1 staining
reactivity with normal peripheral blood lymphocytes of patients.
Both Lym-1 and Lym-2 have different profiles of binding to normal
human peripheral B cells.
[0014] FIGS. 2A-2B show Lym-1 and Lym-2 staining of normal human
tonsil demonstrating membrane positivity in B-cell germinal
centers. Differences in staining patterns are evident between Lym-1
(FIG. 2A) and Lym-2 (FIG. 2B). Only scattered interfollicular
dendritic cells are positive for both antibodies in the T-cell
zones (IHC, frozen sections, .times.325).
[0015] FIGS. 3A and 3B show immunoperoxidase staining of Lym-1 and
Lym-2 monoclonal antibodies with an intermediate grade malignant
B-cell lymphoma. Immunoperoxidase staining of Lym-1 (FIG. 3A) and
Lym-2 (FIG. 3B) monoclonal antibodies with an intermediate grade
malignant B-cell lymphoma (frozen sections, .times.720). Note
prominent membrane staining pattern of majority of cells in the
section.
[0016] FIGS. 4A-4C show binding profiles and Scatchard Plots of
(FIG. 4A) Binding profiles of Lym-1 monoclonal antibodies to Raji
cells and Lym-2 monoclonal antibodies to ARH-77 cells; (FIG. 4B)
Scatchard plot analysis of Lym-1 monoclonal antibodies with Raji
cells; (FIG. 4C) Scatchard plot analysis of Lym-2 monoclonal
antibodies with ARH-77 cells.
[0017] FIGS. 5A and 5B show immunoprecipitation of
.sup.35S-methionine and .sup.14C-leucine-labeled Raji proteins by
Lym-1 (FIG. 5A) and SC-2 anti-HLA-DR antibody (FIG. 5B).
[0018] FIGS. 6A and 6B show a construction schematic of (FIG. 6A)
Lym-1 and (FIG. 6B) Lym-2 CAR T-cells for immunotherapy. FIGS. 6A
and 6B disclose SEQ ID NO: 51.
[0019] FIG. 7 depicts a schematic a non-limiting exemplary Lym-1
gene-transfer vector and transgene. The backbone of the gene
transfer vector is an HIV-based, bicistronic lentiviral vector,
pLVX-IRES-ZsGreen containing HIV-1 5' and 3' long terminal repeats
(LTRs), packaging signal (.PSI.), EF1.alpha. promoter, internal
ribosome entry site (IRES), ZsGreen, a green fluorescent protein,
woodchuck hepatitis virus post-transcriptional regulatory element
(WPRE), and simian virus 40 origin (SV40). Constitutive expression
of the transgene comprising of .alpha.CD8 leader sequence, a scFV
specific to Lym-1, a CD8 hinge and transmembrane region and 4-1BB
and CD3.zeta. signaling domain, is insured by the presence of the
EF-1.alpha. promoter. Expression of the detection protein, ZsGreen
is carried out by the IRES region. Integration of the vector can be
assayed by the presence of ZsGreen in the cells, via fluorescent
microscopy.
[0020] FIG. 8 shows expression of Lym-1 CAR on primary human
T-cells. T-cells were transduced with the Lym-1 CAR and stained
with Biotein-Protein L, followed by Streptavidin-PE. Cells were
analyzed by flow cytometry.
[0021] FIG. 9 shows cytotoxicity of the Lym-1-CAR T-cells.
Cytotoxicity of the Lym-1 CAR expressing T-cells was determined
using an LDH cytotoxicity kit as described in the Methods. Prior to
the assay, T-cells were activated using .alpha.CD3/CD8 beads (Stem
Cell Technologies, 30 ul to 2 ml of media). The activated T-cells
were transduced with Lym-1 CAR lentiviral particles, following
which the T cells were activated using the .alpha.CD3/CD8 beads.
Un-transduced, activated T-cells were used as a control. 15,000
Raji cells were plated per well. Lym-1 CAR transduced T cells were
added in ratios of 20:1, 10:1, 5:1 and 1:1 to the wells. Each data
point represents the average of triplicate measurements.
[0022] FIG. 10 depicts a schematic a non-limiting exemplary Lym-2
gene-transfer vector and transgene. The backbone of the gene
transfer vector is an HIV-based, bicistronic lentiviral vector,
pLVX-IRES-ZsGreen containing HIV-1 5' and 3' long terminal repeats
(LTRs), packaging signal (.PSI.), EF1.alpha. promoter, internal
ribosome entry site (IRES), ZsGreen, a green fluorescent protein,
woodchuck hepatitis virus post-transcriptional regulatory element
(WPRE), and simian virus 40 origin (SV40). Constitutive expression
of the transgene comprising of a CD8 leader sequence, an scFV
specific to Lym-2, a CD8 hinge and transmembrane region and CD28,
4-1BB and CD3.zeta. signaling domain, is insured by the presence of
the EF-1.alpha. promoter. Expression of the detection protein,
ZsGreen is carried out by the IRES region. Integration of the
vector can be assayed by the presence of ZsGreen in the cells, via
fluorescent microscopy.
[0023] FIG. 11 shows expression of Lym-2 CAR on primary human
T-cells. T-cells were transduced with the Lym-2 CAR and stained
with Biotein-Protein L, followed by Streptavidin-PE. Cells were
analyzed by flow cytometry.
[0024] FIG. 12 shows cytotoxicity of the Lym-2-CAR T-cells.
Cytotoxicity of the Lym-2 CAR expressing T-cells was determined
using an LDH cytotoxicity kit as described in the Methods. Prior to
the assay, T-cells were activated using .alpha.CD3/CD8 beads (Stem
Cell Technologies, 30 ul to 2 ml of media). The activated T-cells
were transduced with Lym-2 CAR lentiviral particles, following
which the T cells were activated using the .alpha.CD3/CD8 beads.
Un-transduced, activated T-cells were used as a control. 15,000
Raji cells were plated per well. Lym-2 CAR transduced T cells were
added in ratios of 20:1, 10:1, 5:1 and 1:1 to the wells. Each data
point represents the average of triplicate measurements.
[0025] FIG. 13 demonstrates that Lym-1, Lym-2, and CD19 CAR T-cells
are highly cytotoxic to human lymphoma Raji cells. Raji Burkitt's
lymphoma cells are positive for both HLA-Dr targeted by Lym-1 and
Lym-2 and also CD19 which acted as a positive control for CD19 CAR
T-cells. Negative controls consisted of CD3+ T cells and Zsgreen
cells.
[0026] FIG. 14 demonstrates that Lym-1, Lym-2, but not CD19 CAR are
highly cytolytic against HLA-Dr positive but CD19 negative TLBR-2
human T lymphoma cells in vitro. TLBR-2 human T-lymphoma cells
derived from a breast implant associated lymphoma is positive for
HLA-Dr but not CD19 (Lechner et al. (2012) Clin. Cancer Res. 18
(17):4549-4559). These results demonstrate the specificity of the
Lym-1 and Lym-2 CAR T-cells and their potency in killing HLA-Dr
positive tumors. The percentage of Lym-1 CAR-T and CD19 CAR-T
positive cells were adjusted to 50% using regular un-transduced
primary T cells. The percentage of Lym-2 CAR-T cells was 24%.
[0027] FIG. 15 shows the results of FACs analysis of transfected NK
cells.
DETAILED DESCRIPTION
[0028] It is to be understood that the present disclosure is not
limited to particular aspects described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular aspects only, and is
not intended to be limiting, since the scope of the present
disclosure will be limited only by the appended claims.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this technology belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present technology, the preferred methods, devices and materials
are now described. All technical and patent publications cited
herein are incorporated herein by reference in their entirety.
Nothing herein is to be construed as an admission that the present
technology is not entitled to antedate such disclosure by virtue of
prior invention.
[0030] The practice of the present technology will employ, unless
otherwise indicated, conventional techniques of tissue culture,
immunology, molecular biology, microbiology, cell biology, and
recombinant DNA, which are within the skill of the art. See, e.g.,
Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory
Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current
Protocols in Molecular Biology; the series Methods in Enzymology
(Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A
Practical Approach (IRL Press at Oxford University Press);
MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and
Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)
Culture of Animal Cells: A Manual of Basic Technique, 5th edition;
Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195;
Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson
(1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984)
Transcription and Translation; Immobilized Cells and Enzymes (IRL
Press (1986)); Perbal (1984) A Practical Guide to Molecular
Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for
Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed.
(2003) Gene Transfer and Expression in Mammalian Cells; Mayer and
Walker eds. (1987) Immunochemical Methods in Cell and Molecular
Biology (Academic Press, London); and Herzenberg et al. eds (1996)
Weir's Handbook of Experimental Immunology.
[0031] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 1.0 or
0.1, as appropriate, or alternatively by a variation of +/-15%, or
alternatively 10%, or alternatively 5%, or alternatively 2%. It is
to be understood, although not always explicitly stated, that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
[0032] It is to be inferred without explicit recitation and unless
otherwise intended, that when the present technology relates to a
polypeptide, protein, polynucleotide or antibody, an equivalent or
a biologically equivalent of such is intended within the scope of
the present technology.
Definitions
[0033] As used in the specification and claims, the singular form
"a", "an", and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0034] As used herein, the term "animal" refers to living
multi-cellular vertebrate organisms, a category that includes, for
example, mammals and birds. The term "mammal" includes both human
and non-human mammals.
[0035] The terms "subject," "host," "individual," and "patient" are
as used interchangeably herein to refer to human and veterinary
subjects, for example, humans, animals, non-human primates, dogs,
cats, sheep, mice, horses, and cows. In some embodiments, the
subject is a human.
[0036] As used herein, the term "antibody" collectively refers to
immunoglobulins or immunoglobulin-like molecules including by way
of example and without limitation, IgA, IgD, IgE, IgG and IgM,
combinations thereof, and similar molecules produced during an
immune response in any vertebrate, for example, in mammals such as
humans, goats, rabbits and mice, as well as non-mammalian species,
such as shark immunoglobulins. Unless specifically noted otherwise,
the term "antibody" includes intact immunoglobulins and "antibody
fragments" or "antigen binding fragments" that specifically bind to
a molecule of interest (or a group of highly similar molecules of
interest) to the substantial exclusion of binding to other
molecules (for example, antibodies and antibody fragments that have
a binding constant for the molecule of interest that is at least
10.sup.3 M.sup.-1 greater, at least 10.sup.4M.sup.-1 greater or at
least 10.sup.5 M.sup.-1 greater than a binding constant for other
molecules in a biological sample). The term "antibody" also
includes genetically engineered forms such as chimeric antibodies
(for example, humanized murine antibodies), heteroconjugate
antibodies (such as, bispecific antibodies). See also, Pierce
Catalog and Handbook (1994-1995) (Pierce Chemical Co., Rockford,
Ill.); Kuby, J. (1997) Immunology, 3.sup.rd Ed., W.H. Freeman &
Co., New York. An "antigen binding fragment" of an antibody is a
portion of an antibody that retains the ability to specifically
bind to the target antigen of the antibody.
[0037] As used herein, the term "monoclonal antibody" refers to an
antibody produced by a single clone of B-lymphocytes or by a cell
into which the light and heavy chain genes of a single antibody
have been transfected. Monoclonal antibodies are produced by
methods known to those of skill in the art, for instance by making
hybrid antibody-forming cells from a fusion of myeloma cells with
immune spleen cells. Monoclonal antibodies include humanized
monoclonal antibodies and human antibodies.
[0038] In terms of antibody structure, an immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda (.lamda.) and kappa
(.kappa.). There are five main heavy chain classes (or isotypes)
which determine the functional activity of an antibody molecule:
IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a
constant region and a variable region, (the regions are also known
as "domains"). In combination, the heavy and the light chain
variable regions specifically bind the antigen. Light and heavy
chain variable regions contain a "framework" region interrupted by
three hypervariable regions, also called
"complementarity-determining regions" or "CDRs". The extent of the
framework region and CDRs have been defined (see, Kabat et al.,
Sequences of Proteins of Immunological Interest, U.S. Department of
Health and Human Services, 1991, which is hereby incorporated by
reference). The Kabat database is now maintained online. The
sequences of the framework regions of different light or heavy
chains are relatively conserved within a species. The framework
region of an antibody, that is the combined framework regions of
the constituent light and heavy chains, largely adopts a
.beta.-sheet conformation and the CDRs form loops which connect,
and in some cases form part of, the .beta.-sheet structure. Thus,
framework regions act to form a scaffold that provides for
positioning the CDRs in correct orientation by inter-chain,
non-covalent interactions.
[0039] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located (heavy chain regions labeled CDHR and
light chain regions labeled CDLR). Thus, a CDHR3 is the CDR3 from
the variable domain of the heavy chain of the antibody in which it
is found, whereas a CDLR1 is the CDR1 from the variable domain of
the light chain of the antibody in which it is found. A TNT
antibody will have a specific V.sub.H region and the V.sub.L region
sequence unique to the TNT relevant antigen, and thus specific CDR
sequences. Antibodies with different specificities (i.e., different
combining sites for different antigens) have different CDRs.
Although it is the CDRs that vary from antibody to antibody, only a
limited number of amino acid positions within the CDRs are directly
involved in antigen binding. These positions within the CDRs are
called specificity determining residues (SDRs).
[0040] As used herein, the term "antigen" refers to a compound,
composition, or substance that may be specifically bound by the
products of specific humoral or cellular immunity, such as an
antibody molecule or T-cell receptor. Antigens can be any type of
molecule including, for example, haptens, simple intermediary
metabolites, sugars (e.g., oligosaccharides), lipids, and hormones
as well as macromolecules such as complex carbohydrates (e.g.,
polysaccharides), phospholipids, and proteins. Common categories of
antigens include, but are not limited to, viral antigens, bacterial
antigens, fungal antigens, protozoa and other parasitic antigens,
tumor antigens, antigens involved in autoimmune disease, allergy
and graft rejection, toxins, and other miscellaneous antigens.
[0041] As used herein, the term "antigen binding domain" refers to
any protein or polypeptide domain that can specifically bind to an
antigen target.
[0042] The term "chimeric antigen receptor" (CAR), as used herein,
refers to a fused protein comprising an extracellular domain
capable of binding to an antigen, a transmembrane domain derived
from a polypeptide different from a polypeptide from which the
extracellular domain is derived, and at least one intracellular
domain. The "chimeric antigen receptor (CAR)" is sometimes called a
"chimeric receptor", a "T-body", or a "chimeric immune receptor
(CIR)." The "extracellular domain capable of binding to an antigen"
means any oligopeptide or polypeptide that can bind to a certain
antigen. The "intracellular domain" means any oligopeptide or
polypeptide known to function as a domain that transmits a signal
to cause activation or inhibition of a biological process in a
cell. In certain embodiments, the intracellular domain may
comprise, alternatively consist essentially of, or yet further
comprise one or more costimulatory signaling domains in addition to
the primary signaling domain. The "transmembrane domain" means any
oligopeptide or polypeptide known to span the cell membrane and
that can function to link the extracellular and signaling domains.
A chimeric antigen receptor may optionally comprise a "hinge
domain" which serves as a linker between the extracellular and
transmembrane domains. Non limiting examples of such domains are
provided herein, e.g.:
Hinge domain: IgG1 heavy chain hinge sequence, SEQ ID NO: 42:
TABLE-US-00001 CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG
Transmembrane domain: CD28 transmembrane region SEQ ID NO: 43:
TABLE-US-00002 TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCT
AGTAACAGTGGCCTTTATTATTTTCTGGGTG
Intracellular domain: 4-1BB co-stimulatory signaling region, SEQ ID
NO: 44:
TABLE-US-00003 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG
ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG
AAGAAGAAGAAGGAGGATGTGAACTG
Intracellular domain: CD28 co-stimulatory signaling region, SEQ ID
NO: 45:
TABLE-US-00004 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCC
CCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCAC
GCGACTTCGCAGCCTATCGCTCC
Intracellular domain: CD3 zeta signaling region, SEQ ID NO: 46:
TABLE-US-00005 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA
AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC
TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
[0043] As used herein, the term "HLA-DR" (refers to an MHC class II
cell surface receptor associated with this name and any other
molecules that have analogous biological function that share at
least 80% amino acid sequence identity, preferably 90% sequence
identity, or alternatively at least 95% sequence identity with any
HLA-DR variant, including but not limited to any one of its several
variants, including but not limited to HLA-DR serotypes DR1 to DR
75 comprising a combination of HLA-DRA and HLA-DRB haplotypes.
Examples of the HLA-DR sequences are known in the art and
non-limited examples of such are disclosed in Rose, L. M. et al.
(1996) Cancer Immunol. Immunother. 43:26-30:
TABLE-US-00006 HLA-DRB1*1001 [DR10] SEQ ID NO: 30
GDTRPRFLEEVKFECHFFNGTERVRLLERRVHNQEEYARYDSDVGEYRAV
TELGRPDAEYWNSQKDLLERRRAAVDTYCRHNYGVGESFTVQRRVQPKVT
VYPSKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQN
GDWTFQTLVMLETVPQSGEVYTCQVEHPSVMSPLTVEWRARSESAQSKML
SGVGGFVLGLLFLGAGLFIYFRNQKGHSGLPPTGFLS; HLA-DRB3*0201 D[R52] SEQ ID
NO: 31 GDTRPRFLELLKSECHFFNGTERVRFLERHFHNQEEYARFDSDVGEYRAV
FELGRPDAEYWNSQKDLLEQKRGQVDNYCRHNYGVVESFTVQRRVHPQVT
VYPAKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQN
GDWTFQTLVMLETFPRSGEVYTCQVEHPSVTSPLTVEWSARSESAQSKML
SGVGGFVLGLLFLGAGLFIYFRNQKGHSGLQPTGFLS; HLA-DRB1*0301 [DR17 (3)] SEQ
ID NO: 32 GDTRPRFLEYSTSECHFFNGTERVRYLDRYFHNQEENVRFDSDVGEFRAV
TELGRPDAEYWNSQKDLLEQKRGRVDNYCRHNYGVVESFTVQRRVHPKVT
VYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKTGVVSTGLIQN
GDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKML
SGVGGFVLGLLFLGAGLFIYFRNQKGHSGLQPRGFLS,
as well as equivalents of each thereof.
[0044] Rose et al. also discloses an exemplary epitope to which an
HLA-DR specific antibody may bind and therefore can serve as an
immunogen for the generation of additional antibodies, monoclonal
antibodies and antigen binding fragments of each thereof. The
sequences associated with each of the listed reference(s) and
GenBank Accession Numbers that correspond to the name HLA-DR or its
equivalents including but not limited to the specified HLA-DR
subtypes are herein incorporated by reference as additional
non-limiting examples.
[0045] A "composition" typically intends a combination of the
active agent, e.g., a CAR T cell or a CAR NK cell, an antibody, a
compound, and a naturally-occurring or non-naturally-occurring
carrier, inert (for example, a detectable agent or label) or
active, such as an adjuvant, diluent, binder, stabilizer, buffers,
salts, lipophilic solvents, preservative, adjuvant or the like and
include pharmaceutically acceptable carriers. Carriers also include
pharmaceutical excipients and additives proteins, peptides, amino
acids, lipids, and carbohydrates (e.g., sugars, including
monosaccharides, di-, tri, tetra-oligosaccharides, and
oligosaccharides; derivatized sugars such as alditols, aldonic
acids, esterified sugars and the like; and polysaccharides or sugar
polymers), which can be present singly or in combination,
comprising alone or in combination 1-99.99% by weight or volume.
Exemplary protein excipients include serum albumin such as human
serum albumin (HSA), recombinant human albumin (rHA), gelatin,
casein, and the like. Representative amino acid/antibody
components, which can also function in a buffering capacity,
include alanine, arginine, glycine, arginine, betaine, histidine,
glutamic acid, aspartic acid, cysteine, lysine, leucine,
isoleucine, valine, methionine, phenylalanine, aspartame, and the
like. Carbohydrate excipients are also intended within the scope of
this technology, examples of which include but are not limited to
monosaccharides such as fructose, maltose, galactose, glucose,
D-mannose, sorbose, and the like; disaccharides, such as lactose,
sucrose, trehalose, cellobiose, and the like; polysaccharides, such
as raffinose, melezitose, maltodextrins, dextrans, starches, and
the like; and alditols, such as mannitol, xylitol, maltitol,
lactitol, xylitol sorbitol (glucitol) and myoinositol.
[0046] The term "consensus sequence" as used herein refers to an
amino acid or nucleic acid sequence that is determined by aligning
a series of multiple sequences and that defines an idealized
sequence that represents the predominant choice of amino acid or
base at each corresponding position of the multiple sequences.
Depending on the sequences of the series of multiple sequences, the
consensus sequence for the series can differ from each of the
sequences by zero, one, a few, or more substitutions. Also,
depending on the sequences of the series of multiple sequences,
more than one consensus sequence may be determined for the series.
The generation of consensus sequences has been subjected to
intensive mathematical analysis. Various software programs can be
used to determine a consensus sequence.
[0047] As used herein, the term "CD8 .alpha. hinge domain" refers
to a specific protein fragment associated with this name and any
other molecules that have analogous biological function that share
at least 70%, or alternatively at least 80% amino acid sequence
identity, or alternatively at least 90% sequence identity, or
alternatively at least 95% sequence identity with the CD8 .alpha.
hinge domain sequence as shown herein. The example sequences of CD8
.alpha. hinge domain for human, mouse, and other species are
provided in Pinto, R. D. et al. (2006) Vet. Immunol. Immunopathol.
110:169-177. The sequences associated with the CD8 .alpha. hinge
domain are provided in Pinto, R. D. et al. (2006) Vet. Immunol.
Immunopathol. 110:169-177. Non-limiting examples of such
include:
Human CD8 alpha hinge domain (SEQ ID NO: 33);
PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY Mouse CD8 alpha
hinge domain (SEQ ID NO: 34);
KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY Cat CD8 alpha
hinge domain (SEQ ID NO: 35);
PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY, and
equivalents of each thereof.
[0048] As used herein, the term "CD8 .alpha. transmembrane domain"
refers to a specific protein fragment associated with this name and
any other molecules that have analogous biological function that
share at least 70%, or alternatively at least 80% amino acid
sequence identity, or alternatively at least 90% sequence identity,
or alternatively at least 95% sequence identity with the CD8
.alpha. transmembrane domain sequence as shown herein. The fragment
sequences associated with the amino acid positions 183 to 203 of
the human T-cell surface glycoprotein CD8 alpha chain (NCBI
Reference Sequence: NP_001759.3), or the amino acid positions 197
to 217 of the mouse T-cell surface glycoprotein CD8 alpha chain
(NCBI Reference Sequence: NP_001074579.1), and the amino acid
positions 190 to 210 of the rat T-cell surface glycoprotein CD8
alpha chain (NCBI Reference Sequence: NP_113726.1) provide
additional example sequences of the CD8 .alpha. transmembrane
domain. The sequences associated with each of the listed NCBI are
provided as follows:
Human CD8 alpha transmembrane domain, (SEQ ID NO: 36):
IYIWAPLAGTCGVLLLSLVIT; Mouse CD8 alpha transmembrane domain, (SEQ
ID NO: 37): IWAPLAGICVALLLSLIITLI; Rat CD8 alpha transmembrane
domain, (SEQ ID NO: 38): IWAPLAGICAVLLLSLVITLI, and equivalents of
each thereof.
[0049] As used herein, the term "4-1BB costimulatory signaling
region" refers to a specific protein fragment associated with this
name and any other molecules that have analogous biological
function that share at least 70%, or alternatively at least 80%
amino acid sequence identity, or alternatively 90% sequence
identity, or alternatively at least 95% sequence identity with the
4-1BB costimulatory signaling region sequence as shown herein. The
example sequences of the 4-1BB costimulatory signaling region are
provided in U.S. Patent Application Publication No. 2013/0266551 A1
(filed as U.S. application Ser. No. 13/826,258). The sequence of
the 4-1BB costimulatory signaling region associated disclosed in
the U.S. application Ser. No. 13/826,258 is disclosed as
follows:
The 4-1BB costimulatory signaling region (SEQ ID NO: 39):
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, and equivalents of each
thereof.
[0050] As used herein, the term "CD28 costimulatory signaling
region" refers to a specific protein fragment associated with this
name and any other molecules that have analogous biological
function that share at least 70%, or alternatively at least 80%
amino acid sequence identity, or alternatively 90% sequence
identity, or alternatively at least 95% sequence identity with the
CD28 costimulatory signaling region sequence shown herein. The CD28
costimulatory region comprises an transmembrane domain and an
intracellular domain. The example sequences CD28 costimulatory
signaling domain are provided in U.S. Pat. No. 5,686,281; Geiger,
T. L. et al. (2001) Blood 98:2364-2371; Hombach, A. et al. (2001) J
Immunol. 167:6123-6131; Maher, J. et al. (2002) Nat Biotechnol.
20:70-75; Haynes, N. M. et al. (2002) J Immunol. 169:5780-5786;
Haynes, N. M. et al. (2002) Blood 100:3155-3163. Non-limiting
examples include residues 114-220 of the below CD28 Sequence, (SEQ
ID NO: 40): MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE
FRASLHKGLDSAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY
FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG
GVLACYSLLVTVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS,
and equivalents thereof.
[0051] As used herein, the term "ICOS costimulatory signaling
region" refers to a specific protein fragment associated with this
name and any other molecules that have analogous biological
function that share at least 70%, or alternatively at least 80%
amino acid sequence identity, preferably 90% sequence identity,
more preferably at least 95% sequence identity with the ICOS
costimulatory signaling region sequence as shown herein.
Non-limiting example sequences of the ICOS costimulatory signaling
region are provided in U.S. Publication 2015/0017141A1 the
exemplary polynucleotide sequence provided below.
TABLE-US-00007 ICOS costimulatory signaling region, SEQ ID NO: 47:
ACAAAAAAGA AGTATTCATC CAGTGTGCAC GACCCTAACG GTGAATACAT GTTCATGAGA
GCAGTGAACA CAGCCAAAAA ATCCAGACTC ACAGATGTGA CCCTA
[0052] As used herein, the term "OX40 costimulatory signaling
region" refers to a specific protein fragment associated with this
name and any other molecules that have analogous biological
function that share at least 70%, or alternatively at least 80%
amino acid sequence identity, or alternatively 90% sequence
identity, or alternatively at least 95% sequence identity with the
OX40 costimulatory signaling region sequence as shown herein.
Non-limiting example sequences of the OX40 costimulatory signaling
region are disclosed in U.S. Publication 2012/20148552A1, and
include the exemplary sequence provided below.
TABLE-US-00008 0X40 costimulatory signaling region, SEQ ID NO: 48:
AGGGACCAG AGGCTGCCCC CCGATGCCCA CAAGCCCCCT GGGGGAGGCA GTTTCCGGAC
CCCCATCCAA GAGGAGCAGG CCGACGCCCA CTCCACCCTG GCCAAGATC
[0053] As used herein, the term "CD3 zeta signaling domain" refers
to a specific protein fragment associated with this name and any
other molecules that have analogous biological function that share
at least 70%, or alternatively at least 80% amino acid sequence
identity, or alternatively 90% sequence identity, or alternatively
at least 95% sequence identity with the CD3 zeta signaling domain
sequence as shown herein. The example sequences of the CD3 zeta
signaling domain are provided in U.S. application Ser. No.
13/826,258 (published as US 2013/0266551). The sequence associated
with the CD3 zeta signaling domain is listed as follows (SEQ ID NO:
41): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR, and
equivalents thereof.
[0054] As used herein, the term "T cell," refers to a type of
lymphocyte that matures in the thymus. T cells play an important
role in cell-mediated immunity and are distinguished from other
lymphocytes, such as B cells, by the presence of a T-cell receptor
on the cell surface.
[0055] As used herein, the term "NK cell," also known as natural
killer cell, refers to a type of lymphocyte that originates in the
bone marrow and play a critical role in the innate immune system.
NK cells provide rapid immune responses against viral-infected
cells, tumor cells or other stressed cell, even in the absence of
antibodies and major histocompatibility complex on the cell
surfaces.
[0056] As used herein, the terms "nucleic acid sequence" and
"polynucleotide" are used interchangeably to refer to a polymeric
form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. Thus, this term includes, but is not limited
to, single-, double-, or multi-stranded DNA or RNA, genomic DNA,
cDNA, DNA-RNA hybrids, or a polymer comprising purine and
pyrimidine bases or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases.
[0057] The term "encode" as it is applied to nucleic acid sequences
refers to a polynucleotide which is said to "encode" a polypeptide
if, in its native state or when manipulated by methods well known
to those skilled in the art, can be transcribed and/or translated
to produce the mRNA for the polypeptide and/or a fragment thereof.
The antisense strand is the complement of such a nucleic acid, and
the encoding sequence can be deduced therefrom.
[0058] As used herein, the term signal peptide or signal
polypeptide intends an amino acid sequence usually present at the
N-terminal end of newly synthesized secretory or membrane
polypeptides or proteins. It acts to direct the polypeptide across
or into a cell membrane and is then subsequently removed. Examples
of such are well known in the art. Non-limiting examples are those
described in U.S. Pat. Nos. 8,853,381 and 5,958,736.
[0059] As used herein, the term "vector" refers to a nucleic acid
construct deigned for transfer between different hosts, including
but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a
YAC, etc. In some embodiments, plasmid vectors may be prepared from
commercially available vectors. In other embodiments, viral vectors
may be produced from baculoviruses, retroviruses, adenoviruses,
AAVs, etc. according to techniques known in the art. In one
embodiment, the viral vector is a lentiviral vector.
[0060] As used herein, the term "isolated cell" generally refers to
a cell that is substantially separated from other cells of a
tissue. The term includes prokaryotic and eukaryotic cells.
[0061] "Immune cells" includes, e.g., white blood cells
(leukocytes) which are derived from hematopoietic stem cells (HSC)
produced in the bone marrow, lymphocytes (T cells, B cells, natural
killer (NK) cells) and myeloid-derived cells (neutrophil,
eosinophil, basophil, monocyte, macrophage, dendritic cells). "T
cell" includes all types of immune cells expressing CD3 including
T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells),
natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T
cells. A "cytotoxic cell" includes CD8+ T cells, natural-killer
(NK) cells, and neutrophils, which cells are capable of mediating
cytotoxicity responses.
[0062] The term "transduce" or "transduction" as it is applied to
the production of chimeric antigen receptor cells refers to the
process whereby a foreign nucleotide sequence is introduced into a
cell. In some embodiments, this transduction is done via a
vector.
[0063] As used herein, the term "autologous," in reference to cells
refers to cells that are isolated and infused back into the same
subject (recipient or host). "Allogeneic" refers to non-autologous
cells.
[0064] An "effective amount" or "efficacious amount" refers to the
amount of an agent (e.g., a HLA-DR CAR cell), or combined amounts
of two or more agents, that, when administered for the treatment of
a mammal or other subject, is sufficient to effect such treatment
for the disease.
[0065] The "effective amount" will vary depending on the agent(s),
the disease and its severity and the age, weight, etc., of the
subject to be treated.
[0066] A "solid tumor" is an abnormal mass of tissue that usually
does not contain cysts or liquid areas. Solid tumors can be benign
or malignant. Different types of solid tumors are named for the
type of cells that form them. Examples of solid tumors include
sarcomas, carcinomas, and lymphomas.
[0067] The term "B cell lymphoma or leukemia" refers to a type of
cancer that forms in issues of the lymphatic system or bone marrow,
and has undergone a malignant transformation that makes the cells
within the cancer pathological to the host organism with the
ability to invade or spread to other parts of the body.
[0068] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination for the
intended use. For example, a composition consisting essentially of
the elements as defined herein would not exclude trace contaminants
from the isolation and purification method and pharmaceutically
acceptable carriers, such as phosphate buffered saline,
preservatives and the like. "Consisting of" shall mean excluding
more than trace elements of other ingredients and substantial
method steps for administering the compositions disclosed herein.
Aspects defined by each of these transition terms are within the
scope of the present disclosure.
[0069] As used herein, the term "detectable marker" refers to at
least one marker capable of directly or indirectly, producing a
detectable signal. A non-exhaustive list of this marker includes
enzymes which produce a detectable signal, for example by
colorimetry, fluorescence, luminescence, such as horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase,
glucose-6-phosphate dehydrogenase, chromophores such as
fluorescent, luminescent dyes, groups with electron density
detected by electron microscopy or by their electrical property
such as conductivity, amperometry, voltammetry, impedance,
detectable groups, for example whose molecules are of sufficient
size to induce detectable modifications in their physical and/or
chemical properties, such detection may be accomplished by optical
methods such as diffraction, surface plasmon resonance, surface
variation, the contact angle change or physical methods such as
atomic force spectroscopy, tunnel effect, or radioactive molecules
such as .sup.32P, .sup.35S or .sup.125I.
[0070] As used herein, the term "purification marker" refers to at
least one marker useful for purification or identification. A
non-exhaustive list of this marker includes His, lacZ, GST,
maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP,
cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding
protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct
or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP,
dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin,
Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC
or any other fluorescent dye or hapten.
[0071] As used herein, the term "expression" refers to the process
by which polynucleotides are transcribed into mRNA and/or the
process by which the transcribed mRNA is subsequently being
translated into peptides, polypeptides, or proteins. If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell. The expression level of
a gene may be determined by measuring the amount of mRNA or protein
in a cell or tissue sample. In one aspect, the expression level of
a gene from one sample may be directly compared to the expression
level of that gene from a control or reference sample. In another
aspect, the expression level of a gene from one sample may be
directly compared to the expression level of that gene from the
same sample following administration of a compound.
[0072] As used herein, "homology" or "identical", percent
"identity" or "similarity", when used in the context of two or more
nucleic acids or polypeptide sequences, refers to two or more
sequences or subsequences that are the same or have a specified
percentage of nucleotides or amino acid residues that are the same,
e.g., at least 60% identity, preferably at least 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
higher identity over a specified region (e.g., nucleotide sequence
encoding an antibody described herein or amino acid sequence of an
antibody described herein). Homology can be determined by comparing
a position in each sequence which may be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same base or amino acid, then the molecules are homologous at
that position. A degree of homology between sequences is a function
of the number of matching or homologous positions shared by the
sequences. The alignment and the percent homology or sequence
identity can be determined using software programs known in the
art, for example those described in Current Protocols in Molecular
Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18,
Table 7.7.1. Preferably, default parameters are used for alignment.
A preferred alignment program is BLAST, using default parameters.
In particular, preferred programs are BLASTN and BLASTP, using the
following default parameters: Genetic code=standard; filter=none;
strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50
sequences; sort by=HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs
can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-bin/BLAST. The terms "homology" or
"identical," percent "identity" or "similarity" also refer to, or
can be applied to, the complement of a test sequence. The terms
also include sequences that have deletions and/or additions, as
well as those that have substitutions. As described herein, the
preferred algorithms can account for gaps and the like. Preferably,
identity exists over a region that is at least about 25 amino acids
or nucleotides in length, or more preferably over a region that is
at least 50-100 amino acids or nucleotides in length. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, or alternatively less than 25% identity, with one of the
sequences disclosed herein.
[0073] The phrase "first line" or "second line" or "third line"
refers to the order of treatment received by a patient. First line
therapy regimens are treatments given first, whereas second or
third line therapy are given after the first line therapy or after
the second line therapy, respectively. The National Cancer
Institute defines first line therapy as "the first treatment for a
disease or condition. In patients with cancer, primary treatment
can be surgery, chemotherapy, radiation therapy, or a combination
of these therapies. First line therapy is also referred to those
skilled in the art as "primary therapy and primary treatment." See
National Cancer Institute website at www.cancer.gov, last visited
on May 1, 2008. Typically, a patient is given a subsequent
chemotherapy regimen because the patient did not show a positive
clinical or sub-clinical response to the first line therapy or the
first line therapy has stopped.
[0074] In one aspect, the term "equivalent" or "biological
equivalent" of an antibody means the ability of the antibody to
selectively bind its epitope protein or fragment thereof as
measured by ELISA or other suitable methods. Biologically
equivalent antibodies include, but are not limited to, those
antibodies, peptides, antibody fragments, antibody variant,
antibody derivative and antibody mimetics that bind to the same
epitope as the reference antibody.
[0075] It is to be inferred without explicit recitation and unless
otherwise intended, that when the present disclosure relates to a
polypeptide, protein, polynucleotide, antibody or fragment thereof,
an equivalent or a biologically equivalent of such is intended
within the scope of this disclosure. As used herein, the term
"biological equivalent thereof" is intended to be synonymous with
"equivalent thereof" when referring to a reference protein,
antibody or fragment thereof, polypeptide or nucleic acid, intends
those having minimal homology while still maintaining desired
structure or functionality. Unless specifically recited herein, it
is contemplated that any of the above also includes equivalents
thereof. For example, an equivalent intends at least about 70%
homology or identity, or at least 80% homology or identity and
alternatively, or at least about 85%, or alternatively at least
about 90%, or alternatively at least about 95%, or alternatively at
least 98% percent homology or identity and exhibits substantially
equivalent biological activity to the reference protein,
polypeptide, antibody or fragment thereof or nucleic acid.
Alternatively, when referring to polynucleotides, an equivalent
thereof is a polynucleotide that hybridizes under stringent
conditions to the reference polynucleotide or its complement.
Alternatively, when referring to polypeptides or proteins, an
equivalent thereof is a expressed polypeptide or protein from a
polynucleotide that hybridizes under stringent conditions to the
polynucleotide or its complement that encodes the reference
polypeptide or protein.
[0076] A polynucleotide or polynucleotide region (or a polypeptide
or polypeptide region) having a certain percentage (for example,
80%, 85%, 90%, or 95%) of "sequence identity" to another sequence
means that, when aligned, that percentage of bases (or amino acids)
are the same in comparing the two sequences. The alignment and the
percent homology or sequence identity can be determined using
software programs known in the art, for example those described in
Current Protocols in Molecular Biology (Ausubel et al., eds. 1987)
Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default
parameters are used for alignment. A preferred alignment program is
BLAST, using default parameters. In particular, preferred programs
are BLASTN and BLASTP, using the following default parameters:
Genetic code=standard; filter=none; strand=both; cutoff=60;
expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH
SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs
can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-bin/BLAST.
[0077] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of a PCR reaction, or the enzymatic cleavage of a
polynucleotide by a ribozyme.
[0078] Examples of stringent hybridization conditions include:
incubation temperatures of about 25.degree. C. to about 37.degree.
C.; hybridization buffer concentrations of about 6.times.SSC to
about 10.times.SSC; formamide concentrations of about 0% to about
25%; and wash solutions from about 4.times.SSC to about
8.times.SSC. Examples of moderate hybridization conditions include:
incubation temperatures of about 40.degree. C. to about 50.degree.
C.; buffer concentrations of about 9.times.SSC to about
2.times.SSC; formamide concentrations of about 30% to about 50%;
and wash solutions of about 5.times.SSC to about 2.times.SSC.
Examples of high stringency conditions include: incubation
temperatures of about 55.degree. C. to about 68.degree. C.; buffer
concentrations of about 1.times.SSC to about 0.1.times.SSC;
formamide concentrations of about 55% to about 75%; and wash
solutions of about 1.times.SSC, 0.1.times.SSC, or deionized water.
In general, hybridization incubation times are from 5 minutes to 24
hours, with 1, 2, or more washing steps, and wash incubation times
are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate
buffer. It is understood that equivalents of SSC using other buffer
systems can be employed.
[0079] A "normal cell corresponding to the tumor tissue type"
refers to a normal cell from a same tissue type as the tumor
tissue. A non-limiting example is a normal lung cell from a patient
having lung tumor, or a normal colon cell from a patient having
colon tumor.
[0080] The term "isolated" as used herein refers to molecules or
biologicals or cellular materials being substantially free from
other materials. In one aspect, the term "isolated" refers to
nucleic acid, such as DNA or RNA, or protein or polypeptide (e.g.,
an antibody or derivative thereof), or cell or cellular organelle,
or tissue or organ, separated from other DNAs or RNAs, or proteins
or polypeptides, or cells or cellular organelles, or tissues or
organs, respectively, that are present in the natural source. The
term "isolated" also refers to a nucleic acid or peptide that is
substantially free of cellular material, viral material, or culture
medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized.
Moreover, an "isolated nucleic acid" is meant to include nucleic
acid fragments which are not naturally occurring as fragments and
would not be found in the natural state. The term "isolated" is
also used herein to refer to polypeptides which are isolated from
other cellular proteins and is meant to encompass both purified and
recombinant polypeptides. The term "isolated" is also used herein
to refer to cells or tissues that are isolated from other cells or
tissues and is meant to encompass both cultured and engineered
cells or tissues.
[0081] As used herein, the term "monoclonal antibody" refers to an
antibody produced by a single clone of B-lymphocytes or by a cell
into which the light and heavy chain genes of a single antibody
have been transfected. Monoclonal antibodies are produced by
methods known to those of skill in the art, for instance by making
hybrid antibody-forming cells from a fusion of myeloma cells with
immune spleen cells. Monoclonal antibodies include humanized
monoclonal antibodies.
[0082] The term "protein", "peptide" and "polypeptide" are used
interchangeably and in their broadest sense to refer to a compound
of two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another aspect, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. A protein or peptide must contain at least two
amino acids and no limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's sequence.
As used herein the term "amino acid" refers to either natural
and/or unnatural or synthetic amino acids, including glycine and
both the D and L optical isomers, amino acid analogs and
peptidomimetics.
[0083] The terms "polynucleotide" and "oligonucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides or analogs
thereof. Polynucleotides can have any three-dimensional structure
and may perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: a gene or gene fragment
(for example, a probe, primer, EST or SAGE tag), exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes and primers. A polynucleotide can
comprise modified nucleotides, such as methylated nucleotides and
nucleotide analogs. If present, modifications to the nucleotide
structure can be imparted before or after assembly of the
polynucleotide. The sequence of nucleotides can be interrupted by
non-nucleotide components. A polynucleotide can be further modified
after polymerization, such as by conjugation with a labeling
component. The term also refers to both double- and single-stranded
molecules. Unless otherwise specified or required, any aspect of
this technology that is a polynucleotide encompasses both the
double-stranded form and each of two complementary single-stranded
forms known or predicted to make up the double-stranded form.
[0084] As used herein, the term "purified" does not require
absolute purity; rather, it is intended as a relative term. Thus,
for example, a purified nucleic acid, peptide, protein, biological
complexes or other active compound is one that is isolated in whole
or in part from proteins or other contaminants. Generally,
substantially purified peptides, proteins, biological complexes, or
other active compounds for use within the disclosure comprise more
than 80% of all macromolecular species present in a preparation
prior to admixture or formulation of the peptide, protein,
biological complex or other active compound with a pharmaceutical
carrier, excipient, buffer, absorption enhancing agent, stabilizer,
preservative, adjuvant or other co-ingredient in a complete
pharmaceutical formulation for therapeutic administration. More
typically, the peptide, protein, biological complex or other active
compound is purified to represent greater than 90%, often greater
than 95% of all macromolecular species present in a purified
preparation prior to admixture with other formulation ingredients.
In other cases, the purified preparation may be essentially
homogeneous, wherein other macromolecular species are not
detectable by conventional techniques.
[0085] As used herein, the term "specific binding" means the
contact between an antibody and an antigen with a binding affinity
of at least 10.sup.-6 M. In certain aspects, antibodies bind with
affinities of at least about 10.sup.-7M, and preferably 10.sup.-8
M, 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, or 10.sup.-12 M.
[0086] As used herein, the term "recombinant protein" refers to a
polypeptide which is produced by recombinant DNA techniques,
wherein generally, DNA encoding the polypeptide is inserted into a
suitable expression vector which is in turn used to transform a
host cell to produce the heterologous protein.
[0087] As used herein, "treating" or "treatment" of a disease in a
subject refers to (1) preventing the symptoms or disease from
occurring in a subject that is predisposed or does not yet display
symptoms of the disease; (2) inhibiting the disease or arresting
its development; or (3) ameliorating or causing regression of the
disease or the symptoms of the disease. As understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. For the purposes of the
present technology, beneficial or desired results can include one
or more, but are not limited to, alleviation or amelioration of one
or more symptoms, diminishment of extent of a condition (including
a disease), stabilized (i.e., not worsening) state of a condition
(including disease), delay or slowing of condition (including
disease), progression, amelioration or palliation of the condition
(including disease), states and remission (whether partial or
total), whether detectable or undetectable. When the disease is
cancer, the following clinical end points are non-limiting examples
of treatment: reduction in tumor burden, slowing of tumor growth,
longer overall survival, longer time to tumor progression,
inhibition of metastasis or a reduction in metastasis of the
tumor.
[0088] As used herein, the term "overexpress" with respect to a
cell, a tissue, or an organ expresses a protein to an amount that
is greater than the amount that is produced in a control cell, a
control issue, or an organ. A protein that is overexpressed may be
endogenous to the host cell or exogenous to the host cell.
[0089] As used herein the term "linker sequence" relates to any
amino acid sequence comprising from 1 to 10, or alternatively, 8
amino acids, or alternatively 6 amino acids, or alternatively 5
amino acids that may be repeated from 1 to 10, or alternatively to
about 8, or alternatively to about 6, or alternatively about 5, or
4 or alternatively 3, or alternatively 2 times. For example, the
linker may comprise up to 15 amino acid residues consisting of a
pentapeptide repeated three times. Non-limiting examples of linker
sequences are known in the art, e.g., GGGGSGGGGSGGGG (and
equivalents thereof) (SEQ ID NO: 49); the tripeptide EFM; or
Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO:
50), and equivalents of each thereof. In one aspect, the linker
sequence is a (Glycine4Serine)3 flexible polypeptide linker (SEQ ID
NO: 51) comprising three copies of gly-gly-gly-gly-ser (SEQ ID NO:
52), and equivalents thereof.
[0090] As used herein, the term "enhancer", as used herein, denotes
sequence elements that augment, improve or ameliorate transcription
of a nucleic acid sequence irrespective of its location and
orientation in relation to the nucleic acid sequence to be
expressed. An enhancer may enhance transcription from a single
promoter or simultaneously from more than one promoter. As long as
this functionality of improving transcription is retained or
substantially retained (e.g., at least 70%, at least 80%, at least
90% or at least 95% of wild-type activity, that is, activity of a
full-length sequence), any truncated, mutated or otherwise modified
variants of a wild-type enhancer sequence are also within the above
definition.
[0091] The term "promoter" as used herein refers to any sequence
that regulates the expression of a coding sequence, such as a gene.
Promoters may be constitutive, inducible, repressible, or
tissue-specific, for example. A "promoter" is a control sequence
that is a region of a polynucleotide 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.
[0092] As used herein, the term "WPRE" or "Woodchuck Hepatitis
Virus (WHP) Post-transcriptional Regulatory Element" refers to a
specific nucleotide fragment associated with this name and any
other molecules that have analogous biological function that share
at least 70%, or alternatively at least 80% amino acid sequence
identity, preferably 90% sequence identity, more preferably at
least 95% sequence identity with the WPRE sequence as shown herein.
For example, WPRE refers to a region similar to the human hepatitis
B virus posttranscriptional regulatory element (HBVPRE) present in
the Woodchuck hepatitis virus genomic sequence (GenBank Accession
No. J04514), and that the 592 nucleotides from position 1093 to
1684 of this genomic sequence correspond to the
post-transcriptional regulatory region (Donello, J. E. et al.
(1998) Journal of Virology 72:5085-5092). The analysis using
retroviral vectors revealed that WPRE inserted into the 3'-terminal
untranslated region of a gene of interest increases the amount of
protein produced by 5 to 8 folds. It has also been reported that
the introduction of WPRE suppresses mRNA degradation (Zufferey, R.
et al. (1999) Journal of Virology 73:2886-2892). In a broad sense,
elements such as WPRE that increase the efficiency of amino acid
translation by stabilizing mRNAs are also thought to be
enhancers.
List of Abbreviations
[0093] CAR: chimeric antigen receptor HLA: histocompatibility
lymphocyte antigen Ip: intraperitoneal IRES: internal ribosomal
entry site MFI: mean fluorescence intensity MOI: multiplicity of
infection PBMC: peripheral blood mononuclear cells PBS: phosphate
buffered saline scFv: single chain variable fragment WPRE:
woodchuck hepatitis virus post-transcriptional regulatory
element
MODES FOR CARRYING OUT THE DISCLOSURE
[0094] Due to the unprecedented results being recently obtained in
B-cell lymphomas and leukemia's using autologous treatment with
genetically engineered chimeric antigen receptor (CAR) T-cells
(Maude, S. L. et al. (2014) New Engl. J. Med. 371:1507-1517;
Porter, D. L. et al. (2011) New Engl. J. Med. 365:725-733), a
number of laboratories have begun to apply this approach to solid
tumors including ovarian cancer, prostate cancer, and pancreatic
tumors. CAR modified T-cells combine the HLA-independent targeting
specificity of a monoclonal antibody with the cytolytic activity,
proliferation, and homing properties of activated T-cells, but do
not respond to checkpoint suppression. Because of their ability to
kill antigen expressing targets directly, CAR T-cells are highly
toxic to any antigen positive cells or tissues making it a
requirement to construct CARs with highly tumor specific
antibodies. To date, CAR modified T-cells to human solid tumors
have been constructed against the .alpha.-folate receptor,
mesothelin, and MUC-CD, PSMA, and other targets but most have some
off-target expression of antigen in normal tissues. These
constructs have not shown the same exceptional results in patients
emphasizing the need for additional studies to identify new targets
and methods of CAR T-cell construction that can be used against
solid tumors.
[0095] Thus, this disclosure provides antibodies specific to HLA-DR
and methods and compositions relating to the use and production
thereof. In addition, this disclosure provides as a chimeric
antigen receptor (CAR) comprising an antigen binding domain
specific to HLA-DR, that in some aspects, is the antigen binding
domain of Lym-1 and Lym-2 antibodies and methods and compositions
relating to the use and production thereof.
Antibodies and Uses Thereof
[0096] I. Compositions
[0097] The general structure of antibodies is known in the art and
will only be briefly summarized here. An immunoglobulin monomer
comprises two heavy chains and two light chains connected by
disulfide bonds. Each heavy chain is paired with one of the light
chains to which it is directly bound via a disulfide bond. Each
heavy chain comprises a constant region (which varies depending on
the isotype of the antibody) and a variable region. The variable
region comprises three hypervariable regions (or complementarity
determining regions) which are designated CDRH1, CDRH2 and CDRH3
and which are supported within framework regions. Each light chain
comprises a constant region and a variable region, with the
variable region comprising three hypervariable regions (designated
CDRL1, CDRL2 and CDRL3) supported by framework regions in an
analogous manner to the variable region of the heavy chain.
[0098] The hypervariable regions of each pair of heavy and light
chains mutually cooperate to provide an antigen binding site that
is capable of binding a target antigen. The binding specificity of
a pair of heavy and light chains is defined by the sequence of
CDR1, CDR2 and CDR3 of the heavy and light chains. Thus once a set
of CDR sequences (i.e., the sequence of CDR1, CDR2 and CDR3 for the
heavy and light chains) is determined which gives rise to a
particular binding specificity, the set of CDR sequences can, in
principle, be inserted into the appropriate positions within any
other antibody framework regions linked with any antibody constant
regions in order to provide a different antibody with the same
antigen binding specificity.
[0099] In one aspect, the present disclosure provides an isolated
antibody comprising a heavy chain (HC) immunoglobulin variable
domain sequence and a light chain (LC) immunoglobulin variable
domain sequence, wherein the heavy chain and light chain
immunoglobulin variable domain sequences form an antigen binding
site that binds to an epitope of human HLA-DR.
[0100] In some embodiments, the heavy chain variable region
comprises a CDRH1 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning with any one of the following sequences (i)
GFSLTSYG (SEQ ID NO: 1), (ii) GFTFSNYW (SEQ ID NO: 2), or
equivalents of each thereof, followed by an additional 50 amino
acids, or alternatively about 40 amino acids, or alternatively
about 30 amino acids, or alternatively about 20 amino acids, or
alternatively about 10 amino acids, or alternatively about 5 amino
acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the
carboxy-terminus.
[0101] In some embodiments, the heavy chain variable region
comprises a CDRH2 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning with any one of the following sequences: (i)
IWSDGST (SEQ ID NO: 3), (ii) IRFKSHNYAT (SEQ ID NO: 4), or
equivalents of each thereof, followed by an additional 50 amino
acids, or alternatively about 40 amino acids, or alternatively
about 30 amino acids, or alternatively about 20 amino acids, or
alternatively about 10 amino acids, or alternatively about 5 amino
acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the
carboxy-terminus.
[0102] In some embodiments, the heavy chain variable region
comprises a CDRH3 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning with any one of the following sequences: (i)
ASHYGSTLAFAS (SEQ ID NO: 5), (ii) TRRIGNSDYDWWYFDV (SEQ ID NO: 6),
or equivalents of each thereof, followed by an additional 50 amino
acids, or alternatively about 40 amino acids, or alternatively
about 30 amino acids, or alternatively about 20 amino acids, or
alternatively about 10 amino acids, or alternatively about 5 amino
acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the
carboxy-terminus.
[0103] In some embodiments, the heavy chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the polypeptide encoded by the below noted
polynucleotide sequence:
CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTC
CATCACATGCACCATCTCAGGGTTCTCATTAACCAGCTATGGTGTACACTGGGTTCG
CCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTGATATGGAGTGATGGAAGCA
CAACCTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAG
AGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATATACTAC
TGTGCCAGTCACTACGGTAGTACCCTTGCCTTTGCTTCCTGGGGCCACGGGACTCTG
GTCACTGTCTCTGCA (SEQ ID NO: 7), or an antigen binding fragment
thereof or an equivalent of each thereof.
[0104] In some embodiments, the heavy chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the amino acid sequence:
QLKESGPGLVAPSQSLSITCTISGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSA
LKSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCASHYGSTLAFASWGHGTLVTVSA (SEQ ID
NO: 8), or an antigen binding fragment thereof or an equivalent of
each thereof.
[0105] In some embodiments, the heavy chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the polypeptide encoded by the below noted
polynucleotide sequence:
GAAGTGCAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGCTCCATGAA
ACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTATTGGATGAACTGGGTCCGC
CAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTTAAATCTCATAAT
TATGCAACACATTTTGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGAT
TCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCAT
TTATTACTGTACCAGGAGGATAGGAAACTCTGATTACGACTGGTGGTACTTCGATGT
CTGGGGCGCAGGGACCTCAGTCACCGTCTCCTCAGCTAGC (SEQ ID NO: 9), or an
antigen binding fragment thereof or an equivalent of each
thereof.
[0106] In some embodiments, the heavy chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the amino acid sequence:
EVQLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRFKSHN
YATHFAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRRIGNSDYDWWYFDVW
GAGTSVTVSSAS (SEQ ID NO: 10), or an antigen binding fragment
thereof or an equivalent of each thereof.
[0107] In some embodiments, the light chain variable region
comprises a CDRL1 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning with any one of the following sequences (i)
VNIYSY (SEQ ID NO: 11), (ii) QNVGNN (SEQ ID NO: 12), or equivalents
of each thereof, followed by an additional 50 amino acids, or
alternatively about 40 amino acids, or alternatively about 30 amino
acids, or alternatively about 20 amino acids, or alternatively
about 10 amino acids, or alternatively about 5 amino acids, or
alternatively about 4, or 3, or 2 or 1 amino acids at the
carboxy-terminus.
[0108] In some embodiments, the light chain variable region
comprises a CDRL2 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning with (i) NAK (SEQ ID NO: 13), (ii) SAS (SEQ ID
NO: 14), or equivalents of each thereof, followed by an additional
50 amino acids, or alternatively about 40 amino acids, or
alternatively about 30 amino acids, or alternatively about 20 amino
acids, or alternatively about 10 amino acids, or alternatively
about 5 amino acids, or alternatively about 4, or 3, or 2 or 1
amino acids at the carboxy-terminus.
[0109] In other embodiments, the light chain variable region
comprises a CDRL3 sequence comprising, or alternatively consisting
essentially of, or yet further consisting of, an amino acid
sequence beginning (i) QHHYGTFT (SEQ ID NO: 15), (ii) QQYNTYPFT
(SEQ ID NO: 16), or equivalents of each thereof, followed by an
additional 50 amino acids, or alternatively about 40 amino acids,
or alternatively about 30 amino acids, or alternatively about 20
amino acids, or alternatively about 10 amino acids, or
alternatively about 5 amino acids, or alternatively about 4, or 3,
or 2 or 1 amino acids at the carboxy-terminus.
[0110] In some embodiments, the light chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the polypeptide encoded by the polynucleotide
sequence: GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTC
ACCATCATATGTCGAGCAAGTGTGAATATTTACAGTTATTTAGCATGGTATCAGCAG
AAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCCAAAATCTTAGCAGAAGGT
GTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAAC
AGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACATTC
ACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 17), or an antigen
binding fragment thereof or an equivalent of each thereof.
[0111] In some embodiments, the light chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the amino acid sequence:
DIQMTQSPASLSASVGETVTIICRASVNIYSYLAWYQQKQGKSPQLLVYNAKILAEGVPS
RFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTFTFGSGTKLEIK (SEQ ID NO: 18), or
an antigen binding fragment thereof or an equivalent of each
thereof.
[0112] In some embodiments, the light chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the polypeptide encoded by the polynucleotide
sequence: GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTC
AGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTAATAATGTAGCCTGGTATCAACA
GAAACCAGGGCAATCTCCTAAAGTACTGATTTACTCGGCATCCTACCGGTACAGTGG
AGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAG
TAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACACCTATCC
ATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 19), or an
antigen binding fragment thereof or an equivalent of each
thereof.
[0113] In some embodiments, the light chain variable region
comprises, or alternatively consists essentially of, or yet further
consists of, the amino acid sequence:
DIVMTQSHKFMSTSVGDRVSVTCKASQNVGNNVAWYQQKPGQSPKVLIYSASYRYSG
VPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNTYPFTFGSGTKLEIK (SEQ ID NO: 20),
or an antigen binding fragment thereof or an equivalent of each
thereof.
[0114] In another aspect of the present technology, the isolated
antibody includes one or more of the following characteristics:
[0115] (a) the light chain immunoglobulin variable domain sequence
comprises one or more CDRs that are at least 85% identical to a CDR
of a light chain variable domain of any of the disclosed light
chain sequences;
[0116] (b) the heavy chain immunoglobulin variable domain sequence
comprises one or more CDRs that are at least 85% identical to a CDR
of a heavy chain variable domain of any of the disclosed heavy
chain sequences;
[0117] (c) the light chain immunoglobulin variable domain sequence
is at least 85% identical to a light chain variable domain of any
of the disclosed light chain sequences;
[0118] (d) the HC immunoglobulin variable domain sequence is at
least 85% identical to a heavy chain variable domain of any of the
disclosed light chain sequences; and
[0119] (e) the antibody binds an epitope that overlaps with an
epitope bound by any of the disclosed sequences.
[0120] Exemplary antibodies comprising the disclosed CDR sequences
and heavy and light chain variable sequences are disclosed in Table
1 and Table 2, respectively.
TABLE-US-00009 TABLE 1 ANTIBODY CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3
Lym-1 SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 11 SEQ ID
NO: 13 SEQ ID NO: 15 Lym-2 SEQ ID NO: 2 SEQ ID: NO 4 SEQ ID: NO 6
SEQ ID NO: 12 SEQ ID NO: 14 SEQ ID NO: 16
TABLE-US-00010 TABLE 2 Heavy Chain Light Chain ANTIBODY Variable
Region Variable Region Lym-1 SEQ ID NO: 7 and 8 SEQ ID NO: 17 and
18 Lym-2 SEQ ID NO: 9 and 10 SEQ ID NO: 19 and 20
[0121] In one aspect, the present disclosure provides an isolated
antibody that is at least 85% identical to an antibody selected
from the group consisting of Lym-1 and Lym-2.
[0122] In one aspect, the present disclosure provides an isolated
antibody comprising the CDRs of Lym-1. In one aspect, the present
disclosure provides an isolated antibody that is at least 85%
identical to Lym-1.
[0123] In one aspect, the present disclosure provides an isolated
antibody comprising the CDRs of Lym-2. In one aspect, the present
disclosure provides an isolated antibody that is at least 85%
identical to Lym-2.
[0124] In some aspects of the antibodies provided herein, the HC
variable domain sequence comprises, or consists essentially of, or
yet further consists of, a variable domain sequence of Lym-1 and
the LC variable domain sequence comprises, or consists essentially
of, or yet further consists of a variable domain sequence of
Lym-1.
[0125] In some aspects of the antibodies provided herein, the HC
variable domain sequence comprises, or consists essentially of, or
yet further consists of, a variable domain sequence of Lym-2 and
the LC variable domain sequence comprises, or consists essentially
of, or yet further consists of a variable domain sequence of
Lym-2.
[0126] In some of the aspects of the antibodies provided herein,
the antibody binds human HLA-DR with a dissociation constant
(K.sub.D) of less than 10.sup.-4M, 10.sup.-5M, 10.sup.-6M,
10.sup.-7M, 10.sup.-8M, 10.sup.-9M, 10.sup.-10 M, 10.sup.-11 M, or
10.sup.-12M. In some of the aspects of the antibodies provided
herein, the antigen binding site specifically binds to human
HLA-DR.
[0127] In some of the aspects of the antibodies provided herein,
the antibody is soluble Fab.
[0128] In some of the aspects of the antibodies provided herein,
the HC and LC variable domain sequences are components of the same
polypeptide chain. In some of the aspects of the antibodies
provided herein, the HC and LC variable domain sequences are
components of different polypeptide chains.
[0129] In some of the aspects of the antibodies provided herein,
the antibody is a full-length antibody.
[0130] In some of the aspects of the antibodies provided herein,
the antibody is a monoclonal antibody.
[0131] In some of the aspects of the antibodies provided herein,
the antibody is chimeric or humanized.
[0132] In some of the aspects of the antibodies provided herein,
the antibody is selected from the group consisting of Fab, F(ab)'2,
Fab', scF.sub.v, and F.sub.v.
[0133] In some of the aspects of the antibodies provided herein,
the antibody comprises an Fc domain. In some of the aspects of the
antibodies provided herein, the antibody is a rabbit antibody. In
some of the aspects of the antibodies provided herein, the antibody
is a human or humanized antibody or is non-immunogenic in a
human.
[0134] In some of the aspects of the antibodies provided herein,
the antibody comprises a human antibody framework region.
[0135] In other aspects, one or more amino acid residues in a CDR
of the antibodies provided herein are substituted with another
amino acid. The substitution may be "conservative" in the sense of
being a substitution within the same family of amino acids. The
naturally occurring amino acids may be divided into the following
four families and conservative substitutions will take place within
those families.
[0136] 1) Amino acids with basic side chains: lysine, arginine,
histidine.
[0137] 2) Amino acids with acidic side chains: aspartic acid,
glutamic acid
[0138] 3) Amino acids with uncharged polar side chains: asparagine,
glutamine, serine, threonine, tyrosine.
[0139] 4) Amino acids with nonpolar side chains: glycine, alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan, cysteine.
[0140] In another aspect, one or more amino acid residues are added
to or deleted from one or more CDRs of an antibody. Such additions
or deletions occur at the N or C termini of the CDR or at a
position within the CDR.
[0141] By varying the amino acid sequence of the CDRs of an
antibody by addition, deletion or substitution of amino acids,
various effects such as increased binding affinity for the target
antigen may be obtained.
[0142] It is to be appreciated that antibodies of the present
disclosure comprising such varied CDR sequences still bind HLA-DR
with similar specificity and sensitivity profiles as the disclosed
antibodies. This may be tested by way of the binding assays known
to skill in the art and briefly described herein.
[0143] The constant regions of antibodies can also be varied. For
example, antibodies are provided with Fc regions of any isotype:
IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) or IgM.
Non-limiting examples of constant region sequences include:
[0144] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 21)
APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRR
DSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQA
EGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV
QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLT
LPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC
EVSGESPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYT
CVVSHEDSRTLLNASRSLEVSYVTDHGPMK, and equivalents thereof.
[0145] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 22)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and equivalents thereof.
[0146] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 23)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and equivalents thereof.
[0147] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 24)
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC
DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMH
EALHNRFTQKSLSLSPGK, and equivalents thereof.
[0148] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 25)
GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLR
GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPP
RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV
TSTLTIKESDWLGQSNIFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKS
TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE
RFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD
VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEA
LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY, and equivalents thereof.
[0149] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 26)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHEALHNHYTQKSLSLSLGK, and equivalents thereof.
[0150] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 27)
ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDAS
GDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPS
CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLC
GCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEEL
ALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILR
VAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY, and
equivalents thereof.
[0151] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 28)
ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDAS
GDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPA
LEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGC
AQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLA
RGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDT
FSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY, and equivalents
thereof.
[0152] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO:
29) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, and equivalents
thereof.
[0153] In some aspects, the antibodies comprise a heavy chain
constant region that is at least 80% identical to any one of SEQ ID
NOs: 7 to 10.
[0154] In some aspects, the antibodies comprise a light chain
constant region that is at least 80% identical to any one of SEQ ID
NOs: 17 to 20.
[0155] In some aspects of the antibodies provided herein, the
antibody binds to the epitope bound by Lym-1 and Lym-2
antibodies.
[0156] In some aspects of the antibodies provided herein, the
HLA-DR-specific antibody competes for binding to human HLA-DR with
Lym-1 and Lym-2.
[0157] In some aspects of the antibodies provided herein, the
antibody contains structural modifications to facilitate rapid
binding and cell uptake and/or slow release. In some aspects, the
HLA-DR antibody contains a deletion in the CH2 constant heavy chain
region of the antibody to facilitate rapid binding and cell uptake
and/or slow release. In some aspects, a Fab fragment is used to
facilitate rapid binding and cell uptake and/or slow release. In
some aspects, a F(ab)'2 fragment is used to facilitate rapid
binding and cell uptake and/or slow release.
[0158] The antibodies, fragments, and equivalents thereof can be
combined with a carrier, e.g., a pharmaceutically acceptable
carrier or other agents to provide a formulation for use and/or
storage.
[0159] Further provided is an isolated polypeptide comprising, or
alternatively consisting essentially of, or yet further consisting
of, the amino acid sequence of HLA-DR or a fragment thereof, that
are useful to generate antibodies that bind to HLA-DR, as well as
isolated polynucleotides that encode them. In one aspect, the
isolated polypeptides or polynucleotides further comprise a label
and/or contiguous polypeptide sequences (e.g., keyhole limpet
haemocyanin (KLH) carrier protein) or in the case of
polynucleotides, polynucleotides encoding the sequence, operatively
coupled to polypeptide or polynucleotide. The polypeptides or
polynucleotides can be combined with various carriers, e.g.,
phosphate buffered saline. Further provided are host cells, e.g.,
prokaryotic or eukaryotic cells, e.g., bacteria, yeast, mammalian
(rat, simian, hamster, or human), comprising the isolated
polypeptides or polynucleotides. The host cells can be combined
with a carrier.
[0160] II. Processes for Preparing Compositions
[0161] Antibodies, their manufacture and uses are well known and
disclosed in, for example, Harlow, E. and Lane, D. (1999)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. The antibodies may be generated
using standard methods known in the art. Examples of antibodies
include (but are not limited to) monoclonal, single chain, and
functional fragments of antibodies. Methods for generating such
antibodies are known in the art; see, e.g. Collarini et al. (2009)
J. Immunol. 183(10):6338-6345.
[0162] Antibodies may be produced in a range of hosts, for example
goats, rabbits, rats, mice, humans, and others. They may be
immunized by injection with a target antigen or a fragment or
oligopeptide thereof which has immunogenic properties, such as a
C-terminal fragment of HLA-DR or an isolated polypeptide. Depending
on the host species, various adjuvants may be added and used to
increase an immunological response. Such adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide,
and surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG
(Bacille Calmette-Guerin) and Corynebacterium parvum are
particularly useful. This this disclosure also provides the
isolated polypeptide and an adjuvant.
[0163] In certain aspects, the antibodies of the present disclosure
are polyclonal, i.e., a mixture of plural types of anti-HLA-DR
antibodies having different amino acid sequences. In one aspect,
the polyclonal antibody comprises a mixture of plural types of
anti-HLA-DR antibodies having different CDRs. As such, a mixture of
cells which produce different antibodies is cultured, and an
antibody purified from the resulting culture can be used (see
International Patent Application Publication No. WO
2004/061104).
[0164] Monoclonal Antibody Production. Monoclonal antibodies to
HLA-DR may be prepared using any technique which provides for the
production of antibody molecules by continuous cell lines in
culture. Such techniques include, but are not limited to, the
hybridoma technique (see, e.g., Kohler, G. et al. (1975) Nature
256:495-497); the trioma technique; the human B-cell hybridoma
technique (see, e.g., Kozbor, D. et al. (1983) Immunol. Today 4:72)
and the EBV hybridoma technique to produce human monoclonal
antibodies (see, e.g., Cole et al. (1985) in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., 77-96). Human monoclonal
antibodies can be utilized in the practice of the present
technology and can be produced by using human hybridomas (see,
e.g., Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. U.S.A.
80:2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see, e.g., Cole et al. (1985) in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., 77-96). For
example, a population of nucleic acids that encode regions of
antibodies can be isolated. PCR utilizing primers derived from
sequences encoding conserved regions of antibodies is used to
amplify sequences encoding portions of antibodies from the
population and then reconstruct DNAs encoding antibodies or
fragments thereof, such as variable domains, from the amplified
sequences. Such amplified sequences also can be fused to DNAs
encoding other proteins--e.g., a bacteriophage coat, or a bacterial
cell surface protein--for expression and display of the fusion
polypeptides on phage or bacteria. Amplified sequences can then be
expressed and further selected or isolated based, e.g., on the
affinity of the expressed antibody or fragment thereof for an
antigen or epitope present on the HLA-DR polypeptide.
Alternatively, hybridomas expressing anti-HLA-DR monoclonal
antibodies can be prepared by immunizing a subject, e.g., with an
isolated polypeptide comprising, or alternatively consisting
essentially of, or yet further consisting of, the amino acid
sequence of HLA-DR or a fragment thereof, and then isolating
hybridomas from the subject's spleen using routine methods. See,
e.g., Galfre, G. et al. (1981) Methods Enzymol. 73:3-46. Screening
the hybridomas using standard methods will produce monoclonal
antibodies of varying specificity (i.e., for different epitopes)
and affinity. A selected monoclonal antibody with the desired
properties, e.g., HLA-DR binding, can be (i) used as expressed by
the hybridoma, (ii) bound to a molecule such as polyethylene glycol
(PEG) to alter its properties, or (iii) a cDNA encoding the
monoclonal antibody can be isolated, sequenced and manipulated in
various ways. In one aspect, the anti-HLA-DR monoclonal antibody is
produced by a hybridoma which includes a B cell obtained from a
transgenic non-human animal, e.g., a transgenic mouse, having a
genome comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell. Hybridoma techniques
include those known in the art and taught in Harlow et al. (1988)
Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 349; Hammerling et al. (1981) Monoclonal
Antibodies And T-Cell Hybridomas, 563-681.
[0165] Phage Display Technique. As noted above, the antibodies of
the present disclosure can be produced through the application of
recombinant DNA and phage display technology. For example,
anti-HLA-DR antibodies, can be prepared using various phage display
methods known in the art. In phage display methods, functional
antibody domains are displayed on the surface of a phage particle
which carries polynucleotide sequences encoding them. Phage with a
desired binding property is selected from a repertoire or
combinatorial antibody library (e.g., human or murine) by selecting
directly with an antigen, typically an antigen bound or captured to
a solid surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 with Fab, F.sub.v or
disulfide stabilized F.sub.v antibody domains are recombinantly
fused to either the phage gene III or gene VIII protein. In
addition, methods can be adapted for the construction of Fab
expression libraries (see, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281) to allow rapid and effective identification of
monoclonal Fab fragments with the desired specificity for a HLA-DR
polypeptide, e.g., a polypeptide or derivatives, fragments, analogs
or homologs thereof. Other examples of phage display methods that
can be used to make the isolated antibodies of the present
disclosure include those disclosed in Huston, J. S. et al. (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883; Chaudhary, V. K. et al.
(1990) Proc. Natl. Acad. Sci. U.S.A., 87:1066-1070; Brinkman et
al., J. Immunol. Methods 182: 41-50 (1995); Ames, R. S. et al.
(1995) J. Immunol. Methods 184:177-186; Kettleborough et al., Eur.
J. Immunol. 24: 952-958 (1994); Persic, L. et al. (1997) Gene
187:9-18; Burton, D. R. et al. (1994) Advances in Immunology
57:191-280; International Patent Application No. PCT/GB91/01134;
International Patent Application Publication Nos. WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et
al.); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271
(Affymax); and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727 and 5,733,743.
[0166] Methods useful for displaying polypeptides on the surface of
bacteriophage particles by attaching the polypeptides via disulfide
bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As
described in the above references, after phage selection, the
antibody coding regions from the phage can be isolated and used to
generate whole antibodies, including human antibodies, or any other
desired antigen binding fragment, and expressed in any desired host
including mammalian cells, insect cells, plant cells, yeast, and
bacteria. For example, techniques to recombinantly produce Fab,
Fab' and F(ab').sub.2 fragments can also be employed using methods
known in the art such as those disclosed in International Patent
Application Publication No. WO 92/22324; Mullinax, R. L. et al.
(1992) BioTechniques 12:864-869; Sawai, H. et al. (1995) AJRI
34:26-34; and Better, M. et al. (1988) Science 240:1041-1043.
[0167] Generally, hybrid antibodies or hybrid antibody fragments
that are cloned into a display vector can be selected against the
appropriate antigen in order to identify variants that maintained
good binding activity, because the antibody or antibody fragment
will be present on the surface of the phage or phagemid particle.
See e.g., Barbas III et al., Phage Display, A Laboratory Manual
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2001). However, other vector formats could be used for this
process, such as cloning the antibody fragment library into a lytic
phage vector (modified T7 or Lambda Zap systems) for selection
and/or screening.
[0168] Alternate Methods of Antibody Production. Antibodies may
also be produced by inducing in vivo production in the lymphocyte
population or by screening recombinant immunoglobulin libraries or
panels of highly specific binding reagents (Orlandi, R. et al.
(1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al.
(1991) Nature 349:293-299).
[0169] Alternatively, techniques for the production of single chain
antibodies may be used. Single chain antibodies (scF.sub.vs)
comprise a heavy chain variable region and a light chain variable
region connected with a linker peptide (typically around 5 to 25
amino acids in length). In the scF.sub.v, the variable regions of
the heavy chain and the light chain may be derived from the same
antibody or different antibodies. scF.sub.vs may be synthesized
using recombinant techniques, for example by expression of a vector
encoding the scF.sub.v in a host organism such as E. coli. DNA
encoding scF.sub.v can be obtained by performing amplification
using a partial DNA encoding the entire or a desired amino acid
sequence of a DNA selected from a DNA encoding the heavy chain or
the variable region of the heavy chain of the above-mentioned
antibody and a DNA encoding the light chain or the variable region
of the light chain thereof as a template, by PCR using a primer
pair that defines both ends thereof, and further performing
amplification combining a DNA encoding a polypeptide linker portion
and a primer pair that defines both ends thereof, so as to ligate
both ends of the linker to the heavy chain and the light chain,
respectively. An expression vector containing the DNA encoding
scF.sub.v and a host transformed by the expression vector can be
obtained according to conventional methods known in the art.
[0170] Antigen binding fragments may also be generated, for example
the F(ab').sub.2 fragments which can be produced by pepsin
digestion of the antibody molecule and the Fab fragments which can
be generated by reducing the disulfide bridges of the F(ab').sub.2
fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 256:1275-1281).
[0171] Antibody Modifications. The antibodies of the present
disclosure may be multimerized to increase the affinity for an
antigen. The antibody to be multimerized may be one type of
antibody or a plurality of antibodies which recognize a plurality
of epitopes of the same antigen. As a method of multimerization of
the antibody, binding of the IgG CH3 domain to two scF.sub.v
molecules, binding to streptavidin, introduction of a
helix-turn-helix motif and the like can be exemplified.
[0172] The antibody compositions disclosed herein may be in the
form of a conjugate formed between any of these antibodies and
another agent (immunoconjugate). In one aspect, the antibodies
disclosed herein are conjugated to radioactive material. In another
aspect, the antibodies disclosed herein can be bound to various
types of molecules such as polyethylene glycol (PEG).
[0173] Antibody Screening. Various immunoassays may be used for
screening to identify antibodies having the desired specificity.
Numerous protocols for competitive binding or immunoradiometric
assays using either polyclonal or monoclonal antibodies with
established specificities are well known in the art. Such
immunoassays typically involve the measurement of complex formation
between HLA-DR, or any fragment or oligopeptide thereof and its
specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies specific to two non-interfering
HLA-DR epitopes may be used, but a competitive binding assay may
also be employed (Maddox, D. E. et al. (1983) J. Exp. Med.
158:1211-1216).
[0174] Antibody Purification. The antibodies disclosed herein can
be purified to homogeneity. The separation and purification of the
antibodies can be performed by employing conventional protein
separation and purification methods.
[0175] By way of example only, the antibody can be separated and
purified by appropriately selecting and combining use of
chromatography columns, filters, ultrafiltration, salt
precipitation, dialysis, preparative polyacrylamide gel
electrophoresis, isoelectric focusing electrophoresis, and the
like. Strategies for Protein Purification and Characterization: A
Laboratory Course Manual, Marshak, D. R. et al. eds., Cold Spring
Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed
Harlow and David Lane, Cold Spring Harbor Laboratory (1988).
[0176] Examples of chromatography include affinity chromatography,
ion exchange chromatography, hydrophobic chromatography, gel
filtration chromatography, reverse phase chromatography, and
adsorption chromatography. In one aspect, chromatography can be
performed by employing liquid chromatography such as HPLC or
FPLC.
[0177] In one aspect, a Protein A column or a Protein G column may
be used in affinity chromatography. Other exemplary columns include
a Protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and
the like.
Methods of Use
[0178] General. The antibodies disclosed herein are useful in
methods known in the art relating to the localization and/or
quantitation of a HLA-DR polypeptide (e.g., for use in measuring
levels of the HLA-DR polypeptide within appropriate physiological
samples, for use in diagnostic methods, for use in imaging the
polypeptide, and the like). The antibodies disclosed herein are
useful in isolating a HLA-DR polypeptide by standard techniques,
such as affinity chromatography or immunoprecipitation. A HLA-DR
antibody disclosed herein can facilitate the purification of
natural HLA-DR polypeptides from biological samples, e.g.,
mammalian sera or cells as well as recombinantly-produced HLA-DR
polypeptides expressed in a host system. Moreover, HLA-DR antibody
can be used to detect a HLA-DR polypeptide (e.g., in plasma, a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the polypeptide. The HLA-DR
antibodies disclosed herein can be used diagnostically to monitor
HLA-DR levels in tissue as part of a clinical testing procedure,
e.g., to determine the efficacy of a given treatment regimen. The
detection can be facilitated by coupling (i.e., physically linking)
the HLA-DR antibodies disclosed herein to a detectable
substance.
[0179] In another aspect, provided herein is a composition
comprising an antibody or antigen binding fragment as disclosed
herein bound to a peptide comprising, for example, a human HLA-DR
protein or a fragment thereof. In one aspect, the peptide is
associated with a cell. For example, the composition may comprise a
disaggregated cell sample labeled with an antibody or antibody
fragment as disclosed herein, which composition is useful in, for
example, affinity chromatography methods for isolating cells or for
flow cytometry-based cellular analysis or cell sorting. As another
example, the composition may comprise a fixed tissue sample or cell
smear labeled with an antibody or antibody fragment as disclosed
herein, which composition is useful in, for example,
immunohistochemistry or cytology analysis. In another aspect, the
antibody or the antibody fragment is bound to a solid support,
which is useful in, for example: ELISAs; affinity chromatography or
immunoprecipitation methods for isolating HLA-DR proteins or
fragments thereof, HLA-DR-positive cells, or complexes containing
HLA-DR and other cellular components. In another aspect, the
peptide is bound to a solid support. For example, the peptide may
be bound to the solid support via a secondary antibody specific for
the peptide, which is useful in, for example, sandwich ELISAs. As
another example, the peptide may be bound to a chromatography
column, which is useful in, for example, isolation or purification
of antibodies according to the present technology. In another
aspect, the peptide is disposed in a solution, such as a lysis
solution or a solution containing a sub-cellular fraction of a
fractionated cell, which is useful in, for example, ELISAs and
affinity chromatography or immunoprecipitation methods of isolating
HLA-DR proteins or fragments thereof or complexes containing HLA-DR
and other cellular components. In another aspect, the peptide is
associated with a matrix, such as, for example, a gel
electrophoresis gel or a matrix commonly used for western blotting
(such as membranes made of nitrocellulose or polyvinylidene
difluoride), which compositions are useful for electrophoretic
and/or immunoblotting techniques, such as Western blotting.
[0180] Detection of HLA-DR Polypeptide. An exemplary method for
detecting the level of HLA-DR polypeptides in a biological sample
involves obtaining a biological sample from a subject and
contacting the biological sample with a HLA-DR binding agent, e.g.,
an antibody disclosed herein or known in the art that is capable of
detecting the HLA-DR polypeptides.
[0181] In one aspect, the HLA-DR antibodies Lym-1, or Lym-2, or
fragments thereof are detectably labeled. The term "labeled", with
regard to the antibody is intended to encompass direct labeling of
the antibody by coupling (i.e., physically linking) a detectable
substance to the antibody, as well as indirect labeling of the
antibody by reactivity with another compound that is directly
labeled. Non-limiting examples of indirect labeling include
detection of a primary antibody using a fluorescently-labeled
secondary antibody and end-labeling of a DNA probe with biotin such
that it can be detected with fluorescently-labeled
streptavidin.
[0182] The detection method of the present disclosure can be used
to detect expression levels of HLA-DR polypeptides in a biological
sample in vitro as well as in vivo. In vitro techniques for
detection of HLA-DR polypeptides include enzyme linked
immunosorbent assays (ELISAs), Western blots, flow cytometry,
immunoprecipitations, radioimmunoassay, and immunofluorescence
(e.g., IHC). Furthermore, in vivo techniques for detection of
HLA-DR polypeptides include introducing into a subject a labeled
anti-HLA-DR antibody. By way of example only, the antibody can be
labeled with a detectable marker, e.g. a radioactive marker whose
presence and location in a subject can be detected by standard
imaging techniques. In one aspect, the biological sample contains
polypeptide molecules from the subject.
[0183] Immunoassay and Imaging. A HLA-DR antibody disclosed herein
can be used to assay HLA-DR polypeptide levels in a biological
sample (e.g., human plasma) using antibody-based techniques. For
example, protein expression in tissues can be studied with
classical immunohistochemical (IHC) staining methods. Jalkanen, M.
et al. (1985) J. Cell. Biol. 101:976-985; Jalkanen, M. et al.
(1987) J. Cell. Biol. 105:3087-3096. Other antibody-based methods
useful for detecting protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (MA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes or other radioactive agents, such as iodine
(.sup.125I, .sup.121I, .sup.131I), carbon (.sup.14C), sulfur
(.sup.35S), tritium (.sup.3H), indium (.sup.112In), and technetium
(.sup.99mTc), and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0184] In addition to assaying HLA-DR polypeptide levels in a
biological sample, HLA-DR polypeptide levels can also be detected
in vivo by imaging. Labels that can be incorporated with
anti-HLA-DR antibodies for in vivo imaging of HLA-DR polypeptide
levels include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which can be incorporated into the HLA-DR antibody by labeling of
nutrients for the relevant scF.sub.v clone.
[0185] A HLA-DR antibody which has been labeled with an appropriate
detectable imaging moiety, such as a radioisotope (e.g., .sup.131I,
.sup.112In, .sup.199mTc), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (e.g.,
parenterally, subcutaneously, or intraperitoneally) into the
subject. It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99mTc. The labeled HLA-DR antibody will then
preferentially accumulate at the location of cells which contain
the specific target polypeptide. For example, in vivo tumor imaging
is described in Burchiel, S. W. et al. (1982) Tumor Imaging: The
Radiochemical Detection of Cancer 13.
[0186] In some aspects, HLA-DR antibodies containing structural
modifications that facilitate rapid binding and cell uptake and/or
slow release are useful in in vivo imaging detection methods. In
some aspects, the HLA-DR antibody contains a deletion in the CH2
constant heavy chain region of the antibody to facilitate rapid
binding and cell uptake and/or slow release. In some aspects, a Fab
fragment is used to facilitate rapid binding and cell uptake and/or
slow release. In some aspects, a F(ab)'2 fragment is used to
facilitate rapid binding and cell uptake and/or slow release.
[0187] Diagnostic Uses of HLA-DR antibodies. The HLA-DR antibody
compositions disclosed herein are useful in diagnostic and
prognostic methods. As such, the present disclosure provides
methods for using the antibodies disclosed herein in the diagnosis
of HLA-DR-related medical conditions in a subject. Antibodies
disclosed herein may be selected such that they have a high level
of epitope binding specificity and high binding affinity to the
HLA-DR polypeptide. In general, the higher the binding affinity of
an antibody, the more stringent wash conditions can be performed in
an immunoassay to remove nonspecifically bound material without
removing the target polypeptide. Accordingly, HLA-DR antibodies of
the present technology useful in diagnostic assays usually have
binding affinities of at least 10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9, 10.sup.-10, 10.sup.-11, or 10.sup.-12 M. In certain
aspects, HLA-DR antibodies used as diagnostic reagents have a
sufficient kinetic on-rate to reach equilibrium under standard
conditions in at least 12 hours, at least 5 hours, at least 1 hour,
or at least 30 minutes.
[0188] Some methods of the present technology employ polyclonal
preparations of anti-HLA-DR antibodies and polyclonal anti-HLA-DR
antibody compositions as diagnostic reagents, and other methods
employ monoclonal isolates. In methods employing polyclonal human
anti-HLA-DR antibodies prepared in accordance with the methods
described above, the preparation typically contains an assortment
of HLA-DR antibodies, e.g., antibodies, with different epitope
specificities to the target polypeptide. The monoclonal anti-HLA-DR
antibodies of the present disclosure are useful for detecting a
single antigen in the presence or potential presence of closely
related antigens.
[0189] The HLA-DR antibodies of the present disclosure can be used
as diagnostic reagents for any kind of biological sample. In one
aspect, the HLA-DR antibodies disclosed herein are useful as
diagnostic reagents for human biological samples. HLA-DR antibodies
can be used to detect HLA-DR polypeptides in a variety of standard
assay formats. Such formats include immunoprecipitation, Western
blotting, ELISA, radioimmunoassay, flow cytometry, IHC and
immunometric assays. See Harlow & Lane, Antibodies, A
Laboratory Manual (Cold Spring Harbor Publications, New York,
1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262;
4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074 and 4,098,876. Biological samples can be
obtained from any tissue (including biopsies), cell or body fluid
of a subject.
[0190] In another aspect, the present disclosure provides methods
for determining whether a subject can be effectively treated with
CAR T cell or CAR NK cell composition as described herein. The
method comprises assaying a cancer or tumor sample isolated from
the patient for HLA-DR protein or polypeptide expression using any
appropriate method, e.g., immunohistochemistry using an HLA-DR
antibody or the polymerase chain reaction (PCR). In one aspect, the
expression level of the HLA-DR polypeptide in the biological sample
obtained from the subject is determined and compared with HLA-DR
expression levels found in a biological sample obtained from a
subject or a population of patients free of the disease. Increased
expression of the HLA-DR polypeptide, as compared to the expression
level of the polypeptide or protein in the patient sample(s) from
the patients free of disease indicates that the patient is likely
to be responsive to the CAR T cell or CAR NK cell therapy of this
disclosure, and lack of elevated expression indicates that the
patient is not likely to be responsive to the CAR T cell or CAR NK
cell therapy. Non-limiting examples of samples include, e.g., any
body fluid including, but not limited to, e.g., sputum, serum,
plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid,
ascite fluid or blood and including biopsy samples of body tissue.
The samples are also a tumor cell. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. In a further aspect, an effective amount
of the HLA-DR CAR therapy is administered to the subject or
patient
[0191] In a particular aspect, the present disclosure relates to
methods for determining if a patient is likely to respond or is not
likely to HLA-DR CAR therapy. In specific embodiments, this method
comprises contacting a tumor sample isolated from the patient with
an effective amount of an HLA-DR binding agent, e.g., an HLA-DR
antibody and detecting the presence of any agent or antibody bound
to the tumor sample. In further embodiments, the presence of agent
or antibody bound to the tumor sample indicates that the patient is
likely to respond to the HLA-DR CAR therapy and the absence of
antibody bound to the tumor sample indicates that the patient is
not likely to respond to the HLA-DR therapy. Non-limiting examples
of samples include, e.g., any body fluid including, but not limited
to, e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool,
cerebrospinal fluid, ascite fluid or blood and including biopsy
samples of body tissue. The samples are also a tumor cell. The test
sample used in the above-described method will vary based on the
assay format, nature of the detection method and the tissues, cells
or extracts used as the sample to be assayed. In some embodiments,
the method comprises the additional step of administering an
effective amount of the HLA-DR CAR therapy to a patient that is
determined likely to respond to the HLA-DR CAR therapy. In some
embodiments, the patient a HLA-DR expressing tumor and/or
cancer.
[0192] There are a number of disease states in which the elevated
expression level of HLA-DR polypeptides is known to be indicative
of whether a subject with the disease is likely to respond to a
particular type of therapy or treatment. Non-limiting examples of
such disease states include cancer, e.g., a carcinoma, a sarcoma or
a leukemia. Thus, the method of detecting a HLA-DR polypeptide in a
biological sample can be used as a method of prognosis, e.g., to
evaluate the likelihood that the subject will respond to the
therapy or treatment. The level of the HLA-DR polypeptide in a
suitable tissue or body fluid sample from the subject is determined
and compared with a suitable control, e.g., the level in subjects
with the same disease but who have responded favorably to the
treatment. Non-limiting examples of samples include, e.g., any body
fluid including, but not limited to, e.g., sputum, serum, plasma,
lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascite
fluid or blood and including biopsy samples of body tissue. The
samples are also a tumor cell. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are known in the art and can be readily
adapted in order to obtain a sample which is compatible with the
system utilized.
[0193] In one aspect, the present disclosure provides for methods
of monitoring the influence of agents (e.g., the CART cell or CAR
NK cell compositions of this disclosure, drugs, compounds, or small
molecules) on the expression of HLA-DR polypeptides. Such assays
can be applied in basic drug screening and in clinical trials. For
example, the effectiveness of an agent to decrease HLA-DR
polypeptide levels can be monitored in clinical trials of subjects
exhibiting elevated expression of HLA-DR, e.g., patients diagnosed
with cancer. An agent that affects the expression of HLA-DR
polypeptides can be identified by administering the agent and
observing a response. In this way, the expression pattern of the
HLA-DR polypeptide can serve as a marker, indicative of the
physiological response of the subject to the agent. Accordingly,
this response state may be determined before, and at various points
during, treatment of the subject with the agent. In some
embodiments, the method further comprises the additional step of
administering an effective amount of the HLA-DR CAR therapy to a
patient that is determined to require additional therapy.
[0194] Further method aspects of the present disclosure relate to
methods for determining if a patient is likely to respond or is not
likely to HLA-DR CAR therapy. In specific embodiments, this method
comprises contacting a tumor sample isolated from the patient with
an effective amount of an HLA-DR antibody and detecting the
presence of any antibody bound to the tumor sample. In further
embodiments, the presence of antibody bound to the tumor sample
indicates that the patient is likely to respond to the HLA-DR CAR
therapy and the absence of antibody bound to the tumor sample
indicates that the patient is not likely to respond to the HLA-DR
therapy. In some embodiments, the method comprises the additional
step of administering an effective amount of the HLA-DR CAR therapy
to a patient that is determined likely to respond to the HLA-DR CAR
therapy. In some embodiments, the patient a HLA-DR expressing tumor
and/or cancer.
[0195] III. Kits
[0196] As set forth herein, the present disclosure provides
diagnostic methods for determining the expression level of HLA-DR.
In one particular aspect, the present disclosure provides kits for
performing these methods as well as instructions for carrying out
the methods of the present disclosure such as collecting tissue
and/or performing the screen, and/or analyzing the results.
[0197] The kit comprises, or alternatively consists essentially of,
or yet further consists of, a HLA-DR antibody composition (e.g.,
monoclonal antibodies) disclosed herein, and instructions for use.
The kits are useful for detecting the presence of HLA-DR
polypeptides in a biological sample, e.g., any body fluid
including, but not limited to, e.g., sputum, serum, plasma, lymph,
cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or
blood and including biopsy samples of body tissue. The test samples
may also be a tumor cell, a normal cell adjacent to a tumor, a
normal cell corresponding to the tumor tissue type, a blood cell, a
peripheral blood lymphocyte, or combinations thereof. The test
sample used in the above-described method will vary based on the
assay format, nature of the detection method and the tissues, cells
or extracts used as the sample to be assayed. Methods for preparing
protein extracts or membrane extracts of cells are known in the art
and can be readily adapted in order to obtain a sample which is
compatible with the system utilized.
[0198] In some aspects, the kit can comprise: one or more HLA-DR
antibodies capable of binding and that bind a HLA-DR polypeptide in
a biological sample (e.g., an antibody or antigen-binding fragment
thereof having the same antigen-binding specificity of HLA-DR
antibody Lym-1 or Lym-2); means for determining the amount of the
HLA-DR polypeptide in the sample; and means for comparing the
amount of the HLA-DR polypeptide in the sample with a standard. One
or more of the HLA-DR antibodies may be labeled. The kit
components, (e.g., reagents) can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect the HLA-DR polypeptides. In certain aspects, the kit
comprises a first antibody, e.g., attached to a solid support,
which binds to a HLA-DR polypeptide; and, optionally; 2) a second,
different antibody which binds to either the HLA-DR polypeptide or
the first antibody and is conjugated to a detectable label.
[0199] The kit can also comprise, e.g., a buffering agent, a
preservative or a protein-stabilizing agent. The kit can further
comprise components necessary for detecting the detectable-label,
e.g., an enzyme or a substrate. The kit can also contain a control
sample or a series of control samples, which can be assayed and
compared to the test sample. Each component of the kit can be
enclosed within an individual container and all of the various
containers can be within a single package, along with instructions
for interpreting the results of the assays performed using the kit.
The kits of the present disclosure may contain a written product on
or in the kit container. The written product describes how to use
the reagents contained in the kit.
[0200] As amenable, these suggested kit components may be packaged
in a manner customary for use by those of skill in the art. For
example, these suggested kit components may be provided in solution
or as a liquid dispersion or the like.
[0201] IV. Carriers
[0202] The antibodies also can be bound to many different carriers.
Thus, this disclosure also provides compositions containing the
antibodies and another substance, active or inert. Examples of
well-known carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, agaroses and magnetite. The nature of
the carrier can be either soluble or insoluble for purposes of the
disclosure. Those skilled in the art will know of other suitable
carriers for binding antibodies, or will be able to ascertain such,
using routine experimentation.
Chimeric Antigen Receptors and Uses Thereof
[0203] I. Compositions
[0204] The present disclosure provides chimeric antigen receptors
(CAR) that bind to HLA-DR comprising, or consisting essentially of,
a cell activation moiety comprising an extracellular,
transmembrane, and intracellular domain. The extracellular domain
comprises a target-specific binding element otherwise referred to
as the antigen binding domain. The intracellular domain or
cytoplasmic domain comprises, a costimulatory signaling region and
a zeta chain portion. The CAR may optionally further comprise a
spacer domain of up to 300 amino acids, preferably 10 to 100 amino
acids, more preferably 25 to 50 amino acids.
[0205] Antigen Binding Domain. In certain aspects, the present
disclosure provides a CAR that comprises, or alternatively consists
essentially thereof, or yet consists of an antigen binding domain
specific to HLA-DR. In some embodiments, the antigen binding domain
comprises, or alternatively consists essentially thereof, or yet
consists of the antigen binding domain of an anti-HLA-DR antibody.
In further embodiments, the heavy chain variable region and light
chain variable region of an anti-HLA-DR antibody comprises, or
alternatively consists essentially thereof, or yet consists of the
antigen binding domain the anti-HLA-DR antibody.
[0206] In some embodiments, the heavy chain variable region of the
antibody comprises, or consists essentially thereof, or consists of
SEQ ID NOs: 7 to 10 or an equivalent of each thereof and/or
comprises one or more CDR regions comprising SEQ ID NOs: 1 to 6 or
an equivalent of each thereof. In some embodiments, the light chain
variable region of the antibody comprises, or consists essentially
thereof, or consists of SEQ ID NOs: 17 to 20 or an equivalent
thereof and/or comprises one or more CDR regions comprising SEQ ID
NOs: 11 to 16 or an equivalent thereof.
[0207] Transmembrane Domain. The transmembrane domain may be
derived either from a natural or from a synthetic source. Where the
source is natural, the domain may be derived from any
membrane-bound or transmembrane protein. Transmembrane regions of
particular use in this disclosure may be derived from CD8, CD28,
CD3, CD45, CD4, CDS, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80,
CD86, CD 134, CD137, CD 154, TCR. Alternatively the transmembrane
domain may be synthetic, in which case it will comprise
predominantly hydrophobic residues such as leucine and valine.
Preferably a triplet of phenylalanine, tryptophan and valine will
be found at each end of a synthetic transmembrane domain.
Optionally, a short oligo- or polypeptide linker, preferably
between 2 and 10 amino acids in length may form the linkage between
the transmembrane domain and the cytoplasmic signaling domain of
the CAR. A glycine-serine doublet provides a particularly suitable
linker.
[0208] Cytoplasmic Domain. The cytoplasmic domain or intracellular
signaling domain of the CAR is responsible for activation of at
least one of the traditional effector functions of an immune cell
in which a CAR has been placed. The intracellular signaling domain
refers to a portion of a protein which transduces the effector
function signal and directs the immune cell to perform its specific
function. An entire signaling domain or a truncated portion thereof
may be used so long as the truncated portion is sufficient to
transduce the effector function signal. Cytoplasmic sequences of
the TCR and co-receptors as well as derivatives or variants thereof
can function as intracellular signaling domains for use in a CAR.
Intracellular signaling domains of particular use in this
disclosure may be derived from FcR, TCR, CD3, CDS, CD22, CD79a,
CD79b, CD66d. Since signals generated through the TCR are alone
insufficient for full activation of a T cell, a secondary or
co-stimulatory signal may also be required. Thus, the intracellular
region of a co-stimulatory signaling molecule, including but not
limited CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3, or a ligand that specifically binds with CD83, to may
also be included in the cytoplasmic domain of the CAR.
[0209] In some embodiments, the cell activation moiety of the
chimeric antigen receptor is a T-cell signaling domain comprising,
or alternatively consisting essentially of, or yet further
consisting of, one or more proteins or fragments thereof selected
from the group consisting of CD8 protein, CD28 protein, 4-1BB
protein, and CD3-zeta protein.
[0210] In specific embodiments, the CAR comprises, or alternatively
consists essentially thereof, or yet consists of an antigen binding
domain of an anti-HLA-DR antibody, a CD8 .alpha. hinge domain, a
CD8 .alpha. transmembrane domain, a costimulatory signaling region,
and a CD3 zeta signaling domain. In further embodiments, the
costimulatory signaling region comprises either or both a CD28
costimulatory signaling region and a 4-1BB costimulatory signaling
region.
[0211] In some embodiments, the CAR can further comprise a
detectable marker or purification marker.
[0212] In a further aspect, this disclosure provides complex
comprising an HLA-DR CAR cell bound to its target cell. In a
further aspect, the complex is detectably labeled. Detectable
labels are known in the art and briefly described herein.
[0213] II. Process for Preparing CARs
[0214] Aspects of the present disclosure relate to an isolated cell
comprising a HLA-DR CAR and methods of producing such cells. The
cell is a prokaryotic or a eukaryotic cell. In one aspect, the cell
is a T cell or a NK cell. The eukaryotic cell can be from any
preferred species, e.g., an animal cell, a mammalian cell such as a
human, a feline or a canine cell.
[0215] In specific embodiments, the isolated cell comprises, or
alternatively consists essentially of, or yet further consists of
an exogenous CAR comprising, or alternatively consisting
essentially of, or yet further consisting of, an antigen binding
domain of an anti-HLA-DR antibody, a CD8 .alpha. hinge domain, a
CD8 .alpha. transmembrane domain, a CD28 costimulatory signaling
region and/or a 4-1BB costimulatory signaling region, and a CD3
zeta signaling domain. In certain embodiments, the isolated cell is
a T-cell, e.g., an animal T-cell, a mammalian T-cell, a feline
T-cell, a canine T-cell or a human T-cell. In certain embodiments,
the isolated cell is an NK-cell, e.g., an animal NK-cell, a
mammalian NK-cell, a feline NK-cell, a canine NK-cell or a human
NK-cell.
[0216] In certain embodiments, methods of producing HLA-DR CAR
expressing cells are disclosed comprising, or alternatively
consisting essentially of: (i) transducing a population of isolated
cells with a nucleic acid sequence encoding a HLA-DR CAR and (ii)
selecting a subpopulation of cells that have been successfully
transduced with said nucleic acid sequence of step (i). In some
embodiments, the isolated cells are T-cells, an animal T-cell, a
mammalian T-cell, a feline T-cell, a canine T-cell or a human
T-cell, thereby producing HLA-DR CAR T-cells. In certain
embodiments, the isolated cell is an NK-cell, e.g., an animal
NK-cell, a mammalian NK-cell, a feline NK-cell, a canine NK-cell or
a human NK-cell, thereby producing HLA-DR CAR NK-cells.
[0217] Sources of Isolated Cells. Prior to expansion and genetic
modification of the cells disclosed herein, cells may be obtained
from a subject--for instance, in embodiments involving autologous
therapy--or a commercially available culture.
[0218] Cells can be obtained from a number of sources in a subject,
including peripheral blood mononuclear cells, bone marrow, lymph
node tissue, cord blood, thymus tissue, tissue from a site of
infection, ascites, pleural effusion, spleen tissue, and
tumors.
[0219] Methods of isolating relevant cells are well known in the
art and can be readily adapted to the present application; an
exemplary method is described in the examples below. Isolation
methods for use in relation to this disclosure include, but are not
limited to Life Technologies Dynabeads.RTM. system; STEMcell
Technologies EasySep.TM., RoboSep.TM., RosetteSep.TM., SepMate.TM.;
Miltenyi Biotec MACS.TM. cell separation kits, and other
commercially available cell separation and isolation kits.
Particular subpopulations of immune cells may be isolated through
the use of beads or other binding agents available in such kits
specific to unique cell surface markers. For example, MACS.TM. CD4+
and CD8+ MicroBeads may be used to isolate CD4+ and CD8+
T-cells
[0220] Alternatively, cells may be obtained through commercially
available cell cultures, including but not limited to, for T-cells,
lines BCL2 (AAA) Jurkat (ATCC.RTM. CRL-2902.TM.) BCL2 (S70A) Jurkat
(ATCC.RTM. CRL-2900.TM.), BCL2 (S87A) Jurkat (ATCC.RTM.
CRL-2901.TM.), BCL2 Jurkat (ATCC.RTM. CRL-2899.TM.), Neo Jurkat
(ATCC.RTM. CRL-2898.TM.); for NK cells, lines NK-92 (ATCC.RTM.
CRL-2407.TM.), NK-92MI (ATCC.RTM. CRL-2408.TM.).
[0221] Vectors. CARs may be prepared using vectors. Aspects of the
present disclosure relate to an isolated nucleic acid sequence
encoding a HLA-DR CAR and vectors comprising, or alternatively
consisting essentially of, or yet further consisting of, an
isolated nucleic acid sequence encoding the CAR and its complement
and equivalents of each thereof.
[0222] In some embodiments, the isolated nucleic acid sequence
encodes for a CAR comprising, or alternatively consisting
essentially of, or yet further consisting of an antigen binding
domain of an anti-HLA-DR antibody, a CD8 .alpha. hinge domain, a
CD8 .alpha. transmembrane domain, a CD28 costimulatory signaling
region and/or a 4-1BB costimulatory signaling region, and a CD3
zeta signaling domain. In specific embodiments, the isolated
nucleic acid sequence comprises, or alternatively consisting
essentially thereof, or yet further consisting of, sequences
encoding (a) an antigen binding domain of an anti-HLA-DR antibody
followed by (b) a CD8 .alpha. hinge domain, (c) a CD8 .alpha.
transmembrane domain followed by (d) a CD28 costimulatory signaling
region and/or a 4-1BB costimulatory signaling region followed by
(e) a CD3 zeta signaling domain.
[0223] In some embodiments, the isolated nucleic acid sequence
comprises, or alternatively consists essentially thereof, or yet
further consists of, a Kozak consensus sequence upstream of the
sequence encoding the antigen binding domain of the anti-HLA-DR
antibody. In some embodiments, the isolated nucleic acid comprises
a polynucleotide conferring antibiotic resistance.
[0224] In some embodiments, the isolated nucleic acid sequence is
comprised in a vector. In certain embodiments, the vector is a
plasmid. In other embodiments, the vector is a viral vector. In
specific embodiments, the vector is a lentiviral vector.
[0225] The preparation of exemplary vectors and the generation of
CAR expressing cells using said vectors is discussed in detail in
the examples below. In summary, the expression of natural or
synthetic nucleic acids encoding CARs is typically achieved by
operably linking a nucleic acid encoding the CAR polypeptide or
portions thereof to a promoter, and incorporating the construct
into an expression vector. The vectors can be suitable for
replication and integration eukaryotes. Methods for producing cells
comprising vectors and/or exogenous nucleic acids are well-known in
the art. See, for example, Sambrook et al. (2001) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York).
[0226] In one aspect, the term "vector" intends a recombinant
vector that retains the ability to infect and transduce
non-dividing and/or slowly-dividing cells and integrate into the
target cell's genome. In several aspects, the vector is derived
from or based on a wild-type virus. In further aspects, the vector
is derived from or based on a wild-type lentivirus. Examples of
such, include without limitation, human immunodeficiency virus
(HIV), equine infectious anemia virus (EIAV), simian
immunodeficiency virus (SIV) and feline immunodeficiency virus
(FIV). Alternatively, it is contemplated that other retrovirus can
be used as a basis for a vector backbone such murine leukemia virus
(MLV). It will be evident that a viral vector according to the
disclosure need not be confined to the components of a particular
virus. The viral vector may comprise components derived from two or
more different viruses, and may also comprise synthetic components.
Vector components can be manipulated to obtain desired
characteristics, such as target cell specificity.
[0227] The recombinant vectors of this disclosure are derived from
primates and non-primates. Examples of primate lentiviruses include
the human immunodeficiency virus (HIV), the causative agent of
human acquired immunodeficiency syndrome (AIDS), and the simian
immunodeficiency virus (SIV). The non-primate lentiviral group
includes the prototype "slow virus" visna/maedi virus (VMV), as
well as the related caprine arthritis-encephalitis virus (CAEV),
equine infectious anemia virus (EIAV) and the more recently
described feline immunodeficiency virus (FIV) and bovine
immunodeficiency virus (BIV). Prior art recombinant lentiviral
vectors are known in the art, e.g., see U.S. Pat. Nos. 6,924,123;
7,056,699; 7,07,993; 7,419,829 and 7,442,551, incorporated herein
by reference.
[0228] U.S. Pat. No. 6,924,123 discloses that certain retroviral
sequence facilitate integration into the target cell genome. This
patent teaches that each retroviral genome comprises genes called
gag, pol and env which code for virion proteins and enzymes. These
genes are flanked at both ends by regions called long terminal
repeats (LTRs). The LTRs are responsible for proviral integration,
and transcription. They also serve as enhancer-promoter sequences.
In other words, the LTRs can control the expression of the viral
genes. Encapsidation of the retroviral RNAs occurs by virtue of a
psi sequence located at the 5' end of the viral genome. The LTRs
themselves are identical sequences that can be divided into three
elements, which are called U3, R and U5. U3 is derived from the
sequence unique to the 3' end of the RNA. R is derived from a
sequence repeated at both ends of the RNA, and U5 is derived from
the sequence unique to the 5'end of the RNA. The sizes of the three
elements can vary considerably among different retroviruses. For
the viral genome. and the site of poly (A) addition (termination)
is at the boundary between R and U5 in the right hand side LTR. U3
contains most of the transcriptional control elements of the
provirus, which include the promoter and multiple enhancer
sequences responsive to cellular and in some cases, viral
transcriptional activator proteins.
[0229] With regard to the structural genes gag, pol and env
themselves, gag encodes the internal structural protein of the
virus. Gag protein is proteolytically processed into the mature
proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol
gene encodes the reverse transcriptase (RT), which contains DNA
polymerase, associated RNase H and integrase (IN), which mediate
replication of the genome.
[0230] For the production of viral vector particles, the vector RNA
genome is expressed from a DNA construct encoding it, in a host
cell. The components of the particles not encoded by the vector
genome are provided in trans by additional nucleic acid sequences
(the "packaging system", which usually includes either or both of
the gag/pol and env genes) expressed in the host cell. The set of
sequences required for the production of the viral vector particles
may be introduced into the host cell by transient transfection, or
they may be integrated into the host cell genome, or they may be
provided in a mixture of ways. The techniques involved are known to
those skilled in the art.
[0231] Retroviral vectors for use in this disclosure include, but
are not limited to Invitrogen's pLenti series versions 4, 6, and
6.2 "ViraPower" system. Manufactured by Lentigen Corp.; pHIV-7-GFP,
lab generated and used by the City of Hope Research Institute;
"Lenti-X" lentiviral vector, pLVX, manufactured by Clontech;
pLKO.1-puro, manufactured by Sigma-Aldrich; pLemiR, manufactured by
Open Biosystems; and pLV, lab generated and used by Charite Medical
School, Institute of Virology (CBF), Berlin, Germany.
[0232] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present disclosure, in order to confirm the presence of the
recombinant DNA sequence in the host cell, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays well known to those of skill in the art, such as
Southern and Northern blotting, RT-PCR and PCR; "biochemical"
assays, such as detecting the presence or absence of a particular
peptide, e.g., by immunological means (ELISAs and Western blots) or
by assays described herein to identify agents falling within the
scope of the disclosure.
[0233] Activation and Expansion of Cells. Whether prior to or after
genetic modification of the cells to express a desirable CAR, the
cells can be activated and expanded using generally known methods
such as those described in U.S. Pat. Nos. 6,352,694; 6,534,055;
6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;
7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;
6,797,514 and 6,867,041. Stimulation with the HLA-DR antigen ex
vivo can activate and expand the selected CAR expressing cell
subpopulation. Alternatively, the cells may be activated in vivo by
interaction with HLA-DR antigen.
[0234] Methods of activating relevant cells are well known in the
art and can be readily adapted to the present application; an
exemplary method is described in the examples below. Isolation
methods for use in relation to this disclosure include, but are not
limited to Life Technologies Dynabeads.RTM. system activation and
expansion kits; BD Biosciences Phosflow.TM. activation kits,
Miltenyi Biotec MACS.TM. activation/expansion kits, and other
commercially available cell kits specific to activation moieties of
the relevant cell. Particular subpopulations of immune cells may be
activated or expanded through the use of beads or other agents
available in such kits. For example, .alpha.-CD3/.alpha.-CD28
Dynabeads.RTM. may be used to activate and expand a population of
isolated T-cells
[0235] III. Methods of Use
[0236] Therapeutic Application. Method aspects of the present
disclosure relate to methods for inhibiting the growth of a tumor
in a subject in need thereof and/or for treating a cancer patient
in need thereof. In some embodiments, the tumors/cancer is B-cell
lymphoma or leukemia tumors/cancer. In some embodiments, the tumor
is a solid tumor, e.g. a carcinoma. In some embodiments, the tumor
or cancer expresses HLA-DR. In certain embodiments, these methods
comprise, or alternatively consist essentially of, or yet further
consist of, administering to the subject or patient an effective
amount of an isolated cell. In further embodiments, this isolated
cell comprises a HLA-DR CAR. In still further embodiments, the
isolated cell is a T cell or an NK cell. In some embodiments, the
isolated cell is autologous to the subject or patient being
treated. In a further aspect, the tumor expresses HLA-DR antigen
and the subject has been selected for the therapy by a diagnostic,
such as the one described herein. The therapy can be a first line
therapy, a second line therapy, a third line therapy, or a fourth
line therapy or any additional therapy as determined by the
treating physician. They can be combined with other therapies and
administered sequentially or concurrently.
[0237] The CAR cells as disclosed herein may be administered either
alone or in combination with diluents, other anti-cancer
therapeutics other than the CAR cell, and/or with other components
such as cytokines or other cell populations that are
immunostimulatory.
[0238] Pharmaceutical compositions disclosed herein may be
administered in a manner appropriate to the disease to be treated
or prevented. The quantity and frequency of administration will be
determined by such factors as the condition of the patient, and the
type and severity of the patient's disease, although appropriate
dosages may be determined by clinical trials.
[0239] IV. Carriers
[0240] Additional aspects of the disclosure relate to compositions
comprising a carrier and one or more of the products--e.g., an
isolated cell comprising a HLA-DR CAR, an isolated nucleic acid, a
vector, an isolated cell of any anti-HLA-DR antibody or CAR cell,
an anti-HLA-DR--described in the embodiments disclosed herein.
[0241] Briefly, pharmaceutical compositions disclosed herein
including but not limited to any one of the claimed compositions
may comprise a target cell population as described herein, in
combination with one or more pharmaceutically or physiologically
acceptable carriers, diluents or excipients. Such compositions may
comprise buffers such as neutral buffered saline, phosphate
buffered saline and the like; carbohydrates such as glucose,
mannose, sucrose or dextrans, mannitol; proteins; polypeptides or
amino acids such as glycine; antioxidants; chelating agents such as
EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and
preservatives. Compositions of the present disclosure may be
formulated for oral, intravenous, topical, enteral, and/or
parenteral administration. In certain embodiments, the compositions
of the present disclosure are formulated for intravenous
administration.
EXAMPLES
[0242] The following examples are illustrative of procedures which
can be used in various instances in carrying the disclosure into
effect.
Example 1--Generation of Mouse Anti-Human HLA-DR Monoclonal
Antibodies Antigen
[0243] Raji African Burkitt's lymphoma cell nuclei were used as the
antigen for producing the Lym-1 antibody. CLL biopsy cell nuclei
were used as the antigen for producing the Lym-2 antibody.
Immunization Procedures
[0244] Four week old female BALB/c mice purchased from Harlan
Laboratories were immunized every two weeks .times.4 with 10.sup.7
nuclei emulsified with Complete Freund's Adjuvant (first and second
immunization) or incomplete Freund's Adjuvant (third and fourth
immunization). Mice were injected intradermally with a total of
10.sup.7 nuclei/adjuvant divided into three separate spots on the
back of the mice per immunization. Ten days after the last
immunization, blood samples were obtained and tittered by ELISA
procedures on antigen coated plates. Mice showing the highest
titers then received a fifth immunization boost intravenously
without adjuvant in which 10.sup.6 nuclei were injected via the
lateral tail vein in a 100 .mu.l solution of sterile Phosphate
Buffered Saline.
Generation of Hybridomas
[0245] Four days later, these mice were sacrificed and the spleens
removed for the hybridoma procedure. After dispersing the
splenocytes in a solution of RPMI-1640 medium containing Pen/Strep
antibiotics, the splenocytes were fused with murine NSO cells using
PEG (Hybri MAX, mol wt 1450, Cat. No: p7181, Sigma). HAT selection
was then used to enable only fused cells to grow. Supernatant from
wells with growing hybridoma cells were then screened initially by
ELISA against antigen coated plates and secondarily by flow
cytometry on HLA-DR positive (Raji) and negative human tumor cell
lines (CEM T-cell leukemia). Hybridomas showing a positive and high
mean fluorescent index (MFI) were selected for subcloning by
limiting dilution methods. Subclones were then retested by flow
cytometry, frozen in liquid nitrogen, and expanded in 2 L vessels
to before antibody was purified by tandon Protein A or G and ion
exchange chromatography methods. Purified antibodies were then
vialed and stored at -20.degree. C. until used.
Flow Cytometry Procedures and Data
[0246] Screening methods using flow cytometry were performed on
HLA-DR positive (Raji) and negative (CEM) cell lines using
supernatant from hybridomas found positive by ELISA to antigen
coated plates. Those hybridomas producing high mean fluorescent
indexes (MFI) were then subcloned and rescreened for selective
positivity to HLA-DR. As shown below in FIGS. 1A-1F, Lym-1 and
Lym-2 produced high MFI to the HLA-DR expressing Raji cell line
with a different profile than B1 antibody. From these data, Lym-1
and Lym-2 were selected to generate CAR-T cells as described
below.
Immunohistochemistry with Selected Antibodies
[0247] Antibodies Lym-1 and Lym-2 were found to stain HLA-DR
positive cells in the germinal centers of human tonsil tissue using
standard immunohistochemical procedures and antigen retrieval
methods as shown in FIGS. 2A-2B. Staining in thymus, spleen and
bone marrow was restricted to B-cell or dendritic cells expressing
the HLA-DR antigen (Table 3).
TABLE-US-00011 TABLE 3 Reactivity of Lym-1 and Lym-2 with human
normal lymphoid and hematopoietic tissues in frozen sections or
cytospins Organ Lym-1 Lym-2 Lymph node Germinal center +++.sup.a ++
Mantle zone + +++ T-cell zones - - Interdigitating histiocytes ++
++ Sinus histiocytes - - Endothelium - - Thymus Cortex - - Medulla
++ Dendritic cells - Spleen White pulp ++ B-cell zones ++ B-cell
zones Red pulp - - Bone marrow Myeloid - - Erythroid - -
Megakaryocytes - - .sup.aIntensity of immunoperoxidase staining
from - to +++.
[0248] As shown in FIGS. 3A-3B, HLA-DR positivity was seen on the
cell membrane of antigen positive tumors such as intermediate grade
B-cell lymphomas. Finally, tissue sections from normal tissues and
organs showed restricted reactivity to lymphoid B-cells and
macrophages of the skin (Table 4). The availability of a companion
diagnostic antibody for HLA-DR using immunohistochemistry enables
the identification of patients likely to benefit from HLA-DR CAR
T-cell therapy in upcoming clinical trials.
TABLE-US-00012 TABLE 4 Reactivity of Lym-1 and Lym-2 with normal
non-lymphoid tissues in frozen sections Reactivity Tissue Lym-1
Lym-2 Adrenal -.sup.a - Brain - - Breast - - Cervix - - Colon +
surface epithelium - Duodenum - - Heart - - Kidney - - Liver - -
Lung - - Ovary - - Pancreas - - Salivary glands - - Skin +
macrophages only - Skeletal muscle - - Smooth muscle - - Stomach -
- Testis - - Thyroid - - .sup.aIntensity of immunoperoxidase
staining from - to +++.
Live Cell Radioimmunoassay
[0249] Using Lym-1 or Lym-2, a panel of human lymphoma and solid
tumor cell lines were screened for binding using a live cell
radioimmunoassay procedure. For this assay, suspension cultures and
solid tumor cell lines which were dislodged from their flasks with
EDTA-trypsin were washed twice in cold buffer consisting of PBS,
bovine serum albumin (1 mg/ml), and 0.02% sodium azide. Cells
(5.times.10.sup.5) resuspended in 100 .mu.l of wash buffer were
pipetted into microwells pretreated overnight with BSA (10 mg/ml)
in PBS to prevent antibody binding to the wells. Lym-1 or Lym-2
supernatant were then added (100 .mu.l/well) for a 30 minute
incubation period with continuous shaking using a microshaker
apparatus for 96 well plates at room temperature. After 4 washes,
100,000 cpm of 1-125 goat anti-mouse IgG was then added in 100
.mu.l and incubated with the cells for an additional 30 minute
incubation with continuous shaking. After 4 final washes, the wells
were counted in a gamma counter to determine antibody binding to
each cell preparation. The results of these studies showed that for
a large panel of human lymphoma and leukemia biopsies, reactivity
of Lym-1 and Lym-2 was restricted to tumors of B-cell but not
T-cell origin (Table 5).
TABLE-US-00013 TABLE 5 Reactivity of Lym-1 and Lym-2 with human
malignant lymphoma and leukemia biopsy specimens Diagnosis
Lym-1.sup.a Lym-2.sup.a Lymphomas.sup.b (frozen sections of lymph
node biopsies.sup.c) Well-differentiated lymphocytic 1/3 3/3 Poorly
differentiated lymphocytic, nodular 0/2 2/2 Poorly differentiated
lymphocytic, diffuse 1/3 3/3 Mixed lymphocytic and histiocytic 8/9
7/9 Histiocytic (B-cell) 12/17 12/17 T-cell 0/2 0/2 Leukemias
(cytospins of peripheral blood.sup.d) Chronic lymphocytic B-cell
type 4/10 8/10 T-cell type 0/5 0/5 .sup.aPositive/total.
.sup.bRappaport classification. .sup.cImmunoperoxidase technique.
.sup.dIndirect immunofluorescence.
[0250] Consistent with these results, Lym-1 and Lym-2 was found to
bind to a select number of human lymphoma and leukemia cell lines
as shown in Table 6.
TABLE-US-00014 TABLE 6 Reactivity of Lym-1 and Lym-2 with human
malignant lymphoma cell lines by live cell radioimmunoassay Cell
Line Lym-1 Lym-2 Burkitt's Lymphoma Raji ++++.sup.a ++ EB3 - -
DG-75 ++++ ++++ NK-9 ++ ++++ AL-1 - + Daudi + +++ NU-AmB-1 + ++
SU-AmB-1 - + SU-AmB-2 - - RAMOS - - Chevallier ++++ B46M + + B35M
++++ ++++ DND-39 + - U-698-M + ++ HRIK + - Large Cell Lymphoma
SU-DHL-1 - - SU-DHL-2 - - SU-DHL-4 ++++ SU-DHL-5 + ++ SU-DHL-6 +++
+++ SU-DHL-7 + SU-DHL-8 + SU-DHL-9 + + SU-DHL-10 ++++ SU-DHL-16 - -
NU-DHL-1 ++++ U-937 - - Undifferentiated lymphoma NU-DUL-1 - +
.sup.a-, <2,000 cpm; +, 2,000-6,000 cpm; ++, 6,000-10,000 cpm;
+++, 10,000-15000 cpm; ++++, >15,000 cpm.
[0251] By contrast, Lym-1 and Lym-2 was not found to bind to 35
human solid tumor cell lines using live cell radioimmunoassay
procedures described above (Table 7).
TABLE-US-00015 TABLE 7 Reactivity of Lym-1 and Lym-2 with 35 human
solid tumor cell lines by live cell radioimmunoassay Cell line
Derivation Lym-1 Lym-2 734B Breast carcinoma -.sup.a - 578T Breast
carcinoma - - C-399 Colon carcinoma - - Hutu-80 Colon carcinoma - -
HT-29 Colon carcinoma - - HeLa Cervical carcinoma - - SW 733
Papillary carcinoma of bladder - - SW 780 Transitional cell
carcinoma - - of bladder SW 451 Squamous cell carcinoma of - -
esophagus - - SW 579 Squamous cell carcinoma of - - thyroid SW 156
Hypernephroma - - 60 Small cell carcinoma of lung - - 464 Small
cell carcinoma of lung - - NCI-H69 Small cell carcinoma of lung - -
125 Adenocarcinoma of lung - - A427 Adenocarcinoma of lung - - A549
Adenocarcinoma of lung - - SW 1503 Mesothelioma - - BM 166
Neuroblastoma - - IMR-5 Neuroblastoma - - Y79 Retinoblastoma - -
A172 Astrocytoma - - SW 608 Astrocytoma - - U118 MG Glioblastoma -
- NU-04 Glioblastoma - - CaCl 74-36 Melanoma - - Colo 38 Melanoma -
- SW 872 Liposarcoma - - HS 919 Liposarcoma - - SW 1045 Synovial
sarcoma - - SW 80 Rhabdomyoscarcoma - - SW 1353 Chondrosarcoma - -
4-998 Osteogenic sarcoma - - 4-906 Osteogenic sarcoma - - SU-CCS-1
Clear cell sarcoma - - .sup.a-, <2,000 cpm; +, 2,000-6,000 cpm;
++, 6,000-10,000 cpm; +++, 10,000-15000 cpm; ++++, >15,000
cpm.
Binding Profiles of Lym-1 and Lym-2 Antibodies and Identification
of Lym-1 Antigen
[0252] Binding profiles and Scatchard plot analyses of Lym-1
binding with Raji cells is shown in FIG. 4A. Likewise, Scatchard
plot analyses of Lym-2 binding with the ARH-77 myeloma cell line
are shown in FIG. 4B. These data demonstrated that both antibodies
have 10.sup.8 M.sup.-1 binding affinities to antigen positive tumor
cell lines. As shown in Table 8, when compared to normal peripheral
blood B cells, there was a two to four-fold decrease in binding
affinities compared to that seen with tumor cells. In addition,
metabolic labeling of Raji cells with .sup.35S-methionine and
.sup.14C-leucine showed the characteristic banding pattern seen for
HLA-DR (FIGS. 5A-5B). As a control, the SC-1 anti-HLA-DR antibody
was used in parallel and gave the same banding pattern with
identical protein molecular weights by SDS-gel electrophoresis.
TABLE-US-00016 TABLE 6 Avidity constants of Lym-1 and Lym-2 using
target tumor cell lines (Raji, ARH-77) and tonsil lymphocytes
Monoclonal antibody Tumor cell line Tonsil Lym-1 4.02 .times.
10.sup.8 M.sup.-1 0.88 .times. 10.sup.8 M.sup.-1 Lym-2 2.33 .times.
10.sup.8 M.sup.-1 1.23 .times. 10.sup.8 M.sup.-1
Example 2--Generation of HLA-DR CAR T-Cells
Construction and Synthesis Single Chain HLA-DR Antibody Genes
[0253] The DNA sequences for 2 high binding anti-HLA-DR antibodies
generated in the laboratory (Lym-1 and Lym-2) are obtained from
MCLAB (South San Francisco, Calif.). Both antibodies are tested to
determine which one produces the most effective CAR in assays
described below. As shown below, second or third (FIG. 6)
generation CAR vectors are constructed consisting of the following
tandem genes: a kozak consensus sequence; the CD8 signal peptide;
the anti-HLA-DR heavy chain variable region; a (Glycine4Serine)3
flexible polypeptide linker (SEQ ID NO: 51); the respective
anti-HLA-DR light chain variable region; CD8 hinge and
transmembrane domains; and the CD28, 4-1BB, and CD3.zeta.
intracellular co-stimulatory signaling domains. Hinge,
transmembrane, and signaling domain DNA sequences are ascertained
from a patent by Carl June (see U.S. Patent Application Publication
No. 2013/0287748 A1). Anti-HLA-DR CAR genes are synthesized by
Genewiz, Inc. (South Plainfield, N.J.) within a pUC57 vector
backbone containing the bla gene, which confers ampicillin
resistance to the vector host.
Subcloning of CAR Genes into Lentiviral Plasmids
[0254] NovaBlue Singles.TM. chemically-competent E. coli cells are
transformed with anti-HLA-DR plasmid cDNA. Following growth of the
transformed E. coli cells, the CAR plasmids are purified and
digested with the appropriate restriction enzymes to be inserted
into an HIV-1-based lentiviral vector containing HIV-1 long
terminal repeats (LTRs), packaging signal (.PSI.), EF1.alpha.
promoter, internal ribosome entry site (IRES), and woodchuck
hepatitis virus post-transcriptional regulatory element (WPRE) via
overnight T.sub.4 DNA ligase reaction (New England Biosciences;
Ipswich, Mass.). NovaBlue Singles.TM. chemically-competent E. coli
cells are then transformed with the resulting anti-HLA-DR
containing lentiviral plasmid.
Production of Lentiviral Particles
[0255] Prior to transfection, HEK293T cells are seeded at
4.0.times.10.sup.6 cells/100 mm tissue-culture-treated plate in 10
mL complete-Tet-DMEM and incubated overnight at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. Once 80-90% confluent, HEK293T
cells are co-transfected with
[0256] CAR-gene lentiviral plasmids and lentiviral packaging
plasmids containing genes necessary to form lentiviral envelope
& capsid components, in addition to a proprietary reaction
buffer and polymer to facilitate the formation of
plasmid-containing nanoparticles that bind HEK293T cells. After
incubating transfected-HEK293T cell cultures for 4 hours at
37.degree. C., the transfection medium is replaced with 10 mL fresh
complete Tet DMEM. HEK293T cells are then incubated for an
additional 48 hours, after which cell supernatants are harvested
and tested for lentiviral particles via sandwich ELISA against p24,
the main lentiviral capsid protein. Lentivirus-containing
supernatants are aliquoted and stored at -80.degree. C. until use
for transduction of target CD4.sup.+ and CD8.sup.+ T cells.
Purification, Activation, and Enrichment of Human CD4.sup.+ and
CD8.sup.+ Peripheral Blood T-Cells
[0257] Peripheral blood mononuclear cells (PBMCs) are enriched by
density gradient centrifugation with Ficoll-Paque Plus (GE
Healthcare; Little Chalfont, Buckinghamshire, UK) are recovered and
washed by centrifugation with PBS containing 0.5% bovine serum
albumin (BSA) and 2 mM EDTA. MACS CD4.sup.+ and CD8.sup.+
MicroBeads (Miltenyi Biotec; San Diego, Calif.) kits are used to
isolate these human T-cell subsets using magnetically activated LS
columns to positive select for CD4.sup.+ and CD8.sup.+ T-cells.
Magnetically-bound T-cells are then removed from the magnetic MACS
separator, flushed from the LS column, and washed in fresh complete
medium. The purity of CD4.sup.+ and CD8.sup.+ T-cell populations
are assessed by flow cytometry using Life Technologies Acoustic
Attune.RTM. Cytometer, and are enriched by Fluorescence-Activated
Cell Sorting performed at USC's flow cytometry core facilities if
needed. CD4.sup.+ and CD8.sup.+ T-cells are maintained at a density
of 1.0.times.10.sup.6 cells/mL in complete medium supplemented with
100 IU/mL IL-2 in a suitable cell culture vessel, to which
.alpha.-CD3/.alpha.-CD28 Human T-cell Dynabeads (Life Technologies;
Carslbad, Calif.) are added to activate cultured T cells. T-cells
are incubated at 37.degree. C. in a 5% CO.sub.2 incubator for 2
days prior to transduction with CAR-lentiviral particles.
Lentiviral Transduction of CD4.sup.+ CD8.sup.+ T-Cells
[0258] Activated T-cells are collected and dead cells are removed
by Ficoll-Hypaque density gradient centrifugation or the use of
MACS Dead Cell Removal Kit (Miltenyi Biotec; San Diego, Calif.). In
a 6-well plate, activated T-cells are plated at a concentration of
1.0.times.10.sup.6 cells/mL complete medium. To various wells,
HLA-DR CAR-containing lentiviral particles are added to cell
suspensions at varying multiplicity of infections (MOIs), such as
1, 5, 10, and 50. Polybrene, a cationic polymer that aids
transduction by facilitating interaction between lentiviral
particles and the target cell surface, are added at a final
concentration of 4 .mu.g/mL. Plates are centrifuged at 800.times.g
for 1 hr at 32.degree. C. Following centrifugation,
lentivirus-containing medium are aspirated and cell pellets are
resuspended in fresh complete medium with 100 IU/mL IL-2. Cells are
placed in a 5% CO.sub.2 humidified incubator at 37.degree. C.
overnight. Three days post-transduction, cells are pelleted and
resuspended in fresh complete medium with IL-2 and 400 .mu.g/mL
Geneticin (G418 sulfate) (Life Technologies; Carlsbad, Calif.).
HLA-DR CAR modified T-cells are assessed by flow cytometry and
southern blot analysis to demonstrate successful transduction
procedures. Prior to in vitro and in vivo assays, HLA-DR CAR
T-cells are enriched by FACS and mixed 1:1 for the in vivo
studies.
In Vitro Assessment of CAR Efficacy by Calcein-Release Cytotoxicity
Assays
[0259] HLA-DR antigen positive and negative human cell lines are
collected, washed, and resuspended in complete medium at a
concentration of 1.0.times.10.sup.6 cells/mL. Calcein-acetoxymethyl
(AM) are added to target cell samples at 15 which are then
incubated at 37.degree. C. in a 5% CO.sub.2 humidified incubator
for 30 minutes. Dyed positive and negative target cells are washed
twice and resuspended in complete medium by centrifugation and
added to a 96-well plate at 1.0.times.10.sup.4 cells/well. HLA-DR
CAR T-cells are added to the plate in complete medium at
effector-to-target cell ratios of 50:1, 5:1, and 1:1. Dyed-target
cells suspended in complete medium and complete medium with 2%
triton X-100 serve as spontaneous and maximal release controls,
respectively. The plates are centrifuged at 365.times.g and
20.degree. C. for 2 minutes before being placed back in the
incubator 3 hours. The plates are then centrifuged 10 minutes and
cell supernatants are aliquoted to respective wells on a black
polystyrene 96-well plate and assessed for fluorescence on a
Bio-Tek.RTM. Synergy.TM. HT microplate reader at excitation and
emissions of 485/20 nm and 528/20 nm, respectively.
Quantification of Human Cytokines by Luminex Bioassay
[0260] Supernatants of HLA-DR CAR modified T-cells and HLA-DR
positive and negative tumor cell lines are measured for cytokine
secretion as a measure of CAR T-cell activation using standard
procedures performed routinely in the laboratory. Data are compared
to medium alone and to cultures using non-activated human T-cells
to identify background activity. The concentration of IL-2, IFN-g,
IL-12, and other pertinent cytokines are measured over time during
the incubation process.
In Vivo Assessment of CAR T-Cell Efficacy in Two Xenograft HLA-DR
Positive Cancer Models
[0261] HLA-DR CAR T-cells are further evaluated in vivo using two
different human tumor cell line xenograft tumor models. For both,
solid tumors are established subcutaneously in 6-8 week old female
nude mice by injection of 5.times.10.sup.6 HLA-DR positive or
HLA-DR negative solid tumor cell lines. When the tumors reach 0.5
cm in diameter, groups of mice (n=5) are treated intravenously with
1 or 3.times.10.sup.7 human T-cells as negative controls or HLA-DR
CAR T-cells constructed from the most active HLA-DR antibodies
based upon the in vitro study results. Tumor volumes are then
measured by caliper 3.times./week and volume growth curves are
generated to demonstrate the effectiveness of experimental
treatments over controls.
[0262] HLA-DR is found to be an outstanding target for CAR T-cell
development.
Example 3--Lym-1 CAR Cells
Construction of the CAR Lentiviral Constructs
[0263] The Lym-1 CAR vector contains a CD8 leader sequence followed
by the extracellular antigen binding moiety or scFV, which binds
specifically to Lym-1 antigen. The scFV is connected via a CD8
hinge region to the cytoplasmic signaling domain, comprised of the
CD8 transmembrane region, and the signaling domains from 4-1BB and
CD3 (FIG. 7). The CAR sequence including the signaling domains,
were synthetically synthesized by Genewiz Gene Synthesis services
(Piscataway, N.J.). The plasmids are purified and digested with the
appropriate restriction enzymes to be inserted into an HIV-1-based
lentiviral vector (pLVX-IRES-ZsGreen, Clontech, Signal Hill,
Calif.) containing HIV-1 5' and 3' long terminal repeats (LTRs),
packaging signal (.PSI.), EF1.alpha. promoter, internal ribosome
entry site (IRES), woodchuck hepatitis virus post-transcriptional
regulatory element (WPRE) and simian virus 40 origin (SV40) via
overnight T4 DNA ligase reaction (New England Biosciences; Ipswich,
Mass.), followed by deletion of the IRES-ZsGreen using restriction
enzyme digestion and ligation with T4 DNA ligase. NovaBlue
Singles.TM. chemically-competent E. coli cells are then transformed
with the resulting CAR-containing lentiviral plasmid.
Production of Lentiviral Particles
[0264] Prior to transfection, HEK 293T cells are seeded at
4.0.times.106 cells in a 150 cm2 tissue-culture-treated flask in 20
mL DMEM supplemented with 10% dialyzed FCS and incubated overnight
at 37.degree. C. in a humidified 5% CO2 incubator. Once 80-90%
confluent, HEK 293T cells are incubated in 20 ml DMEM supplemented
with 1-% dialyzed FCS without penicillin/streptamycin for two hours
in at 37.degree. C. in a humidified 5% CO2 incubator. HEK293T cells
are co-transfected with the CAR plasmid and lentiviral packaging
plasmids containing genes necessary to form the lentiviral envelope
& capsid components. A proprietary reaction buffer and polymer
to facilitate the formation of plasmid-containing nanoparticles
that bind HEK 293T cells are also added. After incubating the
transfected-HEK 293T cell cultures for 24 hours at 37.degree. C.,
the transfection medium is replaced with 20 mL fresh complete DMEM.
Lentivirus supernatants are collected every 24 hours for three days
and the supernatants are centrifuged at 1,250 rpm for 5 mins at
4.degree. C., followed by filter sterilization and centrifugation
in an ultracentrifuge at 20,000 g for 2 hrs at 4.degree. C. The
concentrated lentivirus is re-suspended in PBA containing 7%
trehalose and 1% BSA. The lentivirus is then aliquoted and stored
at -80.degree. C. until use for transduction of target CD4+ and
CD8+ T cells. The cell supernatants harvested after 24 hours are
tested for lentiviral particles via sandwich ELISA against p24, the
main lentiviral capsid protein. Transfection efficiency was
estimated between 20%-50%, by staining with a biotin-labeled
Protein L antibody (Genscript, Piscataway, N.J.), followed by
incubation with a streptavidin conjugated to PE, and detection by
FACS analysis.
Purification, Activation, and Enrichment of Human CD4+ and CD8+
Peripheral Blood T-Cells
[0265] Peripheral blood mononuclear cells (PBMCs) enriched by
density gradient centrifugation with Ficoll-Paque Plus (GE
Healthcare; Little Chalfont, Buckinghamshire, UK) are recovered and
washed by centrifugation with PBS containing 0.5% bovine serum
albumin (BSA) and 2 mM EDTA. T-cell enrichment kits (Stem Cell
Technologies) are used to isolate these human T-cell subsets
magnetically using negative selection for CD4+ and CD8+ T-cells.
The purity of CD4+ and CD8+ T-cell populations is assessed by flow
cytometry using Life Technologies Acoustic Attune.RTM. Cytometer,
and are enriched by Fluorescence-Activated Cell Sorting. CD4+ and
CD8+ T-cells mixed 1:1 are maintained at a density of 1.0.times.106
cells/mL in complete 50% Click's medium/50% RPMI-1640 medium
supplemented with 100 IU/mL IL-2 in a suitable cell culture vessel,
to which .alpha.-CD3/.alpha.-CD28 Human T-cell activator beads
(Stem Cell Technologies) are added to activate cultured T cells.
T-cells are then incubated at 37.degree. C. in a 5% CO2 incubator
for 2 days prior to transduction with CAR lentiviral particles.
Lentiviral Transduction of CD4+ CD8+ T-Cells
[0266] Activated T-cells are collected and dead cells are removed
by Ficoll-Hypaque density gradient centrifugation or the use of
MACS Dead Cell Removal Kit (Miltenyi Biotec; San Diego, Calif.). In
a 6-well plate, activated T-cells will be plated at a concentration
of 1.0.times.106 cells/mL in complete medium. Cells will be
transduced with the lentiviral particles supplemented with
Lentiblast, a transfection aid (Oz Biosciences, San Diego, Calif.)
to the cells. Transduced cells were incubated for 24 hours at
37.degree. C. in a humidified 5% CO2 incubator. The cells are spun
down and the media changed, followed by addition of the T-cell
activator beads (Stem Cell Technologies, San Diego, Calif.).
Detection of Lym-1 CAR Expression by Flow Cytometry
[0267] Seven days after Lentivirus transduction, primary T-cells
are washed 3.times. using wash buffer (4% BSA in PBS). Cells are
incubated with Biotein-Protein L (2 ug, Genscript, Piscataway,
N.J.) at 4.degree. C. for 45 min. Cells are again washed 3.times.
with wash buffer, followed by incubation with 2 ul of
Streptavidin-PE (BD Sciences, La Jolla, Calif.) at 4.degree. C. for
45 min. Cells are washed 3.times. and analyzed using flow cytometry
(Attune Cytometer, Applied Biosciences, Carlsbad, Calif.).
Cell Cytotoxicity Assays
[0268] Cytotoxicity of the Lym-1 CAR T-cells are determined using
the lactate dehydrogenase (LDH) cytotoxicity kit (Thermo
Scientific, Carlsbad, Calif.). Activated T-cells are collected and
1.times.106 cells are transduced with the Lym-1 CAR lentiviral
construct as described above. Cells are activated used the T-cell
activator beads (Stem Cell Technologies, San Diego, Calif.) for two
days prior to cytotoxicity assays. The optimal number of target
cells is determined as per the manufacturer's protocol. For the
assays, the appropriate target cells are plated in triplicate in a
96 well plate for 24 hours at 37.degree. C. in a 5% CO2 incubator,
followed by addition of activated CAR T-cells in ratios of 20:1,
10:1, 5:1 and 1:1, and incubated for 24 hours at 37.degree. C. in a
5% CO2 incubator. Cells are lysed at 37.degree. C. for 45 mins and
spun down at 1,250 rpm for 5 minutes. The supernatants are
transferred to a fresh 96 well plate, followed by the addition of
the reaction mixture for 30 minutes. The reaction is stopped using
the stop solution and the plate read at 450 nm with an absorbance
correction at 650 nm.
In Vivo Tumor Regression Assay
[0269] Foxn1 null mice are injected with immortalized B lymphoma
cell line, Raji, which expresses the Lym-1 antigen. Two.times.106
Raji cells with 1.times.106 human fibroblasts in 200 ul of
phosphate buffered saline (PBS) are injected into the left flank of
pre-irradiated mice (400 rads) to reduce the number of circulating
NK cells enabling the heterotransplants to implant at a high
frequency. T-cells are activated for 2 days with the
.alpha.CD3/CD28 activator complex (Stem Cell Technologies, San
Diego, Calif.). The activated T-cells are then transduced with
Lym-1 CAR lentiviral particles, followed by activation with the
.alpha.CD3/CD28 activator complex for an additional 2 days. The
activated T-cells expressing the Lym-1 CAR (2.5.times.106) are
injected intravenously via the lateral tail vein into the mice on
day 7 after tumor inoculation. Tumor sizes are assessed
3.times./week using Vernier calipers and the tumor volumes
calculated.
Detection of Lym-1 CAR Expression
[0270] Analysis of the Lym-1 CAR T-cells for expression of the
Lym-1 CAR, showed 62.5% of the transduced T-cells positive for
Lym-1 (FIG. 8 middle panel). In contrast, only 1% of the
un-transduced T-cells used as a control were positive for CAR
expression (FIG. 8 left panel). CD19 transduced T-cells were used
as a positive control and showed 52% expression of the CD19 CAR
(FIG. 8 right panel).
Cytotoxicity for Lym-1 CAR T-Cells
[0271] The cytolytic activity of the Lym-1 CAR T-cells was examined
using Raji, a B-cell lymphoma cell line. Raji expresses the Lym-1
antigen (HLA-Dr10), as determined by FACS analysis. Lym-1 CAR
T-cells were added to the Raji cells in ratios of 20:1, 10:1, 5:1
and 1:1 of effector to target cells. Lym-1 CAR T-cells showed
increased lysis of the target Raji cells at ratios of 5:1, 10:1 and
20:1 with a lysis rate of 22%. In comparison, untransduced T-cells
did not lyse Raji cells at any of the ratios tested.
Example 4--Lym-2 CAR Cells
Construction of the CAR Lentiviral Constructs
[0272] The Lym-2 CAR vector contains a CD8 leader sequence followed
by the extracellular antigen binding moiety or scFV, which binds
specifically to the Lym-2 antigen (HLA-Dr). The scFV is connected
via a CD8 hinge region to the cytoplasmic signaling domain,
comprised of the CD8 transmembrane region, and the signaling
domains from 4-1BB and CD3. The CAR sequence including the
signaling domains, were synthetically synthesized by Genewiz Gene
Synthesis services (Piscataway, N.J.). The plasmids are purified
and digested with the appropriate restriction enzymes to be
inserted into an HIV-1-based lentiviral vector (pLVX-IRES-ZsGreen,
Clontech, Signal Hill, Calif.) containing HIV-1 5' and 3' long
terminal repeats (LTRs), packaging signal (.PSI.), EF1.alpha.
promoter, internal ribosome entry site (IRES), woodchuck hepatitis
virus post-transcriptional regulatory element (WPRE) and simian
virus 40 origin (SV40) via overnight T.sub.4 DNA ligase reaction
(New England Biosciences; Ipswich, Mass.), followed by deletion of
the IRES-ZsGreen using restriction enzyme digestion and ligation
with T.sub.4 DNA ligase. NovaBlue Singles.TM. chemically-competent
E. coli cells are then transformed with the resulting
CAR-containing lentiviral plasmid.
Production of Lentiviral Particles
[0273] Prior to transfection, HEK 293T cells are seeded at
4.0.times.10.sup.6 cells in a 150 cm.sup.2 tissue-culture-treated
flask in 20 mL DMEM supplemented with 10% dialyzed FCS and
incubated overnight at 37.degree. C. in a humidified 5% CO.sub.2
incubator. Once 80-90% confluent, HEK 293T cells are incubated in
20 ml DMEM supplemented with 1-% dialyzed FCS without
penicillin/streptamycin for two hours in a 37.degree. C. humidified
5% CO.sub.2 incubator. HEK293T cells are co-transfected with the
CAR plasmid and lentiviral packaging plasmids containing genes
necessary to form the lentiviral envelope & capsid components.
A proprietary reaction buffer and polymer to facilitate the
formation of plasmid-containing nanoparticles that bind HEK 293T
cells are also added. After incubating the transfected-HEK 293T
cell cultures for 24 hours at 37.degree. C., the transfection
medium is replaced with 20 mL fresh complete DMEM. Lentivirus
supernatants are collected every 24 hours for 3 days and the
supernatants are centrifuged at 1,250 rpm for 5 mins at 4.degree.
C., followed by filter sterilization and centrifugation in an
ultracentrifuge at 20,000 g for 2 hrs at 4.degree. C. The
concentrated lentivirus is re-suspended in PBA containing 7%
trehalose and 1% BSA. The lentivirus is aliquoted and stored at
-80.degree. C. until use for transduction of target CD4.sup.+ and
CD8.sup.+ T cells. The cell supernatants harvested after 24 hours
are tested for lentiviral particles via a sandwich ELISA against
p24, the main lentiviral capsid protein. Transfection efficiency
was estimated between 20%-50%, by staining with a biotin-labeled
Protein L antibody (Genscript, Piscataway, N.J.), followed by
incubation with a streptavidin conjugated to PE, and detection by
FACS analysis.
Purification, Activation, and Enrichment of Human CD4.sup.+ and
CD8.sup.+ Peripheral Blood T-Cells
[0274] Peripheral blood mononuclear cells (PBMCs) enriched by
density gradient centrifugation with Ficoll-Paque Plus (GE
Healthcare; Little Chalfont, Buckinghamshire, UK) are recovered and
washed by centrifugation with PBS containing 0.5% bovine serum
albumin (BSA) and 2 mM EDTA. T-cell enrichment kits (Stem Cell
Technologies) are used to isolate these human T-cell subsets
magnetically using negative selection for CD4.sup.+ and CD8.sup.+
T-cells. The purity of CD4.sup.+ and CD8.sup.+ T-cell populations
is assessed by flow cytometry using Life Technologies Acoustic
Attune.RTM. Cytometer, and will be enriched by
Fluorescence-Activated Cell Sorting. CD4.sup.+ and CD8.sup.+
T-cells mixed 1:1 are maintained at a density of 1.0.times.10.sup.6
cells/mL in complete 50% Click's medium/50% RPMI-1640 medium
supplemented with 100 IU/mL IL-2 in a suitable cell culture vessel,
to which .alpha.-CD3/.alpha.-CD28 Human T-cell activator beads
(Stem Cell Technologies) are added to activate cultured T cells.
T-cells are then incubated at 37.degree. C. in a 5% CO.sub.2
humidified incubator for 2 days prior to transduction with CAR
lentiviral particles.
Lentiviral Transduction of CD4.sup.+ CD8.sup.+ T-Cells
[0275] Activated T-cells are collected and dead cells removed by
Ficoll-Hypaque density gradient centrifugation or the use of MACS
Dead Cell Removal Kit (Miltenyi Biotec; San Diego, Calif.). In a
6-well plate, activated T-cells are plated at a concentration of
1.0.times.10.sup.6 cells/mL in complete medium. Cells are
transduced with the lentiviral particles supplemented with
Lentiblast, a transfection aid (Oz Biosciences, San Diego, Calif.)
to the cells. Transduced cells are incubated for 24 hours at
37.degree. C. in a 37.degree. C. humidified 5% CO.sub.2 incubator.
The cells are spun down and the media changed, followed by addition
of the T-cell activator beads (Stem Cell Technologies, San Diego,
Calif.).
Cell Cytotoxicity Assays
[0276] Cytotoxicity of the Lym-2 CAR T-cells are determined using
the lactate dehydrogenase (LDH) cytotoxicity kit (Thermo
Scientific, Carlsbad, Calif.). Activated T-cells are collected and
1.times.10.sup.6 cells are transduced with the Lym-2 CAR lentiviral
construct as described above. Cells are activated used the T-cell
activator beads (Stem Cell Technologies, San Diego, Calif.) for two
days prior to cytotoxicity assays. The optimal number of target
cells will be determined as per the manufacturer's protocol. For
the assays, the appropriate target cells will be plated in
triplicate in a 96 well plate for 24 hours at 37.degree. C. in a
37.degree. C. humidified 5% CO.sub.2 incubator, followed by
addition of activated CAR T-cells in ratios of 20:1, 10:1, 5:1 and
1:1, and incubated for 24 as above. Cells will be lysed at
37.degree. C. for 45 mins and centrifuged at 1,250 rpm for 5
minutes. The supernatants are transferred to a fresh 96 well plate,
followed by the addition of the reaction mixture for 30 minutes.
The reaction is stopped using the stop solution and the plate read
at 450 nm with an absorbance correction at 650 nm.
In Vivo Tumor Regression Assay
[0277] Foxn1 null mice are injected with immortalized B lymphoma
cell line, Raji, which expresses the Lym-2 antigen.
Two.times.10.sup.6 Raji cells with 1.times.10.sup.6 human
fibroblasts in 200 ul of phosphate buffered saline (PBS) are
injected into the left flank of the pre-irradiated (400 rads)
BALB/c mice in insure a high take rate of tumor. T-cells are
activated for 2 days with the .alpha.CD3/CD28 activator complex
(Stem Cell Technologies, San Diego, Calif.). The activated T-cells
are then transduced with Lym-2 CAR lentiviral particles, followed
by activation with the .alpha.CD3/CD28 activator complex for an
additional 2 days. The activated T-cells expressing the Lym-2 CAR
(2.5.times.10.sup.6) are injected intravenously into the mice on
day 7 after tumor inoculation. Tumor sizes are assessed
3.times./week using Vernier calipers and the tumor volumes
calculated.
Detection of Lym-2 CAR Expression
[0278] Analysis of the Lym-2 CAR T-cells for expression of the
Lym-1 CAR, showed 28% of the transduced T-cells positive for Lym-2
(FIG. 11 middle panel). In contrast, only 1% of the un-transduced
T-cells used as a control were positive for CAR expression (FIG. 11
left panel). CD19 transduced T-cells were used as a positive
control and showed 52% expression of the CD19 CAR (FIG. 11 right
panel).
Cytotoxicity for Lym-2 CAR T-Cells
[0279] The cytolytic activity of the Lym-2 CAR T-cells was
determined using Raji, a B-cell lymphoma cell line. Raji expresses
the Lym-2 antigen, as determined by FACS analysis. Lym-2 CAR
T-cells were added to the Raji cells in ratios of 20:1, 10:1, 5:1
and 1:1 of effector to target cells. Lym-2 CAR T-cells show
increased lysis of the target Raji cells at ratios of 5:1 and 10:1
with a lysis rate of 22%. In comparison, untransduced T-cells did
not lyse Raji cells at any of the ratios tested.
Example 5--NK Cell Transduction
NK-92MI Transduction
[0280] NK-92Mi cell line was purchased from ATCC (CRL-2408) and
maintained in RPMI-1640 with 10% FBS. Before transduction,
non-tissue treated 24-wells were incubated with 10 .mu.g
RetroNectin (Clontech T100A) in 300 .mu.L Phosphate Buffered Saline
(PBS) at room temperature for 2 hours. One million NK-92Mi cells
and lentivirus (MOI=5) were mixed and added to the RetroNectin
coated plates. The plates were then centrifuged at 28.degree. C.
800 g for 90 min. After centrifugation, the cells were maintained
in a cell culture incubator overnight. After incubation, the cells
were washed with PBS three times the following morning and the
transduced NK-92Mi cells were then transferred to 24 well G-Rex
(Wilson Wolf) plates for expansion. Seven days after Lentivirus
transduction, the cells were washed 3.times. in wash buffer (4% BSA
in PBS), stained with Biotein-Protein L (1 ug/1 million cells.
Genscript) at 4.degree. C. for 45 min, and washed 3.times. with
wash buffer before adding 2 ul Streptavidin-APC (BD science) at
4.degree. C. for 45 min. After a final 3 washes in wash buffer, the
cells were analyzed by FACs (Attune) (FIG. 15).
EQUIVALENTS
[0281] 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 technology belongs.
[0282] The present technology illustratively described herein may
suitably be practiced in the absence of any element or elements,
limitation or limitations, not specifically disclosed herein. Thus,
for example, the terms "comprising," "including," "containing,"
etc. shall be read expansively and without limitation.
Additionally, the terms and expressions employed herein have been
used as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the present technology claimed.
[0283] Thus, it should be understood that the materials, methods,
and examples provided here are representative of preferred aspects,
are exemplary, and are not intended as limitations on the scope of
the present technology.
[0284] The present technology has been described broadly and
generically herein. Each of the narrower species and sub-generic
groupings falling within the generic disclosure also form part of
the present technology. This includes the generic description of
the present technology with a proviso or negative limitation
removing any subject matter from the genus, regardless of whether
or not the excised material is specifically recited herein.
[0285] In addition, where features or aspects of the present
technology are described in terms of Markush groups, those skilled
in the art will recognize that the present technology is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
[0286] All publications, patent applications, patents, and other
references mentioned herein are expressly incorporated by reference
in their entirety, to the same extent as if each were incorporated
by reference individually. In case of conflict, the present
specification, including definitions, will control.
[0287] Other aspects are set forth within the following claims.
Sequence CWU 1
1
5218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Phe Ser Leu Thr Ser Tyr Gly1
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Gly Phe Thr Phe Ser Asn Tyr Trp1
537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ile Trp Ser Asp Gly Ser Thr1 5410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Ile
Arg Phe Lys Ser His Asn Tyr Ala Thr1 5 10512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Ala
Ser His Tyr Gly Ser Thr Leu Ala Phe Ala Ser1 5 10616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Thr
Arg Arg Ile Gly Asn Ser Asp Tyr Asp Trp Trp Tyr Phe Asp Val1 5 10
157354DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7caggtgcagc tgaaggagtc aggacctggc
ctggtggcgc cctcacagag cctgtccatc 60acatgcacca tctcagggtt ctcattaacc
agctatggtg tacactgggt tcgccagcct 120ccaggaaagg gtctggagtg
gctggtagtg atatggagtg atggaagcac aacctataat 180tcagctctca
aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta
240aaaatgaaca gtctccaaac tgatgacaca gccatatact actgtgccag
tcactacggt 300agtacccttg cctttgcttc ctggggccac gggactctgg
tcactgtctc tgca 3548116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Gln Leu Lys Glu Ser Gly
Pro Gly Leu Val Ala Pro Ser Gln Ser Leu1 5 10 15Ser Ile Thr Cys Thr
Ile Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val 20 25 30His Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Val Val 35 40 45Ile Trp Ser
Asp Gly Ser Thr Thr Tyr Asn Ser Ala Leu Lys Ser Arg 50 55 60Leu Ser
Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met65 70 75
80Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Ser His
85 90 95Tyr Gly Ser Thr Leu Ala Phe Ala Ser Trp Gly His Gly Thr Leu
Val 100 105 110Thr Val Ser Ala 1159381DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
9gaagtgcagc ttgaggagtc tggaggaggc ttggtgcaac ctggaggctc catgaaactc
60tcctgtgttg cctctggatt cactttcagt aactattgga tgaactgggt ccgccagtct
120ccagagaagg ggcttgagtg ggttgctgaa attagattta aatctcataa
ttatgcaaca 180cattttgcgg agtctgtgaa agggaggttc accatctcaa
gagatgattc caaaagtagt 240gtctacctgc aaatgaacaa cttaagagct
gaagacactg gcatttatta ctgtaccagg 300aggataggaa actctgatta
cgactggtgg tacttcgatg tctggggcgc agggacctca 360gtcaccgtct
cctcagctag c 38110127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 10Glu Val Gln Leu Glu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser
Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile
Arg Phe Lys Ser His Asn Tyr Ala Thr His Phe Ala Glu 50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr
85 90 95Tyr Cys Thr Arg Arg Ile Gly Asn Ser Asp Tyr Asp Trp Trp Tyr
Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Ser Val Thr Val Ser Ser
Ala Ser 115 120 125116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Val Asn Ile Tyr Ser Tyr1
5126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Gln Asn Val Gly Asn Asn1 5133PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Asn
Ala Lys1143PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Ser Ala Ser1158PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Gln
His His Tyr Gly Thr Phe Thr1 5169PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 16Gln Gln Tyr Asn Thr Tyr
Pro Phe Thr1 517318DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 17gacatccaga tgactcagtc
tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60atcatatgtc gagcaagtgt
gaatatttac agttatttag catggtatca gcagaaacag 120ggaaaatctc
ctcagctcct ggtctataat gccaaaatct tagcagaagg tgtgccatca
180aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag
cctgcagcct 240gaagattttg ggagttatta ctgtcaacat cattatggta
cattcacgtt cggctcgggg 300acaaagttgg aaataaaa 31818106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1
5 10 15Glu Thr Val Thr Ile Ile Cys Arg Ala Ser Val Asn Ile Tyr Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu
Leu Val 35 40 45Tyr Asn Ala Lys Ile Leu Ala Glu Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His
His Tyr Gly Thr Phe Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile
Lys 100 10519321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 19gacattgtga tgacccagtc
tcacaaattc atgtccacat cagtaggaga cagggtcagc 60gtcacctgca aggccagtca
gaatgtgggt aataatgtag cctggtatca acagaaacca 120gggcaatctc
ctaaagtact gatttactcg gcatcctacc ggtacagtgg agtccctgat
180cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagtaa
tgtgcagtct 240gaagacttgg cagagtattt ctgtcagcaa tataacacct
atccattcac gttcggctcg 300gggacaaagt tggaaataaa a
32120107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Val Thr Cys Lys Ala Ser
Gln Asn Val Gly Asn Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Val Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Glu
Tyr Phe Cys Gln Gln Tyr Asn Thr Tyr Pro Phe 85 90 95Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 100 10521384PRTHomo sapiens 21Ala Pro
Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg1 5 10 15His
Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25
30Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser
35 40 45Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr
Tyr 50 55 60Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg
Gln Gly65 70 75 80Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys
Ser Lys Lys Glu 85 90 95Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln
Ala Ser Ser Val Pro 100 105 110Thr Ala Gln Pro Gln Ala Glu Gly Ser
Leu Ala Lys Ala Thr Thr Ala 115 120 125Pro Ala Thr Thr Arg Asn Thr
Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140Glu Lys Glu Lys Glu
Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu145 150 155 160Cys Pro
Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170
175Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val
180 185 190Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val
Ala Gly 195 200 205Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu
Glu Arg His Ser 210 215 220Asn Gly Ser Gln Ser Gln His Ser Arg Leu
Thr Leu Pro Arg Ser Leu225 230 235 240Trp Asn Ala Gly Thr Ser Val
Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255Pro Pro Gln Arg Leu
Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270Val Lys Leu
Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285Ala
Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295
300Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly
Phe305 310 315 320Ala Pro Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr
Thr Phe Trp Ala 325 330 335Trp Ser Val Leu Arg Val Pro Ala Pro Pro
Ser Pro Gln Pro Ala Thr 340 345 350Tyr Thr Cys Val Val Ser His Glu
Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365Ser Arg Ser Leu Glu Val
Ser Tyr Val Thr Asp His Gly Pro Met Lys 370 375 38022330PRTHomo
sapiens 22Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 33023326PRTHomo sapiens 23Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr
Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105
110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser
Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230
235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 245 250 255Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro
Gly Lys 32524377PRTHomo sapiens 24Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105
110Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
115 120 125Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys 130 135 140Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln
Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His225 230
235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 275 280 285Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn305 310 315 320Tyr Asn Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345
350Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln
355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 37525452PRTHomo
sapiens 25Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys
Glu
Asn1 5 10 15Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala
Gln Asp 20 25 30Phe Leu Pro Asp Ser Ile Thr Leu Ser Trp Lys Tyr Lys
Asn Asn Ser 35 40 45Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu
Arg Gly Gly Lys 50 55 60Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser
Lys Asp Val Met Gln65 70 75 80Gly Thr Asp Glu His Val Val Cys Lys
Val Gln His Pro Asn Gly Asn 85 90 95Lys Glu Lys Asn Val Pro Leu Pro
Val Ile Ala Glu Leu Pro Pro Lys 100 105 110Val Ser Val Phe Val Pro
Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125Lys Ser Lys Leu
Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140Gln Val
Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr145 150 155
160Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr
165 170 175Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu
Gly Gln 180 185 190Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu
Thr Phe Gln Gln 195 200 205Asn Ala Ser Ser Met Cys Val Pro Asp Gln
Asp Thr Ala Ile Arg Val 210 215 220Phe Ala Ile Pro Pro Ser Phe Ala
Ser Ile Phe Leu Thr Lys Ser Thr225 230 235 240Lys Leu Thr Cys Leu
Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255Ile Ser Trp
Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270Ile
Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280
285Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr
290 295 300Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile
Ser Arg305 310 315 320Pro Lys Gly Val Ala Leu His Arg Pro Asp Val
Tyr Leu Leu Pro Pro 325 330 335Ala Arg Glu Gln Leu Asn Leu Arg Glu
Ser Ala Thr Ile Thr Cys Leu 340 345 350Val Thr Gly Phe Ser Pro Ala
Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365Gly Gln Pro Leu Ser
Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380Glu Pro Gln
Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val385 390 395
400Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Ala His
405 410 415Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys
Ser Thr 420 425 430Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met
Ser Asp Thr Ala 435 440 445Gly Thr Cys Tyr 45026327PRTHomo sapiens
26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1
5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155
160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys 32527353PRTHomo
sapiens 27Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys
Ser Thr1 5 10 15Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln
Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser
Gly Gln Gly Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala
Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala
Thr Gln Cys Leu Ala Gly65 70 75 80Lys Ser Val Thr Cys His Val Lys
His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys Pro Val
Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110Ser Thr Pro Pro Thr
Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125Leu His Arg
Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135 140Leu
Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe145 150
155 160Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro
Glu 165 170 175Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu
Pro Gly Cys 180 185 190Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr
Cys Thr Ala Ala Tyr 195 200 205Pro Glu Ser Lys Thr Pro Leu Thr Ala
Thr Leu Ser Lys Ser Gly Asn 210 215 220Thr Phe Arg Pro Glu Val His
Leu Leu Pro Pro Pro Ser Glu Glu Leu225 230 235 240Ala Leu Asn Glu
Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser 245 250 255Pro Lys
Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro 260 265
270Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly
275 280 285Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala
Glu Asp 290 295 300Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly
His Glu Ala Leu305 310 315 320Pro Leu Ala Phe Thr Gln Lys Thr Ile
Asp Arg Leu Ala Gly Lys Pro 325 330 335Thr His Val Asn Val Ser Val
Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350Tyr28340PRTHomo
sapiens 28Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp
Ser Thr1 5 10 15Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln
Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser
Gly Gln Asn Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala
Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala
Thr Gln Cys Pro Asp Gly65 70 75 80Lys Ser Val Thr Cys His Val Lys
His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys Pro Val
Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110Arg Leu Ser Leu His
Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125Glu Ala Asn
Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140Ala
Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly145 150
155 160Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val
Leu 165 170 175Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe
Thr Cys Thr 180 185 190Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr
Ala Asn Ile Thr Lys 195 200 205Ser Gly Asn Thr Phe Arg Pro Glu Val
His Leu Leu Pro Pro Pro Ser 210 215 220Glu Glu Leu Ala Leu Asn Glu
Leu Val Thr Leu Thr Cys Leu Ala Arg225 230 235 240Gly Phe Ser Pro
Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255Glu Leu
Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265
270Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala
275 280 285Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val
Gly His 290 295 300Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile
Asp Arg Met Ala305 310 315 320Gly Lys Pro Thr His Val Asn Val Ser
Val Val Met Ala Glu Val Asp 325 330 335Gly Thr Cys Tyr
34029106PRTHomo sapiens 29Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10530237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Gly Asp Thr Arg Pro Arg Phe Leu Glu Glu Val Lys Phe Glu Cys His1
5 10 15Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Arg Val
His 20 25 30Asn Gln Glu Glu Tyr Ala Arg Tyr Asp Ser Asp Val Gly Glu
Tyr Arg 35 40 45Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
Asn Ser Gln 50 55 60Lys Asp Leu Leu Glu Arg Arg Arg Ala Ala Val Asp
Thr Tyr Cys Arg65 70 75 80His Asn Tyr Gly Val Gly Glu Ser Phe Thr
Val Gln Arg Arg Val Gln 85 90 95Pro Lys Val Thr Val Tyr Pro Ser Lys
Thr Gln Pro Leu Gln His His 100 105 110Asn Leu Leu Val Cys Ser Val
Asn Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125Val Arg Trp Phe Arg
Asn Gly Gln Glu Glu Lys Thr Gly Val Val Ser 130 135 140Thr Gly Leu
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met145 150 155
160Leu Glu Thr Val Pro Gln Ser Gly Glu Val Tyr Thr Cys Gln Val Glu
165 170 175His Pro Ser Val Met Ser Pro Leu Thr Val Glu Trp Arg Ala
Arg Ser 180 185 190Glu Ser Ala Gln Ser Lys Met Leu Ser Gly Val Gly
Gly Phe Val Leu 195 200 205Gly Leu Leu Phe Leu Gly Ala Gly Leu Phe
Ile Tyr Phe Arg Asn Gln 210 215 220Lys Gly His Ser Gly Leu Pro Pro
Thr Gly Phe Leu Ser225 230 23531237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Gly Asp Thr Arg Pro Arg Phe Leu Glu Leu Leu Lys Ser Glu Cys His1
5 10 15Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg His Phe
His 20 25 30Asn Gln Glu Glu Tyr Ala Arg Phe Asp Ser Asp Val Gly Glu
Tyr Arg 35 40 45Ala Val Phe Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
Asn Ser Gln 50 55 60Lys Asp Leu Leu Glu Gln Lys Arg Gly Gln Val Asp
Asn Tyr Cys Arg65 70 75 80His Asn Tyr Gly Val Val Glu Ser Phe Thr
Val Gln Arg Arg Val His 85 90 95Pro Gln Val Thr Val Tyr Pro Ala Lys
Thr Gln Pro Leu Gln His His 100 105 110Asn Leu Leu Val Cys Ser Val
Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125Val Arg Trp Phe Arg
Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser 130 135 140Thr Gly Leu
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met145 150 155
160Leu Glu Thr Phe Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu
165 170 175His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Ser Ala
Arg Ser 180 185 190Glu Ser Ala Gln Ser Lys Met Leu Ser Gly Val Gly
Gly Phe Val Leu 195 200 205Gly Leu Leu Phe Leu Gly Ala Gly Leu Phe
Ile Tyr Phe Arg Asn Gln 210 215 220Lys Gly His Ser Gly Leu Gln Pro
Thr Gly Phe Leu Ser225 230 23532237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Gly Asp Thr Arg Pro Arg Phe Leu Glu Tyr Ser Thr Ser Glu Cys His1
5 10 15Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe
His 20 25 30Asn Gln Glu Glu Asn Val Arg Phe Asp Ser Asp Val Gly Glu
Phe Arg 35 40 45Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
Asn Ser Gln 50 55 60Lys Asp Leu Leu Glu Gln Lys Arg Gly Arg Val Asp
Asn Tyr Cys Arg65 70 75 80His Asn Tyr Gly Val Val Glu Ser Phe Thr
Val Gln Arg Arg Val His 85 90 95Pro Lys Val Thr Val Tyr Pro Ser Lys
Thr Gln Pro Leu Gln His His 100 105 110Asn Leu Leu Val Cys Ser Val
Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125Val Arg Trp Phe Arg
Asn Gly Gln Glu Glu Lys Thr Gly Val Val Ser 130 135 140Thr Gly Leu
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met145 150 155
160Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu
165 170 175His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala
Arg Ser 180 185 190Glu Ser Ala Gln Ser Lys Met Leu Ser Gly Val Gly
Gly Phe Val Leu 195 200 205Gly Leu Leu Phe Leu Gly Ala Gly Leu Phe
Ile Tyr Phe Arg Asn Gln 210 215 220Lys Gly His Ser Gly Leu Gln Pro
Arg Gly Phe Leu Ser225 230 2353351PRTHomo sapiens 33Pro Ala Lys Pro
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala1 5 10 15Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 20 25 30Pro Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 35 40 45Asp
Ile Tyr 503449PRTMus musculus 34Lys Val Asn Ser Thr Thr Thr Lys Pro
Val Leu Arg Thr Pro Ser Pro1 5 10 15Val His Pro Thr Gly Thr Ser Gln
Pro Gln Arg Pro Glu Asp Cys Arg 20 25 30Pro Arg Gly Ser Val Lys Gly
Thr Gly Leu Asp Phe Ala Cys Asp Ile 35 40 45Tyr3551PRTFelis catus
35Pro Val Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Gln Ala1
5 10 15Pro Ile Thr Thr Ser Gln Arg Val Ser Leu Arg Pro Gly Thr Cys
Gln 20 25 30Pro Ser Ala Gly Ser Thr Val Glu Ala Ser Gly Leu Asp Leu
Ser Cys 35
40 45Asp Ile Tyr 503621PRTHomo sapiens 36Ile Tyr Ile Trp Ala Pro
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr
203721PRTMus musculus 37Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala
Leu Leu Leu Ser Leu1 5 10 15Ile Ile Thr Leu Ile 203821PRTRattus
norvegicus 38Ile Trp Ala Pro Leu Ala Gly Ile Cys Ala Val Leu Leu
Leu Ser Leu1 5 10 15Val Ile Thr Leu Ile 203942PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1
5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35
4040220PRTUnknownDescription of Unknown CD28 sequence 40Met Leu Arg
Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val1 5 10 15Thr Gly
Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30Asp
Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40
45Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu
50 55 60Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr
Ser65 70 75 80Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu
Ser Val Thr 85 90 95Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp
Ile Tyr Phe Cys 100 105 110Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr
Leu Asp Asn Glu Lys Ser 115 120 125Asn Gly Thr Ile Ile His Val Lys
Gly Lys His Leu Cys Pro Ser Pro 130 135 140Leu Phe Pro Gly Pro Ser
Lys Pro Phe Trp Val Leu Val Val Val Gly145 150 155 160Gly Val Leu
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175Phe
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185
190Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
195 200 205Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215
22041112PRTUnknownDescription of Unknown CD3 zeta signaling domain
41Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1
5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His
Met Gln Ala Leu Pro Pro Arg 100 105 1104248DNAUnknownDescription of
Unknown IgG1 heavy chain hinge sequence 42ctcgagccca aatcttgtga
caaaactcac acatgcccac cgtgcccg 484381DNAUnknownDescription of
Unknown CD28 transmembrane region 43ttttgggtgc tggtggtggt
tggtggagtc ctggcttgct atagcttgct agtaacagtg 60gcctttatta ttttctgggt
g 8144126DNAUnknownDescription of Unknown 4-1BB co-stimulatory
signaling region 44aaacggggca gaaagaaact cctgtatata ttcaaacaac
catttatgag accagtacaa 60actactcaag aggaagatgg ctgtagctgc cgatttccag
aagaagaaga aggaggatgt 120gaactg 12645123DNAUnknownDescription of
Unknown CD28 co-stimulatory signaling region 45aggagtaaga
ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60gggcccaccc
gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120tcc
12346339DNAUnknownDescription of Unknown CD3 zeta signaling region
46agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc
60tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc
120cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg
cctgtacaat 180gaactgcaga aagataagat ggcggaggcc tacagtgaga
ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga tggcctttac
cagggtctca gtacagccac caaggacacc 300tacgacgccc ttcacatgca
ggccctgccc cctcgctaa 33947105DNAUnknownDescription of Unknown ICOS
costimulatory signaling region 47acaaaaaaga agtattcatc cagtgtgcac
gaccctaacg gtgaatacat gttcatgaga 60gcagtgaaca cagccaaaaa atccagactc
acagatgtga cccta 10548108DNAUnknownDescription of Unknown OX40
costimulatory signaling region 48agggaccaga ggctgccccc cgatgcccac
aagccccctg ggggaggcag tttccggacc 60cccatccaag aggagcaggc cgacgcccac
tccaccctgg ccaagatc 1084914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 49Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly1 5 105013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Glu
Phe Gly Ala Gly Leu Val Leu Gly Gly Gln Phe Met1 5
105115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 15525PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 52Gly Gly Gly Gly Ser1 5
* * * * *
References