PD-L1 Antibodies
The present invention relates to the field of medicine. More particularly, the
present invention relates to antibodies that bind human programmed cell death 1 ligand 1
(PD-L1), and may be useful for treating solid and hematological tumors alone and in
combination with chemotherapy and other cancer therapeutics.
Tumor cells escape detection and elimination by the immune system through
multiple mechanisms. Immune checkpoint pathways are used in maintenance of selftolerance
and control of T cell activation, but cancer cells can use the pathways to
suppress the anti-tumor response and prevent their destruction.
The PD-L1 / human programmed cell death 1 (PD-1) pathway is one such
immune checkpoint. Human PD-1 is found on T cells, and the binding of PD-L1 to PD-1
inhibits T cell proliferation and cytokine production. The PD-1/PD-L1 inhibitory axis has
been subjugated by tumors as part of the natural selective process that shapes tumor
evolution in the context of an anti-tumor immune response. PD-L1 also binds B7-1
(CD80); B7-1 is another negative regulator of T cell activation. Accordingly, PD-L1 is
aberrantly expressed by a variety of tumor types, and increased expression of PD-L1 on
tumor cells has been found to be correlated with a worse prognosis in many cancers. PDL1
expression is also up-regulated in the tumor microenvironment in immune and other
cells as a result of immune activation and production of pro-inflammatory cytokines,
further contributing to the establishment of a T-cell immunosuppressive milieu. Blocking
PD-L1 may facilitate the re-activation of tumor-reacting T cells, restoring their ability to
effectively detect and kill tumor cells.
A human IgG1 antibody against human PD-L1, MPDL3280A, has been shown to
block binding to PD-1 and B7-1, and has been tested in human clinical trials (Herbst et
al., Nature (2014) 515:563; Cha et al., Seminars in Oncology (2015) 42(3):484; and U.S.
patent application 2010/0203056). A human IgG1 antibody against human PD-L1,
MEDI4736, has been shown to block binding to PD-1 and B7-1, and has been tested in
human clinical trials (Ibrahim et al., Seminars in Oncology (2015) 42(3):474; and U.S.
patent application 2013/0034559).
3
There remains a need to provide alternative antibodies that bind human PD-L1
and neutralize the PD-L1 interactions with PD-1 and B7-1. In particular, there remains a
need to provide PD-L1 antibodies that bind with more favorable attributes, such as better
association rates. Faster association to the target can translate into better in vivo activity
for a therapeutic antibody. Also, there remains a need to provide PD-L1 antibodies that
better enhance the T cell response to a tumor, as measured by influence on tumor size, by
the level of infiltration of CD3-positive T cells, and by the percentage of T cells that are
CD8-positive in in vivo models.
Certain antibodies of the present invention mediate enhanced T cell response to a
tumor as measured by tumor size compared to certain prior art antibodies in established
and non-established tumor models. Certain antibodies of the present invention mediate
enhanced T cell response to a tumor as measured by CD3-positive T cell infiltration
compared to certain prior art antibodies in a model.
Accordingly, in some embodiments the present invention provides an antibody
that binds human PD-L1 (SEQ ID NO: 1), comprising a light chain (LC) and a heavy
chain (HC), wherein the light chain comprises a light chain variable region (LCVR) and
the heavy chain comprises a heavy chain variable region (HCVR), and wherein the LCVR
comprises light chain complementarity determining regions LCDR1, LCDR2, and
LCDR3 consisting of the amino acid sequences SGSSSNIGSNTVN (SEQ ID NO: 5),
YGNSNRPS (SEQ ID NO: 6), and QSYDSSLSGSV (SEQ ID NO: 7), respectively, and
wherein the HCVR comprises heavy chain complementarity determining regions
HCDR1, HCDR2, and HCDR3 consisting of the amino acid sequences
KASGGTFSSYAIS (SEQ ID NO: 2), GIIPIFGTANYAQKFQG (SEQ ID NO: 3), and
ARSPDYSPYYYYGMDV (SEQ ID NO: 4), respectively.
In some embodiments, the present invention provides an antibody, comprising a
light chain (LC) and a heavy chain (HC), wherein the light chain comprises a light chain
variable region (LCVR) and the heavy chain comprises a heavy chain variable region
(HCVR), wherein the LCVR has the amino acid sequence given in SEQ ID NO: 9, and
the HCVR has the amino acid sequence given in SEQ ID NO: 8.
4
In some embodiments, the present invention provides an antibody, wherein the LC
has the amino acid sequence given in SEQ ID NO: 11, and the HC has the amino acid
sequence given in SEQ ID NO: 10.
In an embodiment, the present invention provides an antibody, comprising two
light chains and two heavy chains, wherein each light chain has the amino acid sequence
given in SEQ ID NO: 11, and each heavy chain has the amino acid sequence given in
SEQ ID NO: 10.
In a further embodiment, the present invention provides an antibody, wherein one
of the heavy chains forms an inter-chain disulfide bond with one of the light chains, and
the other heavy chain forms an inter-chain disulfide bond with the other light chain, and
one of the heavy chains forms two inter-chain disulfide bonds with the other heavy chain.
In a further embodiment, the present invention provides an antibody, wherein the
antibody is glycosylated.
In an embodiment, the present invention provides a mammalian cell, comprising a
DNA molecule comprising a polynucleotide sequence encoding a polypeptide having an
amino acid sequence of SEQ ID NO: 11 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 10, wherein the cell is
capable of expressing an antibody comprising a light chain having an amino acid
sequence of SEQ ID NO: 11 and a heavy chain having an amino acid sequence of SEQ
ID NO: 10.
In a further embodiment, the present invention provides a process for producing
an antibody, comprising a light chain having an amino acid sequence of SEQ ID NO: 11
and a heavy chain having an amino acid sequence of SEQ ID NO: 10, comprising
cultivating a mammalian cell of the present invention under conditions such that the
antibody is expressed, and recovering the expressed antibody.
In a further embodiment, the present invention provides an antibody produced by
a process of the present invention.
In an embodiment, the present invention provides a pharmaceutical composition,
comprising an antibody of the present invention, and an acceptable carrier, diluent, or
excipient.
5
In an embodiment, the present invention provides a method of treating cancer,
comprising administering to a patient in need thereof, an effective amount of an antibody
of the present invention. In a further embodiment, the present invention provides a
method of treating cancer, comprising administering to a patient in need thereof, an
effective amount of an antibody of the present invention, wherein the cancer is
melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer,
gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian
cancer, esophageal cancer, soft tissue sarcoma, or hepatocellular carcinoma.
In a further embodiment, the present invention provides a method of treating
cancer, wherein the cancer is melanoma. In a further embodiment, the present invention
provides a method of treating cancer, wherein the cancer is lung cancer. In a further
embodiment, the present invention provides a method of treating cancer, wherein the
cancer is head and neck cancer. In a further embodiment, the present invention provides
a method of treating cancer, wherein the cancer is colorectal cancer. In a further
embodiment, the present invention provides a method of treating cancer, wherein the
cancer is pancreatic cancer. In a further embodiment, the present invention provides a
method of treating cancer, wherein the cancer is gastric cancer. In a further embodiment,
the present invention provides a method of treating cancer, wherein the cancer is kidney
cancer. In a further embodiment, the present invention provides a method of treating
cancer, wherein the cancer is bladder cancer. In a further embodiment, the present
invention provides a method of treating cancer, wherein the cancer is prostate cancer. In
a further embodiment, the present invention provides a method of treating cancer,
wherein the cancer is breast cancer. In a further embodiment, the present invention
provides a method of treating cancer, wherein the cancer is ovarian cancer. In a further
embodiment, the present invention provides a method of treating cancer, wherein the
cancer is esophageal cancer. In a further embodiment, the present invention provides a
method of treating cancer, wherein the cancer is soft tissue sarcoma. In a further
embodiment, the present invention provides a method of treating cancer, wherein the
cancer is hepatocellular carcinoma.
6
In a further embodiment, these methods comprise the administration of an
effective amount of the antibody of the present invention in simultaneous, separate, or
sequential combination with one or more anti-tumor agents. Non-limiting examples of
anti-tumor agents include ramucirumab, necitumumab, olaratumab, galunisertib,
abemaciclib, cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel,
cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel proteinbound
particles for injectable suspension, ixabepilone, capecitabine, FOLFOX
(leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and
irinotecan), and cetuximab.
In a further embodiment, these methods comprise the administration of an
effective amount of the compound of the present invention in simultaneous, separate, or
sequential combination with one or more immuno-oncology agents. Non-limiting
examples of immuno-oncology agents include nivolumab, ipilimumab, pidilizumab,
pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, and durvalumab.
In an embodiment, the present invention provides an antibody of the present
invention, for use in therapy. In an embodiment, the present invention provides an
antibody of the present invention, for use in the treatment of cancer. In a further
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is melanoma, lung cancer, head and neck
cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer,
prostate cancer, breast cancer, ovarian cancer, esophageal cancer, soft tissue sarcoma, or
hepatocellular carcinoma.
In a further embodiment, the present invention provides an antibody of the present
invention, for use in the treatment of cancer, wherein the cancer is melanoma. In a
further embodiment, the present invention provides an antibody of the present invention,
for use in the treatment of cancer, wherein the cancer is lung cancer. In a further
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is head and neck cancer. In a further
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is colorectal cancer. In a further
7
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is pancreatic cancer. In a further
embodiment, the present invention provides an antibody of the present invention, for use
in the treatment of cancer, wherein the cancer is gastric cancer. In a further embodiment,
the present invention provides an antibody of the present invention, for use in the
treatment of cancer, wherein the cancer is kidney cancer. In a further embodiment, the
present invention provides an antibody of the present invention, for use in the treatment
of cancer, wherein the cancer is bladder cancer. In a further embodiment, the present
invention provides an antibody of the present invention, for use in the treatment of cancer,
wherein the cancer is prostate cancer. In a further embodiment, the present invention
provides an antibody of the present invention, for use in the treatment of cancer, wherein
the cancer is breast cancer. In a further embodiment, the present invention provides an
antibody of the present invention, for use in the treatment of cancer, wherein the cancer is
ovarian cancer. In a further embodiment, the present invention provides an antibody of
the present invention, for use in the treatment of cancer, wherein the cancer is esophageal
cancer. In a further embodiment, the present invention provides an antibody of the
present invention, for use in the treatment of cancer, wherein the cancer is soft tissue
sarcoma. In a further embodiment, the present invention provides an antibody of the
present invention, for use in the treatment of cancer, wherein the cancer is hepatocellular
carcinoma.
In a further embodiment, the present invention provides the antibody of the
present invention for use in simultaneous, separate, or sequential combination with one or
more anti-tumor agents. In a further embodiment, the present invention provides the
antibody of the present invention for use in simultaneous, separate, or sequential
combination with one or more anti-tumor agents selected from the group consisting of
ramucirumab, necitumumab, olaratumab, galunisertib, abemaciclib, cisplatin, carboplatin,
dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin,
navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable
suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and
8
oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), and cetuximab, in the
treatment of cancer.
In a further embodiment, the present invention provides the antibody of the
present invention for use in simultaneous, separate, or sequential combination with one or
more immuno-oncology agents. In a further embodiment, the present invention provides
the antibody of the present invention for use in simultaneous, separate, or sequential
combination with one or more immuno-oncology agents selected from the group
consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab,
urelumab, lirilumab, atezolizumab, and durvalumab, in the treatment of cancer.
In a further embodiment, the present invention provides the use of an antibody of
the present invention for the manufacture of a medicament for the treatment of cancer. In
a further embodiment, the present invention provides the use of an antibody of the present
invention for the manufacture of a medicament for the treatment of cancer, wherein the
cancer is melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic
cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer,
ovarian cancer, esophageal cancer, soft tissue sarcoma, or hepatocellular carcinoma.
In a further embodiment, the present invention provides the use of an antibody of
the present invention in the manufacture of a medicament for the treatment of cancer
wherein said medicament is to be administered simultaneously, separately, or sequentially
with one or more anti-tumor agents. In a further embodiment, the present invention
provides the use of an antibody of the present invention in the manufacture of a
medicament for the treatment of cancer wherein said medicament is to be administered
simultaneously, separately, or sequentially with one or more anti-tumor agents selected
from the group consisting of ramucirumab, necitumumab, olaratumab, galunisertib,
abemaciclib, cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel,
cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel proteinbound
particles for injectable suspension, ixabepilone, capecitabine, FOLFOX
(leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and
irinotecan), and cetuximab.
9
In a further embodiment, the present invention provides the use of an antibody of
the present invention in the manufacture of a medicament for the treatment of cancer
wherein said medicament is to be administered simultaneously, separately, or sequentially
with one or more immuno-oncology agents. In a further embodiment, the present
invention provides the use of an antibody of the present invention in the manufacture of a
medicament for the treatment of cancer wherein said medicament is to be administered
simultaneously, separately, or sequentially with one or more immuno-oncology agents
selected from the group consisting of nivolumab, ipilimumab, pidilizumab,
pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, and durvalumab, in
the treatment of cancer.
An antibody of the present invention is an engineered, non-naturally occurring
polypeptide complex. A DNA molecule of the present invention is a non-naturally
occurring DNA molecule that comprises a polynucleotide sequence encoding a
polypeptide having the amino acid sequence of one of the polypeptides in an antibody of
the present invention.
The antibody of the present invention is an IgG type antibody and has “heavy”
chains and “light” chains that are cross-linked via intra- and inter-chain disulfide bonds.
Each heavy chain is comprised of an N-terminal HCVR and a heavy chain constant
region (“HCCR”). Each light chain is comprised of a LCVR and a light chain constant
region (“LCCR”). When expressed in certain biological systems, antibodies having
native human Fc sequences are glycosylated in the Fc region. Typically, glycosylation
occurs in the Fc region of the antibody at a highly conserved N-glycosylation site. Nglycans
typically attach to asparagine. Antibodies may be glycosylated at other positions
as well.
Optionally, certain antibodies of the present invention contain an Fc portion which
is derived from human IgG1. IgG1 is well known to bind to the proteins of the Fc-gamma
receptor family (FcR) as well as C1q. Interaction with these receptors can induce
antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity
(CDC). Therefore, optionally, certain antibodies of the present invention are a fully
human monoclonal antibody lacking Fc effector function (IgG1, lambda, Fc-null). To
10
achieve an Fc-null IgG1 antibody, selective mutagenesis of residues is necessary within
the CH2 region of its IgG1 Fc region. Amino acid substitutions L234A, L235E, and
G237A are introduced into IgG1 Fc to reduce binding to FcγRI, FcγRIIa, and FcγRIII,
and substitutions A330S and P331S are introduced to reduce C1q-mediated complement
fixation .
To reduce the potential induction of an immune response when dosed in humans,
certain amino acids may require back-mutations to match antibody germline sequences.
Certain antibodies of the present invention contain E1Q and S94R mutations in the
variable heavy chain, and contain T76S and A80S mutations in the variable light chain.
The HCVR and LCVR regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (“CDRs”), interspersed with
regions that are more conserved, termed framework regions (“FR”). Each HCVR and
LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
Herein, the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and
HCDR3” and the three CDRs of the light chain are referred to as “LCDR1, LCDR2 and
LCDR3”. The CDRs contain most of the residues which form specific interactions with
the antigen. There are currently three systems of CDR assignments for antibodies that are
used for sequence delineation. The Kabat CDR definition (Kabat et al., “Sequences of
Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991))
is based upon antibody sequence variability. The Chothia CDR definition (Chothia et al.,
“Canonical structures for the hypervariable regions of immunoglobulins”, Journal of
Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for
the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-
948 (1997)) is based on three-dimensional structures of antibodies and topologies of the
CDR loops. The Chothia CDR definitions are identical to the Kabat CDR definitions
with the exception of HCDR1 and HCDR2. The North CDR definition (North et al., “A
New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology,
406, 228-256 (2011)) is based on affinity propagation clustering with a large number of
11
crystal structures. For the purposes of the present invention, the North CDR definitions
are used.
An isolated DNA encoding a HCVR region can be converted to a full-length
heavy chain gene by operably linking the HCVR-encoding DNA to another DNA
molecule encoding heavy chain constant regions. The sequences of human, as well as
other mammalian, heavy chain constant region genes are known in the art. DNA
fragments encompassing these regions can be obtained e.g., by standard PCR
amplification.
An isolated DNA encoding a LCVR region may be converted to a full-length light
chain gene by operably linking the LCVR-encoding DNA to another DNA molecule
encoding a light chain constant region. The sequences of human, as well as other
mammalian, light chain constant region genes are known in the art. DNA fragments
encompassing these regions can be obtained by standard PCR amplification. The light
chain constant region can be a kappa or lambda constant region. Preferably for antibodies
of the present invention, the light chain constant region is a lambda constant region.
The polynucleotides of the present invention will be expressed in a host cell after
the sequences have been operably linked to an expression control sequence. The
expression vectors are typically replicable in the host organisms either as episomes or as
an integral part of the host chromosomal DNA. Commonly, expression vectors will
contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to
permit detection of those cells transformed with the desired DNA sequences.
The antibody of the present invention may readily be produced in mammalian
cells such as CHO, NS0, HEK293 or COS cells. The host cells are cultured using
techniques well known in the art.
The vectors containing the polynucleotide sequences of interest (e.g., the
polynucleotides encoding the polypeptides of the antibody and expression control
sequences) can be transferred into the host cell by well-known methods, which vary
depending on the type of cellular host.
Various methods of protein purification may be employed and such methods are
known in the art and described, for example, in Deutscher, Methods in Enzymology 182:
12
83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition,
Springer, NY (1994).
In another embodiment of the present invention, the antibody, or the nucleic acids
encoding the same, is provided in isolated form. As used herein, the term “isolated”
refers to a protein, peptide, or nucleic acid which is free or substantially free from any
other macromolecular species found in a cellular environment. “Substantially free” as
used herein means the protein, peptide, or nucleic acid of interest comprises more than
80% ( on a molar basis) of the macromolecular species present, preferably more than
90%, and more preferably more than 95%.
The antibody of the present invention, or pharmaceutical compositions comprising
the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous).
An antibody of the present invention may be administered to a patient alone with
pharmaceutically acceptable carriers, diluents, or excipients in single or multiple doses.
Pharmaceutical compositions of the present invention can be prepared by methods well
known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed.
(2012), A. Loyd et al., Pharmaceutical Press) and comprise an antibody, as disclosed
herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
The term "treating" (or "treat" or "treatment") refers to slowing, interrupting,
arresting, alleviating, stopping, reducing, or reversing the progression or severity of an
existing symptom, disorder, condition, or disease.
“Binds” as used herein in reference to the affinity of an antibody for human PDL1
is intended to mean, unless indicated otherwise, a KD of less than about 1 x10-6 M,
preferably, less than about 1 x 10-9 M as determined by common methods known in the
art, including by use of a surface plasmon resonance (SPR) biosensor at 37oC essentially
as described herein.
"Effective amount" means the amount of an antibody of the present invention or
pharmaceutical composition comprising an antibody of the present invention that will
elicit the biological or medical response of or desired therapeutic effect on a tissue,
system, animal, mammal or human that is being sought by the researcher, medical doctor,
or other clinician. An effective amount of the antibody may vary according to factors
13
such as the disease state, age, sex, and weight of the individual, and the ability of the
antibody to elicit a desired response in the individual. An effective amount is also one in
which any toxic or detrimental effect of the antibody is outweighed by the therapeutically
beneficial effects.
This invention is further illustrated by the following non-limiting example.
Example 1: Antibody expression and purification
The polypeptides of the variable regions of the heavy chain and light chain, the
complete heavy chain and light chain amino acid sequences of Antibody A, and the
nucleotide sequences encoding the same, are listed below in the section entitled “Amino
Acid and Nucleotide Sequences.” In addition, the SEQ ID NOs for the light chain, heavy
chain, light chain variable region, and heavy chain variable region of Antibody A are
shown in Table 1.
The antibodies of the present invention, including, but not limited to, Antibody A
can be made and purified essentially as follows. An appropriate host cell, such as HEK
293 or CHO, can be either transiently or stably transfected with an expression system for
secreting antibodies using an optimal predetermined HC:LC vector ratio or a single vector
system encoding both HC and LC. Clarified media, into which the antibody has been
secreted, may be purified using any of many commonly-used techniques. For example,
the medium may be conveniently applied to a MabSelect column (GE Healthcare), or
KappaSelect column (GE Healthcare) for Fab fragment, that has been equilibrated with a
compatible buffer, such as phosphate buffered saline (pH 7.4). The column may be
washed to remove nonspecific binding components. The bound antibody may be eluted,
for example, by pH gradient (such as 20 mM Tris buffer pH 7 to 10 mM sodium citrate
buffer pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0).
Antibody fractions may be detected, such as by SDS-PAGE, and then may be pooled.
Further purification is optional, depending on the intended use. The antibody may be
concentrated and/or sterile filtered using common techniques. Soluble aggregate and
multimers may be effectively removed by common techniques, including size exclusion,
hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography.
14
The purity of the antibody after these chromatography steps is greater than 95%. The
product may be immediately frozen at -70°C or may be lyophilized.
Table 1: SEQ ID NOs
Antibody A
HCVR 8
LCVR 9
Heavy chain 10
Light chain 11
Assays
In vivo activity - WINN assay
The antibodies of the present invention can be tested for in vivo
immunomodulatory activity with the Winn assay. In the Winn assay, human tumor cells
and human immune cells (allogenic) are injected together at the same into an
immunodeficient mouse, and then followed by dosing with an immunomodulatory agent.
The ability of the immumomodulatory agent to delay or block tumor growth in the model
can be assessed. Tumor volume is measured to determine the effect of the
immunomodulatory agent in the assay and whether there is an enhancement of the
immune response towards the tumor.
As used herein, 2.14H9OPT is a human IgG1 PD-L1 antibody that utilizes the
heavy chain and light chain sequences from U.S. patent application 2013/0034559. As
used herein, S70 is a human IgG1 PD-L1 antibody that utilizes the heavy chain and light
chain sequences from U.S. patent application 2010/0203056. For both 2.14H9OPT and
S70, the heavy chains were fused to the variable region of the human IgG1 constant
region containing residue changes at L234A, L235E, G237A, A330S, and P331S, to
silence effector functions related to Fc gamma receptors and complement cascade. For
both 2.14H9OPT and S70, recombinant protein was expressed in mammalian cells and
purified by standard ProA purification methods.
Enhancement of the immune response to allo-antigens by antibodies of the present
invention may be tested in the NCI-H292 human NSCLC xenograft model. On day 0,
15
NSG mice from Jackson Laboratories (7 weeks of age, female, in groups of 8-10 mice)
are implanted into the flank subcutaneously with either 2 x106 H292 cells, or a mixture of
2 x106 H292 cells and 1 x 106 human PBMCs in HBSS (0.2 ml total volume). Starting on
Day 1, mice are treated with an i.p. injection of antibody at 10 mg/kg, one time per week.
Animal well-being and behavior, including grooming and ambulation are monitored at
least twice per week.
Body weight and tumor volume are measured twice a week. Tumor specimens are
collected on Day 36 from all groups. Immunohistochemical (IHC) staining is performed
on tumor samples using rabbit anti-mouse CD3 antibody (cross-reactive with human,
Abcam), followed by staining with a secondary HRP-conjugated anti-rabbit IgG (Dako).
Tumor volumes are measured twice per week starting on day 4 post cell
implantation using electronic calipers. Tumor Volume (mm3) = π/6 * Length * Width2.
The antitumor efficacy is expressed as T/C ratio in percent and calculated as summarized
below:
%T/C is calculated by the formula 100 ΔT /ΔC if ΔT > 0 of the geometric mean
values. ΔT = mean tumor volume of the drug-treated group on the final day of the study
– mean tumor volume of the drug-treated group on initial day of dosing; ΔC = mean
tumor volume of the control group on the final day of the study – mean tumor volume of
the control group on initial day of dosing. Additionally, % Regression is calculated using
the formula = 100 x ΔT/Tinitial if ΔT < 0. Animals with no measurable tumors are
considered as Complete Responders (CR) and tumors with >50% regressions were Partial
Responders (PR).
Tumor volume data are analyzed through day 35 with a two-way repeated
measures analysis of variance by time and treatment using the MIXED procedures in SAS
software (Version 9.2). The response analyzed is the log transformation of tumor
volume. The log transformation is necessary to equalize the variance across time and
treatment groups. The correlation structure for the repeated measures model is Spatial
Power. Predefined pairwise comparisons of treated group(s) to control group(s) for each
time point are conducted.
16
Tumor sections from the model can also be analyzed for CD3-positive T cell
infiltration by measuring the presence of CD3-positive T cells using staining for CD3 and
analysis with the Aperio Scan scope. The IHC Nuclear Image Analysis macro detects
nuclear staining for a target chromogen for the individual cells in those regions that are
chosen by the user and quantifies their intensities. Three to five annotations are made
from viable tumor area and used in adjusting the parameters until the algorithm results
generate consistent cell identification. The macro is then saved and the slides logged in
for analysis. The % CD3 positive cells as a percent of the total number of cells are
calculated by the Aperio software.
Tumor volume
In experiments performed essentially as described in this assay, Antibody A dosed
at 10 mg/kg, qw, ip is well tolerated as monitored by body weight and clinical
observations. When no immune cells are added to the immunodeficient animal as a
control arm of the experiment (mice implanted with the lung cancer cell line H292, but
with no PBMCs), treatment with Antibody A or 2.14H9OPT dosed at 10 mg/kg, qw, ip
has no effect on H-292 tumor growth compared to treatment with human IgG.
When immune cells and the test antibody are added to the immunodeficient
animal, Antibody A gives a significantly superior result to control IgG, while 2.14H9OPT
does not. Mice co-implanted with NCI-H292 tumors and PBMCs and dosed with
Antibody A at 10 mg/kg qw result in a T/C of 35% that is significantly different from
control IgG treated mice (p = 0.006). Mice co-implanted with NCI-H292 tumors and
PBMCs dosed with 2.14H9OPT at 10 mg/kg, qw, ip result in a T/C of 54 % that is not
significantly different in comparison to treatment with human IgG (P = 0.102).
CD3-positive T cell infiltration
In experiments performed essentially as described in this assay, by IHC analysis,
co-implantation of mice with the lung tumor line H292 and human PBMCs as a control
arm results in a 10% increase of human CD3 T cells present in the tumor as measured on
day 36 following implantation, while animals implanted only with H292 cells have a 3 %
17
increase of CD3 T cells. Treatment with PBMC and Antibody A (dosed at 10 mg/kg qw,
i.p.) results in a 13% increase of human CD3 T cells; the statistical significance of the
PBMC + Antibody A treated group compared to the PBMC + IgG control group has a pvalue
of 0.021. Treatment with PBMCs and 2.14H9OPT (dosed at 10 mg/kg, qw, i.p.)
does not increase the % human CD3 T cell infiltration as compared to the controls treated
with IgG co-implanted with H292 cells and human PBMC (9% CD3 T cells for
2.14H9OPT vs. 10% CD3 T cells for control) .
Established human tumor xenograft model in NSG mice humanized with PBMC
The efficacy of the antibodies of the present invention can be tested in the NCIH827
human NSCLC xenograft model to assess the ability to delay or destroy established
tumors in the model. On day 0, 1x107 H827 cells are implanted subcutaneously into the
flank of NSG mice (7 weeks of age, female, 10 mice per group). With the human
xenograft tumor established, the mice are infused (i.v.) with 5 x106 human PBMCs on
day 34. Starting on day 35, mice are dosed at 10 mg/kg by weekly (3 total doses) i.p.
with either human IgG or the PD-L1 antibody. Animal well-being and behavior,
including grooming and ambulation are monitored at least twice per week. Body weight
and tumor volume are measured twice a week.
In experiments performed essentially as described in this assay, treatment with
Antibody A significantly inhibits tumor growth in the humanized NSG mice, compared to
treatment with human IgG (Table 2).
Table 2: Tumor volume (mm3) in the NCI-H827 human NSCLC xenograft model
Treatment Days 21 28 30 34 36 40 43 47
Hu IgG
Mean 156 290 337 397 445 726 779 883
SEM 15 13 24 34 60 59 75 78
Antibody A
Mean 163 293 336 367 379 433 557 468
SEM 13 26 25 20 51 35 54 41
Treatment Days 50 55 57 62 65 69 72 76
18
Hu IgG
Mean 959 1000 1241 1345 1530 1508 1854 2056
SEM 87 69 102 91 52 90 121 123
Antibody A
Mean 503 593 580 672 625 775 772 691
SEM 76 85 105 154 170 202 221 231
The efficacy of the antibodies of the present invention can also be tested by
measuring the immune response to allo-antigens in the NCI-H292 human NSCLC
xenograft model.
On day 0, 2 x106 H292 cells are implanted subcutaneously into the flank of NSG
mice (7 weeks of age, female, 10 mice per group). After the tumor is established, the
mice are infused (i.v.) with 10 x106 human PBMCs on day 17. Starting on day 18, mice
are dosed at 10 mg/kg by weekly x3 (3 total doses) i.p.s with antibody. Animal wellbeing
and behavior, including grooming and ambulation are monitored at least twice per
week. Body weight and tumor volume are measured twice a week. On days 32-36, mice
are sacrificed and blood is analyzed for peripheral T cell engraftment using TruCount™
tubes to evaluate the impact on peripheral engraftment of the human T-cell compartment,
as well as the exhaustion phenotype of the T-cell subsets.
In experiments performed essentially as described in this assay on two mice per
group, tumor-bearing mice treated with Antibody A display an altered CD4:CD8 ratio
favoring the CD8 compartment compared to the human IgG treated group (Table 3; 63%
of T cells were CD8 for Antibody A, compared to 47% for IgG control). The Antibody A
treated group does not differ significantly from the IgG treated group in terms of absolute
peripheral T-cell counts (CD4 count + CD8 count), but does display higher peripheral T
cell counts than the 2.14H9OPT treated group (43 x 103 cells / μl of blood for hIgG, 45 x
103 cells / μl of blood for Antibody A, and 10 x 103 cells / μl of blood for 2.14H9OPT).
When monitoring T cell activation, PD-1 levels on T cells may be looked at as a
hallmark of exhausted T cells. A smaller number of PD-1+ T cells versus the control,
suggests a greater T cell activation with the PD-L1 antibody. In this study with two mice
per group, a decrease is seen in the PD-1 expression on T-cells in the Antibody A treated
group (15% PD-1+) and the 2.14H9OPT treated group (32% PD-1+) compared to the IgG
treated group (53% PD-1+).
19
Table 3: Effect of Antibody A treatment on peripheral T cell engraftment in the
H292 human NSCLC xenograft model
Treatment Mouse CD8 count CD4 count CD4/CD8 Ratio
10M PBMCs +
hIgG
#1 16801 19591 1.17
#2 23832 26295 1.10
10M PBMCs +
2.14H9OPT
#1 9317 8611 0.52
#2 1577 1419 0.67
10M PBMCs +
Antibody A
#1 28879 15095 0.92
#2 27450 18319 0.90
Mixed Lymphocyte Reaction
The function of blocking of PD-L1 signals by antibodies of the present invention
may be evaluated by measuring the release of cytokines during T cell activation. The
levels of certain cytokines, such as IFN-, are expected to increase if T cell activation is
promoted by treatment with antibodies of the present invention.
CD14+ monocytes are isolated from fresh human PBMC obtained from a healthy
donor (AllCells) with MACS beads (Miltennyi). Immature dendritic cells (DC) are
generated by culturing these monocytes in 12 ml complete RPMI-1640 medium in the
presence of 1000 IU/ml hGM-CSF and 500 IU/ml hIL-4 for 4 days. CD4+ T cells are
purified from fresh human PBMC of a different healthy donor (AllCells) by negative
selection (Milteny). The two types of cells are then mixed in individual wells of a 96-
well plate with 100 l complete AIM-V medium containing 1x105 CD4+ T cells and
4x103 immature DC per well (E:T = 25:1). 100 l complete AIM-V medium is added
containing 2 nM human IgG1 or human PD-L1 antibody in 6 replicates. After incubation
for 2 days at 37oC at 5% CO2, supernatants are harvested and measured for human IFN-γ
with an ELISA kit (R&D Biosystems).
In experiments performed essentially as described in this assay, addition of
Antibody A, S70, or 2.14H9OPT each enhance IFN- production by T lymphocytes in a
20
dose-dependent manner. At the highest concentration tested (33.3 nM), Antibody A has
an increase of 5.71 fold compared to 3.05 fold (S70), and 4.51 fold (2.14H9OPT).
Table 4: IFN-γ secretion fold change vs. IgG control
Antibody Concentration (nM)
0.01 0.05 0.27 1.3 6.7 33.3
Antibody A 1.25 1.95 2.68 4.13 4.11 5.71
S70 1.03 1.40 1.90 2.27 2.82 3.05
2.14H9OPT 1.26 2.27 2.96 4.55 3.24 4.51
Heavy chain dominance of antibody
The structure of the Antibody A/hPD-L1 co-complex is solved for two separate
crystals at 3.7Å and 3.2Å resolutions. When analyzed, each shows the HCDR3 region of
Antibody A directly contacting hPD-L1 while the LCDR3 of Antibody A points away
from the epitope. The CDRs of the light chain of Antibody A have no significant
contacts with either of the hPD-L1 domains. Within a 6Å cutoff, the paratope of
Antibody A is comprised of eighteen heavy chain residues and only seven light chain
residues. The PD-L1/PD-1 binding site amino acids have been reported in Lin et al.
(2008) PNAS 105(8):3011-3016. The contacts made by the variable light chain of
Antibody A on PD-L1 are not the amino acids involved in PD-L1/PD-1 interaction.
To confirm the heavy chain dominance of Antibody A seen in the crystal
structure, the effect on binding is measured for antibodies where the heavy chain of
Antibody A is paired with irrelevant light chains that replace the light chain of Antibody
A. One pairing produced an antibody that bound comparably to PD-L1 by ELISA as
Antibody A; this antibody contained a light chain where none of the Antibody A paratope
amino acids are conserved in variable light chain CDRs. This result suggests that binding
to PD-L1 by Antibody A is almost completely mediated by the heavy chain. The
thermodynamic signature of Antibody A can be analyzed to assess if the heavy chain
dominance positively affects binding of PD-L1.
De-convoluting the thermodynamic signature of Antibody A
21
Superior association by an antibody to the target can be critical in developing a
therapeutic antibody. An antibody that quickly recognizes and binds the target is a
desirable characteristic for a therapeutic antibody. A dominance of binding by the heavy
chain of the antibody can mean less conformational changes need to occur before binding.
To assess the possibility that the heavy chain dominance of Antibody A would
yield desirable binding characteristics, the thermodynamic signature of PD-L1 blockade
is de-convoluted. The thermodynamic studies are completed on Fabs of Antibody A,
S70, and 2.14H9OPT.
The heavy and light chains of the Antibody A Fab, S70 Fab, and 2.14H9OPT Fab
are cloned into the GS vector. Human 293-Freestyle cells (Invitrogen Corp., Carlsbad,
CA) are cultivated and transfected with the GS vectors according to manufacturer's
specifications in suspension shake flask cultures. Briefly, uncut plasmid DNA and 293
fectin are allowed to complex for 25 min. HEK 293 cells are re-suspended in fresh
medium (vortex to remove clumps) and subsequently combined with DNA/fectin
complex before incubation at 37 °C. The conditioned supernatant is harvested after 6
days and assayed for protein expression. The CaptureSelectTM IgG-CH1 Affinity Matrix
(Thermo Fisher Scientific) kit is utilized to purify all Fabs from the HEK 293 expression
supernatant.
Binding of the Fabs is performed by surface plasmon resonance (SPR). Amine
coupling immobilization of human PD-L1 monomer as ligand on to sensor chip surface is
performed at 25°C. Antibody A-Fab, S70-Fab and 2.14H9OPT-Fab are used each as an
analyte, and injected over the human PDL-1 monomer immobilized sensor chip surface.
All sample analytes are run in 3-fold series dilutions (starting concentrations of 3nM for
Antibody A and 9nM for both S70 and 2.14H9OPT), 6 total dilutions with one duplicate
at a middle concentration and a zero. The sample gradients are prepared in the running
buffer HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v surfactant
P20). The binding experiments are repeated at four different temperatures: 20°C, 25°C,
37°C and 42°C. Throughout the kinetics experiments, the flow rate is maintained at 30
μl/min and the association/contact time at 180 sec for all three Fabs. The dissociation
times are 600 sec for S70-Fab and 420 sec for Antibody A-Fab and 2.14H9OPT-Fab.
22
After dissociation, a 0.75 M NaCl, 25 mM NaOH solution regenerates the immobilized
hPD-L1, and then the surface is stabilized for 30 sec with the running buffer. The
regeneration contact times are 18 sec, 24 sec and 30 sec for 2.14H9OPT-Fab, S70-Fab
and Antibody A-Fab consecutively. The binding kinetics are analyzed using the Biacore
T200 Evaluation software (Version 3.0). Data is referenced to a blank flow cell, and the
data is fitted to the 1:1 Langmuir binding model.
Van’t Hoff plots for the interaction of hPD-L1 monomer with the Fabs of
Antibody A, S70 and 2.14H9OPT are used. Steady-state, association, and dissociation
are the three binding phases analyzed. MATLAB is utilized for the linear regression
analysis. R2 measure of goodness of fit of linear regression was ≥ 0.97 for all three Fabs
in all three binding phases.
In experiments performed essentially as described in this assay, the LSMeans
Student’s T demonstrates that the slopes of all the linear plots are statistically different
from each other except for the dissociation phase linear plots of the 2.14H9OPT-Fab and
Antibody A-Fab.
For association, the Antibody A-Fab/hPD-L1 interaction has the most favorable
association phase among all the complexes. Son is a measurement of association; the
more negative the Son value, the more favorable the interaction. Antibody A has a Son
value of -26.0 while S70 and 2.14H9OPT have Son values of -11.7 and -19.4,
respectively. This data demonstrates a more favorable association interaction for
Antibody A with PD-L1 than S70 and 2.14H9OPT under these conditions.
Binding kinetics and affinity
The kinetics and equilibrium dissociation constant (KD) for human PD-L1 is
determined for antibodies of the present invention using surface plasmon resonance
(Biacore).
Immobilization of antibodies of the present invention as ligand on to sensor chip
surface is performed at 25°C. Soluble human PD-L1-Fc fusion protein (and in some
cases, cynomolgus monkey PD-L1-Fc fusion proteins) is injected as analyte at
concentrations ranging from 0.0123 nM - 9 nM. The analysis is performed at 37°C. The
23
contact time for each sample is 180 sec at 30 μl/min. The dissociation time was 240-1500
seconds. The immobilized surface is regenerated for 18 seconds with 0.95 M NaCl / 25
mM NaOH at 30 μl/min, and then stabilized for 30 seconds. Binding kinetics are
analyzed using the Biacore T200 Evaluation software (Version 3.0). Data are referenced
to a blank flow cell, and the data are fit to a 1:1 binding model.
In experiments performed essentially as described in this assay, Antibody A binds
to human PD-L1 with a KD of 82 pM.
Table 5: Binding by SPR of Antibody A
Binding to Antibody A Kon (1/Ms) Koff (1/s) KD (pM)
Human PD-L1 1.40E+06 1.14E-04 82
Cyno PD-L1 1.51E+06 1.84E-04 122
ELISA analysis: Antibody A binds to recombinant PD-L1
The ability for antibodies of the present invention to bind human PD-L1 can be
measured with an ELISA assay. For the PD-L1 binding assay, a 96-well plate (Nunc) is
coated with human PD-L1-Fc (R&D Systems) overnight at 4oC. Wells are blocked for 2
h with blocking buffer (PBS containing 5% nonfat dry milk). Wells are washed three
times with PBS containing 0.1% Tween-20. Anti-PD-L1 antibody or control IgG (100 ul)
is then added and incubated at room temperature for 1 h. After washing, the plate is
incubated with 100 l of goat anti-human IgG F(ab’)2-HRP conjugate (Jackson Immuno
Research) at room temperature for 1 h. The plates are washed and then incubated with
100 l of 3,3’, 5,5’-tetra-methylbenzidine. The absorbance at 450 nm is read on a
microplate reader. The half maximal effective concentration (EC50) is calculated using
GraphPad Prism 6 software.
In experiments performed essentially as described in this assay, Antibody A binds
to human PD-L1 with an EC50 of 0.11 nM. Antibody A retains its binding activities after
4 weeks under all three temperature conditions, 4°C, 25°C and 40°C. Antibody A
showed a similar binding activity to PD-L1 as S70 and 2.14H9OPT.
Flow cytometric analysis: Antibody A binds to cell surface PD-L1
24
The ability for antibodies of the present invention to bind to cell surface expressed
human PD-L1 can be measured with a flow cytometric assay. MDA-MB 231 cells (PDL1-
positive human breast adenocarcinoma cell line) are added to a 96 well U-bottom
plate at 1.5x105 cells per well in 200 l staining buffer and incubated at 4oC for 30 min.
Plate are centrifuged at 1200 rpm for 5 min and supernatant removed. 100 l of
antibody-biotin (serially diluted by 1:4 starting from 10ug/ml) is added. A total of 6
serial dilutions are evaluated. After incubation at 4oC for 30 min, cells are washed twice
with DPBS. 100 l of detection buffer containing 5 l streptavidin-PE is added. After
incubation at 4°C for 30 more min, plate is centrifuged and washed twice with DPBS.
Cells are re-suspended in 200 l DPBS for FACS analysis.
In experiments performed essentially as described in this assay, Antibody A binds
to cell surface PD-L1 on MDA-MB231 cells in a dose dependent manner with an EC50
of 0.14 nM.

We claim:-
1. An antibody that binds human PD-L1 (SEQ ID NO: 1), comprising a light
chain (LC) and a heavy chain (HC), wherein the light chain comprises a
light chain variable region (LCVR) and the heavy chain comprises a heavy
chain variable region (HCVR), and wherein the LCVR comprises light
chain complementarity determining regions LCDR1, LCDR2, and LCDR3
consisting of the amino acid sequences SGSSSNIGSNTVN (SEQ ID NO:
5), YGNSNRPS (SEQ ID NO: 6), and QSYDSSLSGSV (SEQ ID NO: 7),
respectively, and wherein the HCVR comprises heavy chain
complementarity determining regions HCDR1, HCDR2, and HCDR3
consisting of the amino acid sequences KASGGTFSSYAIS (SEQ ID NO:
2), GIIPIFGTANYAQKFQG (SEQ ID NO: 3), and
ARSPDYSPYYYYGMDV (SEQ ID NO: 4), respectively.
2. An antibody, comprising a light chain (LC) and a heavy chain (HC),
wherein the light chain comprises a light chain variable region (LCVR)
and the heavy chain comprises a heavy chain variable region (HCVR),
wherein the LCVR has the amino acid sequence given in SEQ ID NO: 9,
and the HCVR has the amino acid sequence given in SEQ ID NO: 8.
3. The antibody of Claim 2, wherein the LC has the amino acid sequence
given in SEQ ID NO: 11, and the HC has the amino acid sequence given in
SEQ ID NO: 10.
4. The antibody of Claim 3, comprising two light chains and two heavy
chains, wherein each light chain has the amino acid sequence given in
SEQ ID NO: 11, and each heavy chain has the amino acid sequence given
in SEQ ID NO: 10.
5. The antibody of Claim 4, wherein one of the heavy chains forms an interchain
disulfide bond with one of the light chains, and the other heavy chain
forms an inter-chain disulfide bond with the other light chain, and one of
31
the heavy chains forms two inter-chain disulfide bonds with the other
heavy chain.
6. The antibody of any one of Claims 1-5, wherein the antibody is
glycosylated.
7. A mammalian cell comprising a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide having an amino acid
sequence of SEQ ID NO: 11 and a polynucleotide sequence encoding a
polypeptide having an amino acid sequence of SEQ ID NO: 10, wherein
the cell is capable of expressing an antibody comprising a light chain
having an amino acid sequence of SEQ ID NO: 11 and a heavy chain
having an amino acid sequence of SEQ ID NO: 10.
8. A process for producing an antibody comprising a light chain having an
amino acid sequence of SEQ ID NO: 11 and a heavy chain having an
amino acid sequence of SEQ ID NO: 10, comprising cultivating the
mammalian cell of Claim 7 under conditions such that the antibody is
expressed, and recovering the expressed antibody.
9. An antibody produced by the process of Claim 8.
10. A pharmaceutical composition, comprising the antibody of any one of
Claims 1-6 or 9, and an acceptable carrier, diluent, or excipient.
11. A method of treating cancer, comprising administering to a patient in need
thereof, an effective amount of the antibody of any one of Claims 1-6 or 9.
12. The method of Claim 11, wherein the cancer is melanoma, lung cancer,
head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer,
kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian
cancer, esophageal cancer, soft tissue sarcoma, or hepatocellular
carcinoma.
13. The method of Claim 11 or 12, further comprising administering
simultaneous, separate, or sequential combination of one or more antitumor
agents.
14. The antibody of any one of Claims 1-6 or 9, for use in therapy.
32
15. The antibody of any one of Claims 1-6 or 9, for use in the treatment of
cancer.
16. The antibody for use of Claim 15, wherein the cancer is melanoma, lung
cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric
cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer,
ovarian cancer, esophageal cancer, soft tissue sarcoma, or hepatocellular
carcinoma.
17. The antibody of any one of Claims 1-6 or 9 for use in simultaneous,
separate, or sequential combination with one or more anti-tumor agents, in
the treatment of cancer.
18. The antibody for use of Claim 17, wherein the cancer is melanoma, lung
cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric
cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer,
ovarian cancer, esophageal cancer, soft tissue sarcoma, or hepatocellular
carcinoma.