AN ANTIBODY AGAINST PROGRAMMED DEATH 1 (PD-1) RECEPTOR
ITS PREPARATION, NUCLEIC ACID ENCODING IT AND COMPOSITIONS
COMPRISING IT
DESCRIPTION OF THE INVENTION
Field of the Invention
[001] The technical field relates to nrwdulation of immune responses
regulated by the Programmed Death 1 (PD-I) receptor.
Background of the Invention
[002] An adaptive immune response involves activation, selection,
and clonal proliferation of two major classes of lymphocytes termed T cells
and B cells. After encountering an antigen, T cells proliferate and differentiate
into antigen-specific effector cells, while B cells proliferate and differentiate
into antibody-secreting ceils.
[003] T cell activation is a multi-step process requiring several
signaling events between the T ceil and an antigen-presenting cell (APC). For
T cell activation to occur, two types of signals must be delivered io a resting T
ceil. The first type is mediated by the antigen-specific T cell receptor (TcR),
and confers specificity to the immune response. The second, costiimulatory,
tyfje regulates the magnitude of the response and is (jelivered through
accessory' receptors on the T cell.
rnn^l a nrimarv costimulators' sinnal is delivered through the activatina
CD28 receptor upon engagement of its ligands B7-1 or B7-2. In contrast,
engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2
ligands results in attenuation of T cell response. Thus, CTI_A-4 signals
antagonize costimulation mediated by CD28. At high antigen concentrations,
CD28 costimulation overrides the CTLA-4 inhibitory effect. Temporal
regulation of the CD28 and CTLA-4 expression maintains a balance between
activating and inhibitory signafe and ensures the dc!>/elopment of an effective
immune response, while safeguarding against the development of
autoimmunity.
[005] Molecular homologues of CD28 and CTLA-4 and their B-7 like
ligands have been recently Identified. ICOS Is a CD28-like costimulatory
receptor. PD-1 (Programmed Death 1) Is an inhibitory receptor and a
counterpart of CTLA-4. This disclosure relates to modulation of immune
responses mediated by the PD-1 receptor.
[006] PD-1 is a 50-55 kDa type I transmembrane receptor that was
originally identified In a T cell line undergoing activation-induced apoptosis.
PD-1 is expressed on T cells, B cells, and macrophages. The ligands for
PD-1 are the 87 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).
[007] PD-1 Is a member of the immunoglobulin (Ig) superfamily that
contains a single Ig V-IIke domain in Its extracellular region. The PD-1
cytoplasmic domain contains two tyrosines, with the most membrane-proximal
tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immuno-receptor
tyrosine-based inhibitory motif). The presence of an ITIM on PD-1 indicates
that this molecule functions to attenuate antigen receptor signaling by
recruitment of cytoplasmic phosphatases. Human and murine PD-1 proteins
share about 60% amino acid identity with conservation of four potential
N-glycosylation sites, and residues that define the Ig-V domain. The ITIM in
the cytoplasmic region and the mM-like motif surrounding the
carboxy-terminal tyrosine (THYATI in human and mouse) are also conserved
between human and murine orthologues.
[008] PD-1 is expressed on activated T cells, B cells, and
monocytes. Experimental data Implicates the interactions of PD-1 with its
ligands in downregulatlon of central and peripheral immune responses. In
particular, proliferation in wild-type T cells but not in PD-1-deficient T cells is
inhibited in the presence of PD-L1. Additionally, PD-1-deficient mice exhibit
an autoimmune phenotype. PD-1 deficiency in the C57BL/6 mice results in
chronic progressive lupus-like gtomerulonephritis and arthritis. In Balb/c mice,
PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart-
tissue-specific self-reacting antft)odies.
[009] In general, a need exists to provide safe and effective
therapeutic metiiods for immune disorders such as, for example, autoimmune
diseases, inflammatory disorders, allergies, transplant rejection, cancer,
immune deficiency, and other immune system-related disorders. Modulation
of the immune responses involved in these disorders can be accomplished by
manipulation of the PD-1 pathway.
SUMMARY OF THE INVENTION
[010] The present disclosure provides antibodies that can act as
agonists and/or antagonists of PD-1, thereby modulating immune responses
regulated by PD-1. The disclosure further provides anti-PD-1 antibodies that
comprise novel antigen-binding fragments. Anti-PD-1 antibodies of the
invention are capable of (a) specifically binding to PD-1, including human
PD-1; (b) blocking PD-1 interactions with its natural llgand(s); or (c)
performing both functions. Furthermore, the antibodies may possess
immunomodulatory properties, i.e., they may be effective in modulating the
PD-1-associated downregulatlon of immune responses. Depending on the
method of use and the desired effect, the antibodies may be used to either
enhance or inhibit immune responses.
[011] Nonlimiting illustrative embodiments of the antibodies are
refen^ed to as PD1-17, PD1-28, PD1-33. PD1-35, and PD1-F2. Other
embodiments comprise a Vh and/or Vl domain of the Fv fragment of PD1-17,
PD1-28, PD1-33, PD1-35, or PD1-F2. Further embodiments comprise one or
more complementarity determining regions (CDRs) of any of these Vh and Vl
domains. Other embodiments comprise an H3 fragment of the Vh domain of
PD1-17, PD1-28, PD1-33, PD1-35, orPD1-F2.
[012] The disclosure also provides compositions comprising PD-1
antibodies, and their use in methods of modulating Immune response,
including methods of treating humans or animals. In particular embodiments,
anti-PD-1 antibodies are used to treat or prevent Immune disorders by virtue
of increasing or reducing the T cell response mediated by TcR/CD28.
Disorders susceptible to treatment with compositions of tlie invention include
but are not limited to riieumatoid arthritis, multiple sclerosis, inflammatory
bowel disease, Crohn's disease, systemic lupus erythematosis, type I
diabetes, transplant rejection, graft-versus-host disease, hyperprollferative
immune disorders, cancer, and infectious diseases.
[013] Additionally, anti-PD-1 antibodies may be used diagnostically to
detect PD-1 or its fragments in a biological sample. The amount of PD-1
detected may be con-elated with the expression level of PD-1, which, in turn,
is con-elated with the activation status of immune cells (e.g., activated T cells,
B cells, and monocytes) In the subject.
[014] The disclosure also provides isolated nucleic acids, which
comprise a sequence encoding a Vh or Vl domain from the Fv fragment of
PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2. Also provided are isolated
nucleic acids, which comprise a sequence encoding one or more CDRs from
any of the presently disclosed Vh and Vl domains. The disclosure also
provides vectors and host cells comprising such nucleic acids.
[015] The disclosure further provides a method of producing new Vh
and Vl domains and/or functional antibodies comprising all or a portion of
such domains derived from the Vh or Vl domains of PD1-17, PD1-28, PD1-33,
PD1-35,orPD1-F2.
[016] Additional aspects of the disclosure will be set forth in part in the
description which follows, and in part will be obvious from the description, or
may be leamed by practicing the invention. The invention is set forth and
particulariy pointed out in the appended claims, and the present disclosure
should not be construed as limiting the scope of the claims in any way. The
following detailed description includes exemplary representations of various
embodiments of the invention, which are not restrictive of the invention, as
claimed. The accompanying figures constitute a part of this specification and,
together with the description, serve only to illustrate various embodiments and
not limit the invention. Citation of references is not an admission that these
references are prior art to the Invention.
BRIEF DESCRIPTION OF THE FIGURES
[017] Figures 1A and 1B show reactivity of scFv antibodies with
human PD-1 as determined by phage ELISA.
[018] Figures 2A-2C show reactivity of IgG-converted antibodies with
human or mouse PD-1 as determined by ELISA.
[019] Figure 3 shows results of an ELISA demonstrating that selected
PD-1 antibodies inhibit binding of PD-L1 to PD-1.
[020] Figure 4 shows results of an ELISA demonstrating that
immunomodulatory PD-1 antibodies bind to distinct sites on PD-1 as
detemiined by cross-blocking ELISA assays.
[021] Figure 5 shows results of T-cell proliferation assays
demonstrating that co-engagement by TcR and anti-PD-1 antibody PD1-17 or
PD-L1 .Fc reduces proliferation. Co-engagement by TcR and anti-PD-1 J110
has no effect on proliferation.
[022] Figure 6 demonstrates enhanced proliferation of primary T cells
by PD1-17 in a soluble form.
DETAILED DESCRIPTION
Definitions
[023] The term "antibody," as used in this disclosure, refers to an
immunoglobulin or a fragment or a derivative thereof, and encompasses any
polypeptide comprising an antigen-binding site, regardless whether it is
produced in vitro or in vivo. The temn includes, but is not limited to,
polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized,
single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted
antibodies. Unless otherwise modified by the tenm "intact," as in "intact
antibodies," for the purposes of this disclosure, the term "antibody" also
includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and
other antibody fragments that retain antigen-binding function, i.e., the ability to
bind PD-1 specifically. Typically, such fragments would comprise an
antigen-binding domain.
[024] The terms "antigen-binding domain," "antigen-binding fragment,"
and "binding fragment" refer to a part of an antibody molecule that comprises
amino acids responsible for the specific binding between the antibody and the
antigen. In Instances, where an antigen is large, the antigen-binding domain
may only bind to a part of the antigen. A portion of the antigen molecule that
is responsible for specific interactions with the antigen-binding domain is
referred to as "epitope" or "antigenic determinant."
[025] An antigen-binding domain typically comprises an antibody light
chain variable region (Vl) and an antibody heavy chain variable region (Vh),
I
however, it does not necessarily have to comprise both. For example, a
so-called Fd antibody fragment consists only of a Vh domain, but still retains
some antigen-binding function of the intact antibody.
[026] The temri "repertoire" refers to a genetically diverse collection of
1 1
nucleotides derived wholly or partially from sequences that encode expressed
immunoglobulins. The sequences are generated by In vivo rearrangement of,
e.g., V, D, and J segments for H chains and, e.g., V and J segment for L
chains. Alternatively, the sequences may be generated from a cell line by In
vitro stimulation, in response to which the rean^angement occurs.
Alternatively, part or all of the sequences may be obtained by combining, e.g.,
unrearranged V segments with D and J segments, by nucleotide synthesis,
randomised mutagenesis, and other methods, e.g., as disclosed in U.S.
Patent No. 5,565,332.
[027] The temns "specific interaction" and "specific binding" refer to
two molecules forming a complex that is relatively stable under physiologic
conditions. Specific binding is c^racterized by a high affinity and a low to
moderate capacity as distinguished from nonspecific binding which usually
has a low affinity with a moderate to high capacity. Typically, binding is
considered specific when the affinity constant Ka is higher than 10°M"\ or
more preferably higher than 10° M'\ If necessary, non-specific binding can be
reduced without substantially affecting spedlic binding by varying the binding
conditions. The appropriate binding conditions such as concentration of
antibodies, ionic strength of the solution, temperature, time allowed for
binding, concentration of a bioci^ing agent (e.g., serum albumin, milk casein),
etc., may be optimized by a skilled artisan using routine techniques.
Illustrative conditions are set forth in Examples 1, 2,4, 6, and 7.
[028] The phrase "substantially as set out" means that the relevant
CDR, Vh, or Vl domain of the invention will be either Identical to or have only
insubstantial differences in the specified regions (e.g., a CDR), the sequence
of which is set out. Insubstantial differences include minor amino acid
changes, such as substitutions of 1 or 2 out of any 5 amino acids In the
sequence of a specified region.
[029] The term "PD-1 activity" refers to one or more immunoregulatory
activities associated with PD-1. For example, PD-1 is a negative regulator of
the TcR/CD28-mediated immune response. Procedures for assessing the
PD-1 activity in vivo and In vitro are described in Examples 8, 9, and 10.
[030] The terms "modulate," "immunomodulatory," and their cognates
refer to a reduction or an increase in the activity of PD-1 associated with
downregulation of T cell responses due to its Interaction with an anti-PD-1
antibody, wherein the reduction or increase Is relative to the activity of PD-1 in
the absence of the same antitx>dy. A reduction or an increase in activity is
preferably at least about 10%. 20%, 30%. 40%. 50%. 60%. 70%. 80%. 90%,
or more. When PD-1 activity is reduced, the terms "modulatory" and
"modulate" are interchangeable with the temns "inhibitory" and "inhibit.' When
PD-1 activity Is increased, the terms "modulatory" and "modulate" are
interchangeable with the terms "activating" and "activate." The activity of
PD-1 can be determined quantitatively using T cell proliferation assays as
described in Examples 8 and 9.
[031] The temis "treatment" and "therapeutic method" refer to both
therapeutic treatment and prophylactic/preventatlve measures. Those in need
of treatment may Include individuals already having a particular medical
disorder as well as those who may ultimately acquire the disorder (i.e., those
needing preventative measures).
[032] The term "effective amount" refers to a dosage or amount that
is sufficient to reduce the activity of PD-1 to result in amelioration of
symptoms in a patient or to achieve a desired biological outcome, e.g.,
increased cytolytic activity of T cells, Induction of immune tolerance, reduction
or increase of the PD-1 activity associated with the negative regulation of
T-cell mediated immune response, etc.
[033] The temri "isolated" refers to a molecule that is substantially free
of its natural environment. For instance, an isolated protein is substantially
free of cellular material or other proteins from the cell or tissue source from
which It Is derived. TTie term "isolated" also refers to preparations where the
isolated protein is sufficiently pure to be administered as a pharmaceutical
composition, or at least 70-80% (w/w) pure, more preferably, at least 80-90%
(w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least
95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure'.
Anti-PD-1 Antibodies
[034] The disclosure provides anti-PD-1 antibodies that comprise
novel antigen-binding fragments.
[035] In general, antibodies can be made, for example, using
traditional hybrldoma techniques (Kohler and Milstein (1975) Nature, 256:
495-499), recombinant DNA methods (U.S. Patent No. 4,816,567), or phage
display performed with antibody libraries (Clackson et al. (1991) Nature, 352:
624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597). For other antibody
production techniques, see also Antibodies: A Laboratory Manual, eds.
Harlow et al.. Cold Spring Harbor Laboratory, 1988. The invention is not
limited to any particular source, species of origin, method of production.
[036] Intact antibodies, also known as immunoglobulins, are typically
tetrameric glycosylated proteins composed of two light (L) chains of
approximately 25 kDa each and two heavy (H) chains of approximately 50
kDa each. Two types of light chain, designated as the X chain and the k
chain, are found in antiljodies. Depending on the amino acid sequence of the
constant domain of heavy chains, immunoglobulins can be assigned to five
major classes; A, D, E, G, and M, and several of these may be further divided
into subclasses (isotypes), e.g., lgGi, IgGa, IgGa. rgG4. IgAi, and lgA2.
[037] The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are weB known in the art. For a review
of antibody stmcture, see Harlow et al., supra. Briefly, each light chain is
composed of an N-terminal variable domain (Vl) and a constant domain (Cl).
Each heavy chain is composed of an N-terminal variable domain (Vh), three or
four constant domains (Ch), and a hinge region. The Ch domain most
proximal to Vh is designated as Ch1 . The Vh and Vl domains consist of four
regions of relatively conserved sequence called framework regions (FR1,
FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable
sequence called complementarity determining regions (CDRs). The CDRs
contain most of the residues responsible for specific interactions with the
antigen. The three CDRs are referred to as CDR1, CDR2, and CDRS, CDR
constituents on the heavy chain are referred to as H1, H2, and H3, while CDR
constituents on the light chain are referred to as L1, l^, and L3, accordingly.
CDRS and, particularly H3, are the greatest source of molecular diversity
within the antigen-binding domain, H3, for example, can be as short as two
amino acid residues or greater than 26.
[038] The Fab fragment (Fragment antigen-binding) consists of the
Vh-Ch1 and Vl-Q. domains covalently linked by a disulfide bond between the
constant regions. To overcome the tendency of non-covalently linked Vh and
Vu domains in the Fv to dissociate when co-expressed in a host cell, a
so-called single chain (sc) Fv fragment (scFv) can be constructed. In a scFv,
a flexible and adequately long polypeptide links either the C-terminus of the
Vh to the N-termlnus of the Vl or the C-termlnus of the Vl to the N-termlnus of
the Vh- Most commonly, a 15-resldue (Gly4Ser)3 peptide is used as a linker
but other linkers are also known in the art.
[039] Antibody diversity is a result of combinatorial assembly of
multiple germline genes encoding variable regions and a variety of somatic
events. The somatic events include recombination of variable gene segments
with diversity (D) and joining (J) gene segments to make a complete Vh region
and the recombination of variable and joining gene segments to make a
complete Vl region. The recombination process itself is imprecise, resulting In
the loss or addition of amino acids at the V(D)J junctions. These mechanisms
of diversity occur In the developing B cell prior to antigen exposure. After
antigenic stimulation, the expressed antibody genes in B cells undergo
somatic mutation.
[040] Based on the estimated number of germline gene segments, the
random recombination of these segments, and random Vh-Vl pairing, up to
1.6 X 10^ different antibodies could be produced (Fundamental Immunology,
3*^^ ed., ed. Paul, Raven Press, New York, NY, 1993). When other processes
which contribute to antibody diversity (such as somatic mutation) are taken
i
into account, It is thought that upwards of 1 x 10^° different antibodies could be
potentially generated (Immunoglobulin Genes, 2"^ ed., eds. Jonio et al..
Academic Press, San Diego, CA, 1995). Because of the many processes
involved in antibody diversity, it is highly unlikely that independently generated
antibodies will have identical or even substantially similar amino acid
sequences in the CDRs.
[041] The disclosure provides novel CDRs derived from human
immunoglobulin gene libraries. The structure for carrying a CDR will generally
be an antibody heavy or light chain or a portion thereof, in which the CDR is
located at a location corresponding to the CDR of naturally occuning Vh and
Vl. The structures and locations of immunoglobulin variable domains may be
determined, for example, as described in Kabat et al.. Sequences of Proteins
of Immunological Interest, No. 91-3242, National Institutes of Health
Publications. Bethesda, MD, 1991.
[042] DNA and amino acid sequences of anti-PD-1 antibodies, their
scFv fragment, Vh and Vl domains, and CDRs are set forth In the Sequence
Listing and are enumerated as listed in Table 1. Particular nonllmiting
illustrative embodiments of the antibodies are refen-ed to as PD1-17, PD1-28,
PD1-33, PD1-35, and PD1-F2. The positions for each CDR within the Vh and
Vl domains of the illustrative embodiments are listed in Tables 2 and 3.
Table 1: DNA and Amino Acid (AA) Sequences of Vh and Vl Domains and
CDRs
[043] Anti-PD-1 antibodies may optionally comprise antibody constant
regions or parts thereof. For example, a Vl domain may have attached, at its
C terminus, antibody light chain constant domains including human Ck or CA
chains. Similariy, a specific antigen-binding domain based on a Vh domain
may have attached all or part of an immunoglobulin heavy chain derived from
any antibody Isotope, e.g., IgG, IgA, IgE, and IgM and any of the isotope
sub-classes, which include but are not limited to, IgGi and lgG4. In the
exemplary embodiments, PD1-17, PD1-28, PD1-33, and PD1-35, antibodies
comprise C-terminal fragments of heavy and light chains of human IgGix,
while PD1-F2 comprises C-terminal fragments of heavy and light chains of
human IgGiK. The DNA and amino acid sequences for the C-temninal
fragment of are well known In the art (see, e.g. Kabat et al.. Sequences of
Proteins of Immunological Interest, No. 91-3242, National Institutes of Health
Publications, Bethesda, MD, 1991). Nonlimiting exemplary sequences are set
forth in Table 4.
Table 4
[044] Certain embodiments comprise a Vh and/or Vl domain of an Fv
fragment from PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. Further
embodiments comprise at least one CDR of any of these Vh and Vl domains.
Antibodies, comprising at least one of the CDR sequences set out in SEQ ID
NO:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID N0:14, SEQ ID NOs:16-40, SEQ ID NO:47, or SEQ ID NO:49
are encompassed within the scope of this invention. An embodiment, for
example, comprises an H3 fragment of the Vh domain of antibodies chosen
from at least one of PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2.
[045] In certain embodiments, the Vh and/or Vl domains may be
germlined, i.e., the framework regions (FRs) of these domains are mutated
using conventional molecular biology techniques to match those produced by
the germline cells. In other embodiments, the framework sequences remain
diverged from the consensus gennline sequences.
[046] In certain embodiments, the antibodies specifically bind an
epitope within the extracellular domain of human PD-1. The predicted
extracellular domain consists of a sequence from about amino acid 21 to
about amino acid 170 of SEQ ID N0:41 (Swissport Accession No. Q15116).
In certain other embodiments, the antibodies specifically bind an epitope
within the extracellular domain of mouse PD-1, with an affinity of more than
10^ M"\ and preferably more than 10® M'\ The amino add sequence of
mouse PD-1 is set out In SEQ ID NO:56 (Accession No. NM_008798) and is
as a whole about 60% identical to its human counterpart. In further
embodiments, antibodies of the invention bind to the PD-L-binding domain of
PD-1.
[047] It Is contemplated that antit)odies of the inventton may also bind
with other proteins, including, for example, recombinant proteins comprising
all or a portion of the PD-1 extracellular domain.
[048] One of ordinary skill in the art will recognize that the antibodies
of this invention may be used to detect, measure, and inhibit proteins that
differ somewhat from PD-1. The antibodies are expected to retain the
specificity of binding so long as tlie target protein comprises a sequence
which is at least about 60%, 70%, 80%, 90%, 95%, or more Identical to any
sequence of at least 100, 80, 60,40, or 20 of contiguous amino acids In the
sequence set forth SEQ ID N0:41. The percent identity is determined by
standard alignment algorithms such as, for example, Basic Local Alignment
Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410, the
algorithm of Needleman et al. (1970) J. Mol. Biol., 48: 444-453, or the
algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4:11-17.
[049] In addition to the sequence homology analyses, epitope
mapping (see, e.g.. Epitope Mapping Protocols, ed. Monis, Humana Press,
1996) and secondary and tertiary structure analyses can be carried out to
identify specific 3D structures assumed by the disclosed antibodies and their
complexes with antigens. Such methods include, but are not limited to. X-ray
crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13) and computer
modeling of virtual representations of the presently disclosed antibodies
(Fletterick et al. (1986) Computer Graphics and Molecular Modeling, In
Current Communications in Molecular Biology, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY).
Derivatives
[050] This disclosure also provides a method for obtaining an antibody
specific for PD-1. CDRs In such antibodies are not limited to the specific
sequences of Vh and Vl Identified In Table 1 and may include variants of
these sequences that retain the ability to specifically bind PD-1. Such
variants may be derived from the sequences listed in Table 1 by a skilled
artisan using techniques well known in the art. For example, amino acid
substitutions, deletions, or additions, can be made in the FRs and/or in the
CDRs. While changes in the FRs are usually designed to improve stability
and immunogenicity of the antibody, changes in the CDRs are typically
designed to Increase affinity of the antibody for Its target. Variants of FRs
also include naturally occurring immunoglobulin allotypes. iSuch
affinity-Increasing changes may be detemnlned empirically by routine
techniques that involve altering the CDR and testing the affinity antibody for
its target. For example, conservative amiBD acid substitutions can made
within any one of the disclosed CDRs. Vaious alterations can be made
according to the methods described in Anfcody Engineering, 2"^ ed., Oxford
University Press, ed. Borrebaeck, 1995. Titese include but are not limited to
nucleotide sequences that are altered by le substitution of different codons
that encode a functionally equivalent amino acid residue within the sequence,
thus producing a "silent" change. For exa«ple, the nonpolar amino acids
include alanine, leucine, isoleucine, valines, proline, phenylalanine, tryptophan,
and methionine. The polar neutral amino acids Include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The positively
charged (basic) amino acids include argirwe, lysine, and histidine. The
negatively charged (acidic) amino acids indude aspartic acid and glutamic
acid. Substitutes for an amino acid within tie sequence may be selected from
other members of the class to which the anino acid belongs (see Table 5).
Furthermore, any native residue in the polypeptide may also be substituted
with alanine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl.
643:55-67; Sasaki et al. (1998) Adv. Bioplvs. 35:1-24).
[051] Derivatives and analogs of antibodies of the invention can be
produced by various techniques well known in the art, including recombinant
and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory
Manual, 2*^ ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and
Bodansky et al. (1995) The Practice of Peptide Synthesis, 2"^ ed., Spring
Verlag, Berlin, Germany).
[052] In one embodiment a method for making a Vh domain which is
an amino acid sequence variant of a Vh domain of the invention comprises a
step of adding, deleting, substituting, or inserting one or more amino adds in
the amino acid sequence of the presently disclosed Vh domain, optionally
combining the Vh domain thus provided with one or more Vl domains, and
testing the Vh domain or VhA/l combination or combinations for a specific
binding to PD-1 or and, optionally, testing the ability of such antigen-binding
domain to modulate PD-1 activity. The Vl domain may have an amino acid
sequence that is identical or is substantially as set out according to Table 1.
[053] An analogous method can be employed in which one or more
sequence variants of a Vl domain disclosed herein are combined with one or
more Vh domains.
[054] A further aspect of the disclosure provides a method of
preparing antigen-binding fragment that specifically binds with PD-1. The
method comprises:
(a) providing a starting repertoire of nucleic acids encoding a Vh
domain that either includes a CDR3 to be replaced or lacks a CDR3 encoding
region;
(b) combining the repertoire with a donor nucleic acid encoding an
amino acid sequence substantially as set out herein for a Vh CDR3 (i.e., H3)
such that the donor nucleic acid is inserted Into the CDR3 region in the
repertoire, so as to provide a product repertoire of nucleic acids encoding a Vh
domain;
(c) expressing the nucleic acids of the product repertoire;
(d) selecting a binding fragment specific for PD-1; and
(e) recovering the specific binding fragment or nucleic acid
encoding it.
[055] Again, an analogous method may be employed in which a Vl
CDR3 (i.e., L3) of the invention is combined with a repertoire of nucleic acids
encoding a Vl domain, which either include a CDR3 to be replaced or lack a
CDR3 encoding region. The donor nucleic acid may be selected from nucleic
acids encoding an amino acid sequence substantially as set out in SEQ ID
NO:17-40 or SEQ ID NO:50-55.
[056] A sequence encoding a CDR of the invention (e.g., CDR3) may
be introduced into a repertoire of variable domains lacking the respective
CDR (e.g., CDR3), using recombinant DNA technology, for example, using
methodology described by Marks et al. (Bio/Technology (1992) 10: 779-783).
In particular, consensus primers directed at or adjacent to the 5' end of the
variable domain area can be used in conjunction with consensus primers to
the third framework region of human Vh genes to provide a repertoire of Vh
variable domains lacle
attached to antibodies using conventional chemistry. Detectable lat}eis also
include enzyme labels such as horseradish peroxidase or alkaline
phosphatase. Detectable lat>els further include chemical moieties such as
biotin, which may be detected via binding to a specific cognate detectable
moiety, e.g., labeled avidin.
[065] Antibodies, in which CDR sequences differ only insubstantially
from those set out in SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID
N0:8, SEQ ID NO:10, SEQ ID N0:12. SEQ ID N0:14, SEQ ID NOs:16-40,
SEQ ID NO:47, or SEQ ID NO:49 are encompassed within tiie scope of this
invention. Typically, an amino acid is substituted by a related amino acid
having similar charge, hydrophobic, or stereochemical characteristics. Such
substitutions would be within the ordinary skills of an artisan. Unlike in CDRs,
more substantial changes can be made in FRs without adversely affecting the
binding properties of an antibody. Changes to FRs include, but are not limited
to, humanizing a non-human derived or engineering certain framework
residues that are important for antigen contact or for stabilizing the binding
site, e.g., changing the class or subclass of the constant region, changing
specific amino acid residues which might alter the effector function such as Fc
receptor binding, e.g., as described in U.S. Patent Nos. 5,624,821 and
5,648,260 and Lund et al. (1991) J. Immun. 147:2657-2662 and Morgan et al.
(1995) Immunology 86: 319-324, or changing the species fi-om which the
constant region is derived.
[066] One of skill in the art will appreciate that the modifications
described above are not all-exhaustive, and that many other modifications
would obvious to a skilled artisan in light of the teachings of the present
disclosure.
Nucleic Acids. Cloning and Expression Systems
[067] The present disclosure further provides isolated nucleic acids
encoding the disclosed antibodies. The nucleic acids may comprise DNA or
RNA and may be wholly or partially synthetic or recombinant. Reference to a
nucleotide sequence as set out herein encompasses a DNA molecule with the
specified sequence, and encompasses a RNA molecule with the specified
sequence in which U is substituted for T, unless context requires otherwise.
[068] The nucleic acids provided herein comprise a coding sequence
for a CDR, a Vh domain, and/or a Vl domain disclosed herein.
[069] The present disclosure also provides constructs In the form of
plasmlds, vectors, phagemlds, transcription or expression cassettes which
comprise at least one nucleic add encoding a CDR, a Vh domain, and/or a Vl
domain disclosed here.
[070] The disclosure further provides a host cell which comprises one
or more constructs as above.
[071] Also provided are nucleic acids encoding any CDR (HI, H2, H3,
LI, L2, or L3), Vh or Vl domain, as well as methods of making of the encoded
products. The method comprises expressing the encoded product from the
encoding nucleic acid. Expression may beachieved by culturing under
appropriate conditions recombinant liost eels containing the nucleic acid.
Following production by expression a Vh oiVl domain, or specific binding
member may be isolated and/or purified using any suitable technique, then
used as appropriate.
[072] Antigen-binding fragments, Vh and/or Vl domains, and encoding
nucleic acid molecules and vectors may be isolated and/or purified from their
natural environment, in substantially pure or homogeneous form, or, in the
case of nucleic acid, free or substantially free of nucleic add or genes of origin
other than the sequence encoding a polype|*ide with the required function.
[073] Systems for cloning and expression of a polypeptide in a variety
of different host cells are well known in the art. For cells suitable for
producing antibodies, see Gene Expression Systems, Academic Press, eds.
Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant
cells, mammalian cells, and yeast and bacutovims systems. Mammalian cell
i
lines available in the art for expression of a heterologous polypeptide include
Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO
mouse myeloma cells, and many others. A common bacterial host is E. coli.
Any protein expression system compatible with the invention may be used to
produce the disclosed antibodies. Suitable expression systems include
transgenic animals described in Gene Expression Systems, Academic Press,
eds. Femandez et al., 1999.
[074] Suitable vectors can be chosen or constructed, so that they
contain appropriate regulatory sequences, including promoter sequences,
terminator sequences, polyadenylation sequences, enhancer sequences,
marker genes and other sequences as appropriate. Vectors may be plasmids
or viral, e.g., phage, or phagemid, as appropriate. For further details see, for
example, Sambrook et al.. Molecular Cloning; A Laboratory Manual, 2"^ ed.,
Cold Spring Hartx)r Laboratory Press, 1989. Many known techniques and
protocols for manipulation of nucleic acid, for example. In preparatfon of
nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into
cells and gene expression, and analysis of proteins, are described In detail In
Current Protocols in Molecular Biology, 2™* Edition, eds. Ausubel et al., John
Wiley & Sons, 1992.
[075] A further aspect of the disclosure provides a host cell comprising
a nucleic acid as disclosed here. A still furttier aspect provides a method
comprising introducing such nucleic acid into a host cell. The introduction
may employ any available technique. For eukaryotic cells, suitable
techniques may include calcium phosphate transfection, DEAE-Dextran,
eiectroporation, liposome-mediated transfection and transduction using
retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For
bacterial cells, suitable techniques may include calcium chloride
transformation, eiectroporation and transfedion using bacteriophage. The
introduction of the nucleic acid into the cells may be followed by causing or
allowing expression from the nucleic acid, e.g., by culturing host cells under
conditions for expression of the gene.
Methods of Use
[076] The disclosed anti-PD-1 antibodies are capable of modulating
the PD-1-associated downregulation of the immune responses. In particular
embodiments, the Immune response is TcR/CD28-mediated. The disclosed
antibodies can act as either agonists or antagonists of PD-1, depending on
the method of their use. The antibodies can t>e used to prevent, diagnose, or
treat medical disorders in mammals, especially, in humans. Antibodies of the
Invention can also be used for isolating PD-1 or PD-1-expressing cells.
Furthennore, the antibodies can be used to treat a subject at risk of or
susceptible to a disorder or having a disorder associated with aberrant PD-1
expression or function.
[077] Antibodies of the invention can be used in methods for induction
of tolerance to a specific antigen (e.g., a therapeutic protein). In one
embodiment, tolerance is induced against a specific antigen by co-
administration of antigen and an antl-PD-1 antibody of the invention. For
example, patients that received Factor VIII frequently generate antibodies to
this protein; co-administration of an anti-PD-1 antibody of the invention in
combination with recombinant Factor VIII is expected to result In the
downregulation of immune responses to this clotting factor.
[078] Antibodies of the Invention can be used In circumstances where
a reduction In the level of Immune response may be desirable, for example, in
certain types of allergy or allergic reactions (e.g., by inhibition of IgE
production), autoimmune diseases (e.g., rheumatoid arthritis, type I diabetes
mellltus, multiple sclerosis, Inflammatory bowel disease, Crohn's disease, and
systemic lupus erythematosis), tissue, skin and organ transplant rejection,
and graft-versus-hosl disease (GVHD).
[079] When diminished immune response Is desirable, the anti-PD-1
antibodies of the Invention may be used as agonists to PD-1 in order to
enhance the PD-1-associated attenuation of the Immune response. In these
embodiments, co-presentation and physical proximity between positive (i.e.,
mediated by an antigen receptor, e.g., TcR or BcR) and negative (i.e., PD-1)
signals are required. The preferred distance is less than or comparable to the
size of a naturally occurring antigen-presenting cell, i.e., less than about 100
pm; more preferably, less than about 50 pm; and most preferably, less than
about 20 pm.
[080] In some embodiments, the positive (activating) and the negative
(Inhibiting) signals are provided by a llgand or antibodies immobilized on solid
support matrix, or a carrier. In various embodiments, the solid support matrix
may be composed of polymer such as activated agarose, dextran, cellulose,
polyvinylldene fluoride (PVDF). Alternatively, the solid support matrix may be
based on silica or plastic polymers, e.g., as nylon, dacron, polystyrene,
polyacrylates, polyvinyls, teflons, etc.
[081] The matrix can b>e implanted Into the spleen of a patient.
Alternatively, the matrix may be used for the ex vivo incubation of T cells
obtained from a patient, which are then separated and Implanted back into the
patient. The matrix may also be made from a biodegradable material such
polyglycolic acid, polyhydroxyalkanoate, collagen, or gelatin so that they can
be injected into the patient's peritoneal cavity, and dissolve after some tinne
following the injection. The carrier can be shaped to mimic a cell (e.g., bead
or microsphere).
[082] In some embodiments, the positive signal is delivered by a
T-cell-activating antl-CD3 antibody, which l)inds TcR. Activating anti-CD3
antibodies are known in the art (see, for example, U.S. Patent Nos. 6,405,696
and 5,316,763). The ratio between the activating TcR signal and negative
PD-1 signal Is determined experimentally using conventional procedures
known In the art or as described in Examples 8, 9, and 10.
[083] Under certain circumstances. It may be desirable to eircit or
enhance a patient's immune response In order to treat an immune disorder or
cancer. The disorders being treated or prevented by the disclosed methods
include but are not limited to infections with microbes (e.g. bacteria), viruses
(e.g., systemic viral infections such as influenza, viral skin diseases such as
herpes or shingles), or parasites; and cancer (e.g., melanoma and prostate
i
cancers).
[084] Stimulation of T cell activation with anti-PD-1 antibodies
enhances T-T cell responses. In such cases, antibodies act as antagonists of
PD-1. Thus, in some embodiments, the antibodies can be used to Inhibit or
reduce the downreguiatory activity associated with PD-1, I.e., the activity
associated with downregulation of TcR/CD28-mediated immune response. In
these embodiments, the antibodies are not coupled to a positive signal such
as the TcR-mediated stimulation, e.g., the antibodies are in their soluble,
support-unbound, fomi. As denwnstrated in the Examples, a blockade of
PD-1/PD-L Interaction with antagonizing antl-PD-1 antibodies leads to
enhanced T cell proliferative responses, consistent with a downreguiatory role
for the PD-1 pathway In T-T interactions. In various embodiments, the
antibodies inhibit binding of PD-L to PD-1 with an IC50 of less than 10 nM, and
more preferably less then 5 nM, and most preferably less than 1 nM.
Inhibition of PD-L binding can be measured as described In Example € or
using techniques known In the art.
[085] The antibodies or antibody compositions of the present invention
are administered in therapeutically effective amounts. Generally, a
therapeutically effective amount may vary with the subject's age, condition,
and sex, as well as the severity of the medical condition of the subject. A
therapeutically effective amount of antibody ranges from about 0.001 to about
30 mg/kg body weight, preferably from about 0.01 to about 25 mg/kg body
weight, from about 0.1 to about 20 mg/kg body weight, or from about 1 to
about 10 mg/kg. The dosage may be adjusted, as necessary, to suit
observed effects of the treatment. The appropriate dose Is chosen based on
clinical indications by a treating physician.
[086] The antibodies may given as a bolus dose, to maximize the
circulating levels of antibodies for the greatest length of time after the dose.
Continuous infusion may also be used after the bolus dose.
[087] Immune cells (e.g., activated T cells, B cells, or monocytes) can
also be isolated from a patient and incubated ex vivo with antibodies of the
invention. In some embodiments, immune responses can be inhibited by
removing immune cells from a subject, contacting the immune cells in vitro
with an anti-PD-1 antil)Ody of the invention concomitantly with activation of the
immune cells (e.g., by antibodies to the TcR and/or BcR antigen receptor). In
such embodiments, the anti-PD-1 antibody should be used in a multivalent
form such that PD-1 molecules on the surface of an immune cell become
"crosslinked" upon binding to such antibodies. For example, the anti-PD-1
antibodies can be bound to solid support, such as beads, or crosslinked via a
secondary antibody. The immune cells may be then isolated using methods
known in the art and reimplanted into the patient.
[088] In another aspect, the antibodies of the invention can be used
as a targeting agent for delivery of another therapeutic or a cytotoxic agent
(e.g., a toxin) to a cell expressing PD-1. The method includes administering
an anti-PD-1 antibody coupled to a therapeutic or a cytotoxic agent or under
conditions that allow binding of the antibody to PD-1.
[089] The antibodies of the invention may also be used to detect the
presence of PD-1 in biological samples. The amount of PD-1 detected may
be correlated with the expression level of PD-1, which, In tum, is correlated
with the activation status of immune cells (e.g., activated T cells, B cells, and
monocytes) In the subject,
[090] Detection methods that employ antibodies are well known in the
art and include, for example, ELISA, radioimmunoassay, immunoblot,
Western blot, immunofluorescence, immunoprecipitation. The antibodies may
be provided in a diagnostic kit that incorporates one or more of these
techniques to detect PD-1. Such a kit may contain other components,
packaging, instructions, or other material to aid the detection of the protein.
[091] Where the antibodies are intended for diagnostic purposes, it
may be desirable to modify them, for example, with a iigand group (such as
blotin) or a detectable marker group (such as a fluorescent group, a
radioisotope or an enzyme). If desired, the antibodies of the Invention may be
i
labeled using conventional techniques. Suitable detectable labels include, for
example, fluorophores, chromophores, radioactive atoms, electron-dense
reagents, enzymes, and llgands having specific binding partners. Enzymes
are typically detected by their activity. For example, horseradish peroxidase
can be detected by its ability to convert tetramethylbenzldine (TMB) to a blue
pigment, quantifiabJe with a spectrophotometer. For detection, suitable
binding partners include, but are not limited to, blotin and avidin or
streptavidin, IgG and protein A, and the numerous receptor-ligand couples
known in the art. Other permutations and possibilities will be readily apparent
to those of ordinary skill In the art, and are considered as equivalents within
the scope of the instant invention.
[092] Antibodies of the invention can be used in screening methods to
Identify Inhibitors of the PD-1 pathway effective as therapeutics. In such a
screening assay, a first binding mixture is fomned by combining PD-1 and an
antibody of the invention; and the amount of binding in the first binding
mixture (Mo) is measured. A second binding mixture is also formed by
combining PD-1, the antibody, and the compound or agent to be screened,
and the amount of binding in the second twiding mixture (Mi) is measured. A
compound to be tested may be another anti-PD-1 antibody, as illustrated in
the Examples. The amounts of binding In the first and second binding
mixtures are then compared, for example, by calculating the Mi/Mo ratio. The
compound or agent is considered to be capable of modulating a PD-1-
associated downregulation of immune responses if a decrease In binding in
the second binding mixture as compared to the first binding mixture is
observed. The formulation and optimization of binding mixtures is within the
level of skill in the art, such binding mixtures may also contain buffers and
salts necessary to enhance or to optimize binding, and additional control
assays may be included in the screening assay of the invention. Compounds
found to reduce the PD-1-antibody binding by at least about 10% e fluid to the
extent that easy syringability exists. It should be stable under the conditions
of manufacture and storage and nuist be preserved against the contaminating
action of microorganisms such as bacteria and fungi. Prevention of the action
of m/croorgan/sms can be achieved by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like, in many cases, it will be preferable to include
isotonic agents, for example, sugsffs, polyalcohols such as mannitol, sorbitol,
and sodium chloride In the composition. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and/or by the use of
surfactants. Prolonged absorption of the Injectable compositions can be
brought about by including in the composition an agent which delays
absorption, for example, aluminum monostearate, and gelatin.
[099] Oral compositions generally include an Inert diluent or an edible
carrier. They can be enclosed in gelatin capsules or compressed into tablets.
For oral administration, the antibodies can be combined with excipients and
used in the form of tablets, troches, or capsules. Pharmaceutlcally compatible
binding agents, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches, and the like can contain
any of the following ingredients, or compounds of a similar nature; a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent such as
sucrose or saccharin; or a flavoring agent such as peppermint, methyl
salicylate, or orange flavoring.
[0100] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in the
fonmulation. Such penetrants are generally known in the art, and include, for
example, detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration may be accomplished, for example, through the use of
lozenges, nasal sprays, Inhalers, or suppositories. For example, In case of
antibodies that comprise the Fc portion, compositions may be capable of
transmission across mucous membranes in intestine, mouth, or lungs (e.g.,
via the FcRn receptor-mediated pathway as described in U.S. Patent No.
6,030,613). For transdemrial administration, the active compounds may be
formulated into ointments, salves, gels, or creams as generally known in the
art. For administration by inhalation, the antibodies may be delivered in the
form of an aerosol spray from pressured container or dispenser, which
contains a suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer.
[0101] In certain embodiments, the presently disclosed antibodies are
prepared with carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled in the art.
Liposomal suspensions containing the presently disclosed antibodies can also
be used as pharmaceutically acceptable carriers. These can t>e prepared
according to methods known to those skilled in the art, for example, as
described in U.S. Patent No. 4,522,811.
[0102] It may be advantageous to formulate oral or parenteral
compositions in a dosage unit fomi for ease of administration and uniformity
of dosage. The term "dosage unit fomn" as used herein refers to physically
discrete units suited as unitary dosages for the subject to be treated; each unit
containing a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier.
[0103] Toxicity and therapeutic efficacy of the composition of the
invention can be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., for determining the LDjo (the dose
lethal to 50% of the population) and the ED50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio LD50/ED50. Compositions that exhibit large therapeutic indrces are
preferred.
[0104] For any composition used in the present invention, the
therapeutically effective dose can be estimated initially from cell culture
assays. Examples of suitable bioassays include DNA replication assays,
cytokine release assays, transcription-based assays, PD-1/PD-L1 binding
assays, creatine kinase assays, assays based on the differentiation of
pre-adipocytes, assays based on glucose uptake In adipocytes,
immunological assays other assays as, for example, described in the
Examples. The data obtained from the cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. A dose may
be formulated in animal models to achieve a circulating plasma concentration
range that includes the IC50 (i.e., the concentration of the antibody which
achieves a half-maximal inhibition of symptoms). Circulating levels in plasma
may be measured, for example, by high performance liquid chromatography.
The effects of any particular dosage can be monitored by a suitable bioassay.
The dosage lies preferably within a range of circulating concentrations with
little or no toxicity. The dosage may vary depending upon the dosage form
employed and the route of admniistration utilized.
i
[0105] The following Examples do not in any way limit the scope of
the invention. One of ordinary skill in the art will recognize the numerous
modifications and variations that may be performed without altering the spirit
or scope of the present invention. Such modifications and variations are
encompassed within the scope of the invention. The entire contents of all
references, patents, and published patent applications cited throughout this
application are herein incorporated by reference.
EXAMPLES
Example 1: Selection of PD-1 Binding ScFv's
[0106] An scFv phagemid library, which is an expanded version of
10
the 1.38x10 library described by Vaughan et al. (Nature Biotech. (1996) 14:
309-314) was used to select antibodies specific for human PD-1. Soluble
PD-1 fusion protein (at 20 pg/ml h phosphate buffered saline (PBS)) or
control fusion protein (at 50 ng/ml in PBS) was coated onto wells of a
microtiter plate ovemight at 4''C. Wells were washed In PBS and blocked for
1 hour at 37°C in MPBS (3% milk powder in PBS). Purified phage (10^^
transducing units (tu)) was blocl^ed for 1 hour in a final volume of 100 \i\ of 3%
MPBS. Blocked phage was added to blodced control fusion protein wells and
incubated for 1 hour. The blocked and deselected phage were then
transferred to the blocked wells coated with the PD-1 fusion protein and were
incubated for an additional hour. Wells woe washed 5 times with PBST (PBS
containing 0.1% v/v Tween 20), then 5 times with PBS. Bound phage
particles were eluted and used to infect 10 ml exponentially growing E. coll
TGI. Infected cells were grown in 2TY broth for 1 hour at 37°C, then spread
onto 2TYAG plates and incubated overnight at SO^C. Colonies were scraped
off the plates into 10 ml 2TY broth and 15% glycerol added for storage at -
70°C.
[0107] Glycerol stock cultures from the first round of panning selection
were superinfected with helper phage and rescued to give scFv
antibody-expressing phage particles for the second round of panning. A total
of two rounds of panning were carried out In this way for isolation of PD1-17,
except in the second round of panning 20 pg/ml of control protein were used
for deselection. Clones PD1-28, PD1-33, and PD1-35 were selected following
three rounds of selectton. Deselection in the second arvj third rounds was
carried out using 10 pg/ml control fusion protein.
[0108] Antibodies to murine PD-1 were selected by soluble selection
using biotinylated murine PD-1 fusion protein at a final concentration of 100
nM. An scFv phagemid library, as described above, was used. Purified scFv
12
phage (10 tu) in 1 ml 3% MPBS were blocked for 30 minutes, then
biotinylated antigen was added and incubated at room temperature for 1 hour.
Phage/antlgen was added to 250 pi of Dynal M280 Streptavidin magnetk;
beads that had been blocked for 1 hour at 37''C in 1 ml of 3% MPBS and
incubated for a further 15 minutes at room temperature. Beads were captured
using a magnetic rack and washed 4 times in 1 ml of 3% MPBS/ 0.1% (vA^)
Tween 20 followed by 3 washes in PBS. After the last PBS wash, beads were
resuspended in 100 pi PBS and used to infect 5 ml exponentially growing E.
CO// TG-1 cells. Infected cells were incubated for 1 hour at ST^C (30 minutes
stationary, 30 minutes shaking at 250 rpm), then spread on 2TYAG plates and
incubated overnight at 30°C. Output colonies were scraped off the plates and
phage rescued as described above. A second round of soluble selection was
carried out as described above.
Example 2: Specificitv of Antit)0dies for PD-1 by a Phage ELISA
{0109] To determine the specificity of antibodies for PD-1, a phage
ELISA was performed against PD-1 fusion protein and control proteins.
Individual E. coli colonies from selection outputs were picked into 96 well
plates containing 100 \i\ of 2TYAG medium per well. M13K07 helper phage
was added to a multiplicity of infection (moi) of 10 to the exponentially growing
culture and the plates incubated an additional 1 hour at 37°C, Plates were
centrifuged in a benchtop centrlfoge at 2000 rpm for 10 minutes. The
supernatant was removed and cell pellets were r^suspended in 100 pi 2TYAK
and incubated at SO^C overnight with shaking. The next day, plates were
centrifuged at 2000 rpm for 10 minutes and phage-containing supennatant
from each well was transfen'ed to a fresh 96 well plate. Phage samples were
blocked in a final concentration of 3% MPBS prior to ELISA.
[0110] Human or mouse PD-1 fusion protein and control fusion and
non-fusion proteins were coated overnight at 4°C onto 96-well mterotiter
plates at 0.5-2.5 pg/ml In PBS. After coating, the solutions were removed
from the wells, and the plates blodced for 1 hour in 3% MPBS. Plates were
rinsed with PBS and then 50 pi of pre-blocked phage were added to each
well. The plates were incubated for 1 hour and then washed 3 times with
PBST followed by 3 washes with PBS. To each well, 50 pi of a 1:5000
dilution of anti-Mi 3-HRP conjugste (Pharmacia, Peapack, NJ) was added,
and the plates incubated for 40-60 minutes. Each plate was washed three
times with PBST then 3 times with PBS. Fifty pi of TMB substrate was added
to each well, and the samples weie incubated until color developmenL The
reaction was stopped by the addffion of 25 pi of 0.5 M H2SO4. The signal
generated was measured by reading the absorbance at 450 nm using a
microtiter plate reader. Clones showing specific binding to PD-1 fusion
protein but not to control fusion proteins were thus identified and confirmed.
[0111] Specificity data for the PD1 -17 scFv is shown in Figure 1 A.
Reactivity of PD1-28, PD1-33, and PD1-35 scFv's with human PD-1 is shown
in Figure IB (an IgGi control did not bind PD-1).
Example 3: Identification of Antibody Clones
[0112] PD-1-binding scFv £. coli clones were streaked out onto
2TYAG plates and incubated overnight at 30°C. Colonies from these plates
were sequenced using pCANTAB6 vector sequence oligos to amplify the Vh
and Vl regions from the scFv clone. Unique PD-1 binding clones were
assayed for neutralization of PD-L1 binding to PD-1 as described in Example
4. Sequence differences between scFv and IgG formats are due to changes
introduced by PCR primers during the conversion from scFv to IgG.
Example 4: Biochemical Binding Inhibition Assay and Screen
[0113] ScFv production was induced by addition of 1 mM IPTG to
exponentially growing cultures and incubation overnight at 30°C. Crude
scFv-contaJning periplasmic extracts were obtained by subjecting the bacterial
pellets from the overnight induction to osmotic shock. Pellets were
resuspended in 20% (w/v) sucrose, 50mM Tris-HCI, pH 7.5,1 mM EDTA and
cooled on ice for 30 minutes. Cellular debris was removed by centrifugation,
and the scFv was purified by chromatography and buffer-exchanged Into
PBS. Purified scFv's (PD1-17. PD1-28, PD1-33, and PD1-35) were tested for
the ability to inhibit the binding of bfotinylated human PD-L1 fusion protein to
human PD-1 fusion protein Imnfiobllized on plastic in a 96 well microtiter plate
assay. Binding of biotlnylated PD-L1 fusion protein was detected with
AMDEX-aikaline phosphatase, and the signal generated was measured by
reading the absorbance at 405 nm using a microtiter plate reader. Data was
expressed as a percentage of the total binding and a titration of scFv
concentrations was tested to establish clam potency as calculated \C^
values. Clone potency data for the scFv aad IgG antibodies Is shown in Table
5.
[0114] PD1-F2 scFv was produced and purified as described above.
Cells expressing murine PD-1 were addedal 10 cells/well in a final volume of
100 \i\ to a poly-D-lysine-coated 96 well nwotiter plate. Cells were
centrifuged and washed twice in PBS, thenWocked with 300 pi 1% BSA in
PBS for 1 hour at room temperature. Bloded cells were washed three times
in PBST, prior to addition of 25 ^il/well of assay buffer (0.05% BSA, 0.05%
Tween 20 In Dulbecco's PBS) or sample, folowed by 25 pi of biotinylated
murine PD-L1 fusion protein at 300 ng/ml. Binding of biotinylated PD-L1
fusion protein was detected with Amdex aflcailne phosphatase and signals
read as described above. Potencies of PD1-F2 scFv and IgG are shown In
Table 6.
Table 6: Potency of Antl-PD-1 ScFv and IgG Antibodies
Example 5: Conversion of ScFv to IgG
[0115] Heavy and light chain V regions from scFv clones were
amplified by PCR using clone-specific primers. PCR products were digested
with appropriate restriction enzymes and subcloned Into vectors containing
human lgGi heavy chain constant domain (Takahashi et ai. (1982) Cell 29,
671) or vectors containing human lambda or kappa light chain constant
domains (HIeter et al. (1982) Nature 294, 536). Based on the gennlines of the
Vh and Vl segments, It was determined whether kappa or lambda light chain
constant domains were used for conversion (Table 7).
Table 7: Germlines of Vh and Vl Regions of PD-1 Antibody Clones
[0116] The insertion of V region domains into plasmids was verified
by sequencing of plasmid DNA from individual E. coli colonies. Plasmids
were prepared from £. coli cultures by standard techniques and heavy and
light chain constructs cofransfected into eukaryotic cells using standard
techniques. Secreted IgG was purified using Protein A Sepharose
(Pharmacia) and buffer-exchanged into PBS.
[0117] The binding affir% of the anti-mouse PD1 antibody PD1-F2
was determined with a Surface Ptesmon Resonance (SPR) system (BIAcore
3000) (Blacore, Piscataway, NJ) using murine PD-1 fusion Immobilized on a
CMS sensor chip. The concentrafon of PD1-F2 in the flow cell ranged from
7.81 to 125 nM, while the concentetlon of the anti-mouse PD1 antibody J43
(eBiosclence, San Diego, CA) raaged from 25 nM to 500 nM. The equilibrium
constant Kd for PD1-F2 is 6.7x10* M (Ka=1 .5x10® M "^), whereas Ko for J43 is
3.8x10"^ M (Ka=2.6x10® M""").
[0118] The ability of anthfO-l IgG's to bind human or murine PD-1
was determined as follows, ELISAplates were incubated with 2.5 pg/ml
human PD-1/IgG chimera ovemighL Plates were washed with PBS/1 % BSA
and incubated with serial dilutions of a test antibody for 2 hours at room
temperature (RT). After washing, saturating concentrations of
HRP-conjugated goat anti-human antibody or HRP-conjugated rabbit
anti-murine antibody were added, and the samples were incubated for 1 hour
at RT, Unbound goat and rabbit antibodies were washed using PBS/1 %
BSA. The assay was developed using TBM. Results were expressed as OD
405 absorbency values and are presented in Figures 2A-2C. Murine anti-
human PD-1 antibody J110 is commercially available (eBioscience, San
Diego, CA) and was included for comparison.
Examples: Selected PD-1 Antibodies Inhibit Binding of PD-L1 to PD-1
[0119] Inhibition assays were performed to assess the ability of the
antibodies to block binding of PD-L1 to PD-1. ELISA was performed as
described in Example 2 with modifications. After incubation with a primary,
anti-PD-1 antibody for 2 hours at RT, a fixed concentration (1 pg/ml) of
biotin-conjugated PD-LI-lg was added, and ttie samples were further
incubated for 1 hour at RT. After washing, saturating concentrations of
avidin-HRP were added, and incubated for 1 hour at RT. Unbound
avidin-HRP was washed using PBS/1% BSA. The assay was developed
using TMB.
[0120] Results were compared to those obtained with J110 as shown
in Figure 3. Anti-human PD-1 antibodies J110 and PD1-30 did not inhibit the
binding of PD-L1 to PD-1, Anti-human antibodies PD1-17, PD1-2a. PD1-33,
and PD1-35 and anti-mouse antibody PD1-F2 block PD-1/PD-L1 interaction.
Example 7: PD-1 Antibodies Recognize Distinct Sites on PD-1
[0121] Inhibition assays were perfomied to map sites recognize by
the various human anti-human PD-1 antibodies. ELISA was performed as
described in Example 6 with minor modifications. After incubation with
primary antibody for 2 hours at RT, a fixed concentration (0.25 pg/ml) of
biotin-conjugated antl-PD-1 antibody J110 was added, and the samples were
further Incubated for 1 hour at RT. After washing, saturating concentrations of
avidin-HRP were added, and Incubated for 1 hour at RT. Unbound
avidin-HRP was washed using PBS/1% BSA. The assay was developed
using TMB.
[0122] As shown in Figure 4, binding of anti-human PD-1 antibodies
(J110, J116, PD1-17, PD1-28, PD1-33, and PD1-35) defines at least two
distinct sites on PD-1. Cross-blocking results show that J110 and J116, bind
to identical or overlapping sites while PD1-17,28, 33, and 35 bind to another
distinct site. Binding of J116 or J110 to PD-1 blocks the binding of J110. In
contrast, binding of PD1-17, PD1-28, PD1-33, and PD1-35 do not block
binding of J110. This suggests that the tested anti-PD-1 antibodies bind to at
least two distinct epitopes: one recognized by J110 and J116, and the other
one recognized by PD1-17, PD1-28, PD1-33, and PD1-35,
Example 8: PD-1 Engagement Results in Decreased T Cell Responses
[0123] CD4+ T cells (5x10" cells/well) were stimulated with
tosyl-beads (Dynal, Great Neck, NY) coated with anti-hCD3 +/- PD-L1-Fc or
anti-PD-1 (PD1-17 or J110). Concentration of fusion protein or antibody titer
was as indicated in the X-axis of Figure 5. After 72 hours, proliferation was
determined by ^H-thymidine incorporation. Incorporated radioactivity was
determined using a LKB 1205 plate reader.
[0124] As shown In Figure 5, PD-1 engagement by antl-PD-1
antibody PD1-17 or PD-L1 .Fc caused a decrease in T cell proliferation. Thus,
PD1-17 can mimic PD-1 ligands and delivered an Inhibitory signal. As
discussed betow (Example 9), this inhibitory signal results in decreased T cell
proliferation and IL-2 production. Antibodies PD1- 28, PD1-33, and PD1-36
have the same effect as PD1-17. The effect is dose-dependent, as activation
of cells in the presence of Increasing concentrations of PD1-17 or PD-L1 .Fc
results in decreased T cell proliferation. The control antl-PD-1 antibodies,
J110 (Figure 5) or J116 (data not shown), do not inhibit T cell responses and
increasing the concentration of J110 has minimal effect on T cell proliferation.
For comparison, values are represented as percentage of the anti-CD3
response. "100%" represents CPMs obtained when cells were activated with
anti-CD3/murine IgG-coated microspheres. Altogether these results indicate
that some but not all antibodies that recognize PD-1 can act as agonists of the
PD-1 pathway.
[0125] Further experiments were performed to address whether PD-1
downregulation of T cell responses required coordinate TcR/PD-1
engagement on a single (CIS) or a separate (TRANS) cell surfaces. Two sets
of microspheres were prepared: one set contained anti-CD3 and PD-L1 .Fc
(CIS), the other set contained anti-CD3 or PD-LI.Fc (TRANS), Inhibition
through PD-1 was only observed under corKlitions where both PD-1 and TcR
were engaged by ligands on the same surface (CIS). At all beadrcell ratios
tested, no Inhibition was observed in conditions where TCR and PD-1 signals
were delivered on separate surfeces (TRANS).
[0126] To rule out steric hindrance in the TRANS experiments, similar
assays were set up using anti-CD3 antibody and B7.2.Fc. In these assays,
B7 costimulation of T cell responses was observed in both CIS and TRANS
conditions. Altogether, these findings demonstrate that PD-1 proximity to
TCR is required for the receptor modulatory function on T cell activation.
Therefore, to modulate a T cell response, both activating and inhibitory
signals must emanate from the same surface whether the surface is that of a
cell or a tjead.
Example 9: Blockage of PD-1 Engagement by Antibody Results in
Enhanced Proliferation
[0127] For assessing effect of soluble anti-PD-1 antibody on
proliferation, CD4+ T cells were pre-activated for 48 hours with
anti-CD3/anti-CD28-coated beads, harvested, and restimulated with the
indicated concentration of PHA plus 10 ng/ml IL-2 in the presence of PD1-17,
J110, or control IgG. Each of the antibodies was added at various
concentrations at initiation of the culture. Proliferation was measured at 72 hr.
[0128] The results demonstrate that PD1-17 (Figure 6) and PD1-35
(data not shown) enhanced proliferation of primary T cells. The control
antibody J110 did not enliance in vitro T cell responses. Selected anti-PD1
antibodies, as exemplified by PD1-17 and PD-35, Inhibit the interaction of
PD-1 with Its natural Uganda and thereby block delivery of a negative signal.
The blockade of the negative signal also results in enhanced proliferation and
IL-2 production.
Example 10: Treatment of Disorders
[0129] Modulation of Immune response regulated by PD-1 is useful in
instances vi/here an immunosuppressive effect or augmentation of immune
response is desired. This example descrit)es the use of PD-1 antibodies as
PD-1 agonists or antagonists to treat a subject at disease onset or having an
established Immune disorder or cancer, respectively.
[0130} Subjects at risk for or afflicted v^ith cancer may be in need of
immune response augmentation would benefit from treatment with a PD-1
antagonist, such as an anti-PD-1 antibody of the present invention in a soluble
form. Most commonly, antibodies are administered In an outpatient setting by
weekly administration at about 0.1-10 mg/kg dose by slow Intravenous (IV)
infusion. The appropriate therapeutically effective dose of an antagonist Is
selected by a treating clinician and would range approximately from 1 pg/kg to
20 mg/kg, from 1 pg/kg to 10 mg/kg, from 1 pg/kg to 1 mg/kg, from 10 pg/kg
to 1 mg/kg, from 10 pg/kg to 100 pg/kg, from 100 pg to 1 mg/kg, and from 500
pg/kg to 5 mg/kg.
[0131] The antibodies are also used to prevent and/or to reduce
severity and/or symptoms of diseases or conditions that involve an aberrant or
undesirable Immune response, such as In autoimmune disorders exemplified
below.
[0132] Multiple sclerosis (MS) Is a central nervous system disease
that is characterized by inflammation and loss of myelin sheaths. In the
experimental autoimmune encephalitis (EAE) mouse model for multiple
sclerosis (Tuohy et al. (J. Immunol. (1988) 141:1126-1130), Sobel et al. (J.
Immunol. (1984) 132:2393-2401), and Traugott (Cell Immunol. (1989) 119:
114-129), treatment of mice with a PD-1 agonist prior (and continuously) to
EAE induction is expected to prevent or delay the onset of MS.
[0133] Arthritis is a disease characterized by inflammation in the
joints. In the collagen induced arthritis (CJA) mouse model for riieumatoid
arthritis (Courtenay et al. (Nature (1980) 283:666-628) and Williams et al.
(Immunol. (1995) 84:433-439)). treatment with a PD-1 agonist is expected to
prevent or treat rheumatoid arthritis (RA) or other arthritic diseases.
[0134] Systemic Lupus Erythematosis (SUE) is an autoimmune
disease characterized by the presence of autoantibodies. The antibodies and
compositions of this invention can be used as PD-1 agonists to inhibit
activities of autoreactive T cells and B cells, and prevent or treat SLE or
related diseases in NZB X NZW mice (a mouse model for SLE) (Immunologic
Defects in Laboratory Animals, Gershwin et al. eds,, Plenum Press, 1981) or
in humans.
[0135] It is anticipated that PD-1 antibodies of the invention would be
administered as PD-1 agonists in ex vivo therapy with a frequency of one per
month or less. Treatment duration could range between one month and
several years.
[0136] To test the clinical efficacy of antibodies in humans, individuals
with melanoma, prostate cancer, RA, SLE, MS, type I diabetes, are identified
and randomized to a treatment group. Treatment groups include a placebo
group and one to three groups treated with a PD-1 agonist ^different doses).
Individuals are followed prospectively for one to three years. It is anticipated
that individuals receiwng treatment would exhibit an Improvement.
[0137] The specification is most thoroughly understood in light of the
teachings of the references cited within the specification, all of which are
hereby incorporated by reference in their entirety. The embodiments within
the specification provide an illustration of embodiments of the invention and
should not be construed to limit the scope of the invention. The skilled artisan
recognizes that many other embodiments are encompassed by the claimed
invention and that It is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the invention
being indicated by the following claims.
We Claim :
1. An antibody comprising the amino acid sequence as set out in SEQ ID NO : 25, SEQ
ID NO : 31, SEQID NO : 37, or SEQ ID NO : 52.
2. The antibody of claim 1, comprising an amino acid sequence selected from the group
consisting of SEQ ID
NO : 6, SEQ ID NO : 8, SEQ ID NO : 10, SEQ ID NO : 12, SEQ ID NO : 14,
SEQ ID NO ; 16, SEQ ID NO : 47, and SEQ ID NO ; 49.
3. The antibody of claim 1, wherein the antibody specifically binds to an amino acid
sequence that is at least 95% identical to any sequence of at least 100 of contiguous
amino acids of at least one sequence selected from group consisting of SEQ ID NO : 41
and SEQ ID NO : 56.
4. The antibody of claim I, wherein the antibody specifically binds to the extracellular
domain of PD-1 with an affinity constant greater than 10^ M''.
5. The antibody of claim 3, where the antibody inhibits the binding of PD-L to PD-1 with
an IC50 of less than 10 nM.
6. The antibody of claim 1, wherein the antibody is human.
7. The antibody of claim 1, wherein the antibody is IgGi or lgG4.
8. The antibody of claim 7, wherein the antibody is IgGixor IgGu
9. The antibody of claim 1, wherein the antibody is PDl-28, PDl- 33, PDl-35,or PDl-
F2.
10. A pharmaceutical composition comprising the antibody of claim 1.
11. The pharmaceutical composition as claimed in claim 10, wherein the composition is
useful for treatment or prevention of a disorder selected from the group consisting of an
autoimmune disorder, an immune response to a graft, an allergic reaction, and cancer.
i 2. The pharmaceutical composition as claimed in claim 11, wherein it is useful for said
treatment or prevention of a disorder in a human subject.
13. An antibody comprising human framework regions and means for specific binding
to PD-1, wherein the antibody is capable of blocking binding between PD-1 andPD-Ll and
wherein the means comprises a CDR derived from PDl-28, PDl-33. PDl-35, or PDl -F2,
14. An isolated nucleic acid encoding the antibody of claim 1.
15. An expression vector comprising the nucleic acid of claim 14.
16. A bacterial host cell such as E. Coli bacterium, comprising the vector of claim 15.
17. The nucleic acid of claim 14, wherein the nucleic acid encodes the amino acid
sequence set out in SEQID NO : 6, SEQ ID NO : 8, SEQ ID NO : 10, SEQ ID NO : 12,
SEQID NO : 14, SEQ ID NO : 16,SEQID NO : 47, or SEQ ID NO : 49.
18. The nucleic acid of claim 17, wherein the nucleic acid comprises a nucleotide sequence
selected from the group consisting of SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9,
SEQ ID NO :11, SEQ ID NO :13, SEQ ID NO :15, SEQ ID NO : 46, and SEQ ID NO : 48.
19. A method of making an antibody that specifically binds with PD-1, the method
comprises:

(a) providing a starting repertoire of nucleic acids encoding a variable domain that either
includes a CDR3 to be replaced or lacks a CDR3 encoding region;
(b) combining the repertoire with a donor nucleic acid encoding an amino acid sequence
substantially as set out in SEQ ID NO : 25, SEQ ID NO : 31, SEQ
ID NO : 37, or SEQ ID NO : 52, such that the donor nucleic acid is inserted into the
CDR3 region in the repertoire, so as to provide a product repertoire of nucleic acids
encoding a variable domain;
(c) expressing the nucleic acids of the product repertoire;
(d) selecting an antigen-binding fragment specific for PD-1 and
(e) recovering the specific antigen-binding fragment or nucleic acid encoding the
binding fragment.
20. An antibody produced by the method of claim 19.

The disclosure provides antibodies and antigen-binding fragments that can act as agonists
and/or antagonists of PD-1 (Programmed Death 1), thereby modulating immune
responses in general, and those mediated by TcR and CD28. in particular. The disclosed
compositions may be used for example, in treating autoimmune diseases, inflammatory'
disorders, allergies, transplant rejection, cancer, and other immune system disorders.