U.S. patent application number 13/255977 was filed with the patent office on 2012-03-15 for btla antibodies and uses thereof.
This patent application is currently assigned to UNIVERSITE DE LA MEDITERRANEE. Invention is credited to Daniel Olive.
Application Number | 20120064096 13/255977 |
Document ID | / |
Family ID | 40791726 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064096 |
Kind Code |
A1 |
Olive; Daniel |
March 15, 2012 |
BTLA Antibodies and Uses Thereof
Abstract
The invention relates to BTLA antibodies that block BTLA-HVEM
interaction and uses thereof.
Inventors: |
Olive; Daniel; (Marseille,
FR) |
Assignee: |
UNIVERSITE DE LA
MEDITERRANEE
Marseille
FR
|
Family ID: |
40791726 |
Appl. No.: |
13/255977 |
Filed: |
March 16, 2010 |
PCT Filed: |
March 16, 2010 |
PCT NO: |
PCT/EP2010/053356 |
371 Date: |
November 28, 2011 |
Current U.S.
Class: |
424/173.1 ;
435/343.2; 530/389.6 |
Current CPC
Class: |
C07K 16/2878 20130101;
Y02A 50/466 20180101; Y02A 50/41 20180101; A61P 35/00 20180101;
C07K 2317/73 20130101; Y02A 50/412 20180101; Y02A 50/489 20180101;
A61K 2039/505 20130101; Y02A 50/484 20180101; C07K 2317/74
20130101; Y02A 50/386 20180101; A61P 37/04 20180101; Y02A 50/407
20180101; Y02A 50/30 20180101; C07K 16/2827 20130101; C07K 16/2818
20130101; Y02A 50/388 20180101; A61P 31/00 20180101; C07K 2317/54
20130101; C07K 2317/55 20130101; C07K 2317/565 20130101; A61K
39/3955 20130101 |
Class at
Publication: |
424/173.1 ;
435/343.2; 530/389.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/04 20060101 A61P037/04; A61P 35/00 20060101
A61P035/00; A61P 31/00 20060101 A61P031/00; C12N 5/16 20060101
C12N005/16; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 2009 |
EP |
09305246.2 |
Claims
1. A method for treatment of a human or animal body, comprising the
administration of a BTLA antibody that blocks BTLA-HVEM interaction
to said human or animal body.
2. The method of claim 1 wherein said administration is for the
treatment of a cancer or a chronic infection.
3. A vaccine for the treatment of a cancer or a chronic infection
comprising a BTLA antibody that blocks BTLA-HVEM interaction.
4. A kit for the treatment of a cancer or a chronic infection
comprising: a) a BTLA antibody that blocks BTLA-HVEM interaction;
and b) a vaccine for the treatment of a cancer or a chronic
infection.
5. A method of claim 1, wherein said BTLA antibody that blocks
BTLA-HVEM interaction is a BTLA antibody which is obtainable from
the hybridoma accessible under CNCM deposit number I-4123 or a
derivative thereof.
6. A BTLA antibody which is obtainable from the hybridoma
accessible under CNCM deposit number I-4123.
7. A BTLA antibody that blocks BTLA-HVEM interaction which
comprises the CDRs of a BTLA antibody which is obtainable from the
hybridoma accessible under CNCM deposit number I-4123.
8. A hybridoma accessible under CNCM deposit number I-4123.
9. A method of claim 2 wherein said BTLA antibody that blocks
BTLA-HVEM interaction is a BTLA antibody which is obtainable from
the hybridoma accessible under CNCM deposit number I-4123 or a
derivative thereof.
10. A vaccine of claim 3 wherein said BTLA antibody that blocks
BTLA-HVEM interaction is a BTLA antibody which is obtainable from
the hybridoma accessible under CNCM deposit number I-4123 or a
derivative thereof.
11. A kit of claim 4 wherein said BTLA antibody that blocks
BTLA-HVEM interaction is a BTLA antibody which is obtainable from
the hybridoma accessible under CNCM deposit number I-4123 or a
derivative thereof.
Description
FIELD OF THE INVENTION
[0001] The invention relates to BTLA antibodies that blocks
BTLA-HVEM interaction and uses thereof.
BACKGROUND OF THE INVENTION
[0002] Co-receptor signalling is an important mechanism for
coordinating and tightly regulating immune responses. The usual
scheme of activation of .alpha..beta. T cells relies on positive
signals given by peptide antigens presented by HLA class I or II.
Co-receptor signals will either increase or prevent this
activation.
Among the negative signalling molecules, those belonging to CD28/B7
families are by far the most studied. Three members of this family
have been described: CTL-associated antigen-4 (CTLA-4), programmed
death-1 (PD-1) and B and T lymphocyte attenuator (BTLA). They all
play a role in the control of tolerance. They provide negative
signals that limit, terminate and/or attenuate immune
responses.
[0003] BTLA (CD272) is the most recently described member of the
CD28 family, it was first identified as a transcript highly
specific to T helper 1 (T.sub.H1) cells but was later shown to be
expressed by thymocytes. Initially, B7x was suggested as a ligand
of BTLA but it has been recently confirmed that BTLA interacts with
HVEM (herpes virus-entry mediator), a member of the tumour-necrosis
factor receptor (TNFR) family (Gonzalez et al., Proc. Natl. Acad.
Sci. USA, 2005, 102: 1116-21). The interaction of BTLA, which
belongs to the CD28 family of the immunoglobulin superfamily, and
HVEM, a costimulatory tumor-necrosis factor (TNF) receptor (TNFR),
is quite unique in that it defines a cross talk between these two
families of receptors.
[0004] BTLA is constitutively expressed in both B and T cells. Like
PD-1, BTLA contains a membrane proximal immunoreceptor
tyrosine-based inhibitory motif (ITIM) and membrane distal
immunoreceptor tyrosine-based switch motif (ITSM). Disruption of
either the ITIM or ITSM abrogated the ability of BTLA to recruit
either SHP1 or SHP2, suggesting that BTLA recruits SHP1 and SHP2 in
a manner distinct from PD-1 and both tyrosine motifs are required
to block T cell activation.
[0005] The BTLA cytoplasmic tail also contains a third conserved
tyrosine-containing motif within the cytoplasmic domain, similar in
sequence to a Grb-2 recruitment site (YXN). Gavrieli et al recently
showed that a phosphorylated peptide containing this BTLA
N-terminal tyrosine motif can interact with GRB2 and the p85
subunit of PI3K in vitro, although the functional effects of this
interaction remain unexplored in vivo (Gavrieli et al., Bioochem.
Biophysi Res Commun, 2003, 312, 1236-43).
[0006] To date, no satisfactory approach has been proven to induce
potent immune responses against vaccines, especially in cancer
patients. Methods have yet to be devised to overcome the
immunosuppressive mechanisms observed in cancer patients, and
during chronic infections.
SUMMARY OF THE INVENTION
[0007] The invention relates to a BTLA antibody that blocks
BTLA-HVEM interaction for the use in a method for treatment of a
human or animal body by therapy.
[0008] The invention relates to a BTLA antibody that blocks
BTLA-HVEM interaction for the treatment of a cancer or a chronic
infection.
[0009] The invention relates to a vaccine for the treatment of a
cancer or a chronic infection comprising a BTLA antibody that
blocks BTLA-HVEM interaction. The invention relates to a kit for
the treatment of a cancer or a chronic infection comprising:
[0010] a) a BTLA antibody that blocks BTLA-HVEM interaction;
and
[0011] b) a vaccine for the treatment of a cancer or a chronic
infection.
[0012] The present invention also relates to a BTLA antibody
(BTLA8.2) which is obtainable from the hybridoma accessible under
CNCM deposit number I-4123. The invention also relates to a BTLA
antibody which comprises the CDRs of BTLA8.2.
[0013] The invention relates to BTLA8.2 or a derivative thereof for
the use in a method for treatment of the human or animal body by
therapy.
[0014] The invention relates to BTLA8.2 or a derivative thereof for
the treatment of a cancer or a chronic infection.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0015] According to the present invention, "antibody" or
"immunoglobulin" have the same meaning, and will be used equally in
the present invention. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that immunospecifically binds an antigen. As such, the
term antibody encompasses not only whole antibody molecules, but
also antibody fragments or derivatives. Antibody fragments include
but are not limited to Fv, Fab, F(ab').sub.2, Fab', dsFv, scFv,
sc(Fv).sub.2 and diabodies.
[0016] In natural antibodies, two heavy chains are linked to each
other by disulfide bonds and each heavy chain is linked to a light
chain by a disulfide bond. There are two types of light chain,
lambda (.lamda.) and kappa (.kappa.). There are five main heavy
chain classes (or isotypes) which determine the functional activity
of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain
contains distinct sequence domains. The light chain includes two
domains, a variable domain (VL) and a constant domain (CL). The
heavy chain includes four domains, a variable domain (VH) and three
constant domains (CH1, CH2 and CH3, collectively referred to as
CH). The variable regions of both light (VL) and heavy (VH) chains
determine binding recognition and specificity to the antigen. The
constant region domains of the light (CL) and heavy (CH) chains
confer important biological properties such as antibody chain
association, secretion, trans-placental mobility, complement
binding, and binding to Fc receptors (FcR). The Fv fragment is the
N-terminal part of the Fab fragment of an immunoglobulin and
consists of the variable portions of one light chain and one heavy
chain. The specificity of the antibody resides in the structural
complementarity between the antibody combining site and the
antigenic determinant. Antibody combining sites are made up of
residues that are primarily from the hypervariable or
complementarity determining regions (CDRs). Occasionally, residues
from nonhypervariable or framework regions (FR) influence the
overall domain structure and hence the combining site.
Complementarity Determining Regions or CDRs refer to amino acid
sequences which together define the binding affinity and
specificity of the natural Fv region of a native immunoglobulin
binding site. The light and heavy chains of an immunoglobulin each
have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,
H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore,
includes six CDRs, comprising the CDR set from each of a heavy and
a light chain V region. Framework Regions (FRs) refer to amino acid
sequences interposed between CDRs.
[0017] The terms "chimeric antibody" refer to a genetically
engineered fusion of parts of an animal antibody, typically a mouse
antibody, with parts of a human antibody. Generally, chimeric
antibodies contain approximately 33% mouse protein and 67% human
protein. Developed to reduce the Human Anti-animal Antibodies
response elicited by animal antibodies, they combine the
specificity of the animal antibody with the efficient human immune
system interaction of a human antibody.
[0018] According to the invention, the term "humanized antibody"
refers to an antibody having variable region framework and constant
regions from a human antibody but retains the CDRs of the animal
antibody.
[0019] The term "Fab" denotes an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity, in
which about a half of the N-terminal side of H chain and the entire
L chain, among fragments obtained by treating IgG with a protease,
papaine, are bound together through a disulfide bond.
[0020] The term "F(ab').sub.2" refers to an antibody fragment
having a molecular weight of about 100,000 and antigen binding
activity, which is slightly larger than the Fab bound via a
disulfide bond of the hinge region, among fragments obtained by
treating IgG with a protease, pepsin.
[0021] The term "Fab' " refers to an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity,
which is obtained by proteolytic cleavage of an IgG with the
protease, papain.
[0022] A single chain Fv ("scFv") polypeptide is a covalently
linked VH::VL heterodimer which is usually expressed from a gene
fusion including VH and VL encoding genes linked by a
peptide-encoding linker. "dsFv" is a VH::VL heterodimer stabilised
by a disulfide bond. Divalent and multivalent antibody fragments
can form either spontaneously by association of monovalent scFvs,
or can be generated by coupling monovalent scFvs by a peptide
linker, such as divalent sc(Fv).sub.2.
[0023] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
[0024] By "purified" and "isolated" it is meant, when referring to
a polypeptide (i.e. an antibody according to the invention) or to a
nucleotide sequence, that the indicated molecule is present in the
substantial absence of other biological macromolecules of the same
type. The term "purified" as used herein preferably means at least
75% by weight, more preferably at least 85% by weight, more
preferably still at least 95% by weight, and most preferably at
least 98% by weight, of biological macromolecules of the same type
are present. An "isolated" nucleic acid molecule which encodes a
particular polypeptide refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not
encode the polypeptide; however, the molecule may include some
additional bases or moieties which do not deleteriously affect the
basic characteristics of the composition.
[0025] In the context of the invention, the term "treating" or
"treatment", as used herein, means reversing, alleviating,
inhibiting the progress of, or preventing the disorder or condition
to which such term applies, or one or more symptoms of such
disorder or condition. A "therapeutically effective amount" is
intended for a minimal amount of active agent which is necessary to
impart therapeutic benefit to a subject. For example, a
"therapeutically effective amount" to a mammal is such an amount
which induces, ameliorates or otherwise causes an improvement in
the pathological symptoms, disease progression or physiological
conditions associated with or resistance to succumbing to a
disorder.
[0026] As used herein, the term "prevention" refers to preventing
the disease or condition from occurring in a subject which has not
yet been diagnosed as having it.
[0027] As used herein, the term "subject" denotes a mammal, such as
a rodent, a feline, a canine, and a primate. Preferably a subject
according to the invention is a human.
[0028] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. The term is meant to include
all types of cancerous growths or oncogenic processes, metastatic
tissues or malignantly transformed cells, tissues, or organs,
irrespective of histopathologic type or stage of invasiveness. The
terms "cancer" or "neoplasms" include malignancies of the various
organ systems, such as affecting lung, breast, thyroid, lymphoid,
gastrointestinal, and genito-urinary tract, as well as
adenocarcinomas which include malignancies such as most colon
cancers, renal-cell carcinoma, prostate cancer and/or testicular
tumors, non-small cell carcinoma of the lung, cancer of the small
intestine and cancer of the esophagus.
[0029] The inventors have deposited a murine BTLA antibody
(BTLA8.2) producing hybridoma at the Collection Nationale de
Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du
Docteur Roux, 75724 Paris Cedex 15, France), in accordance with the
terms of Budapest Treaty, on Feb. 4, 2009. The deposited hybridoma
has CNCM deposit number I-4123.
[0030] "BTLA8.2" refers to an isolated BTLA antibody which is
obtainable from the hybridoma accessible under CNCM deposit number
I-4123
[0031] The expression "a derivative of BTLA8.2" refers to a BTLA
antibody which comprises the 6 CDRs of BTLA8.2 and its function
conservative fragments.
Antibodies of the Invention and Nucleic Acids Encoding Them
[0032] The inventors have demonstrated that a BTLA antibody that
blocks BTLA-HVEM interaction may be used to overcome the
immunosuppressive mechanisms mediated by HVEM observed in cancer
patients and during chronic infections.
[0033] The invention relates to a BTLA antibody that blocks
BTLA-HVEM interaction for the use in a method for treatment of the
human or animal body by therapy.
[0034] The present invention also relates to an isolated BTLA
antibody (BTLA8.2) which is obtainable from the hybridoma
accessible under CNCM deposit number I-4123.
[0035] The present invention relates to the hybridoma accessible
under CNCM deposit number I-4123.
[0036] The invention relates to an antibody which comprises the 6
CDRs of BTLA8.2.
[0037] In another embodiment, the invention relates to a derivative
of BTLA8.2 which comprises the VL chain and the VH chain of
BTLA8.2.
[0038] In another embodiment, the invention relates to a derivative
of BTLA8.2 which is a chimeric antibody, which comprises the
variable domains of BTLA8.2.
[0039] In an embodiment, an antibody of the invention is a
monoclonal antibody.
[0040] In an embodiment, an antibody of the invention is a chimeric
antibody.
[0041] In an embodiment, an antibody of the invention is a
humanized antibody.
[0042] A further embodiment of the invention relates to a nucleic
acid sequence encoding an antibody of the invention.
[0043] In a particular embodiment, the invention relates to a
nucleic acid sequence encoding the VH domain or the VL domain of an
antibody of the invention. Typically, said nucleic acid is a DNA or
RNA molecule, which may be included in any suitable vector, such as
a plasmid, cosmid, episome, artificial chromosome, phage or a viral
vector.
[0044] The terms "vector", "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g. a foreign
gene) can be introduced into a host cell, so as to transform the
host and promote expression (e.g. transcription and translation) of
the introduced sequence.
[0045] So, a further object of the invention relates to a vector
comprising a nucleic acid of the invention.
[0046] Such vectors may comprise regulatory elements, such as a
promoter, enhancer, terminator and the like, to cause or direct
expression of said antibody upon administration to a subject.
Examples of promoters and enhancers used in the expression vector
for animal cell include early promoter and enhancer of SV40, LTR
promoter and enhancer of Moloney mouse leukemia virus, promoter and
enhancer of immunoglobulin H chain and the like.
[0047] Any expression vector for animal cell can be used, so long
as a gene encoding the human antibody C region can be inserted and
expressed. Examples of suitable vectors include pAGE107, pAGE103,
pHSG274, pKCR, pSG1 beta d2-4- and the like.
[0048] Other examples of plasmids include replicating plasmids
comprising an origin of replication, or integrative plasmids, such
as for instance pUC, pcDNA, pBR, and the like.
[0049] Other examples of viral vector include adenoviral,
retroviral, herpes virus and AAV vectors. Such recombinant viruses
may be produced by techniques known in the art, such as by
transfecting packaging cells or by transient transfection with
helper plasmids or viruses. Typical examples of virus packaging
cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells,
etc. Detailed protocols for producing such replication-defective
recombinant viruses may be found for instance in WO 95/14785, WO
96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S.
Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO 94/19478.
[0050] A further object of the present invention relates to a cell
which has been transfected, infected or transformed by a nucleic
acid and/or a vector according to the invention. The term
"transformation" means the introduction of a "foreign" (i.e.
extrinsic or extracellular) gene, DNA or RNA sequence to a host
cell, so that the host cell will express the introduced gene or
sequence to produce a desired substance, typically a protein or
enzyme coded by the introduced gene or sequence. A host cell that
receives and expresses introduced DNA or RNA bas been
"transformed".
[0051] The nucleic acids of the invention may be used to produce an
antibody of the invention in a suitable expression system. The term
"expression system" means a host cell and compatible vector under
suitable conditions, e.g. for the expression of a protein coded for
by foreign DNA carried by the vector and introduced to the host
cell.
[0052] Common expression systems include E. coli host cells and
plasmid vectors, insect host cells and Baculovirus vectors, and
mammalian host cells and vectors. Other examples of host cells
include, without limitation, prokaryotic cells (such as bacteria)
and eukaryotic cells (such as yeast cells, mammalian cells, insect
cells, plant cells, etc.). Specific examples include E. coli,
Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g.,
Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as
primary or established mammalian cell cultures (e.g., produced from
lymphoblasts, fibroblasts, embryonic cells, epithelial cells,
nervous cells, adipocytes, etc.). Examples also include mouse
SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC
CRL1580), CHO cell in which a dihydrofolate reductase gene
(hereinafter referred to as "DHFR gene") is defective, rat
YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to
as "YB2/0 cell"), and the like.
[0053] The present invention also relates to a method of producing
a recombinant host cell expressing an antibody according to the
invention, said method comprising the steps of: (i) introducing in
vitro or ex vivo a recombinant nucleic acid or a vector as
described above into a competent host cell, (ii) culturing in vitro
or ex vivo the recombinant host cell obtained and (iii),
optionally, selecting the cells which express and/or secrete said
antibody. Such recombinant host cells can be used for the
production of antibodies of the invention.
Methods of Producing Antibodies of the Invention
[0054] Antibodies of the invention may be produced by any technique
known in the art, such as, without limitation, any chemical,
biological, genetic or enzymatic technique, either alone or in
combination.
[0055] Knowing the amino acid sequence of the desired sequence, one
skilled in the art can readily produce said antibodies, by standard
techniques for production of polypeptides. For instance, they can
be synthesized using well-known solid phase method, preferably
using a commercially available peptide synthesis apparatus (such as
that made by Applied Biosystems, Foster City, Calif.) and following
the manufacturer's instructions. Alternatively, antibodies of the
invention can be synthesized by recombinant DNA techniques
well-known in the art. For example, antibodies can be obtained as
DNA expression products after incorporation of DNA sequences
encoding the antibodies into expression vectors and introduction of
such vectors into suitable eukaryotic or prokaryotic hosts that
will express the desired antibodies, from which they can be later
isolated using well-known techniques.
[0056] In particular, the invention further relates to a method of
producing an antibody of the invention, which method comprises the
steps consisting of: (i) culturing a transformed host cell
according to the invention under conditions suitable to allow
expression of said antibody; and (ii) recovering the expressed
antibody.
[0057] In another particular embodiment, the method comprises the
steps of:
[0058] (i) culturing the hybridoma deposited as CNCM I-4123 under
conditions suitable to allow expression of the antibody; and
[0059] (ii) recovering the expressed antibody.
[0060] Antibodies of the invention are suitably separated from the
culture medium by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0061] In a particular embodiment, the human chimeric antibody of
the present invention can be produced by obtaining nucleic
sequences encoding VL and VH domains as previously described,
constructing a human chimeric antibody expression vector by
inserting them into an expression vector for animal cell having
genes encoding human antibody CH and human antibody CL, and
expressing the coding sequence by introducing the expression vector
into an animal cell.
[0062] As the CH domain of a human chimeric antibody, it may be any
region which belongs to human immunoglobulin, but those of IgG
class are suitable and any one of subclasses belonging to IgG
class, such as IgG1, IgG2, IgG3 and IgG4, can also be used. Also,
as the CL of a human chimeric antibody, it may be any region which
belongs to Ig, and those of kappa class or lambda class can be
used.
[0063] Methods for producing chimeric antibodies involve
conventional recombinant DNA and gene transfection techniques are
well known in the art (See patent documents U.S. Pat. No.
5,202,238; and U.S. Pat. No. 5,204,244).
[0064] The humanized antibody of the present invention may be
produced by obtaining nucleic acid sequences encoding CDR domains,
as previously described, constructing a humanized antibody
expression vector by inserting them into an expression vector for
animal cell having genes encoding (i) a heavy chain constant region
identical to that of a human antibody and (ii) a light chain
constant region identical to that of a human antibody, and
expressing the genes by introducing the expression vector into an
animal cell.
[0065] The humanized antibody expression vector may be either of a
type in which a gene encoding an antibody heavy chain and a gene
encoding an antibody light chain exists on separate vectors or of a
type in which both genes exist on the same vector (tandem type). In
respect of easiness of construction of a humanized antibody
expression vector, easiness of introduction into animal cells, and
balance between the expression levels of antibody H and L chains in
animal cells, humanized antibody expression vector of the tandem
type is preferred. Examples of tandem type humanized antibody
expression vector include pKANTEX93 (WO 97/10354), pEE18 and the
like.
[0066] Methods for producing humanized antibodies based on
conventional recombinant DNA and gene transfection techniques are
well known in the art. Antibodies can be humanized using a variety
of techniques known in the art including, for example, CDR-grafting
(EP 239,400; PCT publication W091/09967; U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596), and chain shuffling (U.S. Pat. No. 5,565,332). The
general recombinant DNA technology for preparation of such
antibodies is also known (see European Patent Application EP 125023
and International Patent Application WO 96/02576).
[0067] The Fab of the present invention can be obtained by treating
an antibody which specifically reacts with PD-1 with a protease,
papaine. Also, the Fab can be produced by inserting DNA encoding
Fab of the antibody into a vector for prokaryotic expression
system, or for eukaryotic expression system, and introducing the
vector into a procaryote or eucaryote (as appropriate) to express
the Fab.
[0068] The F(ab').sub.2 of the present invention can be obtained
treating an antibody which specifically reacts with BTLA8.2 with a
protease, pepsin. Also, the F(ab').sub.2 can be produced by binding
Fab' described below via a thioether bond or a disulfide bond.
[0069] The Fab' of the present invention can be obtained treating
F(ab').sub.2 which specifically reacts with human PD-1 with a
reducing agent, dithiothreitol. Also, the Fab' can be produced by
inserting DNA encoding Fab' fragment of the antibody into an
expression vector for prokaryote, or an expression vector for
eukaryote, and introducing the vector into a prokaryote or
eukaryote (as appropriate) to perform its expression.
[0070] The scFv of the present invention can be produced by
obtaining cDNA encoding the VH and VL domains as previously
described, constructing DNA encoding scFv, inserting the DNA into
an expression vector for prokaryote, or an expression vector for
eukaryote, and then introducing the expression vector into a
prokaryote or eukaryote (as appropriate) to express the scFv. To
generate a humanized scFv fragment, a well known technology called
CDR grafting may be used, which involves selecting the
complementary determining regions (CDRs) from a donor scFv
fragment, and grafting them onto a human scFv fragment framework of
known three dimensional structure (see, e. g., W098/45322; WO
87/02671; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,585,089; U.S.
Pat. No. 4,816,567; EP0173494).
[0071] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. It is known that when a humanized antibody is
produced by simply grafting only CDRs in VH and VL of an antibody
derived from a non-human animal in FRs of the VH and VL of a human
antibody, the antigen binding activity is reduced in comparison
with that of the original antibody derived from a non-human animal.
It is considered that several amino acid residues of the VH and VL
of the non-human antibody, not only in CDRs but also in FRs, are
directly or indirectly associated with the antigen binding
activity. Hence, substitution of these amino acid residues with
different amino acid residues derived from FRs of the VH and VL of
the human antibody would reduce of the binding activity. In order
to resolve the problem, in antibodies grafted with human CDR,
attempts have to be made to identify, among amino acid sequences of
the FR of the VH and VL of human antibodies, an amino acid residue
which is directly associated with binding to the antibody, or which
interacts with an amino acid residue of CDR, or which maintains the
three-dimensional structure of the antibody and which is directly
associated with binding to the antigen. The reduced antigen binding
activity could be increased by replacing the identified amino acids
with amino acid residues of the original antibody derived from a
non-human animal.
[0072] Modifications and changes may be made in the structure of
the antibodies of the present invention, and in the DNA sequences
encoding them, and still obtain a functional molecule that encodes
an antibody with desirable characteristics.
[0073] In making the changes in the amino sequences, the
hydropathic index of amino acids may be considered. The importance
of the hydropathic amino acid index in conferring interactive
biologic function on a protein is generally understood in the art.
It is accepted that the relative hydropathic character of the amino
acid contributes to the secondary structure of the resultant
protein, which in turn defines the interaction of the protein with
other molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like. As proposed by Kyte and
Doolittle, J Mol Biol 157:105-132, each amino acid has been
assigned a hydropathic index on the basis of their hydrophobicity
and charge characteristics; these are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0074] A further embodiment of the present invention also
encompasses function-conservative variants of the antibodies of the
present invention.
[0075] "Function-conservative variants" are those in which a given
amino acid residue in a protein or enzyme has been changed without
altering the overall conformation and function of the polypeptide,
including, but not limited to, replacement of an amino acid with
one having similar properties (such as, for example, polarity,
hydrogen bonding potential, acidic, basic, hydrophobic, aromatic,
and the like). Amino acids other than those indicated as conserved
may differ in a protein so that the percent protein or amino acid
sequence similarity between any two proteins of similar function
may vary and may be, for example, from 70% to 99% as determined
according to an alignment scheme such as by the Cluster Method,
wherein similarity is based on the MEGALIGN algorithm. A
"function-conservative variant" also includes a polypeptide which
has at least 60% amino acid identity as determined by BLAST or
FASTA algorithms, preferably at least 75%, more preferably at least
85%, still preferably at least 90%, and even more preferably at
least 95%, and which has the same or substantially similar
properties or functions as the native or parent protein to which it
is compared.
[0076] Two amino acid sequences are "substantially homologous" or
"substantially similar" when greater than 80%, preferably greater
than 85%, preferably greater than 90% of the amino acids are
identical, or greater than about 90%, preferably grater than 95%,
are similar (functionally identical) over the whole length of the
shorter sequence. Preferably, the similar or homologous sequences
are identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison, Wis.) pileup program, or any of sequence comparison
algorithms such as BLAST, FASTA, etc.
[0077] For example, certain amino acids may be substituted by other
amino acids in a protein structure without appreciable loss of
activity. Since the interactive capacity and nature of a protein
define the protein's biological functional activity, certain amino
acid substitutions can be made in a protein sequence, and, of
course, in its DNA encoding sequence, while nevertheless obtaining
a protein with like properties. It is thus contemplated that
various changes may be made in the antibodies sequences of the
invention, or corresponding DNA sequences which encode said
antibodies, without appreciable loss of their biological
activity.
[0078] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein.
[0079] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0080] Another type of amino acid modification of the antibody of
the invention may be useful for altering the original glycosylation
pattern of the antibody.
[0081] By "altering" is meant deleting one or more carbohydrate
moieties found in the antibody, and/or adding one or more
glycosylation sites that are not present in the antibody.
[0082] Glycosylation of antibodies is typically N-linked.
"N-linked" refers to the attachment of the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagines-X-threonine, where X is any
amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites).
[0083] Another type of covalent modification involves chemically or
enzymatically coupling glycosides to the antibody. These procedures
are advantageous in that they do not require production of the
antibody in a host cell that has glycosylation capabilities for
N-or O-linked glycosylation. Depending on the coupling mode used,
the sugar(s) may be attached to (a) arginine and histidine, (b)
free carboxyl groups, (c) free sulfhydryl groups such as thoseof
cysteine, (d) free hydroxyl groups such as those of serine,
threonine, orhydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. For example, such methods are described in
WO87/05330.
[0084] Removal of any carbohydrate moieties present on the antibody
may be accomplished chemically or enzymatically. Chemical
deglycosylation requires exposure of the antibody to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the antibody intact. Enzymatic cleavage of carbohydrate
moieties on antibodies can be achieved by the use of a variety of
endo-and exo-glycosidases.
[0085] Another type of covalent modification of the antibody
comprises linking the antibody to one of a variety of non
proteinaceous polymers, eg., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0086] It may be also desirable to modify the antibody of the
invention with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing inter-chain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and/or antibody-dependent cellular cytotoxicity (ADCC)
(Caron PC. et al. J Exp Med. 1992 Oct. 1;176(4):1191-5 and Shopes
B. J Immunol. 1992 May 1;148(9):2918-22).
Therapeutic Uses of the Antibodies of the Invention
[0087] The invention relates to a BTLA antibody that blocks
BTLA-HVEM interaction for the use in a method for treatment of the
human or animal body by therapy.
[0088] The invention relates to a BTLA antibody that blocks
BTLA-HVEM interaction for the treatment of a cancer or a chronic
infection.
[0089] The invention relates to a vaccine for the treatment of a
cancer or a chronic infection comprising a BTLA antibody that
blocks BTLA-HVEM interaction.
[0090] The invention relates to a kit for the treatment of a cancer
or a chronic infection comprising:
[0091] a) a BTLA antibody that blocks BTLA-HVEM interaction;
and
[0092] b) a vaccine for the treatment of a cancer or a chronic
infection.
[0093] The invention also relates to BTLA8.2 or a derivative
thereof for the use in a method for treatment of the human or
animal body by therapy. As said above, "a derivative of BTLA8.2"
refers to a BTLA antibody which comprises the 6 CDRs of BTLA8.2 and
its function conservative fragments.
[0094] The invention relates to BTLA8.2 or a derivative thereof for
the treatment of a cancer or a chronic infection.
[0095] The invention also relates to a method for treating a cancer
or a chronic infection wherein said method comprises the step of
administering to a subject in need thereof a therapeutically
effective amount of a BTLA antibody that blocks BTLA-HVEM
interaction (e.g., BTLA8.2 or a derivative thereof).
[0096] Examples of cancers include, but are not limited to,
hematological malignancies such as B-cell lymphoid neoplasm, T-cell
lymphoid neoplasm, non-Hodgkin lymphoma (NHL), B-NHL, T-NHL,
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasm and
myeloid cell lineage neoplasm. Examples of non-hematological
cancers include, but are not limited to, colon cancer, breast
cancer, lung cancer, brain cancer, prostate cancer, head and neck
cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone
cancer, cervical cancer, liver cancer, oral cancer, esophageal
cancer, thyroid cancer, kidney cancer, stomach cancer, testicular
cancer and skin cancer.
[0097] Examples of chronic infections include, but are not limited
to, viral, bacterial, parasitic or fungal infections such as
chronic hepatitis, lung infections, lower respiratory tract
infections, bronchitis, influenza, pneumoniae and sexually
transmitted diseases. Examples of viral infections include, but are
not limited to, hepatitis (HAV, HBV, HCV), herpes simplex (HSV),
herpes zoster, HPV, influenza (Flu), AIDS and AIDS related complex,
chickenpox (varicella), common cold, cytomegalovirus (CMV)
infection, smallpox (variola), colorado tick fever, dengue fever,
ebola hemorrhagic fever, foot and mouth disease, lassa fever,
measles, marburg hemorrhagic fever, infectious mononucleosis,
mumps, norovirus, poliomyelitis, progressive multifocal
leukencephalopathy (PML), rabies, rubella, SARS, viral
encephalitis, viral gastroenteritis, viral meningitis, viral
pneumonia, West Nile disease and yellow fever. Examples of
bacterial infections include, but are not limited to, pneumonia,
bacterial meningitis, cholera, diphtheria, tuberculosis, anthrax,
botulism, brucellosis, campylobacteriosis, typhus, gonorrhea,
listeriosis, lyme disease, rheumatic fever, pertussis (Whooping
Cough), plague, salmonellosis, scarlet fever, shigellosis,
syphilis, tetanus, trachoma, tularemia, typhoid fever, and urinary
tract infections. Examples of parasitic infections include include,
but are not limited to, malaria, leishmaniasis, trypanosomiasis,
chagas disease, cryptosporidiosis, fascioliasis, filariasis, amebic
infections, giardiasis, pinworm infection, schistosomiasis,
taeniasis, toxoplasmosis, trichinellosis, and trypanosomiasis.
Examples of fungal infections include, but are not limited to,
candidiasis, aspergillosis, coccidioidomycosis, cryptococcosis,
histoplasmosis and tinea pedis.
[0098] A BTLA antibody that blocks BTLA-HVEM interaction (e.g.,
BTLA8.2 or a derivative thereof) may be used as a vaccine adjuvant
for the treatment of a cancer or a chronic infection.
[0099] The invention relates to a vaccine for the treatment of a
cancer or a chronic infection comprising a BTLA antibody that
blocks BTLA-HVEM interaction (e.g., BTLA8.2 or a derivative
thereof).
[0100] The invention relates to a kit for the treatment of a cancer
or a chronic infection comprising:
[0101] a) a BTLA antibody that blocks BTLA-HVEM interaction (e.g.,
BTLA8.2 or a derivative thereof); and
[0102] b) a vaccine for the treatment of a cancer or a chronic
infection.
[0103] The two elements of the kit may be administered
concomitantly or sequentially over time.
[0104] Examples of vaccine for the treatment of a cancer or a
chronic infection are: include, but are not limited to vaccines
against viral, bacterial, parasitic or fungal infections such as
HIV and HBV and vaccines against viral associated cancers (for
instance HPV or HBV) or anti cancer vaccines for instance used to
treat patients with melanoma, leukemia, breast cancers, lung
cancers.
[0105] The invention also relates to pharmaceutical composition
comprising an antibody of the invention.
[0106] Therefore, an antibody of the invention may be combined with
pharmaceutically acceptable excipients, and optionally
sustained-release matrices, such as biodegradable polymers, to form
therapeutic compositions.
[0107] "Pharmaceutically" or "pharmaceutically acceptable" refers
to molecular entities and compositions that do not produce an
adverse, allergic or other untoward reaction when administered to a
mammal, especially a human, as appropriate. A pharmaceutically
acceptable carrier or excipient refers to a non-toxic solid,
semI-3889 solid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type.
[0108] The form of the pharmaceutical compositions, the route of
administration, the dosage and the regimen naturally depend upon
the condition to be treated, the severity of the illness, the age,
weight, and sex of the patient, etc.
[0109] The pharmaceutical compositions of the invention can be
formulated for a topical, oral, parenteral, intranasal,
intravenous, intramuscular, subcutaneous or intraocular
administration and the like.
[0110] Preferably, the pharmaceutical compositions contain vehicles
which are pharmaceutically acceptable for a formulation capable of
being injected. These may be in particular isotonic, sterile,
saline solutions (monosodium or disodium phosphate, sodium,
potassium, calcium or magnesium chloride and the like or mixtures
of such salts), or dry, especially freeze-dried compositions which
upon addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0111] The doses used for the administration can be adapted as a
function of various parameters, and in particular as a function of
the mode of administration used, of the relevant pathology, or
alternatively of the desired duration of treatment.
[0112] To prepare pharmaceutical compositions, an effective amount
of the antibody may be dissolved or dispersed in a pharmaceutically
acceptable carrier or aqueous medium. The pharmaceutical forms
suitable for injectable use include sterile aqueous solutions or
dispersions; formulations including sesame oil, peanut oil or
aqueous propylene glycol; and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi.
[0113] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0114] An antibody of the invention can be formulated into a
composition in a neutral or salt form. Pharmaceutically acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0115] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. 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 by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0116] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0117] The preparation of more, or highly concentrated solutions
for direct injection is also contemplated, where the use of DMSO as
solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small
tumor area.
[0118] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0119] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0120] The antibodies of the invention may be formulated within a
therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or
about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10
milligrams per dose or so. Multiple doses can also be
administered.
[0121] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g. tablets or
other solids for oral administration; time release capsules; and
any other form currently used.
[0122] In certain embodiments, the use of liposomes and/or
nanoparticles is contemplated for the introduction of antibodies
into host cells. The formation and use of liposomes and/or
nanoparticles are known to those of skill in the art.
[0123] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) are generally designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and such particles may be are easily made.
[0124] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs)). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core. The physical
characteristics of liposomes depend on pH, ionic strength and the
presence of divalent cations.
[0125] The invention will further be illustrated in view of the
following figures and example.
FIGURES
[0126] FIG. 1: BTLA is down regulated during the course of CD8 but
not CD4 T cell differenciation
[0127] CD8 and CD4 T cells were analysed by flow cytometry from
healthy volunteers. [0128] A--gating strategy for identification of
T cells subsets of differenciation. After exclusion of dead cells
(vivid+) and CD14+CD19+cells, T lymphocytes were gated into
CD3.sup.+CD4.sup.+ and CD3.sup.+CD8.sup.+ populations. Cells are
first analyzed for expression of CD45RA and CD27, and then for
expression of CCR7. Naives T lymphocytes (N)
CD45RA.sup.+CD27.sup.+CCR7.sup.+, Central Memory (CM)
CD45RA.sup.-CD27.sup.+CCR7.sup.+, Effector Memory (EM)
CD45RA.sup.-CD27.sup.+CCR7.sup.- and
CD45RA.sup.-CD27.sup.-CCR7.sup.-, Effector Memory RA.sup.+
CD45RA.sup.+CD27.sup.-CCR7.sup.-. [0129] B--BTLA and PD1 expression
on T-CD8 and T-CD4 subsets of differenciation healthy blood donors.
(n=12) The horizontal bar indicates the median and minimum and
maximum values are shown. Percentages of BTLA and PD1 positive
cells are shown within each subset. Each population is compared to
the previous subset of differenciation and the significant P values
are indicated as*: p>0,05, **: 0,001<p<0,01, .sup...*** or
p<0,001(Wilcoxon test). Statistics were calculated in
Prism5.
[0130] FIG. 2: Inhibition of T cells activation by BTLA and PD1
mAbs.
[0131] Purified T CD4 and T CD8 lymphocytes are stimulated by :
[0132] A--beads coated with CD3-CD28 and different combinations of
mAb directed against BTLA, PD1, PDL-1 +PDL-2, and HVEM.
Proliferation is measured by 3Hthymidine incorporation. IFNg and
TNFa release in supernatants are quantified by ELISA. [0133]
B--co-culture with immature allogenic DC in the presence of BTLA,
PD1, PDL1+PDL2, or HVEM mAb [0134] C--co-culture with mature
allogenic DC in the presence of BTLA, PD1, PDL1+PDL2, or HVEM
mAb.
[0135] For DC stimulation, proliferation is measured by the
division index on day 5 after a CFSE labelling into the naive and
the memory T CD4 or T CD8 cells sub-populations. The division index
is calculated with the proliferation tool of FlowJo 8-8-3. The p
value indicated are calculated between each condition and the
isotype control, except for the BTLA+PD1 condition which is always
compared to PD1 alone.
[0136] FIG. 3: BTLA and PD1 expression during CMV specific
activation
[0137] BTLA and PD1 expression were analyzed during CMV re-burst in
2 transplanted patients. The acute and controlled phases of
infection were defined according to CMV-PCR. Acute represents
pooled data from samples taken during the viremia (i.e week 3 to 6
for patient 1 and week 5 to 9 for patient 2), controlled
corresponds to samples taken when viremia was negative (up to week
145 for patient 1 and week 51 for patient 2). [0138] A--Gating
strategy for identification of CD8 subsets of differenciation. CD8
positive and CD8 CMV positive cells were gated from alive
lymphocytes. Population were then defined on CD45RA and CD27
expression, and further sub-divided on CD28 expression. Naive
cells: CD45RA.sup.+CD27.sup.+, CD28.sup.+, Central Memory (CM):
CD45RA.sup.-CD27.sup.+CD28.sup.+, Effector Memory CD27.sup.+:
CD45RA.sup.-CD27.sup.+, CD28.sup.-, Effector Memory CD27-:
CD45RA.sup.-CD27.sup.-CD28.sup.- and Effector memory RA.sup.+
(EMRA): CD45RA.sup.+CD27.sup.-CD28.sup.-. [0139] B--BTLA, PD1 and
HLA-DR expression in total CD8 and CMV tetramer positive CD8 cells
at week 5 after transplantation. [0140] C--BTLA, PD1 and HLA-DR
expression on CD8 and CMV tetramer positive CD8 cells subsets of
differenciation of 2 patients analyzed with 3 different tetramers
of CMV. Expression on total CD8 was measured before CMV re-burst,
acute and controlled phases were defined according to viremia.
Median, minimum and maximum values are shown. Each population is
compared to the previous subset of differenciation *: p>0,05,
**: 0,001<p<0,01, .sup...***: p<0,001(Wilcoxon test).
Statistics were calculated in Prism5.
[0141] FIG. 4: Inhibition of CMV specific activation by BTLA and
PD1 mAb
[0142] Mature or immature DC were stimulated by pp65 CMV peptide,
and co-cultivated with purified T lymphocytes in the presence of
BTLA or PD1 mAb. Activation of total cells, CD8 or CD8+CMV+ T
lymphocytes is analyzed by proliferation measured 7 days after a
CFSE labelling. The division index is calculated with the
proliferation tool of FlowJo.
[0143] FIG. 5: Co-expression of BTLA and PD1 in T CD4 and T CD8
lymphocytes of healthy blood donor.
[0144] FIG. 6: BTLA and PD1 expression after in vitro activation
with CD3/CD28 mAb
[0145] Expression is expressed as the median fluorescence intensity
(mfi) of each marker over the time. CD25 expression is measured as
a marker of activation.
[0146] FIG. 7: BTLA and PD1 expression in CD8+CMV+ T lymphocytes
differenciation subsets in patients.
[0147] Histograms show BTLA and PD1 expression in a patient in T
CD8 subsets of differenciation before CMV infection, and in
CD8+CMV-IE+ subsets of differenciation during acute infection (week
6).
EXAMPLE
Abstract
[0148] PD-1 and BTLA are receptors that negatively regulate T-cell
activation. We have investigated their respective expression on
human T cell subsets and their regulation following activation and
finally compared their role in the regulation of T cell functions
since there is no side by side comparison of their respective
distribution and function. BTLA is expressed on naive CD4 and CD8 T
cells and its expression was down-regulated on Effector-type and
Memory type CD8.sup.+ T cells. In contrast, PD-1 was preferentially
expressed by Effector and Memory type CD4.sup.+ and CD8.sup.+ T
cells rather than Naive T cells. Engagement of PD-1 and/or BTLA by
agonistic specific monoclonal antibodies blocked by the same
strength CD3/CD28-mediated T cell proliferation and Th1 and Th2
cytokine secretion. However, blockade of PD-1 and/or BTLA
engagement following allogenic stimulation of T cells by DCs,
revealed a robust inhibitory effect of PD-1 as compared to BTLA and
no additive effect was detected between the two molecules neither
in weak (iDCs) nor in strong (mDCs) allostimulation. Indeed, PD-1
blockade resulted in memory-type T cell expansion and a major
increase in cytokine secretion including IFN.gamma., TNF and IL-10.
During acute CMV infection, BTLA was overexpressed on CMV specific
T cells especially naive and effector type cells and on memory and
effector type cells after recovery from the CMV infection. We then
monitored the CD8 response to CMV pp65 peptides, our results also
demonstrated that PD-1 expression was up-regulated on CMV-specific
CD8.sup.+ T cells, while BTLA expression was in contrast
down-regulated and in vitro blockade of PD-1 or BTLA pathway by
anti-PD-1 or anti-BTLA antibodies was able to increase the
CMV-specific CD8.sup.+ T cell proliferation. Thus, our results
indicate that like PD-1, BTLA also provides a target for enhancing
the functional capacity of CTLs in viral infections. However, they
differ in their role in the regulation of allogeneic stimulation
and possibly transplantation.
Material and Methods
[0149] Generation of Anti-Human PD-L1, PD-L2, PD-1, BTLA and HVEM
mAbs and Fab Fragmentation
[0150] All mAbs were produced similarly. Female BALB/c mice were
immunised by IP injection with 10 .mu.g of human Ig fusion protein
with 250 .mu.l of Freund adjuvant. Immunisation was repeated three
times at 2 weeks intervals, the fourth immunisation was made by IV
injecting with 10 .mu.g of Ig fusion protein (100 .mu.l) in the
codal tail. Three days later spleen cells were fused with X63Ag8
myeloma cells with PEG 1500 (Roche) and cloned with HAT selection
(Sigma) and Hybridoma cloning factor (HCF from Origen). The
hybridoma supernatants were screened by cell surface staining human
PD1, PD-L1, PD-L2, BTLA or HVEM transfected COS cells line
respectively and for lack of reactivity with untransfected COS
cells. Anti-PD1 (PD1.3, IgG2b; PD1.6, IgG1), anti-PD-L1 (PD-L1.3,
IgG1), anti-PD-L2 (PDL2.1,IgG1), anti-BTLA (BTLA6.4, IgG1; BTLA7.1,
IgG2b; BTLA8.2, IgG1) and anti-HVEM (HVEM 8.5, IgG1) were selected
as reagents for FACS analysis and functional studies. Fab fragments
of blocking mAbs were generated and purified with the ImmunoPure
Fab Preparation Kit following the manufacturer's recommendations
(Pierce). Fragments were concentrated and washed into PBS on a
Centricon-10 (Millipore). Protein purity was assessed by
nonreducing SDS-PAGE, followed by detection with Coomassie blue.
Protein concentration was determined by measurement of the
absorbance at 280 nm.
Analysis by Flow Cytometry of PD-1 and BTLA Expression on T Cell
Subsets and Activated T Cells
[0151] Peripheral blood mononuclear cells (PBMC) were obtained from
healthy donors and isolated by fractionation over Lymphoprep
gradients (Abcys). To facilitate cell surface staining, mAbs to
PD-1 (PD-1.3.1) and BTLA (BTLA7.1) were stained with Alexa Fluor
647 using a commercially available kit (Invitrogen). Briefly, PBMC
were incubated with both the optimized dilution of the Alexa fluor
647-conjugated anti-PD-1 or anti-BTLA Abs and a corresponding
lineage-specific mAb: anti-CD3 PC7, anti-CD4-Pacific Blue,
anti-CD8-Alexa 700, anti-CD27-PE, and biotinylated anti-CD45RA (all
from Becton Dickinson). The use of CD27 and CD45RA as surface
markers allowed us to analyse PD-1 and BTLA expression on different
CD4.sup.+ and CD8.sup.+ subsets: (CD27.sup.+CD45RA.sup.+: Naive
(N), CD27.sup.+CD45RA and CD27.sup.-CD45RA.sup.- Effectors-type T
cells and CD27.sup.-CD45RA.sup.+: Effectors (E),.
[0152] Dead cells were eliminated using LIVE/DEAD Fixable Dead Cell
Stain Kit (Invitrogen). Cells were washed twice in cold PBS with 2%
FCS and 0.02% sodium azide, fixed in 4% paraformaldehyde and
analyzed on FACS Aria flow cytometer (BD Immunocytometry Systems).
To analyse PD-1 and BTLA co-expression, we used the same protocole
with Alexa fluor 647-conjugated anti-PD-1 and Alexa fluor
488-conjugated anti-BTLA in combination with a corresponding
lineage-specific mAbs.
[0153] Expression of PD-1 and BTLA on CMV-CD8.sup.+ T cells was
performed using phycoerythrin (PE) HLA-A*0201 CMV pp65
(NLVPMVATV)MHC tetramers (iTAg.TM., Beckman Coulter Immunotech)
[0154] For T cell expression kinetics of PD-1 and BTLA, CD4.sup.+
and CD8.sup.+ T cells were isolated from PBMC by T cell negative
isolation Kit (Miltenyi Biotec) and cultured in RPMI 1640
supplemented with 10% FCS (GIBCO). CD4.sup.+ or CD8.sup.+ T cells
(1,5.times.10.sup.5 cells/well) were activated in 96-well,
flat-bottom plates (Costar, Cambridge, Mass.) coated with 1
.mu.g/ml anti-CD3 (OKT3) and 2 .mu.g/ml of soluble anti-CD28
(CD28.2); cells were harvested each day for FACS analysis using
Alexa fluor 647-conjugated anti-PD-1 and anti-BTLA. CD25 expression
was used as activation control.
Artificial APC (aAPC) Confection and T Cell Assays
[0155] Human CD4.sup.+ and CD8.sup.+ T cells were purified by
negative selection from peripheral blood mononuclear cells using
magnetic beads (Miltenyi Biotec) and were routinely more than 97%
CD3.sup.+, more than 98% CD4.sup.+ for CD4.sup.+ T cell isolation
and more than 95% CD8.sup.+ for CD8.sup.+ T cell isolation as
determined by flow cytometry. T cells were stimulated with aAPC at
a ratio of 3:1 (cells to beads) comprised of magnetic beads
(Dynabeads M-450 Epoxy, Dynal Biotech) coated with the following
Abs: anti-CD3 (OKT3), anti-human CD28 (CD28.2), anti-human PD-1
(PD-1.6.4), anti-human BTLA (BTLA6.4) and anti-MHC class I (MHC I)
(YJ4). As previously described (28), these aAPCs were coated with
suboptimal anti-CD3 Ab (5%), suboptimal levels of anti-CD28 Ab
(10%), and either anti-MHC class I Ab (CD3/28/MHC I), anti-PD-1 Ab
(CD3/28/PD-1 +CMH I), anti-BTLA Ab (CD3/28/BTLA +CMH I) or
anti-PD-1 +anti-BTLA (CD3/28/PD-1 +BTLA), constituting the
remaining 85% of protein added to the bead. T cells
(1.5.times.10.sup.5 cells/well) were stimulated in round-bottom
96-well. Supernatants were harvested at 48 h to analyse cytokine
secretion by human Th1/Th2 cytokine Kit (BD.TM. Cytometric Bead
Array). Proliferation was measured at day 5 by tritiated thymidine
incorporation for the last 18 h.
Blockade of PD-1-PD-L and BTLA-HVEM Binding
[0156] Competitive binding experiments were performed to test the
binding blockade effect of our mAbs. COS 7 cells transfected to
express PD-L1 or PD-L2 were preincubated with anti-PD-L1
(PD-L1.3.1) or anti-PD-L2 (PD-L2) respectively, followed by the
addition of PD-1-Ig. Cells were washed twice, incubated with goat
anti-human-PE, washed again, fixed with 2% paraformaldehyde, and
analyzed on FACScan. Cells incubated with PD-1-Ig and mouse IgG, or
mouse IgG with no PD-1-Ig were used as positive and negative
controls, respectively. Similarly, PD-1 transfected COS 7 cells
were preincubated with anti-PD-1 (PD-1.3.1) or mouse IgG control,
followed by the addition of PD-L1-Ig or PD-L2-Ig. Anti-PD-1
blockade effect was then evaluated by flow cytometry using goat
anti-human-PE. Blockade of BTLA-HVEM interaction were determined by
two procedures: the first by using anti-BTLA (BTLA8.2) to block the
interaction of HVEM-Ig with BTLA transfectant cells, and the second
by using anti-HVEM (HVEM8.5) that blocks specifically BTLA-HVEM but
not LIGHT-HVEM interaction.
Preparation of Monocyte-Derived DCs
[0157] Peripheral blood mononuclear cells were obtained from
healthy donors and isolated by Lymphoprep (AbCys) density gradient
centrifugation. CD14.sup.+ monocytes were then immunomagnetically
purified with CD14 mAb-conjugated microbeads (Milteniy Biotec).
Purity of the CD14.sup.+ cells by flow cytometry analysis was
>98%. For generation of monocyte-derived DC (Mo-DC), CD14.sup.+
monocytes were cultured for 5 days in 6-well plates at
2.times.10.sup.6 cells/well (Falcon; BD Biosciences) in RPMI 1640
medium containing 10% FCS (GIBCO) and supplemented on days 0, 3 and
5 with 100 ng/ml GM-CSF (AbCys) and 20 ng/ml IL-4 (AbCys). Immature
DCs were harvested at day 5 and their maturation was accomplished
by coculturing them for 2 days with 50-Gy-irradiated
CD40L-transfected cells (2.105/well).
CFSE Labelling and Allogenic Stimulation of T Cells with
Monocyte-Derived DCs
[0158] T cells were isolated from peripheral blood with a Pan
T-negative isolation kit (Miltenyi Biotec) according to the
manufacturer's protocol. CD3.sup.+ T cells were routinely >97%
pure. T cells were labelled with 0.5 .mu.M CFSE (carboxyfluorescein
diacetate, succinimidyl ester) (Invitrogen) for 10 min at
37.degree. C., washed and cultured (2.10.sup.5/well) with immature,
or mature allogenic DCs (2.times.10.sup.4/well) in triplicate in
96-well flat-bottom plates (Falcon; BD Biosciences) in RPMI 1640
medium containing 10% FCS in the presence of blocking Fabs to PD-L1
(PD-L1.3.1), PD-L2 (PD-L2), PD-1 (PD-1.3.1), BTLA (BTLA8.2) and
HVEM (HVEM8.5) or isotype Fab controls. Cultures were incubated for
5 days and then proliferation of CFSE labelled CD4.sup.+ and
CD8.sup.+ T cells were measured by flow cytometry (FACS Canto,
Beckman Coulter). Proliferation also was measured on different T
cell subsets using CD27 and CD45RA as cell surface markers.
ELISA for Cytokine Analysis
[0159] To determine the production of cytokines, cell-free
supernatants were collected at 96 h and assayed for IL-2, IL-10,
IFN.gamma., and TNF by ELISA using OptEIA kits (BD Pharmingen)
according to the manufacturer's instructions.
Induction of Specific Anti-pp65 CD8.sup.+ T Cells
[0160] The iDCs and mature DCs were pulsed for 2 h at 37.degree. C.
in RPMI 1% FCS with a 10 .mu.g/ml CMV pp65 NLVPMVATV peptide. After
two washes, peptide-pulsed DCs (2.times.10.sup.4/well) were
cultured with autologous CFSE labelled- T cells
(2.times.10.sup.5/well) in the presence of anti-PD-1, anti-BTLA
blocking mAbs or isotype control. On day 7, T cells were harvested
and restimulated with 10 .mu.g/ml of CMV pp65 NLVPMVATV peptide for
6 hours. Proliferation was measured by CFSE dilution.
Statistical Analysis
[0161] All data were analysed using GraphPad Prism version 4.00 for
Windows, GraphPad Software. The Wilcoxon matched pairs test was
utilized to compare BTLA expression on matched CD4.sup.+ and
CD8.sup.+ T cells. Kruskal-Wallis ANOVA was used to examine the
variation of PD-1 and BTLA expression on CD4.sup.+ and CD8.sup.+ T
cell subsets. The Mann-Whitney U test was utilized to determine
significance of differences between anti-PD-1, anti-BTLA and
matched isotype controls effect. Differences were considered as
statistically significant when P<0.05.
Results
[0162] PD-1 and BTLA are differently expressed on CD4.sup.+ and
CD8.sup.+ T lymphocytes We first performed the side by side
investigation of the expression of PD-1 and BTLA on human T cell
subsets in the peripheral blood of healthy individuals using CD27
and CD45RA as cell surface markers (FIG. 1A).
[0163] We found a very low expression of PD-1 on both CD4.sup.+ and
CD8.sup.+ T cells, whereas, BTLA was readily expressed on T
lymphocytes at higher levels than PD-1. We observed mean
fluorescence intensity values of for PD-1 and for BTLA in normal
individuals. Our results demonstrated an identical expression of
PD-1 between CD4.sup.+ and CD8.sup.+ cells. In contrast, BTLA was
more expressed on CD4.sup.+ then CD8.sup.+T cells (p=0.0005) (FIG.
1B). Using CD27 and CD45RA markers, we have dissociated CD8.sup.+ T
cells in four subsets (Naives (N) CD27+/CD45RA+, Effector Memory
(EM) CD27-/CD45RA-, Central Memory (CM) CD27+/CD45RA- and Effector
(E) CD27-/CD45RA+). CD4 cells were defined has naive or memory
type. Comparing expression of PD-1 and BTLA, we found a very low
expression of PD-1 on Naive CD4.sup.+ T cells and a highly
significant up-regulation on Effector (p<0.001), and Effector
Memory (p<0.001) CD4.sup.+T cells. However, no change was
detected in the expression of BTLA on different CD4.sup.+ subsets.
On CD8.sup.+ T cells, in addition to significant up-regulation of
PD-1 on Effector and Effector Memory T cells (p<0.001), we found
also a significant up-regulation on Central Memory cells
(p<0.01). In contrast, BTLA demonstrated a significant
down-regulation on Effector (p<0.001), Effector memory
(p<0.01) and Central Memory CD8.sup.+ T cells (p<0.05) as
compared to Naive CD8.sup.+ T cells (FIG. 1B).
[0164] Finally we evaluated the co-expression of PD-1 and BTLA on
different T lymphocyte subsets. Except Naive T cells which express
only BTLA, it appears that PD-1 and BTLA expression are completely
dissociated on Effector CD8.sup.+ T cells. In contrast, high
co-expression was detected on Effector CD4.sup.+ T cells indicating
a strong inhibitory effect of these molecules on this small
sub-population.
[0165] Unlike BTLA which is constitutively expressed on CD4.sup.+
and CD8.sup.+ T cells, the expression of PD-1 is low on resting T
cells. PD-1 is up-regulated on activated CD4.sup.+ and CD8.sup.+ T
lymphocytes and the maximal expression is observed after 48 h.
[0166] Previous reports have shown that BTLA expression decreases
on CD4.sup.+ and CD8.sup.+ T cells stimulated with anti-CD3 and
anti-CD28 mAbs. In our experiments, purified CD4.sup.+ and
CD8.sup.+ T cells were stimulated with anti-CD3 and anti-CD28 and
harvested each day for FACS analysis. Unstimulated T cells
expressed low level of PD-1 whereas BTLA was 4 fold more expressed
than PD-1 on both CD4.sup.+ and CD8.sup.+ T cells. CD8.sup.+ T
cells showed late and very low increase in PD-1 expression. In
contrast, CD4.sup.+ T cells demonstrated early and a progressive
increase in the intensity of PD-1 expression (FIG. 5).
[0167] CD8.sup.+ T cells both demonstrated a progressive decrease
in the intensity of BTLA expression. The level of BTLA expression
diminished at 48 h in CD8.sup.+ T cells, and then there is a
progressive increase to reach the initial resting levels expression
at 96 h (FIG. 5).
PD-1 and BTLA are Potent Inhibitors of CD3/CD28-Mediated
Costimulation
[0168] Previous studies have described agonistic anti-human PD-1
and anti-human BTLA (Bennett etl al., J. Immunol., 2003, 170,
711-8; Chemnitz et al., J. Immunol., 2004, 173, 945-54). PD-1
engagement results in the inhibition of T cell expansion and
CD28-mediated up-regulation of IL-2, IL-10, IL-13, IFN.gamma.and
Bcl-x.sub.L transcripts in primary purified CD4.sup.+ T cells.
However, the relative efficiency of both systems and their
potential cooperation has not been investigated. To investigate
whether PD-1 and BTLA exert a functional cooperation on T cell
inhibition we used artificial antigen presenting cells (aAPC),
purified CD4.sup.+ or CD8.sup.+ T cells were stimulated with aAPC
(3 cells: 1 aAPC) coated with: anti-CD3 (OKT3) and anti-human CD28
(28.2) together with anti-human PD-1 (PD1.6.4), anti-human BTLA
(BTLA6.4) or both and anti-MHC class I (YJ4). For most experiments,
these aAPCs were coated with suboptimal anti-CD3 Ab (5%),
suboptimal levels of anti-CD28 Ab (10%) (Riley et al., Proc. Natl.
Acad. Sci. USA, 2002, 99, 11790-5). Stimulation of primary human
CD4.sup.+ or CD8.sup.+ T cells with anti-CD3-, anti-CD28-, and
anti-MHC class I (designated MHC I)-coated beads for 5 days leads
to robust T-cell proliferation and IL-2 production. In contrast,
PD-1 +MHC I and BTLA+MHC I aAPCs inhibited similarly and
significantly both CD4.sup.+ and CD8.sup.+ T cells
proliferation-CD3/CD28 induced. The inhibitory effect of PD-1 or
BTLA alone was very strong and no additive or synergistc effect was
detected between the two molecules in these conditions (FIG. 2A).
To confirm these results, we tried to evaluate the inhibitory
effect of PD-1 and/or BTLA using high CD3/CD8-mediated T cell
costimulation. In optimal condition of activation, no inhibition
was detected even with anti-PD-1 and anti-BTLA together (data not
shown) indicating that PD-1 and BTLA inhibit by the same strength
only sub-optimal CD3/CD28 mediated activation.
[0169] Both PD-1 and BTLA inhibited CD3 and CD28-mediated
up-regulation of IL-2, IFN.gamma., and TNF by CD4.sup.+ T cells
(FIG. 2A) and CD8.sup.+ T cells (FIG. 2A). IL-4 and IL-5 were also
strongly inhibited by PD-1 and BTLA engagement on both CD4.sup.+
and CD8.sup.+ T cells (data not shown).
[0170] Using sub-optimal conditions of T cell activation, PD-1 and
BTLA mAbs inhibited strongly T cell proliferation and Th1/Th2
cytokines secretion and no functional cooperation was detected
between the two molecules.
PD-1 is More Involved than BTLA in Regulating Allogenic Stimulation
of CD4.sup.+ and CD8.sup.+ T Cells by DC.
[0171] We next investigated the role of PD-1 and BTLA using
allogeneic stimulation, a condition where their ligands PD-L1,
PD-L2 and HVEM respectively, are expressed. We evaluated the
functions of PD-1, BTLA or PD-1 and BTLA using mAbs that block PD-1
PD-L interaction and BTLA-HVEM binding in T cells allogeneic
responses against dendritic cells of week (iDC) vs strong (mDC)
stimulatory capacity.
[0172] To analyse both CD4.sup.+ and CD8.sup.+ T cells,
CFSE-labelled T cells were cultured with allogenic iDCs or mDCs in
the presence of blocking Fab specific to PD-L1, PD-L2,PD-1 and BTLA
or matched isotype Fab controls. Five days later, cells were
harvested and labelled with CD27 and CD45RA to analyse different T
cell subsets proliferation by CFSE dilution. We first compared the
percentage of different subsets in the presence of blocking Fabs or
isotype Fab controls. Interestingly, using weak allostimulation
(iDCs), blockade of PD-1/PD-L interaction by anti-PD-L1 and
anti-PD-L2 together or anti-PD-1 led to highly significant
enrichment in Effector CD4.sup.+ but not CD8.sup.+ T cells.
Blockade of BTLA engagement by anti-BTLA also led to significant
increase in the percentage of Effector CD4.sup.+ T cells. No
significant additive effect was detected between PD-1 and BTLA
(FIG. 2B). Then, we compared the proliferation of allogenic T cells
againstiDCs. Anti-PD-1 Fab and nti-PD-L1+anti-PD-L2 together
resulted in 4-fold increase in CD8.sup.+ T cell proliferation (FIG.
2B) with 3 fold increase in Central (p=0.0068) and Effector memory
(p=0.017) CD8.sup.+ T cells (FIG. 2B). No additive effect was
detected with anti-PD-1 and anti-BTLA together. Anti-PD-1 blockade
as well as anti-PD-L1+anti-PD-L2 blockade resulted in similar
increase in Central Memory and Effector Memory CD8.sup.+ T cells
proliferation.
[0173] Similar results were found with CD4.sup.+ T cells. As shown
in FIG. 2C, mDCs stimulated a robust allogeneic response. Anti-PD-1
Fab, like anti-PD-L1 and anti-PD-L2 Fabs together resulted in 2
fold increase in CD4.sup.+ T cell proliferation with a strong
increase (5-fold) in CD4.sup.+ T cells with surprisingly a membrane
phenotype CD45RA+CD27+Naive phenotype as well as Memory T cells.
The effect of PD-1 blockade was less dramatic on CD8.sup.+ than
with iDCs (data not shown). The treatment of mDC with anti-BTLA
showed no significant increase of CD4.sup.+ or CD8.sup.+ T cell
proliferation compared with that of cultures treated with an
isotype control. Finally, combination of anti-PD-1 and anti-BTLA
showed no additive effect.
[0174] The effect of PD-1 and/or BTLA blockade on cytokine
secretion was examined. PD-1/PD-L interaction blockade using
anti-PD-1 or anti-PD-L1 and anti-PD-L2 Fabs together resulted in
highly significant increase in IFN.gamma. and TNF secretion
following iDCs stimulation and a lesser extent mDCs (FIG. 2B and
2C). Inhibition of BTLA/HVEM interaction using anti-BTLA Fab showed
a weak but significant increase in IFN.gamma. and TNF secretion
using iDCs and mDCs. Similar results were obtained using anti-HVEM
Fab that specifically blocks BTLA/HVEM but not LIGHT/HVEM
interaction confirming the previous observations with anti-BTLA
blockade (FIG. 2B and 2C).
[0175] In all experiments, blockade effect of PD-1/PD-L interaction
on T cell proliferation and cytokine secretion was similar between
anti-PD-1 and anti-PD-L1 and anti-PD-L2 Fabs together, indicating
that PD-L1 and PD-L2 could be the only two ligands for PD-1 on
DCs.
BTLA is Expressed on CD8 T Cells Specific of CMV and EBV Virus
During Acute Viral Infection
[0176] We have tested the expression of BTLA on CMV specific T
cells during reactivation of CMV during the course of kidney
transplantation (FIG. 3). BTLA is overexpressed in CMV specific T
cells during the acute phase of the infection in naive but also
effector type cells (EMRA). After recovery, BTLA expression
decreases and returns to baseline levels except on memory and EMRA
cells. As controls we used the activation antigen HLA-DR that was
also upregulated and indicated the activation status of the
anti-viral specific T cells. An additional marker was evaluated,
PD-1 that proved to be upregulated on activated antiviral specific
T cells on naive as well as memory T cells.
[0177] This observation indicates that BTLA is overexpressed on CMV
specific cells during acute infection and remains elevated on
memory cells after recovery.
PD-1 or BTLA Pathway Blockade Enhances CMV-Specific CD8.sup.+ T
Cell Proliferation
[0178] Recent evidence from lymphocytic choriomeningitis virus
(LCMV) infection in mice, HIV and HCV infection in humans indicates
a crucial role for PD-1 pathway in virus-specific CD8.sup.+ T cells
exhaustion and T cell dysfunction during chronic infection (Barber
et al., Nature, 2006, 439, 682-7; Petrovas et al., J. Exp. MEd.,
2006, 203, 2281-92). In our study, we first compared the expression
of PD-1 and BTLA in CMV-virus-specific CD8.sup.+ T cells. We found
an up-regulation of PD-1 expression on tetramer-positive
(tetramer.sup.+) CD8.sup.+ T cells specific for CMV as compared to
tetramer-negative (tetramer.sup.-) CD8.sup.+ T cells. BTLA in
contrast, was down-regulated on tetramer-positive CD8.sup.+ T cells
(FIG. 4). To address the potential role of PD-1 or BTLA pathways in
the activation of CMV specific CD8.sup.+ T cells we then compared
the effect of PD-1 or BTLA pathway on CMV-specific CD8.sup.+ T cell
proliferation using blocking anti-PD-1 or anti-BTLA in autologous
co-culture of T cells with DCs pulsed with pp65 peptide CMV. DCs of
weak (iDCs) vs strong (mDCs) stimulatory capacity were used and T
cell proliferation were measured by CFSE dilution. Unlike what we
observed in allogenic stimulation of T cells, Anti-PD-1 or
anti-BTLA blocking antibodies enhanced pp65-specific CD8.sup.+ T
cell proliferation to the similar extent suggesting a potential
role of these molecules in restoring CTLs functions (FIG. 4). The
enhancement was generally greatest with the weaker DC populations
(iDC) than with the mDC.
[0179] These data indicate that the cosignaling molecule BTLA is
expressed during infection and is involved in the inhibition of
anti viral T cell function.
REFERENCES
[0180] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
* * * * *