WO2014059251A1 - Enhancement of the immune response - Google Patents

Enhancement of the immune response Download PDF

Info

Publication number
WO2014059251A1
WO2014059251A1 PCT/US2013/064506 US2013064506W WO2014059251A1 WO 2014059251 A1 WO2014059251 A1 WO 2014059251A1 US 2013064506 W US2013064506 W US 2013064506W WO 2014059251 A1 WO2014059251 A1 WO 2014059251A1
Authority
WO
WIPO (PCT)
Prior art keywords
ceacaml
cells
lap
inhibitory agent
cell
Prior art date
Application number
PCT/US2013/064506
Other languages
French (fr)
Inventor
Richard S. Blumberg
Yu-Hwa HUANG
Original Assignee
The Brigham And Women's Hospital, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to KR1020157012338A priority Critical patent/KR102229873B1/en
Priority to JP2015536925A priority patent/JP6356134B2/en
Priority to BR112015008118A priority patent/BR112015008118A2/en
Priority to EP20150606.0A priority patent/EP3679950A1/en
Application filed by The Brigham And Women's Hospital, Inc. filed Critical The Brigham And Women's Hospital, Inc.
Priority to CN201380065223.XA priority patent/CN104853776B/en
Priority to CA2887528A priority patent/CA2887528C/en
Priority to AU2013329083A priority patent/AU2013329083C1/en
Priority to EP13845573.8A priority patent/EP2906241B1/en
Priority to US14/433,828 priority patent/US20150273056A1/en
Publication of WO2014059251A1 publication Critical patent/WO2014059251A1/en
Priority to IL23817115A priority patent/IL238171B/en
Priority to ZA2015/02603A priority patent/ZA201502603B/en
Priority to HK16101811.7A priority patent/HK1213788A1/en
Priority to HK16101826.0A priority patent/HK1213791A1/en
Priority to US16/165,113 priority patent/US20190201524A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the present invention relates to molecular immunology and cell biology. More specifically, the technology described herein relates to the modulation of T-cell tolerance.
  • the immune system exists to protect the body from foreign invaders and diseased cells.
  • a healthy immune system can recognize foreign or aberrant cells and target them for destruction.
  • components of one's immune system e.g. T cells
  • T cell tolerance a phenomenon referred to as "T cell tolerance.” This prevents the immune system from attacking the healthy cells of the host organism itself.
  • T cells that are recognizing and attacking foreign or diseased cells can be "turned off via T cell tolerance. This obviously reduces the host's ability to respond to and recover from the chronic disease.
  • compositions and methods for enhancing the immune response and/or reducing T cell tolerance in a subject are provided herein.
  • the inventors have discovered that certain subpopulations of T cells can coexpress two or more of CEACAMl, PDl, and/or LAP, which promote T-cell tolerance through separate signaling pathways.
  • the discovery that T cells can coexpress these molecules underlies the technology described herein, which relates to administering inhibitors of two or more of these molecules in order to achieve a greater degree of reduction in T cell tolerance than is possible by inhibiting only one pathway.
  • compositions and methods described herein increase the T-cell mediated immune response in a subject (i.e. reduce T-cell tolerance) by concurrently inhibiting the activity of the signaling pathways controlled by at least two of CEACAMl, PDl, and/or LAP. Relieving the suppression of the immune system is highly desirable in, for example, an immunocompromised subject or a subject suffering from cancer.
  • compositions for enhancing the immune response or decreasing T cell tolerance in a subject comprising at least two agents selected from the group consisting of: a CEACAMl inhibitory agent; a PDl inhibitory agent; and a LAP inhibitory agent.
  • the two agents can be a CEACAMl inhibitory agent and a PDl inhibitory agent.
  • the two agents can be a CEACAMl inhibitory agent and a LAP inhibitory agent.
  • the two agents can be a PDl inhibitory agent and a LAP inhibitory agent.
  • the composition can comprise a CEACAMl inhibitory agent, a PDl inhibitory agent, and a LAP inhibitory agent.
  • the composition can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a PDl inhibitory agent.
  • the composition can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a LAP inhibitory agent.
  • the composition can comprise a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PDl inhibitory agent.
  • one or more of the inhibitory agents can be an inhibitory nucleic acid.
  • one or more of the inhibitory agents can be an antibody reagent.
  • the antibody reagent can bind a target selected from the group consisting of: CEACAMl ; PDl ; and LAP. In some embodiments, the antibody reagent can inhibit signaling mediated by the target.
  • the CEACAMl inhibitory agent can bind a CEACAMl molecule having the sequence set forth in SEQ ID NO: 32 or an allelic or splice variant of SEQ ID NO: 32
  • the CEACAMl inhibitory agent can bind a CEACAMl ligand interaction site. In some embodiments, the CEACAMl inhibitory agent can bind to an amino acid residue corresponding to any of amino acid residues 1-429 of SEQ ID NO: 32. In some embodiments, the CEACAMl inhibitory agent can bind an extracellular domain of CEACAMl . In some embodiments, the CEACAMl inhibitory agent can bind the homophilic interaction domain of CEACAMl and inhibits homophilic interaction of CEACAMl molecules.
  • the PDl inhibitory agent can bind a PDl molecule having the sequence set forth in SEQ ID NO: l, or an allelic or splice variant of SEQ ID NO: 1. In some embodiments, the PDl inhibitory agent can bind a PDl ligand interaction site. In some embodiments, the PDl inhibitory agent can bind to an amino acid residue corresponding to any of amino acid residues 41-136 of SEQ ID NO: 1.
  • the LAP inhibitory agent can bind a LAP molecule having the sequence set forth in SEQ ID NO: 34 or an allelic or splice variant of SEQ ID NO: 34. In some embodiments, the LAP inhibitory agent can bind a LAP ligand interaction site. In some
  • the LAP inhibitory agent can bind to an amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 33.
  • described herein is a method for treating a chronic disease, the method comprising administering to a subject in need of treatment thereof an effective amount of a composition described herein.
  • the chronic disease can be selected from the group consisting of: cancer; a persistent infection; and a chronic viral infection.
  • Figure 1 depicts a schematic of the AOM-DSS model in WT mice.
  • Figure 2 depicts the results of FACS analysis examining expression of PDl and TIM-3 in CD4 and CD8 T-cells.
  • Figure 3 depicts the results of FACS analysis examining expression of PDl
  • Figure 4 depicts the results of FACS analysis examining expression of CEACAMl
  • Figures 5A-5B depict graphs demonstrating CEACAMl and PDl are co-expressed on the cell surface of chronically activated CD4 + (Figure 5A) and CD8 + ( Figure 5B) tumor infiltrating T lymphocytes.
  • Figures 6A-6C demonstrate that CEACAMl -4S is preferentially expressed on specific subsets of both circulating and intestinal CD4+ T cells.
  • Figure 6A depicts graphs of FACS analysis demonstrating that expression of CEACAMl is associated with the enrichment of specific cell surface molecules. Percentages on histogram gates indicate the frequency of cells positive for the given marker in the CD4+CEACAM 1 - and CD4+CEACAM 1 + cell populations, respectively, of MLN
  • FIG. 6B depicts graphs of FACS analysis demonstrating that CEACAM1 expression co-segregates with latency associated peptide (LAP) expression and is unrelated to Foxp3 expression in CD4+ T cells from the MLN of naive WT mice.
  • Figure 6C depicts a chart demonstrating that the CEACAM1-S isoform is enriched on CD4+CEAC AM 1 + T cells which express LAP. T cells were isolated from pooled spleen and lymph node (LN) in Figure 6C.
  • FIGS 7A-7F demonstrate that CEACAM1-S directly enhances the generation of
  • FIGS 7A-7D depict graphs of FACS analysis results demonstrating that the percentage of CD4+LAP+ cells is increased in MLN of na ' ive CEACAM1-4S transgenic (4S Tg) mice over- expressing this isoform specifically in T cells (Figure 7A) but not CEACAM1-4L (4L Tg) mice ( Figure 7B).
  • CEACAMl ' ' ' -4S Tg mice ( Figure 7C) but not CEACAMl ' -4L Tg mice ( Figure 7D) are enriched in CD4+LAP+ T cells compared to their littermate controls.
  • Figure 7E depicts graphs of FACS analysis results demonstrating that overexpression of CEACAM1-4S enhances the regulatory function of CD4+LAP+ Treg cells in vitro as shown by the decrease in proliferation of responder T cells when co-cultured with CD4+LAP+ Treg cells from pooled spleen and LN of 4S Tg mice.
  • Figure 7F depicts a chart demonstrating that oral feeding of anti-CD3 antibody significantly increases the percentage of CD4+LAP+ Treg cells in the MLN of WT but not CEACAMl '1' mice. Mice were fed 5 ⁇ g anti-CD3 (or isotype control) for each of 5 consecutive days and CD4+LAP+ T cell frequency was assessed after 7 days.
  • Figure 8 depicts the results of a novel CEACAMl isoform-discriminating qPCR assay which depicts the specificity and efficiency of qPCR primers for mouse CEACAMl -L and -S isoforms amplification verified using mCEACAMl -4L and 4S plasmids. This novel assay was used in Figure 6C.
  • FIGS. 9A-9B demonstrate that CEACAMl -S expression on CD4+ T cells is associated with the expression of molecules which characterize specific subsets of T cells in the
  • Figures 9A and 9B depict graphs of FACS analysis characterizing cell surface molecule expression on CD4+CEACAM1+ and CD4+CEACAM1- cells in
  • Percentages on histogram gates indicate the frequency of cells positive for the given marker in the CD4+CEAC AM 1 - and CD4+CEAC AM 1 + cell populations, respectively.
  • Figure 10 depicts the isoforms of CEACAMl created by alternate splicing.
  • Figure 11 depicts a graph of CEACAMl -S and CEACAMl -L expression using the qPCR assay in Figure 8.
  • Total T cells from spleen were stimulated with plate -bound coated anti-CD3 or anti-CD3 plus CD28 for 2 and subsequently 4 days.
  • CEACAMl -S, -L and total CEACAMl were assessed by qPCR.
  • Figure 11 shows that both of the major CEACAMl isoforms (S and L) which are low in resting T cells are transcriptionally regulated by ligation of T cells through the TCR/CD3 complex (signal 1) but negatively regulated when the TCR/CD3 complex is co-engaged by the classical co-stimulatory signal (signal 2) provided by CD28.
  • Figures 12A-12E demonstrate that CEACAM1-4S is associated with activation of primary T cells and sensitization to activation induced cell death.
  • Figures 12B-12C depict graphs of the results of experiments in which CD3 + T cells isolated from spleen of 4S Tg mice and their WT littermates were cultured with anti-CD3 antibody at indicated concentrations, or 1 ⁇ g/ml anti-CD3 over the indicated time course.
  • FIG. 12B Proliferation assays were performed by [ 3 H] -thymidine incorporation ( Figure 12B) and apoptosis was determined using annexin-V and 7-AAD staining ( Figure 12C).
  • Figure 12D depicts an immunoblot.
  • CD3 + T cells were isolated from the spleen of 4S Tg or WT mice, and cultured with IL-2 (100 U/ml) or anti-CD3 (1 ⁇ ) coated plates. Cell lysates were prepared at the indicated times, and caspase-3 and its activated form were determined using immunoblot.
  • FIGS 13A-13C demonstrate that CEACAM1-S directly enhances the generation of
  • FIGS 13A and 13C depict graphs of the percentage of CD4 + PD1 + CXCR5 T FH cells in the spleen, MLN and PP is increased in 4S Tg mice ( Figure 13A) and decreased in Ceacaml ' ' mice ( Figure 13C) when compared to their littermate controls.
  • Figure 14 demonstrates co-expression of CEACAM1 and PD1 on T cells under physiologic conditions.
  • Lamina limbal lymphocytes were isolated from colon, ileum, jejunum and duodenum as well as mesenteric lymph nodes (MLN) and spleen and after gating on CD3 -positive cells stained for co-expression of PD1 and CEACAMl .
  • the top panel depicts a graph of the summary of quantity of positive cells and the bottom panel depicts the histograms of the flow cytometry.
  • Figure 15 demonstrates co-expression of CEACAMl and PD1 in lamina intestinal lymphocytes infiltrating the colon of mice with colitis induced by adoptive transfer of naive CD4+ T cells in Rag-deficient and CEACAMl -deficient animals. Co-expression of CEACAMl and PD1 is shown after gating on CD3+ lymphocytes obtained from proximal, middle and distal colon.
  • FIG 16 demonstrates that blockade of CEACAMl and PD1 increases TNF-a production from tumor infiltrating lymphocytes (TIL).
  • TILs were isolated from subcutaneous tumors associated with CT26 colorectal carcinoma cell line. TILs were stimulated with anti-CD3 and anti- CD28 monoclonal antibodies in the presence of isotype control antibodies or antibodies that block different co-inhibitory cell surface molecules. Note the synergistic increase of TNF-a production by TILs treated with anti-CEAC AMI and anti-PDl antibodies.
  • T-cell tolerance functions, in part, to provide a population of immune system cells that recognize self-major histocompatibility complex (MHC) molecules but do not recognize self-peptides.
  • MHC self-major histocompatibility complex
  • T cell tolerance occurs when the subject is still in need of a robust immune response.
  • CEACAMl carcinoembryonic antigen related cell adhesion molecule 1
  • PDl programmed cell death 1
  • LAP TGF i latency associated peptide
  • CEACAMl, PDl and LAP have been shown to play in T cell immune regulation, it had not been established or suggested that there exists a population of T cells which expresses detectable levels of two or more of CEACAMl, PDl, and/or LAP prior to the discoveries described herein.
  • T cells co- express detectable levels of (i) CEACAMl and LAP, (ii) CEACAMl and PDl, (iii) PDl and LAP, or (iv) CEACAMl, PDl, and LAP.
  • the discovery of such cells underlies the concept described herein of reducing T cell tolerance by inhibiting at least two of these T cell tolerance-promoting molecules.
  • the ability to target multiple pathways which contribute to T cell tolerance can provide a greater effect upon T cell tolerance, e.g. by synergistic effects, leading to a therapeutic decrease in T cell tolerance. Inhibition of two or more of CEACAMl, PDl, and/or LAP can therefore enhance the responsiveness of the immune system.
  • CEACAMl also known as CD66a or biliary glycoprotein refers to a polypeptide having a single Ig variable domain-like amino terminus, from one to three Ig constant domain-like regions, and a single membrane-spanning segment followed by either a short
  • CEACAMl -S or long (CEACAMl -L) cytoplasmic domain
  • the N-terminal domain has been shown to facilitate homophilic intercellular binding that influences a broad spectrum of cellular processes related to cellular activation and/or cell cycle progression; and is also targeted by the heterophilic adhesins of viral (murine hepatitis virus ) and bacterial ⁇ Neisseria gonorrhoeae and N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae) pathogens, allowing their infection of the diverse array of CEACAMl -expressing human cells and tissues in vivo.
  • CEACAMl When expressed, CEACAMl is characterized by significant alternate RNA splicing leading to eleven isoforms in humans and at least four isoforms in mice. These isoforms differ in the length of the cytoplasmic tail and the number of extracellular Ig-like domains and are named accordingly. As noted, the majority of CEACAMl isoforms possess either a long (CEACAMl -L) CT or a short (CEACAMl -S) cytoplasmic tails (Azuz-Lieberman et al., 17 Intl. Immunol. 837 (2005); Gray-Owen & Blumberg, 2006; Moller et al., 65 Int. J.
  • ITIMs immune -receptor tyrosine-based inhibitory motifs
  • SHP-1 Src homology 2 domain phosphatase 1
  • CEACAMl recruits SHP-1 to the T-cell receptor (TCR) signalsome where SHP-1 blocks ZAP-70 activation via dephosphorylation of CD3 ⁇ , ZAP-70, or both. Indeed, masking the homophilic binding site with a specific Fab causes increased cytotoxicity and lymphocyte degranulation (Chen et al., 180 J. Immunol. 6085 (2008)).
  • TCR T-cell receptor
  • CEACAMl -S isoforms are inhibitory by inducing weak co-stimulatory signals that sensitize cells to activation induced cell death and the emergence of populations of regulatory cells such as CD4+LAP+ T cells (Chen et al., 172 J.
  • CEACAMl functions as a regulatory co-receptor for both lymphoid and myeloid cell types, and is constitutively expressed in a wide range of tissues and cell types. Its expression on natural killer (NK) cells and T-cells is, however, mainly induced by cytokines and membrane-activating receptor activation. As described herein, previous studies have determined that CEACAMl is a ligand for itself (homophilic ligation), and is involved in heterophilic ligation with galectin 3 and selectins.
  • CEACAMl expression which is low on resting T-cells, is transcriptionally regulated by ligation of T-cells through the TCR/CD3 complex (signal 1) but negatively regulated when the TCR/CD3 complex is co-engaged by the classical co-stimulatory signal (signal 2) provided by CD28 and induces both major classes of CEACAMl isoforms (CEACAMl short and CEACAM-long) (see, e.g. US Provisional Application 61/677,596; which is incorporated by reference herein).
  • TCR/CD3 ligation alone in the absence of costimulation
  • CEACAMl the strong induction of CEACAMl by such stimulation may, without wishing to be bound or limited by theory, be part of the tolerogenic program.
  • CEACAMl -4L and CEACAMl -4S which differ only in their cytoplasmic domains, have extracellular domains (ectodomains) comprised of four glycosylated Ig domains.
  • CEACAMl -induced cell signaling is regulated by its intercellular homophilic binding at the cell surface, which is mediated by the N-terminal Ig domain (Dl) in a reciprocal Dl-Dl interaction.
  • the basic structure of the IgV N-terminal domain of CEACAMl is a tertiary fold of a stacked pair of ⁇ -pleated sheets. There are nine component ⁇ strands, with strands A, B, E, and D lying in one sheet and strands C, C, C", F, and
  • the GFCC face of the N-terminal domain of CEACAMl is known to be crucial for mediating homophilic adhesion. Homophilic and heterophilic interactions have been observed for other adhesion receptor-ligand pairs; such as of CD2 with CD58; ICAM-1 with ICAM-1, LFA-1, rhinoviruses or Plasmodium falciparum-mfectcd erythrocytes; or cadherins with cadherins (Watt et al., 98 Blood 1469 (2001)).
  • a CEACAM1 inhibitory reagent can inhibit one or more isoforms of CEACAM1 which can promote T-cell tolerance.
  • a CEACAM1 inhibitory reagent can inhibit one or more isoforms of CEACAM1 which can promote T-cell tolerance and not other isoforms of CEACAM1.
  • Isoforms of CEACAM1 which can promote T-cell tolerance include, but are not limited to the isoforms depicted in Figure 11, e.g.
  • a peptide region responsible for CEACAM1 heterophilic interactions is also on the GFCC'C" face and overlaps partially with the homophilic binding site.
  • binding of a murine CEACAM1 to murine coronavirus requires a uniquely folded CC loop, in which amino acids 34 to 52 play a crucial role (Tan et al., 21 EMBO J. 2076 (2002); Watt et al., 2001).
  • amino acids between residues 27 to 42 (particularly D27L28F29) and S32, Y34, V39, Q44, Q89, and 191 on the GFCC'C" face form differential adhesiotopes for the binding of H. influenzae, and the
  • N. gonorhheae and N. meningiditis Opa proteins are likely a groove; formed by homophilic cis binding that involves V39 and D40 CC loop residues, or formed after disruption of CEACAM1 cis dimerization by cytokine (e.g., TNFa) activation that precedes CEACAM1 /pathogen binding (Watt et al., 2001).
  • cytokine e.g., TNFa
  • CEACAM1 has also been implicated in coronavirus and
  • Immobilized CEACAM1 in which the tertiary structure of this highly flexible molecule is restrained, also exhibits decreased adhesion, further implicating the cytoplasmic regions of the molecule (e.g., intracellular dimerization) in both homophilic and heterophilic interactions and signaling. Further, the formation of homodimers in cis has been characterized for multiple splice variants (isoforms), even those lacking IgC2 domains. [0043] The lack of intradomain disulfide bridges in the N-terminal Dl domain renders the
  • CEACAMl exists in the cell membrane in microclusters; whereas homophilic binding triggers reorganization that results in two different kinds of dimers, as well as trimers and higher-order oligomers.
  • C-dimers antiparallel trans-di srs
  • A-dimers parallel cz -dimers
  • the N- terminal Dl domain participates in both C- and A-dimerization, while the D2-D4 domains are involved only in A-dimerization.
  • Divalent cations decreased ectodomain flexibility and enhanced formation of multimeric complexes, which are further implicated in CEACAMl -mediated cell adhesion.
  • the dimerization of the ectodomains is transduced by the transmembrane domains to the cytoplasmic domains, thus, in turn, directing intracellular signaling.
  • Another binding site might be implicated across the ABED face, depending on the level and flexibility of
  • CEACAMl function is also likely impacted by glycosylation.
  • Carbohydrates account for up to sixty percent of the weight of CEACAMl expressed on the cell surface. The role of this enormous carbohydrate content allows for the construction of "subspecies" of CEACAMl isoforms.
  • the ability of CEACAMl to express sialyl Lewis x modifications can affect leukocyte homing (Chen et al., 86 J. Leuk. Biol. 195 (2009); which is incorporated by reference herein in its entirety).
  • a CEACAMl inhibitory agent can bind a CEACAMl molecule having the sequence set forth in SEQ ID NO: 32 or an allelic or splice variant of SEQ ID NO: 32.
  • Allelic and splice variants of SEQ ID NO: 32 can include those variants described in, e.g. Gaur et al. Molecular Cancer 2008 7:46 Barnett et al. Mol Cell Biol 1993, 13: 1273-1282; and Barnett et al. J
  • a CEACAMl inhibitory agent can bind one or more of the extracellular domains of CEACAMl, e.g. any of amino acids 1-429 of SEQ ID NO: 32.
  • a CEACAMl inhibitory agent can bind the Ig V-set domain of CEACAMl, e.g. any of amino acids 35-140 of SEQ ID NO: 32.
  • a CEACAMl inhibitory agent can bind an Ig C-set domain of CEACAMl, e.g. any of amino acids 140-415 of SEQ ID NO: 32.
  • the extracellular domain structure of CEACAMl variants is further described, for example, in Watt et al.
  • a CEACAMl inhibitory agent can bind a CEACAMl ligand interaction domain.
  • a CEACAMl ligand interaction domain can comprise extracellular residues of a CEACAMl polypeptide.
  • the homophilic ligand interaction domain of CEACAMl can comprise amino acids 73, 74, 98, and 116 of SEQ ID
  • CEACAMl can comprise amino acids 61
  • a CEACAMl inhibitor agent can bind to (or physically interact with) one or more of these homophilic ligand interaction amino acids, e.g. amino acids 73, 74, 98, and 116 of SEQ ID NO: 32.
  • a CEACAMl inhibitory agent can bind to (or physically interact with) one or more of these heterophilic ligand interaction amino acids, e.g. amino acids 61, 62, 63, 66, 68,73, 78, 123, and 125 of SEQ ID NO: 32.
  • physical interaction can encompass steric hindrance of the interaction of one or more of these sites with a ligand involved in tolerogenesis.
  • the CEACAMl inhibitory agent can bind the homophilic interaction domain of CEACAMl and inhibit homophilic interaction of CEACAMl molecules.
  • a CEACAMl inhibitory agent can reduce homophilic binding of CEACAMl .
  • Trans -homophilic CEACAMl binding induces d -dimerization by an allosteric mechanism transmitted by the N terminal immunoglobulin- like domain.
  • CEACAMl -L homodimer formation is reduced by coexpression of CEACAMl -S and modulated by antibody ligation.
  • Transmembrane signaling by CEACAMl can operate by alteration of the monomer/dimer equilibrium. Miiller et al., 187 J. Cell Biol.
  • CEACAMl signaling see, e.g. Markel. EJI 2004 34:2138-2148; which is incorporated by reference herein in its entirety).
  • the CEACAMl inhibitory agent can be a monomeric CEACAMl Fc polypeptide.
  • the CEACAMl inhibitory agent can be a monomeric CEACAMl Fc fusion protein.
  • Monomeric CEACAMl Fc fusion proteins have been described in the art, see, e.g. Biotoni et al. PNAS 2004 101 :9763-8; which is incorporated by reference herein.
  • the CEACAMl inhibitory agent is not a dimeric form of soluble CEACAMl polypeptide or a dimeric form of CEACAMl Fc polypeptide.
  • CEACAMl -4L (isoform 1) has 526 amino acids, having the sequence:
  • the foregoing amino acid sequences include a leader sequence (34 amino acids), and the mature peptide starting at QLT.
  • the DNA and amino acid sequences of CEACAM1 isoforms of various species are available on-line from the NCBI, including human (ID: 634), mouse (ID: 26365), rat (ID: 81613), cow (ID: 404118), zebra fish (ID: 114465), dog (ID: 612435), goat (ID: 100384959), and orangutan (ID: 100172828).
  • the secreted human CEACAM1 isoform 2 (UniProtKB PI 3688-2) has the amino acids:
  • VKTIIVTGK (SEQ ID NO: 30).
  • PD1 (or CD279) is a 288 amino acid type I transmembrane protein composed of one immunoglobulin (Ig) superfamily
  • PD1 is encoded by the Pdcdl and PDCD1 genes on chromosome 1 in mice and chromosome 2 in humans respectively. In both species, Pdcdl is encoded by 5 exons. Exon 1 encodes a short signal sequence, whereas exon 2 encodes an Ig domain. The stalk and transmembrane domains make up exon 3, and exon 4 codes for a short 12 amino acid sequence that marks the beginning of the cytoplasmic domain. Exon 5 contains the C-terminal intracellular residues and a long 3'UTR (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704). PD1 is a member of the B7 family of receptors.
  • Splice variants of PD1 have been cloned from activated human T cells. These transcripts lack exon 2, exon 3, exons 2 and 3, or exons 2 through 4. All these variants, except for the splice variant lacking exon 3 only (PDlAex3), are expressed at levels similar to full-length PD1 in resting peripheral blood mononuclear cells (PBMCs). All variants are significantly induced upon activation of human T cells with anti-CD3 and anti-CD28 (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704).
  • PD1 refers to the 288 amino acid polypeptide having the amino acid sequence of:
  • PD1 refers to human PD1.
  • the term "PD1 " is also used to refer to truncated forms or fragments of the PD1 polypeptide. Reference to any such forms of PD1 can be identified in the application, e.g., by "PD1 (42-136).”
  • PD1 the mature PD1 peptide
  • PD1 IgV domain as PD1 (42-136).
  • Specific residues of PD1 can be referred to as, for example, "PD1 (68).”
  • PD1 has been shown to be expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs). PD1 is not expressed on resting T cells but is inducibly expressed after activation. Ligation of the T cell receptor or B cell receptor can upregulate PD1 on T and B lymphocytes. In normal human reactive lymphoid tissue, PD1 is expressed on germinal center- associated T cells. PD1 compartmentalization in intracellular stores has been described in a regulatory T cell population. PD1 is inducibly expressed on APCs on myeloid CD1 lc+ DCs and monocytes in humans (Keir ME et al, 2008. Annu Rev Immunol.
  • PDl has two known ligands, PD-L1 and PD-L2, which are also members of the B7 family.
  • the binding interface of PDl to PD-L1 is via its IgV-like domain (i.e., PDl (42-136)).
  • Residues important for binding of PDl to its ligands include residues 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136.
  • PD-L1/CD274 has been shown to be constitutively expressed on mouse T and B cells, DCs, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells. CD274/PD-L1 expression is also found on a wide range of nonhematopoietic cells and is upregulated on a number of cell types after activation.
  • PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B 16 melanoma upon treatment with IFN- ⁇ .
  • Residues of PD-L1 important for binding to PDl include PD-L1 (67), PD-L1 (121), PD-L1 (122), PD-L1 (123), PD-L1 (123), PD-L1 (124), and PD-Ll (126).
  • PD-L2 expression is more restricted than PD-L1 expression.
  • PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells.
  • PD-L2 is also expressed on 50% to 70% of resting peritoneal Bl cells, but not on conventional B2 B cells.
  • PD-L2 can also be induced on monocytes and macrophages by GM-CSF, IL-4, and IFN- ⁇ .
  • PD-L2 expression has also been observed on tumor lines.
  • a PDl inhibitory agent can be an anti-PD-Ll antibody reagent.
  • a PDl inhibitory agent can be an anti-PD-L2 antibody reagent.
  • anti-PD-Ll antibody reagent or anti-PD-L2 antibody reagent is one that binds to the target ligand and prevents binding of the ligand to PDl.
  • PDl and its ligands have been shown to have important roles in regulating immune defenses against microbes that cause acute and chronic infections.
  • the PDl : PD-L pathways appear to play important roles in the outcome of infection, and the regulation of the delicate balance between effective antimicrobial immune defenses and immune -mediated tissue damage.
  • a PDl inhibitory agent e.g. an antibody reagent
  • PDl PD-L pathways to evade the immune responses and establish persistent infection.
  • LCMV lymphocytic choriomeningitis virus
  • CD8 T cells Functional dysregulation, also termed herein as "exhaustion,” of CD8 T cells is an important reason for ineffective viral control during chronic infections and is characteristic of chronic LCMV infection in mice, as well as of HIV, HBV, HCV, and HTLV infection in humans and SIV infection in primates.
  • Blocking PD1 :PD-L interactions in vitro has been shown to reverse the exhaustion of HIV-specific, HBV- specific (Boni C et al. 2007. J. Virol. 81 :4215-25), HCV-specific, and SIV-specific (Velu V et al. 2007. J. Virol.81 :5819-28) CD8 and CD4 T cells and restores proliferation and cytokine production (Petrovas C et al. 2006. J. Exp. Med. 203:2281-92; Day CL et al. 2006. Nature 443:350-54;
  • HCV core a nucleocapsid protein
  • C1QBP complement receptor C1QBP
  • the PD1 :PD-L pathway also can play a key role in the chronicity of bacterial infections.
  • Helicobacter pylori causes chronic gastritis and gastroduodenal ulcers and is a risk factor for development of gastric cancer.
  • T cell responses are insufficient to clear infection, leading to persistent infection.
  • Gastric epithelial cells express MHC class II molecules and are thought to have important APC (antigen-presenting cell) function during H. pylori infection.
  • Anti- PD-Ll blocking antibodies enhance T cell proliferation and IL-2 production in cultures of gastric epithelial cells exposed to H.
  • Parasitic worms also have exploited the PD1 :PD-L pathways to induce macrophages with strong suppressive function.
  • PD1 :PD-L pathways to induce macrophages with strong suppressive function.
  • PD1 and PD-L2 are upregulated on activated macrophages.
  • Tumors express antigens that can be recognized by host T cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor
  • PD-L pathways are involved in suppression of anti-cancer/tumor immune responses.
  • PDl expression is upregulated on tumor infiltrating lymphocytes, and this can contribute to tumor immunosuppression.
  • PD-Ll expression has been shown in situ on a wide variety of solid tumors, including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck.
  • PD-Ll expression is inversely correlated with intraepithelial, but not stromal, infiltrating CD8 T cells, suggesting that PD-Ll inhibits the intratumor migration of CD8 T cells.
  • studies relating PD-Ll expression on tumors to disease outcome show that PD-Ll expression strongly correlates with unfavorable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer but not small cell lung cancer (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704).
  • the PDl pathway can also play a role in hematologic malignancies.
  • PDl is highly expressed on the T cells of angioimmunoblastic lymphomas, and PD-Ll is expressed on the associated follicular dendritic cell network.
  • nodular lymphocyte -predominant Hodgkin lymphoma the T cells associated with lymphocytic and/or histiocytic (L&H) cells express PDl .
  • PDl and PD-Ll are expressed on CD4 T cells in HTLV-1 -mediated adult T cell leukemia and lymphoma.
  • PD-L2 has been identified as being highly expressed in mantle cell lymphomas.
  • PD-Ll is expressed on multiple myeloma cells but not on normal plasma cells, and T cell expansion in response to myeloma cells is enhanced in vitro by PD-Ll blockade.
  • PD-Ll is expressed on some primary T cell lymphomas, particularly anaplastic large cell T lymphomas (Keir ME et al, 2008. Annu Rev Immunol. 26:677- 704).
  • a PDl inhibitor agent can bind a PDl molecule having the sequence set forth in SEQ ID NO: 1 or an allelic or splice variant of SEQ ID NO: 1.
  • Splice variants of PDl have been described, for example in Keir et al. Annual Review of Immunology 2008 26:677- 704 and Nielsen et al. Cell Immunol 2005 235: 109-116; which are incorporated herein by reference in their entireties.
  • a PDl inhibitory agent can bind to a PDl ligand interaction site.
  • specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-Ll . In some embodiments, specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-L2. In some embodiments, specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-Ll and PD-L2. In some embodiments, a ligand interaction site of PDl can comprise any of amino acid residues 41-136 of SEQ ID NO: l .
  • a ligand interaction site on PDl can comprise any of the amino acid residues selected from the group consisting of amino acids 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136 of SEQ ID NO: l .
  • the ligand interaction site on PDl comprises any of the amino acid residues selected from the group consisting of amino acids 78, 126, and 136 of SEQ ID NO: 1.
  • a PD1 inhibitory agent can bind to, or physically interact with any of the amino acid residues selected from the group consisting of amino acids 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136 of SEQ ID NO: l .
  • a PD1 inhibitory agent can bind to, or physically interact with, any of the amino acid residues selected from the group consisting of amino acids 41-136 of SEQ ID NO : 1.
  • a PD1 inhibitory agent can bind to, or physically interact with, any of the amino acid residues selected from the group consisting of amino acids 78, 126, and 136 of SEQ ID NO: l .
  • physical interaction can encompass steric hindrance of the interaction of one or more of these sites with a ligand involved in tolerogenesis.
  • Non-limiting examples of anti-PDl antibody reagents can include PD1 binding site sequences from monoclonal antibodies specific for human PD1, such as, MDX-1106 (ONO-4538), a fully human IgG4 anti-PDl blocking antibody (Journal of Clinical Oncology, 2008 Vol 26, No 15S); CT-011 (CureTech, LTD, previously CT-AcTibody or BAT), a humanized monoclonal IgGl antibody (Benson DM et al, Blood.
  • monoclonal antibodies specific for human PD1 such as, MDX-1106 (ONO-4538), a fully human IgG4 anti-PDl blocking antibody (Journal of Clinical Oncology, 2008 Vol 26, No 15S); CT-011 (CureTech, LTD, previously CT-AcTibody or BAT), a humanized monoclonal IgGl antibody (Benson DM et al, Blood.
  • compositions and methods described herein relate to LAP inhibitory agents.
  • latency associated peptide or “LAP” refers to the amino - terminal domain of the TGF 3 precursor peptide. When LAP is cleaved from the precursor peptide, it can remain in noncovalent association with TGF 3, forming the latent complex.
  • LAP can exist as a homodimer of LAP molecules. Sequences of LAP polypeptides are known for a number of species, e.g. human LAP (amino acids 30-278 of SEQ ID NO: 33). Agents that physically interact with, bind to, or sterically occlude ligand binding to these interaction sites can be used to inhibit LAP activity, and particularly LAP activity involved in the establishment of maintenance of tolerogenesis.
  • LAP contains important residues necessary for the interaction with binding partners, e.g. TGF .
  • Cysteines 224 and 226 are important in the mtermolecular disu!phide bond between two
  • the RGB motif (245-247) facilitates the interaction with integrins (Munger et al, Mol Biol Cell,
  • Cysteine 33 is important for the disulphide bridge with the third cysteine-rich repeat of latent ⁇ binding protein (LTBP) (Saharinen et al. The EMBO Journal, 15, 245-253
  • a LAP inhibitor) ' ' agent can bind a LAP molecule having the sequence set forth in SEQ ID NO: 34 or an allelic or splice variant of SEQ ID NO: 34.
  • a LAP inhibitory agent can bind a LAP ligand interaction site, e.g. a site that interacts with mature TGF .
  • LAP ligand interaction site e.g. a site that interacts with mature TGF .
  • Non- limiting examples of such sites include R218 (see, e.g. McGowan et al. The Journal of Clinical Endocrinology and Metabolism 2003 88:3321-6; which is incorporated by reference herein in its entirety).
  • compositions and methods relating thereto which comprise at least two of the following: an inhibitor of CEACAM1 (i.e. a CEACAM1 inhibitory agent), an inhibitor of PD1 (i.e. a PD1 inhibitory agent), and/or an inhibitor of LAP (i.e. a LAP inhibitory agent).
  • an "inhibitory agent” is an agent that reduces the level and/or activity of a target as compared to the level and or activity in the absence of the agent by a statistically significant amount.
  • the level of a target can be the level of a polypeptide target, the level of an isoform or variant of a polypeptide target, or the level of an RNA transcript encoding the polypeptide target.
  • the activity of a polypeptide target can include, but is not limited to, the ability of the target to bind a normal ligand, the ability of the target to interact with other polypeptides (e.g. downstream signaling partners), and/or the ability of the target polypeptide to effect a downstream response (e.g.
  • an inhibitory agent can be an agent that reduces the interaction (e.g. binding, both homophilic and heterophilic) between individual molecules of CEACAM1, PD1, and LAP.
  • an inhibitory agent can reduce the interaction of CEACAM1 and PD1.
  • the compositions described herein can comprise a CEACAM1 inhibitory agent and a PD1 inhibitory agent.
  • the compositions described herein can comprise a CEACAM1 inhibitory agent and a LAP inhibitory agent.
  • the compositions described herein can comprise a LAP inhibitory agent and a PD1 inhibitory agent.
  • the compositions described herein can comprise a
  • CEACAM1 inhibitory agent a LAP inhibitory agent, and a PD1 inhibitory agent.
  • a target-specific inhibitory agent i.e. an inhibitory agent specific for a given target, herein, at least one of CEACAM1, PD1, and/or LAP
  • an "inhibitory agent" can refer to a protein-binding agent that permits inhibition of
  • Such agents include, but are not limited to, antibodies ("antibodies” includes antigen-binding portions of antibodies such as epitope- or antigen- binding peptides, paratopes, functional CDRs; recombinant antibodies; chimeric antibodies; tribodies; midibodies; or antigen-binding derivatives, analogs, variants, portions, or fragments thereof), protein- binding agents, small molecules, recombinant protein, peptides, aptamers, avimers and protein- binding derivatives, portions or fragments thereof.
  • Antisense oligonucleotides represent another class of agents that are useful in the compositions and methods described herein.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity
  • an inhibitory agent can be an inhibitory nucleic acid, an inhibitory peptide, an inhibitory small molecule, an inhibitory antibody reagent, or inhibitory mimics (e.g. peptidomimetics) or combinations thereof.
  • an inhibitory agent can be a mimic or variant of the natural ligand of a target, e.g. a fragment or variant of TGF that will interact with LAP and prevent the binding of LAP to mature TGF .
  • an inhibitory agent can be a dominant negative variant of any of the targets described herein (e.g. CEACAMl, PDl, and/or LAP).
  • a CEACAMl variant which can bind to wild-type CEACAMl via the homodimerization/homophilic interaction domain can reduce or inhibit signaling requiring CEACAMl .
  • an inhibitory agent e.g. an antibody reagent
  • a target e.g. CEACAMl, PDl, and/or LAP
  • an inhibitory agent e.g. an antibody reagent
  • an inhibitory agent specific for a target e.g. CEACAMl, PDl, and/or LAP
  • the inhibitory agent can reduce or inhibit the interaction of a ligand with the target. Specific regions of CEACAMl, PDl and/or LAP that can be targeted by an inhibitory agent are described elsewhere herein.
  • an inhibitory agent can comprise an antibody reagent.
  • antibody reagent refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen (e.g. CEACAMl, PDl, and/or LAP).
  • an antibody reagent can specifically bind to one or more of CEACAMl, PDl, and LAP.
  • an antibody reagent can inhibit the signaling activity of the target molecule, e.g. inhibit signaling activity of CEACAM1, PD1, and/or LAP. Methods for measuring the signaling activity of the target molecule are discussed elsewhere herein.
  • An antibody reagent can comprise an antibody or a polypeptide comprising an antigen- binding domain of an antibody.
  • an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody reagent encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and single domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof).
  • Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non- human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” ("FR").
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties).
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • antigen-binding fragment or "antigen-binding domain”, which are used interchangeably to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term
  • antigen-binding fragment include (i) a Fab fragment, a monovalent fragment consisting of the VL,
  • VH, CL and CHI domains (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
  • a dAb fragment (Ward et al., (1989) Nature 341 :544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.
  • CDR complementarity determining region
  • Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those of skill in the art.
  • the term "monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody” or “monoclonal antibody composition,” which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.
  • the term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or antigen-binding portion thereof can bind.
  • the specificity of an antibody or antigen-binding portion thereof, alone or in the context of a bispecific or multispecific polypeptide agent, can be determined based on affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation (K D ) of an antigen with an antigen- binding protein (such as a bispecific or multispecific polypeptide agent), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K D , the stronger the binding strength between an antigenic determinant and the antigen-binding molecule.
  • the affinity can also be expressed as the affinity constant (K A ), which is 1/ K D ).
  • affinity can be determined in a manner known per se, depending upon the specific antigen of interest.
  • a bispecific or multispecific polypeptide agent as defined herein is said to be "specific for" a first target or antigen compared to a another target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a K D value) that is at least 1 Ox, such as at least 1 OOx, and preferably at least lOOOx, and up to lOOOOx or more better than the affinity with which said amino acid sequence or polypeptide binds to the other target or polypeptide.
  • an affinity as described above, and suitably expressed, for example as a K D value
  • Avidity is the measure of the strength of binding between an antigen-binding molecule
  • Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen- binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule.
  • antigen-binding proteins such as a bispecific polypeptide agent described herein
  • K D dissociation constant
  • KA association constant
  • K D value greater than 10 "4 mol/liter is generally considered to indicate non-specific binding.
  • the K D for biological interactions which are considered meaningful are typically in the range of 10 "10 M (0.1 nM) to 10 "5 M (10000 nM). The stronger an interaction is, the lower is its K D .
  • a binding site on a bispecific or multispecific polypeptide agent described herein may bind to the desired antigen with an affinity less than 500 nM, less than 200 nM, or less than 10 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.
  • selectively binds or “specifically binds” refers to the ability of a polypeptide domain described herein to bind to a target, such as a molecule present on the cell-surface, with a K D of 10 "5 M (10000 nM) or less, e.g., 10 "6 M or less, 10 "7 M or less, 10 "8 M or less, 10 "9 M or less, 10 "10 M or less, 10 "11 M or less, or 10 "12 M or less.
  • a polypeptide agent described herein binds to CEACAMl with a K D of 10 "5 M or lower, but not to another
  • CEACAM molecule such as CEACAM5, or a related homologue
  • the agent is said to specifically bind CEACAMl.
  • Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent.
  • the person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay.
  • humanized or composite human antibody reagents can be used in the compositions and methods described herein.
  • such antibodies are described, e.g., in U.S. Patent App. No. 61/565,640, entitled Anti-CEACAM 1 Recombinant Antibodies for Cancer Therapy, filed 1 December 2011, and the references discussed therein.
  • CEACAMl can comprise a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid residues SSHGMS (SEQ ID NO: 2), a heavy chain CDR2 consisting of the amino acid residues TISSGGTYTYYPDSVKG (SEQ ID NO: 3), a heavy chain CDR3 consisting of the amino acid residues HDFDYDAAWFAY (SEQ ID NO: 4), a light chain CDRl consisting of the amino acid residues SANSSVSYMY (SEQ ID NO: 5), a light chain CDR2 consisting of the amino acid residues LTSNLAS (SEQ ID NO: 6), and a light chain CDR3 consisting of the amino acid residues
  • CDR heavy chain complementarity determining region
  • the CEACAMl -binding antibody or epitope-binding peptide can be constructed to have CDR regions selected from the following: a heavy chain CDR 1 consisting of the amino acid residues SSHGMS
  • a heavy chain CDR2 consisting of the amino acid residues TISSGGTYTYYPDSVKG (SEQ ID NO: l 1), TFSGGGNYTYYPDSVKG (SEQ ID NO: 12) or TISVGGGNTSYPDSVKG (SEQ ID NO: 13); a heavy chain CDR3 consisting of the amino acid residues HDFDYDAAWFAY (SEQ ID NO: 14), or HGGLPFYAMDY (SEQ ID NO: 15), or GLTTGPAWFAY (SEQ ID NO: 16); a light chain CDR1 consisting of the amino acid residues SANSSVSYMY (SEQ ID NO: 17), SVSSSISSSNLH (SEQ ID NO: 18), KSSQSLLNSSNQKNYLA (SEQ ID NO: 19), or RASQKISGYLS (SEQ ID NO: 20); a light chain CDR2 consisting of the amino acid residues SANSSVSYMY (SEQ ID NO: 17), SVSSSISSSNLH (SEQ ID
  • SVSSSISSSNLH SEQ ID NO: 22
  • FASTRES SEQ ID NO: 23
  • AASTLDS SEQ ID NO: 24
  • a light chain CDR3 consisting of the amino acid residues QQWSSNPPT (SEQ ID NO: 25), QQWSSHPFT (SEQ ID NO: 26), QQHYSTPWT (SEQ ID NO: 27) or LQYASSLMYT
  • anti-CEACAMl monoclonal antibodies useful in the compositions and methods described herein include AgBlO, that inhibits CEACAMl -mediated EAE suppression in mice (Fujita et al., 175 Am. J. Pathol. 1116 (2009)); a CEACAMl antibody (Chen et al., 2004); murine D14HD11 (Yu et al., 281 J. Biol. Chem. 39179 (2006)); murine CEACAMl -specific CC1 (Iijima et al., 199 J. Exp. Med.
  • mice anti-human CEACAMl MRG-1 (Ortenberg 2012); mouse anti-human CEACAMl N-domain specific antibodies 5F4, 34B1, and 26H7 (IgGl) (Morales et al., 1999) each of which recognizes the N-terminal domain of CEACAMl (Watt et al., 2001); and 12-140-4,4/3/17, COL-4, YG-C28F2, D14HD11, B18.7.7, D11-AD11, HEA 81, CLB-gran-10, F34-187, T84.1, B6.2, and Bl . l .
  • Monoclonal antibodies 34B1, 26H7, and 5F4 are also discussed in U.S. Patent Pub.
  • CEACAMl -Fc fusion proteins have been described, including, for example, the extracellular portion of CEACAMl fused to human IgFc in a mammalian expression vector (Markel et al., 110 J. Clin. Invest. 943 (2002)); pCEP4-N-CEACAMl-Fc see Gallagher, 71 J. Virol. 3129 (1997)); as well as commercially available sources of CEACAMl fusion proteins, as known to one of ordinary skill in the art.
  • compositions and methods described herein relate to bispecific inhibitory agents, e.g. bispecific polypeptide agents.
  • Bispecific agents comprise a molecule which is able to physically contact and inhibit at least two of CEACAMl, PD1, and/or LAP simultaneously.
  • the term "bispecific polypeptide agent” refers to a polypeptide that comprises a first polypeptide domain which has a binding site that has binding specificity for a first target, and a second polypeptide domain which has a binding site that has binding specificity for a second target, i.e., the agent has specificity for two targets, e.g., CEACAMl and PDl ; PDl and LAP; or CEACAMl and LAP.
  • the first target and the second target are not the same (i.e., are different targets (e.g., proteins)).
  • the different targets can be co-expressed on the same cell.
  • a bispecific polypeptide agent can bind targets present on a single cell (heterophilic binding in cis), and/or bind one target on one cell and the other on another cell
  • a bispecific polypeptide agent as described herein can selectively and specifically bind to a cell that expresses the first target and the second target.
  • a non- limiting example of a bispecific polypeptide agent is a bispecific antibody construct. Bispecific antibody constructs comprising antigen-binding portions of antibodies specific for two different antigens, e.g., PDl and CEACAMl ; or CEACAMl and LAP; or LAP and PDl, can be readily constructed by one of skill in the art.
  • sequences encoding the antigen-binding domain of a first antibody characterized and known to bind a desired epitope on one antigen can be joined, either directly, or through any of a variety of linkers as known to the ordinarily skilled artisan, to sequences encoding the antigen-binding domain of a second antibody characterized and known to bind a desired epitope on a second antigen.
  • Such sequences can be inserted into an appropriate vector and introduced to a cell to produce the bispecific antibody polypeptide by methods known to those of ordinary skill in the art.
  • a composition as described herein can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a PDl inhibitory agent. In some embodiments, a composition as described herein can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a LAP inhibitory agent. In some embodiments, a composition as described herein can comprise a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PDl inhibitory agent.
  • compositions and methods described herein can relate to a trispecific agent, i.e. an agent having specificity for all of CEACAMl, PDl, and LAP.
  • a trispecific agent refers to a polypeptide that comprises at least a first polypeptide domain having a binding site that has binding specificity for a first target, a second polypeptide domain having a binding site that has binding specificity for a second target, and a third polypeptide domain having a binding site that has binding specificity for a third target e.g.,
  • CEACAMl, PDl, and LAP are not the same (i.e., are different targets (e.g., proteins)), but can all be present (e.g., co-expressed) on a cell.
  • a multispecific polypeptide agent as described herein can bind one or more additional targets, i.e., a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target-specific binding sites respectively.
  • a multispecific polypeptide agent has the potential to bind a cell that expresses all the targets the agent is specific for more strongly (e.g., with greater avidity) than a cell that expresses only one target, or less targets than the agent is specific for.
  • a non-limiting example of a multispecific polypeptide agent is a multispecific antibody or antigen-binding fragment thereof.
  • a bispecific polypeptide agent or a trispecific polypeptide agent is a type or subset of multispecific polypeptide agent.
  • a multispecific antibody reagent e.g. a bispecific antibody reagent
  • a bispecific antibody reagent can preferentially bind to a cell expressing each of the target antigens for which the reagent is specific. While, for example, a bispecific antibody reagent can bind to two separate cells, each of which expresses one of the target antigens, it can be more specific for cells which express both of the target antigens on the same cell surface.
  • the antibody reagents provided herein, or used in the methods described herein can further comprise post-translational modifications.
  • post-translational polypeptide modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group.
  • the modified polypeptides can contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates. Such molecules can also be referred to as derivatives.
  • Reagents for use in the compositions and methods described herein include those that are conjugated or associated with polymers, such as PEG, that can enhance the half-life of the peptide in vivo.
  • PEG modification is well-known in the art. See, e.g., PCT US2007/024067.
  • target refers to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain or other moiety (e.g. a small molecule or aptamer among others) which has a binding site can selectively bind.
  • the target can be, for example, an intracellular target (e.g., an intracellular protein target) or a cell surface target (e.g., a membrane protein, a receptor protein).
  • an "antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
  • an "antigen" is a molecule that is bound by a binding site on a polypeptide agent.
  • antigens are bound by antibody ligands and are capable of raising an antibody response in vivo.
  • An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof.
  • the bispecific or multispecific polypeptide agents described herein are selected for target specificity against two or more particular antigens (i.e., CEACAM1, PD1, and/or LAP).
  • the antibody binding site as defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen.
  • antigenic determinant refers to an epitope on the antigen recognized by an antigen-binding molecule (such as bispecific polypeptide agent described herein), and more particularly, by the antigen-binding site of said molecule.
  • an “epitope” can be formed both from contiguous amino acids, or noncontiguous amino acids juxtaposed by folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • An “epitope” includes the unit of structure conventionally bound by an immunoglobulin V H V L pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation.
  • the terms “antigenic determinant” and “epitope” can also be used interchangeably herein.
  • immunoglobulin refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, which comprise two ⁇ sheets and, usually, a conserved disulphide bond.
  • Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signaling (for example, receptor molecules, such as the PDGF receptor).
  • a "target site” or "ligand interaction site” on the target molecule means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homodimerization or
  • a "ligand interaction site" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on a target or antigen to which a binding site of an agent, or a bispecific or multispecific agent described herein can bind such that the target (and/or any pathway, interaction, signaling, biological mechanism or biological effect in which the target or antigen is involved), e.g., the signaling of any of
  • CEACAM1, PD1, and/or LAP is inhibited.
  • a "blocking" antibody or an antibody “antagonist” is one which inhibits or reduces biological activity of the antigen(s) it binds.
  • a CEACAM1/PD1 bispecific antagonist antibody binds CEACAM1 and PD1 and inhibits the ability of CEACAM1 and PD1 to induce or maintain T-cell tolerance.
  • domain refers to a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases can be added, removed or transferred to other proteins without loss of function or properties of the remainder of the protein to which it is added or transferred and/or of the domain itself.
  • binding domain refers to such a domain that is directed against an antigenic determinant.
  • single antibody variable domain is meant a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • variable domains and modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least in part the binding activity and specificity of the full-length domain.
  • a bispecific polypeptide agent will comprise at least two different binding domains
  • Polyclonal antibodies specific for CEACAM1, LAP, and/or PD1 can be produced by various procedures well known in the art.
  • PD1 polypeptides or fragments thereof such as a fragment comprising amino acid residues 42-136 of SEQ ID NO: l
  • polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen, e.g., a PD1 fragment and an adjuvant.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy-bean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxy-succinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI 2 , or R where R and R 1 are different alkyl groups.
  • a bifunctional or derivatizing agent for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxy-succinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI 2 , or R where R and R 1 are different alkyl groups.
  • Animals can be immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g. , the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • Various methods for making monoclonal antibodies described herein are available in the art.
  • the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975) or adaptations thereof, or by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al.
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. It is to be understood that the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with, e.g. PDl, or a fragment or derivative thereof, such as a fragment comprising amino acid residues 42-136 of SEQ ID NO: l, or a cell expressing PDl or a fragment thereof.
  • an immune response e.g., antibodies specific for PDl are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limiting dilution.
  • the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding PDl and exerting a cytotoxic or cytostatic effect on activated lymphocytes.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by injecting mice with positive hybridoma clones.
  • a mouse or other appropriate host animal such as a hamster or macaque monkey
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes can be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); which is incorporated by reference herein in its entirety).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells can be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures ⁇ e.g., by PCR or by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is described in more detail below.
  • antibodies useful in the methods and compositions described herein can also be generated using various phage display methods known in the art, such as isolation from antibody phage libraries generated using the techniques described in McCafferty et al. , Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the nucleic acid sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine).
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the nucleic acid sequences encoding them.
  • DNA sequences encoding V H and V L domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues).
  • the DNA encoding the V H and V L domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • a phagemid vector e.g., p CANTAB 6 or pComb 3 HSS.
  • the vector is electroporated into E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Phage expressing an antigen binding domain that binds to PD1, CEACAM1, or LAP or portions thereof can be selected or identified with antigen e.g. , using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman et al, 1995, J. Immunol. Methods 182:41-50; Ames et al, 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al, 1994, Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • Fab, Fab' and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 1992, 12(6):864-869; and Sawai et al, 1995, AJRI 34:26-34; and Better et al, 1988, Science 240: 1041-1043, the contents of which are herein incorporated by reference in their entireties.
  • Simple binding assays can be used to screen for or detect agents that bind to any of CEACAMl, PDl, and/or LAP, and/or reduce the signaling activity of any of the foregoing. Because CEACAMl and PDl are transmembrane proteins, assays that use the soluble forms of these proteins rather than full-length protein can be used, in some embodiments. Soluble forms include, for example, those lacking the transmembrane domain and/or those comprising the IgV domain or fragments thereof which retain their ability to bind their cognate binding partners.
  • agents that inhibit CEACAMl, PDl, and/or LAP for use in the compositions and methods described herein can be identified by, for example, measuring the activation of the signaling pathways controlled by CEACAMl, PDl and/or LAP.
  • the expression of genes induced or suppressed by the activation of CEACAMl, PDl, and/or LAP signaling can be measured (e.g. by quantitative RT-PCR) or the level of phosphorylation of downstream signaling partners can be measured (e.g. by immunochemistry).
  • markers for each of these pathways, and methods of measuring their levels are known in the art.
  • Exemplary markers include but are not limited to: ZAP-70 phosphorylation and the level of SHP1 bound to CEACAMl as markers for CEACAMl signaling (see, e.g. Chen et al. Journal of Immunology 2008 6085-6093), SHP2 phosophorylation levels as markers for PDl signaling (see, e.g. Yamamoto et al. Blood 2008 111 :3220-4), and the level of phosphorylated SMAD2 and SMAD3 as markers for LAP signaling (see, e.g. Fleisch et al. Endocrine -Related Cancer 2006 13:379-400). All of the foregoing publications are incorporated by reference herein in their entireties.
  • affinity biosensor methods may be based on the piezoelectric effect, electrochemistry, or optical methods, such as ellipsometry, optical wave guidance, and surface plasmon resonance (SPR).
  • efficacy of, e.g. an siRNA on the expression of PDl, LAP, and/or CEACAMl can be monitored using methods known in the art, such as quantitative RT-PCR with specific oligonucleotide primers for each gene respectively, or ELISA for PDl, LAP, and/or
  • CEACAMl a population of T cells
  • the expression of PDl, LAP and/or CEACAMl in a population of T cells can be determined by FACS analysis using the markers characteristic of particular populations and subpopulations known in the art or disclosed herein.
  • methods for enhancing an immune response in vivo comprising administering to the subject a therapeutically effective amount of one or more agents that inhibit CEACAMl, LAP, and/or PDl, as described herein. Modulation of T-cell tolerance by such agents is useful for specifically enhancing an immune response in vivo, which can be useful for the treatment of conditions related to immune function including cancer and chronic infections.
  • Modulation of T-cell tolerance also is useful in in vitro or non-therapeutic applications including determining whether T-cells of a subject are functional (e.g., proliferation and/or cytotoxic functions), to determine if a treatment has rendered T-cells non- functional, in experimental models of cancer, e.g., to determine the effects of increases or decreases in T-cell function on particular organs or physiological processes, and to test for agents which increase or decrease T-cell activity.
  • functional e.g., proliferation and/or cytotoxic functions
  • experimental models of cancer e.g., to determine the effects of increases or decreases in T-cell function on particular organs or physiological processes, and to test for agents which increase or decrease T-cell activity.
  • Other uses will be apparent to one of ordinary skill in the art.
  • CEACAMl can be used alone as a primary therapy or in combination with other therapeutics as a combination therapy to enhance the therapeutic benefits of other medical treatments.
  • CEACAMl, PDl, and/or LAP activity is "decreased” if one or more signaling activities or downstream read-outs of CEACAMl, PDl, and/or LAP activity is reduced or altered by a statistically significant amount, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,a t least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%), or more, up to and including 100%), in the presence of an agent or stimulus relative to the absence of such modulation.
  • CEACAMl, PDl, and/or LAP activity if CEACAMl, PDl, and/or LAP activity is decreased, some downstream read-outs will decrease but others can increase (i.e. things that are normally suppressed by CEACAMl, PDl, and/or LAP activity).
  • a decrease in activity of a target can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen, such as CEACAMl, PDl, or LAP, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen, such as CEACAMl, PDl, or LAP, for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co- factors, etc.), compared to the same conditions but without the presence of an inhibitory agent.
  • a change which can either be an increase or a decrease
  • affinity, avidity, specificity and/or selectivity of a target or antigen such as CEACAMl, PDl, or LAP
  • a decrease in the activity of a target can also mean effecting a change (i.e., in an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen, such as CEACAMl, PDl, and/or LAP, and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signaling pathway or metabolic pathway and their associated biological or physiological effects) is involved.
  • such an action as an agonist or an antagonist can be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se or described herein, depending on the target or antigen involved.
  • a decrease in the activity of a target can, for example, also involve allosteric modulation of the target, (such as CEACAM1, PD1, and/or LAP); and/or reducing or inhibiting the binding of the target to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target.
  • a decrease in the activity of a target can, for example, also involve effecting a change in respect of the folding or confirmation of the target, or with respect to the ability of the target to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate.
  • a decrease in the activity of a target can, for example, also involve effecting a change in the ability of the target to signal, phosphorylate, dephosphorylate, and the like.
  • Immunosuppression of a host immune response plays a role in a variety of chronic immune conditions, such as in persistent infection and tumor immunosuppression. Recent evidence indicates that this immunosuppression can be mediated by immune inhibitory receptors expressed on the surface of an immune cell, and their interactions with their ligands. For example, cytotoxic CD8 T cells can enter a state of "functional exhaustion,” or “unresponsiveness” whereby they express inhibitory receptors that prevent antigen-specific responses, such as proliferation and cytokine production. Accordingly, by inhibiting the activity and/or expression of such inhibitory receptors, an immune response to a persistent infection or to a cancer or tumor that is suppressed, inhibited, or unresponsive, can be enhanced or uninhibited.
  • an "immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus.
  • the response is specific for a particular antigen (an "antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor.
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
  • unresponsiveness or “functional exhaustion” with regard to immune cells includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, for example, because of exposure to
  • immunosuppressants exposure to high or constant doses of antigen, or through the activity of inhibitor receptors or factors, such as CEACAM1, PD1, and/or LAP.
  • inhibitor receptors or factors such as CEACAM1, PD1, and/or LAP.
  • Unresponsiveness includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the antigen has ceased. Unresponsive immune cells can have a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%), or even 100%> in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type.
  • the term "reduces T cell tolerance” means that a given treatment or set of conditions leads to reduced T cell tolerance, i.e. greater T cell activity, responsiveness, and/or ability or receptiveness with regards to activation.
  • Methods of measuring T cell activity are known in the art.
  • T cell tolerance can be induced by contacting T cells with recall antigen, anti-CD3 in the absence of costimulation, and/or ionomycin. Levels of, e.g.
  • LDH-A, RAB10, and/or ZAP70 both intracellular or secreted
  • LDH-A, RAB10, and/or ZAP70 can be monitored, for example, to determine the extent of T cell tolerogenesis (with levels of IL-2, interferon- ⁇ and TNF correlating with increased T cell tolerance).
  • the response of cells pre -treated with, e.g. ionomycin, to an antigen can also be measured in order to determine the extent of T cell tolerance in a cell or population of cells, e.g. by monitoring the level of secreted and/or intracellular IL-2 and/or TNF-a (see, e.g. Macian et al. Cell 2002 109:719-731 ; which is incorporated by reference herein in its entirety).
  • a compound known to reduce T cell tolerance which can be useful as a control, but which has extremely limited applicability in the clinic can include CTLA4. In one embodiment, a compound known to reduce T cell tolerance is not CTLA4.
  • T cells having undergone adaptive tolerance have increased levels of Fyn and ZAP-70/Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element modulator), B lymphocyte-induced maturation protein-1 (Blimp-1), PD1, CD5, and SHP2; increased phosphorylation of ZAP-70/Syk, LAT, PLCyl/2, ERK, PKC- ⁇ / ⁇ ; increased activation of intracellular calcium levels; decreased histone acetylation or hypoacetylation and/or increased CpG methylation at the IL-2 locus.
  • reduction of one or more of any of these parameters can be assayed to determine whether one or more agents that inhibits CEACAM1, LAP, and/or PD-1 enhances an immune response in vivo or decreases immune tolerance.
  • Reduction of T cell tolerance can also be measured by determining the proliferation of T cells in the presence of a relevant antigen assayed, e.g. by a 3 H-thymidine incorporation assay or cell number. Markers of T cell activation after exposure to the relevant antigen can also be assayed, e.g. flow cytometry analysis of cell surface markers indicative of T cell activation (e.g. CD69, CD30, CD25, and HLA-DR). Reduced T cell activation in response to antigen-challenge is indicative of tolerance induction. Conversely, increased T cell activation in response to antigen-challenge is indicative of reduced tolerance.
  • a relevant antigen assayed e.g. by a 3 H-thymidine incorporation assay or cell number. Markers of T cell activation after exposure to the relevant antigen can also be assayed, e.g. flow cytometry analysis of cell surface markers indicative of T cell activation (e.g. CD69, CD30, CD25,
  • Reduction of T cell tolerance can also be measured, in some embodiments, by determining the degree to which the inhibitory agent inhibits the activity of its target.
  • the activity of a CEACAM1 inhibitory agent can be determined using the staphylococcal enterotoxin B (SEB) model. SEB typically stimulates CEACAM1, resulting in upregulation of TIM3. In the presence of a CEACAMl inhibitory agent, the upregulation of TIM3 in response to SEB will be reduced.
  • SEB staphylococcal enterotoxin B
  • the SEB model can also be adapted to measure T cell tolerance and modulation thereof.
  • SEB staphylococcal enterotoxin B
  • TCR reactive T cell receptor
  • T cells expressing reactive TCR V beta regions display a statistically significant greater T cell activity than T cells not contacted with the inhibitory agent, the inhibitory agent is one that reduces T cell tolerance.
  • peripheral tolerance that can be used in some aspects and embodiments to measure reduction(s) in T cell tolerance using the inhibitory agents described herein include, for example, models for peripheral tolerance in which homogeneous populations of T cells from TCR transgenic and double transgenic mice are transferred into hosts that constitutively express the antigen recognized by the transferred T cells, e.g., the H-Y antigen TCR transgenic; pigeon cytochrome C antigen TCR transgenic; or hemagglutinin (HA) TCR transgenic.
  • H-Y antigen TCR transgenic e.g., pigeon cytochrome C antigen TCR transgenic
  • HA hemagglutinin
  • T cells expressing the TCR specific for the antigen constitutively or inducibly expressed by the recipient mice typically undergo an immediate expansion and proliferative phase, followed by a period of unresponsiveness, which is reversed when the antigen is removed and/or antigen expression is inhibited. Accordingly, if, in the presence of a CEACAMl, PDl, and/or LAP inhibitory agent, in such models if the T cells proliferate or expand, show cytokine activity, etc. significantly more than T cells in the absence of the inhibitory agent, than that agent is one that reduces T cell tolerance.
  • Such measurements of proliferation can occur in vivo using T cells labeled with BrDU, CFSE or another intravital dye that allows tracking of proliferation prior to transferring to a recipient animal expressing the antigen, or cytokine reporter T cells, or using ex vivo methods to analyze cellular proliferation and/or cytokine production, such as thymidine proliferation assays, ELISA, cytokine bead assays, and the like.
  • T cell tolerance can also be assessed by examination of tumor infiltrating lymphocytes or T lymphocytes within lymph nodes that drain from an established tumor.
  • T cells exhibit features of "exhaustion” through expression of cell surface molecules such as PDl, TIM-3 or LAG-3, for example, and decreased secretion of cytokines such as interferon- ⁇ . Accordingly, if, in the presence of a CEACAMl, PDl, and/or LAP inhibitory agent, increased quantities of T cells with 1) antigen specificity for tumor associated antigens are observed (e.g.
  • the technology described herein relates to methods for treating a chronic disease.
  • Such methods can comprise administering a composition as described herein (i.e. a composition comprising at least two of (i) a CEACAM1 inhibitory agent, (ii) a PD1 inhibitory agent, and (iii) a LAP inhibitory agent) to a subject in need of treatment for a chronic disease.
  • a chronic disease can be selected from the group consisting of cancer; a persistent infection; and a chronic viral infection.
  • the subject in need of treatment for a chronic disease can be a subject suffering from undesirably high levels of T-cell tolerance, a subject diagnosed as suffering from undesirably high levels of T-cell tolerance, or a subject in need of a reduction or inhibition of T-cell tolerance.
  • an "undesirably high level of T-cell tolerance" can be a level of T-cell tolerance which does not allow a subject's immune system to respond to and/or clear an infection or chronic disease.
  • a subject in need of a reduction or inhibition of T-cell tolerance using the methods described herein includes but is not limited to a subject having cancer or a pre-cancerous condition (e.g. cytological changes indicating a shift from a normal phenotype that is not yet neoplastic but which marks the tendency to progress to a neoplastic phenotype), a subject with a chronic infection, or a subject suffering from infection by a pathogen.
  • a pre-cancerous condition e.g. cytological changes indicating a shift from a normal phenotype that is not yet neoplastic but which marks the tendency to progress to a neoplastic phenotype
  • the subject being administered an agent(s) for inhibiting at least two of CEACAM1, PD1, and/or LAP has a persistent infection with a bacterium, virus, fungus, or parasite.
  • Persistent infections refer to those infections that, in contrast to acute infections, are not effectively cleared by the induction of a naturally occurring host immune response. During such persistent infections, the infectious agent and the immune response reach equilibrium such that the infected subject remains infectious over a long period of time without necessarily expressing symptoms. Persistent infections often involve stages of both silent and productive infection without rapidly killing or even producing excessive damage of the host cells. Persistent infections include for example, latent, chronic and slow infections.
  • Persistent infection occurs with viruses including, but not limited to, human T-Cell leukemia viruses, Epstein-Barr virus, cytomegalovirus, herpesviruses, varicella-zoster virus, measles, papovaviruses, prions, hepatitis viruses, adenoviruses, parvoviruses and papillomaviruses.
  • viruses including, but not limited to, human T-Cell leukemia viruses, Epstein-Barr virus, cytomegalovirus, herpesviruses, varicella-zoster virus, measles, papovaviruses, prions, hepatitis viruses, adenoviruses, parvoviruses and papillomaviruses.
  • a "chronic infection” the infectious agent is present in the subject at all times. However, the signs and symptoms of the disease can be present or absent for an extended period of time.
  • Non-limiting examples of chronic infection include hepatitis B (caused by hepatitis B virus (HBV)) and hepatitis C (caused by hepatitis C virus (HCV)) adenovirus, cytomegalovirus, Epstein- Barr virus, herpes simplex virus 1 , herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, and human T cell leukemia virus II.
  • Parasitic persistent infections can arise as a result of infection by, for example, Leish
  • a chronic infection can be a latent infection.
  • a chronic infection can include periods in which the infection is a latent infection.
  • the infectious agent such as a virus
  • a latent viral infection the virus remains in equilibrium with the host for long periods of time before symptoms again appear; however, the actual viruses cannot typically be detected until reactivation of the disease occurs.
  • latent infections include infections caused by herpes simplex virus (HSV)-l (fever blisters), HSV-2 (genital herpes), and varicella zoster virus VZV (chickenpox-shingles).
  • slow infection the infectious agents gradually increase in number over a very long period of time during which no significant signs or symptoms are observed.
  • Non-limiting examples of slow infections include AIDS (caused by HIV-1 and HIV-2), lentiviruses that cause tumors in animals, and prions.
  • persistent infections that can be treated using the methods described herein include those infections that often arise as late complications of acute infections.
  • SSPE subacute sclerosing panencephalitis
  • regressive encephalitis can occur as a result of a rubella infection.
  • the mechanisms by which persistent infections are maintained can involve modulation of virus and cellular gene expression and modification of the host immune response. Reactivation of a latent infection can be triggered by various stimuli, including changes in cell physiology, superinfection by another virus, and physical stress or trauma. Host immunosuppression is often associated with reactivation of a number of persistent virus infections.
  • the subject being administered an agent(s) for inhibiting CEACAM1 and PD1 or CEACAM1 and LAP for example, has an infection with a pathogen, such as a bacterium, virus, fungus, or parasite.
  • infectious viruses include, but are not limited to: Retroviridae (for example, HIV); Picornaviridae (for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such as strains that cause gastroenteritis);
  • Togaviridae for example, equine encephalitis viruses, rubella viruses
  • Flaviridae for example, dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronaviridae for example,
  • coronaviruses coronaviruses
  • Rhabdoviridae for example, vesicular stomatitis viruses, rabies viruses
  • Filoviridae for example, vesicular stomatitis viruses, rabies viruses
  • ebola viruses for example, ebola viruses
  • Paramyxoviridae for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae for example, influenza viruses
  • Bungaviridae for example, Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses
  • Arena viridae hemorrhagic fever viruses
  • Reoviridae e.g., reoviruses, orbiviurses and rotaviruses
  • papilloma viruses papilloma viruses, polyoma viruses
  • Adenoviridae most adenoviruses
  • Herpesviridae herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses
  • Poxviridae variola viruses, vaccinia viruses, pox viruses
  • Iridoviridae such as African swine fever virus
  • the compositions and methods described herein are contemplated for use in treating infections with these and other viral agents.
  • Examples of fungal infections include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis.
  • infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
  • the compositions and methods described herein are contemplated for use in treating infections with these and other fungal agents.
  • infectious bacteria examples include, but are not limited to: Helicobacterpyloris , Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellular e, M. kansaii, M.
  • compositions and methods described herein are contemplated for use in treating infections with these and other bacterial agents.
  • Other infectious organisms include: Plasmodium falciparum and Toxoplasma gondii.
  • the compositions and methods described herein are contemplated for use in treating infections with these and other agents.
  • the methods further comprise administering an effective amount of a viral, bacterial, fungal, or parasitic antigen in conjunction with an agent(s) for inhibiting PD1, LAP and/or CEACAM1.
  • a viral, bacterial, fungal, or parasitic antigen in conjunction with an agent(s) for inhibiting PD1, LAP and/or CEACAM1.
  • suitable viral antigens include: influenza HA, NA, M, NP and NS antigens; HIV p24, pol, gp41 and gpl20;
  • Metapneumovirus (hMNV) F and G proteins Hepatitis C virus (HCV) El, E2 and core proteins; Dengue virus (DENl-4) El, E2 and core proteins; Human Papilloma Virus LI protein; Epstein Ban- Virus gp220/350 and EBNA-3A peptide; Cytomegalovirus (CMV) gB glycoprotein, gH glycoprotein, pp65, IE1 (exon 4) and pp 150; Varicella Zoster virus (VZV) IE62 peptide and glycoprotein E epitopes; Herpes Simplex Virus Glycoprotein D epitopes, among many others.
  • CMV Cytomegalovirus
  • VZV Varicella Zoster virus
  • the antigenic polypeptides can correspond to polypeptides of naturally occurring animal or human viral isolates, or can be engineered to incorporate one or more amino acid substitutions as compared to a natural (pathogenic or non-pathogenic) isolate.
  • the subject being administered an agent for inhibiting PD1 , LAP and/or CEACAM1 has a cancer or tumor.
  • methods of treating a subject having a cancer or tumor comprising administering an effective amount of an agent(s) for inhibiting PD1 , LAP and/or CEACAM1.
  • a "cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • Hemopoietic cancers such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
  • Metastasis is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • cancers include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.
  • small-cell lung cancer non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocy
  • Macroglobulinemia may be used to determine whether abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • the methods further comprise administering a tumor or cancer antigen to a subject being administered an agent(s) for inhibiting PD1, LAP and/or CEACAM1 described herein.
  • a tumor antigen A number of tumor antigens have been identified that are associated with specific cancers.
  • tumor antigen and “cancer antigen” are used interchangeably to refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells.
  • Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells.
  • cancer antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens.
  • Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g. , activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations.
  • Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.
  • MAGE 1, 2, & 3 defined by immunity
  • MART-l/Melan-A, gplOO carcinoembryonic antigen
  • CEA carcinoembryonic antigen
  • HER-2 HER-2
  • mucins i.e., MUC-1
  • PSA prostate-specific antigen
  • PAP prostatic acid phosphatase
  • viral proteins such as proteins of hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.
  • HBV hepatitis B
  • EBV Epstein-Barr
  • HPV human papilloma
  • the methods further comprise administering a chemotherapeutic agent to the subject being administered an agent(s) for inhibiting
  • Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly crypto
  • pancratistatin a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
  • neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,
  • aminoglutethimide aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • aldophosphamide glycoside aminolevulinic acid
  • eniluracil amsacrine
  • bestrabucil bisantrene
  • edatraxate defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
  • pirarubicin losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton,
  • DMFO difluoromethylornithine
  • leucovorin LV
  • oxaliplatin including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF- A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of treatment can further include the use of radiation.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as an autoimmune disease, a chronic infection or a cancer.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term "effective amount” as used herein refers to the amount of an agent(s) for inhibiting PD1, LAP and/or CEACAM1 needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect, i.e., inhibit T cell tolerance, for example.
  • the term "therapeutically effective amount” therefore refers to an amount of an agent(s) for inhibiting PD1, LAP and/or CEACAM1 using the methods as disclosed herein, that is sufficient to effect a particular effect when administered to a typical subject.
  • an effective amount as used herein would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of disease. Thus, it is not possible to specify the exact "effective amount”. However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the agent(s) for inhibiting PD1, LAP and/or CEACAM1), which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
  • IC50 i.e., the concentration of the agent(s) for inhibiting PD1, LAP and/or CEACAM1
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • agents useful according to the compositions and methods described herein, including antibodies and other polypeptides are isolated agents, meaning that the agents are substantially pure and are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.
  • the agents are sufficiently pure and are sufficiently free from other biological constituents of their host cells so as to be useful in, for example, producing pharmaceutical preparations.
  • an isolated agent may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the agents may comprise only a small percentage by weight of the preparation.
  • agents described herein for inhibiting PD1, LAP and/or CEACAM1 can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject.
  • administering and “introducing” are used interchangeably and refer to the placement of a polypeptide agent into a subject by a method or route which results in at least partial localization of such agents at a desired site, such as a site of inflammation, such that a desired effect(s) is produced.
  • the agents described herein for inhibiting PD1, LAP and/or CEACAM1 are administered to a subject by any mode of administration that delivers the agent systemically or to a desired surface or target, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration.
  • any mode of administration that delivers the agent systemically or to a desired surface or target
  • oral administration forms are also contemplated.
  • injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • the agents for inhibiting PDl, LAP and/or CEACAMl for use in the methods described herein are administered by intravenous infusion or injection.
  • parenteral administration and “administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection.
  • systemic administration refers to the administration of an agent for inhibiting PDl, LAP and/or CEACAMl other than directly into a target site, tissue, or organ, such as a tumor site, such that it enters the subject's circulatory system and, thus, is subject to systemic metabolism and other like processes.
  • administration of an agent for inhibiting PDl, LAP and/or CEACAMl can include formulation into pharmaceutical compositions or pharmaceutical formulations for parenteral administration, e.g., intravenous; mucosal, e.g., intranasal; ocular, or other mode of administration.
  • an agent for inhibiting PDl, LAP and/or CEACAMl described herein can be administered along with any pharmaceutically acceptable carrier compound, material, or composition which results in an effective treatment in the subject.
  • a pharmaceutical formulation for use in the methods described herein can contain an agent for inhibiting PDl, LAP and/or CEACAMl as described herein in combination with one or more pharmaceutically acceptable ingredients.
  • phrases "pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, an agent for inhibiting PDl, LAP and/or CEACAMl .
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in maintaining the stability, solubility, or activity of, an agent for inhibiting PDl, LAP and/or CEACAMl .
  • Each carrier must be
  • compositions which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose,
  • agents for inhibiting PD1, LAP and/or CEACAM1 described herein can be specially formulated for administration of the compound to a subject in solid, liquid or gel form, including those adapted for the following: (1) parenteral administration, for example, by
  • subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation
  • topical application for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin
  • intravaginally or intrarectally for example, as a pessary, cream or foam
  • ocularly (5) transdermally; (6) transmucosally; or (79) nasally.
  • a bispecific or multispecific polypeptide agent can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et ah, Ann. Rev. Pharmacol. Toxicol.
  • Parenteral dosage forms of an agent for inhibiting PD1, LAP and/or CEACAM1 can also be administered to a subject by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.
  • Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • Aerosol formulations An agent for inhibiting PD1, LAP and/or CEACAM1 can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • An agent for inhibiting PD1, LAP and/or CEACAM1 can also be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • An agent for inhibiting PD1, LAP and/or CEACAM1 can also be
  • Suitable powder compositions include, by way of illustration, powdered preparations of an agent for inhibiting PD1, LAP and/or CEACAM1 thoroughly intermixed with lactose, or other inert powders acceptable for intrabronchial administration.
  • the powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which can be inserted by the subject into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation.
  • the compositions can include propellants, surfactants, and co-solvents and can be filled into conventional aerosol containers that are closed by a suitable metering valve.
  • Aerosols for the delivery to the respiratory tract are known in the art. See for example,
  • the formulations of the agents for inhibiting PD1, LAP and/or CEACAM1 described herein further encompass anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, since water can facilitate the degradation of some compounds.
  • water e.g., 5%
  • water is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles &
  • Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • Anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g. , vials) with or without desiccants, blister packs, and strip packs.
  • an agent for inhibiting PD1, LAP and/or CEACAM1 can be administered to a subject by controlled- or delayed-release means.
  • the use of an optimally designed controlled- release preparation in medical treatment is characterized by a minimum of drug substance being employed to address or control the condition in a minimum amount of time.
  • Controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a compound of formula (I) is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the agents for inhibiting PD1, LAP and/or CEACAM1 described herein. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770;
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View,
  • ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug.
  • anion exchangers include, but are not limited to, Duolite® A568 and Duolite® AP143 (Rohm&Haas, Spring House, Pa. USA).
  • an agent for inhibiting PD1, LAP and/or CEACAM1 for use in the methods described herein is administered to a subject by sustained release or in pulses.
  • Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administration of the same dose of the composition at a reduced frequency or administration of reduced doses.
  • Sustained release or pulse administrations are particularly preferred when the disorder occurs continuously in the subject, for example where the subject has continuous or chronic symptoms of a viral infection.
  • Each pulse dose can be reduced and the total amount of the agent for inhibiting PD1, LAP and/or CEACAM1 administered over the course of treatment to the patient is minimized.
  • the interval between pulses when necessary, can be determined by one of ordinary skill in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or the active component of the composition is no longer detectable in the subject prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Intervals can be calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater than the composition half- life.
  • Various methods and apparatus for pulsing compositions by infusion or other forms of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
  • compositions for enhancing the immune response or decreasing T cell tolerance in a subject comprising at least two agents selected from the group consisting of:
  • CEACAM1 inhibitory agent a CEACAM1 inhibitory agent
  • PD1 inhibitory agent a PD1 inhibitory agent
  • LAP inhibitory agent a CEACAM1 inhibitory agent
  • composition of paragraph 1 wherein the two agents are a CEACAM1 inhibitory agent and a PD1 inhibitory agent.
  • composition of paragraph 1, wherein the two agents are a CEACAM1 inhibitory agent and a LAP inhibitory agent.
  • composition of paragraph 1, wherein the two agents are a PD1 inhibitory agent and a LAP inhibitory agent.
  • composition of paragraph 1 comprising a CEACAM1 inhibitory agent, a PD1 inhibitory agent, and a LAP inhibitory agent.
  • composition of any of paragraphs 1-5 comprising a bispecific agent comprising a first portion which is a CEACAM1 inhibitory agent and a second portion which is a PD1 inhibitory agent.
  • composition of any of paragraphs 1-5 comprising a bispecific agent comprising a first portion which is a CEACAM1 inhibitory agent and a second portion which is a LAP inhibitory agent.
  • composition of any of paragraphs 1-5 comprising a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PD1 inhibitory agent.
  • composition of paragraph 10 wherein the antibody reagent binds a target selected from the group consisting of:
  • CEACAM1 PD1 ; and LAP.
  • composition of paragraph 11 wherein the antibody reagent inhibits signaling mediated by the target.
  • the CEACAMl inhibitory agent binds a CEACAMl molecule having the sequence set forth in SEQ ID NO:32, or an allelic or splice variant of SEQ ID NO: 32.
  • composition of paragraph 14 wherein the CEACAMl inhibitory agent binds to an amino acid residue corresponding to any of amino acid residues 1-429 of SEQ ID NO: 32.
  • composition of paragraph 22, wherein the LAP inhibitory agent binds to an amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 33.
  • a method for treating a chronic disease comprising administering to a subject in need of treatment thereof an effective amount of a composition of any of paragraphs 1-23.
  • EXAMPLE 1 Tumor infiltrating lymphocytes co-express CEACAMl together with other accessory molecules that are associated with T cell tolerance and exhaustion.
  • Expression of inhibitory molecules on lymphocytes plays a major role in limiting immune defenses against cancers. Inhibition of these types of molecules is increasingly being shown to permit natural cancer defenses in association with the eradication of tumors.
  • Molecules such as TIM3, CTLA4, LAP, PDl and more recently CEACAMl have been demonstrated to be increased on tumor infiltrating lymphocytes and, in some cases, to be associated with poor prognosis.
  • blockade of these pathways has been demonstrated to be associated with increased cancer responses in mouse models and more recently in humans at least with respect to studies with CTLA4 and PDl . However, blockade of these pathways individually is associated with only limited benefit in humans.
  • CEACAMl and PDl positive (double- positive) T cells are increased in tumor-infiltrating lymphocytes relative to that which is observed on lymphocytes under physiologic circumstances and increased association between CEACAMl and LAP expression on T cells in vivo.
  • Inhibition of two or more of CEACAMl, PDl, and/or LAP together in vivo is a superior therapeutic approach to inhibition of a single factor alone.
  • AOM-DSS model The expression of a tolerance associated phenotype through combinations of cell surface molecules such as PD-1, TIM-3, LAP and CEACAMl was examined using the AOM-DSS model in wild-type mice (see, e.g. De Robertis et al. J Carcinog 2011 10:9; which is incorporated by reference herein in its entirety). Briefly, a dose of azoxymethane (AOM) with subsequent doses of the inflammatory agent dextran sodium sulphate (DSS) ( Figure 1) increases the onset of colorectal cancer, and accordingly, T cell tolerance of the associated tumor infiltrating lymphocytes in approximation to the colorectal cancer cells.
  • AOM azoxymethane
  • DSS dextran sodium sulphate
  • T cells obtained from these mice at Day 44 of the AOM-DSS protocol were subjected to FACS analysis.
  • Tumor infiltrating lymphocytes, tumor intra-epithelial lymphocytes, adjacent tumor infiltrating lymphocytes, adjacent tumor intraepithelial lymphocytes, and cells from the mesenteric lymph nodes were examined for cell surface expression of CD4, CD8, PDl, CEACAMl, and TIM3 ( Figures 2-4).
  • the results of these analyses are summarized in Figures 5A-5B, demonstrating that tumor infiltrating lymphocytes, adjacent tumor infiltrating lymphocytes and adjacent tumor intraepithelial lymphocytes contain significant subpopulations of CEACAMl + PD1 + cells.
  • Figures 5A-5B depict graphs demonstrating CEACAMl and PDl are co-expressed on the cell surface of chronically activated CD4 + ( Figure 5A) and CD8 + ( Figure 5B) tumor infiltrating T lymphocytes.
  • EXAMPLE 2 T lymphocytes with CEACAMl expression are characterized by co-expression of accessory molecules associated with T cell tolerance and a regulatory phenotype.
  • CEACAMl is preferentially expressed on specific subsets of circulating and tissue-resident CD4+ T cells. Attention was focused on defining the accessory molecules expressed by the small subset of CEACAMl + T cells detected under physiologic conditions in blood (data not shown) and mesenteric lymph node (MLN) (see Figure 6A) and which were increased in frequency in PP and LP of WT mice ( Figure 9A and 9B). It was observed that CD4+CEAC AM 1 + T cells from the blood, MLN, PP and LP expressed higher amounts of both CD44 and CD62L than did
  • CD4+CEACAM 1 - T cells ( Figure 6A and 9A and 9B). This is consistent with CEACAMl being associated with effector and/or central memory T cells (Boulton et al., 2002). Additionally, it was noted that CD4+CEACAM1+ T cells from each of these tissue compartments expressed more ⁇ 4 ⁇ 7- integrin than their CD4+CEACAM1 -counterparts, suggesting that CEACAMl expression on T cells, even those within the peripheral immune system, may be associated with enhanced potential to home to intestinal tissues.
  • CEACAMl positivity was associated with preferential expression of PD1, LAP (TGFP Latency Associated Peptide) and B220, all of which have previously been associated with T cell subsets possessing regulatory or accessory potential in the production of IgA (Fagarasan et al., 2001 ; Good-Jacobson et al., 2010; Ochi et al., 2006; Oida et al., 2003).
  • CD4+CEACAM 1 +CD25+ cells (1.52 ⁇ 0.11) isolated from the spleen and LN (data not shown).
  • the relative expression of the L and S isoforms does not differ between CD4+CEACAM1+CD25- and CD4+CEACAM1+CD25+ regulatory T cells with the L isoform predominating in both populations.
  • the L:S ratio was 1.52 for CD4+CEACAM1+CD25- cells and 1.52 for CD4+CEACAM 1 +CD25+ cells.
  • Cells were isolated by flow cytometric sorting from pooled spleen and MLN. These data demonstrate the existence of a close association between the expression of CEACAM1 and LAP.
  • CEACAMl-long and CEACAMl-short isoforms are regulated by tolerogenic signals provided by anti-CD3 stimulation.
  • CEACAM1 Carcinoembryonic antigen cell adhesion molecule 1
  • a member of the carcinoembryonic antigen family is engaged in intercellular binding interactions that affect signal transduction by a number of cell surface receptors.
  • CEACAM1 is constitutively expressed by a variety of cell types including those of endothelial, epithelial and hematopoietic origin such as B cells, polymorphonuclear leukocytes, monocytes, and dendritic cells.
  • CEACAM1 is expressed at minimal levels in circulating natural killer (NK) and T cells but is upregulated in these cell types following a variety of different forms of activation (Azuz-Lieberman et al., 2005; Boulton et al., 2002;
  • CEACAM1 can associate with and modify the function of numerous signaling receptors that are dictated by the
  • CEACAM1 isoforms expressed.
  • the individual CEACAM1 isoforms are generated by alternative splicing and consist of transmembrane proteins that possess a characteristic membrane distal, IgV-like domain (the N-domain) which functions in homophilic binding but differs with respect to the number of membrane proximal extracellular IgC2-like domains and the length of their cytoplasmic (cyt) tail
  • the 11 human and 4 mouse CEACAM1 splice variants encode either a short (S) cyt tail about which little is known or a long (L) cyt tail containing immune receptor tyrosine -based inhibitory motifs (ITIM).
  • ITIM immune receptor tyrosine -based inhibitory motifs
  • CEACAMl-L isoforms is triggered by the phosphorylation of the ITIM tyrosine residues by receptor tyrosine kinases or Src -related kinases, resulting in recruitment of the Src homology 2 (SH2) domain-containing protein-tyrosine
  • CEACAMl-L-mediated recruitment of these phosphatases results in the dephosphorylation of critical tyrosines contained within activating motifs associated with a variety of cell surface receptors such as the B cell receptor, epidermal growth factor receptor and T cell receptor (TCR)/CD3 complex (Abou-Rjaily et al., 2004; Boulton et al., 2002; Chen et al., 2008; Lobo et al., 2009).
  • TCR epidermal growth factor receptor and T cell receptor
  • CEACAMl -L isoforms are functionally inhibitory and are the major isoforms described in mouse and human lymphocytes including NK cells, B cells and T cells.
  • CEACAMl -L and CEACAMl -S isoforms are expressed at varying ratios in different cell types with recent evidence showing that such expression is under the influence of cytokines (e.g. inter feron- ⁇ ), transcription factors (e.g. IRF-1) and splicing regulators (e.g. members of the heterogeneous nuclear ribonucleoprotein family) (Chen et al., 1996; Dery et al. 2011 ; Gencheva et al., 2010). Although not directly demonstrated, this is believed to be the case for T lymphocytes as well.
  • cytokines e.g. inter feron- ⁇
  • transcription factors e.g. IRF-1
  • splicing regulators e.g. members of the heterogeneous nuclear ribonucleoprotein family
  • CEACAMl -S isoforms can be expressed (Donda et al., 2000; Singer et al., 2002).
  • CEACAMl is a 'tunable' receptor system given that CEACAMl -S isoforms may possess weak co-stimulatory function that is capable of ameliorating the inhibitory signals provided by CEACAMl -L isoforms in the context of TCR/CD3 complex signaling when expressed together or novel signals (Chen et al., 2004a; Chen et al., 2008; Lee et al., 2008).
  • CEACAMl -L variants it was found that T cells within the intestines, relative to peripheral tissues such as the spleen, not only exhibit increased overall CEACAMl expression but also uniquely possess a relative overabundance of CEACAMl -S relative to CEACAMl -L isoforms. Moreover, in this environment, it is shown herein that CEACAMl -S isoforms have special physiological functions which at a cellular level enhance T cell activation independently of CEACAMl -L isoforms and are preferentially expressed in cells performing common mucosal immune functions including T cell regulation and the induction of secretory immunity. Specifically, it is demonstrated herein that
  • CEACAMl -S acts as an individual signaling unit which promotes the induction of CD4+LAP+ T regulatory (Treg) cells (Figure 7A-7D), CD4+PD 1 +CXCR5+ follicular helper T cells (3 ⁇ 4) (Figure
  • T cell-associated CEACAMl -S isoforms lead to enhanced mucosal IgA secretion which influences both the composition of commensal microbiota and host defense against exposure to pathogens such as Listeria monocytogenes (data not shown).
  • CEACAMl expression these barrier-protective functions of IgA are diminished. Together, these studies provide tissue-specific evidence for CEACAMl isoform regulation and function.
  • CEACAMl isoforms were assessed by first establishing a novel, quantitative assay for analyzing expression of mouse CEACAMl -L and -S variants as currently available antibodies are not able to distinguish between these two general classes of
  • CEACAMl in a quantitative fashion. Two pairs of real-time PCR primers were designed to specifically quantify these in mouse T cells. The specificity and efficiency of these primers was verified using mouse (m) CEACAMl -4L (4L) and CEACAMl -4S (4S) plasmids ( Figure 8).
  • CEACAMl -S isoforms were detected, CEACAMl -L isoforms predominated in peripheral T cells such that the CEACAMl -L:CEACAM1 -S (L:S) ratio was 1.95 ⁇ 0.34, consistent with previous studies (Singer et al., 2002). Having validated this qPCR assay with splenic T cells, a broad survey of CEACAMl isoform expression in primary T cells isolated from several different tissues was performed.
  • CD3+ T cells isolated from spleen or MLN wherein CEACAMl expression was low and CEACAMl -L isoforms predominated over CEACAMl -S isoforms CD3+ T cells from the intestinal LP of the duodenum, jejunum, ileum or colon and Peyer's patches (PP) were observed to express an overall increase in CEACAMl transcript levels with significantly greater quantities of CEACAMl -S isoforms relative to CEACAMl -L isoforms.
  • LP T cells of the PP and colon contained the highest levels of CEACAMl and the lowest L:S ratios relative to all other compartments examined. This was verified by immunoblotting with the CC1 mAb, further confirming that T cells isolated from PP and colonic LP expressed higher CEACAMl amounts relative to peripheral blood and especially CEACAMl -S isoforms which was most notable in the colonic LP (data not shown).
  • T cells were purified from spleen (SP), PP, colon lamina intestinal (LP) of WT mice and spleen of 4S Tg and 4L Tg mice as well as B cells from spleen of WT mice. These studies both confirm the increased expression of
  • CEACAMl within intestinal T cells and further indicate that CEACAMl -S isoforms are not only expressed by primary T cells but establish that they are a major isoform within intestinal T cells and potentially other tissue compartments.
  • CEACAM1-S independently regulates the expansion and function of CD4+ T cells in which it is preferentially expressed. The observations described herein, of preferential
  • CEACAMl expression on CD4+CD25-LAP+ from WT mice led to a question of the ability of CEACAMl to regulate these cells and, further, to interrogate the relative contribution of CEACAMl - L and CEACAMl -S isoforms in this process.
  • Tg CEACAMl transgenic mice that over- express CEACAMl -specifically in T cells
  • the frequency of CD4+CD25-LAP+ T cells in mucosal tissues was examined under conditions of forced over-expression of the CEACAMl -4L or -4S isoforms.
  • CD4+CD25-LAP+ T cells accumulate in intestinal tissues under steady-state and inflammatory conditions and that orally administered CD3 -specific antibodies induce CD4+CD25-LAP+ regulatory T cells in MLN (Boirivant et al., 2008; Ochi et al.,
  • CEACAMl expression significantly increased the percentage of CD4+CD25-LAP+ T cells in the MLN of WT but not CEACAMl " ' " mice ( Figure 7F).
  • CEACAMl -4S-mediated enrichment of T FH cells occurred independently of CEACAMl-L isoforms since CEACAMV 1 - A Tg mice also contained higher levels of T m cells than their CEACAMV/- counterparts ( Figure 13C).
  • these data are consistent with a role for CEACAM1 and especially the CEACAMl-S isoforms in promoting the development of two types of accessory cells that express LAP (CD4+CD25+LAP+ T cells) and PD1 (T FH cells) which are not only enriched in organized lymphoid structures and mucosal tissues but are also known to be involved in immune tolerance and B cell switching (Cerutti, 2008; Good-Jacobson et al.).
  • CEACAMl-S isoforms provide unique activating signals that sensitize T cells to activation induced cell death and promote the development of regulatory populations of T cells.
  • Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail that signals at least in part via NFAT (data not shown).
  • L long
  • S short
  • CEACAMl-L isoforms predominate with little CEACAMl-S isoforms in mouse and human.
  • CEACAM1-4S functions as an independent signaling unit in the induction of T cell activation.
  • CEACAMl-L isoform expression in T cells is well known to provide important negative regulation of TCR/CD3 complex function after ITIM phosphorylation by the src- related kinase, p56 lck , and recruitment of SHP1 resulting in dephosphorylation of CD3 ⁇ and ZAP70 (Chen et al., 2008), very little is known about CEACAM1-S function in general or in T cells in particular.
  • CEACAMl -S splice variants Previous results in T cells are limited to transfected in vitro model systems and have been controversial with both stimulatory and inhibitory functions assigned to CEACAMl -S splice variants (Chen et al., 2004a; Chen et al., 2004b).
  • CEACAMl -4S (4S) transgenic (Tg) mice were generated, overexpressing the mouse 4S isoform specifically in T cells under the human CD2 (hCD2) promoter in order to explore the function of CEACAMl -S isoforms in T cells in vivo.
  • Intracellular cytokine staining of the CD4+ T cells isolated from spleen, MLN and PP also revealed that IL-2, IL-4, IL-10, IL-17, IFN- ⁇ and TGF- ⁇ were all increased in the T cells of 4S Tg mice when compared to that observed in WT littermates (data not shown).
  • CEACAMl -S induction of cytokines (interferon- ⁇ ) influenced the total CD4+ T cell population (CD4+ T cells isolated from PP of WT or 4S Tg mice) consistent with a broad activating function of the 4S isoform when expressed at high levels (data not shown).
  • CD3+ T cells isolated from the spleen of 4S Tg mice also exhibited a higher proliferation rate and more IFN- ⁇ and IL-2 secretion upon anti-CD3 stimulation ex vivo in comparison to that associated with T cells from WT littermate controls (data not shown).
  • the proliferation rate of splenic CD3+ T cells, as measured by [ 3 H] -thymidine incorporation as well as the production of IFN- ⁇ and IL-2, from the 4S Tg mice was observed to be lower than that detected with the WT littermates after stimulation, consistent with in vitro T cell collapse due to increased activation-induced cell death (AICD) in the presence of the CEACAM1-4S isoform ( Figure 12B).
  • AICD activation-induced cell death
  • mice 4S isoform both co-stimulates primary T cells and sensitizes them to activation induced cell death (AICD), supporting previous in vitro transfection studies that have ascribed activating functions to this CEACAMl isoform in contrast to the inhibitory functions of the L isoform (Chen et al., 2004a; Chen et al., 2004b).
  • CEACAM1-4S The activating function of CEACAM1-4S in T cells is observed independently of CEACAM1-L isoform expression. Since the studies performed in 4S Tg mice were undertaken in the context of endogenous CEACAMl expression, it was possible that the apparent activating and inhibiting functions of the 4S isoform were either the consequence of CEACAMl -mediated neutralization of CEACAMl -L inhibitory function and/or the result of a direct activating signal by CEACAMl -S. To examine this question, 4S Tg mice that lacked expression of any other CEACAMl isoforms were generated by crossing 4S Tg mice with CEACAMl '1' mice on a C57BL/6 background (CEACAMl ' -AS Tg).
  • Ceacaml- ⁇ ' ' -AS Tg mice still contained more activated T cells in vivo as revealed by lower percentages of CD4+(or CD8+) CD62LhiCD441o naive T cells, and higher percentages of CD4+(or CD8+) CD62LloCD44hi effector memory T cells in spleen, MLN, PP and colon LP (data not shown) in comparison to CEACAMl '1' littermates.
  • CEACAMl '1' -AS Tg mice also exhibited more apoptotic, Annexin+ CD3+ T cells within the MLN, blood and PP ( Figure 12E) inversely correlating with decreased levels of CD3+ cells (data not shown).
  • CD4+ T cells isolated from the spleens of CEACAMl '1' -4S Tg mice also proliferated more vigorously and secreted more IL- 2 and IFN- ⁇ upon anti-CD3 stimulation.
  • splenic CD4+ T cells from CEACAMl ' ' mice exhibited increased activation upon anti-CD3 stimulation that was intermediate to the anti-CD3 induced stimulation observed with WT and CEACAMl '1' -A Tg T cells (data not shown).
  • splenic T cells from mice in which only the CEACAMl -4L (4L) isoform was expressed by T cells, generated by crossing previously established 4L Tg mice (Nagaishi et al., 2006) with
  • CEACAMl '1' mice exhibited a blunted response to anti-CD3 stimulation which was less than that observed with WT T cells and is consistent with the direct inhibitory functions of this isoform.
  • loss of dominant endogenous CEACAMl -L inhibitory function in CEACAMl ' mice or gain of CEACAMl -S expression independently of CEACAMl -L isoforms, as observed in CEACAMl '1' -AS, Tg T cells results in enhanced TCR/CD3 complex signaling and sensitization to activation induced cell death in the latter case.
  • gain of CEACAMl -L isoforms independently of CEACAMl -S isoforms leads directly to inhibition of T cell function.
  • CEACAM1-S and CEACAM1-L are able to function as independent activating and inhibitory signaling units in T cells that are in the end both associated with immune tolerance.
  • this activating function is associated ultimately with regulation of immune responses as it results in sensitization to activation induced cell death and the promotion of T cells with regulatory and thus inhibitory function such as CD4+CEACAM1+LAP+ T cells.
  • the CEACAMl -L isoforms directly deliver inhibitory signals to T cells.
  • CEACAMl the association of CEACAMl with specific types of cell surface molecules that are associated with immune tolerance such as TIM3, PD-1 and LAP as well as the regulation of
  • CEACAMl splicing in T cells which demonstrates the important role played by CEACAMl -S isoforms and the unique predominance of CEACAMl -S isoforms in intestinal resident T cells and specific subsets of T cells associated with immune regulation and humoral immunity.
  • CEACAMl is an activation-induced molecule on T cells, the observed tissue-specific splicing was not due to activation of the T cells per se as it was not observed when T cells were stimulated ex vivo.
  • Adoptive transfer experiments demonstrated that CEACAMl -S dominance was gained by naive peripheral T cells upon entry into the intestines, and was lost when mucosal T cells were removed from the intestines and re-stimulated ex vivo.
  • CEACAMl -S facilitates T cell activation, sensitization to activation induced cell death and the induction of secretory IgA immunity and cells with regulatory function.
  • T cell subsets which bear a memory phenotype characteristic of mucosal tissues (Gibbons et al., 2011) and specific subsets of T cells such as CD4+CD25-LAP+ and T m cells which possess regulatory functions and encourage secretory immunity display an enrichment upon CEACAM1-S expression and are driven by its presence.
  • CEACAMl -S expression is promoted and maintained by the mucosal environment and facilitates T cell activation linked to the generation of cell types responsible for mucosal regulation, tolerance and protection.
  • CEACAMl -S is not a minor isoform in primary T cells as was currently thought (Gray-Owen et al., 2006).
  • CEACAMl -S provides unique co-stimulatory signals which promote both activation induced cell death and an effector memory phenotype, whereas CEACAMl -L is directly co-inhibitory for TCR/CD3 complex signaling
  • CEACAMl -S in T cells can elicit this stimulation as an independent signaling unit without CEACAMl -L assistance in vivo.
  • CEACAMl -S regulates specific subsets of T cells.
  • CD4+LAP+ T cells and T m cells which are an integral component of mucosal tissues and organized lymphoid structures, respectively, and are known to regulate immune tolerance and secretory immunity (Linterman et al., 2011). Accumulation of such cell surface molecules such as CEACAMl together with PD1, TIM3 and LAP as well as cell types associated with these cell surface molecules (e.g. CD4+CEACAM 1 +LAP+ or CD4+CEACAM+PD 1 + T cells) amongst tumor infiltrating lymphocytes would be predicted to be deleterious for the host resulting in either direct suppression of effector T cell function or inhibition of the effector T cells through the effects of the accumulated regulatory T cells.
  • CEACAM1-S signaling in T cells, in epithelial cells it has been shown to associate with the cytoskeleton (actin and tropomyosin), calmodulin and annexin II (Edlund et al., 1996; Kirshner et al., 2003; Schumann et al., 2001). Such intracellular associations have interestingly been linked to the promotion of apoptosis of mammary epithelial cells during acini morphogenesis (Kirshner et al., 2003).
  • CEACAM1-S signaling was able to activate NFAT in vivo (Chen et al., 2004b). As shown here, this was associated with the upregulation of NFAT-dependent soluble and cell surface molecules such as PD1 and CD40L (data not shown).
  • these studies not only demonstrate the importance of CEACAM1-S in T cells under physiological conditions but also their role in delivering independent signals that may oppose those provided by CEACAM1-L isoforms and drive the cell to a unique destination that is associated with either cell death or a regulatory function.
  • Wild-type (WT) C57BL/6 (B6) mice were purchased from The Jackson Laboratory.
  • hCD2 promoter controlled CEACAM1-4S transgenic mice were generated by a similar strategy as previously described in the of Brigham and Women's Hospital Transgenic Core Facility (Nagaishi et al., 2006).
  • CEACAM l '1' rmce were previously described (Leung et al., 2006).
  • CC1 antibody for 20 min followed by FITC-conjugated rat anti-mouse IgGl .
  • FITC-conjugated rat anti-mouse IgGl For other cell surface proteins, cells were stained with the indicated fluorescence-conjugated antibodies for 20 minutes, washed and resuspended with 1% BSA/PBS FACS staining buffer containing DAPI (Invitrogen). Cell apoptosis assays were performed according to the Annexin V-FITC Apoptosis Detection Kit manual
  • cytokine staining cells were stimulated with ionomycin (Sigma, 2 tg/ml), PMA (Sigma, 30 ng/ml) and GOLGISTOPTM (BD Bioscience) for 4 hours followed by intracellular cytokine staining using CYTOFIX/CYTOPERMTM solution kit according to the manufacturer's instructions (BD Bioscience). IgA staining was performed according to the instructions provided with the rat anti-mouse IgA antibody (CI 0-3).
  • CD4+LAP+ T cells were stained for 20 min with biotinylated anti-LAP antibody (R&D Systems) followed by APC-conjugated streptavidin (BD Biosciences) as described previously (Ochi et al., 2006).
  • T FH cells were stained for CXCR5 and PD1 as described previously (Linterman et al., 2011). All stained cells were analyzed on an LSRIITM (BD Biosciences) or a MACSQUANTTM (Miltenyi Biotech) flow cytometer, and data were analyzed using FLOWJOTM software.
  • mice were fed with anti-CD3 antibody using a protocol described previously (Ochi et al., 2006). Specifically, groups of paired mice were fed anti-CD3 or IgG isotype control antibody at a dose of 5 ⁇ g/mouse/day for 5 days. After one day's rest, mice were sacrificed at day 7 and the percentage of CD4+LAP+ regulatory T cells was determined in specific lymphoid tissues.
  • Regulatory T cell suppression assays were performed as previously described (Fantini et al., 2007). Briefly, 1x105 freshly isolated splenic CD4+CD25- T cells were stained with CFSE and mixed with an equal number of either CD4+CD25+ or CD4+LAP+ T regulatory cells isolated via magnetic sorting (Miltenyi). T cell mixtures were then activated in the presence of soluble anti-CD3 (1 ⁇ g/ml) and 4x10 5 irradiated total splenocytes as antigen presenters. After 4 days of culture, CFSE dilution of the responder CD4+CD25- T cells was measured as an indication of proliferation.
  • FLUROTM 568 conjugated goat anti-rat IgG secondary antibody Invitrogen
  • FITC-conjugated anti-mouse B220 antibody BD Bioscience
  • NFAI staining the slides were stained with biotin conjugated-anti-mouse CD4 antibody and rabbit anti-NFAll antibodies for 2 hours at RI, followed by incubation with rhodamine red-X-streptavidin (Jackson ImmunoResearch) and ALEXA FLUOR® 488 F(ab')2 fragment of goat anti-rabbit IgG (H+L) (Invitrogen) for 1 hour at RI.
  • the counterstaining was performed using DAPI (Invitrogen).
  • the specimens were analyzed using a NIKON ECLIPSE IITM microscope.
  • GAPDH and 13-actin antibodies were obtained from Sigma- Aldrich.
  • Biotinylated anti-LAP antibody was from R&D Systems (Ochi et al., 2006). All other antibodies used for flow cytometry or immunohistochemistry staining were purchased from BD Biosciences, eBioscience or Biolegend. Human rIL-2 for the stimulation of mouse primary T cells was provided by the National Institutes of Health.
  • Lymphocyte Isolation Single cell suspensions of mononuclear cells from spleen, lymph nodes, blood, Peyer's patches (PP) (Iwasaki and Kelsall, 1999) or intestinal lamina propria (LP) were prepared as previously described (Nakajima et al., 2002).
  • PP or intestines were treated with media containing dithiothreitol 100 ⁇ g/ml (Sigma), 25 mM Hepes, 10% fetal bovine serum (FBS), and 5 mM EDTA in RPMI1640 for 30 min at 37 °C to remove epithelial cells, and were washed extensively with RPMI1640.
  • Tissues were further digested in RPMI 1640 containing 2% FBS, collagenase D (400 U/ml, Roche) and DNase I (0.1 mg/ml, Sigma) in a shaking incubator at 37 °C for 30 min.
  • Cells were then layered on a 40-75%) Percoll gradient (GE Healthcare) and lymphocyte-enriched populations were isolated from the cells at the 40-75%) interface. After washing, single cell suspensions were prepared. Cells were directly used for flow staining, or were stained with PE conjugated anti-CD3 antibody and followed by anti-PE MicroBeads according to the instructions provided by Miltenyi Biotech to further isolate CD3+ T cells.
  • CD3+ or CD4+ T cells were isolated from spleen and/or lymph nodes of mice by pan T cell isolation kit or CD4+ T cell isolation kit for mouse (Miltenyi Biotech) according to the
  • CAGAAGGAGCCAGATCCG-3 ' reverse; SEQ ID NO: 38.
  • hnRNP expression was determined by qPCR, as described previously (Dery et al., 2011).
  • CEACAMl modulates epidermal growth factor receptor— mediated cell proliferation. J. Clin. Invest. 114, 944-952.
  • Boirivant M., Amendola, A., Butera, A., Sanchez, M., Xu, L., Marinaro, M., Kitani, A., Di Giacinto, C, Strober, W., and Fuss, I. J. (2008).
  • a transient breach in the epithelial barrier leads to regulatory T-cell generation and resistance to experimental colitis.
  • TGF-beta receptor signaling is critical for mucosal IgA responses. J. Immunol. 173, 3305-3309.
  • TGF-beta receptor controls B cell responsiveness and induction of IgA in vivo. Immunity 13, 443-451.
  • the cell-cell adhesion molecule carcinoembryonic antigen- related cellular adhesion molecule 1 inhibits IL-2 production and proliferation in human T cells by association with Src homology protein- 1 and down-regulates IL-2 receptor. J. Immunol. 172, 3544- 3552.
  • Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulate human T cell function. J. Immunol. 172, 3535-3543.
  • Biliary glycoprotein (BGPa, CD66a, CEACAMl) mediates inhibitory signals.
  • BGPa, CD66a, CEACAMl Biliary glycoprotein
  • Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70.
  • Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine -protective regulatory T cells. Eur. J. Immunol. 39, 3134-3146.
  • CEACAM1 carcinoembryonic antigen-related cell adhesion molecule- 1
  • Calmodulin binds to specific sequences in the cytoplasmic domain of C-CAM and down-regulates C-CAM self-association. J. Biol. Chem. 271, 1393-1399.
  • XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease.
  • CEACAM1 a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis. J. Biol. Chem. 278, 50338-50345.
  • CEACAMs their role in physiology
  • CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1
  • CEACAM1 is a negative coreceptor for the B cell receptor and promotes CD19-mediated adhesion of B cells in a PI3 Independent manner. J. Leukoc. Biol. 86, 205-218.
  • CD4+CD25- T cells that express latency-associated peptide on the surface suppress
  • CD4+CD45RB high-induced colitis by a TGF-beta-dependent mechanism. J. Immunol. 170, 2516- 2522.
  • Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin. J. Biol. Chem. 276, 47421-47433.
  • Colitis was induced in mice by adoptive transfer of naive CD4+ T cells in Rag- deficient and CEACAM1 -deficient animals and expression of CEACAM1 and PD1 was examined in lamina intestinal lymphocytes infiltrating the colon. Co-expression of CEACAM1 and PD1 is shown in Figure 15, after gating on CD3+, in lymphocytes obtained from proximal, middle and distal colon.
  • TILs Tumor infiltrating lymphocytes
  • TILs were isolated from subcutaneous tumors associated with CT26 colorectal carcinoma cell line. TILs were stimulated with anti-CD3 and anti- CD28 monoclonal antibodies in the presence of isotype control antibodies or antibodies that block different co-inhibitory cell surface molecules. This blockade of CEACAM1 and PD1 increases TNF-a production from tumor infiltrating lymphocytes (TIL). The data demonstrates a synergistic increase of TNF-a production by TILs treated with anti-CEACAMl and anti-PDl antibodies.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Endocrinology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Biotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Steroid Compounds (AREA)

Abstract

The technology described herein relates to compositions and methods for enhancing the immune response and/or reducing T cell tolerance in a subject. In some embodiments, the technology described herein relates to administering inhibitors of two or more of CEACAM1, PD1, and/or LAP to, e.g., synergistically reduce T cell tolerance.

Description

ENHANCEMENT OF THE IMMUNE RESPONSE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional
Application No. 61/713,203 filed October 12, 2012, the contents of which is incorporated herein by reference in its entirety.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant No. DK051362, awarded by the National Institutes of Health. The government has certain rights in the invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 10, 2013, is named 043214-075201_SL.txt and is 29,715 bytes in size.
TECHNICAL FIELD
[0004] The present invention relates to molecular immunology and cell biology. More specifically, the technology described herein relates to the modulation of T-cell tolerance.
BACKGROUND
[0005] The immune system exists to protect the body from foreign invaders and diseased cells.
A healthy immune system can recognize foreign or aberrant cells and target them for destruction. As part of the natural control of the immune system, components of one's immune system (e.g. T cells) that recognize healthy host cells are destroyed or forced to enter an inactive state, a phenomenon referred to as "T cell tolerance." This prevents the immune system from attacking the healthy cells of the host organism itself.
[0006] However, in the case of chronic diseases, the prolonged response of the immune system to the chronic disease can lead to counterproductive T cell tolerance. T cells that are recognizing and attacking foreign or diseased cells can be "turned off via T cell tolerance. This obviously reduces the host's ability to respond to and recover from the chronic disease.
[0007] Chronic immune disorders can wreak havoc, particularly those associated with T-cell tolerance, such as cancers. According to the most recent data from the World Health Organization, ten million people around the world were diagnosed with the cancer in 2000, and six million died from it. Moreover, statistics indicate that the cancer incidence rate is on the rise around the globe. In America, for example, projections suggest that fifty percent of those alive today will be diagnosed with some form of cancer at some point in their lives. Hence, there remains an urgent need for compositions and approaches to treating T-cell tolerance mediated immune disorders. SUMMARY
[0008] Provided herein are compositions and methods for enhancing the immune response and/or reducing T cell tolerance in a subject. As described herein, the inventors have discovered that certain subpopulations of T cells can coexpress two or more of CEACAMl, PDl, and/or LAP, which promote T-cell tolerance through separate signaling pathways. The discovery that T cells can coexpress these molecules underlies the technology described herein, which relates to administering inhibitors of two or more of these molecules in order to achieve a greater degree of reduction in T cell tolerance than is possible by inhibiting only one pathway.
[0009] Accordingly, in some aspects, the compositions and methods described herein increase the T-cell mediated immune response in a subject (i.e. reduce T-cell tolerance) by concurrently inhibiting the activity of the signaling pathways controlled by at least two of CEACAMl, PDl, and/or LAP. Relieving the suppression of the immune system is highly desirable in, for example, an immunocompromised subject or a subject suffering from cancer.
[0010] In one aspect, described herein is a composition for enhancing the immune response or decreasing T cell tolerance in a subject, the composition comprising at least two agents selected from the group consisting of: a CEACAMl inhibitory agent; a PDl inhibitory agent; and a LAP inhibitory agent. In some embodiments, the two agents can be a CEACAMl inhibitory agent and a PDl inhibitory agent. In some embodiments, the two agents can be a CEACAMl inhibitory agent and a LAP inhibitory agent. In some embodiments, the two agents can be a PDl inhibitory agent and a LAP inhibitory agent. In some embodiments, the composition can comprise a CEACAMl inhibitory agent, a PDl inhibitory agent, and a LAP inhibitory agent.
[0011] In some embodiments, the composition can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a PDl inhibitory agent. In some embodiments, the composition can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a LAP inhibitory agent. In some embodiments, the composition can comprise a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PDl inhibitory agent.
[0012] In some embodiments, one or more of the inhibitory agents can be an inhibitory nucleic acid.
[0013] In some embodiments, one or more of the inhibitory agents can be an antibody reagent.
In some embodiments, the antibody reagent can bind a target selected from the group consisting of: CEACAMl ; PDl ; and LAP. In some embodiments, the antibody reagent can inhibit signaling mediated by the target.
[0014] In some embodiments, the CEACAMl inhibitory agent can bind a CEACAMl molecule having the sequence set forth in SEQ ID NO: 32 or an allelic or splice variant of SEQ ID
NO: 32. In some embodiments, the CEACAMl inhibitory agent can bind a CEACAMl ligand interaction site. In some embodiments, the CEACAMl inhibitory agent can bind to an amino acid residue corresponding to any of amino acid residues 1-429 of SEQ ID NO: 32. In some embodiments, the CEACAMl inhibitory agent can bind an extracellular domain of CEACAMl . In some embodiments, the CEACAMl inhibitory agent can bind the homophilic interaction domain of CEACAMl and inhibits homophilic interaction of CEACAMl molecules.
[0015] In some embodiments, the PDl inhibitory agent can bind a PDl molecule having the sequence set forth in SEQ ID NO: l, or an allelic or splice variant of SEQ ID NO: 1. In some embodiments, the PDl inhibitory agent can bind a PDl ligand interaction site. In some embodiments, the PDl inhibitory agent can bind to an amino acid residue corresponding to any of amino acid residues 41-136 of SEQ ID NO: 1.
[0016] In some embodiments, the LAP inhibitory agent can bind a LAP molecule having the sequence set forth in SEQ ID NO: 34 or an allelic or splice variant of SEQ ID NO: 34. In some embodiments, the LAP inhibitory agent can bind a LAP ligand interaction site. In some
embodiments, the LAP inhibitory agent can bind to an amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 33.
[0017] In one aspect, described herein is a method for treating a chronic disease, the method comprising administering to a subject in need of treatment thereof an effective amount of a composition described herein. In some embodiments, the chronic disease can be selected from the group consisting of: cancer; a persistent infection; and a chronic viral infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 depicts a schematic of the AOM-DSS model in WT mice.
[0019] Figure 2 depicts the results of FACS analysis examining expression of PDl and TIM-3 in CD4 and CD8 T-cells.
[0020] Figure 3 depicts the results of FACS analysis examining expression of PDl and
CEACAMl in CD4 and CD 8 T-cells.
[0021] Figure 4 depicts the results of FACS analysis examining expression of CEACAMl and
TIM-3 in CD4 and CD 8 T-cells.
[0022] Figures 5A-5B depict graphs demonstrating CEACAMl and PDl are co-expressed on the cell surface of chronically activated CD4+ (Figure 5A) and CD8+ (Figure 5B) tumor infiltrating T lymphocytes.
[0023] Figures 6A-6C demonstrate that CEACAMl -4S is preferentially expressed on specific subsets of both circulating and intestinal CD4+ T cells. Figure 6A depicts graphs of FACS analysis demonstrating that expression of CEACAMl is associated with the enrichment of specific cell surface molecules. Percentages on histogram gates indicate the frequency of cells positive for the given marker in the CD4+CEACAM 1 - and CD4+CEACAM 1 + cell populations, respectively, of MLN
CD3+ T cells from WT mice. Figure 6B depicts graphs of FACS analysis demonstrating that CEACAM1 expression co-segregates with latency associated peptide (LAP) expression and is unrelated to Foxp3 expression in CD4+ T cells from the MLN of naive WT mice. Figure 6C depicts a chart demonstrating that the CEACAM1-S isoform is enriched on CD4+CEAC AM 1 + T cells which express LAP. T cells were isolated from pooled spleen and lymph node (LN) in Figure 6C. Figures 6A-6C show representative data from 3 independent experiments with n = 5-6 mice per group.
[0024] Figures 7A-7F demonstrate that CEACAM1-S directly enhances the generation of
CD4+LAP+. Figures 7A-7D depict graphs of FACS analysis results demonstrating that the percentage of CD4+LAP+ cells is increased in MLN of na'ive CEACAM1-4S transgenic (4S Tg) mice over- expressing this isoform specifically in T cells (Figure 7A) but not CEACAM1-4L (4L Tg) mice (Figure 7B). CEACAMl''' -4S Tg mice (Figure 7C) but not CEACAMl ' -4L Tg mice (Figure 7D) are enriched in CD4+LAP+ T cells compared to their littermate controls. Figure 7E depicts graphs of FACS analysis results demonstrating that overexpression of CEACAM1-4S enhances the regulatory function of CD4+LAP+ Treg cells in vitro as shown by the decrease in proliferation of responder T cells when co-cultured with CD4+LAP+ Treg cells from pooled spleen and LN of 4S Tg mice. Figure 7F depicts a chart demonstrating that oral feeding of anti-CD3 antibody significantly increases the percentage of CD4+LAP+ Treg cells in the MLN of WT but not CEACAMl'1' mice. Mice were fed 5 μg anti-CD3 (or isotype control) for each of 5 consecutive days and CD4+LAP+ T cell frequency was assessed after 7 days.
[0025] Figure 8 depicts the results of a novel CEACAMl isoform-discriminating qPCR assay which depicts the specificity and efficiency of qPCR primers for mouse CEACAMl -L and -S isoforms amplification verified using mCEACAMl -4L and 4S plasmids. This novel assay was used in Figure 6C.
[0026] Figures 9A-9B demonstrate that CEACAMl -S expression on CD4+ T cells is associated with the expression of molecules which characterize specific subsets of T cells in the
Peyer's patches (PP) and lamina propria (LP). Figures 9A and 9B depict graphs of FACS analysis characterizing cell surface molecule expression on CD4+CEACAM1+ and CD4+CEACAM1- cells in
PP and LP of WT mice. Percentages on histogram gates indicate the frequency of cells positive for the given marker in the CD4+CEAC AM 1 - and CD4+CEAC AM 1 + cell populations, respectively.
Representative data from 3 independent experiments with n = 5-6 mice per group.
[0027] Figure 10 depicts the isoforms of CEACAMl created by alternate splicing.
[0028] Figure 11 depicts a graph of CEACAMl -S and CEACAMl -L expression using the qPCR assay in Figure 8. Total T cells from spleen were stimulated with plate -bound coated anti-CD3 or anti-CD3 plus CD28 for 2 and subsequently 4 days. CEACAMl -S, -L and total CEACAMl were assessed by qPCR. Figure 11 shows that both of the major CEACAMl isoforms (S and L) which are low in resting T cells are transcriptionally regulated by ligation of T cells through the TCR/CD3 complex (signal 1) but negatively regulated when the TCR/CD3 complex is co-engaged by the classical co-stimulatory signal (signal 2) provided by CD28. [0029] Figures 12A-12E demonstrate that CEACAM1-4S is associated with activation of primary T cells and sensitization to activation induced cell death. Figure 12A depicts a graph demonstrating that compared to WT littermates, 4S Tg mice exhibit higher percentages of CD3+ Annexin V+/7AAD" apoptotic cells, (n = 5 mice per group) Figures 12B-12C depict graphs of the results of experiments in which CD3+ T cells isolated from spleen of 4S Tg mice and their WT littermates were cultured with anti-CD3 antibody at indicated concentrations, or 1 μg/ml anti-CD3 over the indicated time course. Proliferation assays were performed by [3H] -thymidine incorporation (Figure 12B) and apoptosis was determined using annexin-V and 7-AAD staining (Figure 12C). (n = 5 mice per group) Figure 12D depicts an immunoblot. CD3+ T cells were isolated from the spleen of 4S Tg or WT mice, and cultured with IL-2 (100 U/ml) or anti-CD3 (1 μ^πιΐ) coated plates. Cell lysates were prepared at the indicated times, and caspase-3 and its activated form were determined using immunoblot. Figure 12E depicts a graph demonstrating that compared to CeacamV'' mice, Ceacaml " "-4S Tg mice have higher percentages of annexin V /7AAD" apoptotic cells (n = 5 mice per group).
[0030] Figures 13A-13C demonstrate that CEACAM1-S directly enhances the generation of
PD-1 bearing Tfollicular helper (T ) regulatory cells. Figures 13A and 13C depict graphs of the percentage of CD4+PD1+CXCR5 TFH cells in the spleen, MLN and PP is increased in 4S Tg mice (Figure 13A) and decreased in Ceacaml '' mice (Figure 13C) when compared to their littermate controls. Figure 13B depicts plots of the results of FACS analysis. CD4+ T cells within the PP or LP, but not in the spleen of 4S Tg mice exhibit increased levels of CD69, PD-1 and CD40L relative to their WT littermate controls. All data are representative of 3 independent experiments with n = 3-6 mice per group.
[0031] Figure 14 demonstrates co-expression of CEACAM1 and PD1 on T cells under physiologic conditions. Lamina propria lymphocytes were isolated from colon, ileum, jejunum and duodenum as well as mesenteric lymph nodes (MLN) and spleen and after gating on CD3 -positive cells stained for co-expression of PD1 and CEACAMl . The top panel depicts a graph of the summary of quantity of positive cells and the bottom panel depicts the histograms of the flow cytometry.
[0032] Figure 15 demonstrates co-expression of CEACAMl and PD1 in lamina propria lymphocytes infiltrating the colon of mice with colitis induced by adoptive transfer of naive CD4+ T cells in Rag-deficient and CEACAMl -deficient animals. Co-expression of CEACAMl and PD1 is shown after gating on CD3+ lymphocytes obtained from proximal, middle and distal colon.
[0033] Figure 16 demonstrates that blockade of CEACAMl and PD1 increases TNF-a production from tumor infiltrating lymphocytes (TIL). TILs were isolated from subcutaneous tumors associated with CT26 colorectal carcinoma cell line. TILs were stimulated with anti-CD3 and anti- CD28 monoclonal antibodies in the presence of isotype control antibodies or antibodies that block different co-inhibitory cell surface molecules. Note the synergistic increase of TNF-a production by TILs treated with anti-CEAC AMI and anti-PDl antibodies.
DETAILED DESCRIPTION
[0034] T-cell tolerance functions, in part, to provide a population of immune system cells that recognize self-major histocompatibility complex (MHC) molecules but do not recognize self-peptides. In chronic diseases (e.g. chronic infections or cancer), T cell tolerance occurs when the subject is still in need of a robust immune response. Previous studies have shown that carcinoembryonic antigen related cell adhesion molecule 1 (CEACAMl), programmed cell death 1 (PDl) and TGF i latency associated peptide (LAP) are important components of immune regulation which increase T cell tolerance. These molecules have been observed to be expressed on separate subpopulations of activated T-cells, especially after prolonged activation. Thus, while CEACAMl, PDl and LAP have been shown to play in T cell immune regulation, it had not been established or suggested that there exists a population of T cells which expresses detectable levels of two or more of CEACAMl, PDl, and/or LAP prior to the discoveries described herein.
[0035] As described herein, the inventors have found that certain subpopulations of T cells co- express detectable levels of (i) CEACAMl and LAP, (ii) CEACAMl and PDl, (iii) PDl and LAP, or (iv) CEACAMl, PDl, and LAP. The discovery of such cells underlies the concept described herein of reducing T cell tolerance by inhibiting at least two of these T cell tolerance-promoting molecules. The ability to target multiple pathways which contribute to T cell tolerance can provide a greater effect upon T cell tolerance, e.g. by synergistic effects, leading to a therapeutic decrease in T cell tolerance. Inhibition of two or more of CEACAMl, PDl, and/or LAP can therefore enhance the responsiveness of the immune system.
[0036] As used herein, "CEACAMl" (also known as CD66a or biliary glycoprotein) refers to a polypeptide having a single Ig variable domain-like amino terminus, from one to three Ig constant domain-like regions, and a single membrane-spanning segment followed by either a short
(CEACAMl -S) or long (CEACAMl -L) cytoplasmic domain (Hinoda et al., 85 PNAS 6959 (1988); which is incorporated by reference herein in its entirety). The N-terminal domain has been shown to facilitate homophilic intercellular binding that influences a broad spectrum of cellular processes related to cellular activation and/or cell cycle progression; and is also targeted by the heterophilic adhesins of viral (murine hepatitis virus ) and bacterial {Neisseria gonorrhoeae and N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae) pathogens, allowing their infection of the diverse array of CEACAMl -expressing human cells and tissues in vivo.
[0037] When expressed, CEACAMl is characterized by significant alternate RNA splicing leading to eleven isoforms in humans and at least four isoforms in mice. These isoforms differ in the length of the cytoplasmic tail and the number of extracellular Ig-like domains and are named accordingly. As noted, the majority of CEACAMl isoforms possess either a long (CEACAMl -L) CT or a short (CEACAMl -S) cytoplasmic tails (Azuz-Lieberman et al., 17 Intl. Immunol. 837 (2005); Gray-Owen & Blumberg, 2006; Moller et al., 65 Int. J. Cancer 740 (1996); Nakajima et al., 168 J. Immunol. 1028 (2002); Singer et al., 168 J. Immunol. 5139 (2002)). The long cytoplasmic tail (-72 amino acids in humans) contains two immune -receptor tyrosine-based inhibitory motifs (ITIMs) (Chen et al., 172 J. Immunol. 3535 (2004)). These isoforms are inhibitory for T-cell responses, which inhibition generally involves the ITIM domains and Src homology 2 domain phosphatase 1 (SHP-1). ITIM phosphorylation and, consequently, its association with SHP-1 requires p56 Lck kinase and the ability to bind homophilically. CEACAMl recruits SHP-1 to the T-cell receptor (TCR) signalsome where SHP-1 blocks ZAP-70 activation via dephosphorylation of CD3^, ZAP-70, or both. Indeed, masking the homophilic binding site with a specific Fab causes increased cytotoxicity and lymphocyte degranulation (Chen et al., 180 J. Immunol. 6085 (2008)). CEACAMl -S isoforms are inhibitory by inducing weak co-stimulatory signals that sensitize cells to activation induced cell death and the emergence of populations of regulatory cells such as CD4+LAP+ T cells (Chen et al., 172 J.
Immunol. 3535 (2004)). The foregoing references are incorporated by reference herein in their entireties.
[0038] Clinical evidence shows that high-level CEACAMl expression on tumors and tumor- infiltrating lymphocytes correlates with poor prognosis and high risk of metastasis. CEACAMl functions as a regulatory co-receptor for both lymphoid and myeloid cell types, and is constitutively expressed in a wide range of tissues and cell types. Its expression on natural killer (NK) cells and T-cells is, however, mainly induced by cytokines and membrane-activating receptor activation. As described herein, previous studies have determined that CEACAMl is a ligand for itself (homophilic ligation), and is involved in heterophilic ligation with galectin 3 and selectins. CEACAMl expression, which is low on resting T-cells, is transcriptionally regulated by ligation of T-cells through the TCR/CD3 complex (signal 1) but negatively regulated when the TCR/CD3 complex is co-engaged by the classical co-stimulatory signal (signal 2) provided by CD28 and induces both major classes of CEACAMl isoforms (CEACAMl short and CEACAM-long) (see, e.g. US Provisional Application 61/677,596; which is incorporated by reference herein). Given the fact that TCR/CD3 ligation alone (in the absence of costimulation) is a common mechanism for the induction of T-cell tolerance, the strong induction of CEACAMl by such stimulation may, without wishing to be bound or limited by theory, be part of the tolerogenic program.
[0039] Further, in regard to the tertiary structure of CEACAMl, the two major isoforms,
CEACAMl -4L and CEACAMl -4S, which differ only in their cytoplasmic domains, have extracellular domains (ectodomains) comprised of four glycosylated Ig domains. CEACAMl -induced cell signaling is regulated by its intercellular homophilic binding at the cell surface, which is mediated by the N-terminal Ig domain (Dl) in a reciprocal Dl-Dl interaction. The basic structure of the IgV N-terminal domain of CEACAMl is a tertiary fold of a stacked pair of β-pleated sheets. There are nine component β strands, with strands A, B, E, and D lying in one sheet and strands C, C, C", F, and
G being antiparallel in the other sheet. The GFCC face of the N-terminal domain of CEACAMl is known to be crucial for mediating homophilic adhesion. Homophilic and heterophilic interactions have been observed for other adhesion receptor-ligand pairs; such as of CD2 with CD58; ICAM-1 with ICAM-1, LFA-1, rhinoviruses or Plasmodium falciparum-mfectcd erythrocytes; or cadherins with cadherins (Watt et al., 98 Blood 1469 (2001)). These interactions indicated that the GFCC faces of the immunoglobulin family members may have evolved, without wishing to be bound or limited by theory, as a sticky patch to recognize a variety of protein-protein interactions (Springer et al., 6 Ann. Rev. Cell Biol. 359 (1990)). The foregoing references are incorporated by reference herein in their entireties.
[0040] In some embodiments, a CEACAM1 inhibitory reagent can inhibit one or more isoforms of CEACAM1 which can promote T-cell tolerance. In some embodiments, a CEACAM1 inhibitory reagent can inhibit one or more isoforms of CEACAM1 which can promote T-cell tolerance and not other isoforms of CEACAM1. Isoforms of CEACAM1 which can promote T-cell tolerance include, but are not limited to the isoforms depicted in Figure 11, e.g. CEACAM1-1L, CEACAMl-l S, CEACAM1-3L, CEACAM1-3S, CEACAM1-4L, CEACAM1-4S, CEACAM1 -3 AL, CEACAM1-3AS, CEACAMl-3, CEACAM1-4C1, and CEACAM1-4C2 including both soluble and trans-membrane isoforms and variants, substitution variants, and conservative substitution variants thereof. Further discussion of CEACAM1 isoforms can be found, e.g. in Beauchemin et al. Exp Cell Res 1999:252-243; which is incorporated by reference herein in its entirety.
[0041] A peptide region responsible for CEACAM1 heterophilic interactions, for example with Neisseria Opa proteins, is also on the GFCC'C" face and overlaps partially with the homophilic binding site. Fedarovich et al., D62 Acta Cryst. 971 (2006). In comparison, binding of a murine CEACAM1 to murine coronavirus requires a uniquely folded CC loop, in which amino acids 34 to 52 play a crucial role (Tan et al., 21 EMBO J. 2076 (2002); Watt et al., 2001). Additionally, amino acids between residues 27 to 42 (particularly D27L28F29) and S32, Y34, V39, Q44, Q89, and 191 on the GFCC'C" face form differential adhesiotopes for the binding of H. influenzae, and the
N. gonorhheae and N. meningiditis Opa proteins. These adhesiotopes are likely a groove; formed by homophilic cis binding that involves V39 and D40 CC loop residues, or formed after disruption of CEACAM1 cis dimerization by cytokine (e.g., TNFa) activation that precedes CEACAM1 /pathogen binding (Watt et al., 2001). The foregoing references are incorporated by reference herein in their entireties.
[0042] Moreover, the IgC2 domain of CEACAM1 has also been implicated in coronavirus and
H. influenzae receptor activity. Immobilized CEACAM1, in which the tertiary structure of this highly flexible molecule is restrained, also exhibits decreased adhesion, further implicating the cytoplasmic regions of the molecule (e.g., intracellular dimerization) in both homophilic and heterophilic interactions and signaling. Further, the formation of homodimers in cis has been characterized for multiple splice variants (isoforms), even those lacking IgC2 domains. [0043] The lack of intradomain disulfide bridges in the N-terminal Dl domain renders the
CEACAMl ectodomain highly flexible. CEACAMl exists in the cell membrane in microclusters; whereas homophilic binding triggers reorganization that results in two different kinds of dimers, as well as trimers and higher-order oligomers. Because of the hinge regions between the Ig domains, antiparallel trans-di srs (C-dimers) and parallel cz -dimers (A-dimers) can be formed. The N- terminal Dl domain participates in both C- and A-dimerization, while the D2-D4 domains are involved only in A-dimerization. Divalent cations decreased ectodomain flexibility and enhanced formation of multimeric complexes, which are further implicated in CEACAMl -mediated cell adhesion. Importantly, the dimerization of the ectodomains is transduced by the transmembrane domains to the cytoplasmic domains, thus, in turn, directing intracellular signaling. Another binding site might be implicated across the ABED face, depending on the level and flexibility of
glycosylations on the CEACAMl molecule (Klaile et al., 187 J. Cell Biol. 553 (2009); which is incorporated by reference herein in its entirety).
[0044] CEACAMl function is also likely impacted by glycosylation. Carbohydrates account for up to sixty percent of the weight of CEACAMl expressed on the cell surface. The role of this enormous carbohydrate content allows for the construction of "subspecies" of CEACAMl isoforms. For example, the ability of CEACAMl to express sialyl Lewis x modifications can affect leukocyte homing (Chen et al., 86 J. Leuk. Biol. 195 (2009); which is incorporated by reference herein in its entirety).
[0045] In some embodiments, a CEACAMl inhibitory agent can bind a CEACAMl molecule having the sequence set forth in SEQ ID NO: 32 or an allelic or splice variant of SEQ ID NO: 32.
Allelic and splice variants of SEQ ID NO: 32 can include those variants described in, e.g. Gaur et al. Molecular Cancer 2008 7:46 Barnett et al. Mol Cell Biol 1993, 13: 1273-1282; and Barnett et al. J
Cell Biol 1989, 108:267-276; which are incorporated by reference herein in their entireties.
[0046] In some embodiments, a CEACAMl inhibitory agent can bind one or more of the extracellular domains of CEACAMl, e.g. any of amino acids 1-429 of SEQ ID NO: 32. In some embodiments, a CEACAMl inhibitory agent can bind the Ig V-set domain of CEACAMl, e.g. any of amino acids 35-140 of SEQ ID NO: 32. In some embodiments, a CEACAMl inhibitory agent can bind an Ig C-set domain of CEACAMl, e.g. any of amino acids 140-415 of SEQ ID NO: 32. The extracellular domain structure of CEACAMl variants is further described, for example, in Watt et al.
Immunobiology 2001 98: 1469-1479; which is incorporated by reference herein in its entirety.
[0047] In some embodiments, a CEACAMl inhibitory agent can bind a CEACAMl ligand interaction domain. In some embodiments, a CEACAMl ligand interaction domain can comprise extracellular residues of a CEACAMl polypeptide. By way of non- limiting example, the homophilic ligand interaction domain of CEACAMl can comprise amino acids 73, 74, 98, and 116 of SEQ ID
NO: 32 and the heterophilic ligand interaction domain of CEACAMl can comprise amino acids 61,
62, 63, 66, 68,73, 78, 123, and 125 of SEQ ID NO: 32. Residues important for ligand binding in a number of CEACAMl variants are further described, for example, in Watt et al. Immunobiology 2001 98: 1469-1479; which is incorporated by reference herein in its entirety. In some embodiments, a CEACAMl inhibitor agent can bind to (or physically interact with) one or more of these homophilic ligand interaction amino acids, e.g. amino acids 73, 74, 98, and 116 of SEQ ID NO: 32. In some embodiments, a CEACAMl inhibitory agent can bind to (or physically interact with) one or more of these heterophilic ligand interaction amino acids, e.g. amino acids 61, 62, 63, 66, 68,73, 78, 123, and 125 of SEQ ID NO: 32. In this context, physical interaction can encompass steric hindrance of the interaction of one or more of these sites with a ligand involved in tolerogenesis.
[0048] In some embodiments, the CEACAMl inhibitory agent can bind the homophilic interaction domain of CEACAMl and inhibit homophilic interaction of CEACAMl molecules. In some embodiments, a CEACAMl inhibitory agent can reduce homophilic binding of CEACAMl . Trans -homophilic CEACAMl binding induces d -dimerization by an allosteric mechanism transmitted by the N terminal immunoglobulin- like domain. CEACAMl -L homodimer formation is reduced by coexpression of CEACAMl -S and modulated by antibody ligation. Transmembrane signaling by CEACAMl can operate by alteration of the monomer/dimer equilibrium. Miiller et al., 187 J. Cell Biol. 569 (2009). Exemplary agents that selectively decrease homophilic CEACAMl binding are described, for example, in U.S. Patents No. 7,132,255 and No. 6,852,320, and the references cited therein, the contents of which are herein incorporated by reference in their entireties.
[0049] Monomeric soluble forms of CEACAMl have been demonstrated to inhibit
CEACAMl signaling (see, e.g. Markel. EJI 2004 34:2138-2148; which is incorporated by reference herein in its entirety). In some embodiments, the CEACAMl inhibitory agent can be a monomeric CEACAMl Fc polypeptide. In some embodiments, the CEACAMl inhibitory agent can be a monomeric CEACAMl Fc fusion protein. Monomeric CEACAMl Fc fusion proteins have been described in the art, see, e.g. Biotoni et al. PNAS 2004 101 :9763-8; which is incorporated by reference herein. In some embodiments, the CEACAMl inhibitory agent is not a dimeric form of soluble CEACAMl polypeptide or a dimeric form of CEACAMl Fc polypeptide.
[0050] The longest isoform, CEACAMl -4L (isoform 1) has 526 amino acids, having the sequence:
1 0 2 0 3 0 4 0 50 60
MGHLSAPLHR VRVPWQGLLL TAS LLTFWNP PTTAQLTTES MPFNVAEGKE VLLLVHNLPQ
7 0 8 0 90 1 0 0 1 1 0 12 0
QLFGYSWYKG ERVDGNRQIV GYAI GTQQAT PGPANS GRET IYPNAS LL IQ NVTQNTGFY
130 1 4 0 15 0 1 60 1 7 0 1 8 0
TLQVIKS DLV NEEATGQFHV YPELPKPS I S SNNSNPVE DK DAVAFTCE PE TQDTTYLWWI
1 90 2 0 0 2 1 0 2 2 0 2 30 2 4 0
NNQS LPVS PR LQLSNGNRTL TLLSVTRNDT GPYECE IQNP VSANRS DPVT LNVTYGPDT P
250 2 60 2 7 0 2 8 0 2 90 30 0 TISPSDTYYR PGANLSLSCY AASNPPAQYS WLINGTFQQS TQELFI PNIT VNNSGSYTCH
310 32 0 330 340 350 360
ANNSVTGCNR TTVKTIIVTE LSPWAKPQI KASKTTVTGD KDSVNLTCST NDTGISIRWF
370 38 0 390 4 00 4 10 42 0
FKNQSLPSSE RMKLSQGNTT LSINPVKRED AGTYWCEVFN PISKNQSDPI MLNVNY ALP
430 44 0 450 4 60 4 70 48 0
QENGLSPGAI AGIVIGVVAL VALIAVALAC FLHFGKTGRA SDQRDLTEHK PSVSNHTQDH
4 90 50 0 51 0 520
SNDPPNKMNE VTYSTLNFEA QQPTQPTSAS PSLTATEIIY SEVKKQ (SEQ ID NO: 32
[0051] The foregoing amino acid sequences include a leader sequence (34 amino acids), and the mature peptide starting at QLT. The DNA and amino acid sequences of CEACAM1 isoforms of various species are available on-line from the NCBI, including human (ID: 634), mouse (ID: 26365), rat (ID: 81613), cow (ID: 404118), zebra fish (ID: 114465), dog (ID: 612435), goat (ID: 100384959), and orangutan (ID: 100172828).
[0052] For example, the secreted human CEACAM1 isoform 2 (UniProtKB PI 3688-2) has the amino acids:
MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQ
LFGYSWYKGERVDGNRQTVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQ
VIKSDLVNEEATGQFHVYPELPKPSISSN SNPVEDKDAVAFTCEPETQDTTYLWWIN QSLP
VSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYYR
PGANLSLSCYAASNPPAQYSWLI GTFQQSTQELFIPNITVN SGSYTCHAN SVTGCNRTTV
KTIIVTELSPWAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFK QSLPSSERMKLSQG
NTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNCK (SEQ ID NO: 31)
[0053] Secreted human CEACAM1 isoform 3 (UniProtKB PI 3688-3) has the amino acids:
MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQ
QLFGYSWYKGERVDGNRQTVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTL
QVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSL
PVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYY
RPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCNRTT
VKTIIVTGK (SEQ ID NO: 30).
[0054] Secreted human CEACAM1 isoform 4 (UniProtKB PI 3688-4) has the amino acids:
MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQ LFGYSWYKGERVDGNRQTVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQ VIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSLP VSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYYR PGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCNRTTV KTIIVTESPVLGEDEAVPGQHHPQHKPCQEGGCWDVLV (SEQ ID NO: 29) [0055] PD1 (or CD279) is a 288 amino acid type I transmembrane protein composed of one immunoglobulin (Ig) superfamily domain, a 20 amino acid stalk, a transmembrane domain, and an intracellular domain of approximately 95 residues containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), as well as an immunoreceptor tyrosine-based switch motif (ITSM). PD1 is encoded by the Pdcdl and PDCD1 genes on chromosome 1 in mice and chromosome 2 in humans respectively. In both species, Pdcdl is encoded by 5 exons. Exon 1 encodes a short signal sequence, whereas exon 2 encodes an Ig domain. The stalk and transmembrane domains make up exon 3, and exon 4 codes for a short 12 amino acid sequence that marks the beginning of the cytoplasmic domain. Exon 5 contains the C-terminal intracellular residues and a long 3'UTR (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704). PD1 is a member of the B7 family of receptors.
[0056] Splice variants of PD1 have been cloned from activated human T cells. These transcripts lack exon 2, exon 3, exons 2 and 3, or exons 2 through 4. All these variants, except for the splice variant lacking exon 3 only (PDlAex3), are expressed at levels similar to full-length PD1 in resting peripheral blood mononuclear cells (PBMCs). All variants are significantly induced upon activation of human T cells with anti-CD3 and anti-CD28 (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704).
[0057] Accordingly, the term " PD1 " as used herein, refers to the 288 amino acid polypeptide having the amino acid sequence of:
MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSES FVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLC GAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLWGWGGLLGSLVLLVW VLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYA TIVFPS GMGTS SP ARRGS ADGPRS AQPLRPED GHC S WPL (SEQ ID NO:l), as described by, e.g., NP 005009, together with any naturally occurring allelic, splice variants, and processed forms thereof. Typically, PD1 refers to human PD1. The term "PD1 " is also used to refer to truncated forms or fragments of the PD1 polypeptide. Reference to any such forms of PD1 can be identified in the application, e.g., by "PD1 (42-136)." For example, the mature PD1 peptide is referred to herein as PD1 (21-288), and PD1 IgV domain as PD1 (42-136). Specific residues of PD1 can be referred to as, for example, "PD1 (68)."
[0058] PD1 has been shown to be expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs). PD1 is not expressed on resting T cells but is inducibly expressed after activation. Ligation of the T cell receptor or B cell receptor can upregulate PD1 on T and B lymphocytes. In normal human reactive lymphoid tissue, PD1 is expressed on germinal center- associated T cells. PD1 compartmentalization in intracellular stores has been described in a regulatory T cell population. PD1 is inducibly expressed on APCs on myeloid CD1 lc+ DCs and monocytes in humans (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704; which is incorporated by reference herein in its entirety). [0059] PDl has two known ligands, PD-L1 and PD-L2, which are also members of the B7 family. The binding interface of PDl to PD-L1 is via its IgV-like domain (i.e., PDl (42-136)).
Residues important for binding of PDl to its ligands include residues 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136. PD-L1/CD274 has been shown to be constitutively expressed on mouse T and B cells, DCs, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells. CD274/PD-L1 expression is also found on a wide range of nonhematopoietic cells and is upregulated on a number of cell types after activation. PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B 16 melanoma upon treatment with IFN-γ. Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modifies phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increases post-transcriptional PD-L1 expression in cancers (Keir ME et al, 2008. Annu Rev
Immunol. 26:677-704). Residues of PD-L1 important for binding to PDl include PD-L1 (67), PD-L1 (121), PD-L1 (122), PD-L1 (123), PD-L1 (123), PD-L1 (124), and PD-Ll (126).
[0060] PD-L2 expression is more restricted than PD-L1 expression. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells. PD-L2 is also expressed on 50% to 70% of resting peritoneal Bl cells, but not on conventional B2 B cells. PD-L2 can also be induced on monocytes and macrophages by GM-CSF, IL-4, and IFN-γ. PD-L2 expression has also been observed on tumor lines.
[0061] In some embodiments, a PDl inhibitory agent can be an anti-PD-Ll antibody reagent.
In some embodiments, a PDl inhibitory agent can be an anti-PD-L2 antibody reagent. In the present context, and anti-PD-Ll antibody reagent or anti-PD-L2 antibody reagent is one that binds to the target ligand and prevents binding of the ligand to PDl.
[0062] PDl and its ligands have been shown to have important roles in regulating immune defenses against microbes that cause acute and chronic infections. The PDl : PD-L pathways appear to play important roles in the outcome of infection, and the regulation of the delicate balance between effective antimicrobial immune defenses and immune -mediated tissue damage. Accordingly, in some embodiments of the aspects described herein, a PDl inhibitory agent (e.g. an antibody reagent) inhibits or blocks binding of PDl to its ligands.
[0063] A number of microorganisms that cause chronic infection appear to have exploited the
PDl : PD-L pathways to evade the immune responses and establish persistent infection. Studies in the lymphocytic choriomeningitis virus (LCMV) model of chronic viral infection were the first to show a role for the PDl :PD-L pathway during chronic infection (Barber DL et al. 2006. Nature 439:682-87). Viruses that cause chronic infections can render virus-specific T cells nonfunctional and thereby silence the antiviral T cell response (Wherry EJ and Ahmed R. 2004. J. Virol. 78:5535-45).
Functional dysregulation, also termed herein as "exhaustion," of CD8 T cells is an important reason for ineffective viral control during chronic infections and is characteristic of chronic LCMV infection in mice, as well as of HIV, HBV, HCV, and HTLV infection in humans and SIV infection in primates.
[0064] In chronic viral infections in humans, several groups have shown that PD1 expression is high on HIV-specific (Petrovas C et al. 2006. J. Exp. Med. 203:2281-92; Day CL et al 2006. Nature 443:350-54; Trautmann L et al 2006. Nat. Med. 12: 1198-202), HBV-specific (Boettler T et al 2006. J. Virol. 80:3532-40; Boni C et al. 2007. J. Virol. 81 :4215-25), and HCV-specific T cells (Bengsch B. et al, 2010 PLoS Pathog. 6(6); Urbani S et al. 2006. J. Virol. 80: 11398-403). Blocking PD1 :PD-L interactions in vitro has been shown to reverse the exhaustion of HIV-specific, HBV- specific (Boni C et al. 2007. J. Virol. 81 :4215-25), HCV-specific, and SIV-specific (Velu V et al. 2007. J. Virol.81 :5819-28) CD8 and CD4 T cells and restores proliferation and cytokine production (Petrovas C et al. 2006. J. Exp. Med. 203:2281-92; Day CL et al. 2006. Nature 443:350-54;
Trautmann L et al. 2006. Nat. Med. 12: 1198-202; Urbani S et al. 2006. J. Virol. 80: 11398-403). Recent work shows that the HCV core, a nucleocapsid protein, can upregulate PD1 and PD-L1 expression on healthy donor T cells and that upregulation of PD1 is mediated by interaction of the HCV core with the complement receptor C1QBP (Yao ZQ et al. 2007. Viral Immunol. 20:276-87). The foregoing references are incorporated by reference herein in their entireties.
[0065] The PD1 :PD-L pathway also can play a key role in the chronicity of bacterial infections. Helicobacter pylori causes chronic gastritis and gastroduodenal ulcers and is a risk factor for development of gastric cancer. During H. pylori infection, T cell responses are insufficient to clear infection, leading to persistent infection. Gastric epithelial cells express MHC class II molecules and are thought to have important APC (antigen-presenting cell) function during H. pylori infection. Anti- PD-Ll blocking antibodies enhance T cell proliferation and IL-2 production in cultures of gastric epithelial cells exposed to H. pylori and CD4 T cells, suggesting that the PD1 :PD-L1 pathway can play an important role in inhibiting T cell responses during H. pylori infection (Das S et al. 2006. J. Immunol. 176:3000-9; which is incorporated by reference herein in its entirety).
[0066] Parasitic worms also have exploited the PD1 :PD-L pathways to induce macrophages with strong suppressive function. During Taenia crassiceps infection in mice, a high percentage of CD4 T cells express PD1, and PD-L1 and PD-L2 are upregulated on activated macrophages.
Blockade of PD-L1, PD-L2, or PD1 significantly decreased suppression of in vitro T cell proliferation by macrophages from Taem'a-infected mice (Terrazas LI et al. 2005. Int. J. Parasitol. 35: 1349-58). Similarly, during Schistosoma mansoni infection in mice, macrophages express high levels of PD-L1 and more modest levels of PD-L2. Anti-PD-Ll completely abrogated the ability of these macrophages to suppress T cell proliferation in vitro, whereas anti-PD-L2 had no effect (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704). The PD1 :PD-L pathways have also been shown to have distinct roles in the immune response to the protozoan parasite Leishmania mexicana (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704). The foregoing references are incorporated by reference herein in their entireties. [0067] Tumors express antigens that can be recognized by host T cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor
microenvironment. Recent work has indicated that the PDl : PD-L pathways are involved in suppression of anti-cancer/tumor immune responses. PDl expression is upregulated on tumor infiltrating lymphocytes, and this can contribute to tumor immunosuppression. PD-Ll expression has been shown in situ on a wide variety of solid tumors, including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck. In addition, in ovarian cancer, PD-Ll expression is inversely correlated with intraepithelial, but not stromal, infiltrating CD8 T cells, suggesting that PD-Ll inhibits the intratumor migration of CD8 T cells. Also, studies relating PD-Ll expression on tumors to disease outcome show that PD-Ll expression strongly correlates with unfavorable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer but not small cell lung cancer (Keir ME et al, 2008. Annu Rev Immunol. 26:677-704).
[0068] The PDl pathway can also play a role in hematologic malignancies. PDl is highly expressed on the T cells of angioimmunoblastic lymphomas, and PD-Ll is expressed on the associated follicular dendritic cell network. In nodular lymphocyte -predominant Hodgkin lymphoma, the T cells associated with lymphocytic and/or histiocytic (L&H) cells express PDl . PDl and PD-Ll are expressed on CD4 T cells in HTLV-1 -mediated adult T cell leukemia and lymphoma. PD-L2 has been identified as being highly expressed in mantle cell lymphomas. PD-Ll is expressed on multiple myeloma cells but not on normal plasma cells, and T cell expansion in response to myeloma cells is enhanced in vitro by PD-Ll blockade. PD-Ll is expressed on some primary T cell lymphomas, particularly anaplastic large cell T lymphomas (Keir ME et al, 2008. Annu Rev Immunol. 26:677- 704).
[0069] In some embodiments, a PDl inhibitor agent can bind a PDl molecule having the sequence set forth in SEQ ID NO: 1 or an allelic or splice variant of SEQ ID NO: 1. Splice variants of PDl have been described, for example in Keir et al. Annual Review of Immunology 2008 26:677- 704 and Nielsen et al. Cell Immunol 2005 235: 109-116; which are incorporated herein by reference in their entireties.
[0070] In some embodiments, a PDl inhibitory agent can bind to a PDl ligand interaction site.
In some embodiments, specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-Ll . In some embodiments, specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-L2. In some embodiments, specific binding to a PDl ligand interaction site modulates interaction of PDl with PD-Ll and PD-L2. In some embodiments, a ligand interaction site of PDl can comprise any of amino acid residues 41-136 of SEQ ID NO: l . In some embodiments, a ligand interaction site on PDl can comprise any of the amino acid residues selected from the group consisting of amino acids 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136 of SEQ ID NO: l . In some embodiments, the ligand interaction site on PDl comprises any of the amino acid residues selected from the group consisting of amino acids 78, 126, and 136 of SEQ ID NO: 1. In some embodiments, a PD1 inhibitory agent can bind to, or physically interact with any of the amino acid residues selected from the group consisting of amino acids 64, 66, 68, 73, 74, 75, 76, 78, 90, 122, 124, 126, 128, 130, 131, 132, 134, and 136 of SEQ ID NO: l . In some embodiments, a PD1 inhibitory agent can bind to, or physically interact with, any of the amino acid residues selected from the group consisting of amino acids 41-136 of SEQ ID NO : 1. In some embodiments, a PD1 inhibitory agent can bind to, or physically interact with, any of the amino acid residues selected from the group consisting of amino acids 78, 126, and 136 of SEQ ID NO: l . In this context, physical interaction can encompass steric hindrance of the interaction of one or more of these sites with a ligand involved in tolerogenesis.
[0071] Non-limiting examples of anti-PDl antibody reagents can include PD1 binding site sequences from monoclonal antibodies specific for human PD1, such as, MDX-1106 (ONO-4538), a fully human IgG4 anti-PDl blocking antibody (Journal of Clinical Oncology, 2008 Vol 26, No 15S); CT-011 (CureTech, LTD, previously CT-AcTibody or BAT), a humanized monoclonal IgGl antibody (Benson DM et al, Blood. 2010 May 11), or those obtained from, clone NAT (Abeam), clone EH12.2H7 (Biolegend), clone Jl 16 (eBioscience), clone MIH4 (eBioscience), clone J105
(eBioscience), or clone 192106 (R& D systems).
[0072] In some embodiments, the compositions and methods described herein relate to LAP inhibitory agents. As used herein, "latency associated peptide" or "LAP" refers to the amino - terminal domain of the TGF 3 precursor peptide. When LAP is cleaved from the precursor peptide, it can remain in noncovalent association with TGF 3, forming the latent complex. In some
embodiments, LAP can exist as a homodimer of LAP molecules. Sequences of LAP polypeptides are known for a number of species, e.g. human LAP (amino acids 30-278 of SEQ ID NO: 33). Agents that physically interact with, bind to, or sterically occlude ligand binding to these interaction sites can be used to inhibit LAP activity, and particularly LAP activity involved in the establishment of maintenance of tolerogenesis.
[0073] LAP contains important residues necessary for the interaction with binding partners, e.g. TGF . Cysteines 224 and 226 are important in the mtermolecular disu!phide bond between two
LAPs. Their mutation to serine renders the molecule "active" (Sanderson et al., Proc. Natl. Acad. Sci.
USA, 92, 2572-2576 ( 1995); Brtmneret al Mol. Endocrinol. 6, 1691 -1700 ( 1992); Brunner et al, J.
Biol. Chem, 264, 13660-13664 (1989); which are incorporated by reference herein in their entireties.
The RGB motif (245-247) facilitates the interaction with integrins (Munger et al, Mol Biol Cell,
9:2627-2638 (1998; DerynckR, TIBS, 19, 54S-553 (1994); which are incorporated by reference herein in their entireties). Cysteine 33 is important for the disulphide bridge with the third cysteine-rich repeat of latent ΤΟΡβ binding protein (LTBP) (Saharinen et al. The EMBO Journal, 15, 245-253
(1996)). Nucleic acid encoding ΤΟΡβ is described in U.S. Pat. No. 5,801,231. The foregoing references are incorporated by reference herein in their entireties. [0074] In some embodiments, a LAP inhibitor)'' agent can bind a LAP molecule having the sequence set forth in SEQ ID NO: 34 or an allelic or splice variant of SEQ ID NO: 34.
[0075] LAP inhibitor)? antibody reagents are known in the art. See, e.g. All et ai. FLOS ONE
2008 :e 19 ! 4; which is incorporated by reference herein in its entirety. Further examples of anti-LAP antibody reagents are described in U.S. Parent No. 8,198,412 and U.S. Patent Publication No, 2008/0206219; which are incorporated by reference herein in their entireties,
[0076] In some embodiments, a LAP inhibitory agent can bind a LAP ligand interaction site, e.g. a site that interacts with mature TGF . Non- limiting examples of such sites include R218 (see, e.g. McGowan et al. The Journal of Clinical Endocrinology and Metabolism 2003 88:3321-6; which is incorporated by reference herein in its entirety).
[0077] Described herein are compositions and methods relating thereto which comprise at least two of the following: an inhibitor of CEACAM1 (i.e. a CEACAM1 inhibitory agent), an inhibitor of PD1 (i.e. a PD1 inhibitory agent), and/or an inhibitor of LAP (i.e. a LAP inhibitory agent). As used herein, an "inhibitory agent" is an agent that reduces the level and/or activity of a target as compared to the level and or activity in the absence of the agent by a statistically significant amount. For the avoidance of doubt, reduction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more would be considered inhibitory. The level of a target can be the level of a polypeptide target, the level of an isoform or variant of a polypeptide target, or the level of an RNA transcript encoding the polypeptide target. The activity of a polypeptide target can include, but is not limited to, the ability of the target to bind a normal ligand, the ability of the target to interact with other polypeptides (e.g. downstream signaling partners), and/or the ability of the target polypeptide to effect a downstream response (e.g.
phosphorylation levels or gene expression). In some embodiments, an inhibitory agent can be an agent that reduces the interaction (e.g. binding, both homophilic and heterophilic) between individual molecules of CEACAM1, PD1, and LAP. For example, an inhibitory agent can reduce the interaction of CEACAM1 and PD1. In some embodiments, the compositions described herein can comprise a CEACAM1 inhibitory agent and a PD1 inhibitory agent. In some embodiments, the compositions described herein can comprise a CEACAM1 inhibitory agent and a LAP inhibitory agent. In some embodiments, the compositions described herein can comprise a LAP inhibitory agent and a PD1 inhibitory agent. In some embodiments, the compositions described herein can comprise a
CEACAM1 inhibitory agent, a LAP inhibitory agent, and a PD1 inhibitory agent.
[0078] A target-specific inhibitory agent (i.e. an inhibitory agent specific for a given target, herein, at least one of CEACAM1, PD1, and/or LAP) can include, by way of non-limiting examples, a protein, an antibody, an antibody reagent which binds specifically to the target and inhibits its signaling activity, or a small interfering RNA specific for or targeted to the target-encoding mRNA. As used herein, an "inhibitory agent" can refer to a protein-binding agent that permits inhibition of
CEACAM1, PD1, and/or LAP as described herein. Such agents include, but are not limited to, antibodies ("antibodies" includes antigen-binding portions of antibodies such as epitope- or antigen- binding peptides, paratopes, functional CDRs; recombinant antibodies; chimeric antibodies; tribodies; midibodies; or antigen-binding derivatives, analogs, variants, portions, or fragments thereof), protein- binding agents, small molecules, recombinant protein, peptides, aptamers, avimers and protein- binding derivatives, portions or fragments thereof. Antisense oligonucleotides represent another class of agents that are useful in the compositions and methods described herein. This class of agents and methods for preparing and using them are all well-known in the art, as are ribozyme and miRNA molecules. See, e.g., PCT US2007/024067 (which is incorporated by reference herein in its entirety) for a thorough discussion.
[0079] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity
[0080] In some embodiments, an inhibitory agent can be an inhibitory nucleic acid, an inhibitory peptide, an inhibitory small molecule, an inhibitory antibody reagent, or inhibitory mimics (e.g. peptidomimetics) or combinations thereof. In some embodiments, an inhibitory agent can be a mimic or variant of the natural ligand of a target, e.g. a fragment or variant of TGF that will interact with LAP and prevent the binding of LAP to mature TGF .
[0081] In some embodiments, an inhibitory agent can be a dominant negative variant of any of the targets described herein (e.g. CEACAMl, PDl, and/or LAP). For example, a CEACAMl variant which can bind to wild-type CEACAMl via the homodimerization/homophilic interaction domain can reduce or inhibit signaling requiring CEACAMl .
[0082] In some embodiments, an inhibitory agent (e.g. an antibody reagent) specific for a target (e.g. CEACAMl, PDl, and/or LAP) can specifically bind to the extracellular region of the target. In some embodiments, an inhibitory agent (e.g. an antibody reagent) specific for a target (e.g. CEACAMl, PDl, and/or LAP) can specifically bind to a ligand interaction region of the target. In some embodiments, the inhibitory agent can reduce or inhibit the interaction of a ligand with the target. Specific regions of CEACAMl, PDl and/or LAP that can be targeted by an inhibitory agent are described elsewhere herein.
[0083] In some embodiments, an inhibitory agent can comprise an antibody reagent. As used herein, the term "antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen (e.g. CEACAMl, PDl, and/or LAP). In some embodiments, an antibody reagent can specifically bind to one or more of CEACAMl, PDl, and LAP. In some embodiments, an antibody reagent can inhibit the signaling activity of the target molecule, e.g. inhibit signaling activity of CEACAM1, PD1, and/or LAP. Methods for measuring the signaling activity of the target molecule are discussed elsewhere herein.
[0084] An antibody reagent can comprise an antibody or a polypeptide comprising an antigen- binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and single domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non- human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
[0085] The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
[0086] The terms "antigen-binding fragment" or "antigen-binding domain", which are used interchangeably to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term
"antigen-binding fragment" include (i) a Fab fragment, a monovalent fragment consisting of the VL,
VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
(v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those of skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.
[0087] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or antigen-binding portion thereof can bind. The specificity of an antibody or antigen-binding portion thereof, alone or in the context of a bispecific or multispecific polypeptide agent, can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen- binding protein (such as a bispecific or multispecific polypeptide agent), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/ KD). As will be clear to the skilled person, affinity can be determined in a manner known per se, depending upon the specific antigen of interest. Accordingly, a bispecific or multispecific polypeptide agent as defined herein is said to be "specific for" a first target or antigen compared to a another target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a KD value) that is at least 1 Ox, such as at least 1 OOx, and preferably at least lOOOx, and up to lOOOOx or more better than the affinity with which said amino acid sequence or polypeptide binds to the other target or polypeptide. For example, when a bispecific or multispecific polypeptide agent is "specific for" a target or antigen compared to another target or antigen, it is directed against said target or antigen, but not directed against such other target or antigen.
[0088] Avidity is the measure of the strength of binding between an antigen-binding molecule
(such as a bispecific polypeptide agent described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen- binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as a bispecific polypeptide agent described herein) will bind to their cognate or specific antigen with a dissociation constant (KD of 10~5 to 10~12 moles/liter or less, such as 10" 1 to 10" 12 moles/liter or less or 10" 8 to 10" 12 moles/liter (i.e., with an association constant (KA) of lO5 to 1012 liter/moles or more, and such as 107 to 1012 liter/moles or 10s to 1012 liter/moles).
Any KD value greater than 10"4 mol/liter (or any KA value lower than 104 M"1) is generally considered to indicate non-specific binding. The KD for biological interactions which are considered meaningful (e.g., specific) are typically in the range of 10"10 M (0.1 nM) to 10"5 M (10000 nM). The stronger an interaction is, the lower is its KD. A binding site on a bispecific or multispecific polypeptide agent described herein may bind to the desired antigen with an affinity less than 500 nM, less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.
[0089] Accordingly, as used herein, "selectively binds" or "specifically binds" refers to the ability of a polypeptide domain described herein to bind to a target, such as a molecule present on the cell-surface, with a KD of 10"5 M (10000 nM) or less, e.g., 10"6 M or less, 10"7 M or less, 10"8 M or less, 10"9 M or less, 10"10 M or less, 10"11 M or less, or 10"12 M or less. For example, if a polypeptide agent described herein binds to CEACAMl with a KD of 10"5 M or lower, but not to another
CEACAM molecule, such as CEACAM5, or a related homologue, then the agent is said to specifically bind CEACAMl. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay.
[0090] In some embodiments, humanized or composite human antibody reagents can be used in the compositions and methods described herein. By way of non-limiting example, such antibodies are described, e.g., in U.S. Patent App. No. 61/565,640, entitled Anti-CEACAM 1 Recombinant Antibodies for Cancer Therapy, filed 1 December 2011, and the references discussed therein. For example, in some embodiments of the compositions and methods described herein, a particular isolated recombinant antibody or antigen-binding portion thereof that binds specifically to
CEACAMl can comprise a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid residues SSHGMS (SEQ ID NO: 2), a heavy chain CDR2 consisting of the amino acid residues TISSGGTYTYYPDSVKG (SEQ ID NO: 3), a heavy chain CDR3 consisting of the amino acid residues HDFDYDAAWFAY (SEQ ID NO: 4), a light chain CDRl consisting of the amino acid residues SANSSVSYMY (SEQ ID NO: 5), a light chain CDR2 consisting of the amino acid residues LTSNLAS (SEQ ID NO: 6), and a light chain CDR3 consisting of the amino acid residues
QQWSSNPPT (SEQ ID NO: 7).
[0091] Alternatively, in some embodiments of the compositions and methods described herein, the CEACAMl -binding antibody or epitope-binding peptide can be constructed to have CDR regions selected from the following: a heavy chain CDR 1 consisting of the amino acid residues SSHGMS
(SEQ ID NO: 8), SFYGMS (SEQ ID NO: 9), or SDYYLY (SEQ ID NO: 10); a heavy chain CDR2 consisting of the amino acid residues TISSGGTYTYYPDSVKG (SEQ ID NO: l 1), TFSGGGNYTYYPDSVKG (SEQ ID NO: 12) or TISVGGGNTSYPDSVKG (SEQ ID NO: 13); a heavy chain CDR3 consisting of the amino acid residues HDFDYDAAWFAY (SEQ ID NO: 14), or HGGLPFYAMDY (SEQ ID NO: 15), or GLTTGPAWFAY (SEQ ID NO: 16); a light chain CDR1 consisting of the amino acid residues SANSSVSYMY (SEQ ID NO: 17), SVSSSISSSNLH (SEQ ID NO: 18), KSSQSLLNSSNQKNYLA (SEQ ID NO: 19), or RASQKISGYLS (SEQ ID NO: 20); a light chain CDR2 consisting of the amino acid residues LTSNLAS (SEQ ID NO: 21),
SVSSSISSSNLH (SEQ ID NO: 22), FASTRES (SEQ ID NO: 23), or AASTLDS (SEQ ID NO: 24); and a light chain CDR3 consisting of the amino acid residues QQWSSNPPT (SEQ ID NO: 25), QQWSSHPFT (SEQ ID NO: 26), QQHYSTPWT (SEQ ID NO: 27) or LQYASSLMYT
(SEQ ID NO: 28). See also U.S. Patent Pub. No. 2004/0047858, for example.
[0092] Other anti-CEACAMl monoclonal antibodies useful in the compositions and methods described herein include AgBlO, that inhibits CEACAMl -mediated EAE suppression in mice (Fujita et al., 175 Am. J. Pathol. 1116 (2009)); a CEACAMl antibody (Chen et al., 2004); murine D14HD11 (Yu et al., 281 J. Biol. Chem. 39179 (2006)); murine CEACAMl -specific CC1 (Iijima et al., 199 J. Exp. Med. 471 (2004)); mouse anti-human CEACAMl MRG-1 (Ortenberg 2012); mouse anti-human CEACAMl N-domain specific antibodies 5F4, 34B1, and 26H7 (IgGl) (Morales et al., 1999) each of which recognizes the N-terminal domain of CEACAMl (Watt et al., 2001); and 12-140-4,4/3/17, COL-4, YG-C28F2, D14HD11, B18.7.7, D11-AD11, HEA 81, CLB-gran-10, F34-187, T84.1, B6.2, and Bl . l . Monoclonal antibodies 34B1, 26H7, and 5F4 are also discussed in U.S. Patent Pub.
No. 2004/0047858, Therapeutic anti-BGP(C-CAMl) antibodies and uses thereof; U.S. Patent No. 7,132,255; WO 99/52552, and Morales et al., 163 J. Immunol. 1363 (1999). Further assessment of specificity was published in Watt et al., 2001, which described that monoclonal antibody 5F4 binds to a domain within the N-Domain of CEACAMl that is involved in homophilic interactions between the N-Domains of different CEACAMl molecules.
[0093] Other CEACAMl -Fc fusion proteins have been described, including, for example, the extracellular portion of CEACAMl fused to human IgFc in a mammalian expression vector (Markel et al., 110 J. Clin. Invest. 943 (2002)); pCEP4-N-CEACAMl-Fc see Gallagher, 71 J. Virol. 3129 (1997)); as well as commercially available sources of CEACAMl fusion proteins, as known to one of ordinary skill in the art.
[0094] Exemplary agents that bind CEACAMl, and methods for identifying such agents and whether such agents enhance or suppress T-cell activity are found, for example, in U.S. Patents No. 7,132,255 and No. 6,852,320, and the references cited therein, the contents of which are herein incorporated by reference in their entireties.
[0095] In some embodiments, the compositions and methods described herein relate to bispecific inhibitory agents, e.g. bispecific polypeptide agents. Bispecific agents comprise a molecule which is able to physically contact and inhibit at least two of CEACAMl, PD1, and/or LAP simultaneously. As used herein, the term "bispecific polypeptide agent" refers to a polypeptide that comprises a first polypeptide domain which has a binding site that has binding specificity for a first target, and a second polypeptide domain which has a binding site that has binding specificity for a second target, i.e., the agent has specificity for two targets, e.g., CEACAMl and PDl ; PDl and LAP; or CEACAMl and LAP. The first target and the second target are not the same (i.e., are different targets (e.g., proteins)). In some embodiments, the different targets can be co-expressed on the same cell. In some embodiments, a bispecific polypeptide agent can bind targets present on a single cell (heterophilic binding in cis), and/or bind one target on one cell and the other on another cell
(heterophilic binding in trans). Accordingly, a bispecific polypeptide agent as described herein can selectively and specifically bind to a cell that expresses the first target and the second target. A non- limiting example of a bispecific polypeptide agent is a bispecific antibody construct. Bispecific antibody constructs comprising antigen-binding portions of antibodies specific for two different antigens, e.g., PDl and CEACAMl ; or CEACAMl and LAP; or LAP and PDl, can be readily constructed by one of skill in the art. Generally, sequences encoding the antigen-binding domain of a first antibody characterized and known to bind a desired epitope on one antigen can be joined, either directly, or through any of a variety of linkers as known to the ordinarily skilled artisan, to sequences encoding the antigen-binding domain of a second antibody characterized and known to bind a desired epitope on a second antigen. Such sequences can be inserted into an appropriate vector and introduced to a cell to produce the bispecific antibody polypeptide by methods known to those of ordinary skill in the art. In some embodiments, a composition as described herein can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a PDl inhibitory agent. In some embodiments, a composition as described herein can comprise a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a LAP inhibitory agent. In some embodiments, a composition as described herein can comprise a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PDl inhibitory agent.
[0096] In some embodiments, the compositions and methods described herein can relate to a trispecific agent, i.e. an agent having specificity for all of CEACAMl, PDl, and LAP. As used herein, the term "trispecific polypeptide agent" refers to a polypeptide that comprises at least a first polypeptide domain having a binding site that has binding specificity for a first target, a second polypeptide domain having a binding site that has binding specificity for a second target, and a third polypeptide domain having a binding site that has binding specificity for a third target e.g.,
CEACAMl, PDl, and LAP. The first, second, and third target are not the same (i.e., are different targets (e.g., proteins)), but can all be present (e.g., co-expressed) on a cell.
[0097] In further embodiments, a multispecific polypeptide agent as described herein can bind one or more additional targets, i.e., a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target-specific binding sites respectively. A multispecific polypeptide agent has the potential to bind a cell that expresses all the targets the agent is specific for more strongly (e.g., with greater avidity) than a cell that expresses only one target, or less targets than the agent is specific for. A non-limiting example of a multispecific polypeptide agent is a multispecific antibody or antigen-binding fragment thereof. For the avoidance of doubt, a bispecific polypeptide agent or a trispecific polypeptide agent is a type or subset of multispecific polypeptide agent.
[0098] It is noted that a multispecific antibody reagent, e.g. a bispecific antibody reagent, can preferentially bind to a cell expressing each of the target antigens for which the reagent is specific. While, for example, a bispecific antibody reagent can bind to two separate cells, each of which expresses one of the target antigens, it can be more specific for cells which express both of the target antigens on the same cell surface.
[0099] In some embodiments, the antibody reagents provided herein, or used in the methods described herein, can further comprise post-translational modifications. Exemplary post-translational polypeptide modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group. As a result, the modified polypeptides can contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates. Such molecules can also be referred to as derivatives.
[00100] Reagents for use in the compositions and methods described herein include those that are conjugated or associated with polymers, such as PEG, that can enhance the half-life of the peptide in vivo. PEG modification is well-known in the art. See, e.g., PCT US2007/024067.
[00101] As used herein, the term "target" refers to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain or other moiety (e.g. a small molecule or aptamer among others) which has a binding site can selectively bind. The target can be, for example, an intracellular target (e.g., an intracellular protein target) or a cell surface target (e.g., a membrane protein, a receptor protein).
[00102] As used herein an "antibody" refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
[00103] As described herein, an "antigen" is a molecule that is bound by a binding site on a polypeptide agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. Generally, the bispecific or multispecific polypeptide agents described herein are selected for target specificity against two or more particular antigens (i.e., CEACAM1, PD1, and/or LAP). In the case of conventional antibodies and fragments thereof, the antibody binding site as defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen. The term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule (such as bispecific polypeptide agent described herein), and more particularly, by the antigen-binding site of said molecule.
[00104] As used herein, an "epitope" can be formed both from contiguous amino acids, or noncontiguous amino acids juxtaposed by folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An "epitope" includes the unit of structure conventionally bound by an immunoglobulin VH VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation. The terms "antigenic determinant" and "epitope" can also be used interchangeably herein.
[00105] As used herein, "immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, which comprise two β sheets and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signaling (for example, receptor molecules, such as the PDGF receptor).
[00106] A "target site" or "ligand interaction site" on the target molecule means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homodimerization or
heterodimerization) of the target; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target, e.g., PD1, LAP and/or CEACAM1. More generally, a "ligand interaction site" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on a target or antigen to which a binding site of an agent, or a bispecific or multispecific agent described herein can bind such that the target (and/or any pathway, interaction, signaling, biological mechanism or biological effect in which the target or antigen is involved), e.g., the signaling of any of
CEACAM1, PD1, and/or LAP is inhibited.
[00107] As used herein, a "blocking" antibody or an antibody "antagonist" is one which inhibits or reduces biological activity of the antigen(s) it binds. For example, a CEACAM1/PD1 bispecific antagonist antibody binds CEACAM1 and PD1 and inhibits the ability of CEACAM1 and PD1 to induce or maintain T-cell tolerance.
[00108] As used herein "domain" refers to a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases can be added, removed or transferred to other proteins without loss of function or properties of the remainder of the protein to which it is added or transferred and/or of the domain itself. In the context of an antibody, or a portion thereof, the term "binding domain" refers to such a domain that is directed against an antigenic determinant. By "single antibody variable domain" is meant a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least in part the binding activity and specificity of the full-length domain. Thus, a bispecific polypeptide agent will comprise at least two different binding domains
[00109] Polyclonal antibodies specific for CEACAM1, LAP, and/or PD1 can be produced by various procedures well known in the art. For example, PD1 polypeptides or fragments thereof, such as a fragment comprising amino acid residues 42-136 of SEQ ID NO: l, can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the protein. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen, e.g., a PD1 fragment and an adjuvant. It can be useful to conjugate the antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy-bean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxy-succinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R where R and R1 are different alkyl groups.
[00110] Animals can be immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g. , the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. [00111] Various other adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
[00112] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Various methods for making monoclonal antibodies described herein are available in the art. For example, the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975) or adaptations thereof, or by recombinant DNA methods (U.S. Pat. No. 4,816,567). For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammer- ling, et al, in: Monoclonal Antibodies and T-Cell Hybrido-mas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. It is to be understood that the term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
[00113] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with, e.g. PDl, or a fragment or derivative thereof, such as a fragment comprising amino acid residues 42-136 of SEQ ID NO: l, or a cell expressing PDl or a fragment thereof. Once an immune response is detected, e.g., antibodies specific for PDl are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding PDl and exerting a cytotoxic or cytostatic effect on activated lymphocytes. Ascites fluid, which generally contains high levels of antibodies, can be generated by injecting mice with positive hybridoma clones.
[00114] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); which is incorporated by reference herein in its entirety). [00115] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[00116] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et ah, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The foregoing references are incorporated by reference herein in their entireties.
[00117] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
[00118] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal.
[00119] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[00120] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures {e.g., by PCR or by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is described in more detail below. [00121] In another example, antibodies useful in the methods and compositions described herein can also be generated using various phage display methods known in the art, such as isolation from antibody phage libraries generated using the techniques described in McCafferty et al. , Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al, Nuc. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. The foregoing references are incorporated by reference herein in their entireties.
[00122] In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the nucleic acid sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine). In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the nucleic acid sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated into E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Phage expressing an antigen binding domain that binds to PD1, CEACAM1, or LAP or portions thereof can be selected or identified with antigen e.g. , using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman et al, 1995, J. Immunol. Methods 182:41-50; Ames et al, 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al, 1994, Eur. J. Immunol. 24:952-958; Persic et al, 1997, Gene 187:9-18; Burton et al, 1994, Advances in Immunology, 191 -280; PCT Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401 ; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108, the contents of which are herein incorporated by reference in their entireties.
[00123] As is described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 1992, 12(6):864-869; and Sawai et al, 1995, AJRI 34:26-34; and Better et al, 1988, Science 240: 1041-1043, the contents of which are herein incorporated by reference in their entireties.
[00124] Simple binding assays can be used to screen for or detect agents that bind to any of CEACAMl, PDl, and/or LAP, and/or reduce the signaling activity of any of the foregoing. Because CEACAMl and PDl are transmembrane proteins, assays that use the soluble forms of these proteins rather than full-length protein can be used, in some embodiments. Soluble forms include, for example, those lacking the transmembrane domain and/or those comprising the IgV domain or fragments thereof which retain their ability to bind their cognate binding partners.
[00125] Further, agents that inhibit CEACAMl, PDl, and/or LAP for use in the compositions and methods described herein, can be identified by, for example, measuring the activation of the signaling pathways controlled by CEACAMl, PDl and/or LAP. By way of non-limiting example, the expression of genes induced or suppressed by the activation of CEACAMl, PDl, and/or LAP signaling can be measured (e.g. by quantitative RT-PCR) or the level of phosphorylation of downstream signaling partners can be measured (e.g. by immunochemistry). Such markers for each of these pathways, and methods of measuring their levels are known in the art. Exemplary markers include but are not limited to: ZAP-70 phosphorylation and the level of SHP1 bound to CEACAMl as markers for CEACAMl signaling (see, e.g. Chen et al. Journal of Immunology 2008 6085-6093), SHP2 phosophorylation levels as markers for PDl signaling (see, e.g. Yamamoto et al. Blood 2008 111 :3220-4), and the level of phosphorylated SMAD2 and SMAD3 as markers for LAP signaling (see, e.g. Fleisch et al. Endocrine -Related Cancer 2006 13:379-400). All of the foregoing publications are incorporated by reference herein in their entireties.
[00126] Another variation of assays to determine binding of an inhibitory agent to, e.g. a CEACAMl protein is through the use of affinity biosensor methods. Such methods may be based on the piezoelectric effect, electrochemistry, or optical methods, such as ellipsometry, optical wave guidance, and surface plasmon resonance (SPR).
[00127] As another approach, efficacy of, e.g. an siRNA on the expression of PDl, LAP, and/or CEACAMl can be monitored using methods known in the art, such as quantitative RT-PCR with specific oligonucleotide primers for each gene respectively, or ELISA for PDl, LAP, and/or
CEACAMl . Alternatively, the expression of PDl, LAP and/or CEACAMl in a population of T cells can be determined by FACS analysis using the markers characteristic of particular populations and subpopulations known in the art or disclosed herein. [00128] In some aspects, provided herein are methods for enhancing an immune response in vivo, comprising administering to the subject a therapeutically effective amount of one or more agents that inhibit CEACAMl, LAP, and/or PDl, as described herein. Modulation of T-cell tolerance by such agents is useful for specifically enhancing an immune response in vivo, which can be useful for the treatment of conditions related to immune function including cancer and chronic infections.
Modulation of T-cell tolerance also is useful in in vitro or non-therapeutic applications including determining whether T-cells of a subject are functional (e.g., proliferation and/or cytotoxic functions), to determine if a treatment has rendered T-cells non- functional, in experimental models of cancer, e.g., to determine the effects of increases or decreases in T-cell function on particular organs or physiological processes, and to test for agents which increase or decrease T-cell activity. Other uses will be apparent to one of ordinary skill in the art. The agents that inhibit PDl, LAP and/or
CEACAMl can be used alone as a primary therapy or in combination with other therapeutics as a combination therapy to enhance the therapeutic benefits of other medical treatments.
[00129] CEACAMl, PDl, and/or LAP activity is "decreased" if one or more signaling activities or downstream read-outs of CEACAMl, PDl, and/or LAP activity is reduced or altered by a statistically significant amount, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,a t least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%), or more, up to and including 100%), in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in some embodiments, if CEACAMl, PDl, and/or LAP activity is decreased, some downstream read-outs will decrease but others can increase (i.e. things that are normally suppressed by CEACAMl, PDl, and/or LAP activity).
[00130] As will be clear to the skilled person, a decrease in activity of a target can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen, such as CEACAMl, PDl, or LAP, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen, such as CEACAMl, PDl, or LAP, for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co- factors, etc.), compared to the same conditions but without the presence of an inhibitory agent. Again, this can be determined in any suitable manner and/or using any suitable assay known per se or described herein, depending on the target involved. A decrease in the activity of a target can also mean effecting a change (i.e., in an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen, such as CEACAMl, PDl, and/or LAP, and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signaling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist can be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se or described herein, depending on the target or antigen involved.
[00131] A decrease in the activity of a target can, for example, also involve allosteric modulation of the target, (such as CEACAM1, PD1, and/or LAP); and/or reducing or inhibiting the binding of the target to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target. A decrease in the activity of a target can, for example, also involve effecting a change in respect of the folding or confirmation of the target, or with respect to the ability of the target to fold, to change its conformation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. A decrease in the activity of a target can, for example, also involve effecting a change in the ability of the target to signal, phosphorylate, dephosphorylate, and the like.
[00132] Immunosuppression of a host immune response plays a role in a variety of chronic immune conditions, such as in persistent infection and tumor immunosuppression. Recent evidence indicates that this immunosuppression can be mediated by immune inhibitory receptors expressed on the surface of an immune cell, and their interactions with their ligands. For example, cytotoxic CD8 T cells can enter a state of "functional exhaustion," or "unresponsiveness" whereby they express inhibitory receptors that prevent antigen-specific responses, such as proliferation and cytokine production. Accordingly, by inhibiting the activity and/or expression of such inhibitory receptors, an immune response to a persistent infection or to a cancer or tumor that is suppressed, inhibited, or unresponsive, can be enhanced or uninhibited.
[00133] As used herein, an "immune response" refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments, the response is specific for a particular antigen (an "antigen-specific response"), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
[00134] As used herein, "unresponsiveness" or "functional exhaustion" with regard to immune cells includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, for example, because of exposure to
immunosuppressants, exposure to high or constant doses of antigen, or through the activity of inhibitor receptors or factors, such as CEACAM1, PD1, and/or LAP. As used herein, the term
"unresponsiveness" includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the antigen has ceased. Unresponsive immune cells can have a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%), or even 100%> in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type.
[00135] As used herein, the term "reduces T cell tolerance" means that a given treatment or set of conditions leads to reduced T cell tolerance, i.e. greater T cell activity, responsiveness, and/or ability or receptiveness with regards to activation. Methods of measuring T cell activity are known in the art. By way of non-limiting example, T cell tolerance can be induced by contacting T cells with recall antigen, anti-CD3 in the absence of costimulation, and/or ionomycin. Levels of, e.g. LDH-A, RAB10, and/or ZAP70 (both intracellular or secreted) can be monitored, for example, to determine the extent of T cell tolerogenesis (with levels of IL-2, interferon-γ and TNF correlating with increased T cell tolerance). The response of cells pre -treated with, e.g. ionomycin, to an antigen can also be measured in order to determine the extent of T cell tolerance in a cell or population of cells, e.g. by monitoring the level of secreted and/or intracellular IL-2 and/or TNF-a (see, e.g. Macian et al. Cell 2002 109:719-731 ; which is incorporated by reference herein in its entirety). A compound known to reduce T cell tolerance which can be useful as a control, but which has extremely limited applicability in the clinic can include CTLA4. In one embodiment, a compound known to reduce T cell tolerance is not CTLA4.
[00136] Other characteristics of T cells having undergone adaptive tolerance is that they have increased levels of Fyn and ZAP-70/Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element modulator), B lymphocyte-induced maturation protein-1 (Blimp-1), PD1, CD5, and SHP2; increased phosphorylation of ZAP-70/Syk, LAT, PLCyl/2, ERK, PKC-Θ/ΙΚΒΑ; increased activation of intracellular calcium levels; decreased histone acetylation or hypoacetylation and/or increased CpG methylation at the IL-2 locus. Thus, in some embodiments, reduction of one or more of any of these parameters can be assayed to determine whether one or more agents that inhibits CEACAM1, LAP, and/or PD-1 enhances an immune response in vivo or decreases immune tolerance.
[00137] Reduction of T cell tolerance can also be measured by determining the proliferation of T cells in the presence of a relevant antigen assayed, e.g. by a 3H-thymidine incorporation assay or cell number. Markers of T cell activation after exposure to the relevant antigen can also be assayed, e.g. flow cytometry analysis of cell surface markers indicative of T cell activation (e.g. CD69, CD30, CD25, and HLA-DR). Reduced T cell activation in response to antigen-challenge is indicative of tolerance induction. Conversely, increased T cell activation in response to antigen-challenge is indicative of reduced tolerance.
[00138] Reduction of T cell tolerance can also be measured, in some embodiments, by determining the degree to which the inhibitory agent inhibits the activity of its target. By way of a non-limiting example, the activity of a CEACAM1 inhibitory agent can be determined using the staphylococcal enterotoxin B (SEB) model. SEB typically stimulates CEACAM1, resulting in upregulation of TIM3. In the presence of a CEACAMl inhibitory agent, the upregulation of TIM3 in response to SEB will be reduced.
[00139] The SEB model can also be adapted to measure T cell tolerance and modulation thereof. In normal mice, neonatal injection of staphylococcal enterotoxin B (SEB) induces tolerance in T cells that express reactive T cell receptor (TCR) V beta regions. If, in the presence of a
CEACAMl, PDl, and/or LAP inhibitory agent, T cells expressing reactive TCR V beta regions (e.g., Vbeta8) display a statistically significant greater T cell activity than T cells not contacted with the inhibitory agent, the inhibitory agent is one that reduces T cell tolerance.
[00140] Other in vivo models of peripheral tolerance that can be used in some aspects and embodiments to measure reduction(s) in T cell tolerance using the inhibitory agents described herein include, for example, models for peripheral tolerance in which homogeneous populations of T cells from TCR transgenic and double transgenic mice are transferred into hosts that constitutively express the antigen recognized by the transferred T cells, e.g., the H-Y antigen TCR transgenic; pigeon cytochrome C antigen TCR transgenic; or hemagglutinin (HA) TCR transgenic. In such models, T cells expressing the TCR specific for the antigen constitutively or inducibly expressed by the recipient mice typically undergo an immediate expansion and proliferative phase, followed by a period of unresponsiveness, which is reversed when the antigen is removed and/or antigen expression is inhibited. Accordingly, if, in the presence of a CEACAMl, PDl, and/or LAP inhibitory agent, in such models if the T cells proliferate or expand, show cytokine activity, etc. significantly more than T cells in the absence of the inhibitory agent, than that agent is one that reduces T cell tolerance. Such measurements of proliferation can occur in vivo using T cells labeled with BrDU, CFSE or another intravital dye that allows tracking of proliferation prior to transferring to a recipient animal expressing the antigen, or cytokine reporter T cells, or using ex vivo methods to analyze cellular proliferation and/or cytokine production, such as thymidine proliferation assays, ELISA, cytokine bead assays, and the like.
[00141] Reduction of T cell tolerance can also be assessed by examination of tumor infiltrating lymphocytes or T lymphocytes within lymph nodes that drain from an established tumor. Such T cells exhibit features of "exhaustion" through expression of cell surface molecules such as PDl, TIM-3 or LAG-3, for example, and decreased secretion of cytokines such as interferon-γ. Accordingly, if, in the presence of a CEACAMl, PDl, and/or LAP inhibitory agent, increased quantities of T cells with 1) antigen specificity for tumor associated antigens are observed (e.g. as determined by major histocompatibility complex class I or class II tetramers which contain tumor associated peptides) and 2 that are capable of secreting high levels of interferon-γ and cytolytic effector molecules such as granzyme-B, relative to that observed in the absence of the inhibitory agent, this would be evidence that T cell tolerance had been reduced. In one aspect, the technology described herein relates to methods for treating a chronic disease. Such methods can comprise administering a composition as described herein (i.e. a composition comprising at least two of (i) a CEACAM1 inhibitory agent, (ii) a PD1 inhibitory agent, and (iii) a LAP inhibitory agent) to a subject in need of treatment for a chronic disease. In some embodiments, a chronic disease can be selected from the group consisting of cancer; a persistent infection; and a chronic viral infection. In some embodiments, the subject in need of treatment for a chronic disease can be a subject suffering from undesirably high levels of T-cell tolerance, a subject diagnosed as suffering from undesirably high levels of T-cell tolerance, or a subject in need of a reduction or inhibition of T-cell tolerance. As used herein an "undesirably high level of T-cell tolerance" can be a level of T-cell tolerance which does not allow a subject's immune system to respond to and/or clear an infection or chronic disease. A subject in need of a reduction or inhibition of T-cell tolerance using the methods described herein includes but is not limited to a subject having cancer or a pre-cancerous condition (e.g. cytological changes indicating a shift from a normal phenotype that is not yet neoplastic but which marks the tendency to progress to a neoplastic phenotype), a subject with a chronic infection, or a subject suffering from infection by a pathogen.
[00142] In some embodiments of the methods described herein, the subject being administered an agent(s) for inhibiting at least two of CEACAM1, PD1, and/or LAP has a persistent infection with a bacterium, virus, fungus, or parasite. "Persistent infections" refer to those infections that, in contrast to acute infections, are not effectively cleared by the induction of a naturally occurring host immune response. During such persistent infections, the infectious agent and the immune response reach equilibrium such that the infected subject remains infectious over a long period of time without necessarily expressing symptoms. Persistent infections often involve stages of both silent and productive infection without rapidly killing or even producing excessive damage of the host cells. Persistent infections include for example, latent, chronic and slow infections. Persistent infection occurs with viruses including, but not limited to, human T-Cell leukemia viruses, Epstein-Barr virus, cytomegalovirus, herpesviruses, varicella-zoster virus, measles, papovaviruses, prions, hepatitis viruses, adenoviruses, parvoviruses and papillomaviruses.
[00143] In a "chronic infection," the infectious agent is present in the subject at all times. However, the signs and symptoms of the disease can be present or absent for an extended period of time. Non-limiting examples of chronic infection include hepatitis B (caused by hepatitis B virus (HBV)) and hepatitis C (caused by hepatitis C virus (HCV)) adenovirus, cytomegalovirus, Epstein- Barr virus, herpes simplex virus 1 , herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, and human T cell leukemia virus II. Parasitic persistent infections can arise as a result of infection by, for example, Leishmania, Toxoplasma, Trypanosoma, Plasmodium,
Schistosoma, and Encephalitozoon.
[00144] In some embodiments, a chronic infection can be a latent infection. In some embodiments, a chronic infection can include periods in which the infection is a latent infection. In a "latent infection," the infectious agent (such as a virus) is seemingly inactive and dormant such that the subject does not always exhibit signs or symptoms. In a latent viral infection, the virus remains in equilibrium with the host for long periods of time before symptoms again appear; however, the actual viruses cannot typically be detected until reactivation of the disease occurs. Non-limiting examples of latent infections include infections caused by herpes simplex virus (HSV)-l (fever blisters), HSV-2 (genital herpes), and varicella zoster virus VZV (chickenpox-shingles).
[00145] In a "slow infection," the infectious agents gradually increase in number over a very long period of time during which no significant signs or symptoms are observed. Non-limiting examples of slow infections include AIDS (caused by HIV-1 and HIV-2), lentiviruses that cause tumors in animals, and prions.
[00146] In addition, persistent infections that can be treated using the methods described herein include those infections that often arise as late complications of acute infections. For example, subacute sclerosing panencephalitis (SSPE) can occur following an acute measles infection or regressive encephalitis can occur as a result of a rubella infection.
[00147] The mechanisms by which persistent infections are maintained can involve modulation of virus and cellular gene expression and modification of the host immune response. Reactivation of a latent infection can be triggered by various stimuli, including changes in cell physiology, superinfection by another virus, and physical stress or trauma. Host immunosuppression is often associated with reactivation of a number of persistent virus infections. In some embodiments of the methods described herein, the subject being administered an agent(s) for inhibiting CEACAM1 and PD1 or CEACAM1 and LAP, for example, has an infection with a pathogen, such as a bacterium, virus, fungus, or parasite.
[00148] Examples of infectious viruses include, but are not limited to: Retroviridae (for example, HIV); Picornaviridae (for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such as strains that cause gastroenteritis);
Togaviridae (for example, equine encephalitis viruses, rubella viruses); Flaviridae (for example, dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (for example,
coronaviruses); Rhabdoviridae (for example, vesicular stomatitis viruses, rabies viruses); Filoviridae
(for example, ebola viruses); Paramyxoviridae (for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza viruses);
Bungaviridae (for example, Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);
Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses);
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as African swine fever virus); and unclassified viruses (for example, the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astro viruses). The compositions and methods described herein are contemplated for use in treating infections with these and other viral agents.
[00149] Examples of fungal infections include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. The compositions and methods described herein are contemplated for use in treating infections with these and other fungal agents.
[00150] Examples of infectious bacteria include, but are not limited to: Helicobacterpyloris , Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellular e, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii. The compositions and methods described herein are contemplated for use in treating infections with these and other bacterial agents. Other infectious organisms (such as protists) include: Plasmodium falciparum and Toxoplasma gondii. The compositions and methods described herein are contemplated for use in treating infections with these and other agents.
[00151] In some embodiments of the aspects described herein, the methods further comprise administering an effective amount of a viral, bacterial, fungal, or parasitic antigen in conjunction with an agent(s) for inhibiting PD1, LAP and/or CEACAM1. Non- limiting examples of suitable viral antigens include: influenza HA, NA, M, NP and NS antigens; HIV p24, pol, gp41 and gpl20;
Metapneumovirus (hMNV) F and G proteins; Hepatitis C virus (HCV) El, E2 and core proteins; Dengue virus (DENl-4) El, E2 and core proteins; Human Papilloma Virus LI protein; Epstein Ban- Virus gp220/350 and EBNA-3A peptide; Cytomegalovirus (CMV) gB glycoprotein, gH glycoprotein, pp65, IE1 (exon 4) and pp 150; Varicella Zoster virus (VZV) IE62 peptide and glycoprotein E epitopes; Herpes Simplex Virus Glycoprotein D epitopes, among many others. The antigenic polypeptides can correspond to polypeptides of naturally occurring animal or human viral isolates, or can be engineered to incorporate one or more amino acid substitutions as compared to a natural (pathogenic or non-pathogenic) isolate. [00152] In other aspects and embodiments of the methods described herein, the subject being administered an agent for inhibiting PD1 , LAP and/or CEACAM1 has a cancer or tumor.
Accordingly, provided herein, in some aspects, are methods of treating a subject having a cancer or tumor comprising administering an effective amount of an agent(s) for inhibiting PD1 , LAP and/or CEACAM1.
[00153] A "cancer" or "tumor" as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
[00154] By "metastasis" is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
[00155] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g. , small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
[00156] In some embodiments described herein, the methods further comprise administering a tumor or cancer antigen to a subject being administered an agent(s) for inhibiting PD1, LAP and/or CEACAM1 described herein. A number of tumor antigens have been identified that are associated with specific cancers. As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g. , activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Many tumor antigens have been defined in terms of multiple solid tumors: MAGE 1, 2, & 3, defined by immunity; MART-l/Melan-A, gplOO, carcinoembryonic antigen (CEA), HER-2, mucins (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, viral proteins such as proteins of hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively. However, due to the immunosuppression of patients diagnosed with cancer, the immune systems of these patients often fail to respond to the tumor antigens.
[00157] In some embodiments of the methods described herein, the methods further comprise administering a chemotherapeutic agent to the subject being administered an agent(s) for inhibiting
PD1, LAP and/or CEACAM1. Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor- free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as d platin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF- A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation.
[00158] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as an autoimmune disease, a chronic infection or a cancer. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
[00159] The term "effective amount" as used herein refers to the amount of an agent(s) for inhibiting PD1, LAP and/or CEACAM1 needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect, i.e., inhibit T cell tolerance, for example. The term "therapeutically effective amount" therefore refers to an amount of an agent(s) for inhibiting PD1, LAP and/or CEACAM1 using the methods as disclosed herein, that is sufficient to effect a particular effect when administered to a typical subject. An effective amount as used herein would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of disease. Thus, it is not possible to specify the exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00160] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the agent(s) for inhibiting PD1, LAP and/or CEACAM1), which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
[00161] The agents useful according to the compositions and methods described herein, including antibodies and other polypeptides, are isolated agents, meaning that the agents are substantially pure and are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the agents are sufficiently pure and are sufficiently free from other biological constituents of their host cells so as to be useful in, for example, producing pharmaceutical preparations. Because an isolated agent may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the agents may comprise only a small percentage by weight of the preparation.
[00162] The agents described herein for inhibiting PD1, LAP and/or CEACAM1 can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject. As used herein, the terms "administering," and "introducing" are used interchangeably and refer to the placement of a polypeptide agent into a subject by a method or route which results in at least partial localization of such agents at a desired site, such as a site of inflammation, such that a desired effect(s) is produced.
[00163] In some embodiments, the agents described herein for inhibiting PD1, LAP and/or CEACAM1 are administered to a subject by any mode of administration that delivers the agent systemically or to a desired surface or target, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration. To the extent that polypeptide agents can be protected from inactivation in the gut, oral administration forms are also contemplated. "Injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In preferred embodiments, the agents for inhibiting PDl, LAP and/or CEACAMl for use in the methods described herein are administered by intravenous infusion or injection.
[00164] The phrases "parenteral administration" and "administered parenterally" as used herein, refer to modes of administration other than enteral and topical administration, usually by injection. The phrases "systemic administration," "administered systemically", "peripheral administration" and "administered peripherally" as used herein refer to the administration of an agent for inhibiting PDl, LAP and/or CEACAMl other than directly into a target site, tissue, or organ, such as a tumor site, such that it enters the subject's circulatory system and, thus, is subject to systemic metabolism and other like processes.
[00165] For the clinical use of the methods described herein, administration of an agent for inhibiting PDl, LAP and/or CEACAMl can include formulation into pharmaceutical compositions or pharmaceutical formulations for parenteral administration, e.g., intravenous; mucosal, e.g., intranasal; ocular, or other mode of administration. In some embodiments, an agent for inhibiting PDl, LAP and/or CEACAMl described herein can be administered along with any pharmaceutically acceptable carrier compound, material, or composition which results in an effective treatment in the subject. Thus, a pharmaceutical formulation for use in the methods described herein can contain an agent for inhibiting PDl, LAP and/or CEACAMl as described herein in combination with one or more pharmaceutically acceptable ingredients.
[00166] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, an agent for inhibiting PDl, LAP and/or CEACAMl . Each carrier must be
"acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen- free water; (16) isotonic saline; (17) Ringer's solution; (19) pH buffered solutions; (20) polyesters, polycarbonates and/or polyanhydrides; (21) bulking agents, such as polypeptides and amino acids (22) serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) other non-toxic compatible substances employed in pharmaceutical formulations. Release agents, coating agents, preservatives, and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.
[00167] The agents for inhibiting PD1, LAP and/or CEACAM1 described herein can be specially formulated for administration of the compound to a subject in solid, liquid or gel form, including those adapted for the following: (1) parenteral administration, for example, by
subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or (79) nasally. Additionally, a bispecific or multispecific polypeptide agent can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et ah, Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.
[00168] Further embodiments of the formulations and modes of administration of an agent for inhibiting PD1, LAP and/or CEACAM1 that can be used in the methods described herein are illustrated below.
[00169] Parenteral Dosage Forms. Parenteral dosage forms of an agent for inhibiting PD1, LAP and/or CEACAM1 can also be administered to a subject by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.
[00170] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[00171] Aerosol formulations. An agent for inhibiting PD1, LAP and/or CEACAM1 can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. An agent for inhibiting PD1, LAP and/or CEACAM1 can also be administered in a non-pressurized form such as in a nebulizer or atomizer. An agent for inhibiting PD1, LAP and/or CEACAM1 can also be
administered directly to the airways in the form of a dry powder, for example, by use of an inhaler.
[00172] Suitable powder compositions include, by way of illustration, powdered preparations of an agent for inhibiting PD1, LAP and/or CEACAM1 thoroughly intermixed with lactose, or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which can be inserted by the subject into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation. The compositions can include propellants, surfactants, and co-solvents and can be filled into conventional aerosol containers that are closed by a suitable metering valve.
[00173] Aerosols for the delivery to the respiratory tract are known in the art. See for example,
Adjei, A. and Garren, J. Pharm. Res., 1 : 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm.,
114: 111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic an diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990);
Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemic delivery of peptides and proteins as well (Patton and Platz, Advanced Drug Delivery Reviews, 8: 179-
196 (1992)); Timsina et. al., Int. J. Pharm., 101 : 1-13 (1995); and Tansey, I. P., Spray Technol.
Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol Sci., 27: 769-783
(1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S. and R. H. Muller, J. Controlled
Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988);
Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-
196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S., et al, Controlled
Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R. W., et al, Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al, Pharm. Res., 13(1): 80-83
(1996), contents of all of which are herein incorporated by reference in their entirety.
[00174] The formulations of the agents for inhibiting PD1, LAP and/or CEACAM1 described herein further encompass anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles &
Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. Anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g. , vials) with or without desiccants, blister packs, and strip packs.
[00175] Controlled and Delayed Release Dosage Forms. In some embodiments of the methods described herein, an agent for inhibiting PD1, LAP and/or CEACAM1 can be administered to a subject by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled- release preparation in medical treatment is characterized by a minimum of drug substance being employed to address or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a compound of formula (I) is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
[00176] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the agents for inhibiting PD1, LAP and/or CEACAM1 described herein. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770;
3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543;
5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View,
Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, Duolite® A568 and Duolite® AP143 (Rohm&Haas, Spring House, Pa. USA).
[00177] In some embodiments, an agent for inhibiting PD1, LAP and/or CEACAM1 for use in the methods described herein is administered to a subject by sustained release or in pulses. Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administration of the same dose of the composition at a reduced frequency or administration of reduced doses. Sustained release or pulse administrations are particularly preferred when the disorder occurs continuously in the subject, for example where the subject has continuous or chronic symptoms of a viral infection. Each pulse dose can be reduced and the total amount of the agent for inhibiting PD1, LAP and/or CEACAM1 administered over the course of treatment to the patient is minimized.
[00178] The interval between pulses, when necessary, can be determined by one of ordinary skill in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or the active component of the composition is no longer detectable in the subject prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Intervals can be calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater than the composition half- life. Various methods and apparatus for pulsing compositions by infusion or other forms of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
[00179] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
[00180] As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. The term "or" is inclusive unless modified, for example, by "either." Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."
[00181] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [00182] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.
[00183] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A composition for enhancing the immune response or decreasing T cell tolerance in a subject, the composition comprising at least two agents selected from the group consisting of:
a CEACAM1 inhibitory agent; a PD1 inhibitory agent; and a LAP inhibitory agent.
2. The composition of paragraph 1, wherein the two agents are a CEACAM1 inhibitory agent and a PD1 inhibitory agent.
3. The composition of paragraph 1, wherein the two agents are a CEACAM1 inhibitory agent and a LAP inhibitory agent.
4. The composition of paragraph 1, wherein the two agents are a PD1 inhibitory agent and a LAP inhibitory agent.
5. The composition of paragraph 1, comprising a CEACAM1 inhibitory agent, a PD1 inhibitory agent, and a LAP inhibitory agent.
6. The composition of any of paragraphs 1-5, comprising a bispecific agent comprising a first portion which is a CEACAM1 inhibitory agent and a second portion which is a PD1 inhibitory agent.
7. The composition of any of paragraphs 1-5, comprising a bispecific agent comprising a first portion which is a CEACAM1 inhibitory agent and a second portion which is a LAP inhibitory agent.
8. The composition of any of paragraphs 1-5, comprising a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PD1 inhibitory agent.
9. The composition of any of paragraphs 1-8, wherein one or more of the inhibitory agents is an inhibitory nucleic acid.
10. The composition of any of paragraphs 1-9, wherein one or more of the inhibitory agents is an antibody reagent.
11. The composition of paragraph 10, wherein the antibody reagent binds a target selected from the group consisting of:
CEACAM1 ; PD1 ; and LAP.
12. The composition of paragraph 11, wherein the antibody reagent inhibits signaling mediated by the target. 13. The composition of any of paragraphs 1-12, wherein the CEACAMl inhibitory agent binds a CEACAMl molecule having the sequence set forth in SEQ ID NO:32, or an allelic or splice variant of SEQ ID NO: 32.
14. The composition of any of paragraphs 1-13, wherein the CEACAMl inhibitory agent binds a CEACAMl ligand interaction site.
15. The composition of paragraph 14, wherein the CEACAMl inhibitory agent binds to an amino acid residue corresponding to any of amino acid residues 1-429 of SEQ ID NO: 32.
16. The composition of any of paragraphs 1-15, wherein the CEACAMl inhibitory agent binds an extracellular domain of CEACAMl .
17. The composition of any of paragraphs 1-16, wherein the CEACAMl inhibitory agent binds the homophilic interaction domain of CEACAMl and inhibits homophilic interaction of CEACAMl molecules.
18. The composition of any of paragraphs 1-17, wherein the PD1 inhibitory agent binds a PD1 molecule having the sequence set forth in SEQ ID NO: l, or an allelic or splice variant of SEQ ID NO: 1.
19. The composition of paragraph 1-12 and 18, wherein the PD1 inhibitory agent binds a PD1 ligand interaction site.
20. The composition of paragraph 19, wherein the PD1 inhibitory agent binds to an amino acid residue corresponding to any of amino acid residues 41-136 of SEQ ID NO: 1.
21. The composition of any of paragraphs 1-12, wherein the LAP inhibitory agent binds a LAP molecule having the sequence set forth in SEQ ID NO: 34, or an allelic or splice variant of SEQ ID NO: 34.
22. The composition of paragraphs 1-12 and 21, wherein the LAP inhibitory agent binds a LAP ligand interaction site.
23. The composition of paragraph 22, wherein the LAP inhibitory agent binds to an amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 33.
24. A method for treating a chronic disease, the method comprising administering to a subject in need of treatment thereof an effective amount of a composition of any of paragraphs 1-23.
25. The method of paragraph 24, wherein the chronic disease is selected from the group
consisting of:
cancer; a persistent infection; and a chronic viral infection.
EXAMPLES
EXAMPLE 1 : Tumor infiltrating lymphocytes co-express CEACAMl together with other accessory molecules that are associated with T cell tolerance and exhaustion. [00184] Expression of inhibitory molecules on lymphocytes plays a major role in limiting immune defenses against cancers. Inhibition of these types of molecules is increasingly being shown to permit natural cancer defenses in association with the eradication of tumors. Molecules such as TIM3, CTLA4, LAP, PDl and more recently CEACAMl have been demonstrated to be increased on tumor infiltrating lymphocytes and, in some cases, to be associated with poor prognosis. In turn, blockade of these pathways has been demonstrated to be associated with increased cancer responses in mouse models and more recently in humans at least with respect to studies with CTLA4 and PDl . However, blockade of these pathways individually is associated with only limited benefit in humans.
[00185] Demonstrated herein is the upregulation of CEACAMl and PDl as well as CEACAMl and LAP together on tumor-infiltrating lymphocytes in models of colon cancer; a previously unreported observation. Accordingly, blockade of both CEACAMl and PDl or CEACAMl and LAP together is more effective than either one alone.
[00186] Described herein are results indicating that CEACAMl and PDl positive (double- positive) T cells are increased in tumor-infiltrating lymphocytes relative to that which is observed on lymphocytes under physiologic circumstances and increased association between CEACAMl and LAP expression on T cells in vivo. Inhibition of two or more of CEACAMl, PDl, and/or LAP together in vivo is a superior therapeutic approach to inhibition of a single factor alone.
[00187] The expression of a tolerance associated phenotype through combinations of cell surface molecules such as PD-1, TIM-3, LAP and CEACAMl was examined using the AOM-DSS model in wild-type mice (see, e.g. De Robertis et al. J Carcinog 2011 10:9; which is incorporated by reference herein in its entirety). Briefly, a dose of azoxymethane (AOM) with subsequent doses of the inflammatory agent dextran sodium sulphate (DSS) (Figure 1) increases the onset of colorectal cancer, and accordingly, T cell tolerance of the associated tumor infiltrating lymphocytes in approximation to the colorectal cancer cells.
[00188] Subpopulations of T cells obtained from these mice at Day 44 of the AOM-DSS protocol were subjected to FACS analysis. Tumor infiltrating lymphocytes, tumor intra-epithelial lymphocytes, adjacent tumor infiltrating lymphocytes, adjacent tumor intraepithelial lymphocytes, and cells from the mesenteric lymph nodes were examined for cell surface expression of CD4, CD8, PDl, CEACAMl, and TIM3 (Figures 2-4). The results of these analyses are summarized in Figures 5A-5B, demonstrating that tumor infiltrating lymphocytes, adjacent tumor infiltrating lymphocytes and adjacent tumor intraepithelial lymphocytes contain significant subpopulations of CEACAMl + PD1+ cells.
[00189] Figures 5A-5B depict graphs demonstrating CEACAMl and PDl are co-expressed on the cell surface of chronically activated CD4+ (Figure 5A) and CD8+ (Figure 5B) tumor infiltrating T lymphocytes. EXAMPLE 2: T lymphocytes with CEACAMl expression are characterized by co-expression of accessory molecules associated with T cell tolerance and a regulatory phenotype.
[00190] CEACAMl is preferentially expressed on specific subsets of circulating and tissue-resident CD4+ T cells. Attention was focused on defining the accessory molecules expressed by the small subset of CEACAMl + T cells detected under physiologic conditions in blood (data not shown) and mesenteric lymph node (MLN) (see Figure 6A) and which were increased in frequency in PP and LP of WT mice (Figure 9A and 9B). It was observed that CD4+CEAC AM 1 + T cells from the blood, MLN, PP and LP expressed higher amounts of both CD44 and CD62L than did
CD4+CEACAM 1 - T cells (Figure 6A and 9A and 9B). This is consistent with CEACAMl being associated with effector and/or central memory T cells (Boulton et al., 2002). Additionally, it was noted that CD4+CEACAM1+ T cells from each of these tissue compartments expressed more α4β7- integrin than their CD4+CEACAM1 -counterparts, suggesting that CEACAMl expression on T cells, even those within the peripheral immune system, may be associated with enhanced potential to home to intestinal tissues. Interestingly, it was also noted that CEACAMl positivity was associated with preferential expression of PD1, LAP (TGFP Latency Associated Peptide) and B220, all of which have previously been associated with T cell subsets possessing regulatory or accessory potential in the production of IgA (Fagarasan et al., 2001 ; Good-Jacobson et al., 2010; Ochi et al., 2006; Oida et al., 2003).
[00191] These observations led to investigation of specific cell subsets on which the identified molecules are known to be enriched. In particular, since the data described herein showed CEACAMl expression to be strongly co-segregating with surface LAP on CD4+ T cells from naive WT animals (Figures 6A and Figure 9A and 9B), CD4+CD25-LAP+ T cells, a subset of Treg that are concentrated in intestinal tissues although functioning throughout the body and provide TGF-β dependent regulatory function and, potentially, support for secretory IgA immunity were carefully examined (Niemir et al., 1995; Ochi et al., 2006; Oida et al., 2003). Whereas on average 43.6 ± 10.9 % of CD4+CD25-LAP+ T cells within MLN co-expressed CEACAMl, only 2.07 ± 0.8 % of CD4+CD25- LAP- cells were also CEACAM1+ under steady-state conditions in WT mice (Figure 6B). This was in stark contrast to the CEACAMl expression in another regulatory T cell subset wherein similar low levels of CEACAMl were observed on CD4+Foxp3+ and CD4+Foxp3- Treg cells from the MLN (Figure 6B). The relative expression levels of the CEACAMl -L and -S isoforms in
CD4+CEACAM1 -LAP-, CD4+CEACAM 1 +LAP-, or CD4+CEACAM 1 +LAP+ cells flow- cytometrically sorted from pooled spleen and MLN of WT mice was examined using the real-time PCR assay described below (Figure 8). Consistent with the findings in the total population of peripheral CD4+ T cells, the ratio of L:S isoforms was 2.78 ± 0.07 in CD4+CEACAM1+LAP- cells, indicating a predominance of the L isoform in this group of T cells (Figure 6C). In contrast, the L:S ratio was decreased to 1.16 ± 0.27 (P < 0.05) in CD4+CEACAM 1 +LAP+ cells indicating an enrichment of the CEACAM1-S isoform in this regulatory cell subset (Figure 6C). No differences were observed in the L:S ratio between CD4+CEAC AM 1 +CD25 - cells (1.52 ± 0.29) and
CD4+CEACAM 1 +CD25+ cells (1.52 ± 0.11) isolated from the spleen and LN (data not shown). The relative expression of the L and S isoforms does not differ between CD4+CEACAM1+CD25- and CD4+CEACAM1+CD25+ regulatory T cells with the L isoform predominating in both populations. The L:S ratio was 1.52 for CD4+CEACAM1+CD25- cells and 1.52 for CD4+CEACAM 1 +CD25+ cells. Cells were isolated by flow cytometric sorting from pooled spleen and MLN. These data demonstrate the existence of a close association between the expression of CEACAM1 and LAP.
EXAMPLE 3: CEACAMl-long and CEACAMl-short isoforms are regulated by tolerogenic signals provided by anti-CD3 stimulation.
[00192] Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), a member of the carcinoembryonic antigen family, is engaged in intercellular binding interactions that affect signal transduction by a number of cell surface receptors. CEACAM1 is constitutively expressed by a variety of cell types including those of endothelial, epithelial and hematopoietic origin such as B cells, polymorphonuclear leukocytes, monocytes, and dendritic cells. In addition, CEACAM1 is expressed at minimal levels in circulating natural killer (NK) and T cells but is upregulated in these cell types following a variety of different forms of activation (Azuz-Lieberman et al., 2005; Boulton et al., 2002;
Coutelier et al., 1994; Nakajima et al., 2002; Singer et al., 2002). On the cell surface, CEACAM1 can associate with and modify the function of numerous signaling receptors that are dictated by the
CEACAM1 isoforms expressed. The individual CEACAM1 isoforms are generated by alternative splicing and consist of transmembrane proteins that possess a characteristic membrane distal, IgV-like domain (the N-domain) which functions in homophilic binding but differs with respect to the number of membrane proximal extracellular IgC2-like domains and the length of their cytoplasmic (cyt) tail
(Gray-Owen et al., 2006). Specifically, the 11 human and 4 mouse CEACAM1 splice variants encode either a short (S) cyt tail about which little is known or a long (L) cyt tail containing immune receptor tyrosine -based inhibitory motifs (ITIM). In the latter case, consistent with the presence of ITIM motifs in their cyt tail, the CEACAMl-L isoforms of mice and humans have been linked to inhibition of many different signaling receptors (Chen et al., 2001 ; Chen et al., 2008; Kammerer et al., 1998;
Pan et al., 2010). In each example, the inhibitory function of CEACAMl-L isoforms is triggered by the phosphorylation of the ITIM tyrosine residues by receptor tyrosine kinases or Src -related kinases, resulting in recruitment of the Src homology 2 (SH2) domain-containing protein-tyrosine
phosphatases (SHP)-l or -2 (Huber et al., 1999; Nagaishi et al., 2006). Thus, CEACAMl-L-mediated recruitment of these phosphatases results in the dephosphorylation of critical tyrosines contained within activating motifs associated with a variety of cell surface receptors such as the B cell receptor, epidermal growth factor receptor and T cell receptor (TCR)/CD3 complex (Abou-Rjaily et al., 2004; Boulton et al., 2002; Chen et al., 2008; Lobo et al., 2009). As such, CEACAMl -L isoforms are functionally inhibitory and are the major isoforms described in mouse and human lymphocytes including NK cells, B cells and T cells.
[00193] CEACAMl -L and CEACAMl -S isoforms are expressed at varying ratios in different cell types with recent evidence showing that such expression is under the influence of cytokines (e.g. inter feron-γ), transcription factors (e.g. IRF-1) and splicing regulators (e.g. members of the heterogeneous nuclear ribonucleoprotein family) (Chen et al., 1996; Dery et al. 2011 ; Gencheva et al., 2010). Although not directly demonstrated, this is believed to be the case for T lymphocytes as well. It has been shown that while mouse and human T cells predominantly express CEACAMl -L isoforms, there is evidence that CEACAMl -S isoforms can be expressed (Donda et al., 2000; Singer et al., 2002). In vitro transfection studies in T cells suggest that CEACAMl is a 'tunable' receptor system given that CEACAMl -S isoforms may possess weak co-stimulatory function that is capable of ameliorating the inhibitory signals provided by CEACAMl -L isoforms in the context of TCR/CD3 complex signaling when expressed together or novel signals (Chen et al., 2004a; Chen et al., 2008; Lee et al., 2008). However, there is no information available on CEACAMl isoform regulation in T cells in vivo and the physiologic function(s) that this might confer.
[00194] By establishing a novel PCR method for quantifying mouse CEACAMl -S and
CEACAMl -L variants, it was found that T cells within the intestines, relative to peripheral tissues such as the spleen, not only exhibit increased overall CEACAMl expression but also uniquely possess a relative overabundance of CEACAMl -S relative to CEACAMl -L isoforms. Moreover, in this environment, it is shown herein that CEACAMl -S isoforms have special physiological functions which at a cellular level enhance T cell activation independently of CEACAMl -L isoforms and are preferentially expressed in cells performing common mucosal immune functions including T cell regulation and the induction of secretory immunity. Specifically, it is demonstrated herein that
CEACAMl -S acts as an individual signaling unit which promotes the induction of CD4+LAP+ T regulatory (Treg) cells (Figure 7A-7D), CD4+PD 1 +CXCR5+ follicular helper T cells (¾) (Figure
13A-13C), and the development of IgA committed B cells within Peyer's patches (PP) (data not shown). Consistent with this, T cell-associated CEACAMl -S isoforms lead to enhanced mucosal IgA secretion which influences both the composition of commensal microbiota and host defense against exposure to pathogens such as Listeria monocytogenes (data not shown). In the absence of
CEACAMl expression, these barrier-protective functions of IgA are diminished. Together, these studies provide tissue-specific evidence for CEACAMl isoform regulation and function.
[00195] The relative expression of CEACAMl isoforms was assessed by first establishing a novel, quantitative assay for analyzing expression of mouse CEACAMl -L and -S variants as currently available antibodies are not able to distinguish between these two general classes of
CEACAMl in a quantitative fashion. Two pairs of real-time PCR primers were designed to specifically quantify these in mouse T cells. The specificity and efficiency of these primers was verified using mouse (m) CEACAMl -4L (4L) and CEACAMl -4S (4S) plasmids (Figure 8).
Quantitative (q)PCR analysis of mouse primary CD3+ T cells isolated from spleen and lymph nodes (LN) demonstrated that CEACAMl -L isoforms predominated in resting peripheral T cells with increased expression upon anti-CD3 stimulation as previously described (Boulton et al., 2002;
Nakajima et al., 2002) (data not shown). In addition, although CEACAMl -S isoforms were detected, CEACAMl -L isoforms predominated in peripheral T cells such that the CEACAMl -L:CEACAM1 -S (L:S) ratio was 1.95 ± 0.34, consistent with previous studies (Singer et al., 2002). Having validated this qPCR assay with splenic T cells, a broad survey of CEACAMl isoform expression in primary T cells isolated from several different tissues was performed. It was noted that, unlike CD3+ T cells isolated from spleen or MLN wherein CEACAMl expression was low and CEACAMl -L isoforms predominated over CEACAMl -S isoforms, CD3+ T cells from the intestinal LP of the duodenum, jejunum, ileum or colon and Peyer's patches (PP) were observed to express an overall increase in CEACAMl transcript levels with significantly greater quantities of CEACAMl -S isoforms relative to CEACAMl -L isoforms. Thus, an average L:S ratio of <1.0 (PP: 0.53 ± 0.08; small intestine: 0.36 ± 0.11 ; and colon; 0.19 ± 0.04) was observed, which was reversed from peripheral T cells (data not shown). Accordingly, it was noted that the quantity and L:S ratio of CEACAMl expression varied cephalocaudally along the axis of the gut. Specifically, the amounts of CEACAMl and L:S ratio were more similar to spleen, MLN and peripheral blood in the proximal intestines. Furthermore, the levels of CEACAMl increased and the L:S ratio progressively decreased from the distal small intestine (ileum) to the colon. As a result, LP T cells of the PP and colon contained the highest levels of CEACAMl and the lowest L:S ratios relative to all other compartments examined. This was verified by immunoblotting with the CC1 mAb, further confirming that T cells isolated from PP and colonic LP expressed higher CEACAMl amounts relative to peripheral blood and especially CEACAMl -S isoforms which was most notable in the colonic LP (data not shown). T cells were purified from spleen (SP), PP, colon lamina propria (LP) of WT mice and spleen of 4S Tg and 4L Tg mice as well as B cells from spleen of WT mice. These studies both confirm the increased expression of
CEACAMl within intestinal T cells and further indicate that CEACAMl -S isoforms are not only expressed by primary T cells but establish that they are a major isoform within intestinal T cells and potentially other tissue compartments.
[00196] Pan T cells from the spleen were stimulated with plate-bound coated CD3, and CD3/CD8 for 2 and subsequently 4 days. CEACAMl -S, -L and total CEACAMl quantitative PCR were analyzed. It was observed that CEACAMl expression, which is low on resting T cells, is transcriptionally regulated by ligation of T cells through the TCR/CD3 complex (signal 1) but negatively regulated when the TCR/CD3 complex is co-engaged by the classical co-stimulatory signal (signal 2) provided by CD28 (Figure 11). These studies show that CEACAM1-S and -L isoforms are positively regulated by tolerogenic signals. EXAMPLE 4: Forced expression of CEACAMl on T cells promotes the generation of cells that co-express LAP (Treg) and PD-1 (Tfollicular helper or TFH).
[00197] CEACAM1-S independently regulates the expansion and function of CD4+ T cells in which it is preferentially expressed. The observations described herein, of preferential
CEACAMl expression on CD4+CD25-LAP+ from WT mice led to a question of the ability of CEACAMl to regulate these cells and, further, to interrogate the relative contribution of CEACAMl - L and CEACAMl -S isoforms in this process. Utilizing CEACAMl transgenic (Tg) mice that over- express CEACAMl -specifically in T cells, the frequency of CD4+CD25-LAP+ T cells in mucosal tissues was examined under conditions of forced over-expression of the CEACAMl -4L or -4S isoforms. These data demonstrated a significant increase in the percentage of CD4+CD25-LAP+ T cells in the MLN from CEACAMl -4S (4S Tg) mice relative to WT controls (Figure 7A and 7B) and m CEACAMl''- S Tg mice compared to CEACAMl '- controls (Figure 7C and 7D), wherein the CEACAMl -4S isoform is the only isoform expressed by T cells. This increase in CD4+CD25-LAP+ cells was specifically detected under conditions of over-expression of the CEACAMl -4S isoform in both WT and CEACAMl '- animals (Figure 7A and 7C) and was notably absent in CEACAMl -4L transgenic mice in which the CEACAMl isoform was overexpressed (Figure 7B and 7D). In stark contrast, the analysis did not reveal any differences in the frequency of CD4+CD25+Foxp3+ T cells upon forced expression of CEACAMl -4S in T cells relative to that observed in WT mice (data not shown). In order to ascertain whether enrichment of activating CEACAMl -S was functionally relevant to CD4+CD25-LAP+ T cells, the in vitro suppressive function of CD4+LAP+ T regulatory cells obtained from the mesenteric lymph node of 4S Tg mice was examined. It was found that these cells possessed enhanced regulatory activity when compared to CD4+CD25-LAP+ T cells from WT controls (Figure 7E). In contrast, no differences were observed in the regulatory potential between 4S Tg and WT CD4+CD25+ Treg cells obtained from the MLN in similar assays (Figure 7E). Taken together, these studies indicate that the relative increase of CEACAMl -4S, in the absence of any contribution from the inhibitory CEACAMl -L isoforms, specifically enhances both the development and functional capacity of regulatory CD4+CD25-LAP+ T cells.
[00198] Previous studies have shown that CD4+CD25-LAP+ T cells accumulate in intestinal tissues under steady-state and inflammatory conditions and that orally administered CD3 -specific antibodies induce CD4+CD25-LAP+ regulatory T cells in MLN (Boirivant et al., 2008; Ochi et al.,
2006). This approach was used in order to determine whether CEACAMl is specifically involved in the induction phase of CD4+CD25-LAP+ T cell development under conditions of physiological
CEACAMl expression. Oral administration of anti-CD3 antibody significantly increased the percentage of CD4+CD25-LAP+ T cells in the MLN of WT but not CEACAMl "'" mice (Figure 7F).
No significant differences were identified in the proportions of CD3+CD25+/-LAP+ Treg cells in spleen, MLN, PP or blood of untreated or isotype-treated CEACAMV ' and WT mice (data not shown). In contrast, oral administration of a CD3-specific antibody was not observed to alter the percentage of CD4+Foxp3+ T regulatory cells in the MLN of WT or CEACAMV1' mice (data not shown). Mice were fed anti-CD3 (5 μg/mouse/day) or an isotype control for 5 consecutive days and tissues were analyzed after 7 days for analysis of regulatory T cell frequency. These studies confirm a physiological role played by CEACAM1 in regulating the expansion of CD4+CD25-LAP+ T cells in mucosal tissues as well as in promoting their suppressive function.
[00199] Given the co-expression of CEACAM1 and PD1 on T cells in multiple tissue compartments, the impact of high levels of CEACAM1-4S on the frequency of cells that co-express PD-1, or so-called TFH cells, in the spleen, MLN and PP of 4S Tg mice was examined. Flow cytometric analysis of each of these tissues revealed an enrichment not only of PD1 itself but also of the PD1+CXCR5+ population of ¾ cells together with CEACAM1 in the 4S Tg mice compared to their WT littermate controls (Figure 13A-13B). Importantly, CEACAMl -4S-mediated enrichment of TFH cells occurred independently of CEACAMl-L isoforms since CEACAMV1- A Tg mice also contained higher levels of Tm cells than their CEACAMV/- counterparts (Figure 13C). Collectively, these data are consistent with a role for CEACAM1 and especially the CEACAMl-S isoforms in promoting the development of two types of accessory cells that express LAP (CD4+CD25+LAP+ T cells) and PD1 (TFH cells) which are not only enriched in organized lymphoid structures and mucosal tissues but are also known to be involved in immune tolerance and B cell switching (Cerutti, 2008; Good-Jacobson et al.).
EXAMPLE 5: CEACAMl-S isoforms provide unique activating signals that sensitize T cells to activation induced cell death and promote the development of regulatory populations of T cells.
[00200] Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail that signals at least in part via NFAT (data not shown). Previous studies on T cells from the periphery have shown that CEACAMl-L isoforms predominate with little CEACAMl-S isoforms in mouse and human. It is demonstrated herein that this is not the case in tissue resident T cells such as in intestines and gut associated lymphoid tissues which demonstrate predominant expression of CEACAMl-S isoforms relative to CEACAMl-L isoforms in both human and mouse. Thus primary mouse and human T cells express both isoforms of CEACAM1.
[00201] CEACAM1-4S functions as an independent signaling unit in the induction of T cell activation. Although CEACAMl-L isoform expression in T cells is well known to provide important negative regulation of TCR/CD3 complex function after ITIM phosphorylation by the src- related kinase, p56lck, and recruitment of SHP1 resulting in dephosphorylation of CD3^ and ZAP70 (Chen et al., 2008), very little is known about CEACAM1-S function in general or in T cells in particular. Previous results in T cells are limited to transfected in vitro model systems and have been controversial with both stimulatory and inhibitory functions assigned to CEACAMl -S splice variants (Chen et al., 2004a; Chen et al., 2004b). Given the impossibility of generating a knockout mouse for a specific CEACAM1-S splice variant without also deleting the alternative CEACAM1-L variant, CEACAMl -4S (4S) transgenic (Tg) mice were generated, overexpressing the mouse 4S isoform specifically in T cells under the human CD2 (hCD2) promoter in order to explore the function of CEACAMl -S isoforms in T cells in vivo. The overexpression of 4S in T cells was confirmed by flow cytometry using the CCl mAb. These studies confirmed that the T cells in the 4S Tg mice uniformly expressed increased CEACAMl as shown by flow cytometry using the CCl monoclonal antibody and anti-CD3 antibody in cells isolated from spleen (data not shown). Overexpression of CEACAMl -S isoforms on T cells in 4S Tg mice as measured by qPCR analysis using the assay described in Figure 8 confirming the dominance of this isoform in the Tg mice (data not shown).
[00202] When examined by flow cytometry, 4S Tg mice exhibited greater quantities of activated CD4+ and CD8+ T cells as shown by lower percentages of CD62LhiCD441o naive T cells, and a higher percentage of CD62LloCD44hi or CD62LhiCD44hi cells consistent with effector memory and potentially central memory T cells (Seder et al., 2003) respectively, in spleen, MLN, PP and colon LP (data not shown). It was noted that 4S Tg mice exhibited increased numbers of apoptotic CD3+ T cells (annexin V+7-AAD-CD3+ cells) within the peripheral blood, spleen, MLN and PP which correlated inversely with decreased levels of CD3+ cells (Figure 12A). In spite of this, total PP and MLN cell counts were found to be very similar between WT and CEACAMl -4S Tg mice (data not shown) indicating that overexpression of CEACAMl -4S was selectively depleting T cells. Intracellular cytokine staining of the CD4+ T cells isolated from spleen, MLN and PP also revealed that IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β were all increased in the T cells of 4S Tg mice when compared to that observed in WT littermates (data not shown). CEACAMl -S induction of cytokines (interferon-γ) influenced the total CD4+ T cell population (CD4+ T cells isolated from PP of WT or 4S Tg mice) consistent with a broad activating function of the 4S isoform when expressed at high levels (data not shown). CD3+ T cells isolated from the spleen of 4S Tg mice also exhibited a higher proliferation rate and more IFN-γ and IL-2 secretion upon anti-CD3 stimulation ex vivo in comparison to that associated with T cells from WT littermate controls (data not shown). However, with longer time -periods of culture, the proliferation rate of splenic CD3+ T cells, as measured by [3H] -thymidine incorporation as well as the production of IFN-γ and IL-2, from the 4S Tg mice was observed to be lower than that detected with the WT littermates after stimulation, consistent with in vitro T cell collapse due to increased activation-induced cell death (AICD) in the presence of the CEACAM1-4S isoform (Figure 12B). Consistent with this, splenic 4S Tg T cells exhibited increased annexin V staining (Figure 12C) and cleavage of caspase 3 (Figure 12D) after 3 hours of anti-CD3 stimulation. These results show that the mouse 4S isoform both co-stimulates primary T cells and sensitizes them to activation induced cell death (AICD), supporting previous in vitro transfection studies that have ascribed activating functions to this CEACAMl isoform in contrast to the inhibitory functions of the L isoform (Chen et al., 2004a; Chen et al., 2004b). Moreover, these observations further support the idea that both the increased total CEACAMl expression and the enrichment of the uniquely activating CEACAMl -S isoform observed on T cells in intestinal tissues is associated with the effector memory T cell phenotype which predominates in the normal intestinal LP under steady- state conditions (Hurst et al., 1999) as well as the selective expansion of T cells with a regulatory phenotype and focused on the promotion of secretory IgA.
[00203] The activating function of CEACAM1-4S in T cells is observed independently of CEACAM1-L isoform expression. Since the studies performed in 4S Tg mice were undertaken in the context of endogenous CEACAMl expression, it was possible that the apparent activating and inhibiting functions of the 4S isoform were either the consequence of CEACAMl -mediated neutralization of CEACAMl -L inhibitory function and/or the result of a direct activating signal by CEACAMl -S. To examine this question, 4S Tg mice that lacked expression of any other CEACAMl isoforms were generated by crossing 4S Tg mice with CEACAMl'1' mice on a C57BL/6 background (CEACAMl ' -AS Tg). Although less than that observed in 4S Tg T cells containing endogenous levels of CEACAMl in all cell types, Ceacaml-\'' -AS Tg mice still contained more activated T cells in vivo as revealed by lower percentages of CD4+(or CD8+) CD62LhiCD441o naive T cells, and higher percentages of CD4+(or CD8+) CD62LloCD44hi effector memory T cells in spleen, MLN, PP and colon LP (data not shown) in comparison to CEACAMl'1' littermates. CEACAMl'1' -AS Tg mice also exhibited more apoptotic, Annexin+ CD3+ T cells within the MLN, blood and PP (Figure 12E) inversely correlating with decreased levels of CD3+ cells (data not shown). CD4+ T cells isolated from the spleens of CEACAMl'1' -4S Tg mice also proliferated more vigorously and secreted more IL- 2 and IFN-γ upon anti-CD3 stimulation. Notably, splenic CD4+ T cells from CEACAMl '' mice exhibited increased activation upon anti-CD3 stimulation that was intermediate to the anti-CD3 induced stimulation observed with WT and CEACAMl'1' -A Tg T cells (data not shown). On the other hand, splenic T cells from mice in which only the CEACAMl -4L (4L) isoform was expressed by T cells, generated by crossing previously established 4L Tg mice (Nagaishi et al., 2006) with
CEACAMl'1' mice, exhibited a blunted response to anti-CD3 stimulation which was less than that observed with WT T cells and is consistent with the direct inhibitory functions of this isoform. Taken together, these studies show that loss of dominant endogenous CEACAMl -L inhibitory function in CEACAMl ' mice or gain of CEACAMl -S expression independently of CEACAMl -L isoforms, as observed in CEACAMl'1' -AS, Tg T cells, results in enhanced TCR/CD3 complex signaling and sensitization to activation induced cell death in the latter case. In comparison, gain of CEACAMl -L isoforms independently of CEACAMl -S isoforms leads directly to inhibition of T cell function. Thus CEACAM1-S and CEACAM1-L are able to function as independent activating and inhibitory signaling units in T cells that are in the end both associated with immune tolerance. [00204] In the case of CEACAMl -S, this activating function is associated ultimately with regulation of immune responses as it results in sensitization to activation induced cell death and the promotion of T cells with regulatory and thus inhibitory function such as CD4+CEACAM1+LAP+ T cells. In contrast, the CEACAMl -L isoforms directly deliver inhibitory signals to T cells.
Discussion
[00205] Described herein is evidence for tissue and cell-type specific expression of
CEACAMl, the association of CEACAMl with specific types of cell surface molecules that are associated with immune tolerance such as TIM3, PD-1 and LAP as well as the regulation of
CEACAMl splicing in T cells which demonstrates the important role played by CEACAMl -S isoforms and the unique predominance of CEACAMl -S isoforms in intestinal resident T cells and specific subsets of T cells associated with immune regulation and humoral immunity. Although CEACAMl is an activation-induced molecule on T cells, the observed tissue-specific splicing was not due to activation of the T cells per se as it was not observed when T cells were stimulated ex vivo. Adoptive transfer experiments demonstrated that CEACAMl -S dominance was gained by naive peripheral T cells upon entry into the intestines, and was lost when mucosal T cells were removed from the intestines and re-stimulated ex vivo. This indicates that the specific mucosal milieu of the intestines promotes CEACAMl -S expression in T cells. Moreover, the results in 4S Tg mice indicate CEACAMl -S facilitates T cell activation, sensitization to activation induced cell death and the induction of secretory IgA immunity and cells with regulatory function. Consistent with this, T cell subsets which bear a memory phenotype characteristic of mucosal tissues (Gibbons et al., 2011) and specific subsets of T cells such as CD4+CD25-LAP+ and Tm cells which possess regulatory functions and encourage secretory immunity display an enrichment upon CEACAM1-S expression and are driven by its presence. Thus, CEACAMl -S expression is promoted and maintained by the mucosal environment and facilitates T cell activation linked to the generation of cell types responsible for mucosal regulation, tolerance and protection.
[00206] Importantly, the studies described herein also demonstrate that CEACAMl -S is not a minor isoform in primary T cells as was currently thought (Gray-Owen et al., 2006). In transgenic mice with conditional expression of CEACAMl in T cells, it was found that CEACAMl -S provides unique co-stimulatory signals which promote both activation induced cell death and an effector memory phenotype, whereas CEACAMl -L is directly co-inhibitory for TCR/CD3 complex signaling
(Nagaishi et al., 2006). Moreover, it is now shown that CEACAMl -S in T cells can elicit this stimulation as an independent signaling unit without CEACAMl -L assistance in vivo. Through these properties, CEACAMl -S regulates specific subsets of T cells. These cell populations include
CD4+LAP+ T cells and Tm cells which are an integral component of mucosal tissues and organized lymphoid structures, respectively, and are known to regulate immune tolerance and secretory immunity (Linterman et al., 2011). Accumulation of such cell surface molecules such as CEACAMl together with PD1, TIM3 and LAP as well as cell types associated with these cell surface molecules (e.g. CD4+CEACAM 1 +LAP+ or CD4+CEACAM+PD 1 + T cells) amongst tumor infiltrating lymphocytes would be predicted to be deleterious for the host resulting in either direct suppression of effector T cell function or inhibition of the effector T cells through the effects of the accumulated regulatory T cells. In view of CEACAM1-S enrichment in these latter cell types irrespective of their localization to peripheral or mucosal tissues as well as with the observations that the intestinal tissues facilitate CEACAM1-S expression, these studies also raise that possibility that CD4+LAP+ T cells and TFH cells may be born in and/or modified remotely by mucosal factors.
[00207] Although little is known about CEACAM1-S signaling in T cells, in epithelial cells it has been shown to associate with the cytoskeleton (actin and tropomyosin), calmodulin and annexin II (Edlund et al., 1996; Kirshner et al., 2003; Schumann et al., 2001). Such intracellular associations have interestingly been linked to the promotion of apoptosis of mammary epithelial cells during acini morphogenesis (Kirshner et al., 2003). In addition, consistent with previous transfection studies in Jurkat T cells, CEACAM1-S signaling was able to activate NFAT in vivo (Chen et al., 2004b). As shown here, this was associated with the upregulation of NFAT-dependent soluble and cell surface molecules such as PD1 and CD40L (data not shown). Thus these studies not only demonstrate the importance of CEACAM1-S in T cells under physiological conditions but also their role in delivering independent signals that may oppose those provided by CEACAM1-L isoforms and drive the cell to a unique destination that is associated with either cell death or a regulatory function.
Experimental Procedures
[00208] Animals. Wild-type (WT) C57BL/6 (B6) mice were purchased from The Jackson Laboratory. hCD2 promoter controlled CEACAM1-4S transgenic mice were generated by a similar strategy as previously described in the of Brigham and Women's Hospital Transgenic Core Facility (Nagaishi et al., 2006). CEACAM l'1' rmce were previously described (Leung et al., 2006). Swiss Webster germ-free (SWGF) mice and C57BL/6 germ-free mice (B6GF) were maintained in germ-free isolators at Taconic. All other mice were maintained under specific pathogen-free conditions at the Harvard Center for Comparative Medicine at Harvard Medical School. All mice were used between 8 and 12 weeks of age.
[00209] Flow Cytometry. For CEACAM1 expression assays, cells were incubated with the
CC1 antibody for 20 min followed by FITC-conjugated rat anti-mouse IgGl . For other cell surface proteins, cells were stained with the indicated fluorescence-conjugated antibodies for 20 minutes, washed and resuspended with 1% BSA/PBS FACS staining buffer containing DAPI (Invitrogen). Cell apoptosis assays were performed according to the Annexin V-FITC Apoptosis Detection Kit manual
(BD Bioscience). For intracellular cytokine staining, cells were stimulated with ionomycin (Sigma, 2 tg/ml), PMA (Sigma, 30 ng/ml) and GOLGISTOP™ (BD Bioscience) for 4 hours followed by intracellular cytokine staining using CYTOFIX/CYTOPERM™ solution kit according to the manufacturer's instructions (BD Bioscience). IgA staining was performed according to the instructions provided with the rat anti-mouse IgA antibody (CI 0-3). CD4+LAP+ T cells were stained for 20 min with biotinylated anti-LAP antibody (R&D Systems) followed by APC-conjugated streptavidin (BD Biosciences) as described previously (Ochi et al., 2006). TFH cells were stained for CXCR5 and PD1 as described previously (Linterman et al., 2011). All stained cells were analyzed on an LSRII™ (BD Biosciences) or a MACSQUANT™ (Miltenyi Biotech) flow cytometer, and data were analyzed using FLOWJO™ software.
[00210] Oral feeding of anti-CD3. Mice were fed with anti-CD3 antibody using a protocol described previously (Ochi et al., 2006). Specifically, groups of paired mice were fed anti-CD3 or IgG isotype control antibody at a dose of 5 μg/mouse/day for 5 days. After one day's rest, mice were sacrificed at day 7 and the percentage of CD4+LAP+ regulatory T cells was determined in specific lymphoid tissues.
[00211] Suppression assays. Regulatory T cell suppression assays were performed as previously described (Fantini et al., 2007). Briefly, 1x105 freshly isolated splenic CD4+CD25- T cells were stained with CFSE and mixed with an equal number of either CD4+CD25+ or CD4+LAP+ T regulatory cells isolated via magnetic sorting (Miltenyi). T cell mixtures were then activated in the presence of soluble anti-CD3 (1 μg/ml) and 4x105 irradiated total splenocytes as antigen presenters. After 4 days of culture, CFSE dilution of the responder CD4+CD25- T cells was measured as an indication of proliferation.
[00212] Total Serum and secretion Immunoglobulin ELISA. The sera, intestinal washes or fecal extracts were collected for ELISA as previously described (Hapfelmeier et al., 2010). Data are presented as mean ± S.D.
[00213] Immunohistochemical analysis. PP were isolated and embedded in OCT compound (Sakura Finetechnical) and snap frozen in dry ice. Frozen sections were prepared on micro slides using a cryostat (Leica), and stored at - 70°C until use. The slides were fixed in 100% acetone for 5 min at 4°C and air dried at room temperature. Cryostat sections were blocked with 10% normal goat serum for 1 hour at room temperature (RI). For B220 and IgA staining, the slides were stained with purified rat anti-mouse IgA antibody for 1 hour at RI, followed by incubation with ALEXA
FLURO™ 568 conjugated goat anti-rat IgG secondary antibody (Invitrogen) and FITC-conjugated anti-mouse B220 antibody (BD Bioscience) for 1 hour at RI. For NFAI staining, the slides were stained with biotin conjugated-anti-mouse CD4 antibody and rabbit anti-NFAll antibodies for 2 hours at RI, followed by incubation with rhodamine red-X-streptavidin (Jackson ImmunoResearch) and ALEXA FLUOR® 488 F(ab')2 fragment of goat anti-rabbit IgG (H+L) (Invitrogen) for 1 hour at RI. The counterstaining was performed using DAPI (Invitrogen). The specimens were analyzed using a NIKON ECLIPSE II™ microscope.
[00214] Statistics. The Mann- Whitney U-test (two-tailed) was used to assess differences in loglO colony forming units (c.f.u.) in quantitative culture assays. The t-test (two-tailed) was used to assess differences between means for other data analyzed. Tests with a P-value less than 0.05 were considered statistically significant. Antibodies and Reagents. A mAb specific for mouse CEACAM1, CC1, was previously described (Dveksler et al., 1993). 5F4 is a mouse anti-human CEACAM1 specific mAb (Morales et al., 1999). Caspase-3 and NFAT1 antibodies were purchased from Cell Signaling. GAPDH and 13-actin antibodies were obtained from Sigma- Aldrich. Biotinylated anti-LAP antibody was from R&D Systems (Ochi et al., 2006). All other antibodies used for flow cytometry or immunohistochemistry staining were purchased from BD Biosciences, eBioscience or Biolegend. Human rIL-2 for the stimulation of mouse primary T cells was provided by the National Institutes of Health.
[00215] Lymphocyte Isolation. Single cell suspensions of mononuclear cells from spleen, lymph nodes, blood, Peyer's patches (PP) (Iwasaki and Kelsall, 1999) or intestinal lamina propria (LP) were prepared as previously described (Nakajima et al., 2002). PP or intestines (removed PP, rinsed with PBS and cut in small pieces) were treated with media containing dithiothreitol 100 μg/ml (Sigma), 25 mM Hepes, 10% fetal bovine serum (FBS), and 5 mM EDTA in RPMI1640 for 30 min at 37 °C to remove epithelial cells, and were washed extensively with RPMI1640. Tissues were further digested in RPMI 1640 containing 2% FBS, collagenase D (400 U/ml, Roche) and DNase I (0.1 mg/ml, Sigma) in a shaking incubator at 37 °C for 30 min. Cells were then layered on a 40-75%) Percoll gradient (GE Healthcare) and lymphocyte-enriched populations were isolated from the cells at the 40-75%) interface. After washing, single cell suspensions were prepared. Cells were directly used for flow staining, or were stained with PE conjugated anti-CD3 antibody and followed by anti-PE MicroBeads according to the instructions provided by Miltenyi Biotech to further isolate CD3+ T cells.
[00216] Single-cell isolations from spleen and mesenteric lymph nodes were performed with a 40 tm cell strainer (BD Bioscience) and red blood lysis buffer (Sigma) for depletion of red blood cells. CD3+ or CD4+ T cells were isolated from spleen and/or lymph nodes of mice by pan T cell isolation kit or CD4+ T cell isolation kit for mouse (Miltenyi Biotech) according to the
manufacturer's instructions. Quantification of Peyer's patch. The entire small intestines were removed from mice. Lipid on the surface was carefully removed, and then the number and size of PPs were determined by macroscopic observation. The PP score was established by combining the number and size, and calculated in a blinded fashion, as described previously (Barreau et al., 2007). For absolute quantification of PP numbers, SI were opened along the mesenteric border, washed by shaking in dH20 for one hour and fixed in 5% glacial acetic acid overnight. After washing in dH20 for another hour, tissues were stained by briefly incubating in Unna's polychrome methylene blue stain (31.3mM methylene blue, 72.4mM potassium carbonate). Tissues were washed once more, then left in dH20 for 48h to allow differentiation of the stain.
[00217] Cell Culture, Proliferation and Determination of Cytokine Production. Cells were resuspended in complete RPMI 1640 medium containing 10% FBS, 1% penicillin and streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 20 mM HEPES, and 1% nonessential amino acids (all from Invitrogen) at a density of 1 x 106 cells/ml, and cultured in 96-well flat bottomed plates with the indicated concentrations of plate -bound anti-CD3 antibodies (eBioscience). Culture supematants were harvested at 24 h and cytokine production determined by ELISA (BD Biosciences) according to the manufacturer's instructions. Proliferation assays were performed by addition of [3H] -thymidine (1.0 μΟΛνβΙΙ) at 48 hours for 8 hours.
[00218] Protein Isolation and Western Blotting. Spleen and MLN mononuclear cells were washed twice in cold PBS. Total protein was isolated with RIPA buffer as previously described (Chen et al., 2008). The nuclear and cytoplasmic protein was isolated using NE-PER Nuclear and
Cytoplasmic Extraction Kit (Thermo Scientific). The concentration of proteins in the supematants was assessed using the BAC Protein Assay (Thermo Scientific). Immunoblotting was performed as previously described by using specific antibodies as indicated in the results and legends (Chen et al., 2008).
[00219] Real-time Quantitative PCR. RNA was extracted using RNEASY™ Mini kit (Qiagen) according to the manufacturer's instructions, and genomic DNA was removed with DNase I. qPCR was performed as described previously using SYBR GREEN™ quantitative PCR master mix (Roche) (Nagaishi et al., 2006). All runs were accompanied by two internal control genes, 13-actin and GAPDH. Samples were normalized using a ΔΔ cycle threshold-based algorithm to provide arbitrary units representing relative expression levels between samples. The following primers were used for mouse CEACAMl :
CEACAMl -L, 5 ' -GCGAGATCTCACAGAGCACA-3 ' (forward; SEQ ID NO: 35) and 5'- GCTGGGAATTGAAGTTCAGG-3 ' (reverse; SEQ ID NO: 36); CEACAM1-S, 5'- CTGGCATCGTGATTGGAGTT-3 ' (forward; SEQ ID NO: 37) and 5'-
CAGAAGGAGCCAGATCCG-3 ' (reverse; SEQ ID NO: 38). hnRNP expression was determined by qPCR, as described previously (Dery et al., 2011).
[00220] References
Abou-Rjaily, G. A., Lee, S. J., May, D., Al-Share, Q. Y., Deangelis, A. M., Ruch, R. J., Neumaier, M., Kalthoff, H., Lin, S. H., and Najjar, S. M. (2004). CEACAMl modulates epidermal growth factor receptor— mediated cell proliferation. J. Clin. Invest. 114, 944-952.
Azuz-Lieberman, N., Markel, G., Mizrahi, S., Gazit, R., Hanna, J., Achdout, H., Gruda, R., Katz, G., Amon, T. I., Battat, S., et al. (2005). The involvement of NK cells in ankylosing spondylitis. Int. Immunol. 17, 837-845.
Barreau, F., Meinzer, U., Chareyre, F., Berrebi, D., Niwa-Kawakita, M., Dussaillant, M., Foligne, B., Ollendorff, V., Heyman, M., Bonacorsi, S., et al. (2007). CARD15/NOD2 is required for Peyer's patches homeostasis in mice. PLoS One 2, e523.
Baumgart, M., Dogan, B., Rishniw, M., Weitzman, G., Bosworth, B., Yantiss, R., Orsi, R. H., Wiedmann, M., McDonough, P., Kim, S. G., et al. (2007). Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J. 1, 403-418.
Bergqvist, P., Stensson, A., Lycke, N. Y., and Bemark, M. (2010). T cell-independent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation. J. Immunol. 184, 3545-3553.
Bjorneklett, A., Fausa, O., and Midtvedt, T. (1983). Bacterial overgrowth in jejunal and ileal disease. Scand. J. Gastroenterol. 18, 289-298.
Boirivant, M., Amendola, A., Butera, A., Sanchez, M., Xu, L., Marinaro, M., Kitani, A., Di Giacinto, C, Strober, W., and Fuss, I. J. (2008). A transient breach in the epithelial barrier leads to regulatory T-cell generation and resistance to experimental colitis. Gastroenterology 135, 1612-1623 el615. Borsutzky, S., Cazac, B. B., Roes, J., and Guzman, C. A. (2004). TGF-beta receptor signaling is critical for mucosal IgA responses. J. Immunol. 173, 3305-3309.
Boullier, S., Tanguy, M., Kadaoui, K. A., Caubet, C, Sansonetti, P., Corthesy, B., and Phalipon, A. (2009). Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits. J. Immunol. 183, 5879-5885.
Boulton, I. C, and Gray-Owen, S. D. (2002). Neisserial binding to CEACAMl arrests the activation and proliferation of CD4+ T lymphocytes. Nat. Immunol. 3, 229-236.
Cazac, B. B., and Roes, J. (2000). TGF-beta receptor controls B cell responsiveness and induction of IgA in vivo. Immunity 13, 443-451.
Cerutti, A. (2008). The regulation of IgA class switching. Nat. Rev. Immunol. 8, 421-434.
Chen, C. J., Lin, T. T., and Shively, J. E. (1996). Role of interferon regulatory factor- 1 in the induction of biliary glycoprotein (cell CAM-1) by interferon-gamma. J. Biol. Chem. 271, 28181- 28188.
Chen, C. J., and Shively, J. E. (2004a). The cell-cell adhesion molecule carcinoembryonic antigen- related cellular adhesion molecule 1 inhibits IL-2 production and proliferation in human T cells by association with Src homology protein- 1 and down-regulates IL-2 receptor. J. Immunol. 172, 3544- 3552.
Chen, D., Iijima, H., Nagaishi, T., Nakajima, A., Russell, S., Raychowdhury, R., Morales, V., Rudd, C. E., Utku, N., and Blumberg, R. S. (2004b). Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulate human T cell function. J. Immunol. 172, 3535-3543.
Chen, T., Zimmermann, W., Parker, J., Chen, I., Maeda, A., and Bolland, S. (2001). Biliary glycoprotein (BGPa, CD66a, CEACAMl) mediates inhibitory signals. J. Leukoc. Biol. 70, 335340. Chen, Z., Chen, L., Qiao, S. W., Nagaishi, T., and Blumberg, R. S. (2008). Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70. J. Immunol. 180, 6085-6093. Cong, Y., Wang, L., Konrad, A., Schoeb, T., and Elson, C. O. (2009). Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine -protective regulatory T cells. Eur. J. Immunol. 39, 3134-3146.
Coutelier, J. P., Godfraind, C, Dveksler, G. S., Wysocka, M., Cardellichio, C. B., Noel, H., and Holmes, K. V. (1994). B lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus. Eur. J. Immunol. 24, 1383-1390. Dery, K. J., Gaur, S., Gencheva, M., Yen, Y., Shively, J. E., and Gaur, R. K. (2011). Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP Al, and hnRNP M. J. Biol. Chem. 286, 16039-16051.
Donda, A., Mori, L., Shamshiev, A., Carena, I., Mottet, C, Heim, M. H., Beglinger, C, Grunert, F., Rochlitz, C, Terracciano, L., et al. (2000). Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response. Eur. J. Immunol. 30, 2593-2603.
Dveksler, G. S., Pensiero, M. N., Dieffenbach, C. W., Cardellichio, C. B., Basile, A. A., Elia, P. E., and Holmes, K. V. (1993). Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor. Proc. Natl. Acad. Sci. U.S.A. 90, 1716- 1720.
Edlund, M., Blikstad, I., and Obrink, B. (1996). Calmodulin binds to specific sequences in the cytoplasmic domain of C-CAM and down-regulates C-CAM self-association. J. Biol. Chem. 271, 1393-1399.
Fagarasan, S. (2008). Evolution, development, mechanism and function of IgA in the gut. Curr. Opin. Immunol. 20, 170-177.
Fagarasan, S., Kinoshita, K., Muramatsu, M., Ikuta, K., and Honjo, T. (2001). In situ class switching and differentiation to IgA-producing cells in the gut lamina propria. Nature 413, 639.
Fagarasan, S., Muramatsu, M., Suzuki, K., Nagaoka, H., Hiai, H., and Honjo, T. (2002). Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298, 1424-1427. Fantini, M. C, Dominitzki, S., Rizzo, A., Neurath, M. F., and Becker, C. (2007). In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc 2, 1789-1794. Fazilleau, N., Mark, L., McHeyzer- Williams, L. J., and McHeyzer- Williams, M. G. (2009a). Follicular helper T cells: lineage and location. Immunity 30, 324-335.
Fazilleau, N., McHeyzer- Williams, L. J., Rosen, H., and McHeyzer- Williams, M. G. (2009b). The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding. Nat. Immunol. 10, 375-384.
Gencheva, M., Chen, C. J., Nguyen, T., and Shively, J. E. (2010). Regulation of CEACAM1 transcription in human breast epithelial cells. BMC Mol. Biol. 11, 79.
Gibbons, D. L., and Spencer, J. (2011). Mouse and human intestinal immunity: same ballpark, different players; different rules, same score. Mucosal Immunol. 4, 148-157. Good-Jacobson, K. L., Szumilas, C. G., Chen, L., Sharpe, A. H., Tomayko, M. M., and Shlomchik, M. J. (2010). PD1 regulates germinal center B cell survival and the formation and affinity of long- lived plasma cells. Nat. Immunol. 11, 535-542.
Gray-Owen, S. D., and Blumberg, R. S. (2006). CEACAM1 : contact-dependent control of immunity. Nat. Rev. Immunol. 6, 433-446.
Greenwald, R. J., Freeman, G. J., and Sharpe, A. H. (2005). The B7 family revisited. Annu. Rev. Immunol. 23, 515-548.
Hapfelmeier, S., Lawson, M. A., Slack, E., Kirundi, J. K., Stoel, M., Heikenwalder, M., Cahenzli, J., Velykoredko, Y., Balmer, M. L., Endt, K., et al. (2010). Reversible microbial colonization of germ- free mice reveals the dynamics of IgA immune responses. Science 328, 1705-1709.
Hashizume, T., Togawa, A., Nochi, T., Igarashi, O., Kweon, M. N., Kiyono, H., and Yamamoto, M. (2008). Peyer's patches are required for intestinal immunoglobulin A responses to Salmonella spp. Infect. Immun. 76, 927-934.
Hermann-Kleiter, N., and Baier, G. (2010). NFAT pulls the strings during CD4+ T helper cell effector functions. Blood 115, 2989-2997.
Huber, M., Izzi, L., Grondin, P., Houde, C, Kunath, T., Veillette, A., and Beauchemin, N. (1999). The carboxyl-terminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells. J. Biol. Chem. 274, 335-344.
Hurst, S. D., Cooper, C. J., Sitterding, S. M., Choi, J., Jump, R. L., Levine, A. D., and Barrett, T. A. (1999). The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements. J. Immunol. 163, 5937- 5945.
lijima, H., Neurath, M. F., Nagaishi, T., Glickman, J. N., Nieuwenhuis, E. E., Nakajima, A., Chen, D., Fuss, I. J., Utku, N., Lewicki, D. N., et al. (2004). Specific regulation of T helper cell 1-mediated murine colitis by CEACAM1. J. Exp. Med. 199, 471-482.
Kammerer, R., Hahn, S., Singer, B. B., Luo, J. S., and von Kleist, S. (1998). Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation. Eur. J. Immunol. 28, 3664-3674.
Kaser, A., Lee, A. H., Franke, A., Glickman, J. N., Zeissig, S., Tilg, H., Nieuwenhuis, E. E., Higgins, D. E., Schreiber, S., Glimcher, L. H., et al. (2008). XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134, 743756.
Kirshner, J., Schumann, D., and Shively, J. E. (2003). CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis. J. Biol. Chem. 278, 50338-50345.
Kuespert, K., Pils, S., and Hauck, C. R. (2006). CEACAMs: their role in physiology and
pathophysiology. Curr Opin Cell Biol 18, 565-571. Lee, H. S., Ostrowski, M. A., and Gray-Owen, S. D. (2008). CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation. J. Immunol. 180, 68276835.
Leung, N., Turbide, C, Olson, M., Marcus, V., Jothy, S., and Beauchemin, N. (2006). Deletion of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) gene contributes to colon tumor progression in a murine model of carcinogenesis. Oncogene 25, 5527-5536.
Linterman, M. A., Pierson, W., Lee, S. K., Kallies, A., Kawamoto, S., Rayner, T. F., Srivastava, M.,
Divekar, D. P., Beaton, L., Hogan, J. J., et al. (2011). Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med 17, 975-982.
Lobo, E. O., Zhang, Z., and Shively, J. E. (2009). Pivotal advance: CEACAM1 is a negative coreceptor for the B cell receptor and promotes CD19-mediated adhesion of B cells in a PI3 Independent manner. J. Leukoc. Biol. 86, 205-218.
Macpherson, A. J., Hunziker, L., McCoy, K., and Lamarre, A. (2001). IgA responses in the intestinal mucosa against pathogenic and non-pathogenic microorganisms. Microbes Infect. 3, 1021-1035. Macpherson, A. J., and Slack, E. (2007). The functional interactions of commensal bacteria with intestinal secretory IgA. Curr. Opin. Gastroenterol. 23, 673-678.
Macpherson, A. J., and Uhr, T. (2004). Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303, 1662-1665.
Manohar, M., Baumann, D. O., Bos, N. A., and Cebra, J. J. (2001). Gut colonization of mice with actA-negative mutant of Listeria monocytogenes can stimulate a humoral mucosal immune response. Infect. Immun. 69, 3542-3549.
Massacand, J. C, Kaiser, P., Ernst, B., Tardivel, A., Burki, K., Schneider, P., and Harris, N. L.
(2008). Intestinal bacteria condition dendritic cells to promote IgA production. PLoS One 3, e2588. Morales, V. M., Christ, A., Watt, S. M., Kim, H. S., Johnson, K. W., Utku, N., Texieira, A. M., Mizoguchi, A., Mizoguchi, E., Russell, G. J., et al. (1999). Regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (CD66a). J. Immunol. 163, 1363- 1370.
Nagaishi, T., Pao, L., Lin, S. H., Iijima, H., Kaser, A., Qiao, S. W., Chen, Z., Glickman, J., Najjar, S. M., Nakajima, A., et al. (2006). SHP1 phosphatase-dependent T cell inhibition by CEACAM1 adhesion molecule isoforms. Immunity 25, 769-781.
Nakajima, A., Iijima, H., Neurath, M. F., Nagaishi, T., Nieuwenhuis, E. E., Raychowdhury, R., Glickman, J., Blau, D. M., Russell, S., Holmes, K. V., et al. (2002). Activation-induced expression of carcinoembryonic antigen-cell adhesion molecule 1 regulates mouse T lymphocyte function. J.
Immunol. 168, 1028-1035.
Niemir, Z. I., Stein, H., Noronha, I. L., Kruger, C, Andrassy, K., Ritz, E., and Waldherr, R. (1995). PDGF and TGF-[beta] contribute to the natural course of human IgA glomerulonephritis. Kidney Int 48, 1530-1541. Ochi, H., Abraham, M., Ishikawa, H., Frenkel, D., Yang, K., Basso, A. S., Wu, H., Chen, M. L., Gandhi, R., Miller, A., et al. (2006). Oral CD3-specific antibody suppresses autoimmune
encephalomyelitis by inducing CD4+ CD25- LAP+ T cells. Nat Med 12, 627-635.
Oestreich, K. J., Yoon, H., Ahmed, R., and Boss, J. M. (2008). NFATcl regulates PD1 expression upon T cell activation. J. Immunol. 181, 4832-4839.
Oida, T., Zhang, X., Goto, M., Hachimura, S., Totsuka, M., Kaminogawa, S., and Weiner, H. L. (2003). CD4+CD25- T cells that express latency-associated peptide on the surface suppress
CD4+CD45RB high-induced colitis by a TGF-beta-dependent mechanism. J. Immunol. 170, 2516- 2522.
Pan, H., and Shively, J. E. (2010). Carcinoembryonic antigen-related cell adhesion molecule- 1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor receptor. Immunity 33, 620-631.
Schumann, D., Chen, C. J., Kaplan, B., and Shively, J. E. (2001). Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin. J. Biol. Chem. 276, 47421-47433.
Seder, R. A., and Ahmed, R. (2003). Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4, 835-842.
Shannon, C. E. (1997). The mathematical theory of communication. 1963. MD Comput. 14, 306317. Singer, B. B., Scheffrahn, I., Heymann, R., Sigmundsson, K., Kammerer, R., and Obrink, B. (2002). Carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes. J. Immunol. 168, 5139-5146. Suzuki, K., Meek, B., Doi, Y., Muramatsu, M., Chiba, T., Honjo, T., and Fagarasan, S. (2004).
Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. Proc. Natl. Acad. Sci. U.S.A. 101, 1981-1986.
Taylor, J. M., Ziman, M. E., Fong, J., Solnick, J. V., and Vajdy, M. (2007). Possible correlates of long-term protection against Helicobacter pylori following systemic or combinations of mucosal and systemic immunizations. Infect. Immun. 75, 3462-3469.
van der Waaij, L. A., Limburg, P. C, Mesander, G., and van der Waaij, D. (1996). In vivo IgA coating of anaerobic bacteria in human faeces. Gut 38, 348-354.
Wijburg, O. L., Uren, T. K., Simpfendorfer, K., Johansen, F. E., Brandtzaeg, P., and Strugnell, R. A. (2006). Innate secretory antibodies protect against natural Salmonella typhimurium infection. J. Exp. Med. 203, 21-26.
Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A. D., Heinz, D. W., Lengeling, A., and Schubert, W. D. (2007). Extending the host range of Listeria monocytogenes by rational protein design. Cell 129, 891-902. Iwasaki, A., and Kelsall, B.L. (1999). Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J Exp Med 190, 229- 239.
EXAMPLE 6
[00221] The expression of CEACAM1 and PD1 on cells was examined under physiological conditions. Lamina propria lymphocytes were isolated from colon, ileum, jejunum and duodenum as well as mesenteric lymph nodes (MLN) and spleen and after gating on CD3 -positive cells stained for co-expression of PD1 and CEACAMl (Figure 14).
[00222] Colitis was induced in mice by adoptive transfer of naive CD4+ T cells in Rag- deficient and CEACAM1 -deficient animals and expression of CEACAM1 and PD1 was examined in lamina propria lymphocytes infiltrating the colon. Co-expression of CEACAM1 and PD1 is shown in Figure 15, after gating on CD3+, in lymphocytes obtained from proximal, middle and distal colon.
[00223] Tumor infiltrating lymphocytes (TILs) were isolated from subcutaneous tumors associated with CT26 colorectal carcinoma cell line. TILs were stimulated with anti-CD3 and anti- CD28 monoclonal antibodies in the presence of isotype control antibodies or antibodies that block different co-inhibitory cell surface molecules. This blockade of CEACAM1 and PD1 increases TNF-a production from tumor infiltrating lymphocytes (TIL). The data demonstrates a synergistic increase of TNF-a production by TILs treated with anti-CEACAMl and anti-PDl antibodies.

Claims

What is claimed herein is:
1. A composition for enhancing the immune response or decreasing T cell tolerance in a subject, the composition comprising at least two agents selected from the group consisting of:
a CEACAMl inhibitory agent; a PD1 inhibitory agent; and a LAP inhibitory agent.
2. The composition of claim 1, wherein the two agents are a CEACAMl inhibitory agent and a PD1 inhibitory agent.
3. The composition of claim 1, wherein the two agents are a CEACAMl inhibitory agent and a LAP inhibitory agent.
4. The composition of claim 1, wherein the two agents are a PD1 inhibitory agent and a LAP inhibitory agent.
5. The composition of claim 1, comprising a CEACAMl inhibitory agent, a PD1 inhibitory agent, and a LAP inhibitory agent.
6. The composition of any of claims 1-5, comprising a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a PD1 inhibitory agent.
7. The composition of any of claims 1-5, comprising a bispecific agent comprising a first portion which is a CEACAMl inhibitory agent and a second portion which is a LAP inhibitory agent.
8. The composition of any of claims 1-5, comprising a bispecific agent comprising a first portion which is a LAP inhibitory agent and a second portion which is a PD1 inhibitory agent.
9. The composition of any of claims 1-8, wherein one or more of the inhibitory agents is an inhibitory nucleic acid.
10. The composition of any of claims 1-9, wherein one or more of the inhibitory agents is an antibody reagent.
11. The composition of claim 10, wherein the antibody reagent binds a target selected from the group consisting of:
CEACAMl ; PD1 ; and LAP.
12. The composition of claim 11, wherein the antibody reagent inhibits signaling mediated by the target.
13. The composition of any of claims 1-12, wherein the CEACAMl inhibitory agent binds a CEACAMl molecule having the sequence set forth in SEQ ID NO:32, or an allelic or splice variant of SEQ ID NO: 32.
14. The composition of any of claims 1-13, wherein the CEACAMl inhibitory agent binds a CEACAMl ligand interaction site.
15. The composition of claim 14, wherein the CEACAMl inhibitory agent binds to an amino acid residue corresponding to any of amino acid residues 1-429 of SEQ ID NO: 32.
16. The composition of any of claims 1-15, wherein the CEACAMl inhibitory agent binds an extracellular domain of CEACAMl .
17. The composition of any of claims 1-16, wherein the CEACAM1 inhibitory agent binds the homophilic interaction domain of CEACAM1 and inhibits homophilic interaction of CEACAM1 molecules.
18. The composition of any of claims 1-17, wherein the PDl inhibitory agent binds a PDl
molecule having the sequence set forth in SEQ ID NO: l, or an allelic or splice variant of SEQ ID NO: 1.
19. The composition of claim 1-12 and 18, wherein the PDl inhibitory agent binds a PDl ligand interaction site.
20. The composition of claim 19, wherein the PDl inhibitory agent binds to an amino acid
residue corresponding to any of amino acid residues 41-136 of SEQ ID NO: 1.
21. The composition of any of claims 1-12, wherein the LAP inhibitory agent binds a LAP
molecule having the sequence set forth in SEQ ID NO: 34, or an allelic or splice variant of SEQ ID NO: 34.
22. The composition of claims 1-12 and 21, wherein the LAP inhibitory agent binds a LAP ligand interaction site.
23. The composition of claim 22, wherein the LAP inhibitory agent binds to an amino acid
residue corresponding to amino acid residue 218 of SEQ ID NO: 33.
24. A method for treating a chronic disease, the method comprising administering to a subject in need of treatment thereof an effective amount of a composition of any of claims 1-23.
25. The method of claim 24, wherein the chronic disease is selected from the group consisting of:
cancer; a persistent infection; and a chronic viral infection.
PCT/US2013/064506 2012-10-12 2013-10-11 Enhancement of the immune response WO2014059251A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
CA2887528A CA2887528C (en) 2012-10-12 2013-10-11 Enhancement of the immune response
BR112015008118A BR112015008118A2 (en) 2012-10-12 2013-10-11 immune response booster
EP20150606.0A EP3679950A1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
EP13845573.8A EP2906241B1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
CN201380065223.XA CN104853776B (en) 2012-10-12 2013-10-11 Enhancement of immune response
JP2015536925A JP6356134B2 (en) 2012-10-12 2013-10-11 Enhanced immune response
AU2013329083A AU2013329083C1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
KR1020157012338A KR102229873B1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
US14/433,828 US20150273056A1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
IL23817115A IL238171B (en) 2012-10-12 2015-04-12 Enhancement of the immune response
ZA2015/02603A ZA201502603B (en) 2012-10-12 2015-04-17 Enhancement of the immune response
HK16101811.7A HK1213788A1 (en) 2012-10-12 2016-02-18 Enhancement of the immune response
HK16101826.0A HK1213791A1 (en) 2012-10-12 2016-02-18 Enhancement of the immune response
US16/165,113 US20190201524A1 (en) 2012-10-12 2018-10-19 Enhancement of the immune response

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261713203P 2012-10-12 2012-10-12
US61/713,203 2012-10-12

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/433,828 A-371-Of-International US20150273056A1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response
US16/165,113 Continuation US20190201524A1 (en) 2012-10-12 2018-10-19 Enhancement of the immune response

Publications (1)

Publication Number Publication Date
WO2014059251A1 true WO2014059251A1 (en) 2014-04-17

Family

ID=50477917

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/064506 WO2014059251A1 (en) 2012-10-12 2013-10-11 Enhancement of the immune response

Country Status (12)

Country Link
US (2) US20150273056A1 (en)
EP (2) EP3679950A1 (en)
JP (1) JP6356134B2 (en)
KR (1) KR102229873B1 (en)
CN (1) CN104853776B (en)
AU (1) AU2013329083C1 (en)
BR (1) BR112015008118A2 (en)
CA (2) CA2887528C (en)
HK (2) HK1213788A1 (en)
IL (1) IL238171B (en)
WO (1) WO2014059251A1 (en)
ZA (1) ZA201502603B (en)

Cited By (83)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015075725A1 (en) 2013-11-25 2015-05-28 Ccam Biotherapeutics Ltd. Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
WO2015090230A1 (en) 2013-12-19 2015-06-25 Novartis Ag Human mesothelin chimeric antigen receptors and uses thereof
WO2015142675A2 (en) 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
WO2015157252A1 (en) 2014-04-07 2015-10-15 BROGDON, Jennifer Treatment of cancer using anti-cd19 chimeric antigen receptor
WO2016014553A1 (en) 2014-07-21 2016-01-28 Novartis Ag Sortase synthesized chimeric antigen receptors
WO2016014530A1 (en) 2014-07-21 2016-01-28 Novartis Ag Combinations of low, immune enhancing. doses of mtor inhibitors and cars
WO2016020538A1 (en) * 2014-08-08 2016-02-11 Transgene Sa Hbv vaccine and antibody combination therapy to treat hbv infections
US9261507B2 (en) 2009-04-30 2016-02-16 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anti CEACAM1 antibodies and methods of using same
WO2016025880A1 (en) 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
WO2016040880A1 (en) 2014-09-13 2016-03-17 Novartis Ag Combination therapies of alk inhibitors
WO2016044605A1 (en) 2014-09-17 2016-03-24 Beatty, Gregory Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
WO2016054555A2 (en) 2014-10-03 2016-04-07 Novartis Ag Combination therapies
WO2016057705A1 (en) 2014-10-08 2016-04-14 Novartis Ag Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof
WO2016090034A2 (en) 2014-12-03 2016-06-09 Novartis Ag Methods for b cell preconditioning in car therapy
WO2016088791A1 (en) * 2014-12-02 2016-06-09 国立大学法人 東京大学 Method for assessing therapeutic effect of anti-cancer agent having anti-cd4 antibody as active ingredient
WO2016100882A1 (en) 2014-12-19 2016-06-23 Novartis Ag Combination therapies
US9394365B1 (en) 2014-03-12 2016-07-19 Yeda Research And Development Co., Ltd Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
WO2016115345A1 (en) * 2015-01-14 2016-07-21 The Brigham And Women's Hospital, Treatment of cancer with anti-lap monoclonal antibodies
WO2016126608A1 (en) 2015-02-02 2016-08-11 Novartis Ag Car-expressing cells against multiple tumor antigens and uses thereof
WO2016138350A1 (en) * 2015-02-26 2016-09-01 The Board Of Trustees Of The University Of Arkansas Recall antigen for promoting t-helper type 1 response
WO2016164580A1 (en) 2015-04-07 2016-10-13 Novartis Ag Combination of chimeric antigen receptor therapy and amino pyrimidine derivatives
WO2016168595A1 (en) 2015-04-17 2016-10-20 Barrett David Maxwell Methods for improving the efficacy and expansion of chimeric antigen receptor-expressing cells
WO2016172583A1 (en) 2015-04-23 2016-10-27 Novartis Ag Treatment of cancer using chimeric antigen receptor and protein kinase a blocker
US9512227B2 (en) 2014-03-12 2016-12-06 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
WO2017015427A1 (en) 2015-07-21 2017-01-26 Novartis Ag Methods for improving the efficacy and expansion of immune cells
WO2017019897A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to tim-3
WO2017040930A2 (en) 2015-09-03 2017-03-09 The Trustees Of The University Of Pennsylvania Biomarkers predictive of cytokine release syndrome
WO2017103895A1 (en) 2015-12-18 2017-06-22 Novartis Ag Antibodies targeting cd32b and methods of use thereof
WO2017106630A1 (en) 2015-12-18 2017-06-22 The General Hospital Corporation Polyacetal polymers, conjugates, particles and uses thereof
WO2017112741A1 (en) 2015-12-22 2017-06-29 Novartis Ag Mesothelin chimeric antigen receptor (car) and antibody against pd-l1 inhibitor for combined use in anticancer therapy
WO2017149515A1 (en) 2016-03-04 2017-09-08 Novartis Ag Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore
US9771431B2 (en) 2011-10-11 2017-09-26 Ccam Biotherapeutics Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
US9771425B2 (en) 2014-10-27 2017-09-26 Agency For Science, Technology And Research Anti-PD-1 antibodies
EP3137502A4 (en) * 2014-04-27 2017-11-29 CCAM Biotherapeutics Ltd. Humanized antibodies against ceacam1
WO2018067992A1 (en) 2016-10-07 2018-04-12 Novartis Ag Chimeric antigen receptors for the treatment of cancer
WO2018102786A1 (en) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Methods for modulation of car-t cells
WO2018106738A1 (en) 2016-12-05 2018-06-14 Massachusetts Institute Of Technology Brush-arm star polymers, conjugates and particles, and uses thereof
US10106608B2 (en) 2015-01-28 2018-10-23 Universitaet Duisburg-Essen Immunostimulatory anti-CEACAM1 antibody
WO2018201056A1 (en) 2017-04-28 2018-11-01 Novartis Ag Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor
WO2018201051A1 (en) 2017-04-28 2018-11-01 Novartis Ag Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor
WO2018223101A1 (en) 2017-06-02 2018-12-06 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
WO2018229715A1 (en) 2017-06-16 2018-12-20 Novartis Ag Compositions comprising anti-cd32b antibodies and methods of use thereof
US10160806B2 (en) 2014-06-26 2018-12-25 Macrogenics, Inc. Covalently bonded diabodies having immunoreactivity with PD-1 and LAG-3, and methods of use thereof
WO2019006427A1 (en) 2017-06-29 2019-01-03 Juno Therapeutics, Inc. Mouse model for assessing toxicities associated with immunotherapies
WO2019075090A1 (en) * 2017-10-10 2019-04-18 Tilos Therapeutics, Inc. Anti-lap antibodies and uses thereof
WO2019081983A1 (en) 2017-10-25 2019-05-02 Novartis Ag Antibodies targeting cd32b and methods of use thereof
WO2019089858A2 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Methods of assessing or monitoring a response to a cell therapy
WO2019090003A1 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Chimeric antigen receptors specific for b-cell maturation antigen (bcma)
WO2019089969A2 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for b-cell maturation antigen
WO2019118937A1 (en) 2017-12-15 2019-06-20 Juno Therapeutics, Inc. Anti-cct5 binding molecules and methods of use thereof
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
WO2019152743A1 (en) 2018-01-31 2019-08-08 Celgene Corporation Combination therapy using adoptive cell therapy and checkpoint inhibitor
WO2019210153A1 (en) 2018-04-27 2019-10-31 Novartis Ag Car t cell therapies with enhanced efficacy
WO2019213282A1 (en) 2018-05-01 2019-11-07 Novartis Ag Biomarkers for evaluating car-t cells to predict clinical outcome
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10519237B2 (en) 2014-03-12 2019-12-31 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10577422B2 (en) 2015-07-30 2020-03-03 Macrogenics, Inc. PD-1-binding molecules and methods of use thereof
WO2020069409A1 (en) 2018-09-28 2020-04-02 Novartis Ag Cd19 chimeric antigen receptor (car) and cd22 car combination therapies
WO2020069405A1 (en) 2018-09-28 2020-04-02 Novartis Ag Cd22 chimeric antigen receptor (car) therapies
US10618963B2 (en) 2014-03-12 2020-04-14 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
WO2020092848A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Methods for treatment using chimeric antigen receptors specific for b-cell maturation antigen
WO2020092854A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Chimeric antigen receptors specific for g protein-coupled receptor class c group 5 member d (gprc5d)
WO2020102770A1 (en) 2018-11-16 2020-05-22 Juno Therapeutics, Inc. Methods of dosing engineered t cells for the treatment of b cell malignancies
EP3660042A1 (en) 2014-07-31 2020-06-03 Novartis AG Subset-optimized chimeric antigen receptor-containing t-cells
WO2020113194A2 (en) 2018-11-30 2020-06-04 Juno Therapeutics, Inc. Methods for treatment using adoptive cell therapy
WO2020160050A1 (en) 2019-01-29 2020-08-06 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for receptor tyrosine kinase like orphan receptor 1 (ror1)
EP3712171A1 (en) 2014-08-19 2020-09-23 Novartis AG Treatment of cancer using a cd123 chimeric antigen receptor
EP3722316A1 (en) 2014-07-21 2020-10-14 Novartis AG Treatment of cancer using a cd33 chimeric antigen receptor
US10954301B2 (en) 2015-12-14 2021-03-23 Macrogenics, Inc. Bispecific molecules having immunoreactivity with PD-1 and CTLA-4, and methods of use thereof
US10995141B2 (en) 2019-04-19 2021-05-04 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
WO2021108613A1 (en) 2019-11-26 2021-06-03 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
US11072653B2 (en) 2015-06-08 2021-07-27 Macrogenics, Inc. LAG-3-binding molecules and methods of use thereof
US11130802B2 (en) 2018-10-10 2021-09-28 Tilos Therapeutics, Inc. Anti-lap antibody variants
WO2021207689A2 (en) 2020-04-10 2021-10-14 Juno Therapeutics, Inc. Methods and uses related to cell therapy engineered with a chimeric antigen receptor targeting b-cell maturation antigen
US11365252B2 (en) 2016-07-20 2022-06-21 University Of Utah Research Foundation CD229 CAR T cells and methods of use thereof
EP3880716A4 (en) * 2018-11-13 2022-08-03 Compass Therapeutics LLC Multispecific binding constructs against checkpoint molecules and uses thereof
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1
WO2022254337A1 (en) 2021-06-01 2022-12-08 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
WO2023250400A1 (en) 2022-06-22 2023-12-28 Juno Therapeutics, Inc. Treatment methods for second line therapy of cd19-targeted car t cells
WO2024031091A2 (en) 2022-08-05 2024-02-08 Juno Therapeutics, Inc. Chimeric antigen receptors specific for gprc5d and bcma
EP4324518A2 (en) 2014-01-31 2024-02-21 Novartis AG Antibody molecules to tim-3 and uses thereof
WO2024129778A2 (en) 2022-12-13 2024-06-20 Juno Therapeutics, Inc. Chimeric antigen receptors specific for baff-r and cd19 and methods and uses thereof

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PE20181322A1 (en) 2015-09-01 2018-08-14 Agenus Inc ANTI-PD1 ANTIBODY AND ITS METHODS OF USE
BR112019011582A2 (en) 2016-12-07 2019-10-22 Agenus Inc. antibodies and their methods of use
CN107099603A (en) * 2017-05-31 2017-08-29 成都克里斯博生物科技有限公司 Tumour immunity T cell detection kit and detection method
US20200240986A1 (en) * 2017-08-07 2020-07-30 Kyushu University, National University Corporation Method for measuring occupancy rate of specific binding substances in cell population
KR102166982B1 (en) * 2019-01-24 2020-10-16 가톨릭대학교 산학협력단 Correlation of CEACAM1 and EpCAM in liver cancer and Methods for providing information for liver cancer therapeutic effect using using the same
WO2023172905A2 (en) * 2022-03-08 2023-09-14 Arizona Board Of Regents On Behalf Of The University Of Arizona Calcimycin as antifungals

Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3536809A (en) 1969-02-17 1970-10-27 Alza Corp Medication method
US3598123A (en) 1969-04-01 1971-08-10 Alza Corp Bandage for administering drugs
US3845770A (en) 1972-06-05 1974-11-05 Alza Corp Osmatic dispensing device for releasing beneficial agent
US3916899A (en) 1973-04-25 1975-11-04 Alza Corp Osmotic dispensing device with maximum and minimum sizes for the passageway
US4008719A (en) 1976-02-02 1977-02-22 Alza Corporation Osmotic system having laminar arrangement for programming delivery of active agent
US4723958A (en) 1986-05-23 1988-02-09 Merck & Co., Inc. Pulsatile drug delivery system
US4747825A (en) 1984-06-29 1988-05-31 Ferring Laboratories, Inc. Apparatus and methodology for pulsed administration of growth promoting agents
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1990002809A1 (en) 1988-09-02 1990-03-22 Protein Engineering Corporation Generation and selection of recombinant varied binding proteins
US4948592A (en) 1986-05-09 1990-08-14 Alza Corporation Pulsed drug delivery
US4965251A (en) 1987-04-03 1990-10-23 The Board Of Regents Of The University Of Washington Pulse treatment of hemoglobinopathies with erythropoietin
WO1991010737A1 (en) 1990-01-11 1991-07-25 Molecular Affinities Corporation Production of antibodies using gene libraries
US5059595A (en) 1989-03-22 1991-10-22 Bioresearch, S.P.A. Pharmaceutical compositions containing 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid and their pharmaceutically acceptable salts in controlled-release form active in the therapy of organic mental disturbances
US5073543A (en) 1988-07-21 1991-12-17 G. D. Searle & Co. Controlled release formulations of trophic factors in ganglioside-lipsome vehicle
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5120548A (en) 1989-11-07 1992-06-09 Merck & Co., Inc. Swelling modulated polymeric drug delivery device
WO1992018619A1 (en) 1991-04-10 1992-10-29 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
WO1992022324A1 (en) 1991-06-14 1992-12-23 Xoma Corporation Microbially-produced antibody fragments and their conjugates
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5354556A (en) 1984-10-30 1994-10-11 Elan Corporation, Plc Controlled release powder and process for its preparation
US5403590A (en) 1992-12-21 1995-04-04 New England Deaconess Hospital Corporation Method of pulsatile drug infusion
WO1995015982A2 (en) 1993-12-08 1995-06-15 Genzyme Corporation Process for generating specific antibodies
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
WO1995020401A1 (en) 1994-01-31 1995-08-03 Trustees Of Boston University Polyclonal antibody libraries
US5516637A (en) 1994-06-10 1996-05-14 Dade International Inc. Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage
US5591767A (en) 1993-01-25 1997-01-07 Pharmetrix Corporation Liquid reservoir transdermal patch for the administration of ketorolac
US5639476A (en) 1992-01-27 1997-06-17 Euro-Celtique, S.A. Controlled release formulations coated with aqueous dispersions of acrylic polymers
US5674533A (en) 1994-07-07 1997-10-07 Recordati, S.A., Chemical And Pharmaceutical Company Pharmaceutical composition for the controlled release of moguisteine in a liquid suspension
US5698426A (en) 1990-09-28 1997-12-16 Ixsys, Incorporated Surface expression libraries of heteromeric receptors
US5733566A (en) 1990-05-15 1998-03-31 Alkermes Controlled Therapeutics Inc. Ii Controlled release of antiparasitic agents in animals
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5750753A (en) 1996-01-24 1998-05-12 Chisso Corporation Method for manufacturing acryloxypropysilane
US5780225A (en) 1990-01-12 1998-07-14 Stratagene Method for generating libaries of antibody genes comprising amplification of diverse antibody DNAs and methods for using these libraries for the production of diverse antigen combining molecules
US5801231A (en) 1985-03-22 1998-09-01 Genentech, Inc. Nucleic acid encoding TGF-β and its uses
US5821047A (en) 1990-12-03 1998-10-13 Genentech, Inc. Monovalent phage display
WO1999052552A1 (en) 1998-04-15 1999-10-21 Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US6365185B1 (en) 1998-03-26 2002-04-02 University Of Cincinnati Self-destructing, controlled release peroral drug delivery system
US20040047858A1 (en) 2002-09-11 2004-03-11 Blumberg Richard S. Therapeutic anti-BGP(C-CAM1) antibodies and uses thereof
WO2008060617A2 (en) 2006-11-15 2008-05-22 The Brigham And Women's Hospital, Inc. Therapeutic uses of tim-3 modulators
US20080206219A1 (en) 2003-08-08 2008-08-28 The Regencts Of The University Of California Novel Indications for Transforming Growth Factor-Beta Regulators
US8198412B2 (en) 2003-09-04 2012-06-12 Riken Antibodies that recognize cutting edge within the TGF-β activation controlling region

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3270960A (en) 1964-09-11 1966-09-06 Sperry Rand Corp Fluid sensor
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US4881175A (en) 1986-09-02 1989-11-14 Genex Corporation Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
EP2262531A1 (en) * 2008-03-08 2010-12-22 Immungene, Inc. Engineered fusion molecules immunotherapy in cancer and inflammatory diseases

Patent Citations (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3536809A (en) 1969-02-17 1970-10-27 Alza Corp Medication method
US3598123A (en) 1969-04-01 1971-08-10 Alza Corp Bandage for administering drugs
US3845770A (en) 1972-06-05 1974-11-05 Alza Corp Osmatic dispensing device for releasing beneficial agent
US3916899A (en) 1973-04-25 1975-11-04 Alza Corp Osmotic dispensing device with maximum and minimum sizes for the passageway
US4008719A (en) 1976-02-02 1977-02-22 Alza Corporation Osmotic system having laminar arrangement for programming delivery of active agent
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4747825A (en) 1984-06-29 1988-05-31 Ferring Laboratories, Inc. Apparatus and methodology for pulsed administration of growth promoting agents
US5354556A (en) 1984-10-30 1994-10-11 Elan Corporation, Plc Controlled release powder and process for its preparation
US5801231A (en) 1985-03-22 1998-09-01 Genentech, Inc. Nucleic acid encoding TGF-β and its uses
US4948592A (en) 1986-05-09 1990-08-14 Alza Corporation Pulsed drug delivery
US4723958A (en) 1986-05-23 1988-02-09 Merck & Co., Inc. Pulsatile drug delivery system
US4965251A (en) 1987-04-03 1990-10-23 The Board Of Regents Of The University Of Washington Pulse treatment of hemoglobinopathies with erythropoietin
US5073543A (en) 1988-07-21 1991-12-17 G. D. Searle & Co. Controlled release formulations of trophic factors in ganglioside-lipsome vehicle
US5571698A (en) 1988-09-02 1996-11-05 Protein Engineering Corporation Directed evolution of novel binding proteins
US5403484A (en) 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1990002809A1 (en) 1988-09-02 1990-03-22 Protein Engineering Corporation Generation and selection of recombinant varied binding proteins
US5059595A (en) 1989-03-22 1991-10-22 Bioresearch, S.P.A. Pharmaceutical compositions containing 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid and their pharmaceutically acceptable salts in controlled-release form active in the therapy of organic mental disturbances
US5120548A (en) 1989-11-07 1992-06-09 Merck & Co., Inc. Swelling modulated polymeric drug delivery device
WO1991010737A1 (en) 1990-01-11 1991-07-25 Molecular Affinities Corporation Production of antibodies using gene libraries
US5780225A (en) 1990-01-12 1998-07-14 Stratagene Method for generating libaries of antibody genes comprising amplification of diverse antibody DNAs and methods for using these libraries for the production of diverse antigen combining molecules
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US5580717A (en) 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
US5733566A (en) 1990-05-15 1998-03-31 Alkermes Controlled Therapeutics Inc. Ii Controlled release of antiparasitic agents in animals
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5969108A (en) 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
US5698426A (en) 1990-09-28 1997-12-16 Ixsys, Incorporated Surface expression libraries of heteromeric receptors
US5821047A (en) 1990-12-03 1998-10-13 Genentech, Inc. Monovalent phage display
US5658727A (en) 1991-04-10 1997-08-19 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
WO1992018619A1 (en) 1991-04-10 1992-10-29 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
WO1992022324A1 (en) 1991-06-14 1992-12-23 Xoma Corporation Microbially-produced antibody fragments and their conjugates
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5639476A (en) 1992-01-27 1997-06-17 Euro-Celtique, S.A. Controlled release formulations coated with aqueous dispersions of acrylic polymers
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5403590A (en) 1992-12-21 1995-04-04 New England Deaconess Hospital Corporation Method of pulsatile drug infusion
US5591767A (en) 1993-01-25 1997-01-07 Pharmetrix Corporation Liquid reservoir transdermal patch for the administration of ketorolac
WO1995015982A2 (en) 1993-12-08 1995-06-15 Genzyme Corporation Process for generating specific antibodies
WO1995020401A1 (en) 1994-01-31 1995-08-03 Trustees Of Boston University Polyclonal antibody libraries
US5516637A (en) 1994-06-10 1996-05-14 Dade International Inc. Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage
US5674533A (en) 1994-07-07 1997-10-07 Recordati, S.A., Chemical And Pharmaceutical Company Pharmaceutical composition for the controlled release of moguisteine in a liquid suspension
US5750753A (en) 1996-01-24 1998-05-12 Chisso Corporation Method for manufacturing acryloxypropysilane
US6365185B1 (en) 1998-03-26 2002-04-02 University Of Cincinnati Self-destructing, controlled release peroral drug delivery system
WO1999052552A1 (en) 1998-04-15 1999-10-21 Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US6852320B2 (en) 1998-04-15 2005-02-08 The Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US7132255B2 (en) 1998-04-15 2006-11-07 The Brigham And Women's Hospital, Inc. Identification of compounds that bind biliary glycoprotein and affect cytotoxic T lymphocyte activity
US20040047858A1 (en) 2002-09-11 2004-03-11 Blumberg Richard S. Therapeutic anti-BGP(C-CAM1) antibodies and uses thereof
US20080206219A1 (en) 2003-08-08 2008-08-28 The Regencts Of The University Of California Novel Indications for Transforming Growth Factor-Beta Regulators
US8198412B2 (en) 2003-09-04 2012-06-12 Riken Antibodies that recognize cutting edge within the TGF-β activation controlling region
WO2008060617A2 (en) 2006-11-15 2008-05-22 The Brigham And Women's Hospital, Inc. Therapeutic uses of tim-3 modulators

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
ADJEI, A; GARREN, J., PHARM. RES., vol. 1, 1990, pages 565 - 569
ALI ET AL., PLOS ONE, 2008, pages EL914
ALL, D. A., DRUG DELIVERY, vol. 2, 1995, pages 10 1 - 20
AMES ET AL., J. IMMUNOL. METHODS, vol. 184, 1995, pages 177 - 186
ANDERSON ET AL., AM. REV. RESPIR. DIS., vol. 140, 1989, pages 1317 - 1324
BARNETT ET AL., J CELL BIOL, vol. 108, 1989, pages 267 - 276
BARNETT ET AL., MOL CELL BIOL, vol. 13, 1993, pages 1273 - 1282
BEAUCHEMIN ET AL., EXP CELL RES, 1999, pages 252 - 243
BENGSCH B ET AL., PLOS PATHOG., vol. 6, no. 6, 2010
BENSON DM ET AL., BLOOD, 11 May 2010 (2010-05-11)
BETTER ET AL., SCIENCE, vol. 240, 1988, pages 1041 - 1043
BIOTONI ET AL., PNAS, vol. 101, 2004, pages 9763 - 8
BOETTLER T ET AL., J. VIROL., vol. 80, 2006, pages 3532 - 40
BONI C ET AL., J. VIROL., vol. 81, 2007, pages 4215 - 25
BRINKMAN ET AL., J. IMMUNOL. METHODS, vol. 182, 1995, pages 41 - 50
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
BRUNNER ET AL., J. BIOL. CHEM, vol. 264, 1989, pages 13660 - 13664
BRUNNER ET AL., MOL. ENDOCRINOL., vol. 6, 1992, pages 1691 - 1700
BRYON, P., ADV. DRUG. DEL. REV., vol. 5, 1990, pages 107 - 132
BURTON ET AL., ADVANCES IN IMMUNOLOGY, 1994, pages 191 - 280
CHEN ET AL., J. IMMUNOL., vol. 172, 2004, pages 3535
CHEN ET AL., J. IMMUNOL., vol. 180, 2008, pages 6085
CHEN ET AL., J. LEUK. BIOL, vol. 86, 2009, pages 195
CHEN ET AL., JOURNAL OF IMMUNOLOGY, 2008, pages 6085 - 6093
CHEN MEI-LING ET AL.: "Latency-Associated Peptide Identifies a Novel CD4+CD25+Regulatory T Cell Subset with TGF-beta Mediated Function and Enhanced Suppression of Experimental Autoimmune Encephalomyelitis.", J IMMUNOL, vol. 180, no. 11, 2008, pages 7327 - 7337, XP055246475 *
CHOTHIA, C. ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
CURRAN MICHAEL A. ET AL.: "PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B 16 melanoma tumors.", PNAS, vol. 107, no. 9, 2010, pages 4275 - 4280, XP055067204 *
DAMMS, B; BAINS, W., NATURE BIOTECHNOLOGY, 1996
DAS S ET AL., J. IMMUNOL., vol. 176, 2006, pages 3000 - 9
DAY CL ET AL., NATURE, vol. 443, 2006, pages 350 - 54
DE ROBERTIS ET AL., J CARCINOG, vol. 10, 2011, pages 9
DE WILDT ET AL., EUR J. IMMUNOL., vol. 26, no. 3, 1996, pages 629 - 39
DERYNCKR, TIBS, vol. 19, 1994, pages 548 - 553
EIR ME ET AL., ANNU REV IMMUNOL., vol. 26, 2008, pages 677 - 704
FEDAROVICH ET AL., D62 ACTA CRYST., vol. 971, 2006
FLEISCH ET AL., ENDOCRINE-RELATED CANCER, vol. 13, 2006, pages 379 - 400
FRENCH, D. L.; EDWARDS, D. A.; NIVEN, R. W., AEROSOL SCI., vol. 27, 1996, pages 769 - 783
FUJITA ET AL., AM. J. PATHOL., vol. 175, 2009, pages 1116
GALLAGHER, J. VIROL., vol. 71, 1997, pages 3129
GAUR ET AL., MOLECULAR CANCER, vol. 7, 2008, pages 46
GODING: "Monoclonal Antibodies: Principles and Practice", 1986, ACADEMIC PRESS, pages: 59 - 103
GONDA, I.: "Aerosols for delivery of therapeutic an diagnostic agents to the respiratory tract", CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, vol. 6, 1990, pages 273 - 313, XP009099233
GRAY JULIET C. ET AL.: "Optimising anti-tumour CD8 T-cell responses using combinations of immunomodulatory antibodies.", EUR. J. IMMUNOL, vol. 38, 2008, ,, pages 2499 - 2511, XP055151985 *
HAMMER-LING ET AL.: "Monoclonal Antibodies and T-Cell Hybrido-mas 563-681", 1981, ELSEVIER
HARLOW ET AL.: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
IIJIMA ET AL., J. EXP. MED., vol. 199, 2004, pages 471
JENS T. CARSTENSEN: "Drug Stability: Principles & Practice", 1995, MARCEL DEKKER, pages: 379 - 80
JOURNAL OF CLINICAL ONCOLOGY, vol. 26, no. 15S, 2008
KABAT, E. A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION NO. 91-3242
KEIR ET AL., ANNUAL REVIEW OF IMMUNOLOGY, vol. 26, 2008, pages 677 - 704
KEIR ME ET AL., ANNU REV IMMUNOL., vol. 26, 2008, pages 677 - 704
KETTLEBOROUGH ET AL., EUR. J. IMMUNOL., vol. 24, 1994, pages 952 - 958
KIM, CHERNG-JU: "Controlled Release Dosage Form Design", 2000, TECHNOMIC PUBLISHING
KLAILE ET AL., J. CELL BIOL., vol. 187, 2009, pages 553
KOBAYASHI, S. ET AL., PHARM. RES., vol. 13, no. 1, 1996, pages 80 - 83
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
MACIAN ET AL., CELL, vol. 109, 2002, pages 719 - 731
MARKEL ET AL., J. CLIN. INVEST., vol. 110, 2002, pages 943
MARKEL, EJI, vol. 34, 2004, pages 2138 - 2148
MARKS ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554
MCGOWAN ET AL., THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 88, 2003, pages 3321 - 6
MELERO IGNACIO ET AL.: "Immunostimulatory monoclonal antibodies for cancer therapy.", NATURE REVIEWS I CANCER, vol. 7, 2007, pages 95 - 106, XP002716002 *
MOLLER ET AL., INT. J. CANCER, vol. 65, 1996, pages 740
MORALES ET AL., J. IMMUNOL., vol. 163, 1999, pages 1363
MULLER ET AL., J. CELL BIOL., vol. 187, 2009, pages 569
MULLINAX ET AL., BIOTECHNIQUES, vol. 12, no. 6, 1992, pages 864 - 869
MUNGER ET AL., MOL BIOL CELL, vol. 9, 1998, pages 2627 - 2638
NAKAJIMA ET AL., J. IMMUNOL., vol. 168, 2002, pages 1028
NIELSEN ET AL., CELL IMMUNOL, vol. 235, 2005, pages 109 - 116
NIVEN, R. W. ET AL., PHARM. RES., vol. 12, no. 9, 1995, pages 1343 - 1349
PATTON, J. S. ET AL., CONTROLLED RELEASE, vol. 15, 1994, pages 79 - 85
PATTON, J.; PLATZ, R., ADV. DRUG DEL. REV., vol. 8, 1992, pages 179 - 196
PATTON; PLATZ, ADVANCED DRUG DELIVERY REVIEWS, vol. 8, 1992, pages 179 - 196
PERSIC ET AL., GENE, vol. 187, 1997, pages 9 - 18
PETROVAS C ET AL., J. EXP. MED., vol. 203, 2006, pages 2281 - 92
RUDT, S; R. H. MULLER, J. CONTROLLED RELEASE, vol. 22, 1992, pages 263 - 272
SAHARINEN ET AL., THE EMBO JOURNAL, vol. 15, 1996, pages 245 - 253
SANDERSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 2572 - 2576
SAWAI ET AL., AJRI, vol. 34, 1995, pages 26 - 34
SINGER ET AL., J. IMMUNOL., vol. 168, 2002, pages 5139
SPRINGER ET AL., ANN. REV. CELL BIOL., vol. 6, 1990, pages 359
TABATA, Y; Y. IKADA, BIOMED. MATER. RES., vol. 22, 1988, pages 837 - 858
TAN ET AL., EMBO J., vol. 21, 2002, pages 2076
TANSEY, I. P., SPRAY TECHNOL. MARKET, vol. 4, 1994, pages 26 - 29
TERRAZAS LI ET AL., INT. J. PARASITOL, vol. 35, 2005, pages 1349 - 58
TIMSINA, INT. J. PHARM., vol. 101, 1995, pages 1 - 13
TRAUTMANN L ET AL., NAT. MED, vol. 12, 2006, pages 1198 - 202
TRAUTMANN L ET AL., NAT. MED., vol. 12, 2006, pages 1198 - 202
URBANI S ET AL., J. VIROL., vol. 80, 2006, pages 11398 - 403
VELU V ET AL., J. VIROL., vol. 81, 2007, pages 5819 - 28
VISSER, J., POWDER TECHNOLOGY, vol. 58, 1989, pages 1 - 10
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WATERHOUSE ET AL., NUC. ACIDS. RES., vol. 21, 1993, pages 2265 - 2266
WATT ET AL., BLOOD, vol. 98, 2001, pages 1469
WATT ET AL., IMMUNOBIOLOGY, vol. 98, 2001, pages 1469 - 1479
YAMAMOTO ET AL., BLOOD, vol. 111, 2008, pages 3220 - 4
YAO ZQ ET AL., VIRAL IMMUNOL., vol. 20, 2007, pages 276 - 87
YU ET AL., J. BIOL. CHEM., vol. 281, 2006, pages 39179
ZANEN, P.; LAMM, J.-W., J. INT. J. PHARM., vol. 114, 1995, pages 111 - 115

Cited By (151)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9261507B2 (en) 2009-04-30 2016-02-16 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anti CEACAM1 antibodies and methods of using same
US11891453B2 (en) 2011-10-11 2024-02-06 Famewave Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
US9771431B2 (en) 2011-10-11 2017-09-26 Ccam Biotherapeutics Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
US10081679B2 (en) 2013-11-25 2018-09-25 Ccam Biotherapeutics Ltd. Compositions comprising anti-CEACAM1 and anti-PD antibodies for cancer therapy
KR102362803B1 (en) * 2013-11-25 2022-02-14 페임웨이브 리미티드 Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
EP3074035A4 (en) * 2013-11-25 2017-06-21 CCAM Biotherapeutics Ltd. Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
KR20160108310A (en) * 2013-11-25 2016-09-19 씨씨에이엠 바이오세라퓨틱스 리미티드. Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
AU2014351308B2 (en) * 2013-11-25 2020-03-05 Ccam Biotherapeutics Ltd. Compositions comprising anti-CEACAM1 and anti-PD antibodies for cancer therapy
WO2015075725A1 (en) 2013-11-25 2015-05-28 Ccam Biotherapeutics Ltd. Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
EP3763387A1 (en) 2013-11-25 2021-01-13 FameWave Ltd Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US11365255B2 (en) 2013-12-12 2022-06-21 Suzhou Suncadia Biopharmaceuticals Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
EP4026909A1 (en) 2013-12-19 2022-07-13 Novartis AG Human mesothelin chimeric antigen receptors and uses thereof
WO2015090230A1 (en) 2013-12-19 2015-06-25 Novartis Ag Human mesothelin chimeric antigen receptors and uses thereof
EP4324518A2 (en) 2014-01-31 2024-02-21 Novartis AG Antibody molecules to tim-3 and uses thereof
US11884728B2 (en) 2014-03-12 2024-01-30 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of amyotrophic lateral sclerosis
US9512225B2 (en) 2014-03-12 2016-12-06 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9394365B1 (en) 2014-03-12 2016-07-19 Yeda Research And Development Co., Ltd Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US10214585B2 (en) 2014-03-12 2019-02-26 Yeda Research And Development Co., Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US9982051B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10618963B2 (en) 2014-03-12 2020-04-14 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10519237B2 (en) 2014-03-12 2019-12-31 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US11884727B2 (en) 2014-03-12 2024-01-30 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of amyotrophic lateral sclerosis
US10961309B2 (en) 2014-03-12 2021-03-30 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US9982048B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9512227B2 (en) 2014-03-12 2016-12-06 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9982050B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US9534052B2 (en) 2014-03-12 2017-01-03 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9982049B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US11643464B2 (en) 2014-03-12 2023-05-09 Yeda Research and Develpment & Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of a retinal degeneration disorder
US11492405B2 (en) 2014-03-12 2022-11-08 Yeda Research And Development Co. Ltd Reducing systemic regulatory t cell levels or activity for treatment of disease and injury of the CNS
US9856318B2 (en) 2014-03-12 2018-01-02 Yeda Research And Development Co., Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US9982047B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US10144778B2 (en) 2014-03-12 2018-12-04 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10981989B2 (en) 2014-03-12 2021-04-20 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
WO2015142675A2 (en) 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
EP4406610A2 (en) 2014-04-07 2024-07-31 Novartis AG Treatment of cancer using anti-cd19 chimeric antigen receptor
WO2015157252A1 (en) 2014-04-07 2015-10-15 BROGDON, Jennifer Treatment of cancer using anti-cd19 chimeric antigen receptor
EP3888674A1 (en) 2014-04-07 2021-10-06 Novartis AG Treatment of cancer using anti-cd19 chimeric antigen receptor
EP3137502A4 (en) * 2014-04-27 2017-11-29 CCAM Biotherapeutics Ltd. Humanized antibodies against ceacam1
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1
EP3766902A1 (en) * 2014-04-27 2021-01-20 FameWave Ltd. Humanized antibodies against ceacam1
US11866509B2 (en) 2014-04-27 2024-01-09 Famewave Ltd. Humanized antibodies against CEACAM1
US10550196B2 (en) 2014-04-27 2020-02-04 Famewave Ltd. Humanized antibodies against CEACAM1
US10160806B2 (en) 2014-06-26 2018-12-25 Macrogenics, Inc. Covalently bonded diabodies having immunoreactivity with PD-1 and LAG-3, and methods of use thereof
US11098119B2 (en) 2014-06-26 2021-08-24 Macrogenics, Inc. Covalently bonded diabodies having immunoreactivity with PD-1 and LAG-3, and methods of use thereof
WO2016014553A1 (en) 2014-07-21 2016-01-28 Novartis Ag Sortase synthesized chimeric antigen receptors
WO2016014530A1 (en) 2014-07-21 2016-01-28 Novartis Ag Combinations of low, immune enhancing. doses of mtor inhibitors and cars
EP3722316A1 (en) 2014-07-21 2020-10-14 Novartis AG Treatment of cancer using a cd33 chimeric antigen receptor
EP3660042A1 (en) 2014-07-31 2020-06-03 Novartis AG Subset-optimized chimeric antigen receptor-containing t-cells
EP4205749A1 (en) 2014-07-31 2023-07-05 Novartis AG Subset-optimized chimeric antigen receptor-containing cells
WO2016020538A1 (en) * 2014-08-08 2016-02-11 Transgene Sa Hbv vaccine and antibody combination therapy to treat hbv infections
WO2016025880A1 (en) 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
EP3712171A1 (en) 2014-08-19 2020-09-23 Novartis AG Treatment of cancer using a cd123 chimeric antigen receptor
EP3659621A1 (en) 2014-09-13 2020-06-03 Novartis AG Combination therapies for cancer
WO2016040892A1 (en) 2014-09-13 2016-03-17 Novartis Ag Combination therapies
EP3925622A1 (en) 2014-09-13 2021-12-22 Novartis AG Combination therapies
WO2016040880A1 (en) 2014-09-13 2016-03-17 Novartis Ag Combination therapies of alk inhibitors
WO2016044605A1 (en) 2014-09-17 2016-03-24 Beatty, Gregory Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
EP3967709A1 (en) 2014-09-17 2022-03-16 Novartis AG Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
EP3662903A2 (en) 2014-10-03 2020-06-10 Novartis AG Combination therapies
WO2016054555A2 (en) 2014-10-03 2016-04-07 Novartis Ag Combination therapies
WO2016057705A1 (en) 2014-10-08 2016-04-14 Novartis Ag Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof
US9771425B2 (en) 2014-10-27 2017-09-26 Agency For Science, Technology And Research Anti-PD-1 antibodies
US11072659B2 (en) 2014-10-27 2021-07-27 Agency For Science, Technology And Research Anti-PD-1 antibodies
US10280224B2 (en) 2014-10-27 2019-05-07 Agency For Science, Technology And Research Anti-PD-1 antibodies
WO2016088791A1 (en) * 2014-12-02 2016-06-09 国立大学法人 東京大学 Method for assessing therapeutic effect of anti-cancer agent having anti-cd4 antibody as active ingredient
JPWO2016088791A1 (en) * 2014-12-02 2017-11-02 国立大学法人 東京大学 Method for determining therapeutic effect of anticancer agent comprising anti-CD4 antibody as active ingredient
WO2016090034A2 (en) 2014-12-03 2016-06-09 Novartis Ag Methods for b cell preconditioning in car therapy
WO2016100882A1 (en) 2014-12-19 2016-06-23 Novartis Ag Combination therapies
AU2016206682B2 (en) * 2015-01-14 2021-11-11 The Brigham And Women's Hospital, Inc. Treatment of cancer with anti-LAP monoclonal antibodies
JP2018508483A (en) * 2015-01-14 2018-03-29 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド Treatment of cancer with anti-LAP monoclonal antibodies
WO2016115345A1 (en) * 2015-01-14 2016-07-21 The Brigham And Women's Hospital, Treatment of cancer with anti-lap monoclonal antibodies
GB2557389B (en) * 2015-01-14 2020-12-23 Brigham & Womens Hospital Inc Treatment of cancer with anti-lap monoclonal antibodies
JP2021020904A (en) * 2015-01-14 2021-02-18 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド Treatment of cancer with anti-lap monoclonal antibodies
IL253462B (en) * 2015-01-14 2022-11-01 The Brigham And Women S Hospital Inc Treatment of cancer with anti-lap monoclonal antibodies
GB2557389A (en) * 2015-01-14 2018-06-20 Brigham & Womens Hospital Inc Treatment of cancer with anti-lap monoclonal antibodies
US10287347B2 (en) 2015-01-14 2019-05-14 The Brigham And Women's Hospital, Inc. Treatment of cancer with anti-lap monoclonal antibodies
JP7152156B2 (en) 2015-01-14 2022-10-12 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド Treatment of cancer with anti-LAP monoclonal antibodies
EP3244926A4 (en) * 2015-01-14 2018-06-27 The Brigham and Women's Hospital, Inc. Treatment of cancer with anti-lap monoclonal antibodies
US11407823B2 (en) 2015-01-14 2022-08-09 The Brigham And Women's Hospital, Inc. Treatment of cancer with anti-LAP monoclonal antibodies
US10017567B2 (en) 2015-01-14 2018-07-10 The Brigham And Women's Hospital, Inc. Treatment of cancer with anti-LAP monoclonal antibodies
IL253462B2 (en) * 2015-01-14 2023-03-01 The Brigham And Women S Hospital Inc Treatment of cancer with anti-lap monoclonal antibodies
US10106608B2 (en) 2015-01-28 2018-10-23 Universitaet Duisburg-Essen Immunostimulatory anti-CEACAM1 antibody
WO2016126608A1 (en) 2015-02-02 2016-08-11 Novartis Ag Car-expressing cells against multiple tumor antigens and uses thereof
WO2016138350A1 (en) * 2015-02-26 2016-09-01 The Board Of Trustees Of The University Of Arkansas Recall antigen for promoting t-helper type 1 response
WO2016164580A1 (en) 2015-04-07 2016-10-13 Novartis Ag Combination of chimeric antigen receptor therapy and amino pyrimidine derivatives
EP4234685A2 (en) 2015-04-17 2023-08-30 Novartis AG Methods for improving the efficacy and expansion of chimeric antigen receptor-expressing cells
WO2016168595A1 (en) 2015-04-17 2016-10-20 Barrett David Maxwell Methods for improving the efficacy and expansion of chimeric antigen receptor-expressing cells
WO2016172583A1 (en) 2015-04-23 2016-10-27 Novartis Ag Treatment of cancer using chimeric antigen receptor and protein kinase a blocker
US11072653B2 (en) 2015-06-08 2021-07-27 Macrogenics, Inc. LAG-3-binding molecules and methods of use thereof
US11858991B2 (en) 2015-06-08 2024-01-02 Macrogenics, Inc. LAG-3-binding molecules and methods of use thereof
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
WO2017015427A1 (en) 2015-07-21 2017-01-26 Novartis Ag Methods for improving the efficacy and expansion of immune cells
EP3878465A1 (en) 2015-07-29 2021-09-15 Novartis AG Combination therapies comprising antibody molecules to tim-3
WO2017019897A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to tim-3
US11623959B2 (en) 2015-07-30 2023-04-11 Macrogenics, Inc. PD-1-binding molecules and methods of use thereof
US10577422B2 (en) 2015-07-30 2020-03-03 Macrogenics, Inc. PD-1-binding molecules and methods of use thereof
WO2017040930A2 (en) 2015-09-03 2017-03-09 The Trustees Of The University Of Pennsylvania Biomarkers predictive of cytokine release syndrome
US10954301B2 (en) 2015-12-14 2021-03-23 Macrogenics, Inc. Bispecific molecules having immunoreactivity with PD-1 and CTLA-4, and methods of use thereof
US11840571B2 (en) 2015-12-14 2023-12-12 Macrogenics, Inc. Methods of using bispecific molecules having immunoreactivity with PD-1 and CTLA-4
WO2017106630A1 (en) 2015-12-18 2017-06-22 The General Hospital Corporation Polyacetal polymers, conjugates, particles and uses thereof
WO2017103895A1 (en) 2015-12-18 2017-06-22 Novartis Ag Antibodies targeting cd32b and methods of use thereof
WO2017112741A1 (en) 2015-12-22 2017-06-29 Novartis Ag Mesothelin chimeric antigen receptor (car) and antibody against pd-l1 inhibitor for combined use in anticancer therapy
WO2017149515A1 (en) 2016-03-04 2017-09-08 Novartis Ag Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore
US11365252B2 (en) 2016-07-20 2022-06-21 University Of Utah Research Foundation CD229 CAR T cells and methods of use thereof
WO2018067992A1 (en) 2016-10-07 2018-04-12 Novartis Ag Chimeric antigen receptors for the treatment of cancer
WO2018102786A1 (en) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Methods for modulation of car-t cells
WO2018106738A1 (en) 2016-12-05 2018-06-14 Massachusetts Institute Of Technology Brush-arm star polymers, conjugates and particles, and uses thereof
WO2018201056A1 (en) 2017-04-28 2018-11-01 Novartis Ag Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor
WO2018201051A1 (en) 2017-04-28 2018-11-01 Novartis Ag Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor
US11944647B2 (en) 2017-06-02 2024-04-02 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
US11413310B2 (en) 2017-06-02 2022-08-16 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
WO2018223101A1 (en) 2017-06-02 2018-12-06 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
WO2018229715A1 (en) 2017-06-16 2018-12-20 Novartis Ag Compositions comprising anti-cd32b antibodies and methods of use thereof
WO2019006427A1 (en) 2017-06-29 2019-01-03 Juno Therapeutics, Inc. Mouse model for assessing toxicities associated with immunotherapies
WO2019075090A1 (en) * 2017-10-10 2019-04-18 Tilos Therapeutics, Inc. Anti-lap antibodies and uses thereof
US11230601B2 (en) 2017-10-10 2022-01-25 Tilos Therapeutics, Inc. Methods of using anti-lap antibodies
WO2019081983A1 (en) 2017-10-25 2019-05-02 Novartis Ag Antibodies targeting cd32b and methods of use thereof
WO2019089969A2 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for b-cell maturation antigen
WO2019089858A2 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Methods of assessing or monitoring a response to a cell therapy
US12031975B2 (en) 2017-11-01 2024-07-09 Juno Therapeutics, Inc. Methods of assessing or monitoring a response to a cell therapy
US11066475B2 (en) 2017-11-01 2021-07-20 Juno Therapeutics, Inc. Chimeric antigen receptors specific for B-cell maturation antigen and encoding polynucleotides
US11623961B2 (en) 2017-11-01 2023-04-11 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for B-cell maturation antigen
WO2019090003A1 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Chimeric antigen receptors specific for b-cell maturation antigen (bcma)
US12006356B2 (en) 2017-12-15 2024-06-11 Juno Therapeutics, Inc. Anti-CCT5 binding molecules and chimeric antigen receptors comprising the same
WO2019118937A1 (en) 2017-12-15 2019-06-20 Juno Therapeutics, Inc. Anti-cct5 binding molecules and methods of use thereof
WO2019152743A1 (en) 2018-01-31 2019-08-08 Celgene Corporation Combination therapy using adoptive cell therapy and checkpoint inhibitor
WO2019210153A1 (en) 2018-04-27 2019-10-31 Novartis Ag Car t cell therapies with enhanced efficacy
WO2019213282A1 (en) 2018-05-01 2019-11-07 Novartis Ag Biomarkers for evaluating car-t cells to predict clinical outcome
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
WO2020069405A1 (en) 2018-09-28 2020-04-02 Novartis Ag Cd22 chimeric antigen receptor (car) therapies
WO2020069409A1 (en) 2018-09-28 2020-04-02 Novartis Ag Cd19 chimeric antigen receptor (car) and cd22 car combination therapies
US11130802B2 (en) 2018-10-10 2021-09-28 Tilos Therapeutics, Inc. Anti-lap antibody variants
WO2020092848A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Methods for treatment using chimeric antigen receptors specific for b-cell maturation antigen
WO2020092854A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Chimeric antigen receptors specific for g protein-coupled receptor class c group 5 member d (gprc5d)
US11970538B2 (en) 2018-11-13 2024-04-30 Compass Therapeutics Llc Multispecific binding constructs against checkpoint molecules and uses thereof
EP3880716A4 (en) * 2018-11-13 2022-08-03 Compass Therapeutics LLC Multispecific binding constructs against checkpoint molecules and uses thereof
TWI840442B (en) * 2018-11-13 2024-05-01 美商坎伯斯治療有限責任公司 Multispecific binding constructs against checkpoint molecules and uses thereof
WO2020102770A1 (en) 2018-11-16 2020-05-22 Juno Therapeutics, Inc. Methods of dosing engineered t cells for the treatment of b cell malignancies
EP4427810A2 (en) 2018-11-30 2024-09-11 Juno Therapeutics, Inc. Methods for treatment using adoptive cell therapy
WO2020113194A2 (en) 2018-11-30 2020-06-04 Juno Therapeutics, Inc. Methods for treatment using adoptive cell therapy
WO2020160050A1 (en) 2019-01-29 2020-08-06 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for receptor tyrosine kinase like orphan receptor 1 (ror1)
US10995141B2 (en) 2019-04-19 2021-05-04 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
US11732046B2 (en) 2019-04-19 2023-08-22 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
WO2021108613A1 (en) 2019-11-26 2021-06-03 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
WO2021207689A2 (en) 2020-04-10 2021-10-14 Juno Therapeutics, Inc. Methods and uses related to cell therapy engineered with a chimeric antigen receptor targeting b-cell maturation antigen
WO2022254337A1 (en) 2021-06-01 2022-12-08 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
WO2023250400A1 (en) 2022-06-22 2023-12-28 Juno Therapeutics, Inc. Treatment methods for second line therapy of cd19-targeted car t cells
WO2024031091A2 (en) 2022-08-05 2024-02-08 Juno Therapeutics, Inc. Chimeric antigen receptors specific for gprc5d and bcma
WO2024129778A2 (en) 2022-12-13 2024-06-20 Juno Therapeutics, Inc. Chimeric antigen receptors specific for baff-r and cd19 and methods and uses thereof

Also Published As

Publication number Publication date
AU2013329083B2 (en) 2018-07-26
EP2906241A4 (en) 2016-06-08
CA3201072A1 (en) 2014-04-17
EP2906241A1 (en) 2015-08-19
IL238171A0 (en) 2015-05-31
CN104853776B (en) 2021-04-09
CA2887528C (en) 2023-08-29
JP2015534949A (en) 2015-12-07
EP2906241B1 (en) 2020-01-08
HK1213788A1 (en) 2016-07-15
AU2013329083C1 (en) 2018-11-08
BR112015008118A2 (en) 2017-12-05
JP6356134B2 (en) 2018-07-11
IL238171B (en) 2019-10-31
CN104853776A (en) 2015-08-19
EP3679950A1 (en) 2020-07-15
US20150273056A1 (en) 2015-10-01
CA2887528A1 (en) 2014-04-17
AU2013329083A1 (en) 2015-05-14
KR102229873B1 (en) 2021-03-19
HK1213791A1 (en) 2016-07-15
ZA201502603B (en) 2018-11-28
KR20150080507A (en) 2015-07-09
US20190201524A1 (en) 2019-07-04

Similar Documents

Publication Publication Date Title
US20190201524A1 (en) Enhancement of the immune response
JP7467527B2 (en) Bispecific antibodies against TIM-3 and PD-1 for immunotherapy of chronic immune diseases
US20200095284A1 (en) Modulation of the immune response
US20170058026A1 (en) Methods for modulating immune responses during chronic immune conditions by targeting il-27 induced pathways
WO2015179799A1 (en) DD1alpha RECEPTOR AND USES THEREOF IN IMMUNE DISORDERS
US20180153986A1 (en) Interactions between ceacam and tim family members
US20180298095A1 (en) Interactions between ceacam and tim family members
US20190290728A1 (en) Methods related to breaking t cell exhaustion

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13845573

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 14433828

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2887528

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2015536925

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 238171

Country of ref document: IL

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2013845573

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20157012338

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2013329083

Country of ref document: AU

Date of ref document: 20131011

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112015008118

Country of ref document: BR

REG Reference to national code

Ref country code: BR

Ref legal event code: B01E

Ref document number: 112015008118

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112015008118

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20150410