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Old problems, new directions - Home - ESMO - European Society for ...
 

Old problems, new directions - Home - ESMO - European Society for ...

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    Old problems, new directions - Home - ESMO - European Society for ... Old problems, new directions - Home - ESMO - European Society for ... Presentation Transcript

    • Old problems, new directions Eli Gilboa ESMO International Symposium on Immunology Nov 15-17, 2007 Athens, Greece
    • Cancer immunotherapy with mRNA transfected dendritic cells A personalized form of cell therapy IL-1  , IL-6, TNF-  , PGE 2 Antigen-Loaded Mature DC DC maturation Cryopreserved DC Vaccine Immature DC Monocytes GM-CSF, IL-4 Tumor antigens (mRNA) Antigen Loading (mRNA transfection) Leukapheresis Tumor Biopsy
    • The underlying premise of developing patient-specific vaccination protocols Added complexity and cost associated with such interventions will be offset by a substantial added benefit to the patient.
    • “ Promising” vaccination strategies
      • DNA vaccines
      • Poxvirus vector-based vaccines
      • GM-CSF transduced tumor vaccines
      • Idiotype + GM-CSF
      • gp96-secreting tumor vaccines
      • Dendritic cell vaccines
      • Listeria vector-based vaccines
      Clinical benefit is minimal, if any.
      • Preclinical murine studies
      • Antitumor effects in murine models
      • No toxicity
      • Clinical trials
      • Immune responses
      • Hints of clinical impact
    • “ Promising” vaccination strategies
      • DNA vaccines
      • Poxvirus vector-based vaccines
      • GM-CSF transduced tumor vaccines
      • Idiotype + GM-CSF
      • gp96-secreting tumor vaccines
      • Dendritic cell vaccines
      • Listeria vector-based vaccines
      • NDV-infected autologous tumor vaccines (Schirrmacher & colleagues)
      Clinical benefit is minimal, if any.
      • Preclinical murine studies
      • Antitumor effects in murine models
      • No toxicity
      • Clinical trials
      • Immune responses
      • Hints of clinical impact
    • Immunological control of cancer Where are we going from here
    • “ Promising” vaccination strategies
      • DNA vaccines
      • Poxvirus vector-based vaccines
      • GM-CSF transduced tumor vaccines
      • Idiotype + GM-CSF
      • gp96-secreting tumor vaccines
      • Dendritic cell vaccines
      • Listeria vector-based vaccines
      • NDV-infected autologous tumor vaccines (Schirrmacher & colleagues)
      Clinical benefit is minimal, if any.
      • Preclinical murine studies
      • Antitumor effects in murine models
      • No toxicity
      • Clinical trials
      • Immune responses
      • Hints of clinical impact
    • Specific Active Immunotherapy of Cancer Cancer Vaccines To engender protective immunity in the cancer patient that will negatively impact on tumor progression.
    • A multi pronged approach to cancer immunotherapy Induction of immunity Persistence of immunity + Immune suppression Immune escape
    • Immune suppression A multi pronged approach to cancer immunotherapy Induction of immunity Persistence of immunity + Immune escape
    • Why is a tumor growing in an immune competent patient not eliminated by an immune response? Lack of immunogenicity The immune system is not activated in response to the growing tumor - “tumor is not sufficiently distinct from normal tissue”
    • Why is a tumor growing in an immune competent patient not eliminated by an immune response? + Lack of immunogenicity The immune system is not activated in response to the growing tumor - “tumor is not sufficiently distinct from normal tissue” Immune suppression Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
    • Why is a tumor growing in an immune competent patient not eliminated by an immune response? Lack of immunogenicity The immune system is not activated in response to the growing tumor - “tumor is not sufficiently distinct from normal tissue” + Immune suppression Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
    • Why is a tumor growing in an immune competent patient not eliminated by an immune response? Immune suppression Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response + Lack of immunogenicity The immune system is not activated in response to the growing tumor - “tumor is not sufficiently distinct from normal tissue”
    • Tumor-induced immune suppression Immune suppressive Cell types Immune suppressive products Regulatory T cells IL-13 secreting NKT cells Immature myeloid cells (ImC) Tolergenic DC (“iDC”, pDC) “ Alternatively activated” M  (AAMs) … and other Cox-2 generated prostanoids B7H1 TGF  IL-10 Decoy receptor 3 (DcR3) STAT3 cAMP Adenosine VEGF Indoleamine deoxygenase (IDO) … and more
    • ...and more evidence
      • Immune mediated tumor rejection in the absence of vaccination, e.g., blocking TGF  signaling in T cells (Gorelik & Flavell, Nat. Med. 2001, 7:1118)
      • Tumor induced immune suppression, not lack of inherent immunogenicity, the main reason for tumor outgowth - in a highly relevant spontaneous tumor model where tumors are heterogenous, multifocal exhibiting different biologies. ( G. Willimsky & T. Blankenstein. Nature, 2005, 437, 141-146 )
      • Vigorous premalignancy-specific effector T cell response in the bone marrow of patients with monoclonal gammopathy. Dhodapkar et al., Exp Med, 2003, 198:1753
      • Inverse correlation between tumor progression in ovarian and colorectal cancer patients and immune infiltrate (Zhang et al., N. Engl. J. Med., 2003, 348:203; Galon et al., Science, 2006, 313:1960)
      • Immune-mediated control of subclinical cancer in murine models (Schreiber, Smyth and colleagues, CRI meeting, Manhattan, NYC, October 2006)
      • Inherent immunogenicity of human cancer – Frequent induction of immune responses which are occasionally associated with better prognosis but ultimately fail to reverse disease course (reviewed by Hodi & Dranoff, Adv. Immunol., 2006, 90:341 )
      Cancer despite immunosurveillance: immunoselection and immunosubversion. Zitvogel, L., Tesniere, A. and G. Kroemer. Can. Rev., Immunol., 2006, 6:715 “ The seventh hallmark of cancer”
    • Tumor-induced immune suppression Immune suppressive Cell types Immune suppressive products Regulatory T cells IL-13 secreting NKT cells Immature myeloid cells (ImC) Tolergenic DC (“iDC”, pDC) “ Alternatively activated” M  (AAMs) … and other Cox-2 generated prostanoids B7H1 TGF  IL-10 Decoy receptor 3 (DcR3) STAT3 cAMP Adenosine VEGF Indoleamine deoxygenase (IDO) … and more
    • Naturally occuring CD4+CD25+ regulatory T cells
      • A distinct lineage of thymic origin, comprise 3-10% of the CD4+ T cell population
      • Immune suppressive - inhibit CD4+ & CD8+ T cell responses
      • Function - Preventing autoimmunity by keeping autoreactive T cells in check.
      • Depletion of Treg in mice with  CD25 antibodies:
        • Induces or exacerbates autoimmune pathology
        • Potentiates tumor immunity, especially in conjunction with vaccination .
    • Elimination of T reg using a diphteria toxin-IL-2 conjugate (ONTAK ® ) in RCC patients vaccinated with tumor RNA transfected DC Dannull et al., J. Clin. Invest. , 2005, 115:3623 7 5 0 5 0 0 2 5 0 0 IFN  /10 5 CD8 + T cells ONTAK® - + n=4 n=6 p=0.019
    • Limitation to targeting CD25 for Treg depletion
      • CD25, a component of the IL-2 receptor complex, is also upregulated on conventional activated (vaccine-induced) T cells.
        • Treg rebound with time
        • The tumor and the vaccination itself can generate Treg
        • Interfere with an ongoing protective immune response against subclinical levels of pathogenic infection
      • A significant fraction of Treg (10-30%), especially recently activated Treg, have downregulated CD25.
      • Depletion is global - risk of autoimmune pathology
      • CD25 depletion constitutes an additional intervention and a reagent not always readily available for clinical testing.
    • Other Treg-specific markers
      • GITR, Lag-3, CTLA-4, CD103 - expressed on the cell surface but, like CD25, not specific.
      • Foxp3
        • Member of the forkhead/wing-helix family of transcription factor repressors
        • Expression exclusively restricted to Treg
        • Master regulator of suppressive phenotype
        • CD4+CD25+foxp3 as well as CD4+CD25-foxp3 Treg.
    • Foxp3 is a nuclear protein - cannot use antibodies or ONTAK ® -like reagents for depletion of foxp3 expressing cells in vivo No additional procedure: Co-vaccination against tumor antigen and foxp3 Stimulate a CD8+ CTL response against foxp3
      • Foxp3 is expressed in the thymus
        • Thymocytes destined to become Treg
          • Fontenot, J.D et al. 2003. Nat Immunol 4:330-336
        • Thymic stroma
          • Chang, X. et al., 2005, J Exp Med 202:1141-1151
    • Immunization (1x) against Foxp3 enhances antitumor immunity in B16 melanoma tumor-bearing mice d3 d0 Tumor Treg Vaccination ( TRP-2 ) Impact on tumor growth Foxp3 vaccination CD25 depletion
    • Repeated depletion of Treg subsequent to tumor vaccination Foxp3 immunotherapy versus CD25 Ab depletion d10 d3 d0 Tumor Treg (1X) Vaccination Treg (2X) Impact on tumor growth 1week
    • Fate of foxp3-expressing cells in mice vaccinated against foxp3 or treated with CD25 Ab Tumor Treg depletion enhances the ratio of conventionalTcells/Treg in the tumor
    • A.  CD25 B. Foxp3 Lymph node Spleen Nair et al. Can. Res., 2007, 67:371 Fate of foxp3-expressing cells in mice vaccinated against foxp3 or treated with CD25 Ab Periphery
    • Why is a tumor growing in an immune competent patient not eliminated by an immune response? Immune suppression Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response + Shift emphasis from inducing immunity to developing methods targeting tumor-induced immune suppression Lack of immunogenicity The immune system is not activated in response to the growing tumor - “tumor is not sufficiently distinct from normal tissue”
    • Tumor-induced immune suppression Immune suppressive Cell types Immune suppressive products Regulatory T cells IL-13 secreting NKT cells Myeloid derived suppressor cells (MDSC) Tolergenic DC (“iDC”, pDC) “ Alternatively activated” M  (AAMs) … and other Cox-2 generated prostanoids B7H1 TGF  IL-10 Decoy receptor 3 (DcR3) VEGF STAT3 cAMP Adenosine Indoleamine deoxygenase (IDO) … and more ???
    • Persistence of immunity + Immune suppression A multi pronged approach to cancer immunotherapy Induction of immunity Immune escape TCR attenuation Costimulation
    • 4-1BB
      • Upregulated on antigen-activated T cells
      • Enhances survival and proliferation of activated CD8 + T cells
      Costimulatory receptors on T cells
      • CD28
      • CD27
      • OX40
      • 4-1BB
      • CTLA-4
      • PD-1
      • HVEM
      • CD30
    • Manipulating costimulation
      • Ectopic expression of ligands in APC or tumor cells
      • Systemic administration of agonistic or blocking antibodies
        • Clinical trials: CTLA-4, PD-1, 4-1BB, OX-40, CD40.
    • Agonistic 4-1BB antibodies enhance proliferation of activated CD8+ T cells and potentiate tumor immunity in mice
        • Complexity & cost of development
        • Regulatory approval process
        • Cost of manufacturing
        • Limited & uncertain access (companies)
      Limitations to use of antibodies (or protein-based ligands) as therapeutic reagents Melero, I., W.W. Shuford, S.A. Newby, A. Aruffo, J.A. Ledbetter, K.E. Hellstrom, R.S. Mittler, and L. Chen. 1997, Nat Med 3:682-685. Antibodies are cell based products
    • Antibody combinations - synergistic antitumor effects Murine studies Antibodies Reference 4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:72764 4-1BB + OX40 Lee et al., 2004, J. Immunol., 173:3002 4-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:1089 4-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693
    • Antibodies Reference 4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:72764 4-1BB + OX40 Lee et al., 2004, J. Immunol., 173:3002 4-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:1089 4-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693 Antibody combinations - synergistic antitumor effects Murine studies
    • Eradication of established 4T1 breast carcinoma tumors in mice by combination therapy with  DR5+  CD40+  CD137 *Uno et al., Nat. Med., 2006, 12:693
    • Antibody combinations - synergistic antitumor effects Murine studies Antibodies Reference 4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:72764 4-1BB + OX40 Lee et al., 2004, J. Immunol., 173:3002 4-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:1089 4-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693
    • In vitro selection of oligonucleotide aptamers An Aptamer Library = A Vast Shape Library Aptamer Library AGGACGAUGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAGACGACUCGC 4 40 possible sequences In vitro selection (SELEX) 40 nucleotide random region
    • Advantages of aptamers vs antibodies as therapeutics
      • Specificity & avidity comparable or better than antibodies
      • Synthesized chemically; they are not cell-based products.
        • Vastly simpler regulatory approval process
        • Development & manufacturing - cost effective
      • Superior pharmacology - tumor penetration
      • Lack of immunogenicity
      • Amenable to chemical modification
    • Aptamers can be made to most any target Target Protein Affinity(Kd) Function Ref. PDGF 0.1nM Inhibitor Green et al., 1996 P-Selectin 0.03nM Inhibitor Jenison et al., 1998 Complement C5 0.03nM Inhibitor Biesecker et al., 1999 MAb to AChR 60nM Inhibitor Lee and Sullenger, 1997 Interferon-Gamma 2.7nM Inhibitor Kubik et al., 1997 VEGF 0.15 Inhibitor Ruckman et al., 1998 Factor VIIa 11nM Inhibitor Rusconi et al., 2000 mCTLA-4 30nM Inhibitor Santulli-Marotto, et al. 2003
    • Isolation of aptamers which bind to murine 4-1BB in solution
    • 4-1BB in vitro costimulation assay Enhancement of proliferation or IFN  release from suboptimally activated CD8+ T cells Isolate CD8+ T cells from BALB/C mice. Incubate o/n with sub-optimal concentration of anti-CD3. Add anti-4-1BB antibody or aptamers coupled to beads. Proliferation assay 48 hrs later (CFSE dilution) and/or IFN  release
    • 4-1BB signaling requires ligand-induced receptor dimerization + MAb +
    • Sequence and computer-predicted secondary structure of a 4-1BB binding aptamer
    • Generating aptamer dimers using complementary 3’ extensions
    • 4-1BB aptamer dimer binds specifically to activated CD8+ T cells
    • Binding of monomeric and dimeric forms of 4-1BB aptamers to 4-1BB expressed on the cell surface
    • 4-1BB Aptamer Dimers Costimulate T Cells (CFSE proliferation assay)
    • 4-1BB Aptamer Dimers Costimulate T Cells (IFN  production)
    • Rejection of P815 mastocytoma tumors injected with 4-1BB aptamer dimers A B C D
    • Summary
      • High affinity 4-1BB binding aptamers can be isolated
      • A subset of which - when multimerized - can function as 4-1BB agonists
      • A low-tech clinically applicable approach
      • Next:
      • Improve aptamer potency by post selection modifications
      • Antitumor potential - stringent murine immunotherapy models
      • Do monomers block 4-1BB signaling?
      • Development of human 4-1BB agonists and clinical studies.
    • The potential of aptamer technology to manipulate immunity
      • Ligands as well as targeting agents for drugs (siRNAs)
      • Feasibility
      • Replace antibodies and expand therapeutic applications?
    • Concluding thoughts
    • or Vaccination : Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
    • Yesterday Vaccination : Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens
    • Today Vaccination : Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
    • Mounting evidence that tumors in cancer patients are capable of stimulating, transiently, protective immunity
      • Frequent induction of immune responses in cancer patients. (reviewed by Hodi & Dranoff, Adv. Immunol., 2006, 90:341 )
      • Correlation between lack of tumor progression in cancer patients and immune infiltrates.
        • Ovarian cancer: Zhang, L., et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med, 2003, 348:203
        • Colorectal cancer : Galon, J., et al.Type, density, and location of immune cells within human colorectaltumors predict clinical outcome. Science, 2006, 313:1960
    • Tomorrow Vaccination : Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
      • Overcoming tumor-induced immune suppression
      • Delivering co-stimulatory ligands to the tumor
      • Promoting “immunogenic” death of the tumor
      • Enhancing the antigenicity of the tumor
      And then... Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
    • 4-1BB aptamers James McNamara Despina Kolonias Fernando Pastor Collaboration Paloma Giangrande Bruce Sullenger Lieping Chen Robert Mittler Jenz Dannull Zhen Su Philip Dahm Doris Coleman Center for Translational Research, Duke University Medical Center Foxp3 vaccination Smita Nair David Boczkowski Martin Fassnacht Sylvia Sichi Benjamin Yang Melinda Malready Donna Yancey Eli Gilboa Johannes Vieweg Duke Cancer Immunotherapy Program
    • 4-1BB aptamers James McNamara Despina Kolonias Fernando Pastor Collaboration Paloma Giangrande Bruce Sullenger Lieping Chen Robert Mittler Jenz Dannull Zhen Su Philip Dahm Doris Coleman Center for Translational Research, Duke University Medical Center Foxp3 vaccination Smita Nair David Boczkowski Martin Fassnacht Sylvia Sichi Benjamin Yang Melinda Malready Donna Yancey Eli Gilboa Johannes Vieweg Duke Cancer Immunotherapy Program