Victor Levitsky

Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors

T cells within non–small cell lung cancer tumors acquire greater numbers, and more diversity, of inhibitory receptors as tumors progress, correlating with a loss in function as well as in their ability to be reactivated after anti-checkpoint treatment. Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non–small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8+ T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1hi T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1hi expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor–specific antibodies. Overall, these data may provide a framework for future personalized T-cell–based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions. Cancer Immunol Res; 3(12); 1344–55. ©2015 AACR.

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

Committing cytomegalovirus-specific CD8 T cells to eliminate tumor cells by bifunctional major histocompatibility class I antibody fusion molecules

Schmittnaegel and colleagues describe the generation of a novel tumor-peptide-MHCI–antibody fusion protein that redirects a highly functional subset of CMV-specific T cells to eliminate tumor cells by engaging a naturally occurring T-cell population in humans that controls cytomegalovirus infection. Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen–specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI–IgG). Here, we show that tumor antigen–expressing cancer cells, which are decorated with pMHCI–IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8+ T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8+ T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI–IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles. Cancer Immunol Res; 3(7); 764–76. ©2015 AACR.

Novel method to characterize immune cells from human prostate tissue

Benign prostatic hyperplasia (BPH) is the most common benign adenoma and prostate cancer is the most frequent malignancy in men over 50 years of age in the Western world, where it remains a significant health problem. Prostate lesions are known to contain immune cells, which may contribute to the immune control of tumor progression. However, due to their low numbers and restricted access to necessary material it is difficult to isolate immune cells from prostate tissue to characterize their immunological features.

Abstract 486: Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer.

IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the described IL-2-based immunocytokines has progressed beyond Phase II trials due to various constraints in their design: 1) pM affinity for IL-2Rαβγ on immune cells and pulmonary vascular endothelium compromising tumor targeting due to the fusion of two wildtype IL-2 moieties to the antibody, together with FcγR binding on the same cells; 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rαβγ binding; 3) Preferential activation of Tregs over immune effectors by wt IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Rα (CD25) binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA- and FAP-IL2v were recombinantly produced and induction of P-STAT5, proliferation, activation induced cell death (AICD), activation markers and cytokines were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting, pharmacodynamics and anti-tumor efficacy were analyzed in SCID and immunocompetent C57Bl/6 mice. FAP- and CEA-IL-2v completely lack binding to CD25, but retain IL-Rβγ binding. They do not bind to CD25 or preferentially activate Tregs, and induce lower degree of AICD. However, IL-2Rβγ bioactivity is retained and they activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers and proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards CD8+ T cells in vivo. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite of higher exposure and circulatory half-life than the corresponding wt IL-2 immunocytokine. MicroSPECT/CT imaging revealed FAP-mediated tumor targeting of FAP-IL2v with low normal tissue uptake with FAP-IL2v tumor targeting being similar to the parental FAP antibody with low accumulation in lymphoid tissues and clearly superior to an FAP-targeted wt IL-2 immunocytokine that shows preferential spleen targeting. Studies in tumor-bearing mice showed dose dependent anti-tumor efficacy of CEA- and FAP-IL2v in established xenograft and syngeneic mouse models. Thus, CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcγR binding, monovalency and high-affinity tumor-targeting as compared to classical immunocytokines. They retain capacity to activate NK and T‐effector cells through IL‐2Rβγ; in particular once targeted to the tumor microenvironment. These data support their investigation for the immunotherapy of CEA/FAP-positive tumors. Citation Format: Christian Klein, Inja Waldhauer, Valeria Nicolini, Claire Dunn, Anne Freimoser-Grundschober, Sylvia Herter, Edwin Geven, Otto Boerman, Tapan Nayak, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Peter Bruenker, Ralf Hosse, Sabine Lang, Sebastian Neumann, Hubert Kettenberger, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Pablo Umana. Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2013-486

Abstract PR8: Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer.

Introduction: IL-2 therapy can lead to durable responses in a modest proportion of cancer patients, but the treatment is associated with significant toxicity. Over the last decades, various IL-2-based immunocytokines have been generated by fusing IL-2 to tumor-targeting antibodies. However, none of these molecules have progressed beyond Phase II trials and they are hampered by various liabilities: 1) High functional affinity (low pM) for IL-2Rabg on immune cells and on pulmonary vascular endothelium (Krieg et al., PNAS, 2010) compromising preferential tumor targeting due to fusion of two IL-2 moieties to the antibody. This is further compounded when the immunocytokine binds to FcgRs on the same cells. 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rabg binding. 3) Preferential activation of Tregs over immune effectors due to use of wildtype IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ra (CD25) binding is fused to the C-terminus of a tumor-specific hIgG1 antibody with a heterodimeric Fc-part. FcgR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were chosen. Experimental procedures: CEA- and FAP-IL2v were recombinantly produced and characterized by surface plasmon resonance. Induction of P-STAT5, proliferation, activation induced cell death (AICD), various activation markers and cytokine release were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting by imaging, immune-pharmacodynamics and anti-tumor efficacy were analyzed in immunocompromised Scid and immunocompetent C57BL/6 mice. Results: IL-2v completely lacks binding to CD25, but retains IL-Rbg binding. In line with this, FAP- and CEA-IL2v do not bind to CD25 or preferentially activate Tregs, and do not cause AICD. However, IL-2Rbg bioactivity is retained and they are still able to activate NK, CD4 and CD8 T cells as shown by concentration dependent increase in activation markers and induction of proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards activated CD8 T cells in vivo. In C57BL/6 mice CEA- and FAP-IL2v demonstrate improved safety despite of ca. 2-fold higher exposure and t1/2 than a wildtype IL-2-based IgG immunocytokine. SPECT/CT imaging revealed FAP-mediated tumor targeting and accumulation of FAP-IL2v with low normal tissue uptake. Notably, FAP-IL2v tumor targeting was similar to the parental FAP antibody with low accumulation in lymphoid tissues; clearly superior to an FAP-targeted wt IL-2 immunocytokine that showed preferential homing to the spleen. Studies in tumor bearing mice showed dose dependent efficacy of CEA- and FAP-IL2v in established xenograft and immunocompetent syngeneic mouse models in terms of survival. Conclusion: CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcgR binding, monovalency and high-affinity tumor-targeting as compared to conventional immunocytokines. They retain the capacity to activate NK and T effector cells through IL 2Rbg; in particular once targeted and immobilized in the tumor microenvironment. These preclinical properties support further investigation for the immunotherapy of CEA/FAP-positive tumors. This abstract is also presented as Poster A23. Citation Format: Christian Klein, Waldhauer Inja, Valeria Nicolini, Dunn Claire, Anne Freimoser, Anne Freimoser, Sylvia Herter, Edwin Geven, Otto Boerman, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Ralf Hosse, Sabine Lang, Sebastian Neumann, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Ekkehard Moessner, Pablo Umana. Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR8.

Activation of cytomegalovirus-specific CD8(+) T-cell response by antibody-mediated peptide-major histocompatibility class I complexes.

Imposing antigenicity on tumor cells is a key step toward successful cancer-immunotherapy. A cytomegalovirus-derived peptide recombinantly fused to a major histocompatibility class I complex and a monoclonal antibody can be targeted to tumor cells by antibody-mediated delivery and activate a strong and specific CD8+ T cell response.

Abstract B69: Novel MHC class I antibody fusions for cancer treatment.

MHC (major histocompatibility complex) class I-restricted CD8 cytotoxic T cells recognize tumor cells or virus infected cells as foreign and consequently initiate a cascade of events resulting in their destruction. An immunotherapeutic strategy based on antibody-mediated targeting of virus-derived peptide-MHC class I complexes to tumor cells aiming at the subsequent elimination of these tumor cells by peptide-specific cytotoxic T cells has been proposed a few years ago. A crucial limitation hampering advances of this therapeutic concept was the recombinant expression of MHC class I fused full IgG immunoglobulins. We have identified novel recombinant fusion formats that allow the expression of full-length peptide-MHC class I IgG fusion molecules. These formats contain a single recombinant human pMHC class I complex consisting of a viral peptide (CMV or EBV derived), beta-2-microglobulin and HLA heavy chain (A*0201, lacking the transmembrane domain) fused to one of the two immunoglobulin heavy chains of a complete antibody molecule. These fusions can be expressed at high levels in standard mammalian expression systems overcoming several former technical hurdles (e.g. prokaryotic expression, low refolding yield, chemical coupling of components). By flow cytometry we show that the new fusion proteins successfully deliver virus-peptide complexed MHC class I molecules to tumor cells. The fusion proteins are potent CD8 T cell recruiters for human donor derived specific T cells or human PBMCs from chronically infected donors. A low frequency of virus peptide specific CD8 T cells in PBMCs (0.25 to 3.8 % of all CD8 positive T cells) effectively triggers in a peptide specific manner the killing of tumor cells at sub-nanomolar concentrations (cytotoxic T cell assay using real time analysis with xCelligence confirmed by classical LDH release). No unspecific activation of T cells was observed even at high concentrations, indicating a favorable safety profile. Confocal time-lapse microscopy showed T cell synapse formation on the tumor cells and serial killing by the T cells. We experimentally compare the killing of tumor cells of the MHC class I fused antibodies with bispecific anti-CD3 mediated T cell recruiters and characterized the virus specific T cells from human donors in detail. Our results show for the first time that recombinant pMHC class I antibody fusions in our novel full human IgG format can be expressed with a significant yield in common mammalian production cell lines. The redirection of MHC class I mediated recruitment of pre-existing virus specific CD8 T cells from human PBMCs is potent mechanism to attack tumor cells with a low risk of unspecific T cell activation making the concept now suitable as a therapeutic option. Citation Format: Martina Schmittnaegel, Eike Hoffmann, Olaf Mundigl, Gerhard Niederfellner, Klaus Bosslet, Pablo Umana, Victor Levitsky, Christian Klein, Hendrik Knoetgen. Novel MHC class I antibody fusions for cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B69.