Dmitry Pankov

Targeting the Intracellular WT1 Oncogene Product with a Therapeutic Human Antibody

A therapeutic monoclonal antibody specific for the intracellular oncoprotein Wilms tumor 1 treats human leukemias in mice. Destroying from Within Anticancer antibody-based drugs have largely targeted proteins on the surface of cancer cells. But, arguably the most important, tumor-specific proteins are on the inside—safely tucked away within the cell. Wilms tumor 1 (WT1) is one of these intracellular oncoproteins. Despite its insider status, degraded WT1 fragments are presented on the surface of leukemia cells and many other cancer tissues, including ovarian. To kill leukemia, Dao and colleagues hypothesized that intracellular WT1 was the perfect target. Dao et al. engineered a monoclonal antibody, named “ESK1,” that recognizes a peptide fragment of WT1, called RMF, complexed with human leukocyte antigen (HLA)–A0201. After demonstrating that ESK1 bound to several WT1+ cell lines in vitro and leukemia patient cells ex vivo, the authors tested their new antibody in two mouse models of human acute lymphoblastic leukemia. They delivered ESK1 alone or along with human “effector” cells (peripheral blood natural killer cells) and saw that the combination therapy killed nearly all leukemia in comparison to control groups, allowing all of the treated mice to have prolonged or even leukemia-free survival. Treating animals with cancers that lacked either HLA-A0201 or WT1 had no effect. With a defined mechanism and no toxicity in mice, this ESK1 antibody is poised for testing in human trials. The authors point out that more than 1 million patients in the world may have a WT1+ cancer, with many of these being HLA-A02+. In this case, ESK1—with its ability to target a cancer protein inside the cell—could help treat many patients that have not responded to antibody-based therapies focused on the cell surface. The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)–A0201 epitope, is a validated target for T cell–based immunotherapy. Using phage display technology, we discovered a fully human “T cell receptor–like” monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201–restricted manner, with high avidity [dissociation constant (Kd) = 0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome–positive leukemia in nonobese diabetic/severe combined immunodeficient γc−/− (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1.

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell–based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor resistance in human BCR-ABL+ leukemias

Acute and chronic leukemias, including CD34(+) CML cells, demonstrate increased expression of the Wilms tumor gene 1 product (WT1), making WT1 an attractive therapeutic target. However, WT1 is a currently undruggable, intracellular protein. ESKM is a human IgG1 T-cell receptor mimic monoclonal antibody directed to a 9-amino acid sequence of WT1 in the context of cell surface HLA-A*02. ESKM was therapeutically effective, alone and in combination with tyrosine kinase inhibitors (TKIs), against Philadelphia chromosome-positive acute leukemia in murine models, including a leukemia with the most common, pan-TKI, gatekeeper resistance mutation, T315I. ESKM was superior to the first-generation TKI, imatinib. Combination therapy with ESKM and TKIs was superior to either drug alone, capable of curing mice. ESKM showed no toxicity to human HLA-A*02:01(+) stem cells under the conditions of this murine model. These features of ESKM make it a promising nontoxic therapeutic agent for sensitive and resistant Ph(+) leukemias.

Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2

ABSTRACT Chimeric antigen receptors (CAR) and bispecific antibodies (BsAb) are two powerful immunotherapy approaches for retargeting lymphocytes toward cancer cells. Despite their success in lymphoblastic leukemia, solid tumors have been more recalcitrant. Identifying therapeutic barriers facing CAR-modified (CART) or BsAb-redirected T (BsAb-T) cells should facilitate their clinical translation to solid tumors. Novel lentiviral vectors containing low-affinity or high-affinity 4-1BB second-generation anti-GD2 (disialoganglioside) CARs were built to achieve efficient T cell transduction. The humanized anti-GD2 × CD3 BsAb using the IgG-scFv platform was described previously. CART and BsAb-engaged T cells were tested for viability, proliferation, and activation/exhaustion marker expression, and in vitro cytotoxicity against GD2(+) tumor cells. The antitumor effect of CAR-grafted and BsAb-T cells was compared in a human melanoma xenograft model. The majority of high CAR density T cells were depleted upon exposure to GD2(+) target cells while the BsAb-T cells survived. The in vitro cytotoxicity of the surviving CART cells was inferior to that of the BsAb-T cells. Using low-affinity CARs, inclusion of the 4-1BB co-stimulatory domain or exclusion of a co-stimulatory domain, or blocking PD1 did not prevent CART cell depletion. Both CART cells and BsAb-T cells penetrated established subcutaneous human melanoma xenografts; while both induced tumor regression, BsAb was more efficient. The fate of T cells activated by BsAb differs substantially from that by CAR, translating into a more robust antitumor effect both in vitro and in vivo.

T cell receptor mimic antibodies for cancer therapy

The major hurdle to the creation of cancer-specific monoclonal antibodies (mAb) exhibiting limited cross-reactivity with healthy human cells is the paucity of known tumor-specific or mutated protein epitopes expressed on the cancer cell surface. Mutated and overexpressed oncoproteins are typically cytoplasmic or nuclear. Cells can present peptides from these distinguishing proteins on their cell surface in the context of human leukocyte antigen (HLA). T cell receptor mimic (TCRm) mAb can be discovered that react specifically to these complexes, allowing for selective targeting of cancer cells. The state-of-the-art for TCRm and the challenges and opportunities are discussed. Several such TCRm are moving toward clinical trials now.

Opportunities and challenges for TCR mimic antibodies in cancer therapy

ABSTRACT Introduction: Monoclonal antibodies (mAbs) are potent cancer therapeutic agents, but exclusively recognize cell-surface targets whereas most cancer-associated proteins are found intracellularly. Hence, potential cancer therapy targets such as over expressed self-proteins, activated oncogenes, mutated tumor suppressors, and translocated gene products are not accessible to traditional mAb therapy. An emerging approach to target these epitopes is the use of TCR mimic mAbs (TCRm) that recognize epitopes similar to those of T cell receptors (TCR). Areas covered: TCRm antigens are composed of a linear peptide sequence derived from degraded proteins and presented in the context of cell-surface MHC molecules. We discuss how the nature of the TCRm epitopes provides both advantages (absolute tumor specificity and access to a new universe of important targets) and disadvantages (low density, MHC restriction, MHC down-regulation, and cross-reactive linear epitopes) to conventional mAb therapy. We will also discuss potential solutions to these obstacles. Expert opinion: TCRm combine the specificity of TCR recognition with the potency, pharmacologic properties, and versatility of mAbs. The structure and presentation of a TCRm epitope has important consequences related to the choice of targets, mAb design, available peptides and MHC subtype restrictions, possible cross-reactivity, and therapeutic activity.

TCR-mimic bispecific antibodies targeting LMP2A show potent activity against EBV malignancies

EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor-like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3-P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV- tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.

In vivo immuno-targeting of an extracellular epitope of membrane bound preferentially expressed antigen in melanoma (PRAME)

Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer/testis antigen that is overexpressed in a broad range of malignancies, while absent in most healthy human tissues, making it an attractive diagnostic cancer biomarker and therapeutic target. Although commonly viewed as an intracellular protein, we have demonstrated that PRAME has a membrane bound form with an external epitope targetable with conventional antibodies. We generated a polyclonal antibody (Membrane associated PRAME Antibody 1, MPA1) against an extracellular peptide sequence of PRAME. Binding of MPA1 to recombinant PRAME was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). Flow cytometry and confocal immunofluorescence microscopy of MPA1 was performed on multiple tumor cell lines. Reverse Transcription Polymerase Chain Reaction (RT-PCR) for PRAME was conducted to compare protein and transcriptional expression levels. We demonstrated a robust proof-of-concept for PRAME targeting in vivo by radiolabeling MPA1 with zirconium-89 (89Zr-DFO-MPA1) and demonstrating high specific uptake in PRAME expressing tumors. To our knowledge, this is the first time a cancer testis antigen has been targeted using conventional antibody technologies. Thus, PRAME can be exploited for multiple clinical applications, including targeted therapy, diagnostic imaging and treatment guidance in a wide-range of malignancies, with minimal off-target toxicity.

Abstract A058: Selective targeting of T regulatory cells by a TCR-mimic monoclonal antibody specific for foxp3-derived epitopes

Immunosuppression by tumor-induced regulatory T (Treg) cells present major obstacles for successful immunotherapy. Treg expansion and its negative prognostic impact represent a general phenomenon seen in multiple types of cancer. Therefore, developing strategies for Treg depletion could enhance the reactivation of immune responses against the malignant tumor cells. However, the strategies of depleting Tregs have been hindered by the lack of specificity, which also leads to the depletion of anti-tumor effector cells. The transcription factor forkhead box p3 (FoxP3) is selectively expressed in and is essential to the suppressive function of Treg cells. FoxP3 thus would be an appealing target for eliminating Treg cells. However, Foxp3 is an undruggable intracellular protein. Here, we took a novel and unconventional approach to target Foxp3 by using a T cell receptor mimic (TCRm) mAb, which recognizes a human Foxp3-derived CD8 T cell epitope, presented by HLA-A*02:01 molecule. We show that the Foxp3 mAb specifically bound to CD4+CD25hiCD127lo Foxp3+ Treg cells from HLA-A0201 positive donors, and tumor cell lines co-expressing Foxp3 and HLA-A*02;01 molecule. Both afucosylated Fc enhanced human IgG1 and bispecific T cell engager formats of the Foxp3 mAb are able to kill in vitro-generated Treg clones from HLA-A0201+ donors and “Treg-like” cutaneous lymphoma cells (HLA-A*02:01+) that have a high level expression of CD4, CD25 and Fox3+. FoxP3-targeting antibodies could potentially be a novel approach in cancer immunotherapy by overcoming immunosuppression caused by Tregs and tumor cells expressing Foxp3. Citation Format: Tao Dao, Casey Jarvis, Andrew C. Scott, Tatyana Korontsvit, Victoria Zakhaleva, Dmitry Pankov, Manuel Direito de Morais Guerrerio, Melissa Mathias, Neal Cheng, Cheng Liu, David A. Scheinberg. Selective targeting of T regulatory cells by a TCR-mimic monoclonal antibody specific for foxp3-derived epitopes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A058.

Abstract A055: Potent therapeutic and immunological effects of the first T-BiTE derived from a TCR-mimic antibody targeting intracellular oncoprotein WT1

Bi-specific T cell engager antibody (BiTE) therapy has recently emerged as an effective immunotherapy by redirecting polyclonal T cell cytotoxicity against cell surface protein on tumor cells. We generated the first BiTE construct derived from a TCR-mimic monoclonal antibody (mAb), ESK1, specific for a peptide from an intracellular oncoprotein, WT1, in the context of HLA-A*02:01 molecules. Despite the low density peptide/HLA-A2 complex on the cell surface, ESK-BiTE was able to selectively activate and induce proliferation of cytolytic human T cells to kill multiple leukemias and cancers in vitro and in mice. Surprisingly, we also discovered that in an autologous setting, ESK-BiTE induced a robust secondary CD8 T cell response specific for antigens other than WT1, including HLA-A2-restricted her2-neu-derived peptide 369-377, in patients with her2- positive ovarian cancer. Therefore, the study demonstrated a new vaccinal mechanism for BiTE mAb action that could contribute to more effective long-term therapeutic activity of BiTE9s and further broaden their reach to other tumor antigens not previously known or originally targeted. Citation Format: Tao Dao, Dmitry Pankov, Andrew Scott, Tatyana Korontsvit, Victoriya Zakhaleva, Manuel Direito de Morais Guerrerio, Yiyang Xu, Jingyi Xiang, Su Yan, Nicholas Veomett, Nicholas Veomett, Leonid Dubrovsky, Michael Curcio, Ekaterina Doubrovina, Cheng Liu, Richard J. O9Reilly, David A. Scheinberg. Potent therapeutic and immunological effects of the first T-BiTE derived from a TCR-mimic antibody targeting intracellular oncoprotein WT1. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A055.