Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

LY6G6D is a selectively expressed colorectal cancer antigen that can be used for targeting a therapeutic T-cell response by a T-cell engager.

Colorectal cancer (CRC) is one of the most common cancers worldwide and demands more effective treatments. We sought to identify tumor selective CRC antigens and their therapeutic potential for cytotoxic T-cell targeting by transcriptomic and immunohistochemical analysis. LY6G6D was identified as a tumor selectively expressed CRC antigen, mainly in the microsatellite stable (MSS) subtype. A specific anti LY6G6D/CD3 T cell engager (TcE) was generated and demonstrated potent tumor cell killing and T cell activation in vitro. Ex vivo treatment of primary patient-derived CRC tumor slice cultures with the LY6G6D/CD3 TcE led to IFNγ secretion in LY6G6D positive tumor samples. In vivo, LY6G6D/CD3 TcE monotherapy demonstrated tumor regressions in pre-clinical mouse models of engrafted human CRC tumor cells and PBMCs. Lastly, 2D and 3D cocultures of LY6G6D positive and negative cells were used to explore the bystander killing of LY6G6D negative cells after specific activation of T cells by LY6G6D positive cells. LY6G6D/CD3 TcE treatment was shown to lyse target negative cells in the vicinity of target positive cells through a combined effect of IFNγ, TNFα and Fas/FasL. In summary, LY6G6D was identified as a selectively expressed CRC antigen that can be utilized to potently re-direct and activate cytotoxic T-cells to lyse LY6G6D expressing CRC using a TcE. This effect can be spread to target negative neighboring tumor cells, potentially leading to improved therapeutic efficacy.

A Novel B7-H6-Targeted IgG-Like T Cell-Engaging Antibody for the Treatment of Gastrointestinal Tumors.

PURPOSE: Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell-engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. EXPERIMENTAL DESIGN: Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures. RESULTS: B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6-positive tumor cells resulted in B7-H6-dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. CONCLUSION: These data highlight the potential of the B7-H6/CD3 ITE to induce T cell-redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

A Novel Antagonistic CD73 Antibody for Inhibition of the Immunosuppressive Adenosine Pathway.

Despite some impressive clinical results with immune checkpoint inhibitors, the majority of patients with cancer do not respond to these agents, in part due to immunosuppressive mechanisms in the tumor microenvironment. High levels of adenosine in tumors can suppress immune cell function, and strategies to target the pathway involved in its production have emerged. CD73 is a key enzyme involved in adenosine production. This led us to identify a novel humanized antagonistic CD73 antibody, mAb19, with distinct binding properties. mAb19 potently inhibits the enzymatic activity of CD73 in vitro, resulting in an inhibition of adenosine formation and enhanced T-cell activation. We then investigated the therapeutic potential of combining CD73 antagonism with other immune modulatory and chemotherapeutic agents. Combination of mAb19 with a PD-1 inhibitor increased T-cell activation in vitro Interestingly, this effect could be further enhanced with an agonist of the adenosine receptor ADORA3. Adenosine levels were found to be elevated upon doxorubicin treatment in vivo, which could be blocked by CD73 inhibition. Combining CD73 antagonism with doxorubicin resulted in superior responses in vivo Furthermore, a retrospective analysis of rectal cancer patient samples demonstrated an upregulation of the adenosine pathway upon chemoradiation, providing further rationale for combining CD73 inhibition with chemotherapeutic agents.This study demonstrates the ability of a novel CD73 antibody to enhance T-cell function through the potent suppression of adenosine levels. In addition, the data highlight combination opportunities with standard of care therapies as well as with an ADORA3 receptor agonist to treat patients with solid tumors.

Discovery Strategies to Maximize the Clinical Potential of T-Cell Engaging Antibodies for the Treatment of Solid Tumors.

T-cell Engaging bispecific antibodies (TcEs) that can re-direct cytotoxic T-cells to kill cancer cells have been validated in clinical studies. To date, the clinical success with these agents has mainly been seen in hematologic tumor indications. However, an increasing number of TcEs are currently being developed to exploit the potent mode-of-action to treat solid tumor indications, which is more challenging in terms of tumor-cell accessibility and the complexity of the tumor microenvironment (TME). Of particular interest is the potential of TcEs as an immunotherapeutic approach for the treatment of non-immunogenic (often referred to as cold) tumors that do not respond to checkpoint inhibitors such as programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) antibodies. This has led to considerable discovery efforts for, firstly, the identification of tumor selective targeting approaches that can safely re-direct cytotoxic T-cells to cancer cells, and, secondly, bispecific antibodies and their derivatives with drug-like properties that promote a potent cytolytic synapse between T-cells and tumor cells, and in the most advanced TcEs, have IgG-like pharmacokinetics for dosing convenience. Based on encouraging pre-clinical data, a growing number of TcEs against a broad range of targets, and using an array of different molecular structures have entered clinical studies for solid tumor indications, and the first clinical data is beginning to emerge. This review outlines the different approaches that have been taken to date in addressing the challenges of exploiting the TcE mode-of-action for a broad range of solid indications, as well as opportunities for future discovery potential.

A Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy-resistant recurrence in the majority of patients. To effectively treat SCLC, we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently redirects T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues. EXPERIMENTAL DESIGN: The antitumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in an SCLC xenograft model reconstituted with human CD3+ T-cells. RESULTS: Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3. CONCLUSIONS: This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells and recruitment of T-cells into noninflamed tumor tissues leading to tumor regression in a preclinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in patients with SCLC.

Antitumor Activity of the IGF-1/IGF-2-Neutralizing Antibody Xentuzumab (BI 836845) in Combination with Enzalutamide in Prostate Cancer Models.

Androgen deprivation therapy and second-generation androgen receptor signaling inhibitors such as enzalutamide are standard treatments for advanced/metastatic prostate cancer. Unfortunately, most men develop resistance and relapse; signaling via insulin-like growth factor (IGF) has been implicated in castration-resistant prostate cancer. We evaluated the antitumor activity of xentuzumab (IGF ligand-neutralizing antibody), alone and in combination with enzalutamide, in prostate cancer cell lines (VCaP, DuCaP, MDA PCa 2b, LNCaP, and PC-3) using established in vitro assays, and in vivo, using LuCaP 96CR, a prostate cancer patient-derived xenograft (PDX) model. Xentuzumab + enzalutamide reduced the viability of phosphatase and tensin homolog (PTEN)-expressing VCaP, DuCaP, and MDA PCa 2b cells more than either single agent, and increased antiproliferative activity and apoptosis induction in VCaP. Xentuzumab or xentuzumab + enzalutamide inhibited IGF type 1 receptor and AKT serine/threonine kinase (AKT) phosphorylation in VCaP, DuCaP, and MDA PCa 2b cells; xentuzumab had no effect on AKT phosphorylation and proliferation in PTEN-null LNCaP or PC-3 cells. Knockdown of PTEN led to loss of antiproliferative activity of xentuzumab and reduced activity of xentuzumab + enzalutamide in VCaP cells. Xentuzumab + enzalutamide inhibited the growth of castration-resistant LuCaP 96CR PDX with acquired resistance to enzalutamide, and improved survival in vivo The data suggest that xentuzumab + enzalutamide combination therapy may overcome castration resistance and could be effective in patients who are resistant to enzalutamide alone. PTEN status as a biomarker of responsiveness to combination therapy needs further investigation.

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro.

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.

Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays.

Detection of disease-associated gene transcripts in primary disease tissues is frequently confounded by the presence of non-involved cell types. Alternative methods of detecting gene expression directly within tissues involve either the generation of antibodies, which can be a lengthy process and may suffer from lack of specificity, or amplification of reverse-transcribed cDNA in tissue sections (in situ RT-PCR). The latter method is highly specific and enables detection of transcripts in the cells originally responsible for their synthesis, but is highly destructive of tissue structures and can be carried out on only one or a few sections per experiment, resulting in low reproducibility. In this study, in situ RT-PCR was applied for the first time to commercially available tissue section microarrays enabling the examination of up to 70 different samples simultaneously. Modifications to the technique are detailed that preserved visible tissue and cellular structures and improved transcript detection whilst preventing significant generation of artefacts.

Pharmacodynamic and antineoplastic activity of BI 836845, a fully human IGF ligand-neutralizing antibody, and mechanistic rationale for combination with rapamycin.

Insulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of multiple cancer types. To date, therapeutic strategies aimed at disrupting IGF signaling have largely focused on antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high affinity to human IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-induced increase in upstream signaling in vitro as well as in human tumor xenograft models in nude mice. Our data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational combinations with other targeted agents in clinical studies.

Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.