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Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3e chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.

Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting >1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival > 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced >2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell–redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596–induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell–dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8 + T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4 − /CD8 + T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential. IMPORTANCE HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed in vitro and ex vivo that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.

T cell engaging bispecific antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein. The molecular weight of this “canonical” BiTE® is ~ 55 kDa, making it susceptible to kidney-mediated clearance and resulting in a short serum half-life (~ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uniform PK profile), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended. A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-containing moieties. Each of these half-life extended (HLE) constructs was evaluated in vitro, in mouse xenograft models and in non-human primates. In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cytotoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were administered IV and activated human T cells were administered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD. However, the albumin fusion–based HLE BiTE® was less efficacious when administered Q4D than the QD- administered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates. The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody constructs showed on-target depletion of CD33-positive monocytes and neutrophils in the blood and depletion of CD33-positive cells in the bone marrow. These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing. Citation Format: Tara L. Arvedson, Mercedesz Balazs, Pamela Bogner, Kurt Black, Kevin Graham, Anja Henn, Matthias Friedrich, Patrick Hoffmann, Roman Kischel, Peter Kufer, Ralf Lutterbuese, Markus Muenz, Tobias Raum, Benno Rattel, Karen Rex, Dan Rock, Oliver Thomas, Joachim Wahl, Andreas Wolf, Angela Coxon. Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2017-55

BiTE antibodies are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They are capable of temporarily mounting a polyclonal T cell response that is not restricted by T cell receptor specificity, presence of MHC class I, generation and presentation of peptide antigen, or the addition of T cell co-stimuli. Examples are the CD19/CD3-bispecific BiTE antibody blinatumomab, which has been reported to show high response rates in leukemia and lymphoma patients, and EpCAM/CD3-bispecific BiTE antibody solitomab (AMG 110), which is in a dose-escalating phase 1 study in patients with solid tumors. Because specific T cell responses against cancer cells are frequently hampered by a variety of immune escape mechanisms, we have here assessed to what extent surface expression of PD-L1 (B7-H1) and CD73, cytoplasmic expression of IDO and serpin PI-9, and secretion of TGF-ß and IL-10 can protect target cells from redirected lysis by AMG 110-engaged T cells. The six human proteins, which are all known to be associated with immune escape of cancer cells by inhibition of T cell function, were stably expressed in human EpCAM-expressing Chinese hamster ovary (CHO) cells. In all cases, expression levels of the six proteins in stable CHO cell lines exceeded those found in human cancer cell lines. Characterized CHO cell lines and the parental EpCAM+ CHO line were used in co-culture assays as target cells for analysis of EC50 values for redirected lysis, and induction of proliferation of resting human peripheral T cells in the presence of various AMG 110 concentrations. High level cytosolic expression of the tryptophan metabolizing enzyme IDO had no effect on redirected lysis but reduced AMG 110-induced T cell proliferation, while expression of the granzyme inhibitor PI-9 increased the EC50 value for redirected lysis but had no effect on T cell proliferation. High level expression of the membrane-bound ligand for PD-1, PD-L1, and of secreted IL-10 and TGF-β all had no impact on AMG 110-induced T cell proliferation. The EC50 value for redirected lysis was however impacted by expression of TGF-ß and PD-L1 as determined by a FACS-based cytotoxicity assays. In no case, transfected CHO cell lines became completely resistant to BiTE-induced lysis despite expressing the immune escape proteins at higher levels than observed in tumor cell lines. Our data suggest that BiTE-engaged T cells are relatively insensitive to proteins expressed by cancer cells to fend off T cells, and that inhibitory effects can be compensated by higher concentrations of the BiTE antibody. Citation Format: Wibke Deisting, Tobias Raum, Peter Kufer, Patrick A. Baeuerle, Markus Muenz. Impact of various immune escape mechanisms on redirected lysis by T cells engaged via EpCAM/CD3 bispecific BiTE® antibody AMG 110 . [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 4734. doi:10.1158/1538-7445.AM2013-4734