Selinexor Sensitizes TRAIL-R2-Positive TNBC Cells to the Activity of TRAIL-R2xCD3 Bispecific Antibody.

Triple-negative breast cancer (TNBC) is an aggressive disease with poor prognosis and limited therapeutic options. Recent advances in the immunotherapy field have enabled the development of new treatment strategies, among which the use of bispecific antibodies (BsAbs), able to redirect T cells against tumors, has shown promising results. In particular, a BsAb that uses TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) as a target was constructed and demonstrated good results in redirecting CD3+ T cells to kill TRAIL-R2-expressing TNBC cells. In the present study, we investigated whether treatment with selinexor, a selective inhibitor of nuclear export (SINE) targeting exportin-1/chromosome maintenance protein 1 (XPO1/CRM1), could potentiate the antitumor activity of this BsAb. In combination experiments, we found that selinexor-exposed TNBC cells exhibited greater growth inhibition when treated with the TRAIL-R2xCD3 BsAb than that expected by simple additivity. Similarly, the apoptosis rate in selinexor/TRAIL-R2xCD3 BsAb-treated TNBC cells was significantly higher than that observed after exposure to either single agent. Together, our results suggest that the combination of selinexor and TRAIL-R2xCD3 BsAb can be a viable anticancer strategy and indicate this treatment as a promising therapeutic option for TNBC patients.

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

PURPOSE: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. EXPERIMENTAL DESIGN: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. RESULTS: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. CONCLUSIONS: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

Novel humanized and highly efficient bispecific antibodies mediate killing of prostate stem cell antigen-expressing tumor cells by CD8+ and CD4+ T cells.

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy.

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.

A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Molecular determinants of inverse agonist activity of biologicals targeting CTLA-4.

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma.

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id x anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id x anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.

Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells.

Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3 epsilon chain of the TCR (alphaCEA x alphaCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-alphaCEA). Together, alphaCEA x alphaCD3 and B7-alphaCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA(+) cancer cells, alphaCEA x alphaCD3 and B7-alphaCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer.

IgG4 breaking the rules.

Immunoglobulin G4 (IgG4) antibodies have been known for some time to be functionally monovalent. Recently, the structural basis for this monovalency has been elucidated: the in vivo exchange of IgG half-molecules (one H-plus one L-chain) among IgG4. This process results in bispecific antibodies that in most situations will behave as functionally monovalent antibodies. The structural basis for the abnormal behaviour of IgG4 seems to be largely the result of a single amino acid change relative to human IgG1: the change of a proline in core hinge of IgG1 to serine. This results in a marked shift in the equilibrium between interchain disulphide bridges and intrachain disulphide bridges, which for IgG4 results in 25-75% absence of a covalent interaction between the H-chains. Because of strong non-covalent interactions between the CH3 domains (and possibly also between the CH1 domain and the trans-CH2 domain) IgG4 is a stable four-chain molecule and does not easily exchange half-molecules under standard physiological conditions in vitro. We postulate that the exchange is catalysed in vivo by protein disulphide isomerase (PDI) and/or FcRn (the major histocompatibility complex (MHC)-related Fc receptor) during transit of IgG4 in the endosomal pathway in endothelial cells. Because IgG4 is predominantly expressed under conditions of chronic antigen exposure, the biological relevance of this exchange of half-molecules is that it generates antibodies that are unable to form large immune complexes and therefore have a low potential for inducing immune inflammation. In contrast to monovalent immunoglobulin fragments, these scrambled immunoglobulins have a normal half-life. The significance of the ensuing bispecificity needs further evaluation, because this will be relevant only in situations where high IgG4 responses are found to two unrelated antigens that happen to be present in the body at the same time and place. In this context the significance of IgG4 autoreactivity might have to be re-evaluated. The main function of IgG4, however, is presumably to interfere with immune inflammation induced by complement-fixing antibodies, or, in the case of helminth infection or allergy, by IgE antibodies.

An engineered bivalent single-chain antibody fragment that increases antigen binding activity.

Bivalent single chain Fv (scFv) was constructed by fusing a polypeptide extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment. The scFv protein was expressed and secreted in a recombinant Pichia pastoris system as a dimer with a C-terminal disulfide bridge, as determined by Western blot analysis under non-reducing conditions. We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer ratio than the two cysteine counterpart (scFv-2Cys). Binding activity measurements performed by means of a competitive radioimmunoassay showed that scFv-1Cys exhibited specific antigen binding activity, which was almost the same as that of the parental MAb, and approximately four- and fortyfold higher than those of the control scFv monomer and scFv-2Cys.

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.