Organ-on-a-chip models for development of cancer immunotherapies.

Cancer immunotherapy has emerged as a promising approach in the treatment of diverse cancer types. However, the development of novel immunotherapeutic agents faces persistent challenges due to poor translation from preclinical to clinical stages. To address these challenges, the integration of microfluidic models in research efforts has recently gained traction, bridging the gap between in vitro and in vivo systems. This approach enables modeling of the complex human tumor microenvironment and interrogation of cancer-immune interactions. In this review, we analyze the current and potential applications of microfluidic tumor models in cancer immunotherapy development. We will first highlight current trends in the immunooncology landscape. Subsequently, we will discuss recent examples of microfluidic models applied to investigate mechanisms of immune-cancer interactions and for developing and screening cancer immunotherapies in vitro. First steps toward their validation for predicting human in vivo outcomes are discussed. Finally, promising opportunities that microfluidic tumor models offer are highlighted considering their advantages and current limitations, and we suggest possible next steps toward their implementation and integration into the immunooncology drug development process.

The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells.

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.

Serum-free production and purification of chimeric IgA antibodies.

Natural IgA antibodies are abundantly produced in vivo to protect serosal surfaces from invading infectious organisms. However, the immunotherapeutic potential of IgA has hardly been explored, although there is evidence that recombinant IgA antibodies may broaden the armentarium to combat certain infectious or malignant diseases. One of the limitations for exploring IgA's therapeutic activity has been the difficulty to obtain enough recombinant material with desired specificity for in vivo studies. Here, we describe the production and purification of monomeric recombinant IgA1 and IgA2 antibodies under serum-free conditions. For antibody production, suspension adapted CHO-K1 cells and a glutamine synthetase selection vector were used, which resulted in specific production rates of up to 2.2 pg/cell/day. Purities of >95% of monomeric antibodies were obtained by a combination of affinity chromatography-using an anti-kappa-light chain matrix-and size exclusion chromatography. Purified antibodies displayed the expected biochemical characteristics and were functionally fully active. Importantly, all required reagents and methods are commercially available and not dependent on the specificity of the desired antibody. In addition, all employed technologies and methodologies are similar to those used for the production of therapeutic IgG antibodies - thus allowing further up-scaling and streamlining according to existing antibody production technologies. In conclusion, the described methodology may assist in the development of recombinant IgA antibodies for therapeutic applications.

Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcgammaRI x anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer.

A phase I study of the bispecific antibody MDX-H210 in combination with granulocyte colony-stimulating factor (G-CSF) was performed in stage IV breast carcinoma patients, overexpressing HER-2/neu. MDX-H210, constructed by crosslinking antigen binding fragments (F(ab') fragments) of monoclonal antibody (mAb) H22 to Fc gamma receptor I (FcgammaRI), and mAb 520C9 to HER-2/neu, respectively, mediates the lysis of tumour cells in vitro, and in human FcgammaRI transgenic mouse models. The proto-oncogene HER-2/neu is overexpressed in approximately 30% of breast cancer patients, and represents a promising target for antibody-based immunotherapy. Fc gamma receptor I (CD64) is an effective trigger molecule, which is expressed on monocytes/macrophages, immature dendritic cells, and G-CSF-primed polymorphonuclear cells (PMN). Patients received G-CSF (Filgrastim) for 8 consecutive days, and cohorts of three patients were treated on day 4 with escalating, single doses of MDX-H210. A total of 30 patients were included, and treatment was generally well tolerated, without reaching dose-limiting toxicity. Side effects consisted mainly of fever and short periods of chills, which were timely related to elevated plasma levels of interleukin 6 and tumour necrosis factor alpha. In the last two cohorts, MDX-H210 plasma levels exceeded 1 microg ml(-1), and on circulating myeloid cells >50% saturation of FcgammaRI was found until day 4. These effector cells were highly effective in antibody-dependent cell-mediated cytotoxicity. Immunohistochemical analyses of tumour biopsies in individual patients documented infiltration of monocytes and PMN after MDX-H210 infusion. Although the clinical course of the disease was not altered by the single dose of MDX-H210, a favourable toxicity profile--even at high doses--and remarkable biological effects were seen when combined with G-CSF. Therefore, the combination of G-CSF and MDX-H210 should be evaluated in further immunotherapeutical strategies.

Bispecific antibodies targeting cancer cells.

In recent years, antibody therapy has become a new treatment modality for tumour patients, although the majority of responses are only partial and not long lasting. Based on evidence that effector-cell-mediated mechanisms significantly contribute to antibody efficacy in vivo, several approaches are currently pursued to improve the interaction between Fc receptor-expressing effector cells and tumour target antigens. These approaches include application of Fc receptor-directed bispecific antibodies, which contain one specificity for a tumour-related antigen and another for a cytotoxic Fc receptor on immune effector cells. Thereby, bispecific antibodies selectively engage cytotoxic trigger molecules on killer cells, avoiding, for example, interaction with inhibitory Fc receptors. In vitro, chemically linked bispecific antibodies directed against the Fc gamma receptors Fc gamma RIII (CD16) and Fc gamma RI (CD64), and the Fc alpha receptor Fc alpha RI (CD89), were significantly more effective than conventional IgG antibodies. Recent animal studies confirmed the therapeutic potential of these constructs. However, results from clinical trials have been less promising so far and have revealed clear limitations of these molecules, such as short plasma half-lives compared with conventional antibodies. In this review, we briefly summarize the scientific background for bispecific antibodies, and describe the rationale for the generation of novel recombinant molecules. These constructs may allow us to more specifically tailor pharmacokinetic properties to the demands of clinical applications.

Epidermal growth factor receptor and g250: useful target antigens for antibody mediated cellular cytotoxicity against renal cell carcinoma?

PURPOSE: Monoclonal antibodies are a novel treatment option for certain tumor patients. We evaluated the potential of antibody derivatives against epidermal growth factor receptor and G250, which are 2 candidate antigens on renal cell carcinoma, to recruit effector cells for killing renal cell carcinoma. MATERIAL AND METHODS: As a measure of cytotoxicity, 51chromium release assays against renal cell carcinoma lines were performed using unseparated blood or isolated cell populations as the source of effectors. Blood was obtained from healthy donors, or from patients receiving granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor for enhancing effector cell function. Parental human IgG1 antibodies against epidermal growth factor receptor and G250 were compared with respective chemically linked bispecific antibodies targeting IgA Fc receptor FcalphaRI (CD89), a novel cytotoxic trigger molecule on polymorphonuclear cells and monocytes/macrophages, which were constructed by chemically crosslinking appropriate F(ab') fragments. RESULTS: Renal cell carcinoma lines were highly resistant to complement dependent lysis. With mononuclear effector cells high levels of renal cell carcinoma killing were observed with a humanized epidermal growth factor receptor directed monoclonal antibody, while the same antibody did not recruit granulocytes (polymorphonuclear cells) for antibody dependent cell mediated cytotoxicity. However, polymorphonuclear cells effectively lysed renal cell carcinoma with [FcalphaRI x epidermal growth factor receptor] bispecific antibody. FcalphaRI mediated killing was significantly enhanced when the blood of patients on granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor therapy was analyzed. However, G250 mediated only low levels of killing with mononuclear cell but not with polymorphonuclear effector cells. CONCLUSIONS: Targeting epidermal growth factor receptor proved to recruit efficiently mononuclear or polymorphonuclear cell mediated killing mechanisms, while G250 directed antibody constructs were significantly less effective. Particularly effective renal cell carcinoma killing was observed with combined [FcalphaRI x epidermal growth factor receptor] bispecific antibody and granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor.

IgA antibodies for cancer therapy.

Antibody-based therapy is a new treatment option for selected tumor patients. Today, human IgG(1) is the most widely used isotype, because it effectively activates human complement, recruits NK cells for ADCC, and has an extended plasma half life. In recent work, however, neutrophils--the most populous cytotoxic cells in humans--were more effectively recruited by human IgA than by IgG antibodies. IgA antibodies may have the additional advantages of forming natural dimers with improved signaling capacity on tumor cells, and being actively transported into mucosal secretions with the potential for improved targeting of certain carcinomas from the luminal surface.

Preclinical and clinical data with bispecific antibodies recruiting myeloid effector cells for tumor therapy.

Bispecific antibodies constitute a novel approach to improve antibody efficacy. In vitro, constructs to recruit myeloid effector cells have been extensively investigated, and first animal data in human Fc receptor transgenic mice confirmed their promising therapeutic potential. Clinical experience with these constructs demonstrated acceptable toxicity, and support therapeutic efficacy in subgroups of patients. However, limited availability, unacceptable immunogenicity, and unfavorable pharmacokinetics of bispecific compared to conventional antibodies often hampered clinical studies. As solutions to these problems are available today, bispecific antibodies hold promise to improve therapeutic efficacy of antibody-based approaches in the near future.

Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies.

Studies with gene-modified mice have recently reinforced the importance of Fc receptor-mediated effector mechanisms for the therapeutic efficacy of rituxan and herceptin - two clinically approved antibodies for the treatment of tumor patients. We investigated Fc receptor-dependent tumor cell killing by mononuclear and granulocytic effector cells - comparing human IgG1 antibodies against CD20 or HER-2/neu with their respective FcgammaRI (CD64)-, FcgammaRIII (CD16)-, or FcalphaRI (CD89)-directed bispecific derivatives. With blood from healthy donors as effector source, human IgG1 and FcgammaRIII (CD16)-directed bispecific antibodies proved most effective in recruiting mononuclear effector cells, whereas tumor cell killing by granulocytes was most potently triggered by FcalphaRI-directed bispecific constructs. Granulocyte-mediated tumor cell lysis was significantly enhanced when blood from G-CSF- or GM-CSF-treated patients was investigated. Interestingly, however, both myeloid growth factors improved effector cell recruitment by different mechanisms, which were furthermore dependent on the tumor target antigen, and on the selected cytotoxic Fc receptor.

Therapeutic efficacy of FcgammaRI/CD64-directed bispecific antibodies in B-cell lymphoma.

CD64 (FcgammaRI) receptors represent highly potent trigger molecules for activated polymorphonuclear cells (PMN) and mediate lysis of a range of tumors in the presence of appropriate monoclonal antibodies. An huCD64 transgenic mouse model designed to analyze the therapeutic activity of a panel of bispecific F(ab')(2) (BsAb) in retargeting granulocyte-colony-stimulating factor (G-CSF)-activated PMN against syngeneic B-cell lymphomas is reported. This model allows careful analysis of the individual elements of the therapeutic process. BsAb were directed against immunoglobulin-idiotype (Id), major histocompatibility class II (MHC II), or CD19 on the tumors and huCD64 on the effectors. In vitro cytotoxicity assays and in vivo tumor tracking showed that, provided effectors were activated with G-CSF, all 3 derivatives destroyed and cleared lymphoma cells, with (huCD64 x MHC II) proving by far the most cytotoxic in vitro. However, though all derivatives delivered some survival advantage, only the [huCD64 x Id] BsAb provided long-term protection to tumor-bearing animals. These results demonstrate that CD64-recruited cytotoxic effectors operate in vivo but that the (huCD64 x Id) conferred an additional anti-tumor function essential for long-term protection. T-cell depletion studies demonstrated that this extra therapeutic activity with [huCD64 x Id] was totally dependent on CD4 and CD8 T cells and that mice, once "cured" with BsAb, were resistant to tumor rechallenge. These findings indicate that CD64 is an effective trigger molecule for delivering cytokine-activated PMN against tumor in vivo and that, provided tumor targets are selected appropriately, CD64-based BsAb can establish long-term T-cell immunity.

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy.

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

Flow cytometric discrimination between viable neutrophils, apoptotic neutrophils and eosinophils by double labelling of permeabilized blood granulocytes.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is frequently used to detect apoptotic cells in tissues, cytospins and suspensions. Here we show that TUNEL staining of freshly isolated granulocytes results in non-specific positivity of a distinct population, which can be observed in the presence or absence of TdT. The morphological features of the false-positive cells examined in fluorescence microscopy suggest that the non-specifically stained cells are eosinophilic granulocytes. Granules of eosinophilic granulocytes were brightly stained by non-specific TUNEL reaction independent of TdT. This staining does not, therefore, indicate apoptosis and most likely reflects 'stickiness' of the permeabilized eosinophils. Immunofluorescence with phycoerythrin (PE)-labelled CD16 antibodies performed simultaneously with conventional TUNEL staining confirmed that the false-positive cells in TUNEL staining were CD16-negative eosinophils. In this report we describe a new procedure that allows: (i) the differentiation of neutrophilic and eosinophilic granulocytes in forward scatter versus log side scatter histograms after permeabilisation; (ii) the reliable discrimination between viable neutrophils, apoptotic neutrophils and eosinophilic granulocytes in cytofluorimetry.

Preclinical studies combining bispecific antibodies with cytokine-stimulated effector cells for immunotherapy of renal cell carcinoma.

BACKGROUND: Bispecific antibodies--consisting of a F(ab')-fragment derived from a monoclonal antibody against a tumor epitope as well as of another antibody against a cytotoxic trigger molecule on immune effector cells--can improve the effectiveness of antibody-based tumor therapy. MATERIALS AND METHODS: We used bispecific antibodies with one specifity against the EGF-receptor, which is overexpressed on the majority of renal cell carcinomas, and another specifity against Fc receptors on human leukocytes (Fc gamma RI/CD64; Fc gamma RIII/CD16 and Fc alpha RI/CD89). As source of effector cells, whole blood from patients treated with G-CSF, GM-CSF or IL2/IFN-alpha was used in 51Cr- release assays using various renal cancer cell lines as tumor targets. Further experiments with Percoll-isolated granulocytes or mononuclear cells from the same donors were performed in order to identify the active effector cell populations. RESULTS: Compared with conventional monoclonal EGF-R directed antibodies (murine IgG2a, humanized IgG1), bispecific antibodies induced significantly enhanced cytotoxicity. Highest amounts of tumor cell killing were observed using whole blood from patients treated with G-CSF or GM-CSF in combination with an [Fc alpha RI x EGF-R] bispecific antibody. Under these conditions, granulocytes constituted the most active effector cell population. CONCLUSION: The combination of myeloid growth factors and bispecific antibodies offer a promising new approach for the treatment of advanced renal cell carcinoma.

Evaluating antibodies for their capacity to induce cell-mediated lysis of malignant B cells.

Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on the antibody isotype. We then compared killing mediated by chimeric IgG1 antibodies with that from Fc gammaRI-directed bispecific antibodies, targeting classical HLA class II, or the Lym-1 and Lym-2 antigens. The latter two are variant forms of HLA class II, which are highly expressed on the surface of malignant B cells but which are found only at low levels in normal cells. With blood from G-CSF-treated donors, bispecific antibodies showed enhanced killing compared to their chimeric IgG1 derivatives, because they were more effective in recruiting Fc gammaRI-expressing PMN cells. G-CSF- and Fc gammaRI-directed bispecific antibodies to HLA class II, therefore, seem to be an attractive combination for lymphoma therapy.