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Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.

The use of a bispecific antibody (BsAb) is a promising and highly specific approach to cancer therapy. In the present study, a fully human recombinant single chain variable fragment BsAb against human epidermal growth factor receptor (HER)2 and cluster of differentiation (CD)3 was constructed with the aim of developing an effective treatment for breast cancer. HER2/CD3 BsAb was expressed in Chinese hamster ovary cells and purified via nickel column chromatography. Flow cytometry revealed that the HER2/CD3 BsAb was able to specifically bind to HER2 and CD3‑positive cells. HER2/CD3 BsAb was able to stimulate T-cell activation and induce the lysis of cultured SKBR‑3 and BT474 cells in the presence of unstimulated T lymphocytes. HER2/CD3 BsAb efficiently inhibited the growth of breast cancer tissue by activating and inducing the proliferation of tumor tissue infiltrating lymphocytes. Therefore, HER2/CD3 BsAb is a potent tool which may be a suitable candidate for the treatment of breast cancer.

Aim: Although prostate cancer is one of the most commonly diagnosed malignancies in men, there is no effective curative therapy for the advanced disease. Therefore, the aim of the present study was to generate prostate-specific membrane antigen (PSMA)×CD3 diabodies as a novel treatment option for this tumor., Methods: A PSMA×CD3 diabody and a covalently linked single-chain diabody were constructed from the anti-PSMA single-chain Fv fragment D7 and an anti-CD3 single-chain Fv fragment. The fusion proteins were periplasmatically expressed in Escherichia coli. The binding properties were tested on PSMA-expressing C4-2 prostate cancer cells and CD3(+) Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability assay was used. T-cell activation was determined by flow cytometry. In vivo activity of the diabody was tested in SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts., Results: Bacterial expression levels were significantly higher for the diabody (1-1.5 mg/l culture) compared with the single-chain diabody (0.2-0.4 mg/l culture). Specific binding on CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown with both diabody formats. In vitro, both diabodies proved to be potent agents for retargeting human CD4(+) and CD8(+) lymphocytes to lyse C4-2 prostate cancer cells. The formation of conjugates between T cells and target cells with clustering of the diabody at sites of interaction could be shown. SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts with the diabody showed an efficient inhibition of tumor growth., Conclusion: Both diabody formats showed a highly efficient and specific T cell-mediated killing of prostate cancer cells and are encouraging for further development in preclinical and clinical studies.

Non-Fc receptor binding anti-CD3 antibodies are in clinical development for the treatment of autoimmune diseases. Results from phase 1/2 clinical trials suggest that teplizumab and otelixizumab preserve residual beta-cell function in patients with recent onset type 1 diabetes. Similarly, encouraging results from phase 1/2 clinical trials have been reported for visilizumab and foralumab in patients with inflammatory bowel disease. However, these CD3-directed therapies have recently suffered setbacks due to the reported inefficacy results observed during phase 2/3 clinical trials due to low dosages or inappropriate clinical endpoints. Due to adverse events observed in the phase 1/2 pilot trials, the dose of anti-CD3 antibodies was reduced in the phase 2/3 confirmatory trials. Thus, these studies reveal a narrow therapeutic window of anti-CD3-based therapies in which low doses are ineffective and higher pharmacologically active doses cause intolerable levels of adverse effects. Combining anti-CD3 antibodies with other drugs may be the most effective way to reduce toxicity while allowing significant therapeutic benefit. Indeed, monotherapy also has its limits from the perspective of targeting only a single arm of the immune process. Notably, several recent experimental studies show potent synergy between anti-CD3 antibodies and various therapeutic modalities for the treatment of autoimmune diseases. In this review we present a review of preclinical studies evaluating combination therapies using anti-CD3 antibodies for the treatment of autoimmune diseases.

The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the 'cloaked' conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients.

The construction and in vitro testing of a photo-activatable anti-tumour immuno-regulatory antibody is described. In this 'cloaked' folated anti-CD3 antibody conjugate, the folate portion of the conjugate is free to bind to folate receptor expressing cancer cells, whilst the anti-CD3 activity is effectively rendered inert by a coating of photo-labile 2-nitrobenzyl groups. On irradiation with UV-A light the activity of the anti-CD3 antibody is restored, not only when it is required, but more importantly, only where it is required. The conjugate can then attract killer T-cells to the surface of the tumour cells and kill them. Unirradiated normal tissues, to which the conjugate has been targeted by specific and non-specific binding, remain unharmed. We believe that these 'photo-switchable' conjugates could be used to markedly improve the targeting of the immune response to folate receptor (FR) expressing ovarian and breast cancers whilst minimising the side effects in the rest of the body.

We describe the first clinical application of T-cell-recruiting bispecific antibodies directly into the tumor without the need to preactivate the effector cells. In a Phase I clinical trial, 10 patients with low-grade B-cell lymphoma were treated by a single locoregional injection of CD3xCD19 bispecific antibodies. Costimulatory signaling, which is required for the optimal activation of resting T cells, was provided by the simultaneous administration of CD28 antibodies. Equal amounts of both antibodies were injected together at 4 different dose levels (30 microg: 3 patients; 270 microg: 3 patients; 810 microg: 3 patients; 1,600 microg: 1 patient). The injection was well tolerated with mild to moderate adverse effects (2/10 patients) consisting of erythema and fever at the third dose level. The maximum tolerated dose was not reached at 810 microg of injected antibodies. Three patients showed a serum peak of TNFalpha on day 2 or 3 after the antibody application, reflecting rather an activation of CD4-positive T cells than an FcR-mediated effect. Five patients developed anti-mouse antibodies after injection of the murine immunoglobulins. Nine patients were evaluable for restaging examinations 6 weeks after the antibody application, with 2 of them (22%) showing a local clinical response. We found that a single locoregional injection of CD3xCD19+CD28 antibodies is feasible up to a dose of at least 1,600 microg of each antibody. However, the development of human anti-mouse antibodies points toward the requirement for new formats of bispecific proteins with reduced immunogenicity., (Copyright 2001 Wiley-Liss, Inc.)

Ten patients with advanced B-cell lymphoma were treated with a single locoregional injection of CD3xCD19 bispecific and costimulating CD28 monospecific antibodies to activate tumor-infiltrating T-lymphocytes. Antibodies were administered at 4 different dose levels (30 microg, 270 microg, 810 microg, 1,600 microg of each antibody) either by intratumoral or intralymphatic injection. Most patients developed responses within different compartments of the immune systems (T cells, NK cells) subsequent to the antibody application. Comparative studies in 2 patients of which treated as well as untreated lymph nodes were available revealed the up-regulation of T-cell activation markers induced by the antibody injection. Additionally, in 1 patient the induction of apoptosis of lymphoma B cells in the antibody-treated lymph node was observed. Specificity analyses of peripheral blood T cells by means of IFN-gamma ELISpot measurement indicated the recruitment of idiotype-specific T cells, as in 1 out of 3 investigated patients an increased T-cell response toward autologous idiotype peptides could be demonstrated. We conclude that a single injection of CD3xCD19 bispecific antibodies is capable to induce an activation of autologous T lymphocytes if simultaneous costimulatory signaling by CD28 antibodies is provided. Furthermore, our data suggest that at least in some patients lymphoma-specific T cells can be recruited by this immunotherapeutic approach toward B-cell lymphoma., (Copyright 2001 Wiley-Liss, Inc.)

Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan-B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 x CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 x CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma. (Blood. 2000;95:2098-2103)

With the aim of developing an effective immunotherapy for malignant glioma, glioma cells were incubated with tumor necrosis factor-alpha (TNF-alpha) to increase their susceptibility to lysis by lymphokine-activated killer (LAK) cells. Treatment with exogenous TNF-alpha induced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells. In addition, the cytolytic activity of LAK cells toward exogenous TNF-alpha treated glioma cells was significantly greater than LAK cell activity toward untreated glioma cells. This increase in cytolytic activity was blocked by anti-ICAM-1 monoclonal antibodies (MAb). Furthermore, co-treatment with a bifunctional antibody (BFA) composed of anti-CD3 (UCHT1) and antiglioma (G-22) antibodies synergistically increased the cytolytic activity of LAK cells towards TNF-alpha-treated glioma cells. These results indicate that a combination of exogenous TNF-alpha and anti-CD3/antiglioma BFA may provide an effective modified adoptive immunotherapy for patients with malignant glioma.

Peripheral blood lymphocytes separated from about 20ml of blood were cultured with immobilized anti-CD3 antibody and IL-2. Total T lymphocytes in the culture expanded about 2,000 times by 2 weeks culture. Expanded T lymphocytes were infused to cancer patients, and safety and feasibility was confirmed. This method will be applicable for prevention of cancer recurrence.

Cytokine release and clinical side effects resulting from the use of OKT3 and BMA 031 monoclonal antibodies in the treatment of kidney graft recipients were evaluated and compared. The rise observed in serum levels of interferon gamma. TNF alpha, and IL-8 was similar after administration of either monoclonal antibody. Furthermore, both OKT3 and BMA 031 resulted in rapid disappearance not only of virtually all T cells, but also of substantial percentages of all major leukocyte populations from the circulation; this effect is probably due to cytokine release activating endothelial cells and thereby causing extravasation even of leukocytes not specifically recognized by the administered antibodies. Evidence has thus been obtained that BMA 031 is as potent as OKT3 in inducing unequivocal signs of T cell activation in vivo. However, while OKT3 therapy was accompanied by adverse side effects in our study as in previous ones, we saw no such reactions in any of the patients receiving BMA 031. This contrast might be due to different mechanisms of leukocyte activation possibly inducing other mediators in the case of OKT3, which then, in combination with the cytokines, could generate treatment-associated morbidity.