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Purpose: Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of beta cells, resulting in a loss of insulin production. Patients with T1D carry a substantial disease burden as well as substantial short-term and long-term risks associated with inadequate glycemic control. Currently, treatment mainly consists of insulin, which only treats the symptoms of T1D and not the root cause. Thus, disease-modifying agents such as anti-CD3 monoclonal antibodies (mAbs) that target the autoimmune destruction of beta cells in T1D would provide significant relief and health benefits for patients with T1D. This review summarizes the clinical evidence regarding the safety and efficacy of anti-CD3 mAbs in the prevention and treatment of T1D., Summary: A total of 27 studies reporting or evaluating data from clinical trials involving otelixizumab and teplizumab were included in the review. Anti-CD3 mAbs have shown significant benefits in both patients at high risk for T1D and those with recent-onset T1D. In high-risk populations, anti-CD3 mAbs delayed time to diagnosis, preserved C-peptide levels, and improved metabolic parameters. In recent-onset T1D, anti-CD3 mAbs preserved C-peptide levels and reduced insulin needs for extended periods. Anti-CD3 mAb therapy appears to be safe, with primarily transient and self-limiting adverse effects and no negative long-term effects., Conclusion: Anti-CD3 mAbs are promising disease-modifying treatments for T1D. Their role in T1D may introduce short-term and long-term benefits with the potential to mitigate the significant disease burden; however, more evidence is required for an accurate assessment., (© American Society of Health-System Pharmacists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021)., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Anti-PD-1/PD-L1 immunotherapy could offer an alternative to traditional chemo- and/or radiotherapy to treat pediatric cancer patients. To unveil the potential benefit of this new therapeutic approach, the prevalence of PD-L1 and other relevant immune markers using quantitative digital image analysis (DIA) could help to clarify this point. A bridging study was first conducted using commercially available normal formalin-fixed paraffin-embedded (FFPE) tonsils to compare immunostaining patterns and intensities from PD-L1, tumor infiltrating lymphocyte (TIL) markers CD3, CD8, FoxP3, CD45RO, and macrophage marker CD68 in adult (n = 5) and pediatric (n = 10) samples. Then, commercially available pediatric FFPE tumor samples from five prevalent pediatric solid tumor indications: ganglioneuroblastoma (n = 7); neuroblastoma (n = 23); nephroblastoma (n = 30); osteosarcoma (n = 24); and rhabdomyosarcoma (n = 25) were immunostained and their images (n = 654) digitally analyzed using predefined algorithms. The qualitative analysis of staining patterns and intensities in all 15 tonsils for all 6 biomarkers was similar regardless of age category. Quantitative DIA showed that PD-L1 values varied across cancer-types, nephroblastoma having the lowest counts. PD-L1 counts in ganglioneuroblastoma, our pediatric indication with the highest average value, was approximately 12-times lower than in a similar nonsmall cell lung cancer study, an indication approved for anti-PD-1/PD-L1 immunotherapies. Variable values were measured for the TIL markers CD3, CD8, and CD45RO. FoxP3 was scant across all indications. The macrophage marker CD68 showed highest values in ganglioneuroblastoma, with lowest levels in nephroblastoma. In conclusion, the low PD-L1 levels uncorrelated with TIL values from the present biomarker morphological study suggest that a PD-L1 immunohistochemistry patient selection strategy used for anti-PD-1/PD-L1 monotherapy in adult tumors may not succeed in these pediatric indications., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

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.

Type 1 diabetes (T1D) is a cell-mediated autoimmune disease. New cases of T1D are on the increase and exogenous insulin therapy is the only intervention regularly initiated for T1D patients. Though tremendous strides have been made in prediction of T1D, prevention and intervention strategies have not experienced the same success. In this review, we will discuss some possible reasons why new intervention therapies for T1D have not been implemented into the mainstream treatment regimen for T1D patients. We will also discuss potential caveats for why prevention and intervention trials in T1D may not have experienced the same success as prediction trials., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Background Aims: Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose., Methods: We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia., Results: Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24-48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10(8), n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia., Conclusions: The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.

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.

T-cell depletion strategies are an efficient therapy for the treatment of acute rejection after organ transplantation and have been successfully used as induction regimens. Although eliminating whole T cells blocks alloreactivity, this therapy challenges the development of regulatory mechanisms because it depletes regulatory cells and modifies the profile of T cells after homeostatic repopulation. Targeting T-cell subpopulations or selectively activated T cells, without modifying Treg cells, could constitute a pro-tolerogenic approach. However, the perfect molecular target that would be totally specific probably still needs to be identified. In this study, we have reviewed the biological activities of broad or specific T-cell depletion strategies as these contribute to the induction of regulatory cells and tolerance in organ transplantation.

One of the approaches to make anti-CD20 antibody more efficient is to express this antibody on the surface of T cells. scFv from anti-CD20 antibody has been expressed on T cell surface to bind to CD20 positive cells and CD3zeta has been expressed as a fusion partner to transduct signals. T cells grafted with this chimeric scFv/CD3zeta were able to redirect grafted T cells to an MHC/Ag-independent antitumor response. To test the effects of CD28 signal on the cellular activation and antitumor effectiveness of chimeric scFv/CD3zeta modified T cells, we constructed a recombinant anti-CD20 scFv/CD28/CD3zeta gene in a retroviral vector. T cells expressing anti-CD20 scFv/CD28/CD3zeta specifically lysed CD20 positive target tumor cells and secreted not only IFN-gamma but also IL-2 after binding to their target cells. Our data indicate that CD3 and CD28 signalling can be delivered in one molecule, which is sufficient for complete T cell activation without exogenous B7/CD28 costimulation.

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.

Haploidentical transplantation is a feasible alternative for patients with life-threatening hematologic diseases who lack a matched donor. Factors affecting the clinical outcomes of haploidentical transplantation remain under investigation. We analyzed 157 consecutive patients with leukemia who underwent transplantation with nonmanipulated granulocyte colony-stimulating factor (G-CSF)-mobilized marrow and peripheral blood cells (G-BMPBs) from haploidentical donors after receiving myeloablative chemotherapy (Ara-C + BuCy + antithymocyte globulin). Follow up observations after transplantation were made from 48 days to 1191 days (median, 448 days). Multivariate analysis indicated that the cohort given higher doses of CD3(+) cells (> or = 177 x 10(6) /kg) in allograft transplantation had a significantly lower treatment-related mortality (TRM) (relative risk [RR] = 0.35; 95% CI = 0.16-0.77; P = .0090), better leukemia-free survival (LFS) (RR = 0.46; 95% CI = 0.26-0.84; P = .0106), and better overall survival (OS) (RR = 0.42; 95% CI = 0.23-0.78; P = .0058). Inversely, advanced-stage disease was a strong predictor of greater posttransplantation relapse (RR = 3.48; 95% CI = 1.26- 9.60; P = .0159), worse LFS (RR = 2.56; 95% CI = 1.33-4.95; P = .0050), and worse OS (RR = 2.77; 95% CI = 1.39-5.53; P = .0038). A high number of CD3(+) cells (> 177 x 10(6)/kg) given to patients resulted in statistically less TRM and more intensive graft versus leukemia effect without producing more severe grades of GVHD, all resulting in a significantly better overall clinical outcome from haploidentical transplantation.

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)

Background: Pancreatic islet transplantation (PIT) is an attractive alternative for patients with type I diabetes mellitus. PIT is not yet an effective clinical reality due in part to the high incidence of rejection and early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. In the present study, the effects of PIT on glycemic parameters were assessed in spontaneously diabetic primates., Methods: Five insulinopenic nonhuman primates (three Macacca fascicularis, one Ceropithecus aethiops, and one Macacca mulatta) were studied. All required twice-daily treatment with 4-10 U of insulin. For immunosuppression, the animals received anti-CD3-immunotoxin (100 microg/kg(initially infused 2 hr before transplantation and again on day +1), cyclosporine (CsA) (20 mg/kg(i.v./2 hr before transplantation), cyclosporine microemulsion (Neoral) 60 mg/kg/b.i.d. on days +1 to +3 with dose adjusted by blood levels, and methylprednisolone (15 mg/kg day 0 to +3). Three recipients were given islets from a single donor (M mulatta). The islets were prepared by a semiautomated technique using Liberase. A mean of 13,136 islet equivalents/kg was infused into the portal vein. Two animals (M fascicularis and M mulatta) were used as a diabetic, nontransplanted control. Several metabolic parameters were evaluated., Results: All monkeys that underwent transplantation experienced reversal of diabetes mellitus with normalization of all diabetic glycemic parameters. In the nontransplanted primates given the same immunosuppression but no PIT, diabetic metabolic parameters were unchanged after 9 months of follow-up. In contrast, all three PIT recipients established fasting and nonfasting euglycemia within 1-2 weeks, and none required exogenous insulin after day 10. Normal intravenous glucose tolerance tests were observed at day 15, and no significant differences in the glucose disappearance rate (Kg) were observed at days 15, 45, 190, and 365 days after transplantation. The acute insulin response to glucose indicated no significant reduction of functional islet mass., Conclusions: PIT in severely insulinopenic type I diabetes mellitus primates resulted in restoration of normal glycemic parameters and durable islet mass. Operational tolerance was achieved with only 4 days of drug administration, sparing the animals from chronic exposure to potentially diabetogenic immunosuppressive drugs. These results offer an exciting new potential for type I diabetes mellitus treatment.

The antitumor effect of PSK was analysed with the "double grafted tumor system" in which BALB/c mice received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with PSK in the right tumor on day 3. PSK inhibited the growth of not only the right but also the left, non-treated tumor. Immunized spleen cells were taken from mice which had been cured by intratumoral administration of 5 mg of PSK. On day 3, one hour after intravenous injection of cyclophosphamide, immunized spleen cells (2 x 10(7) cells/mouse) were injected into the Meth-A tumor. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. However, the intravenous administration of spleen cells showed no antitumor effect. Expansion of peripheral blood lymphocytes was stimulated with immobilized anti CD3 antibody plus IL-2 for use in adoptive immunotherapy. gamma delta T cells proliferated in response to immobilized anti CD3. About 5 x 10(9) BRM-activated killer (BAK) cells were treated in cancer patients who gave their informed consent. One patient with colon cancer and metastatic cancer of the liver was treated with BAK cells by transcatheter arterial infusion without side effect. During the course of BAK treatment, serum IAP CIAE (crossed immunoaffino electrophoresis) pattern of patient changed tumor IAP pattern to normal IAP pattern. Two patients with malignant tumor in maxillary sinus were treated with BAK cells and OK-432 intratumorally. BAK treatment induced more infiltration of T cells, M phi and granulocytes in the tumor than OK-432 treatment alone and showed an antitumor effect with extensive necrosis.

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.