Cytotoxic Monoclonal Antibody Research Articles

Beneficial or detrimental activity of regulatory T cells, indoleamine 2,3-dioxygenase, and heme oxygenase-1 in the lungs is influenced by the level of virulence of Mycobacterium tuberculosis strain infection.

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.

ВОЗМОЖНОСТИ ПРИМЕНЕНИЯ АНТИ-CD20 ИММУНОТЕРАПИИ ДЛЯ ЛЕЧЕНИЯ САХАРНОГО ДИАБЕТА 1 ТИПА У ДЕТЕЙ И ПОДРОСТКОВ (АНАЛИТИЧЕСКИЙ ОБЗОР)

Type 1 diabetes mellitus (DM1) is a world- and widespread disease in children and adolescents caused by the destruction of insulin-producing β-cells in the pancreas. As of today there is only one generally accepted approach to treating DM1, which is the insulin therapy, which in fact is a symptomatic therapy since it is aimed at correcting the outcome of the DM1 pathogenesis through compensation of insulin deficiency and elimination of hyperglycemia. The insulin therapy makes it possible to maintain the acceptable blood glucose levels for decades, but does not prevent the development of severe chronic complications of DM1, which in its turn are the main causes for disability cases and early deaths in such patients. Practitioners and scientists in diabetology are therefore actively searching for the ways to treating DM1 etiologically and pathogenetically. Among such methods there are various promising variants of immunotherapy aimed at prevention or suppression of an autoimmune reaction against β-cells. This review provides the basic information on DM1 and considers the modern approaches to its immunotherapy. The particular attention is paid to immunotherapy using cytotoxic monoclonal antibodies against B-lymphocytes autoreactive towards β-cells.

Immunogenicity of the vaccine against Sars-Cov-2 in cancer patients under active treatment.

e13632 Background: Coranavirus disease 2019 (COVID-19) has affected more than 220 millon people worldwide. The establishment of safe and effective vaccine coverage is crucial in these patients. We aim to investigate the safety, adverse effects and immune response (cellular and humoral) in cancer patients under active treatment and risk criteria. Methods: 56 samples from cancer patients under active treatment for cancer were collected before and after vaccination Demographic, clinical and biochemical data were collected and a post-vaccination symptomatology questionnaire was performed. For the determination of anti-SARS-Cov-2 antibodies (Immunoglobulin G and immunoglobulin M) we used the ELISA technique (LIASON SARS-CoV-2 S1/S2 IgG test and LIASON SARS-CoV-2 IgM test, DiaSorin, Saluggia, Italy). A study of the lymphocyte population was performed by flow cytometry. Results: We enrolled 52 patients with cancer who received mRNA vaccines (mRNA-1273 and BNT162b2), 2 patients with AZD1222vaccine and 2 with Ad26.COV2.S vaccine. All patients were SARS-COV-2 naive as determined by a negative anti-SARS-COVID-2 IgG test baseline. The median follow-up time was 50 days after receip of a second vaccine dosis. All subject received anti- cancer therapy. The most common anti-cancer treatment received by this cohort of patients was cytotoxic chemoterapy (44.6%), immunotherapy (25%), and monoclonal antibody therapy (14.3%). Overall, a high rate of seroconvertion(anti–IgG) (94,5%) was observed in our cohort, 1 patients with chemotherapy (1,8%) and 2 patients with immunotherapy (3,6%) were negatives. No significant differences in antibody titer were observed according to therapy. Thirty percent presented an antibody titer lower than 1000 U/ml, 27 (48%) patients developed an anti-SARS-CoV2 titer between 1000-4000 U/ml, and only 9 (16%) subjects presented a titer higher than 4000 U/ml. Studies of the lymphocyte profile of vaccinated patients showed no significant changes in the subtypes, except for peripheral memory CD3+ CD8+ lymphocytes which were significantly increased (p = 0.0001) after the second dose of anti-SARS-CoV-2 vaccine. Interestingly, cell apoptosis was significantly reduced in almost all T lymphocyte subtypes studied in vaccinated patients. Finally, analysis of blood cells showed a statistically significant increase (p = 0.0402) of eosinophils in vaccinated patients compared to baseline data. Conclusions: A personalized approach to vaccination can be proposed to cancer patients, especially depending on the type of tumor and the specific oncological treatments received. We are currently recruiting patients (n = 150) who have received the third dose of vaccine and plan to follow up at 6 months for humoral and cellular immune response. Final results will be reported at the ASCO meeting.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1636.

“In Vivo Vaccination” Effect in Clinical Responders to Anti-Myeloma Monoclonal Antibody Isatuximab

Background: Isatuximab is a monoclonal antibody directed against surface molecule CD38, which is expressed on the tumor cells of patients with Multiple Myeloma (MM). In clinical trials, isatuximab was well tolerated and demonstrated good efficacy as monotherapy and in combination with standards of care. Isatuximab targets a unique epitope and exhibits anti-myeloma activity primarily through innate immunity via antibody-dependent cellular cytotoxicity (ADCC). It has been proposed that cytotoxic monoclonal antibodies can also induce an adaptive immune response against the antibody's respective target antigen as well as other tumor-associated antigens in the host, a so-called “in vivo vaccination” effect, but this has never been demonstrated in patients with MM undergoing treatment.Methods: Immunomonitoring was performed on 4 patients with relapsed / refractory MM, who had received 3-5 (median: 3) prior therapies, enrolled in a Phase I clinical study (NCT02514668) at the Huntsman Cancer Institute. The patients received 20 mg/kg intravenous single-agent isatuximab weekly for the first cycle (4 weeks) and every 2 weeks for all subsequent cycles (4 weeks) until progression or intolerable toxicity. Blood samples were collected before treatment and at weeks 2, 4, 8, 12 and/or end-of-treatment under isatuximab therapy. Additional samples from clinical responders were obtained monthly. Patients' serum samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) for the presence of autologous antibody responses against a total of 45 myeloma-associated antigens. In addition, we determined the number of peripheral blood T cells specific for CD38, the target for isatuximab, as well as for other myeloma-associated antigens using an Enzyme-Linked ImmunoSpot (ELISPOT) assay.Results: We observed clinical responses in 2 out of 4 patients consecutively enrolled in our immunologic analyses. One of these patients (840002003/ID210), a quadruple refractory patient who had received 5 lines of prior therapy, achieved a complete response (CR) and the other patient (840002002/ID246) who was double refractory after 3 lines of prior therapy achieved a stringent complete remission (sCR, Fig. 1). These two patients with an objective clinical response had new or increasing autologous IgG antibody responses against tumor antigens: one patient (ID210) against MAGE-C2, NY-ESO-1, and against the target for isatuximab CD38; the second patient (ID246) against MAGE-C1 (Fig. 1). Both antibody-positive patients had also developed polyclonal T cell responses against CD38. In addition, one of the clinical responders had T cell responses against 2 other myeloma-associated antigens, MAGE-C2 and PRAME (Fig. 1). We did not observe any new or increasing autologous antibody responses against myeloma-associated antigens in the 2 patients who had not clinically responded to isatuximab.Conclusions: Here, we demonstrate for the first time that treatment with a monoclonal antibody directed against a tumor-associated surface antigen, such as CD38, can lead to an “in vivo vaccination” effect where patients with MM develop autologous T cell responses against the therapeutic target as well as antigen spreading to tumor-associated antigens independent of the target for the monoclonal antibody. Among the limited number of patients analyzed, these autologous adaptive immune responses were observed only in patients with clinical responses to isatuximab treatment.This work was supported by Sanofi. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

COVID-19 related outcomes for hospitalized patients (pts) with solid malignancies (SM).

e18626 Background: Pts with malignancies are at risk for developing severe complications of COVID-19 with high mortality rate. We retrospectively analyzed COVID-19 related outcomes for hospitalized pts with SMs. Methods: We collected data on hospitalized pts with SMs and COVID-19 from 3/1/20 to Jan 1/1/21. Diagnosis COVID-19 was confirmed by RT-PCR of nasopharyngeal swabs. We assessed the association between the 30-day mortality and potential prognostic variables such as tumor types, cancer status, timing of treatment, types of anticancer therapy using logistic regression analyses. Results: A total of 246 hospitalized pts with SMs had COVID-19. Median age was 70 years, 87 (35%) were ≥75 years, 151 (61%) were female. The most common SMs were breast (56 [23%]), non-small cell lung (44 [18%]) and colon (31 [13%]). 154 (63%) pts were on active anticancer therapies. Of those 88 (35.5%) received treatment within 2 weeks, 16 (16.5%) within 4-12 weeks, 99 (39.9%) >3 months prior to COVID-19 diagnosis. 101 (65%) pts received cytotoxic chemotherapies, 26 (16.8%) received immune check point inhibitors (ICIs), 17 (11%) received targeted agents such as anti-EGFR therapy and 11 (7.1%) received monoclonal antibodies. Overall 30-day mortality was 42%, however, all pts with melanoma (7/7) died. The 30-day mortalities for pts who received anticancer treatments within 4 weeks, 4-12 weeks and >12 weeks of COVID-19 diagnosis were 47%, 50%, and 37%. For pts who never received treatment, 30-day mortality was 31%. For pts were in remission, stable disease and progressive disease, the 30-day mortalities were 32%, 35% and 62%. The 30-day mortalities for pts who received cytotoxic therapy, monoclonal antibodies, targeted therapies and ICIs, were 38%, 46%, 41% and 69%. Logistic regression analysis showed that pts who were >80 years of age (OR 3.6, 95% CI 1.6-8.1), had progressive disease (OR 3.4, 95% CI 1.8-6.5) or treated with ICIs (OR 3.6, 95% CI 1.5-8.7) were associated with higher 30-day mortality. Conclusions: COVID-19 associated 30-day mortality is high for hospitalized pts with SMs. Early surveillance of clinical deterioration could be helpful for hospitalized SMs pts with risk factors identified here. Further studies are needed to discern the observed association between ICIs use and worse COVID-19 outcome.[Table: see text]

Perioperative MVT-5873, a fully human monoclonal antibody against a CA 19-9 epitope, for operable CA 19-9 producing pancreatic cancers, cholangiocarcinomas, and metastatic colorectal cancers.

TPS4655 Background: Operable hepatopancreatobiliary (HPB) cancers continue to pose significant challenges. Radical resections are rarely curative, and chemotherapy is able to reduce tumor recurrence for only a fraction of patients. Despite the obvious advantages of extirpation of the identifiable tumor(s), the inflammatory milieu that accompanies surgery and the obligate time off cytotoxic agents allows for activation of remote quiescent disseminated tumor cells, leading to metastatic recurrence. We are conducting a study to determine the safety and efficacy of immediate peri-operative MVT-5873, a cytotoxic monoclonal antibody targeting Carbohydrate Antigen 19-9 (CA 19-9), in patients undergoing resections pancreatic cancer, cholangiocarcinoma or metastatic colorectal cancer to the liver. MVT-5873 is a human IgG1 antibody isolated from a patient following immunization with a sLea-KLH vaccine. MVT-5873 has demonstrated cell surface binding in sLea positive human tumor lines and has been shown to be potent in complement-dependent cytotoxicity assays and antibody-dependent cell mediated cytotoxicity assays. In patients with CA 19-9-producing cancers, MVT-5873 treatment has been shown to decrease serum CA 19-9 levels and prevent tumor progression. This trial may open the door for investigation of additional and/or synergistic agents in the immediate peri-operative period and usher in a new paradigm in the management of surgically treated cancers. Methods: This is a prospective, Phase II trial designed to determine the efficacy (increase in 1-yr DFS) and safety of peri-operative MVT-5873 for subjects with operable pancreatic, liver and bile duct cancers with elevated CA 19-9 levels. Patients may receive any standard neoadjuvant regimen prior to enrollment at the NIH Clinical Center in Bethesda, Maryland. Eligible patients will receive a pre-operative dose of MVT-5873 three days prior to the planned operation to remove all demonstrable disease. Following the operation, patients will receive a total of four doses of MVT-5873; the first two doses on postoperative days four and ten. The third dose will be administered on the normally scheduled postoperative clinic visit, followed by a final dose one month after discharge from the hospital and prior to the start of adjuvant treatment. Clinical trial information: NCT03801915 .

Targeting CA 19-9 with a humanized monoclonal antibody at the time of surgery may decrease recurrence rates for patients undergoing resections for pancreatic cancer, cholangiocarcinoma and metastatic colorectal cancer.

https://clinicaltrials.gov/ct2/show/NCT03801915?term=MVT&rank=3.

Down Regulation of MAPK Signaling by Cytotoxic Human Monoclonal Antibody in Epithelial Ovarian Cancer

Down Regulation of MAPK Signaling by Cytotoxic Human Monoclonal Antibody in Epithelial Ovarian Cancer

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy.

Modern immunotherapy of adult B-lineage acute lymphoblastic leukemia with monoclonal antibodies and chimeric antigen receptor modified T cells.

The introduction of newer cytotoxic monoclonal antibodies and chimeric antigen receptor modified T cells is opening a new age in the management of B-lineage adult acute lymphoblastic leukemia. This therapeutic change must be very positively acknowledged because of the limits of intensive chemotherapy programs and allogeneic stem cell transplantation. In fact, with these traditional therapeutic tools the cure can be achieved in only 40–50% of the patients. The failure rates are particularly high in the elderly, in patients with post-induction persistence of minimal residual disease and especially in refractory/relapsed disease. The place of the novel immunotherapeutics in improving the outcome of adult patients with B-lineage acute lymphoblastic leukemia is reviewed.

Acute Infusional Reactions to Chemotherapy in an Outpatient Unit: Experience from 21,394 Treatment Cycles in Brazil

ABSTRACT Aim: Acute infusional reactions (AIRs) to cancer systemic treatments can occur in varying degrees of frequency and severity, according to the administered drug. We aim to study the frequency and to characterize AIRs in a large Chemotherapy Unit in Sao Paulo, Brazil. Methods: We evaluated and prospectively followed AIRs in patients (pts) with solid tumors treated with intravenous cytotoxic chemotherapies and/or monoclonal antibodies in our Cancer Center from May 15th to October 15th 2013. These therapies were administered following standard guidelines regarding AIR-prophylaxis. AIR was defined as any new sign or symptom during the infusion of above mentioned agents, or any other event treatment-related, in the first 8 hours after therapy. Results: 21,394 chemotherapy infusions were adminstered, and 186 AIRs (0.86%) in 149 pts were diagnosed. Median age was 55 y.o. (24-86), 73% (110 pts) were female and 169 AIRs (91%) occured in patients ECOG-PS 0-1. Breast was the most frequent primary site (62 episodes, 33%) and in 95 episodes (51%) chemotherapy was administered in pts with metastatic disease. Among those pts who presented any AIR, median body mass index was 25.7 kg/m2, median hemoglobin was 12.1 g/dL and median lymphocyte count was 1650 cells/mm3. AIRs were associated to the following agents (>5%): Paclitaxel (39%), Docetaxel (23%), Irinotecan (7,5%) and Oxaliplatin (7,5%). 28 pts had >1 AIRs, being 20 with 2 AIRs, 7 with 3 AIRs and 1 with 4 AIRs. Common signs and symptoms included rash (55%), dyspnea (31%) and back pain (11%). There were 3 episodes of severe reactions (1.6% of all AIRs), leading to definitive treatment interruptions. No AIRs-related deaths were observed. Conclusions: Since AIRs were rare (0.86%) and severe AIRs were extremely uncommon (1.6% of all AIRs) with adequate prophylaxis, no changes in current protocols seem required. Disclosure: All authors have declared no conflicts of interest.

Antibody-drug conjugates targeting CD103-expressing cells

Objective To generate optional antibody-drug conjugates (ADCs) using three different cytotoxic agents and monoclonal antibody (M290) which can efficiently deplete CD103 + cells in vivo with reduced off-target toxicity.The ADCs will be utilized to block the CD103 pathway to determine therapeutic potential in islet allograft rejection.Methods The CD103 monoclonal antibody (M290) was conjugated with MMAF,MMAE or DMI to generate M290-MC-MMAF,M290-vc-PAB-MMAE or M290-MCC-DM1.ADCs were injected intraperitoneally respectively by dose of 2,2,6 mg/kg into C56BL/6 mice,then the depletion efficacy in vivo was evaluated by flow cytometry.The toxicity of ADCs was tested in wild mice by weight monitoring and symptoms.The mice were divided into PBS,M290,M290-SAP and ADCs groups.Results All ADCs resulted in potently depletion of CD103 + cells in vivo with high specificity and less toxicity.The number of intraepithelial lymphocytes of ADCs groups was significantly less than that in PBS group (P ＜0.01).Conclusion The three ADCs were potencial drugs that block CD103/E-cadherin pathway. Key words: Islet transplantation; Antibody-drug conjugates ; Anti-CD103

Recent Developments in Cancer Treatment: A Review

According to the latest cancer statistics presented worldwide, there has been a dramatic increase in the rates of occurrence of some cancers, particularly in the more developed countries. Although many therapeutic strategies to prevent and/or cure this disease have been proposed and evaluated by clinicians and researchers, there remains a need to find more effective approaches. Side effects such as toxicity and drug resistance are two of the most frequent problems faced during chemotherapy. Small - molecule drugs are being intensively pursued as new anticancer therapeutics. Oncology drug discovery has benefited significantly from progress in understanding how to target kinases with small molecules that were found to be correlated with the disease. One reason for this is that many kinases have been found to be intimately involved in the processes leading to tumor cell proliferation and survival. Monoclonal antibodies, that are produced in vitro, can be used in cancer treatment in a number of ways. They may enhance the immune system by reacting with certain types of cancer cells. They can be programmed to act against specific cell growth factors to interfere with the growth of cancer cells. Furthermore, they may be linked to anticancer drugs, radioactive substances, other biologic therapies, or other toxins (antibody – omicsonline.orgdrug conjugates). Finally, the usage of cytotoxic monoclonal antibodies during the process of bone marrow transplantation may be a key to improve the efficacy of the method. The objective of this review was to present some of the new cancer treatment modalities that have been developed. The advantages of each method including its safety and efficacy have been highlighted. The present study may support the improvement and development of new therapeutic approaches.

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.

Staurosporine-induced programmed cell death in Blastocystis occurs independently of caspases and cathepsins and is augmented by calpain inhibition

Previous studies have shown that the protozoan parasite Blastocystis exhibits apoptotic features with caspase-like activity upon exposure to a cytotoxic monoclonal antibody or the anti-parasitic drug metronidazole. The present study reports that staurosporine (STS), a common apoptosis inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, phosphatidylserine (PS) externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. STS-induced PS exposure and DNA fragmentation were abolished by the mitochondrial transition pore blocker cyclosporine A and significantly inhibited by the broad-range cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L, while calpain-specific inhibitors augmented the STS-induced apoptosis response. While the identities of the proteases responsible for STS-induced apoptosis warrant further investigation, these findings demonstrate that programmed cell death in Blastocystis is complex and regulated by multiple mediators.

Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia

C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models. C-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated. C-type lectin-like molecule-1 was expressed in 86.5% (45/52) of cases of acute myeloid leukemia, in 54.5% (12/22) of acute myeloid leukemia CD34(+)/CD38(-) stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemia-derived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to C-type lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. Conclusions Our results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.

CD3 immunotoxin therapy of cutaneous T cell lymphoma

e19511 Background: T cell lymphomas represent 12% of lymphoma cases in the U.S. Cytotoxic chemotherapies, radiation therapies, monoclonal antibodies, transcription modulators and topical therapies yield remissions, but over half of patients relapse and die with progressive disease. Patients ineligible for allogeneic transplant need additional therapeutic options. One class of T cell directed agents are immunotoxins composed of protein synthesis inactivating peptide toxins covalently linked to antibodies or hormone ligands. We prepared a new immunotoxin, A-dmDT390-bisFv(UCHT1) composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with an acidic loop on the extracellular domain of CD3epsilon. We prepared a clinical batch of drug and obtained FDA approval for a phase I trial (IND#100712). Methods: Cohort of three-six CTCL patients were treated with immunotoxin via 15min IV infusion twice daily for four days at 2.5–5ug/kg. CTCAEv3.0 toxicity grading, blood samples counts, chemistries, CMV/EBV PCR, PK, immune response and flow cytometry, and disease assessments by CT scans, skin mapping and bone marrow biopsies were done. Results: Six patients received all eight 2.5ug/kg doses. Toxicities were mild- moderate with fever, chills, nausea, transaminasemia, hypoalbuminemia, lymphopenia and reactivation of EBV and CMV. Side effects responded to antipyretics, anti-emetics, albumin infusions, rituximab and valgancyclovir. Lymphopenia was marked at the end of treatment (99.9% reduction) and was followed by partial recovery at two weeks. Circulating T regulatory cells doubled. Cmax occured 5 min post-infusion and was 18ng/mL. Half-life was 49 min. All patient had anti-immunotoxin antibodies at a median of 1.3ug/mL which increased after 30 days to 9–1700ug/mL. Two of five evaluable patients had PRs lasting one and 6+ months. Conclusions: This novel immunotoxin has dramatic clinical activity even at the lowest dose in CTCL patients and merits applications at higher doses in CTCL and other CD3+ T cell leukemia/lymphoma patients. The lymphodepletion with recovery of T regulatory cells suggests the drug may be beneficial for immunosuppression of T cell autoimmune disorders. No significant financial relationships to disclose.

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (Δ Ψ m), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.

Caspase-3-like protease influences but is not essential for DNA fragmentation in Blastocystis undergoing apoptosis

Blastocystis hominis undergo apoptosis after treatment with a cytotoxic monoclonal antibody (MAb), 1D5, by mechanisms that are not fully understood, although our previous study demonstrated that caspase-3-like protease activity is involved. To elucidate the mechanism of MAb 1D5-induced apoptosis, we inhibited Blastocystis caspase-3-like protease to investigate if there would be a concomitant decrease in in situ DNA fragmentation. However, MAb 1D5-induced apoptosis, evidenced by DNA fragmentation, was not completely blocked by pretreating with specific caspase-3 inhibitor, Ac-DEVD-CHO, indicating that caspase-independent apoptotic pathways might also be involved. Our results also revealed that the treatment with MAb 1D5 resulted in the loss of mitochondrial membrane potential (deltapsim), independent of Ac-DEVD-CHO pretreatment. In conclusion, this study demonstrates that MAb 1D5-induced apoptosis in B. hominis is not wholly dependent on caspase-3-like protease activity and is associated with mitochondrial dysregulation. This is the first report showing evidence for complex apoptotic pathways in a unicellular parasite.