Daudi Burkitt's Lymphoma Cells Research Articles

Sodium diethyldithiocarbamate trihydrate: an effective and selective compound for hematological malignancies.

Myeloid leukemias and lymphomas are among the most common and well-studied hematological malignancies. However, due to the aggressiveness and rapid progression of certain subtypes, treating these diseases remains a challenge. Considering the promising results of diethyldithiocarbamates in preclinical and clinical oncology trials, this study aimed to investigate the potential of sodium diethyldithiocarbamate trihydrate (DETC) as a prototype for developing new drugs to treat hematological malignancies. In silico analysis using SwissADME was conducted to evaluate the physicochemical characteristics and pharmacokinetic properties of DETC. An in vitro investigation utilizing the MTT assay assessed the cytotoxic effects of DETC on neoplastic and non-neoplastic cell lines. Selectivity was determined using a selectivity index and a hemolysis assay, while the mechanism of cell death in neoplastic cell lines was examined through flow cytometry analysis of pro-apoptotic and anti-apoptotic protein levels. The results demonstrated that the physicochemical characteristics of DETC are suitable for oral administration. Furthermore, the compound showed promising cytotoxic activity against human myeloid leukemia (K562) and Burkitt's lymphoma (Daudi) cell lines, with high selectivity for neoplastic cells over non-neoplastic cells of the bone marrow microenvironment (HS-5 cell line). Moreover, hemolysis was observed only at very high concentrations. The cytotoxicity mechanism of DETC against both neoplastic cell lines involved cell cycle arrest and the production of reactive oxygen species. In K562 cells, cell death was induced via apoptosis. Additional experiments are needed to confirm the exact mechanism of cell death in Daudi Burkitt's lymphoma cells.

Abstract 1258: Preclinical characterization of LOXO-338, a novel, oral and selective BCL2 inhibitor

Abstract BCL2 is an important therapeutic target in various hematologic malignancies. Venetoclax is the only FDA-approved BCL2 inhibitor, indicated for use in CLL/SLL and acute myeloid leukemia (AML). LOXO-338 is a novel, oral BCL2 inhibitor, designed to achieve selectivity over BCL-xL (Lin S, et al. AACR, 2019), to avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. Here, we present updated LOXO-338 preclinical characterization of its potency in biophysical and cellular assays, selectivity for BCL2 over other BCL2 family members, and efficacy in murine tumor growth models to support its advancement into human clinical trials. In homogeneous time resolved fluorescence (HTRF) assays measuring the molecular interactions of BCL2 and other BCL2 family members to BAK, LOXO-338 preferentially inhibited the BCL2/BAK interaction with an IC50 value of 4.5 nM; whereas the interactions between BCLxL/BAK, and MCL1/BAK were inhibited by LOXO-338 with IC50 values of 49.6 nM and >10,000 nM, respectively. In cellular immunoprecipitation assays, LOXO-338 showed dose-dependent inhibition of BCL2/BIM interactions in RS4;11 cells (acute lymphoblastic leukemia (ALL) cell line) at concentrations ranging from 30-3000 nM and no inhibition of MCL-1/NOXA interactions in Karpas 299 cells (anaplastic large cell lymphoma cell line) at 3000 nM, the highest concentration tested. Using high-content imaging to measure the effects of LOXO-338 on cellular proliferation we determined LOXO-338 inhibited proliferation of RS4;11 cells with an IC50 value of 2 nM, whereas the IC50 values for the BCL2 independent cell lines H146 (small cell lung cancer (SCLC) cell line), Daudi (Burkitt's lymphoma cell line), and KMS11 (multiple myeloma cell line) cells were greater than 2000 nM. Anti-proliferative effects of LOXO-338 on a panel of cancer cell lines derived from different tissues were assessed using CCK-8 assays. LOXO-338 inhibited the proliferation of cell lines derived from follicular lymphoma (FL), diffuse large B-cell lymphoma, AML, and ALL with IC50 values ranging from 7 nM to 78 nM. In murine tumor xenograft models using FL, AML, and ALL cell lines, LOXO-338 administered once daily demonstrated dose-dependent tumor growth inhibition. LOXO-338 was well-tolerated by the tumor-bearing mice and no significant weight loss of the mice was observed in the studies. The antitumor potency of LOXO-338 is similar to comparable doses of venetoclax in these models. Novel preclinical combination data and comparative profiles to venetoclax will be presented. This preclinical profile of LOXO-338 supports its nomination as a novel BCL2 inhibitor clinical candidate. A first-in-human Phase 1 clinical trial is planned for 2021. Citation Format: Barbara Brandhuber, Karin Ku, Maria J. Lallena, Carmen Baquero, Regina Choy, Kevin Ebata, Sophie Shu Lin, Lihua Jiang, Yanxin Liu, Xiangling Chen, Liguang Lou. Preclinical characterization of LOXO-338, a novel, oral and selective BCL2 inhibitor [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 1258.

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Differential subcellular distribution renders HAI-2 a less effective protease inhibitor than HAI-1 in the control of extracellular matriptase proteolytic activity

The integral membrane, Kunitz-type serine protease inhibitors HAI-1 and HAI-2, can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency. High levels of extracellular matriptase proteolytic activity have, however, been observed in some neoplastic B-cells with high levels of endogenous HAI-2, indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level. The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here. Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells, which naturally express matriptase with very low levels of HAI-2 and no HAI-1, nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme. With increasing HAI-1 expression, cellular matriptase-HAI-1 complex increased, and extracellular active matriptase decreased proportionally. Increasing HAI-2 expression, however, resulted in cellular matriptase-HAI-2 complex levels reaching a plateau, while extracellular active matriptase remained high. In contrast to this differential effect, both HAI-1 and HAI-2, even at very low levels, were shown to promote the expression and cell-surface translocation of endogenous matriptase. The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2. The HAIs, therefore, resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity.

The Antimicrobial Peptide, Nisin, Synergistically Enhances the Cytotoxic and Apoptotic Effects of Rituximab Treatment on Human Burkitt's Lymphoma Cell Lines.

Non-Hodgkin's lymphomas comprise the most common hematological cancers worldwide and consist of a heterogenous group of malignancies affecting the lymphoid system. Treatment of non-Hodgkin's lymphoma has been significantly enhanced with the addition of Rituximab to the standard chemotherapy regimen. However, even with the advancement of treatment patients continue to relapse and develop resistance to Rituximab, rendering subsequent treatments unsuccessful. The use of novel drugs with unique antitumor mechanisms has gained considerable attention. In this study, we explored the in vitro anti-cancer effects of the combined therapy of Rituximab and Nisin on human Burkitt's lymphoma cells. The human Burkitt's lymphoma cells lines, Raji and Daudi, were treated with Nisin, Rituximab, or a combination of the two agents at various concentrations. Cytotoxicity following treatment was determined using cell viability assay. The degree of apoptosis was verified via flow cytometric analysis using FITC annexin V/PI staining. Our findings show that the combined treatment of Rituximab and Nisin results in a more significant reduction in the survival of Raji and Daudi Burkitt's lymphoma cells, compared to Nisin or Rituximab treatment alone. Additionally, our results indicate that Nisin can induce a significant degree of apoptosis in the Burkitt's lymphoma cells compared to the negative controls. However, the addition of Nisin to the Rituximab treatment synergistically enhances the apoptotic antitumor effect. This study demonstrates the synergistic antitumor effect of Nisin treatment in vitro to enhance tumor cell apoptosis and the potential value of Nisin as an adjunct therapy in the treatment of lymphoma.

A Novel Class of Bifunctional Immunotherapeutic That Exploits a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cell Expressing CD38 Demonstrate In Vivo efficacy in Three Distinct Animal Models

Background: Antibody recruiting molecules (ARM) represent a new modality in immunotherapy of cancer. These are bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus can recruit endogenous IgG antibodies independent of their antigen binding specificity representing a breakthrough improvement to previous approaches (Murelli et al.J Am Chem Soc. 2009). We named this active moiety a universal antibody binding terminus (uABT). As a result of antibody recruitment to the cell surface, the target cell is "opsonized" by antibodies which then bring in the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms. Multiple myeloma is a neoplasm that arises from terminally differentiated immunoglobulin producing long-lived plasma cells with 32,000 new cases diagnosed each year. Kleo Pharmaceuticals has developed a series of compounds, CD38-ARM which target human CD38 highly expressed by multiple myeloma cells. CD38 -ARM compounds are therapeutically active in three distinct in vivo models without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab. Methods: CD38-ARM compounds were tested in three independent in vivo models. In the first model, intraperitoneal Daudi xenograft in SCID mice, 20x106 fluorescently labelled Burkitt lymphoma CD38-expressing Daudi cells were injected into SCID mice and treated with 3 mg/kg of compounds. Peritoneal exudates were examined 24 hours later for percentages and absolute numbers of Daudi cells recovered. Activity of compounds was further tested in a MOLP-8 multiple myeloma cell xenograft model in nude mice. Animals received 106 MOLP8 cells subcutaneously, and were treated with a daily dose of 10 mg/kg after tumor volumes of 150 mm3 were reached. Finally, CD38-ARM efficacy was examined in hu IL-15 transgenic NOG mice that have been preconditioned with busulfan and reconstituted i.p. with 2x106human NK cells. Three weeks after reconstitution, 5x106RAJI cells expressing CD38 were implanted s.c. and treatment commenced a week later with 10 mg/kg QDx14. Mice were monitored for NK cell levels and activation status in the blood during the whole study duration by flow cytometry. Results: CD38-uAbt compounds are able to induce clearance of Burkitt's lymphoma Daudi cells expressing high levels of CD38 in a SCID mouse intraperitoneal model. In addition, we show efficacy of one of these compounds in a multiple myeloma xenograft model in nude mice. Using the MOLP8 subcutaneously implanted tumor model we show that administration of our lead molecule is able to induce 50% tumor growth inhibition (TGI) after a daily dosing schedule against this tumor. This activity is comparable to a pharmacologically relevant dose of Daratumumab. Finally, we demonstrate that CD38-ARM treatment shows significant efficacy in humanized mouse model, where IL-15 Tg NOG mice have been reconstituted with human NK cells prior to tumor implantation. In this model daily dosing with the CD38-ARM resulted in up to 70% TGI when compared to untreated control groups. Using this dose/schedule, the molecule did not elicit NK cell depletion as noted in the Daratumumab group. Conclusions: We demonstrated that CD38-ARM compounds are therapeutically active in three distinct in vivo models. Depletion of Daudi cells in a peritoneal SCID model provides strong evidence for the CD38-ARM's capacity to engage macrophage effector functions. Profound inhibition of tumor growth in the NK cell centered humanized hIL-15 Tg NOG mouse model indicates that killing of target cells is executed by human NK cells and provides a foundation to advancing our compounds towards the clinic. Overall, our results demonstrate value of CD38-ARM molecules both as stand-alone therapeutic as well as a platform to develop compounds tailored to a specific indication, by varying target binding moiety of the molecule. Our results also indicate that CD38-ARM compounds engage a variety of effector mechanisms involved in tumor clearance and tumor growth delay, indicating therapeutic potential across a wide range of clinical settings. Disclosures Bunin: Kleo pharmaceuticals: Employment, Equity Ownership. McGrath:Kleo pharmaceuticals: Employment, Equity Ownership. Rossi:Kleo pharmaceuticals: Employment, Equity Ownership. Welsch:Kleo pharmaceuticals: Employment, Equity Ownership. Vidal:Kleo pharmaceuticals: Employment, Equity Ownership. Trinh:Kleo pharmaceuticals: Employment, Equity Ownership. Spiegel:Kleo pharmaceuticals: Equity Ownership. Rastelli:Kleo pharmaceuticals: Employment, Equity Ownership. Alvarez:Kleo pharmaceuticals: Employment, Equity Ownership.

Omipalisib (GSK458), a Dual an-PI3K/mTOR Inhibitor, Exhibits in Vitro and In Vivo activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Reports of recurrent genomic alterations in Burkitt lymphoma (BL) have identified multiple recurrent alterations that result in activation of PI3K highlighting the importance of the PI3K/AKT/mTOR pathway in Burkitt lymphomagenesis. In a cell line model of resistant BL, we have previously identified an increase of PI3K/AKT/mTOR pathway activation suggesting a role in therapy resistance. While inhibition of PI3K-delta using the isoform specific inhibitor idelalisib has demonstrated clinical activity in indolent lymphomas, limited single agent activity has been observed in more aggressive variants. Pre-clinical investigation of idelalisib in BL indicated similar somewhat limited in vitro activity with synergistic activity in combination with chemotherapy. Broader inhibition of both upstream PI3K and downstream mTOR may exhibit more significant anti-lymphoma activity.Objectives: Investigate the in vitro and in vivo activity of the dual pan-PI3K/mTOR inhibitor omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: Experiments were conducted in Raji, Raji 4RH (chemotherapy-rituximab resistant), Ramos, and Daudi BL cells. Cell viability following exposure to omipalisib +/- chemotherapy was analyzed using Cell-Titer Glo and Alamar blu assays. Induction of apoptosis was assessed by flow cytometry for Annexin V (AV)-propidium iodide (PI) staining. Downstream effects of omipalisib on PI3K/Akt/mTOR signaling were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using PI staining. Synergy of combination exposures was determined by calculation of the combination index (CI) using CalcuSyn software. In vivo activity was evaluated using disseminated Raji and subcutaneous Ramos SCID mouse xenograft models. The survival end point was hind limb paralysis in the disseminated model and tumor diameter >2cm in the subcutaneous model. Mice were treated with vehicle or omipalisib daily by oral gavage. Median survival was compared by Kaplan-Meier analysis.Results: Exposure of BL cells to omipalisib for 24-72 hours resulted in a dose- and time-dependent decrease in viable cells at nM concentrations (48h IC50 values: Raji=1.2uM, Raji 4RH=0.02uM, Ramos=0.01uM, Daudi=0.01uM) (Figure 1A). Marked induction of apoptosis occurred following 72h exposure to omipalisib primarily in chemosensitive cells with half-maximal effect noted at approximately 200nM, but requiring significantly higher concentrations to induce apoptosis in therapy resistant Raji 4RH cells (%AV positive at 200nM: Raji=40.7%, Raji 4RH=4.4%, Ramos=59.4% and Daudi=46.9%). Downstream of PI3K/Akt/mTOR, S6 and GSK3β showed reduced phosphorylation after 30 minute omipalisib exposure. G1 cell cycle arrest occurred in all cell lines following exposure to omipalisib for 72 hours; however, chemotherapy-resistant Raji 4RH cells arrested in G2/M at higher concentrations. BL cells exposed to omipalisib in combination with either doxorubicin or dexamethasone, exhibited synergistic anti-tumor activity (CI<0.9) with synergistic induction of apoptosis in therapy sensitive cells exposed to omipalisib and chemotherapy. NOD-SCID mice injected via tail vein with Raji-luc (provided by Dr. Mitchell Cairo) and treated with omipalisib demonstrated decreased luciferase signal compared to controls (Figure 1C) while mice with established subcutaneous Ramos xenografted tumors treated with omipalisib exhibited slower tumor progression compared to controls (Figure 1B), though with only modest prolongation of survival (median 28 vs 34 days, n=15/group, p<0.05).Conclusion: Dual PI3K-mTOR inhibitor omipalisib suppresses the PI3K/Akt/mTOR pathway leading to induction of apoptosis, impaired BL cell proliferation in vitro and in vivo and exhibits synergistic in vitro activity when combined with cytotoxic chemotherapy highlighting the relevance of PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CYT01B, a Novel RAD51 Inhibitor, Act Synergistically with Both Targeted and Chemotherapeutic Anti-Cancer Agents

We have developed CYT01B, a novel RAD51 inhibitor, that sensitizes cells to Activation Induced Cytidine Deaminase (AID) activity. In cancer cells, AID causes significant genotoxic stress through DNA replication fork damage, creating a dependency upon the homologous recombination repair factor, RAD51, for survival. CYT01B acts by destabilizing RAD51 focus formation, leading to its premature nuclear export and degradation. We have shown CYT01B to be effective in AID expressing cells, however, we had yet to address if inhibition of RAD51 could act as a sensitizer to current therapeutics. To determine potential drug combinations, a matrix study was performed with CYT01B (concentration range of 20nM to 5μM) and six different targeted agents or chemotherapeutics in three different cell lines: ARPE19/HPV16 (HPV immortalized normal epithelial cell line), KYSE-70 (head and neck cancer cell line) and Daudi (Burkitt's Lymphoma cell line). We then used the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). The compounds tested were the ATR inhibitor VE-822 (concentration range of 39nM to 2.5μM), the RPA inhibitor TDRL-505 (concentration range of 39nM to 5μM), the proteasome inhibitor Bortezomib (concentration range of 39nM to 2.5μM), Carboplatin (concentration range of 156nM to 10μM), and the PARP inhibitors Olaparib and Niraparib (concentration range of 78nM to 5μM). With VE-822 we observed synergy in the KYSE-70 cell line with ambiguous results in Daudi and ARPE19/HPV16. In ARPE19/HPV16 cell line, synergy was observed with CYT01B at 39nM with all concentrations of VE-822, but antagonistic activity was seen at the high and low concentrations. In Daudi, antagonism was observed at the highest concentrations of VE-822, but an additive effect was noted at the lower concentrations of VE-822. Antagonism was observed at all concentrations of CYT01B with TDRL-505 in both Daudi and ARPE19/HPV16. Weak synergy was observed in KYSE70 cells at 156 and 312nM CYT01B. CYT01B was synergistic with Bortezomib in ARPE19/HPV16 at all concentrations but was consistently antagonistic in KYSE-70 and Daudi. We observed synergy with carboplatin in all cell lines, with the effect consistent across the full concentration range in the cancer cell lines. Synergy was also observed consistently across the full concentration range in all three cell lines with both PARP inhibitors. However, Olaparib showed a stronger synergistic effect than Niraparib. These data suggest that CYT01B may be effective as a combinatorial therapy with platinum based chemotherapeutics and with PARP and ATR inhibitors. Overall, we conclude that there is significant potential for RAD51 inhibition to be used in future combination cancer treatment strategies and warrants further exploration in vivo. DisclosuresMaclay:Cyteir Therapeutics: Employment. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody ‘Ebu-141 Tetra’ Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, ‘EBU-141 Tetra’ was developed.The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma.Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named ‘EBU-141 Tetra’, was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments.The ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the ‘EBU-141 Tetra’ against Daudi Burkitt's lymphoma and U266 plasma cells with EC50 values in the picomolar range and higher maximum lysis rates. In addition, the ‘EBU-141 Tetra’ regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ antibody.Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma. DisclosuresKlausz:Affimed: Research Funding. Otte:Affimed: Research Funding. Klapper:HTG Molecular Diagnostics, Inc.: Research Funding; Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Peipp:Affimed: Research Funding. Gramatzki:Affimed: Research Funding.

A novel hemolytic complement-sufficient NSG mouse model supports studies of complement-mediated antitumor activity in vivo

Monoclonal antibodies (mAbs) have emerged as a mainstream therapeutic option against cancer. mAbs mediate tumor cell-killing through several mechanisms including complement-dependent cytotoxicity (CDC). However, studies of mAb-mediated CDC against tumor cells remain largely dependent on in vitro systems. Previously developed and widely used NOD-scid IL2rγnull (NSG) mice support enhanced engraftment of many primary human tumors. However, NSG mice have a 2-bp deletion in the coding region of the hemolytic complement (Hc) gene, and it is not possible to evaluate CDC activity in NSG mice. To address this limitation, we generated a novel strain of NSG mice—NSG-Hc1—that have an intact complement system able to generate the membrane attack complex. Utilizing the Daudi Burkitt's human lymphoma cell line, and the anti-human CD20 mAb rituximab, we further demonstrated that the complement system in NSG-Hc1 mice is fully functional. NSG-Hc1 mice expressed CDC activity against Daudi cells in vivo following rituximab treatment and showed longer overall survival compared with rituximab-treated NSG mice that lack hemolytic complement. Our results validate the NSG-Hc1 mouse model as a platform for testing mechanisms underlying CDC in vivo and suggest its potential use to compare complement-dependent and complement-independent cytotoxic activity mediated by therapeutic mAbs.

HIgD promotes human Burkitt lymphoma Daudi cell proliferation by accelerated G1/S transition via IgD receptor activity.

The aim of the present study was to investigate the role and molecular mechanism of human IgD (hIgD) on the proliferation of human Burkitt lymphoma Daudi cells in vitro. Logarithmically growing Daudi cells were treated with hIgD for different time periods, and cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. The expressions of Daudi surface markers and IgD receptor (IgDR) as well as cell cycle and apoptosis were measured by flow cytometry analysis. Our results showed that hIgD stimulation induced proliferation and IgDR expression and reduced the apoptosis of Daudi cells. Treatment with hIgD promoted progression of the cell cycle at the G1/S transition, and this was accompanied by upregulation of c-myc, cyclin D3, and CDK6 as well as downregulation of p16 mRNA and protein levels. Moreover, hIgD treatment also upregulated the expression of tyrosine phosphorylation of 70 kDa protein (IgDR) and p-Lyn. Taken together, these results indicate that hIgD can induce Daudi cell proliferation through activating IgDR to initiate the tyrosine phosphorylation signaling cascade to accelerate the G1/S transition.

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

PurposeRGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.Methods661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.ResultsTreatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.Conclusions661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.

Heterodimeric Bispecific Single Chain Variable Fragments (scFv) Killer Engagers (BiKEs) Enhance NK-cell Activity Against CD133+ Colorectal Cancer Cells.

Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse, and express the well-known surface marker CD133. DNA fragments from two fully humanized single chain fragment variable (scFv) antibodies recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigens to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated a unique antibody that recognized the molecular framework and was species cross-reactive. In vitro chromium-51 ((51)Cr) release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133 and that this anti-CD133 scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.

A Novel T-Cell Redirecting Anti-CD19-F(ab)2/CD3scFv Bispecific Antibody Exhibits Potent Lymphoma Cytotoxicity.

AbstractAbstract 2762 Background:The use of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells is a very active area of antibody engineering. Various formats of such agents made recombinantly have shown considerable promise both pre-clinically and clinically. For example, one design termed Bispecific T-cell engager (BiTE) employs a single polypeptide containing 2 antigen-binding specificities (each contributed by a cognate VH and VL) linked in tandem via a flexible linker, and another design termed DART (Dual-Affinity Re-Targeting) utilizes a disulfide-stabilized diabody. Both BiTE and DART, however, exhibit fast blood clearance due to their small size (∼55 kDa). Herein, we describe, for the first time, the generation of a novel T-cell redirecting bsAb, (19)-3s, comprising an anti-CD3 scFv covalently conjugated to a stabilized anti-CD19 F(ab)2. The potential advantages of (19)-3s include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity. Methods and Results:The Dock-and-Lock (DNL) method was used to generate (19)-3s by combining a stabilized anti-CD19 F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Functionally, (19)-3s induced synapse formation between effector and target cells using freshly isolated human T cells mixed with Daudi Burkitt lymphoma cells. Using an E:T ratio of 2.5:1 and 1 μg/mL of (19)-3s, the cell mixture was stained with anti-CD20-APC (for Daudi) and anti-CD7-FITC (for T cells), and cobinding was measured by flow cytometry as the % of CD20+/CD7+ events. After treatment with (19)-3s, 45.5% of events were found to be CD20/CD7 dual-positive, indicating synapsed Daudi and T cells, compared with 2% measured for untreated cells. Gating of the Daudi cell population showed that >90% of Daudi cells were associated with T cells. To access the targeted T-cell killing of Daudi, isolated T cells and Daudi were mixed at an E:T ratio of 12.5:1 and treated with serial dilutions of (19)-3s. After 18-h incubation at 37°C, cytotoxicity was measured using a LDH-release assay. Potent (19)-3s-mediated T-cell killing of Daudi cells was observed at <1 pM, with maximal activity at 10 pM. Similar results were seen with both Ramos and Raji NHL cell lines. In vivo studies to determine Pk and efficacy are underway. Based on DNL constructs of similar design, we expect (19)-3s to have an elimination rate longer than that of MT103, a BiTE comprising scFvs derived from anti-CD19 and anti-CD3, thus perhaps avoiding continuous infusions with this new construct. Conclusions:(19)-3s can bind T cells and NHL cells simultaneously and induce T-cell-mediated killing at pM concentrations in an ex vivo setting. The modular nature of the DNL method will allow the rapid production of a large number of related conjugates for redirected T-cell killing of various malignancies, without the need for additional recombinant engineering and protein production. We are currently evaluating the in vivo activity of (19)-3s, as a prototype, to determine if this novel bsAb format offers additional advantages. Disclosures:Rossi:Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership. Chang:Immunomedics, Inc.: Employment.

Target-Directed Development of a Proposed Biosimilar Rituximab (GP2013): Comparability of Antibody-Dependent Cellular Cytotoxicity Activity and Pre-Clinical Pharmacokinetics and Pharmacodynamics with Originator Rituximab

AbstractAbstract 4867 Background:Biosimilars are biologics approved by highly-regulated markets to be similar to existing agents that aim to offer more affordable treatment, thereby increasing patient access. Development of a biosimilar involves extensive characterization of the originator product over several years and a target-directed iterative development process to ensure a product that is highly comparable to the originator with similar clinical efficacy, safety and quality. Using antibody-dependent cellular cytotoxicity (ADCC), a main mode of action of rituximab, we illustrate how functional/structural relationship can be engineered into a biosimilar to ensure comparability at the in vitro level. Here we also present pre-clinical data confirming in vivo comparability for the proposed biosimilar rituximab GP2013, in terms of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Methods:By employing a highly sensitive glycan quantitation method, relevant post-translational glycosylation patterns were assessed for their impact on in vitro ADCC relative potency data using the Raji and NK3.3 cell lines as target- and effector cells, respectively. Subsequently, bioactivity of GP2013 and originator rituximab were evaluated in a dose-response manner across a wide concentration range against SU-DHL-4 (diffuse large B-cell lymphoma) and Daudi (Burkitt's lymphoma) cell lines using freshly purified human NK cells. In vivo anti-tumor activity was assessed in two xenograft SCID mouse models of non-Hodgkin's lymphoma (SU-DHL-4 and Jeko-1 cell lines). Comparative PK and PD were assessed in single (5 mg/kg, n=14) and multiple (20 or 100 mg/kg, n=8) dose studies in cynomolgus monkeys, the pharmacologically most relevant species. Results:GP2013 and originator rituximab showed similar ADCC potency against both SU-DHL-4 and Daudi cells, with ADCC being reflective of engineered glycosylation patterns and structure-function relationships. In both xenograft mouse models, GP2013 and originator rituximab inhibited tumor growth to a similar extent, including at the more sensitive sub-optimal dose levels that are most likely to identify any potential differences. In primates, PK analysis confirmed bioequivalence between GP2013 and originator rituximab with nearly identical AUC values and 90% CIs entirely within the standard acceptance range of 0.8–1.25. Bioequivalence of PD response (B-cell depletion) was also shown, with 95% CIs of areas under the effect-time curves (AUEC) ratios for relative change from baseline in B-cell populations within the 0.8–1.25 acceptance range. The use of different doses indicated that comparable exposure and PD response can be expected for GP2013 and originator rituximab using indication-specific dosing regimens. Conclusions:This pre-clinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar with regard to ADCC potency, anti-tumor activity, PK exposure (AUC) and B-cell depletion. As such, it is hypothesized that GP2013 will show similar efficacy and safety as the originator product in ongoing clinical trials across different clinical indications. Disclosures:da Silva:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Kronthaler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Meyer:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Papandrikopoulou:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Stangler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Visser:Sandoz Biopharmaceuticals/HEXAL AG: Employment.

Daratumumab, a Novel Human Anti-CD38 Monoclonal Antibody for the Treatment of Chronic Lymphocytic Leukemia and B-Cell Non–Hodgkin Lymphoma

AbstractAbstract 3935Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the present study, we have analyzed the potential of targeting CD38 using DARA in two types of B-cell non-Hodgkin lymphoma (B-NHL) (follicular lymphoma (FL) and mantle cell lymphoma (MCL)), and in chronic lymphocytic leukemia (CLL). Flow cytometry analysis demonstrated that MCL and CLL tumor cells show heterogeneous expression of CD38, while FL cells showed invariable high CD38 levels. CD38 has attracted special attention in CLL where high CD38 expression is a marker of bad prognosis (Hamblin et al, Blood 1999 and 2002) and is expressed preferentially in the proliferating fraction of the tumor (Damle RN, Blood 2007). In addition, we have recently shown that high CD38 expression in MCL was associated with resistance to the proteasome inhibitor bortezomib (Pérez-Galán P, Blood 2011).Here, we tested the cytotoxic activity of DARA in tumor cell lines and in fresh tumor cells obtained from patients. DARA did not induce CDC in MCL cell lines (MINO, REC, HBL2, JEKO), irrespective of CD38 expression levels. Also, FL cell lines (WSU-FSCCL, RL) expressing relatively high CD38 levels were insensitive to DARA-induced CDC. This low CDC was associated with high expression of the complement inhibitors CD55 and CD59. In addition, the number of CD38 molecules per cell in these MCL and FL cell lines was lower than that found on the CDC-sensitive Daudi Burkitt lymphoma cell line, suggesting a threshold for CD38-targeted CDC lysis.In the presence of PBMC effector cells obtained from healthy donors, DARA showed significant levels of ADCC in cells from MCL, FL and CLL. In CLL primary cases (n=8) tested, DARA (14–43% lysis) was generally superior or at least equally effective (mean+/−SD=28,78 +/− 9,78) in inducing ADCC as compared to the anti-CD20 antibodies ofatumumab (mean+/−SD=21,35 +/− 15,71) and rituximab (mean+/−SD=29,30 +/− 15,90).The immunomodulatory agent lenalidomide shows considerable single agent activity in MCL, FL and CLL. Interestingly, it has been shown that lenalidomide may be able to increase ADCC, probably via activation of NK cells. We therefore tested whether the combination of lenalidomide and DARA could enhance ADCC. Noteworthy, DARA-induced ADCC in MCL, FL cell lines and primary CLL cells was significantly (p<0,05) enhanced when effector cells were pretreated with the immunomodulatory agent lenalidomide (3 μM, 72 h) with DARA doses ranging from 0,01–1 μg/ml.Finally, CD38 is important for cell migration and adhesion, especially for CXCR4-CXCL12-induced migration of tumor cells (Vaisitti et al, Leukemia 2010). Our preliminary results suggest that in the CLL subtype with high CD38 and more migratory capacity DARA (10–30 μg/ml) inhibits CXCL12/SDF1α mediated migration up to 70%. These results are of high importance because inhibition of tumor cell trafficking to tissue sites as bone marrow and lymph nodes, may be clinically relevant, as increasing evidence indicates that tumor–microenvironment interactions may play an important role in drug resistance and contribute to clinical failures. We are currently validating this in vitro results in a CLL mouse model.Taken together, these results suggest that DARA may be a promising therapeutic agent both for MCL, FL and CLL, in which DARA exerts its effects mainly via ADCC and for CLL also via inhibition of migration of tumor cells. Interestingly, the cytotoxic activity of DARA by ADCC could be further augmented by addition of lenalidomide in these three models. Disclosures:Lammerts van Bueren:Genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Perez-Galan:Genmab: Research Funding.

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitt's lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 μM, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.

A Novel Ribonuclease-Based Immunotoxin Comprising Quadruple Ranpirnase (Rap) Site-Specifically Conjugated to An Anti-CD22 IgG Showing Potent Anti-Lymphoma Activity.

Abstract 3721Poster Board III-657 BackgroundRap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. The amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap is in advanced Phase IIIb clinical trials against malignant mesothelioma, with reversible dose-limiting renal toxicity and no reported immunogenicity. We have previously shown that effective therapy of human lymphoma xenografts could be achieved with a recombinant fusion protein comprising Rap and a rapidly internalizing anti-CD74 humanized IgG4 antibody (Chang et al., Blood, 2005; 106: 4308-14). In this study, we applied the Dock-and-Lock (DNL) method to generate another novel class of immunotoxins, each of which contains four copies of Rap site-specifically linked to a bivalent IgG. MethodsThe DNL platform technology exploits a pair of distinct protein domains involved in the natural binding of cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of 2 types of modules, one containing the dimerization and docking domain (DDD) of PKA and the other containing the anchoring domain (AD) of an interactive AKAP. We have combined a recombinant Rap-DDD module with a recombinant IgG-AD module derived from the internalizing anti-CD22 humanized antibody, epratuzumab, to generate 22-Rap, in which a dimer of Rap is covalently tethered to the c-terminus of each heavy chain of epratuzumab. The Rap-DDD module was expressed in E. coli as inclusion bodies, which were purified by immobilized metal affinity chromatography and refolded. The epratuzumab-AD module was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography. Results22-Rap was prepared by mixing the epratuzumab-AD module with the Rap-DDD module under mild redox conditions and purified to near homogeneity in a single step using Protein A affinity chromatography. Size exclusion HPLC revealed the presence of a single peak of a retention time consistent with the molecular size of ∼230 kDa, indicative of an IgG and four Rap groups. In vitro transcription/translation assays showed that both the Rap-DDD2 module and 22-Rap retained the activity of Rap. Results of in vitro cytotoxicity assays using Daudi Burkitt lymphoma cells demonstrated that 22-Rap was highly cytotoxic, resulting in nearly 100% cell killing at 1 nM, with an IC50 <50 pM. In comparison, recombinant Rap at 100 nM, either alone or in combination with epratuzumab, resulted in only 50% inhibition of Daudi proliferation. Thus, 22-Rap is about 3 logs more potent than free Rap against Daudi cells. ConclusionThe DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity. Disclosures:Rossi:Immunomedics, Inc: Employment. Chan:Immunomedics, Inc: Employment. Gupta:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.

The LMP1 Promoter Can Be Transactivated Directly by NF-κB

A bioinformatic analysis identified two putative NF-kappaB binding sites in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) promoter. The ability of p65RelA to interact with the LMP1 promoter was shown by in vitro and in vivo assays. Using an EBV-transformed lymphoblastoid cell line as a reporter system for the activity of the +40/-328 LMP1 promoter region, the functional importance of NF-kappaB and other transcription factor binding sites was demonstrated. p65RelA could also induce LMP1 expression from the EBV genome in Daudi and P3HR1 Burkitt's lymphoma cell lines. Finally, it was shown that p65RelA could cooperate with EBNA2 or the aryl hydrocarbon receptor in the transactivation of the LMP1 promoter. Our study established the importance of NF-kappaB and several cis-acting elements in the regulation of the LMP1 promoter in a latency III environment and highlighted a complex interplay between NF-kappaB and other transcription factors in this process.

Class I HLA Folding and Antigen Presentation in β2-Microglobulin-Defective Daudi Cells

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called beta(2)-microglobulin (beta(2)m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in beta(2)m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with beta(2)m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a beta(2)m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of beta(2)m. These findings bear potential implications for tumor immunotherapy.