Antigen-negative Cells Research Articles

Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies.

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

Synthesis and biological evaluation of RGD peptidomimetic-paclitaxel conjugates bearing lysosomally cleavable linkers.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.

MEN1112, a Novel Humanized De-Fucosylated Monoclonal Antibody with High Affinity and Specificity for Bst1/CD157 Antigen and Enhanced CD16 Binding

Bst1/CD157 is a GPI-anchored transmembrane protein belonging to the ADP-ribosyl-cyclase family and it is expressed on some blood cells such as monocytes and neutrophils. Importantly, we demonstrated that this antigen is expressed in blast cells from almost 100% of Acute Myeloid Leukemia patients (either at primary diagnosis or at relapse).MEN1112 is a humanized, de-fucosylated antibody targeting the Bst1/CD157 antigen with high affinity and inducing potent in vitro and ex vivo Antibody Dependent Cell-mediated Cytotoxicity (ADCC) responses against AML cell lines and blasts, respectively. MEN1112 was discovered as a mouse Fab antibody from a phage display library raised against recombinant Bst1/CD157 protein. The Fab was selected base upon its ability to recognize recombinant Bst1/CD157 protein by ELISA and for its ability to recognize Bst1/CD157 positive endogenous cancer cell lines by FACS (fluorescence activated cell sorting). The Fab was also chosen based upon its ability to recognize cynomolgus Bst1/CD157 antigen on recombinant and endogenous cells. The selected Fab sequence was humanized and incorporated into the Biowa Potelligent cell line to produce a de-fucosylated full IgG1 version (MEN1112). The binding affinity of MEN1112 for the Bst1/CD157 antigen showed an EC50 of 1nM as assessed by flow cytometry (FACS) analysis using multiple cell lines. No binding was observed on antigen-negative cell lines demonstrating specificity of MEN1112. High affinity of MEN1112 for the recombinant antigen was confirmed in an ELISA assay (EC50 0.4 nM).Beyond the affinity for the antigen, it is of relevance for its ADCC activity that MEN1112 is characterized by an improved affinity for Fc receptors on effector cells over the parental antibody. In particular, MEN1112, lacking fucose residues in its Fc domain, showed an impressive high affinity binding to the Fc receptor CD16A both high affinity and low affinity alleles.Internalization studies demonstrate the slow internalization of the Bst1/CD157 antigen following binding to MEN1112 which favors the ADCC efficacy of the antibody. Overall our results indicate that MEN1112 has the potential to exert anti-leukemia actions through a powerful ADCC in view of: (i) superior affinity for blast cell membrane antigen Bst1/CD157, (ii) enhanced affinity for all variants of CD16A Fc receptor expressed on effector cells and (iii) minimal internalization following antigen binding. DisclosuresAud:Oxford BioTherapeutics: Employment. Dusek:Oxford BioTherapeutics: Employment. Bisht:Oxford BioTherapeutics: Employment. Swaminithan:Oxford BioTherapeutics: Employment. Awdew:Oxford BioTherapeutics: Employment. Trang:Oxford BioTherapeutics: Employment. Lin Lou:Oxford BioTherapeutics: Employment. Manzini:Menarini Ricerche SpA: Employment. Martinez Mogarra:Menarini Biotech srl: Employment. Bressan:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment.

Immuno-PET of epithelial ovarian cancer: harnessing the potential of CA125 for non-invasive imaging.

BackgroundEpithelial ovarian cancer (EOC) is characterized by the overexpression of cancer antigen 125 (CA125), a mucinous glycoprotein that serves as a tumor biomarker. Early diagnosis of EOC is plagued by its asymptomatic nature of progression and the limitations of currently used immunoassay techniques that detect CA125 as a shed antigen in serum samples. Presently, there is no technique available for the in vivo evaluation of CA125 expression in malignant tissues. Moreover, there could be an unexplored pathophysiological time window for the detection of CA125 in EOC, during which it is expressed on tumor cells prior to being shed into the bloodstream. A method for the in vivo evaluation of CA125 expression on ovarian neoplasms earlier along disease progression and/or recurrence can potentially contribute to better disease management. To this end, the present work utilizes an anti-CA125 monoclonal antibody (MAb) and a single-chain variable fragment (scFv) labeled with the positron-emitting radionuclide 64Cu for preclinical molecular imaging of CA125 expression in vivo.MethodsAnti-CA125 MAb and scFv were prepared and functionally characterized for target binding prior to being tested as radiotracers in a preclinical setting.ResultsImmunoblotting, immunofluorescence, and flow cytometry revealed specific binding of CA125-targeting vectors to NIH:OVCAR-3 cells and no binding to antigen-negative SKOV3 cells. 64Cu-labeled anti-CA125 MAb and scFv were obtained in specific activities of 296 and 122 MBq/mg, respectively. Both radioimmunoconjugate vectors demonstrated highly selective binding to NIH:OVCAR-3 cells and virtually no binding to SKOV3 cells. In vivo radiopharmacological evaluation using xenograft mouse models injected with 64Cu-labeled anti-CA125 MAb provided a standardized uptake value (SUV) of 5.76 (29.70 %ID/g) in OVCAR3 tumors 24 h post-injection (p.i.) versus 1.80 (5.91 %ID/g) in SKOV3 tumors. 64Cu-labeled anti-CA125 scFv provided an SUV of 0.64 (3.21 %ID/g) in OVCAR3 tumors 24 h p.i. versus 0.25 (1.49 %ID/g) in SKOV3 tumors. Results from small-animal PET imaging were confirmed by ex vivo autoradiography and immunohistochemistry.ConclusionsRadiolabeling of anti-CA125 MAb and scFv with 64Cu did not compromise their immunoreactivity. Both radioimmunoconjugates presented specific tumor uptake and expected biological clearance profiles. This renders them as potential immuno-PET probes for targeted in vivo molecular imaging of CA125 in EOC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-014-0060-4) contains supplementary material, which is available to authorized users.

Abstract 4837: Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells

Abstract Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837

Abstract 2660: Targeting an attenuated cytokine to tumor cells via antibody fusion results in enhanced tumor killing with significantly reduced off-target activity

Abstract Many cytokines have potential therapeutic applications and several, including interferon (IFN)α and IFNβ, have been approved for the treatment of cancer and/or autoimmune disease. While these agents are effective, they are associated with significant dose-limiting toxicities that prevent their use at levels sufficient to promote optimal therapeutic benefit. The toxicity of type I interferons, like other cytokines, is likely mediated via cognate receptor activation on cells other than those that mediate their therapeutic effects. For example, IFNα2b, which is indicated for the treatment of multiple myeloma (MM), has a narrow therapeutic index due to significant systemic toxicity. Approaches that seek to minimize dose-limiting systemic toxicity while maximizing tumor-specific cytokine exposure could greatly enhance the therapeutic index and effectiveness of type I IFNs and other cytokines. Other groups have previously shown that a moderate degree of tumor-specificity can be achieved by attaching a cytokine such as IFNα to a tumor-targeting antibody; such first generation antibody-targeted cytokines are highly active but show only moderate tumor-specificity since the cytokine can still potently stimulate its receptor on antigen-negative cells. We sought to improve the therapeutic index of antibody-targeted cytokines by mutating the cytokine portion to significantly reduce affinity for its receptor, thereby making it more dependent on antibody-based cell-targeting. Here we demonstrate that such fusion proteins, consisting of mutant or attenuated cytokines (Attenukines™) attached to tumor-targeting antibodies, are 1,000 to 100,000-fold more potent on target-positive cells compared to target-negative (normal) cells. This is shown for antibody-Attenukine™ fusion proteins based on multiple tumor antigens (CD20, CD38, CD138, HMW-MAA, HLA) and multiple attenuated mutants of IFNα, IFNβ, IL-4 and IL-6. Furthermore, we have evaluated an anti-CD38-attenuated IFNα molecule (anti-CD38-Attenukine™) in various CD38+ MM xenograft models and found that this molecule retains potent tumor-targeting activity and anti-tumor efficacy. Moreover, in non-human primates, we have confirmed that the attenuating mutation in IFNα indeed decreases non-targeted IFNα biomarker responses by greater than 100-fold. Taken together, our findings suggest that the administration of antibody-attenuated cytokine fusion proteins to cancer patients may promote robust cytokine-dependent tumor-killing while minimizing systemic toxicity. Citation Format: Sarah L. Pogue, Tetsuya Taura, Mingying Bi, Glen Mikesell, Yong Yun, Angela Sho, Collette Behrens, Maxwell Stevens, Teresa Domagala, Maya Sokolovsky, Hussein Hallak, Moti Rosenstock, Anthony Doyle, David S. Wilson. Targeting an attenuated cytokine to tumor cells via antibody fusion results in enhanced tumor killing with significantly reduced off-target activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2014-2660

Abstract 2644: Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models

Abstract Most ADCs in the clinic utilize a tubulin-binding small molecule as the cytotoxic payload. Although many cancers are sensitive to tubulin agents, some are more responsive to DNA-interacting agents. We have developed a new class of highly potent cytotoxic agents, IGNs, for use in ADCs that consists of indolino-benzodiazepine dimers with a novel DNA-alkylating mechanism of action. We found that IGNs containing a di-imine moiety act via DNA-crosslinking and -alkylating, whereas IGNs with a mono-imine induce DNA alkylation only. Although ADCs with either form of IGN were highly active, ADCs containing di-imine IGNs caused delayed toxicity in mice, leading to mortality. The mono-imine IGN, DGN462, was selected as our lead IGN as, when conjugated to an antibody via a cleavable linkage, the resultant ADC had favorable tolerability in mice (maximally tolerated dose of 40 mg/kg; 700 µg/kg DGN462 dose) without delayed toxicity. The ability of DGN462 to alkylate DNA was demonstrated by measurement of DNA adduct formation with double-stranded DNA and in cells. DGN462 formed covalent DNA adducts through alkylation of the C2- amino group of guanine. Consistent with the activity of other DNA alkylating agents, DGN462-treated cells progressed slowly through S-phase and were arrested in G2/M phase of the cell cycle in a dose dependent manner. ADCs consisting of ∼3 molecules of DGN462 per antibody, attached via a cleavable linker, had desirable biochemical characteristics, including high monomer content (> 97%), low free drug (< 0.1%) and symmetrical mass distribution profiles. These DGN462 ADCs were highly potent and antigen-specific in vitro against various cell types, including PgP-expressing cells, with IC50 values ranging from 2 pM to 3 nM depending on the target, cell type and antigen density. DGN462 ADCs display bystander activity in vitro, indicating that intracellular processing of conjugate by target cancer cells releases metabolites that can kill antigen-negative cells in direct proximity, which may provide an advantage in solid tumors expressing antigen heterogeneously. A CD33-targeting DGN462 ADC was highly active against AML xenografts, with a minimal efficacious dose (MED) of 0.6 mg/kg (conjugate dose), while a non-targeting control was inactive. A DGN462 ADC targeting epidermal growth factor receptor was highly active and antigen-specific against a head and neck squamous cell carcinoma model, with a 1.6 mg/kg MED. The intact conjugate half-life was approximately 90 hours in mice, and bioactivity of conjugate was maintained at 72 hours post dosing, indicating that intact conjugate remains active in vivo. DGN462 ADCs have an acceptable safety profile and potent antigen-specific DNA-alkylating mechanism of action with the potential for activity in tumors with low sensitivity to tubulin agents, heterogeneous antigen expression and/or PgP-mediated drug resistance. Citation Format: Kathleen Whiteman, Charlene Audette, Andre Dandeneau, Megan Ellis, Nathan Fishkin, Lauren Harvey, Holly Johnson, Yelena Kovtun, Erin Maloney, Michael Miller, Alan Wilhelm, Ravi Chari. Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2014-2644

Limitations of bystander killing in Th1/M1 immune responses against a secreted tumor antigen

T-cell recognition of tumor antigens presented on tumor-infiltrating macrophages (TAMs) induces a tumoricidal M1-like phenotype. Resultant indirect immune responses could eliminate not only antigen secreting (AgPOS), but also antigen negative (AgNEG) tumor cells via bystander killing. Such broad-spectrum response could eliminate antigenically heterogeneous tumors. Using an in vivo model of CD4+ T-cell mediated immunity against MHC II negative myeloma cells, bystander killing of AgNEG cells was ineffective due to strict spatial constraints of Th1-induced TAM cytotoxicity.

Naptumomab Estafenatox: Targeted Immunotherapy with a Novel Immunotoxin

Improvement of cancer therapy by introducing new concepts is still urgent even though there have been major advancements lately. Immunotherapy is well on the way to becoming an established tool in the cancer treatment armory. It seems that a combination of (1) activation of immune effector cells and selective targeting of them to tumors and (2) the inhibition of immune suppression often induced by the tumor itself are necessary to achieve the therapeutic goal. The immunotoxin naptumomab estafenatox was developed in an effort to activate and target the patient’s own T cells to their tumor, by fusing a superantigen (SAg) variant that activates T lymphocytes to the Fab moiety of a tumor-reactive monoclonal antibody. Naptumomab estafenatox targets the 5T4 tumor antigen, a 72-kDa oncofetal trophoblast protein expressed on many carcinomas, including renal cell carcinoma. The therapeutic effect is associated with activation of SAg-binding T cells. The SAg-binding T lymphocytes expand, differentiate to effector cells, and infiltrate the tumor. The therapeutic efficacy is most likely related to the dual mechanism of tumor cell killing: (1) direct lysis by cytotoxic T lymphocytes of tumor cells expressing the antigen recognized by the antibody moiety of the fusion protein and (2) secretion of cytokines eliminating antigen-negative tumor cell variants. Naptumomab estafenatox has been clinically tested in a range of solid tumors with focus on renal cell carcinoma. This review looks at the clinical experience with the new immunotoxin and its potential.

CD8+ T-cell responses rapidly select for antigen-negative tumor cells in the prostate.

Stimulation of patients' immune systems for the treatment of solid tumors is an emerging therapeutic paradigm. The use of enriched autologous T cells for adoptive cell therapy or vaccination with antigen-loaded dendritic cells have shown clinical efficacy in melanoma and prostate cancer, respectively. However, the long-term effects of immune responses on selection and outgrowth of antigen-negative tumor cells in specific tumor types must be determined to understand and achieve long-term therapeutic effects. In this study, we have investigated the expression of a tumor-specific antigen in situ after treatment with tumor-specific CD8(+) T cells in an autochthonous mouse model of prostate cancer. After T-cell treatment, aggregates of dead antigen-positive tumor cells were concentrated in the lumen of the prostate gland and were eventually eliminated from the prostate tissue. Despite the elimination of antigen-positive tumor cells, prostate tumor continued to grow in T-cell-treated mice. Interestingly, the remaining tumor cells were antigen negative and downregulated MHC class I expression. These results show that CD8(+) T cells are effective in eliminating antigen-bearing prostate tumor cells but they also can select for the outgrowth of antigen-negative tumor cells. These findings provide insights into the requirements for an effective cancer immunotherapy within the prostate that not only induces potent immune responses but also avoids selection and outgrowth of antigen-negative tumor cells.

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells—all color-coded in vivo—was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region—before, during and after adoptive T cell therapy—thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies.

IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Abstract 4144: Improvement of tumor cell transduction with lentiviral vectors using chemical and cell targeting approaches.

Abstract Although lentiviral transduction methods are widely used in cancer research their application is frequently hindered by the low transduction efficiency seen for a number of different cell types, in particular primary cells. We have previously reported that a mixture of poloxamer synperonic F108 and polybrene combines reduced toxicity and increased lentiviral gene delivery into a range of different primary cell cultures (1). We now report further improvement in efficiency through a novel combination of the poloxamer-based adjuvant with a modified lentiviral envelope. The envelope was rendered cell specific by fusing monoclonal antibody fragments directed against specific cell-surface epitopes with the viral attachment glycoprotein VSV-G. For specific cellular targeting we inserted chimeric antiCD30 or antiEGFR antibody-VSV-G fusion proteins into the lentiviral genome. The combination of VSV-G-fused antibody fragments against specific tumor cell antigens and the improved adjuvant resulted in a 3-fold higher transduction rate using a GFP reporter containing lentivirus in CD30-positive lymphoma and EGFR-positive epithelial breast cancer cells. The combination of optimized transduction enhancer molecules and retargeted lentiviral vectors enabled quantitative transduction of previously hard-to-transduce cell types. When mixtures of antigen-negative and antigen-positive tumor cells were selected the modified lentivirus particles showed a preference for infecting antigen-positive cells by a factor 4. These results demonstrate that improvements in both chemical charge protecting adjuvants and membrane targeting can lead to increased transduction efficiency without a loss of specificity. The combination of chemical adjuvant and envelope design can lead to the optimization of lentiviral transductions in large-scale systems. *1. Höfig I, Atkinson MJ, Mall S, Krackhardt AM, Thirion C, Anastasov N. Poloxamer synperonic F108 improves cellular transduction with lentiviral vectors. J Gene Med. 2012 Aug;14(8):549-60. Citation Format: Ines Höfig, Michael J. Atkinson, Christian Thirion, Natasa Anastasov. Improvement of tumor cell transduction with lentiviral vectors using chemical and cell targeting approaches. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2013-4144

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Relapse of melanoma after successful adoptive T‐cell therapy: escape through inflammation‐induced phenotypic melanoma cell plasticity

Advanced stage melanomas are refractory to most available therapies including recently approved targeted therapies. This is also true for most biological (IFN-a or IL-2) treatments and therapy involving adoptive transfer of melanoma-reactive T cells (de Souza et al., 2012). A number of mechanisms have been proposed to explain the lack of T-cell functional activity in vivo. Some of these mechanisms include emergence of antigen-loss tumor variants, induction of immune inhibitory molecules such as CTLA4 or PD1 and induction of T-reg (regulatory) cells or myeloid-derived suppressor cells (MDSC) or presence of immune-suppressive cytokines such as TGF-b or IL-10 (Grivennikov et al., 2010). A number of environmental factors can further contribute to genetic instability and induce epigenetic changes of progressing heterogeneous tumors. Presence of inflammatory factors at the tumor site can cause selection of preexisting tumor cell variants resulting in preferential growth of antigen-negative tumor variants or tumor cells that have lost MHC-class I molecules (Khong and Restifo, 2002). Immune editing or alterations of tumor antigens due to immune pressure can also be one of the reasons for lack of T-cell-mediated tumor cell death in vivo (Singh and Paterson, 2007). In clinical trials, adoptive cell therapies (ACT) with melanoma-reactive tumor-infiltrating lymphocytes (TILs) or polyclonal T cells engineered to express chimeric antigen receptor (CAR) with re-directed specificity to melanoma have shown great promise with dramatic regression of bulky tumors (Robbins et al., 2011). However, in most patients, initial regression of tumor cells was followed by appearance of new lesions with therapy resistant melanoma cells (Robbins et al., 2011). Until now, the exact nature of ACT-related tumor resistance and the cause of emergence of antigen-negative melanoma cells were largely unknown. In the current edition of Nature, Landsberg et al. describe a novel mechanism of T-cell-induced loss of melanocytic antigens as a possible cause of tumor cell resistance to ACT. To demonstrate therapy resistance, the authors have used a previously published genetically engineered hepatocyte growth factor (Hgf)-Cdk4 mouse melanoma model and anti-gp100-specific CTLs obtained from a TcR transgenic pmel-1 (gp100) mouse model. TNF-a, a proinflammatory cytokine predominantly secreted by activated T cells caused down-modulation of gp100 antigen expression on the mouse melanoma cells resulting in loss of recognition by anti-gp100-specific CTLs used in ACT. TNF-a also down-modulated pigmentrelated gene expression and tyrosinaserelated protein (TRP)-2 on melanoma cells. Interestingly, the pro-inflammatory environment of TNF-a did not downmodulate the expression of non-melanocytic antigens. On the contrary, there was an increased expression of nerve growth factor receptor (NGFR) on melanoma cells. Down-modulation of gp100 expression was reversible when the activity of TNF-a was neutralized or the resistant tumor cells were re-transplanted into a normal mouse lacking an inflammatory environment. The mechanism of non-genetic reversible antigen down-modulation is novel and has not been described before. This is in contrast to other genetic heterogeneityand immunoselection-related mechanisms described earlier. Understanding inflammation-induced plasticity and the non-genetic heterogeneity of melanoma cells are critical for the development of new therapeutic strategies for melanoma. The authors then corroborated their mouse studies using human melanoma cell lines where they report similar down-modulation of gp100 and MART1/Melan-A expression in the presence of TNF-a. Similar to mouse studies, human melanoma cell lines also showed up-regulation of NGFR expression in the presence of TNF-a. This was confirmed in melanoma patients’ tumor tissue sections with abundant infiltrating immune cells further supporting the clinical relevance of the above findings. The results of Landsberg et al. have important clinical implications if the phenotypic plasticity of melanoma cells in an inflammatory microenvironment is validated in a larger cohort of patients with melanoma after ACT. Down-modulation of gp100 expression and concurrent up-regulation of NGFR expression of melanoma cells in an inflammatory environment raises the possibility of using ACT with a cocktail of CAR-engineered T cells with dual or multiple specificities to target more than one melanoma antigen to ensure complete regression of tumor lesions. For this, a personalized approach of analyzing melanoma antigen expression on tumor cells before and after ACT therapy will be essential for the selection of CARengineered T cells with defined tumor antigen specificity. The proposed approach may enhance the long term disease-free survival of patients with metastatic melanoma.

Clinically significant anti M antibodies – A report of two cases

IntroductionMost anti-M antibodies are not active at 37°C and are thus of no clinical significance. Occasionally these antibodies have a wide thermal range and can lead to hemolytic transfusion reactions or hemolytic disease of the new born. Patient and methodsWe describe two cases of anti-M antibodies, both of which were clinically significant. ResultsThe first case was detected due to crossmatch incompatibility and the second presented as a blood group discrepancy. ConclusionWhen the antibody is active at 37°C, M antigen negative red cell units should be issued.

Retroviral transduction of lineage antigen-negative (Lin−) cells: A valuable alternative for the generation of T cell receptor (TCR) retrogenic mice

Mice with virtually all T cells expressing a single T cell receptor (TCR) on their surface have been instrumental in understanding the development of immature thymocytes. For many years, such an engineering has been achieved essentially by inserting rearranged TCR α and β chain coding sequences into the genome through co-microinjection into fertilized eggs (TCR transgenesis). More recently, a novel methodology relying on the reconstitution of T cell deficient hosts with retrovirally-transduced multipotent bone marrow cells has been developed. Hence, TCR retrogenesis allows for the in vivo study of given TCR specificities in a faster and less expensive manner. While initial procedures were taking advantage of 5-Fluorouracil (5-FU) treatment of RAG-deficient or SCID donor mice as source of haematopoietic stem cells, we used bone marrow cell suspensions enriched in lineage antigen-negative (Lin−) cells from untreated donors for TCR retrogenesis. In contrast to cells from 5-FU-treated donors, transduced Lin− cells consistently generated a sizable retrogenic pool of thymocytes and required less donor mice. In such retrogenic mice, immature thymocytes bearing a major histocompatibility complex (MHC) class II-restricted TCR differentiated into the expected CD4 mature T cell lineage and populated the peripheral lymphoid organs where they retained the capacity to react to their cognate ligand. Lin− cell-enriched BM cells represent therefore, a reliable alternative to 5-FU treatment for retroviral transduction of haematopoietic stem cells and TCR retrogenic derivation.

Abstract 4624: High affinity CD3 RECRUIT-TandAbs for T cell-mediated lysis of malignant CD19+ B cells

Abstract The TandAb technology enables the development of innovative antibody therapeutics for improved treatment of various diseases. This platform has been primarily applied to oncology and comprises of CD3 RECRUIT- and CD16A RECRUIT-TandAbs for activation of T and NK effector cells, respectively, and lysis of target cells expressing specific surface antigens. The CD3 RECRUIT-TandAb AFM11 is a human bispecific tetravalent antibody with two binding sites for the α-chain of CD3 and two binding sites for CD19. CD19 is expressed at early stages of B cell development and persists until the final differentiation into plasma cells. Thus, CD19 represents an attractive target for the treatment of various B cell malignancies including leukemias and lymphomas that lack CD20 expression or are refractory to anti-CD20 antibody therapies. In vitro cytotoxicity assays employing tumor cell lines demonstrate high potency of AFM11 in mediating target cell lysis: EC50 values are in the low to sub-picomolar range depending on CD19+ target cell lines and primary B-CLL tumor cells. Cytotoxic activity of tetravalent AFM11 is superior to those of alternative bivalent antibody formats. High affinity binding of AFM11 to CD3 and CD19 (low to sub-nanomolar range) contributes to efficacious T cell recruitment. This binding was determined by flow cytometry and by a label-free biosensor that measures real-time binding kinetics on live cells. Therapeutic recruitment of T cells has a potential for a devastating cytokine release if it results not in a site-specific but rather systemic activation. Thus, functional in vitro assays were used to demonstrate that high affinity and bivalent binding of AFM11 to CD3 is not sufficient for triggering activation signals in T cells. AFM11 activates T cells only in the presence of CD19+ target cells, and specifically mediates lysis of these cells without affecting antigen-negative bystander cells. In the absence of target cells and at high concentrations AFM11 induces down-modulation of the CD3/TCR complex from the surface of T cells. The latter may contribute additional safety to therapeutic potential of CD3 RECRUIT-TandAbs without affecting the efficacy, since AFM11-treated T cells can be re-engaged for target cell lysis. In vivo AFM11 demonstrated a robust dose-dependent inhibition of tumor growth in a study with Raji xenografts in NOD/scid mice and reconstituted human PBMC. Altogether, these data suggest that AFM11 is a highly efficacious novel drug candidate for the treatment of hematological malignancies and displays an advantageous safety profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4624. doi:1538-7445.AM2012-4624

Generation of a monoclonal antibody reactive to prefusion myocytes

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion–incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell–cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.Electronic supplementary materialThe online version of this article (doi:10.1007/s10974-011-9247-8) contains supplementary material, which is available to authorized users.

Synthesis and Evaluation of Hydrophilic Linkers for Antibody–Maytansinoid Conjugates

The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.