A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy.     

A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen double-positive cells by dual-targeting

To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.     

A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen double-positive cells by dual-targeting

To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.Key words: triplebodies, natural killer cells, dual-targeting, selective cytotoxicity     

Potential of chimeric antigen receptor (CAR)‐redirected immune cells in breast cancer therapies: Recent advances

Despite substantial developments in conventional treatments such as surgery, chemotherapy, radiotherapy, endocrine therapy, and molecular‐targeted therapy, breast cancer remains the leading cause of cancer mortality in women. Currently, chimeric antigen receptor (CAR)–redirected immune cell therapy has emerged as an innovative immunotherapeutic approach to ameliorate survival rates of breast cancer patients by eliciting cytotoxic activity against cognate tumour‐associated antigens expressing tumour cells. As a crucial component of adaptive immunity, T cells and NK cells, as the central innate immune cells, are two types of pivotal candidates for CAR engineering in treating solid malignancies. However, the biological distinctions between NK cells‐ and T cells lead to differences in cancer immunotherapy outcomes. Likewise, optimal breast cancer removal via CAR‐redirected immune cells requires detecting safe target antigens, improving CAR structure for ideal immune cell functions, promoting CAR‐redirected immune cells filtration to the tumour microenvironment (TME), and increasing the ability of these engineered cells to persist and retain within the immunosuppressive TME. This review provides a concise overview of breast cancer pathogenesis and its hostile TME. We focus on the CAR‐T and CAR‐NK cells and discuss their significant differences. Finally, we deliver a summary based on recent advancements in the therapeutic capability of CAR‐T and CAR‐NK cells in treating breast cancer.     

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor γ chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-γ clones, and lysis of erbB-2⁺ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-γ, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL. Received: 23 July 1998 / Accepted: 5 October 1998     

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by ⁵¹Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

Peptide mapping of the epitope on target cells recognized by nonspecific cytotoxic cells (NCC) from channel catfish

Nonspecific cytotoxic cells (NCC) may comprise an important effector population specific for recognition of aberrant (tumour) cells, regulation of cell interactions including antibacterial action and lysis of protozoan parasites. In the present study, peptides were synthesized based on the amino acid sequence of a novel protein (Natural Killer cell Target Antigen, NK Tag) found on the protozoan parasite Tetrahymena pyriformis and on NCC-sensitive tumour target cells. Partially purified NK Tag was obtained from Tetrahymena. It inhibited NCC lysis of a large variety of mammalian tumour target cells. Synthetic peptides composed of short 20 mer sequences obtained from the N-terminal and midregion portions of NK Tag were tested for their ability to inhibit NCC cytotoxicity. Synthetic peptide comprised of aa # 55-74 significantly inhibited NCC lysis of IM-9 target cells. A monoclonal antibody generated against an N-terminal dodecapeptide of NK Tag bound to Tetrahymena and to several mammalian NK-sensitive target cells including K562, YAC-1, U937, NC-37, EL-4, IM-9, HL-60 and MOLT-4. NK Tag sequence comparisons using Swisspro database revealed no significant homologies except in a restricted domain region of several glycolytic pathway enzymes. A supergene family relationship was indicated because of these similarities.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Intranasal administration of Lactobacillus rhamnosus GG protects mice from H1N1 influenza virus infection by regulating respiratory immune responses

Aims: To investigate whether intranasal Lactobacillus administration protects host animals from influenza virus (IFV) infection by enhancing respiratory immune responses in a mouse model. Methods and Results: After 3 days of intranasal exposure to Lactobacillus rhamnosus GG (LGG), BALB/c mice were infected with IFV A/PR/8/34 (H1N1). Mice treated with LGG showed a lower frequency of accumulated symptoms and a higher survival rate than control mice (P < 0·05). The YAC‐1 cell‐killing activity of lung cells isolated from mice treated with LGG was significantly greater than those isolated from control mice (P < 0·01). Intranasal administration of LGG significantly increased mRNA expression of interleukin (IL)‐1β, tumour necrosis factor (TNF) and monocyte chemotactic protein (MCP)‐1 (P < 0·01). Conclusions: These results suggest that intranasal administration of LGG protects the host animal from IFV infection by enhancing respiratory cell‐mediated immune responses following up‐regulation of lung natural killer (NK) cell activation. Significance and Impact of Study: We have demonstrated that probiotics might protect host animals from viral infection by stimulating immune responses in the respiratory tract.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2

Background aimsGraft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts.MethodsAn approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2.ResultsMMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells.ConclusionsThis study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different