Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.     

A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combina-tion are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.     

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 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 single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all three antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (K_D = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (K_D = 29 nM). All three fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC₅₀ for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.Key words: leukemia, natural killer cells, antibody-derivatives, dual targeting, sctb     

Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.     

Intracellular domains of target antigens influence their capacity to trigger antibody-dependent cell-mediated cytotoxicity

Ab-mediated signaling in tumor cells and Ab-dependent cell-mediated cytotoxicity (ADCC) are both considered as relevant effector mechanisms for Abs in tumor therapy. To address potential interactions between these two mechanisms, we generated HER-2/neu- and CD19-derived chimeric target Ags, which were expressed in experimental tumor target cells. HER-2/neu-directed Abs were documented to mediate effective ADCC with both mononuclear cells (MNCs) and polymorphonuclear granulocytes (PMNs), whereas Abs against CD19 were effective only with MNCs and not with PMNs. We generated cDNA encoding HER-2/CD19 or CD19/HER-2 (extracellular/intracellular) chimeric fusion proteins by combining cDNA encoding extracellular domains of HER-2/neu or CD19 with intracellular domains of CD19 or HER-2/neu, respectively. After transfecting wild-type HER-2/neu or chimeric HER-2/CD19 into Raji Burkitt's lymphoma cells and wild-type CD19 or chimeric CD19/HER-2 into SK-BR-3 breast cancer cells, target cell lines were selected for high membrane expression of transfected Ags. We then investigated the efficacy of tumor cell lysis by PMNs or MNCs with CD19- or HER-2/neu-directed Ab constructs. MNCs triggered effective ADCC against target cells expressing wild-type or chimeric target Ag. As expected, PMNs killed wild-type HER-2/neu-transfected, but not wild-type CD19-transfected target cells. Interestingly, however, PMNs were also effective against chimeric CD19/HER-2-transfected, but not HER-2/CD19-transfected target cells. In conclusion, these results demonstrate that intracellular domains of target Ags contribute substantially to effective Ab-mediated tumor cell killing by PMNs.     

Human neutrophil interactions of a bispecific monoclonal antibody targeting tumor and human Fcγ RIII

 2B1 is a bispecific murine monoclonal antibody (bsmAb) targeting the c-erbB-2 and CD16 (FcγRIII) antigens. c-erbB-2 is over-expressed by a variety of adenocarcinomas, and CD16, the low-affinity Fcγ receptor for aggregated immunoglobulins, is expressed by polymorphonuclear leukocytes (PMN), natural killer (NK) cells and differentiated mononuclear phagocytes. 2B1 potentiates the in vitro lysis of c-erbB-2 over-expressing tumors by NK cells and macrophages. In this report, the interactions between 2B1 and PMN were investigated to assess the impact of these associations on in vitro 2B1-promoted tumor cytotoxicity by human NK cells. The peak binding of 2B1 to PMN was observed at a concentration of 10 μg/ml 2B1. However, 2B1 rapidly dissociated from PMN in vitro at 37°C in non-equilibrium conditions. This dissociation was not caused by CD16 shedding. When PMN were labeled with ¹²⁵I-2B1 and incubated at 37°C and the supernatants examined by HPLC analysis, the Fab regions of dissociated 2B1 were not complexed with shed CD16 extracellular domain. While most of the binding of 2B1 to PMN was solely attributable to Fab-directed binding to FcγRIII, PMN-associated 2B1 also bound through Fcγ-domain/FcγRII interactions. 2B1 did not promote in vitro PMN cytotoxicity against c-erbB-2-expressing SK-OV-3 tumor cells. When PMN were coincubated with peripheral blood lymphocytes, SK-OV-3 tumor and 2B1, the concentration of 2B1 required for maximal tumor lysis was lowered. Although PMN may serve as a significant competitive binding pool of systemically administered 2B1 in vivo, the therapeutic potential of the targeted cytotoxicity properties of this bsmAb should not be compromised. Received: 3 May 1995 / Accepted: 6 February 1996     

Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

Background

Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4).

Methodology/Principal Findings

In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4⁺ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G⁺ FcγRIIIa(CD16A)⁺ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4⁺ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A⁺ CD56⁺ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells.

Conclusions/Significance

Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A⁺ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.     

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2⁺ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2⁺ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.     

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.     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

A herpes simplex virus recombinant that exhibits a single-chain antibody to HER2/neu enters cells through the mammary tumor receptor, independently of the gD receptors

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells.     

Human leukocytes kill varicella-zoster virus-infected fibroblasts in the presence of murine monoclonal antibodies to virus-specific glycoproteins.

Seven murine monoclonal antibodies reacting with major glycoproteins of varicella-zoster virus were tested for functional activity in assays for antibody-dependent cellular cytotoxicity (ADCC) and antibody-plus-complement-mediated lysis. Human peripheral blood mononuclear cells killed varicella-zoster virus-infected fibroblasts in the presence of three of four monoclonal antibodies directed against gp98/62 and a single monoclonal antibody directed against gp118. Neither of two monoclonal antibodies directed against gp66 was able to mediate ADCC. In 18-h assays, adherent effector cells were more active than nonadherent effector cells in mediating ADCC. Adherent cells treated with anti-Leu-11b and complement retained their cytotoxic activity, suggesting that monocytes are responsible for most of the adherent-cell-mediated cytotoxicity. Both immunoglobulin G1 and G2a murine monoclonal antibodies were able to participate in ADCC. Of the two immunoglobulin G2a monoclonal antibodies tested, both of which reacted with gp98/62, only one mediated lysis in the presence of complement. These results indicate that some murine monoclonal antibodies against major glycoproteins of varicella-zoster virus have functional activity in cytotoxicity assays.     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.     

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.     

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     

Expression of single-chain antibody-barstar fusion in plants

We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar-barnase system. Based on barstar-barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.     

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer

 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL_17-1A-VH_17-1A-VH_CD3-VL_CD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (⁵¹Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8⁺ as well as CD4⁺ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8⁺ cells induced high ⁵¹Cr release within 4 h while CD4⁺ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997