On-target restoration of a split T cell-engaging antibody for precision immunotherapy.

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

CD33 target validation and sustained depletion of AML blasts in long-term cultures by the bispecific T-cell-engaging antibody AMG 330.

Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells. Here, we evaluated the CD33/CD3-bispecific T cell engaging (BiTE) antibody (AMG 330) for its suitability as a therapeutic agent in acute myeloid leukemia (AML). We first assessed CD33 expression levels by flow cytometry and found expression in >99% of patient samples (n = 621). CD33 was highest expressed in AMLs with NPM1 mutations (P < .001) and lower in AMLs with complex karyotypes and t(8;21) translocations (P < .001). Furthermore, leukemic stem cells within the CD34(+)/CD38(-) compartment displayed CD33 at higher levels than healthy donor stem cells (P = .047). In MS-5 feeder cell-based long-term cultures that supported the growth of primary AML blasts for up to 36 days, AMG 330 efficiently recruited and expanded residual CD3(+)/CD45RA(-)/CCR7(+) memory T cells within the patient sample. Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples and substantially in the remaining samples in a time-dependent manner. This study provides the first correlation of CD33 expression levels with AML genotype in a comprehensive analysis of adult patients. Targeting CD33 ex vivo using AMG 330 in primary AML samples led to T cell recruitment and expansion and remarkable antibody-mediated cytotoxicity, suggesting efficient therapeutic potential in vivo.

IgE knock-in mice suggest a role for high levels of IgE in basophil-mediated active systemic anaphylaxis.

Immunoglobulin E (IgE) production is tightly regulated at the cellular and genetic levels and is believed to be central to allergy development. At least two cellular pathways exist that lead to systemic anaphylaxis reactions in vivo: IgE-sensitized mast cells and IgG1-sensitized basophils. Passive anaphylaxis, by application of allergen and allergen-specific antibodies in mice, indicates a differential contribution of immunoglobulin isotypes to anaphylaxis. However, analysis of a dynamic immunization-mediated antibody response in anaphylaxis is difficult. Here, we generated IgE knock-in mice (IgE(ki) ), which express the IgE heavy chain instead of IgG1, in order to analyze the contribution of IgG1 and IgE to active anaphylaxis in vivo. IgE(ki) mice display increased IgE production both in vitro and in vivo. The sensitization of IgE(ki) mice by immunization followed by antigen challenge leads to increased anaphylaxis. Homozygous IgE(ki) mice, which lack IgG1 due to the knock-in strategy, are most susceptible to active systemic anaphylaxis. The depletion of basophils demonstrates their importance in IgE-mediated anaphylaxis. Therefore, we propose that an enhanced, antigen-specific, polyclonal IgE response, as is the case in allergic patients, is probably the most efficient way to sensitize basophils to contribute to systemic anaphylaxis in vivo.

Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL.

Persistence or recurrence of minimal residual disease (MRD) after chemotherapy results in clinical relapse in patients with acute lymphoblastic leukemia (ALL). In a phase 2 trial of B-lineage ALL patients with persistent or relapsed MRD, a T cell-engaging bispecific Ab construct induced an 80% MRD response rate. In the present study, we show that after a median follow-up of 33 months, the hematologic relapse-free survival of the entire evaluable study cohort of 20 patients was 61% (Kaplan-Meier estimate). The hema-tologic relapse-free survival rate of a subgroup of 9 patients who received allogeneic hematopoietic stem cell transplantation after blinatumomab treatment was 65% (Kaplan-Meier estimate). Of the subgroup of 6 Philadelphia chromosome-negative MRD responders with no further therapy after blinatumomab, 4 are in ongoing hematologic and molecular remission. We conclude that blinatumomab can induce long-lasting complete remission in B-lineage ALL patients with persistent or recurrent MRD. The original study and this follow-up study are registered at www.clinicaltrials.gov as NCT00198991 and NCT00198978, respectively.

Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival.

PURPOSE: Blinatumomab, a bispecific single-chain antibody targeting the CD19 antigen, is a member of a novel class of antibodies that redirect T cells for selective lysis of tumor cells. In acute lymphoblastic leukemia (ALL), persistence or relapse of minimal residual disease (MRD) after chemotherapy indicates resistance to chemotherapy and results in hematologic relapse. A phase II clinical study was conducted to determine the efficacy of blinatumomab in MRD-positive B-lineage ALL. PATIENTS AND METHODS: Patients with MRD persistence or relapse after induction and consolidation therapy were included. MRD was assessed by quantitative reverse transcriptase polymerase chain reaction for either rearrangements of immunoglobulin or T-cell receptor genes, or specific genetic aberrations. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dose of 15 µg/m2/24 hours. RESULTS: Twenty-one patients were treated, of whom 16 patients became MRD negative. One patient was not evaluable due to a grade 3 adverse event leading to treatment discontinuation. Among the 16 responders, 12 patients had been molecularly refractory to previous chemotherapy. Probability for relapse-free survival is 78% at a median follow-up of 405 days. The most frequent grade 3 and 4 adverse event was lymphopenia, which was completely reversible like most other adverse events. CONCLUSION: Blinatumomab is an efficacious and well-tolerated treatment in patients with MRD-positive B-lineage ALL after intensive chemotherapy. T cells engaged by blinatumomab seem capable of eradicating chemotherapy-resistant tumor cells that otherwise cause clinical relapse.

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety. METHODS: We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation. RESULTS: ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells. CONCLUSION: A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Tumor regression in cancer patients by very low doses of a T cell-engaging antibody.

Previous attempts have shown the potential of T cells in immunotherapy of cancer. Here, we report on the clinical activity of a bispecific antibody construct called blinatumomab, which has the potential to engage all cytotoxic T cells in patients for lysis of cancer cells. Doses as low as 0.005 milligrams per square meter per day in non-Hodgkin's lymphoma patients led to an elimination of target cells in blood. Partial and complete tumor regressions were first observed at a dose level of 0.015 milligrams, and all seven patients treated at a dose level of 0.06 milligrams experienced a tumor regression. Blinatumomab also led to clearance of tumor cells from bone marrow and liver. T cell-engaging antibodies appear to have therapeutic potential for the treatment of malignant diseases.

Systemic spread is an early step in breast cancer.

It is widely accepted that metastasis is a late event in cancer progression. Here, however, we show that tumor cells can disseminate systemically from earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. Wild-type mice transplanted with single premalignant HER-2 transgenic glands displayed disseminated tumor cells and micrometastasis in bone marrow and lungs. The number of disseminated cancer cells and their karyotypic abnormalities were similar for small and large tumors in patients and mouse models. When activated by bone marrow transplantation into wild-type recipients, 80 early-disseminated cancer cells sufficed to induce lethal carcinosis. Therefore, release from dormancy of early-disseminated cancer cells may frequently account for metachronous metastasis.

A multimarker real-time RT-PCR for MAGE-A gene expression allows sensitive detection and quantification of the minimal systemic tumor load in patients with localized cancer.

INTRODUCTION: Distant metastases of solid tumors are usually associated with fatal outcome. Disseminated cancer cells are considered early indicators of metastasis. Their sensitive detection and quantification would be a valuable tool for staging of disease and as guidance for therapeutic decisions. EXPERIMENTAL DESIGN: We established a highly sensitive and quantitative multimarker real-time RT-PCR assay for amplification of cancer-related genes MAGE-A1, -A2, -A3/6, -A4, -A10 and -A12 using SYBR green I to detect one single tumor cell in 2 mL of blood or bone marrow. The feasibility of the assay was tested in a large cohort of 177 patients with locally confined prostate carcinoma. RESULTS: Analysis revealed frequent MAGE expression in venous blood and bilateral bone marrow samples (25.5% of all cases) and yielded the first quantitative profile of MAGE expression with a broad range of transcript concentrations for individual markers in the minimal systemic tumor load of patients with localized cancer. CONCLUSIONS: Rare transcripts of different MAGE-A genes can be quantified in clinical samples of cancer patients by a sensitive multimarker real-time RT-PCR. Because of frequent expression of MAGE genes in various types of cancer the multimarker MAGE real-time RT-PCR may be generally useful for detection, quantification and characterization of the individual disseminated tumor load in cancer patients.

Cancer immunotherapy and preclinical studies: why we are not wasting our time with animal experiments.

Experimental research on the immune response to transplanted tumors has led to pioneering discoveries that laid many of the foundations for the current field of immunology. Experimental research in oncology has proven that murine and human tumors have antigens that are truly cancer specific. This article discusses research investigating how can antigens on cancer cells be used to help eradicate cancer.

Preferential targeting of CD4-CCR5 complexes with bifunctional inhibitors: a novel approach to block HIV-1 infection.

Two receptors, CD4 and one of several chemokine receptors, are required for cellular HIV-1 infection, with CCR5 being the main coreceptor for macrophage-tropic strains. We have designed bifunctional fusion proteins, consisting of RANTES/CCL5 and a single-chain Fv Ab fragment against CD4 to simultaneously block CD4 and CCR5. The fusion proteins bind to both receptors, compete with RANTES/CCL5 binding, and induce down-modulation of CCR5 approximately 10 times more efficiently on CD4+ compared with CD8+ T cells. Moreover, after short incubation and subsequent washout, a significant down-modulation of CCR5 was only seen with the fusion proteins and only on CD4+ cells, but not with unmodified RANTES or on CD4- cells, indicating a preferential targeting of CCR5 on CD4+ T cells. The fusion proteins block M-tropic HIV infection more efficiently than RANTES/CCL5 and CD4 Abs alone or in combination. To our knowledge this is the first report of simultaneous blockade of an HIV-1 receptor and coreceptor with bifunctional inhibitors.

Immunomagnetic enrichment, genomic characterization, and prognostic impact of circulating melanoma cells.

PURPOSE: The finding of melanoma cells in the peripheral blood, thus far mainly inferred from the PCR-based demonstration of tyrosinase mRNA, has been associated with metastatic melanoma. Neither the malignant nature nor the prognostic significance of circulating cells could be established. To address this question, we analyzed immunomagnetically isolated circulating melanoma cells for chromosomal aberrations and performed a clinical follow-up study of the enrolled patients. EXPERIMENTAL DESIGN: In a prospective study, blood samples were taken from 164 melanoma patients and 50 donors without malignant disease. Circulating melanoma cells were enriched by immunomagnetic cell sorting using a murine monoclonal antibody against the melanoma-associated chondroitin sulfate proteoglycan. To prove the malignant origin of the positive cells and to define their chromosomal aberrations, we analyzed the genomes of 15 individually isolated cells from seven patients by single-cell comparative genomic hybridization (SCOMP). RESULTS: Absolute and relative frequencies of circulating melanoma cells were associated with stage and with the presence or absence of detectable tumor. The detection of two or more cells correlated significantly with a reduced survival of patients with metastatic melanoma. All of the cells that were analyzed by SCOMP displayed multiple chromosomal changes and carried aberrations typical for melanoma. CONCLUSIONS: Immunomagnetic enrichment enables isolation and genomic characterization of circulating melanoma cells. The prognostic impact on survival of metastatic patients apparently reflects the aggressiveness of an ongoing tumor spread. Direct genomic analysis of the enriched and isolated cells will help to clarify the molecular-genetic basis of the establishment of generalized melanoma.

Natural killer cells activated by MHC class I(low) targets prime dendritic cells to induce protective CD8 T cell responses.

Conserved molecular patterns derived from pathogenic microorganisms prime antigen-presenting dendritic cells (DC) to induce adaptive T cell responses. In contrast, virus-infected or tumor cells that express low levels of major histocompatibility complex (MHC) class I activate natural killer (NK) cells for direct killing. It is unknown whether NK cell recognition of MHC class I(low) targets can also induce adaptive T cell responses. Here, we show that MHC class I(low) targets initiate a cascade of immune responses, starting with the immediate activation of NK cells. The activated NK cells then prime DC to produce IL-12 and to induce highly protective CD8 T cell memory responses. Therefore, sensing of MHC class I(low) targets by NK cells can link innate and adaptive immunity to induce protective T cell responses and may alarm the immune system during early infection with noncytopathic viruses.

From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression.

According to the present view, metastasis marks the end in a sequence of genomic changes underlying the progression of an epithelial cell to a lethal cancer. Here, we aimed to find out at what stage of tumor development transformed cells leave the primary tumor and whether a defined genotype corresponds to metastatic disease. To this end, we isolated single disseminated cancer cells from bone marrow of breast cancer patients and performed single-cell comparative genomic hybridization. We analyzed disseminated tumor cells from patients after curative resection of the primary tumor (stage M0), as presumptive progenitors of manifest metastasis, and from patients with manifest metastasis (stage M1). Their genomic data were compared with those from microdissected areas of matched primary tumors. Disseminated cells from M0-stage patients displayed significantly fewer chromosomal aberrations than primary tumors or cells from M1-stage patients (P < 0.008 and P < 0.0001, respectively), and their aberrations appeared to be randomly generated. In contrast, primary tumors and M1 cells harbored different and characteristic chromosomal imbalances. Moreover, applying machine-learning methods for the classification of the genotypes, we could correctly identify the presence or absence of metastatic disease in a patient on the basis of a single-cell genome. We suggest that in breast cancer, tumor cells may disseminate in a far less progressed genomic state than previously thought, and that they acquire genomic aberrations typical of metastatic cells thereafter. Thus, our data challenge the widely held view that the precursors of metastasis are derived from the most advanced clone within the primary tumor.

Limitations of the immunocytochemical detection of isolated tumor cells in frozen samples of bone marrow obtained from melanoma patients.

We report on a case of a 70-year-old woman with an ocular melanoma, which was diagnosed and treated 14 years ago. The patient was referred to the hospital with a suspected lymphoma. Cytological examination of bone marrow proved a marked infiltration with melanoma cells. Because detection of isolated tumor cells in the bone marrow of patients with various types of tumors was shown to be of prognostic significance and since current tumor-staging techniques are unable to detect single disseminated tumor cells or small aggregates of tumor cells, which might be the seed for subsequent metastatic relapse, we therefore evaluated the feasibility of immunocytochemical screening of bone marrow aspirates of 36 melanoma patients in different clinical stages using three monoclonal antibodies against melanoma-associated antigens in comparison with 43 non-melanoma control patients. Two of these antibodies (HMB45 and NKI-beteb) are directed against the melanoma antigen gp100/pmel17, whereas the third one (TA99) recognizes gp75/Tyrosinase-related protein 1 (TRP-1). None of the patients demonstrated a macroscopic bone marrow infiltration as was present in our patient with metastatic ocular melanoma. Seven (20.6%) of the 34 eligible melanoma patients presented with cells in the bone marrow positive for one or more of the above-mentioned melanosomal markers. Four of the positive patients were clinically free of tumors by the time of puncture, whereas the remaining 3 patients showed overt metastases in the subcutaneous fat (2 patients) and the brain (1 patient). On the other hand, 20 (66%) of the 29 patients with negative bone marrow findings also presented with clinical advanced disease with overt metastasis in the skin, lymph node, spleen, liver, lung, bone and brain. In conclusion, immunocytochemical screening of bone marrow samples is a feasible procedure that allows the detection of micrometastatic tumor cells in a subset of melanoma patients. Massive invasion of bone marrow with melanoma cells is a rare event even in far-advanced metastatic stages and no clear correlation between tumor load and bone marrow infiltration could be established.

T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct.

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.

Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer.

BACKGROUND: Because cancer patients with small tumours often relapse despite local and systemic treatment, we investigated the genetic variation of the precursors of distant metastasis at the stage of minimal residual disease. Disseminated tumour cells can be detected by epithelial markers in mesenchymal tissues and represent targets for adjuvant therapies. METHODS: We screened 525 bone-marrow, blood, and lymph-node samples from 474 patients with breast, prostate, and gastrointestinal cancers for single disseminated cancer cells by immunocytochemistry with epithelial-specific markers. 71 (14%) of the samples contained two or more tumour cells whose genomic organisation we studied by single cell genomic hybridisation. In addition, we tested whether TP53 was mutated. Hierarchical clustering algorithms were used to determine the degree of clonal relatedness of sister cells that were isolated from individual patients. FINDINGS: Irrespective of cancer type, we saw an unexpectedly high genetic divergence in minimal residual cancer, particularly at the level of chromosomal imbalances. Although few disseminated cells harboured TP53 mutations at this stage of disease, we also saw microheterogeneity of the TP53 genotype. The genetic heterogeneity was strikingly reduced with the emergence of clinically evident metastasis. INTERPRETATION: Although the heterogeneity of primary tumours has long been known, we show here that early disseminated cancer cells are genomically very unstable as well. Selection of clonally expanding cells leading to metastasis seems to occur after dissemination has taken place. Therefore, adjuvant therapies are confronted with an extremely large reservoir of variant cells from which resistant tumour cells can be selected.