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CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in several solid malignancies, including gastric, lung, mesothelioma, pancreatic and ovarian cancers. While several MSLN-targeting therapeutic approaches are in development, only limited efficacy has been achieved in patients. A potential shortcoming of several described antibody-based approaches is that they target the membrane distal region of MSLN and, additionally, are known to be handicapped by the high levels of circulating soluble MSLN in patients. We show here, using monoclonal antibodies (mAbs) targeting different MSLN-spanning epitopes, that the membrane-proximal region resulted in more efficient killing of MSLN-positive tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Surprisingly, no augmented killing was observed in antibody-dependent cellular phagocytosis (ADCP) by mAbs targeting this membrane-proximal region. To further increase the ADCP potential, we, therefore, generated bispecific antibodies (bsAbs) coupling a high-affinity MSLN binding arm to a blocking CD47 arm. Here, targeting the membrane-proximal domain of MSLN demonstrated enhanced ADCP activity compared to membrane-distal domains when the bsAbs were used in in vitro phagocytosis killing assays. Importantly, the superior anti-tumor activity was also translated in xenograft tumor models. Furthermore, we show that the bsAb approach targeting the membrane-proximal epitope of MSLN optimized ADCC activity by augmenting FcγR-IIIA activation and enhanced ADCP via a more efficient blockade of the CD47/SIRPα axis.

CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal “don't eat me” signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701–induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20–targeted therapy. Mol Cancer Ther; 17(8); 1739–51. ©2018 AACR.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.

Experiments were performed with human plasma irradiated in vitro or in vivo in order to evaluate the extent to which clastogenic factors might disturb the adaptive response to DNA-damaging factors currently studied in our laboratory. The studies were carried out with plasma isolated from whole blood given 4 Gy of X-rays in vitro and with plasma from people receiving local radiotherapy at a total dose of about 60 Gy gamma rays. Addition of irradiated plasma to culture medium did not result in a statistically significant increase in structural aberrations in chromosomes of non-irradiated normal blood.

With the aim of limiting hospitalisation and a possible loss of autonomy, the paramedical team of the post-emergency and geriatric orientation unit at Nancy general hospital endeavours to anticipate the life programme as soon as the elderly patient arrives. A daily review and close collaboration with the social worker are key elements in the care.

Radiobiological data indicate that fast neutrons could bring a benefit in the treatment of some tumour types, and suggest mechanisms through which this benefit could be achieved. However, radiobiology also clearly indicates that there is a need for patient selection as well as for a high-physical selectivity. The main difficulty when interpreting the results of neutron therapy are the poor technical conditions in which the first treatments were applied. This explains why the value and the place of neutron therapy are not universally recognized, although more than 15,000 patients have been treated so far worldwide. There are, however, clinical indications of fast neutrons bringing a benefit for the following tumour sites: salivary glands, paranasal sinuses, soft tissue sarcomas, prostatic adenocarcinomas, palliative treatment of melanoma and rectum. These tumours represent about 10-15% of all patients currently referred to the radiation therapy departments.

PURPOSE: Between 1978 and 1998, 533 prostate adenocarcinoma patients were treated with mixed photon-neutron radiotherapy. We report on a retrospective series of patients for whom the side effects of the treatment and their impact on quality of life were assessed by a mailed questionnaire. METHODS AND MATERIALS: The European Organization for Research and Treatment of Cancer quality-of-life core questionnaire and a prostate-specific questionnaire were used. Between 1990 and 1996, 308 consecutive patients were treated. Two protocols were used: pelvic fields (50 Gy equivalent photons, 2 Gy/fraction) followed by a prostate boost (66 Gy) or prostate alone. The neutron/photon ratio varied. The questionnaire was mailed to 262 patients presumed to be alive. RESULTS: Of the 262 patients, 230 replied. Of the 230 patients, 73% had no trouble doing strenuous activities and 4% had trouble with taking a short walk. The overall physical condition and quality-of-life questions received a mean score of 5.2 and 5.3 on a 7-point scale, respectively. Twenty-two percent had bowel movements at least four times daily, and 6% did so six times or more. Retaining stool was a problem in 26%, and only 38% reported full continence; 17% urinated four times or more nightly. Urinary incontinence was scored as "quite a bit" or "very much" in 11% and 5%, respectively. Hematuria and dysuria (pain) were reported by 7% and 16%, respectively, mainly as moderate. Only 28% reported easy erections, but 75% judged the sexual change acceptable. A greater neutron/photon ratio was significantly associated with more bowel problems (p = 0.003). CONCLUSION: Mixed photon-neutron therapy for prostate cancer was associated with significant patient-reported side effects. Their significant effect on patients' quality of life is described.

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.

PURPOSE: To retrospectively evaluate survival, progression-free survival (PFS) and biological response in a series of patients irradiated with mixed neutron/photon beams for locally advanced prostate cancer in our institution. PATIENTS AND METHODS: Three hundred and eight patients were treated between January 1990 and December 1996. Fifty-five of these were recruited for pT3 or pN1 tumors after radical prostatectomy. Neoadjuvant androgen deprivation was given in 106 patients. The treatment protocol consisted of a mixed photon/neutron irradiation in a two-to-three proportion, up to a total equivalent dose of 66 Gy (assuming a clinical RBE value of 2.8). Pre- and post-treatment PSA determinations were available in practically all cases. Study endpoints were overall survival (OAS) and progression-free survival (PFS). The Cox proportional hazard regression model was used to investigate the prognostic value of baseline characteristics on survival and progression-free survival were a progression was defined as local, regional, metastatic or biological progression. Mean age was 69 years (49-86); mean pretreatment PSA was 15 (0.5-330) in all patients and 14 (0.5-160) in those receiving neoadjuvant hormonotherapy; seven patients only had an initial PSA < or = 4 ng/mL; 15% were T1, 46% were T2, 28% were T3 or pT3 and 4% were T4 (7% unspecified); WHO grade of differentiation was I in 38%, II in 38% and III in 14% (5% unspecified). RESULTS: The median follow-up was 2.8 years (0-7.8). Five-year overall survival (OAS) was 79% (95% CI: 71-87%) and 5-year progression-free survival (PFS) was 64% (95% CI: 54-74%) for the entire series. PFS in patients with an initial PSA > or = 20 ng/mL was the same. PFS could be predicted by two optimal Cox regression models, one including histological grade (p = 0.003) and initial PSA (p = 0.0009) as cofactors, the other including histological grade (p = 0.003) and T stage (p = 0.02). The main prognostic factors for overall survival were PSA and age. Biological responses with PSA < 1.5 ng/mL, < 1 ng/mL and < 0.5 ng/mL at any time after treatment were documented in 70%, 61% and 47% of the patients, respectively. CONCLUSION: Five-year OAS was 79%, PFS was 64%, and biological response was 70% for prostate cancer patients treated with mixed photon/neutron beams as applied at Louvain-la-Neuve, which are good results as compared with the literature. The usual prognostic factors were confirmed.

The multileaf collimator of the fast neutron therapy facility at Louvain-la-Neuve is described, as well as some of the physics experiments performed in order to evaluate the attenuation of neutron beams in different materials and thus optimize the composition of the collimator leaves. The multileaf collimator consists of two sets of 22 leaves each, which can be moved independently. They are made of iron and their thickness is 95 cm. Seven borated polyethylene disks are located in the distal part of the leaves in order to absorb more efficiently the low-energy component of the neutron spectrum. The width of the leaves is 1 cm at their distal part. The leaves can move 11 cm outwards and 6 cm inwards from their reference position, and field sizes up to 25.7 x 24.8 cm, as well as irregular field shapes, can be obtained. The inner part of the leaves and their two sides are always focused on the target. The complete multileaf collimator can rotate around the beam axis, from -90 degrees to +90 degrees from the reference position. The width of the penumbra (80-20% isodoses) is 0.64 cm and 1.17 cm at the depth of the maximum buildup and at 10 cm in depth respectively, for a 10 x 10 cm field size. The collimator is adequate for the energy of the p(65)+Be neutron beam of Louvain-la-Neuve and has been adapted to the fixed vertical beam. It has been designed following the original plans of Scanditronix, adjusted and fully assembled at the workshop of the Centre de Recherches du Cyclotron (CRC). Systematic measurements were performed in order to optimize the design and the composition of the leaves. In particular the attenuations of the actual beam and of monoenergetic neutron beams were measured in different materials such as iron and polyethylene. Above (upstream) the multileaf collimator, a fixed pre-collimator (iron thickness 50 cm; section 1 x 1 m) defines a conical aperture aligned on the largest opening of the leaves. It contains the two transmission chambers and a 2 cm thick polyethylene layer used for hardening the beam.

A PET camera is used to visualize the positron activity induced during protonbeam therapy in order to verify directly the proton-treatment plans. The positron emitters created are predominantly the 15O and 11C, whose total activity amounts to 12 MBq after an irradiation with 85 MeV protons, delivering 3 Gy in a volume of approximately 300 cm3. Although this method is a useful verification of patient set-up, care must be taken when deriving dose distributions from activity distributions. Correlation between both quantities is difficult, moreover at the last millimeters of their range, protons will no longer activate tissue. Due to the short half-lives the PET camera must be located close to the treatment facility.

We reviewed retrospectively a series of 58 patients with deeply invasive bladder cancer treated with fast neutron therapy (p(65) + Be) in order to evaluate its tolerance and side effects. Patients were divided into three groups according to treatment technique. Patients of group A received whole pelvis irradiation up to 50 Gy photon equivalent followed by a boost to the bladder up to 57-66 Gy photon equivalent (40-56 days). Group B patients were treated by a split course regimen of 30 Gy photon equivalent on the whole pelvis at 3-4 weeks interval (66-108 days). Group C patients, not suitable for radical treatment, received only 40-54 Gy photon equivalent (26-70 days). The overall 5-year actuarial survival rate was 30% (SE 8%). As expected, T stage was a statistically significant prognostic factor. The overall local control rate reached 21% at 4 years. Acute and late side effects were minimal to moderate. These results suggest that high-energy neutron beam treatment is at least as effective as photon beam treatment for bladder carcinoma, without a higher incidence of major side effects.

With conventional photon therapy, radiographs are widely used for the verification of patient positioning. This method cannot be used with neutrons because of the poor sensitivity of standard films to neutrons compared with γ rays. In addition, there is little difference in energy absorption between bone and soft tissue for these neutron beams (Bewley et al, 1973; Catterall & Bewley, 1979). The problem can be solved by introducing into the collimator, at the target position, a diagnostic X-ray tube (Risler & Wootton, 1988). Another approach entails visualizing the neutron-induced activity within the patient using positron emission tomography (PET). At high neutron energies, shortlived positron emitters are produced (Kleck et al, 1988; Vynckier et al, 1988) with activities sufficiently high to be visualized; the dose-equivalent rate close to the patient as a result of this activation is ∼ 30 μSv h−1 immediately after irradiation (Tatcher et al, 1987). The same approach was reported recently for pion radiot...