LekCheck: A Prospective Study to Identify Perioperative Modifiable Risk Factors for Anastomotic Leakage in Colorectal Surgery.

OBJECTIVE: To assess potentially modifiable perioperative risk factors for anastomotic leakage in adult patients undergoing colorectal surgery. SUMMARY BACKGROUND DATA: Colorectal anastomotic leakage (CAL) is the single most important denominator of postoperative outcome after colorectal surgery. To lower the risk of CAL, the current research focused on the association of potentially modifiable risk factors, both surgical and anesthesiological. METHODS: A consecutive series of adult patients undergoing colorectal surgery with primary anastomosis was enrolled from January 2016 to December 2018. Fourteen hospitals in Europe and Australia prospectively collected perioperative data by carrying out the LekCheck, a short checklist carried out in the operating theater as a time-out procedure just prior to the creation of the anastomosis to check perioperative values on 1) general condition 2) local perfusion and oxygenation, 3) contamination, and 4) surgery related factors. Univariate and multivariate logistic regression analysis were performed to identify perioperative potentially modifiable risk factors for CAL. RESULTS: There were 1562 patients included in this study. CAL was reported in 132 (8.5%) patients. Low preoperative hemoglobin (OR 5.40, P < 0.001), contamination of the operative field (OR 2.98, P < 0.001), hyperglycemia (OR 2.80, P = 0.003), duration of surgery of more than 3 hours (OR 1.86, P = 0.010), administration of vasopressors (OR 1.80, P = 0.010), inadequate timing of preoperative antibiotic prophylaxis (OR 1.62, P = 0.047), and application of epidural analgesia (OR, 1.81, P = 0. 014) were all associated with CAL. CONCLUSIONS: This study identified 7 perioperative potentially modifiable risk factors for CAL. The results enable the development of a multimodal and multidisciplinary strategy to create an optimal perioperative condition to finally lower CAL rates.

Ergonomics in handheld and robot-assisted camera control: a randomized controlled trial.

BACKGROUND: Laparoscopic surgery potentially increases the physical burden to operating theater personnel and can cause physical discomfort. This study aims to evaluate if a robotic camera holder (AutoLap™ system) can improve ergonomics for the surgeon and the camera assistant during laparoscopic procedures. METHODS: A total of thirty cases were included and randomized (15 AutoLap™, 15 control). Five types of surgery were included: right hemicolectomy, fundoplication, sigmoid resection, rectopexy, and low anterior resection. The posture of the surgeon and assistant was photographed during predefined steps of the procedure. MATLAB was used to calculate angles relevant for the RULA score. The RULA score is a validated method to evaluate body posture, force and repetition of the upper extremities. Two investigators assessed the RULA score independently. Three subjective questionnaires (SMEQ, NASA TLX, and LED) were used to assess mental and physical discomfort. RESULTS: No differences in patient characteristics were observed. Sixteen fundoplications, seven right hemicolectomies, five sigmoid resections, one rectopexy, and one low anterior resection were included. The mean RULA score of the surgeon was comparable in both groups, 2.58 (AutoLap™) versus 2.72 (control). The mean RULA score of the assistant was significantly different in both groups, with 2.55 (AutoLap™) versus 3.70 (control) (p = 0.001). The inter-observer variability (ICC) was excellent with 0.93 (surgeon) and 0.97 (assistant). The questionnaires showed a significant difference in physical discomfort for the assistant. The LED and SMEQ score were significantly lower in the robotic group. The NASA TLX demonstrated a significant reduction in scores in all domains when using robotics with the exception of the mental domain. CONCLUSION: Use of the AutoLap™ system shows improvement in ergonomics and posture of the first assistant, and ergonomics of the surgeon are not affected. Furthermore, the subjective work load is significantly reduced by using a robotic camera holder. TRIAL REGISTRATION NUMBER: NCT0339960, https://clinicaltrials.gov/ct2/show/study/NCT03339960?term=autolap&rank=5 .

[Acute abdominal pain caused by neuroborreliosis]. / Acute buikpijn bij neuroborreliose.

BACKGROUND: Lyme disease is a multisystem disease which can present itself in several ways. When the nervous system is involved, it is called Lyme neuroborreliosis. Both central and peripheral nervous systems can be affected. CASE DESCRIPTION: A 39-year-old man visited the emergency department multiple times with severe abdominal-pain attacks with motoric unrest. Extensive diagnostic work-up was done, which was initially inconclusive. Lyme neuroborreliosis was suspected when he developed a facial-nerve palsy during admission; the abdominal pain was thought to be caused by thoracic radiculoneuropathy. Serologic testing for antibodies against Borrelia burgdorferi was positive, confirming the diagnosis. The patient was treated with intravenous ceftriaxone. CONCLUSION: This case shows abdominal pain being caused by radiculoneuropathy at thoracic level, an uncommon presentation of Lyme neuroborreliosis. Often, this diagnosis is only made when neurological paralysis occurs. Information regarding skin lesions or a recent tick bite can lead to earlier recognition of the diagnosis.

High turnover of tissue factor enables efficient intracellular delivery of antibody-drug conjugates.

Antibody-drug conjugates (ADC) are emerging as powerful cancer treatments that combine antibody-mediated tumor targeting with the potent cytotoxic activity of toxins. We recently reported the development of a novel ADC that delivers the cytotoxic payload monomethyl auristatin E (MMAE) to tumor cells expressing tissue factor (TF). By carefully selecting a TF-specific antibody that interferes with TF:FVIIa-dependent intracellular signaling, but not with the procoagulant activity of TF, an ADC was developed (TF-011-MMAE/HuMax-TF-ADC) that efficiently kills tumor cells, with an acceptable toxicology profile. To gain more insight in the efficacy of TF-directed ADC treatment, we compared the internalization characteristics and intracellular routing of TF with the EGFR and HER2. Both in absence and presence of antibody, TF demonstrated more efficient internalization, lysosomal targeting, and degradation than EGFR and HER2. By conjugating TF, EGFR, and HER2-specific antibodies with duostatin-3, a toxin that induces potent cytotoxicity upon antibody-mediated internalization but lacks the ability to induce bystander killing, we were able to compare cytotoxicity of ADCs with different tumor specificities. TF-ADC demonstrated effective killing against tumor cell lines with variable levels of target expression. In xenograft models, TF-ADC was relatively potent in reducing tumor growth compared with EGFR- and HER2-ADCs. We hypothesize that the constant turnover of TF on tumor cells makes this protein specifically suitable for an ADC approach.

Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma.

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.

Three-dimensional histologic validation of high-resolution SPECT of antibody distributions within xenografts.

UNLABELLED: Longitudinal imaging of intratumoral distributions of antibodies in vivo in mouse cancer models is of great importance for developing cancer therapies. In this study, multipinhole SPECT with sub-half-millimeter resolution was tested for exploring intratumoral distributions of radiolabeled antibodies directed toward the epidermal growth factor receptor (EGFr) and compared with full 3-dimensional target expression assessed by immunohistochemistry. METHODS: (111)In-labeled zalutumumab, a human monoclonal human EGFr-targeting antibody, was administered at a nonsaturating dose to 3 mice with xenografted A431 tumors exhibiting high EGFr expression. Total-body and focused in vivo tumor SPECT was performed at 0 and 48 h after injection and compared both visually and quantitatively with full 3-dimensional immunohistochemical staining for EGFr target expression. RESULTS: SPECT at 48 h after injection showed that activity was predominantly concentrated in the tumor (10.5% ± 1.3% of the total-body activity; average concentration, 30.1% ± 4.6% of the injected dose per cubic centimeter). (111)In-labeled EGFr-targeting antibodies were distributed heterogeneously throughout the tumor. Some hot spots were observed near the tumor rim. Immunohistochemistry indicated that the antibody distributions obtained by SPECT were morphologically similar to those obtained for ex vivo EGFr target expression. Regions showing low SPECT activity were necrotic or virtually negative for EGFr target expression. A good correlation (r = 0.86, P < 0.0001) was found between the percentage of regions showing low activity on SPECT and the percentage of necrotic tissue on immunohistochemistry. CONCLUSION: Multipinhole SPECT enables high-resolution visualization and quantification of the heterogeneity of (111)In-zalutumumab concentrations in vivo.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

An antibody-drug conjugate that targets tissue factor exhibits potent therapeutic activity against a broad range of solid tumors.

Tissue factor (TF) is aberrantly expressed in solid cancers and is thought to contribute to disease progression through its procoagulant activity and its capacity to induce intracellular signaling in complex with factor VIIa (FVIIa). To explore the possibility of using tissue factor as a target for an antibody-drug conjugate (ADC), a panel of human tissue factor-specific antibodies (TF HuMab) was generated. Three tissue factor HuMab, that induced efficient inhibition of TF:FVIIa-dependent intracellular signaling, antibody-dependent cell-mediated cytotoxicity, and rapid target internalization, but had minimal impact on tissue factor procoagulant activity in vitro, were conjugated with the cytotoxic agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Tissue factor-specific ADCs showed potent cytotoxicity in vitro and in vivo, which was dependent on tissue factor expression. TF-011-MMAE (HuMax-TF-ADC) was the most potent ADC, and the dominant mechanism of action in vivo was auristatin-mediated tumor cell killing. Importantly, TF-011-MMAE showed excellent antitumor activity in patient-derived xenograft (PDX) models with variable levels of tissue factor expression, derived from seven different solid cancers. Complete tumor regression was observed in all PDX models, including models that showed tissue factor expression in only 25% to 50% of the tumor cells. In conclusion, TF-011-MMAE is a promising novel antitumor agent with potent activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous target expression.

IgA EGFR antibodies mediate tumour killing in vivo.

Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

Crosstalk between human IgG isotypes and murine effector cells.

Development of human therapeutic Abs has led to reduced immunogenicity and optimal interactions with the human immune system in patients. Humanization had as a consequence that efficacy studies performed in mouse models, which represent a crucial step in preclinical development, are more difficult to interpret because of gaps in our knowledge of the activation of murine effector cells by human IgG (hIgG) remain. We therefore developed full sets of human and mouse isotype variants of human Abs targeting epidermal growth factor receptor and CD20 to explore the crosstalk with mouse FcγRs (mFcγRs) and murine effector cells. Analysis of mFcγR binding demonstrated that hIgG1 and hIgG3 bound to all four mFcγRs, with hIgG3 having the highest affinity. hIgG1 nevertheless was more potent than hIgG3 in inducing Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis with mouse NK cells, mouse polymorphonuclear leukocytes, and mouse macrophages. hIgG4 bound to all mFcγRs except mFcγRIV and showed comparable interactions with murine effector cells to hIgG3. hIgG4 is thus active in the murine immune system, in contrast with its inert phenotype in the human system. hIgG2 bound to mFcγRIIb and mFcγRIII, and induced potent ADCC with mouse NK cells and mouse polymorphonuclear leukocytes. hIgG2 induced weak ADCC and, remarkably, was unable to induce Ab-dependent cellular phagocytosis with mouse macrophages. Finally, the isotypes were studied in s.c. and i.v. tumor xenograft models, which confirmed hIgG1 to be the most potent human isotype in mouse models. These data enhance our understanding of the crosstalk between hIgGs and murine effector cells, permitting a better interpretation of human Ab efficacy studies in mouse models.

B-cell depletion attenuates white and gray matter pathology in marmoset experimental autoimmune encephalomyelitis.

This study investigated the effect of CD20-positive B-cell depletion on central nervous system (CNS) white and gray matter pathology in experimental autoimmune encephalomyelitis in common marmosets, a relevant preclinical model of multiple sclerosis. Experimental autoimmune encephalomyelitis was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein in complete Freund adjuvant. At 21 days after immunization, B-cell depletion was achieved by weekly intravenous injections of HuMab 7D8, a human-anti-human CD20 antibody that cross-reacts with marmoset CD20. In vivo magnetic resonance imaging showed widespread brain white matter demyelination in control marmosets that was absent in CD20 antibody-treated marmosets. High-contrast postmortem magnetic resonance imaging showed white matter lesions in 4of the 7 antibody-treated marmosets, but these were significantly smaller than those in controls. The same technique revealed gray matter lesions in 5 control marmosets, but none in antibody-treated marmosets. Histologic analysis confirmed that inflammation, demyelination, and axonal damage were substantially reduced in brain, spinal cord, and optic nerves of CD20 antibody-treated marmosets. In conclusion, CD20-postive B-cell depletion by HuMab 7D8 profoundly reduced the development of both white and gray matter lesions in the marmoset CNS. These data underline the central role of B cells in CNS inflammatory-demyelinating disease.

Epidermal growth factor receptor (EGFR) antibody-induced antibody-dependent cellular cytotoxicity plays a prominent role in inhibiting tumorigenesis, even of tumor cells insensitive to EGFR signaling inhibition.

Ab-dependent cellular cytotoxicity (ADCC) is recognized as a prominent cytotoxic mechanism for therapeutic mAbs in vitro. However, the contribution of ADCC to in vivo efficacy, particularly for treatment of solid tumors, is still poorly understood. For zalutumumab, a therapeutic epidermal growth factor receptor (EGFR)-specific mAb currently in clinical development, previous studies have indicated signaling inhibition and ADCC induction as important therapeutic mechanisms of action. To investigate the in vivo role of ADCC, a panel of EGFR-specific mAbs lacking specific functionalities was generated. By comparing zalutumumab with mAb 018, an EGFR-specific mAb that induced ADCC with similar potency, but did not inhibit signaling, we observed that ADCC alone was insufficient for efficacy against established A431 xenografts. Interestingly, however, both zalutumumab and mAb 018 prevented tumor formation upon early treatment in this model. Zalutumumab and mAb 018 also completely prevented outgrowth of lung metastases, in A431 and MDA-MB-231-luc-D3H2LN experimental metastasis models, already when given at nonsaturating doses. Finally, tumor growth of mutant KRAS-expressing A431 tumor cells, which were resistant to EGFR signaling inhibition, was completely prevented by early treatment with zalutumumab and mAb 018, whereas ADCC-crippled N297Q-mutated variants of both mAbs did not show any inhibitory effects. In conclusion, ADCC induction by EGFR-specific mAbs represents an important mechanism of action in preventing tumor outgrowth or metastasis in vivo, even of cancers insensitive to EGFR signaling inhibition.

Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of multiple myeloma and other hematological tumors.

CD38, a type II transmembrane glycoprotein highly expressed in hematological malignancies including multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of action of daratumumab, a novel, high-affinity, therapeutic human mAb against a unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular cytotoxicity in CD38-expressing lymphoma- and MM-derived cell lines as well as in patient MM cells, both with autologous and allogeneic effector cells. Daratumumab stood out from other CD38 mAbs in its strong ability to induce complement-dependent cytotoxicity in patient MM cells. Importantly, daratumumab-induced Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity were not affected by the presence of bone marrow stromal cells, indicating that daratumumab can effectively kill MM tumor cells in a tumor-preserving bone marrow microenvironment. In vivo, daratumumab was highly active and interrupted xenograft tumor growth at low dosing. Collectively, our results show the versatility of daratumumab to effectively kill CD38-expressing tumor cells, including patient MM cells, via diverse cytotoxic mechanisms. These findings support clinical development of daratumumab for the treatment of CD38-positive MM tumors.

Targeted multi-pinhole SPECT.

PURPOSE: Small-animal single photon emission computed tomography (SPECT) with focused multi-pinhole collimation geometries allows scanning modes in which large amounts of photons can be collected from specific volumes of interest. Here we present new tools that improve targeted imaging of specific organs and tumours, and validate the effects of improved targeting of the pinhole focus. METHODS: A SPECT system with 75 pinholes and stationary detectors was used (U-SPECT-II). An XYZ stage automatically translates the animal bed with a specific sequence in order to scan a selected volume of interest. Prior to stepping the animal through the collimator, integrated webcams acquire images of the animal. Using sliders, the user designates the desired volume to be scanned (e.g. a xenograft or specific organ) on these optical images. Optionally projections of an atlas are overlaid semiautomatically to locate specific organs. In order to assess the effects of more targeted imaging, scans of a resolution phantom and a mouse myocardial phantom, as well as in vivo mouse cardiac and tumour scans, were acquired with increased levels of targeting. Differences were evaluated in terms of count yield, hot rod visibility and contrast-to-noise ratio. RESULTS: By restricting focused SPECT scans to a 1.13-ml resolution phantom, count yield was increased by a factor 3.6, and visibility of small structures was significantly enhanced. At equal noise levels, the small-lesion contrast measured in the myocardial phantom was increased by 42%. Noise in in vivo images of a tumour and the mouse heart was significantly reduced. CONCLUSION: Targeted pinhole SPECT improves images and can be used to shorten scan times. Scan planning with optical cameras provides an effective tool to exploit this principle without the necessity for additional X-ray CT imaging.

Late B cell depletion with a human anti-human CD20 IgG1κ monoclonal antibody halts the development of experimental autoimmune encephalomyelitis in marmosets.

Depletion of CD20(+) B cells has been related to reduced clinical activity in relapsing-remitting multiple sclerosis. The underlying mechanism is not understood, because serum IgG levels were unaltered by the treatment. We report the effect of late B cell depletion on cellular and humoral immune mechanisms in a preclinical multiple sclerosis model (i.e., experimental autoimmune encephalomyelitis [EAE] in the common marmoset). We used a novel human anti-human CD20 IgG1κ mAb (HuMab 7D8) that cross-reacts with marmoset CD20. EAE was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein (MOG) in CFA. After 21 d, B cells were depleted in seven monkeys by HuMab 7D8, and seven control monkeys received PBS. The Ab induced profound and long-lasting B cell depletion from PBMCs and lymphoid organs throughout the observation period of 106 d. Whereas all of the control monkeys developed clinically evident EAE, overt neurologic deficits were reduced substantially in three HuMab 7D8-treated monkeys, and four HuMab 7D8-treated monkeys remained completely asymptomatic. The effect of HuMab 7D8 was confirmed on magnetic resonance images, detecting only small lesions in HuMab 7D8-treated monkeys. The infusion of HuMab 7D8 arrested the progressive increase of anti-MOG IgG Abs. Although CD3(+) T cell numbers in lymphoid organs were increased, their proliferation and cytokine production were impaired significantly. Most notable were the substantially reduced mRNA levels of IL-7 and proinflammatory cytokines (IL-6, IL-17A, IFN-γ, and TNF-α). In conclusion, B cell depletion prevents the development of clinical and pathological signs of EAE, which is associated with impaired activation of MOG-reactive T cells in lymphoid organs.

Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage.

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.

Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo.

Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated in humans. Here, we show that natalizumab exchanges Fab arms with endogenous human IgG4 in natalizumab-treated individuals. Gemtuzumab, in contrast, contains an IgG4 core-hinge mutation that blocks Fab-arm exchange to undetectable levels both in vitro and in a mouse model. The ability of IgG4 therapeutics to recombine with endogenous IgG4 may affect their pharmacokinetics and pharmacodynamics. Although pharmacokinetic modeling lessens concerns about undesired cross-linking under normal conditions, unpredictability remains and mutations that completely prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4 antibodies.

Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques.

Neutralizing antibodies are thought to be crucial for HIV vaccine protection, but studies in animal models suggest that high antibody concentrations are required. This is a major potential hurdle for vaccine design. However, these studies typically apply a large virus inoculum to ensure infection in control animals in single-challenge experiments. In contrast, most human infection via sexual encounter probably involves repeated exposures to much lower doses of virus. Therefore, animal studies may have provided an overestimate of the levels of antibodies required for protection in humans. We investigated whether plasma concentrations of antibody corresponding to relatively modest neutralization titers in vitro could protect macaques from repeated intravaginal exposure to low doses of a simian immunodeficiency virus-HIV chimera (SHIV) that uses the CC chemokine receptor 5 (CCR5) co-receptor. An effector function-deficient variant of the neutralizing antibody was also included. The results show that a substantially larger number of challenges is required to infect macaques treated with neutralizing antibody than control antibody-treated macaques, and support the notion that effector function may contribute to antibody protection. Overall, the results imply that lower amounts of antibody than previously considered protective may provide benefit in the context of typical human exposure to HIV-1.

Complement-dependent tumor cell lysis triggered by combinations of epidermal growth factor receptor antibodies.

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.