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PDF file - 83KB, Supplementary Figure S1. Characteristics of purified AMG 330.

Figure S2. Enzalutamide increases PSMA expression levels and may enhance AMG 160-dependent cytotoxicity.

Purpose:Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy.Experimental Design:We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated.Results:AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6–42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68–labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs.Conclusions:The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675

Supplementary methods, tables, and references.

The rules and structure of human culture impact health and disease as much as genetics or the natural environment. To study the origin and evolution of these patterns, we take a multidisciplinary approach combining ancient DNA, skeletal metrics, paleopathology, and stable isotopes. Our analysis focuses on cultural, environmental, and genetic contributions to variation in stature in four populations of Early Neolithic Europe. In Central Europe, low female stature is likely due to male preference in resource allocation under conditions of stress. In contrast, shorter male stature in Mediterranean populations may reflect a lack of preference. Our analysis suggests that biological consequences of sex-specific inequities can be linked to culture as early as 7000 years before present. Understanding these patterns is key to interpreting the evolution of genetic and socio-cultural determinants of health, and our results show that culture, more than environment or genetics, drove height disparities in Early Neolithic Europe.

Inflammatory bowel disease (IBD) is often described as a model for modern civilization diseases in which environmental factors trigger disease manifestation in genetically compromised individuals. Little is known about the evolutionary history of variants associated with IBD in modern Europeans. Here, we analysed 610 IBD-variants in 2445 ancient datasets from human remains spanning the last 12,000 years, including genotypes generated from 172 newly collected individuals from the European Neolithic. We found statistically significant differences in the frequencies of 97 IBD variants between Neolithic and modern populations that can be explained by the adoption of an agricultural lifestyle and behaviour and concomitant possible microbiome changes in the earliest farmers. Later admixture events and selection against pathogens largely influenced the genetic risk architecture of IBD in contemporary Europeans. A better understanding of the evolutionary history of disease variants is an important first step in translating genetic findings into preventive health care.

Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)–blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ∼12,000 y B.P. This shift is hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a decline in physiological health as inferred from skeletal remains. Here, we consider osteological and ancient DNA data from the same prehistoric individuals to study human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared “predicted” genetic contributions to height from paleogenomic data and “achieved” adult osteological height estimated from long bone measurements for 167 individuals across Europe spanning the Upper Paleolithic to Iron Age (∼38,000 to 2,400 B.P.). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −3.82 cm relative to individuals from the Upper Paleolithic and Mesolithic (P = 0.040) and −2.21 cm shorter relative to post-Neolithic individuals (P = 0.068), with osteological vs. expected stature steadily increasing across the Copper (+1.95 cm relative to the Neolithic), Bronze (+2.70 cm), and Iron (+3.27 cm) Ages. These results were attenuated when we additionally accounted for genome-wide genetic ancestry variation: for example, with Neolithic individuals −2.82 cm shorter than expected on average relative to pre-Neolithic individuals (P = 0.120). We also incorporated observations of paleopathological indicators of nonspecific stress that can persist from childhood to adulthood in skeletal remains into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggests that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe. - Introduction - Results -- Archaeological and Anthropological Findings -- The 16th Century Ellwangen Plague Victims Display Genetic Similarity with Modern Inhabitants -- Two Immunity-Related Genes Harbor Strongly Differentiated single nucleotide polymorphisms -- No Evidence for Role of CCR5-D32 in Protection from Y. pestis Infection Discussion -- Natural Selection Has Increased HLA-DRB*13 and Reduced HLA-B*51 and -C*06 Frequencies in Modern Individuals -- Higher Incidence of KIR3DL1 Interaction with HLA-B in Plague Victims Than Modern Inhabitants of Ellwangen - Discussion - Materials and Methods

AMG 794 is a half-life extended BiTE® immune therapy targeting the oncofetal antigen Claudin 6 (CLDN6). AMG 794 redirects T cells to kill CLDN6-expressing tumor cells and is being developed for the treatment of non-small cell lung cancer (NSCLC) and epithelial ovarian cancer (EOC). CLDN6 is a compelling tumor antigen that is expressed during embryonic and fetal development, transcriptionally silenced in adult tissues, and re-expressed on the surface of NSCLC and EOC cells. By immunohistochemistry, CLDN6 staining of the cell membrane was observed in 27% of non-squamous NSCLC (n = 63) and 69% of EOC (n = 92) samples, the majority of which were of the high-grade serous ovarian cancer subtype. Expression of CLDN6 protein was not detected in most normal adult tissues, with rare CLDN6 immunostaining limited to individual cells in the pituitary, pancreas, small intestine, kidney, and female reproductive organs. AMG 794 is a fully human BiTE® molecule that binds both human and cynomolgus monkey CLDN6 and CD3. AMG 794 binds human CLDN6 and CD3 with equilibrium dissociation constant (KD) of 13 nM and 36 nM, respectively. In vitro, AMG 794 redirects human T cells to kill CLDN6-expressing cancer cells with a half-maximal lysis concentration (EC50) of 2.6 ± 1.1 pM to 127.4 ± 53.4 pM. Consistent with the mechanism of action of BiTE® immune therapy, AMG 794 induces T cell activation and transient production of cytokines in co-cultures of T cells and CLDN6-expressing tumor cells. Remarkably, AMG 794 binding and cytotoxic activity is selective for CLDN6 over other claudin family proteins, despite high homology in the extracellular loops with CLDN9. Weekly dosing of AMG 794 significantly inhibited the growth of established lung and ovarian xenograft tumors in immunocompromised mice injected with human T cells. Anti-tumor activity was associated with an increase in tumor-infiltrating T cells. AMG 794 was well tolerated in a one-month repeat-dose toxicology study in cynomolgus monkey, with evidence for target engagement. The potent, selective activity of AMG 794 for CLDN6-expressing NSCLC and EOC cells, together with an acceptable nonclinical safety profile, supported the advancement of AMG 794 into clinical development. A first-in-human study to explore the safety, tolerability, pharmacokinetics, and anti-tumor activity of AMG 794 in patients with CLDN6-positive advanced/metastatic non-squamous NSCLC or EOC will be enrolling patients in March 2022. Citation Format: Elizabeth Pham, Anja Henn, Beate Sable, Joachim Wahl, Kip Conner, Katja Matthes, Shivani Gupta, Rodolfo Yabut, Famke Aeffner, Kristin Lewis Wilson, Jonas Anlahr, Christoph Dahlhoff, Vijay Kale, Matthias Friedrich, Tobias Raum, Peter Kufer, Angela Coxon, Sabine Stienen, Julie M. Bailis. AMG 794, a Claudin 6-targeted half-life extended (HLE) bispecific T cell engager (BITE®) molecule for non-small cell lung cancer and epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5202.

Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting >1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival > 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced >2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ~12,000 years before present (BP). Subsistence shifts from hunting and gathering to agriculture are hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a simultaneous decline in physiological health as inferred from paleopathological analyses and stature reconstructions of skeletal remains. A key component of the health decline inference is that relatively shorter statures observed for early farmers may (at least partly) reflect higher childhood disease burdens and poorer nutrition. However, while such stresses can indeed result in growth stunting, height is also highly heritable, and substantial inter-individual variation in the height genetic component within a population is typical. Moreover, extensive migration and gene flow were characteristics of multiple agricultural transitions worldwide. Here, we consider both osteological and ancient DNA data from the same prehistoric individuals to comprehensively study the trajectory of human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared ‘predicted’ genetic contributions to height from paleogenomic data and ‘achieved’ adult osteological height estimated from long bone measurements on a per-individual basis for n=160 ancient Europeans from sites spanning the Upper Paleolithic to the Iron Age (~38,000-2,400 BP). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −4.47 cm relative to individuals from the Upper Paleolithic and Mesolithic (P=0.016). The average osteological vs. expected stature then increased relative to the Neolithic over the Copper (+2.67 cm, P=0.052), Bronze (+3.33 cm, P=0.032), and Iron Ages (+3.95 cm, P=0.094). These results were partly attenuated when we accounted for genome-wide genetic ancestry variation in our sample (which we note is partly duplicative with the individual polygenic score information). For example, in this secondary analysis Neolithic individuals were −3.48 cm shorter than expected on average relative to individuals from the Upper Paleolithic and Mesolithic (P=0.056). We also incorporated observations of paleopathological indicators of non-specific stress that can persist from childhood to adulthood in skeletal remains (linear enamel hypoplasia, cribra orbitalia, and porotic hyperostosis) into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. Experimental Design: We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. Results: AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6–42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68–labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. Conclusions: The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC. See related commentary by Kamat et al., p. 2675

Genetic studies of Neolithic and Bronze Age skeletons from Europe have provided evidence for strong population genetic changes at the beginning and the end of the Neolithic period. To further understand the implications of these in Southern Central Europe, we analyze 96 ancient genomes from Switzerland, Southern Germany, and the Alsace region in France, covering the Middle/Late Neolithic to Early Bronze Age. Similar to previously described genetic changes in other parts of Europe from the early 3rd millennium BCE, we detect an arrival of ancestry related to Late Neolithic pastoralists from the Pontic-Caspian steppe in Switzerland as early as 2860–2460 calBCE. Our analyses suggest that this genetic turnover was a complex process lasting almost 1000 years and involved highly genetically structured populations in this region., European populations underwent strong genetic changes during the Neolithic. Here, Furtwängler et al. provide ancient nuclear and mitochondrial genomic data from the region of Switzerland during the end of the Neolithic and the Early Bronze Age that reveal a complex genetic turnover during the arrival of steppe ancestry.

We investigated here the novel immunomodulation and anti–multiple myeloma (MM) function of T cells engaged by the bispecific T-cell engager molecule AMG 701, and further examined the impact of AMG 701 in combination with immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide). AMG 701 potently induced T-cell–dependent cellular cytotoxicity (TDCC) against MM cells expressing B-cell maturation antigen, including autologous cells from patients with relapsed and refractory MM (RRMM) (half maximal effective concentration

Helminth infections are among the World Health Organization’s top neglected diseases with significant impact in many Less Economically Developed Countries. Despite no longer being endemic in Europe, the widespread presence of helminth eggs in archaeological deposits indicates that helminths represented a considerable burden in past European populations. Prevalence of infection is a key epidemiological feature that would influence the elimination of endemic intestinal helminths, for example, low prevalence rates may have made it easier to eliminate these infections in Europe without the use of modern anthelminthic drugs. To determine historical prevalence rates we analysed 589 grave samples from 7 European sites dated between 680 and 1700 CE, identifying two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) at all locations, and two food derived cestodes (Diphyllobothrium latum and Taenia spp.) at 4 sites. The rates of nematode infection in the medieval populations (1.5 to 25.6% for T. trichiura; 9.3–42.9% for Ascaris spp.) were comparable to those reported within modern endemically infected populations. There was some evidence of higher levels of nematode infection in younger individuals but not at all sites. The genetic diversity of T. trichiura ITS-1 in single graves was variable but much lower than with communal medieval latrine deposits. The prevalence of food derived cestodes was much lower (1.0–9.9%) than the prevalence of nematodes. Interestingly, sites that contained Taenia spp. eggs also contained D. latum which may reflect local culinary practices. These data demonstrate the importance of helminth infections in Medieval Europe and provide a baseline for studies on the epidemiology of infection in historical and modern contexts. Since the prevalence of medieval STH infections mirror those in modern endemic countries the factors affecting STH decline in Europe may also inform modern intervention campaigns., Author summary Parasitic helminths (worms) are important infections of humans in many less well developed countries, particularly those in tropical and sub-tropical regions. These infections are not a major problem in modern Europe but parasite eggs are readily detected in archaeological contexts. To estimate a key epidemiological parameter, the prevalence of infection, we examined large numbers of single graves from Medieval Europe and found that the rates of infection with two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) were as prevalent as in many modern endemic areas. We also identified two cestodes that humans acquire from eating undercooked red meat (Taenia spp.) or freshwater fish (Diphyllobothrium latum). Using prevalence and ancient DNA data we explored helminth epidemiology in Medieval European populations and factors that may influence infection including age, sex, sanitation, hygiene and culinary practices. The Medieval prevalence rates provide a historical baseline for Europe and an interesting comparator for modern epidemiological studies in other parts of the world. It is noteworthy that helminths were endemic in historical Europe but were eradicated prior to the development of modern drugs. In this sense studying changes in helminth prevalence in historical Europe may provide insights into control efforts in modern endemic regions.

AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell–redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596–induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell–dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.

Previous research found sexual dimorphism in the bony labyrinth of a Greek population sample (Osipov et al. 2013). This study intends to investigate the nature of this structure’s sexual dimorphism across populations of diverse geographic origin and to identify the effect of inter-population variation on the accuracy of determining sex using the bony labyrinth. Three population samples of known sex were analyzed originating from Europe (n = 30), Africa (n = 38), and North America (n = 30). The discriminant function developed in Osipov et al. (2013) was applied, and new function equations for sex estimation were developed. In addition, we used principal component analyses for investigating population differences, while bivariate tests were used to compare across population samples, sexes, and anatomical sides. A significant level of sexual dimorphism was found in all population samples, being driven by both size and shape differences. Discriminant functions for sex estimation were developed for all three population samples combined (71.4% accuracy) as well as separately (70–80% accuracy). The German sample was the least sexually dimorphic, whereas the North American sample exhibited the greatest sexual dimorphism. The size and shape of the bony labyrinth also significantly differed across population samples. The bony labyrinth is found to be sexually dimorphic across distinct population groups. Due to significant differences across our population samples, the accuracy of the previously proposed method for sex determination (Osipov et al., 2013) was relatively low. For this purpose, this study presented new functions, whose accuracy was tested in three distinct population samples.

Shedding light on first encounters between late hunter-gatherers and early farmers in western Europe., Starting from 12,000 years ago in the Middle East, the Neolithic lifestyle spread across Europe via separate continental and Mediterranean routes. Genomes from early European farmers have shown a clear Near Eastern/Anatolian genetic affinity with limited contribution from hunter-gatherers. However, no genomic data are available from modern-day France, where both routes converged, as evidenced by a mosaic cultural pattern. Here, we present genome-wide data from 101 individuals from 12 sites covering today’s France and Germany from the Mesolithic (N = 3) to the Neolithic (N = 98) (7000–3000 BCE). Using the genetic substructure observed in European hunter-gatherers, we characterize diverse patterns of admixture in different regions, consistent with both routes of expansion. Early western European farmers show a higher proportion of distinctly western hunter-gatherer ancestry compared to central/southeastern farmers. Our data highlight the complexity of the biological interactions during the Neolithic expansion by revealing major regional variations.

Genetic studies of Neolithic and Bronze Age skeletons from Europe have provided evidence for strong population genetic changes at the beginning and the end of the Neolithic period. To further understand the implications of these in Southern Central Europe, we analyze 96 ancient genomes from Switzerland, Southern Germany, and the Alsace region in France, covering the Middle/Late Neolithic to Early Bronze Age. Similar to previously described genetic changes in other parts of Europe from the early 3rd millennium BCE, we detect an arrival of ancestry related to Late Neolithic pastoralists from the Pontic-Caspian steppe in Switzerland as early as 2860–2460 calBCE. Our analyses suggest that this genetic turnover was a complex process lasting almost 1000 years and involved highly genetically structured populations in this region. European populations underwent strong genetic changes during the Neolithic. Here, Furtwangler et al. provide ancient nuclear and mitochondrial genomic data from the region of Switzerland during the end of the Neolithic and the Early Bronze Age that reveal a complex genetic turnover during the arrival of steppe ancestry.

Despite great variability, most burials of the Early Iron Age in Central Europe exhibit a high degree of standardization. Richly furnished graves consist of wooden chambers furnished with grave goods like chariots, vessels, and other objects, while less “rich” burials—clustered in “regular” cemeteries—show the same orientation to the south as well as regularly reappearing objects like weapons or ornaments. Because of these strict rules, scholars have accepted such burials as “the norm,” and any other form of deposition of the dead as “abnormal,” hinting at macabre customs like cannibalism or sacrifice. This chapter analyzes one kind of Iron Age deviant burial, those in settlement pits, discussing bioarchaeological and isotopic analyses, a reassessment of archaeological evidence, and a comparison with normative burial practices. The dead in settlements belonged to at least three social categories, each probably considered incomplete in some way and unfit to be buried in regular cemeteries: very small children, adolescents, and other individuals that had suffered an untimely or “bad” death, and individuals of low social standing.

The proper description of former populations is one of the most difficult tasks in anthropology. Archaeological material is often limited due to fragmented and sometimes poorly preserved bone material resulting in incomplete data. Published skeletal raw data are available from the past, but much of this data is either unavailable or not used for scientific studies. The authors seek to elicit more information about prehistoric times by using this dataset to introduce a new method. The purpose is to provide an approach to reconstruct a former population in respect to robusticity and health status. For this in the pilot study the Body Mass Index (BMI) and Frame Index (FI) of early medieval South-West Germany have been analysed. The FI, in contrast to the BMI, has not yet been used for robusticity analysis utilizing only skeletal remains. As far as we know, this is the first time that the FI has been calculated using archaeological material. Due to unknown soft-tissue thickness we introduce the Osseous Frame Index (OFI). The measured OFI reveals new insights in (pre-)historic populations and allows comparisons with modern reference samples. Our OFI calculations are relatively similar to modern calculations. Males have a higher robusticity than females, slightly increasing during life-time compared to females. These calculations provide a better historical understanding of human body composition.

Hypertrophic osteoarthropathy (HOA) is rarely diagnosed in archaeological human skeletons. Here, we report on the well-preserved skeleton of a middle-adult man from the early Medieval settlement site of Lauchheim (Germany) that exhibits pronounced multi-layered shell-like periosteal new bone formation in a bilaterally symmetric fashion on the long bones, the skeletal elements of the pelvis and those of the pectoral girdle. In addition, the two distal phalanges recovered show signs of osteoclastic resorption on their distal tuberosities. The distribution and morphology of the observed lesions are consistent with a diagnosis of HOA. The adult age at death of the individual and the co-occurrence of "healed" and "active" lesions suggest a secondary form of HOA. Given that only skeletal remains were available for study, the underlying (pulmonary or non-pulmonary) primary disease cannot be definitively ascertained in the present case. No osseous changes were found on the ribs, but signs of osteoclastic resorption were observed on the dorsal surface of the sternal body, which might indicate a retrosternal or mediastinal location of the primary disease. Thus far, only a few archaeological case studies of secondary HOA reported signs of the presumed underlying primary disease, which was of a pulmonary nature in each of the individuals.

Objectives The purpose of this study was to put forth a precise landmark-based technique for reconstructing the three-dimensional shape of human entheseal surfaces, to investigate whether the shape of human entheses is related to their size. The effects of age-at-death and bone length on entheseal shapes were also assessed. Materials and methods The sample comprised high-definition three-dimensional models of three right hand entheseal surfaces, which correspond to 45 male adult individuals of known age. For each enthesis, a particular landmark configuration was introduced, whose precision was tested both within and between observers. The effect of three-dimensional size, age-at-death, and bone length on shape was investigated through shape regression. Results The method presented high intra-observer and inter-observer repeatability. All entheses showed significant allometry, with the area of opponens pollicis demonstrating the most substantial relationship. This was particularly due to variation related to its proximal elongated ridge. The effect of age-at-death and bone length on entheses was limited. Discussion The introduced methodology can set a reliable basis for further research on the factors affecting entheseal shape. Using both size and shape, variables can provide further information on entheseal variation and its biomechanical implications. The low entheseal variation by age verifies that specimens under 50 years of age are not substantially affected by age-related changes. The lack of correlation between entheseal shape and bone length or age implies that other factors may regulate entheseal surfaces. Future research should focus on multivariate shape patterns among entheses and their association with occupation.

In recent years increasing interest was drawn towards ionic liquids in analytical separation science, such as capillary electrophoresis. Ionic liquids combining tetrabutylammonium cations with chiral amino acid based anions were prepared and investigated as capillary electrophoresis background electrolyte additives for the enantioseparation of ephedrine, pseudoephedrine, and methylephedrine isomers. For the optimization of buffer pH and ionic liquid concentration a design of experiments approach was performed. The best results for the separation of all enantiomers were achieved using 125 mmol/L tetrabutylammonium l -argininate in a 75 mmol/L phosphate buffer pH 1.5 containing 30 mmol/L β-cyclodextrin.

Objectives In anthropological sciences, entheses are widely utilized as occupational stress markers. However, the reaction of entheseal surfaces to mechanical loading is not well understood. Furthermore, previous studies on entheses relied on the individuals' occupation-at-death. Past research by one of us has identified two patterns among hand entheses, proposing that they reflect two synergistic muscle groups. Here, we investigate the association between these patterns and habitual manual activity using an extensively documented skeletal sample and a three-dimensional system of quantification. Materials and Methods The hand bones utilized belong to 45 individuals from mid-19th century Basel. These were male adults (18 to 48 years old) who were not directly related, showed no manual pathological conditions, and whose occupational activities during their lifetime were clearly documented and could be evaluated according to historical sources. The patterns of entheses were explored using principal component analysis on both raw and size-adjusted variables. The influence of age-at-death, body mass, and bone length was assessed through correlation tests. Results The analysis showed that the previously proposed patterns of entheses are present in our sample. Individuals with the same or comparable occupations presented similar entheseal patterns. These results were not considerably affected by entheseal overall size, age-at-death, body mass, or bone length. Discussion Individuals involved in intense manual labor during their lifetime presented a distinctive pattern of hand entheses, consistent with the application of high grip force. By contrast, individuals with less strenuous and/or highly mechanized occupations showed an entheseal pattern related to the thumb intrinsic muscles.

A richly furnished grave of an elite woman from the Hallstatt period was discovered close to the Heuneburg, the earliest proto-urban settlement north of the Alps. Dendrochronological analysis of timbers from the grave chamber dates the burial to 583 BC, the earliest of a series of such burials north of the Alps and a key anchor in the absolute chronology of the Early Iron Age in Europe. The woman was adorned with gold, bronze, jet and amber jewellery; gold filigree objects, amber fibulae and items of horse-head armour suggest close connections south of the Alps. An infant female burial close to the main grave included gold jewellery made for a child but similar to that of the woman.

Starting from questions about the nature of cultural diversity, this paper examines the pace and tempo of change and the relative importance of continuity and discontinuity. To unravel the cultural project of the past, we apply chronological modelling of radiocarbon dates within a Bayesian statistical framework, to interrogate the Neolithic cultural sequence in Lower Alsace, in the upper Rhine valley, in broad terms from the later sixth to the end of the fifth millennium cal BC. Detailed formal estimates are provided for the long succession of cultural groups, from the early Neolithic Linear Pottery culture (LBK) to the Bischheim Occidental du Rhin Supérieur (BORS) groups at the end of the Middle Neolithic, using seriation and typology of pottery as the starting point in modelling. The rate of ceramic change, as well as frequent shifts in the nature, location and density of settlements, are documented in detail, down to lifetime and generational timescales. This reveals a Neolithic world in Lower Alsace busy with comings and goings, tinkerings and adjustments, and relocations and realignments. A significant hiatus is identified between the end of the LBK and the start of the Hinkelstein group, in the early part of the fifth millennium cal BC. On the basis of modelling of existing dates for other parts of the Rhineland, this appears to be a wider phenomenon, and possible explanations are discussed; full reoccupation of the landscape is only seen in the Grossgartach phase. Radical shifts are also proposed at the end of the Middle Neolithic. Electronic supplementary material The online version of this article (doi:10.1007/s10816-016-9307-x) contains supplementary material, which is available to authorized users.

B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell–dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo. FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo. Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

For enantioseparations of chiral drugs in capillary electrophoresis, chiral ionic liquids (CIL) can be employed instead of traditional running buffer containing a chiral selector. CILs can be applied solely or in addition to the often used cyclodextrin derivatives. Here the separation of phenethylamines, especially of ephedrine, is described using tetrabutylammonium L-argininate (125 mM) in phosphate buffer (75 mM, pH 1.5) in addition to β-cyclodextrin (30 mM). Using this dual-chiral running buffer system ephedrine, pseudoephedrine and methylephedrine, but not norephedrine, could be easily resolved.

Ancient DNA informs on past culturesArchaeology has used analysis of the artifacts and remains of people to uncover their past behaviors and to infer their cultural practices. However, establishing genetic relationships has only recently become possible. Mittniket al.examined the kinship and inheritance of the remains of people from the German Lech River Valley over a time period spanning the Late Neolithic Corded Ware Culture, the Bell Beaker Complex, the Early Bronze Age, and the Middle Bronze Age (see the Perspective by Feinman and Neitzel). From genetic and archaeological analyses, it was revealed that the Early Bronze Age household's burials over multiple generations consisted of a high-status core family and unrelated low-status individuals. Furthermore, women were not related to the men within the household, suggesting that men stayed within their birth communities in this society, but women did not.Science, this issue p.731; see also p.682

For enantioseparations of chiral drugs in capillary electrophoresis, chiral ionic liquids (CIL) can be employed instead of traditional running buffer containing a chiral selector. CILs can be applied solely or in addition to the often used cyclodextrin derivatives. Here the separation of phenethylamines, especially of ephedrine, is described using tetrabutylammonium L-argininate (125 mM) in phosphate buffer (75 mM, pH 1.5) in addition to β-cyclodextrin (30 mM). Using this dual-chiral running buffer system ephedrine, pseudoephedrine and methylephedrine, but not norephedrine, could be easily resolved.

Advanced gastric cancer remains a disease of high unmet medical need. In the United States, most patients present with symptomatic, incurable disease and prognosis is poor, with a 5-year survival rate of less than 10%. BiTE® (bispecific T cell engager) immune therapy activates a patient's own T cells to kill tumor cells and has the potential to overcome common mechanisms of therapy resistance. We generated fully human, half-life extended (HLE) BiTE® molecules against the tumor antigens MUC17 and CLDN18.2 for the treatment of gastric cancer. The mucin MUC17 is a protein normally found in the mucosal layer of intestinal epithelial cells that is delocalized and expressed in 45% of gastric tumors. The claudin CLDN18.2 is a protein normally found in the cellular tight junctions of gastric mucosa and intestinal epithelium that is delocalized and expressed in >60% of gastric tumors. AMG 199 (MUC17 HLE BiTE®) and AMG 910 (CLDN18.2 HLE BiTE®) show potent cytotoxic activity against gastric cancer cell lines that express MUC17 or CLDN18.2, respectively, in vitro, and promote significant tumor growth inhibition against established gastric tumor xenograft models in vivo. In preclinical non-human primate (NHP) toxicology studies, both molecules show evidence for BiTE® target engagement, including T cell activation and proliferation, but demonstrate different effects on target-expressing tissue. Weekly administration of AMG 199 is well tolerated in NHP with minimal findings in MUC17-expressing normal tissues. In contrast, treatment with AMG 910 led to direct cell killing of CLDN18.2-expressing gastric mucosal cells in NHP, a finding which was fully reversible once treatment was stopped. AMG 199 and AMG 910 may offer the potential to improve outcomes in advanced gastric patients worldwide. Citation Format: Julie M. Bailis, Petra Lutterbuese, Oliver Thomas, Kathrin Locher, John Harrold, Michael Boyle, Joachim Wahl, Shyun Li, Alexander Sternjak, Anja Henn, Christoph Dahlhoff, Virginie Naegele, Benno Rattel, Tobias Raum, Angela Coxon. Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3364.

Throughout history, humans have been afflicted by parasitic worms, and eggs are readily detected in archaeological deposits. This study integrated parasitological and ancient DNA methods with a large sample set dating between Neolithic and Early Modern periods to explore the utility of molecular archaeoparasitology as a new approach to study the past. Molecular analyses provided unequivocal species-level parasite identification and revealed location-specific epidemiological signatures. Faecal–oral transmitted nematodes (Ascaris lumbricoides and Trichuris trichiura) were ubiquitous across time and space. By contrast, high numbers of food-associated cestodes (Diphyllobothrium latum and Taenia saginata) were restricted to medieval Lübeck. The presence of these cestodes and changes in their prevalence at approximately 1300 CE indicate substantial alterations in diet or parasite availability. Trichuris trichiura ITS-1 sequences grouped into two clades; one ubiquitous and one restricted to medieval Lübeck and Bristol. The high sequence diversity of T.t.ITS-1 detected in Lübeck is consistent with its importance as a Hanseatic trading centre. Collectively, these results introduce molecular archaeoparasitology as an artefact-independent source of historical evidence.

Neanderthals are commonly depicted as leading dangerous lives and permanently struggling for survival. This view largely relies on the high incidences of trauma that have been reported1,2 and have variously been attributed to violent social behaviour3,4, highly mobile hunter-gatherer lifestyles2 or attacks by carnivores5. The described Neanderthal pattern of predominantly cranial injuries is further thought to reflect violent encounters with large prey mammals, resulting from the use of close-range hunting weapons1. These interpretations directly shape our understanding of Neanderthal lifestyles, health and hunting abilities, yet mainly rest on descriptive, case-based evidence. Quantitative, population-level studies of traumatic injuries are rare. Here we reassess the hypothesis of higher cranial trauma prevalence among Neanderthals using a population-level approach—accounting for preservation bias and other contextual data—and an exhaustive fossil database. We show that Neanderthals and early Upper Palaeolithic anatomically modern humans exhibit similar overall incidences of cranial trauma, which are higher for males in both taxa, consistent with patterns shown by later populations of modern humans. Beyond these similarities, we observed species-specific, age-related variation in trauma prevalence, suggesting that there were differences in the timing of injuries during life or that there was a differential mortality risk of trauma survivors in the two groups. Finally, our results highlight the importance of preservation bias in studies of trauma prevalence. Neanderthals and Upper Palaeolithic modern humans exhibit similar overall incidences of cranial trauma that are higher for males of both taxa; however, there are species-specific, age-related variations in trauma prevalence.

Until the end of the First World War, the investigation of archaeological human remains in Germany followed a typological concept that was dominated by attempts to categorize the morphological variability of human skeletons. Until the end of the Second World War, this approach grew more acute because of the pressure imposed by the National Socialists to conform with politically desired “human races.” After Germany’s division into the FRG and GDR, the scientific landscapes in the two parts of Germany again reflected the different political systems although physical anthropologists remained strongly connected with archaeological sciences in both polities. By the end of the twentieth century, new concepts and methods and explicitly contextual research on human remains slowly gained in importance what is reflected by, e.g., the growing relevance of biomolecular research after the German unification. As a result, however, traditional skeletal biology has turned into a small and endangered academic subject.

Reconstruction of relatedness and ancestry from ancient DNA from Medieval burial provides new insights into kinship behavior., From historical and archeological records, it is posited that the European medieval household was a combination of close relatives and recruits. However, this kinship structure has not yet been directly tested at a genomic level on medieval burials. The early 7th century CE burial at Niederstotzingen, discovered in 1962, is the most complete and richest example of Alemannic funerary practice in Germany. Excavations found 13 individuals who were buried with an array of inscribed bridle gear, jewelry, armor, and swords. These artifacts support the view that the individuals had contact with France, northern Italy, and Byzantium. This study analyzed genome-wide sequences recovered from the remains, in tandem with analysis of the archeological context, to reconstruct kinship and the extent of outside contact. Eleven individuals had sufficient DNA preservation to genetically sex them as male and identify nine unique mitochondrial haplotypes and two distinct Y chromosome lineages. Genome-wide analyses were performed on eight individuals to estimate genetic affiliation to modern west Eurasians and genetic kinship at the burial. Five individuals were direct relatives. Three other individuals were not detectably related; two of these showed genomic affinity to southern Europeans. The genetic makeup of the individuals shares no observable pattern with their orientation in the burial or the cultural association of their grave goods, with the five related individuals buried with grave goods associated with three diverse cultural origins. These findings support the idea that not only were kinship and fellowship held in equal regard: Diverse cultural appropriation was practiced among closely related individuals as well.