An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Intravenous Cetirizine vs Intravenous Diphenhydramine for the Prevention of Hypersensitivity Infusion Reactions: Results of an Exploratory Phase 2 Study.

Pretreatment with antihistamines for the prevention of hypersensitivity infusion reactions is recommended for certain biologics and chemotherapies. Cetirizine is the first injectable second-generation antihistamine recently approved for acute urticaria. A randomized, exploratory phase 2 study evaluated intravenous (IV) cetirizine 10 mg versus IV diphenhydramine 50 mg as pretreatment in patients receiving an anti-CD20 agent or paclitaxel. In the overall population (N = 34) and an elderly subgroup (n = 21), IV cetirizine was as effective as IV diphenhydramine in preventing infusion reactions (primary outcome) and associated with less sedation at all time points, a shorter infusion center stay, and fewer treatment-related adverse events

Abstract CT102: Predictors of response to a modified whole tumor cell immunotherapy in patients with advanced breast cancer from two phase I/IIa trials

Abstract Objectives: SV-BR-1-GM is a GM-CSF secreting breast cancer cell line derived from a Grade II (moderately differentiated) breast tumor that also expresses HLA class I & II antigens and is able to function as an antigen-presenting cell. Irradiated SV-BR-1-GM is used in a regimen including pre-dose low-dose cyclophosphamide and post-dose local IFN-α2b. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 - study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 - study ongoing). The objective is to evaluate potential predictors of response in advanced metastatic breast cancer (aMBC). Methodology: 23 patients with refractory aMBC were treated with the SV-BR-1-GM regimen as monotherapy (cycles every 2 weeks x3 and then monthly). The combination study uses the SV-BR-1-GM regimen with PD-1 inhibitors pembrolizumab or INCMGA00012 with cycles every 3 weeks (12 patients dosed to date). Analyses include circulating tumor cells (CTCs), cancer-associated macrophage-like cells (CAMLs), tumor Grade (well- (Grade I), moderately- (Grade II) or poorly differentiated (Grade III)), HLA-type, and the ability to develop delayed-type hypersensitivity (DTH).Preliminary Data: The patients were heavily pre-treated (median of 4 prior systemic therapies not including hormonal therapy). In the monotherapy study, disease control (including SD, PR or CR) was seen in 6 patients (26%, all with SD). 16 patients (76%) developed measurable DTH (2 patients with no data). In the combination therapy study, disease control was seen in 3 patients (25%, 1 PR and 2 SD) and 10 (91%) developed DTH. In the combined studies, patients with Grade I or Grade II tumors were more likely to achieve disease control (6/9, 67%) compared with those with Grade III tumors (2/20, 10%). Median progression free survival (PFS) on monotherapy was 2.6 months and 4.5 months for patients with Grade I/II disease. Median PFS on the combination study was 4.2 months and 6.9 months for patients with Grade I/II tumors. Patients with 1 or 2 HLA matches with SV-BR-1-GM achieved disease control in 29% (6/21) and 38% (5/13), respectively. In patients with Grade I or II tumors, for those with 1+ or 2+ HLA matches, disease control was seen in 57% (4/7) and 75% (3/4), respectively. None of the 3 patients with high levels of CTCs at baseline had disease control. Mean and median PD-L1 expression levels on CTCs and CAMLS were higher for patients with Grade III tumors compared with Grade I/II tumors, but this was not clearly correlated with clinical response.The SV-BR-1-GM regimen can produce disease control and clinical benefit in very heavily pretreated patients with aMBC refractory to conventional therapies especially if the patients have an intact immune system (DTH), do not have high levels of CTCs, have Grade I or II disease, and have HLA matching with the SV-BR-1-GM cell line. Citation Format: William V. Williams, Markus D. Lacher, Daniel L. Adams, Shaker R. Dakhil, Jarrod P. Holmes, Saveri Bhattacharya, Carmen Calfa, George E. Peoples, Charles L. Wiseman. Predictors of response to a modified whole tumor cell immunotherapy in patients with advanced breast cancer from two phase I/IIa trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT102

Safety and efficacy of a phase I/IIa trial (NCT03066947) of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer

e14026 Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line which expresses HLA class I & II antigens and has functional antigen-presenting cell activity. Prior studies suggest that partial matching of the HLA type of the patient with SV-BR-1-GM may be predictive of tumor regression. Methods: Subjects received low-dose cyclophosphamide 2-3d prior to ID injection of irradiated SV-BR-1-GM (20 million cells divided into 4 sites) and interferon-α into the inoculation sites ~2 & 4 days subsequently. Cycles were q2 weeks x 3 then q mo. Results: A total of 30 patients were screened and 23 inoculated (Table). The patients were heavily pretreated with a median of 4 prior chemo/biological therapy regimens. There were no serious or unexpected adverse events. Local injection-site irritation was the most common toxicity. Objective tumor regression was seen in 3 patients, all of whom matched SV-BR-1-GM at least at one HLA locus: one patient with regression or clearing of 20 lung metastases; one with reduction in cutaneous involvement of the breast from 80% to 30% and one with regression of a breast lesion. Another 3 patients had decreases in circulating cancer-associated macrophage-like cells (CAMLs), which has been shown to correlate with tumor stage. They also all matched at least at one HLA allele. Circulating tumor cells and circulating epithelial cells were present in low numbers and tended to parallel trends in CAMLs which were present in larger numbers. CAMLs in 21/23 patients stained positive for PD-L1. Patients with tumor regression had robust DTH responses to SV-BR-1-GM. Conclusions: SV-BR-1-GM in this regimen appears to be safe and well-tolerated and is associated with objective regression of metastatic breast cancer and/or with decreases in circulating cancer-associated cells in 6/23 (26%) or patients. HLA matching may be a predictor of response. Clinical trial information: NCT03066947. [Table: see text

Abstract P2-14-02: Overall survival following treatment with a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer

Abstract Background: SV-BR-1-GM is a GM-CSF secreting breast cancer cell line derived from a Grade II (moderately differentiated) breast tumor that also expresses HLA class I & II antigens and is able to function as an antigen-presenting cell. Irradiated SV-BR-1-GM is used in a regimen including pre-dose low-dose cyclophosphamide and post-dose injection of IFNα2b into the inoculation sites. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 - study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 - study ongoing). Here we report survival data for patients with advanced metastatic breast cancer (aMBC) treated with the SV-BR-1-GM regimen. Methods: 27 patients with refractory aMBC were treated with the SV-BR-1-GM regimen as monotherapy (cycles every 2 weeks x3 and then monthly). The combination study uses the SV-BR-1-GM regimen with PD-1 inhibitors (PD-1i) pembrolizumab or retifanlimab with cycles every 3 weeks (12 patients dosed to date). Here we report progression free survival (PFS) and overall survival (OS) for patients where that data was collected. Results: A total of 35 patients received the SV-BR-1-GM regimen. The SV-BR-1-GM regimen alone (monotherapy) was given to 27 and 12 received the regimen with a PD-1i checkpoint inhibitor (combination therapy): 4 subjects crossed over from monotherapy. Patients had been heavily pre-treated, median prior regimens = 5. Most patients were estrogen receptor and/or progesterone receptor positive, 18% were Her2/neu positive and 33% were triple negative. The treatment was generally safe and well tolerated. The disease control rate was 30% for the SV-BR-1-GM regimen alone and 33% for the combination with a PD-1i. Several patients had objective complete regression of selected metastases. Median progression free survival was 2.8 months for the SV-BR-1-GM regimen alone and 4.2 months for the PD-1i combination. Median overall survival was 7.0 months for the SV-BR-1-GM regimen alone (data available on 9 patients), and 12.0 months for the PD-1i combination (data available on 7 patients). Conclusions: The median OS compares favorably with published data regarding survival in third line trials (Kazmi Breast Cancer Res Treat. 2020 Aug 17). The protracted OS seen in some subjects suggests some patient subpopulations are more likely to derive clinical benefit. The SV-BR-1-GM regimen alone or in combination with a PD-1i, when administered to heavily pre-treated patients with aMBC, may have elicited effective immune responses in some patients. TablePatients by StudyCharacteristicSV-BR-1-GM Regimen Alone (n=27)SV-BR-1-GM Regimen + PD-1i (n=12)All Patients* (n=35)Age60 ± 1063 ± 1060 ± 10Mean Prior Systemic Regimens5 (range 0-12)6 (range 1-10)5 (range 0-12)% ER/PR +52%75%58%% Her2/neu +15%17%18%% Triple Negative36%25%33%Delayed-type Hypersensitivity81%91%82%Disease Control Rate30%33%29%Median (Range) Progression Free Survival (months)2.8 (0.4-7.4) (n=27)4.2 (0.8-9.4) (n=11)2.8 (0.4-9.4) (n=34)Median (Range) Overall Survival (months)7.0 (1-41) (n=9)12.0 (5.1-21.4) (n=7)10.2 (1-41) (n=14)• Note that 4 patients crossed over from the monotherapy study to the combination therapy study. Citation Format: William Williams, Shaker R Dakhil, Carmen Calfa, Jarrod P Holmes, Saveri Bhattacharya, Jason Lukas, Elizabeth Tan-Chui, George E Peoples, Vivek G Sunkari, Markus D Lacher, Charles L Wiseman. Overall survival following treatment with a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-02

Abstract PS17-20: Response to a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer correlates with tumor grade

Abstract Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line, exceptional for having antigen-presenting capability and expressing both HLA I and II. The parent cell line, SV-BR-1, was derived from a patient with grade II (moderately differentiated) breast cancer. We report molecular characterization of SV-BR-1-GM, noting it retains features of a grade II tumor, and report enhanced disease control in patients with grade I or II breast cancer.Methods: SV-BR-1 and SV-BR-1-GM were characterized molecularly using RNAseq and proteomic analyses. We treated 23 evaluable patients with recurrent and/or metastatic breast cancer refractory to standard therapy. The SV-BR-1-GM regimen included cyclophosphamide 300 mg/m2 2-3d prior to intradermal injection of SV-BR-1-GM (20-40x106 cells divided into 4 sites) and IFNα into the inoculation sites (10,000 IU/site) about 48 and 96 hours subsequently. Cycles were q2 weeks x3 then qmo x 3 (clinical trial NCT03066947). Eleven patients were treated with the above regimen in combination with a PD-1 inhibitor (pembrolizumab or INCMGA00012) (clinical trial NCT03328026). Disease response was evaluated radiographically q3 mo and as clinically indicated. Results: To estimate the tumor grade represented by the SV-BR-1-GM cell line, we developed a score we refer to as Relative Molecular Grade (RMG). SV-BR-1-GM is most similar to the MDA-MB-468 cell line (RMG of 52.1), which was classified as Basal A phenotype. Basal A cancers are less aggressive than Basal B but more aggressive than Luminal, suggesting that SV-BR-1-GM may have retained features of a grade II breast cancer. We also noted that SV-BR-1-GM expresses both Class I (HLA-A, B & C) and Class II (HLA-DR and -DP) molecules, and that the HLA-DR expression is enhanced by treatment with IFNγ. SV-BR-1-GM expressed 31 genes which are overexpressed in breast cancer, 8 cancer-testis antigens and 3 genes expressed in breast tissue. In 30 patients treated with the SV-BR-1-GM regimen (19 with the SV-BR-1-GM regimen alone, 4 who began on the SV-BR-1-GM regimen and transitioned to combination with a PD-1i, and 7 with combination therapy alone) there were 7 with grade II breast cancer and 1 with grade I breast cancer (Table). These patients were heavily pre-treated with an average of 10 prior regimens. While only one patient with grade III cancer showed disease control, 75% of the patients with grade I or II tumors showed disease control. Patients remained on study for up to 259 days.Conclusions: SV-BR-1-GM appears to retain characteristics of a moderately differentiated breast cancer, expresses multiple potential tumor antigens, and can elicit disease control especially in patients with grade I and II breast cancer. TablePatients with Grade I/II TumorsCharacteristicSV-BR-1-GM Regimen Alone(n=6)SV-BR-1-GM Regimen + PD-1i(n=3)All Patients(n=8)Age64 ± 767 ± 465 ± 7Mean Prior Systemic Regimens6 (range 1-20)15 (range 14-15)10 (range 1-20)% ER/PR +80%100%86%% Her2/neu +0%33%14%% Triple Negative20%0%14%Delayed-type Hypersensitivity83%100%88%Disease Control Rate*67%100%75%Days on Study (Range)94 (32-181)189 (133-259)141 (32-259)•Includes CR, PR, SD (including minor responses and mixed responses) Citation Format: William Williams, Shaker R Dakhil, Carmen Calfa, Jarrod P Holmes, Saveri Bhattacharya, Jason Lukas, Elizabeth Tan-Chiu, George E Peoples, Vivek G Sunkari, Markus D Lacher, Charles L Wiseman. Response to a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer correlates with tumor grade [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-20

Abstract P3-09-08: Efficacy and safety of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer alone and in combination with immune checkpoint inhibitors

Abstract Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line, exceptional for having antigen-presenting capability expressing both HLA I and II. We report clinical efficacy, safety, and immunologic correlates of response from our initial Phase I/II trial and initial data from our trial of SV-BR1-GM in combination with immune checkpoint inhibitors. Methods: We enrolled patients with recurrent and/or metastatic breast cancer refractory to standard therapy. Patients received cyclophosphamide 300 mg/m2 2-3d prior to intradermal injection of SV-BR-1-GM (20-40 × 106 cells divided into 4 sites) and IFNα into the inoculation sites (10,000 IU/site) ~2 & 4 days subsequently. Cycles were q2 weeks x3 then qmo x 3. Adverse events (AE) were evaluated after each inoculation. Immunologic responses were measured by delayed type hypersensitivity (DTH) after each inoculation with humoral and cellular responses evaluated ~q3 mo. Disease response was evaluated radiographically q3 mo and as clinically indicated (clinical trial NCT03066947). A similar regimen was used with SV-BR-1-GM in combination with pembrolizumab (200 mg IV) with cycles every 3 weeks (Phase I/II study NCT03328026). Results: In Phase I/IIa (NCT03066947), 23 patients underwent 1 - 8 cycles of treatment. Tumor regression was seen in 3 patients, all of whom matched SV-BR-1-GM at least at one HLA allele. There were no related serious adverse events. The most common adverse event was minor local irritation at the inoculation site. Clinical data are shown in the table. A measurable DTH response was present in 21 patients. Of patients who developed a DTH response and had at least one HLA match, the tumor regression rate was 33% and for those with 2 HLA matches 67%. We saw evidence of antibody responses in 3 of 5 patients evaluated to date. Especially in responders after treatment, blood lymphocytes showed increased cytokine secretion (including ITAC, IFNγ, IL-6 & IL-8) following stimulation with antigens expressed in SV-BR-1-GM. 21/23 patients had expression of PD-L1 in identified circulating cancer-associated cells, and expression levels increased with treatment. Therefore, a combination study with pembrolizumab was initiated. Data on the first 6 patients shows that the regimen is clinically active and safe. One patient with a robust DTH response had evidence of tumor regression in liver metastases. This study is ongoing and is being modified to evaluate combination therapy with the PD-1 inhibitor INCMGA00012 and the IDO inhibitor epacadostat. Conclusions: SV-BR-1-GM appears to be safe and well-tolerated. Contrary to conventional wisdom, SV-BR-1-GM can produce regression of metastatic breast cancer correlating with an immunologic response and HLA matching. Combination therapy with checkpoint inhibitors is ongoing. Citation Format: William Williams, Shaker R Dakhil, Jarrod P Holmes, Saveri Bhattacharya, Carmen Calfa, Ajay Kundra, Daniel L Adams, Diane DaSilva, George E Peoples, Vivek Sunkari, Markus Lacher, Charles L Wiseman. Efficacy and safety of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer alone and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-08

Initial safety analysis of a randomized phase II trial of nelipepimut-S + GM-CSF and trastuzumab compared to trastuzumab alone to prevent recurrence in breast cancer patients with HER2 low-expressing tumors.

The development of HER2-targeted therapy has decreased recurrence rates and improved survival, transforming the natural history of HER2-positive breast cancer. However only a minority of breast cancer patients benefit as these agents are not used in patients with tumors expressing low levels of HER2. Preclinical data suggests a synergistic action of HER2-targeted vaccination with trastuzumab. We report the initial safety interim analysis of a phase II trial that enrolled patients with HER2 low-expressing (IHC 1+/2+) breast cancer who were clinically disease-free after standard therapy. Patients were randomized to receive the HER2-peptide vaccine nelipepimut-S + GM-CSF with trastuzumab (vaccine arm) or trastuzumab + GM-CSF (control arm) and were followed for recurrence. A planned analysis that occurred after enrollment of 150 patients showed no significant differences in toxicity between the two arms, including cardiac toxicity. The clinical efficacy of this combination will be reported 6 months after the final patient was enrolled