When will we have a clone? An industry perspective on the typical CLD timeline.

Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre-transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line.

Critical decisions: A mixed-methods study of decision-making among diverse gynecologic cancer patients considering therapeutic clinical trial enrollment.

OBJECTIVE: Participation in therapeutic clinical trials does not reflect the diversity of gynecologic cancer patients, limiting access to novel therapeutics and generalizability of results. Reasons for inequities in participation among historically underrepresented populations remain undertheorized, as studies have shown equal willingness to participate among groups. We sought to apply a precarity framework to conceptualize the factors that impact patients' desire to enroll, to improve equity in gynecologic oncology clinical trial participation. METHODS: Gynecologic cancer patients at a single tertiary care facility in the Southwestern United States who discussed participation in therapeutic clinical trial with their oncology provider from 2020 to 2021 were identified. Enrolled participants completed surveys and qualitative interviews regarding treatment experiences and decision-making. Oncology providers completed parallel surveys at the time of their patient's enrollment. Descriptive statistics and thematic coding were used to analyze data. RESULTS: 30 patients were enrolled and participated in surveys and interviews. No differences were found in quantitative data assessing shared decision-making and patient-centered communication between those who enrolled and those who did not. Qualitative data demonstrated that patients who declined trial enrollment expressed concerns regarding uncertainty and loss of control, independence in decision-making, and significant resource challenges and financial toxicity of cancer treatment. CONCLUSIONS: We identified a constellation of factors that contribute to desire to enroll in clinical trials, that we describe using the framework of precarity. Through identification of precarious patients and mitigation of burdens, we anticipate improved enrollment and retention in therapeutic clinical trials among diverse gynecologic oncology patients.

Genome-scale functional genomics screening highlights genes impacting protein fucosylation in Chinese hamster ovary cells.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

"Care needs to be integrated" Patient and provider perspectives on a cancer shared-care model.

PURPOSE: Current early-stage breast and gynecological cancer care models often begin with a referral from a primary care provider (PCP) or gynecologist (OB/Gyn) and end with a patient being transitioned back to the referring provider at the completion of treatment. There is frequently little communication between oncologists and the referring provider during treatment, and this pattern continues after the patient completes their treatment. METHODS: We convened a diverse Patient Advisory Board (PAB) to identify areas where breast or gynecological cancer patients felt they could benefit from additional support during and after their cancer care. PAB members attended five Zoom meetings and completed four online surveys. Semi-structured interviews were conducted with primary care or OB/Gyn physicians to collect information on current practices. RESULTS: Patients identified multiple areas in which they needed additional support from their PCP. Providers also identified topics on which they could use additional training. However, there was little overlap between patient and provider priority topics. Both patients and providers agreed that there was inadequate communication between the cancer center and PCPs before, during, and after cancer treatment. CONCLUSIONS: A shared-care model that emphasizes communication between primary care providers, the oncology care team, and patients is urgently needed. Patients indicated the need for additional support from their PCP on specific topics, and PCPs were interested in continuing their education to better serve their patients with cancer. IMPLICATIONS FOR CANCER SURVIVORS: The importance of consistent communication among all parties during the entire cancer journey was emphasized as a key area for improvement.

Improving Sexual and Gender Minority Cancer Care: Patient and Caregiver Perspectives From a Multi-Methods Pilot Study.

Purpose: Up to 1 million lesbian, gay, bisexual, and transgender (i.e., sexual and gender minority, SGM) individuals in the United States have histories of cancer. This medically underserved population is diverse, with complex sexualities and gender identities, and distinct health concerns. SGM persons experience disproportionate risks for, and rates of, anal, breast, cervical, colorectal, endometrial, lung, and prostate cancers, in addition to cancers affecting transgender persons who have undergone sex-reassignment. SGM individuals are linked by shared experiences of stigmatization as a minority population for which little cancer research has been conducted. SGM cancer patients frequently report reluctance to seek healthcare, have poorer outcomes following diagnosis, engage in elevated risk behaviors (i.e. smoking and alcohol use) even after cancer diagnosis, have difficulty making emotional adjustment to illness, and experience higher rates of psychological distress. They report less satisfaction with cancer care, deficiencies in patient-centeredness and shared decision-making, gaps in care, and social isolation. Minority stress resulting from experiences of anti-SGM sentiment and discrimination affects cancer patients and their informal cancer caregivers. Our paper presents findings from a pilot study to identify gaps and opportunities to improve cancer care for SGM patients and caregivers at the University of New Mexico Comprehensive Cancer Center. Methods: Between June 2020 and July 2021, we used a multi-methods research design informed by ecological theory to collect qualitative and quantitative data regarding cancer patient and caregiver quality of life (QoL) and experiences of cancer and survivorship care. We used PROMIS measures distributed via REDCap to assess QoL (i.e., fatigue, pain interference, pain intensity, anxiety, depression, emotional support, social isolation, and companionship), and conducted in-depth semi-structured interviews. We recruited 10 SGM cancer patients and 8 heterosexual, cisgender (H/C) patient matches, and their self-identified informal cancer caregivers (n=36, dyad total n=18). Interviews ranged from 1 to 2 hours, were audio-recorded and transcribed for analysis. The study was approved by the University of New Mexico Human Research Protections Office Institutional Review Board. Results: Results of the PROMIS QoL assessments indicated that SGM patients reported greater anxiety [mean (SD) = 54.5 (8.8)] and depression [mean (SD) = 49.3 (4.8)] than H/C patients [mean (SD)=51.6 (7.5) and 45.4 (6.8) respectively], while heterosexual, cisgender (H/C) patients reported higher fatigue [mean (SD) =52.04 (8.18)] and stronger pain intensity than SGM patients [mean (SD)=48.3 (9.1) and 37.8 (9.1) respectively]. SGM patients reported higher levels of social isolation [mean (SD) = 48.3 (7.3) vs. 42.1 (7.4) for H/C patients, whereas H/C patients reported more emotional support (mean (SD) =57.5 (9.3) vs. 53.0 (6.9)] and companionship [mean (SD) = 55.2 (8.6) vs. 51.5 (11.0)]. SGM and H/C differences in caregiver QoL were most notable with regards to higher levels of fatigue [mean (SD) = 47.1 (6.0) for SGM, and 42.4 (11.5) for H/C] and companionship [mean (SD) = 55.3 (6.0) for SGM, and 50.9 (5.5) for H/C]. Qualitative interviews supported our quantitative results. SGM patients and caregivers articulated experiences of anti-SGM stigma and discrimination contributing to minority stress that influenced their initial cancer care encounters. SGM dyads had more trepidation and/or medical mistrust during initial cancer care encounters when compared to H/C patients and caregivers. SGM patients questioned care that was not culturally responsive to SGM preferences, while H/C patients were more apt to identify gaps in communication and perceived lack of clarity regarding cancer care delivery. Although SGM patients experienced high satisfaction with their cancer care once they developed trust with their providers, they discussed desires to have more direct conversations with their oncologists about their sexual orientation and gender identities and sexual health. All patients and providers in the study (SGM and H/C) appreciated their oncology care teams. All patients and caregivers relied on social networks comprised of friends and family, although SGM patients and caregivers had smaller social networks and relied less on biological family, and single SGM individuals experienced challenges accessing cancer care and struggled with social isolation. We discovered too, that all caregivers, regardless of Sexual Orientation and Gender Identity (SOGI), perceived a lack of support and information pertaining to their loved one's treatment, side effects and best way to provide care. Conclusions: This study demonstrates that prior stigmatizing experiences contribute to minority stress and medical mistrust for SGM cancer patients and their informal caregivers across the cancer care experience. Findings point to specific gaps in SGM cancer patient care, including lack of conversation about patient SOGI, inadequate staff and oncology provider SGM specific knowledge and cultural competence/cultural humility training, and insufficient patient supports for those who lack social support during cancer care treatment. Further, this study reveals inadequacies in SGM specific support, and overall support services for informal cancer caregivers. Additional research is required to develop targeted interventions to address minority stress and clinic environment concerns to improve cancer care for SGM patients. Importantly, while there were differences between SGM and H/C experiences of cancer treatment, significant similarities also emerged. Caregiver expressed consensus about the current lack of support and guidance for informal caregivers of cancer patients. Future work should focus on providing caregiver-specific resources in the clinic setting and facilitating support groups for caregivers to network with one another, as well as for tailoring SGM specific caregiver support services. Our findings highlight areas for improving cancer care for the SGM community, as well as a broader population of patients and caregivers.

Examining Disparities in Route of Surgery and Postoperative Complications in Black Race and Hysterectomy.

OBJECTIVE: To estimate the associations among race, route of hysterectomy, and postoperative complications among women undergoing hysterectomy for benign indications. METHODS: A cohort study was performed. All patients undergoing hysterectomy for benign indications, recorded in the National Surgical Quality Improvement Program and its targeted hysterectomy file in 2015, were identified. The primary exposure was patient race. The primary outcome was route of hysterectomy and the secondary outcome was postoperative complication. Associations were examined using both bivariable tests and logistic regression. RESULTS: Of 15,136 women who underwent hysterectomy for benign indications, 75% were white and 25% were black. Black women were more likely to undergo an open hysterectomy than white women (50.1% vs 22.9%; odds ratio [OR] 3.36, 95% CI 3.11-3.64). Black women had larger uteri (median 262 g vs 123 g; 60.7% vs 25.6% with uterus greater than 250 g), more prior pelvic surgery (58.5% vs 53.2%), and higher body mass indices (32.7 vs 30.4). After adjusting for these and other clinical factors, black women remained more likely to undergo an open hysterectomy (adjusted OR 2.02, 95% CI 1.85-2.20). Black women experienced more major complications than white women (4.1% vs 2.3%; P<.001) and more minor complications (11.4% vs 6.7%; OR 1.78, P<.001). Again these disparities persisted with adjustment (major adjusted OR 1.56, 95% CI 1.25-1.95; minor adjusted OR 1.27, 95% CI 1.11-1.47). CONCLUSIONS: Black women undergo a higher proportion of open hysterectomy and experience more major and minor postoperative complications. These differences persisted even after adjusting for confounding medical, surgical, and gynecologic factors.

Tinkering toward departure: The limits of improvisation in rural Ethiopian biomedical practices.

This paper explores Ethiopian physicians' responses to tensions produced by gaps between ideals of biomedicine and realities of clinical practice in two rural Ethiopian hospitals. Physicians engage in creativity and improvisation, including relying on informal networks and practices and tinkering within diagnoses and procedures, to overcome constraints of lack of resources and limited opportunities to engage in "good medicine." These courageous, but often unsuccessful attempts to mitigate professional and personal conflicts within their medical practices represent improvisation in impossible circumstances. This paper results from ethnographic research conducted in 2013-2014 and includes participant observations and qualitative interviews in two hospitals within the same community. The inherent conflicts among globalized standards, unpredictable transnational medical networks, and innovative practices produce tenuous clinical spaces and practices that rely on a mosaic of techniques and ad hoc connections. Tinkering and improvisation often fail to mediate these conflicts, contributing to physician disenchantment and departure from the community.

The CD100 receptor interacts with its plexin B2 ligand to regulate epidermal γδ T cell function.

γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.

cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αß T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

The junctional adhesion molecule JAML is a costimulatory receptor for epithelial gammadelta T cell activation.

Gammadelta T cells present in epithelial tissues provide a crucial first line of defense against environmental insults, including infection, trauma, and malignancy, yet the molecular events surrounding their activation remain poorly defined. Here we identify an epithelial gammadelta T cell-specific costimulatory molecule, junctional adhesion molecule-like protein (JAML). Binding of JAML to its ligand Coxsackie and adenovirus receptor (CAR) provides costimulation leading to cellular proliferation and cytokine and growth factor production. Inhibition of JAML costimulation leads to diminished gammadelta T cell activation and delayed wound closure akin to that seen in the absence of gammadelta T cells. Our results identify JAML as a crucial component of epithelial gammadelta T cell biology and have broader implications for CAR and JAML in tissue homeostasis and repair.