IL-15/IL-15Rα-Fc fusion protein XmAb24306 potentiates activity of CD3 bispecific antibodies through enhancing T cell expansion.

Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rß (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.

Complex PK-PD of an engineered IL-15/IL-15Rα-Fc fusion protein in cynomolgus monkeys: QSP modeling of lymphocyte dynamics.

XmAb24306 is a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein currently under clinical investigation as an immunotherapeutic agent for cancer treatment. XmAb24306 contains mutations in IL-15 that attenuate its affinity to the heterodimeric IL-15 receptor ßγ (IL-15R). We observe substantially prolonged pharmacokinetics (PK) (half-life ∼ 2.5 to 4.5 days) in single- and repeat-dose cynomolgus monkey (cyno) studies compared to wild-type IL-15 (half-life ∼ 1 hour), leading to increased exposure and enhanced and durable expansion of NK cells, CD8+ T cells and CD4-CD8- (double negative [DN]) T cells. Drug clearance varied with dose level and time post-dose, and PK exposure decreased upon repeated dosing, which we attribute to increased target-mediated drug disposition (TMDD) resulting from drug-induced lymphocyte expansion (i.e., pharmacodynamic (PD)-enhanced TMDD). We developed a quantitative systems pharmacology (QSP) model to quantify the complex PKPD behaviors due to the interactions of XmAb24306 with multiple cell types (CD8+, CD4+, DN T cells, and NK cells) in the peripheral blood (PB) and lymphoid tissues. The model, which includes nonspecific drug clearance, binding to and TMDD by IL15R differentially expressed on lymphocyte subsets, and resultant lymphocyte margination/migration out of PB, expansion in lymphoid tissues, and redistribution to the blood, successfully describes the systemic PK and lymphocyte kinetics observed in the cyno studies. Results suggest that after 3 doses of every-two-week (Q2W) doses up to 70 days, the relative contributions of each elimination pathway to XmAb24306 clearance are: DN T cells > NK cells > CD8+ T cells > nonspecific clearance > CD4+ T cells. Modeling suggests that observed cellular expansion in blood results from the influx of cells expanded by the drug in lymphoid tissues. The model is used to predict lymphoid tissue expansion and to simulate PK-PD for different dose regimens. Thus, the model provides insight into the mechanisms underlying the observed PK-PD behavior of an engineered cytokine and can serve as a framework for the rapid integration and analysis of data that emerges from ongoing clinical studies in cancer patients as single-agent or given in combination.

Surgery Alone (Without Adjuvant Radiation) Adequately Treats Histologic Perineural Basal Cell Carcinomas: A Systematic Review With Meta-Analysis.

BACKGROUND: Histologic perineural invasion (PNI) in basal cell carcinomas (BCC) lacks evidence-based treatment guidelines. OBJECTIVE: Systematically review and analyze treatment outcomes of BCC with histologic PNI (PNBCC). MATERIALS AND METHODS: PubMed, Embase, and Cochrane Reviews were searched through June 25, 2021. Thirteen eligible cohort studies were meta-analyzed. RESULTS: 502 of 713 PNBCC were treated with Mohs Surgery (MMS), wide local excision (WLE), or surgery (MMS or WLE) with adjuvant radiation (Surg + RT). Overall 5-year local control (LC) was 97.2% and cancer-specific survival (CSS) was 99.6%. Surg and Surg + RT did not differ in recurrence (2.1% vs 4.7%; p-value 0.56; RR 1.51 [0.37, 6.20]), LC (97.9% vs 96.2%; p-value 0.19; RR 0.98 [0.96, 1.01]) or CSS (100% vs 99.1%; p-value 0.40; RR 0.99 [0.95, 1.02]). LIMITATIONS: No randomized controlled trials were found. Outcome data were often lacking. CONCLUSION: Overall LC and CSS were high at median 5-year follow-up for surgery alone and Surg + RT. Surgery alone and Surg + RT demonstrated statistically equivalent outcomes. We do not recommend adjuvant radiation therapy for solely histologic PNBCC if clear margins are achieved.

The management of combined ACL and MCL injuries: A systematic review.

Background: The management of combined anterior cruciate ligament (ACL) and medial collateral ligament (MCL) injuries remains contentious. Clinical outcomes of surgical, conservative, and combined approaches have been described in a range of prospective and retrospective studies. The aim of the current systematic review was to evaluate these outcomes and assess the study methodologies. Methods: A comprehensive literature search of the following databases was performed: PubMed, OVID, Cochrane Database of Systematic Reviews and Google Scholar. Studies were assessed using the Coleman Methodology Score. Results: 52 articles were included (3 randomised controlled trials, 8 prospective comparative studies, 17 retrospective comparative studies and 24 case series). Outcome measures were heterogeneous amongst articles. The most common outcomes assessed were AP laxity, Lysholm score and medial/valgus laxity. Complications at varying follow-up times with differing grades of MCL injury were reported in 25 (48%) studies. Evidence was conflicting, with no consensus from the available published literature regarding the best method of treatment for a combined ACL and MCL injury. Conclusions: Heterogeneous outcome measures and limited randomised controlled trials prevent advocacy of a single treatment option. Good outcomes have been reported from repair, reconstruction and conservative management of the MCL together with ACL reconstruction. Further prospective comparative data is required to evaluate MCL management choice and prognostic signs for successful nonsurgical MCL treatment.

Extramedullary Tibial Guide Alignment Is Not Affected by Excess Lower Limb Fat Distribution in Total Knee Arthroplasty.

Background The goal of this study is to investigate whether excess lower limb fat distribution affects tibial guide alignment in conventional total knee arthroplasty (TKA) with extramedullary guides. A thicker soft tissue envelope may affect the accuracy of extramedullary cutting guide placement and subsequent instrumentation. Previous studies have used body mass index (BMI) to stratify patients, a poor proxy of lower limb fat distribution, which may explain conflicting results reported on this topic to date. This study overcomes this issue by using a novel, radiographic anthropometric index to assess lower limb fat distribution. Methodology This is a single-surgeon, single-implant, single-centre retrospective series of 102 consecutive primary TKAs. The suprapatellar fat index (SPFI) and BMI were recorded for all patients, and postoperative tibial component alignment measurements were calculated. Secondary outcome measures included femoral component alignment, femorotibial alignment, length of hospital stay, tourniquet time, blood loss, and complications/reoperations. Results In this study, 102 patients (average age of 69) had an average BMI of 30.8 kg/m2 (19.2-45.5 kg/m2) and an average SPFI of 0.26 (0.09-0.57). Multiple regression analysis demonstrated that increasing leg fat distribution did not affect tibial component alignment in the coronal or sagittal plane. Conclusions Excess lower limb fat distribution, simply measured using the SPFI, does nothave a significant effect on tibial component positioning when extramedullary guides are used in conventional TKA.

Wound Healing on the Dorsal Hands: An Intrapatient Comparison of Primary Closure, Purse-String Closure, and Secondary Intention.

Nonmelanoma skin cancers are common on the dorsal hands where reserve tissue is limited. We highlight the case of an elderly man who had 3 nonmelanoma skin cancers on the left hand that were treated on the same day and left similar wounds. The wounds were repaired by primary closure, secondary intention, and purse-string circumferential closure. All wounds healed with excellent and essentially equivalent cosmetic results. For small shallow wounds on the dorsal hands, dermatologic surgeons should have confidence that secondary intention healing likely will lead to acceptable cosmetic and functional results.

Mechanistic Models of Cellular Signaling, Cytokine Crosstalk, and Cell-Cell Communication in Immunology.

The cells of the immune system respond to a great variety of different signals that frequently reach them simultaneously. Computational models of signaling pathways and cellular behavior can help us explore the biochemical mechanisms at play during such responses, in particular when those models aim at incorporating molecular details of intracellular reaction networks. Such detailed models can encompass hypotheses about the interactions among molecular binding domains and how these interactions are modulated by, for instance, post-translational modifications, or steric constraints in multi-molecular complexes. In this way, the models become formal representations of mechanistic immunological hypotheses that can be tested through quantitative simulations. Due to the large number of parameters (molecular abundances, association-, dissociation-, and enzymatic transformation rates) the goal of simulating the models can, however, in many cases no longer be the fitting of particular parameter values. Rather, the simulations perform sweeps through parameter space to test whether a model can account for certain experimentally observed features when allowing the parameter values to vary within experimentally determined or physiologically reasonable ranges. We illustrate how this approach can be used to explore possible mechanisms of immunological pathway crosstalk. Probing the input-output behavior of mechanistic pathway models through systematic simulated variations of receptor stimuli will soon allow us to derive cell population behavior from single-cell models, thereby bridging a scale gap that currently still is frequently addressed through heuristic phenomenological multi-scale models.

Cutting Edge: Quantitative Determination of CD40L Threshold for IL-12 and IL-23 Production from Dendritic Cells.

Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/µm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.

A hierarchy of affinities between cytokine receptors and the common gamma chain leads to pathway cross-talk.

Cytokines belonging to the common gamma chain (γc) family depend on the shared γc receptor subunit for signaling. We report the existence of a fast, cytokine-induced pathway cross-talk acting at the receptor level, resulting from a limiting amount of γc on the surface of T cells. We found that this limited abundance of γc reduced interleukin-4 (IL-4) and IL-21 responses after IL-7 preexposure but not vice versa. Computational modeling combined with quantitative experimental assays indicated that the asymmetric cross-talk resulted from the ability of the "private" IL-7 receptor subunits (IL-7Rα) to bind to many of the γc molecules even before stimulation with cytokine. Upon exposure of T cells to IL-7, the high affinity of the IL-7Rα:IL-7 complex for γc further reduced the amount of free γc in a manner dependent on the concentration of IL-7. Measurements of bioluminescence resonance energy transfer (BRET) between IL-4Rα and γc were reduced when IL-7Rα was overexpressed. Furthermore, in a system expressing IL-7Rα, IL-4Rα, and γc, BRET between IL-4Rα and γc increased after IL-4 binding and decreased when cells were preexposed to IL-7, supporting the assumption that IL-7Rα and the IL-7Rα:IL-7 complex limit the accessibility of γc for other cytokine receptor complexes. We propose that in complex inflammatory environments, such asymmetric cross-talk establishes a hierarchy of cytokine responsiveness.

Observation of Dog-Ear Regression by Anatomical Location.

BACKGROUND: When an excision is performed by a method other than elliptical excision, direct primary wound closure can result in standing cones or "dog-ears." In 2008, Lee and colleagues noted that dog-ears of <8 mm in height have a statistically greater tendency to resolve without further surgical correction than larger dog-ears. OBJECTIVE: To stratify dog-ears by anatomic location and inform on the need for correction at the time of surgery. MATERIALS AND METHODS: After tumor extirpation, patients were counseled that primary closure of the surgical wound would result in dog-ears at the wound apices. Dog-ears were left uncorrected in participating patients. At 6 months, patients were assessed for resolution of the dog-ears and asked to rate the appearance of the scar. RESULTS: A total of 140 dog-ears were observed in the study period. Anatomical locations included the hand/foot, trunk, limb, and head/neck. Among these dog-ears, 114/140 (81%) showed complete resolution. Patient satisfaction with the scar appearance correlated well with the dog-ear resolution, with most patients rating the appearance of the scar as good to excellent. CONCLUSION: This study suggests that dog-ears on the hand and dog-ears ≤4 mm on the trunk may be observed without any final cosmetic penalty.

Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis-acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance.

Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8(+) T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8(+) T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8(+) T cells and can be manipulated to improve adoptive cancer immunotherapy.

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces.

Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology.

Utility of Wood's light in margin determination of melanoma in situ after excisional biopsy.

BACKGROUND: Margin evaluation of melanoma in situ (MIS) is difficult because of its ill-defined clinical borders. Wood's light examination is commonly used to help delineate MIS margin before excision. OBJECTIVE: To prospectively study the accuracy of preoperative Wood's light examination for margin assessment of MIS. MATERIALS AND METHODS: The authors evaluated 60 patients before excision of MIS under white light and Wood's light. Staged excision was performed using the square procedure technique. After achieving clear margins, they compared final wound size with expected wound size if surgical margins had been based on Wood's light examination. RESULTS: Seven patients (11.7%) had Wood's light enhancement beyond the visible margin of the biopsy site. In all 7, increased wounding would have occurred if the surgical margins had been based on Wood's light examination. In 1 of the 7, use of the Wood's light examination would have reduced the surgical stages needed by 1 stage but would have increased the wound size by 83.3%. CONCLUSION: Wood's light examination has limited utility if complete excisional biopsy of MIS is performed before treatment. In this study, surgical margin based on the Wood's light examination would have resulted in an increased average wound size and would not have reduced the number of stages needed when performing the square procedure.

Ligand-mediated friction determines morphodynamics of spreading T cells.

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.

TCR Microclusters pre-exist and contain molecules necessary for TCR signal transduction.

TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.

Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.

Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.