PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Ibrutinib-based therapy reinvigorates CD8+ T cells compared to chemoimmunotherapy: immune monitoring from the E1912 trial.

ABSTRACT: Bruton tyrosine kinase inhibitors (BTKis) that target B-cell receptor signaling have led to a paradigm shift in chronic lymphocytic leukemia (CLL) treatment. BTKis have been shown to reduce abnormally high CLL-associated T-cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T-cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pretreatment and on-treatment (6 and 12 months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab with fludarabine, cyclophosphamide, and rituximab (FCR). Intriguingly, we report that despite reduced overall T-cell counts; higher numbers of T cells, including effector CD8+ subsets at baseline and at the 6-month time point, associated with no infections; and favorable progression-free survival in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T-cell killing function during ibrutinib-rituximab treatment, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T-cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T-cell cytotoxicity during ibrutinib-rituximab treatment, using the bispecific antibody glofitamab, supporting combination immunotherapy approaches.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

Venetoclax plus R- or G-CHOP in non-Hodgkin lymphoma: results from the CAVALLI phase 1b trial.

Novel strategies, such as chemosensitization with targeted agents, that build on the success of standard immunochemotherapy show promise for the treatment of non-Hodgkin lymphoma (NHL). Here, we report a phase 1b study investigating dose escalation of the BCL2 inhibitor, venetoclax, in combination with rituximab or obinutuzumab and cyclophosphamide, doxorubicin, vincristine, and prednisone (R-/G-CHOP) chemotherapy in B-cell NHL. Objectives included safety assessment and determination of a recommended phase 2 dose (RP2D). Fifty-six patients were enrolled, most with follicular lymphoma (43%) or diffuse large B-cell lymphoma (DLBCL; 32%). Dose-limiting toxicities were reported in 3/14 patients at the first venetoclax dose (200 mg/d), after which dosing was changed from daily to 10 days per cycle and escalated to 800 mg. A further reduction to 5 days per cycle occurred at the 800-mg dose level in the G-CHOP arm. Cytopenias were predominant among grade 3/4 events and reported at a higher rate than expected, particularly in the G-CHOP arm; however, safety was manageable. Overall response rates were 87.5% (R-CHOP and G-CHOP combinations); complete response (CR) rates were 79.2% and 78.1%, respectively. Most double-expressor (BCL2+ and MYC+) DLBCL patients (87.5%; n = 7/8) achieved CR. Although the maximum tolerated dose was not reached, the RP2D for venetoclax with R-CHOP was established at 800 mg days 4 to 10 of cycle 1 and days 1 to 10 of cycles 2 to 8; higher doses were not explored, and this dosing schedule demonstrated an acceptable safety profile. This regimen is subsequently being evaluated in first-line DLBCL in the phase 2 portion of the study. This trial was registered at www.clinicaltrials.gov as #NCT02055820.

The PI3Kδ-Selective Inhibitor Idelalisib Minimally Interferes with Immune Effector Function Mediated by Rituximab or Obinutuzumab and Significantly Augments B Cell Depletion In Vivo.

Idelalisib is a highly selective oral inhibitor of PI3Kδ indicated for the treatment of patients with relapsed chronic lymphocytic leukemia in combination with rituximab. Despite additive clinical effects, previous studies have paradoxically demonstrated that targeted therapies potentially negatively affect anti-CD20 mAb effector mechanisms. To address these potential effects, we investigated the impact of PI3Kδ inhibition by idelalisib on the effector mechanisms of rituximab and obinutuzumab. At clinically relevant concentrations, idelalisib minimally influenced rituximab- and obinutuzumab-mediated Ab-dependent cellular cytotoxicity and phagocytosis on human lymphoma cell lines, while maintaining the superiority of obinutuzumab-mediated Ab-dependent cellular cytotoxicity. Consistent with this, idelalisib did not influence obinutuzumab-mediated B cell depletion in whole-blood B cell-depletion assays. Further, idelalisib significantly enhanced obinutuzumab-mediated direct cell death of chronic lymphocytic leukemia cells. In murine systems, in vivo inhibition of PI3Kδ minimally interfered with maximal rituximab- or obinutuzumab-mediated depletion of leukemic targets. In addition, the duration of rituximab- and obinutuzumab-mediated depletion of leukemia cells was extended by combination with PI3Kδ inhibition. Collectively, these data demonstrate that PI3Kδ inhibition does not significantly affect the effector mechanisms induced by rituximab or obinutuzumab and provides an effective in vivo therapeutic combination. Therefore, combinations of obinutuzumab and idelalisib are currently being assessed in clinical studies.

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.

Antitumour activity of the glycoengineered type II anti-CD20 antibody obinutuzumab (GA101) in combination with the MDM2-selective antagonist idasanutlin (RG7388).

OBJECTIVES: To investigate whether the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) combined with the selective MDM2 antagonist idasanutlin (RG7388) offers superior efficacy to monotherapy in treating B-lymphoid malignancies in preclinical models. METHODS: The combined effect of obinutuzumab or rituximab plus idasanutlin on direct cell death/apoptosis induction and antibody-dependent cellular cytotoxicity (ADCC) was evaluated using p53 wild-type Z-138 and DoHH-2 lymphoma cells. Furthermore, whole blood B-cell depletion was analysed, and tumour growth inhibition was evaluated in subcutaneous xenograft models. RESULTS: Idasanutlin induced concentration-dependent death of Z-138 and DoHH-2 cells. At concentrations >10-100 nm, idasanutlin enhanced obinutuzumab-induced death of DoHH-2 and Z-138 cells without negatively impacting obinutuzumab-mediated ADCC, natural killer cell activation or whole blood B-cell depletion. In the Z-138 xenograft model, a suboptimal dose of obinutuzumab with idasanutlin yielded substantial tumour growth inhibition and prolonged survival in a time-to-event analysis. In the DoHH-2 model, idasanutlin plus obinutuzumab showed superior tumour growth inhibition to idasanutlin plus rituximab. CONCLUSIONS: Obinutuzumab plus idasanutlin enhanced cell death of p53 wild-type tumour cells vs. rituximab plus idasanutlin without affecting obinutuzumab-mediated ADCC or B-cell depletion and showed robust antitumour efficacy in xenograft models, strongly supporting the investigation of this combination in clinical trials.

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Myeloid-T cell interplay and cell state transitions associated with checkpoint inhibitor response in melanoma.

BACKGROUND: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms. METHODS: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed. FINDINGS: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8+ T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function. CONCLUSIONS: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response. FUNDING: This research was funded by Roche Pharma Research and Early Development.

Explainable machine learning for profiling the immunological synapse and functional characterization of therapeutic antibodies.

Therapeutic antibodies are widely used to treat severe diseases. Most of them alter immune cells and act within the immunological synapse; an essential cell-to-cell interaction to direct the humoral immune response. Although many antibody designs are generated and evaluated, a high-throughput tool for systematic antibody characterization and prediction of function is lacking. Here, we introduce the first comprehensive open-source framework, scifAI (single-cell imaging flow cytometry AI), for preprocessing, feature engineering, and explainable, predictive machine learning on imaging flow cytometry (IFC) data. Additionally, we generate the largest publicly available IFC dataset of the human immunological synapse containing over 2.8 million images. Using scifAI, we analyze class frequency and morphological changes under different immune stimulation. T cell cytokine production across multiple donors and therapeutic antibodies is quantitatively predicted in vitro, linking morphological features with function and demonstrating the potential to significantly impact antibody design. scifAI is universally applicable to IFC data. Given its modular architecture, it is straightforward to incorporate into existing workflows and analysis pipelines, e.g., for rapid antibody screening and functional characterization.

Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation.

Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of >45,000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+ T cells with circulating cells identified CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+ T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.

Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies.

PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity.

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition.

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

A Novel Approach for Quantifying the Pharmacological Activity of T-Cell Engagers Utilizing In Vitro Time Course Experiments and Streamlined Data Analysis.

CD3-bispecific antibodies are a new class of immunotherapeutic drugs against cancer. The pharmacological activity of CD3-bispecifics is typically assessed through in vitro assays of cancer cell lines co-cultured with human peripheral blood mononuclear cells (PBMCs). Assay results depend on experimental conditions such as incubation time and the effector-to-target cell ratio, which can hinder robust quantification of pharmacological activity. In order to overcome these limitations, we developed a new, holistic approach for quantification of the in vitro dose-response relationship. Our experimental design integrates a time-independent analysis of the dose-response across different time points as an alternative to the static, "snap-shot" analysis based on a single time point commonly used in dose-response assays. We show that the potency values derived from static in vitro experiments depend on the incubation time, which leads to inconsistent results across multiple assays and compounds. We compared the potency values from the time-independent analysis with a model-based approach. We find comparably accurate potency estimates from the model-based and time-independent analyses and that the time-independent analysis provides a robust quantification of pharmacological activity. This approach may allow for an improved head-to-head comparison of different compounds and test systems and may prove useful for supporting first-in-human dose selection.

Predicting Tumor Killing and T-Cell Activation by T-Cell Bispecific Antibodies as a Function of Target Expression: Combining In Vitro Experiments with Systems Modeling.

Targeted T-cell redirection is a promising field in cancer immunotherapy. T-cell bispecific antibodies (TCB) are novel antibody constructs capable of binding simultaneously to T cells and tumor cells, allowing cross-linking and the formation of immunologic synapses. This in turn results in T-cell activation, expansion, and tumor killing. TCB activity depends on system-related properties such as tumor target antigen expression as well as antibody properties such as binding affinities to target and T cells. Here, we developed a systems model integrating in vitro data to elucidate further the mechanism of action and to quantify the cytotoxic effects as the relationship between targeted antigen expression and corresponding TCB activity. In the proposed model, we capture relevant processes, linking immune synapse formation to T-cell activation, expansion, and tumor killing for TCBs in vitro to differentiate the effect between tumor cells expressing high or low levels of the tumor antigen. We used cibisatamab, a TCB binding to carcinoembryonic antigen (CEA), to target different tumor cell lines with high and low CEA expression in vitro We developed a model to capture and predict our observations, as a learn-and-confirm cycle. Although full tumor killing and substantial T-cell activation was observed in high expressing tumor cells, the model correctly predicted partial tumor killing and minimal T-cell activation in low expressing tumor cells when exposed to cibisatamab. Furthermore, the model successfully predicted cytotoxicity across a wide range of tumor cell lines, spanning from very low to high CEA expression.

FcγRIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies.

Therapeutic antibodies can elicit an immune response through different mechanisms, either cell independent via complement activation (CDC) or through activation of immune-effector cells (such as macrophages and NK cells). After target binding, the Fc part of the antibody will interact with Fc receptors on the surface of effector cells, leading to activation and lysis of the target cells by a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC of an antibody can be increased by modifying the carbohydrates on the Fc part. If the fucose on the first N-acetylglucosamine is absent, the affinity for the FcγRIIIa is increased and the ADCC enhanced. We describe the development of a chromatography method that is based on the differential affinity of the Fc receptor FcγRIIIa (high affinity V158 variant) for fucosylated and a-fucosylated antibodies. Immobilized FcγRIIIa can be used for the separation of immunoglobulins carrying these glycosylation variants for both, analytical and preparative purposes. The biological activity and fucose content of three pools enriched for fully fucosylated, mono-fucosylated or a-fucosylated carbohydrates could be characterized. Mono-fucosylated and a-fucosylated immunoglobulins have the same enhanced biological activity compared to fully fucosylated IgGs. A direct, label- and modification-free analytical method for screening of IgGs from culture supernatant was developed and was amenable to high-throughput screening. Clones producing antibodies with a high content of a-fucosylated oligosaccharides could be successfully selected.

Pharmacodynamics of 2-[4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl]ethan-1-ol (GDC-0879), a potent and selective B-Raf kinase inhibitor: understanding relationships between systemic concentrations, phosphorylated mitogen-activated protein kinase kinase 1 inhibition, and efficacy.

The Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway is involved in cellular responses relevant to tumorigenesis, including cell proliferation, invasion, survival, and angiogenesis. 2-[4-[(1E)-1-(Hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl]ethan-1-ol (GDC-0879) is a novel, potent, and selective B-Raf inhibitor. The objective of this study was to characterize the relationship between GDC-0879 plasma concentrations and tumor growth inhibition in A375 melanoma and Colo205 colon cancer xenografts and to understand the pharmacodynamic (PD) marker response requirements [phosphorylated (p)MEK1 inhibition] associated with tumor growth inhibition in A375 xenografts. Estimates of GDC-0879 plasma concentrations required for tumor stasis obtained from fitting tumor data to indirect response models were comparable, at 4.48 and 3.27 microM for A375 and Colo205 xenografts, respectively. This was consistent with comparable in vitro potency of GDC-0879 in both cell lines. The relationship between GDC-0879 plasma concentrations and pMEK1 inhibition in the tumor was characterized in A375 xenografts after oral doses of 35, 50, and 100 mg/kg. Fitting pMEK1 inhibition to an indirect response model provided an IC(50) estimate of 3.06 microM. pMEK1 inhibition was further linked to A375 tumor volume data from nine different GDC-0879 dosing regimens using an integrated pharmacokinetic-PD model. A simulated PD marker response curve plot of K (rate constant describing tumor growth inhibition) versus pMEK1 inhibition generated using pharmacodynamic parameters estimated from this model, showed a steep pMEK1 inhibition-response curve consistent with an estimated Hill coefficient of approximately equal 8. A threshold of >40% pMEK1 inhibition is required for tumor growth inhibition, and a minimum of approximately 60% pMEK1 inhibition is required for stasis in A375 xenografts treated with GDC-0879.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.