Citations

Type I hypersensitivity reactions (HSR) to dabrafenib are rare but have been previously described. We present a case where a 72-year-old woman with recurrent, metastatic BRAF-mutated melanoma developed a type I HSR to dabrafenib. We, therefore, developed a desensitization protocol with encorafenib, a similar class agent, to allow the patient to continue with treatment. Patients with a history of HSR to dabrafenib may be considered for encorafenib desensitization when other therapeutic options are limited.

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

Abstract The most common adverse event (AE) of dabrafenib and trametinib (DT) is pyrexia, which has been reported to occur in up to 71% of patients. Pyrexia resulted in therapy discontinuation in up to 26% of patients who otherwise were benefiting from the treatment. Little is known about the pathogenesis and optimal management of this common AE. We hypothesized that the etiology of pyrexia in patients treated with DT could be extrapolated from DT-related cutaneous adverse effects in which a paradoxical MAPK pathway activation has been identified. We hypothesized that the mechanism of MAPK pathway activation with DT therapy is similar to that reported in patients with familial Mediterranean fever (FMF), a rare, inherited condition characterized by episodes of fever and rash that responds exceedingly well to colchicine-based therapy. Based on this association, our group explored the use of oral colchicine for the treatment of DT-associated pyrexia in five patients with metastatic melanoma. Citation Format: Jesus Vera, Jonas Paludo, Lisa Kottschade, Matthew Block, Yiyi Yan, Charles Loprinzi, Svetomir Markovic. Dabrafenib-trametinib-induced pyrexia successfully treated with colchicine [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B15.

AbstractThrough our involvement in KEYNOTE‐059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti‐VEGFR‐2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST‐defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression‐free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5‐fold (range 4‐121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post‐treatment versus pre‐treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti‐VEGFR‐2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti‐VEGF(R)/taxane are underway.

Introduction Day 15 absolute lymphocyte count (ALC-15) recovery after autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) is recognized as a prognostic factor for survival. ALC-15 recovery is directly dependent on the amount of collected and infused autograft-absolute lymphocyte count (A-ALC). Our group published a double-blind phase III study which showed that patients infused with an A-ALC ≥ 0.5 x 109 cells/kg experienced superior survival post-APBHSCT. Based on the results from our phase III study, on April 1, 2017, our Bone Marrow Transplant Program changed our standard clinical practice to collect an A-ALC ≥ 0.5 x 109 cells/kg in addition to stem cells for lymphoma patients undergoing APBHSCT. To continue to assess the prognostic ability of A-ALC, we evaluated the overall survival (OS) and progression-free survival (PFS) of diffuse large B-cell lymphoma (DLBCL) patients that underwent APBHSCT after April 1, 2017 compared with a matched-control group in a 1:1:1 ratio with DLBCL patients infused with an A-ALC < 0.5 x 109 cells/kg or an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1, 2017. Methods Eight-five consecutive DLBCL patients after our practice changed on April 1, 2017 received an A-ALC ≥ 0.5 x 109 cells/kg. For each DLBCL patient infused with an A-ALC ≥ 0.5 x 109 cells/kg after April 1, 2017, the set of potential controls was restricted to those DLBCL patients infused with an A-ALC < 0.5 x 109 cells/kg and an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017 . Within this group of potential controls, the subjects were chosen who best matched for the cases in terms of clinical risk variables, including gender, age, stage, lactate dehydrogenase (LDH), performance status, extra-nodal disease, International prognostic index (IPI), and disease status prior to APBHSCT (complete or partial response), using the GREEDY algorithm. Cases were defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg starting from April 1 2017. Control 1 was defined as DLBCL patients infused with an A-ALC < 0.5 x 109 cells/kg prior to April 1 2017. Control 2 was defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017. We truncated the survival follow-up to 3 years starting from the date of transplant to match follow-up time between cases and controls. Both matched cases and control groups patients received BEAM conditioning. This retrospective study was approved by the Mayo Clinic Institutional Review Board according to the regulation of the Declaration of Helsinki. Results Cases, control 1 and control 2 were balanced to the clinical risk variables; age (p = 0.8); gender (p = 0.9); LDH (p = 0.6); stage (p = 0.3); performance status (p = 0.5); extra-nodal disease (p = 0.2); IPI (p = 0.6); and clinical status prior to transplant (p = 0.2). Figure 1A showed the OS and figure 1B the PFS. Both the cases and control 2 showed superior OS and PFS in comparison to control 1. Cases and control 2 showed overall similar OS and PFS. Multivariate analysis including all patients continue to depict the A-ALC ≥ 0.5 x 109 cells/kg as an independent predictor for OS (HR = 0.382, 95%CI=0.241-0.605, p < 0.0001) and PFS (HR = 0.437, 95%CI = 0.279-0.629, p <0.0001). Subgroup analysis, including all patients, shows superior OS and PFS for patients infused with an A-ALC ≥ 0.5 x 109 cells/kg for each of the following clinical variables: age [> 60 years: OS (p <0.0001); PFS (p <0.0001) or ≤ 60 years: OS (p <0.01); PFS (p , 0.02)]; extra-nodal disease [> 1: OS (p <0.008); PFS (p <0.01) or ≤1: OS (p < 0.001): PFS (p <0.0005)]; IPI [> 2: OS (p <0.01); PFS (p <0.02) or ≤ 2: OS (p <0.0008); PFS (p <0.0006)]; LDH [abnormal: OS (p <0.006); PFS (p <0.005) or normal: OS (p <0.002); PFS (p <0.002)]; performance status [> 1: OS (p <0.04); PFS (p <0.03) or ≤ 1: OS (p <0.0001); PFS (p <0.0001)]; stage [I/II: OS (p <0.006); PFS (p <0.006) or III/IV: OS (p <0.002); PFS (p <0.001)]; response prior to transplant [complete response: OS (p <0.0007); PFS (p <0.0001) or partial response: OS (p <0.01); PFS (p <0.02)]. Conclusion Our match-control study continues to support the results of our Phase III study showing that the infusion of A-ALC is a prognostic factor for survival of DLBCL patients undergoing APBHSCT. Our studies support the practice of not only collecting enough stem cells (i.e., CD34) for hematologic engraftment, but also enough immune effector cells (i.e., A-ALC) to improve clinical outcomes in DLBCL patients post-APBHSCT. Figure 1 Disclosures Ansell: Regeneron: Research Funding; Bristol Myers Squibb: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding.

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Abstract CD40 is a co-stimulatory receptor of the TNF receptor superfamily expressed on antigen presenting cells (APCs). Antibodies targeting CD40 may have therapeutic benefit via multiple mechanisms including innate immune activation that can support generation of antigen-specific, antitumor T cell responses, and binding to CD40-expressing cancer cells leading to antibody-mediated target cell killing. Multiple CD40-directed antibodies are in clinical development and differ by immunoglobulin isotype, affinity to CD40, and selectivity for FcγR-binding. These alterations could lead to differences in pharmacodynamic and antitumor activity. SEA-CD40 is an agonistic non-fucosylated, humanized IgG1 monoclonal antibody directed against CD40. SEA-CD40 has enhanced FcγRIIIa binding (~10x greater than parent IgG1 antibody) that drives increased effector function, resulting in more potent immune stimulatory activity than antibodies with muted or selective FcγR binding. The enhanced effector function of SEA-CD40 may confer greater immune stimulation and antitumor activity relative to other CD40-directed therapeutics. Preclinically, SEA-CD40 exposure results in a distinct signature of responses including activation of APCs, CD8+ and CD4+ T cells and NK cells, and targeted depletion of CD40+ B cells. SEA-CD40 demonstrates superior activity compared to other CD40-targeted antibodies in vitro and in vivo, suggesting that the enhanced effector function is critical for optimal immune cell agonism. For example, SEA-CD40 drove in vitro ADCC activity 100-fold above the parent antibody and exhibited robust ADCC with the low and high affinity FcγRIIIA genotype. At matched dose levels in cynomolgus monkeys, SEA-CD40 induced circulating cytokines and sustained B cell depletion that were up to 50-fold above that induced with the parent antibody. The SEA-CD40 signature of activation translates to increased antitumor activity as a single agent and in combination with standard of care treatments in preclinical models, suggesting the potential for beneficial combination therapy in the clinic. The SEA-CD40 immune signature was confirmed by pharmacodynamic changes in an ongoing phase 1 clinical trial in patients with relapsed/refractory metastatic solid tumors (NCT02376699). SEA-CD40 treatment induced dose-dependent increases in circulating cytokines and chemokines associated with myeloid and lymphoid immune activation and trafficking. SEA-CD40 treatment also resulted in activation of CD4+ and CD8+ T cells and CD40-targeted B cell depletion in the periphery. These findings support continued clinical evaluation of SEA-CD40. The ongoing phase 1 clinical trial is actively enrolling and includes a cohort in pancreatic cancer assessing the combination of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab. Citation Format: Haley Neff-LaFord, Juneko E. Grilley-Olson, David C. Smith, Brendan Curti, Sanjay Goel, Timothy M. Kuzel, Svetomir N. Markovic, Olivier Rixe, David L. Bajor, Thomas F. Gajewski, Martin Gutierrez, Elisabeth I. Heath, John Thompson, Sahar Ansari, Shyra Gardai, Celine Jacquemont, Michael Schmitt, Andrew L. Coveler. SEA-CD40 is a non-fucosylated anti-CD40 antibody with potent pharmacodynamic activity in preclinical models and patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5535.

e22124 Background: Therapeutic anti-cancer vaccine strategies have been effective in a broad range of preclinical tumor models, but clinical trials of vaccines in metastatic cancer patients have failed to show clinical benefit. We have previously shown that patients with stage IV melanoma have systemic immunosuppression including elevated plasma levels of immunosuppressive cytokines and impaired DTH responses to recall antigens. Patients with stage IV melanoma also have elevated circulating levels of the vascular growth factors vascular endothelial growth factor A (VEGF A) and placental growth factor (PlGF). Based on these observations, we have hypothesized that vascular growth factors such as VEGF and PlGF mediate systemic immunosuppression. Methods: FVB mice with established ANV6 transplantable breast tumors were bled, and plasma samples were assayed for VEGF, PlGF, and interleukin (IL)-10. Splenocytes taken from tumor-bearing mice were stimulated via the T cell receptor (TCR), and interferon gamma (IFN) production was measured. Splenocytes from normal mice were stimulated in the presence or absence of VEGF; IFN production was measured. Results: Compared with age- and sex-matched controls, tumor-bearing mice had elevated plasma levels of VEGF, PlGF, and the immunosuppressive cytokine IL-10 (see table ). Furthermore, splenocytes from tumor-bearing mice produced less IFN upon TCR stimulation than did splenocytes from control mice. Finally, normal mouse splenocytes produced less IFN when stimulated in the presence of VEGF than in its absence (70.2 ± 37.2 vs. 260.0 ± 130.9 pg/ml). Conclusions: We have shown that mice with advanced tumors exhibit both elevated levels of vascular growth factors and systemic immune inhibition. We have also shown that VEGF leads to impairment of immune function in normal splenocytes. Taken together, these results suggest that excess vascular growth factors may mediate immunosuppression in animals with advanced tumors, which may in turn impair anti-tumor vaccine efficacy. [Table: see text] No significant financial relationships to disclose.