Markovic, Svetomir N.

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.

9082 Background: Human tumors, including melanoma, are complex mixtures of individual, molecularly distinct subpopulations, or clones of cancer cells. Effective cancer therapy will likely require targeting of all tumor subsets within a given cancer. Understanding the tumor complexity and the ability to identify points of therapeutic vulnerability within the individual tumor subsets will be essential for development of effective personalized cancer therapies. Methods: We have developed an approach that combines identification of individual tumor subsets using a multiparameter nuclear flow cytometry coupled with a high-resolution genomic analysis using the array-based comparative genomic hybridization (aCGH). Melanoma nuclei were isolated from tumor tissues and subjected to flow cytomery using melanocyte-specific antibodies (to separate melanoma cells from stroma) and DNA content, to separate individual tumor subpopulations. DNA extracted from isolated nuclear subpopulations was extracted and analyzed by aCGH. This approach was adopted for both fresh-frozen and paraffin-embedded clinical specimens. Results: We initially demonstrate the feasibility of the outlined approach by successful separation of melanoma from stromal nuclei and separation of individual melanoma nuclear subpopulations by DNA content. aCGH analysis of the DNA derived from isolated tumor subpopulations allowed successful identification of potentially targetable molecular aberrations in individual subsets of tumor cells. Notably, such aberrations were often not detected in unsorted, bulk tumors analyzed by the same high-resolution aCGH approach. Conclusions: We demonstrate a feasible approach to in-depth molecular analysis of tumor subpopulations within a clinical cancer tissue. This approach allows identification of potentially targetable molecular aberrations within individual tumor subsets, thus opening a possibility for a broad tumor targeting through design of individually-tailored therapeutic approaches. No significant financial relationships to disclose.

e20013 Background: Recent evidence suggests that patients with advanced cancer exhibit an underlying anti-tumor immune response that is continuously attenuated by regulatory T cells (Treg). Depletion of Treg using conventional chemotherapy may be feasible and clinically beneficial. C-reactive protein (CRP) is reported to rise/fall with initiation/termination of the immune response, and may be a surrogate for the periodicity of Treg proliferation that could be utilized for timing of anti-proliferative therapy. Timed delivery of chemotherapy therapy based on CRP cycling may deplete Treg and yield clinical benefit. Methods: We conducted a pilot clinical for 12 patients with metastatic melanoma who underwent serial CRP measurements (every 2–3 days) for 2 weeks. The CRP oscillation cycle was identified and chemotherapy with temozolomide (200mg/m2 for 5 days, every 28 days) was initiated at the estimated peak of plasma CRP. Patients were evaluated for clinical and immune response endpoints every 8 weeks until progression. Results: All 12 patients (median age 61; 4 female; 7 with M1c disease) exhibited oscillating CRP levels with an average periodicity of 7.8 days. Only 11 patients were treated (1 patient had rapid tumor progression). The two patients who remain progression-free for >2 years (1 PR, 1 CR), were treated in the pre-peak section of the CRP cycle, distinctly separate from the other patients treated post CRP-peak (all progressed <5 months). Presented are peripheral blood immunological laboratory correlates (cellular and cytokine) to CRP oscillation. Conclusions: These data suggest that patient clinical outcome may be dependent on the timing of therapy relative to an individual patient's immune response cycle and outline the dynamic equilibrium of systemic immune homeostasis in patients with advanced melanoma. Further investigations of these observations are under way. [Table: see text]

Background Trans-acting programmed death-ligand 1 (PD-L1) derives from malignant cells in three known forms. High levels of secreted splice variant PD-L1 (sPD-L1), ADAM10/ADAM17-shed sPD-L1, and PD-L1-positive extracellular vesicles (evPD-L1) each predict poor prognosis and limited response to PD-(L)1 checkpoint inhibitors in cancer. To our knowledge, no clinical intervention has reduced any of these circulating forms of extracellular PD-L1. Here, we explore therapeutic plasma exchange (TPE) as a treatment to reduce circulating extracellular PD-L1. Results In patients with melanoma, sPD-L1 levels above 0.277 ng/mL predicted inferior overall survival. In patients undergoing TPE for non-malignant indications, each TPE session removed a mean 70.8% sPD-L1 and 73.1% evPD-L1 detectable in plasma. TPE also reduced total and ADAM10-positive extracellular vesicles. Conclusion Here, we report the first known clinical intervention to remove either sPD-L1 or evPD-L1 from plasma in vivo. TPE reduces plasma sPD-L1 and evPD-L1 in vivo and may have a role in treatment with immunotherapy. TPE may also prove useful in patients with other extracellular vesicle-related conditions.

Background Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, but to the authors' knowledge, limited data exist regarding the safety and efficacy of these agents in transplant recipients. Herein, the authors have reported their experience with 17 patients who were treated with ICIs for metastatic malignancies after undergoing solid organ transplantation. Methods Data were abstracted for solid organ transplant recipients who received ICIs for the treatment of malignancy between January 1, 2016, and September 30, 2019. The authors identified 7 kidney, 8 liver, and 2 heart transplant recipients. Outcomes of interest were adverse drug reactions, cancer progression, and patient survival. Results The most common malignancies treated with ICIs were metastatic squamous cell carcinoma (5 patients; 29%) and hepatocellular carcinoma (5 patients; 29%), which were noted exclusively among liver transplant recipients. The median duration on ICIs was 1.7 months (interquartile range, 0.4‐7.6 months). Five patients (29%) developed adverse reactions, including 4 patients (24%) with immune‐related adverse events(irAEs), 3 patients (18%) with acute allograft rejections, 1 patient (6%) with autoimmune colitis, and 1 patient (6%) with ICI‐induced cardiotoxicity (the patient was a heart transplant recipient). The cumulative incidence of cancer progression was 50% and 69%, respectively, at 6 months and 12 months. Eleven patients (65%) died over the median follow‐up period of 4.6 months (interquartile range, 1.5‐13.2 months) from the time of ICI initiation, with cancer progression being the most common cause of death. Conclusions ICIs can be used as individualized therapy in selected patients who have undergone solid organ transplantation but more studies are needed to determine how best to use these agents to improve outcomes further.