PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Validation of an Immunohistochemistry Assay for Detection of CD155, the Poliovirus Receptor, in Malignant Gliomas

Context.— The oncolytic polio-rhinovirus recombinant (PVSRIPO) has demonstrated promise in currently ongoing phase I/II clinical trials against recurrent glioblastoma and was granted breakthrough therapy designation by the Food and Drug Administration/Center for Biologics Evaluation and Research. A reliable clinical assay to document expression of the poliovirus receptor, CD155, in routinely available patient tumor samples is needed for continued clinical development of PVSRIPO oncolytic immunotherapy in primary brain tumors and beyond. Objectives.— To validate a novel anti-CD155 antibody for immunohistochemistry and develop a robust, reliable, and specific protocol for detecting CD155 expression in glioblastoma formalin-fixed, paraffin-embedded (FFPE) tissue samples. To characterize the expression of CD155 in human glioblastoma cells as well as to evaluate the influence of CD155 expression levels on tumor cell susceptibility to PVSRIPO infection and killing. Design.— Immunohistochemical staining on glioblastoma FFPE tissue sections and immunoblot of corresponding frozen tissues were performed. Positive controls were confirmed sites of poliovirus propagation, spinal cord anterior horn, and tonsils; negative controls were vascular smooth muscle in patient samples and FFPE sections from a confirmed CD155-negative Burkitt lymphoma line (Raji). Results.— We succeeded in developing a reliable assay to specifically detect CD155 by immunohistochemistry in glioblastoma FFPE sections. Our data suggest widespread, virtually universal expression of CD155 in glioblastoma cells at levels commensurate with susceptibility to PVSRIPO infection and killing. Conclusions.— Anti-CD155 antibody D3G7H achieves monospecific detection of CD155 in immunoblots of tumor homogenates and immunohistochemistry of tumor FFPE sections. Our assay has utility in defining appropriate use of PVSRIPO in oncolytic immunotherapy against malignant glioma and other cancer histotypes.

MGD011, Humanized CD19 x CD3 DART® Protein with Enhanced Pharmacokinetic Properties, Demonstrates Potent T-Cell Mediated Anti-Tumor Activity in Preclinical Models and Durable B-Cell Depletion in Cynomolgus Monkeys Following Once-a-Week Dosing

CD19, a lymphocyte-specific marker expressed from early B-lymphocyte development through mature memory B cells, is highly represented in B-cell malignancies, making it attractive for targeted interventions. MGD011 is a Dual-Affinity Re-Targeting (DART®) protein designed to redirect T-cells to eliminate CD19-expressing cells through co-engagement of CD19 and CD3 in a monovalent binding fashion. MGD011 was engineered with a modified Fc domain that is inactivated for binding to FcγRs but retains binding to the neonatal Fc receptor (FcRn) of the immunoglobulin salvage pathway, allowing for extended pharmacokinetic (PK) properties and convenient dosing at a once-a-week or longer interval. To enable preclinical toxicokinetics and dose optimization, MGD00011 was also designed to cross-react with non-human primates. MGD011 demonstrates bispecific binding to B and T cells isolated from human and cynomolgus monkey blood. MGD011 mediates potent in vitro redirected T-cell killing of B-cell lymphoma cell lines and effective autologous B-cell depletion of human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs). MGD011-mediated killing of CD19 target cells was accompanied by target-dependent T-cell expansion and activation, cytokine release and upregulation of perforin and granzyme B, consistent with the redirected T-cell mediated killing mechanism of action endowed in its design. Two tumor models in NOD/SCID/IL2 receptor gamma chain-deficient (NSG) or NSG/beta2-microglobulin-deficient (NSG/Bm2-/-) mice were used to ascertain in vivo activity of MGD011. The growth of newly-implanted tumor xenografts of HBL-2 cells, a mantle cell lymphoma line, or Raji cells, a Burkitt’s lymphoma line, was efficiently inhibited by intravenous (IV) administration of MGD011 following the subcutaneous (SC) implantation of the target cells co-mixed with activated human T cells in Winn’s tumor models. Furthermore, in human PBMC-reconstituted mice bearing pre-established intradermal (ID) HBL-2 xenografts, MGD011 demonstrated potent anti-tumor activity with high complete response rates and no evidence of relapse over the study duration. To assess the safety and pharmacodynamic activity of MGD011, a dose-escalation study of MGD011 administered once weekly by 2-hour IV infusion was conducted in cynomolgus monkeys. MGD011 was well tolerated at doses up to 100 µg/kg, the highest dose tested, with no drug-related adverse findings and modest elevations in serum cytokines. MGD011-related pharmacological effects included marked decrease in circulating B cells at doses as low as 0.5 µg/kg, profound reduction of CD20+ cells in lymphoid organs and only modest recovery 4 weeks after the last dose. As expected, MGD011 displays prolonged PK properties, consistent with that of a human IgG1 in monkeys. In summary, the safety, activity and PK profiles of MGD011 support further investigation as a therapeutic candidate for treatment of B-cell malignancies. Disclosures Liu: Macrogenics Inc.: Employment, Equity Ownership. Lam:Macrogenics Inc.: Employment, Equity Ownership. Alderson:Macrogenics Inc: Employment, Equity Ownership. Yang:Macrogenics Inc: Employment, Equity Ownership. Li:Macrogenics Inc: Employment, Equity Ownership. Long:Macrogenics Inc: Employment, Equity Ownership. Gorlatov:Macrogenics Inc: Employment, Equity Ownership. Burke:Macrogenics Inc: Employment, Equity Ownership. Ciccarone:Macrogenics Inc: Employment, Equity Ownership. Nordstrom:Macrogenics Inc: Employment, Equity Ownership. Johnson:Macrogenics Inc: Employment, Equity Ownership. Moore:Macrogenics Inc: Employment, Equity Ownership. Bonvini:Macrogenics Inc: Employment, Equity Ownership.

Pre-Clinical Activity of Adct-301, a Novel Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Interleukin-2 receptor-α (IL2R-α, CD25), one of a heterotrimer that makes up the IL2R, plays a key role in signal transduction pathways involved in the pathogenesis of autoimmunity and graft rejection (Burchill et al Immunol Lett 2007). In addition, the preponderance of CD25+ cells in hematological malignancies (Srivastava et al Leuk Lymphoma 1994) and the relationship between increased CD25 expression and poor prognosis (Yoshida et al PLoS One 2013) raise the possibility of using an anti-CD25 antibody to deliver a cytotoxin to these cells in patients. Clinical proof of concept for treatment of CD25-positive malignancies has previously been established using radio-immunoconjugates (Dancey et al Clin Cancer Res 2009) and immunotoxins (Kreitman et al J Clin Oncol 2000) utilising antibodies basiliximab and daclizumab. ADCT-301 is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human CD25 attached to a PBD warhead. The drug-antibody ratio is 2.3 ± 0.3. ADCT-301 was potently cytotoxic against CD25-expressing anaplastic large cell lymphoma lines SUDHL1 (341,000 CD25 copies/cell, GI50 0.7 ng/ml) and Karpas 299 (112,000 copies/cell, GI50 3.9 ng/ml) and Hodgkin's lymphoma line L540 (91,000 copies/cell, GI50 3.9 ng/ml). In contrast, CD25-negative Burkitt's lymphoma line, Daudi, gave a GI50 >> 1 mg/ml. The released PBD dimer warhead induces highly cytotoxic interstrand cross-links in the DNA minor groove. Unique to this class of ADCs, the single cell gel electrophoresis (comet) assay can therefore be used as a pharmacodynamic endpoint. For ADCT-301, cross-link formation was dose dependent and the peak of cross-linking occurred 16 to 24 hours after a 2 hour exposure of Karpas 299 cells. In vivo, ADCT-301 demonstrated dose-dependent antitumor activity against SUDHL1 and Karpas 299 xenograft and disseminated models. For example, ADCT-301 at a single dose of 0.2 mg/kg significantly delayed Karpas 299 tumor growth compared to vehicle-treated and isotype control ADC-treated mice, and at 0.4 and 0.6 mg/kg gave 3/10 and 10/10 tumor-free survivors, respectively (Figure A). 10/10 tumor-free survivors were also observed at a single dose of 0.5 mg/kg, whereas Adcetris gave only a modest delay in mean tumor growth at a single dose of 0.5 mg/kg despite this tumor expressing three times the level of Adcetris target CD30 antigen compared to CD25 (Figure B). ADCT-301 was well tolerated with no signs of toxicity at 6 mg/kg, currently the highest dose tested. Together, these data clearly demonstrate the potent antitumor activity of ADCT-301 against CD25-expressing hematological tumors and warrants the rapid development of this agent into the clinic. Figure 1A B Figure 1A B. Disclosures Flynn: Medimmune: Employment. van Berkel:ADC Therapeutics Sarl: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics Sarl: Employment. Levy:Medimmune: Employment. Tiberghien:Medimmune: Employment, Patents & Royalties. Masterson:Medimmune: Employment. D'Hooge:Medimmune: Employment. Adams:Medimmune: Employment. Williams:Medimmune: Employment. Howard:ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties. Hartley:Medimmune: Employment, Equity Ownership, Patents & Royalties; ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties.

SOCS1 Mutation Subtypes Predict Divergent Outcomes in DLBCL Patients

Abstract 419 Suppressor of cytokine signaling 1 (SOCS1) is frequently mutated in Hodgkin, primary mediastinal and diffuse large B-cell lymphomas (DLBCL). In the primary mediastinal B-cell lymphoma line MedB-1, mutated SOCS1 abnormally stabilizes phospho-JAK2, thereby enhances STAT signaling leading to continuous proliferation. Here, we evaluated the prognostic value of SOCS1 mutations by full-length gene sequencing of SOCS1 in 154 comprehensively characterized DLBCL cases. By sequence analysis, we identified 90 SOCS1 mutations in 16% of lymphomas. We defined two distinct subtypes with respect to putative mutational consequences: those predicting the full-length (minor) and a truncated protein (major), respectively. Neither the SOCS1 mutation group, nor minor/major subgroups can be distinguished by clinical phenotype; however, assignment of four established expression-based classifiers revealed significant associations of SOCS1 major cases with germinal center- and specific pathway activation pattern signatures. Above all, SOCS1 major cases had an excellent overall survival, even better than the GCB-like subgroup (see Figure). SOCS1 minor cases had a dismal survival, even worse than the ABC gene signature group (see Figure). SOCS1 mutation subsets retain prognostic significance in uni- and multivariate analyses. Thus, if a SOCS1 mutation is present, the mutation type is an important single gene prognostic biomarker in DLBCL. Disclosures: No relevant conflicts of interest to declare.