ALBERT

Description: Introduction: Adoptive transfer of T cells, genetically engineered to express chimeric antigen receptors (CARs) containing costimulatory domains, such as CD28 or 4-1BB, has yielded impressive clinical results in some blood cancers, but severe toxicities have been observed due to unchecked T cell activation. In contrast, CAR-T cells have demonstrated limited clinical efficacy, associated with poor engraftment, survival and proliferation of adoptively transferred cells when used to target a variety of solid tumors. Thus, technologies that can regulate T cell activation and proliferation in vivo should both mitigate toxicities and maximize anti-tumor efficacy, expanding their clinical utility to a wider range of indications. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), activated by a small molecule chemical inducer of dimerization, rimiducid, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 and transduced with a retrovirus encoding tandem rimiducid-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 signaling elements, and first generation CARs (CAR.ζ) targeting CD19 or PSCA (SFG-iMC-2A-CD19.ζ or SFG-iMC-2A-PSCA.ζ, respectively). iMC activation was measured by treating T cells with and without rimiducid and measuring cytokine production by ELISA and T cell activation markers by flow cytometry. Coactivation through iMC and CAR was tested in coculture assays with or without rimiducid using various tumor cells (CD19+, Raji and Daudi lymphoma; PSCA+, Capan-1 and HPAC pancreatic adenocarcinoma). Efficacy of iMC-modified CAR-T cells were assessed using an immune-deficient NSG mouse tumor model. For CD19-targeted CARs, 1x105 Raji tumor cells were injected i.v. followed on day 7 by a single i.v. injection at various doses of iMC-CD19.ζ-modified T cells. For PSCA-targeted CARs, 2x106 HPAC tumor cells were injected s.c. followed by iMC-PSCA.ζ-modified T cells on day 10. In both models, iMC was activated in vivo by weekly i.p. injections of rimiducid (5 mg/kg). In some experiments, iMC-CAR-modified T cells were engrafted into tumor-free mice. Tumor burden and CAR-T cell expansion in vivo was assessed using luciferase bioluminescent imaging and flow cytometry. Results: T cells transduced with either iMC-CD19.ζ or iMC-PSCA.ζ produce cytokines (e.g., IFN-γ and IL-6) in response to rimiducid; however, the key growth and survival cytokine, IL-2, was only produced when both iMC and CAR were activated simultaneously by rimiducid and tumor antigen, respectively. CD19+ Raji tumor-bearing mice treated with iMC-CD19.ζ-modified T cells with or without rimiducid administration increased survival compared to non-transduced T cells (p = 0.01). However, rimiducid treatment induced a 7.3-fold CAR-T cell expansion compared to mice infused with iMC-CD19.ζ, but untreated with dimer drug (p = 0.02). Additionally, treatment of NSG mice bearing large (〉200 mm3) HPAC tumors with a single dose iMC-PSCA.ζ, resulted in complete elimination in 10/10 mice (100%) of tumors both with and without rimiducid treatment compared to mice receiving non-transduced T cells (p = 0.0003). Rimiducid administration again dramatically increased CAR-T cell levels, resulting in a 23-fold expansion of iMC-PSCA.ζ-modified T cells compared to mice not receiving rimiducid (p = 0.02), justifying ongoing experiments using larger tumors at baseline with fewer T cells. In addition, in tumor-free mice, rimiducid prolonged iMC-PSCA.ζ-modified T cell engraftment and survival for 28 days compared to those mice not treated with dimerizer (p = 0.03). Importantly, following rimiducid withdrawal, CAR-T cell numbers declined, consistent with the requirement of MC-mediated costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3ζ) domain from a potent, regulatable, signal 2 costimulation (iMC) in the novel platform, called "GoCAR-T", allows the expansion of T cells only in response to both rimiducid and tumor antigen, and their decrease in number by withdrawal of rimiducid-induced iMC costimulation. The "GoCAR-T" platform may allow the development of a new generation of more effective CAR-T cell therapies. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Description: Introduction: Promising clinical results with CD19-specific chimeric antigen receptor (CAR)-directed T cells for the treatment of B cell leukemia and lymphoma suggest that CARs may be effective in other hematological malignancies, such as acute myeloid leukemia (AML). CD123/IL-3Rα is an attractive CAR-T cell target due to its high expression on both AML blasts and leukemic stem cells (AML-LSCs). However, the antigen is also expressed at lower levels on normal stem cell progenitors presenting a major toxicity concern should CD123-specific CAR-T cells show long-term persistence. Here, we describe a CAR platform, "GoCAR-T", which uses a proliferation-deficient, first generation, CD123-specific CAR together with a ligand (rimiducid (Rim))-dependent costimulatory switch (inducible MyD88/CD40 (iMC)) to provide physician-controlled eradication of CD123+ tumor cells and regulate long-term CAR-T cell engraftment. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and first generation CAR targeting CD123 (SFG-iMC-CD123.ζ). The effects ofiMC costimulation on CD123-targeted CARs were assessed in coculture assays with CD123+, EGFPluciferase (EGFPluc)-modified leukemic cell lines (KG1, THP-1 and MOLM-13) with and without Rim using the IncuCyte live cell imaging system. IL-2 production was examined by ELISA from coculture supernatants. In vivo efficacy of iMC-CD123.ζ-modified T cells was assessed using an immune-deficient NSG tumor xenograft model. One million EGFPluc-expressing CD123+ THP-1 tumor cells were injected i.v. into the animals, followed by a single i.v. injection on day 7 with 2.5x106 non-transduced or iMC-CD123.ζ-modified T cells. Groups receiving CAR-T cells subsequently received i.p. injections of Rim (1 mg/kg) or vehicle only on days 0 and 15 post-T cell injection. Animals were evaluated for THP-1-EGFPluc tumor burden and weight change on a weekly basis using IVIS bioluminescent imaging (BLI) and for T cell persistence by flow cytometry and qPCR at day 30 post-T cell injection. Results: SFG-iMC-CD123.ζ efficiently transduced activated T cells (66±8%) and showed antigen-specific cytolytic function against CD123+ leukemic cell lines. However, in coculture assays both CAR antigen recognition and Rim-dependent iMC costimulation were required for IL-2 production (285±41 versus 2,541±255 pg/ml for control and 1 nM Rim, respectively), robust CAR-T cell proliferation (87-fold increase with Rim stimulation) and enhanced tumor cell killing. In NSG mice engrafted with CD123+ THP-1-EGFPluc tumor cells, only animals were treated with iMC-CD123.ζ-modified T cells and 1 mg/kg Rim on days 0 and 15 post-T cell injection controlled tumor growth, showing a 2-log (average radiance: NT = 3.0x108; CAR + Vehicle = 2.4x108; CAR + Rim=8.4x105 p/sec/cm2/sr) reduction in tumor burden compared to mice receiving CAR-T cells and vehicle only (Figure 1). Approximately two weeks after the second Rim injection (day 30), CAR-T cells were infrequent (

Description: Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL. Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting. Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells. GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO. Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways. Table A. B. Figure 1. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1B Figure 1B. Figure 2 Cell cycle changes with BCR KO in OCI-Ly7. Figure 2. Cell cycle changes with BCR KO in OCI-Ly7. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) has begun to show impressive clinical results. The efficacy of T cell therapy is dependent not only on tumor recognition, but also on the survival and expansion of T cells following infusion. T cells modified with CAR constructs encoding costimulatory domains such as CD28 or 4-1BB have the capacity to rapidly proliferate in vivo, but severe toxicities have been observed due to unchecked T cell activation. Thus, strategies to regulate T cell activation in vivowould allow physicians to prevent toxicities and maximize anti-tumor efficacy. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), that can be activated by a small molecule chemical inducer of dimerization, AP1903, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a biscistronic retrovirus encoding myristolated tandem AP1903 binding domains (FKBPv36), cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and truncated CD19 to identify transduced T cells (SFG-iMC.2A.ΔCD19). Control vectors without signaling elements, or with only MyD88 or cytoplasmic CD40 were also used to generate gene-modified T cell lines. iMC activation was measured by treating T cells with and without AP1903 and measuring cytokine production by ELISA, and assessing cell surface activation markers by flow cytometry. Co-activation of T cells through CD3ζ in combination with iMC was accomplished using anti-CD3 antibodies, or by co-transducing T cells with first generation CAR constructs recognizing prostate stem cell antigen or CD19 (PSCA.ζ or CD19.ζ, respectively), and coculturing T cells with PSCA+ (Capan-1) or CD19+ tumor cell lines (Raji, Daudi and Nalm-1) with and without AP1903. Efficacy of iMC-modified CAR T cells were assessed using NOD scid gamma (NSG) immune deficient mice engrafted with tumor cell lines followed by intravenous injection of T cells. The iMC costimulatory molecule was subsequently activated in vivo by intraperitoneal injection of AP1903 (5 mg/kg). Tumor burden was assessed and T cell expansion in vivowas measured by bioluminescent imaging using an IVIS instrument. Results: T cells transduced with iMC produce cytokines (e.g. IFN-γ, TNF-α, IL-6) in response to AP1903. iMC activation permits T cell survival in the absence of growth cytokines, such as IL-2, but by itself is not sufficient to induce IL-2 production or autonomous growth. Interestingly, AP1903-induction of MyD88 or cytoplasmic CD40 alone showed minimal T cell activation, suggesting potential synergy of the two signaling molecules. However, co-activation of the T cell receptor (TCR) with soluble anti-CD3 and iMC with AP1903 upregulated CD25 expression, induced IL-2 production and promoted T cell expansion. Importantly, endogenous TCR signaling could be substituted by a PSCA-specific CAR linked to the CD3 ζ endodomain (PSCA.ζ CAR), where co-activation of iMC by AP1903, and CAR by tumor cells expressing PSCA (Capan-1) induced high levels of IL-2 secretion, CD25 upregulation and rapid T cell proliferation. Similar results were achieved using T cells transduced with iMC-enabled CD19 CAR (SFG-iMC.2A.CD19.ζ) when cocultured with CD19+lymphoma cell lines. Treatment of tumor bearing immunodeficient mice with T cells modified with iMC and PSCA.ζ CAR showed enhanced antitumor efficacy when mice were administered with AP1903 dimerizer. Bioluminescence imaging also demonstrated marked proliferation and persistence of iMC-transduced T cells in response to AP1903 administration. Following AP1903 withdrawal, T cell levels declined, consistent with the requirement for costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3 ζ) domain from signal 2 costimulation (iMC) provides a unique mechanism by which T cells can be expanded only in response to both AP1903 and tumor antigen, or reduced in number by withdrawal of AP1903-induced iMC costimulation. Disclosures Foster: Bellicum Pharmaceuticals: Employment, Patents & Royalties. Mahendravada:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Description: Key Points The GCB subtype of DLBCL relies exclusively on tonic BCR signaling via CD79A Y188. PTEN protein expression and BCR surface density determine the contribution of tonic BCR signaling to AKT activity in GCB-DLBCL.