Intratumoral CpG, Local Radiation, and Oral Ibrutinib Combine to Produce Effective in Situ Vaccination in Patients with Low-Grade B-Cell Lymphoma

Introduction: Local treatment with intratumoral CpG (a toll-like receptor 9 agonist, SD-101) and low-dose radiation can elicit antitumor immune responses and global tumor reduction in patients with low-grade lymphoma (Frank, Cancer Discov, 2018). Ibrutinib compromises B-cell survival by inhibiting Bruton's tyrosine kinase, but also modulates T-cells by inhibiting interleukin-2-inducible T-cell kinase. In a mouse model of lymphoma, ibrutinib plus intratumoral CpG was curative of systemic disease, an effect that was T-cell dependent (Sagiv-Barfi, Blood, 2015). Thus, we initiated a phase I/II clinical trial combining oral ibrutinib, intratumoral CpG and local low-dose radiation in adults with recurrent low-grade lymphoma (NCT02927964). Methods: Enrolled patients received intratumoral injections of CpG (SD-101, 3mg) weekly for 5 doses, starting on the second day of a 2-day course of local radiation (4Gy total) to the same site. Daily oral ibrutinib (560mg) began on day 9. Treatment-emergent adverse events (AEs), ibrutinib dose modifications and adherence were recorded at every visit. Revised Lugano criteria (Cheson et al., JCO, 2014) were used to assess response to therapy, based on CT scans at 3, 6, 12, 18, and 24 months. Fine needle aspirates (FNAs) were obtained from CpG-injected and non-injected nodal tumor sites pre- and post-treatment and analyzed by flow cytometry and single-cell RNA sequencing (scRNAseq). When available, viably preserved tumor and peripheral blood cells were used for in vitro immune response assays. Results: As of July 16, 2020, 18 patients had been treated, with a median follow-up of 12 months. Ten were male and 8 were female. All but one had a diagnosis of follicular lymphoma; one patient had marginal zone lymphoma. All were previously treated with an average of 2 prior lines of therapy. AEs were consistent with known effects of ibrutinib (including diarrhea and rash) and of CpG (including fever and flu-like reaction) with no unexpected AEs to suggest synergistic toxicity. There were no grade 4 or 5 events. AEs led to ibrutinib dose reduction or discontinuation in 2 patients and dose interruption in 6 patients. At the time of analysis, 9 of 18 evaluable patients had achieved a partial response (50% ORR) and 12 of 18 patients experienced at least a 30% reduction in the distant uninjected lesions (Figure 1A). Most responses have been maintained for at least 6 months, many longer (Figure 1B). Flow cytometry revealed decreased T follicular helper cells and increased CD4 and/or CD8 effector T-cells, CD137+ activated T-cells, and NK cells in CpG-injected tumors. Abscopal immune effects in distant non-injected lesions included an increase in Granzyme B+ CD8 T-cells, most prominent after the addition of ibrutinib. scRNAseq data showed significant transcriptional shifts in tumor cells and in tumor-infiltrating T-cells, including signatures of interferon response and T cell activation and cytotoxicity. Finally, in vitro assays showed tumor-specific immune responses in peripheral blood T-cells of all 6 evaluable patients tested thus far. Conclusion: Early data suggest that the combination of oral ibrutinib, intratumoral CpG, and local low-dose radiation is safe and can generate systemic antitumor immune responses and systemic tumor shrinkage in low-grade lymphoma. Disclosures Khodadoust: Seattle Genetics: Consultancy; Kyowa Kirin: Consultancy. Levy:Quadriga: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc.: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees.

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Therapeutic and Immunologic Responses Elicited By in Situ Vaccination with CpG, Ibrutinib, and Low-Dose Radiation

Blood ◽

10.1182/blood-2021-154017 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3539-3539

Author(s):

Tanaya Shree ◽

Sarah Haebe ◽

Debra K. Czerwinski ◽

Grady Day ◽

Anuja Sathe ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Low Dose Radiation ◽

Advisory Committees ◽

Tumor Reduction ◽

Dose Radiation

Abstract Introduction: In situ vaccination aims to induce an immune response locally at one tumor site that propagates systemically to all tumor sites. This approach can be effective in indolent lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discov 2018, Hammerich et al., Nat Med 2019). We designed a novel clinical strategy combining in situ vaccination with systemic ibrutinib, a kinase inhibitor that modulates B and T cells. Our preclinical work had shown that combined intratumoral CpG injection and systemic ibrutinib administration was curative of systemic disease in a mouse lymphoma model, an effect that was T cell dependent (Sagiv-Barfi, Blood, 2015). Here we report the results and correlative data from the Phase I/II clinical trial testing this combination along with local low-dose radiation in adults with recurrent low-grade B cell lymphoma (NCT02927964). Methods: Enrolled patients received intratumoral injections of CpG (SD-101, 3 mg) weekly for 5 doses and local radiation (4Gy in two fractions) to the same site. Daily oral ibrutinib (560mg) began after the second intratumoral injection. Revised Lugano criteria (Cheson et al., JCO, 2014) were used to assess overall radiographic responses to therapy. Distal responses were assessed by excluding the injected site and measuring only non-injected sites. Fine needle aspirates (FNAs) were obtained from CpG-injected and non-injected nodal tumor sites pre- and post-treatment and analyzed by flow cytometry and droplet-based single-cell RNA sequencing (scRNAseq). Results: Among the twenty patients treated on study, median age was 64, 55% were male, and all but one had a diagnosis of follicular lymphoma. All patients were previously treated with an average of 2 lines of therapy, and half had previously received chemotherapy. Adverse events (AEs) were consistent with known effects of ibrutinib (including diarrhea and rash) and of CpG (including fever and flu-like reactions). No drug-related grade 4, serious, or unexpected AEs were observed. As anticipated, all patients experienced tumor reduction at the locally treated site (median 84% reduction). Remarkably, all patients experienced some tumor reduction at non-injected non-irradiated index lesions (median 45%, range 13-100%), suggesting the generation of systemic immune responses (Figure 1A). By Cheson criteria, ten patients achieved an objective response, including one complete response (ORR 50%). Despite an overall improvement in tumor burden, three patients had new or progressing non-index lesions and scored as progressive disease. Treatment induced an expansion of naïve and effector memory T cells and reductions in T follicular helper (Tfh) and activated regulatory T cells (Tregs) at the injected site. T cells with high expression of transcripts related to oxidative phosphorylation (Toxphos) increased preferentially in patients with subsequent clinical tumor reduction (Figure 1B), implicating T cell metabolism in successful generation of immune responses. Analysis of single cell T cell receptor (TCR) sequencing data revealed>300 clones that were comprised of at least 2 cells at each timepoint and which expanded or contracted at least two-fold during treatment. Expanding clones were more likely than contracting clones to be activated or memory T cells and less likely than contracting clones to be Tfh or Tregs (Figure 1C-D). Clone dynamics were often similar at the two sampled tumor sites, reflecting systemic immune responses. Finally, in vitro assays showed treatment-induced expansion of tumor-specific T cells in the peripheral blood of all 6 evaluable patients. Conclusion: The combination of oral ibrutinib, intratumoral CpG, and local low-dose radiation is safe and can generate systemic antitumor immune responses and systemic tumor shrinkage in low-grade B cell lymphoma. Figure 1 Figure 1. Disclosures Shree: Gilead: Other: Spouse's employment. Khodadoust: CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alexion, AstraZeneca Rare Disease: Other: Study investigator. Frank: Kite-Gilead: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Research Funding; Allogene Therapeutics: Research Funding. Beygi: Kite/Gilead: Current Employment. Levy: GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Virsti: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

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A Phase I/II Trial of Intratumoral CpG, Local Low-Dose Radiation, and Oral Ibrutinib in Patients with Low-Grade B-Cell Lymphoma

Blood ◽

10.1182/blood-2019-129661 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2825-2825

Author(s):

Tanaya Shree ◽

Michael S. Khodadoust ◽

Debra K. Czerwinski ◽

Matthew J. Frank ◽

Wan X. Hong ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Low Dose ◽

Low Grade ◽

Low Dose Radiation ◽

Advisory Committees ◽

Pre Treatment ◽

Dose Radiation

Background: In situ vaccination for the treatment of cancer aims to trigger an immune response locally that propagates systemically. We have shown that local treatment with intratumoral CpG (a TLR9 agonist) and low-dose radiation can elicit antitumor immune responses and global tumor shrinkage in patients with low-grade lymphoma (Brody, J Clin Oncol, 2010; Frank, Cancer Discov, 2018). Ibrutinib is a small molecule that compromises B cell survival by inhibiting Bruton's tyrosine kinase (BTK) but also modulates T cell phenotypes by inhibiting interleukin-2-inducible T-cell kinase (ITK). Specifically, there is evidence for ibrutinib-induced skewing of CD4 T-cells toward a Th1 phenotype, which would be expected to promote anti-tumor immunity (Dubovsky, Blood, 2013). In a mouse model of lymphoma, systemic ibrutinib plus intratumoral CpG was curative of systemic disease in all treated mice, an effect that was fully T-cell dependent (Sagiv-Barfi, Blood, 2015). Study Design: To test the hypothesis that ibrutinib will enhance the efficacy of in situ vaccination via its effects on T-cells, we initiated a phase I/II clinical trial combining oral ibrutinib (560mg), intratumoral CpG (SD-101, 3mg) and local low-dose radiation in patients with recurrent low-grade lymphoma (NCT02927964). Patients with follicular lymphoma, marginal zone lymphoma, or indolent mantle cell lymphoma relapsed or refractory to at least one line of prior therapy and with at least one superficial disease site accessible for injection and one independent measurable disease site are eligible. Patients with significant autoimmune disease, recent stroke or intracranial hemorrhage, and those requiring anticoagulation with vitamin K antagonists or chronic treatment with strong CYP3A inhibitors are excluded. The primary outcome of the study is determination of safety of the combination treatment. Secondary outcomes include overall response rate, progression-free survival, and correlative studies of tumor and blood immune populations. Fourteen patients have been enrolled thus far, with a goal enrollment of 21-30 patients. Treatment consists of two consecutive days of low-dose (2 Gy) radiation to the chosen treatment site, followed by 5 weekly injections of CpG into that same site. Daily oral ibrutinib is begun one day after the second CpG injection. CT scans are performed at 3, 6, 12, 18, and 24 months. Common Terminology Criteria for Adverse Events (CTCAE) are used to track treatment-emergent safety events, and Lugano criteria (Cheson, J Clin Oncol, 2014) are used to assess response to therapy. Correlative Studies: Fine needle aspirates (FNAs) are obtained from CpG-injected and non-injected sites pre-treatment and at weeks 2 and 6. Peripheral blood samples are obtained pre-treatment and weeks 2, 4, 6, and at all response assessment timepoints. When available, tumor cells from an excisional biopsy pre-treatment are viably preserved. FNAs and peripheral blood samples from pre-treatment and at weeks 2 and 6 are being analyzed by both flow cytometry and single cell RNA sequencing. For patients for whom viably preserved tumor cells are available, these are co-cultured with autologous peripheral blood T cells in an in vitro immune response assays to test for tumor-specific T-cell activation. Summary: This innovative study combines in situ vaccination and ibrutinib-induced T-cell modulation and incorporates serial sampling of multiple tumors and peripheral blood using minimally invasive procedures, analyzed by methods that allow for deep profiling of treatment-induced changes. This study is ongoing and currently accruing at the Stanford Cancer Center. Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Levy:Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Five Prime: Membership on an entity's Board of Directors or advisory committees; Corvus: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees.

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γδ T cells for immune therapy of patients with lymphoid malignancies

Blood ◽

10.1182/blood-2002-12-3665 ◽

2003 ◽

Vol 102 (1) ◽

pp. 200-206 ◽

Cited By ~ 362

Author(s):

Martin Wilhelm ◽

Volker Kunzmann ◽

Susanne Eckstein ◽

Peter Reimer ◽

Florian Weissinger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Low Dose ◽

Γδ T Cells ◽

Low Grade ◽

Treatment Schedule ◽

Γδ T Cell

Abstract There is increasing evidence that γδ T cells have potent innate antitumor activity. We described previously that synthetic aminobisphosphonates are potent γδ T cell stimulatory compounds that induce cytokine secretion (ie, interferon γ [IFN-γ]) and cell-mediated cytotoxicity against lymphoma and myeloma cell lines in vitro. To evaluate the antitumor activity of γδ T cells in vivo, we initiated a pilot study of low-dose interleukin 2 (IL-2) in combination with pamidronate in 19 patients with relapsed/refractory low-grade non-Hodgkin lymphoma (NHL) or multiple myeloma (MM). The objectives of this trial were to determine toxicity, the most effective dose for in vivo activation/proliferation of γδ T cells, and antilymphoma efficacy of the combination of pamidronate and IL-2. The first 10 patients (cohort A) who entered the study received 90 mg pamidronate intravenously on day 1 followed by increasing dose levels of continuous 24-hour intravenous (IV) infusions of IL-2 (0.25 to 3 × 106 IU/m2) from day 3 to day 8. Even at the highest IL-2 dose level in vivo, γδ T-cell activation/proliferation and response to treatment were disappointing with only 1 patient achieving stable disease. Therefore, the next 9 patients were selected by positive in vitro proliferation of γδ T cells in response to pamidronate/IL-2 and received a modified treatment schedule (6-hour bolus IV IL-2 infusions from day 1-6). In this patient group (cohort B), significant in vivo activation/proliferation of γδ T cells was observed in 5 patients (55%), and objective responses (PR) were achieved in 3 patients (33%). Only patients with significant in vivo proliferation of γδ T cells responded to treatment, indicating that γδ T cells might contribute to this antilymphoma effect. Overall, administration of pamidronate and low-dose IL-2 was well tolerated. In conclusion, this clinical trial demonstrates, for the first time, that γδ T-cell–mediated immunotherapy is feasible and can induce objective tumor responses. (Blood. 2003;102:200-206)

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ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽

10.1182/blood-2021-147573 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1204-1204

Author(s):

Bin Cai ◽

Aaron N Nguyen ◽

Songmao Zheng ◽

Jianfeng Shi ◽

Guizhong Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Cytokine Release ◽

Publicly Traded ◽

Advisory Committees ◽

The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

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Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-115319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1886-1886 ◽

Cited By ~ 5

Author(s):

Ehren Dancy ◽

Alfred L. Garfall ◽

Adam D. Cohen ◽

Joseph A Fraietta ◽

Megan Davis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Expansion ◽

Research Funding ◽

Advisory Committees ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

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Repetitive Low-Dose Radiation Therapies As an Alternative Option for Indolent Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood.v128.22.1784.1784 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1784-1784

Author(s):

Khalil Saleh ◽

Jean-Marie Michot ◽

Alina Danu ◽

Julien Lazarovici ◽

Nadine Khalifé-Saleh ◽

...

Keyword(s):

Radiation Therapy ◽

B Cell ◽

Board Of Directors ◽

Low Dose ◽

Progressive Disease ◽

Standard Dose ◽

Low Dose Radiation ◽

Advisory Committees ◽

Dose Radiation

Abstract Introduction:Low-dose radiation therapy (LD-RT) is a therapeutic option in indolent non Hodgkin B-cell lymphomas (iNHL), usually in the palliative setting. If most iNHL are highly sensitive to radiation therapy, with good local control obtained with a dose of 4 Gy in 2 fractions, little is known about the efficacy and outcome of repetitive courses of LD-RT. We report here the results of a study cohort of repetitive LD-RT in iNHL. Methods : We retrospectively reviewed the records of all iNHL patients treated by two or more courses of LD-RT at Gustave Roussy, between January 1990 and December 2015. Patients received LD-RT as palliative treatment for low-bulky disease, patient's comfort or painful adenopathy. Clinical data, histological types, outcome and treatment lines were collected. Overall survival was the time between lymphoma diagnosis and death from any cause. Last LD-RT follow-up period was the time between the last LD-RT session and latest news. Results: Thirty-five pts were analyzed. Among them, 24 pts (69%) had Follicular Lymphoma (FL), 6 pts (17%) Marginal Zone Lymphoma (MZL), 3 pts (9%) had B-cell primitive Cutaneous Lymphoma Follicular Type (CL-FL) and 2 pts (6%) Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL). At lymphoma diagnosis, median age was 57 years [range 20-80]. Ann Arbor stage was I-II in 18 pts (51%), and III-IV in 17 pts (49%). Patients received a median of 4 therapeutics lines (range 2-11), and 2 LD-RT courses (range 2-6). Median overall survival was 146 months [29-298 months]. Four patients had died: 2 of disease progression and 2 others from concomitant illness (1 cardiac disease and 1 hepatocellular carcinoma). No patient had experienced transformation to diffuse large B cell lymphoma after RT-LD treatments. In the vast majority of cases (31/35; 89%), the LD-RT were successively performed to lymphoma relapse outside irradiation fields. Exclusive repetitive courses of LD-RT without chemotherapy were received by 8/35 (23%) of patients; while 24/35 (69%) patients received repetitive LD-RT alternately with immunotherapies or chemotherapies; and 3/35 (9%) others repetitive LD-RT alternately with standard dose RT. After the second course of LD-RT, 12/35 (34%) patients were managed in watch and wait approach, 6/35 (17%) received another LD-RT and 17/35 (49%) patients had experienced a progressive disease and were treated with immunotherapy or chemotherapy or standard dose radiotherapy. The LD-RT was the last treatment modality in 18/35 (51%) patients with histological types distributed in FL (n=10), MZL (n=5) and CT-FL (n=3). With a median last LD-RT follow up of 32 months [7-177 months], 23/35 (66%) patients remained in complete remission, 9/35 patients (26%) had experienced progressive disease and 3/35 (9%) patients had obtained stable disease. Conclusion: As palliative treatment modality, the repetitive low dose radiation therapy 4 Gy in two fractions could provide alternative option treatment in iNHL. This study support further investigations of this simple, well tolerated and not costly therapy in iNHL, especially in the context of new immunotherapeutic agent's area. Disclosures Michot: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ribrag:ArgenX: Research Funding; Esai: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; NanoString: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

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CAR-T Cells with High CAR Expression Intensity Have Improved In Vitro and In Vivo Anti-Lymphoma Effect without Functional Exhaustion

Blood ◽

10.1182/blood-2020-139546 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-34

Author(s):

ANA Carolina Carolina CABALLERO González ◽

Laura Escribà-García ◽

Paula Pujol-Fernández ◽

Eva Escudero-López ◽

Rosanna Montserrat ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Antitumor Effect ◽

Advisory Committees ◽

Expression Intensity ◽

Car T Cells ◽

Car T

Background While immunotherapy with anti-CD19 chimeric antigen receptor (CAR) T cells has shown significant efficacy in B-cell malignancies, CAR T cells directed against CD30 (CAR30) for the treatment of Hodgkin lymphoma (HL) showed modest antitumor effect, with more than 50% of patients being unresponsive. Several factors related to the infused product and persistence may be relevant to increase clinical efficacy, but further investigation is needed. In this way, CAR expression intensity may play an important role on CAR T cell function, but this has not been systematically explored. Aim We have evaluated the impact of CAR expression intensity on T cell function, cell exhaustion and antitumor efficacy against HL and B cell lymphoma. Methods T cells were generated as previously described (Alvarez-Fernández C et al. 2016) and transduced with third generation lentivirus encoding a 4-1BBz CAR (either CAR30 or CAR19). Two populations of CAR+ T cells were sorted according to mean fluorescence intensity (MFI) of CAR: CARHI (MFI> 5x103) and CARLO (MFI <3x103). Cytotoxicity assays were performed using Raji (CD19+) or L540 (CD30+) tumor cell lines. Multiparametric flow cytometry was used to analyze T-cell inhibition and activation markers. CARHI and CARLOin vivo antitumor effect was tested under stringent therapeutic conditions using 5x106 T cells/mice (iv) in a HL NSG model. Results CAR30+ T cells were sorted into CARLO (MFI: 1064±124.7) and CARHI (MFI: 7068±1377) (p=0.01). TSCM were highly represented in CARLO compared to CARHI (CD4+: 70.14±1.78% vs. 55.61±5.5%, CD8+: 83.78±3.8% vs 72.2±5.47%, respectively) (p<0.01). However, these differences disappear after 24h co-culture with tumor cells due to an increase of TSCM in CARHI (CD4+: 72.52±7.54%, CD8+: 80.26±5.3%). CARHI showed a significantly higher in vitro antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio: 96.6±1.86% vs. 89.1±3.83%; 1.25:1 E:T ratio: 84.61±4.7% vs. 31.15±19.79%; CARHI vs. CARLO, respectively) (p<0.0001). No differences were observed in expression of activation markers (i.e.: CD25, CD69, and HLA-DR) among both populations. Generalizability of this finding was studied using a CAR19. Similarly, CAR19+ T cells were arranged into CARLO (MFI: 1610±187) and CARHI (MFI: 10810±1486) subgroups (p<0.01). TSCM represented the most frequent subtype in both populations (CD4+: CARHI 70,22±9,87%, CARLO 69,22±9,33%; CD8+: CARHI 65,1±10,5%, CARLO 60,9±9,5%) and no differences in T cell subset composition between CARHI and CARLO were found. Again, CARHI exhibited superior antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio:59.9±8.72% vs. 28.8±8.7%; 1.25:1 E:T ratio: 21.6±11.4% vs. 2.9±2.9%, CARHI vs. CARLO, respectively) (p<0.0001). At 24h and 72h of antigen encounter, expression of inhibitory markers was determined in both CAR30+ populations. While CD4+ T cells showed significantly higher PD1 and TIM3 co-expression in CARHI compared to CARLO (p<0.05), CD8+ T cells showed similar co-expression (p=0.4 and p=0.8, at 24h and 72h, respectively). A similar kinetics was observed in CAR19+ T cells, suggesting that it could be related to an inhibitory control of activation, but not cellular exhaustion. To confirm this, functional performance of CAR30HI and CAR30LO T cells was evaluated by continuous tumor exposure. CAR30HI function persisted after sequential re-exposition (n=5) to tumor cells; in contrast, the CAR30LO subpopulation showed progressive loss of cytotoxic activity (i.e., tumor death at ratio E:T 5:1 after 4 expositions: 0% vs. 91.96%, CAR30LO and CAR30HI respectively; representative of 2 independent studies with different donors). To assess if these results were consistent in vivo, the antitumor effect of CAR30HI and CAR30LO were evaluated in a xenograft model of HL. Mice treated with CAR30HI T cells showed reduced tumor growth compared to those treated with CAR30LO T cells, which translated into an improved survival. Conclusion We have shown that high expression of a CAR (either CAR30 or CAR19) confers an enhanced in vitro antitumor effect against HL and B cell lymphoma. This effect is maintained after repetitive exposures to tumor cells and is not associated with T cell exhaustion or differentiation. Notably, this enhanced antitumor effect was also found in vivo. Our data shows that CAR expression intensity should be considered as an additional important factor to improve the efficacy of CAR T cells. Disclosures Sierra: Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

Blood ◽

10.1182/blood-2018-99-116843 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4546-4546 ◽

Cited By ~ 1

Author(s):

Raedun Clarke ◽

Sjoukje Van Der Stegen ◽

Chia-Wei Chang ◽

Mushtaq Husain ◽

Yi-Shin Lai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

B Cell Malignancies ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape. Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5' junction, 3' junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16). Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated. In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4). In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. Disclosures Clarke: Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

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Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽

10.1182/blood-2020-139625 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Hong Mu-Mosley ◽

Lauren B Ostermann ◽

Ran Zhao ◽

Challice L. Bonifant ◽

Stephen Gottschalk ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Vehicle Control ◽

Advisory Committees ◽

Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

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Target Expression, Preclinical Activity and Mechanism of Action of EM801: A Novel First-in-Class Bcma T-Cell Bispecific Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.117.117 ◽

2015 ◽

Vol 126 (23) ◽

pp. 117-117 ◽

Cited By ~ 8

Author(s):

Anja Seckinger ◽

Jose Antonio Delgado ◽

Laura Moreno ◽

Brigitte Neuber ◽

Anna Grab ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Mechanism Of Action ◽

Research Funding ◽

Advisory Committees

Abstract Background. T-cell bispecific antibodies (TCBs) simultaneously binding CD3 on T-cells and individual tumor antigens, activate T-cells and destroy tumor antigen carrying cells. B-cell maturation antigen (BCMA), a surface antigen reported to be expressed on normal and malignant plasma cells (PCs), could represent a potentially promising target for TCBs in multiple myeloma (MM). The Aim of our study was to: i) assess expression of BCMA in normal and malignant PCs as well as cells of the bone marrow (BM) microenvironment by gene expression profiling and flow cytometry to validate it as potential clinical target for TCBs; ii) to evaluate activity of EM801 as member of a novel class of BCMA-TCBs in vitro on primary myeloma cells and in vivo in the H929-xenograft reconstituted NOG mouse model; and iii) to delineate its mechanism of action. Results. Expression. We investigated the expression of BCMA in CD138-purified PCs from BM aspirates obtained from 726 patients including MGUS (n=62), asymptomatic (n=59) and symptomatic MM (605), as well as different BM cellular subsets from healthy donors (n=10 PCs; plasmablasts, memory B-cells, T-cells, CD34+, CD14+, CD15+, n=5 each; n=8 mesenchymal stromal cells) using Affymetrix DNA-microarrays. BCMA expression was observed in malignant PC from 723/726 (99.5%) MGUS and MM patients, 10/10 normal PCs and 5/5 plasmablasts; gene expression of BCMA was undetectable in all other normal BM subsets. Using multiparameter flow cytometry, BCMA surface expression on malignant PCs was confirmed in 40/40 patients while being absent on normal BM cells. BCMA is thus a potential target in virtually all myeloma patients. Activity. In vitro, EM801 induced concentration dependent significant cell death in malignant plasma cells in BM-samples of 21/28 (75%) previously untreated and 8/10 (80%) relapsed/refractory MM patients in concentrations ranging from 10pM to 30nM. No or only minor unspecific toxicity on cells of the BM microenvironment was observed. In vivo efficacy of EM801 was studied in a subcutaneous H929 myeloma cell line xenograft model in NOG (NOD/Shi-scid/IL-2Rγnull) mice reconstituted with human PBMCs. Three doses of EM801, i.e. 0.026, 0.26 and 2.6 nM/kg, the same doses of a BCMAxCD3-(scFv)2 and two control groups were investigated (n=9 mice/group). Three weekly intravenous doses were given, starting on day 19 after tumor cell injection when tumor volumes were 293±135 mm3. On day 47, all mice from control groups had their tumors grown beyond 2000 mm3 and were euthanized for ethical reasons. In contrast, at 2.6 nM/kg (0.5 mg/kg) EM801 tumor regression was already observed after the second i.v. injection in 6/9 animals and the tumor regressed to 16±3 mm3 on day 47. BCMAxCD3-(scFv)2 bispecific antibody without Fc did not show any efficacy at all doses studied. Regarding the mechanism of action, we first demonstrated that EM801 effectively binds myeloma cells and T-cells with a strength of 1622±410 pN (5-10 fold of control) as measured by atomic force microscopy. Secondly, increasing concentrations (0.03-30nM) of EM801 led to progressive T-cell activation in primary BM samples, with significantly increased levels of CD69 (P<0.001), CD25 (P<0.001) and HLADR (P=0.001) expression in both CD4 and CD8 T-cells as compared to an unspecific TCB. Thirdly, EM801 induced significant secretion of interferon-γ (19-3000 pg/ml), granzyme B (68-2986 pg/ml), and perforin (145-3712 pg/ml) as measured by ELISA, together explaining the strong in vitro and in vivo activity of EM801. Conclusions. BCMA is selectively expressed at the RNA (723/726) and protein (40/40) levels on malignant PCs from virtually all MM patients, and thus represents a promising TCB-target. The novel BCMA-TCB EM801 was effective in vitro in 29/38 (76%) primary MM patients' BM samples at picomolar to low nanomolar concentrations, easily achievable in vivo in patients, as well as in the H929-xenograft reconstituted NOG mouse model at 0.5 mg/kg once a week. Neither in vitro (the BM microenvironment) nor in vivo the compound shows significant toxicity or side effects. EM801 confers cytotoxicity by effectively coupling T-cells with malignant PCs, inducing T-cell activation, secretion of interferon-γ, granzyme B and perforin, and thereby effectively killing malignant PCs. EM801 is thus a promising new compound for the treatment of multiple myeloma to be investigated in clinical phase I/II trials. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Neuber:EngMab AG: Research Funding. Vu:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Strein:BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees; EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hundemer:EngMab AG: Research Funding. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Paiva:Celgene: Consultancy; Janssen: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Sanofi: Consultancy.

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