P06.01 Bispecific antibody-driven synthetic agonistic receptor – transduced T cells mediate specific and conditional therapy in melanoma cancer models

BackgroundImmunotherapeutic approaches, including immune checkpoint blockade and adoptive T cell therapy (ACT) in the form of tumor-infiltrating lymphocytes (TILs), have had marked success in the treatment of melanoma. Despite these successes, many patients are refractory to treatment or relapse with therapy-resistant disease. To overcome these limitations, we propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target melanoma-associated antigen, and a cross-linking bispecific antibody (BiAb) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. We validated our approach in two murine as well as two human cancer models expressing our melanoma-associated target antigens TYRP (murine) and MCSP (human). We confirmed conditional and specific stimulation and proliferation of our T cells, as well as their tumour-antigen-directed cytotoxicity, in vitro and in vivo.ResultsCrosslinking TYRP-EGFRvIII (murine) and MCSP-EGFRvIII (human) BiAb, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation of SAR-T cells and directed tumour cell lysis with specificity towards two TYRP-expressing murine melanoma and two MCSP-expressing human melanoma cancer models. In vivo, anti-tumoural activity was mediated by the co-administration of SAR-T cells and BiAb, in an A375 melanoma xenograft model. Further, overexpression of IDO (a key immunosuppressive enzyme implicated in the suppression of T cell function in the tumor microenvironment) in a melanoma model did not influence the killing kinetics of SAR T cells.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of synthetic agonistic receptor transduced T cells, and targeted tumour cell lysis. The modularity of our platform is key for a targeting approach in a tumor entity with a high mutational load such as melanoma and is fundamental in our drive towards personalised immunotherapies. Further, the SAR approach has demonstrated resistance to IDO-mediated inhibition in the context of melanoma, an interesting axis that requires further investigation.Disclosure InformationM. Benmebarek: None. J. Keyl: None. F. Märkl: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. C. Karches: None. S. Rausch: None. A. Gottschlich: None. A. Öner: None. M. Feinendegen: None. J. Dörr: None. B. Cadilha: None. S. Endres: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

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P06.03 Bispecific antibody-driven synthetic agonistic receptor engineered T cells lead to specific and conditional therapy in melanoma cancer models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.37 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A20.1-A20

Author(s):

M Benmebarek ◽

F Märkl ◽

J Keyl ◽

B Loureiro Cadilha ◽

M Geiger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumour Cell ◽

Bispecific Antibody ◽

Cell Lysis ◽

Adoptive T Cell Therapy ◽

Melanoma Cancer ◽

Cancer Models ◽

Overlap Extension

BackgroundImmunotherapeutic approaches, including immune checkpoint blockade and adoptive T cell therapy (ACT) in the form of tumor-infiltrating lymphocytes (TIL), have had marked success in the treatment of melanoma. Despite these successes, many patients are refractory to treatment or relapse with therapy-resistant disease. To overcome said limitations, we propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SAR) that are conditionally activated only in the presence of a target melanoma-associated antigen, and a cross-linking bispecific antibody (BiAb) specific for both SAR T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. We validated our approach in two murine as well as two human cancer models expressing our melanoma-associated target antigens TYRP (murine) and MCSP (human). We confirmed conditional and specific stimulation and proliferation of our T cells, as well as their tumour-antigen-directed cytotoxicity, in vitro and in vivo.ResultsCrosslinking TYRP-EGFRvIII (murine) and MCSP-EGFRvIII (human) BiAb, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation of SAR-T cells and directed tumour cell lysis with specificity towards two TYRP-expressing murine melanoma and two MCSP-expressing human melanoma cancer models. In vivo, anti-tumoural activity was mediated by the co-administration of SAR-T cells and BiAb, in A375 and MV3 melanoma xenograft models. Further, we could show that SAR T cells exhibited resistance to MDSC-induced suppression of activation and proliferation.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of SAR transduced T cells, and targeted tumour cell lysis. The modularity of our platform is key for a targeting approach in a tumor entity with a high mutational load such as melanoma and is fundamental in our drive towards personalised immunotherapies. Further, the SAR approach has demonstrated resistance to MDSC-induced suppression, an interesting axis that requires further investigation.Disclosure InformationM. Benmebarek: None. F. Märkl: None. J. Keyl: None. B. Loureiro Cadilha: None. M. Geiger: None. C. Karches: None. S. Endres: None. C. Klein: None. S. Kobold: None. A. Klüver: None. M. Schwerdtfeger: None.

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P07.01 A modular and controllable T cell therapy platform for AML

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.42 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A22.2-A23

Author(s):

M Benmebarek ◽

B Loureiro Cadilha ◽

M Herrmann ◽

S Schmitt ◽

S Lesch ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Activation ◽

Tumour Cell ◽

Cell Activation ◽

Cell Lysis ◽

Target Cells ◽

Single Chain

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). The mutational landscape, heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are equipped with synthetic agonistic receptors (SARs) that are selectively activated only in the presence of a target AML-associated antigen, and a cross-linking tandem single chain variable fragment (taFv) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific taFvs that target AML-associated antigens were designed and expressed in Expi293FTM cells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigens CD33 and CD123.ResultsAnti-CD33-EGFRvIII and anti-CD123 EGFRvIII taFv, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by taFv could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR-taFv combination could also mediate specific cytotoxicity against patient-derived AML blasts and leukemic stem cells whilst driving SAR T cell activation. In vivo, treatment with SAR-taFv combination could efficiently eradicate leukemia and enhance survival in an AML xenograft models. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility and modularity of said activation upon depletion of the T cell engaging molecule, both in vitro and in vivo.ConclusionsHere we apply the SAR-taFv platform in efforts to deliver specific and conditional activation of SAR-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its taFv facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B. Loureiro Cadilha: None. M. Herrmann: None. S. Schmitt: None. S. Lesch: None. S. Stoiber: None. A. Darwich: None. C. Augsberger: None. B. Brauchle: None. M. Schwerdtfeger: None. A. Gottschlich: None. A. Gottschlich Rataj: None. N.C. Fenn: None. C. Klein: None. M. Subklewe: None. S. Endres: None. K. Hopfner: None. S. Kobold: None.

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PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽

10.1182/blood-2019-131325 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1959-1959

Author(s):

Jeong A Park ◽

Hong fen Guo ◽

Hong Xu ◽

Nai-Kong V. Cheung

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Immune Checkpoint ◽

Bispecific Antibody ◽

Ex Vivo ◽

Activated T Cells ◽

The Impact ◽

Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

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L4 Synthetic agonistic receptor-activating BiTEs – a modular platform for the efficient targeting of acute myeloid leukemia

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.4 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A2.2-A3

Author(s):

M Benmebarek ◽

BL Cadilha ◽

M Hermann ◽

S Lesch ◽

C Augsburger ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

T Cell Activation ◽

Tumour Cell ◽

Myeloid Leukemia ◽

Cell Activation ◽

Cell Lysis ◽

Target Cells ◽

Acute Myeloid

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33.ResultsCD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.

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AMG 701 Potently Induces Anti-Multiple Myeloma (MM) Functions of T Cells and IMiDs Further Enhance Its Efficacy to Prevent MM Relapse In Vivo

Blood ◽

10.1182/blood-2019-128528 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 135-135 ◽

Cited By ~ 9

Author(s):

Shih-Feng Cho ◽

Liang Lin ◽

Lijie Xing ◽

Kenneth Wen ◽

Tengteng Yu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd4 T Cells ◽

Xenograft Model ◽

Cell Lysis ◽

Cell Surface Expression ◽

Effector Memory ◽

T Effector Cells

AMG 701 is a half-life extended BiTE® (bispecific T-cell engager) targeting the B cell maturation antigen (BCMA). Here, we confirmed AMG 701-mediated T cell-redirected lysis of MM cells, defined immunomodulatory effects of AMG 701, and investigated combination potential of AMG 701 with immunomodulatory drugs (IMiDs) in human MM. Firstly, AMG 701 induced specific and efficacious T cell-dependent cytotoxicity (TDCC) against all MM cell lines tested, regardless of sensitivity to current anti-MM agents and expression levels of BCMA. AMG 701-induced TDCC was minimally affected in the presence of myeloma-supporting cells and cytokines in the bone marrow (BM) microenvironment, including osteoclasts (OCs), BM stromal cells (BMSCs), and a proliferation-inducing ligand (100 ng/ml). Importantly, AMG 701 induced lysis of autologous patient cells from the relapse and refractory stage of MM (RRMM). AMG 701 rapidly upregulated cell surface expression of CD107a and the production of IFNγ and TNFα, more so in CD8 than CD4 T subsets. It stimulated the proliferation and activation of T cells, to a greater extent in CD8 vs CD4 T cells, leading to significantly increased ratios of CD8/CD4 T cells. Significantly, AMG 701 induced differentiation of naive T cells (CD4 and CD8) to T cells with memory phenotype. This includes central memory (CM), effector memory (EM) T cells, and stem cell like memory cells. Time-course immunophenotyping studies showed that AMG 701 transiently upregulated the expression of key immune checkpoint and costimulatory markers on both CD4 and CD8 T cells. The induced T cells purified from ex vivo co-cultures still effectively lysed MM cells with lower BCMA levels. This may suggest an increased T cell clonality. Furthermore, IMiDs (len or pom) enhanced AMG 701-mediated TDCC against MM cells at earlier time points, lower E/T ratios, lower concentrations, or in the presence of immunosuppressive OCs or BMSCs. The combination AMG 701 and IMiDs maximized MM cell lysis accompanied with a decreased EC50 value. Combined treatments induce a more pronounced immunomodulation than AMG 701 alone in the presence of OCs, as evidenced by higher percentage of CM+EM and CD8/CD4 ratio at d8. AMG 701 with IMiDs combination significantly enhance AMG 701-mediated autologous patient MM cell lysis in a synergistic manner (combination index < 1). In the human NCI-H929 xenograft model reconstituted with human T effector cells, AMG 701 effectively blocked tumor growth 5d after the first injection, regardless of doses (0.02-2 mg/kg). Tumors were completely eradicated following 3 separate injections in the host without weight loss. Next, sub-optimal doses and treatment schedules for AMG 701 and len were then used to investigate in vivo anti-MM effects by the combination vs monotherapy. Mice receiving MM cells were treated, from d15 until the end of the study, with len once daily, AMG 701 once weekly, or combination of AMG 701 and len. Two days after the first drug administration, all three treatments significantly inhibited MM tumor growth in mice (p<0.001). Most importantly, while AMG 701 or len group showed tumor progress eventually, the combination of AMG 701 with len continuously suppressed tumor growth (p<0.05 after d26; p<0.001 after d40 for combination vs either agent alone). Combination of AMG 701 and len significantly induced superior MM cell regression, compared to either monotherapy, resulting in enhanced tumor regression and prevention of disease relapse. Taken together, these results strongly support AMG 701-based clinical studies, both as monotherapy (NCT03287908) and in combination with IMiDs to enhance elimination of residual diseases and prolong long-term durable responses in MM. Disclosures Munshi: Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Wahl:Amgen Research GmbH: Employment. Matthes:Amgen Research GmbH: Employment. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Chapman-Arvedson:Amgen Research: Employment.

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Mechanisms of Resistance and Determinants of Response of the GPRC5D-Targeting T-Cell Redirecting Bispecific Antibody JNJ-7564 in Multiple Myeloma

Blood ◽

10.1182/blood-2020-141187 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Christie P.M. Verkleij ◽

Marloes Broekmans ◽

Amy Wong ◽

Sonja Zweegman ◽

Raluca Verona ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Bispecific Antibody ◽

Cell Lysis ◽

Advisory Committees ◽

Hla Dr ◽

Tnf Α ◽

The Impact

Introduction: New immunotherapies directed against CD38, SLAMF7 or BCMA have significantly improved the outcome of multiple myeloma (MM) patients. However, most patients eventually relapse, underscoring the need for additional immunotherapeutic targets. We have previously shown that expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on MM cells, compared to normal plasma cells or other immune cells. We also showed that the novel GPRC5DxCD3 bispecific antibody (BsAb) JNJ-7564, has promising anti-MM activity in patient-derived BM samples (Verkleij et al., EHA 2019). To elucidate which factors contribute to the observed heterogeneity in ex vivo response, we analyzed the impact of tumor and patient characteristics on efficacy of JNJ-7564. We further investigated whether tumor-intrinsic factors may be determinants of response by also testing in these assays JNJ-7957, a BCMA-targeting BsAb that differs from JNJ-7564 only in the tumor-antigen-binding domain. Methods: Bone marrow (BM) samples obtained from 13 newly diagnosed (ND), 17 daratumumab-naive relapsed/refractory (DARA-naive RR; median of 3 prior therapies) and 15 daratumumab-refractory (DARA-R, median of 6 prior therapies) MM patients were analyzed for tumor- and immune cell composition, and subsequently incubated with JNJ-7564 (0.00128-4.0 µg/mL) or JNJ-7957 (0.8 µg/mL). After 48 hours, MM cell lysis was assessed by flow cytometry. Luciferase-transduced MM cell lines were incubated with JNJ-7564 (0.032-4.0 µg/mL) in the presence of healthy peripheral blood mononuclear cells (PBMCs), purified CD4+CD25- T-cells or regulatory T-cells (Tregs). After 48 hours, MM cell lysis was assessed by bioluminescence assay. Results: We found no difference in JNJ-7564 efficacy with respect to disease stage (NDMM vs DARA-naive RRMM vs DARA-R MM, P=0.48). Importantly, the presence of high-risk cytogenetic abnormalities [del(17p), t(4;14) and t(14;16)] did not impair JNJ-7564 efficacy. The level of target expression was an important determinant of response, as evidenced by superior MM cell lysis in samples with higher than median GPRC5D expression, when compared to lower GPRC5D expression (Fig. 1A). Inferior MM cell lysis was observed in older patients (>67 years), in samples with low T-cell counts or low effector:target (E:T) ratios, and in those with a high frequency of PD-1+ T-cells, HLA-DR+ activated T-cells, or Tregs. These determinants of response also affected JNJ-7564-mediated T-cell activation and degranulation. To further analyze the impact of Tregs, we performed additional cell line experiments. Purified Tregs impaired T-cell proliferation, and were significantly less potent to kill MM cells when redirected by JNJ-7564, compared to CD4+CD25- T-cells (Fig. 1B). This was accompanied by reduced secretion of IFN-γ, TNF-α, IL-2 and granzyme B. To evaluate the impact of BM stromal cells (BMSCs) on JNJ-7564 activity, MM cell lines were co-incubated with PBMCs and patient-derived BMSCs. Direct cell-cell contact hampered MM cell lysis, while indirect contact (transwell) did not affect JNJ-7564 activity. Direct contact also decreased secretion of TNF-α and IL-2, and reduced GPRC5D expression on MM cells, contributing to BMSC-mediated resistance to JNJ-7564. Finally, we simultaneously evaluated the single agent activity of both JNJ-7564 and JNJ-7957 (0.8 µg/mL, dose whereby a plateau in MM cell lysis was observed with both BsAbs) in 40 BM samples. MM cell lysis induced by both agents was strongly correlated (Fig. 1C). In 6 samples, both agents exhibited poor activity (<45% lysis), whereas in 9 samples very good activity was observed (>80% lysis). Comparison of characteristics between these groups showed that a low E:T ratio (Fig. 1D) and high frequency of Tregs (Fig. 1E) significantly impaired efficacy of both BsAbs, suggesting patient-specific factors can determine response to T-cell redirectors targeting different antigens. Conclusion: We show that tumor-related factors, such as GPRC5D expression, as well as differences in the composition of the BM microenvironment, including E:T ratio, frequency of PD-1+ or HLA-DR+ T-cells or immune-suppressing Tregs or BMSCs, contribute to the variability in response to JNJ-7564. Our data indicate that strategies aiming at optimizing E:T ratio (e.g. induction therapy) or Treg depletion, may improve response to T-cell redirecting antibodies in MM. Disclosures Wong: Jhonson & Jhonson: Current Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Verona:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Adams:Johnson & Johnson: Ended employment in the past 24 months. Mutis:Janssen Pharmaceuticals: Research Funding; Genmab: Research Funding; Takeda: Research Funding; Onkimmune: Research Funding; Gadeta: Research Funding. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: 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, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Ferrer: Membership on an entity's Board of Directors or advisory committees.

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P06.06 Enhancing trafficking and resistance to immunosuppression of synthetic agonistic receptor-transduced T cells in solid tumor models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.40 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A21.2-A22

Author(s):

M Schwerdtfeger ◽

M Benmebarek ◽

F Märkl ◽

CH Karches ◽

A Öner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Chemokine Receptors ◽

Cell Activation ◽

Target Cells ◽

Tumor Associated Antigen ◽

Overlap Extension ◽

Part Time

BackgroundChimeric antigen receptor therapy – although very efficacious in B cell malignancies – is facing many challenges which limit its success in solid tumors, e.g. on-target off-tumor toxicities, antigen heterogeneity, lack of T cell migration into tumors and an immunosuppressive tumor microenvironment. To better control on-target off-tumor effects and address antigen heterogeneity we developed a modular approach where we equipped T cells with a synthetic agonistic receptor (SAR). The SAR is only activated in the presence of a bispecific antibody (BiAb) cross-linking the receptor with a tumor-associated antigen. While we could show efficacy of the SAR platform in different models, limited infiltration and immune suppression still hamper its function. We could previously demonstrate that T cell infiltration can be enhanced by transduction with carefully chosen chemokine receptors like CXCR6, CCR4 and CCR8. At the same time, gene silencing of checkpoint molecules like PD-1 can make T cells more resistant to immunosuppression, thus we assumed that combining these approaches might generate a desired T cell product.Materials and MethodsAll constructs had been generated previously by overlap-extension cloning. The EGFRvIII (E3) SAR consists of extracellular EGFRvIII, transmembrane CD28 and intracellular CD28 and CD3ζ. Human CXCR6-GFP, CCR4-GFP and CCR8-GFP are composed of the chemokine receptors fused to GFP via a 2A sequence. Primary human T cells were retrovirally transduced to stably express the SAR and chemokine receptors. We analyzed migration, cytotoxicity and activation of the single and double (E3 SAR and chemokine receptor) transduced T cells. In addition, PD-1 was knocked out using CRISPR-Cas9 and killing kinetics of target cells and T cell activation were assessed.ResultsCo-transduction with chemokine receptors significantly increased migration of E3 SAR T cells to their respective ligand while lysis of target-expressing tumor cell and T cell activation in the presence of BiAb were not affected in vitro. Additionally knocking out PD-1 enhanced killing kinetics and activation of E3 SAR and E3 SAR + CXCR6-GFP transduced T cells compared to corresponding mock electroporated T cells.ConclusionsUsing the controllable and modular SAR – BiAb platform SAR T cell activation can be limited by stopping BiAb dosing if adverse events occur. In addition, SAR T cells can be redirected to an alternative tumor-associated antigen by exchanging the BiAb in the case of antigen escape. Here we present add-ons to this approach for increased tumor infiltration and resistance to immunosuppression. Since migration is enhanced upon co-transduction with chemokine receptors and target cell lysis is accelerated upon PD-1 knockout in vitro these two additional modifications seem very promising options to further improve tumor control in vivo.Disclosure InformationM. Schwerdtfeger: None. M. Benmebarek: None. F. Märkl: None. C.H. Karches: A. Employment (full or part-time); Significant; Daiichi Sankyo Deutschland GmbH. A. Öner: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. B. Cadilha: None. S. Endres: None. V. Desiderio: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

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AFM11, a CD19/CD3 bispecific tandab, to facilitate T-cell-mediated killing of CD19+ cells.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3068 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3068-3068

Author(s):

Eugene Zhukovsky ◽

Uwe Reusch ◽

Carmen Burkhardt ◽

Stefan Knackmuss ◽

Ivica Fucek ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Xenograft Model ◽

Cell Lysis ◽

Full Length ◽

Primary Tumors

3068 Background: CD19, due to broader expression on B cell subtypes, is an attractive alternative to CD20 as a target for treatment of B cell malignancies. T cells are potent tumor-killing effectors that cannot be recruited by full length antibodies, however TandAb technology harnesses their cytotoxic nature for oncology indications. The CD3 RECRUIT TandAb AFM11 enables T cells to potently and specifically kill CD19+ tumors and possesses advantageous PK properties enabling intravenous dosing. Methods: We constructed AFM11, a human bispecific tetravalent antibody with two binding sites for both CD3 and CD19. In vitro efficacy and safety were evaluated on CD19+ cell lines and primary tumors. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: In vitro assays demonstrate higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv. CD8+ T cells dominate early cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 of 0.5 – 5 pM; cytotoxicity is independent of cell CD19 density. AFM11 exhibits similar cytotoxicity at Effector:Target ratios from 5:1 to 1:5 and facilitates T cell serial killing of its targets. AFM11 activates T cells only in the presence of CD19+ cells. In PBMC cultures AFM11 induces CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogates these effects, and indicates strict CD19+ target-dependent T cell activation. Thus, AFM11 should not elicit the devastating cytokine release observed when full length antibodies bind CD3. Cell lysis by AFM11 is restricted to CD19+ targets asCD19- bystanders are not lysed in co-culture assays. Up to one week co-incubation with AFM11 does not inhibit T cell cytotoxicity and thus it does not induce anergy. In vivo AFM11 exhibits a dose-dependent growth inhibition of Raji tumors; a single dose of AFM11 exhibits similar efficacy as 5 daily injections. Conclusions: AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen.

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Preclinical activity and determinants of response of the GPRC5DxCD3 bispecific antibody talquetamab in multiple myeloma

Blood Advances ◽

10.1182/bloodadvances.2020003805 ◽

2021 ◽

Vol 5 (8) ◽

pp. 2196-2215

Author(s):

Christie P. M. Verkleij ◽

Marloes E. C. Broekmans ◽

Mark van Duin ◽

Kristine A. Frerichs ◽

Rowan Kuiper ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibody ◽

Plasma Cells ◽

Cell Lysis ◽

Cell Surface Expression

Abstract Cell surface expression levels of GPRC5D, an orphan G protein–coupled receptor, are significantly higher on multiple myeloma (MM) cells, compared with normal plasma cells or other immune cells, which renders it a promising target for immunotherapeutic strategies. The novel GPRC5D-targeting T-cell redirecting bispecific antibody, talquetamab, effectively kills GPRC5D+ MM cell lines in the presence of T cells from both healthy donors or heavily pretreated MM patients. In addition, talquetamab has potent anti-MM activity in bone marrow (BM) samples from 45 patients, including those with high-risk cytogenetic aberrations. There was no difference in talquetamab-mediated killing of MM cells from newly diagnosed, daratumumab-naïve relapsed/refractory (median of 3 prior therapies), and daratumumab-refractory (median of 6 prior therapies) MM patients. Tumor cell lysis was accompanied by T-cell activation and degranulation, as well as production of pro-inflammatory cytokines. High levels of GPRC5D and high effector:target ratio were associated with improved talquetamab-mediated lysis of MM cells, whereas an increased proportion of T cells expressing PD-1 or HLA-DR, and elevated regulatory T-cell (Treg) counts were associated with suboptimal killing. In cell line experiments, addition of Tregs to effector cells decreased MM cell lysis. Direct contact with bone marrow stromal cells also impaired the efficacy of talquetamab. Combination therapy with daratumumab or pomalidomide enhanced talquetamab-mediated lysis of primary MM cells in an additive fashion. In conclusion, we show that the GPRC5D-targeting T-cell redirecting bispecific antibody talquetamab is a promising novel antimyeloma agent. These results provide the preclinical rationale for ongoing studies with talquetamab in relapsed/refractory MM.

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P06.05 IDO1-deleted CAR T cells show improved therapeutic efficacy in murine pancreatic cancer models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.84 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A43.2-A43

Author(s):

AM Senz ◽

P Metzger ◽

RK Rubens ◽

B Cadilha ◽

M Kirmaier ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Tumor Control ◽

Car T Cells ◽

Cancer Models ◽

Car T

BackgroundIndoleamine-2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that catalyzes the rate limiting reaction in the kynurenine pathway. Dendritic cells, macrophages and several tumor entities have been described to express IDO1. In the tumor microenvironment IDO1 promotes tryptophan starvation and accumulation of kynurenines which result in T effector cell proliferation arrest and T regulatory cell induction. Additionally, IDO1 possesses two immunoreceptor tyrosine-based inhibitory motifs (ITIM) that upon phosphorylation can act as docking sites for the recruitment and activation of the tyrosine phosphatases SHP–1 and SHP–2 and ultimately to an activation of the non-canonical NF-ΚB pathway. Whether IDO1 is expressed in T cells and its potential function is unknown.Materials and MethodsUsing IDO1-deleted splenocytes from CD4-Cre Ido1fl/fl mice and WT controls, we evaluated the induction of IDO1 in T cells, as well as the effect of IDO1 in T cell proliferation, differentiation and metabolism. Additionally, we compared in vitro and in vivo the cytotoxic activity of anti-epithelial cell adhesion molecule (EpCAM) chimeric antigen receptor (CAR) T cells using pancreatic tumor cell lines.ResultsIDO1 is inducible in primary mouse T cells upon T cell activation and type I and type II interferon signaling. Interestingly, the use of IDO1 knockout CAR T cells prolongs survival and improves tumor control compared to WT CAR T cell treatment in subcutaneous and orthotopic pancreatic cancer models. In vitro, T cell proliferation, differentiation and cytotoxic function is comparable in WT and IDO1-deleted T cells. RNA sequencing, metabolic and in vivo tracking studies are currently being performed to pin down IDO1-intrinsic effects on CAR T cells.ConclusionsIDO1 is expressed in T cells upon T cell receptor and IFN stimulation and appears to negatively affect tumor control mediated by CAR T cells. Specific IDO1 deletion may improve therapeutic efficacy of CAR T cells in solid tumors, such as pancreatic cancer.Disclosure InformationA.M. Senz: None. P. Metzger: None. R.K. Rubens: None. B. Cadilha: None. M. Kirmaier: None. S. Lesch: None. M.R. Benmebarek: None. S. Theurich: None. P. Murray: None. S. Endres: None. S. Kobold: None. L.M. König: None. P. Duewell: None. M. Schnurr: None.

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