scholarly journals CD3-Activating Bi-Specific Antibody Targeting CD19 on B Cells in Mono- and Bi-Valent Format

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4169-4169 ◽  
Author(s):  
Yumin CUI ◽  
Zhihua Huang ◽  
Xinfeng Zhang ◽  
Wuzhong Shen ◽  
Hanyang Chen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Specific Antibody ◽  
Cell Activation ◽  
Cell Killing ◽  
Cell Binding ◽  
Dose Dependent

Abstract Immunotherapies targeting B-lineage-specific surface marker CD19 had demonstrated promising clinical results. Two CD19 CAR-T therapies (Kymriah® and Yescarta®) have been approved by FDA to treat patients with B cell malignancies, however, the complicated manufacturing process and low throughput limit its accessibility to more patients, especially in developing countries. The first CD3-activating bi-specific antibody targeting CD19, Blincyto, or CD19 BiTE, was approved to treat relapsed and refractory acute lymphoblastic lymphoma (r/r ALL). The relatively short half-life of Blincyto requires continuous IV infusion for weeks to maintain a steady levels of drug exposure, not to mention the high risk of developing severe cytokine release syndrome in patients. We had established a bispecific antibody platform ITabTM (immunotherapy antibody) for the generation of CD3-activating bi-specific antibodies that could potentially overcome the shortcomings of BiTEs. A CH1 domain was introduced into the ITabTM construct design with the intent to increase the molecular weight thus led to extend the serum half-life of the bispecific antibody. A novel CD3-activating and monkey cross-reactive antibody was generated with a less degree of T cell activation and cytokine release compared to BiTEs. A bi-valent binding to tumor associated antigen (TAA) format was established to target tumor cells and/or stem cells expressing very low levels of TAA. We report here the biological properties of the mono-valent/bi-valent binding of CD19 bi-specific antibody with CD3-activating activity (A-319/A-329). A series of studies were conducted to evaluate the bioactivities of A-319/A-329 in vitro and in vivo including binding to CD3 and CD19 antigens, T-cell and B-cell binding activities, T cell activation and proliferation and B cell killing activities in vitro as well as in vivo efficacy using human PBMC engrafted mouse xenograft models. The in vitro data showed that the mono-valent and bi-valent CD19 binding had little effect on the CD3-associated activities including CD3 antigen binding affinity, T cell binding and T cell activation. In contrast, the bi-valent binding format A-329 showed better potency compared to the mono-valent format A-319 in CD19 binding (KD 0.89 nM vs 19.4 nM); B cell binding (EC50 at 2.3 pM vs 462 pM); in vitro human B cell killing (EC50 0.2 pM vs 3.4 pM). Both A-319 and A-329 were capable of mediating tumor cell lysis with EC50 at 0.03~4 pM. A-329 demonstrated a greater killing activity on Pfeiffer, a human diffuse large B-cell lymphoma (DLBCL) cell line with a low expression of CD19 antigen. In human PBMC engrafted NOG mouse xenograft model, a dose-dependent tumor growth inhibition was observed at 0.5~100 µg/kg in both A-319 and A-329. In monkey studies, when A-319 and A-329 was dosed at 3, 10, 30 µg/kg, twice or three times weekly via IV infusion for A-329 or A-319. Dose-dependent elimination of peripheral blood B cells were observed with both ITabTM. The CD19 bi-valent format of A-329 revealed more complete B cell killing in monkeys. No significant difference of cytokine induction or liver injuries were observed between A-319 and A-329. These results demonstrated that both A-319 and A-329 may benefit patients with B cell malignancies with less dosing frequency and lower cytokine inductions especially, A-329 may have the potential to targeting the low CD19 expressing tumor stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Preclinical Characterization of Combinability and Potential Synergy of Anti-CD20/CD3 T-Cell Dependent Bispecific Antibody with Chemotherapy and PD-1/PD-L1 Blockade

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4168-4168 ◽  
Author(s):  
Liping Laura Sun ◽  
Peiyin Wang ◽  
Robyn Clark ◽  
Maria Hristopoulos ◽  
Diego Ellerman ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Regression ◽  
Single Agent ◽  
Cell Killing ◽  
Killing Activity

Abstract The anti-CD20/CD3 T-cell recruiting bispecific antibody (CD20-TDB) is a full-length, fully humanized IgG1 molecule currently under clinical investigation in B-cell malignancies. Previously we have shown that CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo (Sun et.al. STM 2015). The current standard therapy in B-cell malignancies often contains anti-CD20 based monoclonal antibody and various chemo reagents such as the R-CHOP regimen in Non-Hodgkin's' Lymphoma. Previously we have shown that CD20-TDB can be potentially combined with rituximab as very low level of antigen expression or antigen receptor occupancy is needed for CD20-TDB activity. As many chemo reagents have non-targeted, anti-proliferative activity or immune suppressive activity such as glucocorticoids, it's conceivable that they could potentially interfere with T-cell activation and the subsequent T-cell proliferation and therefore negatively affect CD20-TDB activity. In addition, as a T-cell recruiting bispecific reagent, cell killing activity of CD20-TDB is dependent on T-cell activation which can be subject to negative regulation posed by checkpoint molecules such as PD-1/PD-L1. Here in an effort to better understand the clinical applicability and to improve upon single-agent activity of CD20-TDB, we evaluated the combinability of CD20-TDB with standard-of-care chemo reagents as well as potential synergy of CD20-TDB with PD-1/PD-L1 blockade in vitro and in vivo. B-cell killing activity of CD20-TDB was not significantly impacted by high concentration of chemo reagents including cyclophosphamide, hydroxydaunorubicin, vincristine, and dexamethasone individually in vitro. In vivo in human CD20/CD3 double transgenic mice, no apparent inhibitory effect on CD20-TDB activity in T-cell activation and B-cell depletion was observed with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone either individually or in combination. In vitro, PD-1 and PD-L1 expression appeared to be upregulated on T-cells and B-cells respectively upon CD20-TDB treatment, though the expression of PD-1/PD-L1 didn't appear to inhibit the B-cell killing activity of CD20-TDB significantly. The in vivo anti-tumor activity of the combination of CD20-TDB and anti-PD-L1, as well as CD20-TDB and anti-PD-1, was evaluated in an A20-human CD20 syngeneic mouse lymphoma model. In the A20-human CD20 mouse B-lymphoma tumor model, where the target B lymphoma cells uniformly express high level of PD-L1, single-agent CD20-TDB did not significantly inhibit tumor growth. Treatment with single-agent anti-PD-L1 inhibited tumor growth and resulted in three partial responses (tumor regression of more than 50% but less than 100% of the starting tumor volume) out of nine treated animals. The combination of CD20-TDB and anti-PD-L1 resulted in substantially greater tumor growth inhibition compared to either agent alone and resulted in tumor regression in the majority of the nine animals tested, achieving eight partial responses and one complete response (100% tumor regression, no measurable tumor). Similar results were observed with the combination of CD20-TDB and anti-PD-1. Together, these results suggest that CD20-TDB can have broad clinical applicability, either combining with chemo reagents to enable flexible treatment strategies to incorporate CD20-TDB into current standard of therapy for B cell malignancies or with immune checkpoint inhibitors such as anti-PD-L1/PD-1 to improve upon single-agent efficacy. Disclosures Sun: Genentech Inc.: Employment. Wang:Genentech Inc.: Employment. Clark:Genentech Inc.: Employment. Hristopoulos:Genentech Inc.: Employment. Ellerman:Genentech Inc.: Employment. Mathieu:Genentech Inc.: Employment. Chu:Genentech Inc.: Employment. Wang:Genentech Inc.: Employment. Totpal:Genentech Inc.: Employment. Ebens:NGM: Employment. Polson:Genentech Inc.: Employment. Gould:Genentech Inc.: Employment.

scholarly journals A Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills Lymphoma Cells with Minimal Cytokine Secretion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1671-1671
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Brian Avanzino ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Nog Mice

Abstract Introduction Along with CD20 and CD22, the restricted expression of CD19 to the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates specific to CD19 have been described, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-Ts) have been approved to treat leukemia. To date, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to CD19 circumvent the latter problem but to date have shown similar T-cell over-activation, as well as significant neurotoxicity. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that uses in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a selectively-activating αCD3 that induced potent T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we compared the low-activating (and Fc-containing) CD19 x CD3 to two pan T-cell activating bispecific CD19 x CD3 antibodies (blinatumomab and another developed in-house) in vitro and in vivo for T-cell activation, efficacy in killing lymphoma cells, and toxicity. Methods T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, Ramos, and Nalm6) was measured via calcein release in vitro. In vivo, NOG mice were engrafted with human peripheral blood mononuclear cells (huPBMC) and human lymphoma cell lines, and the mice treated with weekly injections of T-BsAbs. Tumor burden was evaluated via caliper measurement. Pharmacokinetic (PK) studies were performed in NOG mice using ELISA. Results EC50s for cytotoxicity were in the single-digit nanomolar range for the selective T cell activating T-BsAb and sub-nanomolar for the pan T-cell activating controls. The selective T cell activator showed markedly reduced cytokine release for all cytokines tested compared to the pan T-cell controls even at saturating concentrations. In vivo, established CD19 positive B-cell tumors were cleared in NOG mice in the presence of huPBMC. PK profiles of both molecules generated in-house (selective and pan T-cell activators) were consistent with those of an IgG in mice. No activation of T-cells was observed in vitro or in vivo in the absence of CD19 expressing target cells. Conclusions Both the selectively-activating and the pan T-cell activating control bispecific antibodies killed lymphoma cells in vitro and in vivo in a CD19-dependent manner. While the pan T-cell activating controls showed T-cell activation comparable to other CD3-engaging bispecifics, the selective activator induced significantly reduced cytokine secretion by T-cells and demonstrated a half-life consistent with other IgG antibodies. In summary, our selectively activating CD19 x CD3 T-BsAb shows promise as a lymphoma therapeutic differentiated from current T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment. Buelow:Teneobio Inc.: Employment. Avanzino:Teneobio, Inc.: Employment. Balasubramani:Teneobio, Inc.: Employment. Boudreau:Teneobio, Inc.: Employment. Clarke:Teneobio, Inc.: Employment. Dang:Teneobio, Inc.: Employment. Davison:Teneobio, Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Harris:Teneobio, Inc.: Employment. Jorgensen:Teneobio, Inc.: Employment. Li:Teneobio, Inc.: Employment. Medlari:Teneobio, Inc.: Employment. Narayan:Teneobio, Inc.: Employment. Ogana:Teneobio, Inc.: Employment. Pham:Teneobio Inc.: Employment. Prabhakar:Teneobio, Inc.: Employment. Rangaswamy:Teneobio, Inc.: Employment. Sankaran:Teneobio, Inc.: Employment. Schellenberger:Teneobio, Inc.: Employment. Ugamraj:Teneobio, Inc.: Employment. Trinklein:Teneobio, Inc.: Employment. Van Schooten:Teneobio, Inc.: Employment.

A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3721-3721
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Cells ◽  
Cytotoxicity Assays

Abstract Abstract 3721 Background: CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy. In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions: In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment.

Rituximab Directly Decreases T Cell Activation, Both In Vivo and In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3935-3935 ◽  
Author(s):  
Tamar Katz ◽  
Dina Stroopinsky ◽  
Jacob M. Rowe ◽  
Irit Avivi
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Cell Function ◽  
Activation Profile

Abstract Abstract 3935 Rituximab, a chimeric anti-C20 monoclonal antibody, has been extensively used over the last decade for the therapy of B cell malignancies. Recent clinical data suggest that rituximab may affect T cell function, increasing the risk of T cell dependent infections in heavily-treated patients. The current study was designed to investigate the effect of rituximab on T cell activation and assess T cell function following the addition of rituximab to purified T cells. The T cell activation profile, dependent on rituximab administration, was evaluated in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) generated from B-cell non-Hodgkin lymphoma (NHL) patients prior and immediately after the administration of 375 mg/m2 rituximab, were examined for the expression of inflammatory cytokines. The in vitro studies were performed by using CD25 depleted PBMCs or B cell depleted T cells (CD3+CD25-CD19-). The obtained cells were stimulated with allogeneic dendritic cells (DCs), in the absence or presence or 2 mg/ml rituximab. T cell activation was evaluated using immunophenotypic markers, cytokine profile and T cell proliferation assay. Eight NHL patients participated in the study. The level of T cells expressing inflammatory cytokines was significantly decreased following the administration of a single dose of rituximab. T cells expressing IL-2 declined from a mean level of 26.5% to 11.5% and the level of IFN- γ decreased from 22% to 4.2%. Further administration of rituximab, up to 4 weekly doses, resulted in an additional decline in the amount of inflammatory cytokine producing T cells to a level of 1.4% for IL-2 and 3.5% for IFN-g. However, repeated evaluation, performed at 4 months after completing rituximab, showed restoration of the inflammatory population. In accord with this inhibitory effect, in vitro stimulation of T cells with allogeneic DCs, in the presence of rituximab, resulted in a significant decrease in activation markers (CD25, GITR and CTLA-4) (Table 1). These changes were accompanied by a marked reduction in inflammatory cytokine production and proliferative capacity. Of interest, these inhibitory effects were also obtained whilst using B cell depleted T cells (CD3+CD25-CD19-). In conclusion, rituximab administration results in a transient T cell inactivation, demonstrated through the reduction in inflammatory cytokine production and T cell proliferation capacity. This effect appears to be non-B cell dependent, being obtained in the absence of B cell in the culture, and may account for clinical observations in ameliorating T-cell dependent disorders, such as graft-versus-host disease. Table 1. Activation profile depending on rituximab (in vitro) Without rituximab With rituximab *Activation marker (%) CD25 27 9 GITR 15.6 4.7 CTLA4 17.7 7 *Cytokines expression (%) IL-2 22 2 IL12 16 4 IFN-gamma 21 1.8 T cells proliferation (O.D.) DC stimulation 1.528 0.580 CMV stimulation 1.563 0.570 anti CD3/CD28 stimulation 0.705 0.407 * Gated out of lymphocytes Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mouse CD19-Targeted Chimeric Antigen Receptors That Include a 41BB Co-Stimulatory Domain Induce NFkB Signaling to Enhance T Cell Proliferation, Viability, and Leukemia Killing

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 843-843
Author(s):  
Gongbo Li ◽  
Nolan Beatty ◽  
Paresh Vishwasrao ◽  
Justin C. Boucher ◽  
Bin Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Car T Cells ◽  
Apoptosis Protein ◽  
Car T

Abstract CD19 targeted 2nd generation chimeric antigen receptor T (CAR T) cells have been successful against relapsed and/or refractory B cell malignancies. The pending FDA-approval of 2 separate CD19 targeted CAR T products highlight the need to understand the biology behind this novel therapy. CAR design includes a single-chain variable fragment, which encodes antigen-binding, fused to a transmembrane domain, co-stimulatory domain, and CD3ζ activation domain. The two CARs likely to be approved as standard of care include a 41BB or CD28 co-stimulatory domain. CD28 is a critical co-stimulatory receptor required for full T cell activation and persistence, while 4-1BB is a member of the tumor necrosis factor receptor family and also a critical T cell co-stimulatory factor. Early evaluation of the co-stimulatory domains role in CAR design confirmed that they are required to enhance T cell function, but lacked insight regarding their mechanism for this enhancement. Furthermore, clinical outcomes suggest that the co-stimulatory domains in CARs support different T cell functions in patients. For example, while overall outcomes are similar between 41BB (19BBz) and CD28-containing CARs (1928z), 19BBz CAR T cells can persist for years in patients, but functional 1928z CAR T cells rarely persist longer than a month. Recent studies are providing insight to these differences and have demonstrated that 4-1BB-containing CARs reduce T cell exhaustion, enhance persistence, and increase central memory differentiation and mitochondrial biogenesis, while CD28-containing CARs support robust T cell activation and exhaustion, and are associated with effector-like differentiation. However, these studies have been performed mostly in vitro or in immune deficient mice, which limits their ability to model complex immune biology. Therefore, we evaluated murine CD19-targeting CARs with a 4-1BB (m19BBz) or CD28- (m1928z) co-stimulatory domain in relevant animal models of immunity. We directly compared m19BBz and m1928z CAR T cell immune phenotype, cytotoxicity, cytokine production, gene expression, intracellular signaling, and in vivo persistence, expansion, and B cell acute lymphoblastic leukemia (B-ALL) eradication. In vitro assays revealed that m1928z CAR T cells had enhanced cytotoxicity and cytokine production compared to m19BBz CAR T cells. Also, evaluation of m1928z and m19BBz CAR T cells displayed similar immune phenotypes, but markedly different gene expression with m1928z CAR T cells upregulating genes related to effector function and exhaustion, while m19BBz CAR upregulated genes critical for NFkB regulation, T cell quiescence and memory. In vivo, both m1928z and m19BBz CAR T cells supported equivalent protection against B-ALL. Similar to patients, in our mouse models there are functional differences between the mouse CD19-targeted CAR T cells. At 1 week post-infusion m19BBz CAR T cells are present in the blood of mice at significantly greater levels than m1928z CAR T cells. Furthermore, m19BBz CAR T cells enhance proliferation and/or anti-apoptosis protein expression to enhance B cell killing, which is evidenced by our observation that irradiation significantly weakens the in vivo efficacy of m19BBz but not m1928z CAR T cells. Our results suggest that B cell killing by m1928z CAR T cells is not impacted by irradiation because of their efficacious cytotoxicity of B cells. In contrast, m19BBz CAR T cells have enhanced viability and anti-apoptosis protein expression, which allows them to compensate for reduced effector function. We investigated potential mechanisms for the enhanced viability and anti-apoptosis of m19BBz CAR T cells and determined that NFkB signaling is upregulated much greater by m19BBz than m1928z. We have observed this difference in both a reporter cell line and primary mouse T cells. We are now dissecting what cellular components mediate increased NFkB signaling by the m19BBz CAR. Our animal models recapitulate equivalent anti-leukemia efficacy of CD19-targeted CAR T cells regardless of co-stimulatory domain, but underscore that anti-leukemia killing is mediated by different methods depending on the co-stimulatory domain. Our work sheds light on how 4-1BB mechanistically regulates and impacts CAR T function and has implications for future CAR design and evaluation. Disclosures No relevant conflicts of interest to declare.

CD20 Tcb (RG6026), a Novel "2:1" T Cell Bispecific Antibody for the Treatment of B Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1836-1836 ◽  
Author(s):  
Marina Bacac ◽  
Pablo Umaña ◽  
Sylvia Herter ◽  
Sara Colombetti ◽  
Johannes Sam ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Tumor Growth Inhibition ◽  
Tumor Lysis

Abstract Despite the recent advancements in treatment options with the introduction of anti-CD20 monoclonal antibody therapy, approximately 50% of patients with non-Hodgkin's lymphomas (NHL) will not sustain a durable response to standard of care (SOC) treatment. Thus, there remains a continuous need for safer and more effective anti-cancer therapies in this indication. T-cell bispecific antibodies (TCBs) represent a new class of disease targeting agents shown to promote the activation of a patient's own T cells to attack and kill cancer cells. CD20 TCB is a new bispecific antibody with IgG-like pharmacokinetic properties whose unique "2:1" structure leads to increased tumor antigen avidity, T cell activation, and tumor cell killing, as compared to other T cell engaging bispecific antibody molecular formats. The molecule comprises two CD20 binding Fabs (derived from the Type II CD20 IgG1 obinutuzumab), one CD3e binding Fab (fused to one of the CD20 Fabs via a short flexible linker), and an engineered, heterodimeric Fc region with completely abolished binding to FcgRs and C1q. In vitro, CD20 TCB was shown to dose-dependently induce tumor lysis with EC50 values in the range of 0.05 - 3.1 pM. The "2:1"format of CD20 TCB was shown to confer superior potency (up to 10 - 1000x) when compared to CD20 TCBs having the conventional "1:1" IgG-based format (i.e., one binding domain for CD20 and one for CD3). CD20 TCB-mediated tumor lysis resulted in T-cell activation, proliferation and cytokine release with up-regulation of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis upon tumor lysis. CD20 TCB also demonstrated potent ex vivo activity in whole bone marrow aspirate samples of NHL and CLL patients (n=17). Such primary tumor samples preserve the native tumor microenvironment and bear low effector to target cell ratios ranging in this study from 0.02 to 0.8 (average value 0.3). CD20 TCB activity was consistently superior to that of the "1:1" CD20 TCB and demonstrated faster, more profound and more potent B cell depletion with EC50 values ranging from 0.002 to 2.7 nM. In vivo, CD20 TCB displayed potent anti-tumor activity in xenograft models in stem cell humanized mice and induced regression of large, aggressive WSU-DLCL2 lymphoma tumors (0.5 mg/kg, weekly administration). In addition to tumor regression, CD20 TCB treatment led to fast and complete elimination of peripheral blood B cells within 24 h after the first administration (0.05, 0.15 and 0.5 mg/kg, weekly administration) and to a complete elimination of B cells in spleen, bone marrow and lymph nodes after two administrations. B cell depletion was paralleled by transient decrease of T-cell counts in the peripheral blood and by the peak of cytokine release 24 h after the first administration, followed by rapid recovery and return to baseline levels at 72 h post treatment. Tumor growth inhibition mediated by CD20 TCB was accompanied by increase in intra-tumor T-cell infiltration, up-regulation of PD-1 receptor on T cells and PD-L1 in the tumor. Combination studies of CD20 TCB with PD-L1 blocking antibody led to more profound and faster tumor growth inhibition. Taken together, the preclinical data show that CD20 TCB is a novel differentiated CD20-targeting T cell bispecific antibody with promising anti-tumor activity and the ability to modify the tumor microenvironment. CD20 TCB consistently demonstrated superior potency compared to other CD20 TCBs with a conventional "1:1" IgG format. This translated into superior efficacy in vitro, ex-vivo and in vivo, which could not be matched by increasing doses of the "1:1" TCBs. The molecule is now scheduled to start clinical trial by December 2016. Disclosures Bacac: Roche: Employment, Equity Ownership, Patents & Royalties. Umaña:Roche: Employment, Equity Ownership, Patents & Royalties. Herter:Roche: Employment, Patents & Royalties. Colombetti:Roche: Employment. Sam:Roche: Employment. Le Clech:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Richard:Roche: Employment. Nicolini:Roche: Employment. Gerdes:Roche: Employment. Lariviere:Roche: Employment. Neumann:Roche: Employment. Klein:Roche: Employment, Patents & Royalties.

scholarly journals 863 In vitro and in vivo studies establish DuoBody®-CD3xB7H4 as a novel drug candidate for the treatment of solid cancers

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A904-A904
Author(s):  
Louise Koopman ◽  
Laura Smits-de Vries ◽  
Frederikke Lihme Egerod ◽  
Sebastiaan Wubben ◽  
Mischa Houtkamp ◽  
...  
Keyword(s):  
T Cell ◽  
Antitumor Activity ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cynomolgus Monkeys ◽  
Animal Care ◽  
Dose Dependent

BackgroundThe immune checkpoint protein B7H4 is expressed on malignant cells in various solid cancers, whereas its expression is highly restricted in normal tissue. B7H4 is therefore an attractive target for a CD3 bispecific antibody (bsAb) therapeutic. Moreover, its expression is reported to be inversely correlated with PD-L1. Here, we describe the preclinical characterization of two B7H4-targeting CD3 bsAbs with different CD3 affinities, supporting the selection of our clinical lead, DuoBody-CD3xB7H4 (GEN1047).MethodsB7H4 protein expression in patient-derived samples was determined by immunohistochemistry. Controlled Fab-arm exchange of an Fc-silenced B7H4 antibody with two Fc-silenced CD3ε-binding antibodies generated two CD3xB7H4 bsAbs that differ in CD3 binding affinity by approximately 30-fold. In vitro T-cell mediated cytotoxicity, T-cell activation, and cytokine release were assayed using cocultures of B7H4-expressing tumor cells and healthy donor T cells. Nonclinical safety (NCS) of the two CD3xB7H4 bsAbs was assessed in cynomolgus monkeys, and antitumor activity of the clinical lead in vivo was tested in a patient-derived xenograft (PDX) screen in mice with a humanized immune system (HIS).ResultsB7H4 protein expression was confirmed in tumor biopsies from multiple indications, including breast, ovarian and lung cancer. Both bsAbs induced target-specific and dose-dependent tumor cell kill in vitro. Maximal kill and T-cell activation were comparable for both variants, although the potency of the high CD3 affinity bsAb was higher. However, production of inflammatory cytokines at comparable effective concentrations (IC90) was lower for the low CD3 affinity bsAb. Single dose NCS studies in cynomolgus monkeys showed that both CD3xB7H4 bsAbs were well-tolerated. A dose-dependent increase in plasma cytokines IL-6 and MCP-1 2 hours after dosing was observed only with the high CD3 affinity bsAb. Based on these findings, the low CD3 affinity bsAb was selected for follow-up studies and named DuoBody-CD3xB7H4 (GEN1047). DuoBody-CD3xB7H4 demonstrated antitumor activity in vivo in a PDX screen in HIS mice. Repeated dosing of DuoBody-CD3xB7H4 in cynomolgus monkeys confirmed an acceptable safety profile up to the maximal dose tested (30 mg/kg).ConclusionsThese studies describe the preclinical development of DuoBody-CD3xB7H4, a bsAb that induces T-cell mediated cytotoxicity of B7H4-positive tumor cells, which may provide an alternative therapeutic modality in the immune-oncology space for patients with solid cancers.Ethics ApprovalAnimal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). NCS studies were conducted at Citoxlab (Evreux, France) and Charles River Laboratories (Tranent, UK) in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Council of Europe).

scholarly journals 702 Dual blockade of the PD-1 checkpoint pathway and the adenosinergic negative feedback signaling pathway with a PD-1/CD73 bispecific antibody for cancer immune therapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A744-A744
Author(s):  
Tingting Zhong ◽  
Zhaoliang Huang ◽  
Xinghua Pang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Negative Feedback ◽  
Immune Suppression ◽  
Specific Antibody ◽  
Cell Activation ◽  
Immune Therapy ◽  
B Cell Activation

BackgroundCD73 (ecto-5’-nucleotidase) is an ecto-nucleotidase that dephosphorylate AMP to form adenosine. Activation of adenosine signaling pathway in immune cells leads to the suppression of effector functions, down-regulate macrophage phagocytosis, inhibit pro-inflammatory cytokine release, as well as yield aberrantly differentiated dendritic cells producing pro-tumorigenic molecules.1 In the tumor microenvironment, adenosinergic negative feedback signaling facilitated immune suppression is considered an important mechanism for immune evasion of cancer cells.2 3 Combination of CD73 and anti-PD-1 antibody has shown promising activity in suppressing tumor growth. Hence, we developed AK119, an anti- human CD73 monoclonal antibody, and AK123,a bi-specific antibody targeting both PD-1 and CD73 for immune therapy of cancer.MethodsAK119 is a humanized antibody against CD73 and AK123 is a tetrameric bi-specific antibody targeting PD-1 and CD73. Binding assays of AK119 and AK123 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro assays to investigate the activity of AK119 and AK123 to inhibit CD73 enzymatic activity in modified CellTiter-Glo assay, to induce endocytosis of CD73, and to activate B cells were performed. Assay to evaluate AK123 activity on T cell activation were additionally performed. Moreover, the activities of AK119 and AK123 to mediate ADCC, CDC in CD73 expressing cells were also evaluated.ResultsAK119 and AK123 could bind to its respective soluble or membrane antigens expressing on PBMCs, MDA-MB-231, and U87-MG cells with high affinity. Results from cell-based assays indicated that AK119 and AK123 effectively inhibited nucleotidase enzyme activity of CD73, mediated endocytosis of CD73, and induced B cell activation by upregulating CD69 and CD83 expression on B cells, and showed more robust CD73 blocking and B cell activation activities compared to leading clinical candidate targeting CD73. AK123 could also block PD-1/PD-L1 interaction and enhance T cell activation.ConclusionsIn summary, AK119 and AK123 represent good preclinical biological properties, which support its further development as an anti-cancer immunotherapy or treating other diseases.ReferencesDeaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204:1257–65.Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood. 1997; 90:1600–10.Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY, Blackburn MR, Biaggioni I,Carbone DP, Feoktistov I, et al. Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008; 112:1822–31.

107 Effects of IL-2 and IL-15 on the proliferative and antitumor capacities of allogeneic CD20 CAR-engineered γδ T cells in a 3D B cell lymphoma spheroid assay

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A119-A119
Author(s):  
Lu Bai ◽  
Kevin Nishimoto ◽  
Mustafa Turkoz ◽  
Marissa Herrman ◽  
Jason Romero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Car T

BackgroundAutologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells.MethodsMolecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine.ResultsWhen compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production.ConclusionsTogether, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

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