CAP-100: First-in-class antibody for CCR7+ hematological malignancies.

e19008 Background: CCR7 is highly expressed in many hematological malignancies including CLL, several B-cell non-Hodgkin lymphomas (NHL), and various T-cell neoplasias with nodal involvement. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of cells to locations where these chemokines are expressed, such as the lymph node (LN) and central nervous system. In these protective microenvironments CCR7 ligands contribute to tumor cell survival and proliferation. Indeed, both high CCR7 surface expression levels and high migratory responses to CCR7 ligands correlate with LN involvement, adverse prognostic factors, and shorter patient survival. Thus, strategies targeting CCR7 could provide a novel therapeutic approach for CCR7+ hematological malignancies. Methods: We have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21, and evaluated the antibody in various in-vitro and in-vivo preclinical models. Results: CAP-100 effectively inhibited in vitro migration of primary patient samples of CLL, B-cell NHLs and T-cell neoplasias such as T-PLL or T-ALL. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. CAP-100 also abrogated survival elicited by CCR7 in CLL, and showed potent cell killing activity against CLL or CCR7+ T-lymphomas cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed anti-CD20 or anti-CD52 standard-of-care antibodies in B-NHL and T-lymphomas respectively. In all cases, ADCC and migration inhibition were both independent of prognostic markers for high risk disease. Finally, when given as monotherapy in disseminated B-NHL and CLL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition and extended survival significantly. Conclusions: Our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in several CCR7+ hematological malignancies, including relapsed/refractory disease. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in these diseases. CAP-100’s unique propensity to block migration of tumor cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100, either as monotherapy or in combination with novel agents. Clinical trials in CLL and CCR7-expressing NHL will be initiated soon.

Download Full-text

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 ◽

10.1182/blood.v128.22.4168.4168 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4168-4168 ◽

Cited By ~ 1

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.

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-112819 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4169-4169 ◽

Cited By ~ 2

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.

Download Full-text

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

Blood ◽

10.1182/blood.v128.22.1836.1836 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1836-1836 ◽

Cited By ~ 13

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.

Download Full-text

Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

Download Full-text

PD-L1hi plasmablasts limit the T cell response during the acute phase of parasite infections

10.1101/2020.06.10.142067 ◽

2020 ◽

Author(s):

Melisa Gorosito Serrán ◽

Facundo Fiocca Vernengo ◽

Laura Almada ◽

Cristian G Beccaria ◽

Pablo F Canete ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

Cell Activation ◽

Mononuclear Cells ◽

Plasma Cells ◽

Chronic Phase ◽

Surface Expression ◽

T Cell Response

ABSTRACTDuring infections with protozoan parasites or virus, T cell immunosuppression is generated simultaneously with a high B cell activation. Here, we show that in T. cruzi infection, all plasmablasts detected had higher surface expression of PD-L1, than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in an antigen-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection express PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. PD-L1hi plasmablasts suppressed T cell response, via PD-L1, in vitro and in vivo. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, which precede the germinal center (CG) response, are a suppressive population in infections that may influence T cell response.Brief summaryPathogens develop different strategies to settle in the host. We identified a plasmablats population induced by pathogens in acute infections which suppress T cell response.

Download Full-text

Effect of a thymic factor on T lymphocytes in B cell chronic lymphocytic leukemia: in vitro and in vivo studies

Blood ◽

10.1182/blood.v64.3.667.667 ◽

1984 ◽

Vol 64 (3) ◽

pp. 667-671 ◽

Cited By ~ 7

Author(s):

F Lauria ◽

D Raspadori ◽

S Tura

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Proliferative Response ◽

Lymphocytic Leukemia ◽

Before And After ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abnormalities of T lymphocytes in B cell chronic lymphocytic leukemia (B-CLL) have been extensively documented by several immunologic investigations. Following recent studies pointing to the favorable effect of TP-1, a partially purified extract of calf thymus, on the T cell-mediated immunity of several diseases, including Hodgkin's disease, we have used monoclonal antibodies and the enriched T lymphocytes of 16 untreated B-CLL patients to evaluate the proportion of T cell subsets before and after the administration of TP-1. In addition, the proliferative response to phytohemagglutinin (PHA) and the helper function in a pokeweed mitogen (PWM) system were assessed. In ten cases, the effect of TP-1 was also studied in vitro by evaluating the same parameters before and after incubation of B-CLL T cells with the drug. The study demonstrated that in vivo administration of TP-1 increases significantly (P less than .001) the proportion of the defective helper/inducer T cell population (OKT4-positive cells) in B-CLL, leading to a near normal OKT4/OKT8 ratio. Furthermore, the improved phenotypic profile was accompanied by an increased proliferative response to PHA and, in particular, by a significant increase (P less than .01) of T helper capacity; this increase was, however, insufficient to enable the normalization of the serum immunoglobulin levels. The in vitro incubation of B-CLL T lymphocytes did not succeed in producing significant modifications in distribution and function.

Download Full-text

Retroviral Gene Transfer of T Cell Receptors (TCR) Specific for Minor Histocompatibility Antigens to Virus-Specific T Cells as Cellular Immunotherapy of Patients with Relapsed Hematological Malignancies after Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v106.11.5529.5529 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5529-5529

Author(s):

Marieke Griffioen ◽

H.M. Esther van Egmond ◽

Menno A.W.G. van der Hoorn ◽

Renate S. Hagedoorn ◽

Michel Kester ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematological Malignancies ◽

Surface Expression ◽

Retroviral Vector ◽

Cellular Immunotherapy ◽

Minor Histocompatibility Antigens ◽

Vector Coding

Abstract Patients with hematological malignancies can be successfully treated by T cell-depleted allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI). Failure of some relapsed hematological malignancies to respond to DLI is probably due to immune tolerance induced by regulatory and/or anergic T cells. We previously showed that functional T cells with redirected anti-leukemic reactivity can be generated by transfer of TCRs specific for minor histocompatibility antigens (mHags) to total peripheral blood mononuclear cells (PBMC) as well as CMV-specific T cells. By introducing TCRs into CMV or EBV specific T cells, T cells with proper memory/effector phenotypes are targeted, and due to virus persistence these T cells may show prolonged survival in vivo. The purpose of this study is to develop an efficient method for the isolation, retroviral transduction and expansion of TCR-transduced CMV- and EBV-specific T cells for cellular immunotherapy of patients with relapsed hematological malignancies after alloSCT. For clinical application, construction of single retroviral vectors coding for the α as well as β chains of mHag-specific TCRs is required. We used the MP71 retroviral vector for TCR gene transfer, since this vector contains Myeloproliferative Sarcoma Virus LTR sequences and a Mouse Embryonic Stem Cell Virus leader, which has been optimized for use in the clinic. The MP71 vector also contained a Woodchuck Hepatitis Response Element (WPRE). The WPRE, which is used as an element enhancing transgene expression at the post-transcriptional level, has recently been described to encode 60 amino acids of a protein with potential oncogenic activity. Therefore, we reconstructed the MP71 vector by introduction of a multiple cloning site (MCS) and, for safety issues, deleted the WPRE. The TCR α and β genes specific for the hematopoietic-restricted mHag HA-2 were linked by a 50-bp sequence encoding a “self-cleaving” 2A-like peptide and introduced into the MCS of the MP71 vector. Linkage of the TCR α and β genes by the 2A-like sequence allowed additional linkage of the low affinity nerve growth factor receptor (LNGFR) or human CD20 selection marker genes by an IRES sequence. The advantage of the human CD20 gene is that it can also function as suicide gene, allowing elimination of transduced cells in vivo if undesired side effects occur. Introduction of the single MP71 retroviral vector coding for the HA-2 TCR α and β chains as well as LNGFR into a TCR α- and β-deficient Jurkat T cell line led to high levels of TCR surface expression correlating with LNGFR marker gene expression. These data indicate proper cleavage and assembly of the transduced TCR α and β chains. Moreover, removal of the WPRE did not affect the surface expression level of the transduced TCR. Furthermore, CMV- and EBV-specific T cells were isolated from human individuals by the IFN-γ capture assay and subsequently transduced with a single retroviral vector coding for the HA-2-TCR α and β chains as well as LNGFR. CMV- and EBV-specific T cells from different human individuals could be successfully isolated to 60–90% purity and the TCR-transduced CMV- and EBV-specific T cells were shown to be fully functional, recognizing the viral peptides as well as the endogenously-processed HA-2 mHag.

Download Full-text

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽

10.1182/blood.v118.21.1633.1633 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1633-1633

Author(s):

Marcelo A. Navarrete ◽

Benjamin Kisser ◽

Hendrik J. Veelken

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

B Cell Lymphomas ◽

Cd8 Cells ◽

Fab Fragments ◽

The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽

10.1182/blood.v128.22.3017.3017 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3017-3017

Author(s):

Chiara Tarantelli ◽

Eugenio Gaudio ◽

Petra Hillmann ◽

Filippo Spriano ◽

Ivo Kwee ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Mtor Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

Apoptosis Induction ◽

In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

B Cell Activating Factor Inhibition Impairs Bacterial Immunity by Reducing T Cell-Independent IgM Secretion

Infection and Immunity ◽

10.1128/iai.00998-13 ◽

2013 ◽

Vol 81 (12) ◽

pp. 4490-4497 ◽

Cited By ~ 9

Author(s):

Derek D. Jones ◽

Maura Jones ◽

Gregory A. DeIulio ◽

Rachael Racine ◽

Katherine C. MacNamara ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

B Cell ◽

Host Defense ◽

B Cell Differentiation ◽

Content Type ◽

Surface Marker Expression ◽

B Cell Activating Factor

ABSTRACTB cell activating factor of the tumor necrosis factor family (BAFF) is an essential survival factor for B cells and has been shown to regulate T cell-independent (TI) IgM production. DuringEhrlichia murisinfection, TI IgM secretion in the spleen was BAFF dependent, and antibody-mediated BAFF neutralization led to an impairment of IgM-mediated host defense. The failure of TI plasmablasts to secrete IgM was not a consequence of alterations in their generation, survival, or early differentiation, since all occurred normally in infected mice following BAFF neutralization. Gene expression characteristic of plasma cell differentiation was also unaffected by BAFF neutralizationin vivo, and except for CD138, plasmablast cell surface marker expression was unaffected. IgM was produced, since it was detected intracellularly, and impaired secretion was not due to a failure to express the IgM secretory exon. Addition of BAFF to plasmablastsin vitrorescued IgM secretion, suggesting that BAFF signaling can directly regulate secretory processes. Our findings indicate that BAFF signaling can modulate TI host defense by acting at a late stage in B cell differentiation, via its regulation of terminal plasmablast differentiation and/or IgM secretion.

Download Full-text