The IL-15 Superagonist ALT-803 Enhances NK Cell ADCC and in Vivo Clearance of B Cell Lymphomas Directed By an Anti-CD20 Monoclonal Antibody

Abstract Anti-CD20 monoclonal antibodies (mAbs) have provided an important therapeutic passive immunotherapy approach for B cell malignancies. Typically anti-CD20 mAbs are combined with traditional chemotherapy and/or radiation therapy that have the potential for serious long term adverse complications. Novel approaches are needed to improve anti-CD20 mAb anti-lymphoma efficacy with agents that do not result in long term toxicity. ALT-803 is a super agonist IL-15 variant bound to an IL-15Ralpha – Fc fusion and represents a novel immunostimulatory agent for NK and T cells. Notably, ALT-803 exhibits extended in vivo pharmacokinetics and altered biodistribution compared to recombinant human IL-15. We hypothesized that ALT-803 would enhance anti-CD20-mAb-(rituximab) directed NK cell antibody dependent cellular cytotoxicity (ADCC) against B cell lymphomas. ALT-803 at concentrations of 0.35-35 ng/mL potentiated rituximab-triggered NK cell ADCC against the Raji (27% vs. 74%, E:T 25:1, P<0.01) and Daudi (39% vs. 84%, E:T 2:1, P<0.05) B cell lymphoma lines in vitro. Moreover, the activation of NK cells with ALT-803 significantly increased ADCC against primary human follicular lymphoma cells in vitro (11% vs. 33% at a 2.5:1 E:T ratio, P<0.001, N=5 primary follicular lymphomas, N=15 NK cell donors). One mechanism whereby ALT-803 may be modulating human NK cells is via the induction of cytotoxic effector molecules. After 24-48 hour in vitro activation with ALT-803 (0.35-350 ng/mL) an increased expression of granzyme B and perforin were observed in primary human NK cells (P<0.05). The effectiveness of ALT-803 enhancement of NK cell ADCC against B cell lymphoma cell lines was assessed with two in vivo mouse models. First, Daudi cells were engrafted into SCID mice (that have an intact NK cell compartment), and groups were treated with vehicle, rituximab (10 mg/kg), ALT-803 (0.2 mg/kg), or ALT-803+rituximab at day 15 and 18, and assessed for lymphoma percentages in the bone marrow at day 22. Mice treated with ALT-803+rituximab had significantly reduced Daudi B cell burden, compared to rituximab, ALT-803, or vehicle treatment (vehicle versus ALT-803+rituximab, 38% vs. 5%, P<0.01). At doses of 0.2-0.02 mg/kg the effect of ALT-803 on rituximab mediated lymphoma clearance was demonstrated (P<0.02 compared to vehicle treatment). Further, the survival of mice treated with ALT-803+rituximab was significantly longer compared vehicle control, rituximab alone, and ALT-803 alone groups (see KM survival curves, Figure, P<0.05). In our second in vivo model, malignant Raji B cells expressing luciferase were injected into immunodeficient NOD-SCID-gamma-c-/- (NSG) mice (100,000/mouse, day 0) followed by primary human NK cells (4 million / mouse, day 3), and treated (initiated on day 3) with vehicle, ALT-803 (0.05 mg/kg q3-4 days), rituximab (10 mg/kg day 3), or ALT-803+rituximab. At day 16, ALT-803+rituximab exhibited a significant reduction in Raji signal compared to the control groups (P<0.05). These results were confirmed in a second approach where an increased Raji cell numbers were engrafted (1 million / mouse, day 0), primary human NK cells (4 million / mouse) were infused on day 3, and groups of mice were treated with rituximab (5 mg/kg) or ALT-803 (0.2 mg/kg q3-4 days)+rituximab (mean photons per second at day 34, 3.8x109 versus 3.1x108, respectively, P<0.05). ALT-803 was well tolerated at all of the administered dose levels in combination with rituximab. Thus, ALT-803 represents an effective immunostimulatory agent that augments NK cell cytotoxic potential and ADCC against malignant follicular lymphoma and B cell lines in vitro, and significantly increases rituximab-directed clearance of B cell lymphoma by NK cells in two in vivo models. Based on these findings, a phase 1/2 clinical trial of ALT-803 plus rituximab is planned for patients with relapsed/refractory indolent non-Hodgkin lymphomas. Figure 1 Figure 1. Disclosures Liu: Altor BioScience Corporation: Employment. Kong:Altor BioScience Corporation: Employment. Jeng:Altor BioScience Corporation: Employment. Rhode:Altor BioScience Corporation: Employment. Wong:Altor BioScience Corporation: Employment.

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Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽

10.1182/blood.v122.21.377.377 ◽

2013 ◽

Vol 122 (21) ◽

pp. 377-377 ◽

Cited By ~ 3

Author(s):

Shruti Bhatt ◽

Daxing Zhu ◽

Xiaoyu Jiang ◽

Seung-uon Shin ◽

John M Timmerman ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cd20 Antibody ◽

Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

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Acquired Stem Cell Properties In Therapy-Induced Senescence Of Lymphomas and Acute Leukemias In Vitro and In Vivo

Blood ◽

10.1182/blood.v122.21.4193.4193 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4193-4193

Author(s):

J. Henry M. Däbritz ◽

Maja Milanovic ◽

Zhen Zhao ◽

Jan R. Dörr ◽

Yong Yu ◽

...

Keyword(s):

Stem Cell ◽

B Cell ◽

Myeloid Leukemia ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Stem Cell Markers ◽

Hematological Cancer

Abstract Introduction Premature senescence is a permanent proliferative arrest that occurs in response to oncogenic signaling or DNA-damaging chemotherapy. Although tumor cell senescence has been recognized as a prognostically relevant contribution to long-term outcome post-therapy in hematological tumor models, therapeutic utilization of senescence is still hampered by an incomplete understanding of biological properties and long-term fate of senescent tumor cells in patients. Interestingly, senescence-regulating factors have recently been shown to limit reprogramming of somatic cells to pluripotency, and to protect stem cell compartments from premature exhaustion. Hence, we explore here whether cellular senescence and stemness may functionally overlap, thereby potentially equipping arrested cells with latent self-renewing potential. Methods Stem cell-related features (stem cell gene signatures, Sca-1 expression, ALDH and ABC transporter activity) were analyzed in primary apoptosis-blocked Eµ-myc transgenic B-cell lymphomas, which enter treatment-induced senescence (TIS) in response to standard antineoplastic agents. Several (e.g. Suv39h1- or p53-based) genetic models were established, in which TIS occurred in a conditional and reversible fashion. Clonogenicity, proliferative and repopulating assays were performed in vitro and in vivo, comparing individual lymphomas that grew out of senescence (“Previously Senescent”, PS) with matched lymphoma cells that equally received chemotherapy, but were incapable of entering TIS (“Never Senescent”, NS). In a mouse model of T-cell acute lymphoblastic leukemia (T-ALL), stem cell markers and tumor initiating potential were assessed in a flow-sorted non-self-renewing leukemia cell population after senescence induction by chemotherapy. Human hematological cancer cell lines and tumor samples obtained from B-cell lymphoma and acute myeloid leukemia (AML) patients were analyzed for stem cell features after exposure to senescence-inducing chemotherapy in vitro. Results Senescent mouse lymphomas were strongly skewed towards an increased expression of an adult tissue stem cell signature, distinct stem cell markers and functional stemness properties. Upon release from conditional senescence, PS cells resumed proliferation and rapidly exceeded the proliferative, clonogenic and tumor-initiating capacity of NS cells. Interrogation of self-renewal-relevant cascades revealed activation of and dependence on canonical Wnt signaling in senescence, as blocking of this pathway reduced the growth of PS, but not NS cells. Moreover, TIS-related stemness occurred independent of secretable factors. Strikingly, in a murine T-ALL model, temporary senescence enforcement re-programmed non-stem leukemia cells into leukemia stem cells, allowing PS bulk leukemia cells to de novo initiate leukemias in recipient mice. These results were supported by consistent findings in human hematological cancer cell lines as well as primary human B-cell lymphoma and acute myeloid leukemia samples. Conclusions Our findings uncover senescence-associated stemness as a detrimental capability which is latently enriched for by chemotherapy in lymphoma and leukemia. The aggressive growth potential might become evident when senescent cells occasionally acquire alterations that allow them to re-enter the cell-cycle, thereby unleashing the tumor-promoting potential of a biological program so far considered to operate as a tumor-suppressive mechanism. However, targeted intervention at stemness-related signaling cascades in senescence may open novel therapeutic options for apoptosis-resistant lymphoma and leukemia. Disclosures: No relevant conflicts of interest to declare.

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631 Development of highly efficacious and safe targeted cancer immunotherapy via IL12-based TMEkine™ platform

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0631 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A667-A667

Author(s):

Dahea Lee ◽

Donggeon Kim ◽

Soomin Ryu ◽

Byoung Chul Lee

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Cell Lymphoma ◽

Specific Binding ◽

B Cell Lymphoma ◽

Human T Cells ◽

Syngeneic Mouse Model ◽

Anti Cd20

BackgroundWe developed tumor microenvironment-targeting immunocytokine or TMEkine™ utilizing strong anti-tumoral effect of interleukin 12 (IL-12). In this effort, we created a bi-specific 1+1 antibody fusion with conventional knob-in-hole technology where anti-CD20 was paired with IL-12 fc fusion arm. A couple of IL-12 muteins were used in our therapeutic molecules to reduce systemic toxicity. IL-12 has been known for a key orchestrator in immune response. The main actions of IL-12 include the induction of CD4+ Th0 cells toward Th1 type and enhancement of IFN-γ production, stimulation of cytotoxicity and growth of natural killer (NK) cells and CD8+ T cells. For these reasons, IL-12 has long been considered as a potential therapeutic molecule for treating cancers by enhancing immune activity toward tumor cells. However, systemic administration of IL-12 showed poor efficacy and severe adverse effects. With our therapeutic approach of tumor targeting and attenuated IL-12 mutein, we expect that our IL12-based TMEkine™ holds great promise for the future of cancer immunotherapy.In this study, we targeted CD-20 expressing cancers such as B-cell lymphoma with our anti-CD20/IL-12 mutein TMEkine. We evaluated the biological activity of our molecules with in vitro and in vivo efficacy and safety.MethodsThe target specific binding to CD20 and IL-12 receptor was analyzed by FACS and ELISA. Biological activities as signaling transduction and T cell activation were confirmed in vitro using HEKblue IL12 cell line, primary human T cells and NK cells. The anti-tumor efficacy of TMEkine (CD20-IL-12) was assessed in A20 lymphoma syngeneic mouse model. To demonstrate long term protection to A20, the cured five mice after TMEkine administration were re-challenged with A20 and 4T1 cells.ResultsFirst, we analyzed the specific binding of our TMEkine molecules to CD20 expressing B-cell lymphoma cell lines (such as Raji). We showed that TMEkine (CD20-IL-12) binds to Raji and Ramos, which express CD20, but not to Jurkat, which does not express CD20. We also showed that TMEkine molecules bind to IL-12 receptor in a dose-dependent manner. pSTAT4 alphaLISA assay revealed that TMEkine (CD20-IL-12) transduces STAT4 signaling. In our IL-12 mutein, key residues for heparin binding were mutated. The biological activity of our mutein molecule was attenuated due to this change in human PBMC. In addition, our TMEkine molecules significantly induced IFN-γ secretion from primary human T cells and NK cells. An A20 B-cell lymphoma syngeneic mouse model was utilized to investigate the anti-tumor activity of TMEkine (CD20-IL-12). TMEkine molecules were injected three times with Q3D intraperitoneally. Tumor growth was substantially reduced and no cytotoxicity was observed. To further investigate the underlying mechanism, we analyzed tumor infiltrating lymphocytes (TIL) and as expected, we observed the increase in the number of CD8+ T cells in TIL, compared to control group. Interestingly, our tumor re-challenge result demonstrates that TMEkine (CD20-IL-12) protected animals from tumor recurrence implying that immunologic memory response was generated upon our TMEkine (CD20-IL-12) treatment.ConclusionsAltogether, our data suggest that TMEkine (CD20-IL-12) as an efficacious tumor targeting cytokine opening up a new avenue for the treatment of B-cell lymphoma.

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Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽

10.1182/blood.v116.21.3941.3941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3941-3941

Author(s):

David M Goldenberg ◽

Serengulam Govindan ◽

Tom M Cardillo ◽

Robert M Sharkey

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies ◽

Anti Cd20 ◽

Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

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In Vivo Efficacy of Anti-CD20-Interferon-Gamma Fusion Protein Against Syngeneic B Cell Lymphoma Is Mediated By Natural Killer Cells

Blood ◽

10.1182/blood-2019-131890 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1575-1575

Author(s):

Alex Vasuthasawat ◽

Reiko E Yamada ◽

Kham R Trinh ◽

Neiki Rokni ◽

Sherie L Morrison ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

In Vivo Efficacy ◽

Anti Cd20

Background: The interferons, including IFNα/IFNβ (type I) and IFNγ (type II) are essential mediators of anti-cancer immunity. To achieve efficient targeting of IFNs to tumor sites, we have developed antibody (Ab)-IFN fusion protein technology. We previously reported the antigen-specific targeting of IFNα to CD20+ target cells with efficient inhibition of proliferation, induction of apoptosis, and in vivo tumor eradiation dependent upon IFNα receptors on the tumor cell surface (Xuan et al, Blood, 2010). A fusion protein targeting human CD20 (anti-CD20-IFNα) exhibited stronger direct anti-proliferative effects, complement-dependent cytotoxicity (CDC), Ab-dependent cell-mediated cytotoxicity (ADCC), and in vivo potency against B-cell lymphoma xenograft models compared to the parent Ab rituximab (Timmerman et al, Blood 2015). Based on these results, a phase I, first-in-human, dose-escalation trial of anti-CD20-IFNα for B cell non-Hodgkin lymphoma is now underway (NCT02519270). Given the distinct properties of IFNγ from type I IFNs, including upregulation of antigen presentation, control of immune cell trafficking, and activation of T cells, NK cells, and macrophages, we hypothesized that Ab-targeted IFNγ may have anti-tumor effects mechanistically-distinct from those of Ab-IFNα fusions. We now report on the construction and characterization of anti-CD20 fusions containing IFNγ. Methods: The VH and VL regions from antibody 2B8 recognizing human CD20 were engineered in recombinant form with mouse IgG2a constant regions, and fused at the C-terminus with mIFNγ, yielding anti-hCD20-mIFNγ. Tumor cell proliferation in vitro was measured by [3H]-thymidine incorporation, ADCC by LDH release using mouse splenocyte effectors, CDC by propidium iodide (PI) exclusion, and in vivo tumor growth assessed using the huCD20-expressing syngeneic mouse B cell lymphoma 38C13-huCD20. Tumor-infiltrating lymphocytes were measured by flow cytometry. Results: Anti-hCD20-mIFNγ displayed potent IFNγ bioactivity comparable to free recombinant mIFNγ, and suppressed the in vitro proliferation of 38C13-huCD20 lymphoma cells by up to 70% (at 1 nM), though not as potently as anti-hCD20-mIFNα, which inhibited proliferation by 98% (Figure 1). Anti-hCD20-mIFNγ showed enhanced ADCC against lymphoma cells compared with the unfused, parent antibody (16-20% at E:T ratio of 20:1, versus 9-12%, respectively, p=0.0024)(Figure 2), while CDC was identical to unfused antibody. In vivo efficacy was demonstrated in mice bearing established subcutaneous 38C13-huCD20 tumors, with systemic (i.v.) injection of 100 μg anti-hCD20-mIFNγ fusion protein on days 5, 6, 7, or 5, 6, 7, 9 after tumor inoculation resulting in cure of approximately 70-80% of mice in repeated experiments. In contrast, therapy with equimolar doses of unfused, native anti-hCD20 Ab resulted in no cures. Mechanistic studies in anti-hCD20-mIFNγ fusion protein-treated mice showed that depletion of natural killer (NK) cells (using anti-asialo-GM1) significantly abrogated tumor clearance (p=0.01), while depletion of macrophages (clodronate liposomes) had lesser, borderline effects (p= 0.05)(Figure 2), and depletion of complement (cobra venom factor) or T cells (CD4+ or CD8+) had no significant effects on tumor eradication. Subcutaneous mouse B cell lymphomas treated with intratumoral injections of anti-hCD20-mIFNγ displayed increased tumor-infiltrating CD8+ T cells (mean 20.6% versus 5% in PBS-treated controls, p=0.008), and CD4+ T cells (mean 15.3% versus 6.6%). Conclusions: Anti-hCD20-mIFNγ fusion protein has in vitro and in vivo efficacy in a syngeneic, immunocompetent model of B cell lymphoma, with NK cells and possibly macrophages implicated in the mechanism(s) of tumor eradication. Ab-targeted mIFNγ can also promote infiltration of immune cells into the tumor microenvironment. These findings may suggest a novel approach for the immunotherapy of B cell lymphomas and other cancers. Disclosures Vasuthasawat: Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Trinh:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Morrison:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Timmerman:ImmunGene: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support, Research Funding; Merck: Research Funding; Kite, A Gilead Company: Consultancy, Honoraria, Other: travel support, Research Funding; Spectrum Pharmaceuticals: Research Funding.

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Pluripotent stem cell–derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity

Blood ◽

10.1182/blood.2019000621 ◽

2020 ◽

Vol 135 (6) ◽

pp. 399-410 ◽

Cited By ~ 13

Author(s):

Huang Zhu ◽

Robert H. Blum ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Paul Rogers ◽

...

Keyword(s):

Stem Cell ◽

B Cell ◽

Nk Cells ◽

Pluripotent Stem Cell ◽

Nk Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

High Affinity ◽

Induced Pluripotent ◽

Anti Cd20

Abstract Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell–derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood–derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell–mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

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Antibody-Targeted Chemotherapy with Immunoconjugates of Calicheamicin: Differential Anti-Tumor Activity of Conjugated Calicheamicin Targeted to B-Cell Lymphoma Via B-Cell Lineage Specific Molecules CD19, CD20 and CD22.

Blood ◽

10.1182/blood.v104.11.2490.2490 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2490-2490 ◽

Cited By ~ 1

Author(s):

John F. DiJoseph ◽

Douglas C. Armellino ◽

Maureen M. Dougher ◽

Arthur Kunz ◽

Erwin R. Boghaert ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Targeted Chemotherapy ◽

Phase I Clinical Trials ◽

Anti Cd20 ◽

Anti Tumor Activity ◽

Acid Labile

Abstract Antibody-targeted chemotherapy using tumor-targeted immunoconjugates of the cytotoxic agent, calicheamicin, is a clinically validated strategy for the treatment of acute myeloid leukemia. Calicheamicin is a potent cytotoxic natural product that binds DNA in the minor groove and causes double strand DNA breaks. B lymphoid lineage-specific antigens CD19, CD20, and CD22 have been studied extensively as potential targets for therapeutic applications of immunotoxins. In order to determine which one of these three antigens is most suitable for antibody-targeted calicheamicin therapy, we conjugated monoclonal antibodies, BU12 (murine anti-CD19 mAb), rituximab (chimeric anti-CD20 mAb), and m5/44 (murine anti-CD22 mAb) to a hindered disulfide derivative of N-acetyl gamma calicheamicin and evaluated the anti-tumor activity of these conjugates against three human B-cell lymphoma lines (BCL), Ramos, Raji and RL. Each of these three mAb bound to their respective antigens on the surface of BCL and was modulated, indicative of their potential internalization. Immunoconjugates of these mAbs, prepared by covalently linking calicheamicin via either acid-labile or acid-resistant linkers, caused a potent inhibition of BCL growth in vitro (IC50s ranged from 7 pM for the acid-labile linked m544 up to 6.8 nM for the acid-resistant linked anti-CD20 conjugates of calicheamicin). Immunoconjugates with acid-labile linkers were more potent than their counterparts with the acid-stable linker and conjugates targeted to either CD19 or CD22 were more potent than those targeted to CD20 in inhibiting BCL growth in vitro. In contrast, unconjugated mAb to CD19 or CD22 had no effect on BCL growth in vitro whereas anti-CD20 mAb, at concentrations >1 μg/ml, had an inhibitory effect of 30% on in vitro BCL growth. When examined for their effects on the growth of established subcutaneous BCL xenografts in nude mice, calicheamicin conjugated to anti-CD22 was by far the most efficacious conjugate against each of the three BCL xenografts studied. Calicheamicin conjugated to rituximab caused significant inhibition of BCL growth but was less effective than the conjugates of anti-CD22 or anti-CD19 mAb. Interestingly, anti-CD19 conjugates of calicheamicin, while effective in vitro against both Raji and Ramos BCL and effective against Raji BCL xenografts, had no effect on the growth of Ramos BCL xenografts in vivo. The reasons underlying the lack of anti-tumor activity of CD19-targeted calicheamicin conjugate against Ramos xenografts in vivo remain unknown. Based on a number of factors including the potent and consistent anti-tumor activity of the anti-CD22-conjugated calicheamicin, CD22 was selected as the molecular target for further development. A calicheamicin conjugate containing an acid-labile linker of humanized anti-CD22 mAb, CMC-544, is currently being evaluated in phase I clinical trials in non-Hodgkin’s B-cell lymphoma.

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Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

Science Advances ◽

10.1126/sciadv.abd6167 ◽

2021 ◽

Vol 7 (8) ◽

pp. eabd6167

Author(s):

Capucine L. Grandjean ◽

Zacarias Garcia ◽

Fabrice Lemaître ◽

Béatrice Bréart ◽

Philippe Bousso

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Critical Path ◽

B Cell Lymphoma ◽

Antibody Dependent Cellular Cytotoxicity ◽

Fluorescent Reporter ◽

Cd20 Antibody ◽

Anti Cd20 ◽

Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

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Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach

PLoS ONE ◽

10.1371/journal.pone.0208709 ◽

2018 ◽

Vol 13 (12) ◽

pp. e0208709 ◽

Cited By ~ 2

Author(s):

Silvia Da Ros ◽

Luca Aresu ◽

Serena Ferraresso ◽

Eleonora Zorzan ◽

Eugenio Gaudio ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Epigenetic Mechanisms

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Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis

Journal of Experimental Medicine ◽

10.1084/jem.20110325 ◽

2012 ◽

Vol 209 (2) ◽

pp. 291-305 ◽

Cited By ~ 35

Author(s):

Likun Du ◽

Roujun Peng ◽

Andrea Björkman ◽

Noel Filipe de Miranda ◽

Cornelia Rosner ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Class Switch Recombination ◽

B Cell Lymphoma ◽

Dominant Negative ◽

Dominant Negative Effect ◽

End Joining ◽

Class Switch

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.

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