scholarly journals Preclinical Mechanistic Studies Investigating Neutrophil and Lymphoid Cell Depletion By IMGN529, a CD37-Targeting Antibody-Drug Conjugate (ADC)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3119-3119 ◽  
Author(s):  
Jutta Deckert ◽  
Jose F. Ponte ◽  
Jennifer A. Coccia ◽  
Leanne Lanieri ◽  
Sharon Chicklas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Peripheral Blood Cells ◽  
Employment Equity ◽  
Normal Human ◽  
Equity Ownership

Abstract CD37 is a surface antigen widely expressed on malignant B cells in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues, CD37 expression is restricted to lymphoid tissues and blood cells, with high levels of expression on B lymphocytes and low levels on non-B lymphoid and myeloid cells. IMGN529 is a CD37-targeting ADC currently in a Phase I clinical study in adult patients with relapsed or refractory NHL (NCT01534715). This ADC uniquely combines the intrinsic pro-apoptotic and immune effector activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by targeted delivery of its maytansinoid payload, DM1. In the Phase I study, IMGN529 has demonstrated early evidence of clinical activity. A reduction in lymphocyte counts was also observed in the majority of patients after dosing, consistent with the proposed mechanism of action of a CD37-targeted therapy. However, in the initial dose-escalation phase, some patients experienced transient, early-onset neutropenia. To investigate the potential mechanisms of this transient neutropenia observed in patients, different pre-clinical models were considered and utilized to recapitulate clinical findings. In vitro studies with peripheral blood cells from normal human donors demonstrated that incubation with IMGN529 for 1 hour or 24 hours resulted in significant B-cell depletion with no apparent neutrophil depletion detected, similar to observations after rituximab treatment. In contrast, alemtuzumab treatment in vitro resulted in both B-cell and neutrophil depletion. This is consistent with the high level of CD37 expression on target B cells and the relatively low CD37 expression level on other blood cells. Analysis of cytokine release by normal human donor peripheral blood cells incubated with IMGN529 revealed increased levels of IL-8, CCL2 (MCP-1) and CCL4 (MIP-1β), but not IL-6 or TNF, to a similar extent as rituximab but less pronounced than alemtuzumab. An anti-murine CD37 antibody was identified to enable in vivo studies in a murine model and characterize CD37 expression on murine blood cells. Similar to the expression profile of CD37 in human peripheral blood cells, CD37 expression on murine peripheral blood cells was highest in B cells, with much lower expression seen on T cells and granulocytes. In vivo activity of the anti-muCD37 antibody and the corresponding ADC, with the same SMCC-DM1 linker-payload combination as IMGN529, was evaluated to discern antibody and payload-mediated events in comparison to the classic cytotoxic cyclophosphamide (CPA). Treatment of C57/B6 mice with 1-10 mg/kg of anti-muCD37 antibody or anti-muCD37 ADC resulted in a significant decrease in absolute lymphocyte counts (ALC) lasting greater than 7 days and a transient decrease in absolute neutrophil counts (ANC) lasting 1-2 days. A non-targeted control SMCC-DM1 ADC had no effect on ALC or ANC counts, suggesting the decrease is a CD37-mediated effect. In contrast, treatment with CPA resulted in an ALC decrease with similar kinetics but a more pronounced ANC decline. No impact on bone marrow lymphocyte, myeloid or erythroid precursor cell counts was observed in response to the anti-muCD37 antibody or anti-muCD37 ADC, whereas CPA treatment caused reduced cellularity with a decrease in the percentage of mature myeloid precursors and neutrophils in bone marrow. Elevated levels of CCL2 and CCL4 chemokines were detected in mouse plasma after anti-muCD37 ADC treatment, which may contribute to a redistribution of circulating neutrophils into peripheral tissues. Studies are currently underway to assess neutrophil distribution in murine tissues post anti-muCD37 ADC treatment. Current preclinical studies provide no clear evidence for direct IMGN529-mediated depletion of normal human neutrophils in the context of B-cell depletion in vitro. In vivo studies with an anti-muCD37 ADC recapitulate transient peripheral lymphopenia and neutropenia with no impact on bone marrow precursors observed, indicative of a different mechanism than classic chemotherapy-induced bone marrow myelosuppression. These preliminary results suggest a role for chemokine-mediated neutrophil redistribution following CD37 engagement, which is the subject of further studies. Disclosures Deckert: ImmunoGen, Inc.: Employment, Equity Ownership. Ponte:ImmunoGen, Inc.: Employment, Equity Ownership. Coccia:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Chicklas:ImmunoGen, Inc.: Employment, Equity Ownership. Yi:ImmunoGen, Inc.: Employment, Equity Ownership. Watkins:ImmunoGen, Inc.: Employment, Equity Ownership. Ruiz-Soto:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

Allogeneic Donor NK Cell Infusion and IL-2 for Patients with Refractory B-Cell Non-Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3036-3036
Author(s):  
Veronika Bachanova ◽  
Linda J. Burns ◽  
David H. McKenna ◽  
Julie Curtsinger ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Peripheral Blood Cells ◽  
Non Hodgkin Lymphoma ◽  
Pre Treatment

Abstract Abstract 3036 Poster Board II-1012 The potential role of allogeneic natural killer (NK) cells for therapy of refractory lymphoma is supported by the curative potential of allogeneic transplantation for lymphoid malignancies. Haploidentical donor derived NK cells may overcome Class I MHC Ag mediated inhibition and deliver an NK versus lymphoma effect. In a Phase II study we evaluated allogeneic NK cell infusions with Rituximab and IL-2 in a non-transplant setting to determine the expansion of NK cells in vivo and the clinical response in patients with refractory B-cell non-Hodgkin lymphoma (NHL). Six patients with advanced NHL received conditioning with Rituximab 375mg/m2 days -8,-1,+6,+15; Cyclophosphamide 60 mg/kg IV day -5; Fludarabine 25 mg/m2 IV days -6 through -2 as immunosupression to permit homeostatic expansion of allogeneic donor NK cells. Peripheral blood cells were obtained by lymphapheresis from unmobilized, HLA-haploidentical donors and selected for “killer immunoglobulin receptor” (KIR) ligand mismatch when available (3 out of 6 patients). Donor peripheral blood cells were enriched for NK cells with the Miltenyi CliniMACS device by depletion of T (CD3+) cells. The donor NK cells were then activated by overnight incubation with IL-2 (1,000 U/mL) and infused at a median nucleated cell dose of 2.27 ±0.4 × 107/kg. Subcutaneous IL-2 10×106 units (qod x 6 doses) was given to facilitate NK cell survival and expansion. All patients were evaluable for toxicity and efficacy. Patients tolerated the NK infusion well with only transient grade 1-2 toxicity and 5 received all 6 scheduled doses of IL-2. IL-2 activated donor NK cell products showed > 55% cytotoxicity against K562 targets. After IL-2 therapy, we observed a median absolute lymphocyte count of 980 ±440/μL. All cells were of recipient origin with no detectable donor NK cells. Importantly, in all patients the median number of host regulatory T cells (T regs phenotype CD4+Foxp3+CD127−) post treatment was significantly increased compared to pre-treatment (day 14 T regs: 134 ±141 cells/μL versus pre-treatment T regs: 24 ±12 cells/μL; P=0.06). To investigate the possibility of NK trafficking to affected lymph nodes, we performed fine needle aspiration of palpable tumor in 1 patient and demonstrated a low level of donor DNA by RFLP testing (2.5% donor chimerism). Simultaneous absence of NK cells in peripheral blood in the same patient suggested NK cell tissue homing to lymphoma-bearing nodes. Three patients achieved a partial remission (PR), one of whom proceeded to non-myeloablative cord blood allograft 2 month after NK cell infusion; two remain in partial remission after 1 and 4 months of follow-up. The trial failed to achieve prospective statistical parameters established to detect circulating NK cell expansion rate and will be modified. Conclusions This “proof of principle” study demonstrated lack of in vivo expansion of haploidentical NK cells in peripheral blood of patients with lymphoma. However, we identified host factors that interfered with NK cell expansion, including T reg proliferation and possibly inadequate immunosupression, and additionally, the finding of donor DNA in sites of tumor suggested donor NK cell localization to extravascular or tumor sites. Novel approaches to adoptive NK cell therapy trials should incorporate strategies to eliminate or prevent T reg expansion using alternate lymphodepleting regimens. Disclosures No relevant conflicts of interest to declare.

Comparison of cytarabine, mitoxantrone, and CNDAC in vitro treatment of AML bone marrow and peripheral blood cells.

2011 ◽  
Vol 29 (15_suppl) ◽  
pp. 6588-6588
Author(s):  
S. Jagan ◽  
L. A. Paganessi ◽  
S. Gezer ◽  
A. Rizman ◽  
D. Rifai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Peripheral Blood Cells ◽  
In Vitro Treatment

scholarly journals An All Antibody Approach for Conditioning Bone Marrow for Hematopoietic Stem Cell Transplantation with Anti-cKIT and Anti-CD47 in Non-Human Primates

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4428-4428
Author(s):  
Kristopher D Marjon ◽  
James Y Chen ◽  
Jiaqi Duan ◽  
Timothy S Choi ◽  
Kavitha Sompalli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Mast Cell Degranulation ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo

2009 ◽  
Vol 69 (4) ◽  
pp. 1141-1147 ◽  
Author(s):  
JC. Ribeiro ◽  
SF. Andrade ◽  
JK. Bastos ◽  
EL. Maistro
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Chromosome Aberrations ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Chloroform Fraction ◽  
Peripheral Blood Cells ◽  
Swiss Mice ◽  
Marrow Cells

The genotoxic effect of the Austroplenckia populnea chloroform fraction from barkwood extract was tested in vivo on peripheral blood cells of Swiss mice with the comet assay (SCGE), and the clastogenic effect was investigated on peripheral blood cells of Swiss mice and bone marrow cells of Wistar rats, with the micronucleus and chromosome aberrations tests. The animals were treated by gavage with 3 concentrations of the extract: 300, 600 and 900 mg.kg-1. Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment to the SCGE assay and 48 and 72 hours to the micronucleus test. Bone marrow cells of Wistar rats were collected 24 hours after the treatment to the micronucleus and chromosome aberration tests. The results showed that the A. populnea chloroform fraction induced an increase in the average number of DNA damage in peripheral blood cells at the three concentrations tested, but this increase was not statistically significant. In the micronucleus and chromosome aberrations test, no significant increase was observed in the mean number of micronucleated polychromatic erythrocytes (MNPCE) of Swiss mice or MNPCE or chromosome aberrations for the rat bone marrow cells, for any of the tested doses. Our findings enable us to conclude that by the comet assay, A. populnea chloroform fraction from barkwood extract showed no genotoxic effects, and by the micronucleus and chromosome aberration tests, the extract fraction showed no clastogenic/aneugenic effects on the rodent cells tested.

PERIPHERAL BLOOD PLASMACYTOSIS FOLLOWING SYSTEMIC EXPOSURE TO PHYTOLACCA AMERICANA (POKEWEED)

PEDIATRICS ◽  
1966 ◽  
Vol 38 (3) ◽  
pp. 490-493
Author(s):  
B. E. Barker ◽  
P. Farnes ◽  
P. H. LaMarche
Keyword(s):  
Peripheral Blood ◽  
Blood Cells ◽  
Plasma Cells ◽  
Human Peripheral Blood ◽  
Systemic Exposure ◽  
Peripheral Blood Cells ◽  
Phytolacca Americana ◽  
Oral Ingestion

MITOGENIC properties of extracts from Phytolacca americana (pokeweed, scoke, inkberry) for human peripheral blood cells in vitro have been reported from this laboratory. Subsequently, the appearance of leukocytes typical of early and late members of the plasmacytic series was described in the peripheral bloods of two of the authors (P.F. and B.E.B.), who received accidental systemic exposure to the mitogen. Effects of phytomitogens on human cells in vivo are not established, although a number of investigators have administered phytohemagglutinin from Phaseolus vulgaris to patients with aplastic anemia, hoping that the "transformed" lymphocytes might possess hematopoietic potentialities and repopulate the marrow with useful cells. Interpretations of such studies have been complicated by the small numbers of patients studied, the natural course of this group of anemias, simultaneous use of other therapy, and other variables. More information has been accumulated about the effects of phytomitogens on peripheral blood cells in vitro. The process of "transformation" or blastogenesis of lymphocytes, whether induced by plant extracts or specific antigenic stimuli, appears to involve an immune mechanism, although morphologic differentiation toward the plasmacytic series is not seen in the in vitro environment. Recently, we have had the opportunity to study serial blood films from children who received systemic exposure to pokeberry, either through proven oral ingestion (berries recovered from vomitus), or by exposure of fresh cuts and abrasions to pokeberry juice in the course of handling the berries. In each of these instances large cells morphologically typical of plasmablasts and proplasmacytes, and mature plasma cells were found in the peripheral blood films for periods up to 2 weeks following exposure. Mitotic cells were present in the peripheral blood during the first 10 days after exposure (Fig. 1 and 2).

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages 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 low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1731-1736 ◽  
Author(s):  
J ten Hoeve ◽  
RB Arlinghaus ◽  
JQ Guo ◽  
N Heisterkamp ◽  
J Groffen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Catalytic Domain ◽  
Lymphoblastic Leukemia ◽  
Adaptor Protein ◽  
Myelogenous Leukemia ◽  
Peripheral Blood Cells

Abstract The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.

scholarly journals Targeting CD45 with an Amanitin Antibody-Drug Conjugate Effectively Depletes Human HSCs and Immune Cells for Transplant Conditioning

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4526-4526
Author(s):  
Rahul Palchaudhuri ◽  
Bradley R Pearse ◽  
Jennifer L Proctor ◽  
Sharon L. Hyzy ◽  
Sharon Aslanian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Cells ◽  
Half Life ◽  
Immune Cell ◽  
Cell Depletion ◽  
Employment Equity ◽  
P Values ◽  
Equity Ownership

Abstract Introduction Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders and has demonstrated impressive outcomes in autoimmune diseases. Prior to BMT, patients are prepared with high-dose chemotherapy alone or with total body irradiation, and both are associated with early and late morbidities, such as infertility, secondary malignancies and organ toxicity; and substantial risk of mortality. This greatly limits the use of BMT in malignant and non-malignant conditions. To address these issues, we are developing antibody drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) and immune cells to more safely condition patients for BMT. Results To enable simultaneous HSC and immune cell depletion for BMT we investigated targeting human CD45, a protein expressed exclusively on nearly all blood cells including HSCs. Antibody discovery campaigns identified several antibodies with sub-nanomolar affinities for human and non-human primate (NHP) CD45. We then created anti-CD45 ADCs with drug payloads including DNA-damaging, tubulin-targeting and RNA polymerase-inhibiting molecules. An ADC developed with alpha-amanitin (an RNA polymerase II inhibitor) enabled potent in vitro killing of primary human CD34+ HSCs and immune cells (40-120 picomolar IC50s). With this anti-CD45 amanitin ADC (CD45-AM), we explored depletion of HSCs and immune cells in vivo using humanized NSG mice. A single dose of 1 or 3 mg/kg CD45-AM enabled >95% depletion of human CD34+ cells in the bone marrow as assessed 7 or 14 days post-administration (Figure, n = 3/group, p values < 0.05); >95% depletion of human B-, T- and myeloid cells was observed in the periphery and bone marrow (Figure, p values < 0.05). Control non-targeting isotype matched-ADCs and anti-CD45 antibody not bearing a toxin had minimal effect on either HSC or immune cells. In hematopoietic malignancies, an anti-CD45 ADC would ideally reduce disease burden and enable BMT. In a model of acute lymphoblastic leukemia (REH cell line, n = 10 mice/group), and 3 patient-derived models of FLT3+NPM1+ acute myeloid leukemia (n = 4-5 mice/group per model), a single dose of 1 mg/kg CD45-AM more than doubled the median survival and several mice survived disease-free (p values < 0.001). Anti-CD45 antibodies have been investigated for BMT conditioning in patients as naked antibodies that rely on Fc-effector function to deplete lymphocytes (Biol Blood Marrow Transplant. 2003 9(4): 273-81); or as radioimmunotherapy (Blood. 2006 107(5): 2184-2191). These agents demonstrated infusion-related toxicities likely due to effector function elicited by the wild-type IgG backbone. To address this issue, we created anti-CD45 antibodies with reduced Fc-gamma receptor binding that prevented cytokine release in vitro and in humanized mice. As BMT will likely require fast clearing ADCs to avoid depleting the incoming graft, we also created fast-half-life CD45-AM variants with a t½ of 8-15 hours in mice. To determine the safety and pharmacokinetic properties of regular and fast half-life Fc-silent variants in an immune-competent large animal we tested these in cynomolgus monkeys. Single doses (3 mg/kg, iv, n = 3/group) of fast and regular half-life Fc-silent unconjugated anti-CD45 antibodies were both well tolerated in cynomolgus monkeys and displayed pharmacokinetic properties suitable for BMT. Conclusion These results demonstrate that targeting CD45 with an amanitin ADC results in potent in vitro and in vivo human HSC and immune cell depletion. This new CD45-AM ADC also significantly reduced disease burden in multiple leukemia models. Our results indicate Fc-silencing may avoid infusion-related toxicities observed with previous CD45 mAbs. An alpha-amanitin ADC targeted to CD45 may be appropriate for preparing patients for BMT since we hypothesize it may i) be non-genotoxic; ii) effectively deplete both HSC and immune cells; iii) avoid bystander toxicity, due to amanitin's poor cell permeability as a free toxin; and iv) kill cycling and non-cycling cells, the latter being necessary for effective HSC depletion. As our anti-CD45 ADCs are cross-reactive, we are currently investigating their HSC and immune cell depletion activity in vivo in NHPs to enable further preclinical development of these transplant conditioning agents. Disclosures Palchaudhuri: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Bhat:Magenta Therapeutics: Employment. Ladwig:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Gabros:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Gillard:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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