scholarly journals Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Berna Uygur ◽  
Kamran Melikov ◽  
Anush Arakelyan ◽  
Leonid B. Margolis ◽  
Leonid V. Chernomordik
Keyword(s):  
Cell Lines ◽  
Membrane Vesicles ◽  
Retroviral Vectors ◽  
Extracellular Vesicle ◽  
Marker Genes ◽  
Target Cells ◽  
Retroviral Transduction ◽  
Gfp Gene ◽  
Safety Test

AbstractRetroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Here, we show that human cells transduced to stably express GFP transfer GFP gene to non-transduced cells. This horizontal gene transfer was mediated by a fraction of extracellular membrane vesicles that were released by the transduced cells. These vesicles carried endogenous retroviral envelope protein syncytin 1 and essentially acted as replication-competent retroviruses. The ability to transfer the GFP gene correlated with the levels of syncytin 1 expression in the transduced cells and depended on the fusogenic activity of this protein, substantiating the hypothesis that endogenous syncytin 1 mediates fusion stage in the delivery of extracellular vesicle cargo into target cells. Our findings suggest that testing for replication-competent retroviruses, a routine safety test for transduced cell products in clinical studies, should be also carried out for cell lines generated by retroviral vectors in in vitro studies.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3221-3231
Author(s):  
R C Schwartz ◽  
L W Stanton ◽  
S C Riley ◽  
K B Marcu ◽  
O N Witte
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Retroviral Vectors ◽  
Soft Agar ◽  
Lymphoid Cells ◽  
Neoplastic Progression ◽  
Direct Role ◽  
Murine Bone Marrow ◽  
Myc Oncogene

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

Rapid selection of antigen-specific T lymphocytes by retroviral transduction

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Guenther Koehne ◽  
Humilidad F. Gallardo ◽  
Michel Sadelain ◽  
Richard J. O'Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cytotoxic T Lymphocyte ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Transduction ◽  
Alloreactive T Cells ◽  
T Cell Lines ◽  
Selection Of

Abstract Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT+T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT+ fraction relative to the untransduced T-cell population. NIT+ fractions transduced on day 8 possessed more CD4+ T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

scholarly journals Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

2020 ◽  
Vol 21 (15) ◽  
pp. 5249 ◽  
Author(s):  
Anne-Claire Lagrée ◽  
Fabienne Fasani ◽  
Clotilde Rouxel ◽  
Marine Pivet ◽  
Marie Pourcelot ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
In Vitro Studies ◽  
Microvascular Endothelial Cell ◽  
Target Cells ◽  
Microvascular Endothelial

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Exosome: from internal vesicle of the multivesicular body to intercellular signaling device

2000 ◽  
Vol 113 (19) ◽  
pp. 3365-3374 ◽  
Author(s):  
K. Denzer ◽  
M.J. Kleijmeer ◽  
H.F. Heijnen ◽  
W. Stoorvogel ◽  
H.J. Geuze
Keyword(s):  
Dendritic Cells ◽  
B Lymphocyte ◽  
Cytotoxic T Cells ◽  
Membrane Vesicles ◽  
Multivesicular Body ◽  
Cell Types ◽  
Target Cells ◽  
Intercellular Signaling ◽  
Accessory Cells

Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class-II-carrying exosomes that stimulate T cell proliferation in vitro. In addition, dendritic-cell-derived exosomes, when used as a cell-free vaccine, can eradicate established murine tumors. Although the precise physiological target(s) and functions of exosomes remain largely to be resolved, follicular dendritic cells (accessory cells in the germinal centers of secondary lymphoid organs) have recently been shown to bind B-lymphocyte-derived exosomes at their cell surface, which supports the notion that exosomes play an immunoregulatory role. Finally, since exosomes are derived from multivesicular bodies, their molecular composition might provide clues to the mechanism of protein and lipid sorting in endosomes.

In Vitro and In Vivo Efficacy of CD38 Directed Therapy with Daratumumab In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3058-3058 ◽  
Author(s):  
Richard W Groen ◽  
Michael van der Veer ◽  
Frans M Hofhuis ◽  
Berris van Kessel ◽  
Michel de Weers ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Target Cells ◽  
Human Antibody ◽  
Variable Degree ◽  
Expression Levels ◽  
Cd38 Expression

Abstract Abstract 3058 Daratumumab (DARA) is a fully human antibody against CD38, a membrane-associated antigen, which is considered to be a highly relevant target for the treatment of hematological malignancies such as multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). DARA has the ability to effectively kill target cells by CDC, ADCC and by induction of apoptosis. While CD38 is frequently overexpressed on MM cells, it has a variable degree of expression on the malignant CLL cells. To gain detailed insight into the potential therapeutic power of DARA in tumors with different CD38 expression levels, we now investigated the correlation between the level of CD38 expression and DARA-induced kill by CDC and ADCC. To this end, we first used the human MM cell lines L363 and UM9, expressing low and intermediate CD38 levels, respectively. While both cell lines could be lysed via ADCC, they were not susceptible to killing via CDC. To delineate a possible correlation between CD38 expression and induction of CDC, we lentivirally transduced the CD38 gene into L363 and UM9 cells to generate cell lines ranging in CD38 expression from 50,000 to 800,000 molecules per cell. Hereby we covered the range of CD38 expression found in CLL and MM patients. The L363 and UM9 cell lines were also transduced with the luciferase-GFP marker genes to enable their in vivo quantitative monitoring by bioluminescence imaging (BLI). In CDC assays, up to 95% lysis of CD38-transduced L363 and UM9 cells was achieved and showed an excellent correlation with the level of CD38 expression (r2=0.90). In ADCC assays, however, killing already reached maximum levels at DARA concentrations as low as 1 ng/ml at the lowest CD38 expression levels tested. These in vitro assays indicated that cell killing via CDC is enhanced with increasing CD38 expression levels, while CDC resistant, low CD38-expressing tumors could be targeted by DARA via ADCC. To explore this in an in vivo setting, we used a newly developed MM model in Rag2−/−gc−/− mice and we inoculated the mice with nontransduced, CD38low, UM9 cells, which can be lysed by ADCC but not by CDC. Treatment of the mice with DARA one day after tumor inoculation completely prevented the outgrowth of these UM9 cells; treatment of established UM9 tumours at week 3 resulted in a significant delay in tumor growth. These results confirmed that the CDC resistant UM9 could be readily targeted by DARA in vivo, possibly via ADCC mediated by monocytes in this model. In vivo experiments with variants that express high levels of CD38 are in progress. Taken together, these in vitro and in vivo data underscore the potency of DARA as a novel antibody for CD38-targeted therapy in hematological malignancies, and warrant further exploration in clinical trials. Disclosures: de Weers: Genmab bv: Employment. Parren:Genmab: Employment.

Antibody Engineering of Anti-TFPI Bypass Therapeutic BAY 1093884: Isotype Selection and Sequence Optimization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3496-3496 ◽  
Author(s):  
Pedro Paz ◽  
Jinger Xie ◽  
Fred Aswad
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
In Silico ◽  
Target Cells ◽  
T Cell Epitopes ◽  
Serum Complement ◽  
Healthy Donors ◽  
Bayer Healthcare

Abstract Background: BAY 1093884 is a fully human monoclonal antibody against tissue factor pathway inhibitor (TFPI) developed as a bypass agent for patients with hemophilia with or without inhibitors. It restores insufficient thrombin burst, leading to stable clot formation in hemophilic conditions in vitro, and effectively stops bleeding in vivo. Aims: Antibody targeting of TFPI provides the advantages of monoclonal antibodies, such as longer circulation half-life, and the possibility of subcutaneous administration. However, antibody therapeutics can present the challenge of delivering unwanted effects or activities, such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) that target healthy tissues in the patient. Additionally, antibody sequence engineering that optimizes binding to the target protein may introduce immunogenic epitopes that can induce antidrug antibodies and compromise efficacy. Methods: In vitro assays used primary human umbilical vein endothelial cells and EA-hy926 cell lines as target cells. Total peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells isolated from healthy donors were used as effector cells for ADCC. Pooled human serum complement (Quidel®) was used for CDC assays. A proprietary immunogenicity scoring program for T-cell epitopes (Bayer HealthCare) was used to determine immunogenic potential of BAY 1093884 relative to antibodies in the market. Results: In vitro ADCC assessment of BAY 1093884 using PBMCs and NK cells isolated from healthy donors showed no lytic activity against TFPI-expressing primary and immortalized cell lines. Furthermore, no CDC activity using pooled serum complement was detected with BAY 1093884. In silico scoring for T-cell epitopes using a human leukocyte antigen DR-weighted algorithm compared favorably to therapeutic antibodies that have been reported to have low immunogenicity in the clinic. Conclusion: In vitro and in silico immunoprofiling has aided in the design of the bypass anti-TFPI immunoglobulin G2 antibody BAY 1093884 with low toxicity potential and immunogenicity in humans. Disclosures Paz: Bayer HealthCare: Employment. Xie:Bayer HealthCare: Employment. Aswad:Bayer HealthCare: Employment.

Preclinical Evaluation of a Potent Anti-Bcma CD3 Bispecific Molecule for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 383-383 ◽  
Author(s):  
Siler H Panowski ◽  
Tracy Kuo ◽  
Amy Chen ◽  
Tao Geng ◽  
Thomas J Van Blarcom ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Half Life ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Human Igg

Abstract Multiple myeloma (MM) is a debilitating disease characterized by the abnormal accumulation of malignant plasma cells in the bone marrow. Despite recent advances in myeloma therapy, including proteasome inhibitors, immunomodulatory drugs, and targeted antibody therapies, patients relapse and the disease remains incurable and one of high unmet need. T cell redirecting therapies are a new and exciting class of therapeutics that harness the potent cytotoxic activity of T cells and redirect it to target tumor cells. T cell redirecting therapies are only as good as their targeted tumor associated antigen (TAA) and the potent nature of the therapy requires a lack of TAA expression in essential normal tissue. B-cell Maturation Antigen, BCMA, is a tumor necrosis factor superfamily member highly expressed on the surface of myeloma cells. Detectable normal BCMA tissue expression appears limited to plasmablasts and mature plasma cells, making it an ideal T cell redirecting target for the treatment of MM. Other groups have developed T cell redirecting therapies against BCMA, including CAR T and BiTE therapy (a short half-life CD3 bispecific). Here we present preclinical studies on a fully-human IgG CD3 bispecific molecule targeting BCMA (half-life in mice of ~3 days). This molecule utilizes anti-BCMA and anti-CD3 targeting arms paired through hinge mutation technology and placed in an IgG2A backbone. The molecule binds to BCMA-expressing myeloma cell lines and to T cells with affinities of 20pM and ~40nM, respectively. T cells co-cultured with MM cell lines were activated and de-granulated in the presence of BCMA bispecific. In vitro cytotoxicity assays revealed the high potency of the molecule, as it was able to drive lysis of MM target cells with an EC50 of 6± 8 pM (mean ± SD). We also observed strong in vitro potency with the BCMA bispecific in four different MM primary patient samples, EC50 =0.093±0.1 nM (mean ± SD). When the same four samples were targeted with a BCMA antibody drug conjugate (ADC), 3 of the samples gave EC50 values of 1.25±0.7 nM (mean ± SD) - i.e. a 43 fold decrease in potency compared to the CD3 bispecific. The fourth patient did not respond to the ADC. Together, these results illustrate the potential advantages of a CD3 bispecific over an ADC for targeting BCMA. In orthotopic, established, tumor mouse models utilizing three different MM cell lines, (OPM2, MM.1S and MOLP8), a single injection of BCMA bispecific effectively treated tumors in a dose-dependent manner. Re-dosing the bispecific was able to provide additional and prolonged efficacy. The extreme potency of T cell redirecting therapies results in outstanding efficacy, but can also lead to lysis of normal cells expressing even minute levels of target. The species cross-reactivity of the BCMA bispecific allowed for exploratory toxicity studies in cynomologus monkeys. The molecule was able to effectively deplete normal plasma B cells expressing low levels of BCMA, providing evidence of activity. Activity was accompanied by a cytokine spike following initial dosing. No cytokine release was observed following a second bispecific dose. Encouragingly, animals experienced no additional adverse events (AEs), confirming the favorable safety profile of BCMA as a target for MM. In summary, we report on a fully human IgG CD3 bispecific molecule targeting BCMA for the treatment of multiple myeloma. Our BCMA bispecific is expected to have an antibody-like half-life in humans and, taken together, our findings support that the molecule has the potential to be both a potent and safe therapeutic. Disclosures Panowski: Pfizer Inc.: Employment. Kuo:Alexo Therapeutics: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Van Blarcom:Pfizer Inc.: Employment. Lindquist:Pfizer Inc.: Employment. Chen:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

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