Abstract: Abstract 1852 Poster Board I-878 Multiple myeloma patients suffer from infection related complications. Abnormalities in both cellular and humoral immune responses have been considered responsible. Patients have been routinely immunized with vaccinations to prevent infection related problems, however, efficacy of such vaccination in early or stable myeloma remains unclear. Previously, we have shown immunomodulatory and T cell co-stimulatory effects of lenalidomide, which can up-regulate cellular immune responses in myeloma. Based on these results we initiated a study to evaluate the efficacy of lenalidomide compared to placebo on the effect of Hepatitis B (HepB) vaccination in patients with monoclonal gamopathy of undetermined significance (MGUS), smoldering myeloma or stable multiple myeloma (MM) not requiring any therapy. Patients were randomized to lenalidomide or placebo for 14 days with HepB vaccination on day 8. They were given option for 2nd and 3rd HepB vaccinations at 1 month and 6 month. Primary objective was to evaluate antibody response to Hepatitis Surface antigen (HepBSAg) at 1 month after vaccination. We also measured HepBSAg-specific cellular immune responses using HepBSAg protein and HLA-A2 peptide. At the time of data analysis, the study remains blinded. Thirty two patients have completed their initial vaccination (25 MGUS and 7 MM), while 22 patients (16 MGUS, 6 MM) have completed 3 vaccinations with 6 months follow up. None of the 32 patients, with MGUS or MM, had antibody response to vaccination at 1 month; while after 3 vaccination only 30% patients (7 of 24) demonstrated antibody response to HepBSAg (titer values 128.4±36.4). This is significantly below responses reported in literature in healthy individuals (90%). Responses in patients with MGUS (4 of 16) were not significantly different than in patients with MM (3 of 6). No base line patient characteristics predicts responders vs. non-responders. We have further analyzed HepBSAg-specific T cell immune response by detecting the presence of pentamer-positive CD8 cells with HepB surface antigen-peptide in HLA-A2+ samples. Five of seven responders were HLA-A2 positive, and none of them showed T cell response to HbSAg following vaccination as detected by change in pentamer positive cells. Three patients showed T cell-proliferative responses to HepBsAg; one of which had long term response. None of the non-responders tested demonstrated proliferative response to HepBSAg. The randomization remains blinded at the moment and hence effect of lenalidomide on immune response is not available at the present time. These results have very high clinical significance. It suggests that even in MGUS there is significant and profound functional immune suppression. Strategies to prevent infection and improve immune responses needs to be developed for both preventative purposes as well as for anti-MM vaccinations. Disclosures: Laubach: Novartis: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Abstract: We have previously shown that endonuclease activity is deregulated in myeloma and suppression of base excision repair (BER) associated apurinic/apyrimidinic endonuclease (APE) activity, mediated chemically or transgenically, reduces homologous recombination (HR) and genomic instability in multiple myeloma (MM). The purpose of this study was to investigate the role of BER-specific AP nucleases APE1 and APE2, separately or together, in the activation of HR pathway following exposure of MM cells to different DNA damaging agents and unravel possible mechanism/s and translational significance of this cross talk between two repair pathways in MM. We transduced MM cells with lentivirus-based shRNAs, either control (CS) or those targeting APE1, APE2, or both (APE1/2; double knockdown) and selected the transduced cells in puromycin. Knockdowns were confirmed by Western blotting and Q-PCR. Using evaluation by Q-PCR we observed that whereas APE2 was suppressed by 80% in APE2- as well as double-knockdown cells, it was upregulated by 70% in APE1 knock down cells. These data indicate that certain level of AP nuclease activity is probably required by MM cell to function and is consistent with a 25-30% reduced proliferation rate of double-knockdown cells under spontaneous condition. To study the impact of these modulations on ability of cells to activate HR-mediated repair pathway in response to DNA damage, the cells were exposed to either UV (20 J/m2) and incubated for 2 and 48 hrs or melphalan (2.5 µM) treatment for 24 hrs, and then incubation for further 1 and 24 hrs and evaluated for RAD51 and γ-H2AX foci. Following UV treatment, RAD51 foci were detected in 91%, 48%, 49%, and 28% of cells transduced with control, APE1, APE2, or both shRNAs, respectively. Similary melphalan treatment induced RAD51 foci in 76% of control shRNA transduced cells whereas only in 46%, 47%, and 27% of APE1, APE2, and APE1/2-knockdown cells. These data show that AP nuclease activity is involved in DNA damaging agent-induced activation of HR repair pathway. Impact of the suppression of AP nucleases was also assessed on cell proliferation at 48 hrs after treatment with melphalan. Viability of cells lacking APE1, APE2, and APE1/2 relative to control shRNA-transduced cells was reduced by 28%, 26%, and 43% (P 〈 0.00005), respectively, within 48 hrs of treatment. In summary, we show that: 1) AP nuclease activity plays a critical role in the activation of HR-mediated DNA repair and survival of MM cells following DNA damage; 2) Although suppression of APE1 or APE2 alone does not significantly affect spontaneous proliferation rates, simultaneous suppression of both reduces proliferation by ∼25-30%; 3) Suppression of APE1 leads to induction of APE2, indicating that certain level of AP nuclease activity (from either APE1 or APE2) is required by MM cell to function and is consistent with the reduced proliferation rate of double-knockdown cells; 4) Simultaneous suppression of both AP nucleases impairs the activation of HR repair following DNA damage. These data combined with our previous observations conclude that AP nucleases (APE1 and APE2) play critical role in HR-mediated repair and survival of MM cells following DNA damage and are important targets to reduce genomic instability as well as to sensitize MM cells to radio/chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 3986 B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p 〈 0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p 〈 0.05) and in the BM samples (11±4.8, N=4, p 〈 0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM. As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p 〈 0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations. Disclosures: Ghobrial: Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Abstract: We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Abstract: Abstract 4416 We have shown that telomerase activity is significantly elevated in multiple myeloma (MM) cell lines and primary cells, and inhibition of telomerase over a period of 3–5 weeks induces telomere shortening and growth arrest in these and other cancer cells. We recently reported our novel findings, indicating the role of telomerase in repair of DNA breaks and genome maintenance in MM cells. Double-strand breaks (DSBs) in genome are deleterious because if left unrepaired, they may lead to aberrant DNA recombination, instability, or cell death. Using phosphorylated-H2AX staining (a marker for DNA breaks) and the comet assay, a sensitive gel-based technique for detection and assessment of DNA breaks in individual cells, we have shown that telomerase inhibition leads to significantly increased DNA breaks in MM cells. Here we present the direct evidence of repair of DNA breaks by telomerase, in a plasmid substrate. The plasmid (with Kanr) was linearized in a way that cut-site had limited homology with telomeric sequence. The linearized and a circular plasmid (with Ampr; used as internal control) were incubated with myeloma cell extracts in the presence or absence of telomerase inhibitor and transferred to e.coli. The ratio of Kanr and Ampr colonies indicated the extent of repair and re-circularization of linearized plasmid by telomerase in the cell extracts. In two independent experiments, the inhibition of telomerase led to 60% decrease in the repair of DNA breaks in the plasmid substrate. To investigate if the substrate DNA needs to have a specific homology to telomeric sequence, we replaced the TS substrate in Telomerase Detection kit by various substrates designed by us, ending with TTA, TAG, AGG, and GGG. All sequences were utilized by telomerase with similar efficiency and the products of enzyme activity seen on a gel. To investigate the functional significance of these observations, myeloma cells were cultured in the absence or presence of telomerase inhibitor and cell aliquots were collected weekly for three weeks. An aliquot of cells was saved in the beginning of experiment, to be used as (day 0) control. DNA from cultured and day 0 cells was analyzed by SNP6.0 arrays (Affymetrix) and genomewide changes in copy number in cultured cells were identified, using genome of day 0 cells as baseline. Inhibition of telomerase was associated with 43% and 55% increase in the acquisition of copy number changes throughout genome in U266 and RPMI cells, respectively. In both myeloma cell lines as well as other cancer cells tested by us, the inhibition of telomerase was associated with 40% to 50% decrease in the amplification events, whereas 120% to 320% increase in deletions throughout genome, relative to untreated control cells. These data indicate that telomerase-mediated repair prevents copy number changes, especially the deletion events, associated with genomic instability in myeloma cells. As a positive control, the double-stranded breaks were also induced by restriction enzyme; this led to genomewide increase in copy number changes, mostly the deletions. Thus, DNA breaks, whether produced by telomerase inhibition or restriction enzyme, increase the deletion events throughout genome. This is also consistent with our previous observations showing increased instability of Alu sequences following telomerase inhibition. We conclude that telomerase contributes to survival of myeloma cells, not only by preventing telomere attrition, but also the repair of DNA breaks. Inhibition of telomerase, therefore, may increase the efficacy of chemotherapeutic agents targeting DNA repair. Disclosures: No relevant conflicts of interest to declare.

Abstract: Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 456 We have previously demonstrated that IL-17 producing TH17cells, a new subset of T helper cells, are significantly elevated in peripheral blood and bone marrow (BM) from patients with multiple myeloma (MM) and IL-17 produced by these cells promotes MM cell growth and survival, suppresses immune responses and induces osteoclast differentiation both in vitro and in vivo. Based on these observations we have investigated the effects of human anti-IL-17A monoclonal antibody (mAb), AIN-457, in MM. We observed that whereas IL-17A induced proliferation of MM cells (+30.7+2.7%) compared to control; anti-IL-17A mAb AIN-457 significantly inhibited MM cell growth both in presence and absence of BM stromal cells, as measured by thymidine incorporation (−18.7+1.5% and −22.7+2.6% respectively). We have further confirmed these inhibitory effects of anti-IL-17A antibody using MM cell colony forming assay with MethoCult agar plates. While presence of IL-17A increased the colony number from 80 in control plates to 188, presence of AIN-457 reduced the colonies to 〈 40 per unit area (p 〈 0.01). Evaluating the mechanism of action, IL-17A induced IL-6 production (+289.6+38%; p 〈 0.01); while AIN457 significantly down-regulated IL-6 production (−25+7%; p 〈 0.05) in MM-BMSC co-culture. We also observed that AIN-457 significantly reduced adhesion of MM cells to stromal cells (27%, p=0.011). AIN457 significantly inhibited IL-6 production in human fetal bone chips in the presence of MM cells within 24 hours of ex-vivo culture (control − 487+39 pg/ml; IL-17 990+27 pg/ml; p 〈 0.01 and AIN-457 − 326+7 pg/ml; p 〈 0.01). Since IL-17A plays a critical role in bone damage, we further evaluated the effect of this mAb on the generation of osteoclasts. When normal BM cells were cultured for three weeks in osteoclast supporting medium, presence of AIN-457 significantly inhibited TRAP+ multinucleated osteoclast cell numbers by 〉 60%. We next evaluated the efficacy of AIN-457 in vivo in the murine models of human myeloma; in the subcutaneous MM xenograft model, we observed significant reduction in tumor volumes by pre-treatment with AIN457 compared to control (142+77 mm versus 355+56 mm, p=0.019) while IL-17A significantly increased MM cell growth (727+135 mm, p=0.01). More importantly in the SCIDhu model of human myeloma where MM cells grow within the human microenvironment in the mice, administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth as measured by human sIL-6 receptor compared to control mice (5.9±2.2 ng/ml versus 23.2±6.3 ng/ml; n=7; P 〈 0.01). These pre-clinical in vitro and in vivo observations confirm the role of IL-17A produced by TH17 cells in MM and provide the rationale for clinical evaluation of AIN 457 for both anti-myeloma effects as well as to improve bone disease in myeloma. Disclosures: No relevant conflicts of interest to declare.

Abstract: Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)–hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144–52)

Abstract: Introduction: Current therapies for multiple myeloma (MM), such as immunomodulatory agents, proteasome inhibitors, stem-cell transplantation, and monoclonal antibodies against tumor-associated antigens have greatly improved patient survival. However, MM remains an incurable disease as most patients will eventually relapse. Recent advances in targeted T-cell therapies have shown promise in clinical trials but the adaptive immune system may be insufficient to eradicate all MM clones. In contrast, treatments harnessing the innate immune system have been relatively underdeveloped in MM despite evidence suggesting a role of innate immunity in the efficacy of existing therapies. Innate or innate-like cells, such as NK and γδ T cells, have the potential to display strong anti-tumor activity, and strategies aimed to improve or re-direct their cytotoxicity represent a new opportunity for cancer immunotherapies and a complementary approach to existing therapies. Here we describe the preclinical characterization of CTX-8573, a novel multispecific antibody that targets B-cell maturation antigen (BCMA) on tumor cells and promotes potent cytotoxicity by NK and γδ T cells through engagement of the activating receptors NKp30 and CD16a. Method: Bispecific constructs were generated by appending two common-light chain compatible anti-NKp30 Fab fragments to the C-terminus of an anti-BCMA IgG1 antibody containing an afucosylated Fc for enhanced CD16a engagement. To test the effects of targeting NKp30 alone, variants were expressed with an aglycosylated Fc to eliminate CD16a binding. In-vitro assays were performed with primary NK or γδ T cells to determine innate-cell activation, cytokine production, proliferation, and target-cell cytotoxicity against tumor cell lines with a range of BCMA expression levels. In-vivo efficacy studies were performed in multiple humanized mouse models and pharmacokinetics and safety were evaluated in Cynomolgus monkeys. Results: CTX-8573 is highly expressed in CHO cells with minimal aggregation and displays stability, solubility, and binding to BCMA and NKp30 equivalent to the parental monoclonal antibodies. By engaging NKp30 and CD16a, CTX-8573 promotes potent cytotoxicity of BCMA expressing target cells by NK and γδ T cells with 〉 100 fold reduced EC50 compared to the corresponding BCMA monoclonal antibody control. CTX-8573 also demonstrates robust killing of low BCMA expressing cell lines including RPMI-8226 where monoclonal BCMA antibodies lack activity. An aglycosylated variant of CTX-8573 lacking CD16a binding maintains cell killing activity, demonstrating that engagement of NKp30 alone is sufficient to promote innate cell activation and cytotoxicity, although activity is enhanced by CD16A engagement. Furthermore, CTX-8573 maintains its cytotoxic activity in presence of soluble BCMA or BCMA ligands APRIL and BAFF. CTX-8573 does not induce innate cell activation, cytokine production, or killing in the absence of BCMA expressing target cells, supporting a wide therapeutic window. Additionally, unlike daratumumab, CTX-8573 does not induce NK-cell fratricide. In-vivo, CTX-8573 demonstrates anti-tumor efficacy in multiple humanized mouse models including killing of low BCMA expressing cell lines. In Cynomolgus monkeys, CTX-8573 displays standard biphasic pharmacokinetics with a 16 day β-phase half-life and has no evidence of systemic immune activation as measured by C-reactive protein levels. Lastly, NKp30 expression is maintained on bone marrow NK cells from MM patients including the presence of a significant NKp30+CD16a- subpopulation. Conclusion: CTX-8573 represents a novel class of bispecific antibodies that promote potent tumor cell killing by NK and γδ T-cells through engagement of the activating receptors NKp30 and CD16a. CTX-8573 demonstrates strong anti-tumor efficacy in vitro and in vivo, a wide therapeutic window with no evidence of systemic toxicity, and monoclonal-like pharmacokinetics and manufacturability. Together, these data highlight the potential of CTX-8573 as a novel treatment for MM either alone or as a complement to existing therapies. Disclosures Watkins-Yoon: Compass therapeutics LLC: Employment, Equity Ownership. Guzman:Compass therapeutics LLC: Employment, Equity Ownership. Oliphant:Compass therapeutics LLC: Employment, Equity Ownership. Haserlat:Compass therapeutics LLC: Employment, Equity Ownership. Leung:Compass therapeutics LLC: Employment, Equity Ownership. Chottin:University of Louisiana at Lafayette: Employment. Ophir:Compass therapeutics LLC: Employment, Equity Ownership. Vekeria:Compass therapeutics LLC: Employment, Equity Ownership. Nanjappa:Compass therapeutics LLC: Employment, Equity Ownership. Markrush:Compass therapeutics LLC: Employment, Equity Ownership. McConaughy:Compass therapeutics LLC: Employment, Equity Ownership. Wang:Compass therapeutics LLC: Employment, Equity Ownership. Schilling:Compass therapeutics LLC: Employment, Equity Ownership. Kim:Compass therapeutics LLC: Employment, Equity Ownership. Wu:Compass Therapeutics LLC: Employment, Equity Ownership. Liu:Compass therapeutics LLC: Employment, Equity Ownership. Rogers:University of Louisiana at Lafayette: Employment. Villinger:University of Louisiana at Lafayette: Employment. Gong:Compass therapeutics LLC: Employment, Equity Ownership. Hamilton:Compass therapeutics LLC: Employment, Equity Ownership. Bobrowicz:Compass therapeutics LLC: Employment, Equity Ownership. Schuetz:Compass therapeutics LLC: Employment, Equity Ownership. Schmidt:Compass therapeutics LLC: Employment, Equity Ownership. Draghi:Compass therapeutics LLC: Employment, Equity Ownership.