ALBERT

Description: Abstract 609 HM1.24/CD317 or BST2, a cell surface protein highly expressed on malignant plasma cells, represents a potential target of immunotherapy for multiple myeloma (MM). Here we characterized XmAb®5592, a novel Fc-engineered and humanized anti-HM1.24 antibody (Ab), and studied mechanisms of its anti-MM activity. XmAb®5592, with double amino acid substitution in Fc region of the wild type IgG1, has approximately 40-fold and 10-fold increases in affinity for Fc gamma receptor III (FcγRIIIa) and (FcγRIIa), respectively, expressed on effector cells including NK cells. The Fv region of XmAb®5592 was humanized and engineered to achieve high affinity and specificity of binding to HM1.24-expressing target cells. XmAb®5592 reacts against a panel of MM cell lines (n=19) which are both sensitive or resistant to current anti-MM conventional and novel therapies. Importantly, it triggers 10-100-fold higher antibody-dependent cell-mediated cytotoxicity (ADCC) against these MM cell lines than a native/non Fc-engineered version (anti-HM1.24 IgG1) of the Ab. Specifically, the maximum specific lysis of MM1S, MM1R, and RPMI8226 target cells induced by XmAb®5592 is at a concentration of 0.001-0.01 μg/ml, whereas the IgG1 analog did not induce maximum cell lysis until 0.1 μg/ml. The maximum 100% specific lysis of INA-6 target cells occurred at 0.1 μg/ml of XmAb5592, in contrast to 60% maximum lysis induced by10 μg/ml of the IgG1 analog. Since the bone marrow (BM) microenvironment induces resistance in MM cells to conventional therapies, we next asked whether XmAb®5592 induced ADCC against MM cells even in the presence of BM stromal cells (BMSCs). Importantly, XmAb®5592 triggered significant ADCC against MM1S, MM1R, and INA-6 MM cells in the context of BMSCs. XmAb5592 also reacts against patient MM cells, and triggers robust ADCC against CD138-purified patient MM cells in assays using NK effector cells from normal donors. Furthermore, cross-linked XmAb5592 inhibited RMPI 8226 cell growth in the absence of effector cells. The in vivo efficacy of XmAb®5592 was next evaluated in murine subcutaneous (sc) xenograft murine models using RPMI 8226 cells. Administration of XmAb5592 (9mg/kg, ip, 2x/week for 4 weeks) led to a significant reduction in growth of established tumors in vivo compared to a non-engineered IgG1 anti-HM1.24 analog. At termination of the study. 7/15 mice were tumor free in the XmAb- treated group versus only 1/15 tumor free mice in the IgG1 analog treated group. An anti-HM1.24 antibody with Fc region engineered to completely ablate binding to FcγRs (knock-out) behaved equivalent to the PBS vehicle control in these studies, again underlining the significance of interaction with FcγR for anti-tumor efficacy. These results therefore suggest that XmAb5592, an anti-HM1.24 antibody engineered for improved effector function and antitumor potency in vitro and in vivo, is a promising next-generation immunotherapeutic for MM. Disclosures: Muchhal: Xencor: Employment. Horton:Xencor: Employment. Nguyen:Xencor: Employment. Karki:Xencor: Employment. Desjarlais:Xencor: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: XmAb2513 is a novel humanized monoclonal antibody (mAb) that binds to the human cell surface antigen CD30 and demonstrates anti-proliferative activity against CD30-positive (CD30+) cell lines. XmAb2513 also has an engineered Fc region to enhance cell killing activity via recruitment of effector cells through increased binding affinity to Fcγ receptors (FcγRs). Consequently, XmAb2513 exhibits superior antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), when compared to a native IgG1 (unengineered) version of the antibody. To evaluate the potential clinical activity of XmAb2513 in CD30+ diseases such as Hodgkin Lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL), XmAb2513 was tested in murine subcutaneous xenograft models of HL using the CD30+ L540 cell line. In the ICR-SCID mouse strain, intraperitoneal (ip) administration of XmAb2513 at 3 mg/kg every 4 days for 10 doses (q4d ×10), gave a statistically significant reduction in tumor growth and enhanced survival relative to the control. At doses of 10 and 30 mg/kg XmAb2513 (ip, q4d ×10) tumor growth was not only slowed, but elimination of established tumors was observed in 3/9 and 5/9 animals respectively. The treatment was well-tolerated. Preclinical studies were conducted to evaluate the safety and pharmacokinetics of XmAb2513 in large animals. In vitro studies demonstrated that the cynomolgus monkey was the appropriate species for study. Binding affinities of XmAb2513 to both human and cynomolgus monkey CD30 and FcγRs were evaluated by Biacore methods and were found to be similar. Additionally, fluorescein-XmAb2513 gave similar staining patterns in immunohistochemistry cross-reactivity studies with normal human and cynomolgus monkey tissue panels. As evidenced by in-life observations single (0, 1 and 100 mg/kg XmAb2513, intravenous [iv] infusion) and repeat dose (0, 10, 30 and 100 mg/kg XmAb2513, q5d ×6, iv infusion) treatment with XmAb2513 was well-tolerated. Serum cytokines showed no trend that was indicative of an XmAb2513-related effect following single dose administration. The pharmacokinetics of XmAb2513 was also determined after either single or repeat dose administration. In the repeat dose study (0, 3, 10 and 30 mg/kg XmAb2513, q5d ×6, iv infusion) exposure increased in a dose proportional manner, and terminal half-life (t1/2) ranged from 12–17 days. In the single dose study (0, 1 and 100 mg/kg XmAb2513, iv infusion) exposure was also proportional to dose. The exposure and t1/2 data support an every other week dosing interval in the clinic. These preclinical data provide a rationale for the clinical testing of XmAb2513 in patients with hematologic malignancies that express CD30, specifically HL and ALCL, and support the safety of repeat administration of XmAb2513 in humans.

Description: Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Description: Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p 〈 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Description: HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Description: CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40+ malignancies.

Description: XmAb®5574 is an Fc engineered humanized monoclonal antibody (mAb) that binds to the human cell surface antigen CD19 and demonstrates anti-proliferative activity against CD19-positive (CD19+) cell lines. XmAb5574 also has an engineered Fc region to enhance cell killing activity via recruitment of effector cells through increased binding affinity to Fcγ receptors (FcγRs). Consequently, XmAb5574 exhibits superior antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), when compared to a native IgG1 (non Fc-engineered) or an Fc knock out (Fc KO; engineered to ablate FcγR interaction) version of the anti-CD19 antibody. To evaluate the potential clinical activity of XmAb5574 in CD19+ B cell malignancies such as non-Hodgkin’s lymphoma (NHL), XmAb5574 was tested in murine subcutaneous (sc) xenograft models using the CD19+ Ramos and Raji human lymphoma cell lines. XmAb5574, administered by intraperitoneal injection (ip, 2qw ×2), gave dose-related inhibition of tumor growth with these models. The efficacy against established sc Ramos tumors was shown to be FcγR-dependent and enhanced by Fc engineering. Fc KO antibodies had no effect at the doses used. Preclinical studies were conducted to evaluate the safety and pharmacokinetics of XmAb5574 in non-human primates. In vitro studies demonstrated that the cynomolgus monkey was an appropriate species for study. XmAb5574 bound to a CD19-expressing cynomolgus monkey cell line and CD20+ peripheral lymphocytes from either cynomolgus monkey or human whole blood samples. Binding affinities of XmAb5574 to both human and cynomolgus monkey FcγRs were evaluated by Biacore methods and were found to be similar. Additionally, XmAb5574 gave similar staining patterns in immunohistochemistry cross-reactivity studies with normal human and cynomolgus monkey tissue panels. Single dose administration of XmAb5574 (0, 0.3, 1.0, 3.0, and 10.0 mg/kg, intravenous [iv] infusion) to cynomolgus monkeys gave an immediate and sustained depletion of peripheral B cells in a dose-dependent manner. B cells were reduced in the bone marrow and lymph node with the spleen showing involuted germinal centers and decreased CD20 immunostaining. B cell recovery, peripherally evident after 57 days, was observed in lymphoid tissues after 85 days. The native anti-CD19 IgG1 (non Fc-engineered) did not induce B cell depletion at 3 mg/kg, in contrast to almost complete B cell depletion by XmAb5574 at the same dose. The pharmacokinetics of XmAb5574 were determined in cynomolgus monkeys after a single iv infusion at 0.3, 1, 3, or 10 mg/kg. Blood samples were collected throughout the study, processed to serum, and XmAb5574 concentration determined using an ELISA method. Exposure was approximately dose proportional for the 1–10 mg/kg dose levels but decreased at the 0.3 mg/kg level indicating dose-dependent clearance for XmAb5574 in this species. Among the 1–10 mg/kg dose levels, the clearance and half-life ranged from 4.3–5.8 mL/day and 7.7–10.7 days, respectively. Single iv infusions of XmAb5574 (0.3–10 mg/kg) were well tolerated in cynomolgus monkeys. These preclinical data provide a rationale for the clinical testing of XmAb5574 in patients with B cell malignancies.

Description: CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family, is an attractive target for cancers of lymphoid origin since it is expressed on most mature B-cell malignancies, some early B-cell acute lymphocytic leukemias, and multiple myeloma. Finding efficient therapies for multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and rituximab-refractory Non-Hodgkin Lymphoma (NHL) represents an unmet need. Several anti-CD40 antibodies, both agonistic and antagonistic, have demonstrated objective responses in early clinical NHL trials and thus validated this antigen as a target for lymphoproliferative diseases. Here we present the characterization of a novel Fc-engineered and humanized anti-CD40 antibody, XmAb®5485, that was generated using our XmAb antibody engineering technology. This antibody is highly cytotoxic against lymphoma, leukemia and multiple myeloma cell lines as well as primary cancer cells. XmAb5485 is characterized by: i) increased affinity for Fc gamma receptors (FcgR), ii) improved effector function, and iii) significantly increased antitumor potency. We investigated several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity in vitro. The potency (EC50) of XmAb5485 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased up to 150-fold relative to the native non Fc-engineered version (anti-CD40 IgG1) of the antibody in a screen of Burkitt’s lymphoma [BL], CLL and MM-derived cell lines. In the same cell lines, ADCC potency and maximal efficacy (% lysis) of XmAb5485 were also superior to that of rituximab: 74- and 1.3-fold higher in CLL, 12.5- and 1.4-fold higher in BL, and 190- and 1.9-fold higher in MM. In a MM cell line with low density of CD40 expression (~3500 per cell) XmAb5485 facilitated efficient ADCC whereas anti-CD40 IgG1 was virtually ineffective. Furthermore, using a BL cell line (Ramos) XmAb5485 displayed antibody-dependent cellular phagocytosis (ADCP) with potency and efficacy increased relative to rituximab (15- and 1.6-fold) and anti-CD40 IgG1 (5- and 1.2-fold). XmAb5485 also exhibited anti-proliferative apoptotic activity that was similar to that of rituximab. Ex vivo, XmAb5485 mediated potent ADCC of multiple primary patient-derived CLL, MCL, and plasma cell leukemia (PCL, an aggressive form of MM) cells, with substantially increased potency and efficacy relative to rituximab; in contrast, anti-CD40 IgG1 displayed minimal or no activity in these primary tumor cells. In vivo, in an established large (210–350 mm3) sc Ramos tumor xenograft model, 6 mg/kg XmAb5485 cured 80% of mice of detectable tumors and displayed statistically significant superiority over anti-CD40 IgG1. In contrast, only 7% of animals in the rituximab cohort were cured. In summary, our data suggest that XmAb5485, an anti-CD40 Fc variant antibody engineered for increased effector function, is a promising next-generation immunotherapeutic for leukemias, lymphomas, and multiple myeloma.