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: CD30 (also known as Ki-1), a member of the TNF-receptor superfamily, is normally expressed at low levels on activated lymphocytes and has been implicated in cell death and T-cell proliferation. CD30 is highly expressed in Hodgkin’s disease, (HD) and in Anaplastic Large Cell Lymphoma (ALCL). Unmodified CD30 antibodies as well as anti-CD30 based bi-specific antibodies, antibody-toxin conjugates, and radioimmuno-therapeutics have examined CD30 as a therapeutic target in preclinical and clinical studies. Unmodified antibodies have met with limited success and a lack of engagement of immune effector cells may be one of the major short-comings. Although bispecific antibodies proved among the most clinically effective through the recruiting of host effector functions to tumor cells, they pose significant manufacturing challenges. Similarly, toxin- and radio-conjugates require complicated manufacture and handling. Recent advances in antibody engineering have led to the development of “naked” antibodies with greatly enhanced effector function through mutagenesis at the Fc-receptor binding interface. This approach has been applied to a humanized antibody specific for CD30 to produce an antibody with enhanced potency and efficacy coupled with the ease of manufacture and handling of a traditional IgG antibody. The murine:human chimeric antibody cAC10 was humanized using the novel method of human string content optimization. The humanized antibody (hAC10) has an affinity for antigen 4-fold higher than that of the corresponding chimeric antibody. The humanized variable domain was then combined with a modified Fc region and exhibited an approximately 20 fold increase in affinity for the FcγRIIIA receptor resulting in the therapeutic lead XmAb2513. Expression levels from a stable cell-line were close to 1 gm/L in preliminary development. The cytotoxic activity of XmAb2513 was measured by Antibody Dependent Cell-mediated Cytotoxicity (ADCC) assay. ADCC assays used PBMC’s isolated from peripheral blood as effector cells and the human Hodgkin’s cell line L540 which expresses high levels of CD30 as the target at an effector:target ratio of 25:1; cytotoxicity was measured by release of LDH or preloaded TDA. The activity of XmAb2513 was compared to that of cAC10 with no Fc-receptor binding enhancement (IgG1) as well as to the antibody 5F11 (also human IgG1). Significant improvements were observed in both potency (concentration of antibody required to effect 50% of maximal lysis) and efficacy (maximal percent lysis at saturating antibody concentration). The potency of XmAb2513 is ~3 fold higher than that of cAC10-IgG1 and 10-fold higher than 5F11 with an increase in efficacy of 4-fold relative to cAC10-IgG1. XmAb2513 has advantageous properties for a therapeutic compound against CD30-positive lymphomas including high levels of cytotoxicity and ease of manufacture and handling. The promising results reported herein clearly warrant further investigation.

Description: CD19, a B cell–restricted receptor critical for B-cell development, is expressed in most B-cell malignancies. The Fc-engineered anti-CD19 antibody, XmAb5574, has enhanced Fcγ receptor (FcγR) binding affinity, leading to improved FcγR-dependent effector cell functions and antitumor activity in murine xenografts compared with the non–Fc-engineered anti-CD19 IgG1 analog. Here, we use XmAb5574 and anti-CD19 IgG1 to further dissect effector cell functions in an immune system closely homologous to that of humans, the cynomolgus monkey. XmAb5574 infusion caused an immediate and dose-related B-cell depletion in the blood (to

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: FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status. Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo. AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1. Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. Disclosures Jin: Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.

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.