1 Dissociation Constant (KD) Mediates enhanced cytotoxicity PRECLINICAL PHARMACOLOGY OF XmAbTM2513 AN FC ENGINEERED HUMANIZED ANTI-CD30 MAB Phil Hammond, Greg Lazar, Sher Karki, David Carmichael, Seung Chu Xencor, Inc. 111 W. Lemon Ave., Monrovia, CA 91016AbstractXmAb™2513Was humanized with an increase in affinityHas Increased Fc Receptor binding affinityHumanized Version Of Murine Mab AC10Cluster C epitopeXmAb Fc RegionEngineered for high affinity Fc receptor bindingAntiproliferative Against HL And ALCL Cell LinesBlocks CD30 Ligand BindingHas Improved Effector FunctionsCytotoxicity (ADCC)Phagocytosis (ADCP)Exhibits No Complement Dependent Cytotoxicity (CDC) ActivityIs Active In Subcutaneous Xenograft Models Of HLWell Tolerated In Toxicology StudiesPK Suggests at Least Every Other Week DosingXmAbDissociation Constant (KD)Receptor (Allotype)XmAb 2513IgG1 XENP 2477Fold ImprovementFcgRI13FcgRIIA(131H)1479777FcgRIIA(131R)925116FcgRIIIA(158V)917319FcgRIIIA(158F)2359926FcgRIIB120219018XmAb2513 is a new humanized monoclonal antibody (mAb), to the human cell surface antigen CD30, with an engineered Fc region to enhance recruitment of effector cells and potentiate anti-tumor efficacy. It is being developed for CD30-positive (CD30+) diseases such as Hodgkin Lymphoma (HL) and anaplastic large cell lymphoma (ALCL). XmAb2513 was derived from the murine mAb AC10 by humanizing the variable domain using the method of human sequence content optimization while retaining high binding affinity for CD30. In addition, the Fc region was engineered to increase the binding affinity for all Fc receptors (FcRs).Using biacore measurements, XmAb2513 was determined to have a binding affinity of 465 pM for CD30. The Fc engineering increased the binding affinity of XmAb2513 for FcRI, FcRIIa. FcRIIb, and FcRIIIa by between 3- and 26-fold when compared to the binding affinity of an unengineered comparator mAb.XmAb2513 retains the potent anti-proliferative activity exhibited by the parental antibody against HL and ALCL cell lines. In addition, as a result of the Fc engineering, XmAb2513 exhibited superior antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by NK cells that primarily express FcγRIIIa, when compared to the unengineered mAb. The mean efficacy (percentage of cells specifically lysed) improvement was 4.7–fold and the mean potency (concentration giving 50% of maximal lysis) improvement was 2.4-fold over the unengineered mAb. XmAb2513 was also 2.1-fold more efficacious than the unengineered mAb in antibody-dependent cell-mediated phagocytosis (ADCP) assays using FcγRIIa/b and FcγRIIIa expressing macrophages.The in vivo anti-tumor activity of XmAb2513 was evaluated using subcutaneous xenograft models in SCID mice. Statistically significant reductions in tumor growth, together with enhanced survival, were observed at 3 mg/kg while at 10 and 30 mg/kg XmAb2513 was even able to eliminate established tumors.XmAb2513 has been successfully engineered to possess multiple mechanisms of action, including ADCC and ADCP, with significant improvement over those of an unengineered IgG1 mAb comparator. Additionally, XmAb2513 has potent anti-proliferative effects and was efficacious against HL xenografts. These in vitro and in vivo pharmacology data provide a rationale for the clinical testing of XmAb2513 in patients with CD30+ hematologic malignancies.VH, 27 mutationsVL, 15 mutationsMurine = mAC10Chimeric = cAC10Humanized = xAC10IgG1Humanized templateIncreased HSCIncreased affinityLog10 scaleHuman String ContentVH = 0.84%, VL = 0.93%4x increase in antigen binding affinityMeasured by competitive AlphaScreen assayVH HSC ComparisonBinding affinities were determined using a biacore method.Antibodies were immobilized on a Protein A chip and receptors included in the mobile phase.Dissociation constants were determined from Langmuir curve fit.Lazar et al, Mol Immunol. 2007Lazar et al. PNAS 2006Is anti-proliferative against HL and ALCLBlocks CD30 Ligand BindingMediates enhanced cytotoxicityNK cell dependentIncreases cytotoxicity for FcRIIIa allotypes1000200030004000500010010000100000XmAb2513 concentration (ng/mL)Mean Fluorescence Intensity (a.u.)CD30L 1000ng/mLCD30L 300ng/mLCD30L 100ng/mL0.111010010000.250.50.751.01.35F11cAC10xAC10-IgG1XmAb2513BSAhIgGAntibody concentration (ng/ml)Proliferation (RLU x107)Anti-human IgG (10x excess)Efficacy Improved 4.7 ± FoldPotency Improved 2.4 ± FoldKarpas 299(ALCL)Anti-CD30 (100 ng/ml)LDH Release AssayCell Counting AssayCD30+Karpas299Cell0.11101001000515202530cAC105F11XmAb2513Ab Concentration (ng/ml)% Cytotoxicity0.010.1110100100010000515202530XmAb25135F11cAC10Ab Concentration (ng/ml)% CytotoxicityL540 (HD)2.22.01.8Proliferation (RLU x107)Differences in anti-proliferative effects between anti-CD30 antibodies have been attributed to different epitope clusters (Horn-Lohrens et al); AC10 is a cluster C antibody while 5F11 is a cluster A antibody to CD30. To determine antibody effects on cell proliferation, either Karpas299 or L540 cells were grown for 4 days in the presence of antibody at varying concentrations with 10x molar access of cross linking antibody. Cell growth was measured using an ATP dependent luminescence assay.1.6XmAb25131.4cAC10-IgG1Antibody dependent cell-mediated cytotoxicity was measured by both lactate dehydrogenase (LDH) release and cell counting (Guava). Human PBMC effector cells were purified from a Leukopack using a ficoll gradient. L540 Hodgkin Lymphoma target cells were seeded into 96-well plates at 10,000 (LDH) or 15,000 (Sorting) cells/well and opsonized using antibody at the indicated concentration. Effector cells were added at 25x E:T and the plate incubated at 37C for 4 hrs prior to assay. For LDH, data were normalized to maximal (Triton X100 lysis of target cells alone) and minimal (PBMCs alone) lysis. Reported cytotoxicity was derived from LDH data.CD30L binding assay was performed using L540 cells expressing CD30.Fixed concentrations of antibody-crosslinked his-CD30L were added as indicated in the legend.A dose response curve of XmAb2513 was also added as indicated on the X axis.Binding of CD30L to cells was detected using flow cytometry and reagents for the cross-linking antibody.xAC10-IgG15F11Antibody dependent cell-mediated cytotoxicity (ADCC) assays were performed with donor PBMCs isolated from Leukopaks.The target for these data was the HL L540 cell line.Donor allotype was determined by sequencing after PCR from genomic DNA.1.2BSAhIgG1.00.11101001000Antibody concentration (ng/ml)Increases phagocytosis for FcRIIa allotypesIncreased Survival in an L540 XenograftCaused Tumor Regression in L540 XenograftWas well tolerated in pharmacokinetic and toxicology studies% ADCP Improved 2.2 ± FoldLow RangeHigh RangeTumorregressions0/94/95/951015202530354045H/RR/RFcRIIa AllotypePercent phagocytosisXmAb2513XENP2477Was well tolerated on a Q5D X6 dosing regimen in cynomolgus monkeys.Terminal T½ was between 12.6 And 17.1 days.Exposure to XmAb2513 was proportional to dose (after 1st dose).ConclusionsPBS0.3 mg/kg1 mg/kg3 mg/kgThe XmAb™2513 engineered antibody has been shown to have enhanced potency and efficacy as compared to IgG1 antibodies. This was observed both in ADCC assays as well as in antiproliferation assays where the antibody crosslinking required for an antiproliferative effect was mediated by Fc receptor binding. Published results from clinical trials with bispecific antibodies directed to CD30 provide clinical evidence in Hodgkin Lymphoma that enhanced recruitment of effector function is a successful means of generating a cytotoxic antibody (Hartmann, 2001: Borchmann, 2002). However, manufacturing limitations prevent bispecifics from being practical in widespread use. Preclinical results with XmAb™2513 support further testing in the clinic to validate the role of enhanced Fc effector function.PBS3 mg/kg10 mg/kg30 mg/kgFor animal 3503 tumor began to regrow on day 43. On day 45 animal 3508 no longer had measurable tumorReferencesDonor DuplicatesDonor DuplicatesLazar et al. A molecular immunology approach to antibody humanization and functional optimization. Mol Immunol Mar;44(8):Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A Mar 14;103(11):Borchmann et al. Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. Blood Nov 1;100(9):Hartmann et al. Anti-CD16/CD30 bispecific antibody treatment for Hodgkin's disease: role of infusion schedule and costimulation with cytokines. Clin Cancer Res Jul;7(7):Female mice from the ICR-SCID strain were injected subcutaneously with 5x107 cells of the L540 HL line.Animals were randomized to treatment groups when established tumors of mm3 were measured by microcaliper.Dosing was performed by intraperitoneal injection every 4 days for 10 doses (Q4Dx10).Animals were sacrificed when tumors reached 1500 mm3, for humane considerations, or at the completion of the study on day 70.Antibody dependent cell-mediated phagocytosis (ADCP) assays were performed with monocyte-derived macrophages cultured from donor PBMCs isolated from Leukopaks.The target for these data was the HL L540 cell line.Donor allotype was determined by sequencing after PCR from genomic DNA.No H/H donors were identified in the donor populations used for these studies.During the study, the tumors of several animals regressed to the point of being unmeasurable or in fact undetectable.For all except one of the animals, indicated in the 10 mg/kg group, the effect was durable.AcknowledgementWe wish to thank the many Xencor employees who supported this work.