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0 5 10 15 20 25 30 35 40 45 H/R H/R H/R R/R R/R R/R FcRIIa Allotype Percent phagocytosis XmAb2513 XENP2477 PRECLINICAL PHARMACOLOGY OF XmAb TM 2513 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 91016 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 100 Tim e (days) PercentSurvival Abstract Was humanized with an increase in affinity Has Increased Fc Receptor binding affinity Is anti-proliferative against HL and ALCL Mediates enhanced cytotoxicity NK cell dependent Was well tolerated in pharmacokinetic and toxicology studies Acknowledgement XmAb2513 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. Murine = mAC10 Chimeric = cAC10 Humanized = xAC10 V H , 27 mutations V L , 15 mutations Human String Content V H = 0.84%, V L = 0.93% 4x increase in antigen binding affinity 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.60 0.65 0.70 0.75 0.80 0.85 0.90 HSC murine hum an humanized Xencor (optimized) V H HSC Comparison -13 -12 -11 -10 -9 -8 -7 0 10 20 30 40 50 60 70 80 90 100 110 hA C 10_S TD A C 10_control hA C 10_variant hA C 10_variant hA C 10_variant hA C 10_variant hA C 10_variant Log [V ariant],2M % M axim alSignal Humanized template Increased HSC Increased affinity Karpas 299 (ALCL) L540 (HD) 0.1 1 10 100 1000 0 0.25 0.5 0.75 1.0 1.3 5F11 cAC10 xAC10-IgG1 XmAb2513 BSA hIgG Antibody concentration (ng/ml) Proliferation (RLU x10 7 ) 0.1 1 10 100 1000 1.0 1.2 1.4 1.6 1.8 2.0 2.2 5F11 cAC10-IgG1 xAC10-IgG1 XmAb2513 BSA hIgG Antibody concentration (ng/ml) Proliferation (RLU x10 7 ) LDH Release Assay Cell Counting Assay Measured by competitive AlphaScreen assay Anti-human IgG (10x excess) Anti-CD30 (100 ng/ml) CD30+ Karpas299 Cell We wish to thank the many Xencor employees who supported this work. The 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. Antibody 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. 0.1 1 10 100 1000 0 5 10 15 20 25 30 cAC10 5F11 XmAb2513 Ab Concentration (ng/ml) % Cytotoxicity % Cytotoxicity 0.01 0.1 1 10 100 1000 10000 0 5 10 15 20 25 30 XmAb2513 5F11 cAC10 Ab Concentration (ng/ml) 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. 1.00E-10 1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 Dissociation C onstant(K D ) Xm A b2513 XENP2477a Dissociation Constant (K D ) Receptor (Allotype) XmAb 2513 IgG1 XENP 2477 Fold Improvement FcRI 1 3 3 FcRIIA(131H) 147 977 7 FcRIIA(131R) 92 511 6 FcRIIIA(158V ) 9 173 19 FcRIIIA(158F ) 23 599 26 FcRIIB 120 2190 18 XmAb IgG1 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 100 D ay PercentSurvival Increased Survival in an L540 Xenograft PBS 0.3 mg/kg 1 mg/kg 3 mg/kg PBS 3 mg/kg 10 mg/kg 30 mg/kg Low Range High Range Tum orclearance 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 PBS 3 m g/kg 10 m g/kg 30 m g/kg D ay A nim als w ith m easurable tum or For animal 3503 tumor began to regrow on day 43. On day 45 animal 3508 no longer had measurable tumor Tumor regressions 0/9 0/9 4/9 5/9 Caused Tumor Regression in L540 Xenograft 0 5 10 15 20 25 30 35 40 45 F/F F/F F/V F/V F/V V/V V/V FcRIIIa 158 Allotype PercentA D C C Xm Ab2513 XEN P2477 Efficacy Improved 4.7 ± 1.4 - Fold Potency Improved 2.4 ± 1.4 - Fold % ADCP Improved 2.2 ± 0.7 - Fold • 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. Donor Duplicates Donor Duplicates XmAb™2513 Increases cytotoxicity for FcRIIIa allotype creases phagocytosis for FcRIIa allotypes Blocks CD30 Ligand Binding Humanized Version Of Murine Mab AC10 – Cluster C epitope XmAb Fc Region – Engineered for high affinity Fc receptor binding Antiproliferative Against HL And ALCL Cell Lines Blocks CD30 Ligand Binding Has Improved Effector Functions – Cytotoxicity (ADCC) – Phagocytosis (ADCP) Exhibits No Complement Dependent Cytotoxicity (CDC) Activity Is Active In Subcutaneous Xenograft Models Of HL Well Tolerated In Toxicology Studies PK Suggests at Least Every Other Week Dosing • Female mice from the ICR-SCID strain were injected subcutaneously with 5x10 7 cells of the L540 HL line. • Animals were randomized to treatment groups when established tumors of 50-100 mm 3 were measured by microcaliper. • Dosing was performed by intraperitoneal injection every 4 days for 10 doses (Q4Dx10). • Animals were sacrificed when tumors reached 1500 mm 3 , for humane considerations, or at the completion of the study on day 70. • 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. • Antibody 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. • Binding 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. Log 10 scale Conclusions • Was 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). Lazar et al. A molecular immunology approach to antibody humanization and functional optimization. Mol Immunol. 2007 Mar;44(8):1986-98 Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10 Borchmann et al. Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. Blood. 2002 Nov 1;100(9):3101-7. Hartmann et al. Anti-CD16/CD30 bispecific antibody treatment for Hodgkin's disease: role of infusion schedule and costimulation with cytokines. Clin Cancer Res. 2001 Jul;7(7):1873-81 References Lazar et al, Mol Immunol. 2007 Lazar et al. PNAS 2006 0 1000 2000 3000 4000 5000 100 1000 10000 100000 XmAb2513 concentration (ng/mL) Mean Fluorescence Intensity (a.u.) CD30L 1000ng/mL CD30L 300ng/mL CD30L 100ng/mL • 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.
description
PRECLINICAL PHARMACOLOGY OF XmAb TM 2513 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 91016. Humanized template Increased HSC Increased affinity. V H HSC Comparison. 1.3. 30. 30. 1.0. - PowerPoint PPT Presentation
Transcript of H/R
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XmAb2513 XENP2477
PRECLINICAL PHARMACOLOGY OF XmAbTM2513AN FC ENGINEERED HUMANIZED ANTI-CD30 MABPhil Hammond, Greg Lazar, Sher Karki, David Carmichael, Seung Chu
Xencor, Inc. 111 W. Lemon Ave., Monrovia, CA 91016
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Abstract Was humanized with an increase in affinity Has Increased Fc Receptor binding affinity
Is anti-proliferative against HL and ALCL Mediates enhanced cytotoxicityNK cell dependent
Was well tolerated in pharmacokinetic and toxicology studies
Acknowledgement
XmAb2513 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.
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We wish to thank the many Xencor employees who supported this work.
The 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.
Antibody 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 37 C 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.
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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.
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• 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.
Donor DuplicatesDonor Duplicates
XmAb™2513
Increases cytotoxicity for FcRIIIa allotypes
Increases phagocytosis for FcRIIa allotypes
Blocks CD30 Ligand Binding
Humanized Version Of Murine Mab AC10– Cluster C epitope
XmAb Fc Region– Engineered for high affinity Fc receptor binding
Antiproliferative Against HL And ALCL Cell Lines Blocks CD30 Ligand Binding Has Improved Effector Functions
– Cytotoxicity (ADCC)– Phagocytosis (ADCP)
Exhibits No Complement Dependent Cytotoxicity (CDC) Activity Is Active In Subcutaneous Xenograft Models Of HL Well Tolerated In Toxicology Studies PK Suggests at Least Every Other Week Dosing
• 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 50-100 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.
• 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.
• Antibody 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.
• Binding 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.
Log10 scale
Conclusions
• Was 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).
Lazar et al. A molecular immunology approach to antibody humanization and functional optimization. Mol Immunol. 2007 Mar;44(8):1986-98
Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10
Borchmann et al. Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. Blood. 2002 Nov 1;100(9):3101-7.
Hartmann et al. Anti-CD16/CD30 bispecific antibody treatment for Hodgkin's disease: role of infusion schedule and costimulation with cytokines. Clin Cancer Res. 2001 Jul;7(7):1873-81
References
Lazar et al, Mol Immunol. 2007 Lazar et al. PNAS 2006
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• 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.
