Hector Aviña

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody–Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. Mol Cancer Ther; 14(7); 1650–60. ©2015 AACR.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

Development of ASG-15ME, a Novel Antibody–Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

The discovery and preclinical development of ASG-5ME, an antibody drug conjugate targeting SLC44A4 positive epithelial tumors including pancreatic and prostate cancer

Here, we report the development of an antibody–drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line – and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679–87. ©2016 AACR.

Inhibition of Megakaryocyte Differentiation by Antibody–Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia

Thrombocytopenia is a common adverse event in cancer patients treated with antibody–drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877–86. ©2017 AACR. See related article by Zhao et al., p. 1866

Modulation of Macropinocytosis-Mediated Internalization Decreases Ocular Toxicity of Antibody–Drug Conjugates

AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.

Abstract 2709: Evaluation of CD37 expression and binding of AGS67E, an antibody-drug conjugate (ADC) against CD37, on white blood cells (WBCs) collected from phase I non-Hodgkin lymphoma (NHL) patients

AGS67E is an antibody drug conjugate (ADC) against CD37 conjugated to monomethyl auristatin E (MMAE). CD37 is expressed on normal WBCs, but is also highly expressed in NHL, CLL and AML (Pereira et al., 2015). A phase I study is currently evaluating the safety, PK and anti-cancer activity of AGS67E with or without growth factor (GF) in subjects with relapsed/refractory NHL. To assess CD37 expression on WBCs, binding of AGS67E, and potential pharmacodynamic effects, samples from subjects were collected at pre-dose, D2, D8, and D15 and analyzed by flow cytometry. CD37 expression on subject tumor samples was also evaluated by immunohistochemistry (IHC). Our results demonstrated that CD37 was highly expressed in tumor samples and that AGS67E binds to WBCs causing down-regulation of CD37, achieving saturation of binding at 24 hours post-treatment (earliest time measured) at or above 0.9 mg/kg. A dose-dependent decrease in the number of all cell types examined was observed with a nadir occurring at D8, with partial or full recovery at D15, except for neutrophils. NK and T cell counts appeared to be least impacted while neutrophils were most affected. B cell counts were extremely low pre-dose for some patients, presumably from prior therapies. In patients treated at 0.9 mg/kg and higher without GF, recovery of neutrophils was delayed beyond D15. At doses of 1.2 mg/kg and higher, use of GF resulted in a significant recovery of neutrophils by D15. The extent of cell count decreases did not correlate to the proportion of cells expressing CD37. For example, decreases in NK cells, monocytes, and, in some cases, T cells, were much greater than the proportion of cells expressing CD37. Furthermore, mature WBCs are unlikely to be affected by AGS67E. This raises the possibility that the main effect of AGS67E may be on rapidly growing precursor cells and that cells with low, or no, CD37 expression may be impacted by the membrane permeable MMAE through a by-stander effect. The effect of AGS67E on neutrophils was investigated in an in vitro assay where hematopoietic stem cells were differentiated into neutrophils. Using this method, we showed that when AGS67C antibody was conjugated to a non-cleavable, membrane impermeable auristatin (mcMMAF) less cytotoxicity to differentiating neutrophils was observed compared to AGS67E. Previously, we have shown that neutrophils secrete proteases that can liberate MMAE from ADCs (Zhao et al, 2016). These results suggest that AGS67E contributes to neutropenia through a by-stander effect, in addition to the CD37-mediated internalization of the ADC. In conclusion, the results showed that AGS67E bound to its target CD37, modulated its expression, achieved saturation of binding at doses at or above 0.9 mg/kg, and reversibly depleted WBCs, with the exception of neutrophils for which GF administration appeared to significantly improve recovery rate. Citation Format: Sher Karki, Hector Avina, Jacqueline Lackey, Ahmed Sawas, Kerry J. Savage, Raymond Perez, Ranjana Advani, Jasmine Zain, Owen A. O9Connor, Sara Gulesserian, Hui Zhao, Peng Yang, Karen Morrison, Leonard Reyno, Fernando Donate. Evaluation of CD37 expression and binding of AGS67E, an antibody-drug conjugate (ADC) against CD37, on white blood cells (WBCs) collected from phase I non-Hodgkin lymphoma (NHL) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2709. doi:10.1158/1538-7445.AM2017-2709

Abstract 574: AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, Rene Hubert, Mary Brodey, Hector Avina, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Donate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 574.

Abstract 2650: Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin's lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia

We have developed AGS67E, an antibody drug conjugate that targets CD37, a tetraspanin highly expressed on malignant B cells, for the potential treatment of non-Hodgkin9s lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). AGS67E is a fully human anti-CD37 monoclonal IgG2 antibody conjugated to the potent microtubule-disrupting agent, MMAE, via reduced cysteines and the protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl. AGS67E exhibits potent in vitro binding, internalization and cytotoxicity on a variety of NHL, CLL and AML models and patient-derived samples, including CD34+CD38- leukemic stem cells. AGS67E also demonstrates potent anti-tumor responses, including complete tumor regressions in a variety of NHL, CLL and AML xenografts, including Rituxan refractory models and patient-derived samples. In general, CD37 was highly expressed across all models and a strong correlation was observed between the in vitro and in vivo efficacy of AGS67E. To confirm binding of AGS67E in a variety of normal and patient-derived NHL, CLL and AML samples, we developed flow cytometry and immunohistochemistry (IHC) assays which have confirmed reported CD37 expression data in NHL & CLL. In normal hematopoietic cells, AGS67E bound strongly to B cells and to a much lesser extent to monocytes, T cells, neutrophils and NK cells. AGS67E also bound with high and similar affinity to cynomolgus monkey B cells and was equally cytotoxic to these and human B cells. In other normal tissues, AGS67E binding was only evident where lymphoid structures were apparent such as in the spleen and lymph node. With respect to CD37 expression in NHL, CLL and AML, AGS67E was found to bind to >80% of NHL and 100% of CLL and AML samples. Taken together, our findings suggest that AGS67E may serve as a potential therapeutic for NHL, CLL and AML. To our knowledge, this body of work is also the first demonstration that CD37 is well expressed and potentially drug-able in AML. Citation Format: Daniel S. Pereira, Claudia Guevara, Alla Verlinsky, Cyrus Virata, J Hsu Ssucheng, Zili An, Chungying Zhang, Nick Dinh, Hector Avina, Lisa Do, Sher Karki, Joseph Abad, Peng Yang, Jimmy Ou, Karen Morrison, Sing-Ju Moon, Faisal Malik, Liqing Jin, Michael Choi, Christina Wu, Banmeet Anand, Scott Cooper, Ingrid Joseph, Xiao-Chi Jia, Kendall Morrison, Pia Challita-Eid, Fernando Donate, Thomas Kipps, John Dick, David Stover. Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin9s lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2014-2650

Abstract 4566: AGS-1C4D4: A fully human anti-PSCA antibody inhibits tumor formation and metastasis in orthotopic models of pancreatic cancer

Prostate stem cell antigen (PSCA) is a cysteine-rich cell surface glycoprotein expressed in about 50% of pancreatic and prostate cancers. AGS-PSCA is a hybridoma-derived fully human IgG1κ monoclonal antibody (MAb) targeting PSCA previously reported to have anti-tumor efficacy in prostate and pancreatic tumor models. AGS-1C4D4 is a CHO-derived antibody generated from the same gene as AGS-PSCA, with similar specificity and binding affinity to PSCA (Kd = 2.0 × 10-10M). Since hybridoma and CHO-derived MAbs displayed disparate glycosylation patterns by mass spectroscopy, we further evaluated their MAb effector functions. AGS-1C4D4 was found to have more potent ADCC activity in vitro on PSCA-expressing pancreatic cell lines, HPAC and Panc0203, using human PBMCs from multiple normal donors. However, both MAbs were similarly effective in mediating CDC on PSCA-expressing recombinant B300.19 cells. Deglycosylation greatly reduced the relative ADCC and CDC activities of both MAbs compared to their intact versions. Additional studies to elucidate the role of the effector functions of AGS-1C4D4 are currently being conducted in vivo using intact and deglycosylated MAbs and will be presented. While neither intact antibody had any direct cytotoxic activity on HPAC cells in vitro, both MAbs significantly inhibited tumor formation, local invasion and metastases to distant sites in orthotopic HPAC xenograft tumor models in vivo. In addition, AGS-1C4D4 significantly inhibited the growth and metastasis of established orthotopic HPAC tumors in combination with Gemcitabine. AGS-1C4D4 is currently being evaluated in a Phase 2 clinical study for pancreatic cancer in combination with Gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4566. doi:10.1158/1538-7445.AM2011-4566