Antimitotic Agent Monomethyl Auristatin Research Articles

Abstract PO5-27-05: Computational design and validation of a novel peptide-drug conjugate for treatment of triple negative breast cancer

Abstract Sortilin (SORT1) is a member of the vacuolar protein sorting 10 protein (Vps10p) family that functions as a receptor regulating peptide and protein trafficking between the plasma membrane, lysosomes, and trans-golgi network. As a cell surface receptor, SORT1 is able to mediate efficient endocytosis of extracellular ligands to the lysosomal compartment. Numerous reports have identified enriched SORT1 expression in a variety of tumor types, including triple-negative breast cancer (TNBC), a subtype of breast cancer associated with aggressive clinical behavior and poor disease outcomes. We sought to exploit SORT1-dependent internalization of peptides as a platform for rapid and specific chemotherapy delivery into TNBC cells. Using ProteinStudio (our proprietary computation-enabled design capabilities), we generated high-affinity SORT1 targeting peptides that exhibit efficient receptor dependent internalization and lysosomal localization. Alternative computational approaches such as AlphaFold2 failed to recapitulate the peptide design. Peptide drug conjugates (PDCs) were generated via a linkage strategy that combines our designed peptides to the antimitotic agent monomethyl auristatin E (MMAE). Our PDC molecules exhibit potent tumor regression in a MDA-MB-231 TNBC cell derived xenograft model, thereby highlighting the potential of SORT1-engaging PDCs as an efficacious targeted chemotherapeutic delivery strategy. Citation Format: Francine Liu, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Li, Lucas Siow, Christopher Ing, David White. Computational design and validation of a novel peptide-drug conjugate for treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-05.

Efficacy and Safety of Polatuzumab Vedotin in Follicular Lymphoma: A Systematic Review of Clinical Trials

Introduction: Polatuzumab vedotin (Pola) is a humanized anti-CD79b monoclonal antibody linked with antimitotic agent monomethyl auristatin E. On internalization, the linker is cleaved, and antimitotic activity inhibits the cell division and growth of cancer cells. Pola is tested in multiple combinations in relapsed/refractory (RR) follicular lymphoma (FL) in recent years. In this systematic study, we will assess the efficacy and safety of Pola based combinations in RR FL. Methods: A literature search was performed on PubMed with keywords, “Polatuzumab vendotin” and “lymphoma” from the inception of data till 07/01/2023. PRISMA guidelines were followed while conducting this systematic review. We reviewed 160 articles and included 1 randomized clinical trial (RCT, N=41), and 4 non-randomized clinical trials (nRCT, N=163) based on inclusion criteria. Data was extracted for complete response (CR), partial response (PR), overall response (ORR), progression free survival (PFS), overall survival (OS) and ≥grade 3 side effects. Results: In 5 trials (N=204), 13 patients with Pola + atezolizumab (Ate) + obinutuzumab (Obi), 49 patients with Pola + Obi + venetoclax (Ven), 39 patients with Pola + Obi, 62 with Pola + Obi + lenalidomide (Len), and 20 patients with Pola + rituximab (Rit). In an RCT by Morschhauser et al. (N=41), Pola + Rit was compared with pinatuzumab vedotin (Pina) + Rit. ORR, CR, PR, and mPFS was 70%, 45%, 25%, and 15.3 months, respectively with Pola + Rit, as compared to 62%, 5%, 57%, and 12.7 months, respectively, with Pina + Rit. In a trial by Philips et al. (N=39), ORR, CR, PR, and mPFS were 67%, 36%, 31%, and 11.5 months, respectively. In 3 nRCTs (N=124), three drug regimens were used with Pola and Obi as backbone. ORR, CR, PR were 50%-83%, 20%-61%, and 20%-39%. Table 1. ≥ Grade 3 hematological and non-hematological side effects are given in table 1. Conclusion: Pola was well tolerated by most of the patients with FL alone or in combination with Obi, Rit, Len, Ven. Pola had relatively better response and survival rates as compared to Pina in combination with Rit. In early phase clinical trials, three drug regimens of Pola and Obi with Ven, and Len were effective in the treatment of heavily pretreated follicular lymphoma. In an early phase trial, Pola + Obi + Ate had limited efficacy in FL with safety concerns. More randomized double blind multicenter phase III clinical trials are needed to confirm these results.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Synthesis and Evaluation of a Non-Peptide Small-Molecule Drug Conjugate Targeting Integrin αVβ3.

An integrin αVβ3-targeting linear RGD mimetic containing a small-molecule drug conjugate (SMDC) was synthesized by combining the antimitotic agent monomethyl auristatin E (MMAE), an enzymatically cleavable Val-Ala-PABC linker with a linear conjugable RGD mimetic. The structure proposal for the conjugable RGD mimetic was suggested upon the DAD mapping analysis of a previously synthesized small-molecule RGD mimetic array based on a tyrosine scaffold. Therefore, a diversifying strategy was developed as well as a novel method for the partial hydrogenation of pyrimidines in the presence of the hydrogenolytically cleavable Cbz group. The small-molecule RGD mimetics were evaluated in an ELISA-like assay, and the structural relationships were analyzed by DAD mapping revealing activity differences induced by structural changes as visualized in dependence on special structural motifs. This provided a lead structure for generation of an SMDC containing the antimitotic drug MMAE. The resulting SMDC containing a linear RGD mimetic was tested in a cell adhesion and an in vitro cell viability assay in comparison to reference SMDCs containing cRGDfK or cRADfK as the homing device. The linear RGD SMDC and the cRGDfK SMDC inhibited adhesion of αVβ3-positive WM115 cells to vitronectin with IC50 values in the low µM range, while no effect was observed for the αVβ3-negative M21-L cell line. The cRADfK SMDC used as a negative control was about 30-fold less active in the cell adhesion assay than the cRGDfK SMDC. Conversely, both the linear RGD SMDC and the cRGDfK SMDC are about 55-fold less cytotoxic than MMAE against the αVβ3-positive WM115 cell line with IC50 values in the nM range, while the cRADfK SMDC is 150-fold less cytotoxic than MMAE. Hence, integrin binding also influences the antiproliferative activity giving a targeting index of 2.8.

A phase I/II, open-label, dose-escalation, and cohort-expansion study evaluating the safety, pharmacokinetics, and therapeutic activity of OBI-999 in patients with advanced solid tumors.

TPS3657 Background: Aberrant glycosylation is a hallmark of cancer. Glycosphingolipids (GSLs), glycans conjugated to a lipid (ceramide) core, are essential for the recruitment of immune-related proteins to specific membrane microdomains. Globo H (GH) is a GSL found on normal cells but highly overexpressed on various epithelial tumors playing a role in tumor development and progression. GH is a promising target for immunotherapy. OBI-999 is an ADC composed of a human recombinant immunoglobulin G (IgG) monoclonal antibody that selectively and specifically binds to GH, attached by a linker to the antimitotic agent monomethyl auristatin E (MMAE). Its mechanism of action is based on tumor-selective delivery of MMAE to GH-expressing tumors with subsequent tumor cell death. Preclinical studies demonstrated that OBI-999 antibody binds specifically to the GH antigen, and antitumor efficacy was noted in breast, gastric, pancreatic, and lung cancer xenograft models. The pharmacokinetics (PK) of OBI-999 were determined in normal and tumor-bearing mice, rats, and monkeys. Exposure of OBI-999 increased proportionally with dose. No sex difference or accumulation was seen. The primary objectives are to determine dose limiting toxicities (DLTs), the maximum tolerated dose (MTD), and the phase 2 recommended dose (P2RD). The secondary objectives are to assess the rates of objective response and clinical benefit, the duration of progression-free survival, the immunogenicity of OBI-999, and the PK and pharmacodynamics (PD) of OBI-999 and MMAE. Methods: In Part 1, a "3+3" dose-escalation part of the study, up to 30 patients with advanced solid tumors refractory to ≥1 line of systemic therapy, who cannot tolerate standard therapy, or for whom no standard treatment is available, regardless of GH status will be treated. OBI-999 will be administered as a 60-minute IV infusion using a dose range of 0.4, 0.8, 1.2, 1.6, and 2.0 mg/kg on day 1 of every 21-day cycle. In Part 2, the cohort-expansion portion of the study, patients will be treated at the MTD or at a lower RP2D as determined by cumulative toxicities and tolerability profile. The study will determine the preliminary clinical activity and safety of OBI-999 in up to 155 patients with advanced solid tumors, pancreatic, gastric, esophageal, and colorectal cancer according to a Simon two-stage phase 2 design. Patients must have GH overexpression defined as an H-score of ≥100 according to an FDA Investigational Device Exempt (IDE) validated IHC assay. Clinical trial information: NCT04084366 .

Improved targeting of an anti-TAG-72 antibody drug conjugate for the treatment of ovarian cancer.

IntroductionOvarian cancer has only a 17% 5‐year survival rate in patients diagnosed with late stage disease. Tumor‐associated glycoprotein‐72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody‐targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium.MethodsUsing the clinically relevant anti‐TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS‐MMAE), bromoacetamido (Br‐MMAE), and maleimido (mal‐MMAE).ResultsThe drug antibody ratios (DARs) of the three ADCs were 2.3 for VS‐MMAE, 10 for Br‐MMAE, and 9.5 for mal‐MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49‐mal‐MMAE (3.3%ID/g) was low compared to CC49‐Br‐MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49‐VS‐MMAE and CC49‐Br‐MMAE, and 7% for CC49‐mal‐MMAE. CC49‐VS‐MMAE was not further studied due to its low DAR, while CC49‐mal‐MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49‐Br‐MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49‐Br‐MMAE.ConclusionWe conclude that CC49‐Br‐MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model.

Non-toxic fragment of botulinum neurotoxin type A and monomethyl auristatin E conjugate for targeted therapy for neuroendocrine tumors.

Surgical resection is the only cure for neuroendocrine tumors (NETs). However, widespread metastases have already occured by the time of initial diagnosis in many cases making complete surgical removal impossible. We developed a recombinant heavy-chain receptor binding domain (rHCR) of botulinum neurotoxin type A that can specifically target synaptic vesicle 2 (SV2), a surface receptor abundantly expressed in multiple neuroendocrine tumors. Expression of neuroendocrine differentiation markers chromogranin A (CgA) and achaete-scute complex 1 (ASCL1) were signficantly reduced when treated with rHCR. rHCR conjugated to the antimitotic agent monomethyl auristatin E (MMAE) significantly suppressed proliferation of pancreatic carcinoid (BON) and medullary thyroid cancer cells (MZ) at concentrations of 500 and 300 nM respectively, while no growth suppression was observed in pulmonary fibroblasts and cortical neuron control cell lines. In vivo, rHCR-MMAE significantly reduced tumor volume in mouse xenografts with no observed adverse effects. These data suggest recombinant HCR (rHCR) of BoNT/A preferentially targets neuroendocrine cancer without the neurotoxicity of the full BoNT/A and that SV2 is a specific and promising target for delivering drugs to neuroendocrine tumors.

Phase I study of the anti-endothelin B receptor antibody-drug conjugate DEDN6526A in patients with metastatic or unresectable cutaneous, mucosal, or uveal melanoma

Background Endothelin B receptor (ETBR) is involved in melanoma pathogenesis and is overexpressed in metastatic melanoma. The antibody-drug conjugate DEDN6526A targets ETBR and is comprised of the humanized anti-ETBR monoclonal antibody conjugated to the anti-mitotic agent monomethyl auristatin E (MMAE). Methods This Phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity of DEDN6526A (0.3-2.8mg/kg) given every 3weeks (q3w) in patients with metastatic or unresectable cutaneous, mucosal, or uveal melanoma. Results Fifty-three patients received a median of 6 doses of DEDN6526A (range 1-49). The most common drug-related adverse events (>25% across dose levels) were fatigue, peripheral neuropathy, nausea, diarrhea, alopecia, and chills. Three patients in dose-escalation experienced a dose-limiting toxicity (infusion-related reaction, increased ALT/AST, and drug-induced liver injury). Based on cumulative safety data across all dose levels, the recommended Phase II dose (RP2D) for DEDN6526A was 2.4mg/kg intravenous (IV) q3w. The pharmacokinetics of antibody-conjugated MMAE and total antibody were dose-proportional at doses ranging from 1.8-2.8mg/kg. A trend toward faster clearance was observed at doses of 0.3-1.2mg/kg. There were 6 partial responses (11%) in patients with metastatic cutaneous or mucosal melanoma, and 17 patients (32%) had prolonged stable disease ≥6months. Responses were independent of BRAF mutation status but did correlate with ETBR expression. Conclusion DEDN6526A administered at the RP2D of 2.4mg/kg q3w had an acceptable safety profile and showed evidence of anti-tumor activity in patients with cutaneous, mucosal, and uveal melanoma. ClinicalTrials.gov identifier: NCT01522664.

POLARIX: A phase 3 study of polatuzumab vedotin (pola) plus R-CHP versus R-CHOP in patients (pts) with untreated DLBCL.

TPS7571 Background: Rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) remains the standard of care in pts with previously untreated diffuse large B-cell lymphoma (DLBCL) but outcomes remain poor in pts with high-risk disease. Pola is an antibody–drug conjugate targeting CD79b; it delivers the antimitotic agent monomethyl auristatin E. Addition of pola to bendamustine and R in pts with transplant-ineligible DLBCL resulted in improved OS (Sehn et al, 2018). In front-line treatment of DLBCL, pola is being evaluated as a replacement for vincristine within the R-CHOP regimen. In a phase Ib/II study in pts with higher risk DLBCL, pola + R-CHP demonstrated promising efficacy and a safety profile similar to that observed in the R-CHOP arm of the GOYA study (Tilly et al, 2017; Vitolo et al, 2017). The phase 3 POLARIX study investigates pola + R-CHP in untreated DLBCL. Methods: POLARIX is an ongoing, international, randomized, double-blind, active-placebo-controlled, phase 3 study in pts with previously untreated DLBCL. Pts aged 18–80 years with CD20-positive DLBCL (including DLBCL not otherwise specified, GCB, and ABC subtypes), ECOG performance status 0–2, and IPI score 2–5, are stratified by IPI score (2 vs 3–5), bulky disease and geographical region and randomized (1:1). Pts receive 6 cycles of either: pola 1.8 mg/kg on Day 1 plus R-CHP (standard dosing schedule) plus vincristine placebo; or pola placebo plus R-CHOP (standard dosing schedule). R monotherapy is administered in cycles 7 and 8 (both arms). PET-CT and CT scans are obtained at screening, after 4 cycles (planned interim assessment), and 6–8 weeks after end of study treatment. Follow-up will continue for 5 years after treatment. Primary endpoint: investigator-assessed progression-free survival (PFS; Lugano classification). Secondary endpoints: independent review committee-assessed PET-CT complete response rate at end of treatment, event-free survival, 2-year PFS rate, and OS. Enrolment began Nov 2017. This trial is currently recruiting, and plans to enrol 875 patients in 24 countries. Clinical trial information: NCT03274492.

Reductively cleavable polymer-drug conjugates based on dendritic polyglycerol sulfate and monomethyl auristatin E as anticancer drugs

Stimuli-responsive polymer-drug conjugates (PDCs) provide promising approaches in anticancer treatment. Here, we report the synthesis and biological evaluation of PDCs made of the highly potent antimitotic agent monomethyl auristatin E conjugated to dendritic polyglycerol and dendritic polyglycerol sulfate via a reductively cleavable, self-immolative disulfide linker. Cell viability assays with the human cancer cell lines A549 (lung carcinoma) and HeLa (cervix carcinoma) revealed that the drug's cytotoxicity was reduced by conjugation to the polymers, with the sulfated conjugates being more effective than the non-sulfated ones. Kinetic studies using real-time cell analysis indicated a retarded drug release from the polymers, with a much later cytotoxic response after treatment with the non-sulfated conjugates due to less cellular uptake, as confirmed by flow cytometry and confocal laser scanning microscopy. In contrast, the non-cleavable dPGS-MMAE conjugate that was synthesized for comparison was not cytotoxic under the same conditions. Overall, reductively cleavable dPGS-SS-MMAE conjugates showed promising results in vitro and good tolerability in vivo. Further in vivo studies are planned.

Therapeutic Use of Brentuximab Vedotin in CD30+ Hematologic Malignancies.

The CD30 antigen is strongly expressed on neoplastic cells in classical Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL) and other hematologic malignancies (such as DLBCL and cutaneous TCL), while is almost undetectable on healthy tissues, representing an ideal immunotherapeutic target. Since unconjugated anti-CD30 antibody (SGN-30) demonstrated limited clinical activity, researchers' effort aimed to create an antibody-drug conjugate (ADC), leading to discovery of SGN-35 (brentuximab vedotin), in which an anti-CD30 antibody is linked to the antimitotic agent monomethyl auristatin E (MMAE). In the first phase I study in CD30+ hematologic malignancies (the majority of patients with HL), the maximum tolerated dose was fixed respectively at 1.8mg/Kg every 3 weeks, overall response rate (ORR) and complete response (CR) rate were 38% and 24%. In 2 subsequent phase II studies, amazing results were reported, that permitted accelerated FDA approval for relapsed/refractory patients and led to the development of many clinical trials including BV as first-line HL and ALCL treatment. Moreover, as CD30 antigen may be expressed by other malignancies, the potential therapeutic application is increasing, including at least diffuse large B-cell lymphoma, T-cell lymphomas other than ALCL and cutaneous lymphoproliferative disorders. BV is administrated as outpatient regimen and is usually well tolerated; sensorial peripheral neuropathy represents the most common toxic effect, although it is dose-dependent and at least partially reversible in most cases, after dose reduction and/or treatment ending.

Anti-CD22 and anti-CD79b antibody-drug conjugates preferentially target proliferating B cells.

Background and PurposeCD22 and CD79b are cell‐surface receptors expressed on B‐cell‐derived malignancies such as non‐Hodgkin's lymphoma (NHL). An anti‐mitotic agent, monomethyl auristatin E, was conjugated to anti‐CD22 and anti‐CD79b antibodies to develop target‐specific therapies for NHL. The mechanism of action (MOA) and pharmacological and pharmacokinetic (PK) profiles of these antibody‐drug conjugates (ADCs) were investigated in cynomolgus monkeys.Experimental ApproachAnimals were administered anti‐CD22 or anti‐CD79b ADCs, respective unconjugated antibodies or vehicle. Pharmacodynamic effects on total and proliferating B cells and serum PK were then assessed. Antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) of the ADCs were evaluated in vitro.Key ResultsDepletion of B cells was observed after administration of either ADC or the respective unconjugated antibodies. An extended duration of depletion was observed in animals administered ADCs. Similarly, preferential depletion of proliferating B cells in blood and germinal centre B cells in spleen were only observed in animals administered ADCs. Serum PK profiles of ADCs and respective unconjugated antibodies were comparable. In vitro, anti‐human CD22 and anti‐human CD79b antibodies showed no or only moderate ADCC activity, respectively; neither antibody had CDC activity.Conclusions and ImplicationsThe findings support the proposed MOA: initial depletion of total B cells by antibody‐mediated opsonization, followed by preferential, sustained depletion of proliferating B cells by the auristatin conjugate due to its anti‐mitotic action. Delivering potent anti‐mitotic agents to B cells via the specificity of monoclonal antibodies provides a means to eliminate pathogenic B cells in NHL with improved risk–benefit profiles over traditional chemotherapeutics.

Abstract CT233: A first-in-human phase I study of the safety and pharmacokinetic (PK) activity of DEDN6526A, an anti-endothelin B receptor (ETBR) antibody-drug conjugate (ADC), in patients with metastatic or unresectable melanoma

Abstract Background: ETBR, a G-protein coupled receptor that can activate RAF/MEK signaling, is overexpressed in metastatic melanoma compared with normal skin. DEDN6526A is an ADC with the anti-mitotic agent monomethyl auristatin (MMAE) linked to the humanized IgG1 anti-ETBR monoclonal antibody and represents a novel targeted treatment against melanoma. In preclinical models, DEDN6526A shows dose-dependent anti-tumor activity in ETBR-expressing tumor xenografts. Methods: DEDN6526A (0.3-2.8 mg/kg) was given intravenously every 3 weeks (q3w) to pts with metastatic or unresectable cutaneous, mucosal, or ocular (uveal) melanoma in a 3+3 design to determine the maximum-tolerated dose, followed by cohort expansion at the recommended phase II dose (RP2D). PK samples were collected, and archival tumor tissue was assessed for ETBR expression by immunohistochemistry. Clinical activity was evaluated per RECIST v1.1. Results: As of 25 November 2013, 28 pts with median age 65 (range 24-82), ECOG PS 0-1, and median 2.5 prior therapies (range 0-5) enrolled in dose escalation and received median 6 doses of DEDN6526A (range 1-28). Based on cumulative safety data, the RP2D of DEDN6526A is 2.4 mg/kg q3w. Three DLTs occurred: G3 infusion-related reaction (IRR) (1.8 mg/kg), G3 transaminitis (2.4 mg/kg), and G3 elevated ALT and AST and transient G2 elevated bilirubin meeting criteria for drug induced liver injury (2.8 mg/kg). The most common treatment-emergent adverse events (AEs) of any grade (G) in ≥ 20% of pts were fatigue (57%), chills (39%), alopecia (32%), diarrhea (32%), nausea (29%), decreased appetite (25%), headache (25%), IRRs (25%), asthenia (21%), peripheral sensory neuropathy (21%), and vomiting (21%). The protocol was amended to allow steroid pre-medications prior to DEDN6526A administration, which mitigated the IRRs. Treatment-related G≥3 AEs in ≥ 5% of pts, assessed by investigators, were anemia (11%), neutropenia (11%), leukopenia (7%), and white blood count decreased (7%). Exposures of the conjugate (antibody-conjugated MMAE), total antibody, and unconjugated MMAE increased with dose. Among 19 pts treated at doses ≥ 1.8 mg/kg, there were four confirmed partial responses (21%); 6 pts (32%) had stable disease ≥ 6 months. Clinical benefit in pts did not seem to be associated with melanoma origin (cutaneous/mucosal vs. ocular), tumor BRAF mutation status, and/or progression on prior immune therapies. Correlation of ETBR expression with clinical activity will be presented. Conclusions: DEDN6526A administered at the RP2D of 2.4 mg/kg q3w is safe and tolerable. There is early evidence of anti-tumor activity in melanoma pts. Enrollment in the expansion cohort is ongoing. Citation Format: Jeffrey R. Infante, Shahneen K. Sandhu, Catriona M. McNeil, Omar Kabbarah, Chunze Li, Wei Zhong, Jyoti Asundi, Katie Wood, Yu-Waye Chu, Omid Hamid. A first-in-human phase I study of the safety and pharmacokinetic (PK) activity of DEDN6526A, an anti-endothelin B receptor (ETBR) antibody-drug conjugate (ADC), in patients with metastatic or unresectable melanoma. [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 CT233. doi:10.1158/1538-7445.AM2014-CT233

Potent New Weapons in the Oncology Arsenal (Antibody-Drug Conjugates) Require Unique Biological Manufacturing Expertise

Recent advances in coupling antibodies to potent cytotoxic drugs have resulted in stable delivery platforms with improved pharmacokinetics which spares patients from the debilitating systemic toxicity observed with traditional chemotherapy [1]. In 2011 the FDA granted accelerated approval to Seattle Genetics Adcetris® (Bentuximab vedotin) and on Feb. 4th 2013 Health Canada approved the drug which uses a chimeric monoclonal antibody to target CD-30 positive cells. The cytotoxic warhead conjugated to the antibody consists of the potent antimitotic agent monomethyl auristatin E. This is the first new therapy approved since 1977 for treatment of Hodgkin’s lymphoma and it is the first therapy to receive approval against anaplastic large cell lymphoma. Roche-Genentech received approval to offer Kadcyla® (Trastuzumab emtansine) for the treatment of patients with HER2-positive metastatic breast cancer in Feb. 2013. Results from the pivotal EMILIA trial demonstrated significantly improved overall survival compared to current standard of care which uses capecitabine and lapatinib. Kadcyla is the Herceptin antibody conjugated to the maytansine derivative DM1. Manufacturing these highly potent biopharmaceuticals presents a series of unique engineering and chemistry challenges which has resulted in several contract manufacturers constructing facilities to address the unique engineering and chemistry challenges associated with this promising class of therapeutic drugs. By leveraging experience in biopharmaceuticals and potent small molecule drug process development this class of promising new therapies can be reliably and safely manufactured to meet clinical and commercial demands. This article will investigate the experience of Lonza as it synergistically pooled internal resources to address this promising class of therapeutics. Keywords: ADC facilities, ADC linkers, ADC manufacturing, antibody-drug conjugates, cytotoxins.

Anti CD-30 Antibody-Drug Conjugate Brentuximab Vedotin (ADCETRIS®) May Be a Promising Treatment Option for Systemic Mastocytosis (SM).

Abstract 2857▪▪This icon denotes a clinically relevant abstractMastocytosis is a clonal proliferation of abnormal mast cells in single or multiple organs leading to clinical syndromes ranging from a benign self-resolving cutaneous disease to the highly aggressive malignancy, mast cell leukemia (MCL). Mastocytosis is categorized under myeloproliferative neoplasms in the 2008 World Health Organization (WHO) classification of hematopoietic and lymphoid diseases. In almost all cases of SM, the bone marrow (BM) is involved by atypical mast cells. The characteristic Asp816Val (D816V) mutation in the KIT gene present in most cases gives the atypical mast cells a survival advantage that leads to treatment resistance with tyrosine kinase inhibitor (TKIs) like imatinib. According to the 2008 WHO classification, 1 major and 1 minor or 3 minor criterion are required for the diagnosis of systemic mastocytosis (Fig. 1a). Systemic Mastocytosis (SM) is subcategorized into four distinct categories in order of indolent to highly aggressive disease: Indolent Systemic Mastocytosis (ISM), SM with an associated hematologic non-MC-lineage disease (SM-AHNMD), aggressive SM (ASM) and MCL. SM with C findings categorized as ASM (Fig. 1b) is treated with cytoreductive chemotherapy such as interferon 2α or 2-chlorodeoxyadenosine (2-CdA). The overall response rate (ORR) with these agents is 40–50% with significant chemotherapy-related toxicity and short durable responses. The majority of patients with ASM and MCL relapse within a year with a very poor prognosis.The CD30 (Ki-1) antigen is expressed in the majority of ASM cases. Thus, CD30 can serve as a potential therapeutic target as well as a reliable tumor marker to follow disease status. Brentuximab vedotin is an antibody-drug conjugate compound consisting of a chimeric monoclonal antibody against CD30 linked to the antimitotic agent monomethyl auristatin E (MMAE). Here we report evidence of anti-tumor activity in two patients with CD30-positive ASM treated with brentuximab vedotin. Case #1:A 62 year old white male with significant medical history of rheumatoid arthritis, diabetes mellitus and coronary artery disease was evaluated for pancytopenia and hepatomegaly. His initial BM aspiration and biopsy revealed multifocal dense mast cell infiltrate (30–40% involvement with tryptase and CD117 positivity). His initial serum tryptase level was 276 ng/ml. He was treated with 2-CdA continuous infusion for three cycles with no significant response and substantial toxicities. He was subsequently enrolled in a clinical trial with brentuximab vedotin given every 3 weeks at 1.8 mg/kg after confirmation of CD30 positivity on BM aspirate and biopsy (Fig. 2). His peripheral blood counts started to improve after the 5thcycle and he had a durable partial response as assessed by his peripheral blood counts and BM biopsies (Fig 2). Importantly, his sequential bone marrow biopsies showed a decrease in mast cell involvement and CD30 intensity with improved normal marrow cellularity (Fig 2). His only treatment-related toxicities were grade II neuropathy and neutropenia for which his dose was reduced to 1.2 mg/kg. He is currently asymptomatic with ECOG performance status of 0 and does not require any growth factor support at Cycle 12 of this regimen. Case #2:A 79 yr old white male with significant medical history of hypertension, obstructive sleep apnea, coronary artery disease s/p CABG was referred for new diagnosis of ASM on BM biopsy (60–70% marrow involvement with 3+ mast cell density). His initial tryptase level was 224ng/ml. He was enrolled on the same clinical protocol as described above. His sequential BM biopsies revealed significant reduction in mast cell density with mild improvement in overall normal cellularity after 3 cycles of treatment. (Fig 3). Conclusion:Brentuximab vedotin is a promising targeted therapy for SM and needs to be confirmed further by a prospective multicenter clinical trial. In two patients reported here treatment is well tolerated, targets the malignant mast cells and seems to prevent disease progression in a rare disorder with few treatment options and limited response rates. [Display omitted] [Display omitted] [Display omitted] Disclosures:Off Label Use: Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate compound consisting of a chimeric monoclonal antibody against CD30 linked to the antimitotic agent monomethyl auristatin E (MMAE). It is not FDA approved for use in Mastocytosis.

Specific Tumor Targeting and Potent Bystander Killing with SGN-35, an Anti-CD30 Antibody Drug Conjugate.

CD30 is expressed on the surface of Reed Sternberg cells in Hodgkin's Lymphoma (HL) and on anaplastic large cell lymphoma (ALCL) cells. The anti-mitotic agent monomethyl auristatin E (MMAE) has been conjugated to cAC10, the anti-CD30 monoclonal antibody SGN-30, to yield the antibody-drug conjugate SGN-35. SGN-35 is internalized into lysosomes of CD30-positive cells and MMAE is released intracellularly when the protease-sensitive linker is cleaved by lysosomal proteases such as cathepsin B. In mouse xenograft models of HL and ALCL, complete remissions have been observed at well tolerated doses of SGN-35. To characterize further the mode of action in vivo, mice with established CD30-positive xenografts were treated with SGN-35, and the concentration of released MMAE was measured in the tumor and serum. The tumor concentrations of released MMAE achieved were several hundred-fold above the IC50 for MMAE in tumor cell cultures. Peak tumor to serum ratios for released MMAE reached more than 1000:1. In tumors and serum, the peak released MMAE concentrations were achieved 3 days and 1 hour post dose, respectively. As a considerable portion of the tumor volume in HL consists of a CD30-negative cellular infiltrate, the ability of SGN-35 to kill CD30-negative cells in mixed cell populations was investigated. First, cell growth medium from the SGN-35-treated HL cell line L540cy (CD30 positive) was shown to be cytotoxic to the SGN-35-insensitive Ramos cells (CD30 negative). Measurements with mass spectrometry confirmed that MMAE was released into the growth medium of SGN-35-treated L540cy cells, and that the observed concentration of MMAE was consistent with the cytotoxic effect on the Ramos cells. Second, a mixed culture of Karpas 299 cells (ALCL, CD30 positive) and Ramos cells was treated with SGN-35 and examined by FACS. The viability of both cell populations was reduced or eliminated, in contrast to proliferation observed in single Ramos cultures treated with SGN-35 or untreated mixed cultures. These results demonstrate that SGN-35 effectively targets the potent cytotoxic agent MMAE to CD30-positive tumor cells, while free MMAE released by these tumor cells is also available for killing CD30-negative infiltrating bystander cells. SGN-35 is a promising treatment for tumors with homogeneous (ALCL) and heterogeneous (HL) expression of CD30. Clinical trials with SGN-35 are expected to begin by the end of 2006.

Enhanced Activity of Monomethylauristatin F through Monoclonal Antibody Delivery: Effects of Linker Technology on Efficacy and Toxicity

We have previously shown that antibody-drug conjugates (ADCs) consisting of cAC10 (anti-CD30) linked to the antimitotic agent monomethylauristatin E (MMAE) lead to potent in vitro and in vivo activities against antigen positive tumor models. MMAF is a new antimitotic auristatin derivative with a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE, most likely due to impaired intracellular access. In vitro cytotoxicity studies indicated that mAb-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mAb-L1-MMAF) conjugates were >2200-fold more potent than free MMAF on a large panel of CD30 positive hematologic cell lines. As with cAC10-L1-MMAE, the corresponding MMAF ADC induced cures and regressions of established xenograft tumors at well tolerated doses. To further optimize the ADC, several new linkers were generated in which various components within the L1 linker were either altered or deleted. One of the most promising linkers contained a noncleavable maleimidocaproyl (L4) spacer between the drug and the mAb. cAC10-L4-MMAF was approximately as potent in vitro as cAC10-L1-MMAF against a large panel of cell lines and was equally potent in vivo. Importantly, cAC10-L4-MMAF was tolerated at >3 times the MTD of cAC10-L1-MMAF. LCMS studies indicated that drug released from cAC10-L4-MMAF was the cysteine-L4-MMAF adduct, which likely arises from mAb degradation within the lysosomes of target cells. This new linker technology appears to be ideally suited for drugs that are both relatively cell-impermeable and tolerant of substitution with amino acids. Thus, alterations of the linker have pronounced impacts on toxicity and lead to new ADCs with greatly improved therapeutic indices.

Reduction−Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.