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Novel Payloads for Antibody-drug Conjugates

The development of antibody-drug conjugates or ADCs, has resulted in the need of developing novel compounds that can be used as payloads.

Enediynes, represented by are two different classes of antitumor compounds, including the nine-membered cyclic enediynes such as kedarcidin, LDM (Lidamycin, also known as C-1027), maduropeptin, and ten-membered cyclic enediynes such as calicheamicin, esperamycin, and dynemicin, were first discovered in 1987.

Since the discovery of calicheamicin and esperamicin, several more enediynes have been discovered as natural products in bacteria. Synthetic enediynes have also been designed to improve the functionality of the naturally occurring enediynes.

The distinctive strained nine- or ten-member ring system comprising a Z-carbon-carbon double bond and two carbon-carbon triple bonds, are usually arranged with the latter two flanking the former.

Produced by a variety microorganism, including of Actinomadura verrucosospora and Micromonospora echinospora, these compounds present intricate mechanisms of action as well as remarkable biological activities.

Generally speaking, enediynes, which are potent cytotoxic agents (IC50 values in the picomolar range) which damagers of DNA causing single and double strand cuts, are too toxic for clinical use. The potency of these compounds is attributed to their ability to bind to DNA and undergo a Bergmann rearrangement (also known as Bergman cyclization*) in which the strained ring system is converted into a highly reactive 1,4-benzenoid diradical, which damages the DNA by abstracting hydrogens from it.

When the diradical is generated near DNA, it abstracts hydrogen atoms from the sugar backbone of the DNA molecule, which results in single and double strand lesions.

Although this makes enediynes these very toxic, their potent activity can be beneficial when targeting the DNA of cancerous tumors.

Interestingly, most endiynes offer potent activity against the proliferation of various cancer cells including those with resistance to other chemotherapeutic drugs. [1]

Antibody-drug conjugate
The biological evaluation of a select number of enediynes and enediyne analogues has led to the identification of a variety of novel compounds with low picomolar potencies against certain cancer cell lines. Still too toxic as a single agent, these compounds have been used as payloads for antibody-drug conjugates.

Calicheamicin, first discovered in the mid-1980’s, a phenomenally active compound, extremely active against tumor cells was synthesized it in 1992. Linking the compound to an antibody, scientists were able to deliver it to certain cancer types selectively without the side effects of the very toxic compound.

Among the developed therapeutic agents is gemtuzumab ozogamicin (Mylotarg®; Pfizer/Wyeth), an antibody-drug conjugate in which calicheamicin was conjugated with recombinant humanized IgG4 kappa antibody, which binds to CD33 antigens expressed on the surface of leukemia blasts. The drug was approved for the treatment of myeloid leukemia.

Another therapeutic agent in this class is CMC-544 (inotuzumab ozogamicin; Blincyto®; Pfizer/Wyeth) a calicheamicin-conjugated anti-CD22 monoclonal antibody, a highly potent cytotoxic enediyne antibiotics that bind DNA in the minor groove and cause double strand DNA breaks (dsDNAB) leading to cell death. This agent was approved in 2016 for the treatment of adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL).

But not all calicheamicin-conjugated have been successfully developed. CMB-401, for example, an antibody-drug conjugate consisting of the monoclonal antibody hCTM01 directed against polymorphic epithelial mucin covalently bound to the cytotoxic antibiotic calicheamicin by an amide linker.

Although CMB-401 showed targeted killing of MUC1-expressing cells in vitro and produced pronounced dose-related antitumor effects over an eightfold dose range against an MUC1-expressing ovarian carcinoma xenograft (OvCar-3), the drug did not meet the criteria for partial remission. Based on published efficacy of conjugates that deliver calicheamicin via hybrid (bifunctional) linkers [e.g. gemtuzumab ozogamicin, in acute myeloid leukemia, the scientists hypothesize that the amide linker used in CMB-401 may have contributed to its failure to induce a partial response. [2]

Rice University scientists have improved the production of a potent antitumor antibiotics from the enediyne class known as uncialamycin.

Image 1.0: Uncialamycin

, which depending on the epimer and cell line or subline has shown activity against several ovarian tumor cell lines with IC50 values ranging from 9 × 10–12 to 1 × 10–10, has been recognized to be among the rarest and most potent, yet one of the structurally simpler agents, making it attractive for chemical synthesis and potential applications in biology and medicine.

Rice University scientists have developed synthetic strategies and technologies and applied these to the synthesis of a number of uncialamycin analogues. Equipped with suitable functional groups for conjugation to antibodies, uncialamycins are suitable as a payload for antibody-drug conjugates and other delivery systems.

The potency, efficacy and mechanism of action of uncialamycin analogs was demonstrated by scientists at Bristol-Myers Squibb Research & Development with the development of a highly potent uncialamycin analog with a valine-citrulline dipeptide linker conjugated to an anti-mesothelin monoclonal antibody through lysines to generate a novel antibody-drug conjugate. This investigational drug demonstrated subnanomolar potency (IC50 = 0.88 nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. [3]

Lidamycin or LDM (original named C-1027, Lidamycin was isolated from the broth filtrate of Streptomyces globisporus C1027 ) is an antitumor antibiotic which shows extremely potent cytotoxicity toward human cancer cells with IC50 values 1000-fold lower than that of Adriamycin in vitro.

In clinical trials, the compound, which consist of 2 independent parts, an apoprotein moiety (LDP) which forms a hydrophobic pocket to protect the chromophore and a non-protein active enediyne chromophore (AE) responsible for the extremely potent bioactivity, showed remarkable inhibition on a panel of transplantable tumors in mice.

Lidamycin can induce cell damage including apoptosis, cell cycle arrest, and DNA double-strand breaks and is considered to be a desirable cytotoxic payload for antibody-drug conjugates due to its extremely potent cytotoxicity to cancer cells.[4]

Scientists at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, have developed anti-CD30-LDM, a novel ADC which consists of the intact chimeric antibody directed against CD30 and Lidamycin.

The investigational anti-CD30-LDM agent shows attractive tumor-targeting capability and antitumor efficacy both in vitro and in vivo and could be a promising candidate for the treatment of CD30+ lymphomas, including Hodgkin’s lymphoma (HL) and anaplastic large-cell lymphoma (ALCL).[5]

Thailanstatin A
Today, more than 60% of ADCs in clinical trials employ microtubule inhibitors (auristatins or MMAE/MMAF and maytansinoids or DM1/DM4) as their payloads. [6]

Scientists looking for cytotoxic payloads beyond the microtubule inhibitor class, a potent payload for lysine conjugated antibody-drug conjugate called Thailanstatin A was introduced.

A number of variations of Thailanstatin A, a natural product analog of spliceostatin, a complex of proteins and ribonucleoproteins that regulate DNA splicing originally isolated from burkholderia thailandensis bacteria, are being investigated as a potential payload.
The novel, extremely potent, compound fights cancer by inhibiting the machinery in the cell that edits messenger RNA after transcription from DNA but before its translation into proteins.

Scientists at Pfizer’s Oncology-Rinat Research & Development and Pfizer’s Drug Safety Research and Development, in Pearl River, New York prepared a series of thailanstatin-antibody conjugates in order to evaluate their potential in the treatment of cancer.

After exploring a variety of linkers, they found that the most potent antibody-drug conjugates were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (also known “linker-less” conjugates).

Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells.

The researchers noted that the exposure of the thailanstatin-conjugates was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ado-trastuzumab emtansine (Kadcyla®; Genentech/Roche). [7]

Scientists at Pfizer’s showed that there is a high dependence of the potency of thailanstatin based antibody-drug conjugates with the site of conjugation. They showed that site-specific thailanstatin antibody-drug conjugates were very efficacious in an in vivo gastric cancer tumor xenograft model thus demonstrating the suitability of this novel class of payload for consideration in next-generation site-specific ADC programs.

A library of payloads
The development of novel, potent, payloads for antibody-drug conjugates with different mechanisms of action, has, over the last decades, challenged synthetic organic chemists to develop a library of potential payloads designed to address the medical needs for the various types of cancers.

In a recent review, scientists at Rice University describe their work of total synthesis of natural and designed molecules of the calicheamicin, uncialamycin, tubulysin, trioxacarcin, epothilone, shishijimicin, namenamicin, thailanstatin, and disorazole families of compounds. In their review they demonstrate how these novel compounds led to the discovery of analogues of higher potencies, yet some of them possessing lower complexities than their parent compounds as potential payloads for antibody-drug conjugates.[8]

These compounds and others like them may serve as powerful payloads for the development of antibody-drug conjugates intended for personalized targeted cancer therapy.

[1] Abdel-Magid AF. New synthetic enediynes and their conjugates may provide effective treatment for cancer. ACS Med Chem Lett. 2013 Sep 20;4(11):1018-9. doi: 10.1021/ml400362m. eCollection 2013 Nov 14.
[2] Chan SY, Gordon AN, Coleman RE, Hall JB, Berger MS, Sherman ML, Eten CB, Finkler NJ. A phase 2 study of the cytotoxic immunoconjugate CMB-401 (hCTM01-calicheamicin) in patients with platinum-sensitive recurrent epithelial ovarian carcinoma. Cancer Immunol Immunothery 2003 Apr;52(4):243-8. Epub 2003 Feb 26.)
[3] Chowdari NS, Pan C, Rao C, Langley DR, Sivaprakasam P, Sufi B, Derwin D, Wang Y, et al. Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted cancer therapy. Bioorg Med Chem Lett. 2018 Dec 11. pii: S0960-894X(18)30955-7. doi: 10.1016/j.bmcl.2018.12.021. [Epub ahead of print]
[4] Zhang Y, Liu R, Fan D, Shi R, Yang M, Miao Q, Deng ZQ, Qian J, Zhen Y, Xiong D, Wang J. The novel structure make LDM effectively remove CD123+ AML stem cells in combination with interleukin 3. Cancer Biol Ther. 2015;16(10):1514-25. doi: 10.1080/15384047.2015.1071733. Epub 2015 Jul 17.
[5] Wang R, Li L, Zhang S, Li Y, Wang X, Miao Q, Zhen Y. A novel enediyne-integrated antibody-drug conjugate shows promising antitumor efficacy against CD30+ lymphomas. Mol Oncol. 2018 Mar;12(3):339-355. doi: 10.1002/1878-0261.12166. Epub 2018 Jan 26.
[6] Fu Y, Ho M. DNA damaging agent-based antibody-drug conjugates for cancer therapy. Antib Ther. 2018 Sep;1(2):33-43. doi: 10.1093/abt/tby007. Epub 2018 Aug 30.
[7] Puthenveetil S, Loganzo F, He H, Dirico K, Green M, Teske J, Musto S1, Clark T, et al. Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads. Bioconjug Chem. 2016 Aug 17;27(8):1880-8. doi: 10.1021/acs.bioconjchem.6b00291. Epub 2016 Jul 28.
[8] Nicolaou KC, Rigol S. Total Synthesis in Search of Potent Antibody-Drug Conjugate Payloads. From the Fundamentals to the Translational. Acc Chem Res. 2018 Dec 21. doi: 10.1021/acs.accounts.8b00537. [Epub ahead of print]

* The Bergman cyclization or Bergman reaction or Bergman cycloaromatization is an organic reaction and more specifically a rearrangement reaction taking place when an enediyne is heated in presence of a suitable hydrogen donor.

Last Editorial Review: January 3, 2019

Featured Image: Laboratory assistant. Courtesy: © 2010 – 2019 Fotolia. Used with permission. Image 1.0: Uncialamycin Courtesy: © 2010 – 2019 Rice University. Used with permission.

Copyright © 2019 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.



AACR 2018: Highlighting Novel Antibody-Drug Conjugate Technologies and Immuno-Oncology

This year, the American Association for Cancer Research (AACR) will host their annual meeting April 14 – 18, 2018 in the McCormick Place North/South in Chicago, Illinois, USA.

Since the 2017 annual meeting, a dramatic wave of progress in against cancer has come to fruition and basic science findings have resulted in new drug approvals. Much has been accomplished in expanding the  use of genomic data for precision medicine and a greater focus on “big data” has helped accelerate progress in cancer research. ‘Big data’ has become crucial in helping scientists use mathematics, engineering, and artificial intelligence to diagnose cancer at an earlier stage, which helps improves outcomes.

In the last 12 months there has been a more focused and concentrated efforts to help eliminate persistent disparities – helping minorities and the medically underserved.

These developments are definitely reflected in the theme of the American Association for Cancer Research annual meeting: ‘Driving Innovative Cancer Science to Patient Care.’

The meeting is expected to draw more than 20,000 scientists, clinicians, advocates, and others to discuss advances in the field of cancer science. The multidisciplinary program will include an outstanding roster of speakers, hundreds of invited talks.

Antibody-drug Conjugates
Among the many papers and presentations, expect to find data highlights from nine presentations showcasing the Seattle Genetics’ innovative, proprietary antibody-drug conjugate or ADC platform technologies as well as its emerging immuno-oncology pipeline.

The data to be presented includes preclinical and clinical advances with brentuximab vedotin (Adcetris®; Seattle Genetics/Takeda), ladiratuzumab vedotin, SGN-CD48A and SGN-2FF as well as  the first preclinical data describing the novel empowered antibody SEA-BCMA, being developed by Seattle Genetics.

“We are an emerging multi-product company, advancing a substantial pipeline of targeted therapies for patients with solid tumors and blood cancers,” said Dennis Benjamin, Ph.D., Senior Vice President of Research at Seattle Genetics.

“New data featured in nine presentations underscore our commitment to scientific innovation and the needs of patients. These data presentations highlight the potential combination of ADCs with checkpoint inhibitors, novel ADC payloads, antibody masking technologies and progress with our immuno-oncology program, SGN-2FF. We are also presenting preclinical data for our new multiple myeloma program, SEA-BCMA, which has a phase 1 study scheduled to start this year,” Benjamin added.

The information presented includes an oral and poster presentations on Sunday and Monday, April 15 and 16, 2018, respectively (Abstracts #930, 2803), showcasing preclinical data evaluating proprietary NAMPT inhibitors and auristatins as ADC payloads. The data show NAMPT inhibitors have a unique mechanism of action and encouraging therapeutic window. Preclinical data also describe the development of novel auristatin payloads with potential application across multiple tumor types.

An innovative approach to masking antibodies for tumor specific activation will be featured in a poster presentation on Sunday, April 15, 2018 (Abstract #250). Preclinical data demonstrate that coiled-coil masked antibodies and antibody-drug conjugates show improved tolerability and equivalent antitumor activity compared to unmasked counterparts. The data suggest this technology may be applied to a range of antibodies or ADCs and could enable their development against previously inaccessible cancer targets.

The novel preclinical program SEA-BCMA will be highlighted in a poster presentation on Tuesday, April 17, 2018 (Abstract #3833). The cell surface protein BCMA is expressed on cells of several cancer types, including multiple myeloma and other B cell malignancies. SEA-BCMA is an antibody empowered using Seattle Genetics’ proprietary Sugar Engineered Antibody (SEA) technology designed to enhance antibody effector functions. The preclinical data support initiation of a phase I trial for multiple myeloma, which is planned for 2018.

Clinical biomarker data from a phase I trial evaluating the novel immuno-oncology agent SGN-2FF in patients with advanced solid tumors will be shown in a poster presentation on Wednesday, April 18, 2018 (Abstract #5551). The preliminary data demonstrate the biological effects of SGN-2FF and support further development of this novel immuno-oncology agent.

Three poster presentations on Monday and Wednesday, April 16 and 18, 2018 (Abstracts #1789, 2742, and 5619) will highlight preclinical data evaluating the ability of brentuximab vedotin, ladiratuzumab vedotin and SGN-CD48A, each of which are auristatin-based ADCs, to elicit additional mechanisms of action, including immunogenic cell death. These data support clinical evaluation in combination with checkpoint inhibitors. Brentuximab vedotin and ladiratuzumab vedotin are being evaluated in combination with checkpoint inhibitors in multiple ongoing clinical trials.

ADC Therapeutics
Other data includes the latest updated from investigational programs highlight strong preclinical data for its two new investigational programs ADCT-601 targeting AXL and ADCT-701 targeting DLK-1 being developed by ADC Therapeutics. In addition, Jaewoong Lee, Ph.D, of The Beckman Institute of the City of Hope is expected to present the latest update on novel preclinical data for ADCT-301 targeting CD25.

“Our two new investigational programs show compelling efficacy and safety in preclinical studies,” noted Jay M. Feingold, MD, Ph.D, Chief Medical Officer and Senior Vice President of Clinical Development at ADC Therapeutics.

“These results provide an important step to advance ADCT-601 and ADCT-701 into the clinic and enlarge our pipeline of PBD-based ADCs in multiple ongoing clinical trials for the treatment of both solid and hematological cancers,” Feingold added.

Presentations include updated about ADCT-701, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting DLK1-expressing tumors. (Abstract #744, April 15, 1:00 pm – 5:00 pm CT). The ADC is site-specifically conjugated using GlycoConnect™ technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The investigational agent has demonstrated potent and specific in vitro and in vivo anti-tumor activity in DLK1-expressing cancer-derived models and it was stable and well tolerated in rats.

Another updated includes the latest update of the preclinical activity of ADCT-601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting AXL-expressing tumors (Abstract #2792A, April 16, 1:00 pm – 5:00 pm CT). ADCT-601 is an ADC composed of a humanized IgG1 antibody against human AXL, site-specifically conjugated using GlycoConnect™ technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The investigational agent has demonstrated potent and specific in vitro and in vivo anti-tumor activity in various cancer-derived models with different levels of membranous AXL, and it was stable and well tolerated in rats.

CD25 enables oncogenic BCR- and TCR-signaling and represents a therapeutic target in lymphoblastic malignancies (Abstract #2983, April 16, 2018, 4:05 PM – 4:20 PM). Novel data identifies CD25 as a previously unrecognized feedback regulator of oncogenic B/TCR-signaling supporting CD25 as a therapeutic target in refractory lymphoid malignancies. ADC Therapeutics’ ADCT-301 has demonstrated durable remissions in patient-derived Ph+ ALL cells PDX models

Eleven Biotherapeutics
Eleven Biotherapeutics, a late-stage clinical company developing next-generation antibody-drug conjugate therapies is expected to present preclinical data from the company’s novel, next-generation ADC program using an innovative deBouganin cytotoxic protein payload.

“We have uniquely designed our deBouganin payload to address tumor indications that can only be reached through systemic delivery. Our data show that deBouganin exhibits certain advantages over first-generation ADCs, which use more conventional small molecule cytotoxins, with respect to cell killing power, including the ability to kill cancer stem cells, circumvent multi-drug resistance and avoid cross-resistance mechanisms,” said Gregory P. Adams, Ph.D., Chief Scientific Officer of Eleven Biotherapeutics.

“We are pleased to be presenting these promising data highlighting the potential activity and differentiation of our approach compared to first-generation ADCs,” he added.

DeBouganin is a proprietary de-immunized variant of bouganin, a ribosome inactivating protein that when internalized blocks protein synthesis, thereby leading to cell death. Eleven Biotherapeutics will present data from its VB6-845d program, a next-generation ADC comprised of a Fab fragment specific for the epithelial cell adhesion molecule (EpCAM) genetically linked to deBouganin via a furin protease sensitive peptide. Data being presented suggest that VB6-845d mediates tumor cell killing by an immunogenic cell death (ICD) pathway. The potential cross-priming effect initiated by VB6-845d-induced ICD suggests that VB6-845d in combination with immune checkpoint inhibitors may enhance their effectiveness in EpCAM-positive epithelial cancers. Poster presentation: VB6-845d Tumor Cell Killing Elicits Biologic Features of Immunogenic Cell Death (Date and Time: April 16, 2018 from 1:00 to 5:00 p.m. CT)

Targeted protein therapeutics or TPTs are single-protein therapeutics composed of targeting moieties genetically fused via peptide linker to cytotoxic protein payloads. TPTs are designed to overcome efficacy and safety challenges inherent within ADCs. Poster presentation: Engineering and Characterization of Anti-PSMA Humabody-DeBouganin Fusion Proteins (April 18, 2018 from 8:00 a.m. to 12:00 p.m. CT).

Last Editorial Review: April 12, 2018

Featured Image: New Orleans, LA – The AACR 2016 Annual Meeting. Courtesy: © AACR/Scott Morgan. Used with permission.

Copyright © 2013 – 2018 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.


Pfizer and Seattle Genetics Agree on Antibody-Drug Conjugate Collaboration

Pfizer and Seattle Genetics, a clinical-stage biotechnology company focused on the development and commercialization of monoclonal antibody-based therapies for the treatment of cancer , have entered into a collaboration in which Pfizer will pay an upfront fee of $8 million for rights to utilize Seattle Genetics’ antibody-drug conjugate (ADC) technology with antibodies to a single oncology target.

“This collaboration reflects the increasing value of our ADC technology and strong interest in its potential among leaders in the drug development community,” said Eric L. Dobmeier, Chief Business Officer of Seattle Genetics. “We now have ten ongoing ADC collaborations, six collaborator ADCs using our technology are in clinical development, and several additional programs are advancing towards the clinic. We have generated more than $145 million from ADC licensing, and we have the potential to receive significant future milestones and royalties for ADCs developed by our collaborators.”

Under the terms of the agreement Pfizer is responsible for research, product development, manufacturing and commercialization of any ADC products under the collaboration. Seattle Genetics is eligible to receive from Pfizer over $200 million in progress-dependent milestones as well as royalties on worldwide net sales of any resulting ADC products. Seattle Genetics also will receive material supply and annual maintenance fees as well as research support payments for assistance provided to Pfizer under the collaboration.

Antibody-drug conjugate are monoclonal antibodies that selectively deliver potent anti-cancer agents to tumor cells. With over a decade of experience and knowledge in ADC innovation, Seattle Genetics has developed proprietary technology employing synthetic, highly potent cell-killing agents called auristatins (such as MMAE and MMAF) and stable linker systems that attach auristatin to the antibody. Seattle Genetics’ novel linker systems are designed to be stable in the bloodstream and release the potent cell-killing agent once inside targeted cancer cells. This approach is intended to spare non-targeted cells and thus reduce many of the toxic effects of traditional chemotherapy while enhancing the antitumor activity.


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