Teicher, Beverly A.
The challenges posed by development of therapeutic antibody drug conjugates (ADCs) are formidable. Over the past 25 years, many cell surface proteins that have selective aberrant expression on malignant cells or are aberrantly highly expressed on the surface of malignant cells have been identified. In many cases, specific antibodies that bind tightly to malignant cell surface proteins were developed. However, often these antibodies were not active antitumor agents. ADCs may provide an opportunity to make use of antibodies that are specific to cell surface proteins . Successful ADCs have improved tumor specificity and potency compared with traditional drugs [2▪▪,3]. However, heterogeneity of antibody target expression on the tumor surface and expression of the antigen by normal tissues can limit the effectiveness of ADCs. The various antibody conjugate advantages and disadvantages are given as follows:
- targeted therapeutic binding specifically to the target antigen;
- deliver highly potent agents selectively to tumor cells;
- wide therapeutic index;
- prolonged circulation half life: conjugate remaining stable in circulation;
- decreased adverse effects;
- tumor may need to be tested for expression of the antigen;
- molecular target may have some normal tissue expression potentially leading to toxicity;
- toxic payload may have some premature release;
- antibody conjugate may not reach the target cells in sufficient concentration to be lethal;
- antigen expression could be heterogeneous, especially in solid tumors.
For some hematological malignancies, antigen expression is specific and homogeneous, although the actual number of antigens on the cell surface may be lower than that found on solid tumors.
Early ADCs were composed of tumor-specific murine monoclonal antibodies covalently linked to anticancer drugs, such as doxorubicin, vinblastine, and methotrexate. These early conjugates were evaluated in human clinical trials but had limited success because of immunogenicity, lack of potency, and insufficient selectivity for tumor versus normal tissue. The lessons learned from these early explorations led to improvements in essentially all aspects of antibody conjugate therapeutics and hence to renewed interest in ADC technology . Immunogenicity was overcome by replacing murine antibodies with humanized or fully human antibodies. Potency was improved by using drugs that were hundred-fold to thousand-fold more cytotoxic than previously used drugs. Selectivity was addressed by more careful target and antibody selection. As the result of such improvements, in 2000, gemtuzumab ozogamicin (Mylotarg) became the first ADC to be approved by the United States Food and Drug Administration (FDA) for the treatment of acute myelogenous leukemia. However, Mylotarg was withdrawn from the market in 2010 because in postmarketing follow-up clinical trials, it failed to meet the prospective efficacy targets. Two ADCs, trastuzumab emtansine (T-DM1, Kadcyla) and brentuximab vedotin (SGN-35, Adcetris), reached FDA approval in 2013 and 2012 for treatment of metastatic breast cancer and refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma (ALCL), respectively. More than 20 ADCs are in clinical trials (Table 1).
TARGETS AND ANTIBODIES
In selecting cell surface protein targets, whether on malignant cells, malignant disease-associated cells (e.g., tumor endothelial cells), or in the tumor microenvironment, it is important that the antigen expression is abundant on the target cells or in the tumor region and limited on all other cells and normal tissues [5–13]. The patient whose tumor expresses high levels of the target antigen is most likely to benefit from treatment. ADCs are targeted, potent cytotoxic agents. Most of the proteins being targeted with antibody conjugates are normal proteins, as opposed to mutant proteins; therefore, some antigen expression on normal cells is possible and even likely. Technologies for antibody discovery, development, and engineering are well established. Phage display libraries and humanized mice can produce fully human antibodies, and mouse antibody humanization can result in highly specific nonimmunogenic antibodies (Fig. 1). In most cases, the most appropriate antibody for ADC therapeutics requires that the antibody-target complex internalize into the target cells in which the drug is released. The drugs most widely applied to ADCs target tubulin or DNA and are uniformly highly potent cytotoxic agents with half maximal inhibitory concentration (IC50) values in the picomolar range in cell culture.
ADCs are among the most tumor-selective anticancer therapeutics developed to date; however, only a small fraction of the drug reaches the intracellular target. The maytansinoids and dolastatin analogs target tubulin, and both suppress microtubule dynamics [14–16]. The duocarmycins and calicheamicins target the minor groove of DNA. The amatoxin analogs inhibit RNA polymerase II and III and SN-38 targets topoisomerase I resulting in DNA double strand breaks [17▪]. These molecules have in common an extreme potency and lack of tumor selectivity, which limits their use as drugs. Dolastatin 10, the parent molecule of the auristatins, underwent clinical trials in the 1990s . Dolastatin 10 development was terminated in 1995 when it failed to demonstrate efficacy in a phase II trial in prostate cancer patients. The maytansinoids are exquisitely potent cytotoxic agents . Maytansinoids are 19-membered macrocyclic lactams that are related to ansamycin antibiotics. Maytansine was developed and assessed in early clinical trials in the early 1980s. The phase II clinical trials were disappointing, with very little evidence of response . Duocarmycins are members of a small family of antibiotics that also includes yatakemycin and CC-1065 . This class of compounds binds to and alkylates DNA in the A-T-rich regions of the double-helix minor groove. Several semisynthetic derivatives of CC-1065 and duocarmycin, including adozelesin, carzelesin, bizelesin, and KW2189, were evaluated in early clinical trials [22–24]. In each case, dose-limiting toxicities occurred at doses too low to achieve antitumor activity. The calicheamicins bind in the minor grove of DNA in a sequence-specific manner and induce double-strand breaks, causing cell death [25–27]. Calicheamicin has a narrow therapeutic index and serious late toxicities, the development of calicheamicin as a single-agent therapeutic was not pursued. SN-38 is the active metabolite of the camptothecin prodrug, irinotecan, which is a solid cancer therapeutic, particularly colorectal cancer. SN-38 is a highly potent (low nM) camptothecin derivative that targets topoisomerase I, an enzyme that cleaves DNA. SN-38 is the active antitumor species formed from irinotecan. Although SN-38 is potent, it is poorly bioavailable and has a narrow therapeutic index. However, SN-38 can be conjugated to antibodies with coupling on the 20th position of SN-38, thus maintaining SN-38 in the active lactone form [28▪]. Amatoxins are highly cytotoxic cyclopeptides produced by poison mushrooms, such as the death cap (Amanita phalloides) and the destroying angel [29,30]. RNA polymerase II and III are the targets of amatoxins [31–33]. The well recognized normal tissue toxicities of the amantins precluded their clinical exploration. ADCs are an effective method to increase the therapeutic index of these highly potent cytotoxic agents. The drugs used in ADCs must have sufficient water solubility and prolonged stability in aqueous formulations and plasma, a functional group that is suitable for conjugation with a linker and must not be readily susceptible to lysosomal enzyme degradation. Consistent with the potent nature of the drug, ADCs are often scheduled like cytotoxic chemotherapy in clinical regimens, with dosing once every 3 weeks [34,35].
Linkers that are short spacers that covalently couple the drug to the antibody protein must be stable in circulation (Fig. 1). Inside the cell, most linkers are labile; however, some are stable, requiring degradation of the antibody and linker to release the cytotoxic agent. Thus, linkers are a key component of antibody-conjugate structures [36–39]. Many currently used linkers react with Lys side chains throughout the antibody or with the sulfhydryls in the hinge regions of the antibody. Linkers in clinical use include acid-labile hydrazone linkers that are degraded under the low pH conditions found in lysosomes. Disulfide-based linkers are selectively cleaved in the cytosol in the reductive intracellular milieu . Noncleavable thioether linkers release the small-molecule drug after degradation of the antibody in the lysosome, and peptide linkers, such as citrulline-valine, are stable in circulation and degraded by lysosomal proteases in cells. Linkers using l-Ala and d-Ala with attachment to an aniline-bearing maytansinoid are being explored [41▪▪]. Beta-glucuronide linkers have been shown to deliver more intact monomethyl auristatin E (MMAE) into cells [42▪▪]. Linkers with polyethylene glycol spacers have been developed in an effort to increase the solubility of the conjugate [43–45]. Finally, hydrophilic, fully biodegradable polyacetal carrier poly(1-hydroxymethylethylene hydroxymethylformal) with chemically orthogonal linkers can carry 20 drug molecules per antibody . Linkers can influence the circulating half-life and safety of conjugates by minimizing the release of the drug molecule in circulation and by optimizing the delivery of the conjugate to the target tissue. Often during the drug development process, investigators will test several linkers in safety and efficacy assays to select the best candidate conjugate.
Drug-loading stoichiometry and molecular homogeneity are important determinants of the safety and efficacy of antibody conjugates (Fig. 1). The goal is to produce ADCs that are single-chemical species or nearly single-chemical species. Under-conjugated antibodies are avoided because they decrease the potency of the ADC, and highly conjugated species are avoided because they have markedly decreased circulating half-lives and impaired binding to the target protein, thus decreasing the potency and efficacy of the ADC . For most ADCs, linkage of three to four drug molecules per antibody molecule is optimal maintaining the circulating half-life to near that of the naked antibody, preserving antibody binding to the target protein, and delivering sufficient numbers of drug molecules to the target cell to be lethal. Several site-specific conjugation approaches are being explored in an effort to achieve ADCs that are single-chemical species. Antibodies with site-specifically incorporated nonnative amino acids have been very efficiently produced in stable cell lines derived from a Chinese hamster ovary cell line. Site-specific ADCs were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized MMAD with either protease-cleavable or noncleavable linkers [48▪▪]. These conjugates had improved in-vitro efficacy as well as in-vivo efficacy and pharmacokinetic stability in rodent models relative to conventional ADCs.
EXAMPLES: GEMTUZUMAB OZOGAMICIN
CD33 is a 67-kD transmembrane cell-surface glycoprotein of the sialoadhesin family that is expressed by mature and immature myeloid cells and erythroid, megakaryocyte, and multipotent progenitor cells . Gemtuzumab ozogamicin (Mylotarg), a first-generation ADC, is a humanized immunoglobulin G4 (IgG4) anti-CD33 monoclonal antibody conjugated to the antitumor antibiotic calicheamicin . Upon binding of anti-CD33 to the antigen, the complex is rapidly internalized. Intracellularly released calicheamicin binds in the minor groove of DNA and causes double-strand breaks at oligopyridimidineoligopurine tracts. Gemtuzumab ozogamicin was studied in the clinic for over 10 years. In 2010, gemtuzumab ozogamicin was voluntarily withdrawn from the market (Table 1).
CD30 (TNFRSF8), a member of the tumor necrosis factor receptor superfamily, was originally described as a marker of Hodgkin and Reed–Sternberg cells in Hodgkin lymphoma. CD30 is highly expressed on Hodgkin lymphoma and ALCL. Soluble CD30, the extracellular domain of CD30 that is shed, can reduce the effects of CD30-targeting agents by competitive binding. The anti-CD30 antibody designated SGN-30 has potent antitumor activity in vivo, possibly as a mediator of antibody-dependent cellular phagocytosis. SGN-30 has limited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity . The efficacy of SGN-35, an anti-CD30-MMAE conjugate, in Hodgkin lymphoma and ALCL xenograft models as a single agent and in combination with chemotherapeutic regimen, is marked [51,52]. SGN-35 (brentuximab vedotin) was approved in 2012 (Table 1, [52,53▪▪,54]). Brentuximab vedotin was approved by the FDA for two indications: first, patients with Hodgkin lymphoma relapsing after autologous stem-cell transplantation (ASCT), or after two multidrug regimens in patients with Hodgkin lymphoma who are not candidates for ASCT; and second, patients with systemic ALCL who failed at least one prior multidrug chemotherapy regimen. Patients with Hodgkin lymphoma and ALCL treated with brentuximab vedotin showed markedly high response rates for a single agent, exceeding 70 and 80% for Hodgkin lymphoma and ALCL, respectively. The complete response rate was equally as impressive, at 34 and 57% for Hodgkin lymphoma and ALCL, respectively. Cell lines made resistant to brentuximab vedotin became resistant by upregulation of transporter activity (P-gp) which decreased the cytotoxin in the cells or by decreased CD30 on the cell surface along with upregulation of xenobiotic-metabolizing enzymes or by complete loss of CD30 from the cell surface .
CD22 is a B-lymphoid, lineage-specific differentiation antigen that is expressed on both normal and malignant B-cells. Approximately, 85% of acute lymphoblastic leukemia (ALL) cases arise from the B-cell lineage (pre-B-cell or B-ALL). CMC-544 (inotuzumab ozogamicin) is a CD22-targeted anti-CD22-calicheamicin conjugate currently being evaluated in B-cell non-Hodgkin lymphoma patients [49,56]. The anti-CD22 antibody in CMC-544 is a humanized IgG4. Exposure to CMC-544 does not interfere with the antibody-dependent cellular cytotoxicity of rituximab (anti-CD20). Preclinical in-vivo studies explored CMC-544 activity as a single agent and in combination with rituximab . Inotuzumab ozogamicin is in phase III clinical testing (Table 1, ). Although CD22 expression is generally lower on B-ALL cell lines than on B-cell lymphoma cell lines, CMC-544 was a potent cytotoxin toward ALL cells in the same concentration range observed for CD22-positive B-cell lymphoma cells .
CD340 is HER2 (ErbB-2, ERBB2, p185), a member of the epidermal growth factor receptor ErbB protein family of cell surface transmembrane receptor tyrosine kinases. HER2 gene amplification and/or HER2 protein over-expression occurs in 15–25% of breast cancers, as well as in ovarian cancer, stomach cancer, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. Breast cancers are routinely checked for over-expression of HER2 as a diagnostic tool for selecting appropriate patients for treatment with trastuzumab, a humanized mouse HER2-targeted antibody. The anticancer mechanisms attributed to trastuzumab include antibody-dependent cellular cytotoxicity and blockade of HER2 signal transduction, resulting in cell cycle arrest and ultimately cell death. In preclinical studies, the efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid conjugates varied with the linker . Trastuzumab linked to the maytansinoid DM1 showed similar efficacy whether the linker was a nonreducible thioether or a disulfide linker. In trastuzumab-DM1, the noncleavable linker was selected on the basis of the improved in-vivo tolerability of the resulting conjugate. Trastuzumab-DM1 was shown to be an effective anticancer agent even in models refractory to treatment with trastuzumab. T-DM1 was approved by FDA in late 2013 (Table 1, [60,61]). In phase II studies, T-DM1 was active in patients with trastuzumab-refractory and lapatinib-refractory metastatic breast cancer and led to improved progression-free survival, compared with trastuzumab and docetaxel in the first-line setting. In a phase III trial in patients with metastatic breast cancer who previously received trastuzumab and a taxane, T-DM1 resulted in improved progression-free and overall survival, compared with capecitabine and lapatinib. T-DM1 has a favorable toxicity profile; reversible thrombocytopenia and hepatic transaminase elevations are the only grade 3 adverse event present in 5% or more of patients. On the basis of its improved efficacy and toxicity compared with capecitabine or lapatinib, T-DM1 became the standard for patients with HER2+ metastatic breast cancer who have progressed on trastuzumab and a taxane [62▪▪]. The combination of the HER2-directed ADC T-DM1 with the HER2 dimerization inhibitor pertuzumab was explored in cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer, T-DM1 was dose-escalated with a fixed pertuzumab dose. Exposure of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in induction of apoptotic cell death. The presence of the HER3 ligand, heregulin, reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Xenograft studies showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In metastatic breast cancer patients previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be administered at maximal dose with standard-dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity [63,64]. When mechanisms of resistance of cultured cells chronically exposed to T-DM1 were explored, the dominant changes related efflux and metabolism to the cytotoxic small molecule . Exposure to gemcitabine can be used to increase HER2 on the surface of pancreatic cancer cells, thus making them more susceptible to T-DM1 . Other HER2-targeting ADCs are being explored including cleavable linker-duocarmycin bearing ADCs [67,68].
Mesothelin-targeted antibody drug conjugates
Mesothelin (MSLN) is an internalizing tumor differentiation antigen highly expressed by several tumors, including mesothelioma (45%), ovarian (85%), pancreatic (75%), and lung adenocarcinomas. MSLN has limited expression in normal tissues and therefore is an attractive target for an ADC. In fact, there are multiple ADCs directed toward MSLN in development . BAY 94-9343 is a novel ADC consisting of a fully human antimesothelin antibody conjugated to the maytansinoid DM4 via a disulfide containing linker. The antimesothelin antibody binds to human MSLN with high affinity and selectivity, thereby inducing efficient antigen internalization. BAY 94-9343 has potent and selective cytotoxicity of MSLN-expressing cells with an IC50 of 0.72 nM, without affecting MSLN-negative or nonproliferating cells. BAY 94-9343 localized to MSLN-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. BAY 94-9343 has completed phase I clinical trial . Another MSLN-directed ADC includes a high affinity (subnanomolar), humanized antibody conjugated to auristatin antimitotic drugs (MMAE and MMAF) via a noncleavable linker or a cathepsin-cleavable valine-citrulline linker for comparison. Pancreatic, ovarian, and mesothelioma tumor cell lines endogenously expressing MSLN were identified and established as xenografts in mice. A single dose of anti-MSLN ADC was sufficient to inhibit or shrink tumor growth in models of each of the three indications in vivo, as well as inducing complete regressions in primary human pancreatic models, even those expressing low levels of MSLN typical of most human pancreatic tumors. Anti-MSLN-MMAE (at suboptimal doses) appeared to synergize with gemcitabine at clinically relevant doses in a human pancreatic adenocarcinoma (HPAC) xenograft model . A surrogate ADC that cross-reacts with cynomolgus monkey and rat MSLN was generated for nonclinical toxicity studies. The surrogate ADC demonstrated comparable in-vivo efficacy to the lead ADC against human B-cell line (BJAB) xenografts expressing monkey or human MSLN, respectively, thus validating its use in safety studies. In a repeat-dose monkey toxicity study with a clinically relevant dosing schedule, there was no evidence of target-dependent pleuritis, nor any other serositis. A phase I starting dose has been selected .
Prostate-specific membrane antigen targeted antibody drug conjugates
Prostate-specific membrane antigen (PSMA) is a membrane glycoprotein that is expressed in prostate tumors . Androgen receptor, the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway, and PSMA are important targets for prostate cancer therapy. Expression of PSMA, prostate-specific antigen, and androgen receptor was evaluated in cells cultured (LNCaP, C4-2). Cells were tested for susceptibility to PI3K/mTOR inhibitors used alone and in combination with a PSMA ADC, a fully human PSMA monoclonal antibody conjugated MMAE. In androgen-dependent LNCaP cells, rapamycin exerted antiproliferative effects and increased androgen receptor expression without effect on PSMA expression. In androgen-independent C4-2 cells, rapamycin increased androgen receptor expression and PSMA expression without an antiproliferative effect. Androgen receptor inhibitors synergized with PSMA ADC in LNCaP and C4-2 cells. The PSMA ADC synergized with androgen receptor and mTOR inhibitors . The antitumoral activity of a monoclonal antibody targeting the PSMA conjugated to small molecules from the amatoxin family was assessed. Using a series of PSMA-expressing cells, we compared the cytotoxic activity of stable and cleavable linker ADCs and the stability of such constructs in plasma. There was picomolar ADC activity after incubation for 3–5 days with PSMA-positive prostate cancer cells independent of hormone-sensitivity status. Amanitin-based anti-PSMA ADCs demonstrated high activity in prostate cancer xenograft models .
Varied and interesting ADCs directed toward targets on liquid and solid tumors are in clinical trials, and more are nearing clinical trial. With the understanding that ADCs are chemotherapeutics that will be used in combination treatment regimens, the time may have come for this technology to become a major contributor to improving treatment for cancer patients.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
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