The ongoing POLARIX clinical trial (NCT03274492) is investigating the novel investigational antibody—drug conjugate polatuzumab vedotin in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHP) as a first-line therapy for patients with intermediate- or highrisk diffuse large B-cell lymphoma (DLBCL).1 The trial is currently open to enrollment in the United States and multiple global sites, with an estimated primary completion date of December 2019.

: What type of patients are included POLARIX trial and what are some key exclusion criteria?

How are participants randomized? Please describe the arms of the study.

Can you talk more about the study endpoints?

What prompted usage of polatuzumab vedotin in combination with R-CHP and R-CHOP?

What is the mechanism of action of polatuzumab vedotin?

How does polatuzumab vedotin interact with R-CHP?

How are the drugs administered to patients? At what doses and schedules?

In a recent edition of OncLive©, Christopher R. Flowers, MD, MS, a member of the POLARIX: GO39942 Steering Committee and principal investigator of the Emory University School of Medicine study site, shared insights about the trial’s goals and design.2 Following is the complete interview with Dr Flowers, with expanded commentary.

: Patients diagnosed with CD20-positive diffuse DLBCL of any subtype with an Eastern Cooperative Oncology Group performance status of 0 to 2, cardiac ejection fraction of at least 50%, and intermediate- or high-risk (International Prognostic Index [IPI] score of 2-5) are eligible for the study. Full description of the eligibility criteria can be found in the study.

Patients are randomized 1:1 into 2 groups: standard R-CHOP [rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone] given every 21 days for 6 cycles plus polatuzumab vedotin placebo, or R-CHP [rituximab, cyclophosphamide, doxorubicin, and prednisone] plus vincristine placebo and polatuzumab vedotin. Patients in both arms will also receive rituximab monotherapy in cycles 7 and 8.

The investigators will assess the safety and efficacy of polatuzumab vedotin. The primary endpoint is investigator- assessed progression-free survival (PFS). Other endpoints include the rate of PET-negative complete response (CR) at the end of treatment, patient-reported outcomes, and potential biomarkers to predict improved outcomes and as a means for devising new therapeutic approaches in the future.

Polatuzumab vedotin has been demonstrated to have encouraging efficacy when studied as monotherapy and in combination with anti-CD20 antibodies in the treatment of relapsed and refractory DLBCL, both by Palanca-Wessels and colleagues and Morschhauser and colleagues. Also, a doseescalation trial was conducted that revealed acceptable safety of polatuzumab in combination with R-CHP, and it established the dosing regimen for phase II trials [polatuzumab 1.8 mg/kg in combination with R-CHP].

Polatuzumab vedotin is a CD79b-specific antibody conjugated to a microtubule disrupting agent, monomethyl auristatin E, with a stable linker. CD79b is a component of a B-cell receptor restricted to mature B cells, except plasma cells, that is expressed by many B-cell hematologic malignancies. Polatuzumab vedotin is the first CD79b-directed antibody drug conjugated to a potent cytotoxin that is selectively delivered to tumor cells. This combination delivers highly potent cytotoxins directly to tumor cells, which then absorb the cytotoxin.

The standard of therapy for DLBCL is R-CHOP. RCHOP includes vincristine, unlike the studied arm which included polatuzumab vedotin with R-CHP in place of vincristine. Vincristine is a cytotoxic agent, and polatuzumab also works by delivering cytotoxin to tumor cells, but more selectively.

Patients in the R-CHOP plus polatuzumab vedotin placebo group will receive placebo for polatuzumab vedotin, rituximab 375 mg/m

 IV (maximum 2 mg/dose) on day 1, and prednisone 100 mg/day orally on days 1-5 of every 21-day cycle for 6 cycles. Rituximab 375 mg/m

IV will be administered as monotherapy in cycles 7 and 8.

What are the next steps in this research?

What clinical need is the PROLARIX trial seeking to address?

What earlier clinical trial results paved the way for this particular trial?

What are the safety and toxicity concerns with polatuzumab vedotin?

If successful, how would this drug change the standard of care?

Patients in the R-CHP plus vincristine placebo plus polatuzumab vedotin group will receive polatuzumab vedotin 1.8 mg/kg IV, placebo for vincristine IV, rituximab 375 mg/ m

 IV on Day 1, and prednisone 100 mg/day orally on days 1-5 of every 21-day cycle for 6 cycles. Rituximab 375 mg/m

 IV will be administered as monotherapy in Cycles 7 and 8.

Since we are still in the enrollment stage of the trial, next steps are to complete enrollment, and await evaluation of primary endpoint by the statistical group. Then, we will be able to evaluate potential biomarkers to predict improved responses, outcomes, and means for devising new therapeutic approaches.

Currently, patients receiving the first-line treatment for DLBCL, R-CHOP, are still at a 30% to 40% risk of relapse, and early relapse is a poor prognostic sign. PROLARIX focuses on patients with intermediate- to high-risk disease, defined by an IPI score of 2 to 5. PROLARIX is seeking to improve outcomes for these patients with IPI scores of 2 to 5 because these patients have greater risk of relapse and worse survival with standard R-CHOP therapy than does the general patient population with DLBCL.

The phase II study included 65 patients (clinicaltrials. gov, NCT01992653), 40 of whom completed therapy with polatuzumab vedotin with R-CHP and showed promising results. At the end of treatment, 78% of patients had a complete response and 13% had a partial response.

The phase II trial presented by Dr. Tilly showed that 58% of patients in the polatuzumab—R-CHP group experienced grade 3/4 adverse events, including 27% with neutropenia and 11% with febrile neutropenia. One patient experienced grade 5 atrial fibrillation. Serious adverse events, including febrile neutropenia, neutropenia, pneumonia, pulmonary embolism, and influenza A, occurred in 38% of patients. A particular safety concern for polatuzumab vedotin is peripheral neuropathy, because of the conjugated microtubule disrupting agent; it was found to occur in 40% of phase 2 trial patients. Instances of grade 2 or 3 peripheral neuropathy that occurred were during the fifth treatment cycle or later; this is why the POLARIX trial is limited to a maximum of 6 chemotherapy cycles.

The idea behind the PROLARIX trial is to optimize the standard of care already available to further improve patient outcomes and reduce the risk of reccurrence. This treatment regimen will hopefully become the first-line treatment in DLBCL, as it improves the current R-CHOP standard by replacing vincristine with polatuzumab vedotin, a monoclonal antibody designed to selectively target tumor cells.

Forero-Torres A, Kolibaba KS, Lamy T, Jones S, Lee C, Sharma J. Polatuzumab vedotin combined with obinutuzumab, cyclophosphamide, doxorubicin, and prednisone (G-CHP) for patients with previously untreated diffuse large B-cell lymphoma (DLBCL): preliminary results of a phase Ib/II dose-escalation study. Abstract presented at: 58th American Society of Hematology Annual Meeting; December 3-6, 2016; San Diego, CA. bloodjournal.org/content/128/22/1856?sso checked=true.

Katz A. Novel antibody tested in first-line DLBCL therapy. Oncology Live website. onclive.com/publications/oncology-live/2018/vol- 19-no-2/novel-antibody-tested-in-firstline-dlbcl-therapy. Published January 25, 2018. Accessed April 25, 2018.

Articles

Christopher R. Flowers, MD, MS, shares insights about the POLARIX trial in diffuse large B-cell lymphoma.

Polatuzumab Vedotin Plus R-CHOP Being Explored in DLBCL Trial

Antibody-drug conjugates (ADCs) represent a targeted class of agents with an improved therapeutic index over traditional chemotherapy in the treatment of non-Hodgkin lymphoma (NHL). Despite the potential of these agents, the delivery and release of cytotoxic agents to malignant cells through a combination of monoclonal antibody, payload, and linker represent a complex challenge. Nevertheless, because of recent innovations in the treatment of B-cell malignancies, the number of ADCs in clinical trials has increased in recent years. Moreover, these pipeline agents, such as polatuzumab vedotin, offer the potential to change the treatment landscape for NHL and other hematological malignancies through improved targeting and greater potency, compared with early-generation ADCs. This supplement reviews the development of ADCs, assesses the potential of investigational agents, and evaluates their role in the evolving treatment spectrum for lymphoma.

Non-Hodgkin lymphoma (NHL) comprises a heterogeneous group of lymphoid malignancies that originate in B cells, T cells, or natural killer cells and exhibit distinct clinical behavior and variable responses to treatments.

 NHL is the sixth most common cause of cancer death in the United States.

 In 2018, it is estimated that 74,680 new cases and 19,910 deaths due to NHL will occur in the United States.

 NHL is most prevalent in patients ≥70 years and in men.

Mature B-cell neoplasms constitute 80% to 85% of NHLs.5 The most prevalent B-cell lymphomas are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), together representing approximately 65% of all NHL cases.

 Accounting for between 30% and 40% of NHL cases, DLBCL, an aggressive NHL subtype with multiple variants, represents the most common histologic subtype

 In 2016, it was estimated that 27,650 new cases of DLBCL would be diagnosed in the United States.

 Despite improvements in first-line therapy during the past decade, up to 40% of patients with DLBCL will develop relapsed or refractory (R/R) disease, representing a significant cause of morbidity and mortality.

FL is the second most common form of NHL, comprising one-fifth of all NHL cases and accounting for up to 70% of the indolent lymphomas reported in clinical trials in the United States and Europe.

 Although FL usually follows an indolent course, it can lead to poor outcomes with relapse occurring within 2 years of initial treatment.

Treatment of NHL is based on the patient's histologic subtype, extent of disease, and age.

 In addition, most chemotherapy agents target rapidly proliferating cancer cells nonspecifically, and they have toxic effects on normal tissue.

 Monoclonal antibodies, which usually offer inadequate clinical activity when used as monotherapy, are often used in conjunction with chemotherapy to treat lymphoma.

 First approved in the United States in 1997,

 rituximab is an anti-CD20 monoclonal antibody that has transformed the therapeutic landscape of B-cell malignancies, marking a paradigm shift with the beginning of targeted agents to treat lymphoma.

 Other monoclonal antibodies, such as obinutuzumab, a newer glycoengineered type II anti-CD20 monoclonal antibody, have also been incorporated into the treatment armamentarium for lymphoma.

Figure 1. Critical Factors That Influence Antibody-Drug Conjugate Therapeutics.

ADC indicates antibody—drug conjugate; DM1, DM4, derivatives of maytansine; MMAE, monomethyl auristatin E. Reprinted from Panowski S, Bhakta S, Raab H, Polakis P, et al. Site-specific antibody drug conjugates for cancer therapy. 

First- and second-line treatment options for patients with FL vary according to age and include chemotherapy and chemoimmunotherapy combinations or single-agent therapies.

 First-line treatment options for patients with DLBCL typically include anthracycline-based combination chemotherapy regimens comprising cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in combination with the anti-CD20 monoclonal antibody rituximab (R-CHOP).

 Second-line treatment options for patients with DLBCL are stratified according to suitability for high-dose therapy using other chemotherapy combinations with or without rituximab or single-agent therapies.

Chemotherapy is associated with complete response (CR) rates between 40% and 50% in patients with NHL.

 Many patients who initially respond to chemotherapy will eventually relapse, with rates of overall survival (OS) at 3 years reaching approximately 30%.

 Despite recent therapeutic advances in lymphoid malignancies, treatment challenges remain in the management of DLBCL and other R/R NHLs.

Several monoclonal antibodies (eg, bevacizumab, rituximab, and cetuximab) are used in standard treatment regimens for solid tumors and hematological cancers.

 The use of traditional chemotherapies (eg, vinblastine, doxorubicin, and paclitaxel) for cancer treatment is limited due to the nonspecific toxicities of these drugs, resulting in a narrow therapeutic window and increased drug resistance.

 The development of antibody-drug conjugates (ADCs) serves to bridge the gap between the antibody and cytotoxic drug, leading to the introduction of targeted agents with an improved therapeutic index. 

 An ADC consists of a monoclonal antibody conjugated to a cytotoxic agent via a linker.

 The design of ADCs enables the delivery and release of cytotoxins directly to malignant cells, providing high specificity in anticancer activity.

The careful selection of the 3 ADC components (monoclonal antibody, cytotoxic agent, and linker) enables the development of safe, efficacious conjugated therapies. 

 Each component contained within an ADC will be discussed here in detail 

ADCs use high-affinity monoclonal antibodies to deliver and release cytotoxic agents at the tumor site.

 The intravenous administration of ADCs prevents the degradation of the monoclonal antibody by gastric acids and proteolytic enzymes.

 The monoclonal antibody enables the circulation of the ADC in the bloodstream where it binds to the target antigen on the tumor cell surface,

 reducing the likelihood of systemic exposure and therefore limiting toxicity.

 The monoclonal antibody should be specific for the surface antigen on malignant cells to enable the accumulation of the cytotoxic drug at the tumor site and to ensure the targeted delivery of the high-potency cytotoxic agent.

The cytotoxic agent (or payload) impedes cellular mechanisms necessary for tumor survival by disrupting microtubule assembly or inducing DNA damage, causing cell death directly or through apoptosis.

 The cytotoxic agent should be highly potent, with IC50 (ie, the concentration needed to achieve 50% inhibition) values in the nanomolar or picomolar range.

 If it is assumed that all steps involved in the mechanism of ADC action have 50% efficiency, then only 1% to 2% of the administered drug is expected to reach tumor cells.

 Therefore, selecting a cytotoxin that is highly efficacious at very low concentrations is preferable and drugs that would not normally be considered for chemotherapy due to excessive toxicity have a useful role in ADCs.

Linkers serve as short chemical spacers that bind the drug to the antibody, which must be stable in circulation. 

 The properties of the linker influence the therapeutic index, efficacy, and pharmacokinetics of the ADC.

 Stable linkers in ADCs can maintain the antibody concentration in the blood circulation and do not release the cytotoxic drug before reaching the target, limiting nontarget toxicity. At the same time, linkers must be labile enough to allow the efficient release of the cytotoxic agent once the ADC is internalized within the tumor cells.

Important considerations in the design of ADCs are target antigen selection, target internalization, linker stability, conjugation, and cytotoxic payload,

An essential function of ADCs is to promote the efficient internalization of the cytotoxic drug within malignant cells while minimizing toxic effects on healthy cells.

 This function relies on the effective binding of the monoclonal antibody component to the target antigen, which should be tumor specific or tumor associated.

 The monoclonal antibody should display high immuno-affinity for the target antigen.

 In addition, the target antigen should display high penetrance and high expression at the tumor site, with limited expression on healthy tissues to reduce any adverse effects.

Before the cytotoxic drug can be released in its active form, the ADC-target complex must internalize into the target cells via receptor-mediated endocytosis.

 The efficiency of internalization affects drug uptake and release,

 and it is determined by the target itself and the choice of cytotoxin.

 Targets may internalize irrespective of ligand binding, or they may reside permanently on the cell surface. Barriers to reaching the threshold concentration of the cytotoxic drug include inefficient internalization and limited target antigens.

 In addition, the specific epitope that binds to the monoclonal antibody may influence the level of antigen-antibody affinity and play a role in determining internalization efficiency.

Linkers can be modified to impact the biophysical and functional performance in a positive or negative manner by affecting the safety, specificity, potency, and activity of ADCs.

 The stability of the linker has a considerable influence on the toxicities that are exerted by the payload.

 The linker should be stable in circulation and must be labile enough to efficiently release the cytotoxic agent inside the tumor cell.

 There are some linkers that remain stable in the cell, and degradation of the antibody and linker are necessary to release the cytotoxic agent.

 The nature of ADC-associated toxicities is affected by the stability of the linker. Linkers that are more stable behave in a target-specific manner to release their payload and tend to result in specific toxicities; conversely, less stable linkers, which are likely to undergo nonspecific cleavage, tend to have a broader toxicity profile.

Linkers are generally classified into 2 categories: cleavable linkers, which encompass hydrazone, disulfide, and peptide linkers, and noncleavable linkers, which require degradation by lysosomes after internalization.

 Hydrazone linkers are acid sensitive and take advantage of the low pH (4-6) within the endosomes and lysosomes of cancer cells, undergo acid hydrolysis, and release the cytotoxic payload.

 Disulfide linkers are glutathione sensitive and more stable in the circulation. These linkers exploit higher concentrations of glutathione, which are present in cancer and associated with cell survival and tumor growth.

 Peptide linkers are protease sensitive and more stable than the other forms of cleavable linker. These linkers are designed to release the payload upon cleavage by proteases and enable improved control of cytotoxic drug release.

Different mechanisms are utilized to cleave these various linkers from the payload in the cell. These include acidic degradation (hydrazones), thiol-disulfide exchange reactions (disulfide, carbonate), and protease cleavage by cathepsin B (dipeptide).

 Noncleavable linkers (eg, thioether or maleimidocaproyl) use lysosomal enzymes to break down the ADC and release the cytotoxic payload after internalizing at the tumor site.

 For example, ado-trastuzumab emtansine (trastuzumab) utilizes a noncleavable maleimidocaproyl linker that links the maytansinoid toxin to the HER2 antibody.

 This design allows trastuzumab to remain stable in serum for more than 3 days.

Two conjugation approaches are typically used to attach linkers to antibodies.

 The first approach results in heterogeneous payload placement and exploits the reactivity of endogenous lysine or hinge cysteine residues within the antibody. The second approach results in site-specific payload placement and uses reactive chemical handles that are exogenously introduced into particular locations on the antibody through various means. Site-specific conjugation techniques are continuing to evolve, and the functional and analytical advantages of employing site-specific conjugation are being recognized. Nevertheless, heterogeneous conjugates still dominate the ADC pipeline.

An important attribute to consider for any ADC is the amount of drug loading, or the average ratio of conjugated payload to antibody, known as the drug-to-antibody ratio (DAR). This ratio determines the amount of payload per antibody delivered upon internalization and, in turn, affects both efficacy and toxicity.

 Optimizing the DAR is necessary because low drug loading will lead to lower efficacy, whereas high drug loading can negatively affect pharmacokinetics and further increase instability and toxicity.

Different conjugation methods involved in the synthesis of ADCs result in a heterogeneous population of constructs with multiple DARs.21 As such, many current ADCs are characterized by heterogeneous payload conjugation to lysine or cysteine residues. A mixture of ADC species results from the random nature of both conjugation approaches, with DARs ranging from 0 to 8.

 These variations in the DAR can have substantial effects on the disposition of an ADC (eg, tendency to aggregate systemically, efficacy of endocytosis, half-life, and ability to bind target antigens),

Greater toxicity is associated with the more highly conjugated species, which is thought to be caused by increased hydrophobicity and faster clearance by the liver.

 The heterogeneity found in ADCs results in mixtures of inactive (unconjugated) and more toxic isoforms and lowers the therapeutic index.

Extensive research has supported a DAR of approximately 4 as an optimal number of drug molecules per antibody to maximize the efficacy of most ADCs.

 This ratio is generally favored as it reduces the amount of unconjugated antibody, limits the circulation half-life to that of the unmodified antibody, and does not significantly affect antigen binding.

The potency of the cytotoxic payload is a key consideration in the design of ADCs. Highly potent cytotoxic drugs are used as payloads in ADCs and exert their effects on critical cellular processes required for survival. Most ADCs under investigation utilize either maytansine derivatives (DM1/DM4) or auristatins (monomethyl auristatin E and F [MMAE/MMAF]), which are both microtubule inhibitors that are cytotoxic to slowing replicating or nondividing tumor cells. These agents typically induce apoptosis in cells undergoing mitosis by causing cell cycle arrest at G2/M. It has been demonstrated that microtubule inhibitors may also disrupt nondividing cells in interphase.

 Other cytotoxic drugs used in ADCs include enediynes (calicheamicin), duocarmycin derivatives, pyrrolobenzodiazepines, and indolinobenzodiazepines, all of which target the minor groove of DNA, and quinoline alkaloids, which inhibit topoisomerase I. Only a minute amount (<1%) of the injected dose of antibody localizes to the tumors, so cytotoxic payloads must be highly potent, often with IC50 potency values in the picomolar range.

 This potency determines the toxic qualities of ADCs, resulting in the majority of toxicities being characterized by the class of payload.

Early-generation ADCs were constrained by several limitations related to immunogenicity, potency, target selection, and tumor selectivity.

 Limitations of Early-Generation ADCs Immunogenicity. First-generation ADCs consisted of murine monoclonal antibodies covalently linked to anticancer drugs (eg, doxorubicin, methotrexate, vinblastine).

 Murine antibodies caused severe immunogenicity by evoking an immune response and producing human antimouse antibodies in patients.

 The immune response evoked by murine antibodies lowers the effectiveness of the ADC and may occur rapidly, resulting in the formation of human anti-mouse antibodies within 2 weeks of administration.

Early cytotoxins such as doxorubicin (an anthracycline) or methotrexate (an antimetabolite/antifolate) were utilized as payloads in first-generation ADCs.

These cytotoxins have established efficacy when used as standard chemotherapy; however, they are known to have insufficient potency at low concentrations when conjugated to a monoclonal antibody.

Target selection. Lack of specificity to the target antigen in early-generation ADCs contributed to their cytotoxic effects on healthy tissue.

 Other characteristics of the ADC target antigen should be considered, such as poor internalization or inadequate antigen binding.

Advances in Design of New-Generation ADCs

The differences in target antigen requirements for cleavable versus noncleavable linkers suggest that the internalization and trafficking component of ADC processing plays a critical role in its function.

 Cleavable linkers are sensitive to the microenvironment in endosomal compartments, with the trafficking requirements for the release of attached payloads thought to be less stringent than those for ADCs carrying noncleavable linkers, which require complete lysosomal degradation before payload release.

 The nature of the overall relationship between internalization, intracellular trafficking, and ADC potency remains poorly understood.

Based on monoclonal antibodies that did not undergo internalization, early-generation ADCs were engineered to release the cytotoxic payload upon cleavage of the linker by tumor-specific enzymes (such as matrix metalloproteinases) or in tumor-specific environments (such as a lower pH).

 These ADCs failed to undergo internalization and did not improve drug specificity or lower toxicity.

The concept of combining a monoclonal antibody with a cytotoxic drug to form an ADC has existed for more than 4 decades,

 and substantial improvements have been made since the first generation of ADCs.

 New-generation ADCs have several features that are designed to address the limitations of earlier ADCs.

Bioengineering research has enabled the discovery of chimeric, humanized, and fully human antibodies, which have replaced the murine antibodies used in early-generation ADCs.

 These newer antibodies have decreased immunogenicity compared with their earlier counterparts and offer other advantages over murine antibodies, including longer circulation and improved antibodydependent cellular cytotoxicity.

The cytotoxins employed by newer-generation ADCs are considerably more potent than the anticancer agents used in the past (eg, doxorubicin and methotrexate).

 Cytotoxic agents used in ADCs under investigation for the treatment of patients with NHL are discussed here and mainly fall into 2 classes: microtubule inhibitors (DM1, DM4, MMAE, MMAF) or DNA synthesis inhibitors (calicheamicins, duocarmycins, pyrrolobenzodiazepine dimers, and doxorubicin).18 Other cytotoxic agents have been utilized in ADCs with novel mechanisms of action (eg, irinotecan derivatives and alpha-amanitin).

Dolastatin 10 and maytansine are potent antimitotic microtubule-disrupting agents with IC50 potency values in the nanomolar range. DNA-alkylating agents have IC50 potency values in the picomolar range.

 The largest group of ADCs in clinical trials are MMAE and MMAF, which are derivatives of dolastatin 10. Dolastatin 10 failed a phase II study in patients with prostate cancer due to lack of efficacy.

 Auristatins are antimitotic cytotoxins that strongly bind tubulin and inhibit polymerization, leading to cell cycle arrest.

 MMAE and MMAF have been singled out for further study due to several desirable characteristics, including their physiologic stability, potency, aqueous solubility, and ease of attachment of stable linkers.

Clinical trials of maytansine were conducted in the 1970s and later discontinued due to a high level of systemic toxicity.42 Derivatives of maytansine, however, are highly potent cytotoxic agents when linked to a targeting antibody and comprise the largest group of ADCs behind auristatins currently in clinical trials.

Second-generation maytansinoids include DM1 and DM4, which are cytotoxic agents used with trastuzumab and coltuximab, respectively.

Calicheamicins are highly potent enediyne antitumor antibiotics that cleave DNA with high specificity by binding in the DNA minor groove and inducing double-strand breaks.

 Gemtuzumab and inotuzumab utilize calicheamicin payloads, which are associated with narrow therapeutic indices and serious late toxic effects.

 Due to its hydrophobic interactions, only a few molecules of calicheamicin per immunoglobulin can be conjugated before high levels of aggregated protein appear.

Similar to calicheamicins, duocarmycins are potent antitumor antibiotics that are DNA minor groove-alkylating agents. A phase I study investigated BMS&#8209;936561 (also known as MDX&#8209;1203), a conjugate of a human anti&#8209;CD70 antibody (MDX&#8209;1115), and a duocarmycin analogue (MED&#8209;2460), in patients with advanced clearcell renal cell carcinoma and B&#8209;cell NHL. BMS&#8209;936561 was shown to be well tolerated at doses of up to 8 mg/kg.

Pyrrolobenzodiazepines, which are naturally occurring antitumor antibiotics, bind to the DNA minor groove in a sequence-specific manner.43 Pyrrolobenzodiazepine dimers are composed of 2 pyrrolobenzodiazepine units that use different tethers to yield symmetrical and nonsymmetrical dimers. This configuration allows the resulting compound to crosslink DNA by binding to the N2 position of guanine on opposing strands of DNA.

Other approaches are being explored to enhance the potency of ADCs; these include strategies to reduce resistance against the cytotoxin or to increase tumor penetration by using of smaller protein scaffolds.

 In addition to demonstrating efficacy as monotherapy, ADCs have shown synergy when used in conjunction with monoclonal antibodies, including checkpoint inhibitors.

Another important consideration in ADC design is the careful selection of an appropriate target antigen and antibody. Notably, the antibody should bind accurately and efficiently to the antigen on tumor cells while sparing nonmalignant cells. Selective expression of the target antigen on tumor cells and internalization upon antibody engagement are required for successful ADC approaches.

Improved Selectivity and Efficiency of Internalization

The effectiveness of the ADC may be limited by insufficient antigen expression on tumor cells, inappropriate or low-affinity binding, or a lack of internalization following binding.

 To ensure that adequate amounts of the cytotoxic drug are delivered by the ADC, an adequate level of antigen expression is necessary. The level of antigen expression needed for efficient ADC activity depends on the specific antigen in question.

 A minimum threshold of approximately 10,000 antigens per cell has been identified for the delivery of lethal quantities of the cytotoxic drug.

 In B-cell lymphoma, the level of CD19 antigen expression may be limited to as few as 30,000 per cell to elicit ADC activity.

The monoclonal antibody component of the ADC binds to the target antigen and should retain high immunoaffinity, typically in the 0.1- to 1-nanomolar range.

 The internalization process takes place via receptor-mediated endocytosis, which is completed by the formation of a clathrin-coated early endosome containing the ADC antigen complex.

Internalization must be efficient enough to ensure that the necessary threshold concentration of the cytotoxic agent is reached given the level of antigen expression within the cell.

 In addition to antigen affinity, other important properties of antibodies used in ADCs include target specificity, good retention, minimal immunogenicity, low cross-reactivity, and a long circulation in plasma.

The process of internalization and subsequent trafficking through endosomal and lysomal compartments has been shown to vary among target antigens.

 Polson et al studied the in vivo efficacy of ADCs against 7 NHL target antigens and found that ADCs bearing cleavable linkers could mediate tumor growth inhibition against all 7 targets, whereas ADCs bearing noncleavable linkers were efficacious against only 2 of the 7 targets. Thus, the choice of linker is a key consideration that can impact selectivity and internalization of the ADC.

Site-specific conjugation has been used to yield ADCs with higher DARs, specifically between 6 and 8.

 This method may be useful for overcoming low-expression target antigens and slower tumor cell internalization kinetics, while achieving a high therapeutic index.

 However, a higher DAR is associated with deterioration of certain ADC attributes, such as increased systemic clearance, reduction in therapeutic efficacy, lower stability under stressed conditions,

 heterogeneity, and higher propensity to aggregation, presumably due to the hydrophobic nature of the payloads.

 Adverse clinical effects, including immunogenicity, have been associated with protein aggregation.

There has been much interest in exploring how sitespecific conjugation techniques can be used to alter the pharmacokinetics of ADCs.

 Site-specific conjugation techniques currently under investigation include the use of engineered cysteines, unnatural amino acids, and the addition of a selenocysteine, glutamine, or aldehyde tag.

 These technologies can achieve a more homogeneous population of ADCs by engineering conjugates with specific DARs.

 This may result in an improved therapeutic window with enhanced efficacy and decreased toxicity.

Four ADCs are approved in the United States: a CD33- targeting agent indicated for acute myeloid leukemia, an HER2-targeting agent indicated for HER2-positive metastatic breast cancer, and 2 agents indicated for lymphoid malignancies that target CD30 and CD22.

 In addition, several ADCs targeting other B-cell surface antigens are in development for the treatment of NHL 

Initially approved in the United States in 2000 for the treatment of acute myeloid leukemia, gemtuzumab was withdrawn in 2010 due to concerns of treatment-related toxicity.

 In 2017, gemtuzumab was reapproved with a lower recommended dose and altered dosing schedule. Gemtuzumab is a humanized anti-CD33 antibody attached covalently to the cytotoxic agent N-acetyl-gamma-calicheamicin via an acid-labile hydrazone bifunctional linker.

Trastuzumab was approved in the United States in 2013 for treating HER2-positive metastatic breast cancer and is the first ADC approved for a solid tumor indication.

 Trastuzumab is a humanized anti-HER2 antibody attached covalently to the microtubule inhibitor DM1 (a maytansine derivative) via the stable thioether linker MCC (4-[N-maleimidomethyl] cyclohexane-1-carboxylate).

CD30-Targeting Agent. Brentuximab vedotin (brentuximab; SGN-35) was initially approved in the United States in 2011, and it now has multiple indications in the treatment of non-Hodgkin lymphoma and anaplastic large-cell lymphoma.

 Brentuximab is a chimeric anti- CD30 monoclonal antibody attached covalently to the synthetic microtubule disrupting agent MMAE via a cathepsin B-sensitive cleavable dipeptide valine-citrulline linker.

 The CD30 antigen is a transmembrane protein that belongs to the tumor necrosis factor receptor family and is expressed in several lymphoma subtypes, including anaplastic large cell lymphoma and Hodgkin lymphoma, which is characterized by CD30-positive Reed-Sternberg cells.

Upon attachment to the receptor, brentuximab is internalized by CD30-expressing cells. Cathepsin B is a tumor-specific protease that recognizes and cleaves the valine-citrulline dipeptide bond inside the tumor cells.

Table. Selected B-Cell Surface Antigens in NHL

ADC indicates antibody—drug conjugate; BCR, B-cell receptor; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; NHL, non-Hodgkin lymphoma; SEA, sugar-engineered antibody; vcMMAE, valine–citruline monomethyl auristatin E.

Reprinted from: Blood Reviews. Ku M, Chong G, Hawkes EA. Tumour cell surface antigen targeted therapies in B-cell lymphomas:

This action releases MMAE into the cytosol where it inhibits microtubule formation and results in apoptosis.

 Brentuximab has been investigated in phase II studies for the treatment of NHL, including DLBCL. One such study evaluated the safety and efficacy of brentuximab plus R-CHOP in the frontline treatment of patients with high-intermediate or high-risk DLBCL (ClinicalTrials. gov identifier: NCT01925612).

 Of the 51 patients who received the study treatment, 32 were high-intermediate risk (International Prognostic Index [IPI] score of 3, ageadjusted IPI score of 2), and 19 were high risk (IPI of 4 to 5, age-adjusted IPI of 3).

 The overall response rate (ORR) as measured by PET at the end of treatment was 97% (80% CR).

 The rate of CR was 92% in patients who were CD30 positive (n = 13). Four patients (all CD30 negative) had progressed after a median follow-up of 5 months.

 Activated B-cell-like and germinal center B-cell-like subtypes showed similar CR rates.

The most frequent treatment-emergent adverse events (AEs) were nausea, fatigue, peripheral sensory neuropathy, diarrhea, anemia, decreased appetite, febrile neutropenia, and vomiting.

 The most common grade ≥3 AEs were febrile neutropenia (27%), neutropenia (25%), and anemia (24%).

 Neuropathy (mostly grade 1-2) was reported in 55% (38%, 1.2 mg/kg brentuximab plus R-CHOP; 17%, 1.8 mg/ kg brentuximab plus R-CHOP). Treatment discontinuation due to AEs occurred in 10% of patients.

A phase II, open-label, multicenter study evaluated brentuximab in patients with both CD30-expressing and CD30-nonexpressing R/R NHL (ClinicalTrials.gov identifier: NCT01421667).

 The study included 68 patients with CD30-positive (≥1%) B-cell NHL (mostly DLBCL, 49/68).

The ORR was 44% (17% CR) in patients with DLBCL (median duration of response [DOR], 5.6 months) and 26% (16% CR) in those with B-cell NHL.

 In the DLBCL cohort, the median DOR was 16.6 months in patients who achieved CR.

 The rate of CR in patients with DLBCL was 17% in those who were CD30-positive and 10% in those who were CD30-negative.64 Stable disease was maintained in 23% (n = 11), and 33% (n = 16) experienced disease progression.

 No significant correlation was observed between response and CD30 expression level.

The most common treatment-emergent AEs (all grades) in patients with DLBCL included fatigue (55%), diarrhea (43%), neutropenia (41%), nausea (39%), decreased appetite (31%), pyrexia (31%), and peripheral sensory neuropathy (29%).

Antibody-drug conjugates represent a targeted class of agents with an improved therapeutic index over traditional chemotherapy in the treatment of non-Hodgkin lymphoma.

Focus on Pathways: Novel Targeted Antibody-Drug Conjugates in Non-Hodgkin Lymphoma | Special Issues

Polatuzumab Vedotin Plus R-CHOP Being Explored in DLBCL Trial

Novel Targeted Antibody-Drug Conjugates in Non-Hodgkin Lymphoma