Signaling by Receptor Cross-Linking and Receptor Blockade

When the antigen is a cell surface receptor, its clustering by multivalent MAbs can induce apoptosis.² Apoptosis increases with hyper cross-linking (e.g., CD20 target on lymphoma cells).^5–5 Both caspase dependent and independent programmed cell death pathways appear to be involved.⁶ In AIDS-related lymphoma, anti-CD20 MAb diminishes p38MAPK signaling and Bcl-2 expression, whereas in non–AIDS-related lymphoma, signaling through CD20 inhibits AP-1 and nuclear factor (NF)-κB, leading to downregulation of Bcl-xL, thereby sensitizing lymphoma cells to chemotherapy.⁷ Direct receptor blockade by MAbs has also been reported for epidermal growth factor receptor-1 EGF-R1⁸ and human epidermal growth factor receptor-2 (HER-2),⁹ leading to upregulation of the BH3-only protein Bnip3L, thereby sensitizing tumor cells to chemotherapy.¹⁰ MAb inhibition of vascular endothelial growth factor receptor-1 (VEGFR-1)¹¹ or VEGFR-2¹² can also enhance the efficacy of chemotherapy.

Cytophilic MAb and ADCC

The Fc region of immunoglobulin G (IgG) MAb interacts with both activating and inhibitory Fc receptors (FcγR).¹³ Humans have four activating FcγRs: FcγRI (CD64) is a high-affinity FcγR, whereas FcγRIIA (CD32A), FcγRIIIA (CD16A), and FcγRIIIB (CD16B) are low-affinity FcγRs. FcγRIIB (CD32B) is the only known inhibitory FcγR (Table 32-1). All FcγRs are transmembrane glycoproteins except for FcγRIIIB, which is anchored on neutrophils by glycosyl phosphatidylinositol. FcγRI also has an extracellular portion composed of three Ig-like domains, whereas FcγRII and FcγRIII have only two domains. This phenomenon allows FcγRI activation by a single IgG molecule (or monomer), whereas the latter two Fcγ receptors must bind multiple IgG molecules within an immune complex to be activated. FcγRIIA carries the immunoreceptor tyrosine–based activation motif (ITAM) in its intracellular tail for activation. Other FcγRs do not have an ITAM but interact with an adaptor protein called the accessory γ chain (Fcγ subunit), which carries a cytoplasmic ITAM. ITAM becomes tyrosine phosphorylated by members of the Src-kinase family, with subsequent recruitment of kinases containing SH2. These events lead to the activation of phosphatidylinositol 3-kinase and phospholipase-Cγ, followed by protein kinase C activation and sustained calcium elevation.¹³ These biochemical cascades trigger phagocytosis, degranulation, cytokine release, and ADCC. In sharp contrast to activating FcγRs, FcγRIIB is a single-chain receptor that carries the immunoreceptor tyrosine–based inhibitory motif in its cytoplasmic domain. Engagement of this inhibitory receptor activates phosphatase SHP-1 to remove phosphate groups from tyrosine residues, leading to downregulation of both biochemical and cellular functions. The ratio of activating to inhibitory FcγRs on immune cells, such as dendritic cells (DCs), macrophages, and neutrophils, can influence the antitumor properties of MAbs.

Inflammatory mediators (interferon-γ or C5a) increase activating FcγRs and downregulate inhibitory FcγRIIB, whereas interleukin-4 (IL-4), IL-10, and tumor growth factor-β upregulate FcγRIIB, thereby raising the thresholds for cell activation. Removing the inhibitory signals by FcγRIIB-blocking antibodies has shown efficacy in preclinical models.¹³ This finding may be relevant for cross-presentation of antigens, which is acquired endocytically through Fc receptors on DCs during the induction of tumor-specific T-cell responses.¹⁴ In addition to these FcγRs, a unique class of Fc receptor called FcRn (neonatal) is found on endothelial cells and regulates antibody catabolism.¹⁵ FcRn is similar in structure to major histocompatibility complex class I antigen. It binds IgG at an acidic pH of 6.0 to 6.5 but not at neutral or higher pH. When serum IgG is internalized by endothelial cells through pinocytosis, it becomes FcRn-bound in the acidic endosomes and escapes lysosomal degradation by recycling to the cell surface for release back into blood, where the pH is neutral. This regulation of serum half-life by FcRn binding can be exploited in antibody engineering. Although most therapeutic antibodies have been primarily IgGs, both IgA1 and IgA2 can also mediate efficient ADCC by binding to FcαRI (CD89) on human neutrophils and monocytes/macrophages.¹⁶

Certain cancer cells such as colon carcinoma, lymphoma, leukemia, neuroblastoma, and melanoma are effectively killed by natural killer (NK) cells, neutrophils, and activated monocytes in vitro in the presence of specific MAbs. Depending on the affinity of the MAb for the individual FcγR, both NK cells (carrying FcγRII and FcγRIII) and neutrophils (bearing all three FcγR) can mediate efficient ADCC. Because of its high affinity, FcγRI is generally occupied by monomeric IgG in human plasma. Human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) have differential affinities for FcγRII and FcγRIII. Chimeric or humanized IgG1 antibodies (e.g., Lym-1 specific for the human leukocyte antigen–DR subregion, and ch14.18 or hu3F8 for GD2) can exploit FcγRIII for lymphocyte ADCC while using FcγRII for myeloid ADCC.19–19 Among the four IgG subclasses, IgG2 has the lowest affinity for the inhibitory receptor FcγRIIB.¹³ Mouse IgG3 (e.g., 3F8 specific for GD2) can engage both FcγRII and FcγRIII in ADCC,²⁰ despite the low affinity of the monomer for human FcγRs. Polymorphic FcR alleles (FCGR2A^23–23 and FCGR3A^22,24) with higher affinity for human IgG1 mediated more effective ADCC in vitro and were associated with superior clinical responses to rituxan, cetuximab, or Herceptin. In addition to FcγRs, adhesion molecules are critical for MAb-mediated ADCC. These molecules include CR3 (CD11b/Cd18)^17,18,20 and CD66b¹⁷ for neutrophil-mediated ADCC and LFA-1 (CD11a/CD18) for NK-mediated ADCC.²⁵ Because cytokines can increase the expression of adhesion molecules, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ,^18,26–28 and IL-2^29,30 have been used to activate either myeloid- or NK-mediated effector models. Furthermore, because both GM-CSF and IL-2 expand the effector cell pools, they enhance the effector to target ratio, a critical determinant of both in vitro and preclinical models of antibody-based therapy. Optimal combinations of MAbs and cytokines in the appropriate clinical setting are being explored.^31–34

Signaling by Agonistic and Antagonistic Antibodies

Activating signaling receptors on human cytotoxic lymphocyte³⁵ and specifically NK cells³⁶ can enhance ADCC functions. Although most relevant for CD8+ T-cell–mediated tumor lysis (e.g., CD137 [4-1BB], CD134 [OX40], and GITR), agonistic antibodies to CD137 have now been shown to potentiate NK-ADCC in anti-CD20 and trastuzumab therapy.^37,38 Conversely, use of antagonistic antibodies to remove immune checkpoint blockade allows T cells to perform more effective surveillance. The first successful example of such an immune approach was the anti-CTLA4 MAb ipilimumab,³⁹ which was approved by the Food and Drug Administration (FDA) for melanoma. An equally exciting strategy targets the programmed death–1 (PD-1) T-cell coreceptor or its ligand B7-H1/PD-L1 and B7-DC/PD-L2, a pathway that maintains an immunosuppressive tumor microenvironment.⁴⁰ Phase 1 and 2 clinical trials of anti–PD-1⁴¹ or anti–PD-L1⁴² have shown tumor responses in melanoma, non–small cell lung cancer (NSCLC), renal cell carcinoma, and ovarian cancer. Besides turning on T cells, removing inhibitory signals on NK cells could also have clinical potential for both hematologic and solid malignancies.^45–45 Blocking MAbs (e.g., anti-CD47) have also been effective in unleashing macrophages to phagocytose tumor cells in the absence or presence of MAb,⁴⁶ both in vitro and in vivo, in leukemia/lymphoma^47–51 and in solid tumor models.^49,51 In addition, binding of MAb to certain tumor cell surface receptors can be directly cytotoxic, independent of ADCC or CMC. For example, induction of cell death through death receptor family–like TRAIL receptors on tumor cells may have potential for cancer therapy.^52,53

Complement Activation

IgG initiates the classic complement cascade by binding C1q to its CH2 domain. C1q is more avid for human IgG1 and IgG3 than for IgG2 and has no affinity for IgG4.⁵⁴ CMC potency of individual IgG MAb is also dependent on antibody affinity,⁵⁵ antigen density, and nature of the target or the tumor cell. Although some tumor cell lines (e.g., lymphoma and neuroblastoma) are sensitive to CMC, many are resistant to complement because of anticomplement surface proteins such as decay accelerating factor (DAF; CD55),^58–58 homologous restriction factor (CD59),^56,59,60 and membrane cofactor protein (CD46).^57–59,61 The effect of complement extends beyond direct tumor lysis. After complement activation, tumor-bound C3b is cleaved rapidly by plasma protease factor I to iC3b. Through the iC3b receptors CR3 (Mac-1 or alpha_Mbeta₂-integrin) and CR4 (CD11c/CD18, alpha_Xbeta₂-integrin) on leukocytes, tumor cells are opsonized.⁶² C3a and C5a, byproducts of complement activation, are also potent mediators of inflammation⁶³ and are chemotactic for phagocytic leukocytes, drawing them to the tumor sites. C5a can also downregulate the inhibitory receptor FcγRIIB¹³ or induce secondary cytokines to increase vascular permeability for both MAbs and effector cells. Preclinical models of anti-CD20 immunotherapy showed that CMC could interfere with ADCC,⁶⁴ and the adverse clinical outcome correlations with C1qA levels supported this observation.⁶⁵

Solid Tumors

Trastuzumab (Herceptin) is a humanized MAb against the receptor tyrosine kinase ERBB2 (also known as HER2/NEU) on breast cancer cells. Along with inducing CMC and ADCC, it can induce HER2 protein downregulation, prevent HER2-containing heterodimer formation, initiate G1 arrest, induce p27, prevent HER2 cleavage, and inhibit angiogenesis.⁹⁶ Up to 25% of breast cancers are positive for HER2, which is associated with aggressiveness and poor prognosis.^97,98 Based on its synergy with chemotherapy in vitro,^99,100 trastuzumab was tested in a large phase 3 trial of 469 patients with breast cancer, where its combination with chemotherapy produced a longer median response duration (9.1 vs. 6.1 months), higher overall response (OR) (50% vs. 32%), and a lower death rate at 1 year (22% vs. 33%)¹⁰¹ than did chemotherapy alone. On the basis of this trial, the FDA approved the use of trastuzumab and paclitaxel as a first-line treatment of HER2-overexpressing metastatic breast cancer. Subsequent studies showed that 1 year of treatment with trastuzumab after adjuvant or neoadjuvant chemotherapy significantly improved disease-free survival among women with HER2-positive breast cancer.^102,103 A 0.8% incidence of severe congestive heart failure and a 3.6% incidence of significant left ventricular ejection fraction decreases were found in the trastuzumab treatment group compared with 0% and 0.6%, respectively, in the observation group,¹⁰⁴ although both symptomatic and asymptomatic events were largely manageable and reversible.¹⁰⁵ Recent studies also suggest that synergy between trastuzumab and chemotherapy may still be effective beyond disease progression.¹⁰⁶ The combination of trastuzumab and small molecules targeting HER2 in the adjuvant and neoadjuvant settings is being actively pursued.¹⁰⁷ Despite the clinical benefit of trastuzumab, disease in patients with metastatic breast cancer eventually progresses. Pertuzumab, a humanized MAb that inhibits HER2 dimerization, produced an 18% to 24% response when combined with trastuzumab among patients with HER2-positive metastatic breast cancer. In the phase 3 randomized study (n = 808 patients), the combination of pertuzumab + docetaxel + trastuzumab versus docetaxel + trastuzumab as first-line treatment for HER2-positive breast cancer showed significant prolongation of PFS (18.5 vs. 12.4 months) with similar toxicity profiles except for febrile neutropenia and diarrhea.¹⁰⁸ On the basis of this study, pertuzumab (Perjeta) was approved by the FDA. In advanced HER2-positive gastric cancer, a phase 3 trial of trastuzumab plus platinum-based chemotherapy versus chemotherapy alone showed an OS advantage of 2.7 months.¹⁰⁹ However, its benefit when added to standard chemotherapy was uncertain for NSCLC^110,111 or ovarian cancer.¹¹²

Cetuximab (chIgG1 anti-EGFR) is an FDA-approved chimeric antibody designed to induce receptor blockade; it reverses resistance to topoisomerase I inhibitors in addition to having modest monotherapy activity in patients with colorectal cancer (CRC),113,114 with improvements in PFS and OS.¹¹⁵ First-line treatment with cetuximab plus FOLFIRI, compared with FOLFIRI alone, reduced the risk of progression of metastatic CRC, although no significant difference was found in OS.¹¹⁶ An acneiform rash occurred in 82.9% of patients and a grade 3 rash was observed in 4.9%; it was exacerbated by sunlight or secondarily infected by Staphylococcus aureus, which was sometimes preventable by prophylactic topical antibiotics. Serious hypersensitivity-hypomagnesemia reactions requiring H1 antihistamines and steroids developed in only a minority of patients (~1%).¹¹³ Response and survival correlated strongly with the severity of the rash. In contrast, clinical benefit did not correlate with EGFR immunostaining of the tumor.¹¹⁷ Neither EGFR kinase domain mutations nor EGFR gene amplification appeared to be essential for response to cetuximab. Although response to cetuximab was strongly associated with wild-type KRAS,¹¹³ not all KRAS-mutated tumors were resistant to cetuximab.¹¹⁸ Expression of epiregulin and amphiregulin were biomarkers for efficacy.¹¹⁹ Panitumumab, a fully human anti-EGFR antibody generated using human IgG-transgenic mouse technology,¹²⁰ was much less immunogenic, stimulating binding antibodies in only 1.8% and neutralizing antibodies in 0.2% of patients.¹²¹ As first-line treatment in previously untreated patients with metastatic CRC, panitumumab plus FOLFOX4 (fluorouracil, leucovorin, and oxaliplatin) improved median PFS significantly from 8 to 9.6 months for patients with wild-type KRAS tumors in a phase 3 randomized trial, but did not improve their OS.¹²² As second-line treatment, panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) was better than FOLFIRI alone in prolonging median PFS (from 3.9 to 5.9 months) and median OS (from 12.5 to 14.6 months).¹²³ The potential role of anti-EGFR antibody in patients with gastroesophageal cancer is under investigation.¹²⁴

When combined with radiotherapy for locally advanced squamous-cell carcinoma of the head and neck, cetuximab improved the duration of locoregional control from 14.9 to 24.4 months, the median OS from 29.3 to 49 months, and 5-year OS from 36.4% to 45.6%, with Hazard Ratio 0.49 among patients with ≥grade 2 acneiform rash.¹²⁵ As first-line treatment of recurrent or metastatic head and neck cancer, addition of cetuximab to platinum + fluorouracil in a randomized phase 3 trial increased the response rate from 20% to 36%, the prolonged median PFS from 3.3 to 5.6 months, and the median OS from 7.4 to 10.1 months.¹²⁶ Two large randomized phase 3 trials (FLEX and BMS099) demonstrated a small benefit (~1 month) in OS, but not in PFS, with the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC.^127,128 Subgroup analyses suggested a significant association between early occurrence of skin rash¹²⁹ and intensity of EGFR staining¹³⁰ with efficacy.

Despite initial enthusiasm,¹³¹ adjuvant therapy with edrecolomab (17-1A, Panorex), a mouse IgG_2a antibody specific for epithelial cell adhesion molecule (EpCAM) on malignant and normal epithelial cells, did not improve disease-free survival or OS in subsequent phase 3 studies in patients with CRC.^132,133 The story of edrecolomab provided a case study for the clinical failure of MAb on a major scale.¹³⁴ Despite the lack of clinical efficacy, anti-idiotype network and T-cell responses against antibody-modified tumors were found.^135,136 Adecatumumab, the fully human MAb, is undergoing testing in gynecologic cancers.¹³⁷ Catumaxomab (Removab), a rat-mouse bispecific antibody engaging CD3 on T cells and EpCAM on tumor cells, was recently approved in Europe for malignant ascites.^138,139 Oregovomab (MAb B43.13, anti-CA125), a murine IgG₁ antibody for ovarian cancer, did not improve time to relapse in a phase 3 randomized trial of 145 patients.¹⁴⁰ A correlation of improved survival and human anti-mouse antibody (HAMA)/idiotype network response was of biological interest.¹⁴¹

Among the ganglioside antigens on neuroectodermal tumors, GD3 (MAb R24 for melanoma)¹⁴² and GD2 (MAb 3F8 and ch14.18 for neuroblastoma)^34,143 have been studied clinically. GD2 is present on a variety of solid tumors in addition to neuroblastoma, including osteosarcoma, retinoblastoma, some soft tissue sarcomas, brain tumors, mesenchymal stem cells,¹⁴⁴ and cancer stem cells.¹⁴⁵ The clinical effectiveness of anti-GD2 MAb for large, soft tissue masses was minimal. Its utility was more suitable for microscopic marrow disease in the presence of GM-CSF and/or IL-2.^146,147 Activation of CD11b by GM-CSF on myeloid cells correlated with patient PFS.³³ Missing ligands for killer immunoglobulin-like receptors on NK cells significantly affected both PFS and OS.^45,143 Failure to achieve early MRD remission was the strongest predictor of clinical outcome. HAMA response was a strong positive predictor of OS and was associated with the induction of Ab2 and Ab3 through the idiotype network, implicating a potential role of the host immune response in maintaining clinical remission.^148,149 With anti-GD2 therapy, disease relapse was primarily at isolated sites, and isolated central nervous system relapse, previously rare, was salvageable by combining surgical resection, radiation, and intrathecal iodine-131 MAb.¹⁵⁰ Clinical development of anti-GD2 MAb was limited by its adverse effect of pain, thus precluding dose escalation. Humanized forms of 3F8 (hu3F8)¹⁹ and 14.18 (hu14.18)¹⁵¹ are currently undergoing active clinical investigations.

Radioimmunoconjugates

MAbs have the potential to target and ablate tumors in radioimmunotherapy (RIT).¹⁷⁵ Radioimaging can map the biodistribution of MAbs and quantify the relative amounts of MAb deposited in various tissues and organs, thus allowing more precise radiation dose estimates in therapeutic studies. With the advent of single photon emission computed tomography and positron emission tomography, accurate dosimetry is readily achievable. In preclinical models, ablation of established xenografts is possible, although radiation damage to the marrow remains dose-limiting. For patients with lymphoma and leukemia, antitumor activity of RIT is highly reproducible, but major responses in solid tumors are rare. Unlike naked antibodies, the bystander effect of RIT from cross-firing of the radioisotopes accounts for most of the toxicities of radioimmunoconjugates, hence limiting their efficacy.

Most clinical applications of RIT utilize β-emitting radioimmunoconjugates (Table 32-3). β Particles have a relatively long range (0.8 to 5 mm) and low linear energy transfer (approximately 0.2 keV/µm). This long range results in the delivery of radiation not only to the antigen-positive tumor cells but also to surrounding antigen-negative tumor cells, as well as to the neighboring normal tissues. Thus β emitters can treat bulky diseases effectively but are not optimal for eradicating single cells or micrometastasis. Most early human studies of RIT used ¹³¹I, a long-lived β-particle emitter. Because of its γ-particle emission, it is also suitable for dosimetry studies. However, this γ-particle emission poses a radio hazard at high treatment doses, necessitating patient isolation. In vivo dehalogenation of iodinated antibodies can compromise tumor dose with subsequent thyroid damage from the released iodide. Yttrium-90 (⁹⁰Y) is a pure β emitter; its lack of γ radiation allows outpatient treatment. However, ⁹⁰Y has its limitations, including deposition in bone when dissociated from the MAb complex and its lack of γ emissions, thereby requiring the use of indium-111 to estimate biodistribution and dosimetry. Besides ⁹⁰Y, other β emitters recently explored include rhenium-186, rhenium-188, copper-67, and lutetium-177, each with its own limitations.

Alpha particles are helium nuclei; when compared with β particles, they have a shorter range (50 to 80 µm) and a higher linear energy transfer (approximately 100 keV/µm).¹⁷⁶ As few as one or two α particles can destroy a target cell. RIT using α emitters should result in less nonspecific toxicity to normal bystanders, as well as more efficient single-cell killing. This modality is ideal for controlling MRD. Isotopes that emit α particles such as astatine-211 and bismuth-213 have been tested in clinical trials.²¹³ Bi-HuM195 (anti-CD33) administered intravenously for AML¹⁷⁷ and ²¹¹At conjugated to MAb 8C16 and administered intraventricularly or intrathecally for gliomas¹⁷⁸ have been well tolerated and have produced clinical responses. The relative lack of extramedullary toxicities should encourage further development of this targeting technique for micrometastases or neoplasms on the surface of body compartments, such as peritoneal and leptomeningeal metastasis.^181–181

Immunotoxins and Antibody Drug Conjugates

Chemotherapeutic drugs have been conjugated to MAbs for selective tumor delivery. Doxorubicin, melphalan, methotrexate, and vinca alkaloids conjugated to MAbs have limited clinical success. BR96-doxorubicin directed at Lewis Y antigen has shown no clinical benefit in phase 2 trials in patients with breast cancer²⁴⁴ or gastric cancer.²⁴⁵

Agents such as ribosome-inactivating toxins can be potent cancer drugs. One major limitation is the lack of tumor selectivity.²⁴⁶ Two-chain toxins (e.g., ricin and diphtheria toxin [DT]) utilize their B chain for cell binding and their A chain for inhibition of protein synthesis; other toxins (e.g., Pseudomonas exotoxin [PE], Pokeweed antiviral protein, gelonin) have a built-in receptor for cell attachment. When conjugated to MAbs, they become immunotoxins. These toxins can be genetically modified for MAb conjugation and for an improved safety profile.¹⁶⁵ In recombinant toxins (e.g., PE40, PE38, or DT DAB₄₈₆), the cell-binding domains were replaced by scFv.^165,246

MAbs conjugated to different toxins have been actively explored for cancer therapy²⁴⁶: ricin toxin A-chain (RTA) (RTA to anti-CD7, anti-CD22, and anti-CD25); DT (anti-IL2R); and PE (anti-CD25, anti-CD22,^247,248 anti-Lewis Y,²⁴⁹ and anti-HER2). A common toxicity is the vascular leak syndrome, characterized by marked fluid overload, dyspnea, and sensorimotor neuropathies.²⁵⁰ Deglycosylated RTA devoid of mannose and fucose has reduced hepatic sequestration, allowing longer serum half-life. Anti-CD22-dsFv-PE (RFB4[dsFv]-PE38, BL22) demonstrated activity for NHL²⁵¹ and cladribine-resistant hairy cell leukemia.²⁵² Adverse effects include transient hypoalbuminemia and elevated aminotransferase levels; serious but reversible hemolytic-uremic syndrome has also been reported.

An expanding collection of toxic natural compounds, such as calicheamicins²⁵³ and maytansinoids,²⁵⁴ are now candidates for antibody drug conjugates. Gemtuzumab ozogamicin (Mylotarg) is an anti-CD33 antibody conjugated to calicheamicin. Acting like a prodrug, calicheamicin is released from the antibody after internalization, forming a diradical that induces double-strand DNA breaks. Gemtuzumab ozogamicin was active in childhood refractory AML²⁵⁵ and achieved a 30% response rate among patients with refractory AML who were 60 years or older.²⁵⁶ When tested as a low-fractionated dose regimen in addition to frontline chemotherapy in a phase 3 randomized trial, 2-year event-free survival was improved from 17.1% to 40.8% and 2-year OS was improved from 41.9% to 53.2%.²⁵⁷ Toxicities included reversible myelosuppression (especially neutropenia and thrombocytopenia), abnormal liver function tests, and bilirubinemia. The incidence of hepatic toxicity including venoocclusive disease was ~2%.¹⁶⁶ These safety concerns led to U.S. marketing withdrawal. Inotuzumab ozogamicin (CMC-544, anti-CD22 calicheamicin conjugate) has showed activity in NHL,²⁵⁸ and phase 3 results are pending.¹⁶⁶ MAb-calicheamicin conjugates (e.g., anti-Lewis Y²⁵⁹ and anti-Mucin²⁶⁰) have not been successful to date in solid tumors. With most immunotoxins, immunogenicity has been a major constraint, although pegylation may reduce immunogenicity.²⁶¹ Brentuximab Vedotin (Adcetris, SGN-35) is a conjugate of chimeric anti-CD30 MAb SGN-30 and monomethyl auristatin E, which disrupts microtubule when internalized. Monomethyl auristatin E is linked to SGN-30 through a protease-cleavable covalent linker cAC10. Because of its activity and safety profile, it gained FDA approval rapidly for refractory or relapsed Hodgkin disease^262,263 and anaplastic large cell lymphoma.^264,265 Auristatin conjugated through cysteine residues (MEDI-547, human anti-EphA2 MAb) was too toxic,²⁶⁶ and when conjugated to MAb specific for carbonic anhydrase showed early promise²⁶⁷; however, clinical development was halted by Bayer Healthcare. Auristatin conjugated to human anti-GPNMB antibody (Glembatumumab vedotin) is undergoing early-phase clinical trials in patients with breast cancer and melanoma.^268,269 Trastuzumab emtansine (T-DM1) is an antibody conjugate comprising trastuzumab and DM1, a microtubule polymerization inhibitor.²⁷⁰ Among patients with HER2-positive metastatic breast cancer, objective responses were seen with minimal toxicity in a phase 1²⁷¹ and phase 2 study.²⁷² In a randomized phase 2 first-line study, T-DM1 showed at least equivalent efficacy when compared with trastuzumab plus docetaxel.²⁷³ Human antidrug antibody was detected in 4.5% of patients.²⁷⁴

Cellular Immunoconjugates with Bispecific Antibodies

Tumor-selective MAbs can be rendered cytophilic by conjugation with MAbs specific for the trigger molecules on T lymphocytes, NK cells, and granulocytes.169,275,276 These molecules include CD3, CD28, Fc receptors (CD64, CD16) and FcαRI (CD89).¹⁷⁰ Bispecific antibodies (BiAb), which redirect polyclonal T-cells to tumors, seem to be most promising. Here, one binding site of the BiAb engages CD3 on T cells, whereas the other binding site determines tumor specificity, for example, B-NHL (CD19),^91,93 breast cancer (HER2),^279–279 Hodgkin lymphoma (CD30), malignant ascites (EpCAM),^138,139 CRC (EpCAM),²⁸⁰ melanoma (MCSP),²⁸¹ prostate (PSMA),²⁸² gastrointestinal and CRC (EGFR²⁸³ or CEA²⁸⁴). Similar successes have been reported for the trigger molecule CD28 for ALL (CD19 and CD20),^285,286 melanoma (MCSP),²⁸⁷ and Hodgkin disease (CD30),²⁸⁸ although severe cytokine release syndrome associated with anti-CD28 MAb TGN1412¹⁵⁴ may discourage their final clinical translation. BiAb targeting FcγRIII (CD16) has also been explored for Hodgkin disease (CD30),²⁸⁹ CD19,²⁹⁰ CD33,²⁹¹ EGFR,²⁹² and HER2,²⁹³ whereas FcγRI was exploited for CD30,²⁹⁴ EGFR,^295,296 and HER2.²⁹⁷ BiAb has also been made selective for epitopes outside the Fc-binding domain of FcγR to bypass competition with serum IgG.

Immunocytokines

Cell-mediated cytotoxicity has been highly effective against tumors in vitro and in animal models. Immunocytokines171,298 have shown remarkable success in activating and redirecting effectors to human tumors. Most of these studies have focused on NK, natural killer T cells (NKT), or T cells¹⁷¹ and granulocytes.¹⁸ Antibody-IL-2 immunocytokine can eradicate metastatic murine neuroblastoma while inducing long-term antitumor immunity.^171,298 After initial successes with IL-2 immunocytokine,²⁹⁹ constructs containing other cytokines have been explored,¹⁷¹ including IL-12, tumor necrosis factor, and lymphotoxin. This emerging technology has been successfully applied to a number of antigens and clinical models,³⁰⁰ including GD2^299,301 and fibronectin.^302,303 Whereas measurable tumor responses were uncommon in clinical trials, microscopic marrow tumor responses have been reported.²⁹⁹ More recently, the combination of a plasmid DNA vaccine and IL-2 immunocytokine in the mouse model was shown to be more effective than when either one was administered alone.³⁰⁴

Immunoenzymes for ADEPT

Another novel approach (ADEPT) uses MAbs to deliver a covalently conjugated enzyme to the tumor, which can then activate a nontoxic prodrug.¹⁷² Despite preclinical successes, ADEPT has been difficult to translate into clinical benefit. Significant impediments to broaden clinical implementation include immunogenicity of antibody-enzyme conjugate, as well as the presence of endogenous enzymes or endogenous substrates and endogenous inhibitors of these enzymes within the tumors.

Immunoliposomes

With advances in liposome technology, several liposomal agents have been licensed for use in patients with cancer.305,306 When coated with polyethylene-glycol, uptake by the reticuloendothelial system is inhibited, thereby prolonging residence time in the blood. Concurrent developments in drug-loading technology have improved the efficiency and stability of drug entrapment in liposomes. Although there is passive accumulation of liposomes in tumors through enhanced permeability and retention, their uptake can be enhanced when engrafted with surface antibodies or their derivatives. For example, scFv or Fab can target liposomes for uptake into tumors bearing CD19,³⁰⁷ HER2,³⁰⁸ EGFR,³⁰⁹ and GD2.³¹⁰ When liposomes fuse with the tumor targets, their contents can be efficiently delivered intracellularly. Although their potential is high, the clinical benefit of MAb-targeted liposomes in cancer therapy remains to be proven.