Therapeutic Antibodies and Immunologic Conjugates

30 

Therapeutic Antibodies and Immunologic Conjugates Konstantin Dobrenkov and Nai-Kong V. Cheung

S UMMARY

OF

K EY

P OI N T S

• Because of their tumor selectivity, monoclonal antibodies offer exceptional opportunities for targeted therapy. • As naked antibodies they can kill tumors by receptor blockade and by actively inducing apoptosis or by reenergizing endogenous antitumor T-cell immunity. • Tumor cytotoxicity is mediated through white cells by activation of antibody-dependent cell-mediated cytotoxicity, and in the presence of serum by complement-mediated cytotoxicity. • Bispecific or multifunctional constructs can greatly enhance antitumor effect of antibodies.

• As immunoconjugates, antibodies can deliver effector molecules in the form of antibody drug conjugates, radioimmunoconjugates, immunocytokines, immunotoxins, immunoenzymes, immunoliposomes, and cellular immunoconjugates. • In general, antibodies do not have overlapping toxicity profiles with chemotherapy and radiation therapies, and dose-limiting toxicities of immunoconjugates vary depending on the cytotoxic moiety (e.g., myelosuppression in radioimmunoconjugates) being used. • Antibodies are likely to be most beneficial at the time of minimal residual disease, especially when

The clinical development of antibody therapy was accelerated by the introduction of the hybridoma technique in 1975 and the emergence of recombinant technology (Table 30.1).1 Through these innovations, individual plasma cells can be immortalized, and cloning of heavy and light chain repertoires from animals and humans is now routinely done. In the last four decades, monoclonal antibodies (mAbs) have evolved from research tools to inclusion in a rapidly increasing list of licensed pharmaceuticals. Mouse-derived mAbs (*momab) have been chimerized (named as *ximab) and humanized (*zumab), and use of mAbs derived from humans (*umab) is now routine. mAbs have generated excitement on many fronts and will likely play a pivotal role in the history of cancer medicine. Immunoediting using immune checkpoint inhibitors (ICIs) has recently opened new venues in treatment of advanced cancer. The clinical usefulness of mAbs for in vitro diagnosis and ex vivo manipulation of blood or stem cells is well recognized. Their role in the treatment and prophylaxis of graft-versushost disease is detailed elsewhere (see Chapter 28). The use of B-cell idiotype and antiidiotypic antibodies as tumor vaccines is described in Chapter 103. This chapter focuses on the application of naked cancer therapeutic mAbs and their conjugates in cancer therapy.

EFFECTOR MECHANISMS OF MONOCLONAL ANTIBODIES mAbs can mediate highly effective tumoricidal functions both in vitro and in vivo (Fig. 30.1).2 These include signaling through receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC). 486

used in conjunction with standard therapy. • The antibodies listed in Table 30.1 have been licensed in the United States or in the European Union over the last 20 years. In the coming decade, other antibodies currently under review or in various phases of clinical trial research may be added to the list. The prospect for further innovation in this established cancer treatment modality is highly favorable.

Cytophilic Monoclonal Antibodies and Antibody-Dependent Cell-Mediated Cytotoxicity The Fc region of immunoglobulin G (IgG) mAbs interacts with both activating and inhibitory Fc receptors (FcγRs).3 In humans, there are four activating FcγRs: FcγRI (CD64) is a high-affinity FcγR, whereas FcγRIIA (CD32A), FcγRIIIA (CD16A), and FcγRIIIB (CD16B) are low-affinity FcγRs carried on the alpha (α) chain. FcγRIIB (CD32B) is the only known inhibitory FcγR. All FcγRs are transmembrane glycoproteins except for FcγRIIIB, which is anchored on neutrophils by glycosylphosphatidylinositol (GPI). FcγRI also has an extracellular portion composed of three Ig-like domains, whereas FcγRII or FcγRIII has only two domains. This 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 in the alpha chain but interact with an adaptor protein called the accessory gamma (γ) chain (Fcγ subunit), which carries a cytoplasmic ITAM. ITAM becomes tyrosine phosphorylated by members of the Src-kinase family with subsequent recruitment of SH2-containing kinases. These events lead to the activation of phosphatidylinositol 3-kinase (PI3-K) and phospholipase-Cγ (PLCγ), followed by protein kinase C (PKC) activation and sustained calcium elevation.3 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 (ITIM) in its cytoplasmic

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 487

Table 30.1  Antibody Therapy of Human Cancer HISTORICAL MILESTONES • • • • • •

Nobel prize awarded to Emil von Behring for work on serum therapy in collaboration with Shibasaburo Kitasato—1901 Nobel prize awarded to Paul Ehrlich for his work on passive immunization—1908 Serotherapy of chronic myelogenous leukemia—1927 Hybridoma technique of Hans Köhler and César Milstein (winners of 1986 Nobel Prize) mAb therapy of lymphoma—1980 FDA approval of mAbs as standard pharmaceuticals—1986

International Nonproprietary Name

Brand Name

Target; Format

Indications

First EU Approval Year

Rituximab Trastuzumab Gemtuzumab ozogamicin Alemtuzumab

MabThera, Rituxan Herceptin

CD20; chimeric IgG1 HER2; humanized IgG1 CD33; humanized IgG4; ADC

NHL Breast CA AML

1998 2000 Under review

1997 1998 2000a

MabCampath, Campath-1H; Lemtrada

CD52; humanized IgG1

CMLa; multiple sclerosis

2001a; 2013

2001a; 2014

Ibritumomab tiuxetan Tositumomab and iodine-131 Cetuximab Bevacizumab

Zevalin Bexxar

CD20; murine IgG1 CD20; murine IgG2a

NHL NHL

2004 NA

2002 2003a

Erbitux Avastin

EGFR; chimeric IgG1 VEGF; Humanized IgG1

2004 2005

2004 2004

Panitumumab Catumaxomab

Vectibix Removab

2007 2009

2006 NA

Ofatumumab Ipilimumab Brentuximab vedotin Pertuzumab Ado-trastuzumab emtansine

Arzerra Yervoy Adcetris Perjeta

EGFR; Human IgG2 EpCAM/CD3; rat-mouse bispecific CD20; human IgG1 CTLA4; human IgG1 CD30; chimeric IgG1; ADC HER2; humanized IgG1 HER2; humanized IgG1; ADC

CRC, head and neck CA CRC, cervical, ovarian, fallopian tube and primary peritoneal CA CRC Malignant ascites CLL Metastatic melanoma Hodgkin lymphoma, NHL Breast CA Breast CA

2010 2011 2012 2013 2013

2009 2011 2011 2012 2013

Denosumab Obinutuzumab

Xgeva Gazyva

Giant cell tumor of bone CLL, FL

2014 2014

2013 2013

Ramucirumab Pembrolizumab

Cyramza Keytruda

2014 2015

2014 2014

Blinatumomab

Blincyto

2015

2014

Nivolumab

Opdivo

CD19, CD3; murine bispecific tandem scFv PD1; human IgG4

Gastric CA Melanoma, NSCLC, squamous head and neck CA, MSI-H/dMMR solid tumors, urothelial CA, Hodgkin lymphoma, gastric or gastroesophageal junction CA B-cell precursor ALL

2015

2014

Dinutuximab Ramucirumab

Unituxin Cyramza

GD2; chimeric IgG1 VEGFR2; human IgG1

2015 2015

2015 2015

Necitumumab Elotuzumab

Portrazza Empliciti

2015 2016

2015 2015

Daratumumab Olaratumab Atezolizumab

Darzalex Lartruvo Tecentriq

EGFR; human IgG1 CD319 (SLAMF7); humanized IgG1 CD38; human IgG1 PDGFRα; human IgG1 PD-L1; humanized IgG1

Melanoma, NSCLC, renal cell CA, Hodgkin lymphoma, squamous head and neck CA, urothelial CA, MSI-H/dMMR CRC, hepatocellular carcinoma Neuroblastoma NSCLC, gastric or gastroesophageal junction CA, CRC NSCLC Multiple myeloma Multiple myeloma Soft tissue sarcoma Urothelial CA, NSCLC

2016 2016 Under review

2015 2016 2016

Mylotarg

Kadcyla

RANKL; human IgG2 CD20; humanized IgG1; glycoengineered VEGFR2; human IgG1 PD1; humanized IgG4

First US Approval Year

Continued

488 Part I: Science and Clinical Oncology

Table 30.1  Antibody Therapy of Human Cancer—cont’d International Nonproprietary Name

Brand Name

Target; Format

Indications

Avelumab

Bavencio

PD-L1; human IgG1

Durvalumab Inotuzumab ozogamicin

Imfinzi Besponsa

PD-L1; human IgG1 CD22; humanized IgG4; ADC

Merkel cell carcinoma, urothelial CA Urothelial CA B-cell precursor ALL

First EU Approval Year

First US Approval Year

2017

2017

Under review 2017

2017 2017

a

Withdrawn or marketing discontinued for the first approved indication. ADC, Antibody drug conjugate; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CA, cancer; CRC, colorectal cancer; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; dMMR, mismatch repair deficient; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; FDA, US Food and Drug Administration; IgG1, immunoglobulin G1; mAb, monoclonal antibody; MSI-H, microsatellite instability–high; NA, not approved; NHL, non-Hodgkin lymphoma; NSCLC, non–small cell lung cancer; PDGFRα, platelet-derived growth factor receptor alpha; scFv, single-chain variable fragment; VEGFR2, vascular endothelial growth factor receptor–2. Modified from Reichert JM. Antibodies to watch in 2017. MAbs 2017;9(2):167–181.

4. Multistep targeting

1. Native IgG mAb

Biotinylated radioactive ligand Streptavidin

ADCC CMC Radionuclide

DOTA Radioimmunoconjugate Toxin, drug, nanoparticles Cytokine

Tumor cell

Immunocytokine

Immunotoxin

T cell

BiTE

Antibody Drug Conjugate Prodrug ADEPT

2. Immunoconjugates

Drug

Immunoliposome

3. Bispecific mAb

mAb enzyme

Figure 30.1  •  Effector mechanisms of mAbs directed at tumor targets. ADCC, Antibody-dependent cell-mediated cytotoxicity; ADEPT, antibody-directed enzyme prodrug therapy; BiTE, bispecific T-cell engaging mAb; CMC, complement-mediated cytotoxicity; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid; mAb, monoclonal antibody; scFv, single-chain variable fragment. (Modified from Carter P. Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer. 2001;1:118–129.)

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 antigen-presenting cells (APCs), can influence the antitumor properties of mAbs.

Inflammatory mediators (interferon-γ or C5a) increase activating FcγRs and downregulate inhibitory FcγRIIB, whereas interleukin (IL)-4, IL-10, and transforming growth factor–β (TGF-β) upregulate FcγRIIB, thereby raising the thresholds for cell activation. Removing the inhibitory signals by FcγRIIB-blocking antibodies has shown efficacy in preclinical models.3 This may be relevant for cross-presentation

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 489

of antigens that is acquired endocytically through Fc receptors on dendritic cells (DCs) during the induction of tumor-specific T-cell responses.4 In addition to these FcγRs, a unique class of Fc receptors called FcRn (neonatal) is found on endothelial cells and regulates antibody catabolism.5 FcRn is similar in structure to major histocompatibility complex (MHC) class I antigen and also associates with β2-microglobulin. It binds IgG at 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 or macrophages.6 Depending on the affinity of the mAbs for the individual FcγRs, both natural killer (NK) cells (carrying FcγRII and FcγRIII) and neutrophils (bearing all three FcγRs) 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 can exploit FcγRIII for lymphocyte ADCC while using FcγRII for myeloid ADCC.7–9 Among the four IgG subclasses, IgG2 has the lowest affinity for the inhibitory receptor FcγRIIB.3 Mouse IgG3 (e.g., 3F8 specific for GD2) can engage both FcγRII and FcγRIII in ADCC,10 despite the low affinity of the monomer for human FcγRs. Polymorphic FcR alleles (FCGR2A11,12 and FCGR3A12,13) with higher affinity for human IgG1 have been reported to mediate more effective ADCC in vitro, and in some studies were associated with superior clinical responses. In addition to FcγRs, adhesion molecules are critical for mAb-mediated ADCC. These molecules include CR3 (CD11b/Cd18)7,8,10 and CD66b7 for neutrophil-mediated ADCC and LFA-1 (CD11a/CD18) for NKmediated ADCC.14 Because cytokines can increase the expression of adhesion molecules, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ,8,15 IL-2,16 and IL-1517 have been used to activate either myeloid- or NK-mediated effector models. Furthermore, because these cytokines expand the effector cell pools, they could 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.

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 IgG2, and has no affinity for IgG4.18 CMC potency of individual mAbs is also correlated with its slow off-rate.19 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),20 homologous restriction factor (CD59),20,21 and membrane cofactor protein (CD46).22–24 The effect of complement activation extends beyond direct tumor lysis. Following complement activation, tumor-bound C3b is cleaved rapidly by plasma protease factor I to iC3b. Through the iC3b receptors, CR3 (Mac-1 or αMβ2-integrin) and CR4 (CD11c/ CD18, αXβ2-integrin) on leukocytes, iC3b acts as opsonin.25 C3a and C5a, byproducts of complement activation, are also potent mediators of inflammation26 and are chemotactic for phagocytic leukocytes, drawing them to the tumor sites. C5a can also downregulate the inhibitory receptor FcγRIIB3 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,27 and the adverse clinical outcome correlations with C1qA levels supported this observation.28

Signaling by Agonistic and Antagonistic Antibodies When the antigen is a tumor cell surface receptor, its clustering by multivalent mAbs can induce apoptosis.29 Apoptosis increases with hyper-cross-linking (e.g., CD20 target on lymphoma cells).30 Both caspase-dependent and caspase-independent programmed cell death pathways appear to be involved.31 In AIDS-related lymphoma (ARL), 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–κB (NF-κB), leading to downregulation of Bcl-xL, thereby sensitizing lymphoma cells to chemotherapy.32 Direct receptor blockade by mAbs has also been reported for epidermal growth factor receptor 1 (EGFR1)33 and human epidermal growth factor receptor 2 (HER2; EGFR2),34 leading to upregulation of the BH3-only protein Bnip3L, thereby sensitizing tumor cells to chemotherapy.35 In addition, induction of cell death through death receptor family functions such as those of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) receptors on tumor cells can be successfully applied for cancer therapy.36 Activating signaling receptors on human cytotoxic lymphocyte and specifically NK cells37 can enhance ADCC functions. Although most relevant for CD8+ T cell–mediated tumor lysis (e.g., CD137 [4-1BB], CD134 [OX40], and glucocorticoid-induced tumor necrosis factor receptor–related protein [GITR]), agonistic antibodies to CD137 can also potentiate NK-ADCC in anti-CD20 and trastuzumab therapy.38 Conversely, antagonistic antibodies to remove immune checkpoint blockade allow T cells to perform more effective surveillance (Fig. 30.2).39 The first successful example of such an immune approach was the anti-CTLA4 mAb ipilimumab, which was approved by the US Food and Drug Administration (FDA) for use in melanoma. An equally exciting strategy targets the programmed death receptor 1 (PD-1) T-cell coreceptor and its ligands B7-H1/PD-L1 and B7-DC/ PD-L2, a pathway that maintains an immunosuppressive tumor microenvironment.40 In addition to turning on T cells, removing inhibitory signals (KIR-2DL and KIR-3DL) on NK cells could also have clinical potential for both hematologic and solid malignancies.41,42 Blocking mAbs (e.g., anti-CD47) have also been effective in unleashing macrophages to phagocytose tumor cells in the absence or presence of mAbs,43 both in vitro and in vivo, in leukemia and lymphoma44,45 as well as in solid tumor models.45,46

CLINICAL APPLICATION OF NAKED MONOCLONAL ANTIBODIES DIRECTED AT CANCER CELLS (SEE TABLE 30.1) Lymphoma and Leukemia In 1997, the anti-CD20 chimeric antibody rituximab became the first mAb approved by the FDA for the treatment of follicular lymphoma (FL).47 Chemosensitization of rituximab when combined with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) was later demonstrated in diffuse large B-cell lymphoma (DLBCL).48 Addition of rituximab to induction chemotherapy improved survival, but most patients were not cured and experienced relapse after a median of 4 years.49 The use of rituximab maintenance did improve progression-free survival (PFS)50 and overall survival (OS) in a meta-analysis.51 For most patients, rituximab was well tolerated. Severe adverse events thought to be secondary to complement activation often occurred with the first infusion,52 especially if there were high numbers of circulating tumor cells. These infusion-related reactions usually appeared 30 to 120 minutes after mAb injection and could be lethal. B-cell depletion occurred in most patients, although hypogammaglobulinemia appeared in only 14% of all patients, and without clinical morbidity.47 Late-onset neutropenia following rituximab therapy occurred in 5% to 27% of patients and was correlated with high-affinity FCGR3A polymorphism.53 Poor tumor response to rituximab includes low-affinity FcγRIII polymorphic allele in the patient’s white cells, low density of CD20

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Antigen presenting cell CD40

CD40L

TL1A

TNFRSF25

GITR ligand

GITR

4-1BB ligand

4-1BB

OX40 ligand

OX40

CD70

CD27

HHLA2

TMIGD2

ICOS ligand

T cell

Activation

ICOS

CD86 CD28

CD80

MHC class II

TCR

Signal 1

LAG3 CD86

CTLA4

CD80

PDL1

PDL1

PD1

PDL2

CD80

VISTA

?

BTNL2

?

B7-H3

?

B7-H4

?

Butyrophilin family

?

CD48

Inhibition

CD244 Galectin 9 Phosphatidylserine

TIM3 BTLA

HVEM

CD160

Siglec family

LIGHT Activation

Figure 30.2  •  T-cell immune checkpoints: activators and inhibitors. The T-cell receptor (TCR) starts the signaling cascade on interaction with peptide

antigen in the context of the major histocompatibility complex (MHC) (signal 1), but optimal activation of naïve T cells depends on a costimulatory signal through CD28 (signal 2). Additional interactions between ligands and activating (green shading) or inhibitory (red shading) receptors are crucial for further regulating T-cell activation and tolerance. These second signals include costimulatory ligands and receptors (blue), immune checkpoint ligands and receptors (red), tumor necrosis factor (TNF) receptor superfamily (TNFRSF; brown) members and their trimeric TNF superfamily ligands (light brown), and additional members of the immunoglobulin superfamily (purple). Examples of therapeutics targeting these and other pathways are shown in brackets. BTLA, B- and T-lymphocyte attenuator; BTNL2, butyrophilin-like protein 2; CTLA4, cytotoxic T-lymphocyte antigen 4; GITR, glucocorticoid-induced tumor necrosis factor receptor–related protein; HHLA2, HERV-H LTR-associating protein 2; HVEM, herpesvirus entry mediator; ICOS, inducible T-cell costimulator; LAG3, lymphocyte activation gene 3 protein; PD1, programmed cell death protein 1; PDL, programmed cell death 1 ligand; siglec; sialic acid–binding immunoglobulintype lectin; TIM3, T-cell immunoglobulin mucin 3; TL1A, tumor necrosis factor–like ligand 1A; TMIGD2, transmembrane and immunoglobulin domaincontaining protein 2; VISTA, V-domain immunoglobulin suppressor of T-cell activation. (Reprinted with permission from Mahoney KM, Rennert PD, Freeman GJ. Combination cancer immunotherapy and new immunomodulatory targets. Nat Rev Drug Discov. 2015;14:561–584.)

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 491

molecules, or the presence of complement inhibitory molecules (CD55 and CD59) on tumor cells.54 Ofatumumab is a next-generation humanized anti-CD20 antibody with more potent cytolytic potential partly because of membrane proximity of its epitope,19 and was approved for fludarabine refractory chronic lymphocytic leukemia (CLL) resistant to alemtuzumab or with bulky lymphadenopathy. It also showed efficacy when added to chlorambucil in previously untreated patients with CLL55 or Waldenström macroglobulinemia56 and when combined with ibrutinib (an inhibitor of Bruton tyrosine kinase) in patients previously treated for CLL and small lymphocytic lymphoma. However, no survival advantage was found between ofatumumab and rituximab in the setting of autologous stem cell transplantation in patients with relapsed or refractory DLBCL.57,58 Ocrelizumab,59 veltuzumab,60 GA101 (obinutuzumab),61 AME-133v,62 and PRO13192163 are all humanized anti-CD20 antibodies with enhanced binding to low-affinity FcR receptor that are currently undergoing clinical testing.54 Campath-1H (alemtuzumab), a humanized rat IgG1 anti-CD52 mAb, demonstrated antitumor activity in patients with recurrent B-cell CLL in whom treatment with fludarabine had failed.64 Grade 3 or 4 infections were reported in 26.9% of patients.65 Opportunistic infections including bacterial sepsis and viral infections, vasovagal and hypotensive episodes, and marrow aplasia have since been reported.64,66 After treatment with alemtuzumab, cytomegalovirus (CMV) antigenemia responsive to antiviral therapy was found in all patients.67 Epratuzumab is a humanized IgG1 antibody directed at CD22, a cell surface antigen expressed in B-precursor acute lymphoblastic leukemia (ALL), showing modest activity in patients with non-Hodgkin lymphoma (NHL), as single agent68 or in combination with rituximab.69 Most clinical effort has been focused on its immunotoxin or radiolabeled forms.70,71 SGN-30 (chIgG1 anti-CD30), XmAb2513 (humanized SGN-30), and MDX-060 were developed for Hodgkin disease,72 with modest clinical activity as naked mAb. Conjugation of SGN-30 to the antimitotic agent monomethyl auristatin E (MMAE) led to generation of an antibody-drug conjugate, SGN-35 (brentuximab vedotin), which was approved for CD30+ Hodgkin and anaplastic large cell lymphoma.73 Blinatumomab, a tandem single-chain variable fragment (scFv) bispecific antibody (bispecific T-cell engaging antibody [BiTE]) retargets cytolytic T cells to kill leukemia cells through classic cytolytic immune synapse. At extremely low doses of 60 µg/m2/day given as a continuous intravenous injection, for 4 to 8 weeks, major responses were achieved in 82% of patients with mantle cell lymphoma (MCL), FL, and DLBCL,74,75 as well as molecular complete remission (CR) in 80% of patients with adult B-cell acute lymphoblastic leukemia (B-ALL).75 In pediatric patients with relapsed or refractory ALL, blinatumomab achieved a 39% complete remission within the first two cycles; complete minimal residual disease (MRD) response was seen in 52% of these patients. Blinatumomab dosage for children with relapsed or refractory ALL was 5 µg/m2/day for the first 7 days, followed by 15 µg/m2/day thereafter.76 Blinatumomab was approved for adult B-ALL and in pediatric relapsed or refractory B-cell precursor ALL that is Philadelphia chromosome negative.77 Significant side effects include cytokine release syndrome (CRS; especially during the first treatment days) and reversible central nervous system (CNS) symptoms probably due to neuroinflammation (seizures, convulsions, encephalopathy, confusion, and cerebellar symptoms), both preventable by premedication with dexamethasone and stepwise dose escalation.78 In myeloid leukemia, the addition of lintuzumab (HuM195, IgG1, anti-CD33) to salvage induction chemotherapy was found to be safe, but it did not result in a statistically significant improvement in response rate or survival in patients with refractory or relapsed acute myeloid leukemia (AML).79

Solid Tumors Trastuzumab (Herceptin) is a humanized mAb against the receptor tyrosine kinase ERBB2 (HER2/neu) on breast cancer cells. In addition to 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.80 Up to 25% of breast cancers have HER2-positive disease associated with poor prognosis.81 Trastuzumab in combination with paclitaxel improved response duration (median 9.1 versus 6.1 months), objective response rate (ORR; 50% versus 32%), and death rate at 1 year (22% versus 33%),82,83 compared with paclitaxel alone, and was approved by the FDA for 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 in women with HER2-positive breast cancer.84 Severe congestive heart failure occurred at a rate of 0.8% and significant left ventricular ejection fraction decrease was seen in 3.6% of patients in the trastuzumab treatment group versus 0% and 0.6% in the observation group, respectively,85 although both symptomatic and asymptomatic events were largely reversible and manageable.86 Recent studies also have suggested that synergy between trastuzumab and chemotherapy may still be effective beyond disease progression.87 Pertuzumab (Perjeta), a humanized mAb that inhibits HER2 heterodimerization with other HER family members, produced 18% to 24% response when combined with trastuzumab in patients with HER2-positive metastatic breast cancer. When the phase III randomized study (N = 808 patients) of the combination of pertuzumab plus docetaxel plus trastuzumab versus docetaxel plus trastuzumab showed significant prolongation of PFS (18.5 versus 12.4 months) as first-line treatment for HER2-positive breast cancer, it was approved by the FDA. In the neoadjuvant setting, when pertuzumab in combination with trastuzumab and docetaxel in locally advanced, inflammatory, or early-stage HER2-positive breast cancer showed significant improvement in pathologic complete response over trastuzumab plus docetaxel,88,89 it received accelerated FDA approval. In advanced HER2-positive gastric cancer, a phase III trial of trastuzumab plus platinum-based chemotherapy versus chemotherapy alone showed an OS advantage of 2.7 months.90 However, its benefit when added to standard chemotherapy was uncertain for non–small cell lung cancer (NSCLC)91 or ovarian cancer.92 Ado-trastuzumab emtansine, a HER2-targeted antibody and microtubule inhibitor conjugate, significantly improved PFS and OS with less toxicity than lapatinib plus capecitabine in patients with HER2-positive advanced breast cancer.93 This agent was approved by the FDA for the treatment of patients with HER2-positive, metastatic breast cancer who had previously received trastuzumab and a taxane. Cetuximab (chIgG1 anti-EGFR) is an FDA-approved chimeric antibody designed to induce receptor blockade, reversing resistance to topoisomerase I inhibitors in addition to having modest monotherapy activity in colorectal cancer (CRC),94 with improvements in PFS and OS.95 First-line treatment with cetuximab plus FOLFIRI, as compared with FOLFIRI alone, reduced the risk of progression of metastatic CRC, although no significant difference was found in OS.96 An acneiform rash occurred in 82.9% of patients; grade 3 rash was observed in 4.9%. Only a minority of patients (~1%) developed serious hypersensitivity-hypomagnesemia reactions requiring H1 antihistamines and steroids.94 Response and survival correlated strongly with the severity of the rash. In contrast, clinical benefit did not correlate with EGFR immunostaining of the tumor.97 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 (wt) KRAS,94 not all KRAS-mutated tumors were resistant to cetuximab.98 Expression of epiregulin or amphiregulin was a biomarker for efficacy.99 In 2012 the FDA approved cetuximab in combination with FOLFIRI for first-line treatment of patients with KRAS mutation–negative, EGFR-expressing metastatic CRC. Panitumumab, a fully human anti-EGFR antibody generated with human IgG–transgenic mouse technology, was much less immunogenic.100 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 wt KRAS tumors in a phase III randomized trial,

492 Part I: Science and Clinical Oncology

but not their OS.101 As second-line treatment, panitumumab plus FOLFIRI (fluorouracil, leucovorin, and irinotecan) was better than FOLFIRI alone in prolonging median PFS (from 3.9 to 5.9 months) and median OS (12.5 to 14.6 months).102 Addition of panitumumab to epirubicin, oxaliplatin, and capecitabine in patients with previously untreated advanced esophagogastric adenocarcinoma did not increase OS in a phase III clinical trial.103 When combined with radiotherapy for locally advanced squamous cell carcinoma of head and neck, cetuximab improved the duration of locoregional control from 14.9 to 24.4 months, median OS from 29.3 to 49 months, and 5-year OS from 36.4% to 45.6%, with hazard ratio (HR) of 0.49 (P = .002), in patients with grade 2 or greater acneiform rash.104 As first-line treatment of recurrent or metastatic head and neck cancer, addition of cetuximab to platinum plus fluorouracil in a randomized phase III trial increased response rate from 20% to 36% and prolonged median PFS from 3.3 to 5.6 months and median OS from 7.4 to 10.1 months.105 Two large randomized phase III 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.106,107 Subgroup analyses suggested a significant association between early occurrence of skin rash108 and intensity of EGFR staining109 with efficacy. Necitumumab is another EGFR specific human IgG1 antibody that was approved by the FDA in combination with gemcitabine and cisplatin as first-line therapy for metastatic squamous NSCLC.110 However, the addition of necitumumab to pemetrexed and cisplatin was not proven to be of benefit in metastatic nonsquamous NSCLC in another randomized multicenter trial.111 mAb inhibition of vascular endothelial growth factor receptor–1 (VEGFR1)112 or VEGFR2113 can enhance the efficacy of chemotherapy. Bevacizumab (Avastin), a humanized IgG1 specific for vascular endothelial growth factor (VEGF), was approved by the FDA for metastatic renal cancer114; metastatic CRC115; cervical, ovarian, and fallopian tube tumors; and primary peritoneal carcinomatosis. Based on modest improvements in OS, the antibody ramucirumab, specific for VEGFR2, was approved by the FDA for several indications: as a single agent in gastroesophageal junction (GEJ) adenocarcinoma refractory to or progressive following first-line therapy with platinum- or fluoropyrimidine-containing chemotherapy, or in combination with paclitaxel for previously treated advanced gastric or GEJ adenocarcinoma,116 for previously treated metastatic NSCLC in combination with docetaxel,117 in combination with FOLFIRI for metastatic CRC that progressed on a first-line bevacizumab-, oxaliplatin-, and fluoropyrimidine-containing regimen.118 Despite initial enthusiasm, adjuvant therapy with edrecolomab (17-1A, Panorex), a mouse IgG2a antibody specific for epithelial cell adhesion molecule (EpCAM) on malignant and normal epithelial cells, did not improve DFS or OS in subsequent phase III studies in CRC.119,120 The story of edrecolomab provided a case study for clinical failure of mAb on a major scale.121 Despite the lack of clinical efficacy, antiidiotype network and T-cell responses against antibody-modified tumors were found.122 Adecatumumab, the fully human mAb targeting epithelial cell EpCAM, was shown to be safe in combination with docetaxel and demonstrated clinical benefit in 44% of relapsed or refractory advanced-stage breast cancer.123 Catumaxomab (Removab), a rat-mouse bispecific antibody engaging CD3 on T cells and EpCAM on tumor cells, was approved in Europe for malignant ascites.124 Oregovomab (mAb B43.13, anti-CA125), a murine IgG1 antibody for ovarian cancer, did not improve time to relapse in a phase III randomized trial of 145 patients.125 A correlation of improved survival and human antimouse antibody (HAMA)/idiotype network response was of biologic interest.126 There is a paucity of mAbs for the treatment of sarcomas and other solid tumors. To date, only olaratumab, specific for platelet-derived growth factor receptor alpha, is FDA approved for soft tissue sarcoma (STSs) not responsive to radiotherapy, surgery, or anthracycline.127 Among the ganglioside antigens on neuroectodermal tumors, GD3

(mAb R24 for melanoma)128 and GD2 (mAb 3F8 and ch14.18 for neuroblastoma)9,129 have been tested clinically. GD2 is present on a variety of solid tumors in addition to neuroblastoma, including osteosarcoma, retinoblastoma, some STSs, brain tumors, mesenchymal stem cells,130 and cancer stem cells.131 The clinical effectiveness of anti-GD2 mAbs for large soft tissue masses was minimal. Its use was more suitable for microscopic marrow disease in the presence of GM-CSF and/or IL-2.132 Activation of CD11b by GM-CSF on myeloid cells correlated with patient PFS.133 Missing ligands for killer immunoglobulin-like receptors (KIRs) on NK cells significantly affected both PFS and OS.9,134 Failure to achieve early MRD remission was the strongest predictor of clinical outcome.135 Human antimouse antibody (HAMA) response was also a strong positive predictor of OS, 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.136,137 With anti-GD2 therapy, disease relapse was primarily at isolated sites; and isolated CNS relapse, previously rare, was salvageable by combining surgical resection, radiation, and intrathecal iodine-131 (131I) mAb.138 Clinical development of anti-GD2 mAbs was limited by its pain side effects thus precluding dose escalation. Based on a statistical improvement in OS, but not event-free survival (EFS), in patients with metastatic stage 4 neuroblastoma, dinutuximab (Unituxin, ch14.18), in combination with GM-CSF and IL-2, was approved for the treatment of metastatic stage 4 neuroblastoma following stem cell transplantation.129,139 Another form of ch14.18 produced in CHO cells140 instead of mouse SP/2 cells129 as well as humanized forms of 3F8 (hu3F8)141 and 14.18 (hu14.18) are currently under active clinical investigations. The use of ICIs for the treatment of solid tumors is discussed separately in more detail later.

IMMUNE CHECKPOINT INHIBITORS The most recent success in immunotherapy was the introduction of antibodies acting at immune checkpoints (ICIs), producing a novel paradigm in anticancer therapy. Dramatic and sustained responses have been seen in advanced tumors including melanoma, NSCLC, and head and neck cancers. An important attribute of the ICI is the induction of long-term immune surveillance as demonstrated in patients with melanoma.142 The rationale behind ICIs is based on the well-known functions of cytotoxic T cells. Human tumors with somatic mutations carry neoantigens that could stimulate specific T cell–mediated responses. Such responses are regulated by a complex interacting network of stimulatory and inhibitory molecules expressed on immune effectors, cancer cells, and cells in the tumor microenvironment. Agonistic or antagonistic antibodies can now be used to modulate these interactions. In contrast to the antibodies targeting tumor cells directly, ICIs enhance antitumor activity of de novo immune effectors by removing their brakes.143 Normally activated T cells require a sequence of costimulatory signals provided by ligation of T-cell receptors (TCRs) and peptide/ MHC complex followed by interaction of CD28 receptors on T cells with molecules of the B7 family on APCs.144 Cytotoxic T-lymphocyte antigen 4 (CTLA4; CD152), another ligand for the B7 costimulatory molecule, acts as a negative regulator of T cell activation.145 The antitumor therapeutic effect of antibodies blocking CTLA4 was proven in experiments carried out nearly two decades ago.146 Ipilimumab and tremelimumab were the first humanized anti-CTLA4 antibodies tested in patients with advanced, refractory tumors.147,148 With an improvement in OS in previously treated metastatic melanoma, ipilimumab became the first ICI approved by the FDA. Antibodies targeting programmed cell death protein 1 (PD-1; CD279) or its ligand PD-L1 (B7-H1, CD274) constitute another group of ICIs that are actively evaluated in numerous clinical trials. The human PD-1 gene is located on 2q chromosome and encodes a 288–amino acid transmembrane protein.149 The PD-1 receptor is expressed on T cells, B cells, NK T cells, activated monocytes, and

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 493

DCs,150 and its engagement by PD-L1 (or PD-L2) negatively regulates lymphocyte activation.151 Importantly, PD-1 is highly expressed on tumor-infiltrating lymphocytes (TILs).152 Expression of PD-L1 on various tumor cells is upregulated by interferon-γ.153 PD-1/PD-L1 interaction plays a crucial role in the evasion mechanism exploited by tumors to divert T-cell cytotoxicity.154 In a seminal phase I study, a single dose of humanized IgG1 anti-PD-1 antibody, CT-011 (pidilizumab), administered to patients with advanced hematologic malignancies155 was well tolerated and induced one complete response of NHL. Another anti-PD-1 antibody, MDX-1106 (human IgG4), was also safe in patients with refractory solid tumors,156 with only one serious adverse event (inflammatory colitis). CR in one patient with CRC and two partial remissions (PRs) in patients with melanoma and RCC were observed. Tumors from nine patients were studied for B7-H1 (PD-L1) expression. Interesting to note, three out of four patients with B7-H1+ tumors experienced tumor regression after administration of MDX-1106. In contrast, no clinical response was observed in five patients with tumors negative for B7-H1 staining. As of December 2017, two ICIs targeting PD-1 (pembrolizumab, nivolumab) and three ICIs targeting PD-L1 (atezolizumab, durvalumab, avelumab) have been approved by the FDA. Pembrolizumab demonstrated its safety and efficacy in patients with ipilimumab-naïve and ipilimumab-refractory melanoma and was the first FDA-approved anti-PD-1 antibody. Multiple clinical trials generated results that served as a basis for FDA approval of PD-1 inhibitors for treatment of melanoma (Table 30.2).157–161 Anti-PD-1 antibodies (nivolumab, pembrolizumab) soon were approved by the FDA for lung,162–165 renal cell,166 and squamous head and neck cancers167,168 and some others.169,170 Pembrolizumab was the first ICI approved for microsatellite instability–high (MSI-H) or mismatch repair deficient (dMMR) solid tumors. This was the first approval based on a biologic marker and not on tumor site or histology. PD-L1–directed ICIs have been approved for urothelial carcinoma and some other tumors.171–173 Multiple phase III clinical trials are ongoing, and new approved indications are awaited. Combination studies targeting CTLA4 and PD-1/PD-L1 are rapidly accruing.174,175 Nivolumab plus ipilimumab was the first combination approved by the FDA for the treatment of advanced melanoma. Addition of chemotherapy to these combinations is another possible option in order to increase efficacy. Safety and tolerability remain among major concerns for these multiagent therapies. mAbs already established for treatment of various tumors are also being evaluated in combination with ICIs in early-phase clinical trials (e.g., cetuximab [NCT02713373, NCT02124850], trastuzumab [NCT02954536, NCT02605915], ramucirumab [NCT02999295, NCT02443324], necitumumab [NCT02451930]). Clinical trials on combinations of ICIs with other immune modulators, such as anti-CD137 (e.g., NCT02253992), indoleamine 2,3-dioxygenase (IDO) inhibitors (e.g., NCT02073123, NCT02048709), anti-GITR (e.g., NCT01239134, NCT02132754), anti-TLR9 (e.g., NCT02521870) antibodies, and some others are ongoing, and results are awaited. With the success of ICIs, the role of predictive biomarkers has resulted in new challenges. The inflammatory phenotype of tumors can greatly influence antitumor activity of ICIs176; melanomas can be divided into inflammatory and noninflammatory categories, and distinct gene expression profiles of the tumor microenvironment are associated with clinical outcome.177 In general, ICI activity is more pronounced in T cell–inflamed tumors, and the tumor genomic landscape determines PD-1/PD-L1 blockade efficacy. Mutational load predicted tumor response to ICIs in NSCLC178 and some mutations in melanoma were associated with acquired resistance to PD-1 blockade.179 The use of these predictive biomarkers should enrich the frequency of response while reducing unnecessary side effects,180 even though their validity so far remains debatable.181 Of note was pembrolizumab, the first ICI approved in conjunction with a companion diagnostic test (the PD-L1 IHC 22C3). Although PD-L1 expression was associated with clinical response to agents targeting the anti-PD-1/PD-L1 pathway in patients

with melanoma and nonsquamous NSCLC, some patients with PD-L1–negative tumors still benefited from anti-PD-1 therapy.182

Complications and Contraindications Toxicities of mAbs are in general manageable and usually self-limited. Common acute reactions include fever, chills, headache, nausea, fatigue, angioedema, urticaria, pruritus, blood pressure fluctuations, and bronchospasm. Lethal or irreversible side effects unique to some mAbs include autoimmunity (ICIs); cytokine release (antilymphocyte mAbs); complement activation (anti-CD20) syndromes52; immune suppression (anti-CD20, anti-CD52)–associated viral reactivation, opportunistic infections, and progressive multifocal leukoencephalopathy64,66; preexisting IgE responses to galactose-α-1,3-galactose (cetuximab)183; cardiotoxicity (anti-HER2)83; and late-onset neutropenia (rituximab).53 The severe CRS associated with antilymphocyte (e.g., anti-CD28, TGN1412) mAbs is extremely rare,184 although TGN1412 provided a case study for potential catastrophic complications for any first-inhuman biologic. A unique severe self-limited acute side effect is the pain syndrome from cross-reactivity of anti-GD2 mAbs with peripheral pain fibers,185 and occasional transverse myelitis seen with ch14.18 (dinutuximab). The murine-derived N-glyconeuraminic acid (e.g., cetuximab) introduced when mAb is produced in mouse cell lines can react with natural antibodies in humans. HAMA is primarily directed to the murine Fc portion of the antibody, although antiidiotypic responses have also been reported.186 With chimeric, humanized, primatized, and human antibodies, the incidence of neutralizing antibody decreases, but their frequency varies with individual mAbs from minimal (0.2%)100 to substantial (49%).187 The introduction of ICIs in cancer therapy has uncovered a broad spectrum of autoimmune side effects.188 Ipilimumab-related adverse events (irAEs) were often mild to moderate but occurred in more than 70% of patients and correlated with ipilimumab dose,189,190 manifesting as grade 3 or 4 (10%–15% of patients) enterocolitis, hepatitis, dermatitis, and endocrinopathies (e.g., thyroid dysfunction, hypophysitis).191,192 Among these toxicities, skin reactions (rash and itching) were the most common (30%–50% of patients), followed by enterocolitis (~30%),189,190 hepatotoxicity (2%–9%),190 and hypophysitis (1.5%) with vague symptoms of fatigue, headaches, myalgia, loss of appetite, or nausea and vomiting; hypophysitis with adrenal insufficiency or crisis was potentially fatal, requiring urgent attention and treatment.193 Neurologic toxicities were rare, although life-threatening neuropathies, such as Guillain-Barré syndrome, severe motor neuropathy, myasthenia gravis, aseptic meningitis, and optic neuritis have been reported.192 A meta-analysis of 18 clinical trials identified an association of higher dose (10 mg/kg) of anti-CTLA4 mAbs with higher risk of treatment-related mortality (TRM).194 As expected, the development of irAEs was associated with antitumor response and favorable outcome in some studies.190 Most irAEs could be controlled with high-dose corticosteroids, which did not appear to impair the antitumor effects of ipilimumab.195 In general, the toxicity profile of PD-1 and PD-L1 blockade has been less severe than that of anti-CTLA4 mAb in the meta-analysis, and no significant increase in TRM was seen.194 Although the toxicity was less severe, PD-1/PD-L1 inhibitors showed a slightly different organ-specific toxicity profile; inflammatory conditions such as pneumonitis were more common than colitis.196 The incidence of grade 3 or 4 toxicity was 7% to 14%156,157,160,197; fatigue was most common (16%–37% with anti-PD-1 and 12%–24% with anti-PD-L1 mAb), followed by fever, chills, and infusion reactions.196 Dermatologic toxicities included rash, pruritus, and vitiligo (a complication more common than with ipilimumab, 10% versus 2%, respectively).157 Colitis, endocrine toxicities, and hepatic toxicities have been described, but most of a lesser extent than with anti-CTLA4 mAb.157,196 However, pneumonitis, which was rarely reported in the studies of anti-CTLA4 mAb alone, was seen in 10% of patients with anti-PD-1/PD-L1 therapy, leading to three treatment-related deaths in an early-phase study of nivolumab.197

494 Part I: Science and Clinical Oncology

Table 30.2 Clinical Trials of Immune Checkpoint Inhibitors (ICIs) in Patients With Melanoma and Lung

Cancer: Basis for FDA Approval

Clinical Trial MELANOMA

Patient Population

Treatment

Statistical Significant End Points

NCT01866319

Ipilimumab-naïve unresectable or metastatic melanoma

Pembrolizumab every 2 wk or every 3 wk vs ipilimumab every 3 wk

NCT01704287

Melanoma refractory to ipilimumab or BRAF V600–positive melanoma refractory to BRAF or MEK inhibitor Untreated, unresectable, or metastatic melanoma without a BRAF mutation

Pembrolizumab vs investigator-choice chemotherapy

Unresectable or metastatic melanoma, progressed after ipilimumab, or ipilimumab plus a BRAF inhibitor if BRAF V600+ Untreated unresectable stage III or IV melanoma

Nivolumab vs chemotherapy

6-mo PFS: 47.3% (pembrolizumab every 2 wk), 46.4% (pembrolizumab every 3 wk), 26.5% (ipilimumab); 12-mo OS: 74.1%, 68.4%, and 58.2%, respectively ORR: 33.7%, 32.9%, 11.9%, respectively TRAE (grade 3–5) rates lower with pembrolizumab PFS HR: 0.57 (pembrolizumab, 2 mg/kg), 0.50 (pembrolizumab, 10 mg/kg) ORR: 21%, 25%, 4% TRAE (grades 3–4) rates lower with pembrolizumab 12-mo OS: 72.9% (nivolumab), 42.1% (dacarbazine) ORR: 40.0% (nivolumab), 13.9% (dacarbazine) TRAE rates lower with nivolumab ORR: 31.7% (nivolumab) vs 10.6% (chemotherapy) TRAE rate lower with nivolumab

Nivolumab vs nivolumab plus ipilimumab vs ipilimumab every 2 wk

PFS: 11.5 mo (nivolumab plus ipilimumab), 6.9 mo (nivolumab), 2.9 mo (ipilimumab) ORR: 57.6%, 43.7%, 19.0%, respectively In PD-L1–negative tumors, nivolumab plus ipilimumab was more effective than either agent alone

161

NCT01642004

Metastatic squamous NSCLC progressed during or after one prior platinum-based chemotherapy

Nivolumab or docetaxel

162

NCT02142738

Untreated advanced NSCLC PD-L1–positive ≥50% of tumor cells, no EGFR mutation or ALK translocation Metastatic nonsquamous NSCLC with progression on or after platinum-based chemotherapy Treated NSCLC with PD-L1 expression on ≥1% of tumor cells

Pembrolizumab or the investigator’s choice of platinum-based chemotherapy

NCT02008227

Previously treated NSCLC

Atezolizumab or docetaxel

NCT01903993

Previously treated NSCLC

Atezolizumab or docetaxel

1-yr OS: 42% (nivolumab), 24% (docetaxel) ORR: 20% vs 9% OS, PFS, and ORR were better with nivolumab, regardless of PD-L1 expression level TRAE rate lower with nivolumab PFS: 10.3 mo (pembrolizumab), 6.0 mo (chemotherapy) 6-mo OS: 80.2% vs 72.4% ORR: 44.8% vs 27.8% TRAE rate lower with pembrolizumab OS: 12.2 mo (nivolumab) vs 9.4 mo (docetaxel) ORR: 19.2% vs 12.4% If PD-L1 positive, better survival with nivolumab OS: longer for pembrolizumab (2 mg/kg or 10 mg/kg) vs docetaxel The first study showing how PD-L1 tumor proportion score of ≥50% predicts response to pembrolizumab TRAE (grades 3–5) rate lower with pembrolizumab OS: 13.8 mo (atezolizumab), 9.6 mo (docetaxel) If PD-L1 positive, 15.7 mo vs 10.3 mo OS: 12.6 mo (atezolizumab) vs 9.7 mo (docetaxel) Increasing OS associated with increasing PD-L1 expression

NCT01721772

NCT01721746

NCT01844505

Nivolumab vs dacarbazine

Reference 157

158

159

160

LUNG CANCER

NCT01673867

NCT01905657

Nivolumab or docetaxel every 3 wk

Pembrolizumab or docetaxel

163

164

165

171 172

FDA, US Food and Drug Administration; HR, hazard ratio; NSCLC, non–small cell lung cancer; ORR, objective response rate; OS, overall survival; PFS, progression-free survival; TRAE, treatment-related adverse event.

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 495

IMMUNOCONJUGATES The clinical usefulness of naked mAb can be limited by both host (number and activity of effector cells, FcR polymorphism, and interference by inhibitory receptors) and tumor factors (antigen heterogeneity and complement regulatory proteins). Although the CMC and ADCC functions of naked mAb (see Fig. 30.1) can be improved by altering the Fc protein structure198 or by modifying Fc-glycosylation,199 substantial gains in clinical potentials of mAb can derive from immunoconjugates. These include (1) radioimmunoconjugates to deliver beta and alpha emitters,200–202 (2) immunotoxins203 and antibody drug conjugates,204 (3) bispecific mAb (specific for soluble ligands, cell surface antigens, and receptors) to enhance signal blockade or to cross-link effector cells selectively to tumors,205,206 (4) immunocytokines to deliver cytokines to tumor sites while minimizing systemic toxicities,207 (5) antibody-directed enzyme prodrug therapy (ADEPT) to pretarget enzymes to tumor sites for prodrug activation in order for high local concentrations of active drugs to be released without triggering systemic toxicities,208 and (6) immunoliposomes to deliver drugs, isotopes, DNA, or toxins.209

Radioimmunoconjugates202 mAbs have the potential to target and ablate tumors in radioimmunotherapy (RIT). Radioimaging using single-photon emission computed tomography (SPECT) and positron emission tomography (PET) can map the biodistribution of mAbs and quantify the relative amounts of mAbs deposited in various tissues and organs thus allowing more precise radiation dose estimates in therapeutic studies. When imaging and dosimetry are combined with therapy, a theranostic agent is born. In preclinical models, ablation of established xenografts is possible, although radiation damage to the marrow has been dose limiting. For patients with lymphoma and leukemia, antitumor activity of RIT is highly reproducible, but major responses in solid tumors are rare because of the unfavorable therapeutic index (tumor dose versus normal tissue dose). Unlike naked antibodies, the bystander effect of RIT from cross-firing of the radioisotopes could overcome tumor antigen heterogeneity, but at the same time is responsible for most of the dose-limiting toxicities of radioimmunoconjugates. Clinical applications of RIT have generally used beta-emitting radioimmunoconjugates (Table 30.3). Beta emitters, because of their long range, 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 beta-particle emitter. Because of its gamma-particle emission, it is also suitable for dosimetry studies. However, this gamma-particle emission poses a radio-hazard at high treatment doses, necessitating patient isolation. In vivo dehalogenation can compromise tumor dose with subsequent thyroid damage from

the released iodide. Yttrium-90 (90Y) is a pure beta 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 gamma emissions, thereby requiring the use of indium-111 (111In) to estimate biodistribution and dosimetry. In addition to 90Y, other beta emitters recently explored include rhenium-186 (186Re), rhenium-188 (188Re), copper-67 (67Cu), and lutetium-177 (177Lu), each with its own limitations. Alpha particles are helium nuclei; when compared with beta particles, they have a shorter range (50–80 µm) and a higher linear energy transfer (approximately 100 keV/µm).210 As few as one or two alpha particles can destroy a target cell. RIT using alpha emitters should result in less nonspecific toxicity to normal bystanders as well as more efficient single-cell killing. This is ideal for controlling MRD. Alpha particle–emitting isotopes such as astatine-211 (211At) and bismuth-213 (213Bi)210 have been used against AML,211 gliomas,212 and micrometastases or neoplasms on the surface of body compartments, such as ovarian cancer and leptomeningeal metastasis.213,214 In general, the major dose-limiting toxicity of intravenous RIT using IgG is myelosuppression or myeloaplasia requiring stem cell rescue (Table 30.4). Beyond the marrow, significant extramedullary toxicities of kidney and liver have limited further dose escalation. Hypothyroidism is common with 131 I mAb therapy, and secondary myelodysplastic syndrome and AML have also been reported.215 Clinical benefit is most evident for lymphomas and leukemias, whereas results for solid tumors are mostly discouraging. In general, patient referral for RIT was limited because of alternative options in nonradioactive therapies, as well as logistics and medical economic concerns216; for example, despite FDA approval, Bexxar was removed from the market in 2014.

Compartmental Radioimmunotherapy (See Table 30.4) To improve the therapeutic index, compartmental RIT has also been explored. Intraperitoneal 90Y-CC49217 and 90Y-HMFG1 (anti-MUC1)218 in recurrent ovarian cancer were safe but without survival gain. Dosimetry studies following intraperitoneal 211At-MX35 F(ab′)2 (targeting the 95-kD plasma membrane sodium-dependent phosphate transporter protein 2b) in ovarian cancer,219 and therapeutic trials of intraperitoneal lead-212 (212Pb)-trastuzumab appeared safe with no late toxicity at more than 1 year of follow-up.220 Most notable are the intrathecal and intraventricular administration routes for leptomeningeal carcinomatosis, and intratumoral therapy of malignant brain tumors. 131 I-81C6 (antitenascin) and 211At-81C6 have produced objective responses and prolonged patient survival.221,222 Intraventricular 131I-3F8 (anti-GD2)223 in recurrent medulloblastoma and 131I-8H9 (antiB7-H3)138 in relapsed CNS neuroblastoma have both achieved long-term cures. Intratumoral iodine-124 (124I)-8H9 delivered by convention enhanced delivery to diffuse intrapontine glioma (DIPG) was well tolerated with early evidence of prolonged survival.

Table 30.3  Choice of Radioisotopes for Radioimmunotherapy Isotope

Particle(s) Emitted

Iodine-131 (131I) Copper-67 (67Cu) Lutetium-177 (177Lu) Rhenium-188 (188Re)

Beta, gamma Beta Beta Beta (gamma)

Yttrium-90 (90Y) Actinium-225 (225Ac) Astatine-211 (211At) Bismuth-212 (212Bi) (212Pb daughter) Bismuth-213 (213Bi) Radium-223 (223Ra)

Beta Alpha Alpha (gamma) Alpha (beta) Alpha (beta, gamma) Alpha (gamma)

Maximum Energy (keV)

Mean Range of Alpha- or Beta-Particle Emission (µm)

193 62 161 17

610 577 496 2120

800 1800 1500 2400

64 240 7.2 1.0 0.76 274

2280 6000 6000 6000 6000 6000

2700 50 50 50 50 50

Half-Life (h)

496 Part I: Science and Clinical Oncology

Table 30.4  Radioimmunoconjugates and Antibody Drug/Toxin Conjugates Antibody RADIOIMMUNOCONJUGATES

Target

Payload

Cancer Diagnosis

Reference

cG250 C250 B4 Ibritumomab (Zevalin) Tositumomab (Bexxar) Epratuzumab Epratuzumab Epratuzumab   Anti-TAC   Ki-4   P67   HuM195   HuM195

CAIX CAIX CD19 CD20 CD20 CD22 CD22 CD22 CD25 CD30 CD33 CD33 CD33 CD33

131

  MB-1   BC8   Anti-CD66    hMN-14 cT84.66   OKB7   3F8   A33   Lym-1   Anti-id

CD37 CD45 CD66 (a,b,c,d) CEA CEA CR21 (CR2) GD2 GPA33 HLA-DR Idiotypes

Iodine Lutetium 90 Yttrium 90 Yttrium 131 Iodine 131 Iodine 186 Rhenium 90 Yttrium 90 Yttrium 131 Iodine 131 Iodine 213 Bismuth 225 Actinium 90 Yttrium 131 Iodine 131 Iodine 188 Rhenium 131 Iodine 90 Yttrium 131 Iodine 131 Iodine 131 Iodine 131 Iodine 90 Yttrium

Renal CA Renal CA NHL NHL NHL NHL NHL NHL T-cell leukemia NHL AML, MDS, blastic CML AML AML AML NHL AML, ALL, MDS AML, ALL, CML CRC Carcinomas NHL Neuroblastoma CRC NHL NHL

344 345 346 347–349 215,350–352 353 354 355 356 357 358 359 359 360 361 362 363 364 365 366 367 368 369 370

124

Iodine Iodine 131 Iodine 212 Lead 90 Yttrium 211 Astatine 90 Yttrium 131 Iodine 211 Astatine

DIPG CNS neuroblastoma Medulloblastoma Carcinoma Ovarian CA Ovarian CA Ovarian CA Glioma Glioma

138 223 220 218 219 217 221 222

Renal CA NHL NHL, ALL NHL, hairy cell leukemia Hodgkin disease, ALCL AML Myeloma, pediatric solid tumors NHL FRa-positive ovarian, fallopian tube and peritoneal carcinoma Breast CA, melanoma, osteosarcoma Breast CA

177

COMPARTMENTAL RADIOIMMUNOCONJUGATES 8H9 8H9 3F8 Trastuzumab HMFG1 Mx35   Minretumomab (CC49) 81C6 81C6

B7-H3 B7-H3 GD2 HER2 MUC1 SLC34A2 TAG-72 Tenascin-C Tenascin-C

131

ANTIBODY TOXIN/DRUG CONJUGATES G250 Pinatuzumab vedotin Inotuzumab ozogamicin (CMC-544) BL22 Brentuximab vedotin (Adcetris) Gemtuzumab ozogamicin (Mylotarg) Lorvotuzumab mertansine Polatuzumab vedotin Mirvetuximab soravtansine (IMGN853)

CAIX CD22 CD22 CD22 CD30 CD33 CD56 CD79b FRa

Auristatin E Auristatin E Calicheamicin PE38 Auristatin E Calicheamicin Mertansine Auristatin E Maytansinoid DM4

Glembatumumab vedotin Ado-trastuzumab emtansine (T-DM1)

GPNMB HER2

Auristatin E Emtansine

371 372 373 245,246 252,374 249,375–377 378 379 380 381 382–384

ALCL, Anaplastic large cell lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CA, cancer; CML, chronic myeloid leukemia; CNS, central nervous system; CRC, colorectal cancer; DIPG, diffuse intrinsic pontine glioma; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma.

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 497

Multistep Targeting or Pretargeting202 To improve tumor uptake and reduce systemic toxicity, a multistep procedure that pretargets the antibody before the binding of the cytotoxic ligand to the tumor has been used successfully. In general, a tumorspecific antibody is conjugated to a ligand binder such as streptavidin (with high affinity for biotin) or a ligand-specific antibody (binding to metal chelators, such as diethylenetriaminepentaacetic acid [DTPA] or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA]).224,225 In the first step, these bispecific antibodies (BiAbs; 172–200 kDa) are allowed to localize to tumors in vivo, and any excess is cleared from the blood. The small radiolabeled ligand (DOTA-radiometal or its biotinylated form) is then injected intravenously. The ligand penetrates tissues rapidly and, by virtue of the high-affinity interaction, binds tightly to the antibody-conjugate at the tumor site. Unbound ligand is quickly excreted through the kidneys. Because of the short transit time of the toxic ligand (radionuclides or toxins), a substantial improvement in the therapeutic ratio is achievable without sacrificing the percent injected dose per gram in tumor.226 In preclinical studies, DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT) has shown dramatic improvements in therapeutic index (ratio of radiation dose to tumor versus critical normal organs) targeting GD2 in neuroblastoma,227 GPA33 in colon cancer,228 CD20 in NHL,229 and HER2 in breast cancer.230 Alternative pretargeting strategies using antibodies to carcinoembryonic antigen (CEA)231 in CRC,232 SCLC,233 and medullary thyroid carcinoma,231 NT-LU-10 in CRC,234 CC49 in gastrointestinal cancer,235 and anti-CD20 mAb in NHL236 have had variable successes. A three-step approach, which used biotinylated mAb followed by avidin/ streptavidin and then by biotinylated radiometal chelate, was also applied to glioma with encouraging results.237,238 However, immunogenicity of avidin/streptavidin limited repeat injections.

Immunotoxins and Antibody Drug Conjugates (see Table 30.4) Chemotherapeutic drugs have been conjugated to mAb for selective tumor delivery. Doxorubicin, melphalan, methotrexate, and vinca alkaloids conjugated to mAb have limited clinical success.239,240 Twochain toxins (e.g., ricin and diphtheria toxin [DT]) use 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 [PAP], gelonin) have a built-in receptor for cell attachment. When conjugated to mAb, they become immunotoxins. These toxins can be genetically modified for mAb conjugation and for improved safety profile.203 In recombinant toxins (e.g., PE40, PE38, or DT DAB486), the cell-binding domains were replaced by scFv.203,241 Toxins conjugated to mAb include241 ricin toxin A chain (RTA to anti-CD7, anti-CD22, and anti-CD25); DT (anti-IL-2R); and PE (anti-CD25, anti-CD22,242 anti-Lewis Y,243 and anti-HER2). A common toxicity is the vascular leak syndrome, characterized by marked fluid overload, dyspnea, and sensorimotor neuropathies.244 Anti-CD22-dsFv-PE (RFB4[dsFv]-PE38, BL22) demonstrated activity for NHL245 and cladribine-resistant hairy cell leukemia.246 Side effects include transient hypoalbuminemia and elevated aminotransferase levels; serious but reversible hemolytic-uremic syndrome has also been reported. With most immunotoxins, immunogenicity has been a major constraint; pegylation247 and humanization248 should reduce neutralizing antibodies. An expanding collection of toxic natural compounds, such as calicheamicins,249 auristatins,250 and maytansinoids251 are now candidates for ADC. Toxicities included reversible myelosuppression (especially neutropenia and thrombocytopenia), abnormal liver function test results, and bilirubinemia. The incidence of hepatic toxicity including venoocclusive disease for calicheamicin was approximately 2%.204 With auristatins, side effects include peripheral sensory neuropathy, nausea, fatigue, rash, neutropenia, diarrhea, and pyrexia.252,253 The most common adverse effects of maytansinoids (e.g., emtansine, mertansine) are peripheral neuropathy, nausea, fatigue, thrombocytopenia, and diarrhea.

Cellular Immunoconjugates Using Bispecific Antibodies205 Tumor-selective mAb can be rendered cytophilic by conjugation with mAbs specific for the trigger molecules on T lymphocytes, NK cells, and granulocytes. These molecules include CD3, CD28, Fc receptors (CD64, CD16), and FcαRI (CD89).254 BiAb, which redirects polyclonal T cells to tumors, seems to be most promising. Here, one binding site of the BiAb engages CD3 on T cells while the other binding site determines tumor specificity—for example, B-cell non-Hodgkin lymphoma (B-cell NHL) (CD19),74,255 breast cancer (HER2),256,257 B-NHL,258 malignant ascites (EpCAM),124 CRC (EpCAM),259 melanoma (MCSP),260 prostate (PSMA),261 EGFR,262 hepatocellular carcinoma (HCC),263 and metastatic solid tumors (CEA).264 Similar successes have been reported for the trigger molecule CD28 for ALL (CD19 and CD20),265,266 melanoma (MCSP),267 and Hodgkin disease (CD30),268 although severe CRS associated with anti-CD28 mAb TGN1412184 may discourage their final clinical translation. BiAb targeting FcγRIII (CD16) has also been explored for Hodgkin disease (CD30),269 CD19,270 and CD33,271 EGFR,272 and HER2273; FcγRI was exploited for CD30,274 EGFR,275,276 and HER2.277 BiAb has also been made selective for epitopes outside the Fc-binding domain of FcγR to bypass competition with serum IgG.

Immunocytokines278 Cell-mediated cytotoxicity has been highly effective against tumors in vitro and in animal models. Immunocytokines207 have shown remarkable success in activating and redirecting effectors to human tumors. Most of these studies have focused on NK, NKT or T cells207 and granulocytes.8 Antibody–IL-2 immunocytokine can eradicate metastatic murine neuroblastoma while inducing long-term antitumor immunity.207,279 Following initial successes with IL-2 immunocytokine,280 constructs containing other cytokines have been explored.207 These have included IL-12, TNF, and lymphotoxin. This emerging technology has been successfully tested in a number of antigen systems in the clinic,278 including GD2 (IL-2),280 nuclear antigen (IL-2LT and IL-12),281 CD20 (IL-2),282 EpCAM (IL-2),283 fibronectin (IL-2, IL-12, TNF),284–286 and Tn-C (IL-2).287 Although measurable tumor responses were uncommon in clinical trials, microscopic marrow tumor responses have been reported.280 Although immunocytokines have generally shown preferential targeting to tumor sites,278 the therapeutic index achieved by conventional targeting strategies is suboptimal (see the section Radioimmunotherapy), and the antigen specificity can be irrelevant to efficacy.288 Their combinations with vaccines,289 radiation,290 or BiAbs291 are potential future applications.

Immunoenzymes for Antibody-Directed Enzyme Prodrug Therapy Another novel approach (ADEPT) uses mAbs to deliver a covalently conjugated enzyme to the tumor, which can then activate a nontoxic prodrug.208 Despite preclinical successes, ADEPT has been difficult to translate into clinical benefit. Significant impediments to broaden the clinical implementation include immunogenicity of antibodyenzyme 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 cancer patients. When coated with polyethylene glycol (PEG), 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

498 Part I: Science and Clinical Oncology

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 CD19,292 HER2,293 EGFR,294 and GD2,295 When liposomes fuse with the tumor targets, their contents can be efficiently delivered intracellularly. Immunoliposomes have recently been used preclinically to target T cells in vivo after their adoptive transfer.296 While the potential exists, the clinical benefit of mAb-targeted nanoparticles including liposomes in cancer therapy remains to be proven.

IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES297 mAbs have been chimerized and humanized in order to reduce immunogenicity, enhance their binding to human FcR, and prolong their serum half-life. Chimeric mAbs are made by joining the antigencombining variable domains of a mouse mAb to human constant domains: mouse VL to human CL and mouse VH to human CH1-CH2-CH3.198 In humanized mAb, the antigen-binding loops, known as complementarity-determining regions (CDRs) from a mouse mAb are grafted into a human IgG.298 Human antibodies can also be derived from single-chain variable fragments (scFv) or Fab phage display libraries299; this is particularly useful for self-antigens.300 Alternatively, human mAb can be made from hIgG-transgenic mice.301 Because Fc is necessary for antitumor effect, chimerizing or humanizing mouse mAbs with the human IgG1 or IgG3 Fc regions can improve ADCC and CMC functions. Similarly, removing FcγRIIB inhibitory receptor recognition also can enhance antitumor activity.302 Point mutations in the Fc region have increased its affinity for activation receptors or decreased its affinity for the inhibitory receptor.303 Glycosylation of IgG at Asn297 stabilizes the tertiary structure of the CH2 domain, which is critical for effector functions, including ADCC and CMC.304 Glycosylation depends on the cell line that produces the mAbs, and increasing the bisected complex oligosaccharides in the Fc region,305 defucosylation.306 or defucosylation plus increasing mannose-5 residues307 can greatly improve ADCC properties of mAbs.306 CMC can also be improved by Fc region mutations to increase C1q binding308 or decrease by K322A mutation.309 The antigen-binding affinity, molecular architecture, and oligomerization states of mAbs can be reengineered to enhance tumor binding and uptake. For example, affinity can be increased by using phage display libraries,310 ribosome display,311 DNA shuffling,312 or yeast display.313 However, because the binding-site barrier can impede tumor penetration if the mAb has very high affinity,314 the optimal mAb may indeed be a medium-affinity IgG, especially for surface antigens expressed at high density. In addition, the size of the mAb is critical. ScFvs are small (25 kDa) and rapidly cleared by the kidney. On the other hand, mAb forms with molecular weights in the range of 100 to 200 kDa should be ideal for tumor targeting.315 In addition to increasing avidity, oligomerization can increase antitumor activity through a multitude of mechanisms including CMC and ADCC, induction of apoptosis, growth arrest, and synergy with chemotherapy or immunotoxins.30,316 The construction of dual or multispecific mAbs will further expand options.205,206 Although scFv is a powerful building block for polymeric forms or novel fusion proteins,317 single-domain antibodies may further expand the possibilities of antibody-based cancer therapies.318

ALTERNATIVE TARGETS FOR ANTICANCER ANTIBODIES The clinical importance of T cells in cancer therapy is indisputable. Tumor-infiltrating T cells and chimeric antigen receptor (CAR)–modified

T cells are promising cell-based platforms covered in detail in a separate chapter of this textbook. Any tumor-selective mAbs discussed in this chapter can potentially provide the tumor-targeting scFvs for constructing CAR T cells. Similarly, these mAbs or their reengineered forms can provide the building blocks for BiAbs or multispecific antibodies.319 Most tumor-selective antibodies are directed at surface antigens.320 A new class of tumor-specific antigens is the T-cell epitopes, normally recognized by TCRs. These epitopes are tumor-derived peptides presented on specific human leukocyte antigen (HLA) alleles. Highly specific mouse and human antibodies (called T-cell receptor–like or T-cell receptor mimic antibodies) have been made against these epitopes and exploited in cancer therapeutic strategies.321,322 Whereas most of the mAb targeting effort has been focused on individual tumor cells, alternative strategies directed at tumor neovasculature,323 tumor stroma,324 fibronectin,325 tumor stem cells,131,326 tumorinfiltrating macrophages,327 or tumor infiltrating T cells328 (also see the section Immune Checkpoint Inhibitors) are promising approaches. Furthermore, mAbs can be made to inhibit homing of angiogenic progenitors (e.g., anti-VLA4 [natalizumab],329 and anti-VEGFR1330), or to block VEGFR2/KDR (e.g., IMC-1C11, chimeric anti-KDR).331 Targeting tumor vasculature may have significant advantages over direct tumor targeting,332 in that endothelial cells, unlike tumor cells, are less likely to acquire resistance. Another angiogenesis target is αVβ3 integrin, which initiates endothelial proliferation, migration, and matrix remodeling.333 In a phase I trial, chimeric IgG1 (MEDI-522) specific for αVβ3334 was well tolerated, and tumor perfusion was possibly modified. T and NK lymphocytes represent the most important cell effectors in antitumor response. Therefore direct stimulation of these cells using mAbs can enhance their cytotoxic potential against tumors (see Fig. 30.2 for antibody targets). Both cell types are known to express costimulatory receptors that belong to the tumor necrosis factor receptor (TNFR) family. Members of this family with T-cell and NK stimulatory function include CD137 (4-1BB), CD27, CD134 (OX40), and GITR (CD357).335 BMS-663513, a 4-1BB agonist, was tested in a first-in-human phase I clinical trial.336 Patients with melanoma, RCC, or ovarian or prostate cancer were included in this trial. Three PRs in melanoma patients were observed. Further clinical evaluation of this antibody was halted because of liver toxicity at higher doses.337 BMS-663513 and another anti 4-1BB antibody, PF-05082566, are now in early-phase clinical trials (e.g., NCT02253992, NCT02444793). Many clinical studies are currently recruiting patients with various cancers to test other agonistic antibodies—for example, anti-CD27, anti-OX40 or anti–glucocorticoid-induced tumor necrosis factor receptor–related protein (anti-GITR), and anti–inducible T-cell costimulator (anti-ICOS) (see Fig. 30.2). Building on the success of ICI therapy, several checkpoint proteins such as lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain 3 (TIM3), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) have emerged as therapeutic targets for mAbs and entered clinical development (see Fig. 30.2).338,339 Blocking KIRs on NK cells can also enhance their antitumor effect.340 Anti-KIR mAb IPH2101 was shown to be safe in phase I clinical trials in patients with AML and multiple myeloma.341,342 Human anti-KIR mAb lirilumab was tested as a single agent and now is being tested in combination with ICIs in phase II clinical trials (NCT01687387, NCT01714739).343 Monalizumab (IPH2201), a humanized antibody targeting another inhibitory receptor on NK cells—cNKG2A-CD94—also entered the clinic for various cancers, including CLL (NCT02557516) and solid tumors (NCT02643550). The complete reference list is available online at www.ExpertConsult.com.

Therapeutic Antibodies and Immunologic Conjugates  •  CHAPTER 30 499

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