T lymphocytes recognize peptide antigens in the context of MHC molecules. These peptides are derived from two distinct pathways.⁵⁷ Peptides representing proteins sampled from the extracellular world are generally presented in the context of class II MHC proteins, whereas peptides resulting from intracellular synthesis of proteins are presented in the peptide groove of the class I MHC proteins (Fig. 70.2). The binding cleft of MHC molecules has a β-pleated sheet floor and α-helical sides. An immunogenic peptide must be capable of forming noncovalent attachments to key residues along the cleft and interacting with the T cell antigen receptor with other residues. The MHC contact residues of the peptide tend to be near the amino and carboxy terminal ends of the peptide. The cleft of the class I MHC molecule has closed ends and accommodates only a peptide of proper length, 9 to 11 amino acids. The cleft of the class II MHC molecule, on the other hand, is open-ended and can bind peptides of more diverse lengths, 10 to 30 amino acids, with most being 12 to 19.⁵⁸

Peptides located in class II MHC molecules are derived from proteins that have been consumed by APCs⁵⁹ (Fig. 70.2A). The proteins are taken up by phagocytosis, or receptor-mediated endocytosis in clathrin-coated pits, or engulfed by pinocytosis. Once internalized, the antigens are located in membrane-bound vesicles called endosomes. The endosomes then become continuous with lysosomes. The enzymology of the endolysosome has been described in some detail.^59,60 There, in an acidic environment, disulfide bonds in proteins are first reduced by the enzyme GILT (gamma interferon-inducible lysosomal thiol reductase), and the resulting products are subsequently cleaved to peptides by proteases, predominantly cathepsins. The endosome fuses with an exocytic vesicle budding from the Golgi apparatus that contains newly made class II MHC molecules associated with invariant chain, which has been shown to play a critical role in the assembly, intracellular transport, and function of MHC class II molecules.⁶¹ In addition, a chaperone, HLA-DM, plays a critical role in the loading of peptides onto MHC class II molecules. HLA-DM is a peptide exchange factor that binds with empty and peptide-loaded class II molecules in endosomal and lysosomal compartments.⁶¹ In the fused vesicle, peptides are loaded into the class II MHC molecules. Fusion of the endosome with the plasma membrane ultimately displays the class II MHC molecule-peptide complexes on the cell surface.

Peptides are prepared for presentation on class I molecules in a different fashion (Fig. 70.2B). These peptides are derived from intracellular protein synthesis.^62,63 After protein synthesis, proteins introduced into the cytoplasm become the target of the proteasome, a cytoplasmic organelle whose major function is the degradation of proteins tagged for turnover by the addition of ubiquitin.^64,65 During conditions of interferon-γ production such as infection, several proteasome subunits (LMP2, LMP7, and MECL-1) become upregulated and are incorporated into newly assembled proteasomes. Proteasomes that include these IFN-γ-inducible subunits are referred to as immunoproteasomes, whereas those that lack these subunits are called constitutive proteasomes. The IFN-γ-inducible proteasome subunits favor the generation of peptides that have increased affinity for MHC class I molecules.^{66, 67} and ⁶⁸ In addition to the proteasome, several cytoplasmic peptidases (e.g., TPPII, LAP, TOP) have been implicated in the generation of antigenic peptides, although they can cleave some epitopes as well.^65,69 These peptides are then transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) proteins.⁶² Within the ER, peptides may be further trimmed by ER-resident aminopeptidases (i.e., ERAP1 and ERAP2).^65,69,70 Assembly of class I MHC heavy chain molecules with β₂-microglobulin requires the presence of peptides. Within the ER, empty MHC class I molecules are associated with a variety of chaperones, including calnexin, calreticulin, ERp57, and tapasin. Tapasin is a transmembrane protein that tethers empty class I molecules in the ER to TAP.⁷¹ Emerging evidence suggests that tapasin retains unstable MHC class I molecules within peptide-loading compartments until they bind with high-affinity peptides. The assembled MHC class I-peptide complex transits the Golgi apparatus, proceeds in a vesicle to the cell surface, and is displayed on the cell surface after fusion of the vesicle membrane with the plasma membrane.

There has been a lot of interest in the mechanisms whereby tumor cells initiate CD8⁺ T cell responses. Few tumors are derived from professional APCs and, therefore, do not effectively prime naive CD8⁺ T cells. It has now been established that tumor cells can be processed and presented by host APCs, particularly DCs, in a process that is referred to as cross-presentation (Fig. 70.2).^72,73 Tumor antigens are then processed inside the APC, and peptides derived from these antigens are displayed on MHC class I molecules for recognition by CD8⁺ T cells. These APCs also express MHC class II molecules and can prime naive CD4⁺ T cells, which may be important for the generation of effective CD8⁺ memory responses. Once tumor antigen-specific CTLs are generated, they can kill tumor cells without the requirement for costimulation. While the precise mechanisms of cross-presentation remain poorly understood,⁷⁴ the concept of cross-presentation has important applications in the development of tumor vaccines.^72,75

The goal of antigen processing and presentation is the activation of appropriate T lymphocytes to proliferate, produce cytokines, and promote an immunologic reaction or become cytotoxic cells. Although the interaction of the T cell antigen receptor with antigen-MHC provides specificity of response and initiates the crucial events of activation, the interactions are few and have low affinity.⁷⁶ The interaction between T lymphocytes and APCs or target cells is initially stabilized by a number of nonspecific
receptor-counterreceptor interactions, leading to development of an immunologic synapse with its central supramolecular activation cluster.^77,78,79 Chief among these interactions is the coupling of CD2 on the T lymphocyte with lymphocyte function antigen-3 on the APC. Also involved is the interaction of the lymphocyte function antigen-1 molecule with intercellular adhesion molecule-1 and intercellular adhesion molecule-2. Once the cells have been apposed, the specific interaction of the T cell antigen receptor and the antigen-MHC can occur. It now appears that the T cell proceeds toward activation only if certain threshold numbers of TCR-MHC/antigen interactions occur.⁸⁰

The signal transduction pathways that result in T cell activation have been extensively reviewed,^{81, 82} and ^83,84 and we will focus here on the most salient features (Fig. 70.3). Ligation of the TCR with an agonist peptide/MHC complex results in phosphorylation of the cytoplasmic portions of the CD3 and ζ components of the TCR. The cytoplasmic domains of CD3 and ζ contain several conserved peptide sequences called immunoreceptor tyrosine-based activation motifs (ITAMs) that are targets for intracellular protein tyrosine kinases that catalyze the phosphorylation of tyrosine residues in various protein substrates. The tyrosine kinase lck interacts with the cytoplasmic domains of CD4 and CD8 and the tyrosine kinase fyn interacts with the TCR-CD3 complex. Binding of the TCR with peptide/MHC complexes results in receptor clustering, bringing CD4/CD8 and lck in close proximity of the ITAMs within the CD3 and ζ-chains. Lck and fyn subsequently phosphorylate tyrosine residues within the ITAMs, which become docking sites for the ζ-associated protein, ZAP-70, a member of the syk family of protein tyrosine kinases. The bound ZAP-70 then becomes a substrate for lck, and phosphorylation of ZAP-70 results in its activation. Activated ZAP-70 phosphorylates several scaffolding proteins, including LAT (linker of activated T cells) and SLP-76, which, when phosphorylated, serve as docking sites for other proteins that, in turn, activate multiple signaling pathways. One of these signaling pathways involves changes in inositol lipid metabolism. Phospholipase Cγ1 (PLCγ1) becomes tyrosine
phosphorylated and activated as it associates with LAT. Activation of PLCγ1 leads to the hydrolysis of a minor membrane lipid, phosphatidylinositol biphosphate (PIP₂), to yield inositol triphosphate (IP₃) and diacylglycerol (DAG). Each of these products activates downstream events. IP₃ induces a rapid increase of free Ca²⁺ by release from membrane-sequestered Ca²⁺ stores, whereas DAG and Ca²⁺ activate protein kinase C (PKC) θ. T cell activation also results in the activation of the ras and rac signaling pathways. Adapter proteins that are activated by phosphorylated LAT and SLP-76 result in the activation of the guanine nucleotide exchange factors SOS (son of sevenless) and vav, which activate the ras and rac signaling pathways, respectively.

These signaling events ultimately result in the activation of a number of transcription factors, including NFAT, NF-κ B, and AP-1. Cytosolic Ca²⁺ binds with the Ca²⁺-dependent protein calmodulin, and Ca²⁺-calmodulin complexes subsequently activate several enzymes, including the serine/threonine phosphatase calcineurin. Calcineurin then dephosphorylates NFAT, which uncovers a nuclear localization signal that permits NFAT to translocate to the nucleus. Activation of NF-κB is dependent, at least in part, on activated PKCθ. NF-κB is normally found in the cytoplasm in association with a protein called inhibitor of κB (IκB). TCR signals result in phosphorylation of IκB, which is then targeted for degradation by the proteasome. Release of IκB uncovers a nuclear translocation signal in NF-κB that permits its translocation to the nucleus. AP-1 is a transcription factor composed of the proteins Fos and Jun, which are activated by the ras and rac signaling pathways, respectively.

The net result of this extremely complex activation system is the expression of new proteins, the acquisition of functional capacity, or the ability to proliferate. T lymphocyte activation is best understood as a culmination of events leading to IL-2 production.⁸⁵ The constraints on production of this cytokine are more rigorous than those relevant for production of other gene products (such as IL-2 receptor α-chain and transcription factors). The promoter of the IL-2 gene is made up of a number of binding sites for transcription factors, including two NFAT sites, an NF-κB site, and an AP-1 site.⁸⁶ The combination of production of IL-2 receptor α-chain and IL-2 provides an adequate stimulus for the T lymphocyte to successfully proliferate, giving rise to the antigen-specific clonal expansion of lymphocytes characteristic of immunologic responses. However, there are extraordinary controls against inappropriate activation of T lymphocytes.⁸⁷ In addition to a first signal delivered via the T cell antigen receptor complex, full activation of T cells also requires a second signal.⁸⁸ The best characterized origin of these second signals is the interaction of CD28 on the T lymphocyte surface with its cognate ligands CD80 (B7-1) and CD86 (B7-2) on the APC, the most potent of which is the DC. Failure to receive a second signal can lead the T lymphocyte to undergo anergy or apoptosis. Activation of T cells is also regulated by a variety of negative signals, including inhibitory receptors of the CD28 family such as CTLA-4 (cytotoxic T lymphocyte-associated protein 4), which interacts with CD80 and CD86, and PD-1 (programmed death-1), which interacts with PD-L1 and PD-L2.⁸⁸

Unaltered antibodies have been used since the earliest trials of monoclonal antibody therapy in humans.^{140, 141} and ¹⁴² In early trials, success was limited by the absence of suitable tumor cell surface targets, antigenicity of first-generation (murine) mAbs in humans, modulation of the target structure from the tumor cell surface, and poor recruitment of immune effector mechanisms.^143,144 However, enthusiasm for this approach was rekindled by the enormous success of genetically engineered, chimeric, or fully humanized versions of mAbs, most notably rituximab for lymphoid malignancies and trastuzumab in solid tumors.¹⁴⁴

Rituximab is a chimeric monoclonal antibody with humanized framework and Fc regions. It is directed against the CD20, pan B cell antigen. CD20 is present on pre-B cells and mature B cells, but not on precursor cells or terminally differentiated plasma cells. The function of CD20 remains poorly understood,¹⁴⁵ although it has been implicated in B cell activation, regulation of B cell growth, and regulation of transmembrane calcium flux. The antibody fixes human complement and elicits antibody-dependent cellular cytotoxicity (ADCC). Rituximab was approved for use as monotherapy in patients with low-grade or follicular CD20⁺ non-Hodgkin lymphoma (NHL) in 1997. In the pivotal trial involving 166 patients, reported by McLaughlin et al.,¹⁴⁶ patients had relapsed or chemotherapy-resistant disease. Patients received four weekly infusions of rituximab at 375 mg/m². Rituximab produced a tumor response in one-half of the patients, with a median duration of response of 11.8 months, comparable to the patients’ response duration on the last chemotherapy treatment. Human antichimeric antibody responses were uncommon. The most common adverse experience associated with rituximab was a constellation of acute infusion-related events, including chills, fever, headache, rhinitis, pruritus, vasodilation, asthenia, and angioedema. This syndrome can progress to hypotension, urticaria, bronchospasm, and, rarely, death. The risk is particularly great in patients with high circulating white cell counts.¹⁴⁷ Other toxicities were, in general, mild and infrequent.¹⁴⁸ Neutropenia and thrombocytopenia were unusual. Circulating B cells were depleted and remained low until recovery at a median of 12 months. Immunoglobulin levels, however, remained normal. No increased incidence of infections was seen. Rituximab was evaluated again in the relapsed, refractory, low-grade, or follicular NHL patient population, using eight weekly infusions rather than four.¹⁴⁹ This extended regimen produced a response rate of 57% and a time to progression (TTP) in responding patients of more than 19.4 months. Adverse event reporting was commensurate with the longer treatment period.

Davis et al.¹⁵⁰ reported a response rate of 43% and a TTP of 8.1 months in the patients with a significant poor prognostic factor, importantly in patients with low-grade or follicular NHL who had bulky disease, using the standard 4-infusion regimen. These investigators also showed an overall response rate (ORR) of 40% in patients who had progressed after an initial response to rituximab, with an estimated median TTP for responding patients of 17.8 months.¹⁵¹

Rituximab has been used in the first-line treatment of patients with indolent lymphoma, both as monotherapy and in combination with chemotherapy. Hainsworth¹⁵⁰ reported the results of rituximab as monotherapy in 39 previously untreated patients. Patients received rituximab × 4 at the usual dose and schedule, with an ORR of 54%. Patients who had responded or who had stable disease were treated with an additional four weekly treatments of rituximab at 6 months intervals to a maximum of 4 treatment cycles. The ORR rose to 72% after the second course of treatment. Progression-free survival (PFS) at 1 year was 77%. The addition of rituximab to cyclophosphamide, hydroxyldaunomycin, oncovin (vincristine), and prednisone (CHOP) chemotherapy produced impressive results. Patients were treated with six infusions of rituximab, one associated with each of six cycles of CHOP. In 40 patients with newly diagnosed (n = 31) or relapsed/refractory (n = 9) low-grade or follicular NHL, rituximab plus CHOP produced an ORR of 95% and complete response (CR) rate of 55%. With a median follow-up of 29 months, median duration of response and median TTP had not been reached. Eight of 18 patients tested for the BCL-2 [t(14;18)] translocation by PCR testing were positive at initiation of therapy. Seven of these 8 patients, after therapy, became negative for the translocation. The authors concluded that the addition of rituximab to CHOP produced benefits in efficacy parameters without significant additional toxicity. Elimination of PCR positivity for the t(14;18) translocation had not been previously reported with CHOP alone.

Rituximab also has been used with success in patients with more aggressive lymphomas. Vose et al.¹⁵¹ reported the results of rituximab plus CHOP chemotherapy (again using 6 infusions of rituximab in association with 6 cycles of CHOP) in 33 previously untreated patients with advanced aggressive B cell NHL. The combination produced an ORR of 94% and a CR rate of 61%. With a median observation time of 26 months, 29 of 31 patients achieving a remission were in continuing remission at the time of the report. Thirteen patients were BCL-2 positive at study entry. Eleven of these 13 patients became BCL-2 negative after treatment, and 10 of the 11 remained BCL-2 negative. The authors concluded that the results were achieved without significant added toxicities above those expected with CHOP. The Groupe d’Etude des Lymphomes de l’Adulte undertook a study to compare the utility of CHOP with that of rituximab plus CHOP in elderly patients with diffuse B cell lymphoma.¹⁵² Patients between the ages of 60 and 80 years with untreated, diffuse large B cell lymphoma were eligible for the study. Patients were randomly assigned to receive 8 cycles of CHOP chemotherapy (197 patients) or to receive 8 cycles of CHOP, each given after an infusion of rituximab (202 patients). Rituximab plus CHOP produced a superior rate of remission, 76% versus 63% (P = 0.005). With
a median follow-up of 2 years, event-free and overall survivals (OSs) were significantly higher in the rituximab plus CHOP group (P < 0.001 and P = 0.007, respectively). These results were achieved without a significant incremental increase in toxicity. The addition of rituximab to CHOP reduced the risk of treatment failure (risk ratio, 0.54; 95% confidence interval [CI]: 0.44, 0.77) and the risk of death (risk ratio, 0.64; CI: 0.45, 0.89). The addition of immunotherapy to standard chemotherapy had accomplished what 25 years of chemotherapy manipulation had failed to do (i.e., improve on the results of CHOP chemotherapy).^153,154 Maintenance rituximab has also been found to prolong event-free survival (EFS) and response duration in follicular lymphoma.¹⁵⁵ In subsequent ongoing randomized phase III studies,^156,157 rituximab maintenance regimen provided significant PFS and OS at 3-year¹⁵⁶ and 4-year¹⁵⁷ follow-up assessments in both previously treated¹⁵⁶ and untreated¹⁵⁷ patients with follicular NHL, compared with no further treatment, though no difference in OS has been reported by other investigators.¹⁵⁸

The mechanism by which rituximab produces these impressive results is less clear. A number of possible mechanisms have been considered: initiation of complement-mediated cell lysis, induction of ADCC, and signaling via CD20 leading to programmed cell death and/or sensitization to cytotoxic drugs. Pretreatment lymphoma cells from 29 patients were examined by flow cytometry for expression of complement inhibitors CD46, CD55, and CD59.¹⁵⁹ Expression of these cell surface inhibitors of complement activation was not predictive of outcome to rituximab therapy. Considerable evidence suggests that induction of ADCC plays an important role in rituximab’s antilymphoma effects. A rituximab-like antibody for which an IgG4γ framework was substituted for the IgG1 framework of rituximab was incapable of producing B cell depletion in primates.¹⁶⁰ Rituximab was relatively ineffective in eliminating Raji B cell implants in FcRγ-/-/nu/nu knockout mice compared to nu/nu mice.¹⁶¹ These mice lack the activating receptor for Fc portions of antibodies, a critical component of the antibody-dependent cell-mediated cytotoxicity mechanism. In patients, response to rituximab has been shown to be associated with homozygosity for the high-affinity allotype of the FcγRIIIa receptor.¹⁶² Evidence also exists that rituximab signaling or interference with normal signaling via CD20 may directly induce apoptosis or sensitize cells to the deleterious effects of chemotherapeutic agents.¹⁶³ A direct, growth inhibitory effect of rituximab, with accompanying apoptosis, on cell lines cultured in the absence of complement was demonstrated.¹⁶⁴ Anti-CD20-associated apoptosis has been associated with upregulation of the proapoptotic protein, Bax¹⁶⁵ and downregulation of antiapoptotic protein BCL-2 through inactivation of STAT3.¹⁶⁶ Downregulation of STAT3 appears to be a result of downregulation of an IL-10 autocrine pathway.¹⁶⁷ These changes and/or others may be responsible for increased sensitivity to chemotherapeutic agents.¹⁶⁸

In the wake of the success of rituximab, a number of other antilymphoma mAbs have entered the clinic.¹⁶⁹ Alemtuzumab is a humanized IgG1κ monoclonal antibody directed against the CD52 cell surface antigen.¹⁴⁴ CD52 is expressed on normal and malignant lymphocytes of B- and T cell lineage, as well as NK cells, monocytes, and macrophages. Alemtuzumab is indicated for the treatment of B cell chronic lymphocytic leukemia (CLL) in patients who have been treated with alkylating agents and who have failed fludarabine therapy. The pivotal clinical trial was carried out in 93 patients with fludarabine-refractory CLL .¹⁷⁰ Alemtuzumab produced a response rate of 33%. Virtually all of the responders were partial responders; the CR rate was 2%. Median duration of response was 7 months. Median TTP was 4.7 months for the group as a whole; 9.5 months for responders. The most common adverse events were infusion related—most were grade 1 or 2 in severity, including rigors in 90% of patients (grade 3 in 14%), fever in 85% of patients (grade 3 or 4 in 20%), nausea in 53% of patients, and vomiting in 38% of patients. Infusion-associated side effects declined with subsequent infusions. During the study, 28% of patients experienced dyspnea, 17% experienced hypotension, and 3% experienced hypoxia. Overall, 55% of patients developed an infection during the study. Approximately one-half of these infections were considered serious (grade 3 or 4). Septicemia occurred in 15% of patients, and two deaths resulted. Opportunistic infections occurred in 12% of patients. Ten percent of patients died during or within 30 days of treatment—one-third of these were attributed to progressive disease. Twenty-four percent of patients discontinued treatment because of a drug-related side effect. Most patients who discontinued had not responded to therapy. Serious infusion-related events associated with alemtuzumab appear to result from ligation of CD16 on NK cells resulting in what has been termed cytokine storm—release of IL-6, TNF-α, and interferonγ.¹⁷¹ Prolonged immunosuppression after use of alemtuzumab can result in opportunistic infections.¹⁷² Treatment schemas now include the routine use of prophylaxis with both antibiotics and antivirals.

To improve on the immunogenicity and efficacy of rituximab, the last few years have seen the development of new generations of anti-CD20 monoclonal antibodies (mAbs) with enhanced antitumor activity resulting from increased complement-dependent cytotoxicity (CDC) and/or ADCC and increased Fc binding affinity for the low-affinity variants of the FcγRIIIa receptor (CD16) on immune effector cells. These second-generation mAbs, such as ofatumumab, veltuzumab, and ocrelizumab, are in clinical development. They are humanized or fully human to reduce immunogenicity, but with an unmodified Fc region. Ofatumumab is a fully human anti-CD20 IgG1 mAb in clinical development for hematologic malignancies and autoimmune diseases. Ofatumumab specifically recognizes an epitope encompassing both the small and large extracellular loops of the CD20 molecule, and is more effective than rituximab at CDC induction and killing target cells. Veltuzumab (IMMU-106, hA20) is a humanized anti-CD20 mAb with complementarity-determining regions similar to rituximab. This antibody has enhanced binding avidities and a stronger effect on CDC compared to rituximab. Ocrelizumab is a humanized mAb with the potential for enhanced efficacy in lymphoid malignancies compared to rituximab because of increased binding affinity for the low-affinity variants of the FcγRIIIa receptor. Third-generation mAbs are also in clinical development. They are also humanized mAbs, but in addition they have an engineered Fc to increase their binding affinity for the FcγRIIIa receptor. Third-generation mAbs also in clinical development include AME-133v, PRO131921, and GA-101 (Table 70.2), with enhanced affinity for the FcγRIIIa receptor and an enhanced ADCC activity compared to rituximab.¹⁷³

Two other mAbs with potential utility in the treatment of lymphoma are in early clinical development.^169,174 Epratuzumab is a humanized IgG1 monoclonal antibody directed against the CD22 antigen. CD22 is a pan-B cell antigen with distribution similar to that of CD20. Epratuzumab has a favorable safety profile in early trials. Approximately 50% of follicular lymphoma patients and 25% of diffuse large-cell lymphoma patients responded in a small phase II trial. Some of the responses have been long-lived. A recent phase II trial testing the safety and efficacy of combining epratuzumab with R-CHOP (ER-CHOP) in untreated DLBCL showed that the addition to standard R-CHOP, E 360 mg/m² intravenously, administered for 6 cycles in 107 patients, showed similar toxicity to standard R-CHOP. ORR in the 81 eligible patients was 96% (74% CR/CR unconfirmed [Cru]) by computed tomography scan and 88% by positron emission tomography. By intention to treat analysis, at a median follow-up of 43 months, the EFS and OS at 3 years in all 107 patients were 70% and 80%, respectively. Comparison with a cohort of 215 patients who were treated with R-CHOP showed improved EFS in the ER-CHOP
patients. ER-CHOP was well tolerated and results appear promising as a combination therapy.¹⁷⁵

Apolizumab is a humanized IgG1 monoclonal antibody that binds to a variant of the HLA-DR β-chain. The antibody induces complement-mediated lysis, ADCC, and tyrosine phosphorylation signaling events in cell lines in vitro. The antibody binds to approximately 70% of lymphoma specimens. Administration of the antibody to patients results in typical infusion-related side effects. Four of 8 patients with follicular lymphoma responded to apolizumab. A phase I/II dose-escalation study of thrice-weekly apolizumab (1.5, 3.0, 5.0 mg/kg/dose) for 4 weeks in relapsed CLL resulted in significant toxicity and lack of efficacy; thus, further clinical trials of apolizumab were discontinued, as were other trials in lymphoma and solid tumors.¹⁷⁶ Milatuzumab (hLL1, IMMU-115; Immunomedics) is a fully humanized mAb specific for CD74, a cell surface-expressed epitope of the HLA class II-associated invariant chain. CD74 plays an important role as an accessory signaling molecule and survival receptor in the maturation and proliferation of B cells by activating the PI3K/Akt and the NF-κB pathways.¹⁷⁷ Milatuzumab demonstrated antiproliferative activity in transformed B cell lines, improved survival in preclinical models, and is presently being evaluated for the treatment of several hematologic malignancies.

Inroads are being made in other hematologic cancers. In multiple myeloma, it was recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Preclinical studies showed that elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model and that the CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated ADCC, both for OPM2 myeloma cells using NK cells or peripheral blood mononuclear cells from healthy donors and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo antitumor activity. Elotuzumab is currently in a phase I clinical trial in relapsed/refractory myeloma.¹⁷⁸ In AML, one strategy for the development of mAbs targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC (leukemia stem cell) compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, mAbs targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.¹⁷⁹

Much time and energy were expended searching for tumor antigens, particularly after the development of mAb techniques.¹⁷ These brute-force immunization, hybridization, and screening procedures yielded little. They defined only a single truly tumor-specific antigen. That was the Id of clonally distributed antibody expressed on the surface of certain B cell lymphomas. Because it represents a unique protein structure within the combining site of the antibody, the Id can serve as an antigen for antibody production. This fact was demonstrated by Sirisinha and Eisen¹⁸⁰ in the early 1970s. Furthermore, they demonstrated that an immunologic response to Id could lead to tumor protection.¹⁸¹

Levy and Miller¹⁸² have explored the utility of anti-Id strategies in indolent B cell lymphoma over many years. Initially, these investigators raised custom-made anti-Id mAbs¹⁸³ for passive administration. A first patient with far advanced,
chemotherapy-refractory disease received anti-Id mAbs. Gradual reductions in serum Id and tumor volume were noted. The patient then entered a long-term complete remission.¹⁸⁴ In an initial series of patients treated with anti-Id therapy, 11 patients were reported.¹⁸⁵ In this group, a second near-complete remission, four partial remissions, and five insignificant responses were seen. There was little toxicity associated with administration of the anti-Id antibodies. The most common side effects were chills and fever. Transient shortness of breath, headache, nausea, emesis, diarrhea, and myalgias were occasionally observed. Unusual toxicities included transient leukopenia or thrombocytopenia and transient elevations of hepatic enzymes. Several interesting problems of anti-Id therapy were noted in this early series: the interfering effect of circulating Id, the development of human antimouse antibody, and the emergence of Id-negative tumor cell variants.^{186, 187, 188} and ¹⁸⁹ An attempt to reduce the incidence of emergence of Id-negative lymphoma variants with a short course of chlorambucil was unsuccessful.¹⁹⁰ The cumulative experience suggests that anti-Id therapy can result in a 15% CR rate and a 50% partial response rate. The mechanism of tumor response in these trials remains unclear. However, response in these trials correlated with anti-Id-induced signal transduction events in the lymphoma cells, suggesting that activation of apoptotic pathways may lead to lymphoma cell death after interaction of the anti-Id antibody with the lymphoma surface-bound immunoglobulin receptor.¹⁹¹ This group has turned to active immunization strategies in indolent lymphoma (see section “Immunization Strategies”).

The use of immunoglobulin-radionuclide conjugates in cancer treatment represents appropriation of a classic guided-missile strategy. In theory, the antibody homes to its antigenic target and delivers a cytotoxic assault on the cell to which it attaches. Radionuclides offer certain advantages over other cytotoxic agents. They do not have to be internalized. Radioactive particles can deliver their effects over distances of 1 to 5 mm, thus limiting collateral damage to normal tissues while still potentially providing antitumor effects against antigen-negative variants in the vicinity in what has been termed a cross-fire effect. The principles of radiation physics underlying radioimmunotherapy (RIT) have been reviewed by Press and Rasey.¹⁹² Radiolabeled antibodies deliver continuous, exponentially decreasing, low-dose-rate radiation. Traditional external beam radiotherapy delivers intermittent, fractionated radiation at higher dose rates. The most commonly used isotopes for RIT have been iodine 131 (I-131) and yttrium 90 (Y-90). These radionuclides kill cells primarily through emission of β particles, resulting in DNA strand breaks. The β particles of Y-90 are more energetic than those of I-131. They affect cells in a radius of approximately 5 mm compared to approximately 1 mm for those of I-131. I-131 also emits γ rays. This allows direct imaging of the distribution of the radioconjugate but raises issues regarding shielding and health care worker and family member safety.

Several recent reviews attest to the evolution of this field.^193,194 A number of theoretical and experimentally generated concerns with RIT appear to have been overcome in the successful clinical studies described below. There had been concern that effective delivery of radioimmunoconjugates would be impeded by heterogeneous tumor vasculature, slow diffusion of these large molecules in interstitial spaces, heterogeneous biodistribution in tumor nodules, and high intratumoral pressures.

The two products in clinical use presently, Y-90 ibritumomab tiuxetan (Zevalin) and I-131 tositumomab (Bexxar), are both directed against the anti-CD20 antigen of B lymphocytes, the same structure targeted by rituximab. Both products are based on murine mAbs. Both are administered after infusion of unconjugated anti-CD20 antibodies—rituximab in the case of Zevalin and tositumomab in the case of Bexxar. Both have used nuclear medicine imaging as a preparatory step to administration, but it is no longer required for Y-90 ibritumomab. Simple dosimetry is accomplished for I-131 tositumomab by capturing whole-body gamma counts after infusion of a 5 mCi “dosimetric dose” of the agent. Imaging was carried out in Y-90 ibritumomab tiuxetan-treated patients to assure normal biodistribution. Whole-body dosimetry is carried out in I-131 tositumomab-treated patients to allow calculation of a patient-specific activity (mCi) to deliver a desired total-body dose of radiation (cGy). Both have been studied most extensively in indolent lymphoma and have been approved for treatment of patients with relapsed or refractory follicular, including rituximab refractory, or transformed B (CD20⁺) NHL.

The approval for ibritumomab tiuxetan rested primarily on two clinical studies. The first was a randomized controlled comparison of the effectiveness of Y-90 ibritumomab tiuxetan to that of rituximab in patients with relapsed or refractory, follicular, or transformed B cell NHL.¹⁹⁵ The study involved 143 patients; 73 randomized to Y-90 ibritumomab tiuxetan (single administration), and 70 randomized to rituximab (weekly × 4). The median number of prior therapies was 2. Approximately one-half of the patients failed to respond to or had a TTP of less than 6 months to their last chemotherapy regimen. Y-90 ibritumomab tiuxetan produced a statistically superior response rate (using the response definitions of the International Workshop), 80% versus 56% (P = 0.002). The number of durable responders at 6 months favored Y-90 ibritumomab tiuxetan-treated patients, but the significance of the observation was lost at 9 months and 12 months. Median TTP (estimated by Kaplan-Meier methods) was 11.2 months for patients treated with Y-90 ibritumomab tiuxetan and 10.1 months for patients treated with rituximab (P = 0.173). Grade 3 and 4 nonhematologic adverse events were unusual in both groups. Y-90 ibritumomab tiuxetan produced grade 3 or 4 neutropenia in 57% of patients, grade 3 or 4 thrombocytopenia in 60% of patients, and grade 3 or 4 anemia in 2% of patients. One patient in the Y-90 ibritumomab tiuxetan-treated group developed myelodysplasia. One patient in the rituximab-treated group developed pancreatic carcinoma. The second trial was a phase II experience in 57 patients who had failed to respond to rituximab or had a TTP of ≤6 months.¹⁹⁶ These patients had a median of 4 prior therapies, and 74% had bulky tumors (greatest diameter ≥ 5 cm). In this patient population, Y-90 ibritumomab tiuxetan produced a response rate of 74% and CR rate of 15%. The median TTP was estimated at 6.8 months. Grade 4 neutropenia occurred in 35% of patients, grade 4 thrombocytopenia in 9% of patients, and grade 4 anemia in 4% of patients.

The pivotal study for I-131 tositumomab enrolled 60 patients with refractory or transformed low-grade NHL who had been treated with at least two different qualifying chemotherapy regimens.¹⁹⁷ Patients must also have failed to achieve an objective response or relapsed within 6 months after completion of their last qualifying chemotherapy (LQC) regimen. Median age was 60 years, and other poor prognostic features included: median of 4 prior therapies, bulky disease, bone marrow involvement, elevated serum lactate dehydrogenase, advanced stage, and transformation from an initial low-grade histology to a higher-grade histology in 38% of the patients. A statistically significant improvement in the primary endpoint was achieved, with a longer duration of response (>30 days) after I-131 tositumomab therapy (n = 26) compared to patients after their LQC (n = 5; P < 0.001). Improvements in secondary efficacy endpoints after I-131 tositumomab compared to those after LQC were also achieved: overall response (47% vs. 12%; P < 0.001), duration of response (11.7 vs. 4.1 months; P < 0.001), and CR (22% vs. 2%; P = 0.002). Fifteen of 60 patients (25%) were classified as long-term responders (patients with a MIRROR Panel-assessed TTP of a year or more). Nine (15%) of the 60 patients remained in CR, with TTP ranging from 41+ to 57+ months.

A second trial examined the incremental benefit of the radioconjugate (I-131 tositumomab) compared to the nonradioactive antibody (tositumomab).¹⁹⁸ This study was a randomized, twoarm, open-label, multicenter study that enrolled patients with chemotherapy-relapsed or refractory low-grade or transformed low-grade NHL. Patients were randomized to receive either I-131 tositumomab therapy or unlabeled tositumomab alone. The primary endpoint was a comparison of the rates of CR. Secondary endpoints included ORR, duration of responses, and TTP. A total of 78 patients (18% with transformation) participated in the study. Patients had been previously treated with one to three chemotherapy regimens. One or more therapies must have included an anthracycline, anthracenedione, or alkylating agent. A significant difference was observed for the primary efficacy endpoint. The CR rate was 33% (14 of 42 patients) for the patients treated with I-131 tositumomab compared to 8% (3 of 36) for patients treated with unlabeled tositumomab (P = 0.012). In addition, the ORR was greater after treatment with I-131 tositumomab: 23 of 42 patients (55%) compared to 7 of 36 patients (19%; P = 0.002). Nineteen patients initially treated with the unlabeled antibody crossed over to receive I-131 tositumomab after disease progression. A CR then was observed in 42% (8 of 19 patients) and an ORR in 68% (13 of 19 patients) in the crossover patient population. A total of 20 patients (33%) from the I-131 tositumomab-treated populations, including patients in the crossover arm, were classified as having a long-term response, including ten patients continuing in CR, with TTP ranging from 23+ to 59+ months.

The efficacy of I-131 tositumomab was also evaluated in patients who had progressed after rituximab.^199,200 Patients must have had prior treatment with at least four doses of rituximab without an objective response, or to have progressed during or after treatment. Twenty-four patients did not respond to their last treatment with rituximab, and, of the 16 patients who did respond to rituximab, five patients had a duration of response exceeding 6 months. A response occurred in 27 of 40 patients (68%), with a median duration of response of 14.7 months (95% CI; 10.6 months no response). A CR occurred in 12 of 40 patients (30%); the median duration of CR had not been reached (95% CI; 11 months no response). Twenty-four patients had a longer (at least 30 days) duration of response after I-131 tositumomab than after rituximab; 5 patients had a longer duration of response after rituximab than after I-131 tositumomab; 9 patients had equivalent durations of response; and 2 patients were censored (P < 0.001). A total of 14 patients (35%) had a TTP of 12 months or longer. The median PFS was 10.4 months (95% CI, 5.7 to 8.6) for all patients and 24.5 months for confirmed responders (95% CI, 16.8 to not reached [NR]). PFS for 15 confirmed CR patients was NR with an estimated 3 years PFS of 73%. Prior response to rituximab did not significantly affect the confirmed OR rate, duration of response, or median PFS.

Radioiodinated tositumomab and Y-90 ibritumomab tiuxetan have also been used at myeloablative doses, with stem cell rescue in patients with relapsed B cell lymphomas.^{201,202,203,204} Radioiodinated tositumomab was used initially as a single agent.²⁰² Twenty-five patients were imaged after a tracer dose of radioiodinated tositumomab. Twenty-two of these 25 patients achieved favorable biodistributions (i.e., had tumor doses in excess of doses to normal organs). These 21 patients received therapeutic infusions of radioiodinated tositumomab (345 to 785 mCi) followed by reinfusion of autologous hematopoietic stem cells. All patients achieved bone marrow engraftment (19 with bone marrow stem cells, two with peripheral blood stem cells). However, two patients died before full neutrophil recovery; one of sepsis, one of progressive lymphoma. Nonhematologic toxicities included nausea in most patients, mild mucositis in five patients, and partial alopecia in four patients. One patient experienced reversible cardiomyopathy and interstitial pneumonitis. Eighteen patients responded to this therapy; 16 patients experienced a CR. With a median follow-up of 2 years, 2 years PFS was estimated at 62%, with OS estimated at 93%. Press et al. then combined radioiodinated tositumomab with chemotherapy and autologous stem cell transfusion in a series of patients with relapsed B cell lymphomas.²⁰³ Fifty-two patients received the planned therapy. Patients were again given tracer doses of radioiodinated tositumomab and underwent sequential gamma camera imaging. Absorbed doses of radiation to tumor sites and normal organs were determined. Thereafter, patients received a therapeutic infusion of radioiodinated tositumomab calculated to deliver between 20 and 27 Gy to normal organs (e.g., liver, kidneys, and lungs). Patients then received etoposide, 60 mg/kg, and cyclophosphamide, 100 mg/kg, followed by reinfusion of autologous hematopoietic stem cells. The maximal tolerated dose of radioiodinated tositumomab to be combined with chemotherapy was determined to be that dose that delivered 25 Gy to normal organs. Eight patients experienced 13 grade 3 or 4 toxic events. These included 3 patients with acute respiratory distress syndrome, 3 patients with severe mucositis or gastrointestinal toxicity, 1 patient with venoocclusive disease, and 4 patients with fatal infections. At 2 years, the Kaplan-Meier estimates of OS and PFS for all treated patients were 83% and 68%, respectively. These findings were considered superior to results previously observed in patients who had undergone conventional external beam total-body radiation with etoposide/cyclophosphamide preparation for transplantation in the same institution. Thirty-one patients with CD20⁺ NHL were treated with high-dose Y-90 ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide and were followed by autologous hematopoietic cell transplantation (HCT).²⁰⁴ Treatment also was well tolerated; there were 2 deaths and 5 relapses. At a median follow-up of 22 months, the 2 years estimated OS rates are 92% and 78%, respectively. Retreatment with tositumomab and I-131 tositumomab, has also been found possible in patients with progressive disease after treatment with I-131 tositumomab, who were able to receive subsequent therapy, including cytotoxic chemotherapy and stem cell transplantation.²⁰⁵ In patients with prior response, I-131 tositumomab can produce second responses that can be durable.²⁰⁶