Newer Treatments for Non-Hodgkin’s Lymphoma: Monoclonal Antibodies
Significant advances have been made in the application of monoclonal antibody-based therapies to the treatment of patients with lymphoma. The most promising areas appear to be the use of unconjugated monoclonal
ABSTRACT: Significant advances have been made in the application of monoclonal antibody-based therapies to the treatment of patients with lymphoma. The most promising areas appear to be the use of unconjugated monoclonal antibodies and the use of radiolabeled monoclonal antibodies. The recent approval by the US Food and Drug Administration (FDA) of rituximab (Rituxan), an unconjugated chimeric antibody against the CD20 antigen for the treatment of relapsed low-grade or follicular B-cell non-Hodgkins lymphoma marked a milestone in the development of these antibody-based treatments. Other new drug applications to the FDA are pending using both unconjugated and radiolabeled monoclonal antibodies, and it is anticipated that further new treatment options based on monoclonal antibody technology will soon be available for the treatment of patients with non-Hodgkins lymphoma. Forthcoming clinical trial results combining these new agents with current therapies are needed to determine if the addition of these new biologic agents to our armamentarium against lymphoma will alter the natural history of this disease for our patients. The most promising of these treatments and the comparison of these strategies are reviewed here. [ONCOLOGY 12(Suppl 8):63-76, 1998]
Introduction
Nearly 100 years ago, Ehrlich described the concept of harnessing the immune system to treat cancer.[1] This idea motivated many scientists and clinicians with the appeal of developing treatments for patients with cancer that are more tumor-specific and less toxic to the host. This did not become possible until the development of hybridoma technology by Kohler and Milstein, which allowed for the production of large quantities of a single antibody with defined specificity (monoclonal antibody).[2] Many clinical trials quickly ensued, however, the application of antibody-based treatments from clinical trials into accepted clinical practice has been discouragingly slow.
The recent approval by the US Food and Drug Administration (FDA) of rituximab (Rituxan), an unconjugated chimeric antibody against the CD20 antigen, for the treatment of relapsed low-grade or follicular B-cell non-Hodgkin’s lymphoma marked a milestone in the development of these antibody-based treatments. Other new drug applications to the FDA are pending using both unconjugated and radiolabeled monoclonal antibodies, and it is anticipated that further new treatment options based on monoclonal antibody technology will soon be available for the treatment of patients with non-Hodgkin’s lymphoma. The most promising of these treatments and the comparison of these strategies are reviewed here.
Two types of treatment have emerged and have been explored widely. These are based on the use of either native or modified unconjugated monoclonal antibodies, or on the use of monoclonal antibodies, to target radionuclides, drugs, or toxins to the tumor. The first approach utilizes unconjugated monoclonal antibodies. Early trials employed murine or rat antibodies, whereas more recent studies have used chimeric or humanized monoclonal antibodies. These monoclonal antibodies contain mostly human antibody sequences with only the variable region or the actual antibody binding sites coming from the original murine antibody structure. These modified monoclonal antibodies have a longer half-life in vivo, are less immunogenic, and have increased clinical activity. In general, treatment with unconjugated monoclonal antibodies is well-tolerated with minimal infusion-related symptoms and often no dose-limiting toxicity (certain exceptions apply). A closely related approach to unconjugated monoclonal antibody therapy is the use of a tumor vaccine based on the idiotype to induce tumor-specific immunity directly in B-cell non-Hodgkin’s lymphoma patients, eliminating the need to produce and administer a custom anti-idiotype monoclonal antibody
The second strategy uses the monoclonal antibody as a carrier to specifically target a radionucleotide or toxin to the tumor cells. This approach has clearly demonstrated increased antitumor activity but must be dosed carefully and is associated with dose-limiting toxicity. Results from clinical trials of both approaches using unlabeled or radionucleotide conjugated monoclonal antibody-based therapies are strongly affected by the types of monoclonal antibody used, the characteristics of the target antigen, and the type of non-Hodgkin’s lymphoma treated.
Monoclonal antibody-based treatments have resulted in documented antitumor responses in many patients with B- and T-cell non-Hodgkin’s lymphoma. However, there have also been many failures and anecdotal responses that have not led to treatments that are applicable for general therapy. Indeed, over the past 20 years we have learned that there are important antigen and antibody as well as tumor characteristics that are critical in the successful application of monoclonal antibody-based therapies for the treatment of cancer. Progress has been made, and the approval of rituximab as the first of these new agents ushers in the beginning of a new era in cancer therapy by providing tumor specificity with less host toxicity.
General Principles of Monoclonal Antibody-Based Therapy
Characteristics of Tumor Antigens
Identification and characterization of the cell-surface antigen for immunotherapeutic attack is critical. Desirable antigen characteristics are different for each type of monoclonal antibody-based therapy and are detailed in Table 1. In general, the ideal tumor antigen should be present in high density on the surface of all of the tumor cells and not be expressed on normal cells. In reality, true tumor specificity is rare and often makes it difficult to apply the treatment to more than a single patient. As an example, the antigen receptor “idiotype” is tumor-specific but requires a custom antibody to be made for each patient with lymphoma, whereas antigens such as CD19 or CD20 are “lineage-specific”, and expression is limited to malignant and normal B lymphocytes. Thus, although absolute tumor specificity is often a goal of immunotherapy, most antigens are only relatively tumor-specific with the antigen expressed on some normal host cells. Expression on critical host cells or tissues must be avoided.
Other important antigen characteristics that should be considered include:
secretion or shedding of antigen from the cell into the circulation
modulation or internalization of the antigen into the cell upon monoclonal antibody-binding
antigen mutation or inhomogeneous expression on the malignant clone, and
the biologic function of the antigen that may be blocked, augmented, or triggered by monoclonal antibody-binding.
Treatments with unconjugated monoclonal antibodies or monoclonal antibodies targeting toxins or radionuclides differ in which of these characteristics are critical for the successful application of antibody therapy.
Mechanisms of Anti-Tumor Effect
Unconjugated monoclonal antibodies depend on either immune-mediated effects due to complement or antibody-dependent cell-mediated cytotoxicity (ADCC) or direct effects to cause tumor cell kill or growth inhibition. Over the past years there has been an increasing recognition of the direct effects of monoclonal antibodies binding to tumor cells. These effects range from the blocking of a growth factor receptor (eg, the interleukin-2 [IL-2] receptor),[3,4] to the stimulation of the immunoglobulin (Ig) receptor (anti-idiotype monoclonal antibodies).[5] These effects are often diverse and depend on the antigen that is targeted and on the stage of differentiation of the tumor cell. Monoclonal antibodies may effect cells directly by growth inhibition and cell-cycle arrest and by the induction of apoptosis. Synergy of monoclonal antibodies with conventional treatments such as chemotherapy drugs and irradiation may also occur and is currently a major focus of investigation.
The mechanism of tumor cell kill using conjugated monoclonal antibodies may include the antitumor activity of unconjugated monoclonal antibodies (if sufficient monoclonal antibody is administered), but also the antitumor effects due to the targeting of the drug, toxin, or radionuclide to the tumor tissues. In most trials there is insufficient evidence to identify the true contribution of tumor cell kill to the monoclonal antibody alone vs the specific and nonspecific antitumor effect of the monoclonal antibody and the targeted agent. However, it appears that the majority of the effect is due to targeted effects of the radioisotope.
Lymphoma Tumor Antigens
A large number of antigens have been targeted for monoclonal antibody-based therapy for non-Hodgkin’s lymphoma. The general characteristics of selected tumor antigens are shown in Table 2. For the most part, these antigens are lineage-specific and expressed by B-cell or T-cell malignancies as well as the normal host lymphocytes at a given stage of differentiation. Issues such as modulation, antigen shedding, and secretion have been identified as critical negative characteristics for unconjugated monoclonal antibody-based treatments, while modulation and internalization is required for the success of immunotoxin-based approaches. To date, few monoclonal antibody-based treatments have been able to reliably induce complete remissions in the majority of patients, and the generation of antigen-negative tumor cell variants has not been a problem. As treatments continue to improve, this is likely to become a greater issue.
Antigens range from truly tumor-specific (anti-idiotype) to lineage-specific (such as anti-CD19, 20, or 22 for B cells or anti-CD3, 4, or 8 for T cells) or more broadly expressed antigens (such as CD52 [target for CAMPATH]) that may be expressed on multiple cell lineages. The selection of the target antigen plays a major role in the success of monoclonal antibody-based therapy. Over the past years, the CD20 antigen has emerged as an excellent target antigen for unconjugated and radiolabeled monoclonal antibody therapy. In retrospect, this may be predicted by many of the characteristics of this antigen. In a similar fashion, the failure of many monoclonal antibody-based treatments can be traced to shortcomings of the target antigen and the selected monoclonal antibody-based treatment.
Types of Monoclonal Antibodies
Equally important to antigen selection are the physical properties of the antibody used for therapy. Specificity and affinity for the target antigen may differ even between antibodies directed against the same cell surface antigen. The species of the antibody also affects interaction with the human immune system and largely determines the immunogenicity of the antibody. Clinical trials using murine or rat monoclonal antibodies may generate human anti-mouse antibody responses (HAMA), limiting their effect and repeat use. Genetic modification of the antibodies by grafting human Ig constant regions onto murine variable regions containing the antibody-binding specificity results in “chimeric” antibodies. Further “humanization” of the antibody can by accomplished by grafting the complementary determining regions onto a human antibody variable region framework. Chimeric and humanized antibodies have decreased immunogenicity, longer serum half-life, and a greater ability to interact with human effector mechanisms such as complement-mediated cytotoxicity (CDC), and ADCC.
Antibody fragments, including F(ab)2 constructs, in which the antibody Fc portion is removed and F(ab)’ constructs in which only a monovalent antibody-binding site remains have also been used, generally to target toxins or radionuclides. Even smaller fragments can be constructed such as monovalent single-chain Fv which contains variable heavy and variable light chain sequences linked by a short peptide. These antibody fragments generally have a shorter half-life with greater and more rapid tissue penetration in vivo, but lack the ability to mediate CDC or ADCC. Additional constructs in clinical trials include bispecific antibodies containing two different antigen specificities as well as antibodies containing multiple Fc regions to either target cell-mediated cytotoxicity or augment immune-effector function.
Each of these antibody characteristics are important in determining the outcome of antibody therapy, however, the relative merits of antibody selection and antigen selection are largely unknown and are often empirically evaluated using murine tumor models and early phase clinical trials.
Unconjugated Monoclonal Antibody Therapy
Many monoclonal antibodies have now been evaluated in the treatment of B- and T-cell non-Hodgkin’s lymphoma.( Table 3) Detailed enumeration of each of these approaches is beyond the scope of this article, but those with significant anti-tumor activity or illustrative of the problems of this approach will be considered here.
Several basic principles are important to the success of this approach. The target antigen must be expressed on all of the tumor cells, and not shed or secreted into the serum where it blocks the binding to the tumor cell. The antigen needs to be expressed in high level on the surface of the tumor cell, and in an ideal situation would have a critical biologic function with no antigen mutation or loss variants. Antigens with minimal modulation or internalization have demonstrated the greatest anti-tumor effect. The mechanism of action of unconjugated monoclonal antibodies depends on the antibody binding to each tumor cell and killing it through immune or direct effects. Bystander cells are generally not affected. We have reviewed several approaches that have demonstrated success.
Targeting the Antigen Receptor with Anti-Idiotype Antibodies
The immunoglobulin that is clonally expressed on the surface of most B-cell malignancies can be exploited as a tumor-specific target. At Stanford University, Levy and colleagues used anti-idiotype monoclonal antibodies as a therapy for patients with relapsed B-cell non-Hodgkin’s lymphoma.[6-9] This approach was tumor-specific and nontoxic, but required a custom antibody to be made for each patient. In a series of clinical trials, significant antitumor activity was noted with objective clinical responses seen in more than 68% of patients, with some patients continuing in clinical remission for more than 10 years. Other investigators have utilized polyclonal anti-idiotype antisera or monoclonal antibodies for a variety of B-cell non-Hodgkin’s Lymphoma.[10-16]
The mechanism of action of anti-idiotype antibodies appears to include direct effects of the antibody on the tumor cells in addition to immune functions of CDC or ADCC. The ability of the treatment monoclonal antibody to induce tyrosine phosphorylation in the tumor correlated with the antitumor effect observed in the patient, suggesting that the biologic function of the target molecule (surface Ig) and the state of differentiation of the tumor cell were important factors in the outcome of antibody therapy.[5] These effects were observed even though the tumors expressed high levels of the bcl-2 protein, which normally induces resistance to the induction of apoptosis by a variety of agents. Interestingly, this treatment may also induce a form of tumor dormancy, as patients in complete remission for many years were still found to have cells bearing the tumor-specific sequences using sensitive polymerase chain reaction (PCR)-based assays.[17]
A number of problems were identified with the use of anti-idiotype monoclonal antibodies, including antigen modulation, tumor cell secretion of blocking idiotype into the serum, and the development of human anti-mouse antibody. However, the most critical issue was the escape or relapse of patients with idiotype negative tumor cell variants due to somatic mutation of the idiotype structure.[18] This activity is a characteristic of the stage of differentiation of follicular lymphoma cells and while the emergence of these variants demonstrates the powerful selective antitumor activity of this approach, it limits the use of single anti-idiotype monoclonal antibodies.
Immunoglobulin Idiotype Vaccination
Levy and colleagues at Stanford University have extended their earlier clinical trials of anti-idiotype monoclonal antibody therapy to the use of a custom idiotype vaccine to induce a tumor-specific immune response in the patient. In this approach, the immunoglobulin (idiotype) is isolated from the tumor cell and used to vaccinate the patient while in remission following conventional chemotherapy.[19,20] The generation of an immune response required a carrier protein (KLH) and a vaccine adjuvant (SAF-1).
In a recent summary of their work, immune responses were observed in the majority of patients in complete remission but were less common in patients with only partial remissions.[20] Interestingly, the immune responses to the carrier protein did not differ between the groups. Those patients who made an immune response (usually a polyclonal anti-idiotype antibody response or a T-cell proliferative response) to the idiotype had a lower risk of relapse and a longer survival when compared to the immune non-responders. This was true for patients without and for those with residual disease. Although this has not yet been subjected to a randomized study, the preliminary results are very promising.
The use of the idiotype vaccine stimulates a polyclonal immune response that appears capable of covering most of the variant immunoglobulin mutations that were observed to escape following therapy with monoclonal anti-idiotype antibodies.[21] Promising data have now been presented by a number of different investigators for the treatment of Ig expressing malignancies such as B-cell non-Hodgkin’s lymphoma and plasma cell myeloma. Obstacles to this approach include the requirement for a custom vaccine in each patient and the low level immune responses that are usually generated. However, newer techniques allowing isolation of the idiotype-specific tumor genes from needle biopsies and direct immunization with DNA containing the idiotype sequences alone or combined with immunomodulatory sequences may shorten production time and increase immunogenicity.[22] These approaches hold promise and are underway at a number of institutions both in the United States and in Europe.
Anti-CD20 Therapy with Rituximab
Early work identified the CD20 antigen as a potential target for immunotherapy although responses to murine monoclonal antibodies were only observed in a minority of patients.[23] Rituximab (IDEC-C2B8, Rituxan or MabThera in Europe) is an anti-CD20 monoclonal antibody that was the first monoclonal antibody approved by the FDA for the treatment of cancer. This chimeric monoclonal antibody contains murine IgG1-kappa variable regions and human IgG1-kappa heavy- and light-chain constant regions.[24]
The human IgG1 heavy chain constant region confers augmented ability to mediate cell killing using human sources of complement and antibody-dependent, cell-mediated cytotoxicity (ADCC) effector cells compared with the murine parent monoclonal antibody[24]. The monoclonal antibody binds with high affinity to the human CD20 antigen that is expressed in high copy number on nearly all B-cell non-Hodgkin’s lymphomas. In addition, some B-cell tumor cell lines are directly growth inhibited by the antibody and undergo apoptosis upon antibody binding.[25,26] The CD20 antigen is nearly an ideal tumor antigen for unconjugated monoclonal antibody-based therapy. It is expressed on a wide variety of B-cell non-Hodgkin’s lymphoma, is lineage-specific, expressed in high copy number, has a biologic function (calcium channel), does not modulate or internalize and has not been described to mutate.
Single infusions of rituximab in doses ranging from 10 to 500 mg/m2 were given to 15 patients with relapsed B-cell non-Hodgkin’s lymphoma. Infusion-related side effects were observed, but were generally mild grade I and II and most often consisted of fever, chills, skin rash, and occasional bronchospasm or mild hypotension.[27] Depletion of normal and malignant B cells in the peripheral blood was rapidly observed in nearly all patients, but without subsequent immunodeficiency.
Tumor biopsies taken from patients 2 weeks following the dose of monoclonal antibody (at the 50 mg/m² dose levels and above) demonstrated antibody bound to tumor cells throughout the lymph node in most patients. Despite the fact that patients only received a single infusion of the antibody, partial remissions were observed in 2 patients and minor responses in other patients. There was no effect on serum immunoglobulin levels or complement levels. B cells were depleted and began to recover in the peripheral blood 3 to 6 months following therapy. However, there was no increase in the type or severity of infections observed during or following treatment. Normal B cells were observed to recover in the peripheral blood in 3 to 6 months.
Subsequent studies utilized four infusions of the monoclonal antibody administered at one dose each week over a total treatment course of 22 days. An initial phase I/II trial evaluated 125 mg/m², 250 mg/m² and 375 mg/m² doses and had a 33% response rate at each dose level.[28,29]
Most patients had relapsed follicular low-grade non-Hodgkin’s lymphoma. There were no dose-limiting side effects observed, and no increase in side effects noted with the multiple-dosing schedule. Similar infusion-related adverse events were noted with the first infusion, but decreased markedly with subsequent infusions.
The 375 mg/m² dose was selected for the phase II study. This phase II trial in 37 patients [29] and a larger multiple center trial in 166 patients [30] with relapsed or refractory low-grade or follicular B-cell non-Hodgkin’s lymphoma produced nearly identical results and formed the basis for application for drug approval in the United States and in Europe. Overall on an intent-to-treat basis, 48% to 50% of patients had a remission, with minimal adverse effects, in response to the four monoclonal antibody infusions. The majority of patients were judged to have partial remissions, using strict criteria for complete remissions (neck, chest, abdomen and pelvis CT scans with all nodes < 1 x 1 cm) and evaluated by an independent third-party panel of lymphoma experts.
The median time-to-progression for the responding patients in the smaller trial was 10.2 months, and it has not yet been reached in the larger trial at greater than 12 months. No cumulative toxicity has been observed. CD20 antigen negative variants have not been observed in relapsing tumors, and early experience suggests that retreating of responding patients can induce a second remission in about 40% to 50%. Immune responses to the chimeric antibody are rare (< 1%) and do not appear to be clinically significant.
There were no changes in mean serum Ig levels and no apparent increase in infections during the treatment course or in the 1 year follow-up. Importantly, tumor responses were observed in patients who had failed anthracycline-containing regimens, patients aged greater than 60 years, patients with bulky disease (greater than 7 cm masses), and those with splenomegaly and bone marrow involvement.
A lower response rate was observed in patients with a very short remission to prior chemotherapy, but a surprisingly high response rate (78%) was observed in patients who had failed prior high dose chemotherapy with autologous bone marrow or stem-cell transplantation.
A lower response rate was observed for patients with Working Formulation group A histologies (small, well-differentiated lymphocytic lymphoma) compared with patients with any of the follicular histologies, possibly due to the lower level of expression of CD20 that is characteristic of some of these tumor histologies. Additional data presented in abstracts from Europe demonstrated a 33% response rate in patients with aggressive B-cell histologies and with mantle-cell lymphoma. Multiple studies are currently ongoing in a number of institutions.
Rituximab With or Following Chemotherapy
A supporting study has also been presented in abstract form using rituximab (Rituxan) combined with a standard course of cyclophosphamide (Cytoxan), doxorubicin (Adriamycin), vincristine (Oncovin), and prednisone (CHOP) chemotherapy for patients with low grade non-Hodgkin’s lymphoma[31]. This treatment consisted of six doses of the monoclonal antibody given throughout the six cycles of CHOP (two initially, one dose following cycles 2 and 4, and two doses following cycle 6 of CHOP).
The rationale for this study was to combine the independent activity and low toxicity of the monoclonal antibody with chemotherapy and to take advantage of possible synergy with chemotherapy.[25] The toxicity of the treatment appeared to be comparable to that observed with the antibody alone and that expected from treatment with CHOP. Tumor responses were observed in all patients, including the conversion from PCR positive to negative for the bcl-2/Ig translocation in the peripheral blood and bone marrow of 6 of 8 patients. This has rarely been reported using CHOP alone. Whether this will translate into improved disease-free survival and prolonged remissions, or just reflect the particular ability of the antibody to clear the peripheral blood and bone marrow of tumor cells remains to be determined.
Additional trials are evaluating rituximab combined with or following chemotherapy in many B-cell tumors, both low-grade and aggressive histologies. Rituximab is the first of a new class of agents that provides greater tumor specificity with less host toxicity. Randomized clinical trials evaluating the role of this new agent in the treatment of patients with B-cell non-Hodgkin’s lymphoma alone and in combination with or following standard therapies are eagerly awaited.
CD-52 and CAMPATH-1H
CD-52 is a 12 amino acid , 21-28 K Da glycosylphosphatidyl-inositol (GPI)-linked glycoprotein, expressed on both T- and B-lymphocytes and at lower levels on monocytes. CAMPATH-1H is a humanized IgG1 monoclonal antibody. While initial reports demonstrated limited activity in the treatment of patients with nodal-based B-cell non-Hodgkin’s lymphoma[32], significant activity against chronic lymphocytic leukemia (CLL) has been observed. Interestingly, marked infusional toxicity has been observed with this antibody, including fever, chills and rigors, and hypotension and appears to be related to the secretion of cytokines TNF-alpha, IFN-gamma, and IL-6 through cross-linking of CD16 (low affinity Fc-receptor to IgG) on natural killer cells.[33] Also, due to the extensive expression of the antigen on normal lymphocytes, marked and prolonged lymphopenia with significant immunosuppression has been observed.
However, significant antitumor activity has been observed in patients with CLL or PLL. In a recent report, 11 of 15 patients with T-cell PLL had a major remission including 9 patients with a complete response.[34] In B-cell CLL 29 patients with previously treated CLL (8 with a prior response and 21 refractory) were treated with doses escalating to 30 mg three times each week for 12 weeks.[35]
Overall there was a 42% response rate with one complete response. As seen previously, 28 of 29 patients cleared their blood, 36% cleared the bone marrow, 32% resolved splenomegaly but only 7% cleared lymph nodes. The antibody has also been used to purge bone marrow in vivo prior to ABMT.[36,37] In previously untreated CLL, the response rate appears higher, with 8 of 9 patients having a partial response (N = 5) or complete response (N = 3), with responses lasting 8 to 24 months plus.[38] These patients were treated with intravenous or subcutaneous CAMPATH-1H at doses up to 30 mg, three times each week for 18 weeks. All patients cleared the peripheral blood and 7 of 9 cleared their bone marrow. Serious infections with CMV were reported in only 1 patient.
Therapy with CAMPATH-1H appears to rapidly lyse tumor cells due to complement fixation. While the antigen does not modulate from the cell surface, antigen negative variant normal T-cells have been observed to emerge in rheumatoid arthritis patients treated with low levels of the antibody.[39] Neutralizing immune responses to CAMPATH-1H have been observed in patients with rheumatoid arthritis treated with low-dose subcutaneous injections.[40] Additional ongoing studies are exploring further uses of the lymphocyte depleting ability of the antibody for bone marrow purging, the treatment of graft vs host disease, and in the treatment of patients with auto-immune diseases.
Additional Unconjugated Monoclonal Antibody Trials
A large number of additional B- and T-cell antigens have been exploited as antibody targets. Unfortunately, the majority of these approaches have demonstrated unimpressive antitumor activity and have been abandoned or modified to target toxins or radionucleotides to augment antitumor activity. In many cases, at least in retrospect, this was often due to undesirable characteristics of many of the target antigens such as modulation. However, clinical activity was seen in clinical trials of unconjugated murine monoclonal antibodies against the Lym-1 antigen (a DR variant) [41], CD19 [42], CD5 [43-47], and CD4.[48]
One area of interest has been in the blocking of biologic receptors. The most extensively studied has been the use of monoclonal antibodies against the IL-2 receptor for the treatment of patients with T-cell malignancies. This pioneering work demonstrated the ability of the monoclonal antibody to block the interaction of IL-2 with the receptor complex, and in tumor cells that were driven by IL-2 this resulted in antitumor effects. These effects were observed in patients with adult T-cell leukemia and with some cutaneous T-cell malignancies.[3,4] Unfortunately, these tumor cells often became resistant to this blockade and acquired growth independence from IL-2. Subsequent studies have utilized radiolabeled monoclonal antibodies or immunotoxins.
Antibody-Based Immunotoxin Therapy
The use of antibodies to target drugs or toxins was undertaken to augment the antitumor activity observed using unmodified murine monoclonal antibodies. For these conjugates to be active, the complex must enter the cell, be cleaved from the monoclonal antibody, and enter the sensitive compartment of the cell to invoke cell damage and eventual cell death. A number of toxin-conjugated antibodies have been used in clinical trials in patients with non-Hodgkin’s lymphoma. In vitro, these agents have demonstrated impressive and selective antitumor effects. What has also become clear is that the majority of these agents also have potent nonspecific toxicity associated with their use in vivo, which is dose-limiting.
Ricin-Conjugated Monoclonal Antibodies
The most clinical experience using immunotoxins for the treatment of lymphoma has been with monoclonal antibodies coupled to toxins based on ricin. This potent toxin consists of an A chain, and a B chain that binds to galactose sugar molecules on the cell surface, but also facilitates translocation of the A chain across the endosomal membrane to the cell cytoplasm were the A chain is catalytically active in inactivating the 60S ribosome. Strategies using ricin-conjugated monoclonal antibodies have focused on coupling the isolated ricin A chain or intact ricin with blocked B chain galactose binding sites to monoclonal antibodies.
A variety of lymphoid antigens have been the target of immunotoxin-directed therapy. These have included CD5, CD10, CD19, CD21, CD22, and CD25. These antigens all undergo capping and modulation into the cell cytoplasm. Stable cell surface antigens such as CD20 do not internalize and are not effective targets for immunotoxin directed therapy.
The largest experience has been with B4-blocked ricin, an immunotoxin made of intact ricin (A and B chains) with chemically blocked galactose binding sites conjugated to the anti-CD19 monoclonal antibody (B4). Phase I and II clinical trials identified the dose-limiting toxicity as hepatotoxicity, with elevation of transaminases and thrombocytopenia as the major toxicities.[49-51] Pharmacokinetic analysis demonstrated a short half-life and required a slow infusion. The maximal tolerated dose was 50 µg/kg/day x 5 days for patients with relapsed B-cell non-Hodgkin’s lymphoma, and was lower when used as an adjuvant following high-dose therapy and ABMT.[51] Administration by continuous infusion allowed dose escalation to 7 days with a similar toxicity profile. Although responses were observed in the treatment of patients with measurable disease, these were typically of short duration and were more often observed in patients with lower amounts of tumor burden.
A phase II trial among patients in remission following ABMT demonstrated feasibility of treatment with repeated courses of the immunotoxin. Based on this, a large multicenter, randomized Phase III clinical trial was initiated which evaluated the effect of the immunotoxin as an adjuvant following high-dose therapy and ABMT. This trial proved to be difficult to complete and was stopped at an interim analysis that demonstrated no benefit from the immunotoxin treatment.[52]
Vitetta and colleagues have used an alternate ricin construct based on isolated deglycosylated ricin A chain, which exhibits minimal binding to normal cells due to the absence of B chain and diminished uptake by hepatic carbohydrate receptors due to deglycosylation. The deglycosylated ricin A chain was coupled to intact or Fab’ fragments of anti-CD19 (HD37) and anti-CD22 (RFB4) monoclonal antibodies for the treatment of patients with B-cell non-Hodgkin’s lymphoma. In phase I clinical trials, the dose-limiting toxicity was identified to be the development of a capillary leak syndrome characterized by pulmonary edema, weight gain, and hypoalbuminemia.[53-56] This appeared to be related to the total dose of toxin administered. Immune responses to the murine monoclonal antibody and to the ricin A chain were observed in the majority of patients. In these clinical trials of the immunotoxins, short-lived clinical responses were observed in up to 30% of patients with relapsed B-cell non-Hodgkin’s lymphoma. Responses were more frequent in patients with minimal disease compared with those with lymph nodes greater than 2 cm in size. Evaluation of these agents as adjuvant treatment following standard induction chemotherapy is being planned.
Additional clinical trials have utilized immunotoxins against the CD5 antigen that is expressed on some T-cell and B-cell malignancies [57] as well as the IL-2 receptor expressed on T-cell malignancies and in Hodgkin’s disease.[58] In the majority of these trials, similar toxicity was dose-limiting and responses were generally limited to the blood with minimal effects observed in areas of more bulky disease.
Problems common to trials utilizing immunotoxins include the potent immunogenicity of all of the toxins, both in inducing antibody responses to the toxin, but also to the carrier immunoglobulin. Both the human anti-mouse antibody and the human anti-ricin antibody (HARA), or another toxin-immune response are nearly universal and limit repeated exposure. In addition, all clinical trials have demonstrated dose-limiting toxicity most often consisting of a vascular leak syndrome or of hepatic toxicity. More serious toxicities, including aphasia and cardiac events, have been observed. While the exact mechanism of this toxicity is not clear, it is likely due in part to the nonspecific removal of the immunotoxin by the reticuloendothelial system of the liver and possibly in vessels. Despite these serious in vivo toxicities, the immunotoxins provide very potent tumor-specific killing potential and may play an important role in the in vitro treatment of bone marrow or stem-cell collections prior to treatment with high-dose therapy and stem-cell support.
Radiolabeled Antibody Therapy
General Principles of Radioimmunotherapy
Radiolabeled antibodies possess several potential theoretical advantages for the treatment of non-Hodgkin’s lymphoma. Since radioimmunoconjugates kill tumor cells primarily by emission of radioactive particles, they are therapeutically effective even in situations where defective immune effector systems compromise the utility of unmodified antibodies (Table 1).
Second, the beta particles emitted by commonly used radioisotopes (iodine-131 [131I], yttrium-90 [90Y]) are tumoricidal over a distance of many cell diameters, allowing eradication of antigen-negative tumor cells by radioactive “cross-fire” from neighboring antigen-positive, antibody-coated cells. Furthermore, this “cross-fire” phenomenon minimizes the deleterious impact of inhomogeneous penetration of antibody into tumor sites, since tumor cells with limited access to radiolabeled antibodies can still be killed by radioactive cross-fire from nearby labeled cells. The exquisite radiosensitivity of hematologic malignancies renders them particularly susceptible to radioimmunotherapeutic approaches. A relative disadvantage of this approach is that there is nonspecific and specific toxicity to normal cells due to the radiation of non-tumor tissue. Thus, dosing is critical and dose-limiting toxicity has been observed with all radiolabeled monoclonal antibody-based treatments.
The most desirable radionuclide for radioimmunotherapeutic trials is a subject of great debate, and the ideal choice may depend on the antigenic target selected and the size of the antibody construct employed (see Table 4). Iodine-131 has been used in the majority of reported clinical trials because of its accessibility, simple radiochemistry, relative low cost, and its long track record of clinical success in treating thyroid carcinoma (and, more recently, non-Hodgkin’s lymphoma).[59-62] A disadvantage of 131I-conjugates include their tendency to be degraded after internalization into tumor cells with subsequent release of 131I-tyrosine and free 131I to the bloodstream.[63,64] In addition, the energetic gamma rays emitted by 131I pose a radiation risk to health care personnel.
Yttrium-90 is a pure beta particle emitter that is the second most commonly used radioisotope for radioimmunotherapy. Its advantages include its favorable half-life (64 hours), highly energetic beta emissions, absence of significant gamma emissions (making it a safer reagent for medical personnel), and its stable retention inside tumor cells after endocytosis. Unfortunately, the absence of gamma emissions renders 90Y problematic for radioimmunoscintigraphy, and it is more expensive and less accessible than 131I.
A surrogate radiometal, indium-111 (111In) has been substituted as an imaging reagent for patients scheduled for 90Y therapy, on the assumption that its biodistribution will reliably mimic that of 90Y. Free 90Y accumulates non-specifically in the liver and bones, but the development of newer, stabler chelation methods for radioimmunotherapy (eg, “DOTA”: 1,4,7,10-tetraazocyclododecane-N, N’, N’’ ‘- tetracrotic acid) has minimized this problem. Other beta-emitting isotopes such as copper-67 (67Cu) also appear promising in preliminary clinical studies.[65]
Alpha-emitting radionuclides such as astatine-211 (211At) and Bismuth-212 (212Bi) offer high linear energy transfer to tumor targets, but their short half-lives, difficult radiochemistry, and lack of accessibility have limited their clinical applicability. Iodine-125 emits energetic Auger electrons by electron capture which theoretically should possess antitumor efficacy. Auger electrons have very short pathlengths, however, and are only cytotoxic if radioimmunoconjugates are deposited in a nuclear or perinuclear location.
Clinical Trials of Radioimmunotherapy for Hodgkin’s Disease
Lenhard and Order pioneered radioimmunotherapy for hematologic malignancies using 131I-labeled polyclonal antiferritin antibodies to treat patients with relapsed Hodgkin’s disease.[66] Objective tumor responses were observed in 15 of 37 patients (40%) with refractory Hodgkin’s disease treated with 50 to 100 mCi 131I-antiferritin, including one complete remission (3%). In addition, 17 of 22 patients (77%) with “B symptoms” (fever, weight loss, nightsweats) experienced symptomatic relief after “isotopic immunoglobulin” therapy. A subsequent trial investigated the utility of 90Y-labeled polyclonal antiferritin antibodies in 44 patients with relapsed, advanced Hodgkin’s disease.[67] Thirty-nine of the 44 patients exhibited satisfactory tumor-targeting with a trace-labeled dosimetric infusion of 111In-antiferritin and were subsequently treated with 1 to 5 cycles of 10 to 50 mCi 90Y-antiferritin conjugated to 2 to 5 mg polyclonal antiferritin. Twenty of the 39 patients (51%) had objective responses, including 10 complete remissions and 10 partial remissions (Table 5). Hematologic toxicity was dose-limiting, and required autologous bone marrow transplantation in approximately half the patients. Remissions were more commonly observed in patients with small tumor volumes (< 30 cc) than in those with large tumors (> 500 cc).[68]
Bierman and co-workers extended these observations by treating patients with poor prognosis Hodgkin’s disease with 90Y-labeled antiferritin followed by high-dose chemotherapy and autologous bone marrow transplantation.[69] Fourteen patients were tested with trace-labeled infusions of 111In-labeled polyclonal antiferritin antibodies (2 to 5 mg, 5 mCi), followed by planar gamma camera imaging to assess antibody biodistributions. Patients with masses > 5 cm were irradiated with 1.5 Gy on 2 consecutive days before 111In injection to augment tumor vascular permeability and increase tumor uptake.
All patients exhibited positive tumor imaging with 111In-antiferritin rendering them eligible for the subsequent infusion of 90Y-antiferritin (18 to 33 mCi on day -12), followed by cyclophosphamide (Cytoxan, Neosar) (1.5 g/m² IV on days -6 through -3), carmustine (BiCNU) (300 mg/m² IV on day -6), etoposide (VePesid) (125 mg/m² IV bid on days -6 through -4), and marrow infusion on day 0. Five patients died of early transplant-related toxicities between days -2 to +19 (eg, fungal infection, respiratory failure, etc.). Two patients did not receive the full therapeutic regimen because of the development of early life-threatening complications. Four patients were alive at the time of publication, including three who were progression-free for periods of 24+, 25+, and 28+ months.
At 12 months, the overall survival rate was projected to be 36% with a progression-free survival of 21%. The authors concluded that the outcome of these poor-prognosis patients with this novel, combined modality approach compared favorably to the expected outcome with conventional transplant regimens.
Vriesendorp has recently published a brief summary of five consecutive studies treating 134 patients with recurrent Hodgkin’s disease with radiolabeled antiferritin, including the three trials described above plus preliminary results of two additional studies. Overall a 60% response rate is reported in patients with recurrent Hodgkin’s disease treated with 90Y-antiferritin.[70] Responses were more common in patients with longer disease histories (> 3 years), smaller tumor burdens (< 30 cc), and in patients receiving at least 0.4 mCi per kg of body weight.
Clinical Trials of Radioimmunotherapy for B-Cell Lymphoma
The most promising trials in the radioimmunotherapy field have been conducted using radiolabeled anti-CD20 antibodies to treat patients with relapsed B-cell lymphomas (Table 6). In a Phase I/II trial, Kaminski administered trace-labeled infusions of 131I-anti-B1 (anti-CD20) antibody (Bexxar) to 34 patients with recurrent B-cell lymphomas.[59,71] Quantitative gamma camera imaging was used to calculate individual therapeutic doses of 131I-anti-B1 that would deliver whole-body radiation doses varying from 25 cGy to 85 cGy in a Phase I dose escalation manner.[59,60]
Prior to trace-labeled and therapeutic infusions, varying doses of unlabeled B1 antibody (0, 135, or 685 mg) were infused to test the hypothesis that blocking nonspecific Fc receptor sites and normal B-cell “antigen sinks” (eg, the spleen) would improve the biodistribution of subsequently administered 131I-anti-B1 antibody. This proved to the case, with the best radiolabeled antibody biodistributions occurring after infusion of 685 mg of “cold”, non-radioactive B1 antibody.
Twenty-eight evaluable patients were treated with 34 to 161 mCi of 131I-anti-B1.[60] Fourteen patients (50%) achieved complete remissions, and eight (29%) had partial responses, for a total objective response rate of 79%. Interestingly, all of the low-grade lymphoma patients responded, with 77% achieving complete remissions (median duration > 16 months). Myelosuppression was dose-limiting, with a maximally tolerated whole body radiation dose of 75 cGy. Nonhematologic toxicities were mild, consisting of low-grade fevers, chills, fatigue, and nausea. Six of the 34 patients enrolled developed human anti-mouse antibody.
Knox et al administered 90Y-labeled anti-CD20 antibodies to 18 patients with relapsed B-cell lymphoma (4 treated with the anti-B1 antibody and 14 with the IDEC Y2B8 antibody) in single doses of 13.5 to 53.4 mCi [72]. Six complete responses and seven partial responses occurred (overall response rate, 72%), with a median response duration of 6 months. Four patients developed human anti-mouse antibody. Doses of 50 mCi 90Y produced severe hematologic suppression in all four patients treated, with two requiring reinfusion of autologous, peripheral blood stem cells. No other serious toxicities were observed.
Press et al explored the maximal single-agent potential of 131I-labeled anti-B-cell antibody therapy in a series of trials using autologous stem-cell support to circumvent the dose-limiting hematologic toxicity observed in most radioimmunotherapy trials.[61,62,73] In a Phase I dose-escalation trial, the biodistribution of 131I-labeled anti-CD20 (anti-B1) and anti-CD37 (MB-1) antibodies were studied in 43 patients with relapsed B-cell non-Hodgkin’s lymphoma.[61] Weekly trace-131I-labeled infusions (5 to 10 mCi) were given at successive doses of 0.5, 2.5, and 10 mg/kg of antibody.
Twenty-four of the 43 patients had “favorable antibody biodistributions,” defined by absorption of higher radiation doses by all assessable tumor sites than by any critical normal organ (eg, liver, kidneys, lungs). A protein dose of 2.5 mg/kg achieved optimal biodistributions with the anti-CD20 (anti-B1) antibody, whereas 10 mg/kg was required to achieve “favorable biodistributions” with the anti-CD37 antibody. Patients with tumor volumes > 500 cc or significant splenomegaly rarely had biodistributions favorable enough for entry to the therapeutic arm of this protocol.
Nineteen patients received therapeutic infusions, including 12 with the anti-B1 (anti-CD20) antibody, one with the 1F5 (anti-CD20) antibody, and six with the MB-1 (anti-CD37) antibody. 131I doses were calculated to deliver radiation ranging from 10 to 31 Gy (234 to 777 mCi) in an escalating dose manner to critical normal organs. Fifteen patients required autologous bone marrow reinfusion. Nonhematologic toxicities were mild at doses less than 23 Gy and consisted of nausea, fever, thyroid dysfunction, and transient, mild elevations of liver function tests. At doses > 27 Gy delivered to the lung, 2 of 4 patients developed reversible cardiopulmonary toxicity, resulting in termination of the dose-escalation study. Eighteen patients (95%) had objective responses, including 16 complete responses (84% of treated patients). Currently, eight patients remain in continuous remission 46 to 95 months following therapy.
In a subsequent Phase II trial, Press et al administered trace-labeled infusions of 131I-labeled anti-B1 (anti-CD20) antibody to 25 patients with relapsed B-cell non-Hodgkin’s lymphoma.[62] Twenty-two patients had “favorable antibody biodistributions,” as defined above. One of these patients formed human anti-mouse antibodies after the trace-labeled infusion, but the other 21 were treated with therapeutic infusions of 2.5 mg/kg of 131I-anti-B1 with individualized doses of 131I (345 to 785 mCi), calculated to deliver ~ 27 Gy to the dose-limiting normal organ (generally the lungs) with higher doses (27 to 92 Gy) to tumor sites.
All patients had previously stored autologous hematopoietic stem cells reinfused (19 bone marrow and 2 peripheral blood stem-cell transplants). Seventeen of the 21 patients (81%) eventually achieved complete remissions and one achieved a partial remission for a total response rate of 86%. (One patient converted from a partial response to a complete response after the original publication.)[62] Eleven patients from this group currently have no evidence of disease 24 to 48 months after radioimmunotherapy.
Remission durations appear much longer for patients with follicular lymphomas than for intermediate and high-grade non-Hodgkin’s lymphoma. Current efforts are aimed at improving this promising results by integrating 131I-anti-B1 with high-dose VP-16 (60 mg/kg), and cyclophosphamide (100 mg/kg) in an autologous stem-cell transplant program (Press, unpublished results). Thirty-four patients have been treated since initiation of the trial and 27 are free from progression after a median follow-up of 15 months.
Studies targeting B lymphocyte surface antigens other than CD20 have also achieved promising clinical results. DeNardo et al treated 25 patients with relapsed non-Hodgkin’s lymphoma and CLL with low, fractionated doses (30-60 mCi) of an 131I-labeled antibody recognizing a DR variant antigen (Lym-1) given at 2 to 6 week intervals.[74-76] Three patients (12%) achieved complete remissions and 10 patients achieved partial remissions (40%), for an overall response rate of 52%. Hematologic toxicity, particularly thrombocytopenia was dose-limiting.
A subsequent trial investigated the efficacy of administering escalating doses of 131I-Lym-1 (40 to 100 mCi/m²) at 4 week intervals to 20 non-Hodgkin’s lymphoma patients.[76] Seven complete responses (35%) and 4 partial responses (20%) were observed (overall response rate of 55%).
Goldenberg and colleagues administered repetitive doses of 131I-labeled anti-CD22 antibody LL2 to 21 patients with relapsed B-cell non-Hodgkin’s lymphoma.[77,78] Six patients received intact 131I-LL2 IgG, 14 received 131I-LL2 F(ab’)2 fragments, and 1 received chimeric 131I-LL2. Seventeen patients were assessable, and four had objective responses (24%), including one complete response.
In a separate trial, the same investigators treated 3 patients with high doses (90 mCi/m2) of 131I-LL2 IgG followed by autologous bone marrow support.[78] Both evaluable patients had partial responses lasting 2 and 8 months, respectively. Radiolabeled tumor-specific anti-idiotypic antibodies were administered to non-Hodgkin’s lymphoma patients by Parker, Royston, and co-workers.[79,80] Nine patients were treated with 1 to 4 courses of 10 to 54 mCi (1,000-2,320 mg) of 90Y-anti-idiotypic antibody. Two complete responses and one partial response were observed. Other trials [71,81] are summarized in Table 6.
Multi-center trials of 131I-anti-B1 (anti-CD20, Bexxar), 90Y-B28 (anti-CD20, Idec Y2B8), 131I-Lym-1 (anti-DR variant,), and 131I-LL2 (anti-CD22) are currently underway in the United States to confirm the single institution experiences reported above.
Clinical Trials of Radioimmunotherapy for T-Cell Lymphoma
T-cell lymphomas constitute only 15% of the cases of non-Hodgkin’s lymphomas in the United States. For this reason, few radioimmunotherapy trials have been conducted targeting T-cell lymphomas (Table 7). Rosen et al evaluated 7 patients with cutaneous T-cell lymphomas with an 131I-labeled anti-CD5 antibody (T101).[82,83] Six patients received therapeutic infusions of 100 to 150 mCi of 131I conjugated to 9.9 to 16 mg of T101 antibody. Two patients (33%) achieved partial responses lasting 2 months each, and the other 4 patients had minor responses lasting 3 weeks to 3 months.
All patients experienced resolution of pruritis following treatment. Transient infusional toxicities included mild fever and pruritis in all patients and brief dyspnea in two patients. Three patients experienced myelosuppression, and one patient treated with 150 mCi required platelet transfusions. All patients demonstrated modulation (internalization) of the CD5 antigen after antibody therapy with resultant intracellular degradation (“dehalogenation”) of the radioimmunoconjugate. All patients also developed human anti-mouse antibodies, which interfered with repetitive antibody infusions.
Waldmann treated 18 patients with human T-cell lymphotropic virus-I-associated adult T-cell leukemia/lymphoma with 90Y-labeled anti-Tac monoclonal antibody targeting the interleukin 2a receptor.[10] The first 9 patients were treated on a Phase I dose-escalation protocol with cohorts of three patients each receiving 5, 10, and 15 mCi of Y-90 conjugated to 2 to 10 mg of anti-Tac (with the antibody protein dose determined by circulating interleukin 2a receptor levels).
The second group of 9 patients were treated on a Phase II fixed dose trial of 10 mCi 90Y-anti-Tac. Retreatment was performed in 12 responding patients with up to nine cycles administered at 6+ week intervals. Nine of 16 evaluable patients responded (56%) including two complete responses (13%) and seven partial responses (44%). Remissions lasted from 1.6 to 22.4 months (median 9.2 months). Granulocytopenia and thrombocytopenia were dose-limiting, though transient hepatic toxicity (three cycles) and proteinuria (4 patients) were also observed.
Conclusions
Significant advances have been made in the application of monoclonal antibody-based therapies to the treatment of patients with lymphoma. The most promising areas appear to be the use of unconjugated monoclonal antibodies and the use of radiolabeled monoclonal antibodies. The approval of the chimeric monoclonal antibody rituximab brings these advances from clinical trials to the patient’s bedside. It is anticipated that radiolabeled monoclonal antibodies will also soon be available.
It remains to be determined what the exact role each of these new agents will play in the ultimate management of patients with lymphoma, however, they will provide exciting new options. Each approach has its benefits and limitations. These range from ease of use, toxicity, side effects, immunogenicity, to degree of antitumor effect and effect on bone marrow.
Forthcoming results from clinical trials combining these new agents with current therapies are needed to determine if the addition of these new biologic agents to our armamentarium against lymphoma will alter the natural history of this disease for our patients.
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