Basic Science / ANTICANCER ANTIBODIES

Anticancer Antibodies
Jeffrey S. Ross, MD,1,2 Karen Gray, PhD,1 Gary S. Gray, PhD,1 Peter J. Worland, PhD,1 and Mark Rolfe, PhD1
Key Words: Cancer; Antibodies, review; Conjugation; Humanization; Herceptin; Rituxan; Zevalin; Mylotarg; Prostate-specific membrane antigen; PSMA; Campath; Avastin
DOI: 10.1309/Y6LPC0LR726L9DX9

Abstract
The recent clinical and commercial success of anticancer antibodies such as rituximab and trastuzumab has created great interest in antibodybased therapeutics for hematopoietic malignant neoplasms and solid tumors. Given the likelihood of lower toxic effects of antibodies that target tumor cells and have limited impact on nonmalignant bystander organs vs small molecules, the potential increased efficacy by conjugation to radioisotopes and other cellular toxins, and the ability to characterize the target with clinical laboratory diagnostics to improve the drugs clinical performance, current and future antibody therapeutics are likely to find substantial roles alone and in combination therapeutic strategies for treating patients with cancer. It also is likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan and gemtuzumab ozogamicin. This review considers the structure of anticancer therapeutic antibodies and the techniques used to reduce their antigenicity. Efficacy and toxic effects, conjugation with isotopes and toxins, and validation of the antibody targets also are discussed. Antibodies approved by the Food and Drug Administration are described in detail, as are antibodies in late and early stages of clinical development.

The modern era of targeted therapy for cancer was launched in 1975 with the discovery of monoclonal antibodies by Kohler and Milstein.1 Therapeutic antibodies have become a major strategy in clinical oncology owing to their ability to bind specifically to primary and metastatic cancer cells with high affinity and create antitumor effects by complement-mediated cytolysis and antibody-dependent, cell-mediated cytotoxicity (naked antibodies) or by the focused delivery of radiation or cellular toxins (conjugated antibodies).2-7 As of late 2002, there were 6 anticancer therapeutic antibodies approved for sale in the United States Table 1. A large number of additional antibodies targeting solid tumors and hematologic malignant neoplasms are in the early or late stage of clinical development Table 2.

Antibody Structure
Therapeutic monoclonal antibodies are typically of the IgG class containing 2 heavy and 2 light chains Figure 1. The heavy chains form a fused Y structure with 2 light chains running parallel to the open portion of the heavy chain. The tips of the heavy-light chain pairs form the antigen-binding sites with the primary antigen recognition regions known as the complementarity-determining regions (CDRs). The early promise of mouse monoclonal antibodies for the treatment of human cancers was not realized owing to a combination of factors that included the following8: 1. Unfocused target selection that led to the identification of target antigens that were not critical for cancer cell survival and progression;
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Table 1 Approved Anticancer Antibodies

Drug Name FDA Approval Alemtuzumab May (Campath) 2001 Source (Partners)* Type Target CD52 Approved and Investigational Indications Chronic lymphocytic and chronic myelogenous leukemia; multiple sclerosis, chronic progressive

Daclizumab (Zenapax)

March 2002

BTG, West Conshohocken, Monoclonal antibody, PA (ILEX Oncology, humanized; anticancer, Montville, NJ; Schering immunologic; multiple AG, Berlin, Germany) sclerosis treatment; immunosuppressant Protein Design Labs, Monoclonal IgG1 chimeric; Fremont, CA (Hoffmannimmunosuppressant; La Roche, Nutley, NJ) antipsoriatic; antidiabetic; ophthalmologic; multiple sclerosis treatment

Rituximab (Rituxan)

November 1997

Trastuzumab (Herceptin) Gemtuzumab (Mylotarg) Ibritumomab (Zevalin) Edrecolomab (Panorex)

IDEC Pharmaceuticals, San Monoclonal IgG1 chimeric; Diego, CA (Genentech, anticancer, immunologic; South San Francisco, CA; antiarthritic, immunologic; Hoffmann-La Roche; Zen- immunosuppressant yaku Kogyo, Tokyo, Japan) September Genentech (Hoffmann-La Monoclonal IgG1 humanized; p185neu 1998 Roche; ImmunoGen, anticancer, immunologic Cambridge, MA) May Wyeth/AHP Collegeville, PA Monoclonal IgG4 humanized CD33/cali, 2000 cheamicin February IDEC Pharmaceuticals Monoclonal IgG1 murine; CD20/ 2002 anticancer yttrium 90 GlaxoSmithKline, London, England Monoclonal IgG2A murine; anticancer

IL-2 receptor, Transplant rejection, general and bone CD25 marrow; uveitis; multiple sclerosis, relapsing-remitting and chronic progressive; cancer, leukemia, general; psoriasis; diabetes mellitus, type 1; asthma; ulcerative colitis CD20 Non-Hodgkin lymphoma, B-cell lymphoma, chronic lymphocytic leukemia; rheumatoid arthritis; thrombocytopenic purpura Cancer: breast, nonsmall cell of the lung, pancreas

January 1995

Acute myelogenous leukemia (patients older than 60 y) Low-grade lymphoma, follicular lymphoma, transformed non-Hodgkin lymphoma (relapsed or refractory) Epithelial cell Cancer: colorectal adhesion molecule

FDA, US Food and Drug Administration. * Partners are companies that participated in drug development or that distribute the drug. Approved in Germany; not approved by the FDA.

2. Low overall potency of naked mouse antibodies as anticancer drugs; 3. Poor tumor cell penetration of antibodies; 4. Limited success in producing radioisotope and toxin conjugates; and 5. The development of human antimouse antibodies (HAMAs) preventing the use of multiple dosing schedules.

Chimeric, Humanized, and Deimmunized Antibodies

The next advance in antibody therapeutics began in the early 1980s when recombinant DNA technology was applied to antibody design to reduce the antigenicity of murine and other rodent-derived monoclonal antibodies (Figure 1). By this approach, antigenicity could be reduced to permit multiple dosing, affinity could be maintained, the half-life of injected doses could be optimized, and host immunologic effector function could be accessed. Chimeric antibodies were developed in which the constant domains of the human IgG molecule were combined with the murine variable regions by transgenic fusion of the immunoglobulin genes; the chimeric monoclonal antibodies were produced from
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engineered hybridomas and Chinese hamster ovary (CHO) cells.9,10 The use of chimeric antibodies substantially reduced the HAMA responses but did not eliminate them. 11,12 Although several chimeric antibodies achieved regulatory approval, certain targets required fully humanized antibodies to achieve appropriate dosing. Partially humanized antibodies then were developed in which the 6 CDRs of the heavy and light chains and a limited number of structural amino acids of the murine monoclonal antibody were grafted by recombinant technology to the CDR-depleted human IgG scaffold.13 Although this process further reduced or eliminated the HAMA responses, in many cases, substantial further antibody design procedures were needed to reestablish the required specificity and affinity of the original murine antibody.14,15 A second approach to reducing the immunogenicity of monoclonal antibodies has been to replace immunogenic epitopes in the murine variable domains with benign amino acid sequences, resulting in a deimmunized variable domain. The deimmunized variable domains are linked genetically to human IgG constant domains to yield a deimmunized antibody (Biovation, Aberdeen, Scotland). In addition, primatized antibodies subsequently were developed that featured a chimeric antibody structure of human and monkey that, as a near exact copy of a human antibody, further reduced immunogenicity
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Table 2 Selected Anticancer Antibodies in Clinical Trials

Clinical Trial Status/Drug Name
Phase 3 Tositumomab (Bexxar) CeaVac Epratuzumab (LymphoCide) Mitumomab Bevacizumab (Avastin) Cetuximab (C-225; Erbitux) Edrecolomab Lintuzumab (Zamyl) MDX-210 IGN-101 Phase 2 MDX-010 MAb, AME ABX-EGF EMD 72 000 Apolizumab Labetuzumab ior-t1 MDX-220 MRA H-11 scFv Oregovomab huJ591 MAb, BZL Visilizumab TriGem TriAb R3 MT-201 G-250, unconjugated ACA-125 Onyvax-105 Phase 1 CDP-860 BrevaRex MAb AR54 IMC-1C11 GlioMAb-H ING-1 Anti-LCG MAbs MT-103 KSB-303 Therex KW-2871 Anti-HMI.24 Anti-PTHrP 2C4 antibody SGN-30 TRAIL-RI MAb, CAT Prostate cancer antibody H22xKi-4 ABX-MA1 Imuteran Clinical trial Monopharm-C

Source
Corixa, Seattle, WA Titan Pharmaceuticals, South San Francisco, CA Immunomedics, Morris Plains, NJ ImClone Systems, New York, NY Genentech, South San Francisco, CA ImClone Systems Johnson & Johnson, New Brunswick, NJ Protein Design Labs, Fremont, CA Medarex, Princeton, NJ; ImmunoDesigned Molecules, Havana, Cuba Igeneon, Vienna, Austria Medarex Applied Molecular Evolution, San Diego, CA Abgenix, Fremont, CA Merck KGaA, Darmstadt, Germany Protein Design Labs Immunomedics Center of Molecular Immunology, Havana, Cuba Immuno-Designed Molecules Chugai Pharmaceutical, Tokyo, Japan Viventia Biotech, Toronto, Canada AltaRex, Waltham, MA Millennium Pharmaceuticals, Cambridge, MA; BZL Biologics, Framingham, MA Protein Design Labs Titan Pharmaceuticals Titan Pharmaceuticals Center of Molecular Immunology Micromet, Munich, Germany Johnson & Johnson CellControl Biomedical, Martinsried, Germany Onyvax, London, England Celltech, Slough, England AltaRex AltaRex ImClone Systems Viventia Biotech Xoma, Berkeley, CA eXegenics, Dallas, TX Micromet KS Biomedix, Guildford, England Antisoma, London, England Kyowa Hakko, Tokyo, Japan Chugai Chugai Genentech Seattle Genetics, Seattle, WA Cambridge Antibody Technology, Cambridge, England Biovation, Aberdeen, Scotland Medarex Abgenix Nonindustrial source Viventia Biotech

Features
Anti-CD20 murine monoclonal antibody with iodine 131 conjugation Anti-CEA murine monoclonal antibody; anticancer immunologic vaccine Chimeric monoclonal antibody; anticancer immunologic; immunosuppressant Murine monoclonal antibody; anticancer immunologic Anti-VEGF humanized monoclonal antibody; anticancer immunologic; antidiabetic; ophthalmologic Anti-EGFR chimeric monoclonal antibody; anticancer immunologic Murine monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Bispecific chimeric monoclonal antibody; antiHER-2/neuanti-Fc gamma RI; anticancer immunologic Murine monoclonal antibody; anticancer immunologic Humanized anti-HER-2 monoclonal antibody; anticancer immunologic; immunostimulant Chimeric monoclonal antibody; anticancer immunologic; imaging agent; antiarthritic immunologic; ophthalmologic; cardiovascular Monoclonal antibody, human; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; immunoconjugate; anticancer immunologic Murine monoclonal antibody; anticancer immunologic; antipsoriatic; antiarthritic immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; antiarthritic immunologic; anticancer immunologic; GI inflammatory and bowel disorders Humanized monoclonal antibody; anticancer immunologic Monoclonal antibody, murine; anticancer immunologic; immunoconjugate Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; immunosuppressant; anticancer immunologic; GI inflammatory and bowel disorders Murine monoclonal antibody; anticancer immunologic Murine monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic; imaging agent; immunoconjugate Humanized monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer immunologic Humanized monoclonal antibody; anticancer immunologic; cardiovascular Murine monoclonal antibody; anticancer immunologic Murine monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Humanized monoclonal antibody; imaging agent; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer; imaging agent Murine monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic Chimeric monoclonal antibody; anticancer immunologic; osteoporosis Chimeric monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer immunologic; multiple sclerosis treatment; immunosuppressant; immunoconjugate Humanized monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer Chimeric monoclonal antibody; anticancer immunologic Humanized monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer immunologic Monoclonal antibody; anticancer immunologic; imaging agent

CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; GI, gastrointestinal; VEGF, vascular endothelial growth factor.

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Investigational Indications
Non-Hodgkin lymphoma Cancer: colorectal, nonsmall cell of the lung, breast, liver Non-Hodgkin lymphoma Small cell cancer of the lung; melanoma Cancer: colorectal, breast, nonsmall cell of the lung; diabetic retinopathy Cancer: head and neck, nonsmall cell of the lung, colorectal, breast, pancreas, prostate Cancer: colorectal and breast Acute myelogenous leukemia; myelodysplastic syndrome Cancer: ovarian, prostate, colorectal, renal, breast Cancer: nonsmall cell of the lung, liver, colorectal, esophageal, stomach Cancer: prostate, melanoma; infection, general Cancer: sarcoma, colorectal; rheumatoid arthritis; psoriatic arthritis Cancer: renal, nonsmall cell of the lung, colorectal, prostate Cancer: stomach, cervical, nonsmall cell of the lung, head and neck, ovarian Non-Hodgkin lymphoma; chronic lymphocytic leukemia Cancer: colorectal, breast, small cell of the lung, ovarian, pancreas, thyroid, liver T-cell lymphoma; psoriasis; rheumatoid arthritis Cancer: prostate, colorectal Rheumatoid arthritis; cancer, myeloma; Crohn disease; Castleman disease Non-Hodgkin lymphoma, melanoma Cancer: ovarian Cancer: prostate and general Transplant rejection, bone marrow; cancer, T-cell lymphoma; ulcerative colitis; myelodysplastic syndrome; systemic lupus erythematosus Cancer: melanoma, small cell of the lung, brain Cancer: breast, nonsmall cell of the lung, colorectal Cancer: head and neck; diagnosis of cancer Cancer: prostate, colorectal, stomach, nonsmall cell of the lung Cancer: renal Cancer: ovarian Cancer: colorectal; sarcoma, general Cancer: general; restenosis Cancer: myeloma, breast Cancer: ovarian Cancer: colorectal Diagnosis of cancer; cancer, brain Cancer: breast, lung (general), ovarian, prostate Cancer: lung, general; diagnosis of cancer B-cell lymphoma, non-Hodgkin lymphoma, chronic myelogenous leukemia, acute myelogenous leukemia Diagnosis of cancer; cancer, colorectal Cancer: breast Melanoma Myeloma Hypercalcemia of malignancy; cancer, bone Cancer: breast Hodgkin lymphoma Cancer: general Cancer: prostate Hodgkin lymphoma Melanoma Cancer: breast, ovarian Cancer: colorectal; diagnosis of cancer

and enabled the capability for continuous repeated dosing and long-term therapy.16 Finally, fully human antibodies have been developed using murine sources and transgenic techniques.16

Efficacy and Toxic Effects

By using modern antibody design and deimmunization technologies, scientists and clinicians have strived to improve the efficacy and reduce the toxic effects of anticancer antibody therapeutics.2,8,17-19 The bacteriophage antibody design system has facilitated the development of highaffinity antibodies by increasing antigen-binding rates and reducing corresponding detachment rates.19 In the bacteriophage antibody design technique, genes coding for the variable regions of antibody molecules isolated from normal human B cells are engineered genetically into the DNA of a bacteriophage. The phage particles then specifically encode the synthesis of specific antibody fragments by the infected bacteria, which ultimately are displayed on the surface of the phages forming the phage display library. By using affinity purification, the phage particles are recovered from the column and cloned to produce the antibody fragments. This method has been used extensively to increase the production of antibodies to the increasing numbers of potential tumor antigen targets recently discovered by modern genomic and proteomic techniques. Increased antigen binding also is achieved in bivalent antibodies with multiple attachment sites, a feature known as avidity. Modern antibody design has strived to create small antibodies that can penetrate to cancerous sites but maintain their affinity and avidity. A variety of approaches have been used to increase antibody efficacy.2 Recent clinical trials combining anticancer antibodies with conventional cytotoxic drugs have yielded promising results.2-5 The application of radioisotope, small molecule cytotoxic drug, and protein toxin conjugation has resulted in promising results in clinical trials and achieved regulatory approval for several drugs now on the market (see Conjugated Antibody Therapeutics). Antibodies also have been designed to increase their enhancement of effector functions of antibody-dependent cellular cytotoxicity. Another cause of the toxic effects of conjugated antibodies has been the limitations of the conjugation technology, which can restrict the toxin molecule/antibody molecule ratio.2,3,5 Methods designed to overcome the toxic effects of conjugated antibodies include the use of antibody-targeted liposomal small molecule drug conjugates and the use of antibody conjugates with drugs in nanoparticle formats, to enhance bonding strength, that enable controlled release of the cytotoxic agent. Another technique that uses site-selective prodrug activation to reduce toxic effects on bystander tissue is the antibody-directed enzyme prodrug therapy in which an
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antibody-bound enzyme is targeted to tumor cells, which permits selective activation of a nontoxic prodrug to a cytotoxic agent at the tumor site for cancer therapy. A variety of factors can reduce antibody efficacy.8 These include the following: (1) limited penetration of the antibody into a large solid tumor or into vital regions such as the brain; (2) reduced extravasation of antibodies into target sites owing to decreased vascular permeability; (3) cross-reactivity and nonspecific binding of antibody to normal tissues, reducing the targeting effect; (4) heterogeneous tumor uptake resulting in untreated zones; (5) increased metabolism of injected antibodies, reducing therapeutic effects; and (6) rapid formation of HAMA and human antihuman antibodies, inactivating the therapeutic antibody. Toxic effects have been a major obstacle in the development of therapeutic antibodies for cancer.2-5 Cross-reactivity with healthy tissues can cause substantial side effects for unconjugated (naked) antibodies, which can be enhanced when the antibodies are conjugated with toxins or radioisotopes. Immune-mediated complications include dyspnea from pulmonary toxic effects, occasional central and peripheral nervous system complications, and decreased liver and renal function. On occasion, unexpected toxic complications can be seen, such as the cardiotoxic effects associated with the HER-2/neu targeting antibody trastuzumab (Herceptin) (see Trastuzumab). Radioimmunotherapy with isotopicconjugated antibodies also can cause bone marrow suppression (see Conjugated Antibody Therapeutics).

Figure 1 Types of monoclonal antibodies. Left, A fully humanized monoclonal antibody with the antigen binding murine complementarity-determining regions (black) interspersed within the variable regions of the light (light gray) and heavy (dark gray) chains of the Fab portion of the engineered antibody. Right, A chimeric antibody in which the antigen-binding murine component (black) of the variable region of the Fab section is maintained integrally.

Unconjugated Antibodies
Unconjugated or naked antibodies include a variety of targeting molecules on the market and in early and late clinical development (Tables 1 and 2). A variety of mechanisms have been cited to explain the therapeutic benefit of these drugs, including enhanced immune-effector functions and direct inactivation of the targeted pathways as seen in the antibodies directed at surface receptors such as HER-1 (epidermal growth factor receptor [EGFR]) and HER-2.2-5 Surface receptor targeting can reduce intracellular signaling, resulting in decreased cell growth and increased apoptosis.16

variety of methods to complex the isotope, toxin, or cytotoxic agent to the antibody.2,3 Cytotoxic small molecule drug conjugates have been tested widely, but enthusiasm for this approach has been limited by the relatively low potency of these compounds.2 Fungal-derived potent toxins have yielded greater success with the calicheamicin-conjugated anti-CD33 antibody gemtuzumab ozogamicin approved for the treatment of acute myelogenous leukemia and a variety of antibodies conjugated with the fungal toxin maytansinoid (DM-1) in preclinical development and early clinical trials (see Antibodies Designed for Hematologic Malignant Neoplasms in Early-Stage Clinical Trials and Selected Antibodies for Solid Tumors in Early-Stage Clinical Trials). The interest in radioimmunotherapy increased substantially in 2001 with approval by the US Food and Drug Administration (FDA) of the 90Y-conjugated anti-CD20 antibody ibritumomab tiuxetan (see Ibritumomab Tiuxetan). A variety of isotopes are under investigation in addition to 90Y as potential conjugates for anticancer antibodies.3 Radioimmunotherapy features the phenomenon of the bystander effect, ie, if antigen expression is heterogeneous, extensive tumor cell killing can still take place, even on nonexpressing cells, but also can lead to substantial toxic effects when the neighboring cells are vital nonneoplastic tissues, such as the bone marrow and liver.

Conjugated Antibody Therapeutics

As shown in Table 1, of the 7 anticancer antibodies on the market, 1 (ibritumomab tiuxetan [Zevalin]) is conjugated to a radioisotope (yttrium 90 [90Y]), and 1 (gemtuzumab ozogamicin [Mylotarg]) is conjugated to a complex natural product toxin. Conjugation procedures have been designed to improve the efficacy of antibody therapy and have used a
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Target Validation
The ideal cancer target Image 1 can be defined as a macromolecule (1) that is crucial to the malignant phenotype and is not expressed substantially in vital organs and tissues, (2) that can be measured reproducibly in readily obtained clinical samples, (3) that is correlated definably with clinical outcome, and (4) in which interruption, interference, or inhibition yields a
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Image 1 HER-2/neu testing in breast cancer. A, Immunohistochemical analysis (HercepTest, DAKO, Glostrup, Denmark) with continuous membranous 3+ positive immunostaining for HER-2/neu protein. B, HER-2/neu gene amplification detected by fluorescence in situ hybridization (Ventana Inform System, Ventana Medical Systems, Tucson, AZ). C, HER-2/neu gene amplification detected by chromogenic in situ hybridization (Zymed System, Zymed Laboratories, South San Francisco, CA).

clinical response in a substantial proportion of patients whose tumors express the target and a minimal to absent response in patients whose tumors do not express the target. For antibody therapeutics, additional important criteria include the use of cell surface targets that, when complexed with the therapeutic naked or conjugated antibody, internalize the antigen-antibody complex by reverse pinocytosis, thus facilitating tumor cell killing.

and higher affinity monoclonal antibodies with reduced immunogenicity after humanization or deimmunization and the emerging conjugation capabilities, antibody therapeutics have become a major weapon in the treatment of leukemias and lymphomas.20-23 Rituximab (Rituxan) Approved in 1997, rituximab is arguably the most commercially successful anticancer drug of any type since the introduction of the taxanes. Rituximab sales exceeded $700 million in the United States in 2001.4 By targeting the CD20 surface receptor common to many B-cell nonHodgkin lymphoma (NHL) subtypes, rituximab, a chimeric monoclonal IgG1 antibody, induces apoptosis, antibodydependent cell cytotoxicity, and complement-mediated cytotoxicity.16 Rituximab originally was used in aggressive recurrent or refractory NHL in combination with standard of
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Antibody Therapeutics for Hematologic Malignant Neoplasms

The earliest and most successful clinical use of antibodies in oncology has been for the treatment of hematologic malignant neoplasms.2-5,16,20-23 By taking advantage of improved recombinant technologies generating more specific
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care chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone) and achieved significantly improved disease-free survival rates compared with those for patients receiving cytotoxic agents alone.24-27 In the 5 years after its approval, numerous clinical studies were performed that were designed to expand on the original NHL indication for rituximab. These studies investigated the use of rituximab in more poorly differentiated aggressive lymphomas and lymphoid leukemias, in combination with other anticancer antibodies and with existing and novel small molecule cytotoxic agents, antichemoresistance gene therapies, and biologic response modifiers.25 More recently, rituximab has been tried with apparent modest success in nonmalignant conditions, including inflammatory bowel disease and rheumatoid arthritis.23-27 It is too early to tell whether rituximab will be approved for treatment of these conditions. Ibritumomab Tiuxetan Ibritumomab tiuxetan consists of the murine version of the anti-CD20 chimeric monoclonal antibody, rituximab, that has been linked covalently to the metal chelator, MD-diethylenetriamine pentaacetic acid (or DTPA), permitting stable binding of indium 111 when used for radionuclide tumor imaging and 90Y when used to produce enhanced targeted cytotoxity.28-30 In early 2002, ibritumomab tiuxetan became the first radioconjugated antibody therapeutic for cancer approved by the FDA. The 90Y isotope in ibritumomab tiuxetan is a pure beta-emitter, and the requisite safety precautions for health care workers, patients, and their families are not overly burdensome.30 Ibritumomab tiuxetan is dosed on the basis of the patients body weight and baseline platelet count and was approved for virtually the identical indications as rituximab.28-30 The toxic effects of ibritumomab tiuxetan are primarily hematologic and have been found to be transient and reversible. 28 In a randomized phase 3 trial comparing ibritumomab tiuxetan with a standard dose of rituximab in patients with relapsed indolent or follicular transformed NHL, the overall response rate was 80% in the ibritumomab arm compared with 56% in the rituximab arm (P = .002), with complete remission rates of 30% and 16%, respectively (P = .04).28 Since its FDA approval, numerous patients who have received ibritumomab tiuxetan after their disease became refractory to a rituximab-based regimen have achieved clinically significant responses.29,30 Medical oncologists likely will drive the use of ibritumomab tiuxetan, referring patients to nuclear medicine and radiation oncology departments for treatment of CD20+ B-cell lymphoid malignant neoplasms.31 Gemtuzumab Ozogamicin The approval of gemtuzumab ozogamicin by the FDA in 2000 marked the introduction of the first plant toxinconjugated
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antibody therapeutic. 32-36 Gemtuzumab ozogamicin is targeted against CD33, a surface marker expressed by 90% of myeloid leukemic blasts but absent from stem cells, and is armed with calicheamicin, a potent cytotoxic antibiotic that inhibits DNA synthesis and induces apoptosis. 32 In combined phase 2 studies of 142 patients with CD33+ acute myeloid leukemia in first relapse, gemtuzumab ozogamicin monotherapy was associated with a 30% overall response rate. In another study, the overall remission rate was 28%, with complete remission in 13% of patients, complete remission with incomplete platelet recovery in 15%, and a median survival of 14.5 months for patients achieving complete remission.36 Major side effects typical of conventional cytotoxic chemotherapy, including myelosuppression, hepatotoxic effects, severe mucositis, and infections, are comparably low in gemtuzumab ozogamicintreated patients.34 In patients with leukocyte counts of more than 30,000/L (30 109/L), treatment with gemtuzumab ozogamicin has been associated with tumor lysis and adult respiratory distress syndrome.33 The current indication for use of gemtuzumab ozogamicin is for patients with acute myelogenous leukemia (AML) who are older than 60 years, with the recommendation that, before the initiation of therapy, the leukemic blast count be below 30,000/L.33-35 Alemtuzumab (Campath) for Injection Alemtuzumab, a humanized monoclonal antibody, was approved in mid-2001 for the treatment of B-cell chronic lymphocytic leukemia (CLL) in patients who have been treated with alkylating agents and in whom fludarabine therapy failed.37,38 Alemtuzumab targets CDw52, a small, anchored glycoprotein that is expressed highly on normal T and B lymphocytes and in a large proportion of lymphoid cell malignant neoplasms. 34 Alemtuzumab causes complement and antibody-dependent, cell-mediated cytotox-icity.37,38 In previously untreated patients with Bcell CLL, the overall response rate was approximately 90% with a median response duration of 9 to 12 months, whereas in previously treated patients with B-cell CLL, response rates were on the order of 40%, with a 2% to 4% complete remission rate.38 Clinical research studies also have shown that alemtuzumab is active against T-cell prolymphocytic leukemia and low-grade NHL and may prevent graft-vs-host disease associated with bone marrow transplantation.38 The side effects of alemtuzumab include fever, rigor, nausea, vomiting, and hypotension, believed to be the results of tumor necrosis factor alpha and interleukin (IL)-6 releases.38 These complications usually were less severe with subsequent infusions, responded to treatment with corticosteroids, and were prevented by administration of acetaminophen and antihistamines. Immunosuppression resulting from normal B- and T-lymphocyte
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depletion was frequent, resulting in an increased risk for opportunistic infections.38 Daclizumab (Zenapax) Daclizumab is a chimeric monoclonal antibody that targets the IL-2 receptor. This antibody is used primarily to prevent and treat organ transplant rejection, but it also has been used in a wide variety of chronic inflammatory conditions, including psoriasis, multiple sclerosis, ulcerative colitis, asthma, type 1 diabetes mellitus, and uveitis and in a variety of leukemias.39 The IL-2 Tac receptor is expressed in adult T-cell leukemia and hairy cell leukemia, and studies indicate that this target can be inactivated by daclizumab treatment.40 Large clinical studies in NHL and lymphoid leukemias combining this antibody with chemotherapy and other antibodies have not been performed. Selected Antibodies Designed for Hematologic Malignant Neoplasms in Advanced-Stage Clinical Trials Epratuzumab (LymphoCide) Epratuzumab is a chimeric monoclonal antibody that binds to the CD22 antigen and is in phase 3 clinical trials for the treatment of NHL. This novel therapeutic agent also is being evaluated for combination therapy for lymphoma.41 Lintuzumab (Zamyl) This humanized antibody targets the CD33 receptor on myeloid blasts and is in phase 3 clinical trials for the treatment of AML and myelodysplastic syndromes.42 Iodine 131 Tositumomab (Bexxar) Iodine 131 (131I)-tositumomab is a radiolabeled antiCD20 murine monoclonal antibody in phase 3 clinical trials for the treatment of relapsed and refractory follicular or lowgrade and transformed NHL.43 Treatment with 131I-tositumomab in clinical trials has produced high response rates and durable complete remissions in patients who have received previous chemotherapy or rituximab. Complications related to treatment with 131I-tositumomab include myelosuppression, secondary acute leukemia, myelodysplasia, and hypothyroidism.43 The FDA approval of 131I-tositumomab is pending over safety issues associated with the relatively long half-life of the 131I isotope.44 Antibodies Designed for Hematologic Malignant Neoplasms in Early-Stage Clinical Trials A substantial number of monoclonal antibodies designed to treat hematologic malignant neoplasms are in early-stage clinical trials (Table 2). Popular targets for these antileukemia and antilymphoma drugs include CD20, CD22, CD33, HLA-DR, and ferritin.3-5,16,21-22,43
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Antibody Therapeutics for Solid Tumors

Interest in the development of antibody therapeutics for solid tumors among many commercial organizations and universities has been influenced substantially by the technologic advances in antibody engineering and the approval and recent clinical and commercial success of trastuzumab, the only therapeutic antibody approved by the FDA for the treatment of solid tumors (edrecolomab [Panorex] is approved in Germany, but not in the United States). Trastuzumab The HER-2/neu growth factor receptor is a member of a 4-member receptor tyrosine kinase signaling family that includes EGFR. In 1987, Slamon and coworkers45 first described the association of an amplification of the HER2/neu gene with an unfavorable outcome in breast cancer. During the next 12 years, more than 100 major studies involving more than 16,000 patients confirmed the adverse prognostic significance of HER-2/neu gene amplification and the overexpression of the p183HER-2 protein.46 By using recombinant technologies, trastuzumab, a monoclonal IgG1 class humanized murine antibody, was developed by Genentech (South San Francisco, CA) and targeted specifically for patients with advanced relapsed breast cancer that overexpressed the HER-2/neu protein.47 In a clinical trial in which an immunohistochemical assay was used to select patients for the phase 3 pivot trial, the addition of trastuzumab to chemotherapy (either anthracycline plus cyclophosphamide or paclitaxel) was associated with a longer time to disease progression (median, 7.4 vs 4.6 months; P < .001), a higher rate of objective response (50% vs 32%; P < .001), a longer duration of response (median, 9.1 vs 6.1 months; P < .001), a lower rate of death at 1 year (22% vs 33%; P = .008), longer survival (median, 25.1 vs 20.3 months; P = .01), and a 20% reduction in the risk of death.48 Trastuzumab has been combined with multiple cytotoxic drugs, forming new strategies for the treatment of metastatic breast cancer.49 Class III or IV cardiac dysfunction occurred in 27% of the group treated with anthracycline and cyclophosphamide plus trastuzumab compared with 8% of the group given an anthracycline and cyclophosphamide alone.48 Cardiac toxic effects have remained a significant limiting factor for the use of trastuzumab since its FDA approval in late 1998.50 The best method to identify patients for trastuzumab therapy has been a source of controversy. The original immunohistochemical technique used as the clinical trial assay was succeeded by the commercial HercepTest (DAKO, Glostrup, Denmark). This assay originally was criticized for yielding false-positive results,51 although better performance ultimately was achieved when the test was performed exactly according to the manufacturers instructions. Concern over
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immunohistochemical accuracy using standard formalinfixed, paraffin-embedded tissue sections52 has encouraged the evaluation of the fluorescence in situ hybridization (FISH) assay53 for its ability to predict trastuzumab response rates. Reports that FISH could outperform immunohistochemical analysis in predicting trastuzumab response54 and well-documented lower response rates of 2+ immunohistochemical staining vs 3+ staining tumors55 resulted in an approach that uses immunohistochemical analysis as a primary screen with FISH testing of all 2+ cases or primary FISH-based testing. In a recently published study in which trastuzumab was used as a single agent, the response rate in 111 assessable patients with 3+ immunohistochemical staining was +35%, and the response rate for 2+ cases was 0%; the response rates in patients with and without HER2/neu gene amplification detected by FISH were 34% and 7%, respectively.55 The FISH approach has been approved by the FDA for inclusion in the trastuzumab package insert. More recently, the chromogenic in situ hybridization technique (Image 1) has been introduced; it combines the best aspects of immunohistochemical analysis and FISH and shows potential to become the major approach to HER-2/neu testing.56 The FDA approval of trastuzumab, the integrated use of an on-slide diagnostic test guiding patient selection that has become part of the standard of care for evaluating patients with breast cancer,57 and the demonstration of efficacy as a single agent and in combination with a variety of chemotherapeutic agents have opened a new era of development of antibody therapeutics directed against solid tumors. At present, a wide variety of strategies and clinical trials are underway to expand the use of trastuzumab, including adjuvant and neoadjuvant trials and novel combination trials for advanced breast cancer, as well as attempts to achieve responses in HER-2/neu overexpressing nonbreast cancers.49,58,59 Selected Antibodies for Solid Tumors in Late-Stage Clinical Trials During the 4 years after the FDA approval of trastuzumab, there were no additional antibodies approved for the treatment of solid tumors. Nevertheless, substantial progress has been made in this field, and a number of latestage and early-stage products show substantial promise. Cetuximab (Erbitux) The EGFR (HER-1) is the target of a variety of small molecule drugs and the antibody cetuximab, which is in latestage clinical trials.60 Cetuximab, a chimeric monoclonal antibody, binds to the EGFR with high affinity, blocking growth factor binding, receptor activation, and subsequent signal-transduction events leading to cell proliferation.61
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Cetuximab enhanced the antitumor effects of chemotherapy and radiotherapy in preclinical models by inhibiting cell proliferation, angiogenesis, and metastasis and by promoting apoptosis.61 Cetuximab has been evaluated alone and in combination with radiotherapy and various cytotoxic chemotherapeutic agents in a series of phase 2 and 3 studies that primarily treated patients with head and neck or colorectal cancer. 61,62 Breast cancer trials also are underway.63 The FDA approval process for cetuximab has been slowed owing to concerns about clinical trial design and outcome data management.64 In addition, FDA approval of oxaliplatin for the treatment of metastatic colorectal cancer that is refractory to standard 5-fluorouracil and irinotecan treatment may have an impact on the regulatory approval of cetuximab by requiring that the antibody add additional benefit to oxaliplatin-based regimens. Nevertheless, it is anticipated that, based on promising observations in the phase 3 trials, this antibody ultimately will achieve FDA approval. Bevacizumab (Avastin) Bevacizumab (rhuMAb-VEGF [vascular endothelial growth factor]), a humanized murine monoclonal antibody targeting the VEGF ligand, is in phase 3 clinical trials for colorectal and breast cancer. VEGF regulates vascular proliferation and permeability and functions as an antiapoptotic factor for newly formed blood vessels.65-67 Patients treated with bevacizumab alone have shown significant tumor responses. Patients treated with bevacizumab in combination with conventional chemotherapy have had greater responses.65-67 The phase 2 study evaluating bevacizumab in metastatic renal cell carcinoma reached its prespecified efficacy end point earlier than expected. Bevacizumab in combination with capecitabine (Xeloda), a form of chemotherapy, received fast-track approval status from the FDA for the treatment of metastatic breast cancer. Bevacizumab has been combined with trastuzumab in a 2-antibody therapeutic strategy for HER-2/neu overexpressing breast cancer.68 Finally, late-stage clinical trials using bevacizumab with 5fluorouracil, leucovorin, and irinotecan in advanced colorectal cancer are underway.69 Edrecolomab Edrecolomab is a murine IgG2A monoclonal antibody that targets the human tumor-associated antigen epithelial cell adhesion molecule (17-1A). Edrecolomab has been approved in Germany since 1995, but to date has not been approved by the FDA. In a study of 189 patients with resected stage III colorectal cancer, treatment with edrecolomab resulted in a 32% increase in overall survival compared with no treatment (P < .01).70 Edrecolomabs antitumor effects are mediated through antibody-dependent
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cellular cytotoxicity, complement-mediated cytolysis, and the induction of an anti-idiotypic network.71 Edrecolomab also is being tested in large multicenter adjuvant phase 3 studies in stage II and stage III rectal cancer and stage II colon cancer. Edrecolomab was well tolerated when used as monotherapy and added little to chemotherapy-related side effects when used in combination. Sequential treatment of patients with metastatic breast cancer with edrecolomab, after adjuvant chemotherapy, reduced the levels of disseminated tumor cells in the bone marrow and eliminated epithelial cell adhesion moleculepositive micrometastases.72 CeaVac Active specific immunotherapy of patients with advanced colorectal cancer with the anti-idiotypic antibody CeaVac has entered phase 3 testing.73-75 CeaVac is designed to stimulate the immune system to create specialized antibodies that mimic specific antigens found on the surface of cancer cells and act as a trigger for the immune system to recognize and destroy targeted cancer cells. In clinical testing, CeaVac was the first monoclonal antibody to consistently break immune tolerance to carcinoembryonic antigen (CEA) in patients with cancer, and it generated high levels of antibodies and helper T cells to CEA. In a study of 32 patients with colorectal cancer, CeaVac generated a potent immune response in all patients, with 19 showing no evidence of disease. In addition, 7 of 8 patients with advanced disease exhibited no disease recurrence for 1 to 3 years.73

MDX-210 MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for IgG (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 is designed to direct Fc gamma RIpositive effector cells, such as monocytes and macrophages, to phagocytose or kill tumor cells that overexpress HER-2/neu.76 MDX-210 was immunologically active at well-tolerated doses.76,77 MDX-210 has advanced to phase 3 testing for the treatment of HER-2/neu overexpressing ovarian cancer and continues in phase 2 trials for the treatment of renal cell, breast, colorectal, and prostatic carcinomas. Selected Antibodies for Solid Tumors in Early-Stage Clinical Trials More than 70 unconjugated and conjugated monoclonal antibodies are in early-phase clinical trials for the treatment of solid tumors (Table 2). huJ591 (Anti-PSMAEXT) Prostate-specific membrane antigen (PSMA) is a membrane-bound glycoprotein highly restricted to normal prostatic epithelial cells. PSMA expression increases dramatically in primary and metastatic prostate cancer Image 2.78 PSMA expression is increased further in association with clinically advanced prostatic cancer, particularly in hormonerefractory disease, and is an ideal sentinel molecule for use in targeting prostatic cancer cells. In addition, significant PSMA expression has been identified in the tumor vasculature of a variety of nonprostate cancers Image 3.79 Humanized and

Image 2 Validation of an antibody target: prostate-specific membrane antigen (PSMA). A, Localization of the PSMA target by immunohistochemical analysis in a case of metastatic poorly differentiated prostate cancer (7E11 antibody; peroxidase antiperoxidase). B, Indium 111huJ591EXT diagnostic immunoscintiscan of a patient with prostate cancer metastatic to the left cervical lymph nodes. In the phase 1 clinical trial of an yttrium 90conjugated anti-PSMA monoclonal antibody (huJ591EXT), the patients were scanned with a diagnostic indium 111conjugate to localize the disease before treatment.

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fully human antibodies specific for the extracellular domain of PSMA have been developed. A phase 1 clinical trial of one of these antibodies, huJ591 conjugated with 90Y, has yielded promising results.80 Programs using toxin conjugates with anti-PSMA antibodies are in preclinical development.81 Finally, antibodies to PSMA have been used as diagnostic imaging agents (Image 2), including the commercially available ProstaScint (Cytogen, Princeton, NJ).82 G-250 The chimeric monoclonal antibody cG250 targets the G250 antigen, a transmembrane protein that is expressed on renal carcinoma cells.83,84 Preclinical and early clinical testing have shown promising results. TriGem TriGem is an anti-idiotype antibody that mimics the disialoganglioside GD2. This agent is being tested in the treatment of metastatic malignant melanoma.85 Other Agents A substantial number of pharmaceutical companies, biotechnology firms, and universities are developing monoclonal antibodies for the treatment of solid tumors (Table 2). A variety of epithelial surface antigens, including HER-2/neu, MUC1, TAG 72, CEA, CA-125, tenascin, and others, have become the preferred targets of these organizations.4 For some groups, antibodies attacking tumor angiogenesis are favored, whereas others have favored the development of additional EGFR-targeting antibodies such as EMD 72 00086 and ABXEGF.87 Sb-408075 is an immunoconjugate composed of the microtubule inhibitor DM-1 linked to the monoclonal antibody C242 directed against CanAg, an antigen found on tumor epithelium. Phase 1 testing of this compound in colon, pancreatic, and lung cancers evaluated the safety and pharmacokinetics of doses from 22 to 245 mg/m2.88 BB-10901 is an immunoconjugate composed of the microtubule inhibitor DM1 linked to the monoclonal antibody huN901 directed against CD56 found on small cell lung cancer and other neuroendocrine tumors. Phase 1 testing of this compound in small cell lung cancer and other neuroendocrine tumors evaluated the safety and pharmacokinetics of repeated doses ranging from 5 to 40 mg/m2.81

Image 3 Expression of prostate-specific membrane antigen (PSMA) in nonprostate cancer. Left, Endothelial cells are identified by red immunofluorescence (rhodamine conjugate) using an antibody to platelet endothelial adhesion molecule (PECAM). Center, PSMA expression is identified by green immunofluorescence (fluorescein isothiocyanate [FITC] conjugate) in the endothelium of tumor blood vessels of a neuroblastoma. Right, Dual immunofluorescent staining using FITC-conjugated anti-PSMA and rhodamine-conjugated anti-PECAM confirms the colocalization of PSMA and PECAM expression in the neuroblastoma tumor endothelium, as evidenced by the summation yellow.

Potential Future Roles for Pathologists and Clinical Laboratories in Anticancer Antibody Development and Clinical Use
Given the major roles of pathologists and clinical laboratories in the selection of patients for eligibility to receive trastuzumab for the treatment of metastatic breast
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cancer,46,48,54,55 there is considerable interest in the potential that similar activities will develop for other anticancer antibodies as they are approved and marketed. For hematologic malignant neoplasms, flow cytometry and molecular diagnostics laboratories commonly are used by hematopathologists to correctly classify and phenotype patients with NHL for eligibility to receive rituximab21-25 and ibritumomab tiuxetan,29,30 as well as patients with acute leukemia for eligibility to receive gemtuzumab ozogamicin.36 As new antiNHL and antileukemia antibodies are developed that target specific chromosomal translocations, issues will arise including whether to use cytogenetics or FISH as the detection technique. The emerging chromogenic in situ hybridization approach also will be evaluated to learn whether it can be used to detect subtle translocations in addition to gene amplifications such as for HER-2/neu. For solid tumors, the antibodies in development also may require increased efforts by pathologists and laboratories to select patients and monitor their responses to the treatments. Should the C-225 (cetuximab) antibody be approved by the FDA, quantitative immunohistochemical analysis may prove beneficial for the detection of EGFR expression to
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guide its clinical use.62,63 Interestingly, a gene amplification detection strategy is unlikely to be successful for EGFR because, in contrast with HER-2/neu, EGFR protein overexpression frequently occurs in the absence of gene amplification.60,61 Similarly, for angiogenesis inhibitors such as bevacizumab, it is unclear whether measurements of the VEGF receptors by immunohistochemical analysis or microvessel density determinations will be able to improve the current efficacy levels for the antibody now in late-stage clinical trials.66-70 Finally, it is not known whether even more detailed molecular testing will be needed for early-stage antibody development, including improving targeting by sequencing antibody binding sites and obtaining better pharmacodynamic assays to adjust dosing in phase 1 clinical trials such as immunohistochemically measured downstream biomarkers in the target pathway89; whether high-throughput genomic transcriptional profiling pharmacogenomic testing will impact antibody therapeutics90; or whether a proteomics approach in serum may prove to be the best method of predicting anticancer antibody efficacy and toxic effects.91 The types of tissues (eg, frozen vs paraffinembedded) that will be needed for clinical testing and how and where the tests will be done (eg, routinely performed in pathology departments or commonly referred to esoteric reference laboratories) also are unknown.

long-term future of this promising form of targeted therapy for cancer will be decided.
From 1Divisions of Molecular Medicine, Molecular Pathology, and Oncology Therapeutics, Millennium Pharmaceuticals, Cambridge, MA; and 2Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY. Address reprint requests to Dr Ross: Dept of Pathology and Laboratory Medicine, Albany Medical College, 47 New Scotland Ave, Albany, NY 12208.

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Conclusions
Despite the recent clinical and commercial success of the currently used anticancer antibodies, a substantial number of important questions remain concerning the future of this therapeutic strategy: (1) Can antibody engineering technology be advanced to improve pharmacodynamics and increase target affinity and antigen-antibody internalization? (2) Can humanization, deimmunization, and human antibody strategies be accelerated and improved to limit human antihuman antibody responses in treated patients? (3) Will radiolabeled antibodies be accepted in the clinical and commercial marketplace, given the inconvenience associated with their administration and requirements for nuclear medicine oversight? (4) Will plant and fungal toxins such as calicheamicin and maytansinoid substantially improve antibody efficacy and become major approaches to cancer therapy? (5) Will future combinations of multiple antibodies and small molecule drugs become capable of producing prolonged disease-free remissions in high-grade hematologic malignant neoplasms and aggressive solid tumors? (6) Can current international regulatory agencies respond to the large number of anticancer antibodies in clinical development and facilitate their evaluation, especially when they are combined with other agents? During the next several years, the answers to these questions will become known, and the
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