SURGICAL PHARMACOLOGY

Novel Antibody Drug Products Joseph T. DiPiro, PharmD,Augusta,Georgia,Robert G. Hamilton, PhD, Baltimore,Maryland, John P. Wei, MD, Augusta,Georgia

Recent developments in protein and genetic engineering methods have allowed the production of antibody-derived molecules that have important potential as therapeutic agents. Although monoclonal antibodies of murine origin have been used for therapeutic purposes, limitations due to anti-antibody responses and suboptimal effectiveness for some indications, such as tumor cell killing, have led to the development of human monoelonal antibodies, chimeric and complementarity determiningregion grafted antibodies, immunotoxins, and other engineered products. These novel antibodies are being tested for the treatment and prevention of infections diseases and for the diagnosis and treatment of cancers, as well as for indications considered nontraditional for antibodies (e.g., as antithrombotics or inhibitors of neutrophil adherence). The availability of antibody drug products raises a number of issues for clinicians. Among these are new patterns of adverse effects, immunogenicity (development of anti-antibody response), important questions regarding administration and dosage, and substantial cost implications.

ntibodies have been used as therapeutic agents since A Emil A. yon Behring's demonstration more than 100 years ago that serum from animals resistant to diphtheria toxin was protective when given to nonexposed animals. Since then, a range of antibody drugs have been developed, from crude human and animal antisera specific for snake or spider venom and infectious agents to purified polyclonal intravenous human immunoglobulin G (IgG) and monoclonal antibodies. At present, there are dozens of monoclonal antibody products being tested clinically for a variety of indications that have been developed by methods involving novel cell and molecular biology techniques. This review presents an overview of the methods of antibody production, proposed applications of antibodies and antibody-derived products, and a discussion of issues relevant to these agents as drug products.

ANTIBODY CONCEPTS: STRUCTURE AND FUNCTION The antibody molecule, as shown in Figure 1, is composed of two heavy and two light polypeptide chains that are linked by disulfide bonds. Three hypervariable or complementarity-determining regions (CDRs) in each of the variable regions of the heavy and light chains form the antigen-binding pocket that interacts specifically with antigen. The crystallizable fragment (Fc) contains sites that elicit biologic effector functions (see Figure 1, Table I) by interacting directly with complement components and cell surface receptors. Fab fragments (or the dimer F(ab)'2) of antibody that bind antigen but do not contain an Fc region can be useful for selected applications that do not require the invoking of effector functions. Most antibody-derived drugs are of the immunoglobulin G (IgG) or immunoglobulin M (IgM) class. IgG antibodies possess the longest biologic half-life in blood, bind maximally to phagocytic and placental cell rer~ptots, and activate complement (Clq, C4b). These functions allow antibody to bind to toxins, to enhance the opsonic function of phagocytes, to promote antibody-dependent cell-mediated cytotoxicity (ADCC), and to potentially regulate immune function through binding to the variable region or idiotype of other antibodies. It is the From The Universityof Georgia College of Pharmacy (JTD), Augusta, combination of the antibody's specificity and affinity for Georgia, Department of Surgery (JTD, JPW), Medical College of Georgia, Augusta, Georgia, and The Asthma and Allergy Center a particular antigen and its ability to clear antigen and (RGH), Johns Hopkins University School of Medicine, Baltimore, promote complement and phagocytic protective mechaMaryland. nisms of the immune system that confer great potential as Requests for reprints should be addressed to Joseph T. DiPiro, a drug. Table II summarizes the attributes and applicaPharmD, Clinical Pharmacy Program, 1120 15th Street, H-1087, Autions of antibody drugs that involve both Fab and Fc gnsta, Georgia 30912-2390. Manuscript submitted October 24, 1991, and accepted in revised interactions. A number of these are described in detail form March 24, 1992. below.

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DIPIRO El' AL

TABLE I Effector Functions of Human I m m u n o g l o b u l i n s

Property

IgM

IgD

IgG1

IgG2

IgG3

IgG4

IgA

IgE

Placental transfer Biologic half-life (days)l" Elicit immediate hypersensitivity Blocks immediate hypersensitivity Complement activation (bacterial lysis) Binding to phagocytic cells (ADCC)

-* 5 . +++ -

2-8

++ 23 . + ++ ++

+ 23

++ 7 . ++ ++

23

6

++ +/-

?

1-5 ++ -

.

. -

.

. + +

-

Ig = immunoglobulin;ADCC = antibody-dependentcell-mediatedcytotoxicRy. * ' - ' indicatesno to minimal activityor function; '+' to' + + +' indicatesrelativelygreateractivityor function. tit cannot be assumed that the haft-life in serum of in vitro generated monoclonal and human-mouse engineered antibodies will be the same as unprocessed immunoglobulin. Adapted from [1], with permission.

VL L Fab

/ V H ~ i

Fc / '-

Hvpervariable Regions "(Antigen binding)

~

C ~ I 2 ~ - S S F

0,3

I!!l

[il

!:~1

!"!

C~

binding

Membrane binding

Figure 1. A structural diagram o f the immunoglobulin G molecule. Fab is the antigen-binding fragment. Fc is the crystalline fragment. VH and VL are the variable regions. CH1-3 are the constant regions, and SS indicates disulfide bonds.

ANTIBODY DRUG PRODUCTION METHODS The ideal antibody drug should be highly specific (monospecific) and avid for its target antigen. Moreover, it should be nonreactive with irrelevant human tissues, cells, and plasma components so that it maximizes the target-to-nontarget ratio of localization [I ]. Ideally, its constant region domains should be of human origin so that the antibody induces a minimal immune response that may shorten biologic half-life. Its constant region domains should possess the class and subclass that maximize the antibody's diagnostic and/or therapeutic action, while minimizing undesirable side effects. Such an antibody should be produced with no aggregates, contaminating proteins, or viral DNA, bacteria, mycoplasma, virus, or endotoxin. Current antibody production methods have resulted from simultaneous developments in immunochemistry, cellular biology, and molecular biology. Immunoehemistry: Early attempts to prepare large amounts of human antibodies for use as drugs involved the screening of paraproteins from the serum of myeloma patients for antibody with specificity to defined antigens [2]. The screening process was inefficient, costly, and rarely identified myeloma proteins with a desired antigen specificity. Isolation of human antibodies from the serum of immunized individuals has been accomplished by affinity chromatography. This approach, however, remains largely unrewarding because large quantities of human 78

T H E A M E R I C A N J O U R N A L OF S U R G E R Y

serum are required, and, in addition, it is unethical to inject desired drugs, cancer antigens, or infectious agents into humans to produce hyperimmunized sera. Antibodies have been successfully purified from the serum of immunized animals using these approaches; however, animal proteins are highly immunogenic in man. Immunochemistry methods alone have not resulted in the desired homogeneous human antibody in the quantities, specificity, and quality (e.g., purity, affinity) required for use as antibody drugs. Cellular biology: Alternative approaches for producing homogeneous human antibody have involved cell biology techniques in which antibody-secreting human B cells were immortalized by Epstein-Barr virus (EBV), a known B-lymphotropic herpes virus. Cells secreting antibody specific for the antigen of interest were then selected and expanded by cloning methods [3]. EBV-transformed or lymphoblastoid B-cell lines often display undesirable growth characteristics, and it is difficult to select B cells secreting antibody with the desired antigen specificity and isotype. In addition, the availability of ideal human specimens containing B cells (spleen, lymph nodes, tonsils, and bone marrow) have remained limited in supply. Some of these difficulties have been addressed by cell hybridization methods based on the techniques of Kohler and Milstein [4]. Classically, cell hybridization involves the fusion of primed B lymphocytes from an immunized host with immortalized lymphocytes (e.g., mouse or human myeloma cells) [4,5]. The result of this fusion process is an immortalized hybrid (hybridoma) that continuously secretes antibody of the desired specificity. To date, most hybridomas have been murine in origin. The production of human hybridomas has been complicated by the lack of suitable human myeloma fusion partners. Some success has been achieved with cross-species and heterohybridomas that employ mouse myeloma and F1 crosses of mouse myeloma with human lymphocytes as fusion partners [6]. Unfortunately, most of these hybrids continue to exhibit instability, sporadically release chromosomes during division, and produce unpredictable levels of antibody. Details of the methods and the associated limitations in producing human monoclonal antibodies are described elsewhere in several reviews [7-9].

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Molecular biology: The newest approaches for the production of complete human and human-like antibodies have involved the engineering of heavy- and light-chain immunoglobulin genes by molecular biology methods and transfection of these into the genome of immunoglobulin-nonproducing lymphoid cells (a transfectoma) [10,11]. These methods are based on the knowledge of which genes code for the major structural domains of the heavy- and light-chain polypeptides of the antibody. Exons or genes that code for the heavy- and light-chain variable regions are in juxtaposition, proximal to the exons that encode the immuneglobulin constant regions (CIql, hinge, CH2, and C83). A procedure called interspecies isotype switching employs recombinant DNA techniques to link mouse VH and VL exons to human immunoglobulin constant region exons (Figure 2). The result is a chimeric antibody that is produced with mouse antibody V region and human constant regions. Intraspecies isotype switching can subsequently be accomplished if different effector functions from pre-existing human antibody are desired. For example, VH and VL region exons from an IgG1 antibody can be linked to a different set of human constant region exons (IgG2, IgG3, or IgG4). The transfectoma differs from a hybridoma that receives its immunoglobulin genes as a result of the fusion between a unique B lymphocyte and an immortal myeloma cell rather than active insertion. Cited advantages of the transfectoma over the human hybridoma include an apparent increased stability of the resultant clones, an increase in the amount of antibody secreted due to possible repetitive autologous transfections that amplify the number of immunoglobulin genes within a given B cell, and the ability to insert engineered genes into a variety of cells (eukaryotic and prokaryotic) for subsequent expression [10,12]. The production of a mouse-human chimeric antibody transfectoma begins with the identification of a mouse hybridoma that generates an antibody with the desired specificity and avidity. The V region genes are cloned and inserted into a mammalian cell (expression vector) conmining appropriate constant region genes. Both immunoglobulin heavy- and light-chain genes must eventually be transfected into the same recipient cell. Several cell types have been used for expression of recombinant immunoglobulin DNA. Procaryotic (e.g., Escherichia coli) and low eukaryotic (yeast) organisms are able to receive and synthesize both antibody heavy and light chains encoded by engineered genes. Although these two organisms are easy to maintain, neither produces large quantities of "functional" antibody, due primarily to the lack of biosynthetic pathways required for proper folding, glycosylation, and assembly oftbe polypeptides [13-15]. Production of functional chimeric antibodies by transfection has been most successfully performed by gone transfer into mouse myeloma cells that normally synthesize immunoglobulins. The CDR-grafted method has been developed in an attempt to produce an antibody that is more human-like and, therefore, less immunogenic than the chimeric of mouse V region with human C region [16,17]. In this process, just the DNA that codes for the hypervariable

T A B L E lI

Applications and Attributes of Antibody Drugs I. Diagnostic antibodies A. Applications 1. Site-specific delivery of radionuclides 2. Deposition of toxins to specific tissues B. Principal attributes 1. Used in low concentrations (ng to p,g per dose) 2. Generally given as a single dose 3. No expected pharmacologic effect 4. Rapid clearance desired to improve target-to-nontarget ratio 5. Can use Fab or F(ab):'zfragments I1. Scavenger and blocking antibodies A. Applications 1. Bind inflammatory mediator and growth factors 2. Block mediator and growth factor receptors 3. Neutrophil adherence inhibitors 4. Scavenge drug toxins (e.g., antidigoxin) 5. Block antibody Fc receptors (pentapeptide from IgE) B. Principal attributes 1. Antibody availability must be maintained until drug/antigen/cells have been cleared from the blood 2. Generally used in moderate concentrations (mg per dose) 3. Unlabeled or conjugated with toxins 4. Several doses are frequently required, which pose concerns of immunogenicity 5. Long biologic half-life desired 6. Immune complex-related adverse reactions may occur III. Prophylactic antibodies A. Applications 1. Vaccines for infectious agents 2. Antithrombotics (inhibit platelet activation) 3. Selective cell clearance a. Elimination of T-helper cells with OKT-3 to reduce graff rejection in kidney transplantation (muromonab-OKT-3) b. Clearance of IgE-bearing B cells for treatment of allergies. 4. Elicit a specific enzymatic activity 5. Site-specific drug activation (e.g., for cytotoxic drugs) 6. Enhancer of phagocytosis (tufsin from IgG) 7. Supplement to antibodies in immunodeficient patients B. Principal attributes 1. Used in high concentrations (mg to g per dose) 2. Used in unlabeled form, frequently with multiple doses 3. Long biologic half-life desired IV. Carriers of receptors (immunoadhesins: anti-CD4 and antiIgG Fc) V. Anti-idiotype regulatory antibodies Reproducedfrom [1], with permission.

regions or CDRs is transplanted into DNA coding for the remainder of the framework of human variable regions. The resultant hybrid genes can then be used in place of the mufine V genes in the expression vector. This allows the production of transfectoma cell lines that secrete a second-generation chimeric antibody that is composed of greater than 90% human framework and the remainder, mouse heavy- and light-chain CDRs, that form the antigen binding site. The cited advantage of the CDR-grafted

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GENE

GEN

GENE

GENE

I

Protein , /

,'i v-|, CL x

'

Protein /

t"

"1

CH3I[

/

Human L Chain

Mouse L Chain

VH --'."'1 CH1Hmg e CH2

"/Ljc.3

"',

~lumanH Chain

Mouse H Chain

Mouse-Human chimeric L Chain Gene

Protein

Mouse-Human chimeric H Chain Gene

Cotrans fection into nonproduclng rnyeloma cell line

Figure 2. Schematic representation of chimeric antibody production, First, a chimeric gene with mouse variable and human constant regions is ligated into an expression vector containing a selectable marker. The heavy-chain and light-chain vectors are then cotransfected into a recipient cell line. With two independent selectable markers, stable cell lines synthesizing both heavy and light chains can be isolated. Finally, the chimeric antibodies are purified from tissue culture supernatant. Reproduced with permission from [ 1].

I exoanslon

transfectofna cell llne l

culture

Mouse-Humanchimeric antibody

antibody over the mouse-human chimeric antibody is a prolonged half-life in blood due to reduced immunogenicity. CLINICAL USES FOR NEW ANTIBODY PRODUCTS A striking aspect of antibody drugs is the wide spectrum of potential clinical uses. Although their most common applications to date have been in the treatment of infectious and neoplastic diseases, antibodies are being considered for the treatment of autoimmune disorders (e.g., rheumatoid arthritis and myasthenia gravis), atherosclerosis, graft-versus-host disease, and conditions with ischemia-reperfusioninjury (e.g., myocardial infarction or stroke), among others. In the following section is presented an overview of selected clinical conditions for which antibody-derived drugs are being studied, with a focus on their use for detection and treatment of infectious diseases and solid tumors. ANTIBODY DRUGS FOR SOLID TUMORS Detection and treatment of cancer with monoclonal antibodies presupposes that the tumor expresses a determinant that is recognized as being different from other 80

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host cells. A cancer cell may be immunologically different from the host if there is (1) an alteration in the expression of the major histocompatibility complex (MHC), (2) mutation and alteration in the expression of some normal molecule such that an antigenic epitope is produced, or (3) disregulation of normal cell inhibitory processes such that molecules that are only expressed during embryonic and fetal development and not during adult life are no longer suppressed and presented on the cell surface. Unfortunately, because of the diversity of human malignancies and inherent intratumoral heterogeneity, most monoclonal antibodies being developed today lack complete sensitivity (i.e., capability of binding to 100% of the cancer cells) and specificity (i.e., capability of distinguishing normal tissue from cancerous tissue). Cancer immunotherapy with monoclonal antibodies alone is still in its infancy. Clear delineation of action with regard to kinetics and distribution of the antibody and to mechanisms of optimal cell kill is still lacking [18]. Ultimately, more lethal antibody-derived drugs created by recombinant genetic engineering may be required, or possibly, a combination of antibody with other active biologic agents (e.g., tumor necrosis factor or interleukin-2) may be needed.

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Monoclonal antibodies have been used alone in several trials against various solid tumors, and, with rare exceptions, these trials have not yielded durable anti-tumor effects. Treatment of the hematologically derived malignancies has held more promise because of the differential expression of the various leukocyte antigens and cell determinants on those cells and the availability of antibodies against those epitopes. For the more-common solid tumors, the lack of specific antibodies and the murine derivation of most of the antibodies used (which will induce an anti-antibody response on retreatment) have hampered therapy to cure advanced disease. Monoclonal antibodies have been used in experimental trials preoperatively to localize solid tumors. Protocols with radionuclide-tagged antibody have demonstrated a high rate of localization in various tumors either preoperatively or intraoperatively using hand-held scintillation devices [19]. The most widely tested antibodies in this regard have been the anticarcinoembryonic antigen antibody and B72.3, an antibody directed against the TAG72 glycoprotein expressed by colorectal, gastric, ovarian, and breast malignancies. Anticarcinoembryonic monoclonal antibody had a sensitivity of 76% for primary colorectal tumors and 44% for hepatic metastases in one study [20]. Radiolabeled anticarcinoembryonic antibody preoperative imaging was also capable of detecting occult metastases not identified by standard radiologic techniques [21]. Monoclonal antibody B72.3 used in preoperative imaging identified disease in 70% of patients with colorectal tumors [22,23]. Radiolabeled murine monoclonal antibody has been used successfully to image malignant melanoma with 79% to 88% sensitivity [24,25]. Therapeutic trials using monoclonal antibodies against the solid tumors are still in the early phases, although results with the antibodies 17-1A and L6 in the treatment of solid malignancies demonstrate potential usefulness. In a phase I trial using the monoclonal antibody L6, one patient with breast cancer obtained a complete remission [26]. Treatment of patients with malignant melanoma with monoclonal antibodies against GD2 and GD3 gangliosides has not demonstrated significant durable responses, and the overall trend has been to use genetically engineered antibodies in therapeutic trials

[27,281. In the future, monoclonal antibody conjugates, e.g., an immunotoxin formed by attachment to the ricin A chain [29], radionuclide chelation with yttrium-90 or rbenium-188 as #-particle emitters, or conjugates with tumor cell growth hormones [30], may be more effective. Second-generation antibodies, e.g., chimeric or CDRgrafted molecules, may improve the immunologic interaction with the human host-defense system. ANTIBODIES FOR THE PREVENTION AND TREATMENT OF INFECTIOUS DISEASES To the present, most antibody products used to prevent or treat infectious diseases were either crude sera from animal or human sources, relatively purified immunoglobulins from immunized humans, or purified human, polyclonal IgG. Monoclonal antibodies have been investigated for a number of infectious problems; however, most

T A B L E III

Anti-Lipid A Antibody P r o d u c t s Product HA-1A T88 E5

Generic Name

Source

Dose Studied

Manufacturer

Nebacurnab -Edobacornab

Human Human Mouse

100 mg 300 rng 2 x 2 mg/kg

Centocor Cetus Xorna/Pfizer

attention has been focused on their use for the treatment of gram-negative bacterial sepsis. Antibodies for gram-negative sepsis: Since the pathogenic mechanisms have been more clearly recognized, and with the availability of monoclonal antibodies, new approaches to the treatment of gram-negative sepsis have been developed. Antibodies proposed for treatment of gram-negative sepsis have been directed against the lipid-A portion of endotoxin from gram-negative bacteria, other bacterial cell-wall antigens, mediators (such as interleukin-1 and tumor necrosis factor) that are responsible for the effects of endotoxin, or the inflammatory cells that participate in the response to gram-negative bacteria. Studies of antiserum from humans immunized with the heat-inactivated E. coil J5 mutant demonstrated a reduction in mortality from gram-negative bacteremia [31] and protection of high-risk surgical patients from septic shock [32]. The limitations of antisera (limited availability, potential transmission of viral infection, and variation in anti-endotoxin activity) led to the investigation of anti-endotoxin monoclonal antibodies. At present, three monoclonal IgM anti-lipid-A antibody products have been studied in clinical trials for the treatment of gram-negative sepsis (Table HI). It appears that isotype selection may be important for these antibodies since polyclonal IgG with anti-E, coil J5 activity was not effective for treatment of gram-negative septic shock [33]. Presently, there are few published clinical reports involving the use of anti-endotoxin antibodies. Zeigler and associates [34] described the use of HA-1A (an antibody with lipid-A specificity) in patients with sepsis and suspected gram-negative infection. Their randomized, double-blinded, single-dose, placebo-controlled (3.5 g albumin) trial included 543 patients. Overall, 28-day mortality was not different between patients who received HA-1A or placebo. However, when the analysis was limited to the 200 patients with sepsis and gramnegative bacteremia, there was a significant reduction in mortality in patients given HA-1A (49% versus 30% with placebo). Of 101 patients with gram-negative bacteremia and shock, mortality was reduced with HA-1A from 57% to 33%. There was no benefit in nonbacteremic, septic patients. Greenman and associates [35] reported their clinical experience with E5, a murine IgM anti-lipid-A monoclonal antibody. In a blinded, randomized trial, 486 patients with suspected gram-negative sepsis received either E5 antibody or placebo in two doses (2 mg/kg) 24 hours apart. When all patients with gram-negative sepsis were

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included, the antibody did not reduce 30-day mortality. may be effective in inhibiting platelet function. Coller However, when only patients who had gram-negative and associates [44] reported the use of a murine monosepsis without shock were considered, a survival advan- clonal antibody F(ab')2 that blocks the platelet receptor tage for the E5 group was demonstrated with a relative for glycoprotein IIb/IIIa. In a newly dead subject, it was risk of 2.3. Also, resolution of associated morbidities (dis- determined to be a potent, immediate-acting inhibitor of seminated intravaseular coagulation, adult respiratory platelet aggregation. The relative effectiveness of antidistress syndrome, and acute renal failure) occurred more body antithrombotic agents in comparison with traditionfrequently in the antibody group. E5 was not effective in al agents remains to be determined. reducing mortality for patients experiencing shock. Antibodies inhibiting neutrophil adherence: NeuOther investigators have demonstrated the effective- trophils are important mediators of microvascular injury ness of anticytokine antibodies in animals. Anti-tumor- during inflammatory processes such as that which occurs necrosis-factor (anti-TNF) antibodies have been studied after ischemia and reperfusion. Several investigators have in animal models of gram-negative infection [36-38]. In studied antibodies that reduce neutrophil participation in two of the studies, the anti-TNF antibody was adminis- inflammatory reactions [45-47] by binding to the tered at the time of bacterial challenge or shortly thereaf- CD1 lb/CD18 glycoprotein complex on the neutrophil ter [36,37]. Tracey and associates [38] demonstrated surface. The CD1 lb/CD18 complex is important in neuthat anti-TNF F(ab')2 fragments protected baboons trophil adhesiveness to endothelial cells. from shock and organ failure when administered prior to Mileski and associates [45] demonstrated that the bacterial challenge. An anti-TNF IgG monoclonal anti- murine monoclonal antibody 60.3 was beneficial when body (CB 0006) has been studied in a few patients with administered to rhesus monkeys after hemorrhagic shock encouraging results [39]. In another animal study, pre- in that it reduced fluid requirements and weight gain, treatment with anti-interleukin-6 antibody was effective maintained higher hematocrit, and prevented gastritis. in reducing mortality from lethal bacterial or TNF chal- Using the same antibody and a rabbit-hemorrhagiclenge [40]. shock model, Vedder and associates [46] demonstrated Antibodies as vaeeines: The recognition that the an increased 5-day survival rate from 29% in controls to antibody-binding site (paratope) has a complimentary 100% in rabbits with the antibody. A different monostructural relationship with the antigen epitope has led to clonal antibody that also binds to the CD 11b/CD 18 comthe development of anti-idiotype vaccines. These anti- plex was effective in reducing reperfusion injury in a dog idiotype vaccines may be useful in generating an immune model of myocardial ischemia [47]. response for antigens that are too toxic to administer asa vaccine or that are T-cell independent and do not gener- ISSUES IN THE USE OF ANTIBODIES AS ate an adequate antibody response (e.g., lipopolysaecha- DRUGS ride). An anti-idiotype vaccine can be generated by inAntibodies as a class of drugs may present new proboculation of an animal with the substance (antigen) lems for clinicians, including novel patterns of adverse against which the vaccine isto be directed. A monoclonal reactions, tolerance through the production of anti-antiantibody to the antigen can then be produced by tradi- body antibodies, unique considerations in dosage and adtional methods. The antibody, which has the structural ministration, and the potential for dramatically increased features complimentary to the antigen, is then given to a treatment costs. naive animal, and a monoclonal antibody that reacts with Adverse effects: In the limited clinical trials to date, the paratope of the first antibody is generated. The sec- most monoclonal antibody drugs have demonstrated a ond antibody may then possess structural features that relatively low frequency of adverse effects, even with muare similar to the initial antigen and, when it is adminis- fine-derived monoclonal antibodies. Allergic reactions tered as a vaccine, can cause the in vivo production of occurred in 1.6% of 479 patients who received the murine antibodies that are cross-reactive with the antigen. E5 antibody for gram-negative sepsis [351. DiUman [18] Although not yet introduced into clinical trials, anti- summarized the tolerance of 177 patients with cancer to idiotype vaccines have demonstrated benefits in animal 19 different murine monoclonal antibodies. The most models. Kato and associates [41] reported protection frequent adverse effects were fever (15% of patients), from lethal exposure to E. coli lipopolysaccharide after increased serum transaminases (14%), rigors and chills administration of an anti-idiotype vaccine. Using a dental (13%), diaphoresis (11%), urticaria (11%), and pruritus caries model in rats, the investigators were able to demon- (9%). Anaphylaxis was reported in 1 of the 177 patients. strate that an anti-idiotype vaccine was able to induce a At present, only one monoclonal antibody product protective immune response to pathogens of mucosal sur- (muromonab OKT-3, used for the treatment and prevenfaces [42]. Also, an anti-idiotype vaccine to Clostridium tion of graft rejection) has been administered to sufficient perfringens type D toxin was protective in mice and rab- numbers of patients to provide a valid profile of adverse bits challenged with the toxin or live organisms [43]. effect potential. With muromonab OKT-3, a characteristic group of symptoms (fever, headache, myalgia, tachyO T H E R POTENTIAL USES FOR ANTIBODY cardia) is observed in most patients on initial administraDRUGS tion [481. Aseptic meningitis and pulmonary edema have Antiplatelet antibodies: Since the activation of also been observed [48]. These effects may be characterplatdets requires the interaction of mediators with sur- istic of antibodies that result in destruction of large numface receptors, antibodies that block platelet receptors bers of lymphocytes. Recently, an increased risk of lym82

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phoproliferative disorders has been observed in cardiac transplant patients who received muromonab OKT-3

[49]. Anti-antibody response: As recognized for some time, the administration of foreign proteins may generate anti-protein antibodies, and xenogeneic proteins are most likely to generate these responses. One of the important limitations of non-human monoclonal antibodies is the generation of an anti-antibody response that reduces antibody effectiveness by binding the administered antibody. With murine monoelonal antibodies, the response is termed HAMA, or human anti-mouse antibody, and has been reported to occur in up to 86% of patients who receive muromonab OKT-3 [50] and 40% of 104 patients who receive a murine antibody for colon cancer [51]. These anti-antibodies may be anti-isotype and could potentially confer crossover inhibition of other murine antibodies, or they may be idiotype specific and potentially inhibit antibodies from other species with thesame epitope specificity. Administration and dosage: A major issue for the clinical use of monoclonal antibodies is the selection of a dose and administration regimen. The antibody doses chosen for clinical trials have been determined empirically, and the resources required for clinical trials inhibit defining dose-response relationships in humans. With increased clinical experience, we expect that optimal doses and regimens will become more apparent. With the antilipopolysaceharide antibodies, for example, it can be foreseen that controversy will develop as to the dose administered, whether multiple doses are necessary, whether the antibodies are best administered for prophylaxis or treatment, and finally, whether "cocktails" of different antibodies will be more effective than single antibodies. There will be important pharmacokinetic issues relating to the use of monoelonal antibodies. In contrast to typical drugs, antibody pharmacokinetics can be influenced more by the antigen burden than by major organ function. In a mouse tumor model of chronic lymphocytic leukemia (CLL), in which the animals were administered a murine monoelonal antibody to common CLL antigen, the blood clearance and volume of distribution were increased three- and six-fold, respectively, compared with non-tumor-bearing controls [52]. Also, the isotype and structural form of the antibody can affect the observed pharmacokineties. Fab fragments and immunoconjugates may differ considerably from intact antibodies. Changes in pharmacokineties will influence the dosage and regimen needed for treatment. Antibody product selection: As new antibody products are approved for treatment, clinicians will be faced with the need to consider a number of new factors in the choice of product, in addition to those discussed above. Potential selection factors include the source of the antibody (human versus animal), isotype, antigen-binding specificity, potential for cross-reactivity, and cost, It is not clear if human antibodies will be preferable to murine antibodies for all indications, particularly if there are substantial differences in product cost. Factors such as antibody isotype (usually IgG versus IgM) or antigenbinding specificity have uncertain relationships to effica-

cy for most indications. Also, there is the risk that any administered antibody may cause harm by unintended binding to tissues with features similar to the intended antibody target. Finally, cost will be a major issue in antibody product selection since a single antibody dose may cost a few thousand dollars. CONCLUSION Monoclonal antibody technology and novel methods for antibody engineering have resulted in the development of many new antibody-like products that have potential applications in the clinical setting. Although the most common uses for antibody drugs will be the treatment of infectious diseases, cancer, and organ transplant rejection, other important indications will emerge. A thorough understanding of the structure and functions of novel antibodies and antibody-like products will aid the clinician in the optimal application of these products. A number of new issues will be introduced by antibody drugs. Among them is the source of the antibody (human or animal) and the potential for anti-antibody response. Factors such as pharmacokinetics, binding specificity, and isotypo have, as yet, undetermined importance for antibody selection. The large number of different antibodies being studied in clinical trials suggests that, in the near future, clinicians may have many new and effective treatments available for a variety of diseases. Clearly, these new agents will pose substantial cost burdens, and there will be a need to carefully assess costs versus benefits. REFERENCES 1. Hamilton RG, Sun L, Bcall PT. Production and quality control of monoclonal antibody reagents and drugs. In: Moulds JM, Masouredis SP, editors. Monoclonal antibodies. Arlington, VA: American Association of Blood Banks, 1989: 92. 2. Krausr RM. The search for antibodies with molecular uniformity. Adv Immunol 1970; 12: 1-56. 3. Kozbor D, Roder JC. Requirements for the establishment of high titcred human monoclonal antibodies using the Epstein-Burr virus technique. J Immunol 1981; 127: 1275-80. 4. Kohlcr G, Milstein C. Continuous cultures of fused cells secreting antibody of predr specificity. Nature 1975; 256: 495-7. 5. Goding JW. Theory and production of monoclonal antibodies. In: Goding JW, oditor. Monoclonal antibodies: principles and practice. New York: Academic Press, 1983; 5-97. 6. Osbcrg L, Purch E. Human X (mouse X human) hybridomas stably producing human antibodies. Hybridoma 1983; 2: 361-7. 7. Englcman EG, Foung SKH, Larrick J, Raubitschek A, editors. Human hybridomas and monoclonal antibodies. New York: Plenum Press, 1985. 8. Strdkauskas A J, editor. Human hybridomas. Diagnostic and therapeutic applications. New York: Marcel Dekker, 1987. 9. Ringcrtz NR, Savage RE, editors. Cell hybrids. New York: Academic Press, 1976. lO. Borrebaeck CAK, editor. Antibody engineering: a practical guide. New York: WH Freeman Co., 1992. 11. Strdkauskas A J, Taylor CL, Smith MR, Bear PD. Transfection of human ceils: an alternative method for the establishment of human hybrid clones. In: Strdkauskas A J, editor. Human hybridomas. Diagnostic and therapeutic applications. New York: Marcel Dckker, 1987: 95-120. 12. Morrison SL, Oi VT. Transfer and expression of immunoglobulin genes. Ann Rev Immunol 1984; 2: 239-56.

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THE AMERICAN JOURNAL OF SURGERY VOLUME 164 JULY 1992

Novel antibody drug products.

Recent developments in protein and genetic engineering methods have allowed the production of antibody-derived molecules that have important potential...
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