David A. Scheinberg
Deborah A. Mulford
Joseph G. Jurcic
Richard P. Junghans
Monoclonal antibodies (mAbs) are remarkably versatile agents with potential therapeutic applications in a number of human diseases, including cancer. There are now eight FDA-approved mAbs for the treatment of cancer. MAbs have long promised to offer a safe, specific approach to therapy. Over more than a decade, preclinical evaluation and human clinical trials have identified new strategies for the use of mAbs, as well as a number of obstacles to their effective application. Although the use of antibodies as targeting agents dates to the 1950s,1 clinical investigation of antibodies as a potential treatment for cancer could not be initiated until the mid-1970s, when efficient and reliable methods for the production of mAbs were developed.2
Five approaches to mAb therapy are used in humans. First, mAbs can be used to focus an inflammatory response against a target cell. The binding of a mAb to a target cell can result in the fixation of complement, which yields cell lysis or results in opsonization that marks the cell for lysis by various effector cells, such as natural killer (NK) cells, neutrophils, and monocytes. Second, mAbs may be used as carriers to deliver another small molecule, atom, radionuclide, peptide, or protein to a specific site in vivo. Third, mAbs may be directed at critical hormones, growth factors, interleukins, or other regulatory molecules or their receptors to control growth or other cell functions. Fourth, anti-idiotypic mAbs may be used as vaccines to generate an active immune response. Finally, mAbs may be used to speed the clearance of other drugs or toxins or fundamentally alter the pharmacokinetic properties of other therapeutic agents. For example, mAbs may be fused to drugs or factors to increase their plasma half-life, change their biodistribution, or render them multivalent. Alternatively, mAbs may be used to clear previously infused mAbs from the circulation.
Despite the diversity of approaches, significant problems remain that are peculiar to mAbs. The mAbs are large, immunogenic proteins, often of rodent origin, that rapidly generate neutralizing immune responses in patients within days or weeks after their first injection. The sheer size of mAbs—150 kd for immunoglobulin G to 950 kd for immunoglobulin M, 100 times larger than that of typical drugs—makes their pharmacology (particularly diffusion into bulky tumors or other extravascular areas) problematic for effective use. Many early mAbs or mAb constructs were either poorly cytotoxic or relatively nonspecific, which rendered them ineffective. Moreover, the high degree of mAb specificity that is routinely achievable may allow tumor cells that do not bear the specific antigen target to escape from cytotoxic effects.
Nonetheless, mAbs still have great potential to be safe and effective anticancer agents. Recent clinical investigations have highlighted several areas in which mAbs can be effective, either alone or in combination with other, more conventional agents.
This chapter reviews the basic biochemical and biologic properties of mAbs and the most commonly used derivatives (immunotoxins, radioimmunoconjugates, mAb fragments), discusses the pharmacologic issues peculiar to mAbs, and outlines some of the important clinical results obtained with mAbs. Potential solutions to the most difficult issues in the use of mAbs are presented. Because mAbs and conjugates of mAbs represent many different drugs with characteristics that result from their origin (rodent or human), their isotypes, their structure, or the various conjugated toxic agents, generalizations about the properties of mAbs often may not be possible. Treatment of cancer with mAbs is a new and rapidly changing field, and readers are encouraged to consult other reviews for more comprehensive discussions of individual areas.3
Immunoglobulins are separated into five classes or isotypes based on structure and biologic properties: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin A (IgA), and immunoglobulin G (IgG). IgM is the primordial antibody whose expression by the B cell on its surface represents the commitment of that cell to a particular but broad recognition space that subsequently narrows as part of the maturation response induced by antigen interactions.4 In some cases, the antibodies interact with specialized receptors that link their action to host cellular defenses; in others, the antibodies interact with the humoral complement system. IgG is further divided into four subclasses, and IgA into two subclasses. Heritable deficiencies in individual immunoglobulin classes or IgG subclasses are associated with susceptibility to particular infections and autoimmune disorders.5 Table 31.1 summarizes various features of the antibodies discussed in this section.
The fundamental structural elements of all antibodies are indicated by size as heavy and light chains of 55 to 75 kd and 22 kd, respectively (Fig. 31.1). Light chains are either κ or λ and are each distributed among all immunoglobulin subclasses. Heavy chains are µ, δ, γ, ε, and α, corresponding to IgM, IgD, IgG, IgE, and IgA and the biologic characteristics of each antibody class. The amino-terminal domain of each chain is the variable (VH or VL) region that mediates antigen recognition; the remaining domains are constant regions designated CL for light chain and CH1, CH2, and CH3 for heavy chain (and CH4 for µ and ε). Between CH1 and CH2 is the hinge region, which confers flexibility on the antibody “arms” and susceptibility to proteases (see later), except in IgM and IgE, in which the CH2 domain itself serves this role.
Heavy (H) and light (L) chains are normally paired 1:1 with each other, but the smallest stable unit is a four-chain (HL)2 structure (Fig. 31.1), for a nominal total mass of 150 to 160 kd for IgG and higher for other isotypes (Table 31.1). IgE and IgG are composed of a single (HL)2 unit, whereas IgM exists as a pentamer of (HL)2 units joined by disulfide bonding with a third J-chain component. IgA exists mainly as a monomer in serum, but in secretions it exists primarily as a dimer plus trimer and higher forms in which the oligomers are linked by J chain as well as the fragment of secretory chain (secretory piece) that is involved in the mucosal transport.
The V region itself is composed of subdomains—relatively conserved framework regions interdigitated with the complementarity-determining regions (CDRs; also termed “hypervariable segments” [HVSs]) that make primary contact with antigen (Fig. 31.1).6 Three CDRs are found in each heavy and light chain that may participate in antigen binding. The V regions should be seen as juxtaposed three-fingered gloves, with the CDRs covering the tips (Fig. 31.2), arrayed in a broad contact surface with antigen (Fig. 31.3).
TABLE 31.1 PROPERTIES OF ANTIBODY CLASSES
Figure 31.1 Antibody structure. The structural relationships and functions of domains of immunoglobulin G. (Reproduced with permission from Wasserman RL, Capra JD. Immunoglobulins. In: Horowitz MI, Pigman W, eds. The Glycoconjugates. New York: Academic Press, 1977:323.)
Antibodies are glycoproteins. Glycosylation of proteins plays various roles related to solubility, transport, conformation, function, and stability. Carbohydrate is located mainly in antibody C domains, with a lower frequency in V regions (see data on M195 later).7 IgG contains a major conserved glycosylation site in CH2 that contributes to the conformation of this domain, which is crucial to the functional ability to bind to complement and to Fcγ receptors.
Figure 31.2 Space-filling model of human immunoglobulin G1 antibody with complementarity-determining regions in color representing anti–Tac-H; human myeloma protein Eu with complementarity-determining regions grafted from murine anti-Tac. (Photo provided courtesy of Dr. C. Queen.) (Please see color insert.)
The IgG antibody “unit” has been defined in terms of susceptibility to proteases that cleave in the exposed, nonfolded regions of the antibody (Fig. 31.1). A summary of antibody fragments and engineered or synthetic products is presented in Table 31.2. Fab contains the V region and first C domain of the heavy chain (VH + CH1 = Fd) and the entire light chain (L); Fab' includes in addition a portion of the H chain hinge region and one or more free cysteines (Fd'); Fabc2 is a dimer of Fab' linked through hinge disulfide(s); and Fv is a semistable antibody fragment that includes only VH + VL, the smallest antigen-binding unit. Fc is the C-terminal crystallizable fragment that includes the complement and Fc receptor–binding domains (see later). Genetically engineered products include the δ constructs; these lack the second C domain of heavy chain and behave like Fab'2, with bivalence, abbreviated survival and lack of interaction with host effector systems, but they do not require enzymic processing.8 Another genetically engineered product, sFv (single-chain Fv), is Fv with a peptide linkage engineered to join the C-terminus of one chain to the N-terminus of the other for improved stability. More advanced products have been designed that conceptually represent the antigen-binding domain in a single peptide product9; this is not related structurally to an antibody and is therefore considered an antibody mimic.
Antibodies possibly represent the most strikingly evolved, adaptive system in all of biology. The most diverse representation of classes and functions is found in Mammalia.
The power of antigen recognition begins with an inherited array of duplicated and diversified germ-line V genes, a random mutational process that creates novel CDRs, a combinatorial selection process that amplifies the germ-line capabilities, and a controlled and directed mutational process that hones the specificity and matures the antibody into a high-affinity, antigen-specific reagent.
Figure 31.3 Antigen-antibody binding surface juxtaposition. The variable (V) region (Fv) of antibody (right) binds to influenza virus protein neuraminidase (left) in the top panel. The VH (red) and VL (blue) regions are separately colored to show their respective binding contributions. The bottom panel offsets the two molecules by 8 Å to show the complementarity of surfaces that promotes the binding interaction. The stippled surface of the neuraminidase defines the antigen “epitope.” (Photo provided courtesy of Drs. P.M. Colman and W.R. Tulip, CSIRO Australia.) (Please see color insert.)
The biologic expression of antibody begins with the B-cell progenitor, which undergoes a series of maturation steps that begin with V gene selection for heavy chain followed by light chain V selection that yields surface expression and secretion by the mature B cell. On interaction with antigen, the B cells are activated to proliferate, secrete antibody, undergo CDR mutagenesis and affinity maturation, and finally undergo chain switch and plasma cell conversion. Plasma cells remain in tissues, spleen, or lymph nodes and secrete large quantities of antibody, which is the sole function of this terminally differentiated cell.4
The genes of heavy and light chains share important features of structure and maturation. Each gene locus contains widely separated V, C, and minigene domains that are placed into juxtaposition by DNA recombination mechanisms. The minigenes—diversity (D) and joining (J) regions for heavy chain and J regions for light chain—contribute to or constitute, with modifications, the CDR3.10 The κ and λ light chain loci are located on chromosomes 2 and 22, respectively, but all heavy chains are contained within a single massive locus on chromosome 14.
Germ-line diversity is essential to the generation of the antibody repertoire. On the heavy chain locus are an estimated 80 functional VH genes, 12 D regions, and 6 J regions for a potential of 6,000 combinations (Fig. 31.4).10, 11, 12 Roughly 80 Vκ light chain and 5 Jκ domains are found, which, randomly associated, can generate 400 combinations (the λ locus contains a smaller number of distinct V genes). A simple arithmetical calculation suggests that VκVH combinations alone could generate a diversity of approximately 2 × 106. Yet even this number is conservative, because this diversity is amplified in turn by errors in recombination and processes called N and P nucleotide addition in CDR3, which add enormously to the potential complexity, in theory exceeding the total lifetime B-cell output by several orders of magnitude.10Many authors, however, have cautioned that the mathematical diversity does not allow for the redundancy in configurations that could provide equivalent binding domains; in terms of antigen binding, the practical diversity is probably in range of 1×107.
TABLE 31.2 ANTIBODY FRAGMENT DEFINITIONS
V gene selection is based on random expression followed by specific amplification. The argument has been made on physicochemical grounds that 105 different antibody molecules are sufficient to create a topologic set that recognizes any antigen surface with an affinity of 105 to 106 M-1,13 a weak but biologically important number that corresponds to recognition affinities of naive antibody-antigen contacts that are often broadly polyreactive. B cells express antibody, principally IgM and IgD, on their membranes. On contacting antigen, these cells are stimulated to divide and undergo CDR mutations. Subsequent binding and stimulation are in proportion to the strength of the binding reaction; hence, an in vivo selection occurs for mutations that enhance the affinity of the antibody for the antigen, a process termed “affinity maturation.”14 Simultaneous with this increased affinity is a narrowing of the specificity, with the antibody shedding its early polyreactive phenotype. The cell then undergoes “class switch” to one of the mature antibodies (IgG, IgA, IgE) by deleting DNA between the VDJ region and the new C region of the heavy chain, which brings this new C domain in juxtaposition with the V region (Fig. 31.4). (The light chain is unchanged.) Some time after commitment to a mature antibody, the cell ceases its CDR mutagenesis, affinity maturation is completed, and the B cell undergoes morphogenesis to a tissue-resident plasma cell.4
Affinity is a quantitative measure of the strength of the interaction between an antibody and its cognate antigen, analogous to the equilibrium constant in the chemical mass action equation:
Figure 31.4 Generation of diversity. VJ and VDJ joining occur in L chain and H chain by excision of intervening DNA in the genome. Class switch involves deletion of intervening constant (C) domains (Cµ, Cδ, etc.) and transcription through the new proximal C region. C is finally joined to the V gene by splicing of the messenger RNA. (C, constant; J, joining; V, variable.) (Reproduced with permission from Cooper MD. Current concepts: B lymphocytes—normal development. N Engl J Med 1987;317:1452.)
The equilibrium or affinity constant (Ka) is represented in units of M-1. In most instances studied by x-ray crystallography, contacts between antibody and protein antigen are dominated by noncovalent hydrogen bonds (O—H), with a lower frequency of salt bridges (COO— + H3N), for a total of 15 to 20 contacts. The effect of adding a new H–bond can be estimated from the free energy gain (0.5 to 1 kcal per mole α10°C) and from ΔG =–RT ln Ka to yield affinity increases of threefold to tenfold. Therefore, the affinity maturation that takes place (or affinity that may be lost in antibody engineering) changes quickly with a relatively small change in the number of bonds; that is, creating as few as three new hydrogen bonds may generate an affinity enhancement of more than 100-fold. This has been borne out by affinity changes that accompanied productive amino acid substitutions in V region engineering (see later). Of note, antibody affinities are generally much higher for protein antigens than for carbohydrate antigens, which may have less opportunity for hydrogen-bonding interactions (but are also “T-independent” antigens).
Although affinity and Ka directly express the binding potential of the antibody and are the most suitable measures for comparing affinities, the inverse of Ka', termed Kd or the dissociation constant, is expressed in molar units and indicates the concentration that is the middle of the range for the biologic action of the antibody:
That is, Kd is the concentration of free antibody at which antigen is 50% saturated; if the antibody is in large excess, the input antibody concentration approximates the free concentration. Kd is a frequently used term, but its relationship to affinity must always be borne in mind; that is, low affinity equals high Kd, and high affinity equals low Kd. For example, a Ka of 2 × 109 M-1 implies a Kd of 0.5 × 10-9 mol/L (0.5 nmol/L), or approximately 0.1 µg/mL antibody concentration for IgG. If antigen is in the picomolar (10-12 mol/L) range, this concentration of antibody will have half of the antigen saturated, and half of the antigen will remain “free.” At 10-fold higher antibody concentration (1 µg/mL, 10 × Kd), antigen will be 90% saturated and 10% free, and at 10-fold higher concentration (10 mg/mL, 100 × Kd), antigen will be 99% saturated and only 1% unbound. A key point of understanding is that the ratio of antibody to antigen has very little impact on the degree of antigen saturation when antibody is in excess. If antibody concentration is 1 nmol/L with a Kd of 1 nmol/L, it does not matter whether antigen is 0.1 nmol/L at the Kd, 0.1 pmol/L, or 0.1 fmol/L; antigen in each case is 50% bound, although the ratio of antibody to antigen is 10, 104, and 107, respectively. Only the relation of free antibody to its Kd determines the degree of antigen saturation.
The affinity constant Ka is itself composed of two terms that describe the on-rate (forward; units of M-1 S-1) and off rate (back; units of S-1) of the reaction:
To a first approximation, the forward rate is diffusion limited and is comparable for many antibodies reacting with macromolecules or cell-bound structures. Reactions of antibodies with haptens and other small molecules in solution are dominated by the faster linear and rotational diffusion rates of the smaller component.15 For example, when 0.1 nmol/L of dinitrophenyl-lysine (0.1 ng/mL) or 0.1 nmol/L of cell-bound HLA-A2 (50 ng/mL) is mixed with specific IgG antibody at 10 µg/mL (65 nmol/L), 0.1 second is required for the antibody to react with 50% of the antigen for the hapten but 4 minutes is required to react with the surface protein. Yet they have virtually the same affinity constant.15 This is due to the fact that the fast association rate is balanced by a fast dissociation rate for the hapten (clearance half-time [t½] = 0.7 seconds) whereas stability is longer for the protein antigen (t½ = 6 minutes).
Although exceptions exist, the on-rates of antibodies to protein and cell-bound antigens are primarily in this range and inversely proportional to antibody concentration for antibody in excess of antigen (i.e., at 1 µg/mL, the 50% on time would be on the order of 0.5 to 1 hour). Accordingly, differences in affinity between antibodies to the same cellular antigen are in many instances reflective of the off-rate (κb). For most purposes, an antibody is generally considered of “good” affinity if its Ka is equal to or greater than 109 M-1, where off-rate t½ values of an hour or more at 4°C are common. Association and dissociation times at 37°C are both accelerated relative to the times at 4°C, on the order of 5 or more, frequently with a net decrease in antibody affinity of 2-fold to 10-fold. This must be explicitly tested, however, because in some instances protein-ligand affinities have been found to be enhanced by higher temperature.16
The foregoing expresses basic principles of binding processes. A further important feature of antibodies is their multivalent structures. Although the on-rates for monovalent Fab and bivalent Fab'2 constructs are comparable, the bivalent off-times may be 10-fold longer than for the monovalent constructs, which yield affinities that are similarly enhanced.15 To discriminate the affinity that is intrinsic to the V region antigen interaction from the effective affinity in a bivalent or multivalent interaction, the latter is often referred to as avidity. For monovalent interactions, avidity equals affinity; for multivalent interactions, avidity is greater than or equal to affinity. Theory predicts avidity enhancements that vastly exceed observed numbers, but structural constraints undoubtedly restrain the energy advantage of multivalent binding.17, 18 In the extreme, steric factors constrain some bivalent antibodies (e.g., anti-Tac)19 to bind only monovalently to antigens on cell surfaces although they will bind bivalently to antigen in solution. When antigens are presented multivalently on the surfaces of cells, viruses, or other pathogens, even the low-affinity IgM interactions can yield a high-avidity, stable binding to such targets in vivo.
PHARMACOKINETICS AND PHARMACODYNAMICS
The metabolism of immunoglobulins determines the duration of usefulness of antibodies in vivo. Under normal conditions, the serum levels of endogenous immunoglobulins are determined by a balance between synthetic and catabolic rates.20 When antibodies are administered as therapeutics, these catabolic rates effectively specify the dose and schedule necessary to maintain therapeutic blood levels when steady-state exposures are targeted. Table 31.1 lists the half-lives of human antibody survival in humans. IgG has the longest survival, 23 days (this value is for IgG1, IgG2, and IgG4; IgG3 survival is 7 days). A key element in the regulation of IgG catabolism is the Brambell receptor (FcRB), named after its discoverer, F. W. R. Brambell, who described this receptor more than 30 years ago (see ref. 21 for a review). This receptor is located in endosomes of endocytically active tissues, primarily vascular endothelium, which is mainly responsible for the catabolism of plasma proteins, including IgG. There, FcRB binds IgG, recycling it to the cell surface and diverting it from the pathway to lysosomes and the catabolism that is the fate of other, nonprotected proteins. In this role, FcRB is also termed the IgG protection receptor (FcRp). Yet FcRB is also responsible for transmission of IgG from mother to young, via yolk sac, placenta, or newborn intestine; in this manifestation, it is termed the IgG neonatal transport receptor (FcRn) for neonatal intestine, the tissue from which the receptor was initially cloned.
A substantial body of knowledge exists on the metabolism of immunoglobulins in various disease states. Conditions of protein wasting (enteropathies, vascular leak syndromes, burns), febrile states, hyperthyroidism, hypergammaglobulinemia, and inflammatory disorders are accompanied by significant acceleration of immunoglobulin catabolism.20 This information is of importance for understanding in vivo survival data in various clinical applications. In fact, the controlled conditions of testing immunoglobulin metabolism are rarely duplicated in practice, with antibody survivals typically shorter than suggested by the numbers above. Typically, murine antibody survival t½ values are in the range of less than 1 to 3 days, and antibodies with human gamma Fc domains (chimeric or humanized) have t½ values in the range of 1 to 15 days. Some of this acceleration in clearance is clearly due to disease-associated catabolic factors and to antigen binding in vivo, but subtle changes in the drug structure during product preparation may have a role in this acceleration as well. The influence of antigen expression on antibody clearance in vivo is considered later.
Antibody fragments have been studied because of their abbreviated survival and because their small size may translate into better tissue penetration. Fab and Fab'2 have survivals in vivo of 2 to 5 hours in mice, with comparable values in humans, and survival is dependent largely on kidney filtration mechanisms.20This is not based on size alone, because the Fc fragment, which is comparable in size to Fab, is not filtered and has an in vivo survival of 10 days in humans. These rapidly catabolized fragments, like other filtered proteins, are largely absorbed in the proximal tubule and degraded to amino acids, which are returned to circulation. No intact immunoglobulin or fragments reenter circulation once filtered.22 In normal kidney, less than 5% of filtered light chain is excreted intact, whereas this fraction increases in the setting of renal tubular disease.23
A recently active area of investigation has been the role of circulating antigen in the setting of antitumor therapies. This was first encountered in anti-idiotype therapies directed at the surface Ig on B-cell lymphomas, some of which secreted high levels of idiotype.24 This prevented access of administered antibody to the idiotype on tumor cells, which effectively neutralized the drug, unless very high doses were given to overwhelm the secreted quantities of idiotype. Subsequent further studies showed that other tumor antigens, including carcinoembryonic antigen (CEA),25 gangliosides GD2 and GD3,26, 27 and Tac,28 could achieve significant levels that might require adjustments to therapy.
Key observations include that there is (a) a direct relationship between the soluble antigen levels and the dose necessary to attain 50% (or 90% or 99%) bindability of administered antibody and (b) a predictable relationship between the rate of antigen synthesis and the time to antibody saturation.28 The actual partitioning of antibody between soluble and cellular antigen depends on several features, including the effective avidity of antibody for antigen on cells (which may be higher than for soluble antigen) and other factors influencing tumor penetration.
A special concern in this setting is that many soluble forms of antigen have short t½ values once shed from tumor cell surfaces, and the interaction with antibody may prolong their in vivo survival and increase their level in the whole body. When the target itself is a cytokine or cytokine receptor, adverse consequences conceivably could derive from the antibody treatment if the antigen retains activity in the antibody complex, as shown for interleukin-3, interleukin-4, and interleukin-7 complexes in vivo.29 If the antigen does not retain activity in complex, then this problem causes no concern because the free concentration of antigen cannot be increased by the presence of antibody, even after antibody is fully saturated with excess antigen. A different potential consequence of antigen load is that it may reduce transport of radioantibody to tumor for imaging or therapy. Studies have shown, however, that antibody can partition sufficiently between soluble and cellular antigen to yield targeting adequate for tumor-imaging purposes (CEA,25 Tac28).
Antibodies mediate several actions of potential therapeutic interest, some of which are part of the normal biologic function of antibodies and some of which are adapted in novel ways to the needs of specific settings.
The complement (C') system is at least as primitive evolutionarily as antibodies. One view is that the alternate (antibody-independent) pathway is the primordial system, which diversified to create the classic pathway to collaborate with antibody to direct the attack complex (C5–C9) against antibody-coated targets. The most effective mediator of complement-dependent cytotoxicity (CDC) is IgM. Single IgM molecules can fix and activate complement on cell surfaces. In contrast, IgG-mediated CDC depends on the juxtaposition of pairs of IgG molecules to bind complement to cells,30 with substantially lower complement fixation and reduced killing efficiency relative to IgM. Human IgG1 and IgG3; mouse IgG2a, IgG2b, and IgG3; rat IgG2a; and rabbit IgG all fix and activate complement, whereas human IgG2 fixes C' poorly, and human IgG4 and murine IgG1 normally do not fix C'.31 (Complement fixation depends on conserved residues in the CH2 domain; short hinge regions are thought to hinder C1q access and binding with these latter isotypes.) Although human IgG3 fixes human C' better than IgG1, actual target lysis may be better with IgG1 due to more efficient activation of C4.32 For the most part, human and murine antibodies function comparably well in directing CDC with rabbit C' in in vitro tests and in fixing human or rabbit C'. Some cases may exist, however, in which murine IgG3 is more potent owing to the unique feature of Fc polymerization on cell surfaces apparently not being similarly available to human IgG.33
Despite these considerations, the impact of C' fixation and CDC in therapeutic applications against cancer is uncertain. Two considerations may be relevant: First, the complete first component of human complement is very large (approximately 800 kd) and, like IgM,20 probably has limited extravascular penetration. Second, the cells to which complement has ready access (e.g., cells of the hematopoietic system and vascular endothelium) are endowed with potent phosphoinositol-linked membrane protease activities, decay-accelerating factor (DAF, CD55), and homologous restriction factor (HRF, CD59), which act as steps subsequent to C1 fixation to inactivate the human complement cascade.34, 35 Rabbit, guinea pig, or other heterologous sera can kill cells in vitro that are resistant to human C' because they bypass the human species restriction of these protease activities.
Antibody-Dependent Cellular Cytotoxicity
In antibody-dependent cellular cytotoxicity (ADCC), target cells are coated with antibody and engage effector cells equipped with Fc receptors (FcRs) that bind to the Fc region of IgG, release cytolysins, and lead to cell killing. The only classes demonstrated to mediate ADCC are IgE and IgG. Because of the dangers of anaphylaxis that would be associated with IgE use, antitumor IgE antibodies are not likely to be developed, and further discussion therefore focuses on IgG.
Classically, cells that mediated ADCC were called “K (killer) cells,” and all bear FcRs on their surfaces. Among these, there are at least three IgG FcRs and two IgE FcRs. Table 31.3 lists the FcRs for IgG and their properties. 36, 37, 38 All FcRs are capable of directing the ADCC of effectors against appropriate antibody-coated targets. Cytolytic mechanisms include perforins, a system closely related to the C9 protein of the complement attack complex (C5-C9), serine proteases (granzymes) in large granular lymphocytes (LGLs); superoxides, free radicals, proteases, and lysozymes in monocytes-macrophages and granulocytes; tumor necrosis factor; and other components. As far as is known, the lytic mechanisms of LGLs and cytotoxic lymphocytes are similar or identical, and the monocyte-macrophage and granulocyte mechanisms also share numerous features. The monocyte-granulocyte mechanisms are probably adapted to killing and engulfment of microorganisms, whereas T cells, the closest lineage relative of LGL-NK cells, have the role of killing cells of self-origin that express foreign or neoantigens. In general, the most potent of the mediators of cellular killing in circulation have been the LGLs. These cells also perform natural killing (NK cells), which is an antibody-independent lectin-like ligand interaction system.39 Other effectors, for example, monocytes and granulocytes, have been shown to mediate ADCC against nucleated targets,40 but in most direct comparisons with LGLs, the LGLs were more potent than these numerically more dominant cells.41 Nevertheless, the most effective approaches may include a multipronged attack that enlists the collaboration of more than a single cellular system to kill antibody-coated tumor targets.
TABLE 31.3 FCγ RECEPTORS: PROPERTIES AND BINDING CHARACTERISTICS
Among the features that influence the amount of killing in ADCC are (1) the species origin of the antibody, (2) the IgG subclass, (3) the number of antibody molecules bound per target cell, (4) the ratio of effector cells to targets, (5) the activation state of effectors, (6) the presence of irrelevant IgG, and, perhaps, (7) the presence of tumor cell protective factors and (8) different classes of Fc receptors that enhance or inhibit effective activity.42, 43, 44 These are discussed in turn.
1. The species origin appears to have a significant influence on the ability of an antibody to recruit human effectors to kill human tumors. Although human and rat antibodies mediate ADCC with human effectors, murine antibodies are often less potent in this role. Long ago, isologous antiserum was determined to be more effective with any species' effector cells,45 which suggests that the match of antibody to effector cell Fc receptor is a significant feature of ADCC.
2. In principle, all IgG subclasses are capable of ADCC. However, the IgG1 subclass of humans, the IgG2a and IgG3 subclasses of the mouse, and the IgG2b and IgG3 subclasses of the rat have been inferred to be the most active with human cells. This does not always parallel the order of FcR binding affinity, and other factors of Fc-FcR binding must be postulated that influence the induction of cytolysis.46
3. The selection of highly expressed target antigens has a direct impact on the likelihood that the cell can be killed with ADCC. The control of the number of antibody molecules bound reveals that a nearly linear relationship exists with cytolysis.47 A corollary of this phenomenon is that the modulation of antigen by antibody binding (antigen-antibody complex internalization or shedding) reduces target susceptibility even when baseline antigen is highly expressed.
4. Higher effector-to-target ratios yield increased killing, although a plateau in efficacy typically is achieved at higher ratios.45, 48 In vivo, the ratio of effector cells to targets is not so readily controlled, except by stimulating proliferation or supplementing effectors, but this effect may provide a stronger rationale for treatment in adjuvant settings when the tumor burden is small, that is, after debulking surgery or induction chemotherapy.
5. The activation state of effectors has been shown in several systems, both in vitro and in vivo, to play an important role in the lysis of targets. This activation is achieved with any of several agents and by the expression of different classes of activating or inhibiting Fc receptors. 42, 43, 44 Evaluation of the application of cytokines specific to the range of potential effectors is beyond the scope of this review, but in each instance cytolytic capacity has been strongly correlated with the degree of effector cell activation. Only LGLs (NK cells) appear to have significant antitumor potency in ADCC in the absence of cytokine activation, but here, too, activation with cytokines also increases ADCC killing (Fig. 31.5).48 Interleukin-2 (IL-2) activation of LGLs has been the most widely applied in clinical trials to date (see Chapter 36).
6. The presence of circulating IgG is probably the most problematic of features for exploiting ADCC in vivo. Clearly, the very existence of FcRs and the presence of cytolytic granules are teleologic indications of the relevance of this capability to biology. Although one might argue that monocytes-macrophages and granulocytes are adapted to combat microorganisms, the sole role of T cells is to kill nucleated cells of self that present viral or other abnormal peptides, for which they use distinct cytolytic mechanisms. As stated above, LGLs apparently duplicate the cytolytic mechanisms of T cells but in addition possess FcRs to enable them to interact with antibodies that will direct them to these targets through non–major histocompatibility complex mechanisms. The problem with this interaction is that monomeric Fc of IgG has an affinity of approximately 5 × 105 M-1 (Kd = 2 mmol/L) for the dominant FcR on LGLs (Table 31.3). At a 1 g/dL in vivo concentration, IgG is 65 mmol/L and 30-fold above this Kd, which implies that more than 95% of the FcR on LGLs is occupied with IgG Fc. (The occupancy fraction on monocytes-macrophages with higher-affinity FcR type I is still higher.) Countering this in the biologic interaction is that the affinity of specific IgG for antigen is typically much higher than this, which yields a stable multivalent surface presentation of IgG Fc on the target that in turn may interact in a multivalent manner with the effector cell FcR. In practice, however, most ADCC assays are markedly inhibited by added human serum. (Assays using fetal calf serum are not so affected because IgG is absent due to lack of placental transport in ungulates.)20 Whether the longer-term in vivo incubations of days versus the brief duration (approximately 3 hours) of in vitro assays allow a therapeutic effect in a treatment program requires further study. However, observed clinical responses to antibody therapies (see later) suggest that ADCC may in some instances be operative in vivo.
Figure 31.5 Impact of interleukin 2 (IL-2) on antibody-dependent cellular cytotoxicity (ADCC) after 16 hours of activation of peripheral blood lymphocytes. (NK, natural killer cells.) (Reproduced with permission from Junghans RP. A strategy for evaluating lymphokine activation and novel monoclonal antibodies in antibody-dependent cell-mediated cytotoxicity and effector cell retargeting assays. Cancer Immunol Immunother 1990;31:207.)
7. Interest has recently focused on tumor-based factors that may mediate resistance to ADCC. One such factor is the complement regulatory protein HRF. HRF was originally defined as acting at C2 and C9 of the complement cascade. The cytolytic protein perforin I that is released by LGL-NK cells and cytotoxic lymphocytes is also referred to as C9-related protein and is likewise subject to proteolysis by HRF, which thereby neutralizes the lytic power of the killer cell.49 (The observation has been made, however, that HRF-related protein Ly6 [CD59] does not protect against perforin lysis.) HRF is present at high levels on activated T cells and NK cells and is thought to play a role in protecting these cells from autolysis during lysis of intended targets. Cells that are resistant to ADCC could be induced to become sensitive by blocking with anti-HRF antibodies.34 Another factor that may contribute to cellular resistance is the secretion of mucins, which inhibit the penetration of antibodies and other macromolecules to the cell surfaces. Other issues of tumor penetration are discussed later.
8. Finally, the relevance of the in vitro ADCC assay to in vivo function is much discussed. Only in a few instances has this been examined by comparing ADCC-competent and ADCC-in competent antibodies.44, 50 In a complement-deficient leukemic AKR mouse model, a leukemia-specific IgG monoclonal antibody suppressed tumor, whereas an IgM antibody of the same specificity was ineffective.51 This suggested that binding to antigen was not sufficient and that C' played no role. Other reports of studies in mice showed that the only antibody to induce an in vivo response was that which showed ADCC activity in vitro.52 Several studies have shown that antibody plus IL-2 activation of effectors was much more effective than either alone, which implicates cooperation between the cellular and humoral immune systems that is the sine qua non of ADCC. Human trials in which a leukemic patient received human IgG Fc coupled to a murine antibody showed a more effective response than when the antibody was without human Fc.53 A further human trial with class/isotype-switched CAMPATH-1 antilymphocyte antibody showed a dramatic response in patients with B-cell chronic lymphocytic leukemia only for the one isotype that mediates ADCC in vitro.54
Antibody-dependent phagocytosis may be performed by cells of the granulocytic and monocyte-macrophage lineages. Furthermore, these cells have receptors for C3 fragments, which enhance binding of antibody-coated targets that also activate complement, leading to C3 fixation. Only activated macrophages, however, are capable of engulfing antibody-coated erythrocytes. The ability to engulf larger tumor cells has been uncertain, but one in vitro evaluation of activated monocytes demonstrated phagocytosis of melanoma and neuroblastoma targets when assays were appropriately monitored.55 Nevertheless, phagocytic cells in the liver (i.e., Kupffer cells) and spleen probably are the primary mediators of circulatory clearance of antibody-coated platelets in alloimmune and autoimmune settings56 and in the instances in which rapid clearance of leukemic cells was observed during antibody therapies. Whether these cells are trapped and then lysed by ADCC mechanisms rather than phagocytosis is uncertain.
Antibody binding occurs without cooperation of other elements of the immune system. Therefore, just as antitoxin can prevent a toxin from acting at its target site in the body, antibody can also deny access of growth factors to tumors whose proliferation is factor dependent. This approach has been applied more widely in nonmalignant settings for the suppression of immune responses in autoimmune and alloimmune settings.57 This approach is limited in malignancy because most tumors appear to be autonomous. In principle, an antibody directed against a cytokine should have the same result. However, the short half-lives of most cytokines and the locally high antibody concentrations required may make this approach more difficult. Such autocrine or paracrine loops may be better interrupted by an antireceptor than by an anticytokine antibody. One report has documented a marked enhancement of cytokine activity by antibody to cytokine via t½ prolongation, which runs counter to the goal of suppressing cytokine activity.29
Design of such applications also must consider the receptor occupancy that is necessary for cell survival and proliferation. Only 10% occupancy of the receptor for granulocyte-macrophage colony-stimulating factor is sufficient to induce maximal activation of granulocytes.58 Similarly, one must block more than 90% of the α chain of IL-2 receptor with antibody to have a significant impact on IL-2–dependent, antigen-induced T-cell proliferation.47
Apoptosis is a process by which signals are transmitted through cell surface receptors to induce autoenzyme-mediated cell death or by which downstream events are accessed to achieve the same result. This has been demonstrated most persuasively during development and in the programming of T-cell precursors in the thymus. The Fas antigen is probably the natural membrane receptor for this process and is expressed in liver, heart, thymus, lung, and ovary, although other antigens may exert similar effects. Anti-Fas antibody administration resulted in an extraordinarily complete and rapid tissue destruction in animal studies.59 One report suggests that part of the killing mechanism of T cells is to engage this receptor on target cells.60 Some hematologic malignant cells, like their normal counterparts, are Fas-positive and are potential targets of antibody therapy (a) if these antibodies are not cross-reactive for normal tissue, (b) if ways of engaging the receptor can be selectively achieved (i.e., bifunctional anti-Fas antitumor antibody), or (c) if other antigens unique to tumors can be found that also access this cellular process. To date, such tumor-specific, apoptosis-inducing antigens have not been described, but lineage-associated, apoptosis-inducing antigens have been targeted in the treatment of lymphoma (see later section).
Antibody recognition of antigen entails the presentation of a molecular surface that is the complement in space of the antigen (Fig. 31.3), termed a “mirror image.” In the Jerne network nomenclature, the designation of antigen and antibody becomes arbitrary. The antigen is Ab0, the antibody is Ab1, the antibody to the antibody idiotype is Ab2, and so forth. Although antibody can react with idiotype in many ways, a subset of Ab2 is still considered to exist that mimics Ab0 (antigen), and a fraction of Ab3 raised against Ab2 mimics Ab1 and reacts with Ab0 (antigen).61 Therefore, a tumor antigen may not be immunogenic in the human host that carries it, but a murine antibody (Ab1) can be raised to this antigen, and a goat antibody (Ab2) can be raised to this. This Ab2 antibody includes epitopes that mimic antigen but presents them in a novel context in which they may be immunogenic in the original host. Such Ab2s have been used as vaccines to induce Ab3 antibody responses in the host that can cross-react with antigen (Ab0) on tumor. Antibody therapy in this sense is applied to induce an endogenous antibody and occasionally a T-cell response against tumor.62, 63
ANTIBODY MODIFICATIONS FOR THERAPY
As discussed later, despite all the functions that antibodies perform in vivo, not all antibodies are therapeutically successful. Two major factors have been the focus of research efforts considered in this section: (a) immunogenicity and (b) lack of therapeutic and cytolytic potency. The approaches to improve potency have themselves been twofold: (a) to improve the collaboration of antibodies with the other components of the immune system and (b) to use the antibody as a vector to deliver toxic agents (toxins or radioactivity) to tumor cells. This latter approach essentially abandons the immunologic collaboration of antibodies with the remainder of the immune system.
Reduction of Immunogenicity
Immunogenicity derives from the fact that most antibodies are of non-human origin and as such are foreign proteins in the human host. The human antiglobulin response to mouse antibodies is directed mainly against the C domains of the murine antibody, with typically lower titers against the V domains. To address this problem, three versions of the foreign protein have been prepared. First, there is a chimeric version, in which the mouse C domains are replace by human C domains. Second, there is a “humanized” or hyperchimeric version, in which the murine framework regions are replaced by human framework sequences (Fig. 31.4). Third, entirely “human” IgG produced in vivo (in transgenic mice).64, 65
The first humanized antibody for cancer therapy was the panlymphocyte antibody CAMPATH-1H.66 Since that time, many others have been prepared and tested in humans. The hyperchimeric and humanized antibodies have been much more successful in avoiding antiglobulin67 responses, with an incidence of 4% with CAMPATH-1H and comparably low antiglobulin response rates with anti–Tac-H68 and HuM195.69, 70 The less extensively substituted chimeric antibodies have been more widely applied, with anti–V-region responses observed.61, 71, 72 Further experience is required to ascertain the rules governing these responses. The innovation of human combinatorial phage display libraries70, 73 is also being applied for deriving antiself and antitumor human antibodies. Efforts to suppress human antimurine antibody with immunosuppressive drugs have not been successful to date.
Human IgG C domains (specifically CH2) can confer a longer in vivo t½ on the order of 23 days, for human IgG in humans versus the 1- to 3-day t½ of murine antibody in humans.20 One study compared a chimeric anti-idiotype antibody with the parental murine antibody, demonstrating a prolonged in vivo survival (with t½ longer than 10 days for a nonreactive chimeric construct).47 Other studies involving chimeric antibodies against colon carcinoma yielded survival t½ values of 3 to 6 days and 4 to 12 days.71, 72 The humanized CAMPATH-1H had survival t½ values of 1 to 6 days,67 and anti–Tac-H had values of 2 to 15 days.68 Thus, observed survivals fall short of those expected from controlled studies in normal volunteers. Some of this acceleration in clearance is clearly due to disease-associated catabolic factors,20 but in vivo antigen binding and product preparation may also play a role in rapid clearance of the antibody. Further studies are necessary to better understand the various factors that determine the t½ of the antibody in vivo.
Binding Affinity of Engineered Antibodies
The affinity for substrate (antigen) is an intrinsic characteristic of an antibody conferred by the particular amino acid sequence and spatial presentation of the CDRs. In the past, the affinity that was retrieved from a given hybridoma was an immutable feature of the antibody; it could be altered only by reducing the valence of the product (i.e., Fab vs. Fab'2, IgG vs. IgM), which only reduced affinity. More recent efforts in the preparation of IgG dimers, however, have shown a marked increase in affinity of up to 1,000-fold,74 which may accentuate the improvement in Fc-dependent functions with such constructs. With CDR manipulation in the humanization of antibodies, a major disturbance sometimes occurred in the affinity for antigen. Search for causes of this affinity loss revealed the importance of single, critical residues or carbohydrates to total affinity.75, 76, 77
Novel procedures using phage display technology offer powerful new procedure to improve affinities by mutating the CDR's. Fab molecules are expressed on the surface of phage and are selected against immobilized antigen and enriched in proportion to their affinity. This enabled a random CDR mutagenesis-selection procedure that recapitulates in vitro the in vivo process of affinity selection and maturation.78 By this procedure, the affinity for antigen of any low-affinity antibody can be enhanced 1,000-fold or more in a simple selection procedure. Finally, the issue of valence has been addressed to reduce the likelihood of antigen modulation during therapy by preparing univalent IgG that still retains the Fc effector domains and Fc-dependent functions.79, 80
The opportunities and problems of CDC as a means of killing tumors were outlined earlier. The humanization of antibodies has in some instances shown an improvement in cellular killing with heterologous complement, but for the most part, the effect has not been dramatic and is not sufficiently potent to kill human tumor cells with human complement when murine antibodies failed. On the other hand, the principle of the relative advantage of human over mouse antibodies has been clearly violated by occasional observations of failure to fix C' with chimeric human IgG1 and IgG3 molecules when the murine antibodies have fixed C', first noted by Junghans et al.47 and since corroborated by others.77, 81 In any case, the impact of humanization of IgG antibodies is not expected to render them as effective as IgM antibodies for CDC killing. In addition, the possibility exists that the murine IgG3 may be more potent than any human IgG given its capacity for polymerization on cell membranes with enhanced C' fixation and lysis.33 Finally, dimeric forms of human IgG1 antibodies have been engineered that are far more effective in fixing complement than monomeric versions,82, 83 but none has yet been tested for these effects in humans.
Antibody-Dependent Cellular Cytotoxicity
In contrast to CDC, ADCC with human antibodies show a fairly consistent advantage of over mouse antibodies by their improved efficacy of their interaction with human effector cells. In several tests, a marked increase is seen in the potency of cellular killing in the chimeric constructs with human Fc domains. Of the human isotypes, IgG1 has been consistently the most effective, and chimeric and humanized antibodies are equivalent when normalized to molecules bound per target cell.47 Bifunctional antibodies (BFAs), discussed later, appear to have the best opportunity to enhance this killing activity, although clinical data are very limited at this point. Another approach has been to apply anti–homologous restriction factor (anti-HRF) antibodies to block the inhibitory effects of HRF on ADCC (see earlier), which has been demonstrated in vitro34 but to date has not been not tested in in vivo models.
FEATURES OF SPECIFIC MODIFICATIONS
Winter and collaborators showed that the CDRs of antibodies could be transferred from murine to human frameworks with maintenance of binding specificity,84,85 albeit with losses in affinity of 1.6- to 15-fold. Such affinity losses became a recurrent concern with these constructs (Fig. 31.6). Subsequently, this group humanized a rat antibody, CAMPATH-1, which is directed at a human panlymphocyte antigen, with the intention of therapeutic application in leukemias and lymphomas (see “Clinical Trials of Monoclonal Antibodies in Cancer Therapy” later in the chapter). The second antibody to be humanized for therapy, anti–Tac-H, was derived from the murine anti–human IL-2 receptor antibody anti-Tac for use in alloimmune and autoimmune settings and in Tac-expressing leukemias and lymphomas (Fig. 31.2).47, 86 The manipulations to create this antibody also had a modest impact on affinity, which was reduced from 9 × 109 to 3 × 109M-1 47, but another humanized antibody, HuM195, actually showed increased affinity after engineering.75 Since that time, several antibodies have been humanized, and humanization effectively reduces immunogenicity.
Another type of chimeric antibody genetically couples a natural ligand with an immunoglobulin Fc domain to confer in vivo survival characteristics of antibodies and recruitment of host effector functions. The prototypical immunoadhesin was CD4IgG, which was used to target gp120-expressing cells infected with human immunodeficiency virus87 but may plausibly be extended to human tumor antigens for which ligands can be derived or for which effector cell antigens can be recruited (see the following section).
(BFAs) were devised to address two problems of ADCC. First, effector-target conjugate formation is inefficient due to competition with circulating Fc for FcR binding. Second, mouse antibodies, as noted, often fail to promote ADCC with natural human targets, even in the absence of competing IgG,45 a difficulty partially addressed by “humanization” of antibodies.
BFAs improve conjugate formation by creating molecules that have dual specificities, one to the target cell and one to an “activator” antigen of the killer cell (Fig. 31.7).88 BFAs direct killer cells—both NK cells and T cells—to lyse targets dictated by the antibody specificity in a function called “effector cell retargeting.” In the case of NK cells, the BFA substitutes through its anti-FcR (anti-CD16) moiety a high-affinity antigen-antibody interaction for a low-affinity, nonspecific Fc-FcR interaction, causing a marked improvement in conjugate formation and target cell lysis, even in the presence of human serum. In the case of T cells, the BFA recruits an entirely new cell class into antibody-directed killing. With the binding of anti-CD3 to the CD3 antigen, the T cell is stimulated into a killing mode, bypassing the normal major histocompatibility complex and antigen-specific restrictions of T-cell killing.
CD16 and CD3 each involve proteins that are central to the killing mechanisms of LGLs and cytotoxic T lymphocytes, respectively. The CD3 antibodies are mainly directed against the ε chain of the CD3 complex. Other surface antigens on these cells also have been used, generally to less effect,88, 89 although promising results with tri functional antibodies that are also anti-CD28 have been obtained.90 Other FcRs have been targeted on monocytes-macrophages.40
Figure 31.6 Schematic of chimeric versus hyperchimeric (humanized) antibodies. See also Figure 31.2. (Reproduced with permission from Junghans RP, Waldmann TA, Landolfi NF, et al. Anti Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders. Cancer Res 1990; 50:1495.)
Figure 31.7 Schematic of bifunctional antibodies as conjugates versus quadroma (hybrid hybridoma) products.
Bifunctional constructs are generated by chemical processes, by chemical cross-linking (heteroaggregates) of complete IgG, by chain shuffling, by cross-linking of Fab' molecules, or by hybrid hybridoma (quadroma) technology.91 These latter products are the most useful for therapy because they provide a continuous supply of monomeric IgG products with normal in vivo survivals; however, because 10 combinations of heavy and light chains are predicted from the parental antibodies in the mix, of which only one is the desired BFA, purification by standard chromatographic methods may not be able to provide a pure BFA preparation.92 On the other hand, aggregates and fragments prepared with high yield by other methods have abbreviated survivals in vivo. Newer genetic constructs are in preparation—single-chain BFAs, fos-jun–linked Fabs, immunoadhesin with antitumor antibody, and so on—whose efficacy and advantages will be determined in the near future. Bifunctional immunoadhesin antibody constructs have linked anti-CD3 antibody with hormones or cytokines to interact with receptor-bearing cells on tumor targets,93, 94 and other work is underway using B7 as an immunoadhesin in BFAs to recruit effector cells through CD28.
Typically, these constructs are markedly more active than the parental antibodies in net lysis of tumor cell targets (the difference is particularly great in the presence of human serum). BFAs can cure or prevent tumors in animal hosts in which unmodified antitumor antibodies are inactive.95, 96, 97 Although this approach is promising, clinical trials have been limited.
Antibody as Vector for Toxic Agents
Many of the deficiencies of antibodies in recruitment of host effector functions are being addressed by the methods described previously. Other approaches have bypassed this effort by exploiting the specificity and affinity of antibodies to direct cytotoxic agents against tumor cells. These toxic agents include chemotoxins, biotoxins, and radioisotopes. Radioimmunotherapies are discussed later in this chapter.
Conjugates of antibody with anthracyclines and other chemotherapy agents have the potential to permit delivery of high doses of drug to antigen-expressing tumor cells while sparing nonexpressor normal cells from the toxic effects of the drug. Several studies have shown efficacy in animal models in which antibody alone and drug alone were ineffective.98, 99 As a rule, chemotoxins and biotoxins are expected to be more effective in cases in which the antigen is known to be internalized after antibody binding, as with the Lewis Y antigen targeted by BR96-doxorubicin conjugates.98 This approach has been taken furthest with the anti-CD33 targeting of leukemia cells in patients using a humanized Ig conjugated to calicheamicin, a small-molecule toxin. (see “Clinical Trials of Monoclonal Antibodies in Cancer Therapy”).
A merging of BFA technology with chemotherapy delivery has been developed that displays an enzymatic activity that converts prodrug to active agent at the site of the tumor,100 called “antibody-dependent enzyme-prodrug therapy” (ADEPT). The design of this therapy is dictated by a problem common to all antibody-toxin/radioisotope approaches: the high body burden of toxin and nonspecific toxic effects at sites away from tumor. To circumvent this problem, antibody in the form of enzyme-conjugated antibody (ECA) is allowed to equilibrate to obtain optimal tumor penetration (Fig. 31.8). When antigen saturation is achieved and the tumor to normal tissue ratio of ECA is optimal, then a low molecular weight prodrug is injected. Rapid tumor penetration occurs, and prodrug is then converted to active drug, thereby killing tumor cells. To accentuate this effect, the unbound ECA may be cleared from circulation by a second antibody to exaggerate the tumor to plasma ratio of ECA and minimize prodrug conversion except at sites of antibody binding. Optimizations of this strategy have been analyzed by Jain and coworkers.101 Analogous approaches with radiolabeled antibodies are under development.102
Figure 31.8 Schematic of antibody-dependent enzyme-prodrug therapy (ADEPT). (Reproduced with permission from Senter PD. Activation of prodrugs by antibody-enzyme conjugates: a new approach to cancer therapy. FASEB J 1990;4:188.)
Several animal studies have validated the principles of this approach, and clinical studies have been initiated.103 In pioneering work by Pastan, Vitetta, and others, the application of extremely potent biotoxins to clinical cancer therapy was explored. The first-generation products were chemically cross-linked conjugates of antibody with unmodified toxins. Early studies determined that whole toxin molecules coupled with antibody were too toxic due to nonspecific uptake.104 Molecular analysis of toxin domains was done for the purpose of designing molecules that were less nonspecifically toxic. Three domains corresponded to specific functions, as exemplified by work with the Pseudomonas exotoxin (PE): adherence, causing nonspecific binding and uptake, mainly by liver; translocation, responsible for moving the toxin from the endosomic vesicle to the cytoplasm; and adenosine diphosphate ribosylation, the enzymic activity that is responsible for inactivating elongation factor 2 and arresting protein synthesis.105 In principle, a single molecule of toxin is sufficient to kill the cell. Studies were performed with diphtheria toxin, which acts like PE, and with ricin, which inhibits protein synthesis by acting on the ribosome directly.
Modifications were made to eliminate or block the adherence domain, which led to a marked reduction in nonspecific toxicity. In addition, the discovery was made that the chemical coupling of toxins to antibody could be accompanied by a loss of specific activity of the toxin due to preferential use of key active-site lysines by the linkage. To bypass this, a genetic construct of PE was prepared that put into a single chain the antigen-recognition domain of the antibody (single-chain Fv) and a truncated version of the toxin (Fig. 31.9).106 This construct, anti-Tac (single-chain Fv)–PE40, had high toxicity for antigen-expressing cells and no toxicity for antigen-negative cells; the concentration causing 50% inhibition (IC50) was higher for lower-expressing cells, but in each case the IC50 corresponded to binding of approximately 100 antibody molecules per cell. Another construct was designed with truncated PE and an anti-CD22 variable domain (Fv).107 Sixteen patients with cladribine-refractory hairy-cell leukemia (a leukemia whose malignant cells are strongly positive for CD22) were treated with this immunotoxin, BL22. Two patients experienced hemolytic uremic syndrome, but otherwise the treatment was well tolerated. Despite the poor prognosis of these heavily pretreated and refractory patients, 11 patients (69%) achieved a complete remission, and 10 of these had elimination of minimal residual disease by immunohistochemical analysis of the bone marrow. Several additional toxin constructs have been prepared with similar in vitro profiles of activity, including B4-blocked ricin108 and CD22-ricin A for treating B-cell chronic lymphocytic leukemia and purging leukemic cells from autologous marrow grafts.109, 110 Excellent summaries of these applications are available.104, 111
Figure 31.9 Single-chain Fv-PE40 toxin. (PE40, truncated version of Pseudomonas exotoxin; VH, variable region of the heavy chain; VL, variable region of the light chain.) (Reproduced with permission from Chaudhary VK, Queen C, Junghans RP, et al. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 1989; 339:394.)
Antibody as a Vector for Radioactivity
Labeling of antibodies with radioactive atoms is another major approach to antibody-based cancer treatment that has received considerable attention. This approach remains promising and benefits from some inherent advantages over direct (passive) or toxin-based immunotherapy. These advantages result from the use of radiation as the primary cytotoxic agent. The cytotoxic activity of radiolabeled antibodies is the result of electron (beta-particle) or alpha-particle emissions that occur when the radionuclide decays while attached to an antibody that is bound to tumor-associated antigen. These emissions travel a distance that depends on their type (electron or alpha particle) and energy. As they travel, they deposit energy along their paths. If an adequate number of such emissions traverse a given tumor cell nucleus, the total energy deposited within the nucleus will kill the cell by causing an irreparable number of DNA strand breaks. A radiolabeled antibody attached to a particular tumor cell may also, because of the range and random direction of its emissions, kill adjacent tumor cells that do not express the antigen or that have not been reached by the radiolabeled antibody. Because of the range of the emissions, tumor cell kill is not necessarily dependent on internalization of the antigen-bound antibody, nor does it depend on any specific metabolic process of the tumor cell.
As a further distinguishing feature of radiolabeled antibody therapy, the loss of cytotoxic activity over time due to radioactive decay has important implications for its use in comparison with the use of passive or toxin-based immunotherapy. Depending on the clearance kinetics of the antibody and of the free radionuclide after detachment from the antibody, radionuclide loss due to decay may be either advantageous or disadvantageous. If the half-life of the radionuclide matches the uptake kinetics of the target tissue, and the antibody remains tumor-associated for a sufficient period of time, the decrease in radioactivity reduces normal tissue exposure once the antibody dissociates or is catabolized. A radionuclide that is cleared too rapidly will deliver the largest fraction of its decay energy to normal tissue before reaching the tumor. Alternatively, a radionuclide with a prolonged residence time will deliver a small fraction of its decay energy to the tumor, because the time during which it is tumor-associated is a small proportion of its total decay lifetime.
The radionuclide half-life also determines the rate at which radiation is delivered to the target cells (the dose rate). Depending on the repair rate of the target cells, this value may be more important than the total dose. A very long-lived radionuclide (i.e., one with a very low dose rate) may be incapable of eradicating tumor cells that exhibit a rapid repair capacity, because the cells may be capable of repairing the radiation-induced damage as it occurs. This phenomenon does not apply to alpha-particle emitters, because the damage to DNA from the energy deposited along each alpha-particle track is far greater than the repair capacity of cells.
Although it is not essential for tumor cell kill, an additional feature of some radionuclides that has permitted a detailed investigation of the in vivo pharmacokinetics of radiolabeled antibodies is the release of photons in addition to electrons and alpha particles during decay. Relative to electrons and alpha particles, photons deposit very little energy within tissue, and those that emit sufficient energy, between 100 and 400 keV, can be imaged externally. Images of the spatial distribution of radiolabeled antibody within a patient may be obtained using nuclear medicine scanners. After a low level of radioactivity is administered, such pharmacokinetic information can be used to assess antibody targeting and to determine the likelihood of tumor eradication or normal tissue morbidity before a therapeutic dose is given.
This brief introduction to the unique aspects of antibody therapy combined with radionuclides reveals some of the complexity of radioimmunotherapy. A large number of parameters must be chosen for the implementation of this approach. The optimal treatment strategy for one set of circumstances (disease type, location, radiosensitivity, patient treatment history) may not apply under a different set of conditions. The following section reviews some of the pharmacologic and dosimetric considerations associated with the implementation of radioimmunotherapy.
Pharmacology and Pharmacokinetics of Radiolabeled Monoclonal Antibodies
The pharmacokinetics of radiolabeled antibodies is difficult to describe in terms of average or typical kinetics. In general, in vivo behavior depends on the antibody, the target antigen, the radiolabel, and the patient's prior treatment history (e.g., exposure to mouse-derived antibody). The kinetics may change depending on whether the antibody is an intact IgG or a fragment and whether it cross-reacts with normal tissue. Antigens shed into the circulation also may affect antibody kinetics by complexing with and increasing the clearance rate of circulating antibody. Internalization of a cell-bound antibody-antigen complex may lead to catabolism of the complex, followed by release of the radionuclide. Depending on the technique used to label the antibody, radionuclide also may be released while the antibody is in circulation. The kinetics of the free radionuclide reflect the pharmacokinetics of the parent element rather than that of the administered antibody. Additionally, if previous antibody exposure produces a human immune reaction, the pharmacokinetics are profoundly affected. Mouse-derived antibodies elicit human antimouse activity (HAMA), and humanized antibodies may elicit human antihuman activity (HAHA). The effect in both cases is usually rapid complexing with administered antibody, followed by rapid clearance of the complex from the circulation. This phenomenon may eliminate the therapeutic activity of radiolabeled antibody and thereby greatly reduce the applicability of radioimmunotherapy. A human immune reaction (HAMA or HAHA) usually precludes the use of multiple courses of radioimmunotherapy or the retreatment of patients who have undergone radioimmunotherapy.
As indicated earlier, the photon emissions of most radionuclides used in radioimmunotherapy allow for a detailed assessment of antibody pharmacokinetics. Measurement of the radioactivity in sequential blood samples is generally combined with gamma camera imaging and with whole-body, urine measurements, or both. Imaging is rarely performed after a therapeutic administration, however, because the radioactivity in the patient exceeds the imaging (or counting) capacity of most nuclear medicine imaging devices. Imaging information obtained from a trace-labeled administration of antibody is used instead to project the kinetics of the therapeutic administration.
The distinction between a therapeutic and a tracer administration of antibody lies in the amount of radioactivity and the type of radionuclide administered. Radioactivity, expressed in megabecquerels (MBq, or millions of decays per second), may vary from a diagnostic administration of 20 to 200 MBq to a therapeutic administration of 2,000 to 15,000 MBq for iodine-131 (131I), one of the most commonly used radionuclides in radioimmunotherapy. The very large range of values for therapy reflects the diversity of antibodies used and the range of patient responses. Doses as low as 1,000 MBq have elicited patient responses in B-cell lymphoma.112 Alternatively, doses as high as 22,000 MBq have been used in the treatment of B-cell lymphoma. Therapeutic doses of the radiolabeled conjugate depend on the choice of antibody and antigen target.
A number of studies have examined the relationship between the amount of antibody administered and the resulting pharmacokinetics.113, 114, 115, 116, 117, 118,119 In most cases, a (non–statistically significant) trend toward longer plasma clearance half-times is observed with increasing milligram dose. In those cases in which a statistically significant effect is observed,25, 113, 119, 120 the results may be due to diminished binding and removal of antibody from the circulation once target antigen sites are saturated. The relationship between serum pharmacokinetics and antibody dose is therefore likely to depend on the available antigen sites and their distribution relative to the milligram amount of administered antibody. Antibodies that exhibit such dependence generally yield improved diagnostic119 and therapeutic results120 when the dose is increased.
The plasma clearance half-times of antibodies that target hematologic malignancies are generally shorter than those of solid tumor antibodies. In hematologic malignancies, the half-time of the first component of plasma clearance is on the order of 1 to 3 hours.112, 117, 121 This is thought to reflect rapid binding of antibody to circulating and therefore readily available target antigen sites. Except for antibodies that target antigen that is also released in circulation, the plasma clearance curves for solid tumor antibodies are generally fit by a single-exponential or double-exponential curve in which the half-time of the rapid component is on the order of 3 to 10 hours.
Due to the high molecular weight of most radioimmunoconjugates, the initial distribution of intravenously administered antibody is generally confined to the vascular space and to the extracellular space of tissues that lack a fully developed capillary basal lamina (e.g., marrow, liver, spleen, and other tissues of the reticuloendothelial system).122, 123 In a 70-kg man, this volume is approximately 4 L; that is, it modestly exceeds plasma volume. In this way, significantly higher “apparent” distribution volumes may be observed when targeting tumor cells that are rapidly accessible to intravenously administered radiolabeled antibody.121 In such cases, antibody binding to tumor cells provides an alternative mechanism for the rapid reduction in plasma concentration.
The larger molecular weight of antibodies raises issues that are not generally relevant to the targeting of chemotherapeutic agents. The molecular weight of the IgG antibody, the isotype most commonly used for cancer treatment, is 150 kd, which is 150 times heavier than most chemotherapeutic agents. In order to target antigen-positive cells, intravenously administered antibody must cross the capillary basal lamina and then traverse the extravascular space of solid tumor.
In most normal organs, the capillary basal lamina presents a substantial barrier to traversal of a 150-kd protein.124 Due to the “leaky” nature of tumor vasculature,124, 125126 this barrier is greatly diminished in tumors, and passage of antibodies is close to that of low molecular weight chemotherapeutic agents. The vascular permeability coefficient of tumor capillaries for methotrexate, for example, ranges from 1 to 10 × 10-6 cm/second127; the corresponding value for IgG is 0.6 × 10-6 cm/second.124 Once the antibody reaches the extravascular side of the capillary, it must cross the interstitial space to bind antigen-positive cells. For most chemotherapeutic agents, such transport occurs by diffusion.127 A typical low molecular weight cytotoxic agent (350 Da) has a diffusion coefficient in the interstitial space of tumor of approximately 6.4 × 10-6 cm2/second.128 The corresponding value for IgG antibody ranges from 0.005 to 0.015 × 10-6 cm2/second.129 These values translate into 4 seconds (for the low molecular weight cytotoxic agent) versus 0.5 to 1 hour (for the antibody) to achieve 16% of the source concentration at a 100-mm distance. Antibody transport across the interstitial space is therefore primarily dependent on bulk fluid flow or convection. Such flow relies on a positive pressure difference between the periphery and the tumor center.
Interstitial pressure is consistently higher in solid tumors than in normal tissues.130, 131 This pressure is thought to arise because solid tumors do not have completely developed lymphatic drainage. In normal tissues, vascular fluid that filters into the interstitial space and is not reabsorbed into the microvascular network is taken up by lymphatic vessels. In solid tumors, such vessels may not exist or may be inadequate to reabsorb excess interstitial fluid rapidly.132 Additionally, interstitial pressure increases with the size of the tumor. Such interstitial fluid pressure presents a significant physiologic barrier to antibody penetration of large solid tumors and may help explain the highly nonuniform distribution of antibody in solid tumors as well as observations of increased antibody uptake in smaller tumor cell clusters.133
A further barrier to antibody penetration of a cluster of antigen-positive cells is the binding-site barrier phenomenon.134, 135 The binding-site barrier arises as a result of the low antibody concentration in the tumor interstitial space relative to the local antigen concentration. (The local concentration of antigen sites depends on the number of antigen sites per cell [typically 104 to 106] and the number of cells per unit volume.) The antibody is, in effect, prevented from diffusing to the interior of the solid tumor until the antigen sites in the periphery are occupied. In systems in which interstitial pressure is not of concern (e.g., in vitro tumor cell spheroids or micrometastases), the binding-site barrier may, depending on the concentration of antibody, yield a highly nonuniform distribution of antigen-bound, radiolabeled antibody, with very high concentrations in the periphery and negligible amounts in the center.136, 137
CONSIDERATIONS SPECIFIC TO RADIOIMMUNOTHERAPY
Dose in the context of chemotherapy is fundamentally different from the therapeutically relevant quantity in radioimmunotherapy. The quantity of a cytotoxic agent that is delivered to a patient in the chemotherapeutic context is generally the amount in milligrams or the area under a blood time × concentration curve. The latter provides a measure of the drug's residence time in the circulation. The radioimmunotherapeutic equivalents are activity (also often referred to as dose) in megabecquerels and cumulated activity in megabecquerels × seconds (total number of radionuclide decays). The cumulated activity need not be limited to blood circulation. For solid tumors in particular, one is interested in the cumulated activity in the tumor. Using external imaging at various times after antibody administration to obtain a time-activity curve that is then integrated over time, one may obtain the cumulated activity for tumor or other tissues. The therapeutically relevant quantity for radioimmunotherapy, however, is the absorbed dose (also often referred to as the dose) in grays (energy absorbed per unit mass of tissue). This value is obtained by multiplying the total number of decays that have occurred in a given tissue (i.e., the cumulated activity) by the total energy released per radionuclide decay and by a factor that accounts for the fraction of emitted energy that is absorbed within the tissue. This fraction depends on the tissue's geometry and the energy (or range) of each radionuclide emission. Dividing by the mass of the target tissue yields the absorbed dose. The resulting absorbed-dose estimate only accounts for emissions that occur within the given tissue. Depending on the range and type of radionuclide emissions, radioactivity in other organs also may contribute to the total absorbed dose of a given target organ. Contributions from other organs are calculated as described earlier, except that the geometric factor reflects the fraction of emitted energy in a source organ that is absorbed by the given target organ. The total target tissue absorbed dose is then obtained by adding the absorbed dose contributions from all the source organs to the target tissue self-dose. The procedure described here was developed by the Medical Internal Radiation Dose Committee.138 The absorbed dose to a given organ is a much more precise measure of cytotoxic potential than the administered activity or the cumulated activity, because the pharmacokinetics and the radionuclide properties are accounted for by the absorbed-dose value.
The red marrow is the dose-limiting organ in most implementations of intravenously administered radiolabeled antibody.139, 140 Marrow vasculature, unlike that of most normal organs, is fenestrated and does not present a significant barrier to antibody penetration. The red marrow is composed of cells that are continuously undergoing cell division and that are therefore more radiosensitive.141 These two factors—easy accessibility and enhanced radiosensitivity—account for the marrow toxicity that is observed in almost all radiolabeled-antibody dose-escalation studies.
The marrow absorbed dose from radiolabeled antibody that does not bind to components of the marrow, blood, or bone is generally obtained by assuming that marrow kinetics is the same as that of blood and multiplying by a factor to account for the different antibody concentrations in the two volumes.139, 142 In hematologic disease, antibody binding to specific blood or marrow components occurs and must be considered in determining the red marrow absorbed dose.143 In this case, imaging of a marrow-rich, low-background region (e.g., head and neck of the femur), in combination with one or more bone marrow biopsies, is generally used to obtain a time-activity curve for red marrow dosimetry.143, 144
Several of the radioimmunotherapy protocols for patients with hematologic diseases include hematopoietic stem cell transplantation. In such protocols, the red marrow is no longer the dose-limiting organ, and preliminary results suggest that lung, liver, or renal toxicity may limit the total dose that can be administered.69, 145, 146 In such cases, determination of the spatial distribution of absorbed dose and a dose-volume histogram for the actual organ volume of each patient are necessary to assess the probability of normal tissue morbidity. Although techniques for determining the spatial distribution of absorbed dose by performing three-dimensional dosimetry have been developed,147, 148 a key obstacle to their clinical implementation has been the difficulty of obtaining accurate, patient-specific, three-dimensional biodistribution data. Ongoing improvements in quantitative imaging with single-photon emission computed tomography may eventually overcome this difficulty.
The choice of an appropriate isotope depends on a variety of factors, including the physical and biological half-life of the nuclide and its emission characteristics, the labeling efficiency of the isotope, and the pharmacology of the immunoconjugate. Because of their long range, beta particles can destroy target cells without antigen-antibody complex internalization and may also kill antigen-negative tumor cells. The gamma emissions from 131I allow dosimetry studies to be performed easily, but treatment at high doses requires patient isolation and can result in significant radiation exposure to hospital staff. Additionally, the 8-day half-life poses a waste hazard. Radiolabeling with 131I can also cause loss of biological function, particularly at high specific activities. This decrease in immunoreactivity is directly related to the number of tyrosine residues in the hypervariable region of the mAb to which radioiodine attaches.149
Yttrium-90 (90Y) is a pure β-emitter; its lack of gamma emissions allows outpatient administration of high doses and reduces radiation exposure risk to hospital staff, and the high-energy beta allows a low effective dose.150, 151 If the targeted antigen undergoes modulation, 90Y is more likely to be retained intracellularly than 131I.152 Moreover, if 90Y dissociates from the mAb complex in vivo, the isotope is likely to be deposited in bone, potentially delivering additional radiation to leukemic cells in the marrow. Therapy with 90Y, however, poses several difficulties: (a) unlike 131I, 90Y cannot be directly conjugated to a mAB but must be linked to the antibody by a bifunctional chelate, and (b) because of the absence of gamma emissions, biodistribution and dosimetry studies require administration of mAb trace-labeled with a second isotope, usually Indium-111 (111In). The biodistribution of 90Y and 111In is not identical; studies have shown, however, that it is similar enough that the prediction of safe doses of radiolabeled antibody with maximal antitumor effect is possible.153 The use of quantitative positron emission tomography (PET) following the administration of 86Y-labeled mAb may permit more precise dose estimates, thereby maximizing the antitumor effect and minimizing toxicity.154 Other radiometals, such as rhenium-186, rhenium-188, and copper-67, have been studied.
Alpha particle–emitting isotopes, such as 212Bi, 213Bi, and 211At, have also displayed potent antitumor effects.155 Because of their high linear energy transfer, as few as one to two alpha particles can destroy a target cell. The 50 to 80 µm range of these particles could result in decreased toxicity to surrounding normal bystander cells. The potential for specific antitumor effects makes targeted alpha-particle therapy an attractive approach for the treatment of micrometastatic disease or minimal residual disease.
TABLE 31.4 INCIDENCE OF HUMAN ANTIMOUSE ACTIVITY (HAMA) RESPONSES IN CLINICAL TRIALS OF MONOCLONAL ANTIBODIES (mAbs)
The safety and feasibility of targeted alpha-particle therapy was first demonstrated using humanized anti-CD33 HuM195 labeled with213Bi in patients with myeloid leukemia.156 Fourteen of the 18 patients had a reduction in the percentage of bone marrow blasts after therapy; however, there were no complete remissions, demonstrating the difficulty of targeting one or two 213Bi atoms to each leukemic blast at the specific activities used in this trial. Subsequently, remissions have been observed in some patients treated with 213Bi-HuM195 after partial cytoreduction with cytarabine.157
225Ac decays by alpha emission, with a 10-day half-life through three atoms, each of which also emits an alpha particle, yielding a total of four alpha particles, and it can be conjugated to a variety of antibodies using derivatives of the macrocyclic ligand 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA). Therefore, 225Ac-DOTA can act as an atomic nanogenerator, delivering an alpha-particle cascade to a cancer cell when coupled to an internalizing antibody. As a result of these properties, 225Ac immunoconjugates are approximately 1,000 times more potent than 213Bi-containing conjugates.158 Although this increased potency could make 225Ac more effective than other α-emitters, the possibility of free daughter radioisotopes in circulation after decay of 225Ac raises concerns about the potential toxicity of this isotope. In nude mice bearing human prostate carcinoma and lymphoma xenografts, single nanocurie doses of 225Ac-labeled, tumor-specific antibodies prolonged survival compared with controls and cured a substantial proportion of animals.158
Produced by the bombardment of bismuth with alpha particles in a cyclotron, the halogen 211At emits two alpha particles in its decay to stable 107Pb.159 Due to its long 7.2-hour half-life, 211At-labeled constructs can be used even when the targeting molecule does not gain immediate access to tumor cells. Additionally, its daughter, polonium-211 (211Po), emits K x-rays that permit photon counting of samples and external imaging for biodistribution studies.
Investigators at Duke University have extensively studied 211At-81C6, a chimeric astatine-labeled antibody that targets tenascin, a glycoprotein overexpressed on gliomas relative to normal brain tissue. Early results of a phase I dose-escalation trial of 211At-81C6 in patients with malignant gliomas following surgical resection of the tumor suggest that adjuvant therapy with 211At-81C6 prolongs survival in these patients compared with historic controls.160
Like 225Ac, radium-223 (223Ra) emits four alpha particles over its decay scheme. Because of its bone-seeking properties, unconjugated cationic 223Ra is a promising candidate for delivery of high-LET radiation to cancer cells on bone surfaces. Preliminary results of a clinical phase I study demonstrated reduction in pain intensity and tumor marker levels in the treatment of skeletal metastases in patients with prostate and breast cancer.161
In an effort to reduce radiation doses to normal organs and improve tumor to normal organ dose ratios, pretargeted methods of radioimmunotherapy have been developed. First, a monoclonal antibody or engineered targeting molecule conjugated to streptavidin is given. After administration of a biotinylated N-acetylgalactosamine–containing “clearing agent” to remove excess circulating antibody, therapeutically radiolabeled biotin is infused. The radiolabeled biotin can bind specifically to “pretargeted” streptavidin at the tumor, while unbound radiolabeled biotin is rapidly excreted in the urine.162
This approach has been applied to a mouse model of adult T-cell leukemia (ATL) that expresses the cell-surface marker CD25.163 After treatment with a streptavidin-labeled, humanized anti-CD25 antibody and the clearing agent, immunodeficient mice with human ATL received DOTA-biotin labeled with either 213Bi or 90Y. Treatment with 213Bi reduced the levels of the surrogate tumor markers human β2-microglobulin and soluble CD25 and improved survival compared with controls. Treatment with 90Y, however, did not improve survival compared with controls. Mice treated with 213Bi using the pretargeting approach survived longer than those treated with 213Bi labeled directly to anti-Tac. This approach was also studied using an anti-CD25 single-chain Fv-streptavidin fusion protein, followed by radiolabeled biotin. Significant antitumor effects were seen after administration of 213Bi-DOTA-biotin to leukemic mice, and when the 213Bi-DOTA-biotin was combined with unconjugated anti-Tac, 7 of 10 mice were cured. In a recent phase I trial, 15 patients with relapsed or refractory CD20-positive B-cell NHL received an anti-CD20 second-generation fusion protein (B9E9FP), followed by a synthetic clearing agent 48 or 72 hours later to remove the unbound B9E9FP.164 Twenty-four hours later, 90Y-DOTA-biotin or 111In-DOTA-biotin was injected. In this trial, the clearing agent was very effective in removing the B9E9FP, and the radioimmunoconjugate was rapidly absorbed at the tumor sites. Both the nonhematologic and hematologic toxicities were minimal, and three patients had a response.
Another pretargeting approach that has yielded promising results in animal studies and clinical trials uses a bifunctional antibody in which one arm binds to tumor antigen and the other to a radiolabeled ligand. In some implementations, the radiolabeled ligand is designed to be multivalent and therefore capable of attaching to the ligand-binding arm of two different bifunctional antibodies.165
TOXICITY OF ANTIBODY THERAPIES
Most native Abs, whether rodent or human, are remarkably nontoxic. Maximum tolerated doses (MTDs) are generally not reached in therapeutic trials of mAbs. However, reaching an MTD may be irrelevant, since the goal of mAb therapy is usually to saturate available target sites, thereby achieving the maximum biologic response in the tumor. Hence, further increases in delivered doses may not improve cell kill and may have the theoretical adverse consequences of increased immunogenicity and rapid modulation (loss) of cell surface protein targets.
The mAbs with the most potent activities in vitro (CDC or ADCC) tend to have the most prominent toxicities. Administration of CAMPATH-1H and rituximab (anti-CD20), each a potent activator of the human immune system, results in fever, chills, and rigors in a dose-dependent manner. Usually the infusion-related reaction follows the first dose of antibody only.67 Release of cytokines from targeted cells also may contribute to toxicity. In principle, one would expect that toxicities of mAbs should relate to targeted tissues, neoplastic or normal. Hence, the more specific a mAb, the fewer toxic effects expected. For example, the antibody 3F8, a potent activator of human complement, targets GD2 on neuroblastoma but also binds to GD2 on peripheral nerve, which results in a severe pain syndrome.166 CAMPATH-1H, which targets both normal as well as malignant lymphoid cells, is associated with a high rate of opportunistic infections in treated patients, including infections with herpes viruses, cytomegalovirus, and Pneumocystis carinii.67
HAMA is sometimes characterized as an adverse effect, but it is generally not defined as a treatment-related toxicity (Table 31.4). Treatment of patients with an active HAMA response does not appear to increase toxicity; it can lead to adverse consequences for pharmacokinetics due to rapid clearing of antibody and the development of serum sickness.167 Anaphylaxis has been reported in fewer than 1% of infused patients.
Toxicities associated with conjugated mAbs are generally a consequence of the cytotoxic agent carried by the mAb. With radioimmunoconjugates, myelosuppression is prominent in all studies in which dose escalation is applied.145, 151, 167, 168, 169, 170, 171 Autologous or allogeneic bone marrow transplantation is often required as a rescue in treated patients.145, 167 Toxin conjugates pose a special problem, and some unusual toxicities have been observed. Temporary hepatic injury, as evidenced by elevations in liver enzyme function test results, and vascular leak syndromes, characterized by weight gain, edema, and hypoalbuminemia, are also seen.105, 108, 109 Neurologic toxicity has been observed, but this effect may be due to targeting of neural tissues.172 The long-term consequences of therapy with mAb constructs are entirely unknown but are not expected to differ from those of the cytotoxic agent carried by the mAb.
CLINICAL TRIALS OF MONOCLONAL ANTIBODIES IN CANCER THERAPY
Numerous therapeutic and radioimmunodiagnostic trials with mAbs or mAb constructs have been reported. Those that illustrate important aspects of mAb therapy or describe pivotal trials that have altered the standard of care for a certain malignancy are addressed in this chapter (Table 31.5).
In early trials, the majority of the monoclonal antibodies were of murine origin. These antibodies were safe, even at high doses. However, the usefulness of rodent mAb was limited due to the high rate of HAMA, which developed in most patients following the first dose of antibody. Additionally, murine isotypes are sometimes not capable of effector activity. Genetic engineering has led to the production of partially human (chimeric), more fully human (humanized), and fully human antibodies in order to improve therapeutic efficacy. Dozens of these chimeric or humanized mAbs have been investigated in clinical trials. Several showed significant activity against their targets, minimal toxicity, and a marked reduction in HAMA compared with their murine counterparts.
TABLE 31.5 FDA-APPROVED MONOCLONAL ANTIBODIES, TARGETS, INDICATIONS, AND OTHER USES
To date, eight mAbs have been approved by the US Food and Drug Administration for human use in the treatment of specific malignancies. Rituximab (Rituxan) was the first mAb approved by the FDA in 1997 for the treatment of relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma (B-NHL). Today, rituximab is used in patients with many types of CD20-positive B-NHL. Based on an overall response rate of 33% in heavily pretreated chronic lymphocytic leukemia (CLL) patients, alemtuzumab (Campath-1H) was approved by the FDA for the treatment of fludarabine-refractory B-cell CLL. Patients with metastatic Her-2 Neu overexpressing metastatic breast carcinoma can receive targeted therapy with trastuzumab (Herceptin). Bevacizumab and cetuximab recently received FDA approval for the treatment of metastatic colorectal carcinoma. Gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate, was approved by the FDA in 2000 for the treatment of CD33-positive, relapsed acute myeloid leukemia (AML) patients who are 60 years of age or older. 90Y-ibritumomab tiuxetan (Zevalin) was the first radiolabeled antibody approved for cancer therapy. Subsequently, 131I-tositumomab (Bexxar) was approved by the FDA for the treatment of relapsed or refractory low-grade follicular or transformed CD20-positive B-cell NHL.
Rituximab, a chimeric IgG1 anti-CD20 antibody, represents an important advance in the treatment of B-cell NHL. Multiple mechanisms of action of rituximab have been proposed, including complement fixation, ADCC, and direct cytotoxicity via apoptotic pathways activated by binding to CD20.173 In the phase II trial that led to its approval by the FDA, the overall rate of response to single-agent rituximab in patients with heavily pretreated, relapsed low-grade lymphoma was 48%, with a 12-month median duration of response.174 Similarly, phase II studies of relapsed or refractory intermediate and high-grade lymphoma demonstrated a 32% overall rate of response to rituximab alone. Patients with untreated indolent NHL achieved an overall response rate of 73%, a complete response rate of 37%, and a median progression-free survival of 34 months with rituximab therapy.175 Hainsworth et al. administered rituximab weekly for 4 weeks at 6-month intervals. A Swiss research group investigated the benefit of maintenance rituximab.176 In this study, all patients received four weekly doses of rituximab. At week 12, patients with responding or stable disease were then randomized to no further treatment or a prolonged course of rituximab (one dose of antibody at 2-month intervals for a total of four times). The median event-free survival was increased from 12 months in the control arm to 23 months in the study group.
The impressive single-agent activity of rituximab led to the exploration of its use in combination with standard chemotherapy for treatment of indolent and aggressive NHL. Czuczman et al. established the safety and efficacy of rituximab and cyclophosphamide, doxorubicin, vincristine sulfate, and prednisone (CHOP) chemotherapy in a phase II trial of patients with previously treated and newly diagnosed low-grade NHL.177 In a large multicenter, randomized phase III trial, the addition of rituximab to cyclophosphamide, vincristine, and prednisone (CVP) significantly improved the overall response rate (81% vs. 57%), complete response rate (40% vs. 10%), and time to treatment failure (27 vs. 7 months).178 The benefit of rituximab in combination with chemotherapy for elderly patients with untreated diffuse large B cell lymphoma (DLBCL) was investigated in a randomized phase III trial.179 With a median follow-up of 3 years, those who were treated with rituximab and CHOP (R-CHOP) had significantly higher rates of complete response (76% vs. 63%), event-free survival (53% vs. 35%, P = .00008), and overall survival (62% vs. 51%, P = .008) than those treated with chemotherapy alone. In another phase III multicenter trial,180 a similar patient population (N = 632) was randomized to R-CHOP versus CHOP, followed by a second randomization to maintenance (Hainsworth schedule)175 or no further therapy. There were no statistically significant differences in overall survival. However, the patients who received CHOP and maintenance rituximab had a statistically significant improvement in time to treatment failure. The Mabthera International trial (MinT study), a randomized phase III trial investigating the efficacy of R-CHOP versus CHOP in untreated, younger (less than 60 years), good-prognosis patients with DLBCL was closed to patient accrual in December 2003. The trial ended early because a preplanned interim analysis demonstrated a significant improvement in time to treatment failure, a primary endpoint of the study. In all of the aforementioned studies, the combination of rituximab and standard chemotherapy did not result in an increase in toxicity. As a result of these and other studies, rituximab has become a common part of many lymphoma treatment regimens.
Many studies, some mentioned in earlier in this section, have focused on the issue of maintenance therapy in patients with lymphoma. In the Hainsworth trial, untreated patients with indolent NHL received four weekly doses of rituximab, followed by maintenance rituximab.175 Patients who had stable disease or an objective tumor response received weekly rituximab for four doses at 6-month intervals (maximum of four courses of rituximab). At 6 weeks, only 7% of these patients achieved a complete response, whereas 37% had a complete response following at least one course of maintenance treatment. A Swiss research group also investigated the benefit of maintenance rituximab.176 As in the previous trial, all patients received four weekly doses of rituximab. At week 12, patients with responding or stable disease were then randomized to no further treatment versus maintenance rituximab (one dose of antibody at 2-month intervals for a total of four times). The median EFS was increased from 12 months in the control arm to 23 months in the study group.
A number of studies using weekly rituximab at 375 mg/m2 demonstrated an inferior response rate for patients with small lymphocytic leukemia (SLL) or CLL compared with the 48% response rate associated with relapsed advanced follicular NHL.174, 181, 183, 184 One possible explanation for the inferior results in this malignancy is due to the low density of CD20 expression on tumor cells. A previous study discovered a correlation between CD20 antigen density and response rates in patients with NHL.184 Pharmacokinetics may also point to another reason for its lack of activity in CLL and SLL. Since mean plasma antibody concentration is strongly correlated with response to rituximab, the high intravascular tumor burden in CLL and SLL may result in rapid intravascular clearance of the antibody, leading to decreased efficacy. Because of this finding, a thrice weekly dosing schedule was further explored for rituximab monotherapy. A phase I/II trial demonstrated that this schedule was safe and more effective than the weekly administration.185 Alternatively, O'Brien et al. demonstrated that higher doses of weekly rituximab, ranging from 500 to 2,250 mg/m2, were associated with an overall response rate of 36%. Response rates from 44% to 75% were seen at the higher dose levels.186 Data from these two trials have established that single-agent rituximab is both a safe and an effective treatment for patients with CLL or SLL as long as a modified dose or schedule is utilized. Currently, the use of rituximab in combination with cyclophosphamide and a nucleoside analog is currently being investigated in both upfront and relapsed CLL.187, 188
The use of rituximab for the treatment of mantle-cell lymphoma appears more useful in the setting of minimal residual disease. Progression-free survival was not prolonged in patients with untreated mantle-cell lymphoma who received R-CHOP.189 However, the number of molecular remissions in patients with mantle-cell lymphoma seems to increase following each antibody administration in the post–autologous bone-marrow transplantation setting.190
Rituximab has also shown some activity in other malignancies or autoimmune disorders. Patients with refractory hairy-cell leukemia can achieve complete remissions following rituximab.191 A response rate of 20% has been demonstrated in patients with multiple myeloma whose tumors overexpress CD20.192 An overall response rate of approximately 50% has been observed in several small studies of patients with relapsed or refractory immune thrombocytopenic purpura. Similarly, rituximab is used to treat relapsed or refractory pure red-cell aplasia and autoimmune hemolytic anemia.193, 194
Alemtuzumab is humanized monoclonal antibody that targets CD52, a glycoprotein highly expressed on both B-CLL cells and normal B and T cells. The mechanism of action of alemtuzumab is not well understood, but it is presumed to work via ADCC, CDC, and/or apoptosis. Initial studies demonstrated that this agent was effective at clearing tumor cells from the blood and bone marrow of patients with NHL but did not result in significant diminution of bulky lymphadenopathy.66 Therefore, CLL seemed the optimal setting for this new agent, as it is primarily a malignancy of the peripheral blood and bone marrow.
Based on the results of a multicenter study, alemtuzumab (Campath-1H) was approved by the FDA for the treatment of B-cell CLL unsuccessfully treated by alkylating agents and fludarabine. In a pivotal multicenter phase II trial,195 heavily pretreated patients in whom fludarabine had failed (N = 93) achieved an overall response rate of 33% and a complete response rate of 2% with 12 weeks of intravenous alemtuzumab. The median time to progression was 4.7 months (9.5 months for responders), and the median survival was 16 months (32 months for responders). Side effects were frequent and included infusion-related reactions (occurring mostly within the first week of therapy) and infection (26% of patients had a grade 3 or 4 infection). Nine deaths (9.6%) occurred on study, which compares favorably with the death rate of 22% reported in fludarabine trials.196
In a phase II trial, untreated, symptomatic patients with B-cell CLL were treated with subcutaneous alemtuzumab.197 The overall response rate and complete remission rate were 87% and 19%, respectively, which were similar to published results for treatment with fludarabine in untreated patients. Significant clearance of tumor cells was noted in the blood, bone marrow, and nonbulky lymphadenopathy. None of the patients with a large lymph node (measuring greater than 5 cm) achieved a complete response. Using a dose-escalation approach for the first week of therapy, alemtuzumab was well tolerated. Although 20% of patients had grade 4 neutropenia (ANC < 0.5 × 109/L), none of the 38 patients had fever and neutropenia. Ten percent of patients had CMV reactivation, which responded promptly to intravenous antibiotics.
In order to increase response rates, time to progression, and median survival, alemtuzumab is also being investigated in combination with other agents, such as purine analogs and rituximab. A German group treated relapsed or refractory CLL patients (N = 14) with alemtuzumab and fludarabine. Preliminary results from 11 evaluable patients revealed a 64% response rate (8 complete responses; 1 partial response) and an acceptable safety profile.198 In a phase II study of previously treated patients with lymphoid malignancies, combination therapy with rituximab was also well tolerated and produced an improved overall response rate of 52% (complete response, 8%; nodular partial response, 4%; partial response, 40%).199 Further investigation of combined treatment programs using one or more monoclonal antibodies with fludarabine and/or alkylator-based chemotherapeutics in CLL are ongoing and may continue to demonstrate improved response rates and overall survival.
Alemtuzumab is active against T-prolymphocytic leukemia (T-PLL), a disease that is resistant to most chemotherapeutic agents. In a retrospective analysis of 76 patients with heavily pretreated T-PLL, Keating et al. demonstrated a 51% overall response rate and a 39.5% complete response rate following alemtuzumab treatment.200 The median time to progression (4.5 months) was double that of the first-line therapy. In a prospective phase II trial, patients with relapsed T-PLL who were treated with alemtuzumab achieved a 76% overall response rate and a 60% complete response rate.201 The median disease-free interval was 7 months, a significant improvement over the brief response (3 months) typically associated with conventional CHOP therapy or pentostatin. Further prospective studies are needed to define how alemtuzumab should be utilized in T-PLL, whether as a single agent or in combination with cytotoxic chemotherapeutics in the upfront, relapsed, or peritransplant setting.
Graft versus host disease (GVHD) is a major contributor to transplant-related mortality following allogeneic stem cell transplantation. Alemtuzumab is also utilized for both in vitro and in vivo depletion of T-cells in order to reduce the incidence of acute GVHD. Since it also eliminates host T cells, graft rejection is minimized. Sixty-five patients with lymphoproliferative disorders were treated with a nonmyeloablative allogeneic stem cell transplantation following BEAM-alemtuzumab conditioning.202 The incidence of acute GVHD was 17%, and only grade I-II acute GVHD was observed. Sustained donor engraftment occurred in most patients (97%), and a high rate of complete remission (70%) was seen. In a retrospective analysis comparing the results from two prospective nonmyeloablative transplants, the incidence of acute and chronic GVHD was significantly reduced in those patients receiving alemtuzumab/cyclosporin compared with the cyclosporin A/methotrexate group.203 Many of the alemtuzumab-treated patients required donor-lymphocyte infusions, but there were no significant differences between the two groups in both event-free and overall survival. Further studies with alemtuzumab are ongoing, with the goal of minimizing acute and chronic GVHD without compromising the overall response rate.
Trastuzumab (Herceptin) is a humanized IgG1 antibody that targets the HER2/neu antigen, which is overexpressed in 25 to 30% of breast carcinomas. In 1998, it was approved by the FDA for treatment of her-2/Neu positive metastatic breast carcinoma. Like rituximab, trastuzumab appears to act via multiple mechanisms, including down-regulation of her-2/Neu expression, induction of G1 arrest and downstream cell regulatory signals, initiation of ADCC and CDC, and promotion of apoptosis.204 Trastuzumab is well tolerated and has single-agent activity in her-2/Neu overexpressors lasting approximately 9 months.205,206 In combination with a chemotherapeutic agent, such as paclitaxel, docetaxel, or doxorubicin, the response rate, time to progression, and overall survival are significantly improved.207, 208 However, this survival benefit was not observed in patients with her-2 negative tumors assessed by fluorescence in situ hybridization or immunohistochemical analysis (score less than 3, NE+). Based on these positive findings, trastuzumab is being studied in combination with chemotherapy in the adjuvant setting or in conjunction with other targeted therapies. Its use in other her-2/Neu–overexpressing, epithelial tumors is another active research focus. Further discussion of trastuzumab is covered in Chapter 30.
Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has shown promising activity in a variety of solid tumors in early phase I and II trials and was approved by the FDA in 2004 for the treatment of metastatic colorectal cancer. VEGF is overexpressed in cancer cells of many solid tumors and hematologic malignancies. Increased expression of VEGF is commonly observed in tumors associated with poorer prognosis and decreased rates of disease-free and overall survival.209 Antibodies that target the VEGF receptor and/or neutralize VEGF were developed in the hope of blocking this important regulator of tumor angiogenesis. Preclinical studies confirmed that infusion of VEGF neutralizers resulted in an inhibition of tumor growth in multiple human cancer xenograft models.
A randomized phase III trial comparing irinotecan, bolus fluorouracil, and leucovorin (IFL) with and without bevacizumab was conducted in 813 patients with untreated metastatic colorectal cancer.210 Patients who received bevacizumab and IFL therapy had a significant improvement in median duration of survival (20.3 vs. 15.6 months, P < .001) and progression-free survival (10.6 vs. 6.2 months, P < .001). Overall, the treatment was not associated with more toxicity than treatment with IFL, except for an increased incidence of grade 3 hypertension (11% vs. 2.3%). Based on these data, bevacizumab plus fluorouracil-based chemotherapy has become a standard treatment regimen for metastatic colorectal cancer.
Clinical development of bevacizumab is being investigated in numerous solid tumors and hematologic malignancies as monotherapy and in combination with other cytotoxic agents. In a prospective, double-blind, placebo-controlled phase II trial, an interim analysis demonstrated a significant prolongation of time to progression in patients with metastatic renal cell carcinoma who received bevacizumab compared with placebo (P < .001).211 Preliminary data from a phase II trial in pancreatic cancer shows an impressive response rate of 24%.212 A phase I dose-escalation study is ongoing to evaluate the use of bevacizumab in combination with chemoradiation for locally advanced or recurrent head and neck cancer. Other trials are planned or ongoing in sarcoma, melanoma, hepatocellular carcinoma, esophageal carcinoma, multiple myeloma, myelodysplastic syndrome, AML, NHL, and CML. There has been one negative trial thus far, using bevacizumab with capecitabine in patients with previously treated metastatic breast cancer.213 It remains to be seen whether this agent will be an effective treatment for the multiple solid and hematologic malignancies that overexpress VEGF. Refer to Chapter 30 for a more detailed discussion of bevacizumab.
Overexpression of the epidermal growth factor receptor (EGFR), a regulator of cellular growth and survival, has been observed in multiple epithelial tumors and is a potential target for cetuximab, a chimeric monoclonal antibody against EGFR. In a recent phase II trial,214 patients with irinotecan-refractory, EGFR-positive metastatic colorectal cancer were randomized to receive either cetuximab and irinotecan or cetuximab monotherapy. The response rate (23% vs. 10.8%, P = .007) and median time to progression (4.1 vs. 1.5 months, P < .001) were significantly improved in the combination therapy arm versus the monotherapy arm. There was also a trend towards an increase in median survival in the cetuximab and irinotecan group (8.6 vs 6.9 months). Unlike the trastuzumab data, which demonstrated a relationship between Her-2/ Neu overexpression and efficacy, the level of EGFR expression did not correlate with response. However, the previously reported association between skin reactions following cetuximab and higher response rates was confirmed in this study. Based on these data and previous studies, cetuximab was approved by the FDA for the treatment of irinotecan-refractory metastatic colorectal cancer.
The activity of cetuximab in head and neck cancer with radiation therapy is very encouraging.215 In a phase III study of radiation therapy with or without cetuximab in locally advanced squamous cell carcinoma of the head and neck, the combined modality treatment arm was not associated with increased toxicity compared with the control group, except for a higher incidence of grade 3/4 skin reactions. Median survival was prolonged from 28 to 54 months by adding cetuximab to high doses of radiation therapy. The combination of cetuximab and platinum-based chemotherapy in platinum-refractory advanced head and neck cancer resulted in an 11% response rate. These data suggest that cetuximab can reverse platinum-resistance in some head and neck cancer patients. Based on phase II data, cetuximab also enhances the activity of standard chemotherapy in patients with pancreatic cancer and non–small cell lung cancer. The use of cetuximab is currently being investigated in multiple epithelial tumors. Chapter 30 contains a more detailed discussion of cetuximab.
Gemtuzumab ozogamicin (GO, Mylotarg) consists of a recombinant, humanized anti-CD33 monoclonal antibody conjugated to calicheamicin, a potent antitumor antibiotic. Within the acidic environment of lysosomes after internalization, calicheamicin dissociates from the antibody and migrates to the nucleus, where it binds within the minor groove of DNA and causes double-stranded DNA breaks. In a phase I trial, 8 of 40 patients with relapsed or refractory AML treated with escalating doses of gemtuzumab ozogamicin had reductions in the percentage of bone marrow blasts to below 5%, and complete remission was achieved in three patients.216
Subsequently, 142 patients with AML in first relapse were treated with two doses of gemtuzumab ozogamicin (9 mg/m2) 2 weeks apart in three phase II trials.217, 218, 219 Patients with secondary AML or prior MDS were excluded. Complete remission was achieved in 23 patients (16%), and 19 patients (13%) had a complete response without complete platelet recovery. The response rate was not significantly influenced by age or duration of prior remission. Response durations, however, were brief unless patients received additional chemotherapy or hematopoietic stem cell transplantation. Grade 3 or 4 hyperbilirubinemia developed in 23% of patients, and elevated levels of serum transaminases were seen in 17%. Hepatic veno-occlusive disease (VOD) developed in 4% of patients. Other toxicities included pulmonary edema and acute respiratory distress syndrome, particularly in patients with high leukocyte counts.
Recent phase II studies have reported higher rates of VOD associated with gemtuzumab ozogamicin. Among 46 patients who received gemtuzumab ozogamicin in first relapse and then underwent stem cell transplantation, 8 (17%) developed VOD, and there were 5 fatal cases.220 In a report from the Dana-Farber Cancer Institute, 9 of 14 patients (64%) who received gemtuzumab ozogamicin before allogeneic stem cell transplantation developed VOD, compared with 4 of 48 patients (8%) without prior exposure to gemtuzumab ozogamicin (P < .0001).221 Another study noted that 11 of 23 patients (48%) treated with gemtuzumab ozogamicin after stem cell transplantation developed liver injury suggestive of VOD.222 In a series of 119 patients treated with gemtuzumab ozogamicin at the MD Anderson Cancer Center, 14 (12%) developed VOD in the absence of stem cell transplantation, but the majority of these patients received gemtuzumab ozogamicin in combination with other agents or at more frequent intervals than originally described.223
Combination therapy that includes gemtuzumab ozogamicin is now under investigation for newly diagnosed AML. Because of its toxicity profile, however, administration of full-dose gemtuzumab ozogamicin with other agents has been difficult. In a study conducted by the Medical Research Council, 64 patients with newly diagnosed AML received one of three standard induction regimens along with gemtuzumab ozogamicin.224 The maximum tolerated dose of gemtuzumab ozogamicin was a single infusion of 3 mg/m2. Hepatotoxicity and delayed hematopoietic recovery prevented delivery of higher doses for induction or repeated administration during subsequent chemotherapy courses. Overall, the complete remission rate was 86%. Similarly, DeAngelo and colleagues found that a 6 mg/m2 dose of gemtuzumab ozogamicin could be given safely with cytarabine and daunorubicin in younger patients with newly diagnosed AML.225Grade 3 or 4 liver function abnormalities were seen in 16% of patients, and none of the patients developed VOD. Among 43 evaluable patients, 36 (84%) achieved a complete remission.226 Since CD33 is universally expressed by APL cells, including the leukemic stem cell, the use of gemtuzumab ozogamicin is particularly attractive for treating this disease.227 Preliminary results suggest that gemtuzumab ozogamicin in combination with ATRA can produce high molecular remission rates as first-line therapy and that its use as consolidation could potentially eliminate the need for standard anthracycline-based consolidation.228
Radioimmunotherapy for Hematopoietic Cancers
Hematopoietic cancers have been treated successfully with radiolabeled mAbs in a number of systems. This is due to the accessibility of the cells in the vasculature, their relative radiosensitivity, and the large number of differentiation antigens available as cell surface targets. Current technology and pharmacologic issues have limited the success of this approach for the treatment of solid tumors or for intraperitoneal (regional) infusions.229, 230Alternatively, antitumor activity was demonstrated in the majority of patients with leukemia and lymphoma in the early radioimmunotherapeutic trials.
The isotope used most widely has been 131I, not because it is the most effective but because it is inexpensive and is readily conjugated to mAbs through simple chemistry. It also emits a gamma particle for imaging and quantitative dosimetry, in addition to its cytotoxic beta emission. The agent 131I-BC8 (anti-CD45) appears to be effective at clearing the bone marrow of normal and leukemic cells before allogeneic bone marrow transplantation.231 Long-term survival has been seen in a significant fraction of patients treated in this manner. A high-dose approach also has been taken with 131I-M195 (anti-CD33) and HuM195151 and with another anti-CD33 mAb, p67,232 in the treatment of myeloid leukemia. 131I-M195 mAb specifically targets the marrow and kills 99% of leukemia cells at high doses, even with tumor burdens as high as 1 kg. In early phase I trials in which conventional allogeneic bone marrow transplantation was performed after 131I-M195 ablation in patients with refractory leukemia, high response rates (90%) were achieved, with little apparent toxicity above that expected from an allogeneic transplant.151 At lower doses, this agent appears active against minimal disease in myeloid leukemia as well.233
Nonmyeloablative use of an 131I anti-CD20 murine antibody, 131I-tositumomab (Bexxar), is well tolerated and highly effective in the treatment of relapsed or refractory low-grade, follicular, or transformed B-cell NHL (B-NHL). Based on promising data from two earlier trials, a multicenter, nonrandomized phase III study was conducted to evaluate the efficacy of 131I-tositumomab in patients with relapsed (relapse within 6 months of completion of last chemotherapy) or refractory low-grade or transformed B-NHL.234 Although the primary toxicity was hematologic, only one patient was hospitalized for fever and neutropenia. Four heavily pretreated patients developed myelodysplasia, but it is uncertain whether this condition was secondary to the 131I-tositumomab or due to previous alkylating agent exposure. The overall and complete response rates were 65% and 20%, respectively. Five 131I-tositumomab trials involving 250 patients have been conducted since 1990.235 Thirty percent of these patients with relapsed, refractory, and transformed B-NHL have had durable responses lasting 60 months. The majority of these long-term responders had many high-risk features, including advanced stage, poor response to last treatment, bulky disease, and bone marrow involvement. Based on these data, 131I-tositumomab was approved by the FDA for the treatment of CD20-positive follicular NHL (with or without transformation) in patients whose disease is refractory to rituximab and has relapsed following chemotherapy. Additionally, encouraging results have been seen with myeloablative doses of 131I-tositumomab in relapsed B-cell lymphoma.236
Administration of the radioimmunoconjugate 90Y-ibritumomab tiuxetan (Zevalin) is safe and leads to a significantly prolonged time to progression in patients with newly diagnosed or pretreated low-grade or transformed NHL. Over the past 9 years, over 770 patients have tolerated this treatment, with minimal side effects. Since there is an increased rate of myelosuppression with increased lymphomatous bone marrow involvement, use of this radioimmunoconjugate is restricted to patients who have less than 25% malignant infiltration of the bone marrow.237 The annual rate of MDS/AML is 0.62% per year following 90Y-ibritumomab tiuxetan. This rate is similar to the incidence of MDS/AML for patients with NHL who are being treated with standard dose chemotherapy.237, 238 The results from a phase I/II trial conducted in patients with relapsed or refractory follicular or relapsed aggressive NHL were recently updated with a median follow-up of 63 months for responders.239 Fifty-one patients were treated with escalating doses of 90Y-ibritumomab tiuxetan. The median time to progression for patients who achieved either a complete response or an unconfirmed complete response was 28.3 months. The subset of complete responders (confirmed or unconfirmed complete response) who received the maximum tolerated dose (0.4 mCi/kg) had a median time to progression of 45 months. Overall response rates in the range of 74 to 83% were even seen in rituximab-refractory patients.240 A phase III trial was designed to compare 90Y-ibritumomab tiuxetan to rituximab in the same patient population. One hundred forty-three patients were randomized to either the study arm or the control arm. The study group had a better overall response rate (80% vs. 56%; P = .002) than the control group. The complete response rate of the study group was nearly double that of the control group (30% vs. 16%; P = .04). The differences in time to progression, however, were not statistically significant. Randomized trials comparing 131I-tositumomab and 90Y-ibritumomab tiuxetan have not been done.
Conclusions Drawn from Clinical Studies
Many mAbs have been proven to be both safe and effective anticancer therapies. More antineoplastic mAbs are still at the phase I and early phase II stages of investigation. Several generalizations can be made regarding their use and efficacy from published data. First, many mAbs can be administered safely and can reach their target tissues. The most efficient delivery appears to occur with hematopoietic neoplasms and with small tumor burdens. Second, and perhaps more important, rodent mAbs are highly immunogenic, and neutralizing human antibody responses develop in most patients except those who are very immunosuppressed. The advent of humanized and chimeric antibodies has enabled more effective delivery of mAbs.
Third, mAbs without potent effector functions in vitro are not likely to be active against tumors in vivo. As a corollary, mAbs that are highly active work via ADCC, CDC, or apoptosis or via a conjugate, such as a radioisotope or toxin. Fourth, the pharmacodynamics and kinetics of the large IgG structure are significant obstacles to the effective use of radiolabeled mAbs to treat solid tumors. Use of mAbs in solid tumors may be most appropriate in settings of minimal residual disease or as an adjuvant. These studies will require large randomized studies to confirm activity. In contrast, radioimmunoconjugates can reduce or eliminate large tumor burdens consisting of leukemia or lymphoma cells.
CURRENT OBSTACLES TO MONOCLONAL ANTIBODY CANCER THERAPY
A number of significant obstacles have slowed successful therapeutic applications of mAbs (Table 31.6). Better chemical methods for attaching radionuclides or toxins to mAbs appear to be resolving some of the issues of biochemical stability. New approaches using antibody fragments or genetically engineered single-chain binding proteins may improve delivery to tumors, but the pharmacologic difficulties may still be significant. Rapid modulation of cell surface immune complexes, a phenomenon that reduces ADCC and CDC, can be used to advantage by coupling toxins or isotopes that require entry into the cell. Efficient delivery of the toxin to the appropriate subcellular compartment and retention of radionuclides within cells still pose problems. New methods of engineering rodent mAbs into humanized mAbs or of producing true human mAbs resolves many of the issues related to HAMA (Table 31.4), but it is unclear whether anti-idiotype responses will be seen after repeated doses. One of the paradoxes of mAb-based therapies is that increasing the specificity of the agents may yield more avenues of tumor cell escape. Because native mAbs target and kill individual cells based on the presence of antigen, tumor cells that have little or no antigen may be spared any cytocidal effects. Antigen-negative cells are thus selected for later relapse.241 In contrast, radioimmunoconjugates with long-range beta emissions may kill antigen-negative bystander cells242 but will consequently have greater toxicity.
TABLE 31.6 CURRENT OBSTACLES TO MONOCLONAL ANTIBODY (mAb) TREATMENT OF CANCER
Monoclonal antibodies are versatile anticancer agents with wide-ranging potential for therapy. As of late 2004, eight mAbs have already been approved by the FDA for the treatment of specific malignancies. These mAbs are also being investigated for use in other hematologic and solid tumors. Many other mAbs or their constructs remain in the early stages of clinical development. MAbs are of great interest primarily because of their specificity and potential for reduced toxicity compared with cytotoxic chemotherapy. In addition, their long half-lives and ability to kill cells via a variety of mechanisms also make them attractive drugs. Nonimmunogenic, humanized, and chimeric mAbs of appropriate specificity have been genetically engineered to block a certain receptor or to lyse tumor cells via ADCC or CDC. When the antibody is conjugated to radioisotopes or toxins, this treatment can reduce or eliminate bulky tumors. Unconjugated mAbs are ideally suited for the treatment of minimal residual disease, in either the consolidation or adjuvant setting. Finally, mAbs are likely to be most effective when integrated into combination therapeutic strategies involving chemotherapy, radiation therapy, and biologic therapy.
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