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`
`
`Cancer Chemotherapy and Biotherapy:
`Principles and Practice
`
`2
`
`

`

`
`
`
`
`EDITED BY
`
`Bruce A. Chabner, M.D.
`Chief, Hematology/Oncology
`Clinical Director
`Massachusetts General Hospital Cancer Center
`Boston, Massachusetts
`
`Dan L. Longo, M.D.
`Director, Biological Response Modifiers Program
`Division of Cancer Treatment
`National CancerInstitute
`Frederick, Maryland
`
`Cancer Chemotherapy
`and Biotherapy:
`Principles and Practice
`
`SECOND EDITION
`
`
`SSSSSeS
`
`ecieeeaaSTNae
`
`
`
`LY Lippincott - Raven
`
`S$
`
`H
`
`E
`
`R
`
`S
`
`P
`
`U
`
`B
`
`L
`
`§
`
`Philadelphia «© New York
`
`3
`
`

`

`
`
`Copyright © 1996 by Lippincott—Raven Publishers. All rights reserved. This book is protected by copy-
`right. No part of it may be reproduced, stored in a retrieval system, or transmitted, in any form or by
`any means—electronic, mechanical, photocopy, recording, or otherwise—without the prior written
`permission ofthe publisher, except for brief quotations embodiedin critical articles and reviews. Printed
`in the United States of America. For information write Lippincott—Raven Publishers, 227 East Wash-
`ington Square, Philadelphia, PA 19106.
`
`Library of Congress Cataloging-in-Publications Data
`
`Cancer chemotherapy and biotherapy : principles and practice / edited
`by Bruce A. Chabner, Dan L. Longo —~ 2nd ed.
`p.
`cm.
`Rev.ed. of: Cancer chemotherapy.
`Includes bibliographical references and index.
`ISBN 0-397-51418-2 (hard : alk. paper)
`1. Cancer—Chemotherapy.
`2. Cancer—Immunotherapy.
`(Dan Louis), 1949-
`.
`II. Cancer chemotherapy.
`[DNLM: 1. Neoplasms—drug therapy. 2. Biological Products—therapeutic use.
`3. Antineoplastic Agents—therapeutic use. 4. Chabner, Bruce. QZ 267 C21515 1996}
`RC271.C5C32219
`1996
`616.99'4061—dc20
`DNLM/DLC
`for Library of Congress
`
`I. Longo, Dan L
`
`95-38920
`CIP
`
`The material contained in this volume was submitted as previously unpublished material, except in
`the instances in which credit has been given to the source from which someoftheillustrative material
`wasderived.
`Great care has been taken to maintain the accuracy ofthe information contained in the volume. How-
`ever, neither Lippincott~Raven Publishers nor the editors can be held responsible for errors or for any
`consequencesarising from the use of the information herein.
`The authors and publisher have exerted every effort to ensure that drug selection and dosage set forth
`in this text are in accord with current recommendations and practice at the time ofpublication. How-
`ever, in view of ongoing research, changes in government regulations, and the constant flow of infor-
`mationrelating to drug therapy and drug reactions, the reader is urged to check the packageinsert for
`each drug for any change in indications and dosage and for added warnings and precautions. This is
`particularly important when the recommendedagentis a new orinfrequently employed drug.
`Materials appearing in this book prepared by individuals as part of their official duties as U.S.
`Government employees are not covered by the above-mentioned copyright.
`
`987654321
`
`4
`
`

`

`Cancer Chemotherapy and Biotherapy, second edition,
`edited by Bruce A. Chabner and Dan L. Longo.
`Lippincott-Raven Publishers, Philadelphia ©1996
`
`
`CHAPTER
`
`
`
`Antibody-Based Immunotherapies
`for Cancer
`
`_
`
`Richard P. Junghans, George Sgouros,
`and David A. Scheinberg
`
`Monoclonal antibodies (mAbs) are remarkably versatile
`agents with potential therapeutic applications in a num-
`ber ofhuman diseases, including cancer. mAbs have long
`promised to offer a safe, specific approach to therapy.
`Morethan a decadeofpreclinical evaluation and human
`clinical trials have identified new strategies for the use of
`mAbs,as well as a numberofdifficult obstacles to their
`effective application. Although the use of antibodies as
`targeting agents dates to the 1950s,’ it was not until
`methods for production of mAbs appeared in the late
`1970s, whereby reproducible lots of a defined molecule
`could be produced in quantities adequate for clinical
`study, that the properties of antibodies as therapeutic
`agents for cancer could be studied appropriately.
`Five approaches to therapy are used in the application
`of mAbs in humansin vivo. First, mAbs can be used to
`focus an inflammatory response againsta targetcell. Bind-
`ing of a mAbto a target cell can result in fixation of
`complement,yielding cell lysis or can result in opsoniza-
`tion, which marks the cell for lysis by variouseffectorcells
`such as natural killer (NK)cells, neutrophils, or 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 bedirected atcrit-
`ical hormones, growth factors, interleukins, or other regu-
`latory molecules or their receptors in order to control
`growth or othercell functions. Fourth,anti-idiotypic mAbs
`maybeused as vaccines to generate an active immunere-
`sponse.Finally, mAbs may be used to speed the clearance
`of other drugs or toxins or can fundamentally alter the
`pharmacokinetic properties of other therapeutic agents.
`For example, mAbs may be fused to drugsorfactors to in-
`crease their plasma half-life, change their biodistribution,
`or render them multivalent. Alternatively, mAbs may be
`usedto clear previously infused mAbsfrom thecirculation.
`Yet despite the diversity of approaches, significant
`problems remain that are peculiar to mAbs. mAbsare
`large, immunogenic proteins, often of rodentorigin,that
`rapidly generate neutralizing immune responses in pa-
`tients within days to weeks after theirfirst injection. The
`sheersize ofmAbs, 150 kDafor IgG to 900 kDafor IgM,
`100 timeslarger than typical drugs, makes their pharma-
`cology (particularly diffusion into bulky tumorsor other
`
`extravascular areas) problematic for effective use. Many
`early mAbs or mAb constructs were either poorly cy-
`totoxic or relatively nonspecific, rendering them ineffec-
`tive. Moreover, the high degree of mAb specificity thatis
`routinely achievable now can work against mAbs,since
`tumorcells that do not bear the specific antigen target
`mayescape from cytotoxic effects.
`Within this context,it is still clear that mAbs have great
`potential to be safe andeffective anticancer agents; recent
`clinical investigations have highlighted several areas where
`mAbscan beeffective, either alone or in combination with
`other, more conventional agents.
`This chapter reviews the basic biochemical andbiologic
`properties of mAbs and the most commonly used deriva-
`tives
`(immunotoxins,
`radioimmunoconjugates, mAb
`fragments), discusses the pharmacologic issues peculiar to
`mAbs, and outlines some of the importantclinical results
`with mAbs. Potential solutions to the most difficult issues
`in the use ofmAbswill be presented. Since mAbs and con-
`jugates ofmAbsrepresent manydifferent drugs, with char-
`acteristics that result from their origin (rodent or human),
`their isotypes, their structure, or the various conjugated
`toxic agents, generalizations about the properties ofmAbs
`often may not be possible. mAb therapy of canceris a new
`and rapidly changingfield, and readers are encouraged to
`consult other reviews for more comprehensive discussions
`ofindividual areas.3*
`
`IMMUNOGLOBULIN (Ig) CLASSES
`
`Immunoglobulins are separated into five classes or iso-
`types based on structure and biologic properties: IgM,
`IgD, IgE, IgA, and IgG. For reasons discussed under
`“Ontogeny” (below), IgM is the primordial antibody
`whose expression bytheB cell onits surface represents the
`commitmentof that cell to a particular but broad recog-
`nition space that subsequently narrowsas part of the mat-
`uration response induced by antigen interactions.’ IgD is
`normally coexpressed with IgM on B cells and may play a
`signaling role in B-cell development. IgE, IgA, and IgG
`are mature immunoglobulinsthat are expressed after mat-
`uration of the response and class switch have occurred.
`655
`
`a
`
`eG
`
`5
`
`

`

`
`
`
`
`
`
`V,CH1-3
`—
`
`V,CH1-4
`=
`
`_—
`190,000
`8S
`13%
`0.03
`0.003
`
`2.5
`2
`
`_ F
`
`eeR-I,FceR-I
`
`Mastcells
`
`_
`175,000
`78
`9%
`4
`0.3
`
`2.8
`lor2
`_
`
`Hi chain domains
`Otherchains
`Subclasses
`
`Heavy chain allotypes
`Molecular weight
`Sedimentation constant
`Carbohydrate content
`Serum level (mg/100 ml)
`Percentage oftotal
`serum Ig
`Half-life (days)
`Antibody valence
`Complementfixation
`(classic)
`Fe receptors
`
`Bindingto cells
`
`V,CH1-3
`_
`IgGl IgG2,
`IgG3,IgG4
`Gm (ca. 30)
`150,000
`6.6S
`3%
`1250 + 300
`75-85
`
`V,CHI-3
`J chain, S piece
`IgAl IgA2
`
`Am (2)
`160,000
`78,98,118,148
`7%
`210 + 50
`7-15
`
`23 (IgG3 7d)
`2
`+ (IgG1,2,3)
`
`5.8
`DACs cas
`=
`
`V,CH1-4
`J chain
`
`Mm (2)
`950,000
`19S
`10%
`125 + 50
`5-10
`
`5.1
`10
`+e
`
`FeyR-LFeyR-H,
`FeyR-II
`Monocyte
`macrophages,
`neutrophils,
`LGLs
`
`_
`
`?
`
`eso
`PLPESES:
`7212
`
`Primary Ab response; B-cell
`Homocytotropic
`response;
`B-cell surface Ig,
`surface lg
`Ab; anaphylaxis;
`placental
`rheumatoid factor
`allergy
`transfer
`
`Otherbiologic properties|Secondary Ab Secretory antibody
`
`656
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`Each of these antibodies participates in specialized func-
`tions: IgE in immediate-type hypersensitivity reactions
`and parasite immunity, IgA in mucosal immunity, and
`IgG in humoral immunity. In somecases, the antibodies
`interact with specialized receptors that link their action to
`host cellular defenses; in others, the antibody interacts
`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.}°
`Table 28-1 summarizesvarious features of the antibodies
`that will be discussed in this section.
`
`STRUCTURE
`
`The fundamental structural elements ofall antibodiesare
`indicated by size as heavy and light chains of 55-75 and
`22 kDa(Fig. 28-1). Light chains are either kappa (k) or
`lambda (A) and are each distributed among all
`im-
`munoglobulin subclasses. Overall, kappa comprises 60%
`oflight chain in humans versus 95% of light chain in
`mouse. Heavy chains are pt, 5, y, €, and a, corresponding
`
`
`
`Table 28-1. Properties of Antibody Classes.
`
`to IgM,D,G, E, and A, and conferring the. biologic char-
`acteristics of each antibodyclass. Each chain is composed
`of so-called immunoglobulin (Ig)—like domains of an-
`tiparallel beta-pleated sheets, twoforlight chain and four
`such domainsfor heavy chain, excepting IgM and IgE,
`which have five. 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 p and e€). Be-
`tween CH] and CH2is the so-called hinge region, which
`confers flexibility on the antibody “arms” and suscep-
`tibility to proteases (below) excepting IgM and IgE in
`which the CH, domain itself serves this role.
`Heavy (H)andlight (L) chains are normally paired 1:1]
`with each other, but the smallest stable unit is a four-
`chain (HL), structure (see Fig. 28-1), for a nominal total
`mass of 150 to 160 kDafor IgG and higherfor otheriso-
`types (see Table 28-1). While isolated light chain (Bence
`Jones protein) exists in small amounts as monomers or
`dimers in normal individuals, the isolated heavy chain is
`stable only in association with another heavy chain to
`mask the hydrophobic surface on the carboxy-terminal
`CH3 domain (CH4 in IgM) and to generate a high-
`affinity noncovalent interaction between the molecular
`
`
`PROPERTY
`
`IgG
`
`IgA
`
`IgM
`
`IgD
`
`Monomer
`
`Monomer
`
`Monomer
`
`IgE Pentamer
`
`Usual molecular form
`Monomer, dimer,etc.
`Molecular formula
`"y2«2 or y2A2
`(a22)n or (a2A2)n
`(W2«2)5 or (w2A2)5 82K20r82A2=22. or €2A2
`
`6
`
`

`

`
`
`Antibody-Based Immunotherapies for Cancer
`
`657
`
`
`
`Figure 28-1. Antibody structure. The structuralrelationships and functions of domainsof IgG. (Reprinted with permission from Wasser-
`man RL, and Capra JD, Immunoglobulins. In Horowitz MI, Pigman W,eds, The glycoconjugates. NY: Academic Press, 1977: 323.)
`
`
`
`LIGHT CHAIN ;__
`REGIONS |
`
`LIGHT CHAIN
`
`
`HYPERVARIABLE {
`
`
`
`HYPERVARIABLE
`REGIONS
`
`
`
`
`
`
`
`ANTIGENered Fah
`
`
`
`INTERCHAIN
`JY DISULFIDE
`j
`BONDS
`
`
`
`CARBOHYDRATE
`
`INTRACHAIN
`DISULFIDE
`BONDS
`
`BIOLOGICAL
`ACTIVITY|Fc
`MEDIATION
`
`VL AND Vy: VARIABLE REGIONS
`C, AND Cy. CONSTANT REGIONS
`
`__|
`
`halves." It is notable that the inter-heavy chain disulfides
`and heavy-light chain disulfides are not required for as-
`sembly, which is mediated through primary noncovalent
`interchain interactions. IgE and IgG are composed ofa
`single (HL). unit, whereas IgM exists as a pentamer of
`(HL), units joined by disulfide bonding with a third J-
`chain component. IgA exists mainly as a monomerin
`serum butin secretions exists primarily as a dimer plus
`trimer and higher formsin which thé oligomersare linked
`by J chain as well as the fragment ofsecretory chain (se-
`cretory piece) that is involved in the mucosal transport.
`The V regionitself is composed of subdomains: rela-
`tively conserved framework regions interdigitated with
`the
`so-called
`complementarity-determining regions
`(CDRs)[also termed hypervariable segments (HVSs)] that
`make primary contact with antigen"? (see Fig. 28-1).
`There are three CDRs in each heavy andlight chain that
`mayparticipate in antigen binding. The V regions should
`be seen as juxtaposed three-finger gloves, with the CDRs
`covering the tips (Fig. 28-2), arrayed in a broad contact
`surface with antigen (Fig. 28-3).
`Antibodies are glycoproteins. Glycosylation ofproteins
`plays variousroles includingsolubility, transport, confor-
`mation, function and stability. Carbohydrate is located
`mainly in antibody constant domains, with a lower fre-
`quency in V regions (see data on M195 below).4 IgG
`contains a major conserved glycosylation site in CH,
`which contributes to the conformation ofthis domain that
`is crucial to the functional ability to bind to complement
`and to Fey receptors.
`The IgG antibody “unit” has been definedin terms of
`susceptibility to proteases that cleave in the exposed, non-
`
`folded regions of the antibody (see Fig. 28-1). A tabula-
`tion of antibody fragments and engineered or synthetic
`products is presented in Table 28-2. Fab contains the
`V regionandfirst constant domain ofthe heavy chain (VH
`+ CH] = Fd)andtheentire light chain (L); Fab’ includes
`additionally a portion ofthe H chain hinge region and one
`or more free cyteines (Fd’); Fab’2 is a dimer ofFab’ linked
`through hinge disulfide(s); and Fv is a semistable antibody
`fragmentthat includes only VH + VL, the smallest anti-
`gen-binding unit. Fc is the C-terminal crystallizable frag-
`mentthat includes the complement and Fe receptor-bind-
`ing domains (below). Genetically engineered products
`include the ACH2constructs, lacking the second constant
`domain ofheavy chain, which behavelike a Fab’2,with bi-
`valency, abbreviated survival, and lack of interaction with
`host effector systems, but which do not require enzymic
`processing.'** sFv is Fv with a peptide linkage engineered
`to join the C-terminusof one chain to the N-terminus of
`the other for improvedstability. More advanced products
`have been designed that conceptually represent the anti-
`gen-binding domain in a single peptide product!*;thisis
`not related structurally to an antibody and is therefore
`considered an antibody mimic.
`
`ONTOGENY
`
`Antibodies represent the moststrikingly evolved, adaptive
`system possibly in all of biology. It is both an ancient and
`evolved system, present
`in mammals, birds, reptiles,
`amphibians, teleosts, elasmobranchs(sharks), and possi-
`bly cyclostomes(hagfish, lampreys), which,iftrue, would
`
`7
`
`

`

`658
`
`
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`
`“class switch”to oneofthe mature antibodies (IgG, IgA,
`
`tissues, spleen, or lymph nodesand secrete large quanti-
`ties of antibody, the sole function of this terminally dif-
`ferentiated cell.’
`'
`The genes of heavy andlight chains share important
`features of structure and maturation. Each gene locus
`contains widely separated variable, constant, and so-called
`minigene domains that are placed into juxtaposition by
`DNA recombination mechanisms. The minigenes—
`diversity (D) andjoining (J) regions for heavy chain and
`J regions for light chain—contribute to or constitute,
`with modifications, the CDR3.!5 The kappa and lambda
`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.
`To understand the nature of the generation of the
`antibody repertoire,it is instructive to recapitulate what
`is known about germ-line diversity. On the heavy chain
`locus, there are an estimated 80 functional VH genes, 12
`D regions, and 6 J regions for a potential of 6000 com-
`binations!*"? (Fig. 28-4). There are roughly 80 V kappa
`[Vx] light chain and 5 J kappa [Jk] domains, which,ran-
`domly associated, can generate 400 combinations (the
`lambda locus contains a smaller number of distinct V
`genes). A simple arithmetic calculation suggests that
`VkVH combinations alone could generate a diversity of
`approximately 2 X 10°. Yet even this numberis conserv-
`ative, 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 poten-
`tial complexity, in theory exceeding thetotal lifetime B-
`cell output by several orders of magnitude.”° However,
`many authors have cautioned that the mathematical di-
`versity does not allow for the redundancy in configura-
`tions that could provide equivalent binding domains; in
`terms of antigen binding, the practical diversity is proba-
`bly in the 1 to 10 X 10° range. The smallest “complete”
`immune systém is that of the young tadpole with 10° B
`cells, which suggests that repertoires of 10° to 10° con-
`stitute a sufficiently complete topologic set for meaning-
`fully diverse, if not exhaustive, antigen recognition.”
`V gene selection is based on random expression
`followed byspecific amplification. It has been argued on
`physicochemical grounds that 10° different antibody
`molecules are sufficient to create a topologic set that
`recognizes any antigen surface with an affinity of 10° to
`10°M-!,22 a weak butbiologically important numberthat
`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. Oncontacting antigen, these cells are
`stimulated to divide and undergo CDR mutations. Sub-
`sequentbindingandstimulation are in proportion to the
`strength ofthe binding reaction; hencethereis an in vivo
`selection for mutations that enhances the affinity of the
`antibody for the antigen, a process termed affinity mat
`uration2® Simultaneous with this increased affinity is 4
`narrowingofthe specificity, with the antibody shedding
`its early polyreactive phenotype. Thecells then undergo
`
`|
`
`Figure 28-2. Space-filling model of human \gG1 antibody
`with CDRsin color representing anti-Tac-H; human myeloma
`protein Eu with CDRs grafted from murine anti-Tac. (Photo
`
`provided courtesy of Dr. C. Queen.) (See ColorPlate 1.)
`
`include all chordates.' Its most diverse representation of
`classes and functions is found in mammalia. The power of
`antigen recognition begins with an inherited array of du-
`plicated and diversified germ-line V genes, a random mu-
`tational 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.
`The biologic expression of antibody begins with the
`B-cell progenitor, which undergoesa series ofmaturation
`steps that begins with V gene selection for heavy chain
`followed by light chain V selection that yields surface
`expression andsecretion by the mature B cell. Upon in-
`teraction with antigen, the B cells are activated to prolif-
`erate, secrete antibody and undergo CDR mutagenesis
`and affinity maturation, and finally to undergo chain
`switch and plasmacell conversion. Plasma cells remain in
`
`%£
`
`fai
`
`OPRrSeres
`
`B*SPSLIEE
`TELATurrss
`aaa
`
`8
`
`

`

`Antibody-Based Immunotherapies for Cancer
`
`
`659
`
`Figure 28-3. Antigen-antibody binding surface juxtaposition. The V region (Fy) of antibody (right) bindsto influenza virus protein
`neuraminidase(left) in the top panel. The VH (red) and VL(blue) are separately colored to show their respective binding contribu-
`tions. The bottom panel offsets the two molecules by 8 A to show the complementarity of surfaces that promotesthe binding interac-
`tion. The stippled surface of the neuraminidasedefines the antigen “
`epitope.” (Photo provided courtesy of Drs. P.M. Colman and W.R.
`Tulip, CSIRO Australia.) (See Color Plate 2.)
`
`|
`
`
`
`
`
`
`IgE) by deleting out DNA between the VDJregion and
`the new C regionof the heavy chain, which bringsthis
`new constant domain in juxtaposition with the V region
`(see Fig. 28-4). (Light chain is unchanged.) Some time
`after commitment to a mature antibody,thecell will cease
`its CDR mutagenesis, affinity maturation will have been
`completed, andthe B cell will undergo morphogenesis to
`a tissue-resident plasma cell.°
`
`ANTIGEN-ANTIBODY INTERACTIONS
`
`Affinity is a quantitative measure ofthe strength of the
`interaction betweenantibody andits cognate antigen and
`
`is intendedin the samesense as the equilibrium constant
`in the chemical mass action equation:
`
`[AB] = K,[A] [B]
`
`(28-1)
`
`The equilibrium or affinity constant is represented in
`units of M-', In mostinstances studied by x-ray crystal-
`lography, contacts between antibodyandprotein antigen
`are dominated by noncovalent hydrogen bonds (O—H),
`with a lowerfrequency ofsalt bridges (COO-— + HN),
`with a total of about 15 to 20 contacts. The effect of
`adding a new H- bondcan beestimated from the free
`energy gain (0.5 to 1 kcal/mol-°C) and from AG =
`—RTIn K,to yield affinity increases of approximately
`
`9
`
`

`

`660
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`
`
` Table 28-2. Antibody FragmentDefinitions
`DESCRIPTION
`REPRESENTATION
`DESIGNATION
`
`Ly
`
`Fab
`
`Enzyme-generated products
`
`Fab
`
`Fab’
`
`Fab’2
`
`Fy
`
`Fe(or Fe’)
`
`Fe
`
`(or
`
`pFc (or pFc’
`
`pFc’)
`
`NY
`
`Fd
`
`olie
`
`Ed’
`
`BS
`
`SAa
`
`esi
`
`
`CH,
`
`CH;
`
`
`
`Lee
`
`|CHs
`
`|
`
`Genetically engineered products
`
`delta CH,
`
`sFv
`
`ae
`CH;
`
`V8
`ZeNu
`
`Complete IgG
`
`Papain digest; Fd + L
`
`Pepsin digest monomer; Fd’ + L
`
`Pepsin digest dimer
`
`V region digestion fragment; VH + VL
`
`C region digestion fragment; crystallizable fragment
`
`Smaller fragments of Fe
`
`Deleted CH, domain; dimer of V - CH, — CH; + L
`
`Single-chain Fv; VH and VL joined by peptide linker
`
`
`
`which antigen is 50% saturated;if the antibodyis in large
`
`
`aeenerAEBBBc
`ae
`(RHEE,RT
`
`
`Synthetic products
`
`ABU emy
`Antigen-binding unit; peptide mimic
`ee
`
`3- to 10-fold. Therefore, the affinity maturation that
`takes place (or affinity that may be Jost in antibody engi-
`neering) changes quickly with a relatively small change in
`the numberofbonds. Thatis, creating as few as three new
`hydrogen bonds maygenerate an affinity enhancementof
`more than 100-fold. This has been borne outby affinity
`changes that accompanied productive amino acid substi-
`tutions in V region engineering (below). It is notable that
`antibody affinities for protein antigens are generally much
`higher than for carbohydrate antigens, which may have
`less opportunity for hydrogen-bondinginteractions (but
`are also “T-independent”antigens).
`
`10
`
`Although affinity and K,directly express the binding
`potential of the antibody and are the most suitable mea-
`sures for comparing affinities, the inverse of the K,,
`termed K, or dissociation constant, is expressed in molar
`units and indicates the concentration thatis the middle of
`the range for the biologic action of the antibody:
`
`K,.=2
`
`(28-2)
`
`That is, the K, is the concentration of free antibody at
`
`10
`
`

`

`Antibody-Based Immunotherapies for Cancer
`
`661
`
`
`
`Figure 28-4. Generation of diversity. VJ and VDJ joining occurin L chain and H chain by excision ofintervening DNA in the genome.
`Class switch involves deletion ofintervening constant (C) domains(C, Cd,etc.) and transcription through the new proximal C region.
`C is finally joined to the V gene bysplicing of the mRNA.(Reprinted with permission from Cooper MD, Current concepts: b lympho-
`cytes—normal development. N Engl J Med 317:1452, 1987.)
`
`
`
`
`rs }otyfopot bey 4% bots fo{22]
`
`
`
`A. Heavy chain genes on chromosome 14 p32
`
`
`
`
`
`
`v(>50)
`
`D> 20}
`eo
`
`al
`
`B. Kappa light chain genes
`on chromosome 2 p11
`V(>S80)
`J
`———— eo” OK
`By
`t}-
`
`Hie EH
`
`C. Class switching
`by DNA deletion
`
`«Suh CuO
`VDJ
`Ze Se
`
`he %
`
`cy.
`
`y%
`
`
`
`cS
`Histones
`
`CY, % a,
`
`cd
`su Cu
`VDJ
`Ai =)
`
`Sy,
`
`CY,
`
`
`
`Sass
`
`Recombinase
`
`voy Su Sy,
`
`Cy;
`
`
`
`excess, the input antibody concentration approximates
`the free concentration. The Ky is a frequently used term,
`but its relationship to affinity must always be borne
`in mind: i.e., low affinity = high K,; high affinity = low
`K,. For example, a K, of 2 X 10° M™ implies a K, of
`0.5 X 10°? (0.5 nM), or about 0.1 pg/ml antibody
`concentration for IgG. If antigen is in the picomolar
`(107? M) range, this concentration of antibody will have
`half of antigen saturated and half of antigen will remain
`“free.” At 10-fold higher antibody concentration (1 pg/ml,
`10 X K,), antigen will be 90% saturated and 10% free,
`
`and at 100-fold higher concentration (10 pg/ml, 100 x
`K,), antigen will be 99% saturated and only 1% unbound.
`It is a key point ofunderstanding that the ratio ofantibody
`over antigen has very little impact on the degree ofantigen
`saturation where antibody is in excess. If antibody concen-
`tration is 1 nMf with a K, of 1 nM,it does not matter
`whetherantigen is 0.1 nM at the Ky, 0.1 pM, or 0.1 £¥,;
`antigen in each case is 50% bound,although theratio of
`antibody to antigen is 10, 10*, and 107. It cs only the re-
`lation offree antibody to its K, that determines the degree
`ofantigen saturation.
`
`11
`
`=
`
`11
`
`€
`

`

`662
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`antibody in comparing Fab with higher-valency homo-
`logues. When antigens are presented multivalently on sur-
`faces ofcells, viruses, or other pathogens, even the low-
`affinity IgM interactions can yield a high-avidity, stable
`bindingto suchtargets in vivo.
`
`The affinity constant K, is itself composed of two
`terms that describe the on (forward; units ofM-*-s~*) and
`off (back; units of s~*) rates of the reaction:
`
`K,=—2
`
`(28-3)
`
`To a first approximation, the forward rate is diffusion
`limited and comparable for many antibodies reacting
`with macromolecules orcell-boundstructures; reactions —
`of antibodies with haptens and other small moleculesin
`solution will be dominated by the faster linear and rota-
`tional diffusion rates of the smaller component.” Forex-
`ample, 0.1 nM of DNP-lysine (0.1 ng/ml) or 0.1 nM
`cell-bound HLA-A2 (50 ng/ml) mixed with specific IgG
`antibody at 10 pg/ml (65 nM) requires 0.1 second to
`react with 50% of the antigen for the hapten but requires
`4 minutes for the surface protein. Yet they havevirtually
`the sameaffinity constant.This is due to the fact that
`the fast association rate is balanced bya fast dissociation
`rate for the hapten (2 = 0.7 s) versus a longerstability
`for the protein antigen (4. = 6 min).
`While there are exceptions, the on rates of antibodies
`to protein and cell-bound antigensare primarily in this
`range andinversely proportional to antibody concentra-
`tion for antibody in excess of antigen (ic., at 1 pg/ml,
`the 50% on time would beof the order of 0.5 to 1 hour).
`Accordingly, differences in affinity between antibodies to
`the samecellular antigen will in manyinstances be seen to
`be reflective of the off rate (,). For most such purposes,
`an antibody is generally considered of “good” affinity if
`its K,= 10° M"}, where off rate 4, values of an hour or
`more at 4°C are common.Association and dissociation
`times at 37°Care both acceleratedrelative to 4°C, on the
`order of 5 or more, frequently with a net decrease in
`antibody affinity of2- to 10-fold. This-:must be explicitly
`tested, however, since there are instances of protein-
`ligandaffinities that are enhanced by higher temperature.Ӥ
`The foregoing expresses basic principles of binding
`processes. A further importantfeature ofantibodiesis em-
`bodied in their multivalent structures. While the on rates
`for monovalent Fab and bivalent Fab’2 constructs are
`comparable, the bivalentofftimes may be 10-fold or more
`longer than the monovalentconstructs,yielding affinities
`that are similarly enhanced.” Todiscriminate the affinity
`that is intrinsic to the V region antigeninteraction from
`theeffective affinity in a bi- or multivalentinteraction, the
`latter is often referred to as avidity. For monovalentinter-
`actions, avidity = affinity; for multivalent interactions,
`avidity = affinity. Theory predicts avidity enhancements
`that vastly exceed observed numbers, but structural con-
`straints undoubtedlyrestrain the energy advantage ofmul-
`tivalent binding.” In its extreme,steric factors constrain
`somebivalent antibodies (e.g., anti-Tac”*) to bind only
`monovalently to antigens on cell surfaces but which will
`bind bivalently to antigen in solution. Yet even where anti-
`gen onthesurface is not bivalently bound by antibody,
`andforall solution interactions, careful treatmentofthese
`settings will note the molarity of binding site rather than
`
`oepees
`PaeS
`avaEe
`
`
`
`humans, and is dependent largely on kidney filtration
`
`PHARMACOKINETICS/PHARMACODYNAMICS
`
`Metabolism of immunoglobulins determines the duration
`ofusefulness in vivo of antibodies. Under normal condi-
`tions, the serum levels of endogenous immunoglobulins
`are determined by a balance between synthetic and cata-
`bolic rates.2 When antibodies are administered as thera-
`peutics, these catabolic rates effectively specify the dose
`and schedule necessary to maintain therapeutic blood lev-
`els where steady-state exposures are targeted. Table 28-1
`lists the half-lives ofhuman antibody survivals in humans,
`with IgG having the longest survival, 23 days (IgG1,2,4;
`IgG3 survival is 7 days.) Autologous IgG survivals are
`correlated with animal size, with IgG survival in mouse of
`4 days, in dog 8 days, in baboon 12 days, in cow 21 days,
`etc.2! Ofinterest, however,is the observation ofsurvivals
`in heterologous systems in which the shorter of the sur-
`vival of the IgG in the host orin the donor is dominant.
`Thatis, murine IgG will survive the same in humansor in
`mice, whereas human IgG will survive shorter in mice at
`a rate compatible with their own catabolism ofIgG.” The
`site of catabolism ofintact IgG remains controversial, al-
`though recent work with slowly mobilized radioisotope
`conjugates suggests that the reticuloendothelium (RE)
`system is a prominentsite ofthis catabolism.*°*™
`A substantial body of knowledge exists on the metab-
`olism of immunoglobulins in various disease states.
`Conditions of protein wasting (enteropathies, vascular
`leak syndromes, burns), febrile states, hyperthyroidism,
`hypergammaglobulinemia, and inflammatory disorders
`are accompaniedby significant acceleration of immuno-
`globulin catabolism.” This information is of importance
`to understanding in vivo survival data in variousclinical
`applications. In fact, the controlled conditionsof testing
`immunoglobulin metabolism are rarely duplicated in
`practice, with antibody survivals typically shorter than
`suggested by the numbers above. Typically, murine anti-
`body survival 4,2 valuesare in the rangeofless than 1 to
`3 days, and antibodies with human gamma Fc domains
`(chimeric or humanized) have f,/2 values in the range of
`1 to 15 days. Some ofthis 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. Influence on antibody clearance
`by antigen expression in vivo is considered below.
`Antibodyfragments have been studied because oftheir
`abbreviated survival and because small size may translate
`into bettertissue penetration. Fab and Fab’2 have survivals
`in vivo of 2 to 5 hours in mice, with comparable values in
`
`12
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`

`Antibody-Based Immunotherapiesfor Cancer
`
`663
`
`mechanisms.’ This is not based on size alone, because the
`Fe fragment, which is comparable in size to Fab,is notfil-
`tered 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 that are returned to circulation.
`Nointact immunoglobulin or fragments reenter circula-
`tion once fi