`
`
`
`
`
`-
`
`Hospira v. Genentech
`Hospira v. Genentech
`IPR2017-00737
`IPR2017—00737
`Genentech Exhibit 2031
`
`Genentech Exhibit 2031
`
`

`

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

`

`
`
`Cancer Chemotherapy
`and Biotherapy:
`Principles and Practice
`
`SECOND EDITION
`
`
`
`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 Cancer Institute
`
`Frederick, Maryland
`
`
`
`
`n Lippincott - Raven
`
`
`
`Philadelphia - New York
`
`_It______
`
`

`

`
`
`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 embodied in 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—397-514184 (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.CSC32219
`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`The material contained in this volume was submitted as previously unpublished material, except in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 fi'om which some of the illustrative material
`was derived.
`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
`consequences arising 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 of publication. How-
`ever, in view of ongoing research, changes in government regulations, and the constant flow of infor-
`mation relating to drug therapy and drug reactions, the reader is urged to check the package insert for
`each drug for any change in indications and dosage and for added warnings and precautions. This is
`particularly important when the recommended agent is a new or infrequently employed drug.
`Materials appearing in this book prepared by individuals as part of their official duties as US.
`Government employees are not covered by the above-mentioned copyright.
`
`987654321
`
`

`

`Cancer Chemotherapy and Biothcrapy, second ulitim,
`edited by Bruce A. Chabner and Dan L. Longo.
`Lippincott—Raven Publishers, Philadelphia ©1996
`
`
`CHAPTER
`
`Antibody-Based lmmunotherapies
`for Cancer
`
`
`
`.
`‘
`
`Richard P. Junghons, George Sgouros,
`and David A. Scheinberg
`
`Monoclonal antibodies (mAbs) are remarkably versatile
`agents with potential therapeutic applications in a num—
`ber of human diseases, including cancer. mAbs have long
`promised to offer a safe, specific approach to therapy.
`More than a decade of preclinical evaluation and human
`clinical trials have identified new strategies for the use of
`mAbs, as well as a number of difl‘icult obstacles to their
`effective application. Although the use of antibodies as
`targeting agents dates to the 19503,1 it was not until
`methods for production of mAbs appeared in the late
`19705,2 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 humans in vivo. First, mAbs can be used to
`focus an inflammatory response against a target cell. Bind-
`ing of a mAb to 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 various effector cells
`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 be directed at crit—
`ical hormones, growth factors, interleukins, or other regu—
`latory molecules or their receptors in order to control
`growth or other cell fimctions. Fourth, anti-idiotypic mAbs
`may be used as vaccines to generate an active immune re—
`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 drugs or factors to in-
`crease their plasma half-life, change their biodistribution,
`or render them multivalent. Alternatively, mAbs may be
`used to clear previously infused mAbs from the circulation.
`Yet despite the diversity of approaches, significant
`problems remain that are peculiar to mAbs. mAbs are
`large, immunogenic proteins, often of rodent origin, that
`rapidly generate neutralizing immune responses in pa-
`tients within days to weeks after their first injection. The
`sheer size ofmAbs, 150 kDa for IgG to 900 kDa for IgM,
`100 times larger than typical drugs, makes their pharma-
`cology (particularly diffusion into bulky tumors or 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 that is
`routinely achievable now can work against mAbs, since
`tumor cells that do not bear the specific antigen target
`may escape from cytotoxic effects.
`Within this context, it is still clear that mAbs have great
`potential to be safe and effective anticancer agents; recent
`clinical investigations have highlighted several areas where
`mAbs can be eEective, 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 deriva-
`tives
`(immunotoxins,
`radioimmunoconjugates, mAb
`fragments), discusses the pharrnacologic issues peculiar to
`mAbs, and outlines some of the important clinical results
`with mAbs. Potential solutions to the most difficult issues
`
`in the use of mAbs will be presented. Since mAbs and con—
`jugates ofmAbs represent many different 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 of mAbs
`often may not be possible. mAb therapy of cancer is a new
`and rapidly changing field, and readers are encouraged to
`consult other reviews for more comprehensive discussions
`of individual areas?‘8
`
`IMMUNOGLOBULIN (lg) 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 by the B cell on its surface represents the
`commitment of that cell to a particular but broad recog-
`nition space that subsequently narrows as 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 immunoglobulins that are expressed after mat-
`uration of the response and class switch have occurred.
`
`655
`
`th-
`
`

`

`
`
`656 .
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`Each of these antibodies parn'cipates in specialized func-
`tions: IgE in immediate-type hypersensitivity reactions
`and parasite immunity, IgA in mucosal immunity, and
`IgG in humoral immunity. In some cases, 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.10
`Table 28—1 summarizes various features of the antibodies
`that will be discussed in this section.
`
`STRUCTURE
`
`The fundamental structural elements of all antibodies are
`
`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
`irn—
`munoglobulin subclasses. Overall, kappa comprises 60%
`of light chain in humans versus 95% of light chain in
`mouse. Heavy chains are u, 8, y, e, and a, corresponding
`
`
`
`Table 28-1. Proper’ries of Antibody Classes.
`
`to IgM, D, G, E, and A, and conferring the biologic char-
`acteristics of each antibody class. Each chain is comp0sed
`of so-called irnmunoglobulin (Ig)—like domains of an-
`tiparallel beta-pleated sheets, two for light chain and four
`such domains for heavy chain, excepting IgM and. IgE,
`which have five. The amino-terminal domain of each
`chain is the variable (VI-I 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 6). Be-
`tween CH1 and CH2 is 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 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); structure (see Fig. 28-1), for a nominal total
`mass of 150 to 160 kDa for IgG and higher for other iso-
`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
`
`IgE
`
`Usual molecular form
`Molecular formula
`
`Monomer
`
`-y2K2 or 72x2
`
` Pentamer
`
`Monomer, dimer, etc.
`(a2K2)n or (u2k2)n
`
`Monomer
`
`Monomer
`
`(u2K2)5 or (u2)\2)5
`
`82nd or 82m did or 52x2
`
`V,CH1-3
`—
`
`V,CH1 -4
`. ..
`
`—
`190,000
`88
`13%
`0.03
`0.003
`
`2.5
`2
`
`— F
`
`ceR—I,FceR—II
`
`Mast cells
`
`—
`175,000
`78
`9%
`4
`0.3
`
`2.8
`1 or 2
`—
`
`H chain domains
`Other chains
`Subclasses
`
`Heavy chain allotypes
`Molecular weight
`Sedimentation constant
`Carbohydrate content
`Serum level (mg/100 ml)
`Percentage of total
`serum lg
`Half-life (days)
`Antibody valence
`Complement fixation
`(classic)
`Fc receptors
`
`Binding to cells
`
`V,CH1-3
`—
`IgG1,IgG2,
`IgG3,IgG4
`Gm (ca. 30)
`150,000
`668
`3%
`1250 i 300
`75—85
`
`23 (IgGS 7d)
`2
`+ (IgG1,2,3)
`
`V,CHl-3
`I chain, 8 piece
`IgA1,IgA2
`
`Am (2)
`160,000
`7S,9S,1 18,148
`7%
`210 i 50
`7—15
`
`5.8
`2,4,6, .
`—
`
`.
`
`.
`
`Fc'yR-I,Fc'yR-II,
`Fc-yR-III
`Monocyte
`macrophages,
`neutrophils,
`LGLs
`
`—
`
`V,CH1~4
`I chain
`
`Min (2)
`950,000
`19S
`10%
`125 1- 50
`5-—10
`
`5.1
`10
`+ +
`
`P
`
`Other biologic properties
`
`Secondary Ab
`response;
`placental
`transfer
`
`Secretory anfibody
`
`Primary Ab response;
`B-cell surface Ig,
`rheumatoid factor
`
`B—cell
`surface 1g
`
`Homocytotropic
`Ab; anaphylaxis;
`allergy
`
`
`
`é
`:fi‘l7gig
`
`'is
`
`_-sdsééE-‘Jf
`
`
`Sigma"-'‘
`
`

`

`
`
`
`
`|_
`
`Antibody-Based Immunotherapies for Cancer
`
`Figure 28-1. Antibody structure. The structural relationships and Functions of domains of IgG. (Reprinted with permission From Wasser-
`man RL, and Capra JD, Immunoglohulins. ln Horowitz MI, Pigman W, eds, The glycoconjugates. NY: Academic Press, 1977: 323.)
`
`
`
`
`
`
`
`LIGHT CHAIN ___
`HYPERVARIABLE{
`REGIONS
`
`LIGHT CHAIN
`
`HEAVY CHAIN
`
`HEAVY CHAIN
`HYPERVARIABLE
`REGIONS
`
`CARBOHYDRATE
`
`
`
`ANTIGEN
`(BINDING) F°b
`
`BIOLOGICAL
`ACTIVITY Fc
`MEDIATION
`
`
`
`INTERCHAIN
`L DISULFIDE
`I
`BONDS
`
`INTRACHAIN
`DISULFI DE
`BONDS
`
`VL AND VH1VARIABLE REGIONS
`CL AND CH: CONSTANT REGIONS
`
`
`
`halves.n 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 of a
`single (HL); unit, Whereas IgM exists as a pentamer of
`(HL); units joined by disulfide bonding with a third I-
`chain component. IgA exists mainly as a monomer in
`serum but in secretions exists primarily as a dimer plus
`trirner and higher forms in which the oligomers are linked
`by I chain as well as the fi'agment of secretory chain (se-
`cretory piece) that is involved in the mucosal transport.
`The V region itself is composed of subdomains: rela—
`tively conserved framework regions interdigitated with
`the
`so-called
`complementarity—determining regions
`(CDRs) [also termed hypermriable segments (HVSs)] that
`make primary contact with antigen”,12 (see Fig. 28-1).
`There are three CDRs in each heavy and light chain that
`may participate 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 various roles including solubility, 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).13 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 Pay receptors.
`The IgG antibody “unit” has been defined in terms of
`susceptibility to proteases that cleave in the exposed, non-
`
`_I
`
`folded regions of the antibody (see Fig. 28-1). A tabula-
`tion of anu'body firagments and engineered or synthetic
`products is presented in Table 28-2. Fab contains the
`V region and first constant domain ofthe heavy chain (VH
`+ CH1 = Pd) and the entire light chain (L); Fab’ includes
`additionally a portion ofthe H chain hinge region and one
`or more flee cyteines (Fd'); Fab’2 is a dimer ofFab’ linked
`through hinge disulfide(s); and Fv is a semistable antibody
`fragment that includes only VH + VL, the smallest anti-
`gen-binding unit. Fe is the C-terminal crystallizable frag-
`ment that includes the complement and Fe receptor—bind-
`ing domains (below). Genetically engineered products
`include the ACHZ constructs, lacking the second constant
`domain ofheavy chain, which behave like a Fab’2, with bi-
`valency, abbreviated survival, and lack of interacu'on with
`host effector systems, but which do not require enzymic
`processing.13a st 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 anti-
`gen—binding domain in a single peptide productm’; this is
`not related structurally to an antibody and is therefore
`considered an antibody mimic.
`
`ONTOGENY
`
`Antibodies represent the most strikingly 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, if true, would
`
`

`

`658
`
`
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`
`Figure 28-2. Space-filling model of human [96] antibody
`with CDRs in color representing anti-Tac-H; human myeloma
`protein Eu with CDRs grafted from murine anti-Toe. (Photo
`provided courtesy of Dr. C. Queen.) (See Color Plate 1.)
`
`I
`
`
`
`include all chordates.14 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 undergoes a series ofmaturation
`steps that begins with V gene selection for heavy chain
`followed by light chain V selection that yields surface
`expression and secretion 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 plasma cell conversion. Plasma cells remain in
`
`tissues, spleen, or lymph nodes and secrete large quanti-
`ties of antibody, the sole functiOn of this terminally dif.
`ferentiated cell.9
`.
`The genes of heavy and light 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) and joining (I) regions for heavy chain and
`I regions for light chain—contribute to or constitute,
`with modifications, the CDR3.15 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 I regions for a potential of 6000 com-
`binationsm‘19 (Fig. 28-4). There are roughly 80 V kappa
`[VK] light chain and 5 I kappa [In] 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 106. Yet even this number is 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 the total 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‘5 range. The smallest “complete”
`immune system is that of the young tadpole with 106 B
`cells, which suggests that repertoires of 105 to 10‘5 con-
`stitute a sufficiently complete topologic set for meaning-
`fully diverse, if not exhaustive, antigen recognition.21
`V gene selection is based on random expression
`followed by specific amplification. It has been argued on
`physicochemical grounds that 105 diEerent antibody
`molecules are sufficient to create a topologic set that
`recognizes any antigen surface with an affinity of 105 t0
`10'5M‘1,22 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. Sub-
`sequent binding and stimulation are in proportion to the
`strength ofthe binding reaction; hence there is an in ViVO
`selection for mutations that enhances the affinity of the
`antibody for the antigen, a process termed afi‘inity 14W“
`“ration.23 Simultaneous with this increased affinity is a
`narrowing of the specificity, with the antibody shedding
`its early polyreactive phenotype. The cells then undergo
`
`“class switch” to one ofthe mature antibodies (IgG, 13A?
`
`-a
`
`Wyn-cs
`rOenigma:
`"Fm3”
`
`.....Inm-"_I___.__._w
`
`.1"
`
`

`

`Antibody-Based lmmunotherapies For Cancer
`
`
`Figure 28-3. Antigen-antibody binding surtace luxtaposition. The V region (Fv) oi antibody (right) binds to 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 ottsets the two molecules by 8
`to show the complementarity of surfaces that promotes the binding interac-
`tion. The stippled surface of the neuraminidase defines the antigen
`"epitope." (Photo provided courtesy of Drs. P.M. Colman and W.R.
`Tulip, CSIRO Australia.) (See Color Plate 2.)
`
`l
`
`
`
`
`
`IgE) by deleting out DNA between the VD] region and
`the new C region of the heavy chain, which brings this
`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, the cell will cease
`its CDR mutagenesis, affinity maturation will have been
`completed, and the B cell will undergo morphogenesis to
`a tissue-resident plasma cell.9
`
`ANTIGEN-ANTIBODY INTERACTIONS
`
`Afi‘inity is a quantitative measure of the strength of the
`interaction between antibody and its cognate antigen and
`
`is intended in the same sense as the equilibrium constant
`in the chemical mass action equation:
`
`[AB] = KalA] [B]
`
`(28—1)
`
`The equilibrium or aflinity constant is represented in
`units of M‘l. In most instances studied by x-ray crystal-
`lography, contacts between antibody and protein antigen
`are dominated by noncovalent hydrogen bonds (0—H),
`with a lower frequency ofsalt bridges (COO'— + H3N),
`with a total of about 15 to 20 contacts. The effect of
`adding a new H- bond can be estimated from the free
`energy gain (0.5 to l kcal/mol-°C) and from AG =
`—RTln Ka to yield affinity increases of approximately
`
`

`

`660
`CANCER CHEMOTHERAPY AND BlOTHERAPY
`
` L Table 28-2. Antibody Fragment Definitions
`
`Ji
`
`DESCRIPTION
`
`Complete IgG
`
`Papain digest; Pd + L
`
`Pepsin digest monomer; Fd’ + L
`
`Pepsin digest dimer
`
`which antigen is 50% saturated; if the antibody is in large
`
`
`V region digestion fragment; VH + VL
`
`C region digestion fragment; crystallizable fragment
`
`Smaller fragments of PC
`
`Deleted CH2 domain; dimer of V — CH1 - CH3 + L-
`
`Single-chain Fv; VH and VL joined by peptide linker
`
`Antigen-binding unit; peptide mimic
`
`___J
`
`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 that is the middle of
`the range for the biologic action of the antibody:
`
`K4 = 1
`
`(28-2)
`
`That is, the K4 is the concentration of free antibody at
`
`DESIGNATION
`
`REPRESENTATION
`
`Fat
`
`\, /
`
`Enzyme-generated products
`
`Fd
`
`\\L
`
`Fd’
`k
`
`VFW
`
`/4 C
`
`H2
`
`CH3
`
`
`
`
`
`IICH2
`llCHs
`
`\ /
`CH3
`
`\Ia
`¢‘1»
`
`w
`
`Fab
`
`Fab’
`
`Fab’2
`
`Fv
`
`Fc(och')
`
`F (
`F )
`p c orp c'
`
`Genetically engineered products
`
`delta CH2
`
`st
`
`Synthetic products
`
`ABU
`
`
`
`3- to 10—fold. Therefore, the affinity maturation that
`takes place (or affinity that may be lost in antibody engi-
`neering) changes quickly with a relatively small change in
`the number ofbonds. That is, creating as few as three new
`hydrogen bonds may generate an aflinity enhancement of
`more than 100—fold. This has been borne out by 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-bonding interactions (but
`are also “T-independent” antigens).
`
`_‘aM‘Aghiis“5;?I
`
`
`
`

`

`
`
`Antibody-Based Immunotherapies for Cancer
`
`Figure 28-4. Generation of diversity. VJ and VDJ ioining occur in Lchain and H chain by excision of intervening DNA in the genome.
`Class switch involves deletion of intervening constant (C) domains (C, Cd, etc.) and transcription through the new proximal C region.
`C is Finally joined to the V gene by splicing of the mRNA. (Reprinted with permission from Cooper MD, Current concepts: b lympho-
`cytes-normal development. N Engl J Med 31721452, 1987.)
`
`A. Heavy chain genes on chromosome 14 p32
`
`C 3
`
`am
`
`vi>50l
`
`D(> 20)
`
`J
`
`3. Kappa light chain genes
`on chromosome 2 p11
`Vl>50l
`
`J
`
`C. Class switching
`
`by DNA deletion VDJ
`
`S};
`
`m
`
`CPL
`08
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`
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`
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`VDJ
`
`
`excess, the input antibody concentration approximates
`the free concentration. The Kd is a frequently used term,
`but its relationship to aflinity must always be borne
`in mind: i.e., low affinity = high Kd; high affinity = 10w
`Kd. For example, a IQ of 2 X 109 M‘1 implies a IQ of
`0.5 X 10“9 M(0.5 nM), or about 0.1 (Lg/ml antibody
`concentration for IgG. If antigen is in the picomolar
`(10‘12 M) range, this concentration of antibody will have
`half of antigen saturated and half of antigen will remain
`“free.” At lO-fold higher antibody concentration (1 ug/ml,
`10 X IQ), antigen will be 90% saturated and 10% free,
`
`and at 100-fold higher concentration (10 (Lg/ml, 100 X
`IQ), 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 nM with a Kd of 1 nM, it does not matter
`whether antigen is 0.1 nM at the IQ, 0.1 pM, or 0.1 HM;
`antigen in each case is 50% bound, although the ratio of
`antibody to antigen is 10, 104, and 107. It is only the re-
`lation offree antibody to its K4 that determines the degree
`ofantigen saturation.
`
`

`

`662
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`The afl‘inity constant K, is itself composed of two
`terms that describe the on (forward; units ofM‘l-s‘l) and
`off (back; units of 5“) rates of the reaction:
`
`K. = ikl,
`
`(28-3)
`
`To a first approximation, the forward rate is diffusion
`limited and comparable for many antibodies reacting
`with macromolecules or cell-bound structures; reactions '
`of antibodies with haptens and other small molecules in
`solution will be dominated by the faster linear and rota—
`tional difliision rates of the smaller component.24 For ex-
`ample, 0.1 nM of DNP-lysine (0.1 ng/ml) or 0.1 nM
`cell-bound HIA—AZ (50 ng/ml) mixed with specific IgG
`antibody at 10 pig/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 have virtually
`the same affinity constant.“ This is due to the fact that
`the fast association rate is balanced by a fast dissociation
`rate for the hapten (t1/2 = 0.7 5) versus a longer stability
`for the protein antigen (1.1/2 = 6 min).
`While there are exceptions, the on rates of antibodies
`to protein and cell—bound antigens are primarily in this
`range and inversely proportional to antibody concentra-
`tion for antibody in excess of antigen (i.e., at 1 ug/ml,
`the 50% on time would be of the order of 0.5 to 1 hour).
`Accordingly, diflerences in aflinity between antibodies to
`the same cellular antigen will in many instances be seen to
`be reflective of the off rate (kg). For most such purposes,
`an antibody is generally considered of “good” affinity if
`its K, 2 109 M", Where off rate 271/2 values of an hour or
`more at 4°C are common. Association and dissociation
`times at 37°C are both accelerated relative to 4°C, on the
`order of 5 or more, frequently with a net decrease in
`antibody affinity of 2- to 10-fold. Thismust be explicitly
`tested, however, since there are instances of protein—
`ligand affinities that are enhanced by higher temperature.25
`The foregoing expresses basic principles of binding
`processes. A further important feature ofantibodies is em—
`bodied in their multivalent structures. While the on rates
`for monovalent Fab and bivalent Fab’2 constructs are
`comparable, the bivalent 01ftimes may be 10-fold or more
`longer than the monovalent constructs, yielding affinities
`that are similarly enhanced.24 To discriminate the affinity
`that is intrinsic to the V region antigen interaction from
`the effective aflinity in a bi- or multivalent interaction, the
`latter is often referred to as avidity. For monovalent inter-
`actions, avidity : affinity; for multivalent interactions,
`avidity 2 affinity. Theory predicts avidity enhancements
`that vastly exceed observed numbers, but structural con-
`straints undoubtedly restrain the energy advantage ofmul-
`tivalent binding.”27 In its extreme, steric factors constrain
`some bivalent antibodies (e.g., anti-Tacza) to bind only
`monovalently to antigens on cell surfaces but which will
`bind bivalently to antigen in solution. Yet even where anti-
`gen on the surface is not bivalently bound by antibody,
`and for all solution interactions, careful treatment ofthese
`settings will note the molarity of binding site rather than
`
`antibody in comparing Fab with higher-valency homo-
`logues. When antigens are presented multivalently on sur-
`faces of cells, viruses, or other pathogens, even the low-
`affinity IgM interactions can yield "a high-avidity, stable
`binding to such targets in vivo.
`
`humans, and is dependent largely on kidney filtration
`
`PHARMACOKINETICS/PHARMACODYNAMICS
`
`Metabolism of immunoglobulins determines the duration
`of usefulness 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.29 When antibodies are administered as thera—
`peutics, these catabolic rates efi'ectively 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.21 Of interest, however, is the observation of survivals
`in heterologous systems in which the shorter of the sur-
`vival of the IgG in the host or in the donor is dominant.
`That is, murine IgG will survive the same in humans or in
`mice, whereas human IgG will survive shorter in mice at
`a rate compatible with their own catabolism ofIgG.21 The
`site of catabolism of intact IgG remains controversial, al—
`though recent work With slowly mobilized radioisotope
`conjugates suggests that the reticuloendothelium (RE)
`system is a prominent site of this catabolism."’°’3°a
`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 accompanied by significant acceleration of immuno-
`globulin catabolism.29 This information is of importance
`to 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 anti-
`body survival t1/2 values are in the range of less than 1 to
`3 days, and antibodies with human gamma Fc domains
`(chimeric or humanized) have t1/2 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. Influence on antibody clearance
`by antigen expression in vivo is considered below.
`Antibody fragments have been studied because oftheir
`abbreviated survival and because small size may translate
`into better tissue penetration. Fab and Fab’Z have survivalS
`in vivo of 2 to 5 hours in mice, with comparable values in
`
`55
`
`
`
`__...m.n1".—
`
`R .
`
`
`
`
`
`mutt_'!'."'""2.'!'
`
`

`

`Antibody-Based lmmunotherapies for Cancer
`
`Soluble versus Surface Antigen
`
`mechanisms.29 This is not based on size alone, because the
`Fc fragment, which is comparable in size to Fab, is not fil—
`tered and has an in vivo survival of 10 days in humans.
`These rapidly catabolized fragments,
`like other