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`REPORTS IN
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`MEDICINAL
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`CHEMISTRY
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`Volume29
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`Sponsored by the Division of Medicinal Chemistry
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`EDITOR-IN-CHIEF:
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`PARKE-DAVIS PHARMACEUTICAL RESEARCH
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`Chapter 32. Humanized Monoclonal Antibodies
`
`Leonard Presta
`Genentech, Inc., 460 Point San Bruno Blvd.
`South San Francisco, CA 94080
`
`lntrodyction - Almost two decades ago, the development of monoclonal hybridoma
`technology - the ability to make cultured cells produce antibodies of predefined specificity
`- promised a new weapon in the arsenal of molecules able to combat disease (1 ).
`Antibodies against a specific antigen (or target molecule) could be generated, incorporated
`into a hybridoma for production, and then used in diagnosis or therapy. However, only
`rodent monoclonal antibodies could be made due to technological limitations.
`In most
`clinical applications, these rodent monoclonal antibodies exhibit properties which severly
`limit their utility. First, they may induce an immunogenic response in humans, referred to as
`HAMA (human anti-mouse antibodies), when the human immune system recognizes them as
`foreign substances. Second, the therapeutic efficacy of the rodent antibody may also be
`reduced because a rodent antibody is cleared from serum more rapidly than human ones.
`Third, in humans, rodent antibodies generally exhibit only weak recruitment of effector
`functions, such as antibody-dependent cell-mediated cytotoxicity and complement fixation,
`which may be requisite for the function of the antibody. Use of a human antibody would
`circumvent these three problems.
`
`With advances in molecular biology and mammalian tissue culture, it became
`possible to obtain useful amounts of any antibody, thereby surmounting the limitations of
`hybridoma technology and use of only rodent antibodies. But one problem still existed -
`how does one obtain human antibodies against a particular human antigen? Even if one
`could ethically use a human subject as the biological factory, the human immune system, in
`general, does not produce antibodies against human proteins. Two techiques have been
`developed to address this problem. Earliest of these was the construction of chimeric
`antibodies in which entire antigen-binding domains from a rodent antibody are substituted
`for those of a human antibody (Fig. 1) (2).
`In many cases, these molecules still exhibited
`some, albeit reduced, HAMA. The next step was generation of humanized antibodies in
`which a significantly reduced number of rodent residues are incorporated into a human
`antibody such that the humanized antibody has the same binding characteristics and
`specificity as the original rodent antibody (3-5).
`In order to understand the technique of
`humanization and the difference between a chimeric and humanized antibody, the structure
`of antibodies must be appreciated.
`
`Antjbody Structure - An antibody consists of four peptide chains -- two identical light
`chains and two identical heavy chains - which form a 'Y' shape. The functions of the
`antibody reside in different domains. Antigen binding occurs at the ends of the arms of the
`'Y', each arm being referred to as a Fab or antigen binding fragment. Hence each antibody
`can bind two antigen molecules. The effector functions reside in the base of the 'Y',
`referred to as the Fe portion. Each light chain consists of one variable domain and one
`constant domain; each heavy chain consists of one variable domain and three constant
`domains (Fig. 1 ). The 'constant' notation refers to the fact that for a particular species and
`immunoglobulin class (e.g. lgG, lgE, lgA) the amino acid sequence for the constant domain
`
`ANNUAL REPOR'I'S IN MEDICINAL CHEMISTRY -29
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`Copyright © 1994 by Aoa.demjc Press, Inc
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`is, for our purposes, invariant. The 'variable' notation refers to the fact that certain portions
`of variable domains differ extensively in sequence among antibodies and are used in the
`binding and specificity of each particular antibody for its particular antigen. These portions
`are referred to as complementarity-determining regions (CDRs). Both light and heavy chain
`variable domains contain three CDRs to give a total of six CDRs involved in binding antigen.
`Each CDR is a contiguous sequence of amino acids which form a loop connecting two �
`strands of the framework. Note though that the amino acid sequence of the non-CDR
`portion of the 'variable' domain is relatively invariant and is used to categorize these
`domains into subgroups (6).
`
`0.
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`. ,
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`Mouse
`
`Chimera
`
`�
`�
`Humanized
`Human
`Figure 1. Chimeric and Humanized Antibodies. CDRs are represented by 'fingers'
`on variable domains of the light chain (VL) and heavy chain (VH). Note that only the mouse,
`chimera and humanized antibodies bind antigen. Disulfide bonds between the two heavy
`chains and between the light and heavy chains are denoted by thicker, dark lines.
`
`In one type of chimeric antibody all of the variable domains from a rodent antibody
`are fused onto the constant domains of a human antibody (Fig. 1 ). This type of chimeric
`antibody is comprised of four rodent domains and eight human domains and consequently
`retains approximately 33% rodent residues. In the corresponding humanized antibody, only
`the six rodent CDRs replace the six human CDRs {Fig. 1 }. The resulting antibody contains
`only about 5-10% rodent residues and is still chimeric in that it has residues derived from
`different species. From the perspective of the human immune system, the humanized
`antibody is the more 'human' of the two types as it contains fewer rodent residues.
`Moreover one must appreciate that the amino acids in CDRs are highly variable and this
`variance is independent of species. The nature of the CDRs is dependent upon the target,
`whereas the framework is species dependent. Hence even though the humanized antibody
`still retains about 5-10% rodent residues, these residues would also be variant in human as
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`HumaniZed Monoclonal Antibodies
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`Presta illJl
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`well as rodent and thus the humanized antibody could well be very similar to a human
`antibody directed against the same site on the same target as the original rodent antibody.
`
`Humanjzed Antibody Constructjon - At first glance constructing a humanized antibody is
`rather simple and straightforward. One chooses a human antibody, clips off the six human
`CDRs and replaces them with their structurally analogous rodent CDRs.
`Indeed some
`humanized antibodies have approached this simplicity in method (7,8). However, there are
`both theoretical and technical difficulties. Among the former is the exact definition of a
`CDR, i.e. exactly which residues comprise the CDRs and which comprise the framework.
`Two definitions have been proposed. The first relies solely on analyses of the amino acid
`sequences of a multitude of antibodies (6). The variance of amino acid types at each
`residue position of antibodies was evaluated and those positions which exhibited a
`relatively large variance were included in a CDR as long as they were contiguous to other
`positions with high variance. Residue positions showing relatively low variance were termed
`framework. The second definition was derived from structures of antibodies (9). When the
`two definitions are compared some striking differences are apparent. While the sequence
`based CDRs are generally larger than, and encompass, the structure-based CDRs, a
`notable discrepancy occurs in the definition of CDR-H1 (i.e. the first CDR loop in the heavy
`chain sequence) where the two definitions overlap by only two residues. Generally, the
`sequence-based definition of CDAs has been used when an antibody is humanized.
`
`The technical difficulties fall into three categories. First, the choice of constant
`domains must be made. To date almost all antibodies humanized have been those of the
`lgG class.
`In humans there are four recognized subclasses, denoted lgG 1, lgG2, lgG3 and
`lgG4, the classification of which is based on the sequence of their constant domains. Each
`subclass also exhibits differences in effector, or biological, functions mediated by the
`constant domains. The intended therapeutic use of the antibody must be considered when
`choosing the lgG subclass, e.g. is complement fixation an advantage or disadvantage for
`the particular application?
`
`Second, the choice of human variable domains, both light and heavy, must be
`In the homology or 'best-fit' method, the sequence of the rodent variable domain is
`made.
`screened against the entire library of known human variable domain sequences. The human
`sequence which is closest to that of the rodent is then accepted as the human framework
`(8, 1 O}. Another method uses a particular framework derived from the consensus sequence
`of all human antibodies of a particular subgroup of light or heavy chains. The same
`framework may be used for several different humanized antibodies (11, 12). Remembering
`that the purpose of humanization is to trick the human immune system into recognizing the
`humanized antibody as one of its own, is the 'best-fit' or the consensus framework better for
`this trickery? Unfortunately there are very limited in vivo data on the reaction of the human
`immune system to humanized antibodies and no comparative study has been performed on
`In the final analysis both may function well with regard to acceptance by the
`the two types.
`human immune system, with perhaps an occasional aberration.
`
`Third is the most imposing technical difficulty -- designing the humanized
`antibody such that it has the same binding affinity and specificity as the original rodent
`antibody. When the rodent CDRs are simply grafted onto a human framework the resultant
`humanized antibody may exhibit signficiantly reduced binding compared to the original
`rodent antibody (11, 1 2). This reduction in binding may be caused by certain buried
`framework residues. Each CDR is a loop and these loops may be anchored to the
`framework not only covalently at their ends but through noncovalent interactions with the
`sidechains of buried framework residues. For example, in CDR-L 1 the sidechain at position
`29 (residue numbering is according to ref. 6) is usually hydrophobic, buried and contacts
`hydrophobic sidechains at framework positions 2, 25, 33, 71 and 90 (13).
`ff an incorrect
`amino acid is chosen for any of these framework positions the packing of CDR-L 1 against
`the protein might be altered, resulting in an incorrect presentation of the exposed CDR-L 1
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`sidechains and consequent reduction in binding to antigen. Such a case may occur when
`the sidechains at position 29 in the rodent CDR and positions 2, 25, 33, 71 and 90 in the
`rodent framework differ from those in the human antibody.
`In order to retain proper binding
`the offending framework residues must be altered from human to murine so that the CDR
`packs against the protein properly. Alternatively, the buried sidechain in the CDR itself,
`such as at position 29 in CDR-L 1, might be changed from murine to human to evaluate
`whether proper CDR conformation can be attained. This regimen must be evaluated for
`each of the six CDRs because if just one CDR has an improper conformation it may reduce
`the binding of the humanized antibody to the point that it is useless as a therapeutic
`replacement for the rodent antibody. While buried framework residues can affec t the
`binding of the humanized antibody through an indirect mechanism, i.e. by influencing the
`conformation of a CDR, occassionally an exposed framework residue is involved in direct
`binding to the antigen and must be included in the humanized form (11, 14).
`
`While performing this regimen for six CDAs may seem a daunting task, especially
`since crystal structures of the rodent and human antibodies are usually not available,
`nature has provided some guidance. Even though CDRs vary widely in sequence and to a
`lesser extent in size, analysis of crystal structures of Fab fragments shows that for each
`CDR (except CDR-H3) only a limited number of conformations may be utilized (13). These
`canonical conformations are dependent on size and sequence of the CDR. Hence if one
`inspects the sequence and size of the particular rodent CDR the canonical conformation
`
`can then be assigned. This provides information as to which CDR residues will be exposed,
`which will be buried and which framework residues may play a role in stabilizing the CDR
`conformation.
`
`How has this technique fared regarding the ability of a humanized antibody to
`
`reproduce the binding affinity of its parent rodent antibody? The first humanization involved
`transferring only the CDRs of the heavy chain from a murine to human antibody. When
`expressed with the murine light chain, the humanized heavy chain exhibited binding less
`than 2-fold down from the parent murine (15). Two years later another two humanizations
`were published in which both the light and heavy chain CDRs were transferred. In the first, a
`humanized anti-lysozyme antibody, binding was reduced by 10-fold (16). The second was
`more interesting since it was potentially clinically relevant, underscored the importance of a
`single buried residue for maintaining proper CDR conformation, and noted the difference
`between the sequence-based and structure-based CDA definitions (17). Directed against
`the CAMPATH-1 antigen on human lymphocytes and monocytes, the initial humanized
`version was 40-fold down in binding. Changing the human Ser27 to murine Phe27 in the
`
`heavy chain resulted in a version which exhibited only a 3-fold reduction in affinity. Was
`this a framework or a CDR residue? According to the structure-based definition of CDRs
`used to design the humanized anti-CAMPATH-1 antibody, heavy chain position 27 was part
`of the framework while according to the sequence-based definition position 27 was part of
`the CDR. Regardless of the definition, the marked improvement in binding when the murine
`Phe was substituted for the human Ser emphasized the need to pay attention to framework,
`as well as CDR, sequence in future humanizations.
`
`Since 1988 the technique has developed and become increasingly utilized for
`clinically relevant antibodies. The 'best-fit' method, used first in 1989 (10), has remained
`the more popular method for designing the sequence of the humanized antibody than the
`later consensus method (11 ). More recently, in the design of an anti-lgE humanized
`both methods were evaluated (14). The study concluded that although the
`antibody,
`consensus sequence showed the best overall binding, no clear advantage in binding was
`evident for the consensus antibody versus the 'best-fit' antibody. Another humanization of
`an anti-lgE antibody, utilizing only the consensus method, was reported earlier that year
`(12).
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`Humanized Monoclonal Antibodies
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`Presta. �
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`Both of the anti-lgE humanizations used another recent technology in the design
`of the antibody - molecular modeling. As the database of antibody crystal structures
`grows (which consists predominanly of structures of Fab fragments), information
`accumulates on the three-dimensional structure of variable domains and the relationship
`between CDRs and framework. Since the structure of the rodent antibody of interest will
`usually not be available, a computer graphical model based on crystal structures can be
`generated and used to determine which framework residues might influence CDR
`conformation. Many of the humanizations published of late include use of molecular
`modeling or crystal structure information (7,12,14,18,19). Only recently, however, have
`crystal structures of humanized antibodies been determined and compared to the molecular
`model used to design the humanized antibody (20,21 ).
`In this case, the model correctly
`predicted the conformation of the framework and five of the CDR loops. The conformation of
`the sixth CDR in the model differed from that in the crystal structure, but this same CDR also
`varied in conformation among the crystal structures of three closely related humanized
`variants (21 ).
`
`Though some humanizations have been successful without any alteration of the
`chosen human framework (7,8), other studies have found it necessary to alter some human
`framework residues to murine in order to attain binding equivalent to the parent murine
`antibody.
`In both of the aforementioned anti-lgE humanizations, murine f ramework
`residues were important (12, 14).
`In humanization of the Mikj31 antibody, eighteen
`framework changes from the 'best-fit' sequence were incorporated (18). Another version of
`MikJ31 with just six changes bound only about 2-fold less well than the version with eighteen
`changes. However the latter was superior in biological activity and it was noted that the
`bioactivities of these two versions could not be predicted solely from their binding affinities
`to the target molecule, in this case the IL2 receptor J3-chain. Such a lack of correlation
`between binding affinity and bioactivity had been noted previously for another humanized
`antibody (11 ). The importance of a particular framework residue may vary from antibody to
`antibody. For example, among six humanizations reported very recently, two retained the
`human amino acid at heavy chain position 71 (7,8), three changed to the murine amino acid
`(14, 18, 19) and in the last the murine and human consensus residues at position were
`identical and no change was required (12). This emphasizes the importance of considering
`each murine antibody as idiosyncratic, i.e. each antibody may possess its own unique CDR
`framework interactions which are sensitive to changes in amino acid constitution. As more
`humanizations are performed and antibody crystal structures become increasing available,
`perhaps nature's rules for determining these characteristic packing interactions may
`become more evident so that they can be ascertained during the initial design of the
`humanized antibody. Some effort in this area has already been accomplished (13,22,23).
`
`While buried framework residues have been shown to be important, less attention
`has been focused on exposed (or semi-exposed) framework residues. Crystal structures of
`Fab-antigen complexes have shown that on occassion a framework residue will be involved
`in direct binding to the antigen (24). The first mention of an exposed framework residue
`important for retention of binding and bioactivity in a humanized antibody was for the anti
`p185HER2 antibody (11 ). The murine Arg at position 66 in the light chain, predominantly Gly
`in human kappa light chains, was shown to improve binding by 4-fold and improve
`bioactivity. More recently, having the murine amino acids at light chain residues 1 and 3
`were shown to enhance binding (14). Though the involvement of exposed framework
`residues in binding antigen may not be common, they deserve consideration when
`attempting to further improve the binding of a Jess than competent humanized antibody.
`
`The focus of humanized antibodies has been on the variable domains since these
`bind antigen, but some studies have begun to concentrate on the rest of the humanized
`molecule, namely the Fe portion which possesses the effector functions. While some
`humanized antibodies have exhibited the ability to effect antibody-dependent cell-mediated
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`In other studies the differences between the
`cytolysis (11, 18,25,26) others have not (8).
`various classes of lgG have been evaluated (27) and, recently, the removal of conserved
`carbohydrate in the Fe has been investigated (28). As humanizations become more
`commonplace (or are replaced by human antibodies) and clinical data become available, the
`necessity for understanding and tailoring the Fe for a desired effector function will increase.
`
`Therapeytjc Use of Chjmerjc and Humanized Aotjbodjes - By the mid-1980's, it was
`recognized that rodent antibodies used as therapeutics would be problematic in that they
`may elicit a HAMA response, are more rapidly cleared from serum and less efficacious at
`eliciting effector functions (29). The HAMA response was also categorized into at least two
`types of anti-antibodies: those directed against the CDRs, referred to as anti-idiotypic
`antibodies. and those directed against the remainder of the molecule, referred to as non
`anti-idiotypic antibodies (30).
`Initially chimeric antibodies were thought be the solution.
`In
`one interesting study the immunogenicity of chimeric antibodies was evaluated by
`introducing human antibodies and hurnan/rnurine chimeric antibodies into mice (31 ). The
`murine response to fully human antibodies was robust, with 90% of the anti-human
`antibodies directed against the constant domains and 10% directed against the variable
`domains. The chimeric antibodies also elicited a response directed against the human
`variable domains, whereas fully murine antibodies gave no response or only an anti
`allotypic response. The latter arise from variation in the sequence of constant (and
`sometimes variable) domains among individuals and is akin to blood group antigens (A, B,
`AB, O}.
`In humans, chimeric antibodies have given mixed results. For example, the first
`chimeric antibody in a clinical trial, directed against a surface antigen on adenocarcinoma
`cells of gastrointestinal origin, elicited a detectable but modest HAMA response in only 1 of
`1 O patients (32}.
`In contrast, the same investigators later reported that a different chimeric
`antibody, directed against a different surface antigen on adenocarcinoma cells, elicited a
`HAMA response in 7 of 12 patients after only one infusion (33}. For both antibodies the
`HAMA response was directed against the murine variable region.
`
`The first humanized antibody used in humans, CAMPATH-1H, induced remission in
`two patients with non-Hodgkin lymphoma and showed no detectable HAMA (34). CAMPATH-
`1 H was evaluated a few years later for rheumatoid arthritis (35).
`In this study, 7 of 8
`patients showed significant clinical benefit and all 8 patients exhibited no HAMA after one
`course of treatment. However upon retreatment, 3 of 4 patients did show a response. Of
`these, two had only an anti-idiotypic response while the third had both an anti-idiotypic and
`anti-allotypic response. Another antibody which was among the earliest humanized, the
`anti-Tac antibody (10), has been shown to be less immunogenic in cynomolgus monkeys
`than the parent murine antibody (36). As expected the response in the cynomolgus
`monkeys to the humanized antibody was anti-idiotypic while that against the murine was
`directed against the rest of the imrnunoglobulin. These anti-idiotypic antibodies have
`recently been shown to be directed primarily against combinations of CDRs H1, H2 and L3
`rather than against a single CDR (37).
`In a similar study, murine and humanized OKT4A
`antibodies were evaluated in cynomolgus monkeys (38). All 17 monkeys which received
`murine OKT 4A showed a response which was both idiotypic and non-idiotypic. While all 8
`monkeys which received the humanized OKT4A also showed a response, it was only anti
`idiotypic,
`In addition, higher serum levels of the humanized antibody were maintained for a
`longer period following treatment than were serum levels of the murine OKT4A.
`
`Finally, during the past year a study was published in which mice injected with
`murine antibodies were used as a model for estimating potential patient sensitization
`against humanized antibodies (39). As in a similar earlier study (31 ), anti-idiotypic and anti
`al!otypic antibodies were elicited. That both types of anti-antibodies have been found
`underscores the need to determine the biological and therapeutic consequences of each.
`Anti-idiotypic antibodies directed against the CDRs may prove to be a necessary evil when
`humanized antibodies are used as therapeutics since the CDRs are the part of the
`immunoglobulin which bind antigen and therefore cannot be altered. Anti-idiotypic
`
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`Humanized Monoclonal Antibod1es
`
`Presta. �
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`antibodies could present a problem in that they would block the function of the humanized
`antibody after a repeated application, thereby requiring an increased dose and/or
`significantly reducing the efficacy of the humanized antibody.
`If this is the only problem
`caused by anti-idiotypic antibodies, then one possible solution would be to have several
`bioactive humanized antibodies which bind the same antigen but have different CDRs. After
`a course of therapy with the first, the second (or even third) could then be used if a chronic
`therapy was required. Anti-allotypic antibodies, directed against idiosyncratic sequences
`of an individual, will be more problematic. Chronic therapy may require matching of a
`recipient's allotype prior to therapy (39).
`If the allotype of the recipient differs from that of
`the available humanized antibody then treatment might have to be abandoned or,
`alternatively, the constant portion of the humanized antibody would have to be redesigned
`for the patient's allotype. While such a 'tailored-to-fit' antibody is technically feasible, it
`would undoubtedly significantly increase the cost of treatment. Since no studies have been
`published on the biological consequences of either anti-idiotypic or anti-allotypic antibodies
`in humans, these questions await further clinical trial.
`
`Conclusion - Although several successful humanizations of antibodies have been
`published, clinical data at this point in time are scarce due to the relatively recent
`development of the technique. What is available supports the contention that antibodies
`hold great promise as therapeutics (3,4). Most likely, however, humanization as a
`technique will be replaced by novel methods of generating human antibodies. Two such
`technologies are the production of human antibody repertoires in transgenic mice (40) and
`from phage display libraries (41 ,42). Though it is destined for replacement, humanization of
`antibodies has stimulated research on the structure-function of antibodies and provided an
`important intermediary step in utilization of monoclonal antibodies as therapeutics.
`
`References
`1. G. Kohler and C. Milstein, Nature,� 52 (1975).
`2. S.L Morrison, M.J. Johnson, LA. Herzenberg and V.T. Oi, Proc.Natl.Acad.Sci.USA, fil, 6851 {1984).
`3. G. Winter and W.J. Harris, Trends Pharrnacol.Sci.,
`14, 139 (1993).
`
`
`4. LK. Jolliffe, lnU.Rev.lmmunol., .lQ, 241 (1993).
`5. LG. Presta, Curr.Opinion StruclBiol., 2. 593 (1992).
`6. E.A. Kabat, T.T. Wu, H.M. Peny, K.S. Gottesman and C. Froeller, 'Sequences of Proteins of
`Immunological Interest', Vol. 1, 5th Ed., NIH Publ. No. 91-3242 (1991).
`7. M.E. Vemoeyen, J.A. Saunders, M.A. Price, J.D. Marugg, S. Bnggs, !:.L. Broderick, S.J. Eida, AT.A.
`Mooren and A.A Badley, Immunology, :za. 364 (1993).
`8. M.J. Sims, D.G. HassaJ, S. Brett, W. Bowan, M.J. Lockyer, A Angel, A.P. Lewis, G. Hale, H. Waldmann
`and S.J. Crowe, J.lmmunol., ill, 2296 (1993).
`9. C. Chothia and A.M. Lask, J.Mol.Biol., �. 901 (1987).
`10. C. Queen, W.P. Schneider, H.E. Selick, P.W. Payne, N.F. Landolfi,
`J.F. Duncan, N.M. Avdalovic, M.
`Levitt, R.P. Junghans and T.A. Waldmann, Proc.Natl.Acad.Sci.USA, .§2, 10029 (1989).
`11. P. Carter, L. Presta, C.M. Gorman, J.B.B. Ridgway, D. Hanner, W.L.T. Wong, A.M. Bowland, C. Kotts,
`M.E. Carver and H.M. Shepard, Proc.Natl.Acad.Sci.USA. �. 4285 (1992).
`12. LG. Presta, S.J. Lahr, A.L Shields, J.P. Porter, C.M. Gorman, B.M. Fancily and P.M. Jardieu,
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