Engineering antibodies for imaging and therapy

449

Engineering antibodies for imaging and therapy Paul Carter* and A Margaret Merchant

Several advances made during the past year will probably

facilitate the development of therapeutic antibodies. Most

notably, significant progress has been made in the rapid

isolation of high affinity human antibodies from phage

display libraries and by immunization of transgenic mice.

The therapeutic potential of bispecific antibody fragments

and Fc-containing proteins has been greatly enhanced by

improved production methods. The utility of radiolabeled

antibody fragments has been improved by the development

of site-specific labeling methods and by the advent of the

‘minibody: an engineered fragment that has proved to be

highly successful for tumor imaging in mice.

Addresses Department of Molecular Oncology, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94060, USA *e-mail: [email protected]

Current Opinion in Biotechnology 1997, 9449-454

http://biomednet.com/elecref/0956166900600449

0 Current Biology Ltd ISSN 0956-l 669

Abbreviations CDR complementarity determining region

CH3 heavy chain third constant

CL light chain constant SC single chain

VL light chain variable

&I heavy chain variable

VA h light chain variable

VK K light chain variable

Introduction In this review we focus on very recent work in engineering antibodies for imaging and therapy. In particular we highlight direct routes to human antibodies, immunotoxins and immunoconjugates, immunoliposomes, bispecific antibodies, minibodies and site-specific radiolabeling of antibody fragments. We have attempted to minimize the overlap with many excellent reviews on related topics: antibody engineering [1,2], humanization [3], human antibodies from phage libraries [4](C Rader, CF Barbas III, this issue pp 503-508) and transgenic mice [5], immunotoxins [6], bispecific antibodies [7,8], immunoadhesins [9], antibody-based therapy [lo], disulfide-stabilized Fv fragments [ll], targeted delivery of prodrugs to tumors [ 12,131, expression of antibody fragments in Escherichia co/i [14] and intracellular antibodies [ 151.

Humanized and human antibodies The clinical utility of rodent monoclonal antibodies is limited by their immunogenicity and often inefficient secondary immune functions. Over the past decade ‘humanization’ has been the strategy most widely adopted to enhance the clinical utility of antibodies. Humanized

antibodies are commonly created by transplanting the antigen binding segments, known as complementarity determining regions (CDRs), from rodent antibodies into human antibodies. In most cases transplanting the CDR residues is not sufficient to obtain efficient antigen binding. In addition it is also necessary to recruit one or more framework-region residues from the rodent antibody. More than 100 antibodies have been humanized world wide, including 20 or so that have been clinically evaluated (reviewed in [3]) with some tangible successes. For example, a humanized antibody directed against the 185 kDa product of the HERZitaeu proto-oncogene has shown some efficacy in a Phase II trial for metastatic breast cancer, with minimal side effects and no detectable immunogenicity [ 16.1.

Although very often successful, humanization is rather laborious and is being superseded by the rapid and direct isolation of human antibodies from phage display libraries and transgenic mice. The development of each of these human antibody technologies was a technical tour de force that built upon extensive knowledge of the human immunoglobulin heavy and light chain loci.

In the phage display strategy, human antibody VH and VL domains are PCR-cloned and displayed on the surface of filamentous phage as single-chain (SC) Fv or Fab fragments (Figure 1). Antigen-specific phage are then selectively recovered by in vitro panning against the target antigen of interest. The advent of huge (>lO*o clones) human antibody phage libraries has allowed the isolation of high affinity (nanomolar [17] or even subnanomolar [ 18”] range) antibodies to a plethora of different antigens within a few weeks. Alternative strategies have been used to obtain a very diverse repertoire of antigen binding sites. Griffiths et a/. [17] developed the first very large antibody phage library using germline VH, V, and Vk gene segments and synthetic DNA to randomize the third CDR of the light and heavy chains. Their work represented a watershed in the development of antibody phage as it was the first broadly useful method for obtaining high (nanomolar) affinity human antibodies to a wide range of antigens. More recently, Vaughan ef a/. [ 18**] PCR-cloned the naturally rearranged VH, V, and VI genes from 43 different donors to create a human scFv library.

High affinity human antibodies have also been obtained from transgenic mice containing human antibody genes and disrupted endogenous immunoglobulin loci. Immu- nization leads to the production of human antibodies, which can then be recovered using standard hybridoma technology [19*,20”]. The megabase size range of the human immunoglobulin loci together with the complexity of the segmented gene structure represents a formidable

450 Protein engineering

Figure 1

scFv Bispecific d&body

n Ag VL VI-I vH

VL VL

CL

Fab

1 1997 Current Optn~on I” Bmchnology

Minibody

Antibody fragments highlighting the antigen (Ag) binding sites.

Heavy chains are shown in black and light chains in white. A linker

connecting the carboxyl terminus of VH with the amino terminus of

VL is shown for scFv and diabody fragments. The alternative linker

connectivity, carboxyl terminus of VL to the amino terminus of VH, has

also been used successfully but is not shown here for the sake of

simplicity.

obstacle to the development of transgenic mice that utilize the full human immunoglobulin gene repertoire. Transgenic mice have been created that contain a nearly complete set of diversity and joining segments, plus a small subset of human VH and V, genes. These mice, although containing only a limited selection of human immunoglobulin genes, are still capable of yielding high affinity antigen-specific antibodies [19*]. Mice containing a more complete set of human V, (but not Vk) and VH genes closely recapitulate antibody production seen in humans, including gene rearrangement, diverse gene usage, and somatic mutation. These mice have yielded high affinity, fully human IgGz antibodies to multiple antigens, including human proteins [ZO”].

Human antibodies, like the humanized antibodies, will doubtless be optimized for therapeutic applications, probably by phage display. For example, it may be desirable to mature the affinity of antibody fragments from the nanomolar to the picomolar range. This is possible by randomly mutagenizing individual CDR segments and combining enhanced affinity mutants as demonstrated for anti-gpl20 [Zl] and anti-p18SHER2 antibodies [ZZ**](reviewed by C Rader, CF Barbas III, this issue pp 503-508). It may be possible to select for antibodies that bind both human and corresponding rodent antigens. This would permit preclinical and clinical studies to be

conducted with the same antibody (or closely related antibodies), thereby streamlining the drug development process. In addition, the antigen-binding variable domains of human antibodies will probably be optimized for particular therapeutic indications by recasting into either traditional (IgG, F(ab’)z and Fab) or engineered (such as minibody, diabody, miniantibody and linear F(ab’)z) formats. Such domain shuffling permits tailoring of pharmacokinetic and pharmacodynamic properties of the antibody, as well as recruitment of desired secondary immune functions.

lmmunotoxins and immunoconjugates Towards clinical utility Immunotoxins are chimeric molecules comprising a cell- binding moiety, such as an antibody or ligand, linked to a toxin or its subunit (reviewed in [6]). Over the past two decades clinical evaluation of immunotoxins has been beset by problems of immunogenicity, which often precludes multiple dosing, and by toxicity, which is sometimes life threatening. This syndrome is characterized by extravasation of serum proteins and by retention of water, particularly in peritoneal tissues and sometimes in the lungs. In spite of these problems, objective antitumor responses by an immunotoxin, anti-Lewisy-PJez&omonas exotoxin, were recently described for the first time in epithelial tumors [23]. This immunotoxin binds to the carbohydrate antigen, Lewisv, found on many human tumors. Following internalization Pseudomonas exotoxin then kills the cells by ADP-ribosylation of elongation factor 2, which inactivates protein synthesis.

The immunogenicity of the murine antibody component of immunotoxins can probably be minimized by replacing it with a humanized or human antibody. Human enzymes, such as nucleases, are being explored as potentially less immunogenic toxins. For example, a human ribonuclease, angiogenin, has been fused to an anti-transferrin receptor F(ab’)z [24] or scFv [25]. These immunotoxins are cytotoxic in o&-o to antigen-positive, but not antigen-negative, cells [24,25].

Ferociously toxic small molecules have been conjugated to antibodies as an alternative to protein toxins. These include calicheamicins [26] and maytansinoids [27], which are lOO-lOOO-fold more toxic than conventional chemo- therapeutic drugs. Calicheamicins are derived from the soil microorganism Micromonospora echnospot-a calichenisis,

and can give rise to sequence-specific DNA cleavage. Maytanisoids are ansa macrolides from aerobic acti- nomycetes of the genus Nor-cad that block tubulin polymerization. Maytansinoid-antibody conjugates have cured mice bearing subcutaneous COLO 205 human colon tumor xenografts, albeit at doses close to the maximum tolerated dose [28’*]. Complete regression (but not cure) was even obtained by treating mice with large (260-500mm3) COLO 205 tumors that show homogeneous antigen expression. Similar success was

Engineering antibodies for imaging and therapy Carter and Merchant 451

obtained in treating subcutaneous LoVo and HT-29 tumors that express antigen at heterogeneous levels. It remains to be seen if serum shedding of maytansinoid [27], potential toxicity or immunogenicity will limit the utility of antibody-maytansinoid conjugates. Certainly these conjugates warrant clinical evaluation.

Vasculature targeting

The potential benefit of attacking tumors by targeting the vasculature that supplies them has long been appreciated. Firstly, the vascular endothelial cells, unlike the tumor itself, are directly accessible to administered therapeutic agents. Secondly, judicious vasculature damage is expected to translate into widespread tumor cell death because each capillary nourishes a large number of tumor cells.

The concept of tumor vasculature targeting is supported by the observation of tumor shrinkage upon infarcting the corresponding vasculature [29]. This was accomplished by targeting a model endothelial antigen, class II major histocompatibility complex, with an antibody-ricin immunotoxin. Following internalization of the immunotoxin, ricin A chain kills cells by enzymatically removing a critical adenine residue from the 60s ribosomal subunit, thus blocking protein synthesis. Subsequent tumor regrowth probably reflects survival of a small proportion of the cancer cells at the tumor-host interface. Significantly greater efficacy was achieved by combining the anti-endothelial immunotoxin with a second immunotoxin directed against the tumor cells themselves [29]. More recently, tissue factor was successfully targeted to tumor vasculature using a bispecific antibody. This triggered local thrombosis that resulted in significant anti-tumor efficacy [30*]. A prerequisite for clinical evaluation of vasculature targeting is the identification of suitable tumor vasculature (or neovasculature) restricted antigens (see [30*] for a list of candidate molecules).

lmmunoliposomes Liposomes are lipid bilayers that have self-assembled into small colloidal particles, thereby encapsulating some of the surrounding medium. The therapeutic potential of liposomes for the delivery of drugs, toxins and DNA has long been appreciated; however, translation of liposomes into clinical practice has been hampered by their poor stability and rapid clearance from circulation. Technological innovations have revitalized interest in liposomes as therapeutic agents. In particular, improved methods have been developed for loading and retaining drugs, and the advent of sterically stabilized (StealthTRf) liposomes has increased the serum permanence time [31].

Liposomes can be targeted to specific tissues by the attachment of an antibody or other ligand. The construction of sterically stabilized immunoliposomes [32,33’] has been greatly facilitated by high level (gram per liter) expression of humanized Fab’ fragments in Esderichiu co/i [34]. Fab fragments are readily attached through their free thiol

group to liposomes containing a maleimide-derivatized lipid [32] or polyethylene glycol [33*]. In v&o coupling is preferable to biosynthetic attachment of lipid in E. co/i

[35,36] because of the greater versatility of molecules that can be attached. The blood-brain barrier represents a formidable challenge to tissue-specific drug delivery by liposomes. Coupling sterically-stabilized liposomes to an anti-transferrin receptor antibody allows passage across the blood-brain barrier [37]. Extending this approach will likely benefit from, and may require, targeting of antigens that are more restricted in their tissue distribution than is the transferrin receptor.

Diabodies Bispecific antibodies have shown some promise in several small scale clinical trials in tumor imaging and therapy (reviewed in [8]). The emergence of efficient recombinant routes to bispecific antibody fragments (reviewed in [7,8]), will facilitate more extensive clinical evaluation of such molecules.

Particularly promising is the diabody [38], an antibody fragment which has two antigen binding sites and can be a bivalent or bispecific fragment. Bispecific diabodies are heterodimers of two ‘crossover’ scFv fragments in which the VL and VH domains of the two antibodies are present on different polypeptide chains. High level diabody secretion from E. cofi is possible, as demonstrated by the recovery of a humanized bispecific diabody in yields approaching one gram per liter [39*]. In addition, the fraction of diabody preparation that is functional heterodimer (as opposed to inactive homodimers) has been improved by domain interface engineering [40]. Bispecific diabodies have been used successfully to direct cytotoxic T cells to kill target breast tumor cells [39*] and B-cell lymphoma cells [41*] in vitro. Such diabodies are strong candidates for clinical evaluation. Phage display of diabodies allows for the selection of antibodies with dual antigen-binding specificities [42] and perhaps even allosteric binding properties.

Humanized or human diabodies pose little risk of immunogenicity since the only non-natural sequences are the short linkers between variable domains; even these can be ‘humanized’ by recruiting residues from CL [41*]. Nev- ertheless, non-natural linear or conformational epitopes are a potential source of immunogenicity. Clinical trials will determine whether immunogenicity is a significant limitation for diabodies as therapeutic agents.

Fc-containing bispecific antibodies Bispecific antibodies containing an Fc region are likely to be preferred over antibody fragments, for some clinical applications, in order to obtain long serum half-life and/or to recruit effector functions. Since the advent of hybrid hybridoma technology little progress has been made in developing more efficient routes to Fc-containing bispecific antibodies [43]. In hybrid hybridomas, heavy


chains typically form homodimers as well as the desired heterodimers; additionally, light chains frequently mispair with non-cognate heavy chains. Hence, coexpression of two antibodies may produce up to ten heavy and light chain pairings. The unwanted chain pairings often compromise the yield of the bispecific antibody and impose significant purification challenges [44].

In an effort to control heavy chain pairing, antibody heavy chains have been engineered for heterodimerization using stericahy complementary mutations at the CH3 domain interface [45”]. A ‘knob’ mutation was first created by the replacement of a small residue by a large one: Thr366+Trp. The ‘knob’ was designed to fit into a ‘hole’ on the partner CH3 domain. The hole was created by replacing a larger residue with a smaller one: Tyr407-+Thr. ‘Knobs-into-holes’ engineering permitted the production of an antibody-immunoadhesin hybrid with >90% mutant yield, compared to <60% for chains containing wild- type CH3 domains [45**]. Knobs-into-holes mutations are directly applicable to the construction of bispecific immunoadhesins, thereby expanding immunoadhesins as a class of novel therapeutics (reviewed in [9]). In addition, the mutations identified are anticipated to facilitate the production of Fc-containing bispecific antibodies by reducing the complexity of the mixture of products from a possible ten major species [44] down to four or fewer.

Minibodies The ‘minibody’ comprises an scFv joined to a CH3 domain via a linker [46**]. The minibody is readily expressed in SpZ/O cells and a readily prepared version forms a disulfide-linked dimer by virtue of the CH3 domain and a cysteine-containing linker. Minibodies have proved to be excellent imaging agents in tumor-bearing mice [46**]. An anti-carcinoembryonic antigen minibody localized very efficiently to a tumor xenograft (33% injected dose per gram of tumor, six hours after injection). Tumor uptake is most likely favored by the small size of the minibody (80kDa) compared with that of an IgG (150kDa), enabling more efficient extravasation. Retention in the tumor is probably facilitated by the high antigen-binding affinity (Kd = 0.5 nM) and slow dissociation rate from the antigen. The remaining circulating minibody was cleared rapidly to give a tumor: blood ratio of 65 at 48 hours after administration. These favorable pharmacokinetic properties are likely to be significantly influenced by the minibody size. Minibodies are well suited to the construction of bispecific antibodies by using CH~ domains with appropriate interface mutants ([45”]).

Site-specific radiolabeling of antibody fragments Antibodies are commonly radiolabeled by coupling ra- dionuclides to solvent accessible lysine or tyrosine residues. This inevitably leads to heterogeneous populations of molecules and risks impairing antigen binding, as tyrosines are very common in CDRs. Ideally one would like to

define both the stoichiometry and site of attachment of the radionuclide. An elegant way of accomplishing this is by appending the sequence Gly&ys, a chelation site for 99mTc, to the carboxyl terminus of a protein of interest [47]. Efficient labeling of an scFv fragment and imaging of a tumor xenograft in nude mice was demonstrated. This fusion chelate technology is amenable to imaging with WmTc and perhaps radioimmunotherapy with 186Re or 188Re.

A second strategy for site-specific radiolabeling of proteins is by radiophosphorylation of an engineered kinase recognition site. The scFv fragments fused to a peptide tag that included a substrate site for casein kinase II were purified following secretion from E. cob [48*]. These scFv were then efficiently and site-specifically labeled with casein kinase II and [@‘PI-ATP in vitro. Such phosphorylated peptides are useful reagents, for example radioimmunoassay, and may have value in the clinic.

Toward drugs derived from antibodies An emerging strategy in biotechnology is the development of small organic drugs that mimic protein-protein interactions. In most cases whole antibodies are poorly suited starting points for such endeavors as they have six (or more) loops that can potentially be involved in binding antigen. In contrast, VH domains are more suitable starting points for peptidomimetic drug development since they have only three CDR segments [49]. VH domains are sometimes capable of binding antigen ]50] but are unfortunately prone to aggregation because of the exposed hydrophobic residues at the VI,/VH interface. This problem has been addressed by taking advantage of the camel, which has an extensive repertoire of functional antibodies that lack light chains [51]. The recruitment of camel residues into a human VH, ‘camelization’, has largely overcome the limitations of human VH. Antigen-specific VH were recovered by panning libraries of camelized VH [52,53*]. An ideal starting point for the development of peptidomimetic drugs would be a binding interaction dominated by a single loop. This sometimes occurs with camel antibodies, as shown by the structure of a camel antibody complexed with the antigen lysozyme [54”]. In this case the interaction between VH and antigen was found to be dominated by a CDR (H3).

Conclusions and future directions Humanization has greatly enhanced the utility of antibodies for therapy, but is nevertheless already being superseded by the more rapid approach of directly obtaining high affinity human antibodies. Phage display libraries offer several advantages over transgenic mice as a route to human antibodies. Antibody phage libraries are the fastest route to obtaining human antibodies: two weeks to obtain antigen-positive clones and two months to obtain a panel of well-characterized antibody fragments. In addition, the phage format is ideal for optimization of the isolated variable domains. The phage, but not

Engineering antibodies for imaging and therapy Carter and Merchant 453

mice, allow direct selection for antibodies that bind closely related antigens, or, alternatively, that do not bind closely related antigens. The phage libraries overcome tolerance encountered when animals are immunized with self (or closely related) antigens. Finally, antibody phage technology is more widely available than transgenic mice and is evolving much more rapidly. Together, these merits of antibody phage libraries lead us to believe that this technology will dominate the development of the next generation of therapeutic antibodies.

Acknowledgement We thank Alice Bodtkc-Roberts for invaluable help with literature database searching.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

. of special interest

. . of outstanding interest

1. Nilsson B: Antibody engineering. Gun Opin Struct Biol 1995, 5450-456.

2.

3.

4.

Kelley RF: Engineering therapeutic antibodies. In Protein engineering: principles and practice. Edited by Cleland JL, Craik CS. New York: Wiley-Liss; 1996:399-434.

Adair JR, Bright SM: Progress with humanized antibodies-an update. Exp Opin /west Drugs 1995, 4663-070.

Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR: Making antibodies by phage display technology. Ann Rev lmmunol 1994, 12~433-455.

5. Neuberger M, Brtiggemann M: Mice perform a human repertoire. Nature 1997, 386:25-26.

6.

7.

6.

Ghetie MA, Vitetta ES: Recent developments in immunotoxin therapy. Curr Opin lmmunol 1994, 6:707-714.

Holliger P, Winter G: Engineering bispecific antibodies. Curr Opin Biotechnoll993, 4:446-449.

Carter P, Ridgway J, Zhu Z: Toward the production of bispeciftc antibody fragments for clinical applications. J Hematotherapy 1995, 41463-470.

9.

10.

11.

12.

Ashkenazi A, Chamow S: lmmunoadhesins as research tools and therapeutic agents. Curr Opin lmmunol 1997, 9:195-200.

Von Mehren M, Weiner LM: Monoclonal antibody-based therapy. Curr Opin Oncoll996, 6:493-496.

Reiter Y, Brinkmann U, Lee B, Pastan I: Engineering antibody Fv fragments for cancer detection and therapy: disulfide- stabilized Fv fragments. Nat Biotech 1996, 14:1239-l 245.

Melton RG, Sherwood RF: Antibody-enzyme conjugates for cancer therapy. J Nat/ Cancer lnst 1996, 66:153-l 65.

13.

14.

Bagshawe KD: Antibody-directed enzyme prodrug therapy: a review. Drug Dev Res 1995, 34:220-230.

Plijckthun A, Krebber A, Krebber C, Horn U, Kniipfer U, Wenderoth R, Nieba L, Proba K, Riesenberg D: Producing antibodies in Escherkhie co/i: from PCR to fermentation. In Antibody Engineering: a Practical Approach. Edited by Hoogenboom H, McCafferty J, Chiswell D. Oxford: IRL Press; 1996:203-252.

15. Chen SY, Marasco WA: Novel genetic immunotoxins and intracellular antibodies for cancer therapy. Semin Oncoll996, 23:146-l 53.

16. .

Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, Sklarin NT, Seidman AD, Hudis CA, Moore J et a/: Phase II study of weakly intravenous recombinant humanized anti-pl66Raaa monoclonal antibody in patients with HERl/neu- overexpressing metastatic breast c&er. J C/in Oncoll996, 14:737-744.

This ia the first report of objective responses in solid tumors treated with a humanized antibody.

1 7 Griffiths AD, Williams SC, Hartley 0, Tomlinson IM, Waterhouse P, Crosby WL, Kontennann R, Jones PT, Low NM, Allison TJ et a/: Isolation of high affinity human antibodies directly from large synthetic repertoires. EMS0 J 1994, 13:3245-3260.

16. Vaughan TJ, Williams Al, Pritchard K, Osbourn JK, Pope AR, . . Eamshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS:

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 1996. 141309-314.

A very large ‘(1 .I x 1010) natural human scFv phage library is constructed and used for the rapid identification of nanomolar and subnanomolar affinity antibodies to haptens and protein antigens.

19. Fishwild DM, O’Donnell SL, Bengoechea T, Hudson DV, Harding F, . Bernhard SL, Jones D, Kay RM, Higgins KM, Schramm SR,

Lonberg N: High avidity human IgGx monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 1996, 14:645-651.

Human antibodies with high apparent affinities are obtained following immunization of transgenic mice containing a small fraction of the human immunoglobulin gene repertoire.

20. . .

Mendez MJ, Green LL, Corvalan JRF, Jia XC, Maynard-Currie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL et al: Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 1997, 16:146-l 56.

Transgenic mice containing a significant fraction of the human immunoglobulin gene loci are immunized and shown to produce high affinity human antibodies to several different human antigens.

21. Yang W-P, Green K. Pinz-Sweeney S, Briones, AT, Burton DR, Barbas CF Ill: CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-l antibody into the picomolar range. J MO/ Biol 1995, 264:392-403.

22. . .

Schier R, McCall A, Adam GP, Marshall KW, Merritt H, Yim M, Crawford RS, Weiner LM, Marks C, Marks JD: Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J MO/ Bioll996, 263:55 l-567.

A phage display approach is used to enhance the affinity (K& of a human antibody from 16 nM to 13 pM. The affinity enhancement strategies described together with those in (211 will probably be broadly useful in engineering antibodies for human therapy.

23.

24.

25.

26.

27.

26. . .

Pai LH, Wittes R, Setser A, Willingham MC, Pastan I: Treatment of advanced solid tumors with immunotoxin LMB-1 : an antibody linked to Pseudomonas exotoxin. Nat Med 1996, 2:350-353.

Rybak SM, Hoogenboom HR, Meade HM, Raus JC, Schwartz D, Youle RJ: Humanization of immunotoxins. Proc Nat/ Acad Sci USA 1992,69:3165-3169.

Newton DL, Xue Y, Olson KA, Fett JW, Rybak SM: Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains. Biochemistry 1996, 35:545-553.

Hinman LM, Hamann PR, Wallace R, Menendez AT, Dun FE, Upeslacis J: Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 1993, 53:3336-3342.

Chari RVJ, Martell BA, Gross JL, Cook SB, Shah SA, Blattler WA, McKenzie SJ, Goldmacher VS: lmmunoccmjugates containing novel maytansinoids: promising anticancer drugs. Cancer Res 1992, 52:127-l 31.

Liu C, Tadayoni BM, Bourret LA, Mattocks KM, Derr SM, Widdison WC. Kedersha NL. Ariniello PD. Goldmacher VS. Lambert JM et’al.: Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Nat/ Acad Sci USA 1996,93:6616-8623.

An antibody-maytansinoid conjugate is found to cure mice bearing small tumor xenografts and to give complete regressions for mice with large tumors or for mice in which only a small fraction of the tumor cells is antigen positive.

29. Burrows FJ, Thorpe PE: Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature. Proc Nat/ Acad Sci USA 1993, 90:6996-9000.

30. Huang X, Molema G, King S, Watkins L, Edgington TS, . Thorne PE: Tumor infarction in mice by antibody-directed

targ&lng of tissue factor to tumor va&ulature.cience 1997 275:547-550.


A bispecific antibody is used to deliver tissue factor to tumor vasculature, triggering local thrombosis and complete tumor regression in some mice.

31. Lasic DD, Papahadjopoulos D: Liposomes revisited. Science 1995, 267:1275-l 276.

32. Park JW, Hong K, Carter P, Asgari H, Guo LY, Keller GA, Wirth C, Shalaby R, Kotts C, Wood WI et al.: Development of anti- pl65HER2 immunoliposomes for cancer therapy. Proc Nat/ Acad Sci USA 1995, 92:1327-l 331.

33. Kirpotin D, Park JW, Hong K, Zalipsky S, Li WL, Carter P, . Benz CC, Papahadjopoulos D: Sterically stabilized anti-HER2

immunoliposomes: design and targeting to human breast cancer cells in vifro. Biochemistry 1997, 36:66-75.

Cell binding and internalization of anti-p1 65 HER2 immunoliposomes is shown to increase concomitantly with the surface density of the Fab’ fragment used for targeting, reaching plateaus at approximately 40 Fab’/liposome for binding and 1 O-l 5 Fab’/liposome for internalization.

34.

35.

36.

37.

36.

39. .

Carter P, Kelley RF, Rodrigues ML, Snedecor B, Covarrubias M, Vellioan MD, Wong WLT, Rowland AM, Kotts CE, Carver ME et al. High level Escherichia co/i expression and production of a bivalent humanized antibody fragment Bio-Technology 1992, 10:163-167.

Laukkanen ML, Teeri TT, Keinanen K: Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein- single-chain antibody fusion protein. Protein fng 1993 6449-454.

Laukkanen ML, Alfthan K, Keinanen K: Functional immunoliposomes harboring a biosynthetically lipid-tagged single-chain antibody. Biochemistry 1994, 33:11664-l 1670.

Huwyler J, Wu D, Pardridge WM: Brain drug delivery of small molecules using immunoliposomes. Proc War/ Acad Sci USA 1996, 93:14164-l 4169.

Holliger P, Prosper0 T, Winter G: ‘Diabodies’: small bivalent and bispecific antibody fragments. froc Nat/ Acad Sci USA 1993, 90:6444-6448.

Zhu Z, Zapata G, Shalaby MR, Snedecor B, Chen H, Carter P: High level secretion of a humanized bispecific diabody from Escherichia co/i. &o-Technology 1996, 14:192-l 96. . _

A humanized blspectftc dlabody IS recovered from t. co/! In yields approaching a gram per liter. This is the first demonstration of bispecific antibody fragment production that is practical for large-scale human therapy. See also [41’].

40. Zhu Z, Presta LG, Zapata G, Carter P: Remodeling domain interfaces to enhance heterodimer formation. Protein Sci 1997, 6:761-766.

41. Holliger P, Brissinck J, Williams RL, Thielemans K, Winter G: . Specific killing of lymphoma cells by cytotoxic T-cells

mediated by a bispecific diabody. Protein Eng 1996, 9:299-305.

52. Davies J, Riechmann L: Antibody V, domains as small recognition units. Bio-Technology 1995, 13:475-479.

53. Davies J, Riechmann L: Single antibody domains as small . recognition units: design and in vitro antigen selection of

camelized, human VR domains with improved protein stability. Protein Eng 1996, 9:531-537.

This work, together with [39’1, demonstrates that diabodies can be used to retarget cytotoxic T cells to kill tumor cells in vitro.

42. McGuinness BT, Walter G, FitzGerald K, Schuler P, Mahoney W, Duncan AR, Hoogenboom H: Phage diabody repertoires for selection of large numbers of bispecific antibody fragments. Nat Biotechnoll996, 14:1149-l 154.

Antigen-specific VH fragments were isolated from a phage library in which the unfavorable stability and aggregation properties of human VH were overcome by recruiting residues from natural VH antibodies from camels.

54. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F, . . Hamers R, Muyldermans S, Wyns L: Crystal structure of a

camel single-domain VH antibody fragment in complex with lysoxyme. Nat Struct 6iol 1996, 3:603-611.

43. Milstein C, Cuello AC: Hybrid hybridomas and their use in The structure of a camel VH-lysozyme complex revealed that the contacts immunohistochemistry. Nature 1963, 305:537-540. with antigen were dominated by the heavy chain CDR3.

44. Suresh MR, Cuello AC, Milstein C: Bispecific monoclonal antibodies from hybrid hydridomas. Methods Enzymoll966, 121:21 O-226.

45. Ridgway JBB, Presta LG, Carter P: ‘Knobs-into-holes’ . . engineering of antibody CR3 domains for heavy chain

heterodimerization. Protein Eng 1996, 9:617-621. A rational design strategy is used to direct the assembly of antibody heavy chains. This technology is anticipated to be broadly useful in the production of Fc-containing bifunctional therapeutics.

46. Hu S, Shively L, Raubitschek A, Sherman M, Williams LE, . . Wong JY, Shively JE, Wu AM: Minibody: a novel engineered anti-

carcinoembryonic antigen antibody fragment (single-chain Fv- CR3) which exhibits rapid, high-level targeting of xenografts. Cancer /?es 1996, 56:3055-3061.

A designer bivalent antibody fragment, the minibody, is shown to be a highly effective imaging agent in tumor-bearing mice. Thus the minibody is a very promising candidate for clinical radioimmunoimaging trials.

47. George Al, Jamar F, Tai MS, Heelan BT, Adams GP, McCartney JE, Houston LL, Weiner LM, Oppermann H, Peters AM, Huston JS: Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide. Proc Nat/ Acad Sci 1995, 926356-6362.

48. Neri D, Petrul H, Winter G, Light Y, Marais R, Britton KE, . Creighton AM: Radioactive labeling of recombinant antibody

fragments by phosphorylation using human casein kinase II and [y-s2PI-ATR Nat Biotechnoll996, 14:465-490.

An SCFV fragment is appended with a recognition site for casein kinase, permitting site-specific radiophosphorylation in vitro. Site-specific labeling is superior to traditional ‘random’ labeling in that the resultant molecular population is much more homogeneous with well-defined radionuclide stoichiometry and is likely close to full immunoreactivity.

49. Sheriff S, Constantine KL: Redefining the minimal antigen- binding fragment Nat Struct Biol 1996, 3:733-736.

50. Ward ES, Gussow D, Griffiths AD, Jones PT, Winter G: Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia co/i. Nature 1969, 3411544-546.

51. Hamers-Casterman C. Atarhouch T. Muvldermans S. Robinson G. Hamers C, Songa EB; Bendahman’N, Garners R: Naturally occurring antibodies devoid of light chains. Nature 1993, 363:446-446.

Engineering antibodies for imaging and therapy

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