Bispecific antibody conjugates in therapeutics

Advanced Drug Delivery Reviews 55 (2003) 171–197 www.elsevier.com / locate / addr

Bispecific antibody conjugates in therapeutics Ying Cao a , *, Laura Lam b a

b

Abbott Laboratories, Dept. 04 A6, Bldg. AP8 B, 100 Abbott Park Rd., Abbott Park, IL 60064, USA Department of Pharmaceutical Services, University of Chicago Hospitals, 5841 South Maryland Ave., Chicago, IL 60637, USA Received 10 April 2002; accepted 25 June 2002

Abstract Bispecific monoclonal antibodies have drawn considerable attention from the research community due to their unique structure against two different antigens. The two-arm structure of bsMAb allows researchers to place a therapeutic agent on one arm while allowing the other to specifically target the disease site. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc. Furthermore, bsMAb may redirect the cytotoxicity of immune effector cells towards the diseased cells or induce a systemic immune response against the target. BsMAb holds great promise for numerous therapeutic needs in the light of: (1) recent breakthroughs in recombinant DNA technology, (2) the increased number of identified disease targets as the result of the completion of human genomic map project, and (3) a better understanding of the mechanism of human immune system. This review focuses on therapeutic applications and production of bsMAb while providing the up-to-date clinical trial information.  2002 Elsevier Science B.V. All rights reserved. Keywords: bsMAb; Bispecific monoclonal antibody; Radioimmunotherapy; Immunotherapy; ADEPT; Gene therapy; Vaccine

Contents 1. Introduction ............................................................................................................................................................................ 2. Applications of BsMAb ........................................................................................................................................................... 2.1. Drug / Toxin / Cytokine targeting ........................................................................................................................................ 2.2. Antibody directed enzyme prodrug therapy ........................................................................................................................ 2.3. Radioimmunotherapy ....................................................................................................................................................... 2.4. Immunotherapy................................................................................................................................................................ 2.5. Anti-vascular therapy ....................................................................................................................................................... 2.6. Gene therapy ................................................................................................................................................................... 2.7. Therapeutic vaccine ......................................................................................................................................................... 3. Production of BsMAb.............................................................................................................................................................. 3.1. Chemical method ............................................................................................................................................................. 3.2. Biological method ............................................................................................................................................................ 3.3. Genetic engineering ......................................................................................................................................................... 4. BsMAb Humanization ............................................................................................................................................................. 5. Clinical trial information.......................................................................................................................................................... *Corresponding author. Tel.: 1 1-847-935-1113 (office). E-mail address: [email protected] (Y. Cao). 0169-409X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 02 )00178-3

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6. Conclusion.............................................................................................................................................................................. Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................

1. Introduction Since the beginning of hybridoma technology 27 years ago [1], considerable effort has been expended in adding monoclonal antibodies (MAbs) to the armamentarium of therapeutics. MAbs are now in the stage of rapid growth, thanks to advances in recombinant DNA technology. This technology permits design flexibility in size, configuration, valence and effector functions in the construction of humanized MAb or its fragments. There are now 11 FDAapproved MAb-based pharmaceuticals routinely used for various therapeutic strategies such as inhibition of alloimmune and autoimmune reactivity, antitumour therapy, antiplatelet therapy and antiviral therapy (Table 1). Bispecific monoclonal antibody (bsMAb), first introduced by Nisonoff and Rivers into the academia 40 years ago, is a unique type of MAb with two different binding specificities within a single molecule [2]. Today, bsMAb has been extensively studied in both diagnostic [3,4] and therapeutic areas [5–12]. In therapeutic areas (see Fig. 1), bsMAb has been used to specifically recruit a variety of different

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effector mechanisms including cell-mediated cytotoxicity by targeting CTLs, NK cells, neutrophils and monocytes / macrophages [5–12], as well as other cytotoxic mechanisms by recruiting toxins, drugs, enzymes, DNA or radionuclides [13,14]. Recently, bsMAb has been introduced in cancer vaccine development [15–18]. Some of these exciting explorations have already been expanded to redirecting cytotoxicity to tumor cells, HIV and other infectious diseases; targeting enzymes to achieve site-specific activating anti-cancer prodrugs; localizing fibrinogen activators to dissolve fibrin clots, and delivering antigen specifically to antigen-presenting cells as vaccines. Due to its great potential for new therapeutic applications, enormous research efforts have been devoted to this area. Although current knowledge of bsMAb is limited, a number of therapeutic products are in phase II / III clinical trials. With mixed results from clinical trials, this area warrants further attention and open discussions. This paper investigates the various therapeutic applications and the production method of bsMAb currently available while presenting recent progresses from the clinical frontier.

Table 1 Monoclonal antibodies on the market Generic name

Trade name

Target

Type

Indication

Company

Approval date

Muromonab Abciximab Rituzimab Daclizumab Basiliximab Palivizumab Infliximab

OKT3 ReoPro Rituxan Zenapax Simulect Synagis Remicade

CD3 platelet CD20 CD25 CD25 RSV TNFa

Murine Chimeric Chimeric CDR-grafted Chimeric CDR-grafted Chimeric

Jonhnson & Johnson Eli Lilly Genentech Roche Novartis Medimmune Centocor / J & J

06 / 86 12 / 94 12 / 97 12 / 97 05 / 98 06 / 98 08 / 98

Trastuzumab Gemtuzumab Alemtuzumab Ibritumomab tiuxetan

Herceptin Mylotarg Campath Y-90-Zevalin

HER-2 / neu CD33 CD52 CD20

CDR-grafted CDR-grafted CDR-grafted Murine

Organ rejection Antiplatelet NHL Organ rejection Organ rejection RSV Rheumatoid arthritis Crohn’s disease Breast cancer AML CLL NHL

Genentech American Home Products Millennium / ILEX IDEC

09 / 98 05 / 00 07 / 01 02 / 02

NHL, Non-Hodgkin’s lymphoma; AML, Acute myeloid leukemia; CLL, Chronic lymphocytic leukemia; RSV, Respiratory syncytial virus.

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Fig. 1. Schematic representation of different applications of BsMAbs in therapeutics.

2. Applications of BsMAb

2.1. Drug /Toxin /Cytokine targeting An expanding field in MAb-based therapeutics is the use of MAbs to direct selective cytotoxic agents. Chemical drugs, toxins and cytokines have all been conjugated to MAbs and are at various stages of development in clinical trials. The most exciting result was the first FDA approved Mylotarg for the treatment of acute myeloid leukemia (AML). Mylotarg (Gemtuzumab) is a recombinant humanized antibody linked with a potent anti-tumor antibiotic called calicheamicin. The antibody specifically binds to the CD33 antigen expressed by myeloid leukemia cells. One of the limitations of using chemical conjugates is the generation of complex aggregates of the two entities resulting in a batch-tobatch variation. BsMAb with intrinsic binding sites to any two antigens has the capability to form homogeneous and reproducible immunoconjugates. It therefore can overcome the problem of chemical conjugation needed for linking MAb and cytotoxic agents. BsMAb has been constructed to deliver various drugs, e.g. doxorubicin [19–21], epirubicin [22], methotrexate [23] or vinca alkaloids [24–27]. Ford et al. have successfully demonstrated a bsMAb

targeting of doxorubicin to colon cancer cells expressing carcinoembryonic antigen (CEA) in vitro and in vivo [19]. In the in vitro study, three human colon cancer cell lines (COLO320DM, LS174T and SKCO1) have been used with low, medium and high CEA expression, respectively. The IC 50 values for doxorubicin with COLO320DM, LS174T and SKCO1 were 1163, 324 and 28.5 ng / ml, respectively. All bsMAb at concentration of 1, 0.1 and 0.01 g / ml resulted in significant reductions in doxorubicin IC 50 values with the CEA-expressing cell lines SKCO1 and LS174T, but not with COLO320DM. In the in vivo study, bsMAb also significantly inhibited the growth of CEA-expressing LS174T cells in nude mice [19]. BsMAb has been expanded to target toxins, e.g. ricin A [28], saporin [29–32] and gelonin [33] to tumors. Clinical studies have successfully utilized a synergistic effect of two bsMAbs to deliver saporin (ribosome-inactivating protein) for the treatment of B-cell lymphoma. Both bsMAbs have one arm directed at saporin and another arm at the CD22 on target B cells. However, the two bsMAbs recognized different, non-overlapping epitopes on saporin. This strategy allowed high-avidity double attachment of saporin to the target. In a small-scale clinical trial, five patients were treated with weekly doses of 2 to 4

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mg of saporin for up to 6 weeks. All patients showed a rapid and beneficial response with minimal toxicity [31,32]. Another pair of bsMAbs (anti-gelonin / anti-CD30) has been found to have a similar beneficial synergistic effect on Hodgkin’s lymphoma cell lines [33]. Gelonin is a ribosome-inactivating protein that displays a lower toxicity compared to other ribosomeinactivating proteins. These two bsMAbs were produced using the same anti-CD30 MAb and two different anti-gelonin MAbs, directed to unrelated epitopes of the gelonin molecule. In the in vitro study, both bsMAbs enhanced gelonin toxicity against the CD30 positive L540 Hodgkin’s lymphoma cell line. In the presence of either of the bsMAbs, protein synthesis was inhibited with an IC 50 of 5 3 10 210 M or 8 3 10 211 M, respectively, compared to IC 50 5 3 10 28 M in the absence of bsMAb. Two bsMAb combinations had shown a synergistic effect with IC 50 of 6 3 10 212 M. Among CD30-positive tumor cells, the Hodgkin’s lymphoma L428 was also sensitive to gelonin delivered by bsMAb with IC 50 of 6 3 10 211 M [33]. This result further indicates the beneficial effect of combining two bsMAbs to deliver toxins. Tumor necrosis factor a (TNFa) used in cancer immunotherapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, a bsMAb (anti-CEA 3 anti-TNFa) has been constructed to target this cytokine to the tumor [34]. A two-step injection protocol was used to target TNFa to the human colorectal carcinoma T380 in nude mice. First, a variable dose of 125 I-labeled bsMAb was injected, followed by 1 mg of 131 I-labeled TNFa 24 or 48 h later. Mice pretreated with 3 mg of bsMAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFa showed a 1.5- to 2-fold increased localization of 131 I-labeled TNFa in the tumor as compared to control mice receiving TNFa only. With pretreatment of 25 mg bsMAb, mice showed a better targeting of TNFa with a 3.2-fold increased concentration of 131 I-labeled TNFa in the tumor. In a one-step injection protocol using a pre-mixed bsMAb / TNFa preparation, similar results were obtained 6 h post-injection (3.5-fold increased TNFa tumor concentration). Furthermore, a longer retention time of TNFa was observed

leading to a 8.1-fold increased concentration of TNFa in the tumor 14 h post injection [34]. The advantage of using bsMAb to deliver high molecular weight toxins or drugs is that it avoids the complex chemistry involved in directly linking the antibody to the cytotoxic agent. This provides a more uniform cross-linking between the target and the effector molecule. However, if the toxin or drug is a low molecular weight effector molecule, the advantage may be outweighed by the dose limitation of this approach. To circumvent this limitation, a strategy combining bsMAb with drug carriers such as polymeric biodegradable carriers or liposomes may be adopted.

2.2. Antibody directed enzyme prodrug therapy Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme specifically to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Since each enzyme molecule has the capacity to convert a large number of nontoxic prodrugs to cytotoxic drugs, ADEPT has the potential to increase the concentration of cytotoxic drugs at tumor sites. ADEPT has better tumor penetration than antibody-drug conjugate by generating small, toxic molecules within a tumor mass. The favorable diffusion characteristics of small molecules addresses the issue of heterogeneity of antigen expression by tumor cells because their access to cells was not limited by antigen expression, hence giving rise to the so-called bystander effect. Furthermore, ADEPT circumvents the problem associated with releasing drugs from the carrier molecules [35]. BsMAb or bifunctional MAb provides an alternative approach to antibody-enzyme conjugate in ADEPT system [36–38]. A recombinant bifunctional fusion protein, comprised of an anti-CEA single chain Fv (scFv) antibody and the amino-terminus of the enzyme carboxypeptidase G2 (CPG2), has been constructed and tested in nude mice bearing CEApositive LS174T human colon adenocarcinoma xenografts [36]. The bifunctional antibody was cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor-to-plasma ratios of 1.5:1 (6 h), 10:1 (24 h), 19:1 (48 h) and 12:1 (72 h). 125 I-bifunctional antibody was retained in the kidney,

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liver and spleen but its catalytic activity was not, resulting in excellent tumor-to-normal tissue ratios 48 h post-injection. They were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen). Favorable tumor-to-normal tissue ratios occurred at early time points when there was still 21% (24 h) and 9.5% (48 h) of the injected activity present per gram of tumor tissue. The high tumor concentration and selective tumor retention of the active enzyme delivered by bifunctional antibody demonstrate that bifunctional fusion protein promises improved clinical efficiency for ADEPT [36]. Sahin et al. developed a bsMAb HRS-3 /AP-1 with reactivity against the Hodgkin’s- and Reed–Sternberg cell-associated CD30 antigen and alkaline phosphatase, respectively. After an active incubation with alkaline phosphatase, purified bsMAb and F(ab9)2 fragments were equally effective in converting a noncytotoxic prodrug, mitomycin phosphate (MOP), into cytotoxic mitomycin alcohol. The cytotoxicity of MOP was unaffected when the cells were pretreated with either the bsMAb or the enzyme alone. The bsMAb HRS-3 /AP-1 did not bind to CD30negative HPB-ALL cells and was unable to activate MOP on these cells. However, in co-cultivation experiments with HPB-ALL and L540 cells, the activation of MOP by the bsMAb HRS-3 /AP-1 and alkaline phosphatase led to considerable cytotoxicity against the antigen-negative bystander cells. Thus, this immunotherapeutic approach might be effective in cases where not all the tumor cells express the respective tumor antigen [38]. The advantage of bsMAb / enzyme approach over bifunctional fusion protein is that the pharmacokinetics and the targeting-site accumulation of each component could be controlled separately. Obviously, multi-step injection is the limitation of this approach, though it may be argued that multi-step injection is still less complex than many currently-used chemotherapy protocols. In addition to cancer therapy, bsMAb can be used in thrombosis therapy to improve fibrinolytic efficacy of tissue plasminogen activator (TPA). TPA triggers the conversion of plasminogen to the fibrinolytic enzyme plasmin, which causes the dissolution of thrombi. Branscomb et al. have demonstrated that a bsMAb against human fibrin and TPA could enhance

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fibrinolysis 10-fold than that with TPA only in vitro [39]. Urokinase plasminogen activator (UPA) has also been used to dissolve fibrin-containing clots by bsMAb targeting. This resulted in 13-fold and 6-fold more fibrinolysis in vitro and in vivo respectively than that with urokinase only. Another study using baboon bearing 125 I-fibri clots in femoral vein has showed that the continuous intravenous infusion of a bsMAb resulted in a 5-fold enhanced thrombolytic effect of UPA than that of UPA alone [40,41].

2.3. Radioimmunotherapy Conventional radioimmunotherapy (RIT) using systemically administered MAb linked to radionuclides is a promising approach to metastatic cancer treatment. However, conventional RIT with MAb conjugates damages critical organs due to exposure to high radiation dose from long circulating radiolabeled antibody and non-specific accumulation of radiolabeled antibody in exposed organs. Pretargeted radioimmunotherapy with bsMAb is a multi-step strategy that allows quick and specific delivery of radioisotope ( 90 Y, 131 I, 188 Re and 32 P) to a tumor with minimal radiation exposure, hence lowered toxicity to normal organs [42–48] (see Fig. 2). Pretargeting involves the administration of a bsMAb that has high affinity for both a tumor antigen and a small, rapidly-excreted radionuclide. After the bsMAb has been concentrated in the target tumor, a clearing agent is administered to remove the excess bsMAb from the circulation. Following the clearing agent, the radionuclide is given and the maximum tumor concentration and maximum tumor-to-blood ratio is achieved in 1–3 h. Unbound radiolabel is rapidly excreted via the kidney by glomerular filtration mechanism [48]. Gautherot et al. have compared pretargeting bsMAb to the directly radiolabeled F(ab9)2 fragment for the treatment of LS174T colorectal xenografts [44]. A total of 6 groups of tumor-bearing mice were treated with anti-CEA 3 anti-diethylenetriamine pentaacetic acid (DTPA) bsF(ab9)2 and 131 I-labeled di-DTPA bivalent hapten. Three groups of mice were injected with various activities of 131 I-labeled bivalent hapten (75, 96, and 112 MBq) at 20 h after administration of bsF(ab)92. Three other groups were

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Fig. 2. Schematic representation of three-step pretargeting radioimmunotherapy. Pretargeting approach involves the administration of bsMAbs with high affinity for both a tumor antigen and a small rapidly excreted radionuclide molecule. After the bsMAbs have concentrated in the target tumor, a clearing agent is administered to remove the excess bsMAbs from the circulation. Following the clearing agent, radionuclide molecules are given, and the maximum tumor concentration and tumor-to-blood ratio is achieved in 1–3 h. Unbound radiolabel is rapidly excreted via the kidney by glomerular filtration mechanism.

injected with an almost constant activity of labeled hapten (102 MBq) at 3 time points (15, 30 and 48 h) after bsF(ab9)2 administration. For conventional RIT, mice were treated with 96 MBq 131 I-labeled antiCEA F(ab9)2. Conventional RIT induced severe toxicity and resulted in the death of several treated mice. Nevertheless, all surviving mice treated with 131 I-labeled anti-CEA F(ab9)2 relapsed shortly after treatment (tumor growth delay 5 48613 day). As for mice treated with pretargeted bsMAb, toxicity varied with the pretargeting time interval and the administered activity. For 20 h pretargeting time, the maximum tolerated dose was 96 MBq. For all pretargeting groups except one (with 48 h pretargeting time interval and growth delay of 82626 day), no tumor growth was observed over a period of 8 months. Furthermore, 33% of the treated mice were consid-

ered cured based on clinical and histologic criteria. Clinical data demonstrated that bsMAb could deliver large radiation doses to tumor cells, resulting in an excellent therapeutic index. In a dose-escalation study, 23 patients with recurrent or metastatic medullary thyroid carcinoma received 131 I-labeled bivalent hapten ranging from 40 to 200 mCi on the fourth day after receiving bsMAb, of which five had minor responses, five had stable disease and five had a significant decrease in bone pain. In 13 small-cell lung carcinoma patients, three had partial responses and one had stable disease [44]. Although pretargeted radioimmunotherapy using 131 I shows encouraging results, 131 I is not an optimal radionuclide for human use because of its long halflife, strong g emission, poor specific activity and low b particle energy. 188 Re, though unsuitable for direct

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antibody labeling, could be used with two-step pretargeting strategy because 188 Re has a greater range than 131 I. This would allow the eradication of solid tumors around 1 cm in diameter. One study has compared the distribution and dosimetry of a bivalent hapten labeled with 188 Re or 125 I [43]. After administration of a pretargeting bsMAb (anti-CEA / anti-hapten), AG 8.1 or AG 8.0 hapten radiolabeled with 188 Re or 125 I respectively was administered into a nude mouse model grafted subcutaneously with a CEA positive human colon carcinoma cell line (LS174T). This study indicates that 188 Re can be used for radiolabeling of hapten in pretargeted RIT. Although the method used for hapten radiolabeling did not provide optimal tumor uptake, the use of a bifunctional chelating agent associated with AG 8.1 should solve this problem. Apart from b-emitting radionuclides, bsMAb can be used to deliver a-emitting radionuclides such as polyhedral boron anion to tumor sites. After a large number of stable boron atoms are localized in the tumor by bsMAb, the boron atoms are irradiated with low energy thermal neutrons. This irradiation will allow boron atoms to release a particles to destroy tumors locally [49,50]. Clinical success of using a-emitting radionuclide 213 Bi has already been seen in conventional RIT, and hopefully this success will be extended to the pretargeted approach [51,52]. Pretargeting RIT combines long-circulating bsMAb and small, rapidly-excreted molecules to achieve a high tumor-to-blood ratio. Pretargeting reduces the total radiation exposure to normal tissue because of the absence of radiation during the localization phase of bsMAb and the rapidly-excreted small radiolabeled molecules from the renal system. The pretargeting approach allows the control of hematological toxicity, which is dose-limiting toxicity with conventional RIT. However, from perspective of drug development, this multi-step strategy places additional manufacturing burden and complicates the design of clinical trials. Furthermore, since the internalized bsMAb will fail to capture the radiolabeled small molecules, it cannot be used in this approach. NeoRx Corporation in Seattle has come up with an alternative three-step delivery system. In the first step, an antibody-streptavidin conjugate is injected and accumulates at the tumor sites. Secondly, a

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synthetic clearing agent binds and clears the circulating conjugates from the circulation via the liver. The final step exploits the ultra high affinity interaction between biotin and steptavidin in order to deliver a biotin-radionuclide conjugate to the tumor. Since biotin-radionuclide conjugate is a small molecule, it localizes quickly to the antibody-receptor on the tumor while minimizing exposure of non-targetorgan to radiation [53]. Although immunogenicity of streptavidin is the major hurdle to human use, this system has already been tested in phase II clinical trial for treatment of non-Hodgkin’s lymphoma. Since the interior of a tumor mass usually lacks radiosensitivity due to low level of oxygenation, antibody fragments should be more useful than whole immunoglobulin in tumor RIT. As the field of antibody engineering comes of age, new antibody fragments such as single chain or diabody have a great potential to open up exciting opportunities for new therapy.

2.4. Immunotherapy BsMAb is able to activate and target the cellular immune system to kill tumor cells or other pathogens. A number of different effector cells have been studied extensively in this application, including CTL (CD8 1 ) cells, natural killer cells, macrophages and polymorphonuclear cells [8] (see Table 2). Initially, CTL cells were believed to be the most suitable candidate for retargeting cytotoxicity since they participate in the recognition and the subsequent killing of tumor cells, viral infected cells and allogeneic targets. Two possible mechanisms have been proposed. First, CTL cells can express death ligands on their surfaces that can bind to the death receptors on the target cells, thereby initiating the target cell’s apoptosis; Second, CTL cells can release perforins and granzymes in the narrow intercellular space between CTLs and target cells. Granzymes can enter the target cell to induce apoptosis of the target through the pores, which are created by perforins on the target cell membrane [6,54]. The primary cytotoxic trigger on CTL is the TcR / CD3 complex, which is antigen-specific and major histocompatibility complex (MHC)-restricted. However, a bsMAb can react with TcR / CD3 complex to initiate retargeted cytotoxicity bypass MHC restriction. Many

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Table 2 Effector molecules and target molecules in BsMAb-based therapeutics Diseases

Target molecules

Effector cells

Triggers

Colorector carcinoma

CEA 17-1A / EGP2 EGF-R 17-1A / EGP2 HER-2 / neu HER-2 / neu Mov-18 EGF-R HER-2 / neu EGF-R EGP2 HER-2 / neu CD19 CD20 CD22 HLA CD25 CD30 CD52 TAG-72 CD33 CD52 CD19 Tissue factor Alpha(v) integrin VEGFR-2 Endoglin Endosialin Aminopeptidase A

T cells

TcR / CD3 CD2

NK cells

CD16 CD38 CD44 CD56 CD69 NK-TR NKRP-1 Ly-49D KIRs

Neutrophils

CD64 CD89 CR3

Monocytes / Macrophages

CD64 CD89 CR3 Mannose receptor

Dendritic cells

CD64 Mannose receptor

Erythrocytes

CR1

Squamous cell carcinoma Renal cell cancer Breast cancer Ovarian cancer

Prostate cancer Lung cancer B cell lymphoma

T cell lymphoma

AML CLL Tumor vasculature

groups have demonstrated lyses of target cells by CTLs using bsMAb [55–61]. Unfortunately, the targeting of TcR can produce disparate outcomes depending on the subset and differentiation state of T ¨ cell being stimulated. The naıve CTLs (CD8 1 ) cannot lyse target cells since they require pre-activation by cytokines, such as IL-2, in order to gain cytotoxic power. Furthermore, additional costimulatory signals are often needed for the full activation of CTL that involves CD28, CD2, LFA-1, ICAM-1, CD5, CD40 and the presence of cytokines, e.g. IL-2 or TNFa [9,62,63]. Conversely, in the absence of cytokines, TcR targeting can induce T cell apoptosis, which could be a major challenge for bsMAb therapy [12,64]. In addition to CD8 1 CTLs, TcR-specific bsMAb will also trigger CD4 1 helper T cells. Under such circumstances, co-administration of cytokines will again influence the outcome of the TcR-targeting response. Inflammatory cytokines can

¨ CD4 1 T cells to differentiate into Th1 induce naıve cells, whereas cytokines such as IL-4 and IL-10 will promote Th2 cell differentiation. It is still unknown which type of response would be most beneficial to immunotherapy. In addition to TcR / CD3, other trigger molecules on CTLs include CD2 [65,66] and TcgR / Tc d R [67]. Cytotoxicity is not due to bystander lyses, because direct contact between an effector and a target cell is required [68]. Since CTLs require complicated interactions to be fully activated, the use of CTLs as immune effector cells for bsMAb immunotherapy is more complex compared to NKs and myeloid effector cells.Recently, NKs, macrophages and neutrophils have gained much attention because they can readily be activated and mobilized in vivo. NK cells, unlike T cells, are preferable to the decreased MHC class I molecules, which reduce NK cell inhibition by killing cell inhibitory receptors (KIR). Myeloid cells are not generally considered as

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effector cells since they do not specifically recognize tumor cells. However, in the presence of bsMAb, myeloid cells were able to kill tumor cells. Furthermore, myeloid cells are found to infiltrate tumors engineered to secrete IL-2, IL-4, IL-7, IFNg, TNFa, G-CSF and the induction of tumor immunity requires the communication between neutrophils and T cells [5]. On NKs and myeloid cells, FcgRs are the most widely investigated trigger molecules [69]. FcgRI (CD64) is an activating receptor, which is found on monocytes, macrophages and dendritic cells and can be induced on neutrophils by G-CSF or IFNg [70,71]. It has been successfully used in bsMAbmedicated immunotherapy [72–77]. Due to high affinity for IgG, all CD64 molecules are occupied by serum IgG. BsMAb are therefore designed to bind to the outside of the Fc-binding domain of CD64. So far at least four bsMAbs using CD64 as trigger molecule have been tested in human clinical trials, of which MDX-210 (CD64 3 HER-2 / neu) is already in phase III trial. FcgRIII (CD16) has also been extensively studied, but it may not be a good trigger molecule since it is present on neutrophils as a glycosyl-phosphatidylinositol-anchored receptor that cannot trigger cytotoxicity. In addition, it exists as a soluble molecule in serum and may trigger side effect by forming immune complex [78,79]. Although there is a disadvantage of using CD16 as a trigger molecule, at least two bsMAbs, 2B1 (HER-2 / neu 3 CD16) and HRS-3 /A9 (CD30 3 CD16) have been tested in phase II clinical trials. FcaRI (CD89) is present on monocytes, macrophages and neutrophils. It is involved in the first line of humoral defense on mucosal surfaces of the body and is one of the most potent FcRs for induction of tumor cytotoxicity [80,81]. Most cellular components of immune system express FcRs, including NKs, monocytes, macrophages and granulocytes. These cells tend to exist in a constitutively activated state, although their activities can be increased by cytokines. They may therefore require less costimulation than resting T cells to initiate cytotoxicities [12]. Other triggers have been studied for retargeted cytotoxicity, including complement receptor (CR3), mannose-binding receptor, scavenger receptor, Tolllike receptors, as well as a number of adhesion molecules (CD2, CD38, CD44, CD69, NKRP-1, NK-TR, Ly-49D, KIRs) [12,82–88]. Cancer im-

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munotherapies involving bsMAb mediated-killing have been widely explored. These include: antiepidermal growth factor receptor for breast cancer, anti-sialyl Lewis a for colon cancer, anti-ovarian cancer, anti-renal cell carcinoma, anti-lung small cell carcinomas, anti-CD19 for B lymphoma, anti-CD13 for acute myeloid leukemia and anti-tenascin for gliomas [89–97]. Because of the problems of delivering sufficient number of effector cells and bsMAbs to the tumor, bsMAb may be reserved as adjuvant treatment for minimal residual disease [11]. BsMAb has also been tested for its immunotherapeutic applications in viral infections [98]. Chamow et al. have developed a humanized bsMAb (CD4 / anti-CD3) to target the HIV-infected cells. This bsMAb can first target HIV-infected cells by the natural affinity of CD4 for gp120 on the HIV virus, then recruit and activate CTLs to lyse target cells through their anti-CD3 moiety in a non-MHC restricted manner. They demonstrated that bsMAb could specifically lyse HIV-infected cells using purified CTL and whole peripheral blood lymphocyte (PBL). In contrast, a human anti-gp120 MAb can lyse HIV-infected target cells only with PBL fractions, but not with purified CTL. Moreover, while the cytotoxicity of anti-gp120 MAb was completely blocked by the human serum with abundant IgG, which competes for FcgR binding, bsMAb-mediated lyses of target cells were not affected. This offers a potential advantage of bsMAb over MAb for HIV immunotherapy [99,100].

2.5. Anti-vascular therapy Most immunotherapeutic strategies require direct contact between immune effector cells and diseased cells. Whereas in human body, cells of the normal organs are almost all closely aligned to blood supply, in solid tumors often up to 6–8 layers of tumor cells are fed by one blood vessel. All cytotoxic drugs, antibodies and CTLs need to transverse great distance before reaching the tumor cells [101]. In addition, there are no ‘traffic signs’ at the luminal side of the tumor blood vessels directing the drugs, bsMAb or cells towards the tumor cells. The masking of the tumor cells from blood born entities by the vascular endothelial cells, the decreased expression of lymphocytes-guiding adhesion molecules by

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tumor endothelium, irregular blood flow and vascular permeability and increased intratumor pressure, all make the accessibility of the target cells a cumbersome event [102]. To overcome these barriers, tumor vascular endothelial cells have been chosen as a target to kill tumor cells by cutting off their blood supply. BsMAb has been used to achieve this goal by cross-linking tumor vascular endothelial cells and immune effector cells to activate the coagulation process. One bsMAb has been developed to direct against the MHC class II molecule, an artificially induced tumor endothelial cell antigen as well as tissue factor that is one of the initiating receptors of the blood coagulation cascades. A single intravenous administration of bsMAb / tissue factor mixture into tumor bearing mice allowed rapid blood coagulation in the tumor blood vessels. Intravenous administration of bsMAb / tissue factor complex led to complete tumor regressions and partial remissions in 38% and 24% of the mice bearing subcutaneous tumors with 0.8–1.0 cm in diameter at the start of the treatment, respectively [103,104]. BsMAb-based therapies targeting vascular compartments not only greatly reduce the impact of physical barriers of solid tumors but also avoid developing the classical drug-resistance in tumor cells, for endothelial cells are highly regulated and genetically stable cells. In conclusion, therapy directed against the tumor vasculature should have broad-spectrum activity, since angiogenesis is required for tumor progression. More research and investigations are called for in this promising field.

2.6. Gene therapy Adenovirus-mediated gene therapy depends on the expression of the coxsackievirus / adenovirus receptor (CAR) and a (v)-integrins on target cells. However, normal cells also express these molecules and result in vector-related toxicity. MAb-aided adenoviral gene therapy may decrease this toxicity by lowering the administration dose of adenovirus. In addition, it may enhance the infection efficiency of adenoviral vectors on specific cell population with even low expression of viral receptors. Hence, targeting the adenoviral vector has important in vivo applications. Since most of human adenoviruses bind to the CAR via carboxyl-terminal knob domain of their fiber

protein, the targeting of adenovirus to specific cell lines can be achieved by using bsMAb to redirect the binding of knob domain to new cellular receptors on target cells [105–111]. Nettelbeck et al. have developed a bispecific scFv (anti-CD105 3 anti-knob) targeting the adenovirus to the surface protein endoglin (CD105) of the vascular endothelial cells. Endoglin (CD105) is a component of the transforming growth factor b-receptor complex. This bsMAb fragment enhanced the selective adenoviral transduction of vascular endothelial cells, signaling a promising direction for bsMAb in anti-vascular cancer gene therapy [105]. Reynolds et al. have used a bsMAb to target the angiotensin-converting enzyme (ACE), which is preferentially expressed on pulmonary capillary endothelium [106]. Administration of bsMAbtargeted vector complex into rats resulted in at least a 20-fold increase in both adenoviral DNA localization and luciferase transgenic expression in the lungs, compared to the untargeted vector. Furthermore, bsMAb-targeting led to an 80% reduction of transgenic expression in non-target organs. These studies so far show that bsMAb can specifically modify the pharmacokinetics of adenoviral vector in vivo and thus encourage the further development of injectable adenoviral vectors. In order to achieve a high therapeutic efficacy, bsMAb fragments may be better candidates than whole immunoglobulins since they can circumvent the problem of viral clustering, which may occur with bivalent MAb by trapping in the reticulo-endothelial system. Another bsMAb (anti-knob 3 anti-TAG72) has been used clinically in gene therapy to treat ovarian cancer [107]. This bsMAb augmented gene transfer to primary ovarian cancer cells 2-to-28 fold relative to untargeted gene transfer, while decreasing gene transfer to autologous cultured mesothelial cells 4-to9 fold. In vivo data demonstrated that bsMAb retargeting improved the selectivity of adenoviral gene transfer for ovarian tumors 8-to-252 fold on intraperitoneal injection. These results suggest that bsMAb retargeting may improve the therapeutic index of gene therapy for ovarian cancer in clinical trials [107]. Besides a viral vector, other vectors such as liposome can also be targeted by bsMAb to the specific tumor or disease sites. Further exploration of bsMAb-redirected gene therapy could potentially

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open up a new horizon for the treatment of cancer and other deadly diseases.

2.7. Therapeutic vaccine Therapeutic vaccine can cost-effectively induce a systemic immune response specifically against the target antigen to either treat existing disease or prevent it. To augment this response, costimulatory molecules are often needed. In this application, bsMAb can initiate or enhance the immune response by binding target to either T cells or costimulatory molecules such as CD28 or B7 [112–120]. Haas et al. have modified a tumor vaccine with bsMAb to promote the binding of costimulatory molecules to enhance the immune response [112]. Following the infection of tumor cells with a nonvirulent strain of Newcastle disease virus, the cells were further modified by incubation with a bsMAb (anti-HN 3 anti-CD28). This bsMAb binds specifically to the viral hemagglutinin-neuraminidase (HN) molecule on the infected tumor cells and to the CD28 molecule. In the in vitro study, the bsMAb up-regulated early (CD69) and late (CD25) T-cell activation markers on CD4 and CD8 T cells from either normal healthy donors or cancer patients and induced tumor cytostasis in non-modified bystander tumor cells. In addition, in combination with the bsMAb (anti-HN 3 anti-CD3), the study showed an augmented antitumor cytotoxicity and T-cell proliferate responses. Guo et al. reported a nonviral way to develop a cancer vaccine that relies on simple cell culture techniques. In a two-step process, tumor cells removed from a patient are first stimulated by cytokines to enhance the expression of tumor antigens and the immune activating molecules. Then treated tumor cells are bound with bsMAb recognizing a tumor antigen and CD28 on T cells. By specifically targeting CD28 on T cells and tumor antigen, bsMAb facilitates their interactions, which greatly enhances T cell activity and the immune response. Experiments were performed on mice susceptible to fast-growing tumors. Disease-free mice were vaccinated with the modified cell vaccine, and 2 weeks later, injected with cancer cells. No tumors were detected in the vaccinated mice, whereas non-vaccinated mice had rapid tumor growth. Another experiment showed that T cells harvested from the vacci-

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nated mice acquired the ability to kill cancer cells in the cell culture. This provided evidence that an enhanced T cell immune response had been stimulated [113]. The human MAb repertoire to the Haemophilus influenza type b (Hib) polysaccharide (PS) is dominated by MAbs expressing an idiotype—Hibld-1. Reason et al. prepared a vaccine consisting of bispecific F(ab9)2 fragment with one specific for Hibld-1 and another for CD3. This bispecific idiotypic vaccine stimulated production of human MAbs to Hib PS in severe combined immunodeficient mice engrafted with normal human adult PBLs. The induced MAbs uniformly expressed Hibld-1 and protected neonatal rats from Hib bacteremia [114]. Mocikat et al. explored a B-cell lymphoma vaccine that conferred a long lasting tumor immunity [115]. B-cell lymphoma cells were fused to xenogeneic hybridoma cells that secrete a MAb against a surface molecule on antigen presenting cells (APC). The resulting ‘trioma’ cell produces a bsMAb containing both lymphoma idiotype and APC-binding arms. The bsMAb can direct the B-cell lymphoma idiotype to APCs, which process and present lymphoma idiotype leading to T-cell activation with a long lasting tumor immunity. BsMAb that targets tumor antigens and cellular FcgR cannot only induce cytotoxicity to tumor cells but also trigger adoptive immune responses. Wallace et al. have generated a fusion protein containing prostate cancer antigen (PSA) and scFv of antiFcgRI antibody. The fusion protein can be internalized and processed by myeloid cells and presented by MHC class-I molecule resulting in the tumor cell lyses by PSA-specific CTLs. This immunization protocol may offer a potential advantage if it proves capable of effectively inducing antigen presentation in vivo. It requires no therapeutic vectors and utilizes the entire antigen molecules, thus simplifying the clinical development and the process for sourcing immunogens [119,120].

3. Production of BsMAb

3.1. Chemical method Forty years ago, two different polyclonal anti-

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bodies were chemically linked together generating bispecific polyclonal antibodies leading to the earliest concept of bsMAb [2]. The chemical reaction in general dissociates two different antibodies and reassociates them to allow a portion of the reassembled antibodies to be bispecific in nature. With the advent of monoclonal technology and bifunctional reagents, the chemical reaction can be further tailored in favor of heteroconjugates formation. Since then, homogeneous bispecific monoclonals have been tested in human clinical trials. So far, the several successful bsMAb tested in clinical trials are chemically conjugated bispecific F(ab9) fragments. Two groups of bifunctional reagents, homobifunctional and heterobifunctional, have been studied. Homobifunctional reagents react with the free thiols generated upon reduction of inter-heavy chain disulfide bonds. 5, 59-dithiobis(2-nitrobenzoic acid) (DTNB) or o-phenylenedimaleimide (O-PDM) can activate thiol groups on Fab9 fragments of MAb [121–123]. DTNB acts to regenerate disulfide bonds between the two Fab9s, whereas O-PDM acts to form a thioether bond between the two Fab9. In general, O-PDM-produced thioether bond is more stable than the disulfide bond regenerated by DTNB [122]. Furthermore, O-PDM has been used to generate a variety of antibody constructs beyond the concept of bsMAb, e.g. trimeric and tetrameric antibodies with two or three specificities. These multi-valent and multi-specific antibodies could be more potent in redirecting effector cell for tumor cytotoxicity than corresponding dimeric antibodies [124]. Heterobifunctional reagents, on the other hand, can introduce a reactive group onto a protein, which enables it to react with a second protein. N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) is an example that can react with primary aminogroups and generate free thiol groups on specific protein [125,126]. SPDP has shown the capability of combining any two proteins with exposed amino groups, such as antibodies and Fab9 fragments, regardless of class or isotype [127]. Although chemical crosslinking is a fast and straightforward method for producing bsMAb and the favorable higher yields make the final products easy to purify, this approach causes random cross-linking of protein molecules and hence exhibits significant batch-to-batch variations. Furthermore, the chemical reaction can directly

cause protein denaturation, which inactivates the antibody. Although the advancing and challenging research has moved further toward genetic engineering of antibody molecules, traditional methods are not completely out of date. Chaudri et al. devised a method of making bsMAb through hybridization of complementary oligonucleotides that were covalently linked to two different Fab9fragments respectively [128].

3.2. Biological method The biological method using somatic hybridization to produce bsMAb has been adopted through generation of either quadroma or trioma. These quadromas or triomas are generated either by the fusion of two established hybridomas, or by the fusion of one established hybridoma with lymphocytes derived from a mouse [129–131]. Modification of hybridoma cells such as inserting a drug resistant gene has greatly facilitated making functional-stable quadromas [132,133] (see Fig. 3). However, the induction of cell mutants and drug selection are always laborintensive and time-consuming. The method without drug selection therefore has been explored, which utilizes direct fluorescein cell labeling and fluorescence-activated cell sorter (FACS) selection technology [134,135]. In detail, two hybridomas are prelabeled with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) respectively via the cell membrane marker octadecylamine. Labeled hybridomas are fused and the cells staining for both fluochromes selected by FACS. Electrofusion or microelectrofusion has also been intensively studied and proved superior to the traditional chemical fusion reagents such as polyethylene glycol (PEG) [136,137]. Biologically produced bsMAb is synthesized, assembled and secreted in the same way as native immunoglobins [138]. Individual light chain and heavy chain are transcribed separately and assembled randomly, resulting in only a small fraction of the final product to be desirable bsMAb. Intensive efforts are therefore required to remove impaired heavy and light chains from the mixture, which significantly raises the overall cost for antibody production.

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Fig. 3. Production of quadroma. (1) A HAT-sensitive hybridoma A is isolated from the parental hybridoma A by culturing in 8-azaguanine. This hybridoma A is subsequently made resistant to ouabain by mutant selection. After fusion with a wild-type hybridoma B, a quadroma is selected by culturing the cells in HAT medium supplemented with ouabain. The unfused parental cells will die due to sensitivity to HAT and lack of resistant to ouabain, respectively. (2) Hybridoma A and B are pre-labeled with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) via the cell membrane marker octadecylamine. Labeled hybridomas are fused and a fluorescence-activated cell sorter (FACS) selects the quadroma staining with both fluochromes.

3.3. Genetic engineering The high cost and difficulty of producing clinicalgrade bsMAb have hindered its development. This production problem has been largely overcome recently, for bsMAb fragments at least, with the advent of a plethora of formats for recombinant production by secretion from E. coli. Genetic engineering can produce rationally designed protein structures to meet all the needs in their clinical applications [139– 141]. Fig. 4 demonstrates many varieties of bsMAb generated by traditional methods and genetic engineering (see Fig. 4). Songsivilai et al. first described this using transfectants to secret chimeric bsMAb [142]. Generally, the antibodies or their fragments of different specificities are expressed as distinct polypeptides from the same transcript or from separate plasmid constructs, followed by com-

bining different antibody fragments through a binding motif. Recently, bsMAb molecules have been made as a single covalent structure by combining two single chain Fv (scFv) fragments using a polypeptide linker [143–145]. Despite the functionality of these molecules, the wrong domain association between the two scFv and the constraint between the two paratopes, created by additional oligopeptide linker, are the concerns in its clinical applications. Hence, the length and sequence of the linker may determine the flexibility and correct folding of the molecule [146]. Ridgway et al. reported a ‘knobs into holes’ method of making bsMAb [147]. In this method, the knobs were created by replacing small amino acid side chains with larger ones at the interface between C H 3 domains, and large side chains with smaller ones at the same interface that generated the holes.

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Fig. 4. Schematic representation of bsMAb structures generated by different methods. (*) This has also been constructed by chemical and genetic method.

The protein engineering of the heavy chain C H 3 domains leads to the formation of heterodimers (Ab1-knob-hole-Ab2) which are more favorable than homodimers (Ab1–Ab1 and Ab2–Ab2). The studies showed that knobs into holes strategy can make 90% of secreted IgG to be heterodimeric. This elegant method has been extended to make full-length bispecific human IgG which could be used in immunotherapeutic strategies [148]. Leucine zipper-based dimerization of MAb fragments has also been introduced [149]. Leucine zipper is a sequence derived from transcription factors fos and jun. A murine anti-CD3 Fab9-fos and anti-Tac Fab9-jun were individually expressed as homodimers in SP2 / 0 cells. When they were reduced, mixed and reoxidized, the formation of fos and jun leucine zipper resulted in generation of bsMAb fragment Fab9 (anti-CD3 3 anti-Tac). De Kruif and Logtenberg described the fusion of fos or jun leucine zipper and a truncated mouse IgG3 hinge region to scFv proteins [150]. In their method, two cysteine residues

were engineered into the zipper domains to produce disulfide-stabilized homodimers. These modified zipper cassettes and scFv isolated from a phage display library have produced functional and stable homodimers in E. coli. After reduction, mixing and reoxidation of the homodimers, functional bispecific scFv molecules were developed. Rheinnecker also utilized a similar approach to create scFv dimers by fusing an artificial helix-turn-helix dimerization domain of p53 to a long IgG3 hinge-scFv construct [151]. The extremely high affinity between streptavidin and biotin has been utilized to construct bispecific molecules. Linear fusion of anti-neuraminidase scFv with streptavidin has been linked to biotinylayted anti-ferritin Fab9 to form bispecific reagents, which can react with immobilized neuraminidase and free ferritin when analyzed in a BIAcore姠 biosensor [152]. The Fab fragment of immunoglobulins composed of two polypeptide chains with variable and constant regions generating a stable protein structure, which

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has been used as a platform of constructing bispecific miniantibody. Muller et al. have replaced variable domains of Fab with two different scFv to generate a bispecific miniantibody [153,154]. A design using adjacent dicistronic gene arrangement driven by a single promoter has been used for this miniantibody production in E. coli. Holliger et al. have created a new bsMAb fragment named diabody which has shown superior properties in the immunotherapy [155–160]. Diabodies comprise a heavy chain variable (VH ) domain and a light chain variable (VL ) domain, connected by a peptide linker that is too short to allow pairing between the two domains. This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen-binding sites. In detail, the V-domains of antibody A and antibody B are fused to create the two chains VH A–VL B and VH B–VL A, which are inactive in binding to antigen. Upon pairing to each other, it resumes the functional antigen-binding sites of antibody A and B. Diabodies can be expressed in bacteria (E. coli) and yeast (Pichia pastoris) in functional forms with high yields up to 1 g / l. Recently, a phage display approach was developed to generate and screen diabodies [161]. Although diabody appears more favorable than scFv, construction of diabody libraries from large antibody repertoires requires several cloning steps and the stability of the library in bacteria has not yet been thoroughly evaluated [162]. In order to increase the valence of diabody, two diabodies were linked tandemly to form a tetravalent diabody (Tandab). Four VH and VL domains may be joined together in an orientation that prevents intermolecular pairing. Tandab specific against human CD3 and CD19 turned out to be more favorable for therapeutics than diabody or bispecific scFv of the same specificities [163]. This study has also shown that Tandab has longer blood retention than diabody in mice. Treatment of SCID mice bearing Burkitt’s lymphoma with human peripheral blood lymphocytes and anti-CD28 MAb resulted in complete elimination of tumors in all of the mice within 10 days. These findings could mark the future in the development of multivalent MAbs. In clinical applications, a potential risk of treating patients with engineered proteins is to elicite an

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immune response since the engineered protein contains some foreign peptide sequences such as linker or heterodimerization domains. Unfortunately, such immunogenicity issues can only be addressed through costly and time-consuming clinical trials. In addition, the choice of expression systems can be critical if glycosylated recombinant antibodies are to be generated. Although bacterial system is efficient for scFv expression, but the nonglycosylated antibodies contaminated with toxic lipopolysacharide could reduce the yield of antibodies during purification. Glycosylated yeast products with increased mannose content may be different from mammalianexpressed glycoproteins, leading to rapid removal form the circulation. Under these circumstances, mammalian expression system may be more desirable, provided that stable, high level of expression of the desired construct is achieved [164].

4. BsMAb Humanization Immunogenicity of therapeutic antibodies has a significant impact on their extensive applications to treat many diseases. The human anti-mouse antibody (HAMA) response reduces the effectiveness of antibodies by neutralizing their binding activities and rapidly clearing the antibodies from circulation. Furthermore, HAMA response could cause significant toxicities through immune complex formation with subsequent administrations of mouse antibodies. Unfortunately, it is difficult to generate human MAbs by hybridoma technology or using Epstein–Barr virus transformation of human B-cells secreting MAbs [165]. To reduce HAMA response, genetically engineered antibodies containing fewer mouse but more human origins are pursued to address immunogenic complications. A chimeric antibody is the first generation of engineered antibody whose mouse constant region is replaced by human immunoglobulin counterpart, [166]. Although chimeric antibody is less immunogenic than murine antibody, a human anti-chimeric antibody response is still mounted by the immune system. CDR-grafting or humanized antibody is the second generation, in which the complementary determine regions (CDR) of the murine variable genes are grafted on a

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structurally complementary scaffold of human origin [167]. Humanized antibodies retain the antigen-binding properties of the original murine antibodies but dramatically reduce immunogenicity, though an antiidiotype response is still induced [168]. The last few years have seen a rapid growth in the area of generating complete human antibodies. Phage display could offer almost unlimited sequence variability, which makes it possible to produce all the human antibodies with specificities unaccounted for by the use of conventional chemical or hybridoma technology [169]. Phage display offers a number of distinct advantages: First, geno-phenotype linkage of phages display is a time-saving process for a rapid subcloning of V genes into other prokaryotic vectors; Second, bacterial expression system allows for massproduction of antibody fragments at a reasonable cost. However, the availability of B cells from naturally immunized individuals has been sporadic and limited, and protocols for in vitro immunization of human B cells are not yet established. In addition, intensive efforts are often required to gain the high affinity human antibodies using chain shuffling or mutagenesis followed by re-selection. Transgenic mouse carrying parts of the human Ig loci is an alternative choice for generating human antibodies [170–175]. The mouse genes in these transgenic mice for creating antibodies have been inactivated and replaced by human antibody genes that contain key gene sequences for both the heavy and light chains of human antibodies. The major advantage of transgenic mice over phage display is that the wellestablished hybridoma technology can be utilized directly and the access to affinity maturation machinery of mice can result in truly high affinities. However, the fusion partner to immortalize the B cell repertoire is still mouse myeloma. The resulting antibodies contain mouse Galal–3Gal residue that can be recognized by the human immune system [176]. Although all the bsMAb can be humanized by the technologies described above and the pre-clinical results are promising [177–179], they have not yet been evaluated in the clinical studies. In addition, targeting antigen-presenting receptors such as FcR may increases the immunogenicity of bsMAb including the generation of anti-idiotype antibodies [180]. The human anti-idiotype response may also cause a

rapid clearance of the antibody prior to reaching the targets, which may not be prevented, since it is part of natural feedback mechanisms of the immune system. On the other hand, humanized or human MAbs have proved themselves to be less immunogenic than murine-derived MAbs, which is expected to extend to the bsMAb.

5. Clinical trial information OC / TR (anti-CD3 3 anti-folate receptor) recognizes the folate receptor on ovarian cancer cells and the CD3 antigen on T lymphocytes. A phase II clinical trial was conducted on advanced ovarian cancer patients who received the murine form of OC / TR labeled T cells and IL-2. All evaluated patients had HAMA responses [181]. Compared with the chimeric form, the murine OC / TR displayed higher immunogenicity in those patients. A separate phase II study has investigated the efficacy of the intraperitoneal immunotherapy using murine OC / TR, activated and expanded T cells, and IL-2 in the advanced ovarian cancer patients. It manifested impressive local anti-tumor responses in this study. Of 16 patients, five had complete responses, three had partial responses, two had stable disease, and six had disease progression. One of the patients classified as having a complete response had retroperitoneal lymph node metastases that progressed during treatment. After removal of resectable tumor, seven heavily pretreated patients with ovarian cancer received daily intraperitoneal treatment with activated and expanded OC / TR labeled T cells and IL-2 for 10 days. One patient had a partial response, four had stable disease and two had disease progression. This study proposed possible beneficial effects of OC / TR locally administrated with effector cells and cytokines [181–183]. The most frequent adverse events were fever, tachycardia, ascites, abdominal pain, fatigue and nausea. Although local administration of OC / TR produced mild side effects, low doses of OC / TR F(ab9)2 administered systemically could lead to nonspecific T cell activation, TNFa secretion and toxicity, which limited its clinical utility. Nonspecific T cell activation and the resulting toxicity may have been caused by the bsMAb-mediated cross-linking of T cells with nonmalignant cells expressing folate

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receptors or the impurity of F(ab9)2 such as intact IgG with Fc portion bound to locally displayed FcgRs [12]. To this author’s knowledge, this OC / TR has been discontinued for the further development. SHR-1 (anti-CD3 3 anti-CD19) is a bsMAb derived from a quadroma that retargets T cells to the CD19 positive non-Hodgkin’s lymphoma (NHL). Phase I study has been conducted on this bsMAb [95]. In the dose-escalation study from 10 mg to 5 mg, three NHL patients were observed with low toxicity. T and B cells disappeared from peripheral blood with concomitant thrombocytopenia. Pharmacokinetic analysis indicated that the C max value was 200–300 ng / ml immediately after intravenous infusion of 2.5 mg SHR-1 with a half-life of 10.5 h. Phase I / II study failed to show any clinical response on chronic lymphocytic leukemia (CLL) patients with the intravenous administration of SHR-1 in conjunction with the subcutaneous administration of IL-2. Furthermore, bsMAb-coated T cells did not actively localize in the tumor. One of the explanations is that activated endothelium throughout the body may form a temporary sink for T-cells, thereby preventing T-cells from reaching the tumor [6]. BIS-1 (anti-CD3 3 anti-EGF-2) is a bsMAb specifically against EGF-2 present on carcinomas and CD3 on T cells. In a pilot clinical study, patients with malignant ascites or pleural exudates were treated locally with autologous T cells activated ex vivo and redirected towards tumor cells with BIS-1. The data suggested that the treatment induced both antitumour activity and a strong local inflammatory reaction. This was accompanied by no or minimal local and systemic toxicity such as mild fever. However, patients treated with intravenous administration of F(ab9)2 fragments in combination with subcutaneous administration of IL-2 demonstrated no antitumor response, though proinflammatory cytokines were induced and isolated peripheral blood leukocytes of treated patients showed high killing potential in vitro [184,185]. Hu1D10 (anti-CD3 3 anti-1D10) is a humanized bsMAb that recognizes the CD3 antigen on T cells and the 1D10 antigen present on the majority of B cell and pre-B-cell malignancies. This bsMAb was developed by genetic engineering using leucine zipper technology. A phase I study on the patients with relapsed NHL demonstrated positive results. Of

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the five patients with relapsed lymphoma, three had stable disease, one had progressed and one had a 40% reduction in tumor size on the 50th day. In a phase II study, among eight patients with relapsed NHL receiving Hu1D10 (0.15–1.5 mg / kg), minor toxicities were observed, including fever, chills, dyspnea, headache, myalgia, nausea and vomiting. One patient had transient grade III hypoxia with the first infusion only. No B cell depletion was detected during the study. Result showed that five patients receiving weekly infusions of Hu1D10 had objective responses to treatment. Three of these patients had not responded to prior therapy with rituximab [186,187]. The advanced recombinant DNA technology makes Hu1D10 more suitable for human use than anti-CD3 bsMAb produced by conventional methods. The reduced toxicities may be due to humanization, lack of Fc portion, or less nonspecific expression of tumor antigen in nonmalignant cells. This is the first anti-CD3 bsMAb showing positive response in systemic administration. Overall, most of the anti-CD3 bsMAb have impressive local anti-tumor responses. However, they fail to present therapeutic efficacy by systemic administration. Based on the clinical experiences, we can conclude that the purity of bsMAb, target antigen selection, HAMA response, T cell pre-activation as well as the function of cytokines are all critical issues in this therapeutic approach. A glimmer of hope from the Hu1D10 clinical trial will allow us to resume our initial interest to further optimize its fundamental aspects. Compared to those of anti-CD3 bsMAb, the clinical trials of anti-FcgRs bsMAb (especially anti-CD64 bsMAb) have lighted on more positive responses with limited toxicities. 2B1 (anti-CD16 3 anti-HER-2 / neu) is a mouse bsMAb specifically against HER2 / neu and FcgRIII (CD16). It is created from a quadroma. 2B1 promotes the targeted lysis of malignant cells overexpressing HER2 / neu proto-oncogene by NKs and mononuclear phagocytes expressing the FcgRIII. A phase I study demonstrated that the dose-limiting toxicity was thrombocytopenia and the principal nondose-limiting toxicities were fever, rigor, nausea, vomiting and leukopenia. The pharmacokinetics of this murine antibody were described by nonlinear kinetics with a half-life of 20 h. HAMA responses were induced in 14 of the 15 patients. The initial

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2B1 treatment brought forth more than 100-fold increases in circulating levels of TNFa, IL-6 and IL-8 and slight rise in GM-CSF and IFNg. The maximum tolerated dose (MTD) for patients with extensive prior myelosuppressive chemotherapy was 2.5 mg / m 2 . Although 2B1 yielded minor positive responses, it induced massive cytokine release by cross-linking of FcgRs on circulating leukocytes. The toxicity makes this bsMAb decidedly inauspicious for human use, part of the rationale for the production of bispecific scFv fragment [12,188,189]. HRS-3 /A9 (anti-CD16 3 anti-CD30) is a mouse bsMAb, which is directed against the CD16 antigen and the Hodgkin’s-associated CD30 antigen. The first phase I / II trial on patients with refractory Hodgkin’s disease established some therapeutic efficacy of HRS-3 /A9. Of the 15 patients on the trial, adverse events consisted of fever, pain and allergic exanthema. Nine patients developed HAMA responses and four patients developed an allergic reaction. MTD was not reached at 64 mg / m 2 / dose and no dose-limited toxicity was observed. Of the 15 patients, one complete, one partial, three mixed responses were achieved and two patients had a stable state and eight experienced disease progression [190,191]. Second randomized pilot trial has confirmed the antitumor efficacy and the minor toxicity of HRS-3 /A9. Also it suggested that coadministration of cytokines might contribute to an augmented antitumor activity. Of the 16 patients, one had complete remission and three partial remissions lasting 5–9 months, and four stable disease for 3 to 6 months. IL-2 pretreatment resulted in a significant

increase of circulating NK cells in all patients treated. This has converted two cases of stable disease into one complete remission and one partial remission. The side effects consisted of mild fever in only six patients [192]. MDX-210 (anti-CD64 3 anti-HER-2 / neu) is the most advanced bsMAb product in clinical trials (see Table 3). It targets HER-2 / neu over expressing cancer cells and human FcgRI (CD64). It is a chemically linked bsFab9 fragment, which consists of murine anti-HER-2 / neu Fab9 and humanized antiCD64 Fab9. Since it is not a complete humanized antibody, all patients exhibited HAMA responses in different phases of clinical trials. A phase I study showed that MDX-210 primarily induced grade I and II ‘flu like’ symptoms. Other toxicities included grade I or II chest pain and dyspnea, grade II creatinine elevation, transient grade IV thrombocytopenia and grade III hypotension. MDX-210 at dose of 0.35–18 mg / m 2 was well tolerated in patients with refractory breast or ovarian cancer. One patient had a partial remission and another had a mixed response. Since MDX-210 lacks Fc domain, the overall toxicity of MDX-210 is more limited than those of whole IgG bsMAb. In a phase II study, one prostate cancer patient with advanced metastatic disease had a 90% drop in prostate specific antigen (PSA). The levels of PSA remain reduced for 6 months. Nine of 22 patients experienced a reduction of PSA and seven had 50– 99% reduction. Another phase II study showed that two of four patients with refractory kidney cancer achieved a response; one patient with a large liver

Table 3 Bispecific monoclonal antibodies at various stages of clinical trials BsMAb

Sponsors

Targets

Status

Indications

MDX-210 MDX-447 MDX-260 Hu1D10 HRS-3/A9 PENTACEA OC/TR 2B1 MDX-220 BIS-1 SHR-1 251 3 22

Medarex Medarex/Genmab/Merck Medarex Protein Design Labs Biotest IBC/Immunomedics Centocor/GlaxoSmithKline National Cancer Institute Medarex/Genmab University Hospital Groningen University of Cologne University of Pittsburgh

CD64 3 HER-2/neu CD64 3 EGF-R CD3 3 glioma CD3 3 1D10 CD16 3 CD30 CEA 3 drug/radioisotope CD3 3 folate receptor CD16 3 HER-2/neu CD64 3 TAG-72 CD3 3 EGP2 CD3 3 CD19 CD64 3 CD33

phase III phase II phase II phase II phase II phase II phase II phase II phase I phase I phase I phase I

Prostate, ovarian, breast, lung, pancreatic, kidney Bladder, cervical, lung, head/neck, ovarian, prostate Melanoma, glinoma, neurablastoma non-Hodgkin’s lymphoma Hodgkin’s lymphoma Small cell lung cancer Ovarian cancer Ovarian, breast, lung, pancreatic, kidney, prostate AML Lung cancer non-Hodgkin’s lymphoma AML

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tumor achieved a partial response and one patient’s pulmonary metastases was reduced by 49%. Furthermore, thirteen of 20 patients with refractory kidney cancer experienced disease stabilization. Of these 13 patients, five patients achieved 120-day stable period while receiving ongoing treatment. Reductions in PSA lasted over 160 days in some cases. In order to further enhance cytotoxicity, increase effector-to-target ratio, and improve antigen presentation, G-CSF, GM-CSF or IFNg have been added to the MDX-210 therapy. Overall, these treatments were well tolerated and received some positive responses. In late stage breast cancer patients, multiple doses of MDX-210 in conjunction with G-CSF for 6 weeks induced stabilization or regression of disease in 3 of 12 patients. Relief of bone pain, shrinkage of a lymph node tumor and clearance of marked amounts of bone marrow metastases were also evident. Another study was conducted on 25 patients with HER2 1 advanced prostate cancer treated with MDX-H210 along with GM-CSF for 6 weeks. Seven out of twenty patients had a . 50% PSA response with a median duration of 128 days. Seven of twelve patients had some degree in pain relief. The PSA related toxicity decreased in 15 of 18 patients [193–198]. MDX-447 (anti-CD64 3 anti-EGF-R) is the first humanized bsMAb Fab9 fragment made by CDRgrafting, which is also a chemical conjugate. It directly targets FcgRI (CD64) and EGF-R. Phase I study was conducted for MDX-447 in 64 patients with renal cell or head and neck cancer. All patients received increased doses from 1 to 40 mg / m 2 of weekly infusions of MDX-447 with or without GCSF (3 mg / kg). The hypotension was the doselimiting toxicity in this study. The MTD of MDX447 alone was 30 mg / m 2 , whereas 3.5 mg / m 2 was for MDX-447 with G-CSF. The clinical toxicity related to the release of IL-6 and TNFa. The addition of G-CSF aggravated toxicity considerably. A total of 21 patients showed stable disease for at least 12 weeks and one patient had 24% reduction of mucoepidermoid parotid tumor [199]. Compared to MDX-210, MDX-447 appeared to have more side effects. This may be explained by high levels of EGF-R expression on normal tissue [5]. BsMAb continues to undergo clinical evaluations. It is important to understand the pharmacological and

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biological properties of these novel therapeutic proteins. To this author’s knowledge, 12 bsMAb have been tested in different phases of clinical trials (see Table 3). Eleven out of twelve are for cancer immunotherapy, the other is for pretargeted radioimmunotherapy for the treatment of small cell lung carcinoma. It is anticipated that other clinical applications of bsMAb will follow up in the future. Clinical data have shed light on a multitude of issues that underlay the progress toward a more advanced clinical stage. The most prominent clinical problems encountered by bsMAb-based therapeutics are immunogenicity from mouse-derived bsMAb, lack of therapeutic efficacy, toxicity associated with cytokine storm and unfavorable pharmacokinetics. HAMA response has been noted with the use of murine antibodies in 90% of patients after multiple doses, which leads to rapid clearance and inactivation of the antibodies administered. Clinical results show a clear benefit using local administration of bsMAb only or in combination with autologous effector cells and cytokines. However, they demonstrate no or inconsistent therapeutic efficacy by systemic administration. Direct intravenous administration of bsMAb can cause significant toxicity as a result of the release of cytokines such as IL-2, IL-6, IFNg and TNFa. Pharmacokinetic profile of bsMAb with serum half-lives of several hours seems less favorable than that of MAbs.

6. Conclusion With its unique two-arm structures, bsMAb provides powerful tools for targeted-delivering drugs, toxins, cytokines, enzymes, DNAs and radionuclides. Besides, bsMAb is able to activate the immune defense system by artificially combining humoral and cellular components to retarget them to tumors or viral infected cells. The unique biological properties of bsMAb warrant enthusiasm for their continued development. The recent FDA-approved MAb therapeutics also underscored the potentials of bsMAb. The extensive pre-clinical and clinical experiences have set the stage for the development of ideal bsMAb model in clinical applications: (1) it has to be a human or at least a humanized antibody to avoid immunogenicity. (2) it can be robustly

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mass-produced at a reasonable cost. (3) its size can be control-designed to obtain a favorable pharmacokinetic profile and an easy access to a specific target. Recombinant antibodies and their fragments have become the paradigm for meeting these requirements. Fledgling therapeutic applications of bsMAb will mature continuously as we constantly expand our knowledge about protein engineering, new disease targets, drug delivery systems and immune systems. For example, bsMAb cancer vaccine can be explored through activation of humoral or cellular components by retargeting FcR-positive antigen presenting cells. BsMAb-directed drug carriers such as liposomes, micelles or other polymeric drug delivery systems are also worthwhile to pursue. The dependence of tumor growth on the tumor’s blood supply also renders tumor endothelial cells an attractive target for therapeutic purposes. Cutting-edge molecular biology and protein engineering have added new dimensions to our quest for the far-reaching implications of these molecules. The recently completed human genome project has dramatically increased the number of potential therapeutic targets. BsMAb will definitely play a significant role in therapeutics directed toward some of these novel targets. BsMAbbased therapy, although holding great promises, does come with great cost. Patience and continual effort are needed to fulfill this once promising idea into one that provides cures for many devastating diseases.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10] [11]

[12] [13]

[14]

Acknowledgements The author would like to dedicate this article to his father, Professor G.Y. Cao, on his retirement from CAAS after a brilliant career in biological research. He also would like to thank Claire Mei and Jeff Sivik for reviewing the manuscript.

[15]

[16]

[17]

References [1] G. Kohler, C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256 (1975) 495–497. [2] A. Nisonoff, M.M. Rivers, Recombination of a mixture of

[18]

[19]

univalent antibody fragments of different specificity, Arch. Biochem. Biophys. 93 (1961) 460–462. M.R. Suresh, A.C. Cuello, C. Milstein, Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays, Proc. Natl. Acad. Sci. USA 83 (1986) 7989– 7993. Y. Cao, S. Christian, M.R. Suresh, Development of bsMAb anti-biotin 3 anti-HRPO as a universal immunoprobe for detecting biotinylated macromolecules, J. Immunol. Methods 220 (1998) 85–91. H.H. Van Ojik, T. Valerius, Preclinical and clinical data with bispecific antibodies recruiting myeloid effector cells for tumor therapy, Crit. Rev. Oncol. Hematol. 38 (2001) 47–61. S. Withoff, W. Helfrich, L.F.M.H. de Leij, G. Molema, Bi-specific antibody therapy for the treatment of cancer, Curr. Opin. Mol. Ther. 3 (2001) 53–62. R. Talac, H. Nelson, Current perspectives of bispecific antibody-based immunotherapy, J. Biol. Regul. Homeost. Agents 14 (2000) 175–181. A.B. van Spriel, H.H. van Ojik, J.G. van De Winkel, Immunotherapeutic perspective for bispecific antibodies, Immunol. Today 21 (2000) 391–397. B.J. Kroesen, W. Helfrich, G. Molema, L. de Leij, Bispecific antibodies for treatment of cancer in experimental animal models and man, Adv. Drug Deliv. Rev. 31 (1998) 105–129. H. Wang, Y. Liu, L. Wei, Y. Guo, Bi-specific antibodies in cancer therapy, Adv. Exp. Med. Biol. 465 (2000) 369–380. R. Koelemij, P.J. Kuppen, C.J. van de Velde, G.J. Fleuren, M. Hagenaars, A.M. Eggermont, Bispecific antibodies in cancer therapy, from the laboratory to the clinic, J. Immunother. 22 (1999) 514–524. D.M. Segal, G.J. Weiner, L.M. Weiner, Bispecific antibodies in cancer therapy, Curr. Opin. Immunol. 11 (1999) 558–562. B. Bodey, B.J. Bodey, S.E. Siegel, H.E. Kaiser, Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents, Curr. Pharm. Des 6 (2000) 261–276. Y. Cao, M.R. Suresh, Bispecific antibody as novel bioconjugates, Bioconjugate Chem. 9 (1998) 635–644. V. Schirrmacher, C. Haas, Modification of cancer vaccines by virus infection and attachment of bispecific antibodies. An effective alternative to somatic gene therapy, Adv. Exp. Med. Biol. 451 (1998) 251–257. C. Haas, C. Herold-Mende, R. Gerhards, V. Schirrmacher, An effective strategy of human tumor vaccine modification by coupling bispecific costimulatory molecules, Cancer Gene Ther. 6 (1999) 254–262. C. Haas, G. Strauss, G. Moldenhauer, R.M. Iorio, V. Schirrmacher, Bispecific antibodies increase T-cell stimulatory capacity in vitro of human autologous virus-modified tumor vaccine, Clin. Cancer Res. 4 (1998) 721–730. R. Mocikat, M. Selmayr, S. Thierfelder, H. Lindhofer, Trioma-based vaccination against B-cell lymphoma confers long-lasting tumor immunity, Cancer Res. 57 (1997) 2346– 2349. C.H. Ford, P.A. Osborne, B.G. Rego, A. Mathew, Bispecific antibody targeting of doxorubicin to carcinoembryonic an-

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

tigen-expressing colon cancer cell lines in vitro and in vivo, Int. J. Cancer 92 (2001) 851–855. D. Morelli, A. Sardini, E. Villa, M.L. Villa, S. Menard, M.I. Colnaghi, A. Balsari, Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin, Cancer Immunol. Immunother. 38 (1994) 171–177. V.S. Reddy, C.H. Ford, Production of hybrids secreting bispecific antibodies recognising CEA and doxorubicin, Anticancer Res. 13 (1993) 2077–2083. N.G. Papadopoulos, A.D. Gritzapis, G.V. Dedoussis, G. Spanakos, C.N. Baxevanis, M. Papamichail, Production and characterization of a monoclonal antibody against epirubicin, Hybridoma 14 (1995) 593–596. K. Affleck, M.J. Embleton, Monoclonal antibody targeting of methotrexate (MTX) against MTX-resistant tumour cell lines, Br. J. Cancer 65 (1992) 838–844. K. Kuus-Reichel, C. Knott, P. Sam-Fong, E. Petrella, J.R. Corvalan, Therapy of streptozotocin induced diabetes with a bifunctional antibody that delivers vinca alkaloids to IL-2 receptor positive cells, Autoimmunity 22 (1995) 173–181. K. Kuus-Reichel, C.L. Knott, P. Sam-Fong, R.A. Jue, D.G. Mackensen, J.R. Corvalan, Production and in vivo characterization of a bifunctional antibody (IVA039.1) with specificity for the mouse interleukin-2 receptor and vinca alkaloids, Hybridoma 13 (1994) 115–122. W. Smith, V.A. Gore, D.R. Brandon, D.N. Lynch, S.A. Cranstone, J.R. Corvalan, Suppression of well-established tumour xenografts by a hybrid-hybrid monoclonal antibody and vinblastine, Cancer Immunol. Immunother. 31 (1990) 157–163. J.R. Corvalan, W. Smith, V.A. Gore, D.R. Brandon, Specific in vitro and in vivo drug localization to tumour cells using a hybrid–hybrid monoclonal antibody recognising both carcinoembryonic antigen (CEA) and vinca alkaloids, Cancer Immunol. Immunother. 24 (1987) 133–137. M.V. Pimm, R.A. Robins, R.W. Baldwin, Capture of recombinant ricin A chain by a bispecific anti-RTA:anti-CEA monoclonal antibody pre-targeted to a human gastric carcinoma xenograft in nude mice, J. Cancer Res. Clin. Oncol. 118 (1992) 367–370. M.A. Bonardi, A. Bell, R.R. French, Initial experience in treating human lymphoma with a combination of bispecific antibody and saporin, Int. J. Cancer Suppl. 7 (1992) 73–77. M.A. Bonardi, R.R. French, P. Amlot, G. Gromo, D. Modena, M.J. Glennie, Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing, Cancer Res. 53 (1993) 3015–3021. R.R. French, A.J. Bell, T.J. Hamblin, A.L. Tutt, M.J. Glennie, Response of B-cell lymphoma to a combination of bispecific antibodies and saporin, Leuk. Res. 20 (1996) 607–617. R.R. French, T.J. Hamblin, A.J. Bell, A.L. Tutt, M.J. Glennie, Treatment of B-cell lymphomas with combination of bispecific antibodies and saporin, Lancet 346 (1995) 223–224.

191

[33] S. Sforzini, A. Bolognesi, R. Meazza, Differential sensitivity of CD30 1 neoplastic cells to gelonin delivered by antiCD30 / anti-gelonin bispecific antibodies, Br. J. Haematol. 90 (1995) 572–577. [34] B. Robert, J.P. Mach, J.C. Mani, M. Ychou, S. Folli, J.C. Artus, A. Pelegrin, Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha, Cancer Res. 56 (1996) 4758–4765. [35] K.D. Bagshawe, S.K. Sharma, P.J. Burke, R.G. Melton, R.J. Knox, Developments with targeted enzymes in cancer therapy, Curr. Opin. Immunol. 11 (1999) 579–583. [36] J. Bhatia, S.K. Sharma, K.A. Chester, Catalytic activity of an in vivo tumor targeted anti-CEA scFv / carboxypeptidase G2 fusion protein, Int. J. Cancer 85 (2000) 571–577. [37] K. De Sutter, W.A. Fiers, Bifunctional murine / human chimeric antibody with one antigen-binding arm replaced by bacterial beta-lactamase, Mol. Immunol. 31 (1994) 261–267. [38] U. Sahin, F. Hartmann, P. Senter, C. Pohl, A. Engert, V. Diehl, M. Pfreundschuh, Specific activation of the prodrug mitomycin phosphate by a bispecific anti-CD30 / anti-alkaline phosphatase monoclonal antibody, Cancer Res. 50 (1990) 6944–6948. [39] E.E. Branscomb, M.S. Runge, C.E. Savard, K.M. Adams, G.R. Matsueda, E. Haber, Bispecific monoclonal antibodies produced by somatic cell fusion increase the potency of tissue plasminogen activator, Thromb. Haemost. 64 (1990) 260–266. [40] J.R. Charpie, M.S. Runge, G.R. Matsueda, E. Haber, A bispecific antibody enhances the fibrinolytic potency of single-chain urokinase, Biochemistry 29 (1990) 6374–6378. [41] Y. Imura, J.M. Stassen, T. Kurokawa, S. Iwasa, H.R. Lijnen, D. Collen, Thrombolytic and pharmacokinetic properties of an immunoconjugate of single-chain urokinase-type plasminogen activator (u-PA) and a bispecific monoclonal antibody against fibrin and against u-PA in baboons, Blood 79 (1992) 2322–2329. [42] M.R. Patrick, K.A. Chester, G.A. Pietersz, In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody, Cancer Immunol. Immunother. 46 (1998) 229–237. [43] J.F. Gestin, A. Loussouarn, M. Bardies, Two-step targeting of xenografted colon carcinoma using a bispecific antibody and 188Re-labeled bivalent hapten: biodistribution and dosimetry studies, J. Nucl. Med. 42 (2001) 146–153. [44] E. Gautherot, E. Rouvier, L. Daniel, Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and 131I-labeled bivalent hapten, J. Nucl. Med. 41 (2000) 480–487. [45] J. Barbet, F. Kraeber-Bodere, J.P. Vuillez, E. Gautherot, E. Rouvier, J.F. Chatal, Pretargeting with the affinity enhancement system for radioimmunotherapy, Cancer Biother. Radiopharm. 14 (1999) 153–166. [46] L.E. Dillehay, R. Mayer, Y.G. Zhang, Y. Shao, S.Y. Song, D.G. Mackensen, J.R. Williams, Prediction of tumor response to experimental radioimmunotherapy with 90Y in nude mice, Int. J. Radiat. Oncol. Biol. Phys. 33 (1995) 417–427.

192

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197

[47] M.H. Kranenborg, O.C. Boerman, J.C. Oosterwijk-Wakka, M.C. de Weijert, F.H. Corstens, E. Oosterwijk, Development and characterization of anti-renal cell carcinoma 3 antichelate bispecific monoclonal antibodies for two-phase targeting of renal cell carcinoma, Cancer Res. 55 (1995) 5864–5867. [48] D.A. Goodwin, C.F. Meares, N. Watanabe, Pharmacokinetics of pretargeted monoclonal antibody 2D12.5 and 88Y-Janus2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecanetetraaceti c acid (DOTA) in BALB / c mice with KHJJ mouse adenocarcinoma: a model for 90Y radioimmunotherapy, Cancer Res. 54 (1994) 5937–5946. [49] L. Liu, R.F. Barth, D.M. Adams, A.H. Soloway, R.A. Reisfeld, Bispecific antibodies as targeting agents for boron neutron capture therapy of brain tumors, J. Hematother. 4 (1995) 477–483. [50] R.H. Pak, F.J. Primus, K.J. Rickard-Dickson, L.L. Ng, R.R. Kane, M.F. Hawthorne, Preparation and properties of nidocarborane-specific monoclonal antibodies for potential use in boron neutron capture therapy for cancer, Proc. Natl. Acad. Sci. USA 92 (1995) 6986–6990. [51] J.G. Jurcic, M.R. McDevitt, G. Sgouros, A. Ballangrud, R.D. Finn, D. Ma, K. Hamacher, M.W. Geerlings, J.L. Humm, M.W. Brechbiel, Phase I trial of targeted alpha-particle therapy for myeloid leukemias with bismuth-213-HuM195 (anti-CD3), Proc. Am. Soc. Clin. Oncol. 18 (1999) 7. [52] T.M. Behr, M. Behe, M.G. Stabin, E. Wehmann, C. Apostolidis, R. Molinet, F. Strutz, A. Fayyazi, E. Wieland, S. Gratz, High-linear energy transfer (LET) alpha versus lowLET beta emitted in radioimmunotherapy of solid tumors: therapeutics efficacy and dose-limiting toxicity of 213Biversus 90Y-labeled CO10-1A Fab9 fragments in a human colonic cancer model, Cancer Res. 59 (1999) 2635–2643. [53] P.L. Weiden, H.B. Breitz, Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin’s lymphoma (NHL), Crit. Rev. Oncol. Hematol. 40 (2001) 37–51. [54] S. Shresta, C.T. Pham, D.A. Thomas, T.A. Graubert, T.J. Ley, How do cytotoxic lymphocytes kill their targets?, Curr. Opin. Immunol. 10 (1998) 581–587. [55] S. Canevari, S. Menard, D. Mezzanzanica, S. Miotti, S.M. Pupa, A. Lanzavecchia, M.I. Colnaghi, Anti-ovarian carcinoma anti-T3 heteroconjugates or hybrid antibodies induce tumor cell lysis by cytotoxic T-cells, Int. J. Cancer Suppl. 2 (1998) 18–21. [56] J. De Jonge, C. Heirman, M. De Veerman, S. Van Meirvenne, M. Moser, O. Leo, K. Thielemans, In vivo retargeting of T cell effector function by recombinant bispecific single chain Fv (anti-CD3 3 anti-idiotype) induces long-term survival in the murine BCL1 lymphoma model, J. Immunol. 161 (1998) 1454–1461. [57] B.K. Link, S.A. Kostelny, M.S. Cole, W.P. Fusselman, J.Y. Tso, G.J. Weiner, Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen-independent mechanisms, Int. J. Cancer 77 (1998) 251–256. [58] P. Holliger, O. Manzke, R. Hawkins, M. Span, B. Fleischmann, L. Qinghua, V. Diehl, O. Cochet, G. Winter, H. Bohlen, CEA-specific activation in colon carcinoma by anti-

[59]

[60]

[61]

[62] [63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

CD3 3 anti-CEA bispecific diabodies and B7 3 anti-CEA bispecific fusion proteins, Cancer Res. 59 (1999) 2909– 2916. O. Manzke, K.J. Fitzgerald, P. Holliger, J. Klock, M. Span, B. Fleischmann, J. Hescheler, L. Qinghua, K.S. Johnson, V. Diehl, H.R. Hoogenboom, H. Bohlen, Anti-CD3 3 anti-nitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T-cells to tumors, Int. J. Cancer 82 (1999) 700–708. P. Holliger, J. Brissinck, R.L. Williams, K. Thielemans, G. Winter, Specific killing of lymphoma cells by cytotoxic T-cells mediated by a bispecific diabody, Protein Eng. 9 (1996) 299–305. L. Davico Bonino, L.B. De Monte, G.C. Spagnoli, Bispecific monoclonal antibody anti-CD3 3 anti-tenascin: an immunotherapeutic agent for human glioma, Int. J. Cancer 61 (1995) 509–515. C.A. Chambers, J.P. Allison, Costimulatory regulation of T-cell function, Curr. Opin. Cell Biol. 11 (1999) 203–210. C. Demanet, J. Brissinck, J. de Jonge, K. Thielemans, Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation, Blood 87 (1996) 4390–4398. P.J. Noel, L.H. Boise, C.B. Thompson, Regulation of T cell activation by CD28 and CTLA4, Adv. Exp. Med. Biol. 406 (1996) 209–217. R.L. Bolhuis, E. Sturm, E. Braakman, T cell targeting in cancer therapy, Cancer Immunol. Immunother. 34 (1991) 1–8. A. Tutt, G.T. Stevenson, M.J. Glennie, Trispecific F(ab9)3 derivatives that use cooperative signaling via the TCR / CD3 complex and CD2 to activate and redirect resting cytotoxic T cells, J. Immunol. 147 (1991) 60–69. S. Ferrini, I. Prigione, S. Mammoliti, M.I. Colnaghi, S. Menard, A. Moretta, L. Moretta, Retargeting of T-cell receptor gamma / delta 1 lymphocytes against tumor cells by bispecific monoclonal antibodies, Int. J. Cancer Suppl. 4 (1989) 53–55. I.G. Barr, H.R. MacDonald, F. Buchegger, V. von Fliedner, Lysis of tumor cells by the retargeting of murine cytolytic T lymphocytes with bispecific antibodies, Int. J. Cancer 40 (1987) 423–429. Y.M. Deo, R.F. Graziano, R. Repp, J.G.J. van de Winkel, Clinical significance of IgG Fc receptors and FcgR-directed immunotherapies, Immunol. Today 18 (1997) 126–135. I. Heijnen, L.J. Rijks, A. Schiel, B. Stockmeyer, H.H. van Ojik, M. Dechant, T. Valerius, T. Keler, A.L. Tutt, M.J. Glennie, E.A. van Royen, P.J. Capel, J.G. van de Winkel, Generation of HER-2 / neu-specific cytotoxic neutrophils in vivo: Efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcg receptor I bispecific antibodies, J. Immunol. 159 (1997) 5629–5639. R. Repp, T. Valerius, G. Wiegland, W. Becker, H. Steininger, Y.M. Deo, G. Helm, M. Gramatzki, J.G. van de Winkel, N. Lang, J.R. Kalden, G-CSF-Stimulated PMN in immunotherapy of breast cancer with a bispecific antibody to FcgRI

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83] [84]

and to HER-2 / neu (MDX-210), J. Hematother. 4 (1995) 415. T. Keler, R.F. Graziano, A. Mandal, P.K. Wallace, J. Fisher, P.M. Guyre, M.W. Fanger, Y.M. Deo, Bispecific antibodydependent cellular cytotoxicity of HER2 / neu-overexpressing tumor cells by Fcg receptor type I-expressing effector cells, Cancer Res. 57 (1997) 4008–4014. T. Keler, P.K. Wallace, L.A. Vitale, C. Russoniello, K. Sundarapandiyan, R.F. Graziano, Y.M. Deo, Differential effect of cytokine treatment on FcaRI and FcgRI mediated tumorcytotoxicity by mionocyte, J. Immunol. 164 (2000) 5746–5752. R.F. Graziano, J. Goldstein, K. Sundrapandiyan, C. Somasundaram, T. Keler, Y.M. Deo, Targeting tumor cell destruction with CD64-directed bispecific fusion proteins, Cancer Immunol. Immunother. 45 (1997) 124–127. J. Goldstein, R.F. Graziano, K. Sundarapandiyan, C. Somasundaram, Y.M. Deo, Cytolytic and cytostatic properties of an anti-human FcgRI (CD64) 3 epidermal growth factor bispecific fusion protein, J. Immunol. 158 (1997) 872–879. C. Russoniello, C. Somasundaram, J. Schlom, Y.M. Deo, T. Keler, Characterization of a novel bispecific antibody that mediates Fcg receptor type I-dependent killing of tumorassociated glycoprotein-72-expressing tumor cells, Clin. Cancer Res. 4 (1998) 2237–2243. R.T. Curnow, Clinical experience with CD64-directed immunotherapy, Cancer Immunol. Immunother. 92 (1997) 210–215. S.T. Hsieh-Ma, A.M. Eaton, T. Shi, D.B. Ring, In vitro cytotoxic targeting by human mononuclear cells and bispecific antibody 2B1, recognizing c-erbB-2 protooncogene product and Fc gamma receptor III, Cancer Res. 52 (1992) 6832–6839. L.M. Weiner, J.I. Clark, D.B. Ring, R.K. Alpaugh, Clinical development of 2B1, a bispecific murine monoclonal antibody targeting c-erbB-2 and Fc gamma RIII, J. Hematother. 4 (1995) 453–456. T. Valerius, B. Stockmeyer, A.B. van Spriel, R.F. Graziano, I.E. van den Herik-Oudijk, R. Repp, Y.M. Deo, J. Lund, J.R. Kalden, M. Gramatzki, J.G.J. van de Winkel, FcaRI (CD89) as a novel trigger molecule for bispecific antibody therapy, Blood 90 (1997) 4485–4492. T.M. Deo, K. Sundarapandiyan, T. Keler, R.F. Graziano, Bispecific molecules directed to the Fc receptor for IgA (FcaRI, CD89) and tumor antigens efficiently promote cellmediated cytotoxicity of tumor targets in whole blood, J. Immunol. 160 (1998) 1677–1686. C. Somasundaram, R. Arch, S. Matzku, M. Zoller, Development of a bispecific F(ab9)2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity, Cancer Immunol. Immunother. 42 (1996) 343–350. L.L. Lanier, NK cell receptors, Annu. Rev. Immunol. 16 (1998) 359–393. W.M. Yokoyama, Natural killer cell receptors, Curr. Opin. Immunol. 10 (1998) 298–305.

193

[85] P.D. Stahl, R.A. Ezekowitz, The mannose receptor is a pattern recognition receptor involved in host defense, Curr. Opin. Immunol. 10 (1998) 50–55. [86] N.C. Franc, K. White, R.A. Ezekowitz, Phagocytosis and development: back to the future, Curr. Opin. Immunol. 11 (1999) 47–52. [87] J.A. Hoffmann, F.C. Kafatos, C.A. Janeway, R.A. Ezekowitz, Phylogenetic perspectives in innate immunity, Science 284 (1999) 1313–1318. [88] A. Cesano, S. Visonneau, S. Deaglio, F. Malavasi, D. Santoli, Role of CD38 and its ligand in the regulation of MHCnonrestricted cytotoxic T cells, J. Immunol. 160 (1998) 1106–1115. [89] S. Ohta, H. Tsukamoto, K. Watanabe, Tumor-associated glycoantigen, sialyl Lewis (a) as a target for bispecific antibody-directed adoptive tumor immunotherapy, Immunol. Lett. 44 (1995) 35–40. [90] R.L. Bolhuis, C.H. Lamers, S.H. Goey, Adoptive immunotherapy of ovarian carcinoma with bs-MAb-targeted lymphocytes: a multicenter study, Int. J. Cancer Suppl. 7 (1992) 78–81. [91] S. Canevari, G. Stoter, F. Arienti, Regression of advanced ovarian carcinoma by intraperitoneal treatment with autologous T lymphocytes retargeted by a bispecific monoclonal antibody, J. Natl. Cancer Inst. 87 (1995) 1463–1469. [92] J. Van Dijk, S.T. Zegveld, G.J. Fleuren, S.O. Warnaar, Localization of monoclonal antibody G250 and bispecific monoclonal antibody CD3 / G250 in human renal-cell carcinoma xenografts: relative effects of size and affinity, Int. J. Cancer 48 (1991) 738–743. [93] A. Azuma, H. Yagita, H. Matsuda, K. Okumura, H. Niitani, Induction of intercellular adhesion molecule 1 on small cell lung carcinoma cell lines by gamma-interferon enhances spontaneous and bispecific anti-CD3 3 antitumor antibodydirected lymphokine activated killer cell cytotoxicity, Cancer Res. 52 (1992) 4890–4894. [94] P.M. Anderson, W. Crist, D. Hasz, A.J. Carroll, D.E. Myers, F.M. Uckun, G19.4 (a-CD3) 3 B43 (a-CD19) monoclonal antibody heteroconjugate triggers CD19 antigen-specific lysis of t(4;11) acute lymphoblastic leukemia cells by activated CD3 antigen-positive cytotoxic T cells, Blood 80 (1992) 2826–2834. [95] G.C. De Gast, A.A. Van Houten, I.A. Haagen, S. Klein, R.A. de Weger, A. van Dijk, J. Phillips, M. Clark, B.J. Bast, Clinical experience with CD3 3 CD19 bispecific antibodies in patients with B cell malignancies, J. Hematother. 4 (1995) 433–437. [96] T. Kaneko, Y. Fusauchi, Y. Kakui, A bispecific antibody enhances cytokine-induced killer-mediated cytolysis of autologous acute myeloid leukemia cells, Blood 81 (1993) 1333–1341. [97] F.H. Valone, P.A. Kaufman, P.M. Guyre, Clinical trials of bispecific antibody MDX-210 in women with advanced breast or ovarian cancer that overexpresses HER-2 / neu, J. Hematother. 4 (1995) 471–475. [98] J. Berg, E. Lotscher, K.S. Steimer, D.J. Capon, J. Baenziger, H.M. Jack, M. Wabl, Bispecific antibodies that mediate

194

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197

killing of cells infected with human immunodeficiency virus of any strain, Proc. Natl. Acad. Sci. USA 88 (1991) 4723– 4727. [99] S.M. Chamow, D. Zhang, X.Y. Tan, A humanized, bispecific immunoadhesin-antibody that retargets CD3 1 effectors to kill HIV-1-infected cells, J. Hematother. 4 (1995) 439–446. [100] S.M. Chamow, D.Z. Zhang, X.Y. Tan, A humanized, bispecific immunoadhesin-antibody that retargets CD3 1 effectors to kill HIV-1-infected cells, J. Immunol. 153 (1994) 4268–4280. [101] R.K. Jain, Delivery of molecular and cellular medicine to solid tumors, Adv. Drug Deliv. Rev. 26 (1997) 71–90. [102] G. Molema, B.J. Kroesen, W. Helfrich, D.K.F. Meijer, L.F.M.H. de Leij, The use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy, J. Controlled Release 64 (2000) 229–239. [103] F.J. Burrows, Y. Watanabe, P.E. Thorpe, A murine model for antibody-directed targeting to vascular endothelial cells in solid tumors, Cancer Res. 52 (1992) 5954–5962. [104] X. Huang, G. Molema, S. King, L. Watkins, T.S. Edgington, P.E. Thorpe, Tumor infaction in mice by antibodydirected targeting of tissue factor to tumor vasculature, Science 275 (1997) 547–550. [105] D.M. Nettelbeck, D.W. Miller, V. Jerome, Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105), Mol. Ther. 3 (2001) 882– 891. [106] P.N. Reynolds, K.R. Zinn, V.D. Gavrilyuk, A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo, Mol. Ther. 2 (2000) 562– 578. [107] F.J. Kelly, C.R. Miller, D.J. Buchsbaum, J. Gomez-Navarro, M.N. Barnes, R.D. Alvarez, D.T. Curiel, Selectivity of TAG-72-targeted adenovirus gene transfer to primary ovarian carcinoma cells versus autologous mesothelial cells in vitro, Clin. Cancer Res. 6 (2000) 4323–4333. [108] J. Grill, V.W. Van Beusechem, P. van Der Valk, Combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids, Clin. Cancer Res. 7 (2001) 641–650. [109] C. Ebbinghaus, A. Al-Jaibaji, E. Operschall, A. Schoffel, I. Peter, U.F. Greber, S. Hemmi, Functional and selective targeting of adenovirus to high-affinity Fcgamma receptor I-positive cells by using a bispecific hybrid adapter, J. Virol. 75 (2001) 480–489. [110] H.J. Haisma, J. Grill, D.T. Curiel, S. Hoogeland, V.W. van Beusechem, H.M. Pinedo, W.R. Gerritsen, Targeting of adenoviral vectors through a bispecific single-chain antibody, Cancer Gene Ther. 7 (2000) 901–904. [111] T.J. Wickham, D.M. Segal, P.W. Roelvink, M.E. Carrion, A. Lizonova, G.M. Lee, I. Kovesdi, Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies, J. Virol. 70 (1996) 6831–6838. [112] C. Haas, C. Herold-Mende, R. Gerhards, V. Schirrmacher, An effective strategy of human tumor vaccine modification by coupling bispecific costimulatory molecules, Cancer Gene Ther. 6 (1999) 254–262.

[113] Y.J. Guo, X.Y. Che, F. Shen, Effective tumor vaccines generated by in vitro modification of tumor cells with cytokines and bispecific monoclonal antibodies, Nat. Med. 3 (1997) 451–455. [114] D.C. Reason, M.Y. Kitamura, A.H. Lucas, Induction of a protective human polysaccharide-specific antibody response in hu-PBL SCID mice by idiotypic vaccination, J. Immunol. 152 (1994) 5009–5013. [115] R. Mocikat, M. Selmayr, S. Thierfelder, H. Lindhofer, Trioma-based vaccination against B-cell lymphoma confers long-lasting tumor immunity, Cancer Res. 57 (1997) 2346– 2349. [116] S. Wu, J. Ma, X. Che, Treatment of hepatocellular carcinoma with the cellular tumor vaccines generated by in vitro modification of tumor cells with non gene transfer approach, Adv. Exp. Med. Biol. 451 (1998) 283–293. [117] V. Schirrmacher, C. Haas, Modification of cancer vaccines by virus infection and attachment of bispecific antibodies. An effective alternative to somatic gene therapy, Adv. Exp. Med. Biol. 451 (1998) 251–257. [118] C. Haas, G. Strauss, G. Moldenhauer, R.M. Iorio, V. Schirrmacher, Bispecific antibodies increase T-cell stimulatory capacity in vitro of human autologous virusmodified tumor vaccine, Clin. Cancer Res. 4 (1998) 721– 730. [119] P.K. Wallace, K.Y. Tsang, J. Goldstein, P. Correale, T.M. Jarry, J. Schlom, P.M. Guyre, M.S. Ernstoff, M.W. Fanger, Exogenous antigen targeted to FcgRI on myeloid cells is presented in association with MHC class I, J. Immunol. Methods 248 (2001) 183–194. [120] D.M. Segal, G.J. Weiner, L.M. Weiner, Introduction: bispecific antibodies, J. Immunol. Methods 248 (2001) 1–6. [121] M.B. Brenner, I.S. Trowbridge, J.L. Strominger, Crosslinking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit, Cell 40 (1985) 183–190. [122] M.J. Glennie, H.M. McBride, A.T. Worth, G.T. Stevenson, Preparation and performance of bispecific F(ab9 gamma)2 antibody containing thioether-linked Fab9 gamma fragments, J. Immunol. 139 (1987) 2367–2375. [123] M.R. Shalaby, H.M. Shepard, L. Presta, M.L. Rodrigues, P.C. Beverley, M. Feldmann, P. Carter, Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene, J. Exp. Med. 175 (1992) 217–225. [124] A. Tutt, J. Greenman, G.T. Stevenson, M.J. Glennie, Bispecific F(ab9gamma)3 antibody derivatives for redirecting unprimed cytotoxic T cells, Eur. J. Immunol. 21 (1991) 1351–1358. [125] D.M. Segal, J.H. Qian, M.A. Garrido, P. Perez, D.F. Winkler, J.R. Wunderlich, D.P. Snider, M.J. Valdayo, J.A. Titus, Targeting of cytotoxic cells against tumors with heterocrosslinked, bispecific antibodies, Princess Takamatsu Symposia 19 (1988) 323–331. [126] G. Jung, U. Freimann, Z. Von Marschall, R.A. Reisfeld, W. Wilmanns, Target cell-induced T cell activation with biand trispecific antibody fragments, Eur. J. Immunol. 21 (1991) 2431–2435.

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197 [127] J. Van Dijk, T. Tsuruo, D.M. Segal, R.L. Bolhuis, R. Colognola, R.J. van de Griend, G.J. Fleuren, S.O. Warnaar, Bispecific antibodies reactive with the multidrug-resistancerelated glycoprotein and CD3 induce lysis of multidrugresistant tumor cells, Int. J. Cancer 44 (1989) 738–743. [128] Z.N. Chaudri, M. Bartlet-Jones, G. Panayotou, T. Klonisch, I.M. Roitt, T. Lund, P.J. Delves, Dual specificity antibodies using a double-stranded oligonucleotide bridge, FEBS Lett. 450 (1999) 23–26. [129] R.G. Cotton, C. Milstein, Letter: Fusion of two immunoglobulin-producing myeloma cells, Nature 244 (1973) 42– 43. [130] M.R. Suresh, A.C. Cuello, C. Milstein, Bispecific monoclonal antibodies from hybrid hybridomas, Methods Enzymol. 121 (1986) 210–228. [131] O. Nolan, O.R. Kennedy, Bifunctional antibodies: concept, production and applications, Biochim. Biophys. Acta 1040 (1990) 1–11. [132] W.B. De Lau, A.E. Van Loon, K. Heije, D. Valerio, B.J. Bast, Production of hybrid hybridomas based on HAT(s)neomycin(r) double mutants, J. Immunol. Methods 117 (1989) 1–8. [133] R. Bos, W. Nieuwenhuizen, Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines, Hybridoma 11 (1992) 41–51. [134] L. Karawajew, O. Behrsing, G. Kaiser, B. Micheel, Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP), J. Immunol. Methods 111 (1988) 95–99. [135] L. Karawajew, B. Micheel, O. Behrsing, M. Gaestel, Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter, J. Immunol. Methods 96 (1987) 265–270. [136] Y. Cao, T. Vinayagamoorthy, A.A. Noujaim, M.R. Suresh, A rapid non-selective method to generate quadromas by microelectrofusion, J. Immunol. Methods 187 (1995) 1–7. [137] F.T. Kreutz, D.Z. Xu, M.R. Suresh, A new method to generate quadromas by electrofusion and FACS sorting, Hybridoma 17 (1998) 267–273. [138] S. Songsivilai, P.J. Lachmann, Bispecific antibody: a tool for diagnosis and treatment of disease, Clin. Exp. Immunol. 79 (1990) 315–321. [139] A. Pluckthun, P. Pack, New protein engineering approaches to multivalent and bispecific antibody fragments, Immunotechnology 3 (1997) 83–105. [140] P. Carter, Bispecific human IgG by design, J. Immunol. Methods 248 (2001) 7–15. [141] P.J. Hudson, Recombinant antibody constructs in cancer therapy, Curr. Opin. Immunol. 11 (1999) 548–557. [142] S. Songsivilai, P.M. Clissold, P.J. Lachmann, A novel strategy for producing chimeric bispecific antibodies by gene transfection, Biochem. Biophys. Res. Commun. 164 (1989) 271–276. [143] D.M. Kranz, M. Gruber, E.R. Wilson, Properties of bispecific single chain antibodies expressed in Escherichia coli, J. Hematother. 4 (1995) 403–408.

195

[144] S. Thirion, K. Motmans, H. Heyligen, J. Janssens, J. Raus, C. Vandevyver, Mono- and bispecific single-chain antibody fragments for cancer therapy, Eur. J. Cancer Prev. 5 (1996) 507–511. [145] M. Gruber, B.A. Schodin, E.R. Wilson, D.M. Kranz, Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli, J. Immunol. 152 (1994) 5368–5374. [146] W.D. Mallender, S.T. Ferreira, E.W. Voss, T. Coelho-Sampaio, Inter-active-site distance and solution dynamics of a bivalent-bispecific single-chain antibody molecule, Biochemistry 33 (1994) 10100–10108. [147] J.B. Ridgway, L.G. Presta, P. Carter, ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Eng. 9 (1996) 617–621. [148] A.M. Merchant, Z.P. Zhu, J.Q. Yuan, A. Goddard, C.W. Adams, L.G. Presta, P. Carter, An efficient route to human bispecific IgG, Nature Biotechnol. 16 (1997) 677–681. [149] S.A. Kostelny, M.S. Cole, J.Y. Tso, Formation of a bispecific antibody by the use of leucine zippers, J. Immunol. 148 (1992) 1547–1553. [150] J. De Kruif, T. Logtenberg, Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library, J. Biol. Chem. 271 (1996) 7630–7634. [151] M. Rheinnecker, C. Hardt, L.L. Iiag, P. Kufer, R. Gruber, A. Hoess, A. Lupas, C. Rottenberger, A. Pluckthun, P. Pack, Multivalent antibody fragments with high functional affinity for a tumor associated carbohydrate antigen, J. Immunol. 157 (1996) 2989–2997. [152] L.A. Pearce, G.W. Oddie, G. Coia, A.A. Kortt, P.J. Hudson, G.G. Lilley, Linear gene fusion of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents, Biochem. Mol. Biol. Int. 42 (1997) 1179–1188. [153] K.M. Muller, K.M. Arndt, A. Pluckthun, A dimeric bispecific miniantibody combines two specificities with avidity, FEBS Lett. 432 (1998) 45–49. [154] M. Alt, R. Muller, R.E. Kontermann, Novel tetravalent and bispecific IgG-like antibody molecules combining singlechain diabodies with the immunoglobulin g1 Fc or CH3 region, FEBS Lett. 454 (1999) 90–94. [155] P. Holliger, T. Prospero, G. Winter, ‘Diabodies’: small bivalent and bispecific antibody fragments, Proc. Natl. Acad. Sci. USA 90 (1993) 6444–6448. [156] P. Holliger, O. Manzke, R. Hawkins, M. Span, B. Fleischmann, L. Qinghua, V. Diehl, O. Cochet, G. Winter, H. Bohlen, CEA-specific activation in colon carcinoma by anti-CD3 3 anti-CEA bispecific diabodies and B7 3 antiCEA bispecific fusion proteins, Cancer Res. 59 (1999) 2909–2916. [157] O. Manzke, K.J. Fitzgerald, P. Holliger, J. Klock, M. Span, B. Fleischmann, J. Hescheler, L. Qinghua, K.S. Johnson, V. Diehl, H.R. Hoogenboom, H. Bohlen, Anti-CD3 3 antinitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T-cells to tumors, Int. J. Cancer 82 (1999) 700–708.

196

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197

[158] A. Todorovska, R.C. Roovers, O. Dolezal, A.A. Kortt, H.R. Hoogenboom, P.J. Hudson, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol. Methods 248 (2001) 47–66. [159] K. Fitzgerald, P. Holliger, G. Winter, Improved tumor targeting by disulfide-stabilised diabodies expressed in Pichia pastoris, Protein Eng. 10 (1997) 1221–1225. [160] G.P. Adams, R. Schier, A.M. McCall, R.S. Crawford, E.J. Wolf, L.M. Weiner, J.D. Marks, Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2 / neu, Br. J. Cancer 77 (1998) 1405–1412. [161] B.T. McGuinness, G. Walter, K. Fitzgerald, P. Schuler, W. Mahoney, A.R. Duncan, H.R. Hoogenboom, Phage diabody repertoires for selection of large numbers of bispecific antibody fragments, Natl. Biotechnol. 14 (1996) 1149– 1153. [162] M.S. Hayden, L.K. Gilliland, J.A. Ledbetter, Antibody engineering, Curr. Opin. Immunol. 9 (1997) 201–212. [163] B. Cochlovius, S.M. Kipriyanov, M.J. Stassar, J. Schuhmacher, A. Benner, G. Moldenhauer, M. Little, Cure of Burkitt’s lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 3 CD19 tandem diabody and CD28 costimulation, Cancer Res. 60 (2000) 4336–4341. [164] J. Kriangkum, B. Xu, L.P. Nagata, R.E. Fulton, M.R. Suresh, Bispecific and bifunctional single chain recombinant antibodies, Biomol. Eng. 18 (2001) 31–40. [165] K. James, G.T. Bell, Human monoclonal antibody production: current status and future prospects, J. Imunol. Methods 100 (1987) 5–40. [166] S.L. Morrison, M.J. Johnson, L.A. Herzenberg, V.T. Oi, Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains, Proc. Natl. Acad. Sci. USA 81 (1984) 6851–6855. [167] G.L. Boulianne, N. Hozumi, M.J. Shulman, Production of functional chimeric mouse / human antibody, Nature 312 (1984) 643–646. [168] S.L. Morrison, In vitro antibodies: strategies for production and application, Ann. Rev. Immunol. 10 (1992) 239–265. [169] H. De Haard, P. Henderikx, H.R. Hoogenboom, Creating and engineering human antibodies for immunotherapy, Adv. Drug Deliv. Rev. 31 (1998) 5–31. [170] A. Jakobovits, Production and selection of antigen-specific fully human monoclonal antibodies from mice engineered with human Ig loci, Adv. Drug Deliv. Rev. 3 (1998) 33–42. [171] D. Fishwild, S. O’Donnell, T. Bengoechea, D. Hudson, F. Harding, S. Bernhard, D. Jones, R.M. Kay, K. Higgins, S. Schramm, N. Lonberg, High-avidity human IgG K monoclonal antibodies from a novel strain of minilocus transgenic mice, Nat. Biotechnol. 14 (1996) 845–851. [172] N. Lonberg, L. Taylor, F. Harding, M. Trounstine, K. Higgins, S. Schramm, C. Kuo, R. Mashayekh, K. Wymore, J. McCabe, D. Munoz-O’Regan, S. O’Donnell, E.S.G. Lapachet, T. Bengoechea, D. Fishwild, C. Carmack, R. Kay, D. Huszar, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368 (1994) 856–859.

[173] S. Morrison, Success in specification, Nature 368 (1994) 812–813. [174] M. Neuberger, Generating high-avidity human Mabs in mice, Nat. Biotechnol. 14 (1996) 826. [175] K. Tomizuka, T. Shinohara, H. Yoshida, H. Uejima, A. Ohguma, S. Tanaka, K. Sato, M. Oshimura, I. Ishida, Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and k loci and expression of fully human antibodies, Proc. Natl. Acad. Sci. USA 97 (2000) 722–727. [176] C.A.K. Borrebaeck, Human monoclonal antibodies: The emperor’s new clothes?, Nat. Biotech. 17 (1999) 621. [177] T.J. Vaughan, J.K. Osbourn, P.R. Tempest, Human antibodies by design, Nat. Biotech. 16 (1998) 535–539. [178] R.F. Graziano, P.R. Tempest, P. White, T. Keler, Y.M. Deo, H. Ghebremariam, K. Coleman, L.C. Pfferkorn, M.W. Fanger, P.M. Guyre, Construction and characterization of a humanized anti-g-immunoglobulin receptor type I (FcgRI) monoclonal antibody, J. Immunol. 155 (1995) 4996–5002. [179] J. Goldstein, J.L. Boxter, J. Zhang, Fully human singlechain bispecific antibodies directed to the Fc receptor for IgA (FcaRI, CD89) and tumor antigens promote cellmediated cytotoxicity of tumor targets, Proc. Am. Ass. Cancer Res. 41 (2000) 286. [180] M. Clark, Antibody humanization: a case of the ‘‘Emperior’s new clothes’’?, Immunol. Today 21 (2000) 397– 402. [181] R.L.H. Bolhuis, C.H.J. Lamers, S.H. Goey, A.M.M. Eggermont, J.B.M.Z. Trimbos, Adoptive immunotherapy of ovarian carcinoma with bs-MAb-targeted lymphocytes—a multicenter study, Int. J. Cancer, Suppl. 7 (1992) 78–81. [182] T. Bauknecht, J. Hernando, Z. Rajabi, Phase I / II trial of adoptive immunotherapy with bispecific antibodies (OC / TR: MOV18 / CD3) in recurrent ovarian cancer, Gyn. Oncol. 60 (1996) 130–131. [183] R.M. Luiten, S.O. Warnaar, D. Sanborn, Chimeric bispecific OC / TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients, J. Immunother. 20 (1997) 496–504. [184] P.J. Hudson, Recombinant antibodies: a novel approach to cancer diagnosis and therapy, Exp. Opin. Invest. Drugs 9 (2000) 1231–1242. [185] B.J. Kroesen, A. Ter Haar, H. Spakman, P. Willemse, D.T. Sleijfer, E.G. de Vries, N.H. Mulder, H.H. Berendsen, P.C. Limburg, T.H. The, Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells, Cancer Immunol. Immunother. 37 (1993) 400–407. [186] B.K. Link, H. Wang, J.C. Byrd, J.P. Leonard, T.A. Davis, Phase I trial of humanized 1D10 monoclonal antibody targeting class II molecules in patients with relapsed lymphoma, 36th Annual Meet., Am. Soc. Clin. Oncol. 19 (2000) 24. [187] B.K. Link, G.J. Weiner, Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells, Blood 81 (1993) 3343–3349.

Y. Cao, L. Lam / Advanced Drug Delivery Reviews 55 (2003) 171–197 [188] L.M. Weiner, M. Holmes, A. Richeson, A. Godwin, G.P. Adams, S.T. Hsieh-MA, D.B. Ring, R.K. Alpaugh, Binding and cytotoxicity characteristics of the bispecific murine monoclonal antibody 2B1, J. Immunol. 151 (1993) 2877– 2886. [189] L.M. Weiner, J.I. Clark, M. Davey, W.S. Li, I. Garcia De Palazzo, D.B. Ring, R.K. Alpaugh, Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII, Cancer Res. 55 (1995) 4586–4593. [190] F. Hartmann, C. Renner, W. Jung, C. Deisting, M. Juwana, Treatment of refractory Hodgkin’s disease with an antiCD16 / CD30 bispecific antibody, Blood 89 (1997) 2042– 2047. [191] L. da Costa, C. Renner, F. Hartmann, M. Pfreundschuh, Immune recruitment by bispecific antibodies for the treatment of Hodgkin disease, Cancer Chemother. Pharmacol. 46 (2000) 33–36. [192] F. Hartmann, C. Renner, W. Jung, L. da Costa, S. Tembrink, G. Held, A. Sek, J. Konig, S. Bauer, M. Kloft, M. Pfreundschuh, Anti-CD16 / CD30 bispecific antibody treatment for Hodgkin’s disease: role of infusion schedule and costimulation with cytokines, Clin. Cancer Res. 7 (2001) 1873–1881. [193] N.D. James, P.J. Atherton, J. Jones, A.J. Howie, S. Tchekmedyian, R.T. Curnow, A phase II study of the bispecific antibody MDX-H210 (anti-HER2 3 CD64) with GM-CSF in HER2 1 advanced prostate cancer, Br. J. Cancer 85 (2001) 152–156. [194] P.A. Kaufman, P.M. Guyre, L.D. Lewis, F.H. Valone, V. Memoli, W. Wells, M.S. Ernstoff, Y.M. Deo, J. Fisher, M. Mrozek-Orlowski, K. Phipps, M.W. Fanger, HER-2 / neu targeted immunotherapy: a pilot study of multi-dose MDX-

[195]

[196]

[197]

[198]

[199]

197

210 in patients with breast or ovarian cancers that overexpress HER-2 / neu overexpression in metastatic breast cancer, Tumor Target. 2 (1996) 17–28. J.A. Posey, R. Raspet, U. Verma, Y.M. Deo, T. Keler, J.L. Marshall, J. Hodgson, A. Mazumder, M.J. Hawkins, A pilot trial of GM-CSF and MDX-H210 inpatients with erbB-2 positive advanced malignancies, J. Immunother. 22 (1999) 371–375. T. Schwaab, L.D. Lewis, B.F. Cole, Y.M. Deo, M.W. Fanger, P. Wallace, P.M. Guyre, P.A. Kaufman, J.A. Heaney, A.R. Schned, R.D. Harris, M.S. Ernstoff, Phase I pilot trial of the bispecific antibody MDXH210 (anti-FcgRI X anti-HER-2 / neu) in prostate patients whose cancer overexpresses HER-2 / neu, J. Immunother. 24 (2001) 79– 87. F.H. Valone, P.A. Kaufman, P.M. Guyre, V. Lewis, V. Memoli, Y.M. Deo, R.F. Graziano, J.L. Fisher, L. Meyer, M. Mrozek-Orlowski, K. Wardwell, V. Guyre, T.L. Morely, C. Arvizu, M.W. Fanger, Phase Ia / Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpressed the proto-oncogene HER2 / neu, J. Clin. Oncol. 13 (1995) 2281–2292. M. Watanabe, P.K. Wallace, T. Keler, Y.M. Deo, D.F. Hayes, Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX210, Breast Cancer Res. Treatment 53 (1999) 199–207. D.G. Pfister, A. Lipton, R. Belt, A phase I trial of the epidermal growth factor receptor-directed bispecific antibody MDX-447 in patients with solid tumors, Proc. Am. Soc. Clin. Oncol. 18 (1999) 1667.