Redirection of cellular cytotoxicity

�9 1995 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4992/95/261153-165/$6.60

Redirection of Cellular Cytotoxicity

A Two-Step Approach Using Recombinant Single-Chain Fv Molecules

A. J. T. GEORGE, *'1'2 J. A. TITUS, 2 C. R. JOST, 2 I. KtJRtJCZ, 2 P. PEREZ, 2

S. M. ANDREW, 2 P . J . NICHOLLS, 3 J. s . H U S T O N , 4 A N D D. M. lEGAL 2

1Department of Immunology, Royal Postgraduate Medical School, Hammersrnith Hospital, Du Cane Road, London

W12 ONN, UK; 2Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; 3Surgical Neurology Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda,

MD; 4Creative BioMolecules Inc., Hopkinton, MA

ABSTRACT

In this article the authors discuss an indirect system for redirecting cellular cytotoxicity, which utilizes a "universal" bispecific antibody to redirect T-cells to kill cells targeted with single-chain Fv (sFv) fusion proteins that carry a peptide tag recognized by the bispecific antibody. This approach has a number of theoretical advantages in the immunotherapy of cancer.

Index Entries: Bispecific antibody; single-chain Fv; redirected cellular cytotoxicity; immunotherapy.

*Author to whom all correspondence and reprint requests should be addressed. Current affiliations: P. Perez, Department of Microbiology, University of Salamanca, Spain; S. M. Andrew, Biological Sciences Division, Lancaster University, Lancaster, UK; P. J. Nicholls, Celltech Limited UK, Slough, UK.

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154 George et al.

I N T R O D U C T I O N

The use of bispecific antibodies to redirect cytotoxic cells to kill tumor cells has great potential in the treatment of cancer (1,2). Thus the use of antibodies with anti-CD3 x antitumor specificity has been shown to be capable of redirecting both cytotoxic T-cells, as well as cytokine-secreting helper T-cells, to kill tumor cells (3,4). Similarly, the use of bispecific antibodies with anti-Fc receptor specificity can be used to direct Fc receptor-positive cells to kill suitable targets (5). The strength of the bispecific approach is that it offers the possibility of conferring a novel specificity on an entire popula- tion of effector cells, redirecting them to kill their novel targets. There are a number of clinical trials of bispecific antibodies currently underway, both in the treatment of glioma and ovarian carcinoma, with some encouraging results (6, 7).

There are several drawbacks to the use of bispecific antibodies. Some of these are common to all forms of antibody therapy (e.g., immunogenicity, lack of suitable target antigens) (8). However, there are additional obstacles to the widespread use of bispecific antibodies, including the difficulty in manufacturing large amounts of clinical grade reagents. Currently, the majority of bispecific antibodies are made either by fusing two hybridomas to make a hetero- hybridoma (9,10), or by chemical conjugation of antibody fragments (5,11). Both of these approaches have a number of disad- vantages, and it is likely that they will be superseded by the use of recombinant bispecific molecules (12,13).

The in vivo delivery of bispecific molecules to their target also presents a major challenge. Currently, the localization of conven- tional, monospecific, antibodies to a tumor is low, with typically less than 0.01% of the injected dose localizing per gram of tumor (8,14). The problem is likely to be compounded in the case of bispecific antibodies, because the molecules will bind to circulating effector cells and will have difficulty in localizing to the tumor. In addition, the binding of bispecific antibody to effector cells at sites distant to the tumor could, following crosslinking by circulating antigen or an immune response, lead to activation of the cells and consequent toxicity. In the clinical trials reported to date these problems have been largely circumvented by using local administration of activated T-cells precoated with bispecific antibody, either in the space left by resection of the primary tumor (6) or into the peritoneal cavity (7). This is not an option in the majority of cancers, where systemic administration is required.

Cell Biophysics Volume 26, 1995

Redirection of Cellular Cytotoxicity 155

A final problem to be considered is the need to activate the effector cells. This is especially necessary in the case of T-cells. In experimental settings, or for local administration as outlined earlier, it is possible to activate the T-cells ex vivo, using a number of pro- tocols. In addition, it is possible to design strategies that activate the T-cells, for example, by use of trispecific antibodies that cross- link activating molecules on the T-cells (e.g., CD2) with the "trig- gering" CD3 (15). However, once again, these approaches run the risk of activating cells at sites distant to the tumor.

In this work the authors describe a two-step approach that may overcome some of these problems. This approach has the pharmacokinetic advantages of two-step strategies, which can be used to increase the localization of a therapeutic agent to a cell (8). In addition, it utilizes recombinant single-chain Fv molecules (sFv), that consist of the VH and VL domains of an antibody linked by a flexible peptide linker (16,17). These molecules have a number of advantages in tumor targeting, including increased solid tissue pene- trance and clearance, as well as a low immunogenicity (18,19). They can also be created using phage display technology, allowing novel specificities to be isolated (12,20-22). As most of the experimental background to this work has already been published (23), we concentrate in this work on the theoretical advantages that might accrue to this approach.

MATERIALS A N D METHODS

sFv Constructs The sFv used in this study have been previously described

(23-25). U7.6 sFv-myc has specificity for the hapten, dinitrophenol (DNP), whereas OKT9 sFv-H6-myc has specificity for the human transferrin receptor. The constructs were cloned into the vector pHEN 1 (26), and expressed following induction with IPTG. The sFv are in both cases made as a fusion protein with a short peptide tag derived from the c-myc oncogene, which is recognized by the monoclonal antibody Mycl 9E10.2 (27). OKT9 sFv-H6-myc, in addition, incorporates a six-histidine sequence that allows for its purification by metal chelate affinity chromatography. The pHEN I vector incorporates the pelB leader sequence that directs synthesis of the proteins to the periplasm of the cells.


156 George et al.

Re folding Although small quantities of U7.6 sFv-myc could be found in

soluble form in the bacterial culture supernatant, the yields were low. In addition no OKT9 sFv-H6-myc could be found in the culture supernatant. The authors therefore developed a refolding protocol that utilizes the ability of the disulphide bonds to correctly form in the periplasm of the Escherichia coll. This method is described in detail elsewhere (23,28), but in brief, the bacteria are lysed, the in- soluble pellet is then taken up in high molarity of denaturing agent (either 7.5M guanidine hydrochloride or 9M urea). The protein is allowed to renature by dialysis against 0.1M Tris-HC1, 2 mM EDTA, 0.4M arginine, pH 8.0. The material was purified either by affinity chromatography after refolding (U7.6 sFv-myc) or be metal chelate affinity chromatograhy in guanidine hydrochloride (OKT9 sFv- H6-myc).

Enzyme-Linked Immunosorbent Assay (ELISA) The ability of the U7.6 sFv-myc to bind to its antigen was tested

by ELISA. DNP-BSA was coated onto the plastic wells of a microtiter plate, which were then blocked with BSA. Suitable dilutions of the purified U7.6 sFv-myc were added to the wells, and the bound material detected with the antibody Mycl 9E10.2, followed by alkaline phosphatase conjugated goat anti-mouse Ig.

Recognition by sFv of Cel l S u r f a c e Antigens The ability of the sFv to recognize cell surface antigens was

tested by flow cytometry. In the case of the U7.6 sFv-myc the murine methyl cholanthrene induced sarcoma cell line, B6 MC1, was coated with trinitrophenol (TNP). In the case of OKT9 sFv-H6- myc a variety of cell lines expressing the human transferrin receptor were used. In all cases, the sFv was detected using FITC-conjugated Myc 1 9E10.2.

Retargeting Experiments Human peripheral blood lymphocytes were activated with im-

mobilized anti-CD3 and IL2, and coated with bispecific heteroconjugate antibody (anti-CD3 x antipeptide). The heteroconjugate was prepared by crosslinking of the anti-CD3 antibody OKT3 and Myc 1 9E10.2 (anti-myc peptide) as previously described (29). TNP modified L-cells, that had been transfected with the human transferrin



pelB V L L V H myc

U7.6 sFv-myc

pelB V L L V H H 6 myc " / / / / / ~ m ~ l l

OKT9 sFv-H6-myc

Fig. 1. The U7.6 sFv-myc and OKT9 sFv-H6-myc constructs. The nature of the constructs of the two sFv used in this project are shown in the figure. Both sFv utilize the ([Gly]4Ser)3 linker (L) to bridge the V~ and VH domains, and are made as a fusion protein with myc. In addition, the OKT9 sFv-H6-myc con- tains a six histidine sequence (H6) to allow for metal chelate affinity chromatography purification.

receptor gene, were used as target cells. The targets were labeled with [51Cr] and incubated with the sFv fusion proteins. They were then mixed with the effector cells, at appropriate ratio, and after a 3-4-h incubation, the specific lysis was determined.

RESULTS

Construction of U7.6 sFv-myc and OKT9 sFv-H6-myc Construction of the U7.6 sFv-myc and OKT9 sFv-H6-myc have

been described in detail elsewhere (23-25). In brief, the VH and VL domains of the antibodies were amplified by the polymerase chain reaction (PCR) from hybridoma derived cDNA. The primers for the amplification incorporated not only suitable restriction cloning sites, but also, in the case of the primers complementary to the regions adjacent to the linker, sequences encoding the ([Gly]4Ser)3 linker of the sFv. The products of the two amplifications were then mixed together and a second PCR performed, using primers from the extreme ends of the construct (5' end of the Vt and 3' end of VH). This led to the generation of a single gene fragment of the size expected for an sFv. These fragments were then cloned into pBluescript prior to subcloning into pHEN I to give the gene constructs shown in Fig. 1.


158 George et al.

0.2

0.15-

0,1-

,~- s 0 0.05-

0-

-0.05

I I I I I I

<. U7.6 sFv-myc di lut ion (log2)

Fig. 2. ELISA analysis of U7.6 sFv-myc. Refolded U7.6 sFv-myc was tested for binding to either DNP-BSA (squares) or BSA alone (circles) on ELISA microtiter plates. The binding was detected with Myc 1 9E10.2 followed by alkaline phosphatase conjugated goat antimouse Ig. The first dilution cor- responds to a concentration of U7.6 sFv-myc of 12 #g/mL.

Expression and Characterization of the sFv The gene constructs were expressed in the E. coli strain HB2151

by overnight induction at room temperature with I mM IPTG. The sFv were refolded and purified from cell lysates, and the resulting proteins characterized by SDS-PAGE and Western blotting, demon- strating that a protein of the expected molecular weight had been isolated (data not shown).

The molecules for tested for immunoreactivity by ELISA (U7.6 sFv-myc) and FACS (U7.6 sFv-myc and OKT9 sFv-H6-myc) analyses. As shown in Fig. 2, dilutions of the refolded U7.6 sFv-myc bound to DNP-BSA in ELISA, this binding was specific in as much as the antibody did not bind to BSA on its own, and the binding was inhibited by free 1 mM DNP hapten (data not shown).

Both sFv were capable of binding to cell surfaces bearing the appropriate antigen, as demonstrated in the case of U7.6 sFv-myc in Fig. 3. The U7.6 sFv-myc bound specifically to B6MC1 cells coated with TNP, but not to B6MC1 cells alone. This binding was inhibited by free DNP hapten. Similar results were seen in the case of OKT9 sFv-H6-myc binding to both K562, HUT 102, and murine L-cells transfected with the human transferrin receptor, but not to L-cells alone. This binding could be inhibited by the parental OKT9 IgG.



Binding of (~-DNP sFv to TNP-modified cells

IX ILl 3 to ~E

z . . I . . I I I l l o

TNP-MCl , :i~, l

I I

2/ r~,

Mcl t

4 ~ ~,

LOG FLUORESCENCE 4

Fig. 3. FACS analysis of the binding of U7.6 sFv-rnyc to cell surface antigens. The binding of 12/~g/mL U7.6 to TNP-coated B6MC1 cells is shown (A). The negative control (secondary antibody alone) is shown as a dotted line. (B) The binding is shown to cells not coated with TNP. The sFv was detected with a secondary FITC labeled Myc 1 9E10.2.

Redirected Cellular Cytotoxicity Following characterization of the fusion proteins the authors

next determined whether they could be used to redirect cytotoxicity. Human peripheral blood lymphocytes were activated by a combination of IL-2 and immobilized anti-CD3. They were coated with a "universal" bispecific antibody, consisting of anti-CD3 conjugated to Myc I 9E10.2, which recognizes the peptide tag of the fusion proteins. They were then tested for their ability to redirect lysis to target cells, that had been coated with the Fv-myc fusion proteins. As is shown in Fig. 4, this indirect method is capable of redirecting cytotoxic T-cells to kill target cells. In the presence of the universal bispecific antibody, the U7.6 sFv-myc is capable of redirecting the T-cells to TNP-modified murine L-cells transfected with the human transferrin receptor gene. This killing is specific to TNP- modified cells, and can be blocked by free DNP hapten (data not shown). Similarly, a combination of the OKT9 sFv-H6-myc and the universal bispecific is capable of causing lysis of cells bearing the human transferrin receptor. The cytotoxicity in all cases is depen- dent on the presence of both the sFv-myc fusion protein and the universal bispecific.


160 George et al.

25

d~ 20

.._1 15

10 ,..-.,

O9 s

0

Fig. 4. Indirect redirected cellular cytotoxicity. Activated human peripheral blood lymphocytes were coated with the 0.3 #g/mL of the universal anti-CD3 x antipeptide bispecific antibody. They were then incubated, at a 21:1 effector:target ratio, with plCr]-Iabeled, TNP-modified, murine L-cells transfected with the gene encoding the human transferring receptor together with the appropriate sFv (U7.6 sFv-myc [12 #g/mL] or OKT9 sFv-H6-myc [15 #g/mL]). The cells were incubated for 3-4 h and the specific [slCr]-released determined. Data taken from ref. 23.

CONCLUSIONS

The authors have shown that it is possible to use fusion proteins, containing sFv directed at cell surface molecules and a peptide derived from the c-myc oncogene, in combination with a "universal bispecific" antibody to redirect T-cells to kill novel target cells (23). Previously, Gilliand et al. used a universal hybrid hybridoma with anti-CD3 and antirat specificities to target cytotoxic T-cells against tumor cells coated with rat antitumor antibodies (30). The redirected lysis is specific, in as much as it depends on the presence of both an appropriate sFv and the universal bispecific antibody, and on the target cell bearing the appropriate antigen. In addition, in the case of the U7.6 sFv-myc the lysis could be blocked by free, soluble, DNP hapten.

In our experiments, the universal targeting system was a con- ventional bispecific antibody, formed by crosslinking two IgG molecules. However, in practice, the antibody could be replaced by any



molecule capable of recognizing both CD3 on the T-cell and the sFv. These could include fusion proteins between anti-CD3 and streptavidin (employing an biotinylated sFv) and genetically engi- neered bispecific molecules (13,31-35). In addition, this system could be used to redirect cells other than T-cells, such as NK-cells, by targeting a molecule such as CD16 rather than CD3.

There are a number of potential advantages to this approach. In the first place, it provides a universal system whereby novel sFv can be tested for their ability to target lysis to unwanted cells prior to the production of a recombinant bispecific molecule. This could save time and money in the development of recombinant molecules.

Second, the indirect approach allows one to take advantage of the indirect methods of administering therapeutic agents. Thus, the sFv fusion proteins could be administered first, allowed to clear from the body, prior to administration of the bispecific antibody. This could be important in reducing nonspecific toxicity, as any free sFv (or indeed sFv bound to soluble antigen found in the cir- culation) could be allowed to clear, thereby reducing the activation of the T-cells at sites distant from the tumor. Such indirect, multi- stage, approaches have been shown to have advantages in the localization of radiolabels to tumors. Thus, the administration of a biotinylated antibody, followed by avidin, and subsequent detection with radiolabeled biotin, can increase the tumor localization index and reduce nonspecific uptake of radiolabel in other organs (36).

A further potential benefit to this approach is that it would allow the use of a cocktail of sFv, with specificity for the tumor cell, in combination with one universal antibody. This could lead to multisite recognition of the tumor, which could enhance the specificity and affinity of the interaction between the cytotoxic cell and the tumor cell (37). The use of multiple specificities might also reduce the ability of the tumor cells to develop antigen loss variants (38).

The use of recombinant sFv as a targeting agent may also have pharmacokinetic advantages. Thus the rapid clearance, the ability to penetrate solid tumors, and low immunogenicity are attractive features of the molecule for tumor immunotherapy (39).

We have demonstrated that it is possible to use recombinant sFv molecules to redirect cellular cytotoxicity in an indirect man- ner. This approach may have a number of advantages for tumor therapy. Currently work is underway to test the indirect system, and to prove its efficacy.


162 George et al.

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Redirection of cellular cytotoxicity