The improved lytic function and in vivo efficacy of monovalent monoclonal CD3 antibodies

Eur. J. Immunol. 1989.19: 381-388 Monovalent CD3 antibodies 381

Mike Clark,’ Carol Bindon,’ Martin Dyer’, Peter FriendA, Geoff Hale,’ Steve Cobbold,’ Roy CalneA and Herman Waldmann’

Department of Pathology’, University of Cambridge, and Departments of Haematology’ and SurgeryA, Addenbrooke’s Hospital, Cambridge

1 Introduction

The improved lytic function and in vivo efficacy of monovalent monoclonal CD3 antibodies*

A series of hybrid-hybridomas were derived by the cell fusion of a CD3 antibodysecreting hybridoma with other Ig-producing cell lines. The Ig molecules secreted by these hybrid-hybridomas were fractionated by ion-exchange chromatography, and fractions containing monovalent CD3 antibodies were tested for complement-mediated lysis of T cells. Two monovalent CD3 antibodies with mixed heavy chain isotypes were very poor in lysis but, in contrast, a monovalent antibody possessing two identical rat YZb heavy chains but two non-identical light chains was found to be more lytic with human complement than the parental bivalent CD3 antibody. The difference in lysis was not readily explicable in terms of a difference in complement activation at the level of Clq binding or cell-associated C3. A highly purified batch of this lytic monovalent CD3 antibody was prepared in a form suitable for in vivo therapy. In a preliminary clinical study in one patient with a T lymphoma in leukemic phase this monovalent CD3 antibody was found to be very effective in depleting CD3+ tumor cells in the peripheral blood and bone marrow. The cells in the peripheral blood were completely cleared and there was no evidence for antigenic modulation. In addition the antibody was able to reverse cell-mediated kidney rejection in three kidney graft patients. These results suggest that monovalent antibody may be effective in vivo as well as in vitro and that a fuller clinical evaluation is justified.

Generally antibodies are multivalent for antigen binding, IgM having a valency of 10 and IgG a valency of 2. This is due to the allelic exclusion for Ig genes at the clonal B cell level resulting in the symmetrical arrangement of pairs of identical heavy and light chains in each antibody molecule. The natural role of IgG antibody in the body is to recognize and attach to foreign pathogens and toxins, and to neutralize them with the assistance of various other humoral and cellular effector mechanisms. When exploiting monoclonal antibodies (mAb) for immunosuppressive or anti-tumor therapy we are, in contrast, directing these effector mechanisms at self tissues. These multivalent antibodies when directed to cell surface antigens are often able to cross-link the molecules, and in many in- stances this can lead to a patching and eventual loss of surface expression of antigen [l]. This phenomenon, known as antigenic modulation, can occur both in vitro and in vivo and results in the cells becoming insensitive to lysis by antibody- mediated effector mechanisms [2-4]. CD3 is an example of a modulating antigen but another more extensively studied sys- tem has been the induction of modulation of surface Ig on B lymphoma cells by anti-idiotypic antibodies [5 ] . A method of avoiding modulation was found to be the use of anti-idiotypic

[I 72621

* This work was supported by grants from the U.K. Medical Research Council and the British Technology Group. “CAMPATH” is a trademark of the Wellcome Foundation.

Correspondence: Mike Clark, Division of Immunology, Department of Pathology, University of Cambridge, Tennis Court Road, Cam- bridge CB2 lQP, GB

Abbreviations: C’ML.: Complement-mediated lysis IMDM Iscove’s modification of Dulbecco’s medium FCS: Fetal calf serum PBS: Phosphate-buffered saline PBMC: Peripheral blood mononuclear cells BSA Bovine serum albumin mAb Monoclonal antibody(ies) ELISA: Enzyme-linked immunosorbent assay FPLC: Fast performance liquid chromatography

antibodies which had been rendered monovalent by pro- teolytic and chemical modification [6-8].

For mAb it is possible to produce monovalent antibodies without a need for chemical modification. When two antibody- producing cells are induced to fuse to form a new hybrid cell the Ig chains are codominantly expressed and are able to associate with each other to give rise to various novel hybrid antibody molecules in addition to the two parental types [9]. For example the combination of two IgG antibodies can generate up to ten different antibody molecules [lo, 111. In general, only the correct combination of heavy and light chain will retain specificity for antigen and so within the mixture of IgG molecules there will be some which only have a single active binding specificity and these will be monovalent. Using affinity chromatography based on differences in Ig light chain allotype Cobbold and Waldmann were able to enrich for this form of monovalent mAb [12]. In vitro these antibodies proved more effective than the parental mixture in promoting complement- mediated lysis (C’ML). Affinity purification is inappropriate for large scale production of therapeutic antibody. A better approach would be to generate monovalent antibody which could be purified to homogeneity by ion-exchange chromatography. Using this approach we have isolated a number of fractions from hybrid antibodies with CD3 specificity and tested them for human C’ML of T cells in vitro. The antibodies were compared for terminal lysis as well as for Clq binding and C3 binding. Clinical grade antibody was then prepared from the best of these and evaluated in vivo in one patient with a CD3’ T lymphoma in leukemic phase and in four kidney graft patients with biopsy-proven cell-mediated rejection of the transplanted organ.

2 Material and methods

2.1 Antibodysecreting cell lines

Details of the specificities of the hybridoma cell lines used are given in Table 1. Further details of YTH12 and YTH67 have

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 0014-2980/89/0202-0381$02.50/0

382 M. Clark, C. Bindon, M. Dyer et al. Eur. J. Immunol. 1989.19: 381-388

Table 1. Details of parental cell lines and hybrid hybridomas

Parental cell lines Name Isotype Specificity

YTHl2 Rat IgG?b. h YTH67 Rat IgG2,,n MGlCD19 Mouse IgG,,x J558L Mouse, A

Hybrid hybridoma Name Parental cells

CD3 CD5 CD19 None

Ig production

SHK5 YTHl2 X YTH67 SHL45 YTH12 X J558L Rat fib, rat h, mouse A SHRl YTH12 X MGlCD19 Rat Y2br rat A. mouse y,,

Rat Y&, rat yza. rat x , rat A

mouse x

been given elsewhere [13-151. Although the original isolates of these cell lines (clones YTH12.5 and YTH67.1) secreted a rat x-la light chain derived from the parental myeloma cell line [16] the particular clones used here were myeloma light chain loss variants isolated during recloning (clones YTH12.5.14.2 and YTH67.1.1TG6). These clones will be referred to by abbreviations of the full names. YTH12 was included in the CD3 panel of the Third International Workshop on human leukocyte differentiation antigens [17]. Cells were maintained in sodium hydrogen carbonate buffered Iscove's modification of Dulbecco's medium (IMDM) supplemented with 2% (v/v) fetal calf serum (FCS).

2.2 Cell fusion

The method for production of hybrid hybridomas was previously described [ l l , 131. Briefly, one parental cell line had been preselected for resistance to the drug 6-thioguanine and sensitivity to counter selection by the drug combination of hypoxanthine, aminopterin and thymidine (HAT). This cell line was then fused using polyethylene glycol treatment with the second cell line pretreated with a superlethal dose (5-10 mM) of the irreversible biochemical inhibitor iodoacetamide. Hybrid-hybridoma cells were then selected for in IMDM supplemented with 5% FCS and HAT. Culture supernatant from growing cultures was analyzed using antibodies specific for the different Ig isotypes in enzyme-linked immunosorbant assays (ELISA) or red cell-linked assays as previously described [13, 181. Specificity for cell surface antigens CD3, CD5 and CD19 was assessed by indirect immunofluorescence assays on suitable target cells bearing these antigens. Hybrids from positive cultures were recloned twice on semisolid agar and retested at each stage.

2.3 Antibody purification

The antibody present in the spent culture supernatant from cells in stationary phase was fractionated as follows. Antibody was first concentrated by precipitation with 50% saturation of ammonium sulfate and the precipitate was then redissolved in the minimum volume of water and dialyzed into 50 m M sodium malonate buffer at pH 5.8 containing 0.1% (w/v) betaine. Ion exchange chromatography was carried out on a Pharmacia (Uppsala, Sweden) Mono S fast performance liquid

chromatography (FPLC) column using the malonate buffer and eluting with a linear 0-1 M NaCl gradient. The fractions obtained were desalted into phosphate-buffered saline (PBS) on a Sephadex-G25 column and were analyzed using the isotype-specific assays as above. In addition the fractions were also analyzed by reducing sodium dodecyl sulfate and by native polyacrylamide gel electrophoresis as well as isoelectric focusing using a Pharmacia "Phastsystem" and "Phastgels" following the recommended protocols.

Antibody for clinical use was fractionated similarly except that all steps were carried out aseptically and the buffers and solu- tions were prepared sterile from pyrogen-free water. In addition the antibody was first fractionated in bulk by ion- exchange chromatography on a large volume Pharmacia Fast Flow S column and the monovalent antibody fraction was then rechromatographed on the Mono S FPLC column, desalted into PBS, and human plasma protein fraction was added at an equal protein concentration as a stabilizer before aliquots were dispensed into sterile nitrogen-filled Mallinckrodt vials. These aliquots were then stored at - 20 "C before use. Sample aliquots were tested for sterility and for pyrogens.

2.4 C'ML

C'ML was carried out as previously described [15]. Peripheral blood mononuclear cells (PBMC) were isolated from defibrin- ated blood by density gradient centrifugation over Ficoll- Hypaque (density e = 1.077 kg/l). T cell blasts were pre ared by culturing these cells in IMDM + 5% (v/v) FCS at 10 cells/ ml in plastic culture flasks coated with CD3 mAb [ l l , 141. After 3 days the blasts were washed and transferred to new flasks and expanded for a further 7 to 14 days in IMDM supplemented with 5% (v/v) FCS and 20 unitslml recombinant interleukin 2. For lysis fresh PBMC or T cell blasts were washed in Hepes-buffered IMDM and labeled with 'lCr by incubating approx 2 x lo7 cells with 150 pCi = 5.55 MBq of Na:'Cr04 in a volume of 200 pl of Hepes-buffered IMDM for 1 h at 37 "C. The labeled cells were then washed and added at 5 x lo4 cells/well to dilutions of antibody in U-shaped microtiter plates. Autologous serum was added as a complement source at a final concentration of 25% (vh) and the plates were incubated for 1 h at 37 "C. Cell lysis was estimated from specific radioactivity release determined by counting a 100-pl aliquot of the supernatant after pelleting cells at 100 x g for 2 min. The percentage specific "Cr release was calculated from the formula:

?

Specific release (%) = (test-spontaneous) x lOO/(total-spontaneous)

2.5 Complement-binding studies

Complement-binding studies were carried out using methods previously described [15, 191. Clq was purified and then labeled with "'I by the lactoperoxidase method. Purified antibody to C3d was radiolabeled with 12'1 using the iodogen method. These radiolabeled reagents were titrated before use. C lq binding studies were carrried out in IMDM containing 1% (wh) bovine serum albumin (BSA). Cells and antibody at different concentrations were mixed and radiolabeled Clq was added. After incubation for 1 h at room temperature the cells and supernatant were separated by spinning through oil in


microfuge tubes. The tubes were frozen in solid COz and the tubes clipped in half. The radioactivity in the upper and lower half of the tubes was then determined. C3 binding was mea- sured by incubating cells and antibody in the presence of C6- deficient serum for 30 min at 37 “C. The cells were washed and then resuspended in medium containing radiolabeled anti-C3d antibody. After 30 min at room temperature bound and free counts were determined by separating the cells and supernatant by centrifugation over oil as above.

2.6 Clinical studies

Approval for the use of mAb was given by the ethical commit- tee of Addenbrooke’s Hospital and written consent was obtained from each patient. Antibody was diluted in 500 ml of saline and infused over a 2- to 4-h period. For the kidney graft patients the antibody was administered under the cover of steroids. Blood samples were collected at intervals for conventional biochemical and hematological assays. In addition the leukemic T cells were isolated on Ficoll-Hypaque (Pharmacia) and tested for sensitivity to monovalent CD3 antibody by autologous C’ML, and the surface expression of CD3 antigen was determined by indirect immunofluorescence and flow cytometry.

The presence of excess functional CD3 antibody in patients’ serum samples was determined by incubating fresh PBMC from an untreated normal donor with patients’ serum for 45 min on ice. Bound antibody was detected by indirect immunofluorescence and flow cytometry and the amount estimated by comparison with a standard curve of artificially prepared antibody concentrations in normal serum.

Anti-globulin responses were detected by an ELISA. The antibody used for therapy or control antibodies was adsorbed to the wells of plastic microtiter plates which were then blocked for further nonspecific binding with 5% BSA in PBS. Various dilutions of patients’ sera were incubated in these plates for 1 h at room temperature and the plates were then washed with 0.1% BSA in PBS. Bound antibody was detected using biotinylated anti-human Ig followed by streptavidin peroxi- dase and finally the substrate o-phenylenediamine.

3 Results

3.1 Derivation of hybrids

Using a simple cell fusion method previously described [ l l , 13, 151 we derived a number of cell hybrids between a rat IgGzb CD3-secreting hybridoma cell line and several different antibody-secreting cell lines (Table 1, Sect. 2.1). Cultures of hybrid cells secreting mixed molecular species were identified using Ig isotype-specific assays as previously described and positive cultures were cloned. Different antibody-containing fractions were separated by ion-exchange chromatography on a Mono S column using FPLC. Under the conditions used (50 mM sodium malonate, pH 5.8) the parental CD3 antibody was bound to the column but the bulk of the contaminating serum proteins such as BSA was in the breakthrough fractions. Elution profiles for the different hybrids are summarized in Fig. 1.

Analysis of the fractions indicated in Fig. 1 was carried out using gel electrophoresis and isotype-specific assays as

described (data not shown). In theory a cell line producing one heavy chain and two light chains can assemble three different antibody molecules and a cell line producing two heavy and two light chains can assemble ten different antibody molecules [lo, 11, 201. In practice, however, there may be preferential association between some chains resulting in only certain of the theoretical forms being observed.

The results obtained with the fractions from the various cell lines (Table 1 and Fig. 1) suggested that the antibody profile of each was as follows. Hybrid YTH12, the parental bivalent CD3, yielded a single antibody fraction “a” while the bovine serum proteins were found in the breakthrough and trailing fractions. The other hybrids all yielded fractions eluting in a similar position containing this bivalent CD3; they were SHKS “b”, SHL45 “a” and SHR1“b”. For hybrids SHKS and SHRl the other parental antibody forms (rat IgG2, CD5 and mouse IgGl CD19, respectively) were both found in the unbound breakthrough from the columns (not shown in the profiles).

SHL45

E l u t l o n Volume

Figure 1. Ion-exchange chromatography of CD3 antibodies. The elution profiles for the four different CD3-producing cell lines separated by ion-exchange chromatography on a Pharmacia Mono-S FPLC column are shown. The salt gradient from 0 to 400 mM NaCl is indicated as well as the UV absorbance at 280 nm on a 0-2 scale. The fractions which were collected are indicated by the letters a, b, c and d. The constituents of the relevant fractions as determined by gel electrophoresis and ELISA are indicated by the schematic antibody molecules where the heavy and light chains from the CD3 parent are in black and from the other parental cell tine in white.

384

SHKS yielded one additional antibody fraction “a” which con- tained a bispecific antibody monovalent for CD3 and CD5 with a mixed rat y2&, Fc region. SHK5 thus represents an example of a cell line which shows preferential association of the correct heavy and light chain pairs which reduced the ten theoretical forms to three observed forms. SHRl showed a greater number of poorly resolved fractions, “b, c and d containing different combinations of the rat and mouse heavy and light chains. The leading edge of the SHRl profile, fraction “a”, was, however, the bispecific antibody monovalent for CD3 and CD19 with a mixed mouse yl/rat Y2b Fc region. The hybrid-hybridoma SHLA5 which secreted a single heavy chain and two light chains gave the theoretical three forms of antibody in well-resolved fractions, the two additional forms of antibody being the monovalent antibody with two different light chains (“b”) and the inactive antibody with two myeloma- derived light chains (“c”).

M. Clark, C. Bindon, M. Dyer et al. Eur. J . Immunol. 1989.19: 381-388

3.2 C’ML of T lymphocytes

The fractions were desalted into PBS on a Sephadex G-25 column and were then titrated for their ability to induce autologous C’ML of human T cell blasts (Fig. 2). The monovalent fraction “b” from hybridoma SHL45 gave more lysis than the parental bivalent CD3 antibody YTH12 or the equivalent bivalent fraction “a” from SHL45. However, a monovalent fraction from hybrid S W (“a”), containing a mixed rat Y2a/Y2b Fc region, and a similar fraction from SHRl (“a”) containing a mixed mouse yl/rat y2b Fc region, both gave very poor lysis. Thus, monovalency of binding was not sufficient to demonstrate improved lysis. SHKS “a” is of particular interest because it could bind to T cells simultaneously through its monovalent specificities for both CD3 and CD5, possibly resulting in a higher affinity of binding to cells and an increase in the site density of antibody molecules at the cell surface. However, this did not seem to offer any advantage in lysis.

70 1 6 o 1

A

10

0 1 2 5 1 0 20 50 100

Antibody concentration I p q d ’

Figure 2. C‘ML by antibody fractions. The different fractions indicated in Fig. 1 were titrated in autologous C’ML of human T cell blasts. Included as a positive control was a rat IgG2b antibody CAM- PATH-1G which is potently lytic for human lymphocytes (0). The other fractions were; bivalent CD3 YTH12 “a” (0) and SHL45 “a” (0); monovalent CD3 SHKS “a” (A), SHL45 “b” (M) and SHRl “a” (V); inactive antibody SHL45 “c” (0).

Thus the most efficient form of monovalent CD3 in lysis had a rat region composed of two identical Y2b heavy chains associated with two different light chains. SHRl has monovalent specificities for the antigens CD3 and CD19 but as CD19 is a B cell and not a T cell antigen it can be regarded as similar in its binding to T cells to SHL45 “b”. In similar experiments this bispecific antibody was also found to be non-lytic for a CD19’ B lymphoma cell line.

3.3 Interference in CML

The original unfractionated SHL45 antibody supernatant was not lytic with autologous complement (data not shown). To find out at what ratio the different forms would interfere with each other, fractions SHL.45 “a” and “c” were titrated from 50 pg/ml and a constant concentration of 50 &ml of the lytic fraction SHL45 “b” was added. The results shown in Fig. 3a demonstrate that at a ratio of 1 part fraction “a” to 1 part fraction “b” lysis was completely abolished. Thus, at the ratio of fraction “a” to “b” found in the SHL45 supernatant (Fig. 1) there would be complete interference in lysis. As would be expected, fraction “c” which is the inactive fraction had no effect on lysis by fraction “b”.

Fig. 3b shows the abilities of the parental bivalent CD3 antibody YTH12, the monovalent mixed rat y2dmouse yl SHRl fraction “a” and the bispecific monovalent rat Yh/Y2b S H U fraction “a”, to inhibit lysis by the monovalent SHL45 fraction “b” antibody. All of the non-lytic CD3 antibodies were ca- pable of interfering with lysis by the lytic monovalent CD3 antibody SHL45 fraction “b”. However, in a similar experi- ment none of these antibodies had any effect in inhibiting lysis of the T cell blasts by the CAMPATH-1G antibody (data not shown).

3.4 Complement activation by CD3 antibodies

In a previous study of C’ML by mAb it was found that the ability to lyse cells depended very much on the antigen

50-

LO-

2 3 0 - u c !n - ., g 2 0 - s

10-

LO-

30-

2Q-

10-

1 2 5 10 20 50 1 2 5 10 20 50

Antibody concentrotion I@-’

Figure 3. Interference of lysis by non-lytic CD3 fractions. The ability of various fractions to interfere with the lysis of T cell blasts by the monovalent CD3 antibody SHL45 “b” was investigated. The fractions were titrated in twofold dilutions starting at 50 &ml and a constant 50 pg/d of SHL45 “ b was added. The arrows indicate the control level of lysis given by SHL45 “b” alone. (A) shows the bivalent CD3 fraction SHL45 “a” (0) and the inactive fraction SHL45 “c” (0). (B) shows the monovalent CD3 antibodies SHKS “a” (A) and SHRl “a” (V) as well as the parental bivalent CD3 YTH12 “a” (0).


specificity of the antibodies as well as their isotypes [15]. Anti- bodies to some antigens were found to be very inefficient in activating the complement pathway despite binding high levels of Clq. In order to determine what might be the rate limiting step in C’ML by CD3 antibodies, we studied the ability of cells to bind radiolabeled human Clq and also radiolabeled anti- C3d antibody (Fig. 3). Complement activation and binding was studied at various antibody concentrations and for comparison cell lysis of these same PBMC was also determined (Fig. 4). It is interesting to note that the efficiency of lysis of resting lymphocytes was less than for activated T cells (c.f. Fig. 1). Two antibodies for which complement activation has previously been studied were included as controls [15]. The first, CAMPATH-lG, is an example of a very lytic rat lgGSb antibody directed against the CAMPATH-1 antigen while the second is an example of a poorly lytic rat IgGSb antibody directed against the leukocyte common antigen (“200, CD45).

The bivalent (YTH12) and monovalent (SHL45 “b”) CD3 antibodies showed very slightly lower levels of Clq binding compared to the poorly lytic CD45 control antibody and much lower binding than the very lytic CAMPATH-1G antibody. Monovalent CD3 (SHL45 “b”) gave slightly higher Clq binding compared with bivalent CD3 at all concentrations of antibody. The non-lytic monovalent CD3 antibody, containing a mixed mouse yl/rat Y2b Fc region SHRl “a”, was also very poor at binding Clq.

At the level of C3 binding there is a dramatic change with the monovalent (SHL45 “b”) and bivalent (YTH12) CD3, both showing high levels, binding just slightly less than the potently lytic CAMPATH-1G antibody. Thus the paradox is that both the monovalent and bivalent CD3 antibodies seem to be very efficient in activating C3 from the modest levels of Clq bound but that lysis is poor when compared to that obtained with similar levels of C3 fixed by CAMPATH-1G. Differences in lysis between CAMPATH-1G monovalent CD3 (SHL45 “b”) and bivalent CD3 (YTH12) must therefore result from the stages subsequent to C3 activation and binding. Despite this the presence of high levels of cell bound C3 is likely to be of significance for in vivo opsonization by C3bi receptor-bearing effector cells. In contrast to the other two CD3 antibodies the non-lytic mixed Fc monovalent CD3 antibody SHRl “b” was found to be relatively ineffecient in fixing C3.

3.5 Treatment of lymphoma patient

A therapeutic batch of monovalent antibody was prepared from SHL45 as described. This antibody was used to treat a 48-year-old male with a cutaneous T cell lymphoma. The lymphoma had previously failed to respond to treatment with cyclophosphamide, hydroxydaunarubicin, vincristine, pre- dnisolone and radiotherapy, and 3 months after presentation he had progressed rapidly into the leukemic phase. His peripheral white cell count at the start of antibody treatment was 48.3 X 109/liter, consisting of 96% leukemic blasts. A bone marrow aspirate taken at the same time showed 92% blasts, 6% myeloid cells and 2% erythroid cells. By indirect immunofluorescence and flow cytometry the leukemic blasts were found to have the phenotype 100% positive for CD2, CD3, and CD7 and negative for CAMPATH-1, CD1, CD4, CD5, CD6 and CD8. In vitro, these cells were lysed by the monovalent SHL45 but not the bivalent CD3 antibody and autologous complement (data not shown).

Table 2. Treatment of T lymphoma patient in leukemic phase

Day Antibody No x lo-’ blastn (mg) Pre Post

1 2 45.4 20.1 2 4 21.0 3.8 3 4 0.4 0.0 4 2 0.0 nda) 6 2 nd nd 8 8 0.0 nd

a) Not determined.

The patient was treated with monovalent CD3 antibody over an 8-day period followed by blood cell counts and the collec- tion of serum to determine antibody levels (Table 2). No adverse reactions to the antibody were seen and leukemic blasts were completely and rapidly cleared from the peripheral blood. At the end of the course of treatment, the cutaneous nodules had regressed substantially; a repeat bone marrow aspirate showed 71% leukemic blasts, 18% erythroid cells and 11% myeloid cells. Leukemic blasts in the marrow remained CD3’ with an antigen density comparable to that of the original population, suggesting that no significant antigenic modulation had occurred (data not shown). Five days after stopping antibody treatment leukemic blasts began to reappear in the peripheral blood, and although a further course of chemotherapy was given the patient developed a pneumonia and died 3 weeks later.

3.6 Treatment of organ graft rejection

CD3 antibodies and in particular the mAb OKT3 have been of value in the prevention of graft rejection in organ transplant patients [21-231. Having demonstrated that the monovalent CD-3 antibody was effective in depleting CD3’ tumor cells in a T lymphoma patient, it was of interest to evaluate the immunosuppressive potential of the same antibody in organ graft rejection. Patients were considered for treatment with the monovalent CD3 antibody if cell-mediated rejection was demonstrable by biopsy and if there were no indications of humoral involvement. Four patients with biopsy-proven acute cellular rejection of renal allografts were eventually treated. Brief details are given in Table 3.

The first three patients treated all showed a satisfactory resolu- tion of the graft rejection following the course of monovalent CD3 antibody as indicated by a drop in the serum creatinine and urea levels to normal. Fig. 5 shows a more detailed time course of therapy for patient 2. The fourth patient treated developed a severe chest infection and treatment was stopped after seven doses of antibody and subsequently he was found to have an abscess, containing staphylococcus aureus, between the graft and the bladder necessitating the removal of the kidney. The two patients treated with 5 mglday both showed reactions to the early doses of antibody analogous to those reported for OKT3 treatment [22, 231. Neither of the two patients receiving 2 mg daily doses or a fifth patient who received only a single dose of 2 mg showed any reactions to the antibody.

386 M. Clark, C. Bindon, M. Dyer et al. Eur. J. Immunol. 1989.19: 381-388

7 -

a i6- Y - m 5-

4 -

Y

&

“i N

9 3 -

: 2 - u

‘1

Y&!?z!- 0 1 2 5 10 20 50 100 0 l U % L - - - - 1 2 5 10 2 0 5 0 100

Antibody concentrotion Ipg.mlil

Figure 4. Determination of the limiting steps in complement activation. Selected CD3 antibodies were compared for their abilities to activate human complement. (A) shows the autologous C‘ML of fresh human PBMC while (B) shows the amount of radioactive Clq and in (C) the amount of radioactive anti-C3d which could be bound to these cells. Two control antibodies were included: the potently lytic antibody CAMPATH-1G (0) and the poorly lytic CD45-specific rat IgG2b antibody YTH24.5 (A). The CD3 antibodies were the parental bivalent antibody YTH12 “a” (0) and the monovalent antibodies SHL45 “b” (W) and SHRl “a” (V).

Table 3. Summary of details of kidney organ transplant patients

Patient Age Sex Transplanted for Treatment (Yeam) with CD3

1 67 Male hypertensive renal failure 8 days at 5 mg/day

2 18 Female renal failure secondary to 10 days at glomerulonephritis 5 rng/day

3 19 Female renal failure secondary to systemic 9 days at lupus erythematosus 2 mg/day

4 29 Male diabetic renal failure 7 days at 2 mg/day

3.7 Serum antibody levels Table 5. Anti-globulin titers in kidney transplant patients

Serum samples were collected and stored for each patient at regular intervals during the course of treatment. The levels of free CD3 antibody in the serum were detected as described. Antibody levels in the serum were determined for samples taken prior to the daily dose of antibody and thus represent the level of antibody remaining from the previous daily dose. The results shown in Table 4 indicate that the doses of antibody given were sufficient to maintain a detectable level of serum antibody between the daily doses. As expected, higher levels were maintained in the patients given 5 mg daily doses but in all patients the serum levels were sufficient to have

Patient Titer Monovalent CD3 Irrelevant

(SHL45 b) Rat IgG&

1 28 42 2 1377 110 3 35 32 4 1025 72

Controls 4. 9, 22, 46, 79 21, 28, 34, 67, 83

achieved detectable C3 activation at the equivalent concentrations in vitro. Table 4. Serum Ig levels in kidney organ transplant patients

Patient Daily dose (mg) Serum CD3 antibody level”)

(CLg/~)

1 5 2 5 3 2 4 2

a) Mean f SD.

1.2 k 0.4 1.6 f 0.3 0.5 f 0.3 1.1 f 0.7

3.8 Anti-globulin response to monovalent CD3

The anti-globulin responses were determined for each patient. Two patients gave a demonstrable anti-globulin response which was more than tenfold higher than any of the control sera from normal donors, the other two patients showed no significant response above controls (see Table 5) . The anti- globulin responses in both patients were specific for the monovalent antibody used for therapy and only cross-reacted weakly on an irrelevant rat IgG2,, antibody. For both patients

Eur. J. Irnmunol. 1989.19: 381-388 Monovalent CD3 antibodies 387

20-

I: E

0

5 9 10-

1250

1000

f ’3 g

._ c + ._

500 u 0

250

0

0 10 20 220 Days Post Transplant

Figure 5. Reversal of kidney graft rejection with monovalent CD3 antibody. Shown is a flow chart for the treatment of patient 2 for cell- mediated rejection of a kidney graft. The serum levels of creatinine and urea are given at various time points up to 220 days following the transplant operation. Doses 5 mg of antibody were given on the days indicated by arrows at the top of the chart.

the anti-globulin was just detectable above background at 10 days after starting therapy and reached a peak at 20-30 days. In addition the sera from these two patients reacted with both the parental type bivalent CD3 (SHLA5 “a”) as well as the inactive form with two J558L-derived light chains (SHL45 “c”), suggesting that the response was predominately directed towards the CD3 heavy chain variable region. Anti-globulin responses of this type biased towards the idiotype have been reported by other groups using CD3 and T cell receptor- specific antibodies for therapy [24-271.

4 Discussion

In hybridoma cells expressing multiple Ig light and/or heavy chain genes, antibody molecules of different functional valen- cies for antigen are generated by the pairing of the different chains. In a previous study it was shown that from the culture supernatant of such a cell line a monovalent antibody could be enriched which was more lytic with autologous human complement than the conventional bivalent antibody. A simple explanation of this observation would have been the prediction that monovalent antibodies were more efficient at binding and at activating the early stages of the complement cascade. In this study we have described a number of experiments aimed at testing these predictions. A series of CD3 antibodies were generated by the cell fusion of the cell line YTHl2 (rat IgG2b CD3) with other Ig chain-producing cell lines. Different Ig- containing fractions were prepared by ion-exchange chromatography and tested in complement lysis and complement fixation. The best of these fractions was then prepared in a form suitable for the in vivo administration to a patient with a T lymphoma and also to patients with cell-mediated kidney graft rejection.

The first observation was that the valency of binding was not a sufficient criteria for improved complement lysis. Thus, two monovalent CD3 antibodies containing a mixed Fc region made up of two heavy chains of different isotypes were no more lytic than the parental bivalent CD3. One of these was

tested for complement fixation and the result indicated that while the antibody bound Clq very little bound C3 was detected. In contrast, a monovalent CD3 antibody with two identical rat Y2b heavy chains but two different light chains was found to be more potent than the parental bivalent CD3 in lysis. A study of complement fixation by these two antibodies led to an interesting conclusion.

In previous studies on human CML by mAb the amount of C3 associated with the cell correlated with lysis [15, 191. Here we found a very poor correlation between bound C3 and lysis. From the data presented several conclusions can be drawn. (a) With regard to C lq binding there was little difference between the CD3 antibody with one rat Y2b and one mouse y1 heavy chain compared with the antibodies with two rat Y2b heavy chains. Thus, only one rat Y2b heavy chain is essential for c l q binding. (b) Only the antibodies with two rat Y2b heavy chains went on to fix substantial quantities of C3 indicating a more stringent requirement than for C lq binding. (c) Despite the similar levels of cell-bound C3 the monovalent antibody with two rat Y2b heavy chains gave more lysis than the parental bivalent antibody. This argues for a step subsequent to C3 being the rate-limiting step in lysis for example the formation of the membrane attack complex itself. An alternative explanation is that it was not the rate of activation of complement which was rate limiting but instead the cells may have been triggered by antibody binding to the T cell receptor complex to be more resistant to complement lysis. A number of cell surface inhibitors of the complement cascade as well as membrane turnover have been implicated in the observed resistance of some leukocytes to CML [28-311. The CD3 molecule forms part of the T cell antigen receptor complex and is involved in the triggering of T cell responses which may also invoke the activation of mechanisms for the protection of the T cell from any nonspecific lysis. However, any explanation of the resistance of T cells to lysis by these CD3 antibodies must also take account of the observation that these same cells are simultaneously susceptible to lysis by the CAMPATH-1G antibody and, in addition, that the lysis by CAMPATH-1G is more efficient. Further experiments will be necessary to resolve these questions.

We conclude that the antibody with two similar rat Y2b heavy chains but two different light chains was the most effective in lysis and complement fixation. In view of the therapeutic rele- vance of CD3 antibodies for tumor therapy of T cell malignan- cies and also for immunosuppression of T cell-mediated graft rejection, a batch of this antibody suitable for in vivo administration was prepared. In a patient with a T lymphoma in leukemic phase a short course of antibody totalling 22 mg over 8 days was sufficient to eliminate circulating blasts from a starting cell count of 45 x 109/l. An improvement in the ratio of leukemic blasts to normal hemopoietic blasts was also seen in an examination of pretreatment vs. post-treatment marrow aspirates. There was no evidence for a return of CD3 antigen- modulated cells in this patient, suggesting that the cells were indeed being destroyed during the course of treatment.

The antibody was also used to treat biopsy-proven acute cellular kidney graft rejection. Rejection was reversed in all three patients who were evaluable for reversal of organ graft rejection. Two patients who received 5 mg daily doses both showed some reactions to the antibody on the 1st dose. These reactions were similar in nature to those reported for the majority of patients treated with the CD3 antibody OKT3. A larger

388

series of patients would determine if 2 mg or a lower dose would result in reliable immunosuppression without any of these classical early dose side effects associated with OKT3. The two patients who received 2 mg daily doses did not show such reactions, neither did a fifth patient who received just a single dose of 2 mg. Excess active antibody could be detected in the serum collected from patients 20 h after the previous daily dose implying that the doses given even at 2 mg/day were sufficient. Peripheral blood samples taken during the course of treatment showed no evidence for antigen modulation as there were no lymphocytes expressing the T cell markers CD5 and CD7 but lacking CD3. This together with the data from the lymphoma patient suggests that the monovalent antibody was very effective at killing the cells, and that this was responsible for its immunosuppressive function rather than any modulation or blocking of the T cell receptor.

M. Clark, C. Bindon, M. Dyer et al. Eur. J. Immunol. 1989.19: 381-388

The results of the in vitro studies suggest that events late in the pathway of complement activation may be rate limiting for lysis with some forms of mAb and that these merit further study. The in vivo studies with the most effective of these monovalent mAb were very encouraging and would seem to justify a more extensive trial.

We wkh to thank Mark Frewin and Pat Maher for expert technical assistance.

Received November 6, 1988.

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The improved lytic function and in vivo efficacy of monovalent monoclonal CD3 antibodies