Cancer ImmunolImmunother(1991) 32:364-372 034070049100014M

~

ancer mmunol9gy mmunotherapy

© Springer-Verlag 1991

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody* A. Hekman, A. Honselaar, W. M. J. Vuist, J. J. Sein, S. Rodenhuis, W. W. ten Bokkel Huinink, R. Somers, Ph. Rümke, and C. J. M. Me|ief The NetherlandsCancerInstitute,Antonivan LeeuwenhoekHuis, Plesmanlaan121, 1066 CX Amsterdam,The Netherlands Received 19 June 1990/Accepted30ctober 1990

Sulnmary. Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAh) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15-30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody-coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow, mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment. Key words: Immunotherapy - B cell lymphoma Monoclonal antibodies

Introduction Clinical trials have shown that administration of mouse or rat monoclonal antibodies (mAb) can induce tumour regression of lymphoid malignancies. However, responses were generally limited and of short duration [9, 11, 15]. This has focused attention on the use ofmAbs as carriers of * Supportedby the DurchCancer Society(grantNKI 84-14) Offprint requests to: A, Hekman,Divisionof Immunology,The Netherlands CancerInstitute,Antonivan Leeuwenhoekhuis,Plesmanlaan 121, 1066 CX Amsterdam,The Netherlands

cytotoxic agents, such as bacterial or plant toxins or radioisotopes, but the potential of unconjugated antibodies, dependent on physiological effector mechanisms, has certainly not yet been fully explored. Many factors may influence the effectiveness of mAb therapy, among them the nature of the target antigen, its density, distribution and tendency to modulate, the species and isotype of the antibody and the competence of host effector mechanisms. The relative importance of these factors and their interactions are still largely unknown. Malignancies of B lymphoid origin are unrivalled for evaluating the various aspects of mAb serotherapy, because these tumours express a number of well-characterized antigens, seem to be sensitive to immune manipulation and are more accessible than solid tumours, and patients are less likely to make anti-(mouse Ig) antibodies. The potential of unconjugated mAbs is exemplified by the 50% response rate (complete and partial remissions) of patients with B cell lymphoma treated with mAbs against the tumour idiotype [3], and by the impressive anti-tumour effects seen in two non-Hodgkin lymphoma patients after treatment with a "humanized" mAb, CAMPATH-1H, in which the Fc region of a routine antibody is replaced by a human Fc region [10]. This paper reports a study in which six patients with B cell lymphoma were treated with a mouse IgG2a mAb (designated CLB-CD19 [5], against the B-lymphocytespecific antigen CD19. This antigen, a 90-kDa glycoprotein, is present on the cell surface of normal as well as malignant B lymphocytes. It is one of the earliest B-cellspecific antigens to appear during B cell differentiation [32]. Plasma cells do not carry CD19 antigen, which may play an important role in B cell differentiation and proliferation, since anti-CD19 antibodies inhibit pokeweedmitogen-driven in vitro immunoglobulin production as well as in vitro B cell proliferation induced by anti-IgM antibodies [5, 20, 29]. These characteristics led us to investigate CD19 as a target for immunotherapy. Its early expression in B cell development makes it likely that less mature clonogenic tumour cells will express the target antigen [29]. At the other end of the spectrum of B cell differentiation, its absence from plasma cells will moderate

365 the e x p e c t e d e f f e c t o f t h e r a p y w i t h a m A b against a B cell a n t i g e n o n n o r m a l B c e l l f u n c t i o n and i m m u n o g l o b u l i n l e v e l s . C D 1 9 is n o t e x p r e s s e d o n h e m o p o i e t i c p r o g e n i t o r cells [ 2 7 - 29], e n s u r i n g that r e p o p u l a t i o n o f n o r m a l B l y m p h o c y t e s w i l l be p o s s i b l e . T h e m A b C L B - C D I 9 u s e d in this c l i n i c a l s t u d y has b e e n s h o w n to i n h i b i t t u m o u r g r o w t h o f D a u d i B u r k i t t l y m p h o m a x e n o g r a f t e d in n u d e m i c e [30]. T h e a i m o f this s t u d y w a s to d e t e r m i n e the k i n e t i c s , p e n e t r a t i o n to e x t r a v a s c u l a r t u m o u r sites, t o x i c i t y and p o s sible a n t i - t u m o u r e f f e c t s o f the m A b C L B - C D 19. A n initial g r o u p o f f o u r p a t i e n t s w a s treated a c c o r d i n g to a d a i l y escalating 4-day schedule. The dosage was based on our p r e v i o u s e x p e r i e n c e w i t h a n t i - i d i o t y p e m A b s [25] and d a t a r e p o r t e d f o r o t h e r m A b s [2, 16, 21]. I n i t i a l l y a d o s e was c h o s e n that c o u l d be e x p e c t e d to result in m A b e x c e s s in the c i r c u l a t i o n . T h i s d o s e w a s a d m i n i s t e r e d in an e s c a l a t i n g f a s h i o n , in o r d e r to start w i t h a l o w d o s e in the c a s e o f u n e x p e c t e d s i d e - e f f e c t s . O n the basis o f the results o b t a i n e d w i t h this s c h e d u l e the m A b w a s a d m i n i s t e r e d to later p a t i e n t s in l a r g e r d o s e s d i s t r i b u t e d o v e r a l o n g e r t i m e , since it is l i k e l y that l o n g e r c i r c u l a t i o n o f the m A b w i l l i n c r e a s e the o p p o r t u n i t y o f b i n d i n g to t u m o u r cells and result in b e t t e r p e n e t r a t i o n to e x t r a v a s c u l a r tissue.

Materials and methods mAb CLB-CD19. The mAb CLB-CD19 is directed against the B tymphocyte cell-surface antigen defined by the CD 19 cluster of mAbs [ 14]. The mAh does not react with tissues other than those rich in B lymphocytes. Myeloid and erythroid progenitor cells were not affected by preincubation of normal bone marrow with CLB-CD 19 and rabbit complement or depletion by immuno-rosetting [27]. Details of the characterization of mAb CLB-CD 19 have been published elsewhere [5]. From the series of isotype switch variants of the original IgG1 antibody the IgG2a isotype was used throughout this study. Large-scale production by hollow-fiber technology (Acusyst P, Endotronics, Coon Rapids, Minn.) and purification by ion-exchange chromatography was done under GMP conditions by Euroclone BV, Amsterdam. The purified mAb was sterile and free of pyrogens as tested by injection into rabbits of l mg/kg body weight. The mAb was diluted to 1 mg/tal in phosphate-buffered saline (PBS) with 5 mg/tal human serum albumin and stored at -70 ° C until use. lmmunofluorescence. Mononuclear cells were isolated from peripheral blood and bone marrow and from cell suspensions of metastatic lymph hode and other tumour tissues by centrifugation over Ficoll/Hypaque (Nyegaard, Oslo). Standard immunofluorescence techniques were used, with incubations of 30 min at 0 ° C and PBS containing 0.1% bovine serum albumin and 0.02% sodium azide as incubation and washing medium. The immunological phenotype was determined by means of the following antibodies: CLB-CD19, fluorescein-conjugated rabbit anti-(human IgM, IgD, IgG, ~; or ~) (Dako, Copenhagen) or mAbs against these immunoglobulins (CLB, Amsterdam) and mAbs against CD20, CD21 (B1 and B2, Coulter), CD24 and CD9 (BA1 an BA2, Hybritech) and CD56 (Leul9, Becton Dickinson), CD45, CD25, CD27, CD28, CD22 (CLB), CD3, CD4, CD8 (Ortho or CLB), CD14 (LeuM3, Becton Dickinson). As second antibody for indirect immunofluorescence fluorescein-conjugated goat F(ab)2 anti-(mouse lgG) (absorbed with human Ig, Tago, Burlingame, Calif.) (GAM-FITC) was used. Staining was observed with a Leitz fluorescence microscope and flow cytometry was carried out on a Becton Dickinson FACS IV or FACSCAN apparatus. Coating of tumour cells and normal B cells in vivo by infused mAb was detected by incubating ceI! suspensions with GAM-FITC. To assess the degree of saturation the mean fluorescence intensity obtained with GAM-FITC alone was compared to the intensity of cells incubated with

excess CLB-CD 19 in vitro followed by GAM-FITC. Changes in antigen density during mAb administration were determined by comparing the mean fluorescence intensity of cells taken during treatment after incubation with CLB-CD19 in vitro with the mean fluoresceuce intensity of pretreatment cells. Modulation in vivo was expressed as the percentage reduction of mean fluorescence intensity, using cryopreserved cells in a single experiment. A fluorescence-inhibition assay was used to test for CD 19 antigen in soluble form: CLB-CD 19 (2 gg/tal) was incubated with an equal volume of patient serum s amples for 30 min at 4 ° C before being added to CD 19+ cells (Daudi or Jiyoye cell tines) in an immunofluorescence test. In the case of patient 6 a mAb against the idiotype of the tumour cell immunoglobulin [24] was used to follow the serum concentration of idiotypic lg. A finorescence-inhibition assay was performed by incubating a nearly saturating amount of anti-idiotype mAb with a series of dilutions of serum samples of the patient for 30 min at 4°C before addition to idiotype-bearing tumour cells for indirect immunofluorescence. Relative concentrations of idiotypic Ig were detennined from the serum dilutions causing 50% reduction of mean fluorescence intensity. Cell culture. Cell lines were cultured in Dulbecco's modified minimal essential medium (Flow) supplemented with 10% fetal calf serum and antibiotics (penicillin 100 IU/ml and kanamycin 100 gg/tal). The same medium was used for proliferation and cytotoxicity assays. Immunohistology and immunocytology. Tissue sections prepared from frozen tissues and cell smears were stained with mAbs against B and T cell markers listed above, followed by alkaline-phosphatase-labelled anti-(mouse Ig) by Dr. T. Vroom, Dept of Pathology, Slotervaart Hospital, by methods previously described [8]. Concentration of fi'ee CLB-CD19 in the serum. Unbound mAb concentrations in serum were determined by two methods. (a) Indirect immunofluorescence was pefformed by incubating several dilutions of the test serum with Jiyoye cells, a Burkitt lymphoma cell line with high CD19 expression, and staining with GAM-FITC. Mean fluorescence intensity was interpolated on a standard curve of intensities obtained with known concentrations of CLB-CD19. (b) A double-determinant enzyme-linked immunosorbent assay (ELISA) was performed by incubating test serum samples in microtiter plates (Falcon 3911) coated with goat anti-(mouse Ig) (Tago, 10 gg/tal in carbonate buffer pH 9.6) and developing with peroxidase-conjugated goat anti-(mouse Ig) (Tago) followed by 0.1 mg/tal 3,3',5,5'-tetramethytbenzoic acid with 0.03 mg/tal H202 in sodium acetate/ciüic acid buffer pH 6.0. Between incubations plates were washed with PBS containing 0.1% (w/v) gelatin and 0.05% (v/v) Tween 20. Absorbances were measured in a Titertek Multiscan ELISA reader (Flow) at 450 um. Concentrations of mAb were calculated from the absorbance at 450 nm and a reference curve of known concentrations of CLB-CD19. The'anti-(mouse Ig) antibodies used in these assays had been absorbed with human immunoglobulins. Antibodies against mouse immunog lobulin. Microtiter plates were coated with CLB -CD 19 (10 gg/tal in PB S). After incubatiou with patient's sera, binding of human antibodies was detected with peroxidase-conjugated sheep anti-(human Ig) absorbed with mouse immunoglobulin (Amersham) and tetramethylbenzoic acid as described above. Patient characteristics. Patients were eligible for this trial if they had histologically confirmed, progressive and measurable B cell lymphoma and had fafled previous conventional therapy (chemotherapy or radiotherapy). They were not required to be resistant to standard chemotherapy. Patients had to have a performance status ~2.5x109/1, and platelets >50 × 109/]. They sho~ld have no concomitant serious medical problems. Details of the patients in this study are shown in Tables 1 and 2. All patients had stage IV non-Hodgkin's lymphoma and two had high leukemic levels of circulating lymphocytes (Table 2). Previous treatment included chemotherapy, radiotherapy or both (details in Table 1), but all treatment had stopped at least 4 weeks before entry into this study.

366 Pretherapy evaluation included physical examination with measurement of all accessible lesions, serum biochemistry, differential blood counts and immunological phenotyping, coagulation tests, urinalysis, complement and immunoglobulin levels, chest X-ray, and ECG. Tumour mass was evaluated by clinical measurement of accessible lymph nodes and skin lesions and by computed tomography, and expressed as the sum of the projected surface areas. Bone marrow aspiration and fine-needle biopsy of lymph node and other organs when appropriate were pefformed for immunopathology and surface marker determination. Written informed consent was obtained from all patients prior to entry in the smdy, which was approved by the Ethical Committee of the Netherlands Cancer Institute.

Study design, mAb was administered by i.v. infusion over 4 h, initially daily for 4 days and at 3 - 7 days intervals in the later treatments, Before each infusion the patients were skin-tested with 2 gg mAb in 0.1 ml PBS for immediate hypersensitivity to the mouse protein and received 500 mg acetaminophen and 25 mg diphenhydramine. Patients were closely monitored during treatment for evidence of anaphylaxis or complications of cell destruction, such as hypotension, fever, disseminated vascular coagulation etc. Appropriate clinical chemistry determinations were done at regular intervals. Differential blood cell counts were performed before and after each mAb infusion and at regular intervals thereafter, when blood samples were also obtained for surface marker determina-

tions and free serum mAb assays. Bone marrow aspiration and fineneedle biopsies of lymph nodes or other tumour biopsies were done after the end of the last infusion. Tumour mass was evaluated regularly. Standard criteria of response were used [31 ]. Serum immunoglobulin and complement levels were determined before and at the end of the treatment period and then at approximately monthly intervals for 6 months.

Results T h e m A b C L B - C D 19 has b e e n a d m i n i s t e r e d to six patients w i t h B cell n o n - H o d g k i n ' s l y m p h o m a stage I V , one o f w h o m r e c e i v e d t w o t r e a t m e n t s w i t h m A b w i t h an i n t e r v a l o f 8 m o n t h s . Details o f the patients are s u m m a r i z e d in T a b l e s 1 and 2. A l l p a t i e n t s h a d w i d e s p r e a d disease; t w o w e r e l e u k e m i c w i t h w h i t e c e l l c o u n t s a b o v e 90 × 109/1. T u m o r cells in all c a s e s e x p r e s s e d the B cell a n t i g e n s C D 1 9 and C D 2 0 , as w e l l as s u r f a c e i m m u n o g l o b u l i n . Surf a c e Ig was a l w a y s I g M , w i t h a s i n g l e light chain. T u m o u r cells f r o m f o u r patients also e x p r e s s e d I g D w i t h the s a m e l i g h t chain.

Table 1. Summary of patient characteristics Patient age (years)/sex

Histological classificationa

Previous therapy u

Tumour localization

1 (67/F)

Diffuse small cleaved cell

CVP, chlorambucil prednisone

Lymph nodes, bone marrow, colon, rectum/sigmoid, tonsils, spleen

2 (67/M)

Diffuse small cleaved cell

CVP, ara-C, iphosphamide + VP- 16 cytosar + mithoxanthrone, spirogeranium, chlorambucil, radiotherapy

Lymph nodes, bone marrow, conjunctivae, tonsils, spleen

3 (68/F)

Follicular, predominantly small cleaved cell

Radiotherapy

Lymph nodes, bone marrow, skin

4 (63/M)

Small lymphocytic consistent with B-CLL

Chlorambucil, gastrectomy, radiotherapy

Lymph nodes, bone marrow, stomach, spleen

5 (6 l/F)

Small lymphocytic plasmacytoid

Chlorambucil

Lymph nodes, bone marrow, stomach, conjunctivae, skin

6 (52/M)

Follicular and diffuse predominantly small cleaved cell

CHOP, CVP, splenectomy chlorambucil

Lymph nodes, bone marrow

a Working formulation B-CLL, B cell chronic lymphatic leukemia u CVP: cyclophosphamide, vincristine, prednisone; ara-C: cytosine arabinoside; CHOP: cyclophosphamide, doxorubicin, vincristine, prednisone

Table 2. Pretherapy laboratory data Patient

1 2 3aa 3bd 4 5 6

WBC 109/1 (1-1)

Lymph 109/1 (1-1)

Gran 109/1 (1-I )

Platelets 109/] (1-1)

Hb (mmol/1)

2.4 2.9 2.9 5.1 96.2 7.9 138.0

1.4 0.7 0.6 1.0 86.6 1.2 129.0

0.8 0.8 1.7 3.2 7.7 4.9 5.5

58 128 123 130 115 335 249

6.3 7.4 7.7 7.9 7.8 6.7 8.1

a Normal value 45-335 IE/ml b Normal value 80-220 IE/ml

Serum immunoglobulin

CD19 + cells (%)

IgM a

IgGb (IE/ml)

IgAc

Blood

Bone marrow

110 30 690 475 100 5200 314

200 110 110 87 270 130 103

210 80 110 84 140 30 38

27 25 22 16 90 20 94

87 65 7 8 95 64 72

c Normal value 40-265 IE/ml d First (a) and second (b) course of treatment

367 Table 3. Schedules of treatment with anti-CD19 monoclonal antibody Patient

Duration of therapy (days)

Days on which therapy was given

Individual doses (mg)

Total dose (mg)

1 2 3aa 4 5 6 3ba

4 4 4 4 18 22 i1

1,2,3,4 1,2,3,4 1,2,3,4 1,2,3,4 1,4,8,11,15,18 1,8,15,22 1,4,8,11

15,30,60, 120 15,30,60,120 15,30,60,120 15,30,60, 120 60 250 250

225 225 225 225 360 1000 1000

a First and second course of treatment

,o.

Absence of soluble CD19 antigen

UUII 15

40-

30

[J 60

A 1 2 0 mg C L B - C D 1 9

30-

20-

10

0

m 0

2

~'°11

4

U

U U

!

i

i

6

8

10

i

12 days 14

11 U

6 x 60 mg CLB-CD19

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Treatment schedules and serum levels of CLB-CD19

20

\

10

i

0

_ E

The occurrence of CD19 antigen in soluble form in the serum was tested by a fluorescence-inhibition assay, in which a nearly saturating amount of CLB-CD 19 was incubated with patient's serum before indirect immunofluorescent staining was performed. This test mimicks the potential inhibiting effect of soluble antigen on binding of infused mAb to target cells. No decrease in mean fluorescence intensity was seen using serum from the patients in this study. Similarly, no free CD19 antigen was detected in the serum of ten other patients with different B cell malignancies, including some with massive tumour or with high levels of idiotypic immunoglobulin in the serum (data not shown).

150

10

U II

II

20

days

, x~~o~~ c~~.co~~

30

c

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100

50

0 T0



~ 10

~ 20

~" days

3O

Fig. 1. Concentration of CLB-CD19 in the serum resulting from different treatment schedules determined by the fluorescence-inhibition test (A-C) and by enzyme-linked immunosorbent assay (. . . . , A). A Patient 4, doubling doses on 4 days; B patient 5, 60 mg twice a week; C patient 3, second treatment, 250 mg twice a week

m A b treatment was given in different dosages and time schedules (see Table 3) aimed at saturation of binding sites, also in extravascular tumour localizations [3, 21]. Two assays were used to determine mAb concentrations in the patients' serum during and after treatment: indirect immunofluorescence and a sandwich ELISA. Whereas the immuno-fluorescence test only determines antibody capable of binding to antigen, the ELISA detects immune complexes and inactive or partially degraded mouse immunoglobulins as well. The two assays yielded similar results, as illustrated in Fig. 1 A, indicating the absence of immune complexes and inactive mAb. In patients 1, 2, 3 and 4, who received 15 mg, 30 mg, 60 mg and 120 mg mAb on 4 consecutive days, free m A b was detected in the serum after the first 15-mg infusion. The concentration of m A b increased with higher doses, reached a peak after the fourth infusion and then declined to undetectable levels in 7 - l 3 days with serum elimination half-lives varying from 22 h to 60 h. A typical serum concentration curve is shown in Fig. 1 A. The maximum concentrations reached and the estimated serum elimination half-lives are summarized in Table 4. Based on these results other schedules were devised, attempting to prolong the exposure to mAb. Patient 5 was treated with 60 mg mAb twice a week during 3 weeks. This proved to be just sufficient to keep the m A b continuously detectable in the circulation (Fig. 1 B). Two patients received a total dose of 1000 mg CLB-CD19 divided into 250 mg given once every week (patient 6) or

368 Table 4. Serum concentrations of CLB-CD19 ~o

Patient

1

2 3ab 4 5 6 3b~

Max.a on day 4 (gg/ml)

Peak concentrations after infusions (~tg/ml)

68 45 47 41 13,14,18,21,12,13 25,38,43,25 64,98,75,130

LI Ull U

100 '

15 30 60 120 mg CLB-CD19

tl/2 (h) 38 38 6O 24 22 21 48

Maximalconcentration was reached 2-4 h after the end of the infusion b First and second course of treatment a

twice a week (patient 3 at the second course of treatment). One week after a dose of 250 mg the m A b was barely detectable in the circulation of patient 6, so that no dose-accumulative effect was obtained. When 250 mg mAb was given twice a week (patient 3, second treatment) a cumulative effect was seen (Fig. 1 C). Results of these extended administration schedules are also shown in Table 4.

80'

ô

g

60'

40'

20'

Fig. 2, Effect of infusions of CLB-CD19 on blood lymphocytecounts of patient 4 (lymphocyteswere >90% CD 19+ CD20+ ~+)

-~~ ";

60 120 mg CLB-CD19

~~o o0

E

20.

I

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0

0

Effect of CLB-CD19 on peripheral blood lymphocytes The effect of mAb infusions on circulating tumour cells could be determined most clearly in the leukemic patients 4 and 6. Patient 4 was treated with the escalating schedule. The first mAb infusion of 15 mg reduced the number of peripheral lymphocytes (>90% lymphoma cells by phenotype) by 85%, but the higher doses on subsequent days had progressively smaller effects (Fig. 2). Cells remaining in the blood after mAb infusion were incubated with GAMFITC to detect in vivo binding of mAb, and with excess CLB-CD19 and G A M - F I T C to determine total CD19 antigen density. From the moment after the 30-mg infusion on day 2 there was no difference between the fluorescence intensity obtained with or without incubation with CLBC D I 9 in vitro, indicating that all available binding sites had been saturated in vivo (see Fig. 3). Even on day 11, cells coated with mAb (but not to saturation) were still present in the circulation. Mean fluorescence intensity as a measure of CD 19 antigen density rapidly decreased during the first days to 30% of the original value, but then remained stable and did not decrease further. These partially modulated, antibody-coated cells remained present in the circulation for 6 days. On day 11, when free mAb was no longer detected in the serum, antigen density started to increase. These results are illustrated in Fig. 3 and in the left column of Fig. 4. Patient 6 received a total of 1000 mg CLB-CD19 in four doses of 250 mg at weekly intervals. Before mAb treatment the peripheral blood cell count was rising rapidly, from 34.9 x 109/1 to 138.0 × 109/1 in 5 weeks. Infusions of CLB-CD19 resulted in the coating of all B cells to saturation during the period from the first infusion to 1 week after the last dose, but had only minimal effect on the number of cells. Only the rate of increase seemed to

;2

;

;

"

ä

"

l'e

"

1'2

" days

Fig. 3. CD19 expression and antibody binding in vivo determined by immunofluorescence on blood lymphocytes of patient 4 during mAb treatment. - - - - , Mean fluorescence intensity of cells incubated with fluorescein-conjugated goat F(ab)2 anti-(mouse Ig) (GAM-FITC) only (to detect mouse antibody bound in vivo), - - - , mean fluorescence intensity of cells after additional incubation in vitro with CLB-CD19 followed by GAM-FITC (to determine total binding capacity)

slow during the 4 weeks of treatment. In this case antigen density did not change significantly. The other patients had low to normal lymphocyte counts with 2 0 % - 3 0 % CD19 ÷ and CD20 ÷ cells. Determination of changes in these small numbers of cells were considered too inaccurate for meaningful interpretation.

Penetration of CLB-CD19 to extravascular tumour Cells from bone marrow and tumour lymph nodes (by fine-needle biopsy) were obtained before and on the last day of treatment. Patients had various degrees of bone marrow involvement (Table 2). Cell suspensions were tested for the presence of in vivo bound mAb, degree of saturation and antigen density. After infusion of 225 mg mAb antibody-coated cells were found in the bone marrow. Penetration to lymph nodes of this dose was more variable. Patient 1 had no lymph node cells coated with mAb, but mAb was detected on most lymph node B cells of patients 2 and 4. No lymph node material was obtained from patients 3 and 5. Data from patient 4 are shown in Fig. 4, illustrating the different degrees of penetration to bone marrow and lymph node after a total dose of 225 mg mAb. Whereas nearly all CD 19-binding sites on cells from

369 BLOQO

BONEMARROW

LYMPHNODE i

~L

?,

,Il I f'uorescence ,ntens,~y

-'r

cell suspension consisted of 60% tumour B cells, staining both with anti-CD20 and with a mAb raised against the idiotype of this lymphoma (Fig. 5 B, C) and 40% T cells reacting with anti-CD3 mAb (Fig. 5 D). All B cells stained with GAM-FITC, and addition of CLB-CD 19 to the lymph node cells in vitro did not increase the fluorescence intensity beyond that obtained with GAM-F1TC alone, indicating saturation of all CD19 binding sites. Similarly, in patient 3, 1000 mg CLB-CD19 given over 2 weeks in the second treatment essentially saturated the lymph hode tumour cells. Patients 3 and 5 had tumour cell infiltrates in the skin, and patient 2 mmour involvement of conjunctivae. Biopsies of these tumours showed penetration of mAb to these sites (data not shown).

,

Fig. 4. Irnmunofluorescence test with cells from blood, bone marrow and lymph node of patient 4 on day 0 (before treatment), day 4 (after the last infusion) and day 11. CLB-CD19 infusions were given on days 1, 2, 3, and 4. Cells were incubated in vitro with GAM-FITC alone to detect mouse antibody bound in vivo (. . . . ), or with excess CLB-CD19 and GAM-F1TC to determine total binding capacity ( --). Fluorescence intensity is given on the horizontal axis on a logarithmic scale

c1

IJ

D

fluorescence

intensity

Fig. 5. Immunofluorescence test with lymph hode cells from patient 6, obtained on day 22 after the fourth infusion of 250 mg CLB-CD l 9. Cells were incubated in vitro with GAM-FITC alone to detect mouse antibody bound in vivo ( - - - - , repeated in all panels for comparison), or with excess CLB-CD19 (A), anti-CD20 (B), anti-tumour idiotype (C) or anti-CD3 (D). Fluorescence intensity is given on the horizontal axis on a logarithmic scale

borte marrow were saturated, cells from the lymph node were only partially coated. CD19 density on bone marrow and lymph node cells was slightly less than before treatment, but the difference was less marked than on the blood lymphocytes. With patient 6, who received four weekly doses of 250 mg CLB-CD19, this quantity was found to saturate essentially all CD19-binding sites in a lymph node obtained after the fourth infusion, despite the very high number of circulating B cells. As shown in Fig. 5, the lymph node

Toxicity No clinically significant toxicity was seen, although most patients complained of feelings of fatigue. A minor rise in temperature (0.3-0.5 °C) occurred frequently, but only on two occasions did fever to grade I develop (according to the WHO toxicity grades) and once to grade Il (38.2 ° C). Infusion of mAb was associated with thrombocytopenia only in patient 4, where the platelet count decreased from 115 x 109/1 to 76× 109/1 with the first infusion. Platelets had retumed to normal values 1 month after treatment. Patient 1 started with a thrombocytopenia (58 x 1091), which did not change during treatment. Except for lymphocytes all other hematological parameters remained constant. Liver and kidney function remained invariably stable in all patients. In patients 1 and 2 we observed a disappearance of an existing grade I proteinuria. Patient 4 twice showed a transient drop in blood pressure (grade I), accompanied by feelings of lightheadedness. There were no heart problems. During treatment the two leukemic patients 4 and 6 got erythematous macular skin lesions with pruritis. A biopsy of a skin lesion of patient 6 showed diffuse small-cleaved cell lymphoma and a deposition of human IgM complexes around the blood vessels of the skin. No biopsy was done of patient 4. No allergic reactions were observed. Tests of complement components, and CH50 and Clq binding showed some fluctuations over time, but no significant changes correlated with therapy. The patients treated according to the 4-day escalating schedule were hospitalized during this period, but the later patients who received the m a h once or twice a week were treated on an out-patient basis. None of the patients made antibodies against mouse IgG detectable in ELISA with CLB-CD19-coated plates. Before the second treatment of patient 3 her serum was also tested in a fluorescence-inhibition assay, to detect possible antibodies directed against the idiotype of CLB-CD 19 that might inhibit the binding of CLB-CD 19 to target cells, and could be below the detection limit of the ELISA. The assay was similar to that used in the tests for soluble CD19 antigen. No inhibition of the binding of CLB-CD19 to Jiyoye cells was observed with any serum sample from patient 3.

370 Table 5. Results of therapy Patient

1

Total dose mAb (mg)

Duration of Clinical response treatment (days)

225

4

2 3aa 3ba 4 5

225 225 1000 225 360

4 4 11 4 18

6

1000

22

Blood B lymphocyteswere decreasedfor 6 weeks Progression after 1 month Partial remission3 months Partial remission9 months No change for 4 weeks Regression of lymphomas of the skin during 3 months Progressionduringtreatment

long the exposure to mAb. All evaluable lymph nodes regressed again to an average of 50%. At that level disease remained stable for 9 months. Concomitant with the decrease of lymph node size, the relative number of monoclonal B cells in the lymph node decreased from 60% before treatment to 25 % 1 month after the end of treatment, as determined by cytofluorography on cell suspensions from fine-needle biopsies of the same lymph node. T lymphocytes in the post-treatment lymph node consisted of 80% CD8 + and 20% CD4 + cells. Patient 5 had a regression of skin lesions, but no reduction of tumour at other sites. None of the other patients showed an objective response (Table 5).

a First and secondcourseof treatment

Discussion Effect on immunoglobulin leveIs Immunoglobulin levels were followed as a measure of the effect of CLB-CD19 on the function of the normal B cell compartment. Serum concentrations of IgM, IgG and IgA were determined before and at the end of treatment and then at monthly intervals for 4 - 6 months. Some of the patients had low or slightly below normal levels before treatment, but with one exception no significant decline of normal Ig was seen. In the serum of patient 3 the elevated polyclonal IgM concentration of 690 IE/ml (normal value 4 5 - 3 3 5 IE/ml) steadily decreased over the period of both mAb treatments to 330 IE/ml. No infections occurred. In two of the five patients who had a serum paraprotein the concentration of the paraprotein decreased. In the case of patient 6 the Ig produced by the tumour could be detected by means of a mAb against the idiotype of the tumour cell surface Ig. In a fluorescence-inhibition assay the relative levels of idiotypic Ig were determined from the serum concentrations causing 50% reduction of mean fluorescence intensity of the anti-idiotype mAb. Shortly after treatment the concentration of idiotypic Ig was reduced to 50% of the pretreatment level. Interestingly, this decrease continued, until 6 weeks later the serum contained only 20% of the pretreatment concentration. Longer follow-up was not possible because other treatment became necessary. Over the same period the concentration of serum IgM decreased by 70% and the Bence Jones protein in the urine became undetectable. In the case of patient 5 the paraprotein component, as determined by gel electrophoresis, and the Bence Jones protein in the urine decreased by 20%.

Clinical response One of the six patients in this trial achieved a partial remission. After the first treatment of patient 3 with 225 mg mAb the lymph nodes regressed during 4 weeks, but began to increase in size after a stable period of 2 months. It was decided to treat the patient, who had received no other therapy in the mean time, again with CLB-CD19, 8 months after the first mAb treatment. In the second course of treatment 1000 mg mAb was given over 2 weeks, to pro-

This paper reports our initial clinical experience with a mAb against the B-cell-specific antigen CD19 in patients with advanced B cell lymphoma. CD19 antigen does not seem to be shed or secreted by normal or malignant B cells, since no free CD19 antigen could be detected in the serum. As a result, doses as low as 1 5 - 3 0 mg were sufficient to coat circulating tumour cells and to yield low but detectable levels ( 1 - 5 gg/ml) of free mAb. The final serum concentrations reached depended on the dose and the administration schedule. The dose-dependent serum levels of free CLB-CD19 and the estimated serum elimination halflives of 2 1 - 6 0 h are in the same range as those reported by Press et al. [21], who used a mAb against another B-celldifferentiation antigen, CD20. The initial 4-day doubling schedule resulted in reproducible peak blood levels and circulating mAb for 7 - 1 0 days. At this dose, mAb was found on bone marrow cells, but penetration into lymph nodes was variable and never saturating. Instead of increasing the dose in the same schedule we explored the effects of giving the higher dose over a longer period to prolong the exposure to mAb. The continuous presence of circulating mAb was obtained by a doses of 60 mg twice a week or 250 mg once a week, with better penetration into lymph nodes. Variations of this extended schedule were only tested in single patients, but the results are in reasonable agreement and provide a basis for larger trials. Patients differed in total tumour burden and in the number of circulating cells binding the mAb, which complicates the comparison of the dose levels, but from the degree of extravascular tissue penetration it may be concluded that a dose of at least 1000 mg seems to be necessary to saturate all tumour localisations. This observation is in agreement with other mAb trials in lymphoma [2, 3, 16, 21, 25]. However, most trials did not show a correlation between dose of mAb and clinical response. Antibodies against CD 19 can induce modulation of the antigen, as has been shown in vitro for CLB-CD19 [4] as well as for other anti-CD19 mAbs [23, 29]. However, the observations on the peripheral lymphocytes and lymph node cells of patients 4 and 6 indicate that this does not imply that complete modulation of CD19 antigen on tumour cells will necessarily occur in vivo, even in considerable antibody excess. Clearly, tumour cells of individual

371 patients can differ considerably in rate of modulation and/or reexpression of the antigen. Only one patient in this study (patient 3) achieved a partial remission. A second treatment with CLB-CD19 after relapse again resulted in a partial remission, indicating that no resistance to the action of the m A b had developed. Although no conclusions can be drawn from this small group of patients, it is interesting that the responding patient was the only case of follicular lymphoma without diffuse elements in this group. Moreover, this patient had the smallest total tumour mass and had received no previous chemotherapy. These factors may all have contributed to the response to m A b treatment. Also patient 5, who had a response in the tumour infiltrates in the skin, had received only limited prior chemotherapy, in contrast to the other four patients. Treatment with CLB-CD19 induced no serious toxicity, an experience similar to that in most other trials of mAh therapy. No direct assays of the effect on normal B cell function could be performed, because of the very low numbers of normal B lymphocytes that could be obtained from these patients, even before the m A b infusions. Levels of normal immunoglobulins did not change during or after treatment. In contrast, the idiotypic IgM of patient 6 decreased steadily over 6 weeks, and the paraprotein of patient 5 diminished to a lesser extent. This could reflect a reduction of tumour mass, but in the case of the rapidly progressive patient 6 this seems unlikely. Alternatively, CLB-CD19, which has been shown to inhibit pokeweedmitogen-driven immunoglobulin synthesis in vitro [5], may have inhibited the production of immunoglobulin by the tumor cells in vivo. None of the patients made antibodies against the mouse immunoglobulin. This agrees with the observation in other m A b trials that patients with B cell malignancies develop anti-(mouse Ig) antibodies much less frequently than patients with T cell malignancies or with solid tumours [9]. The absence of an anti-(mouse Ig) response makes it possible to repeat or to extend the treatment over longer periods. With a few notable exceptions [3, 10, 16], most immunotherapy trials with unmodified mAbs have so far yielded only limited responses. Various approaches are being tested to improve the activity of mAbs (recently reviewed in [9, 11]. Large doses of a 131I-labelled m A b against a B cell antigen (CD37) resulted in four complete and one partial remission in five patients treated after careful dosimetry studies [22]. However, severe myelosuppression occurred in all patients. CD19 may be a suitable target for toxin conjugates, becanse it internalizes after modulation [4, 23]. Recombinant D N A technology has opened the possibility of altering the biological effects of unconjugated mAbs by replacing the Fc region. The humanized mAb CAMPATH-1 proved to be superior to the original rat m A b in its therapeutic effects in non-Hodgkin lymphoma [6, 10]. Combination with other biological response modifiers may be another was to augment the therapeutic effect. Intefleukin-2 has been shown to enhance the antibody-dependent cell-mediated cytotoxicity of Fc-receptorbearing lymphocytes at much lower concentrations than are required for lymphokine-activated killer cell induction [12, 1 7 - 1 9 , 26]. In vivo combination of mAb with recom-

binant interleukin-2 (rIL-2) showed a synergistic effect in therapy of several murine tumours [1, 7, 13]. We recently reported that the activity of CLB-CD19 in the Daudi xenograft model was strongly enhanced by combination with rIL-2, which by itself had no effect on tumour growth [30]. It may thus be expected that the combination of mAb and rIL-2 can be effective with lower doses of rIL-2 than are currently used, with correspondingly lower toxicity. The findings of this trial with CLB-CD 19 alone will form the basis of a larger trial of the combination of this m A b with rIL-2 for B cell lymphoma.

Acknowledgements.We thank Dr. Menno A. de Rie (CLB) for providing the monoclonal antibody CLB-CD19 and for many stimulating discussions. We are grateful to Dr. S. P. Israels and Dr. J. M. V. Burgers for referring patients and to the staff of the Clinical Research Unit for their assistance in this trial. We thank Dr. Thea Vroom for performing the immunohistology,Mark Dessing for help with the flow cytometry,Marie Anne van Halem for secretarial assistance and Dr. Elaine Rankin for critically reading the manuscript. We also acknowledgethe help of many members of the departments of Haematology (in particular Mieke Egbers), Clinical Chemistry and Pathology.

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Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody.

Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differe...
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