processing of antibodies bound to b-cell lymphomas and other hematological … · and other...

[CANCER RESEARCH 56. 3062-3068. July 1. 1996]

Processing of Antibodies Bound to B-Cell Lymphomas and OtherHematological Malignancies1

Rafik Hanna, Gaik Lin Ong, and M. Jules Mattes2

Garden State Cancer Center. Center for Molecular Medicine and Immunology, Newark, New Jersey 07103

ABSTRACT

In an attempt to recognize general patterns in the processing of Abs(antibodies) bound to the surface of tumor cells, eight monoclonal antibodies were tested on 10 hematological malignancies of various histolÃ³gica! types. The results were compared with previous findings obtainedwith carcinomas, melanomas, and gliomas, using some of the same antibodies. The data demonstrated that some B-cell lymphomas appear to be

unusual in that Abs were unable to bind to them irreversibly; except forthose Abs that were rapidly internalized, none of the Abs tested was ableto bind irreversibly to the B-cell lymphomas Raji or RL. In contrast, most

Abs bound irreversibly to the tumors of other histolÃ³gica! types, includingother hematological tumors. Irreversible Ab binding to B-cell lymphomaswas achieved by cross-linking the Abs on the cell surface. Such differences

between cell lines may be due to differences in the supramolecular structure of the surface membrane, which affect the frequency or stability ofbivalent Ab binding. The Ab binding interaction could not be described interms of "functional affinity." These results may lead to improvements in

the use of Abs for tumor immunotherapy and for other purposes.

INTRODUCTION

To optimize the use of Abs3 for cancer therapy, it is essential to

understand the fate of Abs after binding to the cell surface. This isparticularly true for radioimmunotherapy, in which the radiation dosedelivered to the tumor depends on the length of time the radiolabelremains at the tumor site. We previously investigated the fate of Absbinding to adherent tumor cells in vitro, including cell lines derivedfrom carcinomas of various histological types, melanomas, and gliomas (1, 2). The data demonstrated that most Abs bind irreversibly tothese target cells and remain on the cell surface until they are internalized and catabolized. The half-life for catabolism was 2-3 days and

was similar for many different Abs; it seemed reasonable to attributethis process to the normal turnover of the cell surface constituents. Asmall fraction of the bound Ab, generally 10-20%, dissociated intact

from the cell. This dissociation was near complete in 4 h, and wesuggested that it might be due to monovalently bound Ab. In thisreport, we describe the extension of these studies to B-cell lymphomasand other hematological malignancies. Our interest in B-cell lympho

mas is based on two factors: (a) this tumor type has been treated moreeffectively by Ab-based therapy than any other tumor type to date(3-7); it is, therefore, important to optimize the therapeutic approach.

This study includes Abs that have been widely used for immunotherapy, i.e., Abs to CD19, CD20, and CD22 and the Ab Lym-1 reacting

with the MHC class II antigen; and (Â¿>)previous investigations in otherlaboratories (8, 9) have addressed the processing of Abs by B-cell

lymphomas and have reached conclusions that appear to be discordantwith our results (using adherent tumor cell lines). One major difference is that, with B-cell lymphomas, many Abs were considered to be

Received 2/5/96; accepted 4/26/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by NIH Grants CA63624 and RR05903.2 To whom requests for reprints should be addressed, at Center for Molecular Medicine

and Immunology. 1 Bruce Street. Newark. NJ 07103.' The abbreviations used are: Ab, antibody; ATCC. American Type Culture Collec

tion; TCA. trichloroacetic acid.

not significantly internalized. Therefore, it seemed interesting to investigate this tumor type, using methods similar to those we had usedpreviously. This comparison was facilitated by the use of broadlyreactive Abs that react with tumors of most or all histological types.Ab MA 103 is extremely useful in investigations of this type, since itreacts strongly with every human cell line that has been tested (>200;Ref. IO).4

MATERIALS AND METHODS

Cell Lines and Antibodies. The B-cell lymphoma RL (11) was obtainedfrom Dr. J. Gribben (Harvard Medical School, Boston, MA), and the pre-B-cellleukemia Nalm-6 was obtained from Dr. R. Stein (Center for Molecular

Medicine and Immunology). All other cell lines were obtained from the ATCC(Rockville, MD). These included the B-cell lymphomas Raji and Daudi. theT-cell leukemias MOLT-4 and CCRF CEM, the myeloid leukemias K-562 andHL-60, the monocytoid lymphoma U-937, and the myeloma U266B1. They

were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY)supplemented with 12.5% fetal bovine serum, penicillin, streptomycin, gluta-

mine, and sodium pyruvate (Life Technologies).IgG2a Abs W6/32, reacting with a nonpolymorphic determinant of MHC

class I, and MA103 were described previously (1). Ab 1F5, an IgG2a reactingwith CD20 (12), was provided by Dr. E. A. Clark (University of Washington,Seattle, WA) and was also prepared from hybridoma cells obtained fromATCC (HB#9645). YTH24.5, a rat IgG2b reacting with CD45, was purchasedfrom Bioproducts for Science (Indianapolis, IN). Ab LL2 (13, 14), an IgG2areacting with CD22 (originally named EPB-2), was provided by Dr. H. J.Hansen (Immunomedics, Inc., Morris Plains, NJ). Ab Lym-1 (15), an IgG2a

reacting with the MHC class II antigen, was provided by Dr. G. DeNardo(University of California-Davis Medical Center, Sacramento, CA). Ab 5E9, an

IgGl reactive with the transferrin receptor, was produced by hybridoma cellsobtained from ATCC. Anti-CD 19# 1, an IgGl, was purchased from Biosource(Camarillo, CA); and anti-CD 19#2. HD37. an IgGl. was provided by Dr. E.

Vitella (Southwestern Medical Center, Dallas, TX). If required, IgG Abs werepurified from ascites fluid by protein A affinity chromatography by melhodsthat have been described ( 1). Second Abs used for cross-linking were either apolyclonal rabbit anti-mouse IgG (#Z109; DAKO, Burlingame, CA) or amonoclonal rat anti-mouse IgG2a (#LO-MG2A-7-C; Biosource).

Radiolabeling. Procedures for labeling Abs with I25I by chloramine T

were described previously (1). The specific activities were 10-20 mCi/mg.Conjugated Abs were analyzed by gel filtration HPLC on a Bio-Sil SEC-250column (BIO-RAD, Hercules, CA), and >95% of the radioactivity migrated as

the expected IgG.Standard Ab Processing Experiments. All incubations were performed in

tissue culture medium at 37Â°C,except where noted. Cells (IO7) were centri-fuged, washed once, and then incubated in 1 ml containing IO7 cpm of Ab for

1 h. To ensure the specificity of Ab binding, controls were set up in everyexperiment, containing 10-fold smaller samples (cells. Ab, and medium), withand without excess unlabeled Ab (50 Â¿ilat 50-250 /xg/ml). In all experiments,

at least 90% of the bound cpm was specifically inhibited by the unlabeled Ab.After the binding incubation, cells were washed four times with 5 ml tissueculture medium by centrifugation. The specificity controls were counted, andthe large aliquot was suspended in 30 ml, then dispensed in a 24-well plate

(Falcon; VWR Scientific, Piscataway, NJ) with 1.5 ml/well. Samples of 1.5 mlwere saved for determination of the radioactivity, which was the "initiallybound" cpm. The plate was incubated in a humid incubator containing 5% CO2

and at 3, 21, 45, and 69 h; or at other times noted, the cells and supernatant

4 M. J. Mattes, unpublished data.

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Ab PROCESSING BY B-CELL LYMPHOMAS

were collected as follows. Cells were suspended by repeated pipetting andtransferred into conical tubes. The wells and pipette were rinsed with 1 ml,which was added to the initial cell suspension collected. The tube was centri-

fuged 10 min at 2000 rpm, and 1 ml of supernatant was carefully collected(40% of the total supernatant). The supernatant was counted for radioactivity,then analyzed by TCA precipitation as described ( 1). cpm not precipitated byTCA were considered degraded, and precipitated cpm were considered intact.We realize that large Ab fragments would also be precipitated, but such

partially degraded Ab is unlikely to appear in the supernatant, although it canbe detected inside the cell (16, 17). In some experiments, the "intact" cpm in

the supernatant was also analyzed by gel filtration HPLC, and found to migratelike the original Ab, a result supported also by data of Press et al. (18). Theoriginal cell pellet was washed once with 5 ml, and the pellet was counted forradioactivity, cpm are expressed as the percentage of initially bound cpm.

Binding Parameters. A final volume of 0.15 ml contained \0h cells andapproximately 5 X IO5 cpm I25l-labeled Ab mixed with unlabeled Ab to obtain

the desired protein concentration, which was determined in preliminary experiments to produce 10-90% saturation of antigenic sites. The incubation was forl h at 37Â°C, which was determined in preliminary experiments to allow

equilibrium to be reached. The methods used were described previously (19),except that cell-bound Ab was collected by centrifugation over a phthalate oil

mixture (20).Cross-Linking with a Second Ab. In two-step experiments, the above

method was followed, except that the final medium used for plating the cellscontained the second Ab, at the concentrations given in "Results." To prepare

soluble complexes for the one-step method, following Lansdorp et al. (21), the

mouse Ab at 15 Â¿ig/mlwas mixed with the rat monoclonal second Ab at 30/Â¿g/mlin PBS, 1.0% human albumin, and incubated overnight at 37Â°C.The

mixture was then diluted to the usual concentration of radioactivity for Abprocessing experiments, as described above. The formation of complexes wasmonitored by gel filtration HPLC analysis: approximately 91% of the IgG cpmwere converted from monomers to high molecular weight complexes. The highrecovery of cpm from the column indicated that insoluble immune precipitateswere not formed.

RESULTS

Ab Processing by B-Cell Lymphomas. Nine Abs have beentested on three B-cell lymphoma cell lines: Raji, Daudi, and RL. Some

Table 1 The fate of Abs bound io B-cell Ivmpliomas

l B W6/32 & CD45 1 C CD20 & CD19

l l ' 'Ã‡T' l ' 'r-rr' i i i i i i i

0 20 40 60 0 20 40 60 0 20 40 60

HoursFig. l. Processing of Abs bound to B-cell lymphomas. Target cells were coated with

I25l-labeledAbs, washed, and then cultured for 3 days. â€¢¿�and O. retained cell-bound cpm;

â€¢¿�and D. Ab released intact into the supernatant (TCA precipitatale!; A and A. Abdegraded and released into the supernatant. A: â€¢¿�,â€¢¿�.and A. MA103 on Raji; O, D, andA, MA103 on the renal cell carcinoma SK-RC-18, tested previously (1). shown forcomparison. B: â€¢¿�,â€¢¿�.and A, W6/32; O. D. and A, anti-CD45 on Raji. C: â€¢¿�.â€¢¿�and A,anti-CD20; O, Q and A, anti-CD19 on Raji. D: â€¢¿�.â€¢¿�and A. Lym-1; O, D. and A, 5E9on Raji. E: â€¢¿�.â€¢¿�and A. MA103; O, D. and A. anti-CD45 on Daudi. F: â€¢¿�.â€¢¿�and A.anti-CD20 on Daudi: O. D, and A. anti-CD20 on RL. Values shown are means ofduplicates. Standard deviations are omitted for clarity but were always <5% of boundcpm. Results shown are representative of two to three experiments.

CelllineRajiRajiRajiRajiRajiRajiRajiRajiRajiRLRLRLRLRLRLDaudiDaudiDaudiDaudiDaudiAbMA

103W6/32CD45CD20CD19#1CD19#2Lym-1CD225E9MA

103W6/32CD20CD19#15E9CD22MA103CD45CD20CD1902CD22Peak

%releasedintact54-66*'48-4945^668-6952-557060-6718-2136^1162-7147-4857-6248-6037-3914-2118-2132-3753-5740-4518-20After

3days%

retained13-2110-1124-2613-1412-142119-259-1612-159-166-1216-1813-1710-1516-2250-6856-5730-3331-3624-36%degraded"11-2634-3522-2816-2033-344710-1263-7252-5415-2035-4118-2129-3451-5550-517-1013-1710-1430-3437-53

" Degraded and released into the medium.h Values shown are the range obtained in two or more experiments, each performed in

duplicate.

but not all of the Abs were tested on all three cell lines. Representativeresults are shown in Fig. 1, and the data are summarized in Table 1.Table 1 lists the peak percentage released intact, which occurredsometimes on day 1, 2, or 3. A gradual, small decrease in the intact Abin the supernatant at later time points was frequently seen, as demonstrated in Fig. 1, B-F; this decrease can be attributed to the re-

binding and gradual catabolism of released Ab by the cells. In controlexperiments, Abs incubated in medium without cells showed nodetectable deterioration in 3 days. Results with Raji and RL were verysimilar and will be presented first; differences observed with Daudiwill be discussed below. Two patterns of Ab processing were seen.Two Abs, anti-CD22 and 5E9 (anti-transferrin receptor), were inter

nalized and catabolized rapidly. For these Abs, there was relativelylittle release of intact Ab, since the bound Ab was internalized so fast.The second pattern was more common, applying to 7 of the 10 Abstested. With these Abs, the majority of the bound Ab dissociatedintact, and the dissociation was quite rapid, being complete in 21 hand usually near-complete in 4 h. With these Abs, there was a

relatively small but significant amount of Ab catabolism, approximately 10-30% in 3 days, and a similar fraction of the bound Ab,10-25%, remained on the cell after 3 days. Fig. \A shows results with

Ab MA 103 bound to Raji, and for comparison results with the sameAb bound to SK-RC-18, a renal cell carcinoma, from an earlier study

is shown. This Ab is handled very differently by the two cell lines.Although the retention of cpm by the two cell types does not differvery much, the mechanism of Ab loss from the cell is very different;MA103 largely dissociates intact from Raji but is catabolized bySK-RC-18. In previous tests on approximately 20 cell lines (1, 2),

MA 103 always bound at least as avidly as any other Ab, as judged bylow levels of Ab dissociation. Hence, its extensive dissociation fromRaji is striking. Results with other Abs bound to Raji are shown inFig. 1, B-D, and demonstrate that extensive dissociation is also seenwith Abs W6/32 (anti-MHC class I), anti-CD45, anti-CD20, anti-CD 19, and Lym-1. Like MA103, W6/32 also reacts with tumors of

diverse histolÃ³gica! types and was previously tested on a large panelof adherent tumor cells. W6/32 also displayed a high level of dissociation from Raji (48-49%), which is markedly higher than the levels

observed previously with adherent tumor cells (<28%; Refs. I and 2).W6/32 and anti-CD 19 showed somewhat greater catabolism than the

3063




15 20

HoursFig. 2. Processing of Abs bound to B-cell lymphomas: early time points. The symbols

are defined as in Fig. 1. â€¢¿�.â€¢¿�and A. MA103 on Raji; O, D, and A, anti-CD20 on RL.

Values shown are means of duplicates. Standard deviations are omitted for clarity butwere always <5% of bound cpm. Results shown are representative of two to threeexperiments.

other Abs, approximately 35% in 3 days, but still displayed >50%dissociation of bound Ab. Experiments shown in Fig. 1 used the CD 19Ab from Biosource, but essentially identical results were obtainedwith a second CD 19 Ab, HD37 (Table 1). Fig. ID also shows resultswith an anti-transferrin receptor Ab, 5E9, for comparison. This Ab

was rapidly internalized and catabolized, as expected (22). Most ofthese Abs were also tested on RL, another B-cell lymphoma, and

produced very similar results (Table 1); Fig. IF shows results with1F5 (anti-CD20) binding to RL.

The Daudi cell line, as noted above, produced results that weresignificantly different. With Daudi (Fig. IE), MA103 was releasedintact in relatively small amounts, with 17% released intact in 3 days,although this is still higher than the level observed with most othercell lines (see below). The catabolic rate was also very low; therefore,as a consequence, MA103 was retained by the cells for prolongedperiods. However, with other Abs bound to Daudi, a higher percentage of the bound cpm was released intact. With YTH24.5 (anti-CD45;

Fig. IE), 37% was released intact in 3 days, which, again, is highrelative to the values obtained with other cell lines (see below). With1F5 (Fig. IF), 57% was released intact, which is similar to the resultsseen with Raji and RL. With anti-CD19, 40-45% was released intact.W6/32 and Lym-1 did not react with Daudi, at least not strongly

enough for experiments of this type to be performed. Thus, Abs bindmore avidly to Daudi than to Raji or RL, but Daudi still displayssomewhat more dissociation than most other cell lines, as will beshown below.

It is important to note that dissociation was quite fast. As shown inFig. 2, approximately 20% of the bound Ab dissociated in 30 min,with both MA103 bound to Raji and 1F5 bound to RL. A consideration of the methods used in these experiments strongly suggests thatthe fraction of dissociated Ab must be significantly underestimated,due to loss of Ab during the washing procedure. That is, after the Abincubation, cells are washed four times by centrifugation, each timefor 5 min; the entire washing process, until the final resuspension,requires a minimum of 30 min, and generally 40-50 min. Clearly, any

Ab that dissociates during this time period will be lost in the washes,and it is also clear that substantial Ab will dissociate within thisinterval. This can be estimated by assuming that the dissociation rateduring this first 40 min is the same as the dissociation rate measuredfrom 0-30 min after plating the cells. Making this correction, we canestimate that approximately 35-40% of the bound MA103 had dis

sociated and been lost during the washes, and that, in all, approximately three-fourths of the bound MA103 dissociated intact. The loss

of specifically bound cpm during washing was demonstrated directlyby using the phthalate oil separation method, which requires nodilution of the sample and is virtually instantaneous. Using MA 103bound to RL, the cpm specifically bound was 57% higher by using thephthalate oil method than the conventional washing procedure, whichimplies a 36% loss of bound Ab during the washing procedure.

Variations in our standard protocol were tested for their effect onAb processing, using Ab MA 103 and Raji cells. Increasing the lengthof the Ab incubation, from 1 to 4 h, had no effect on the subsequenthandling of the bound Ab (data not shown). Increasing the incubationvolume from 1.5 ml (the standard volume) to 24 ml did result in asignificant increase in the percentage released intact (an indicationthat dissociated Ab can significantly re-bind), but this increase was

relatively small, <10% of the total bound cpm, a level too low toaffect the conclusions of these studies. Given that such a high percentage of Ab is released intact and this release is quite rapid, it isinteresting to consider whether the remaining bound Ab persists onthe cell surface. At 2 days, approximately 20% of MA 103 remainedon the cell, but this could be intracellular and thus protected fromdissociation. To investigate this possibility, excess cold Ab was addedat various times: this method has been found effective at releasingbound Ab from the cell surface* (1, 23).4 In these experiments, the

cells were incubated in medium with excess unlabeled Ab overnight,since complete elution of bound Ab requires several hours (1). Resultsdemonstrated that MA 103 remaining on the cell for as long as 3 dayswas, in fact, predominantly present on the cell surface (data notshown).

Ab Processing by Other Hematological Tumor Cell Lines. Itseemed possible that the difference between B-cell lymphomas and

adherent tumor cells might be related in some way to their growthcharacteristics, i.e., their growth as nonadherent or adherent cell lines.To investigate this point, experiments were performed with othernonadherent tumor cell lines, including T-cell leukemias, myeloidleukemias, a monocytoid lymphoma, a pre-B-cell leukemia, and a

myeloma. At least three Abs were tested on each tumor cell line, i.e.,MA 103, W6/32, and anti-CD45 (YTH24.5), which react with nearly

all of the nonadherent cells tested. These three Abs had been testedpreviously on the B-cell lymphomas. The results are summarized in

Table 2, and Fig. 3 shows results with four representative cell lines.The data demonstrate that these nonadherent target cells are more

Table 2 The fate of Abs bound to other hematological tumors

AbMA

103MA103MA103MA103MA103MA

103MA103W6/32W6/32W6/32W6/32W6/32W6/32CD45CD45CD45CD45CD45CD45Cell

lineK-562HL-60CCRF

CEMMOLT-4U-937U-266Nalm-6HL-60CCRF

CEMMOLT-4U-937U-266Nalm-6K-562HL-60CCRF

CEMMOLT-4U-937U-266Peak

%releasedintact7-89-1034-4012-1310-1114-1910-1214-1534-3537-3815-1723-2825-2821-2218-2138^Â»

123-2514-1524-30After

3%

retained44-4840-5829-3535^1729-3159-6761-7139^74-59-1029-3721-2224-2723-2732-3428-3233-3624-2529-30days%degraded43-4834-4924-2537-5062-6315-1619-2440-4967-6854-6347-5843-5653-5761-6248-5125-2644-4664-6536-41

3064




40 60

HoursFig. 3. Processing of Abs bound to non-B-cell hemalological tumors. The symbols are

defined as in Fig. !.â€¢.â€¢.and A, M A103; O. Q and A. anti-CD45. The target cells wereU-937 (Ai. U266 (ÃŸ),CCRF CEM (O. and MOLT-4 (D). Values shown are means ofduplicates. Standard deviations are omitted for clarity but were always <5% of boundcpm. Results shown are representative of two to three experiments.

similar to the adherent target cells tested previously and are differentfrom the B-cell lymphomas in the percentage of bound Ab released

intact. Thus, the differences observed between cell lines are not due tothe adherent/nonadherent distinction but rather indicate an unusualproperty of B-cell lymphomas. Although none of the non-B-cell

hematological tumors displayed the high level of dissociation seenwith Raji and RL, these tumors were diverse in another respect, thecatabolic rate. Some cell lines, such as U-937, K-562, and HL-60,

showed fairly rapid catabolism, with 50% or more of the bound Abcatabolized in 3 days (Fig. 3, A and D), which was similar to thecatabolic rate observed with adherent tumor cells (1, 2). In contrast,some nonadherent tumors, including U266 (Fig. 3ÃŸ)and Nalm-6

displayed much lower catabolic rates. Therefore, these cells retainedthe Ab for prolonged periods. It should be noted, however, that all celllines did catabolize a significant fraction of the bound Ab in 3 days,with the lowest level being approximately 10%. The two Abs MA103and YTH24.5 were always handled similarly by each cell line, whichsupports our previous hypothesis that such catabolism reflects theturnover rate of cell surface constituents. The T-cell line CCRF CEM

displayed one unusual property in that the release of intact Ab continued at an almost constant rate for 3 days (Fig. 3C). The explanationfor this is not known. It was not observed with the second T-cell linetested, MOLT-4 (Fig. 3D).

Validity of "Functional Affinity." We attempted to determine the"functional affinity" of Ab MA 103 binding to Raji as an approach to

further characterize the binding interaction. Bound and free Ab wereseparated instantly by centrifugation into phthalate oil to avoid Abdissociation during standard washing procedures. As shown in Fig. 4,a Scatchard-type plot for MA103 binding to Raji did not produce a

straight line but instead a curve that was concave upward. We notethat a straight line could be drawn through the eight points shown inFig. 4, by linear regression analysis, with a reasonable correlationcoefficient of â€”¿�0.825,but clearly this would not provide an accurate

description of the data. Moreover, dissociation did not proceed at aconstant rate, meaning that a semi-log plot of %Retained cpm versus

Hours did not produce a straight line but rather a curve concaveupward (data not shown). We conclude that the dissociation rate is notconstant, and hence that dissociation is not a first-order reaction. On

the basis of these two discrepancies, we conclude that the equation

defining affinity does not adequately describe the interaction of Abswith these target cells.

Effect of Cross-Linking Abs on the Cell Surface. We previouslydemonstrated that Ab cross-linking was very effective at preventing

the dissociation of intact Ab bound to the cell surface (23). WithB-cell lymphomas, because dissociation is so great, it might beexpected that cross-linking would have a dramatic effect. Fig. 5A

shows results of an experiment using MA 103 and Raji in whichmonoclonal rat anti-mouse IgG was included in the incubation me

dium at a final concentration of 6.7 /xg/ml. This second Ab stronglyreduced the amount of Ab released intact, from 59 to 13%. It had onlya slight effect, if any, on the catabolic rate; therefore, it resulted inmarkedly prolonged retention of the Ab by the target cell. Four-fold

less concentrated second Ab, at 1.9 fig/ml, was only slightly lesseffective. Use of the second Ab at 0.4 /xg/ml still had a significanteffect, although considerably less than at higher concentrations. Apolyclonal second Ab, rabbit anti-mouse IgG, was very similar in its

effect when used at 1:7500. We note that increased catabolism did notresult from cross-linking, which is different from results obtained

previously using the same Ab, and second Abs, on adherent tumorcells (23). Very similar results were obtained with Ab 1F5 bound toRL cells (Fig. 5ÃŸ).

Ab cross-linking at the cell surface is most readily performed byadding the cross-linking reagent to cells that have been coated with

Ab, then washed. However, such a protocol cannot be readily performed in vivo. Therefore, other experiments were performed in amanner more directly applicable to the in vivo situation. Thus, Abaggregates were pre-formed, then applied in one step to target cells.

These aggregates were formed with the mouse mAb 1F5 and a ratanti-mouse IgG monoclonal Ab. It would be expected that such

aggregates would be predominantly tetramers, containing two mouseAbs and two rat Abs, and therefore would be tetravalent in terms ofantigen binding (21). The binding of the aggregates to target cells wasstill antigen specific and not via Fc receptors, since it was effectivelyinhibited by excess unlabeled 1F5. The processing of such aggregateswas markedly different from that of control 1F5 (Fig. 5C), withreduced dissociation of intact Ab, although the reduction in dissociation was much less than when the second Ab was added sequentially(in much larger amounts), as seen in Fig. 5ÃŸ.In addition, acceleratedcatabolism was observed, which did not occur when the second Abwas added sequentially. The accelerated catabolism partly offset the

3 -

I"3

1 -

0.20 0.25 0.30 0.35 0.40 0.45 0.50

Bound nM

Fig. 4. A Scatchard-type plot for binding of Ab MA103 to Raji. The two symbols showresults of two separate experiments, each performed in duplicate. The points form a curveconcave upward, rather than a straight line; therefore, the affinity cannot be determinedfrom the slope of the line.

3065




D.Ã¼T3

13g25

10 20 30 40 50

HoursFig. 5. The effect of cross-linking on the processing of Abs bound to the surface of

B-cell lymphomas. The symbols are denned as in Fig. I â€¢¿�.â€¢¿�.and A. effect of a secondAb, monoclonal rat anti-mouse IgG. as a cross-linking reagent; O, D, and A. control

medium. A. MA103 binding to Raji. B, 1F5 binding to RL. C. complexes of 1F5 and thesecond Ab were pre-formed, then added to Raji target cells. Values shown are means of

duplicates. Standard deviations are omitted for clarity but were always <5% of boundcpm. Results shown are representative of two to three experiments.

decreased dissociation; therefore, the retention of the Ab by the cellswas not increased dramatically.

DISCUSSION

Whereas many investigators previously analyzed the processing ofindividual Abs on one or a few B-cell lines, the first broad approach

to these questions was provided by Press et al. (8), who tested sevenAbs on Daudi. Their results are generally compatible with thosepresented here, but it seems useful to point out certain differences inthe methods used: (a) we tested many target cell lines, which revealedthe difference between individual target cells; (b) our experimentsextended for 3 days, instead of 1 day, which allowed long-term

patterns of Ab processing to be more clearly seen; (c) Press et al. (8)used a 4Â°CAb incubation, rather than 37Â°C,for their processing

experiments. We have investigated the effect of this temperaturedifference in a series of experiments in which six Abs were tested onRaji and/or RL cells. The change in temperature significantly affectedthe results obtained, with the magnitude of the effect dependingstrongly on the particular Ab used. Using a low-temperature Ab

incubation, the subsequent release of intact Ab was markedly increased, and the catabolism of bound Ab was markedly decreased.5

However, the maximum difference observed was approximately 20%of the total bound cpm, which, although significant, did not alter thegeneral conclusions of the experiments. We note that the 37Â°Cincu

bation used here would tend to decrease the percentage of bound Abreleased intact; and (d) the time required for washing will cause asignificant, unavoidable underestimate of the percentage dissociated,as discussed in "Results," and the magnitude of this effect may differ

substantially, depending on minor experimental details. Pulczynski etal. (24) previously compared the processing of two Abs, anti-CD 10

5 Unpublished data.

and anti-CD 19, by Raji and Nalm-6, two cell lines that were used

herein and that processed Abs very differently in our experiments.They reported that both of the Abs were capped and that the processwas similar in both cell lines; however, their assay was immunoflu-

orescence, which is difficult to directly compare with the catabolismassay used here.

Two major conclusions are suggested from these studies: (a) manyB-cell lymphomas appear to be rather unusual in that Abs are unableto bind to them irreversibly. This "slippery" characteristic of some

B-cell lymphomas must be confirmed and extended by testing additional cell lines, including EB virus-transformed, nonmalignant B-cells. We have recently evaluated four additional B-cell lymphomas:SU-DHL-4 was similar to Raji and RL in its processing of AbsMA103 and YTH24.5; Namalwa was like Daudi; and SU-DHL-6 andBJAB were intermediate, with 30-40% release of intact MA103.5

The two cell lines tested here that are related to B cells, the Nalm-6pre-B-cell leukemia and the U266 myeloma, displayed very differentprocessing patterns from most B-cell lymphomas.

The dissociation of the majority of bound Ab from B-cell lympho

mas would not be considered surprising, unless it is recognized thatthis does not occur with tumors of other histological types. Theunusual property of many B-cell lymphomas is most clearly demon

strated by the results with MAI03 and YTH24.5, since these Abs reactwith a wide variety of cell types. Could the results be due to lowdensity of these antigens on B-cell lymphomas? This is unlikely, since

irreversible binding does not appear to require high antigen density.Although we did not determine the number of binding sites for eachAb at saturation, some indication of antigen density can be derivedfrom the cpm specifically binding under our standard experimentalconditions: IO6 cells and 5 X IO5 cpm. The cpm binding to B-cell

lymphomas, using MA 103 or YTH24.5, was at least as high as forsome of the other cell lines. We conclude that, using Abs that bind toboth B-cells and non-B-cells, dissociation is consistently much higherwith many B-cells than with other cell lines. For the Abs which arespecific for B-cell lymphomas, it is clearly difficult to reach conclu

sions regarding differences among cell lines. However, the total bodyof data strongly supports the conclusions described above. Thus, of 17Abs previously tested on adherent tumor cells, 15 bound essentiallyirreversibly, with only a small percentage dissociating. (Two unusualAbs did largely dissociate intact, which can probably be attributed tolow avidity). In contrast, we have not identified any Ab that binds thisstrongly to B-cell lymphomas. To further confirm our conclusions, we

are continuing to test additional Abs.Data published from other laboratories are not inconsistent with our

conclusions. Some apparent discrepancies can be attributed to the factthat Press et al. (8) used Daudi target cells, which appears to bind Abssomewhat more avidly than Raji or RL cells. Of the Abs tested byPress et al. (8), those which bound most avidly to Daudi were CD37and MHC class II, and even with these cells, approximately 30% ofthe bound Ab was rapidly released intact (probably dissociated),judging from the loss of cpm from the cell. We did not test CD37, andour anti-MHC class II Ab, Lym-1, did not bind sufficiently to Daudi

to allow the assay to be performed.The second major conclusion is that different cell lines may vary

greatly in their processing of bound Ab, and the processing route oftendepends less on the particular Ab than on the particular cell line.Moreover, it may be that no cell line is similar to tumor cells in vivoin patients. Even freshly derived tumor specimens, grown in tissueculture for short periods, may be very different in their metabolismfrom in situ tumors. However, it still seems useful to analyze cell linesin vitro, at least to determine the range of processing behavior that ispossible.

Considering the clinical importance of CD20 Abs, it seems useful3066




to discuss more fully results with this Ab. The Ab used here was 1F5;this same Ab as well as B l was used by Press et al. (4, 8). Judgingfrom direct comparisons by Press et al. (4), the two Abs are verysimilar, and in fact 1F5 appeared to have a higher "avidity." The data

of Press et al. (8) demonstrated substantial dissociation of CD20 Absfrom Daudi, but cell retention was still relatively high, compared tothe other Abs tested. However, recent results from Press et al (18)demonstrated higher level of dissociation of CD20 Abs from B-cell

lymphoma specimens freshly obtained from patients. Our results mayprovide insight into this situation. In fact, Daudi displayed considerably less dissociation of most Abs than the two other B-cell lympho-

mas tested, Raji and RL. Thus, Raji and RL may be more similar tothe freshly obtained lymphoma specimens tested by Press et al. (18).

In explaining the high level of dissociation of 1F5 Abs, the possibility should also be considered that 1F5 might be a relatively low-

avidity Ab. We would emphasize that even with carcinoma targetcells, to which most Abs bind irreversibly, a few Abs do bind weakly,with high levels of dissociation. Relatively low avidity of 1F5 is, infact, suggested by results with Daudi, since this B-cell lymphoma,

unlike the others, bound MA 103 quite well, yet 1F5 largely dissociated. In any case, the dissociation of 1F5, like the dissociation of allother Abs tested, could be greatly reduced by cross-linking. The effectof cross-linking may be partly due to interaction with Fc receptors, as

well as due to increased Ab valency, although initial binding of 1F5aggregates to the cell surface was shown to be antigen specific. In anycase, the functional consequence of cross-linking is reduced dissociation. In some cases, cross-linking also enhanced the catabolic rate.

This would tend to decrease radioisotope retention in tumor cells, butwe have shown previously that this effect can be countered by usingradiolabels that are trapped within lysosomes after catabolism of theAbs to which they were conjugated (residualizing radiolabels; Ref.23). Other investigators have developed modified Abs having highervalency, including trivalent and tetravalent, and benefits of thesemodifications have been described in terms of Ab avidity (25-28).However, this approach has not yet been applied to B-cell lymphomas

where, we predict, the greatest benefit will occur.Another important issue in regard to immunotherapy is whether

most or all Abs are internalized by B-cell lymphomas, since Ab

conjugates with toxins or drugs (5, 29, 30) require internalization to beactive. Our data demonstrate that the level of Ab internalization andcatabolism by B-cell lymphomas is low relative to most tumor cell

lines of other histological types. However, in consideration of the factthat some of the Ab conjugates are extremely potent, it seems usefulto emphasize that in fact every Ab tested was significantly catabolizedby every target cell tested in our study, to a level of at least 10%, andusually more, within 3 days. Thus, a high density antigen that isinternalized poorly may in fact deliver more Ab intracellularly than arapidly internalized Ab that is present at a lower density on the cellsurface. However, it is also known that the toxicity of a particulartoxin-conjugate will not be directly correlated with the amount inter

nalized, since other factors also are important, such as the route takeninside the cell (29, 30). In interpreting these data, it is helpful toconsider that dissociation and catabolism are, to some extent, competing processes. That is, if an Ab rapidly dissociates, it cannot beinternalized and catabolized. Similarly, if an Ab is rapidly internalizedand catabolized, it cannot dissociate intact.

Vervoordeldonk et al. (31) demonstrated that the processing of Absbound to B-cell lymphomas was affected by the Fc receptors present

on these cells and thus depended, in part, on the subclass of IgG used.This factor must be taken into consideration and is likely to haveaffected some of our experiments, but it does not affect the interpretation or conclusions of this study, for the following reasons: (a)mouse IgGl but not IgG2a was recognized by the Fc receptor present

on human B-cell lymphomas (31); most of the key Abs used in ourexperiments were IgG2a; (b) many non-B-cell hematological tumors

express the same Fc receptor (31), yet processed the same Abs verydifferently from B-cell lymphomas; and (c) Fc receptors would be

expected to increase the Ab avidity and were found to enhancecatabolism (31), which cannot explain the high level of dissociationobserved.

These experiments have provided additional evidence that "functional affinity" cannot adequately described the interaction between

most Abs and the cell surface. In a previous study, using erythrocytesas target cells to eliminate any role of Ab internalization, similarconclusions were reached (32). The validity of "functional affinity"

was also questioned by Kaufmann and Jain (33). The concave upwardcurve obtained in the Scatchard-type plot is precisely the shape

predicted from theoretical analyses of bivalent Ab binding (32, 33)and has been observed previously (34), albeit rarely.

Although we have emphasized the therapeutic implications of theseresults, they also raise a basic question of cell physiology: why areAbs unable to bind irreversibly to B-cell lines? We cannot currently

answer this question, but we can outline some possibilities. It is clearthat irreversible binding requires bivalent binding and that bivalentlybound Ab is continuously wobbling, such that it becomes transientlymonovalently bound. Any factor that affects the frequency or stabilityof bivalent binding will affect the irreversibility of Ab binding. Suchfactors may include: (a) membrane fluidity, which can be affected bythe cytoskeleton as well as other factors; (b) the density and size ofcarbohydrate chains on the cell surface, which could interfere with theability of a monovalently bound Ab to reach a second antigen molecule; and (c) membrane morphology, such as the occurrence ofmicrovilli. In fact, B-cell lymphomas have been described as having

an unusually high density of microvilli, as detected by both scanningand transmission electron microscopy (35). Since it seems likely thatthe occurrence of dense microvilli would affect the processing ofbound Ab (although the precise mechanism is obscure), we are currently investigating a possible correlation between microvilli andbound-Ab processing.

ACKNOWLEDGMENTS

We are grateful to many investigators who provided antibodies and celllines, who are listed in "Materials and Methods." We also thank Philip

Andrews for assistance with radiolabeling and Dr. David M. Goldenberg forhis support.

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1996;56:3062-3068. Cancer Res Rafik Hanna, Gaik Lin Ong and M. Jules Mattes Other Hematological MalignanciesProcessing of Antibodies Bound to B-Cell Lymphomas and

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