isolation and characterization of canine venereal tumor … · canine tumor-inhibitory and blocking...

[CANCER RESEARCH 39, 3920-3927, October 1979]0008-54 72/79 /0039-OOOOS02.00

Isolation and Characterization of Canine Venereal Tumor-associatedInhibitory and Blocking Factors1

William E. Beschorner,2 Allan D. Hess,3 Samuel T. Nerenberg, and Robert B. Epstein

Veterans Administration West Side Hospital and Departments oÃMedicine [R. B E.Â¡and Pathology Â¡W.E. B., A. D. H., S. T. N.J. Abraham Lincoln School ofMedicine. University of Illinois. Chicago. Illinois 606 i 2

ABSTRACT

Spontaneous regression of the canine venereal tumor isassociated with the production of a serum factor which inhibitsin vitro tumor colony-forming units in agar. Logarithmic or

persistent tumor growth, on the other hand, is characterizedby a serum factor which protects cells against in vitro inhibition(blocking factor). These factors have been characterized byimmunochemical methods. Whole regressor and blocking serawere fractionated by Sephadex G-200 filtration and immunoab-sorption with rabbit antiserum specific for canine immunoglob-ulin G2a. Fractions were characterized by immunoelectropho-

resis, radial immunodiffusion, and disc gel electrophoresis. Invitro inhibitory and blocking activity of the whole serum wasaccounted for by the purified immunoglobulin G2a.

Blocking activity was also found in protein eluted from logarithmically growing tumors. Preparative polyacrylamide electrophoresis revealed five major fractions with blocking activityonly in the immunoglobulin G fraction. Tumor eluates andimmunoglobulin G isolated from serially removed tumors demonstrated relative inhibitory and blocking activities that correlated with the clinical course of the tumor.

Using ultrafiltration and sodium dodecyl sulfate electrophoresis of tumor-associated immunoglobulin G at low pH, it was

not possible to identify an antigen complexed to the blockingantibody.

INTRODUCTION

Following a period of logarithmic growth, naturally occurringor induced canine TVT's4 show spontaneous regression, in

dolent local tumor growth, or progression with mÃ©tastases(3,6, 30, 33). The tumor is unique because of its successfulcellular transplantation between unmodified random recipientsunder either natural or laboratory conditions (4, 8). The hom-

ograft nature of the tumor is supported by cytogenetic evidence, by the presence of histocompatibility antigens as determined by serological and by mixed lymphocyte-tumor cell

culture testing (8, 17, 23). The TVT is therefore of considerablebiological interest with respect to transplantation and tumorimmunogenetics (37). Development of an in vitro culture systemfor cloning TVT cells which respond to either humoral inhibitory

1This work was supported by Medical Research Service of the Veterans

Administration.2 To whom requests for reprints should be addressed, at the Department of

Pathology, The Johns Hopkins Hospital, Baltimore. Md. 21205.3 Present address: The Bone Marrow Transplant Unit, The Johns Hopkins

Hospital, Baltimore. Md. 21205.' The abbreviations used are: TCFUâ€ž,tumor colony-forming units in agar; TVT,

transmissible venereal tumor; IEP, immunoelectrophoresis; SRID, single radialimmunodiffusion; SDS. sodium dodecyl sulfate; NDS, normal dog serum; PBS.phosphate-buffered saline (0.01 M phosphate, 0.9% NaCI, pH 7.2).

Received January 5. 1979; accepted July 6, 1979.

factors from dogs that have undergone tumor regression orblocking factors (protecting cells against the inhibitory factors)and from dogs with progressive tumor growth has been described (3). These in vitro inhibition and blocking effects correlate with in vivo events.

The present study was undertaken to characterize serum-and tumor-associated factors responsible for inhibition and

blocking.

MATERIALS AND METHODS

The Tumor System. Canine venereal tumors are regularlytransplanted in outbred dogs by injecting 1 x 108 viable tumor

cells s.c. as described previously (4, 8). All dogs initiallydevelop palpable tumors within 7 to 14 days which continuedto grow for at least 1 month (3). Tumor cells were obtainedduring logarithmic growth (21 to 28 days following inoculation);at which time they averaged 5 cu cm in volume. Histologicalexamination at this stage reveals the tumor to be composedalmost entirely of large undifferentiated tumor cells with prominent irregular nucleoli.

Assay of Serum Inhibitory and Blocking Factors. Seruminhibitory and blocking activity was assayed in vitro by theeffects on TCFUa as previously described by Bennett ef al. (3).Briefly, tumor cells harvested from logarithmically growing tumors (20 to 27 days posttransplant) were mechanically dispersed and washed 3 times in a standard medium consistingof TC-199, 75 fig DEAE-dextran per ml, 40 jug sodium pyruvate

per ml, 50 units penicillin per ml, and 50 fig streptomycin perml. Duplicate cultures of 2 x 105 tumor cells were plated in the

standard medium in 0.3% agar over 0.1 ml of pooled, heat-

inactivated NDS. Examination of the cell suspensions just before plating revealed them to consist of a single-cell suspension

with greater than 90% viability by trypan blue exclusion. Immediately after plating, examination of the plates failed to revealany cell aggregates. Retri dishes were incubated at 37Â°in 5%

CO2 for 48 to 72 hr, after which 20 to 50 colonies consistingof 20 to 25 tumor cells were observed in each plate. Serataken from unrelated animals during the progressive phase oftumor growth regression were fractionated as described belowand assayed for blocking and inhibitory activities. Serum, serum fractions, and tumor eluates were sterilized by passagethrough a 13-mm diameter, 0.45-fim Millipore filter (Millipore

Corp., Bedford, Mass.). Inhibitory activity was assayed byplating tumor cells over a feeder layer of 0.1 ml of the respectivefraction and 0.1 ml of heat-inactivated NDS. Inhibition wasdefined as a significant reduction (p < 0.01, Student's f test)

or absence of tumor colonies as compared to tumor colonygrowth obtained with NDS. Blocking activity was assayed bypreincubation of equal volumes of serum or eluate fractionsand tumor cells at 37Â° for 30 min. The tumor cells were

3920 CANCER RESEARCH VOL. 39

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Canine Tumor-inhibitory and Blocking Factors

centrifugea at 1000 x g for 10 min, and the supernatant wasdiscarded. The cells were resuspended in complete media andplated over a feeder layer of 0.1 ml of a standard inhibitoryserum (titer, 1:1054) that was pooled for hyperimmunizedrÃ©gresser animals. Blocking activity was defined as an increased number of colonies (p < 0.01, Student's t test) as

compared to cells preincubated in normal dog sera. All fractions were coded before assaying, and tests were read in asingle blind manner with no knowledge of the sample source.

Sephadex Fractionation of Whole Serum. Two-mi samplesof canine serum were chromatographed on a 1.8- x 60-cmcolumn of Sephadex G-200. This study included serum samples from 2 normal dogs, 2 rÃ©gresserdogs, and 2 dogs withprogressively growing tumors. The column was eluted withPBS at the rate of 6 to 7 ml/hr, and 2.5-ml fractions were

collected. The absorbance was automatically monitored at 280nm with an LKB No. 7000 monitor.

Development of an Antiserum Specific for Canine IgG. Toproduce a rabbit antiserum with a high specificity for caninelgG2a.b,whole-dog serum was subjected to IEP in 1% agaroseusing commercial rabbit anti-canine immunoglobulin (National

Biochemical Corp., Cleveland, Ohio) to develop the precipitinarcs. The arcs in the "slow" y region corresponding to lgG2a.b

(20) were removed and washed thoroughly in 0.9% NaCIsolution for 10 days to remove unreacted proteins. Eight arcswere then homogenized in an equivalent volume of Freund's

complete adjuvant and injected i.d. into multiple sites on thebacks of rabbits. The animals were "boosted1 i.v. 3 weeks

later and at 1 week intervals thereafter with 2 arcs homogenized in 0.9% NaCI solution. Rabbits were routinely bled at 2and 4 days following each booster injection. The specificity ofthis antiserum was tested by IEP and SRID.

An antiserum specific for canine lgG2a was produced byadsorbing the anti-lgG2a-b with titered amounts of lgG2b-en-riched serum fraction. This was the fraction eluted from DEAE-Sephadex A-50 between 0.05 and 0.10 M sodium phosphate,pH 8.0 (45).

Purification of IgG from Serum Using a Monospecific An-

tiserum. An insoluble polymer of rabbit antiserum (specific forlgG2a) was prepared by using the method of Avrameas andTernyck (1). Whole-serum samples (6 dogs) taken at different

times in the course of tumor growth, in addition to SephadexG-200-treated serum (7S fractions), were adsorbed with thepolymerized anti-lgG2a by using a batch technique (1 g poly

mer/2 ml serum). Adsorbed protein was eluted twice from thepolymer with 0.5 M glycine buffer, pH 3.5, for 10 min at roomtemperature using 2 ml/g of adsorbent. Following centrifuga-

tion, the supernatant was then neutralized with a 0.5 M Trisbase and dialyzed for 2 to 3 days against PBS with 4 to 6buffer changes.

Purification of IgG from Tumor Eluates. Tumor cells fromfresh canine venereal tumors were prepared after a thoroughwashing and debridement by forcing the tissue through a No.60 steel mesh screen followed by suspension in 20 volumes ofPBS. The cells were washed 7 times and eluted with 0.5 Mglycine, pH 3.0 (2 volumes per volume packed cells), at 37Â°

for 2 hr followed by centrifugation at 3000 x g for 10 min. Theresulting supernatant was neutralized with Tris base, dialyzedagainst PBS for 2 to 3 days with 4 to 6 buffer changes, andconcentrated on a Amicon PM-10 filter (Amicon Corp., Lexington, Mass.).

Canine IgG was isolated from the eluate by use of preparativepolyacrylamide electrophoresis (26). Thirty mg of eluted protein were mixed with sucrose and bromphenol blue and layeredon a 4- x 30-cm 5% polyacrylamide (3% bisacrylamide) gel in0.04 M Tris-glycine buffer, pH 9.3. Protein bands were local

ized by staining a thin longitudinal slice of the gel in Coomasieblue (31). Corresponding bands from the unstained gel wereremoved as a disc by a fine wire, allowing 0.5-cm margins.

Protein was eluted from each disc with PBS and dialyzed.Protein concentration was estimated by the difference in UVabsorbance at 215 and 225 nm (43) and by absorption at 278nm, assuming an extinction coefficient (E278 0.1%, 1 cm) of1.34(42).

IEP. The protein content of fractions were evaluated qualitatively by IEP against commercial goat or rabbit anti-dogserum (Cappell Industries, Downington, Pa.) or rabbit anti-dog

lgG2a.b as described above, using 1% agarose or 1% Nobleagar as the supporting medium (34).

SRID. The relative content and purity of IgG in each fractionwere determined by radial immunodiffusion (24) against mon-ospecific rabbit anti-lgG2a.b or rabbit anti-dog serum.

Polyacrylamide Disc Electrophoresis. The relative purity ofthe isolated IgG was demonstrated with polyacrylamide gelelectrophoresis by using 4 or 8% acrylamide monomer concentration (3% cross-link) in 0.04 M Tris-glycine, pH 9.3 and

stained with Coomassie Brilliant Blue (5, 43).Polyacrylamide Electrophoresis in SDS. The molecular

weight and purity of isolated fractions were determined by theuse of SDS-polyacrylamide gel electrophoresis using Fair-bank's technique (9, 44). To explore the possibility of a bound

antigen, the fractions were also acidified to pH 3.0 with 0.05M glycine to separate antigen-antibody complex prior to de-

naturation in 1% SDS, followed by neutralization with Tris base.The bands were stained with Coomassie Brilliant Blue, and themigration distance was compared to that of similarly treatedstandards (myosin, bovine serum albumin, ovalbumin, chymo-trypsinogen, and RNase).

Separation of Antibody-Antigen Complex. An attempt wasmade to separate a small-molecular-weight antigen from puri

fied blocking factor by ultrafiltration method at low pH asdescribed by SjÃ¶gren ef al. (35). Under the conditions used,more than 98% of IgG was retained on the XM-100 filter,compared to less than 35% of bovine serum albumin (M. W.68,000).

RESULTS

Sephadex Fractionation of Blocking and RÃ©gresserSera.Chart 1 shows the Sephadex G-200 chromatography of NDS

as compared with serum from a dog with a logarithmicallygrowing tumor 14 days posttransplantation (blocking serum,Dog 5540) and from a dog 2 months after tumor rejection(regression serum, Dog 5301 ). Although there was little changein the relative absorbance of Fraction 1, the blocking andregressor sera had considerably more protein in Fraction 2than did NDS. Good separation of the 19S immunoglobulinsfrom the 7S immunoglobulins was demonstrated by IEP anddisc gel electrophoresis. No IgM was detected in Fraction 2,while only a trace of 7S immunoglobulin was found in Fraction1.

OCTOBER 1979 3921



W. E. Beschorner et al.

6 i

IOoo(M

oCo

-O

-O<t

3-

2-

Froction 1 Fraction 2 â€” Normal Serumâ€¢â€¢â€¢Blocking Serum

â€” RÃ©gresser Serum

20 30 6040 50

Fraction Number

Chart i. Sephadex G-200 chromatography of normal, blocking (Dog 5540). and rÃ©gresser(Dog 5301 ) sera

TO

Whole sera and Sephadex fractions were tested for their

ability to inhibit colony growth in vitro (inhibitory TCFUa) as

well as to block tumor inhibition (blocking TCFUa). InhibitoryTCFUa's were demonstrated by plating tumor cells with either

whole serum or a serum fraction mixed with heat-inactivated

NDS. Inhibition was determined by a marked decrease in

colonies compared to cells plated in NDS. Blocking activity

was demonstrated by preincubating cells with a test fractionand then plating them with a feeder layer of known inhibitory

serum.

Both blocking and inhibitory activities were found in Fraction

2 of the respective sera (Table 1). Although the blocking activitywas present exclusively in the 7S fraction, some inhibitory

activity was present in the void volume fraction as well as

Fraction 2. Since the inhibitory activity in the 19S fraction was

resistant to reduction and acetylation (12), it is probably due to7S or intermediate-S immunoglobulin contaminants rather than

to IgM.

Blocking and Inhibitory Activity of Serum IgG. Immuniza

tion of rabbits with precipitin arcs (containing a specific antigen-antibody complex) produced an antiserum specific for

canine IgG lgG2a.b. In Noble agar, the resulting antiserum

produced an IEP arc in the ÃŸto slow y region with a separation

near the well (Fig. 1). In agarose, the antiserum produced an

arc in the a? to y region with lgG2a that was easily distinguished

as an anodal spur. No cathodal spurs or arcs corresponding to

IgGjc or lgG2d were evident.

By absorbing the antiserum with a titered amount of a serumfraction eluted from DEAE-Sephadex between 0.05 and 0.10

M phosphate, pH 8.0, an antiserum was obtained which pro

duced a single precipitin arc against NDS in the slow y region.

This arc extends to and becomes continuous with the anodalspur produced by the anti-lgG2a.b (Fig. 1 ).

lgG2a was purified from 3 rÃ©gresser sera (including one

pooled serum) and 3 blocking sera by immunoadsorption withpolymerized anti-lgG?a. In Experiments 1 and 2 (Table 2), wholeserum was adsorbed; whereas in Experiment 3, the 7S Seph-

Table 1TCFU, 's of Sephadex fractions of rÃ©gresserand blocking sera

Sera from dogs with growing or stationary tumor (blocking sera) or from dogsspontaneously rejecting their tumors (rÃ©gressersera) were fractionated by Sephadex G-200 chromatography. The fractions (see Chart 1) were tested for theirinhibitory effect on tumor colony formation in agar or their ability to protectagainst known inhibitory serum by preincubation with tumor cells.

TCFUa assay"

Inhibitory Blocking

Serum

% of in- % of

Fraction Colonies hibition Colonies blocking

NormalBlocking

(Dog5540)RÃ©gresser

(Dog 5301 )Whole12Whole12Whole12282928241519Oc8C1C000144632100719601026C030C41503093010714318

" Mean colony count for 2 plates, counted in single-blind manner. Variability

of replicate assay was less than 5%.6 Tumor cells were preincubated with the test fraction and then plated with

known inhibitory serum.c p < 0.01 (Student's r test) compared to colonies present with whole normal

dog serum.

adex G-200 fraction was adsorbed. Eluting the polymer with

glycine buffer at pH 2.0 to 4.0 produced a maximum yield of

200 to 400 Â¿ig/g polymer at pH 3.5. Along with the low yield of

specific lgG2a, a varying proportion of albumin contaminant

was nonspecifically bound. The albumin was largely elin.mated

(to a trace contaminant) by prefractionating the whole serum

on Sephadex. Fig. 2 shows that purified lgG2a from blockingserum reacts against anti-dog serum and anti-lgG2a.b to pro

duce a single precipitin arc in the y region. A similar pattern

was observed with lgG2a that was purified from regressor

serum. Disc polyacrylamide electrophoresis in gels containing8% acrylamide monomer (3% cross-link) revealed a finely split

band with an RF of 0.25. Using Ouchterlony double diffusion

against a panel of reference antisera, Dr. H. Y. Reynolds





Table 2

Inhibitory and blocking activity of whole serum and purified lgG2,

lgG2a was purified from serum or 7S Sephadex fractions by immunoadsorption with polymerized rabbitanti-dog lgG2a. The effect of whole serum and purified lgG2a from NDS rÃ©gressersera and blocking serawere tested on the inhibitory and blocking TCFUa as described in Table 1.

TCFUa assay

Inhibitory Blockingtxpen-

ment123DogNDS1666,

16805154NDS

10485301NDS

5301uourse

ot tumorgrowthRegression

LoggrowthRegression

LoggrowthLog

growthWhole29

10a (65)"

31(0)52

6a (88)

50(4)28

23 (18)igG2.28

(3)6a (79)

28(3)50

(4)14a (73)

51(2)Not

done15a (46)Whole09

(31)30a(103)4

(0)2 (0)

24a(42)0

25a (89)lgG2c4

(14)5 (17)

37a(127)4

(0)2 (0)

40a(75)Not

done23a (82)

p < 0.01 compared to TCFUa with whole normal dog serum (Student's f test).' Numbers in parentheses, percentage of inhibition or blocking.

confirmed the IgG nature of our preparations by demonstratinga reaction against antiserum specific for lgG2a.bbut no reactionagainst antisera specific for lgG2c, lgG2d, IgA, or IgM. Finally,a precipitin ring produced by our preparation in SRID againstanti-human IgG but not by anti-human IgA or IgM further

supports the identity of the purified protein as a subclass ofIgG.

Table 2 summarizes the results of 3 experiments in whichlgG2a purified from 3 different regressor sera and 3 differentblocking sera were shown to account for the inhibitory andblocking activities, respectively. Although considerable day-to

day variation in the total number of colonies was noted, in eachexperiment the extent of inhibition or blocking in the wholeserum was comparable to the inhibition or blocking found inthe respective purified lgG2a.

Isolation and Characterization of the Blocking Antibodyfrom Tumor Eluates. In the TCFUa, no effector cells are added;therefore, the soluble factors probably express their activitythrough direct interaction with the tumor cells. If these antibodies also interacted directly with tumor cells in vivo, then itshould be possible to elute blocking antibody from washedtumor cells. Protein was eluted from washed tumor cells duringlog or stable growth (25 to 40 days posttransplantation) withglycine buffer, pH 3.0. IEP with rabbit antitumor eluate anddisc gel polyacrylamide electrophoresis demonstrated 5 principal proteins in these eluates. Fig. 3 shows a typical patternproduced by electrophoresis of 30 mg of tumor eluate. Proteinextracted from the second band (130 mm migration) reactedwith anti-lgG2a.b and anti-whole-dog serum on IEP to produce

a single short arc in the slow y region.Due to the wide separation of proteins by electrophoresis,

preparative electrophoresis was an effective method for purification of the eluate IgG. The protein produced a single bandon disc gel electrophoresis with 4 or 8% polyacrylamide (3%cross-link) and a single ring in radial immunodiffusion againstanti-whole dog serum. The IgG identity was confirmed by anidentity reaction with serum lgG2a as tested by Ouchterlonydouble diffusion against anti-lgG2a.b or anti-whole dog serum.

Electrophoresis of eluate IgG which was denatured in SDS atpH 3.0 produced a single band having a molecular weight of160,000 Â±10% (S.D.).

The IgG purified from 3 early tumors accounted for the

Table 3

Effect of tumor eluates and respective purified IgG on blocking TCFUa

Tumor cell suspensions were washed with PBS and eluted with 0.5 M glycine.pH 3.0. IgG was purified from the respective eluate by preparative polyacrylamideelectrophoresis. The blocking activity of the eluate and IgG was tested by theblocking TCFUa as described in Table 1.

Experiment1

23Dog5154

55395422NDS0

11Blocking

TCFUa"Whole

eluate EluateIgG23a(45)6 31 (61)

22 (82) 20(74)18 (67) 15(55)

In each experiment for both whole eluate and eluate IgG, p <compared to blocking with whole NDS (Student s ( test).

Numbers in parentheses, percentage of blocking.

0.01,

blocking produced by each eluate (Table 3). None of the other4 proteins obtained by preparative electrophoresis producedmeasurable blocking or inhibitory activity in the TCFUa.

In Chart 2, the blocking and inhibitory activities of tumoreluates and their respective IgG preparations are comparedwith the clinical course of Dog 5540. Tumor cells were transplanted s.c. into multiple sites, and the average tumor size wascalculated periodically. Tumors were removed at Days 22, 35,and 64. After an initial lag phase, the average tumor sizeincreased logarithmically until Day 35. The tumors were relatively stable until Day 56 when early rejection was noted by adecrease in volume. Tumors removed on Day 22 were firm andfree of necrosis or hemorrhage. The eluate and IgG fractionfrom this tumor demonstrated predominantly blocking activity(80 to 90%). Tumor removed on Day 35 demonstrated centralnecrosis. The corresponding eluate and IgG fractions showeda decrease in the blocking activity (55% blocking) with aconcomitant increase in inhibitory activity (45% inhibition). Thefinal tumor, removed on Day 64, exhibited both central andperipheral necrosis consistent with rejection. The corresponding eluate and IgG fraction had predominantly inhibitory activity(65%), although some blocking persisted (30%). The fractionswere all tested at the same time in the TCFUa against washedtumor cells that were harvested from an unrelated dog duringlogarithmic growth.

Fractionation of the Blocking Factor. The possibility thatthe blocking factor was an immune complex was investigatedby attempting to separate a small antigen from blocking IgG

OCTOBER 1979 3923




200

Chart 2. Correlation of tumor growth in Dog 5540 with blocking and inhibitory activity of purified IgG from tumor eluate.Tumors were biopsied on Days 22. 35, and 64 as shown. Blockingand inhibition are indicated as percentages of the activity ascompared to known high-titered blocking sera and inhibitory sera,respectively

TCFUa of Eluate IgG

I Percent Inhibition

Percent Blocking

that was purified from the eluates of logarithmically growingtumors (25 to 35 days posttransplantation). At pH 3.0, the IgGwas fractionated on an XM-100 filter as described above.

Under these conditions, antigens with a molecular weight lessthan 70,000 would pass into the filtrate, but the antibody wouldremain in the retained material. Table 4 demonstrates that,when IgG from 2 different tumors was fractionated in thismanner, no blocking activity was lost. No blocking activity wasrecovered in the filtrate, and recombination of the filtrate andretained material did not lead to an increase in blocking.

DISCUSSION

Much evidence has been adduced demonstrating that tumorsare antigenic in man and inbred rodent tumor models as revealed by induction of a potent cellular immune responsedemonstrable by lymphocyte-dependent cytotoxicity, lymphocyte-dependent colony inhibition, and mixed lymphocyte tumor

cultures (39). Moreover, serum from animals with progressivelygrowing tumors has been shown to block cellular immunity astested by in vitro and in vivo methods. Blockage of cellularimmunity by serum factors has been proposed as a mechanismwhereby tumors escape the cell-mediated immune response

(15, 16). On the other hand, humoral inhibition of tumor growthas measured in vitro has also been described in the mouseMoloney sarcoma, gross viral-induced tumors, and methyl-cholanthrene-induced tumors (10, 14, 28, 29).

The canine venereal tumor is a unique model for the study oftumor immunology. It is a naturally occurring cancer that canbe readily transplanted among outbred animals. It is also highlyantigenic, consistently stimulating the production of both inhibitory and blocking factors. The relative activities of these 2factors in serum have recently been shown to correlate withthe clinical course of the canine tumor (3). Sera from dogs thatspontaneously reject their tumor inhibit tumor colony formationin agar, whereas sera from dogs with progressively growingtumors will protect tumor cells against inhibition. With the invitro conditions used, the TCFUa has a poor plating efficiencywith less than 1 cell in 1000 forming colonies. Nonetheless,

Table 4

Blocking activity of tumor eluate IgG, fractionated by acidic Ultrafiltration

IgG from tumor eluate of a progressively growing tumor was purified as inTable 3. acidified to pH 3.0 with glycine buffer, and pressure filtered through anAmicon XM-100 filter. Antigens with a molecular weight of less than 70,000

would be expected to pass into the filtrate with the antibody remaining in theretentate. Blocking activity was tested by the blocking TCFUa as in Table 1.

Blocking TCFUa after preincubation with

Dog5539

5422Eluate

IgG19

15Retentate1416Filtrate0 2Retentate8

and filtrate1414

Concentration adjusted to be equivalent to assays with untreated IgG.

we believe that the cells that do form colonies represent across-section of the tumor as a whole because of the close

clinical correlation of serum and eluate inhibitory and blockingactivities as determined by counting the colonies in a single-

blind manner. Furthermore, as predicted from this assay, invivo growth of the canine venereal tumors was markedly alteredwhen humoral factors were removed by repeated plasmapher-

esis shortly after the tumor was transplanted (8).We have shown that the blocking factor is restricted to the

7S immunoglobulins and that both the inhibitory and the blocking activity could be found in lgG2a purified from the respectivesera. The possibility that other 7S immunoglobulins possessthese activities has not been excluded. These findings areconsistent with the preliminary characterization of blockingfactors in other tumor systems as well as homograft enhancement and rejection factors in mice (18, 19, 22, 40, 41).

Previous investigators of canine immunoglobulins, unable toseparate lgG2a from lgG2b, described them (lgG2a.b) as themajor y-globulin arcs in agar separating near the specimen well(19, 20). Further characterization by these investigators hasshown lgG2a.bto be (a) the major class of antigen precipitatingantibodies, (b) able to bind complement, and (c) the predominant canine IgG. Recently, the 2 subclasses have been partiallyseparated by anion-exchange chromatography (45). We wereable to produce an antiserum specific for lgG2a by absorptionof the rabbit anti-lgGja.b with a limited amount of lgG2b-enriched





fraction. The monospecificity of the antiserum (lgG2a) wasconfirmed by 1ERand SRID. The arc produced by this antiserumagainst NDS and IEP formed an identity reaction with theanodal spur produced by anti-lgG2a.b. Using the convention of

lettering the subclasses from the most cathodal to the mostanodal, the antiserum is specific for the 2a subclass of canineIgG.

IgG has been identified on the cell surface of tumor cells byindirect immunofluorescence (4) and by its presence in tumoreluate (8). The present study is in agreement with these observations and has extended them to demonstrate that inhibitingand blocking activity resides solely in the IgG fraction purifiedfrom tumor eluates. The antigen specificity of the eluted IgG isnot known, and possibly the difference between inhibitory andblocking IgG could be related to Idiotypic differences. However,because the factors were consistently effective in vitro withtumor cells harvested from unrelated dogs, the data suggestthat they are directed against antigens (including possiblehistocompatibility antigens) present in the tumor. Because theTCFUa were performed with washed tumor cells harvestedfrom young logarithmically growing tumors, the observed inhibitory or blocking activity was dependent on the added factorand apparently not related to differences in the tumor itself orthe profile of infiltrating inflammatory cells. Using differentmethods for determining blocking activity, others have foundblocking factors in the eluates of human (33) and ofbenzo(a)pyrene and methylcholanthrene-induced mouse sar

comas (36).Evidence has been presented (2, 7, 35, 36) that shows the

blocking factors as defined by in vitro and in vivo methods arean antigen-antibody complex. These conclusions were made

on the basis of either addition or removal of tumor transplantation antigens to blocking serum or tumor eluates. However,their results are also consistent with the hypothesis that competitive inhibitory antibodies are activated or inactivated by theremoval or addition of antigens. In the canine TVT system, wehave been unable to identify an antigen-antibody complex in

the purified blocking IgG. Fractionation of a purified blockingfactor from a logarithmically growing TVT by pressure ultrafil-

tration at acid pH to separate small antigens did not reduce theblocking activity. SDS electrophoresis of blocking antibodydenatured at pH 3.0 failed to identify an intermediate (M.W.50,000 to 160,000) size antigen. Finally, the localization ofblocking activity in the 7S Sephadex fraction (Table 1), with noactivity in the void volume fraction suggests that the blockingantibody is not complexed to a large antigen (M.W. >160,000).

A major question evolving from these studies concerns thedifference between inhibitory and blocking antibodies. Usingthe rat kidney sarcoma, Steele ef al. (38) have suggested onthe basis of differential adsorption with Staphylococcus aureus,Cowan I, that the blocking and inhibitory antibodies belong thedifferent subclasses of IgG. In the TVT system, the activitiescould not be differentiated by subclass; both blocking andinhibition being found in the 2a subclass of IgG.

In summary, we have purified the inhibitory and blockingfactors associated with the canine venereal tumor from theserum and from tumor eluate. The serum factors were purifiedby the Â¡mmunoadsorption technique using an antisera specificfor a single subclass of IgG, lgG2a, whereas the eluate factorswere purified by preparative electrophoresis. The differencesbetween blocking and inhibitory factors could not be explained

on the basis of IgG subclass as both activities were present inthe 2a subclass of IgG. The inhibitory or blocking activity onthe in vitro growth of a canine venereal tumor from an unrelateddog (the TCFUa) closely correlated with the growth characteristics of the host tumor. Finally, using a purified blocking factor,we were unable to demonstrate any acid-dissociable antigen

complexed to the factor.

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Fig. 1. Immunoelectrophoresis of normal dog serum in Noble agar (7) and agarose (2) demonstrating the specificity of rabbit anti-lgG2, b W and rabbit anti-lgG.(ÃŸ).

Fig. 2. Immunoelectrophoresis of normal dog serum (C S.) and blocking serum (B.S., Dog 5540). lgG2a was purified from progresser (blocking) serum byimmunoadsorption. IEP was performed with rabbit anti-canine serum (A) or rabbit anti-canine lgG2ab (B).

Fig. 3. Preparative polyacrylamide gel electrophoresis of eluate from a 35-day-old tumor (Dog 5539) on a 5% gel (3% cross-link). The anode is at the bottom.

OCTOBER 1979 3927



1979;39:3920-3927. Cancer Res William E. Beschorner, Allan D. Hess, Samuel T. Nerenberg, et al. Tumor-associated Inhibitory and Blocking FactorsIsolation and Characterization of Canine Venereal

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