1460(8) is consistent with its important role in
the immune response and in disease. As
shown in the present study, however, theimmunologic milieu, rather than the antigen, may be more important in determining
the development of a successful immune
response. A search for factors that modify
the developmental regulation of CD4' subsets might ultimately be more productive
than the hope that universally protective
microbial antigens can be reliably identified.
REFERENCES AND NOTES
1. F. P. Heinzel et al., Proc. Nati. Acad. Sci. U.S.A.
88, 7011 (1991); F. P. Heinzel et al., J. Exp. Med.
169, 59 (1989).
2. M. Lehn et al., J. Immunol. 143, 3020 (1989); C.
Bogdan etal., J. Exp. Med. 174, 1549 (1991); R.
de Waal Malefyt et al., ibid., p. 915.
3. R. G. Titus, R. Ceredig, J.-C. Cerottini, J. A. Louis,
J. Immunol. 135, 2108 (1985).
4. M. D. Sadick et at., J. Exp. Med. 171, 115 (1990).
5. B. J. Holaday etal., J. Immunol. 147,1653 (1991);
P. Scott, P. Natovitz, R. L. Coffman, E. Pearce, A.
Sher, J. Exp. Med. 168, 1675 (1988).
6. M. Belosevic et al., J. Immunol. 143, 266 (1989);
R. Chatelain, K. Varkila, R. L. Coffman, ibid. 148,
1182 (1992).
7. We selected clones 9.1-2 and H27 and U2, U3,
and U4 by limiting dilution from characterized
Leishmania-specific TH1 and TH2 CD4+ cell linesthat adoptively transfer protection or exacerbation, respectively (5). Clone 9.1-2 is a protective
CD4+ clone derived from an immunized BALB/cmouse (8). The remaining TCR sequences weregenerated from hybridomas established by fusionof CD4+ lymph node cells from healing (that is,previously treated with anti-CD4) BALB/c mice or
BALB/c mice with progressive infection with the
TCRa- and TCRP-negative mutant thymoma cellline, BW 5147 1100.129.237 (20). Viable clones
were selected for analysis only if they expressed
CD4 and TCR, as assessed by fluorescenceanalysis, and released either IFN-y or IL-4 in
response to stimulation with a sonicate of Leishmania antigen plus antigen-presenting cells. The

cytokine response was only transiently expressedin these hybridomas but permitted the assignment of one-third of the viable hybridomas to a
TH1 or TH2 phenotype.
8. P. Scott, P. Caspar, A. Sher, J. Immunol. 144,
1075 (1990).
9. G. Del Prete et al., J. Clin. Invest. 88, 346 (1991);
H. Yssel etal., J. Exp. Med. 174, 593 (1991); J. B.
A. G. Haanen et al., ibid., p. 583; M. Yamamura etal., Science 254, 277 (1991); P. Salgame et al.,ibid., p. 279; L. Romani etat., J. Exp. Med. 176,19(1992); A. Sher and R. L. Coffman, Annu. Rev.
Immunol. 10, 385 (1992).
10. N. E. Street and T. R. Mosmann, FASEB J. 5, 171(1991).
11. B. D. Evavold et al., J. Immunol. 148, 347 (1992).
12. M. Rocken, J.-H. Saurat, C. Hauser, ibid., p. 1031.
13. R. A. Seder, W. E. Paul, M. M. Davis, B. F. de St.
Groth, J. Exp. Med. 176,1091 (1992).
14. J. L. Jorgensen, P. A. Reay, E. W. Ehrich, M. M.
Davis, Annu. Rev. Immunol. 10, 835 (1992).
15. B. Boitel etal., J. Exp. Med. 175, 765 (1992); J.-L
Casanova, P. Romero, C. Widmann, P. Kourilsky,
J. L. Maryanski, ibid. 174,1371 (1991).
16. S. L. Reiner, unpublished data.
17. S. Adams, P. LaBlanc, S. K. Datta, Proc. Nati.
Acad. Sci. U.S.A. 88,11271 (1991).
18. J. L. Jorgensen et al., Nature 355, 224 (1992).
19. S. Candeias, C. Waltzinger, C. Benoist, D. Mathis,J. Exp. Med. 174, 989 (1991).
20. J. White et al., J. Immunol. 143, 1822 (1989).
21. K. Tomonari, E. Lovering, S. Fairchild, S. Spencer,
Eur. J. Immunol. 19, 1131 (1989).
22. K. Tomonari, E. Lovering, S. Spencer, Immunogenetics 31, 333 (1990).
23. Y. Choi et al., Proc. Natt. Acad. Sci. U.S.A. 86,
8941 (1989).
24. A. H. Taylor, A. M. Haberman, W. Gerhard, A. J.
Caton, J. Exp. Med. 172,1643 (1990).
25. H. S. Chou et al., EMBO J. 5, 2149 (1986).
26. We thank M. Sadick and B. Holaday for providing
T cell clones; 0. Kanegawa, F. Fitch, D. Loh, J.
Kappler, P. Marrack, K. Wang, L. Hood, I. Weissman, and G. Fathman for reagents; N. Killeen, A.
Theofilopoulos, and A. Caton for technical advice;and D. Littman, A. DeFranco, and J. Boothroyd for
critical comments. Supported by NIH grantA130663 and the Burroughs Wellcome Fund.
7 October 1992; accepted 1 1 January 1993

Inhibition of Human Colon Cancer Growth by
Antibody-Directed Human LAK Cells in SCID Mice

Downloaded from www.sciencemag.org on June 14, 2013that have been implicated in many bacterial,
mycobacterial, fungal, and parasitic diseases(9). There are two models for the development of TH1 and TH2 responses: the firstsuggests that they derive from distinct precursors (10), whereas the second suggeststhat they arise from a common lineage. The
use of peptide-specific clones (I 1), limiting
dilution analysis (12), and transgenic miceexpressing a single TCR (13) support thesecond possibility, and the findings in thispaper extend these previous observations bylinking subset maturation to biologically important antigens implicated in the immune
response to an infectious organism.
Evidence supporting an oligoclonal immune response was obtained by flow cytofluorometric analysis, direct sequencing ofreceptors from parasite-specific CD4' T
cell clones, and amplification of genomic
rearrangements as extracted from infected
mice. The response had unusual features.
First, several of the clones used common
variable region genes but expressed distinct
junctional sequences. The junctional diversity created by N region additions andexonucleotytic nibbling generates the
CDR3 domain of the TCR implicated in
the interaction with peptide (14). Although this implies that these different
clones should recognize distinct epitopes,
instances in which conserved V regions
seem to contribute substantially to finespecificity despite CDR3 differences havebeen noted (15). Second, 90% of the deduced junctional sequences of the Leishnania-specific clones contain anionic residues in this region (16), perhaps indicating
recognition of a cationic parasite antigen(17). The importance of charged residues
within the CDR3 domain in shaping theTCR-peptide interaction with MHC has
been delineated with the use of transgenic
mice expressing single-chain TCRs (18).
Third, the occurrence of relatively conserved TCR sequences among different animals and across MHC class II boundaries
was not predicted. The generation of therearranged functional TCR is not random;bias in the use of junctional nucleotide
additions contributes to constraints on
CDR3 length (19). The frequent use of the
Va8-JjTA72 rearrangement suggests that
the CDR3 region of the TCRa chain may
interact with a monomorphic parasite determinant, whereas the variation in pairingwith V,34 or in the V4-D-J. recombination
may provide the necessary polymorphism tobind different MHC molecules.
The nature of the antigen constitutes animportant area for further investigation.
Experiments to date have identified reactivity in association with a 10-kD protein that
stimulates the protective BALB/c-derived
clone, 9.1-2 (8). The identification of thisantigen in all Leishmaunia species analyzed

Hiroshi Takahashi,* Tetsuya Nakada, Isabelle Puisieux
Advanced human colon cancer does not respond to lymphokine-activated killer (LAK) cells.
In order to direct cytotoxic cells to the tumor, human LAK cells linked with antibodies to atumor cell surface antigen were tested with established hepatic metastases in severe
combined immunodeficient (SCID) mice. These cells had increased uptake into the tumor
and suppression of tumor growth as compared with LAK cells alone, thereby improving thesurvival of tumor-bearing mice. Thus, tumor growth can be inhibited by targeted LAK cells,and SCID mice can be used to test the antitumor properties of human effector cells.

Lymphokine-activated killer cells generat-

ed by interleukin-2 (IL-2) are cytotoxic tocolon cancer cells in vitro (1). However, inclinical trials, advanced human colon cancer has been refractory to adoptive immunotherapy using LAK cells (2). This limitation of LAK therapy may be due in part toan ineffectiveness of LAK cell cytotoxicity(3) or a lack of recruitment of LAK cells to
SCIENCE * VOL. 259 * 5 MARCH 1993

the tumor mass (4). In this report, wedemonstrate that human LAK cells can betargeted to the tumor by antibodies to atumor cell surface antigen. This targeting
augments the LAK cell effectiveness against
hepatic metastases of human colon cancer
in SCID mice (5).
SCID mice have no functional T and B
cells and therefore will not reject xenografts

INE
30h

80.

SCID mice. C.B-17 SCID mice were purchased

El hour* 3 hours
from Taconic (Germantown, New York). All ani- 0
1 day
mal experiments were approved by the commitX 20
E
tee on research animal care protocol review
n 2 days
0
E7 daysgroup and carried out according to Massachusetts General Hospital guidelines. Mononuclear
l*14days
cells were isolated from healthy volunteers by*
gradient density centrifugation and incubated in *EI
e 0 i
the plastic plate coated with fibronectin to pre_i
Blood Lung Liver Spleen Kidneypare adherent cell-free human PBLs. LAK cells
were generated by incubation of these PBLs with recombinant human IL-2 (100 U/mI; Shionogi
Pharmaceutical Co., Osaka, Japan) for 48 hours at 370C. The biodistribution was examined byintravenous injection of human LAK cells labeled with [3H]uridine and human IL-2 (500 U permouse) into SCID mice. The radioactivity of each organ was examined after tissues were digested
by Solvable (Du Pont) and was expressed as the percentage of injected dose per organ. Three
animals were used to determine each value (mean ± SD).

of human lymphocytes or human tumors(5). These mice can be repopulated withhuman T or B cells, and human tumors
with infiltrating lymphocytes have been
propagated (6). SCID mice have been used
to examine the growth of human lymphoma
cells after exposure to cytokine-activatedhuman killer cells in vitro (7). However,the use of the SCID mouse model to test
the antitumor effects of exogenous humaneffector cells by intravenous administration
has not been established. The short-term
survival and biodistribution of human LAK
cells labeled with I3Hluridine was examined
in SCID mice after intravenous injection(Fig. 1). Human LAK cells were detectable
up to 14 days, and most cells were in theliver after being temporarily trapped in thelung.
Injection of LS 180 human colon adenocarcinoma cells into the portal vein via thespleen of SCID mice establishes in thesemice a model for human colon cancer (8,9). Although this animal model does not
represent all the steps of the metastaticcascade (10), it simulates the metastatic
growth of tumor cells in the liver after
vascular spread. All mice eventually die
from extensive hepatic parenchymal involvement of tumors within 6 to 7 weeks
after tumor cell injection.
Although tumor-specific antibodies may
be useful for targeting cytotoxic effector
cells to tumor cells, it is not clear whether
in vivo immunolocalization is improved(11). The monoclonal antibody (MAb)
SF-25 recognizes a 125-kD cell surface antigen (Ka = 2.36 x 109 M- 1) that isexpressed on human colon adenocarcinomas (12). MAb SF-25 injected intravenously immunolocalizes to human colon

adenocarcinomas previously established in
the liver of nude mice (8). The MAb SF-25
alone does not inhibit tumor cell growth invitro, and SF-25 antigen is neither internalized nor shed from the tumor cell surface(13). We used MAb SF-25 to augment
LAK cell effectiveness.
The immunological defect of SCID miceis limited to a stem cell progenitor of T and
B lymphocytes (5); macrophage and naturalkiller (NK) cell functions are intact (14).
Therefore, such effector cell populations
might inhibit tumor growth through interactions with injected antibodies (15). To
assess if MAb SF-25 would augment the invivo antitumor effect of human LAK cells
without interfering with the murine immune system in this animal model, we
linked a modified MAb SF-25 to human
LAK cells with polyethylene glycol (PEG)(16). We used a recombinant chimericantibody construct of MAb SF-25 (MAbc-SF-25) that has the variable region ofmurine MAb SF-25 and constant region ofhuman immunoglobulin (Ig) GI (17). Most
LAK cells express an Fc receptor for IgG(FcyR type III) (CD16) (1, 18), and human IgGl binds to this Fc-yR (19). Therefore, the antigen-combining site of MAb
c-SF-25 is more likely to be exposed in an
outward orientation because the Fc region
should bind to FcyR on the cell surface of
LAK cells. PEG treatment then enhances
the binding of MAb to LAK cells. Tentimes more antibodies were conjugated to
LAK cells by PEG treatment than by incu-

C~~~~~~~~~~~~~~C
40 .

1200
0

.0

*

a

3.13 1.56 0.78

Efetor: ta

ratio

Fig. 2. Cytotoxicity produced by human PBLs
and LAK cells conjugated to MAb c-SF-25.
Circles, PBLs; squares, LAK cells; empty symbols, cells alone; filled symbols, cells conjugated to c-SF-25. Cytotoxicity was assessed by
4-hour 51Cr-release assay with LS 180 cells astargets at different effector/target cell ratios (E/T
ratio) (21). Results are the mean SD of values
obtained in triplicate experiments. Although
PEG treatment did not affect the viability ofeffector cells, it was slightly detrimental to LAK
cell cytotoxicity.

bation with the MAb c-SF-25 alone (-0.1
and 0.01 pmol of MAb per 107 LAK cells,
respectively) (20).
The availability of the antigen-combining site to bind with target cells was examined with the use of a cytotoxicity assay ofmacrophage-depleted human peripheral
blood lymphocytes (PBLs); NK cells inhuman PBLs express Fc-yR type III andinitiate antibody-dependent cell-mediatedcytotoxicity (ADCC) on binding to theantibody (21) (Fig. 2). Human PBLs conjugated to MAb c-SF-25 (c-SF-25-PBL)
were cytotoxic toward LS 180 cells. In
contrast, human PBLs alone under thesame conditions were not. The ADCC
response requires simultaneous binding of
the antigen-combining site of an antibody
with the antigen and the Fc region to FcyRs
on the effector cell (21). Therefore, MAb
c-SF-25 is functionally available to bind to
LS 180 tumor cells after conjugation of theantibody to cells. Human LAK cells withand without conjugated c-SF-25 were cytotoxic (Fig. 2). However, when these c-SF25-LAK cells were labeled with [3H~uridine
and injected into the SCID-human coloncancer model, increased uptake into thetumor site was observed (Table 1). Tumor
uptake of c-SF-25-LAK was about threetimes higher than that of LAK cells alone,

Table 1. Tumor uptake of LAK cells conjugated to MAb c-SF-25.

Percentage of injected dose per gram of tissue*
3 hourst

Molecular Hepatology Laboratory, Massachusetts
General Hospital Cancer Center, Charlestown, MA
02129, and Department of Medicine, Harvard Medical
School, Boston, MA 02115.
*To whom correspondence should be addressed at
Molecular Hepatology Laboratory, Massachusetts
General Hospital Cancer Center, Charlestown, MA
02129.

+

CD

6 hours

24 hours

6.24 + 0.221
5.09 ± 2.70 0.65174

22.19 ± 1.36 0.0001
26.22 + 4.98 0.0061
6.57 ± 0.73
8.91 + 3.51
*Human LAK cells were labeled with [3H]uridine and intravenously injected into SCID mice bearing LS1 80 hepatic
metastases. Tumor uptake was determined by measurement of radioactivity in tumors dissected from surrounding
normal liver and was expressed as the percentage of injected dose per gram of tumor tissue. Two to four animals
tHours after intravenous injection of [3H]uridine-labeledwere used to determine each value (mean - SD).
tStatistical significance was examined by two-sided Student's unpaired t test.
effector cells.

c-SF-25-LAK
LAK alone

SCIENCE*VOL. 259*

5 MARCH 1993

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Downloaded from www.sciencemag.org on June 14, 2013

Fig. 1. Biodistribution of human LAK cells inre moffiffi.......
...........................

........

....TreatedSin

A Multiple, >2_.
2-so-

Untreated-,

le1251-SF-25

X
.
E 1 .
:
2
*

125-SF-25 +Cold SF-25

a(Kruskal-Wallis test, P = 0.0011) is given aso U * . .a..

1251-SF-25+

Cold B2TT

D
L
CL

c

0.

2e n
2 U) 3follows: untreated mice versus mice treated withL, ,LAK cells alone (LAK), LAK cells incubated with+,
U. oc-SF-25 without PEG treatment (LAK + c-SF-25),c-SF-254.AK; CO ,
c-SF-25-LAK, or LAK cells conjugated to human
IgG1 isolated from myeloma plasma (huigGi-LAK) (P= 0.5755, P= 0.0749, P= 0.0002, and P =0.9165, respectively); c-SF-25-LAK versus LAK, LAK + c-SF-25, or hulgG1 -LAK (P = 0.0020, P =0.2783, and P = 0.0040, respectively). The statistically significant a level is 0.05/7 = 0.0071. The
effect of multiple injections of c-SF-25-LAK cells (Kruskal-Wallis test, P = 0.0005) is given as follows:
untreated mice versus mice receiving single or multiple injections (P = 0.0030 and P = 0.0005,
respectively) and multiple versus single injections (P = 0.9840). The statistically significant a levelis 0.05/3 = 0.0167. Most of the mice treated with c-SF-25-LAK were free of detectable hepatic
metastasis.

E

Negative control/4

Multiple treatment
Single treatment

Untreated-..............

and the increased uptake was statisticallysignificant (Table 1).
Five days after the injection of LS 180
cells into the portal vein and when micrometastatic disease was present, c-SF-25LAK cells, LAK cells alone, LAK cells
incubated with MAb c-SF-25 without PEG
treatment (20), or human IgGl-LAK cells
were injected intravenously into SCID
mice [107 cells with 500 U of IL-2 in 200 pulof phosphate-buffered saline (PBS) per
mouse, respectively] (Fig. 3). The weight ofhepatic tumor masses from each mouse wasdetermined 5 weeks after tumor cell injection; that of untreated mice was 1.07
1.60 g (mean SD, n = 23). A single
intravenous injection of c-SF-25-LAK cells
substantially inhibited the tumor growth
0.23 g, n = 20). In contrast,(0.08
human LAK cells alone, LAK cells incubated with c-MAb SF-25, and human
IgGl-LAK cells were less effective (0.58
0.76 g, n = 20; 0.25 0.41 g, n = 9; and0.54 0.80 g, n = 9, respectively). The
decrease in tumor weight was statisticallysignificant only in the c-SF-25-LAK-treated group as compared with the untreatedgroup. The anti-tumor effect of multiple
injections was also examined by the injection of c-SF-25-LAK cells once weekly forthree consecutive weeks. Inhibition of tumor growth was more prominent with multiple injections (untreated mice, 2.76
1.12 g, n = 9; single injection, 0.51 0.95
g, n = 9; and multiple injections, 0.11
0.27 g, n = 9).
The duration of survival of SCID mice
injected with c-SF-25-LAK cells (107) 7
days after the tumor cell injection wasdetermined. All untreated mice died within
7 weeks after tumor cell injection as a resultof massive parenchymal spread of tumor in
the liver (median survival 30 days, n = 10).

In contrast, the treated animals survived
longer than untreated controls (49 days, n= 9) (P = 0.002 versus untreated mice by
the Mantel-Haenszel test, statistically significant at a level of 0.05/6 = 0.0083 after
Bonferroni correction for multiple comparisons). Single or multiple injections (once aweek to a total of four injections) of c-SF25-LAK cells beginning at 14 days after thetumor cell injection (when there are macrometastases in the liver) improved thesurvival rate of the mice (single injection,
45 days, n = 9, P = 0.034 versus untreated
mice; and multiple injections, 54 days, n =4, P = 0.006 versus untreated mice).
Under our experimental conditions,however, the in vivo purging of tumor cellsby c-SF-25-LAK was not perfect. Therefore, the expression of SF-25 antigen in thetumors from mice treated with c-SF-25LAK cells was examined to investigate
whether treatment selected for the growthof tumor cells that no longer bore the SF-25
antigen. The SF-25 antigen was expressedin c-SF-25-LAK-treated tumor cells, as
shown by histological examination (Fig.
4A) as well as by flow cytometry (Fig. 4B).
Furthermore, LS 180 cells derived fromboth c-SF-25-LAK-treated and untreatedmice were lysed by LAK cells in vitro.
Therefore, the outgrowth of tumor in treated mice was not the result of selection of
SF-25-negative or LAK-resistant tumor
cells but may have resulted from an insufficient dose of c-SF-25-LAK for the tumor
burden.
Adoptive immunotherapy using tumorinfiltrating lymphocytes (TILs) cultured
with IL-2 has been reported to be more
effective than LAK immunotherapy withrespect to tumor response rates (22). TILs
contain tumor-specific T lymphocytes (23).
The cellular basis for successful adoptive

1462

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Fig. 3. Tumor growth after c-SF-25-LAK treatment. Tumors were carefully separated fromsurrounding normal liver after formaldehyde fixation and evaluated by an independent investigator (M. Nakaki). A vertical axis denotes theweight of hepatic metastases. The differences intumor weights were tested by Kruskal-Wallis
statistic followed by Mann-Whitney rank sum testwith Bonferroni correction to conduct multiple
comparisons. The effect of different treatments

Fig. 4. Expression of SF-25 antigen in LS 180
turnor after treatment with c-SF-25-LAK. (A)Histological examination of SF-25 antigen. The
cryostat sections of hepatic metastases derived from treated (single or multiple injection ofc-SF-25-LAK cells) or untreated SCID mice
were incubated with '251-labeled MAb SF-25
and autoradiographed as described (8). SF-25
antigen was highly expressed in both c-SF-25LAK-treated and untreated tumors as shownby the dense and homogenous radioactive
spots. This reaction was specific because it
was inhibited only by unlabeled specific antibody and not by an irrelevant MAb (B2TT) totetanus toxoid. (B) Flow cytometric analysis of
SF-25 antigen. A single cell suspension of LS
180 cells was prepared from hepatic turnors
derived from the c-SF-25-LAK-treated (single
or multiple) and untreated mice. The expression of SF-25 antigen at the cellular level was
examined by flow cytometry with the use offluorescein isothiocyanate (FITC)-labeled MAb
SF-25. FITC-labeled irrelevant MAb (B2TT)
served as a negative control. The antigen was
uniformly expressed at high density in most of
the cell populations (>99%) from both untreated and treated mice.

immunotherapy of tumors by TIL may relate to the ability of tumor-specific T cells
to traffic to and target tumor cells in vivo
(24). Human TILs, but not human LAK
cells (4), have been shown to immunolocalize to tumors in vivo (24). The present
studies demonstrate the ability of MAbdirected human LAK cells to inhibit thegrowth of human colon cancer establishedin the liver of SCID mice. Because no
increased cytotoxicity was observed inc-SF-25-LAK cells as compared with LAK
cells alone in vitro, this augmentation of
the anti-tumor effect must be largely due tothe specific targeting of LAK cells to the

REFERENCES AND NOTES
1. E. A. Grimm, A. Mazumder, H. Z. Zhang, S. A.
Rosenberg, J. Exp. Med. 155,1823 (1982); J. H.
Phillips and L. L. Lanier, ibid. 164, 814 (1986).
2. S. A. Rosenberg et al., N. Engi. J. Med. 313, 1485
(1985); W. H. West et al., ibid. 316, 898 (1987).
3. I. Kawase et al., Cancer Res. 48, 1173 (1988).
4. B. Mukherji et al., Int. J. Radiat. Appl. Instrum. B
15, 419 (1988).
5. G. C. Bosma, R. P. Custer, M. J. Bosma, Nature
301, 527 (1983); M. J. Bosma and A. M. Carroll,Annu. Rev. Immunol. 9, 323 (1991).
6. D. E. Mosier, R. J. Gulizia, S. M. Baird, D. B.
Wilson, Nature 335, 256 (1988); J. M. McCune etat., Science 241, 1632 (1988); R. B. Bankert etal.,
Curr. Top. Microbiol. Immunol. 152,201 (1989); K.
Pfeffer, K. Heeg, R. Bubeck, P. Conradt, H. Wagner, ibid., p. 212; E. Simpson, J. Farrant, P.
Chandler, Immunol. Rev. 124, 97 (1991).
7. I. G. H. Schmidt-Wolf, R. S. Negrin, H.-P. Kiem, K.
G. Blume, I. L. Weissman, J. Exp. Med. 174,139

mg/mi) in RPMI 1640 for 30 min at 40C. An equalvolume of 30% PEG 8000 (Sigma) in RPMI 1640
was added to this cell mixture and incubated foran additional 90 min at 4'C. Finally, the cells were
washed with PBS three times. As a control, human
LAK cells were incubated with MAb c-SF-25 (2mg/mi) for 90 min at 40C and washed three times.
21. E. Vivier et al., J. Immunol. 146, 206 (1991).
22. S. A. Rosenberg etal., N. Engi. J. Med. 319, 1676
(1988).
23. J. T. Kumick, R. L. Kradin, R. Blumberg, E. E.
Schneeberger, L. A. Boyle, Clin. Immunol. Immunopathol. 38, 357 (1986); L. M. Muul, P. J. Spiess,
E. P. Director, S. A. Rosenberg, J. Immunol. 138,
989 (1987).

24. B. Fisher etal., J. Clin. Oncol. 7, 250 (1989); K. D.
Griffith et al., J. Nati. Cancer Inst. 81, 1709 (1989).
25. R. J. Dearman et aL, Blood 72, 1985 (1988); A. W.
Tong, J. C. Lee, R.-M. Wang, G. Ordonez, M. J.
Stone, Cancer Res. 49, 4103 (1989).
26. K. Nishihara et at., Cancer Res. 48, 4730 (1988);
S. A. Rosenberg et al., N. EngI. J. Med. 323, 570(1990); T. Friedmann, Cancer Cells 3, 271 (1991).
27. Supported by grants CA-57584 and CA-35711
from the National Institutes of Health. We thank J.
R. Wands and K. J. Isselbacher for review of themanuscript and for advice and support and R.
Carlson and M. Nakaki for assistance.
2 October 1992; accepted 17 December 1992

Optical Time-of-Flight and Absorbance Imaging
of Biologic MediaDavid A. Benaron and David K. Stevenson
Imaging the interior of living bodies with light may assist in the diagnosis and treatment of
a number of clinical problems, which include the early detection of tumors and hypoxic

cerebral injury. An existing picosecond time-of-flight and absorbance (TOFA) opticalsystem has been used to image a model biologic system and a rat. Model measurements
confirmed TOFA principles in systems with a high degree of photon scattering; rat images,
which were constructed from the variable time delays experienced by a fixed fraction ofearly-arriving transmitted photons, revealed identifiable internal structure. A combinationof light-based quantitative measurement and TOFA localization may have applications incontinuous, noninvasive monitoring for structural imaging and spatial chemometric analysis in humans.

(1991).
8. H. Takahashi et al., Gastroenterology 96, 1317

(1989).
9. The SCID-human colon cancer model was established according to the method described in (8),with modification. SCID mice anesthetized byintraperitoneal injection of 2% chloral hydrate (0.4
ml per mouse) were injected with 1 06 LS 180 cells
and 20 1Lg of rabbit antibodies to asialo GM,(Wako) in 0.25 ml of PBS (injection into the portal
vein via splenic hilus with a 27-gauge needle).
After 1 min, the portal vein was ligated and thespleen removed.
10. I. J. Fidler, Cancer Res. 50, 6130 (1990).
11. H. Nelson, Cancer Cells 3, 163 (1991).
12. B. Wilson et al., Proc. Natl. Acad. Sci. U.S.A. 85,
3140 (1988); H. Takahashi et al., Cancer Res. 48,
6573 (1988).
13. H. Takahashi, unpublished data.
14. K. Dorshkind, S. B. Pollack, M. J. Bosma, R. A.
Phillips, J. Immunol. 134, 3798 (1985); G. J.
Bancroft, R. D. Schreiber, E. R. Unanue, Immunol.
Rev. 124, 5 (1991).
15. D. Herlyn and H. Koprowski, Proc. Nati. Acad. Sci.
U.S.A. 79, 4761 (1982); Z. Steplewski et al., ibid.
85, 4852 (1988); W. J. Johnson etal., J. Immunol.
136, 4704 (1986).
16. J. F. Jones and D. M. Segal, J. Immunol. 125,926(1980).
17. MAb c-SF-25 was produced and provided byCentocor Inc. (Malvem, PA). We constructed ahuman-mouse chimeric 1g gene by joining variable
region genes of light and heavy chains isolated
from MAb SF-25 hybridoma cells to constant region genes of human K light and y 1 heavy chains.
This construct was then transfected into Sp2/0
myeloma cells to produce the chimeric MAb.
18. J. R. Ortaldo, A. Mason, R. Overton, J. Exp. Med.
164, 1193 (1986); K. Roberts, M. T. Lotze, S. A.
Rosenberg, Cancer Res. 47, 4366 (1987).
19. J. C. Unkeless, J. Clin. Invest. 83, 355 (1989); J. V.
Ravetch and J. P. Kinet, Annu. Rev. Immunol. 9,457 (1991).
20. LAK cells were incubated with MAb c-SF-25 (2The principles that underlie the imaging ofMedical Spectroscopy and Imaging Laboratory Section, Neonatal and Developmental Biology Laboratory,
Department of Pediatrics, Stanford University Schoolof Medicine, Palo Alto, CA 94305.

form transillumination "shadowgrams" ofsuch tumors (13), to detect cerebral bleeding(14), and to reconstruct two-dimensional
steady-state tomographs (15). Temporalvariations in absorbance, which can becaused by optical changes in certain proteinsas oxygen pressure varies (16), have beenused to estimate changes in the oxygenationof the brain (8, 12, 17) and extremities (18)and to measure blood flow and volume intissues (19). Spectral analysis may also permit in vivo quantitation of substances suchas glucose and cholesterol (20).
Optical imaging (7) and spectroscopy(20) in vivo have been difficult. With
conventional radiological methods, photon
scattering is minimal and, therefore, radiation travel is linear. In contrast, light takes
highly irregular, diffusive paths through tissue, degrading image quality; detail is lostmuch as it is with distance in a fog. Scattering is the major attenuator of the intensity of transmitted photons in tissue (21).
On average, a photon travels only millimeters into tissue, or less, before it scatters(22). Multiple scattering events occur for
virtually all photons that propagate throughtissue (22, 23), which causes them to take awide range of paths as they traverse thetissue, each requiring a different amount oftime. Because path lengths vary with absorbance and scattering, they are difficult topredict (12).

5 MARCH 19931463

the interior of living bodies with light haveapplication to a variety of situations thatinvolve radiation transmission throughopaque scattering media. Images can beformed with x-ray tomography (1), magnetic resonance (2), ultrasound (3), positronemission (4), thermal emission (5), electrical impedance (6), and other probes; butthese have drawbacks that limit their use in
the continuous, noninvasive monitoring of
humans (7). Light, on the other hand, isnonionizing, relatively safe, and known tofunction well as a medical probe (8-13). In
particular, red and near-infrared light passeasily through structures such as the skull(9), penetrate deeply into many tissues(10), are well tolerated in large doses (1 1),
and can be used to quantitate chemical
concentrations if an optical path length is
known or estimated (12).
Variations in tissue absorbance and scattering over space and time influence photon
travel through tissue. For example, breast
tumors often attenuate light to a greater
degree than neighboring tissues, possibly because of the presence of numerous bloodvessels and mitochondria (11, 13). Spatial
variations in absorbance have been used to

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tumor mass rather than the cytotoxicity
induced by an ADCC mechanism (25). We
conclude that the targeting of human LAK
cells to colon tumors is an important factor
in the inhibition of tumor growth in vivo
and that the SCID mouse-human xenogeneic chimeric model is useful for the studyof anti-tumor properties of human effector
cells. As demonstrated by the results of thepresent studies, it will be possible to test
genetically modified human effector cells by
conjugating the cells with tumor-specificantibodies to further increase their antitumor effects (26).