Cancer Treahent
(1990)
Review
17, 357-37
1
Pitfalls in the clinical application of monoclonal antibodies in malignant melanoma: modulation impaired accessibility of antigens to monoclonal antibodies Wolfgang
Tilgen*
and
Siegfried
by and
Matzku
Department of Dermatology, University of Heidelberg and the Institute of Radiology and Pathophysiology, Division of Applied Immunology, German Cancer Research Center, II-6900 Heidelberg, FRG
Introduction New diagnostic and therapeutic possibilities were introduced by the hybridoma technique (21), which enables production of monoclonal antibodies against tumor-associated antigens (1-4, 7, 11, 14, 16, 22, 30, 3 1,40,45). Modest results of the pioneering clinical studies in radioimaging (5, 6, 23, 34, 35), serotherapy (8, 9, 12, 17, 32, 43) and the use of monoclonal antibodies (MAbs) as carriers for cytotoxic agents (33, 36, 44) have now shown the limitations of the approach (10, 13, 15, 18-20, 24-28, 37-42). It has become increasingly clear that the specificity of MAbs, i.e. the ability of an antibody to reveal an antigen which is expressed selectively on the tumor cells, cannot ensure effective tumor targeting. We therefore wanted (1) to evaluate modulation of cell surface associated antigens by MAbs, i.e. internalization or shedding ofimmune complexes and (2) to study the accessibility of melanoma cells within solid tumor tissue. We have addressed these questions by following the pathways of radionuclide (rZ51)- and goldlabeled antibodies in the living cell in vitro with immunoelectron microscopy (IEM) and with a radioantibody binding assay (RBA) and by analyzing the biodistribution of labeled MAbs in human xenograft tumors with the paired label assay (PLA) and whole body autoradiography (WBA). We also performed some clinical pilot studies with immunoscintigraphy and serotherapy.
Antigen
modulation
by monoclonal
antibodies
in vitro
Initially we tested a panel of MAbs looking for subcellular localization (37, 38). Studies were carried out in vitro on metabolically inactive fixed melanoma cells grown as monolayer. Two binding types were identified: Antigen-antibody complexes either covered the whole cell surface [Figure 1 (a)] or were localized intracellularly outlining mitochon* Corrcspondencr 0305.-7372/90/2&3357
address:
W. ‘l‘ilgen,
Universitats-Hautklinik,
Voss-Str.
2, 6900 Hridelberg, G 1990 Acadrmic
+ 15 $03.00/0 357
F.K.G. Press Limited
&we 1. Localization ofantibody binding by immunoelcctron microscopyofmelanoma is restricted to the cell surfacr (arrowheads) ( x 3525). (b) MAb binds to a cytoplasmic on mitochondriae, Golgi apparatus, the nuclear envelope and also to the cell Cy, cytoplasm; N, nucleus; NC, nucleolus ( x 3525).
cells in uitro.(a) Reaction antigen which is localized membrane (arrowheads).
PITFALLS
IN
THE
MONOCLONAL
ANTIBODY
APPROACH
359
dria, Golgi apparatus, rough endoplasmic reticulum and the nuclear envelope [Figure 1 (b)]. In living cells, three binding modalities for immune complexes (IC) could be demonstrated by radioimmunoelectron microscopy (RIEM) and radioantibody binding assay (39). In the RBA (24-27) it turned out that the binding of e.g. MAb LlO, which is directed against an epitope of the integral membrane protein gp95 (11, 16) was not greatly influenced by the metabolic activity of the target cell, showing roughly the same amount of bound radioactivity at 0” and 37°C. After exposing the cells to an iso-osmolar buffer of pH 2.8, which dissociates the ICs from the cell surface, most of the radioactivity was eluted, indicating a predominant binding of the MAb to the cell surface. This nondesorbable radioactivity corresponded to the few ICs localized in the cytoplasm as shown by RIEM. Labeled antigen-antibody complexes covering the entire cell membrane were seen after 30 min and most of the immune complexes remained stable on the cell surface even after 120 min. It appears therefore that the first prototype ofbinding leads to a stable, membrane anchored immune complex. The second prototype of binding was observed with MAb R24 directed against the glycolipid antigen GD3 (11, 30). In the RBA, uptake of MAb was highest with fixed cells at O”, intermediate with living cells at 0” and lowest with cells kept at 37°C. This pointed to a release of the immune complexes from the cell surface. Due to the low binding level, RIEM could demonstrate only few labeled immune complexes in living cells. Immunoelectron microscopy solved the problem. Glutaraldehyde-fixed cells exhibited uniform staining along the whole cell surface. In contrast, metabolically active unfixed cells showed only localized reactivity, with patchy deposits of immune complexes, or a complete lack of immunostaining. Taken together this points to a shedding process. The third prototype of binding behaviour is illustrated by MAb M.2.9.4 (4). RBA data showed that cells exhibited markedly higher uptake at 37°C than at 0°C. The portion of binding resistant to pH 2.8 buffer treatment corresponded to the increased incorporation of labeled MAb into the living cell. The latter component of uptake was completely lacking in metabolically inactive fixed cells. RIEM also showed a rapid binding to viable cells with a pronounced deposition of decay tracks over the whole cell surface. After 2 h substantial amounts of immune complexes had migrated into the cells. The ‘antigen route’ seemed to be directed to the nuclear region [Figure 21. Internalization of individual MAbs may follow different pathways (29). One of them is constitutive endocytosis by endocytotic vesicles. Another pathway is shown here for the 5 nm colloidal gold-labeled mAb HD-MEL 3, which was distributed all over the cell surface, but grouped in clusters both in the region of non-coated plasma membrane invaginations and in coated pits or coated vesicles, pointing to a mechanism of antibody uptake reminiscent of receptor-mediated endocytosis (Figure 3). The third binding type therefore represents an internalization process. Accessibility The problem
of target
cells
within
solid
tumor
tissue
of accessibility of target cells within solid tumor tissue was previously shown we processed melanoma cells growing to small tumor nodules in culture dishes. IEM showed that, in contrast to the monolayer culture, only the outermost layer of melanoma cells was stained by monoclonal antibodies and virtually no labeling was seen within the tumor cell cluster (Figure 4). In the paired label assay (Table 1) no clear
in vitro when
360
W. TILGEN
AND
S. MATZKU
Figure 2. Radioimmunoelectron microscopy of melanoma cells in vitro with the ‘251-labeled MAb M.2.9.4. After 120 min substantial amounts of immune complexes (arrowheads) had migrated into the cytoplasm of the metabolically active cells kept at 37°C and only few remained on the cell surface. (N, nucleus; PM, plasma membrane) ( x 13 200).
correlation was observed between types ofMAb binding and gross uptake, since significant accumulation was noted throughout. The localization indices were variable, neither high internalization ratios nor high antigen densities being reproducibly linked to high specific uptake. An exception was the shedding-related binding type being associated with low localization indices. Diagnostic tumor targeting with MAbs, and even more so immunotherapy with antibody conjugates, ideally requires antibody-binding to every tumor cell with saturation of all available binding sites. In view of the basic architecture of solid
ANTIBODY
.Wl’KOAC:H
melanoma, access ofmacromolecules may be limited in the steps oftranscapillary or interstitial diffusion/convection (18-20). In fact, relative accumulation, scinti,graphic contrast is greatly enhanced when using MAb fragments. In intact IgG, impressive localization is achieved in a shorter period of time with of fragments as shown in the PTA for MAb HWMel3 (Table 2). However, this of fragments in immunoscintigraphy does not offer a universal solution to the access, because it is not clear whether every melanoma cell&provided it
361
transport resulting in contrast to both types application problem of carries the
W.
362 Table
1. Accumulation melanoma
cell
of MAbs properties
TILGEN
in nude mice in vitro
AND
S. MATZKU
with
MML-I
melanoma
xenografts:
Localization Antigen” density
MAh LlO HD-Mel HD-Mel3 225.28 M.2.7.6 M.2.9.4 R24
1
34.0 19.0 16.0 1.3 0.5 16.0 0.2
Type
of binding
Tumor/blood
Surface binding Surface binding Surface binding Surface binding Internalization Internalization Shrdding
Tumor/liver
0.5 1.8 3.3 3.4 5.5 2.0 1.2
Tumor/hone
Tumor/musclr
5.3 1.4 3.0 3.5 6.3 1.8 0.8
anti-melanoma
to
index”
1.6 1.9 2.9 3.2 5.7 2.9 1.1
“Apparent antibody bindin! sites per cell x 10’. “Paired label assay, comparison being made between “S1-laheled antibody (B40) injected into the same animals (n = 3-6).
Comparison
MAbs
6.9 2.0 2.6 3.4 5.1 3.4 1.1
and “‘I-laheled
control
antigen-is reached by the antibody or its fragment. With macro-autoradiography large size sections are exposed on X-ray film. The resulting autoradiographs give an overview of a tumor cross-section, and in the case of whole body sections of tumor-bearing rodents they also give a comprehensive image of radioactivity distribution in normal tissues. Whole body autoradiography impressively showed thedifferential access and clearance of monoclonal antibodies and their fragments. The common pattern was a predominant accumulation in the periphery of the tumor nodes (Fig. 5a). The central part was stained to a different degree depending on the transplanted melanoma cell line (Fig. 5b). MAb R24 was diffusely distributed throughout the tumor cross-sections at a non-significant level pointing to a shedding process (Fig. 5~). HD-Mel3 showed a non-uniform distribution pattern all over the melanoma node, but radioactivity was restricted to a few “hot” spots (Fig. 5d). Thus, WBA may bridge the gap between microscopic examination and the low resolution techniques of scintigraphy or paired label assay.
Table
2. Accumulation of intact IgG
of HD-Mel3 and fragments”
in nude
mice
with
MML-I
melanoma
Localization Time Fah fragments: 6h 12 h 24 h F(ab’), fragments: 12 h 24 h 48 h Intact IgG: 24 h 48 h 96 h
xenografts:
Comparison
Muscle
BOtW
8.42 39.88 15.98
indices
Blood
Spleen
Kidney
9.88 26.92
17.86 38.33 36.43
9.78 20.59
25.26
33.12 76.93 53.26
23.13
8.32 18.00 12.78
8.81 17.51 49.08
7.82 15.31 33.50
13.81 25.83 55.83
7.88 17.58 34.36
8.38 12.93 5.47
8.20 11.11 10.94
2.00 3.32 1.96
1.95 3.27 1.77
1.74 2.73 2.00
1.92 2.93 2.02
1.61 2.62 1.59
1.80 2.95 1.98
*Paired label assay, comparison being (B40) injected into the same animal.
made
between
12”1-laheled
Liver
HD-Mel
3 and ‘s’I-labeled
control
antibody
PI’I‘E‘ALLS
IN
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MONOCLONAL
AN1‘IBODY
APPROACH
363
d
@ure 5. Whole body autoradiographs performed 48 h after application of “‘I-lahcled MAbs: Distribution pattern of difkent anti-melanoma MAhs in representative melanoma xenografts. (a) M.2.10.15 in MeWo, peripheral staining. (b) M.2.7.6 in SK-MEL-28, peripheral and intratumoral staining. (c) R24 in Co10 38, no significant staining. (d) HD-Mel3 in MML-I, intratumoral staining. ‘I’hc insets show additional sections through the same tumor.
W.
364
‘I‘ILGEN
AND
S. MA’I‘ZKU
b b’igm 6. Immunoscintigraphy in the melanoma patient. ia) Clinical aspect of hundreds of cuta~~cous and suhcutanrous metastases of different size on the thigh. (b) Immunoscintigram with the ““““l‘r-labeled hlAb 225.28% Radioactivity acrumulation was only detected in the region of two big ulccratrd melanoma nod&s iarrows), thr ahundancc ofsmall mctastases was not detected. (K = background radioactivity in thr kidncys.1
Immunoscintigraphy
of melanoma
patients
At this point, experimental models seemed to be limited, and further information could only be reached by clinical studies. We therefore began immunoscintigraphy in melanoma patients with the MAb 225.28s directed against a high molecular weight melanomaassociated antigen ( 14, 3 1, 45). H owever, this method, which was started with the aim of specifically imaging tumor lesions, offered no advantages over conventional diagnostic procedures; on the contrary, radioimaging could only recognize tumor nodules greater than 2 cm in diameter (Fig. 6a, b). In addition, radioimmunotherapy with I-labeled mAbs, which was clearly successful in the nude mouse model (24), has not been useful in the clinic. We therefore have to return to an experimental model to define and clarify the problem. Autoradiography
vs. immunohistology solid tumor
The question was raised whether MAb and zones of high antigen expression
for
MAb-distribution
in
tissue
accumulation coincided or whether nonuniform
with viable distribution
tumor tissue was due to
a
b
366
W.
TILGEN
AND
S. MATZKU
Figure 7. Access of MAb to the melanoma-assoriatrd antigen: Immunohistology, macroautoradiography and homogetwous distribution of conventional histology of melanoma xrnografts (arrows). (a) I mmunohistology: thr target antigen as detected by the biotinylated MAb HD-Mel 3 followed by streptavidin-biotinylated peroxidase complex. (h) Control section stained with a sheep anti-mouse Ig showing a rim of immunoglohulinpositive murine cells around the xcnograft nodules; no indication of intra-tumoral foci of immunoglohulin accumulation. ir) Autoradiography demonstrating a non-uniform distribution pattern ofMAh HD-MCI 3 which is not due to variations in the antigen expression as shown in Fig. 7a. (d) Hematoxylin and eosin-stained paraffin sections revealed small necroses (arrows) corresponding the hot spots in Fig. 7~.
diflerential access to viable or necrotic tumor tissue. To analyse this situation we compared immunohistology, autoradiography, and conventional hematoxylin (HE) staining on neighbouring sections of xenografts. It was found that the typical spotty pattern of radioactivity accumulation observed after injection of lz51-labeled HD-Mel3 (Fig. 7c) was paralleled by virtually homogeneous distribution of the target antigen (Fig. 7a). A control section stained with sheep antimouse IgG showed a rim of immunoglobulin-positive murine cells around the xenograft nodules, but gave no indication of intra-tumoral foci of immunoglobulin accumulation (Fig. 7b). The most revealing observation, however, was made on HE-stained sections adjacent to the section processed by autoradiography, since hot spots in the latter coincided a series of sections, no with micro-necroses in the former (Fig. 7d). Wh en inspecting difference in the frequency of patent microvessels was observed between necrotic and viable regions of MML-I tumor tissue. According to the autoradiographic analysis, nonuniform MAb distribution in melanoma tissue was the dominant feature. This points to regional differences in either permeability ofcapillaries or interstitial diffusion/convection, or antigen density, or antigen modulation rather than to a dominance of one single parameter throughout tumor tissue. To get insight into the mechanisms leading to focal accumulation of radioactive MAbs in MML-I xenografts, quantitative information was needed. It was gained by computeraided densitometry, a method for objective evaluation of radioactivity distribution in contrast to the visual impression obtained from the X-ray film. Representative images were obtained after 24 h and 48 h, respectively. The radioactivity profile was placed in a position which visualized the highest peak-to-background ratios, ranging from 1540.
PI’I‘FALLS
IN
‘I‘HE
MONOCLONAL
ANTIBODY
APPROACH
367
F&we 8. Computer-aided densitometry ofautoradiographs obtained with MAb fragments: 48 h after application of “‘I-lab&d MAb HD-Mel 3, tumors were excised and autoradiographs were performed. These W-K subjcctcd to scanning densitomctrp to obtain an estimate of radioactivity levels in hot and cold awas. ‘l‘hc horizontal lint through thr tumor nodule indicates the location of the prolile.
The high ratios were mainly (Fig. 8).
due to surprisingly
Conclusions
therapeutic
and first
low levels of accumulation
results
with
in ‘cold’
areas
MAb-serotherapy
Assuming that these model experiments reflect the actual situation in the melanoma patient, our data suggested that MAb access was determined both by the mode of antigen presentation and by the texture of tumor tissue. Although a few questions were answered, every answer seemed to pose a new question. Nevertheless, MAbs do work in clinical application as shown by patients treated with a serotherapy. Beside an inflammatory reaction and local pain in the area of subcutaneous tumor nodules, we observed concomitant regression of lung and lymph node metastases in one patient (Fig. 9). Thus, our studies may help to overcome the inherent limitations of the monoclonal antibody
368
\2’.
‘I‘ILGEN
AND
S. MATZKU
Figure 9. Sonographic follow-up of lymph node metastascs under scrotherapy with MAb R24. Sonography of a rcprcscntativr metastasis showed a characteristic low echogcnicity j a). After a singlr low dose infusion of MAh R24, the metastasis grew up for 3 months to a volume of 31 x 22 mm (b, c); after a further period of 6 weeks significant changes in structure and volume (reduction to 21 x 16 mm) occurred (d).
approach in clinical diagnostics idea and its realization.
and therapy
and may shorten
the distance
between
the
Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft, SFB 136~ Cancer Research, and the Tumorzentrum HeidelberglMannheim. The generous gift of monoclonal antibodies by Professors W. Dippold, Medical Clinic, University of Mainz; S. Ferrone, New York Medical College, Valhalla, NY; K. E. Hellstrom, Seattle and C. Sorg, Department of Experimental Dermatology, University of Miinster is gratefully acknowledged. The authors would like to thank Professor P. Georgi and Dr. U. Mende, Department of Radiology, University of Heidelberg performing immunoscintigraphy in melanoma patients and sonography, respectively. M. Engstner,
B. Gamer, J. Schmid
H. KirchgeBner for performing
and M. Thome are thanked computer-aided densitometry.
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(1990)
Review
17, 357-37
1
Pitfalls in the clinical application of monoclonal antibodies in malignant melanoma: modulation impaired accessibility of antigens to monoclonal antibodies Wolfgang
Tilgen*
and
Siegfried
by and
Matzku
Department of Dermatology, University of Heidelberg and the Institute of Radiology and Pathophysiology, Division of Applied Immunology, German Cancer Research Center, II-6900 Heidelberg, FRG
Introduction New diagnostic and therapeutic possibilities were introduced by the hybridoma technique (21), which enables production of monoclonal antibodies against tumor-associated antigens (1-4, 7, 11, 14, 16, 22, 30, 3 1,40,45). Modest results of the pioneering clinical studies in radioimaging (5, 6, 23, 34, 35), serotherapy (8, 9, 12, 17, 32, 43) and the use of monoclonal antibodies (MAbs) as carriers for cytotoxic agents (33, 36, 44) have now shown the limitations of the approach (10, 13, 15, 18-20, 24-28, 37-42). It has become increasingly clear that the specificity of MAbs, i.e. the ability of an antibody to reveal an antigen which is expressed selectively on the tumor cells, cannot ensure effective tumor targeting. We therefore wanted (1) to evaluate modulation of cell surface associated antigens by MAbs, i.e. internalization or shedding ofimmune complexes and (2) to study the accessibility of melanoma cells within solid tumor tissue. We have addressed these questions by following the pathways of radionuclide (rZ51)- and goldlabeled antibodies in the living cell in vitro with immunoelectron microscopy (IEM) and with a radioantibody binding assay (RBA) and by analyzing the biodistribution of labeled MAbs in human xenograft tumors with the paired label assay (PLA) and whole body autoradiography (WBA). We also performed some clinical pilot studies with immunoscintigraphy and serotherapy.
Antigen
modulation
by monoclonal
antibodies
in vitro
Initially we tested a panel of MAbs looking for subcellular localization (37, 38). Studies were carried out in vitro on metabolically inactive fixed melanoma cells grown as monolayer. Two binding types were identified: Antigen-antibody complexes either covered the whole cell surface [Figure 1 (a)] or were localized intracellularly outlining mitochon* Corrcspondencr 0305.-7372/90/2&3357
address:
W. ‘l‘ilgen,
Universitats-Hautklinik,
Voss-Str.
2, 6900 Hridelberg, G 1990 Acadrmic
+ 15 $03.00/0 357
F.K.G. Press Limited
&we 1. Localization ofantibody binding by immunoelcctron microscopyofmelanoma is restricted to the cell surfacr (arrowheads) ( x 3525). (b) MAb binds to a cytoplasmic on mitochondriae, Golgi apparatus, the nuclear envelope and also to the cell Cy, cytoplasm; N, nucleus; NC, nucleolus ( x 3525).
cells in uitro.(a) Reaction antigen which is localized membrane (arrowheads).
PITFALLS
IN
THE
MONOCLONAL
ANTIBODY
APPROACH
359
dria, Golgi apparatus, rough endoplasmic reticulum and the nuclear envelope [Figure 1 (b)]. In living cells, three binding modalities for immune complexes (IC) could be demonstrated by radioimmunoelectron microscopy (RIEM) and radioantibody binding assay (39). In the RBA (24-27) it turned out that the binding of e.g. MAb LlO, which is directed against an epitope of the integral membrane protein gp95 (11, 16) was not greatly influenced by the metabolic activity of the target cell, showing roughly the same amount of bound radioactivity at 0” and 37°C. After exposing the cells to an iso-osmolar buffer of pH 2.8, which dissociates the ICs from the cell surface, most of the radioactivity was eluted, indicating a predominant binding of the MAb to the cell surface. This nondesorbable radioactivity corresponded to the few ICs localized in the cytoplasm as shown by RIEM. Labeled antigen-antibody complexes covering the entire cell membrane were seen after 30 min and most of the immune complexes remained stable on the cell surface even after 120 min. It appears therefore that the first prototype ofbinding leads to a stable, membrane anchored immune complex. The second prototype of binding was observed with MAb R24 directed against the glycolipid antigen GD3 (11, 30). In the RBA, uptake of MAb was highest with fixed cells at O”, intermediate with living cells at 0” and lowest with cells kept at 37°C. This pointed to a release of the immune complexes from the cell surface. Due to the low binding level, RIEM could demonstrate only few labeled immune complexes in living cells. Immunoelectron microscopy solved the problem. Glutaraldehyde-fixed cells exhibited uniform staining along the whole cell surface. In contrast, metabolically active unfixed cells showed only localized reactivity, with patchy deposits of immune complexes, or a complete lack of immunostaining. Taken together this points to a shedding process. The third prototype of binding behaviour is illustrated by MAb M.2.9.4 (4). RBA data showed that cells exhibited markedly higher uptake at 37°C than at 0°C. The portion of binding resistant to pH 2.8 buffer treatment corresponded to the increased incorporation of labeled MAb into the living cell. The latter component of uptake was completely lacking in metabolically inactive fixed cells. RIEM also showed a rapid binding to viable cells with a pronounced deposition of decay tracks over the whole cell surface. After 2 h substantial amounts of immune complexes had migrated into the cells. The ‘antigen route’ seemed to be directed to the nuclear region [Figure 21. Internalization of individual MAbs may follow different pathways (29). One of them is constitutive endocytosis by endocytotic vesicles. Another pathway is shown here for the 5 nm colloidal gold-labeled mAb HD-MEL 3, which was distributed all over the cell surface, but grouped in clusters both in the region of non-coated plasma membrane invaginations and in coated pits or coated vesicles, pointing to a mechanism of antibody uptake reminiscent of receptor-mediated endocytosis (Figure 3). The third binding type therefore represents an internalization process. Accessibility The problem
of target
cells
within
solid
tumor
tissue
of accessibility of target cells within solid tumor tissue was previously shown we processed melanoma cells growing to small tumor nodules in culture dishes. IEM showed that, in contrast to the monolayer culture, only the outermost layer of melanoma cells was stained by monoclonal antibodies and virtually no labeling was seen within the tumor cell cluster (Figure 4). In the paired label assay (Table 1) no clear
in vitro when
360
W. TILGEN
AND
S. MATZKU
Figure 2. Radioimmunoelectron microscopy of melanoma cells in vitro with the ‘251-labeled MAb M.2.9.4. After 120 min substantial amounts of immune complexes (arrowheads) had migrated into the cytoplasm of the metabolically active cells kept at 37°C and only few remained on the cell surface. (N, nucleus; PM, plasma membrane) ( x 13 200).
correlation was observed between types ofMAb binding and gross uptake, since significant accumulation was noted throughout. The localization indices were variable, neither high internalization ratios nor high antigen densities being reproducibly linked to high specific uptake. An exception was the shedding-related binding type being associated with low localization indices. Diagnostic tumor targeting with MAbs, and even more so immunotherapy with antibody conjugates, ideally requires antibody-binding to every tumor cell with saturation of all available binding sites. In view of the basic architecture of solid
ANTIBODY
.Wl’KOAC:H
melanoma, access ofmacromolecules may be limited in the steps oftranscapillary or interstitial diffusion/convection (18-20). In fact, relative accumulation, scinti,graphic contrast is greatly enhanced when using MAb fragments. In intact IgG, impressive localization is achieved in a shorter period of time with of fragments as shown in the PTA for MAb HWMel3 (Table 2). However, this of fragments in immunoscintigraphy does not offer a universal solution to the access, because it is not clear whether every melanoma cell&provided it
361
transport resulting in contrast to both types application problem of carries the
W.
362 Table
1. Accumulation melanoma
cell
of MAbs properties
TILGEN
in nude mice in vitro
AND
S. MATZKU
with
MML-I
melanoma
xenografts:
Localization Antigen” density
MAh LlO HD-Mel HD-Mel3 225.28 M.2.7.6 M.2.9.4 R24
1
34.0 19.0 16.0 1.3 0.5 16.0 0.2
Type
of binding
Tumor/blood
Surface binding Surface binding Surface binding Surface binding Internalization Internalization Shrdding
Tumor/liver
0.5 1.8 3.3 3.4 5.5 2.0 1.2
Tumor/hone
Tumor/musclr
5.3 1.4 3.0 3.5 6.3 1.8 0.8
anti-melanoma
to
index”
1.6 1.9 2.9 3.2 5.7 2.9 1.1
“Apparent antibody bindin! sites per cell x 10’. “Paired label assay, comparison being made between “S1-laheled antibody (B40) injected into the same animals (n = 3-6).
Comparison
MAbs
6.9 2.0 2.6 3.4 5.1 3.4 1.1
and “‘I-laheled
control
antigen-is reached by the antibody or its fragment. With macro-autoradiography large size sections are exposed on X-ray film. The resulting autoradiographs give an overview of a tumor cross-section, and in the case of whole body sections of tumor-bearing rodents they also give a comprehensive image of radioactivity distribution in normal tissues. Whole body autoradiography impressively showed thedifferential access and clearance of monoclonal antibodies and their fragments. The common pattern was a predominant accumulation in the periphery of the tumor nodes (Fig. 5a). The central part was stained to a different degree depending on the transplanted melanoma cell line (Fig. 5b). MAb R24 was diffusely distributed throughout the tumor cross-sections at a non-significant level pointing to a shedding process (Fig. 5~). HD-Mel3 showed a non-uniform distribution pattern all over the melanoma node, but radioactivity was restricted to a few “hot” spots (Fig. 5d). Thus, WBA may bridge the gap between microscopic examination and the low resolution techniques of scintigraphy or paired label assay.
Table
2. Accumulation of intact IgG
of HD-Mel3 and fragments”
in nude
mice
with
MML-I
melanoma
Localization Time Fah fragments: 6h 12 h 24 h F(ab’), fragments: 12 h 24 h 48 h Intact IgG: 24 h 48 h 96 h
xenografts:
Comparison
Muscle
BOtW
8.42 39.88 15.98
indices
Blood
Spleen
Kidney
9.88 26.92
17.86 38.33 36.43
9.78 20.59
25.26
33.12 76.93 53.26
23.13
8.32 18.00 12.78
8.81 17.51 49.08
7.82 15.31 33.50
13.81 25.83 55.83
7.88 17.58 34.36
8.38 12.93 5.47
8.20 11.11 10.94
2.00 3.32 1.96
1.95 3.27 1.77
1.74 2.73 2.00
1.92 2.93 2.02
1.61 2.62 1.59
1.80 2.95 1.98
*Paired label assay, comparison being (B40) injected into the same animal.
made
between
12”1-laheled
Liver
HD-Mel
3 and ‘s’I-labeled
control
antibody
PI’I‘E‘ALLS
IN
THE
MONOCLONAL
AN1‘IBODY
APPROACH
363
d
@ure 5. Whole body autoradiographs performed 48 h after application of “‘I-lahcled MAbs: Distribution pattern of difkent anti-melanoma MAhs in representative melanoma xenografts. (a) M.2.10.15 in MeWo, peripheral staining. (b) M.2.7.6 in SK-MEL-28, peripheral and intratumoral staining. (c) R24 in Co10 38, no significant staining. (d) HD-Mel3 in MML-I, intratumoral staining. ‘I’hc insets show additional sections through the same tumor.
W.
364
‘I‘ILGEN
AND
S. MA’I‘ZKU
b b’igm 6. Immunoscintigraphy in the melanoma patient. ia) Clinical aspect of hundreds of cuta~~cous and suhcutanrous metastases of different size on the thigh. (b) Immunoscintigram with the ““““l‘r-labeled hlAb 225.28% Radioactivity acrumulation was only detected in the region of two big ulccratrd melanoma nod&s iarrows), thr ahundancc ofsmall mctastases was not detected. (K = background radioactivity in thr kidncys.1
Immunoscintigraphy
of melanoma
patients
At this point, experimental models seemed to be limited, and further information could only be reached by clinical studies. We therefore began immunoscintigraphy in melanoma patients with the MAb 225.28s directed against a high molecular weight melanomaassociated antigen ( 14, 3 1, 45). H owever, this method, which was started with the aim of specifically imaging tumor lesions, offered no advantages over conventional diagnostic procedures; on the contrary, radioimaging could only recognize tumor nodules greater than 2 cm in diameter (Fig. 6a, b). In addition, radioimmunotherapy with I-labeled mAbs, which was clearly successful in the nude mouse model (24), has not been useful in the clinic. We therefore have to return to an experimental model to define and clarify the problem. Autoradiography
vs. immunohistology solid tumor
The question was raised whether MAb and zones of high antigen expression
for
MAb-distribution
in
tissue
accumulation coincided or whether nonuniform
with viable distribution
tumor tissue was due to
a
b
366
W.
TILGEN
AND
S. MATZKU
Figure 7. Access of MAb to the melanoma-assoriatrd antigen: Immunohistology, macroautoradiography and homogetwous distribution of conventional histology of melanoma xrnografts (arrows). (a) I mmunohistology: thr target antigen as detected by the biotinylated MAb HD-Mel 3 followed by streptavidin-biotinylated peroxidase complex. (h) Control section stained with a sheep anti-mouse Ig showing a rim of immunoglohulinpositive murine cells around the xcnograft nodules; no indication of intra-tumoral foci of immunoglohulin accumulation. ir) Autoradiography demonstrating a non-uniform distribution pattern ofMAh HD-MCI 3 which is not due to variations in the antigen expression as shown in Fig. 7a. (d) Hematoxylin and eosin-stained paraffin sections revealed small necroses (arrows) corresponding the hot spots in Fig. 7~.
diflerential access to viable or necrotic tumor tissue. To analyse this situation we compared immunohistology, autoradiography, and conventional hematoxylin (HE) staining on neighbouring sections of xenografts. It was found that the typical spotty pattern of radioactivity accumulation observed after injection of lz51-labeled HD-Mel3 (Fig. 7c) was paralleled by virtually homogeneous distribution of the target antigen (Fig. 7a). A control section stained with sheep antimouse IgG showed a rim of immunoglobulin-positive murine cells around the xenograft nodules, but gave no indication of intra-tumoral foci of immunoglobulin accumulation (Fig. 7b). The most revealing observation, however, was made on HE-stained sections adjacent to the section processed by autoradiography, since hot spots in the latter coincided a series of sections, no with micro-necroses in the former (Fig. 7d). Wh en inspecting difference in the frequency of patent microvessels was observed between necrotic and viable regions of MML-I tumor tissue. According to the autoradiographic analysis, nonuniform MAb distribution in melanoma tissue was the dominant feature. This points to regional differences in either permeability ofcapillaries or interstitial diffusion/convection, or antigen density, or antigen modulation rather than to a dominance of one single parameter throughout tumor tissue. To get insight into the mechanisms leading to focal accumulation of radioactive MAbs in MML-I xenografts, quantitative information was needed. It was gained by computeraided densitometry, a method for objective evaluation of radioactivity distribution in contrast to the visual impression obtained from the X-ray film. Representative images were obtained after 24 h and 48 h, respectively. The radioactivity profile was placed in a position which visualized the highest peak-to-background ratios, ranging from 1540.
PI’I‘FALLS
IN
‘I‘HE
MONOCLONAL
ANTIBODY
APPROACH
367
F&we 8. Computer-aided densitometry ofautoradiographs obtained with MAb fragments: 48 h after application of “‘I-lab&d MAb HD-Mel 3, tumors were excised and autoradiographs were performed. These W-K subjcctcd to scanning densitomctrp to obtain an estimate of radioactivity levels in hot and cold awas. ‘l‘hc horizontal lint through thr tumor nodule indicates the location of the prolile.
The high ratios were mainly (Fig. 8).
due to surprisingly
Conclusions
therapeutic
and first
low levels of accumulation
results
with
in ‘cold’
areas
MAb-serotherapy
Assuming that these model experiments reflect the actual situation in the melanoma patient, our data suggested that MAb access was determined both by the mode of antigen presentation and by the texture of tumor tissue. Although a few questions were answered, every answer seemed to pose a new question. Nevertheless, MAbs do work in clinical application as shown by patients treated with a serotherapy. Beside an inflammatory reaction and local pain in the area of subcutaneous tumor nodules, we observed concomitant regression of lung and lymph node metastases in one patient (Fig. 9). Thus, our studies may help to overcome the inherent limitations of the monoclonal antibody
368
\2’.
‘I‘ILGEN
AND
S. MATZKU
Figure 9. Sonographic follow-up of lymph node metastascs under scrotherapy with MAb R24. Sonography of a rcprcscntativr metastasis showed a characteristic low echogcnicity j a). After a singlr low dose infusion of MAh R24, the metastasis grew up for 3 months to a volume of 31 x 22 mm (b, c); after a further period of 6 weeks significant changes in structure and volume (reduction to 21 x 16 mm) occurred (d).
approach in clinical diagnostics idea and its realization.
and therapy
and may shorten
the distance
between
the
Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft, SFB 136~ Cancer Research, and the Tumorzentrum HeidelberglMannheim. The generous gift of monoclonal antibodies by Professors W. Dippold, Medical Clinic, University of Mainz; S. Ferrone, New York Medical College, Valhalla, NY; K. E. Hellstrom, Seattle and C. Sorg, Department of Experimental Dermatology, University of Miinster is gratefully acknowledged. The authors would like to thank Professor P. Georgi and Dr. U. Mende, Department of Radiology, University of Heidelberg performing immunoscintigraphy in melanoma patients and sonography, respectively. M. Engstner,
B. Gamer, J. Schmid
H. KirchgeBner for performing
and M. Thome are thanked computer-aided densitometry.
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