Download 1975–1995 Revised anti-cancer serological response: Biological

Annals of Oncology 7: 227-232, 1996.
O 1996 Kluwer Academic Publishers. Printed in the Netherlands.
Review
1975—1995 Revised anti-cancer serological response: Biological significance
and clinical implications
S. Canevari, S. M. Pupa & S. Menard
Division of Experimental Oncology E, lstituto Nazionale Tumori, Milan, Italy
Summary
In the 1970s a considerable amount of work was carried out
in an attempt to identify an anti-tumor serological response
in cancer patients. These analyses have not been very informative due to the complexity and heterogeneity of the response. More recently, the availability of recombinant molecules, synthetic peptides and analytic and semi-quantitative
assays has enabled a better dissection of humoral immunity.
Antibodies against intracellular antigens (c-myb, c-myc, p53
and p21 ras) have been found in a significant, albeit varying,
proportion of patients bearing various tumors. Association
with a poor prognosis is documented for anti-p53 antibodies
in breast carcinoma patients. A number of cell surface antigens, including mucins, oncoproteins and carbohydrate antigens have been found to elicit a humoral immune response
Background
In animal models it has been convincingly demonstrated that the immune system can recognize tumor cells
[1-3]. In contrast to animal models, the existence of tumor-specific antigens that are recognized by the autologous tumor-bearing host was not unequivocally demonstrated in human neoplasms until recently [4].
Whereas only a very limited number of tumor rejection
antigens involved in animal model systems have been
identified through a serological immune response, most
of tumor-associated antigens (TAA) present on human
cancer cells have been detected by using antibodies.
These TAA, expressed by cancer cells either to greater
extent that in normal cells or as new molecules completely absent in normal cell counterparts, appear as a
result of aberrant gene or oncogene expression or as a
consequence of errors in glycosylation [5,6].
As for other cellular antigens, the immune system
can turn on against TAA either one of its arms: the one
that relies on T lymphocytes, which directly or indirectly kill tumor cells also by recruitment of macrophages,
polymorphonuclear cells and natural killer (NK) cells,
or the one that depends on antibodies. Table 1 lists the
possible mechanisms of action of antibodies. The majority of these mechanisms were demonstrated to be
active in experimental models and some of them have
been proven effective also in the clinic [7-9].
and, in some instances, circulating immune complexes were
observed. A protective role for or, on the other hand, masking effects of such antibodies is still controversial.
An indication that a serologkal response can be beneficial
comes from vaccination studies. A significant association between the development of an anti-tumor antigen antibody
response and prolonged survival was observed following vaccination of melanoma patients with GM2 or anti-idiotypic
antibodies which molecularly mimic tumor-associated antigens. It is to be hoped that in the near future the numerous
ongoing immunization trials and prognostic studies demonstrate whether antibody response can exert a protective role
in vivo.
Key words: prognosis, serological response, TAA, therapy,
vaccination
The purpose of this presentation is to review the
data obtained in recent years regarding serological response of cancer patients, with special emphasis on its
biological relevance and the possibility of exploiting it
for therapeutic purposes.
Antibody response in tumor-bearing patients
In the 1970s, at the beginning of the development of
tumor immunology, a considerable amount of work
was carried out in an attempt to identify an anti-tumor
humoral immune response, and the sera of a thousand
Table 1. Mechanisms of anti-tumor antibodies.
Activity
Antibody region involved*
Direct:
• inhibition of proliferation
• inhibition of metastatic spread
Only variable
Only variable
Indirect
• activation of idiotypic network
• complement-mediated lysis
• antibody-dependent cellular
cytotoxicity
• targeting of toxic agents
Only variable
Both constant and variable13
Both constant and variable11
Both constant and variable11
• For antibody structure [37].
b
The variable region is involved in the antigen recognition.
228
cancer patients were analyzed for the presence of antibodies reacting with their own or with allogenic tumors
[10].
However, since the patient's humoral immune response, as identified by the methodologies utilized at
that time, was likely to be polyclonal, it soon became
evident from the complexity of the immune response
that molecular techniques were needed to identify
'altered' molecules expressed by tumor cells. Although
antibodies were quite common, an extensive survey of
sera from normal as well as non-cancer and cancer patients indicated that only a small proportion of them
were restricted to TAA, whereas the majority of serological responses were against artifactual antigens
(Fetal Calf Serum, HLA antigens) or undefined molecules. As reviewed by L. J. Old [11], two major problems limited the development of tumor immunology:
the need to identify cancer-associated antigens and
the need for means by which to monitor immune
responses. More recently, new approaches, such as
molecular biology and hybridoma technology, made
possible to identify a series of molecules, all of them
having the characteristic of behaving as 'tumor antigens'.
At that point, become easier to evaluate the possible
presence in cancer patients' sera of antibodies specifically directed against these altered molecules. The
availability of recombinant molecules, synthetic peptides and analytic and semi-quantitative immunochemical assays guarantees the specificity of observed reactions.
More convincing evidence of anti-tumor serological
response in cancer patients is listed in Table 2. Note
that the majority of them were obtained after 1990,
when new biotechnological tools, previously mentioned, became widely available.
Antibodies against intracellular antigens such as
c-myb, c-myc, p53 and p21 ras proteins have been
found in a significant, although varying, proportion of
patients with different tumors. The greater incidence of
antibody in cancer patients compared to normal sera
suggests that immunization to intracellular proteins
occurred as a result of the malignancy. However the
issue of controls representative of tissue destruction
and inflammation was probably not enough critically
dealt with and further studies are needed to exclude the
role of the surrounding tissues in the release of intracellular components.
Forthy-three percent of breast carcinoma patients
were found positive for the presence of anti-c-myb antibodies [12]. p53 was also found to be immunogenic
even though the patients' antibodies recognized both
the wild type and the mutated proteins. The release of
intracellular molecules by necrotic tumor cells seems
the most likely explanation of how such proteins become immunogenic. A possible role for these antibodies in the control of tumor progression is highly
unlikely, since they do not react against live tumor
cells. Detection of anti-p53 antibodies at the time of
diagnosis in breast and colorectal carcinoma patients
appears to be associated with a poor prognosis [13,14],
and it has been suggested that this plasma assay be used
for prognostic purposes [13]. The finding that anti-p53
antibodies are mainly elicited in patients with an
altered p53 in their tumors indicates that detection of
such humoral immune response might be a simpler
assay than p53 primary tumor characterization by molecular sequencing. In lung carcinoma development,
alteration of p53 was found to be a preneoplastic event
which occurs already in the normal bronchus epithelium. Detection of anti-p53 antibodies in a population
at high risk for lung cancer might be a useful indicator
for early diagnosis [15]. Moreover, an antibody response to wild type and mutated p21 ras proteins even
occurs in 32% of the examined colon cancer patients.
Whether humoral immune response to ras p21 protein
in cancer patients correlates with clinical or pathological variables is still under investigation [16].
Antibodies against cell-surface proteins might have
a completely different impact on the control of tumor
growth. Therefore, much more attention has been paid
to the identification of a humoral immune response to
cell-surface TAA. As shown in Table 2, a number of
cell-surface antigens, including mucins, oncoproteins
and carbohydrate antigens have been found to elicit a
humoral immune response in cancer-bearing patients.
Mucins, like PEM/MUC1, were found to induce antibodies, and in early-stage breast carcinomas these antibodies were observed as immune complexes with the
circulating mucins [17]. The finding that patients with
mucin/antibody complexes have a better prognosis
than patients with circulating free mucins suggests a
protective role for the anti-mucin antibodies against
tumor progression. In breast cancer patients, a humoral
Table 2. Antibody response in cancer patients.
Antigen
Tlimor
Intracellular localization
c-myb
Breast, colorectal, ovarian
c-myc
Colorectal, breast, ovarian,
osteosarcoma
p53
Breast, lung, colorectal, prostate, bladder, ovarian, thyroid,
pancreatic, B-cell lymphoma,
multiple-myeloma
p21 ras
Colorectal
Cell-surface localization
HER2/neu
Breast
PEM/MUC1
Anonymous proteins
T, Tn, sialyl Tn
Gangliosides (GM1,
GM2, GD2)
Melanoma antigen
Ovarian, breast, colorectal,
pancreatic
Lung
Breast, lung, pancreatic
Melanoma
Melanoma
References
[121
[38, 39)
(13-15,
40-50]
(161
[18', 51,
52)
(53b-551
(20]
[21]
(211
[561
EBV-immortalized B-cell clones from "breast and bovarian cancer
patients.
229
immune response directed against the HER2/neu
oncoprotein was found [18]. The presence of circulating antibodies correlates with oncoprotein overexpression on primary tumors and with advanced-stage disease. The finding that the soluble extracellular domain
of the oncoprotein is also detectable in the sera of
advanced-stage patients [19] raises the question of
whether immune complexes are formed and if so what
their relevance is to tumor progression. In small-cell
lung cancer patients, the presence of antibodies directed against autologous tumor cell proteins was found to
be associated with improved survival but the actual
target molecule recognized by these antibodies is still
unknown [20].
Finally, it has been reported that antibodies to carbohydrate structures are quite commonly found in
human sera. In particular, in many melanoma patients
humoral immune response to gangliosides (GM1,
GM2 and GD2) is present either as 'natural' antibodies
or as a result of immunogenic stimulation by the tumor
[21].
Very recently the expression of cDNA libraries from
human rumors of different oncotypes in Escherichia
coli and screening for clones reactive with high-titer
IgG antibodies in autologous patient serum lead to the
discovery of several antigens with a restricted expression pattern in each tumor [22].
Antibody response upon vaccination
Approaches to the control of cancer by manipulating
the host immune response date back to the turn of this
century [23, 24]. 'Active specific immunotherapy' was
the term originally used to describe the approaches
which involve immunization with a specified antigen to
induce a specific immune response.
The field, now termed tumor vaccinology, was the
object of a renewed surge of interest based on recent
advances in biotechnology and the much more detailed
understanding of cancer immunology [4]. About one
half of human cancer vaccines on trial used purified,
synthetic or recombinant TAA [25], and the majority of
these studies relied on the possibility of inducing or increasing a preexisting antibody response.
Table 3 lists the more relevant ongoing studies, some
of which have already reached the phase HI level.
Two pilot studies were conducted in ovarian and
breast cancer patients with T or sialyl-Tn. All patients
developed antigen specific antibodies of both IgM and
IgG isotypes. Complement mediated cytotoxic antibodies for cancer cells were also induced, which were
partially inhibited by the immunizing carbohydrate.
However, no correlation between antibody titers and
clinical outcome was found. Over 300 patients with
melanoma have been immunized with purified gangliosides. The antibody response induced by these immunizations was primarily an IgM response of moderate
titer and short duration. A double blind randomized
Table 3. Ongoing clinical trials in which antibody response becomes
evident upon vaccination.
Immunizing antigen
Tumor
References
TAA
Synthetic T, sialyl-Tn
Purified gangliosides (GM2,
GD2, GD3)
Ovarian, breast
Melanoma
[57, 58]
[26, 59]
Melanoma
Colorectal
Colorectal
[8]
[601
[61]
Colorectal
[31]
Anti-idiotypic antibody (Ab2fi)'
Mouse Ab2p anti-HMW-MAA mAb
Mouse Ab2p" anti-CEA mAb
Polyclonal goat Ab2p" anti-CCAA
mAb
Human Ab2p" anti-CCAA mAb
* See Figure 1 for the rationale and terminology of the approach.
Abbreviations: HMW-MAA - high molecular weight melanomaassociated antigen; CEA - carcinoembryonic antigen; CCAA colon carcinoma-associated antigen.
trial of vaccination with GM2 plus BCG was conducted in 122 tumor-free patients after surgery. Anti-GM2
antibody-positive patients showed a highly significant
increase in disease-free survival and a 17% increase in
overall survival [26]. Three approaches to augment the
immunogenicity of gangliosides and carbohydrate TAA
in general have been tested: 1) the use of immunological adjuvants; 2) the synthesis of derivates (congeners)
of these saccharides; and 3) the chemical coupling to
immunogenic carrier proteins [27].
An attractive alternative to the TAA for efficient
immunization is represented by ann'-idiotypic antibodies (Ab2P) which molecularly mimic TAA (for the
rationale and the terminology of the approach see
legend of Figure 1). Anti-idiotype vaccination has already been used successfully to prevent or suppress the
growth of experimental tumors [28, 29], and it has the
advantage of not being genetically restricted. Moreover,
by presenting the antigen in a different molecular environment it is possible to break immune tolerance [30].
Several clinical studies, listed in Table 3, are underway.
A study in melanoma patients aimed to evaluate the
relationship between the development of humoral
immune response following Ab2 p1 vaccination and
survival time was conducted in 58 sequential tumorbearing patients. Kaplan-Meier survival analysis
showed that the Ab3 antibody level (in 60% of cases)
and development of anti-TAA antibodies (in 35% of
cases) displayed a significant association with survival
prolongation [8]. Various approaches to enhancing cellular and humoral responses to Ab2 vaccines have
been tested, such as the use of different carriers, adjuvants and immunomodulators and there appear to have
been interesting results in colorectal and melanoma
patients from the association of Ab2 vaccines with IL-2
(M. Maio, personal communication). In a preliminary
series of 6 colorectal cancer patients it was recently
demonstrated that a human Ab2 vaccine induces both
230
Ab1
Ab2p
Ab3 = Ab1'
(anti-TAA)
(anti-idiotype)
(anti-anti-idiotype)
tin
TAA
tumor cell
Figure 1. The idiotypic network and the molecular mimicry of antigen. According to the network hypothesis [62] immunization with a specific TAA gives rise to antibody against this TAA, termed Abl; Abl, which, in turn, induces a series of anti-idiotypic antibodies, termed
Ab2. Some of these Ab2 molecules can effectively mimic the three-dimensional structure of the TAA identified by the Abl. These particular
anti-idiotypes called Ab28, due to the antigen molecular mimicry can induce specific antibody responses (Ab3) similar to those induced by
the original TAA. Thus Ab3 are also termed Abl-like or Abl'. Idiotype - collection of the unique antigenic sites (idiotopes) of an antibody.
a cellular and a humoral immune response against the
nominal TAA [31].
Since the entire idiotypic network can be mounted
in immunocompetent cancer patients and murine
monoclonal antibodies (mAb) to several TAA have
been administered in a thousand patients for diagnostic
and therapeutic studies over the past decade, a reevaluation of the possible beneficial effect of the
human anti-mouse antibody (KAMA) response is warranted. Indeed, a large survey of literature data [32]
indicated that following just a single injection of murine
mAb, more than 60% of patients are able to develop a
humoral immune response against the foreign protein.
The specificity of HAMA response in regard to reactivity with different regions of the murine immunoglobulin molecule was analyzed in less than one fourth
of the study. A consistent proportion (from 30% to
60%) of cases with positive HAMA was found to produce anti-idiotypic antibodies, however only in few
instances the development of Ab2 p anti-idiotypic antibodies which mimic TAA was sought. Up to now, there
have been only anecdotal reports of positive influence
of HAMA response on the disease course [33]. In contrast, shortening of mAb pharmacokinetics and interference in in vitro tumor marker evaluation in patient
sera are well documented [32,34,35].
Concluding remarks
On the basis of the data reported, it is not yet possible
to assert that humoral immune response to any immunogenic tumor antigen truly represents a protective
immune response in vivo. The finding that seropositivity in tumor-bearing patients is frequently compatible
with progressive growth of immunogenic tumors can be
explained, assuming that the mechanism of antibodydependent epitope masking, a phenomenon envisaged
in experimental models, is operating and therefore,
indicative of a population with poor prognosis [36].
However, it is not possible at present, to rule out the
possibility that its association with poor prognosis is an
epi-phenomenon. Some investigators suggest that
alteration of TAA (i.e., p53, HER2/neu) becomes evident only in advanced disease and evokes a humoral
response which, in turn, is too late to control the
growth of very aggressive tumor cells. Moreover, an
indication that a serological response can be beneficial
comes from the vaccination studies.
The majority of the antibodies against TAA were of
the IgG subclass, implying that cognate helper T-cell
immunity was present and operative in patients with a
B-cell response. While much emphasis has been placed
on the role of CD 8+ T-cells in the TAA recognition
and the effector phase of the immune response against
tumors, evidence has accumulated that CD4+ T-cells
also play a crucial role in the anti-tumor response by
mediating critical priming and effector functions. The
recent evidence that tumor antigens defined by T-cell
responses such as MAGE-1 and tyrosinase can also be
detected by a serological approach [22] suggests that an
integrated immune response against TAA may exist
that involves both CD8+ and CD4+ T-cells as well as
B-cells.
There are numerous immunization trials in progress
which offer virtually unlimited opportunities to test for
231
the presence of anti-tumor antibodies. These studies as
well as the numerous prognostic studies which have
accumulated in recent years, are expected to provide an
answer in the near future to the question of whether
anti-tumor antibodies are protective per se or their
presence simply reflects the development of a specific
CD4+ T-cell response.
Acknowledgements
Partially supported by CNR-ACRO and AIRC/FIRC.
We are grateful to Dr. Jose Garcia Puche for his constructive suggestions, Laura Mameli for manuscript
preparation and Mario Azzini for figure preparation.
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Received 13 December 1995; accepted 6 February 1996.
Correspondence to:
Silvana Canevari, Ph X).
Division of Experimental Oncology E
Istituto Nazionale Tumori
Via Venezian 1
20133 Milan
Italy