707764

research-article2017
TUB0010.1177/1010428317707764Tumor BiologySell

Review
Tumor Biology

Cancer immunotherapy: Breakthrough June 2017: 1­–14 

© The Author(s) 2017
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DOI: 10.1177/1010428317707764
https://doi.org/10.1177/1010428317707764
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Stewart Sell

Abstract
From the application of Coley’s toxin in the early 1900s to the present clinical trials using immune checkpoint regulatory
inhibitors, the history of cancer immunotherapy has consisted of extremely high levels of enthusiasm after anecdotal case
reports of enormous success, followed by decreasing levels of enthusiasm as the results of controlled clinical trials are
available. In this review, this pattern will be documented for the various immunotherapeutic approaches over the years.
The sole exception being vaccination against cancer causing viruses, which have already prevented thousands of cancers.
We can only hope that the present high level of enthusiasm for the use of immune stimulation by removal of blocks to
cancer immunity will be more productive than the incremental improvements using previous immunotherapies.

Keywords
Cancer immunotherapy, history of immunotherapy, Coley’s toxins, checkpoint inhibitors, cancer vaccines

Date received: 3 February 2017; accepted: 8 April 2017

Introduction inhibitors) will allow the immune response to individual

The idea that the immune response could not only protect
and defend us against infectious diseases but also provide
a defense against cancer was developed in the late 1800s
with concepts such as the side chain theory and targeted
therapy (magic bullet) by Paul Ehrlich.1 The approached
championed by Ehrlich was applied to cancer chemother-
apy as well as to cancer immunology.2 The direction of the
immune response to infectious diseases using vaccines
(vaccination) has contributed immeasurably to the health
and longevity of humans because of protection not only
against viral infections3 but also against bacteria,4 fungi,5
and worms.6 As presented in the following, vaccines
against viral infections that cause cancer such as hepatitis
B and human papillomaviruses are very effective in pre-
venting cancers of the liver and cervix (see below). In this
review, many of the attempts to use the immune system
against cancer are presented. These varied and diverse
approaches first produced striking anecdotal results with
reports of individual patients with complete cures or sub-
stantial remissions. This pattern has been repeated many
times with the results of disappointing but incremental
advances in the treatment of cancer. There are high expec-
tations that removing blocks to immunity (checkpoint
cancers to attack and either cure or reduce the growth of
the cancer. The question remains will this be the long
anticipated breakthrough or will we again see an incre-
mental advance in cancer therapy.
Coley’s toxin
The saga of cancer immunotherapy begins in the 1890s
with the development of Coley’s toxin by Dr William B.
Coley described in detail by Stephen Hall in “A commo-
tion in the blood”7 (see also review by McCarthy8). Shortly
after receiving his license to practice medicine in New
York in 1890, Coley observed that occasional patients with
inoperable cancers had remissions of their tumors during
life-threatening infections such as severe erysipelas
Wadsworth Center, New York State Department of Health and
Albany College of Pharmacy and Health Sciences, Albany, NY, USA
Corresponding author:
Stewart Sell, Wadsworth Center, New York State Department of
Health and Albany College of Pharmacy and Health Sciences, Albany,
NY 12201-2002, USA.
Email: stewart.sell@health.ny.gov

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2 Tumor Biology
Figure 1.  Projected enthusiasm levels for various cancer
immunotherapies. As early anecdotal reports of spectacular
cures of patient with advanced cancer appear (usually in the
press), there is high enthusiasm, remarkable press coverage,
and large amounts of money raised for clinical trials. With
phase I–II clinical trials, the response rate substantially
decreases and enthusiasm begins to decline, but press reports
continue to be high. After phase III trials, a relatively modest
response for limited type of cancer is reported (see Table 1).
Finally, because of dangerous side effects and limited long-term
remissions, general clinical application is limited.
(Streptococcus pyogenes). In 1891, he intentionally
infected a patient with S. pyogenes, who had a large inop-
erable neck tumor. After a life-threatening systemic infec-
tion, Mr Zola recovered and his tumor showed marked
regression. By 1891 he had treated three patients with
large tumors; all had noticeable reduction in tumor size,
but two of the three patients died.9 During the next few
years, Coley intentionally infected 12 additional cancer
patients: 4 developed severe infections, 2 died, and 2
appeared to be cured of their tumors. Eight other patients
did not develop severe infections and tumor reduction was
seen in none of these.10 In 1893, Coley produced tissue
extracts of cultures of S. pyogenes and Bacillus prodigio-
sus (an endotoxin producing organism) that became known
as Coley’s toxin. Although not known at the time, this
toxin acts as a superantigen and activates release of
cytokines, including tumor necrosis factor (TNF), interfer-
ons (IFN), and a number of interleukins (ILs). Treatment
of a 13-year-old boy with inoperable metastatic tumors by
direct injection of the toxin into the tumors resulted in
severe fever and chills and the tumors shrank. This patient
lived to the age of 47 years and died of causes other than
cancer. In 1910 in a lecture at the Royal Society of
Medicine in London, Coley showed photographs of a
patient with a large tumor on the face that became much
smaller after 63 injections of his toxin.11 By 1914 Coley12
had treated over 500 patients, claiming to have induced
over 150 remissions. However, other physicians could not
reproduce his results. Because of this failure, the poten-
tially fatal side effects of toxin administration, and the
introduction of irradiation for cancer treatment, the toxin
approach was discontinued. Coley’s positive experience
with early positive results within a limited number of
patients, followed by an inexplicable inability to duplicate
the results, establishes a pattern that besets those working
in cancer immunotherapy to this day (Figure 1). It is likely
that Coley induced what is today known as a cytokine
storm.11 The severe fever and chills are reproduced today
after treatment in some patients, and the specific cytokines
that appear to be involved include IL-2, TNF-β, IL-12,
IFN-α, and TNF-α. Each of these was used in clinical trials
in the 20th century as is reviewed below. The next approach
extends the use of bacterial stimulation to activate the cell-
mediated arm of the immune response to become active
against cancer.
Bacille de Calmette de Guerin
Bacille de Calmette de Guerin (BCG) is an attenuated
form of tubercle bacillus used with limited success as a
vaccine for tuberculosis. A large-scale epidemiologic
study in Europe suggested a slight decrease in the inci-
dence of cancer in population immunized with BCG, com-
pared with populations not immunized. This application of
BCG is not recommended in the United States at this time.
In the early 1960s, Bast et al.13 at National Institutes of
Health (NIH) demonstrated complete remission of trans-
plantable hepatomas in guinea pigs treated with BCG.
Injection of the tumor cells into the flank led not only to
local growth of the transplanted tumor but also metastasis
to the draining lymph node. Injection of BCG into the pri-
mary transplant site resulted in infiltration of lymphocytes
around the tumor sites with regression of both the primary
and metastatic lesions. It was then reported that this
approach could be used to induce regression of leukemia
and melanoma lesions in children, but such results were
again not reproducible. On the one hand, repeated injec-
tions of BCG into the bladder of humans with superficial
bladder cancer have led to significant remissions.14 This
application via the bladder produces recruitment of acti-
vated lymphocytes and macrophages into the bladder wall,
increases ILs in the urine, and induces systemic immunity
to BCG. Long-term remission is achieved in up to 50% of
patients; about 35% show no response and 5% have severe
adverse effects.15 Overall, as with Coley’s toxin, the pat-
tern of response of cancer to BCG did not meet the enthu-
siasm of the original reports but at least an incremental
advance was made by this approach in the treatment of a
specific type of tumor, superficial bladder cancer. The non-
specific activation of cell-mediated immunity was then
attempted using contact sensitivity haptens that would
activate T-cell cytotoxicity to superficial cancer cells
exposed to the hapten.
Sell 3
Dinitrochlorobenzene
Dinitrochlorobenzene (DNCB) acts as a hapten that binds
to tumor cells in the skin and induces a T-cell cytotoxic
response analogous to poison ivy (contact dermatitis).
Painting superficial skin metastases with DNCB induced a
marked local inflammatory reaction at the lesion sites.16 At
first, it was reported that treatment of the skin lesions was
followed by reduction in the size of internal metastases.
However, this was not reproducible. While DNCB treat-
ment appears to have some cosmetic effect, there is little to
no evidence to indicate that there is an effect of overall
survival. Treatment of therapy-resistant warts with DNCB
appears to have a beneficial effect.17 If non-specific cell-
mediated immune did not work, perhaps the effector
cytokines could be used.
Non-immune-specific cytokines
IFN-α
Viral interference, discovered in the 1930s, results in pro-
tection of cells or animals against a viral infection, if there
has been a recent previous infection with a less severe,
unrelated virus. Alex Isaacs and Jean Lindenmann18 found
that the interference phenomenon could be transferred
from treated cells to uninfected cells by tissue culture
fluid. They called the active factor IFN and later showed
that it was produced by the cultured cells and not by the
virus. Later, this was known as IFN-α when Gresser et al.19
in 1969 found a new IFN made by lymphocytes, IFN-γ. In
1966, Gressner found that IFN-γ not only inhibited virus-
induced tumors but also reduced growth of epithelial
tumors. Initial reports of substantial remissions or different
cancers were followed by more complete studies that
showed little or no effect. In 1986, Jorge Quesada et al.20 at
the MD Anderson Cancer Center treated a 25-year-old
man with advanced hairy cell leukemia with IFN-α. At that
time, there was no effective treatment for this disease.
Daily doses of 3 million units achieved complete remis-
sions. By 1986, IFN-α had been used to treat 30 other
patients with this disease, achieving 9 complete and 17
partial remissions. IFN-α was subsequently approved for
treatment of hairy cell leukemia by the U.S. Food and
Drug Administration (FDA).21,22 Although not effective
for other cancers, the action of this agent reinvigorated the
field of cytokine treatment of cancer.
TNF
In 1975, Carswell et al.23 found that a novel factor appeared
in the serum of mice after treating with BCG and endo-
toxin had induced necrosis of tumors; they called this sub-
stance TNF. Another effect of this factor, independently
discovered by others, was to cause wasting disease in
treated animals with large tumors. Because of this effect,
TNF was also called cachectin. In other trials, TNF pro-
duced sever systemic effects (cytokine storm) including
fever, chills, and systemic shock. At the maximum toler-
ated dose, there was little, if any, effect on tumor growth.
Thus, the prospects for using this very interesting and
potent factor for treating cancer were not promising.
In 2006, Ferdy Lejeune et al.24 infused 10× the maxi-
mum tolerated dose of TNF into the isolated limb of a
patient with multiple melanoma lesions and the lesions
faded away. The systemic shock that was caused by TNF
that escaped into the systemic circulation had to be aggres-
sively treated. Later, the treatment effect was found to be
enhanced by adding low-dose IFN-γ and chemotherapy
(Mephalan).25 This was known as “triple therapy.” This
approach has limited applications because it can only be
used on isolated limbs26. Lejeune stated that “You can cure
the limb, but you can’t cure the patient. The current use of
TNF in cancer is in the regional treatment of locally
advanced soft tissue sarcomas and metastatic melanomas
or other non-operable tumors in order to avoid amputation
of the limb.27 These results led to further cytokine/cell-
mediated approaches.
IL-2 and natural killer cells
In the 1960s, it was observed that supernatants from cultures
of lymphocytes stimulated with mitogens, such as phytohe-
magglutinin (PHA), produced factors that had biological
effects on other cells (conditioned medium). These factors
were given names that reflected their biological effects,
such as migration inhibitory factor (MIF) for macrophages,
macrophage activating factor (MAF), lymphocyte activat-
ing factor (LAF), and lymphocyte migration inhibitory fac-
tor (LIF). In 1974, supernatants of PHA were used as
conditioned medium to stimulate growth of cultured leuke-
mic cells. The stimulatory factor in the conditioned medium
was first named LAF and then as T-cell growth factor and
IL-2. In 1986, Steve Rosenberg and Lotze28 at NIH began to
organize a team to develop immunotherapy of cancer. He
started with passive transfer of spleen lymphocytes from
pigs inoculated with tumor cells from selected patients
(refer adoptive transfer explained in the following). As no
positive results were found, he looked for a new approach.
Also at NIH, Elizabeth Grimm29 attempted to culture T-cells
with cancer cells and IL-2 in vitro to obtain a tumor-reactive
T-cell line. She did obtain lymphocytes that could kill tumor
cells, but these did not have the properties of T-cells. These
were called lymphokine activated killer cells (LAKs). Many
years earlier, Karl-Eric and Ingegard Hellstrom et al.,30 in
Seattle, had cultured pediatric neuroblastoma cells with
lymphocytes from the same patients to identify immune
cells from the patients that would inhibit growth of the
tumor cells (colony inhibition assay). Their preliminary
findings suggested that some patients with remission had
lymphocytes that inhibited colony formation. However, this
4 Tumor Biology
inhibition was significant only when compared to a group of
control lymphocyte donors whose lymphocytes had no
effect on colony formation. When other normal donors were
used as a cell source, their lymphocytes did inhibit tumor
growth even though there was no evidence that they had
been previously exposed to the tumors. This effect was pro-
posed to be due to non-immunized normal lymphocytes that
were named natural killer cells (NKs).31 Grimm29 found that
IL-2 exposure increased the activity of NKs, designating
these cells as LAKs.
In the meantime, Rosenberg and Michael Lotze28
attempted to use large amounts of Il-2 for immunotherapy.
By 1984, 75 patients had been treated with IL-2 plus LAKs
with little or no response. If the dose of IL-2 was increased to
the maximum tolerated by the patients, several spectacular
cures were found. However, these patients developed severe
side effects, including vascular leak syndrome and life-
threatening pulmonary edema. These effects were so severe
that treatment had to be stopped. Two patients given up for
dead actually recovered and were judged to be free of cancer.
Biopsies from tissues of successfully treated patients showed
lymphocytic infiltration and necrosis, and over the next
3 months, the tumor disappeared. In studies of an additional
45 patients, a response rate of up to 45% was reported with
LAK/IL-2 treatment. Again it appeared that a breakthrough
in cancer therapy had been made. Observations by Rosenberg
and co-workers received national lay press coverage. When
more complete studies were available, complete responses to
this regimen in 8% of patients and partial responses in
another 10% of patients were found. However, severe side
effects including shock (capillary leak syndrome), chills,
fever, nausea, vomiting, diarrhea, cutaneous erythema, ane-
mia, and moderate-to-severe liver and kidney dysfunction as
well as thyroiditis cause most centers to discontinue this
approach.32,33 As of today, IL-2 is only approved for treat-
ment of mycosis fungoides and Sézary syndrome, tumors of
mature T-helper cells with less than satisfactory results. A
recent study of these conditions recommends IFNs, reti-
noids, and chemotherapy (doxorubicin and gemcitabine) as
well as a monoclonal antibody, alemtuzumab (anti-CD52),
but not IL-2.34 A more recent approach is the use of IL-2
fused with the diphtheria toxin, a chain which delivers the
toxin to the IL-2 receptor on the T-helper cell (CD25).35 IL-2
is still used in trials in combination with other therapies such
as adoptive transfer of lymphocytes to stimulate T-cell prolif-
eration (see in the following) in otherwise untreatable cases.
Overall, these results are very similar to those previously
reported for Coley’s toxin, and in fact, it is likely that Coley’s
toxin did produce high levels of IL-2. Now, treatment
approaches were then extended to the use of other ILs.
IL-12
IL-12 is produced by activated antigen-presenting cells
and has pleiotropic effects.36 Originally called natural
killer cell stimulatory factor (NKSF),37 IL-12 not only
activates NK cells but also has other actions. These include
activation of CD4+ Th1 helper cells and CD8+ T-cytotoxic
cells, stimulation of B-cells, inhibition of angiogenesis,
and blockade of growth of human tumors in immune-defi-
cient mice, particularly if the IL-12 gene is introduced into
fibroblasts.38 In early FDA phase I clinical trials (testing
for toxicity and dose level that can be given), no problems
were found when patients were primed with low doses of
IL-12 and then given daily doses of IL-12 2 weeks later.
However, in phase II trials in 1995, higher doses of IL-2
were given. Of 15 patients treated with high doses of IL-2,
all became seriously ill (cytokine storm) and 2 died. Because
of high toxicity, IL-2 is now used in clinical trials as an
addition to gene therapy and only as local injections.39,40
The robust response seen in animal studies has not been
translated for humans. If one cytokine gave less than satis-
factory results, perhaps more than one cytokine would be
more effective.
Cytokine mixtures
Doses of toxic cytokines, such as IL-2 and TNF, may be
given in mixtures at lower than toxic levels. An example of
such a mixture is a commercially available preparation of
natural cytokines called “leukocyte IL” or “Multikine”
(Cel-Sci Corporation, Vienna, VA, USA) that includes
IL-2, granulocyte-macrophage colony-stimulating factor
(GM-CSF), IFN-γ, and TNF-α.41 Phase II trials suggest
that Multikine may stimulate a healthy immune system.
However, a phase III clinical study in head and neck can-
cer has been put on hold by the FDA. While cytokines
were being extensively used, there was an increasing inter-
est and application of transfer of immune active cells.
Adoptive cell transfer
Tumor-infiltrating lymphocytes
In 1894, the preeminent American surgeon at Johns Hopkins,
William Halsted,42 noted that the breast cancer patients
whose tumors had a lymphocytic infiltrate had a better post-
operative prognosis than those patients whose tumor did not
have an infiltrate. He postulated that these lymphocytes
were reacting against the tumor and were inhibiting growth.
However, an alternative hypothesis was presented that the
reason that the tumors with infiltrates did not metastasize as
readily as those without was because such tumors required
stimulation by the lymphocytes, and thus, they were more
dependent on the lymphocytes for their growth than those
without lymphocytes (immune stimulation).43,44 The effect
of lymphocytes on tumors may depend on the type of infil-
trating lymphocytes. For example, infiltrates of Th1 cells
indicate a good prognosis, whereas infiltrate with Th2 sug-
gests a poor one.45 In any case, such lymphocytes became
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known as tumor-infiltrating lymphocytes (TILs). In 1984,
Alexander Knuth et al.46 extracted lymphocytes infiltrating
a tumor, cultured them with IL-2, and showed that they
could lyse tumor cells. These cells were T-cytotoxic cells
and were specific for the tumor from which they were iso-
lated. Rosenberg at NIH soon reported that reinfusion of
TILs back into the patient from induced “substantial remis-
sion” in eight or nine patients with melanoma. After treat-
ment of more patients, a partial response rate of 55% was
claimed,28,47 and in subsequent studies, a much lower
response was found. This approach has been abandoned. A
next step was to transfect the TILs with cytokine genes that
might increase the efficiency of TILs. This has not proven
effective. A more recent approach being tested is to insert
chimeric tumor antigen receptors on peripheral blood lym-
phocytes cultured from the patient.48
Chimeric antigen receptor T-cells
In this approach, T-cells from the blood of patients with
refractory B-cell tumors are transfected with anti-CD19,
so that they recognize essentially all stages of B-cell devel-
opment.49,50 Upon transfusion into the patient, these cells
can effectively attack B-cells. The more recent versions of
these engineered cells contain extracellular domains that
recognize CD19 linked to intracellular activation domains
which when the receptor binds to CD19 stimulates prolif-
eration and cytokine production by the transplanted cells.
They have been used effectively for management of acute
lymphoblastic leukemia and are proposed for use in
Hodgkin’s disease and blast crisis of chronic lymphocytic
leukemia. The transfused cells rapidly expand in the
patient.51 This is desirable because the result is that there
are more effector cells to attack the cancer. This is undesir-
able because these cells also attack normal B-cells, caus-
ing severe clinical complications including B-cell aplasia,
neurologic toxicity, cytokine release syndrome, allergy,
and anaphylaxis. This has led to consideration of how to
diminish or prevent the side effects.52 An ingenious
approach is to construct the chimeric receptor T-cell so that
the activation domains of the engineered receptor require a
small molecule to join them.53,54 Thus, control of the CAR
T-cells can be manipulated, so that the activity can be
turned on when necessary. It remains to be seen how much
of an impact this approach will eventually have. First, it is
presently applied to a small proportion of leukemia patients
because about 80% of patients with B-cell cancers respond
well with prolonged complete remissions to standard
chemotherapy and/or monoclonal antibody treatment
without CAR T-cells (see in the following). Second, the
serious side effects may eventually prove to be worse than
the treatment for many patients. Third, obtaining periph-
eral blood lymphocytes from each patient, rapidly transfer-
ring them to production laboratories for culture, and
genetically engineering the cells are prohibitively
expensive, unless some advance in methodology occurs.
Thus, this approach is an example of impressive biomedi-
cal engineering, but it may simply be impractical for more
than occasional use (Figure 1). For more information, see
the article on the front page of the New York Times (2
August 2016).
Immune RNA
The rationale for this approach was that the RNA could be
extracted from immune cells of immunized animals
(sheep) and used to transfer immunity to cancers to which
the sheep was immunized.55 There are problems with this
approach. First, if the immune RNA is active, then immu-
nity to the normal cells of the patients would most likely be
transferred as well. However, if specific tumor immunity
could be induced, the injected RNA would be rapidly
destroyed by the potent ribonucleases present in blood and
tissue fluids.56 An interesting sidelight of this work is that
chest X-rays of lung cancers before and after treatment
were used to evaluate the size of the tumor seen. However,
not only the size of the tumor was smaller but also the size
of the heart and the density of the ribs were lower.55 Thus,
it has been suggested that the size of the tumor on the chest
X-ray was determined by the extent of radiological expo-
sure. Better success will result through the use of active
stimulation of the immune response to tumor-causing
viruses.
Tumor vaccines
Viral vaccines
With considerably less fanfare than other immunological
approaches, vaccination against cancers caused by viruses
has been remarkably successful.57 Thousands, if not mil-
lions, of serious cancers have been prevented by this
approach. This list of viruses and virus-induced cancers
includes Epstein–Barr virus (Burkett’s lymphoma, etc),
hepatitis B virus (HBV, hepatocellular carcinoma (HCC)),
hepatitis C virus (HCV, hepatocellular carcinoma, human
papillomavirus (cervical cancer, etc.)), Kaposi’s sarcoma
virus, and Merkel cell carcinoma virus.57 Worldwide, over
2 billion people are infected with HBV. HBV infection is
believed to be responsible for up to 60% of HCC.58 In
endemic areas of HBV infection, introduction of HBV
vaccines has reduced the prevalence of chronic infection in
children by more than 90%.59 Thus, vaccination against
HBV has already had a significant effect in reduction of
HCC and cirrhosis. There is no doubt that vaccination
against viruses causing cancers has had more impact on
preventing cancers than any other immunological
approach. If vaccination against tumor-causing viruses
works, then maybe vaccination against tumor antigens
would be effective.
6 Tumor Biology
Figure 2.  (a) Gross photograph of self-healing malignant
melanoma. The growing margins of the cancer are advancing
at the same time that the center has “healed.” (b) Lymphocyte
infiltrate underlying the cancer. (c) Histocytes (macrophages)
containing melanin underlying the “healed” zone.
Vaccines against tumor-specific
antigens
Self-healing malignant melanoma
The occasional observation of spontaneous remissions of
cancer gives rise to speculations of the development of a
cancer-specific immune response in such patients. Figure 2
shows the expanding growth of a self-healing malignant
melanoma, while that the central zone of the tumor shows
healing with no melanoma lesion. Histologically, lympho-
cyte infiltration of the tumor may be observed along the
regressing margins, with collections of melanin-contain-
ing macrophages underlying the normal appearing skin in
the center of the lesions. Since 1866, there have been 76
reported cases of spontaneous regression of metastases of
malignant melanomas.60 The mechanism of these regres-
sions could be immunological, endocrine related, or related
to loss of tumor vasculature.61 However, the histological
findings are consistent with a delayed-type cellular
response to the tumor and suggests tumor immunity. Most
studies of tumor-specific immunity in patients address
malignant melanoma or renal cell carcinoma, two tumors
noted for their relatively high incidence of spontaneous
remission of metastases as compared to other cancers.
Direct evidence for tumor-specific transplantation anti-
gens was originally derived from tumor transplantation
studies in mice.
Tumor-specific transplantation antigens
In the 1890s, Paul Ehrlich62 studied transplantation of
tumor cells in animals and noted that growth of meta-
static lesions increased after removal of the primary
tumor. He attempted to generate immunity to cancer by
injection of “weakened” cancer cells. During the early
part of the 20th century, it was found that tumors trans-
planted from one mouse to another were rejected. This,
of course, is because of histocompatibility differences
(graft rejection). In 1943, Ludwig Gross63 observed that
tumors were rejected more slowly in partially inbred
mice, and a decade later, Foley64,65 showed that tumor
grafts growing in inbred mice were rejected by mice
immunized to the tumors. In 1960, George Klein et al.66,67
did the definitive study to show that tumors did have
tumor-specific transplantation antigens (Figure 3).68
Several fundamental concepts were observed from stud-
ies of specific tumor transplantation.
Concomitant immunity.  A tumor bearing individual may be
immune to his/her own tumor cells. Tumor cells taken
from an individual’s own tumor will not grow if injected
into another site even though the tumor continues to grow
at the primary site.69
Immunosurveillance.  Newly arising tumors are recognized
by the immune system and held in check by immune
mechanisms.70,71 Of interest, however, there is no increase
in tumor incidence in immunodeficient mice, which can-
not mount an immune response to cancer.72
Immunoediting (also known as immunoselection). The
immune systems select for destruction immunogenic
tumor cells. The result is that the tumors that do arise are
weakly antigenic or non-antigenic.73
Human tumor-specific vaccines
The first human tumor-specific antigen vaccine was
developed after the identification of a tumor antigen on
malignant melanoma cells by Theiry Boon in 1993.74 In
1981, Alexander Knuth in Germany sent cancer cells from
a patient with multiple metastases to Boon in Belgium.
Boon cultured the cells in vitro and treated them with
mutagens. After irradiation, the non-viable melanoma
cells were injected back into the patient. After three injec-
tions of such cells over 3 months, the patient’s tumors dis-
appeared and with repeated injections did not recur.
Lymphocytes harvested from the patient very effectively
killed the cultured tumor cells in vitro. Boon then used
IL-2 to stimulate growth of a T-cell line from the patient.
When the T-cell line was tested against a DNA library
from tumor cells transfected into tissue-cultured cells, the
cloned T-cell line killed some of the transfected cells.
From one colony of susceptible cells, a 9-amino acid
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Figure 3.  Demonstration of tumor-specific transplantation antigens by Geroge Klein et al. (a) Primary methylcholanthrene–
induced cancers from inbred mice were excised and the cells maintained in vitro. (b) When these cells were injected into another
mouse of the same strain, they grew to produce tumors. However, when injected back into mouse A from which the primary
tumor had been removed they did not grow. (c) Then, when the primary mouse was injected with cells from a tumor arising in a
different mouse of the same strain, these cells grew. The conclusion is that the original mouse developed transplantation immunity
to the primary tumor that is specific for that tumor and not for other tumors of the same type: tumor-specific transplantation
immunity.

peptide was identified, which was presented as an antigen Monoclonal antibodies

by class II major histocompatibility complex (MHC).
This peptide was designated as melanoma-associated
antigen-1 (MAGE-1). Since then, a number of putative
tumor antigens have been identified from melanoma,
prostate, pancreas, and colon tumors.75 Unfortunately,
most of these have had very little effect on tumor growth,
but some positive effects have been obtained in combina-
tion with chemotherapy.76 For example, vaccines for
­prostate cancer increase survival in metastatic castra-
tion-resistant cancer (Sipuleucel 4.1 months; Prostvac
8.5 months).75 Glycoprotein 100 (gp-100) or melanocyte
protein PMEL is a 661 amino acid transmembrane gly-
coprotein that is enriched in melanosomes. When applied
topically, it permeates mouse and human skin and
induces T-cell cytotoxic responses.76 It is currently being
tested as an adjunct to chemotherapy in some clinical
trials. It appears that high levels of antigen-specific
T-cells do not necessarily prevent progression of human
melanomas.77 Thus, although there may be some benefi-
cial effects in combination therapies, tumor vaccines do
not play a prominent role in immunotherapy. Paul
Ehrlich’s1 original hypothesis on the side chain theory
predicted antibodies as the magic bullet for immunother-
apy. This theory is being tested using monoclonal
antibodies.
In 1975, Georges Kohler and Cesar Milstein78 fused anti-
body-producing B-cells with cells of multiple myeloma to
form an immortalized cell line that produced high levels of
the specific antibody. This Nobel Prize winning discovery
allowed for the production of large amounts of a single
antibody (monoclonal antibody, -mab). In 1984, Masui
et al.79 at University of California, San Diego (UCSD)
demonstrated that administration of a mouse monoclonal
antibody to the human epidermal growth factor receptor
(EGFR) could markedly inhibit the growth of human cancer
cells in immunodeficient mice. The mouse model proved
the principle, but mouse monoclonal antibodies cannot be
used in humans due to their high immunogenicity.
Subsequently, a massive and incredibly productive effort
was made to genetically engineer antibody-producing cells
using recombinant DNA to create constructs capable of
expression in mammalian cell lines.80 Gene segments
capable of producing antibodies were isolated and cloned
into tissue culture cells that could be grown in a bioreactor.
The antibody proteins produced from the DNA clones
were then harvested in large amounts. This was a major
advance, but even more astounding was that the DNA
could be engineered to express different parts of the anti-
body molecule of mice and humans. Thus, the genetically
8 Tumor Biology

Figure 4.  Genetically engineered monoclonal antibodies. Because mouse monoclonal antibodies cannot be used for human
therapy, various parts of the antibody are replaced by human immunoglobulin chains. These are identified by suffixes: -omab is a
mouse monoclonal; -ximab has human constant heavy and light chains and mouse variable heavy and light chains; zumab (humanized)
has human chains except for mouse complementary binding regions (antibody binding sites); -umab is a fully human antibody.

engineered antibodies may have human constant chains Herceptin (trastuzumab)

and mouse variable chains; it may have human heavy and
light chains, but the complementary determining regions
(antigen binding sites) of the mouse antibody. In addition,
fully human antibodies have been produced (see Figure 4).
These products have been coded by specific suffixes. That
is, -mab refers to monoclonal antibody, -omab refers to
mouse monoclonal antibody, and -zumab refers to human-
ized mouse monoclonal antibodies.
Rituximab
One of the first anticancer antibodies to be used was the
human B-cell marker CD20 (rituximab) for treatment of
B-cell malignancies. Rituximab has significantly improved
the treatment of diffuse large-cell B lymphoma and follicu-
lar lymphoma.81 When combined with fludarabine and
cyclophosphamide, rituximab treatment has achieved long-
term disease-free survival in chronic lymphocytic leukemia
associated with mutated immunoglobulin heavy chain vari-
able region.82 About 70% of patients with acute B-cell
tumors have complete remissions with the combination of
cyclophosphamide, doxorubicin, vincristine, and pred-
nisone therapy (CHOP) plus rituximab, whereas the remis-
sion rate is 40% when CHOP is used without Rituximab.83
As is the case in other immunotherapy approaches, success
of this regimen is limited to selected cancers.
Herceptin is a humanized mouse monoclonal antibody to
the human epidermal growth factor receptor 2 (HER2)
present on about 30% of human breast cancers and certain
colon cancers.84 This antibody is used in conjunction with
chemotherapeutic regimen of anthracycline, cyclophos-
phamide, or paclitaxel. Herceptin blocks the cell signaling
necessary for continued growth of the cancer cells. Adding
herceptin to chemotherapy increases disease-free survival
about 10% over chemotherapy alone (84% vs 75%). This
is achieved at a cost of increased side effects, such as con-
gestive heart failure and acute leukemia.85
Other monoclonal antibodies
Overall, over 300 genetically engineered monoclonal
antibodies have been approved by the FDA for human use
in a variety of diseases in addition to cancer.86 Among the
other cancer-directed monoclonal antibodies are alerntu-
zumab (targeted at CD53) for chronic lymphocytic leuke-
mia, gemtuzumab (directed at CD33) for acute
myelogenous leukemia, and cetuximab, panitumuab
(EGFR), and bevacizumab (vascular endothelial growth
factor (VEGF)) for colon and lung cancer.86 In general,
FDA phase II trials showed promise for anti-EGFR thera-
pies, which was not the case in phase III trials. Trials
Sell 9
continue to test this anticancer approach in combination
with chemotherapy. Results of monoclonal antibody treat-
ment of cancer fit the pattern of high initial enthusiasm
and eventual limited effectiveness in clinical trials. The
field of tumor immunity continues to move to new ideas.
The next idea is that there is an immune response to can-
cer, but it is inhibited by immune checkpoint regulators. If
these checkpoints can be removed or attenuated, then an
individual’s own anticancer response may be able to block
growth of the tumor.
Immune checkpoint regulation
inhibitors
Cytotoxic T-lymphocyte-associated protein 4
Editorial comments in the scientific and lay press have
recently emphasized breakthrough developments in the
very expensive trials of immunotherapy. These articles
referred initially to discoveries that immune checkpoint
regulation inhibitors (CPRIs) could be manipulated by
specific antibodies.87,88 The rationale for this approach is
that cancers can turn off the immune effector T-cytotoxic
cells. Specific T-cells are activated by tumor-specific
antigenic peptides presented by dendritic cells to the
T-cell receptor (TCR) and by co-stimulation by reaction
of B7 on the dendritic cells with CD28 on the T-cell.87
However, IFN-γ production by the T-cell upregulates
production of cytotoxic T-lymphocyte-associated protein
4 (CTLA-4) in the cancer cell causing displacement of
CD28 on the T-cell. Reaction of the TCR with MHC and
CTLA-4 instead of CD28 with B7 on the dendritic cell
results in death of the reactive T-cell and protection of the
cancer cell. Treatment with anti-CTLA-4 blocks this
inhibition and allows the CD28 to reappear on the T-cell,
so that it can develop an immune response to the cancer.
An antibody that can accomplish this is ipilimumab. The
preclinical model for this approach was developed by
Leach et al.,89 who showed that treatment of mice injected
with anti-CTLA-4 greatly inhibited the growth of trans-
planted tumor cells. Early trials using anti-CTLA-4 (ipil-
imumab) showed that after 3 months of treatment, there
was increased survival of patients treated with ipili-
mumab compared to patients treated with tumor antigen
gp 100. Anti-CTLA-4 treatment achieved a plateau of
survival of about 20% versus none in patients treated
with gp 100 alone.90 It should be pointed out that these
were advanced cases for which other therapies had failed
or showed little effect. In one study, treatment with tras-
tuzumab increased responses to CTLA-4 blockade lead-
ing to increased T-cell responses in mice with primary
breast cancers; there was, as well with cytotoxic
T-lymphocyte antigen-4 (CTLA-4) blockade, an increase
in Tregs, which are essential to protect mice from
immune-mediated autoimmune reactions during tumor
rejection. This effect is not so obvious in human subjects
who may have decreased Treg activity and lymphocytic
infiltration of various organs and symptoms of cytokine
storm.91 Predilection to severe side effects was supported
by findings in the mouse model that included depigmen-
tation and deaths of older mice that were apparently due
to autoimmune reactions.92
Programmed cell death protein-1/programmed
cell death ligand-1
A second costimulatory immune checkpoint inhibitor is
programmed cell death ligand-1 (PD-L1).93 When anti-
gen-specific T-cells react with antigen on tumor cells via
the TCR, tumor-derived PD-L1 protein reacts with pro-
grammed cell death protein-1 (PD-1) and eliminates
T-cell killer effects. Like anti-CTLA-4, anti-PD-L1 blocks
this tumor defensive reaction and allows T-cells to attack
the cancer cells. Zou et al.94 recently reviewed the results
of 22 ongoing clinical trials using variations of this
approach. The clinical response rate varied from 14.5% in
refractory non-small-cell lung cancer to 87% in refractory
Hodgkin’s lymphoma (Table 1). In a comparison with
chemotherapy alone, anti-PD-1 (Keytruda) treatment
resulted in an increase of 17% in the 1-year survival rate
of non-small-cell lung cancer.95 Active investigation is
underway to predict which patients will respond.96 There
is good correlation between the immunohistological iden-
tification of high expression of PD-L1 on the cancer cells
and clinical response to anti-PD-1.97 Other possible
response markers are high levels of Th1 cytokines, the
presence of tumor antigens,98 lack of immunosuppression,
high levels of PD-L1-high cytotoxic T-cells in tumors,99
low cancer stem cell–like properties,82 and mismatch
repair deficiency.98 The largely anecdotal cases of com-
plete lasting clinical responses have achieved notoriety
(New York Times, 31 July, 1 August 2016). Is this a true
breakthrough or another incremental improvement in can-
cer immunotherapy? It is far from clear. The treatment is
extremely costly, up to US$250,000 per year per patient.
This approach has works in only a minority of patients,
and there are potentially severe side effects.100 This side
effect profile is the result of blocking of controls on the
immunoreactive T-cells, so that the T-cells also attack nor-
mal organs. As a result, a significant number of patients
successfully treated for cancer develop life-threatening
diabetes, cytokine storm, and a variety of autoimmune
diseases.100,101 A major effort is now underway to try to
prevent or limit these untoward reactions. However, they
do present serious problems for the general application of
CPRI therapies particularly outside of major medical
centers. It would clearly be desirable to target the PD-1/
PD-L1 checkpoint exclusively for cancers or non-immu-
nologically downregulated expression of the PD-L1 gene
in tumor cells.102
10 Tumor Biology
Table 1.  Some examples of response rates in ongoing clinical
trials with anti-PD-1 Nivolumab (Optivo).
Type of cancer Response rate (%)
Advanced melanoma 28
Treatment-refractory melanoma 12.8
Melanoma without BRAF mutation 40
Non-small-cell lung carcinoma (NSCLC) 14.5
Previously treated advanced NSCLC 17
Advanced NSCLC 20
Relapsed or refractory Hodgkin’s 87
Renal cell carcinoma (RCC) 27
Previously treated RCC 34
Nivolumab (Optivo) followed by ipilimumab  
 Melanoma 40
  Stage III or IV melanoma 57.6
Source: Modified from Thompson et al.82
These are ongoing trials; final conclusive results are not yet available.
Frequently, an early promising complete response is followed by resis-
tance to treatment.
Combinatorial therapies
In the last 5 years, the number of clinical trials including
combinations of chemotherapy, monoclonal antibody,
tumor antigen, and immunotherapy has increased dramati-
cally and is projected to continue to rise even further.102 In
fact, many of the early clinical trials of CPRI therapy were
done on patients whose cancers had resisted conventional
chemotherapy.94 The projected potential combinations will
keep clinical trials ongoing for many years. This includes
combining chemotherapy and CPRIs with tumor antigen
(gp-100), adoptive cell transplantation, targeting cancer
stem cells and tumor stroma, microbiota alteration (select-
ing beneficial bacteria), activation of antigen-presenting
cells, and signal induction inhibitors, such as BRAF and
MEK inhibitors.103,104
Oncolytic virus immunotherapy
In this new variation on an old theme of viral oncolysis,
viruses are engineered to infect specific tumors and cause
their destruction.105 The FDA approved in 2015 an injecta-
ble form of T-VEC, Imlygic, for the treatment of melanoma
in patients with inoperable tumors.106 T-VEC is a re-engi-
neered herpes simplex virus 1 (HSV-1), a relatively innocu-
ous virus that normally causes cold sores. Genetic
modifications have been made to attenuate the virus (so, it
could no longer cause herpes), to increase selectivity for
cancer cells (so, it destroys cancer cells while leaving
healthy cells unharmed), and to secrete the cytokine
GM-CSF (a protein naturally secreted in the body to initiate
an immune response). Inside a healthy cell, the modified
virus is unable to replicate. Inside a cancer cell, the virus
replicates and produces GM-CSF until cell lysis, releasing
more virus, granulocyte-macrophage colony-stimulating
factor (GM-CSF), and tumor antigens. GM-CSF attracts
dendritic cells to the tumor site and activates them. These
dendritic cells process and present the tumor antigen to
T-cells. The T-cells then identify and destroy the cancer
cells throughout the body. This approach is now in FDA
phase II clinical trials.
Therapy directed to the tumor
microenvironment
This therapeutic approach is based on the concept that
tumor stem cells are protected from chemotherapy by their
microenvironment. In the case of multiple myeloma, for
example, the myeloma cells form junctions with the bone
marrow stromal cells that support the interchange of signal-
ing molecules that protect the myeloma cells. It was postu-
lated that such junctions could be broken by the proteasome
inhibitor bortezomib (PS-341; Velcade) which also pre-
vents degradation of pro-apoptotic factors and thus allows
programmed cell death.107,108 Treatment of multiple mye-
loma with combinations of bortezomib and thalidomide
were approved by the FDA in 2008. In phase III trials, the
combination of bortezomib and melphalan–prednisone is
superior to melphalan–prednisolone in the treatment of
older myeloma patients who were not eligible for high-dose
chemotherapy; the complete response rate achieved was
32%.109 In another study, bortezomib added to other treat-
ment regimens significantly increased response rates.110,111
There are frequent side effects, such as neuropathy, fevers,
hypoxia, and hypotension. Recently, a humanized mono-
clonal antibody (elotuzumab) to the signaling lymphocytic
activation molecule F7 (SLAMF7) receptor has been
developed.112 This binds to the Fc receptor (CD-16),
activates NK, and blocks interaction between SLAMF7 on
myeloma cells and stromal cells. In preliminary trials, this
has increased progression-free survival rates by 14% over
lenalidomide plus dexamethasone therapy alone.113
Significant side effects are also associated with this regi-
men, including fatigue, diarrhea, and fever.
Summary and conclusion
The immunotherapeutic approaches to cancer and their
proved and projected results are listed in Table 2. The
results of application of various cancer immunotherapeu-
tics have repeatedly resulted in impressive anecdotal cures
that cause optimism and stimulate hyperbolic press reports.
Decreasing enthusiasm ensues as the results of controlled
clinical trials are reported. Finally, an incremental result is
obtained, usually limited to a relatively small number of
patients with a highly specific type of cancer. In the case of
Coley’s toxin, the first reported successful immunotherapy,
no treatment using this approach currently remains. This is
due to the inability to reproduce early anecdotal results and
because of important and severe side effects (cytokine
Sell 11

Table 2.  Summary of cancer immunotherapy approaches.

1. Non-specific immunity
  Coley’s toxins (1890–1926) Various cancers Not used
  BCG (1060s) Superficial bladder cancer 50% increase in 5-year survival
  DNCB (1960s) Superficial skin cancer Cosmetic only
  NK/LAK (1970s) Various (melanoma, etc.) Clinical trials ineffective
 IFN-α (1970s) Hairy cell leukemia <50% effective
Tumor necrosis factor Melanoma Isolated limb
 IL-2 Various Stimulate T-cell proliferation
 IL-12 Experimental Too toxic
2. Specific passive immunity (cellular)
  TILs (1980s) Various Discontinued
  Cart-cells (2010) B-cell tumors To be determined
3. Specific passive immunity (humoral)
  Monoclonal antibodies 1980s  
 Alemtuzumab-anti-cd52 Sézary’s syndrome 10% effective
 Herceptin—anti-EGFR Breast, colon 6-month increase in survival
 Rituximab—anti-CD20 B-cell tumors 20% increase in 5-year survival
4. Specific active immunity—vaccination
  Viral (HPV, HBV, HCV, etc.) Various Up to 100% effective
  Melanoma T-CTL peptides Melanoma Anecdotal results
5. Checkpoint inhibitors 2000s
 CTL-4 Various To be determined
 PD-1 Various To be determined
6. Oncolytic virus immunotherapy Experimental
7. Microenvironment
 Bortezomib Multiple myeloma Up to 30% partial response
 Elotuzumab Multiple myeloma To be determined

BCG: Bacille de Calmette de Guerin; DNCB: dinitrochlorobenzene; NK/LAK: natural killer cells/lymphokine activated killer cell; IFN-α: interferon-
α; IL: interleukin; TILs: tumor-infiltrating lymphocytes; EGFR: epidermal growth factor receptor; HPV: human papillomavirus; HBV: hepatitis B virus;
HCV: hepatitis C virus; CTL: cytotoxic T-lymphocyte.

storm). Non-immune-specific approaches including BCG
and IFN-α have resulted in successes in small groups of
patients. Use of IL-2 and IL-2-expanded tumor-infiltrating
lymphocytes was first reported to be a major breakthrough,
but like Coley’s toxin did not stand the test of time. The use
of adoptive cell transfer is now being tested using geneti-
cally engineered chimeric antigen receptor T-cells (CAR-T-
cells) with the receptor linked to cell activation molecules.
This approach is presently applied to a very small number
of treatment-resistant B-cell tumors and is enormously
expensive as it requires expansion and engineering of lym-
phocytes in vitro from each individual patient. In addition,
it is associated with severe long-term side effects.
In 1980s, engineered monoclonal antibodies attracted
attention for the treatment of various cancers. Again, after
extensive clinical trials, this treatment is now limited to
certain kinds of cancer, for example, herceptin for EGF
receptor-positive breast cancer and anti-CD-20 for B-cell
tumors. Vaccines using tumor-specific transplantation-
type antigens again have also shown promise for a few
patients, but not as a general application. The most suc-
cessful approach that has received less attention is the use
of cancer virus vaccines. These have had an incredible role
in preventing and even treating many cancers including
liver, lymphoma, cervical, and other cancers. However,
cancer causing viruses have not been identified for most
human cancers. The latest immunotherapeutic emphasis is
on checkpoint regulatory inhibitors. Several of these
agents are FDA approved (an anti-CTLA-4 and two PD-1
antibodies) for specific oncologic conditions, and multiple
additional CPRIs are under development. We are in the
euphoric phase of the application of these therapies. There
are many anecdotal cures that have been reported with this
approach, but we are still in the mid-phase of larger clini-
cal trials. This is a work in progress and just exactly how
much overall impact this approach will have remains to be
seen. However, the results so far are highly encouraging.
These agents may work in a minority of cancer patients
and are associated with severe and long-term side effects,
but in those that respond this is clearly a welcome result.
Hopefully, this approach will prove to be the exception to
the rule of slight incremental improvement achieved with
other immunotherapies for cancer.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
12 Tumor Biology
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: Dr
Sell’s laboratory is supported by NIH grant CA161694.
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