17
`
`lmmunoconjugates
`
`Walter A. Blattler, DrScNat, Ravi V. ]. Chari, PhD,
`and john M. Lambert, PhD
`
`CONTENTS
`
`INTRODUCTION
`TuMOR-SPECIFIC ANTIBODIES
`RADIOIMMUNOCONJUGATES
`lMMUNOTOXINS
`ANTIBODY-DRUG CONJUGATES
`CoNCLUSIONS
`
`1. INTRODUCTION
`Successful anticancer drugs must exploit known or unknown, gross or ever so
`subtle, differences between normal and malignant cells. The development of immuno(cid:173)
`toxins is one of the first attempts to develop rationally anticancer drugs that are based
`on known cellular differences associated with cancer cells. Much immunological evi(cid:173)
`dence had accumulated that transformed cells express tumor-specific antigens. How(cid:173)
`ever, it was difflcult to generate heterosera with well-defined antitumor reactivity.
`The isolation in 1967 of an agglutinin from wheat germ that identified a tumor-specific
`determinant on neoplastic cell surfaces (J) marked the first time that a pure molecular
`species was available for targeting of tumors.
`Further probing of cell surfaces with lectins and agglutinins, however, was hampered
`by the availability of only a small number of lectins with an even smaller number of
`different binding specificities. This situation changed dramatically with the advent of
`the monoclonal antibody (MAb) technology (2). The potential for generating a nearly
`unlimited reservoir of reagents each with its own binding specificity for an antigen
`was rapidly exploited in creating MAbs that bound to novel tumor cell-speciflc anti(cid:173)
`gens. Although some naked antibodies were used in clinical tests for the treatment of
`cancer, many immunologists doubted that the humoral part of the immune system
`would have sufflcient cytotoxic potential to eliminate millions of tumor cells. MAb
`were, therefore, armed with extraneous cytotoxic effector functions and became
`delivery vehicles that imparted tumor speciflcity to otherwise nonselective cytotoxic
`effector molecules.
`
`From: Cancer Therapeutics: Experimental and Clinical Agents
`Edited by: B. Teicher Humana Press Inc., Totowa, NJ
`
`371
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`Effector
`
`Conjugate
`
`Radioisotope
`
`Radioimmunoconjugate
`
`~ Linker
`
`Toxin
`
`Immunotoxin
`
`Cytotoxic Drug Antibody-Drug Conjugate
`
`Antibody
`
`Fig. 1. Schematic representation of immunoconjugates.
`
`The covalent binding of an effector molecule to an MAb yields an immunoconjug(cid:173)
`ate (Fig. 1), which is called an immunotoxin, when the effector molecule is a toxin, an
`antibody-drug conjugate when cytotoxic drugs are used as effectors, and a radio(cid:173)
`immunoconjugate in the case of linked radioisotopes. Common to all three method(cid:173)
`ologies is their reliance on the tumor-specific binding of their MAb component. There(cid:173)
`fore, we shall first discuss the generation of "tumor-specific" MAbs and then describe
`the development and testing of radioimmunopharmaceuticals, of immunotoxins, and
`of antibody-drug conjugates.
`
`2. TUMOR-SPECIFIC ANTIBODIES
`The ideal MAb for the generation of immunoconjugates would bind to an antigen
`exclusively present on the surface of tumor cells, and would further be expressed
`homogeneously on all tumor cells or at least on all tumor stem cells (the latter, how(cid:173)
`ever, is difficult to assay). In addition, the antigen should not be shed from cells,
`should not be present in the serum of patients, and ideally, for practical medical and
`commercial reasons, should be present on the tumors of all patients with the same
`type of cancer.
`In the infancy of immunotoxin development, several MAb were claimed to be
`tumor-specific. However, the development and use of more thorough analytical
`methods, such as analysis with a fluorescence activated cell sorter (FACS), sensitive
`immunohistochemical staining techniques using large panels of fresh-frozen tissue
`sections, and modern biochemical and molecular biological techniques, contributed
`to today's generally accepted view that most antibodies recognize tumor-associated
`antigens that are expressed only preferentially on tumors. Some antigens may be found
`on only a limited number of tissues, whereas others are on only one specific tissue
`type and are, therefore, tissue-specific. In the best case, some tumor-associated anti(cid:173)
`gens may be expressed only during a particular developmental stage of a certain cell
`type. Some degree of tumor specificity often presents itself by the overexpression of
`certain surface antigens on transformed cells, such as erbB-2/HER-2 on breast tumor
`cells of a subgroup of patients (3), or certain carbohydrate antigens on epidermoid
`carcinomas (4). The only surface antigens that are absolutely tumor-specific are the
`surface immunoglobulin or idiotype present on the cells of B-cellleukemia and lym(cid:173)
`phomas, and the clonotypic T -cell receptor on T -cell leukemia and lymphoma cells.
`Not only are these structures tumor-specific, but individualized, patient-specific
`MAbs have been created (5).
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`To generate MAb with antihuman tumor reactivity, typically mice or rats were
`immunized with whole cells or cell membrane preparations from tumor cell lines or
`from tumor biopsies. The .spleens of the immunized animals were then used to generate
`and select antibody-producing hybridomas. However, many MAbs used in immuno(cid:173)
`conjugates, in particular, antibodies reactive with hematopoietic cells, were originally
`developed as research tools to differentiate between various normal cell types and
`were, therefore, generated by injecting animals with normal human cells, such as the
`various cell types from blood.
`MAb that have the potential to be used in anticancer immunoconjugates are con(cid:173)
`veniently grouped into those that react with hematopoietic tumors and those that
`bind to antigens on solid tumors. Because of the rapid renewal of hematopoietic cells
`and the experience of regeneration of blood cells after bone marrow transplantation,
`tissue-specific antibodies were widely used in immunoconjugates against leukemias
`and lymphomas. T -cell malignancies were treated, for example, with conjugates bind(cid:173)
`ing to the T-cell markers CD5, CD7, or the IL-2 receptor /3-chain (CD 25); B-cell
`malignancies with antibody conjugates against the B-cell differentiation antigens
`CD19, CD20, and CD22; and analogously, myeloid malignancies with conjugates
`against the myeloid marker CD33 (6). Most of these antigens are differentiation anti(cid:173)
`gens that are expressed throughout the ontogeny of a particular cell type starting at
`the earliest lineage restricted stage to ensure that the conjugates were able to treat the
`yet unidentified clonogenic tumor cells.
`It has been much more difficult to identify cell-surface markers useful for immuno(cid:173)
`conjugates against solid tumors. The principle of tissue specificity is not as easily
`applied as in the hematopoietic area, except possibly for tumors of nonessential tissues,
`where the temporary removal of certain cell populations may be tolerated. In the
`absence of tumor specificity and tissue specificity, the selection of antigens was largely
`based on their overexpression on tumor cells relative to normal tissues. For lists of
`possible candidate surface antigens for immunoconjugate targeting, the reader is
`referred to two comprehensive reviews (7,8).
`For the development of highly cytotoxic immunoconjugates that bind to antigens
`also expressed on some normal tissues, although hopefully at lower levels, it was
`essential to find animal models for toxicity studies, where similar crossreactivity was
`observed. Fortunately, many of the antigenic determinants were found to be preserved
`in nonhuman primates where they were expressed with a similar tissue distribution as
`in humans. A good example is the data presented for the anti-LeY antibody in ref. (4).
`A problem commonly encountered in solid tumors is the heterogeneous expression
`of an antigen on cells of a given tumor. Although some cells may express large numbers
`of an antigen on their surface, other cells in the same biopsy sample, equally having a
`transformed phenotype, may be antigen-negative. If transformation is a clonogenic
`event, then these different cell populations may represent differentiation stages that are
`not necessarily all tumorigenic. Heterogeneous expression of an antigen may, there(cid:173)
`fore, not necessarily disqualify it from being a target for therapeutic immunoconjugates.
`If one surveys the known antigenic cell-surface markers for human solid tumors,
`(see, e.g., 7,8), one is struck by the paucity of such known markers. Also, when anti(cid:173)
`bodies were generated with different tumor tissues or tumor cell lines, often antibodies
`to the same antigens were generated. For example, when mice were immunized with
`the breast tumor line MCF-7, MAb Bl and B3 were obtained that reacted with the
`LeY carbohydrate chain (4), and immunization with cell line H3396 derived from a
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`metastatic breast adenocarcinoma yielded antibodies BR64 and BR96, both of which
`also react with the LeY carbohydrate chain (9). These results are a reflection of the
`limitations of the immunological methodology used to identify these antigens. They
`probably represent the most immunodominant markers recognized by the murine
`immune system, and only the screening of much larger panels of hybridomas, a work(cid:173)
`intensive and time-consuming undertaking, might allow the discovery of further novel
`antigens with this technology. This realization, far from being discouraging, pre(cid:173)
`dicts that we have barely scratched the surface for the discovery of tumor cell-surface
`markers for therapeutic targeting, and it has spawned the development of several new
`methodologies. The most promising techniques might be the phage display of the
`entire murine or human immunological repertoire and its use in the probing of cell
`surfaces (10), or the searching for interactions on cell surfaces with combinatorial
`libraries of peptides that carry their genetic information in the form of amplifiable
`DNA sequences (JI).
`In most patients treated with murine MAb, a prompt human antimurine antibody
`(HAMA) response was observed, which led to the development of several "humaniza(cid:173)
`tion" technologies. Humanization is the attempt to give murine antibodies an appear(cid:173)
`ance that is not recognized as foreign by the human immune system while preserving
`their specificity and binding avidity.
`It was well known that heterosera against xenogeneic immunoglobulins largely
`reacted with the constant region or Fe portion of the molecule, and the first approach
`at "humanization" was therefore the genetic construction of chimeric antibodies,
`comprising the murine variable region and the human constant region of lgG (12).
`Most chimeric antibodies displayed much reduced immunogenecity, but a response to
`the murine Fv portion could ultimately be observed. In reshaped or CDR-grafted
`antibodies, the murine content was further reduced by grafting the murine comple(cid:173)
`mentary determining regions (CDRs) or hypervariable region onto a human variable
`region framework (13). These antibodies were generally found not to be immuno(cid:173)
`geneic, but it was often difficult to maintain the binding affinity of the parent murine
`antibodies. Further amino acid changes in the framework region are generally necessary
`to maintain the original conformations of the CDRs. These changes need to be deduced
`for each antibody through computer model building, and the ultimate success-pres(cid:173)
`ervation of full binding-is often difficult to achieve even with extensive changes that
`potentially negate the advantage of CDR grafting over chimerization. In the newest
`approach, called variable domain resurfacing (14), the affmity is maintained by retain(cid:173)
`ing the CDRs and the core of the murine variable region framework. Only the surface
`residues of the murine variable region framework are replaced by those from a human
`variable region. A simple algorithm predicts the necessary changes in the framework
`region, and when this method was applied to two murine antibodies, their affinities
`were unaffected (14). This approach assumes that the immunogenecity of murine
`antibody variable regions is determined by the accessible surface residues only, an
`assumption not yet tested with globulins, but generally accepted for the antigenecity
`of proteins (15,16).
`
`3. RADIOIMMUNOCONJUGATES
`Ever since the appreciation of the cytocidal effects of high doses of radiation, oncol(cid:173)
`ogists have attempted to harness the energy of radioactivity to eradicate tumors in
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`patients afflicted with cancer. The goal of radiotherapy is to deliver a sufficiently high
`dose of radiation locally to the tumor in order to sterilize the tumor without causing
`lethal damage to the surrounding tissues. Successful killing of all tumor cells requires
`radiation doses of at least 60 gy to be concentrated at the tumor site, which is at the
`limit of the dose that can be delivered by external beam radiation while sparing normal
`tissue. Unfortunately, the wide application of external beam radiotherapy, while
`improving survival, has rarely resulted in cure. The notion that the ability of oncolo(cid:173)
`gists to eradicate tumors could be improved by in vivo administration of a radio(cid:173)
`nuclide was first developed using iodine-131 to treat thyroid carcinomas, which con(cid:173)
`centrate radioiodine from blood resulting in delivery of local tumoricidal doses of
`80-300 gy (17).
`Radioimmunoconjugate therapy, which exploits the availability of specific anti(cid:173)
`bodies that can localize to tumor cells, has been under investigation for a number of
`years as one way of improving radiotherapy. The hope of radioimmunoconjugate
`therapy is that targeting of radioactivity by antibodies could overcome two drawbacks
`of external beam radiotherapy: (1) specific targeting by radio labeled antibodies should
`allow more precise delivery of the radiation dose to the tumor with concomitant sparing
`of a greater amount of the surrounding normal tissue; and (2) radiolabeled antibody
`will deliver a radiation dose to small undetected areas of tumor or micrometastases.
`Radionuclides that are useful for radioimmunoconjugate therapy must emit particles
`whose energy can be deposited locally, ideally within a radius that encompasses one
`or a few cells. Furthermore, such radionuclides should have relatively short half-lives,
`so that radioactivity incorporated into the patient decays within a reasonable period
`of time, and in addition, they should be isotopes of elements whose chemistry allows
`them to be readily conjugated to antibodies. Several radioisotopes that may meet
`these criteria and that have been used in trials of radioimmunoconjugate therapy are
`shown in Table 1.
`Chemically, the radioisotopes shown in Table 1 comprise two groups, the radio(cid:173)
`metals and radioactive isotopes of iodine. Iodine (and astatine) is generally conju(cid:173)
`gated directly to tyrosine residues in antibodies simply by mixing the protein with
`sodium iodide in the presence of an oxidizing agent, such as Chloramine T or related
`compounds (20). The reaction is extremely rapid, even at 0°C, although one must
`take care to avoid damage to the antibody by excessive oxidation. Alternatively,
`radioiodine can be conjugated to antibodies using iodinated compounds that allow
`labeling without exposing the protein to oxidative conditions, and furthermore, allow
`the possibility of utilizing iodinated compounds that are not subject to enzymatically
`catalyzed dehalogenation (21-23).
`The radioactive metals are conjugated to antibodies by the use of chelating agents
`that are in turn chemically linked to the protein. Although the early chelates have
`high stability constants, they are kinetically labile, and in vivo, the radiometal readily
`exchanges into metal-transport proteins, such as transferrin, thereby losing any target
`specificity. Once lost from a conjugated chelate, a radiometal, such a yttrium-90, can
`ultimately be deposited in bone, resulting in prolonged irradiation of bone marrow.
`Recently, chelating agents that "cage" the metal and are far more stable have been
`developed for diagnostic and therapeutic applications with antibodies (24,25). Figure
`2 illustrates the structure of two such antibody-conjugated macrocyclic chelators,
`which are ideal reagents for binding copper-67 and yttrium-90. In vivo studies show
`that radiometals targeted by antibodies linked to caged chelating agents have greatly
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`Table 1
`Radionuclides with Potential for Radioimmunotherapya
`Particle
`energy,
`maximum
`energy, MeV
`
`Path length,
`mmb
`
`Radioisotope Half-life
`7.2 h
`Astatine-211
`Bismuth-212
`1 h
`Copper-67
`2.4 d
`
`Decay particle
`
`Q
`
`Q
`{3
`
`Iodine-125
`
`60.1 d
`
`Auger electron
`(electron capture)
`
`5.9
`6.1
`0.57
`
`7.5
`
`0.81
`
`Iodine-131
`
`8.1 d
`
`Rhenium-186
`
`Rhenium-188
`
`3.5 d
`3.7 d
`17 h
`
`Yttrium-90
`
`2.5 d
`
`{3
`
`{3
`
`{3
`
`1.1 and 0.93
`
`2.1
`
`2.7
`
`Comments
`Iodine chemistry
`
`0.04-0.08
`0.04-0.08
`0.6
`
`-y-Emission for
`imaging
`0.001-0.02 Requires
`internalization
`for cytocidal
`effect
`High-energy
`-y-emission for
`imaging
`-y-Emission for
`imaging
`-y-Emission for
`imaging
`
`0.8
`
`1.8
`
`4.4
`
`5.3
`
`°Compiled from published data (6,18,19).
`b The path length is defined as the radius of a sphere within which 90"7o of the energy emitted by a
`radionuclide is absorbed (19).
`
`improved tumor localization of the radioactivity, with less deposition into bone and
`less marrow toxicity (26).
`The 13-emitters, yttrium-90 and iodine-131, have been the radioisotopes used most
`extensively in therapeutic studies to date, more because of their ready availability
`than because they have the most ideal characteristics for therapy (27,28). Iodine-131
`is a medium-range {3-emitter whose energy is absorbed within one or two cell diameters,
`whereas the more energetic {3-particle of yttrium-90 can penetrate several cell diameters.
`This is the basis for the theoretical benefit of using radionuclides as the effector killing
`moiety for antibody-directed therapy, namely that the antigen targeted by the antibody
`need not be expressed on all of the tumor cells in a tumor mass. Thus, antigen-negative
`tumor cells may also be killed by the radiation concentrated at the tumor by antigen(cid:173)
`positive tumor cells (a "bystander" killing effect). The a-emitters may not share this
`potential advantage because of the extremely short range of a-particles. However, this
`property could be an advantage when targeting an antigen expressed homogeneously
`on all tumor cells and that is internalized by the cells, in that a higher proportion of
`the energy of the radiation is deposited in the target cell. Unfortunately, the two a(cid:173)
`emitters with appropriate chemical properties for conjugation, bismuth-212 (29) and
`astatine-211 (30), have very short half-lives, which may reduce their effectiveness in
`vivo (27), and which presents logistical difficulties in their use.
`The fate of the antigen/antibody complex on the surface of the tumor cell will
`influence the best choice of radioisotope or method of linking it to the antibody.
`Radioiodine is retained better in tumor tissues if it is targeted by an antibody that is
`not internalized. Otherwise, on internalization, radioimmunoconjugates are enzy(cid:173)
`matically degraded and dehalogenated with the consequence that the radioactivity
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`Conjugate Structure
`
`Macrocycle Metal Ion
`
`(')
`::r
`J:ll
`~ .....
`
`--I
`-..
`
`I'D .. ......
`...... s
`~
`==' 0
`(')
`0
`.!:!.
`t::
`
`OQ a I'D en
`
`Cu2+
`
`< r--\ >
`/~'(V"•'lf""-o-c..,-C: :) TET A
`
`coo· coo·
`
`(LJ)
`coo· coo·
`
`NH
`+ 2
`
`0
`
`{oo· coo·
`
`H
`
`,-N....../"../'.
`
`n - s']( ~ctt,
`~H2
`
`Nr--\ )
`NH __J=\_ _(
`N
`)
`( \_ ) \
`coo· coo·
`
`0
`
`DOTA
`
`y3+
`
`Fig. 2. Structural formula of conjugated macrocyclic chelators for copper and yttrium ions.
`
`~
`--I
`--I
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`rapidly diffuses away and is cleared (6,18). Internalizing antibodies are better suited
`for targeting radiometals, such as yttrium-90 and copper-67, which are retained by the
`target cells on internalization and eventual degradation of the radioimmunoconjugate
`(6), since cellular proteins are generally good chelators of transition metals. Antibodies
`that target antigens that readily internalize are necessary for targeting iodine-125 whose
`decay produces Auger electrons of extremely short path length requiring proximity to
`the nucleus to elicit cell killing. Iodine-125 is therefore best conjugated via nonmetab(cid:173)
`olizable adducts (21-23).
`There has been some debate about what are the most desirable properties for the
`antibody component of a radioimmunoconjugate, given a high specificity for an anti(cid:173)
`gen selectively expressed on tumor tissue. In contrast to diagnostic uses of radio(cid:173)
`immunoconjugates, where the most important parameters are {1) a high ratio of radio(cid:173)
`isotope delivered to the tumor compared with that delivered to normal tissue and (2)
`rapid clearance of radioisotope from the blood pool, which otherwise masks the radio(cid:173)
`activity concentrated at the tumor (31), the most important factor for the radiothera(cid:173)
`peutic is the total amount of radioisotope delivered to the tumor and its residence
`time in the tumor (i.e., dose deposited at the tumor), provided toxicity to normal
`tissues is tolerated. Although intact IgG penetrates from blood vessels more slowly than
`Fab or genetically engineered antibody fragments, most studies show that a greater
`dose of radioactivity is deposited at tumor sites when using radioimmunoconjugates
`containing intact IgG, suggesting that its slower clearance from blood, and the pos(cid:173)
`sibility for bivalent binding to target cells, are the most important parameters for a
`therapeutic application. Most clinical experience to date has been with mouse IgG in
`radioimmunoconjugates, which means that the generation of HAMA has been a factor
`that may limit the ability of patients to receive multiple doses of conjugate. The advent
`of humanized antibodies may overcome this limitation. Genetic engineering can also
`be used to make small fragments of humanized antibodies where the single binding
`domain can have very high affinity, and may, therefore, both penetrate into tumor
`tissue quickly and be well retained by the tumor, thereby increasing the dose delivered
`to the tumor.
`What is the clinical experience in the evaluation of radioimmunoconjugate therapy
`in clinical trials? Can a sufficiently high dose of radiation be delivered to tumor in
`vivo to kill enough tumor cells to effect a therapeutic response? The clinical studies to
`date can be divided into two general groups, those treating tumors that are particu(cid:173)
`larly radiosensitive, such as lymphomas and leukemias (6), and those treating solid
`tumors {18).
`Clinical evaluation of radioimmunoconjugate therapy for non-Hodgkin's lym(cid:173)
`phoma (NHL) has been facilitated by the availability of a variety of B-cell-specific
`MAb, such as anti-idiotype antibodies, LYM-1 (anti-HLA-DR), anti-B1 (anti-CD20),
`MB-1 (anti-CD37), and OKB7 (anti-CD21) (6,32-34). These antibodies have been
`coupled to iodine-131 and have been used in cumulative doses of up to 750 mCi/pa(cid:173)
`tient. These large doses are well tolerated with the important exception of severe myelo(cid:173)
`suppression. Even though this severe side effect can be ameliorated by fractionating
`the dose of radiotherapeutic into multiple smaller doses given over several weeks, it
`would appear that the best clinical results are obtained in those trials that employ
`massive myeloablative doses of the radioimmunotherapeutic (6). In the studies from
`the Fred Hutchinson Cancer Center in Seattle, Washington, 16 of 19 patients who
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`were administered therapeutic doses achieved a complete remission (34). However,
`the cost of this therapeutic benefit is that 15 of the patients required an autologous
`bone marrow transplant (ABMT). These investigators suggest that the only possibility
`for complete eradication of the tumor is to use massive doses of radioimmunotherapy
`that are so high as to require bone marrow transplant support. It remains to be deter(cid:173)
`mined whether the therapeutic benefit of systemic delivery of massive doses of radia(cid:173)
`tion with ABMT support is superior to other protocols utilizing chemotherapy and
`total body irradiation (external beam) as ablative regimens for ABMT protocols in
`the treatment of patients with relapsed lymphoma.
`Other leukemias and lymphomas that have been targeted in trials of radioimmuno(cid:173)
`therapy are acute myelogenous leukemia (AML), T-cell malignancies, and Hodgkin's
`disease (6, and references therein). Iodine-131-labeled anti-CD33 and anti-CD45 anti(cid:173)
`bodies have been used to target AML (35), whereas patients with chronic lymphocytic
`leukemia (CLL) or cutaneous T -cell lymphoma have been treated with anti-CD5
`labeled with iodine-131 or yttrium-90, and those with adult T-cellleukemia have been
`treated with anti-CD25 (IL-2 receptor) conjugated with yttrium-90 (6). The most
`promising responses in these studies were also achieved at dose levels that caused severe
`myelosuppression as the major side effect (6).
`In studies where Hodgkin's disease was treated with antiferritin antibodies coupled
`to yttrium-90 (the tumor cells are rich in ferritin), impressive response rates have been
`reported (36), although again at doses that were also myeloablative so that 17 of 37 pa(cid:173)
`tients required ABMT rescue (3 patients died of bone marrow aplasia). The yttrium-90
`was conjugated to antibody using diethylenetriamine penta-acetic acid as the chelator,
`from which yttrium-90 is known to escape in vivo to be taken up by bone, which thus
`contributes to hematopoietic toxicity (26). It may be that the ferritin-rich tumor can
`also take up the released radiometal by chelation, which may contribute to the thera(cid:173)
`peutic efficacy of this conjugate.
`The clinical experience with the treatment of solid tumors by radioimmunoconjugate
`therapy has generally been disappointing (18). Indeed, if optimal therapeutic effects
`in relatively radiosensitive neoplasms, such as NHL, can only be achieved at doses of
`radioimmunoconjugate that are myeloablative, then it is unlikely that therapeutic
`efficacy in solid tumors can be achieved at doses that are not also myeloablative. Fur(cid:173)
`thermore, the highest doses delivered via radioimmunoconjugates are usually esti(cid:173)
`mated to be in the range of 10-20 gy, although it is generally accepted that doses of at
`least 60 gy are needed to eradicate solid tumors (27,37). A Phase II clinical trial of
`radioimmunotherapy with iodine-131-labeled CC49 antibody in colorectal cancer
`exemplifies the lack of therapeutic efficacy in the treatment of solid tumors. Despite
`an antibody of relatively high affinity for the target tumor-associated glycoprotein 72,
`no tumor responses were observed, and the doses delivered to the tumor were only in
`the range of 0.2-6. 7 gy (38). Recent Phase I studies with iodine-131-labeled A33 anti(cid:173)
`body were similarly disappointing (39). A Phase II trial of the CC49 radioimmuno(cid:173)
`conjugate in metastatic prostate cancer also failed to demonstrate any efficacy with
`maximal tumor doses estimated in the range of only 2-10 gy (40).
`One approach to increasing the dose delivered to the tumor, while maintaining the
`total body dose at tolerable levels, is to treat locally tumors that are confined to par(cid:173)
`ticular body cavities. Intralesional radioimmunotherapy of malignant glioma may offer
`one compartmentalized setting where cytocidal doses of radiation may be delivered to
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`380
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`Part II I Newer Strategies and Targets
`
`the tumor without significant toxicity to bone marrow, liver, or kidney. Treatment
`of 17 patients with iodine-131-labeled antitenascin antibodies resulted in 3 partial
`responses and 3 complete remissions (41). Intraperitoneal infusion of yttrium-90-
`labeled HMFG1 antibody was given to 52 patients with ovarian cancer (42). The results
`were encouraging, with 19 of 21 patients that were regarded as receiving treatment in
`an adjuvant setting still alive (median followup, 35 mo). However, even in this intra(cid:173)
`compartment setting, the authors doubt that the therapeutic effect was due to a cyto(cid:173)
`cidal effect of the radiation dose, and suspect that the HMFG1 murine antibody in(cid:173)
`duced immunological reactivity against the tumor, an observation also noted by
`others when treating breast cancer with iodine-131-labeled L6 antibody (43).
`What are the future prospects for radioimmunotherapy? Several investigators are
`beginning to think of this modality as a complement to conventional external beam
`radiotherapy. For example, several patients with AML achieved complete remission
`when given iodine-131-labeled antibody together with 12 gy of external beam irradia(cid:173)
`tion and cytoxan (6,44). A similar approach may be appropriate in treatment of certain
`solid tumors in order to achieve a sterilizing total dose of radiation at the tumor (45).
`The early results in the use of radioimmunotherapy for treating relapsed leukemias
`and lymphomas have been encouraging, although the therapy is far from optimized
`and may generally require concomitant ABMT. In particular, the optimal radionu(cid:173)
`clide and method of linkage to antibody still need to be defined (27,28), and human(cid:173)
`ized antibodies need to be tested in the clinic to overcome the limitations on multiple(cid:173)
`course therapy imposed by the generation of HAMA (35). Nevertheless, even with
`these improvements, it may be that the long-term prospects for radioimmunotherapy
`may be confmed to treating radiosensitive tumors utilizing myeloablative doses together
`with bone marrow rescue, or as an adjunct to external beam irradiation, owing to the
`intrinsic limitations of radiolabeled antibodies to deliver a sterilizing dose of radiation
`to tumor (46). Radioimmunotherapy will likely remain confmed to specialized clinical
`centers with facilities for performing ABMT and for coping with issues, such as radia(cid:173)
`tion exposure of medical staff and handling radioactive waste, which are problematic
`with systemic administration of radioactivity.
`
`4. IMMUNOTOXINS
`The limited expression of antigens suitable as targets for immunoconjugates on the
`surface of tumor cells_ (in general 104-10' and very rarely more than 106 antigens/ cell)
`coupled with the pharmacodynamics of large molecules, such as ')'-immunoglobulins,
`compelled scientists to search for the most potent cytotoxic agents to be used as effec(cid:173)
`tors in immunoconjugates. Known protein toxins from plants, such as ricin, abrin,
`volkensin, and viscumin, and from bacteria, such as diphtheria toxin and pseudomonas
`exotoxin A, fit into this category. This spurred research into a better understanding of
`the mechanism by which these toxins destroy cells, so as to be able to harness their
`deadly power for the selective killing of tumor cells.
`The above-listed toxins kill cells by catalytically inactivating cellular protein synthe(cid:173)
`sis. The plant toxins, also called ribosome-inactivating proteins (RIPs) are N-glyco(cid:173)
`sidases that remove the adenine base of residue 4324 of the 28S ribosomal RNA of the
`60S subunit of eukaryotic cells (47). The bacterial toxins use NAD+ to ADP-ribosylate
`elongation factor 2 (48). Because the final targets for the toxic action are cytoplasmic,
`the process of intoxication involves, therefore, at least three functions:
`
`IMMUNOGEN 2006, pg