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`PHIGENIX
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`[CANCER RESEARCH 54. 5301—5309. October 15. 1994]
`
`Review
`ion
`Wted
`:ell
`
`Monoclonal Antibodies as Agomsts: An Expanded Role for
`Their Use in Cancer Therapy1
`
`Ellen S. Vitetta2 and Jonathan W. Uhr
`
`Cancer Immunobt'ology Center and Department of Microbiology. University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 7523549576
`Introduction
`
`er.
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`on
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`I'11
`8:
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`ct
`r-
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`In
`C
`d
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`The development of technologies to generate MAbs3 (1) created
`considerable excitement in oncology because of their potential use for
`tumor therapy. The initial rationale was to harness the exquisite
`specificity of antibodies to bind to tumor-specific antigens and,
`thereby, to kill tumor cells by means of conventional effector mech—
`anisms that have been perfected during the long evolution of the
`mammalian immune system, e.g., opsonization, ADCC, or comple-
`ment-mediated lysis (2, 3). When MAbs were evaluated for cross-
`reactivity with normal tissues, however, it became apparent that the
`majority of tumor—associated antigens were not tumor specific (4),
`thus creating an apparent obstacle to the above strategy. However, the
`density of such antigens (e.g., interleukin receptors and carbohydrate
`moieties peculiar to tumors) was often increased on cells from par-
`ticular tumors (5—7),
`thereby providing an operational window of
`specificity. In addition, lineage-specific antigens can serve as targets,
`provided that stem cells in the lineage are antigen~negative and,
`hence, able to reconstitute the cellular compartment or the tissue
`involved. Subsequent to selection for MAbs of suitable specificity. the
`isotypes of such MAbs were then selected to maximize effector
`functions such as ADCC (8).
`Despite this initial intellectual appeal, the general therapeutic effi-
`cacy of tumor-reactive MAbs has been disappointing. In particular,
`the results of clinical studies in patients with solid tumors showed
`little efficacy (9—13), except in the setting of minimal disease (14).
`This relates in part to the fact that patients in Phase I trials usually
`have large tumors with poor access to circulating MAb. In addition,
`the above criteria for selecting MAbs may not have been optimal. as
`will be discussed in this article.
`
`In contrast to results with carcinomas, significant success has been
`reported in treating NHL and T-cell leukemias with tumor-reactive
`antibodies. Levy and Miller (15) and Hamblin er a]. (16) have used
`anti-idiotope MAbs to treat NHL and chronic lymphocytic leukemia,
`respectively. In the majority of cases of NHL, there have been partial
`or complete remissions using single anti-idiotopic antibodies. Re-
`lapses frequently indicated the emergence of idiotope-negative vari-
`ants (17, 18). Dyer et al. (2) have also obtained impressive anti-tumor
`effects in NHL with anti-CD52. Finally, anti-CD25 has shown some
`efficacy in the treatment of human T-cell lymphotrophic virus l-in-
`duced adult T-cell
`leukemia (19). These results have demonstrated
`both the effectiveness of some antibodies in eliminating neoplastic
`
`Received 6/14/94; accepted 8/15/94.
`The costs of publication of this article were defrayed in pan by the payment of page
`charges. This aniclc must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`' This study was supported by NIH Grants CA-28149 and CA-4IOS7.
`2 To whom requests for reprints should be addressed, at the Cancer lmmunobiology
`Center and Department of Microbiology, 6000 Harry Hines Boulevard. Dallas, Texas
`75235-8576.
`
`3 The abbreviations used are: MAb, monoclonal antibody; ADCC, antibody-dependent
`cellular cytotoxicity; NHL, non-Hodgkin's lymphoma; CCA, cell cycle arrest; DLC,
`dormant lymphoma cell; IT, immunotoxin; erbB-ZR, erbB-2 receptor; lL, interleukin.
`
`There is considerable experimental and some clinical evidence to
`indicate that the effector functions of MAbs can play a major role in
`tumor immunity. This issue has been studied by two approaches: (a)
`the use of class switch variants of tumor-reactive antibodies in hu-
`mans, human tumor/nude mouse models, or murine tumor models as
`well as in in vitro cytotoxic assays; and (b) analysis of the effects of
`different human immunoglobulin isotypes involving large panels of
`tumor-reactive antibodies, both for their experimental in viva antitu-
`mor effects and their cytotoxic effects in vitro. Both approaches have
`led to the same conclusion, i.e.. that in vivo, opsonization and ADCC
`can play critical roles in antitumor activity (20, 21) and that murine
`lgGZa (8, 22, 23) is by far the most effective isotype. The effective-
`ness of lgGZa correlates with its capacity to interact with host effector
`cells. Similar results were obtained in mice using switch variants of
`tumor-reactive antibodies (24),
`i.e.,
`lgGZa was the most effective.
`However, all isotypes showed some antitumor activity. Although in
`vitro assays indicated that the ability of MAbs to bind complement
`was important (25), no evidence was provided to indicate that this
`effector function operated in vivo. Indeed, complement depletion of
`Nude mice bearing human xenografts did not affect the antitumor
`function of lgGZa MAbs (8). Macrophages were thought to play an
`important role in the antitumor effects of lgGZa because agents that
`damaged macrophages abolished the tumoricidal effect of the MAb
`(8). In the studies by Dyer et a1. (2), rat or humanized MAb specific
`to CD52 was used to treat NHL. By studying a switch variant of the
`rat MAb, they showed that lgGZb was far superior to lgGZa in treating
`NHL in a study in which two patients were each treated with one of
`the antibody isotypes. Both MAbs were able to remove peripheral
`neoplastic lymphocytes, but only the lgGZb antibody produced a
`long-lasting depletion of
`lymphocytes from blood and tissues,
`whereas the lgGZa tumor cells rebounded rapidly. Additional clinical
`observations support the role of lgGZb in killing lymphoma cells (2).
`ln extensive experimental studies, rat lgGZb was more effective than
`5301
`
`8- and T—cells and the problems associated with the generation of
`antigen-negative variants.
`Despite the results of clinical studies in patients with epithelial
`tumors and the above mentioned limitations of MAb therapy, we
`believe that the potential of MAbs as therapeutic agents has not been
`thoroughly explored. The purpose of this review is to reevaluate the
`prevalent concept that the major antitumor effects of these antibodies
`are due to the harnessing of conventional effector mechanisms of the
`host. We will review evidence supporting an alternative interpretation,
`i.e., that antibodies directed against cell surface molecules on many
`types of tumor cells can act as ligands, resulting in powerful antitumor
`effects mediated by signal
`transduction. If MAbs are selected by
`virtue of this characteristic, they may serve as important adjuncts to
`conventional chemotherapy. We will use 8— and T~cell tumors as the
`major example but will also discuss breast carcinoma.
`
`Role of Effector Functions of MAbs
`
`THIS MATERIAL MAY BE PROTECTED
`
`BY COPYRIGHT b‘iW (17 US. (2095)
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`PHIGENIX
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`MONOCLONAL ANTIBODIES AS AGONISTS
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`other isotypes in killing lymphoid cells in vivo or mediating cellular
`cytotoxicity (ADCC) in vitro (20). With regard to human MAbs, lgGl
`and igGB are most effective in inducing ADCC (26), but lgG4 from
`some patients can demonstrate such function in vitro (27).
`in summary, there is evidence that antibody-induced opsonization
`and cellular cytotoxicity can induce antitumor effects, but their im-
`portance in antibody-induced tumor immunity is not clear. Certain
`immunoglobulin isotypes, depending upon the species of origin and
`the host, appear to be most efficacious with regard to these effector
`functions. There are additional issues that remain unsolved. Thus, the
`relative contributions of ADCC, opsonization, and cytostasis to these
`antitumor effects are not known. importantly, there appears to be
`variation among individuals in regards to the role that the different
`isotypes can play in MAb-induced tumor immunity (27).
`indeed,
`MAbs display isotypic polymorphisms in the human that may account
`for these differences.“ if so,
`these immunoglobulin isotypes with
`minimal amino acid differences may provide critical clues to the
`structural motifs involved in ADCC and other effector functions.
`
`
`
`Ca“
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`O
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`-CaMkinase 11
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`Signaling Functions of Antibodies
`
`>"P_.
`
`Ets-‘l
`
`NFkB
`
`The immunoglobulin Receptor Complex on B Cells (Fig. l). B
`cells usually express cell surface lgM and lgD with identical variable
`regions (28). By themselves, the heavy chains of these molecules are
`incapable of signaling because of very short cytoplasmic tails (29. 30).
`However, each H chain is associated with an lgor and lgB (or lgy)
`molecule (31, 32) that contains structural motifs that can bind mem-
`bers of the src-famiiy protein tyrosine kinases, e.g.. Lyn (33). Fyn.
`Bik (34—36), Lck (36), and SYK (37—39). After cross-linking lgM or
`lgD on mature B-celis, protein tyrosine kinases become enzymat-
`ically active, resulting in the phosphorylation of [go and lgB, the
`kinases themselves, and several other intracellular protein targets
`(32, 40—43). This initiates a cascade of biochemical events along
`two major pathways, resulting in cellular proliferation and/or dif-
`ferentiation (41, 44—46). These include phosphoinositide metabo-
`lism with activation of PLCy and generation of ins P3 and diacyl—
`glycerol
`that
`result
`in elevations
`in
`intracellular Ca2+ and
`activation of protein kinase C (47). Protein kinase C (a serine/
`threonine kinase) then regulates transcriptional activity of the AP-]
`complex (48). A second pathway involves activation of Ras and
`another series of intermediate messengers (GAP, Grb2, Raf,
`MAPKK, and MAPK; Refs. 49—51), resulting in phosphorylation
`of c-Jun, a transcriptional regulator (52). Other possible pathways
`include phosphatidylinositol 3-kinase (42, 53—55), which may act
`downstream on NF-KB (56) and the recently described Jak proteins
`involved in signaling by cytokine receptors (57).
`in summary, the major pattern for B-ceii receptor signaling is that
`cross-linking of membrane immunoglobulin stimulates activation of
`protein tyrosine kinases as proximal events which, via a series of
`intermediates, stimulate serine/threonine kinases, which in turn regu-
`late gene transcription (distal events). However, the precise sequence
`of reactivities, the relationships between the components of this com—
`plex signaling cascade, and their regulation are not yet well defined.
`The lg signaling complex is not limited to membrane immunoglob-
`ulin, lga, and lgB (Fig. 1). There are additional molecules on the
`B-cell surface, e.g., CD19 (58—60), CD20 (60—62), CD21 (58),
`CD22 (63), CD24 (44), CD32 (44), CD45 (64), leu13 (65), and CD81
`(TAPA-l; Refs. 44, 46, 66, and 67), which can interact with or affect
`signaling by the immunoglobulin complex. Interactions with these
`molecules (with the exception of CD32; Refs. 68 and 69) usually
`enhance the
`immunoglobulin-mediated signals that
`lead to the
`
`‘ H. Waldmann. personal communication.
`
`Fig. 1. Model of negative signaling in B-cells by cross—linking lgM [adapted from
`Cambier er
`(II.
`(157) and Fearon or al.
`(158)]. This model
`illustrates some of the
`components of the membrane lgM signaling cascade that may be involved in the induction
`of cell cycle arrest and/or apoptosis. The model shows surface lgM cross-linked by an lgG
`anti—u chain antibody. The igu and igB (or lgy) chains associate with the ji chains. For
`simplicity. a number of molecules have been depicted as part of the lg signaling complex.
`although in some cases. the evidence is incomplete. The small number of arrows versus
`the large number of intracellular second messengers reflects a lack of understanding of the
`precise interactions and sequence of events that
`take place after membrane lgM is -
`cross-linked.
`
`activation of normal B-cells. in addition, antibodies directed against
`these molecules can induce signals in normal B-cells in the absence of
`lgM cross-linking (66, 70—72). The outcome of signaling after the
`cross-linking of surface immunoglobulin or the other molecules in the
`receptor complex depends upon the stage of differentiation of the
`B-lymphocyte. Mature B-cells proliferate and differentiate into im-
`munoglobulin-secreting and memory cells; immature B-ceils and cer-
`tain B-cell lymphomas undergo CCA or apoptosis (73—79), as will be
`discussed.
`I
`Negative Signaling in Neoplastic B-Celis. The earliest studies in
`vivo indicating that antibodies against surface immunoglobulins on
`B-cells had antitumor effects were those of Lynch et a1. (80) who
`showed that immunization of mice with myeloma proteins induced an
`antitumor response following challenge with myeloma cells. This
`response was attributed to T-ceiis, which were thought to suppress
`tumor cell growth. Krolick et al. (81) described the induction of a
`dormant tumor state in mice bearing an aggressive lymphoma (BCL,)
`if mice with advanced tumor were treated with irradiation, spienec-
`tomy, and an anti-id or anti-5 immunotoxin. Although treated mice
`appeared clinically normal for 1 year following treatment, after sac-
`rifice, many of their tissues were able to transfer tumor to naive
`recipients. The antitumor effect in the donor mice was presumed to be
`an anti-id response to the initially massive tumor.
`in this regard,
`George et al. (82) and Stevenson er a1. (83) showed that immunization
`with the monoclonal BCLl immunoglobulin induced an anti-idiotypic
`response that led to a state of dormancy in mice challenged with the
`BCLl
`tumor cells.
`injection of anti-idiotype-containing serum into
`naive mice that were challenged with BCL1 tumor cells resulted in a
`state of dormancy in some of the recipients (82, 83). We have used
`this model to demonstrate that the major outcome of such immuniza-
`tion in over 500 mice is a dormant state and that such mice carry
`DLCs for as long as 2 years (79). Dormancy was also induced in the
`5302
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`MONOCLONAL ANTIBODIES AS AGONISTS
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`-
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`induce CCA depended both on their affinity and the epitope on the
`CD19 molecule which they recognized (71). This is an important
`point operationally because it means that,
`to determine signaling
`potential, 3 panel of MAbs against a given molecule should be studied
`to have a reasonable chance of detecting one that reacts with the
`appropriate epitope and has the necessary affinity to deliver a signal.
`In contrast to anti-CD19, treatment of Daudi cells in vitra with anti-p.
`induced both CCA and apoptosis (78).
`Taken together with other findings, these observations raise the
`possibility that CCA and apoptosis may involve two distinct signaling
`pathways in neoplastic B-cells. In this regard, anti-sense lyn experi-
`ments in Daudi cells treated with anti-CD19 or anti-p. were carried out
`to determine if src-family kinases are critical for inducing CCA and/or
`apoptosis. The selection of anti-sense-lyn was based on previous
`findings that lyn is associated with CD19 (59) as well as membrane
`immunoglobulin (33). Pretreatment with anti-sense lyn before the
`addition of anti-CD19 or anti—u completely prevented the induction of
`CCA by the cross-linking of CD19 or lgM. In contrast, induction of
`apoptosis by anti-p. was not inhibited (78). These results are similar to
`those obtained with a cell line (3B3) derived from the mouse BCL1
`tumor cells (78). These results suggest that there are two signaling
`pathways, a CCA-pathway that
`is lyn-dependent and an apoptosis
`pathway that is lyn-independent. Anti-sense studies by Yao and Scott
`(91) suggest that Blk may be critical for inducing apoptosis.
`Additional evidence suggests but does not prove that other B-cell-
`reactive antibodies can deliver negative signals to tumor cells. In the
`report of a recent clinical trial conducted by Kaminski er a1. (95),
`anti-CD20, coupled to well-tolerated amounts of 131I, was adminis-
`tered to patients with NHL. Durable remissions were achieved in a
`significant number of patients with few side effects, and the marrow
`was unaffected.
`In a human SClD/B-lymphoma model,
`the cold
`anti-CD20 MAb was a more potent antitumor agent
`than its 13"I-
`conjugate (96). Hence, the antibody itself may have played a major
`role in the antitumor activity observed clinically, although the mech-
`anisms are unclear (97). The above interpretation is consistent with an
`earlier study which showed that unlabeled anti-CD20 administered to
`patients with NHL induced dose-dependent tumor regressions with a
`partial response (over 50% tumor reduction) in a patient receiving the
`largest amount of antibody (1 g; Ref. 98). Anti-TAPA-l (CD81) can
`also induce a reversible antiproliferative effect on a human lymphoma
`cell line (66, 99). There is also evidence to suggest that anti-CD21,
`anti—CD23, and anti-CD24 can down-regulate the growth of Epstein-
`Barr virus-positive tumors in SCID mice (100) and humans (101). As
`mentioned before,
`these molecules are part of the immunoglobulin
`signaling complex (44, 46, 58—62, 67, 102—106);
`therefore,
`it
`is
`possible that their antitumor effects result from signal transduction. To
`distinguish between effector function and signaling, however, exper-
`iments comparing lgG antibody and its l-‘(ab’)2 fragments in vivo will
`be necessary.
`
`Effector Functions versus Signaling
`
`vast majority of ASCID mice receiving antibodies directed against
`epitopes on the immunoglobulin molecule of the BCLl tumor cells,
`proving that antibody can induce dormancy (79). Although insuffi-
`cient by themselves, Id-immune T—cells could enhance the induction
`
`and the duration of dormancy induced by antibody.5 Thus, just as in
`humans with B-cell lymphoma (15, 16), anti-Id can be highly effec-
`tive as an antitumor agent in mice with the BCLl lymphoma. Mul—
`tiparameter flow cytometry was used to isolate and characterize the
`DLCs. The DLCs were physiologically different from BCLI cells
`growing in naive mice;
`they were smaller in size, appeared less
`“malignant” morphologically, had a different profile of oncogene
`expression and a proportion of these cells were in CCA (79). Since the
`size of the population of DLC was stable for many months, it was
`presumed that cell death was balancing residual cell replication.
`There is a large body of in vitro evidence to support the notion that the
`in viva results may depend heavily on signal transduction mechanisms.
`Indeed,
`it has been shown that there is a correlation between clinical
`responses to anti-Id and the capacity'of anti-Id to induce phosphorylation
`of proteins in tumor cells freshly prepared from patients with NHL (84).
`It is known that the cross—linking of lgM on many but not all murine
`lymphoma cells can result in growth arrest in G1 followed by apoptosis
`(40, 41, 46, 73, 74, 76, 77, 85—87). Both phosphorylation of tyrosine
`residues on the src-like kinases and phosphoinositide hydrolysis (47) are
`important components. It has been postulated that the above events reflect
`a physiological mechanism underlying tolerance to self by which normal
`immature B-cells undergo clonal anergy or deletion following interaction
`with self antigens (88, 89). The generation of CCA can also be induced
`in mature B-cells when surface lgM is cross-linked in the absence of a
`T—cell signal (89). However, in some B-lymphomas, cross-linking does
`not appear to signal negatively (90, 91), either because the cells represent
`a more advanced stage of maturation or signaling can no longer override
`the uncontrolled growth signals inherent in these particular tumor cells.
`Other Molecules in the lg-Signaling Complex. CD19 is part of
`the multimolecular immunoglobulin receptor complex on the B-cell
`and can associate with membrane immunoglobulin, leul3, CD81, and
`CD21 (58, 66). Although CD19 associates with membrane immuno-
`globulin, signaling through CD19 is apparently distinct, since it dis-
`plays differences in Ca2+ flux, PIP2 turnover kinetics, phosphoprotein
`patterns,
`involvement of protein kinase C (92), and apoptotic re-
`sponses (71). The importance of CD19 signaling in the antitumor
`activity of anti-CD19 antibody was suggested by experimental studies
`on the efficacy of an CD19-ricin A chain IT in SCID mice with Daudi
`cell xenografts (93). It was demonstrated that the anti-CD19 antibody
`(HD37) alone was as effective as its respective IT in inhibiting growth
`of several human B-lymphomas in SCID mice. The inhibition was
`immunologically specific because isotype-matched control lgGl or an
`anti—CD22 (RFB4) antibody alone (although potent as an IT) had no
`antitumor activity. When HD37 was administered with the RFB4 IT,
`the combination cured mice of minimal disease; neither IT nor anti-
`body alone (even at high doses) was curative (94). More importantly,
`the F(ab’)2 fragments of HD37 were as effective as the intact antibody
`when doses were adjusted for the 10-fold longer half-life of the latter
`in SCID mice (71). These experiments indicate that the antitumor
`effect of HD37 is not mediated by conventional effector mecha-
`nisms in the host and, therefore, suggests that the beneficial results
`involve signal transduction. This interpretation is fully supported
`by in vitro studies, which demonstrate that both intact anti-CD19
`antibody and its F(ab')2 fragments induce CCA in several human
`lymphoma cell lines (71).
`Using a panel of anti-CD19 MAbs, it was found that their ability to
`
`How can one reconcile the data indicating an important role in
`tumor immunity for effector functions of MAb with the data indicat-
`ing that agonist activity is critical and that effector functions may play
`a minor role? In the past, MAbs were selected as antitumor reagents
`by virtue of their specificity for the tumor and then, secondarily, for
`their effector function. The effectiveness of a particular antibody was
`entirely dependent on its ability to recruit conventional effector func-
`tion(s), unless it coincidentally possessed negative signaling capacity.
`It is not surprising, therefore, that the agonist function of antibodies
`was inadvenently obscured by this biased process of selection. Only
`when MAbs are selected for negative signaling functions will it be
`5303
`
`5 E. Racila, R. Scheuermann, L. Picker. E. Yefenof, T. Tucker, W. Chang, R. Marches.
`N. Street, E. S. Vitetta, and J. W. Uhr, submitted for publication, 1994.
`
`PHIGENIX
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`MONOCLONAL ANTIBODIES AS AGONISTS
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`possible to assess whether or not there is an additive role for MAbs
`acting through effector functions. Indeed, it is likely that this will be
`the case. If so, a signaling antibody could be altered by genetic
`engineering to introduce the desired Fc portion. Moreover, a nonsig-
`naling antibody could be used on the basis of its effector function, and
`another antibody with a different specificity could be used simulta-
`neously for its signaling ability. New information regarding signaling
`will facilitate the design of experiments to address the relative con—
`tributions of these two antitumor effects of antibody and to explore
`various regimens to optimize their therapeutic use.
`
`Anti-erbB-ZR Signaling of Carcinoma
`
`Negative signaling of B-cell tumors by cross-linking lgM or other
`molecules on the cell surface could be considered a phenomenon
`
`unique to lymphocytes since the normal cellular counterpart, an im-
`mature B-cell,
`is destined to become anergic or deleted following
`interaction of its membrane immunoglobulin with self antigens as a
`means of establishing self tolerance. The question then arises as to
`whether negative signaling from MAbs can occur in nonlymphoid
`neoplasms. In this regard, there is considerable evidence that epider-
`mal growth factor
`receptor (107) and erbB-ZR (also known .as
`p185”ERR2 oncoprotein) on breast, ovarian, and several other types of
`carcinomas (108, 109) can also function as a suitable target for
`negative signaling by MAbs (109—113). For simplicity, we will dis-
`cuss only the erbB-ZR. erbB-2R is a member of the epidermal growth
`factor receptor family (114) and is presumed to act as a signaling
`receptor for a yet-to-be-identified ligand concerned with regulation of
`growth and differentiation of breast cells and other cell types. Over-
`expression of erbB—2R on breast cancer cells is associated with a poor
`prognosis (115—122). If a MAb with sufficient affinity against a
`particular epitope on erbB-2R is added to erbB-ZR-overexpressing
`breast or ovarian carcinoma cells, a strong antiproliferative effect can
`be induced (109, 111, 123, 124). One example is the MAb anti-
`erbB-2R 4D5, which in ng concentrations can inhibit proliferation of
`breast cancer cells that overexpress erbB-ZR (108, 123) and is pres-
`ently being evaluated in clinical trials. It is presumed to act via signal
`transduction because of its antiproliferative effect in vitro and the
`accumulating body of evidence that cross-linking of erbB-ZR induces
`a series of biochemical changes associated with a signaling cascade
`(125). We have recently shown that 4D5 induces both CCA and cell
`death in erbB-ZR overexpressing breast cancer cells and that these
`effects require functional
`tyrosine kinases.6 Thus, as with B-lym-
`phoma cells, in breast cancer cells, there appear to be two pathways,
`one for CCA and another for induction of cell death.
`
`is not surprising that nonlymphocytic neoplastic cells can be
`It
`signaled by cross-linking particular surface molecules since these may
`play major roles in the regulation of cellular growth and differentia-
`tion. This does not imply that all tumor cells will be susceptible to
`such regulation. However, we would speculate that a proportion of
`tumors of many cell lineages express surface molecules which, when
`extensively cross-linked, may deliver sufficiently strong signals to
`override the malignant phenotype and induce either CCA or death.
`
`Additive Effects
`
`in an increase in the proportion of apoptotic cells.7 Besides enhancing
`negative signaling, an additional benefit
`to using two (or more)
`antibodies specific for molecules on the same tumor cell
`is the
`inhibition of emergence of antigen- or epitope-negative variants.
`Thus, Levy and Miller (15) and Kwak er al. (126) have combined two
`or more anti-idiotopes to lessen the possibility that idiotope—negative
`NHL cells will escape inhibition.
`Similar results have been obtained with anti-erbB-ZR antibodies.
`
`There are reports of additive antitumor effects when two anti-erbB-ZR
`MAbs are used simultaneously in vitro (127, 128) or in nude mice
`(112, 127). These experiments,
`therefore, support
`the strategy of
`searching for combinations of MAbs that display such additive ef-
`fects. There are several types of combinations to consider, none of
`which are mutually exclusive: (a) a MAb directed against a surface
`molecule that induces CCA and another directed against a different
`surface molecule that induces cell death; (17) MAbs that bind to two
`different molecules, resulting in signaling via the same intracellular
`pathway; and (c) MAbs that bind to different epitopes on the same
`molecule. lnfonnation concerning these combinations will be critical
`in developing an in \‘ier paradigm for predicting the efficacy of
`antitumor MAbs in vim.
`
`Are the Negative Signals Physiological?
`
`It seems reasonable to assume that cross—linking of receptors by
`MAbs that signal CCA and apoptosis are imitating the signals induced
`by physiological agonists. For example, tolerance induction to self I
`antigens on immature B-cells requires signaling through the immu;
`noglobulin receptor complex as discussed above. However, there are
`theoretical considerations as well as experimental data that suggest a
`more complicated interpretation. Thus, the interaction of a physiolog-
`ical ligand with a small percentage of receptors on a cell and cross-
`linking a proportion of them should be sufficient to deliver a signal.
`Nature would be expected to provide a considerable excess of such
`receptors to ensure signaling when required. In contrast,
`if all
`the
`receptors are cross-linked and, indeed, clustered into large aggregates
`by MAbs, a different signal, quantitatively or qualitatively, might be
`expected. These theoretical considerations are indirectly supported by
`a number of studies. Thus, optimal negative signaling of B—cell tumors
`requires concentrations of antibody that exceed the number of lgM
`molecules on the cell surface, indicating that both saturation of surface
`lgM and cross-linking of newly expressed IgM during the incubation
`period of one or more days is required for a maximum effect (129—
`131). As previously mentioned, the same is true for studies of cells
`overexpressing erbB-ZR in which increased cross-linking increases
`the negative signaling (112, 127) and the proportion of cells that die.6
`In addition, the frequency and magnitude of elevations of intracel—
`lular calcium ions change markedly as concentrations of ligand
`(132) (including anti—rt) (133) are increased from physiological to
`pharmacological levels. It is possible, therefore, that the antitumor
`effects induced by MAbs acting as agonists may be different in
`intensity and/or quality from those induced by the physiological
`ligands at their usual concentrations.3 lndeed, such abnormal sig-
`naling may by responsible for inducing apoptosis. In this regard, it
`has recently been shown that anti—p. and anti—5 bound to plastic can
`induce apoptosis in normal B-cells (134).
`
`is of particular interest that, in virro, simultaneous addition of
`It
`anti-CD19 (which by itself induces CCA) and anti-n (which can
`induce both CCA and apoptosis) to human B-lymphoma cells results
`
`° R. Marches, R. H. Scheuermann. L. Picker. T. T. Tucker. E. Racila. N. E. Street, G.
`Shen.J. Li. B. Wei. A. llgen, E. S. Vitetta. and l. W. Uhr. submitted for publication.
`
`7 E. Racila. R. Scheuermann. L. Picker, T. Tucker, R. Marches. N. Street, E. S. Vitetta.
`and l. W. Uhr. unpublished results.
`ligand concentrations are
`3It is possible.
`that at particular stages of development.
`markedly increased in order to induce apoptosis in a particular cell lineage and. in that
`sense. are physiological.
`5304
`
`PHIGENIX
`
`Exhibit 1010-04
`
`

`

`There are other potential uses of MAb in tumor therapy aside from
`inducing negative signaling or conventional effector mechanisms.
`Some MAbs may block critical interactions between the tumor cells
`and neighboring cells, stroma, or matrix that are necessary for either
`tumor growth or development of metastases or both. For example, a
`Mab could inhibit one step in the multistep process by which tumors
`establish metastases. A recent example is the therapeutic use of
`anti-CD44, an antibody directed against a surface glycoprotein in-
`volved in cell migration and adhesion. Injection of this MAb or its
`F(ab')2 fragment 1 week after inoculation of a human melanoma cell
`line into SCID mice prevented metastases but not the development of
`the primary tumor (135). The antibody showed no effect on growth of
`the tumor cells in vitro. Thus, blocking the interaction between CD44
`and its ligand is presumed to have interrupted an interaction critical to
`the metastatic process. There are a large number of other candidate
`molecules that could play similar roles. For example, CD54 has been
`reported to be expressed on metastatic melanoma (136), and its
`expression on primary lesions is associated with a poor prognosis,
`suggesting that it also can contribute to tumor dissemination (137).
`Anti-CD54 evoked a marked antitumor effect when injected into
`SCID mice with human myeloma cells (138), perhaps because CD54/
`CD11a/CD18 interactions are important
`in homotypic growth of
`myeloma (139).
`MAbs to growth factors or their receptors can also have significant
`antitumor activity. Thus, antibodies against IL-6 and the 1L~6 receptor
`(140) show efficacy in treatment of a human myeloma in SCID mice
`(138) and transient responses in patients, if the particular tumor cells
`are dependent on IL-6 for growth (14]). This approach is complicated
`by the high levels of IL-6 produced in some patients by the bone
`marrow stroma, as well as the myeloma cells, and the close proximity
`of the secreted cytokine to the high affinity receptors on neighboring
`myeloma cells (142). MAbs against the IL-ZR (CD25) have been used
`to treat adult T-cell leukemia with some partial or complete transient
`remissions (19). These antitumor effects could be due entirely to
`_, blocking binding of growth factors necessary for tumor cell growth,
`but the roles of signal transduction and effector functions have not
`been excluded. In these examples, one must avoid activation of the
`receptor with the antibody used. Again, epitope-specific screening is
`required