ed antigen-specific activity in vitro and was
`8- to 25-fold more potent than nonbinding
`(IgG-DOX or SN7-DOX) conjugates against
`carcinoma lines that expressed the BR96
`antigen. BR96-DOX was much less active
`against cells that did not bind BR96 (27).
`We evaluated the antitumor activity of
`BR96-DOX against human lung adenocar-
`cinoma L2987, colon carcinoma RCA, and
`breast carcinoma MCF7 growing as subcu-
`taneous transplants in athymic mice (Fig.
`2). According to immunohistology, the
`degree of binding of BR96 to cells from
`these carcinoma lines was similar to that of
`biopsy material from human carcinomas of
`the same respective types (21). Controls
`included untreated mice and mice that
`received DOX (at doses optimized to pro-
`duce maximal antitumor activity in each
`model), unconjugated BR96, mixtures of
`BR96 and DOX, and DOX conjugated to
`either normal human IgG or the control
`mAb SN7. Doses of DOX and mAb are
`presented as milligrams per kilogram of
`body weight per injection. Therapy (three
`treatments 4 days apart) started 14 to 28
`days after tumor transplantation when tu-
`mors were well established. Treatment with
`BR96-DOX consistently cured most mice
`bearing L2987 (Fig. 2A) or RCA (Fig. 2B)
`tumors and complete and partial tumor
`regressions were produced against MCF7
`tumors (Fig. 2C). Equivalent doses of non-
`binding IgG-DOX or SN7-DOX had no
`effect against these tumors. Although opti-
`mal doses of DOX delayed the growth of
`small L2987 tumors (50 to 100 mm3) and
`MCF7 tumors, regressions or cures were not
`observed. Alone, DOX was not active
`against established RCA tumors either in
`terms of tumor growth delay or regressions;
`however, BR96-DOX cured 78% of the
`treated mice. BR96-DOX also produced
`56% cures and 22% complete and 22%
`partial regressions of lung tumors which
`were 250 to 800 mm3 in size at the start of
`therapy (Fig. 2D). In contrast, antitumor
`activity was not observed after treatment
`with an optimal dose of DOX.
`Table 1 summarizes the tumor regression
`rates after treatment with various doses of
`BR96-DOX, IgG-DOX, DOX, and mix-
`tures of mAb and DOX against established
`L2987 and RCA tumor xenografts. When
`administered at equivalent DOX doses of
`.5 mg/kg (three injections 4 days apart),
`BR96-DOX produced long-term cures in 72
`to 100% of mice (n = 281) bearing L2987
`tumors. In the RCA colon tumor model,
`which was not sensitive to unconjugated
`DOX, BR96-DOX administered at equiva-
`lent DOX doses of 2 10 mg/kg (three injec-
`tions 4 days apart) cured 72 to 100% of
`mice (n = 48). Mice cured of L2987 or
`RCA tumors remained alive and tumor-free
`for more than 1 year with no indication of
`
` on May 21, 2014
` on May 21, 2014
` on May 21, 2014
` on May 21, 2014
`
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`
`Downloaded from
`Downloaded from
`Downloaded from
`Downloaded from
`
`--
`
`Cure of Xenografted Human Carcinomas by
`BR96-Doxorubicin Immunoconjugates
`P. A. Trail,* D. Willner, S. J. Lasch, A. J. Henderson, S. Hofstead,
`A. M. Casazza, R. A. Firestone, 1. Helistrom, K. E. Hellstrom
`Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody
`BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen
`related to Lewis Y that is abundantly expressed at the surface of cells from many human
`carcinomas; it has a high degree of tumor selectivity and is internalized after binding.
`BR96-DOX induced complete regressions and cures of xenografted human lung, breast,
`and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of
`mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured
`94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats,
`like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.
`
`Although chemotherapy is an effective
`treatment for selected human tumors, only
`modest progress has been made for the
`majority of carcinomas, including carcino-
`mas of the breast, lung, and colon. The
`introduction of the monoclonal antibody
`(mAb) technology in the 1970s raised
`hopes that tumor-specific mAbs could be
`used to target antitumor agents and provide
`more effective therapy (1). Various immu-
`noconjugates, in which antibodies were
`used to target chemotherapeutic drugs (2-
`7), or plant and bacterial toxins (8-10)
`have been evaluated in preclinical models
`and found to be active in vitro and in vivo.
`However, activity was usually
`assessed
`against newly implanted rather than estab-
`lished tumors and was typically superior
`only to matching, but not optimal, doses of
`unconjugated drug. Although conjugates
`have been described with antitumor activ-
`ity against established tumors that was su-
`perior to that of an optimal dose of uncon-
`jugated drug, the therapeutic index was
`low, and superior activity was achieved
`only at or near the maximum tolerated dose
`(MTD) of the conjugate (7). The results of
`clinical studies of drug and toxin conjugates
`have also been disappointing, particularly
`for solid tumors (11-14).
`As an attempt to improve antibody-
`directed therapy of human carcinomas, we
`have conjugated the mAb BR96 with the
`anticancer drug doxorubicin (DOX). As
`used in the studies described here, BR96 is
`a chimeric (mouse-human) variant of the
`murine BR96 mAb (15), is of the human
`immunoglobulin Gi (IgG1) isotype, and
`S. J. Lasch, A. J. Henderson, A. M.
`P. A. Trail,
`Casazza, Bristol-Myers Squibb Pharmaceutical Re-
`search Institute, Princeton, NJ 08543.
`D. Willner, S. Hofstead, R. A. Firestone, Bristol-Myers
`Squibb Pharmaceutical Research Institute, Walling-
`ford, CT 06492.
`I. Hellstrom and K. E. Hellstrom, Bristol-Myers Squibb
`Seattle, WA
`Research
`Pharmaceutical
`Institute,
`98121.
`*To whom correspondence should be addressed.
`
`212
`
`was produced by homologous recombina-
`tion (16). BR96 binds to a tumor-associated
`antigen that is closely related to Lewis Y
`expressed
`abundantly
`and
`(LeY)
`is
`(>200,000 molecules per cell) on human
`carcinoma lines. According to immunohis-
`tology, BR96 binds the majority of human
`carcinomas of the breast, lung, and colon
`(15). Although BR96, like essentially all
`mAbs to human tumors, is not truly tumor-
`specific, it offers advantages over most other
`antibodies to Ley (17-20). First, BR96 is
`more tumor selective and the normal tissues
`to which it binds primarily comprise differ-
`entiated cells of the esophagus, stomach,
`and intestine as well as acinar cells of the
`pancreas (15, 21). Second, BR96 is rapidly
`internalized into lysosomes and endosomes
`after binding to cells expressing the antigen
`(15, 22). We used the latter characteristic
`to design conjugates that rapidly release
`DOX after antigen-specific internalization.
`We prepared the conjugates by linking the
`DOX derivative maleimidocaproyl doxoru-
`bicin hydrazone to BR96 or control immu-
`noglobulins (Fig. 1). These conjugates in-
`corporate a thioether linker, which imparts
`acceptable stability in plasma (23, 24), and
`an acid-labile hydrazone bond (7, 25, 26)
`which liberates DOX once it is internalized
`into the acidic environment of lysosomes
`and endosomes.
`The BR96-DOX conjugate demonstrat-
`
`O
`
`OH
`
`N-NHCOO()
`
`0
`
`N
`
`3CO
`
`ON
`
`H3
`
`0
`
`OH
`Fig. 1. Structure of the BR96-DOX immunocon-
`jugate.
`
`SCIENCE * VOL. 261
`
`*
`
`9 JULY 1993
`
`IMMUNOGEN 2093, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`produced a delay in tumor growth but no
`tumor regressions, and if the dose was re-
`duced to 50% of the MTD, DOX had no
`effect (7). In contrast, activity equivalent
`to that of an optimal dose of DOX (8
`mg/kg) was achieved at a dose of BR96-
`DOX of 1 mg/kg; the BR96-DOX conjugate
`produced antitumor activity comparable to
`that of an optimal dose of unconjugated
`DOX at 1/8th of the equivalent DOX dose.
`In summary, the BR96-DOX conjugate was
`more active, had a much broader range of
`therapeutic doses, and was more potent
`than unconjugated DOX.
`Although
`many
`immunoconjugates
`have been described that are active against
`subcutaneous tumors (2-7), activity against
`established disseminated disease has not,
`for the most part, been observed (2, 28).
`Cells from L2987 lung carcinoma were se-
`lected in vitro for their ability to grow as
`multicellular spheroids. When we injected
`these cells iv into athymic mice, tumors
`developed at various sites, including lymph
`nodes, lung, spleen, liver, brain, and sub-
`cutaneously, and ascites was formed in
`some animals., Cells obtained from these
`disseminated tumors expressed the BR96
`antigen, as shown by indirect immunofluo-
`rescence, in amounts similar to those of the
`L2987 cell line and subcutaneous tumors.
`Mice inoculated with L2987 spheroids and
`treated (14 mice per group) with BR96-
`DOX (DOX equivalent of 8 mg/kg admin-
`istered as three injections 4 days apart) had
`an increased median survival time (MST)
`(MST of >200 days) relative to that of
`control mice (MST of 85 days) or mice
`treated with an optimal dose of DOX (MST
`of 140 days).
`In another experiment, therapy was de-
`layed until mice displayed extensive dissem-
`inated disease, defined as >0.5 g of visible
`tumor burden (Fig. 3). The disease in these
`
`....-
`
`side effects. Administered at equivalent
`doses, BR96 was not active against estab-
`lished tumors (either in terms of tumor
`growth delay or regressions), and the delay
`of tumor growth produced by mixtures of
`BR96 and DOX was equivalent to that of
`DOX administered alone.
`Contrary to our expectations, cells lack-
`ing BR96 expression were not detected after
`treatment with BR96-DOX (27). Also,
`cells obtained from tumors that grew back
`after BR96-DOX induced regression were as
`sensitive in vitro to DOX as the parental
`cell line; the IC50 (concentration required
`to produce 50% inhibition of [3Hlthymi-
`dine incorporation) was 0.4 + 0.1 FLM and
`0.3 ± 0.2 pM of DOX for treated and
`parental, respectively. These cells were also
`as sensitive to BR96-DOX as the parental
`cell line with IC50 values of 2.6 ± 0.8 pLM
`and 2.7 ± 0.5 liM equivalent DOX for
`treated and parental, respectively. These
`data suggest that it may be possible to
`successfully re-treat tumors with several
`rounds of BR96-DOX therapy.
`The MTD (equivalent DOX dose) of the
`
`BR96-DOX conjugate (three injections 4
`days apart) was 20 mg/kg administered in-
`traperitoneally (ip). When administered in-
`travenously (iv), the MTD was 210 mg/kg.
`This was the maximum dose that could be
`given iv because of the constraints of injec-
`tion volume. At the doses tested, there was
`no difference in the antitumor activity of
`BR96-DOX whether administered ip or iv.
`At doses of BR96-DOX (Table 1) equiva-
`lent to .5 mg/kg of DOX [BR96 (.250
`mg/kg)], more than 70% of treated animals
`were cured of established L2987 tumors. In
`fact, BR96-DOX was active at doses as low
`as a DOX equivalent of 1 mg/kg. The
`BR96-DOX conjugate was, therefore, ac-
`tive at a dose equivalent to 1/20th of its
`MTD. These data demonstrate the broad
`range
`of therapeutic
`doses
`that were
`achieved with BR96-DOX. The MTD of
`unconjugated DOX (8 mg/kg iv and 4
`mg/kg ip) was lower than that of the BR96-
`DOX conjugate. Unconjugated DOX, ad-
`ministered ip at the MTD, did not inhibit
`the growth of established L2987 tumors.
`When administered iv at the MTD, DOX
`
`A
`
`B
`
`7/8 Cures
`11/8CR
`
`7/9 Cures
`1/9 PR
`
`-
`
`02
`
`;
`
`100
`40
`.0
`80
`60
`120
`Days after tumor Implantation
`Days after tumor Implantation
`Fig. 2. Antigen-specific antitumor activity of BR96-DOX conjugates. Partial tumor regression (PR)
`reflects a decrease in tumor volume to <50% of the initial tumor volume; complete tumor regression
`(CR) refers to a tumor that for a period of time is not palpable; and cure is defined as an established
`tumor that is not palpable for a period of time > 10 tumor volume doubling delays (the time in days
`that it takes for control tumors to double in size). Data are presented as median tumor size. (A)
`Xenografts from L2987 lung tumor 50 to 100 mm3 at the initiation of therapy. Control animals (a);
`animals treated with BR96-DOX [DOX (5 mg/lkg)] (0), IgG-DOX [DOX (5 mg/kg)] (A), or optimized
`DOX (8 mg/kg) (o) 14, 18, and 22 days after the tumor was implanted. (B) Xenografts from RCA
`colon tumor 50 to 100 mm3 at the initiation of therapy. Control animals (-); animals treated with
`BR96-DOX (10 mg/kg) (0), IgG-DOX (10 mg/kg) (A), or optimized DOX (8 mg/kg) (O) 16, 20, and
`24 days after the tumor was implanted. (C) Xenografts from MCF7 breast tumor 100 to 125 mm3 at
`the initiation of therapy. Control animals (-); animals treated with BR96-DOX (5 mg/kg) (0),
`IgG-DOX (5 mg/kg) (A), or DOX (6 mg/kg) () 14,18, and 22 days after the tumor was implanted.
`(D) Xenografts from L2987 lung tumor 250 to 800 mm3 at the initiation of therapy. Control animals
`(-); animals treated with BR96-DOX (8 mg/kg) (-) or DOX (8 mg/kg) (0) 28, 32, and 36 days after
`the tumor was implanted.
`
`80
`
`200
`
`120
`100
`160
`140
`180
`Days after tumor cell Inoculation
`Fig. 3. Conjugates of BR96-DOX cure athymic
`mice of large disemminated tumors. Mice were
`inoculated iv with L2987 spheroids and select-
`ed approximately 12 weeks later on the basis of
`visible tumor burden. Selected animals were
`randomized and left as untreated controls (A)
`or treated with BR96-DOX (8 mg/kg) (0) or
`DOX (8 mg/kg) (m) 82, 86, and 90 days after
`inoculation of tumor cells.
`
`SCIENCE * VOL. 261
`
`*
`
`9 JULY 1993
`
`213
`
`IMMUNOGEN 2093, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`animals was so far advanced that 50% of
`control animals died during the first 6 days of
`the experiment. The MST of the control
`group was 90 days and 100% of the mice
`were dead by day 102. Optimal doses of
`DOX (8 mg/kg) had no effect on the large
`disseminated tumors; the MST was 94 days
`and 100% of the mice were dead by day 140.
`In contrast, mice treated with BR96-DOX
`(8 mg/kg) had a MST of >200 days and
`eight of the ten animals survived for the
`duration of the experiment. Surviving mice
`were killed 200 days after cell inoculation,
`and sections of the lung, lymph nodes,
`spleen, colon, jejunum, kidney, liver, brain,
`and heart were examined by histology. Sev-
`en of the eight surviving mice were free of
`detectable tumor (70% cures by combined
`life-span and histologic examination) -
`Although tissues from athymic mice do
`not bind BR96, normal tissues from several
`strains of rats, including athymic Rowett
`rats (Harlan Sprague-Dawley), were shown
`by immunohistology to bind BR96. The
`binding of BR96 to rat tissues was similar to
`that of normal human tissues (21), that is,
`BR96 bound to cells in the esophagus,
`
`stomach, and intestine and acinar cells of
`the pancreas (15). We implanted L2987
`tumors subcutaneously in athymic rats and
`initiated therapy when the tumors were 50
`to 100 mm3 in size (Fig. 4). The MTD of
`unconjugated DOX (4 mg/kg administered
`as three injections 4 days apart) resulted in
`a delay in tumor growth and 25% cures.
`However, BR96-DOX given at a matching
`DOX dose [DOX (4 mg/kg) and BR96 (140
`mg/kg)] cured 100% of the rats, and a dose
`equivalent to DOX (2 mg/kg) [BR96 (70
`mg/kg)] cured 88% of the rats. Of the rats
`treated with BR96-DOX, 94% (15 out of
`16) remained alive and tumor-free with no
`evidence of toxicity 150 days after the last
`dose of BR96-DOX.
`The BR96-DOX conjugate demonstrat-
`ed strong antitumor activity in all preclin-
`ical models evaluated. The efficacy and
`potency of BR96-DOX are likely a result of
`several factors. The antigen to which BR96
`binds is abundantly expressed at the tumor
`cell surface and the active drug is released
`after antigen-specific binding and internal-
`ization of the conjugate into the acidic
`environment of lysosomes or endosomes.
`
`Table 1. Antitumor activity of BR96-DOX against established human tumor xenografts.
`
`Injection dose
`(mg/kg)
`
`Tumor regressions (%)*
`
`Acid-labile immunoconjugates, in which a
`less stable disulfide linker was used, have
`been investigated (7, 26). Although these
`conjugates were active in an antigen-specific
`manner, they had poor potency in vivo (7).
`The use ofa more stable thioether linker and
`a mAb with higher avidity and more rapid
`rates of internalization improved the activity
`and potency of BR96-DOX and also in-
`creased the range of therapeutic doses.
`In the studies reported here, administra-
`tion of BR96-DOX at cumulative doses of
`at least 15 mg of DOX per kilogram of body
`weight and 700 mg of mAb per kilogram of
`body weight [equivalent to DOX (45 mg per
`square meter of body surface area) and mAb
`(2100 mg/m2) (29)] resulted in more than
`70% cures of established lung tumors. This
`dose of mAb in mice is approximately
`equivalent to a cumulative dose in humans
`of 3 g of mAb per patient and is only
`required
`slightly
`higher than that
`to
`achieve saturation of human carcinomas in
`patients given L6, another anticarcinoma
`mAb (30). The optimal schedule for ad-
`ministering BR96-DOX was not deter-
`mined in the studies reported here, and the
`dose of BR96-DOX required to achieve
`cures may be further reduced with schedule
`optimization.
`The demonstration of tumor cures in
`animals in which BR96 binds to normal
`tissues highlights the fact that the appropri-
`ate combination of mAb, drug, and linker
`chemistry are critical aspects to successful
`antibody-directed therapy. The use of drugs
`
`140
`
`80
`100
`120
`60
`40
`20
`Days after tumor Implantation
`Fig. 4. Conjugates of BR96-DOX cure human
`lung tumors implanted in athymic rats. Athymic
`rats that expressed the BR96 antigen in several
`normal tissues were implanted subcutaneously
`with L2987 human lung tumors. Therapy was
`administered 14, 18, and 22 days after the
`tumor was implanted when tumors were 50 to
`100 mm3 in size. Control animals (m), animals
`treated with BR96-DOX (4 mg/kg) (0), BR96-
`DOX (2 mg/kg) (A), or DOX (4 mg/kg) (0). Data
`points (0) and (A) superimpose. The BR96-
`DOX conjugate at 4 mg/kg cured 100% of the
`animals, whereas doses of 2 mg/kg cured 88%
`of the animals and induced complete tumor
`regression in the other 12%.
`
`Complete
`
`Partial
`
`0
`
`0.9
`3.3
`8.0 ± 0.7
`1.0
`3.7
`17.9 + 1.5
`33.7 + 2.4
`11.6 ± 1.2
`50.0
`30.0
`20.0
`22.0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`0
`
`0.9
`7.0
`2.0 ± 0.4
`0
`5.6 ± 0.7
`2.6
`21.3
`11.9 + 2.1
`0
`40.0
`10.0
`44.0
`0
`3.7 +±1.0
`0.8 + 0.8
`0
`0
`0
`0
`0
`0
`
`Num-
`ber
`of
`mice
`
`8
`29
`100
`27
`117
`62
`44
`10
`10
`10
`9
`19
`27
`125
`8
`8
`8
`9
`9
`9
`
`0
`10.0
`10.7 + 0.1
`11.0
`5.5 +±1.3
`0
`0
`0
`10
`0
`0
`0
`
`0
`10.0
`0
`22.0
`0
`0
`0
`10
`0
`0
`0
`0
`*Single injection.
`
`10
`10
`28
`9
`18
`10
`10
`10
`10
`10
`29
`10
`§Three injec-
`
`Treat-
`ment
`
`Sched-
`ule
`
`BR96-DOX
`
`q4dx3t
`
`BR96-DOX
`
`q1dx1t
`
`IgG-DOX
`
`DOX
`BR96
`
`q4dx3
`
`q4dx3
`q4dx3
`
`BR96+DOX q4dx3
`
`BR96-DOX
`
`q4dx3
`
`BR96-DOX
`
`q7dx3§
`
`DOX
`
`20.0
`15.0
`10.0
`8.0
`5.0
`2.5
`1.25
`30.0
`25.0
`20.0
`15.0
`10.0
`5.0
`8.0
`
`8.0
`8.0
`8.0
`
`400
`200
`100
`400
`200
`100
`
`903
`625
`376 + 5.4
`176
`90
`900
`625
`420
`210
`405
`
`Cures
`mAb BR96*
`Tumor L2987
`100
`689
`711 ±36
`83.0
`0.8
`83.0 ± 1.1
`513 + 12
`0.1
`88.5
`317
`3
`72.3 ± 2.2
`246 + 5
`30.4
`3
`3.4
`109
`49 ± 1
`6.9 ± 0.9
`50.0
`1078
`30.0
`930
`60.0
`735
`540
`11.0
`403 ± 5.2
`0
`0
`202
`3.2
`0
`0
`0
`0
`0
`0
`0
`Tumor RCA
`100
`20.0
`80.0
`15.0
`71.7 + 0.9
`10.0
`11.0
`5.0
`0
`2.5
`100
`20.0
`100
`15.0
`80
`10.0
`10
`5.0
`IgG-DOX
`q4dx3
`0
`10.0
`0
`DOX
`8.0
`q4dx3
`0
`DOX
`q7dx3
`10.0
`tThree injections administered with a 4-day interval.
`*Mean + SEM.
`tions administered with a 7-day interval.
`
`214
`
`SCIENCE * VOL. 261
`
`*
`
`9 JULY 1993
`
`IMMUNOGEN 2093, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`such as DOX, with an established clinical
`profile, may offer a safety advantage over
`more potent but less defined agents. The
`toxic effects of DOX are dose-related and it
`is likely that increasing the intratumoral
`concentration of DOX will produce a sig-
`nificant increase in antitumor activity (31,
`32). Although studies on human tumors
`growing in immunocompromised animals
`are not ideal to predict anticancer activity
`in humans, our findings support the clinical
`evaluation of BR96-DOX.
`
`REFERENCES AND NOTES
`
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`5. R. 0. Dillman, D. E. Johnson, D. L. Shawler, J. A.
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`9 February 1993; accepted 3 May 1993
`
`Separable Regulatory Elements Governing myogenin
`Transcription in Mouse Embryogenesis
`Tse-Chang Cheng, Mia C. Wallace, John P. Merlie,
`Eric N. Olson*
`Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell pre-
`cursors within somites and limb buds is among the earliest events associated with myo-
`genic lineage determination in vertebrates. Mutations in the myogenin promoter that
`abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2)
`suppressed transcription of a linked lacZtransgene in subsets of myogenic precursors in
`mouse embryos. These results suggestthat myogenic HLH proteins and MEF-2 participate
`in separable regulatory circuits leading to myogenin transcription and provide evidence for
`positional regulation of myogenic regulators in the embryo.
`
`The formation of skeletal muscle during
`development involves a series of events in
`which multipotential mesodermal stem
`cells give rise to myoblasts, which ultimate-
`ly undergo terminal differentiation in re-
`sponse to external cues. Analysis of muscle
`determination and differentiation in tissue
`culture has revealed a family of muscle-
`specific HLH proteins including MyoD,
`myogenin, Myf-5, and MRF-4, each of
`which can activate myogenesis in a variety
`of cell types in vitro (1). These myogenic
`
`regulators are expressed only in skeletal
`muscle and are first detected during em-
`bryogenesis within myogenic precursor cells
`in the myotomal compartment of the
`somites and in the limb buds, localizations
`consistent with their involvement in myo-
`genic lineage specification (2-4).
`Activation of muscle-specific transcrip-
`tion by myogenic HLH proteins is mediated
`by their direct binding to the E box con-
`sensus DNA sequence CANNTG in the
`control regions of most muscle-specific
`
`SCIENCE * VOL. 261
`
`*
`
`9 JULY 1993
`
`11
`
`genes (1). These regulatory factors also
`positively auto- and cross-regulate their
`own transcription in transfected cells (5,
`6). Whether autoregulatory interactions
`among these genes contribute to their ex-
`pression during embryogenesis or whether
`this is simply a tissue culture phenomenon
`is unknown.
`An indirect pathway for muscle gene
`activation has also been described in which
`myogenin and MyoD induce myocyte en-
`hancer factor-2 (MEF-2) (7, 8), which
`binds an AT-rich DNA sequence associated
`with numerous muscle-specific genes (9).
`MEF-2 can activate muscle transcription in
`the absence of the E box consensus se-
`quence (10-12) and is up-regulated when
`myoblasts enter into the differentiation
`pathway (9). Paradoxically, activation of
`myogerin gene transcription in cultured
`muscle cells requires binding of MEF-2 to
`the myogenn promoter (6). These results
`suggest that myogenin and MEF-2 partici-
`pate in a complex regulatory circuit involv-
`ing positive feedback loops that amplify
`their expression and stabilize the myogenic
`program.
`Whereas much has been learned about
`the mechanisms through which myogenic
`HLH proteins activate muscle-specific tran-
`scription in cultured cells, little is known of
`the mechanisms that regulate muscle gene
`expression during embryogenesis or of the
`regulatory circuits that control expression
`of the myogenic regulators themselves. Be-
`cause myogenin is the only myogenic HLH
`protein expressed in all skeletal muscle lines
`(13, 14), analysis of the mechanisms that
`control its expression should reveal up-
`stream regulators of myogenic lineage spec-
`ification. Here, we used lacZ transgenes
`linked to wild-type and mutant myogemnn
`promoters to begin to define the regulatory
`networks that direct myogenn transcription
`in the mouse embryo.
`The reporter gene Myol5651acZ, which
`contains lacZ linked to the region extend-
`ing from +18 to -1565 base pairs (bp)
`relative to the myogenin transcription initi-
`ation site (Fig. 1), was expressed in the
`same embryonic cells as the endogenous
`gene (Fig. 2, A and D) and therefore serves
`as a marker for activation of myogemnn tran-
`scription in individual cells (4). Expression
`of lacZ from this transgene can be detected
`in rostral somites by day 9.0 after coitus
`(p.c.); expression progresses caudally over
`the next several days concomitant with
`somite maturation (2, 4, 13, 15). In em-
`
`T.-C. Cheng and E. N. Olson, Department of Biochem-
`istry and Molecular Biology, University of Texas M. D.
`Anderson Cancer Center, Houston, TX 77030.
`M. C. Wallace and J. P. Merlie, Department of Molec-
`ular Biology and Pharmacology, Washington Universi-
`ty School of Medicine, St. Louis, MO 63110.
`*To whom correspondence should be addressed.
`
`215
`
`IMMUNOGEN 2093, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676