Striker.
Son And Grandson Of Striker.
CONTACT: originalbulldogclub@gmail.com
Father Of Striker: World Champion, Italian Champion, International Champion, Social Champion Ocobo Pearly Boy, Son Of Ch.Ocobo Tully. Mother: Tuffnuts Snow Angel, Daughter Of Ch. Tuffnuts Striker, Son Of Living Legend
Care Of The New Born French Bulldog & English Bulldog, Bullies & Frenchies
Early spring is far the most favorable season for whelping, since ere the youngsters have reached the weaning they can be put out of doors for a few hours at least on all pleasant days, where they are sure to gain in health, strength and vitality with infinitely greater rapidity than when between walls. Breeders of long experience fully appreciate this fact, also, that where open and pure air and sunshine are denied during the early days of life the future is greatly prejudiced; consequently they have a decided preference for those brood animals that come in use during the first three or four months of the year. And where the rule, fixed with some, to mate only in this period is observed, the puppies not only do not require such extreme care and attention but thrive infinitely better, while being far on the road to maturity they are well fortified against cold weather before it sets in.
If warm, clean and well-ventilated quarters are provided it is, of course, possible to rear winter puppies, yet even in the presence of these conditions they scarcely ever do as well and develop into as hardy specimens as those much beyond walls; and it can safely be accepted that the exceptions are seldom met with among the largest and heaviest varieties, which are ever likely to prove flat failures unless under the most healthful influences from the very first.
In the fact that for several days they are very feeble and extremely sensitive to cold appears one of the first great dangers encountered by puppies born in inclement seasons; hence the advice in the foregoing chapter, that during the whelping the temperature of the room be about 80° Fahr. And this degree should be maintained until the little ones - very wet when they come into the world have thoroughly dried and gained measurably in vitality. Then the temperature may be allowed to fall to 75 °, where it should stand until the following day. After that, if the puppies are vigorous they ought to be able to bear a temperature of 700. But no lower degree will be safe for the next four weeks; nor will any very considerable fall be allowable before the fifth or sixth month.
These limits are set for fairly hardy breeds, yet there are some, black-and-tans, Italian greyhounds and other toys, for instance, which require a higher degree of warmth. In fact these varieties can scarcely be too warm during the earliest days of life.
It can safely be accepted that if a puppy becomes chilled during the first two or three days the chances are nearly all against recovery, also that when this happens in a room kept as warm as it ought to be the victim is naturally weakly, and even were warmth restored it would scarcely be possible to rear him.
Doubtless inferring as much from the limitations of the human mother, whose milk secretion is often as long delayed without injury to her child, some who have publicly discussed the subject of breeding have stated that the secretion of milk in the canine mother may be delayed from twenty-four to forty-eight hours and yet be within the normal limits. But the obvious method of reasoning is not justified in this instance, and certainly the assumption is wholly at variance with experience, which has shown that in the majority of cases milk forms in the breasts of the latter before birth; also, that new-born puppies must be put to them as soon as possible, for where they have failed to nurse within the first few hours of life they have very generally died.
While milk is usually present at birth the supply is seldom abundant in the first twenty-four hours, during which period, fortunately, puppies are easily satisfied; but, as a rule, under their vigorous nursing, which acts as a potent stimulant, the quantity soon becomes greater, and afterwards keeps pace with their increasing demands. Cases are not infrequent, however, where the breasts fail to respond as they ought and their supply remains scanty; in which event the mother must be encouraged to drink freely of liquids, as milk, gruel, water, etc., for the purpose of securing an increase. And such efforts must not be delayed until they are clearly demanded, but be made as soon as there appears the barest reason for suspecting that the supply will be insufficient.
This treatment, while of value in some cases, is, however, far more often ineffectual than effectual, no matter how faithfully applied, consequently to rely upon it solely would be extremely hazardous, and in all instances preparations should be promptly made to nourish the puppies artificially in the event their mother fails them.
Unfortunately it is impossible even twenty-four hours after whelping to determine positively whether the quantity of milk will be large or small; but still products of experience, far from being certain, however, enable breeders to form a probable diagnosis.
The outlook may be considered favorable if at this time the breasts are large and a fairly good flow of milk has been established. The chances are also increased somewhat where puppies have been previously born, for in many instances the quantity of milk is notably greater after the second and third litters; and it would seem that in certain mothers it grew more abundant after every whelping, up to the fourth or fifth year.
The kind and amount of food also bear upon this question, - more heavily even than the age, - and in all cases where the mother has not been wisely and generously fed during gestation a scanty supply of milk is the rule, while in exceptions to it, in which there is an abundance of milk after whelping, its early decline in quantity or quality, and generally both, may be confidently expected. Again, the health of the nursing mother is a matter of infinite importance, and the fact need not be urged that if her constitution is poor or she is a victim of harassing and debilitating disease her milk supply can never be abundant; moreover, that what little she has will be vitiated and unwholesome if not absolutely poisonous.
Summarizing briefly, for the purpose of emphasizing the first essentials brought out in the foregoing : The puppies should be put to the breasts and made to nurse if possible as soon as the whelping is over and the bedding has been changed. Duly considering the facts stated, an estimate should be made of the chances of having a goodly supply of milk, and if they seem poor everything needful should be at once obtained and kept in readiness to nourish artificially as soon as indications for such treatment appear.
As for signs manifested by little ones that are denied sufficient milk, their abdomens instead of being well rounded out and somewhat resistant to pressure are quite flat, the walls being relaxed and flaccid; they sleep much of the time and seem unwilling to make any attempt to nurse, and when taken in the hands are limp and feeble. These symptoms in the main are those of weakness and may be occasioned by a large variety of influences, yet the conditions of the mother's breasts being questionable they must point to starvation as the cause.
French Bulldog & English Bulldog, Bullies & Frenchies Breeders generally seem impressed with the idea that most canine mothers suffer from milk fever, yet this is by no means the rule where puppies are born alive and continue to live and nurse vigorously during the next forty-eight hours. And the reason why they so often escape appears in the fact that they begin to suckle their young very soon after whelping, consequently their breasts are seldom very much swollen even when the milk is abundant.
Where French Bulldog & English Bulldog, Bullies & Frenchies puppies that have reached an advanced stage of gestation are born dead or die shortly after delivery, usually, but not invariably, all the phenomena of milk fever manifest themselves, i.e., the breasts become much swollen and evidently painful, the skin is hot and dry and the pulse quickened; there are thirst, loss of appetite and a decided disinclination to activity. Of these symptoms generally the fever and acceleration of pulse are the first to disappear, and in the course of from twenty-four to forty-eight hours, at which time the breasts, although still greatly distended, are evidently less acutely painful. The rapidity with which the swelling in them subsides depends much upon the treatment, but even when none is applied the natural state of things is generally restored ere the end of a week.
The dangers of this and other fevers after whelping have been very greatly exaggerated, and in consequence the notion is prevalent that all mothers should be kept on low diet, and especially "sloppy foods," for the first two or three days at least, as a preventive measure; whereas milk fever is not in the slightest degree dangerous, and deserves consideration only where the puppies have been lost. And even then it occasions discomfort merely. As for puerperal fever, the only fever peculiar to the whelping state which need disturb the minds of apprehensive breeders, that is very rare in canine mothers, and a person may breed extensively for years without seeing a case of it.
Where the French Bulldog & English Bulldog, Bullies & Frenchies are lost and milk fever occurs it is eminently right and proper that treatment be applied for the purpose of lessening the mother's discomforts. But "sloppy foods," which are so generally supposed to be the least favorable to inflammation and fever, are the very ones that must not be given in this instance, for they would surely tend to increase the secretion of milk and so intensify the existing trouble. And the same can be said of water merely. Consequently for several days the patient should have such foods as boiled rice, broken dog cakes or well-baked bread crusts, - in limited quantity always, softened by a little milk or broth, and be allowed water only at intervals of five or six hours, and then even but a little at a time.
If her breasts are badly swollen and what is popularly termed "caked," the mother should be quartered in the kitchen or other convenient, well-warmed place until her discomfort has abated. During the day hot, dry flannels should be applied to her breasts for fifteen or twenty minutes at a sitting, and the application be repeated as often as possible; while at night they should be gently rubbed with camphorated oil, the same being used generously.
Notwithstanding the notion that cathartics are always required in cases of this sort, neither these nor any other drugs need be given, for under the simple treatment advised very considerable improvement will take place within forty-eight hours, and the milk "dry up" as quickly as under dosing. And it is well to add that the breast pump must not be used in these cases, for while it would afford temporary relief it would greatly retard recovery.
Only rarely are mothers wholly destitute of milk in the first days after whelping, but not infrequently it happens that the supply is far from sufficient for all the puppies, and in no small number of instances the flow is abundant at first and then lost altogether in the course of a week or two; in which cases it is necessary either to provide a foster mother or nourish artificially - alternatives that are extremely vexatious and generally difficult to meet.
Those who have large kennels and breed extensively are to some extent fortified against these accidents, for very often they have two or more bitches come in at about the same time and if the milk of one is insufficient or lost she is assisted or her entire duty assumed by the fortunate mother or mothers. But in the absence of such happy conditions it is extremely difficult and more often than otherwise impossible to obtain a suitable foster mother.
If a mother lost her last litter because of failure of her milk supply it by no means follows that she will be as unfortunate with her next, still her owner should have a foster at hand to prevent this accident if again threatened. And he who has a bitch of great value and has paid a large price for "service" can wisely do likewise, for the chances are many that even were the assistant not an imperative necessity she might yet be used to very great advantage and prove a profitable investment, - results, by the way, that may confidently be expected where this provision is made for first litters.
French Bulldog & English Bulldog, Bullies & Frenchies Foster mothers can generally be obtained for trifling sums, but very rarely indeed within a day or even a week; therefore they should be sought for early; and the course that suggests itself as the easiest and surest is to advertise the want in the kennel papers at least a month before the bitches they are to assist are expected to whelp.
Quality need not enter into the considerations in choosing a foster mother, for a mongrel will do as well as a pure breed, and perhaps better. Nor is the question of size a very important one, notwithstanding some breeders insist that the two mothers should be of about the same size; and while it is better, of course, that they be nearly so, or the foster the larger, a difference in the other direction need not weigh heavily unless very great.
But it is of the highest importance that the foster mother be in good health, and imperatively necessary is it that she be not only absolutely free from mange and other contagious affections but that there exist not the slightest danger of her being the carrier of disease. For instance, did she belong to a kennel in which there were or had recently been cases of distemper, although she herself might be perfectly safe from the disease she would yet be ineligible, since she would likely transmit the germs in her coat.
With due regard to possible irregularities in the duration of gestation, it is advisable that the foster mother be due to whelp about a day earlier than the bitch whose duties she may be called upon to assume. Yet nice adjustments need not be attempted, for milk three or four days older than that of the adopted is generally well borne, and, in fact, instances are not infrequent where puppies in the first or second days of life do well on milk from a week to ten days old.
As for French Bulldog & English Bulldog, Bullies & Frenchies puppies that are in the last part of the first week, milk even three or four weeks old is often kindly received by them. And in one case the writer put puppies of this age to the breasts of a mother and brought back her milk, which had nearly all disappeared, after she had weaned her own litter.
Another case of even greater interest than this was that of a Gordon. Some five months after whelping, one of her mates, a toy, had a litter of puppies, of which this setter at once - became very fond. And as she was entente cor-diale with their mother she was permitted to pass much of her time with them and snuggle them as she would her own. Very strangely, indeed, ere the end of the second week - and doubtless in consequence of frequent tugging by the puppies - milk appeared in her breasts, which were absolutely dry when these little ones were born; and she nursed them regularly until weaned. More than this, after the toys had been weaned, two mastiffs about a week old were put to her breasts, and these, also, she carried to the weaning.
Considering that delay is extremely liable to prove fatal, obviously the transfer of the puppies to the foster should be made as soon as signs appear indicating that their mother will be unable to nourish them. And the fact deserves emphasis that to wait for positive proof in this instance is always hazardous.
As to disposal of the foster's puppies, if they are worth raising efforts should be made to nourish them artificially, otherwise they should be given to the suspected mother, and in case her milk takes on an increase some of her own little ones can be returned to her.
In discussing this subject it is quite the custom to intimate that it is a hard task to induce foster mothers to do their duty by their charges, yet the writer's experience is at variance, and only in one instance has he encountered any difficulty, although he has made many transfers, while in this exceptional case the introduction was so abrupt and due formalities so slighted the results occasioned no surprise.
A wise course to pursue in making the transfer is to remove both litters, put all the puppies into the same basket and keep them together for several hours. In the mean time the breasts of the foster mother will fill up, and the distension causing her some discomfort, she will generally receive the strangers as cordially as she would her own. If, however, to delay is dangerous they should simply be put to her breasts and she be made to understand that she has no choice in the matter. Careful watching will, of course, be advisable for a time, but if all goes well the first day no uneasiness need be felt thereafter.
French Bulldog & English Bulldog, Bullies & Frenchies Puppies can be fed by means of a spoon or nourished in the same way as infants deprived of their mother's milk. And in event of the latter, which is the easier, the apparatus used should consist of merely a small bottle and rubber nipple, in the top of which has been placed a small piece of perfectly clean sponge, which fits easily without compression. But as most of the nipples found in shops have such small holes, and the milk does not flow as freely as it ought, it will be necessary to enlarge one or more of them to about the size of a "darning needle."
Puppies that are nourished artificially require feeding nights as well as days, and during the first week once every hour and a half is none to often; while every two hours should be the rule in the second week, and three hours in the third.
All this time they must be kept in a very warm place, and in a basket lined with a piece of flannel or soft blanket. And during the first few days a fold of this should be laid over them lest draughts strike them and they become chilled.
As for the quantity of food to be given at each feeding, it should be sufficient to fill out their abdomens, but not distend them - merely enough to overcome that flabbiness of the abdominal muscles which is so apparent when the stomach is empty.
Milk is the food required, and it may be direct from the cow or in condensed form. In the first instance it should be scalded, allowed to cool until merely "blood warm," and then to each feeding should be added a "pinch" of the saccharated pepsin, an agent which contains the digestive principles of the gastric juice. By this means cow's milk will be rendered nearly, if not quite, as easily digestible as the canine mother's, and dilution will not be necessary, nor would it be advisable, for the former is not so rich as the latter.
Milk properly condensed has essentially the same composition as before evaporation, minus the water, - that is, unless cane sugar has been added or the heat has been carried too high, - but that which is commonly used for domestic purposes is not above suspicion of sophistication and indifferent methods of preservation, therefore only the brands intended largely for infant feeding should be used. As for the dilutions required, they vary with the preparations, but as a rule half a teaspoonful of milk to eight tea-spoonfuls of water is about the correct proportion.
While feeding artificially it is highly important that the apparatus be kept perfectly clean, also that the intestinal discharges be constantly watched, and in the event they become diarrhoea-like it may be accepted that the milk is too rich and requires dilution, while if they have a sour odor it will be necessary to give at each feeding as much lime-water as milk. And the discharges being watery and sour-smelling, colic either exists or is imminent, hence one drop of laudanum should be given between each feeding until a change for the better shall have occurred.
If a French Bulldog & English Bulldog, Bullies & Frenchies puppy takes kindly to the bottle the first day the chances of carrying him through are fairly good. But only an occasional one will do this, and even he is likely to fall victim to diarrhoea, colic or other digestive disturbances. However, this unfavorable outlook ought not to deter any one from endeavoring to save valuable puppies when this is the last resource.
In some countries infants deprived of their mother's milk are often nursed by animals, and most frequently by the she-goat, which, besides being docile and easily trained to yield nourishment to the child, has teats of a very favorable shape and size, and this animal has proved as convenient a foster mother for puppies of large breeds. The sheep has also been pressed into like service, but being generally wild and intractable she has rarely proved an eminent success. As for the small breeds, to secure a foster for them is easier, as cats can as a rule be readily persuaded to adopt them; and their milk seems well suited to puppies, for in many instances they have been nourished by it from birth and thrived as well as they could have done on the milk of their natural mother.
Returning to the real mother, even if she has a goodly supply of milk she can rarely do justice to more than eight puppies, and were it the rule to reduce all large litters to six, breeding would be far more profitable than it is now; moreover, there would be a marked improvement in the canine race. But the man who has paid a large price for his bitch and a heavy fee for "service" is generally anxious to "get his money back" as soon as possible, and the larger the litter the greater his delight. As he views the matter, every puppy has a fixed value, and to sacrifice one is simply to throw away so many hard dollars; therefore, he looks to the mother to nurse them all, even if there are ten or more - that is, unless he has learned from experience that he is expecting altogether too much.
The infinite majority of those who have yielded to such irrational promptings have met with bitter disappointment and seen puppy after puppy drop off with alarming frequency. And oftentimes within such experiences the few remaining at the end of the third week have scarcely been worth raising; whereas had the litters been properly reduced in size at first, very many of the puppies would doubtless have been saved and reached the weaning strong and healthy.
While nursing a litter of good size a mother is under a strain that may affect her vitality. Unless a puppy has an abundant supply of milk to draw from he cannot thrive and develop well; and were one deprived of it to live he must be more or less weakly and stunted. Acting in accordance with these important facts the intelligent breeder who has too large a litter will promptly reduce its size, either by providing a foster-mother, by artificial feeding or drowning. If the latter means must be resorted to, as a rule he need not apply it the first day unless there are several females in the litter that he does not care to raise; in which event the sooner he disposes of them the better.
Other weedings may be left to the second day, because it is rarely possible on the first to distinguish between the weakly and the healthy and vigorous, and of course the former are to go if a sacrifice is demanded. And even with a litter reduced to fair size the chances are that more than one member of it will drop out before the weaning period has been reached.
Bearing strongly upon this point is the important fact already stated, that puppies should have ample nourishment in the first few hours of life. Now, if the milk supply is scanty during the first week, even if abundant afterward, the deprivation will very generally have a permanent effect. In other words, the puppies will as a rule have experienced a set-back, from the ill effects of which they will never recover, and if they live they will be but indifferent specimens of their breeds. The results from this cause, not unnaturally, are the most pronounced in the largest breeds, and are exhibited by impaired growth, malformations, etc.
Notwithstanding every precaution is taken, mothers will sometimes be found with puppies under them; and while many or the little victims of this accident are crushed and at once beyond all hope, now and then one is suffocated without other injury; in which instance recovery is possible if treatment is applied promptly. Consequently, if a puppy that has been lain on is warm when found, efforts should always be made to restore him, even if there are no signs of life. Enveloped in hot flannel, his head only left uncovered, one thickness of clean cloth should be spread over his gaping mouth, and to the upper side the holder should put his own mouth and slowly inflate the unfortunate's lungs. That done he should remove his mouth and gently compress the chest, so as to force out the air that he has driven in. And this artificial respiration should be persisted in for at least ten minutes.
Care Of The New Born French Bulldog & English Bulldog, Bullies & Frenchies - Conclusions
While fortunate results from this treatment may be rare, in consequence of too great delay, it should always be tried when there appears "a living chance," the fact being kept in mind that in these cases, as in other experiences, success sometimes attends when least expected.
Impediments to nursing due to temporary or permanent malformations of the teats are not common, still that they sometimes occur justifies their consideration here.
Where the breasts are very much distended with milk it may be difficult for new-born puppies to seize and hold the teats, in which case it is merely necessary to draw them out with the fingers and press them into the little ones' mouths.
In extremely rare instances a teat is so much shorter than usual the fingers cannot grasp it, and the following means - often resorted to in like cases in human mothers must be employed: Take a beer or ginger ale bottle and rinse it with very hot water. Dip the neck, merely, in cold water and allow it to remain in the same for about ten seconds; then surround the depressed teat with the mouth of the bottle and maintain gentle but firm pressure against the breast. As the bottle cools the teat will be drawn up into the neck, and after being fixed there for ten or fifteen minutes it will when released very generally project enough to enable a puppy to obtain a firm hold.
Extremely long and misshapen teats are sometimes encountered, and with these nothing can be done in the way of treatment. Fortunately, however, seldom more than two or three such are found on the same mother; and although the puppies may refuse them at first, as a rule they take kindly to them in the course of a few days that is, unless, as is sometimes the case, the breasts from which they project are incapable of secreting milk.
About the third week, in consequence of being bitten and scratched by the puppies, the teats as well as the breasts often become quite sore, and in fact with some Varieties the mothers generally require much persuasion at this period before they will allow themselves to be nursed, so painful is the operation.
In such cases weaning should be effected as soon as possible consistent with the welfare of the puppies; and until then the treatment should be the application of the so-called apple-butter, which can be made as follows. Remove the skin and core of an apple of medium size. Slice and crush in a mortar, and put into a frying-pan, together with a lump of perfectly fresh butter - but just churned and yet without salt - about the size of a hen's egg. Stew these with gentle heat until the apple has all disappeared, and then pour them into a bowl and set away to cool.
This mixture, which cools to about the consistency of vaseline, should be freely and often applied to the sores; and being of innocent ingredients the puppies cannot be endangered by it, as they would be by medicinal applications having any considerable action.
After the weaning this remedy can be persisted in until the sores are healed, or the ointment of the oxide of zinc, which is generally more easily obtainable, can be used, and it will speedily effect a cure.
As for "drying up the milk" after weaning, but rarely will it be necessary to make any efforts to this end, for mothers very generally do well when left entirely to themselves. If, however, it so. happens that the breasts are much swollen and painful, warm camphorated oil may be freely applied; and beyond this no other treatment will be required.
Instances are numerous in which mothers have had an abundance of milk yet their puppies, apparently healthy and vigorous at birth, have all died in the course of three or four days, and the popular assumption has been that the milk was either lacking in essential nutritive elements or contained noxious properties. The writer has found the former the rule, yet he has encountered cases where the milk was up to the standard quality, and although no poisons could be detected he felt convinced that they were there, or had been there, to account for the fatality. And in several of these he made experiments, the results of which led up to the belief that the harmful properties, if there were any in the milk, were limited to that furnished during the first day.
In three cases where puppies were dropping off quickly on the third day, he took from free-milking mothers puppies of about the same age as those dying and put them to the suspected breasts, and kept them there until the weaning, which they reached in good condition; while in two other cases he was fortunate enough to be able to try the same experiment on the second day with the same good results. And in this way he narrowed the poisons down to the first day.
A short time before these experiments were made a well-known and highly intelligent breeder advanced the theory that it was the very first milk that contained the noxious properties, and he advised that the breast-pump be applied the day before whelping and all the milk be drawn out.
In discussing this treatment shortly afterward the writer said in substance as follows : Were it possible to interpret correctly the wants of a bitch in whelp, to know just what forms of nourishment are essential to her support and to maintain her in the highest state of health, with all her functions unimpaired, then, if consonant with this knowledge proper care was administered, it would seem like an unwarrantable interference to attempt to nurse her artificially previous to whelping. This perfect familiarity with her wants, however, is denied, and doubtless there is often something wanting in her composition, and its loss prevents her reaching perfection of health. Nor would slight or even considerable deviation from the normal be inconsistent with the outward evidences of health, for functions might be disordered to a considerable extent and yet the fact remain concealed from even the most careful observer. And did any weakness or disorder of the system exist the milk would likely be more or less vitiated. But it is not alone sufficient to consider mere abnormalities of this nature, and one must go farther and include psychical influences which have a most decided bearing.
For instance, in the human race the disturbing passions will cause certain secretions to become corrupted and even acquire poisonous properties. Beyond doubt the same causes are nearly if not quite as active in the lower orders, for the bite of an animal goaded to desperation heals less rapidly and is attended with greater inflammation than one administered when the system is uninfluenced by excited passion. And if great mental disturbances in the human mother diminish or vitiate the secretion of milk and it becomes hurtful and even deadly to the nursing child, assuredly it is reasonable to assume that the same effects would follow like cause in the canine mother. Another fact to be considered is, that seldom are valuable bitches in the last few days of pregnancy permitted to follow their own instinctive promptings, but very generally they are kept under rigid restraint, lest accidents occur; and if they have been allowed constantly liberty, as they ought, they must be more or less nervous, fretful, and discontented.
All of which goes to show that the influences which may affect the milk of the dam are many and varied, and each and every one must be considered and given due weight in discussing this important question.
Experience acquired since giving expression to the foregoing has led the writer to believe that where a large number of dogs are kept together, are much restrained, and the preparation of the food and the feeding and watering are left to hired help, - in which instance such duties are scarcely likely to be attended to with absolute faithfulness, - it is a wise procedure to use the breast-pump and draw out all the milk on the day previous to whelping. If, on the other hand, the expectant mother is alone or has but one or two mates, has been given ample exercise, fed from her master's table and otherwise treated with the consideration she deserves, to empty the breasts as advised is rarely necessary. Also, that the operation can do no harm if it fails to do good.
An abnormal condition of the milk that is responsible for not a few deaths among puppies is excessive acidity. Now, the milk of a healthy dam is either slightly alkaline or else neutral, and a slight acidity is not appreciable to the eye, but when this reaction is a decided one the milk is thicker than usual or distinctly curdled, and gives rise to colic and diarrhoea, which are speedily fatal unless prompt relief is afforded.
This peculiar trouble is often caused by fermented foods, as meal puddings that have been too long kept. There are some mothers, also, that exhibit it no matter how they are fed, and in them it is attributed to some functional derangement, possibly in the organs concerned in digestion. And it may be suspected if the puppies, healthy at birth, begin on the third or fourth day to moan and cry, grow cold and clammy to the touch, and at the same time emit a sour and otherwise offensive odor.
As soon as these signs are noted a piece of blue litmus paper should be obtained of the nearest druggist for the purpose of testing the milk, and if the same turns it red it is very acid, and the puppies must be taken from the mother and vigorous treatment applied to her at once.
By means of a breast-pump all the milk must be drawn out, and this operation repeated three or four times during the next twelve hours. As soon as possible, also, the mother must be given an antacid in the form of bicarbonate of soda, of which the dose for other than toys is one-half a teaspoonful, to be dissolved in about one-half a teacupful of water and poured down the patient's throat, while for toys it should be decreased one-half. And this medicine, in like doses, should be repeated at intervals of three hours during the first day, once in six hours the next, and three times daily throughout the remainder of the week.
During the period in which the breast-pump is in use the puppies must not be allowed to nurse the mother, but kept near a fire, in a basket lined with well-warmed flannel or blanket, and treated as follows: -
Put four tablespoonfuls of lime-water into a cup and add to the same sixteen drops of laudanum. Of this mixture give each puppy one teaspoonful. Half an hour later, to every one that is still crying and moaning give another teaspoonful of the mixture. After that, until they become quiet, give one teaspoonful every hour to all that moan or cry.
No effort should be made to nourish them until the fourth or fifth hour after they have been taken from their mother. Then they should be given a mixture of cow's milk and lime-water in equal parts, to which should be added boiling water in quantity merely sufficient to make the whole "blood warm." And of this four or five tea-spoonfuls should be administered every two hours, by means of a spoon.
On the day following that on which the acidity was detected, the milk in the mean time having all been drawn out three or four times as advised, the puppies may be returned to their mother and permitted to nurse, provided always her milk is no longer acid. If, however, it is still sufficiently so to turn litmus paper red the puppies must be kept from her and nourished artificially until the soda has rendered the milk secretion alkaline or neutral; in which condition it will no longer affect the color of this paper.
If the bicarbonate of soda is faithfully given the mother for a week there will be but little danger of her milk again becoming excessively acid; but still it will be best to continue to use means of prevention, and lime-water is the antacid to be relied upon after the first week. This should be added to milk in the proportion of one teacupful to every pint; and of the mixture she should be encouraged to drink freely several times daily. But in event she refuses it, it may be accepted that she tastes the lime-water and the proportion of the same must be lessened.
The means of anticipating excessive acidity has been advised in chapter "Before Whelping," and that - the precipitated phosphate of lime - should be given daily to every expectant mother that has ever experienced this accident.
Evaluation of the anti-HER2 C6.5 diabody as a PET radiotracer to monitor HER2 status and predict response to trastuzumab treatment
Smitha Reddy,1 Calvin C. Shaller,1 Mohan Doss,2 Irina Shchaveleva,1 James D. Marks,3 Jian Q. Yu,2 and Matthew K. Robinson1Abstract
Purpose
The rapid tumor targeting and pharmacokinetic properties of engineered antibodies make them potentially suitable for use in imaging strategies to predict and monitor response to targeted therapies. This study aims to evaluate C6.5 diabody (C6.5db), a non-covalent anti-HER2 single chain-Fv dimer, as a radiotracer for predicting response to HER2-targeted therapies such as trastuzumab.
Experimental Design
Immunodeficient mice bearing established HER2-positive tumor xenografts were injected with radioiodinated C6.5db and imaged using PET/CT. Radiotracer biodistribution was quantified using biopsied tumor and normal tissues. Potential competition between trastuzumab and C6.5db was examined in vitro by flow cytometry and co-immunoprecipitations.
Results
Biodistribution analysis of mice bearing xenografts with varying HER2 density revealed that the tumor uptake of 125I-C6.5db correlates with HER2 tumor density. In vitro competition experiments suggest that the C6.5db targets an epitope on HER2 that is distinct from that bound by trastuzumab. Treatment of SK-OV-3-tumored mice with trastuzumab for 3 d caused a 42% (P=0.002) decrease in tumor uptake of 125I-C6.5db. This is consistent with a dramatic decrease in the tumor PET signal of 124I-C6.5db after trastuzumab treatment. Furthermore, BT-474-tumored mice showed a ?60% decrease (P=0.0026) in C6.5db uptake after 6 d of trastuzumab treatment. Immunohistochemistry of excised xenograft sections and in vitro flow cytometry revealed that the decreased C6.5db uptake upon trastuzumab treatment is not associated with HER2 downregulation.
Conclusions
Introduction
Our understanding of the molecular processes that drive both cancer formation and progression has increased dramatically in recent years. This has led to development of targeted therapeutics designed to disrupt specific cancer-associated processes. Development of companion diagnostics is hypothesized to aid in stratifying patients based on the molecular underpinnings of their disease and thus improve the clinical implementation of these targeted therapies. Molecular imaging agents capable of either detecting the levels of biomarkers or monitoring changes in the biomarkers in response to therapy have the potential to make an important contribution to effective disease management.
The human epidermal growth factor receptor family of receptor tyrosine kinases (RTKs) is known to play a critical role in the normal development and homeostasis of a variety of tissues (1). As such, inappropriate signaling through this family of RTKs is associated with formation and progression of a number of cancers (2). This is exemplified by the role of the human epidermal growth factor receptor 2 (HER2) RTK in breast cancer (3, 4). Overexpression of the HER2 protein leads to inappropriate activation of signaling pathways downstream of the RTK (5) and is associated with poor clinical outcome and a high risk of relapse (6). This aggressive subtype of breast cancer (BrCa) accounts for approximately 20 30 % of all BrCa. The anti-HER2 monoclonal antibody (mAb) trastuzumab blocks unregulated signaling associated with HER2 overexpression (7). Diagnosis of HER2-positive BrCa is made through measuring either overexpression of HER2 protein by immunohistochemical staining (IHC) or gene-amplification via fluorescence in situ hybridization (FISH) techniques in biopsied primary tumor. Despite meeting current diagnostic criteria for HER2-positive BrCa, only a third of eligible patients in the metastatic setting respond to single-agent trastuzumab treatment (8). Although combining trastuzumab with chemotherapy increased response rates in both the adjuvant (9-11) and metastatic (12-14) setting, responders are seen to relapse despite continued treatment. This intrinsic and acquired resistance can in principle be due to a number of reasons including discordance in the HER2 expression in primary versus metastatic lesions as was seen by Zidan et al (15).
Whole-body, non-invasive, molecular imaging strategies have the potential to extend the analysis of HER2-status to biopsy-inaccessible lesions. To that end, the diagnostic potential of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with radiolabeled trastuzumab is being investigated in multiple phase I clinical trials (16, 17). The pharmacokinetics (PK) of intact IgG molecules, although appropriate for therapeutic strategies, is not optimal for imaging. Their long half-life requires that imaging be performed multiple days post-injection in order to achieve sufficient blood clearance and optimal tumor:blood ratios. Advances in antibody engineering have facilitated the development of engineered antibody fragments that retain the antigen-binding specificity of mAbs but have tumor targeting and pharmacokinetic properties optimized for use as targeting vehicles for payloads, such as diagnostic or therapeutic radionuclides, chemotherapeutics, or toxins, based on the cell surface expression of tumor associated markers, like HER2 (18-23). We previously demonstrated that an anti-HER2 single chain Fv (scFv)-based antibody molecule, called C6.5db, can function as an effective PET radiotracer in xenograft models of HER2-positive disease (19). In this study, we expand our analysis of the C6.5db to understand both how antigen expression and trastuzumab therapy impact on the function of the C6.5db in targeting HER2-positive tumors in our mouse models.
Materials and Methods
Cell culture
SK-OV-3 cells (ATCC# HTB-77), MDA-MB231 (ATCC# HTB-26), and BT-474 (ATCC# HTB-20) cells were purchased from the American Type Culture Collection. MDA-MB361/DYT2 was a kind gift from Dr. Dajun Yang (Georgetown University, Washington DC). SK-OV-3 cells were cultured in DMEM/HEPES, and MDA-MB361/DYT2, MDA-MB231, and BT-474 cells were cultured in DMEM/F12 medium under 5% CO2 at 37°C. All media were supplemented with 10% fetal bovine serum (FBS).
Production and radioiodination of C6.5db
C6.5db was expressed in TG1 Escherichia coli, and purified by immobilized metal ion chromatography followed by high performance liquid chromatography size-exclusion chromatography over a Superdex 75 column (Amersham Pharmacia, Piscataway, NJ) as previously described (19). Protein was stored at -70°C at 0.5 mg/mL in PBS containing 10% glycerol until ready for use.
C6.5db was radioiodinated with Na125I (cat # NEZ033H, DuPont NEN, Wilmington, DE) or Na124I (RITVERC Isotope Products, St. Petersburg Russia) using either Iodogen-coated glass beads or with the water-soluble form of Bolton-Hunter reagent Sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate (SHPP, cat # 27712, Pierce Biotechnology) as described in Robinson et al (19). Immunoreactivity of the radiolabeled preparations were assayed by live-cell binding assays with SK-OV-3 cells under conditions of antigen excess as described previously (24).
Flow cytometry
SK-OV-3 and BT-474 cells were grown to subconfluence in T75 flasks, rinsed with Hank's solution containing 1 mM EDTA, and harvested with trypsin. Cells were pelleted down by centrifugation at 100 g for 5 min, resuspended in ice- cold FACS buffer (PBS containing 1% bovine and 0.1% sodium azide), and counted on a hemocytometer. About 250,000 cells were used per condition. Cells were exposed to 5 ug of rituximab, trastuzumab, or pertuzumab for 30 min on ice, washed with 1 mL of ice-cold FACS buffer, and centrifuged at 100 Χg for 5 min. Cells were then treated with 5 ug of C6.5db for 30 min on ice, washed with 1 mL of ice-cold FACS buffer, recovered by centrifugation and treated with secondary antibody for 30 min on ice, washed, centrifuged, and resuspended in 100 uL of 1% paraformaldehyde in PBS. In the reciprocal experiment, cells were first treated with either C6.5db or PBS on ice followed by treatment with rituximab, trastuzumab, or pertuzumab. C6.5db was detected with Alexa Fluor 488 conjugated anti-6ΧHis secondary antibody (Qiagen, Inc, Valencia, CA) while human IgGs were detected with FITC-conjugated anti-human IgG (Milipore, Billerica, MA). Flow cytometry was conducted using a FACScan flow analyzer (BD BioSciences). Non-stained cells and cells stained with secondary antibody alone (no primary antibody) were included as negative controls in each experiment. Each experiment was repeated at least three times. Data were analyzed using FlowJo software (version 8.8.6; Stanford University).
Immunoprecipitation
HER2 ECD was expressed and purified from stable clones of HEK 293 cells as described previously (25). HER2 was coupled to Aminolink resin following the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). HER2-coupled beads were mixed with 10-fold molar excess of rituximab, trastuzumab, or an equal volume of PBS, and rocked at room temperature for 1 h to allow binding to occur. Beads were then washed four times with vendor provided wash buffer, followed by incubation with 10-fold molar excess of C6.5db for 1 h. Beads were washed four times with wash buffer, pelleted down, mixed with 4Χ LDS sample buffer (Invitrogen, Carlsbad, CA) containing 40 mM dithiothreitol (DTT), and boiled at 70°C for 10 min. Samples were then spun down, equal volumes of the supernatants were loaded onto 4-12% gradient Bis-Tris gels (Invitrogen, Carlsbad, CA), and transferred onto nitrocellulose membranes. Blots were blocked with 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline), and C6.5db was detected by sequentially probing the membrane with anti-His antibody (#34670 Qiagen, Valencia, CA) and horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (#NA931V Amersham, Piscataway, NJ). Proteins were detected using Supersignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Blots were then stripped with Restore western blot stripping buffer (Pierce Biotechnology, Rockford, IL), and reprobed with HRP-conjugated sheep anti-human antibody (#NA933V Amersham, Piscataway, NJ) to detect rituximab and trastuzumab. Biodistribution and PET/CT imaging
CB.17/ICR severe combined immunodeficient (SCID) mice aged 6-8 weeks were obtained from the Fox Chase Cancer Center Laboratory Animal Facility. SK-OV-3, MDA-MB361/DYT2, and MDA-MB231 cells were implanted s.c in the inguinal region of mice at a density of 3 Χ 106 cells per mouse. At ? 6 weeks post-implantation, 0.2% Lugol's solution was added to the drinking water to block thyroid accumulation of radioiodine and biodistribution studies were conducted as described previously (23). Cohorts of mice (n = 5) received radioiodinated C6.5db via tail-vein injection and blood samples (? 70 ?L) were collected at 5 minutes p.i. and just prior to euthanasia. Animals were dissected and major organs were weighed and counted in a ?-well counter (Cobra Quantum, Packard Instruments, Meriden, CT). The retention of the radiolabel in tumor and normal tissues was expressed as a percentage of the injected dose per gram of tissue (% ID/gm). Values are reported as the mean and standard error of mean (SEM).
Individual cohorts of mice, injected with 124I-C6.5db, were imaged on a clinical Discovery LS clinical PET/computed tomography (CT) scanner (GE Healthcare, Milwaukee, WI) using a custom-built acrylic holder mounted on a patient bed as previously described (19). In addition, to the CT scan for attenuation correction, a CT scan with 0.6 mm slice thickness was also acquired for image registration. PET was acquired for 10 minutes in 2-D mode. PET images were reconstructed on a 128Χ128 matrix for a 30 cm-diameter field of view using ordered subsets expectation maximization (OSEM) algorithm. The PET, CT and fused images were visualized with the MIM software package (MIMvista Corp., Cleveland, OH).
For trastuzumab/rituximab blockade experiments with SK-OV-3 xenografts, cohorts of mice (n = 5) were implanted s.c. with 3 Χ 106 cells per mouse. Mice were treated with trastuzumab (10 mg/ Kg) or rituximab (10 mg/ Kg) combined with excess IgG2A (50 mg/ Kg) using the following two dosing regimens: A) one dose administered 3 d prior to injection of radioiodinated C6.5db or B) two doses administered 6 d and 3 d prior to injection of radioiodinated C6.5db. Tumors were subjected to IHC in formalin-fixed paraffin-embedded sections using antigen retrieval and staining with the anti-HER2 mAb, CB 11 (Biogenex, San Ramon, CA). Statistics
Statistical analyses to determine outliers (Grubbs test) and comparisons between different cohorts of mice (unpaired t test) were carried out using the online version of GraphPad (GraphPad Software, Inc.). Animals with uptake values that were considered outliers by the Grubbs test were removed from the analysis.
Results
Radiotracer preparations
The C6.5db was radiolabeled directly on tyrosine residues using Iodogen or indirectly on lysine residues using SHPP with efficiencies of 20 38% and 13 - 16%, respectively. Preparations ranged from 95% - 97.5% radiochemical purity as measured by instant thin layer chromatography (TLC) and the purified radiotracer had a specific activity of 0.5 0.57 ?Ci/?g protein. Immunoreactivity of all the preps, except that used for Figure 1, ranged from 69% - 78% active. The 124I-SHPP-C6.5db preparation used to evaluate the impact of tumor size on targeting had a 43.5% immunoreactivity.
Tumor size and impact on imaging with the C6.5db C6.5db uptake correlates with antigen expression
Effective use of the C6.5db as an immunoPET radiotracer requires that it accumulate to sufficiently high levels within a tumor to be detectable by the PET scanner, while maintaining a high tumor: normal tissue contrast. Antibody-based radiotracers target and are retained in tumor based on their binding to their cognate antigen on the tumor cell surface. Here, we examined the impact of two variables, tumor size and the density of HER2 on the tumor cell surface, on the ability of C6.5db to target and image subcutaneous xenografts in a SCID mouse model.
SCID mice received either two or four s.c. injections of SK-OV-3 tumor cells (1 Χ 106 copies HER2 / cell) at discrete anatomical locations at combinations of 2, 4, 6, and 8 weeks prior to intravenous (i.v.) injection of radioiodinated C6.5db. These implantations resulted in tumor sizes that ranged from 25 - 701 mg. In order to minimize non-specific uptake in iodine metabolizing tissues such as the thyroid and stomach, C6.5db was indirectly radiolabeled with 124I-SHPP and administered to animals and mice were then imaged 24 or 48 hours (h) post injection (p.i.) on a clinical PET/CT scanner (Discovery LS PET/CT, GE Healthcare) as described previously (19). As seen in Figure 1, at 48 h post-injection [124I-SHPP]-C6.5db demonstrated sufficient targeting of tumors in this model to allow for effective imaging of tumors weighing at least 50 mg (? 4 mm3). Quantification of tumor uptake, based on biodistribution analysis and the specific activity of the radiotracer, showed that 0.02, 0.06, 0.12, and 0.58 ?g [124I-SHPP]-C6.5db accumulated in the 25, 50, 95, and 360 mg tumors, respectively, at the time of imaging.
The impact of antigen density on tumor targeting of C6.5db was examined in a series of experiments using three different tumor cell lines (SK-OV-3, MDA-MB361/DYT2, and MDA-MB231) that differ in their levels of HER2 expression. The IHC staining patterns of tumor xenografts grown in SCID mice corresponds with the level of HER2 found on the surface of these cells when grown in vitro (data not shown). At 24 h p.i., tumor uptake of the 125I-C6.5db into each of the xenografts positively correlated with levels of HER2 expressed on the tumor cell surface (Table 1). At 24 h p.i. the approximate 3-fold higher level of HER2 on the surface of SK-OV-3 (1 Χ 106 HER2 per cell) as compared to MDA-MB361/DYT2 (3.7 Χ 105 HER2 per cell) positively correlated with a statistically significant 1.7-fold higher uptake (P = 0.002) of C6.5db in SK-OV-3 xenografts. The tumors in these two groups did not differ significantly (P = 0.06) in size. Consistent with the very low level of HER2 expression on the surface of MDA-MB231 cells (2.3 Χ 104 HER2 per cell), tumor uptake of the C6.5db in these xenografts did not differ significantly from that seen in blood, and was 6.2-fold (P < 0.0001) and 3.6-fold lower (P < 0.001) than that seen in SK-OV-3 and MDA-MB361/DYT2 xenografts, respectively. Using blood levels as a measure of clearance rates, no significant difference was seen between the clearance rates of the C6.5db from animals in the three groups. The increased tumor uptake in SK-OV-3 (0.76 ± 0.25 %ID/g) as compared to MDA-MB361/DYT2 (0.41 ± 0.04 %ID/g) was maintained at 48 h p.i., but was not statistically significant (P = 0.20), perhaps due to the fact that the MDA-MB361/DYT2 tumors were larger than the SK-OV-3 tumors analyzed at this time point (0.69 ± 0.2 g vs. 0.25 ± 0.1 g). Consistent with uptake seen at 24 h p.i., uptake into MDA-MB231 tumors at 48 h p.i. did not differ from that seen in blood (0.13 ± 0.01 vs 0.1 ± 0.01 %ID/g, P = 0.07) and was 7.6-fold (P = 0.02) and 4.1-fold (P = <0.001) lower than uptake into SK-OV-3 and MDA-MB361/DYT2 tumors, respectively.
Biodistribution of 125I-C6.5 diabody in SCID mice bearing s.c. xenografts of tumor cells with varying HER2 density The C6.5db Binds to an Epitope on HER2 Distinct from that Bound by Trastuzumab
Because of trastuzumab's role in the treatment of HER2-positive disease, its HER2-binding activity was taken into account as part of the development of the C6.5db for use as either a diagnostic radiotracer or therapeutic. We examined the ability of C6.5db and trastuzumab to compete for binding to HER2 in a series of in vitro experiments. Pretreatment of either SK-OV-3 or BT-474 cells (Figure 2A) with a saturating concentration of trastuzumab at 4°C did not inhibit the ability of C6.5db to bind to the cells as compared to treatment with an equal concentration of rituximab, the FDA-approved anti-CD20 mAb (26). In a reciprocal experiment, pretreatment with C6.5db also failed to compete for subsequent trastuzumab binding (Figure 2B). Co-immunoprecipitation experiments using purified HER2 extracellular domain (ECD; (25)) provided further support for this finding (Figure 2C). Immobilized HER2 ECD pretreated with a saturating concentration of trastuzumab was able to bind to and co-precipitate C6.5db at levels equal to that seen with HER2 ECD beads pretreated with either an equal amount of rituximab or vehicle as controls. In contrast, the anti-HER2 mAb pertuzumab (27) effectively competed the binding of C6.5db to both SK-OV-3 and BT-474 cells (Figure 2D) as compared to the rituximab control. In the reciprocal experiment C6.5db was able to partially compete the binding of pertuzumab (Figure 2E). Taken together, these data suggest that the C6.5db binds to an epitope distinct from that bound by trastuzumab (28). Rather C6.5db appears to bind to HER2 near, but probably distinct from, the epitope recognized by pertuzumab (27). This interpretation is further supported by the observation that intact IgG molecules based on the C6.5 scFv do not exhibit the therapeutic activity associated with pertuzumab (29).
C6.5db does not compete with trastuzumab for binding to HER2 in vitro Trastuzumab Treatment Inhibits Tumor Targeting by the C6.5db In Vivo
Based on our in vitro experiments that indicate trastuzumab and the C6.5db do not compete for HER2 binding we hypothesized that the C6.5db could effectively target HER2-positive tumors in mice pretreated with trastuzumab. Cohorts of SCID mice (n = 5) bearing SK-OV-3 tumor xenografts (mean tumor size=147 mg) were pretreated with a single dose of trastuzumab three days prior to administration of radioiodinated C6.5db. A second cohort of animals was pre-treated with the anti-CD20 IgG rituximab to control for non-specific effects associated with bulk IgG levels in the SCID mice. Tumor regression was not seen in response to trastuzumab-therapy over the 3-day treatment regimen (data not shown) but biodistribution analysis of 125I-C6.5db at 24 h p.i. (Table 2) revealed a statistically significant 42% decrease (P= 0.002) in tumor uptake of the radiotracer in animals pretreated with trastuzumab (1.58 ± 0.08 %ID/g) as compared to those pretreated with rituximab (2.71 ± 0.24 %ID/g). This decrease in radiotracer tumor targeting is evident in PET images of mice receiving 124I-C6.5db and imaged 24 h p.i. (Figure 3). Tumor uptake in animals treated with either trastuzumab or rituximab for six days showed the same statistically significant decrease (P = 0.002) in tumor uptake in the trastuzumab treated cohort (1.16 ± 0.20 %ID/g) as compared to the rituxumab-treated controls (2.29 ± 0.16 %ID/g). This decrease in C6.5db uptake was also seen in SCID mice bearing s.c. BT-474 BrCa xenografts pretreated with trastuzumab for six days (Table 3). Animals pretreated with trastuzumab showed a ?60% decrease in tumor uptake of 125I-C6.5db at 24 h p.i. as compared to rituximab treated controls (%ID/g of 1.42 ± 0.18 for trastuzumab vs. 3.55 ± 0.39 for rituximab, p=0.003). This loss of tumor targeting does not correlate with a large decrease in HER2 levels on the surface of the tumor cells. SK-OV-3 tumor xenografts from mice treated for up to one week with either trastuzumab or rituximab were subjected to IHC. As seen in Figure 4A, SK-OV-3 and BT-474 tumors from trastuzumab treated animals exhibited similar HER2 staining patterns to those from animals treated with rituximab. Consistent with these results, treatment of SK-OV-3 and BT-474 (Figure 4B) cells in vitro with trastuzumab (10 ?g/mL) over a time course of 72 hours failed to decrease the levels of HER2 on the cell surface when measured by FACS with a non-competing anti-HER2 antibody. Interestingly, despite a lack of physical competition for binding epitopes, trastuzumab treatment inhibited C6.5db binding, as measured by FACS, compared to non-treated controls over the same 72-hour time frame. The exact mechanism by which trastuzumab treatment inhibits C6.5db binding is not yet understood, and studies are underway to investigate this further.
Discussion
The quantitative nature of PET facilitates the accurate measurement of tracer concentration within a lesion and such measurements correlate well with those obtained through standard biodistribution studies (19, 30, 31). Monoclonal antibodies, combined with flow cytometry, have long been used to quantitatively measure the expression of cell surface proteins. This has led us, and others, to hypothesize that antibody-based radiotracers, coupled with PET, can be used to measure antigen expression in vivo. In addition, significant data in the literature suggests that smaller antibody fragments, affibodies, or engineered antibody fragments are poised to be more effective than intact mAbs as PET radiotracers due to their faster blood clearance and higher tumor:background ratios (32-34)).
At the most basic level, response to mAb-based therapies requires that the target protein be expressed on the surface of tumor cells, and that the therapeutic mAb effectively target and accumulate to sufficient levels within the tumor. The ability to monitor each of these variables has the potential to guide patient selection and treatment plans. In the setting of HER2-positive breast cancer, response to trastuzumab positively correlates with the level of HER2 expression. High-level overexpression in biopsied tumor tissue, as measured by IHC or FISH, is the critical criteria for treatment eligibility. Thus we and others (34) have speculated that a molecular imaging based approach to evaluate HER2 expression across a patient's entire tumor burden could provide a more complete analysis of HER2 expression, potentially providing a better prediction of initial response to trastuzumab-based therapy or even obviating the need for invasive biopsy procedures. The data we present here demonstrates that tumor uptake of the C6.5db is dependent upon antigen density on the surface of the tumor cells and suggest that C6.5db-based radiotracers may be useful for evaluating the levels of HER2 expression on tumor cells in vivo, and by extension predicting initial response to trastuzumab therapy. Data from Cai et al (35) suggests that this approach may be applicable to target antigens beyond HER2. Uptake of [64Cu-DOTA]-cetuximab correlated with the level of EGFR expression across a number of tumor models, suggesting that immunoPET-based determination of antigen density could be applied to a broader range of target antigens.
Biological properties of the target antigen, the strategy employed to radiolabel the tracer, and the intended imaging application are all critical components in radiotracer design. ImmunoPET images obtained with a residualizing radionuclide, such as the 64Cu or 89Zr used to label cetuximab by Cai (35) and Aerts (36), depict the cumulative antibody bound to, and internalized by the cells over the course of the experiment. This is ideal for the purpose of lesion detection but potentially suboptimal for monitoring antigen levels. When targeting rapidly internalizing antigens, such as EGFR or HER2, the long half-life of intact mAbs coupled with residualizing radionuclides would be predicted to obscure internalization rates, and thereby provide an inaccurate estimate of level of antigen expression. The radiohalogen 124I has a physical half-life that pairs appropriately with the biological half-lives of mAb-based tracers (33). However, it has been speculated that 124I is inappropriate for labeling of mAbs because internalization and degradation leads to rapid loss of the iodine from cells resulting in both insufficient tumor:normal tissue contrast for imaging (37) and unwanted uptake by tissues, such as the thyroid, that express the Na/I symporter. Engineered antibodies such as the C6.5db have the potential to function as effective radiotracers, in part, because their rapid systemic clearance leads to positive tumor-to-blood ratios early after administration (19). Our results demonstrate that sufficient uptake of 124I-C6.5db is achieved to afford PET detection of tumors as small as 50 mg at 48 h p.i. in our preclinical model. In addition, we argue that 124I-C6.5db, and by extension other 124I-mAbs, provide a representation of the antibody bound to the tumor cell surface at the time of imaging, thus decreasing the impact of internalization rates on tumor signal and potentially providing a more quantitative approach to measuring either inherent differences in antigen expression between tumors or changes in antigen expression within a tumor in response to therapy. However, the positive impact of the rapid clearance of C6.5db is balanced by its negative effect on the limiting time available for the antibody to accumulate to high levels in the tumor (38). This rapid clearance could in principle be exploited to enable the use of residualizing isotopes with similar quantitative results. Although thyroid uptake was not quantified in this study we have demonstrated in previous work that the use of 124I in conjunction with a partially residualizing labeling strategy (e.g. SHPP) does not dramatically alter the performance of the C6.5db as a radiotracer and decreases thyroid uptake (19). This suggests that such a labeling strategy could be used in conjunction with thyroid blocking to reduce thyroid exposure in patients and still provide quantitative analysis of HER2 levels.
Accumulation of antibody-based therapeutics to sufficient levels within a tumor is essential for therapeutic efficacy. The decrease in overall tumor uptake seen with the C6.5db upon trastuzumab treatment implies that trastuzumab is effectively targeting tumor in our preclinical models to induce this effect. It is intriguing to speculate that molecular imaging with agents such as the C6.5db, when used in the clinical setting, could potentially shed light on whether and how trastuzumab is targeting lesions in a patient. In addition, significant effort is ongoing in the preclinical setting to understand both how physical properties of mAbs (e.g. intrinsic affinity, molecular size, PK) dictate tumor targeting and how those properties can be modified to improve antibody targeting (for review see (39). Imaging strategies that can function as companion diagnostics during the development process have the potential to aid in translation of new therapeutic antibodies during translation into the clinic.
Antibody-based cancer therapeutics can induce anti-tumor effects through a number of mechanisms of action including inhibiting signal transduction and/or focusing the anti-tumor effects of the immune system (40). Since its initial approval more than a decade ago trastuzumab has become standard-of-care for HER2-postive breast cancer. Despite this fact, trastuzumab's mechanism of action has yet to be definitively identified. It most likely functions through multiple processes including antibody-dependent cellular cytotoxicity (ADCC), inhibiting HER2 shedding, and blocking signaling (for review see (41)). Although somewhat controversial (42) and its relevance to the clinical setting not yet fully demonstrated (43-45), trastuzumab-induced down-regulation of HER2 has also been reported in both in vitro cell culture (46, 47) and xenograft models (48, 49). Our IHC and FACS results are in alignment with clinical findings, in that trastuzumab treatment failed to induce detectable levels of HER2 down-regulation in our model systems. Consistent with our findings, McLarty et al report that trastuzumab treatment (4 mg/kg) of athymic mice bearing MDA-MB361 xenografts followed by SPECT imaging (3 d p.i. of trastuzumab) with 111In-diethylenetriaminepenta-acetic acid-pertuzumab (111In-DTPA-pertuzumab) showed a significant decrease in the tumor uptake of 111In-DTPA-pertuzumab, despite no apparent decrease in HER2 levels by IHC (32). Interestingly, chronic treatment (3 weeks) induced a significant decrease in HER2 levels by IHC, and was associated with loss of HER2-positive tumor cells. In our studies, chronic treatment of mice bearing SK-OV-3 or BT-474 xenografts failed to induce an obvious change in HER2 expression (data not shown), similar to the situation seen in the clinic (43-45). It is worthy of note in this context that when McLarty et al compared trastuzumab-induced changes in HER2 density between SKBR-3 (high HER2 expression) and MDA-MB361 (moderate HER2 expression) BrCa cells in culture, they found that the effects of trastuzumab on HER2 density was more profound in MDA-MB361 cells than SKBR-3 cells (32). One possible explanation for the apparent differences in HER2 down-regulation seen in these studies may be cell line-dependent variability in receptor down-regulation.
Despite an inability to detect down-regulation of HER2 by FACS and IHC, our results are consistent with those of other groups (42, 50) and suggest that in vivo targeting of 124I-C6.5db is perturbed as an early response to trastuzumab-based therapy. This is particularly true in the context of the results obtained with 111In-DTPA-pertuzumab and our data demonstrating that the C6.5db binds to HER2 near the epitope bound by pertuzumab. The mechanism by which trastuzumab treatment inhibits the targeting of both pertuzumab and C6.5db-based PET radiotracers is unclear. The epitopes for C6.5db and pertuzumab are located in domain II of the HER2 extracellular domain, distinct from the domain IV epitope bound by trastuzumab (51). This, coupled with the inability of trastuzumab to compete with C6.5db for HER2 binding, suggested that the therapeutic levels of trastuzumab circulating in the animals, or by extension patients, should not compete for HER2 binding and therefore should not have resulted in the decreased in vivo targeting, nor the time-dependent decrease in binding to cells treated with trastuzumab in culture. In light of trastuzumab's complicated mechanism of action, it is interesting to speculate that trastuzumab treatment results in a physical change to the receptor, such as altered clustering or dimerization patterns. Data from Kani et al (52) demonstrating that binding of antibodies to HER2 alters its partitioning in the membrane, particularly with regards to localization with HER3, coupled with recent data from Junttila et al (53) demonstrating that trastuzumab inhibits ligand-independent signaling through the HER2/HER3 heterodimer supports this hypothesis. The manner in which this would result in decreased binding of C6.5db is not yet clear. Altered packing of the receptor may result in steric inhibition of C6.5db binding to domain II. Alternatively, binding of trastuzumab to HER2 may prevent cross-linking of two HER2 molecules by C6.5db, forcing monovalent association of the radiotracer. Monovalent binding of the C6.5db is predicted to lower its functional affinity 40-fold, decrease its cell surface residence from 5 hr to 5 min, and significantly lower tumor uptake in vivo (54). Efforts to more fully address the basis for the decreased uptake and determine whether this decreased binding can function as a measure of therapeutic response are underway.
Beyond treatment with trastuzumab, agents such as the hsp-90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-demethoxygeldanamycin (17-DMAG) have been reported to induce rapid and transient degradation of HER2 as part of their proposed mechanisms of action and this down-regulation has been imaged with trastuzumab-based radiotracers (20, 55, 56). The ability to monitor the efficacy of this type of agent in a robust manner, with dedicated radiotracers such as the C6.5db, has the potential to improve the development and clinical outcome associated with its use. SmithJones (20) demonstrated that 68Ga-trastuzumab F(ab')2 can detect a 50% decrease in HER2 expression in BT-474 xenografts treated with Hsp-90 inhibitors and that the change in HER2 density can be detected before subsequent tumor inhibition is apparent by FDG-based imaging (57). Interestingly, and consistent with our inability to observe down-regulation of HER2 upon trastuzumab treatment, IHC was unable to detect less than a 70% reduction in HER2 expression in BT-474 BrCa xenografts in athymic mice treated with the heat shock protein-90 (Hsp-90) inhibitor 17-demethoxygeldanamycin (17-DMAG) (55). Thus, differences in trastuzumab-based HER2 down-regulation seen between preclinical and clinical studies may be due, at least in part, to the inability of IHC to sensitively detect those changes.
In conclusion, we hypothesize that molecular imaging with antibody-based radiotracers has the potential to make a positive impact in both guiding the development and use of targeted therapies that inhibit either the activity or expression of cell-surface proteins. The targeting properties of engineered antibody fragments, such as the C6.5db, are well suited for PET imaging and can provide specific information regarding the expression and modulation of targets in a non-invasive manner, regardless of their location. One important future goal is to test C6.5db in transgenic mouse models that express HER2 antigen on normal tissues and shed those antigens into the bloodstream, similar to the clinical setting (58). The development of trastuzumab has revolutionized the treatment of both early and advanced stage HER2-positive BrCa, but acquired resistance to treatment is frequently encountered in advanced disease, and in a small proportion of early stage patients after adjuvant therapy. It is interesting to speculate that molecular imaging, as with the C6.5db or similar antibody-based agents, may serve as an effective method to monitor patients for initial response as well as for development of resistance to trastuzumab.
Statement of Translational Relevance
Strategies to both predict and monitor patient response are critical for the effective development and clinical implementation of targeted therapies. Molecular imaging strategies, such as positron emission tomography (PET) are well suited to this role. We have previously described an antibody-based PET radiotracer, C6.5 diabody (C6.5db), which selectively binds to HER2. Here we demonstrate that imaging with the C6.5db has the potential to quantify HER2 levels in vivo thus predicting response to trastuzumab therapy. We also provide data to suggest that C6.5db-based PET imaging may be an effective strategy for monitoring patient response to trastuzumab or other HER2-directed therapies.
Acknowledgments
We are grateful to Drs. Gregory P. Adams and Louis M. Weiner for expert advice. We thank the Imaging Facility, Histopathology Facility and Laboratory Animal Facility at the Fox Chase Cancer Center for their expert technical assistance.
Financial Support: HRSA grant to the American Russian Cancer Alliance (MKR), NIH postdoctoral training grant CA09035 (SR) NIH CORE grant CA06927 and through support by an appropriation from the Commonwealth of Pennsylvania (MKR).
Footnotes
Conflict of Interest: Matthew K. Robinson is a member of the scientific advisory board of Avipep Pty, Ltd. Avipep has licensed the C6.5db from University of California, San Francisco. James D. Marks is an inventor of the C6.5db that was licensed to Avipep Pty, Ltd
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