Immunological materials

The present invention is concerned with a cytotoxic agent comprising an immunoglobulin specific for antigens on the surface of the cell to be killed having 1-10 polymer carrier molecules covalently bonded thereto and the polymer carrier having 5-500 molecules of a cytotoxic drug covalently bound. The compounds of the present invention are useful as anti tumor agents.

The present invention encompasses a cytotoxic agent comprising an 
immunoglobulin specific for antigens on the surface of cells to be killed 
having 1-10 polymer carrier molecules covalently bound thereto, said 
polymer carrier having about 5-500 molecules of a cytotoxic drug 
covalently bound thereto and said polymer carrier having a molecular 
weight of 5000-500,000 and free carboxyl, amino or cycloimidocarbonate 
groups for covalently bonding, said cytotoxic drug also having amino or 
carboxyl groups available for covalent bonding. 
Cytotoxic agents of the present invention are represented by the formula A 
##STR1## 
WHEREIN Ig represents an immunoglobulin specific to antigens on the 
surface of cells to be killed; the polymer carrier has a molecular weight 
of 5000-500,000 and is selected from the group of polymers comprising: 
A. DEXTRAN ACTIVATED WITH CYANOGEN BROMIDE TO FORM CYCLOIMIDOCARBONATE AND 
FUNCTIONALIZED WITH --NH(CH.sub.2).sub.x CO.sub.2 H, or 
--NH--(CH.sub.2).sub.x --NH.sub.2, wherein x is 3-8, 
B. AMINOETHYLATED DEXTRAN, 
C. POLYGLUTAMIC ACID 
D. POLYASTIC ACID, 
E. POLYARGININE, 
F. SERUM ALBUMEN, 
G. FIBRINOGEN, 
H. .gamma.-GLOBULIN, 
I. POLYLYSINE (SOLUBILIZED BY REACTION WITH CITRACONIC ANHYDRIDE), 
J. COPOLYMERS OF LYSINE-PHENYLALANINE, LYSINE-TYROSINE, AND GLUTAMIC 
ACID-TYROSINE; 
Cd represents a covalently bound cytotoxic drug selected from the group 
comprising: 
P-[N,N-bis(2-chloroethyl)]phenylenediamine, 
P-[bis(2-chloroethyl)amino]phenylalanine, 
4-p-[bis(2-chloroethyl)amino]phenyl butyric acid, 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide, 
2-amino-N-[p-bis(2-chloroethyl)amine]phenyl-3- hydroxy-2-hydroxymethyl 
propionamide, 
N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}- benzyl}-L-glutamic 
acid, 
Cytosine arabinoside, cytosine arabinoside monophosphate, 
0-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid, 
Daunomycin, doxorubicin, and diphtheria toxin; 
B.sub.1 represents a covalent bond between Ig and polymer carrier of the 
formula 
##STR2## 
--N .dbd. CH--(CH.sub.2).sbsb.3--CH .dbd. N--; 
B.sub.2 represents a covalent bond between the polymer carrier and CD of 
the formula 
##STR3## 
m = 5-500; 
n = 1-10. 
Polymer carriers of the present invention have a molecular weight range of 
between 5000 and 500,000 and are functionalized so as to react with both 
cytotoxic drugs and amino or carboxylic acid groups of immunoglobulins. 
Suitable polymer carrier and functional binding groups are polypeptides 
having free amino or carboxyl group for bonding such as: polyglutamic acid 
(carboxyl), polylysine (amino), polyethyleneimine(amino), polyaspartic 
acid (carboxyl), polyarginine (amino); copolymers such as: 
glutamic acid-tyrosine (carboxyl) 
lysine-phenylalanine (amino) 
lysine-tyrosine amino; 
polypeptidyl proteins and multichain polyamino acids (carboxyl and amino) 
as described by Sela et al., Biochem J. 85 223 (1962); 
proteins such as: 
______________________________________ 
human serum albumin 
human fibrinogen carboxyl and amino 
human .gamma.-globulin 
______________________________________ 
Also suitable are functionalized dextrans such as aminoethylated dextran as 
described by Harding, Am. N.Y. Acid. Sci. 186, 270 (1971) (amino). Dextran 
activated with cyanogan (amino or carboxyl) broxmide and functionalized 
with H.sub.2 N--(CH.sub.2).sub.x --NH.sub.2 or H.sub.2).sub.x --CO.sub.2 H 
to 
form dextrans represented by: 
##STR4## 
wherein x is 3-8, preferably 6. The cyanogen bromide forms a reactive 
cycloimidocarbonate 
##STR5## 
when reacted with dextran. 
It is generally preferred to have only 1-10 polymer carriers covalently 
linked to the Ig so as not to disturb the antibody activity of the Ig. In 
this manner the immunoglobulin specifically directs or homes large volumes 
of cytotoxic drug to the surface of the cell to be killed. 
Cytotoxic drugs useful for practicing the present invention are as follows: 
##STR6## 
The above are preferred agents against neoplastic disease. 
5-Fluorodeoxyuridine, o-(N,N-bis-2-chloropropyl)-carbamoylylycollic acid, 
daunomycin, doxorubicin, and diptheria toxin are likewise useful. It is, 
however, to be recognized that other cytotoxic drugs with suitable 
functional groups can be bound to polymer carriers which, in turn, are 
bound to an immunoglobulin. The latter serving as a means of directing the 
drug to the cell to be killed. 
The bond between the cytotoxic drug and the polymer carrier should be such 
that the cytotoxic drug is cleaved enzymatically on the target cell 
surface. The peptide bond is especially preferred since proteases are on 
cell surfaces and therefor the cytotoxic drug which is peptide bound to a 
polymer carrier which in turn is bound to Ig can be selectively delivered 
to the surface of the cell to be killed and then released to provide a 
high concentration of cytotoxic drug at the target cell. 
Structures of immunoglobulins are remarkably similar, Science 189, 1075 
(1975), in that gross structural features and amino acid content varies 
only slightly from one immunoglobulin to another. 
Immunoglobulins specific to antigens on the surface of cells to be killed 
and techniques for their production are known. Some representative 
immunoglobulins are: 
a. goat anti-CEA Ig from human colonic adenocarcinoma, Hsu-Fu Chao et al., 
Res. Comm. in Chem., Path, and Pharmacol 9, 749 (1974). 
b. Ig from rabbit anti-acute lymphoblastic leukemia serum, Greaves et al., 
Clin. Immunol. and Immunopath. 4, 67 (1975). 
c. Ig from various primate anti-leukemia antisera, anti-acute lymphoblastic 
leukemia, acute myleoblastic leukemia, chronic lymphoblastic leukemia, and 
chronic granulocytic leukemia, all as described by Mohanakumar, et al., J. 
Nat. Cancer Inst. 52, 1435 (1974). 
d. human anti-human meningioma Ig., Winters and Rich, Int. J. Cancer 15, 
815 (1975). 
e. rabbit anti-human prostate Ig, Moncure et al., Cancer Chemotherapy 
Reports 59, 105 (1975). 
f. rabbit anti-human chorionic gonadotropin serum 
g. goat anti-human lymphoid cell Ig. 
h. rabbit anti-mouse (EL4) lymphoid cell Ig. 
i. Ig against lymphocyte surface antigens for example Ia antigens. Staines 
et al., Tissue Antigens 393 (1975). 
j. Ig against surface antigens on antibody producing cells. 
k. Ig against fungal cell surface antigens. Fukazawa et al., J. Bact. 95 
754 (1968). 
l. Ig against Bacterial cell surface antigens. 
m. Ig against antigen on the surface of cells involved in inflammatory 
response. 
Polymer carriers are covalently bound to the above set out immunoglobulins 
by way of free carboxyl or amino groups on the immunoglobulin. Water 
soluble carbodiimides such as 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide conveniently forms a 
peptide bond between a carboxyl group on the immunoglobulin and an amino 
group on a polymer carrier or between an amino group on the immunoglobulin 
and a carboxyl containing polymer carrier. Alternatively immunoglobulins 
(Ig) are covalently linked to polymer carriers having free amino groups by 
condensation with glutaraldehyde, a diimide linkage as shown in the 
following scheme. 
##STR7## 
Some preferred embodiments of the present invention are set out in the 
formula B: 
##STR8## 
wherein m is 5-100, n is 1-10 and dextran molecular weight, 500-500,000, 
preferably 5000-50,000 and most preferably 15,000-20,000, activated by 
cyanogen bromide and functionalized with 1,6-diaminohexane. 17,000 Is a 
very desirable molecular weight and H.sub.2 N--(CH.sub.2).sub.6 --NH.sub.2 
can be replaced with --(CH.sub.2).sub.x --x = 3-8. 
In a similar manner cytotoxic drugs, CD(b), CD(d), and CD(e) are bound to 
dextran as polymer carrier and m, n, x and molecular weight variables are 
the same as in B. 
C represents another preferred embodiment: 
##STR9## 
wherein m is 5-100, n is 1-10, the polymer carrier is polyglutamic acid 
having a molecular weight of 5,000-500,000, preferably 5,000-100,000, and 
most preferably about 35,000; or C' using CD(b) is preferred: 
##STR10## 
wherein m, n and molecular weight of the polyglutamic acid is as described 
above in C. 
In a similar manner cytotoxic drugs (d) and (e) (C"and C'") are bound to 
polyglutamic acid wherein the molecular weight parameters of the polymer 
carrier, m, and n, are as earlier defined in C. The peptide linkages are 
preferably achieved by use of 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Generally 10-100 CD are 
bound to each Ig by way of the polymer carrier. 
##STR11## 
D represents another preferred embodiment 
##STR12## 
wherein m is 5-100, n is 1-10, and the molecular weight of the polylysine 
polymer carrier is 5,000-500,000, preferably 5000-100,000. The cytotoxic 
drug (b) or (f) may be used in place of 
4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid. In addition a preferred 
embodiment is obtained fixing the parameters in formula D as shown and 
replacing polylysine with polyethylenimine of the above set out molecular 
weight range with molecular weight 20,000 being especially preferred. Free 
amino groups on the polymer carrier can be reacted further with citraconic 
acid anhydride to function the amide and aid in solubility of the 
cytotoxic agent. 
Table I illustrates preferred embodiment of the present invention. 
TABLE 1 
__________________________________________________________________________ 
Ig B.sub.1 B.sub.2 
Polymer Carrier 
CD 
__________________________________________________________________________ 
##STR13## 
##STR14## 
Polyglutamic acid 
a,b,d,e 
##STR15## 
##STR16## 
Polylysine M.W. 5000-100,000 solabilized by reaction 
ith citraconic anhydride 
b,c,f 
or 
NCH(CH.sub.2).sub.3CHN 
##STR17## 
or 
NCH(CH.sub.2 .sub.3CHN 
##STR18## 
Polyethyleneimine M.W. 5,000-100,000 
b,c,f 
##STR19## 
##STR20## 
aminoethylaled dextran 
b,c,f 
or 
NCH(CH.sub.2).sub.3CHN 
##STR21## 
##STR22## 
dextran activated with CNBr and functionalized 
b,c,f 
or 
NCH(CH.sub.2).sub.3CHN 
H.sub.2N(CH.sub.2).sub.6NH.sub.2 
##STR23## 
##STR24## 
dextran activated with CNBr and functionalized with 
H.sub.2N(CH.sub.2 .sub.6CO.sub.2 H 
a,b,d,e 
__________________________________________________________________________ 
The present invention also encompasses polymer carriers suitable for 
binding to immunoglobulin having a molecular weight between 5,000 and 
500,000 and having 5-500 molecules of cytotoxic drug covalently bound 
thereto. These embodiments of the present invention are represented by the 
following formula: 
##STR25## 
where the terms polymer carrier, B.sub.2, CD, and m are as previously 
defined. 
A preferred embodiment of polymer carrier is polyglutamic acid having a 
molecular weight of 5,000-100,000, most preferably 35,000; 
B 2 is 
##STR26## 
CD represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, 
p-[bis[2-chloroethyl)amino]phenylalamine, 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide, or 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl 
propionamide; 
Another preferred embodiment is dextran activated with cyanogen bromide and 
functionalized with 1,6-diaminohexane having a molecular weight of 
5,000-100,000, most preferably 17,000, 
B.sub.2 is 
##STR27## 
CD represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, 
p-[bis(2-chloroethyl)amino]phenylalanine, 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide, or 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl 
propionamide; and 
m = 5-100. 
Another preferred embodiment is aminoethylated dextran having a molecular 
weight of 5,000-500,000 in which 
CD represents p-[bis(2-chloroethyl)amino]phenylalanine or 
4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid, 
##STR28## 
and 
m = 5-100 
Another preferred embodiment is polylysine, molecular weight of 
5,000-100,000, reacted with citraconic acid anhydride in which 
CD represents p-[bis(2-chloroethyl)amino]phenylalanine, 
4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid, or 
N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}benzyl}-L-glutamic 
acid; 
##STR29## 
and 
m = 5-100. 
Also preferred is polyethyleneimine having a molecular weight of 
5,000-100,000, most preferably 20,000 in which 
CD represents p-[bis(2-chloroethyl)amino]phenylalanine, 
4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid, or 
N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}benzyl}-L-glutamic 
acid; 
##STR30## 
and 
m = 5-100. 
Compounds of the present invention are preferably administered 
intravenously and they are especially advantageous by reason of their low 
toxicity. For example, p-[N,N-bis(2-chloroethyl)]phenylenediamine unbound 
has an LD.sub.50 of 5 compared to an LD.sub.50 of 200 for that compound 
bound to polyglutamic acid. Dosages of compounds of the present invention 
can be based on dosages of known cytotoxic agents. For example (Melphalan) 
p-[N,N-bis(2-chloroethyl)amino]-phenylalanine is recommended at a dose of 
6 mg daily for 2-3 weeks whereas 1-20 times of this drug bound to a 
polymer carrier according to the present invention for a like period of 
time would constitute an effective dose. In a similar manner 
(chloroambacil) 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid is 
recommended at 0.1-0.2 mg/kg/day for 3-6 weeks. That dosage can be 
increased up to 20 times when bound according to methods of the present 
invention. Compounds of the present invention preferably administered in a 
suitable manner by intraperitaneal injection of a dose having 0.1-0.2 
mg/kg/day of cytotoxic agent bound to the polymer carrier and 
immunoglobulin to a warm blooded animal. The utility of the present 
compound is further disclosed in the hereinafter set forth examples.

The following examples are presented to further illustrate the present 
invention. They should not be construed as limiting it either in spirit or 
in scope. In these examples quantities are indicated in parts by weight 
unless parts by volume are specified, and temperatures are indicated in 
degrees Centigrade (.degree. C.). The relationship between parts by weight 
and parts by volume is the same as that existing between grams and 
milliliters. 
EXAMPLE 1 
500 Mg of dextran (Molecular Weight 17,700) is dissolved in 500 ml of 
distilled water and the pH is adjusted to 11.0 with sodium hydroxide. 400 
Mg of cyanogen bromide in 1.6 ml of acetonitrile is added to the dextran 
solution dropwise with rapid stirring at 23.degree. C. This mixture is 
stirred for 10 minutes with the pH maintained at 11.0 by addition of 
sodium hydroxide and then 100 mg of diaminohexane dissolved in 2.5 ml of 
water is added with stirring; the pH is lowered to 9.0 with hydrochloric 
acid and stirring is continued for 5 minutes. This procedure provides 
dextran having three hydroxyl groups functionalized with 
##STR31## 
To this functionalized dextran solution is added dropwise a solution of 250 
mg of p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride in 5 ml of 
ethanol containing 2% w/v hydrochloric acid and diluted with 20 ml of 60% 
aqueous polylene glycol containing 1.2% w/v potassium hydrogen phosphate. 
During the addition the pH is allowed to fall to 6.5 and is maintained at 
that level by the addition of sodium hydroxide. The mixture is stirred for 
15 minutes at 23.degree. C. cooled to 4.degree. C., and transferred to 
membrane ultra filtration apparatus for diafiltration with water. 
Diafiltration is continued until the effluent is free of uncombined 
p[N,N-(bis(2-chloroethyl)]phenylenediamine. The solution is concentrated 
by ultrafiltration to 1/5 the reaction volume and then freeze dried to 
provide dextran having 3 hydroxyls functionalized with 
##STR32## 
and about 15 molecules of p-[N,N-(di-2-chloroethyl)phenylelediamine 
mustard bound as 
##STR33## 
This functionalized dextran polymer having 
N,N-[bis(2-chloroethyl)]phenylenediamine covalently linked thereto is then 
bound to immunoglobulin (Ig) specific to the cell to be killed--for 
example, rabbit antiserum against mouse lymphoma cells (EL4) made specific 
by repeated absorption with normal mouse spleen cells, Br.J. Cancer 30 
297-304 (1974). 
6 Ml of phosphate buffered saline pH 7.2 containing 180 mg of 
functionalized dextran polymer carrier is mixed with 6 ml of the same 
buffer containing 18 mg of rabbit anti mouse EL4 lymphoma Ig. To this 
solution is added 1.2 ml of 0.1% w/v glutaraldehyde in 12 .times. 0.1 ml 
increments over a 5 minute time period. The mixture is rotated at 
4.degree. C. for 30 minutes, then centrifuged at 35,000 .times. g for 10 
minutes and the supernate discarded. The precipitate is resuspended in 25 
ml of 40% ammonium sulfate and recentrifuged as above. The precipitate is 
redissolved in 4 ml of phosphate buffered saline and dialysed exhaustively 
to remove ammonium sulfate. 
The resulting cytotoxic agent -- polymer carrier -- Ig, is characterized as 
having 31 mg/ml of protein Folin and Cioculteau, J. Biol. Chem. 73 627 
(1927), and the alkylating activity is determined by the method of 
Espstein et al. J. Analyt. Chem. 27 1435-1439 (1955), to be 2.25 mg/ml for 
the dextran carrier-cytotoxic agent moiety. Thus the complex contains 36 
p-[N,N-bis(2-chloroethyl)]phenylenediamines per Ig, indicating about 2 
dextran carriers per Ig. 
To show the effectiveness of the complex as an antitumor agent in vivo 
several groups of five C56BL/6 mice were inoculated with 5 .times. 
10.sup.4 EL4 cells intraperitoneally. This is approximately 10,000 times 
the LD.sub.50 challenge dose. After 24 hours the mice receive the first of 
four daily injections of the complex or controls. The results are shown in 
the following table. 
__________________________________________________________________________ 
Dose per Injection 
Drug Alkylating 
Ig Median % Mice Free 
activity in Survival 
of tumor at 
Treatment .mu.g/injection 
(mg) 
Time (Days) 
day 60 
__________________________________________________________________________ 
Saline -- -- 13 0 
RIg alone -- 6.2 
25 20 
PDM-DEX 450 -- 16 0 
+ 
PDM-DEX 450 
RIg unlinked 6.2 
28 0 
PDM-DEX-RIg 
450 6.2 
&gt;60 100 
__________________________________________________________________________ 
P M = p-[N,N-bis[2-chloroethyl)]phenylenediamine (CDa) 
DEX = dextran polymer carrier activated with CNBr and functionalized with 
H.sub.2 N-(CH.sub.2).sub.6 -NH.sub.2 
RIg = rabbit antiserum against mouse EL4 lymphoma 
EXAMPLE 2 
Following the procedure set out in Example 1 and replacing 
p-[N,N-bis(2-chloroethyl)]phenylenediamine with an equivalent quantity of 
p-[bis(2-chloroethyl)amino]phenylalanine provides a polymer carrier having 
a cytotoxic drug bound thereto as 
##STR34## 
In a similar manner and using an equivalent quantity of 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide provides 
dextran having a cytotoxic drug bound thereto as 
##STR35## 
Using an equivalent quantitity of 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl 
propionamide provides a dextran polymer carrier having cytotoxic drug 
bound as: 
##STR36## 
Also following the procedures in Examples 1 and 2 for binding rabbit anti 
sera against mouse EL4 any of the following known Ig may be bound to a 
like manner using equivalent quantities. 
a. Goat anti-CEA Ig from human colonic adenocarcinoma, Hsu. Fu Chao et al., 
Res. Comm. in Chem. Path. and Pharmacol. 9, 749 (1974). 
b. Ig from rabbit anti-acute lymphobastic leukemia serum, Greaves et al., 
Clin. Immunol. and Immunopath. 4 67 (1975). 
c. Ig from various primate anti-leukemia antisera, anti-acute lymphoblastic 
leukemia, acute myleoblastic leukemia, chronic lymphoblastic leukemia, and 
chronic granulocytic leukemia cell as described by Mohanakumar et al., J. 
Nat. Cancer Inst. 52, 1435 (1974). 
d. Human anti-Human meningioma Ig. Winters and Rich, Int. J. Cancer 15, 815 
(1975). 
e. Rabbit anti-human prostrate Ig, Moncure et al., Cancer Chemotherapy 
Reports 59 105 (1975). 
f. Rabbit anti-human chorionic gonadotropin serum. 
EXAMPLE 3 
Preparation of Drug-Carrier (PDM - PGA) 
p-[N,N-(bis-2-chloroethyl)]-phenylenediamine -- polyglutamic acid 
1. 250 mg polyglutamic acid PGA(m.wt. 35,000) is dissolved in water by 
addition of 1N NaOH dropwise until all of the PGA is in solution. The pH 
is adjusted to 7.0 by the addition of 1N NaOH dropwise until all of the 
PGA is in solution. The pH is adjusted to 7.0 by the addition of 1N 
hydrochloric acid if necessary. (The PGA used is polyL- glutamic acid). 
2. 1g of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is added to 
the PGA solution at room temperature. 
3. 140 mg of PDMp-[N,N.(bis-2-chloroethyl)]-phenylenediamine-HCl is 
dissolved in 3.5 ml ethanol containing 2% w/v hydrochloric acid and is 
diluted with 10.5 ml 60% aqueous propylene glycol containing 1.2% w/v 
K.sub.2 HPO.sub.4. 
4. The above PDM solution is added dropwise to the stirred PGA and EDC 
mixture, the pH being maintained at from 6 to 7 by the addition of sodium 
hydroxide where necessary. 
5. The above mixture is cooled to 4.degree. C. and an equal volume of cold 
0.2 M Na-acetic acid buffer [pH 4.0] is added. 
6. The white gelatinous precipitate formed is centrifuged cold and washed 
by three repeated suspensions and centrifugations in cold acetate buffer. 
7. The washed precipitate is re-dissolved in 1N NaOH to a final volume of 5 
ml, adjusting the pH to 7 where necessary with 1N hydrochloric acid. 
The drug/carrier combination has the following characteristics: 
1. The preparation is soluble in water above pH 4.5. 
2. When scanned in the U.V. range the preparation has an absorption maximum 
at 277 nm due to the presence of covalently bound PDM (.gamma.max of free 
PDM is 258 nm). From the extinction of PDM the degree of substitution of 
PDM molecules per PGA molecule is calculated at approximately 50:1. 
3. The `mustard` moiety is shown to be present by determination of 
alkylating activity of the preparation. This is done by reacting the 
preparation with nitrobenzylpyridine in acetone at 80.degree. C. and 
measuring the intensity of blue dye formed, by spectroscopy (Epstein J, 
Rosenthal, R. W. and Ess, R. J. Anal. Chem. 27, 1435 (1955). 
4. When chromatographed on ion-exchange resins the preparation shows 
polyanionic behavior indicating the presence of free carboxyl residues 
remaining on the carrier. E.G. on DEAE Sephadex A50 or QAE Sephadex A50 
eluted with a gradient from 0.1 M Tris-HCl pH 6.5 to 1M sodium chloride, 
the preparation shows a peak elution profile at a concentration of 0.43 M 
sodium chloride. 
Method of Linking Drug-Carrier to immunoglobulin 
1. The PDM-PGA solution is concentrated by membrane ultrafiltration to 200 
mg/ml. 
2. 0.6 ml is allowed to react with 20 mg EDC at room temperature for 2 
minutes, then diluted to 20 ml with phosphate-buffered saline pH 7.2. 
3. The diluted solution is gradually added to an equal volume of a stirred 
solution of rabbit immunoglobulin (RIg) as described in Example 1 at 5 
mg/ml in 0.9% sodium chloride. This immunoglobulin preparation is obtained 
from antiserum to mouse lymphoma cells (EL4) extensively absorbed to 
remove antibody to normal mouse tissues. 
4. 1.2 ml of 5M dodium acetate is added to quench any excess EDC remaining. 
5. The PDM-PGA-RIg preparation is dialysed overnight against 0.9% sodium 
chloride at 4.degree. C. 
6. The preparation is concentrated by membrane ultrafiltration to a final 
volume of 10 ml. 
The drug-carrier-globulin preparation has the following chemico-physical 
properties: 
1. Ethanol precipitability. 0.1 ml of ethanol is added to 0.1 ml of the 
PDM-PGA-RIg preparation, to 0.1 ml PDM-PGA at the same concentration. 
After 20 minutes at room temperature the samples are centrifuged at 2500 
rpm for 10 minutes on a bench centrifuge and the supernatants diluted for 
U.V. spectroscopy. From the U.V. readings, the percentages of PDM-PGA and 
RIg precipitated are calculable: 
Pdm-pga alone -- 0% 
R-ig alone -- 80% 
Pdm in PDM-PGA-RIg -- 61% 
Hence 61% of the drug-carrier is precipitated with 80% of the RIg. 
suggesting 76.5% of the drug-carrier is linked. 
2. Behavior on ion-exchange resins. An unlinked mixture of RIg and PDM-PGA 
chromatographed on QAE or DEAE Sephadex with a gradient from 0.1 M 
Tris-HCL pH 6.5 to 1 M sodium chloride produces two elution peaks (a) 
excluded from the gel, the RIg peak (b) eluted at a peak of 0.43 sodium 
chloride, the PDM-PGA. In contrast, the PDM-PGA-RIg preparation shows a 
much diminished RIg peak and a shift of the second peak to 0.35 M. 
3. Immunoelectrophoretic behavior. Electrophoresis of 2 l of PDM-PGA-RIg on 
an agar-coated slide followed by diffusion with goat-anti-rabbit antiserum 
gives a precipitin line showing rabbit immunoglobulin present in material 
having marked anionic characteristics consistent with PDM-PGA. PDM-PGA 
itself gives no precipitin line. 
4. Alkylating activity. By testing the material with nitrobenzyl pyridine 
in the manner described above, it is shown that the full alkylating 
activity of the drug-carrier is retained following linkage to globulin. 
5. Analysis. The preparation thus contains the following amounts of drug, 
carrier and globulin: 
______________________________________ 
PDM 3.1 mg/l by U.V. spectroscopy 
1.9 mg/ml in terms of alkylating activity 
PGA 8 mg/ml 
calculated from starting quantities 
RIg 10 mg/ml 
______________________________________ 
The drug/carrier/globulin shows the following biological properties: 
1. Antibody activity. Complement-dependent cytotoxicity is determined by 
the release of radioactive chromium from labelled mouse lymphoma cells. 
The titre of the PDM-PGA-RIg is 1 in 20 compared with 1 in 30 for unlinked 
RIg (both at 10 mg/ml starting concentration. 
2. Cytostatic activity in vitro. The mouse lymphoma cells are maintained in 
tissue culture for two days in the presence of a range of concentrations 
of PDM-PGA-RIg. Cytostasis is determined by measuring inhibition of 
cellular tritiated thymidine incorporation during the last four hours of 
culture, in comparison with cells in the absence of drug complexes. The 
results of a typical experiment shown below give the percentage inhibition 
at four concentrations of the components. 
__________________________________________________________________________ 
Concentrations % Inhibition with various preparations 
Drug (in terms of PDM-PGA+ 
alkylating) activity 
Globulin 
RIg normal rabbit 
PDM-PGA+ 
PDM-PGA-RIg 
.mu.g/ml .mu.g/ml 
alone 
Globulin 
RIg complex 
__________________________________________________________________________ 
120 624 56 58 76 83 
60 312 28 25 58 67 
30 156 14 18 38 54 
15 78 8 9 28 43 
__________________________________________________________________________ 
For any given concentration, the PDM-PGA-RIg complex demonstrates greater 
cytostatic properties than the other materials. 
3. Anti-tumor activity in vivo. Groups of five C57B1/6 strain mice are 
injected with 5 .times. 10.sup.4 EL4 lymphoma cells intraperitoncally on 
day 0. At 24 hour intervals on days, 1,2,3 and 4; the mice are injected 
i.p with the drug-carrier-globulin complex and compared with mice 
similarly treated with various components of the complex. The survival 
times of the various groups are given below. 
__________________________________________________________________________ 
% increase 
% mice 
in survival 
surviving 
Dose per injection 
Median time com- 
without 
Drug (alkylating) 
Survival 
pared with 
evidence 
activity Globulin 
time(MST) 
saline con- 
tumour at 
Treatment 
.mu.g mg days trols. days 45 
__________________________________________________________________________ 
saline -- -- 13 -- 0 
RIg alone -- 4 19 47% 0 
PDM-PGA 765 -- 25 92% 20 
PDM-PGA + 
765 
38 192% 40 
RIg unlinked 4 
PDM-PGA-RIg 765 4 &gt;60 &gt;360% 100 
__________________________________________________________________________ 
Thus doses of PDM-PGA and RIg when linked in the complex have greater 
antitumor activity than either component alone or the two components 
together but unlinked. 
EXAMPLE 4 
Goat anti-human melanoma Ig is prepared from a melanoma cell suspension 
containing 2.8 .times. 10.sup.7 cells injected intraperitoneally into a 
young adult female goat. This injection is repeated four times at weekly 
intervals and then the animal is bled out one week after the final 
injection. Serum is separated from the blood and this is absorbed with 3g 
wet weight pooled human spleen cells/ml for 90 mins. at 4.degree.. This 
absorption is repeated twice. The absorbed serum is then fractionated to 
yield Ig by a standard ammonium sulphate precipation method followed by 
dialysis. 
P-[bis(2-chloroethyl)amino]phenylalanine-polyglutamic acid (m.w. 35,000) is 
prepared by methods described in Example 3 replacing 
p-[N,N-(bis-2-chloroethyl)]-phenylenediamine with 
p-[bis(2-chloroethyl)amino]phenylalanine and using equivalent quantities. 
To this polymer carrier cytotoxic drug is bound the above goat anti-human 
melanoma Ig. 
Freeze dried p-[bis-(2-chloroethyl)amino]phenylalanine-polyglutamic acid 
(M.W. 35,000) is bound to the goat anti-human melanoma Ig by dissolving 
the polymer-carrier-cytotoxic drug complex in water to give a 
concentration of 120 mg/ml. 200 Mg of 
ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to 10 ml of the 
polymer carrier-cytotoxic drug solution and when dissolved, the solution 
is diluted to 400 ml. with phosphate buffered saline, pH 72. 
The dilute solution is added to 400 ml of a rapidly stirring solution of 
goat anti-melanoma Ig at 5 mg/ml containing 28.8 m 5M sodium acetate. The 
preparation is dialysed against sterile 0.9% NaCl for 19 hours with one 
change of saline after 16 hours. The volume after dialysis is 890 ml. The 
preparation is membrane-filter sterilized and analysed for drug and 
protein concentration: 
Drug concentration by U.V. spectroscopy 697 .mu.g/ml 
Drug concentration (alkylating activity) 317 .mu.g/ml 
Protein concentration 2.25 mg/ml 
Substitution ratio (active drug molecules per Ig molecule) 70:1. 
EXAMPLE 5 
Using the procedure set out in Example 4 and using equivalent quantities, 
p-[N,N-bis(2-chloroethyl)]-phenylenediamine, 
2-amino-N-[p-bis(2-chloroethyl)amino]-phenyl-3-hydroxypropionamide, and 2 
-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropio 
namide provides goat-anti-human melanoma Ig labeled with the respective 
cytotoxic agent. For instance, using 
p-[N,N-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide and 
polyglutamic acid molecular weight 35,000 provides a cytotoxic agent 
having 20 molecules of cytotoxic drug/Ig. 
EXAMPLE 6 
40 Mg of polylysine M.W. 16,000 dissolved in 4 ml of 0.9% sodium chloride 
and 40 mg of methotrexate is dissolved in a similar amount of 0.9% sodium 
chloride by addition of 1N sodium hydroxide until a clear solution is 
obtained. The pH is adjusted to 7.0 and the methotrexate solution is added 
dropwise to the polylysine solution and then 40 mg of 
1-ethyl-3(3-dimethylaminopropyl)carbodiimide is stirred in. The pH is 
lowered to 4 with hydrochloric acid and then returned to 7 with sodium 
hydroxide. The methotrexate-polylysine precipitates and 0.1 of citraconic 
anhydride is added and the pH maintained above 8 with sodium hydroxide. 
The methotrexate-polylysine precipitate gradually redissolves as 
citraconylation proceeds. The polymer carrier-cytotoxic drug complex is 
bound to Ig as described in Example 3. 
EXAMPLE 7 
Using equivalent quantities of polyethyleneimine (M.W. 20,000), 
4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid and 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as in Example 6, the 
cytotoxic drug is linked to the polymer carrier. Reaction with citraconic 
anhydride is not carried out with polyethyleneimine as it was with 
polylysine. To 40 mg of goat anti-human lymphoma Ig in 40 ml of buffer 
with the pH adjusted to 3 with hydrochloric acid is added dropwise 32 mg 
of the above polymer carrier-cytotoxic drug in 8 ml of buffer. The pH is 
maintaineed below 4. 20 Ml of 5% glutaraldehyde in saline is added and the 
pH is raised to 9.5 with 1N sodium hydroxide. The solution is held at that 
pH for 3-20 seconds and then returned to pH 3 by the addition of 1N 
hydrochloric acid. The process of addition and pH adjustment is repeated 
three times. The pH is then adjusted to 6 and the preparation dialysed 
against saline. 
EXAMPLE 8 
10 Mg. of polyethyleneimine (M.W. 20,000) is dissolved in 0.25 ml of water 
and the pH is adjusted to 7 with hydrochloric acid and 20 mg of cytosine 
arabinoside monophosphate is added and the pH is readjusted to 8, and then 
20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added and the 
volume brought to 1 ml. This solution is allowed to stand for 16 hours at 
20.degree. C and protected from light. The preparation is dialysed against 
saline buffered to pH 7.3 with tris-acetate buffer (0.02M). This polymer 
carrier having cytotoxic drug bound to it may in turn be bound to Ig 
mentioned in Example 1 by the carbodiimide method or the glutaraldehyde 
method. 
EXAMPLE 9 
Cytosine arabinoside is linked through the amino group to polymer carriers 
such as polyglutamic acid and dextran activated with cyanogen bromide by 
methods set out in the earlier examples. 
EXAMPLE 10 
2.39 Parts of N-carbobenzyloxy-L-serine and 2.69 parts of 
p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride were stirred 
together at room temperature and in the dark as a suspension in 50 parts 
by volume of dry methylene chloride. Then distilled 1.01 parts of 
distilled triethylamine was added and stirring was continued for 30 
minutes. A solution of 2.16 parts of N,N-dicycohexylcarbodiimide in 50 
parts by volume of dry methylene was added over 10 minutes. Stirring was 
continued for 24 hours to provide a dark solution and a white precipitate. 
The reaction mixture was filtered and the filtrate was successively washed 
with aqueous sodium bicarbonate, 2 molar hydrochloric acid, and water. The 
organic layer was then dried over sodium sulfate and filtered and removal 
of solvent in vacuo provided a crude solid which after recrystallization 
from acetone/hexane or toluene provided 2 
-carbobenzyloxyamino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropiona 
mide, melting at 145.degree.-146.degree. and having the following 
structural formula 
##STR37## 
0.85 Parts of this material was dissolved in 85 parts by volume of 
distilled methanol containing 0.2 parts by volume of concentrated 
hydrochloric acid and the solution hydrogenated at room temperature and 
atmospheric pressure over 0.17 parts of a 5% palladium-on-charcoal 
calalyst. The catalyst was removed by filtration, the solvent removed in 
vacuo, and the product was precipitated upon addition of dry ether. 
2-Amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide 
hydrochloride is isolated as a hydroscopic solid having the formula 
##STR38## 
Replacement hydrochloric acid with an equivalent amount of sulfuric, 
phosphoric, hydrobromic, hydriodic, sulfamic, citric, lactic, maleic, 
malic, russinic, tartaric, cinnamic, acetic, benzoic, gluconic, ascorbic 
acid provide the corresponding acid addition salt. Neutralization of the 
acid salt with base and extraction with ether provides the free base, 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3 -hydroxypropionamide. 
##STR39## 
EXAMPLE 11 
5.9 Parts of DL-serine was dissolved in 1100 parts by volume of 0.2 molar 
sodium carbonate. 5.6 parts by volume of a 1.0 molar cupric sulfate 
solution was then added, followed by 34 parts by volume of 40% aqueous 
formaldehyde solution. The solution was then heated at 
95.degree.-100.degree. C. for 20 minutes and a precipitate of copper 
resulted. 
The reaction mixture was allowed to cool to room temperature and then 
filtered to remove the precipitated copper. After acidification with 
glacial acetic acid, the solution was concentrated under reduced pressure 
to around 100 parts by volume and then poured onto a Zeolite 225 ion 
exchange column, H form. This was washed with water until the acid band 
disappeared, when the column was eluted with 2M ammonium hydroxide, 
collecting and combining those fractions which gave a position ninhydrin 
reaction. These fractions were then concentrated in vacuo, when IMS (95% 
ethanol) was added to precipitate the required product. After standing at 
0.degree. C for 3 days the crude product was filtered off, washed with 
IMS, and then recrystalized from IMS/water to afford 
2-amino-3-hydroxy-2-hydroxymethylpropionic acid, melting at 
253.degree.-254.degree. C. and having the following structural formula 
##STR40## 
19.20 parts of this propionic acid is reacted with 21.56 parts by volume 
of N-benzylchloroformate in 236 parts by volume of N-benzylchloroformate 
in 236 parts by volume of sodium bicarbonate containing 29.8 parts of 
sodium carbonate. Following the procedure set out in Example 
1,2-carbobenzyloxyamino-3-hydroxy-2-hydroxymethylpropionic acid, melting 
at 109.degree.-112.degree. C. (lit 112.degree.-114.degree.) is isolated. 
This compound has the following structural formula 
1.0 parts of this material were placed with 1.1 parts of 
p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride in 20 parts by 
volume of methylene chloride. 0.418 Parts of distilled triethylamine was 
added with continuing stirring and after stirring for 10 minutes 0.85 
parts of N,N-dicyclohexylcarbodiimide in 20 parts by volume of dry 
methylene chloride was added over a 10 minute period. The reaction was 
worked up as in Example 1 to provide 
2-carbobenzyloxyamino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydr 
oxymethylpropionamide, melting at 138.degree.-141.degree. C, and having the 
following structural formula 
##STR41## 
Using equivalent quantities and following the procedures in Example 1, 0.5 
parts of this material is catalytically hydrogenated over 5% 
palladium-on-charcoal catalyst to provide 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropi 
onamide hydrochloride having the following structural formula 
##STR42## 
Other pharmaceutically acceptable acid addition salts and the free base 
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropi 
onamide are prepared as described in Example 1. 
Alternatively, 5.20 parts of 
2-carbobenzyloxyamino-3-hydroxy-2-hydroxymethylpropionic acid and 5.95 
parts of 1-hydroxy benzotriazole in 130 parts by volume of dry methylene 
chloride are reacted. To this reaction mixture was added 4.03 parts of 
N,N-dicyclohexylcarbodimide and stirring continued for 16 hours. Then 5.7 
parts of p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride and 2.99 
parts by volume of triethylamine are added and stirred for 65 hours and 
worked up as earlier described to provide 
2-carbobenzyloxyamino-N-[p-bis(2-chloroethyl)amino]-phenyl-3-hydroxymethyl 
propionamide.