CA2014459C - Targeted forms of methyltrithio antitumor agents - Google Patents

Abstract

This disclosure describes a method for constructing carrier-drug conjugates from the family of methyltrithio antibactarial and antitumor agents.

Description

_ 1 -Title: TARGETED FORMS OF METHYLTRITHIO
ANTITtJMOR AGENTS
SUMMARY OF THE INVENTION
The invention describes carrier-drug conjugates of the disulfide analogs of the al, a2, a3' a4' ~1' R2' Y1' ~1 and pseudoaglycone components of the LL-E33288 complex and derivatives thereof, as well as the disulfide analogs of BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724 antitumor antibiotics and derivatives thereof. The carrier portion of the conjugate is a mono- or ~~olyclonal antibody, their fragments, chemically or genetically manipulated counterparts, or growth factors or steroids. The invention includes the method of using the carrier-drug conjugates as well as their process of manufacture.
DESCRIPTION OF THE DRAWINGS
Figure I : The ult raviolet spect rum of the ant itumor antibiotic designated as LL-E33288y1I.
Figure II: The proton magnetic resonance spectrum of the antitumor antibiotic designated as LL-E33288~1I.
Figure III : The inf rayed spect rum of the ant itumor antibiotic designated as LL-E33288y1I.

- 2 -Figure Iv: The proton magnetic resonance spectrum of the ant:itumor antibiotic designated as LL-E33288a2I.
Figure V: The carbon--13 nuclear magnetic resonance spectrum of the antitumor antibiotic designated as LL-E33288a2I.
Figure VI: The ultrav:Lolet spectrum of the antitumor ~intibiotic designated as LL-E33288a:~I
Figure VII: The infraread spectrum of the antitumor antibiotic designated as LL-E33288a3I.
Figure VIII: The proton magnetic resonance spectrum of the antitumor antibiotic designated as LL-E332F38a3I.
Figure IX: The carbon-~13 nuclear magnetic resonance spectrum o1= the antitumor antibiotic designated as LL-E33288a3I-2o Figure X: The ultraviolet spectrum of the antitumor antibiotic designated as N-acetyl Lh-E33288b1I.
Figure XI: The infrarEad spectrum of the antitumor antibiotic designated as N-acetyl LL-E33288b1I
Figure XII: The proton magnetic resonance spectrum of the anti.tumor antibiotic designated as N-acetyl. LL-E33288S1I.
Figure XIII: The carbon-~13 nuclear magnetic resonance spectrum o!: the antitumor antibiotic designated as N-acetyl LL-E33288d1I.
Figure XIV: The ultraviolet spectrum of the antitumor antibiotic designated as iodo LL-E33288 Frseudoaglycone.
Figure XV: The infrared spectrum of the antitumor

- 3 -antibiotic designated as iodo LL-E33288 pseudoaglycone.
Figure XVI: The proton magnetic resonance spectrum of the antitumor antibiotic designated as iodo LL-E33288 psewdoaglycone.
Figure XVIII: The carbon-13 magnetic resonance spectrum of the antitumor .antibiotic designated as iodo LL-E33288 pseudoaglycone.
DETAILED DESCRIPTION OF THE INVENTION
Members of the family of antibacterial and antitumor agents, known collectively as the LL-E33288 complex are used to prepare the disulfur antitumor agents which are some of the starting materials for targeted forms of the antitumor agents of our invention.
The LL-E33288 complex, the components thereof, namely, LL-E33288a1Hr, LL-E33288a1I, LL-E33288a2Br, LL-E33288a2I, LL-E33288a3Br, LL-E33288a3I, LL-E33288a4Br~
LL-E33288p1Hr, LL-E33288/31I, LL-E33288/32Hr, LL-E33288~32I, LL-E33288Y1Br, LL-E33288Y1I a:nd LL-E3328851I, and methods for their production by aerobic fermentation utilizing a new strain of Micromonospora echi:nospora ssp calichensis or natural or derived mutants thereof are described elsewhere.
Proposed structures for some ~of the above named components are reproduced in Table 1.
A

.- , _ Table 1: Proposed Structures ~tor CHg-SSS-W isolated from natural sources (wherein W is the substituent attached to CHg-SSS- below) H o ~,L~
C~ R~ OCH~
OCH~ ~~
'10087 O H H R~. s C H~
~jfO
C !4~ O C Hip p- JLI~/' A~= s '~ ' Ah s C H O'~~
C H~ O
!!a~ s H C~.., CHs O~OCH~ OCH
OH
OC14~ C
DssiBnatlon R~ R2 R3 R4 Rs Rg R7 X

E33288as~ Ark RZ; H H C2HS I

E33288as~ Ar1 H H R4 I

E33288~~~ Ar1 R~; H R4 (CH3)2C H i E33288~~1 Ark RZ; H R4 C2H5 I

E332888~~ Ark R2 H R4 CH3 I

E33288~~sr Ark R~; H R4 (CH3)2CH Br E3328871Br Ark R~; H R4 C2H5 Br E33288a28r Ark R~; H H C2H5 Br E33288assr Ark H H R4. Br Esporamicin A~ CH3 R~; R3 (CH3)2CH H Ar2 Esporamlcin Az CH3 R'; R3 (CH3)2C H Ar2 H

Esparam~lo A CH3 R'; R3 CH3CH2 H Ar2 . 201 4459 Additional members of the LL-E33288 complex are described in Canadian Patent 1,301,167 and are likewise useful for preparing the targeted forms of the antitumor agents of our invention. This application describes the LL-E33288bromo-and iodo-pseudoaglycones of the series, which have been prepared by chemical means. The application also describes dihydro derivatives accessible from all the above-named antitumor antibiotics through sodium borohydride reduction of the ketone at C11 to a hydroxyl group. These latter proposed structures are reproduced in Table 2.
Still other members of the LL-E33288 family of antitumor antibiotics are described and claimed in our copending Canadian Patent Application No. 2,014,472, filed on April 12th, 1990, and also are useful for preparing additional targeted forms of the antitumor agents of our invention. This application describes N-acyl derivatives of several members of the LL-E33288 complex which have been prepared by chemical means. These proposed structures are likewise reproduced in Table 2.

20'! 4459 -~ 6 -Table 2: Proposed Structures for CHg~SSS-W derived from chemical manipulation of the compounds of Table 1 (wherein W is the substituent attached to CH;3-SSS- below R~ ~
110.~n 11 m H O R:. .
i R,S K
O C ~Clt O
Oll H N O N I WOOR
ORS its..
CK O
C tl~ O C ~ C O-~ O
I
A~ .
A5 ' C ~ N
R o~w ~o~;w w-OCt~ ON
DeaIBnatlon Rt R2 R3 R4 RS Rs~ Rd Ry Rg Rg X
Dihydro LL-E33288az~ Ark R2~ H H C2H5 H OH H I
N~Acyl LL-E33288as1 Ark R2~ H H C2H5 RCO =_O I
Dihydro LL-E33288as~ Art H H R~~ OH H I
Dihydro LL-E33288~~1 Ark R2~ H R4~ (CH3)2CH H OH H I
N-Acyl LL-E33288S~t Ark R2~ H R4~ (CH3)2CH RCO =_O I
Dihydro LL-E33288Y~1 Art R2~ H R4~ C2H5 H OH H I
N-Acyl LL-E332887~t Ark R2~ H R4~ C2H5 RCO =_O I
Dihydro LL-E332888~~ Art R2~ H R~~ CH3 H OH H 1 N-Acyl LL-E3328tt8~t Ark R2~ H R4- CH3 RCO =_O I
lodo LL-E33288 Art H H H ~ _O 1 pseudoa~lycona Dlhydro~lodo LL-E33288 Ark H H H OH H I
paeudoaplycone Dihydro LL-E33288~~Br Ark R2~ H R4~ (CH3)2CH H OH H Br N-Acyl LL-E33288Ster Ark R2~ H R8~ (CH3)2CH RCO =_O Br Dihydro LL~E332887~er Ark R2~ H R,t~ C2H5 H OH H Br N-Acyl LL-E33288yt~r Ark R2~ H R,~ C2HS RCO =_O Br Dihydro LL-E33288aZSr Ark R2~ H H C2H5 H OH H Br N-Acyl LL-E33288asar Ark R2~ H H C2HS RCO =_O Br Dihydro-E33288a~sr Ark H H R~~ O H H Br Bromo LL-E332e8 Ark H H H -_O Br pseudoaplycone Dlhydro-bromo LL-E33288 Ark H H H OH H Br paeudoaplyoone N-Acetyl Eaperamicin A~ CH3 R2~ R3~ (CH3)2CH CH3C0 H Ar2 N-Acetyl Eaperamicin A2 CH3 R2~ R3~ (CH3)2CH CH3C0 Ar2 H
N~Acetyl Eaperamlcln Alb CH3 R2~ R3~ CH3CH2 CH3C0 H Ar2 R s hydroEen or a branched or unbrsnched alkyl (C, - C") or alkylene (C, ~ C,a group, an aryl or heteroaryl group, or an aryl-slkyl (C, ~ C~ or beteroaryl-alkyl (C, ~ Cx) Eroup, all optionally subatltuted by om or swore hydroxy, amino, carboxy, halo, nitrc lower (C, - C~) alkoxy, or lower (C, - C,) thloalkos)r groups.

_7_ Certain other antibiotics are useful in our invention, namely:
1) Esperamicin HBM-1675, a novel class of potent antitumor antibiotics. I. Physico-chemical data and partial structure. M. Ronishi, et. al., J.
Antibiotics, 38, 1605 (1985). A new antitumor antibiotic complex, M. Ronishi, et. al., U.R. Patent Application GB 2,141,425A, May 15, 1984.
2) New antitumor antibiotics, FR-900405 and FR-900406.I. Taxonomy of the producing strain. M.
Iwami, et. al., J. Antibiotics 38, 835 (1985). New antitumor antibiotics FR-900405 and FR-900406. II.
Production, isolation, characterization and antitumor activity. s. Riyoto, et. al., J. Antibiotics, 38, 340 (1985).
3) PD 114759 and PD 115028, novel antitumor antibiotics with phenomenal potency. I. Isolation an8 characterization. R.H. Hunge, et. _a~., J. Antibiotics, 37, 1566 (1984). Biological and biochemical activities of the novel antitumor antibiotic PD 114759 and related derivatives. D.W. Fry et. al., Investigational New Drugs, 4, 3 (1986).

4) New antibiotic complex CL-1577A, CL-15778 produced by Stre~yces sp. ATCC 39363. European Patent Application 0,132,082, A2.

5) CL-1577D and CL-1577E Antibiotic antitumor compounds, their production and use. U.s. Patent 4,539,203.

6) CL-1724 Antibiotic compounds, their production and use. U.s. Patent 4,554,162.

7) New antitumor antibiotics HBM-1675-A3 and HHM-1675-A4, obtained by fermentation of actinomadura verrucosospora strains 8964-92 (ATCC 39334) or AB27Y
(ATCC 39638). U.B. Patent 4,675,187.

8) New N-acetyl-esperamicin Al, A2 and Aib derivatives with antimicrobial and antitumor activities. European Patent 289,030.

The complete structures of esperamicins A1, A2, and Alb (the BBM-1675 complex) and their respective N-acetyl derivatives have been reported, and these are included in Tables 1 and 2. The physir_al characteristics of the other above-named antitumor antibiotics indicate that they all are identical or very similar in structure to the esperamicins, and all contain a methyltrithio functional group.
As can be seen from the structures disclosed above, the al, a2, a3, a4, p1' ~2' Y1' 6' and pseudoaglycone components of the LL-E33288 complex their dihydro and N-acyl counterparts, as well as the BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-15778, CL-1577D, CL-1577E
and CL-1724 antibiotics and their N-acyl counterparts, each contain a methyltrithio group in their structure. The methyltrithio moiety of the above-named antibiotics is subject to displacement by a variety of thiol-containing organic molecules resulting in the formation of a new class of anticancer and antibacterial agents as described in our copending Canadian application No. 2,014,486 filed on April 12th, 1990.
It has now been discovered that the displacement of the rnethyltrithio unit of the compounds listed in Tables 1 and 2 as depicted in Scheme I can be used to introduce a spacer (Sp), the Judicious choice of which enables the introduction of targeting units into the compounds of the above-named patents and applications.

20'1 ~~~~
-~ 9 CH3888-W Q-Sp-SH a Q-Sp-SS-W
Schemes I
wherein 8p is a straight or branched-chain divalent or trivalent (C1-C18) radica7~~, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C3-C18) cycloalkyl or heterocycloatlkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (Cl-C18) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C»-C18) radical, or divalent or trivalent (CZ-C18) unsaturated alkyl radical, wherein it 8p is a trivalent radical, it can be additionally substituted by amino, alk~tlamino, arylamino, heteroarylamino, carboxyl,. lower alkoxy, hydroxy, thiol or lower alkylthio groups;; Q is, or can be subsequently converted to, halogen, am»no, alkylamino, carboxyl, carboxaldehyde, hydroxy, i:hiol, a-haloacetyloxy, lower alkyldicarboxyl, -CONHNH2" -NHCONHNH2, -NHCSN8NH2, -ONH2, -CON3, -SS N
-C02N , , ( N-, -C02 , '0 F F
O

, -C02 O F~ -C02 O -i~IO 2 ~ O 0 O
F F

2014~~9 a. - 10 -and W is as shown in Tables 1 and 2, above.
As long as the product from Scheme I contains at least one functional <~roup which can be converted to, or is directly reactive with a targeting unit (Tu), targeted forms of the ania tumor antibiotics of the above-named patents and applications can be generated, as shown in Scheme II be:Low:
Q-$p-8$-W ~~u- (Y) i u- ( Z-gp-88-W) m (Y) n-m Scheme II
wherein Q, Sp, and W are as hereinbefore defined, Tu is a mono- or polyclonal aniabody, its fragments, its chemically or geaeticall;~ manipulated counterparts, or growth factors or steroicls; Y is a side-chain amine, carboxy, or thiol group of a protein, an aldehyde de-rived from carbohydrate residues, or an amidoalkylthio group; n is an integer off.' from 1 to 100; Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reducaion and 3 is -CONH-, -CONHN=CH-, -CONHNHCH2-, -HHCONBN=CH-, -NHCONHNHCH2-, -NHCSNHN=CH-, -NHCSNHNHC~i2-, -ON=CH-, -NH-, -NHCH2-, -N=CH-, -COZ-, -NHCH2C02..~ -gg-~
O _S O.,~C_ I
-S N '~~' -S ~ CH3 -NHCOCH2 -CH
r N ' ~ ' ' CH2 . CH2 or -NHCOCH~-CH
I

and m is 0.1 to 15.

2~ 1 4459 .. 11 -As an example, ~snd with reference to Scheme II, above, the 3-mercaptopropionic acid derivative of E-3328871= (Q=C02H, Sp=-CFI2CH2-), when converted to its activated hydroxysuccinim:Lde form (Q=C028u, Sp=-CH2-CH2-) can be used to react with some of the e-amino groups of lysine residues (e. g., Tu=monoclonal antibody, Y=-NH2 wherein n=50-l00 from available lysine residues), at a pH between 7.0 and 9.5 in aqueous buffered solutions at temperatures between 4°C to 40°C
l0 to produce targeted forms of the antibiotics attached at random sites along the protein backbone (Tu=monoclonal antibody, ~Z=-NHCO-, 8p=-CH2CH2-, , m=i-10). only a fraction of the available lysine residues are substituted :Ln this manner, since high loading is generally not considered compatible with preserving the antibody immunoreactivity. The same randomly-substituted immunoconjugates can also be prepared from the 3-mercaptopropionic acid derivative using other carboxyl group activating agents such as a 2o variety of carbodiimides, or the corresponding acyl azide. Alternatively, a :3-mercaptopropionyl hydrazide derivative of E-3328871= I;Q=H2NNHC0-, Sp=-CH2CH2-), when reacted with a periodate-oaidized antibody (Tu=monoclonal antibody, 'if=-CHO, n=i-15) as described in U.B. Patent No. 4,67i,!~58 at a pH between 4 and 7, in a buffered aqueous solution at a temperature of between 4°C and 40°C, rea~~ts only at the aldehyde functionality (derived fr~~m cleavage of vic-diols of carbohydrate residues situated on the Fc portion of the antibodies) to generate monoclonal antibody conjugates containing the drug substituted at specific sites along the backbone of the protein (Tu~onoclonal antibody, Z=-CH=NNHCO-, Sp=-CH2CH2-, m=0.5-10). In order to block unreacted aldehyde groups on the antibody and thus avoid crosslinking, ~ns well as stabilize the hydrolytically labile schiff~s base linkages, it is preferable (though not essential) to react the latter 20~ 1 4459 -~ 12 -conjugate first with a compound such as acetyl hydra-zide or tyrosine hydrazidea, then reduce with sodium cyanoborohydride or sodiuao borohydride to produce the stabilized constructs of i:his invention (Tu=monoclonal antibody, Z=-CH2NHNHCO-, E3p=-CH2CH2-, m=0.5-10). Other aldehyde-reactive groups :~s part of the drug construct are within our invention i:o generate the products of Scheme II. Such functional groups are preferably, though not limited to, those which react with aldehydes under acidic aqueous condjltions. The reactivity of protein lysines under basic conditions is sufficiently great such that their amides compete with the products of Scheme II for available aldehydes of the monoclonal antibody. Alternative aldehyde-reactive groups are, for example, the semicarbazide, the thiosemicarbazide, and the O-substituted hydroxylamine functionalities.
Assembly of targeted forms of the compounds listed in Tables 1 and 2 :Ls not restricted to the sequence outlined in Scheme II. The targeting unit (Tu) can be first modified to contain a thiol group, which is then reacted with the compounds of Tables 1 and 2 in accordance with l3cheme IIi below:
Tu (Y) n + Q-SP-S-p ---~ i u- (Z-SP-SH) m (Y) n-m Tu-(Z-Sp-SS-W)m (Y) n-m Scheme III
wherein Tu, Y, Q, Sp, W, n, and m are as hereinbefore defined, and P is hydrogen or 2-(pyridylthio), with the proviso that when Y is a thiol derived from a backbone ~.z~ 1 4459 amino acid residue of Tu, Z-Bp taken together is a covalent bond.
As an example, amd with references to Scheme III, above, a monoclonal antibody can be reacted with 3-(2-dithiopyridyl)propionic acid hydroxysuccin-imide ester to modify the protein through lysine residues (Tu=monoclonal antibody, Y=N82, n=50-100, Q=-C028u, 8p=-C82-CH2-, P=,2-pyridylthio). Following reduction with, for example, dithiothreitol, an in-to termediate is generated (z~u=monoclonal antibody, Z=-NHCO-, Sp=-C82C82-, m=1. to 15) which can be reacted with the compounds of Tables 1 and 2 to generate the subject immunoconjugates. Similarly, 2-iminothiolane can be reacted with a monoclonal antibody to introduce thiol groups onto the surface of the protein directly, without requiring a reduction step (Tu~onoclonal antibody, Z=-NSCO-, 8p=-(C;H2)3-, m=i to 15), and this intermediate can be reacta~d with the compounds of Tables 1 and 2 as before. Alternatively, sulfhydryl 2o groups inherent within th~~ structure of monoclonal antibodies in dimeric form as cystine residues can be used to participate in they reaction of scheme III
directly. Such sulfhydryl.s are traditionally exposed by a combination of enzymatic digestion and reduction of native monoclonal antibodies (Tu=Fab~ fragment, Z-Bp=bond, Y=8H), but the use of genetically-altered constructs of monoclonal antibodies containing unpaired cystine residues is likewise contemplated.
A preferred embodiment of this invention is a 3o protein-drug conjugate of the formula:
Tu- ( Z-8p-sit-W) m I
( Y ) n-m ,-prepared from the class of antitumor antibiotics designated LL-E33288 (CH3888-W) comprising:
displacing the dithiomethyl!moiety with a compound of formula Q-8p-SH, wherein'8p is straight or branched-chain divalent .or trivalent (C2-Cl0) radicals or divalent or trivalent (C2-C5) arylalkyl or heteroarylalkyl radicals, wherein if 8p is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups: and Q is l0 carbonyl, lower alkyldic;drboxyl aahydride, -C028u, -CONHNH2, or to produce an intermediate of general formula Q-8p-88-W, wherein Q, 8p, and W are as hereinbefore defined, reacting Q-8p-is8-W with a molecule of the formula Tu-(Y)n wherein !L'u is a monoclonal antibody which e$hibits preferent:Lal reactivity with a human tumor-associated antigen.. Y is a side-chain amino group on the antibody, or an a:ldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from i to 100,, to produce a compound of the formula:
Tu-(Z-8p-88-W)m ( Y ) n-m wherein Tu, Y, 8p, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONBNHCH2-, t -NFICOCH2-fi H
CH2 or -NHCOCH2-CH
~:o H

and m is 0.1 to 15.
A number of di:~ferent monoclonal antibodies (MoAbs) are used to exemplify targeting of the methyltrithio anticancer compounds. MoAbs Lym i and Lym 2 recognize differeai: antigens on mature io B-lymphocytes and their ~~roduct lymphomas. The production and characterLstics of these MoAbs are described by A. L. Epsteis~, et, al . , ~~Cancer Research~~
47, 830 (1987). MoAb 87::.3 targets primarily to carcinomas of the breast and colon, though reactivity With pancreatic, ovarian,, and lung carcinomas has also bean noted. The antibod~~ has been described by T.L.
Rlug, et. al. , ~~Int. J. ~;ancer~~ 38, bbi ( 1986 ) . MoAb CT-M-01, which recognizes: primarily breast tumors is described in EPO application Sb X01482.4 filed July 3, 2o 1986 and MAC-68 is produced by a sub-clone of the hybridoma ~rhich produces CT-M-01, and recognizes both breast and colon carcinomas. Intermediates of the subject compounds useful for, and conjugates with these antibodies, ars deseribe~l in the experimental section.
It should not, however, tie construed that this patent is limited to or restricted by the aforementioned antibodies. Instead, thE: methodology is sufficiently general that it can be a~~plied to all antibodies regardless of their class or isotype, their enzymatically-derived fragments, their chemically manipulated and stabilizE:d fragments, as well as their respective chimeric and humanized counterparts. Nor are the targeting units restricted only to monoclonal antibodies. Other proteins, as well as small molecules foz which receptors exist: on target issues, are within the purview of our disco~rery as targeting entities.

'20~445~

The methods of this invention used to produce monoclonal antibody conjugates from the compounds of Tables 1 and 2 yield constructs which retain good immunoreactivity with target cell lines, as determined by the following in vitro, assays:
Tarq_et Cells All target calls were maintained in RPMI 1640 media supplemented with 5% Fetal Calf Serum (FCS), ITs (Collaborative Research, Cats 40351), streptomycin l0 (50 ug/ml), penicillin (50 units/ml), gentamycin sulfate (50 ~g/ml) and glutamine (.03%). The cells were maintained in a humidified 5% C02 incubator at 37°C.
I. Immunoreactivity Assay Procedure I - Elise Apprapriata target cells were harvested, counted and suspended in Dulbscco~s Phosphate Buffered Saline (DPBS) at an optimal concentration for monoclonal antibody (MoAb~) being tasted. 0.1 ml of cells was aliquotad in each well of.a sterile tissue culture polystyrene 96-well plats. Tha plates were centrifuged far 5 minutes at 1,000 RPM~s and the supernatant was flicked off. Plates ware air-dried overnight and may be stored at 4°C for up to 3 months.
Non-specific binding sites were blocked by adding 200 ~l of i% gelatin in DPBB per well and incubating the plate for i hour at 37°c is a humid incubator. (All subseque~at incubations are done under similar conditions). They plates were washed once with 250 ~l of 0.05% TWEEN-20 in DPBS (washing solution) using the automated ELI81~, washing system from Dynatech (Ultrawash II'~. Samples to be tested were diluted to make a final concentration of 3 ~g/ml MoAb equivalents in 0.1% gelatin-DPBB. Si,x additional threefold serial dilutions were prepared from each 3 ~g/ml sample and 100 ~1 was added to appropriate wells in triplicate.
The bottom row of wells only received 100 ~1 of 0.1%
*Trade-mark ~414~59 -~~-gelatin as background. Plates were incubated for 45 minutes and then washed three times. Alkaline phosphatase conjugated affinity purified goat anti-mouse immunoglobulins (Cappel Cats 8611-0231) was diluted 1:125 in 0.1% gelatin and 100 ~1 was added to each well. Plates were incubated for 45 minutes and then washed three times. 200 ~l of p-nitrophenyl phosphate substrate solution (see below) was added to each well. After 45 minutes at room temperature the reaction was stopped by the addition of 50 gel of 3M
NaOH. The absorbance of the contents of each well was read at 405 nm in the automated spectrophotometer from Dynatech (EIA Autoreader ~ EL-310).
Substrate Diethanolamine Suffer !10%) 97 ml diethanolamine 800 ml water 0.2 grams NaN3 100 mg MgCl2 6820 The reagents were dissolved by continuous stirring and iM HCl was added until the pH was 9.8.
The total volume was made up to 1 liter with water and filter sterilized with a 0.2 ~ filter. The buffer was stored in the dark at 4°C. Immediately before use, p-nitrophenyl phosphate (sigma, Cats 104-40) was dissolved in the 10% dietb~anolamine buffer (must be at room temperature) to give a final concentration of 1 mg/ml.
Calculation of O. D. yalues The percentage Minding of each sample was 3o calculated by the following equation:
A-H
x 1.00 - % Binding c-a A = Average O.D. of test sample B = Average O.D. of background C = Average O.D. of 3 ~q/m.l unmanipulated MoAb control 2y 1 4459 .. l8 _ The % binding was plotted on the non-log scale of a semi-log graph and the MoAb concentration was plotted on the log scale. The HDSO (i.e. dose of antibody needed to give 50% binding) of each test sample was derived from the graph and the amount of retention of immunoreactivity was calculated by the following equation:
HDSO of MoAb control io a 100 = % Immunoreactivity retained HD50 of test sample Procedure 2 - Indirect RIA
Appropriate amounts of target cells in o.2 ml of 10% FCS media were aliquoted into 4 ml polystyrene tubes. samples to be tesi:ed were diluted to a concentration of 2 ~g/ml MoAb equivalents in io% FCs media. Five three-fold aerial dilutions were prepared from each 2 ~g/ml sample :end 0.2 ml was added to each 2o tube in duplicate. Background samples received only cells and media. Cells were incubated at 4°C for 1 hour, then washed 2 times (all RIA washes were done with a 3 ml volume) with a% FC8 media. 0.05 ml of sheep F(ab~)Z anti-mouse ;CgG [125I] (DuPont, Cats NEX
162-0142) containing approximately 500,000 CPM~s was added to each tube: cells were incubated an additional hour at 4oC, washed once with 2% FC8 and twice with PHB. 0.5 ml of PHS was added to each tube, cells were vortexed, transferred to clean tubes and counted for i 3o minute in a Packard Gamma 500.
The % binding o:E each value was determined and graphed like the preceding ELISA equation, except CPM~s were substituted fo:c O.D. units and C = Average CPM~s of 1 ~g/ml unmanipu7Lated MoAb control. The %
immunoreactivity retained of each sample was calculated as previously discussed.
procedure 3 - Direct RIA

.. ig -Appropriate amounts of target cells in 1 ml of 10% FCS media were alic,~uoted into 4 ml polystyrene test tubes, centrifuged and supernatant was discarded.
Samples to be tested were diluted to make a concentration of 200 ~g/ml MoAb equivalents in 10% FCS
media. Five additional five-fold serial dilutions were prepared from each 200 ~g/'ml sample and 0.05 ml was added to each tube in dup7.icate. 0.05 ml of 1251-MoAb was added to each tube (oF~timal amount is individually determined for each MoAb and batch). Positive control samples contained cells, ~~edia and 1251-MoAb. Back-ground samples contained non-specific cells, media and 1251-MoAb. Cells were incubated for i hour at 4oC, washed once with 2% FCS media, twic~ with PBB, transferred and counted as previously mentioned.
The % 1251-MoAb binding inhibition of each sample was calculated by t:he following formula:
A H 1i:5 x 100 - % I-MoAb Binding inhibition C-B
A = Averag. CPM~s of samp7.~
B = Averag~ CPM~s of backcrround C = Average CPM~s of positive control The plot and % jLmmunoreactivity retained by each sample was calculateZ! as previously discussed except the HD50 is actually BID50 (Dose of MoAb needed to give 50% inhibition of the binding of 1251-MoAb).
o s:
1) Tubes were always vigorously vorteued immediately after the addition of every reagent in the RIA~s.
2) An internal control sample equalling 50% of the unmanipulated MoAb control was included in each set of assays to confirm whether each procedure was quantitative in predicaing the conjugates retention of immunoreactivity.

-~ 20 -The results from these assays are tabulated below in Table 3.
Table 3 Immunoreacti.vity of MoAb Conjugates Non-specific conjugates Immunoreactivity using the product of % of unmodified MoAb Exaa~le 3 with: ~~eparation control Lym 1 ~ 1 15 B72.3 ~1 70 ~2 10 xydrazide of 3-mercap-topropionic acid di-sulfide of LLE3328871I (Example 4) con~uqated to:
Lym 1 ~1 100 ~2 87 ~3 64 #4 80 ~5 100 2() 1 4459 ~~ 21 -Table a (continued) Immunoreactivity of MoAb Conjugates Hydrazide of 3-mercapto-propionic acid disulfide Immunoreactivitv of E3328871I % of unmodified (Example 4) conjugated to: Preparation MoAb control Lym 2 #1 57 #2 85 #3 39 #~ 70 H72.3 #1 100 #2 90 CT-M-of #1 60 MAC-68 #1 40 #2 28 2o Hydrazide conjugates prepared using the product of Example 5 with:
Lym i #1 ioo #2 100 Hydrazide of 3-mercapto-propionic acid disulfide of LL-E33288a3= (Example 6~~
3p conjugated to:
Lym 1 78 ~ p ~I 4 4 5 9 -~ 22 -Table a Lcontinued) Immunoreactivity of MoAb Conluqates Hydrazide of 3-mercapto-propionic acid disulfide of N-acetyl LL-E3328861I ImmunoreactivitY
(Example 7) % of unmodified conjugated to: 1?reparation MoAb control Lym 1 82 872.3 100 Hydrazide of 3-mercapto-propionic acid disulfide of LL-E33288a2I
(Example 8) con j ug~ated to Hydrazide of 3-mercapto-propionic acid disulfide of iodo LL-E33288 pseudoaglycone (Example 9) conjugated to:

8ydrazid~ of 3-mercapto-butyric acid disulfide of LL-E33288b1I
(Example 10) conjugated to:
Lym-1 73 2p 1 4459 Table 3 1'continued) Immunoreactivitv of MoAb Coniucates Hydrazide of 3-mercapto-isovaleric acid disulfide of LL-E33288b1I Immunoreactivity (Example 11) % of unmodified conjugated to: Preparation MoAb control Lym-1 64%
Hydrazide of p-mercapto-dihydrocinnamic acid disulfide of LL-E33288b1I (Example 12) conjugated to:
Lym-1 61%
The monoclonal a~atibody conjugates of this invention are active as anticancer agents. The assay described below for assessing ~ vitro cytotoxicity shows the dramatic prefers:nce of the constructs for target cell lines as oppo.~sd to non-target cells, and provides a measure of uti7.ity of targeted forms of the compounds compared to thej.r noa-targeted counterparts.
Cvtotoxicity Assays ~$ O tro Samples to be tested were diluted to a con-ceatration of 0.2 or 0.02 ~g/ml of parent drug equi-valents (starting concentration is dependent on cell line to be tested and poteency of the parent drug).
3o Three or four additional !'ive-fold dilutions were prepared from each original sample dilution and 0.2 ml was added to sterile 15 m7L polystyrene tubes. At least one similar conjugate consisting of parent drug and an irrelevant MoAb was included in each assay to determine specificity of the relevant conjugate. 105 appropriate target cells is 0.2 ml of 10% P'CS media were aliquoted into the tubes and vortex~ad. In addition, an identical 2 p 1 4 4 59 .. 24 -test was performed utilizing irrelevant cells as tar-gets to further confirm s~~ecificity of relevant conjugate. MoAb controls received only equivalent amounts of MoAb and posit~Lve control samples received only 10% FC8 media.
Cells were incubated at 37°C for 7 minutes then washed 4 times with Et ml of 2% FCS media. 0.1 ml of 10% FCS was added to each tube, cells were vortexed and 0.2 ml was aliquoted 1:o each well of a sterile io 96-well polystyrene tissues culture plate.
Plates were incubated for 2 days in a humidified 37°C incubator with 5% C02. One half of the media was removed and replaced with fresh media containing 2 ~Ci/ml 38 thhmidine (DuPont, NEN, Cat# NET-027). Incubation was continued for 24 hours, cells were frozen, thawed and harvested by a P8D cell harvester (Cambridge Technology, Inc.). Each sample was counted for i minute in a Beckman L8 5800 scintil-lation counter on channel i.
The % growth inhibition was calculated as follows:
Average CPM of test va7lue x 100 - % Growth Average CPM of media control l00 - % Growth - % Inhibition The % inhibition was plotted on the non-log scale of a semi-log graph and the parent drug concentration was plotted on the log scale. The IC50 (concentration of parent drug needed to give 50%
inhibition) of each test sample was derived from the graph and the amount of retention of cytotoxicity was calculated by the following equation:
IC50 of parent drug x ;L00 = % Cytotoxicity Retained IC50 of test sample zc~ ~ 459 -~ 25 The results from the in vitro cytotoxicity assay are tabulated below in Table 4.
Tab a 4 In yitro Cytotoxicit:y of MoAb Conjugates MoAb Preparation Cytotoxicity % product of %E3328871I Example 1 Non-specific con jugates prepared to using product of Example 3 with:
Lym 1 ~l .9 11.3 H72.3 1.4 3.8 % product of %E33288~1I Example 4 Hydrazide conju-2o gates prepared using product of Example 4 with:
Lym 1 ~1 80 ~2 56 191 ~3 40 60 2p ~I 459 -~ 26 -Table ~! I; continued) MoAb Preearation Cytotoxicity % product of %E3328871I Example 4 Hydrazide conju-gates prepared using product of Example 4 with:
Lym 1 I;1~3 Against0 0 noa-tar-c~eted cells) Lym 2 111 2 9 ~3 2 55 H72.3 ~1 0 0 #2 0 0 MAC-68 ~1 90 CT-M-O Il)' 1 111 8 3 0 %E3328871I

Hydrazide conju-gates prepared using product of Example 5 with:

Lym 1 IlE l 3 0 0 #2 100 -, 2~ -Table 4 ccontinued) Mo~,b Cytotoxicit~

Hydrazide conjugate prepared using product of Example 6 with: % LL-E33288a3I

LyW 1 5 0 0 CT-~M-O 1 3 0 0 Hydrazide conjugate prepared using product % N-acetyl of Example 7 with: LL-E33288b1I

Lya1 1 4 0 0 CT-~M-01 7 0 0 8ydrazide conjugate prepared using product % product of of Example 8 with: Example 8 CT-~M-Ol -18 Hydrazide conjugate prepared using product % product of of EBample 9 with: Example 9 Hydrazide conjugate prepared using product % product of of Example 1o with: Example 10 Lym 1 400 2 ~ ~ 4 4 5~
_. 2 8 Table ~ ('continued) MoAb C~totoxicitv Hydrazide conjugate prepared using product % product of of Example 11 with: Example 11 Lym 1 320 Hydrazide conjugate prepared using product % product of of Example 12 with: Example 12 Lym 1 5 6 0 The following a.~say system was used to measure the in vivo activity of the conjugates of this invention.
In vivo tests for aatitumor activity on drug-monoclonal antibody conjugates were done using human tumor genographs in athymj.c laude) mice.
2o Hurkitt lymphom:: (Raji) aid myeloma tH8 Sultan) cells were harvesl:ed from culture flasks and inoculated subcutaneously t> 80 t 106 Raji cells or 40 s 106 H8 Sultan cells) inl:o test mice. Solid tumors, ovarian carcinomas tCA73, Ovcar-3), breast carcinoma tMZ-1) and colon carcinom:~ tL8171T) were propagated in athymic mice, removed, cul: into 2 mm3 fragments and implanted subcutaneously jLnto test mice t5-8 fragments per mouse).
Drugs, monoclonal antibodies and 3o drug-monoclonal antibody <:onjugates were administered intraperitoneally once each 3 days for 3 or 5 total injections starting on da!~ 2, 3, 4, 6, 7 or 8 days after tumor implantation. Tumor measurements tthe length and width of the t~imor) were made by means of a Fowler ultra CAL II electronic caliper each 7 days for 4 to 6 weeks post tumor implantation. Tumor mass in mg was estimated from the formula:

2p' 4459 .. 2 g _ Length(mm~~ x Width(mm) Tumor growth inhibition was calculated for each test group on a percent of control [mean mg of treated (T) divided by mean mg of control (C) x 100].
A T/C value < ~2% in grou~~s with >_ 65% surviving animals is considered nec~assary to demonstrate activity.
io The results from this assay appear in Table 5.

-~ 30 -Table 5 In Vivo.Ant3,tumar Testing Results Dosaqe(mca) Tumor Size S T
MoAb Drua ITJC)%control 8ydrazide of 3-mercaptopro-~ 14.5 0.26 12 5/6 pionic acid disulfide of LL-E3328871I conjugated to Lym 2 8ydrazide alone - 0.26 34 4/6 MoAb Lym 2 alone 14.5 - 32 6/6 Mixture, hydrazide +
MoAb Lym 2 14.5 0.26 20 5/6 ip treatment against human melanoma cell line 8.8. Sultan, 3 injections starting on day 5 after tumor implantation, measurements given made on day 35 post-implantation 2p~ 459 -~ 31 -Table 5 (:continued) Dosag~e(mcq) Tumor Size 8 T

Mob Druc IT(C)%control Hydrazide of 3-mercapto- 15.5 0.25 39 7/7 propionic acid disulfide of LL-E3328871I

conjugated to MAC-68 Hydrazide alone - 0.25 - 0/6 MoAb MAC-68 alone 31 - 78 6/6 Mixture, hydrazide + 15.5 0.25 - 0/6 MoAb MAC-68 Melphalan (as positive - 200 43 6/6 control) ip treatment against human ovarian cancer line CA73, three injections started 3 daps after tumor implantation, measurements given made on dap 35 post-implantation .. i'014459 Table 5 (continued) Dosagetmc~ Tumor Size S T
MoAb Druc (T/C)%control Hydrazide of 3-mercapto- 8.75 0.25 14 4/6 propionic acid disulfide of LL-E3328871I
conjugated to CT-M-01 Hydrazide alone - 0.25 - 0/6 MoAb CT-M-01 alone 8.75 - 75 5/6 Mixture, hydrazide + 8.75 0.25 - 0/6 MoAb CT-M-01 vincristine (positive - 20 0 4/4 control) ip treatment against human breast cancer cell line MX-i, 2o three injections started on day 2 following tumor implanta-tion, measurements given made oa day 35 post-implantation 2 p1445'~

Table 5 (continued) Dosaqe(mcqy Tumor Size MoAb D~ (T,/C) %control 8ydrazide of 3-mercapto- 6.2 0.125 62 6/6 propionic acid disulfide of LL-E3328871I con-jugated to 872.3 8ydrazide alone - 0.125 85 6/6 MoAb H72.3 alone b.2 - 96 6/6 Mixture, hydrazide + 6.2 0.125 105 5/6 MoAb H72.3 LL-E3328871= - 0.005 111 5/6 (3 treatments Cis platinum (positive - 6o 6 s/s control, 3 treatments) ip treatments against human ovarian cell line ovCAR-3, five injections starting on day a after tumor implantation (unless otherwise noted), measurements given made on day 35 post-implantation 35 _ .. g,~ -Table 5 (continued Dosaqe(mcg) Tumor Size s T
MoAb Drua (T/C)%control Hydrazide of 3-mercapto- 27 0.26 6 3/6 propionic acid disulfide of LL-E3328871I con-jugated to Lym 1 Hydrazide alone - 0.26 72 6/6 MoAb Lym 1 alone 27 - 72 6/6 Mixture, hydrazide + 13 0.13 61 4/6 MoAb Lym 1 ip treatment against human Hurkitt lymphoma cell line Raji TC, three injections startesd on dap 7 after tumor implanta-tion, measurements given made on day 2s post-implantation 201 ~~59 Table 5 (continued) Dosaqe(mcg,~ Tumor Size S T
MoAb Drua (T/C)%control Hydrazide of 3-mercapto- 28 0.75 1.3 6/6 propionic acid disulfide of LL-E33288a3I
conjugated to Lym 1 LL-E33288a3I alone - 0.75 365 6/6 Hydrazide alone - 0.75 330 6/6 MoAb Lym alone 28 - 68 6/6 ip treatment against human Hurkitt lymphoma cell line Raji TC, three injections started on dap 7 after tumor implantation, measurements given made oa day 28 post-implantation Hydrazide of 3-mercapto- 83 2.0 0.02 6/6 propionic acid disulfide of LL-E33288a3I
conjugated to CT-M-O1 LL-E33288a3I alone - 2.0 - 0/6 MoAb CT-M-O1 alone 83 - 75 5/6 vincristine (positive - 20 1.2 5/5 control ip treatment against human breast cancer cell line MX01, three injections started on day 2 following tumor implantation, measurements given made on day 35 post-implantation.

Z~'' 459 -~ 3 6 -Table 5 (:continued) Dosaq e (mc9~) Tumor sizes T

MoAb Drug (TfC) %control Hydrazide of N-[[(~- 50 0.22 23 7/7 methylcoumarin-7-yl)-amino]acetyl]cysteine disulfide of LL-E33288b1I

conjugated to Lym i Hydrazide alone - 0.22 228 4/7 MoAb Lym 1 alone 50 - 61 7/7 Mixture, hydrazide plus 50 0.22 56 7/7 MoAb Lym 1 ip treatment against human Burkitt lymphoma cell line Raji TC, three injections starti~d 7 after tumor on day implantation, measurements gives made on day post-implantation Hydrazide of 3-mercapto- 125 1.0 0 6/6 propionic acid disulfide of N-Acetyl LL-E33288b1I
conjugated to CT-M-O1 Hydra$ide alone - 2.0 71 3/6 Mixture, N-acetyl 125 1.0 5 1/6 LL-E3328851= plus MoAb CT-M-O1 2i0 1 4459 Table 5 (continued) Dosage(mcg) Tumor Size S
T

MoAb Druc (T,/ C)%control N-acetyl LL-E332886 I

- 1.0 6 iv treatment against human breast cancer cell line MX-1, three injections starting on day 2 following tumor implantation, measurements given made on day 42 post-implantation Hydrazide of 3-mercapto- 37 1.0 11 6/6 propionic acid disulfide of N-acetyl LL-E33288b1I

conjugated to Lym 1 is Hydrazide alone - i.o 517 6/6 N-acetyl - 0.5 226 6/6 LL-E3328861I alone MoAb Lym 1 alone 37 - 178 6/6 iv treatment against human Hurxftt lymphoma cell line Raji TC, three injections starting on dap 7 after tumor implantation, measurements given made on day 35 post-implantation _~ 2~I14~59 _. 3 g -Table 5 I: continued) Dosaqe(mcg) Tumor size s T
MoAb Drua (T/C)%control Hydrazide of 3-mercapto- 127 2.7 61 6/6 propionic acid disulfide of N-acetyl LL-E3328861I
conjugated to 872.3 N-acetyl LL-E33288S1I - 2.0 32 1/6 io alone MoAb B72.3 alone 127 - 125 6/6 vincristine (positive - 2o 23 6/6 control) iv treatment against human colon cancer cell line Ls174T, three injections started on day 8 following tumor implantation, measurements given made on~day 21 2o post-implantation Table 5 (continued) Doss e(mcq) Tumor Size MoAb Drug (TJC)%control Hydrazide of 3-mercapto- 112 2.7 40 5/6 propionic acid disulfide of N-acetyl LL-E33288d1I
conjugated to CT-M-O1 N-acetyl LL-E33288b1I alone - 2.0 32 1/6 Vincristine (positive - 20 23 6/6 control) iv treatment against human colon carcinoma line L8174T, three injections started s days after tumor implantation, measurements given made on day 21 post-implantation -~o Table 5 (continued) Dosaqe(mcg) Tumor Size 8 T
MoAb Drug (TJC)%control Hydrazide of 3-mercapto- 24 1.0 0.33 2/6 propionic acid disulfide of LL-E33288a2I
conjugated to CT-M-O1 Hydrazide alone - 1.0 - 0/s iv treatment against human breast cancer cell line Mx-i, three injections started on day 2 following tumor implantation, measurements given made on day 35 post-implantation 20'14459 _. ,1 _ The invention will be further described in conjunction with the following non-limiting examples.
Example 1 3-Mercaptopropionic Acid Disulfide of To a solution o!' 90 mg of LL-E3328871I in 90 ml of acetonitrile was added 10.6 mg of 3-mercaptopropionic acid i.n 1 ml of acetonitrile. The solution was vortexed and then stored at -20°C for 6 l0 days. The solvent was removed ~ vacuo and the residue chromatographed over io m7. of silica gel in methylene chloride. The column was developed with 50 ml of methylene chloride, 50 ml of 4% methanol in methylene chloride and finally 100 ml of 8% methanol in methylene chloride. Evaporation of this last fraction gave a residue which was taken up in ethyl acetate with the aid of a little acetone and added dropwise to an excess of hexane. The precipitate was collected and dried, giving 39 mg of the desired product (FABMB, M+H 1394).
Exam, Dle Z
p-Nitrophenyl a-mercaptopropionic acid disulfide of LL-E33288~1I
(A) Preparation of p-nitrophenyl ester of 3-mercaptopropionic acid Commercial 3-mercaptopropionic acid in methylene chloride containing a catalytic amount of concentrated sulfuric acic! was treated with isobutylene for 20 minutes. The soluiaon pas then extracted with iN sodium bicarbonate solution after which the methylene chloride solution was dried using anhydrous magnesium sulfate. The solution was then evaporated to a colorless mobile liquid which NMR and mass spectral data indicated was the 8-~t-butylmercaptopropionic acid, t-butyl ester.

~0 1 ~~459 _. ,~ 2 -An aliquot of this ester was refluxed with 6N
hydrochloric acid in dioxame for 2.5 hours. The solvent was evaporated, ethyl acetate was added and this solution was extracted with sodium carbonate. The sodium carbonate extract eras treated with 6N
hydrochloric acid until the pH of the suspension was 2Ø The suspension was then extracted with ethyl acetate, the extract driedl over anhydrous magnesium sulfate and the solvent evaporated to a colorless liquid which iH NMR and mass spectral data indicated was 8-t-butylmercaptopropi.onic acid.
This compound ways converted to the p-nitrophenyl ester by treatment with equimolar amounts of p-nitrophenol and dicyc:lohexylcarbodiimide in tetrahydrofuran for 4 hours. The dicyclohexyl urea by-product was removed by filtration and the filtrate was evaporated to an oil ~irhich was purified by passage over neutral silica gel usring the solvent system hexane:methylene chloride (50:50). The pure p-nitropheayl ester deriva~tivs was a faintly yellow, mobile oil.
Ths free mercapt:an was unmasked by the following procedure. The 8-~-butylmercaptopropionic acid p-nitrophenyl ester was dissolved in trifluoroacetic acid and :~ slight molar excess (l0%) of mercuric acetate was added. The mixture was stirred for 30 minutes, then the t:rifluoroacetic acid was evaporated and the residuE: taken up in di-methylformamids. This so~.ution was treated with hydrogen sulfide gas for »5 minutes, then the black mercuric sulfide was filtE:red off and the filtrate evaporated under reduced ~~ressure to eliminate up to 99% of the dimethylformamide. The resultant slightly brownish mobile liquid was purified over neutral silica gel using hexane:methylenea chloride (50:50). The major component was shown by iH NMR to contain a small amount of the t-butyl mercapto derivative. Analytical HPLC

2p ~ 459 -~ 43 -over two Perkin-Elmer Pecosphere Cis columns in tandem (4.6 x 33 mm and 4.6 x 83 mmJ using a gradient system of 37.5/62.5 to 47.5/52.5 of acetonitrile and O.iM
ammonium acetate buffer at: pH 6.5 (acetic acid) over a 12 minute span indicated that the product was 88% of the p-nitrophenyl ester of 3-mercaptopropionic acid and 10% of the less polar s-t-~butylmercaptopropionic acid p-nitrophenyl ester. There was also a small amount of free p-nitrophenol present:.
(B) Reaction of p-nitrophenyl ester of 3-mercaptopro-pionic acid with LL-~:3328871I
A 100 mg portio:~ of LL-E3328871I was dissolved in 50 ml of acet:onitrile. To this was added a solution of 25.7 mg of ~~-nitrophenyl ester of 3-mercaptopropionic acid i.n i ml of acetonitrile. The reaction was left at -20oC; for 48 hours. HPLC
indicated the reaction was: complete. The solution was evaporated to dryness and the residue taken up in 4-5 ml of ethyl acetate u::ing sonication to effect solution. The mixture was filtered and the filtrate dripped into 45 ml of stirred hexane. The resultant faintly yellow solid was c;ollected and dried under ' reduced pressure, giving ~~3 mg of the g-nitrophenyl ester of propionic acid derivative of LL-E3328871I as established by 18 NMR. By FABMB the (M+8] ion appeared at m/z=1515.
Retention time on Cis reverse phase HPLC:is min. with 50% acetonitrilca/O.iM aqueous ammonium acetate (LL-E33288b1I:8.0 min., ester hydrolysis product: l.5 min.).

2~U 1 4459 .. 44 _ Exam a 3 N-Hydroxvsuccinimidyl 3-mercaptopropionate disulfide of LL~~E3328871I
TO a SOlutiOn 07:' 5 mg Of the 3-mercaptopropionic acid disulfide analog of LL-E3328871I from Example 1 in 0.5 ml of tetrahydrofuran was added o.45 mg of N-hydroxysuccin-imide in 0.1 ml of tetrah~tdrofuran and then 1.8 mg of dicycloheuylcarbodiimide in 0.2 ml of tetrahydrofuran.
1o The reaction was allowed i:o stir at room temperature for 4 hours and was then <;uenched with a large excess of hexanes. The solid waa3 isolated by filtration and dissolved in ethyl acetate. The resulting solution was washed three times with brine, dried with magnesium sulfate, and evaporated to 5 mg of the desired product as a tan powder which Was used without further purification. Retention mime on reverse phase C18 HFLC: 15 minutes with 40% acetonitrile/0.1 M aqueous ammonium acetate (startin<~ material: 6.0 minutes).
Exam a 4 3-Mercaptogropiony;,~ hydrazide disulfide of L1L-E3328871I
To 5.4 ml (3 eq;l of anhydrous hydrazine in 100 ml of refluuing tetral~ydrofuran under argon was added dropwise 9.2 ml (83 mmol) of methyl 3-mercaptopropionate in 50 ml tetrahydrofuran over 2 hours. The solution was :refluxed an additional two hours, evaporated, and then diluted and evaporated twice from 300 ml of toluene. The product was applied to a plug of silica gel with 5% ethyl acetate/chloroform and eluted from the plug with 20%
methanol/chloroform. The resultant 3-mercaptopropionyl hydrazide was a faintly pink oil which solidified when cooled but melted at room temperature.
To 50 mg of LL-;E33288~1I in 50 ml of acetonitrile at -15OC was added 6.6 mg of ~ ~ ~ 4 4 59 3-mercaptopropionyl hydrazide in 1 ml tetrahydrofuran.
One equivalent of triethyl,amins or one equivalent of triethylamine and one equ3,valent of acetic acid was added. The reaction was allowed to stir at 0°C for one hour and the solvent was then evaporated. The residue was chromatographed on silica gel with a l0-15%
methanol-in-chloroform gradient to yield 26 mg of the desired product. FABMB, m/z=1408 (M+H)t retention time on reverse phase C18 BFLC: 5.0 minutes in ~1%
1o acetonitrile/o.i M aqueous ammonium acetate.
Exaaaple 55 N-[[(4-Methyl-coumarin-7-yl)amino]acetyl]cysteine hydrazide disulfide of LL-E3328871I
A mixture of i.0~ g (5.7 mmol) of ~-methyl-7-aminocoumarin, 3.0 ml of ethyl bromoacetate (5 eq), 90 mg (0.1 eq) of sodium iodide, and 30 ml dimethylformamide was heated under argon at 8o°C for 5 hours. The mixture was cooled, diluted with ethyl ether, washed three times with 50% brine, dried with magnesium sulfate, and evaporated to dryness. The crude product was dissolv~~d in chloroform containing 1%
ethyl acetate and filtered through a plug of silica gel. Recryatallization from diethyl ether containing a trace of chloroform yielded pure ethyl N-[(4-methyl-coumarin-7-yl.)amino]acetate.
To 1.96 g (7.5 a~mol) of the above ester in 15 ml of methanol and 15 a~l of tetrahydrofuran was added 10 ml of iN aqueous sodium hydroxide. After 30 minutes, l ml of 10% aqueous hydrochloric acid was added. The organic solvents were evaporated and the resultant crystalline product was filtered and washed with cold ethanol and the=~ ether. This material was dissolved in 20 ml of tet:~ahydrofuran and 4 ml of dimethylformamide. Dicycl.ohexylcarbonyldiimidazole (1,3 g, 2.2 eq) was added and the reaction allowed to stir for 15 minutes. Cyst:eine ethyl ester ~- 46 -hydrochloride (1.6 g, 2.5 eq) aid triethylamine (1.2 ml) were then added. After a further three hours, the reaction was diluted with ethyl ether containing 5%
methylene chloride and washed once with lo% aqueous hydrochloric acid and twice with brine. After drying with magnesium sulfate and evaporating the solvents, the crude product was cry:3tallized by dissolving in chloroform containing a minimal amount of ethanol and then adding an excess of ~~ther. The crystals were 1o filtered and dried to give pure ~1-[[(4-methyl-coumarin-7-yl)amino]acetyl]cysteina ethyl ester.
A mixture of 5 tnl of chloroform, 20 ml of methanol, and 0.4 ml of h~~drazine hydrate was heated to reflex under argon. To this was added 550 mg of N-I[(4-methyl-coumarin-7-;~1)amino]acetyl]cysteine ethyl ester. After refluxing for 9 hours the mixture was cooled and the solid product was filtered and washed with chloroform and then ~:thyl ether. The crude product (which contained ~thiol and disulfide) was dis-2o solved in dimethylformamide containing dithiothreitol and triethyl amine. After 3o minutes the product was precipitated with excess inthyl ether and collected by filtration. This material was purified further by recrystallization from degassed acetonitrile containing dithiothreitol and a trac~~ of triethyl amine to give pure ~1-[[(4-methyl-coumaran-7-yl)amino]acetyl]cysteine hydrazide.
To 12 mg of LL-1~3328871I in 12 ml aceto-nitrile at 0oC was added .1 mg of ~T-[[(4-methyl-3o coumarin-7-yl)amino]acety:l]cysteine hydrazide in 1.2 ml dimethylformamide. After stirring overnight another 2 mg of ~1-[[(4-methyl-cowaarin-7-yl)amino]acetyl]-cysteine hydrazide in o.6 ml dimethylformamide was added. The reaction was atirred for 3 days at 0oC and filtered. The acetonitri;le was evaporated and the resultant dimethylformami~de solution was diluted with an excess of 1:1 hexanes/~ether. The product was _. 4 7 _ isolated by filtration and further purified by chromatography on silica gel with a 15-20% gradient of methanol in chloroform to yield 3 mg of the desired product. Retention time on reverse phase C18 HPLC: 3.5 minutes using 45% acetonit:rile/0.1 M aqueous ammonium acetate (LL-E33288d1I:i5.5 min. in the same system).
Example 6 3-Mercaptopropionyl h~drazide disulfide of LL-E3'~3288a3I
To 10 mg of LL-F;33288a3I in 9 ml of acetonitrile at -15°C was added 6.6 mg of 3-mercaptopropionyl hydraz;ide in 1 ml acetonitrile.
one equivalent of triethyl.amine or one equivalent of triethylamine and one equivalent of acetic acid were added. The reaction was allowed to stir at 0°C for one hour and the solvent was than evaporated. The residue was chromatographed on silica gel with a 10-15%
methanol-in-chloroform gradient to give the desired product. FA8M8. m/g-1251 (M+H): retention time on reverse phase C18 HPLC: 2.1 minutes in the system 45%
acetonitrile/o.i M aqueou~~ ammonium acetate (LL-E33288a5I: 5.7 min. in. the same system).
Example 7 3-Mercaptopropionyl hydrazide disulfide of N-acetyl LL-E3328871I
To 10 mg of N-acetyl LL-E3328871= in 10 ml of acetonitrile at -15°C was added 1.5 eq of 3-mercaptopropionyl hydraz;ide in s5 ~l acetonitrile.
one equivalent of triethy7.amine was added. The reaction was allowed to star at 0°C for two hours and the solvent was then evaporated. The residue was chromatographed on silica gel with a 10-15%
methanol-in-chloroform gradient to yield the desired pr°duct. FAHMB, m/z=1450(M+H): retention time on C18 reverse phase HPLC:2.5 min. with 50%

2 0~ 1 4 4 5 9 ..48_ acetonitrile/o.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E3a28871I:6.6 min. in the same system).
Exam a 8 3-Mercaptopropionyl hydrazide disulfide of LL-E33288a2I
To 10 mg of LL-F~33288a2I in 10 ml of acetonitrile at -lSoC pas added 1.5 eq of 3-mercapto-propionyl hydrazide in 1 a~l acetoaitrile. One l0 equivalent of triethylamine was added. The reaction was allowed to stir at 0oC- for one hour and the solvent was then evaporated. The residue was chromatographed on silica gel with a 5-15~c methanol-in-chloroform gradient to yield the desired product. FAHMB, m/z=1248 15 (M+H): retention time on CllB r~verse phase BPLC:2.6 min. with 58% acetonitrilEr/o.o5 M aqueous ammonium dihydrogen phosphate (LL-~:33288a2=:7.5 min. in the same system).

.._ 2p 1, 4459 .. 4g -Example 9 3-Mercaptopropionyl hydrazide disulfide of iodo LL-E33288 pseudoaqlycone To 10 mg of iodo LL-E33288 pseudoaglycone in

9 ml of acetonitrile at -~.SoC was added 1.5 eq of 3-mercaptopropionyl hydraz;ide in 1 ml acetonitrile.
one equivalent of triethy7.amine was added as a catalyst. The reaction was allowed to stir at 0°C for one hour and the solvent was then evaporated. The io residue was chromatographed on silica gel with a 10-15%
methanol-in-chloroform gradient to yield the desired product. FAHMB, m/z=1091 (M+H); retention time on C18 reverse phase HPLC:2.8 mi:~. with 50%
acetonitrile/o.o5 M aqueous ammonium dihydrogen phosphate (iodo LL-E33288 pseudoaglycone: 7.9 min. in the same system).
E,~l a 0 3-Mercaptobutyryl t.ydrazide disulfide of LL-E3328871=
2o To 17.2 g (0.2 aroles) of crotonic acid was added 18 ml (0.26 moles) of thioacetic acid. This mixture was heated at ref7.ux under argon for 6 hours.
The excess thioacetic acid was removed under aspirator vacuum and the resultant oil was dissolved in 10o ml solute ethanol containing 2001 of concentrated sulfuric acid. This reacl:ion was refluxed for 10 hours and then reduced in volumE: under aspirator vacuum.
Hexanes were added and the resultant solution washed successively with two portions of saturated sodium bicarbonate and one portion of water. This solution was then dried with magne.~ium sulfate, filtered, and reduced in volume to an oil. This crude product was dissolved in 250 ml of mei:hanol containing 12 ml of hydrazine and the resultant mixture was refluxed for 1o hours under argon. The reaction mixture was reduced in volume and then distilled rapidly by Rugelrohr and crystallized from a mixture of chloroform-hexanes to give 3-meraaptobutyryl byd~razide.
To 5 mg of LL-E3328871= in 5 ml o!
acetonitrile at -15°C was added 1.5 eq of 3-mercaptopropionyl bydraside in 1 ml acetonitrile.
One equivalent of triethylamine was added. The reaction vas allowed to stir at o°c for one hour and the solvent was then evaporated. The residue was cbromatographed on silica gel with a l0-as~C
methanol-in-chloroform gradient to yield the desired product. FAHMB, m/e=1~22 (M+8): retention time on C18 reverse phase APLC: 3.5 min. vitb 13'~ aceto-nitrile/0.05 M aqueous ammonium dihydrogen phosphate (LL-E3328871I:13.1 min. in the same system).
Ez~~le 11 3-Mercaptoisovalerv~l hydrazide disu~"~~de of LL-~E33288~1=
To 10 g (0.1 moles) of 3,3-dimethyl acrylic acid was added 9 ml (0.13 moles) of thioacetic acid.
2o Thia mixture was heated at: retlux under argon for 6 hours. The excess thioacetic acid was removed under aspirator vacuum and the resultant oil was dissolved in 100 ml absolute ethanol ~~or~~~irt~r~~S 200 yl of concentrated sulfuric acid. This reaction was refluxed for 34 hours before addia5t is ml of bydrazine. The resultant mixture was refl.used for 21 hours under argon. The reaction mixture was reduced in volume and then dissolved in a mixture of brine and saturated sodium bicarbonate. The ~~roduct was extracted with several volumes o! chloroform. The combfned chloroform layers were dried with magnesium sulfate, filtered, and reduced is volume to an oi.l. This oil vas purified by flash chromatography with a methanol-chl~orofortn gradient and then crystallized from chloroform-hexanes to give 3-mercaptoisovale:ql hydrazide.

To 15 mg of LL-E33288Y1I in 5 ml of acetonitrile at -lSoC was added 1.5 eq of 3-mercaptoisovaleryl hydrazide in 100 ul acetonitrile. One equivalent of triethylamine was added as a catalyst. The rea~~tion was allowed to stir at ambient temperature for 3 hours and the solvent Was then evaporated. The residue was chromatographed on silica gel with a 10-15~ methanol-in-chloroform gradient to yield the desired product. FABMS, m/z=:1436 (M+H)s retention time on C18 reverse phase HPLC:3.9 min, w th 43$ acetonitrile/0.05 M
aqueous ammonium dihydrogen phosphate (LL-E33288y1I:13.4 min.
in the same system).
Example 12 p-Mercaptodihydrocinnamoyl hydrazide disulfide of LL--E33288Y1I
To 500 mg (2.75 mmol) of p-mercaptodihydrocinnamic acid was added 15 ml methanol containing one drop of concentrated sulfuric acid. 'The reaction was refluxed for 5 hours and then cooled to ambient temperature. Hydrazine (1.5 ml) was added and the re;aultant mixture was refluxed for 2 hours under argon and then ;stirred for 10 hours at ambient temperature. A 200 mg portion of dithiothreitol was added to reduce any disulfides present and the reaction mixture was cooled to -15°. The resultawt crystals were filtered, washed with a mixture of ether and methanol, and then dried in a vacuum oven (50°/5 microns/10 hours) to give p-mercapto-dihydrocinnamoyl hydrazide.

- 51a -To 25 mg of LL-E33288Y1I in 25 ml of acetonitrile at -15°C was added 1.5 eq of p-mercaptodihydrocinnamoyl hydrazide in 1 ml acetonitrile. The reaction was allowed to stir at ooC
for 10 hours and the solvent 'was then evaporated. The residue was chromatographed on silica gel with a 10-15~ methanol-in-chloroform gradient to yield 'the desired product. FABMS, m/z=1484 (M+H); retention time on C18 reverse phase A

BPLC:5.4 min. with 43% ace~tonitrile/0.05 M aqueous ammonium dihydrogen phosphate (LL-E3328871I: 13.4 min.
in the same system).
Exam a 3 3-Mercaptoisovalery~l hydrazide disulfide of N-acetyl LL-E3328871I
To 20 mg of N-acetyl LL-E33288~1I in 15 ml of acetonitrile at -lSoC was added 3 eq of 3-mercaptoisovaleryl hydra,zide in 6.2 ml acetonitrile.
one equivalent of triethylamine was added. The reaction was allowed to stair at ambient temperature for 2 hours and the solvent was then exaporated. The residue was chromatographe~d on silica gel with a l0-15%
methanol-in-chloroform gradient to yield the desired product. Retention time am C18 reverse phase HPLC:2.5 min. with 50% acetonitrile~/o.o5 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E3328871I: 6.6 min.
is the same system).
Example 1!
3-Mercaptobutyryl hvdrazide disulfide of N-acetyl. LL-E33288~1=
To 10 mg of N-acetyl LL-E3328871I in 7.5 ml of acetonitrile at -15°C arcs added 3 eq of 3-mercaptobutyryl hydrazide in 5 ml acetonitrile. one equivalent of triethylamine was added. The reaction was allowed to stir at amt~ient temperature for to hours and the solvent was then evaporat~d. The residue was chromatographed on silica gel with a 10-15%
methanol-in-chloroform gradient to yield the desired product. Retention time on C18 reverse phase HPLC:7.3 min. with 43% acetonitrile/0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E3328871I:5.6 min. in the same system).

,. 2 p 1 4 4 5 9 - s3 -,~~ple is b-.Kercabto inydrQc#t:na~~~r~.hydrazide disulfide of N-acetyl LL-E3328871 To 10 mg of N-acetyl LL-E3328871I in 7.5 mi of acetoaitrile at -lSoC ras added 3.o eq o!
p-maraap~sdihydrt: ~ln~~~"~~~yt~ -bydraside in 2 . 0 ml acetonitrile. one equivalent of triethylamiae was added, The reaction was allowed to skit at ambient temperature for 2 hours and the solvent was then to evaporated. The residue was cbromatographed oa silica gel with a 10-ls~k methanol-ia-chloroform gradient to yield the desired product. retention time on c18 reverse phase HpLC:~.3 min. with 43~ saetonitrile/o.05 M aqueous ammonium dihydrogen phosphate QN-acetyl 15 LL-E3328871I:5.6 min. in t;he same syatem~.
$~Q a 16 Non-specific conj,uaatioa to rroteins The hydroxysuccinimide eater described in asaaple 3 was covaleatly attached to antibodies under 20 slightly alkaline conditions. The following is a general procedure used to make the antibody conjugates listed is Table 6. Antibody at a concentration of 3-5 mg/ml in phosphate buffer containing O.iM sodium chloride, p8 7.5 was reacted with a 5-20-fold molar 25 ucess of the product from E:ample 3 with stirring, at room temperature for from i-4 hours. Tha conjugated protein was desalted chromatographicaliy and aggregated protein was separated from monomeric material by gel filtration 8PLC. Honomeric fractions were pooled and 3o concentrated.

~~abls 6 Non-specific can~uaatea prepared using' the praduct of ~;xample 3 MoAb Drug Loading Lym 1 5.2 872.3 6.0 B72.3 2.9 l0 Exasmple 17 Bite-saecific c:oniugate preparation Ths general mai:hod far attaching hydrazide derivatives of drugs to oxidized antibodies is described in T. J. MclCearn, at al., in U.B. Patent No. 4,671,958. Ths proc~~dura has bean applied to preparing antibody conjuc~atas from the products of Examples 4 to 15 with spcjcific modifications as described below. The products from these reactions and their characteristics are summarized in Table 7.
(A) Antibody,Oxidation Antibody at a concentration of 5 to 10 mg/ml was dialyz~gd ovaraight against a 200 fold valume of 50mM sodium ac~atate buffer, pH 5.5 containing O.1M sodium chloride (Bu:Efar A). After dialysis, the MoAb was oaidized with 1!5mM to 200mM periodic acid in 0.2M sodium acetate. Th~~ oxidation was allowed to proceed in the dark, wit; stirring, at 4°C for ~5 minutes after which time the oxidized MoAb was desalted on a >_3 bed volume sepha~~ea G-25 column. The degree of oxidation of the antibody was assessed by reaction with 3o g-nitrophenylhydrazina a:nd comparing absorbanca of the protein at 2sOmm vs. p-nitrophenylhydrazina at 395mm.
(H) Drua Hydrazide Coniuqation The oxidized MoAb was reacted with 10 to 200-fold molar eacass of drug hydrazide. The hydrazides were dissolved into dimethylformamide and added to the aqueous solution of MoAb. To avoid precipitation of MoAb, the final volume of dimethylfarmamida added did not eaceed 10% of the *Trade-mark ,61109-7770 ~;.~ x total reaction volume. Reaction was allowed to proceed for 3 hours at room temperature, with stirring. To prevent crosslinking of unreacted aldehydes and subsequent aggregation, a blocking agent, acetyl hydrazide was added in 100-fold molar excess three hours after addition of tb,e drug hydrazide. To stabi-lize the Schiff~s base linkage between aldehyde and drug hydrazide (a hydrazone), the product generally was reduced to an alkyl hydrazine by the addition of lomM
1o sodium cyanoborohydride, allowing the reaction to pro-ceed for one more hour (total conjugation time - 4 hours). The conjugate was chromatographically desalted and exhaustively dialyzed (minimum time 48 hours) into pH 6.5 phosphate buffer for storage and testing.
Conjugates were analyzed for the presence of aggregates by gel filtration BPLC and for free drug by reverse phase HPLC. Drug loading vas determined spectroacopically using thus extinction coefficients of both the antibody and the drug to estimate molar 2o concentrations of drug in conjugates.

20'14459 -~ 5 6 -Table 7 Hydrazide conjugates,prepared from the product of Example 4 MoAb Preparat~ Druq Loadinq M/M

Lym 1 #1 1.4 ~2 2.4 ~3 1.0 ~4 6.7 ~5 3.3 Lym 2 ~l 2.9 ~2 1.9 ~3 2.0 ~4 2.8 872.3 ~l 2.3 ~2 1.3 #3 2.5 CTM-Ol il 3.1 ~2 2.3 ~3 2.9 MAC-68 ~1 1.7 #2 3.1 ~3 2.4 Hvdrazide Conjugates pr~a~aredfrom the product of Example 5 Lym 1 ~l 0.15 ~2 0.76 ~3 3.2 2~ 1 4459 _. 5 ~ -Table 7 fcontinuedl Hydrazide conjugates prepared from the product of Exaatple 6 MoAb Preparat3.on Druq Loading M/M

Lym 1 3.0 CT-M-O1 ~1 2.4 #2 2.9 l0 Hvdrazide conj uqates prepa~ced from the product of ExautDlB 77 Lym 1 2.8 Lym 2 1.4 B72.3 ~1 2.1 ~2 2.4 CT-M-O1 ~1 1.6 ~2 3.6 ~3 2.5 ~4 2.4 Hydrazide conj ugates prepared from the product of Exam a 8 CT-M-01 4.8 ~Ydrazide conj uqates prepared from the product of Exan~_ple 9 CT-M-O1 3.0 Hydrazide conjugates ~preraared from the groduct of Examt~le 10 Lym 1 3.7 201 4~a9 _. gg _ Table 7 (continued) Hydrazide coniuqates prepared from the,product of Exam~~le 11 MoAb Preparati,o~ Druq Loading MOM
Lym 1 6 . 2 Hvdrazide conjugates prepared from the product of ERamLile 12 Lym 1 3.5

Claims (54)

1. We claim:
   
   i. A carrier-drug conjugate of the formula prepared from a compound of formula CH3SSS-W wherein CH3SSS-W is an antitumor antibiotic designated as LL-E33288.alpha.1Br, a1I, .alpha.2Br, .alpha.2I, .alpha.3Br, .alpha.4Br, .beta.1 Br, .beta.1I, .beta.2Br, .beta.2I, .gamma.1Br, .gamma.1I, .delta.1I, the iodo or bromo pseudoaglycones, their dihydro or N-acyl counterparts, BBM-1675, FR-900405,-FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl counterparts comprising:
   reacting CH2SSS-W with a compound of general formula Q-Sp-SH, wherein S P is a straight or branched-chain divalent or trivalent (C1-C18) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C3-C18) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl-or heteroaryl-alkyl (C1-C18) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or divalent or trivalent (C2-C18) unsaturated alkyl radical, wherein if S P is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; and Q is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, .alpha.-haloacetyloxy, lower alkyldicarboxyl, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, -ONH2, -CON3, to produce an intermediate of formula Q-Sp-SS-W, wherein Q and Sp are as hereinbefore defined and W is as defined in Tables I
   and II, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y)n wherein the carrier Tu is defined as a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from glycoprotein carbohydrate residues, or an amidoalkylthio group; and n is an integer of from 1 to 100, to produce a compound of the formula:
   
   wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-, -NHCONHNHCH2-, -NHCSNHN=CH-, -NHCSNHNHCH2-, -ON-CH-, -NH-, -NHCH2-, -N=CH-, -CO2-, -NHCH2CO2-, -SS-, and m is 0.1 to 15.
2. 2. A carrier-drug conjugate according to Claim 1 of the formula prepared from the class of antitumor antibiotics designated LL-E33288 (CH3SSS-W) comprising:
   displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C2-C10) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C2-C5) radicals, wherein it Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups: and Q is, or can be subsequently converted to, carbonyl, lower alkyldicarboxylanhydride, -CONHNH2, or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y)n Wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula:
   
   wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH2-, and m is 0.1 to 15.
3. 3. A protein-drug conjugate according to Claim 2 wherein CH3SSS-W is the antitumor antibiotic designated LL-E33288Y1I having:
   a) ultraviolet spectrum as shown in Figure I:
   b) a proton magnetic resonance spectrum as shown in Figure II; and c) an infrared spectrum as shown in Figure III.
4. 4. A protein-drug conjugate according to Claim 2 wherein CH3SSS-W is the antitumor antibiotic designated LL-E332SS.alpha.2I having:
   a) a proton magnetic resonance spectrum as shorn in Figure IV; and b) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure V.
5. 5. A protein-drug conjugate according to Claim 2 wherein CH3SSS-W is the antitumor antibiotic designated LL-E332SS.alpha.3I having:
   a) ultraviolet spectrum as shown in Figure VI;
   b) an infrared spectrum as shown in Figure VII;
   c) a proton magnetic resonance spectrum as shown in Figure VIII; and d) a carbon-13 nuclear magnetic resonance spectrum as shorn in Figure IX.
6. 6. A protein-drug conjugate according to Claim 2 Wherein CH3SSS-W is the antitumor antibiotic designated N-acetyl LL-E33288~1I having:
   a) ultraviolet spectrum as shown in Figure X;
   b) an infrared spectrum as shown in Figure XI;
   c) a proton magnetic resonance spectrum as shown in Figure XII; and d) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure XIII.
7. 7. A protein-drug conjugate according to Claim 2 wherein CH3SSS-W is the antitumor antibiotic designated iodo LL-E33288 pseudoaglycone having:
   a) ultraviolet spectrum as shows in Figure XIV;
   b) an infrared spectrum as shown in Figure XV;
   c) a proton magnetic resonance spectrum as shown in Figure XVI; and d) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure XVII.
8. 8. A protein-drug conjugate according to Claim 2 wherein Tu is the monoclonal antibody designated Lym 1.
9. 9. A protein-drug conjugate according to Claim 2 wherein Tu is the monoclonal antibody designated Lym 2.
10. 10. A protein-drug conjugate according to claim 2 wherein Tu is the monoclonal antibody designated CT-M-01.
11. 11. A protein-drug conjugate according to Claim 2 wherein Tu is the monoclonal antibody designated B72.3.
12. 12. A protein-drug conjugate according to Claim 2 wherein Tu is the monoclonal antibody designated B72.3.
13. 13. A protein-drug conjugate according to claim 2 wherein Q is the hydroxysuccinimide ester of a carboxyl group, Sp is -CH2CH2-, Y is -YH2, Z is -CONH-.
    and m is 0.5 to 15.
14. 14. A protein-drug conjugate according to claim 2 wherein Q is the hydroxysuccinimide ester of a carboxyl group, Sp is -CH2CH(CH3)-, Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
15. 15. A protein-drug conjugate according to claim 2 wherein Q is the 4-nitrophenyl ester of a car-boxyl group, sp is -CH2CH2-, Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
16. 16. A protein-drug conjugate according to claim 2 wherein Q is the hydrosysuccinimide ester of a carboxyl group, Sp is -CH2C(CH3)2-, Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
17. 17. A protein-drug conjugate according to claim 2 wherein Q is the hydroxysuccinimide ester of a carboxyl group, sp is Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
18. 18. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is -CH2CH2-, Y is -CHO, Z is -CONHN=CH-, and m is 0.1 to 10.
19. 19. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is -CH2CH2-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
20. 20. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is -CH2CH(CH3)-, Y is -CHO, Z is -CONHN=CH-, and m is 0.1 to 10.
21. 21. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is -CH2C(C83)-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
22. 22. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNC2, Sp is -CH2C(CH3)2-, Y is -CHO, Z is -CONHN=CH-, and m is 0.1 to 10.
23. 23. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is -CH2C(CH3)2-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
24. 24. A protein-drug conjugate according to Claim 2 wherein Q is -CON8NH2, Sp is Y is -CHO, Z is -CONHN=CH-, and m is 0.1 to 10.
25. 25. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
26. 26. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is Y is -CHO, Z is -CONHN=CH-, and m is 0.1 to 10.
27. 27. A protein-drug conjugate according to Claim 2 wherein Q is -CONHNH2, Sp is Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
28. 28. A protein-drug conjugate according to Claim 8 wherein CH3SSS-W is LL-E33288.delta.1I, Q is -CONHNH2, Sp is -CH2CH2-, Y is -CHO, Z is -CONHNHCH2, and m is O.1 to 10.
29. 29. A protein-drug conjugate according to Claim 10 wherein CH3SSS-W is LL-E33288.alpha.3I, Q is -CONHNH2, Sp is -CH2CH2-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
30. 30. A protein-drug conjugate according to Claim 10 wherein CH3SSS-W is N-acetyl LL-E33288.delta.1I, Q
    is -CONHNH2, Sp is -CH2CH2-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
31. 31. A protein-drug conjugate according to Claim 8 wherein CH3SSS-W is LL-E33288.delta.1I, Q is -CONHNH2, Sp is -CH2CH(CH3)-, Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
32. 32. A protein-drug conjugate to Claim 10 wherein CH3SSS-W is LL-E33288.alpha.3I, Q is -CONHNH2, Sp is -CH2C(CH3)2-, Y is -CHO-, Z is -CONHNHCH2-, and m is 0.1 to 10.
33. 33. A protein-drug conjugate according to Claim 10 wherein CH3SSS-W is N-acetyl LL-E33288.delta.1I, Q
    is -CONHNH2, Sp is , Y is -CHO, Z is -CONHNHCH2-, and m is 0.1 to 10.
34. 34. A process for preparing the targeted derivatives of compounds of formula CH3SSS-W, wherein CH3SSS-W is an antitumor antibiotic LL-E332SS.alpha.1Br, .alpha.1I, .alpha.2BR, .alpha.2I, .alpha.3Br, .alpha.3 I, .alpha.4 Br, .beta.1 BR, .beta.1I, .beta.2Br, .beta.2I, .gamma.1Br, .gamma.1I, .delta.1I, the iodo or bromo pseudoaglycones, their dihydro or N-acetyl counterparts, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, or their N-acetyl counterparts, comprising reacting CH3SSS-W with a compound of formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C1-C18) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C3-C18) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C1-C18) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or divalent or trivalent (C2-C18) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups: and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, lower alkyldicarboxyl anhydride, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, -ONH2, or in acetonitrile in the presence of one equivalent of triethylamine or one equivalent of triethylamine and one equivalent of acetic acid at -10° to -30°C for 1-48 hours, isolating the intermediate of formula Q-Sp-SS-W, wherein Q and Sp are as hereinbefore defined and W is as defined in Tables I and II, reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, or lower alkyldicarboxylic anhydride with a molecule of the formula Tu-(Y) n wherein Tu is a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino or carboxy functionality; n is 1-100, in aqueous buffer at a pH of between 6.5 and 9, at 4° to 40°C either directly or in the presence of a water-soluble carbodiimide, to generate the compound wherein Tu, Sp, W, n, and Y are as hereinbefore defined, m is 1-15 and Z is formed from covalent reaction of the groups Q and Y and is -CONH-, -NH-, -N=CH=, or -CO2- ~or reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is a carboxylic acid, with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol, or 4-nitrophenol in the presence of a carboxyl activating agent to generate a compound of formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is with a molecule of formula Tu-(Y) n, where Tu and n are as hereinbefore defined, and Y is a side-chain amino group, in an aqueous buffered solution at a pH between 6.5 and 9, at a temperature of between 4° and 40°C, inclusive, to generate compounds of the formula:
    wherein Tu, Sp, W, Y, and n are as hereinbefore defined, m is 1-15, and Z is formed from covalent reaction between Q and Y and is defined as -CONH-or reacting a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CONHNH2 with nitrous acid in aqueous acetonitrile to generate a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CON3 with a compound of formula Tu-(Y) n, wherein Tu, Y, and n are as hereinabove defined to produce a compound of the formula wherein Tu, Z, Sp, W, m, Y, and n are as hereinabove defined;
    
    or reacting a compound of formula Q-Sp-SS-W
    wherein Sp and W are as hereinbefore defined and Q is hydroxy, with an alpha-haloacetic anhydride to produce a compound wherein Q is a-haloacetyloxy, and reacting the .alpha.-haloacetyloxy-Sp-SS-W, or a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is with a molecule of the formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is a side-chain thiol of a protein, or an amidoalkylthio group introduced on an amine of Tu using reagents for introducing thiol groups followed by reduction with an agent which generates the thiol group, above, or an amidoalkylthio group introduced on an amine of Tu using 2-imiaothiolane, and n is 1-10, under aqueous buffered conditions at a pH between 4.5 and 7, at a temperature between 4° and 40°C, inclusive, to produce a compound of formula:
    
    wherein Tu, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q
    and Y and Z is and n is 1 to 10:
    or reacting a compound of the formula Q-Sp-SS-W
    wherein Sp and W are as hereinbefore defined and Q is -NH2, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, or -ONH2 with a molecule of formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is an aldehyde generated from carbohydrate residues on Tu by ozidation in the presence of an alkaline earth periodate, in an aqueous buffer at a pH between 4.0 and 6.5, at 4° to 40°C, inclusive, and n is 1 to 20 to generate a compound of formula:
    wherein Tu, Sp, W, Y, and n are as hereinbefore defined and Z is formed from the covalent reaction of Q and Y
    and is -ON=CH-, -N=CH-, -CONHN=CH-, -NHCONHN=CH-, or -NHCSNHN=CH-, and m is 0.1 to 15; or treating the compound immediately hereinabove of formula:
    
    wherein Tu, Z, Sp, W, Y, n, and m are as immediately hereinabove defined with acetylhydrazine or tyrosine hydrasine in an aqueous buffer at a pH between 4.0 and 6.5, at 4° to 40°C, inclusive, to generate a compound of formula:
    wherein Tu, Z, Sp, W, n, and m are as immediately hereinabove defined and Y is -CH=NNHCOCH3 or and reacting this compound with sodium cyanoborohydride or sodium borohydride, in an aqueous buffer at a pH of 4.0 to 6.5, at a temperature of 4° to 40°C inclusive, to generate a compound of formula:
    Wherein Tu, Sp, W, m, and n are as hereinabove defined, Z is -NH-CH2-, -CONHNHCH2-, -NHCONHNHCH2-, or -NHCSNHNHCH2-, and Y is -CH2NHNHCOCH3 or
35. 35. A process according to claim 34 wherein the carboxyl activating agent is carbodiimide.
36. 36. A process according to claim 34 wherein the agent for introducing thiol groups is 3-(2-dithio-pyridyl)propionic acid hydroxysuccinimide ester.
37. 37. A process according to claim 34 wherein the agent which generates the thiol group is dithiothreitol.
38. 38. A process according to claim 34 for producing a target compound from CH3SSS-W comprising reacting CH3SSS-W with .beta.-mercapto-propionic acid at -20°C in acetonitrile in the presence of a tertiary amine base, isolating the material, and reacting the intermediate with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in tetrahydrofuran at ambient temperature for about four hours, isolating the intermediate, and reacting it with Tu in a buffered solution at pH 7.5 for 1 to 4 hours, to produce the compound wherein Tu is a monoclonal antibody, Z is -NHCO-, Sp is -CH2-CH2-, Y is -NH2, and m is 2 to 15.
39. 39. A process according to Claim 34 for producing a targeted compound from CH3SSS-W comprising reacting CH3SSS-W with 3-mercaptobutyric acid at -20°C in acetonitrile in the presence of a tertiary amine base, isolating the material, and reacting the intermediate with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in tetrahydrofuran at ambient temperature for about four hours, isolating the intermediate, and reacting it with a monoclonal antibody in a buffered solution at pH 7.5 for 1 to 4 hours, to produce the compound wherein Tu is the monoclonal antibody, Z is -NHCO-, Sp is -CH2-CH(CH3)-, Y is -NH2, and m is 2 to 15.
40. 40. A process according to Claim 34 for producing a targeted compound from CH3SSS-W comprising reacting CH3SSS-W with 3-mercaptoisavaleric acid at -20°C in acetonitrile in the presence of a tertiary amine base, isolating the material, and reacting the intermediate with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in tetrahydrofuran at ambient temperature for about four hours, isolating the intermediate, and reacting it with a monoclonal antibody in a buffered solution at pH 7.5 for 1 to 4 hours, to produce the compound wherein Tu is the monoclonal antibody, Z is -NHCO-, Sp is -CH2C(CH3)2-, Y is -NH2, and m is 2 to 15.
41. 41. A process according to claim 34 for producing a targeted compound from CH3SSS-W comprising reacting CH3SSS-W with p-mercaptodihydrocinnamic acid at -20°C in acetonitrile in the presence of a tertiary amine base, isolating the material, and reacting the intermediate with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in tetrahydrofuran at ambient temperature for about four hours, isolating the intermediate, and reacting it with a monoclonal antibody in a buffered solution at pH 7.5 for 1 to 4 hours, to produce the compound wherein Tu is the monoclonal antibody, Z is -NHCO-, Sp is , Y is -NH2-, and m is 2 to 15.
42. 42. A process according to Claim 34 for producing a targeted compound from CH3SSS-W comprising reacting CH3SSS-W with N[[(4-methylcoumarin-7-yl)amino]acetyl]cysteine at -20°C in acetonitrile in the presence of a tertiary amine base, isolating the material, and reacting the intermediate with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in tetrahydrofuran at ambient temperature for about four hours, isolating the intermediate, and reacting it with a monoclonal antibody in a buffered solution at pH 7.5 for 1 to 4 hours, to produce the compound wherein Tu is the monoclonal antibody, Z is -NHCO-, Sp is Y is -NH2, and m is 2 to 15.
43. 43. A process according to Claim 34 for producing a targeted compound of the formula from CH3SSS-W comprising reacting CH3SSS-W with .beta.-mercaptopropionic acid hydrazide at -15° in tetrahydrofuran in the presence of a tertiary amine base for from one to 36 hours, isolating the material, and reacting the intermediate with a monoclonal antibody which was oxidized with sodium periodate in acetate buffer, at a pH of 5.5 to 7.0 at 4°C for about 45 minutes and dialyzed to remove excess sodium perio-date, to produce the compound wherein Tu is the monoclonal antibody, Z is -CH=NNHCO-, Sp is -CH2-CH2-, Y is CHO, and m is 1 to 10.
44. 44. a process according to Claim 34 for producing a targeted compound of the formula from CH3SSS-W comprising reacting CH3SSS-W with 3-mercaptobutyric acid hydraside at -15° in tatrahydrofuran in the presence of a tertiary amine base for from one to 36 hours, isolating the material, and reacting the intermediate with a monoclonal antibody which was ozidized with sodium periodate in acetate buffer, at a pH of 5.5 to 7.0 at 4°C for about 45 minutes and dialyzed to remove excess sodium perio-date, to produce the compound wherein Tu is the monoclonal antibody, 2 is -CH=NNHCO-, Sp is -CH2C(CH3)-, Y is CHO, and m is 1 to 10.
45. 45. A process according to claim 34 for producing a targeted compound of the formula from CH3SSS-W comprising reacting CH3SSS-W with 3-mercaptoisovaleric acid hydrazide at -15° in tetrahydrofuran in the presence of a tertiary amine base for from one to 36 hours, isolating the material, and reacting the intermediate with a monoclonal antibody which was ozidized with sodium periodate in acetate buffer, at a pH of 5.5 to 7.0 at 4°C for about 45 minutes and dialysed to remove excess sodium perio-date, to produce the compound wherein Tu is the monoclonal antibody, 3 is -CH=NNHCO-, Sp is -CH2C(CH3)2-, Y is CHO, and m is 1 to 10.
46. 46. A process according to Claim 34 far producing a targeted compound of the formula from CH3SSS-W comprising reacting CH3SSS-W with p-mercaptodihydrocinnamic acid hydrazide at -15° in tetrahydrofuran in the presence of a tertiary amine base for from one to 36 hours, isolating the material, and reacting the intermediate with a monoclonal antibody which was oxidised with sodium periodate in acetate buffer, at a pH of 5.5 to 7.0 at 4°C for about 45 minutes and dialyzed to remove excess sodium peri-odat~, to produce the compound therein Tu is the mono-clonal antibody, Z is -CH=HNHCO-, Sp is Y is CHO, and m is 1 to 10.
47. 47. A process according to Claim 34 for producing a targeted compound of the formula from CH3SSS-W comprising reacting CH3SSS-W with N-[[(4-methylcoumaryl-7-yl)amino]acetyl]cysteine hydrazide at -15° in tetrahydrofuran in the presence of a tertiary amine base for from one to 36 hours, isolating the material, and reacting the intermediate with a monoclonal antibody which was oxidized with sodium periodate in acetate buffer, at a pH of 5.5 to 7.0 at 4°C for about 45 minutes sad dialyzed to remove excess sodium periodate, to produce the compound therein Tu is the monoclonal antibody, Z is -CH=NNHCO-, Sp is Y is CHO, and m is 1 to 10.
48. 48. A process according to Claim 43, therein unreacted aldehyde groups are blocked by reaction with acetyl hydrazine for about 3 hours followed by reduction with sodium cyanoborohydride to produce the compound wherein Tu is the monoclonal antibody, Z is -CH2NHNHCO-, Sp is -CH2CH2-, Y is -CH2NHNHCOCH3, and m is 1 to 10.
49. 49. A process according to claim 44, wherein unreacted aldehyde groups are blocked by reaction with acetyl hydrazine for about 3 hours followed by reduction with sodium cyanoborohydride to produce the compound wherein Tu is the monoclonal antibody, Z is -CH2NHNHCO-, Sp is -CH2CH(CH3)-, Y is -CH2NHNHCOCH3, and m is 1 to 10.
50. 50. A process according to Claim 45, wherein unreacted aldehyde groups are blocked by reaction with acetyl hydrazine for about 3 hours followed by reduction with sodium cyanoborohydride to produce the compound wherein Tu is the monoclonal antibody, Z is -CH2NHNHCO-, Sp is -CH2C(CH3)2, Y is -CH2NHNHCOCH3, and m is 1 to 10.
51. 51. A process according to Claim 46, wherein unreacted aldehyde groups are blocked by reaction with acetyl hydrazine for about 3 hours followed by reduction with sodium cyanoborohydride to produce the compound wherein Tu is the monoclonal antibody, Z is -CH2NHNHCO-, Sp is Y is -CH2NHNHCOCH3, and m is 1 to 10.
52. 52. A process according to Claim 47, wherein unreacted aldehyde groups ere blocked by reaction with acetyl hydrazine for about 3 hours followed by reduction with sodium cyanoborohydride to produce the compound wherein Tu is the monoclonal antibody, Z is -CH2NHNHCO-, Sp is Y is -CH2NHNHCOCH3, and m is 1 to 10.
53. 53. Use of an oncolytic amount of a product prepared from a compound of general formula CH3SSS-W
    wherein CH3SSS-W is an antitumor antibiotic designated as LL-E33288.alpha.1Br, .alpha.1I, .alpha.2Br,.alpha.2I, .alpha.3Br, .alpha.3Br, .alpha.3I, .alpha.4Br, .beta.1Br, .beta.1I, .beta.2Br, .beta.2I, ~1I, .sigma.1I, the iodo or bromo pseudoaglycones, their dihydro or N-acyl counterparts, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, or their N-acetyl counterparts comprising:
    reacting CH3SSS-W with a compound of general formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C1-C18) radical, divalent or trivalent aryl or het~roaryl radical, divalent or trivalent (C3-C18) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl-or heteroaryl-alkyl (C1-C18) radical divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or divalent or trivalent (C2-C18) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carbonyl, lower alkoxy, hydroxy, thiol, a lower alkylthio groups; and Q
    is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, .alpha.-haloacetyloxy, lower alkyldicarboxyl, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, -ONH2, -CON3, to produce an intermediate of formula Q-Sp-SS-W, wherein Q and Sp are as hereinbefore defined and W is as defined in Tables I
    and II, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is defined as a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, growth factors, or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from glycoprotein carbohydrate residues, or an amidoalkylthio group; and n is an integer of from 1 to 100, to produce a compound of the formula:
    wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q
    and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-, -NHCONHNHCH2-, -NHCSNHN=CH-, -NHCSNHNHCH2-, -ON=CH-, -NH-, -NHCH2-, -N=CH-, -CO2-, -NHCH2CO2-, -SS-, -a5- and m is 0.1 to 15, as a tumor growth inhibitor in a mammal.
54. 54. Use of an effective oncolytic amount of a protein-drug conjugate of the formula prepared from the antitumor antibiotic designated LL-E33288~1I (CH3SSS-w) having a) ultraviolet spectrum as shown in Figure I;
    b) a proton magnetic resonance spectrum as shown in Figure II;
    and c) an infrared spectrum as shorn in Figure III; or prepared from the antitumor antibiotic designated LL-E332SS.alpha.2I (CH3SSS-W) having a) a proton magnetic resonance spectrum as shown in Figure IV; and b) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure V: or prepared from the antitumor antibiotic designated LL-E33288.alpha.3I (CH3SSS-W) having a) ultraviolet spectrum as shown in Figure VI;
    b) an infrared spectrum as shown in Figure VII;
    c) a proton magnetic resonance spectrum as shown is Figure VIII; and d) a carbon-13 nuclear magnetic resonance spectrum as shows in Figure IX; or prepared from the antitumor antibiotic designated N-acetyl LL-E33288.delta.1I (CH3SSS-W) having a) ultraviolet spectrum as shown in Figure X;
    b) an infrared spectrum as shown in Figure XI;
    c) a proton magnetic resonance spectrum as shown in Figure XII; and d) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure XIII; or prepared from the antitumor antibiotic designated iodo LL-E33288 pseudoaglycone (CH3SSS-W) having a) ultraviolet spectrum as show in Figure XIV;
    b) as infrared spectrum as shown in Figure XV;
    c) a proton magnetic resonance spectrum as shown in Figure XVI; and d) a carbon-13 nuclear magnetic resonance spectrum as shown is Figure XVII;
    comprising:
    displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C2-C5) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C2-C5) radicals, wherein it Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; and Q is, or can be subsequently converted to, carboxyl, lower alkyldicarboxylanhydride, -CONHNH2, or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q and Sp are as hereinbefore defined and W is as defined in Tables I and II, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y)n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula:
    
    wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q
    and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH2-, and m is 0.1 to 15, as an oncolytic agent in a mammal.