J . Med. Chem. 1989,32, 548-555
`548
`New Antitumor Monoclonal Antibody-Vinca Conjugates LY203725 and Related
`Compounds: Design, Preparation, and Representative in Vivo Activity'
`
`Bennett C. Laguzza,*p+ Cynthia L. Nichols, Stephen L. Briggs, George J. Cullinan, David A. Johnson,
`James J. Starling, A. Leroy Baker, Thomas F. Bumol, and Jose R. F. Corvalant
`
`Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, and Lilly Research Centre, Erl Wood
`Manor, Windlesham, Surrey, England GU20 6PH. Received February 26, 1988
`
`A method has been developed to allow the direct coupling of the cytotoxic vinca alkaloid 4-desacetylvinblastine-
`3-carbohydrazide (DAVLB hydrazide) to a variety of murine monoclonal antibodies directed against human solid
`tumors. Periodate oxidation of carbohydrate residues on the antibodies, followed by reaction with DAVLB hydrazide
`in aqueous acid affords, in most cases, conjugates with conjugation ratios of 4-6 vincas per antibody in high yield
`without significantly impairing antigen binding or solubility. The outcome of the conjugation reaction is highly
`dependent on the concentration of, and time of exposure of the protein to, the oxidant. These conjugates exhibit
`potent antitumor activity in vivo against a number of human solid tumor-nude mouse xenografts, with efficacy and
`safety increased over unconjugated DAVLB hydrazide. This antitumor activity is also superior to that of similarly
`prepared but nontarget tumor binding antibody-DAVLB hydrazide conjugates. MoAb-DAVLB hydrazide conjugates
`release DAVLB hydrazide in solution in a temperature- and pH-dependent manner. Hydrolytic release of unmodified
`DAVLB hydrazide from tumor-localized MoAb-DAVLB hydrazide conjugates in vivo may be an important factor
`in their antitumor activity.
`
`Monoclonal antibody-drug conjugates and monoclonal
`antibody-toxin conjugates have gained considerable at-
`tention as potentially useful tools for the treatment of
`human
`The promise of these new therapeutics
`is the prediction that the monoclonal antibody (MoAb),
`selected to specifically bind a particular tumor-associated
`antigen, will preferentially concentrate the attached on-
`colytic agents or toxins at or within a tumor mass. By
`targeting the drug to the tumor site, the conjugate should
`boost tumor cell killing by the drug or toxin, while di-
`minishing unwanted side effects.
`Numerous conjugates of antitumor MoAbs with a variety
`of cytotoxic agents, including adriamycin, vindesine, me-
`thotrexate, radionuclides, and the protein toxin ricin, have
`
`already been r e p ~ r t e d . ~ , ~ In the majority of these exam-
`ples, the drug is coupled to the antibody lysine amino
`groups via amide, alkyl, or imine carbon-nitrogen bonds,
`while toxins such as ricin are best attached by way of
`heterobifunctional disulfide reagents. Several of these
`conjugates have been reported to inhibit the growth of
`tumor cells in vitro and in vivo. For example, KS1/4S2-
`DAVLB (LY256787, 1)' is a conjugate comprised of the
`adenocarcinoma-reactive MoAb KS1/4S28 coupled
`through its lysine amino groups to the 4-OH of the cyto-
`toxic vinca alkaloid 4-de~acetylvinblastine~ (DAVLB, 7a)
`via a succinate bridge.1° This conjugate exhibited sig-
`nificant antitumor effects in vivo against human lung and
`colorectal adenocarcinoma xenografts in Nu/Nu (nude)
`mice.
`
`0
`1 KS114 - DAVLB (LY256787)
`We were interested in the design and development of
`unique, second generation anticancer MoAb-drug conju-
`
`0
`
`'Address correspondence to this author at GL307, B242/1 Lilly
`Research Laboratories, P.O. Box 708, Greenfield, IN 46140.
`t Lilly Research Centre.
`
`gates. Our chemical objectives were to (1) choose from an
`established family of cytotoxic vinca alkaloids'l a drug
`candidate bearing conjugatable functional groups, (2) ex-
`plore an alternative site and method of attachment of the
`chosen drug to the MoAb, and (3) preserve the MoAb's
`antigen binding and specificity while retaining solubility.
`Through such a construction we hoped to achieve the
`biological objectives of (1) selective in vivo delivery of a
`potent antitumor agent to target tumor masses and (2) a
`significant increase in the safety and efficacy of conjugated
`drug over unconjugated drug. These goals appear to have
`been realized with KS1/4S2-4-desacetylvinblastine-3-
`carbohydrazide conjugate (LY203725,g) and related con-
`jugates (MoAb-DAVLB hydrazide, 2). This paper de-
`
`(1) Presented in part at the UCLA Symposium on the Pharma-
`cology and Toxicology of Proteins, Lake Tahoe, CA, Feb 21-27,
`1987. J. Cell Biochem. 1987, Supp IlB, 189, 191, and 192.
`(2) Frankel, A. F.; Houston, L. L.; Issell, B.; Fathman, G. Annu.
`Reo. Med. 1986, 37, 125-142.
`(3) Reisfeld, R. A.; Cherish, D. A. Cancer Surueys 1985, 4(1),
`271-290.
`(4) Ghose, T. I.; Blair, A. H.; Vaughan, K.; Kulkarni, P. Targeted
`Drugs; Goldberg, E. P., Ed.; J. Wiley and Sons: New York,
`1983; pp 1-22.
`(5) Pietersz, G. A.; Kanellos, J.; Smkyth, M. J.; Zalcberg, J.;
`McKenzie, I. F. C. Immunol. Cell Bid. 1987, 65 (PtZ), 111.
`(6) Ghose, T.; Blair, A. H. Crit. Reu. Ther. Drug Carrier Syst.
`1987, 3(4), 263.
`(7) Bumol, T. F.; Parrish, J.; DeHerdt, S. V.; Spearman, M. E.;
`Pohland, R.; Borowitz, M. J.; Briggs, S. L.; Baker, A. L.;
`Marder, P.; Apelgren, L. D. Breast Cancer; Ceriani, R. L., Ed.;
`Plenum Press: New York, 1987; pp 205-215.
`(8) Bumol; et al. Targeted Diagnosis and Therapy; Marcel Dek-
`ker: New York, in press.
`(9) (a) Barnett, C. J.; Cullinan, G. J.; Gerzon, K.; Hoying, R. C.;
`Jones, W. E.; Newlon, W. M.; Poore, G. A.; Robinson, R. L.;
`Sweeney, M. J.; Todd, G. C.; Dyke, R. W.; Nelson, R. L. J.
`Med. Chem. 1978, 21, 88. (b) Conrad, R. A.; Cullinan, G. J.;
`Gerzon, K.; Poore, G. A. J. Med. Chem. 1979, 22, 391. (c)
`Cullinan, G. J.; Gerzon, K. U S . Patent 1980, No. 4,203,898.
`(10) For earlier examples of vinca-succinate conjugates, see: Row-
`land, G. F.; Axton, C. A.; Baldwin, R. W.; Brown, J. P.; Cor-
`valan, J. R. F.; Embleton, M. J.; Gore, V. A.; Hellstrom, I.;
`Hellstrom, K. E.; Jacobs, E.; Marsden, C. H.; Pimm, M. V.;
`Simmonds, R. G.; Smith, W. Cancer Immunol. Immunother.
`1985, 19, 1.
`(11) Johnson, I. S.; Armstrong, J. G.; Gorman, M.; Burnett, J. P.
`Cancer Res. 1963, 23, 1390.
`
`0022-2623/89/l832-0548$01.50/0
`
`0 1989 American Chemical Society
`
`IMMUNOGEN 2089, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Antitumor Monoclonal Antibody-Vinca Conjugates
`
`Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3 549
`
`Rinrlino
`
`Antigen Bin
`
`A. MoAb OXIDATION
`
`","i;;S
`
`Carbohydrate
`
`P O - 3
`
`oxidative cleavage occurs
`here
`
`ol/ "$
`
`
`
`M O A T ) o n
`
`OXIDIZED MoAb
`
`FCarbohydrate
`
`B. REACTIONS WITH AMINES
`
`Figure 1. 2-D representation of an immunoglobulin G (IgG)
`molecule.
`
`scribes the design, synthesis, and representative biological
`activity12 of these new conjugates.
`
`OXIDIZED MoAb
`
`un
`
`CH30
`
`2 MoAb - DAVLB HYDRAZIDE
`Conjugate Design: A. MoAb Site of Drug Attach-
`ment. IgG immunoglobulins are symmetrical, bivalent
`glycoproteins of approximately MW 15oo00, composed of
`two identical, short, amino acid chains ("light" (L) chains)
`paired with two identical, longer, amino acid chains
`("heavy" (H) chains).13 The carbohydrate content of these
`macromolecules averages 3-5 '?& by weight and is usually
`comprised of branched chains of N-acetylglucosamine,
`D-mannose, L-fucose, and D-galactose, with N-acetylneur-
`aminic acid capping the nonreducing ends.14J5 Similar
`carbohydrate structures have been found on monoclonal
`IgG's.16 These polysaccharide chains are usually N-linked
`at the N-acetylglucosamine reducing ends to Asn residues
`in the CH2 domain on the heavy chains of the IgG mole-
`cule, a site remote from the antigen binding regions (see
`Figure 1). However, examples of IgGs bearing carbohy-
`drate in the antigen binding half of the protein have been
`found.16
`In contrast, lysine residues are much more widely dis-
`tributed throughout the antibody primary amino acid se-
`quence and are often found in or near the antigen binding
`domains. It is the 6 amino groups of these lysine residues
`
`(12) Also see: '(a) Johnson, D. A.; Laguzza, B. C. Cancer Res. 1987,
`47, 3118. (b) Bumol, T. F.; Laguzza, B. C.; DeHerdt, S. V.;
`Parrish, J. E.; Andrews, E. L.; Zimmerman, J. L.; Baker, A. L.;
`Marder, P.; Nichols, C. L.; Apelgren, L. D. Proc. Am. Assoc.
`Cancer Res. 1987,28, 393. (c) Bumol, T. F.; Laguzza, B. C.;
`DeHerdt, S. V.; Andrews, E. L.; Baker, A. L.; Mader, P.; Ni-
`chols, C. L.; Apelgren, L. D. Second International Conference
`on Monoclonal Antibody Immunoconjugates for Cancer, 1987.
`(d) Starling, J. J.; Maciak, R. S.; Hinson, N. A.; Nichols, C. L.;
`Laguzza, B. C. J. Cell. Biochem. 1987, Supp. llB, 192.
`(13) Benacerraf, B.; Unanue, E. R. Textbook of Immunology;
`Williams and Wilkins: Baltimore, MD, 1983; Chapter 3.
`(14) Kabat, E. A. Structural Concepts in Immunology and Zmmu-
`nochemistry; Kabat, E. A,, Ed.; Holt, NY, 1976; p 28.
`(15) Rademacher, T. W.; Homans, S. W.; Pareck, R. B.; Bwek, R.
`A. Biochem. SOC. Symp. 1986,51, 131.
`
`RNHNHz
`
`RCONHNH,
`
`(hydrazides)
`
`5
`Figure 2. Periodate oxidation of MoAbs and coupling with
`amines.
`
`~~~~
`
`Table I. In Vitro Biological Comparison of Several Vinca
`Derivatives"
`P3UCLA cellsc
`vinca
`CEM cellsd
`IC,.
`rrelmL
`derivativeb
`IC,, u d m L
`0.03
`<O.oo03*
`~ 6
`ND f
`0.0003
`7a
`7b
`0.10
`ND f
`1.4
`0.003
`7c
`Also see ref 9a. Compounds were tested as their sulfate salts.
`ICB, concentration demonstrating 50% inhibition of cell prolifer-
`ation as determined by the MTT colorimetric proliferation assay
`(ref 33) with 48-h constant exposure of drug utilizing P3UCLA (ref
`12a) human lung adenocarcinoma cell cultures. Reported ICw's
`are means and are within 10% of the experimental values (N = 3).
`72-h constant exposure to CCRF-CEM human leukemic lym-
`phoblasts at 37 "C at a concentration of 2.4 X 10' cells/mL, with
`% inhibition of cell proliferation per given dose determined by
`Coulter Counter measurement (ref 34). Reported ICm's are means
`and are within 3% of the experimental values ( N = 2). eLowest
`dose tested. fND = not done.
`that are the traditional sites of drug attachment, as is the
`case with 1. Consequently, lysine-linked antibody-drug
`conjugates can show significant reduction in antigen re-
`activity and affinity.16J7 On the other hand, coupling
`drugs to the antibody carbohydrate groups could afford
`conjugates with little or no obstruction of antigen bind-
`ing.1eJ8 Furthermore, antibody hydrophilicity, and
`therefore solubility, should be better retained in carboh-
`ydrate-linked conjugates, as ionizable lysine t amino groups
`would not be consumed by the coupling of the drug to the
`antibody.
`Conjugate Design: B. Attachment of Drugs to
`Antibody Carbohydrate Groups. The native carbohy-
`drate groups on an antibody are relatively inert as sites
`for drug attachment. However, numerous studies have
`
`(16) Ghose, T. I.; Blair, A. H.; Kulkarni, P. N. Methods Enzymol.
`1983, 93, 280.
`(17) Marder, P.; Apelgren, L. D.; Bumol, T. F. J. Zmmunol. Meth-
`ods 1987, 96, 165.
`(18) Blair, A. H.; Ghose, T. I. J. Zmmunol. Methods 1983, 59,
`129-143.
`
`IMMUNOGEN 2089, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`550 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3
`
`Lagutza et al.
`
`Table 11. MoAbs, Clinical Targets, and Antigen-Positive Human Tumor Cell Lines
`
`MoAb:"
`KS1/4S2c
`
`isotype
`IgG2a
`
`clinical targets
`adenocarcinoma (lung, colon, breast, prostate);
`squamous carcinoma (lung)
`all KS1/4 antigen-positive
`targets
`melanomas
`squamous carcinomas
`(head, neck, lung)
`
`antigen-positive-human
`tumor cell linesb
`P3/UCLA (lung adenocarc)
`HT29 (colon adenocarc)
`
`same as for KS1/4S2
`
`M14 (melanoma)
`
`T222 (lung squamous carc)
`
`LIKSd
`IgG2b
`L2KSd
`IgGl
`L4KSd
`IpG2a
`IgG2a
`9.2.27'
`IgG2a
`PF4IAf
`IgG3
`PFl/Bf
`IgG3
`PFl/D/
`LS174T (colon adenocarc)
`CEA-bearing tumors (colon, lung, breast, and othersIh
`IgG2a
`1495.558
`none
`none (control myeloma IgG)
`IgGl
`X63AG8Sl'
`All MoAbs listed are murine-derived. *For background on the origin and nature of these cell lines, see ref 12a and references therein.
`cSee ref 8. dSee ref 12d. OSee ref 31. /See ref 12a and 32. gSee ref 10. hCEA = carcinoembryonic antigen.
`
`shown that glycol units on these carbohydrate moieties can
`be oxidatively cleaved with sodium metaperiodate (NaI04)
`under mild conditions, generating aldehyde sites that are
`suitable for coupling with amine-bearing reagents (Figure
`2a).16J9-22 Reaction of the oxidized antibody with lo
`amines hypothetically provides Schiff bases 3, while cou-
`pling with hydrazine and hydrazide reagents affords the
`more stable hydrazone adducts 4 and 5 (see Figure 2b).23
`The synthesis of an antimouse lymphoma antibody-dau-
`norubicn hydrazone conjugate represents an early appli-
`cation of this conjugation strategy.24 More recently,
`preparation of a tumor-imaging conjugate of an oxidized
`monoclonal antibody coupled to an amine-bearing "lIn-
`chelate has been reported.25
`Conjugate Design: C. Choice of Drug. The cytotoxic
`vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (6)
`(DAVLB h y d r a ~ i d e ) , ~ a derivative of vinblastine 7b,26
`appeared to be a good first choice as a drug candidate for
`building a carbohydrate-linked MoAb-vinca conjugate. It
`significantly inhibited the growth of human tumor cells
`in vitro (see Table I) and appeared superior in this activity
`to the related compounds desacetylvinblastine 7a9 the
`4-hemisuccinate derivative 7c7 (used to prepare conjugate
`I), and vinblastine 7b26 (see Table I). Another attractive
`feature of this compound was the unique hydrazide
`functionality at carbon 3. This seemed well suited for
`coupling the potent vinca 6 directly to oxidized MoAb
`carbohydrate aldehyde sites via acylhydrazone bonds, as
`shown in structure 2, obviating the need for any additional,
`and perhaps biologically deactivating, chemical bridge.
`An important feature of the hydrazone linkages of the
`proposed MoAb-DAVLB hydrazide conjugates 2 is that
`they were expected to be susceptible to acid-catalyzed
`hydrolysis, such as could occur during endocytosis, once
`
`I I
`
`I
`
`,
`
`,
`
`
`
`Andersen, B. R.; Abele, D. C.; Vannier, W. E. J . Zmmunol.
`1966, 97, 913.
`Willan. K. J.: Goldinp. B.: Givol. D.: Dwek. R. A. FEBS Lett.
`1977, 80, 133.
`Muravama. A.: Shimada. K.: Yamamoto, T. Immunochemistry
`.
`.
`
`1978,-15, 523.'
`O'Shanessv. D. J.: Dobbersen. M. J.: Quarles. R. H. Zmmunol.
`Lett. 1984; 8, 273.
`h i n d structures are also possible. See: Hansske, F.; Sprinzl,
`M.; Cramer, F. Bioorg. Chem. 1974, 3, 367.
`Hurwitz, E.; Wilchek, M.; Pitha, J. J. Appl. Biochem. 1980, 2,
`25.
`Rodwell, J. D.; Alvarez, V. L.; Lee, C.; Lopes, A. D.; Goers, J.
`W. F.; King, H. D.; Powsner, H. J.; McKearn, T. J. Proc Natl.
`Acad. Sci. U.S.A. 1986,83, 2632.
`(a) Neuss, N.; Gorman, M.; Hargrove, W.; Cone, N. J.; Biem-
`ann, K.; Buchi, G.; Manning, R. E. J. Am. Chem. 5". 1964,86,
`1440. (b) Moncrief, J. W.; Lipscomb, W. N. J. Am. Chem. Soc.
`1965, 87, 4963.
`
`I
`
`-
`
`
`
`the conjugate had localized at the target cancer site.27
`Significant hydrolysis might also occur a t the tumor cell
`surface, in the absence of internalization, as the intratu-
`moral pH of tumor tissue in several human patients has
`been determined to be acidic.28 Therefore, it was pre-
`dicted that DAVLB hydrazide might be released from the
`conjugate at the tumor site, unmodified and fully active.
`Conjugate Design: D. Choice of MoAb. Monoclonal
`antibodies that recognize antigens on human solid tumor
`cells including adenocarcinomas, melanomas, and squa-
`mous carcinomas were considered highly desirable as drug
`targeting agents. These classes of cancers encompass most
`of the major types of human solid tumors such as lung,
`breast, colon, rectal, and skin.29 A representative listing
`of the MoAbs employed in the construction of MoAb-
`DAVLB hydrazide conjugates described in this paper,
`some clinically important antigen-positive human tumor
`targets, and some examples of antigen-positive human
`tumor cells lines useful for biological testing of the MoAbs
`and their conjugates are shown in Table 11.
`Results and Discussion
`Chemistry. DAVLB hydrazide (6) was prepared by
`treating vinblastine 7b26 with anhydrous hydrazine in
`absolute methanol? Chromatography and recrystallization
`of the crude reaction product provided the desired com-
`pound in greater than 95% purity. MoAb-DAVLB hy-
`drazide conjugates 2 were synthesized as follows: The
`various monoclonal antibodies examined in this study were
`oxidized by sodium metaperiodate in cold sodium acetate
`buffer and the products isolated by Sephadex size-exclu-
`sion column chromatography. Treatment of the oxidized
`proteins with 6 or its sulfate salt in acetate buffer provided
`the desired MoAb-DAVLB hydrazide conjugates. Con-
`jugates 2 were mostly separated from unconjugated
`DAVLB hydrazide 6 by gravity flow Sephadex-size ex-
`clusion chromatography in phosphate buffered saline a t
`pH 7.4. Residual unconjugated 6 (-510%) was removed
`by exhaustive dialysis; absence of free 6 in the final
`products was confirmed by reverse-phase HPLC. Alter-
`natively, conjugate was purified from the crude reaction
`mixture by automated size exclusion and ion exchange
`chromatography. In this way MoAb-DAVLB hydrazide
`
`(27) Schneider, Y.-J.; Octave, J.-N.; Trouet, A. Current Topics in
`Membranes and TransDort: Academic Press. Inc.: New York.
`.
`.
`
`1985; Chapter 10.
`(28) (a) Lavie. E.: Hirschbern. D.: Hellstrom. K. E.: Hellstrom, I.
`Proc, Am. Assoc. Cancer Res. 1987, 28, 388. (b) Thistleth-
`waite, A. J.; Leeper, D. B.; Moylan, D. J. 3rd; Nerlinger, R. E.
`Znt. J. Radiat. Oncol. Biol. Phys. 1985, 11 (9), 1647.
`(29) American Cancer Society, 1985 Cancer Facts and Figures;
`1985.
`
`IMMUNOGEN 2089, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Antitumor Monoclonal Antibody- Vinca Conjugates
`
`Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3 551
`
`Table 111. Summary of Conjugation Results
`
`70
`yield"
`name
`comDd
`CRb
`5.0
`9
`84
`KSlI4SB-DAVLB hydrazide
`5.8
`76
`LlKS-DAVLB hydrazide
`10
`5.4
`64
`L2KS-DAVLB hydrazide
`11
`100
`4.3
`76
`L4KS-DAVLB hydrazide
`12
`92
`4.6
`84
`9.2.27-DAVLB hydrazide
`13
`82
`3.9
`86
`PF4/A-DAVLB hydrazide
`14
`92
`5.2
`a5
`PFl/B-DAVLB hydrazide
`15
`109
`4.6
`PFl/D-DAVLB hydrazide
`16
`77
`100
`3.8
`65
`14.95.55-DAVLB hydrazide
`17
`0 (control)
`5.5
`84
`X63AGSSl-DAVLB hydrazide
`18
`a Reported yields are overall protein yields after oxidation and conjugation, using method A as reported in the Experimental Section.
`bConjugation ratio, mmol 6/mmol MoAb; results averaged over several runs. 'The degree of binding of conjugates to antigen-positive tumor
`cell lines (see Table I1 for cell line descriptions) was compared to that of native, conjugated parent MoAb by several different methods.
`Conjugates 9, 13, 18: FACCS (see ref 17); conjugates 10-12, 17: RIA (see ref 13, p 72); conjugates 14-16: ELISA (see ref 35).
`
`% immunoreactivity
`remaining
`100
`100
`
`Table IV. Effect of Various Reaction Parameters on
`Conjugation Ratio"
`
`studv
`1
`
`2
`
`3
`
`DAVLBHYD
`oxidn
`NaI04,
`concn,
`time,
`CRb
`mM
`min
`mM
`5.0
`21
`0.3
`0
`21
`1.9
`5.0
`10
`21
`2.0
`40
`5.0
`21
`5.4
`160
`5.0
`14
`3.1
`160
`5.0
`5.0
`21
`4.3
`160
`5.0
`5.2
`28
`160
`6.4
`5.0
`35
`160
`42
`7.6
`160
`5.0
`21
`0.2
`160
`0.0
`3.1
`1.0
`21
`160
`2.0
`21
`4.0
`160
`3.0
`21
`3.9
`160
`4.0
`21
`4.8
`160
`5.0
`4.9
`160
`21
`"All data obtained for preparation of conjugate 9 (KS1/4-
`DAVLB hydrazide), using method A described in the Experimen-
`tal Section; qualitatively similar results obtained for the prepara-
`tion of all of the conjugates listed in Table I. Conjugation ratio;
`expressed as mmol DAVLB hydrazide (6)/mmol KS1/4S2.
`
`conjugates 2 containing an average of 4-6 mol of DAVLB
`hydrazide (6) per mole of antibody, as determined by dual
`wavelength UV spectroscopy, were routinely obtained.
`These results are summarized in Table 111. The amount
`of aggregated protein in the conjugates was usually low
`(5-1570).
`
`rp.p'
`
`HO
`
`-73
`
`6 R=H,R'=NHNH,
`7 E R=H,R'=OCH,
`7 b R=COCH,,R'=OCH,
`
`7 C R = CO(CH,),COOH, R' = OCH,
`
`CHsOOC
`
`In developing the oxidation-coupling procedure de-
`scribed above, several reaction parameters were explored.
`The ability to conjugate DAVLB hydrazide (6) to the
`MoAbs was clearly dependent on the extent of prior oxi-
`dation of the protein. Conjugation ratio (mmol vinca/
`mmol MoAb) increased with increasing periodate con-
`centration and time of exposure of the MoAb to the oxi-
`dant (Table IV, studies 1 and 2). The temperature of the
`oxidation step also significantly influenced the outcome
`of the vinca coupling reaction (data not shown). The
`degree of coupling of DAVLB hydrazide (6) to oxidized
`MoAb was also dependent on the concentration of vinca
`
`-
`E
`0
`N
`0
`
`E m P P
`9
`
`1 N
`
`c
`
`A
`
`'-
`-..--__ - 1
`180 360 540 720 900 1080 1260 1440 1620 1800
`Time (seconds)
`
`I
`
`0
`
`B
`
`I
`
`'
`
`
`
`
`/
`i
`1;
`I /
`p"-' - - - - - - - - ---- _______--_
`--,
`
`0
`
`180 360 540 720 900 1080 1260 1440 1620 1800
`Time (seconds)
`Figure 3. (a) HIC profiles of KS1/4SZ-DAVLB hydrazide (9)
`vs KSl/S2 MoAb. TSK-phenyl column; 0.1 M PO4 buffer, p H
`6.5,2-0 M NaCl gradient. Sample elution was monitored by UV
`at 280 nm. (-) KS1/4SP-DAVLB hydrazide (9); (---) KS1/4S2.
`Conjugate 9 was prepared by method A (Experimental Section).
`(b) RPC profiles of KS1/4SB-DAVLB hydrazide (9) vs KS1/
`4S2-DAVLB (1). TSK phenyl column; 0.1 M PO4, pH 6.5,O-30%
`CH,CN gradient. Elution was monitored by UV a t 280 nm. (- - -)
`KS1/4SB-DAVLB hydrazide (9); (-) KS1/4SB-DAVLB
`(1).
`Conjugate 9 was prepared by method A (see Experimental Sec-
`tion).
`employed (Table IV, study 3) but appeared insensitive to
`pH in the range of pH 4-6 (not shown).
`The hydrophilicities of the DAVLB hydrazide conju-
`gates in this study, relative to their parent MoAbs, were
`determined by Hydrophobic Interaction (HIC) and Re-
`verse Phase chromatography (RPC). Figure 3a compares
`the HIC elution profiles of KSlI4SB-DAVLB hydrazide
`conjugate (9) to KS1/4S2. These data show that conjugate
`9 is only slightly less hydrophilic than the unmodified
`MoAb. In contrast, the much more hydrophobic, lysine-
`linked conjugate KS1/4SB-DAVLB 1 does not elute under
`these conditions (not shown). Figure 3b shows the results
`of a comparison of the RPC elution profiles of conjugate
`9 and conjugate 1 using a stronger eluting solvent. In this
`system, 1 elutes slowly from the column, even in the
`presence of the organic cosolvent CH3CN. In marked
`contrast, 9, being much more hydrophilic, elutes near the
`void volume under these conditions, as does the parent
`
`IMMUNOGEN 2089, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`552 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3
`
`Laguzza et al.
`
`Table V. In Vivo Efficacy of MoAb-DAVLB Hydrazide Conjugates in Human Tumor-Nude Mouse Xenografts: Tumor Initiation
`Model
`
`human tumor xenografts:" EDm (mg/kg)b
`P3UCLA
`compd
`T222
`HT29
`LS174
`M14
`name
`DAVLB hydrazide
`6
`2.0
`0.5
`1.0
`1.0
`1.0
`KS 1 / 4S2-DAVLB hydrazide
`>2.0e
`<0.25c
`NT
`<0.0625c
`NTd
`9
`LlKS-DAVLB hydrazide
`NT
`NT
`0.05
`0.15
`0.0625
`10
`<0.0625'
`9.2.27-DAVLB hydrazide
`NT
`NT
`NT
`NT
`13
`0.3
`PFl/D-DAVLB hydrazide
`NT
`NT
`NT
`NT
`16
`0.15
`NT
`14.95.55-DAVLB hydrazide
`NT
`NT
`0.6
`17
`NT
`>1.0
`NT
`X63AG8Sl-DAVLB hydrazide
`18
`3.0
`>2.0e
`"See Table I1 for descriptions of cell lines and corresponding MoAb reactivity. *Dose of DAVLB hydrazide (6), given alone or as a
`conjugate, required.to cause 50% suppression of tumor growth as compared to controls, 28 days post tumor implantation. % suppression
`determined as the ratio of average tumor mass of the treatment group ( N = 5) to that of the control group (N = 10). The total amount of
`conjugate (vinca + MoAb) administered to achieve the ED,, dose of conjugated 6 is approximately 30-40 times the value shown. See
`Experimental Section for dosing protocols and details. Also see ref 7 and 12. 'Lowest dose tested. d N T = not tested. eHighest dose tested.
`21CO
`
`1
`
`- 1500
`E 1200
`
`600
`
`I-
`
`300
`
`T
`
`c O s o s m #a
`
`Figure 4. Release of DAVLB hydrazide (6) from KS1/4S2-
`DAVLB hydrazide (9). Conjugate was prepared by method B (see
`Experimental Section); CR = 5.3. % Free DAVLB hydrazide (6)
`is that % of the total amount of 6 in the conjugate sample
`(protein-bound + unbound) that elutes as unbound 6 on
`RPHPLC. This material represented 84-95% of all eluting
`substances combined.
`MoAb KS1/4S2 (not shown). These findings are con-
`sistent with the fact that concentrated (>lo mg/mL),
`homogeneous solutions of conjugate 9 are more easily
`prepared than for 1.
`The potential for MoAb-DAVLB hydrazide conjugates
`to release free DAVLB hydrazide (6) by hydrolysis was
`evaluated by subjecting several of the conjugates listed in
`Table I11 to storage in buffers at different pH's and tem-
`peratures. The amount of free DAVLB hydrazide (6)
`released over time under each set of conditions was then
`measured by Reverse Phase HPLC analysis. Figure 4
`shows data obtained from a 7-day stability study of
`KSlI4SB-DAVLB hydrazide (9); qualitatively similar re-
`sults were obtained with conjugates 12 and 13. While little
`free DAVLB hydrazide (6) was formed in the sample of
`conjugate 9 stored at physiological pH and 4 "C, release
`of 6 was much more significant a t acidic pH. At physio-
`logical temperature (37 "C), release of 6 was accelerated
`and enhanced a t both pH's, with the greatest release by
`far occurring a t pH 5.2. In all samples, the majority of
`release occurred in the first 24 h, except in the case of the
`sample stored a t pH 5.2 and 4 "C, which continued to
`exhibit slow, sustained release of 6 from the conjugate over
`the entire time course of the study.
`Biological Results: A. Conjugate Immunoreactiv-
`ity. The degree of antibody immunoreactivity remaining
`post conjugation was determined by measuring the binding
`of each conjugate to an antigen-positive target tumor cell
`and comparing the result to that obtained with the parent
`unconjugated MoAb. Results for several conjugates are
`summarized in Table 111. All of the conjugates listed in
`Table I11 showed excellent retention of antibody target cell
`
`23
`
`16
`
`I
`I
`I
`58
`30
`37
`44
`51
`Days of Tumor Growth
`Figure 5. Effect of KS1/4SB-DAVLB hydrazide (9) vs uncon-
`jugated DAVLB hydrazide (6) on growth of established P3UCLA
`lung adenocarcinoma xenografts in nude mice. (-n-) 9 at 2
`control.
`mg/kg (vinca content); (--m--) 6 at 2 mg/kg; (-@-)
`Each point is the mean f the S.E. of N = 5 mice (treatment group)
`or N = 10 mice (control group).
`binding. For example, by indirect fluorescence techniques,
`KS1/4S2-DAVLB hydrazide 9 gave a mean of 99.4% (S.E.
`= 5.549; N = 16) retention of immunoreactivity of the
`parent KS1/4S2 MoAb (see Table 111).
`Biological Results: B. In Vivo Antitumor Activi-
`ty.12 The DAVLB hydrazide conjugates were evaluated
`in vivo for their ability to suppress growth of antigen-
`bearing, solid human tumors subcutaneously implanted
`in nude (Nu/Nu) mice. Table V gives, for each MoAb-
`DAVLB hydrazide conjugate listed, the minimum effective
`dose (mg/kg) of DAVLB hydrazide (6), given as a conju-
`gate, required to achieve 50% suppression of growth of
`each type of tumor xenograft indicated. In this "Tumor
`Initiation" model,7 in which the tumor load prior to con-
`jugate administration was relatively small, all of the hy-
`drazide conjugates caused significant growth suppression
`of their respective tumor targets. This antitumor activity
`of each conjugate was superior to that of parent, uncon-
`jugated antibody (data not shown), unconjugated DAVLB
`hydrazide (6), and to control, nontumor binding DAVLB
`hydrazide conjugate 18 (note: conjugate 9 served as a
`control for conjugate 13 against M14 xenograft; a control
`conjugate was not run for conjugate 17 against LS174
`xenograft). For example, KS1/4S2-DAVLB hydrazide (9),
`had an EDb0 (<0.0625mg/kg) at least 16 times less than
`that of free DAVLB hydrazide (6) (1.0 mg/ kg) and control
`conjugate 18 (>l.Omg/kg) against P3UCLA lung adeno-
`carcinoma xenografts. The therapeutic index (lethal
`dose/EDbo) of this conjugate (TI = 64), based on DAVLB
`hydrazide (6) content, was also significantly increased over
`that of free 6 (TI = 4). Interestingly, the in vitro activity
`of these conjugates was often slightly less than that of
`unconjugated DAVLB hydrazide (e.g., IC50 = 3.4 ng/mL
`
`IMMUNOGEN 2089, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Antitumor Monoclonal Antibody-Vinca Conjugates
`
`Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3 553
`
`for LlKS-DAVLB hydrazide (10) vs 28 ng/mL for un-
`conjugated hydrazide; [3H]leucine uptake, P3/UCLA cells;
`J. J. Starling, unpublished results).
`The MoAb-DAVLB hydrazide conjugates were also
`effective against well-established tumors. Figure 5 displays
`the in vivo activity of conjugate 9 against 16-day estab-
`lished P3UCLA human lung adenocarcinoma xenografts.
`At the dose indicated (mg/kg vinca content of 9), not only
`was tumor growth suppressed by conjugate administration
`but actual regression of >90% of the original tumor mass
`occurred with no apparent toxicity.
`In contrast, an
`equivalent vinca dose of unconjugated DAVLB hydrazide
`(6) only plateaued growth, with no regressions observed.
`Furthermore, this dose of unconjugated 6 was lethal to the
`entire treatment group, prior to completion of the exper-
`iment. DAVLB hydrazide conjugates of nontumor binding
`MoAbs as well as KS1/4S2 alone were inactive in this
`model (not shown).12b
`. Discussion. The chemistry used to construct the
`MoAb-DAVLB hydrazide conjugates described in this
`paper is reproducible and applicable to a wide variety of
`MoAbs of varying isotype. Overall protein recoveries are
`high, and acceptable conjugation ratios are consistently
`obtained. Although the conjugation conditions prescribed
`in this paper generally do not provide the maximum con-
`jugation ratio possible per given MoAb, they do assure a
`reasonable balance of yield, minimal aggregate content,
`conjugation ratio, product solubility, and retention of im-
`munoreactivity.
`The conjugation ratio achieved for a particular MoAb
`is highly dependent on exposure of the protein to oxidant.
`This is consistent with our postulate that DAVLB hy-
`drazide (6) is condensing with periodate-generated aldeh-
`yde sites on the carbohydrate chains of the MoAb. Further
`evidence that aldehyde sites are present following oxidation
`is provided by the ready reaction of the oxidized antibody
`with the classical aldehyde determination reagent, 2,4-
`dinitrophenylhydrazine, and by the incorporation of tri-
`tium when exposed to [3H]s~dium borohydride30 (data not
`shown). Other experiments are underway to further elu-
`cidate the exact chemical nature of these conjugates.
`Most MoAbs conjugated by this method showed little
`alteration in antigen binding, as predicted. However, this
`was not the case for all MoAbs that were evaluated. Al-
`though it conjugated well (CR = 6.5, 78% yield), one
`MoAb’s ability to bind to antigen-positive tumor cells was
`essentially abolished by the conjugation procedure. Even
`very mild oxidation of this MoAb resulted in complete loss
`of antigen binding (J. J. Starling and B. C. Laguzza, un-
`published results). Thus, oxidation of and coupling
`amine-bearing reagents to MoAb carbohydrate sites does
`not guarantee per se that the antibody will retain its an-
`tigen recognition and binding characteristics, contrary to
`popular expectations.16J8
`One of the anticipated advantages of a MoAb-DAVLB
`hydrazide conjugate was the potential for acid-catalyzed
`release of unmodified, fully active DAVLB hydrazide (6)
`from the conjugate, once the conjugate had localized at the
`target tumor site. Our data shows that in solution, free
`
`(30) Van Lenten, L.; Ashwell, G. J. Biol. Chem. 1971, 246, 1889.
`(31) Bumol, T. F.; Walker, L. E.; Resifeld, R. A. J. Biol. Chem.
`1984,259, 12733.
`(32) Fernsten, P. D.; Pekny, K. W.; Reisfeld, R. A.; Walker, L. E.
`Cancer Res. 1986, 46, 2970.
`(33) Mosman, T. J. Zmmunol. Methods 1983, 65, 55.
`(34) Kinahan, J. J.; Otten, M.; Grindey, G. B. Cancer Res. 1979,39,
`3531.
`(35) Goding, J. W. Monoclonal Antibodies: Principles and Prac-
`tice; Academic Press, Inc.: New York, 1983; Chapter 3, p 78.
`
`DAVLB hydrazide can be released from the MoAb-
`DAVLB hydrazide conjugates and that the release is