WO2013068874A1

WO2013068874A1 - Antibody-drug conjugates

Info

Publication number: WO2013068874A1
Application number: PCT/IB2012/055940
Authority: WO
Inventors: Russell Dushin; Eric Feyfant; Puja Sapra; Lioudmila Gennadievna Tchistiakova; Feng Tian
Original assignee: Pfizer Inc.; Ambrx Inc.
Priority date: 2011-11-11
Filing date: 2012-10-27
Publication date: 2013-05-16

Abstract

Disclosed are anti-5T4 antibody drug conjugates with an improved pharmacokinetic profile and methods for preparing and using the same. In one embodiment, the antibody-drug conjugate has the formula Ab-(D)p, wherein Ab is an anti-5T4 antibody comprising a non- natural amino acid; D is a dolastatin linker derivative; and p is from about 1 to about 8.

Description

ANTIBODY-DRUG CONJUGATES

FIELD

The present invention generally relates to anti-5T4 antibody-drug conjugates for the treatment of cancer.

BACKGROUND OF THE INVENTION

Antibody-drug conjugates (ADCs) combine the binding specificity of monoclonal antibodies with the potency of chemotherapeutic agents. The technology associated with the development of monoclonal antibodies to tumor associated target molecules, the use of more effective cytotoxic agents, and the design of chemical linkers to covalently bind these components, has progressed rapidly in recent years (Ducry L, et a/. Bioconjugate Chemistry, 21 :5-13, 2010).

Once a tumor associated antigen is identified as a target, numerous challenges remain. Each monoclonal antibody must be characterized separately, an appropriate linker designed, and a suitable cytotoxic agent identified that retains its potency upon delivery to tumor cells. The drug moiety must be linked to specific amino acid residues on the antibody without compromising the pharmacokinetics of the ADC, binding activity of the antibody, and/or the potency of the drug. Cysteine, lysine , or histidine residues have functional groups that are commonly used as a site to link the drug moiety, but the location within the tertiary structure of the antibody may present limitations on availability for conjugation. Recently, a new technology in the protein sciences has evolved that overcomes the limitations associated with site-specific modifications of proteins [US Patent No. 7,045,337]. This technology incorporates non- genetically encoded amino acids (i.e., "non-natural amino acids") into proteins, permitting a site-specific introduction of chemical functional groups that are not found in proteins and provide an alternative to the naturally-occurring functional groups.

The human 5T4 tumor associated antigen is the target antigen of the present invention. An antibody to the 5T4 antigen previously described [US 2007/0231333] has been modified by the incorporation of non-natural amino acids as sites for conjugation of dolastatin linker derivatives. As a result, the novel anti-5T4 ADCs of the present invention overcome the challenges associated with ADC technology. The resulting ADCs have an improved pharmacokinetics profile compared to anti-5T4 ADCs without non-natural amino acid conjugation sites. Moreover, the ADCs of the present invention retain 5T4 antigen specificity and drug potency, thus delivering sufficient cytotoxic drug to the target cells, providing an innovative and effective treatment for cancer.

SUMMARY OF THE INVENTION

In one embodiment, an antibody-drug conjugate of the present invention has the formula: Ab-(D)p wherein, Ab is an anti-5T4 antibody comprising a non-natural amino acid; D is a dolastatin linker derivative; and p is from about 1 to about 8.

The present invention further provides anti-5T4 antibody-drug conjugates wherein (a) said anti-5T4 antibody comprises a non-natural amino acid at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain and further comprises a heavy chain variable region having (i) a VH CDR1 region as shown in SEQ ID NO: 5, (ii) a VH CDR2 region as shown in SEQ ID NO: 6, and (iii) a VH CDR3 region as shown in SEQ ID NO: 7; (b) said dolastatin linker derivative is selected from the group consisting of NC-D-1 , HC-D-1 , NCA-D-2, PHC-D-2, SC-D-1 , and SHC-D-1 ; and (c) p is from about 1 to about 8.

The present invention further provides an anti-5T4 antibody-drug conjugate wherein said anti-5T4 antibody comprises one or more non-natural amino acids at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain and further comprises a light chain variable region having (a) a VL CDR1 region as shown in SEQ ID NO: 8, (b) a VL CDR2 region as shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown in SEQ ID NO: 10.

The present invention further provides an anti-5T4 antibody-drug conjugate wherein said anti-5T4 antibody comprises one or more non-natural amino acids at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain and further comprises a heavy chain variable region having (a) a VH CDR1 region as shown in SEQ ID NO: 5, (b) a VH CDR2 region as shown in SEQ ID NO: 6, and (c) a VH CDR3 region as shown in SEQ ID NO: 7 and a light chain variable region having (a) a VL CDR1 region as shown in SEQ ID NO: 8, (b) a VL CDR2 region as shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown in SEQ ID NO: 10. The present invention further provides an anti-5T4 antibody-drug conjugate wherein said anti-5T4 antibody comprises one or more non-natural amino acids at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain and comprises the VH region of SEQ ID NO: 3 and the VL region of SEQ ID NO: 4.

The present invention further provides anti-5T4 antibody-drug conjugates wherein said non-natural amino acid at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain is p-acetyl-L-phenylalanine.

The present invention further provides an anti-5T4 antibody-drug conjugate wherein said anti-5T4 antibody comprises one or more non-natural amino acids at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain, and wherein said anti-5T4 antibody-drug conjugate has an improved pharmacokinetic profile compared to anti-5T4 ADCs without non-natural amino acid conjugation sites.

The present invention further provides an anti-5T4 antibody-drug conjugate wherein said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO: 1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is NC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 121 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 120 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is NC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 121 is serine, Xaa 178 is serine, and Xaa 164 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is NC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 121 is serine, Xaa 164 is alanine, and Xaa 178 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is NC-D-1 , and p is about 2. The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 121 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 120 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is SC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 121 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 120 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is SC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is SC-D-1 , and p is about 2.

The present invention further provides an antibody-drug conjugate wherein: said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine, said dolastatin linker derivative is SHC-D-1 , and p is about 2.

The present invention provides a pharmaceutical composition comprising an antibody-drug conjugate indicated above and a pharmaceutically acceptable carrier.

The present invention further provides a method of treating a 5T4-positive cancer in a patient in need thereof, comprising administering to said patient an antibody-drug conjugate indicated above.

The present invention further provides a method of treating a 5T4-positive cancer wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testes.

More preferably, the present invention provides a method of treating a 5T4- positive cancer wherein said cancer is selected from the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.

The invention further provides an antibody-drug conjugate indicated above for use in therapy. The invention further provides the use of an antibody-drug conjugate indicated above for the manufacture of a medicament.

The invention further provides the use indicated above, wherein said use is for the treatment of a 5T4-positive cancer and wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testes.

More preferably, the invention further provides the use indicated above, wherein said use is for the treatment of a 5T4-positive cancer wherein said cancer is selected from the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.

The invention further provides a nucleic acid that encodes an anti-5T4 antibody comprising a non-natural amino acid at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain, a vector comprising said nucleic acid, and a host cell comprising said vector.

The invention further provides a process for producing an anti-5T4 antibody comprising one or more non-natural amino acids at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain wherein said process comprises cultivating the host cell comprising the above mentioned vector and recovering the antibody from the cell culture.

The invention further provides a process for producing an anti-5T4 antibody-drug conjugate comprising: (a) chemically synthesizing a dolastatin linker derivative selected from the group consisting of NC-D-1 , HC-D-1 , NCA-D-2, PHC-D-2, SC-D-1 , and SHC- D-1 ; (b) conjugating said dolastatin linker derivative to the anti-5T4 antibody recovered from the cell culture, and; (c) purifying the antibody-drug conjugate.

DETAILED DESCRIPTION

The present invention provides anti-5T4 antibody-drug conjugates for the treatment of cancer. In order that the present invention is more readily understood, certain terms and general techniques are first defined.

All amino acid abbreviations used in this disclosure are those accepted by the

United States Patent and Trademark Office as set forth in 37 C.F.R. § 1 .822 (B)(J).

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual^ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

5T4 refers to the 5T4 oncofetal antigen, a 72 kDa highly glycosylated

transmenbrance glycoprotein comprising a 42 kDa non-glycosylated core (see

US5,869,053). Human 5T4 is expressed in numerous cancer types, including carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung,

esophagus, ovary, prostate, pancreas, skin, stomach, and testes. Highly tumorigenic cells, also called cancer stem cells or tumor-initiating cells have been shown to have high levels of 5T4 expression (WO2010/1 1 1659). Anti-5T4 antibodies of the invention include antibodies that specifically bind the human 5T4 antigen (see US

2007/0231333).

An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding fragment (i.e., "antigen-binding portion") or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site including, for example without limitation, scFv, single domain antibodies {e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1 126-1 136). An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "antigen binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen {e.g., target X). Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include Fab; Fab'; F(ab')2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341 :544-546), and an isolated complementarity determining region (CDR).

A "variable region" of an antibody refers to the variable region of the antibody light chain (LCVR) or the variable region of the antibody heavy chain (HCVR), either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FRs) connected by three

complementarity determining regions (CDRs) also known as hypervariable regions, contribute to the formation of the antigen binding site of antibodies. If variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (i.e., in the framework region), appropriate amino acid substitution, preferably, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canoncal class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901 -917, 1987). When choosing FR to flank subject CDRs, e.g., when humanizing or optimizing an antibody, FRs from antibodies which contain CDR1 and CDR2 sequences in the same canonical class are preferred. A "CDR" of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions or methods of CDR determination well known in the art. For example, antibody CDRs may be identified as the

hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., 1989, Nature 342:877-883. Other approaches to CDR identification include the "AbM definition," which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to herein as the

"conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1 156-1 166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches.

The term "monoclonal antibody" (mAb) refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Preferably, a monoclonal antibody of the invention exists in a homogeneous or substantially homogeneous population.

"Humanized" antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50 (1999) and Fellouse, F.A., et al, J. Mol. Biol., 373(4):924-40 (2007)).

A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-natural amino acid" is "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and variously hyphenated and non-hyphenated versions thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally occurring amino acids that are not naturally-encoded include, but are not limited to, N- acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O- phosphotyrosine. Additionally, the term "non-natural amino acid" includes, but is not limited to, amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of natural or other non-natural amino acids.

The monoclonal antibodies of the present invention are derived from the A1 anti-

5T4 antibody (a humanized anti-5T4 lgG1 antibody) wherein said anti-5T4 antibody comprises a non-natural amino acid at a position selected from the group consisting of positions 120, 121 , 164, or 178 of the heavy chain. The preferred monoclonal antibodies of the invention are selected from the group consisting of A1 -120, A1 -121 , A1 -164, and A1 -178, wherein a non-natural amino acid is at the indicated numbered position of the heavy chain of the A1 antibody. The most preferred antibody of the present invention is A1 -121 .

All of the preferred monoclonal antibodies of the invention comprise: (a) a LCVR of SEQ ID NO: 4, further comprising: a LCDR1 of SEQ ID NO: 8, a LCDR2 of SEQ ID NO: 9, and a LCDR3 of SEQ ID NO: 10; and, b) a HCVR of SEQ ID NO: 3, further comprising: a HCDR1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 6, and a HCDR3 of SEQ ID NO: 7.

In addition, all of the preferred antibodies of the present invention comprise a LC of SEQ ID NO: 2 and a HC of SEQ ID NO: 1 , wherein the Xaa positions of SEQ ID NO: 1 are indicated in Table 1 for the anti-5T4 monoclonal antibodies A1 -120, A1 -121 , A1 - 164, and A1 -178.

Table 1

*pAF designates the non-natural amino acid p-acetyl-L-phenylalanine

The term "specifically binds" as used herein in reference to the binding between an antibody and a 5T4 antigen refers to an antibody that only binds to cells expressing the 5T4 antigen.

The term "pharmaceutically acceptable", as used herein, refers to a material, including but not limited to, a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term "potency" is a measurement of biological activity and may be designated as IC₅₀, or the effective concentration of antibody needed to inhibit 50% of growth of a 5T4 positive cell line as described in Example 2. Alternatively, potency may refer to anti-tumor activity as determined in an in vivo tumor xenograft model as shown in Example 3.

Pharmacokinetics refers to the study of absorption, distribution, metabolism and excretion (ADME) of bioactive compounds in a higher organism. An improved pharmacokinetics profile generally refers to increased in vivo half-life (e.g. plasma half life) allowing more effective tissue distribution.

The terms "polynucleotide" or "nucleic acid molecule", as used herein, are intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

The polynucleotides that encode the antibodies of the present invention may include the following: only the coding sequence for the variant, the coding sequence for the variant and additional coding sequences such as a functional polypeptide, or a signal or secretory sequence or a pro-protein sequence; the coding sequence for the antibody and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the antibody. The term 'polynucleotide encoding an antibody" encompasses a polynucleotide which includes additional coding sequence for the variant but also a polynucleotide which includes additional coding and/or non- coding sequence. It is known in the art that a polynucleotide sequence that is optimized for a specific host cell/expression system can readily be obtained from the amino acid sequence of the desired protein (see GENEART AG, Regensburg,

Germany).

The polynucleotides encoding the antibodies of the present invention will typically include an expression control polynucleotide sequence operably linked to the antibody coding sequences, including naturally-associated or heterologous promoter regions known in the art. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host cell line, the host cell is propagated under conditions suitable for expressing the nucleotide sequences, and, as desired, for the collection and purification of the antibodies. Preferred eukaryotic cell lines include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells, or human embryonic kidney cell lines. The most preferred host cell is a CHO cell line. Methods for in vivo incorporation of non-natural amino acids into proteins and methods for expressing the proteins are described in US 7,045,337, herein incorporated by reference.

The term "linkage," as used herein to refer to bonds or chemical moieties formed from a chemical reaction between the functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, carbamates, imines phosphate esters, oximes, hydrazones, acetals, orthoesters, amides, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood.

Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are a reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.

Dolastatins and their peptidic analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents

Chemother. 42:2961 -2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin F (MMAF), monomethylauristatin-D (MMAD), monomethyl auristatin E

(MMAE), and 5-benzoylvaleric acid-AE ester (AEVB). The preferred dolastatin derivative or analog is MMAD. Described herein are dolastatin linker derivatives or analogs comprising at least one non-natural amino acid or modified non-natural amino acid with an oxime, aromatic amine, or heterocycle (e.g.indole, quinoxaline,phenazine, pyrazole, triazole, etc.).

Such dolastatin linker derivatives comprising non-natural amino acids may contain further functionality, including but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality. Indeed, there will be overlap depending upon the particular circumstances. By way of example only, a water-soluble polymer overlaps in scope with a derivative of

polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.

Dolastatin derivatives with linkers containing a hydroxylamine ether (also called an aminooxy) group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers). Like hydrazines, hydrazides and semicarbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain carbonyl- or dicarbonyl-groups, including but not limited to, ketones, aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tarn, J., J. Arn. Chern. Soc. 1 17:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chern. Res. 34(9): 727-736 (2001 ). The result from the reaction of an aminooxy group with a carbonyl- or dicarbonyl-containing group such as, by way of example, ketones, aldehydes or other functional groups with similar chemical reactivity, is an oxime.

The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1 ) at least one functional group on the sidechain of the non-natural amino acid has at least one characteristics and/or activity and/or reactivity orthogonal to the chemical reactivity of the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in the polypeptide that includes the nonnatural amino acid; (2) the introduced non-natural amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability commensurate with the naturally-occurring amino acids or under typical physiological conditions, and further preferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acid includes a functional group that can be transformed into an oxime group by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide that includes the non-natural amino acid (unless of course such a destruction of biological properties is the purpose of the

modification/transformation), or where the transformation can occur under aqueous conditions at a pH between about 4 and about 8, or where the reactive site on the non- natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide.

Preferably, non-natural amino acids of the present invention include, but are not limited to, compounds selected from a group consisting of O-methyl-L-tyrosine, L-3-(2- naphthyl)-alanine, 3-methyl-L-phenylalanine, fluorinated phenylalanine, p-benzoyl-L- phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, p-amino-L- phenylalanine, 3,4-dihydroxy-L-phenylalanine, and isopropylL- phenylalanine, p-azido- L-phenylalanine, p-acetyl-L-phenylalanine, m-acetyl-L-phenylalanine, 4-(2-oxo- propoxy)-L-phenylalanine. More preferably, the non-natural amino acids of the present invention are selected from the group consisting of p-azido-L-phenylalanine, p-acetyl-L- phenylalanine, m-acetyl-L-phenylalanine, and 4-(2-oxo-propoxy)-L-phenylalanine. The most preferred non-natural amino acid of the present invention is p-acetyl-L- phenylalanine.

Non-natural amino acids of the present invention may include protected or masked carbonyl or dicarbonyl groups, which can be transformed into a carbonyl or dicarbonyl group after deprotection of the protected group or unmasking of the masked group and thereby are available to react with hydroxylamine ethers or oximes to form oxime groups.

Non-natural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids comprising novel functional groups, amino acids that covalently or noncovalently interact with other molecules, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, aldehyde containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically deavable and/or photodeavable amino acids, amino acids with elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox- active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.

The chemical moieties incorporated into polypeptides via incorporation of non- natural amino acids into such polypeptides offer a variety of advantages and

manipulations of polypeptides. For example, the unique reactivity of a carbonyl or dicarbonyl functional group (including a keto- or aldehyde- functional group) allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine containing reagents in vivo and in vitro.

Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl- or dicarbonyl- group (including a keto- or aldehyde group), a carbonyl-like or dicarbonyl-like-group (which has reactivity similar to a carbonyl- or dicarbonyl-group and is structurally similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or masked dicarbonyl-group (which can be readily converted into a carbonyl- or dicarbonyl-group), or a protected carbonyl- or protected dicarbonyl-group (which has reactivity similar to a carbonyl- or dicarbonyl-group upon deprotection).

Non-natural amino acid dolastatin linked derivatives containing an oxime group allow for reaction with a variety of reagents that contain certain reactive carbonyl- or dicarbonyl- groups (including but not limited to, ketones, aldehydes, or other groups with similar reactivity) to form new non-natural amino acids comprising a new oxime group. Such an oxime exchange reaction allows for the further functionalization of dolastatin linked derivatives. In some embodiments, the number of drug moieties, p, conjugated per antibody molecule via the oxime linkage ranges from an average of about 1 to about 8; about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from an average of about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or about 2 to about 3. In other embodiments, p is an average of 1 , 2, 3, 4, 5, 6, 7, or 8. In certain embodiments described herein are non-natural amino acid dolastiatin linked derivatives with side chains comprising a masked oxime group (which can be readily converted into an oxime group), or a protected oxime group (which has reactivity similar to an oxime group upon deprotection).

For comparison purposes, the ADC A1 -mc-MMAD and/or A1 -vc-MMAD were used. The linker payload, mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D) was conjugated to the A1 anti-5T4 monoclonal antibody through a cysteine residue at a ratio of approximately 4 drug moieties per antibody molecule. The linker payload mc- Val-Cit-PABA-MMAD or vc-MMAD ( maleimidocapronic -valine-citruline-p- aminobenzyloxycarbonyl- monomethylauristatin-D) was conjugated to the A1 anti-5T4 monoclonal antibody through a cysteine residue at a ratio of approximately 4 drug moieties per antibody molecule (see Sun, et al., Bioconjugate Chem. 16: 1282-1290, 2005, herein incorporated by reference).

Immunotherapy

For immunotherapy, an antibody can be conjugated to a suitable drug, such as a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like. The conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The conjugate can be used accordingly in a variety of settings for the treatment of animal cancers. The conjugate can be used to deliver a drug to a tumor cell or cancer cell. Without being bound by theory, in some embodiments, the conjugate binds to or associates with a cancer-cell or a tumor-associated antigen, and the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor- mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell.

Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in a linker) are hydrolytically cleaved by one or more tumor-cell or cancer-cell- associated proteases, resulting in release of the drug. The released drug is then free to migrate within the cell and induce cytotoxic or cytostatic or other activities.

Therapy for Cancer

As discussed above, cancers positive for the 5T4 antigen, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by

uncontrolled cell growth, can be treated or prevented by administration of an antibody- drug conjugate. The present invention provides a method of treating a 5T4-positive cancer wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin (i.e. melanoma), stomach, and testes. More preferably, the present invention provides a method of treating a 5T4-positive cancer wherein said cancer is selected from the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.

In other embodiments, methods for treating or preventing cancer are provided, including administering to a patient in need thereof an effective amount of a conjugate and a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In some embodiments, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The conjugate can be administered to a patient that has also undergone a treatment, such as surgery for treatment for the cancer. In another embodiment, the additional method of treatment is radiation therapy.

Multi-Drug Therapy for Cancer

Methods for treating cancer include administering to a patient in need thereof an effective amount of an antibody-drug conjugate and another therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to,

methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, Cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

Pharmaceutical Compositions

The ADCs of the present invention can be in the form of a pharmaceutical composition for administration that are formulated to be appropriate for the selected mode of administration, and pharmaceutically acceptable diluent or excipients, such as buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, carriers, and the like. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 18^th ed., 1995, incorporated herein by reference, provides a

compendium of formulation techniques as are generally known to practitioners.

These pharmaceutical compositions may be administered by any means known in the art that achieve the generally intended purpose to treat cancer. The preferred route of administration is parenteral, defined herein as referring to modes of administration that include but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of the invention include all compositions wherein an ADC is present in an amount that is effective to achieve the desired medical effect for treating cancer. While individual needs may vary from one patient to another, the determination of the optimal ranges of effective amounts of all of the components is within the ability of the clinician of ordinary skill.

Example 1

Synthesis of Dolastatin Linked Derivatives

Synthesis of NC-D-1

Compound 1a: Tetra (ethylene glycol) (10mg, 51 .5mmol), N- hydroxyphthalimide (8.4g, 51 .15mmol) and triphenylphosphine (17.6g, 67mmol) are dissolved in 300mL of tetrahydrofuran followed by addition of diisopropyl

azodicarboxylate (DIAD) (12.8 mL, 61 .78 mmol) at 0°C. The resulting solution is stirred at room temperature overnight, and then concentrated to dryness. The residue is purified by flash column chromatography to give 5.47g (31 %) of compound 1 a.

Compound 1 b: to a solution of compound 1 a (200mg, 0.59mmol) in 15mL, dichloromethane is added Dess-Martin Periodinane (300mg, 0.71 mmol). The reaction mixture is stirred at ambient temperature overnight. The reaction is quenched with the solution of sodium bisulfate in15 ml of saturated sodium bicarbonate. The mixture is separated. The organic layer is washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue is purified by flash column chromatography to give 150mg (75%) of compound 1 b.

Compound 1c: To a solution of monomethyldolastin hydrochloride salt (50mg, 0.062 mmol) in 1 mL of Ν,Ν-dimethylformamide (DMF) is added compound 1 b (63mg, 0.186 mmol) and 70 L of acetic acid, followed by addition of 8 mg of sodium

cyanoborohydride. The resulting mixture is stirred at ambient temperature for 2 hours. The reaction mixture is diluted with water and purified by HPLC to give 60mg (80%) of compound 1 c. MS (ESI) m/z 547 [M+2H], 1092 [M+H]. Compound 1d: compound 1 c (60mg, 0.05mmol) is dissolved in 1 ml_ of DMF, and 32 μΙ_ of hydrazine is added. The resulting solution is stirred at ambient temperature for 1 hour. The reaction is quenched with I N hydrochloric acid (HCI) solution. The reaction is purified by HPLC to give 33 mg (55%) of compound 1 d. MS (ESI) m/z 482 [M+2H], 962 [M+H].

Conjugation to A1 -121 mAb

Compound 1 d is conjugated to A1 -121 mAb via the non-natural amino acid p- acetyl-L-phenylalanine resulting in the compound A1 -121 -NC-D-1 .

The conjugation reaction mixture contains 15-20 mg/ml A1 -121 mAb, molar ratio of drug-linker: A1 -121 = 10:1 , 1 % acetic hydrazide. The amount of drug-linker needed is calculated using the equation below:

Drug - lin ker(mg) = x ratioiDrug - lin ker : mAb) x MWiDrug - lin ker)

Assume the ratio of Drug-linker : mAb is 10; and, the MW of mAb is 150,000.

The molecular weights of dolastatin linked derivatives are listed below:

Dilute the mAb stock solution with 50mM Na Acetate, 5% Trehalose, pH 4.0, if necessary. Add the required volume (required mg divided by 20) of drug-linker stock solution to the mAb. Add 1/10 of the final volume of the catalyst stock solution, 10% acetic hydrazide in 50mM Na Acetate/pH4 (1 .35M). Add 20% DMSO final (PHC-D-2 only). Mix well with the pipette. The tube holding the reaction mixture is securely capped and covered with aluminum foil. The tube is placed in a rocker with gentle rotation (50-70 rpm). The temperature is controlled at 28°C. Allow the conjugation reaction to proceed for 18-48 hrs. The conjugation efficiency is accessed by RP-HPLC analysis after overnight incubation and at the end of the reaction. ynthesis of NC-D-1

Structure of NC-D-1 conjugated to pAF residue on an anti-5T4 mAb:

Synthesis of HC-D-1

Compound 2a: To a solution of tetra (ethylene glycol) (40.6 mL, 235 mmol) in

100 mL of tetrahydrofuran is added 47 mg of sodium. 12 mL of tert-butylacrylate is added after sodium is dissolved. The reaction mixture is stirred at room temperature for 24 hours. The reaction mixture is concentrated in vacuo and quenched with 2 mL of 1 N HCI. The residue is suspended in brine and extracted with ethyl acetate (100 mLX1 , 50 mL X2). The organic layer is combined and washed with brine, dried over sodium sulfate and concentrated in vacuo to give 6.4 g (23%) of compound 2a. Compound 2b: Compound 2a (1 .0 g, 3.12 mmol), N-hydroxyphthalimide (61 1 mg, 3.744 mmol) and triphenylphosphine (1 .23 g, 4.68 mmol) are dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mmol) at 0°C. The resulting solution is stirred at room temperature overnight, and then concentrated to dryness. The residue is purified by flash column chromatography using SiliaSep Cartridges (80g), eluting with 0-100% ethyl acetate/hexanes, to give 1 .0 g (100%) of compound 2b.

Compound 2c: Compound 2b (1 g) is dissolved in 15 mL 4N HCI/Dioxane. The reaction mixture is stirred at room temperature for 2 hours and concentrated in vacuo to give 1 .0 g of compound 2c.

Compound 2d: To a solution of compound 6 (1 .93 g, 4.68 mmol) and N- hydroxysuccinimide (646 mg, 5.616 mmol) in 20 mL of tetrahydrofuran is added 1 .062 g (5.148 mmol) of Ν,Ν-dicyclohexylcarbodiimide (DCC). The reaction mixture is stirred at room temperature overnight and filtered. The filtration is concentrated and purified by flash column chromatography using SiliaSep Cartridges (80g), eluting with 0-100% ethyl acetate/hexanes to give 2.37g (100%) of compound 2d.

Compound 2e: Compound 2e is made according to the literature (Bioconjugate Chem. 2002, 13 (4), 855-869).

Compound 2f: To a solution of compound 2e (200 mg, 0.527 mmol) in 2 mL of DMF is added 295 mg (0.58 mmol) of compound 2d. The reaction mixture is stirred at room temperature overnight and concentrated in vacuo. The residue is treated with ether, filtered, washed with ether and dried in vacuo to give 402 mg (98%) of compound 2f.

Compound 2g: To a solution of compound 2f (406 mg, 0.527 mmol) and bis(p- nitrophenol) carbonate (481 mg, 1 .58 mmol) in 10 mL of DMF is added 0.186 mL(1 .054 mmol) of diisopropylethylamine. The reaction mixture is stirred at room temperature for 5 hours. The solvent is removed in vacuo. The residue is treated with ether, filtered, washed with ether, 5% citric acid, water, ether and dried in vacuo to give 350 mg (72%) of compound 2g.

Compound 2h: To a solution of 50 mg (0.062 mmol) of monomethyldolastatin hydrochloride, 87.2 mg (0.093 mmol) of compound 2g and 4.7 mg (0.031 mmol) of 1 - hydroxybenzotriazole (HOBt) in 1 mL of DMF is added 22 μί (0.124 mmol) of diisopropylethylamine. The reaction mixture is stirred at room temperature for 16 hours. The reaction mixture is purified by HPLC to give 41 mg (42%) of compound 2h. MS (ESI) m/z 785 [M+2H].

Compound 2i: Compound 2h (41 mg, 0.026 mmol) is dissolved in 1 ml_ of DMF. 17 μΙ_ (0.52 mmol) of anhydrous hydrazine is added. The resulting solution is stirred at room temperature for 1 hour. The reaction is quenched with 1 N hydrochloride solution. The reaction mixture is purified by preparative HPLC, eluting with 20-70%CH3CN/H20 in 20 min at 254 nm, to give 22 mg (58%) of HC-D-1 . MS (ESI) m/z 720 [M+2H].

HC-D-1 is conjugated to A1 -121 mAb via the non-natural amino acid p-acetyl-L- phenylalanine, as described above, resulting in the compound A1 -121 -HC-D-1 .

Synthesis of HC-D-1.

Structure of HC-D-1 Conjugated to pAF residue on an anti-5T4 mAb:

Synthesis of NCA-D-2 and PHC-D-2

The synthesis of the dolastatin linked derivatives NCA-D-2 and PHC-D-2 is described in US Provisional Application (WSGR Docket No. 31362-727.101 ), herein incorporated by reference. Conjugation of the dolastatin linked derivatives to A1 -121 mAb containing the non-natural amino acid p-acetyl-L-phenylalanine is described above, and results in compounds NCA-D-2-A1 -121 mAb and PHC-D-2-A1 -121 mAb, respectively.

Structure of NCA-D-2 Conjugated to pAF residue on an anti-5T4 mAb:

Structure of PHC-D-2 Conjugated to pAF residue on an ati-5T4 mAb:

Example 2

Cytotoxicity Assay

Cell lines expressing 5T4, and the negative control Raji cell line, are cultured with increasing concentrations of ADC. After four days, viability of each culture is assessed. IC₅₀ values are calculated by logistic non-linear regression and are presented as Ab ng/mL. A1 -121 -NC-D-1 , A1 -121 -HC-D-1 , A1 -121 -NCA-D-2, and A1 - 121 -PHC-D-2 are shown to inhibit the growth of 5T4 expressing cell lines that express a high level of the 5T4 antigen (MDAMB435/5T4) or express moderate levels of the 5T4 antigen (MDAMB468, and MDAMB361 DYT2), while being relatively inactive on 5T4 negative cells (Raji), Table 2.

Table 2

Example 3

Subcutaneous Xenograft Model

Female, athymic (nude) mice are injected s.c. with MDAMB435/5T4 cells or MDAMB361 DYT2 cells. Mice with staged tumors, approximately 0.1 to 0.3 g (n=6 to 10 mice/treatment group) are administered intravenously Q4Dx4 with normal saline

(vehicle), A1 -121 -NC-D-1 , A1 -121 -HC-D-1 , A1 -121 -NCA-D-2, A1 -121 -PHC-D-2, A1 - mcMMAD (A1 IgG without a non-natural amino acid at position 121 ), or a nonbinding control antibody, 2h6, conjugated to mc-MMAD at the doses of 1 , 3, and/or 10 mg Ab/kg. All ADCs are dosed based on Ab content. Tumors are measured at least once a week and their size (mm² ± SEM) is calculated as mm² = 0.5 x (tumor width²) x (tumor length) . The data in Table 3 indicates that A1 -121 -NCA-D-2 inhibits the growth of MDAMB361 DYT2 xenografts at all dose levels while A1 -mcMMAD was not active in this model at doses of 1 , 3, or 10 mg/kg. Table 3

GT= group terminated due to large tumor size

The data in Table 4 indicates that A1 -121 -NCA-D-2 inhibits the growth of MDAMB435/5T4 xenografts at the 3 mg/kg and 10 mg/kg dose levels while Al mcMMAD was not active in this model at doses of 1 , 3, or 10 mg/kg. Table 4

GT= group terminated due to large tumor size The data in Table 5 indicates that A1 -121 -NC-D-1 inhibits the growth of

MDAMB435/5T4 xenografts at the 10 mg/kg dose while A1 -mcMMAD was not active in this model at doses of 1 , 3, or 10 mg/kg. Table 5

GT= group terminated due to large tumor size

The data in Table 6 indicates that A1 -121 -NC-D-1 inhibits the growth of MDAMB361 DYT2 xenografts at the 3 mg/kg and 10 mg/kg dose levels while A1 - mcMMAD was not active in this model at doses of 1 , 3, or 10 mg/kg. Table 6

GT= group terminated due to large tumor size

The data in Table 7 indicates that A1 -121 -HC-D-1 inhibits the growth of

MDAMB435/5T4 xenografts at the 10 mg/kg dose and A1 -121 -PHC-D-2 inhibits the growth of MDAMB435/5T4 xenografts at the 10, 3, and 1 mg/kg doses. The comparator ADC, A1 -vcMMAD was effective in this model at the 3, 1 , and 0.3 mg/kg doses. Table 7

Example 4

Selecting Mutation Sites on the

CH1 Domain of the A1 mAb

Amino acid positions of the anti-5T4 antibody, A1 , selected for mutation and subsequent location of non-natural amino acids as conjugation sites of dolastatin linked derivatives are selected based on structural information. The desired mutations sites have certain characteristics: (1 ) the mutations sites have to be accessible for conjugation i.e. the calculated solvent accessible surface is above 40%. (2) The mutation sites cannot be involved in the integrity of the 3D structure of the antibody. A preference is given to residues outside secondary structure. (3) All residues in interface with other domains (VH-CH1 , VL-CL, CH1 -CL, and CH1 -CH2) are not considered. (4) Finally, proline and glycine are deselected for their role in the structural integrity of the protein.

Modeling of the 3D structure of the anti-5T4 antibody of the present invention is done using other antibodies with high sequence homology with the anti-5T4 antibody. This provides comparative modeling to predict the 3D structure of the anti-5T4 Fab. This modeling is subsequently validated with the X-ray structure of the anti-5T4 Fab.

The selection of a homologous human antibody is completed using the algorithm Psi-Blast against the Protein Data Base, using the most homologous template considering both heavy and light chains. Three templates are selected, all of which are humanized murine antibodies with sequence identity between 85% and 90%.

Protein Data Base codes: 1 T3F: Humanized murine anti-IFN-gamma Fab (HuZAF); 1 B2W: humanized murine anti-gamma-interferon antibody; 1 L7I: humanized murine anti-ErbB2antibody.

The sequences are aligned using the Salign algorithm implemented in the comparative modeling software MODELER 9v7 (A. Sali and T. L. Blundell, J. Mol. Biol. 234, 779-815, 1993). Using the three templates and the sequence alignment described above, 50 models are built and selected based on PDF score and DOPE evaluation. All algorithms are implemented in MODELER 9v7.

The calculation of the solvent accessible surface area (SASA) is implemented in Discovery Studio (Accelrys, Inc). All residues with SASA more that 40% are considered as solvent accessible. Detection of secondary structure on the model is conducted by the Kabsch and Sander method implemented in Discovery Studio (Accelrys, Inc). Mutation sites are selected and prioritized based on the different criteria: SASA > 40%; no Proline or Glycine residues; no involvement of the residue in the fold stability; no involvement of the residue in the different interface domains; and, side chain orientation (toward or outward the protein). Examples of mutation sites selected in the CH1 domain of the anti-5T4 antibody of the present invention are listed and prioritized in Table 8.

Table 8

Example 5

Pharmacokinetic Analysis of ADCs

Pharmacokinetic analysis is done with the ADC's containing the non-natural amino acid p-acetyl-L-phenylalanine at positions 120, 121 , 164 or 178 of the A1 antibody heavy chain conjugated with the dolastatin linked derivative NC-D-1 . For comparison, the ADC's A1 -mc-MMAD and A1 -vc-MMAD, which do not contain a non- natural amino acid, are used.

Female nu/nu mice (5 per dose group) are injected with a single IV bolus at 3 mg/kg for each ADC tested. Whole blood (10μΙ_) is serially obtained at T= 0, 4, 8, 24, 48, 96, 168, 240, and 336 hours post dosing (hpd). The whole blood sample is diluted to 200 μΙ_ with buffer and centrifuged. The supernatant is then transferred to a 96 well plate for ELISA analysis. The ELISA is a standard capture assay utilizing the 5T4 antigen as the capturing reagent and biotinylated anti-dolastatin antibody/ streptavidin- HRP as the detection reagents.

The data in Table 9 indicates that all of the ADC's containing the non-natural amino acid p-acetyl-L-phenylalanine conjugated with the dolastatin linked derivative NC-D-1 have a longer t1/2 (plasma half life) and a greater area under the curve (AUC) or exposure than the comparator ADCs, A1 -mc-MMAD and A1 -vc-MMAD. This data unexpectedly demonstrates that the insertion of a single non-natural amino acid into the heavy chain of an antibody conjugated to a dolastatin linked derivative provides the advantage of an improved pharmacokinetics profile indicated by a longer plasma half life and higher AUC compared to a similar ADC lacking a non-natural amino acid.

Table 9

ELISA.

*^* AUC (0-last) is the Area Under the Curve calculated from T=0 to T= 336hpd.

^*** AUC (0-inf) is the Area Under the Curve calculated from T=0 to a theoretical infinity.

The data in Table 10 indicates that the ADCs containing the non-natural amino acid p-acetyl-L-phenylalanine at position 121 conjugated with the dolastatin linked derivatives NC-D-1 , NCA-D-2, HC-D-1 , or PHC-D-2 have a longer t1/2 (plasma half life) and a greater AUC compared with the controls A1 IgG antibody or the A1 -121 mutant IgG antibody. This data unexpectedly demonstrates that the insertion of a single non-natural amino acid into the heavy chain of an antibody conjugated to various dolastatin linked derivatives provides the advantage of an improved pharmacokinetics profile indicated by a longer plasma half life and greater AUC compared to a control IgG antibody with or without a non-natural amino acid at position 121 .

Table 10

ELISA.

^** AUC (0-last) is the Area Under the Curve calculated from T=0 to T= 336hpd.

^*** AUC (0-inf) is the Area Under the Curve calculated from T=0 to a theoretical infinity. Example 6

A1 -5T4 ADC Plasma Stability

Stability analysis of A1 -5T4-D-M-1 ADCs in mouse, rat, and human plasma is done with the ADCs containing the non-natural amino acid p-acetyl-L-phenylalanine at positions 120 or 121 of the A1 antibody heavy chain conjugated with the dolastatin linked derivatives HC-D-1 , SHC-D-1 , or SC-D-1 , which are synthesized as described in Example 1 under the synthesis of HC-D-1 .

HC, SHC, and SC designate a linker derivative of polyethylene glycol (PEG) in decreasing order of chain length of the PEG derivative i.e. HC PEG chain length > SHC PEG chain length > SC PEG chain length, as shown in the following structures of the dolastatin linked derivatives HC-D-1 , SHC-D-1 , and SC-D-1 :

The stability analysis of the A1 -5T4 -D-M-1 ADCs (A1 -5T4-120-HC-D-1 , A1 -5T4- 120-SHC-D-1 , A1 -5T4-120-SC-D-1 , A1 -5T4-121 -HC-D-1 , A1 -5T4-121 -SHC-D-1 , and A1 -5T4-121 -SC-D-1 ) as measured by the release of MMAD from the cleavable linker, is performed in fresh nu/nu female mouse, rat, or human plasma. The A1 -5T4 -D-M-1 ADCs are added at a concentration of 0.2mg/mL with aliquots removed at T= 5min, 4hr, 8hr, 24hr, 48hr, and 72hrs, with each time point taken triplicate. An organic extraction is performed to remove free MMAD which is then quantitated by LC/MS/MS.

Stability analysis of the ADCs in nu/nu female mouse plasma is shown in Table 1 1 . It is evident that the dolastatin derivative SC-D1 demonstrates the greatest stability in mouse plasma when conjugated at both the 120 and 121 positions. SHC-D1 has greater stability at position 120 than at position 121 . Table 11

Stability analysis of the ADCs in rat plasma is shown in Table 12. All 3 cleavable linkers show greater stability at position 120 vs 121 in rat plasma. Shortened cleavable linkers (SHC-D1 and SC-D1 ) show greater stability in rat plasma at site 121 when compared to linker HC-D1 .

Table 12

Stability analysis of the ADCs in human plasma is shown in Table 13. All cleavable linkers show stability in human plasma up to 72hrs when conjugated at positions 120 and 121 . Table 13

The above data unexpectedly shows that the shortened cleavable linker SC-D-1 is stable at both positions 120 and 121 in mouse, rat and human plasma. Cleavable linker SHC-D-1 is more stable when conjugated at position 120 than position 121 in both mouse and rat plasma. Finally, all three cleavable linkers show stability in human plasma up to 72hrs when conjugated at positions 120 and 121 .

The importance of both the chain length of the PEG derivative of the cleavable linker and the position at which it is conjugated to the antibody are important parameters to be considered for an ADC. A person skilled in the art could not predict such effectiveness on stability of a short cleavable linker such as SC-D-1 . Moreover, by moving the conjugation position only one amino acid position, i.e. position 121 to position 120, imparts surprising stability, in some instances, as demonstrated with both the SC-D-1 and the SHC-D-1 cleavable linkers.

SEQUENCE LISTING

SEQ ID NO:l Humanized Al human IgGl heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEF KGRF I SRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSXaa¹²⁰

Xaa^I2ITKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGXaa^Iff4LTSGVHTFPAVL QXaa^I7SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMI SR PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK I SKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

120

Xaa is alanine or non-natural amino acid p-acetyl-L- phenyalanine

121

Xaa is serine or non-natural amino acid p-acetyl-L- phenyalanine

Xaa¹⁶⁴ is alanine or non-natural amino acid p-acetyl-L- phenyalanine

178

Xaa is serine or non-natural amino acid p-acetyl-L- phenyalanine

SEQ ID NO: 2 Humanized Al human Kappa light chain:

DIQMTQSPSSLSASVGDRV I CKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSG SGYGTDFTL ISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLS S PVTKSFNRGEC

SEQ ID NO: 3 Al-VH

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEF KGRF I SRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSS

SEQ ID NO: 4 Al-VL

DIQMTQS PS SLSASVGDRV I CKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSG SGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIK SEQ ID NO: 6 Al-HC CDR2

WINTNTGEPRYAEEFKG

SEQ ID NO: 7 Al-HC CDR3

DWDGAYFFDY

SEQ ID NO: 8 A1-LC-CDR1

KASQSVSNDVA

SEQ ID NO: 9 A1-LC-CDR2

FATNRYT

SEQ ID NO: 10 A1-LC-CDR3

QQDYSSPWT

SEQ ID NO: 11 Human 5T4 antigen MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASS FSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNR NLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAEL AALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPF AFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVV AALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLD LSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAE LQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDR LTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLG IVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYH YRYEINADPRLTNLSSNSDV

Claims

We Claim:

1 . An antibody-drug conjugate of the formula:

Ab-( D)p

or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-5T4 antibody comprising a non-natural amino acid; D is a dolastatin linker derivative; and p is from about 1 to about 8.

2. The antibody-drug conjugate of claim 1 wherein:

(a) Ab is an anti-5T4 antibody comprising a non-natural amino acid at a position selected from the group consisting of positions 120, 121 ,164, or 178 of the heavy chain and further comprising a heavy chain variable region having:

(i) a VH CDR1 region as shown in SEQ ID NO: 5,

(ii) a VH CDR2 region as shown in SEQ ID NO: 6, and

(iii) a VH CDR3 region as shown in SEQ ID NO: 7;

(b) D is a dolastatin linker derivative selected from the group consisting of NC-D- 1 , HC-D-1 , NCA-D-2, PHC-D-2, SC-D-1 , and SHC-D-1 ; and

(c) p is from about 1 to about 4.

3. The antibody-drug conjugate of Claims 1 or 2 wherein said anti-5T4 antibody comprises a non-natural amino acid at a position selected from the group consisting of positions 120, 121 ,164, or 178 of the heavy chain and further comprises a light chain variable region having:

(a) a VL CDR1 region as shown in SEQ ID NO: 8,

(b) a VL CDR2 region as shown in SEQ ID NO: 9, and

(c) a VL CDR3 region as shown in SEQ ID NO: 10.

4. The antibody-drug conjugate of Claims 1 -3 wherein said anti-5T4 antibody comprises a non-natural amino acid at a position selected from the group consisting of positions 120, 121 ,164, or 178 of the heavy chain and further comprises:

a) a heavy chain variable region having

(i) a VH CDR1 region as shown in SEQ ID NO: 5,

(ii) a VH CDR2 region as shown in SEQ ID NO: 6, and

(iii) a VH CDR3 region as shown in SEQ ID NO: 7. b) a light chain variable region having

(i) a VL CDR1 region as shown in SEQ ID NO: 8,

(ii) a VL CDR2 region as shown in SEQ ID NO: 9, and

(iii) a VL CDR3 region as shown in SEQ ID NO: 10.

5. The antibody-drug conjugate of any one of Claims 1 -4, wherein said anti-5T4 antibody comprises a non-natural amino acid at a position selected from the group consisting of positions 120, 121 ,164, or 178 of the heavy chain and further comprises the VH region of SEQ ID NO: 3 and the VL region of SEQ ID NO: 4.

6. The antibody-drug conjugate of any one of Claims 1 -5, wherein said anti-5T4 antibody conjugate has an improved pharmacokinetics profile compared to an anti-5T4 antibody conjugate without a non-natural amino acid in the antibody heavy chain.

7. The antibody-drug conjugate of any one of Claims 1 -6 wherein said non-natural amino acid at a position selected from the group consisting of positions 120, 121 ,164, or 178 of the heavy chain of said anti-5T4 antibody consists of p-acetyl-L- phenylalanine.

8. The antibody-drug conjugate of any one of Claims 1 -7 wherein said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine.

9. The antibody-drug conjugate of any one of Claims 1 -8, wherein D is NC-D-1 .

10. The antibody-drug conjugate of any one of Claims 1 -9, wherein p is about 2.

1 1 . The antibody-drug conjugate of Claim 1 wherein:

(a) said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine,

(b) said dolastatin linker derivative is NC-D-1 , and

(c) p is about 2.

12. The antibody-drug conjugate of Claim 1 wherein:

(a) said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 121 is serine, Xaa 164 is alanine, Xaa 178 is serine, and Xaa 120 is the non-natural amino acid p-acetyl-L-phenylalanine,

(b) said dolastatin linker derivative is NC-D-1 , and

(c) p is about 2.

13. The antibody-drug conjugate of Claim 1 wherein:

(a) said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 121 is serine, Xaa 178 is serine, and Xaa 164 is the non-natural amino acid p-acetyl-L-phenylalanine,

(b) said dolastatin linker derivative is NC-D-1 , and

(c) p is about 2.

14. The antibody-drug conjugate of Claim 1 wherein:

(a) said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 121 is serine, Xaa 164 is alanine, and Xaa 178 is the non-natural amino acid p-acetyl-L-phenylalanine, (b) said dolastatin linker derivative is NC-D-1 , and

(c) p is about 2.

15. The antibody-drug conjugate of Claim 1 wherein:

(b) said dolastatin linker derivative is SC-D-1 , and

(c) p is about 2.

16. The antibody-drug conjugate of Claim 1 wherein:

(b) said dolastatin linker derivative is SC-D-1 , and (c) p is about 2.

17. The antibody-drug conjugate of Claim 1 wherein:

(a) said anti-5T4 antibody consists of a light chain having SEQ ID NO: 2 and a heavy chain having SEQ ID NO:1 wherein Xaa 120 is serine, Xaa 164 is alanine, Xaa

178 is serine, and Xaa 121 is the non-natural amino acid p-acetyl-L-phenylalanine,

(b) said dolastatin linker derivative is SC-D-1 , and

(c) p is about 2.

18. The antibody-drug conjugate of Claim 1 wherein:

(b) said dolastatin linker derivative is SHC-D-1 , and

(c) p is about 2.

19. A pharmaceutical composition comprising the antibody-drug conjugate of any one of Claims 1 -18 and a pharmaceutically acceptable carrier.

20. A method of treating 5T4-positive cancer in a patient in need thereof, comprising administering to said patient the antibody-drug conjugate according to any one of Claims 1 -19.

21 . The method of Claim 20 wherein said 5T4-positive cancer is selected from the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.

22. The antibody-drug conjugate of any one of Claims 1 -19 for use in therapy.

23. The use according to Claim 22, wherein said use is for the treatment of 5T4- positive cancer.

24. The use of Claim 23 wherein said 5T4-positive cancer is selected from the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.

25. A nucleic acid that encodes the anti-5T4 antibody of any one of Claims 1 -8.

26. A vector comprising the nucleic acid of Claim 25.

27. A host cell comprising the vector according to Claim 26.

28. A process for producing an antibody comprising cultivating the host cell of Claim 27 and recovering the antibody from the culture.

29. A process for producing an anti-5T4 antibody-drug conjugate comprising:

(a) chemically synthesizing a dolastatin linker derivative selected from the group consisting of NC-D-1 , HC-D-1 , SC-D-1 , SHC-D-1 , NCA-D-2, and PHC-D-2;

(b) conjugating said dolastatin linker derivative to the antibody recovered from the cell culture of Claim 28, and;

(c) purifying the antibody-drug conjugate.