HK1193052B - Antibody-drug conjugates - Google Patents

Description

Antibody-drug conjugates

Technical Field

The present invention relates generally to anti-5T 4 antibody-drug conjugates for the treatment of cancer.

Background

Antibody-drug conjugates (ADCs) combine the binding specificity of monoclonal antibodies with the efficacy of chemotherapeutic agents. The technology associated with the development of monoclonal antibodies against tumor-associated target molecules, the use of more potent cytotoxic agents, and the design of chemical linkers covalently binding these components has progressed rapidly in recent years (Ducry L., et al. bioconjugate C chemistry,21:5-13,2010).

Very potential ADCs such as SGN-75(US2009/148942) and trastuzumab (trastuzumab) -DM1(US2009/0226465) are currently undergoing clinical trials. However, many challenges remain due to the consideration of other tumor-associated antigens as targets. Each monoclonal antibody must be individually characterized and designed with the appropriate linker and identify the appropriate cytotoxic agent that retains its potency when delivered to tumor cells. The density of antigen on the cancer target and whether normal tissue expresses the target antigen must be considered. Other considerations include whether the entire ADC is internalized when bound to a target, the choice of a cytostatic or cytotoxic agent is preferred in view of possible normal tissue exposure and/or the type and stage of cancer to be treated, and whether the linker linking the antibody to the drug-loaded linker is cleavable or non-cleavable. In addition, the conjugation ratio of the antibody to the drug moiety must be sufficient without compromising the binding activity of the antibody and/or the efficacy of the drug. Clearly, ADCs are complex biologies and the challenge of developing effective ADCs remains high.

The human 5T4 tumor associated antigen is the target antigen of the present invention. Recent data have shown that this 5T4 antigen is expressed in high amounts on certain highly tumorigenic cells, also known as tumor initiating cells (WO 2010/111659). The initiating tumor cells show resistance to standard therapies, which are thought to be responsible for tumor recurrence and metastasis, and therefore represent another obstacle to ADC development.

The novel anti-5T 4 ADCs of the present invention overcome these challenges associated with ADC technology, providing highly specific and potent ADCs that bind to tumor cells expressing the 5T4 antigen and deliver sufficient cytotoxic drugs to the cells, thus providing innovative and effective cancer therapy.

Disclosure of Invention

In one embodiment, the antibody-drug conjugate of the invention has the formula: ab- (LU-D)_pOr a pharmaceutically acceptable salt thereof, wherein Ab is an anti-5T 4 antibody or antigen-binding portion thereof, the anti-5T 4 antibody or antigen-binding portion thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, VH CDR1 region as set forth in SEQ ID NO: 6 and the VH CDR2 region as set forth in SEQ ID NO: 7, LU is a linker unit selected from maleimidocaproyl (maleimidocaproyl) and maleimidocaproyl-valine (Val) -citrulline (Cit) -PABA, p is an integer from about 1 to about 8, and D is a pharmaceutical unit selected from MMAE, MMAF and MMAD.

The invention further provides an anti-5T 4 antibody-drug conjugate, wherein the anti-5T 4 antibody or antigen-binding portion thereof comprises a heavy chain variable region having (a) the amino acid sequence set forth in SEQ ID NO: 5, (b) a VH CDR1 region as set forth in SEQ ID NO: 6, and (c) a VH CDR2 region as set forth in SEQ ID NO: 7, and a VH CDR3 region.

The invention further provides an anti-5T 4 antibody-drug conjugate, wherein the anti-5T 4 antibody or antigen-binding portion thereof comprises a heavy chain variable region having (a) the amino acid sequence set forth in SEQ ID NO: 8, (b) a VL CDR1 region as set forth in SEQ ID NO: 9, and (c) a VL CDR2 region as set forth in SEQ ID NO: 10, a light chain variable region of the VL CDR3 region.

The present invention further provides an anti-5T 4 antibody-drug conjugate, wherein the anti-5T 4 antibody or antigen-binding portion thereof further comprises a heavy chain variable region having (a) the amino acid sequence set forth in SEQ ID NO: 5, (b) a VH CDR1 region as set forth in SEQ ID NO: 6, and (c) a VH CDR2 region as set forth in SEQ ID NO: 7 and the light chain variable region has (a) the VH CDR3 region shown in SEQ ID NO: 8, (b) a VL CDR1 region as set forth in SEQ ID NO: 9, and (c) a VL CDR2 region as set forth in SEQ ID NO: 10, the VL CDR3 region.

The invention further provides an anti-5T 4 antibody-drug conjugate, wherein the anti-5T 4 antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:3 and the VH region of SEQ ID NO: 4, VL region.

The present invention also provides an anti-5T 4 antibody-drug conjugate, wherein the anti-5T 4 antibody is a polypeptide consisting of a sequence having SEQ id no:1 and a light chain having the sequence of SEQ ID NO:2, light chain.

The present invention further provides an anti-5T 4 antibody-drug conjugate, wherein: (a) the anti-5T 4 antibody is a peptide consisting of a peptide having seq id NO:1 and a light chain having the sequence of SEQ ID NO:2, (b) the LU is maleimidocaproyl, (c) the drug is MMAF, and (d) p is an integer of about 4.

The present invention further provides an anti-5T 4 antibody-drug conjugate, wherein: (a) the anti-5T 4 antibody is a peptide consisting of a peptide having seq id NO:1 and a light chain having the sequence of SEQ ID NO:2, (b) the LU is maleimidocaproyl-Val-Cit-PABA, (c) the drug is MMAE, and (d) p is an integer of about 4.

The present invention further provides an anti-5T 4 antibody-drug conjugate, wherein: (a) the anti-5T 4 antibody is a peptide consisting of a peptide having seq id NO:1 and a light chain having the sequence of SEQ ID NO:2, (b) the LU is maleimidocaproyl-Val-Cit-PABA, (c) the drug is MMAD, and (d) p is an integer from about 1 to about 8.

The present invention further provides an anti-5T 4 antibody-drug conjugate, wherein: (a) the anti-5T 4 antibody is a peptide consisting of a peptide having seq id NO:15 and a light chain having the sequence of SEQ ID NO:2, (b) the LU is maleimidocaproyl-Val-Cit-PABA, (c) the drug is MMAE, and (d) p is an integer from about 1 to about 8.

The present invention provides an anti-5T 4 antibody-drug conjugate, wherein the antibody recognizes an epitope on human 5T4 antigen, wherein the epitope comprises SEQ ID NO:11, amino acid residues 173 to 258 and 282 to 361.

The invention provides a pharmaceutical composition, which comprises the antibody-drug conjugate and a pharmaceutically acceptable carrier.

The present invention further provides a method of treating 5T4 positive cancer in a patient in need of such treatment, the method comprising administering to the patient an antibody-drug conjugate as described above.

The present invention further provides a method of treating a 5T 4-positive cancer, wherein the cancer is selected from the group consisting of carcinoma of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, liver, skin, stomach, and testes.

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

The present invention further provides an antibody-drug conjugate as described above for use in therapy.

The invention also provides the application of the antibody-drug conjugate in the preparation of medicines.

The present invention further provides the use as described above, wherein the use is for the treatment of a 5T 4-positive cancer, and wherein the cancer is selected from the group consisting of bladder, breast, cervix, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testes.

More preferably, the present invention further provides the use as described above, wherein the use is for the treatment of 5T4 positive cancer, and wherein the cancer is selected from colorectal cancer, breast cancer, pancreatic cancer and non-small cell lung cancer.

The invention further provides nucleic acids encoding anti-5T 4 antibodies, vectors comprising the nucleic acids, and host cells comprising the vectors.

The present invention also provides a method for producing an anti-5T 4 antibody, the method comprising culturing a host cell comprising the vector described above and recovering the antibody from the cell culture.

The present invention further provides a method of preparing an anti-5T 4 antibody-drug conjugate, the method comprising: (a) taking the antibody recovered from the cell culture, (b) chemically linking the antibody to a pharmaceutical unit selected from MMAE, MMAD and MMAF via a linker unit selected from maleimidocaproyl or maleimidocaproyl-Val-Cit-PABA, and (c) purifying the antibody-drug conjugate.

Detailed Description

The present invention provides anti-5T 4 antibody-drug conjugates for use in the treatment of cancer. In order to understand the present invention more clearly, some terms are first defined.

All amino acid abbreviations used in this disclosure are those accepted by the U.S. patent and trademark office as set forth in 37 c.f.r. § 1.822(B) (I).

"5T 4" refers to the 5T4 tumor fetal antigen, which is a 72kDa highly glycosylated transmembrane glycoprotein that contains a 42kDa non-glycosylated core (see US 5,869,053). Human 5T4 is expressed in a variety of cancer types including carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, liver, skin, stomach, and testes. High tumorigenic cells (also known as cancer stem cells or tumor-initiating cells) have been shown to have high amounts of 5T4 expression (WO 2010/111659). The anti-5T 4 antibodies of the invention include antibodies that specifically bind to the human 5T4 antigen (see US 2007/0231333).

An "antibody" is an immunoglobulin molecule that specifically binds to a target, such as a carbohydrate, polynucleotide, fat, polypeptide, etc., through at least one recognition region located in the variable region of the immunoglobulin molecule. "antibody" as used herein includes 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 antibodies, and any other modified conformation of immunoglobulin molecules comprising an antigen recognition region, including, for example, but not limited to, Fab ', F (ab')₂Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of a single arm of an antibody, isolated Complementarity Determining Regions (CDRs), scFv, single domain antibodies (e.g., shark antibodies (shark antibodies) and camelid antibodies (camelid antibodies)), large antibodies (maxiboidies), miniantibodies (minibodies), antibodies (variants,intracellular antibodies (intrabodies), diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.

Antibodies include any type of antibody, such as IgG, IgA, or IgM (or subtypes thereof), and the antibody need not be of any particular type. Immunoglobulins can be classified into different types according to the antibody amino acid sequence of the constant region of their heavy chains. There are five major immunoglobulin types: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subtypes (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to the different types of immunoglobulins are designated α, γ and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known.

The "variable region" of an antibody refers to either the variable region of an antibody light chain or the variable region of an antibody heavy chain (either alone or in combination). As is known in the art, the variable regions of the heavy and light chains each consist of four Framework Regions (FRs) and three Complementarity Determining Regions (CDRs), also known as hypervariable regions, connecting the four framework regions to form the antigen-binding portion of the antibody. If it is desired to have a variant of the subject variable region, particularly an amino acid residue substitution outside of the CDR (i.e., the framework region), the appropriate amino acid substitution (preferably a conservative amino acid substitution) can be identified by comparing the subject variable region to the variable regions of other antibodies comprising the CDR1 and CDR2 sequences of the canonical class (canonical class) that is identical to the subject variable region (Chothia and Lesk, Jmol Biol 196(4): 901. 917, 1987). When the FRs are selected to flank the subject CDR, e.g., when humanizing or optimizing an antibody, FRs derived from an antibody comprising the same canonical types of CDR1 and CDR2 sequences are preferred.

The "CDRs" of a variable domain are amino acid residues located within the variable region that are identified according to the Kabat (Kabat) definition, the cauchy (Chothia) definition, the cumulative definition of both Kabat and cauchy, the AbM definition, the contact definition, and/or the conformation definition, or any CDR determination method well known in the art. Antibody CDRs may be recognized as hypervariable regions originally defined by kappa et al. See, for example, 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 structural loops originally described by Cauchy et al. See, e.g., Chothiaet al, 1989, Nature 342: 877-883. Other methods of identifying CDRs include "AbM definition" (which is a combination of the kappa and Cauchy methods, derived from the AbM antibody model software using Oxford molecules (Oxford Molecular)) Or "contact definition" of CDRs based on observed antigen contact as described by maccall et al, 1996, j.mol.biol.,262: 732-. In another approach, referred to herein as "conformation definition" of CDRs, the position of the CDRs may be identified as residues that contribute enthalpically to antigen binding. See, e.g., Makabe et al, 2008, journal Biological Chemistry,283: 1156-1166. Other CDR boundary definitions may not strictly follow one of the above methods, but will overlap with at least part of the kappa CDR, although they may be based on specific residues or groups of residues or even the whole CDR does not significantly affect antigen binding prediction or experimental results are shortened or prolonged. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment that includes more than one CDR, the CDR may be defined according to any of the kabat, cauchy, elongation, AbM, contact and/or conformation definitions.

The term "monoclonal antibody" (Mab) refers to an antibody derived from a single cell or cell strain, including, for example, any eukaryotic, prokaryotic, or phage clone, and does not refer to a method of making the same. Preferably, the monoclonal antibodies of the invention are present in a homogeneous or substantially homogeneous population.A white chain or fragment thereof (such as Fv, Fab ', F (ab') 2) or other antigen binding subsequence of an antibody). Preferably, a human beingA humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a recipient-derived Complementarity Determining Region (CDR) are replaced with 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 used to refer to an antibody 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 prepared using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques or combinations thereof, or other techniques known in the art (see, e.g., Jayasena, s.d., clin.chem.,45:1628-50(1999) and Fellouse, f.a., et al, j.mol.biol.,373(4):924-40 (2007)).

Tables 1 and 2 below illustrate preferred CDRs for antibodies of the invention.

TABLE 1

TABLE 2

The invention includes an antibody, or antigen-binding portion thereof, comprising:

a) a light chain variable region comprising:

i) has a sequence selected from SEQ ID NO: LCDR1 of the amino acid sequence of 8 or 17,

ii) has an amino acid sequence selected from SEQ ID NO: 9 or 18, and

iii) has an amino acid sequence selected from SEQ ID NO: 10 or 19, and

b) a heavy chain variable region comprising:

i) has a sequence selected from SEQ ID NO: 5 or 22, HCDR1,

ii) has an amino acid sequence selected from SEQ ID NO: 6 or 23, and

iii) has an amino acid sequence selected from SEQ ID NO: 7 or 24, HCDR 3.

Preferred antibodies or antigen-binding portions thereof of the invention comprise:

a) an LCVR comprising SEQ ID NO: LCDR1 of SEQ ID NO: LCDR2 of 9 and SEQ ID NO: LCDR3 of 10, and

b) an HCVR comprising SEQ ID NO: 5 HCDR1, SEQ ID NO: HCDR2 of SEQ ID NO: HCDR3 of 7.

Preferred monoclonal antibodies of the invention are referred to herein as A1 (humanized anti-5T 4IgG1 antibody), A1-IgG4 (humanized anti-5T 4IgG4 antibody), A3 (mouse/human chimeric antibody), and A3hu (humanized anti-5T 4IgG1 antibody). The SEQ ID NOs encoding the amino acid sequences of monoclonal antibodies a1, a1-IgG4 and A3 are provided in table 3 below:

TABLE 3

Mab	LC	HC	LCVR	LCDR1	LCDR2	LCDR3	HCVR	HCDR1	HCDR2	HCDR3
											A1	2	1	4	8	9	10	3	5	6	7
A1-IgG4	2	12	4	8	9	10	13	5	6	7
											A3	2	15	21	22	23	24	16	17	18	19
A3hu	30	25	31	32	33	34	26	27	28	29

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody specifically binding to an antigen".

By "anti-5T 4 antibody-drug conjugate" is meant an anti-5T 4 antibody or antigen-binding portion thereof linked to a cytotoxic drug moiety (D) via a linker unit molecule (LU) as described herein.

Joint unit (LU): LU describes the direct or indirect linkage between the antibody and the drug. Attachment of the linker to the mAb can be accomplished via a number of means, such as via surface lysines, reductive coupling to oxidized carbohydrates, and reduction of cysteine residues released via interchain disulfide bonds. A variety of ADC ligation systems are known in the art, including hydrazone, disulfide and peptide-based ligation.

Drug (D): a drug is any substance with biological or detectable activity (e.g., therapeutic agents, detectable labels, binding agents, etc.) and prodrugs that are metabolized to an active agent in vivo. The terms "drug" and "drug-loaded" are used interchangeably. In some embodiments, the drug is auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin may be, for example, an ester formed from auristatin E and a keto acid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. patent nos. 6,884,869, 7,098,308, 7,256,257, 7,423,116, 7,498,298 and 7,745,394, each of which is incorporated by reference in its entirety for all purposes.

Auristatins have been shown to interfere with microtubule dynamics and nuclear and cell division and have anti-cancer activity. The auristatins of the invention bind to tubulin and exhibit cytotoxic or cytostatic effects on 5T4 expressing cells or cells. There are several different assays known in the art that can be used to determine whether auristatin or the antibody-drug conjugate formed therefrom exhibits a cytostatic or cytotoxic effect on the desired cell or cells. Methods for determining whether a compound binds to tubulin are known in the art. See, e.g., Muller et al, anal. chem 2006,78, 4390-: 965 + 976, and Hamel et al, The Journal of biological Chemistry,1990265:28,17141 + 17149.

Examples of drugs or drug-carriers are selected from DM1 (maytansine), N2 '-deacetyl-N2' - (3-mercapto-1-oxopropyl) -or N2 '-deacetyl-N2' - (3-mercapto-1-oxopropyl) -maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valinyl-N- [ (1S,2R) -2-methoxy-4- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-3-oxo-3- [ [ (1S) -2-phenyl-1- (2-thiazolyl) ethyl ] amino ] propyl ] -1-methyl-3- [ [ (1S) -2-phenyl-1- (2-thiazolyl) ethyl ] amino ] propyl ] -1 -pyrrolidinyl ] -1- [ (1S) -1-methylpropyl ] -4-oxobutyl ] -N-methyl- (9Cl) -L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl ] -N-methyl-L-valyl- (3R,4S,5S) -3-methoxy-5-methyl-4- (methylamino) heptanoyl- (. alpha.R,. beta.R, 2S) -beta-methoxy-alpha-methyl-2-pyrrolidinopropionyl- L-phenylalanine), or mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-valcic- (p-aminobenzyloxycarbonyl) -monomethylotilin E or N- [ [ [4- [ [ N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl ] -L-valyl-N5- (aminocarbonyl) -L-ornyl ] amino ] phenyl ] methoxy ] carbonyl ] -N-methyl-L-valyl-N- [ (1S,2R) -4- [ (2S) -2- [ (1R,2R) -3- [ [ (1R,2S) -2-hydroxy-1-methyl-2-phenylethyl ] amino ] -1-methoxy-2-methyl-3-oxopropyl ] -1-pyrrolidinyl ] -2-methoxy-1- [ (1S) -1-methylpropyl ] -4-oxobutyl ] -N-methyl-L-valinamide). DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE and MMAF are auristatin derivatives. Preferred loading agents of the invention are selected from mc-MMAF or mc-Val-Cit-PABA-MMAE.

The term "epitope" refers to a portion of a molecule that is recognized and bound by one or more antigen binding regions of an antibody. Epitopes are usually composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term "antigenic epitope" as used herein is defined as a portion of a polypeptide that is specifically bound by an antibody as determined by any method well known in the art, such as conventional immunoassays. A "nonlinear epitope" or "conformational epitope" comprises discrete polypeptides (or amino acids) within an antigenic protein that are bound by an antibody specific for the epitope.

The term "binding affinity (K) as used herein_D) "is intended to refer to the dissociation constant for a particular antigen-antibody interaction. K_DIs the off-rate (k)_off) ") versus binding rate (or" on-rate (k) "_on)”) The ratio of (a) to (b). Thus, K_DIs equal to k_off/k_onAnd expressed in molar concentration (M). From this, it is known that K is_DThe smaller the affinity of the binding is, the stronger. Thus, K_DEqual to 1. mu.M denotes relative to K_DEqual to 1nM has weak binding affinity. K of antibody_DValues can be determined using methods well established in the art. K for determining antibody_DBy using Surface Plasmon Resonance (SPR), generally using methods such asA biosensor system of the system.

The term "specific binding" as used herein relates to the binding between an antibody and 5T4 antigen and the antibody has a K of less than about 30nM as measured by SPR at 25 ℃_DBinds to the 5T4 antigen.

As used herein, "pharmaceutically acceptable salts" refers to pharmaceutically acceptable organic or inorganic salts of molecules or macromolecules.

The term "potency" is a measure of biological activity, which can be IC₅₀Indicates, or inhibits 50% as described in example 3, that 5T4 positive cells are an effective concentration of antibody required for growth. Alternatively, efficacy may refer to anti-tumor activity as measured in an in vivo tumor xenograft model as shown in example 4.

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

Polynucleotides encoding the antibodies of the invention may include the following: only the coding sequence of the variant, the coding sequence of the variant and additional coding sequences such as a functional polypeptide or signal or secretory sequence or a proprotein sequence, the coding sequence of the antibody and non-coding sequences such as introns or 5 'and/or 3' non-coding sequences of the coding sequence of the antibody. The term "antibody-encoding polynucleotide" is intended to include additional variantsThe coding sequence of (a), but also includes additional coding and/or non-coding sequences of the polynucleotide. It is known in the art that polynucleotide sequences optimized for a particular host cell/expression system can be readily derived from the amino acid sequence of the desired protein (see, e.g., SEQ ID NO: S)AG, riegen burg, Germany).

Polynucleotides encoding the antibodies of the invention will generally include expression control polynucleotide sequences operably linked to the antibody coding sequence, including naturally-associated or heterologous promoter regions known in the art. Preferably, the expression control sequence will be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Once the vector is incorporated into an appropriate host cell line, the host cell is propagated under conditions suitable for expression of the nucleotide sequence, and the antibody is collected and purified, as desired. Preferred eukaryotic cells include CHO cell lines, various COS cell lines, Hela (HeLa) cells, myeloma cell lines, transformed B cells, or human embryonic kidney cell lines. The most preferred host cell line is the CHO cell line.

The invention encompasses antibodies, or antigen-binding portions thereof, that bind to a particular epitope on the 5T4 antigen. The recognized epitope is a nonlinear or conformational epitope comprising a first contact between amino acid residues 173 and 252 and a second contact between amino acid residues 276 and 355 of the human 5T4 antigen (SEQ ID NO: 11) (see example 7). Thus, the CDRs and the variable regions of the heavy and light chains described herein were used to prepare full-length antibodies as well as functional fragments and analogs that maintain the binding affinity of the protein using CDRs specific for the above-described epitope of 5T4 antigen.

The binding affinity of the antibody of the present invention was measured by SPR (example 6). In these experiments, the 5T4 antigen was immobilized at low densityOn the chip, the antibody flows through the chip. The accumulation of material on the chip surface is measured. This assay allows for the real-time determination of both the on-rate and off-rate to obtain the affinity (K) of the binding_D). The humanized antibodies of the invention have a KD of between about 0.30 to about 30nM, about 0.30 to about 20nM, about 0.30 to about 10nM, about 0.5 to about 7nM, about 1.0 to about 5nM, and about 1.0 to about 3 nM.

Conjugation of drugs to antibodies

The drug has been or is modified to include a group reactive with the conjugation site on the antibody. For example, drugs can be attached by alkylation (e.g., the-aminolysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification of hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation with thiols. In some embodiments, the number p of drug moieties conjugated to each antibody molecule is an average of 1 to 8,1 to 7,1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an average value of 2 to 8,2 to 7,2 to 6,2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6,7, or 8. In some embodiments, p is an average value 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 is between 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. See, for example, Current protocols Protein Science (John Wiley & Sons, Inc.), Chapter 15 (chemical modification of proteins) (the disclosure of which is incorporated herein by reference in its entirety).

For example, when chemically activating a protein results in the formation of free thiol groups, the protein may be conjugated using a thiol reactant. In one aspect, the reagent is a reagent that is substantially specific for free thiol groups. Such agents include, for example, maleimide, haloacetamides (e.g., iodoacetamide, bromoacetamide, or chloroacetamide), haloesters (e.g., iodoester, bromoester, or chloroester), halomethyl ketones (e.g., iodomethyl ketone, bromomethyl ketone, or chloromethyl ketone), benzyl halides (e.g., benzyl iodide, benzyl bromide, or benzyl chloride), vinyl sulfones, and pyridyl sulfides.

Joint

The drug may be linked to the antibody via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the linker may be a double peptide linker, such as a valine-citrulline (Val-Cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproyl-valine-citrulline-p-aminomethoxycarbonyl (mc-Val-Cit-PABA) linker. The other linker was sulfosuccinimidyl-4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimido group which reacts with a thiol group (thiol group, -SH), while its sulfo-NHS ester is reactive towards a primary amine (as seen in lysine and the N-terminus of the protein or peptide). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that can be hydrolyzed at a particular pH or pH range, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. The linker may be covalently linked to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released, e.g., mc linkers and the like.

The linker may comprise a group for attachment to the antibody. For example, the linker can include an amino, hydroxyl, carboxyl, or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodoacetamide, bromoacetamide, or chloroacetamide), a haloester (e.g., iodoester, bromoester, or chloro ester), a halomethyl ketone (e.g., iodomethyl ketone, bromomethyl ketone, or chloromethyl ketone), a benzyl halide (e.g., benzyl iodide, benzyl bromide, or benzyl chloride), a vinyl sulfone, and a pyridyl sulfide). See Wong, Chemistry of Protein Conjugation and Cross-linking; CRC Press, Inc., Boca Raton, 1991.

Immunotherapy

For immunotherapy, the antibody may be conjugated to a suitable drug, such as a cytotoxic agent, a cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like. The conjugates can be used to inhibit proliferation of tumor or cancer cells, cause apoptosis of tumor or cancer cells, or to treat cancer in a patient. The conjugates can accordingly be used in different conditions to treat cancer in an animal. The conjugates can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds or associates with a cancer cell or tumor-associated antigen, and the conjugate and/or drug can enter the tumor cell or cancer cell via receptor-mediated pinocytosis. The antigen may be attached to a tumor cell or cancer cell, or may 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 the linker) are hydrolytically cleaved by one or more tumor cell or cancer cell associated proteases, resulting in the release of the drug. The released drug is then free to move within the cell to induce cytotoxicity or cytostatic or other activity. In some embodiments, the drug is cleaved from the antibody outside of the tumor or cancer cell, and the drug subsequently enters the cell or acts on the cell surface.

Cancer treatment

As described above, cancers (including but not limited to tumors, metastases, or other diseases or conditions characterized by uncontrolled cell growth) can be treated or prevented by administering the protein-drug conjugates.

In other embodiments, the invention provides methods of treating or preventing cancer comprising administering to a patient in need of treatment or prevention of cancer an effective amount of a conjugate and a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is one with which no unreactivity has been found for treating cancer. In some embodiments, the chemotherapeutic agent refers to an agent with which cancer has been found to be unreactive. The conjugate can be administered to a patient who is also receiving treatment, such as surgery for the treatment of cancer. In another embodiment, the additional treatment is radiation therapy.

Multi-drug therapy for cancer

A method of treating cancer comprises administering to a patient in need of treatment an effective amount of an antibody-drug conjugate and another therapeutic agent that is an anti-cancer agent. Suitable anti-cancer agents include, but are not limited to, methotrexate (methotrexate), paclitaxel (taxol), L-asparaginase, mercaptopurine (mertepurine), thioguanine, hydroxyurea, cytarabine (cytarabine), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), nitrosourea, cisplatin (cissplatin), carboplatin (carboplatin), mitomycin, dacarbazine (dacarbazine), procarbazine (procarbazine), topotecan (topotecan), mechlorethamine, cyclophosphamide (cytoxan), etoposide (etoposide), 5-fluorouracil (5-fluorouracil), BCNU, irinotecan (irinotecan), camptothecin (camptothecin), bleomycin (bleomycin), doxorubicin (doxin), idarubicin (birubicin), vincristine (vincristine), vincristine (vincristine), vincristine (vincrist, Vinorelbine (vinorelbine), paclitaxel (paclitaxel), calicheamicin (calicheamicin), and docetaxel (docetaxel).

The ADCs of the present invention may be in the form of a pharmaceutical composition for administration which is formulated to be suitable for the selected mode of administration, together with pharmaceutically acceptable diluents or excipients such as buffers, surfactants, preservatives, co-solvents, isotonicity agents, stabilisers, carriers and the like. Remington's pharmaceutical sciences, Mack Publishing Co., Easton Pa.,18th ed.,1995, incorporated herein by reference, provides a summary of modulation techniques generally well known in the art.

These pharmaceutical compositions may be administered by any method known in the art to achieve the general purpose of treating cancer. Preferred routes of administration are non-oral, defined herein as modes of administration including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion. The dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the effect desired.

Compositions within the scope of the present invention include all compositions wherein the ADC is present in an amount effective to achieve the desired medical effect of treating cancer. Although individual requirements may vary from patient to patient, the ideal range for determining the effective amount of all of the ingredients is within the ability of a skilled clinician.

Example 1

Preparation of anti-5T 4ADC

5T4-A1 Antibody Drug Conjugates (ADCs) were prepared by partial reduction of the mAb with tris (2-carboxyethyl) phosphine (TCEP), followed by reaction of the reduced Cys residue with the desired maleimide end linker-loading drug. Specifically, 5T4-A1mAb was partially reduced by addition of 2.8 molar excess of tris (2-carboxyethyl) phosphine (TCEP) and 1mM diethylenetriaminepentaacetic acid (DTPA) at 37 ℃ for 2 hours in 100mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid buffer) pH 7.0. The desired linker-loading drug was then added to the reaction mixture at a molar ratio of linker-loading drug/mAb-thiol of 5.5 (maleimidocaproyl-monomethylauristatin F [ mc-MMAF ]) or 8 (maleimidocaproyl-valine-citrulline-p-aminomethoxycarbonyl-monomethylauristatin E [ mc-Val-Cit-PABA-MMAE ]), and reacted in the presence of 15% by volume of Dimethylacetamide (DMA) at 25 ℃ for a further 1 hour. After an incubation period of 1 hour, N-ethylmaleimide (4.5-fold excess over mc-MMAF and 2-fold excess over mc-Val-Cit-PABA-MMAE) was added to cap the unreacted thiol groups, allowed to react for 15 minutes, followed by addition of 6-fold excess L-Cys to quench any unreacted linker-loaded drug. The reaction mixture was dialyzed overnight at 4 ℃ in Phosphate Buffered Saline (PBS) pH 7.4 and purified by SEC (AKTA explorer protein purifier, Superdex 20010/30GL column). The ADC was then analyzed for purity using Size Exclusion Chromatography (SEC), load was calculated with Hydrophobic Interaction Chromatography (HIC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS), and concentration was determined via UV spectrophotometer.

Example 2

Binding test

Cells expressing the 5T4 antigen and negative control Raji cells were seeded at a density of 500,000 cells/well into non-tissue culture treated 96-well plates and stored on ice. Dilutions of A1 and A1-IgG4 antibody or A1-mcMAMAF ADC were prepared in Dulbecco's Phosphate Buffered Saline (DPBS) with 3% bovine serum albumin BSA and added to the plates to a final concentration of 10. mu.g/mL. The well plates were then incubated on ice for 1 hour and then washed 2 times. Secondary antibody (goat anti-human IgG Fc conjugated to PE (phycoerythrin)) was added to the wells. After incubation at 4 ℃ for 30 minutes, the average fluorescence intensity was measured using a flow cytometer.

The data in table 4 show that the a1 antibody binds to a variety of 5T4 positive cells. The data in table 5 show that a1 and a1-IgG4 antibodies and a1-mcMMAF ADC express similar binding to a variety of different cells.

TABLE 4

TABLE 5

Example 3

Cytotoxicity assays

Cells expressing 5T4 and negative control Raji cells were cultured with increasing concentrations of ADC.

After four days, each culture was examined for viability. IC (integrated circuit)₅₀Values were calculated by logistic nonlinear regression and expressed as ng Ab/mL. A1-mcMAF, A1-vcMAE, A3-mcMAF and A3-mcMAE were shown to inhibit the growth of 5T4 expressing cells (MDAMB435/5T4, MDAMB468 and MDAMB361DYT2), but were non-responsive to 5T4 negative cells (Raji) (Table 6).

TABLE 6

In addition, 5T4+ primary lung tumor 37622a cells were isolated and grown in culture. Cells were incubated with increasing concentrations of ADC. After ten days, each culture was tested for viability using the MTS method. IC (integrated circuit)₅₀Values were calculated by logistic nonlinear regression and expressed as ng Ab/ml. A1-mcMAF, A1-vcMAE, A3-mcMAF, and A3-vcMAE inhibited primary lung tumor cell growth (Table 7).

TABLE 7

Example 4

Subcutaneous xenograft model

Athymic (nude) dams (or another immunosuppressed mouse strain) are injected subcutaneously with MDAMB435/5T4, MDAMB361DYT2, or H1975 tumor cells. Mice with staged tumors of approximately 0.1 to 0.3 grams (n ═ 6 to 10 mice/treatment group) were administered intravenously Q4Dx4 saline (vehicle), a1-mcMMAF, a1-vcMMAE, a1-mcMMAD, a1-smccDM1, A3-mcMMAF, A3-vcMMAE, or a non-binding control antibody conjugated to mcMMAF or vcMMAE at a dose of 3 mgAb/kg. All ADCs were administered according to Ab content. Tumor size (mm) was measured at least once a week²± SEM) is calculated from the formula: mm is²0.5x (tumor width)²) x (tumor length).

The data in Table 8 show that A1-mcMAF, A1-vcMAE, A1-vcMAD, A3-mcMAF, and A3-vcMAE inhibit the growth of MDAMB435/5T4 xenografts, whereas A1-mcMAD and A1-smccDM1 are not active in this mode.

The data in Table 9 show that A1-mcMAF, A1-vcMAE, A1-vcMAD, A1-smccDM1, A3-mcMAF, and A3-vcMAE inhibit the growth of MDAMB361DYT2 xenografts, whereas A1-mcMAD is inactive in this mode.

The data in Table 10 show that A1-mcMAF, A1-vcMAE, A1-vcMAD, A3-mcMAF, and A3-vcMAE inhibit the growth of H1975 xenografts, whereas A1-mcMAD and A1-smccDM1 are not active in this mode.

TABLE 8

GT is the group that terminated the test due to large tumor size

TABLE 9

GT is the group that terminated the test due to large tumor size

Watch 10

GT is the group that terminated the test due to large tumor size

Alternatively, nude mice with subcutaneously established primary tumor cell xenografts of 37622a were treated with a dose of 3mg Ab/kg of a1-mcMMAF, a1-mcMMAD, a1-vcMMAD or A3-mcMMAF by intravenous injection of Q4Dx4, and tumor growth was monitored over a 96 day period. Table 11 shows that a1-mcMMAF, a1-vcMMAD, and A3-mcMMAF inhibited the growth of 37622a primary tumor xenografts relative to vehicle control treated animals, whereas a1-mcMMAD was not active in this model.

TABLE 11

GT is the group that terminated the test due to large tumor size

Unexpectedly, the data in tables 8 to 11 show that ADCs with the same antibody and drug loading but different linkers have different therapeutic profiles, i.e., a1-mcMMAD and a1-vcMMAD in all four xenograft models. In addition, the data show that ADCs with the same antibody and linker but with different drug loadings also had different therapeutic profiles, i.e., a1-mcMMAF and a1-mcMMAD in all four xenograft models. Thus, the drug MMAD was effective in all four xenograft models when linked to the a1 antibody via a vc linker, but was not active in any of the xenograft models tested when linked via a mc linker. In contrast, the drug MMAF was highly effective in all four xenograft models when linked to the a1 antibody via the mc linker, whereas the chemically related drug MMAD was not active in all four xenograft models when linked to the same antibody via the same linker.

Yet another unexpected observation appeared in ADC a1-smccDM1 (tables 8 to 10). This ADC was very effective for MDAMB361DYT2 xenografts, but was not substantially effective for MDAMB435/5T4 and H1975 xenografts, even though all xenografts highly expressed the 5T4 target antigen. This data shows that the efficacy of the linker-loaded drug cannot be predicted even with the same high affinity antibody or even with the same ADC.

Example 5

Antibody-dependent cell-mediated cytotoxicity (ADCC)

ADCC assay:

blood from healthy volunteers was collected into BD Vacutainer CPT cell preparation tube containing heparin sodium peripheral blood mononuclear spheres (PBMC) were resuspended in detection buffer (RPMI 1640 supplemented with 10mM HEPES) to 2.5 × 10⁷Target cells (MDAMB435/5T4 or MDAMB435/neo) at a density of 1 × 10⁴Cells/well seeded in 96-well test plates A1 antibody or A1-mcMAF was added followed by human PBMC effector cells (5 × 10)⁵) Into the well so that the effect: the target cell ratio (E: T) was 50: 1. the test plate was incubated at 37 ℃ for 4 hours to test ADCC activity. The discs were harvested by adding an equal volume of CytoTox-One reagent (Promega) to the discs. A stop solution (Promega; 50ul) was added to each well and the fluorescence intensity was measured to quantify the release of lactate dehydrogenase. For the positive control, 2 μ l of lysis buffer was added per well to produce maximum LDH release (100% cytotoxicity) in the control well wells. Percent cytotoxicity was calculated using the formula:

when "experimental" corresponds to a signal measured under one of these experimental conditions, "effector spontaneity" corresponds to a signal measured in the presence of PBMC only, "target spontaneity" corresponds to a signal measured in the presence of target cells only, and "target maximum" corresponds to a signal measured in the presence of target cells only lysed by detergent.

ADCC activities of A1-IgG1Ab and A1-mcMAF relative to A1-IgG4Ab are shown in Table 12. Both the a1 antibody and a1-mcMMAF showed comparable ADCC activity, indicating that ADCC activity of a1-mcMMAF may promote antitumor activity.

TABLE 12

Compound (I)	Cytotoxicity%
		A1-IgG1	37±8
A1-mcMMAF	34±1
		A1-IgG4	9±5

Example 6

Binding affinity

Surface Plasmon Resonance (SPR) analysis is performed usingThis was performed to determine the affinity constants for binding of A1-IgG1 and A1-IgG4 to human or Malayan (cynomolgus)5T4 at pH6.0 and pH 7.4.The technique exploits the change in refractive index of the surface layer when the huA1 antibody variant binds to human 5T4 protein immobilized on the surface layer. In combination with detection by SPR, i.e., detection of laser light refracted from the surface. Analysis of signal kinetics such as association and dissociation rates allows discrimination between non-specific and specific interactions. The 5T4 protein used in this assay consisted of a human or macaque (cynomolgus)5T4 extracellular domain fused to a human IgG1-Fc domain and was immobilized at low density (45.1 and 45.4RU for human and macaque, respectively) on a CM5 chip to measure the affinity constant correctly.

The measurement of specific binding to the 5T4 ectodomain was obtained by subtracting the binding to a reference surface, which was immobilized on a CM5 chip with only human IgG1-Fc protein at the same density as immobilized on the 5T4-Fc surface. Subsequently, different concentrations of A1, A1-IgG4 or A3 antibody in HBS-EP pH 7.4 or MES-EP pH6.0 buffer were injected onto the surface. The surface was regenerated twice between injection cycles with glycine pH 1.7+ 0.05% surfactant P20(GE healthcare group, BR-1000-54).

The results show that a1 has a slightly higher affinity for human 5T4 than a1-IgG4 at pH6.0 and pH 7.4 using a low density 5T4 surface (1.5-fold and 1.2-fold, respectively, table 13). In addition, a1 exhibited slightly better binding to marmoset 5T4 than a1-IgG4 at both pH6.0 and pH 7.4 (1.7-fold and 1.2-fold, respectively), and both a1 and a1-IgG4 bound human 5T4 3-to 4-fold more than marmoset 5T4 (table 12).

Watch 13

Antibodies	Antigens	pH	ka(1/Ms)	kd(1/s)	KD(nM)
						A1-IgG1	hu5T4	6.0	4.31E+05	4.59E-04	1.06
A1-IgG4	hu5T4	6.0	6.26E+05	8.93E-04	1.43
						A1-IgG1	cyno5T4	6.0	2.33E+05	6.41E-04	2.76
A1-IgG4	cyno5T4	6.0	2.02E+05	9.50E-04	4.70
						A1-IgG1	hu5T4	7.4	2.75E+05	1.32E-04	0.48
A1-IgG4	hu5T4	7.4	3.28E+05	1.72E-04	0.52
						A1-IgG1	cyno5T4	7.4	1.51E+05	2.73E-04	1.80
A1-IgG4	cyno5T4	7.4	1.81E+05	3.82E-04	2.11

Comparing the a1 and A3 antibodies, it is clear that the binding of the a1 antibody to human and marumand 5T4 was better at pH 7.4 than at pH6.0, whereas the A3 antibody exhibited improved binding at pH6.0 relative to pH 7.4 (table 14).

TABLE 14

Antibodies	Antigens	pH	ka(1/Ms)on	kd(1/s)off	KD(nM)
						A1	hu5T4	6.0	4.31E+05	4.59E-04	1.06
A3	hu5T4	6.0	3.51E+05	4.17E-05	0.12
						A1	cyno5T4	6.0	2.33E+05	6.41E-04	2.76
A3	cyno5T4	6.0	4.58E+05	1.87E-04	0.41
						A1	hu5T4	7.4	2.75E+05	1.32E-04	0.48
A3	hu5T4	7.4	1.79E+05	3.06E-05	0.17
						A1	cyno5T4	7.4	1.51E+05	2.73E-04	1.80
A3	cyno5T4	1.98E+05	1.62E-04	0.82	1.98E+05

Example 7

Epitope mapping using 5T4 chimeras

To recognize the epitope bound by the a1 and A3 antibodies, an enzyme-linked immunosorbent assay (ELISA) was performed using (1) the 5T4 extracellular domain Fc construct and (2) the human/mouse 5T4 chimeric construct transiently expressed in COS-1 cells. The extracellular domain comprises an amino-terminal region, two leucine-rich repeats, and a hydrophilic region therebetween. The mouse and rat 5T4 ectodomain comprises a 6 amino acid direct repeat within its hydrophilic region.

Fusion proteins comprising the extracellular domain of 5T4 and an Fc constant region derived from human IgG1 were prepared using human 5T4 (amino acids 1-355), mouse 5T4 (amino acids 1-361), rat 5T4 (amino acids 1-361), marmoset 5T4 (amino acids 1-355), chimpanzee 5T4 (amino acids 1-355), and marmoset 5T4 (amino acids 1-355). The results of binding to the human/mouse 5T4 chimeric construct are listed in table 15, which show specific binding, partial binding, or lack of binding of the a1 and A3 antibodies.

Table 15 refers to the binding capacity of the antibodies to different human/mouse chimeras, the name given by the content of mouse 5T 4. When no binding was observed, this indicates that this is where the antibody binds to human 5T4, as these antibodies do not bind to mouse 5T 4. For example, the A3 antibody has the most N-terminal binding epitope (between 83-163) and as a result shows lack of binding to the 5T4 chimera of residues 83-163 substituted with mouse 5T4, and thus A3 cannot bind. From these results, it was found that the humanized a1 antibody had a first contact between human 5T4 at amino acid residues 173 and 252 and a second contact between human 5T4 at amino acid residues 276 and 355. The a3 antibody binds to the first leucine rich repeat region of human 5T4 between amino acid residues 83 to 163. The number of amino acid residues corresponds to SEQ ID NO:11, human 5T4 antigen amino acid sequence.

Watch 15

Example 8

Comparison of A1-mcmMAF ADC with A1-IgG4-CM ADC

Compare the safety and efficacy of A1-mcMAF with A1-IgG4-AcBut calicheamicin (calicheamicin) (A1-IGG 4-CM). A1-4-CM consisted of an A1-IgG4 antibody conjugated to a calicheamicin-loaded drug with the linker AcBut [ - (4' acetylphenoxy) butanoic acid ]. Calicheamicin is a potent antineoplastic agent which is an enediyne antibiotic derived from the bacterium Micromonospora echinospora (Micromonospora echinospora).

The cell binding activities of A1Ab, A1-IgG4Ab, A1-mcMAF ADC and A1-IgG4-CM ADC were compared using several 5T4 positive cells (see example 2, Table 5). The data show that similar binding was observed at the a1 antibody, the a1-IgG4 antibody, and the a1-mcMMAF ADC, all of which had higher mean fluorescence intensity relative to a1-IgG4-CM for all 5T4 positive cells tested.

A1-mcMAF and A1-IgG4-CM were tested in parallel in MDAMB435/5T4 subcutaneous xenograft mode. Both ADCs reached approximately 200mm in tumor volume²Administered intravenously (Q4dx 2). The antitumor activity of A1-IgG4-CM at a dose of 3mg/kg was similar to that of A1-mcMAF administered at a dose of 10mg/kg (Table 16). According to these results, the antitumor activity of A1-IgG4-CM was approximately 3.3 times higher than that of A1-mcmMAF.

TABLE 16

It is expected that the 3.3-fold higher potency of a1-IgG4-CM relative to a1-mcMMAF would be read as a 3.3-fold higher safety range of a1-mcMMAF in animal toxicity tests than that of a1-IgG 4-CM. However, when reviewing the safety of A1-IgG4-CM in Malay monkeys (cynomolgus macque), it was found that A1-IgG4-CM was at least 100-fold more toxic than A1-mCMMAF in Malay monkeys. Toxicity was observed at each dose when a1-IgG4-CM was administered to male (n-3) and female (n-3) mares at 0.032, 0.095 and 0.32mg Ab/kg/cycle (2, 6, 20 μ g calicheamicin/kg/cycle). After 2 cycles (2 doses) of administration, 4 of 6 animals in the 0.095 treatment group were euthanized or found dead. In contrast, in the A1-mcMAF group (247. mu.g mcMAF/kg/cycle) at doses up to 10mg/kg, no mortality was observed during the same 4 cycles after 2 cycles (2 doses). To summarize, a 1-mcmaf was safe in the 10mg/kg dose group when both were administered to the mare twice during the 4-week observation period, whereas a1-IgG4-CM was considered toxic in the 0.096mg/kg dose group.

Unexpectedly, these results show that the safety range of a1-mcMMAF is 105 times (10/0.095 ═ 105) that of a1-IgG4-CM, rather than the 3.3 times safety range expected from the relative antitumor potency of each ADC. This data shows that antibody-drug conjugates using antibodies directed against the same antigen target but conjugated with different loading drugs have unpredictable properties.

Example 9

A1-mcMMAF mouse PK/PD patterns and clinical dose prediction

The PK/PD model has been used in mouse xenograft experiments to quantify the tumor response of a1-mcMMAF to determine effective concentrations on different cells. The PK/PD model for tumor killing by compartment (transit component) as used herein was previously described by Simeoni et al (Simeoni et al, Cancer Res,64:1094, (2004)). This model has been modified to apply to linear, exponential and sigmoidal growth of tumors and saturation killing of drugs. The PK/PD pattern parameters include:

kg ex exponential growth

Growth of kg S-shaped curve

w0 initial tumor volume

Rate of tau transduction

k_maxMaximum kill rate

kC₅₀Concentration of half maximal kill rate

The PK/PD pattern results were used to calculate tumor resting concentration (TSC, equation 1). This is the drug concentration when the tumor growth rate is equal to the tumor death rate and the tumor volume remains unchanged. TSC may be defined as the minimum concentration required for therapeutic efficacy. TSC is a reference for the clinical dosage selection given at concentrations greater than (>) TSC for clinical efficacy.

Mouse PK was determined in a separate experiment as A1-mcMAF (3mg/kg IV, athymic nu/nu mother). Mouse xenograft experiments were performed using 3 different 5T4 cells administered at doses ranging from 1 to 30mg/kg a1-mcMMAF every four days: cell line MDAMB435/5T4 (doses 1, 3, 10 and 30mg/kg), cell line H1975 (doses 1, 3 and 10mg/kg) and cell line 37622A (doses 1 and 10 mg/kg). The PK/PD patterns were performed as follows, with the TSC shown in Table 17.

The mouse PK/PD parameters for each xenograft cell line were combined with the expected human PK of a1-mcMMAF to mimic the dose required to achieve clinical efficacy. Using this approach, a1-mcMMAF had the expected minimum effective clinical dose of about 0.22 to about 2.3mg/kg Q3 weeks [ every three weeks ] (table 17).

In embodiments of the invention, the dosage range may be from about 0.18mg/kg to about 2.7mg/kg, about 0.22mg/kg to about 2.6mg/kg, about 0.27mg/kg to about 2.5mg/kg, about 0.32mg/kg to about 2.3mg/kg, about 0.37mg/kg to about 2.15mg/kg, about 0.42mg/kg to about 2.10mg/kg, about 0.47mg/kg to about 2.05mg/kg, about 0.52mg/kg to about 2.00mg/kg, about 0.57mg/kg to about 1.95mg/kg, about 0.62mg/kg to about 1.90mg/kg, about 0.67mg/kg to about 1.85mg/kg, about 0.72mg/kg to about 1.80mg/kg, about 0.82mg/kg to about 1.70mg/kg, about 0.67mg/kg to about 1.02mg/kg, about 1.50mg/kg, From about 1.12mg/kg to about 1.40mg/kg, or from about 1.20mg/kg to about 1.30 mg/kg. Preferably, the dosage range may be from about 0.22mg/kg to about 2.3 mg/kg.

Equation 1

1.1

If it is

1.2

If it is

TABLE 17

1 humanized A1 human IgG1 heavy chain of SEQ ID NO

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

2 humanized A1 human kappa light chain

DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:3 A1-VH

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSS

SEQ ID NO:4 A1-VL

DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIK

SEQ ID NO:5 A1-HC CDR1

NFGMN

SEQ ID NO:6 A1-HC CDR2

WINTNTGEPRYAEEFKG

SEQ ID NO:7 A1-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

11 human 5T4 antigen

MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLGIVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYHYRYEINADPRLTNLSSNSDV

12 humanized A1 human IgG4m heavy chain of SEQ ID NO

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO 13A 1 human IgG4m VH (A1-IGG4-VH)

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSS

SEQ ID NO:14A1-IgG4-VH-CDR1

NFGMN

15 chimeric A3 heavy chain (muA3-huIgG1) SEQ ID NO

EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

16 chimeric A3 VH

EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSS

17 chimeric A3 VH-CDR1 SEQ ID NO

TYAMN

18 chimeric A3 VH-CDR2 SEQ ID NO

RIRSKSNNYATYYADSVKD

19 chimeric A3 VH-CDR3 of SEQ ID NO

QWDYDVRAMNY

20 chimeric A3 light chain (muA3-huKappa) SEQ ID NO

DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSGSSTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

21 chimeric A3 VL of SEQ ID NO

DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQGTKLEIK

22 chimeric A3 VL-CDR1 of SEQ ID NO

KASQDVDTAVA

23 chimeric A3 VL-CDR2 of SEQ ID NO

WASTRLT

24 chimeric A3 VL-CDR3 of SEQ ID NO

QQYSSYPYT

25 humanized A3 human IgG1 heavy chain of SEQ ID NO

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

26 humanized A3 VH of SEQ ID NO

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSS

27 humanized A3 VH-CDR1

TYAMN

28 humanized A3 VH-CDR2

RIRSKSNNYATYYADSVKD

29 humanized A3 VH-CDR3

QWDYDVRAMNY

30 humanized A3 human kappa light chain

DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

31 humanized A3 VL of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIK

32 humanized A3 VL-CDR1 of SEQ ID NO

KASQDVDTAVA

33 humanized A3 VL-CDR2 of SEQ ID NO

WASTRLT

34 humanized A3 VL-CDR3 of SEQ ID NO

QQYSSYPYT

Claims (12)

1. An antibody-drug conjugate of the formula:

Ab-(LU-D)_p

or a pharmaceutically acceptable salt thereof, wherein

(a) Ab is an anti-5T 4 antibody, or antigen-binding portion thereof, comprising:

(i) as shown in SEQ ID NO: the VH CDR1 region shown in FIG. 5,

(ii) as shown in SEQ ID NO: the VH CDR2 region shown in FIG. 6,

(iii) as shown in SEQ ID NO: the VH CDR3 region shown in FIG. 7,

(iv) as shown in SEQ ID NO: the VL CDR1 region shown in FIG. 8,

(v) as shown in SEQ ID NO: 9, and the VL CDR2 region, and

(vi) as shown in SEQ ID NO: 10, the VL CDR3 region shown in FIG. 10,

(b) LU is a linker unit selected from maleimidocaproyl and maleimidocaproyl-Val-Cit-PABA,

(c) p is an integer of 1 to 8, and

(d) d is a drug unit selected from MMAE and MMAF.

2. The antibody-drug conjugate of claim 1, wherein the anti-5T 4 antibody or antigen-binding portion thereof comprises the amino acid sequence of seq id NO:3 and the VH region of SEQ ID NO: 4, VL region.

3. The antibody-drug conjugate of claim 1, wherein the anti-5T 4 antibody or antigen-binding portion thereof consists of a heavy chain variable region having the sequence of seq id NO:1 and a light chain having the sequence of SEQ ID NO:2, light chain.

4. The antibody-drug conjugate of claim 1, wherein the antibody or antigen binding portion thereof recognizes an epitope on the human 5T4 antigen, wherein the epitope is located on the amino acid sequence of SEQ ID NO:11, at amino acid residues 173 to 258 and 282 to 361.

5. The antibody-drug conjugate of claim 1, wherein the LU is maleimidocaproyl.

6. The antibody-drug conjugate of claim 1, wherein the drug is MMAF.

7. The antibody-drug conjugate of claim 1, wherein p is an integer from 1 to 4.

8. The antibody-drug conjugate of claim 1, wherein:

(a) the anti-5T 4 antibody consists of a heavy chain variable region having SEQ ID NO:1 and a light chain having the sequence of SEQ ID NO:2, and (b) a light chain component of,

(b) the LU is maleic imidocaproyl,

(c) the drug is MMAF, and

(d) p is an integer from 1 to 8.

9. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. An antibody-drug conjugate according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for use in therapy.

11. Use of an antibody-drug conjugate of any one of claims 1 to 8 in the manufacture of a medicament for the treatment of a 5T4 positive cancer.

12. The antibody-drug conjugate for use according to claim 11, wherein the 5T4 positive cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer.