Patent Application
20170326249
Incorporated herein by reference in its entirety is a Sequence Listing named “20170322_SEQT_12526USNP_YC.txt,” comprising SEQ ID NO:1 through SEQ ID NO:28, which include nucleic acid and/or amino acid sequences disclosed herein. The Sequence Listing has been submitted herewith in ASCII text format via EFS-Web, and thus constitutes both the paper and computer readable form thereof. The Sequence Listing was first created using Patent In 3.5 on Mar. 22, 2017, and is approximately 32 KB in size.
This disclosure relates to an antibody-drug conjugate of an anti-glypican-3 antibody and a tubulysin analog, and its preparation and uses.
Antibody-drug conjugates (ADCs, also referred to as immunoconjugates) are anti-cancer agents that are generating intense current interest. In an ADC, a therapeutic agent (also referred to as the drug, warhead, or payload) is covalently linked, or conjugated, to an antibody whose antigen is expressed by a cancer cell. The antibody, through its binding to the antigen, directs the ADC to the cancer—that is, the antibody acts as a targeting agent specifically delivering the ADC to the cancer cell. Once there, cleavage of the covalent link (referred to as the linker) or degradation of the antibody results in the release of the therapeutic agent at the cancer site. Conversely, while the ADC is circulating in the blood system, the therapeutic agent is held inactive because of its covalent linkage to the antibody. Thus, the therapeutic agent in an ADC can be much more potent (i.e., cytotoxic) than ordinary chemotherapy agents because of its localized release. Recently, two ADCs have received marketing approval: ADCETRIS™, in which an anti-CD30 antibody is conjugated to an auristatin, and KADCYLA™, in which the anti-Her2 antibody trastuzumab is conjugated to a maytansinoid. For a review on the mode of action of ADCs, see Schrama et al. 2006. (The full bibliographic citation for this and other documents cited herein by first author or inventor and year are listed at the end of this specification.)
Glypican-3 is an oncofetal antigen that belongs to the glypican family of glycosyl-phosphatidylinositol-anchored heparin sulfate proteoglycans. Glypicans are characterized by a covalent linkage to complex polysaccharide chains called heparinsulphate glycosaminoglycans. Glypicans are involved in cell signaling at the cellular-extracellular matrix interface (Sasisekharan et al. 2002). To date, six distinct members of the human glypican family have been identified. Cell membrane-bound glypican-3 is composed of two subunits, linked by one or more disulfide bonds.
Glypican-3 is expressed in fetal liver and placenta during development and is down-regulated or silenced in normal adult tissues. Mutations and depletions in the glypican-3 gene are responsible for the Simpson-Golabi-Behmel or Simpson dysmorphia syndrome in humans. Glypican-3 is expressed in various cancers and, in particular, hepatocellular carcinoma (HCC, the most common form of liver cancer), melanoma, Wilm's tumor, and hepatoblastoma (Jakubovic and Jothy 2007; Nakatsura and Nishimura 2005).
HCC is the third leading cause of cancer-related deaths worldwide. Each year, HCC accounts for about 1 million deaths (Nakatsura and Nishimura 2005). Hepatitis B virus, hepatitis C virus, and chronic heavy alcohol use leading to cirrhosis of the liver remain the most common causes of HCC. Its incidence has increased dramatically in the United States because of the spread of hepatitis C virus infection and is expected to increase for the next 2 decades. HCC is treated primarily by liver transplantation or tumor resection. Patient prognosis is dependent on both the underlying liver function and the stage at which the tumor is diagnosed (Parikh and Hyman 2007). Thus, effective HCC treatment strategies are needed.
There are various disclosures of the uses of anti-glypican-3 antibodies in cancer therapy, either as a therapeutic antibody or in a conjugate. Smith et al. 2007 disclose conjugates of an anti-glypican-3 antibody and an auristatin. Zhang et al. 2014 disclose ADCs of an anti-glypican-3 antibody and a DNA minor groove binder-alkylator of the cyclopropabenzindole (CBI) type. Terrett et al. 2014 disclose anti-glypican-3 antibodies and their use for treating glypican-3 related conditions, including HCC and, generically, their use in immunoconjugates. Other disclosures relating to immunoconjugates of anti-glypican-3 antibodies include Ho et al., 2015 and 2015.
This disclosure provides an antibody-drug conjugate (ADC) comprising an anti-glypican-3 antibody as the targeting agent and a tubulysin analog, which ADC has an unexpectedly desirable combination of potency, therapeutic index, and pharmacokinetic properties and which can be used to treat a variety of cancers, including HCC, lung cancer, and ovarian cancer. The antibody-drug conjugate has a structure represented by formula I
wherein
As reflected by the subscript m, each antibody Ab can conjugate with more than one drug moiety, depending on the number of sites antibody Ab has available for conjugation and the experimental conditions employed. Those skilled in the art will appreciate that, while each individual antibody Ab is conjugated to an integer number of drug moieties, a conjugate preparation of the conjugate may analyze for a non-integer ratio of drug moieties to antibody Ab, reflecting a statistical average. This ratio is referred to as the substitution ratio (SR) or, synonymously, the drug-antibody ratio (DAR). Preferably, each antibody Ab is conjugated to 3 or 4 drug moieties (i.e., m is 3 or 4). The average m for a conjugate preparation preferably is between 3 and 3.5 (i.e., the DAR is 3 to 3.5).
In a preferred embodiment, the antibody Ab has a heavy chain variable region amino acid sequence according to SEQ ID NO:7 and a kappa light chain variable region amino acid sequence according to SEQ ID NO:8.
In another preferred embodiment, antibody Ab has a heavy chain constant region comprising comprising SEQ ID NO:9 and a kappa light chain constant region comprising SEQ ID NO:10. The heavy chain constant region may further have a lysine at its C-terminus.
In yet another preferred embodiment, antibody Ab has a heavy chain comprising SEQ ID NO:11 and a kappa light chain comprising SEQ ID NO:12. Such antibody is referred to herein as GPC3.1. Correspondingly, the ADC of formula I where the antibody is GPC3.1 is referred to herein as ADC3.1.
The heavy chain of antibody GPC3.1 optionally may further have a lysine at its C-terminus.
This disclosure also provides a method of treating a cancer in a human subject suffering from such cancer, comprising administering to the human subject a therapeutically effective amount of an antibody-drug conjugate of this disclosure, where the cancer is hepatocellular carcinoma, ovarian, or lung cancer, especially liver or lung cancer. The antibody-drug conjugate preferably is administered intravenously, at a dose of between 0.1 and 20 mg/kg, preferably between 0.5 and 15 mg/kg, and more preferably between 1.0 and 5 mg/kg.
This disclosure also provides a pharmaceutical formulation comprising an antibody-drug conjugate of this disclosure and a pharmaceutically acceptable excipient.
This disclosure also provides an isolated nucleic acid molecule encoding an antibody heavy chain comprising SEQ ID NO:11, which nucleic acid molecule preferably comprises SEQ ID NO:13.
This disclosure also provides an expression vector comprising the nucleic acid molecule of SEQ ID NO:13, and a host cell comprising such expression vector.
This disclosure also provides an isolated nucleic acid molecule encoding an antibody kappa chain comprising SEQ ID NO:12, which nucleic acid molecule comprises SEQ ID NO:15.
This disclosure also provides an expression vector comprising the nucleic acid molecule of SEQ ID NO:15, and a host cell comprising such expression vector.
“Antibody” means whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chain variants thereof. A whole antibody is a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region comprising three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (VL or Vk) and a light chain constant region comprising one single domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions contain a binding domain that interacts with an antigen. The constant regions may mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. An antibody is said to “specifically bind” to an antigen X if the antibody binds to antigen X with a KD of 5×10−8 M or less, more preferably 1×10−8 M or less, more preferably 6×10−9 M or less, more preferably 3×10−9 M or less, even more preferably 2×10−9 M or less. The antibody can be chimeric, humanized, or, preferably, human. The heavy chain constant region can be engineered to affect glycosylation type or extent, to extend antibody half-life, to enhance or reduce interactions with effector cells or the complement system, or to modulate some other property. The engineering can be accomplished by replacement, addition, or deletion of one or more amino acids or by replacement of a domain with a domain from another immunoglobulin type, or a combination of the foregoing.
“Antigen binding fragment” and “antigen binding portion” of an antibody (or simply “antibody portion” or “antibody fragment”) mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody, such as (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab′ fragment, which is essentially an Fab with part of the hinge region (see, for example, Abbas et al., Cellular and Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Preferred antigen binding fragments are Fab, F(ab′)2, Fab′, Fv, and Fd fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv, or scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding portion” of an antibody.
An “isolated antibody” means an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds antigen X is substantially free of antibodies that specifically bind antigens other than antigen X). An isolated antibody that specifically binds antigen X may, however, have cross-reactivity to other antigens, such as antigen X molecules from other species. In certain embodiments, an isolated antibody specifically binds to human antigen X and does not cross-react with other (non-human) antigen X antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
“Monoclonal antibody” or “monoclonal antibody composition” means a preparation of antibody molecules of single molecular composition, which displays a single binding specificity and affinity for a particular epitope.
“Human antibody” means an antibody having variable regions in which both the framework and CDR regions (and the constant region, if present) are derived from human germ-line immunoglobulin sequences. Human antibodies may include later modifications, including natural or synthetic modifications. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, “human anti-body” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
“Human monoclonal antibody” means an antibody displaying a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
At a first glance, the role to be performed by an antibody in an ADC appears to be a simple one: lead the conjugated drug to the target cell, and once there, release its drug payload, either inside the target cell or in the environs thereof. However, the selection of a suitable antibody for a successful ADC entails many variables beyond merely binding to the antigen of interest. Multiple factors may affect the overall efficacy of an ADC, including stability in circulation prior to reaching the target cell, binding affinity the antigen, safety vis-a-vis non-target cells that also express the antigen, and pharmacokinetics. The interplay among these factors is difficult to predict. As the data presented hereinbelow demonstrates, not all antibodies binding to glypican-3 produce an ADC as efficacious as antibody GPC3.1.
CDR1, CDR2, and CDR3 of the heavy chain of antibody GPC3.1 comprise the amino acids of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. CDR1, CDR2, and CDR3 of the light (kappa) chain of antibody GPC3.1 comprise the amino acids of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The amino acid sequences of the heavy and kappa chain variable regions are given by SEQ ID NO:7 and SEQ ID NO:8, respectively.
The heavy chain constant region of antibody GPC3.1 is of the IgG1 isotype, comprising the R214, E356, and M358 allotypes (numbering per EU index as set forth in Kabat et al., “Sequences of proteins of immunological interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991; hereinafter “Kabat”). Its amino acid sequence is set forth in SEQ ID NO:9. This allotype combination has a high prevalence in the Caucasian population.
The kappa light chain constant region of antibody GPC3.1 has an amino acid sequence as set forth in SEQ ID NO:10.
The complete heavy and kappa light chain amino acid sequences of antibody GPC3.1 are set forth in SEQ ID NO:11 and NO:12, respectively.
Terrett et al. 2014 disclose an anti-glypican-3 antibody 4A6 that has the same heavy and light chain variable regions as antibody GPC3.1, of the IgG1 or IgG4 isotype. It further generically discloses that antibody 4A6 can be used in ADCs, but does not provide any working examples.
Antibody GPC3.1 can be produced by recombinant expression of its heavy and kappa chains in a suitable host cell. SEQ ID NO:13 shows a DNA sequence, inclusive of a signal peptide, that can be used for recombinant production of the heavy chain, while SEQ ID NO:14 shows the amino acid sequence encoded thereby. The alignment between the DNA and amino acid sequences is shown in
Those skilled in the art will know that, when an antibody is produced recombinantly with a heavy chain C-terminal lysine group, such lysine is often removed by endogenous carboxypeptidases during cell culture production (Luo et al. 2012). Therefore antibody GPC3.1 can also be produced employing a DNA sequence corresponding to SEQ ID NO:13 but with an added codon for lysine at the C-terminal position and then allowing post-translational enzymatic removal of the lysine.
This disclosure also provides nucleic acids encoding antibody GPC3.1, in particular a nucleic acid (SEQ ID NO:13) encoding its heavy chain (SEQ ID NO:11), and conservative modifications of such nucleic acids. A “conservative modification” means, in respect of a nucleic acid sequence, a modification that replaces a nucleic acid therein with another but the modification results in the modified nucleic acid sequence encoding the same or a conservatively modified amino acid sequence compared to the one encoded by the original nucleic acid sequence or, where the original nucleic acid does not encode an amino acid sequence, the resultant modified nucleic acid sequence is essentially the same as the original nucleic acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acid sequences may encode any given protein. A nucleic acid sequence may have plural conservative modifications.
Where a polypeptide or nucleic acid molecule is associated with a particular SEQ ID NO:, preferably such polypeptide or nucleic acid molecule consists of the amino acid or nucleic acid sequence of the associated SEQ ID NO:.
The therapeutic agent in the conjugate of this disclosure is a synthetic tubulysin analog and has a structure represented by formula (II) (Cheng et al. 2013):
The tubulysins are potent naturally occurring cytotoxins, which act as anti-mitotic agents that interfere with mitosis by preventing the assembly of the tubulins into microtubules. The affected cells to accumulate in the G2/M phase and undergo apoptosis.
To conjugate a therapeutic agent to an antibody, a linker moiety is needed. In the instance of the present invention, the linker moiety has a structure represented by formula (III):
It comprises a valine-citrulline dipeptide (Val-Cit, recited in the conventional N-to-C direction), which is designed to be cleaved by the intracellular enzyme cathepsin B after the ADC has reached a target cancer cell and has been internalized by it, thus releasing the therapeutic agent to exert its cytotoxic effect. See Dubowchik et al. 1998a, 1998b, and 2002.
In the preparation of the conjugate of this disclosure, drug (II) and linker (III) are coupled to produce a therapeutic agent-linker compound having a structure represented by formula (IV), by forming an amide bond between the —CO2H group of the citrulline in linker (II) and the aromatic —NH2 of compound (II).
Compound (IV) is then conjugated to the antibody to prepare an ADC of formula (I). An ε-amino group in the side chain of a lysine residue of antibody GPC3.1 is reacted with 2-iminothiolane to introduce a free thiol (—SH) group. The thiol group can react with the maleimide group in compound (IV) to effect conjugation:
Typically, a thiolation level of two to four thiols per antibody is achieved. For a representative procedure, see Cong et al. 2015, the disclosure of which is incorporated herein by reference.
In addition to the naturally occurring tubulysins, synthetic tubulysin analogs with potent cytotoxic activity are known, for example as disclosed in Cheng et al. 2013 and Cong 2015. These references further disclose that such tubulysin analogs can be used in ADCs.
In particular, Cheng et al. 2013 discloses the preparation of a tubulysin analog-linker compound referred to there as formula (VI-t) (i.e., identical to formula (IV) above excepting the racemic methyl group alpha to the carboxylic acid) and, at Table 4 therein, its conjugates with an anti-CD70 antibody or anti-mesothelin antibody.
The practice of this invention can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation.
The antibody GPC3.1 VH and VK sequences were cloned into expression vectors containing the osteonectin signal sequence and the human IgG1 and kappa constant regions. The resulting heavy and light chain expression vectors were co-transfected into CHO cells and stable clones were selected and screened for IgG expression. One clone was chosen and expanded for antibody production.
This general procedure can be used to make ADC3.1 and other antibody-drug conjugates disclosed herein. Initially the antibody is buffer exchanged into 0.1 M phosphate buffer (pH 8.0) containing 50 mM NaCl and 2 mM diethylene triamine pentaacetic acid (DTPA) and concentrated to 5-10 mg/mL. Thiolation is achieved through addition of 2-iminothiolane to the antibody. The amount of 2-iminothiolane to be added can be determined by a preliminary experiment and varies from antibody to antibody. In the preliminary experiment, a titration of increasing amounts of 2-iminothiolane is added to the antibody, and following incubation with the antibody for 1 h at RT (room temperature, circa 25° C.), the antibody is desalted into 50 mM HEPES, 5 mM Glycine, 2 mM DTPA, pH 5.5 using a SEPHADEX™ G-25 column and the number of thiol groups introduced determined rapidly by reaction with dithiodipyridine (DTDP). Reaction of thiol groups with DTDP results in liberation of thiopyridine, which can be monitored spectroscopically at 324 nm. Samples at a protein concentration of 0.5-1.0 mg/mL are typically used. The absorbance at 280 nm can be used to accurately determine the concentration of protein in the samples, and then an aliquot of each sample (0.9 mL) is incubated with 0.1 mL DTDP (5 mM stock solution in ethanol) for 10 min at RT. Blank samples of buffer alone plus DTDP are also incubated alongside. After 10 min, absorbance at 324 nm is measured and the number of thiol groups is quantitated using an extinction coefficient for thiopyridine of 19,800 M−1.
Typically a thiolation level of about two to three thiol groups per antibody is desirable. For example, with some antibodies this can be achieved by adding a 15-fold molar excess of 2-iminothiolane followed by incubation at RT for 1 h. The antibody is then incubated with 2-iminothiolane at the desired molar ratio and then desalted into conjugation buffer (50 mM HEPES, 5 mM glycine, 2 mM DTPA, pH 5.5)). The thiolated material is maintained on ice while the number of thiols introduced is quantitated as described above.
After verification of the number of thiols introduced, the dimer-linker of formula (IV) is added at a 2.5-fold molar excess per thiol. The conjugation reaction is allowed to proceed in conjugation buffer containing a final concentration of 25% propylene glycol and 5% trehalose. Commonly, the drug-linker stock solution is dissolved in 100% DMSO. The stock solution is added directly to the thiolated antibody.
The conjugation reaction mixture is incubated at RT for 2 h with gentle stirring. A 10-fold molar excess of N-ethyl maleimide (100 mM stock in DMSO) is then added to the conjugation mixture and stirred for an additional hour to block any unreacted thiols.
The sample is then filtered via a 0.2μ filter The material is buffer exchanged via TFF VivaFlow 50 Sartorius 30 MWCO PES membrane into 10 mg/mL glycine, 20 mg/mL sorbitol, 15% acetonitrile (MeCN) pH 5.0 (5× TFF buffer exchange volume), to remove any unreacted drug. The final formulation is carried out by TFF into 20 mg/mL sorbitol, 10 mg/mL glycine, pH 5.0.
A 3H thymidine assay, where the inhibition of incorporation of 3H thymidine indicates inhibition of proliferation of the tested cell line, was used to assess the dose-dependent inhibitory effect of ADC3.1 on the proliferation of Hep3B and H446 cells. The human tumor cell lines were obtained from the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, USA, and cultured according to instructions from ATCC. Cells were seeded at 1.0×104 cells/well in 96-well plates. 1:3 serial dilutions of ADC3.1 were added to the wells. Plates were allowed to incubate for 72 h. The plates were pulsed with 1.0 μCi of 3H-thymidine per well for the last 24 hours of the total incubation period, harvested, and read on a Top Count Scintillation Counter (Packard Instruments, Meriden, Conn.). The EC50 values—the ADC concentration at which cell proliferation was reduced by 50%—were determined using PRISM™ software, version 4.0 (GraphPad Software, La Jolla, Calif., USA).
The EC50 so values, along with those against HepG2 and HuH7D12 hepatocellular carcinoma cell lines, are shown in Table 1, plus data for the drug alone (formula (II)). These results show that, generally, ADC3.1 is effective in delivering the drug to the target cell, at a level comparable to that of unconjugated drug. That is, release of the drug from the conjugate is efficient.
The data show that, at 0.1 μmol/kg, ADC3.1 was highly effective in causing tumor regression (
The efficacy of ADC3.1 in a xenograft study of the single does efficacy of ADC3.1 against HuH7D12 cells is shown in
The corresponding split dosing study is shown in
The results indicate that the single dosing regimen was more efficacious, with tumor regression noted with a single dose of 0.3 μmol/kg (seven of eight mice becoming tumor-free).
A similar xenograft study was performed, comparing single dose (
Again, the single dose regimen was somewhat more efficacious, although exhibiting higher transient body weight loss. In the single dose study, tumor regression was observed in eight of eight mice at 0.3 μmol/kg and in four of eight mice at 0.1 μmol/kg. In the split dose study, tumor regression was observed in four of eight mice at 0.1 μmol/kg.
This example provides results of patient derived xenograft (PDX) studies.
This example describes a study seeking to identify variants of antibody GPC3.1, which might be better targeting agents for an ADC.
Anti-glypican-3 antibodies internalize with similar efficiencies into target cells such as cancerous cells that express high levels of glypican-3, regardless of whether they are low affinity binders (fast koff, KD ≈10 nM) or high affinity binders (slow koff, KD≦1 nM). Hypothetically, it is possible that an ADC of anti-glypican-3 antibody with relatively low affinity may exhibit reduced toxicity against normal cells, which have a lower expression level of glypican-3. Further, an ADC of an anti-glypican-3 antibody with relatively low affinity may distribute more facilely into distant tumor tissues. To evaluate this hypothesis, 83 variants of Antibody GPC3.1 were prepared, containing modifications in the variable region. Of these, the three most promising (designated antibodies A, B, and C) were selected for head-to-head comparisons against antibody GPC3.1.
Compared to Antibody GPC3.1, Antibody A has the same heavy chain CDR1 and light (kappa) chain CDR1 and CDR2 (SEQ ID NO;1, NO:4, and NO:5, respectively) but different heavy chain CDR2 and CDR3 (SEQ ID NO:17 and NO:18, respectively) and kappa chain CDR3 (SEQ ID NO:19). Also, Antibody A differs from Antibody GPC3.1 in certain heavy chain framework amino acids, as noted in its heavy chain variable region sequence (SEQ ID NO:23). Its kappa chain variable region sequence is provided in SEQ ID NO:24. Its heavy and kappa chain constant regions have the same sequence as those in Antibody GPC3.1 (SEQ ID NO:9 and NO:10, respectively).
Compared to Antibody GPC3.1, Antibody B has the same heavy chain CDR3 and light (kappa) chain CDR1 and CDR2 (SEQ ID NO:3, NO:4 and NO:5, respectively) but different heavy chain CDR1 and CDR2 (SEQ ID NO:20 and NO:21, respectively) and kappa chain CDR3 (SEQ ID NO:22). Also, Antibody B differs from Antibody GPC3.1 in certain heavy chain framework amino acids, as noted in its heavy chain variable region sequence (SEQ ID NO:25). Its kappa chain variable region sequence is provided in SEQ ID NO:26. Its heavy and kappa chain constant regions have the same sequence as those in Antibody GPC3.1 (SEQ ID NO:9 and NO:10, respectively).
Compared to Antibody GPC3.1, Antibody C has the same heavy chain CDR3 and light (kappa) chain CDR1 and CDR2 (SEQ ID NO:3, NO:4 and NO:5, respectively) but different heavy chain CDR1 and CDR2 (SEQ ID NO:20 and NO:17, respectively) and kappa chain CDR3 (SEQ ID NO:19). Also, Antibody C differs from Antibody GPC3.1 in certain heavy chain framework amino acids, as noted in its heavy chain variable region sequence (SEQ ID NO:27). Its kappa chain variable region sequence is provided in SEQ ID NO:28. Its heavy and kappa chain constant regions have the same sequence as those in Antibody GPC3.1 (SEQ ID NO:9 and NO:10, respectively).
The respective koff and KD values for antibodies A, B, C and GPC3.1 are shown in Table 2.
Compared to antibody GPC3.1, variant antibodies A, B, and C exhibited between 10-and 30-fold improvement in KD and koff according to Biacore™ assays. They also exhibited faster clearance in mice.
Thus, in view of the foregoing in vitro results, ADCs A, B, and C were viewed as promising candidates for in vivo comparative studies against ADC3.1. The results of such studies are presented and discussed in Example 10 hereinbelow.
This example describes a different study with a similar objective, that is, to identify other anti-glypican-3 antibodies, which might be more efficacious as a targeting agent in an ADC than antibody GPC3.1.
Rather than modifying antibody GPC3.1, anti-glypican-3 antibodies were made de novo by immunizing HuMab® transgenic mice. Methods for raising of human antibodies by immunizing HuMab® transgenic mice are disclosed in Terrett et al., U.S. Pat. No. 8,680,247 B2 (2014), the disclosure of which is incorporated herein by reference. The binding properties of four antibodies so raised and antibody GPC3.1 are shown in Table 3 below.
Binning studies showed that antibody E bound to a different epitope than antibody GPC3.1 and was non-blocking vis-a-vis it. The epitope grouping of antibody D was not determined.
In view of the above results antibody E was selected for ADC comparative studies against ADC 3.1.
ADCs were prepared combining drug linker compound (IV) and antibodies A, B, C, and E, to produce ADCs respectively designated ADC A, ADC B, ADC C, and ADC E. In vivo xenograft studies were performed for these four ADCs alongside ADC 3.1, a vehicle (formulation buffer) control, and an ADC control (an ADC of an anti-CD70 antibody with drug-linker (IV)), using Hep3B cells. Dosing was 0.1 μmol/kg (5 mg/kg) in one case (
For comparative purposes, an ADC of anti-GPC3 antibody GPC3.1 and drug-linker compound (V) was prepared.
The drug moiety in compound (V) belongs to the class of cytotoxins known as cyclopropabenzindoles (CBIs) and have been used in ADCs (Zhang et al. 2015).
When tested against Hep3B cancer cells by the 3H thymidine assay, the ADC of compound (V) had a potency (EC50 0.079 nM) that compared favorably against that of ADC3.1 (EC50 0.15 nM). However, the former's pharmacokinetic (PK) properties were not as desirable as that of ADC3.1.
The foregoing detailed description of the invention includes passages that are chiefly or exclusively concerned with particular parts or aspects of the invention. It is to be understood that this is for clarity and convenience, that a particular feature may be relevant in more than just the passage in which it is disclosed, and that the disclosure herein includes all the appropriate combinations of information found in the different passages. Similarly, although the various figures and descriptions herein relate to specific embodiments of the invention, it is to be understood that where a specific feature is disclosed in the context of a particular figure or embodiment, such feature can also be used, to the extent appropriate, in the context of another figure or embodiment, in combination with another feature, or in the invention in general.
Further, while the present invention has been particularly described in terms of certain preferred embodiments, the invention is not limited to such preferred embodiments. Rather, the scope of the invention is defined by the appended claims.
Full citations for the following references cited in abbreviated fashion by first author (or inventor) and date earlier in this specification are provided below. Each of these references is incorporated herein by reference for all purposes.
Cheng et al., U.S. Pat. No. 8,394,922 B2 (2013).
Cong et al., U.S. Pat. No. 8,980,824 B2 (2015).
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Table 4 below provides a short summary of the sequence listings filed together with this specification.
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 62/333,944, filed May 10, 2016; the disclosure of which is incorporated herein by reference
| Number | Date | Country | |
|---|---|---|---|
| 62333944 | May 2016 | US |
