Patent Application
20250235548
The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 7, 2023, is named 2023 11 15 ZYME094WO.xml, and is 90,074 bytes in size.
The present disclosure relates to the field of immunotherapeutics and, in particular, to antibody-drug conjugates targeting human sodium-dependent phosphate transporter 2B (hNaPi2b).
Sodium-dependent phosphate transporter 2B (NaPi2b) is a transmembrane protein encoded by the SLC34A2 gene. The NaPi2b polypeptide is 690 amino acids in length, with a limited extracellular domain of amino acids 188-361 exposed on the surface of cells. It is widely expressed in normal tissues and overexpressed in a variety of cancers including ovarian cancer, endometrial cancer, and lung cancer.
Given the overexpression of NaPi2b in certain types of cancers, NaPi2b-targeted agents have been studied in clinical trials for the treatment of cancer but have returned mixed results. A Phase I/II clinical trial to study upifitamab rilsodotin, an antibody-drug conjugate (ADC) of the NaPi2b-targeting antibody MX35 with an auristatin-F payload (Dolaflexin platform) in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC) was undertaken by Mersana Therapeutics. The NSCLC arm of the study was discontinued due to lack of efficacy, while upifitamab rilsodotin was granted Fast Track Designation for the treatment of platinum-resistant ovarian cancer patients who have received three to four prior lines of therapy. Mersana also completed a Phase I/II clinical trial of XMT-1592 in ovarian cancer; XMT-1592 is a site specific ADC, comprised of antibody MX35 conjugated to an auristatin-F payload using their Dolasynthen platform. The development of this ADC has been discontinued. Lifastuzumab vedotin, an ADC of lifastuzumab with an MMAE payload was studied in a clinical trial sponsored by Genentech in patients with ovarian cancer or NSCLC, but this trial has since been discontinued
Camptothecin analogues have been developed as payloads for ADCs. Two such ADCs have been approved for treatment of cancer. Trastuzumab deruxtecan (Enhertu™) in which the camptothecin analogue, deruxtecan (Dxd), is conjugated to the anti-HER2 antibody, trastuzumab, via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™) in which the camptothecin analogue, SN-38, is conjugated to the anti-Trop-2 antibody, sacituzumab, via a hydrolysable, pH-sensitive linker.
Other camptothecin analogues and derivatives, as well as ADCs comprising them have been described. See, for example, International (PCT) Publication Nos. WO 2019/195665; WO 2019/236954; WO 2020/200880 and WO 2020/219287.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention.
Described herein are antibody-drug conjugates (ADCs) targeting human NaPi2b and methods of use. One aspect of the present disclosure relates to an antibody-drug conjugate having Formula (X):
T-[L-(D)m]n (X)
—CO2R8, -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl;
R4 is selected from:
Another aspect of the present disclosure relates to an antibody-drug conjugate having the structure:
wherein n is 4, and T is an anti-NaPi2b antibody construct as described herein.
Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier or diluent.
Another aspect of the present disclosure relates to a method of inhibiting the proliferation of cancer cells comprising contacting the cells with an effective amount of the antibody-drug conjugate as described herein.
Another aspect of the present disclosure relates to a method of killing cancer cells comprising contacting the cells with an effective amount of the antibody-drug conjugate as described herein.
Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the antibody-drug conjugate as described herein.
Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy.
Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in the treatment of cancer.
Another aspect of the present disclosure relates to a use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.
Another aspect of the present disclosure relates to a kit comprising the antibody-drug conjugate as described herein and a label and/or package insert containing instructions for use.
The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct that binds sodium-dependent phosphate transporter 2B (NaPi2b) (an anti-NaPi2b antibody construct) conjugated to a camptothecin analogue of Formula (I) as described herein. The ADCs of the present disclosure may find use, for example, as therapeutics, in particular in the treatment of cancer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
As used herein, the term “about” refers to an approximately +/−10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”
Where a range of values is provided herein, for example where a value is defined as being “between” an upper limit value and a lower limit value, it is understood that the range encompasses both the upper limit value and the lower limit value as well as each intervening value.
As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term “consisting essentially of” when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of” when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen-binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. Often, the three heavy chain CDRs and the three light chain CDRs are required to bind antigen. However, in some instances, even a single variable domain can confer binding specificity to the antigen. Furthermore, as is known in the art, in some cases, antigen-binding may also occur through a combination of a minimum of one or more CDRs selected from the VH and/or VL domains, for example HCDR3.
A number of different definitions of the CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol, 196:901-917), as well as the IMGT, AbM (University of Bath) and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745) definitions. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Accordingly, as would be readily apparent to one skilled in the art, the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it is to be understood that the disclosure herein of a VH includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, disclosure herein of a VL includes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.
1Either the Kabat or Chothia numbering system may be used for HCDR2, HCDR3 and the light chain CDRs for all definitions except Contact, which uses Chothia numbering
2Using Kabat numbering. The position in the Kabat numbering scheme that demarcates the end of the Chothia and IMGT CDR-H1 loop varies depending on the length of the loop because Kabat places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loop can be unambiguously defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.
The term “identical” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, or about 98% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which a test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).
The term “acyl,” as used herein, refers to the group —C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “acyloxy” refers to the group —OC(O)R, where R is alkyl.
The term “alkoxy,” as used herein, refers to the group —OR, where R is alkyl, aryl, heteroaryl, cycloalkyl or cycloheteroalkyl.
The term “alkyl,” as used herein, refers to a straight chain or branched saturated hydrocarbon group containing the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, t-pentyl, neo-pentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.
The term “alkylaminoaryl,” as used herein, refers to an alkyl group as defined herein substituted with one aminoaryl group as defined herein.
The term “alkylheterocycloalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one heterocycloalkyl group as defined herein.
The term “alkylthio,” as used herein, refers to the group —SR, where R is an alkyl group.
The term “amido,” as used herein, refers to the group —C(O)NRR′, where R and R′ are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “amino,” as used herein, refers to the group —NRR′, where R and R′ are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “aminoalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one or more amino groups, for example, one, two or three amino groups.
The term “aminoaryl,” as used herein, refers to an aryl group as defined herein substituted with one amino group.
The term “aryl,” as used herein, refers to a 6- to 12-membered mono- or bicyclic hydrocarbon ring system in which at least one ring aromatic. Examples of aryl include, but are not limited to, phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8-tetrahydro-naphthalenyl, indanyl, and the like.
The term “carboxy,” as used herein, refers to the group —C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl or cycloheteroalkyl.
The term “cyano,” as used herein, refers to the group —CN.
The term “cycloalkyl,” as used herein, refers to a mono- or bicyclic saturated hydrocarbon containing the specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, and the like.
The term “haloalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one or more halogen atoms.
The terms “halogen” and “halo,” as used herein refer to fluorine (F), bromine (Br), chlorine (Cl) and iodine (I).
The term “heteroaryl,” as used herein, refers to a 6- to 12-membered mono- or bicyclic ring system in which at least one ring atom is a heteroatom and at least one ring is aromatic.
Examples of heteroatoms include, but are not limited to, O, S and N. Examples of heteroaryl include, but are not limited to: pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.
The term “heterocycloalkyl,” as used herein, refers to a mono- or bicyclic non-aromatic ring system containing the specified number of atoms and in which at least one ring atom is a heteroatom, for example, O, S or N. A heterocyclyl substituent can be attached via any of its available ring atoms, for example, a ring carbon, or a ring nitrogen. Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.
The terms “hydroxy” and “hydroxyl,” as used herein, refer to the group —OH.
The term “hydroxyalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one or more hydroxy groups.
The term “nitro,” as used herein, refers to the group —NO2.
The term “sulfonyl,” as used herein, refers to the group —S(O)2R, where R is H, alkyl or aryl.
The term “sulfonamido,” as used herein, refers to the group —NH—S(O)2R, where R is H, alkyl or aryl.
The terms “thio” and “thiol,” as used herein, refer to the group —SH.
Unless specifically stated as being “unsubstituted,” any alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group referred to herein is understood to be “optionally substituted,” i.e. each such reference includes both unsubstituted and substituted versions of these groups. For example, reference to a “—C1-C6 alkyl” includes both unsubstituted —C1-C6 alkyl and —C1-C6 alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group referred to herein is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl and sulfonamido.
A chemical group described herein as “substituted,” may include one substituent or a plurality of substituents up to the full valence of substitution for that group. For example, a methyl group may include 1, 2, or 3 substituents, and a phenyl group may include 1, 2, 3, 4, or 5 substituents. When a group is substituted with more than one substituent, the substituents may be the same or they may be different.
The term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment. The animal may be a human, a non-human primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like). In certain embodiments, the subject is a human.
It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein, and vice versa.
Particular features, structures and/or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and/or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.
It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.
The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analogue having Formula (I). In certain embodiments, the ADC has Formula (X):
T-[L-(D)m]n (X)
Components of Formula (X) are described below.
The ADCs of the present disclosure comprise an anti-NaPi2b antibody construct. In this context, the term “antibody construct” refers to a polypeptide or a set of polypeptides that comprises one or more antigen-binding domains, where each of the one or more antigen-binding domains specifically binds to an epitope or antigen. Where the antibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind the same epitope or antigen (i.e. the antibody construct is monospecific) or they may bind to different epitopes or antigens (i.e. the antibody construct is bispecific or multispecific). The antibody construct may further comprise a scaffold and the one or more antigen-binding domains can be fused or covalently attached to the scaffold, optionally via a linker.
In accordance with the present disclosure, the anti-NaPi2b antibody construct of the ADC comprises at least one antigen-binding domain that specifically binds to human NaPi2b (hNaPi2b). By “specifically binds” to hNaPi2b, it is meant that the antibody construct binds to hNaPi2b but does not exhibit significant binding to NaPi2a or NaPi2c. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure may be capable of binding to an NaPi2b from one or more non-human species. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure are capable of binding to cynomolgus monkey NaPi2b.
Human NaPi2b is also known as human “solute carrier family 34 member 2” or “SLC34A2.” The protein sequences of hNaPi2b from various sources are known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. Examples of hNaPi2b sequences include for example those provided under NCBI reference numbers NP_006415.3, NP_001171470.2, and NP_001171469.2. An exemplary hNaPi2b protein sequence is provided in Table 2 as SEQ ID NO: 1 (UniProt ID: 095436). An exemplary cynomolgus monkey NaPi2b protein sequence is also provided in Table 2 (SEQ ID NO: 2; UniProt ID: A0A2K5UHY1), as is an exemplary mouse NaPi2b protein sequence (SEQ ID NO:3; UniProt ID: Q9DBP0).
Specific binding of an antigen-binding domain to a target antigen or epitope may be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry or a traditional binding assay (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding may be defined as the extent of binding to a non-target protein (such as hNaPi2a or hNaPi2c) being less than about 5% to less than about 10% of the binding to hNaPi2b as measured by ELISA or flow cytometry, for example.
The term “dissociation constant (KD or Kd)” as used herein, is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, ligand-protein interactions refer to, but are not limited to protein-protein interactions or antibody-antigen interactions. The KD measures the propensity of two proteins complexed together (e.g. AB) to dissociate reversibly into constituent components (A+B), and is defined as the ratio of the rate constant of dissociation, also called the “off-rate (koff)”, to the association rate constant, or “on-rate (kon)”. Thus, KD equals koff/kon and is expressed as a molar concentration (M). It follows that the smaller the KD, the stronger the affinity of binding, and thus a decrease in KD indicates an increase in affinity. Therefore, a KD of 1 mM indicates weak binding affinity compared to a KD of 1 nM. Affinity is sometimes measured in terms of a KA or Ka, which is the reciprocal of the KD or Kd. KD between antibody and its antigen can be determined using methods well established in the art. One method for determining such KD is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. Isothermal titration calorimetry (ITC) is another method that can be used to measure KD. The Octet™ system may also be used to measure the affinity of antibodies for a target antigen.
In certain embodiments, specific binding of an antibody construct for NaPi2b may be defined by a dissociation constant (Kd or KD) of ≤1 μM, for example, ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, or ≤10 nM. In certain embodiments, specific binding of an antibody construct for a particular antigen or an epitope may be defined by a dissociation constant (KD) of 10−6 M or less, for example, 10−7 M or less, or 10−8 M or less. In some embodiments, specific binding of an antibody construct for a particular antigen or an epitope may be defined by a dissociation constant (KD) between 10−6 M and 10−9 M, for example, between 10−7 M and 10−9 M. As is known in the art the numerical value of the dissociation constant obtained may vary depending on how it is tested. For example, the expression level of NaPi2b in the cell line, format of the antibody construct (i.e. monovalent or bivalent), and type of assay (i.e. ELISA or flow cytometry), may affect the numerical value of the dissociation constant when measured in a cell-based assay. The data provided in the Examples illustrate this general point, as shown in Examples 10, 11, and 16.
In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure have a Kd that is lower than that of reference antibody lifastuzumab, and comparable to that of reference antibody MX35, when measured by flow cytometry in cells that express NaPi2b at high levels. Accordingly, in these embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain having an affinity for human NaPi2b that is greater than that of reference antibody lifastuzumab and comparable to that of reference antibody MX35.
In certain embodiments the anti-NaPi2b antibody constructs exhibit comparable levels of internalization to the reference antibody MX35 and exhibit greater levels of internalization compared to reference antibody lifastuzumab in high and mid NaPi2b-expressing cells. In some embodiments, internalization is measured after 4 hours, after 5 hours or after 24 hours of treatment.
Antibody internalization may be measured using art-known methods, for example, by a direct internalization method according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as the pHAb Dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dyes (Thermofisher Scientific Corporation, Waltham, MA) and Incucyte® Fabfluor-pH Antibody Labeling Reagent (Sartorius AG, Göttingen, Germany) and analysis techniques such as microscopy, FACS, high content imaging or other plate-based assays.
NaPi2b expression varies depending on cell type as indicated throughout the disclosure and the level of NaPi2b expression is referred to herein as “high”, “mid,” “low” or “negative.” These terms are used for reference to describe levels of expression in general according to the designations shown in Table 15.1 in Example 15 and are not intended to be limited to the specific numerical values for average NaPi2b protein per cell included therein. Alternatively, expression level of NaPi2b in cells or tumors may be assessed by immunohistochemistry (IHC) according to methods known in the art. For example, IHC may be used to stain for NaPi2b in tumor tissue samples from xenograft models, cell-derived (CDX) or patient-derived (PDX). Tissue samples may be examined, and an H-score calculated as known in the art and described, for example in Example 35, herein. The higher the H-score, the higher the expression of NaPi2b in the tissue sample.
The anti-NaPi2b antibody constructs of ADCs of the present disclosure comprise at least one antigen-binding domain that is capable of binding to hNaPi2b. The at least one antigen-binding domain capable of binding to hNaPi2b typically is an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of an antigen-binding antibody fragment include, but are not limited to, a Fab fragment, a Fab′ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb).
A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab′ fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C-terminus to the N-terminus of a VH by a polypeptide linker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).
An “sdAb” format refers to a single immunoglobulin domain. The sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.” An sdAb comprises three CDR/hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).
In those embodiments in which the anti-NaPi2b antibody constructs comprise two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain, such as an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, a natural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, fynomers, affimers, alphabodies, DARPins and avimers.
The present disclosure describes herein the identification of a mouse antibody that specifically binds hNaPi2b; a mouse-human chimeric variant of this antibody is identified as variant 23855. The anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen-binding domain derived from this mouse antibody or humanized antibody variants of same. Representative humanized antibody variants (v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460) of the mouse antibody are also described. In certain embodiments, the anti-NaPi2b antibody constructs described herein specifically bind human NaPi2b having the sequence as set forth in SEQ ID NO:1.
In certain embodiments, the anti-NaPi2b antibody construct of the ADC competes with any one of humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or with parental chimeric antibody v23855, for binding to human NaPi2b. In assessing competition as described below, each of variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, and v23855, are referred to as a competition reference antibody.
One can determine whether antibody constructs compete with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or parental chimeric antibody v23855 for binding to hNaPi2b using competition assays known in the art. For example, the competition reference antibody is first allowed to bind to hNaPi2b under saturating conditions and then the ability of the test antibody construct to bind to hNaPi2b is measured. If the test antibody construct is able to bind to hNaPi2b at the same time as the competition reference antibody, then the test antibody construct is considered to bind to a different epitope than the competition reference antibody. Conversely, if the test antibody construct is not able to bind to hNaPi2b at the same time as the competition reference antibody, then the test antibody construct is considered to bind to the same epitope, to an overlapping epitope, or to an epitope that is in close proximity to the epitope bound by the competition reference antibody. Such competition assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), bio-layer interferometry, flow cytometry and the like. An “antibody that competes with” a competition reference antibody refers to an antibody that blocks binding of the reference antibody to its epitope in a competition assay by 50% or more.
In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to hNaPi2b, where the antigen-binding domain comprises a set of CDRs based on the CDRs of parental chimeric antibody v23855 described herein. The CDR sequences of the parental chimeric antibody v23855 and representative humanized antibody variants are shown in. Table 3.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 7, 8, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 19, 20, and 18.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 4, 5, and 6, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences as set forth in SEQ ID NO: 17 and SEQ ID NO:18 and the LCDR sequence YTS.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 10, 11, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 19, 20, and 18.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 12, 13, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 19, 20, and 18.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 14, 15, and 16, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 21, 22, and 23.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADC of the present disclosure comprise an antigen-binding domain having:
In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) selected from the light chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.
In certain embodiments, the anti-NaPi2b antibody constructs of ADCs of the present disclosure comprise an antigen-binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.
In certain embodiments, the anti-NaPi2b antibody constructs of ADCs of the present disclosure comprise an antigen-binding domain having a VH sequence comprising the CDR sequences of the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain having a VL sequence comprising the CDR sequences of the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.
One skilled in the art will appreciate that a limited number of amino acid substitutions may be introduced into the CDR sequences or into the VH or VL sequences of known antibodies without the antibody losing its ability to bind its target. Candidate amino acid substitutions may be identified by computer modeling or by art-known techniques such as alanine scanning, with the resulting variants being tested for binding activity by standard techniques. Accordingly, in certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a set of CDRs (i.e. heavy chain HCDR1, HCDR2 and HCDR3, and light chain LCDR1, LCDR2 and LCDR3) that have 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the % sequence identity is calculated across all six CDRs and where the antigen-binding domain retains the ability to bind hNaPi2b.
In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the variant comprises between 1 and 10 amino acid substitutions across the set of CDRs (i.e. the CDRs may be modified by up to 10 amino acid substitutions with any combination of the six CDRs being modified), and where the antigen-binding domain retains the ability to bind hNaPi2b. In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the variant comprises between 1 and 7 amino acid substitutions, between 1 and 5 amino acid substitutions, between 1 and 4 amino acid substitutions, between 1 and 3 amino acid substitutions, between 1 and 2 amino acid substitutions, or 1 amino acid substitution, across the set of CDRs, and where the antigen-binding domain retains the ability to bind hNaPi2b.
In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the antigen-binding domain retains the ability to bind hNaPi2b. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the antigen-binding domain retains the ability to bind hNaPi2b.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v23855, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v23855, where the antigen-binding domain retains the ability to bind hNaPi2b.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v29456, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29456, where the antigen-binding domain retains the ability to bind hNaPi2b.
In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v29452, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29452, where the antigen-binding domain retains the ability to bind hNaPi2b.
In some embodiments, the anti-NaPi2b antibody construct of the ADCs of the present disclosure comprises the VH and VL sequences of any one of v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. The SEQ ID NOs: of the VH and VL sequences of these variants are provided below in Table 4. The sequences themselves are provided in Table 7.4 of the Examples.
In some embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises the VH sequence and the VL sequence of v29456. In some embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises the VH sequence and the VL sequence of v29452.
In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises a) a VH sequence having the 3 HCDRs of v29456 and having at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29456, and b) a VL sequence having the 3 LCDRs of v29456 and having at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29456, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.
In certain other embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprise a) a VH sequence having the 3 HCDRs of v29452 and having at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29452, and b) a VL sequence having the 3 LCDRs of v29452 and having at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29452, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.
In one embodiment, the anti-NaPi2b antibody construct of the ADCs of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO:63 and two light chains having the amino acid sequence as set forth in SEQ ID NO:62. In one embodiment, the anti-NaPi2b antibody construct of the ADCs of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO:61 and two light chains having the amino acid sequence as set forth in SEQ ID NO:62. In still other embodiments, the anti-NaPi2b antibody construct of the ADCs of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO:66 and two light chains having the amino acid sequence as set forth in SEQ ID NO:67. In yet other embodiments, the anti-NaPi2b antibody construct of the ADCs of the present disclosure comprises two heavy chains having the amino acid sequence as set forth in SEQ ID NO:68 and two light chains having the amino acid sequence as set forth in SEQ ID NO:67.
The anti-NaPi2b antibody constructs of the ADC may have various formats. The minimal component of the anti-NaPi2b antibody construct is an antigen-binding domain that binds to hNaPi2b. The anti-NaPi2b antibody constructs may further optionally comprise one or more additional antigen-binding domains and/or a scaffold. In those embodiments in which the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within hNaPi2b, may bind to a different epitope within hNaPi2b, or may bind to a different antigen. Thus, the anti-NaPi2b antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.
In certain embodiments, the anti-NaPi2b antibody construct comprises at least one antigen-binding domain that binds to hNaPi2b and a scaffold, where the antigen-binding domain is operably linked to the scaffold. The term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner. Suitable scaffolds are described below.
In certain embodiments, the anti-NaPi2b antibody construct comprises two antigen-binding domains optionally operably linked to a scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise three or four antigen-binding domains and optionally a scaffold. In these formats, when comprising a scaffold, at least a first antigen-binding domain is operably linked to the scaffold and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or to the first antigen-binding domain or, when more than two antigen-binding domains are present, to another antigen-binding domain.
Anti-NaPi2b antibody constructs that lack a scaffold may comprise a single antigen-binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such anti-NaPi2b antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through substitution of non-cysteine residues to cysteine residues in each chain (for example, at position 44 in VH and 100 in VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. Biol., 399:436-449).
Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of anti-NaPi2b antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab2 and F(ab′)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.
Combinations of antigen-binding domains in different forms may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv/sdAb) construct.
In certain embodiments, the anti-NaPi2b antibody construct may be in an antibody format that is based on an immunoglobulin (Ig). This type of format is referred to herein as a full-size antibody format (FSA) or Mab format and includes anti-NaPi2b antibody constructs that comprise two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-NaPi2b antibody construct may be based on an IgG class immunoglobulin, for example, an IgG1, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the anti-NaPi2b antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-NaPi2b antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti-NaPi2b antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-NaPi2b antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the anti-NaPi2b antibody constructs may also comprise hybrids of isotypes and/or subclasses in some embodiments. It is also to be understood that the Fc region and/or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art. Thus, in certain embodiments, the anti-NaPi2b antibody construct comprises a VH amino acid sequence fused to IgG1 constant domain amino acid sequences (i.e. CH1, hinge, CH2, CH3 amino acid sequences) and a VL amino acid sequence fused to kappa or lambda constant amino acid sequences domain (i.e. CL amino acid sequences). Exemplary amino acid sequences are provided in the Examples and Sequence Tables.
In some embodiments, the anti-NaPi2b antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the anti-NaPi2b antibody construct may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody” and “humanized antibody” both refer generally to antibodies that combine immunoglobulin regions or domains from more than one species.
A “chimeric antibody” typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.
A “humanized antibody” is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen. This technique for creating humanized antibodies is often referred to as “CDR grafting.”
In some instances, additional modifications are made to further refine antibody performance. For example, some framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. In general, a variable domain in a humanized antibody will comprise all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more detail in Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.
A number of approaches are known in the art for selecting the most appropriate human frameworks in which to graft the non-human CDRs. Early approaches used a limited subset of well-characterised human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach). More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs (“homology matching” or “best-fit” approach). An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by back-mutating some of the residues of human origin to the corresponding non-human ones. Methods for preparing humanized antibodies by these approaches are well-known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).
Alternatively, or in addition to, these traditional approaches, more recent technologies may be employed to further reduce the immunogenicity of a CDR-grafted humanized antibody. For example, frameworks based on human germline sequences or consensus sequences may be employed as acceptor human frameworks rather than human frameworks with somatic mutation(s). Another technique that aims to reduce the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues required for antigen-binding activity (the “SDRs”) are grafted into a human germline framework. This method improves the “humanness” (i.e. the similarity to human germline sequence) of the humanized antibody and thus may help reduce the risk of immunogenicity of the variable region. These techniques have been described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872, and Kashmiri, et al., 2005, Methods, 36:25-34).
In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure comprises humanized antibody sequences, for example, one or more humanized variable domains. In some embodiments, the anti-NaPi2b antibody construct can be a humanized antibody. Non-limiting examples of humanized antibodies based on the anti-NaPi2b antibody v23855 are described herein (see Examples and Sequence Tables and sequences for v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460).
In certain embodiments, the anti-NaPi2b antibody constructs comprise one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of the forms described above (for example, scFvs, Fabs and/or sdAbs). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CH1 and CL domains or barnase-barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).
A scaffold may be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. Where the scaffold is a polypeptide, each antigen-binding domain of the anti-NaPi2b antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold. Anti-NaPi2b antibody construct comprising a polypeptide scaffold in which one or more of the antigen-binding polypeptide constructs are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.
In embodiments where the anti-NaPi2b antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.
A number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold. An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287: 43331-43339 (2012)). Other selectively pairing molecular pairs include, for example, the barnase-barstar pair (Deyev, et al., Nat Biotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(1-2):23-26 (1999)) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011/135040).
Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues and derivatives, toxins, cytokines, chemokines and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Müller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U.S. Patent Application Publication No. 2009/0285816).
For example, fusing antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Müller et al., ibid.). Antigen-binding moieties may be fused at the N- and/or C-termini of albumin, optionally via a linker.
Derivatives of albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Patent Application Publication Nos. WO 2012/116453 and WO 2014/012082). As a result of the segmentation of albumin, the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moieties, optionally via linkers.
In certain embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.
The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
In certain embodiments, the anti-NaPi2b antibody constructs may comprise a scaffold that is based on an immunoglobulin Fc region. The Fc region may be dimeric and composed of two Fc polypeptides or alternatively, the Fc region may be composed of a single polypeptide.
An “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. When referring to a dimeric Fc region, the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.
An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, an Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.
In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a human IgG1 Fc region.
In certain embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the first and second Fc polypeptides are different. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.
In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the same position. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids on each sequence at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.
In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.
Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96/027011 (“knobs into holes”), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange). Other examples include approaches combining positive and negative design strategies to produce stable asymmetrically modified Fc regions as described in International Publication Nos. WO 2012/058768 and WO 2013/063702. In certain embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012/058768 or WO 2013/063702.
Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO:16), corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgG1 heavy chain. Also shown in Table 5 are CH3 domain amino acid modifications that promote formation of a heterodimeric Fc as described in in International Patent Application Publication Nos. WO 2012/058768 and WO 2013/063702.
In certain embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 5.
1Sequence from positions 231-447 (EU numbering)
In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to the Fc, such as receptors of the FcγRI, FcγRII and FcγRIII subclasses.
In some embodiments, the anti-NaPi2b antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII and FcγRIII receptors.
A number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fcγ receptors are known in the art. Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcγRIIIa (an activating receptor) may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcγRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcγRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fcγ receptors (“knock-out” variants) may be useful.
Examples of amino acid modifications to the CH2 domain that alter binding of the Fc by Fcγ receptors include, but are not limited to, the following: S298A/E333A/K334A and S298A/E333A/K334A/K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L/R292P/Y300L/V305I/P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90); F243L/R292P/Y300L/L235V/P396L (increased affinity for FcγRIIIa) (Nordstrom J L, et al., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A/E333A/K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D/I332E/A330L and S239D/I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D/S267E and S267E/L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter binding of the Fc by FcγRIIb are described in International Publication No. WO 2021/232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).
In certain embodiments, the anti-NaPi2b antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domain comprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all of the Fcγ receptors (i.e. a “knock-out” variant).
Various publications describe strategies that have been used to engineer antibodies to produce “knock-out” variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, “Antibody Fc engineering for optimal antibody performance” In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include reduction of effector function through modification of glycosylation, use of IgG2/IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 domain of the Fc (see also, U.S. Patent Publication No. 2011/0212087, International Publication No. WO 2006/105338, U.S. Patent Publication No. 2012/0225058, U.S. Patent Publication No. 2012/0251531 and Strop et al., 2012, J. Mol. Biol., 420: 204-219).
Examples of mutations that may be introduced into the hinge or CH2 domain to produce a “knock-out” variant include the amino acid modifications L234A/L235A, and L234A/L235A/D265S.
In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise a scaffold based on an IgG Fc in which native glycosylation has been modified. As is known in the art, glycosylation of an Fc may be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine (i.e. N297A, Q, K or H) results in an aglycoslated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).
Conversely, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC, based on improved binding to FcγRIIIa (see, for example, Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low fucose antibodies may be produced, for example in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in the variant CHO cell line, Lee 13, that has a reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03/035835), or in other cells that generate afucosylated antibodies (see, for example, Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, International Publication No. WO 2009/135181 describes the addition of fucose analogues to culture medium during antibody production to inhibit incorporation of fucose into the carbohydrate on the antibody.
Other methods of producing antibodies with little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, the GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Pat. No. 8,409,572).
Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and/or improved ADCC function (see, for example, International Publication No. WO 2003/011878, U.S. Pat. No. 6,602,684 and US Patent Application Publication No. US 2005/0123546). Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, for example, International Publication Nos. WO 1997/030087, WO 1998/58964 and WO 1999/22764).
The anti-NaPi2b antibody constructs described herein may be produced using standard recombinant methods known in the art (see, for example, U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).
Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-NaPi2b antibody construct is generated and inserted into one or more vectors for further cloning and/or expression in a host cell. Polynucleotide(s) encoding the anti-NaPi2b antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and “Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As would be appreciated by one of skill in the art, the number of polynucleotides required for expression of the anti-NaPi2b antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold. For example, when an anti-NaPi2b antibody construct is in a monospecific mAb format or FSA format, two polynucleotides each encoding one polypeptide chain will be required. When multiple polynucleotides are required, they may be incorporated into one vector or into more than one vector.
Generally, for expression, the polynucleotide or set of polynucleotides is incorporated into an expression vector or vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. One skilled in the art will appreciate that the choice of regulatory elements is dependent on the host cell selected for expression of the antibody construct and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal-affinity tags, histidine tags, avidin/streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.
Suitable host cells for cloning or expression of the anti-NaPi2b antibody constructs include various prokaryotic or eukaryotic cells as known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.
In certain embodiments, the anti-NaPi2b antibody construct may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U.S. Pat. Nos. 5,648,237; 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003.
Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).
Suitable host cells for the expression of glycosylated anti-NaPi2b antibody constructs are usually eukaryotic cells. For example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension may be particularly useful for expression of antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse sertoli TM4 cells (see, for example, Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumour (MMT 060562), TRI cells (see, for example, Mather et al., 1982, Annals N.Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR− CHO cells, see Urlaub et al., 1980. Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0 and Sp2/0). Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003).
In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.
The host cells comprising the expression vector(s) encoding the anti-NaPi2b antibody construct may be cultured using routine methods to produce the anti-NaPi2b antibody construct. Alternatively, in some embodiments, host cells comprising the expression vector(s) encoding the anti-NaPi2b antibody construct may be used therapeutically or prophylactically to deliver the anti-NaPi2b antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.
Typically, the anti-NaPi2b antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni+2 affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used. The degree of purification necessary will vary depending on the use of the anti-NaPi2b antibody constructs. In some instances, no purification may be necessary.
In certain embodiments, the anti-NaPi2b antibody constructs are substantially pure. The term “substantially pure” (or “substantially purified”) when used in reference to an anti-NaPi2b antibody construct described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct. In certain embodiments, an anti-NaPi2b antibody construct that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.
Certain embodiments of the present disclosure relate to a method of making an anti-NaPi2b antibody construct comprising culturing a host cell into which one or more polynucleotides encoding the anti-NaPi2b antibody construct, or one or more expression vectors encoding the anti-NaPi2b antibody construct, have been introduced, under conditions suitable for expression of the anti-NaPi2b antibody construct, and optionally recovering the anti-NaPi2b antibody construct from the host cell (or from host cell culture medium).
In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or they be conducted in vitro after isolation of the anti-NaPi2b antibody construct from the host cell.
Post-translational modifications include various modifications as are known in the art (see, for example, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N.Y. Acad. Sci., 663:48-62). In those embodiments in which the anti-NaPi2b antibody constructs comprise one or more post-translational modifications, the constructs may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.
Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4.
Other examples of post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.
Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
The camptothecin analogue comprised by the ADCs of the present disclosure is a compound having Formula (I):
wherein:
—CO2R8, -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl;
In some embodiments, the camptothecin analogues are compounds of Formula (I), with the proviso that when R1 is NH2, R2 is other than H.
In some embodiments, in compounds of Formula (I), R1 is selected from: —CH3, —CF3, —OCH3, —OCF3 and NH2.
In some embodiments, in compounds of Formula (I), R1 is NH2.
In some embodiments, in compounds of Formula (I), R1 is selected from: —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (I), R1 is selected from: —CH3, —CF3, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (I), R2 is selected from: —H, —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (I), R2 is selected from: —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (I), R2 is selected from: —H, —F, —Br and —Cl.
In some embodiments, in compounds of Formula (I), R2 is selected from: —F, —Br and —Cl.
In some embodiments, in compounds of Formula (I), R3 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5,
—CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (I), R4 is selected from:
In some embodiments, in compounds of Formula (I), R5 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (I), R6 and R7 are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5, —C3-C8 heterocycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (I), R8 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (I), each R9 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), each R9 is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), each R9 is independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (I), each R10 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), each R10 is independently selected from: —C1-C6 alkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), each R10 is independently selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, —NR14R14′, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), R10′ is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), R11 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (I), R12 is selected from: —H, —C1-C6 alkyl, —CO2R8, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16.
In some embodiments, in compounds of Formula (I), R12 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl, —S(O)2R16 and
In some embodiments, in compounds of Formula (I), R13 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (I), R14 and R14′ are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (I), R16 is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (I), R16 is selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (I), R17 is selected from: unsubstituted C1-C6 alkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —C3-C8 heterocycloalkyl, —(C1-C6 alkyl)-C3-C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (I), R18 and R19 taken together with the N atom to which they are bonded form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —(C1-C6 alkyl)-O—R5.
In some embodiments, in compounds of Formula (I), Xa and Xb are each independently selected from: NH and O.
Combinations of any of the foregoing embodiments for compounds of Formula (I) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
In certain embodiments, the compound of Formula (I) has Formula (II):
wherein:
—CO2R8, -aryl, -heteroaryl, —(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (II), R2 is selected from: —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (II), R2 is selected from: —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (II), R2 is selected from F and Cl.
In some embodiments, in compounds of Formula (II), R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (II), R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (II), R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
In some embodiments, in compounds of Formula (II), R20 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5,
—CO2R8, unsubstituted aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl,
In some embodiments, in compounds of Formula (II), R2 is selected from: —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (II), R2 is selected from: —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—
R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (II), R2 is selected from: —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R20 is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
In some embodiments, in compounds of Formula (II), R5 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (II), R6 and R7 are each independently selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (II), R6 is H, and R7 is selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5, —C3-C8 heterocycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (II), R6 is H, and R7 is selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (II), R6 and R7 are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5, —C3-C8 heterocycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (II), R8 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (II), each R9 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), each R9 is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), each R9 is independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (II), each R10 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), each R10 is independently selected from: —C1-C6 alkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), each R10 is independently selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, —NR14R14′, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), R10′ is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), R11 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (II), R12 is selected from: —H, —C1-C6 alkyl, —CO2R8, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16.
In some embodiments, in compounds of Formula (II), R12 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl, —S(O)2R16 and
In some embodiments, in compounds of Formula (II), R13 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (II), R14 and R14′ are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (II), R16 is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (II), R16 is selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (II), R17 is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (II), R17 is selected from: unsubstituted C1-C6 alkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —C3-C8 heterocycloalkyl, —(C1-C6 alkyl)-C3-C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (II), R18 and R19 taken together with the N atom to which they are bonded form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —(C1-C6 alkyl)-O—R5.
In some embodiments, in compounds of Formula (II), Xa and Xb are each independently selected from: NH and O.
Combinations of any of the foregoing embodiments for compounds of Formula (II) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
In certain embodiments, the compound of Formula (I) has Formula (III):
wherein:
In some embodiments, in compounds of Formula (III), R2 is selected from: —H, —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (III), R2 is selected from: —H, —F and —Cl.
In some embodiments, in compounds of Formula (III), R15 is selected from: —CH3, —CF3, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (III), R15 is selected from: —CH3 and —OCH3.
In some embodiments, in compounds of Formula (III), R2 is selected from: —H, —F and —Cl, and R15 is selected from: —CH3, —CF3, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (III), R2 is selected from: —H, —F and —Cl, and R15 is selected from: —CH3 and —OCH3.
In some embodiments, in compounds of Formula (III), R4 is selected from:
In some embodiments, in compounds of Formula (III), R5 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (III), R8 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (III), each R9 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), each R9 is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), each R9 is independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (III), each R10 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), each R10 is independently selected from: —C1-C6 alkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), each R10 is independently selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, —NR14R14′, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), R10′ is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), R11 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (III), R12 is selected from: —H, —C1-C6 alkyl, —CO2R8, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16.
In some embodiments, in compounds of Formula (III), R12 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl, —S(O)2R16 and
In some embodiments, in compounds of Formula (III), R13 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (III), R14 and R14′ are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (III), R16 is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (III), R16 is selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (III), R18 and R19 taken together with the N atom to which they are bonded form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —(C1-C6 alkyl)-O—R5.
In some embodiments, in compounds of Formula (III), Xa and Xb are each independently selected from: NH and O.
Combinations of any of the foregoing embodiments for compounds of Formula (III) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl group as defined in any one of Formulae (I), (II) or (III) is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl group as defined in any one of Formulae (I), (II) or (III) is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl and sulfonamido.
In certain embodiments, the camptothecin analogue comprised by the ADC according to the present disclosure is a compound having Formula (I) and is selected from the compounds shown in Tables 6 and 7.
In certain embodiments, the camptothecin analogue is a compound having Formula (II). In some embodiments, the camptothecin analogue is a compound having Formula (II), in which R2 is F, and R20 is H, —(C1-C6)—O—R5 or
In some embodiments, the camptothecin analogue is a compound having Formula (II), in which R2 is F; R20 is H, —(C1-C6)—O—R5 or
R5 is H, and R18 and R19 taken together with the N atom to which they are bonded form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, the camptothecin analogue is a compound having Formula (II), in which R2 is F; R20 is —(C1-C6)—O—R5, and R5 is H. In certain embodiments, the camptothecin analogue is a compound having Formula (II) and is selected from the compounds shown in Table 6.
In certain embodiments, the camptothecin analogue is a compound having Formula (III). In certain embodiments, the camptothecin analogue is a compound having Formula (III), in which R2 is F; R15 is —CH3; R4 is
R9 is —C1-C6 hydroxyalkyl, and Xa and Xb are each O. In certain embodiments, the camptothecin analogue is a compound having Formula (III) and is selected from the compounds shown in Table 7.
In certain embodiments, the camptothecin analogue comprised by the ADC according to the present disclosure is Compound 139, Compound 140, Compound 141 or Compound 148. In some embodiments, the camptothecin analogue comprised by the ADC according to the present disclosure is Compound 139 or Compound 141.
It is to be understood that reference to compounds of Formula (I) throughout this disclosure, includes in various embodiments, compounds of Formula (II) and Formula (III), as well as the individual compounds shown in Tables 6 and 7, to the same extent as if embodiments reciting each of these Formulae or compounds individually were specifically recited.
The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analogue having Formula (I). In certain embodiments, the ADC has Formula (X):
T-[L-(D)m]n (X)
In certain embodiments, in conjugates of Formula (X), m is between 1 and 2. In some embodiments, m is 1.
In some embodiments, in conjugates of Formula (X), n is between 1 and 8, for example, between 2 and 8. In some embodiments, n is between 4 and 8.
In certain embodiments, in conjugates of Formula (X), m is between 1 and 2, and n is between 2 and 8, or between 4 and 8. In some embodiments, in conjugates of Formula (X), m is 1, and n is between 2 and 8, or between 4 and 8.
As noted above and reflected by parameters m and n in Formula (X), the anti-NaPi2b antibody construct, “T,” can be conjugated to more than one compound of Formula (I), “D.” Those skilled in the art will appreciate that, while any particular anti-NaPi2b antibody construct T is conjugated to an integer number of compounds D, analysis of a preparation of the conjugate to determine the ratio of compound D to anti-NaPi2b antibody construct T may give a non-integer result, reflecting a statistical average. This ratio of compound D to targeting moiety T may generally be referred to as the drug-to-antibody ratio, or “DAR.” Accordingly, conjugate preparations having non-integer DARs are intended to be encompassed by Formula (X).
In certain embodiments, in the conjugates of Formula (X), D is a compound of Formula Formula (II) or Formula (III). In certain embodiments, in the conjugates of Formula (X), D is a compound selected from the compounds shown in Tables 6 and 7. In certain embodiments, in the conjugates of Formula (X), D is Compound 139, Compound 140, Compound 141 or Compound 148. In some embodiments, in the conjugates of Formula (X), D is Compound 139 or Compound 141.
Certain embodiments of the present disclosure relate to ADCs having Formula (X), in which D is a compound of Formula (IV):
wherein:
wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or
In some embodiments, in compounds of Formula (IV), R1a is selected from: —CH3, —CF3, —OCH3, —OCF3 and —NH2.
In some embodiments, in compounds of Formula (IV), R1a is selected from: —CH3, —CF3, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (IV), Ria is selected from: —CH3, —OCH3 and NH2.
In some embodiments, in compounds of Formula (IV), R1a is selected from: —CH3 and —OCH3.
In some embodiments, in compounds of Formula (IV), R2a is selected from: —H, —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (IV), R2a is selected from: —H, —F and —Cl.
In some embodiments, in compounds of Formula (IV), R2a is —F.
In some embodiments, in compounds of Formula (IV), X is —O—, —S— or —NH—, and R4a is selected from:
In some embodiments, in compounds of Formula (IV), X is —O— or —NH—.
In some embodiments, in compounds of Formula (IV), each R9a is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (IV), each R9a is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (IV), each R10a is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl, —(C1-C6 alkyl)-aryl and
In some embodiments, in compounds of Formula (IV), each R10a is independently selected from: —C1-C6 alkyl, -aryl, —(C1-C6 alkyl)-aryl and
In some embodiments, in compounds of Formula (IV), R12a is selected from: —C1-C6 alkyl, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16.
In some embodiments, in compounds of Formula (IV), R13a is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (IV), R14a′ is selected from: H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (IV), R16a is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (IV), R22 and R23 are each independently selected from: —H, -halogen, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 aminoalkyl, —C1-C6 hydroxyalkyl and —C3-C8 cycloalkyl.
In some embodiments, in compounds of Formula (IV), Xa and Xb are each independently selected from: NH and O.
In some embodiments, in compounds of Formula (IV), Xa and Xb are each O.
In some embodiments, in compounds of Formula (IV), X is O; R4a is
Xa and Xb are each O, and R9a is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (IV), R1a is —CH3 or —OCH3; X is O; R4a is
Xa and Xb are each O; and R9a is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (IV), R1a is —CH3 or —OCH3; R2a is H or F; X is O; R4a is
Xa and Xb are each O; and R9a is —C1-C6 alkyl.
Other combinations of any of the foregoing embodiments for compounds of Formula (IV) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
Certain embodiments of the present disclosure relate to ADCs having Formula (X), in which D is a compound of Formula (V):
wherein:
—CO2R8, -aryl, -heteroaryl, —(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (V), R2a is selected from: —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (V), R2a is selected from: —CF3, —F, —Cl and —OCH3.
In some embodiments, in compounds of Formula (V), R2a is F.
In some embodiments, in compounds of Formula (V), R20a is selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5,
—CO2R8, -aryl, -heteroaryl, —(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (V), R20a is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (V), R20a is selected from: —H, —C1-C6alkyl, —(C1-C6 alkyl)-O—R5,
—(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (V), R20a is selected from: —H, —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5,
In some embodiments, in compounds of Formula (V), R20a is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5,
—CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl,
In some embodiments, in compounds of Formula (V), R6 and R7 are each independently selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (V), R6 is H, and R7 is selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5, —C3-C8 heterocycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (V), R6 is H, and R7 is selected from: —H, —C1-C6 alkyl, —C3-C8 cycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (V), R6 and R7 are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl, —(C1-C6 alkyl)-O—R5, —C3-C8 heterocycloalkyl and —C(O)R17.
In some embodiments, in compounds of Formula (V), R8 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (V), each R9 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (V), each R9 is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (V), each R9 is independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (V), each R10 is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (V), each R10 is independently selected from: —C1-C6 alkyl, —NR14R14′, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (V), R11 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (V), R12 is selected from: —H, —C1-C6 alkyl, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16.
In some embodiments, in compounds of Formula (V), R12 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —CO2R8, unsubstituted -aryl, -aminoaryl, -heteroaryl, —(C1-C6 alkyl)-aminoaryl, —S(O)2R16 and
In some embodiments, in compounds of Formula (V), R13 is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (V), R14 and R14′ are each independently selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (V), R16 is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (V), R16 is selected from: unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (V), R17 is selected from: unsubstituted —C1-C6 alkyl, —C3-C8 cycloalkyl, —C3-C8 heterocycloalkyl, —(C1-C6 alkyl)-C3-C5 heterocycloalkyl, unsubstituted -aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and —(C1-C6 alkyl)-aminoaryl.
In some embodiments, in compounds of Formula (V), R18 and R19 taken together with the N atom to which they are bonded form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 aminoalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl and —(C1-C6 alkyl)-O—R5.
In some embodiments, in compounds of Formula (V), R17 is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (V), Xa and Xb are each independently selected from: NH and O.
In some embodiments, in compounds of Formula (V), Xa and Xb are each O.
In some embodiments, in compounds of Formula (V), R20a is —(C1-C6 alkyl)-O—R5.
In some embodiments, in compounds of Formula (V), R20a is —(C1-C6 alkyl)-O—R5, and R5 is H.
In some embodiments, in compounds of Formula (V), R2a is F; R20a is —(C1-C6 alkyl)-O—R5, and R5 is H.
Other combinations of any of the foregoing embodiments for compounds of Formula (V) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
Certain embodiments of the present disclosure relate to ADCs having Formula (X), in which D is a compound of Formula (VI):
wherein:
wherein *is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or
In some embodiments, in compounds of Formula (VI), R2a is selected from: —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (VI), R2a is selected from: —CH3, —CF3, —F, —Cl, —OCH3 and —OCF3.
In some embodiments, in compounds of Formula (VI), R2a is selected from: F and Cl.
In some embodiments, in compounds of Formula (VI), R2a is F.
In some embodiments, in compounds of Formula (VI), X is —O—, —S— or —NH—, and R25 is selected from: —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5a, —(C1-C6 alkyl)-aryl,
or X is O, and R25—X— is selected from:
In some embodiments, in compounds of Formula (VI), X is —O—, —S— or —NH—, and R25 is selected from: —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5a, —(C1-C6 alkyl)-aryl,
In some embodiments, in compounds of Formula (VI), X is —O—, —S— or —NH—, and R25 is selected from: —C1-C6 alkyl, —(C1-C6 alkyl)-O—R5a,
In some embodiments, in compounds of Formula (VI), X is —O—, —S— or —NH—, and R25 is selected from:
In some embodiments, in compounds of Formula (VI), X is —O— or —NH—.
In some embodiments, in compounds of Formula (VI), R6a is H.
In some embodiments, in compounds of Formula (VI), R6a is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (VI), R7a is selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl and —C(O)R17a.
In some embodiments, in compounds of Formula (VI), each R9a is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (VI), each R9a is independently selected from: —C1-C6 alkyl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (VI), each R10a is independently selected from: —C1-C6 alkyl, —C3-C8 cycloalkyl, -aryl, —(C1-C6 alkyl)-aryl and
In some embodiments, in compounds of Formula (VI), each R10a is independently selected from: —C1-C6 alkyl, -aryl, —(C1-C6 alkyl)-aryl and
In some embodiments, in compounds of Formula (VI), R12a is selected from: —C1-C6 alkyl, -aryl, —(C1-C6 alkyl)-aryl and —S(O)2R16a.
In some embodiments, in compounds of Formula (VI), R13a is selected from: —H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl and —C1-C6 aminoalkyl.
In some embodiments, in compounds of Formula (VI), R14a′ is selected from: H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl and —C3-C8 heterocycloalkyl.
In some embodiments, in compounds of Formula (VI), R16a is selected from: -aryl, -heteroaryl and —(C1-C6 alkyl)-aryl.
In some embodiments, in compounds of Formula (VI), R17a is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (VI), R22 and R23 are each independently selected from: —H, -halogen, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl and —C3-C8 cycloalkyl.
In some embodiments, in compounds of Formula (VI), Xa and Xb are each independently selected from: NH and O.
In some embodiments, in compounds of Formula (VI), Xa and Xb are each O.
In some embodiments, in compounds of Formula (VI), X is O, and R25 is —C1-C6 alkyl.
In some embodiments, in compounds of Formula (VI), R2a is F; X is O, and R25 is —C1-C6 alkyl.
Other combinations of any of the foregoing embodiments for compounds of Formula (VI) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl group as defined in any one of Formulae (IV), (V) or (VI) is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl group as defined in any one of Formulae (IV), (V) or (VI) is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl and sulfonamido.
In certain embodiments, in ADCs having Formula (X), D is a compound of Formula (IV), in which R1a is —CH3, and R2a is F. In some embodiments, in ADCs having Formula (X), D is a compound of Formula (IV), in which R1a is —CH3; R2a is F; X is —O—; R4a is
R9a is —C1-C6 alkyl, and Xa and Xb are each O.
In certain embodiments, in ADCs having Formula (X), D is a compound of Formula (V), in which R2a is F, and R20a is H, —(C1-C6)—O—R5 or
In some embodiments, in ADCs having Formula (X), D is a compound of Formula (V), in which R2a is F; R20a is H, —(C1-C6)—O—R5 or
R5 is H, and R18 and R19 taken together with the N atom to which they are bonded form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, in ADCs having Formula (X), D is a compound of Formula (V), in which R2a is F; R20a is —(C1-C6)—O—R5, and R5 is H.
In certain embodiments, in ADCs having Formula (X), D is a compound of Formula (VI), in which R2a is F; X is —O—, and R25 is —C1-C6 alkyl.
The conjugates of Formula (X) include a linker, L, which is a bifunctional or multifunctional moiety capable of linking one or more camptothecin analogues, D, to the anti-NaPi2b antibody construct, T. A bifunctional (or monovalent) linker, L, links a single compound D to a single site on the anti-NaPi2b antibody construct, T, whereas a multifunctional (or polyvalent) linker, L, links more than one compound, D, to a single site on the anti-NaPi2b antibody construct, T. A linker that links one compound, D, to more than one site on the anti-NaPi2b antibody construct, T, may also be considered to be multifunctional.
Linker, L, includes a functional group capable of reacting with the target group or groups on the anti-NaPi2b antibody construct, T, and at least one functional group capable of reacting with a target group on the camptothecin analogue, D. Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). Groups on the anti-NaPi2b antibody construct, T, and the camptothecin analogue, D, that may serve as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde and ketone groups.
Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “self-stabilizing” maleimides as described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062.
Non-limiting examples of functional groups capable of reacting with amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut's reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the use of succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to convert a carboxyl group to an activated ester, which may then be reacted with an amine.
Non-limiting examples of functional groups capable of reacting with an electrophilic group such as an aldehyde or ketone carbonyl group include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
In certain embodiments, linker, L, may include a functional group that allows for bridging of two interchain cysteines on the anti-NaPi2b antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).
Alternatively, the anti-NaPi2b antibody construct, T, may be modified to include a non-natural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker. For example, conjugation of the linker to the anti-NaPi2b antibody construct may make use of click chemistry reactions (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97), such as the azide-alkyne cycloaddition (AAC) reaction, which has been used successfully in the development of antibody-drug conjugates. The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling of an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling of an azide with a cyclooctyne.
Linker, L, may be a cleavable or a non-cleavable linker. A cleavable linker is a linker that is susceptible to cleavage under specific conditions, for example, intracellular conditions (such as in an endosome or lysosome) or within the vicinity of a target cell (such as in the tumor microenvironment). Examples include linkers that are protease-sensitive, acid-sensitive or reduction-sensitive. Non-cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-drug moiety.
Examples of cleavable linkers include, for example, linkers comprising an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by a cell, for example, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix in the vicinity of a target cell, such as a cancer cell, may be employed. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteases (MMPs), elastase and kallikrein-related peptidases.
For conjugates intended to be internalized by a cell, linker, L, may comprise an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease. Examples of such proteases include, for example, cathepsins B, C, D, H, L and S, and legumain.
Cleavage recognition sequences may be, for example, dipeptides, tripeptides or tetrapeptides. Non-limiting examples of dipeptide recognition sequences that may be included in cleavable linkers include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys. Examples of tri- and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.
Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimydyl-4-(2-pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Other cleavable linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.
A further example of a cleavable linker is a linker comprising a β-glucuronide, which is cleavable by β-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007/011968). β-glucuronide may also function to improve the hydrophilicity of linker, L.
Another example of a linker that is cleaved internally within a cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, for example, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).
In certain embodiments, the linker, L, comprised by the conjugate of Formula (X) is a cleavable linker. In some embodiments, linker, L, comprises a cleavage recognition sequence. In some embodiments, linker, L, may comprise an amino acid sequence that is recognized and cleaved by a lysosomal protease.
Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative and self-elimination groups, stretchers, or hydrophilic moieties.
Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylene diamine (MED) and hemi-aminal groups. Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB or PABC group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Pat. No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-immolative/self-elimination groups are typically attached to an amino or hydroxyl group on the compound, D. Self-immolative/self-elimination groups, alone or in combination are often included in peptide-based linkers, but may also be included in other types of linkers.
Stretchers that find use in linkers for drug conjugates include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers.
PEG and mPEG stretchers can also function as hydrophilic moieties within a linker. For example, PEG or mPEG may be included in a linker either “in-line” or as pendant groups to increase the hydrophilicity of the linker (see, for example, U.S. Patent Application Publication No. US 2016/0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that may optionally be incorporated into linker, L, include, for example, β-glucuronide, sulfonate groups, carboxylate groups and pyrophosphate diesters.
In certain embodiments, ADCs of Formula (X) may comprise a cleavable linker. In some embodiments, ADCs of Formula (X) may comprise a peptide-containing linker. In some embodiments, ADCs of Formula (X) may comprise a protease-cleavable linker.
In some embodiments, in ADCs of Formula (X), m is 1, and linker, L, is a cleavable linker having Formula (XI):
In some embodiments, in linkers of Formula (XI), q is 1.
In some embodiments, in linkers of Formula (XI), s is 1. In some embodiments, in ADCs of Formula (XI), s is 0.
In some embodiments, in linkers of Formula (XI), r is 1. In some embodiments, in ADCs of Formula (XI), r is 3.
In some embodiments, in linkers of Formula (XI):
where # is the point of attachment to T, and * is the point of attachment to the remainder of the linker.
In some embodiments, in linkers of Formula (XI), Str is selected from:
In some embodiments, in linkers of Formula (XI), Str is selected from:
In some embodiments, in linkers of Formula (XI), AA1-[AA2]r is a dipeptide (i.e. r=1).
In some embodiments, in linkers of Formula (XI), AA1-[AA2]r has a sequence selected from: Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.
In some embodiments, in linkers of Formula (XI), AA1-[AA2]r is a tripeptide (i.e. r=2). In some embodiments, in linkers of Formula (XI), AA1-[AA2]r has a sequence selected from: Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.
In some embodiments, in linkers of Formula (XI), AA1-[AA2]r is a tetrapeptide (i.e. r=3). In some embodiments, in linkers of Formula (XI), AA1-[AA2]r has a sequence selected from: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.
In certain embodiments, in ADCs of Formula (X), m is 1, and linker, L, is a cleavable linker having Formula (XII):
In some embodiments, in linkers of Formula (XII), q is 1.
In some embodiments, in linkers of Formula (XII), v is 0. In some embodiments, in ADCs of Formula (XII), s is 1.
In some embodiments, in linkers of Formula (XII), r is 1. In some embodiments, in ADCs of Formula (XII), r is 3.
In some embodiments, in linkers of Formula (XII):
where # is the point of attachment to T, and * is the point of attachment to the remainder of the linker.
In some embodiments, in linkers of Formula (XII), Str is selected from:
In some embodiments, in linkers of Formula (XII), Str is selected from:
In some embodiments, in linkers of Formula (XII), AA1-[AA2]r is a dipeptide (i.e. r=1). In some embodiments, in linkers of Formula (XII), AA1-[AA2]r has a sequence selected from: Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.
In some embodiments, in linkers of Formula (XII), AA1-[AA2]r is a tripeptide (i.e. r=2). In some embodiments, in linkers of Formula (XII), AA1-[AA2]r has a sequence selected from: Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val.
In some embodiments, in linkers of Formula (XII), AA1-[AA2]r is a tetrapeptide (i.e. r=3). In some embodiments, in linkers of Formula (XII), AA1-[AA2]r has a sequence selected from: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.
In some embodiments, in linkers of Formula (XII), Y is —NH—CH2. In some embodiments, in linkers of Formula (XII), v is 1 and Y is —NH—CH2.
In some embodiments, ADCs of Formula (X) may comprise a disulfide-containing linker. In some embodiments, in ADCs of Formula (X), m is 1, and linker, L, is a cleavable linker having Formula (XIII):
In some embodiments, ADCs of Formula (X) may comprise a β-glucuronide-containing linker.
Various non-cleavable linkers are known in the art for linking drugs to targeting moieties and may be useful in the ADCs of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the anti-NaPi2b antibody construct, as well as a maleimido- or haloacetyl-based moiety for reaction with the camptothecin analogue, or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols, —SH) on the camptothecin analogue, while the sulfo-NHS ester is reactive toward primary amines (as found in lysine and at the N-terminus of proteins or peptides) on the anti-NaPi2b antibody construct. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (“long chain” SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N—(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those comprising a haloacetyl-based functional group such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).
Non-limiting examples of drug-linkers comprising camptothecin analogues of Formula (I) are shown in Table 8, Table 9, and Table 10. Non-limiting examples of conjugates comprising these drug-linkers are shown in Table 11, Table 12 and Table 13. In certain embodiments, the ADC of Formula (X) comprises a drug-linker selected from the drug-linkers shown in Tables 8, 9 and 10. In certain embodiments, the ADC of Formula (X) is selected from the conjugates shown in Tables 11, 12 and 13, where T is the anti-NaPi2b antibody construct and n is between 1 and 10. In some embodiments, the ADC of Formula (X) is selected from the conjugates shown in Tables 11, 12 and 13, where T is the anti-NaPi2b antibody construct and n is between 2 and 8. In some embodiments, the ADC of Formula (X) is selected from the conjugates shown in Tables 11, 12 and 13, where T is the anti-FRα antibody construct and n is between 4 and 8.
In certain embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)m) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148 and MC-GGFG-Compound 148, and n is 4 or 8. In some embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)m) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148 and MC-GGFG-Compound 148, and n is 8.
ADCs of Formula (X) may be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press)). In addition, various antibody drug conjugation services are available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).
Typically, preparation of the ADCs comprises first preparing a drug-linker, D-L, comprising one or more camptothecin analogues of Formula (I) and linker L, and then conjugating the drug-linker, D-L, to an appropriate group on the anti-NaPi2b antibody construct, T. Ligation of linker, L, to the anti-NaPi2b antibody construct, T, and subsequent ligation of the anti-NaPi2b antibody construct-linker, T-L, to one or more camptothecin analogues of Formula (I), D, remains however an alternative approach that may be employed in some embodiments.
Suitable groups on compounds of Formula (I), D, for attachment of linker, L, in either of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups and hydroxyl groups. In some embodiments of the present disclosure, linker, L, is attached to a compound of Formula (I), D, via a hydroxyl or amine group on the compound.
Suitable groups on the anti-NaPi2b antibody construct, T, for attachment of linker, L, in either of the above approaches include sulfhydryl groups (for example, on the side-chain of cysteine residues), amino groups (for example, on the side-chain of lysine residues), carboxylic acid groups (for example, on the side-chains of aspartate or glutamate residues), and carbohydrate groups.
For example, the anti-NaPi2b antibody construct T may comprise one or more naturally occurring sulfhydryl groups allowing the anti-NaPi2b antibody construct, T, to bond to linker, L, via the sulfur atom of a sulfhydryl group. Alternatively, the anti-NaPi2b antibody construct, T, may comprise one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”) and 2-iminothiolane hydrochloride (Traut's Reagent). Alternatively, the anti-NaPi2b antibody construct, T, may comprise one or more carbohydrate groups that can be chemically modified to include one or more sulfhydryl groups.
Carbohydrate groups on the anti-NaPi2b antibody construct, T, may also be oxidized to provide an aldehyde (—CHO) group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which could subsequently be reacted with linker, L, for example, via a hydrazine or hydroxylamine group on linker, L.
The anti-NaPi2b antibody construct, T, may also be modified to include additional cysteine residues (see, for example, U.S. Pat. Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, for example, Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow for site-specific conjugation. Alternatively, the anti-NaPi2b antibody construct, T, may be modified to include a non-natural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker, for example, for example, by click chemistry (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect™ technology (Synaffix B V, Nijmegen, Netherlands), which involves enzymatic remodelling of the antibody glycans to allow for attachment of a linker by metal-free click chemistry (see, for example, European Patent No. EP 2 911 699).
Other protocols for the modification of proteins for the attachment or association of linker, L, are known in the art and include those described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002).
Alternatively, ADCs may be prepared using the enzyme transglutaminase, in particular, bacterial transglutaminase (BTG) from Streptomyces mobaraensis (see, for example, Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of a glutamine (the amine acceptor, typically on the antibody) and an alkyleneamino group (the amine donor, typically on the drug-linker), which can be, for example, the ε-amino group of a lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a glutamine containing peptide, or “tag,” which allows BTG conjugation to be used to conjugate the antibody to a drug-linker (see, for example, U.S. Patent Application Publication No. US 2013/0230543 and International (PCT) Publication No. WO 2016/144608).
A similar conjugation approach utilizes the enzyme sortase A. In this approach, the antibody is typically modified to include the sortase A recognition motif (LPXTG, where X is any natural amino acid) and the drug-linker is designed to include an oligoglycine motif (typically GGG) to allow for sortase A-mediated transpeptidation (see, for example, Beerli, et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature:Scientific Reports, 6:31899).
Once conjugation is complete, the average number of compounds of Formula (I) conjugated to the anti-NaPi2b antibody construct, T, (i.e. the “drug-to-antibody ratio” or DAR) may be determined by standard techniques such as UV/VIS spectroscopic analysis, ELISA-based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS. In addition, distribution of drug-linked forms (for example, the fraction of the anti-NaPi2b antibody construct, T, containing zero, one, two, three, etc. compounds of Formula (I), D) may also optionally be analyzed. Various techniques are known in the art to measure DAR distribution, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al., 2011, mAbs, 3:161-172).
For therapeutic uses, the ADCs of the present disclosure are typically formulated as pharmaceutical compositions. Certain embodiments of the present disclosure thus relate to pharmaceutical compositions comprising an ADC as described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions may be prepared by known procedures using well-known and readily available ingredients.
Pharmaceutical compositions may be formulated for administration to a subject by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spray. The term “parenteral” as used herein includes subcutaneous injection, and intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions may be provided as unit dosage formulations.
In certain embodiments, the pharmaceutical compositions comprising the ADCs are formulated for parenteral administration, for example as lyophilized formulations or aqueous solutions. Such pharmaceutical compositions may be provided, for example, in a unit dosage injectable form.
Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).
In certain embodiments, the compositions comprising the ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and/or suspending agent that are known in the art. The sterile injectable solution or suspension may comprise the ADC in a non-toxic parentally acceptable diluent or carrier. Acceptable diluents and carriers that may be employed include, for example, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a carrier. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and/or buffering agents may also be included in the injectable solution or suspension.
In certain embodiments, the composition comprising the ADC may be formulated for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and/or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
Certain embodiments of the present disclosure relate to the therapeutic use of the ADCs described herein. Some embodiments relate to the use of the ADCs as therapeutic agents.
Certain embodiments of the present disclosure relate to methods of inhibiting abnormal cancer cell or tumor cell growth; inhibiting cancer cell or tumor cell proliferation, or treating cancer in a subject, comprising administering an ADC described herein. In certain embodiments, the ADCs described herein may be used in the treatment of cancer. Some embodiments of the present disclosure thus relate to the use of the ADCs as anti-cancer agents.
Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells comprising contacting the cells with an ADC as described herein, for example, an ADC of Formula (X). Some embodiments relate to a method of killing cancer or tumor cells comprising contacting the cells with an ADC as described herein, for example, an ADC of Formula (X).
Some embodiments relate to methods of treating a subject having a cancer by administering to the subject an ADC as described herein, for example, an ADC of Formula (X). In this context, treating the subject may result in one or more of a reduction in the size of a tumor, the slowing or prevention of an increase in the size of a tumor, an increase in the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of a subsequent occurrence of a tumor (for example, metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumor, and/or an increase in the overall survival time of a subject having cancer.
Certain embodiments relate to the use of an ADC as described herein, for example, an ADC of Formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC as described herein, for example, an ADC of Formula (X), in a method of inhibiting proliferation of and/or killing cancer cells in vitro. Some embodiments relate to the use of an ADC as described herein, for example, an ADC of Formula (X), in a method of inhibiting proliferation of and/or killing cancer cells in vivo in a subject having a cancer.
Examples of cancers which may be treated in certain embodiments are carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as “solid tumors.” Examples of commonly occurring solid tumors that may be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, uterine cancer, non-small cell lung cancer (NSCLC) and colorectal cancer. Various forms of lymphoma also may result in the formation of a solid tumor and, therefore, may also be considered to be solid tumors in certain situations. Typically, the cancer to be treated is an NaPi2b-expressing cancer.
Certain embodiments relate to methods of inhibiting the growth of NaPi2b-positive tumor cells comprising contacting the cells with an ADC as described herein, for example, an ADC of Formula (X). The cells may be in vitro or in vivo. In certain embodiments, the ADCs may be used in methods of treating an NaPi2b-positive cancer or tumor in a subject.
Cancers that overexpress NaPi2b are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, and lung cancers (such as non-small cell lung cancer (NSCLC). In one embodiment, an ADC as described herein may be used in a method of treating ovarian cancer or lung cancer. In one embodiment, an ADC as described herein may be used in a method of treating NSCLC.
Certain embodiments relate to pharmaceutical kits comprising an ADC as described herein, for example, an ADC of Formula (X).
The kit typically will comprise a container holding the ADC and a label and/or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.
In addition to the container holding the ADC, the kit may optionally comprise one or more additional containers comprising other components of the kit. For example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution), other buffers or diluents.
Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. If appropriate, one or more components of the kit may be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).
The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.
The following Examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.
Examples 1-3 below illustrate various methods of preparing camptothecin analogues of Formula (I). It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known in the art. It is also understood that one skilled in the art would be able to make, using the methods described below or similar methods, other compounds of Formula (I) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (Thermo Fisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or synthesized according to sources known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013) or prepared as described herein.
The following abbreviations are used throughout the Examples section: BCA: bicinchonic acid; Boc: di-tert-butyl dicarbonate; CE-SDS: capillary electrophoresis sodium dodecyl sulfate; DCM: dichloromethane; DTPA: diethylenetriamine pentaacetic acid; DIPEA: N,N-diisopropylethylamine; DMF: dimethylformamide; DMM™: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-rnethyl-morpholinium chloride; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc: fluorenylmethyloxycarbonyl; HATU: hexafluorophosphate azabenzotriazole tetramethyl uronium; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HPLC: high-performance liquid chromatography; LC/MS: liquid chromatography mass spectrometry; MC: maleimidocaproyl; MT: maleimidotriethylene glycolate; NMM: N-methylmorpholine; PNP: p-nitrophenol; RP-UPLC-MS: reversed-phase ultra-high performance chromatography mass spectrometry; SEC: size exclusion chromatography; TCEP: tris(2-carboxyethyl) phosphine; Tfp: tetrafluorophenyl; TLC: thin layer chromatography; TFA: trifluoracetic acid.
To a stirring solution of chloride compound in dimethylformamide (0.05-0.1 M) was added the appropriate secondary amine (3 eq.). Upon completion (determined by LC/MS, typically 1-3 h), the reaction mixture was purified by reverse-phase HPLC to provide the desired product after lyophilization.
To a stirring solution of amine compound in dimethylformamide (0.05-0.1 M) was added triethylamine (1.2 eq.), the appropriate carboxylic acid (1.1 eq.) followed by a solution of DMM™ (2 eq.) in water (1 M). Upon completion (determined by LC/MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to provide the desired product after lyophilization.
To a stirring solution of amine compound in dimethylformamide (0.05-0.1 M) was added DIPEA (3 eq.) followed by the appropriate sulfonyl chloride. Upon completion (determined by LC/MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to provide the desired product after lyophilization.
Step 1: To a stirring solution of amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 eq.) then triethylamine (2 eq.). Upon completion (determined by LC/MS typically 1-4 h), the reaction mixture was concentrated to dryness then purified by reverse-phase HPLC to provide the desired PNP-carbamate intermediate after lyophilization. This intermediate can either used to generate a single analog or be divided into multiple batches in order to generate multiple analogs in the second step. Step 2: To the PNP-carbamate intermediate in dimethylformamide (0.1-0.2 M) was added the appropriate primary amine (3 eq.). Upon completion (determined by LC/MS, typically 1 h), the reaction mixture was purified by reverse-phase HPLC to provide the desired product after lyophilization.
To a stirring solution of amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 eq.) then triethylamine (2 eq.). Upon completion (determined by LC/MS, typically 1-4 h), the appropriate alcohol was added to the resultant PNP-carbamate intermediate. Upon completion (determined by LC/MS, typically 1-16 h), the reaction mixture was purified by reverse-phase HPLC to provide the desired product after lyophilization.
To a stirring solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20% by volume). Upon completion (determined by LC/MS, typically 1 h), the reaction mixture was concentrated in vacuo to provide a crude solid or was purified as described in General Procedure 9.
To a rapidly stirring solution of Boc-GGFG-OH (3 eq.) and HOAt (3 eq.) in a 10% v/v mixture of dimethyl formamide in dichloromethane (0.02 M) was added EDC (HCl salt, 3 eq.). After 5 min, a solution of the amine containing payload (1 eq.) in a 10% v/v mixture of dimethyl formamide in dichloromethane (0.02 M) was added, followed immediately by the addition of CuCl2 (4 eq.). Upon completion (determined by LC/MS, typically 1-16 h), the reaction mixture was concentrated in vacuo to provide a crude solid or was purified by preparative HPLC to provide the desired product after lyophilization.
To a stirring solution of amine compound (1 eq.) in dimethylformamide (˜0.02 M) was added a solution of MT-OTfp (1.2-1.5 eq.) in acetonitrile (˜0.02 M) then DIPEA (10 μL, 4 eq.). Upon completion (determined by LC/MS, typically 1-16 h), the reaction mixture was concentrated in vacuo to provide a crude solid which was purified by preparative HPLC to provide the desired product after lyophilization.
Flash Chromatography: Crude reaction products were purified with Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC), eluting with linear gradients of ethyl acetate/hexanes or methanol/dichloromethane on a Biotage® Isolera™ automated flash system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was conducting using Biotage® Snap Ultra C18 columns (12, 30, 60, or 120 g), eluting with linear gradients of 0.1% TFA in acetonitrile/0.1% TFA in water. Purified compounds were isolated by either removal of organic solvents by rotavap or lyophilization of acetonitrile/water mixtures.
Preparative HPLC: Reverse-phase HPLC of crude compounds was performed using a Luna® 5-μm C18 100 Å (150×30 mm) column (Phenomenex, Torrance, CA) on an Agilent 1260 Infinity II preparative LC/MSD system (Agilent Technologies, Inc., Santa Clara, CA), and eluting with linear gradients of 0.1% TFA in acetonitrile/0.1% TFA in water. Purified compounds were isolated by lyophilization of acetonitrile/water mixtures.
LC/MS: Reactions were monitored for completion and purified compounds were analyzed using a Kinetex® 2.6-μm C18 100 Å (30×3 mm) column (Phenomenex, Torrance, CA) on an Agilent 1290 HPLC/6120 single quad LC/MS system (Agilent Technologies, Inc., Santa Clara, CA), eluting with a 10 to 100% linear gradient of 0.1% formic acid in acetonitrile/0.1% formic acid in water.
NMR: 1H NMR spectra were collected with a Bruker AVANCE III 300 Spectrometer (300 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are reported in parts per million (ppm).
The title compound was prepared according to the procedure provided in Li, et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.
The title compound was prepared according to the procedure provided in Li, et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).
LC/MS: Calc'd m/z=479.2 for C26H26FN3O5, found [M+H]+=480.4.
1H NMR (300 MHz, CDCl3) δ 8.20 (d, J=8.0 Hz, 1H), 7.82 (d, J=10.4 Hz, 1H), 7.67 (s, 1H), 5.77 (d, J=16.4 Hz, 1H), 5.42 (s, 2H), 5.33 (d, J=16.4 Hz, 1H), 4.26 (s, 2H), 3.81 (t, J=4.7 Hz, 4H), 2.82-2.76 (m, 4H), 2.57 (d, J=1.7 Hz, 3H), 1.99-1.82 (m, 2H), 1.06 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).
LC/MS: Calc'd m/z=618.2 for C32H31FN4O6, found [M+H]+=619.4.
1H NMR (300 MHz, CDCl3) δ 8.07 (d, J=7.9 Hz, 1H), 7.88-7.44 (m, 7H), 5.73 (d, J=16.4 Hz, 1H), 5.33 (s, 2H), 5.33-5.26 (m, 1H), 4.19 (s, 2H), 3.12 (s, 4H), 2.80 (s, 4H), 2.54 (s, 3H), 1.90 (dt, J=11.6, 7.0 Hz, 2H), 1.04 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).
LC/MS: Calc'd m/z=633.2 for C32H32FN5O6, found [M+H]+=634.4.
1H NMR (300 MHz, MeOD) δ 8.32 (d, J=8.0 Hz, 1H), 7.85 (d, J=10.5 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=8.7 Hz, 2H), 6.74 (d, J=8.7 Hz, 2H), 5.61 (d, J=16.5 Hz, 1H), 5.44 (s, 2H), 5.41 (d, J=16.5 Hz, 1H), 4.51 (s, 2H), 3.22-3.07 (m, 8H), 2.58 (s, 3H), 2.03-1.93 (m, 2H), 1.02 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).
LC/MS: Calc'd m/z=492.2 for C27H29FN4O4, found [M+H]+=493.4.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).
LC/MS: Calc'd m/z=569.2 for C32H32FN5O4, found [M+H]+=570.4.
1H NMR (300 MHz, MeOD) δ 8.39 (d, J=8.1 Hz, 1H), 7.79 (d, J=10.6 Hz, 1H), 7.21 (d, J=9.0 Hz, 2H), 7.14 (d, J=9.0 Hz, 2H), 5.62 (d, J=16.4 Hz, 1H), 5.49 (s, 2H), 5.41 (d, J=16.4 Hz, 1H), 4.45 (s, 2H), 3.44-3.38 (m, 4H), 3.06-3.00 (m, 4H), 2.58 (d, J=1.8 Hz, 3H), 2.00-1.89 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).
LC/MS: Calc'd m/z=477.2 for C27H28FN3O4, found [M+H]+=478.2.
1H NMR (300 MHz, MeOD) δ 8.34 (d, J=7.6 Hz, 1H), 7.94 (d, J=10.3 Hz, 1H), 7.70 (s, 1H), 5.63 (d, J=16.4 Hz, 1H), 5.52 (s, 2H), 5.44 (d, J=16.5 Hz, 1H), 4.99 (s, 2H), 3.73-3.46 (m, 4H), 2.64 (s, 3H), 2.03-1.90 (m, 2H), 1.90-1.84 (m, 6H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).
LC/MS: Calc'd m/z=578.2 for C31H35FN4O6, found [M+H]+=579.4.
The title compound was prepared according to General Procedure 6 starting from Compound 111 (5.0 mg) to give the title compound as an off-white solid (TFA salt, 4.4 mg).
LC/MS: Calc'd m/z=478.2 for C26H27FN4O4, found [M+H]+=479.2.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and (R)-morpholin-2-yl methanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).
LC/MS: Calc'd m/z=509.2 for C27H28FN3O6, found [M+H]+=510.4.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).
LC/MS: Calc'd m/z=525.6 for C27H28FN3O5S, found [M+H]+=526.5.
1H NMR (300 MHz, 10% D2O/CD3CN) 8.36 (d, J=8.1 Hz, 1H), 7.83 (d, J=10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J=16.4 Hz, 1H), 5.52-5.29 (m, 3H), 5.02 (d, J=14.6 Hz, 1H), 4.71-4.54 (m, 1H), 4.27 (dd, J=12.4, 5.0 Hz, 1H), 3.98 (dd, J=12.3, 3.4 Hz, 1H), 3.55 (s, 1H), 3.30-3.03 (m, 4H) 2.97-2.72 (m, 3H), 2.62 (s, 1H), 2.55 (s, 3H), 0.95 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-yl methanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).
LC/MS: Calc'd m/z=521.5 for C28H28FN3O6, found [M+H]+=522.5.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.36 (d, J=7.9 Hz, 1H), 7.86 (dd, J=10.6, 5.0 Hz, 1H), 7.50 (d, J=1.8 Hz, 1H), 5.63-5.49 (m, 2H), 5.37 (dd, J=17.8, 14.1 Hz, 2H), 5.05 (s, 2H), 4.63 (d, J=2.5 Hz, 1H), 4.55 (d, J=10.7 Hz, 1H), 4.33 (s, 2H), 3.92 (d, J=10.7 Hz, 1H), 3.36 (s, 2H), 2.57 (s, 3H), 2.41-2.13 (m, 2H), 1.97-1.85 (m, 2H), 0.95 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ6-thiomorpholine-1,1-dione. Purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).
LC/MS: Calc'd m/z=557.6 for C27H28FN3O7S, found [M+H]+=558.4.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.44 (d, J=8.2 Hz, 1H), 7.80 (d, J=11.0 Hz, 1H), 7.50 (s, 1H), 5.58 (d, J=16.5 Hz, 1H), 5.45-5.26 (m, 3H), 4.60 (d, J=14.9 Hz, 1H), 4.33 (d, J=14.7 Hz, 1H), 3.88 (d, J=4.8 Hz, 2H), 3.41-2.85 (m, 4H), 2.53 (s, 2H), 2.19 (p, J=2.5 Hz, 2H), 1.74 (p, J=2.5 Hz, 2H), 1.27 (s, 2H), 0.95 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).
LC/MS: Calc'd m/z=505.5 for C28H28FN3O5, found [M+H]+=506.6.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.25 (d, J=7.9 Hz, 1H), 7.87 (d, J=10.6 Hz, 1H), 7.50 (s, 1H), 5.65-5.27 (m, 4H), 4.98 (s, 2H), 4.24 (s, 1H), 3.83-3.57 (m, 4H), 2.54 (s, 5H), 2.01-1.86 (m, 2H), 1.70 (s, 2H), 0.95 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 3-fluoroazetidin-3-yl methanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).
LC/MS: Calc'd m/z=497.5 for C26H25F2N3O5, found [M+H]+=498.4.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.24 (d, J=7.9 Hz, 1H), 7.85 (d, J=10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J=16.5 Hz, 1H), 5.48-5.28 (m, 3H), 4.98 (s, 2H), 4.44-4.14 (m, 4H), 3.78 (d, J=14.9 Hz, 2H), 2.01-1.86 (m, 2H), 0.95 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and azetidin-3-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).
LC/MS: Calc'd m/z=479.5 for C26H26FN3O5, found [M+H]+=480.4.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.23 (d, J=7.8 Hz, 1H), 7.90 (d, J=10.6 Hz, 1H), 7.53 (s, 1H), 5.58 (d, J=16.5 Hz, 1H), 5.50-5.28 (m, 3H), 5.01 (s, 2H), 4.31-4.17 (m, 2H), 4.15-4.00 (m, 2H), 3.62 (d, J=3.9 Hz, 2H), 2.58 (s, 3H), 2.01-1.86 (m, 2H), 0.96 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-yl methanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).
LC/MS: Calc'd m/z=543.5 for C28H28F3N3O5, found [M+H]+=544.4.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.25 (d, J=8.0 Hz, 1H), 7.77 (dd, J=10.7, 1.4 Hz, 1H), 7.47 (s, 1H), 5.55 (d, J=16.5 Hz, 1H), 5.42-5.25 (m, 3H), 4.66 (d, J=3.2 Hz, 2H), 3.90-3.77 (m, 1H), 3.71-3.45 (m, 4H), 2.24 (q, J=11.8, 9.2 Hz, 2H), 2.01-1.86 (m, 2H), 0.94 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).
LC/MS: Calc'd m/z=519.6 for C29H30FN3O5, found [M+H]+=520.4.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.22 (s, 1H), 7.92 (d, J=10.7 Hz, 1H), 7.54 (s, 1H), 5.59 (dd, J=17.6, 7.6 Hz, 2H), 5.33 (t, J=17.4 Hz, 2H), 4.98-4.81 (m, 1H), 4.67-4.44 (m, 2H), 4.28-3.93 (m, 4H), 2.73 (s, 2H), 2.34-2.03 (m, 4H), 1.91 (d, J=14.0 Hz, 5H), 0.96 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 1.2 (10 mg) and methane sulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (0.8 mg, 7% yield).
LC/MS: Calc'd m/z=487.1 for C23H22FN3O6S, found [M+H]+=488.2.
1H NMR (300 MHz, MeOD) δ 8.33 (d, J=8.1 Hz, 1H), 7.83 (d, J=10.8 Hz, 1H), 7.68 (s, 1H), 5.62 (d, J=16.3 Hz, 1H), 5.52 (s, 2H), 5.42 (d, J=16.4 Hz, 1H), 4.87 (s, 2H), 3.06 (s, 3H), 2.59 (s, 3H), 2.06-1.93 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (5.0 mg, 17% yield).
LC/MS: Calc'd m/z=608.1 for C29H25FN4O8S, found [M+H]+=609.2.
1H NMR (300 MHz, CDCl3) δ 8.02-7.92 (m, 3H), 7.74 (d, J=10.5 Hz, 1H), 7.65 (s, 1H), 7.33 (d, J=8.6 Hz, 2H), 5.66 (d, J=16.8 Hz, 1H), 5.28 (d, J=16.5 Hz, 1H), 5.14 (d, J=5.4 Hz, 2H), 4.67 (s, 2H), 4.28 (d, J=6.3 Hz, 2H), 3.39 (s, 3H), 2.03-1.83 (m, 2H), 1.04 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 1.2 (10 mg) and benzenesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (9.8 mg, 73% yield).
LC/MS: Calc'd m/z=549.6 for C28H24FN3O6S, found [M+H]+=550.6.
1H NMR (300 MHz, DMSO-d6) δ 8.60 (t, J=6.2 Hz, 1H), 8.17 (d, J=8.1 Hz, 1H), 7.83 (d, J=10.8 Hz, 1H), 7.71 (dd, J=7.1, 1.7 Hz, 2H), 7.66-7.48 (m, 2H), 7.46 (dd, J=8.3, 6.8 Hz, 2H), 7.40-7.27 (m, 2H), 7.18 (s, 1H), 7.01 (s, 1H), 5.45 (s, 2H), 5.33 (s, 2H), 4.63 (d, J=6.2 Hz, 2H), 2.48 (s, 3H), 1.98-1.76 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. Purification of the title compound was accomplished as described in General Procedure 9, using a 12 g C18 column and eluting with a 5 to 75% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (37.8 mg, 47% yield).
LC/MS: Calc'd m/z=594.6 for C28H23FN4O8S, found [M+H]+=595.2.
To a solution of Compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL) was added platinum 1% vanadium 2% on carbon (75 mg). The flask was purged with H2 then stirred at room temperature under an H2 atmosphere for 45 min. The mixture was filtered through a pad of celite, washed with DMF, and the filtrate evaporated to give the title compound as a pale yellow solid (30 mg, 84% yield).
LC/MS: Calc'd m/z=564.6 for C28H24FN4O6S, found [M+H]+=565.2.
1H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J=8.2 Hz, 1H), 8.02 (t, J=6.2 Hz, 1H), 7.88 (d, J=10.8 Hz, 1H), 7.48-7.35 (m, 2H), 7.31 (d, J=8.4 Hz, 1H), 6.63-6.45 (m, 2H), 5.45 (s, 2H), 5.36 (s, 2H), 4.50 (d, J=6.3 Hz, 2H), 1.98-1.75 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 1.2 (20 mg) and 2-hydroxymethanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (1.3 mg, 13% yield).
LC/MS: Calc'd m/z=517.1 for C24H24FN3O7S, found [M+H]+=518.2.
1H NMR (300 MHz, DMSO-d6) δ 8.30 (d, J=8.4 Hz, 1H), 7.91 (d, J=10.9 Hz, 1H), 7.84 (t, J=6.3 Hz, 1H), 7.33 (s, 1H), 5.50-5.33 (m, 4H), 5.07 (t, J=5.4 Hz, 1H), 4.78 (d, J=6.0 Hz, 2H), 4.07 (s, 3H), 3.80 (dt, J=6.3 Hz, J=5.8 Hz, 2H), 1.86 (m, 2H), 0.87 (d, J=7.3 Hz, 3H).
To a solution of chlorosulfonyl isocyanate (3 μL) in dichloromethane (1 mL) was added tert-butanol (3 μL). This solution was stirred for 1 h, then Compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added followed by triethylamine (13 μL). The reaction was stirred for 1 hr then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient. To the purified solid in dichloromethane (1 mL) was added trifluoroacetic acid (200 μL). The reaction was stirred for 16 h then concentrated to dryness to provide the title compound as an off-white solid (7.5 mg, 48% yield).
LC/MS: Calc'd m/z=488.1 for C22H21FN4O6S, found [M+H]+=489.0.
1H NMR (300 MHz, MeOD) δ 8.25 (d, J=8.1 Hz, 1H), 7.73 (d, J=10.7 Hz, 1H), 7.62 (s, 1H), 5.59 (d, J=16.4 Hz, 1H), 5.45 (s, 2H), 5.39 (d, J=16.4 Hz, 1H), 4.81 (s, 2H), 2.55 (d, J=1.7 Hz, 3H), 2.07-1.89 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4 starting from Compound 1.2 (24 mg). Purification was accomplished as described in General Procedure 9, using a 12 g column C18 column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (14 mg, 53% yield).
LC/MS: Calc'd m/z=574.2 for C29H23FN4O8S, found [M+H]+=575.2.
The title compound was prepared according to General Procedure 4 starting from Compound 1.2 (25 mg) and aqueous methyl amine (500 μL, 40 wt. % in water) as the primary amine. In this instance, the intermediate PNP-carbamate was used crude. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (8.9 mg, 31% yield).
LC/MS: Calc'd m/z=466.2 for C24H23FN4O5, found [M+H]+=467.2.
1H NMR (300 MHz, MeOD) δ 8.26 (d, J=8.2 Hz, 1H), 7.79 (d, J=10.7 Hz, 1H), 7.66 (s, 1H), 5.61 (d, J=16.3 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J=16.4 Hz, 1H), 4.97 (s, 2H), 2.73 (s, 3H), 2.57 (s, 3H), 2.08-1.93 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to the second step of General Procedure 4 using Compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (0.6 mg, 12% yield).
LC/MS: Calc'd m/z=557.2 for C30H28FN5O5, found [M+H]+=558.4.
1H NMR (300 MHz, MeOD) δ 8.25 (d, J=8.1 Hz, 1H), 7.80 (d, J=10.8 Hz, 1H), 7.67 (s, 1H), 7.43 (d, J=8.2 Hz, 2H), 7.24 (d, J=8.3 Hz, 2H), 5.63 (d, J=16.4 Hz, 1H), 5.48 (s, 2H), 5.43 (d, J=16.4 Hz, 1H), 5.01 (s, 2H), 4.37 (s, 2H), 2.56 (d, J=1.7 Hz, 3H), 2.05-1.94 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
The title compound was prepared according to the second step of General Procedure 4 using Compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (2.4 mg, 66% yield).
LC/MS: Calc'd m/z=496.2 for C25H25FN4O6, found [M+H]+=497.2.
1H NMR (300 MHz, MeOD) δ 8.08 (d, J=8.0 Hz, 1H), 7.74 (d, J=10.5 Hz, 1H), 7.68 (s, 1H), 5.64 (d, J=16.4 Hz, 1H), 5.41 (s, 2H), 5.31 (d, J=16.4 Hz, 1H), 4.96 (s, 2H), 3.63 (t, J=5.2 Hz, 2H), 3.29 (t, J=5.3 Hz, 2H), 2.54 (s, 3H), 1.98-1.87 (m, 2H), 1.01 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 5 starting from Compound 1.2 (50 mg) and reacting methanol with the intermediate PNP-carbamate. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (3.5 mg, 6% yield).
LC/MS: Calc'd m/z=467.2 for C24H22FN3O6, found [M+H]+=468.2.
1H NMR (300 MHz, MeOD) δ 8.17 (d, J=8.2 Hz, 1H), 7.77 (d, J=10.5 Hz, 1H), 7.69 (s, 1H), 5.65 (d, J=16.5 Hz, 1H), 5.48 (s, 2H), 5.33 (d, J=16.4 Hz, 1H), 4.86 (d, J=5.6 Hz, 2H), 3.65 (s, 3H), 2.56 (s, 3H), 2.02-1.89 (m, 2H), 1.02 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 5 starting from Compound 1.2 (18 mg) and reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (4.2 mg, 19% yield).
LC/MS: Calc'd m/z=497.2 for C25H24FN3O7, found [M+H]+=498.2.
1H NMR (300 MHz, DMSO) δ 8.23 (d, J=8.2 Hz, 1H), 7.78 (d, J=10.7 Hz, 1H), 7.40 (s, 1H), 5.47 (d, J=16.5 Hz, 1H), 5.42 (s, 2H), 5.34 (d, J=16.4 Hz, 1H), 4.77 (s, 2H), 3.99 (t, J=4.9 Hz, 2H), 3.64-3.38 (m, 2H), 2.48 (s, 3H), 2.02-1.67 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).
A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0° C. To this solution was first added a 1 M BCl3 in DCM (71 mL, 71 mmol), followed by a 1 M chloro(diethyl)alumnae in DCM (71 mL, 71 mmol), then finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated at reflux for 3 h, cooled to room temperature, and quenched by the addition of an aqueous 2 M HCl solution. The resulting heterogenous mixture was heated to reflux for 1 h, cooled to room temperature, then the pH was adjusted to ˜12 with Na2CO3. The layers were separated, and the aqueous layer extracted with DCM (3×100 mL). The combined organic layers were dried over Na2SO4, concentrated, and flash purified as described in General Procedure 9, eluting with 0 to 20% EtOAc/Hexanes to give the title compound (6 g, 28 mmol, 39% yield).
LC/MS: Calc'd m/z=217.1 for C9H9ClFNO2, found [M+H]+=218.1.
1H NMR (400 MHz, CDCl3) δ 7.19 (d, J=9.2 Hz, 1H), 6.44 (d, J=12.8 Hz, 1H), 4.59 (s, 2H), 3.86 (s, 3H)
To a solution of Compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene (200 mL) was added toluene-4-sulfonic acid (157 mg, 0.9 mmol). This solution was heated at 140° C. for 3 h then cooled to room temperature. The product as yellow precipitate was collected by filtration to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).
LC/MS: Calc'd m/z=445.2 for C22H18ClFN2O5, found [M+H]+=445.1.
1H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J=12.0 Hz, 1H) 7.80 (d, J=9.2 Hz, 1H) 7.27 (s, 1H), 6.50 (s, 1H), 5.45 (s, 2H), 5.41 (s, 2H), 5.33 (s, 2H) 4.08 (s, 3H), 1.87-1.83 (m, 2H), 0.87 (t, J=7.2 Hz, 3H)
The title compound was prepared according to General Procedure 1 starting from Compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (5.6 mg, 41% yield).
LC/MS: Calc'd m/z=495.2 for C26H26FN3O6, found [M+H]+=496.4.
1H NMR (300 MHz, MeOD) δ 7.84-7.70 (m, 2H), 7.59 (s, 1H), 5.62 (d, J=16.3 Hz, 1H), 5.45-5.36 (m, 3H), 4.29 (s, 2H), 4.12 (s, 3H), 3.58-3.48 (m, 2H), 3.28-3.09 (m, 2H), 2.75-2.61 (m, 2H), 2.05-1.91 (m, 2H), 1.02 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (2.5 mg, 14% yield).
LC/MS: Calc'd m/z=634.2 for C32H31FN4O7S, found [M+H]+=635.4.
The title compound was prepared according to General Procedure 1 starting from Compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (4.0 mg, 23% yield).
LC/MS: Calc'd m/z=649.2 for C32H32FN5O7S, found [M+H]+=650.4.
1H NMR (300 MHz, DMSO) δ 8.08 (s, 2H), 7.90-7.67 (m, 2H), 7.35 (s, 1H), 7.32-7.26 (m, 2H), 6.67-6.57 (m, 2H), 5.46 (d, J=16.5 Hz, 1H), 5.33-5.22 (m, 3H), 3.92 (s, 3H), 3.02-2.72 (m, 4H), 2.75-2.58 (m, 4H), 1.97-1.70 (m, 2H), 0.90 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (2.1 mg, 19% yield).
LC/MS: Calc'd m/z=508.2 for C27H29FN4O5, found [M+H]+=509.4.
The title compound was prepared according to General Procedure 1 starting from Compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (3.2 mg, 20% yield).
LC/MS: Calc'd m/z=585.2 for C32H32FN5O5, found [M+H]+=586.4.
1H NMR (300 MHz, MeOD) δ 7.83-7.74 (m, 2H), 7.62 (s, 1H), 7.06 (d, J=8.9 Hz, 2H), 6.98 (d, J=8.9 Hz, 2H), 5.65 (d, J=16.4 Hz, 1H), 5.36 (s, 2H), 5.27 (d, J=16.4 Hz, 1H), 4.13 (s, 2H), 4.06 (s, 3H), 3.26 (br s, 4H), 2.79 (br s, 4H), 1.97-1.83 (m, 2H), 1.00 (t, J=7.4 Hz, 3H).
To a solution of Compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL) was added hexamethylenetetramine (236 mg, 1.7 mmol) followed by iPr2NEt (100 μL, 0.56 mmol). This solution was heated at reflux for 5 h, cooled to room temperature and quenched with 12 M aqueous HCl (60 μL). This solution was concentrated to ½ H volume and 1 M aqueous HCl (1.5 mL) was added, stirred for 5 min, then concentrated to give a brown residue. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5 to 40% CH3CN/H2O+0.1% TFA gradient to give the title compound as pale yellow solid (179 mg, 75% yield).
LC/MS: Calc'd m/z=425.4 for C22H20FN3O5, found [M+H]+=426.2.
The title compound was prepared according to General Procedure 3 starting from Compound 2.8 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 5 to 65% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (8.5 mg, 91% yield).
LC/MS: Calc'd m/z=503.1 for C23H22FN3O7S, found [M+H]+=504.2.
1H NMR (300 MHz, DMSO-d6) δ 7.98 (d, J=12.1 Hz, 1H), 7.89 (t, J=6.4 Hz, 1H), 7.80 (d, J=9.1 Hz, 1H), 7.28 (s, 1H), 5.42 (s, 2H), 5.39 (s, 2H), 4.77 (d, J=6.4 Hz, 2H), 4.06 (s, 3H), 3.06 (s, 3H), 1.95-1.73 (m, 2H), 0.88 (d, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 3 starting from Compound 2.8 (7.5 mg) and benzenesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 5 to 70% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (4.6 mg, 46% yield).
LC/MS: Calc'd m/z=565.6 for C28H24FN3O7S, found [M+H]+=566.2.
1H NMR (300 MHz, DMSO-d6) δ 8.59 (t, J=6.3 Hz, 1H), 7.94 (d, J=12.2 Hz, 1H), 7.82-7.68 (m, 2H), 7.62-7.46 (m, 1H), 7.51-7.40 (m, 1H), 7.28 (d, J=8.3 Hz, 1H), 6.52 (s, 1H), 5.44 (s, 1H), 5.36 (s, 1H), 4.64 (d, J=6.3 Hz, 1H), 4.09 (s, 2H), 1.95-1.81 (m, 1H), 0.89 (t, J=7.3 Hz, 2H).
The title compound was prepared according to General Procedure 3 starting from Compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and eluting with a 5 to 75% CH3CN/H2O+0.1% TFA gradient to give the title compound as pale yellow solid (9.7 mg, 71% yield).
LC/MS: Calc'd m/z=610.6 for C28H23FN4O9S, found [M+H]+=611.5.
To a solution of Compound 2.11 (9.7 mg, 0.016 mmol) in methanol (1.6 mL) was added platinum 1% vanadium 2% on carbon (15 mg). The flask was purged with H2 then stirred at room temperature under an H2 atmosphere for 45 min. The mixture was filtered through a pad of celite, washed with DMF, then the filtrate was evaporated to give the title compound as a pale yellow solid (1.5 mg, 16% yield).
LC/MS: Calc'd m/z=580.6 for C28H25FN4O7S, found [M+H]+=581.4.
1H NMR (300 MHz, MeOD) δ 7.77 (d, J=11.0 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J=8.6 Hz, 1H), 6.61 (d, J=8.6 Hz, 1H), 5.59 (d, J=16.3 Hz, 1H), 5.39 (d, J=16.4 Hz, 1H), 5.30 (s, 1H), 4.56 (s, 1H), 4.10 (d, J=3.7 Hz, 3H), 2.04-1.91 (m, 2H), 1.31 (s, 1H), 1.02 (t, J=7.3 Hz, 3H), 0.90 (s, 1H).
The title compound was prepared according to General Procedure 3 starting from Compound 2.8 (8 mg) and 2-hydroxymethanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 15 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (2.2 mg, 22% yield).
LC/MS: Calc'd m/z=533.1 for C24H24FN3O8S found [M+H]+=534.2.
1H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J=12.2 Hz, 1H), 7.89-7.79 (m, 2H), 7.29 (s, 1H), 5.43 (s, 2H), 5.40 (s, 2H), 4.76 (d, J=6.4 Hz, 2H), 4.06 (s, 3H), 3.81 (t, J=6.3 Hz, 2H), 3.34 (t, J=6.3 Hz, 2H), 1.94-1.75 (m, 2H), 0.87 (d, J=7.4 Hz, 3H).
The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4 starting from Compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as the solvent. Flash purification was accomplished as described in General Procedure 9, using a 12 g C12 column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (61 mg, 86% yield). This intermediate was divided and used to generate the following compounds.
LC/MS: Calc'd m/z=590.1 for C29H23FN4O9, found [M+H]+=591.2.
The title compound was prepared according to second step of General Procedure 4 using Compound 2.14 (15 mg) as the PNP-carbamate and aqueous methyl amine (500 uL, 40 wt. % in water) as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (5.8 mg, 47% yield).
LC/MS: Calc'd m/z=482.2 for C24H23FN4O6, found [M+H]+=483.2.
1H NMR (300 MHz, DMSO-d6) δ 8.00-7.87 (m, 2H), 7.31 (s, 1H), 5.48-5.39 (m, 3H), 4.81 (s, 3H), 2.56 (s, 3H), 1.93-1.81 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).
The title compound was prepared according to the second step of General Procedure 4 using Compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).
LC/MS: Calc'd m/z=573.2 for C30H28FN5O6, found [M+H]+=574.2.
1H NMR (300 MHz, MeOD) δ 7.79 (d, J=11.9 Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 7.59 (s, 1H), 7.43 (d, J=8.2 Hz, 2H), 7.25 (d, J=8.2 Hz, 2H), 5.61 (d, J=16.3 Hz, 1H), 5.52-5.35 (m, 3H), 4.98 (s, 2H), 4.39 (s, 2H), 4.01 (s, 3H), 2.03-1.93 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to the second step of General Procedure 4 using Compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (1.5 mg, 12% yield).
LC/MS: Calc'd m/z=512.2 for C25H25FN4O7, found [M+H]+=513.2.
1H NMR (300 MHz, MeOD) δ 7.93 (d, J=12.1 Hz, 1H), 7.88 (d, J=9.2 Hz, 1H), 7.56 (s, 1H), 5.62 (d, J=16.2 Hz, 1H), 5.52 (s, 2H), 5.45 (d, J=16.3 Hz, 1H), 4.98 (s, 2H), 4.17 (s, 3H), 3.59 (t, J=5.6 Hz, 2H), 3.28 (t, J=5.6 Hz, 2H), 2.10-1.91 (m, 2H), 1.05 (t, J=7.3 Hz, 3H).
To a stirring solution of HNO3 (121.2 mL, 67% purity, 2.0 eq.) in H2SO4 (500 mL) at 0° C. was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 eq.). After the addition was complete, the ice bath was removed, and the reaction was allowed to stir for 5 h at 25° C. The mixture was poured into ice (5 L), filtered and then dried under vacuum. The title compound was obtained as a yellow solid (219 g).
1H NMR (400 MHz, CDCl3) δ 10.39 (s, 1H), 8.23 (d, J=6.8 Hz, 1H), 7.91 (d, J=7.6 Hz, 1H).
A mixture of Compound 3.1 (219 g, 1.0 eq.), tert-butyl carbamate (124 g, 1.2 eq.), Cs2CO3 (575 g, 2.0 eq.), Pd2(dba)3 (40 g, 0.05 eq.) and XPhos (84 g, 0.2 eq.) in toluene (2000 mL) was degassed and purged with N2 for three cycles. The mixture was then stirred at 90° C. for 15 h under N2 atmosphere. The reaction mixture was diluted with H2O (800 mL) and extracted with EtOAc (300 mL×2). The combined organic layers were washed with brine (200 mL×2), then dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=100: 1 to 20:1) to afford the title compound as a yellow solid (140 g, 56% yield).
1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.94 (s, 1H), 8.42 (d, J=7.6 Hz, 1H), 8.16 (d, J=10.8 Hz, 1H), 1.50 (s, 9H)
To a solution of Compound 3.2 (100 g, 1.0 eq.) in H2O (300 mL) and EtOH (1200 mL) was added NH4Cl (30.5 g, 1.62 eq.). Iron (78.6 g, 4.0 eq.) was added in portions at 80° C. The mixture was stirred at 80° C. for 6 h. The mixture was filtered, water was added to the filtrate, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether:ethyl acetate=1:0 to 0:1), TLC (petroleum ether) to afford the title compound as a yellow solid (19.0 g, 21% yield).
LC/MS: Calc'd m/z=254.1 for C12H15FN2O3, found [M+H]+=255.0.
1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.57 (s, 1H), 7.58 (d, J=4.8 Hz, 1H), 7.21 (s, 2H), 6.53 (d, J=12.8 Hz, 1H), 1.43 (s, 9H).
A mixture of Compound 3.3 (4.20 g, 1.2 eq.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 eq.) and TsOH (monohydrate, 253 mg, 0.1 eq.) in toluene (350 mL) was stirred at 110° C. for 2 hrs. The reaction solution was cooled to 25° C., filtered, the solid was washed with methyl-t-butyl ether (30 mL) and then dried under vacuum. The title compound was obtained as a yellow solid (4.5 g, 62% yield).
LC/MS: Calc'd m/z=481.2 for C25H24FN3O6, found [M+H]+=482.1.
1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.65 (s, 1H), 8.43 (d, J=8.4 Hz, 1H), 7.95 (d, J=12.0 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.25 (s, 2H), 1.80-1.92 (m, 2H), 1.52 (s, 9H), 0.88 (t, J=7.2 Hz, 3H)
To a mixture of Compound 3.4 (4.00 g) in MeOH (360 mL) was added a solution of FeSO4 (heptahydrate, 1.2 g), H2SO4 (280 L) in H2O (4 mL). The reaction mixture was heated at 65° C. while H2O2(24 mL, 30% purity) was added dropwise over 30 min and then stirred 0.5 h. The reaction solution was cooled to 25° C., then filtered to provide the title compound as a yellow solid (1.53 g, 33.2% yield). To the filtrate was added H2O (400 mL), then quenched with saturated aqueous Na2S203. The pH was adjusted to 7-8 with saturated aqueous Na2CO3 then the solution was concentrated and filtered. The solid was triturated with MeOH (30 mL) at 55° C. for 1 h, then filtered, to provide a second batch of the title compound as a brown solid (1.09 g, 26% yield).
LC/MS: Calc'd m/z=511.2 for C26H26FN3O7, found [M+H]+=512.2.
1H NMR (300 MHz, d6-DMSO) δ 9.47 (s, 1H), 8.47 (d, J=7.6 Hz, 1H), 7.94 (d, J=12.0 Hz, 1H), 7.29 (d, J=1.6 Hz, 1H), 6.49 (s, 1H), 5.86-5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J=4.4 Hz, 2H), 1.90-1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J=6.4 Hz, 3H).
In a 50 mL round-bottom flask containing Compound 3.5 (150 mg, 0.293 mmol) was added DCM (2.9 mL) followed by Dess-Martin periodinane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). This solution was stirred at room temperature for 18 h then diluted with DCM, washed with saturated aqueous NaHCO3 and brine. The layers were separated, and the combined organic layers were evaporated onto celite. Flash purification was accomplished as described in General Procedure 9, using a 10 g silica column and eluting with 0 to 10% DCM/MeOH to give the title product as an orange powder (42.5 mg, 28%).
LC/MS: Calc'd m/z=509.2 for C26H24FN3O7, found [M+H]+=510.4.
1H NMR (300 MHz, Acetone-d6) δ 11.10 (s, 1H), 9.68 (d, J=8.6 Hz, 1H), 8.81 (s, 1H), 8.04 (d, J=11.9 Hz, 1H), 7.63 (s, 1H), 5.73 (s, 2H), 5.69 (d, J=16.2 Hz, 1H), 5.42 (d, J=16.2 Hz, 1H), 2.02-1.95 (m, 2H), 8.47 (d, J=7.6 Hz, 1H), 7.94 (d, J=12.0 Hz, 1H), 7.29 (d, J=1.6 Hz, 1H), 6.49 (s, 1H), 5.86-5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J=4.4 Hz, 2H), 1.90-1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J=6.4 Hz, 3H).
The title compound was prepared according to General Procedure 6 starting from Compound 3.4 (40 mg) to give the title compound as a red solid (TFA salt, 36 mg, 87% yield).
LC/MS: Calc'd m/z=381.1 for C20H16FN3O4, found [M+H]+=382.2.
1H NMR (300 MHz, DMSO) δ 8.28 (s, 1H), 7.72 (d, J=12.5 Hz, 1H), 7.21 (d, J=7.3 Hz, 1H), 5.43 (d, J=16.2 Hz, 1H), 5.34 (d, J=16.2 Hz, 1H), 5.17 (s, 2H), 1.92-1.74 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 6 starting from Compound 3.5 (5 mg) to give the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).
LC/MS: Calc'd m/z=411.2 for C21H18FN3O5, found [M+H]+=412.2.
1H NMR (300 MHz, MeOD) δ 7.71 (d, J=12.2 Hz, 1H), 7.60 (s, 1H), 7.29 (d, J=9.5 Hz, 1H), 5.61 (d, J=16.3 Hz, 1H), 5.47 (s, 2H), 5.40 (d, J=16.3 Hz, 1H), 5.25 (s, 2H), 2.03-1.94 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
To a stirring solution of Compound 3.5 (100 mg) in dichloromethane (5 mL) was added a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL). After 1 h, additional thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added. After another 1 h the reaction was diluted with dichloromethane (10 mL) and toluene (1 mL) then concentrated in vacuo to provide the title compound as a red solid that was used in subsequent reactions without additional purification.
LC/MS: Calc'd m/z=529.1 for C26H25ClFN3O6, found [M+H]+=530.2.
To Compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg) then DIPEA (99 μL). This solution was heated at 60° C. for 16 h then concentrated to dryness in vacuo. Flash purification was accomplished as described in General Procedure 9, using a 12 g C18 column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).
LC/MS: Calc'd m/z=510.2 for C26H27FN4O6, found [M+H]+=511.4.
1H NMR (300 MHz, MeOD) δ 8.88 (d, J=8.2 Hz, 1H), 7.96 (d, J=11.9 Hz, 1H), 7.62 (s, 1H), 5.60 (d, J=16.4 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J=16.4 Hz, 1H), 4.80 (s, 2H), 2.07-1.89 (m, 2H), 1.64 (s, 9H), 1.02 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 6 starting from Compound 3.10 (2.1 mg) to give the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).
LC/MS: Calc'd m/z=410.1 for C21H19FN4O4, found [M+H]+=411.2.
1H NMR (300 MHz, MeOD) δ 7.82 (d, J=12.1 Hz, 1H), 7.60 (s, 1H), 7.37 (d, J=9.1 Hz, 1H), 5.61 (d, J=16.3 Hz, 1H), 5.42 (s, 2H), 5.41 (d, J=16.3 Hz, 1H), 4.69 (s, 2H), 2.08-1.94 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 1 starting from Compound 3.9 (150 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to give the title compound as a red solid (TFA salt, 103 mg, 52% yield).
LC/MS: Calc'd m/z=580.2 for C30H33FN4O7, found [M+H]+=581.4.
1H NMR (300 MHz, MeOD) δ 9.06 (d, J=8.3 Hz, 1H), 7.93 (d, J=12.0 Hz, 1H), 7.66 (s, 1H), 5.63 (d, J=16.3 Hz, 1H), 5.51 (s, 2H), 5.43 (d, J=16.4 Hz, 1H), 4.92 (s, 2H), 3.84 (s, 4H), 3.10 (s, 4H), 1.99 (d, J=5.5 Hz, 2H), 1.63 (s, 9H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 6 starting from Compound 3.12 (45 mg) to give the title compound as a red solid (TFA salt, 37 mg, 99% yield).
LC/MS: Calc'd m/z=480.2 for C25H25FN4O5, found [M+H]+=481.4.
1H NMR (300 MHz, MeOD) δ 7.73 (d, J=12.0 Hz, 1H), 7.54 (s, 1H), 7.48 (d, J=9.2 Hz, 1H), 5.60 (d, J=16.3 Hz, 1H), 5.47-5.34 (m, 3H), 4.65 (s, 2H), 3.91-3.85 (m, 4H), 3.30-3.24 (m, 4H), 2.08-1.91 (m, 2H), 1.02 (t, J=7.3 Hz, 3H).
To a 5 mL flask containing Compound 3.6 (37 mg, 0.067 mmol) was added dichloromethane (1.45 mL) followed by acetic acid (18.69 μL, 0.327 mmol), piperidine (21.52 μL, 0.218 mmol), and sodium triacetoxyborohydride (23.0 mg, 0.109 mmol). This solution was then stirred at room temperature for 2 h, quenched by the addition of water+0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5 to 40% CH3CN/H2O+0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to give the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).
LC/MS: Calc'd m/z=478.2 for C26H27FN4O4, found [M+H]+=479.4.
1H NMR (300 MHz, MeOD) δ 7.78 (d, J=12.1 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J=9.1 Hz, 1H), 5.60 (d, J=16.4 Hz, 1H), 5.47-5.35 (m, 3H), 4.86 (s, 2H), 3.80-3.68 (m, 2H), 3.28-3.19 (m, 2H), 2.02-1.68 (m, 8H), 1.01 (t, J=7.4 Hz, 3H).
To a 2 mL vial containing Compound 3.6 (15 mg, 0.029 mmol) was added dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and N-methylpiperazine (4.90 μL, 0.044 mmol). This solution was stirred at room temperature for 4 h then sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) was then added and stirred for an additional 45 min. Excess hydride was then quenched by the addition of a 0.1% aqueous TFA solution (0.5 mL). Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and eluting with a 5 to 40% CH3CN/H2O+0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was deprotected according to General Procedure 6 to give the title product as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).
LC/MS: Calc'd m/z=493.2 for C26H28FN5O4, found [M+H]+=494.4.
1H NMR (300 MHz, MeOD) δ 7.68 (d, J=12.2 Hz, 1H), 7.56 (s, 1H), 7.53 (d, J=9.5 Hz, 1H), 5.60 (d, J=16.3 Hz, 1H), 5.45-5.30 (m, 3H), 4.15 (s, 2H), 3.55-3.44 (m, 2H), 3.18-3.07 (m, 2H), 2.93 (s, 3H), 2.70-2.51 (m, 2H), 2.03-1.89 (m, 2H), 1.02 (t, J=7.4 Hz, 3H).
The Boc-protected precursor of the title compound was prepared according to General Procedure 1 starting from Compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC was accomplished as described in General Procedure 9, eluting with a 35 to 44% CH3CN/H2O+0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to give the title compound (TFA salt, 2.4 mg, 17% yield over 2 steps).
LC/MS: Calc'd m/z=619.2 for C31H30FN5O6S, found [M+H]+=520.4.
1H NMR (300 MHz, MeOD) δ 7.81-7.60 (m, 7H), 7.34 (s, 1H), 5.51 (d, J=16.4 Hz, 1H), 5.35 (d, J=16.4 Hz, 1H), 5.22 (s, 2H), 4.10 (s, 2H), 3.15-3.02 (m, 4H), 2.79-2.71 (m, 4H), 2.00-1.93 (m, 2H), 1.00 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 2 followed by General Procedure 6 starting from Compound 3.10 (8 mg) and acetic acid. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a red solid (4.0 mg, 56% yield).
LC/MS: Calc'd m/z=452.2 for C23H21FN4O5, found [M+H]+=453.2.
1H NMR (300 MHz, MeOD) δ 7.69 (d, J=12.1 Hz, 1H), 7.56 (s, 1H), 7.38 (d, J=9.3 Hz, 1H), 5.59 (d, J=16.3 Hz, 1H), 5.44-5.33 (m, 3H), 4.85 (s, 3H), 2.03 (s, 3H), 2.00-1.84 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 3 followed by General Procedure 6 starting from Compound 3.10 (8 mg) and methane sulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a red solid (4.4 mg, 57% yield).
LC/MS: Calc'd m/z=488.1 for C22H21FN4O6S, found [M+H]+=489.2.
1H NMR (300 MHz, MeOD) δ 7.74 (d, J=12.2 Hz, 1H), 7.60 (s, 1H), 7.49 (d, J=9.3 Hz, 1H), 5.61 (d, J=16.2 Hz, 1H), 5.45 (s, 2H), 5.40 (d, J=16.2 Hz, 1H), 4.78 (s, 2H), 3.05 (s, 3H), 2.08-1.94 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 3 followed by General Procedure 6 starting from Compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a red solid (1 mg, 16% yield).
LC/MS: Calc'd m/z=518.5 for C23H23FN4O7S, found [M+H]+=519.5.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 7.77-7.61 (m, 1H), 7.48-7.30 (m, 2H), 5.53 (d, J=16.3 Hz, 1H), 5.31 (d, J=15.4 Hz, 3H), 4.69 (s, 2H), 3.97 (dd, J=6.6, 4.9 Hz, 2H), 3.39 (t, J=5.8 Hz, 2H), 2.93 (s, 1H), 1.99-1.83 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).
To a solution of Compound 3.10 (10 mg, 0.02 mmol) in DMF (400 μL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 μL, 0.04 mmol). This solution was stirred at room temperature for ˜30 min, then used directly in subsequent reactions.
The title compound was prepared by addition of MeOH (100 μL) to 200 ul of the solution of Compound 3.20. This solution was stirred at room temperature for 30 min. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained according to General Procedure 6 as a red solid (2.1 mg, 47% yield).
LC/MS: Calc'd m/z=468.4 for C23H21FN4O6, found [M+H]+=468.3.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 7.72 (d, J=12.2 Hz, 1H), 7.41 (d, J=18.1 Hz, 1H), 6.96 (s, 1H), 5.52 (d, J=3.6 Hz, 1H), 5.39-5.23 (m, 3H), 4.82 (s, 1H), 4.73 (s, 1H), 3.63 (d, J=1.2 Hz, 3H), 1.56 (s, 3H), 1.27 (s, 2H), 0.94 (t, J=7.4 Hz, 3H).
The title compound was prepared by addition of methylamine hydrochloride (10 mg) to 200 ul of the solution of Compound 3.20, followed by iPr2NEt (5 μL). This solution was stirred at room temperature for 30 min. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained according to General Procedure 6 as a red solid (2.9 mg, 64.5% yield).
LC/MS: Calc'd m/z=467.5 for C23H21FN5O5, found [M+H]+=468.5.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.13 (d, J=9.2 Hz, 1H), 7.92 (s, 1H), 7.73 (d, J=12.3 Hz, 1H), 7.52-7.35 (m, 2H), 6.94 (d, J=9.2 Hz, 2H), 5.55 (d, J=16.5 Hz, 2H), 5.44-5.27 (m, 4H), 4.85 (s, 2H), 4.78 (s, 1H), 1.56 (d, J=2.5 Hz, 3H), 1.27 (s, 2H), 0.93 (q, J=11.7, 9.5 Hz, 3H).
The title compound was prepared by addition of ethanolamine (100 μL) to 200 ul of the solution of Compound 3.20. This solution was stirred at room temperature for 30 min. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained according to General Procedure 6 as a red solid (0.5 mg, 8.5% yield).
LC/MS: Calc'd m/z=497.5 for C24H24FN5O6, found [M+H]+=498.5.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 7.77-7.61 (m, 1H), 7.48-7.30 (m, 2H), 5.53 (d, J=16.3 Hz, 1H), 5.31 (d, J=15.4 Hz, 1H), 5.19 (s, 2H), 4.69 (s, 2H), 3.97 (dd, J=6.6, 4.9 Hz, 2H), 3.39 (t, J=5.8 Hz, 2H), 2.93 (s, 1H), 2.01-1.83 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).
To a stirring solution of Compound 3.5 (100 mg) in 2 mL dichloromethane was added thionyl chloride (35 μL, 2.5 eq.). The solution was stirred at room temperature for 20 min, then additional thionyl chloride (35 μL, 2.5 eq.) was added. After 20 minutes, toluene (1 mL) was added, and the reaction mixture was concentrated in vacuo. The crude solid was suspended in DMSO (1 mL) and sodium azide (19 mg, 1.5 eq.) was added. This solution was stirred at room temperature for 16 h. Purification was accomplished as described in General Procedure 9, eluting with a 5 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (20 mg, 23% yield).
LC/MS: Calc'd m/z=436.1 for C21H17FN6O4, found [M+H]+=437.2.
1H NMR (300 MHz, MeOD) δ 7.75 (d, J=12.2 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J=9.3 Hz, 1H), 5.61 (d, J=16.3 Hz, 1H), 5.46-5.35 (m, 3H), 5.07 (s, 2H), 2.03-1.97 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
The title compound was prepared according to General Procedure 2 starting from Compound 145 (10 mg) and glycolic acid. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 45% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).
LC/MS: Calc'd m/z=468.1 for C23H21FN4O6, found [M+H]+=469.2.
1H NMR (300 MHz, MeOD) 7.70 (d, J=12.2 Hz, 1H), 7.60 (s, 1H), 7.42 (d, J=9.4 Hz, 1H), 5.62 (d, J=16.3 Hz, 1H), 5.43 (s, 2H), 5.36 (d, J=16.2 Hz, 1H), 4.95 (d, J=5.9 Hz, 2H), 4.08 (s, 2H), 2.04-1.90 (m, 1H), 1.03 (t, J=7.4 Hz, 3H).
To a solution of Compound 145 (9 mg, 1.0 eq.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 eq.) then DIPEA (8 μL, 2.0 eq.). The resulting solution was stirred at 25° C. for 2 h, after which complete conversion to the isothiocyanate intermediate was observed. Methylammonium chloride (3 mg, 2.0 eq.) was then added and the reaction mixture was heated at 60° C. for 30 min. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 45% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a yellow solid (2.3 mg, 22% yield).
LC/MS: Calc'd m/z=483.1 for C23H22FN5O4S found [M+H]+=484.2.
1H NMR (300 MHz, MeOD) δ 7.70 (d, J=12.0 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J=9.3 Hz, 1H), 5.62 (d, J=16.2 Hz, 1H), 5.36 (s, 2H), 5.31 (d, J=16.2 Hz, 1H), 5.30 (s, 2H), 3.04 (s, 3H), 1.99-1.90 (m, 2H), 1.02 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 5 starting from Compound 145 (10 mg) and 2-mercaptoethanol. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 45% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).
LC/MS: Calc'd m/z=514.1 for C24H23FN4O6S found [M+H]+=515.2.
1H NMR (300 MHz, MeOD) δ 7.71 (d, J=12.1 Hz, 1H), 7.60 (s, 1H), 7.36 (d, J=9.4 Hz, 1H), 5.62 (d, J=16.3 Hz, 1H), 5.42 (s, 2H), 5.35 (d, J=16.2 Hz, 1H), 4.88 (d, J=4.6 Hz, 2H), 3.68 (t, J=6.4 Hz, 2H), 3.03 (t, J=6.5 Hz, 2H), 2.04-1.92 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
To a 5 mL flask containing Compound 140 (50 mg) was added water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg) and propionaldehyde (74 μL). The obtained suspension was cooled to −15° C. using an ice brine bath, then sulfuric acid (0.40 mL) was added dropwise. Hydrogen peroxide (95 μL) was then added dropwise. This mixture was stirred at −15° C. for 10 min then allowed to warm up to room temperature and stirred for 2 h. The reaction mixture was diluted with water (30 mL) and the obtained suspension was extracted with DCM (3×30 mL). The organic phase was then evaporated to dryness. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 70% CH3CN/H2O+0.1% TFA gradient to give the title compound as a dark orange solid (2.4 mg, 4.4% yield).
LC/MS: Calc'd m/z=410.1 for C22H20FN3O4 found [M+H]+=410.2.
1H NMR (300 MHz, MeOD) δ 7.63 (d, J=12.3 Hz, 1H), 7.55 (s, 1H), 7.36 (d, J=9.4 Hz, 1H), 5.57 (d, J=16.4 Hz, 1H), 5.37 (d, J=16.4 Hz, 1H), 5.21 (s, 2H), 3.13 (q, J=7.7 Hz, 2H), 2.02-1.90 (m, 2H), 1.38 (t, J=7.7 Hz, 3H), 1.01 (t, J=7.3 Hz, 3H).
In a 5 mL conical flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL), at −20° C., was added Compound 3.5 (15 mg). The obtained suspension was stirred at −20° C. for 5 min. Water (59 μL) was added, and the reaction mixture was allowed to warm up to room temperature and stirred for 2 h, then heated at 70° C. for 1 h. The reaction mixture was allowed to cool down to room temperature and partially evaporated. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 40 to 55% CH3CN/H2O+0.1% TFA gradient to give the title compound as a dark orange solid (5.1 mg, 31% yield).
LC/MS: Calc'd m/z=555.2 for C27H27FN4O8 found [M+H]+=555.2.
1H NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.56 (d, J=8.5 Hz, 1H), 8.00 (d, J=12.0 Hz, 1H), 7.31 (s, 1H), 7.11-6.62 (m, 2H), 6.52 (s, 1H), 5.58 (s, 2H), 5.49-5.27 (m, 4H), 1.94-1.77 (m, 2H), 1.52 (s, 9H), 1.38 (t, J=7.7 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H).
The title compound was prepared according to General Procedure 6 starting from Compound 3.29 (5.1 mg) to give the title compound as yellow powder (TFA salt, 3.8 mg, 73% yield).
LC/MS: Calc'd m/z=455.1 for C22H19FN4O6 found [M+H]+=455.2.
1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J=12.4 Hz, 1H), 7.29 (d, J=9.7 Hz, 1H), 7.21 (s, 1H), 7.0-6.50 (m, 2H), 5.45 (s, 2H), 5.40 (s, 2H), 5.33 (s, 2H), 1.95-1.77 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).
In a 50 mL flask containing Compound 3.5 (30 mg) was added MeOH/Dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL). The reaction mixture was then stirred at reflux for 24 h. The reaction mixture was concentrated, poured into water (30 mL), and extracted with DCM (3×50 mL). The organic phases were combined and dried over MgSO4. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 40% CH3CN/H2O+0.1% TFA gradient to give the title compound as a dark orange solid (5.1 mg, 16% yield).
LC/MS: Calc'd m/z=426.1 for C22H20FN3O5 found [M+H]+=426.2.
1H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J=12.3 Hz, 1H), 7.24 (d, J=9.9 Hz, 1H), 7.20 (s, 1H), 6.47 (s, 1H), 6.30-5.92 (brs, 2H), 5.40 (s, 2H), 5.24 (s, 2H), 4.93 (s, 2H), 3.43 (s, 3H), 1.95-1.75 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).
In a 5 mL conical flask containing Compound 3.6 (15 mg) was added dichloromethane (0.6 mL) followed by 3-azabicyclo[3.1.1]heptan-6-ol (10 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by addition of water+0.1% TFA and diluted with DMF. The reaction mixture was then partially evaporated. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the Boc-protected title compound as a yellow powder. Deprotection was performed according to General Procedure 6, and the obtained residue was purified by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as yellow powder (TFA salt, 7.1 mg, 39% yield).
LC/MS: Calc'd m/z=507.2 for C27H27FN4O5 found [M+H]+=507.4.
1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=12.1 Hz, 1H), 7.46 (d, J=9.4 Hz, 1H), 7.23 (s, 1H), 6.64-5.85 (m, 3H), 5.60-5.25 (m, 4H), 4.85 (s, 1H), 4.10-3.95 (m, 1H), 3.68 (s, 2H), 2.45-2.33 (m, 2H), 1.96-1.72 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).
In a 5 mL conical flask containing Compound 3.6 (15 mg) was added dichloromethane (0.6 mL) followed by (3-fluoroazetidin-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by addition of water+0.1% TFA, diluted with DMF, then partially evaporated. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the Boc-protected title compound as a yellow powder. Deprotection was then performed according to General Procedure 6. The obtained residue was purified by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to give the title compound as yellow powder (TFA salt, 1.8 mg, 10% yield).
LC/MS: Calc'd m/z=499.2 for C25H24F2N4O5 found [M+H]+=499.4.
1H NMR (300 MHz, DMSO-d6) δ 7.82 (d, J=12.4 Hz, 1H), 7.45 (d, J=9.5 Hz, 1H), 7.21 (s, 1H), 5.45-5.33 (m, 4H), 3.75-3.61 (m, 2H), 1.93-1.78 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).
To a stirring solution of Compound 3.9 (210 mg) in DMF (5 mL) was added sodium iodide (5.9 mg) followed by methylammonium chloride (107 mg). The reaction mixture was then stirred at room temperature overnight. Reverse phase purification was accomplished as described in General Procedure 9 using a 30 g C18 column and eluting with a 10 to 65% CH3CN/H2O+0.1% TFA gradient to give the title compound as a yellow solid (15.0 mg, 7.2% yield).
LC/MS: Calc'd m/z=524.2 for C27H29FN4O6, found [M+H]+=525.4.
The Boc-protected version of the title compound was prepared according to General Procedure 2 starting from Compound 3.34 (6.4 mg) and glycolic acid. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient. Deprotection was then performed according to General Procedure 6 to give the title compound as yellow powder (TFA salt, 2.0 mg, 28% yield).
LC/MS: Calc'd m/z=482.2 for C24H23FN4O6, found [M+H]+=483.2.
1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J=12.3 Hz, 1H), 7.27 (d, J=9.5 Hz, 1H), 7.22 (s, 1H), 6.48 (s, 1H), 6.28-6.02 (m, 2H), 5.40 (s, 2H), 5.21 (s, 2H), 5.06-4.93 (m, 2H), 4.18 (s, 2H), 2.80 (s, 3H), 1.92-1.78 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).
The Boc-protected version of the title compound was prepared according to General Procedure 3 starting from Compound 3.34 (8.0 mg) and methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient. Deprotection was then performed according to General Procedure 6 to give the title compound as yellow powder (TFA salt, 2.6 mg, 34% yield).
LC/MS: Calc'd m/z=502.1 for C23H23FN4O6S, found [M+H]+=503.2.
1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J=12.3 Hz, 1H), 7.41 (d, J=9.4 Hz, 1H), 7.23 (s, 1H), 6.63-5.84 (m, 2H), 5.42 (s, 2H), 5.29 (s, 2H), 4.81-4.64 (m, 2H), 3.14 (s, 3H), 2.67 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).
To a 10 mL round bottom flask containing Compound 3.4 (62.0 mg) was added water (0.89 mL), FeSO4 (heptahydrate, 18.0 mg), and 3-methoxypropanal (113.0 mg). To the obtained suspension was added sulfuric acid (0.495 mL) dropwise while stirring at −15° C. in an ice salt bath. Hydrogen peroxide (0.118 mL) was then added dropwise. The mixture was stirred at −15° C. for 10 min and was then allowed to warm up to room temperature and stirred for 1 h. The reaction mixture was then diluted with water (30 mL) and the obtained suspension was extracted with DCM (3×30 mL). The organic phase was evaporated to dryness. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 45% CH3CN/H2O+0.1% TFA gradient to give the title compound as a dark orange solid (TFA salt, 3.1 mg, 4.4% yield).
LC/MS: Calc'd m/z=440.2 for C23H22FN3O5, found [M+H]+=440.2.
1H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J=12.4 Hz, 1H), 7.33 (d, J=9.4 Hz, 1H), 7.20 (s, 1H), 6.60-6.42 (m, 2H), 5.40 (s, 2H), 5.25 (s, 2H), 3.69 (t, J=6.5 Hz, 2H), 3.24 (s, 3H), 3.23 (t, J=6.5 Hz, 2H), 1.96-1.76 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).
To a 25 mL round bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL) was added N-methylmorpholine (0.343 mL), HOAt (0.142 g), and HATU (0.435 g). After stirring at room temperature for 5 min, this solution was added to a 10 mL cone-shaped flask containing Compound 140 (0.127 g). This solution was stirred at room temperature for 24 h then directly purified by preparative HPLC as described in General Procedure 9, eluting with a 25 to 45% CH3CN/H2O+0.1% TFA gradient to give the title compound as a bright yellow powder (43.0 mg, 38% yield).
LC/MS: Calc'd m/z=424.1 for C22H18FN3O5, found [M+H]+=424.2.
1H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.73 (d, J=8.5 Hz, 1H), 8.61 (s, 1H), 7.96 (d, J=912.1 Hz, 1H), 7.29 (s, 1H), 6.60-6.42 (m, 2H), 5.41 (s, 2H), 5.21 (s, 2H), 2.20 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).
To a solution of Compound 3.2 (1.3 g, 1.0 eq.) in MeOH (12 mL) at 0° C. was added sodium methoxide (0.74 g, 3.0 eq.). After the addition was complete, the ice bath was removed and the resulting solution was stirred at room temperature for 72 h. The reaction was then quenched with ice water (50 mL) and extracted with DCM (3×100 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to yield the title compound as an orange solid (1.2 g, 89% yield).
LC/MS: Calc'd m/z=296.10 for C13H16N2O6, found [M+H]+=297.1.
1H NMR (300 MHz, MeOD) δ 10.29 (s, 1H), 8.61 (s, 1H), 7.73 (s, 1H), 4.08 (s, 3H), 1.57 (s, 9H)
To a solution of Compound 3.39 (500 mg, 1 eq.) in MeOH (10 mL) and H2O (1 mL) was added B2(OH)4 (454 mg, 3 eq.). The resulting mixture was cooled to 0° C. and an aqueous 5 M NaOH solution (2.75 mL) was added with stirring over the course of 10 min. The reaction mixture was stirred for an additional 5 min then quenched by pouring the solution into ice (40 mL). The resulting mixture was extracted with DCM (3×50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was accomplished as described in General Procedure 9, using a 25 g silica column and eluting with 10 to 50% hexanes/EtOAc to give the title compound as an orange solid (386 mg, 86%).
LC/MS: Calc'd m/z=266.1 for C13H18N2O4, found [M+H]+=297.2.
A mixture of Compound 3.40 (385 mg, 1.0 eq.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (362 mg, 0.95 eq.), TsOH (monohydrate, 25 mg, 0.1 eq.) and toluene (30 mL) in a 250 mL round bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 h. The reaction mixture was then cooled to 25° C. and concentrated in vacuo. Purification was accomplished as described in General Procedure 9, using a 25 g silica column and eluting with a 0 to 50% DCM/MeOH gradient to provide the Boc-protected intermediate as a red solid. This material was then deprotecting according to General Procedure 6 followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CH3CN/H2O+0.1% TFA gradient to give the title compound as a red solid (TFA salt, 300 mg, 53% yield).
LC/MS: Calc'd m/z=393.2 for C21H19N3O5, found [M+H]+=393.2.
1H NMR (300 MHz, MeOD) δ 8.27 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 7.11 (s, 1H), 5.61 (d, J=16.2 Hz, 1H), 5.38 (d, J=16.2 Hz, 1H), 5.24 (s, 2H), 4.11 (s, 3H), 2.06-1.91 (m, 2H), 1.04 (t, J=7.4 Hz, 3H).
To a stirring solution of HNO3 (2.0 g, 1.4 mL, 67% purity, 2 eq.) in H2SO4 (8 mL) at 0° C. was added 3-bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 eq.). After the addition was complete, the ice bath was removed, and the reaction was allowed to stir for 5 h at room temperature. The mixture was poured into ice (100 mL) and the precipitate extracted with DCM (3×100 mL). The combined organic fractions were then washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo to yield the title compound as a yellow solid (4.4 g, 93% yield).
LC/MS: Calc'd m/z=296.90 for C8H3BrF3NO3, found [M+H]+=298.0.
1H NMR (300 MHz, MeOD) δ 10.35 (s, 1H), 8.29 (s, 1H), 8.23 (s, 1H).
A mixture of Compound 3.42 (800 mg, 1 eq.), tert-butyl carbamate (378 mg, 1.2 eq.), Cs2CO3 (1.7 g, 2 eq.), Pd2(dba)3 (122 mg, 0.05 eq.), and dicyclohexyl[2′,4′,6′-tris(propan-2-yl)[1,1′-biphenyl]-2-yl]phosphane (XPhos) (256 mg, 0.2 eq.) in toluene (5 mL) was degassed and purged with N2 for three cycles. The mixture was then stirred at 90° C. for 15 h under N2 atmosphere. The reaction mixture was diluted with H2O (25 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (2×25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was achieved according to General Procedure 9, using a 25 g silica column and eluting with 0 to 25% DCM/MeOH to give the title compound as an orange solid (750 mg, 84% yield).
LC/MS: Calc'd m/z=334.1 for C13H13FN2O5, found [M−H]−=333.1.
To a solution of Compound 3.43 (750 mg, 1 eq.) in MeOH (16 mL) and H2O (1.6 mL) was added B2(OH)4 (603 mg, 3 eq.). The resulting mixture was cooled to 0° C. and an aqueous 5 M NaOH solution (2.75 mL) was added with stirring over the course of 10 min. The reaction mixture was stirred for an additional 5 min then quenched by pouring the solution into ice (50 mL). The resulting mixture was extracted with DCM (3×75 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was accomplished as described in General Procedure 9, using a 25 g silica column and eluting with 10 to 50% hexanes/EtOAc to give the title compound as an orange solid (460 mg, 67%).
LC/MS: Calc'd m/z=304.1 for C13H15F3N2O3, found [M+H]+=305.2.
A mixture of Compound 3.44 (460 mg, 1 eq.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (378 mg, 0.95 eq.), TsOH (monohydrate, 26 mg, 0.1 eq.) and toluene (35 mL) in a 250 mL round bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 h. The reaction mixture was then cooled to 25° C. and concentrated in vacuo. Purification was accomplished as described in General Procedure 9, using a 25 g silica column and eluting with a 0 to 50% DCM/MeOH gradient to provide the Boc-protected intermediate as a red solid. This material was then deprotecting according to General Procedure 6 followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CH3CN/H2O+0.1% TFA gradient to give the titled compound as a yellow solid (6.2 mg, 48%).
LC/MS: Calc'd m/z=431.1 for C21H16F3N3O4, found [M+H]+=432.2.
1H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.27 (s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 5.59 (d, J=16.3 Hz, 1H), 5.39 (d, J=16.3 Hz, 1H), 5.28 (s, 2H), 2.00-1.89 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to the procedure described in Chinese Patent Publication No. CN105218644.
To L-phenylalanine (965 mg) in acetonitrile (10 mL) and dimethyl formamide (0.5 mL) was added DIPEA (1.51 mL) then Compound 4.1 (1.3 g). After 1 h the reaction was concentrated to dryness. Flash purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (430 mg, 30% yield).
LC/MS: Calc'd m/z=501.2 for C28H71N3O6S, found [M+H]+=502.4.
1H NMR (300 MHz, DMSO) δ 8.16 (d, J=8.1 Hz, 1H), 8.04 (t, J=5.8 Hz, 1H), 7.90 (d, J=7.5 Hz, 2H), 7.72 (d, J=7.4 Hz, 2H), 7.59 (t, J=6.0 Hz, 1H), 7.54-7.39 (m, 2H), 7.33 (t, J=7.6 Hz, 2H), 7.28-7.13 (m, 5H), 4.44 (td, J=8.5, 5.1 Hz, 1H), 4.33-4.13 (m, 3H), 3.83-3.59 (m, 4H), 3.06 (dd, J=13.7, 5.1 Hz, 1H), 2.88 (dd, J=13.8, 9.0 Hz, 1H).
The title compound was prepared according to the procedure described in International Patent Publication No. WO 2017/054080.
To a solution of Compound 4.3 (1.61 g, 3.58 mmol) in DMF (35 mL) was added Gly-Gly-Phe (1 g, 3.58 mmol) as a single portion followed by iPr2NEt (1.25 mL, 7.2 mmol). This solution was stirred at room temperature for 1 h, then evaporated to dryness. Purification was accomplished as described in General Procedure 9 using a 30 g C18 flash column and eluting with a 10 to 90% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (400 mg, 20% yield).
LC/MS: Calc'd m/z=562.6 for C26H34N4O10, found [M−H]−=561.5.
1H NMR (300 MHz, CDCl3) δ 7.60 (t, J=5.6 Hz, 2H), 7.41 (d, J=7.7 Hz, 1H), 7.32-7.07 (m, 5H), 6.70 (s, 2H), 6.33-6.07 (m, 3H), 4.72 (td, J=7.6, 5.3 Hz, 1H), 4.12-3.78 (m, 4H), 3.72 (ddd, J=15.2, 6.9, 4.8 Hz, 5H), 3.60 (dd, J=11.6, 6.1 Hz, I0H), 3.12 (ddd, J=48.2, 14.0, 6.5 Hz, 2H), 2.52 (d, J=11.7 Hz, 2H).
The title compound was prepared according to the procedure described in US Patent Publication No. US 2017/021031.
The title compound was prepared according to the procedure described in US Patent Publication No. US 2017/021031 using Fmoc-GGFGG-OH as the starting peptide.
The title compound was prepared according to General Procedure 7 starting from Compound 104 (20 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (14 mg, 42% yield).
LC/MS: Calc'd m/z=1051.4 for C52H58N9O12S, found [M+H]+=1052.6.
The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from Compound 4.7 (14 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (9.1 mg, 56% yield).
LC/MS: Calc'd m/z=1234.4 for C60H67FN10O16S, found [M+H]+=1235.8.
The title compound was prepared according to General Procedure 7 starting from Compound 108 (12 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (13 mg, 62% yield).
LC/MS: Calc'd m/z=987.4 for C52H58N9O10, found [M+H]+=988.6.
The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from Compound 4.9 (13 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (3.1 mg, 20% yield).
LC/MS: Calc'd m/z=1170.5 for C60H67FN10O14, found [M+H]+=1171.6.
To a solution of Compound 1.2 (31 mg, 0.076 mmol) in DMF (750 μL) was added (9H-fluoren-9-yl)methyl (2-(((2-(((4-nitrophenoxy)carbonyl)amino)ethoxy)methyl)amino)-2-oxoethyl)carbamate (41 mg, 0.076 mmol) followed by iPr2NEt (26 μL, 0.15 mmol). This solution was stirred at room temperature for 2 h and then applied directly to 12 g C18 column. Purification was accomplished as described in General Procedure 9, eluting with a 10 to 100% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (21 mg, 35% yield).
LC/MS: Calc'd m/z=804.87 for C43H41FN6O9, found [M+H]+=805.6.
Compound 4.11 (21 mg, 0.026 mmol) was taken up in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, the resulting residue was redissolved in DMF (5 mL), and then evaporated to dryness once more. To this residue was added DMF (50 μL) and DCM (450 μL) followed by Compound 4.4 (15 mg, 0.026 mmol), NMM (10 μL) and HATU (10 mg, 0.026 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 30 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (7.6 mg, 26% yield).
LC/MS: Calc'd m/z=1127.1 for C54H63FN10O16, found [M+H]+=1128.2.
To a solution of Compound 4.5 (50 mg, 0.14 mmol), in DCM (800 μL) was added 2-hydroxyethane-1-sulfonyl chloride (100 mg, 0.7 mmol) followed by TFA (200 μL). This solution was stirred at room temperature for 30 min then evaporated to dryness. Purification was accomplished as described in General Procedure 9, using a 10 g silica column and eluting with a 10 to 100% EtOAc/Hexanes gradient to provide the title compound as a clear film (31 mg, 50% yield).
LC/MS: Calc'd m/z=452.1 for C20H21ClN2O6S, found [M+Na]+=472.9.
The title compound was prepared as described in General Procedure 3, using Compound 1.2 (28 mg, 0.07 mmol) and Compound 4.13 (31 mg, 0.07 mmol). Purification was accomplished as described in General Procedure 9, using a 12 g C18 column and eluting with a 10 to 100% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (22 mg, 39% yield).
LC/MS: Calc'd m/z=825.9 for C42H40FN5O10S, found [M+H]+=826.7.
Compound 4.14 (22 mg, 0.027 mmol) was taken up in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, the resulting residue was redissolved in DMF (5 mL), and then evaporated to dryness once more. To this residue was added DMF (50 μL) and DCM (450 μL) followed by Compound 4.4 (30 mg, 0.053 mmol), NMM (10 μL) and HATU (18 mg, 0.048 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 30 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (6.4 mg, 21% yield).
LC/MS: Calc'd m/z=1148.2 for C53H62FN9O17S, found [M+H]+=1148.6.
1H NMR (300 MHz, MeOD) δ 8.60 (t, J=6.5 Hz, 1H), 8.36 (t, J=8.6 Hz, 2H), 8.13 (d, J=6.6 Hz, 1H), 7.77 (d, J=10.6 Hz, 1H), 7.65 (d, J=4.8 Hz, 1H), 7.28-7.00 (m, 6H), 6.80 (s, 2H), 5.69-5.50 (m, 3H), 5.45-5.33 (m, 2H), 4.44 (dd, J=8.7, 5.7 Hz, 1H), 3.96 (t, J=5.3 Hz, 2H), 3.90-3.76 (m, 5H), 3.76-3.57 (m, 7H), 3.09-2.81 (m, 3H), 2.61-2.45 (m, 5H), 2.04-1.90 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
To a solution of Compound 4.5 (100 mg, 0.27 mmol) in DCM (800 μL) was added (S)-morpholin-2-ylmethanol (160 mg, 1.36 mmol) followed by TFA (200 μL). This solution was stirred at room temperature for 1 h then evaporated to dryness. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 10 to 90% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (TFA salt, 105 mg, 72% yield).
LC/MS: Calc'd m/z=425.2 for C23H27N3O5, found [M+Na]+=448.0.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (50 mg, 0.117 mmol) and Compound 4.16 (63 mg, 0.117 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 100% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 33 mg, 35% yield).
LC/MS: Calc'd m/z=817.9 for C45H44FN5O9, found [M+H]+=818.7.
Compound 4.17 (33 mg, 0.04 mmol) was taken up in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, the resulting residue was redissolved in DMF (5 mL), and then evaporated to dryness once more. To this residue was added DMF (100 μL) and DCM (900 μL) followed by Compound 4.4 (45 mg, 0.08 mmol), NMM (20 μL) and HATU (28 mg, 0.073 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 30 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (22 mg, 48% yield).
LC/MS: Calc'd m/z=1140.2 for C56H66FN9O16, found [M+H]+=1141.1.
1H NMR (300 MHz, MeOD) δ 8.35 (d, J=7.5 Hz, 2H), 7.74-7.61 (m, 1H), 7.53 (s, 1H), 7.34-7.10 (m, 6H), 6.81 (s, 2H), 5.65-5.30 (m, 4H), 4.64 (t, J=3.4 Hz, 2H), 4.42 (tt, J=6.3, 2.5 Hz, 1H), 4.09 (d, J=12.3 Hz, 1H), 3.98-3.76 (m, 8H), 3.72 (t, J=6.0 Hz, 2H), 3.69-3.44 (m, 17H), 3.21-2.85 (m, 3H), 2.64-2.42 (m, 5H), 2.03-1.84 (m, 2H), 0.98 (t, J=7.3 Hz, 3H).
Compound 3.5 (55 mg, 0.11 mmol) was dissolved in TFA (500 μL) and stirred at room temperature for 20 min, then hexafluoroisopropanol (2 mL) was added followed by Compound 4.5 (40 mg, 0.11 mmol). This solution was stirred at room temperature for ˜16 h then concentrated to dryness. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (11 mg, 14% yield).
LC/MS: Calc'd m/z=719.7 for C39H34FN5O8, found [M+H]+=720.6.
Compound 4.19 (11 mg, 0.015 mmol) was taken up in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, the resulting residue was redissolved in DMF (5 mL), and then evaporated to dryness once more. To this residue was added DMF (50 μL) and DCM (450 μL) followed by Compound 4.4 (26 mg, 0.045 mmol), NMM (5 μL) and HATU (18 mg, 0.045 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 32 to 45% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (4.6 mg, 29% yield).
LC/MS: Calc'd m/z=1142.0 for C50H56FN9O15, found [M+H]+=1143.1.
1H NMR (300 MHz, MeOD) δ 8.36 (s, 1H), 8.28 (d, J=6.1 Hz, 1H), 8.16 (dd, J=20.1, 6.8 Hz, 3H), 7.59-7.44 (m, 2H), 7.31-7.08 (m, 6H), 6.79 (s, 2H), 5.58 (d, J=16.1 Hz, 1H), 5.37 (d, J=16.1 Hz, 1H), 5.30-5.16 (m, 3H), 4.56-4.39 (m, 1H), 4.07-3.90 (m, 2H), 3.85 (dt, J=11.5, 5.4 Hz, 4H), 3.79-3.67 (m, 4H), 3.67-3.55 (m, 7H), 3.54 (d, J=6.5 Hz, 8H), 3.10 (dd, J=14.0, 6.1 Hz, 1H), 2.92 (dd, J=13.9, 9.1 Hz, 1H), 2.53 (t, J=6.0 Hz, 2H), 1.98 (q, J=7.2 Hz, 2H), 1.31 (s, 1H), 1.04 (t, J=7.3 Hz, 3H).
Compound 4.19 (25 mg, 0.035 mmol) was taken up in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, the resulting residue was redissolved in DMF (5 mL), and then evaporated to dryness once more. To this residue was added DMF (50 μL) and DCM (450 μL), followed by MC-GGF-OH (33 mg, 0.07 mmol), NMM (20 μL) and HATU (25 mg, 0.066 mmol). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 30 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (4.3 mg, 13% yield).
LC/MS: Calc'd m/z=952.0 for C47H50FN9O12, found [M+H]+=952.9.
1H NMR (300 MHz, CD3CN) δ 7.96-7.72 (m, 1H), 7.39-7.07 (m, 8H), 6.94 (d, J=9.1 Hz, 1H), 6.73 (s, 2H), 5.44 (d, J=16.2 Hz, 1H), 5.25 (d, J=16.2 Hz, 1H), 5.06 (d, J=4.4 Hz, 2H), 4.81 (d, J=26.1 Hz, 4H), 4.61 (s, 1H), 3.96 (s, 1H), 3.77 (d, J=8.1 Hz, 7H), 3.02 (d, J=5.6 Hz, 5H), 2.19 (t, J=7.7 Hz, 3H), 1.50 (dp, J=14.8, 7.4 Hz, 6H), 1.32-1.12 (m, 3H), 0.96 (t, J=7.2 Hz, 3H).
The title compound was prepared according to Procedure 7 starting from Compound 140 (28 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (10 mg, 17% yield).
LC/MS: Calc'd m/z=799.3 for C40H42N7O10, found [M+H]+=800.6.
The title compound was prepared according to General Procedure 6 followed by General Procedure 8 starting from Compound 4.22 (10 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (6.8 mg, 55% yield).
LC/MS: Calc'd m/z=982.4 for C48H51FN8O14, found [M+H]+=983.6.
The title compound was prepared according to General Procedure 7 starting from Compound 142 (TFA salt, 45 mg). Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (13 mg, 22% yield).
LC/MS: Calc'd m/z=898.4 for C45H51N8O11, found [M+H]+=899.6.
The title compound was prepared according to General Procedure 6 followed by General Procedure 8 starting from Compound 4.24 (13 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (2.6 mg, 17% yield).
LC/MS: Calc'd m/z=1081.4 for C53H60FN9O15, found [M+H]+=1082.6.
1H NMR (300 MHz, MeOD) δ 9.34 (d, J=8.5 Hz, 1H), 7.87 (d, J=11.8 Hz, 1H), 7.62 (s, 1H), 7.33-7.19 (m, 5H), 6.80 (s, 2H), 5.62 (d, J=16.3 Hz, 1H), 5.51 (s, 2H), 5.47-5.35 (m, 3H), 4.73 (dd, J=9.6, 5.1 Hz, 1H), 4.61 (s, 3H), 4.30-4.15 (m, 2H), 4.11 (s, 2H), 4.00-3.82 (m, 4H), 3.82-3.70 (m, 7H), 3.70-3.50 (m, 13H), 3.18-3.04 (m, 1H), 2.88 (s, 1H), 2.64 (d, J=5.8 Hz, 4H), 2.54 (t, J=6.0 Hz, 2H), 2.09-1.92 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
To a stirring solution of Compound 4.6 (60 mg) in dichloromethane (2 mL) was added ethylene glycol (100 μL) followed by trifluoracetic acid (0.4 mL). After 30 min the reaction was concentrated in vacuo. Purification of the intermediate compound was accomplished as described in General Procedure 9, using a 10 g flash column and eluting with a 0 to 20% dichloromethane/methanol gradient. To the purified intermediate in tetrahydrofuran (0.5 mL) was added bis-nitrophenol carbonate (58 mg) followed by DIPEA (50 μL). The solution was stirred for 16 h, quenched with acetic acid (˜100 μL) then concentrated to dryness. Purification was accomplished as described in General Procedure 9, using a 10 g flash column and eluting with a 0 to 20% dichloromethane/methanol gradient to provide the title compound as a white solid (40 mg, 53% yield from Compound 4.6).
LC/MS: Calc'd m/z=796.3 for C40H40N6O12, found [M+Na]+=819.4.
To a solution of Compound 4.26 (40 mg) in dimethylformamide (1 mL) was added DIPEA (26 μL) then a solution of Compound 1.2 (24 mg) in dimethylformamide (0.5 mL). This solution was stirred for 4 h at room temperature then quenched with a 20% piperidine in dimethylformamide solution (0.5 mL) and stirred for an additional 20 min. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (TFA salt, 19 mg, 39% yield).
LC/MS: Calc'd m/z=844.3 for C41H45FN8O11, found [M+H]+=845.6.
The title compound was prepared according to General Procedure 8 starting from Compound 4.27 (10 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (8.8 mg, 75% yield).
LC/MS: Calc'd m/z=1127.4 for C54H62FN9O17, found [M+H]+=1128.6.
1H NMR (300 MHz, MeOD) δ 8.27 (d, J=8.1 Hz, 1H), 7.81 (d, J=10.7 Hz, 1H), 7.65 (s, 1H), 7.32-7.16 (m, 5H), 6.81 (s, 2H), 5.62 (d, J=16.4 Hz, 1H), 5.53 (s, 2H), 5.42 (d, J=16.4 Hz, 1H), 4.93 (s, 2H), 4.67 (s, 1H), 4.51 (dd, J=9.3, 5.6 Hz, 1H), 4.18 (t, J=4.7 Hz, 2H), 4.01-3.44 (m, 19H), 3.17 (dd, J=13.9, 5.8 Hz, 1H), 2.97 (dd, J=13.9, 9.0 Hz, 1H), 2.57 (s, 3H), 2.52 (t, J=6.0 Hz, 2H), 2.03-1.91 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
To a stirring solution of Fmoc-glycine (217 mg) in dimethylformamide (2.5 mL) was added HATU (254 mg), HOAt (83 mg) then NMM (188 μL). This solution was stirred for 10 min then Compound 141 (50 mg) was added and the reaction was stirred at room temperature for 16 h. Lithium hydroxide (2.5 mL, 1 M in water) was added, and the reaction mixture was stirred for 2 h. This solution was partially concentrated, then a solution of 20% piperidine in dimethylformamide (0.5 mL) was added and was stirred for another 20 min. The reaction was then evaporated onto celite and purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 0 to 40% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (TFA salt, 44 mg, 62% yield).
LC/MS: Calc'd m/z=468.1 for C23H21FN4O6, found [M+H]+=469.4.
1H NMR (300 MHz, MeOD) δ 8.99 (d, J=8.3 Hz, 1H), 7.99 (s, 1H), 7.87 (d, J=12.0 Hz, 1H), 7.55 (s, 1H), 5.60 (d, J=16.3 Hz, 1H), 5.46-5.35 (m, 3H), 5.30 (s, 2H), 3.53-3.45 (m, 1H), 3.43-3.38 (m, 1H), 2.03-1.87 (m, 2H), 1.02 (t, J=7.3 Hz, 3H).
To a stirring solution of Compound 4.4 (23 mg) in a mixture of dimethylformamide (0.1 mL) and dichloromethane (0.9 mL) was added HATU (14 mg), a solution of Compound 4.29 (20 mg) in dimethyl formamide (0.1 mL) and dichloromethane (0.9 mL), and DIPEA (24 μL). The mixture was stirred for 15 min, then the reaction was partially concentrated. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 0 to 40% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (7.1 mg, 20% yield).
LC/MS: Calc'd m/z=1012.4 for C49H53FN8O15, found [M+H]+=1013.6.
1H NMR (300 MHz, MeOD) δ 9.89 (s, 1H), 8.75 (d, J=8.3 Hz, 1H), 8.44-8.32 (m, 1H), 8.27-8.14 (m, 2H), 7.78 (d, J=11.9 Hz, 1H), 7.53 (s, 1H), 7.39-7.20 (m, 5H), 6.82 (s, 2H), 5.57 (d, J=16.3 Hz, 1H), 5.39 (d, J=16.3 Hz, 1H), 5.34-5.25 (m, 2H), 5.22 (s, 2H), 4.32-4.09 (m, 2H), 3.96-3.83 (m, 3H), 3.76 (t, J=6.0 Hz, 2H), 3.69-3.62 (m, 2H), 3.62-3.47 (m, 9H), 3.40-3.33 (m, 1H), 3.08 (dd, J=14.0, 9.6 Hz, 1H), 2.56 (t, J=6.1 Hz, 2H), 2.03-1.91 (m, 2H), 1.04 (t, J=7.3 Hz, 3H).
To a stirring solution of Compound 145 (32 mg) in dichloromethane (2 mL) and acetonitrile (0.5 mL) was added di-tert-butyl dicarbonate (20 μL) followed by DIPEA (42 μL). The reaction mixture was stirred at room temperature for 3 h then concentrated to dryness to provide the title compound as a red solid (34 mg, 87%).
LC/MS: Calc'd m/z=510.2 for C26H27FN4O6, found [M+H]+=511.2.
To a stirring solution of Fmoc-glycine (98 mg) in dimethylformamide (1 mL) was added HATU (115 mg), HOAt (37 mg) then NMM (85 μL). This solution was stirred for 10 min, then Compound 4.31 (28 mg) was added. The reaction was stirred at room temperature for 16 h then quenched with a solution of 20% piperidine in dimethylformamide (1 mL) and stirred for an additional 20 min. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5 to 40% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (TFA salt, 25 mg, 67% yield).
LC/MS: Calc'd m/z=567.2 for C28H30FN6O7, found [M+H]+=568.4.
1H NMR (300 MHz, MeOD) δ 9.01 (d, J=8.3 Hz, 1H), 7.83 (d, J=11.9 Hz, 1H), 7.52 (s, 1H), 5.57 (d, J=16.4 Hz, 1H), 5.38 (d, J=16.3 Hz, 1H), 5.27 (d, J=3.1 Hz, 2H), 4.80 (s, 2H), 4.10 (s, 2H), 1.97 (q, J=7.4 Hz, 2H), 1.50 (s, 9H), 1.02 (t, J=7.3 Hz, 3H).
To a stirring solution of Fmoc-GGF-OH (28 mg) and HATU (20 mg) in a mixture of DMF (0.2 mL) and dichloromethane (1.8 mL) was added Compound 4.32 (25 mg) followed by DIPEA (32 μL). This solution was stirred for 15 min at room temperature, quenched with a solution of 20% piperidine in dimethylformamide (0.250 mL), stirred for an additional 20 min, then partially concentrated in vacuo. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 10 to 45% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (TFA salt, 22 mg, 64% yield).
LC/MS: Calc'd m/z=828.3 for C41H45FN8O10, found [M+H]+=829.6.
The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from Compound 4.33 (15 mg). Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient. The title compound was obtained post Boc-deprotection as a white-solid (TFA salt, 8.5 mg, 52% yield).
LC/MS: Calc'd m/z=1011.4 for C49H53FN8O15, found [M+H]+=1012.6.
1H NMR (300 MHz, MeOD) δ 9.04 (d, J=8.0 Hz, 1H), 8.40 (d, J=5.7 Hz, 1H), 8.21 (d, J=7.7 Hz, 1H), 8.05 (d, J=11.5 Hz, 1H), 7.67 (s, 1H), 7.42-7.03 (m, 5H), 6.81 (s, 2H), 5.63 (d, J=16.4 Hz, 1H), 5.51 (s, 1H), 5.43 (d, J=16.5 Hz, 1H), 4.81 (s, 2H), 4.75-4.58 (m, 1H), 4.29-4.10 (m, 2H), 3.98-3.81 (m, 4H), 3.78-3.71 (m, 2H), 3.71-3.63 (m, 2H), 3.62-3.53 (m, 9H), 3.14-2.98 (m, 1H), 2.54 (t, J=6.0 Hz, 2H), 2.08-1.93 (m, 2H), 1.03 (t, J=7.3 Hz, 3H).
To a solution of Fmoc-Gly-OH (100.9 mg, 0.34 mmol) in dimethylformamide (550 μL) was added NMM (0.112 mL, 1.02 mmol) and HATU (0.103 g, 0.272 mmol). This solution was stirred at room temperature for 20 min, then a solution of Compound 148 (32.5 mg, 0.068 mmol) in DMF (250 μL) was added, and the reaction mixture was stirred for 16 h. Purification was accomplished as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5 to 40% CH3CN/H2O+0.1% TFA gradient. The obtained residue was re-purified according to General Procedure 9, using a 10 g flash column and eluting with a 0 to 10% MeOH/DCM gradient to provide the title compound as a yellow powder (15.3 mg, 30% yield).
LC/MS: Calc'd m/z=757.3 for C43H40FN5O7, found [M+H]+=758.6.
To a 50 mL flask containing Compound 4.35 (15.3 mg, 0.02 mmol) was added a solution of 20% piperidine in DMF (2.0 mL). This solution was stirred at room temperature for 5 min then evaporated to dryness. The obtained residue was then dissolved in 10% DMF/DCM (1.0 mL), then NMM (5.50 μL, 0.05 mmol), Compound 4.4 (11.2 mg, 0.02 mmol) and HATU (8.7 mg, 0.02 mmol) were added. This solution was stirred for 45 min, then partially evaporated. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 15 to 45% CH3CN/H2O+0.1% TFA gradient to give the title product as a yellow powder (7.8 mg, 33% yield).
LC/MS: Calc'd m/z=1079.4 for C54H62FN9O14, found [M+H]+=1080.8.
1H NMR (300 MHz, MeOD) δ 8.99 (d, J=8.2 Hz, 1H), 7.52 (d, J=12.3 Hz, 1H), 7.39-7.25 (m, 5H), 7.25-7.17 (m, 1H), 6.79 (s, 2H), 5.53 (d, J=16.4 Hz, 1H), 5.33 (d, J=16.5 Hz, 1H), 4.80-4.72 (m, 1H), 4.32-4.11 (m, 2H), 3.98-3.79 (m, 6H), 3.76 (t, J=6.0 Hz, 2H), 3.66-3.60 (m, 2H), 3.62-3.49 (m, I0H), 3.15-3.03 (m, 1H), 2.65-2.47 (m, 6H), 1.96 (q, J=7.4 Hz, 2H), 1.72-1.57 (m, 4H), 1.57-1.42 (m, 2H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared according to General Procedure 7 starting from Compound 127 (46 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (7.2 mg, 21% yield).
LC/MS: Calc'd m/z=983.0 for C48H51N8O12S, found [M+H]+=983.9.
The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from Compound 4.37 (7.2 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 10 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (1 mg, 12% yield).
LC/MS: Calc'd m/z=1166.2 for C56H60FN9O16, found [M+H]+=1167.1.
To a stirring solution of Compound 4.6 (44 mg) in dichloromethane (2 mL) was added 3-azabicyclo[3.1.1]heptan-6-ol (5.3 mg) followed by trifluoracetic acid (0.4 mL). After 30 min the reaction was concentrated in vacuo. Purification was accomplished as described in General Procedure 9, using a 10 g flash column and eluting with a 0 to 20% dichloromethane/methanol gradient to provide the title compound as a white solid (14.7 mg, 46% yield).
LC/MS: Calc'd m/z=682.8 for C37H42N6O7, found [M+H]+=683.6.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (3 mg, 0.007 mmol) and Compound 4.39 (14.7 mg, 0.022 mmol) and utilizing 200 μL DMF. Following complete consumption of Compound 1.1, a solution of 20% piperidine in DMF (200 μL) was added and this solution was stirred at room temperature for 10 min. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 37% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 1.8 mg, 29% yield).
LC/MS: Calc'd m/z=852.9 for C44H49FN8O9, found [M+H]+=853.7.
The title compound was prepared according to Procedure 8 starting from Compound 4.40 (1.8 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 45% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white-solid (TFA salt, 0.5 mg, 22% yield).
LC/MS: Calc'd m/z=1135.5 for C57H66FN9O15, found [M+H]+=1136.3.
To a stirring solution of Compound 4.6 (144 mg) in dichloromethane (2 mL) was added (3-fluoroazetidin-3-yl)methanol (16 mg) followed by trifluoracetic acid (0.4 mL). After 30 min the reaction was concentrated in vacuo. Purification was accomplished as described in General Procedure 9, using a 10 g flash column and eluting with a 0 to 20% dichloromethane/methanol gradient to provide the title compound as a white solid (55 mg, 54% yield).
LC/MS: Calc'd m/z=674.7 for C35H39N6FO7, found [M+H]+=675.6.
The title compound was prepared according to General Procedure 1 starting from Compound 1.1 (11.6 mg, 0.027 mmol) and Compound 4.42 (55 mg, 0.082 mmol) and utilizing 500 μL DMF. Following complete consumption of Compound 1.1, a solution of 20% piperidine in DMF (500 μL) was added and this solution was stirred at room temperature for 10 min. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 32% CH3CN/H2O+0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 8.1 mg, 28% yield).
LC/MS: Calc'd m/z=844.3 for C42H46F2N8O9, found [M+H]+=845.3.
The title compound was prepared according to Procedure 8 starting from Compound 4.43 (8.1 mg). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 45% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white-solid (TFA salt, 2.9 mg, 28% yield).
LC/MS: Calc'd m/z=1127.4 for C55H63F2N9O15, found [M+H]+=1128.8.
To Compound 4.27 (450 mg) was added a solution of 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (130 mg) and N-ethyldiisopropylamine (250 μL) in DMF (10 mL). This solution was stirred at room temperature for 30 min then concentrated to −1 mL volume. Purification was accomplished as described in General Procedure 9 first using a 60 g C18 flash column and eluting with a 10 to 60% CH3CN/H2O+0.1% TFA gradient followed by preparative HPLC of impure fractions using a 20 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (320 mg, 66% yield).
LC/MS: Calc'd m/z=1037.4 for C51H56FN9O14, found [M+H]+=1038.6.
1H NMR (300 MHz, MeOD) δ 8.10 (d, J=8.1 Hz, 2H), 8.01 (s, 1H), 7.95 (d, J=7.0 Hz, 1H), 7.74 (d, J=10.4 Hz, 1H), 7.66 (s, 1H), 7.56 (s, 1H), 7.32-7.10 (m, 5H), 6.69 (s, 2H), 5.63 (d, J=16.4 Hz, 1H), 5.46 (s, 2H), 5.32 (s, 1H), 5.28 (d, J=16.5 Hz, 1H), 4.88 (s, 2H), 4.67 (d, J=6.4 Hz, 2H), 4.48 (d, J=7.1 Hz, 2H), 4.15 (t, J=4.2 Hz, 2H), 3.92 (dd, J=17.1, 6.2 Hz, 2H), 3.83-3.57 (m, 6H), 3.46 (t, J=7.1 Hz, 2H), 3.16 (dd, J=14.0, 5.9 Hz, 1H), 2.95 (dd, J=13.9, 8.9 Hz, 1H), 2.53 (s, 3H), 2.21 (t, J=7.6 Hz, 2H), 1.97-1.79 (m, 2H), 1.58 (dp, J=15.0, 7.6 Hz, 4H), 1.29 (dd, J=16.6, 9.3 Hz, 3H), 1.01 (t, J=7.3 Hz, 3H).
A solution of Compound 140 (860 mg, 1.7 mmol, TFA salt), Boc-Gly-OH (760 mg, 4.3 mmol), HATU (1.6 g, 4.1 mmol), and N-ethyldiisopropylamine (0.6 mL) in DMF (4 mL) was stirred at room temperature for 24 h then poured into water (50 mL). The resulting solid was collected by filtration, redissolved in 10% MeOH/DCM and purification was accomplished as described in General Procedure 9, using a 30 g silica column and eluting with a 0 to 10% MeOH/DCM to provide the title compound as a yellow solid (750 mg, 80% yield).
LC/MS: Calc'd m/z=538.5 for C27H27FN4O7, found [M+H]+=539.4.
1H NMR (300 MHz, MeOD) δ 8.84 (d, J=8.4 Hz, 1H), 8.52 (s, 1H), 8.00 (s, 1H), 7.87 (d, J=12.1 Hz, 1H), 7.62 (s, 1H), 5.60 (d, J=16.3 Hz, 1H), 5.40 (d, J=16.4 Hz, 1H), 5.27 (s, 2H), 4.02 (s, 2H), 1.99 (dt, J=8.7, 6.7 Hz, 2H), 1.52 (s, 9H), 1.03 (t, J=7.4 Hz, 3H).
The title compound was prepared in three steps from Compound 4.46 (750 mg). The Boc protecting group was cleaved in neat TFA (2 mL) followed by precipitation in Et2O (50 mL). The solid was collected by filtration and added to a solution of 2,5-dioxopyrrolidin-1-yl (2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoate (340 mg, 1.1 equiv) and N-ethyldiisopropylamine (300 μL) in DMF (1.7 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (50 mL). The precipitate was collected by filtration, dried under vacuum then dissolved in neat TFA (2 mL). After 20 min, Et2O (50 mL) was added and the precipitate collected by filtration to provide the title compound as a yellow solid (531 mg, 54% yield).
LC/MS: Calc'd m/z=585.2 for C31H28FN5O6, found [M+H]+=586.1.
To Compound 4.47 (490 mg) was added a solution of Boc-gly-gly-NHS (250 mg, 1.1 equiv) and N-ethyldiisopropylamine (250 μL) in DMF (3 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (50 mL). The precipitate was collected by filtration then dissolved in neat TFA (2 mL). After 20 min, Et2O (50 mL) was added and the precipitate collected by filtration to provide the title compound as a yellow solid (500 mg, 88% yield).
LC/MS: Calc'd m/z=699.2 for C35H34FN7O8, found [M+H]+=700.4.
To Compound 4.48 (500 mg) was added a solution of 2,5-dioxocyclopentyl 6-(2,5-dioxopyrrol-1-yl)hexanoate (210 mg, 1.1 equiv) and N-ethyldiisopropylamine (215 μL) in DMF (4 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (50 mL). The precipitate was collected by filtration then dissolved in DMF (2 mL). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 24 to 38% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (190 mg, 40% yield).
LC/MS: Calc'd m/z=892.9 for C45H45FN8O11, found [M+H]+=893.6.
1H NMR (300 MHz, CD3CN) δ 8.67 (d, J=8.4 Hz, 1H), 8.44 (s, 1H), 7.78 (d, J=12.1 Hz, 1H), 7.41 (s, 1H), 7.30 (d, J=4.3 Hz, 4H), 7.26-7.16 (m, 1H), 6.72 (s, 2H), 5.52 (d, J=16.4 Hz, 1H), 5.31 (d, J=16.4 Hz, 1H), 5.12 (s, 2H), 4.64 (dd, J=9.7, 5.0 Hz, 1H), 4.11 (d, J=3.2 Hz, 2H), 3.87-3.68 (m, 4H), 3.37 (t, J=7.1 Hz, 2H), 3.00 (dd, J=14.0, 9.7 Hz, 1H), 2.20 (t, J=7.6 Hz, 2H), 1.49 (dq, J=19.5, 7.4 Hz, 4H), 1.22 (p, J=7.6, 7.1 Hz, 2H), 0.94 (t, J=7.3 Hz, 3H).
A solution of Compound 4.46 (1.8 g), iron (II) sulfate heptahydrate (1.4 g, 1.5 equiv), and sulfuric acid (450 μL, 2.5 equiv) in MeOH (33 mL) was heated to 60° C. and hydrogen peroxide (1.25 mL, 12 equiv) was added dropwise over 10 min. This solution was heated for another 20 min then cooled to room temperature and poured into ice water (˜200 mL). The brown precipitate was collected by filtration and the filtrate was quenched with saturated aqueous Na2S203. MeOH was evaporated and the solution allowed to stand for 2 h while a second brown precipitate formed. This precipitate was collected by filtration and the combined precipitates were purified as described in General Procedure 9 using a 50 g silica column and eluting with a 0 to 15% MeOH/DCM gradient to provide the title compound as a yellow solid (860 mg, 45% yield).
LC/MS: Calc'd m/z=568.5 for C28H29FN4O8, found [M+H]+=569.7.
The title compound was prepared in three steps from Compound 4.50 (750 mg). The Boc protecting group was cleaved in neat TFA (2 mL) followed by precipitation in Et2O (100 mL). The solid was collected by filtration and added to a solution of 2,5-dioxopyrrolidin-1-yl (2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoate (600 mg, 1.1 equiv) and N-ethyldiisopropylamine (300 μL) in DMF (7 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (100 mL). The precipitate was collected by filtration, dried under vacuum then dissolved in neat TFA (2 mL). After 20 min, Et2O (100 mL) was added and the precipitate collected by filtration to provide the title compound as a yellow solid (756 mg, 78% yield).
LC/MS: Calc'd m/z=615.2 for C32H30FN5O7, found [M+H]+=616.3.
To Compound 4.51 (756 mg) was added a solution of Boc-gly-gly-NHS (375 mg, 1.1 equiv) and N-ethyldiisopropylamine (400 μL) in DMF (5 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (75 mL). The precipitate was collected by filtration then dissolved in neat TFA (4 mL). After 20 min, Et2O (100 mL) was added and the precipitate collected by filtration to provide the title compound as a yellow solid (826 mg, 95% yield).
LC/MS: Calc'd m/z=729.2 for C36H36FN7O9, found [M+H]+=730.2.
To Compound 4.52 (826 mg) was added a solution of 2,5-dioxocyclopentyl 6-(2,5-dioxopyrrol-1-yl)hexanoate (382 mg, 1.1 equiv) and N-ethyldiisopropylamine (300 μL) in DMF (5.5 mL). This solution was stirred at room temperature for 30 min then pipetted into Et2O (100 mL). The precipitate was collected by filtration then dissolved in DMF (2 mL). Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 40% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (370 mg, 35% yield).
LC/MS: Calc'd m/z=922.9 for C46H47FN8O12, found [M+H]+=923.8.
1H NMR (300 MHz, CD3CN) δ 8.63 (d, J=8.4 Hz, 1H), 7.67 (d, J=11.9 Hz, 1H), 7.38-7.27 (m, 5H), 7.24 (d, J=4.3 Hz, 1H), 6.72 (s, 2H), 5.48 (d, J=16.4 Hz, 1H), 5.28 (d, J=16.3 Hz, 1H), 5.24-5.01 (m, 4H), 4.65 (dd, J=9.7, 4.9 Hz, 1H), 4.13 (s, 2H), 3.85-3.75 (m, 3H), 3.37 (t, J=7.1 Hz, 2H), 3.00 (dd, J=14.0, 9.8 Hz, 1H), 2.21 (t, J=7.6 Hz, 2H), 1.51 (dp, J=22.0, 7.4 Hz, 4H), 1.22 (p, J=7.4, 7.0 Hz, 2H), 0.94 (t, J=7.3 Hz, 3H).
To Compound 3.4 (500 mg, 1.0 mmol) was added TFA (4 mL) and this solution was allowed to stand at rt for 1 h, then Et2O (100 mL) was added, and the precipitate was collected by filtration. This solid was taken up in DMF (3.4 mL) and Boc-Ala-OH (590 mg, 3.1 mmol, 3 equiv) and HATU (1.2 g, 3.1 mmol, 3 equiv) were added followed by N-ethyldiisopropylamine (0.9 mL, 5.2 mmol, 5 equiv). This solution was stirred at rt for 3 days then poured into ice water (50 mL) and the precipitate was collected by filtration to give the title compound as a brown solid (125 mg, 22% yield).
LC/MS: Calc'd m/z=552.6 for C28H29FN4O7, found [M+H]+=553.7.
To Compound 4.54 (125 mg, 0.225 mmol) in a 100 mL round bottom flask was added TFA (2 mL). This solution was allowed to stand for 10 min, then Et2O (50 mL) was added, and the precipitate collected by filtration. The resulting orange solid was added to a solution of Boc-Val-NHS (78 mg, 0.25 mmol, 1.1 equiv) and N-ethyldiisopropylamine (80 μL, 0.45 mmol, 2 equiv) in DMF (2 mL). This solution was stirred at rt for 30 min, then pipetted into Et2O (40 mL) in a 50 mL falcon tube and the precipitate was collected by centrifugation and decanting of the Et2O. The pellet was dissolved in TFA (2 mL) and allowed to stand for 10 min prior to the addition of Et2O (40 mL). The precipitate was collected by centrifugation and decanting the Et2O. The pellet was dried under high vacuum to give the title compound as an orange solid (135 mg, 90% yield over 3 steps).
LC/MS: Calc'd m/z=551.2 for C28H30FN5O6, found [M+H]+=552.2.
To Compound 4.55 (20 mg, 0.03 mmol) was added a solution of 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (11 mg, 0.036 mmol) and N-ethyldiisopropylamine (10 μL) in DMF (1 mL). This solution was stirred at rt for 30 min then purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 60% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (8.8 mg, 40% yield).
LC/MS: Calc'd m/z=744.8 for C38H41FN6O9, found [M+H]+=745.6.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.60 (d, J=8.5 Hz, 1H), 8.31 (s, 1H), 7.96 (d, J=6.5 Hz, 1H), 7.65 (d, J=12.0 Hz, 1H), 7.37-7.26 (m, 2H), 6.75 (s, 2H), 5.45 (d, J=16.6 Hz, 1H), 5.25 (d, J=16.3 Hz, 1H), 5.04 (d, J=4.0 Hz, 2H), 4.78-4.58 (m, 1H), 4.30-4.13 (m, 1H), 2.32-2.16 (m, 2H), 2.10 (dt, J=13.6, 6.8 Hz, 1H), 1.88 (q, J=7.4 Hz, 2H), 1.57 (dq, J=15.5, 7.6 Hz, 4H), 1.45 (d, J=7.1 Hz, 3H), 1.26 (tt, J=10.1, 6.1 Hz, 2H), 1.05-0.83 (m, 9H).
To Compound 4.55 (20 mg, 0.03 mmol) was added a solution of bis(2,5-dioxopyrrolidin-1-yl) adipate (30 mg, 0.09 mmol, 3 equiv) and N-ethyldiisopropylamine (10 μL) in DMF (1 mL). This solution was stirred at rt for 30 min then purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 25 to 35% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (4.1 mg, 18% yield).
LC/MS: Calc'd m/z=776.8 for C38H41FN6O11, found [M+H]+=777.6.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.65 (dd, J=8.4, 2.3 Hz, 1H), 8.38 (s, 1H), 7.95 (d, J=6.5 Hz, 1H), 7.72 (d, J=12.0 Hz, 1H), 7.37 (d, J=11.8 Hz, 2H), 5.58-5.19 (m, 2H), 5.10 (s, 2H), 4.78-4.56 (m, 1H), 4.23 (dd, J=8.4, 7.0 Hz, 1H), 2.80 (s, 4H), 2.65 (t, J=6.9 Hz, 2H), 2.39-2.22 (m, 2H), 2.11 (q, J=6.8 Hz, 1H), 1.94-1.81 (m, 2H), 1.79-1.57 (m, 4H), 1.45 (d, J=7.1 Hz, 3H), 1.10-0.78 (m, 9H).
A solution of Compound 4.55 (20 mg, 0.03 mmol), 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanoic acid (17 mg, 0.03 mmol), and HATU (13 mg, 0.03 mmol) in DMF (300 μL) was cooled to 0° C. and N-ethyldiisopropylamine (16 μL, 0.09 mmol) was added. This solution was stirred for 30 min the purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 50% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (13.7 mg, 42% yield).
LC/MS: Calc'd m/z=1088.2 for C50H70FN9O17, found [M+H]+=1088.8.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 8.62 (d, J=8.5 Hz, 1H), 8.33 (s, 1H), 7.66 (d, J=12.2 Hz, 1H), 7.35 (s, 1H), 5.52-5.18 (m, 2H), 5.04 (s, 2H), 4.72 (q, J=7.1 Hz, 1H), 4.32 (d, J=7.3 Hz, 1H), 4.15-3.98 (m, 4H), 3.68-3.48 (m, 35H), 3.41-3.34 (m, 6H), 2.18 (h, J=6.8 Hz, 1H), 1.88 (q, J=7.4 Hz, 2H), 1.47 (d, J=7.1 Hz, 3H), 1.13-0.84 (m, 9H).
The title compound was prepared as described in Wang, et al., Nano Lett., 2014, 14(10):5577-5583.
To a solution of Compound 4.58 (200 mg, 0.7 mmol) in DCM (1.4 mL) was added β-mercaptoethanol (50 μL, 0.7 mmol) and this solution was stirred at rt for 5 h. The solution was diluted with DCM (10 mL), washed with a water (3×10 mL), dried over Na2SO4, and concentrated to an oil. Purification was accomplished as described in General Procedure 9, using a 10 g silica column, and eluting with a 0 to 10% MeOH/DCM to give the title compound as a colorless solid (212 mg, 82% yield).
LC/MS: Calc'd m/z=253.1 for C11H23NO3S2, found [M+H, −Boc]+=154.0.
1H NMR (300 MHz, Chloroform-d) δ 4.94 (s, 1H), 3.91 (t, J=5.7 Hz, 2H), 3.49 (q, J=6.4 Hz, 2H), 2.86 (dt, J=23.7, 6.1 Hz, 4H), 2.15 (s, 2H), 1.47 (s, 9H).
To Compound 4.59 (212 mg, 0.837 mmol) in a 25 mL round bottom flask was added a 4 M HCl/dioxane solution (5 mL) and the solution was stirred at rt for 30 min, then evaporated to dryness. The residue was suspended in EtOAc (10 mL) and evaporated to dryness to give the amine as the HCl salt and as a white powder. To this solid was added a solution of 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxopyrrol-1-yl)propanoate (245 mg, 0.92 mmol, 1.1 equiv.) and N-ethyldiisopropylamine (0.438 mL, 2.51 mmol) in DMF (1.7 mL). This solution was stirred at rt for 20 min then 4-nitrophenyl carbonate (280 mg, 0.92 mmol) was added and the reaction was then left to stir overnight. Purification of the crude reaction mixture was accomplished as described in General Procedure 9, using a 12 g C18 flash column, and eluting with a 10 to 100% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a white solid (141 mg, 36% yield).
LC/MS: Calc'd m/z=469.5 for C18H19N3O8S2, found [M+H]+=470.2.
1H NMR (300 MHz, Chloroform-d) δ 8.37-8.25 (m, 2H), 7.46-7.35 (m, 2H), 6.71 (d, J=2.1 Hz, 2H), 6.32 (s, 1H), 4.55 (t, J=6.6 Hz, 2H), 3.83 (t, J=7.0 Hz, 2H), 3.65-3.50 (m, 2H), 3.09-2.99 (m, 2H), 2.84 (q, J=6.1 Hz, 2H), 2.52 (td, J=7.1, 3.1 Hz, 2H).
A solution of Compound 4.60 (18 mg, 0.038 mmol) and N-ethyldiisopropylamine (15 μL, 0.087 mmol) in DMF (300 μL) was added to Compound 145 (13 mg, 0.029 mmol) and this solution was stirred at rt for 20 min. The solution was acidified with an aqueous 1 M HCl solution (100 μL) and purified directly. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a 20 to 45% CH3CN/H2O+0.1% TFA gradient to provide the title compound as a yellow solid (6.8 mg, 32% yield).
LC/MS: Calc'd m/z=740.8 for C33H33FN6O9S2, found [M+H]+=741.5.
1H NMR (300 MHz, 10% D2O/CD3CN) δ 7.63 (d, J=12.1 Hz, 1H), 7.39-7.22 (m, 2H), 6.74 (d, J=6.7 Hz, 2H), 5.50 (d, J=16.2 Hz, 1H), 5.26 (d, J=16.2 Hz, 1H), 5.20 (s, 2H) 4.69 (s, 2H), 4.28 (t, J=6.3 Hz, 2H), 3.62 (t, J=7.0 Hz, 2H), 3.31 (t, J=6.6 Hz, 2H), 2.74-2.64 (m, 2H), 2.35 (t, J=7.0 Hz, 2H), 1.90 (dd, J=15.5, 8.1 Hz, 2H), 1.23-1.04 (m, 6H), 0.93 (t, J=7.4 Hz, 3H).
Cytotoxicity of the camptothecin analogues was assessed in vitro as follows.
In vitro potency was assessed on multiple cancer cell lines: SK-BR-3 (breast cancer), SKOV-3 (ovarian cancer), Calu-3 (lung cancer), ZR-75-1 (breast cancer) and MDA-MB-468 (breast cancer). Serial dilutions of camptothecin analogues were prepared in RPMI 1640+10% FBS, and 20 μL of each dilution was added to 384-well plates. Cells cultured in log-phase growth were detached by brief incubation in 0.05% Trypsin and resuspended in respective culturing media at 20,000 cells/mL (with the exception of ZR-75 cells, which were resuspended at 10,000 cells/mL). 50 μL of cell suspension was then added to the plates containing test articles. Cells were incubated with test articles for 4 d at 37° C. (with the exception of ZR-75 cells, which were incubated for 5 d). Growth inhibition was assessed by CellTiter-Glo® (Promega Corporation, Madison, WI) and luminescence was measured on a plate reader. IC50 values were determined by GraphPad Prism (GraphPad Software, San Diego CA).
The results are shown in Table 5.1.
Antibody constructs that specifically bind human NaPi2b were generated by immunizing mice with human cells over-expressing NaPi2b as summarized below.
HEK293-6E cells (National Research Council of Canada) were transiently transfected with a pTT5-based expression plasmid (National Research Council of Canada) encoding human NaPi2b (pTT5-huNaPi2b, expressing the sequence of NaPi2b as set forth in SEQ ID NO:1), according to manufacturer's instructions for Lipofectamine 2000 (Thermo Fisher Scientific). Ten B6x129 mice were subcutaneously immunized with transfected HEK293-6E cells over 63 days, after which blood was drawn and spleens harvested.
Anti-human NaPi2b antibody titers were determined by flow cytometry using CHO—S cells expressing human NaPi2b. All ten mice mounted a significant response against human NaPi2b.
Splenocytes from all mice were subsequently pooled and used for hybridoma generation. P3X63Ag8.653 cells (ATCC cat #CRL-1580) were mixed with IgG+ B cells isolated from spleens and fused using an ECM 2001 electrofusion instrument (BTX, Harvard Bioscience) with optimized settings. Following overnight recovery, hybridomas were diluted and plated in selection media containing the following final concentrations of substituents: 100 M hypoxanthine, 0.4 M aminopterin and 16 M thymidine. Following 14 days of selection, hybridomas were diluted to an average of one cell per well and plated into 96-well plates. Cell supernatants containing secreted antibodies were assessed for binding on CHO—S cells transfected with the same plasmid used to transfect HEK293-6E cells (pTT5-huNaPi2b). Hybridoma cells from wells containing supernatants having antibodies that bound NaPi2b were harvested for sequencing.
The murine VH and VL sequences for one of the anti-NaPi2b antibodies identified were used to prepare a mouse-human chimeric IgG1/kappa antibody construct, v23855, as follows. Coding sequences for antibody variable regions were cloned in frame into a human IgG1 expression vector (with human IgG1 constant region starting with alanine 118 according to Kabat numbering) or a human C kappa expression vector (with human C kappa constant region starting at arginine 108 according to Kabat numbering), both expression vectors based on pTT5. The activities of the resultant recombinant chimeric antibody construct were confirmed in specificity binding assays and were found comparable to the parental antibodies (data not shown).
The chimeric anti-human NaPi2b antibody construct, variant v23855, generated as described in Example 6, was humanized. The CDR sequences of v23855 are provided in Table 7.1, and the mouse VH and VL sequences are provided in Table 7.2. Humanization was conducted as described below.
Sequence alignment of the mouse VH and VL sequences of v23855 to respective human germline sequences identified IGHV1-46*03 and IGKV1D-39*01 as the closest, as well as most frequent, human germline sequences (and respectively IGHJ4*03 and IGKJ2*04 joining region germline sequences were selected). CDR sequences according to the AbM definition (see Table 7.1) were ported onto the framework of these selected human germline sequences as shown in
This process resulted in four variable heavy chain humanized sequences and three variable light chain humanized sequences. Full heavy chain sequences containing humanized heavy chain variable domain (VH) and hIgG1 heavy chain constant domains (CH1, hinge, CH2, CH3), and full light chain sequence containing humanized light chain variable domain (VL) and human kappa light chain constant domain (kappa CL) were assembled. Monoclonal antibody (mAb) variants were then assembled such that each of the humanized heavy chains was paired with each of the humanized light chains to provide 12 humanized variants to be evaluated experimentally.
Each of the 12 humanized antibody constructs, as well as the parental v23855 construct, were produced in full-size antibody (FSA) format containing two identical full-length heavy chains and two identical kappa light chains.
The full-length heavy chain contained the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 [SEQ ID NO:33]; see Table 7.3). The light chain contained human kappa CL sequence of IGKC*01 [SEQ ID NO:34]; see Table 7.3).
Each of the humanized VH domain sequences as well as the mouse VH domain sequence was appended to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide four humanized full heavy chain sequences and one parental mouse-human chimeric full heavy chain sequence. Each of the humanized VL domain sequences or mouse VL domain sequence was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences and one parental mouse-human chimeric light chain sequence. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.
Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO:35](Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842)) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA. Sequences of the humanized VH and VL sequences are provided in Table 7.4 below.
The heavy and light chains of each of the humanized antibody variants and parental mouse-human chimeric antibody variant were expressed in 300 mL cultures of CHO-3E7 cells. Briefly, CHO-3E7 cells, at a density of 1.7-2×106 cells/mL, viability >95%, were cultured at 37° C. in FreeStyle™ F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (Hyclone SH30034.01) and 0.1% Pluronic® F-68 (Gibco/Thermo Fisher Scientific, Waltham, MA). A total volume of 300 mL CHO-3E7 cells+1× antibiotic/antimycotics (GE Life Sciences, Marlborough, MA) was transfected with a total of 300 ag DNA (150 ag of antibody DNA and 150 μg of GFP/AKT/stuffer DNA) using PEI-MAX® (Polyscience, Inc., Philadelphia, PA) at a DNA:PEI ratio of 1:4 (w/w). Twenty-four hours after the addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration)+1% w/v Tryptone (final concentration) were added to the cells, which were then transferred to 32° C. and incubated for 6 more days prior to harvesting.
Protein-A purification was performed using 1 mL HiTrap™ MabSelect™ SuRe™ columns (Cytiva, Marlborough, MA). Clarified supernatant samples were loaded on equilibrated columns in Dulbecco's PBS (DPBS). The columns were washed with DPBS. Proteins were eluted with 100 mM sodium citrate buffer pH 3.0. The eluted fractions were pH adjusted by adding 10% (v/v) 1 M HEPES (pH ˜10.6-10.7) to yield a final pH of 6-7. Samples were buffer exchanged into DPBS using 5 mL Zeba™ Spin columns (Thermo Scientific). Protein was quantitated based on absorbance at 280 nm (A280 nm).
Following purification, purity of samples was assessed by SDS-PAGE under non-reducing and reducing conditions. Protein sample was mixed with NuPAGE® LDS Sample Buffer and NuPAGE® Sample Reducing Agent (for reducing condition only) according to manufacturer's protocol, after which sample was heated at 70° C. for 15 min. Treated protein samples containing 1.5 g of protein and Molecular Weight (MW) Precision Plus Protein™ Dual Color (Bio-Rad) standards for MW estimation were loaded on the NuPAGE 4-12% Bis-Tris gel (15 wells). XCell SureLock© Mini-Cell system from Life Technologies (Thermo Fisher Scientific) as well as NuPAGE® MOPS SDS Running Buffer, were used to perform gel electrophoresis at 200 V for 50 minutes. Gels were stained with a Biosafe Coomassie solution and ChemiDoc™ MP Imaging system (Bio-Rad) was used to capture images of gels.
The yields for each of the twelve humanized antibody variants were similar, ranging from approximately 23-30 mg (or ˜77-100 mg/L of culture) and approximately 2-fold higher yield than the parental mouse-human chimeric antibody, v23855 (14 mg yield). The SDS-PAGE results for these antibody samples are shown in
Species homogeneity of the humanized antibody variants and parental mouse-human chimeric antibody variant samples was assessed by UPLC-SEC after protein-A purification.
UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6×150 mm, stainless steel, 1.7 m particles) (Waters LTD, Mississauga, ON) set to 30° C. and mounted on a Waters Acquity UPLC™ H-Class Bio system with a photodiode array (PDA) detector. The mobile phase was Dulbecco's phosphate buffered saline (DPBS) with 0.02% Tween 20 pH 7.4 and the flow rate was 0.4 mL/min. Total run time for each injection was 7 min with a total mobile phase volume of 2.8 mL. Elution was monitored by UV absorbance in the range 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing the Apex Track™ and detect shoulders features.
The apparent purity of the humanized antibody variants was assessed using mass spectrometry and non-denaturing deglycosylation.
10 μg of each sample were incubated with 1 μg of deglycosylation mix (NEB, P6044) for 1 hour at room temperature and transferred to the incubator at 37° C. for 16 hours.
After deglycosylation, 5 μL of the eluted sample were transferred into glass inserts in LC-MS vials. For LC-MS analysis, 1 μL of sample were injected into using an Agilent PLRP-S column (1000 Å, 2.1×50 mm, 8 m) using an Agilent 1290 Infinity II LC system coupled to Agilent 6545 QTOF with Dual Jet Stream electrospray ionization source with a column temperature of 70° C. and a flow rate of 0.3 mL/mi. Mobile phases consisted of A: LC-MS grade water with 0.1% v/v formic acid, 0.025 v/v trifluoroacetic acid and 10% v/v isopropyl alcohol in, and B: acetonitrile with 0.1% v/v formic acid and 10% v/v isopropyl alcohol. The column was pre-equilibrated in 20% mobile phase B before injection. Then, a 20 minute 20 to 40% mobile phase B gradient was applied, followed by a 2 minute 27 to 90% mobile phase B gradient and a column wash of 2 minutes at 99% mobile phase B.
The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.
400 μL of purified samples primarily at concentrations of 0.4 mg/mL in PBS were used for DSC analysis with a VP-Capillary DSC (Malvern Panalytical Inc., Westborough, MA). At the start of each DSC run, 5 buffer blank injections were performed to stabilize the baseline, and a buffer injection was placed before each sample injection for referencing. Each sample was scanned from 20° C. to 100° C. at a 60° C./hr rate, with low feedback, 8 sec filter, 3 min pre-scan thermostat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine melting temperature (Tm) as an indicator of thermal stability.
The Fab Tm values determined for the humanized variants are shown in Table 8.1. All humanized variants exhibited increased thermal stability compared to the parental antibody, v23855 (Fab Tm of ˜72.4° C.), with Fab Tm values ranging from ˜78-83° C.
To characterize the binding of parental chimeric anti-NaPi2b antibody v23855 to NaPi2b, competition binding or epitope binning assays were carried out against anti-NaPi2b reference antibodies MX-35 (v18992) and lifastuzumab (v18993). Binding was assessed by flow cytometry using HEK293-6e cells as described below.
Each of the anti-NaPi2b detection antibodies: v23855, v18992, v18993, and Palivizumab (anti-RSV, v16955) was conjugated with AF647 fluorophores using Zenon Human IgG labeling kit (ThermoFisher Scientific Corporation, Waltham, MA; Cat. No. Z25408 Lot. No. 1937175). HEK293-6e cells were transfected for ˜24 hours to transiently express human NaPi2b (1 μg of pTT5-NaPi2b per 1 million cells) or transfected with GFP (ATUM, Menlo Park, CA; pD2610-v23, also 1 μg of DNA per 1 million cells). Following transfection, human NaPi2b-expressing HEK296-6e cells and transfected GFP-expressing HEK296-6e cells were mixed at 4:1 ratio. Each well of a V-bottom 96-well plate was seeded with 100,000 cells of the mixture and incubated with 100 μg/mL of unlabeled competitor anti-NaPi2b antibody for an hour on ice. Post incubation, cells were washed and stained with 1 μg/mL of AF647-conjugated anti-NaPi2b detection antibodies for an hour on ice. Following staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 10,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the FITC/GFP negative live cell population and using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ). Percentage inhibition was calculated using the following formula:
The competition binding results of parental chimeric anti-NaPi2b antibody v23855 against v18992 (MX35) and v18993 (lifastuzumab) are shown in Table 9.1.
96
97
96
0
Each of the tested anti-NaPi2b antibodies competed against itself (>95% inhibition, see data in bold text) as expected. The chimeric anti-NaPi2b antibody v23855 competed for binding to v18992 and v8993, demonstrated by comparable % inhibition to itself (>94%). No competition binding against negative control palivizumab (v16955) was observed, as expected.
The binding cross-reactivity of humanized antibody variant v29456 to human, cynomolgus and mouse NaPi2b was assessed by flow cytometry using HEK293-6e transfected cells. Reference anti-NaPi2b antibodies MX-35 (v18992) and lifastuzumab (v18993) were included as comparators and the anti-RSV antibody palivizumab (v22277) was included as a negative control.
Briefly, HEK293-6e cells were transfected for ˜24 hours to transiently express human NaPi2b, cynomolgus or mouse NaPi2b, at 1 μg of DNA per 1 million cells. Following transfection, 50,000 cells per well were seeded in V-bottom 96-well plates and incubated with 200 nM of primary antibody for 18-24 hours at 4° C. to prevent internalization. Post-incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, Cat. No. 109-605-098, Lot. No. 124868) at 4° C. for 1 hour. Following staining and washing, fluorescence was detected by flow cytometry on BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 10,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live singlet cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ). The Bmax and Kd of each primary antibody were calculated using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA)
The binding results of humanized antibody variant v29456, MX-35 (v18992) and lifastuzumab (v18993) are shown in Table 10.1 and
v29456 and v18992 showed binding to human, cynomolgus and mouse NaPi2b on transfected HEK296-6e cells. Lifastuzumab (v18993) displayed binding to human and cynomolgus NaPi2b and showed minimal binding to mouse NaPi2b transfected HEK293-6e cells.
v29456 had comparable apparent Kd value to v18992 and v18993 on human NaPi2b binding and exhibited the greatest binding to cynomolgus NaPi2b, yielding apparent Kd 10-fold and 2-fold lower than v18992 (MX35) and v18993 (lifastuzumab), respectively. Negative control palivizumab (v22277) did not bind to any species tested, as expected.
The binding affinity of anti-NaPi2b antibody constructs was assessed by Kinexa in the endogenous NaPi2b-expressing cell line, IGROV-1. Affinity of antibody constructs was assessed in monovalent format to reduce the effects of avidity and internalization and compared directly to the parental chimeric antibody variant, also in monovalent format. v29814 (monovalent format of parental chimeric variant 23855), v36123 (monovalent format of humanized antibody variant 29452, and v36124 (monovalent format of humanized antibody variant 29456) were assessed. The experiment was performed as described below.
IGROV-1 cells were cultured in RPMI 1640 Medium, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA) in T175 culture flasks (Corning, Corning, NY) and incubated at 37° C. with 5% CO2 until achieving 80% confluency. Cells were detached from culture vessels by incubation with Cell Dissociation Buffer (Invitrogen, Waltham, MA) for 30-60 minutes at 37° C. with 5% CO2, collected by neutralizing Cell Dissociation Buffer with at least 5 times volume of RPMI 1640 Medium, ATCC modification supplemented with 10% FBS, and maintained on ice until use. Cells were counted using the Vi-Cell™ XR Cell Viability Analyzer (Beckman Coulter, Brea, California).
The solid phase was prepared by coating one vial of PMMA (polymethyl methacrylate) beads (Sapidyne, Boise, Idaho) with 1 mL of 20 μg/mL BSA-biotin (Sigma-Aldrich, St. Louis, Missouri) in PBS pH 7.4. The beads were incubated for 2 hours at room temperature with gentle rotation. Beads were settled, supernatant removed, and beads rinsed five times with PBS pH 7.4. The beads were then coated with 1 mL of 100 μg/mL of streptavidin (Jackson ImmunoResearch, West Grove, PA) in PBS pH 7.4 with 10 mg/mL BSA (Sigma-Aldrich, St. Louis, Missouri) with rotation at room temperature for 1 hour. Beads were settled, supernatant removed, and beads rinsed five times with PBS pH7.4. The final step of the solid phase preparation was done by coating with 30 μg/mL of biotin goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) for 1 hour with rotation at room temperature.
The cell-binding assay was set up using the antibody constructs or variants as constant binding partner at two different concentrations of 50 μM and 500 μM. For the titration curve with antibody variants fixed at 50 μM, at least 10 million IGROV-1 cells were used as titrant. For the titration curve with antibody variants fixed at 500 μM, at least 5 million cells were used as titrant. The antibody variants and cells were mixed in PBS pH 7.4, 1 mg/mL BSA, 0.2% NaN3 and incubated at 4° C. for 7 days with gentle rotation until equilibrium was reached. After incubation, the mixture of antibody variants and cells was centrifuged to separate the cells from the unbound free antibody variants. The free antibody variants were loaded onto the KinExA 3200 (Sapidyne, Boise, Idaho) with biotinylated—anti-human IgG PMMA as solid phase and 0.5 μg/mL of Alexa 647 goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) as detection antibody.
Results for parental chimeric monovalent antibody variant (v29814) and two humanized, monovalent antibody variants are shown in Table 11.1. N-curve analysis was used to calculate the affinity and receptor expression level with the concentrations of antibody variant as reference point. Narrow 95% confidence intervals were obtained for both the affinity and receptor expression level and % error of the fit was less than 1.5%. The N-curve analysis is shown in
# Parentheses indicates 95% confidence interval
All humanized anti-NaPi2b antibody variants displayed similar binding profiles to each other and approximately 2-fold lower binding affinity compared to chimeric parental antibody construct. The calculated receptor expression level was between 0.9-1.3 million per cell.
Internalization of the chimeric parental anti-NaPi2b antibody v23855 and a representative humanized variant, v29456 (H1L2), in NaPi2b-expressing cell lines (HCC-78 and NCI-H441) was determined by flow cytometry as described below. The NaPi2b-targeting antibodies lifastuzumab (v18993) and MX35 (v18992) were used as positive controls, and the anti-RSV antibody palivizumab (v22277) was used as a negative control.
Briefly, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting anti-Human IgG Fc (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4° C. Cells were seeded at 50,000 cells/well in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) in 48-well plates and incubated overnight under standard culturing conditions (37° C./5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 10 nM and incubated under standard culturing conditions for 5-24 hours to allow for internalization. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 100 nM for 30 minutes at 4° C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488/FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results are shown in
Chimeric parental antibody v23855 and the humanized antibody variant v29456 showed comparable levels of internalization to the humanized antibody MX35 (v18992) and much greater levels of internalization compared to humanized antibody lifastuzumab (v18993) across all time points (5 hours and 24 hours) at 10 nM antibody treatment on both HCC-78 and NCI-H441 cells. For instance, following a 5-hour incubation in HCC-78, v23855 and v29456 showed 21.9 and 25.3-fold increase in internalized fluorescence compared to negative control palivizumab, respectively. Similarly, following a 24-hour incubation in HCC-78 cells, v23855 and v29456 showed 50.9- and 59.9-fold increase in internalized fluorescence compared to negative control palivizumab, respectively.
The isoelectric point, propensity for self-aggregation, and non-specific binding of anti-NaPi2b antibodies v23855 (parental chimeric), v29452 (H1L3) and v29456 (H1L2), were determined in order to assess the developability of these antibodies. The isoelectric point was measured by capillary isoelectric focusing (cIEF), the propensity for self-aggregation was measured by Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and non-specific binding was measured by NS-ELISA, as described below.
Capillary Isoelectric Focusing (cIEF)
cIEF was carried out using Maurice C. (ProteinSimple©) system, System Suitability Kit and Method Development Kit. System suitability standard, fluorescence calibration standard, cartridge and samples were prepared according to vendor's recommendations. The capillary was automatically calibrated with a fluorescence standard preconditioned with Maurice cIEF System Suitability Kit to ensure the capillary was functioning properly. The antibody samples were diluted to a concentration of 0.5 mg/mL in a final volume of 40 μL in Gibco™ Distilled Water, and mixed Maurice cIEF Method Development Kit Samples. The samples were then vortexed, centrifuged and the supernatant pipetted into individual wells of a 96-well plate. All electropherograms were detected with UV absorbance at 280 nm. All data analyses were performed using vendor software Compass for iCE (ProteinSimple©). The Compass software aligns each electropherogram using the pI markers so that the x-axis is displayed as a normalized pI for each injection.
AC-SINS method was carried out in a 384-well plate format (Corning® #3702). Initially, 20 nm gold nanoparticles (Ted Pella, Inc., #15705) washed with 0.22 μm filtered Gibco™ Distilled Water were coated with a mixture of capture antibody—80% AffiniPure Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories© #109-005-088), and the non-capture antibody −20% ChromPure Goat IgG, whole molecule (Jackson ImmunoResearch Laboratories© #005-000-003), that were initially buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg/mL. The mixture of gold nanoparticles, capture antibody and non-capture antibody was incubated in the dark for 18 h at room temperature. Sites unoccupied on the gold nanoparticles were blocked with 1 μM thiolated polyethylene glycol (2 kD) in 20 mM sodium acetate, pH 4.3 to a final concentration of 0.1 μM, followed by 1 h incubation at room temperature. The coated nanoparticles were then concentrated by centrifugation at 21,000×g for 7 min, at 8° C. 95% of the supernatant was removed and the gold pellet was resuspended in the remaining buffer. 5 μL of concentrated nanoparticles were added to 45 μL of antibody at 0.05 mg/mL in Gibco™ PBS pH 7.4 in a 384-well plate. The coated nanoparticles were incubated with the antibody of interest for 4 h at room temperature in the dark. The absorbance was read from 450-700 nm at 1 nm increments, and a Microsoft Excel macro was used to identify the max absorbance, smooth the data, and fit the data using a second-order polynomial. The Δlambda (nm) was calculated based on the smoothed max absorbance of the average blank (PBS alone) subtracted from the smoothed max absorbance of the antibody sample to determine the antibody AC-SINS score. Antibody-antibody interactions directly correlate with the shift in maximum absorbance wavelength of gold nanoparticles coated with the antibody of interest. The cutoff of Δlambda 10 nm was set as high self-aggregation propensity of the antibody.
NS-ELISA was used to measure the propensity of the antibodies to bind to a range of biomolecules to emulate the undesirable non-specific interactions to biological matrices in vivo as described below.
NS-ELISA was carried out in Corning@ 96-well EIA/RIA Easy Wash™ Clear Flat Bottom Polystyrene High Bind Microplate coated overnight at 4° C. with 50 μL of Heparin (Sigma, H3149) diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 250 μg/mL. The plate was incubated for 2 days at room temperature, wells that were coated with heparin were not covered to allow air dry. Insulin (Sigma-Aldrich®, 19278) and KLH (Sigma-Aldrich®, H8283) were each diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 5 μg/mL. ssDNA (Sigma-Aldrich®, D8899) and dsDNA (Sigma-Aldrich®, D4553) was diluted with Gibco™ PBS pH7.4 to a final concentration of 10 μg/mL. 50 μL each of insulin, KLH, dsDNA and ssDNA were added to a 96 well plate, followed by the incubation at 37° C. for 2 h. The coating materials were removed, and the plate was blocked with 200 μL of Gibco™ PBS pH7.4, 0.1% Tween®20, and incubated for 1 h at room temperature with shaking at 200 rpm. The plate was washed 3 times with Gibco™ PBS pH7.4, 0.1% Tween 20. 50 μL of each mAb at 100 nM (15 mg/mL) in Gibco™ PBS pH 7.4, 0.1% Tween®20 was added in duplicate to the wells and incubated for 1 h at room temperature with shaking at 200 rpm. Plates were washed three times with Gibco™ PBS pH7.4, 0.1% Tween 20, and 50 μL of 50 ng/mL anti-human IgG HRP (Thermofisher Scientific©, H10307) was added to each well. Plates were incubated for 1 h at room temperature, with shaking at 200 rpm. The plate was washed three times with Gibco™ PBS pH7.4, 0.1% Tween 20, and 100 μL of TMB substrate (Cell Signaling Technology©, 7004P6) added to each well. Reactions were stopped after approximately 10 minutes by adding 100 μL of 1 M HCl to each well, and absorbance was read at 450 nm. Binding scores were calculated as the ratio of the ELISA signal of the antibody to the signal of a well containing buffer instead of the primary antibody. The cutoffs considered for each binding molecule (ssDNA. KLH, Insulin, dsDNA and Heparin) were internally calculated, based on the average of Zymeworks Inc. produced antibodies and antibodies benchmarks published in the literature.
The results of all three assays are shown in Table 13.1. In these assays a score higher than the cutoff was taken to indicate potentially less desirable biophysical characteristics.
The pI values determined for the main isoform for the variants v23855; v29452 and v29456 are 8.35, 8.53 and 8.53 respectively, which all fall within the typical range for therapeutic antibodies. The analysis of Δlambda showed no potential issues on AC-SINS for all variants. Additionally, there were no potential issues identified for NS-ELISA.
The objective of this experiment was to assess if fragmentation of antibody variant v29456 and reference antibody MX35 (v18992) occurred over time after incubation in mouse plasma or in PBS pH 7.4 at 37° C.
Briefly, v29456 or v18992 were each diluted into either PBS or mouse plasma to a final concentration of 0.5 mg/ml and incubated at 37° C. Samples were removed after 0, 7 and 14 days and stored at −80° C. until characterization. For characterization, samples were thawed at room temperature and 50 μg were incubated with 5 μg of recombinant EndoS endoglycosidase for one hour at room temperature. An immunoprecipitation slurry was generated by a 45 min incubation of 95 μL/sample of magnetic Sepharose streptavidin-coated beads with 15 μg/sample of biotinylated goat anti-Human IgG Fc capture antibody, followed by 4 washes with PBS pH 7.4 with the aid of a DynaMag™-2 magnet (Invitrogen™).
After deglycosylation, plasma-incubated samples were incubated with 95 μL of the immunoprecipitation slurry for 1.5 hrs at room temperature. Then, the slurry underwent 6 washes with PBS pH 7.4 and 2 washes with LC-MS-grade water with the aid of a DynaMag™-2 magnet (Invitrogen™). A final wash with PBS pH 7.4 was performed before elution. Protein was eluted by incubating beads with 35 μL of LC-MS grade water with 20% acetonitrile and 0.1% formic acid for one hour at room temperature. PBS samples were not immunoprecipitated.
5 μL of the eluted sample were transferred to glass inserts in LC-MS vials. For LC-MS analysis, 1 μL of sample were injected into a Waters™ BioSuite Phenyl Column, 1000A, 10 μm, 4.6 mm×75 mm using a Waters™ ACQUITY™ UPLC I-Class HPLC system coupled to a Waters™ Synapt™ G2-Si HDMS with a column temperature of 7° C. and a flow rate of 0.3 mL/min. Mobile phases consisted of A: LC-MS grade water with 0.1% v/v formic acid, 0.025 v/v trifluoroacetic acid and 10% v/v isopropyl alcohol in, and B: acetonitrile with 0.1% v/v formic acid and 10% v/v isopropyl alcohol. The column was pre-equilibrated in 10% mobile phase B before injection. Then, a 20 min 10 to 27% mobile phase B gradient was applied, followed by a 2 min 27 to 90% mobile phase B gradient and a column wash of 2 min at 99% mobile phase B.
The column was re-equilibrated to 10% mobile phase B for 2 minutes between runs. ESI was performed in positive mode with 3 kV of capillary voltage, 12° C. source temperature, 100 V sampling cone voltage, source offset of 80 V, source gas flow of 0 ml/min, desolvation temperature 50° C., cone gas flow 0 L/Hr, desolvation gas flow 800 L/hr, nebuliser gas flow 6.5 bar. Data format was continuum with analyser set in sensitivity mode, with a m/z range from 500 to 7000.
Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos® v4.0 using a deconvolution window of 60000-160000 Da with an m/z range of 1000-4000. For all time points, the highest intensity deconvoluted mass was assigned as the reference mass of v29456. The reference mass was defined as the average mass of v29456 or v18992 with two 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stubs arising from EndoS activity on N-glycans, 16 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Mass assignments had a mass tolerance of ±10 Da. Other assigned mAb proteoforms were: mAb reference mass with a phosphoric acid adduct, mAb reference mass with the loss of one fucose unit, mAb reference mass with the addition of a hexose unit. A pentasaccharide adduct (likely penta-mannose) relative to the reference mass of v29456 without 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] was also identified. Apparent purity was calculated as the ratio of all v29456 or v18992 mAb proteoforms deconvoluted peak intensities divided by all observed deconvoluted peak intensities. Mouse plasma proteins present in the day 0 mouse plasma control, but not observed in the PBS controls, were not considered in the apparent purity calculations.
The data is provided in Table 14.1 and shows that there was no evidence of fragmentation of v29456 incubated at 37° C. for 7 and 14 days in mouse plasma or PBS pH 7.4, as apparent purity after 7 and 14 days was similar to day 0 controls. v18992 showed fragmentation after 14 days in PBS based on appearance of low molecular weight species and a decrease in apparent purity of >10% relative to day 0 control. v18992 fragmentation was not observed in plasma.
Surface NaPi2b protein was measured on tumor cell lines by quantitative flow cytometry using a set of beads with known levels of antibody binding capacity (ABC) as described below. Reference humanized antibody MX35 (v18992) conjugated to Alexa Fluor® AF647 and control anti-RSV antibody palivizumab (v21995) conjugated to Alexa Fluor® AF647 were used to fluorescently label tumor cells and beads. Variant 22277 differs from anti-RSV antibody v21995 used in previous examples in that it has a heterodimeric Fc. This does not affect the function of this antibody. The representative cell lines evaluated were OVCAR-3, IGROV-1, HCC-78, TOV-21G, NCI-H441, HCT116, and EBC-1.
Conjugations of v18992 and v21995 to Alexa Fluor® AF647 were performed as follows: v18992 and v21995 were each reacted with 8 equivalents of NHS-AF647 (Thermo Fisher #A20006, 10 mM) in PBS. The reaction was protected from light at room temperature, and was allowed to proceed for 200 and 150 minutes, respectively. Following incubation, the reactions were purified through four and two rounds of purification, respectively, using a 40 kDa Zeba column (Thermo Fisher, Waltham, MA) pre-equilibrated with PBS, pH 7.4. Confirmation of conjugation and quantification of unconjugated NHS-AF647 were measured by SEC chromatography (Ex: 650 nm, Em: 665 nm).
Cells were detached from culture vessels using Cell Dissociation Buffer (Invitrogen, Waltham, MA) and seeded at 50,000 cells/well in conical-bottom 96-well plates in triplicate. Cells and anti-Human QSC® beads (Bangs Laboratories, Inc., Fishers, IN) were stained with v18992-AF647 with a pre-determined excess level of conjugated antibody or negative control v21995-AF647 at the same concentration and incubated for 30 minutes at 4° C. Following incubation, cells and beads were washed in FACS buffer and analyzed on the BD™ Fortessa HTS and processed using FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ).
The median AF647 fluorescence intensity for all bead populations were plotted against relevant ABC values using Bangs Laboratories QuickCal v 2.3 calibration line template for QSC® anti-Human IgG Lot #14490.
Surface protein expression for cell lines was calculated based on a monovalent binding model and therefore equivalent to the background subtracted ABC (SABC). The median fluorescence intensity of v21995-AF647-stained cells from each of the respective cell lines was used as a background value for determination of SABC.
Results are shown in Table 15.1. Reported NaPi2b proteins per cell are an average of at least two biological replicates. Tumor cell lines were designated with high, mid, low, or negative expression of the target; cell lines were designated as “high” expressors if the average number of NaPi2b proteins detected was greater than 900,000 per cell; “mid” if the number was between 40,000 and 900,000 per cell; “low” if the number was between 500 and 40,000 per cell; and “negative” if the number was negative (below limit of quantitation of the calibration beads).
The ability of the parental chimeric antibody construct v23855 and the humanized antibody variants described in Examples 7 and 8 to bind to NaPi2b expressed on cells was assessed on the endogenous NaPi2b-expressing cell line, IGROV-1, by flow cytometry. IGROV-1 cells express endogenous NaPi2b at a high level.
Briefly, cells were seeded at 50,000 cells/well in conical-bottom 96-well plates and treated with test antibody for 24 hours at 4° C. to prevent internalization. Palivizumab (anti-RSV antibody, v22277) was included as a negative control. Reference anti-NaPi2b antibodies lifastuzumab (v18993) and MX35 (v18992) conjugated to a maleimide functionalized auristatin drug linker (DL2) were included as comparators; conjugation of this drug linker has shown no impact to the antibody binding capability (data not shown). Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4° C. for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results for parental chimeric construct (v23855) and all humanized antibody variants are tabulated in Table 16.1. Full dose-response binding curves are represented for parental chimeric antibody (v23855) and two representative humanized antibody variants (v29452, v29456) in
All humanized antibody variants bound to IGROV-1 cells similarly, yielding apparent Kd values within 2-fold and comparable Bmax values. Reference antibody MX35-DL2 ADC showed comparable binding to chimeric v23855 and humanized antibodies. Humanized antibody lifastuzumab-DL2 ADC showed lower binding compared to all other targeted antibodies, with a lower Bmax value and greater apparent Kd value. Negative control palivizumab (v22277) showed no cellular binding (NB), as expected.
Antibody-drug conjugates shown in Table 17.1 below were prepared. Exemplary protocols are provided below.
v29456-MC-GGFG-AM-DXd1 DAR8: A solution (2.47 mL) of the humanized variant v29456 (54 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (1.00 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.90 mL in PBS, pH adjusted to 7.4) and 25 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (134 μL, 9.0 eq.). After 4 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 1 mM DTPA in PBS, pH 7.4. An aliquot of the reduced antibody solution (13.5 mg, 1.32 mL) was diluted with 1 mM DTPA (33.5 μL in PBS, pH adjusted to 7.4). To the antibody solution was added 38.3 μL of DMSO and an excess of MC-GGFG-AM-DXd1 (111.7 μL; 12 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 min. At that time, additional drug-linker (14 μL, 1.5 eq.) was added. The conjugation reaction proceeded at room temperature with mixing for an additional 60 min. An excess of a 10 mM N-acetyl-L-cysteine solution (76.8 μL, 8 eq.) was added to quench the conjugation reaction.
v29456-MC-GGFG-AM-Compound 139 DAR8, v29456-MC-GGFG-Compound 141 DAR8: A solution (1.14 mL) of humanized variant v29456 (25 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (252 μL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.4 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (207 μL, 12 eq.). After 180 minutes at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 10 mM NaOAc pH 5.5. To the antibody solution was added 667 μL of 10 mM NaOAc, pH 5.5, 197 μL of DMSO and an excess of either MC-GGFG-AM-Compound 139 or MC-GGFG-Compound 141 (243 μL; 16 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 minutes. An excess of 10 mM N-acetyl-L-cysteine solution (971 μL, 64 eq.) was added to quench each conjugation reaction.
v29456-MC-GGFG-AM-Compound 139 DAR4, v29456-MC-GGFG-Compound 141 DAR4: A solution (2.01 mL) of humanized variant v29456 (44 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (734 μL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (704 μL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (72.8 μL, 2.4 eq.). After 120 minutes at 37° C., 1.76 mL of the reduced antibody was diluted with 1.76 mL of PBS, pH 7.4 and 0.44 mL of 100 mM NaOAc, pH 5.5. To the antibody solution was added 288 μL of DMSO and an excess of either MC-GGFG-AM-Compound 139 or MC-GGFG-Compound 141 (152 μL; 10 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 minutes. An excess of 10 mM N-acetyl-L-cysteine solution (607 μL, 40 eq.) was added to quench each conjugation reaction.
v29456-MT-GGFG-AM-Compound 139, v29456-MT-GGFG-AM-Compound 141 DAR8: A solution (2.47 mL) of the humanized variant v29456 (54 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (1.00 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.90 mL in PBS, pH adjusted to 7.4) and 25 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (134 μL, 9.0 eq.). After 4 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 1 mM DTPA in PBS, pH 7.4. An aliquot of the reduced antibody solution (13.5 mg, 1.32 mL) was diluted with 1 mM DTPA (33.5 μL in PBS, pH adjusted to 7.4). To the antibody solution was added 38.3 μL of DMSO and an excess of either MT-GGFG-AM-Compound 139, or MT-GGFG-AM-Compound 141 (111.7 μL; 12 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 180 min. An excess of a 10 mM N-acetyl-L-cysteine solution (55.8 μL, 6 eq.) was added to quench the conjugation reaction.
v29456-MT-GGFG-AM-Compound 136, v29456-MT-GGFG-AM-Compound 129 DAR8 Å solution (456 μL) of the humanized variant v29456 (10 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (1.06 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.40 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (82.7 μL, 12.0 eq.). After 3 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 4.5. To an aliquot of the reduced antibody solution (1 mg, 200 μL) was added 20 μL of DMSO and an excess of MT-GGFG-AM-Compound 136 or MT-GGFG-AM-Compound 129 (13.8 μL; 20 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 2.5 h.
v29456-MT-GGFG-Compound 141 DAR8: A solution (2.78 mL) of the humanized variant v29456 (61 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (3.92 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (1.80 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (505 μL, 12 eq.). After 3 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (7 KDa MWCO; Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 4.5. To an aliquot of the reduced antibody solution (20 mg, 2.95 mL) was added 295 μL of DMSO and an excess of MT-GGFG-Compound 141 (206.8 μL; 15 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 2.5 h. An excess of a 10 mM N-acetyl-L-cysteine solution (165.5 μL, 12 eq.) was added to quench the conjugation reaction.
v29456-MT-GGFG-Compound 140 DAR8: A solution (1.51 mL) of the humanized variant v29456 (33 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (0.61 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.55 mL in PBS, pH adjusted to 7.4) and 25 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (81.9 μL, 9.0 eq.). After 3.5 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 1 mM DTPA in PBS, pH 7.4. To an aliquot of the reduced antibody solution (10 mg, 1.55 mL) was added 48.5 μL of DMSO and an excess of either MT-GGFG-Compound 140 (124.1 μL; 11 eq.) from a 10 mM DMSO stock solution. Additional drug-linker (73.1 μL; 7 eq) was added. The conjugation reaction proceeded at room temperature with mixing for 3.5 h. An excess of a 10 mM N-acetyl-L-cysteine solution (180.9 μL, 16.5 eq.) was added to quench the conjugation reaction.
v29456-MT-GGFG-Compound 148 DAR8: A solution (1.51 mL) of the humanized variant v29456 (33 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (0.61 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.55 mL in PBS, pH adjusted to 7.4) and 25 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (81.9 μL, 9.0 eq.). After 3.5 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 1 mM DTPA in PBS, pH 7.4. To an aliquot of the reduced antibody solution (10 mg, 1.48 mL) was added 164.7 μL of DMSO and an excess of MT-GGFG-Compound 148 (82.7 μL; 9 eq.) from a 10 mM DMSO stock solution. Additional drug-linker (31.7 μL; 3 eq.) was added. The conjugation reaction proceeded at room temperature with mixing for 3.5 h. An excess of a 10 mM N-acetyl-L-cysteine solution (57.1 μL, 6 eq.) was added to quench the conjugation reaction.
v22277-MC-GGFG-AM-DXd1 DAR8: A solution (2.18 mL) of the control variant v22277 (10 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (4.1 μL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (563 μL in PBS, pH adjusted to 6.7) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (68.9 μL, 10.0 eq.). After 3 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 5.5. To the reduced antibody solution was added 230.0 μL of DMSO and an excess of MC-GGFG-AM-DXd1 (51.7 μL; 15 eq.) from a 20 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 60 min. An excess of a 20 mM N-acetyl-L-cysteine solution (51.7 μL, 15 eq.) was added to quench the conjugation reaction. The quenching reaction proceeded at 0-4° C. for 30 min.
v21995-MC-GGFG-AM-DXd, v21995-MC-GGFG-Compound 141 DAR8: A solution (1.76 mL) of the humanized variant v21995 (32 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (2.77 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (1.20 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (264 μL, 12.0 eq.). After 3 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 5.5. To the reduced antibody solution was added 600 μL of DMSO and an excess of either MC-GGFG-AM-DXd or MC-GGFG-Compound 141 (308.5 μL; 14 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 90 min. An excess of a 10 mM N-acetyl-L-cysteine solution (198 μL, 9 eq.) was added to quench the conjugation reaction. The quenching reaction proceeded at 0-4° C. for 30 min.
v21995-MT-GGFG-Compound 140, MT-GGFG-AM-Compound 141 DAR8: A solution (2.07 mL) of the humanized variant v21995 (30 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (2.48 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (1.20 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (248 μL, 12.0 eq.). After 3 hours at 37° C., the reduced antibody was purified by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with pH 5.5PBS, pH 7.4. To an aliquot of the reduced antibody solution (3.00 mL, 15 mg) was added 150 μL of DMSO and an excess of either MT-GGFG-Compound 140 or MT-GGFG-AM-Compound 141 (77.5 μL; 7.5 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 30 min. 150 μL of DMSO and an excess of either MT-GGFG-Compound 140 or MT-GGFG-AM-Compound 141 (77.5 μL; 7.5 eq.) from a 10 mM DMSO stock solution were added again, and the conjugation reaction proceeded at room temperature with mixing for another 30 min.
ADCs prepared as described in Example 17 were purified on an AKTA™ pure chromatography system (Cytiva Life Sciences, Marlborough, MA) using a 53 mL HiPrep 26/10 Desalting column (Cytiva Life Sciences, Marlborough, MA) and a mobile phase consisting of 10 mM NaOAc, pH 4.5 with 150 mM NaCl and a flow rate of 10 mL/min. The purified ADC was then sterile filtered (0.2 μm).
Alternatively, ADCs prepared as described in Example 17 were purified by two to three passages over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™) pre-equilibrated with PBS, pH 7.4 for the first passage, and with 10 mM NaOAC, pH 4.5 or pH 5.5 for the subsequent passages. The purified ADC was then sterile filtered (0.2 μm).
Following purification, the concentration of the ADCs was determined by a BCA assay with reference to a standard curve generated using the humanized variant v29456. Alternatively, concentrations were estimated by measurement of absorption at 280 nm using extinction coefficients taken from the literature (European Patent No. 3 342 785, for MC-GGFG-AM-DXd1) or determined experimentally (for the remaining drug-linkers). ADCs were also characterized by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described below.
Antibody and ADCs were analyzed by HIC to estimate the drug-to-antibody ratio (DAR). Chromatography was performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a TSKgel® Butyl-NPR column (2.5 μm, 4.6×35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) and employing a gradient of 95/5% MPA/MPB to 5/95% MPA/MPB over a period of 12 minutes at a flow rate of 0.5 mL/min (MPA=1.5 M (NH4)2SO4, 25 mM NaxPO4, pH 7 and MPB=75% 25 mM NaxPO4, pH 7, 25% isopropanol). Detection was by absorbance at 280 nm.
The extent of aggregation of the antibody and ADCs (˜15-150 g, 5 μL injection volume) was assessed by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using an AdvanceBio SEC column (300 angstroms, 2.7 μm, 7.8×150 mm) (Agilent, Santa Clara, California) and a mobile phase consisting of 150 mM phosphate, pH 6.95 and a flow rate of 1 mL/min. Detection was by absorbance at 280 nm.
The individual contributions of the DARO, DAR2, DAR4, DAR6 and DAR8 species to the average DAR of the purified ADCs were assessed by integration of the HPLC-HIC chromatogram. The average drug to antibody ratio (DAR) of each ADC was determined by the weighted average of each DAR species. The average DAR for each ADC, when rounded to the nearest integer, was the same as the target DAR shown in Table 18.1.
The extent of aggregation and monomer content was assessed by integration of the HPLC-SEC chromatogram. The monomer peak of each ADC was identified as the peak with the same retention time as the unconjugated antibody from which each ADC was derived from. All peaks with an earlier retention time relative to the monomer species was determined to be aggregated species. Percent monomer species determined for each ADC is shown in Table 18.1. All ADC preparations showed >95% monomer species.
aPurified using Zeba ™ Spin Desalting Columns
bPurified using AKTA ™ pure chromatography system
The ability of v29456 ADCs to exert a bystander killing effect on cancer cells was assessed according to the method described below. Bystander killing can occur after target-specific uptake of an ADC into an antigen-positive cell. In this case, catabolism of the ADC results in release of payload or an active catabolite, which then crosses the cell membrane of nearby cells to elicit their death.
The ADCs tested were v29456-MT-GGFG-AM-Compound 139, v29456-MT-GGFG-AM-Compound 141, v29456-MT-GGFG-Compound 141, v29456-MT-GGFG-Compound 140, v29456-MT-GGFG-Compound 148. Positive controls v29456-MC-GGFG-AM-DXd1 and v29456-MCvcPABC-MMAE, whose drug linkers have been known to be active in bystander activity, and negative (non-NaPi2b targeting) controls palivizumab (v22277) MC-GGFG-AM-DXd and palivizumab (v22277) MCvcPABC-MMAE, were included. Cell lines used were HCC-78 (high NaPi2b expression) and EBC-1 (negative NaPi2b expression).
NaPi2b-positive HCC-78 and NaPi2b-negative EBC-1 cells were seeded either as mono-cultures or co-cultures in a 48-well plate at 15,000 cells and 5,000 cells, respectively, in 100 μL assay media (RPMI1640+10% FBS). ADCs were diluted to 10 nM in assay media and 100 μL was added to the cell-containing plates (5 nM final ADC concentration). Cells were incubated with ADCs for 4 days under standard culturing conditions (37° C./5% CO2). Following incubation, cells were dissociated, washed, and stained using a viability dye, YO—PRO®-1 (ThermoFisher Scientific, Waltham, MA), and an anti-NaPi2b antibody, MX35, conjugated to Alexa Fluor®647, for 20 minutes at 4° C. After incubation, cells were washed in FACS buffer, resuspended in 70 μL FACS buffer per well, and 35 μL per well were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ). Dead cells were excluded by gating on YO—PRO®-1 staining. The number of HCC-78 and EBC-1 cells were determined by the number of events in the Alexa Fluor®647 positive (NaPi2b-positive) and Alexa Fluor®647 negative (NaPi2b-negative) gates, respectively. Percent viability was calculated as the number of cells in treatment condition divided by the number of cells in the no-treatment condition.
The results are provided in Table 19.1 below and in
The cell growth inhibition (cytotoxicity) capabilities of the humanized variant v29456 conjugated to various drug-linkers, conjugated at an antibody-to-drug ratio of 8.0, were assessed in a panel of NaPi2b-expressing cell lines as described below. Cell lines used were HCC-78 (lung carcinoma), IGROV-1 (ovarian adenocarcinoma), and HCT116 (colorectal carcinoma; NaPi2b-negative). ADCs with antibody palivizumab (anti-RSV) (v22277) were used as non-targeted controls.
Briefly, cells were seeded in 384-well plates and treated with a titration of test article prepared in cell growth medium. Cells were incubated for 4 days under standard culturing conditions. After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI) was spiked in all wells and luminescence corresponding to ATP present in each well was measured using a Synergy™ H1 plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values were calculated based on blank wells (no test article added), and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.
The results are shown in Table 20.1 and representative curves are plotted in
All v29456 ADCs displayed significant cytotoxicity in NaPi2b expressing cell lines HCC-78 and IGROV-1, yielding single-digit nanomolar or lower EC50 values after 4-day treatment. In NaPi2b-negative cell line HCT116, v29456 and palivizumab ADCs yielded comparable potency, indicating NaPi2b-targeted ADCs did not show target-dependent cytotoxicity, as expected.
The cell growth inhibition (cytotoxicity) capabilities of the humanized variant v29456 conjugated to select camptothecin drug-linkers conjugated at an antibody-to-drug ratio of 8 and 4 were assessed against NaPi2b-expressing cell lines IGROV-1 (ovarian adenocarcinoma) and TOV-21G (ovarian adenocarcinoma), according to the methods described in Example 20. ADCs with antibody palivizumab (anti-RSV) (v21995) were used as non-targeted controls.
The results are shown in Table 21.1 and representative curves are plotted in
Humanized variant v29456 ADCs displayed targeted cytotoxicity against high NaPi2b-expressing cell line IGROV-1 and moderate NaPi2b-expressing cell line TOV-21G, with higher potency against IGROV-1 than TOV-21G, thereby demonstrating NaP2ib-dependent killing. ADCs with a DAR of 8 showed greater potency than ADCs with a DAR of 3.4. As expected, palivizumab ADC control showed less activity against both cell lines compared to NaPi2b-targeted ADCs.
The 3D cytotoxicity capabilities of the humanized variant v29456 conjugated to various drug linkers were assessed in a panel of NaPi2b-expressing cell line spheroids as described below. Cell lines used were HCC-78 (lung carcinoma) and IGROV-1 (ovarian adenocarcinoma). ADCs with antibody palivizumab (anti-RSV) (v22277, v21995) were used as non-targeted controls.
Briefly, cells were seeded in Ultra-Low Attachment 384-well plates at 3,000 cells/well, centrifuged, and incubated for 3 days under standard culturing conditions to allow for spheroid formation and growth. Monoculture cell line spheroids were then treated with a titration of test article, generated in cell growth medium. Spheroids were incubated for 6 days under standard culturing conditions. After incubation, CellTiter-Glo® 3D reagent (Promega Corporation, Madison, WI) was spiked in all wells. Plates were incubated in the dark at room temperature for 1 hour and luminescence was quantified using a BioTek Cytation 5 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values were calculated based on blank wells (no test article added), and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.
The results are shown in Table 22.1 and representative curves are plotted in
All v29456 ADCs showed comparable or improved potency over v29456 conjugated to DXd1, with EC50 values at sub-nanomolar range, against both NaPi2b-expressing cell lines. As expected, palivizumab ADC controls showed less activity against both cell lines compared to NaPi2b-targeted ADCs.
The cell growth inhibition (cytotoxicity) capabilities of the humanized variant v29456 conjugated to select camptothecin drug-linkers conjugated at an antibody-to-drug ratio of 8 and 4 were assessed against NaPi2b-expressing cell line spheroids according to the methods described in Example 22. Cell lines used were IGROV-1 (ovarian adenocarcinoma) in monoculture and TOV-21G (ovarian adenocarcinoma) in co-culture with HDFa (adult human dermal fibroblasts, Thermo Fisher Scientific, Waltham, MA) at a 1:1 seeding density ratio (3,000 TOV-21G cells +3,000 HDFa cells per well). ADCs with antibody palivizumab (anti-RSV) (v21995) were used as non-targeted controls.
The results are shown in Table 23.1 and representative curves are plotted in
All v29456 ADCs showed targeted killing against NaPi2b-expressing spheroids, with sub-nanomolar to single-digit nanomolar EC50, while negative control palivizumab ADC showed double-digit nanomolar potency. ADCs with a DAR of 8 showed greater potency than ADCs with a DAR of 3.4.
In vivo anti-tumor activities of humanized variant v29456 ADCs were assessed in the OVCAR3 xenograft model of ovarian cancer (one study) and the NCI-H441 xenograft model of lung cancer (two studies), which both express high levels of NaPi2b. The studies were carried out as described below.
For the high Napi2b expressing OVCAR3 ovarian cancer model, tumor fragments (˜1 mm3) were implanted subcutaneously into female CB.17 SCID mice. When mean tumor volume reached ˜100-150 mm3 the animals were assigned to groups, n=8 per group, and administered a single IV dose of test articles on study day 1. Tumor volume and body weight were measured twice weekly with a study duration of 60 days. Treatment groups are described in Table 24.1.
For the NCI-H441 model of lung cancer, 5×106 cells in 0.1 ml 1:1 PBS:Matrigel were implanted subcutaneously into male NU-Foxn1nu mice. When mean tumor volume reached ˜145 mm3 the animals were assigned to groups, n=6 per group, and administered a single IV dose of test articles on study day 0. Tumor volume and body weight were measured twice weekly with a study duration of 28-35 days. Treatment groups for NCI-H441 studies are described in Table 24.2 and Table 24.3.
For statistical analyses, a linear mixed effects model was fit to log-transformed tumor volumes, followed by F-test for the null hypothesis that mean growth rates are equal and post-hoc pairwise comparisons.
For the OVCAR3 model, the results are shown in
For the NCI-H441 model, Study 1, the data is provided in
In the NCI-H441 model, Study 2,
In vivo anti-tumor activity of humanized variant v29456 ADCs was assessed in two patient-derived (PDX) models of ovarian cancer, CTG-2025, and CTG-0958. An ADC of reference antibody v18993 (lifastuzumab) conjugated to MCvcPABC-MMAE at DAR4 was also tested as a comparator. These studies were carried out as described below.
For the ovarian cancer PDX model CTG-2025, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜240 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 25.1.
For the ovarian cancer PDX model CTG-0958, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜225 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 25.2.
The results for the CTG-2025 study are shown in
The results for the CTG-0958 study are shown in
The pharmacokinetics (PK) of v29456 and ADCs comprising v29456 conjugated to Compound 139 or Compound 141 at a DAR4 and DAR8 was assessed in humanized FcRn Tg32 mice. This mouse model can be predictive of the pharmacokinetics of a drug in humans (see Avery et al. (2016) Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies, mAbs, 8:6, 1064-1078). PK of the MX35 antibody (v18992) and of v18993 (lifastuzumab)-MCvcPABC-MMAE DAR4 were assessed for comparison.
All test articles were administered at 5 mg/kg to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; Stock #014565) by intravenous injection as shown in Table 26.1. For each test article, blood was collected from n=4 animals by retro-orbital bleed at 1, 3, and 6 hours and 1, 3, 7, 10, 14, 21 days post-dose. Blood was processed to serum and stored frozen at −80° C. in 96-well storage plates prior to pharmacokinetic analysis.
Mouse serum containing Napi2b-TOPO ADC was captured onto a 384 well plate coated with Goat anti-Human IgG Fc antibody (Jackson 109-005-098). The total antibody was detected with a goat anti-Human IgG Fab Biotin antibody (Jackson 109-065-097), followed by streptavidin SULFO-TAG (Mesoscale). After addition of MSD GOLD Read Buffer A, electrochemiluminescence (ECL) signal by the SULFO-TAG label was measured using the plate reader (Mesoscale).
The PK profiles obtained are shown in
Internalization of humanized anti-NaPi2b antibody v29456 (H1L2) conjugated to drug linker MT-GGFG-AM-Compound 139 at DAR 8, in NaPi2b-expressing cell lines (IGROV-1 and OVCAR-3) was determined by flow cytometry as described in Example 12. The NaPi2b-targeting antibodies lifastuzumab (v18993) and MX35 (v18992) were used as positive controls, and palivizumab (anti-RSV) (v22277) was used as a negative control.
Results are shown in
Humanized antibody variant v29456 covalently conjugated to drug linker MT-GGFG-AM-Compound 139 showed comparable levels of internalization to the humanized antibody MX35 (v18992) and much greater levels of internalization compared to humanized antibody lifastuzumab (v18993) across all time points (4 hours and 24 hours) at 10 nM antibody treatment on both IGROV-1 and OVCAR-3. Following a 4-hour incubation in IGROV-1, v29456-MT-GGFG-AM-Compound 139 showed 16.6 and 16.7-fold increase in internalized fluorescence compared to negative control palivizumab, respectively. Similarly, following a 4-hour incubation in OVCAR-3 cells, v29456-MT-GGFG-AM-Compound 139 showed 23.0- and 24.6-fold increase in internalized fluorescence compared to negative control palivizumab, respectively.
Internalization and co-localization of parent antibody v29456 with lysosomes was also assessed in IGROV-1 cells by high content imaging. The data obtained showed that anti-NaPi2b antibody v29456 co-localized with lysosomes after a 24 hour incubation period (data not shown).
The cell growth inhibition (cytotoxicity) capabilities of the humanized variant v29456 conjugated to MT-GGFG-AM-Compound 139, at DAR8, was assessed against NaPi2b-expressing cell line spheroids according to the methods described in Example 22. Cell lines used were IGROV-1 (ovarian adenocarcinoma), NCI-H441 (lung carcinoma), and TOV-21G (ovarian adenocarcinoma) in monoculture (3,000 cells per well). Reference antibody lifastuzumab conjugated to MMAE was tested as a comparator. ADCs with anti-RSV antibody palivizumab (v21995) were used as non-targeted controls.
The results are shown in Table 28.1 and representative curves are plotted in
v29456-MC-GGFG-AM-Compound 139 showed targeted killing against NaPi2b-expressing spheroids, with sub-nanomolar to single-digit nanomolar EC50, while negative control palivizumab ADC showed double-digit nanomolar or lower potency. v29456-MC-GGFG-AM-Compound 139 showed greater potency than the lifastuzumab-MCvcPABC-MMAE comparator in TOV21-G and NCI-H441 tumor spheroids.
A Membrane Proteome Array™ (Integral Molecular, Philadelphia, PA, USA) was used to screen for specific off-target binding interactions for antibody, humanized v38591 anti-NaPi2b (SLC34A2) variant. This anti-NaPi2b humanized antibody variant has amino acid sequences that are identical to v29456, except that the heavy chains of v38591 include a C-terminal lysine.
Briefly, the study consisted of three phases: phase (1) determination of assay screening conditions, phase (2) membrane proteome array (library) screen and phase (3) protein target validation. In phase (1), conditions appropriate for detecting v38591 binding by high-throughput flow cytometry were determined, including the optimal antibody concentration and cell type for screening (two cell types were tested, HEK293T and avian QT6). In phase (2), using optimal conditions determined in phase 1, v38591 was screened against the library of over 6000 human membrane proteins (individually expressed in unfixed HEK293T cells), including 94% of all single-pass, multi-pass, and GPI-anchored proteins, including GPCRs, ion channels and transporters. In phase (3), each protein target hit from the screen stage (potential off-target interactions) was assessed in titration experiment using flow cytometry.
Phase (1) determined that the HEK293T cell type and an antibody concentration of g/mL were optimal for library screening. As shown in
The ability of the humanized antibody variant v38591 and the antibody drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 8 and v38591-MC-GGFG-AM-Compound 139 DAR 4 to bind to NaPi2b expressed on cells was assessed on the endogenous NaPi2b-expressing tumor cell lines IGROV-1 (ovarian adenocarcinoma) and OVCAR-3 (ovarian adenocarcinoma) by flow cytometry. IGROV-1 and OVCAR-3 cells express endogenous NaPi2b at high levels, as described in Example 15. v38591-MC-GGFG-AM-Compound 139 DAR 8 and v38591-MC-GGFG-AM-Compound 139 DAR 4 were prepared similarly to the methods described for v29456-MC-GGFG-AM-Compound 139 DAR 8 and v29456-MC-GGFG-AM-Compound 139 DAR 4 in Example 17.
Cells were maintained under standard culture conditions (37° C./5% CO2) until assay set-up; IGROV-1 cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA); OVCAR-3 cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), and 0.01 μg/mL human insulin (Sigma-Aldrich, Oakville, Canada); Cells were removed from culture vessels using Cell Dissociation Buffer (Invitrogen, Waltham, MA), seeded at 50,000 cells/well in conical-bottom 96-well plates, and treated with test antibody or antibody-drug conjugate for 24 hours at 4° C. to prevent internalization. Reference anti-NaPi2b antibodies MX35 (v18992) and lifastuzumab (v18993) were included as positive controls; palivizumab (anti-RSV antibody, v22277) was included as a negative control. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4° C. for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody or antibody-drug conjugate using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results are shown in Table 30.1 and plotted in
The binding cross-reactivity of humanized antibody variant v38591 and the antibody drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 8 and v38591-MC-GGFG-AM-Compound 139 DAR 4 to cynomolgus monkey and mouse NaPi2b was assessed by flow cytometry using HEK293-6e transfected cells. Reference anti-NaPi2b antibodies MX35 (v18992) and lifastuzumab (v18993) were included as positive controls; palivizumab (anti-RSV antibody, v22277) was included as a negative control.
HEK293-6e cells were maintained under standard culture conditions (37° C./5% CO2) with shaking at 115 rpm for suspension and cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 1% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1× Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were transfected with human NaPi2b (pTT5-huNaPi2b) (CL_#13432), cynomolgus (CL_#13433) or mouse (CL_#13476) NaPi2b (all from GenScript Biotech, Piscataway, NJ), 1 μg of DNA per 1 million cells, using 293Fectin™ Transfection Reagent (Thermo Fisher Scientific, Waltham, MA) and Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA), and incubated for 24 hours under standard culture conditions (37° C./5% CO2/115 rpm). Following transfection, cells were seeded at 50,000 cells/well in conical-bottom 96-well plates and treated with test antibody or antibody-drug conjugate for 24 hours at 4° C. to prevent internalization. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4° C. for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody or antibody-drug conjugate using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results are shown in Table 31.1 and plotted in
The binding cross-reactivity of humanized antibody variant v38591 and the antibody drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 8 and v38591-MC-GGFG-AM-Compound 139 DAR 4 to human NaPi2b, NaPi2a, and NaPi2c was assessed by flow cytometry using HEK293-6e transfected cells. Reference anti-NaPi2b antibodies MX35 (v18992) and lifastuzumab (v18993) were included as positive controls; reference anti-NaPi2a (polyclonal rabbit anti-human SLC34A1; Atlas Biotechnologies Inc, Edmonton, AB; Cat. No. HPA051255) and anti-NaPi2c (polyclonal rabbit anti-human SLC34A3; Thermo Fisher Scientific, Waltham, MA; Cat. No. PA5-50762) antibodies were included; palivizumab (anti-RSV antibody, v22277) was included as a negative control.
HEK293-6e cells were maintained under standard culture conditions (37° C./5% CO2) with shaking at 110 rpm for suspension and cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 1% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1× Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were transfected with human NaPi2b (pTT5-huNaPi2b) (CL_#13432), human NaPi2a (CL_#13435), or human NaPi2c (CL_#13436) (all from GenScript Biotech, Piscataway, NJ), 1 μg of DNA per 1 million cells, using 293Fectin™ Transfection Reagent (Thermo Fisher Scientific, Waltham, MA) and Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA), and incubated for 24 hours under standard culture conditions (37° C./5% CO2/110 rpm). Following transfection, cells were seeded at 50,000 cells/well in conical-bottom 96-well plates and treated with test antibody or antibody-drug conjugate for 24 hours at 4° C. to prevent internalization. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) or anti-Rabbit IgG F(ab″)2 AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 111-605-047). Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody or antibody-drug conjugate using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results are shown in Table 32.1 and plotted in
Internalization of the anti-NaPi2b ADC v38591-MC-GGFG-AM-Compound 139 DAR 8 and the unconjugated humanized antibody v38591 was determined by flow cytometry in a high NaPi2b-expressing ovarian adenocarcinoma cell line, OVCAR-3, as described below. The NaPi2b-targeting antibodies lifastuzumab (v18993), MX35 (v18992), and palivizumab (anti-RSV) (v22277) were included as controls.
Briefly, cells were seeded at 50,000 cells/well in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) in 48-well plates and incubated overnight under standard culturing conditions (37° C./5% CO2) to allow attachment. Antibodies were fluorescently labeled by coupling to anti-Human IgG Fc Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4° C. Coupled antibodies were added to cells the following day and incubated under standard culturing conditions for 15 minutes, and 4, 16, and 24 hours to allow for internalization. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 200 nM for at least 45 minutes at 4° C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488/FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
Results are shown in Table 33.1 and
The ability of the antibody drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 8 and v38591-MC-GGFG-AM-Compound 139 DAR 4 to exert a bystander killing effect on cancer cells was assessed according to the method described below. Bystander killing can occur after target-specific uptake of an ADC into an antigen-positive cell, as described in Example 19. Cell lines used were HCC-78 (high NaPi2b expression) and EBC-1 (negative NaPi2b expression). Surface NaPi2b expression for these cell lines were measured as described in Example 15.
All test articles were assessed at a concentration of 5 nM. The unconjugated human antibody variant v38591, negative controls palivizumab (v21995) MC-GGFG-AM-Compound 139 DAR 8 and DAR 4, palivizumab (v22277) MC-GGFG-AM-DXd1, and palivizumab (v22277) MCvcPABC-MMAE, were included as controls. The human antibody variant v29456 conjugated to MC-GGFG-AM-DXd1 was included as a positive control. Reference antibody drug conjugate v18993 (lifastuzumab)-MCvcPABC-MMAE was included as a positive control at both 5 nM and 20 nM concentrations (EC99 on HCC-78 cells=20 nM).
Briefly, NaPi2b-positive HCC-78 and NaPi2b-negative EBC-1 cells were seeded either as mono-cultures or co-cultures in a 48-well plate at 15,000 cells and 5,000 cells, respectively, in 100 μL assay media (RPMI1640+10% FBS). ADCs were diluted to 10 nM or 40 nM in assay media and 100 μL was added to the cell-containing plates (5 nM or 20 nM final ADC concentration). Cells were incubated with ADCs for 4 days under standard culturing conditions (37° C./5% CO2). Following incubation, cells were dissociated, washed, and stained using a viability dye, YO—PRO®-1 (ThermoFisher Scientific, Waltham, MA), and an anti-NaPi2b antibody, MX35, conjugated to Alexa Fluor®647, for 20 minutes at 4° C. After incubation, cells were washed in FACS buffer, resuspended in 60 μL FACS buffer per well, and 35 μL per well were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ). Dead cells were excluded by gating on YO—PRO®-1 staining. The number of HCC-78 and EBC-1 cells were determined by the number of events in the Alexa Fluor®647 positive (NaPi2b-positive) and Alexa Fluor®647 negative (NaPi2b-negative) gates, respectively. Percent viability was calculated as the number of cells in each treatment condition divided by the number of cells in the no-treatment condition.
The results are tabulated in Table 34.1 and shown in
In vivo anti-tumor activities of humanized variant v29456 ADCs were assessed in several patient-derived (PDX) xenograft models. For all ovarian cancer patient-derived xenograft (PDX) models, research level immunohistochemistry (IHC) assessment on same study tissue was performed by staining with a commercial anti-NaPi2b antibody (Clone D6W2G, Rabbit mAb #42299, Cell Signaling Technology). NaPi2b H-scores were determined by a pathologist for all PDX models, as summarized in Table 35.2. H-scores were calculated on a scale from 0-300 using the formula: H-score=(0×P0)+(1×P1)+(2×P2)+(3×P3), where P0, P1, P2, and P3 represent the percentage of cells with absent, weak, moderate or strong staining, respectively.
For all ovarian cancer PDX models, anti-tumor activity was determined by % tumor growth inhibition (% TGI) calculated as [(1−TVtreatment/TVvehicle)×100] at Day 28, or at the closest evaluable time point, as summarized in Table 35.2.
For the ovarian cancer PDX model CTG-0703, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜240 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 35.1.
As shown in
For the ovarian cancer PDX model CTG-1301, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜230 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 35.1. As shown in
For the ovarian cancer PDX model CTG-3718, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜200 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 35.1. As shown in
For the ovarian cancer PDX model CTG-1703, tumor fragments were implanted subcutaneously into female athymic Nude-Foxn1nu mice. When mean tumor volume reached ˜260 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 1 with a single IV dose of test articles as shown in Table 35.1. As shown in
The ovarian cancer PDX models CTG-2025 and CTG-0958 described in Example 25 used the treatment groups shown in Table 35.1. Updated versions of
As shown in
As shown in
The data provided in Table 35.2 indicates that v29456-MC-GGFG-AM-Compound 139 has anti-tumor activity in ovarian cancer xenograft models with a range of NaPi2b expression levels.
The objective of this study was to determine the maximum tolerated dose of test articles v38591-MC-GGFG-AM-Compound 139 (DAR4), and v38591-MC-GGFG-AM-Compound 139 (DAR8) in male cynomolgus monkeys following three repeat slow intravenous bolus injections. In addition, the toxicokinetic (TK) profiles of these ADCs in cynomolgus monkeys was characterized.
In this study, vehicle and test article ADCs were administered by slow intravenous injection over 3 minutes on Day 1, Day 22, and Day 43 to male cynomolgus monkeys (n=3/group). Study design, dose levels and dose volume details are summarized in the Table 36.1. All the animals were evaluated for moribundity/mortality, clinical signs, body weight, food consumption, clinical pathology (hematology, serum chemistry and coagulation), changes in organ weight, and macroscopic and microscopic changes in organs/tissues. Blood samples were collected for toxicokinetic analysis following the first and second dose. The test article concentrations in all dose formulations were analyzed using UV-Vis assay. Scheduled necropsy was conducted on study Day 50.
Total antibody in cynomolgus monkey serum samples was measured by MSD (Mesoscale Discovery), assay details as described in Example 26. Note for cynomolgus monkey serum samples, Goat anti-human IgG (H+L) Biotin antibody was used for antibody capture in MSD assay.
v38591-MC-GGFG-AM-Compound 139, DAR8: All animals survived to their scheduled euthanasia (Study Day 50). No abnormal functional observational battery, or detailed clinical observations were noted. Test article-related but non-adverse cage-side clinical observations included soft and/or loose feces intermittently throughout the study at 45 mg/kg/day.
Preterminal Animals: Mean body weight gain and mean body weights were comparable to controls and no effect on qualitative food consumption was noted. As compared to animal baseline values and/or historical control data, fibrinogen (FIB) levels were transiently increased on Day 8 for animals administered 45 mg/kg/dose; however, values returned to baseline throughout the remainder of the study. Increases in alanine aminotransferase (ALT) were observed from Day 21 for all dose levels.
Terminal Animals: A single animal administered 30 mg/kg/dose was noted with the macroscopic observation of thickened intestinal wall of the duodenum. Dose-responsive decrease in absolute, organ to body weight, and organ to brain weight ratios were noted in the testes and epididymides of animals administered 15, 30, or 45 mg/kg/dose. Microscopic examination results are currently pending.
v38591-MC-GGFG-AM-Compound 139, DAR4: All animals survived to their scheduled euthanasia (Study Day 50). No abnormal functional observational battery or detailed clinical observations were noted. Test article-related but non-adverse cage-side clinical observations included soft and/or loose feces intermittently throughout the study at 90 mg/kg/day.
Preterminal Animals: Mean body weight gain and mean body weights were comparable to controls and no effect on qualitative food consumption was noted. As compared to animal baseline values and/or historical control data, fibrinogen (FIB) and lactate dehydrogenase (LDH) levels were transiently increased on Day 4; however, values returned to baseline throughout the remainder of the study.
Terminal Animals: A single animal administered 90 mg/kg/dose was noted with the macroscopic observation of thickened intestinal walls of the cecum, duodenum, and colon. No test article-related organ weight changes were observed. Microscopic examination results are currently pending.
Pharmacokinetic (PK) profile: The PK profiles obtained are shown in
In vivo anti-tumor activity of ADCs of humanized variant v38591 (with heavy chains including C-terminal lysine) was assessed in eight patient-derived (PDX) models of NSCLC cancer: LU5213, LU5245, LU6802, LU6904, LU11692, LU11796, LU11870 and LU11876. An ADC of reference antibody v18993 (lifastuzumab) conjugated to MCvcPABC-MMAE at DAR4 was also tested as a comparator in some models. These studies were carried out as described below.
For all NSCLC PDX models, anti-tumor activity was determined by % tumor growth inhibition (% TGI) calculated as [(1−TVtreatment/TVvehicle)×100] at Day 28, or at the closest evaluable time point, as summarized in Table 37.9.
For the NSCLC cancer PDX model LU5213, tumor fragments were implanted subcutaneously into NOD/SCID mice. When mean tumor volume reached ˜180 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.1.
For the NSCLC PDX model LU5245, tumor fragments were implanted subcutaneously into female NOD/SCID mice. When mean tumor volume reached ˜160 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.2.
For the NSCLC PDX model LU6802, tumor fragments were implanted subcutaneously into female BALB/c nude mice. When mean tumor volume reached ˜160 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.3.
For the NSCLC PDX model LU6904, tumor fragments were implanted subcutaneously into female BALB/c nude mice. When mean tumor volume reached ˜160 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.4.
For the NSCLC PDX model LU11692, tumor fragments were implanted subcutaneously into female BALB/c nude mice. When mean tumor volume reached ˜175 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.5.
For the NSCLC PDX model LU11796, tumor fragments were implanted subcutaneously into female NOD/SCID mice. When mean tumor volume reached ˜195 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.6.
For the NSCLC PDX model LU11870, tumor fragments were implanted subcutaneously into female NOD/SCID mice. When mean tumor volume reached ˜155 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.7.
For the NSCLC PDX model LU11876, tumor fragments were implanted subcutaneously into female NOD/SCID mice. When mean tumor volume reached ˜190 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 37.8.
The results for the LU5213 study are shown in
The results for the LU5245 study are shown in
The results for the LU6802 study are shown in
The results for the LU6904 study are shown in
The results for the LU11692 study are shown in
The results for the LU11796 study are shown in
The results for the LU11870 study are shown in
The results for the LU11876 study are shown in
The data provided in Table 37.9 demonstrates that v38591-MC-GGFG-AM-Compound 139 has anti-tumor activity in NSCLC xenograft models.
For all NSCLC patient-derived xenograft (PDX) models, research level immunohistochemistry (IHC) assessment on same study tissue will be performed according to the method described in Example 35.
In vivo anti-tumor activity of ADCs of humanized variant v38591 was assessed in four patient-derived (PDX) models of endometrial cancer, UT14026, UT5318, UT5326 and UT5321. An ADC of reference antibody v18993 (lifastuzumab) conjugated to MCvcPABC-MMAE at DAR4 was also tested as a comparator in some models. These studies were carried out as described below.
For all endometrial cancer PDX models, anti-tumor activity was determined by % tumor growth inhibition (% TGI) calculated as [(1−TVtreatment/TVvehicle)×100] at Day 28, or at the closest evaluable time point, as summarized in Table 38.5.
For the NSCLC cancer PDX model UT14026, tumor fragments were implanted subcutaneously into NOD/SCID mice. When mean tumor volume reached ˜140 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 38.1.
For the NSCLC PDX model UT5318, tumor fragments were implanted subcutaneously into female NOD/SCID mice. When mean tumor volume reached ˜180 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 38.2.
For the NSCLC PDX model UT5326, tumor fragments were implanted subcutaneously into female BALB/c nude mice. When mean tumor volume reached ˜170 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 38.3.
For the NSCLC PDX model UT5321, tumor fragments were implanted subcutaneously into female BALB/c nude mice. When mean tumor volume reached ˜155 mm3 the animals were assigned to groups (n=3 per group) and treated on study day 0 with a single IV dose of test articles as shown in Table 38.4.
The results for the UT14026 study are shown in
The results for the UT5318 study are shown in
The results for the UT5326 study are shown in
The results for the UT5321 study are shown in
The data provided in Table 38.5 demonstrates that v38591-MC-GGFG-AM-Compound 139 has anti-tumor activity in endometrial cancer xenograft models.
For all endometrial cancer patient-derived xenograft (PDX) models, research level immunohistochemistry (IHC) assessment on same study tissue was performed by staining with a commercial anti-NaPi2b antibody (Clone D6W2G, Rabbit mAb #42299, Cell Signaling Technology). NaPi2b H-scores will be determined by a pathologist for all PDX models. H-scores will be calculated on a scale from 0-300 using the formula: H-score=(0×P0)+(1×P1)+(2×P2)+(3×P3), where P0, P1, P2, and P3 represent the percentage of cells with absent, weak, moderate or strong staining, respectively.
A modified anti-NaPi2b antibody construct based on the sequence of v38591 was constructed, having mutations at positions L234A, L235A and D265S (EU numbering) of the Fc region. This modified construct is referred to herein as v40502, or v40502 (LALADS) and was constructed as follows.
Humanized VH and VL sequences of v38591 (corresponding to the VH and VL sequences of v29456 provided in Examples 7 and 8 as SEQ ID NO:24 (VH) and SEQ ID NO:29 (VL)) were used to construct v40502 (LALADS), such that the coding sequence of the antibody variable regions were cloned in frame into a human IgG1 expression vector (with human IgG1 constant region starting with alanine 118 according to Kabat numbering and bearing L234A, L235A and D265S mutations (EU numbering) [SEQ ID NO:64]; see Table 39.1) or a human C kappa expression vector (with human C kappa constant region starting at arginine 108 according to Kabat numbering: [SEQ ID NO:65]; see Table 39.1), both expression vectors based on pTT5. The v40502 (LALADS) variant includes C-terminal lysines in both heavy chains, similar to the v38591 antibody; these C-terminal lysines are cleaved from the majority of the expressed antibody once it is secreted from the cell it is produced in.
Two identical full-length heavy chains and two identical kappa light chains described above were assembled into full-size antibody (FSA) format.
39 Expression and Purification of v40502 (LALADS)
The antibody construct v40502 (LALADS) was prepared as follows in 2 batches (400 ml each).
ExpiCHO™ cells were cultured at 37° C. in ExpiCHO™ expression medium (Thermo Fisher, Waltham, MA) on an orbital shaker rotating at 120 rpm in a humidified atmosphere of 8% CO2, 400 mL expression volumes were used. Each 1 mL of cells at a density of 6×106 cells/mL was transfected with a total of 0.8 μg DNA. Prior to transfection the DNA was diluted in 76.8 μL OptiPRO™ SFM (Thermo Fisher, Waltham, MA), after which 3.2 μL of ExpiFectamine™ CHO reagent (Thermo Fisher, Waltham, MA) was directly added to make a total volume of 80 μL. After incubation for 1-5 minutes, the DNA-ExpiFectamine™ CHO Reagent complex was added to the cell culture (80 uL complex per 1 mL of cell culture) then incubated in a 120 rpm shaking incubator at 37° C. and 8% CO2. Following incubation at 37° C. for 18-22 hours, 6 μL of ExpiCHO™ Enhancer and 240 μL of ExpiCHO™ Feed (Thermo Fisher, Waltham, MA) were added per 1 mL of culture. Cells were maintained in culture at 37° C. for a total of 8 days, after which each culture was harvested by transferring into appropriately sized centrifuge tubes and centrifuging at 4200 rpm for 15 minutes. Supernatants were filtered using a 0.2 m polyethersulfone membrane (Thermo Fisher, Waltham, MA), then analyzed by non-reducing SDS-PAGE and Octet (ForteBio).
Protein purification was performed in either batch mode or with the use of an AKTA™ Pure purification system. In batch mode, supernatants from transient transfections were applied to slurries containing 50% MabSelect SuRe™ resin (Cytiva, Marlborough, MA) and incubated at room temperature for 1 hr on an orbital shaker at 150 rpm. The slurries were transferred into chromatography columns and supernatants were allowed to flow through while resins remained in the column. The resins were then washed with at least 5 Bed Volumes (BV) of resin Equilibration buffer (PBS). To elute the targeted proteins, 2.5 BV of Elution Buffer (100 mM sodium citrate buffer pH 3.5) was added to the columns and collected. Elutions were then neutralized by adding 20% (v/v) 1 M Tris pH 9 to reach a final pH of 7.0. In AKTA™ Pure purification mode, supernatants from transient transfections were loaded onto HiTrap MabSelect SuRE LX columns (Cytiva, Marlborough, MA) that were pre-equilibrated with 5 Column Volume (CV) of PBS. After the proteins were captured, the columns were then washed with 10 CV of PBS. The captured proteins were eluted with 5 CV of Elution Buffer (100 mM sodium citrate buffer pH 3.5) in fractions. Pooled fractions were neutralized with 20% (v/v) if 1 M Tris pH 9. Samples were then buffer exchanged into PBS pH 7.4. The protein content of samples was determined by 280 nm absorbance measurement using a Nanodrop™.
The purity of protein samples was assessed by non-reducing and reducing LabChip™ CE-SDS. LabChip™ GXII Touch (Perkin Elmer, Waltham, MA). Analysis was carried out according to Protein Express Assay User Guide (PerkinElmer, Waltham, MA), with the following modifications. Samples at a concentration range of 5-2000 ng/μL were added to separate wells in 96 well plates (#MSP9631, BioRad, Hercules, CA) along with 7 μL of HT Protein Express Sample Buffer (#CLS920003, Perkin Elmer) and denatured at 90° C. for 5 mins. The LabChip™ instrument was operated using the LabChip™ HT Protein Express Chip (Perkin Elmer #760528) with HT Protein Express 200 assay setting.
Final yields from two production batches were ˜57 and 64 mg (˜142-160 mg/L of culture). The purified antibody displayed Caliper profiles reflective of expected antibody composition reflecting a single species corresponding to full-size antibody (NR Caliper) and intact heavy and light chains for all antibodies (R Caliper) (data not shown).
39.2 Quality Assessment of Modified h12A10 Antibody
Species homogeneity of the antibody was assessed by UPLC-SEC after protein-A purification (final purification step).
Samples were analyzed as follows: UPLC-SEC was performed using an Agilent Technologies AdvanceBio SEC300 Å SEC column (7.8×150 mm, 1.7 m particles) (Agilent Technologies, Santa Clara, California) set to 25° C. and mounted on an Agilent Technologies 1260 infinity II system with a DAD detector. Run times consisted of 7 min and a total volume per injection of 5 μL with a running buffer of 200 mM K3PO4, 200 mM KCl, pH 7. Elution was monitored by UV absorbance in the range 190-400 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB™ CDS ChemStation™ software. The profile reflected high species homogeneity (data not shown).
SPR analysis was carried out to characterize binding of the v40502 (LALADS) anti-NaPi2b antibody to human Fc gamma receptors (FcγR) reagents using a Cytiva Biacore™ T200 instrument. The binding characteristics of this antibody were compared to the wildtype (v38591) and commercial Trastuzumab (Roche Diagnostics).
Binding kinetics was carried out on a Series S Protein A Chip (Cytiva©, 29127556) with PBS-T (PBS+0.05% (v/v) Tween 20, pH7.4) running buffer at a temperature of 25° C. Approximately 400-600 RUs of each antibody (2 μg/mL) were captured on the protein A surface on flow cell 2, 3 and 4 by injecting at 10 μL/min for 60s. Flow cell 1 was used as a reference surface. Six types of human FcγRs were tested, including a commercial FcγRI CD64 (Sino Biological Inc. 10256-H08H) and five in-house produced FcγRs (Table 40.1). Five concentrations of a 3-fold dilution series of FcγRs were prepared starting at 12 μM, 5 μM, or 30 nM, depending on the FcγR proteins. Each in-house FcγR with five concentrations were flowed as analyte using single-cycle kinetics strategy. The flow rate of FcγRs was 50 μL/min with contact time of 40 sec and dissociation time of 120 sec. For the high affinity FcγRI CD64, multicycle kinetics were applied. The flow rate of the analyte was 100 μL/min with contact time of 100 sec and dissociation time of 650 sec. Blank buffer control was injected using the same flow rate and contact time for each cycle. Surface regeneration was accomplished using 10 mM glycine-HCl, pH 1.5 (Cytiva©, BR100354) for 30 sec with a flow rate of 50 L/min after each cycle. Binding constants and the maximum observed binding signal (Rmax) were determined using the Biacore™ T200 Evaluation software (version 3.0; GE Healthcare). For single-cycle kinetics, blank-subtracted sensorgrams were analyzed using steady state affinity fitting model. For multicycle kinetics, results were fit to the 1:1 Langmuir binding model. Binding percentage was calculated from observed Rmax and theoretical Rmax (Rmax/Rmax_theo), based on antibody capture density.
Binding results of SPR analysis are shown in Table 40.1. Six FcγRs reagents were tested, covering type I, II and III human FcγR5. Rmax values and binding percentage indicated that the LALADS mutations fully abrogated all binding to human FcγR with nearly no binding detected for v40502 (LALADS). Trastuzumab and the wildtype v38591 without the Fc LALADS mutation showed nearly full binding to the tested FcγR5.
The isoelectric point, propensity for self-aggregation, and non-specific binding of LALADS anti-NaPi2b antibody v40502 was determined and compared to its wildtype v38591 as an assessment of developability. The isoelectric point was measured by capillary isoelectric focusing (cIEF), the propensity for self-aggregation was measured by affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and non-specific binding was measured by NS-ELISA, as described below.
Capillary Isoelectric Focusing (cIEF)
cIEF was conducted using Maurice C. system, System Suitability Kit and Method Development Kit (ProteinSimple©). System suitability standard, fluorescence calibration standard, cartridge and samples were prepared according to vendor's recommendations. The capillary was automatically calibrated with a fluorescence standard preconditioned with Maurice cIEF System Suitability Kit to ensure the capillary was functioning properly. The antibody samples were diluted to a concentration of 0.5 mg/mL in a final volume of 40 μL in Gibco™ Distilled Water, and mixed Maurice cIEF Method Development Kit Samples. The samples were then vortexed, centrifuged and the supernatant pipetted into individual wells of a 96-well plate. All electropherograms were detected with UV absorbance at 280 nm and protein native fluorescence. All data analyses were performed using vendor software Compass for iCE (ProteinSimple©). The Compass software aligns each electropherogram using the pI markers so that the x-axis is displayed as a normalized pI for each injection.
The pI values were determined for the main isoform for the variants. The measured pI was compared to in-house made Trastuzumab and anti-NaPi2b antibody v38951 having a wildtype Fc region. The measured pI of LALADS anti-Napi2b antibody was expected to be slightly higher its wildtype.
AC-SINS method was carried out in a 384-well plate format (Corning®#3702). Initially, 20 nm gold nanoparticles (Ted Pella, Inc., #15705) washed with 0.22 μm filtered Gibco™ Distilled Water were coated with a mixture of capture antibody—80% AffiniPure Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories© #109-005-088), and the non-capture antibody—20% ChromPure Goat IgG, whole molecule (Jackson ImmunoResearch Laboratories© #005-000-003), that were initially buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg/mL. The mixture of gold nanoparticles, capture antibody and non-capture antibody was incubated in the dark for 18 h at room temperature. Sites unoccupied on the gold nanoparticles were blocked with 1 μM thiolated polyethylene glycol (2 kD) in 20 mM sodium acetate, pH 4.3 to a final concentration of 0.1 μM, followed by 1 h incubation at room temperature. The coated nanoparticles were then concentrated by centrifugation at 21,000×g for 7 min, at 8° C. 95% of the supernatant was removed and the gold pellet was resuspended in the remaining buffer. 5 μL of concentrated nanoparticles were added to 45 μL of antibody at 0.05 mg/mL in Gibco™ PBS pH 7.4 in a 384-well plate. The coated nanoparticles were incubated with the antibody of interest for 4 h at room temperature in the dark. The absorbance was scanned from 450-700 nm at 1 nm increments, and a Microsoft Excel macro was used to identify the max absorbance, smooth the data, and fit the data using a second-order polynomial.
The Δlambda (nm) was calculated based on the smoothed max absorbance of the average blank (PBS alone) subtracted from the smoothed max absorbance of the antibody sample to determine the antibody AC-SINS score. Antibody-antibody interactions directly correlate with the shift in maximum absorbance wavelength of gold nanoparticles coated with the antibody of interest. The cutoff of Δlambda 10 nm was set as high self-aggregation propensity of the antibody, based on the literature.
NS-ELISA was used to measure the propensity of the antibodies to bind to a range of biomolecules to emulate the undesirable non-specific interactions to biological matrices in vivo as described below.
NS-ELISA was carried out in a Corning@ 96-well EIA/RIA Easy Wash™ Clear Flat Bottom Polystyrene High Bind Microplate coated overnight at 4° C. with 50 μL of Heparin (Sigma, H3149) diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 250 μg/mL. The plate was incubated for 2 days at room temperature, wells that were coated with heparin were not covered to allow air dry. Insulin (Sigma-Aldrich®, 19278) and KLH (Sigma-Aldrich®, H8283) were each diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 5 μg/mL. ssDNA (Sigma-Aldrich®, D8899) and dsDNA (Sigma-Aldrich®, D4553) was diluted with Gibco™ PBS pH7.4 to a final concentration of 10 μg/mL. 50 μL each of insulin, KLH, dsDNA and ssDNA were added to a 96 well plate, followed by the incubation at 37° C. for 2 h. The coating materials were removed, and the plate was blocked with 200 μL of Gibco™ PBS pH7.4, 0.1% Tween®20, and incubated for 1 h at room temperature with shaking at 200 rpm. The plate was washed 3 times with Gibco™ PBS pH7.4, 0.1% Tween 20. 50 μL of each mAb at 100 nM (15 mg/mL) in Gibco™ PBS pH 7.4, 0.1% Tween®20 was added in duplicate to the wells and incubated for 1 h at room temperature with shaking at 200 rpm. Plates were washed three times with Gibco™ PBS pH7.4, 0.1% Tween 20, and 50 μL of 50 ng/mL anti-human IgG HRP (Thermofisher Scientific©, H10307) was added to each well. Plates were incubated for 1 h at room temperature, with shaking at 200 rpm. The plate was washed three times with Gibco™ PBS pH7.4, 0.1% Tween 20, and 100 μL of TMB substrate (Cell Signaling Technology©, 7004P6) added to each well. Reactions were stopped after approximately 10 minutes by adding 100 μL of 1 M HCl to each well, and absorbance was read at 450 nm.
Binding scores were calculated as the ratio of the ELISA signal of the antibody to the signal of a well containing buffer instead of the primary antibody. The cutoffs considered for each binding molecule (ssDNA. KLH, Insulin, dsDNA and Heparin) were internally calculated, based on the average of Zymeworks Inc. produced antibodies and antibodies benchmarks published in the literature.
The results of all three assays are shown in Table 41.1. For cIEF, the LALADS variant showed a measured pI of 9.229, which is slightly higher but close to pI of the wildtype (9.160) and Trastuzumab (8.902). In AC-SINs and NS-ELISA assays, a score higher than the cutoff was taken to indicate potentially less desirable biophysical characteristics. AC-SINs and NS-ELISA did not identify any potential issues for any of the tested antibodies.
To investigate the stability of variant 40502, the following studies were performed: 4° C. and 40° C. thermal stability, freeze and thaw, photo stability, and low pH stability, in which samples were characterized at specific timepoints by UPLC-SEC and RPLC-MS.
For thermal stability study, variant 40502 was buffer exchanged into either 20 mM Histidine pH 5.0 with 6% w/v sucrose (His 5Su) or 20 mM Sodium Succinate pH 4.5 with 6% w/v sucrose (Succ5Su) and incubated at 1 mg/ml at either 4° C. or 40° C., protected from light. Samples were evaluated by uPLC-SEC and RPLC-MS at timepoints 0, 7 and 13 days. The 40° C. samples underwent an additional incubation of 15 min at 60° C.
For low pH stability study, variant 40502 was buffer exchanged into 20 mM sodium citrate pH 3.5 and incubated at 1 mg/ml at room temperature. Sample was evaluated by uPLC-SEC and RPLC-MS at timepoint 3 hours.
For photo stability study, variant 40502 was buffer exchanged into either His 5Su or Succ5Su and incubated at 1 mg/ml exposed directly to the light of a biological safety cabinet, at room temperature, and evaluated by uPLC-SEC and RPLC-MS at timepoint 7 days.
For the freeze and thaw study, variant 40502 was buffer exchanged into either His 5Su or Succ5Su and transferred to −20° C. After 16 hours at −20° C., the sample underwent 4 cycles of transfer to room temperature for 6 min, followed by transfer to −20° C. for 10 minutes. After the final room temperature transfer, the protein was evaluated by uPLC-SEC and RPLC-MS.
For RPLC-MS, the photo, freeze-thaw, and low pH stability samples were diluted 1:1 v:v with PBS. Thermal stability samples were not diluted. Then, 10 μL samples were deglycosylated with 1 μg of recombinant EndoS endoglycosidase per 10 μg of antibody for one hour at room temperature. Also, thermal stability samples were reduced with 1 μl of 0.5 M TCEP for one hour after the intact RPLC-MS runs were finished and re-injected for RPLC-MS analysis. For RPLC-MS analysis, 1 μL of sample were injected into a Waters™ BioSuite Phenyl Column, 1000 Å, 10 μm, 4.6 mm×75 mm using an Agilent 1290 Infinity II Liquid Chromatography System coupled with Agilent 6545 Quadrupole Time of Flight (Q-TOF) with a column temperature of 70° C. and a flow rate of 0.3 ml/min. Mobile phases consisted of A: LC-MS grade water with 0.1% v/v formic acid, 0.025 v/v trifluoroacetic acid and 10% v/v isopropyl alcohol in, and B: acetonitrile with 0.1% v/v formic acid and 10% v/v isopropyl alcohol. The column was pre-equilibrated in 10% mobile phase B before injection. Then, a 5 min 10 to 60% mobile phase B gradient was applied, followed by a 2 min 60 to 90% mobile phase B gradient and a column wash of 2 min at 99% mobile phase B. The column was re-equilibrated to 10% mobile phase B for 2 minutes between runs. ESI (electro spray ionization) was performed in a Dual AJS source in positive mode with 5 kV of VCap voltage, 2 kV Nozzle voltage, 170 V fragmentor voltage, gas temperature of 300° C., gas flow of 13 L/min, nebulizer pressure of 45 psig, sheath gas temperature of 400° C. and a sheath gas flow of 12 L/min. Data format was continuum with analyser set in sensitivity mode, with a m/z range from 500 to 7000 with a scan rate of 1 spectra per sec.
Peak integration, mass spectrum deconvolution and mass assignments were performed in Protein Metrics Byos® v4.6 using a deconvolution window of 20000-163000 Da with an m/z range of 1000-4000. For all time points, the highest intensity deconvoluted mass was assigned as the reference mass of variant 40502 or a corresponding N-glycan adduct of the reference mass. The reference mass was defined as the average mass of variant 40502 with two 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stubs arising from EndoS activity on N-glycans, 16 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Mass assignments had a mass tolerance of ±10 Da. Other variant 40502 adducts assigned based on mass shifts relative to the reference mass were: variant 40502 reference mass with the loss of one fucose unit, variant 40502 reference mass with the addition of a hexose unit. N-glycan adducts relative to the reference mass of variant 40502 without 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stubs were also identified.
Analytical SEC (Size Exclusion Chromatography) was performed using an Agilent Infinity 111290 HPLC with Advance Bio SEC column (300 Å, 2.7 μm, 7.8×150 mm) equilibrated with 5 column volumes of Mobile Phase A (200 mM KPO4, 200 mM KCl, pH 7.0) at room temperature. 5 μl of incubated sample was injected and eluted isostatically for 7 mins at 1 mL/min with absorbance monitored at A280. Chromatograms were exported and integrated in PMI Byos v4.6 to provide complete, baseline-to-baseline integration of each peak. The peak corresponding to the major component for IgG (approximate peak apex time 3.5 min, integration window from 3.1 to 4.2 min) was reported as the monomer based on the SEC profile of control trastuzumab. A time window from 2 to 3.1 min was designated as high molecular weight species (HMWS), and a time window from 4.2 to 5.0 min was designated as low molecular weight species (LMWS), excluding solvent peaks (over 5.0 min).
The uPLC-SEC results are shown in Table 42.1. The protein was stable at all conditions as evaluated by uPLC-SEC, based on the similarity of monomer % relative to day 0 controls (<2% decrease). RPLC-MS results are shown in Table 42.2. The protein was stable at most conditions as evaluated by RPLC-MS, based on the similarity of desired proteoform % relative to day 0 controls (<10% decrease).
Antibody-drug conjugates shown in Table 43.1 were prepared. Exemplary protocols are provided below.
v40502-MC-GGFG-AM-Compound 139 DAR4: A solution (5.07 mL) of the v40502 variant (29 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (0.53 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (1.42 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (55.0 μL, 2.75 eq.). After 3 hours at 37° C., the reduced antibody was diluted with PBS (2.21 mL), and pH adjusted with a 100 mM NaOAc solution at pH 5.0 (1.16 mL). To the antibody solution was added 980 μL of DMSO and an excess of MC-GGFG-AM-Compound 139 (179.9 μL; 9 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 min. An excess of a 100 mM N-acetyl-L-cysteine solution (23.95 μL, 12 eq.) was added to quench the conjugation reaction. Purification and characterization were performed as described in Example 18.
Of note, the DAR8 version of the ADC described above could be generated following the protocol used to generate v29456-MC-GGFG-AM-Compound 139 DAR8 described in Example 17.
v38591-MC-GGFG-AM-Compound 139 DAR4: A solution (0.69 mL) of the v38591 variant (15 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (0.48 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (0.3 mL in PBS, pH adjusted to 7.4) and 10 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (23.2 μL, 2.25 eq.). After 2 hours at 37° C., the reduced antibody was diluted with PBS (0.5 mL), and pH adjusted with a 100 mM NaOAc solution at pH 5.0 (0.25 mL). To the antibody solution was added 167 μL of DMSO and an excess of MC-GGFG-AM-Compound 139 (82.7 μL; 8 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 min. Purification was performed using Amicon® Ultra centrifugal filter (30 kDa MWCO) pre-equilibrated with 10 mM NaOAc pH 4.5. Characterization was performed as described in Example 18.
v38591-MC-GGFG-AM-Compound 139 DAR8: A solution (2.31 mL) of the v38591 variant (50 mg) in PBS, pH 7.4 was diluted in PBS, pH 7.4 (1.27 mL) and reduced by addition of 5 mM diethylenetriamine pentaacetic acid (DTPA) (1.0 mL in PBS, pH adjusted to 7.4) and 25 mM of an aqueous tris(2-carboxyethyl)phosphine (TCEP) solution (165.2 μL, 12.0 eq.). After 2 hours at 37° C., the reduced antibody was purified using Amicon® Ultra centrifugal filter (30 kDa MWCO) pre-equilibrated with 10 mM NaOAc pH 5.5. The reduced antibody was then diluted with 10 mM NaOAc solution at pH 5.5 (0.75 mL). To the antibody solution was added 126 μL of DMSO and an excess of MC-GGFG-AM-Compound 139 (124.1 μL; 12 eq.) from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature with mixing for 120 min. Purification was performed using Amicon® Ultra centrifugal filter (30 kDa MWCO) pre-equilibrated with 10 mM NaOAc pH 4.5. Characterization was performed as described in Example 18.
The ability of the humanized antibody variants v38591 and v40502 and the antibody-drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 4 and v40502-MC-GGFG-AM-Compound 139 DAR 4 to bind to NaPi2b on the surface of cells was assessed on the endogenous NaPi2b-expressing cancer cell lines IGROV-1 (ovarian carcinoma), HCC-78 (lung adenocarcinoma), H441 (lung adenocarcinoma), and the NaPi2b-negative cancer cell line EBC-1 (lung carcinoma) by flow cytometry. IGROV-1, HCC-78, and H441 cells express endogenous NaPi2b at high to moderate levels, as described in Example 15.
Cells were maintained under standard culture conditions (37° C./5% CO2) until assay set-up. IGROV-1, HCC-78, H441 and EBC-1 cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). Cells were removed from culture vessels using Cell Dissociation Buffer (Invitrogen, Waltham, MA), seeded at 50,000 cells/well in conical-bottom 96-well plates, and treated with test antibody or antibody-drug conjugate for 24 hours at 4° C. to prevent internalization. Palivizumab (anti-RSV antibody, v21995) was included as a negative control. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4° C. for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647/APC-A GeoMean (fluorescence signal area geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody or antibody-drug conjugate using GraphPad Prism Software Version 9 (GraphPad Software, La Jolla, CA).
Results are shown in Table 44.1 and plotted in
The NaPi2b-mediated internalization of the anti-NaPi2b antibodies v38591 and v40502 and the antibody-drug conjugates v38591-MC-GGFG-AM-Compound 139 DAR 4 and v40502-MC-GGFG-AM-Compound 139 DAR 4 was evaluated in a human cancer cell lines expressing endogenous NaPi2b at high to moderate amounts, as described in Example 15. The cell lines used included IGROV-1 (ovarian carcinoma), HCC-78 (lung adenocarcinoma), and H441 (lung adenocarcinoma) cells and internalization was assessed by flow cytometry following 4 and 24 hours treatment at 2.5 nM of antibody or ADC. The anti-RSV antibody palivizumab (v21995) was used as a negative control.
Briefly, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting anti-Human IgG Fc (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4° C. Cells were seeded at 50,000 cells/well in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) in 48-well plates and incubated overnight under standard culturing conditions (37° C./5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 2.5 nM and incubated under standard culturing conditions for 4-24 hours to allow for internalization. An untreated control (cells incubated with complete growth medium only, no test article) was included for each cell line at each treatment time point. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 100 nM for 30 minutes at 4° C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488/FITC-A GeoMean (fluorescence signal area geometric mean, proportional to anti-Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ). Internalized Fluorescence Fold-Over Untreated values were calculated by dividing FITC-A GeoMean values of treatment wells by the FITC-A GeoMean values of the untreated wells. Internalized Fluorescence Fold-Over Untreated values were plotted using GraphPad Prism Software Version 9 (GraphPad Software, La Jolla, CA).
Results are shown in Table 45.1 and plotted in
The cell growth inhibition (cytotoxicity) capabilities of the humanized variant v38591 and v40502 conjugated to the drug linker MC-GGFG-AM-Compound 139 at DAR 4 were assessed against NaPi2b-expressing cell line spheroids according to the methods described below. Cell lines used were H1781 (lung adenocarcinoma), and H441 (lung adenocarcinoma). Lifastuzumab vedotin (v18993-MCvc-PABC-MMAE) and ADCs with non-targeted antibody palivizumab (anti-RSV) (v21995 or v22277) were used as controls.
Briefly, cells were seeded in Ultra-Low Attachment 384-well plates at 1,000 cells/well, centrifuged, and incubated for 3 days under standard culturing conditions to allow for spheroid formation and growth. Spheroids were then treated with a titration of test article, generated in cell growth medium, and incubated for 7 days under standard culturing conditions. After incubation, CellTiter-Glo® 3D reagent (Promega Corporation, Madison, WI) was spiked in all wells. Plates were incubated in the dark at room temperature for 1 hour and luminescence was quantified using a BioTek Cytation 5 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values were calculated based on untreated wells (cells with complete growth medium only, no test article added), and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.
The results are shown in Table 46.1 and representative curves are plotted in
The ability of v38591-MC-GGFG-AM-Compound 139, v38591 antibody, v40502-MC-GGFG-AM-Compound 139, v40502 antibody, v18993 (lifastuzumab) antibody and v21995-MC-GGFG-AM-Compound 139 to mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of NaPi2b-expressing IGROV-1 tumor cells was assessed using flow cytometry with human peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophage (macs) as effector cells, respectively.
In ADCC, Fcγ receptor (FcγR) on the surface of immune effector cells binds to the Fc region of an IgG1 antibody already bound to a target cancer cell. Activation of the FcγR triggers the killer effector cell population within the PBMCs to release perforin, which creates a pore within the target cancer cell plasma membrane, as well as granzyme B, which passes through the created pores and induces target cancer cell death. Similarly, in ADCP, upon activation of FcγR via the Fc region, macs engulf target cancer cells and trigger their death via phagocytosis.
For ADCC assay, 1 human PBMC donor was rested overnight in RPMI+10% ultra-low FBS+100 U/mL recombinant human IL-2. Tumor cells were stained with Cell Tracker™ Green following the manufacturer's protocol (Invitrogen) and mixed with human PBMC effector cells at an effector:target ratio of 25:1.
The cell mixture was added to a serial dilution of v38591-MC-GGFG-AM-Compound 139, v38591 antibody, v40502-MC-GGFG-AM-Compound 139, v40502 antibody, v18993 (lifastuzumab) antibody and v21995-MC-GGFG-AM-Compound 139 or to RPMI+10% ultra-low FBS as untreated control at 37° C. and 5% CO2 for 4 h. Viability staining was performed using a fixable LIVE/DEAD™ violet dye following the manufacturer's protocol (Invitrogen). Cells were washed once with PBS, centrifuged at 400×g for 5 min and resuspended in FACS buffer (PBS+2% FBS). Cancer cell cytotoxicity was assessed using the BD LSRFortessa™ Cell Analyzer (X-20) HTS (BD Biosciences) flow cytometer by gating for single, dead cancer cells that expressed green fluorescence compared to the total number of cancer cells that expressed green fluorescence. The percent ADCC (% Cytotoxicity) was determined by subtracting the percent cytotoxicity of the untreated cocultures from the percent cytotoxicity of the antibody-treated cocultures.
For ADCP assay, human monocytes were differentiated into macrophages, from human PBMC isolated from 1 healthy donor, by culturing in RPMI+ultra-low 10% FBS+10 ng/mL recombinant human M-CSF for 8 days; culture medium was supplemented every 2-3 days. After 8 days of cultivation, macrophage cellular phenotype (CD14, CD16, and CD11b expression) and morphology were confirmed by flow cytometry and microscopy, respectively. Differentiated macrophages were stained with Cytolight Rapid Red and cancer cells were stained with Cell Tracker Green. Cytolight Rapid Red-stained human-macrophages (red) and Cell Tracker™ Green-labeled cancer cells (green) were mixed at an effector (macrophage cell) to target (cancer cell) ratio of 2:1. The cell mixture was added to a serial dilution of v38591-MC-GGFG-AM-Compound 139, v38591 antibody, v40502-MC-GGFG-AM-Compound 139, v40502 antibody and v21995-MC-GGFG-AM-Compound 139 or to RPMI+10% ultra-low FBS as untreated control and incubated at 37° C. and 5% CO2 for 2 h. A live/dead stain was performed using the violet fixable viability dye following the manufacturer's protocol. Cells were washed once with PBS with 2% FBS, centrifuged and resuspended in FACS buffer. Phagocytic activity was assessed using the BD LSRFortessa™ Cell Analyzer (X-20) HTS (BD Biosciences) flow cytometer by gating for live, single, cells that were double positive for green and red fluorescence. ADCP (%) was determined by subtracting the percent double positive cells of the untreated cocultures from the percent double positive cells of the antibody-treated cocultures. Data analysis for ADCC and ADCP assay was performed with FlowJo™ 10 software. The results are shown in Table 47.1. v38591-MC-GGFG-AM-Compound 139, v38591 antibody, and v18993 (lifastuzumab) antibody induced dose-dependent ADCC in NaPi2b-expressing IGROV-1 cells with comparable activity after 4 hours of treatment at 12, 48 and 0.0192 nM, with a maximum of 17.21%, 19.96% and 14.41% of ADCC respectively. In contrast, v40502-MC-GGFG-AM-Compound 139, v40502 antibody and v21995-MC-GGFG-AM-Compound 139 did not significantly induce ADCC, with a maximum of 5.32%, 6.56% and 7.01% respectively. Similarly, v38591-MC-GGFG-AM-Compound 139 and v38591 antibody induced ADCP in NaPi2b-expressing IGROV-1 cells with comparable activity after 2 hours of treatment at 100 and 1 nM, with a maximum of 12.3% and 9.7%, respectively. v40502-MC-GGFG-AM-Compound 139, v40502 antibody and v21995-MC-GGFG-AM-Compound 139 did not induce ADCP, with a maximum of 0.2% and 0.4%, respectively.
The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.
Modifications of the specific embodiments described herein that would be apparent to those skilled in the art are intended to be included within the scope of the following claims.
This application is a continuation of International Application No. PCT/CA2023/051385 filed on Oct. 19, 2023, which claims the benefit of U.S. Provisional Application No. 63/459,205, filed Apr. 13, 2023, and U.S. Provisional Application No. 63/417,488, filed Oct. 19, 2022, which is hereby incorporated in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63459205 | Apr 2023 | US | |
| 63417488 | Oct 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CA2023/051385 | Oct 2023 | WO |
| Child | 19172273 | US |
