WO2023010057A1 - Atrc-101 target expression assay - Google Patents

Atrc-101 target expression assay Download PDF

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Publication number
WO2023010057A1
WO2023010057A1 PCT/US2022/074218 US2022074218W WO2023010057A1 WO 2023010057 A1 WO2023010057 A1 WO 2023010057A1 US 2022074218 W US2022074218 W US 2022074218W WO 2023010057 A1 WO2023010057 A1 WO 2023010057A1
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Prior art keywords
antibody
seq
atrc
tumor
subject
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PCT/US2022/074218
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French (fr)
Inventor
Iraz T. AYDIN
Jonathan BENJAMIN
Shaun M. Lippow
Amy MANNING-BOG
Carl MILLWARD
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Atreca, Inc.
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Publication of WO2023010057A1 publication Critical patent/WO2023010057A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This application relates to methods and compositions for selecting patients for treatment with the ATRC-101 antibody.
  • a method of determining whether a subject with a tumor is likely to respond to treatment with ATRC-101 comprising: contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises a heavy chain variable region comprising: an HCDR1 comprising a sequence (SEQ ID NO:l); an HCDR2 comprising a sequence (SEQ ID NO:2); and an HCDR3 comprising a sequence (SEQ ID NO:3).
  • the antibody further comprises a light chain variable region comprising: an LCDR1 comprising a sequence (SEQ ID NO:4); an LCDR2 comprising a sequence (SEQ ID NO:5); and an LCDR3 comprising a sequence (SEQ ID NO:6), wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
  • the method further comprises detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay and determining that the subject will be responsive to treatment of ATRC-101 if the binding of the antibody as determined by the immunohistochemistry assay corresponds to an H-score that is equal to or greater than an H-score cutoff. [0004] In some embodiments, the method further comprises selecting the subject for treatment with ATRC-101 if the subject is determined to be responsive to treatment of ATRC-101
  • a method of selecting a subject for treatment with ATRC-101 comprising detecting binding of an antibody with a tumor sample derived from the subject in an immunohistochemistry assay, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3.
  • the antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO: 5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
  • the method further comprises determining an H-score that corresponds to the binding and selecting the subject for treatment with ATRC101 if the H-score that is equal to or greater than an H-score cutoff.
  • a method of treating a tumor with ATRC-101 comprising selecting a subject with the tumor suitable for treatment with ATRC101, wherein the binding of an antibody to a target of ATRC-101 in a tumor sample obtained from the subject corresponds to an H-score that is equal to or greater than an H-score cutoff.
  • the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3.
  • the antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
  • the method further comprises administering an effective amount of ATRC-101 to the subject.
  • a method of detecting expression of a target of ATRC-101 in a tumor of a subject comprising contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3.
  • the antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
  • the method further comprises detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, thereby detecting expression of the target of ATRC-101.
  • the H-score cutoff is selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the H-score cutoff is 50.
  • the antibody has a heavy chain variable region comprising a sequence SEQ ID NO: 19 and a light chain variable region comprising a sequence SEQ ID NO: 20, In some embodiments, the antibody has an Fc region comprising SEQ ID NO: 24, or wherein the antibody has an Fc region comprising a sequence that is at least 80% identical to SEQ ID NO: 24, provided that the sequence comprises a N297A mutation in the heavy chain.
  • a method of determining whether a subject with a tumor is likely to respond to treatment with ATRC-101 comprises contacting a tumor sample obtained from the subject with the antibody described above, detecting the binding of the antibody to the tumor sample in an immunoassay, and predicting the subject is responsive to the treatment with ATRC-101 if the binding of the antibody corresponds to an H-score that is greater than an H-score cutoff.
  • the H-score cutoff is selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, or 100.
  • the tumor is selected from the group consisting of non-small cell lung, breast, ovarian, colorectal, acral melanoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, and urothelial carcinoma.
  • the method further comprises administering an effective amount of ATRC-101 to the subject.
  • the tumor is selected from the group consisting of non-small cell lung carcinoma, breast cancer, ovarian cancer, colorectal cancer, and acral melanoma.
  • the method further comprises administering an effective amount of pembrolizumab.
  • the method further comprises administering an effective amount of Doxorubicin or pegylated liposomal Doxorubicin,
  • kits used to identify a subject that is likely to respond to treatment with ATRC-101 comprising the antibody disclosed above.
  • the antibody comprises a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3.
  • the antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
  • said kit may further comprise one or more reagents used in an assay (e.g ., an immunohistochemistry assay) to detect the binding of the antibody to a tumor sample from the subject.
  • said one or more additional assay reagents provided in one or more vials, containers, or compartments of said kit.
  • the kit further comprises an isotype-matched negative control (e.g., Mouse IgG2a).
  • FIG. 1A-1C shows examples of baseline IHC staining of the target of ATRC-101 using mATRClOl N297A antibody in three tumor samples.
  • the IHC results in FIG. 1A-1C were determined to have H-scores of 0, 30, and 80, respectively.
  • FIG 2A shows best RECIST response in patients treated with ATRC-101 is associated with ATRC-101 target expression as measured by H-score.
  • the study population is defined as individuals with valid IHC H-score data at screening and the best overall response according to RECIST 1.1 criteria.
  • FIG. 2B is a Kaplan- Meier curve showing the probability of Progression Free Survival (PFS) is associated with ATRC- 101 target expression as measured by H-score in patients treated with ATRC-101 administered as a monotherapy Q3W.
  • PFS Progression Free Survival
  • FIG. 3A-B shows the results of patients who have received ATRC-101 at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg (FIG. 3A).
  • Stable disease (SD) was observed in eight (8) patients, four (4) of which showed tumor reduction (FIG. 3B).
  • FIG. 4 shows the baseline characteristics, including the H-score of IHC results, of 18 patients at baseline.
  • the patients have a variety of tumor types, including breast, colorectal, non small cell lung, acral melanoma, and ovarian tumors. These patients were grouped and received ATRC-101 at doses as indicated. The results indicate that expression levels of ATRC-101 target in patients with different tumor types were highly variable.
  • FIG. 5A shows target lesion response in target-positive tumor patients treated with ATRC-101 or ATRC-101 and pembrolizumab over time, as reflected in a change in summary of diameters (SOD).
  • Target-positive patients have tumors with H-scores >50 when analyzed using the methods disclosed herein.
  • FIG. 5B shows durable responses observed in 12 patients with H-scores of > 50 carrying different tumor types.
  • OVC ovarian cancer
  • CRC colorectal cancer
  • BRC breast cancer
  • SmB head and neck cancer
  • MEL melanoma
  • FIG. 6 shows tumor response in target-positive NSCLS patients treated with ATRC-101 as Q3W monotherapy at either 3 mg/kg ( ⁇ ) or 30 mg. kg (®) over time.
  • the results showed that 60% of NSCLC patients with an H-score >50 demonstrate Stable Disease or Partial Response as a best overall response.
  • FIG. 7A-B show the ATRC-101 clinical trial design for monotherapy (FIG. 7A) and combination therapies (FIG. 7B).
  • FIG. 8A-8E show the results of tumor growth of BALB/c mice with established EMT6 tumors after various monotherapy or combination therapy as indicated.
  • mATRClOl if used, was administered intraperitoneally twice weekly.
  • Doxorubicin (Dox) if used, was administered intravenously once weekly.
  • FIG. 8 A shows the tumor growth measurements from mice administered with i) mATRC at 3 mg/kg (“3 mpk”) or ii) PBS.
  • FIG. 8B shows tumor growth measurements from mice administered with i) Dox at 5 mg/kg or ii) PBS.
  • FIG. 8C shows tumor growth measurements from mice administered with i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) PBS as a control.
  • FIG. 8D shows tumor growth measurements from mice treated with either i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) Dox at 5mg/kg.
  • FIG. 8E shows tumor growth measurements from mice treated with either i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) mATRClOl at 3 mg/kg.
  • FIG. 9 shows the probability of survival of BALB/c mice with established EMT6 tumors after undergoing various monotherapy or combination therapy: i) Dox at 5mg/kg, ii) mATRClOl antibody at 3 mg/kg, or iii) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg. Mice administered with PBS were included as controls.
  • FIG. 10A-10D show immunohistochemical analyses of mATRClOl reactivity in EMT6 tumors from mice treated with Pegylated Doxil (PLD).
  • FIG. 10A and 10B show the immunohistochemistry staining performed on the tumors in mice treated with PBS, lmg/kg PLD, 2 mg/kg PLD, or 10 mg/kg PLD. The stainings were performed using mATRClOl or an isotype control antibody.
  • FIG. IOC and 10D show the results of immunohistochemistry stainings performed on tumors from mice treated with PLD at indicated doses, i.e., 2 mg/kg, 5 mg/kg, or 10 mg/kg.
  • FIG. 10C and FIG. 10D show the results of immunohistochemistry stainings performed on the tumor samples at one week and four weeks, respectively, after the mice received the first dose of PLD.
  • an “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g ., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g, bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g, Zapata el al, Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g, scFv); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site and a residual “Fc” fragment, reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment with two antigen combining sites and is still capable of cross-linking antigen.
  • a “tumor-binding antibody” as used herein refers to an antibody that binds tumor tissue.
  • the tumor-binding antibody binds the tumor tissue through a binding interaction with an extracellular RNA-protein complex.
  • Extracellular RNA-protein complex refers to a complex that is detected on the outside of tumor cells. The complex need not be integrated into the external surface of the cells, but in some embodiments, it may be associated with the outside of the tumor cells as a conglomeration or aggregate of RNA and protein molecules interacting with the cell membrane or otherwise present outside of the tumor cell.
  • the extracellular RNA-protein complex is external to tumor cells, it may additionally be present internally in a cell.
  • tumor-binding antibody exhibits preferential binding to tumor tissue compared to tumor-adjacent tissue (TAT), e.g., a specific signal above the noise is noted with enhanced reactivity apparent in tumor vs. adjacent tissues.
  • TAT tumor-adjacent tissue
  • the tumor-binding antibody is an “anti-tumor antibody” that decreases tumor growth rate, tumor size, invasion, and/or metastasis via direct or indirect effects on tumor cells.
  • V-region refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4.
  • the heavy chain V-region, VH is a consequence of the rearrangement of a V-gene (HV), a D-gene (HD), and a J-gene (HJ), in what is termed V(D)J recombination during B-cell differentiation.
  • the light chain V-region, VL is a consequence of the rearrangement of a V-gene (LV) and a J-gene (LJ).
  • CDR complementarity-determining region
  • HVR hypervariable regions
  • the CDRs are the primary contributors to binding to an epitope of an antigen.
  • the CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the particular CDR is located.
  • HCDR3 VH CDR3
  • LCDR3 VL CDR3
  • CDR CDR3 from the variable domain of the light chain of the antibody in which it is located.
  • CDR is used interchangeably with “HVR” in this application when referring to CDR sequences.
  • the amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g. , Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g. , Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C.
  • CDRs as determined by Kabat numbering is based, for example, on Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • an “Fc region” refers to the constant region of an antibody, excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy.
  • Fc region may vary; however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering according to the EU index as in Kabat et al , (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • the term “Fc region” may refer to this region in isolation or in the context of an antibody or antibody fragment.
  • Fc region includes naturally occurring allelic variants of the Fc region and modified Fc regions, e.g., that are modified to modulate effector function or other properties such as pharmacokinetics, stability, or production properties of an antibody. Fc regions also include variants that do not exhibit alterations in biological function.
  • one or more amino acids can be deleted from the N-terminus or C- terminus of the Fc region of an immunoglobulin without substantial loss of biological function.
  • Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie et al, Science 247:306-1310, 1990).
  • a single amino acid substitution S228P according to Kabat numbering; designated IgG4Pro
  • IgG4Pro a single amino acid substitution
  • IgG4Pro an amino acid substitution
  • an “EC50 as used herein in the context of an Fc receptor engagement assay refers to the half maximal effective concentration, which is the concentration of an antibody that induces a response (signal generated in engagement assay) halfway between the baseline and maximum after a specified exposure time. Fc receptor engagement assays are further described herein in the “Variant Binding Activity” section. In some embodiments, the “fold over EC50” is determined by dividing the EC50 of a reference antibody by the EC50 of the test antibody.
  • the term “monovalent molecule” herein refers to a molecule with one antigen-binding site, e.g, a Fab or scFv.
  • bivalent molecule herein refers to a molecule with two antigen-binding sites.
  • a bivalent molecule of the present invention is a bivalent antibody or a bivalent fragment thereof.
  • a bivalent molecule of the present invention is a bivalent antibody.
  • a bivalent molecule of the present invention is an IgG.
  • monoclonal antibodies have a bivalent basic structure.
  • IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and thus have higher valencies. This bivalency increases the avidity of antibodies for antigens.
  • the terms “monovalent binding” or “monovalently binds to” as used herein refer to the binding of one antigen-binding site to its antigen.
  • bivalent binding or “bivalently binds to” as used herein refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. In some embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.
  • identity in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (e.g, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, e.g, the length of the two sequences, when compared and aligned for maximum correspondence over a comparison window or designated region.
  • Alignment for determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.
  • BLAST 2.0 algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST 2.0 algorithms, described in Altschul etal. , Nuc. Acids Res. 25:3389-3402 (1977) and Altschul etal., J. Mol. Biol. 215:403-410 (1990).
  • BLAST 2.0 can be used with the default parameters to determine percent sequence identity.
  • the terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence.
  • an amino acid residue in a VH region polypeptide “corresponds to” amino acid in the VH region of SEQ ID NO: 19 when the residue aligns with the amino acid in SEQ ID NO: 19 when optimally aligned to SEQ ID NO: 19.
  • the polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.
  • a “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and/or size of the side group chain is maintained.
  • Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at a pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Val, Leu and lie; (vi) hydrophobic sulfur-containing amino acids Met and Cys, which are not as hydrophobic as Val, Leu, and lie; (vii) small polar uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide-comprising amino acids
  • nucleic acid and “polynucleotide” are used interchangeably and, as used herein, refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide and combinations thereof.
  • the terms also include, but are not limited to, single- and double-stranded forms of DNA.
  • a polynucleotide e.g ., a cDNA or mRNA
  • a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • the nucleic acid molecules may be modified chemically or biochemically or contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, the substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, and the like), charged linkages (e.g, phosphorothioates, phosphorodithioates, and the like), pendent moieties (e.g, polypeptides), intercalators (e.g, acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g, alpha anomeric nucleic acids, and the like).
  • uncharged linkages e.g, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, and the like
  • charged linkages e.g, phosphorothioates, phosphorodithioates, and the like
  • pendent moieties
  • the above term also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hair pinned, circular, and padlocked conformations.
  • a reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand with its complementary sequence.
  • the term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure and the vector incorporated into the host cell genome as into which it has been introduced.
  • a “vector” refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector.
  • Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • substitution denotes replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.
  • An “isolated” nucleic acid refers to a nucleic acid molecule separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule. Still, the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an antibody or fragment thereof refers to one or more nucleic acid molecules encoding antibody heavy or light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • a host cell is a recombinant host cell and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages.
  • a polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions, and/or insertions.
  • cancer cell or “tumor cell” as used herein refers to a neoplastic cell.
  • the term includes cells from tumors that are benign as well as malignant. Neoplastic transformation is associated with phenotypic changes of the tumor cell relative to the cell type from which it is derived. The changes can include loss of contact inhibition, morphological changes, and unregulated cell growth,
  • “Inhibiting growth of a tumor” and “inhibiting growth of cancer” as used herein are interchangeable and refer to slowing growth and/or reducing the cancer cell burden of a cancer patient. “Inhibiting growth of cancer” thus includes killing cancer cells, as well as decreasing the rate of tumor growth, tumor size, invasion, and/or metastasis by direct or indirect effects on tumor cells.
  • therapeutic agent refers to an agent that, when administered to a patient suffering from a disease, in a therapeutically effective dose, will cure or at least partially arrest the symptoms of the disease and complications associated with the disease.
  • treatment refers to the administration of an agent to impede a disease, such as the growth of cancer, to cause a cancer tumor to shrink as determined by measuring at least one dimension of the tumor, to extend the expected survival time of the subj ect and/or time to progression of the tumor or the like. Treatment may also refer to any course that one skilled, such as a treating physician, deems expedient.
  • tissue sample or “tissue sample” or “patient sample” or “patient cell or tissue sample” or “specimen” each refer to a collection of similar cells obtained from a tissue of a subject or patient.
  • the source of the tissue sample may be solid tissue from fresh tissue, frozen and/or preserved organ or tissue or biopsy or aspirate; blood or any blood constituents, bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid or cells from any time in gestation or development of the subject.
  • the tissue sample may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Cells may be fixed in a conventional manner, such as in an FFPE manner.
  • RECIST or “Response Evaluation Criteria in Solid Tumours” refers to a set of published rules that define when cancer patients improve (“respond”), stay the same (“stable”), or worsen (“progression”) during treatments.
  • Response as defined by RECIST 1.1 criteria, have been published, for example, Eisenhauer et al ., European Journal of Cancer 45 (2009) 228-247.
  • Respond to treatment, and other forms of this verb, as used herein, refer to the reaction of a subject to treatment with an agent.
  • a subject responds to treatment if growth of a tumor in the subject is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • a subject responds to treatment if a tumor in the subject shrinks by about 5%, 10%, 20%, 30%, 40%, 50%, or more as determined by any appropriate measure, e.g ., by mass or volume. Tumor size can be measured, for example, by radiologic measurements.
  • a subject responds to treatment with an anti -turn or antibody disclosed herein if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50%, or more beyond the life expectancy predicted if no treatment is administered.
  • a subject responds to treatment with an agent if the subject has an increased overall survival or increased time to progression.
  • the subject responds to treatment with an agent if it demonstrates stable disease as opposed to progressive disease in the absence of the treatment.
  • the term “likely to respond to treatment with ATRC-101” means that a patient will likely have a Best Overall Response, for example, a CR, PR, SD, or PD, after receiving treatment of ATRC-101.
  • a patient with an H-score, tumor proportion score, or intensity score that is greater than or equal to a cutoff score as disclosed herein is more likely to respond to treatment than a patient with an H-score, tumor proportion score, or intensity score that less than the cutoff score.
  • the H-score, tumor proportion score, or intensity score is determined based on the binding of an ATRC-101 diagnostic antibody disclosed herein to a tumor sample derived from the patient in an immunohistochemistry assay.
  • a cutoff refers to the value of a predetermined measure on subjects exhibiting certain attributes that allow the best discrimination between two or more categories of an attribute. For example, a cutoff that will enable one to discriminate between two categories, such as target expression and low target expression, may be used to determine the suitability for the ATRC-101 treatment for. These cutoffs may be used to separate the subjects with values lower than or higher than the cutoff to evaluate the treatment plan for the patient.
  • the term “effective amount” refers to an amount of a pharmacological agent effective in treating, eliminating, or mitigating at least one symptom of the disease being treated.
  • the effective amount can be an amount effective to invoke an anti-tumor response is to evoke a humoral and/or cellular immune response in the recipient subject leading to growth inhibition or death of target tumor cells or reduce tumor load or bring about other desired beneficial clinical outcomes related to cancer improvement.
  • baseline herein refers to the disease status before a patient has been treated with ATRC-101. Accordingly, the term “baseline H-score” refers to the H-score determined for a tumor sample obtained from the patient prior to ATRC-101 treatment.
  • ATRC-101 is a fully human immunoglobulin G, subclass 1 (IgGl)/lambda monoclonal antibody that is an engineered version of an antibody expressed by a plasmablast B cell that was originally isolated from a patient with NSCLC adenocarcinoma undergoing treatment with a checkpoint inhibitor.
  • the parental antibody's variable fragment (Fv) region was optimized to generate ATRC- 101.
  • ATRC-101 targets a complex containing human polyadenylate-binding protein family member(s) and polyadenylated ribonucleic acid (poly(A) RNA) (“RNP complex”).
  • poly(A) RNA polyadenylated ribonucleic acid
  • RNP complex polyadenylated ribonucleic acid
  • WO 2020/168231 the entire content of which is herein incorporated by reference.
  • ATRC-101 binds highly selectively to tumor cells.
  • ATRC-101 does not show any appreciable binding to normal human tissues. This indicates that the ATRC-101 target is a tumor- associated version of a complex containing a polyadenylate-binding protein family member and poly(A) RNA.
  • ATRC-101 In preclinical proof of concept studies, a chimeric version of ATRC-101 harboring the mouse IgG2a Fc region (AB6042-mIgG2a) demonstrated significant anti-tumor activity in syngeneic mouse models of breast carcinoma and colon carcinoma. Based on preclinical observations, ATRC-101 is proposed to cause tumor growth inhibition and regression, in part, by (a) altering the composition of the tumor microenvironment to be more anti-tumorigenic and proinflammatory and (b) inducing an adaptive immune response that recruits effector CD8+ T cells to attack the tumors. Consistent with that model, administration of low-dose AB6042- mIgG2a enhanced the antitumor activity of anti -mouse PD-1 in two syngeneic models of breast carcinoma.
  • ATRC-101 is an engineered version of an antibody identified by IRC® as disclosed in, e.g., WO 2012148497A2, the entire content of which is herein incorporated by reference.
  • ATRC- 101 is being evaluated as monotherapy or combination therapy (e.g, with Pembrolizumab) for the treatment of patients with advanced solid malignancies, including breast cancer (BC), non small cell lung cancer (NSCLC), colorectal cancer (CRC), ovarian cancer, acral melanoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, and urothelial carcinoma.
  • Tables 1-5 below show the ATRC-101 sequences.
  • Table 1 shows the heavy and light chain variable region sequences
  • Table 2 shows the heavy chain and the light chain sequences
  • Tables 3-5 show the CDR sequences.
  • the VH CDRS as listed in Table 3, are defined as follows: HCDR1 is defined by combining Rabat and IMGT; HCDR2 is defined by Rabat, and the HCDR3 is defined by IMGT.
  • the VL CDRS as listed in Table 3, are defined by Rabat.
  • the numbering and placement of the CDRs can differ depending on the numbering system employed. It is understood that disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated CDRs, regardless of the numbering system employed.
  • Table 4 shows the CDR sequences defined using the IMGT numbering system
  • Table 5 shows the CDR sequences defined using the Rabat numbering system.
  • an ATRC-101 diagnostic antibody can have the same sequence structures as the corresponding ATRC-101 therapeutic antibody or different sequence structures; that is, an ATRC-101 diagnostic antibody may also have a therapeutic effect.
  • an ATRC-101 diagnostic antibody has the same antigen-binding sequences (e.g ., the same VH and VL sequences) as the corresponding ATRC-101 therapeutic antibody but has a different isotype.
  • the ATRC-101 diagnostic antibody is a mouse IgG2a with a mutation at the N297 position in the heavy chain, such as N297A, N297G, or N297Q.
  • an ATRC-101 diagnostic antibody of the present invention has one, two, or three CDRs of a VH sequence, as shown in Table 1.
  • the ATRC-101 diagnostic antibody has at least one mutation and no more than 10, 20, 30, 40, or 50 mutations in the VH amino acid sequences compared to a VH sequence set forth in any one of SEQ ID NOS: 19.
  • the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in any one of SEQ ID NOS: 19.
  • an ATRC-101 diagnostic antibody of the present invention has one, two, or three CDRs of a VL sequence, as shown in Table 1.
  • the tumor binding antibody has at least one mutation and no more than 10, 20, 30, 40, or 50 mutations in the VL amino acid sequences compared to a VH sequence set forth in any one of SEQ ID NOS: 19.
  • the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in any one of SEQ ID NOS:19.
  • the VH amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, or 5 amino acid deletions or insertions, relative to an HCDR sequence shown in Tables 3-5.
  • the VH region comprises an HCDR1 with 1, 2, 3, or 4 substitutions relative to an HCDR1 sequence of any anof SEQ ID NOS:l, 7, or 13.
  • the VH region comprises an HCDRwith 1, 2, 3, or 4 substitutions relative to an HCDR2 sequence of any ane of SEQ ID NOS: 2, 8, or 14.
  • the VH region comprises a HCDR3 with 1, 2, 3, or 4 substitutions relative to a HCDR3 sequence of any anof SEQ ID NOS: 3, 9, or 15.
  • the VL amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, or 5 amino acid deletions or insertions, relative to an LCDR sequence shown in Tables 3-5.
  • the VL region comprises a LCDR1 with 1, 2, 3, or 4 substitutions relative to a LCDR1 sequence of any of SEQ ID NOS:4, 10, or 16.
  • the VL region comprises a LCDR2 with 1, 2, 3, or 4 substitutions relative to a CDR2 sequence of any of SEQ ID NOS: 5, 11, or 17.
  • the V L region comprises a CDR3 that has 1, 2, 3, or 4 substitutions relative to a CDR3 sequence of any one of SEQ ID NOS: 5, 11, or 17.
  • the ATRC-101 diagnostic antibody has an Fc region comprising the sequence of SEQ ID NO: 24.
  • the ATRC diagnostic antibody has a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 24 and comprises an alanine at position 180 of SEQ ID NO: 24 (corresponding to position 297 in the heavy chain).
  • mATRClOl the mIgG2a antibody
  • mATRClOl can be used to evaluate the expression of the ATRC-101 target in tumor samples.
  • mATRClOl comprises a VH and a VL sequences as set forth in Table 1 and CDRs as set forth in Table 3 (or Table 4 or Table 5).
  • the mATRClOl comprises an Fc region set forth in SEQ ID NO: 23.
  • the diagnostic antibody is mATRClOl N297A. Similar to mATRClOl, mATRClOl N297A also comprises VH and VL sequences as set forth in Table 1 and CDRs as set forth in Table 3 (or Table 4 or Table 5). But mATRC 101 N297A differs from mATRC in that it has an N297A mutation in the heavy chain Fc region. The Fc region of mATRC 101 N297A comprises a sequence set forth in SEQ ID NO: 24. As described below, the mATRClOl N297A antibody can be used to select patients likely to respond to ATRC-101.
  • mATRC 101 N297 A showed a broader range of staining with higher H-Score averages by cancer indication.
  • An array of negative, low, moderate, and high cases was observed for mATRClOl N297A, with H-Scores ranging from 0 to 270 across the 142 tumors.
  • mATRClOl N297A is more sensitive in determining a patient’s likelihood of responsiveness to the ATRC therapeutic antibody.
  • the diagnostic antibody is mATRClOl comprising alternative mutations that disrupt signaling involving the Fc region of the antibody.
  • alternative mutations include T299A, E233P, L234A, L234F, L234V, L235A, L235E, G236del, S267K, P329G, P329A, P331S, and combinations thereof, including but are not limited to, (1) L234A and L235A; (2) L234A, L235A, and P329G; (3) L234A, L235A, and P329A; (4) L234A, L235E; L234A, L235A, and G237A; (5) L234A, G237A; L235A, and G237A; (6) L234F, L235E, and P331S; and (7) E233P, L234V, L235A, G236del, and S267K.
  • ATRC-101 targets an extracellular RNA- protein complex that comprises mRNA and further comprises an mRNA binding protein.
  • the ATRC-101 diagnostic antibodies bind to the same target protein complex as the ATRC-101 and variants thereof, as disclosed in WO 2020/168231.
  • an ATRC-101 diagnostic antibody in accordance with the disclosure, may be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
  • the antibody is a substantially full-length antibody, e.g, an IgG antibody or other antibody class or isotype defined herein.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody and production by recombinant host cells.
  • an ATRC-101 diagnostic antibody by the present disclosure is in a monovalent format.
  • the anti-tumor antibody is in a fragment format, e.g, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
  • an ATRC-101 diagnostic antibody of the present invention may be conjugated or linked to imaging/detectable moieties.
  • the ATRC-101 diagnostic antibody may be conjugated to a detectable marker, an imaging agent, or an oligonucleotide.
  • Methods for conjugating or linking antibodies to the desired molecule are well known in the art.
  • the moiety may be linked to the antibody covalently or by non-covalent linkages.
  • the method of determining whether a subject is likely to respond to treatment of ATRC-101 comprises measuring the amount of the ATRC-101 target and comparing the amount with a cutoff. If the amount of target in the tumor sample is equal to or greater than the cutoff, the patient is likely to respond to the treatment. If the amount of the ATRC- 101 target is not greater than the cutoff then patient is not likely to respond to the treatment.
  • a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein e.g. , mATRClOl N297A.
  • the method comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining an H-score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the binding corresponds to an H-score that is equal to or greater than an H-score cutoff.
  • a method of treating a tumor with ATRC-101 comprises selecting a subject with a tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101.
  • the subject is selected if the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to an H-score that is equal to or greater than an H-score cutoff.
  • a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein e.g ., mATRClOl N297A.
  • the method comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining a tumor proportion score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the tumor proportion score corresponds to a tumor proportion score that is equal to or greater than a tumor proportion score cutoff.
  • a method of treating a tumor with ATRC-101 comprises selecting a subject with the tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101.
  • the subject is selected based on the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to a tumor proportion score equal to or greater than a tumor proportion score cutoff.
  • a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein, e.g. , mATRClOl N297A comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining an intensity score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the intensity score corresponds to an intensity score that is equal to or greater than an intensity score cutoff.
  • a method of treating a tumor with ATRC-101 comprises selecting a subject with the tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101. The subject is selected based on that the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to an intensity score equal to or greater than an intensity score cutoff.
  • a cutoff can be determined based on the readout from the assay used.
  • the cutoff may be a value from a normal sample determined using the same assay.
  • a cutoff may be a mean or a median value of assays conducted to detect the presence of the target (e.g ., the RNP complex) on a set of reference samples.
  • the cutoff may be a percentile value (e.g., 20%, 25%, 30%, 50%, 60%, 75%, or 80%) of the results of the assays conducted to detect the presence of the target in a set of reference samples.
  • x can be used as the cutoff: a test tumor sample is considered to have a positive expression of the target if it shows a value equal to or greater than X when assayed under the same conditions as the reference samples.
  • the reference samples may be non-tumor samples with the same tissue origin as the tumor samples to be evaluated for the likelihood of responsibilities to the ATRC-101 antibody.
  • the reference samples are tumor samples of a variety of tissue origins.
  • the method may comprise obtaining a tumor sample from a patient having the tumor.
  • the tumor sample is an FFPE sample.
  • the tumor sample is a fresh frozen sample.
  • the tumor sample comprises a breast tumor sample, a non-small cell lung cancer sample, an ovarian tumor sample, a colorectal tumor sample, an acral melanoma sample, an esophageal tumor sample, a squamous cell carcinoma tumor sample, head and neck squamous cell carcinoma tumor sample, hepatocellular carcinoma tumor sample, and a urothelial carcinoma tumor sample.
  • a method of detecting and/or quantifying the expression of a target of ATRC-101 in a tumor of a subject comprises contacting a tumor sample obtained from the subject with an ATRC-101 diagnostic antibody disclosed herein, e.g ., mATRClOl N297A, and detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, thereby detecting and/or quantifying the expression of a target of ATRC-101.
  • an ATRC-101 diagnostic antibody disclosed herein, e.g ., mATRClOl N297A
  • the target’s expression can be assessed by detecting the binding of an ATRC-101 diagnostic antibody (e.g, mATRClOl N297A) to the tumor sample using various assays.
  • the detection is performed by an immunohistochemistry staining (IHC); illustrative examples are described in Example 1 and Example 3.
  • the method comprises contacting the tumor sample with an ATRC-101 diagnostic antibody and detecting the binding of the diagnostic antibody to the target by adding a secondary antibody. Said secondary antibody binds to the ATRC diagnostic antibody.
  • the secondary antibody is conjugated to a detectable moiety.
  • the signal from the detectable moiety can be quantified, which corresponds to the binding of the ATRC-101 diagnostic antibody to the ATRC-101 target in the tumor.
  • the detectable moiety is an enzyme (e.g, a horseradish peroxidase, or “HRP”), which upon contacting its substrate, (e.g, DAB), produces a discrete insoluble reaction product at the site of antigen in the presence of the enzyme (e.g. , HRP).
  • HRP horseradish peroxidase
  • the expression of the ATRC-101 target in the tumor sample is detected by an immunohistochemistry (IHC) assay, and the amount is quantified by an H-score or a tumor proportion score (also referred to herein as a standard percent score). Both the H-score and the tumor proportion percent score are described in Example 1.
  • an H-score is generated by summing the percentages of cells with the intensity of expression multiplied by their corresponding differential intensity on a four-point semi-quantitative scale (0, 1+, 2+, 3+).
  • H-score [(% at ⁇ 1) x 0] + [(1% at 1+) xl] + [(% at 2+) x2]+ [(% at 3+) x3].
  • the scores range from 0 to 300.
  • the cutoff used to determine the likelihood of a patient’s responsiveness is an H-score cutoff
  • the H-score cutoff may be 10, 20, 30, 40, 50, 60, 70, 80, or 100 according to the scale described above.
  • the H-score cutoff is 40, 50, or 60.
  • the H-score cutoff is 50.
  • a tumor proportion score can be assigned by summing the percentages of intensities at either >1+, >2+, or >3+.
  • scores range from 0 to 100.
  • the cutoff used to determine the likelihood of a patient’s responsiveness is a tumor proportion cutoff
  • the tumor proportion score cutoff may be 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80%. In one embodiment, the tumor proportion score cutoff is 30%, 40%, or 50%. Kits.
  • kits for carrying out the methods of the invention.
  • a kit comprises an ATRC-101 diagnostic antibody described above.
  • the kit may further comprise a T cell-binding agent, e.g ., a T cell-binding agent that specifically binds to CD3 or CD8.
  • the kit may further comprise assay diluents, standards, controls, and/or detectable labels.
  • the assay diluents, standards, and/or controls may be optimized for a particular sample matrix.
  • the diluents, standards, and controls may include i) human blood, serum, or plasma; ii) animal blood, serum, or plasma, or iii) artificial blood, serum or plasma substitutes.
  • ATRC-101 diagnostic antibodies are commonly produced using vectors and recombinant methodology well known in the art (see, e.g, Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology).
  • Commercial vendors offer reagents, cloning vectors, and genetic manipulation kits.
  • isolated nucleic acids encoding a VH and/or VL region, or fragment thereof, of any of the anti tumor antibodies as described herein; vectors comprising such nucleic acids and host cells into which the nucleic acids are introduced that are used to replicate the antibody-encoding nucleic acids and/or to express the antibodies.
  • nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the anti-tumor antibody e.g ., the antibody’s light and/or heavy chains).
  • the host cell contains (1) a vector containing a polynucleotide that encodes the VL amino acid sequence and a polynucleotide that encodes the VH amino acid sequence, or (2) a first vector containing a polynucleotide that encodes the VL amino acid sequence and a second vector containing a polynucleotide that encodes the VH amino acid sequence.
  • the invention provides a method of making an ATRC-101 diagnostic antibody as described herein.
  • the method includes culturing a host cell, as described in the preceding paragraph, under conditions suitable for the expression of the antibody.
  • the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally can self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g, pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl plasmids, pCRl, RP4, phage DNAs, and shuttle vectors. These and many other cloning vectors are available from commercial vendors.
  • Expression vectors are generally replicable polynucleotide constructs containing a nucleic acid of the present disclosure.
  • the expression vector may be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
  • Suitable expression vectors include but are not limited to plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and other vectors.
  • Suitable host cells for expressing an anti-tumor antibody as described herein include both prokaryotic and eukaryotic cells.
  • an anti-tumor antibody may be produced in bacteria, particularly when glycosylation and Fc effector function are not needed.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • the host cell may be a eukaryotic host cell, including eukaryotic microorganisms, such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern, vertebrate, invertebrate, and plant cells.
  • invertebrate cells include insect cells. Numerous baculoviral strains that may be used in conjunction with insect cells have been identified. Plant cell cultures can also be utilized as host cells.
  • vertebrate host cells are used for producing an anti-tumor antibody of the present disclosure.
  • mammalian cell lines such as a monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al, ./. Gen Virol. 36:59,1977; baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g ., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g, in Mather et al, Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells may be used to express anti -turn or antibodies.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al, Proc. Natl. Acad.
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • a CHO cell line e.g., the CHO-K1 cell line.
  • One or more expression plasmids can be introduced that encode heavy and light chain sequences.
  • an expression plasmid encoding a heavy chain, e.g, SEQ ID NO: 1723, and an expression plasmid encoding a light chain, e.g, SEQ ID NO: 1724 are transfected into host cells as linearized plasmids at a ratio of 1:1 in the CHO-K1 host cell line using reagents such as Freestyle Max reagent.
  • Fluorescence-activated cell sorting (FACS) coupled with single-cell imaging can be used as a cloning method to obtain a production cell line.
  • FACS Fluorescence-activated cell sorting
  • a host cell transfected with an expression vector encoding an anti-tumor antibody of the present disclosure, or fragment thereof, can be cultured under appropriate conditions to allow the expression of the polypeptide.
  • the polypeptides may be secreted and isolated from a mixture of cells and a medium containing the polypeptides. Alternatively, the polypeptide may be retained in the cytoplasm on a membrane fraction, and the cells harvested, lysed, and the polypeptide isolated using the desired method.
  • an anti-tumor antibody of the present disclosure can be produced by in vitro synthesis (e.g, Sutro Biopharma biochemical protein synthesis platform).
  • a method of generating variants of an anti tumor antibody as disclosed herein.
  • a construct encoding a variant of a VH CDR3 as described herein can be modified, and the VH region encoded by the modified construct can be tested for binding activity to the ATRC-101 target in the context of a VH region as described herein, that is paired with a VL region or variant region as described herein.
  • a construct encoding a variant of a VL CDR3 as described herein can be modified.
  • the VL region encoded by the modified construct can be tested for binding to an ATRC-101 target comprising polyadenylated RNA.
  • Such an analysis can also be performed with other CDRs or framework regions, and an antibody having the desired activity can then be selected.
  • ATRC-101 may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents, and/or immunotherapeutic agents.
  • additional therapeutic agents e.g., radiation therapy, chemotherapeutic agents, and/or immunotherapeutic agents.
  • ATRC- 101 can be administered in conjunction with an agent that enhances the presence or activity of T cells.
  • ATRC-101 is administered in combination with an immune checkpoint agent.
  • the agent is a biologic therapeutic or a small molecule.
  • the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein, or a combination thereof.
  • the agents inhibit, e.g, by blocking ligand binding to the receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDOl, ID02, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK1, CHK2, A2aR, B-7 family ligands or their receptors, or a combination thereof.
  • a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDOl, ID02, B7-H3, B7-H4, BTLA, HVEM, TIM
  • the agent targets PD-1, e.g, an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1.
  • the agent is pembrolizumab.
  • the agent targets CTLA-4.
  • the agent targets LAG3.
  • the agent targets TIM3.
  • the agents target ICOS.
  • ATRC-101 is administered in conjunction with a therapeutic antibody, such as an antibody that targets a tumor cell antigen.
  • therapeutic antibodies include as rituximab, trastuzumab, tositumomab, ibritumomab, alemtuzumab, atezolizumab, avelumab, durvalumab, pidilizumab, AMP -224, AMP-514, PDR001, cemiplimab, BMS-936559, CK-301, epratuzumab, bevacizumab, elotuzumab, necitumumab, blinatumomab, brentuximab, cetuximab, daratumumab, denosumab, dinutuximab, gemtuzumab ibritumomab ipilimumab, nivolumab, obinut
  • ATRC-101 is administered with a chemotherapeutic agent.
  • cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosf
  • alkylating agents such
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, mifepristone, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • cancer therapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors.
  • protein kinase inhibitors such as afatinib, axitinib, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib,
  • chemotherapeutic agents include topoisomerase I inhibitors (e.g ., irinotecan, topotecan, camptothecin, and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g, melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g, cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g, 5-fluorouracil, capecitabine, gemcitabine, fluorubic
  • Illustrative chemotherapeutic agents additionally include paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (e.g, CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g, imatinib mesylate and gefitinib); proteasome inhibitors (e.g, bortezomib); NF-KB inhibitors, including inhibitors of IKB kinase and other inhibitors of proteins or enzymes known to be upregulated, overexpressed or activated in cancers, the inhibition of which down-regulates cell replication. Additional agents include asparaginase and a Bacillus Calmete-Guerin preparation.
  • ATRC-101 is administered with a chemotherapeutic agent that increases the expression of the ATRC-101 target.
  • the agent that increases the level of ATRC-101 target is Doxorubicin or pegylated liposomal Doxorubicin.
  • ATRC-101 and the additional agent can be administered following the same or different dosing regimen.
  • ATRC-101 and the additional agent is administered sequentially in any order during the entire or portions of the treatment period.
  • ATRC-101 and the additional agent is administered simultaneously or approximately simultaneously ( e.g ., within about 1, 5, 10, 15, 20, 30, 45, or 60 minutes of each other).
  • the additional agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before administration of ATRC-101.
  • the additional agent is administered from 1 to 4 weeks, or longer before ATRC-101 is administered.
  • Immunohi stochemi cal (IHC) stainings are performed as follows. mATRClOl or mATRClOl N297A as disclosed above, were used as the primary antibodies to detect the presence of ATRC-101 target in formalin-fixed, paraffin-embedded (FFPE) human tissues according to the below procedures. The concentrations of the antibodies and some assay conditions used in the assay are disclosed in Table 8.
  • FFPE tissue blocks are first cut at 4-5 pm thickness, and sections are mounted onto positively charged, capillary gap glass slides. Slides are baked (60°C, dry heat) prior to use. Tissue sections are then de-waxed using organic solvents (xylene, 100%, four changes) and an alcohol series (100%, 70%, 30% ethanol) descending to distilled water to sufficiently hydrate the tissues and allow proper binding of the primary antibody and other detection reagents.
  • organic solvents xylene, 100%, four changes
  • alcohol series 100%, 70%, 30% ethanol
  • antigen retrieval is performed after tissue sections are dewaxed.
  • This step uses a steam heat-indiJced epitope recovery (SHIER) solution (Tris-EDTA, pH 9) that is drawn into the capillary gap formed between paired microscope slides with a commercial steamer or a pressure cooker (20 minutes above 97°C) as a heat source (for description retrieval with heat and SHIER solutions does not contribute to antigen unmasking and is not used (referred to as NO SHIER).
  • SHIER steam heat-indiJced epitope recovery
  • Sequential detection of antibodies, a primary antibody and a detection antibody (or a secondary antibody) are employed during IHC.
  • the location of the primary antibody is ultimately visualized by applying a colorimetric chromogen (DAB) that precipitates a discrete insoluble reaction product at the site of antigen in the presence of horseradish peroxidase (HRP).
  • DAB colorimetric chromogen
  • Nuclei are counterstained using hematoxylin (blue stain) to assess cell and tissue morphology.
  • slides are unpaired, rinsed in distilled water, dehydrated in an alcohol series (70%, 95%, 100% ethanol) and inorganic solvent (xylene, 100%, four changes), then permanently coverslipped, using CytoSeal (or equivalent), for interpretation and storage. Slides are examined under a microscope to assess staining.
  • Reagent Manufacturing Buffer (Discovery Life Sciences, Santa Barbara, CA) with goat serum is used to prepare working dilutions of primary antibody and mouse isotype control serum.
  • Target recognition for the primary antibodies at the site of antigen-primary antibody interaction in FFPE sections uses reagents from Polink-2 Plus HRP kits (GBI Labs, Bothell, WA).
  • Percent scores are calculated by summing the percentages of intensities at either >1+, >2+, or >3+ Thus, scores range from 0 to 100.
  • the H-scores are calculated by summing the percentages of cells with the intensity of expression multiplied by their corresponding differential intensity all of four points; dictate if scale (0, 1+, 2+, 3+). Thus, the scores range from 0 to 300.
  • H-score [(% at ⁇ 1) x 0] + [(1% at 1+) xl] + [(% at 2+)x2]+ [(% at 3+) x3]
  • FIG. 1A-1C shows the images of IHC staining using the mATRClOl N297A antibody of the ATRC-101 target results corresponding to H-scores of 0, 30, and 180, respectively.
  • the image in FIG. 1 A, where H-score 0, showed a faint amount of non-specific staining.
  • MATRClOl N297A IS SENSITIVE IN DETECTING ATRC-101 TARGET [0124]
  • Tumor tissue sections from various tumor types were stained with mATRClOl N297A or mATRClOl in IHC, as described above.
  • mATRClOl showed higher binding to the same tissue section as compared to the mATRC across all tumor types.
  • H-scores were used to represent signal intensity.
  • Table 10 Average Tumor H-Scores [0125] When comparing the mATRClOl (original) and mATRClOl N297A (mutant) sensitivity scoring data, mATRClOl N297A showed a broader range of staining with higher H- Score averages by cancer indication. An array of negative, low, moderate, and high cases was observed for mATRClOl N297A, with H-Scores ranging from 0 to 270 across the 142 tumors. Further sensitivity analysis and validation used only mATRClOl N297A, as shown in Examples 3 and 4 below.
  • This example describes using mATRClOl N297A in automatic IHC staining assays performed on LEICA BOND-III Instrument to detect the ATRC-101 target.
  • H-Score >10 cut-off for mATRClOl (N297A) the 95% Cl was 100.0% ⁇ 0.0%.
  • the lower bound of these values is above 85%, demonstrating acceptable assay concordance for mATRClOl (N297A) using the TechMate IHC assay and the Leica IHC assay.
  • mATRClOl N297A was prepared using Therm oFi sher BSA Block and Diluent (Cat #: 003218 Ready -to-use diluent with BSA for stabilizing the antibody and preservative). The same antibody diluent was also used to prepare working dilutions of the species-matched standard control antibody, and isotype-matched negative control (Mouse IgG2a).
  • the Leica BOND Polymer Refine Detection Kit (Cat #: DS9800) was used for mATRClOl N297A target recognition and binding at the site of antigen-primary antibody interaction in FFPE sections. Samples were processed using a Leica BOND-III Automated IHC Stainer platform by selecting Leica validated IHC Protocol F.
  • the Leica BOND-III Instrument (Leica Biosystems) is a fully automated IHC commercial staining platform designed to perform clinical IHC tests using pre-programmed, and user validated programmed staining protocols.
  • the instrument is controlled using Leica’ s BOND software.
  • This automated platform uses a capillary gap process for all reagent changes, including antibody incubation, detection steps up to and including counterstaining, and intervening washes. All procedures were carried out at room temperature ( ⁇ 25°C) unless otherwise specified.
  • the BOND-III instalment software assigned a unique barcode identifier to each tissue slide during the slide set up process that is encoded with the staining protocol.
  • the instrument Prior to each staining run, the instrument performed an operation verification by scanning each unique barcode to ensure adequate reagent availability for staining completion of protocols assigned to all tissue slides loaded onto the instrument. Each staining run was logged into an internal database file on the operating desktop for all tests performed on each Leica BOND-III instrument. For each slide, the instrument retained a time-stamped record of all operations performed and any errors encountered during the staining procedure are retained for each tissue slide.
  • FFPE tissue specimens were prepared less than 30 minutes after the biopsy tissue is removed from the patient prior to immersion in fixative, and the specimens were subject to 24- 48 hours fixation time in 10% neutral buffered formalin. Tissue specimens were cut into sections of 4-5 pm and mounted onto positively-charged glass. Unstained slides were placed in the dark at 2-8 °C for long term storage.
  • Antigen retrieval was performed using an enzymatic epitope retrieval (Dako Proteinase K, S3020, diluted to 1:120 in TBST) for 10 minutes at 37°C on the Leica BOND-III platform, followed by three rinses of BOND wash solution.
  • IHC was performed following the Customer Specific Protocol IHCS6-84 for mATRClOl N297A.
  • Peroxide Block part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800
  • the primary antibody was detected by incubating the slides with Post-Primary reagent (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 8 minutes, rinsing the slides three times with BOND wash solution, and then incubated with Polymer (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 8 minutes. Following two washes with BOND wash solution and one deionized water rinse, mATRClOl N297A was visualized by incubating in DAB chromogen (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 10 minutes. Slides were counterstained with hematoxylin (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 5 minutes, rinsed with deionized water with an intervening BOND wash solution step.
  • Post-Primary reagent part of the Leica BOND Polymer Re
  • mATRClOl N297A is reactive in a subset of tumor cells where it primarily localizes to the cytoplasm.
  • H&E hematoxylin and eosin
  • mATRClOl N297A staining was scored semi-quantitatively by a board-certified pathologist for diffuse cytoplasmic staining in tumor cells.
  • the main components to scoring ribonucleoprotein(s) expression in tumor cells include percentages at differential intensities to determine H-Score (described below).
  • H-Score H-Score
  • numeric scoring excludes any surrounding staining in stroma, areas of non-tumor, and adjacent normal tissue. Ischemic or necrotic areas are not scored in any cancer indication. Percentages of cells expressing ribonucleoprotein(s) are captured at each intensity (brown DAB (3, 3’-diaminobenzidine staining) on a four-point semi-quantitative scale:
  • the percentage at each intensity was estimated directly and reported as one of the following: 0, 1, 2, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%.
  • the H-Score was calculated by summing the percentage of cells with intensity of expression (brown staining) multiplied by their corresponding differential intensity on a four-point semi- quantitative scale (0, 1+, 2+, 3+), with the H-scores in a range from 0 to 300.
  • H-Score [ (% at ⁇ 1) x 0] + [ (% at 1+) x 1] + [ (% at 2+) x 2] + [ (% at 3+) x 3]
  • Tumor tissue samples are defined as positive if mATRClOl N297A tumor cytoplasmic H-score> 50.
  • AUC area under the curve
  • H-score H-score
  • the assay was used retrospectively to date, in Clinical Trial ATRC-101 A01(NCT04244552), conducted under IND 142779. IHC cut-off H-score >50 was chosen as the initial threshold for eligibility with the objectives of minimizing the false negative rate and gaining more precision around the optimal threshold.
  • Intra-assay consistency (precision) and inter-assay consistency (reproducibility) were determined using a 3 -run series with 3 replicate sections (per run) of each of the 5 selected samples in five indications for mATRClOl (N297A), resulting in a set of 9 replicates for each sample, and 45 total replicates per indication.
  • Two CLIA-certified operators ran the assays using different Leica BOND-III instruments (Operator 1, Run 1; Operator 2, Run 2; Operator 2, Run 3). Positive standard and negative controls were included in each run and reacted as expected.
  • H-Scores were low ( ⁇ 50), differences between replicates translated into higher CV values by nature of the test. In such cases, the sample can be considered acceptable at the pathologist’s discretion by considering all data points.
  • ATRC-101 Phase lb trial is a first-in-human, open label study of ATRC-101 in patients with selected solid tumor cancers. Enrollment is limited to patients with tumor types reactive to ATRC-101 in over 50% of patient samples evaluated preclinically, which includes non-small cell lung, breast, ovarian, colorectal, and acral melanoma.
  • Tumor lymphocyte infiltration and other potential biomarkes of activity in tumors, plasms, and PBMCs were analysed.
  • Some of the objectives of the Phase lb trial include determining Patients’ maximum tolerated dose (MTD), determining recommended phase 2 dose (RP2D), and characterizing safety profile of the antibody.
  • Tumor response evaluation include determining Patients’ maximum tolerated dose (MTD), determining recommended phase 2 dose (RP2D), and characterizing safety profile of the antibody.
  • the overall tumor burden was evaluated at the baseline, i.e., before beginning treatment, and at a subsequent time in point, e.g ., more than 28 days from the initiation of the treatment, according to Response Evaluation Criteria in Solid Tumours (RECIST): standard as discussed in Eisenhauer, et al ., European Journal of Cancer 45 (2009) 228-247, the relevant disclosure is herein incorporated by reference.
  • RECIST Response Evaluation Criteria in Solid Tumours
  • “Complete Response” or “CR” refer to the disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to ⁇ 10 mm.
  • Partial Response refers to that at least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.
  • Progressive Disease or “PD”: At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is thesmallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions).
  • Non-Progressive Disease or “Non-PD” refers to a combination of Stable Disease, Partial, and Complete Responses.
  • a patient is considered as having Best Overall Response (BOR) if having any response to the treatment of ATRC-101 assessed, for example if the patient is assessed with a CR, PR, SD, or PD. Evaluation of nontarget lesions
  • CR Complete Response
  • Non-CR/Non-PD Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.
  • Progressive Disease Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of “non-target” lesions only is exceptional, the opinion of thetreating physician should prevail in such circumstances, and the progression status should be confirmed at a later time.
  • a patient is considered as having Best Overall Response (BOR) if having any response to the treatment of ATRC-101 assessed, for example if the patient is assessed with a CR, PR, SD, or PD.
  • BOR Best Overall Response
  • Results shown in FIG. 3 A-B indicate that eight (8) of the 20 patients (40%) evaluable experienced stable disease (SD) as their best RECIST response, four (4) of which showed tumor reduction, with the remaining patients having progressive disease (PD).
  • Eighteen (18) clinical trial participants with a variety of tumor types, including breast, colorectal, non-small cell lung, acral melanoma, and ovarian tumors were analyzed for target expression using the assay described in Example 1. These patients were grouped and received ATRC-101 at doses as indicated. The results indicate that expression levels of ATRC-101 target in patients with different tumor types were highly variable.
  • Example 3 The IHC assay as described in Example 3 was used to investigate target expression associated with response to treatment with ATRC-101. Forty-two (42) RECIST-evaluable patients with H-scores determined at screening were evaluated for association of H-scores to response to treatment with ATRC-101. Results of the analysis showed that fourteen (14) of the twenty-three (23) patients (61%) having an H-score of > 50 experienced non-progressive disease. FIG. 2A.
  • SOD sum of diameters
  • SOD was calculated according to RECIST1.1 and is described in the Eisenhauer et al (2009). In brief, up to 5 target lesions are identified per patient, but no more than 2 per organ. The longest diameters for each target lesion (with the notable exception of lymph node target lesions) are summed. For lymph nodes, the shortest diameter is used.
  • the change in SOD at timepoint x is SOD(x)-SOD (baseline)/SOD (baseline). Baseline is from the last pretreatment scan.
  • FIG. 5A show that the majority of twenty (20) patients with H-scores >50 (target-positive) exhibited a decrease in SOD during the course of the treatment.
  • FIG. 5B shows durable responses observed in twelve (12) of the patients carrying different tumor type, including patients with ovarian cancer, non-small cell lung carcinoma, colorectal cancer, breast cancer, head and neck cancer, and melanoma.
  • mice with established EMT6 tumors were dosed with mATRClOl (1 or 3 mg/kg) or vehicle intraperitoneally (IP) twice weekly plus Doxorubicin (2 or 5 mg/Kg) or vehicle (saline) IV once weekly following randomization on Day 6.
  • IP vehicle intraperitoneally
  • Doxorubicin (2 or 5 mg/Kg) or vehicle (saline) IV once weekly following randomization on Day 6.
  • Statistical analyses of tumor volumes were performed using the normalized area above the curve and the normalized growth rate metrics developed at Atreca.
  • One-sided log-rank (Mantel-Cox) test was used to assess survival advantage relative to the indicated reference group. P-values ⁇ 0.05 were considered significant.
  • EMT6 tumor and non-tumor bearing mice were dosed with vehicle or Doxorubicin (2 or 10 mg/kg) or PLD (1 ,2, 5 and 10 mg/kg).
  • Normal mouse tissues were collected at 24 hours and 2 weeks after the last dose. Reactivity for mATRClOl in EMT6 tumor and normal mouse tissues was evaluated by immunohi stochemi stry .
  • the increase in immunoreactively is positively related to the dosage of the PLD, i.e., the higher the PLD dosage, the higher the immunoreactively to mATRC- 101

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Abstract

Disclosed herein are methods and compositions for selecting patients that are likely to respond to treatment with ATRC-101. Also disclosed are methods and compositions that are used to evaluate the efficacy of ATRC-101 in treating patients. The methods and compositions can be used for diagnosis, prognosis and determining the optimal treatment plans for cancer patients.

Description

ATRC-101 TARGET EXPRESSION ASSAY
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 63/203,711, filed July 28, 2021, and U.S. Provisional Application No. 63/277,073, filed November 8, 2021. The entire contents of said provisional applications are herein incorporated by reference for all purposes.
FIELD OF CANCER THERAPEUTICS
[0002] This application relates to methods and compositions for selecting patients for treatment with the ATRC-101 antibody.
BRIEF SUMMARY
[0003] In one aspect, disclosed herein is a method of determining whether a subject with a tumor is likely to respond to treatment with ATRC-101, the method comprising: contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises a heavy chain variable region comprising: an HCDR1 comprising a sequence (SEQ ID NO:l); an HCDR2 comprising a sequence (SEQ ID NO:2); and an HCDR3 comprising a sequence (SEQ ID NO:3). The antibody further comprises a light chain variable region comprising: an LCDR1 comprising a sequence (SEQ ID NO:4); an LCDR2 comprising a sequence (SEQ ID NO:5); and an LCDR3 comprising a sequence (SEQ ID NO:6), wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain. The method further comprises detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay and determining that the subject will be responsive to treatment of ATRC-101 if the binding of the antibody as determined by the immunohistochemistry assay corresponds to an H-score that is equal to or greater than an H-score cutoff. [0004] In some embodiments, the method further comprises selecting the subject for treatment with ATRC-101 if the subject is determined to be responsive to treatment of ATRC-101
[0005] In yet another aspect, disclosed herein is a method of selecting a subject for treatment with ATRC-101. the method comprising detecting binding of an antibody with a tumor sample derived from the subject in an immunohistochemistry assay, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3. The antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO: 5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain. The method further comprises determining an H-score that corresponds to the binding and selecting the subject for treatment with ATRC101 if the H-score that is equal to or greater than an H-score cutoff.
[0006] In yet another aspect, disclosed herein is a method of treating a tumor with ATRC-101, the method comprising selecting a subject with the tumor suitable for treatment with ATRC101, wherein the binding of an antibody to a target of ATRC-101 in a tumor sample obtained from the subject corresponds to an H-score that is equal to or greater than an H-score cutoff. The antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3. The antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain. The method further comprises administering an effective amount of ATRC-101 to the subject.
[0007] In yet another aspect, disclosed herein is a method of detecting expression of a target of ATRC-101 in a tumor of a subject, the method comprising contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3. The antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain. The method further comprises detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, thereby detecting expression of the target of ATRC-101.
[0008] In some embodiments, the H-score cutoff is selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the H-score cutoff is 50.
[0009] In some embodiments, the antibody has a heavy chain variable region comprising a sequence SEQ ID NO: 19 and a light chain variable region comprising a sequence SEQ ID NO: 20, In some embodiments, the antibody has an Fc region comprising SEQ ID NO: 24, or wherein the antibody has an Fc region comprising a sequence that is at least 80% identical to SEQ ID NO: 24, provided that the sequence comprises a N297A mutation in the heavy chain.
[0010] In another aspect, disclosed herein is a method of determining whether a subject with a tumor is likely to respond to treatment with ATRC-101, wherein the method comprises contacting a tumor sample obtained from the subject with the antibody described above, detecting the binding of the antibody to the tumor sample in an immunoassay, and predicting the subject is responsive to the treatment with ATRC-101 if the binding of the antibody corresponds to an H-score that is greater than an H-score cutoff. In some embodiments, the H-score cutoff is selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the tumor is selected from the group consisting of non-small cell lung, breast, ovarian, colorectal, acral melanoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, and urothelial carcinoma.
[0011] In some embodiments, the method further comprises administering an effective amount of ATRC-101 to the subject. In some embodiments, the tumor is selected from the group consisting of non-small cell lung carcinoma, breast cancer, ovarian cancer, colorectal cancer, and acral melanoma. In some embodiments, the method further comprises administering an effective amount of pembrolizumab. In some embodiments, the method further comprises administering an effective amount of Doxorubicin or pegylated liposomal Doxorubicin,
[0012] In yet another aspect, disclosed herein is a kit used to identify a subject that is likely to respond to treatment with ATRC-101, wherein the kit comprises the antibody disclosed above. In some embodiments, the antibody comprises a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3. The antibody further comprises a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain. In some embodients, said kit may further comprise one or more reagents used in an assay ( e.g ., an immunohistochemistry assay) to detect the binding of the antibody to a tumor sample from the subject. In some embodiments, said one or more additional assay reagents provided in one or more vials, containers, or compartments of said kit. In some embodiments, the kit further comprises an isotype-matched negative control (e.g., Mouse IgG2a).
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A-1C shows examples of baseline IHC staining of the target of ATRC-101 using mATRClOl N297A antibody in three tumor samples. The IHC results in FIG. 1A-1C were determined to have H-scores of 0, 30, and 80, respectively.
[0014] FIG 2A shows best RECIST response in patients treated with ATRC-101 is associated with ATRC-101 target expression as measured by H-score. The study population is defined as individuals with valid IHC H-score data at screening and the best overall response according to RECIST 1.1 criteria. The p-value of p=0.011 reported is calculated from the exact Wilcoxon rank sum test for comparing the IHC H-score distribution at screening among the two levels: progressive disease (PD) and non-progressive disease (Non-PD) of response. FIG. 2B is a Kaplan- Meier curve showing the probability of Progression Free Survival (PFS) is associated with ATRC- 101 target expression as measured by H-score in patients treated with ATRC-101 administered as a monotherapy Q3W.
[0015] FIG. 3A-B shows the results of patients who have received ATRC-101 at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg (FIG. 3A). Stable disease (SD) was observed in eight (8) patients, four (4) of which showed tumor reduction (FIG. 3B).
[0016] FIG. 4 shows the baseline characteristics, including the H-score of IHC results, of 18 patients at baseline. The patients have a variety of tumor types, including breast, colorectal, non small cell lung, acral melanoma, and ovarian tumors. These patients were grouped and received ATRC-101 at doses as indicated. The results indicate that expression levels of ATRC-101 target in patients with different tumor types were highly variable.
[0017] FIG. 5A shows target lesion response in target-positive tumor patients treated with ATRC-101 or ATRC-101 and pembrolizumab over time, as reflected in a change in summary of diameters (SOD). Target-positive patients have tumors with H-scores >50 when analyzed using the methods disclosed herein.
FIG. 5B shows durable responses observed in 12 patients with H-scores of > 50 carrying different tumor types. Abbreviations: OVC: ovarian cancer; NSCLC non-small cell lung carcinoma; CRC: colorectal cancer; BRC: breast cancer; SmB, HNC: head and neck cancer; and MEL: melanoma.
[0018] FIG. 6 shows tumor response in target-positive NSCLS patients treated with ATRC-101 as Q3W monotherapy at either 3 mg/kg (©) or 30 mg. kg (®) over time. The results showed that 60% of NSCLC patients with an H-score >50 demonstrate Stable Disease or Partial Response as a best overall response.
[0019] FIG. 7A-B show the ATRC-101 clinical trial design for monotherapy (FIG. 7A) and combination therapies (FIG. 7B).
[0020] FIG. 8A-8E show the results of tumor growth of BALB/c mice with established EMT6 tumors after various monotherapy or combination therapy as indicated. In these studies, mATRClOl, if used, was administered intraperitoneally twice weekly. Doxorubicin (Dox), if used, was administered intravenously once weekly. FIG. 8 A shows the tumor growth measurements from mice administered with i) mATRC at 3 mg/kg (“3 mpk”) or ii) PBS. FIG. 8B shows tumor growth measurements from mice administered with i) Dox at 5 mg/kg or ii) PBS. FIG. 8C shows tumor growth measurements from mice administered with i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) PBS as a control. FIG. 8D shows tumor growth measurements from mice treated with either i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) Dox at 5mg/kg. FIG. 8E shows tumor growth measurements from mice treated with either i) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg or ii) mATRClOl at 3 mg/kg.
[0021] FIG. 9 shows the probability of survival of BALB/c mice with established EMT6 tumors after undergoing various monotherapy or combination therapy: i) Dox at 5mg/kg, ii) mATRClOl antibody at 3 mg/kg, or iii) Dox at 5 mg/kg in combination with mATRClOl at 3 mg/kg. Mice administered with PBS were included as controls.
[0022] FIG. 10A-10D show immunohistochemical analyses of mATRClOl reactivity in EMT6 tumors from mice treated with Pegylated Doxil (PLD). FIG. 10A and 10B show the immunohistochemistry staining performed on the tumors in mice treated with PBS, lmg/kg PLD, 2 mg/kg PLD, or 10 mg/kg PLD. The stainings were performed using mATRClOl or an isotype control antibody. FIG. IOC and 10D show the results of immunohistochemistry stainings performed on tumors from mice treated with PLD at indicated doses, i.e., 2 mg/kg, 5 mg/kg, or 10 mg/kg. FIG. 10C and FIG. 10D show the results of immunohistochemistry stainings performed on the tumor samples at one week and four weeks, respectively, after the mice received the first dose of PLD.
[0023] FIG. 11 shows the distribution of the H-scores in the corresponding CV value for each set of replicates (n=9) of IHC assays for tumor samples derived from a variety of tumors using mATRClOl N297A. The graph is plotted using data shown in Table 11 DETAILED DESCRIPTION TERMINOLOGY
[0024] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules and the like.
[0025] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field; for example, ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.
[0026] As used herein, “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g ., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g, bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.
[0027] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g, Zapata el al, Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g, scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site and a residual “Fc” fragment, reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment with two antigen combining sites and is still capable of cross-linking antigen.
[0028] A “tumor-binding antibody” as used herein refers to an antibody that binds tumor tissue. In one embodiment, the tumor-binding antibody binds the tumor tissue through a binding interaction with an extracellular RNA-protein complex. “Extracellular RNA-protein complex” refers to a complex that is detected on the outside of tumor cells. The complex need not be integrated into the external surface of the cells, but in some embodiments, it may be associated with the outside of the tumor cells as a conglomeration or aggregate of RNA and protein molecules interacting with the cell membrane or otherwise present outside of the tumor cell. Although the extracellular RNA-protein complex is external to tumor cells, it may additionally be present internally in a cell. A “tumor-binding antibody” of the present disclosure exhibits preferential binding to tumor tissue compared to tumor-adjacent tissue (TAT), e.g., a specific signal above the noise is noted with enhanced reactivity apparent in tumor vs. adjacent tissues. In some embodiments, the tumor-binding antibody is an “anti-tumor antibody” that decreases tumor growth rate, tumor size, invasion, and/or metastasis via direct or indirect effects on tumor cells.
[0029] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4. The heavy chain V-region, VH, is a consequence of the rearrangement of a V-gene (HV), a D-gene (HD), and a J-gene (HJ), in what is termed V(D)J recombination during B-cell differentiation. The light chain V-region, VL, is a consequence of the rearrangement of a V-gene (LV) and a J-gene (LJ).
[0030] As used herein, “complementarity-determining region (CDR)” refers to the three hypervariable regions (HVRs) in each chain that interrupt the four “framework” regions established by the light and heavy chain variable regions. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the particular CDR is located. Thus, for example, a VH CDR3 (HCDR3) is located in the variable domain of the heavy chain of the antibody in which it is found. In contrast, a VL CDR3 (LCDR3) is the CDR3 from the variable domain of the light chain of the antibody in which it is located. The term “CDR” is used interchangeably with “HVR” in this application when referring to CDR sequences. [0031] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g. , Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g. , Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al, 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. etal. , 1992, the structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani etal. , J. Mol. Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al ., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al. , Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al, Methods Enzymok, 203, 121— 153, (1991); Pedersen etal. , Immunomethods, 1, 126, (1992); and Rees etal. , In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs as determined by Kabat numbering is based, for example, on Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0032] An “Fc region” refers to the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, e.g, for human immunoglobulins, “Fc” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy. It is understood in the art that the boundaries of the Fc region may vary; however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering according to the EU index as in Kabat et al , (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The term “Fc region” may refer to this region in isolation or in the context of an antibody or antibody fragment. “Fc region” includes naturally occurring allelic variants of the Fc region and modified Fc regions, e.g., that are modified to modulate effector function or other properties such as pharmacokinetics, stability, or production properties of an antibody. Fc regions also include variants that do not exhibit alterations in biological function. For example, one or more amino acids can be deleted from the N-terminus or C- terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie et al, Science 247:306-1310, 1990). For example, for IgG4 antibodies, a single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibodies (see, e.g., Angal et al., Mol Immunol 30:105-108, 1993).
[0033] An “EC50 as used herein in the context of an Fc receptor engagement assay, refers to the half maximal effective concentration, which is the concentration of an antibody that induces a response (signal generated in engagement assay) halfway between the baseline and maximum after a specified exposure time. Fc receptor engagement assays are further described herein in the “Variant Binding Activity” section. In some embodiments, the “fold over EC50” is determined by dividing the EC50 of a reference antibody by the EC50 of the test antibody.
[0034] The term “monovalent molecule” herein refers to a molecule with one antigen-binding site, e.g, a Fab or scFv.
[0035] The term “bivalent molecule” herein refers to a molecule with two antigen-binding sites. In some embodiments, a bivalent molecule of the present invention is a bivalent antibody or a bivalent fragment thereof. In some embodiments, a bivalent molecule of the present invention is a bivalent antibody. In some embodiments, a bivalent molecule of the present invention is an IgG. In general, monoclonal antibodies have a bivalent basic structure. IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and thus have higher valencies. This bivalency increases the avidity of antibodies for antigens. [0036] The terms “monovalent binding” or “monovalently binds to” as used herein refer to the binding of one antigen-binding site to its antigen.
[0037] The terms “bivalent binding” or “bivalently binds to” as used herein refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. In some embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.
[0038] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (e.g, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, e.g, the length of the two sequences, when compared and aligned for maximum correspondence over a comparison window or designated region. Alignment for determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST 2.0 algorithms, described in Altschul etal. , Nuc. Acids Res. 25:3389-3402 (1977) and Altschul etal., J. Mol. Biol. 215:403-410 (1990). Thus, for purposes of this invention, BLAST 2.0 can be used with the default parameters to determine percent sequence identity.
[0039] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a VH region polypeptide “corresponds to” amino acid in the VH region of SEQ ID NO: 19 when the residue aligns with the amino acid in SEQ ID NO: 19 when optimally aligned to SEQ ID NO: 19. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence. [0040] A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and/or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at a pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Val, Leu and lie; (vi) hydrophobic sulfur-containing amino acids Met and Cys, which are not as hydrophobic as Val, Leu, and lie; (vii) small polar uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide-comprising amino acids Asn and Gin; and (xi) beta-branched amino acids Thr, Val, and He. Reference to the charge of an amino acid in this paragraph refers to the charge at pH 6-7.
[0041] The terms “nucleic acid” and “polynucleotide” are used interchangeably and, as used herein, refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide and combinations thereof. The terms also include, but are not limited to, single- and double-stranded forms of DNA. In addition, a polynucleotide, e.g ., a cDNA or mRNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, the substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, and the like), charged linkages (e.g, phosphorothioates, phosphorodithioates, and the like), pendent moieties (e.g, polypeptides), intercalators (e.g, acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g, alpha anomeric nucleic acids, and the like). The above term also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hair pinned, circular, and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.
[0042] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure and the vector incorporated into the host cell genome as into which it has been introduced. A “vector” refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
[0043] A “substitution,” as used herein, denotes replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.
[0044] An “isolated” nucleic acid refers to a nucleic acid molecule separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule. Still, the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
[0045] “Isolated nucleic acid encoding an antibody or fragment thereof’ refers to one or more nucleic acid molecules encoding antibody heavy or light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
[0046] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cell and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages. [0047] A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions, and/or insertions.
[0048] The term “cancer cell” or “tumor cell” as used herein refers to a neoplastic cell. The term includes cells from tumors that are benign as well as malignant. Neoplastic transformation is associated with phenotypic changes of the tumor cell relative to the cell type from which it is derived. The changes can include loss of contact inhibition, morphological changes, and unregulated cell growth,
[0049] “Inhibiting growth of a tumor” and “inhibiting growth of cancer” as used herein are interchangeable and refer to slowing growth and/or reducing the cancer cell burden of a cancer patient. “Inhibiting growth of cancer” thus includes killing cancer cells, as well as decreasing the rate of tumor growth, tumor size, invasion, and/or metastasis by direct or indirect effects on tumor cells.
[0050] As used herein, “therapeutic agent” refers to an agent that, when administered to a patient suffering from a disease, in a therapeutically effective dose, will cure or at least partially arrest the symptoms of the disease and complications associated with the disease.
[0051] As used herein, “treatment,” or “therapy,” and other forms of these words refer to the administration of an agent to impede a disease, such as the growth of cancer, to cause a cancer tumor to shrink as determined by measuring at least one dimension of the tumor, to extend the expected survival time of the subj ect and/or time to progression of the tumor or the like. Treatment may also refer to any course that one skilled, such as a treating physician, deems expedient.
[0052] As used herein, “Sample” or “tissue sample” or “patient sample” or “patient cell or tissue sample” or “specimen” each refer to a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue sample may be solid tissue from fresh tissue, frozen and/or preserved organ or tissue or biopsy or aspirate; blood or any blood constituents, bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid or cells from any time in gestation or development of the subject. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Cells may be fixed in a conventional manner, such as in an FFPE manner.
[0053] As used herein, “RECIST” or “Response Evaluation Criteria in Solid Tumours” refers to a set of published rules that define when cancer patients improve (“respond”), stay the same (“stable”), or worsen (“progression”) during treatments. Response, as defined by RECIST 1.1 criteria, have been published, for example, Eisenhauer et al ., European Journal of Cancer 45 (2009) 228-247.
[0054] As used herein, “Respond” to treatment, and other forms of this verb, as used herein, refer to the reaction of a subject to treatment with an agent. As an example, a subject responds to treatment if growth of a tumor in the subject is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In another example, a subject responds to treatment if a tumor in the subject shrinks by about 5%, 10%, 20%, 30%, 40%, 50%, or more as determined by any appropriate measure, e.g ., by mass or volume. Tumor size can be measured, for example, by radiologic measurements. In another example, a subject responds to treatment with an anti -turn or antibody disclosed herein if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50%, or more beyond the life expectancy predicted if no treatment is administered. In another example, a subject responds to treatment with an agent if the subject has an increased overall survival or increased time to progression. In some cases, the subject responds to treatment with an agent if it demonstrates stable disease as opposed to progressive disease in the absence of the treatment. Several methods may be used to determine if a patient responds to treatment, including the RECIST criteria, as set forth herein. In one embodiment, the threshold for a response is a reduction in the sum of diameters >=30% relative to baseline as per RECISTvl.l.
[0055] The term “likely to respond to treatment with ATRC-101” means that a patient will likely have a Best Overall Response, for example, a CR, PR, SD, or PD, after receiving treatment of ATRC-101. In general, a patient with an H-score, tumor proportion score, or intensity score that is greater than or equal to a cutoff score as disclosed herein is more likely to respond to treatment than a patient with an H-score, tumor proportion score, or intensity score that less than the cutoff score. In some embodiments, the H-score, tumor proportion score, or intensity score is determined based on the binding of an ATRC-101 diagnostic antibody disclosed herein to a tumor sample derived from the patient in an immunohistochemistry assay.
[0056] As used herein, “a cutoff,” as in, e.g ., “an H-score cutoff,” refers to the value of a predetermined measure on subjects exhibiting certain attributes that allow the best discrimination between two or more categories of an attribute. For example, a cutoff that will enable one to discriminate between two categories, such as target expression and low target expression, may be used to determine the suitability for the ATRC-101 treatment for. These cutoffs may be used to separate the subjects with values lower than or higher than the cutoff to evaluate the treatment plan for the patient.
[0057] As used herein, the term “effective amount” refers to an amount of a pharmacological agent effective in treating, eliminating, or mitigating at least one symptom of the disease being treated. In some cases, the effective amount can be an amount effective to invoke an anti-tumor response is to evoke a humoral and/or cellular immune response in the recipient subject leading to growth inhibition or death of target tumor cells or reduce tumor load or bring about other desired beneficial clinical outcomes related to cancer improvement.
[0058] The term “baseline” herein refers to the disease status before a patient has been treated with ATRC-101. Accordingly, the term “baseline H-score” refers to the H-score determined for a tumor sample obtained from the patient prior to ATRC-101 treatment.
ATRC-101
[0059] Disclosed herein are methods and compositions for selecting patients likely to respond to the treatment with ATRC-101. Also disclosed are methods and compositions used to evaluate the efficacy of ATRC-101 in treating patients. The methods and compositions can be used for diagnosis, prognosis, and determining the optimal treatment plans for cancer patients.
[0060] ATRC-101 is a fully human immunoglobulin G, subclass 1 (IgGl)/lambda monoclonal antibody that is an engineered version of an antibody expressed by a plasmablast B cell that was originally isolated from a patient with NSCLC adenocarcinoma undergoing treatment with a checkpoint inhibitor. The parental antibody's variable fragment (Fv) region was optimized to generate ATRC- 101.
[0061] ATRC-101 targets a complex containing human polyadenylate-binding protein family member(s) and polyadenylated ribonucleic acid (poly(A) RNA) (“RNP complex”). WO 2020/168231, the entire content of which is herein incorporated by reference. Although poly(A) RNA and polyadenylate-binding protein family members are widely expressed across normal human tissues, ATRC-101 binds highly selectively to tumor cells. ATRC-101 does not show any appreciable binding to normal human tissues. This indicates that the ATRC-101 target is a tumor- associated version of a complex containing a polyadenylate-binding protein family member and poly(A) RNA.
[0062] In preclinical proof of concept studies, a chimeric version of ATRC-101 harboring the mouse IgG2a Fc region (AB6042-mIgG2a) demonstrated significant anti-tumor activity in syngeneic mouse models of breast carcinoma and colon carcinoma. Based on preclinical observations, ATRC-101 is proposed to cause tumor growth inhibition and regression, in part, by (a) altering the composition of the tumor microenvironment to be more anti-tumorigenic and proinflammatory and (b) inducing an adaptive immune response that recruits effector CD8+ T cells to attack the tumors. Consistent with that model, administration of low-dose AB6042- mIgG2a enhanced the antitumor activity of anti -mouse PD-1 in two syngeneic models of breast carcinoma.
[0063] ATRC-101 is an engineered version of an antibody identified by IRC® as disclosed in, e.g., WO 2012148497A2, the entire content of which is herein incorporated by reference. ATRC- 101 is being evaluated as monotherapy or combination therapy (e.g, with Pembrolizumab) for the treatment of patients with advanced solid malignancies, including breast cancer (BC), non small cell lung cancer (NSCLC), colorectal cancer (CRC), ovarian cancer, acral melanoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, and urothelial carcinoma. [0064] Tables 1-5 below show the ATRC-101 sequences. Table 1 shows the heavy and light chain variable region sequences, Table 2 shows the heavy chain and the light chain sequences, and Tables 3-5 show the CDR sequences. The VH CDRS, as listed in Table 3, are defined as follows: HCDR1 is defined by combining Rabat and IMGT; HCDR2 is defined by Rabat, and the HCDR3 is defined by IMGT. The VL CDRS, as listed in Table 3, are defined by Rabat. As known in the art, the numbering and placement of the CDRs can differ depending on the numbering system employed. It is understood that disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated CDRs, regardless of the numbering system employed. Table 4 shows the CDR sequences defined using the IMGT numbering system, and Table 5 shows the CDR sequences defined using the Rabat numbering system.
Table 1 VH and VL
Figure imgf000019_0001
Table 2 Heavy and Light Chain
Figure imgf000019_0002
Figure imgf000020_0004
Table 3 CDRs
Figure imgf000020_0001
Table 4 CDRs as defined using the IMGT numbering system
Figure imgf000020_0002
Table 5 CDRs as defined using the Rabat numbering system
Figure imgf000020_0003
DIAGNOSTIC ANTIBODY
[0065] In one aspect, provided herein are antibodies that can be used to detect the expression of the levels of an ATRC-101 target, determine whether a subject with a tumor is likely to respond to treatment of the ATRC-101 therapeutic antibody disclosed above, and/or select a subject for treatment with the ATRC-101 therapeutic antibody. These antibodies are referred to as ATRC- 101 diagnostic antibodies in this application. An ATRC-101 diagnostic antibody can have the same sequence structures as the corresponding ATRC-101 therapeutic antibody or different sequence structures; that is, an ATRC-101 diagnostic antibody may also have a therapeutic effect. In some embodiments, an ATRC-101 diagnostic antibody has the same antigen-binding sequences ( e.g ., the same VH and VL sequences) as the corresponding ATRC-101 therapeutic antibody but has a different isotype. In some embodiments, the ATRC-101 diagnostic antibody is a mouse IgG2a with a mutation at the N297 position in the heavy chain, such as N297A, N297G, or N297Q.
[0066] In some embodiments, an ATRC-101 diagnostic antibody of the present invention has one, two, or three CDRs of a VH sequence, as shown in Table 1. In some embodiments, the ATRC-101 diagnostic antibody has at least one mutation and no more than 10, 20, 30, 40, or 50 mutations in the VH amino acid sequences compared to a VH sequence set forth in any one of SEQ ID NOS: 19. In some embodiments, the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in any one of SEQ ID NOS: 19.
[0067] In some embodiments, an ATRC-101 diagnostic antibody of the present invention has one, two, or three CDRs of a VL sequence, as shown in Table 1. In some embodiments, the tumor binding antibody has at least one mutation and no more than 10, 20, 30, 40, or 50 mutations in the VL amino acid sequences compared to a VH sequence set forth in any one of SEQ ID NOS: 19. In some embodiments, the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in any one of SEQ ID NOS:19.
[0068] In some embodiments, the VH amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, or 5 amino acid deletions or insertions, relative to an HCDR sequence shown in Tables 3-5. In some embodiments, the VH region comprises an HCDR1 with 1, 2, 3, or 4 substitutions relative to an HCDR1 sequence of any anof SEQ ID NOS:l, 7, or 13. In some embodiments, the VH region comprises an HCDRwith 1, 2, 3, or 4 substitutions relative to an HCDR2 sequence of any ane of SEQ ID NOS: 2, 8, or 14. In some embodiments, the VH region comprises a HCDR3 with 1, 2, 3, or 4 substitutions relative to a HCDR3 sequence of any anof SEQ ID NOS: 3, 9, or 15.
[0069] In some embodiments, the VL amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, or 5 amino acid deletions or insertions, relative to an LCDR sequence shown in Tables 3-5. In some embodiments, the VL region comprises a LCDR1 with 1, 2, 3, or 4 substitutions relative to a LCDR1 sequence of any of SEQ ID NOS:4, 10, or 16. In some embodiments, the VL region comprises a LCDR2 with 1, 2, 3, or 4 substitutions relative to a CDR2 sequence of any of SEQ ID NOS: 5, 11, or 17. In some embodiments, the V L region comprises a CDR3 that has 1, 2, 3, or 4 substitutions relative to a CDR3 sequence of any one of SEQ ID NOS: 5, 11, or 17.
[0070] In some embodiments, the ATRC-101 diagnostic antibody has an Fc region comprising the sequence of SEQ ID NO: 24. In some embodiments, the ATRC diagnostic antibody has a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 24 and comprises an alanine at position 180 of SEQ ID NO: 24 (corresponding to position 297 in the heavy chain).
Table 6. Fc region sequences
Figure imgf000022_0001
[0071] In some cases, the mIgG2a antibody, referred to as mATRClOl in this disclosure, can be used to evaluate the expression of the ATRC-101 target in tumor samples. mATRClOl comprises a VH and a VL sequences as set forth in Table 1 and CDRs as set forth in Table 3 (or Table 4 or Table 5). The mATRClOl comprises an Fc region set forth in SEQ ID NO: 23.
[0072] In some embodiments, the diagnostic antibody is mATRClOl N297A. Similar to mATRClOl, mATRClOl N297A also comprises VH and VL sequences as set forth in Table 1 and CDRs as set forth in Table 3 (or Table 4 or Table 5). But mATRC 101 N297A differs from mATRC in that it has an N297A mutation in the heavy chain Fc region. The Fc region of mATRC 101 N297A comprises a sequence set forth in SEQ ID NO: 24. As described below, the mATRClOl N297A antibody can be used to select patients likely to respond to ATRC-101.
[0073] Compared to mATRC 101, mATRC 101 N297 A showed a broader range of staining with higher H-Score averages by cancer indication. An array of negative, low, moderate, and high cases was observed for mATRClOl N297A, with H-Scores ranging from 0 to 270 across the 142 tumors. As such, mATRClOl N297A is more sensitive in determining a patient’s likelihood of responsiveness to the ATRC therapeutic antibody.
[0074] In some embodiments, the diagnostic antibody is mATRClOl comprising alternative mutations that disrupt signaling involving the Fc region of the antibody. Examples of these alternative mutations include T299A, E233P, L234A, L234F, L234V, L235A, L235E, G236del, S267K, P329G, P329A, P331S, and combinations thereof, including but are not limited to, (1) L234A and L235A; (2) L234A, L235A, and P329G; (3) L234A, L235A, and P329A; (4) L234A, L235E; L234A, L235A, and G237A; (5) L234A, G237A; L235A, and G237A; (6) L234F, L235E, and P331S; and (7) E233P, L234V, L235A, G236del, and S267K.
ATRC-101 TARGET
[0075] As known and disclosed in WO 2020/168231, ATRC-101 targets an extracellular RNA- protein complex that comprises mRNA and further comprises an mRNA binding protein. The ATRC-101 diagnostic antibodies bind to the same target protein complex as the ATRC-101 and variants thereof, as disclosed in WO 2020/168231. ANTIBODY FORMATS
[0076] In a further aspect of the invention, an ATRC-101 diagnostic antibody, in accordance with the disclosure, may be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g, an IgG antibody or other antibody class or isotype defined herein. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody and production by recombinant host cells.
[0077] In some embodiments, an ATRC-101 diagnostic antibody by the present disclosure is in a monovalent format. In some embodiments, the anti-tumor antibody is in a fragment format, e.g, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
ANTIBODY CONJUGATES/ IMAGING AGENTS
[0078] Further, an ATRC-101 diagnostic antibody of the present invention may be conjugated or linked to imaging/detectable moieties. For example, the ATRC-101 diagnostic antibody may be conjugated to a detectable marker, an imaging agent, or an oligonucleotide. Methods for conjugating or linking antibodies to the desired molecule are well known in the art. The moiety may be linked to the antibody covalently or by non-covalent linkages.
DETECTING THE INTERACTION BETWEEN THE ATRC-101 TARGET AND THE ATRC- 101 DIAGNOSTIC ANTIBODIES
[0079] In some embodiments, the method of determining whether a subject is likely to respond to treatment of ATRC-101 comprises measuring the amount of the ATRC-101 target and comparing the amount with a cutoff. If the amount of target in the tumor sample is equal to or greater than the cutoff, the patient is likely to respond to the treatment. If the amount of the ATRC- 101 target is not greater than the cutoff then patient is not likely to respond to the treatment.
[0080] In some embodiments, disclosed herein is a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein, e.g. , mATRClOl N297A. The method comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining an H-score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the binding corresponds to an H-score that is equal to or greater than an H-score cutoff.
[0081] In some embodiments, disclosed herein is a method of treating a tumor with ATRC-101. The method comprises selecting a subject with a tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101. The subject is selected if the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to an H-score that is equal to or greater than an H-score cutoff.
[0082] In some embodiments, disclosed herein is a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein, e.g ., mATRClOl N297A. The method comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining a tumor proportion score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the tumor proportion score corresponds to a tumor proportion score that is equal to or greater than a tumor proportion score cutoff.
[0083] In some embodiments, disclosed herein is a method of treating a tumor with ATRC-101. The method comprises selecting a subject with the tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101. The subject is selected based on the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to a tumor proportion score equal to or greater than a tumor proportion score cutoff.
[0084] In some embodiments, disclosed herein is a method of selecting a subject for treatment with ATRC-101 using an ATRC-101 diagnostic antibody disclosed herein, e.g. , mATRClOl N297A. The method comprises detecting the binding of the antibody with a tumor sample derived from the subject in an immunohistochemistry assay and determining an intensity score that corresponds to the binding and selecting the subject for treatment with ATRC-101 if the intensity score corresponds to an intensity score that is equal to or greater than an intensity score cutoff. [0085] In some embodiments, disclosed herein is a method of treating a tumor with ATRC-101. The method comprises selecting a subject with the tumor suitable for treatment with ATRC-101 and treating the subject with ATRC-101. The subject is selected based on that the binding of an ATRC-101 diagnostic antibody to a tumor sample obtained from the subject corresponds to an intensity score equal to or greater than an intensity score cutoff.
[0086] A cutoff can be determined based on the readout from the assay used. In some cases, the cutoff may be a value from a normal sample determined using the same assay. In some cases, a cutoff may be a mean or a median value of assays conducted to detect the presence of the target ( e.g ., the RNP complex) on a set of reference samples. In some cases, the cutoff may be a percentile value (e.g., 20%, 25%, 30%, 50%, 60%, 75%, or 80%) of the results of the assays conducted to detect the presence of the target in a set of reference samples. For example, if 75% of the reference samples express the target corresponding to a value X, then x can be used as the cutoff: a test tumor sample is considered to have a positive expression of the target if it shows a value equal to or greater than X when assayed under the same conditions as the reference samples.
[0087] In some embodiments, the reference samples may be non-tumor samples with the same tissue origin as the tumor samples to be evaluated for the likelihood of responsibilities to the ATRC-101 antibody. In some embodiments, the reference samples are tumor samples of a variety of tissue origins.
[0088] The method may comprise obtaining a tumor sample from a patient having the tumor. In some embodiments, the tumor sample is an FFPE sample. In some embodiments, the tumor sample is a fresh frozen sample. In some embodiments, the tumor sample comprises a breast tumor sample, a non-small cell lung cancer sample, an ovarian tumor sample, a colorectal tumor sample, an acral melanoma sample, an esophageal tumor sample, a squamous cell carcinoma tumor sample, head and neck squamous cell carcinoma tumor sample, hepatocellular carcinoma tumor sample, and a urothelial carcinoma tumor sample.
[0089] In some embodiments, disclosed herein is a method of detecting and/or quantifying the expression of a target of ATRC-101 in a tumor of a subject. The method comprises contacting a tumor sample obtained from the subject with an ATRC-101 diagnostic antibody disclosed herein, e.g ., mATRClOl N297A, and detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, thereby detecting and/or quantifying the expression of a target of ATRC-101.
[0090] The target’s expression can be assessed by detecting the binding of an ATRC-101 diagnostic antibody (e.g, mATRClOl N297A) to the tumor sample using various assays. In some embodiments, the detection is performed by an immunohistochemistry staining (IHC); illustrative examples are described in Example 1 and Example 3. In one exemplary assay, the method comprises contacting the tumor sample with an ATRC-101 diagnostic antibody and detecting the binding of the diagnostic antibody to the target by adding a secondary antibody. Said secondary antibody binds to the ATRC diagnostic antibody. In some embodiments, the secondary antibody is conjugated to a detectable moiety. The signal from the detectable moiety can be quantified, which corresponds to the binding of the ATRC-101 diagnostic antibody to the ATRC-101 target in the tumor. In some embodiments, the detectable moiety is an enzyme (e.g, a horseradish peroxidase, or “HRP”), which upon contacting its substrate, (e.g, DAB), produces a discrete insoluble reaction product at the site of antigen in the presence of the enzyme (e.g. , HRP).
[0091] In some embodiments, the expression of the ATRC-101 target in the tumor sample is detected by an immunohistochemistry (IHC) assay, and the amount is quantified by an H-score or a tumor proportion score (also referred to herein as a standard percent score). Both the H-score and the tumor proportion percent score are described in Example 1. In some embodiments, after the IHC is performed and the reaction product is formed, an H-score is generated by summing the percentages of cells with the intensity of expression multiplied by their corresponding differential intensity on a four-point semi-quantitative scale (0, 1+, 2+, 3+).
H-score= [(% at <1) x 0] + [(1% at 1+) xl] + [(% at 2+) x2]+ [(% at 3+) x3]. The scores range from 0 to 300.
[0092] In some embodiments, the cutoff used to determine the likelihood of a patient’s responsiveness is an H-score cutoff, and the H-score cutoff may be 10, 20, 30, 40, 50, 60, 70, 80, or 100 according to the scale described above. In some embodiments, the H-score cutoff is 40, 50, or 60. In some embodiments, the H-score cutoff is 50.
[0093] Similarly, a tumor proportion score can be assigned by summing the percentages of intensities at either >1+, >2+, or >3+. Thus, scores range from 0 to 100.
Percent Score >1+ = (% at 1+) + (% at 2+) + (% at 3+)
Percent Score >2+ = (% at 2+) + (% at 3+)
Percent Score >3+ = (% at 3+)
[0094] In some embodiments, the cutoff used to determine the likelihood of a patient’s responsiveness is a tumor proportion cutoff, and the tumor proportion score cutoff may be 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80%. In one embodiment, the tumor proportion score cutoff is 30%, 40%, or 50%. Kits.
[0095] The disclosure also includes reagents and kits for carrying out the methods of the invention. In one embodiment, a kit comprises an ATRC-101 diagnostic antibody described above. The kit may further comprise a T cell-binding agent, e.g ., a T cell-binding agent that specifically binds to CD3 or CD8. The kit may further comprise assay diluents, standards, controls, and/or detectable labels. The assay diluents, standards, and/or controls may be optimized for a particular sample matrix. For example, for measurements in blood, serum, or plasma samples, the diluents, standards, and controls may include i) human blood, serum, or plasma; ii) animal blood, serum, or plasma, or iii) artificial blood, serum or plasma substitutes.
GENERATION OF ANTIBODIES
[0096] ATRC-101 diagnostic antibodies, as disclosed herein, are commonly produced using vectors and recombinant methodology well known in the art (see, e.g, Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology). Commercial vendors offer reagents, cloning vectors, and genetic manipulation kits. Accordingly, in a further aspect of the invention, provided herein are isolated nucleic acids encoding a VH and/or VL region, or fragment thereof, of any of the anti tumor antibodies as described herein; vectors comprising such nucleic acids and host cells into which the nucleic acids are introduced that are used to replicate the antibody-encoding nucleic acids and/or to express the antibodies. Such nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the anti-tumor antibody e.g ., the antibody’s light and/or heavy chains). In some embodiments, the host cell contains (1) a vector containing a polynucleotide that encodes the VL amino acid sequence and a polynucleotide that encodes the VH amino acid sequence, or (2) a first vector containing a polynucleotide that encodes the VL amino acid sequence and a second vector containing a polynucleotide that encodes the VH amino acid sequence.
[0097] In a further aspect, the invention provides a method of making an ATRC-101 diagnostic antibody as described herein. In some embodiments, the method includes culturing a host cell, as described in the preceding paragraph, under conditions suitable for the expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).
[0098] Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof, include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally can self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g, pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl plasmids, pCRl, RP4, phage DNAs, and shuttle vectors. These and many other cloning vectors are available from commercial vendors.
[0099] Expression vectors are generally replicable polynucleotide constructs containing a nucleic acid of the present disclosure. The expression vector may be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and other vectors. [0100] Suitable host cells for expressing an anti-tumor antibody as described herein include both prokaryotic and eukaryotic cells. For example, an anti-tumor antibody may be produced in bacteria, particularly when glycosylation and Fc effector function are not needed. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. Alternatively, the host cell may be a eukaryotic host cell, including eukaryotic microorganisms, such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern, vertebrate, invertebrate, and plant cells. Examples of invertebrate cells include insect cells. Numerous baculoviral strains that may be used in conjunction with insect cells have been identified. Plant cell cultures can also be utilized as host cells.
[0101] In some embodiments, vertebrate host cells are used for producing an anti-tumor antibody of the present disclosure. For example, mammalian cell lines such as a monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al, ./. Gen Virol. 36:59,1977; baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g ., in Mather, Biol. Reprod. 23:243-251, 1980 monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g, in Mather et al, Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells may be used to express anti -turn or antibodies. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0, NS0, and Sp2/0. Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003. [0102] In some embodiments, an antibody of the present invention is produced by a CHO cell line, e.g., the CHO-K1 cell line. One or more expression plasmids can be introduced that encode heavy and light chain sequences. For example, in one embodiment, an expression plasmid encoding a heavy chain, e.g, SEQ ID NO: 1723, and an expression plasmid encoding a light chain, e.g, SEQ ID NO: 1724, are transfected into host cells as linearized plasmids at a ratio of 1:1 in the CHO-K1 host cell line using reagents such as Freestyle Max reagent. Fluorescence-activated cell sorting (FACS) coupled with single-cell imaging can be used as a cloning method to obtain a production cell line.
[0103] A host cell transfected with an expression vector encoding an anti-tumor antibody of the present disclosure, or fragment thereof, can be cultured under appropriate conditions to allow the expression of the polypeptide. The polypeptides may be secreted and isolated from a mixture of cells and a medium containing the polypeptides. Alternatively, the polypeptide may be retained in the cytoplasm on a membrane fraction, and the cells harvested, lysed, and the polypeptide isolated using the desired method.
[0104] In some embodiments, an anti-tumor antibody of the present disclosure can be produced by in vitro synthesis (e.g, Sutro Biopharma biochemical protein synthesis platform).
[0105] In some embodiments, provided herein is a method of generating variants of an anti tumor antibody as disclosed herein. Thus, for example, a construct encoding a variant of a VH CDR3 as described herein can be modified, and the VH region encoded by the modified construct can be tested for binding activity to the ATRC-101 target in the context of a VH region as described herein, that is paired with a VL region or variant region as described herein. Similarly, a construct encoding a variant of a VL CDR3 as described herein can be modified. The VL region encoded by the modified construct can be tested for binding to an ATRC-101 target comprising polyadenylated RNA. Such an analysis can also be performed with other CDRs or framework regions, and an antibody having the desired activity can then be selected. COMBINATION THERAPY
[0106] ATRC-101 may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents, and/or immunotherapeutic agents.
[0107] In some embodiments, ATRC- 101 can be administered in conjunction with an agent that enhances the presence or activity of T cells. In one aspect, ATRC-101 is administered in combination with an immune checkpoint agent. In one aspect, the agent is a biologic therapeutic or a small molecule. In another aspect, the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein, or a combination thereof. In certain embodiments, the agents inhibit, e.g, by blocking ligand binding to the receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDOl, ID02, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK1, CHK2, A2aR, B-7 family ligands or their receptors, or a combination thereof. In some embodiments, the agent targets PD-1, e.g, an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1. In one embodiment, the agent is pembrolizumab. In some embodiments, the agent targets CTLA-4. In some embodiments, the agent targets LAG3. In some embodiments, the agent targets TIM3. In some embodiments, the agents target ICOS.
[0108] In some embodiments, ATRC-101 is administered in conjunction with a therapeutic antibody, such as an antibody that targets a tumor cell antigen. Examples of therapeutic antibodies include as rituximab, trastuzumab, tositumomab, ibritumomab, alemtuzumab, atezolizumab, avelumab, durvalumab, pidilizumab, AMP -224, AMP-514, PDR001, cemiplimab, BMS-936559, CK-301, epratuzumab, bevacizumab, elotuzumab, necitumumab, blinatumomab, brentuximab, cetuximab, daratumumab, denosumab, dinutuximab, gemtuzumab ibritumomab ipilimumab, nivolumab, obinutuzumab, ofatumumab, ado-trastuzumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, and ranibizumab. [0109] In some embodiments, ATRC-101 is administered with a chemotherapeutic agent. Examples of cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2”- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens, including for example tamoxifen, raloxifene, mifepristone, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Other cancer therapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors. Examples of additional chemotherapeutic agents include topoisomerase I inhibitors ( e.g ., irinotecan, topotecan, camptothecin, and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g, melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g, cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g, 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea). Illustrative chemotherapeutic agents additionally include paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (e.g, CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g, imatinib mesylate and gefitinib); proteasome inhibitors (e.g, bortezomib); NF-KB inhibitors, including inhibitors of IKB kinase and other inhibitors of proteins or enzymes known to be upregulated, overexpressed or activated in cancers, the inhibition of which down-regulates cell replication. Additional agents include asparaginase and a Bacillus Calmete-Guerin preparation.
[0110] In some embodiments, ATRC-101 is administered with a chemotherapeutic agent that increases the expression of the ATRC-101 target. In some embodiments, the agent that increases the level of ATRC-101 target is Doxorubicin or pegylated liposomal Doxorubicin.
[0111] Various combinations described herein may be employed to treat a cancer patient. By “combination therapy” or “in combination with,” it is not intended to imply that the therapeutic agents must be administered at the same time and/or formulated for delivery together. However, these methods of delivery are within the scope described herein. ATRC-101 and the additional agent can be administered following the same or different dosing regimen. In some embodiments, ATRC-101 and the additional agent is administered sequentially in any order during the entire or portions of the treatment period. In some embodiments, ATRC-101 and the additional agent is administered simultaneously or approximately simultaneously ( e.g ., within about 1, 5, 10, 15, 20, 30, 45, or 60 minutes of each other). In still other embodiments, the additional agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before administration of ATRC-101. In some embodiments, the additional agent is administered from 1 to 4 weeks, or longer before ATRC-101 is administered.
[0112] The following examples are offered for illustrative purposes and are not intended to limit the invention. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same results.
EXAMPLES
EXAMPLE 1. ATRC-101 TARGET EXPRESSION IHC ASSAY
Immunohi story chemical staining (aka immunohistochemistry staining)
Assay I
[0113] Immunohi stochemi cal (IHC) stainings are performed as follows. mATRClOl or mATRClOl N297A as disclosed above, were used as the primary antibodies to detect the presence of ATRC-101 target in formalin-fixed, paraffin-embedded (FFPE) human tissues according to the below procedures. The concentrations of the antibodies and some assay conditions used in the assay are disclosed in Table 8.
[0114] FFPE tissue blocks are first cut at 4-5 pm thickness, and sections are mounted onto positively charged, capillary gap glass slides. Slides are baked (60°C, dry heat) prior to use. Tissue sections are then de-waxed using organic solvents (xylene, 100%, four changes) and an alcohol series (100%, 70%, 30% ethanol) descending to distilled water to sufficiently hydrate the tissues and allow proper binding of the primary antibody and other detection reagents.
[0115] Next, antigen retrieval is performed after tissue sections are dewaxed. This step uses a steam heat-indiJced epitope recovery (SHIER) solution (Tris-EDTA, pH 9) that is drawn into the capillary gap formed between paired microscope slides with a commercial steamer or a pressure cooker (20 minutes above 97°C) as a heat source (for description retrieval with heat and SHIER solutions does not contribute to antigen unmasking and is not used (referred to as NO SHIER). After the antigen retrieval, samples are tested by IHC according to the general procedure outlined in Table 8 using the TechMate instrumentation platform and the MIP program (which does not include enzymatic digestion). After heat-induced epitope retrieval, the process steps are automated using a TechMate Instrument (Roche Diagnostics) running QML workmate software v3.96. This automated platform uses a capillary gap process for all reagent changes, up to and including counterstaining and intervening buffer washes. All steps are carried out at room temperature (25°C).
[0116] Sequential detection of antibodies, a primary antibody and a detection antibody (or a secondary antibody) are employed during IHC. The location of the primary antibody is ultimately visualized by applying a colorimetric chromogen (DAB) that precipitates a discrete insoluble reaction product at the site of antigen in the presence of horseradish peroxidase (HRP). Nuclei are counterstained using hematoxylin (blue stain) to assess cell and tissue morphology. After the detection, slides are unpaired, rinsed in distilled water, dehydrated in an alcohol series (70%, 95%, 100% ethanol) and inorganic solvent (xylene, 100%, four changes), then permanently coverslipped, using CytoSeal (or equivalent), for interpretation and storage. Slides are examined under a microscope to assess staining.
[0117] Reagent Manufacturing Buffer (Discovery Life Sciences, Santa Barbara, CA) with goat serum is used to prepare working dilutions of primary antibody and mouse isotype control serum. Target recognition for the primary antibodies at the site of antigen-primary antibody interaction in FFPE sections uses reagents from Polink-2 Plus HRP kits (GBI Labs, Bothell, WA).
Table 8 Antibody Specifications and IHC Assay Conditions for mATRClOl
Figure imgf000037_0001
Table 9. IHC Procedure Protocol
Figure imgf000038_0001
Scoring of ATRC-101 target expression
[0118] The results of IHC in formalin-fixed, paraffin-embedded (FFPE) samples were evaluated using an H-score method. Target expression was scored semi-quantitatively by a board- certified pathologist for cytoplasmic staining in tumors. Percentages at differential intensities are recorded to determine Percent Scores and H-Scores (described below). When scoring tumor tissues for RNP complexes, numeric scoring excludes any surrounding staining in the stroma, areas of non-tumor, and adjacent normal tissue. Ischemic or necrotic areas are not scored in any indication.
[0119] Both tumor proportion Score and H-Score approaches can be used to assess target expression in tumor cells. Both approaches require recording the percentage of tumor cells with riboprotein staining at a corresponding differential intensity on a four-point semi-quantitative scale (0, 1+, 2+, 3+). On this scale: 0= null, negative, or non-specific staining, l+= low or weak staining, 2+= medium or moderate staining, and 3+= high or strong staining.
[0120] The percentage of tumor cells staining at each intensity is estimated directly and typically reported as one of the following; though other increments can also be used at the pathologist’s discretion: 0, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%. [0121] Percent scores are calculated by summing the percentages of intensities at either >1+, >2+, or >3+ Thus, scores range from 0 to 100.
Percent Score >1+ = (% at 1+) + (% at 2+) + (% at 3+)
Percent Score >2+ = (% at 2+) + (% at 3+)
Percent Score >3+ = (% at 3+)
[0122] The H-scores are calculated by summing the percentages of cells with the intensity of expression multiplied by their corresponding differential intensity all of four points; dictate if scale (0, 1+, 2+, 3+). Thus, the scores range from 0 to 300.
H-score= [(% at <1) x 0] + [(1% at 1+) xl] + [(% at 2+)x2]+ [(% at 3+) x3]
[0123] FIG. 1A-1C shows the images of IHC staining using the mATRClOl N297A antibody of the ATRC-101 target results corresponding to H-scores of 0, 30, and 180, respectively. The image in FIG. 1 A, where H-score=0, showed a faint amount of non-specific staining.
EXAMPLE 2. MATRClOl N297A IS SENSITIVE IN DETECTING ATRC-101 TARGET [0124] Tumor tissue sections from various tumor types were stained with mATRClOl N297A or mATRClOl in IHC, as described above. mATRClOl showed higher binding to the same tissue section as compared to the mATRC across all tumor types. H-scores were used to represent signal intensity.
Table 10: Average Tumor H-Scores
Figure imgf000039_0001
[0125] When comparing the mATRClOl (original) and mATRClOl N297A (mutant) sensitivity scoring data, mATRClOl N297A showed a broader range of staining with higher H- Score averages by cancer indication. An array of negative, low, moderate, and high cases was observed for mATRClOl N297A, with H-Scores ranging from 0 to 270 across the 142 tumors. Further sensitivity analysis and validation used only mATRClOl N297A, as shown in Examples 3 and 4 below.
EXAMPLE 3. ATRC-101 TARGET EXPRESSION LEICA IHC ASSAY
Assay 2 (Leica IHC Assay)
[0126] This example describes using mATRClOl N297A in automatic IHC staining assays performed on LEICA BOND-III Instrument to detect the ATRC-101 target.
Validation of Assay 2 (Leica IHC Assay) based on Assay 1 (IHC)
[0127] For inter-laboratory concordance testing from Assay 1 to the Leica BOND III IHC staining platform of Assay 2, formalin-fixed, paraffin-embedded (FFPE) samples were utilized that represented a range of mATRClOl (N297A) staining. The samples included two colon cancer, one colon tubular adenoma, four melanomas, one breast cancer, one uterine cancer, and one lung cancer. While the colon tubular adenoma sample does not represent the colon cancer indication, it was retained in this analysis because it supports laboratory concordance. In this sample, denomatous cells were scored as opposed to the tumor.
[0128] The ten selected tissues were run in serial sections (one section of tissue per slide with minimal loss of material between preparations) by different operators at DLS-NT on the TechMate IHC staining platform and at DLS-SB on the Leica BOND III IHC staining platform. A board-certified pathologist reviewed and scored both laboratory sets of slides by recording the percentage of tumor cells with cytoplasmic tumor staining at differential intensities to calculate H-Scores (Section 2e). Each sample was assigned positive or negative status using H-score cut offs of >10 as positive and >50 as positive. [0129] Acceptable concordance requires that the lower bounds of the selected 95% confidence interval (Cl), computed by percent agreement, meet or exceed 85%. Concordant scoring was observed between all samples run on the TechMate of Assay and on the Leica BOND III by demonstrating an appropriate 95% Cl when considering positive/negative agreement using the described positivity cut-offs for mATRClOl (N297A).
[0130] The 95% Cl for assay concordance samples (10 pairs = 20 Total N) is calculated as follows: Mean ± [Z-Value x (Standard Deviation/Square Root of Total N)]. Using the H-Score >10 cut-off for mATRClOl (N297A), the 95% Cl was 100.0% ± 0.0%. Using the H-Score >50 cut-off, the 95% Cl was 95.0% ±- 6.9% [where 1.96 x (15.8%/V20) = 6.9%]. The lower bound of these values is above 85%, demonstrating acceptable assay concordance for mATRClOl (N297A) using the TechMate IHC assay and the Leica IHC assay.
Assay Procedure
[0131] mATRClOl N297A was prepared using Therm oFi sher BSA Block and Diluent (Cat #: 003218 Ready -to-use diluent with BSA for stabilizing the antibody and preservative). The same antibody diluent was also used to prepare working dilutions of the species-matched standard control antibody, and isotype-matched negative control (Mouse IgG2a). The Leica BOND Polymer Refine Detection Kit (Cat #: DS9800) was used for mATRClOl N297A target recognition and binding at the site of antigen-primary antibody interaction in FFPE sections. Samples were processed using a Leica BOND-III Automated IHC Stainer platform by selecting Leica validated IHC Protocol F.
Leica BOND-III Instrument
[0132] The Leica BOND-III Instrument (Leica Biosystems) is a fully automated IHC commercial staining platform designed to perform clinical IHC tests using pre-programmed, and user validated programmed staining protocols. The instrument is controlled using Leica’ s BOND software. This automated platform uses a capillary gap process for all reagent changes, including antibody incubation, detection steps up to and including counterstaining, and intervening washes. All procedures were carried out at room temperature (~25°C) unless otherwise specified. The BOND-III instalment software assigned a unique barcode identifier to each tissue slide during the slide set up process that is encoded with the staining protocol. Prior to each staining run, the instrument performed an operation verification by scanning each unique barcode to ensure adequate reagent availability for staining completion of protocols assigned to all tissue slides loaded onto the instrument. Each staining run was logged into an internal database file on the operating desktop for all tests performed on each Leica BOND-III instrument. For each slide, the instrument retained a time-stamped record of all operations performed and any errors encountered during the staining procedure are retained for each tissue slide.
Specimen Preparation
[0133] FFPE tissue specimens were prepared less than 30 minutes after the biopsy tissue is removed from the patient prior to immersion in fixative, and the specimens were subject to 24- 48 hours fixation time in 10% neutral buffered formalin. Tissue specimens were cut into sections of 4-5 pm and mounted onto positively-charged glass. Unstained slides were placed in the dark at 2-8 °C for long term storage.
Antigen Retrieval
[0134] Antigen retrieval was performed using an enzymatic epitope retrieval (Dako Proteinase K, S3020, diluted to 1:120 in TBST) for 10 minutes at 37°C on the Leica BOND-III platform, followed by three rinses of BOND wash solution.
Staining and Detection
[0135] IHC was performed following the Customer Specific Protocol IHCS6-84 for mATRClOl N297A. To reduce staining from endogenous peroxidases, slides were incubated with Peroxide Block (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 5 minutes followed by three washes with BOND wash solution and incubation with the anti- mATRClOl N297A clone for 15 minutes. Slides were then rinsed three times with BOND wash solution. The primary antibody was detected by incubating the slides with Post-Primary reagent (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 8 minutes, rinsing the slides three times with BOND wash solution, and then incubated with Polymer (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 8 minutes. Following two washes with BOND wash solution and one deionized water rinse, mATRClOl N297A was visualized by incubating in DAB chromogen (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 10 minutes. Slides were counterstained with hematoxylin (part of the Leica BOND Polymer Refine Detection Kit Cat #: DS9800) for 5 minutes, rinsed with deionized water with an intervening BOND wash solution step.
[0136] Upon completion of the automated protocol, slides were removed from the BOND-III Automated staining instrument, dehydrated through alcohols (70%, 95%, 100%), cleared in xylene, and sealed by application of a coverslip.
Evaluation Scoring scheme
[0137] mATRClOl N297A is reactive in a subset of tumor cells where it primarily localizes to the cytoplasm. The approach used for scoring mATRClOl N297A as detected by IHC in FFPE tumor tissue samples, is described below. All samples are also stained with H&E (hematoxylin and eosin) for morphological assessment to assist in scoring.
[0138] mATRClOl N297A staining was scored semi-quantitatively by a board-certified pathologist for diffuse cytoplasmic staining in tumor cells. The main components to scoring ribonucleoprotein(s) expression in tumor cells include percentages at differential intensities to determine H-Score (described below). When scoring tumor tissues for ribonucleoprotein(s) staining, numeric scoring excludes any surrounding staining in stroma, areas of non-tumor, and adjacent normal tissue. Ischemic or necrotic areas are not scored in any cancer indication. Percentages of cells expressing ribonucleoprotein(s) are captured at each intensity (brown DAB (3, 3’-diaminobenzidine staining) on a four-point semi-quantitative scale:
0 = null, negative or non-specific staining 1+ = low or weak staining 2+ = medium or moderate staining 3+ = high or strong staining. [0139] The percentage at each intensity was estimated directly and reported as one of the following: 0, 1, 2, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. The H-Score was calculated by summing the percentage of cells with intensity of expression (brown staining) multiplied by their corresponding differential intensity on a four-point semi- quantitative scale (0, 1+, 2+, 3+), with the H-scores in a range from 0 to 300.
H-Score = [ (% at <1) x 0] + [ (% at 1+) x 1] + [ (% at 2+) x 2] + [ (% at 3+) x 3]
[0140] Under this scoring scheme, an adequate sample must have at least 100 viable neoplastic cells overall. If this condition is not met, the sample is reported as “not evaluable.” Tumor tissue samples are defined as positive if mATRClOl N297A tumor cytoplasmic H-score> 50.
Cut-off of the ATRC-101 Target Expression IHC Assay
[0141] Participants from Q3W dosing cohorts that were postulated to yield biologically relevant serum concentrations of ATRC-101 (3, 10, and 30 mg/kg), had available pretreatment (baseline) ATRC-101 target expression IHC results, and response assessments (RECIST vl.l) were included in an analysis to determine the eligibility criterion initial cut-off. Nineteen (19) participants met these conditions, 11 with progressive disease (PD) and 8 with stable disease (SD) as best outcome (data not shown). The pretreatment tumor samples available for IHC analysis were obtained during or prior to the screening window for 14 and 5 participants, respectively. Receiver Operating Characteristic (ROC) analysis of disease control was performed and demonstrated an area under the curve (AUC) of 0.8011 (P=0.0287) optimal cutoff (Youden’s J- statistic) H-score=88 with sensitivity of 75% and specificity of 82%. The assay was used retrospectively to date, in Clinical Trial ATRC-101 A01(NCT04244552), conducted under IND 142779. IHC cut-off H-score >50 was chosen as the initial threshold for eligibility with the objectives of minimizing the false negative rate and gaining more precision around the optimal threshold.
Precision and reproducibility of the ATRC-101 Target Expression IHC Assay
[0142] To assess precision and reproducibility of the Leica IHC Assay, 5 tumor samples from each of the five indications were selected for use. These samples showed a range of high, moderate, low, and absent mATRClOl (N297A) cytoplasmic staining in tumor cells. Each sample was run in triplicate for mATRClOl (N297A) in a single run (precision). In two separate runs, performed on non-consecutive days, the same samples were run in triplicate with mATRClOl (N297A) by both the same and a different operator (reproducibility). All replicate slides were prepared as serial sections (one section of tissue per slide with minimal loss of material between slide preparations).
[0143] Intra-assay consistency (precision) and inter-assay consistency (reproducibility) were determined using a 3 -run series with 3 replicate sections (per run) of each of the 5 selected samples in five indications for mATRClOl (N297A), resulting in a set of 9 replicates for each sample, and 45 total replicates per indication. Two CLIA-certified operators ran the assays using different Leica BOND-III instruments (Operator 1, Run 1; Operator 2, Run 2; Operator 2, Run 3). Positive standard and negative controls were included in each run and reacted as expected. All replicates stained during validation were reviewed and scored by Discovery’s board-certified pathologist for mATRClOl (N297A) using tumor cytoplasmic H-scores. According to the cutoffs established by Atreca, a sample with a tumor cytoplasmic H-score of 50 or more was called positive (POS).
[0144] Each sample for mATRClOl (N297A) precision and reproducibility was deemed acceptable by the pathologist as compared to the sensitivity test. Similar cellular patterns of mATRClOl (N297A) reactivity were observed in all replicates in the pattern, percent, and intensity of tumor staining. Any minor or graded changes observed in mATRClOl (N297A) staining between replicates were attributable to slight changes in the amount of tumor in each serial section. Summarized validation scoring results for mATRClOl (N297A) H-Score analysis is presented in Table 11 and includes the mean, coefficient of variation (CV), standard deviation (Std Dev), and standard error of the mean (SEM), for each indication and the corresponding set of replicates. Table 11. Summary of mATRClOl (N297A) Tumor Staining Precision & Reproducibility
Figure imgf000046_0001
Figure imgf000047_0001
[0145] The data in Table 11 are visualized in FIG. 11, which displays the distribution of the H- Scores and the corresponding CV value for each set of replicates (n=9). For example, the sample replicated with H-Scores ranging between 40-79, included CV values of 9% -53%. In general, when H-Scores were low (<50), differences between replicates translated into higher CV values by nature of the test. In such cases, the sample can be considered acceptable at the pathologist’s discretion by considering all data points.
[0146] Acceptance criteria for mATRClOl (N297A) IHC assay validation was determined through evaluation of consistency in staining patterns and the percent of agreement/concordant estimates. Acceptance of the precision and reproducibility testing requires that the lower bounds of the selected 95% confidence interval (Cl), computed by percent agreement, meets, or exceeds 85%.
[0147] Confidence interval assessments for binomial proportions were performed, which includes overall positive/negative staining agreement (p), alpha (a), and Zcrit (Z). Wilson Score method was used to calculate the upper limit and the lower limit of 95% Cl. For this analysis, the H-Score cutoff >50 was the reference point used to categorize the staining result (positive or negative), and to generate the overall agreement (p) for all replicates within each indication (N=45). Testing included nine replicates for 5 tissues per indication. For example, 9/9 replicates for NSCLC sample QMTB494-3 were positive for mATRClOl (N297A) {aka, mATRClOl N297A). If a QMTB494-3 replicate had been negative, it would have lowered the overall agreement proportion (p). [0148] Overall, the results for the mATRClOl (N297A) in breast cancer, colon cancer, and ovarian cancer resulted in 45 concordant and 0 discordant outcomes. As such, 45/45 mATRClOl (N297A) tests agreed with the appropriate majority in these cancer indications for a lower bound value of 92.1%, and an upper bound 100.0%. For melanoma and NSCLC, 43 concordant and 2 discordant outcomes resulted to yield a lower bound value of 85.2%, and an upper bound 98.8%. The lower bounds of all 95% Cl values for mATRClOl (N297A) using the H-Score >50 cut-off exceeded 85% and therefore also met the acceptance criteria for validation. All tumor types met the acceptance criteria for validation. Based on these results, the mATRClOl (N297A) IHC assay for detection in various human solid tumors is considered successfully validated for use in clinical testing by DLS’s Medical Director (a board-certified pathologist).
EXAMPLE 4 HISTOLOGICAL ANALYSIS OF ANTIBODY BINDING TO ATRC-101 TARGET IN ATRC-101 PHASE IB TRIAL Trial design
[0149] ATRC-101 Phase lb trial is a first-in-human, open label study of ATRC-101 in patients with selected solid tumor cancers. Enrollment is limited to patients with tumor types reactive to ATRC-101 in over 50% of patient samples evaluated preclinically, which includes non-small cell lung, breast, ovarian, colorectal, and acral melanoma.
[0150] A total of 26 patients had been dosed across five cohorts through intravenously infusion of ATRC-101. See FIG. 7A-B. 24 patients received a median of 6.1 weeks of therapy and were dosed every 21 days at the following dose regimen: 0.3 mg/kg (n = 3), 1 mg/kg (n = 3), 3 mg/kg (n = 9), 10 mg/kg (n = 6), and 30 mg/kg (n = 3). Tumor types enrolled include colorectal (n = 11), ovarian (n = 5), breast (n = 3) and acral melanoma (n=l). Patients enrolled in the study had received a median of 5 prior lines of treatment. N represents the number of patients in each of the groups. Target expression was analyzed retrospectively. Tumor lymphocyte infiltration and other potential biomarkes of activity in tumors, plasms, and PBMCs were analysed. Some of the objectives of the Phase lb trial include determining Patients’ maximum tolerated dose (MTD), determining recommended phase 2 dose (RP2D), and characterizing safety profile of the antibody. Tumor response evaluation
[0151] The overall tumor burden was evaluated at the baseline, i.e., before beginning treatment, and at a subsequent time in point, e.g ., more than 28 days from the initiation of the treatment, according to Response Evaluation Criteria in Solid Tumours (RECIST): standard as discussed in Eisenhauer, et al ., European Journal of Cancer 45 (2009) 228-247, the relevant disclosure is herein incorporated by reference. Specifically, each patient tumor response to ATRC101 treatment is evaluated and recorded into the following categories:
Evaluation of the target lesions
[0152] “Complete Response” or “CR” refer to the disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to < 10 mm.
[0153] “Partial Response” or “PR” refers to that at least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.
[0154] “Progressive Disease” or “PD”: At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is thesmallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions).
[0155] “Stable Disease” or “SD”: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
“Non-Progressive Disease” or “Non-PD” refers to a combination of Stable Disease, Partial, and Complete Responses.
[0156] A patient is considered as having Best Overall Response (BOR) if having any response to the treatment of ATRC-101 assessed, for example if the patient is assessed with a CR, PR, SD, or PD. Evaluation of nontarget lesions
[0157] Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (< 10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a participant to be considered in complete clinical response.
[0158] Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.
[0159] Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of “non-target” lesions only is exceptional, the opinion of thetreating physician should prevail in such circumstances, and the progression status should be confirmed at a later time.
[0160] A patient is considered as having Best Overall Response (BOR) if having any response to the treatment of ATRC-101 assessed, for example if the patient is assessed with a CR, PR, SD, or PD.
[0161] Results shown in FIG. 3 A-B, indicate that eight (8) of the 20 patients (40%) evaluable experienced stable disease (SD) as their best RECIST response, four (4) of which showed tumor reduction, with the remaining patients having progressive disease (PD). Eighteen (18) clinical trial participants with a variety of tumor types, including breast, colorectal, non-small cell lung, acral melanoma, and ovarian tumors were analyzed for target expression using the assay described in Example 1. These patients were grouped and received ATRC-101 at doses as indicated. The results indicate that expression levels of ATRC-101 target in patients with different tumor types were highly variable. FIG. 4.
[0162] The IHC assay as described in Example 3 was used to investigate target expression associated with response to treatment with ATRC-101. Forty-two (42) RECIST-evaluable patients with H-scores determined at screening were evaluated for association of H-scores to response to treatment with ATRC-101. Results of the analysis showed that fourteen (14) of the twenty-three (23) patients (61%) having an H-score of > 50 experienced non-progressive disease. FIG. 2A.
[0163] Patients treated with ATRC-101 monotherapy Q3W at doses 3, 10, or 30 mg/kg were analyzed for time to progression of disease as associated with target expression. Results of the analysis indicated that patients with an H-score >50 exhibited a median time to progression of 77 days while patients with an H-score <50 exhibited a median time to progression of 77 days (p = 0.01). FIG. 2B.
[0164] This correlation between ATRC-101 target expression and responsiveness to the ATRC- 101 (as indicated by being able to control disease progresson) supports that this target is of clinical therapeutic importance. This also indicates that the expression of the target protein is correlated with efficacy, as such, mATRClOl N297A can be used to select a patient who will benefit from ATRC-101 treatment.
[0165] Tumor response over time for patients with H-scores >50 as determined by the assay described in Example 3 was further examined by evaluating change in the SOD (sum of diameters) of the patient’s lesions over the course of therapy. SOD was calculated according to RECIST1.1 and is described in the Eisenhauer et al (2009). In brief, up to 5 target lesions are identified per patient, but no more than 2 per organ. The longest diameters for each target lesion (with the notable exception of lymph node target lesions) are summed. For lymph nodes, the shortest diameter is used. The change in SOD at timepoint x is SOD(x)-SOD (baseline)/SOD (baseline). Baseline is from the last pretreatment scan. Patients included in these analyses were treated with either ATRC-101 Q3W monotherapy or a combination of ATRC-101 and pembrolizumab. FIG. 5A show that the majority of twenty (20) patients with H-scores >50 (target-positive) exhibited a decrease in SOD during the course of the treatment. FIG. 5B shows durable responses observed in twelve (12) of the patients carrying different tumor type, including patients with ovarian cancer, non-small cell lung carcinoma, colorectal cancer, breast cancer, head and neck cancer, and melanoma. [0166] In particular, 60% of NSCLC patients with H-score >50 treated with ATRC-101 as Q3W monotherapy at either 3 mg/kg or 30 mg. kg demonstrated Stable Disease or Partial Response. FIG. 6.
EXAMPLE 3 COMBINATION THERAPY WITH DOXORUBICIN AND PEGYLATED LIPOSOMAL DOXORUBICIN
[0167] In order to assess the potential utility of ATRC-101 in combination with chemotherapeutic agents, non-clinical studies were performed to assess the efficacy of mATRClOl in combination with chemotherapeutic agents, including Doxorubicin and pegylated liposomal Doxorubicin (PLD), and the impact of these anti-tumor small molecules on mATRClOl immunoreactivity in mouse tumor and normal tissues.
[0168] Female BALB/c mice with established EMT6 tumors were dosed with mATRClOl (1 or 3 mg/kg) or vehicle intraperitoneally (IP) twice weekly plus Doxorubicin (2 or 5 mg/Kg) or vehicle (saline) IV once weekly following randomization on Day 6. Statistical analyses of tumor volumes were performed using the normalized area above the curve and the normalized growth rate metrics developed at Atreca. One-sided log-rank (Mantel-Cox) test was used to assess survival advantage relative to the indicated reference group. P-values <0.05 were considered significant. In monotherapy studies with Doxorubicin and PLD, EMT6 tumor and non-tumor bearing mice were dosed with vehicle or Doxorubicin (2 or 10 mg/kg) or PLD (1 ,2, 5 and 10 mg/kg). Normal mouse tissues were collected at 24 hours and 2 weeks after the last dose. Reactivity for mATRClOl in EMT6 tumor and normal mouse tissues was evaluated by immunohi stochemi stry .
[0169] The combination of 3 mg/kg mATRClOl and 5 mg/kg Doxorubicin demonstrated significant tumor growth inhibition compared to either monotherapy, or vehicle (p<0.05) through Day 22. See FIG. 8C, 8D, and 8E. Moreover, the combination resulted in a significant survival benefit compared to vehicle and 5 mg/kg Doxorubicin monotherapy (p<0.0005). See FIG. 9. [0170] Immunohistochemical analyses of mATRClOl reactivity in EMT6 tumors from mice treated with chemotherapy alone showed an apparent dose-dependent increase in immunofluorescence intensity with increasing concentrations of Doxorubicin or PLD
[0171] In one such study, tumors that have been treated with a single dose of PLD at 24 hours (FIG. 10 A) or at 2 weeks (FIG. 10B), demonstrated increased immunoreactivity to mATRC-101 as compared to the isotype control. The increase in immunoreactively is positively related to the dosage of the PLD, i.e., the higher the PLD dosage, the higher the immunoreactively to mATRC- 101
[0172] In another study, immunohistochemistry stainings were performed on tumors 1 week (FIG. IOC) or 4 weeks (FIG. 10D) after the mice received the first dose of PLD as indicated. The mice were treated with PLD once weekly. The results show that administration of 2mg/kg of PLD increased the immune reactivity to mATRClOl as compared to that of the isotype control after a treatment period of one (1) week (FIG. IOC), and similar results were observed for mice that received 5mg/kg or lOmg/kg for a treatment period of four (4) weeks (FIG. 10D).
[0173] Furthermore, tissue cross reactivity studies in selected normal organs from mice treated with Doxorubicin or PLD demonstrated no reactivity above vehicle.
[0174] This study provides non-clinical evidence that administration of mATRClOl in combination with Doxorubicin increases anti-tumor activity in the EMT6 model. Exposure to Doxorubicin or PLD in EMT6 tumor-bearing mice increased mATRClOl immunoreactivity in the tumor, in a dose-dependent manner. Moreover, mATRClOl immunoreactivity in normal tissues was not influenced by Doxorubicin or PLD. Taken together, these findings support the clinical evaluation of the combination of ATRC-101 and doxorubicin or DLP n solid tumors.

Claims

WHAT IS CLAIMED IS:
1. A method of determining whether a subject with a tumor is likely to respond to treatment with ATRC-101, wherein the method comprises contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain, detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, and determining that the subject will be responsive to treatment of ATRC-101 if the binding of the antibody as determined by the immunohistochemistry assay corresponds to an H-score that is equal to or greater than an H-score cutoff.
2. The method of claim 1, wherein the method comprises selecting the subject for treatment with ATRC-101 if the subject is determined to be responsive to treatment of ATRC-101.
3. A method of selecting a subj ect for treatment with ATRC- 101 , the method comprising detecting binding of an antibody with a tumor sample derived from the subject in an immunohistochemistry assay, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain, determining an H-score that corresponds to the binding, and selecting the subject for treatment with ATRC101 if the H-score that is equal to or greater than an H-score cutoff.
4. A method of treating a tumor with ATRC-101, the method comprising: selecting a subject with the tumor suitable for treatment with ATRC101, wherein the binding of an antibody to a target of ATRC-101 in a tumor sample obtained from the subject corresponds to an H-score that is equal to or greater than an H-score cutoff, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising a sequence (SEQ ID NO:l), an HCDR2 comprising a sequence (SEQ ID NO:2), and an HCDR3 comprising a sequence (SEQ ID NO:3); and a light chain variable region comprising: an LCDR1 comprising a sequence (SEQ ID NO:4), an LCDR2 comprising a sequence (SEQ ID NO:5), and an LCDR3 comprising a sequence (SEQ ID NO:6); wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain, and administering an effective amount of ATRC-101 to the subject.
5. A method of detecting expression of a target of ATRC-101 in a tumor of a subject, wherein the methods comprise: contacting a tumor sample obtained from the subject with an antibody that binds to a target of ATRC-101, wherein the antibody comprises: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain, detecting the binding of the antibody to the tumor sample in an immunohistochemistry assay, thereby detecting expression of the target of ATRC-101.
6. The method of any one of claims 1-5, wherein the antibody has a heavy chain variable region comprising a sequence SEQ ID NO: 19 and a light chain variable region comprising a sequence SEQ ID NO: 20.
7. The method of any one of claims 1-6, wherein the antibody has an Fc region comprising SEQ ID NO: 24, or wherein the antibody has an Fc region comprising a sequence that is at least 80% identical to SEQ ID NO: 24, provided that the sequence comprises a N297A mutation in the heavy chain.
8. The method of any one of claims 1-7, wherein the H-score cutoff is selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, or 100.
9. The method of claim 8, wherein the H-score cutoff is 50.
10. The method of any of claims 1-3 or 5-9, wherein the method further comprises administering an effective amount of ATRC-101 to the subject.
11. The method of any of claims 1-10, wherein the tumor is selected from the group consisting of non-small cell lung carcinoma, breast cancer, ovarian cancer, colorectal cancer, and acral melanoma.
12. The method of any of claims 1-11, wherein the method further comprises administering an effective amount of pembrolizumab.
13. The method of any of claims 1-12, wherein the method further comprises administering an effective amount of Doxorubicin or pegylated liposomal Doxorubicin.
14. A kit used to identify a subject that is likely to respond to treatment with ATRC-101, wherein the kit comprises an antibody comprising: a heavy chain variable region comprising: an HCDR1 comprising SEQ ID NO:l, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a light chain variable region comprising: an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; wherein the antibody is a mouse IgG2a, and wherein the antibody comprises a N297A mutation in the heavy chain.
15. The kit of claim 14, wherein said kit further comprises one or more reagents used in an assay to detect the binding of the antibody to a tumor sample from the subject, said one or more additional assay reagents provided in one or more vials, containers, or compartments of said kit.
16. The kit of claim 15, wherein the assay is an immunohistochemistry assay.
17. The kit of claim 15, wherein the one or more reagents used in the assay include isotype-matched negative control (Mouse IgG2a).
PCT/US2022/074218 2021-07-28 2022-07-27 Atrc-101 target expression assay WO2023010057A1 (en)

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Citations (3)

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US20180080938A1 (en) * 2013-04-02 2018-03-22 Merck Sharp & Dohme Corp. Immunohistochemical assay for detecting expression of programmed death ligand 1 (pd-l1) in tumor tissue
US20190194342A1 (en) * 2016-02-18 2019-06-27 Elanco Us Inc. Chimeric canine anti-cd20 antibody
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Publication number Priority date Publication date Assignee Title
US20180080938A1 (en) * 2013-04-02 2018-03-22 Merck Sharp & Dohme Corp. Immunohistochemical assay for detecting expression of programmed death ligand 1 (pd-l1) in tumor tissue
US20190194342A1 (en) * 2016-02-18 2019-06-27 Elanco Us Inc. Chimeric canine anti-cd20 antibody
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