Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • 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
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/54—F(ab')2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • 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
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

• ADCs Anti-cancer antibody-drug conjugates
• drugs which may be toxins or other cell growth inhibitors (termed here as drug or payload molecules) to cancer cells.
• Twelve ADCs are currently marketed in the US, and approximately 100 ADCs are in development (Chau et al., Lancet. 2019;394(10200):793-804; Coats et al., Clin Cancer Res. 2019. Epub 2019/04/14. doi: 10.1158/1078-0432.CCR-19- 0272; Wolska-Washer et al., Drug Saf. 2019;42(2):295-314; Beck et al., Nat Rev Drug Discov. 2017; 16(5):315-37).
• compositions and methods for reducing off-target toxicity of ADCs can be used for treatment of tumors with ADCs, while reducing off-target toxicity of the ADCs.
• the drug in the ADC may be referred to herein as “payload”.
• the compositions comprise an ADC and an agent targeted to the payload that is delivered by or derived from the ADC.
• the agent targeted to the payload is termed herein as “payload-binding agent” or PBA.
• PBA payload-binding agent
• the ADC and the payload-binding agent may be provided in the same composition or in different compositions.
• the payload binding agent may be a peptide or an antibody or a fragment or an antibody mimetic or a modification thereof directed to the ADC payload, and which binds to the payload. If the payload binding agent is an antibody or a fragment or modification thereof, it may be referred to as “anti-payload antibody”.
• this disclosure provides a method for inhibiting or preventing the growth of one or more tumors comprising administering to an individual in need of treatment, an ADC and a payload binding agent, wherein the payload binding agent has specific affinity for the ADC payload.
• the ADC and the payload binding agent may be administered in the same composition or different compositions, via the same routes or via different routes, or using the same regimen or different regimen.
• this disclosure provides peptides or antibodies or antibody fragments or modifications, which are specific for an ADC payload molecule.
• the antipayload antibody may be whole immunoglobulin molecules such as polyclonal or monoclonal antibodies or chimeric antibodies including humanized antibodies.
• the antibody fragments or modification can be antigen-binding fragments thereof, including, but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, CDR fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, or single domain antibodies (nanobodies) and the like.
• Antibody mimetics may include affibodies, nanofitins, or the like.
• the fragments of the antibodies may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or may be genetically engineered by recombinant DNA techniques. These techniques are well known in the art.
• the antibody or fragments or modifications thereof may be modified so as to impart longer half-life, stability and the like.
• the disclosure provides antibodies or fragments or derivatives thereof directed to several ADC payload, including full length antibodies, scFv, Fab and other fragments directed specifically to auristatins (e.g., monomethyl auristatin E [MMAE], monomethyl auristatin F [MMAF], monomethyl auristatin F methyl ester, monomethyl auristatin D, and the like.)
• auristatins e.g., monomethyl auristatin E [MMAE], monomethyl auristatin F [MMAF], monomethyl auristatin F methyl ester, monomethyl auristatin D, and the like.
• the anti-payload specifically binds to MMAE and/or MMAF.
• Figure 1 Provided are the top 5 scFv clones that were identified following biopanning and screening of the immunized murine scFv phage library.
• the fraction bound represents the ELISA signal for phage bound to MMAF-biotin-streptavidin immobilized on an ELISA plate, for each clone with or without MMAE or T-vc-MMAE spiked in. Bars represent the mean of duplicate samples with standard deviation error bars.
• Clones 1B3, 1H2, 3B8, 2E8 and 2C2 had a >50% decrease in the binding fraction with the addition of 30-100 nM free MMAE, with no change in binding with the addition of 100 nM T-vc-MMAE.
• Figure 2 Provided are the sequences from the top 5 clones identified from the 1 st panning. Complementarity determining regions identified using the North definition are shown in boxed regions. Clone 1H2 has an identical sequence to clones 2E8 and 2C2 from figure 1. The sequence in the top line is SEQ ID NO:31.
• Figure 3 A random mutagenesis scFv library was built from clones 1B3, 1H2 and 3B8 (figure 1 and 2) to identify clones with increased MMAE binding affinity. Shown is the fractional binding signal for four 96-well plates from two different panning dissociation steps (3-hour and 24-hour). The fractional binding signal for the 3-hour plates is the signal observed with 10 nM free MMAE spiked in divided by a control well with no MMAE. The fractional signal for the 24-hour plates is with 1 nM free MMAE spiked in.
• Figure 4 Clones from the screening results in figure 3 were sequenced.
• the parent scFv sequences that were used to build the mutagenesis library are provided (1B3, 1H2, 3B8) with 1H2 set as the reference sequence. Complementarity determining regions identified using the North definition are shown in boxed regions.
• the amino acid sequence for the variable domains of clone MA24E2 were used to inform the development of the humanized variant ABC3315.
• the sequence of the 1B3 construct in the top line is SEQ ID NO:31.
• Figure 5 The binding of ABC3315 to MMAF-biotin-streptavidin was decreased with increasing concentrations of free MMAE and MMAF (IC50: ⁇ 1 nM), whereas trastuzumab-vc-MMAE (T-vc-MMAE) did not compete for binding. Points represent the mean of samples in triplicate with standard deviation error bars.
• Figure 6 Shown are kinetic titration binding SPR sensorgrams for ABC3315 to MMAF-peg 11 -biotin neutravidin (left), MMAE to ABC3315-pegl2-biotin-streptavidin (middle) and polatuzumab vedotin (PV) to ABC3315-pegl2-biotin-streptavidin (right).
• Best fit values for the association rate constant (Kon) dissociation rate constant (koff) and equilibrium dissociation rate constant (KD) are provided in the insets. No binding signal was observed for PV to ABC3315-pegl2-biotin-streptavidin.
• FIG. 7 ABC3315 selectively inhibits MMAE toxicity.
• A RAMOS cells were incubated with MMAE (10 pM - 100 nM), with or without co-incubation with 500 nM ABC3315.
• ABC3315 increased the IC50 of free MMAE by 800-fold.
• B Polatuzumab vedotin (PV), a clinically-approved anti-CD79b ADC that incorporates MMAE as the payload molecule, was incubated with CD79b+ Ramos cells at concentrations of 3 pM - 30 nM, with or without coincubation with 500 nM ABC3315.
• PV Polatuzumab vedotin
• ABC3315 did not alter the on- target cellular cytotoxicity of PV (IC50: 0.12 nM alone vs 0.13 nM with ABC3315).
• C ABC3315 (500 nM) increased the IC50 of free MMAE against SKBR3 cells by greater than 500-fold.
• D HER2+ SKBR3 cells were incubated with T-vc-MMAE with and without anti- MMAE fab with negligible changes in cellular cytotoxicity (IC50: 0.03 nM alone vs 0.04 nM ABC3315). Points represent the mean of triplicate wells with standard deviation error bars.
• FIG. 8 ABC3315 does not alter the efficacy of PV.
• A The tumor volumes over time for each group are provided with standard deviation error bars. Mice were sacrificed at a tumor volume of 2000 mm 3 .
• Figure 10 Evaluation of ABC3315-WEDD HSA binding. Graphs showing ABC3315-WEDD injected over a SPR chip with immobilized human serum albumin at a range of concentrations. Top: The observed association and dissociation curves were fit to a 1 : 1 Langmuir binding model to obtain binding rate constants. (Bottom) The observed Rmax for each concentration was used to estimate an equilibrium dissociation rate constant. Fit values for binding parameters are provided in the insets of each panel.
• FIG. 11 Evaluation of ABC3315-WE human serum albumin (HSA) binding.
• HSA human serum albumin
• Figure 12 Affinity matured scFv sequences using ABC3315 as the reference sequence.
• the sequence of the top line is SEQ ID NO:31.
• FIG. 13 ELISA comparison of ABC3315 Mutants. ABC3315 mutants were evaluated for retained binding activity using an indirect ELISA method. All mutants, except for VH F37V, showed a similar binding signal for MMAF-pegll-biotin-neutravidin across the concentrations of Fab that were tested.
• Figure 14 Competitive Cell Cytotoxicity assay. The VH F27L mutants’ inhibition of MMAE mediated cellular cytotoxicity in comparison to ABC3315 was evaluated. Greater cell survival was observed across the range of MMAE concentrations with VH F27L co-treatment relative to ABC3315 co-treatment.
• Figure 15 Graph showing the concentration of free MMAE in plasma and red blood cells following administration of 100 mg/kg TvcMMAE alone or in combination with ABC3315 was evaluated. ABC3315 decreased the free concentration of MMAE in plasma by 85% and red blood cells by 73%.
• FIG. 16 Graphs showing results from TvcMMAE administration at a dose of 80 mg/kg alone or in combination with ABC3315.
• TvcMMAE administered alone resulted in a significant decrease in the white blood cell count and red blood cell count relative to mice administered a PBS vehicle.
• Mice that were administered TvcMMAE in combination with ABC3315 did not exhibit a significant decrease in white blood cells or red blood cells relative to control mice.
• Figure 17 Data showing the impact of ABC3315 on the efficacy of TvcMMAE in a HER2+/HER2- bystander xenograft model was evaluated. No significant difference was observed between mice administered 3 mg/kg TvcMMAE alone or in combination with ABC3315. A representative tumor is provided on the right.
• FIG. 18 Graph showing ABC3320 decreases MMAE ADC toxicity against differentiating neutrophils.
• Human peripheral blood mononuclear cells were incubated with trastuzumab-vc-MMAE (TvcMMAE) alone or in combination with ABC3320.
• TvcMMAE trastuzumab-vc-MMAE
• Flow cytometry was used to evaluate the number of neutrophils following TvcMMAE treatment using CD66b+ as a neutrophil marker.
• ABC3320 increased the IC50 of TvcMMAE from 1.9 nM to 27 nM.
• Figure 19 Graph showing ABC3320 co-treatment decreased the body weight loss that was observed in rats treated with 25 mg/kg trastuzumab-vc-MMAE (ADC) relative to rats treated with TvcMMAE alone.
• Figure 20 Graphs showing ABC3320 decreases hematologic toxicity in rats following a 25 mg/kg dose of TvcMMAE.
• Figure 21 Graphs showing ABC3320 decreases trastuzumab-vc-MMAE (25 mg/kg) mediated liver toxicity in rats.
• Figure 22 Graphs showing ABC3320 does not decrease the efficacy of polatuzumab vedotin (PV) in mice bearing Ramos xenografts and does not decrease the efficacy of trastuzumab-vc-MMAE (TvcMMAE) in mice bearing a mixed xenograft model of HER2+ NCI-N87 cells and HER2- MCF7 cells.
• PV polatuzumab vedotin
• TvcMMAE trastuzumab-vc-MMAE
• FIG. 23 Graph showing ABC3320 decreases free MMAE concentration in plasma following TvcMMAE administration.
• PBS phosphate buffered saline
• Plasma samples 100 pl were placed into wells of a rapid equilibrium dialysis device (Thermo ScientificTM, 90006) and free MMAE was separated from bound MMAE (plasma protein bound or ABC3320 bound) following manufacturer recommendations. Free and bound MMAE concentrations were determined using LC-MS/MS. Free concentrations of MMAE were on average 200x lower for samples obtained from mice treated with ABC3320 when compared to samples obtained from mice administered TvcMMAE with PBS.
• the present disclosure provides compositions and methods for treatment of diseases (e.g., cancer) using ADC, while reducing off-target toxicity associated with the ADC.
• the method comprises administering to an individual in need of treatment an ADC and one or more payload binding agents directed to the drug portion of the ADC.
• the payload binding agent is effective in reducing the non-target toxicity of the ADC or the non-target toxicity derived from the free drug dissociated therefrom.
• the compositions comprise antibodies (including fragments or modifications thereof), which are directed to the drug of the ADC.
• Compositions are also provided that comprise the ADC and an agent (such as an antibody) which is directed to the drug that makes up the ADC.
• the agents bind to free drug and may or may not bind to the drug when it is part of the ADC.
• the present disclosure relates in part to PCT application PCT/US2020/063453, and its publication, published as PCT publication WO 2021/113740, published June 10, 2021, the entire disclosure of which is incorporated herein by reference.
• An ADC comprises an antibody group, a linking group, and a drug group.
• the antibody group targets an antigen, such as a tumor cell antigen
• the linking group is used to attach the drug group to the antibody group
• the drug group is an agent that is cytotoxic to the targeted cell.
• the antibody group may be referred to as an antibody or by the name of the antibody.
• the drug group may be referred to as a drug or by the name of the drug.
• treatment refers to reduction or delay in one or more symptoms or features associated with the presence of the particular condition being treated. Treatment does not necessarily mean complete cure and does not preclude relapse, but may be used in connection with any such relapse.
• terapéuticaally effective amount is the amount sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The exact amount desired or required will vary depending on the mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art (such as a clinician) with the benefit of the present disclosure.
• a general reference to an antibody in this disclosure is also intended to include all full-length antibodies, antibody fragments containing antigen binding domains, as well as modified antibodies or fragments containing substitutions or modifications of the amino acid residues, and including where antibody fragments, modified or not, may be linked together via covalent linkages with or without linkers.
• a reference is made to an antibody being “targeted to payload” or “directed to payload” or similar phrases it means the antibody has specific affinity for the payload, when the payload is in the form of free payload, including when it is cleaved from the ADC.
• the anti-payload antibody may or may not bind to the payload when the payload is conjugated to the antibody part of the ADC.
• off-targef ’ and “non-targef ’ in reference to toxicity refer to toxicity that accompanies the administration of many chemotherapeutic agents. While the intended purpose of administration of a chemotherapeutic agent is to reduce or inhibit the growth of tumors or any accompanying metastases, the growth, function, and/or physiology of normal cells are often adversely affected in the course of treatment of cancer. A reduction of off-target or non-target toxicity is intended to reduce any adverse effects on the non-tumor or non-metastatic cells.
• the term “payload binding agent” (PBA) as used in this disclosure refers to an agent that specifically binds to a payload (drug) portion of an ADC.
• the PBA may be an antibody, a fragment or modification thereof, a peptide, an aptamer, a Spiegelmer, a fibronectin, a DARPin, a cyclodextrin, or an affitin.
• the PBA is an antibody
• the PBA may be referred to herein as an anti-drug antibody or anti-payload antibody.
• the anti-payload antibody may be whole immunoglobulin molecules such as polyclonal or monoclonal antibodies or chimeric antibodies including humanized antibodies or may be antigen-binding fragments thereof, including, but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, CDR fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, single domain antibodies (nanobodies) and the like.
• the fragments of the antibodies may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or may be genetically engineered by recombinant DNA techniques. These techniques are well known in the art.
• chimeric antibody refers to an antibody that has framework residues from one species, such as human, and complementarity-determining regions (CDRs, (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds a payload.
• CDRs complementarity-determining regions
• some portions of the heavy and/or light chains may be identical or homologous to sequences from a particular species while other portions may be identical or homologous to sequences from a different species.
• Chimeric antibodies generally exhibit decreased immunogenicity and increased stability. Techniques for cloning murine immunoglobulin variable domains known in the art - such as, for example, see Orlandi et al., Proc. Natl Acad. Sci.
• polynucleotides encoding the variable domains of the light chain or the heavy chain of an antibody derived from an animal (e.g., mouse, rat, or chicken) other than human can be linked to polynucleotides encoding the constant domains of the light chain or the heavy chain derived from a human antibody to produce a polynucleotide (such as DNA) encoding a chimeric antibody.
• an animal e.g., mouse, rat, or chicken
• a “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a single or different human immunoglobulins.
• frameworks from one human antibody can be engineered to include CDRs from a different human antibody.
• Methods for producing human antibodies are known in the art - such as, for example, see Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8: 117-26.
• a “humanized antibody” is typically a human antibody that has one or more amino acid residues imported into it (i.e., introduced into it) from a source that is non-human.
• a humanized antibody is a recombinant protein in which the CDRs of an antibody from a species such as rodent, rabbit, dog, goat, or horse are imported into human heavy and light variable domains.
• the constant domains (also referred to as framework regions) of the antibody molecule are generally the same as those of a human antibody.
• the non-human immunoglobulin providing the CDRs can be termed as “donor” and the human immunoglobulin providing the framework can be termed as “acceptor”.
• all the CDRs can be from the donor immunoglobulin in a humanized immunoglobulin.
• Constant regions need not be always present, but if they are, they can be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
• a humanized antibody binds to the same payload as the donor antibody that provides the CDRs.
• the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
• Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.
• Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S.
• Antibody fragments can be produced by enzymatic digestion. For example, papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a “Fc” fragment.
• the Fab fragment contains an entire L chain and the variable region domain of the H chain (VH), and the first constant domain of one heavy chain.
• VH variable region domain of the H chain
• Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
• Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site and single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
• a suitable linker may be 3-20 amino acids and may comprise G, S, or a combination thereof.
• any binding partner described herein, including but not necessarily limited to the binding partners referred to herein as ABC3319, ABC3320, and ABC3315, that contain amino acid residues at the unchanged positions shown in Table 1 i.e., in L H55Y the H is an unchanged position and Y is it’s changed position in the light chain
• a component of a described binding agent comprises an amino acid sequence that binds to albumin, further representative and nonlimiting examples of which include the following sequences: QRLIEDICLPRWGCLWEDDF (SEQ ID NO: 6); QRLMEDICLPRWGCLWEDD (SEQ ID NO: 7);
• QRLMEDICLPRWGCLWE SEQ ID NO:8
• DICLPRWGCL SEQ ID NO:9
• a binding partner described herein is adapted to bind to a red blood cell.
• the adaptation may be in the form of another binding partner that becomes a component of an anti-payload agent.
• a binding partner of this disclosure thus comprises an amino acid sequence that imparts to the binding partner the ability to bind to a red blood cell.
• the red blood cell binding component comprises a single domain antibody.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
• the pharmaceutical composition may comprise buffer components and stabilizers, including, but not limited to, sucrose, polysorbate 20, NaCl, KC1, sodium acetate, sodium phosphate, arginine, lysine, trehalose, glycerol, and maltose.
• buffer components and stabilizers including, but not limited to, sucrose, polysorbate 20, NaCl, KC1, sodium acetate, sodium phosphate, arginine, lysine, trehalose, glycerol, and maltose.
• the ADCs and the antipayload antibodies or fragments or modifications thereof are the only protein molecules present in the compositions.
• the ADCs and the anti-payload antibodies or fragments or modifications thereof are the only antibodies present in the composition.
• compositions comprising the ADC and the payload binding agent may be administered together, or separately and independently using any suitable route including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the administration(s) may be carried out in a continuous manner or may be intermittent. Appropriate dosage will depend upon the particular tumor being treated, the specifics and condition of the individual patient, the mode of administration etc.
• the ADC may be delivered locally, while the PBAis delivered so as to be available systemically.
• the PBA may be delivered i.v.
• the ADC may be delivered systemically, and the anti-payload antibody is delivered so as to be available systemically.
• an ADC may be delivered i.v., while the anti-payload antibody may be delivered subcutaneously.
• the ADC and the PBA may be administered as a single composition or may be administered as separate compositions. When administered as separate compositions, they may be administered sequentially or concurrently.
• the two compositions may be administered at the same or different times, by the same or different routes, for same or different lengths of time, on the same or different regimens.
• the amount of the ADC and the PBA separately or together are in amounts sufficient enough to reduce the non-target toxicity of the ADC by at least 5% relative to that expected with the ADC alone at the same concentration as used in the combination.
• the reduction in toxicity may be 10%, 20%, 30%, 40%, 50% or more.
• Reduction in non-target toxicity can be evaluated by methods known in the art. For example, reduction could be classified as a reduction in the percent of patients at a specific dose that experience a grade 3 or higher adverse reaction with and without the PBA (Clin. Invest. (2013) 3(12), 1157-1165). Additionally, within an individual patient, reduction in non-target toxicity could be classified as a reduction in the severity of an adverse reaction (e.g., neutropenia) with, versus without the PBA.
• an adverse reaction e.g., neutropenia
• the ADC and the PBA can be administered to an individual in need of treatment at dose(es) that is/are effective to treat a solid tumor.
• suitable dosages of the PBA and the ADC can range from about 0.1 mg/kg to 100 mg/kg, including all 0.1 mg/kg values and ranges therebetween. Examples of dosages include 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 mg/kg.
• the amount of ADC and the anti-payload antibody can be administered to an individual in need of treatment at a dose that is effective to treat a solid tumor.
• suitable dosages of the antibodies or fragments thereof — for both the ADC and the antipayload antibody — can range from about 0.1 mg/kg to 100 mg/kg, including all 0.1 mg/kg values and ranges therebetween. Examples of dosages include 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 mg/kg.
• a variety of dosage regimens are contemplated including dosage regimens in which the ADC and the antipayload antibody may be administered repeatedly, e.g., on a daily, weekly or monthly schedule, over a short period or an extended period of time, e.g., months to years (e.g., maintenance therapy).
• the range for administration of the anti-payload antibody may be from 0.01-100 mg/kg, including all 0.01 mg/kg values and ranges therebetween.
• a suitable ratio of the ADC to the anti-drug antibody may be determined by one skilled in the art. In an embodiment, the molar ratio can be from 1 : 1 to 1 : 100 ADC: anti -Drug antibody.
• half-life extension strategies can be used to increase plasma half-life of the anti-payload antibodies including fusion of moieties to the sdAb or Fab that bind blood components such as albumin, red blood cells (e.g., Band 3 of RBCs) or endogenous IgG as well as PASylation and PEGylation.
• the anti-payload antibody may be fused to moieties that bind to albumin, red blood cells or endogenous IgG, or fragments thereof, or may be PASylated and/or PEGylated.
• a bispecific antibody may have an arm that binds to the payload and another arm that binds to albumin or red blood cells (e.g., Band 3 of RBCs).
• the antibodies or fragments do not contain a polyhistidine tag. It is therefore considered that sdAb-sdAb fusion proteins that combine anti-payload binding activity and anti-albumin or anti-red cell binding activity may exhibit desirable pharmacokinetic attributes (restricted distribution within tissues and tumors relative to distribution within blood, elimination via renal filtration due to low molecular weight, long half-life) and can provide optimal enhancement of ADC therapeutic selectivity (increasing the ratio of efficacy to off-site toxicity).
• sdAb-peptide or Fab-peptide fusion proteins that combine anti-payload binding affinity (via the sdAb or Fab) and albumin binding affinity (via the peptide) may exhibit desirable pharmacokinetic attributes (restricted distribution within tissues and tumors relative to distribution within blood, elimination via renal filtration due to low molecular weight, long half-life) and can provide optimal enhancement of ADC therapeutic selectivity (increasing the ratio of efficacy to off-site toxicity).
• composition/compositions may be administered alone or in combination with other types of treatments (e.g., surgical resection, radiation therapy, chemotherapy, hormonal therapy, immunotherapy, or other anti -turn or agents).
• treatments e.g., surgical resection, radiation therapy, chemotherapy, hormonal therapy, immunotherapy, or other anti -turn or agents.
• compositions may be used for any type of cancer, including carcinoma, lymphoma, sarcoma, melanoma, and leukemia.
• Non-limiting examples include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, myeloma (including multiple myeloma), hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma/glioma (e.g., anaplastic astrocytoma, glioblastoma multiforme, anaplastic oligodendroglioma, anaplastic oligodendroastrocytoma), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, brain cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer,
• this disclosure provides a method for generating and identifying anti-payload antibodies that are suitable to be used in combination with an ADC therapy to reduce non-target cytotoxicity.
• the method comprises generating or obtaining a library of antibodies, identifying specific antibodies based upon positive binding to the payload molecule by ELISA and/or by surface plasmon resonance techniques, identifying antibodies that have the desired affinity (such as KD equal to or less than 50 nM, determining binding kinetic parameters, and determining in vivo efficacy.
• Any auristatin described herein may be a monomethyl auristatin.
• PBA sequences provided by the present disclosure are as follows.
• Example 5 The method of Example 1, wherein the drug is auristatin E or auri statin F.
• Example 6 The method of Example 1, wherein the KD of the PBA is less than or equal to 50 nM.
• Example 6a The method of Example 1, wherein the KD of the anti-drug antibody is less than or equal to 50 nM.
• Example 9 A composition comprising an ADC and a PBA, which is directed to the drug portion of the ADC.
• Example 9a The composition of Example 9, wherein the PBA is an antibody of a fragment or modification thereof, which is directed to the drug portion of the ADC (antidrug antibody).
• Example 10 A kit comprising: i) a composition comprising an ADC; ii) a composition comprising an anti-drug antibody which is specific for the drug in the ADC; iii) optionally, instructions for use including instructions for administration of i) and ii).
• MMAF Monomethyl auristatin F
• KLH keyhole limpet hemocyanin
• BSA BSA
• EDC keyhole limpet hemocyanin
• 0.5 mg MMAF was dissolved in MES buffer (0.1 M MES, pH 4.7) / 30% DMF and mixed with 2 mg of KLH or BSA in MES buffer, then 0.5 mg of EDC dissolved in water was added. The solution was incubated at room temperature overnight. Approximately 50 pg of KLH-MMAF immunogen emulsified in Freund’s incomplete adjuvant was used for single animal immunization.
• Female Balb/c mice were subcutaneously injected with 200 pL of emulsion and given a booster every three weeks.
• Anti -MMAF -B SA plasma titers were evaluated with ELISA using an anti-mouse Fc secondary antibody conjugated to alkaline phosphatase.
• Spleens were surgically removed from immunized mice and teased apart with forceps and a needle.
• the released splenocytes were collected in a 15-ml conical tube.
• the lymphocyte samples were lyzed and homogenized in 5 ml of TRIzol reagent, then incubated for 5 minutes before adding 1 mL of chloroform.
• the mixture was vortexed for 30 seconds, incubated for 3 minutes, and centrifuged for 15 minutes at 12,000*g 4 °C, after which the mixture was separated into 3 layers: a lower red phenol-chloroform, interphase, and a colorless upper aqueous phase.
• RNA-primer mixture 4 pl of 5*SSIV Buffer, 1 pl of 100 mM DTT, 1 pl of RNaseOUTTM Recombinant RNase Inhibitor, and 1 pl of SuperScript® IV Reverse Transcriptase (200 U/pL).
• the combined reaction mixture was incubated at 50- 55°C for 15 minutes and then inactivated at 80°C for 10 minutes.
• amplified PCR product (1 pg/replicate) and pADL-lOb phagemid vector (2 pg/replicate) were digested in triplicate with Sfil restriction enzyme according to the manufacturer’s recommendation. Reactions were incubated at 50 °C for 16 h. Cut products were recovered through DNA gel electrophoresis using a 1% agarose gel.
• a test library was first developed by ligating digested DNA into the phagemid vector. Ligated vector was purified with an E.Z.N.A cycle pure kit.
• Transformed bacteria were serially diluted to 1 :10000 on LB agar plates containing 100 pg/ml ampicillin and 2% (wt/v) glucose and grown overnight at 37°C. Library size was evaluated by colony counting. Fragmentation PCR and sequencing were conducted as described above. The remaining bacteria suspension was plated onto four 245 mm square LB agar dishes containing 100 pg/ml ampicillin and 2% (wt/v) glucose and grown overnight at 37 °C. This process was repeated until the library size was >10 7 . The library was recovered by first scraping bacteria from the 245 mm dishes in 8 mL of LB medium and combined into a 50 ml conical. The collected cell suspension was mixed with sterile glycerol (20%, v/v). Cell library aliquots were stored at -80 °C.
• Phage particles were pelleted by centrifugation for 10 minutes at 3,200 x g and 4 °C. Precipitated phage particles were re-suspended in 1 mL of ice-cold PBS, and transferred to a microcentrifuge tube. Phage particle suspension was centrifuged for 90 seconds at 16,000 x g and 4 °C to pellet any residual bacteria. Phage particles from the supernatant were reprecipitated by adding 250 pl of ice-cold 20% (wt/v) PEG6000/2.5 M NaCl solution, mixed by inversion, and incubated on ice for 10 minutes.
• Phage particles were pelleted by centrifugation at 16,000 xg at 4 °C for 20 minutes and re-suspended in 0.5 mL of ice-cold PBS. The remaining bacterial debris was removed by centrifugation for 90 seconds at 16,000 xg and 4 °C. Phage concentration was determined before panning via a titration method. Briefly, phage was serially diluted by factors of 10 in PBS. To a single well on a low-binding 96-well round-bottom culture plate, 10 pL of each dilution to a well with 90 pL of TGI cells (pre-grown suspension). Plates were incubated for 15 min at 37 °C to infect TGI cells.
• MMAF was conjugated to biotin-PEG2-amine and used for bio-panning and screening of anti-MMAE scFvs.
• 0.5 mg MMAF was dissolved in MES buffer (0.1 M MES, pH 4.7) / 30% DMF and mixed with 100 pg of biotin-PEG2-amine in MES buffer, then 1.5 mg of EDC dissolved in water was added. The reaction was incubated overnight at room temperature and stored at 4 °C. Streptavidin magnetic beads were washed three times with PBST (PBS + 0.05% Tween 20) and blocked with MPBS (PBS + 5% non-fat dry milk) for 2 h.
• PBST PBS + 0.05% Tween 20
• the streptavidin beads were incubated with 1 mL of 1 pM biotin- MMAF for 15 minutes, followed by three washes with PBST.
• the stock phage was diluted to 10 12 c.f.u. (colony forming unit)/mL in blocking buffer (2% milk PBS), and 1 ml of the diluted phage was added to the MMAF -biotin coated streptavidin beads and incubated for 2 hours.
• the stock phage was diluted 1 : 1 in blocking buffer.
• phage-infected TGI cells were grown overnight, serially diluted in 2xYT media, spread over individual culture plates containing selective medium (LB agar + 100 pg/mL ampicillin + 2% wt/v glucose), and incubated overnight at 37 °C.
• selective medium LB agar + 100 pg/mL ampicillin + 2% wt/v glucose
• a master plate was generated by inoculating a single colony into wells of a 96-well round-bottom culture plate filled with 100 pL of 2xTY supplemented with 100 pg/mL ampicillin, 2% (wt/vol) glucose and 15% (vol/vol) glycerol, and grown overnight at 37 °C, 300 rpm.
• Genes for scFv clones 3B8, 1H2 and 1B3 were codon optimized for A. coli and synthesized by GeneArt.
• the heavy and light chains for each clone were amplified by PCR and subsequently mutated using a PCR based random mutagenesis method.
• the purified mutagenesis product was combined, and the heavy and light chains ligated using an overlap extension PCR method.
• the full library was constructed following the methods used to construct the immunized phage library with the phagemid vector pComb3XSS used in place of the pADL-lOb phagemid vector.
• the phage containing supernatant was diluted 1 : 10 in 2% milk-PBS in individual wells of a 96-well plate with or without 1 nM free MMAE for 1-hour. Subsequently, the phage containing solution was transferred to the wells of a Nunc Maxisorb plate containing immobilized MMAF-pegl 1- biotin-neutravidin and incubated for 2-hours at room temperature on a shaking incubator. Plates were washed and bound phage detected. Colonies with a signal knockdown >70% were sent for DNA sequencing.
• the amino acid sequence for the variable heavy and variable light chain for clone MA24E2 were input into Abysis.
• Murine framework residues that occur with low frequency in human antibodies were conservatively mutated to an amino acid with high frequency.
• the murine and humanized sequences were input into Abodybuilder to create a structural model prediction for the human and murine sequences. Predicted structures were overlayed in ChimeraX to ensure the human sequence was predicted to have a similar structure as the murine sequence.
• the humanized sequence was expressed as a fab fragment (ABC3315) in ExpiCHO-S cells and purified using CaptureSelectTM CH1-XL affinity resin following manufacturer recommendations.
• Anti-MMAE fab was diluted to 1 nM and incubated with a range of concentrations of MMAE, MMAF and trastuzumab-vc-MMAE. Solutions were added in triplicate to individual wells of an ELISA plate with MMAF-pegl 1-biotin-streptavidin immobilized. The plate was incubated for two hours at room temperature on a shaking platform set at 300 rpm. Wells were washed 4x with PBST and 250 pL of a 1 :1,000 dilution of an anti -Human AP secondary added to each well and incubated for 1.5 hours. Wells were washed 2x with PBST and 2x with distilled water.
• a SR7500DC surface plasmon resonance was utilized for binding assessments of anti-MMAE fab.
• MMAF-peg 11 -biotin was flowed over the left channel of a neutravidin immobilized SPR chip. Subsequently unbound sites on both channels were blocked by injection of free biotin.
• ABC3315 fab was serially injected for 3 minutes at concentrations of 1.23, 3.70, 11.11, 33.33 and 100 nM with a 3-hour dissociation step after the final 100 nM injection.
• To evaluate ABC3315 binding to free MMAE and PV ABC3315 was conjugated to nhs-pegl2-biotin and injected over the left channel of a streptavidin SPR chip.
• MMAE was serially injected for 3 minutes at concentrations of 0.37, 1.11, 3.33, 10, and 30 nM with a 3-hour dissociation step after the final 30 nM injection.
• a second kinetic titration was completed with PV injected at conjugated MMAE concentrations of 1.23, 3.70, 11.11, 33.33 and 100 nM with a 3-hour dissociation step after the final 100 nM injection.
• Observed sensorgrams were fit using the kinetic titration module of ClampXP to obtain the association rate constant, dissociation rate constant and equilibrium dissociation rate constant.
• Ramos cells at a density of 50,000 cells/mL were split into individual wells of a 96-well flat-bottom culture plate.
• Media containing dilutions of MMAE or PV with or without 500 nM anti-MMAE fab was added to individual wells and the cells incubated for 4- days at 37 °C in a humified incubator with 5% CO2.
• 25 pL of 4 mg/ml MTT was added to individual wells and the plates incubated for 2-hours. Subsequently, MTT was solubilized overnight following the addition of 100-pL of 10% SDS, 0.1M HCL.
• the absorbance of individual wells was read at a wavelength of 550 nm and 690 nm.
• SKBR3 were trypsinsed and diluted to a concentration of 40,000 cells/mL. 100 pL of the cell suspension was added to individual wells of a 96-well u-bottom culture plate and cells allowed to attach overnight. The following day the culture media was aspirated and replaced with fresh media containing MMAE or trastuzumab-vc-MMAE with and without 500 nM anti-MMAE fab. Cells were incubated for 6-days with fresh media and drug dilutions added on days 3 and 5.
• variants of ABC3315 with improved affinity or humanness have been generated.
• a mutant scFv phage library was generated based on an ABC3315 scFv sequence using error prone PCR.
• Phage displaying scFv mutants were bound to MMAF-pegll-biotin-streptavidin magnetic beads for one hour and subsequently scFv displaying phage was dissociated from the beads for 24hr, 72hr and 120hr for the 1 st , 2 nd ’ and 3 rd round of panning, with 1 pM of free ABC3315 included in the dissociation buffer to prevent scFv phage from rebinding during the dissociation. Following each round of dissociation, remaining scFv displaying phage was eluted using 10 mg/mL trypsin for 20 minutes.
• Eluted phage was amplified following each round and amplified phage was used as the input titer for the following round of panning. Following the third round of panning a small amount of the phage eluate was titered and the remaining phage amplified. From the titered phage, 2-96 well master plates were generated, and phage screened using a dissociation ELISA. Briefly, 10 ug/ml streptavidin was immobilized onto wells of a Nunc® ImmobilizerTM Amino (Thermo Scientific) plate overnight.
• Bound phage with 1 pM MMAF was normalized to the bound phage for each clone with PBS only treatment. Wells that were observed to have minimal change with MMAF incubation were predicted to have slow dissociation rates and were sequenced.
• phagemid DNA was isolated, digested using Ndel and Xhol restriction enzymes, scFv DNA isolated using agarose gel electrophoresis, scFv DNA ligated into pet22b and transformed into SHuffle E. Coli cells. The following day, individual transformed cells were inoculated into the inner wells of an ELISA plate and grown overnight.
• ABC3315 mutants showed a similar binding signal to immobilized MMAF-pegl 1 -biotin as ABC3315, indicating retained binding function following mutagenesis.
• the VH F37V clone showed a greatly decreased binding signal relative to ABC3315 demonstrating that the phenylalanine residue at position 37 of the heavy chain is necessary for high affinity binding.
• ABC3315 and VH F27L were compared in a competitive SKBR3 cell cytotoxicity assay as described, however, the concentration of Fab was kept in a 5-fold excess relative to MMAE in-place of a constant 500 nM concentration of Fab for all MMAE dilutions.
• VYYCLASQFTTDYFEYWGQGNLVTVSS (SEQ ID NO:91)
• TKLTVL (SEQ ID NO: 121)
• TKLTVL (SEQ ID NO: 117)
• TKLTVL (SEQ ID NO: 119)
• TKLTVL (SEQ ID NO: 121)
• TKLTVL (SEQ ID NO: 121)
• TKLTVL (SEQ ID NO : 131 )
• TLVTVSS (SEQ ID NO: 100)
• TKLTVL (SEQ ID NO: 148)
• TLVTVSS (SEQ ID NO: 163)
• GQGTMVTVSS (SEQ ID NO: 165)
• IPDRFSGSKSGTSATLDITGLRTGDEADYYCGTWDSSLDTDVVFGGGTKLTVL SEQ ID NO: 167
• IHGDNNRPSGVPDRFSGSKSGTSAYLAISGLQAEDEADYYCQTYDSRSSGSRVFGGGT KVTVL (SEQ ID NO: 139)

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Peptides Or Proteins (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicinal Preparation (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=90014109

Family Applications (1)

Country Status (15)

Families Citing this family (2)

Family Cites Families (5)

• 2023
  • 2023-08-24 IL IL319054A patent/IL319054A/en unknown
  • 2023-08-24 AU AU2023329378A patent/AU2023329378A1/en active Pending
  • 2023-08-24 PE PE2025000438A patent/PE20251523A1/es unknown
  • 2023-08-24 JP JP2025511575A patent/JP2025528390A/ja active Pending
  • 2023-08-24 EP EP23858316.5A patent/EP4577568A2/en active Pending
  • 2023-08-24 GE GEAP202516711A patent/GEAP202516711A/en unknown
  • 2023-08-24 CA CA3265572A patent/CA3265572A1/en active Pending
  • 2023-08-24 CR CR20250060A patent/CR20250060A/es unknown
  • 2023-08-24 WO PCT/US2023/072852 patent/WO2024044709A2/en not_active Ceased
  • 2023-08-24 CN CN202380069240.4A patent/CN120035610A/zh active Pending
  • 2023-08-24 KR KR1020257009590A patent/KR20250060232A/ko active Pending
• 2025
  • 2025-02-21 MX MX2025002127A patent/MX2025002127A/es unknown
  • 2025-02-21 DO DO2025000040A patent/DOP2025000040A/es unknown
  • 2025-02-24 CO CONC2025/0002048A patent/CO2025002048A2/es unknown
  • 2025-02-24 JO JOJO/P/2025/0041A patent/JOP20250041A1/ar unknown

Also Published As

Similar Documents

Legal Events