Patent Application
20240261445
Not applicable.
Not applicable.
The present disclosure generally relates to the biomedical field, particularly to the methods of administering therapeutic antibodies which provide better clinical outcomes.
In one aspect of the present disclosure, a method of treating disseminated human lymphoma in a subject in need thereof is provided. The method comprises applying a theranostic treatment comprising at least one of [225Ac]Ac-Ofatumumab and [177Lu]Lu-ofatumumab.
In another aspect of the present disclosure, a method of treating disseminated human lymphoma in a subject in need thereof is provided. The method comprises applying a theranostic treatment comprising a combination of [225Ac]Ac-Ofatumumab and [89Zr]Zr-Ofatumumab.
In a further aspect of the present disclosure, a method of treating a CD20 disease in a subject in need thereof is provided. The method comprises applying a theranostic treatment comprising at least one of [225Ac]Ac-Ofatumumab, [177Lu]Lu-ofatumumab, and a combination of [225Ac]Ac-Ofatumumab and [89Zr]Zr-Ofatumumab.
Those of skill in the art will understand that the drawings disclosed herein are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Ac225 anti CD20 is a highly active cancer therapeutic and has been shown to cure lymphoma in mice if tumor burden is low. Lu177 anti CD20 can cure lymphoma, though best suited to high tumor burden situations. Zr89 anti CD20 images lymphoma well. Disclosed herein is Ac225 anti CD20 as a cancer therapeutic with doses and sequences defined by Zr89 anti CD20, as well as Lu177 anti CD20 imaging. While Ac 225 can only be imaged poorly, the present disclosure contemplates situations in which patients are imaged with low count anti CD20 images/quantitation, including a combination of Ac225 and Zr89.
In one aspect of the present disclosure, Ac225 anti CD20 is utilized as a cancer therapeutic with Zr89 anti cD20 to predict Ac225 anti CD20 biodistribution. In another aspect of the present disclosure, Ac225 anti CD20 is utilized as a cancer therapeutic with Lu177 anti cD20 to predict Ac225 anti CD20 biodistribution. In yet another aspect of the present disclosure, Ac225 anti CD20 is utilized as a cancer therapeutic with both Zr89 and Lu177 anti cD20 to predict Ac225 anti CD20 biodistribution. In some embodiments, treatment of non-malignant disease is conducted with a sequential imaging and dosing method.
In the present disclosure, a precision radiopharmaceutical therapy of NHL is developed which includes Zr89 ofatumomab (for imaging and predicting dosimetry), Lu177 ofatumomab for therapy and dosimetry, and ultimately Ac225 ofatumomab for the optimal treatment of low tumor burden foci. Aim 1 explores both disseminated and solid tumor lymphoma models (as well as the combination of both disease sites model) the efficacy of Lu 177 ofatumomab and Ac225 ofatumomab as monotherapies, either as single dose or multiple dose regimens, including combined and sequential dosing to determine which regimen is most curative. Aim 1 accordingly informs Aim 2, which is a first in man study of Zr89 ofatumomab for imaging and predicted tumor dosimetry. Aim 2 is conducted in patients with recurrent diffuse large B cell lymphoma and includes “no rituximab pre-dose” and rituximab pre-dose groups, to determine if and how pre-dosing with unlabeled antibody changes/improves radiation absorbed dose. In Aim 3, patients from Aim 2 receive a therapy dose of Lu177 ofatumomab in a standard phase I dose escalation trial with dose escalation based on predicted bone marrow radiation absorbed dose. These therapy doses are followed by post treatment dosimetry images to measure actual radiation absorbed dose to tumors (from Lu177) vs predicted dose from Zr89 PET. These aims and studies inform subsequent Ac225 ofatumomab clinical studies once stable Ac225 supplies for human use are available. Overall, the disclosed regimens have highly curative potential of otherwise incurable NHL.
The disclosed regimens can be varied with differing imaging, beta therapy and alpha therapy emitters given sequentially or in combination. The disclosed regimens are applicable to malignant and benign diseases with a variety of targets, and are shown using the CD20 targeting space. The disclosed approach has broad utility to CD20 positive non-malignant diseases as well as to other targets.
As described herein, Non-Hodgkin Lymphoma (NHL) is curable with the disclosed approach. Using patient specific dosimetry and sequencing of therapy, precise patient specific radiation dosing can be delivered. Further, the anti CD20 radiolabeled antibody labeled with Ac225 and developed herein has broad applicability in treating autoimmune diseases. Further, the sequencing approach described herein also has broad applicability (e.g. imaging/high beta energy therapy/alpha emitter therapy).
In some aspects of the present disclosure, Ac225 therapy (alone or in combination with Zr89 is utilized as a cancer therapeutic for NHL as well as other CD20 positive diseases, including multiple sclerosis (MS). In some embodiments, administration of treatment therapy is sequential with precision imaging, beta and alpha emitters, given sequentially or simultaneously.
Immunotherapies that target the CD20 protein expressed on most non-Hodgkin lymphoma cells have improved clinical outcomes, but relapse is common. 225Ac-labeled anti-CD20 ofatumumab was prepared and evaluated for in vitro characteristics and therapeutic efficacy in a murine model of disseminated human lymphoma.
225Ac was chelated by DOTA-ofatumumab, and radiochemical yield, purity, immunoreactivity, stability, and chelate number were determined. In vitro cell killing of CD20-positive, human B-cell lymphoma Raji-Luc cells was assayed. Biodistribution was determined as percentage injected activity per gram (% IA/g) in mice with subcutaneous Raji-cell tumors (n=4). [225Ac]Ac-ofatumumab biodistribution in C57BL/6N mice was performed to estimate projected human dosimetry. Therapeutic efficacy was tested in mice with systemically disseminated Raji-Luc cells, tracking survival, bioluminescence, and animal weight for a targeted 200 d, with single-dose therapy initiated 8, 12, or 16 d after cell injection, comparing no treatment, ofatumumab, and low (3.7 kBq/mouse) and high (9.25 kBq/mouse) doses of [225Ac]Ac-IgG and [225Ac]Ac-ofatumumab (n=8-10/cohort).
Radiochemical yield and purity were 32%±9% and more than 95%, respectively. Specific activity was more than 5 MBq/mg. Immunoreactivity was preserved, and more than 90% of the 225Ac remained chelated after 10 d in serum. Raji-Luc cell killing in vitro was significant, specific, and dose-dependent. In tumor-bearing mice, [225Ac]Ac-ofatumumab displayed low liver (7% IA/g) and high tumor (28% IA/g) uptake. Dosimetry estimates indicated that bone marrow is likely the dose-limiting organ. When therapy was initiated 8 d after cell injection, untreated mice and mice treated with cold ofatumumab or low- or high-dose [225Ac]Ac-IgG showed indistinguishable median survivals of 20-24 d, with extensive cancer-cell burden before death. Low- and high-dose [225Ac]Ac-ofatumumab profoundly (P<0.05) extended median survival to 190 d and more than 200 d (median not determinable), with 5 and 9 of 10 mice, respectively, surviving at study termination with no detectable cancer cells. Surviving mice treated with high-dose [225Ac]Ac-ofatumumab showed reduced weight gain versus naïve mice. When therapy was initiated 12 d, but not 16 d, after cell injection, high-dose [225Ac]Ac-ofatumumab significantly extended median survival to 40 d but was not curative.
In an aggressive disseminated tumor model, [225Ac]Ac-ofatumumab was effective at cancer-cell killing and curative when administered 8 d after cell injection. [225Ac]Ac-ofatumumab has substantial potential for clinical translation as a next-generation therapeutic for treatment of patients with non-Hodgkin lymphoma.
Although immunotherapies that target CD20 on most non-Hodgkin lymphoma (NHL) cells have improved patient outcomes, current therapies are inadequate because many cases are, or become, refractory or undergo relapse. Here, the third-generation human anti-CD20 antibody ofatumumab was labelled with 177Lu, determined the in vitro characteristics of [177Lu]Lu-ofatumumab, estimated human dosimetry, and assayed tumor targeting and therapeutic efficacy in a murine model of disseminated NHL.
CHX-A99-diethylenetriaminepentaacetic acid-[177Lu]Lu-ofatumumab was prepared. Radiochemical yield, purity, in vitro immunoreactivity, stability, (n 5 7), affinity, and killing of CD20-expressing Raji cells (n5 3) were evaluated. Human dosimetry was estimated from biodistribution studies as percentage injected activity per gram using C57BL/6N mice. Tissue and organ biodistribution was determined in R2G2 immunodeficient mice with subcutaneous Raji-cell tumors. Therapy studies used R2G2 mice with disseminated human Raji-luc tumor cells (n 5 10 mice/group). Four days after cell injection, the mice were left untreated or were treated with ofatumumab, 8.51 MBq of [177Lu]Lu-IgG, or 0.74 or 8.51 MBq of [177Lu]Lu-ofatumumab. Survival, weight, and bioluminescence were tracked.
Radiochemical yield was 93% 6 2%, radiochemical purity was 99% 6 1%, and specific activity was 401 6 17 MBq/mg. Immunoreactivity was substantially preserved, and more than 75% of 177Lu remained chelated after 7 d in serum. [177Lu]Lu-ofatumumab specifically killed Rajiluc cells in vitro (P, 0.05). Dosimetry estimated that an effective dose for human administration is 0.36 mSv/MBq and that marrow may be the dose-limiting organ. Biodistribution in subcutaneous tumors 1, 3, and 7 d after [177Lu]Lu-ofatumumab injection was 11, 15, and 14 percentage injected activity per gram, respectively. In the therapy study, median survival of untreated mice was 19 d, not statistically different from mice treated with 8.51 MBq of [177Lu]Lu-IgG (25 d). Unlabeled ofatumumab increased survival to 46 d, similar to 0.74 MBq of [177Lu]Lu-ofatumumab (59 d), with both being superior to no treatment (P, 0.0003). Weight loss and increased tumor burden preceded death or killing of the animal for cause. In contrast, treatment with 8.51 MBq of [177Lu]Lu-ofatumumab dramatically increased median survival (0.221 d), permitted weight gain, eliminated detectable tumors, and was curative in 9 of 10 mice.
[177Lu]Lu-ofatumumab shows favorable in vitro characteristics, localizes to tumor, and demonstrates curative therapeutic efficacy in a disseminated lymphoma model, showing potential for clinical translation to treat NHL.
The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
The term “formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
The term “pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc., may also be used.
The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
A “stable” formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0° ° C. and about 60° C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.
Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing disseminated human lymphoma or non-Hodgkin's lymphoma. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.
According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
When used in the treatments described herein, a therapeutically effective amount of a theranostic treatment agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to cure disseminated human lymphoma or non-Hodgkin's lymphoma.
The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as in some embodiments the necessary therapeutically effective amount is reached by administration of a number of individual doses.
Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes reversing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.
Administration of theranostic treatment agents can occur as a single event or over a time course of treatment. For example, theranostic treatment agents can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Depending upon the embodiment, certain conditions extend treatment from several days to several weeks. For example, in some embodiments treatment extends over one week, two weeks, or three weeks. For more chronic conditions, treatment extends from several weeks to several months or even a year or more.
Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for disseminated human lymphoma.
A theranostic treatment agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a theranostic treatment agent can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a theranostic treatment agent, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a theranostic treatment agent an antibiotic, an anti-inflammatory, or another agent. A theranostic treatment agent can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a theranostic treatment agent can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.
Active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.
An effective dose range of a therapeutic can be extrapolated from effective doses determined in animal studies for a variety of different animals. In general, a human equivalent dose (HED) in mg/kg can be calculated in accordance with the following formula (see e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, which is incorporated herein by reference):
Use of the Km factors in conversion results in more accurate HED values, which are based on body surface area (BSA) rather than only on body mass. Km values for humans and various animals are well known. For example, the Km for an average 60 kg human (with a BSA of 1.6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Km of 25. Km for some relevant animal models are also well known, including: mice Km of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Km of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).
Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment, and the potency, stability, and toxicity of the particular therapeutic formulation.
The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage may be adjusted by the individual physician in the event of any complication.
In some embodiments, the theranostic treatment agents may be administered in an amount from about 1 mg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 25 mg/kg, or about 1 mg/kg to about 15 mg/kg, or about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg to about 5 mg/kg, or about 3 mg/kg. In some embodiments, theranostic treatment agents may be administered in a range of about 1 mg/kg to about 200 mg/kg, or about 50 mg/kg to about 200 mg/kg, or about 50 mg/kg to about 100 mg/kg, or about 75 mg/kg to about 100 mg/kg, or about 100 mg/kg.
The effective amount may be less than 1 mg/kg/day, less than 500 mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It may alternatively be in the range of 1 mg/kg/day to 200 mg/kg/day.
In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
Cells generated according to the methods described herein can be used in cell therapy. Cell therapy (also called cellular therapy, cell transplantation, or cytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting T-cells capable of fighting cancer cells via cell-mediated immunity can be used in the course of immunotherapy, grafting stem cells can be used to regenerate diseased tissues, or transplanting beta cells can be used to treat diabetes.
Stem cell and cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.
Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogeneic strategy is that unmatched allogenic cell therapies can form the basis of “off the shelf” products.
Autologous cell therapy or autologous transplantation uses cells that are derived from the subject's own tissues. Some embodiments involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.
Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig derived cells can be transplanted into humans. Xenogeneic cell therapies can involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.
Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.
Also provided are screening methods. The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.
Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).
Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about −2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.
When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.
Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.
The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 Å to about 15 Å.
Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to theranostic treatment agents. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.
In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.
Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the found to function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
This Example describes a novel therapeutic approach for treating cancer and other diseases, including lymphoma and MS, with [225Ac]Ac-Ofatumumab alone or in combination with [89Zr]Zr-Ofatumumab.
Non-Hodgkin lymphoma (NHL) will be diagnosed in approximately 80,000 patients and cause over 20,000 deaths in the United States in 2023. Although chemotherapy is initially effective, many patients, even with low-grade lymphomas, relapse. This has driven development of therapeutic antibodies that target the CD20 protein expressed on the surface of mature B cells and most NHL cells, as most are of B-cell origin. Anti-CD20 immunotherapy has a highly favorable safety profile, significantly improves the outcomes of most patients, and, along with chemotherapy, is now part of the standard of care for many cases of NHL.
The chimeric mouse-human monoclonal antibody, rituximab, was the first Food and Drug Administration-approved anti-CD20 therapeutic, with others subsequently developed for improved biologic and pharmacologic properties, including fully human ofatumumab. Ofatumumab binds CD20 with high affinity, allowing targeting of cells with low CD20 expression, including those with resistance to rituximab. As a type I anti-CD20 antibody, ofatumumab is effectively internalized, which benefits imaging and therapy using residualizing radiometals, such as 89Zr and 225Ac.
Lymphoma is highly susceptible to ionizing radiation; however, external-beam irradiation is used sparingly in the disseminated setting. To overcome the limitations of external-beam radiotherapy, systemically administered β-particle-emitting radioimmunotherapies to anti-CD20 have been translated into 2 Food and Drug Administration-approved drugs: murine 131I-tositumomab (Bexxar; GlaxoSmithKline) and 90Y-ibrutumomab (Zevalin; Acrotech Biopharma LLC). Although studies show long-term safety and effectiveness, Bexxar has been discontinued commercially in the United States, and Zevalin is used infrequently.
225Ac has gained increased use as a therapeutic radionuclide, with its 10-d half-life matching well the pharmacokinetics of intact antibodies. In its decay pathway, 225Ac yields 4 net α-particles with high linear energy transfer and short pathlengths, providing effective radiotoxicity to targeted tumor cells while relatively sparing nontargeted cells.
It has been demonstrated that [89Zr]Zr-ofatumumab has excellent uptake into human lymphoma xenografts and enables in vivo localization using PET as well as, or better than, [89Zr]Zr-rituximab. As disclosed herein, [225Ac]Ac-ofatumumab was prepared and demonstrated to have potent cytotoxicity to CD20-expressing cells in vitro and in vivo. Exemplary embodiments of the present disclosure include [225Ac]Ac-ofatumumab and combination [225Ac]Ac-ofatumumab+[89Zr]Zr-ofatumumab treatments for cancer and other CD20 diseases such as MS In therapeutic studies using an aggressive, disseminated murine model of human lymphoma, [225Ac]Ac-ofatumumab shows excellent, often curative, efficacy.
Chemicals and reagents are listed in TABLE 2. Water (MilQ Integral 5 system; Millipore) was treated with a 50 g/L concentration of Chelex 100 (Bio-Rad Laboratories, Inc.). Raji and Raji-Luc cells were cultured in RPMI medium with 10% fetal bovine serum.
DOTA was dissolved in H2O and conjugated to antibodies as previously described. For 225Ac chelation, 1.85 MBq of 225Ac in 0.2 M HCl was added to 2 M tetraethyl-ammonium-acetate to obtain pH 6.0. One hundred micrograms of DOTA antibody prepared at an 8:1 DOTA-to-antibody molar ratio were added, and the reaction was brought to 150 μL with 20 mM sodium acetate, 150 mM NaCl (pH 7.0), and 15 μL of a 10 mg/mL solution of sodium ascorbate. After 4 h at 37° C., diethylenetriaminepentaacetic acid (pH 7.0) was added to 5 mM final concentration for 10 min followed by size-exclusion column purification into saline with sodium ascorbate added to 10 μg/mL. All quantifications were at secular equilibrium, using a Capintec CRC 55tW dose calibrator and a Beckman 8000 γ-counter with a 250- to 480-keV energy window or by scanning of thin-layer chromatography strips. Fast protein liquid chromatography, thin-layer chromatography, and mass spectrometry were performed as previously described.
Immunoreactivity was assayed by Raji-cell binding as previously described, without or with cold ofatumumab blocking. To assay stability, [225Ac]Ac-ofatumumab or 225Ac was added to human serum, incubated at 37° C., and assayed by thin-layer chromatography as previously described.
To assay cell killing, 5×105 Raji-luciferase cells in 1 mL of RPMI medium with 10% heat-inactivated fetal bovine serum were added per well, followed by no antibody, native ofatumumab, [225Ac]Ac-IgG, or [225Ac]Ac-ofatumumab. When used, the antibody mass was 0.1 μg/well. Medium was exchanged after 24 h, and viability assays were performed 48 h later by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) or bioluminescence imaging as previously described.
The Washington University in St. Louis Institutional Animal Care and Use Committee approved the animal studies. Eight- to 10-wk-old R2G2 mice (no. 021; Envigo) were inoculated subcutaneously with 5×106 Raji-Luc cells. When palpable tumors were present, 8-11 μg (4.07 kBq) of [225Ac]Ac-ofatumumab were administered intravenously and biodistribution analyzed 7 d later. The femur was measured after marrow extraction.
R2G2 mice were injected intravenously with 1×106 Raji-Luc cells. In a first study, 8 d later, the mice either were untreated or were treated with ofatumumab or 3.7 or 9.25 kBq/mouse of [225Ac]Ac-IgG or [225Ac]Ac-ofatumumab. In a second study, 12 or 16 d after cell injection, the mice either were untreated or were treated with 9.25 kBq of [225Ac]Ac-ofatumumab. The administered antibody mass was adjusted to 20 μg/mouse of IgG or ofatumumab. Bioluminescent images were acquired and quantified as previously described and normalized to the first imaging time point. The mice were humanely euthanized if they had hind-limb paralysis (HLP), more than a 20% weight loss, or morbidity or reached the scheduled study termination point.
Biodistribution and dosimetry studies were performed on 6- to 8-wk-old female C57BL/6N mice (no. 556; Charles River) injected intravenously with 3 μg (˜3.7 kBq) of [225Ac]Ac-ofatumumab. At selected time points after injection of [225Ac]Ac-ofatumumab, the mice were euthanized and organs γ-counted at secular equilibrium to determine decay-corrected percentage injected activity per gram (% IA/g). Bone (tibia and fibula) was counted after marrow separation.
Integrated time-activity curves from the murine data and the mean absorbed dose (D) were calculated according to MIRD methodology using the formula D=A×Δ×ϕ, where A is the cumulated activity, Δ is the mean α-particle energy, and ϕ is the absorbed fraction, with extrapolation to infinity, yielding a maximally conservative estimate. α particles were assumed to deposit all their energy locally (ϕ=1). The trapezoidal rule was used to integrate the time-activity curve of the α-particles emitted from the decay of 225Ac and its α-particle—emitting daughters (221Fr, 217As, 213Bi, and 213Po) using values from International Commission on Radiological Protection publication 107, with all daughter decays assumed to occur in the same organ as the 225Ac decay, yielding a Δ of 4.42−12 J/(Bq·s) for 225Ac and its daughters. These mouse data were extrapolated to the adult female human model using the relative organ mass scaling method. Equilibrium in the decay chain and no translocation during the decay between succeeding disintegrations were assumed. Thus, the same estimated integrated time-activity curve obtained for 225Ac was applied to its daughters. The absorbed dose of [225Ac]Ac-ofatumumab was summed after applying weighting factors in the 2 possible pathways, 2% for 209Ti and 98% for 213Po. A relative biological effectiveness of 5 for α-particles was used in the calculation of sieverts.
GraphPad Prism software, version 8.4.3, was used for statistical analyses. A P value of less than 0.05 was considered statistically significant, with statistical tests noted in the text or figure legends. Data are shown as mean±SD.
[225Ac]Ac-DOTA-ofatumumab was prepared using a 1-step method and characterized (
Biodistribution with Subcutaneous Raji-Cell Tumors
Immunodeficient R2G2 mice were used (B6;129-Rag2tm1Fwa II2rgtm1Rsky/DwlHsd) that are proficient in double-strand DNA-break repair. Prkdcscid mice that lack double-strand DNA repair because of the scid mutation are known to show artifactual radiosensitivity to DNA damage, such as that induced by α-particles. Compared with Prkdescid mice, it was expected that the nontargeted (nontumor) cells in R2G2 mice will better reflect the response of nontargeted (nontumor) cells in humans to α-particle transit, as both tumor and nontumor cells are proficient in double-strand DNA-break repair.
The biodistribution of [225Ac]Ac-ofatumumab was evaluated 7 d after injection in mice bearing subcutaneous Raji tumors (
To investigate in vitro cytotoxicity, Raji-Luc cells were incubated with medium only, native (cold) ofatumumab, [225Ac]Ac-IgG, or [225Ac]Ac-ofatumumab for 24 h, followed by medium exchange and, 48 h later, viability assays (
The biodistribution of [225Ac]Ac-ofatumumab was evaluated using non-tumor-bearing, wild-type C57BL/6N mice (TABLE 3). Blood [225Ac]Ac-ofatumumab levels were high (43±6% IA/g) at 4 h after injection and slowly fell to 15% IA/g at 12 d after injection. Uptake of 225Ac-ofatumumab was 5-8 IA/g in bladder, 8-11% IA/g in kidney, and 6-12% IA/g in marrow, with a gradual diminution in marrow over time. Liver uptake was 10-15% IA/g, with perhaps some contribution by dechelated 225Ac, which accumulates in the liver. Fecal 225Ac levels were consistent with [225Ac]Ac-ofatumumab or metabolites being excreted via the gastrointestinal route.
[225Ac]Ac-ofatumumab human radiation dosimetry estimates were then determined for a human female model (TABLE 1). As free 213Bi in the kidney was not evaluated, this absorbed dose may be somewhat underestimated. Extrapolated human radiation dose estimates reveal heart wall (1,919 mSv/MBq) as the organ with the highest predicted dose, followed by liver, spleen, and red marrow at 1,833, 1,803, and 1,620 mSv/MBq, respectively. Marrow is likely the dose-limiting organ. The calculated effective dose equivalent was 1,496 mSv/MBq.
The magnitude, duration, and tumor targeting of the radiopharmaceutical in the biodistribution studies, as well as the in vitro tumoricidal activity, motivated an in vivo lymphoma treatment study. Therapeutic efficacy was evaluated in R2G2 mice with intravenously injected Raji-Luc cells, which become widely disseminated. This model recapitulates many features of clinical NHL, as it invades multiple organs, including the hematopoietic compartment. HLP is a frequent cause for censoring.
First, the maximal tolerated dose of [225Ac]Ac-ofatumumab in naïve R2G2 mice was identified. Eighty days after injection, 3.7, 11.1, 18.5, and 37 kBq/mouse yielded 5 of 5, 4 of 5, 3 of 5, and 0 of 5 survivors, respectively. Thus, for therapy single injections were used for the nonmyeloablative doses of 3.7 kBq/mouse (low dose) and 9.25 kBq/mouse (high dose).
In the first therapy study, 8 d after cell injection the mice were randomized to remain untreated or to be treated with native ofatumumab or low or high doses of [225Ac]Ac-IgG or [225Ac]Ac-ofatumumab (n=10 mice per cohort). Survival, tumor burden, and weight were monitored for 200 d. In untreated mice, median survival was 21 d, with 9 of 10 mice succumbing before 29 d (
Control radiolabeled nonspecific antibody, [225Ac]Ac-IgG, at low and high doses yielded median survivals of 20 and 24 d, respectively (
In contrast, low- or high-dose [225Ac]Ac-ofatumumab treatment increased median survival to 190 d and more than 200 d (median survival was nondeterminable as there were <50% deaths), respectively (
All mice treated with low- or high-dose [225Ac]Ac-ofatumumab that survived had effective cancer-cell suppression with no detectable Raji-Luc cells at study termination (
Because of its 10-d half-life, it was desirable to determine how rapidly [225Ac]Ac-ofatumumab treatment kills cancer cells. Comparison of bioluminescence from study day 3 to day 20 (5 d before and 12 d after starting therapy) revealed a continued increase in cancer-cell numbers in all cohorts except if treated with [225Ac]Ac-ofatumumab (
To investigate systemic toxicity of [225Ac]Ac-ofatumumab, animal weights in the therapy study initiated 8 d after cell injection were determined (
Mice in the low-dose [225Ac]Ac-ofatumumab-treated cohort that survived to study termination showed a continuous gradual weight gain (
Next, it was determined whether tumor burden at the time of therapy is a relevant parameter in treatment outcome. Treatment was delayed from 8 d until either 12 or 16 d after cell injection (untreated mice typically die at 19-21 d after cell injection) to provide a larger pretreatment disseminated disease burden, comparing no treatment with high-dose (9.25 kBq) [225Ac]Ac-ofatumumab. To assay tumor-cell burden over time, bioluminescence in untreated mice 8, 12, and 16 d after cell injection was compared (
When therapy was initiated 16 d after cell injection, [225Ac]Ac-ofatumumab provided no survival benefit versus no treatment (
In contrast, a single treatment with [225Ac]Ac-ofatumumab 12 d after cell injection increased median survival to 40 d (
The present disclosure further validates the use of anti-CD20 antibodies for radioimmunotherapy of NHL. [225Ac]Ac-ofatumumab was produced with high immunoreactivity, radiochemical yield, and purity and with stable chelation of 225Ac in vitro and in vivo. [225Ac]Ac-ofatumumab specifically killed CD20-expressing cells in vitro and showed high tumor uptake in vivo. In therapeutic studies using a murine model of disseminated human lymphoma, a single [225Ac]Ac-ofatumumab treatment showed excellent efficacy, with curative ability except for very advanced disease.
[225Ac]Ac-ofatumumab was prepared using a mild 1-step procedure in which 225Ac is directly chelated to DOTA-conjugated antibody at 37ºC. Other investigators also have used this approach. 225Ac was stably chelated, and uptake of [225Ac]Ac-ofatumumab uptake by CD20-expressing subcutaneous tumors (28% IA/g) was similar to the 33% IA/g of [89Zr]Zr-DFO-ofatumumab. The approximately 5 MBq/mg specific activity obtained for [225A]Ac-ofatumumab was sufficient for preclinical use and should enable scaling up for clinical use.
The major finding of the current study was the high therapeutic potency of [225Ac]Ac-ofatumumab in mice with an aggressive murine model of disseminated human lymphoma, similar to micrometastatic disease in humans. When administered 8 d after cell injection, control unlabeled ofatumumab and low (3.7 kBq/mouse) or high (9.25 kBq/mouse) doses of nontargeted [225Ac]Ac-IgG did not improve median survival compared with untreated mice or inhibit cancer-cell growth. In contrast, both low and high doses of [225Ac]Ac-ofatumumab rapidly and significantly inhibited cancer-cell growth and increased median survivals to 190 and more than 200 d, respectively. Half the mice in the low-dose group survived, and none of the animals in the high-dose group had disease- or treatment-related mortality. All mice in these 2 cohorts that survived until study termination showed apparent complete elimination of cancer cells, as no cancer cells were detected at study completion on day 200. Also, the potential for systemic anticancer effects was tested in disease settings of further progression. Twelve days after cell injection, high-dose [225Ac]Ac-ofatumumab killed cancer cells and significantly extended median survival to 40 d, showing therapeutic benefit even with a high pretreatment burden of cancer cells, although the therapy was not curative. Not surprisingly, there was a limit to therapeutic efficacy; therapy initiated 16 d after cell injection was unable to improve survival. This lack of benefit was likely because approximately 3 d before expected death was not enough time to allow for sufficient targeting and absorbance of the dose of intact radiolabeled antibody, and possibly also because of altered pharmacokinetics due to the high cancer-cell burden.
Dosimetry of organs from [225Ac]Ac-ofatumumab-injected C57BL/6N mice extrapolated to an adult female human model suggests that marrow will be the dose-limiting organ, as is common with unsealed, intact antibody-based radiotherapeutics, including Bexxar and Zevalin. It is worth noting that uptake in nontumor target organs can often be significantly reduced by predosing with unlabeled antibody, which improves lesion-to-background-organ ratios, as used in the Zevalin and Bexxar therapeutic regimens and in other applications. Fully human ofatumumab is unlikely to induce an immune response, increasing the potential for fractionated dosing of [225Ac]Ac-ofatumumab to ameliorate myelotoxicity. In addition, autologous stem-cell transplantation after radioimmunotherapy, as used in some Zevalin and Bexxar protocols, may be useful. Development of a theranostic partner, such as [89Zr]Zr-ofatumumab, for personalized image-based dosimetry is also considered herein.
Notably, low-dose [225Ac]Ac-ofatumumab treatment 8 d after cell injection was curative in half the mice, which displayed weight gains similar to those of control naïve mice without tumor cells or therapy. The 5 nonsurviving mice in this cohort showed effective repression of cancer-cell growth for approximately 50-140 d and gains of weight over this time of remission. Thus, fractionated cycles of this low-dose therapy may induce cancer-cell elimination, as well as curative efficacy in even more mice while maintaining reduced toxicity. Mice treated with high-dose [225Ac]Ac-ofatumumab showed reduced weight gains compared with naïve mice, indicating some systemic toxicity. Some of this may result from kidney distribution of the 225Ac daughter radionuclide, 213Bi, although this possibility remains to be confirmed. However, if so, there are approaches that may ameliorate such toxicity.
As described herein, the therapeutic efficacy of β-particle-emitting [177]Lu-ofatumumab was tested using the murine disseminated Raji-Luc lymphoma model of the present disclosure. When therapy was started 4 d after cell injection, 8.51 MBq of [177Lu]Lu-ofatumumab showed remarkable therapeutic efficacy, with apparent complete elimination of tumor cells and no disease-related deaths. As disclosed herein, it was further indicated that when therapy was initiated 8 d after cell injection, [177Lu]Lu-ofatumumab was ineffective, indicating potential superiority of [225Ac]Ac-ofatumumab under this condition. Similar observations were noted previously using a murine disseminated multiple-myeloma model, comparing [177Lu]Lu- and [225Ac]Ac-daratumumab.
Therapeutic efficacy of rituximab (an internalizing anti-CD20 antibody, like ofatumumab) radiolabeled with 213Bi (half-life [t½], 45.6 min; 1 net α-particle) has been reported, applying a similar disseminated Raji-Luc model using Prkdcscid mice. Cancer-cell killing was effective, and cures were common when the single-dose treatment was started 4 d after injection of 1×106 Raji-Luc cells. However, single-dose [213Bi]Bi-rituximab treatment was much less effective when initiated 7 d after cell injection, being markedly less effective than single-dose treatment with [225Ac]Ac-ofatumumab initiated 8 d after cell injection as found in the current study, although in a somewhat different animal system. These results may in part relate to the decay rates of 213Bi versus 225Ac and the localization times for intact antibodies to tumor. However, if targets are readily accessible, 213Bi can deliver a high dose rate, but if there exist groups of cells, or even solid tumors, with reduced antibody accessibility, sufficient penetration or dose deposition may not occur before radioactive decay, rendering the therapy less effective and yielding superior results with the longer-lived 225Ac. In this view, for antibody-mediated radioimmunotherapy for larger tumors, 225Ac may be a more appropriate choice for α-particle-mediated therapy. Further evaluation of the comparative therapeutic and off-target effects of 213Bi-versus 225Ac-labeling for radioimmunotherapy with intact antibodies is of interest.
Other investigators have studied α-emitters for radioimmunotherapy of NHL using preclinical models. Previous therapeutic benefit had been found using a pretargeted approach with a 213Bi-labeled anti-CD20 1F5(ScFv)4SA molecule. Similarly, [211At]At-1F5 (211At t½, 7.2 h; 1 net α-particle) was 80% curative 6 d after intravenous cell injection with supporting stem cell transplantation but only poorly effective on subcutaneous tumors, perhaps limited by radioactive decay before tumor penetration. [212Pb]Pb-rituximab (212Pb t½, 10.6 h; 1 net α-particle) in a disseminated model improved survival times with therapy initiated at both low- and high-tumor burdens. [227Th]Th-rituximab (227Th t½, 18.7 d; 5 net α-particles) was often curative of small subcutaneous tumors and was superior to [90Y]Y-ibritumomab-tiuxetan, although targeting of the 223Ra daughter to bone adds complexity. Similarly, treatment with [149Tb]Tb-rituximab (149Tb t½, 4.2 h, 1 α-particle) shortly after intravenous injection of Daudi cells significantly increased survival. The surprising and excellent results with 225Ac labeling disclosed herein likely reflect a very good match between antibody-specific targeting times to tumor and the half-life of this α-particle emitter. In conclusion, a single dose of [225Ac]Ac-anti-CD20 ofatumumab showed excellent therapeutic efficacy in a murine model of human disseminated lymphoma and was often curative. Fractionated dosing may improve efficacy. With increasing use of targeted radiotherapies, a renewed application of radioimmunotherapy targeting CD20 for NHL seems warranted, given the exceptional therapeutic efficacy of [225Ac]Ac-anti-CD20 ofatumumab and remaining unmet clinical needs in this disease.
[225Ac]Ac-ofatumumab shows good in vitro characteristics and effectively targets CD20-expressing tumor xenografts. [225Ac]Ac-ofatumumab showed curative efficacy in a murine model of disseminated human lymphoma.
This Example describes a novel therapeutic approach for treating cancer and other diseases, including lymphoma and MS, with [177Lu]Lu-Ofatumumab.
Non-Hodgkin lymphoma (NHL) is a common hematologic malignancy, with over 80,000 new cases and 20,000 deaths estimated for the United States in 2022 (1). The standard of care for many cases of NHL involves chemotherapy and immunotherapy targeting the CD20 protein, which is highly expressed on most NHL cells, with murine/human chimeric rituximab used most commonly. Although this chemotherapy-with-immunotherapy combination is usually initially effective, many cases are refractory or undergo relapse, indicating the need for improved therapies.
Radioimmunotherapy joined clinical practice 2 decades ago with Food and Drug Administration approval of 2 anti-CD20 radioimmunotherapies for lymphoma: Zevalin ([90Y]Y-ibritumomab tiuxetan; Acrotech Biopharma, Inc.) and Bexxar (tositumomab and 131I-tositumomab; GlaxoSmithKline), which use murine-derived anti-bodies radiolabeled with b-particle-emitting radioisotopes. Because of potential immune reactions, these antibodies were approved for only a single therapeutic dose. 90Y (half-life [t1/2], 2.7 d) emits high-energy β-particles (average, 934 keV), whereas 131I emits lower-energy β-particles (average, 187 keV), with average ranges in tissue of 3,800 mm and 360 mm, respectively, enabling killing over many cell diameters. Thus, in addition to working against individual tumor cells, b-particles may work against larger tumors, tumor-cell aggregates with imperfect antibody access, and heterogeneous tumors, although with potential off-target damage. Despite long-term safety and clinical effectiveness, Bexxar has been discontinued in the United States and Zevalin is applied infrequently, in part because of economic and logistic concerns that were present when they were introduced and because of competing nonradioactive therapies.
Some concerns that limited the use of Bexxar and Zevalin have been overcome with greater integration of radiopharmaceutical therapy into medicine (5), as exemplified by the Food and Drug Administration approval of 177Lu-labeled agents for prostate cancer treatment (Pluvicto; Advanced Accelerator Applications (6)) and neuroendocrine tumors (Lutathera; Advanced Accelerator Applications (7)). 177Lu (t1/2, 6.6 d) emits β-particles of 149 keV on average, with an average tissue range of 220 μm. Emission of low-abundance γ-particles by 177Lu permits imaging by SPECT.
Recently, ofatumumab, a third-generation anti-CD20 fully human antibody, was developed. Ofatumumab is a type I antibody that is internalized after CD20 binding (8). We showed by biodistribution and PET imaging studies that [89Zr]Zr-DFO-ofatumumab targets CD20-positive subcutaneous xenograft tumors as well as [89Zr]Zr-DFO-rituximab (9).
Here, we describe the synthesis and evaluation of [177Lu]Lu-ofatumumab. We present in vitro characteristics, dosimetry estimation, and subcutaneous tumor targeting. We also show that [177Lu]Lu-ofatumumab therapy results in long-term survival and elimination of tumor cells in a murine model of disseminated human lymphoma.
Ofatumumab (IgG1 κ; Novartis) was purchased from the Washington University clinical pharmacy, and human IgG1 κ was purchased from BioXcel. Raji cells and Raji-luc cells stably expressing luciferase (10) were cultured as previously described (9). SCN-CHX-A″-DTPA ({[(R)-2-amino-3-(4 isothiocyanatophenyl)propyl]-trans(S,S)-cyclohexane-1,2-diamine-pentaacetic acid}) was from Macrocyclics, size-exclusion chromatography columns from Fisher Scientific, and d-luciferin from GoldBio. Sigma provided human serum, sodium acetate, diethylenetriamine pentaacetate, tetramethylammonium acetate, and I-sodium ascorbate. 177Lu from the University of Missouri was dissolved in 0.2 M HCl. Silica gel thin-layer chromatography paper was from Agilent, and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) salt assay was from Promega.
Antibody was incubated with SCN-CHX-A″-DTPA in 0.1 M sodium carbonate, pH 9.0, at a chelator-to-antibody molar ratio of 8:1 for 1 h at 37° C. and purified by size-exclusion chromatography into 0.5 M NH4OAc, pH 7.0. A 477-MBq quantity of 177Lu was added to 400 μg of CHX-A″-DTPA-antibody with 20 mM NH4OAc, pH 7.0. After 2 h at 37° C., DTPA was added to 5 mM final concentration, followed by size-exclusion chromatography purification into saline and the addition of a 10 mg/mL concentration of I-sodium ascorbate. Thin-layer chromatography and fast-performance liquid chromatography were done as previously described (9). Radiochemical yield was assayed with a CRC55-tW dose calibrator. Chelate number was determined using a Fisher Scientific Exactive Plus EMR mass spectrometer operating at a mass (m)-to-charge (z) range from 800 to 12,000 and a resolving power of 8,750 or 17,500 at 300 m/z. Data were analyzed using Protein Metric Intact software.
To assay stability, 14.8 MBq of [177Lu]Lu-ofatumumab or 177Lu were added to 10% human serum in 20 mM NaOAc 150 mM NaCl pH 7.0 with 10 mg/mL L-SA and incubated at 37° C. Another aliquot of [177Lu]-ofatumumab was incubated at 4° C. in buffer without serum and with 10 mg/mL L-SA. Aliquots were analyzed by thin-layer chromatography at 0, 1, 5, and 7 d. Immunoreactivity was assayed as previously described (9). To assay affinity, 2.5×106 Raji cells without or with 10 μg of ofatumumab were incubated with [177Lu]Lu-ofatumumab, washed after 4 h at 23° C., and γ-counted. To assay cell killing, 2×106 Raji-luc cells in 1 mL of RPMI medium with 10% heat-treated fetal bovine serum were exposed to no treatment, ofatumumab, [177Lu]Lu-IgG, or [177Lu]Lu-ofatumumab, with cognate unlabeled antibody added to 20 μg total. After 14 h at 37° C., the cells were washed and 20% were resuspended in fresh medium for an additional 168 h followed by MTS assay.
Biodistribution of [177Lu]Lu-Ofatumumab in Mice with Subcutaneous Raji Tumors
The Washington University in St. Louis Animal Care and Use Committee approved the animal studies. Biodistribution with tumor-bearing mice used female 6- to 8-wk-old immunodeficient Rag2-IL2rg (R2G2, B6; 129-Rag2tm1FwaII2rgtm1Rsky/DwlHsd) mice (Envigo) injected subcutaneously with 5×106 Raji-luc cells. Mice with palpable tumors were injected intravenously with 10-20 μg of [177Lu]Lu-ofatumumab and killed 1, 3, or 7 d later. Distribution was calculated as decay-corrected percentage injected activity per gram (% IA/g) using a Beckman 8000 γ-counter and a 1- to 500-keV window.
Naïve 5- to 6-wk-old C57BI6/N mice injected intravenously with 370 kBq (10 μg) of [177Lu]Lu-ofatumumab were killed 4 h, 1 d, 2 d, 5 d, or 11 d later, and tissue and organs were γ-counted. Bone was counted after marrow separation. Urine and feces were collected at 4 h, 1 d, and 2 d. Organ residence times were calculated by analytic integration of single or multiexponential fits of the time-activity curve and scaled to human organ weight by relative organ mass scaling (11), which was not applied to the gastrointestinal tract organs. To estimate human radiation dose, residence times were entered into OLINDA, version 2.2, using the MIRD adult-female model and organ weights from International Commission on Radiological Protection publication 106 (12). The calculated radiation dose includes contributions from β- and γ-rays from 177Lu within the organ, neighboring organs, and remainder of the body.
R2G2 mice (10 per group) injected intravenously with 1×106 Raji-luc cells and either left untreated or injected 4 d later with ofatumumab, [177Lu]Lu-IgG, or [177Lu]Lu-ofatumumab. When used, 20 μg of antibody were injected per mouse. Bioluminescent images were acquired as previously described (13). Mice were killed if they experienced hind-limb paralysis, lost more than 20% of their body weight, or had other signs of morbidity.
Statistical analyses used Prism software (version 9.0; GraphPad).
SCN-CHX-A″-DTPA was conjugated to ofatumumab and purified. Mass spectrometry indicated an average of 3.2 chelators per antibody. After 177Lu radiolabeling, [177Lu]Lu-ofatumumab was purified (n=7). Radiochemical purity was more than 99%±1%, radiochemical yield was 93%±2%, and specific activity was 401±17 MBq/mg. Immunoreactivity was 49%±3% and 2%±1% after blocking with unlabeled ofatumumab.
After 7 d, over 90% of 177Lu remained chelated in buffer at 4° C., and over 75% remained chelated in human serum at 37° C. (
[177Lu]Lu-ofatumumab biodistribution was determined in C57BI6/N mice 4 h, 1 d, 2 d, 7 d, and 11 d after injection (TABLE 4) as % IA/g. Blood % IA/g was 38% at 4 h and 19% after 11 d. Bone distribution was less than 4%, indicating stable chelation because free 177Lu is a bone-seeking radionuclide (15). Liver was 9% IA at 4 h and 5% IA/g at 11 d, and marrow was 14% IA at 4 h and 9% IA/g at 11 d. Spleen was 8-9% IA/g. Approximately 13% of the injected activity was excreted.
To estimate human dosimetry, integrated time-activity curves for [177Lu]Lu-ofatumumab were calculated (TABLE 5). The longest (59.7 h) was in the blood, with extended time-activity curves seen in the blood-rich heart cavity, lung, and liver. Because of its large mass, muscle had the second longest time-activity curve, at 39 h. The adult human female model (Table 6) showed estimated dosimetry of 0.2-0.5 mSv/MBq in most organs, with the largest dose being to the heart wall (1.02 mSv/MBq) and lesser doses found for liver, spleen, and kidney (0.36, 0.48, and 0.43 mSv/MBq, respectively). Estimated doses to the osteogenic cells (bone surfaces) and red marrow were 0.82 and 0.54 mSv/MBq, respectively. The estimated effective dose was 0.36 mSv/MBq.
Biodistribution of [177Lu]Lu-Ofatumumab in Mice with Subcutaneous Raji-Cell Tumors
Biodistribution was investigated in R2G2 mice with subcutaneous Raji-cell tumors (
To evaluate [177Lu]Lu-ofatumumab therapeutic efficacy, R2G2 mice were injected intravenously with Raji-luc cells, and tumor cells were quantified by bioluminescent imaging (13). After injection, these cells disseminate to many organs (10,13,17,18), with hind-limb paralysis being a typical cause for killing of the animal due to growth in and around the spine.
Four days after cell injection, the mice either were left untreated or were treated with native ofatumumab, 8.51 MBq of [177Lu]Lu-human IgG1 (345±27 MBq/kg), 0.74 MBq (30±2.2 MBq/kg) of [177Lu]Lu-ofatumumab, or 8.51 MBq (345±25.1 MBq/kg) of [177Lu]Lu-ofatumumab (10 mice per group). Survival (
The median survival of untreated mice was 19 d, with none surviving beyond 22 d. Unlabeled ofatumumab yielded a median survival of 46 d, superior to untreated mice (Mantel-Cox, P<0.0001), with 1 mouse surviving without weight loss or increased bioluminescence. An 8.51-MBq dose of [177Lu]Lu-IgG yielded 0 of 10 surviving mice and a median survival of 25 d, which was not different from that of untreated mice. For all 3 groups, increased bioluminescence and weight loss occurred before death or killing for cause.
A 0.74-MB dose of [177Lu]Lu-ofatumumab yielded median survival of 59 d (9/10 mice not surviving), with increased bioluminescence and weight loss before death or killing for cause. This survival was superior to that of untreated mice (Mantel-Cox, P<0.0001) but not to that of mice receiving treatment with unlabeled ofatumumab. Hind-limb paralysis was frequently associated with death or killing for cause (TABLE 7).
Notable therapeutic efficacy resulted from treatment with 8.51 MBq of [177Lu]Lu-ofatumumab, with 9 of 10 mice surviving with continuous low bioluminescence (
To determine how quickly therapy affected tumor cells, bioluminescence slopes from 1 to 18 d after initiation of therapy were compared (
Our preclinical studies add to prior work demonstrating the potential of radiolabeled anti-CD20 antibodies to treat NHL. We show that [177Lu]Lu-ofatumumab can be produced with high radiochemical yield and purity, excellent affinity, good stability and immunoreactivity, and potent cell killing. Additional advances include using a fully human anti-CD20 and 177Lu, which have broad applicability in radiotherapy of cancer. In a model of rapidly progressing disease, we evaluated [177Lu]Lu-ofatumumab therapy using dose-response studies and bioluminescence monitoring of tumor-cell burden. A single 8.51-MBq dose of [177Lu]Lu-ofatumumab displayed curative efficacy.
Human dosimetry estimates predict that the highest dose from [177Lu]Lu-ofatumumab (1.02 mSv/MBq) will be to the heart wall. The relatively radiation-resistant liver and spleen showed 0.36 and 0.48 mSv/MBq, respectively. The predicted dose to red marrow is 0.54 mSv/MBq, and hematologic toxicity likely will be dose limiting in clinical use, as was found with Bexxar, Zevalin, [177Lu]Lu-J591 (19), [177Lu]Lu-G250 anti-CAIX (20), and [177Lu]Lu-rituximab (21). As 2 Sv is a typical maximal dose for acceptable hematologic toxicity without stem cell support, delivering this radiation to the marrow would be tolerable. As there may be patient-to-patient variability with [177Lu]Lu-ofatumumab due to cross reactivity with normal CD20-positive cells, our dosimetry data provide guidance for activity administration to humans. Dosimetric estimation could also potentially be obtained using a PET imaging surrogate, such as [89Zr]Zr-ofatumumab (9,22).
The stable in vivo chelation of 177Lu by CHX-A″-DTPA-ofatumumab agrees with the results of others using this chelator-radionuclide combination (23,24). Although it has been suggested that, for stable 177Lu chelation, macrocyclic DOTA requires high temperatures incompatible with maintaining antibody function (24,25), experiments show that this is not the case (26,27). Thus, CHX-A″-DTPA and DOTA both appear practical for chelation of 177Lu to antibodies and antibody fragments.
Others have used [177Lu]Lu-anti-CD20 intact antibodies or 177Lu-labeled antibody-based radiopharmaceuticals for preclinical and clinical therapy. Ertveld et al. (23), using a single-domain anti-CD20 antibody in immunocompetent mice with CD20-expressing subcutaneous tumors, found a modest therapeutic effect at 140 MBq/mouse; 50 MBq/mouse induced expression of proinflammatory genes, whereas 140 MBq/mouse increased the percentage in the tumor of PD-L1-positive myeloid cells and alternatively activated macrophages. Krasniqi et al. (28) compared a single-domain anti-CD20 antibody with unlabeled rituximab and [177Lu]Lu-CHX-A″-DTPA-rituximab in mice with CD20-expressing subcutaneous tumors. All treatments increased survival over no treatment, but [177Lu]Lu-CHX-A″-DTPA-rituximab was only slightly better than rituximab. In a phase I/II study of [177Lu]Lu-DOTA-rituximab in 31 patients with relapsed or refractory CD20-positive lymphoma, mainly hematologic toxicity was observed, with frequent tumor responses and 8 of 11 patients with follicular lymphoma alive after an 84-mo median follow-up (29).
A major finding of the current study is the high therapeutic efficacy of [177Lu]Lu-ofatumumab in a murine model of disseminated lymphoma. Therapy was initiated 4 d after intravenous cell injection, when tumor cells are present individually or as small groups, comparable to micrometastatic or minimal residual disease in humans. An 8.51-MBq dose of [177Lu]Lu-ofatumumab reduced tumor burden within about 2 d and eliminated bioluminescence-detectable tumors, with 9 of 10 mice still alive 221 d later. This response was dose-dependent and specific, as 0.74 MBq of [177Lu]-Lu ofatumumab and 8.51 MBq of [177Lu]-IgG did not extend survival or prevent tumor-cell proliferation. Although attenuation from tissue, skin, and fur means that bioluminescent imaging may not detect a low tumor-cell burden (13), the durability of the response suggests complete elimination of tumor cells by 8.51 MBq of [177Lu]Lu-ofatumumab. After initial weight loss, these mice gained weight, suggesting no or low whole-body toxicity. The internalization of ofatumumab after CD20 binding (30) and the residualization of 177Lu within the cell may contribute to its therapeutic efficacy. Moreover, the lack of murine sequences in [177Lu]Lu-ofatumumab suggests a potential for fractionated therapy or repeated treatments. In an interesting approach, with relatively small subcutaneous tumors of rituximab-resistant Raji cells, Malenge et al. (26) combined [177Lu]Lu-lilotomab (anti-CD37) and unlabeled rituximab, with good therapeutic results.
α-particle therapy is another potential approach to treating lymphoma. Using a murine Raji-cell disseminated lymphoma model, [213Bi]Bi-rituximab (t1/2, 45.6 min) was typically curative when tumor burden was low (4 d after cell injection) but not when it was higher (18), perhaps because of lack of time to target larger tumor masses before decay. Similarly, [149Tb]Tb-rituximab (t1/2, 4.2 h) therapy initiated 2 d after Daudi-cell intravenous injection increased survival (31). A 1F5 anti-CD20 antibody with chelated 211At (t1/2, 7.2 h) was 80% curative when injected 6 d after intravenous cell injection with supporting stem-cell transplantation but only slowly reduced the growth of subcutaneous tumors (32). On the basis of these results and on the multiday tumor-targeting pharmacokinetics of intact antibodies, radioimmunotherapy of larger tumor masses with intact antibodies will likely be most successful using radioisotopes that permit tumor localization before decay, including 177Lu or α-particle-emitting 225Ac with its 10-d half-life.
Our studies add to the literature demonstrating the effectiveness of 177Lu-radiopharmaceuticals in cancer therapy. We found remarkable effectiveness in micrometastatic disease, and the 6.6-d half-life and multiple-cell-diameter killing range of 177Lu suggests that [177Lu]Lu-ofatumumab may be effective against larger tumors.
Although initial anti-CD20 radioimmunotherapies showed limited commercial success for several reasons, we suggest that a reevaluation of next-generation β- and α-particle therapies is in order. [177Lu]Lu-ofatumumab CD20-targeted radioimmunotherapy may be an effective approach for therapy of NHL or other CD20-expressing diseases.
Chx-A″-DTPA-ofatumumab stably chelates 177Lu in vitro and in vivo, and [177Lu]Lu-Chx-A″-ofatumumab effectively targets CD20-expressing tumor xenografts. In a mouse model of disseminated human lymphoma, therapy with [177Lu]Lu-ofatumumab showed curative therapeutic efficacy.
This application claims priority from U.S. Provisional Application Ser. No. 63/483,532 filed on 6 Feb. 2023, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63483532 | Feb 2023 | US |
