Patent Application
20230009161
The present disclosure relates to a gene transfer system comprising a viral gene delivery vector particle and a bispecific polypeptide configured to bind a viral gene delivery vector particle and target cell-specific receptor protein.
Selective transduction of only target cells and tissues represents a major goal of therapeutic gene delivery. To do so, gene vectors must avoid binding to off-target cells while quickly binding to target cells with high specificity, and efficiently deliver DNA to the nucleus following cell entry. Among common viral vectors, lentivirus (LV) is one of the most efficient gene transduction system for stable, long-term transgene expression. Importantly, the safety of LV has greatly improved since adverse effects were first observed in patients with X-clinical severe combined immunodeficiency (SCID) who underwent retrovirus-mediated gene therapy. As a result, LV vectors are now routinely used in CAR-T cell therapies (i.e. T-cells modified to possess a chimeric antigen receptor) for B-cell malignancies where cells are selected, transduced with LV vectors, expanded, and reinfused into patients; two such therapies have already received regulatory approval.
Despite the routine in vivo delivery of cells transduced with LV vectors ex vivo, LV vectors are rarely used directly for in vivo gene therapy. This is because common LV vectors lack cell specificity: wildtype LV envelope proteins generally bind proteins ubiquitously present on the surface of most cells, leading to extensive off-target effects. Strategies to alter or restrict the natural tropism of LV vectors include either pseudotyping LV with different viral envelope proteins possessing altered tropism and biodistribution, or genetically inserting ligands, peptides, and single-chain antibodies into viral envelope glycoprotein domains to confer new cellular specificity. Unfortunately, introducing large proteins can be deleterious to the structure of viral proteins, impede proper folding of the incorporated peptide that diminishes cell binding, and may hinder viral infectivity by altering normal functions of viral attachment proteins or preventing conformational changes necessary for fusion. Indeed, modified vectors can suffer from inconsistent specificity, reduced fusion activity, and low viral titers. Not surprisingly, the success of modifying viral envelope glycoproteins domains depends on the size, structure, and binding activity of ligand.
Disclosed herein is a gene delivery system comprising a viral gene delivery vector particle comprising a polynucleotide encoding at least one gene-of-interest and a bispecific polypeptide configured to bind a viral gene delivery vector particle and target cell-specific receptor protein, wherein the viral gene delivery vector particle is a lentivirus. In some embodiments, the lentivirus comprises a modified Sindbis virus envelope protein unable to bind a cell surface protein.
Also disclosed herein is a composition comprising a viral gene delivery vector particle comprising a polynucleotide encoding at least one gene-of-interest and a bispecific polypeptide configured to bind a viral gene delivery vector particle and target cell-specific receptor protein, wherein the viral gene delivery vector particle is a lentivirus. In some embodiments, the lentivirus comprises a modified Sindbis virus envelope protein unable to bind a cell surface protein.
Further disclosed are methods of transducing a cell with at least one gene-of-interest, methods of targeting at least one gene-of-interest to a cell or tissue, methods of generating CAR cells (CAR-T cells), and methods of treating a disease or disorder using the compositions or gene delivery systems.
Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentiviral (LV) vectors are efficient gene delivery vehicles but offer little specificity. To enable highly specific transduction, cell-specific receptor binding must be robust while minimizing off-target binding. With wildtype viral vectors that are either pseudotyped with Ab or mixed with adaptor molecules, the resulting vectors can still bind and transduce off-target cells/tissues via the native viral envelope proteins.
Described herein is a versatile redirection platform combining modified Sindbis (mSindbis)-pseudotyped LV with bispecific antibodies (bsAb) that bind both mSindbis E2 and specific cell receptors. A E2- and HER2-targeted bsAb provided the specificity required to redirect mSindbis LV to transduce HER2+ cells, thus enabling the use of LV with an unmodified viral envelope that likely maximizes stability, high titer production, and efficient transduction. A longstanding challenge in bsAb engineering has been the proper pairing of heavy and light chains leading to high purity and yield of the final product. As an example method to generate bispecific antibodies, orthogonal mutation pairs were introduced into heavy and light chains to yielded high fidelity pairing of the correct heavy and light chains for functional bsAb (See Lewis et al., Nat Biotechnol 2014 February; 32(2):191-8, incorporated herein by reference in its entirety). Here, the versatile gene carrier system, combining bsAb with a mutated LV that abrogates its native receptor binding tropism, facilitated highly potent and specific gene delivery.
A single dose of a targeted lentiviral vector administered in vivo, as described herein, generated CAR-T cells from circulating T lymphocytes in a humanized tumor mouse model of B cell leukemia. The in vivo engineered CAR-T cells greatly suppressed CD19+ tumor cell growth and prolonged the overall survival time of mice, despite the highly aggressive nature of the tumor model.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
A “bispecific polypeptide,” as used herein, refers to a polypeptide having binding specificities for at least two different moieties or targets.
The term “viral vector particle” as used herein refers to a recombinant virus which carries a polynucleotide encoding at least one gene-of-interest, which is generally flanked by viral LTRs.
The term “transducing” denotes the delivery of a polynucleotide to a recipient cell either in vivo or in vitro, via a replication-defective viral vector, for example, a viral gene delivery vector particle.
The term “chimeric antigen receptor” and “CAR” are used interchangeably herein to refer to molecules that combine antibody-based specificity for a desired antigen (e.g., tumor antigen) with a cell receptor (e.g. T cell receptor)-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity.
“Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, mRNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double-stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide”, and “protein,” are used interchangeably herein.
As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of the compounds and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization of the compound and/or composition to a desired site. The compound and/or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compounds and/or compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one aspect of the methods provided herein, the mammal is a human.
As used herein, “treat,” “treating” and the like means a slowing, stopping or reversing of progression of a disease or disorder when provided a composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the cell proliferation. As such, “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or symptoms of the disease. “
A “lentivirus” refers to a retroviral genus capable of infecting dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus: HIV type 1 and HIV type 2), etiologic agent of human acquired immunodeficiency syndrome (AIDS); Visna-maedi, a causative agent of encephalitis (bizna) or pneumonia (medi), caprine arthritis-causing encephalitis, encephalitis virus); Equine infectious anemia virus which causes autoimmune hemolytic anemia and brain disease in horses; Feline immunodeficiency virus (FIV), which causes immune system deficiency in cats; Bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis and possible central nervous system infections in cattle; And simian immunodeficiency virus (SIV), an ape-like virus that causes immune system deficiency and brain disease in subhuman primates. As used herein, the term “lentivirus” also includes lentiviruses that are pseudotyped with a glycoprotein derived from another virus, such as lentiviruses pseudotyped with measles, lentiviruses pseudotyped with nipah viruses, etc.
The lentiviral genome is generally composed of 5′long terminal repeat (LTR), gag gene, pol gene, env gene, additional genes (nef, vif, vpr, vpu) and 3′ LTR. The virus LTR is divided into three regions called U3, R and U5. The U3 region includes an enhancer and a promoter element. The U5 region contains a polyadenylation signal. The R (repeat) region separates the U3 and U5 regions, and the sequence of the transcribed R region appears at both the 5′ and 3′ ends of the viral RNA. See, for example, “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)), O Narayan and Clements J. Gen. Virology 70: 1617-1639 (1989), Fields et al. Fundamental Virology Raven Press. (1990), Miyoshi H, Blomeru, Takahashi M, Gage F H, Verma I M. J Virol. 72 (10): 8150-7 (1998), and U.S. Pat. No. 6,013,516.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The present disclosure provides gene delivery systems comprising a viral gene delivery vector particle comprising a polynucleotide encoding at least one gene-of-interest and a bispecific polypeptide configured to bind a viral gene delivery vector particle and a target cell-specific receptor protein.
Viral particles, also known as virions, consist of a nucleic acid(s) surrounded by a capsid coat. The viral particles can be enveloped or nonenveloped depending on the presence or absence of an envelope comprised of host cell membranes, as well as viral glycoproteins. The viral particle may be a member of the Retroviridae (retrovirus) family, or a derivative thereof. The viral particle may be a pseudotyped viral particle or a pseudovirus comprising a heterologous envelope protein or an envelope protein originating for a different virus. In some embodiments, the viral particle is a lentivirus. In some embodiments, the viral gene delivery vector particle is a lentivirus. Lentiviruses are a subtype of retroviruses that are capable of infecting non-dividing and actively dividing cell types. In select embodiments, the viral gene delivery vector particle is a lentivirus comprising a modified Sindbis virus envelope protein unable to bind a cell surface protein.
In some embodiments, the viral particle comprises at least one protein (such as an envelope protein (e.g., gp160 protein, gp4l protein, etc.), capsid protein, matrix protein, etc.) or glycoprotein that has been modified in such that the virus does not bind to its target cell. For example, modifications can be made to block the interactions between a viral envelope glycoprotein and a specific target cell surface receptor which determines the cellular target for the virus. For example, modifications can include in Sindbis, one or more mutations and/or deletions in (a) the E3 leader sequence (e.g., amino acid residues 61-64 can be deleted (deletion of these amino acid residues is known to reduce tropism and result in higher titer production)), (b) the E2 glycoprotein where, mutations such as SLKQ68-71AAAA or KE159-160AA (which are known to reduce natural tropism while retaining higher titer production) can be made (See, Morizono et al., Nat Med. 2005 March; 11(3):346-52. Epub 2005 Feb. 13), and (c) the E1 glycoprotein, where mutations such as AK226-227SG (which are believed to allow E1 to mediate fusion in absence of cholesterol in target membrane”, see for example, Pariente et al Mol Ther. 2007 November; 15(11):1973-81. Epub 2007 Jul. 24.) can be made. In lentiviruses pseudotyped with measles, mutations can be made in the H (hemagglutinin) protein, such as at amino acids Y481A, R533A, SF548-549LS (which abrogate native receptor binding to SLAM and CD46 typically found on immune cells; see Vongpunsawad S, et al., J Virol. 2004 January; 78(1):302-13 and Nakamura T, et al., Nat Biotechnol. 2005 February; 23(2):209-14. In lentiviruses pseudotyped with nipah, mutations can be made in glycoprotein G, such as, at amino acids, E501A, W504A, Q530A, and E533A (see Bender et al. PLOS Pathogens. 2016 June journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005641).
The bispecific polypeptide is any polypeptide capable of interacting with two different binding partners at the same time. In some embodiments, the bispecific polypeptide comprises at least one binding domain configured to bind the viral gene delivery vector particle and at least one binding domain configured to bind the target cell-specific receptor protein. In some embodiments, the bispecific polypeptide binds an envelope protein of the virus particle (e.g. the modified Sindbis virus envelope protein). In select embodiments, the bispecific polypeptide binds the modified Sindbis virus envelope protein in the E2 domain.
In some embodiments, the bispecific polypeptide further comprises a flexible linker covalently joining the two binding domains. The linkers may be flexible such that they do not constrain either of the two components they link together in any particular orientation. The linkers may comprise any amino acid sequence. The linkers may essentially act as a spacer. In some embodiments, the linkers are glycine-rich and/or serine-rich (e.g. (G4S)6). In some embodiments, the bispecific polypeptide can comprise one flexible linker, two flexible linkers, three flexible linkers, four flexible linkers, five flexible linkers, six flexible linkers, seven flexible linkers, eight flexible linkers nine flexible linkers, ten flexible linkers, eleven flexible linkers, twelve flexible linkers, etc. When multiple flexible linkers are used the flexible linkers may be the same, or the flexible linkers can be different.
The bispecific polypeptide may be an antibody, fragment, or derivative thereof. In some embodiments, the antibody, fragment or derivative thereof is two or more Fab-fragments, two or more F(ab2)′-fragments, single domain antibodies, an IgG with Fc, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a humanized antibody, a human synthetic antibody, or a chemically modified derivative thereof, a multispecific antibody, a diabody (e.g., two or more scFv fragments covalently linked together), tandem scFv fragments, bivalent (or bispecific) (scFv)2, so-called miniantibody, VHH nanobodies, another type of a recombinant antibody, or the like as known in the art (See Spiess et al., Mol Immunol 2015 October; 67(2):95-106, incorporated herein by reference in its entirety). By the term “recombinant antibody” as used herein, is meant an antibody or antibody fragment which is generated using recombinant DNA technology, such as, for example, an antibody or antibody fragment expressed by a bacterial system, a yeast expression system, a fungus-based expression system, a plant-based expression system, or a mammalian cell expression system. The term should also be construed to mean an antibody or antibody fragment which has been generated by the synthesis of a DNA molecule encoding the antibody or antibody fragment and which DNA molecule expresses an antibody or antibody fragment protein, or an amino acid sequence specifying the antibody or antibody fragment, wherein the DNA or amino acid sequence has been obtained using recombinant or synthetic DNA or amino acid sequence technology which is available and well known in the art. In select embodiments, the bispecific polypeptide comprises two or more Fab domains individually configured to bind the viral gene delivery vector particle and the target cell-specific receptor protein. In other select embodiments, the bispecific polypeptide comprises at least three or more Fab domains individually configured to bind the viral gene delivery vector particle and the target cell-specific receptor protein. In other select embodiments, the bispecific polypeptide comprises at least four or more Fab domains individually configured to bind the viral gene delivery vector particle and the target cell-specific receptor protein. In other select embodiments, the bispecific polypeptide comprises at least five or more Fab domains individually configured to bind the viral gene delivery vector particle and the target cell-specific receptor protein.
In some embodiments, the antibody is a human or humanized antibody. The term “humanized antibody”, as used herein, is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the presently disclosed subject matter may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The target cell-specific receptor protein may be any protein known in the art to be associated with a particular subset of cells. The cell-specific receptor protein may be associated with cells from a certain tissue or cells from a certain state of disease, including but not limited to, CD3, CD4, CD8 for T-cells, CD19 for B-cells, cancer cell markers (e.g., HER2), or the like. In some embodiments, the target cell-specific receptor protein is selected from the group consisting of a T cell receptor, a B cell receptor, and a cancer cell marker. The target cell-specific receptor protein may be exogenous or endogenous to the cell type. For example, recombinant cells may comprise an exogenous receptor protein targeted by the bispecific polypeptide.
The gene-of-interest may comprise one or more fully functioning genes. The gene may comprise any gene encoding a functioning protein, a fragment, or derivative thereof. The gene may comprise a marker protein, a therapeutic protein, elements required for genomic editing or gene silencing. In some embodiments, the gene-of-interest comprises a chimeric antigen receptor. The gene-of-interest may comprise genetic elements that aid in targeted integration of therapeutic transgenes of interest or targeted knockout of genes-of-interest (e.g. components of CRISPR/Cas9).
The present disclosure also provides a composition (e.g. a pharmaceutical composition) comprising a viral gene delivery vector particle comprising a polynucleotide encoding at least one gene-of-interest and a bispecific polypeptide configured to bind a viral gene delivery vector particle and target cell-specific receptor protein. Descriptions provided above for the viral gene delivery vector particle, the polynucleotide, the at least one gene-of-interest, and the bispecific polypeptide provided above are maintained for the composition.
The compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The route by which the disclosed compositions are administered and the form of the composition will dictate the type of carrier to be used.
The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis). Techniques and formulations may generally be found in “Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.
The present disclosure provides methods of transducing a cell with at least one gene-of-interest, comprising contacting a cell expressing the target cell-specific receptor protein with the gene delivery system or the composition described in section 2. The present systems or compositions may be delivered to a cell with any suitable means. In certain embodiments, the system is delivered in vivo. In other embodiments, the system is delivered to isolated/cultured cells in vitro to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition.
The present disclosure also provides methods of targeting at least one gene-of-interest to a cell or tissue, comprising administering to a subject having a cell or tissue expressing the target cell-specific receptor protein the gene delivery system or the composition described in section 2.
The present disclosure further provides methods of generating CAR-T cells. In some embodiments, the methods generative CAR T cells in vivo and comprise administering to a subject the gene delivery system or the composition described in section 2, wherein the at least one gene of interest comprises a chimeric antigen receptor and the target cell-specific receptor protein is a T cell receptor. In some embodiments, the T cell receptor is selected from the group consisting of CD3, CD4, CD8, or a combination thereof.
The present disclosure further provides methods of treating a disease or disorder comprising administering to a subject an effective amount of the gene delivery system or the composition described in section 2, wherein the at least one gene of interest comprises a chimeric antigen receptor, a therapeutic protein, or a combination thereof.
Essentially any disease treatable with a therapeutic protein or genome editing may be used with the methods disclosed herein to target the protein to a cell or tissue as described herein. Furthermore, essentially any disease that involves the specific or enhanced expression of a particular antigen can be treated by targeting CAR cells to the antigen, as known in the art. For example, autoimmune diseases, infections, and cancers can be treated with methods, systems, and/or compositions of the invention. These include cancers, such as primary, metastatic, recurrent, sensitive-to-therapy, refractory-to-therapy cancers (e.g., chemo-refractory cancer). The cancer may be of the blood, lung, brain, colon, prostate, breast, liver, kidney, stomach, cervix, ovary, testes, pituitary gland, esophagus, spleen, skin, bone, and so forth (e.g., B-cell lymphomas or a melanomas) or any disease characterized as a cancer due to uncontrollable cell division. In the case of cancer treatment CAR cells typically target a cancer cell antigen (also known as a tumor-associated antigen (TAA)).
The specific dose level may depend upon a variety of factors including the age, body weight, and general health of the subject, time of administration, and route of administration. An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which achieves a medically desirable effect. For therapeutic purposes, and effect amount is the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject and/or that reduces or inhibits one or more symptoms to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of the disease or disorder. For prophylaxis purposes, an effective amount is that amount which induces a protective result without significant adverse side effects.
The frequency of dosing the effective amount can vary, but typically the effective amount is delivered daily, either as a single dose, multiple doses throughout the day, or depending on the dosage form, dosed continuously for part or all of the treatment period.
The composition or systems described herein may be formulated for any appropriate manner of administration, and thus administered, including for example, topical, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, intratumoral, or via inhalation.
A wide range of second therapies may be used in conjunction with the systems and compositions of the present disclosure. The second therapy may be a therapeutic agent or may be a second therapy not connected to administration of an agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, or a second chemotherapeutic agent.
In one aspect, the disclosure provides kits comprising at least one or all of the components of the disclosed system as described elsewhere herein (e.g. a viral gene delivery vector particle, a polynucleotide encoding at least one gene-of-interest, and/or a bispecific polypeptide). The components of the kit may be packaged separately or individually.
The disclosed kits can be employed in connection with disclosed methods of use.
The kits may further include information, instructions, or both for use of the kit in transducing a cell with at least one gene-of-interest, targeting at least one gene-of-interest to a cell or tissue, generative CAR cells, or treating a disease or disorder. The information and instructions may be in the form of words, pictures, or both, and the like.
Cell lines 293T cells were cultured in DMEM containing 10% FBS. Human SKBR3 cells were purchased from the University of North Carolina at Chapel Hill (UNC-CH) Tissue Culture Facility, and A2780 cells were provided by Michael Jay (UNC-CH). SKBR3 cells were cultured in McCoy's medium containing 15% fetal bovine serum (FBS), and A2780 cells were cultured in RPMI 1640 containing 10% FBS and 1% L-glutamine. For co-culture studies, SKBR3 and A2780 cells were both cultured in McCoy's medium with 15% FBS. All cells were maintained at 37° C. and 5% CO2.
B cell lymphoma tumor cell lines (BV-173 and Daudi) and T cell lymphoma tumor cells (Sup-T1) were purchased from ATCC and cultured in RPMI-1640 medium (Gibco) supplemented with 10% HyClone FBS (GE Healthcare), penicillin (100 U mL−1; Gibco), and streptomycin (100 U mL−1, Gibco). All cells were maintained at 37° C. and 5% CO2 for growth. All cell lines are regularly tested for Mycoplasma, and the identity of each cell line was validated via flow cytometry for relevant surface markers and also monitored for morphological drift in culture. Cell lines were maintained in culture no longer than 30 days and then replaced with an earlier passage of cells thawed from cryopreservation. BV-173 cells were transduced with a gamma retroviral vector encoding the Firefly-Luciferase (FFluc) gene. Sup-T1 cells were engineered with a TCR construct to express full-length human CD3. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Gulf Coast Regional Blood Center) using Lymphoprep medium (Accurate Chemical and Scientific Corporation). PBMCs were then activated for 48 hours in bioreactors with soluble anti-CD3 (200 ng mL−1; Miltenyi Biotec) and anti-CD28 (200 ng mL−1; BD Biosciences) mAbs. Activated PBMCs were washed with PBS and allowed to rest at 37 C and 5% CO2 in growth culture medium for at least 24 hours prior to lentiviral transduction or in vivo studies. Primary T cells were activated, cultured, and transduced in complete medium consisting of 45% Click's Medium (Irvine Scientific), 45% RPMI-1640 (Gibco), 10% HyClone FBS (GE Healthcare), 2 mmol L−1 GlutaMax (Gibco), penicillin (100 U mL−1; Gibco), and streptomycin (100 U mL−1; Gibco) with 10 ng mL IL-7 and 5 ng mL−1 IL-15 (PeproTech).
Preparation and characterization of fluorescent Sindbis pseudotyped lentivirus WT Sindbis and mSindbis pseudotyped lentiviruses (LV) were internally labeled with a GFP reporter gene. Particles were prepared by transfecting 293T cells with packaging plasmids pMDLg/pRRE and pRSV-Rev, transfer plasmid eGFP, and WT Sindbis or mSindbis envelope plasmid at a 1:1:1:1 ratio in serum-free media. The cell supernatant was collected 48 h later, and lentiviruses were purified from cell supernatant by ultracentrifugation using 25% (w/v) sucrose in HEPES-NaCl buffer. Lentiviruses were resuspended in 10% sucrose in HEPES-NaCl buffer, divided into aliquots, and stored at −80° C. Viral titer was quantified by qPCR-based lentivirus titration kit according to manufacturer's protocol (Applied Biological Materials, Inc., Richmond, British Columbia, Canada). Packaging plasmids pMDLg/pRRE (Addgene plasmid #12251) and pRSV-Rev (Addgene plasmid #12253) were provided by Didier Trono.
Lentiviral vector design, production, and titration Mutant Sindbis pseudotyped lentiviruses (SINV-LV) were generated via four plasmid transfection in 293T packaging cells. The mutant Sindbis envelope plasmid was constructed by cloning the Sindbis virus glycoprotein insert from plasmid 2.2 (Addgene plasmid no. 34885) into an expression vector plasmid backbone under the CAG promoter. The ZZ domains of Protein A were removed from the mutant E2 domain of the new mammalian expression plasmid via Gibson Assembly cloning. Negative control envelope plasmids for antibody binding specificity studies were kind gifts of Bob Weinberg (pCMV-VSV-G, Addgene plasmid no. 8454) and Jakob Reiser (pCG-HcΔ18, Addgene plasmid no. 84817). To generate functional pseudotyped LV vectors with measles virus glycoproteins, Jakob Reiser also provided the sequence for cloning the measles virus fusion (F) protein envelope plasmid (pCG-FcΔ30). The pLL3.7 transfer plasmid (Addgene plasmid no. 11795) was a gift from Luk Parijs and used as the transgene cassette for expressing eGFP as a reporter of transduction in SINV-GFP. Using Nod and BspEI in a restriction enzyme double digest, we generated a new transfer plasmid, pLL CD19 CAR, from the pLL3.7 plasmid backbone for producing SINV-CAR. The new gene cassette for pLL CD19 CAR consisted of an EF-1α internal promoter, anti-CD19 scFv, CD8 flexible hinge domain, CD8 transmembrane domain, CD28 costimulatory endodomain, CD3C chain, and WPRE post-transcriptional regulatory element all flanked by the original LTRs of the pLL3.7 plasmid backbone. Third generation lentiviral packaging plasmids pMDLg/pRRE (Addgene plasmid no. 12251) and pRSV-Rev (Addgene plasmid no. 12253) were gifts of Didier Trono.(30)
LV were produced via transient transfection of LV-MAX cells according to manufacturer protocols for the LV-MAX lentiviral production system kit (Gibco). Briefly, 1.2×108 viable cells were seeded in a vented shaker flask for a final production volume of 30 mL. A 3:2 ratio of packaging plasmids (envelope, gag/pol, and rev) to transfer plasmid was combined with LV-MAX Transfection Reagent in serum-free medium and subsequently added to cells in shaker flask after 10 minutes of incubation. At ˜48 hours following transfection, cells were collected from suspension culture along with their medium and centrifuged at 1,300×g for 15 mins to pellet cells. Supernatant containing LV vectors was harvested and filtered through a 0.45 μm low protein binding filter to further remove cell debris. Filtered supernatant was added carefully dropwise to a sucrose cushion (25% w/v sucrose in HEPES-NaCl buffer) and subjected to ultracentrifugation at 36,000 rpm for 2.5 hrs at 4° C. Following ultracentrifugation, supernatant and sucrose cushion were carefully aspirated leaving LV pellet at bottom center of tubes. LV pellets were resuspended overnight at 4° C. with 10% w/v sucrose in HEPES-NaCl buffer, aliquoted, and frozen at −80 C for long-term storage. In vivo grade LV was prepared by the Duke University Viral Vector Core (Boris Kantor Lab) using calcium phosphate-based transfection of adherent HEK-293T cells and subsequent double-sucrose gradient purification. All LV were tittered immediately after thawing a fresh aliquot on ice using a qPCR lentiviral titration kit according to manufacturer protocols (Applied Biological Materials Inc., Cat #LV900).
Bispecific antibody construction, expression, and characterization Sequences for chimeric anti-Sindbis E1 or E2 and anti-HER2 antibodies (Ab) were generated by combining the VH/VL regions of commercially available humanized anti-HER2 (Trastuzumab) and murine anti-Sindbis with the CH1/CL and Fc regions of human IgG1 Ab. Mouse anti-Sindbis E1 and E2 VH/VL sequences were provided by Diane Griffin (Johns Hopkins University; unpublished results). To generate bispecific IgG antibodies (bsIgG1) that recognized both Sindbis E1 or E2 and anti-HER2, separate orthogonal mutation sets were incorporated into anti-HER2 and anti-Sindbis Fab domains. Orthogonal mutation sets provided high fidelity pairing of heavy and light chains. These mutations were also incorporated into the chimeric monoclonal antibody, IgG1HER2.
Heavy and light chain antibody constructs were generated on separate mammalian expression vectors, each with the same backbone and CAG promoter sequence. Twist Bioscience performed the molecular cloning of antibody gene constructs for mammalian expression. Following an albumin signal peptide for protein secretion, the bispecific antibody (BsAb) tandem Fab (tFab) heavy chain construct consisted of a murine anti-Sindbis E2 variable heavy domain (VH) and human IgG1 constant heavy 1 domain (CHI) covalently linked with a humanized anti-CD3 VH and human IgG1 CH1 by a flexible glycine-serine peptide linker (G4S)6. The C-terminus of this VH-CH1-Linker-VH-CH1 bispecific heavy chain construct contained an 8× polyhistidine tag for purification purposes. A separate construct was designed for each of the two different light chains of the tFab. The anti-Sindbis E2 light chain consisted of a variable light domain and human constant lambda light chain domain (VL-Cλ), while the anti-CD3 light chain consisted of a variable light domain and human constant kappa light chain domain (VL-Cκ). The murine anti-Sindbis E2 VH/VL sequences were kindly provided by Diane Griffin (Johns Hopkins University; unpublished results), and the anti-CD3 VH/VL sequences were publicly available from a humanized version of the mAb clone UCHT1. To generate the bispecific tFab (Fabα-E2-Linker-Fabα-CD3), separate orthogonal amino acid mutation sets were incorporated into the separate anti-E2 and anti-CD3 Fab domains. These orthogonal mutation sets provided high-fidelity pairing of antibody heavy and light chains for correct assembly of desired BsAb molecule. This OrthoMab technology to generate high-fidelity BsAbs was licensed through a partnership between Dualogics and UNC-CH. A humanized anti-CD3 IgG1 mAb (IgG1α-CD3) was also generated with the same set of orthogonal mutations from the tFab's anti-CD3 portion and used as a control molecule for in vitro experimentation.
Plasmids encoding chimeric heavy and light chains were cotransfected into Expi293F cells (Thermo Fisher Scientific, Grand Island, N.Y.) using the ExpiFectamine 293 transfection kit based on manufacturer protocols (Gibco) and grown. IgG1HER2, bsIgG1E2×HER2, and bsIgG1E1×HER2 were purified after 72 hours from expression supernatant using protein A agarose (Thermo Fisher Scientific). BsIgG1 antibodies were separated via size exclusion chromatography (ENnrich SEC 650 10×300 column, Bio-Rad Laboratories, Inc., Hercules, Calif.). The tandem Fab was designed to include a polyhistidine tag on its C-terminus and was purified from expression supernatant using Ni-NTA agarose (Qiagen Inc, Germantown, Md.). tFab required co-transfection of three separate plasmids at equimolar ratios (heavy chain plasmid, anti-E2 light chain plasmid, and anti-CD3 light chain plasmid), while IgG1α-CD3 only required co-transfection of two separate plasmids at equimolar ratios (anti-CD3 heavy chain plasmid including an IgG1 Fc and anti-CD3 light chain plasmid). After ˜5 days of recombinant protein expression, suspension cells were pelleted by centrifugation at 8,000×g, and the supernatant containing expressed antibodies was harvested and filtered through a 0.2 μm PEG filter. tFabα-CD3×α-E2 was purified from cell culture supernatant via immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose (Qiagen). IgG1α-CD3 was purified from cell culture supernatant via affinity chromatography using protein A plus agarose (ThermoFisher Scientific).
Purified proteins were simultaneously concentrated and buffer exchanged into PBS using ultrafiltration (MWCO 30 K, Amicon Ultra). Antibody concentration was determined by spectrophotometry measurements using calculated protein extinction coefficients (A280 NanoDrop™ One/One©). The size and purity of purified antibodies were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were detected with Coomassie stain (Imperial Protein Stain, Thermo Scientific).
Antibody binding assays HER2-specific ELISAs were performed to confirm binding of purified antibodies to HER2 as well as compare dissociation constants of bispecific antibodies relative to parental monoclonal control, IgG1HER2. Briefly, recombinant human ErbB2/HER2 Fc chimera protein (R&D Systems, cat no. 1129-ER, Minneapolis, Minn.) was coated onto high-binding half-area 96-well Costar plates (Corning) at 1 μg/ml in bicarbonate buffer overnight at 4° C. After blocking plate with 5% nonfat milk in PBS with 0.05% Tween (PBST), purified antibody samples were diluted in 1% nonfat milk in PBST at various concentrations and incubated for 1 h, followed by washes with PBST. Bound antibodies were detected using goat anti-human kappa light chain HRP (Sigma-Aldrich, cat no. A7164, 1:10,0000 dilution) for 1 h followed by 1-step Ultra TMB (Thermo Fisher Scientific). After stopping the HRP reaction with 2N sulfuric acid, the absorbance at 450 nm and 570 nm was measured using a Spectramax M2 plate reader (Molecular Devices).
Indirect enzyme-linked immunosorbent assay (ELISA) was used to characterize and compare binding affinities of purified antibodies to both target antigens: human CD36 and mutant Sindbis E2 glycoprotein. Briefly, either human CD3ε protein (Novus Biologicals, Cat #NBP2-22752) or SINV-LV particles, purified from in-house recombinant production (see above), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat #3690) overnight at 4° C. Human CD3ε protein was diluted to 1 μg mL−1 in carb-bicarb buffer (pH 9.6, Sigma C3041) for overnight coating, while purified SINV-LV stocks were diluted 100-fold in the same carb-bicarb buffer for overnight coating. The next morning, plates were washed 5× with PBS-0.05% Tween (PBST) and subsequently blocked for 1-2 hours at room temperature with 5% w/v non-fat milk in PBST. Purified antibody samples and controls were serially diluted in 1% w/v milk-PBST, spanning at least three orders of magnitude in concentration, and added to the blocked plates for 1-2 hour incubation at room temperature. Following 5×PBST washes of the plates, bound antibodies were detected using goat anti-human kappa light chain HRP conjugated secondary antibody (Sigma-Aldrich, Cat #A7164) at 1:1,000 dilution in 1% w/v milk-PBST for 1 hour incubation at room temperature. Following 5×PBST washes to remove unbound secondary detection antibody, 1-Step Ultra TMB-ELISA substrate solution (Thermo Scientific) was added for up to 10 mins to detect HRP activity. The enzymatic reaction was quenched by adding equal volume of 2 N sulfuric acid, and the color development was immediately determined by taking absorbance measurements at 450 nm (signal) and 570 nm (background) wavelengths using a SpectraMax M2 microplate reader (Molecule Devices). Negative control wells, including antigen coated, blocked wells without primary antibody incubation and uncoated, blocked wells with primary antibody incubation, both revealed negligible signal development in the assay. Background subtracted absorbance values for each sample condition, run in triplicate, were imported into GraphPad Prism 8 software for calculating the binding affinity of each antibody titration curve and presented as equilibrium dissociation constants (KD). A nonlinear curve fit with one site-specific binding was used to determine the KD values.
To evaluate the specificity of tFab binding to mutant Sindbis glycoproteins, purified LVs, made from the same passage of LV-MAX packaging cells, with different envelopes (SINV, VSV-G, and Measles) were blotted directly onto a nitrocellulose membrane for dot blot immunoassay. Briefly, nitrocellulose membranes were blotted directly with 1 μL of purified LV samples of different envelope pseudotypes. Once samples were dry, the membranes were washed 5× with PBST before blocking the membranes for 1 hour at room temperature in 5% w/v milk-PBST with gentle agitation. IgG1α-CD3 negative control or tFabα-CD3×α-E2 were diluted separately to 3 μg mL-1 concentration in 1% w/v milk-PBST. The blocked membranes were transferred separately to these primary antibody solutions and incubated for 1 hour at room temperature with gentle agitation for antibody binding. After 5× washes with PBST, primary antibodies bound to the membranes were detected using goat anti-human kappa light chain HRP conjugated secondary antibody (Sigma-Aldrich, Cat #A7164) at 1:1,000 dilution in 1% w/v milk-PBST for 1 hour incubation at room temperature with gentle agitation. After 5× washes with PBST, the membranes were imaged together with identical exposure times using a ChemiDoc XRS+ imaging system (Bio-Rad). Chemiluminescent signal of secondary antibody binding was detected using Clarity Western ECL substrate (Bio-Rad, Cat #1705061).
Viral infectivity assay SKBR3 (HER2−) and A2780 (HER2−) cells were seeded at 3×104 cells per well in 96-well tissue culture treated plate. Sindbis pseudotyped lentiviruses (multiplicity of infection, MOI=3) were premixed with antibodies at 1 nM concentration for 1 h at room temperature, and then incubated with cells at 37° C. in 5% CO2. Twenty-four hours later, the transduction mixture was removed from cells and cells were washed three times with PBS. Cells were allowed to grow for 72 h in fresh cell culture media at 37° C. in 5% CO2. Cells were washed and the percentage of transduced cells (GFP+) in each well was quantified using iQue Screener PLUS flow cytometer (Intellicyt, Albuquerque, N. Mex.). Additionally, to confirm that viral infectivity was dependent upon HER2 specificity of the bsAb, the viral infectivity assay was repeated with increasing concentrations of bsIgG1E2×HER2 in the presence and absence of excess IgG1HER2 (100 nM).
To test the selectivity of targeted viral systems for HER2+ cells, a co-culture model of SKBR3 and A2780 cells that were maintained in McCoy's 5A medium supplemented with 15% FBS was established. Cells in the co-culture were infected with nontargeted or redirected LV vectors as described above. Seventy-two hours post-infection, treated cells were washed and labeled with IgG1HER2 followed by goat anti-human IgG-Alexa Fluor 594 (Thermo Fisher Scientific) to generate two key cell populations: cells double positive for GFP and HER2 expression and cells double negative for GFP and HER2 expression. The percentages of GFP+ cells of all HER2+ cells and GFP+ cells of all HER2− cells in each well were quantified using iQue Screener PLUS flow cytometer. Data were analyzed using ForeCyt software and BD FACSDiva software.
In vitro transduction assays The CD3+ Sup-T1 tumor cell line was transduced with SINV-GFP in the presence of increasing concentrations of tFab to demonstrate BsAb-mediated enhanced transduction of target cells. Sup-T1 cells were seeded in sterile 96-well tissue culture treated plates (Corning Costar Cat #3599) at 1×105 cells/well. SINV-GFP at a multiplicity of infection (MOI) of 25, based on qPCR tittering, was premixed with various concentrations of tFab in serum-containing growth culture medium for 1 hour at room temperature to allow tFab to bind onto the surface of SINV-GFP particles before directly adding this transduction mixture to the plated cells. Each tFab concentration tested (1, 10, 30, and 50 nM) is reported as the final concentration of the tFab once diluted and added to cells for transduction in 96-well plates. To confirm enhanced transduction was dependent on the CD3 specificity of the tFab, excess IgG1α-CD3 (300 nM) was added to replicate sample wells at each tFab concentration to competitively block binding of CD3 as entry receptor for targeted transduction with SINV-GFP plus tFab. After 24 hours of transduction at 37° C. and 5% CO2, cells were washed twice with cold growth culture medium using low-speed plate centrifugation (300×g) to remove residual antibody and LV prior to resuspension in fresh growth culture medium. Cells were allowed to grow and express GFP transgene for 72 hours at 37° C. and 5% CO2 prior to washing them into PBS and analyzing their GFP expression via flow cytometry using an Attune N×T flow cytometer with plate autosampler (Applied Biosystems).
A similar transduction assay with CD3+ Sup-T1 and CD3− BV-173 tumor cell lines was established to demonstrate specificity and selectivity of SINV-GFP plus tFab transduction to CD3+ target cells. Sup-T1 and BV-173 cells were seeded together at a 1:1 ratio in each well of sterile 96-well tissue culture treated plates (Corning Costar Cat #3599) at 1×105 total cells/well. SINV-GFP at a MOI of 25, based on qPCR tittering, was premixed with 30 nM final concentration of tFab in serum-containing growth culture medium for 1 hour at room temperature before directly adding this transduction mixture to the plated co-culturing cells. A control transduction of SINV-GFP at MOI 25 without addition of tFab was also dosed to co-culturing cells. After 24 hours of transduction at 37° C. and 5% CO2, cells were washed twice with cold growth culture medium using low-speed plate centrifugation (300×g) to remove residual antibody and LV prior to resuspension in fresh growth culture medium. Cells were allowed to grow and express GFP transgene for 72 hours at 37° C. and 5% CO2 prior to washing them into PBS for surface marker phenotype staining with anti-CD3 APC (BD Cat #340440) and anti-CD19 PE (BD Cat #340364). Phenotypic antibody staining was allowed to proceed for 30 mins at 4° C. followed by two PBS washes of samples to remove unbound antibodies. Washed cells were resuspended into PBS and analyzed for their GFP expression via flow cytometry using an Attune N×T flow cytometer with plate autosampler (Applied Biosystems).
Activated primary human PBMCs were transduced with SINV-CAR at a MOT of 10, based on qPCR, with and without addition of tFab to demonstrate functional CAR expression and subsequent cytotoxic activity of CAR-T cells in vitro. In brief, 2.5×105 activated PBMCs were transduced in 250 uL final volume per well of growth culture medium supplemented with IL-7 and IL-15 cytokines in 48-well tissue culture treated plates. SINV-CAR at a MOI of 10, based on qPCR tittering, was premixed with 50 nM final concentration of tFab in serum-containing growth culture medium for 1 hour at room temperature before directly adding this transduction mixture to the plated PBMCs. SINV-CAR at MOI 10 was also dosed directly without addition of tFab for targeting along with other non-transduced control PBMC sample wells. After 6 hours of transduction at 37° C. and 5% CO2, PBMC samples were washed twice with cold growth culture medium to remove residual antibody and LV prior to resuspension in fresh growth culture medium and transfer to a new, sterile 24-well tissue culture treated plate for 84 hours of growth and CAR expression at 37° C. and 5% CO2. A portion of each sample well was collected and washed into PBS for phenotypic surface marker staining by a panel of antibodies and subsequent CAR expression analysis using an LSR Fortessa flow cytometer (BD Biosciences). The remaining PBMCs in each sample well were resuspended and counted by trypan blue dye exclusion for subsequent plating with CD19+ tumor B cells to demonstrate CAR functionality by a co-culture cytotoxicity assay described in more details below.
In vitro co-culture tumor cytotoxicity assay Transduced and non-transduced control PBMCs (1.5×105 cells/well or 3×105 cells/well) were cocultured with tumor cell lines (BV-173 or Daudi, 1.5×105 cells/well in 24-well plates), in complete medium, in the absence of cytokines (E:T=1:1 or E:T=2:1). The effector-to-target (E:T) ratio was not corrected for the percentage of CAR+ T cells but was calculated based on the total number of T cells in culture. After 4-5 days of culture, cells were harvested and stained with CD3 (APC-H7, clone SK7 from BD Biosciences) and CD19 (FITC, clone SJ25C1 from BD Biosciences) monoclonal Abs to detect T cells and tumor cells, respectively. Residual tumor cells in culture were enumerated by flow cytometry. Culture supernatants were harvested after 24 or 48 hours of culture and IFN-γ and IL-2 measured using the DuoSet Human IFN-γ and DuoSet Human IL-2 ELISA kits respectively (R&D Systems). Data acquisition was performed on a Synergy2 microplate reader (BioTek) using the Gen5 software.
Tumor mouse model for testing efficacy of in vivo generated CAR-T cells All tumor mouse model experiments were performed in accordance with UNC Animal Husbandry and Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by UNC IACUC (Protocol #: 18-251). Female NSG mice (7-9 weeks of age, obtained from the UNC Animal Services Core) were used to establish the chronic myeloid leukemia xenograft tumor mouse model. Mice were irradiated at a low dose (100 rad) by a cesium irradiator on Day −6 of the study prior to any cell engraftments. The following day (Day-5), 5×105 FFLuc BV-173 tumor B cells were injected in 150 μL sterile PBS via i.v. tail vein. After allowing 5 days for tumor cell engraftment, 5×106 activated PBMCs were injected on Day 0 in 150 uL sterile PBS via i.v. tail vein. 30 minutes after infusing the PBMCs, mice were randomly separated into two different treatment groups: (1) SINV-CAR without tFab or (2) SINV-CAR with premixed tFab. In both groups, SINV-CAR was dosed at 2.5×107 infectious units (IU), based on qPCR, in 150 uL sterile PBS per mouse via i.v. tail vein injection. This dosage equated to 5×1010 viral particles per mouse, based on absolute particle counts of SINV-CAR using NanoSight NS500 (Malvern Panalytical) nanoparticle tracking analysis. tFab (5 μg/mouse) was premixed with SINV-CAR for 1 hour at room temperature in 150 μL sterile PBS prior to i.v. injections. B cell tumor growth was monitored weekly by bioluminescent imaging (BLI; total flux, photons/second) using an Ami HT optical imaging system (Spectral Instruments Imaging). Peripheral blood samples were taken weekly from mice via the submandibular route. Peripheral blood was subjected to red blood cell lysis followed by antibody staining and flow cytometry to assess number of human T cells (CD3+) and tumor B cells (CD19+) in circulation. Mice were sacrificed according to UNC guidelines for either tumor growth or occurrence of signs of discomfort, such as tumor-mediated paralysis. Upon sacrifice, peripheral blood was collected from cardiac puncture of the heart, and spleens were measured and weighed prior to smashing over cell strainers into single cell suspensions. Blood and spleen were subjected to red blood cell lysis, antibody staining, and flow cytometry using an LSR Fortessa flow cytometer (BD Biosciences) to detect and quantify CAR+ T cells and CD19+ tumor B cells in isolated tissues. Antibodies used for phenotypic staining of in vivo samples included CD3 (APC-H7, clone SK7), CD8 (Alexa Fluor 700, clone RPA-T8), CD45 (APC, clone 2D1) and CD19 (FITC, clone SJ25C1) along with CountBright absolute counting beads (Invitrogen). All flow cytometry data analysis was performed with FlowJo v10 software.
Immunophenotyping T cells were stained with Abs against CD3 (APC-H7, clone SK7), CD8 (Alexa Fluor 700, clone RPA-T8) and CD45 (APC, clone 2D1) from BD Biosciences. Tumor cells were stained with Ab against CD19 (FITC, clone SJ25C1) from BD Biosciences. The expression of the anti-CD19 CAR was assessed using specific anti-idyotipic Ab, followed by the staining with a secondary rat anti-Mouse Ab (PE, clone X56) from BD Biosciences. Data acquisition was performed on BD LSRFortessa or Canto H flow cytometer using the BD FACS-Diva software or on a MACSQuant (Miltenyi Biotec). Data analyses was performed with the FlowJo software (Version 9 or 10).
Transmission electron microscopy (TEM) of lentivirus Purified SINV-LV was incubated on a glow discharged CF300Cu grid. Excess sample was wicked away from the grid and rinsed with washing buffer (lx PBS). The grid was blocked in 1% w/v BSA-PBS, rinsed with washing buffer, and incubated with tFab (10 μg mL−1) at room temperature. Following another buffer rinse, secondary gold bead conjugated antibody (Abcam, Cat #ab39596) was incubated with the grid at a final stock dilution of 1:50 at room temperature. The grid was rinsed with washing buffer prior to addition of 4% PFA for fixation. Following a final buffer rinse, negative staining was performed. The grid was rinsed with DI water followed by addition of 1% uranyl acetate solution to the grid for 10 minutes. A final rinse with DI water was performed. The entire process took place in a 150×15 mm petri dish to prevent evaporation of solutions. Images were captured using an FEI Tecnai T12 transmission electron microscope at 120 kV.
Statistical analysis All data are presented as mean±SD. All graphs and statistical tests were performed using GraphPad Prism 7 or 8 software. Either a post hoc Tukey's test or Bonferroni correction was performed to correct for multiple comparisons after two-way ANOVA. Survival analysis was performed using the Kaplan-Meier method with a log-rank test to determine statistical significance. All p values less than 0.05 were considered statistically significant.
A chimeric bsAb was engineered against both (i) HER2 overexpressed on breast cancer cells and (ii) Sindbis Env glycoproteins displayed on LV. This was accomplished by merging human IgG1 backbones with HER2− and Sindbis envelope-binding VH and VL domains previously isolated from mouse IgG. Bispecific antibodies were prepared that bound either Sindbis Env glycoprotein E1 (responsible for pH-dependent endo-lysosomal membrane fusion and escape) or E2 domain (responsible for binding high-affinity laminin receptors or heparin sulfate for cellular entry) (
The specificity and affinity of the bsAb was confirmed using antigen-specific ELISAs against HER2. Both bispecific bsIgG1E2×HER2 and bsIgG1E1×HER2 possessed similar binding affinities to HER2 as the monoclonal anti-HER2 IgG1 (Trastuzumab; IgG1HER2 control): the KD for bsIgG1E2×HER2, bsIgG1E1×HER2, and IgG1HER2 were 0.32±0.05 nM, 0.26±0.02 nM, and 0.72±0.08 nM, respectively (
Using flow cytometry, the transduction efficiency of native, nontargeted WT and mSindbis lentiviruses expressing GFP in HER2+ SKBR3 cells was measured using a low vector-to-cell ratio (commonly referred to as multiplicity of infection, or MOI) of three. As expected, mSindbis had markedly lower transduction efficiency compared to WT Sindbis, transducing only ˜1% of target HER2+ cells vs ˜4% for WT Sindbis, with two-fold lower mean fluorescence intensity (MFI) than WT Sindbis (
Whether increasing the concentration of bsIgG1E2×HER2 could further enhance the transduction efficiency of both redirected LV was assessed next. At the highest bsIgG1E2×HER2 concentration tested, redirected WT Sindbis and mSindbis LV transduced ˜32% and ˜17% of SKBR3 cells, increasing the fraction of GFP+ SKBR3 cells by ˜10-fold and ˜22-fold, respectively, compared to their corresponding nontargeted LVs (
To assess whether bsIgG simply need to engage the LV or if efficient transduction is dependent on bsIgG binding to specific viral epitopes, the transduction potencies of LVs pre-mixed with bsIgG1E1×HER2 were evaluated in parallel. Interestingly, bsIgG1E1×HER2 did not improve the transduction efficiency of either LV at all, with comparable percentages of GFP+ cells and MFI of transduced cells to that of nontargeted LV alone (
To evaluate the specificity of bsAb-mediated LV for target cells relative to off-target cells, their transduction potencies on HER2+ (SKBR3) and HER2− (A2780) cells were separately compared, where A2780 represented a nonspecific cell control with little to no HER2 expression. As expected, a comparable transduction of HER2 cells with either WT and mSindbis LV alone (5% and 0.2% of A2780 cells, respectively) as with HER2+ cells (7% and 1.7% of SKBR3 cells, respectively) (
To further assess the specificity of gene transfer, bsIgG1E2×HER2-targeted LV were assessed for selectively transducing HER2+ cells in co-cultures of both HER2+ and HER2− cells. In good agreement with its broad transduction nature and results from mono-culture experiments, nontargeted WT Sindbis had very poor selectivity, transducing ˜8% of HER2+ cells (
For in vivo applications, FcRn recycling and non-specific uptake by Fc receptors on immune cells present a challenge for in vivo efficiency of targeted viral vectors via systemic administration. A Fc-free tandem Fab that similarly binds Sindbis E2 and HER2 (
The transduction efficiency of targeted LV with bsIgG1E2×HER2 versus tandem FabE2×HER2 using flow cytometry was compared. As expected, bsIgG1E2×HER2 transduced ˜5-fold more SKBR3 cells compared to WT Sindbis LV, and ˜10-fold vs mSindbis (
Adoptive transfer of CD19-specific CAR-T cells has demonstrated considerable success for the treatment of B cell malignancies in patients with relapsed or refractory diseases, providing the basis for three cell therapies approved by the U.S. Food and Drug Administration (FDA) to date. However, the generation of CAR-T cell products in all instances involves time consuming and complex manufacturing processes that delay the immediate availability of these cellular therapies for patients with aggressive disease, and also lead to exorbitant costs (
Direct in vivo engineering of CAR-T cells, based on transducing T cells circulating in the peripheral blood with viral vectors as described herein, may bypass the need for ex vivo manufacturing of patient-derived T cells entirely (
Bispecific binders in a tandem Fab format (tFab), comprised of two distinct Fab domains linked via a glycine-serine flexible linker and lacking the Fc antibody domain (
To evaluate the capacity of the SINV/tFab platform in targeting human T cells, SINV-LV encoding an eGFP fluorescent reporter transgene (denoted as SINV-GFP) were generated, mixed with different amounts of tFab, and the level of induced eGFP expression in a CD3 human cell line was quantified. A tFab dose-dependent transduction enhancement saturated at ˜50 nM concentration of tFab (
A second-generation CD19-specific CAR encoding the CD28 costimulatory endodomain was cloned into the SINV-LV (SINV-CAR;
The efficacy of the SINV-CAR/tFab vector system was evaluated in a xenograft mouse model (
The aggressive BV-173 B cell lymphoma model appears to result in accumulation and spread of tumor cells in the spleen: at the time of sacrifice, very enlarged spleens were discovered in mice treated with SINV-CAR alone (
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.
This application claims the benefit of U.S. Provisional Application No. 62/946,202, filed on Dec. 10, 2019, the contents of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/064340 | 12/10/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62946202 | Dec 2019 | US |
